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(Hypertension. 1997;30:1431-1439.)
© 1997 American Heart Association, Inc.

Articles

Perivascular Sensory Nerve Ca²⁺ Receptor and Ca²⁺-Induced Relaxation of Isolated Arteries

Richard D. Bukoski; Ka Bian; Yanlin Wang; ; Maria Mupanomunda

From the Section of Hypertension and Vascular Research, Departments of Internal Medicine (R.D.B., K.B., M.M.), Physiology and Molecular Biophysics (R.D.B.), and Pharmacology and Toxicology (R.D.B., Y.W.), University of Texas Medical Branch, Galveston Island, Tex.

Correspondence to Richard Bukoski, PhD, Professor of Medicine and Physiology, 8.104 Medical Research Building, University of Texas Medical Branch, Galveston, TX 77555-1065. E-mail rbukoski{at}impo1.utmb.edu

	Abstract

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Abstract The present study tested two hypotheses: (1) that a receptor for extracellular Ca²⁺ (Ca²⁺ receptor [CaR]) is located in the perivascular sensory nerve system and (2) that activation of this receptor by physiological concentrations of extracellular Ca²⁺ results in the release of vasodilator substance that mediates Ca²⁺-induced relaxation. Reverse transcription-polymerase chain reaction using primers derived from rat kidney CaR cDNA sequence showed that mRNA encoding a CaR is present in dorsal root ganglia but not the mesenteric resistance artery. Western blot analysis using monoclonal anti-CaR showed that a 140-kD protein that comigrates with the parathyroid CaR is present in both the dorsal root ganglia and intact mesenteric resistance artery. Immunocytochemical analysis of whole mount preparations of mesenteric resistance arteries showed that the anti-CaR–stained perivascular nerves restricted to the adventitial layer. Biophysical analysis of mesenteric resistance arteries showed that cumulatively raising Ca²⁺ from 1 to 1.25 mol/L and above relaxes precontracted arteries with an ED₅₀ value of 2.47±0.17 mmol/L (n=12). The relaxation is endothelium independent and is unaffected by blockade of nitric oxide synthase but is completely antagonized by acute and subacute phenolic destruction of perivascular nerves. A bioassay showed further that superfusion of Ca²⁺ across the adventitial surface of resistance arteries releases a diffusible vasodilator substance. Pharmacological analysis indicates that the relaxing substance is not a common sensory nerve peptide transmitter but is a phospholipase A₂/cytochrome P450–derived hyperpolarizing factor that we have classified as nerve-derived hyperpolarizing factor. These data demonstrate that a CaR is expressed in the perivascular nerve network, show that raising Ca²⁺ from 1 to 1.25 mol/L and above causes nerve-dependent relaxation of resistance arteries, and suggest that activation of the CaR induces the release of a diffusible hyperpolarizing vasodilator. We propose that this system could serve as a molecular link between whole-animal Ca²⁺ balance and arterial tone.

Key Words: calcium • receptors, sensory • muscle, smooth, vascular • resistance • hyperpolarizing factor

	Introduction

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The observation that Ca²⁺ ion, acting at the cell exterior, modulates vascular tone and resistance to blood flow has long intrigued smooth muscle physiologists. In 1911, Cow¹ showed that elevation of Ca²⁺ above normal levels depresses reactivity of isolated arteries. More than 40 years later, Holman,² using isolated guinea pig taenia coli, reported that raising extracellular Ca²⁺ transiently decreases electrical activity of the guinea pig taenia coli, contrary to the predicted augmentation. Bohr³ subsequently showed that elevated concentrations of extracellular Ca²⁺ depress the early rapid response of rat aorta to epinephrine, whereas the same levels of Ca²⁺ increase the steady state response. Nearly a decade later, Holloway and Bohr⁴ reported a method for quantifying Ca²⁺-induced relaxation of isolated arteries and suggested that Ca²⁺ induces relaxation via a membrane stabilizing action. Subsequent work by Bohr and colleagues^{5 6} has further characterized the vessel response to extracellular Ca²⁺.

Although these experiments established the fact that extracellular Ca²⁺ suppresses vascular force generation, acceptance of this phenomenon as a physiological regulatory mechanism has been limited for two reasons. One is the fact that it has generally been assumed that the concentrations of Ca²⁺ required to alter vascular function cannot be achieved in vivo. New evidence suggests that this is not true and indicates that in tissues involved in transcellular Ca²⁺ movement (eg, intestine, kidney, and bone), the concentration of Ca²⁺ in the interstitial space can be significantly higher than is present in the mixed venous plasma.⁷

A second reason for limited acceptance is that no viable molecular mechanism explaining Ca²⁺-induced relaxation has been demonstrated. The identification of a cellular mechanism for this action has been a focus of our laboratory,^{8 9} and we recently demonstrated that Ca²⁺-induced relaxation of the rat mesenteric resistance artery is attenuated by blockade of Ca²⁺-activated K⁺ channels and is associated with a decrease in myofilament Ca²⁺ sensitivity.¹⁰ Because extracellular Ca²⁺ should not directly activate K⁺ channels and regulation of myofilament Ca²⁺ sensitivity is a G protein-coupled process,¹¹ we proposed that extracellular Ca²⁺ induces relaxation by activating a membrane spanning receptor for extracellular Ca²⁺ that is similar to that which has been reported in other tissues, including parathyroid, thyroid, and kidney.^{12 13 14 15}

Our initial attempts at identifying the CaR in arterial tissue using RT-PCR amplification of CaR mRNA failed to show the appropriate transcript¹⁶ and confirmed earlier reports.^{12 13 15} More recently, however, it was demonstrated that message for the CaR is expressed in neural tissue.¹⁷ With the knowledge that small arteries of the rat contain a dense perivascular sensory nerve network that is functionally linked with vasodilation^{18 19} and that the cell bodies of the sensory fibers that are the sites of mRNA processing and protein synthesis are located in the DRG,²⁰ we postulated that the perivascular sensory nerve network houses a vascular CaR that mediates Ca²⁺-induced relaxation of small arteries. We now demonstrate that a receptor for extracellular Ca²⁺ is present in sensory nerves of the mesenteric resistance artery wall and present physiological and pharmacological evidence that is consistent with the hypothesis that Ca²⁺ modulates vascular reactivity by activation of this receptor. Preliminary reports of this work have been published in abstract form.^{21 22}

	Methods

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Materials
Peptide antagonists, including CGRP_8–37, Spantide II, galantide, 4 Cl-D-Phe,⁶Leu¹⁷-VIP, and -helical CRF 9–41, were purchased from Phoenix Pharmaceuticals; nor-binaltorphimine was purchased from Research Biochemicals; and SR48968 and SR140333 were generously supplied by Sanofi Recerche. Unless otherwise noted, all other chemicals used in these studies were of analytical grade and were purchased from the Sigma Chemical.

Animals
All procedures involving animals were performed in accordance with approval of the Institutional Animal Care and Use Committee. Male Wistar rats (6 to 10 weeks of age) were obtained from Harlan-Sprague Dawley and on arrival in our animal care facility were maintained in colony rooms with fixed light/dark cycles and constant temperature and humidity and provided with Purina rodent chow and water ad libitum. Vascular or other tissue was isolated while the rats were anesthetized with a mixture of ketamine and xylazine (100/5 mg/kg). Subchronic phenolic denervation of mesenteric arteries was achieved by applying 10% ethanolic phenol onto branch I mesenteric arteries through a midline abdominal incision while the animals are under anesthesia. The wound was then closed, and arteries distal to the site of phenol application were isolated 72 hours later.

RT-PCR
Total RNA was isolated as recently described,²³ and 2 µg was reverse-transcribed using AMV reverse transcriptase and deoxynucleotides provided in a kit from Boehringer-Mannheim Biochemicals that was used according to the manufacturer's instructions. Resulting cDNA was amplified through 35 cycles using Taq DNA polymerase (Promega) with an annealing temperature of 55°C and a polymerization period of 7 minutes at 72°C. The products were size-separated on 1.0% agarose gels and stained with ethidium bromide. The sequence of the primers, which were designed to amplify CaR cDNA corresponding to the region 941 to 1993, were 5'-GAACCTGGACGAGTTCTG-3' for the forward primer and 5'-CCATGTTGTTGGTGAAG-3' for the reverse primer. Sequence analysis of the 1052-bp PCR product from both DRG and kidney was performed using cycle sequencing with a dye terminator (Perkin-Elmer Cetus) and a 3703A Applied Biosystems DNA sequence analyzer and showed complete sequence homology with the published rat kidney sequence.¹⁵

Western Blot Analysis
Protein was extracted and size-separated using 8% SDS-PAGE and then electroblotted onto nitrocellulose membrane as recently described.²³ The membrane was incubated overnight with a monoclonal mouse anti-CaR (0.13 µg/mL) raised against the conserved parathyroid CaR sequence ADDDYGRPGIEKFREEAEERDI (provided by NPS Pharmaceuticals in conjunction with Drs Allen Spiegel and Paul Goldsmith, NIDDK). In some experiments, anti-CaR was preabsorbed with 50 mg/mL excess antigen. The membrane was then incubated with horseradish peroxidase-coupled anti-mouse IGG (Amersham), and the resulting protein/antibody complex was visualized on x-ray film using the enhanced chemiluminescence method (ECL; Amersham).

Immunocytochemistry
Vessels were cleaned of fat and connective tissue, and luminal blood was removed by flushing with cold PSS. After brief fixation in ice-cold methanol, segments were rinsed in TBS, intrinsic peroxidase activity was quenched using a mixture of H₂O₂ and NaN₃ (DAKO), and nonspecific binding was blocked using a serum-free blocker (DAKO). The tissues were then incubated overnight with TBS alone (negative control) or anti-CaR (20 µg/mL). After washing, intrinsic biotin was blocked using a biotin blocking kit (DAKO), and the segments were incubated with biotin-conjugated secondary antibody (1:250) in the presence of the 2% serum from the appropriate species. The segments were then stained using horseradish peroxidase-conjugated avidin (Vector Labs) using 3–3'-diaminobenzene (Sigma) as substrate, counterstained with hematoxylin, dehydrated through sequential ethanol and xylene, and permanently mounted. The preparations were then visually examined using a Nikon Microphot FXA research microscope, and photographs were taken with the focus on either the adventia, media, or intima as judged by the pattern of nuclear staining.

Biophysical Measurements
Isometric force generation was measured using previously described methods.¹⁰ Branch II mesenteric resistance arteries were isolated, cleaned of fat and connective tissue, and placed in ice-cold PSS of the following composition (in mol/L) NaCl 140, KCl 4.7, MgSO₄7H₂O 1.17, NaHCO₃ 5, KH₂PO₄ 1.15, Na₂HPO₄ 1.10, CaCl₂ 1.0, HEPES 20, and glucose 5, pH 7.4. The vessels were then mounted on a wire myograph warmed to 37°C and gassed with 95% air/5% CO₂, stretched to a predetermined length that was equivalent to an internal diameter of 200 to 225 µm, and allowed to equilibrate for a 30-minute period. After the equilibration period, the vessels were induced to contract with 5 µmol/L norepinephrine until reproducible contractile responses were obtained (three or four times).

Some vessels were studied after removal of the endothelium using a human hair, and functional denudation was verified in subsequent protocols by the absence of a relaxation response to the addition of 1 µmol/L acetylcholine.²⁴ Other vessels underwent a procedure to chemically damage perivascular nerves.²⁵ PSS was drained away from the segment, and 50 µL of a 0.75% solution of ethanolic phenol (90:10) in PSS was directly applied for a 15-second period (time empirically derived in preliminary experiments) followed by extensive washing over a 30-minute period. This treatment results in a nearly complete loss of a contractile response to activation of perivascular nerves using electrical field stimulation at 30 Hz, 70 V, and 2 ms duration.

Ca²⁺-induced relaxation was assessed by cumulatively adding Ca²⁺ to vessels that were precontracted with 5 µmol/L norepinephrine; the magnitude of relaxation was expressed as the percentage of the initial tension. When phasic activity was present, measurements were made from the precontraction baseline to the trough of the phasic transition.

Bioassay
A bioassay was used to determine whether a diffusible vasodilator substance is elicited by Ca²⁺ from the adventitial surface of mesenteric resistance arteries. The bioassay segment was a 1.5- to 2-mm-long ring of mesenteric resistance artery mounted on a wire myograph; donor tissue consisted of a section of the mesenteric resistance artery arcade with a mass 20 to 30 times that of the bioassay segment that was affixed above the preparation by means of a cannula placed in a section of the mesenteric trunk. PSS that was gassed with 95% air/5% CO₂ and kept at 35°C was allowed to drop onto the bioassay segment at a flow rate of 1.5 mL/min either directly from a cannula or after superfusion across the donor segment. To quantify the relaxation response, the experimental trace was digitized using UN-SCAN-IT software (Silk Scientific Corp), and the area under the relaxation curve was determined for each treatment.

Statistical Analysis
All data are presented as mean±SEM, and statistical analysis was performed using the SYSTAT software package. Comparisons among groups were performed using ANOVA with a repeated measures design when appropriate. A value of P<.05 was taken to indicate a statistically significant difference.

	Results

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CaR Gene Expression
To assess CaR gene expression, total RNA was isolated from rat kidney (positive control, dorsal root ganglia, and mesenteric resistance artery). Complementary DNA was synthesized by RT and amplified by PCR with primers designed to amplify a 1052 bp region of the rat kidney CaR.¹⁵ mRNA encoding the CaR was present in the kidney and DRG but not the mesenteric resistance artery (Fig 1A). Control experiments in which PCR was performed in the absence of the RT step revealed no product, indicating that there was no contamination of the sample with genomic DNA. Sequence analysis of the amplimer isolated from both the kidney and DRG indicated complete homology with the published rat kidney sequence. These data indicate that mRNA for the CaR is present in cell bodies of sensory nerves but is not detectable in the arterial wall. Possible explanations are that CaR protein is synthesized in the DRG cell body and transported peripherally to perivascular neurons or that the concentration of CaR mRNA is too low to be detected in nerves of the arterial wall using the RT-PCR method.

View larger version (40K): [in this window] [in a new window]   Figure 1. Tissue distribution of CaR mRNA and protein. A, A 1052-bp RT-PCR product homologous with rat kidney CaR was found in kidney and DRG but not in the mesenteric resistance artery. +, PCR of the reverse-transcribed RNA product; -. PCR of non–reverse-transcribed RNA showing the absence of contamination with genomic DNA; ladder is 1 kb DNA (Stratagene). B, Western blot analysis using monoclonal anti-CaR. Lanes contain 20 µg protein from thyroparathyroid and DRG and 75 µg from mesenteric resistance artery. Protein migrating at 140 and 160 kD is CaR. -, Monoclonal anti-CaR alone; +, antibody preabsorbed with excess antigen.

CaR Protein Expression
Western blot analysis was used to test the hypothesis that protein immunoreactive with the parathyroid CaR is present in DRG and the mesenteric resistance artery wall. Protein extracted from thyroparathyroid (positive control, dorsal root ganglia, and mesenteric resistance arteries) was used in Western blot analysis as described in "Methods." Consistent with previous reports,^{12 13} the parathyroid CaR migrated as a doublet with molecular masses of 140 and 160 kD (Fig 1B). Immunoreactive protein from DRG and mesenteric resistance arteries appeared as a single band that comigrated with the lower band of the parathyroid doublet (Fig 1B). Preabsorption of the anti-CaR with excess antigen significantly reduced CaR signal from all three preparations.

Immunocytochemical Localization
Immunocytochemical analysis was used to determine the site of expression of the CaR in the mesenteric resistance artery wall. Whole-mount preparations were immunostained with anti-CaR as described in "Methods" and counterstained with hematoxylin to visualize nuclei in the various layers of the artery. A fine nerve-like pattern of staining was present in the adventitial layer (Fig 2A) but not in the medial (Fig 2B) or endothelial (Fig 2C) layers. Moreover, no staining was observed when secondary antibody was used alone (Fig 2D).

View larger version (157K): [in this window] [in a new window]   Figure 2. Photomicrographs of whole-mount preparations of incubated mesenteric resistance artery stained with monoclonal anti-CaR (1:100) and stained with DAB, which yields a brown stain. Counterstaining was performed using hematoxylin, which provides a purple nuclear stain. A, Adventitial layer with CaR-positive nerve fibers; randomly oriented nuclei are adventitial cells. B, Medial layer showing oblique pattern of nuclear staining of smooth muscle cells without distinct DAB staining. C, Intimal layer with ordered nuclear staining of endothelial cells. D, Adventitial layer incubated with biotinylated anti-mouse IGG alone and counterstained with hematoxylin. Vessels measure 283 µm from edge to edge.

Biophysical Measurements
Experiments were also performed to test the hypothesis that activation of the perivascular CaR is linked with Ca²⁺-induced modulation of arterial reactivity. Because there are no known antagonists of the CaR, these experiments were designed to establish the Ca²⁺ sensitivity and neuronal dependence of the Ca²⁺ relaxation event. As illustrated in Fig 3A, the addition of 5 µmol/L norepinephrine to the mesenteric resistance artery induces an increase in tone that persists at a stable level for 30 minutes and exhibits vasomotion. When extracellular Ca²⁺ is cumulatively added, dose-dependent relaxation was observed (Fig 3B) with an ED₅₀ value of 2.47±0.17 mmol/L determined from vessels from 12 separate animals. Moreover, the addition of 3 or 5 mmol/L Ca²⁺ often caused a transient suppression of rhythmic activity, which returned after a 3- to 5-minute delay.

View larger version (12K): [in this window] [in a new window]   Figure 3. A, Time course of the isometric force response of an isolated mesenteric artery to 5 µmol/L norepinephrine. B, Response of the same artery to cumulative addition of extracellular Ca2+ after induction of contraction with 5 µmol/L norepinephrine.

Because it is possible that Ca²⁺-induced relaxation is mediated by an effect of Ca²⁺ to induce the production of NO, from either the endothelium^{6 26} or nitroxidergic nerves,²⁷ the effect of inhibition of NO synthase on Ca²⁺-induced relaxation was assessed by pretreatment with 0.3 mmol/L L-NAME, which is a dose²⁴ that effectively inhibits acetylcholine-induced relaxation of segments precontracted with 100 mmol/L K⁺ (control, 32.5±8% relaxation; L-NAME, 14.5±7.2%; P<.05, n=6). Blockade of NO synthase had no effect on the sensitivity to Ca²⁺ or the magnitude of relaxation induced by Ca²⁺, indicating that neither endothelial nor neuronal NO mediates the relaxation (Fig 4).

View larger version (14K): [in this window] [in a new window]   Figure 4. Response of isolated mesenteric resistance arteries after precontraction with 5 µmol/L norepinephrine under control conditions, after 15-minute preincubation with 0.3 mmol/L L-NAME, or after mechanical disruption of the endothelium (denuded). No effect of either endothelial denudation or blockade of NO synthase was detected. Values are mean±SEM (n=5 or 6).

Endothelial cells release vasodilator substances other than NO(ie, cyclooxygenase products such as prostacyclin and the putative endothelium-derived hyperpolarizing factor).²⁸ A role for the endothelium was therefore assessed by studying vessel segments that were mechanically denuded of endothelium. Removal of the endothelium significantly reduced acetylcholine-induced relaxation of vessels precontracted with 5 µmol/L norepinephrine (relaxation_intact=90.5±3.9% versus relaxation_denuded=8.3±2.5, P<.005, n=5) but had no effect on relaxation induced by Ca²⁺ (Fig 4).

To test the hypothesis that Ca²⁺-induced relaxation is dependent on functionally intact perivascular nerves, the effect of phenolic nerve destruction was assessed.²⁵ Acute denervation was achieved by applying a 0.75% solution of phenol directly to the vessel for 15 seconds followed by extensive washing. After treatment with phenol, there was generally a reduction in the force response to norepinephrine and in contrast to the relaxation response observed before the addition of phenol, cumulative addition of Ca²⁺ resulted in an incremental increase in active force (Fig 5A). To test whether the intrinsic ability of the vessel to relax was affected by phenol, the relaxation responses were assessed to the NO donor sodium nitroprusside and the K⁺ channel opener (pinacidil). In contrast with the response to Ca,²⁺ both agonists relaxed precontracted arteries. The response to nitroprusside was intact compared with prior results,²⁴ whereas the response to pinacidil was shifted to the right (pD2 [mol/L] for pinacidil_cont=-5.81±0.2 versus pinacidil_phenol=-4.67±0.19, P<.05, n=5], indicating a slight inhibitory effect on the K⁺ channel agonist.

View larger version (14K): [in this window] [in a new window]   Figure 5. A, Effect of acute denervation by topical application of 0.75% phenol to isolated segments on relaxation induced by cumulative addition of Ca2+, sodium nitroprusside (SNP), and pinacidil. Vessel segments were precontracted with 5 µmol/L norepinephrine. The effect of phenol treatment on relaxation induced by Ca2+ was significant at P=.007 (n=5). B, Effect of subchronic phenolic denervation (n=4, P=.026) performed as described in the text on relaxation of norepinephrine precontracted arteries induced by Ca2+ Values are mean±SEM.

As a test for possible nonspecific phenol-induced damage to the artery, the effect of applying phenol 3 days before harvesting the vascular tissue was assessed as described in "Methods." As with the acute application of phenol, increasing Ca²⁺ caused contraction of vessels that were taken from animals treated with phenol, whereas vessels from sham/vehicle–treated animals relaxed in response to Ca²⁺ (Fig 5B). It should be noted that the relaxation response of sham-treated animals was decreased relative to the responses shown in Figs 3 to 5, and we suspect that this is the result of a neurotoxic effect of the vehicle (100% ethanol).

Because these data indicated that extracellular Ca²⁺ induces relaxation through a nerve-dependent mechanism, a bioassay was used to test the hypothesis that extracellular Ca²⁺ elicits the release of a diffusible vasodilator substance from the adventitial surface of mesenteric resistance arteries. Direct addition of norepinephrine to the bioassay vessel through a cannula caused an increase in force; and increasing concentrations of extracellular Ca²⁺ caused a graded decrease in tension (Fig 6). When the procedure was repeated, allowing the solution to first superfuse the donor arcade, two effects were observed. The first was that the contractile response of the bioassay vessel to norepinephrine was significantly attenuated by superfusion of the medium across the donor (peak response was 68±8.9% of control, P=.009), suggesting the presence of a basal vasodilator factor; the second was that the relaxation response to elevated Ca²⁺ was significantly enhanced, particularly at the lowest concentrations of Ca²⁺ (Fig 6).

View larger version (17K): [in this window] [in a new window]   Figure 6. Bioassay. A, Top, Response of a bioassay mesenteric resistance artery to 5 µmol/L norepinephrine and subsequent direct addition Ca2+ through a cannula. Bottom, Response of the same artery to the same concentrations of norepinephrine and Ca2+ that were first superfused over an isolated mesenteric artery donor arcade. B, Line graph showing the difference in the magnitude of the relaxation response to Ca2+ with and without superfusion over the donor vessel. Responses are presented as areas under the curve for a given time period; values are mean±SEM, P<.05 (n=9).

Experiments were also performed to test the hypothesis that the vasodilator substance that mediates Ca²⁺-induced relaxation is one of the primary sensory nerve transmitters (ie, either CGRP or a member of the tachykinin family).²⁹ Blockade of CGRP receptors by preincubation with CGRP_8–37 (1 µmol/L), which is a selective CGRP antagonist³⁰ that significantly depresses CGRP-induced relaxation in this preparation (data not shown), was without effect on Ca²⁺-induced relaxation (Fig 7). Similarly, blockade of the substance P (neurokinin 1) receptor with either the peptide antagonist Spantide II (10 µmol/L)³¹ or the nonpeptide antagonist SR140333 (0.3 µmol/L)³² was without effect (Fig 7). Moreover, blockade of the neurokinin 2 (tachykinin A) receptor with the nonpeptide antagonist SR48968 (0.3 µmol/L)³² also was without effect on Ca²⁺-induced relaxation (Fig 7). Because these data and preliminary experiments using peptide antagonists for other possible neurotransmitters, including 4 Cl-D-Phe,⁶Leu¹⁷-VIP, nor-binaltorphimine (dynorphin), -helical CRF, and galantide (galanin),³³ indicated that the mediator might not be a peptide transmitter, we tested the hypothesis that the mediator is a hyperpolarizing factor.

View larger version (19K): [in this window] [in a new window]   Figure 7. Response of norepinephrine-contracted mesenteric resistance arteries to Ca2+ after preincubation with CGRP8–37 (1 µmol/L; spantide II [10 µmol/L], SR140333 [3 µmol/L], or SR 48968 [3 µmol/L]). No effect of any of the antagonists was detected. Values are mean±SEM (n=6 or 7 per group).

Pretreatment with 10 mmol/L TEA, which is a broad range K⁺ channel blocker,³⁴ completely inhibited Ca²⁺-induced relaxation (Fig 8). Similarly, when mesenteric resistance arteries were precontracted with 100 mmol/L K⁺ in the presence of 1 µmol/L phenotolamine, the relaxation response to cumulative addition of Ca²⁺ was completely blunted (Fig 8). Because these data supported the hypothesis that Ca²⁺ may induce the production of a hyperpolarizing factor and there is considerable evidence that endothelium-derived relaxing factors are cytochrome P450–derived metabolites of arachidonic acid,^{35 36} we examined the effect of miconazole, which is a cytochrome P450 blocker.³⁷ Pretreatment of the mesenteric resistance arteries with 3 µmol/L miconazole, which had no effect on relaxation induced by pinacidil (data not shown), significantly inhibited Ca²⁺-induced relaxation (Fig 8). Finally, because arachidonic acid is liberated in many cells by activation of phospholipase A₂, we assessed the effect of 5 µmol/L quinacrine, which is a phospholipase A₂ antagonist.³⁸ This concentration of quinacrine, which had no effect on pinacidil-induced relaxation (data not shown), antagonized Ca²⁺-induced relaxation of isolated mesenteric resistance arteries (Fig 8).

View larger version (19K): [in this window] [in a new window]   Figure 8. Response of mesenteric resistance arteries to cumulative addition of Ca2+ after precontraction induced by 100 mmol/L K+ in the presence of 1 µmol/L phentolamine, pretreatment with 10 mmol/L TEA, miconazole (3 µmol/L), or quinacrine (5 µmol/L) before precontraction with 5 µmol/L norepinephrine. *Significant difference from control at P<.05 (n=5 or 6 per group).

	Discussion

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The present report describes several new and potentially important findings regarding the regulation of vascular reactivity by extracellular Ca.²⁺ One finding is that DRG, which house cell bodies of sensory nerve fibers, express mRNA for the CaR, and this message is apparently processed such that CaR protein is transported from DRG to the perivascular nerve network of peripheral arteries. The second finding is that raising Ca²⁺ from 1.0 to 1.25 mmol/L and above relaxes isolated resistance arteries, and the relaxation is completely eliminated by phenolic destruction of perivascular nerves. The third finding, based on the results of the bioassay, is that the adventitial surface of resistance arteries releases a diffusible vasodilator substance in response to increasing concentrations of Ca.²⁺ The fourth is that Ca²⁺-induced relaxation appears to be mediated by production or release of a nerve-derived hyperpolarizing factor, which is a cytochrome P450–generated metabolite of arachidonic acid. These findings have been collectively synthesized to form a novel hypothesis that states that alterations in extracellular Ca²⁺ modulate vascular reactivity via activation of a perivascular CaR that is localized to sensory nerves and responds to changes in extracellular Ca²⁺ with the release of a hyperpolarizing vasodilator.

Our findings concerning perivascular localization of the CaR confirm the report of Ruat et al,¹⁷ who showed that cerebral arteries stain positively for a perivascular CaR. Moreover, our data provide an important extension of this finding by demonstrating that the peripheral/perivascular sensory nerve system expresses a Ca²⁺-sensing receptor. A key implication of this observation is that in contrast with the current view that the distribution of the CaR is limited to a few tissues (parathyroid, thyroid, kidney, and brain), the receptor might be present in an extensive network throughout the organism. Our findings also mesh neatly with the concept that sensory nerve fibers have a sensory efferent motor function through which neurotransmitter is released in response to either reflex activation or local stimuli, including cytokines and hydrogen ions.^{19 29 39} These observations should have important implications in terms of our understanding of how whole-animal Ca²⁺ homeostasis modulates regional function.

Several aspects of our experiments merit further discussion, including the data that support the perivascular localization of the CaR. The demonstration is based on two independent pieces of evidence. One is the RT-PCR analysis that shows that message for the CaR is present in the DRG but not in tissue from the arterial wall. This is important because it explains why prior attempts at identifying the CaR using Northern blot analysis were not successful. The second set of supportive data are the results of the Western blot and immunocytochemical analyses. Interpretation of the RT-PCR data are strengthened significantly by the demonstration that antibody specific for the CaR revealed a perivascular nerve localization of the protein.

One point that needs to be addressed is the data that link activation of the CaR with modulation of vascular tone. The primary evidence that serves to establish the linkage are the fact that Ca²⁺-induced relaxation is not dependent on an intact endothelium, the finding that Ca²⁺-induced relaxation is abolished by acute and subchronic phenolic denervation, and the results of the bioassay that showed that raising extracellular Ca²⁺ induces the release of a diffusible vasodilator substance. Although provocative, we recognize that these findings are all indirect, and absolute proof of a linkage between the CaR and relaxation will depend on specific pharmacological blockade or tissue-specific deletion of the CaR in a relevant animal model. To our knowledge, however, no selective antagonists of the CaR are available, and the murine deletion mutant that has been developed does not grow past the neonatal stage and thus cannot be used for this type of cardiovascular study.⁴⁰

A related question is the identity of the vasodilator substance that mediates Ca²⁺-induced relaxation; the present study indicates that it is neither NO nor a common sensory nerve peptide transmitter such as CGRP or a member of the tachykinin peptide family but rather a nerve-derived hyperpolarizing factor. Among the data that support this conclusion are our findings that two maneuvers that antagonize the action of endothelium-derived hyperpolarizing factors (eg, precontraction of the artery with a depolarizing concentration of K⁺ and pretreatment with TEA) completely block Ca²⁺-induced relaxation (Fig 8). In addition, the results of the pharmacological analysis suggest further that Ca²⁺-induced production of the hyperpolarizing factor is associated with the release of arachidonic acid via activity of phospholipase A₂ and subsequent metabolism by a cytochrome P450 enzyme. Of note, the brain CaR has been shown to be coupled to the release of arachidonic acid when expressed in Chinese hamster ovary cells,⁴¹ and epoxyeicosatrienoic acids, which are cytochrome P450–generated compounds, have been shown to be hyperpolarizing vasodilators.^{42 43} To our knowledge, this is the first demonstration that an exogenous ligand (ie, Ca²⁺) can elicit the production of a nerve-derived hyperpolarizing factor from sensory nerves.

Another point that warrants discussion is the relationship between the apparent Ca²⁺ sensitivity of Ca²⁺ relaxation and the Ca²⁺ sensitivity of the CaR in other tissues. The CaR of the parathyroid gland is thought to regulate PTH secretion in response to changes in serum-ionized Ca²⁺ across the range of 0.8 to 2 mmol/L. The Ca²⁺ relaxation event is also sensitive over this range, but relaxation clearly occurs at concentrations as high as 5 mmol/L. There are two possible explanations. One is that the sensitivity of the perivascular CaR is similar to that of the parathyroid and a separate, non-CaR–linked mechanism is operative at the higher levels of Ca.²⁺ The second possibility is that the Ca²⁺ sensitivity of the perivascular CaR exhibits a broader range than that for PTH.

Aside from the fact that our results provide a mechanism by which increases in extracellular Ca²⁺ relax isolated arteries—a phenomenon that has intrigued vascular physiologists for >85 years^{1 2 3} —they may also provide insight into the physiological mechanisms by which alterations in whole-animal Ca²⁺ balance may be linked with blood pressure regulation. Although there is abundant evidence supporting the hypothesis that Ca²⁺ supplementation lowers blood pressure in hypertensive humans and in animal models of high blood pressure,^{44 45 46} the mechanism remains unclarified.¹⁶ The present data permit the formulation of a testable theoretical paradigm that describes a mechanism by which Ca²⁺ supplementation might modulate vascular reactivity and blood pressure. Specifically, we propose that increased Ca²⁺ intake causes elevations in interstitial Ca²⁺ in tissues involved in transcellular Ca²⁺ movement (eg, the intestine and kidney), and this induces the release of a local hyperpolarizing vasodilator, which in turn decreases vascular resistance and lowers blood pressure.

Other potential implications of this work are based on the observations that mutations in the CaR have already been linked with familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism.^{47 48} If gene polymorphisms exist in the perivascular CaR, they could contribute to the inherited component of human essential and experimental genetic hypertension.⁴⁹ Finally, because the CaR is a proven target for pharmacological manipulation,⁵⁰ our data also provide the rationale for exploring the application of CaR agonists and antagonists to a variety of human pathologies, including hypertension, and the management of pain and inflammation.

	Selected Abbreviations and Acronyms

 

CaR = Ca2+ receptor CGRP = calcitonin gene–related peptide CRF = corticotrophin-releasing factor DRG = dorsal root ganglia L-NAME = nitro-L-arginine methyl ester NO = nitric oxide PCR = polymerase chain reaction PSS = physiological salt solution RT = reverse transcription TBS = Tris-buffered saline TEA = tetraethyammonium VIP = vasoactive intestinal peptide

	Acknowledgments

 
This work was supported by grants from the National Institutes of Health (HL-54901) and NPS Pharmaceuticals (Salt Lake City, Utah) and we acknowledge the thoughtful input of Drs Edward Nemeth and Kim Rogers of NPS Pharmaceuticals.

Received May 28, 1997; first decision June 30, 1997; accepted July 9, 1997.

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