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Mechanisms by which BK Ca (large-conductance calcium-sensitive potassium) channels are involved in vascular remodeling in hypertension are not fully understood. Vascular smooth muscle cell (VSMC) proliferation and vascular morphology were compared between hypertensive and normotensive rats. BK Ca channel activity, protein expression, and interaction with IP3R (inositol 1,4,5-trisphosphate receptor) were examined using patch clamp, Western blot analysis, and coimmunoprecipitation. On inside-out patches of VSMCs, the Ca 2+ -sensitivity and voltage-dependence of BK Ca channels were similar between hypertensive and normotensive rats. In whole-cell patch clamp configuration, treatment of cells with the IP3R agonist, Adenophostin A (AdA), significantly increased BK Ca channel currents in VSMCs of both strains of rats, suggesting IP3R-BK Ca coupling; however, the AdA-induced increases in BK Ca currents were attenuated in VSMCs of hypertensive rats, indicating possible IP3R-BK Ca decoupling, causing BK Ca dysfunction. Co-immunoprecipitation and Western blot analysis demonstrated that BK Ca and IP3R proteins were associated together in VSMCs; however, the association of BK Ca and IP3R proteins was dramatically reduced in VSMCs of hypertensive rats. Genetic disruption of IP3R-BK Ca coupling using junctophilin-2 shRNA dramatically augmented Ang II-induced proliferation in VSMCs of normotensive rats. Subcutaneous infusion of NS1619, a BK Ca opener, to reverse BK Ca dysfunction caused by IP3R-BK Ca decoupling significantly attenuated vascular hypertrophy in hypertensive rats. In summary, the data from this study demonstrate that loss of IP3R-BK Ca coupling in VSMCs induces BK Ca channel dysfunction, enhances VSMC proliferation, and thus, may contribute to vascular hypertrophy in hypertension.

Apelin-13 causes vasoconstriction by acting directly on APJ receptors in vascular smooth muscle (VSM) cells; however, the ionic mechanisms underlying this action at the cellular level remain unclear. Large-conductance Ca(2+)-activated K(+) (BKCa) channels in VSM cells are critical regulators of membrane potential and vascular tone. In the present study, we examined the effect of apelin-13 on BK(Ca) channel activity in VSM cells, freshly isolated from rat middle cerebral arteries. In whole-cell patch clamp mode, apelin-13 (0.001-1 μM) caused concentration-dependent inhibition of BK(Ca) in VSM cells. Apelin-13 (0.1 µM) significantly decreased BK(Ca) current density from 71.25 ± 8.14 pA/pF to 44.52 ± 7.10 pA/pF (n=14 cells, P<0.05). This inhibitory effect of apelin-13 was confirmed by single channel recording in cell-attached patches, in which extracellular application of apelin-13 (0.1 µM) decreased the open-state probability (NPo) of BK(Ca) channels in freshly isolated VSM cells. However, in inside-out patches, extracellular application of apelin-13 (0.1 µM) did not alter the NPo of BK(Ca) channels, suggesting that the inhibitory effect of apelin-13 on BKCa is not mediated by a direct action on BK(Ca). In whole cell patches, pretreatment of VSM cells with LY-294002, a PI3-kinase inhibitor, markedly attenuated the apelin-13-induced decrease in BK(Ca current density. In addition, treatment of arteries with apelin-13 (0.1 µM) significantly increased the ratio of phosphorylated-Akt/total Akt, indicating that apelin-13 significantly increases PI3-kinase activity. Taken together, the data suggest that apelin-13 inhibits BK(Ca) channel via a PI3-kinase-dependent signaling pathway in cerebral artery VSM cells, which may contribute to its regulatory action in the control of vascular tone.

Little is known about the vasomotor effects of sirolimus, and preliminary studies using animal models have provided conflicting results. The present study was designed to determine the effects of sirolimus on vasomotor tone in human blood vessels. Human radial artery segments were cut into rings, denuded of endothelium, and placed into organ chambers for isometric tension recording. Sirolimus (10(-10) to 10(-6) M) caused concentration-dependent relaxation of human arteries contracted with U46619 (9,11-dideoxy-11alpha,9alpha-epoxymethano-prostaglandin F(2alpha); 10(-8) M) [-log (M) EC(50) (pD(2)) = 7.28 +/- 0.1; E(max) = 57 +/- 6%] or phenylephrine (10(-6) M) (pD(2) = 7.16 +/- 0.4; E(max) = 45 +/- 9%). Sirolimus-induced relaxation was unaffected by treatment with indomethacin (10(-5) M) but was nearly abolished in tissues contracted by depolarization with elevated K(+) (60 mM). In U46619-contracted rings, the response to sirolimus was markedly inhibited in the presence of the specific ATP-sensitive potassium (K(ATP)) channel blocker, glyburide (10(-6) M), but was unaffected by treatment with blockers of large conductance, calcium-activated potassium channel (iberiotoxin, 10(-7) M), small conductance, calcium-activated potassium channel (apamin, 10(-6) M), or voltage-gated potassium channel (4-aminopyridine, 10(-3) M). The K(ATP) channel opener, aprikalim (10(-7) to 10(-5) M), caused concentration-dependent relaxations that were inhibited by glyburide (10(-6) M) and abolished in tissues contracted with elevated K(+) (60 mM), thus confirming that K(ATP) channel opening causes relaxation of these arteries. These data suggest that sirolimus, at concentrations attained in vivo, causes relaxation of human arteries, and this effect is mediated by opening of K(ATP) channels in vascular smooth muscle. Reduced vasomotor tone is a heretofore unrecognized action of sirolimus that could potentially contribute to its efficacy in drug-eluting stents.

The present study was designed to determine if endogenous calcitonin gene-related peptide (CGRP) affects the process of nitrate tolerance development in blood vessels. Rat aortic rings were suspended in organ chambers and relaxations to nitroglycerin (10(-9) -10(-6) M) were obtained in nitrate tolerant and nontolerant rings contracted with norepinephrine (10(-7) M). Tolerance was induced by incubating the rings with (tolerant) or without (nontolerant) nitroglycerin (10(-4) M) for 90 minutes, followed by repeated rinsing for 1 hour. Some rings were treated with CGRP8-37 (10(-6) M), glyburide (10(-6) M), or iberiotoxin (10(-7) M) during the 90-minute desensitization period with nitroglycerin (10(-4) M), and were then washed out during the 1-hour rinsing period. Other rings were treated with capsaicin (10(-5) M) prior to the 90-minute desensitization period. Calcitonin gene-related peptide release was measured by radioimmunoassay. Relaxation to nitroglycerin was markedly reduced in tolerant rings, as compared with nontolerant. Incubation with CGRP8-37 (10(-6) M) specifically during the 90-minute desensitization period with nitroglycerin resulted in even greater impairment in the response to nitroglycerin in tolerant rings, even though the calcitonin gene-related peptide antagonist had been washed out before completion of the nitroglycerin dose-response curve. Similar results were obtained following depletion of calcitonin gene-related peptide stores in sensory nerves by treatment with capsaicin (10(-5) M) prior to the 90-minute desensitization period with nitroglycerin. Prior treatment with CGRP8-37 or capsaicin had no effect on the response to nitroglycerin in nontolerant rings. Incubation with glyburide (10(-6) M), but not iberiotoxin (10(-7) M), specifically during the 90-minute desensitization period, mimicked the effect of CGRP8-37 and capsaicin in tolerant rings, suggesting a role for KATP channels in the effect of calcitonin gene-related peptide. Nitroglycerin (10(-4) M) caused a greater than twofold increase over basal levels in calcitonin gene-related peptide release in nontolerant rings, which was abolished in rings treated with capsaicin and in nitrate tolerant rings. These results suggest that nitroglycerin releases calcitonin gene-related peptide from sensory nerves during the process of desensitization to nitrovasodilators, and that interference with either the release or action of endogenous calcitonin gene-related peptide during this period enhances the extent to which nitrate tolerance occurs. The finding that nitroglycerin-induced release of calcitonin gene-related peptide from sensory nerves attenuates the desensitizing effect of nitroglycerin represents a heretofore unknown event in the development of nitrate tolerance, and demonstrates a novel role for calcitonin gene-related peptide in the vasculature.

The present study was designed to determine the effects of melatonin on coronary vasomotor tone. Porcine coronary arteries were suspended in organ chambers for isometric tension recording. Melatonin (10(-10)-10(-5) M) itself caused neither contraction nor relaxation of the tissues. Serotonin (10(-9)-10(-5) M) caused concentration-dependent contractions of coronary arteries, and in the presence of melatonin (10(-7) M) the maximal response to serotonin was increased in rings with but not without endothelium. In contrast, melatonin had no effect on contractions produced by the thromboxane A(2) analog U-46619 (10(-10)-10(-7) M). The melatonin-receptor antagonist S-20928 (10(-6) M) abolished the potentiating effect of melatonin on serotonin-induced contractions in endothelium-intact coronary arteries, as did treatment with 1H-[1, 2,4]oxadiazolo[4,3-a]quinoxalin-1-one (10(-5) M), methylene blue (10(-5) M), or N(G)-nitro-L-arginine (3 x 10(-5) M). In tissues contracted with U-46619, serotonin caused endothelium-dependent relaxations that were inhibited by melatonin (10(-7) M). Melatonin also inhibited coronary artery relaxation induced by sodium nitroprusside (10(-9)-10(-5) M) but not by isoproterenol (10(-9)-10(-5) M). These results support the hypothesis that melatonin, by inhibiting the action of nitric oxide on coronary vascular smooth muscle, selectively potentiates the vasoconstrictor response to serotonin in coronary arteries with endothelium.

We compared the tolerance-inducing effects of nitroglycerin (NTG) and nicorandil (NIC) in porcine isolated coronary arteries and assessed the role of KATP channels in the response to NIC in nitrate-tolerant and nontolerant preparations. In coronary arteries contracted with U46619 (1-3 x 10(-9) M), NTG, NIC, sodium nitroprusside (SNP), and cromakalim produced concentration-dependent relaxations. The rank order of potency was NTG > or = SNP > cromakalim > nicorandil. Exposure of the rings to NTG (10(-4) M) for 90 min, followed by repeated rinsing for 1 h, produced a parallel, rightward shift of the subsequent concentration-response curves to NTG and SNP; a slight but significant reduction in the maximal response to NTG was also observed. Previous exposure to NTG had no effect on the NIC or cromakalim concentration-response curves. When the tissues were exposed to NIC (3 x 10(-4) M) for 90 min, followed by repeated rinsing for 1 h, there was no effect on the subsequent concentration-response curves to NTG, NIC, SNP, or cromakalim. In both nitrate-tolerant and nontolerant coronary arteries, glibenclamide (GLI 10(-6) M), a selective KATP channel blocker, caused a parallel rightward shift in the concentration-response curve to cromakalim, but had no effect on responses to NTG or SNP. In nontolerant coronary arteries, GLI had no effect on NIC-induced relaxation, but in nitrate-tolerant preparations, GLI produced a significant rightward shift in the NIC concentration-response curve. The results demonstrate that prolonged exposure to NTG, but not NIC, causes tolerance in isolated porcine coronary arteries and that the response to NIC is not affected by nitrate tolerance. The data also suggest that NIC-induced relaxation of nitratetolerant, but not nontolerant, coronary arteries is mediated by activation of KATP channels.

The autonomic nervous system plays a vital role in the regulation of vasomotor tone. Previous studies of systemic arteries and veins have shown that the classic autonomic neurotransmitters, norepinephrine and acetylcholine, act on smooth muscle, adrenergic nerve endings, and endothelial cells in the blood vessel wall to regulate vasomotor tone. Similar studies with isolated tissues indicate that bronchial arteries are innervated by adrenergic neurons and that norepinephrine activates postjunctional alpha 1-adrenoceptors to cause smooth muscle contraction. Isolated canine bronchial arteries fail to relax in response to beta-adrenoceptor agonists, and they do not contract when exposed to acetylcholine. This lack of responsiveness may be species-specific, however, since isolated bronchial arteries from other species respond to these agonists. Acetylcholine causes endothelium-dependent relaxation of bronchial arteries in vitro; this response is mediated by endothelial M3-muscarinic receptors. The role of the endothelium in mediating responses to adrenergic agonists, as well as the prejunctional effects of norepinephrine and acetylcholine on adrenergic nerve endings, remain to be explored in isolated bronchial arteries.

Experiments were designed to study the potential mechanisms underlying the vasodilator effect of celiprolol. Rings of canine left circumflex coronary artery and rat mesenteric artery, with and without endothelium, were suspended in organ chambers for isometric tension recording. In both blood vessels, celiprolol (10(-9)-10(-4) M) failed to produce relaxation in rings with and without endothelium; these same tissues relaxed in an endothelium-dependent manner to acetylcholine (10(-6) M). All tissues relaxed completely in the presence of papaverine (10(-4) M). In the coronary artery, isoproterenol (10(-9)-10(-4) M) produced endothelium-independent relaxations which were inhibited in a competitive fashion by celiprolol (pA2 = 7.52 +/- 0.14; slope = 0.98, 95% confidence limits = 0.80-1.15). In other experiments, strips of canine saphenous veins were incubated with [3H]norepinephrine [( 3H]NE) and suspended for superfusion. Electrical stimulation (2 Hz, 4 V, 2 ms for 6 min) produced an increase in [3H]NE overflow. Isoproterenol (2 X 10(-6) M) augmented the evoked release of [3H]NE. Treatment of the strips with celiprolol (up to 5 X 10(-6) M) did not inhibit isoproterenol-induced facilitation of [3H]NE release. Thus, although celiprolol is a potent antagonist of postjunctional beta-adrenoceptors in the coronary artery, no evidence was obtained for a direct or indirect vasodilator effect of celiprolol on isolated blood vessels.

Experiments were designed to characterize pre- and postjunctional muscarinic receptors in the canine saphenous vein. In rings contracted by 2-Hz electrical stimulation, acetylcholine produced concentration-dependent relaxations that result from prejunctional inhibition of norepinephrine release from adrenergic nerve endings (prejunctional effect). In quiescent preparations, acetylcholine caused concentration-dependent contractions (postjunctional effect). Both responses to acetylcholine were inhibited in a competitive manner by atropine, pirenzepine and gallamine. The affinity of atropine for pre- and postjunctional muscarinic receptors was similar (pKB = 8.7 and 9.2, respectively). However, gallamine displayed higher affinity for the prejunctional than for the postjunctional receptor (pKB = 6.1 and 4.7, respectively), whereas the converse was true for pirenzepine (pKB = 6.5 and 8.1). The data support the presence in the canine saphenous vein of two muscarinic receptor subtypes, M1 postjunctionally and M2 prejunctionally, which are distinguishable by selective antagonists.