Indian Journal of Health Sciences and Biomedical Research KLEU

: 2020  |  Volume : 13  |  Issue : 3  |  Page : 178--196

Biochemical features of blood vessel extracellular calcium-sensing receptor and their physiological mechanism of action in the regulation of vascular tone and blood pressure

Leta Melaku, Lemma Amsalu 
 Department of Biomedical Sciences, College of Health Sciences, Arsi University, Assela, Oromia, Ethiopia

Correspondence Address:
Mr. Leta Melaku
Department of Biomedical Sciences, College of Health Sciences, Arsi University, Assela, Oromia


The extracellular calcium-sensing receptor (CaR) is a G protein coupled receptor superfamily that has ability to sense very small changes in extracellular Ca2+ with primary physiological function of maintenance of constant blood Ca2+ levels. In addition to being expressed in tissues involved in [Ca2+]o homeostasis, it is also expressed in tissues not involved in mineral homeostasis, suggestive of additional physiological functions such as control vasomotor tone and blood pressure. However, the exact cellular mechanisms involved in these responses remain unclear. Stimulation of CaRs on perivascular nerves is proposed to evoke synthesis and release of nitric oxide (NO) from pre-synaptic terminals, which activate large conductance Ca2+-activated K+ channels in adjacent VSMCs that induce membrane hyperpolarisation and subsequent vasorelaxation. Plus, activation of CaRs on endothelial cells is reported to generate NO or vasoactive lipids or induce activation of intermediate conductance Ca2+-activated K+ channels to induce vasorelaxations. In contrast, CaR-induced activation of Gq and MAPK kinase-mediated pathways in VSMCs are linked to vasoconstriction and cell proliferation respectively. Taken together, these findings indicate that stimulation of vascular CaRs is reported to induce both constrictions and relaxations. In the endothelium intact artery, CaR mediated vasorelaxations are the dominant physiological phenomenon.

How to cite this article:
Melaku L, Amsalu L. Biochemical features of blood vessel extracellular calcium-sensing receptor and their physiological mechanism of action in the regulation of vascular tone and blood pressure.Indian J Health Sci Biomed Res 2020;13:178-196

How to cite this URL:
Melaku L, Amsalu L. Biochemical features of blood vessel extracellular calcium-sensing receptor and their physiological mechanism of action in the regulation of vascular tone and blood pressure. Indian J Health Sci Biomed Res [serial online] 2020 [cited 2021 Dec 1 ];13:178-196
Available from:

Full Text


Humphry Davy first recognized calcium as an element in 1808 and the name was given after the Latin for lime: calx.[1] Calcium is the fifth most abundant element, 1000 – 1200g, in adult human.[2] Approximately 99% of body calcium resides in the bones and teeth as the hydroxyapatite [Ca10(PO4)6(OH)2] crystal.[3],[4] From remaining 1%, about 0.9% is found intracellularly within the soft tissues and less than 0.1% is present in the ECF.[5] The normal serum calcium level is ~8.8 to 10.4 mg/dl (2.2 to 2.6 mmol/L)[6] and it is present in three forms: about 50% is free ionized,[7] about 10% is combined with various anions (bicarbonate, citrate, phosphate, lactate, and sulphate)[8] and the remaining 40% is bound to serum proteins, mainly serum albumin.[9] The importance of calcium ions in the regulation of physiological functions has been known since the 19th century.[10] The free ionized calcium is responsible for a variety of physiological processes including neuromuscular transmission, muscle contraction, cardiac automaticity, nerve function, hormone secretion, cell division and movement, intercellular adhesion, and cell motility and certain oxidative processes.[11],[12],[13],[14] It is also a co-factor for many steps during blood coagulation.[15] Calcium is also a major intracellular messenger in many intracellular responses to chemical and electrical stimuli and required by many enzymes for full activity.[16],[17] The extracellular fluid (ECF) ionized calcium (1.0 – 1.25 mmol/L) concentration is 10,000 times higher than the concentration of the intracellular fluid (ICF) ionized calcium.[18] Intracellular fluid ionized calcium is at low level because cell membrane permeability to Ca 2+ is low and cells also have powerful mechanisms for extrusion, sequestration and buffering of calcium.[16] Nevertheless, total calcium concentration in the cell interior varies during normal function by up to 10-fold (e. g. from 10-4 to 10-3 mmol/L).[19] The plasma ionized calcium concentration is tightly maintained mainly by action of three main calciotropic hormones (parathyroid hormone, calcitriol and calcitonin) [Figure 1] and other nutrients, most notably magnesium and phosphorus.[20],[21],[22]{Figure 1}

The way body maintains and regulates extracellular calcium levels is a complex puzzle that has intrigued researchers for decades.[13],[15],[23] One large piece of this puzzle that is falling into place concerns a cell-surface receptor that responds to, or “senses,” extracellular calcium-ion concentrations in parathyroid, kidney and other tissues. Experiments using a RIA to detect PTH levels in whole animals revealed that an increase in the level of free ionized calcium in the serum resulted in a decrease in PTH and that a decrease in serum Ca 2+ caused an increase in PTH levels.[24],[25] Further studies revealed that increase in extracellular Ca 2+ increased the production of both inositol 1, 4, 5 triphosphate and diacylglycerol, which are recognized as components associated with receptor-mediated intracellular Ca 2+ mobilization.[26],[27],[28]

Finally, Nemeth and Scarpa, using the fluorescent indicator fura-2, eloquently demonstrated that extracellular divalent cations evoked rapid and transient increases in intracellular calcium mobilization in bovine parathyroid cells in the absence of Ca 2+ transmembrane influx.[29] This observation, in conjunction with the others mentioned above, provided compelling evidence that there was a receptor at the cell surface of parathyroid cells sensitive to extracellular Ca 2+ that regulated PTH secretion through the mobilization of intracellular Ca 2+. In 1993, a receptor exhibiting molecular sensor of free ionized serum calcium, calcium-sensing receptor (CaSR/CaR) was first cloned from bovine parathyroid gland [30] and then from human parathyroid cells [31] as well as from rat kidney cells.[32] Detection of the CaR by Western blotting revealed that the receptor produced three different protein bands between 100 and 200 kDa that represent different monomeric forms of the CaR.[33],[34] The lowest band, at approximately 120 kDa, is a non-glycosylated form of the CaR that is expressed at a much lower level than the other two forms and is not always detected in Western blots. Two additional bands equivalent to molecular masses of approximately 130 – 140 and approximately 150 – 160 kDa correspond respectively to the immature form of CaR, which is glycosylated with carbohydrates containing high mannose content, and the mature form of the receptor, which is glycosylated with complex carbohydrates. Only the mature form of the receptor is expressed at the cell surface.[34],[35],[36] It has been demonstrated that the CaR is normally expressed at the cell surface as a homodimer, although there have been instances where the CaR has been detected in heterodimeric complexes with another receptor, such as the metabotropic glutamate receptor (mGluR).[34],[37],[38] Although the central role of the CaR is regulation of calcium homeostasis, it is also expressed in non-calciotropic tissues as well to regulate a multitude of cellular processes unrelated to mineral ion homeostasis.[39],[40],[41],[42]

 The Structural Biology of Calcium-Sensing Receptor

The gene encoding humans CaR is located on the 3q13.3 – 21 that consists of seven exons [Figure 2].[43],[44] It is under the control of two promoters (the upstream P1 containing a TATA box and a CAAT box, and the downstream P2, which is GC rich) that result in alternative transcripts of exon 1 (exon 1A and exon 1B). It appears that exon 1A has more impact on CaR expression than exon 1B.[45],[46],[47]{Figure 2}

The presence of the two promoter regions suggests tissue-specific CaR promoter regulation and alternatively spliced mRNA transcripts.[48] Exons 2 to 7 encode CaR amino acid.[49]

The human CaR consists of 1078 amino acid residues and belong to glutamate family C of the G protein-coupled receptors (GPCR) or 7 transmembrane receptors.[17],[42],[50] Family C also includes the metabotropic glutamate receptors, GABAB receptors, receptors for taste and pheromones, and an amino acid-and divalent cation-sensing receptor called GPRC6A.[51],[52]

Like all GPCR's, CaR is comprised of the three main structural domains: extracellular domain (ECD); seven transmembrane domain (7TMD); and intracellular carboxyl-terminal domain (ICD) [Figure 3][30],[53],[54] the G proteins, CaR also activates phosphatidylinositol{Figure 3}

The extracellular domain

Like other subfamilies of glutamate family C, CaR contains a very large extracellular domain, which covers 612 amino acids that includes two clusters of acidic amino acid residues that probably interact with Ca 2+ through electrostatic interactions.[55],[56],[57] From total 612 amino acids of ECD, amino acid residue 36-513 forms two adjacent monomeric lobes, lobe 1 and lobe 2, that are connected by two disulfide bonds involving cysteines 129 and 131 [Figure 4][22],[31],[58] and it's molecular modelling exhibits a bilobed, venus flytrap (VFT)-like structure [Figure 5].[55],[59],[60]{Figure 4}{Figure 5}

The ECD of CaR contains 11 potential N linked glycosylation sites on venus flytrap domain and a total of 19 cysteines, nine of them being highly conserved and located at the end of the ECD and constituting the so-called cysteine-rich domain [Figure 3] and [Figure 6].[61],[62],[63],[64] The eight conserved N linked glycosylation sites, Asn90, Asn130, Asn261, Asn287, Asn446, Asn468, Asn488, and Asn541, was demonstrated to be important for cell surface expression whereas the conserved cysteine rich domain is important for receptor dimerization, cell surface expression, and signal transduction from the corresponding venus flytrap domain to the seven-transmembrane domain.[62],[63] Immediately following the cysteine-rich domain are 14 amino acids, residues 599 – 612, that have been described as a peptide linker connecting the extracellular domain to the seven-transmembrane domain.[65] Insertion or deletion of amino acids within the peptide linker of the CaR had a negative impact on the cell surface expression of the receptor and abrogated its signaling capacity in response to extracellular ligands.[65] Recent findings have suggested that the CaR has at least two distinct types of motifs mediating dimerization and functional interactions: covalent interactions involving intermolecular disulfide bonds and non-covalent, possibly hydrophobic, interactions.[66]{Figure 6}

It has been proposed that the extracellular domain of the CaR contains the sites of various ligands thereby initiating conformational changes in the TMD and intracellular domains that initiate signal transduction.[67] Ca 2+ plays the role of first messenger for the CaR [17] and the main importance of extracellular domain of the CaR is highlighted by the fact that it controls the concentrations of extracellular free ionized Ca 2+ [Ca 2+o [Figure 3].[56],[68] Several groups have generated models to identify the Ca 2+-binding pocket. Both Huang and Millerl [69] and Silve et al.[70] have proposed that residues Ser147, Ser170, Asp190, Tyr218, and Glu297 are critical to the binding of Ca 2+ on the extracellular domain of CaR. In separate experiments, both groups performed mutational studies on the receptor that revealed that these five amino acids were positioned in a crevice between the two lobes of the extracellular domain and necessary for full CaR activity.[69],[70] It has been hypothesized that based on the Hill coefficient, three to five Ca 2+ ions can bind cooperatively to the extracellular calcium-sensing receptor.[71],[72],[73] Using the aforementioned strategy, Huang and Miller [69] also identified two additional Ca 2+-binding sites, one in lobe I, containing residues Glu378, Glu379, Thr396, Asp398, and Glu399, and the other in lobe II containing residues Glu224, Glu228, Glu229, Glu231, and Glu232. Therefore, CaR is sensitive to relatively small changes in Ca 2+ in extracellular fluid having a very high concentration of extracellular Ca 2+ (1.1 – 1.3 mM).[68] It has been proposed that the binding of calcium ions at the cleft between lobes in the extracellular domain induces rotation along the dimer interface. Such a conformational change could thus alter the spatial configuration of the membrane spanning portion of the receptor and modulate signal transduction.[74] The N-terminus of ECD also contains a signal peptide cleavage site.[75]

The transmembrane domain

The transmembrane domain (TMD) of the human CaR spans is formed from amino acid residues 613 – 862 and it includes seven hydrophobic regions, referred to as TM1 – TM7 [Figure 3] and [Figure 6].[31],[76] The TMD is central to the transduction of signals from the ECD into a cellular response.[73],[75],[77] It has been hypothesized that ligand binding to the extracellular domain of a CaR leads to conformational changes in TM6 and TM7 and, particularly, a proline residue (P823) in TM6 has been identified as a key structural element involved in calcium signaling.[77],[78],[79] Puzzling, it has been shown that a mutant CaR lacking the ECD also responds to [Ca 2+]o and other polyvalent cations, indicating that TMD also participates in sensing of [Ca 2+]o and other ligands.[76] These data indicate that the actual calcium-sensing domain of the CaR is complex and includes both the ECD and TMD.[44],[53]

In addition to its role in signal transduction, the transmembrane domain is believed to be involved in receptor dimerization through non-covalent interactions.[66] This is consistent with the identification of a consensus dimerization motif for non-covalent hydrophobic interactions in TM5 of the CaR, suggesting that TM5 may be a critical region for receptor dimerization.[80]

The intracellular tail

Unlike both extracellular and transmembrane domains, very few naturally occurring mutations have been identified in the 216 amino acids that comprise the intracellular tail of the CaR.[81],[82] The intracellular tail of the CaR is the least conserved domain between species.[51] However, there remain portions of the intracellular tail that are highly homologous between species, including a membrane proximal region spanning residues 863 – 925 and a region comprised of amino acids 960 – 984. The former has been shown to be essential to the cell surface expression and receptor's ability to initiate signaling cascades, whereas the latter has been shown to be involved in binding to accessory proteins.[82],[83],[84],[85] The nature of an intracellular pathway activated by the CaR depends markedly on the cell type in which the receptor is expressed.[53] Most studies on CaR signalling have been performed in parathyroid cells and human embryonic kidney (HEK-293) cells stably transfected with the CaR. Like other members of GPCRs, CaR acts mainly through G proteins.[73] Experiments using an antibody specific for the CaR phosphorylated at Thr888 revealed that an increase in extracellular Ca 2+ or acute treatment of the CaR with a calcimimetic increased phosphorylation of the CaR at Thr888, an effect ablated by treatment with a calcilytic.[86] It was also observed that after phosphorylation, the Thr888 residue could be dephosphorylated, a process that was inhibited in the presence of the phosphatase inhibitors calyculin or endothall thioanhydride, suggesting that protein phosphatase 2 is responsible for the dephosphorylation of the CaR.[86] This suggests that the CaR is able to activate PKC, which in turn phosphorylates the CaR, leading to inhibition of CaR activity, forming a negative feedback loop. In addition to the PKC phosphorylation sites, there are two predicted protein kinase A (PKA) phosphorylation sites, Ser899 and Ser900 [Figure 3] and [Figure 6], but inhibition studies suggest that PKA has only a minor role in the regulation of the CaR.[87] In human embryonic kidney (HEK-293) and bovine parathyroid cells, the CaR interacts with Gαq/11 subunits of heterotrimeric G proteins, resulting in activation of phospholipases C, A2 and D [Figure 7].[88],[89],[90]{Figure 7}

The CaR also interacts with a pertussis-toxin-sensitive inhibitory G protein, Gαi, which results in inhibition of adenylyl cyclase (AC).[91] In parallel independent of the G proteins, CaR also activates phosphatidylinositol 4-kinase (PI4K) that catalyses the first step of the inositol lipid biosynthesis by a Rho-dependent mechanism in various cell types.[92],[93],[94] Kifor et al.[95] demonstrated activation of extracellular-signal-regulated kinase (ERK) 1 and 2 in response to CaR agonist treatment in HEK-293 cells and in bovine parathyroid cells. In rat Leydig cancer (H-500) cells expressing CaR, calcium has been shown to activate ERK, p38 MAPK and Jun amino-terminal kinase (JNK).[96] In addition, the CaR has been shown to trans-activate epidermal growth factor receptor (EGFR) in rat Leydig cancer (H-500) cells, HEK-293 cells and human prostate cancer (PC3) cells.[97],[98],[99] Thus, MAPKs can be activated directly by the CaR or indirectly mediated by EGFR transactivation. Activation of MAPKs has been shown to be important for many distal effects of the CaR, such as proliferation, differentiation, regulation of peptide secretion and ion channel activity.[53],[90] Generally, upon changes in [ECF]o, several intracellular signaling pathways are affected, including the cAMP protein kinase A, phospholipase C-protein kinase C, and inositol phosphate pathways.

 Interacting Protein Partners of the Calcium-Sensing Receptor

As with all GPCRs families, the CaR signals through interacting with heterotrimeric G proteins. Recently, several other proteins have been also identified that interact with the CaR and influence its signaling characteristics.[69],[84],[100]


Filamin A was the first discovered protein interacts with CaR [83],[84] and the sites interaction was residues 962 – 981 of the CaR and amino acids 1566 – 1719 filamin A.[100] Silencing of the filamin A gene in HEK293 cells stably expressing the CaR with small interfering RNA (siRNA) caused a significant decrease in CaR-mediated JNK activation, indicating the necessity of the interaction between the CaR and filamin A for the induction of the JNK signaling cascade by the CaR.[101],[102],[103] Experiments that examined the expression levels of the CaR in M2 cells, a cell line that does not express filamin A, revealed that CaR expression was almost doubled when filamin A was transfected into the M2 cells.[100] Inhibition of CaR expression with CaR antisense cDNA 48 h after the transfection of CaR with or without filamin A resulted in a lower level of CaR expression in cells not expressing filamin A, suggesting that filamin A also protects the CaR against degradation.

Potassium channels

The inwardly rectifying potassium channel 4 family of proteins, consisting of Kir4.1 and Kir4.2, are believed to be involved in the regulation of membrane potential and the recycling of K + for Na +-K +-ATPases.[104] In addition to filamin A, the inwardly rectifying potassium channel Kir4.2 was identified as a binding partner for the CaR intracellular tail.[105] Because filamin A has been shown to interact with the potassium channel Kir2.1,[106] it is possible that filamin A may act as a scaffolding protein that brings the CaR and either Kir4.1 or Kir4.2 together.[73]


Dorfin was identified as another interacting protein partner of the CaR and sites interaction was residues 880 – 900 of the CaR and amino acids 660 – 838 of dorfin.[107] In HEK293 cells transiently expressing the CaR, an increase in the level of dorfin led to an increased rate of CaR degradation. Dorfin-mediated degradation of the CaR via proteasome.[73]

Coimmunoprecipitation experiments indicated that the CaR and dorfin are part of a protein complex that includes the valosin-containing protein, suggesting that the dorfin-mediated degradation of the CaR through the ER associated degradation pathway.[73]

Associated molecule with SH3 domain of signal-transducing adaptor molecule

AMSH is an ubiquitin isopeptidase that is a key regulatory component of endosomal sorting of the epidermal growth factor receptor.[108] A more recent study revealed AMSH as an interacting partner of the CaR.[109] A reduction in CaR expression was observed in response to the increasing levels of transfected AMSH.[110]


β-Arrestins are ubiquitously expressed proteins that are involved in the desensitization and internalization of most GPCRs including CaR into clathrin-coated pits.[111] Both β-arrestin 1 and 2 isoforms were found to interact with a region of the CaR intracellular tail between amino acids 877 and 1079 and they reduce the level of CaR activity.[112] Lorenz et al.[113] showed that overexpression of both β-arrestin 1 and 2 had a negative influence on CaR-mediated inositol phosphate production. It is interesting that aside from its important role in receptor desensitization, β-arrestin is one of at least three CaR-interacting protein partners (dorfin, AMSH, and filamin) that promote CaR degradation or stabilization of the receptor.[73] And control of CaR expression by these proteins may be regulated in a tissue-specific and/or situation specific manner.

Receptor activity-modifying proteins

The receptor activity-modifying protein (RAMP) family consists of single-transmembrane spanning proteins that have been shown to affect receptor trafficking, glycosylation, ligand specificity, and second messenger production in several cell types.[114] When the CaR was coexpressed with either RAMP1 or RAMP3, the receptor was delivered to the cell surface, but when coexpressed with RAMP2, the CaR was still not able to be detected at the cell surface.[115]

RAMP3 had a greater influence on the cell surface expression of the CaR than RAMP1, but both had similar effects on receptor trafficking and glycosylation of CaR.[115] Therefore, CaR-interacting partner protein can influence the functionality of the receptor in a tissue-specific manner.

 Unique Features of Extracellular Calcium-Sensing Receptor

There are several unique features that set CaR aside from other GPCRs. The CaR is a multifaceted receptor with multiple ligands, such as cations, metabolites, nutrients, and activates a plethora of signalling pathways [Table 1].[116],[117] As the receptor's name suggests, the principal physiological ligand of the CaR is calcium and it is the first G protein-coupled receptor found to have an inorganic ion, Ca 2+, as the first (extracellular) and second (intracellular) messengers.[17] Unique in comparison to many GPCR receptors, which are activated by nanomolar quantities of agonist, the CaR is somewhat resistant to desensitization and allows to detect very small fluctuations in the [Ca 2+]o at very high concentration (1.1 – 1.3 mM).[68] Another feature of the CaR is lack of specificity. Although the main ligand is Ca 2+, CaR is also stimulated in vitro by other divalent cations, such as magnesium and barium, by the trivalent elements, gadolinium and lanthanum, and by polycationic compounds, such as neomycin and spermine.[118] The affinity of cations to the CaR is variable and they rank as follows: La 3+ > Gd 3+ > Ca 2+ = Ba 2+ > Mg 2+.[71],[93] In vivo Mg 2+ is present at a sufficient concentration to activate CaR in an additive fashion and it also been shown to contribute to the regulation of CaR mRNA and protein expression.[17] However, the physiological concentrations of other cations are insufficient to activate CaR under normal circumstances and they contribute to heavy metal toxicity due to high affinity of CaR for them.[119],[120],[121],[122] In addition to these direct agonists, there are several agents, such as L-amino acids, polypeptides and endogenous polyamines, that can act as allosteric activators of the CaR.[53],[123] Amino acids have a greater influence on the CaR mediated mobilization of Ca 2+ from intracellular stores than on CaR-induced phosphorylation of ERK.[124] However, the physiological concentrations of endogenous polyamines, based on order of their potency: spermine > spermidine >> putrescine, are insufficient to activate CaR under normal circumstances.[125] This suggest that a modest rise in polyamines might contribute to CaR signaling physiologically.[126]{Table 1}

Amyloid-β peptide, which is excessively produced in the brain of patients with Alzheimer's disease, has also been shown to stimulate the CaR and is proposed to act on the receptor in a fashion similar to spermine because both molecules have a similar spacing of positive charges.[127] In a screen of a di-and tri-peptide library, the CaR was found to be activated by 46 different γ-glutamyl peptides, including γ-Glu-Cys-Gly and γ-Glu-Val-Gly, which have been categorized as taste-enhancing substances that can elicit the kokumi taste response.[128],[129] There are also many polyvalent aminoglycoside antibiotics that act via the CaR.[130] The order of potency of the aminoglycoside antibiotics tested is as follows: neomycin > tobramycin > gentamicin > kanamycin, suggesting that their efficacies positively correlate with the number of attached amino groups.[131] The affinity of CaR was not only influenced by ionic strength but also changes in pH, which suggests alterations in CaR agonist sensitivity by pH are partially the result of conformational changes to the receptor.[117] Therefore, in many cells and tissues, CaR also acts as a coincidence detector, sensing both changes in extracellular Ca 2+ plus the presence of various allosteric activators including amino acids, polyamines, and/or peptides. Such plasticity with respect to both agonist and signaling outcomes allows CaR to uniquely contribute to the physiology of organs and tissues where it is expressed.[22]

 The Role of Vascular Calcium Sensing Receptor in the Regulation of Vascular Tone and Blood Pressure

It is well known that calcium is an important second messenger that acts as an important regulator of vascular contractility.[53] Vascular tone and hence blood pressure are determined by the contractile state of vascular smooth muscle cells (VSMCs) within the blood vessel wall, which is regulated by intracellular calcium concentration ([Ca 2+]i).[132],[133] The major pathways for an increase in myocytes [Ca 2+]i are voltage-dependent Ca 2+ channels (VDCC) and nonselective cation channels at the plasmalemmal membrane or the internal store release channels, i. e., the ryanodine (RyR) and the inositol trisphosphate (IP3R) receptors found in the sarcoplasmic reticulum (SR) membrane.[134],[135] Therefore, vasoconstrictors act through increasing [Ca 2+]i as well as on the apparent calcium sensitivity of the contractile process in VSMCs, whereas relaxing factors have the opposite effect.[133],[134] The vascular endothelium is a cell monolayer that also plays a key role in the regulation of vasomotor tone through the release of vasoconstrictors (endothelin) and through the tonic production and release of NO, PGI2, and EDHF.[136] In contrast to the VSMCs, an increase in endothelial [Ca 2+]i results in vascular relaxation through endothelium-derived relaxing factors such as nitric oxide (NO), prostacyclin (PGI2), and endothelium-derived hyperpolarizing factor (EDHF).[133] These findings suggesting that the nature of calcium signals in endothelium and smooth muscle are fundamentally different.[137],[138] Therefore, an increase in [Ca 2+]i in endothelium or in VSMCs exerts opposing influences on blood vessel diameter for precise regulation of organ and tissue perfusion. However, recent studies suggest that, in addition to its many functions as an intracellular messenger, Ca 2+ may also be an extracellular first messenger through the calcium-sensing receptor (CaR) that are expressed in ECs,[139],[140],[141] VSMCs [142],[143] and on perivascular nerves [144],[145],[146] of blood vessels. In 1997, 4 years after the cloning of the CaR, Bukoski et al.[147] suggested that [Ca 2+]o induces relaxation of the isolated arteries by activating CaR in perivascular nerves, resulting in the release of a nerve-derived hyperpolarizing vasodilator, possibly a cannabinoid. However, Wang and Bukoski reported coronary arteries to contract at [Ca 2+]o levels of 1.5 – 3 mM, and relax only to a small degree with greater [Ca 2+]o concentrations (5 mM).[148] Likewise, in another studies binding of Ca 2+ ( first messenger) to CaR mediate biphasic effects with an initial endothelium-independent vasoconstrictions [149],[150],[151] followed by endothelium-dependent relaxations.[141],[152],[153]

An increase [Ca 2+]o from 1 mM to 6 mM potentiates pre-contracted tone in endothelium-removed vessel segments by affecting Gα protein subunits [17],[30],[154] In contrast, increasing [Ca 2+]o from 1 mM to 6 mM evokes the endothelial membrane hyperpolarization in a functionally intact endothelium by activating of potassium channels.[155],[156] Likewise, it also results in vascular relaxation through endothelium-derived relaxing factors such as nitric oxide (NO), prostacyclin (PGI2), and endothelium-derived hyperpolarizing factor (EDHF)[133],[141] Under physiological condition, CaR-mediate vasorelaxations more dominantly than vasoconstrictions [117],[157] In support of this hypothesis, increasing dietary calcium levels have been reported to have lowering effects on blood pressure in models of hypertension.[158] A calcimimetic, NPS R-568, has been reported to significantly decrease blood pressure in uremic and spontaneously hypertensive rats but not in normotensive rats [159],[160] Furthermore, it is speculated that CaR has non-trivial effects on vascular tone and blood pressure in response to systemic as well as local changes in [Ca 2+]o.[161]

 The Major Proposed Mechanisms of Vasodilation

The vascular endothelium CaR activation mostly results in an activation of the G proteins (Gq/11) that causes hydrolysis of membrane phospholipid PIP2 through the stimulation of phospholipase C (PLC) [Figure 8].[162] The stimulation of PLC results in generation of IP3, which promotes the release of Ca 2+ into the cytoplasm by activation of endoplasmic or sarcoplasmic reticulum IP3-gated calcium release channels.[163],[164],[165] Another molecule generated upon PLC activation, diaglycerol, provides the signals for activation of the serine/threonine kinase protein kinase C.[166] Due to the absence of VDCC, activation of PKC causes calcium influx into endothelial cells through transient receptor potential channels (TRPC1, TRPC3, TRPC4, TRPC6, TRPV4, etc.) from the extracellular milieu, which causes feature increase in cytosolic free Ca 2+ within endothelial cells.[156],[167],[168] In parallel, CaR also activates PI4K, which is an enzyme that carries out the first step in inositol lipids biosynthesis, independently of heterotrimeric G proteins, but by a Rho-dependent mechanism [Figure 8].[166] Endothelial calcium ''waves'' and “puffs” (small punctate and local increases of calcium) is an initial step required for endothelium-dependent vasorelaxations.{Figure 8}

One consequence of increases in [Ca 2+]i in vascular endothelium,300 – 500 nM, is the activation of calmodulin, which in turn activates the opening small and intermediate conductance calcium-activated potassium channels (SKCa and IKCa) on endothelium [Figure 9].[169] Opening of the small and intermediate conductance calcium-activated potassium channels pore is independent of the membrane potential but strictly depends on [Ca 2+]i. Thus, calcium sensitivity of both channels depends on CaM and the binding of calcium ions to the coupled calmodulin (CaM) induces a conformational change of the complex involving an interlacing of cytoplasmic loops that leads to the channel pore opening.[170],[171] Then after, intracellular K + moves down its electrochemical gradient through the opened SKCa and IKCa channels to the extracellular space and results in the hyperpolarization of the endothelial cells [Figure 9].[136] The hyperpolarization of the endothelial cells is transmitted to the smooth muscle cells by direct electrical coupling through myoendothelial junctions and/or by the accumulation of potassium ions in the intercellular myoendothelial space.[172]{Figure 9}

An efflux of K + in the lumen of the blood vessel from endothelium would be washed away by the flowing blood and be most likely without physiological consequences. However, the membrane potential hyperpolarization induced by SKCa and IKCa channels opening would further increase calcium influx in endothelial cells by increasing the electrochemical gradient for calcium.[133] An efflux of K + toward the abluminal side can accumulate in the intercellular space between endothelial and smooth muscle cells and when it reach sufficient levels (from ~4 mM to 12 mM) there will be activation of both inwardly rectifying potassium channel (Kir) and Na +/K + ATPase in the membrane of the vascular smooth muscle on the smooth muscle cells in the immediate vicinity of the endothelial cells releasing K +.[173],[174],[175] A unique feature of Kir channels is the effect of extracellular potassium on its gating. A moderate increase in potassium concentration, in the range of 1 to 15 mM, enhances potassium efflux through Kir at physiologically relevant potentials.[176],[177] Whereas, at the expense of ATP, Na +/K + ATPase exchanges three intracellular Na + s for two extracellular K + s.[178] Thus, the net result is loss of positively charged K + from vascular smooth muscle cells, which results in hyperpolarization and subsequent dilation of the artery [Figure 10].[169],[179],[180],[181],[182],[183] It is also possible that other K + channels such as Kv7 and KATP channels expressed in VSMCs may be involved in mediating CaR induced vasorelaxations.[184],[185]{Figure 10}

Although it has been known that gap junctions, the minute tunnels with internal diameter of 1.5nm, exist between endothelial cells and between vascular smooth muscle cells, they also seem to exist between endothelium and vascular smooth muscle (myoendothelial gap junctions).[186],[187],[188],[189] These myoendothelial gap junctions are thought to have a major role in electrical spread of hyperpolarization from the endothelial cells to the smooth muscle cells through myoendothelial gap junctions.[190],[191] Therefore, K + could be an EDHF or contribute to the mechanism of EDHF-mediated responses. In addition, it is well recognised that SKCa and IKCa channels that are expressed in ECs have pivotal roles in mediating endothelium-derived hyperpolarisations which couple to relaxation of VSMCs.[192],[193],[194],[195] In addition, the endothelial CaR can also modulate the diameter of blood vessels via another type of endothelium derived hyperpolarizing factor (EDHF).[136] The endothelial CaR activation via Gq subunits of heterotrimeric G proteins activation, also result in activation of phospholipases A2 (PLA2) that translocated from the cytoplasm to cellular membranes.[196] There is also evidence that CaR-induced activation of ERK can lead to the phosphorylation and activation of PLA2.[95] However, PKC activity is only partially responsible for CaR-mediated activation of PLA2.[162] Because PLA2 is constitutively active, the translocation to the membrane places it in contact with the phospholipid substrate and promotes the release of arachidonic acid within the cell.[164],[165],[197],[198]

The liberated arachidonic acid has several possible fates. It either reincorporated into plasma membrane phospholipids, where it can act as a messenger or metabolized further by cyclooxygenase, epoxygenase, lipoxygenase, or Ω-hydroxylase [Figure 11].[144],[145],[146]{Figure 11}

The first mechanism proposed for EDHF dilations involves the metabolism of arachidonic acid through the epoxygenase pathway to form epoxyeicosatrienoic acids (EETs).[199],[200],[201],[202] The EETs diffuse from the endothelium to the vascular smooth muscle and activate large conductance calcium-activated K + channel (BKCa) that results in further K + efflux from the smooth muscle cell and causes VSMC hyperpolarization and vasorelaxation [Figure 12].[203],[204],[205] In addition, the opening of the large-conductance Ca 2+-activated K + channel (BKCa) also promotes the closure of VDCC and thus opposing vasoconstriction.[133] Additionally, EETs activate smooth muscle vanilloid transient receptor potential channel (TRPV4), which increase the frequency of calcium sparks and subsequently that of spontaneous transient outward currents. The implication is that BKCa channels are clustered in a plasmalemmal region close to the RyR calcium SR release channels and so are exposed to a high concentration of store-released calcium early after SR calcium channel opening.[206],[207]{Figure 12}

In arterial smooth muscle and intact arteries, Ca 2+ sparks are observed just under the cell membrane consistent with a predominant subsarcolemmal localization of the RyR Ca 2+ release channels in the sarcoplasmic reticulum.[208],[209] This EET-dependent activation of a calcium-signaling complex (TRPV4-ryanodine receptors-BKCa) hyperpolarizes and relaxes the smooth muscle cells.[210] In addition, EETs may also regulate the activity of endothelial SKCa and IKCa.[211] Although EETs activate BKCa through a G protein–signaling cascade, the existence of a specific cell membrane receptor(s) for EETs that stimulate production of cAMP and activate protein kinase A that hyperpolarization through facilitate opening of both BKCa and KATP in vascular smooth muscle has been also established [Figure 12].[212],[213]

Furthermore, metabolism of arachidonic acid through the lipoxygenase pathway results in released of 12-(S)-HETE from the endothelium that also activates BKCa on the smooth muscle cells.[214],[215],[216] Prostacyclin is the major metabolite of arachidonic acid produced by cyclooxygenase in endothelial cells.[217] It activates IP3 receptors on vascular smooth muscle and, in most normal arteries, produces relaxation. Depending on the artery and/or the species, a hyperpolarization can occur, which involves the opening of one or more of types of potassium channels.[218] Thus, ATP-sensitive potassium channels (KATP), large conductance calcium-activated potassium channels (BKCa), inwardly rectifying potassium channels (Kir) and/or voltage activated potassium channels (KV) can be associated with the prostacyclin-induced relaxation.[219] In addition to transmitting state of hyperpolarization, myoendothelial gap junctions can also allow passage of small water soluble molecules (<1,000 Da) including cyclic guanosine monophosphate, inositol triphosphates, and inorganic ions, thus providing smooth muscle cell relaxation.[220],[221],[222],[223] The main source of NO• in the vasculature is the microvascular endothelium, from where it can contribute to cGMP activation in vascular smooth muscle cells and thus influence vascular reactivity.[224],[225] Endothelium-derived NO is synthesized by endothelial nitric oxide synthase (eNOS) which converts semi-essential amino acid L-arginine to L-citrulline and NO [Figure 13].[226],[227],[228],[229] The eNOS stimulation is due to the activation of calmodulin, as consequence of increases in [Ca 2+]i in vascular endothelium.[230] Thus, high [Ca 2+]i-induced eNOS upregulation is indeed CaR mediated.{Figure 13}

NO regulates vascular tone by different signaling pathways.[231],[232],[233] First, the classic NO-sGC-cGMP pathway vasodilator effects [Figure 14].[234],[235],[236] The NO• formed diffuses to underlying vascular smooth muscle, where its actions include stimulation of soluble guanylate cyclase (sGC), which induces formation of cyclic guanosine monophosphate (cGMP).[237],[238],[239] Cyclic GMP activates protein kinase G, which prevents the calcium influx from VDCC and calcium release mediated by IP3R.[233],[234] PKG also acts on sarco/endoplasmic reticulum calcium ATPase (SERCA) to promote the reuptake of cytosolic calcium into the sarcoplasmic reticulum (SR) and the opening of large conductance calcium activated potassium channel (BKCa) activation in vascular smooth muscle cells [Figure 14].[240],[241],[242] As a result, the intracellular concentration of calcium decreases and calmodulin is inactivated which no longer able to activate Myosin light chain kinase (MLCK). Calcium depletion also increases the activity of myosin light chain phosphatase (MLCP). The actin-myosin cross-bridge is broken and smooth muscle relaxation ensues.[243],[244],[245] Secondly, independent of the classic NO-sGC-cGMP pathway, NO• can undergo reactions, in the presence of an electron acceptor, with cysteine thiol containing compounds to form biologically active S-nitrosylated molecules.[246],[247],[248] Like PKG, NO• derived peroxynitrite (ONOO•) can directly acts on sarco/endoplasmic reticulum calcium ATPase (SERCA) to enhance the reuptake of cytosolic calcium into the sarcoplasmic reticulum by S-glutathiolation [Figure 14].[249] The S-nitrosylated molecules can also regulates the expression and functions of G protein coupled receptors (GPCRs).[250],[251] For instance, NO and S-nitrosothiols modulate the activity of GPCR kinase 2 (GRK2) which phosphorylates β-adrenoceptors and induce receptor desensitization and internalization to prevent the loss of β-adrenergic signaling in blood vessels to induce vasodilatation.[252],[253],[254] Likewise, S-nitrosoglutathione inhibits α1-adrenoceptor-mediated vasoconstriction and ligand binding.[255] In addition, S-nitrosylation of cysteine 289 of the AT1 receptor decreases its binding affinity for angiotensin II.[256] Cytosolic β-arrestin binding to CaR and GRK-phosphorylated GPCRs sterically impedes the interaction of G-proteins with activated GPCRs, resulting in GPCR signaling termination.[257],[258],[259] Also, β-arrestin 2, can be S-nitrosylated on cysteine 410 by endogenous NO and S-nitrosogluthathione, which promotes binding of β-arrestin 2 to clathrin heavy chain/β-adaptin, thereby accelerating receptor internalization and induces vasorelaxation.[233],[260]{Figure 14}

In generally, NO can also induces vasorelaxation by facilitating formation ofS-nitrosylated that has many functions:- firstly:-S-nitrosylation increases the activity of SERCA, which accelerates calcium depletion and induces relaxation. Secondly, GPCR can be directly S-nitrosylated by NO, which impedes the binding of ligands for the receptor or G-protein coupling. Thirdly, S-nitrosylation of GRK2 prevents the desensitization and internalization of β-adrenoceptors. Fourthly, S-nitrosylation of β-arrestin 2 increases receptor internalization.

Finally, the recent evidence suggests that the CaR is expressed in JG cells and that activation of the receptor decreases renin secretion, as well as Ang II, by suppressing the activity of the Ca 2+-inhibitable type V adenylate cyclase (AC-V),[261] and through activation of the PLC/IP3 pathway and the ryanodine receptor that results in stimulation of calcium/calmodulin by phosphodiesterases.[262],[263],[264]


Calcium is a crucial signal molecule that acts via changes in intracellular Ca 2+ levels through the actions of calcium channels and pumps. However, it is now well known that calcium may also be an extracellular first messenger through a G-protein-coupled receptor that senses extracellular Ca 2+ concentration, the calcium sensing receptor (CaR). It consists of three structural domains – a large extracellular domain in the N-terminal portion of the receptor, a seven-transmembrane region that anchors it in the plasma membrane and an intracellular carboxyl terminal tail with regulatory proteins phosphorylation sites. CaR function as a disulfide-linked dimer that likely can be driven to distinct active conformations by various Ca 2+ plus modulator combinations, which can drive preferential coupling to divergent signaling pathways. Binding of extracellular Ca 2+ or other ligands to the ECD of CaR triggers a number of intracellular signalling systems like the activation of Gαq proteins result in stimulation of phospholipase C, which leads to the generation of second messengers (DAG and IP3) and intracellular Ca 2+ release; inhibition of adenylate cyclase activity result in suppression of intracellular cAMP; activation of PKC and MAPK – p38, JNK/SAPK and MEK1/ERK1,2 etc., In addition to the G proteins, the CaR binds the scaffolding protein filamin A, G-protein coupled kinases (GRKs) and β-arrestins, which add the complexity of the downstream signalling mechanism of the receptor. Therefore, it is not surprising that CaR is involved in the regulation of such diverse processes as hormone secretion, gene expression, ion channel activity, modulation of inflammation, proliferation, differentiation and apoptosis. It is now evident that the presence of the CaR in animal blood vessels of many types, in perivascular nerves, endothelial cells and vascular smooth muscle cells, suggests it may regulate the vascular tone. This could provide a mechanism for the almost 100-year-old observation that [Ca 2+]o induces vasodilation. In particular, these novel results indicate that stimulation of CaRs induces endothelium-dependent vasorelaxations which are mediated by opening of the Ca 2+-sensitive potassium channels, NO production and inhibit renin production. All together, these results indicate that the CaR may have a physiological role in the modulation of blood pressure.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


1Ramon D, Thomas A. Handbook of Chemistry and Physics. 64th ed. Boca Raton, Florida: CRC Press; 1984. p. 21-46.
2Peacock M. Calcium metabolism in health and disease. Clin J Am Soc Nephrol 2010;5 Suppl 1:S23-30.
3Baker SB, Worthley LI. The essentials of calcium, magnesium and phosphate metabolism: Part I. Physiology. Crit Care Resusc 2002;4:301-6.
4Favus M, Bushinsky D, Lemann J. Regulation of calcium, magnesium, and phosphate metabolism. In: Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism. Ch. 13; 2006.
5Blaine J, Chonchol M, Levi M. Renal control of calcium, phosphate, and magnesium homeostasis. Clin J Am Soc Nephrol 2015;10:1257-72.
6Robertson WG, Marshall RW. Calcium measurements in serum and plasma--total and ionized. CRC Crit Rev Clin Lab Sci 1979;11:271-304.
7Moore E. Ionised calcium in normal serum, ultrafiltrates, and whole blood determined by ion exchange electrodes. J Clin Investig 1970;49:318-34.
8Toffaletti J, Gitelman HJ, Savory J. Separation and quantitation of serum constituents associated with calcium by gel filtration. Clin Chem 1976;22:1968-72.
9Toffaletti J, Savory J, Gitelman HJ. Use of gel filtration to examine the distribution of calcium among serum proteins. Clin Chem 1977;23:2306-10.
10Ringer S. A further contribution regarding the influence of the different constituents of the blood on the contraction of the heart. J Physiol 1883;4:29-42.
11Pietrobon D, Di Virgilio F, Pozzan T. Structural and functional aspects of calcium homeostasis in eukaryotic cells. Eur J Biochem 1990;193:599-622.
12Brown EM. Extracellular Ca2+ sensing, regulation of parathyroid cell function, and role of Ca2+ and other ions as extracellular ( first) messengers. Physiol Rev 1991;71:371-411.
13Kurokawa K. The kidney and calcium homeostasis. Kidney Int Suppl 1994;44:S97-105.
14Bushinsky DA, Monk RD. Electrolyte quintet: Calcium. Lancet 1998;352:306-11.
15Stewart A, Broadus A. Mineral metabolism. In: Felig P, Baxter JD, Broadus AE, Frohman LA, editors. Endocrinology and Metabolism. New York, USA: McGraw-Hill; 1987. p. 1317-453.
16Aguilera IM, Vaughan RS. Calcium and the anaesthetist. Anaesthesia 2000;55:779-90.
17Brown EM, MacLeod RJ. Extracellular calcium sensing and extracellular calcium signaling. Physiol Rev 2001;81:239-97.
18Carafoli E. The regulation of the cellular functions of Ca2+. In: Bronner F, Coburn JW, editors. Disorders of Mineral Metabolism. New York, USA: Academic Press; 1981. p. 1.
19Wacker W, Williams R. Magnesium/calcium balances and states of biological systems. J Theoretical Biol 1968;20:65-78.
20Carafoli E. The calcium-signalling saga: tap water and protein crystals. Nat Rev Mol Cell Biol 2003;4:326-32.
21Manoj P, Stephen T. Cations: Potassium, calcium, and magnesium. Continuing Education in Anaesthesia. Crit Care Pain 2012;12:195-8.
22Zhang C, Miller CL, Brown EM, Yang JJ. The calcium sensing receptor: From calcium sensing to signaling. Sci China Life Sci 2015;58:14-27.
23Austin LA, Heath H 3rd. Calcitonin: Physiology and pathophysiology. N Engl J Med 1981;304:269-78.
24Sherwood LM, Potts JT Jr., Care AD, Mayer GP, Aurbach GD. Evaluation by radioimmunoassay of factors controlling the secretion of parathyroid hormone. Nature 1966;209:52-5.
25López-Barneo J, Armstrong CM. Depolarizing response of rat parathyroid cells to divalent cations. J Gen Physiol 1983;82:269-94.
26Brown E, Enyedi P, LeBoff M, Rotberg J, Preston J, Chen C. High extracellular Ca-2+ and Mg-2+ stimulate accumulation of inositol phosphates in bovine parathyroid cells. FEBS Lett 1987;218:113-8.
27Kifor O, Brown E. Relationship between diacylglycerol levels and extracellular Ca-2+in dispersed bovine parathyroid cells. Endocrinology 1988;123:2723-9.
28Shoback DM, Membreno LA, McGhee JG. High calcium and other divalent cations increase inositol trisphosphate in bovine parathyroid cells. Endocrinology 1988;123:382-9.
29Nemeth E, Carafoli E. The role of extracellular calcium in the regulation of intracellular calcium and cellfunction–introduction. Cell Calcium 1990;11:319-21.
30Brown EM, Gamba G, Riccardi D, Lombardi M, Butters R, Kifor O, et al. Cloning and characterization of an extracellular Ca (2+)-sensing receptor from bovine parathyroid. Nature 1993;366:575-80.
31Garrett JE, Capuano IV, Hammerland LG, Hung BC, Brown EM, Hebert SC, et al. Molecular cloning and functional expression of human parathyroid calcium receptor cDNAs. J Biol Chem 1995;270:12919-25.
32Riccardi D, Park J, Lee WS, Gamba G, Brown EM, Hebert SC. Cloning and functional expression of a rat kidney extracellular calcium/polyvalent cation-sensing receptor. Proc Natl Acad Sci U S A 1995;92:131-5.
33Bai M, Quinn S, Trivedi S, Kifor O, Pearce S, Pollak M, et al. Expression and characterization of inactivating and activating mutations in the human Ca-0(2+)-sensing receptor. J Biol Chem 1996;271:19537-45.
34Bai M, Trivedi S, Brown EM. Dimerization of the extracellular calcium-sensing receptor (CaR) on the cell surface of CaR-transfected HEK293 cells. J Biol Chem 1998;273:23605-10.
35Mentaverri R, Yano S, Chattopadhyay N, Petit L, Kifor O, Kamel S, et al. The calcium sensing receptor is directly involved in both osteoclast differentiation and apoptosis. FASEB J 2006;20:2562-4.
36Brown EM. The calcium-sensing receptor: physiology, pathophysiology and CaR-based therapeutics. Subcell Biochem 2007;45:139-67.
37Gama L, Wilt SG, Breitwieser GE. Heterodimerization of calcium sensing receptors with metabotropic glutamate receptors in neurons. J Biol Chem 2001;276:39053-9.
38Chang W, Tu C, Chen TH, Bikle D, Shoback D. The extracellular calcium-sensing receptor (CaSR) is a critical modulator of skeletal development. Sci Signal 2008;1:ra1.
39Bruce JI, Yang X, Ferguson CJ, Elliott AC, Steward MC, Case RM, et al. Molecular and functional identification of a Ca2+ (polyvalent cation)-sensing receptor in rat pancreas. J Biol Chem 1999;274:20561-8.
40Rácz GZ, Kittel A, Riccardi D, Case RM, Elliott AC, Varga G. Extracellular calcium sensing receptor in human pancreatic cells. Gut 2002;51:705-11.
41Hodgkin MN, Hills CE, Squires PE. The calcium-sensing receptor and insulin secretion: a role outside systemic control 15 years on. J Endocrinol 2008;199:1-4.
42Ward DT, Riccardi D. New concepts in calcium-sensing receptor pharmacology and signalling. Br J Pharmacol 2012;165:35-48.
43Sarkar P, Kumar S. Tissue specific role of calcium sensing Receptor. Atlas Genet Cytogenet Oncol Haematol 2014;18:532-9.
44Tennakoon S, Aggarwal A, Kállay E. The calcium-sensing receptor and the hallmarks of cancer. Biochim Biophys Acta 2016;1863:1398-407.
45Chikatsu N, Fukumoto S, Takeuchi Y, Suzawa M, Obara T, Matsumoto T, et al. Cloning and characterization of two promoters for the human calcium-sensing receptor (CaSR) and changes of CaSR expression in parathyroid adenomas. J Biol Chem 2000;275:7553-7.
46Canaff L, Hendy GN. Human calcium-sensing receptor gene. Vitamin D response elements in promoters P1 and P2 confer transcriptional responsiveness to 1,25-dihydroxyvitamin D. J Biol Chem 2002;277:30337-50.
47Kállay E, Bonner E, Wrba F, Thakker RV, Peterlik M, Cross HS. Molecular and functional characterization of the extracellular calcium-sensing receptor in human colon cancer cells. Oncol Res 2003;13:551-9.
48Hendy GN, Canaff L, Cole DE. The CASR gene: Alternative splicing and transcriptional control, and calcium-sensing receptor (CaSR) protein: Structure and ligand binding sites. Best Pract Res Clin Endocrinol Metab 2013;27:285-301.
49Hendy GN, Canaff L. Calcium-sensing receptor gene: Regulation of expression. Front Physiol 2016;7:394.
50Aida K, Koishi S, Tawata M, Onaya T. Molecular cloning of a putative Ca(2+)-sensing receptor cDNA from human kidney. Biochem Biophys Res Commun 1995;214:524-9.
51Pin JP, Galvez T, Prézeau L. Evolution, structure, and activation mechanism of family 3/C G-protein-coupled receptors. Pharmacol Ther 2003;98:325-54.
52Bräuner-Osborne H, Wellendorph P, Jensen AA. Structure, pharmacology and therapeutic prospects of family C G-protein coupled receptors. Curr Drug Targets 2007;8:169-84.
53Smajilovic S, Tfelt-Hansen J. Calcium acts as a first messenger through the calcium-sensing receptor in the cardiovascular system. Cardiovasc Res 2007;75:457-67.
54D'Souza-Li L. Position of Mutations in the CASR, CaSR Database; 2015. Available from: 2&v=new. [Last accessed on 2019 Dec 16].
55Ray K, Hauschild B, Steinbach P, Goldsmith P, Hauache O, Spiegel A. Identification of the cysteine residues in the amino-terminal extracellular domain of the human Ca2+receptor critical for dimerization. Implications for function of monomeric Ca2+receptor. J Biol Chem 1999;274:27642-50.
56Hu J, Spiegel AM. Structure and function of the human calcium-sensing receptor: Insights from natural and engineered mutations and allosteric modulators. J Cell Mol Med 2007;11:908-22.
57Sharan K, Siddiqui JA, Swarnkar G, Chattopadhyay N. Role of calcium-sensing receptor in bone biology. Indian J Med Res 2008;127:274-86.
58Kunishima N, Shimada Y, Tsuji Y, Sato T, Yamamoto M, Kumasaka T, et al. Structural basis of glutamate recognition by a dimeric metabotropic glutamate receptor. Nature 2000;407:971-7.
59Goldsmith P, Fan G, Ray K, Shiloach J, McPhie P, Rogers K, et al. Expression, purification and biochemical characterization of the aminoterminal extracellular domain of the human calcium receptor. J Biol Chem 1999;274:11303-9.
60Khan M, Conigrave A. Mechanisms of multimodal sensing by extracellular Ca2+-sensing receptors: A domain-based survey of requirements for binding and signalling. Br J Pharmacol 2010;159:1039-50.
61Fan G, Goldsmith PK, Collins R, Dunn CK, Krapcho KJ, Rogers KV, et al. N-linked glycosylation of the human Ca2+ receptor is essential for its expression at the cell surface. Endocrinology 1997;138:1916-22.
62Ray K, Clapp P, Goldsmith PK, Spiegel AM. Identification of the sites of N-linked glycosylation on the human calcium receptor and assessment of their role in cell surface expression and signal transduction. J Biol Chem 1998;273:34558-67.
63Fan GF, Ray K, Zhao XM, Goldsmith PK, Spiegel AM. Mutational analysis of the cysteines in the extracellular domain of the human Ca2+receptor: Effects on cell surface expression, dimerization and signal transduction. FEBS Lett 1998;436:353-6.
64Hu J, Hauache O, Spiegel AM. Human Ca2+ receptor cysteine-rich domain. Analysis of function of mutant and chimeric receptors. J Biol Chem 2000;275:16382-9.
65Ray K, Adipietro KA, Chen C, Northup JK. Elucidation of the role of peptide linker in calcium-sensing receptor activation process. J Biol Chem 2007;282:5310-7.
66Zhang Z, Sun S, Quinn SJ, Brown EM, Bai M. The extracellular calcium-sensing receptor dimerizes through multiple types of intermolecular interactions. J Biol Chem 2001;276:5316-22.
67Reyes-Cruz G, Hu J, Goldsmith PK, Steinbach PJ, Spiegel AM. Human Ca (2+) receptor extracellular domain. Analysis of function of lobe I loop deletion mutants. J Biol Chem 2001;276:32145-51.
68Lopez-Fernandez I, Schepelmann M, Brennan SC, Yarova PL, Riccardi D. The calcium-sensing receptor: One of a kind. Exp Physiol 2015;100:1392-9.
69Huang C, Miller RT. The calcium-sensing receptor and its interacting proteins. J Cell Mol Med 2007;11:923-34.
70Silve C, Petrel C, Leroy C, Bruel H, Mallet E, Rognan D, et al. Delineating a Ca2+ binding pocket within the venus flytrap module of the human calcium-sensing receptor. J Biol Chem 2005;280:37917-23.
71Hofer AM, Brown EM. Extracellular calcium sensing and signalling. Nat Rev Mol Cell Biol 2003;4:530-8.
72Quinn SJ, Bai M, Brown EM. pH Sensing by the calcium-sensing receptor. J Biol Chem 2004;279:37241-9.
73Aaron L, Bryan K, Thomas R. The calcium-sensing receptor: A molecular perspective. Endocrine Rev 2011;32:3-30.
74Brown EM. Clinical utility of calcimimetics targeting the extracellular calcium-sensing receptor (CaSR). Biochem Pharmacol 2010;80:297-307.
75Hu J, Spiegel AM. Naturally occurring mutations of the extracellular Ca2+-sensing receptor: Implications for its structure and function. Trends Endocrinol Metab 2003;14:282-8.
76Ray K, Northup J. Evidence for distinct cation and calcimimetic compound (NPS 568) recognition domains in the transmembrane regions of the human Ca2+ receptor. J Biol Chem 2002;277:18908-13.
77Hu J, McLarnon SJ, Mora S, Jiang J, Thomas C, Jacobson KA, et al. A region in the seven-transmembrane domain of the human Ca2+ receptor critical for response to Ca2+. J Biol Chem 2005;280:5113-20.
78Wess J. G-protein-coupled receptors: Molecular mechanisms involved in receptor activation and selectivity of G-protein recognition. FASEB J 1997;11:346-54.
79Zhao X, Hauache O, Goldsmith P, Collins R, Spiegel A. A missense mutation in the seventh transmem brane domain constitutively activates the human Ca2+receptor. FEBS Lett 1999;448:180-4.
80Hebert T, Moffett S, Morello J, Loisel T, Bichet D, Barret C, et al. A peptide derived from alpha (2)-adrenergic receptor transmembrane domain inhibits both receptor dimerization and activation. J Biol Chem 1996;271:16384-92.
81Heath H 3rd, Odelberg S, Jackson CE, Teh BT, Hayward N, Larsson C, et al. Clustered inactivating mutations and benign polymorphisms of the calcium receptor gene in familial benign hypocalciuric hypercalcemia suggest receptor functional domains. J Clin Endocrinol Metab 1996;81:1312-7.
82Ray K, Fan GF, Goldsmith PK, Spiegel AM. The carboxyl terminus of the human calcium receptor. Requirements for cell-surface expression and signal transduction. J Biol Chem 1997;272:31355-61.
83Awata H, Huang C, Handlogten ME, Miller RT. Interaction of the calcium-sensing receptor and filamin, a potential scaffolding protein. J Biol Chem 2001;276:34871-9.
84Hjälm G, MacLeod RJ, Kifor O, Chattopadhyay N, Brown EM. Filamin-A binds to the carboxyl-terminal tail of the calcium-sensing receptor, an interaction that participates in CaR-mediated activation of mitogen-activated protein kinase. J Biol Chem 2001;276:34880-7.
85Chang W, Pratt S, Chen T, Bourguignon L, Shoback D. Amino acids in the cytoplasmic C terminus of the parathyroid Ca2+-sensing receptor mediate efficient cellsurface expression and phospholipase C activation. J Biol Chem 2001;276:44129-36.
86Davies SL, Ozawa A, McCormick WD, Dvorak MM, Ward DT. Protein kinase C-mediated phosphorylation of the calcium-sensing receptor is stimulated by receptor activation and attenuated by calyculin-sensitive phosphatase activity. J Biol Chem 2007;282:15048-56.
87Bösel J, John M, Freichel M, Blind E. Signaling of the human calcium-sensing receptor expressed in HEK293-cells is modulated by protein kinases A and C. Exp Clin Endocrinol Diabetes 2003;111:21-6.
88Kifor O, Diaz R, Butters R, Brown EM. The Ca2+-sensing receptor (CaR) activates phospholipases C, A2, and D in bovine parathyroid and CaR-transfected, human embryonic kidney (HEK293) cells. J Bone Miner Res 1997;12:715-25.
89Handlogten M, Huang C, Shiraishi N, Awata H, Miller R. The Ca2+-sensing receptor activates cytosolic phospholipase A2 via a Gq alpha dependent ERK-independent pathway. J Biol Chem 2001;276:13941-8.
90Colella M, Gerbino A, Hofer A, Curci S. How to cite this article: Recent advances in understanding the extracellular calcium-sensing. Faculty Rev 2016;F1000:25-35.
91Brennan SC, Mun HC, Leach K, Kuchel PW, Christopoulos A, Conigrave AD. Receptor expression modulates calcium-sensing receptor mediated intracellular Ca2+mobilization. Endocrinology 2015;156:1330-42.
92Huang C, Handlogten ME, Miller RT. Parallel activation of phosphatidylinositol 4-kinase and phospholipase C by the extracellular calcium-sensing receptor. J Biol Chem 2002;277:20293-300.
93Tfelt-Hansen J, Brown EM. The calcium-sensing receptor in normal physiology and pathophysiology: A review. Crit Rev Clin Lab Sci 2005;42:35-70.
94Tfelt-Hansen J, Hansen JL, Smajilovic S, Terwilliger EF, Haunso S, Sheikh SP. Calcium receptor is functionally expressed in rat neonatal ventricular cardiomyocytes. Am J Physiol Heart Circ Physiol 2006;290:H1165-71.
95Kifor O, MacLeod RJ, Diaz R, Bai M, Yamaguchi T, Yao T, et al. Regulation of MAP kinase by calcium-sensing receptor in bovine parathyroid and CaR-transfected HEK293 cells. Am J Physiol Renal Physiol 2001;280:F291-302.
96Tfelt-Hansen J, MacLeod RJ, Chattopadhyay N, Yano S, Quinn S, Ren X, et al. Calcium-sensing receptor stimulates PTHrP release by pathways dependent on PKC, p38 MAPK, JNK, and ERK1/2 in H-500 cells. Am J Physiol Endocrinol Metab 2003;285:E329-37.
97Yano S, Macleod RJ, Chattopadhyay N, Tfelt-Hansen J, Kifor O, Butters RR, et al. Calcium-sensing receptor activation stimulates parathyroid hormone-related protein secretion in prostate cancer cells: Role of epidermal growth factor receptor transactivation. Bone 2004;35:664-72.
98MacLeod RJ, Yano S, Chattopadhyay N, Brown EM. Extracellular calcium-sensing receptor transactivates the epidermal growth factor receptor by a triple-membrane-spanning signaling mechanism. Biochem Biophys Res Commun 2004;320:455-60.
99Tfelt-Hansen J, Yano S, John Macleod R, Smajilovic S, Chattopadhyay N, Brown EM. High calcium activates the EGF receptor potentially through the calcium-sensing receptor in Leydig cancer cells. Growth Factors 2005;23:117-23.
100Zhang M, Breitwieser GE. High affinity interaction with filamin A protects against calcium-sensing receptor degradation. J Biol Chem 2005;280:11140-6.
101Pi M, Spurney RF, Tu Q, Hinson T, Quarles LD. Calcium-sensing receptor activation of rho involves filamin and rho-guanine nucleotide exchange factor. Endocrinology 2002;143:3830-8.
102Rey O, Young S, Yuan J, Slice L, Rozengurt E. Amino acid-stimulated Ca2+ oscillations produced by the Ca2+-sensing receptor are mediated by a phospholipase C/inositol 1,4,5-trisphosphate-independent pathway that requires G (12), Rho, filamin-A, and the actin cytoskeleton. J Biol Chem 2005;280:22875-82.
103Huang C, Wu Z, Hujer KM, Miller RT. Silencing of filamin A gene expression inhibits Ca2+-sensing receptor signaling. FEBS Lett 2006;580:1795-800.
104Lourdel S, Paulais M, Cluzeaud F, Bens M, Tanemoto M, Kurachi Y, et al. An inward rectifier K(+) channel at the basolateral membrane of the mouse distal convoluted tubule: Similarities with Kir4-Kir5.1 heteromeric channels. J Physiol 2002;538:391-404.
105Huang C, Sindic A, Hill CE, Hujer KM, Chan KW, Sassen M, et al. Interaction of the Ca2+-sensing receptor with the inwardly rectifying potassium channels Kir4.1 and Kir4.2 results in inhibition of channel function. Am J Physiol Renal Physiol 2007;292:F1073-81.
106Sampson LJ, Leyland ML, Dart C. Direct interaction between the actin-binding protein filamin-A and the inwardly rectifying potassium channel, Kir2.1. J Biol Chem 2003;278:41988-97.
107Huang Y, Niwa J, Sobue G, Breitwieser GE. Calcium-sensing receptor ubiquitination and degradation mediated by the E3 ubiquitin ligase dorfin. J Biol Chem 2006;281:11610-7.
108McCullough J, Clague MJ, Urbé S. AMSH is an endosome-associated ubiquitin isopeptidase. J Cell Biol 2004;166:487-92.
109Herrera-Vigenor F, Hernández-García R, Valadez-Sánchez M, Vázquez-Prado J, Reyes-Cruz G. AMSH regulates calcium-sensing receptor signaling through direct interactions. Biochem Biophys Res Commun 2006;347:924-30.
110Reyes-Ibarra AP, García-Regalado A, Ramírez-Rangel I, Esparza-Silva AL, Valadez-Sánchez M, Vázquez-Prado J, et al. Calcium-sensing receptor endocytosis links extracellular calcium signaling to parathyroid hormone-related peptide secretion via a Rab11a-dependent and AMSH-sensitive mechanism. Mol Endocrinol 2007;21:1394-407.
111DeWire S, Ahn S, Lefkowitz R, Shenoy S. β-Arrestins and cell signaling. Annu Rev Physiol 2007;69:483-510.
112Pi M, Oakley R, Gesty-Palmer D, Cruickshank R, Spurney R, Luttrell L, et al. β-Arrestin and G protein receptor kinase-mediated calcium-sensing receptor desensitization. Mol Endocrinol 2005;19:1078-87.
113Lorenz S, Frenzel R, Paschke R, Breitwieser G, Miedlich S. Functional desensitization of the extracellular calcium-sensing receptor is regulated via distinct mechanisms: role of G protein-coupled receptor kinases, protein kinase C and β-arrestins. Endocrinology 2007;148:2398-404.
114Morfis M, Christopoulos A, Sexton PM. RAMPs: 5 years on, where to now? Trends Pharmacol Sci 2003;24:596-601.
115Bouschet T, Martin S, Henley JM. Receptor-activity-modifying proteins are required for forward trafficking of the calcium-sensing receptor to the plasma membrane. J Cell Sci 2005;118:4709-20.
116Miedlich SU, Gama L, Seuwen K, Wolf RM, Breitwieser GE. Homology modeling of the transmembrane domain of the human calcium sensing receptor and localization of an allosteric binding site. J Biol Chem 2004;279:7254-63.
117Smajilovic S, Tfelt-Hansen J. Novel role of the calcium-sensing receptor in blood pressure modulation. Hypertension 2008;52:994-1000.
118Coburn JW, Elangovan L, Goodman WG, Frazaõ JM. Calcium-sensing receptor and calcimimetic agents. Kidney Int Suppl 1999;73:S52-8.
119Ikaria A, Nakajima K, Kawano K, Suketa Y. Polyvalent cation sensing mechanism increases Na +- independent Mg2+ transport in renal epithelial cells. Biochem Biophys Res Commun 2001;287:671-4.
120Faurskov B, Bjerregaard HF. Evidence for cadmium mobilization of intracellular calcium through a divalent cation receptor in renal distal epithelial A6 cells. Eur J Physiol 2002;445:40-50.
121McLarnon SJ, Riccardi D. Physiological and pharmacological agonists of the extracellular Ca2+-sensing receptor. Eur J Pharmacol 2002;447:271-8.
122Brown EM. Is the calcium receptor a molecular target for the actions of strontium on bone? Osteoporos Int 2003;14 Suppl 3:S25-34.
123Mun H, Culverston E, Franks A, Collyer C, Clifton Bligh R, Conigrave A. A double mutation in the extracellular Ca2+-sensing receptor's Venus flytrap domain that selectively disables L-amino acid sensing. J Biol Chem 2005;280:29067-72.
124Lee HJ, Mun HC, Lewis NC, Crouch MF, Culverston EL, Mason RS, et al. Allosteric activation of the extracellular Ca2+-sensing receptor by L-amino acids enhances ERK1/2 phosphorylation. Biochem J 2007;404:141-9.
125Quinn SJ, Ye CP, Diaz R, Kifor O, Bai M, Vassilev P, et al. The Ca2+-sensing receptor: A target for polyamines. Am J Physiol 1997;273:C1315-23.
126Riccardi D. Wellcome Prize Lecture. Cell surface, ion-sensing receptors. Exp Physiol 2002;87:403-11.
127San Gabriel A, Uneyama H, Maekawa T, Torii K. The calcium-sensing receptor in taste tissue. Biochem Biophys Res Commun 2009;378:414-8.
128Bystrova MF, Romanov RA, Rogachevskaja OA, Churbanov GD, Kolesnikov SS. Functional expression of the extracellular-Ca2+-sensing receptor in mouse taste cells. J Cell Sci 2010;123:972-82.
129Ohsu T, Amino Y, Nagasaki H, Yamanaka T, Takeshita S, Hatanaka T, et al. Involvement of the calcium-sensing receptor in human taste perception. J Biol Chem 2010;285:1016-22.
130McLarnon S, Holden D, Ward D, Jones M, Elliott A, Riccardi D. Aminoglycoside antibiotics induce pH-sensitive activation of the calcium-sensing receptor. Biochem Biophys Res Commun 2002;297:71-7.
131White E, McKenna J, Cavanaugh A, Breitwieser GE. Pharmacochaperone-mediated rescue of calcium-sensing receptor loss-of-function mutants. Mol Endocrinol 2009;23:1115-23.
132Hai CM, Murphy RA. Ca2+, crossbridge phosphorylation, and contraction. Annu Rev Physiol 1989;51:285-98.
133Ledoux J, Werner ME, Brayden JE, Nelson MT. Calcium-activated potassium channels and the regulation of vascular tone. Physiology (Bethesda) 2006;21:69-78.
134Boittin F, Macrez N, Halet G, Mironneau J. Norepinephrine-induced Ca2+ waves depend on InsP3 and ryanodine receptor activation in vascular myocytes. Am J Physiol Cell Physiol 1999;277:C139-51.
135Kasai Y, Yamazawa T, Sakurai T, Taketani Y, Iino M. Endothelium-dependent frequency modulation of Ca2+ signaling in individual vascular smooth muscle cells of the rat. J Physiol 1997;504:349-57.
136Haddy FJ, Vanhoutte PM, Feletou M. Role of potassium in regulating blood flow and blood pressure. Am J Physiol Regul Integr Comp Physiol 2006;290:R546-52.
137Watanabe H, Vriens J, Prenen J, Droogmans G, Voets T, Nilius B. Anandamide and arachidonic acid use epoxyeicosatrienoic acids to activate TRPV4 channels. Nature 2003;424:434-8.
138Ahmmed GU, Malik AB. Functional role of TRPC channels in the regulation of endothelial permeability. Pflugers Arch 2005;451:131-42.
139Romani B, Raqeeb A, Laforenza U, Scaffino M, Moccia F, Avelino-Cruz J. Cardiac microvascular endothelial cells express a functional Ca2+-sensing receptor. J Vasc Res 2009;46:73-82.
140Bonomini M, Giardinelli A, Morabito C, Di Silvestre S, Di Cesare M, Di Pietro N, et al. Calcimimetic R-568 and its enantiomer S-568 increase nitric oxide release in human endothelial cells. PLoS One 2012;7:e30682.
141Loot AE, Pierson I, Syzonenko T, Elgheznawy A, Randriamboavonjy V, Zivković A, et al. Ca2+-sensing receptor cleavage by calpain partially accounts for altered vascular reactivity in mice fed a high-fat diet. J Cardiovasc Pharmacol 2013;61:528-35.
142Molostvov G, Fletcher S, Bland R, Zehnder D. Extracellular calcium-sensing receptor mediated signalling is involved in human vascular smooth muscle cell proliferation and apoptosis. Cell Physiol Biochem 2008;22:413-22.
143Li GW, Xing WJ, Bai SZ, Hao JH, Guo J, Li HZ, et al. The calcium-sensing receptor mediates hypoxia-induced proliferation of rat pulmonary artery smooth muscle cells through MEK1/ERK1,2 and PI3K pathways. Basic Clin Pharmacol Toxicol 2011;108:185-93.
144Alam MU, Kirton JP, Wilkinson FL, Towers E, Sinha S, Rouhi M, et al. Calcification is associated with loss of functional calcium-sensing receptor in vascular smooth muscle cells. Cardiovasc Res 2009;81:260-8.
145Chow J, Estrema C, Orneles T, Dong X, Barrett K, Dong H. Calcium-sensing receptor modulates extracellular Ca2+ entry via TRPC-encoded receptor-operated channels in human aortic smooth muscle cells. Am J Physiol Cell Physiol 2011;301:C461-8.
146Yamamura A, Guo Q, Yamamura H, Zimnicka AM, Pohl NM, Smith KA, et al. Enhanced Ca(2+)-sensing receptor function in idiopathic pulmonary arterial hypertension. Circ Res 2012;111:469-81.
147Bukoski RD, Bian K, Wang Y, Mupanomunda M. Perivascular sensory nerve Ca2+receptor and Ca2+-induced relaxation of isolated arteries. Hypertension 1997;30:1431-9.
148Wang Y, Bukoski RD. Distribution of the perivascular nerve Ca2+ receptor in rat arteries. Br J Pharmacol 1998;125:1397-404.
149Ohanian J, Gatfield KM, Ward DT, Ohanian V. Evidence for a functional calcium-sensing receptor that modulates myogenic tone in rat subcutaneous small arteries. Am J Physiol Heart Circ Physiol 2005;288:H1756-62.
150Li GW, Wang QS, Hao JH, Xing WJ, Guo J, Li HZ, et al. The functional expression of extracellular calcium-sensing receptor in rat pulmonary artery smooth muscle cells. J Biomed Sci 2011;18:16.
151Thakore P, Ho W. Vascular actions of calcimimetics: role of Ca2+-sensing receptors versus Ca2+influx through L-type Ca2+ channels. Br J Pharmacol 2011;162:749-62.
152Weston AH, Absi M, Harno E, Geraghty AR, Ward DT, Ruat M, et al. The expression and function of Ca (2+)-sensing receptors in rat mesenteric artery; comparative studies using a model of type II diabetes. Br J Pharmacol 2008;154:652-62.
153Awumey E, Bridges L, Williams C, Diz D. Nitric-oxide synthase knockout modulates Ca2+-sensing receptor expression and signalling in mouse mesenteric arteries. J Pharmacol Exp Ther 2013;346:38-47.
154Molostvov G, James S, Fletcher S, Bennett J, Lehnert H, Bland R, et al. Extracellular calcium-sensing receptor is functionally expressed in human artery. Am J Physiol Renal Physiol 2007;293:F946-55.
155Mulvany MJ, Halpern W. Contractile properties of small arterial resistance vessels in spontaneously hypertensive and normotensive rats. Circ Res 1977;41:19-26.
156Jackson WF. Potassium channels in the peripheral microcirculation. Microcirculation 2005;12:113-27.
157Greenberg HZ, Shi J, Jahan KS, Martinucci MC, Gilbert SJ, Vanessa Ho WS, et al. Stimulation of calcium-sensing receptors induces endothelium-dependent vasorelaxations via nitric oxide production and activation of IKCa channels. Vascul Pharmacol 2016;80:75-84.
158Hatton DC, McCarron DA. Dietary calcium and blood pressure in experimental models of hypertension. A review. Hypertension 1994;23:513-30.
159Ogata H, Ritz E, Odoni G, Amann K, Orth SR. Beneficial effects of calcimimetics on progression of renal failure and cardiovascular risk factors. J Am Soc Nephrol 2003;14:959-67.
160Odenwald T, Nakagawa K, Hadtstein C, Roesch F, Gohlke P, Ritz E, et al. Acute blood pressure effects and chronic hypotensive action of calcimimetics in uremic rats. J Am Soc Nephrol 2006;17:655-62.
161Ziegelstein RC, Xiong Y, He C, Hu Q. Expression of a functional extracellular calcium-sensing receptor in human aortic endothelial cells. Biochem Biophys Res Commun 2006;342:153-63.
162Handlogten ME, Huang C, Shiraishi N, Awata H, Miller RT. The Ca2+-sensing receptor activates cytosolic phospholipase A2 via a Gqalpha -dependent ERK-independent pathway. J Biol Chem 2001;276:13941-8.
163Fukao M, Hattori Y, Kanno M, Sakuma I, Kitabatake A. Sources of Ca2+ in relation to generation of acetylcholine-induced endothelium-dependent hyperpolarization in rat mesenteric artery. Br J Pharmacol 1997;120:1328-34.
164Hutcheson IR, Chaytor AT, Evans WH, Griffith TM. Nitric oxide-independent relaxations to acetylcholine and A23187 involve different routes of heterocellular communication. Role of Gap junctions and phospholipase A2. Circ Res 1999;84:53-63.
165You J, Marrelli SP, Bryan RM Jr. Role of cytoplasmic phospholipase A2 in endothelium-derived hyperpolarizing factor dilations of rat middle cerebral arteries. J Cereb Blood Flow Metab 2002;22:1239-47.
166Smajilovic S, Yano S, Jabbari R, Tfelt-Hansen J. The calcium-sensing receptor and calcimimetics in blood pressure modulation. Br J Pharmacol 2011;164:884-93.
167Taylor HJ, Chaytor AT, Edwards DH, Griffith TM. Gap junction-dependent increases in smooth muscle cAMP underpin the EDHF phenomenon in rabbit arteries. Biochem Biophys Res Commun 2001;283:583-9.
168Tomioka H, Hattori Y, Fukao M, Watanabe H, Akaishi Y, Sato A, et al. Role of endothelial Ni2 -sensitive Ca2+ entrypathway in regulation of EDHF in porcine coronary artery. Am J Physiol Heart Circ Physiol 2001;280:H730-7.
169Edwards G, Dora KA, Gardener MJ, Garland CJ, Weston AH. K+is an endothelium-derived hyperpolarizing factor in rat arteries. Nature 1998;396:269-72.
170Ishii TM, Silvia C, Hirschberg B, Bond CT, Adelman JP, Maylie J. A human intermediate conductance calcium-activated potassium channel. Proc Natl Acad Sci U S A 1997;94:11651-6.
171Bond CT, Maylie J, Adelman JP. Small-conductance calcium-activated potassium channels. Ann N Y Acad Sci 1999;868:370-8.
172Busse R, Edwards G, Félétou M, Fleming I, Vanhoutte PM, Weston AH. EDHF: Bringing the concepts together. Trends Pharmacol Sci 2002;23:374-80.
173Johnson TD, Marrelli SP, Steenberg ML, Childres WF, Bryan RM Jr. Inward rectifier potassium channels in the rat middle cerebral artery. Am J Physiol 1998;274:R541-7.
174Marrelli SP, Johnson TD, Khorovets A, Childres WF, Bryan RM Jr. Altered function of inward rectifier potassium channels in cerebrovascular smooth muscle after ischemia/reperfusion. Stroke 1998;29:1469-74.
175Weston A, Richards G, Burnham M, Feletou M, Vanhoutte P, Edwards G. K+-induced hyperpolarization in rat mesenteric artery: Identification, localization and role of Na +/K + -ATPases. Br J Pharmacol 2002;136:918-26.
176Knot H, Zimmermann P, Nelson M. Extracellular potassium induced hyperpolarization and dilatations of rat coronary and cerebral arteries involve inward rectifier potassium channels. J Physiol 1996;492:419-30.
177Quignard J, Harley E, Duhault J, Vanhoutte P, Feletou M. K + channels in bovine retinal pericytes: Effects of -adrenergic stimulation and elevated glucose. J Cardiovasc Pharmacol 2003;42:379-88.
178Edwards G, Richards G, Gardener M, Feletou M, Vanhoutte P, Weston A. Role of the inward-rectifier K + channel and Na +/K +-ATPase in the hyperpolarization to K in rat mesenteric arteries. In: Vanhoutte PM, editor. EDHF 2002. London, UK: Taylor and Francis; 2003. p. 309-17.
179Doughty JM, Plane F, Langton PD. Charybdotoxin and apamin block EDHF in rat mesenteric artery if selectively applied to the endothelium. Am J Physiol 1999;276:H1107-12.
180Burnham M, Bychkov R, Feletou M, Richards G, Vanhoutte P, Weston A, et al. Characterization of an apamin-sensitive small-conductance Ca2+-activated K+channel in porcine coronary artery endothelium: Relevance to EDHF. Br J Pharmacol 2002;135:1133-43.
181Bychkov R, Burnham MP, Richards GR, Edwards G, Weston AH, Félétou M, et al. Characterization of a charybdotoxin-sensitive intermediate conductance Ca2+-activated K+channel in porcine coronary endothelium: Relevance to EDHF. Br J Pharmacol 2002;137:1346-54.
182Eichler I, Wibawa J, Grgic I, Knorr A, Brakemeier S, Pries AR, et al. Selective blockade of endothelial Ca2+-activated small- and intermediate-conductance K +-channels suppresses EDHF-mediated vasodilation. Br J Pharmacol 2003;138:594-601.
183Marrelli SP, Eckmann MS, Hunte MS. Role of endothelial intermediate conductance KCa channels in cerebral EDHF-mediated dilations. Am J Physiol Heart Circ Physiol 2003;285:H1590-9.
184Brayden JE. Functional roles of KATP channels in vascular smooth muscle. Clin Exp Pharmacol Physiol 2002;29:312-6.
185Jepps TA, Olesen SP, Greenwood IA. One man's side effect is another man's therapeutic opportunity: Targeting Kv7 channels in smooth muscle disorders. Br J Pharmacol 2013;168:19-27.
186Christ GJ, Spray DC, el-Sabban M, Moore LK, Brink PR. Gap junctions in vascular tissues. Evaluating the role of intercellular communication in the modulation of vasomotor tone. Circ Res 1996;79:631-46.
187Emerson GG, Segal SS. Electrical coupling between endothelial cells and smooth muscle cells in hamster feed arteries: Role in vasomotor control. Circ Res 2000;87:474-9.
188Sandow SL, Hill CE. Incidence of myoendothelial gap junctions in the proximal and distal mesenteric arteries of the rat is suggestive of a role in endothelium-derived hyperpolarizing factor-mediated responses. Circ Res 2000;86:341-6.
189Emerson GG, Segal SS. Electrical activation of endothelium evokes vasodilation and hyperpolarization along hamster feed arteries. Am J Physiol Heart Circ Physiol 2001;280:H160-7.
190Coleman HA, Tare M, Parkington HC. K + currents underlying the action of endothelium-derived hyperpolarizing factor in guinea-pig, rat and human blood vessels. J Physiol 2001;531:359-73.
191Coleman H, Tare M, Parkington H. EDHF is not K + but may be due to spread of current from the endothelium in guinea pig arterioles. Am J Physiol Heart Circ Physiol 2001;280:H2478-83.
192Weston AH, Absi M, Ward DT, Ohanian J, Dodd RH, Dauban P, et al. Evidence in favor of a calcium-sensing receptor in arterial endothelial cells: Studies with calindol and Calhex 231. Circ Res 2005;97:391-8.
193Dora KA, Gallagher NT, McNeish A, Garland CJ. Modulation of endothelial cell KCa3.1 channels during endothelium-derived hyperpolarizing factor signaling in mesenteric resistance arteries. Circ Res 2008;102:1247-55.
194Edwards G, Félétou M, Weston AH. Endothelium-derived hyperpolarising factors and associated pathways: A synopsis. Pflugers Arch 2010;459:863-79.
195Garland CJ, Hiley CR, Dora KA. EDHF: Spreading the influence of the endothelium. Br J Pharmacol 2011;164:839-52.
196Aiello EA, Walsh MP, Cole WC. Phosphorylation by protein kinase A enhances delayed rectifier K+current in rabbit vascular smooth muscle cells. Am J Physiol 1995;268:H926-34.
197Fulton D, McGiff JC, Quilley J. Role of phospholipase C and phospholipase A2 in the nitric oxide-independent vasodilator effect of bradykinin in the rat perfused heart. J Pharmacol Exp Ther 1996;278:518-26.
198Adeagbo AS, Henzel MK. Calcium-dependent phospholipase A2 mediates the production of endothelium-derived hyperpolarizing factor in perfused rat mesenteric prearteriolar bed. J Vasc Res 1998;35:27-35.
199Gebremedhin D, Ma YH, Falck JR, Roman RJ, VanRollins M, Harder DR. Mechanism of action of cerebral epoxyeicosatrienoic acids on cerebral arterial smooth muscle. Am J Physiol 1992;263:H519-25.
200Bauersachs J, Hecker M, Busse R. Display of the characteristics of endothelium-derived hyperpolarizing factor by a cytochrome P450-derived arachidonic acid metabolite in the coronary microcirculation. Br J Pharmacol 1994;113:1548-53.
201Fisslthaler B, Popp R, Kiss L, Potente M, Harder DR, Fleming I, et al. Cytochrome P450 2C is an EDHF synthase in coronary arteries. Nature 1999;401:493-7.
202Campbell WB, Gebremedhin D, Pratt PF, Harder DR. Identification of epoxyeicosatrienoic acids as endothelium-derived hyperpolarizing factors. Circ Res 1996;78:415-23.
203Bolz SS, Fisslthaler B, Pieperhoff S, De Wit C, Fleming I, Busse R, et al. Antisense oligonucleotides against cytochrome P450 2C8 attenuate EDHF-mediated Ca (2+) changes and dilation in isolated resistance arteries. FASEB J 2000;14:255-60.
204Imig JD, Falck JR, Wei S, Capdevila JH. Epoxygenase metabolites contribute to nitric oxide-independent afferent arteriolar vasodilation in response to bradykinin. J Vasc Res 2001;38:247-55.
205Tanaka Y, Koike K, Toro L. MaxiK channel roles in blood vessel relaxations induced by endothelium-derived relaxing factors and their molecular mechanisms. J Smooth Muscle Res 2004;40:125-53.
206ZhuGe R, Fogarty K, Tuft R, Walsh J. Spontaneous transient outward currents arise from microdomains where BK channels are exposed to a mean Ca2+concentration on the order of 10 μm during a Ca2+spark. J Gen Physiol 2002;120:15-27.
207Karkanis T, DeYoung L, Brock G, Sims S. Ca2+-activated Cl − channels in corpus cavernosus smooth muscle: A novel mechanism for control of penile erection. J Appl Physiol. 2003;94:301-13.
208Jaggar JH, Porter VA, Lederer WJ, Nelson MT. Calcium sparks in smooth muscle. Am J Physiol Cell Physiol 2000;278:C235-56.
209Gordienko D, Greenwood I, Bolton T. Direct visualization of sarcoplasmic reticulum regions discharging Ca2+ sparks in vascular myocytes. Cell Calcium 2001;29:13-28.
210Earley S, Heppner TJ, Nelson MT, Brayden JE. TRPV4 forms a novel Ca2+ signaling complex with ryanodine receptors and BKCa channels. Circ Res 2005;97:1270-9.
211Baron A, Frieden M, Bény JL. Epoxyeicosatrienoic acids activate a high-conductance, Ca (2+)-dependent K + channel on pig coronary artery endothelial cells. J Physiol 1997;504 (Pt 3):537-43.
212Li PL, Campbell WB. Epoxyeicosatrienoic acids activate K + channels in coronary smooth muscle through a guanine nucleotide binding protein. Circ Res 1997;80:877-84.
213Gauthier KM, Falck JR, Reddy LM, Campbell WB. 14,15-EET analogs: Characterization of structural requirements for agonist and antagonist activity in bovine coronary arteries. Pharmacol Res 2004;49:515-24.
214Pfister SL, Spitzbarth N, Nithipatikom K, Edgemond WS, Falck JR, Campbell WB. Identification of the 11,14,15- and 11,12, 15-trihydroxyeicosatrienoic acids as endothelium-derived relaxing factors of rabbit aorta. J Biol Chem 1998;273:30879-87.
215Faraci FM, Sobey CG, Chrissobolis S, Lund DD, Heistad DD, Weintraub NL. Arachidonate dilates basilar artery by lipoxygenase-dependent mechanism and activation of K(+) channels. Am J Physiol Regul Integr Comp Physiol 2001;281:R246-53.
216Campbell WB, Spitzbarth N, Gauthier KM, Pfister SL. 11,12,15-Trihydroxyeicosatrienoic acid mediates ACh-induced relaxations in rabbit aorta. Am J Physiol Heart Circ Physiol 2003;285:H2648-56.
217Moncada S, Vane JR. Pharmacology and endogenous roles of prostaglandin endoperoxides, thromboxane A2, and prostacyclin. Pharmacol Rev 1978;30:293-331.
218Félétou M, Vanhoutte PM. Endothelium-derived hyperpolarizing factor: Where are we now? Arterioscler Thromb Vasc Biol 2006;26:1215-25.
219Feletou M, Vanhoutte P. EDHF: The complete story. In: Taylor & Francis. Boca Raton, FL, USA: CRC Press; 2006. p. 1-298.
220Chadha PS, Liu L, Rikard-Bell M, Senadheera S, Howitt L, Bertrand RL, et al. Endothelium-dependent vasodilation in human mesenteric artery is primarily mediated by myoendothelial gap junctions intermediate conductance calcium-activated K+channel and nitric oxide. J Pharmacol Exp Ther 2011;336:701-8.
221Hannah RM, Dunn KM, Bonev AD, Nelson MT. Endothelial SK (Ca) and IK (Ca) channels regulate brain parenchymal arteriolar diameter and cortical cerebral blood flow. J Cereb Blood Flow Metab 2011;31:1175-86.
222Hasenau AL, Nielsen G, Morisseau C, Hammock BD, Wulff H, Köhler R. Improvement of endothelium-dependent vasodilations by SKA-31 and SKA-20, activators of small- and intermediate-conductance Ca2+ -activated K+ -channels. Acta Physiol (Oxf) 2011;203:117-26.
223Behringer EJ, Segal SS. Tuning electrical conduction along endothelial tubes of resistance arteries through Ca (2+)-activated K(+) channels. Circ Res 2012;110:1311-21.
224McCarron RM, Chen Y, Tomori T, Strasser A, Mechoulam R, Shohami E, et al. Endothelial-mediated regulation of cerebral microcirculation. J Physiol Pharmacol 2006;57 Suppl 11:133-44.
225Berra Romani R, Raqeeb A, Laforenza U, Scaffino MF, Moccia F, Avelino-Cruz JE, et al. Cardiac microvascular endothelial cells express a functional Ca + -sensing receptor. J Vasc Res 2009;46:73-82.
226Palmer RM, Ashton DS, Moncada S. Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature 1988;333:664-6.
227Venema RC, Sayegh HS, Arnal JF, Harrison DG. Role of the enzyme calmodulin-binding domain in membrane association and phospholipid inhibition of endothelial nitric oxide synthase. J Biol Chem 1995;270:14705-11.
228Michel JB, Feron O, Sacks D, Michel T. Reciprocal regulation of endothelial nitric-oxide synthase by Ca2+-calmodulin and caveolin. J Biol Chem 1997;272:15583-6.
229Alderton WK, Cooper CE, Knowles RG. Nitric oxide synthases: Structure, function and inhibition. Biochem J 2001;357:593-615.
230Tfelt-Hansen J, Ferreira A, Yano S, Kanuparthi D, Romero JR, Brown EM, et al. Calcium-sensing receptor activation induces nitric oxide production in H-500 Leydig cancer cells. Am J Physiol Endocrinol Metab 2005;288:E1206-13.
231Hussain MB, Hobbs AJ, MacAllister RJ. Autoregulation of nitric oxide-soluble guanylate cyclase-cyclic GMP signalling in mouse thoracic aorta. Br J Pharmacol 1999;128:1082-8.
232Derbyshire ER, Marletta MA. Structure and regulation of soluble guanylate cyclase. Annu Rev Biochem 2012;81:533-59.
233Chen Z, Zhang X, Ying L, Dou D, Li Y, Bai Y, et al. cIMP synthesized by sGC as a mediator of hypoxic contraction of coronary arteries. Am J Physiol Heart Circ Physiol 2014;307:H328-36.
234Chan CK, Mak J, Gao Y, Man RY, Vanhoutte PM. Endothelium-derived NO, but not cyclic GMP, is required for hypoxic augmentation in isolated porcine coronary arteries. Am J Physiol Heart Circ Physiol 2011;301:H2313-21.
235Gao Y, Vanhoutte PM. Tissues cIMPly do not lie. Naunyn Schmiedebergs Arch Pharmacol 2014;387:901-3.
236Gao Y, Chen Z, Leung SW, Vanhoutte PM. Hypoxic vasospasm mediated by cIMP: When soluble guanylyl cyclase turns bad. J Cardiovasc Pharmacol 2015;65:545-8.
237Furchgott R. Studies on relaxation of rabbit aorta by sodium nitrite: The basis for the proposal that acid-activable inhibitory factor from bovine retractor penis is inorganic nitrite and the endothelium-derived relaxing factor is nitric oxide. In: Vanhoutte PM, editor. Vasodilatation: Vascular Smooth Muscle Peptides, Autonomic Nerves and Endothelium. New York, USA: Raven Press; 1988. p. 401-14.
238Furchgott RF, Vanhoutte PM. Endothelium-derived relaxing and contracting factors. FASEB J 1989;3:2007-18.
239Denninger JW, Marletta MA. Guanylate cyclase and the.NO/cGMP signaling pathway. Biochim Biophys Acta 1999;1411:334-50.
240Weisbrod RM, Griswold MC, Yaghoubi M, Komalavilas P, Lincoln TM, Cohen RA. Evidence that additional mechanisms to cyclic GMP mediate the decrease in intracellular calcium and relaxation of rabbit aortic smooth muscle to nitric oxide. Br J Pharmacol 1998;125:1695-707.
241Cohen R, Weisbrod R, Gericke M, Yaghoubi M, Bierl C, Bolotina V. Mechanism of nitric oxideeinduced vasodilatation refilling of intracellular stores by sarcoplasmic reticulum Ca2+ATPase and inhibition of storeoperated Ca2+ influx. Circ Res 1999;84:210-9.
242Carvajal JA, Germain AM, Huidobro-Toro JP, Weiner CP. Molecular mechanism of cGMP-mediated smooth muscle relaxation. J Cell Physiol 2000;184:409-20.
243Lee MR, Li L, Kitazawa T. Cyclic GMP causes Ca2+desensitization in vascular smooth muscle by activating the myosin light chain phosphatase. J Biol Chem 1997;272:5063-8.
244Webb RC. Smooth muscle contraction and relaxation. Adv Physiol Educ 2003;27:201-6.
245Mizuno Y, Isotani E, Huang J, Ding H, Stull JT, Kamm KE. Myosin light chain kinase activation and calcium sensitization in smooth muscle in vivo. Am J Physiol Cell Physiol 2008;295:C358-64.
246Ignarro LJ, Lippton H, Edwards JC, Baricos WH, Hyman AL, Kadowitz PJ, et al. Mechanism of vascular smooth muscle relaxation by organic nitrates, nitrites, nitroprusside and nitric oxide: Evidence for the involvement of S-nitrosothiols as active intermediates. J Pharmacol Exp Ther 1981;218:739-49.
247Myers PR, Minor RL Jr., Guerra R Jr., Bates JN, Harrison DG. Vasorelaxant properties of the endothelium-derived relaxing factor more closely resemble S-nitrosocysteine than nitric oxide. Nature 1990;345:161-3.
248Stamler JS, Simon DI, Osborne JA, Mullins ME, Jaraki O, Michel T, et al. S-nitrosylation of proteins with nitric oxide: Synthesis and characterization of biologically active compounds. Proc Natl Acad Sci U S A 1992;89:444-8.
249Adachi T, Weisbrod R, Pimentel D, Ying J, Sharov V, Schoneich C. S-€ Glutathiolation by peroxynitrite activates SERCA during arterial relaxation by nitric oxide. Nat Med 2004;10:1200-7.
250Whalen EJ, Foster MW, Matsumoto A, Ozawa K, Violin JD, Que LG, et al. Regulation of beta-adrenergic receptor signaling by S-nitrosylation of G-protein-coupled receptor kinase 2. Cell 2007;129:511-22.
251Daaka Y. S-nitrosylation-regulated GPCR signaling. Biochim Biophys Acta 2012;1820:743-51.
252Whalen E, Johnson A, Lewis S. b-adrenoceptor dysfunction after inhibition of NO synthesis. Hypertension 2000;36:376-82.
253Chruscinski A, Brede ME, Meinel L, Lohse MJ, Kobilka BK, Hein L. Differential distribution of beta-adrenergic receptor subtypes in blood vessels of knockout mice lacking beta (1)- or beta (2)-adrenergic receptors. Mol Pharmacol 2001;60:955-62.
254Guimarães S, Moura D. Vascular adrenoceptors: An update. Pharmacol Rev 2001;53:319-56.
255Nozik-Grayck E, Whalen E, Stamler J, McMahon T, Chitano P, Piantadosi C. S-nitrosoglutathione inhibits a1-adrenergic receptormediated vasoconstriction and ligand binding in pulmonary artery. Am J Physiol Lung Cellular Mol Physiol 2006;290:L136-43.
256Leclerc PC, Lanctot PM, Auger-Messier M, Escher E, Leduc R, Guillemette G. S-nitrosylation of cysteine 289 of the AT1 receptor decreases its binding affinity for angiotensin II. Br J Pharmacol 2006;148:306-13.
257Pitcher JA, Freedman NJ, Lefkowitz RJ. G protein-coupled receptor kinases. Annu Rev Biochem 1998;67:653-92.
258Pierce KL, Premont RT, Lefkowitz RJ. Seven-transmembrane receptors. Nat Rev Mol Cell Biol 2002;3:639-50.
259Reiter E, Lefkowitz RJ. GRKs and beta-arrestins: Roles in receptor silencing, trafficking and signaling. Trends Endocrinol Metab 2006;17:159-65.
260Ozawa K, Whalen E, Nelson C, Mu Y, Hess D, Lefkowitz R. S-nitrosylation of b-arrestin regulates b-adrenergic receptor trafficking. Mol Cell 2008;31:395-405.
261Ortiz-Capisano MC, Ortiz PA, Garvin JL, Harding P, Beierwaltes WH. Expression and function of the calcium-sensing receptor in juxtaglomerular cells. Hypertension 2007;50:737-43.
262Watanabe S, Fukumoto S, Chang H, Takeuchi Y, Hasegawa Y, Okazaki R, et al. Association between activating mutations of calcium-sensing receptor and Bartter's syndrome. Lancet 2002;360:692-4.
263Ortiz-Capisano MC, Liao TD, Ortiz PA, Beierwaltes WH. Calcium-dependent phosphodiesterase 1C inhibits renin release from isolated juxtaglomerular cells. Am J Physiol Regul Integr Comp Physiol 2009;297:R1469-76.
264Beierwaltes WH. The role of calcium in the regulation of renin secretion. Am J Physiol Renal Physiol 2010;298:F1-11.