The perivascular sensory nerve Ca2+ receptor and blood pressure regulation *1A hypothesis

During this era of rapidly expanding information about the physiology of biologic systems, it remains important that we attempt to assimilate new discoveries with past observations, particularly when the historical findings are implicated in disease processes. As we proceed with the assimilation process and make previously unrecognized connections between old facts and new discoveries, we sometimes find that they mesh neatly together and provide a new framework for understanding phenomena that were, previously, only poorly understood. With this in mind, the goal of the present paper is to describe how our knowledge of historical data linking whole-animal Ca2+ homeostasis with vascular reactivity and blood pressure (BP) takes on new dimensions when considered in terms of recent advances showing that Ca2+ serves as a primary signal to modulate cell function. Specifically, we have formulated a novel hypothesis that potentially explains the molecular connection between Ca2+ homeostasis and BP, clarifies decades-old observations regarding vascular reactivity that were poorly understood, and provides new avenues for critical hypothesis testing. Of note, as this paper is not intended to be a literature review, no attempt has been made to be inclusive in citation of the literature; rather, in most instances, only seminal reports or other pertinent advances are cited. The hypothesis developed in this paper states that changes in the concentration of ionized Ca2+ in the interstitial compartment modulate vascular reactivity by inducing the release of a vasodilator substance from perivascular sensory nerves secondary to activation of a Ca2+ receptor present on the surface of the sensory nerve. Significant changes in interstitial Ca2+ would occur primarily in tissues involved in transcellular Ca2+ movement, ie, intestine, kidney, and bone, and would thus be linked to Ca2+ balance of the whole animal and the endocrine factors that regulate it (Figure 1). Within the framework of this hypothesis, we predict that changes in Ca2+ balance associated with altered Ca2+ intake or pathologic conditions (eg, hyperparathyroidism) would be associated with changes in intestinal and renal blood flow and would have effects on total peripheral resistance (small bowel) and salt and water handling (kidney) that could ultimately affect blood pressure. This hypothesis was formulated using information taken from several independent areas. One is the theoretical construct that holds that BP regulation is linked with whole-animal Ca2+ homeostasis. The second is basic research that has been carried out investigating the effects of extracellular Ca2+ on vascular reactivity. The third is the study of the cellular and molecular mechanisms by which extracellular Ca2+ modulates signal transduction and function of nonvascular cell types, particularly the parathyroid gland. Three lines of evidence link whole-animal Ca2+ homeostasis with blood pressure. Perhaps the most convincing body of data are the results of studies using animal models of hypertension. The earliest work in this area was performed by Bethelot and colleagues in the mid-70s and examined the effect of parathyroidectomy on the development of hypertension in mineralocorticoid hypertensive rats.1,2 At the end of this decade, Ayachi published the first report showing that elevated dietary Ca2+ intake prevents the development of hypertension in the spontaneously hypertensive rat (SHR) model of genetic hypertension.3 Since that report, numerous studies have shown that there is an inverse correlation between Ca2+ intake and BP in several animal models of hypertension, including the SHR,4,5 DOCA-salt rat and dog,6,7 and Dahl S rat.8 A second line of evidence showing an association between Ca2+ homeostasis and BP are the results of epidemiologic studies that show an inverse correlation between Ca2+ intake and blood pressure in human populations. Among the first studies were post-hoc analyses of the National Health and Nutrition Examination Survey (NHANES) database, which showed an inverse correlation between dietary calcium intake and blood pressure.9,10 Subsequent studies have been performed in several different cultures and the results generally support this inverse correlation between calcium intake and BP.11,12 The third line of evidence are the results of clinical trials that assessed the effect of Ca2+ supplementation on BP in humans. The results of the first placebo-controlled trial that measured the effect of Ca2+ on blood pressure were reported by McCarron and Morris in 1985.13 This study assessed the effect of 1 g calcium or placebo per day over an 8-week period on 48 hypertensive patients and found that there was a small (3.8-mm Hg), but significant, decrease in BP. Moreover, a significant number of subjects responded with either a large increase or decrease in pressure, suggesting that subgroups of Ca2+-sensitive individuals may exist. Since this first report, additional trials have been performed and the results have been conflicting; some reported a small decrease in pressure associated with Ca supplementation14–16 and others reported no change.17,18 In an effort to better understand these data, metaanalyses have been performed, and the general conclusion is that Ca2+ supplementation causes a small (< 2 mm Hg) decrease in pressure in hypertensive humans.19,20 Of potential importance to the question of the effect of Ca2+ on human BP are two recent clinical trials. One was a large (n = 4589) multicenter trial that examined the effect of 2 g elemental Ca2+ or placebo on blood pressure and incidence of preeclampsia in young nulliparous women in the US21 The study concluded that there was no effect of Ca2+ on any parameter of BP in this group. The reasons for the disparity between this study and prior reports that showed effects of Ca2+ during pregnancy22,23 are not clear, but they may reflect differences in the industrialized and nondeveloped cultures that were studied. In contrast with this negative data obtained with a pregnant population, the results of another large (n = 459) trial, Dietary Approaches to Stop Hypertension (DASH), were recently published and showed that supplementation of a fruit and vegetable diet with dairy products, raising calcium intake from approximately 400 mg to 1200 mg per day, caused a significant reduction in pressure in both normotensive (3.5 mm Hg) and hypertensive subjects (11.4 mm Hg).24 These latter data imply that additional nutritional factors that are present in dairy products may interact with Ca2+ to lower blood pressure. Taken together, the data from the three different areas summarized above support a connection between Ca2+ intake (and hence Ca2+ homeostasis) and BP. Because of the potentially important basic science and therapeutic implications, there have been many attempts to understand the physiologic mechanisms by which Ca2+ intake modifies blood pressure.25,26 The working hypothesis that my laboratory has pursued for nearly a decade is that whole-animal Ca2+ homeostasis is linked with BP through vascular actions of serum ionized Ca2+ or the calciotropic hormones parathyroid hormone (PTH) and 1,25-(OH)2-vitamin D3 (1,25 vitD). This hypothesis was based on observations that serum ionized Ca2+ is depressed and PTH and 1,25 vitD are elevated in the subgroup of humans with essential hypertension that respond to Ca2+ with a decrease in blood pressure.27–29 To date, we and others have examined the vascular actions of PTH and 1,25 vitD and have reached the conclusion that, consistent with early reports,30 PTH is a vasodilator and would thus be expected to exert a depressor effect on the vasculature. In contrast, we have found that 1,25 vitD acts via a genomic mechanism to modulate contractile protein expression and enhance contractility of vascular smooth muscle, and could thus have a long-term pressor action.31–34 In contrast with the results for PTH and 1,25 vitD, however, there has been little serious consideration of the possibility that changes in ionized Ca2+ could alter vascular reactivity and modulate blood pressure. Two factors have, in large part, contributed to this skepticism. One is that it has been thought that only Ca2+ in the serum would have access to, and affect, the muscle cell and it is accepted that the concentration of Ca2+ in the serum is tightly regulated within narrow limits.35 The second is that, whereas Ca2+ has been shown to alter vascular reactivity, most studies have found effects only at very high, nonphysiologic concentrations.36,37 On the basis of recent experiments performed in my laboratory, however, we argue that there is now sufficient evidence to propose that shifts in extracellular Ca2+ concentration can occur and do modify vascular function. In 1911, Cow published the results of experiments in which he examined the reactivity of arteries isolated from several species and found that elevation of Ca2+ above normal depresses reactivity.38 A half-century later, Bohr36 reported that elevated concentrations of extracellular Ca2+ depress the early rapid response of the aorta to epinephrine, whereas they increase the steady-state response. Hollaway and Bohr then reported a method for quantifying Ca2+-induced relaxation of isolated femoral arteries and attributed the relaxing action of Ca2+ to a stabilizing effect of the cation acting at the cell membrane.37 Webb and Bohr then characterized further the relaxation response of rat tail arteries and in 1978 reported that the effect is antagonized by pretreatment with ouabain, incubation in low extracellular Na+, or decreasing temperature, and concluded that Ca2+ relaxes smooth muscle by activating the Na+ pump.39 Wu and Bohr subsequently showed that K+-induced contraction of rat aorta is depressed as extracellular Ca2+ is raised from 1.6 to 2, 5, and 10 mmol/L, and is partially dependent upon an intact endothelium.40 Our own work in this area initially tested the hypothesis that more physiologically relevant concentrations of extracellular Ca2+ modulate arterial function and demonstrated that raising Ca2+ from 1.0 to 1.5 to 2 mmol/L significantly depresses the contractile response of isolated arteries to norepinephrine.41 Subsequent experiments were designed to expand upon our discovery made using Ca2+-depleted vessels that raising extracellular Ca2+ from 0.8 to 1.6 mmol/L causes a reproducible relaxation. As shown in Figure 2, raising extracellular Ca2+ bathing norepinephrine-precontracted arteries from 1.0 to ≥ 1.25 mmol/L induces significant relaxation. Moreover, we have shown that the relaxation is blocked by precontracting the artery with depolarizing concentrations of K+ and by K+ channel blockade, and is associated with a decrease in myofilament Ca2+ sensitivity.42 The latter observations had important implications, as it was known that myofilament Ca2+ sensitivity is modulated via a G-protein–coupled mechanism and suggested that Ca2+-induced relaxation was associated with a receptor-linked event. At that time, the best candidate for a receptor-based mechanism linking changes in extracellular Ca2+ with alterations in G-protein–coupled function was the Ca2+ receptor cloned from bovine parathyroid by Brown and colleagues.43 The cDNA encoding the bovine parathyroid Ca2+ receptor predicts a 120-kDa protein with the seven-membrane spanning domain that characterizes G-protein–linked receptors. The receptor has a large extracellular domain and multiple glycosylation sites and is believed to contain the low-affinity Ca2+ binding site(s) and a shorter intracellular tail containing four consensus protein kinase (PKC) phosphorylation sites. The receptor shares approximately 40% homology with the metabotropic glutamate receptor, but is otherwise unique.43 An increase in ionized Ca2+ at the surface of the parathyroid cell is believed to activate the Ca2+ receptor, which is then coupled with inositol triphosphate (IP3)-mediated release of intracellular Ca2+ and a decrease in cAMP. These signals, in turn, work to decrease PTH secretion.44 Although messages encoding the Ca2+ receptor have been cloned from bovine43 and human parathyroid,45 human thyroid,46 and rat kidney47 and brain,48 until our recent work, there was no evidence that it could be expressed in the wall of the artery or anywhere else in the peripheral vascular system. Original attempts to localize the Ca2+ receptor to the vascular system using Northern blot analysis,43,47 and by our group using reverse-transcriptase-polymerase chain reaction (RTPCR),25 failed to show the appropriate transcript. On the basis of reports that local neurogenic vasodilation is mediated by the release of vasodilator peptides from sensory nerves,49,50 and the report of Ruat et al that brain tissue contains messages encoding the Ca2+ receptor,48 we formulated the hypothesis that a Ca2+ receptor is present in the perivascular sensory nerve network. Moreover, as cell bodies of the sensory afferent nerves are located in dorsal root ganglia51 we postulated that messages encoding the Ca2+ receptor (CaR) would be restricted to dorsal root ganglia (DRG), whereas protein would be transported to and expressed in the peripheral nerve processes. Both of these postulates have been supported by experimental data,52 ie, we have demonstrated that CaR mRNA is present in DRG and have shown using immunocytochemical methods that CaR protein is both in the DRG and in the perivascular nerve network.52 These findings not only established that a CaR is present in the arterial wall, but had two additional consequences. One was that they caused a shift in our thinking from the restricted view that only Ca2+ in the serum would be available to alter vascular reactivity, to considering the possibility that Ca2+ in the interstitial space could serve as a vascular effector. The second was that they supported the novel concept that extracellular Ca2+ could modulate vascular function through an action on perivascular nerves. In support of the latter we have performed a series of experiments addressing the hypothesis that Ca2+ relaxes isolated arteries by inducing the release of a vasodilator substance from perivascular sensory nerves. The results support this concept and show that acute and subacute phenolic denervation ablate Ca2+-induced relaxation,52 chronic denervation of rats by neonatal treatment with capsaicin significantly impairs Ca2+-induced relaxation,53 Ca2+ induces the release of a diffusible vasodilator substance from the adventitial surface of donor mesenteric arteries in a bioassay system, and the dilator substance is not a typical sensory nerve peptide transmitter such as calcium gene-related peptide (CGRP); rather it has the pharmacologic properties of a hyperpolarizing factor.52 The latter finding is of potential importance because it provides the first description of a nerve-derived hyperpolarizing vasodilator. We have thus far reviewed data that link blood pressure with whole-body Ca2+ homeostasis and discussed the results of our experiments indicating that Ca2+ can relax arteries via activation of a perivascular CaR. Equally as important for our hypothesis, however, is the question of whether the concentration of Ca2+ in the fluid in contact with the perivascular sensory nerve CaR can achieve levels high enough to activate the receptor. Although it is established that serum ionized Ca2+ in the mixed venous blood is tightly within narrow limits (1.1–1.3 mmol/L) by endocrine mechanisms, the concentration of Ca2+ in serum is ultimately a function of the amount of Ca2+ that diffuses into and out of the blood from the interstitial space at specific vascular/parenchymal interfaces. These interfaces are bone, where deposition and resorption of Ca2+ are under the influence of calcitonin and PTH; the small bowel, where active absorption of Ca2+ takes place and is modulated by 1,25 vitD; and the kidney, where reabsorption of Ca2+ from the urine takes place and is enhanced by PTH and 1,25 vitD. An important, albeit simple, concept when considering Ca2+ homeostasis is that for Ca2+ to move from the interstitial space into the serum compartment, it must diffuse down a chemical gradient. Thus, in tissues where Ca2+ enters the blood, the concentration of ionized Ca2+ in the interstitium must be greater than that in the plasma. Due to the fact that the measurement of any ion in the interstitial fluid of any tissue is technically challenging, the precise concentration of Ca2+ in the interstitium, and thus the degree to which it is elevated above serum levels, has not been well established. When it has been measured, however, ultrafiltrate or ionized Ca2+ in the interstitium of subcutaneous tissue has been shown to be greater than serum Ca2+.54,55 To our knowledge, the concentration of interstitial Ca2+ in the submucosal space or the renal interstitium has not been measured. Direct measurements, have, however, been made beneath resorbing osteoclasts and surprisingly high values (20–30 mmol/L) were recorded.56 Whereas such dramatic increases almost certainly do not occur in other tissues, it is likely that modest elevations in interstitial Ca2+ do occur in the submucosa and renal interstitium during periods of high Ca2+ intake or during states in which there is excess Ca2+ mobilization, ie, hyperparathyroidism or exposure to microgravity. This Ca2+ would then be available to activate the perivascular sensory nerve CaR and induce release of vasodilator (Figure 1). The sequence of events postulated here is of potential significance for several reasons. One is that it provides a novel mechanism of autoregulation linking changes in local transcellular Ca2+ transport with changes in blood flow. For example, during periods of high Ca2+ intake in which Ca2+ absorption from the gut and reabsorption from the kidney would be elevated, one would predict vasodilation and an attendant decrease in resistance and an increase in blood flow. Evidence supporting the prediction that Ca2+ intake modulates intestinal blood flow was provided by Schleiffer and colleagues, who showed that chronic intestinal administration of Ca2+ to the rat causes a significant increase in mesenteric blood flow.57 Additionally, changes in renal blood flow might well be linked with blood pressure, as Cowley and Roman have shown, through an elegant series of experiments, that chronic local administration of vasoactive drugs to the renal medulla modulates papillary blood flow, Na+ and H2O reabsorption, and BP of the whole animal.58 Another reason why our hypothesis may have important consequences is that it describes a role for the sensory nerve CaR as cardiovascular regulator and, as such, this protein will need to be considered as a potential site for pathologic activity. Of interest, mutations in the human parathyroid CaR have already been described; they are linked with inherited diseases including familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism.59 It will be of interest to determine whether a similar lesion might be present in the sensory nerve CaR and contribute to cardiovascular disease processes. Finally, because the parathyroid CaR has served as a successful receptor for drug targeting,60 the perivascular sensory nerve CaR should also serve as a unique molecular target for the development of novel vasodilator compounds, ie, selective CaR agonizes that could then be used in the treatment of hypertensive disease. I am grateful for the thoughtful input of the following individuals during the development of these ideas: Drs. Don DiPette, Scott Supowit, and Donna Wang from UTMB, and Dr. Edward Nemeth, of NPS Pharmaceuticals.