Calcium acts as a first messenger through the calcium-sensing receptor in the cardiovascular system

Abstract

It is well known that calcium is an important second messenger in the cardiovascular system. However, recent studies suggest that, in addition to its many functions as an intracellular messenger, Ca²⁺ may also be an extracellular first messenger through the calcium-sensing receptor (CaR). The CaR belongs to family C of the G-protein-coupled receptors, which are also known as seven transmembrane domain receptors. The CaR receptor is expressed in all major organs involved in Ca²⁺ homeostasis. Furthermore, increasing evidence suggests that the CaR is also involved in regulating various cellular functions in tissues not involved in Ca²⁺ homeostasis. Recently, expression of a functional CaR has also been reported in crucial components of the cardiovascular system. It has previously been shown that the CaR is functionally expressed in the atria and ventricle of the rat heart. In blood vessels, the CaR protein was first reported in perivascular nerves of rat mesenteric resistance arteries, and was proposed to modulate myogenic tone in the arteries. Since then, the CaR has been detected in homogenates of whole vessels from rat subcutaneous small arteries and in endothelial cells from rat mesenteric and porcine coronary arteries. Furthermore, a recent report demonstrated that the CaR is present in endothelial cells from human aorta and that it stimulates production of nitric oxide in these cells. Taken together, these results indicate that the CaR present in blood vessels may have a physiological role in modulation of arterial blood pressure. This review discusses CaR expression and function, with a focus on the role of the CaR in the cardiovascular system.

Calcium signaling, Calcium-sensing receptor, Cardiovascular system, G protein, Seven transmembrane domain receptor

Time for primary review 28 days

1 Introduction

The pivotal role of calcium in the cardiovascular system is well known. Ca²⁺ acts as a second messenger via rapid, as well as more sustained, changes in intracellular calcium levels through the actions of calcium channels, exchangers and pumps. However, it is conceivable that calcium may also act as a first messenger through a G-protein-coupled receptor (GPCR) that senses extracellular Ca²⁺ (Ca_o²⁺), the calcium-sensing receptor (CaR, also known as CaSR) [1]. Activation of the CaR elicits complex intracellular signals through modulation of a wide range of intracellular signalling proteins, including G proteins and phospholipase C (PLC), which in turn stimulate inositol triphosphate production, and thereby increase intracellular Ca²⁺ (Ca_i²⁺) release. Downstream of or in parallel with PLC, the CaR also activates mitogen-activated protein kinases (MAPKs) and phosphatidylinositol-4-kinase (PI4K). The CaR is important in maintaining and regulating mineral ion homeostasis. However, the receptor is also widely expressed in tissues not involved in calcium homeostasis and modulates various cellular functions, including secretion of peptides, ion-channel/transporter activity, gene expression, proliferation, differentiation, apoptosis and chemotaxis [2]. In the cardiovascular system, a functional CaR has been shown to be present in the heart as well as in blood vessels [3–8]. Bukoski et al. reported the presence of CaR in perivascular nerves of isolated rat arteries and showed that increased Ca_o²⁺ causes nerve-dependent relaxation of the pre-contracted arteries [7]. Ohanian et al. has detected the CaR protein in rat subcutaneous small artery homogenates and reported that it modulates myogenic tone [8]. Recently, a functional CaR protein has been reported in endothelial cells from rat mesenteric and porcine coronary arteries [4]. The same study also showed that stimulation of the receptor induces hyperpolarization of vascular smooth muscle cells (VSMCs) [4]. Recently, the CaR has been reported in endothelial cells from human aorta, where it mediates nitric oxide (NO) production [9]. Thus, all these studies indicate a physiological role of the CaR in the cardiovascular system. Therefore, this review aims to provide a brief background on the CaR structure, signalling and function, followed by a more detailed discussion of the role of CaR in the cardiovascular system.

2 Calcium as first messenger mediated by the CaR

2.1 Molecular structure of the CaR

The human CaR consists of 1078 amino acid residues [10]. The receptor has three structural domains: a large, extracellular amino (N)-terminal domain (ECD) of 612 amino acid residues; a seven transmembrane domain (7TMD) of 250 amino acid residues; and a 216-residue intracellular carboxyl (C)-terminal domain (ICD) (Fig. 1).

Fig. 1

Schematic representation of the main predicted topological features of the extracellular Ca²⁺-sensing receptor cloned from human parathyroid glands. The extracellular domain contains approximately 600 amino acids, and the transmembrane and intracellular domains each contain approximately 200 amino acids. Also illustrated are the protein kinase C (PKC) phosphorylation sites, N-glycolysation sites and cysteine residues.

The ECD of the CaR is the main Ca_o²⁺ binding site [11], and contains several N-linked glycosylation sites, which are important for cell-surface expression but do not seem to be critical for signal transduction [12]. The ECD also contains several cysteine residues essential for proper trafficking and function of the receptor [13]. The CaR is expressed on the cell surface as a dimer, and at least two distinct types of dimerization-mediating motifs have been identified: covalent interactions involving disulfide bonds between cysteine residues 129 and 131, and non-covalent, possibly hydrophobic interactions [14,15]. The disulfide bonds are thought to constrain the CaR in its inactive form, as disrupting these bonds increases the CaR sensitivity to Ca_o²⁺. The TMD with the characteristic seven transmembrane helices of the GPRCs is central to the transduction of signals from the CaR ECD into a cellular response, and several natural activating and inactivating mutations have been identified in this region [16,17]. Puzzling, it has been shown that a mutant CaR lacking the ECD also responds to Ca_o²⁺ and other polyvalent cations, indicating that TMD also participates in sensing of Ca_o²⁺[18]. These data indicate that the actual calcium-sensing domain of the CaR is complex and includes both the ECD and TMD. In the ICD, the CaR has two protein kinase A (PKA) sites and five protein kinase C (PKC) phosphorylation sites. The PKC phosphorylation sites seem to be involved in downregulation of the receptor activity and thereby are a negative feedback mechanism [19]. The ICD also contains several amino acids that are involved in trafficking of the receptor to the cell surface and signal transduction [20]. Moreover, a stretch of amino acids in the ICD has been shown to be critical for binding of the receptor to the scaffolding protein filamin-A [21]. Formation of this complex seems to protect the receptor against degradation and to participate in CaR-mediated activation of MAPKs [22].

2.2 Ligands of the CaR

The CaR was originally named owing to its ability to function as a sensitive ‘detector’ of changes in Ca_o²⁺ concentrations. The first impression might be that Ca_o²⁺ is not a particularly potent agonist of the CaR, half-maximal activation of the bovine parathyroid CaR is about 1.25 mM Ca_o²⁺in vitro[23]. However, Ca_o²⁺ binds to the CaR with a high positive cooperativity, with a Hill coefficient of 3–4 [23]. This allows the CaR to detect very small fluctuations in the Ca_o²⁺ at the range of extracellular calcium levels. Unlike many other GPCRs, the CaR is thought to be somewhat resistant to desensitization, allowing it to continuously monitor Ca_o²⁺ levels.

Although the main ligand of the CaR is Ca_o²⁺, the CaR also responds to many other cations, naturally occurring polyamines and some antibiotics (Table 1) [2,24]. The affinity of these molecules to the CaR is variable. In general, molecules with high-charge density are more efficient agonists of the CaR than those with lower charge density; for example, spermine, which has four positively charged amino groups, is more potent then spermidine, which has only three positively charged amino groups [23]. Inorganic di- and trivalent cations have been tested for their potency on the CaR, and they rank as follows: La³⁺>Gd³⁺>Be²⁺>Ca²⁺=Ba²⁺>Sr²⁺>Mg²⁺[25]. Furthermore, the CaR has also been shown to be sensitive to changes in ionic strength and pH [26,27].

Table 1

A list of known agonists and antagonists of the CaR

Direct agonists (type I agonists)	Positive allosteric modulators (type II agonists)	Positive allosteric modulators (antagonists)
Ca2+	L-type amino acids	Calcilytics
Other cations (e.g. Gd3+, Mg2+)	Calcimimetics
Polyamines (e.g. spermine)
Antibiotics (e.g. neomycin)
pH
Ionic strength

In addition to the direct agonists, which are known as type I agonists, several other agents function as allosteric activators of the CaR and are classified as type II agonists (Table 1). They modulate the receptor by allosteric action, changing its affinity to Ca_o²⁺ and other type I agonists. The most biologically relevant of these are the L-type amino acids, the most potent of which are those with aromatic side chains [28]. Several pharmacological CaR modulators, which bind to the TMD and increase receptor sensitivity, have also been developed and are known as calcimimetics [24]. They are now used in treatment of uremic hyperparathyroidism. Negative allosteric modulators of the CaR are named calcilytics.

Based on the list of known agonists, the CaR seems to be a promiscuous receptor that senses changes in multiple physiological parameters, and its name ‘calcium-sensing receptor’ is likely to be an oversimplification. It functions as an integrator of the stimuli to the cells and is probably always activated to some extent. Stimulation of the receptor can be converted into activation of many signalling pathways known to be used by the CaR, as discussed below.

2.3 CaR signalling

The nature of an intracellular pathway activated by the CaR depends markedly on the cell type in which the receptor is expressed. Most studies on CaR signalling have been performed in parathyroid cells and human embryonic kidney (HEK-293) cells stably transfected with the CaR. The CaR, like other GPCRs, acts mainly through G proteins. In HEK-293 and bovine parathyroid cells, the CaR interacts with Gα_q/11 subunits of heterotrimeric G proteins, resulting in activation of phospholipases C and A₂[29,30] (Fig. 2). This interaction also induces activation of protein kinase C (PKC), which in turn modulates the activity of the receptor [19,31]. In parallel, the CaR activates phosphatidylinositol 4-kinase (PI4K), which catalyzes the first step of the inositol lipid biosynthesis, independently of the G proteins, by a Rho-dependent mechanism [32]. In some cells (such as HEK-293, parathyroid cells and kidney cells), the CaR interacts with a pertussis-toxin-sensitive inhibitory G protein, Gα_i, which results in inhibition of adenylyl cyclase (AC).

Fig. 2

Signalling pathways activated by the CaR. The CaR is activated by Ca_o²⁺, calcimimetics and numerous other agents. The signalling pathways have been reported in various cell types and the relationships among the pathways are often unknown, as indicated. Please refer to text for detailed information. Black and blue arrows signify stimulation, and red arrows signify inhibition. Abbreviations: arachidonic acid (AA), adenylyl cyclase (AC), protein kinase B (AKT), activating transcription factor-2 (ATF-2), adenosine triphosphate (ATP), cyclic adenosine monophosphate (cAMP), diacylglycerol (DAG) extracellular regulated kinase (ERK), alpha subunit of i and q subtypes of the heterotrimeric G proteins (Gi and Gq₁₁), inositol-1,4,5-triphosphate (IP₃), Jun amino terminal kinase (JNK), mitogen-activated protein kinase (MAPK), MAPK kinase (MEK), p38 MAPK (p38), phosphatidylinositol-4-kinase (PI4K), phosphatidylinositol 3-kinase (PI3K), phospholipase A2 (PLA2), phospholipase c (PLC) protein kinase C (PKC), phosphatidylinositol-4,5-biphosphate (PIP₂) and stress-activated protein kinase ERK kinase 1 (SEK1).

The CaR has also been shown to activate several MAPK cascades in various cell types. Kifor et al. 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 [33]. ERK activation by the CaR has now been demonstrated in many cell types, including rat neonatal cardiomyocytes [2,6]. In rat Leydig cancer (H-500) cells expressing CaR, calcium has been shown to activate ERK, p38 MAPK and Jun amino-terminal kinase (JNK) [34]. In addition, the CaR has been shown to transactivate epidermal growth factor receptor (EGFR) in rat Leydig cancer (H-500) cells, HEK-293 cells and human prostate cancer (PC3) cells [35–37]. 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.

In addition to the G proteins, the CaR binds the scaffolding protein filamin A in HEK-293 and parathyroid cells [21]. This interaction is functionally important, because its impairment attenuates CaR-mediated ERK activation. Furthermore, it protects against degradation of the CaR, which might explain the seemingly low level of internalization of the CaR secondary to ligand binding [22]. In parathyroid cells, the CaR has been found to be localized within caveolae, which are small cholesterol-rich structures in the plasma membrane that are highly enriched with multiple signalling molecules and the structural protein caveolin-1 [38]. Caveolin-1 also interacts with filamin-A [39], so filamin-A may also be important for localization of CaR to the caveolae. Other important molecules, recently found to interact with the CaR in heterologous cell culture models (including HEK-293 cells; transformed monkey kidney fibroblast, COS-7, cells; and human osteosarcoma, U20S, cells) are G-protein-coupled kinases (GRKs) and β-arrestins [40]. β-arrestins and GRKs are typically involved in homologous desensitization of GPCRs. Although the CaR is believed to desensitize very slowly, β-arrestins and GRKs seem to mediate CaR desensitization in vitro. In addition, this study elegantly showed that β-arrestin2-null mice seem to have a leftward shift in the calcium–parathyroid hormone relationship. Thus, β-arrestins are also important for regulating CaR function in the parathyroid glands in vivo. Recently, β-arrestins and GRKs have also been shown to act as signal transducers themselves [41]. This is mediated by activation of MAPKs, adding to the complexity of the downstream signalling mechanism of the GPCR. Future studies are needed to investigate whether this is also the case for the CaR.

2.4 The function of the CaR in tissues that regulate calcium homeostasis

Ca²⁺ has a key role in many physiological processes, so Ca²⁺ homeostasis is tightly regulated. The coefficient of variation of the serum ionized calcium concentration about its mean value is 2% or less in healthy individuals. There are three so-called calciotropic hormones, PTH, calcitonin (CT) and 1.25(OH)₂ vitamin D₃, which regulate Ca²⁺ transport processes. The inverse sigmoidal relationship between Ca_o²⁺ and secretion of the most potent Ca_o²⁺-elevating hormone, PTH, and the positive relationship between Ca_o²⁺ and secretion of the Ca_o²⁺-lowering hormone CT are both mediated by the CaR [42]. CaR in the parathyroid glands has a central role in Ca²⁺ homeostasis. A reduction in Ca²⁺ plasma concentration results in a CaR-mediated increase in PTH secretion from the parathyroid cells. The increased PTH level promotes distal renal tubular Ca_o²⁺ reabsorption and bone resorption by the lining cells, both of which increase Ca_o²⁺ levels [43]. Furthermore, the relative hypocalcemia also leads to reduced secretion of calcitonin from thyroid C cells mediated by the CaR, preventing inhibition of bone resorption by calcitonin [44]. Both PTH and a low Ca²⁺ level induce synthesis of 1.25(OH)₂ vitamin D₃ in the proximal tubular cells of the kidney. The vitamin D metabolite stimulates intestinal Ca_o²⁺ absorption. The reverse of these events occurs in hypercalcemia.

In addition to the parathyroid glands, the kidney, bone and intestine are important organs in the maintenance of Ca²⁺ homeostasis. The body maintains a constant Ca_o²⁺ concentration by the concerted actions of Ca_o²⁺ absorption in the intestine, reabsorption in the kidney and exchange from the bone. In vitro studies have reported the presence of CaR in cells of the kidney, bone and intestine [45]. In the kidney, the CaR is expressed in many segments of the nephron [46]. In brief, the functions of the CaR along the nephron are: (1) to diminish the inhibitory effect of PTH on renal phosphate reabsorption in the proximal tubule [47]; (2) to inhibit renal calcium excretion in the cortical thick ascending limb of the loop of Henle [48]; and (3) to reduce urinary concentrating capacity in the inner medullary collecting duct [49]. For an extended review see [2]. In the bone, the CaR is expressed in bone-degrading cells, osteoclasts and their precursors [45]. However, there are discrepant reports about the presence of the CaR in bone-forming cells, osteoblasts, although most reports support the presence of the receptor (see [45] for review). CaR expression has also been demonstrated in several cell types of the intestine [50], and a recent study has shown that it abrogates intestinal net fluid secretion induced by cyclic-nucleotide-generating secretagogues [51]. However, exact roles for the CaR in the gastrointestinal tract are now only beginning to be determined. Therefore, the presence of the CaR in these four organs, all of which are important in Ca²⁺ homeostasis, may allow Ca_o²⁺ to act as a fourth calciotropic hormone, or as a “first messenger”.

2.5 The function of the CaR in tissues not involved in calcium homeostasis

The importance of calcium in many organs has led to speculation whether the CaR may have a role in tissues that are not directly involved in systemic mineral ion metabolism. Expression of the CaR is now well documented in many tissues such as brain, pancreas, blood vessels, and heart, as well as in many cell types throughout the body. It has elegantly been shown that agonist-induced release of Ca_i²⁺ in a stimulated cell results in active secretion of Ca²⁺ across the cell membrane after stimulation, resulting in increases in the local Ca_o²⁺ concentration sufficient to be detected by the CaR on the cell membrane of adjacent cells in vitro[52,53]. Thus, a Ca_i²⁺ signal may induce generation of a Ca_o²⁺ signal that is sensed by the CaR on neighbouring cells (paracrine) or even on the same cell (autocrine). In addition to its role in regulation of PTH and CT release, the CaR has also been shown to regulate secretion of parathyroid-hormone-related peptide [34,54], gastrin [55] and insulin [56], as well as gene expression of the pituitary-tumor-transforming gene [57] and, possibly, vascular endothelial growth factor [57]. In multiple cells in the brain, the CaR activates various ion channels, including non-selective cation channels [58,59] and calcium-sensitive K⁺ channels [60,61]. Furthermore, Weston et al. recently showed that activation of the CaR opens intermediate conductance calcium-sensitive K⁺ channels (IK_Ca) in arterial endothelial cells [4]. By contrast, a recent report demonstrated that expression of the CaR with an inwardly-rectifying K⁺ channel, Kir4.1 or Kir4.2, in Xenopus oocytes inhibits the channel function [62]. These discrepancies seem to be due to the different mechanisms by which the CaR modulates activity of the K⁺ channels: activation of the channels seems to be due to an indirect, CaR-activated transduction pathway, whereas inhibition may require a direct interaction between the channel and the CaR [61,62].

The roles of the CaR in cell growth, apoptosis and differentiation have been reported in various cell types (see [45] for review). Furthermore, the CaR stimulates chemotaxis of pre-osteoblastic cells [63] and macrophages [64], both of which cell types are probably involved in the pathogenesis of atherosclerosis. Recently, haematopoietic stem cells from CaR-knock-out mice are reported to have reduced ability to lodge at the endosteal niche, which is important for bone development, thereby indicating that CaR functions in the engraftment of haematopoietic stem cells in adult bone marrow [65].

Therefore, although it is now well known that Ca_o²⁺ acts as an extracellular first messenger to regulate various cellular functions, future challenges will be to investigate the roles of the CaR in CaR-expressing tissues that are not involved in Ca²⁺ homeostasis. Below, we will discuss the role of the CaR in the cardiovascular system.

3 The CaR in the cardiovascular system

3.1 The CaR in the heart

The evidence that calcium is a first messenger in cardiomyocytes was obtained from two studies, in which we and others showed that a functional CaR is expressed in neonatal as well as adult rat cardiomyocytes [5,6]. CaR messenger RNA (mRNA) and protein were detected in both atrial and ventricular myocytes. Challenging isolated ventricular myocytes with Ca_o²⁺ and other type 1 CaR agonists induced concentration-dependent increases in Ca_i²⁺ concentrations and intracellular inositol phosphate (IP) concentrations, indicating that the CaR is linked to the PLC pathway. A positive allosteric modulator of the CaR, AMG 073, shifted the calcium–IP curve to the left [6]. Furthermore, expression of a non-functional CaR mutant significantly inhibited the Ca_o²⁺-induced IP response, strongly supporting CaR as a mediator of the Ca_o²⁺-induced increase in IP levels. We also reported that CaR in cardiomyocytes activates ERK1/2, which are components of the MAPK signalling pathway and are known to function downstream of the CaR. Interestingly, ERK1/2 activation by Ca_o²⁺ was more rapid in the presence of AMG 073. Different kinetics of ERK1/2 activation with Ca_o²⁺ and calcimimetics have previously been observed in HEK-293 cells [66]. Stimulation of the CaR with AMG 073 induced a reduction in DNA synthesis in neonatal cardiomyocytes, suggesting that CaR is involved in regulation of the cell cycle. Although adult cardiomyocytes lose the ability to proliferate, cell proliferation may take place in neonatal cardiomyocytes [67,68]. Moreover, DNA synthesis is observed in neonatal cardiomyocytes undergoing hypertrophy, perhaps due to a partial progression through the cell cycle [68,69]. The CaR-mediated reduction in DNA synthesis observed in our studies was not correlated with changes in cell number, indicating that CaR protects against cardiac hypertrophy. Recently, increased expression of the CaR protein in the heart was reported during ischemia/reperfusion [70]. These reports indicate that the CaR is an active player on the cell membrane of the cardiomyocytes. Further studies are needed to determine the exact expression pattern of the CaR and its signalling mechanisms to understand its role in heart physiology and pathophysiology.

3.2 The CaR in blood vessels

Over several decades, a number of studies have shown that increased Ca_o²⁺ concentrations induce relaxation of arteries (for an extended review see [71]). The first clue was in 1911 when Cow showed that increased Ca_o²⁺ concentrations reduces reactivity of isolated arteries [72]. More than 50 years later, Bohr reported that Ca_o²⁺ inhibits the early rapid response of the rat aorta to epinephrine, whereas it increases the late steady-state response [73]. Hollaway and Bohr have since suggested that increased Ca_o²⁺ induces relaxation via a membrane-stabilizing effect [74]. Several studies have subsequently characterized the vessel response to Ca_o²⁺[75–77]. However, the molecular mechanism for this phenomenon has remained unknown. Such a Ca_o²⁺-mediating molecular mechanism could involve the CaR. Four years after cloning of the CaR from bovine parathyroid cells, Bukoski et al. proposed that Ca_o²⁺ induces relaxation of isolated arteries by activating the CaR in perivascular nerves [7]. They detected CaR protein in rat mesenteric resistance arteries, and immunocytochemical analysis showed a fine nerve-like pattern of anti-CaR staining in the adventitial layer of the vessel [7,78]. The authors did not detect CaR mRNA in the arteries, but did detect it in dorsal root ganglia, and suggested that the CaR protein might be transported from the dorsal root ganglia to the perivascular nerve network of peripheral arteries. In addition, Ca_o²⁺ induced endothelium-independent relaxation of arteries that had been pre-contracted with norepinephrine. A bioassay showed that superfusion of Ca_o²⁺ across the adventitial surface of resistance arteries results in release of a diffusible, relaxing substance, which was identified as a hyperpolarizing factor not a peptide transmitter. Further studies demonstrated that this was probably a cannabinoid receptor agonist [79]. Phenolic nerve destruction ablated the relaxing effect of Ca_o²⁺, and Ca_o²⁺-induced relaxation of mesenteric resistance arteries isolated from rat neonates treated with the neurotoxin capsaicin was significantly reduced compared with controls [7,80]. Furthermore, capsaicin treatment significantly reduced the density of neurons containing the CaR. Bukoski et al. suggested that Ca_o²⁺ induces relaxation of the isolated mesenteric arteries by activating the CaR in perivascular sensory nerves, resulting in the release of a nerve-derived hyperpolarizing vasodilator, and proposed cannabinoid as a candidate. They have since reported that the CaR protein is found in perivascular nerves in renal, coronary and cerebral arteries, and that Ca_o²⁺ also induced relaxation in these vessels [78]. However, the same authors reported coronary arteries to contract at Ca_o²⁺ levels of 1.5–3 mM, and relax only to a small degree with greater Ca_o²⁺ concentrations (5 mM). This observation was consistent with a relatively low level of the CaR protein in the coronary artery. In 2004, Ohanian et al. found a similar biphasic effect of Ca_o²⁺ in rat subcutaneous small arteries that had been shown to express the CaR [8]. The study was performed with a pressure myograph under isobaric conditions. Under these conditions, arteries develop myogenic tone and relaxation can be studied without pharmacological pre-contraction. Concentrations of 0.5–2 mM Ca_o²⁺ induced some vasoconstriction, and 3–10 mM Ca_o²⁺ induced vasorelaxation. In addition, Mg_o²⁺ and neomycin (both of which are CaR agonists) induced concentration-dependent relaxation similar to that observed with Ca_o²⁺, indicating involvement of the CaR. The localization of the CaR on the arteries was not investigated.

Although the CaR was initially not observed in medial and endothelial layers of the blood vessels, subsequent studies by several other groups have provided evidence supporting a functional role for CaR in these cells. In 2000, two studies reported conflicting results on the presence of the CaR in VSMCs. Wonneberger et al. reported the presence of CaR transcripts in the gerbilline spiral modiolar artery and found a biphasic increase in Ca_i²⁺ in response to increased Ca_o²⁺ concentrations [81]. Furthermore, they showed that stimulation of the CaR with type I agonists caused a biphasic vasoconstriction of the artery. Since the increase in Ca_i²⁺ was paralleled by the biphasic vasoconstriction, they concluded that the CaR is most likely to be localized in the VSMCs. By contrast, Farzaneh-Far et al. did not detect CaR transcripts in VSMCs cultured from rat aorta. They suggested the presence of a distinct calcium-sensing receptor, closely related to the CaR [82]. Recently, we have demonstrated expression of CaR mRNA and protein in cultured VSMCs from rat aorta. We observed increased cell proliferation in response to increased Ca_o²⁺[3]. However, we were not able to conclusively state that the CaR is a mediator of the Ca_o²⁺ effect. In addition to expression of the CaR in the VSMCs, its presence was recently demonstrated in endothelial cells from rat mesenteric arteries and porcine coronary arteries [4]. Stimulation of the receptor with the specific positive allosteric modulator calindol, but not its less potent S-enantiomer, induced endothelium-dependent hyperpolarization of the VSMCs (Fig. 3). The hyperpolarization to calindol was significantly reduced in the presence of a negative allosteric modulator of the CaR, Calhex. Moreover, a specific inhibitor of the intermediate conductance Ca²⁺-sensitive potassium channels (IK_Ca) abolished calindol-induced hyperpolarization. The results therefore indicate that the CaR in the endothelial layer of rat mesenteric and porcine coronary arteries activates IK_Ca, resulting in K⁺-induced hyperpolarization of the VSMCs [4,83]. Although hyperpolarization is usually associated with relaxation, calindol did not induce relaxation of phenylephrine-pre-contracted mesenteric arteries. This could be due to phenylephrine-induced ‘K⁺ clouds’, which prevent hyperpolarizing after K⁺ efflux from endothelial-cell K⁺ channels [84]. Nevertheless, these results, supported by reports of relaxant effects of Ca_o²⁺ and other CaR agonists on basal vascular tone in the rat subcutaneous arteries, indicate that the CaR may have a physiological role in the modulation of blood pressure. In support of this hypothesis, increasing dietary calcium levels have been reported to have lowering effects on blood pressure in models of hypertension [85].

Fig. 3

Calcimimetic (calindol) induces endothelium-dependent hyperpolarization of rat mesenteric artery smooth muscle. a) In endothelium-intact segments calindol produced concentration-dependent hyperpolarization. The S-enantiomer of calindol had no effect. b) After removal of endothelium, calindol had no effect. Absence of endothelium was confirmed by the lack of response to acetylcholine (Ach). A potassium-channel opener, levcromakalim (LK), was added at the end of the experiment to confirm the integrity of the tissue. (Figure reproduced with permission from Ref. [4]).

More recently, evidence for the presence of the CaR in human cardiovascular tissue, namely aortic endothelial cells, has been provided [9]. Although stimulation of the receptor with Ca_o²⁺, neomycin and gadolinium did not increase Ca_i²⁺, the CaR agonist spermine stimulated an increase in Ca_i²⁺. Moreover, spermine stimulated production of NO in the cells. The responses to spermine were reduced or absent in the cells transfected with small-interfering RNA specifically targeted to the CaR. Thus, the production of NO in response to CaR stimulation in the aortic endothelial cells further suggests a possible role of the CaR in vasodilation. So, the overall picture is that the CaR is present in the perivascular nerves of the adventitia, and probably also in the VSMCs and endothelial cells. Stimulation of the receptor induces opening of the Ca²⁺-sensitive potassium channels and NO production. Furthermore, Ca_o²⁺ has non-trivial effects on myogenic tone experimentally. We speculate that the CaR modulates the vascular tone of the vessels in response to systemic as well as local changes in Ca_o²⁺ concentrations.

4 Conclusion and future issues

It is now evident that several components of the cardiovascular system express the CaR. The CaR is modulated by Ca_o²⁺ levels that are relevant to the milieu outside the cells, making the receptor a candidate for effecting changes in systemic and possibly more local calcium concentrations. Initial animal studies in heart indicate that the receptor is present in atrial and ventricular myocytes. The CaR induces stimulation of IP- and ERK-signalling pathways. We speculate that the receptor may have a role in regulating hypertrophy of the cardiomyocytes. The receptor is expressed in neonatal and adult cardiomyocytes, and could therefore be important in the development of the normal myocardium. Furthermore, it has been shown that the CaR can regulate activity of ion channels in other cells. Therefore, it is possible that the CaR in the cardiomyocytes may also regulate ion channels, and thereby the membrane potential. A future challenge will be to investigate possible roles of the CaR in the development and electrophysiology of the heart. The presence of the CaR has been reported in animal blood vessels of many types, in perivascular nerves, endothelial cells and vascular smooth muscle cells, and we suggest that it may regulate the vascular tone (Fig. 4). This could provide a mechanism for the almost 100-year-old observation that Ca_o²⁺ induces vasodilation. In support of this hypothesis it has been shown that the calcimimetic NPS R-568 lowers blood pressure and improves cardiac morphology (capillary density, fibrosis) in subtotally nephrectomized rats in vivo[86,87]. Although parathyroidectomy had a similar effect, it is currently unclear whether all of the effects of the calcimimetic are due to reduced PTH concentrations or whether the calcimimetics directly affect target structures such as vessels. The calcimimetics have also been shown to have beneficial effects on cardiovascular risk factors compared with standard care of secondary hyperparathyroidism [88]. Here, it should be noted that there are several other possible mechanisms for these effects other than direct modulation of the CaR by the calcimimetics in the cardiovascular system. More recently, it has been reported that administration of the calcilytic NPS 2143 increases blood pressure in rats in the presence of parathyroid glands [89]. Owing to the increasing clinical use of calcimimetics in treatment of hyperparathyroidism and a potential use of calcilytics in the treatment of osteoporosis, understanding the role of CaR in blood pressure and cardiac function is essential. Future studies using knock out mice should reveal the importance of the CaR in vascular tone regulation. We conclude that the discovery of the CaR in key components of the cardiovascular system is indicative of a possible role for the receptor in heart and vascular physiology. In particular, these novel results indicate that the CaR may modulate myogenic tone through activation of NO production and K-channels in the vascular tree, thus making calcium a first messenger that modulates the system.

Fig. 4

Schematic figure of known effects of calcium and the CaR in the vessels. Black text and arrows signify known effects, red text and arrows signify proposed effects. See text for details.

Glossary

• AC

  adenylyl cyclase

• Ca_o²⁺

  extracellular calcium

• Ca_i²⁺

  intracellular calcium

• cAMP

  cyclic adenosine monophosphate

• CaR

  calcium-sensing receptor

• CT

  calcitonin

• DAG

  diacylglycerol

• ECD

  extracellular amino (N)-terminal domain

• EGFR

  epidermal growth factor receptor

• ERK

  extracellular signal-regulated kinase

• GRK

  G protein-coupled kinase

• HEK

  human embryonic kidney

• ICD

  intracellular carboxy (C)-terminal domain

• IP

  inositol phosphate

• IP3

  inositol triphosphate

• JNL

  Jun amino-terminal kinase

• GDP

  guanosine diphosphate

• GPCR

  G-protein-coupled receptor

• MAPK

  mitogen-activated protein kinase

• NO

  nitric oxide

• PI4K

  phosphatidylinositol-4-kinase

• PKA

  protein kinase A

• PKC

  protein kinase C

• PLC

  phospholipase C

• PTH

  parathyroid hormone

• TMD

  transmembrane domain

• Vitamin D

  1.25(OH)₂ vitamin D₃

• VSMCs

  vascular smooth muscle cells

Acknowledgments

This work was supported by grants from the Danish National Research Foundation, the Danish Medical Research Council, and the Danish Heart Association to J.T.H., and the Villadsen Family Foundation and Copenhagen University to S.S.