Richard D. Bukoski

Endocannabinoids Acting at Cannabinoid-1 Receptors Regulate Cardiovascular Function in Hypertension

Background—Endocannabinoids are novel lipid mediators with hypotensive and cardiodepressor activity. Here, we examined the possible role of the endocannabinergic system in cardiovascular regulation in hypertension. Methods and Results—In spontaneously hypertensive rats (SHR), cannabinoid-1 receptor (CB1) antagonists increase blood pressure and left ventricular contractile performance. Conversely, preventing the degradation of the endocannabinoid anandamide by an inhibitor of fatty acid amidohydrolase reduces blood pressure, cardiac contractility, and vascular resistance to levels in normotensive rats, and these effects are prevented by CB1 antagonists. Similar changes are observed in 2 additional models of hypertension, whereas in normotensive control rats, the same parameters remain unaffected by any of these treatments. CB1 agonists lower blood pressure much more in SHR than in normotensive Wistar-Kyoto rats, and the expression of CB1 is increased in heart and aortic endothelium of SHR compared with Wistar-Kyoto rats. Conclusions—We conclude that endocannabinoids tonically suppress cardiac contractility in hypertension and that enhancing the CB1-mediated cardiodepressor and vasodilator effects of endogenous anandamide by blocking its hydrolysis can normalize blood pressure. Targeting the endocannabinoid system offers novel therapeutic strategies in the treatment of hypertension.

Induction of contraction in isolated rat aorta by cyclosporine

Cyclosporine (CsA) is a new immunosuppressive agent that has adverse effects of nephrotoxicity and de novo appearance of hypertension. It has been hypothesized that the mechanism of the contrary effects is through an action of CsA on vascular smooth muscle. To test this hypothesis, thoracic aortas were isolated from Wister Kyoto rats and ring segments prepared for measurement of tension. CsA (5 ± 10-6M) induced a slow increase in tone of the isolated rings with a response at 3 hr of 0.70 ± 0.17 N/m2 × 10-4 This contraction was significantly inhibited by the Ca2+ channel blocker verapamil (0.30 ± 0.08 N/m2 × 104 after 3 hr, P<0.05) and by the noncompetitive a-antagonist phenoxybenzamine (0.06 ± 0.07 N/m2 × 104 after 3 hr, P<0.05). The competitive a-antagonist phentolamine had mixed effects. CsA does not irreversibly alter vascular smooth muscle contractile ability since a 3 hr exposure to the agent had no effect on either the maximal contractile response or sensitivity to KC1. We conclude that CsA can directly induce contraction in vascular smooth muscle, perhaps by inducing a release of norepinephrine from adrenergic nerve terminals. The data are consistent with the hypothesis that CsA induces nephrotoxicity and de novo hypertension through a contractile effect on vascular smooth muscle.

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

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.

Chronic alcohol consumption lowers blood pressure but enhances vascular contractility in Wistar rats

Objective: To determine the effect of chronic alcohol consumption upon blood pressure, blood pressure reactivity and vascular contractility in Wistar rats. Design: Wistar rats were fed a liquid diet containing 36% ethanol; control rats were pair fed. Methods: Rats were maintained on diets for 18 weeks. Indirect systolic blood pressure was measured weekly. Catheters were implanted for assessment of direct arterial pressure and blood pressure reactivity to norepinephrine, angiotensin II and ethanol injections. In a subgroup of rats, contractility of isolated mesenteric resistance vessels was measured. Results: In comparison with simultaneously pair-fed controls, ethanol-treated rats developed significantly lower blood pressure within 3 weeks of exposure to alcohol; this continued throughout the study. Despite the reduction in blood pressure, in vitro assessment of vascular contractility in mesenteric resistance vessels indicated that ethanol consumption significantly enhanced vascular contractility to norepinephrine and attenuated the vasodepressive effects of ethanol. Measurement of blood pressure reactivity to infused pressor agents showed no difference between controls and ethanol-treated rats in response to norepinephrine but a significantly attenuated pressor response to angiotensin II was observed in ethanol-treated rats. Conclusions: The blood pressure results contrast with reports of elevated blood pressure in Wistar rats given ethanol in drinking water. This disparity may be due to nutritional factors. Increased vascular contractility combined with hypotension suggests that cardiovascular regulatory systems offset the direct effects of ethanol upon the vasculature. This view is reinforced by the lack of difference between groups in blood pressure reactivity to norepinephrine. The attenuated angiotensin II responses in the ethanol-treated rats suggests altered levels of circulating angiotensin II in this group.

Arachidonic acid activates jun N-terminal kinase in vascular smooth muscle cells

We have previously demonstrated that arachidonic acid activates extracellular signal-regulated protein kinases (ERKs) group of mitogen-activated protein kinases (MAPKs) in vascular smooth muscle cells (VSMC). To understand the role of arachidonic acid in cellular signaling events, we have now studied its effect on jun N-terminal kinases (JNKs) group of MAPKs in VSMC. Arachidonic acid activated JNK1 in a time- and concentration-dependent manner with maximum effects at 10 min and 50 μM. Induced activation of JNK1 by arachidonic acid is specific as other fatty acids such as linoleic and stearic acids had no such effect. Indomethacin and nordihydroguaiaretic acid (NDGA), potent inhibitors of the cyclooxygenase (COX) and the lipoxygenase (LOX)/monooxygenase (MOX) pathways, respectively, had no effect on arachidonic acid activation of JNK1 suggesting that the observed phenomenon is independent of its metabolism through either pathway. However, 12-hydroperoxyeicosatetraenoic acid (12-HpETE), the LOX metabolite of arachidonic acid significantly induced JNK1 activity. Protein kinase C (PKC) depletion by prolonged treatment of VSMC with phorbol 12-myristate 13-acetate (PMA) resulted in partial decrease in the responsiveness of JNK1 to arachidonic acid suggesting a role for both PKC-dependent and -independent mechanisms in the activation of JNK1 by this important fatty acid. On the other hand, the responsiveness of JNK1 to 12-HpETE was completely abolished in PKC-depleted cells, suggesting a major role for PKC in 12-HpETE-induced JNK1 activation. IL-1β and TNF-α activated JNK1 in a time-dependent manner with maximum effect at 10 min. Desensitization of JNK1 by arachidonic acid significantly reduced its responsiveness to both the cytokines. In addition, 4-bromophenacyl bromide (4-BPB), a potent and selective inhibitor of phospholipase A2 (PLA2), significantly attenuated the cytokine-induced activation of JNK1. Together, these results show that (1) arachidonic acid and its LOX metabolite, 12-HpETE, activate JNK1 in VSMC, (2) PKC-dependent and -independent mechanisms play a role in the activation of JNK1 by arachidonic acid and 12-HpETE, and (3) arachidonic acid mediates, at least partially, the cytokine-induced activation of JNK1.

Distribution of the perivascular nerve Ca2+ receptor in rat arteries

1 We recently showed that perivascular sensory nerves of mesenteric branch arteries express a receptor for extracellular Ca2+ (CaR), and reported data indicating that this CaR mediates relaxation induced by physiologic levels of Ca2+. We have now tested whether the perivascular sensory nerve CaR‐linked dilator system is a local phenomenon restricted to the mesentery, or is present in other circulations. 2 Vessels from the mesenteric, renal, coronary, and cerebral circulations were studied. Immunocytochemical analysis was performed using anti‐CaR and anti‐neural cell adhesion molecule (NCAM) antibodies. Wire myography was used to assess contracation and relaxation. 3 Although perivascular nerves of all arteries stained for CaR protein, there were regional differences. A morphometric method used to estimate CaR positive nerve density revealed the following rank order: mesenteric branch artery>basilar artery=renal interlobar artery>main renal trunk artery>left anterior descending coronary artery. 4 Vessels from the mesentery, renal, coronary, and cerebral circulations showed nerve‐dependent relaxation in response to electrical field stimulation (EFS) when precontracted with serotonin in the presence of guanethidine. The degree of Ca2+‐induced relaxation of mesenteric, renal, and cerebral arteries positively correlated with the magnitude of EFS‐induced relaxation. In contrast, coronary arteries contracted at Ca2+ levels between 1.5 and 3 mmol L−1, and relaxed to a small degree to 5 mmol L−1 Ca2+. 5 Thus, a functional perivascular sensory nerve CaR‐linked dilator system is present to varying degrees in the mesenteric, renal, and cerebral circulations, but only to a very limited extent in the coronary circulation.

Interstitial Ca2+ undergoes dynamic changes sufficient to stimulate nerve-dependent Ca2+-induced relaxation

We recently described a perivascular sensory nerve-linked dilator system that can be activated by interstitial Ca2+([Formula: see text]). The present study tested the hypothesis that [Formula: see text] in the rat duodenal submucosa varies through a range that is sufficient to activate this pathway. An in situ microdialysis method was used to estimate [Formula: see text]. When the duodenal lumen was perfused with Ca2+-free buffer, [Formula: see text] was 1.0 ± 0.13 mmol/l. [Formula: see text] increased to 1.52 ± 0.04, 1.78 ± 0.10, and 1.89 ± 0.1 when the lumen was perfused with buffer containing 3, 6, and 10 mmol/l Ca2+, respectively ( P < 0.05).[Formula: see text] was 1.1 ± 0.06 mmol/l in fasted animals and increased to 1.4 ± 0.06 mmol/l in free-feeding rats ( P < 0.05). Wire myography was used to study isometric tension responses of isolated mesenteric resistance arteries. Cumulative addition of extracellular Ca2+-relaxed serotonin- and methoxamine-precontracted arteries with half-maximal effective doses of 1.54 ± 0.05 and 1.67 ± 0.08 mmol/l, respectively ( n = 5). These data show that duodenal[Formula: see text] undergoes dynamic changes over a range that activates the sensory nerve-linked dilator system and indicate that this system can link changes in local Ca2+ transport with alterations in regional resistance and organ blood flow.

Cytochrome P-450 metabolites of 2-arachidonoylglycerol play a role in Ca2+-induced relaxation of rat mesenteric arteries.

The perivascular sensory nerve (PvN) Ca(2+)-sensing receptor (CaR) is implicated in Ca(2+)-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 A(2) (PLA(2)), and cytochrome P-450 (CYP) on Ca(2+)-induced relaxation of PE-contracted rat mesenteric arteries. Our findings indicate that Ca(2+)-induced vasorelaxation is not dependent on the endothelium. The DAGL inhibitor RHC 802675 (1 microM) and the CYP and PLA(2) inhibitors quinacrine (5 microM) (EC(50): 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 (AACOCF(3), 1 microM) reduced Ca(2+)-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) (EC(50): control 0.96 +/- 0.14 nM, IBTX 1.3 +/- 0.5 microM) and miconazole (48 +/- 3%), and 11,12-GEET responses were blocked by IBTX (EC(50): control 55 +/- 9 nM, IBTX 690 +/- 96 nM) and SR-141716A. The data suggest that activation of the CaR in the PvN network by Ca(2+) 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 Ca(2+)-induced relaxation of resistance arteries; therefore this receptor may be a potential target for the development of new vasodilator compounds for antihypertensive therapy.

Effect of chronic sensory denervation on Ca2+-induced relaxation of isolated mesenteric resistance arteries

We recently reported that Ca(2+)-induced relaxation could be linked to a Ca2+ receptor (CaR) present in perivascular nerves. The present study assessed the effect of chronic sensory denervation on Ca(2+)-induced relaxation. Mesenteric resistance arteries were isolated from rats treated as neonates with capsaicin (50 mg/kg), vehicle, or saline. The effect of cumulative addition of Ca2+ was assessed in vessels precontracted with 5 microM norepinephrine. Immunocytochemical studies showed that capsaicin treatment significantly reduced the density of nerves staining positively for calcitonin gene-related peptide (CGRP) and for the CaR (CGRP density: control, 51.1 +/- 3.9 microns2/mm2; capsaicin treated, 31.4 +/- 2.8 microns2/mm2, P = 0.01; control CaR density, 46 +/- 4 microns2/mm2, n = 7; capsaicin-treated CaR density, 24 +/- 4 microns2/mm2, n = 8, P = 0.002). Dose-dependent relaxation to Ca2+ (1-5 mM) was significantly depressed in vessels from capsaicin-treated rats (overall P < 0.001, n = 6 or 7), whereas the relaxation response to acetylcholine remained intact. These data support the hypothesis that Ca(2+)-induced relaxation is mediated by activation of the CaR associated with capsaicin-sensitive perivascular neurons.We recently reported that Ca2+-induced relaxation could be linked to a Ca2+ receptor (CaR) present in perivascular nerves. The present study assessed the effect of chronic sensory denervation on Ca2+-induced relaxation. Mesenteric resistance arteries were isolated from rats treated as neonates with capsaicin (50 mg/kg), vehicle, or saline. The effect of cumulative addition of Ca2+ was assessed in vessels precontracted with 5 μM norepinephrine. Immunocytochemical studies showed that capsaicin treatment significantly reduced the density of nerves staining positively for calcitonin gene-related peptide (CGRP) and for the CaR (CGRP density: control, 51.1 ± 3.9 μm2/mm2; capsaicin treated, 31.4 ± 2.8 μm2/mm2, P = 0.01; control CaR density, 46 ± 4 μm2/mm2, n = 7; capsaicin-treated CaR density, 24 ± 4 μm2/mm2, n = 8, P = 0.002). Dose-dependent relaxation to Ca2+ (1-5 mM) was significantly depressed in vessels from capsaicin-treated rats (overall P < 0.001, n = 6 or 7), whereas the relaxation response to acetylcholine remained intact. These data support the hypothesis that Ca2+-induced relaxation is mediated by activation of the CaR associated with capsaicin-sensitive perivascular neurons.

The perivascular sensory nerve Ca2+ receptor and blood pressure regulation ☆: A 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 Ca homeostasis with vascular reactivity and blood pressure (BP) takes on new dimensions when considered in terms of recent advances showing that Ca serves as a primary signal to modulate cell function. Specifically, we have formulated a novel hypothesis that potentially explains the molecular connection between Ca 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