The perivascular sensory nerve (PvN) Ca2+-sensing receptor (CaR) is implicated in Ca2+-induced relaxation of isolated, phenylephrine (PE)-contracted mesenteric arteries, which involves the vascular endogenous cannabinoid system. We determined the effect of inhibition of diacylglycerol (DAG) lipase (DAGL), phospholipase A2 (PLA2), and cytochrome P-450 (CYP) on Ca2+-induced relaxation of PE-contracted rat mesenteric arteries. Our findings indicate that Ca2+-induced vasorelaxation is not dependent on the endothelium. The DAGL inhibitor RHC 802675 (1 μM) and the CYP and PLA2 inhibitors quinacrine (5 μM) (EC50: RHC 802675 2.8 ± 0.4 mM vs. control 1.4 ± 0.3 mM; quinacrine 4.8 ± 0.4 mM vs. control 2.0 ± 0.3 mM; n = 5) and arachidonyltrifluoromethyl ketone (AACOCF3, 1 μM) reduced Ca2+-induced relaxation of mesenteric arteries. Synthetic 2-arachidonoylglycerol (2-AG) and glycerated epoxyeicosatrienoic acids (GEETs) induced concentration-dependent relaxation of isolated arteries. 2-AG relaxations were blocked by iberiotoxin (IBTX) (EC50: control 0.96 ± 0.14 nM, IBTX 1.3 ± 0.5 μM) and miconazole (48 ± 3%), and 11,12-GEET responses were blocked by IBTX (EC50: control 55 ± 9 nM, IBTX 690 ± 96 nM) and SR-141716A. The data suggest that activation of the CaR in the PvN network by Ca2+ leads to synthesis and/or release of metabolites of the CYP epoxygenase pathway and metabolism of DAG to 2-AG and subsequently to GEETs. The findings indicate a role for 2-AG and its metabolites in Ca2+-induced relaxation of resistance arteries; therefore this receptor may be a potential target for the development of new vasodilator compounds for antihypertensive therapy.
- Ca2+-sensing receptor
- arachidonic acid
ionized calcium (Ca2+) is a second messenger involved in a number of cellular functions, such as secretion of hormones, muscle contraction, endocytosis, enzyme control, regulation of gene expression, as well as cell proliferation, differentiation, and apoptosis. The involvement of Ca2+ in coronary and vascular smooth muscle (VSM) reactivity has a bearing on vessel tone and blood pressure control. High Ca2+ intake has been shown to lower blood pressure in animal models of hypertension (31, 32, 40, 50), and diets high in fruits and vegetables, supplemented with predominantly low-fat milk, significantly reduced blood pressure compared with fruit- and vegetable-only diets (1, 3, 4, 57). Current evidence suggests that adequate Ca2+ intake (1,000–1,500 mg/day) is critical to optimal blood pressure regulation, and randomized controlled trials have revealed significant reductions in hypertension risk and blood pressure levels in humans (48, 49). It is now clear that Ca2+ availability to organs that participate in cardiovascular control is a key factor in blood pressure regulation. Although there is abundant clinical evidence to support the blood pressure-lowering effect of dietary Ca2+, mechanisms underlying this phenomenon are not clear, and no plausible explanation as to how Ca2+ lowers blood pressure exists.
In a series of studies, Ca2+-induced relaxation of mesenteric arteries was found to be dependent on an intact perivascular sensory nerve (PvN) network that expresses a Ca2+-sensing receptor (CaR), providing strong evidence that the CaR may serve as the missing link between Ca2+ intake and vascular function (8–10, 36, 51). We have demonstrated that dorsal root ganglion (DRG) expresses a CaR (8, 62, 63) and also provided further evidence that Ca2+-induced relaxation of isolated precontracted mesenteric arteries is mediated in part by a hyperpolarizing endocannabinoid vasodilator transmitter (36). On the basis of this cumulative evidence, we hypothesized that activation of the PvN CaR releases endocannabinoids, 2-arachidonoylglycerol (2-AG) and anandamide (AEA), that are metabolized by cytochrome P-450 (CYP) and phospholipase A2 (PLA2) to generate hyperpolarizing vasodilator compounds (8, 11, 36). This conclusion was based largely on the finding that the CB1 receptor antagonist SR-141716A and the novel anandamide/abnormal cannabidiol receptor antagonist O-1918 partially inhibited Ca2+-induced relaxation, thus suggesting a role for a hyperpolarizing vasodilator with activity at an endocannabinoid receptor. Furthermore, we demonstrated that iberiotoxin (IBTX), a Ca2+-activated K+ (KCa) channel blocker, reduced Ca2+-induced relaxation of isolated rat mesenteric arteries (36). The involvement of KCa channels is consistent with a role for endothelium-derived factors that modulate myogenic tone. Resistance artery function may be dependent on the balance between localized generation of vasoconstrictor and vasodilator substances (23–25). In the present study, we provide additional evidence to support the linkage between activation of the PvN CaR and vascular relaxation and propose that the receptor mediates the generation of glycerated epoxyeicosatrienoic acids (GEETs), which serve as vasodilators in this system.
MATERIALS AND METHODS
All procedures were approved by the Institutional Animal Care and Use Committee. Male Wistar rats, 10–12 wk of age (Harlan Sprague Dawley, Indianapolis, IN), were maintained at constant temperature and humidity with fixed light-dark cycles and provided standard rodent chow (Harlan Tekland, Madison, WI) and water ad libitum.
Acetylcholine (ACh) hydrochloride, phenylephrine (PE), 1,6-bis(cyclohexyloximino-carbonylamino)hexane (RHC 80267), miconazole, quinacrine, IBTX, and SR-141716A were from Sigma (St. Louis, MO); arachidonyltrifluoromethyl ketone (AACOCF3) were from EMD Chemicals (San Diego, CA); and 2-AG and GEET compounds were supplied by Dr. J. R. Falck of the University of Texas Southwestern Medical School.
Mesenteric arteries were dissected from rats that were deeply anesthetized with isoflurane and killed by open-chest cardiac puncture. The small intestine and its feeding vessels were removed in block and placed in physiological salt solution (PSS; mM: 115 NaCl, 4.7 KCl, 1.4 MgSO4·7H2O, 5 NaHCO3, 1.2 KH2PO4, 1.1 Na2HPO4, 1.0 CaCl2, 20 HEPES, and 5 glucose, pH 7.4). Branch I and II arteries were carefully dissected from the surrounding fat and mesenterium, taking care to leave a portion of the omental membrane attached to the vessel. A 40-μm-diameter stainless steel wire was then inserted into the lumen to facilitate easy handling of the vessel segment between solutions and for mounting in the myograph chamber.
Isometric force generation in isolated arteries was determined by established methods (11). Briefly, 2-mm-long segments of mesenteric branch arteries were mounted in a small-vessel Mulvany-Halpern 510A Auto Dual Wire Myograph (DMT-USA, Marietta, GA) by means of tungsten-free stainless steel wires inserted through the lumen, maintained in PSS with 100 μM ascorbic acid, and gassed with a mixture of 95% air and 5% CO2. The myograph is designed with an automated normalization function controlled from the interface “Normalization” menu, and a standardized procedure was carried out according to the manufacturer's protocol after 30-min equilibration at 37°C. The following normalization parameters for the mesenteric artery were used: target transmural pressure 13.3 kPa (100 mmHg); time 60 s; IC1/IC100 = 0.9 (IC100 = internal circumference corresponding to target pressure, IC1 = normalized internal circumference); eyepiece calibration 2*Δ (mm/division). The procedure defines the lumen diameter (d100) that the artery would have had in vivo when relaxed and under a transmural pressure of 100 mmHg (2, 30). The arteries were then set to the lumen diameter of d1 = 0.9 × d100, where active force development is maximal. Active force development of ≥10 mN in arteries was considered optimum for the experiment to proceed. The vessels were then challenged with 5 μM phenylephrine (PE) until reproducible contractions were observed. Relaxation was assessed by addition of graded concentrations of Ca2+ and test compounds cumulatively to vessels that were precontracted to maximal tone. When inhibitors were used, tissues were preincubated with the compounds in the bath for 20 min and present during relaxation assays. The inhibitors had no effect on basal tensions. Except for experiments with ACh, where endothelium-replete and endothelium-denuded arteries were used, all studies were carried out with intact mesenteric arteries.
Concentration-response data were analyzed with SigmaStat 3.1 and SigmaPlot 9.0 statistics programs from Systat Software (Point Richmond, CA). Comparisons between groups and within groups were done by analysis of variance (ANOVA), and differences with P < 0.05 were considered significant. EC50 values were determined from fitting data to a four-parameter logistic function in the Pharmacology menu of the SigmaPlot program.
To determine whether Ca2+-induced relaxation was dependent on the intact endothelium, responses were measured in endothelium-replete and endothelium-denuded arteries. As shown in Fig. 1, removal of the endothelium only reduced Ca2+-induced relaxation by ∼20% (Fig. 1A) compared with controls, while ACh-induced responses were reduced by ∼80% (Fig. 1B; P < 0.05) at higher concentrations of agonists. A large component of Ca2+-induced relaxation was retained after removal of the endothelium. Figure 2 shows the effect of the diacylglycerol (DAG) lipase (DAGL) inhibitor RHC 80267 on Ca2+-induced relaxation, which was reduced by inhibitor and reversed by washing. The EC50 for Ca2+-induced relaxation was 2.8 ± 0.4 mM in the presence of the inhibitor compared with 1.4 ± 0.3 mM for control, a 50% reduction in Ca2+ sensitivity (P < 0.05). To further explore the possible involvement of DAG and its downstream metabolites in Ca2+-induced relaxation, we examined the effects of the CYP inhibitors miconazole and quinacrine (also an inhibitor of PLA2) as well as AACOCF3 and SR-141716A on Ca2+-induced vascular relaxation. In the presence of 5 μM miconazole, Ca2+-induced relaxation was completely blocked, whereas quinacrine and AACOCF3 partially blocked this response (Figs. 3 and 4). The EC50 in the presence of quinacrine was 4.8 ± 0.4 mM compared with 2.0 ± 0.3 mM for control, about a 2.5-fold reduction in Ca2+ sensitivity (P < 0.05). The CB1 receptor antagonist SR-141716A also blocked Ca2+-induced relaxation of PE-contracted arteries in a concentration-dependent manner as shown in Fig. 5.
To determine whether 2-AG and its CYP metabolites are able to relax PE-contracted mesenteric arteries, we tested synthetic 2-AG and GEET compounds (5,6-GEET, 8,9-GEET, 11,12-GEET, and 14,15-GEET) derived from CYP metabolism of 2-AG as well as the effect of CB1 and KCa channel blockade on responses. Figure 6A shows relaxation of PE-contracted mesenteric artery segments following graded additions of Ca2+ or 2-AG. 2-AG induced concentration-dependent relaxation with an EC50 value of 0.96 ± 0.14 nM. The curve was shifted to the right in the presence of 100 nM IBTX (EC50 = 1.3 ± 0.5 μM) (Fig. 6B). Miconazole (1 μM) also blocked 2-AG relaxation by ∼48 ± 3% (Fig. 6C). Similar concentration-response curves were obtained for the GEET compounds (Fig. 7A). The 5,6-, 11,12-, and 14,15-GEET regioisomers relaxed precontracted arteries with EC50 values ranging from 18 to 55 nM, and the EC50 for 8,9-GEET was 450 nM. The data show that isomers of GEET induced relaxation of PE-contracted arteries, which was blocked by IBTX, a KCa channel blocker (Table 1, Fig. 7B, and Fig. 8). The concentration-response curve was shifted to the right by the inhibitor. The EC50 for the 11,12-GEET response in the presence of 100 nM IBTX was ∼690 ± 96 nM compared with 55 ± 4 nM for control (P < 0.001), about a 12-fold reduction in sensitivity. The 14,15-GEET response was also attenuated by 1 μM SR-141716A, a CB1 antagonist with an EC50 of 20 ± 3 nM, but a large component of this response (≈40% at the highest concentration) was unaffected (Fig. 8).
Earlier studies from our laboratory demonstrate that Ca2+-induced relaxation of PE-contracted mesenteric arteries is reduced by two different KCa channel blockers, IBTX (36) and O-1918 (11), and a CB1 antagonist. These findings have led to the hypothesis that extracellular Ca2+ (Ca) evokes the release of a hyperpolarizing vasodilator with activity at an endocannabinoid receptor. The results of the present study confirm that Ca2+-induced relaxation of isolated, PE-contracted rat mesenteric arteries is largely independent of a functional endothelium, indicating a mechanism that involves vasodilators apart from endothelium-derived factors such as nitric oxide. A major component of the relaxation is due to endothelium-independent factors, consistent with a role for GEET, and possibly CYP epoxygenase metabolites of arachidonic acid (AA). Thus agonist activation of the perivascular CaR leads to the generation of hyperpolarizing factors from 2-AG, and possibly AA, that activate KCa channels, resulting in the relaxation of smooth muscles.
The CaR signals through the Gαq-phospholipase C pathway (7); thus downstream DAG production is involved. DAG is a strong activator of protein kinase C (PKC) and is metabolized by enzymes such as DAGL to 2-AG (27, 61). In neuronal cells, activation of the metabotropic glutamate receptor, a C family G protein-coupled receptor (GPCR) similar to the CaR, leads to the breakdown of DAG to 2-AG and subsequently to AA (20, 21, 38). The reduction in relaxation observed in the presence of RHC 80267, a selective inhibitor of DAGL and a tool for investigating the role of DAG and AA in physiological processes, suggests that Ca2+-induced relaxation involves the conversion of DAG to 2-AG, a known vasodilator, and its downstream metabolites, AA by PLA2 and GEET by CYP epoxygenase. RHC 80267 has been shown to potentiate ACh-induced relaxation of contracted mesenteric arteries via inhibition of acetylcholinesterase in the vascular wall (28), an effect independent of inhibition of nitric oxide synthase, PKC, or PLA2. Inhibition of CYP by miconazole and CYP/PLA2 with quinacrine as well as blockade of Ca2+-induced relaxation by the specific PLA2 inhibitor AACOCF3 in the present study suggest that Ca2+-induced relaxation is mediated by CYP metabolites of 2-AG and/or its PLA2 metabolites that may have been converted to vasodilators downstream. Synthetic GEET metabolites of 2-AG relaxed PE-contracted mesenteric arteries, which was blocked by IBTX, and the 2-AG effect was blocked by IBTX and miconazole, indicating that the process involves hyperpolarization. The CB1 receptor antagonist SR-141716A also partially blocked Ca2+-induced and GEET-induced relaxation, suggesting involvement of the endocannabinoid system. Therefore, these results support the notion that Ca2+-induced relaxation of isolated mesenteric arteries is mediated by DAG metabolites. Thus activation of the sensory nerve CaR leads to release of 2-AG and its metabolism to the CYP metabolite GEET that contribute to Ca2+-induced relaxation. The higher EC50 for 8,9-GEET was unexpected and suggests isomerization, decomposition, or a lower potency for this isomer. The GEET regioisomers are labile compounds and degrade over time.
CYP metabolites of the AA monoxygenase pathway are major players in the bioactivation and physiological actions of AA (15–17). CYP hydroxylase-derived 20-hydroxyeicosatetraenoic acid (20-HETE) is a potent vasoconstrictor and CYP epoxygenase-derived EET a natriuretic vasodilator in the kidney (24, 25). In rat mesenteric artery, involvement of ACh-induced CYP-derived metabolites of AA in hyperpolarization is minimal (26), and chronic hypoxia was shown to enhance CYP2C9 expression in association with increased 11,12-EET production leading to KCa-dependent hyperpolarization of VSM and attenuated reactivity (22). Furthermore, CYP metabolites of AA may be important mediators of angiotensin II-induced constriction of rat mesenteric arteries in vivo (18). In the peripheral vasculature, AA is metabolized via CYP-dependent pathways to EET or 20-HETE, the latter being metabolized further to prostaglandins (56). EETs (5,6-, 8,9-, 11,12-, and 14,15-EET) are vasodilators that show no predictable stereoselectivity in their dilator responses (64, 65). Apart from their properties as powerful vasodilators, these compounds have also been shown to be powerful and selective angiogenic lipids, suggesting a physiological role for them in angiogenesis and de novo vascularization (53). The inhibition of Ca2+-induced relaxation of PE-contracted mesenteric arteries by quinacrine and AACOCF3 suggests that a pathway through PLA2 is also involved. Thus it appears that stimulation of the perivascular nerve CaR leads to PLA2 activation and release of AA from membrane phospholipids and metabolism by CYP to vasodilatory compounds. The CYP epoxygenases metabolize endogenous pools of AA to EETs, and several isoforms of CYP have been identified as predominant stereoselective epoxygenases in human, rat, and mouse tissues (19, 20, 56, 58). There is abundant evidence to indicate that EETs are vasodilators produced from AA in the endothelium by CYP epoxygenase and hyperpolarize VSM cells by opening KCa channels (5, 12–14, 22). Several studies have also shown that CYP metabolites regulate cellular Ca2+ responses by exerting their effects on store-operated calcium entry and voltage-gated Ca2+ entry channels; however, the nature of some of these Ca2+ entry pathways has not been fully determined. The mechanism by which CYP metabolites of AA activate intracellular second messenger systems is not clear, although specific GPCRs may be involved. Specific CYPs have been shown to localize in VSM and endothelium and contribute to the regulation of vascular tone and homeostasis. However, nerve-derived hyperpolarizing factors may also play a role. Studies on astrocytes, which express glutamatergic and γ-aminobutyric acid receptors, have shown the involvement of increased Ca2+ and EET production in the regulation of cerebrovascular blood flow (29, 35, 39). Furthermore, increases in intracellular Ca2+ (Ca) concentration in astrocytic processes precede vasodilation of arterioles within cortical slices (59). It is therefore reasonable to assume from the present studies that CaR-mediated generation of EETs in perivascular nerves may also play a role in Ca2+-induced relaxation. This mechanism may therefore be important in counteracting the effects of vasoconstrictor CYP metabolites that are associated with increased vascular tone as in hypertension. A recent study by Ohanian et al. (52) demonstrated a functional role for the CaR in regulating myogenic tone in rat subcutaneous small arteries, which was dependent on PKC.
Endocannabinoids such as AEA are membrane-derived signaling molecules released “on demand” from nerves (54), blood cells, and endothelial cells. AEA is synthesized from its precursor N-arachidonoylphosphatidylethanolamine from membrane phospholipids by hydrolysis through multiple pathways and plays a role in the regulation of numerous physiological and pathological processes (42–44). These findings indicate that biosynthetic and degrading enzymes are key regulators of lipid signaling in the vasculature (41, 47). Therefore, signaling pathways that lead to the activation of these regulatory enzymes will be important in maintaining normal vascular tone, and this process may be altered under disease conditions, such as in hypertension, where vascular reactivity is increased. Our earlier finding (36) that Ca2+-induced relaxation is reduced by the CB1 antagonist SR-141716A and the present data indicating attenuation of the 14,15-GEET-induced response in the presence of SR-141716A support the involvement of a CB1 receptor pathway in the GEET response. A large component of the response, however, was unaffected by the CB1 antagonist, suggesting the involvement of an alternate pathway. If AEA is produced after Ca2+ addition to precontracted arteries it could activate AEA receptors (11) or be metabolized by PLA2 to AA and then by CYP to vasodilatory compounds.
A number of studies have established associations between genetically controlled alterations in blood pressure and the activity and/or transcriptional regulation of CYP enzymes that are linked to the pathophysiology of hypertension (45, 46), a leading cause of cardiovascular, cerebral, and renal morbidity/mortality. Zhao and Imig (66) have shown that EETs and 20-HETE levels in the kidney are altered in diabetes, pregnancy, and animal models of hypertension, and blockade of EET formation is associated with salt-sensitive hypertension (34, 37). Thus abnormalities in the CYP system may contribute to the pathogenesis of cardiovascular diseases such as hypertension. A preponderance of CYP hydroxylase-derived AA metabolites in the vasculature will likely contribute to hypertension (60), while CYP epoxygenase-derived AA metabolites will reduce it. Desensitization of the CaR occurs with repeated stimulation (6), further suggesting that an understanding of the regulation of the signaling pathways for this receptor may shed light on altered vasodilator release in hypertension.
In conclusion, the present study demonstrates that Ca2+-induced relaxation of both intact and endothelium-denuded mesenteric arteries is mediated by a mechanism involving the synthesis of CYP metabolites of 2-AG and AA, GEETs and EETs, respectively, and that GEET-induced relaxation operates through activation of KCa channels. Furthermore, the results indicate that hyperpolarizing compounds of the endocannabinoid pathway may also play a role. On the basis of these findings, we propose that agonist activation of the perivascular nerve CaR stimulates Ca2+-dependent PLA2 and N-acetyl transferase to release AEA/AA from membrane phospholipids as well as 2-AG from DAG as substrates for CYP-mediated synthesis of nerve-derived vasodilators, which then diffuse into the underlying smooth muscle cells to induce relaxation (Fig. 9). We showed in an earlier study (6) that activation of the DRG CaR, stably expressed in HEK293 cells, leads to the mobilization of Ca. The DRG houses cell bodies of sensory nerves that send efferent processes to tissues such as the perivascular adventitia. Therefore activation of the CaR at the perivascular nerve terminal can lead to the release of hyperpolarizing vasodilator compounds, such as CYP metabolites of AA and 2-AG, that can then diffuse into and relax adjacent smooth muscle cells (8, 9, 11, 36).
This study was supported by National Heart, Lung, and Blood Institute Grants R01-HL-064761, UH1-HL-059868, and R25-HL-059868.
↵† Deceased 2 March 2004.
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- Copyright © 2008 by the American Physiological Society