Protective effect of melatonin against homocysteine-induced vasoconstriction of human umbilical artery

Hyperhomocysteinemia is a major and independent risk factor for vascular disease. Oxidative stress is a possible mechanism for homocysteine (Hcy)-induced endothelial dysfunction. Herein, we evaluated the antioxidant property of melatonin (MLT) in relation to the vasoconstrictive effect of Hcy on the human umbilical artery. In an initial experiment in a cell-free system, a micromolar concentration of iron was found to catalyze oxygen-dependent oxidation of Hcy. MLT (10 or 100 microM) did not affect oxygen-dependent oxidation of Hcy. Next, smooth muscle contraction induced by prostaglandin F(2alpha) (10 microM) was measured in arterial strips. Hcy (10 to 500 microM) increased this vascular tension in a concentration-dependent manner (P < 0.0001). Addition of Fe(2+) (10 microM) significantly potentiated the Hcy effect. Removal of endothelium (P < 0.05), pretreatment with a nitric oxide (NO) synthesis inhibitor (l-N(G)-monomethylarginine, 200 microM, P < 0.001), or pretreatment with a hydroxyl radical ((*)OH) scavenger (mannitol, 10 mM, P < 0.001) significantly attenuated contraction potentiated by Hcy plus Fe(2+). At a much lower concentration than mannitol, MLT (1 to 100 microM) significantly reduced the contractile effect of Hcy and Fe(2+) in a concentration-dependent manner. Hcy plus Fe(2+) significantly impaired calcium ionophore A 23187-induced relaxation (P < 0.0001), while MLT restored this relaxation in a concentration-dependent manner. These findings suggest that Hcy potentiates vascular tension in human umbilical artery, possibly by suppressing bioavailable NO. MLT protects against the vasoconstrictive effect of Hcy, most likely by scavenging (*)OH arising from Hcy autooxidation.     

Tyrosine phosphorylation increases Ca2+ sensitivity of vascular smooth muscle contraction

In order to elucidate the role of tyrosine phosphorylation in vasoconstriction, we investigated the effects of inhibitors of tyrosine kinase (genistein, 30 microM) and phosphatase (sodium o-vanadate, 5 microM) on the contraction of aorta isolated from guinea pig. Genistein significantly inhibited norepinephrine-induced contraction, but it did not affect that induced by KCI. Thus, tyrosine phosphorylation may not be involved in the contractile response to KCI alone. The aortic contraction elicited by KCl was significantly augmented by sodium o-vanadate, which increased both the maximum force and pD2 values of KCl contraction. In the presence of verapamil, KCl-induced contraction was abolished even after pretreatment with sodium o-vanadate. Sodium o-vanadate also augmented Ca2+-induced contraction in the aortic strips depolarized with KCl, increasing both its maximum force and pD2 values. Neither basal 45Ca2+ uptake nor verapamil-sensitive 45Ca2+ uptake induced by KCl were affected by pretreatment with sodium o-vanadate. These results suggest that tyrosine phosphorylation is involved in the contraction of guinea-pig aorta not through transplasmalemmal Ca2+ entry but through increased Ca2+ sensitivity of the intracellular contractile pathway.     

The frequency of calcium oscillations induced by 5-HT, ACH, and KCl determine the contraction of smooth muscle cells of intrapulmonary bronchioles

Increased resistance of airways or blood vessels within the lung is associated with asthma or pulmonary hypertension and results from contraction of smooth muscle cells (SMCs). To study the mechanisms regulating these contractions, we developed a mouse lung slice preparation containing bronchioles and arterioles and used phase-contrast and confocal microscopy to correlate the contractile responses with changes in [Ca(2+)](i) of the SMCs. The airways are the focus of this study. The agonists, 5-hydroxytrypamine (5-HT) and acetylcholine (ACH) induced a concentration-dependent contraction of the airways. High concentrations of KCl induced twitching of the airway SMCs but had little effect on airway size. 5-HT and ACH induced asynchronous oscillations in [Ca(2+)](i) that propagated as Ca(2+) waves within the airway SMCs. The frequency of the Ca(2+) oscillations was dependent on the agonist concentration and correlated with the extent of sustained airway contraction. In the absence of extracellular Ca(2+) or in the presence of Ni(2+), the frequency of the Ca(2+) oscillations declined and the airway relaxed. By contrast, KCl induced low frequency Ca(2+) oscillations that were associated with SMC twitching. Each KCl-induced Ca(2+) oscillation consisted of a large Ca(2+) wave that was preceded by multiple localized Ca(2+) transients. KCl-induced responses were resistant to neurotransmitter blockers but were abolished by Ni(2+) or nifedipine and the absence of extracellular Ca(2+). Caffeine abolished the contractile effects of 5-HT, ACH, and KCl. These results indicate that (a) 5-HT and ACH induce airway SMC contraction by initiating Ca(2+) oscillations, (b) KCl induces Ca(2+) transients and twitching by overloading and releasing Ca(2+) from intracellular stores, (c) a sustained, Ni(2+)-sensitive, influx of Ca(2+) mediates the refilling of stores to maintain Ca(2+) oscillations and, in turn, SMC contraction, and (d) the magnitude of sustained airway SMC contraction is regulated by the frequency of Ca(2+) oscillations.     

Acute actions of marine toxin latrunculin A on the electrophysiological properties of cultured dorsal root ganglion neurones

The effects of latrunculin A, isolated from the nudibranch Chromodoris sp., on the excitability of neonatal rat cultured dorsal root ganglion neurones were investigated using patch-clamp recording and Ca(2+) imaging techniques. Under current-clamp conditions, acute application of latrunculin A (100 microM) reversibly induced multiple action potential firing and significantly increased action potential duration. No significant effects on action potential peak amplitude, threshold of action potential firing, resting membrane potential and input resistance were observed. Under voltage-clamp conditions, significant and dose-dependent suppression of K(+) current was seen with 10-100 microM latrunculin A. Additionally, a significant difference between inhibition of the current measured at the peak and the end of a 100 ms voltage step was seen with 100 microM latrunculin A. Fura-2 fluorescence Ca(2+) imaging revealed that latrunculin A (100 microM) significantly inhibited Ca(2+) transients evoked by KCl-induced depolarisation in all neurones. In 36% of DRG neurones, latrunculin A alone had no effect on intracellular Ca(2+). In 64% of neurones, latrunculin A alone evoked a transient rise in intracellular Ca(2+). Moreover, latrunculin A (10-100 microM) significantly inhibited the mean high voltage-activated Ca(2+) current. The effects of latrunculin A on action potential firing and K(+) currents were attenuated by intracellular phalloidin, an indication that these effects are mediated through actin disruption.     

Altered calcium sensitivity contributes to enhanced contractility of collateral-dependent coronary arteries.

Coronary arteries distal to chronic occlusion exhibit enhanced vasoconstriction and impaired relaxation compared with nonoccluded arteries. In this study, we tested the hypotheses that an increase in peak Ca(2+) channel current density and/or increased Ca(2+) sensitivity contributes to altered contractility in collateral-dependent coronary arteries. Ameroid occluders were surgically placed around the proximal left circumflex coronary artery (LCX) of female miniature swine. Segments of epicardial arteries ( approximately 1 mm luminal diameter) were isolated from the LCX and nonoccluded left anterior descending (LAD) arteries 24 wk after Ameroid placement. Contractile responses to depolarization (10-100 mM KCl) were significantly enhanced in LCX compared with size-matched LAD arterial rings [concentration of KCl causing 50% of the maximal contractile response (EC(50)); LAD = 41.7 +/- 2.3, LCX = 34.3 +/- 2.7 mM]. However, peak Ca(2+) channel current was not altered in isolated smooth muscle cells from LCX compared with LAD (-5.29 +/- 0.42 vs. -5.68 +/- 0.55 pA/pF, respectively). Furthermore, whereas half-maximal activation of Ca(2+) channel current occurred at nearly the same membrane potential in LAD and LCX, half-maximal inactivation was shifted to a more positive membrane potential in LCX cells. Simultaneous measures of contractile tension and intracellular free Ca(2+) (fura 2) levels in arterial rings revealed that significantly more tension was produced per unit change in fura 2 ratio in LCX compared with LAD in response to KCl but not during receptor-agonist stimulation with endothelin-1. Taken together, our data indicate that coronary arteries distal to chronic occlusion display increased Ca(2+) sensitivity in response to high KCl-induced depolarization, independent of changes in whole cell peak Ca(2+) channel current. Unaltered Ca(2+) sensitivity in endothelin-stimulated arteries suggests more than one mechanism regulating Ca(2+) sensitization in coronary smooth muscle.     

Mg2+ blocks myogenic tone but not K+-induced constriction: role for SOCs in small arteries

The effects of Mg(2+) and nifedipine (Nif) on vasoconstriction and Ca(2+) transients were studied in intact, pressurized rat mesenteric arteries with myogenic tone. Changes in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) were measured with confocal microscopy in fluo 4-AM loaded, individual myocytes. Myogenic tone was abolished by 10 mM Mg(2+) or 0.3 microM Nif. Contractions induced by 75 mM K(+) depolarization were blocked by 0.3 microM Nif, but not by 10 mM Mg(2+). Phenylephrine (PE; 5 microM) evoked sustained [Ca(2+)](cyt) elevation and vasoconstriction with superimposed Ca(2+) oscillations and vasomotion. The subsequent addition of 10 mM Mg(2+) or 0.3 microM Nif reduced [Ca(2+)](cyt) and abolished plateau vasoconstriction. When added before PE, both Mg(2+) and Nif abolished the PE-evoked Ca(2+) oscillations and vasomotion. Mg(2+) dilated the PE-constricted arteries after a brief (< or =180-240 s) vasoconstriction, but Nif did not. Both agents also abolished the vasoconstriction attributed to Ca(2+) entry through store-operated channels (SOCs) during internal Ca(2+) store refilling that followed store depletion. The data suggest that Ca(2+) entry through SOCs helps maintain both myogenic tone and alpha(1)-adrenoceptor-induced tonic vasoconstriction.     

Vascular contractile effect of urotensin II in young and aged rats: influence of aging and contribution of endothelial nitric oxide

To elucidate whether aging influences the vascular contractile effect of urotensin II in rat thoracic aorta, and to evaluate the contribution of endothelial vasodilating substances in mediating the effect of urotensin II, the effect of urotensin II was examined in the vessels of young (2-3-month-old) and aged rat. Isolated rat aortic rings incubated in Krebs-Henseleit solution gassed with 95% O2/5% CO2 were stimulated with urotensin II, and the developed tension was measured. Urotensin II increased the developed tension, which was decreased by aging. In 2-3-months-old young aorta without endothelium, urotensin II (10(-10) to 10(-7)) elicited a concentration-dependent aortic contraction to the maximal response almost equivalent to high KCl-induced contraction (79.4+/-11.3% of KCl(max)). In the presence of endothelium, the urotensin II-induced vasoconstriction in young aorta was significantly attenuated to 33.3+/-4.6% of KCl(max). However, the contractile response was greater in the pretreatment with N(G)-nitro-L-arginine (L-NNA) (100 microM) (50.3+/-8.4% of KCl(max) in endothelial denuded aorta), suggesting the vasorelaxing role of endothelial nitric oxide. In 25-27-months-old aged rat aorta, the urotensin II-mediated contraction was remarkably decreased, both in the presence (6.3+/-2.0% of KCl(max)) and absence (11.7+/-3.0% of KCl(max)) of endothelium. A cyclooxygenase inhibitor, diclofenac (10 microM), did not have any effect on the urotensin II-induced contraction. These results suggest that urotensin II can induce vascular smooth muscle contraction in rat aorta, and there was an aging-related decline in the urotensin II-induced contraction. Endothelial production of nitric oxide in response to urotensin II but not cyclooxygenase metabolites such as prostacyclin may play a role in reducing the vascular constriction especially in young aorta.     

Acute relaxant effects of 17-beta-estradiol through non-genomic mechanisms in rabbit carotid artery

Estrogens could play a cardiovascular protective role not only by means of systemic effects but also by means of direct effects on vascular structure and function. We have studied the acute effects and mechanisms of action of 17-beta-estradiol on vascular tone of rabbit isolated carotid artery. 17-Beta-estradiol (10, 30, and 100 microM) elicited concentration-dependent relaxation of 50 mM KCl-induced active tone in male and female rabbit carotid artery. The stereoisomer 17-alpha-estradiol showed lesser relaxant effects in male rabbits. Endothelium removal did not modify relaxation induced by 17-beta-estradiol. The NO synthase inhibitor L-NAME (100 microM) only reduced significantly relaxation produced by 30 microM 17-beta-estradiol. Relaxation was not modified by the estrogen receptor antagonist ICI 182,780 (1 microM), the protein synthesis inhibitor cycloheximide (1 microM), and the selective K(+) channel blockers charybdotoxin (0.1 microM) and glibenclamide (1 microM). CaCl(2) (30 microM -10 mM) induced concentration-dependent contraction in rabbit carotid artery depolarized by 50 mM KCl in Ca(2+) free medium. Preincubation with 17-beta-estradiol (3, 10, 30, or 100 microM) or the L-type Ca(2+) channel blocker nicardipine (0.01, 0.1, 1, or 10 nM) produced concentration-dependent inhibition of CaCl(2)-induced contraction. In conclusion, 17-beta-estradiol induces endothelium-independent relaxation of rabbit carotid artery, which is not mediated by classic estrogen receptor and protein synthesis activation. The relaxant effect is due to inhibition of extracellular Ca(2+) influx to vascular smooth muscle, but activation of K(+) efflux is not involved. Relatively high pharmacological concentrations of estrogen causing relaxation preclude acute vasoactive effects of plasma levels in the carotid circulation.     

Vascular effects of caffeic acid phenethyl ester (CAPE) on isolated rat thoracic aorta

This study was aimed to investigate the vascular activity of caffeic acid phenethylester (CAPE), one of the major components of honeybee propolis. Experiments were performed on rat thoracic aortic rings, mounted in an isolated organ bath and connected to an isometric force transducer. The effect of CAPE (0.1-300 microM) was evaluated on tissue pre-contracted with phenylephrine (PE, 1 microM) or with KCl (100 mM). In another set of experiments, tissue was incubated with CAPE (1-100 microM) and responses to PE (0.01-3 microM) or KCl (60 mM) were evaluated. The effect of CAPE on cytosolic Ca(2+) concentration in aortic smooth muscle cells stimulated with PE or KCl was also evaluated. CAPE (0.1-300 microM) caused a concentration-dependent relaxation (pEC(50) 4.99 +/- 0.19; Emax 100.75 +/- 1.65%; n = 4) of tissue pre-contracted with PE that was reduced by endothelium removal or by incubation with N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 microM). CAPE also relaxed KCl-precontracted tissue (pEC(50) 4.40 +/- 0.08; n = 4). CAPE inhibited contractile responses to PE or to KCl, and also inhibited the contractile response to PE obtained in a Ca(2+)-free medium. In addition, CAPE inhibited the increase in cytosolic Ca(2+) concentration triggered by stimulation of aortic smooth muscle cells with PE or KCl. Our results demonstrate a vascular activity for CAPE, that is only partially dependent on nitric oxide. Indeed, at high concentrations, CAPE vasorelaxant effect occurs also in absence of endothelium and it is likely due to an inhibitory effect on calcium movements through cell membranes.     

Nitric oxide inhibits Ca(2+) mobilization through cADP-ribose signaling in coronary arterial smooth muscle cells

The present study was designed to determine whether the cADP-ribose-mediated Ca(2+) signaling is involved in the inhibitory effect of nitric oxide (NO) on intracellular Ca(2+) mobilization. With the use of fluorescent microscopic spectrometry, cADP-ribose-induced Ca(2+) release from sarcoplasmic reticulum (SR) of bovine coronary arterial smooth muscle cells (CASMCs) was determined. In the alpha-toxin-permeabilized primary cultures of CASMCs, cADP-ribose (5 microM) produced a rapid Ca(2+) release, which was completely blocked by pretreatment of cells with the cADP-ribose antagonist 8-bromo-cADP-ribose (8-Br-cADPR). In intact fura 2-loaded CASMCs, 80 mM KCl was added to depolarize the cells and increase intracellular Ca(2+) concentration ([Ca(2+)](i)). Sodium nitroprusside (SNP), an NO donor, produced a concentration-dependent inhibition of the KCl-induced increase in [Ca(2+)](i), but it had no effect on the U-46619-induced increase in [Ca(2+)](i). In the presence of 8-Br-cADPR (100 microM) and ryanodine (10 microM), the inhibitory effect of SNP was markedly attenuated. HPLC analyses showed that CASMCs expressed the ADP-ribosyl cyclase activity, and SNP (1-100 microM) significantly reduced the ADP-ribosyl cyclase activity in a concentration-dependent manner. The effect of SNP was completely blocked by addition of 10 microM oxygenated hemoglobin. We conclude that ADP-ribosyl cyclase is present in CASMCs, and NO may decrease [Ca(2+)](i) by inhibition of cADP-ribose-induced Ca(2+) mobilization.     

Histaminergic effect of crude papaya latex on isolated guinea pig ileal strips.

In the present study, we investigated the effect of the crude latex of Carica papaya L. (CPX) on isolated guinea pig ileal strips. CPX (0.5-512 microg/ml) caused concentration-dependent contraction of ileal strips suspended in Tyrode solution. The concentration of atropine (0.69 microM) that significantly blocked the contractile effect of acetylcholine on the isolated guinea pig ileum showed no significant effect on CPX- and histamine-induced contractions of the ileal strips. Mepyramine (87.6 nM) significantly blocked the contractile effect of histamine and CPX on the ileum. The same concentration of mepyramine, however, had no significant effect on acetylcholine-induced contraction of the isolated ileal strips. Removal of Ca2+ from the bathing medium abolished ileal contractions induced by acetylcholine, histamine and CPX. All the test substances were able to provoke ileal contractions after replacement of the Ca(2+)-free solution with Tyrode solution. Furthermore, 10(-5) M of nifedipine, a Ca(2+)-entry antagonist, reversibly inhibited the contractile effect of all the test substances on the ileal strips. Results of this study together appear to show that CPX-induced contraction of the isolated guinea pig ileum is mediated via H1-receptors and dependent on extracellular Ca2+ influx.     

Effects of econazole on receptor-operated and depolarization-induced contractions in rat isolated aorta

In our previous study, econazole caused a decrease in serum nitrite levels in septic mice in vivo, but it enhanced the mortality rate. The aim of the study was to investigate the in vitro effects of econazole on receptor-operated and depolarization-induced contractions on endothelium-intact and -denuded rat isolated aorta. Econazole (0.1, 1 and 10 microM) significantly inhibited receptor-operated (phenylephrine, Phe) and depolarization (KCl)-induced contractions of endothelium-intact or -denuded rings in a noncompetitive and concentration-dependent manner. Removal of endothelium changed the pD'2 values only for KCl-induced responses. The pD'2 values of L-type calcium channel blocker nifedipine were significantly higher than the econazole on Phe concentration-response curves in endothelium-intact and -denuded rings. Econazole caused a biphasic response in precontracted by Phe or KCl in endothelium-intact and -denuded rings, first a transient contraction following sustained relaxation. Removal of endothelium did not affect the contractile responses induced by Phe. The contractile responses induced by 10 microM econazole in the KCl-precontracted rings were antagonized by the treatment of alpha-adrenergic receptor antagonist, phentolamine (10 microM). Deendothelization was significantly increased the IC50 values of econazole obtained from Phe- and KCl-precontractions. The relaxations induced by 10 microM econazole in endothelium-intact rings precontracted with Phe or KCl were not changed by NO synthase inhibitor, L-N(G)-nitroarginine (100 microM). The IC50 values of econazole were significantly higher than nifedipine in endothelium-intact and -denuded rings. These results suggest that econazole is a noncompetitive antagonist on alpha1-adrenoceptor-mediated and depolarization-induced contractions in rat isolated aorta by inhibiting Ca2+ entry through L-type calcium channels, and the endothelium seems to modulate vascular responses induced by this agent. The vascular effects of econazole may limit the usage of this agent in septic shock.     

Vasorelaxant effect of the flavonoid galangin on isolated rat thoracic aorta

Here we investigated the effect of the flavonoid galangin in isolated rat thoracic aortic rings. Galangin (0.1-100 microM) induced relaxation in rings pre-contracted with phenylephrine (PE 1 microM) or with KCl (100 mM) or pre-treated with the nitric oxide synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME, 100 microM), the cyclooxygenase inhibitor indomethacin (10 microM) and the adenylate cyclase inhibitor, SQ 22,536 (100 microM). In another set of experiments, rat aortic rings were incubated with galangin (1-100 microM) and the contractile responses to PE (0.001-3 microM) or to KCl (60 mM) were evaluated. We also evaluated the effect of galangin (100 microM) on PE (10 microM)-induced contraction in a Ca2+-free medium. Galangin relaxed aortic rings with or without endothelium. Galangin effect was significantly inhibited by L-NAME. Galangin inhibited the contractile response to PE, either in presence or in absence of external calcium, and to KCl. In the end, we also found that galangin caused nitric oxide (NO) release from aortic rings and abolished the increase in [Ca2+]i triggered by PE or KCl in aortic smooth muscle cells, either in presence and in absence of external Ca2+. Our results suggest that galangin reduces the contractility of rat aortic rings through an endothelium-dependent mechanism, involving NO, and also through an endothelium-independent mechanism, inhibiting calcium movements through cell membranes.     

Inhibitory effects of tetramethyl and tetraethyl derivatives of pyrazine on dog saphenous vein.

The effects of two structurally similar pyrazine derivatives, tetramethylpyrazine (TMP) and tetraethylpyrazine (TEP) on the contractile responses of dog saphenous vein to KCl (via membrane depolarization), phenylephrine (PHE, alpha 1-adrenergic agonist), and B-HT 920 (alpha 2-adrenergic agonist) were investigated. The relaxant or inhibitory effect of TMP and TEP was most potent on KCl-induced responses and least potent on PHE-induced responses. Their effect on KCl-induced responses was more prominent at 30 mM KCl than at 100 mM KCl. In Ca(2+)-free medium, PHE and B-HT 920 elicited transient responses, which were also markedly and reversibly inhibited by TMP and TEP. Similar results were also obtained when prostaglandin F2 alpha was used as an agonist. In all four types of contractile responses involving different receptors, the inhibitory effect of TEP was consistently more potent than that of TMP. We conclude that both TMP and TEP behave as a nonselective smooth muscle relaxant having similar and multiple actions including their general interference with the processes involving both Ca2+ entry and intracellular Ca2+ release.     

Tyrosine kinase-dependent calcium signaling in airway smooth muscle cells

Contractile agonists may stimulate mitogenic responses in airway smooth muscle by mechanisms that involve tyrosine kinases. The role of contractile agonist-evoked activation of tyrosine kinases in contractile signaling is not clear. We addressed this issue using cultured rat airway smooth muscle cells. In these cells, serotonin (5-HT, 1 microM) caused contraction (quantitated by a decrease in cell area), which was blocked by the tyrosine kinase inhibitor genistein (40 microM). Genistein and tyrphostin 23 (40 and 10 microM, respectively) significantly decreased 5-HT-evoked peak Ca(2+) responses, and the effect of genistein could be observed in the absence of extracellular Ca(2+). The specific inhibitor of mitogen-activated protein kinase kinase PD-98059 (30 microM) had no significant effect on peak Ca(2+) levels. Western analysis of cell extracts revealed that 5-HT caused a significant increase in tyrosine phosphorylation of proteins with molecular masses of approximately 70 kDa within 10 s of stimulation but no measurable tyrosine phosphorylation of the gamma isoform of phospholipase C (PLC-gamma). Tyrosine phosphorylation was inhibited by genistein. Furthermore, genistein (40 microM) significantly attenuated 5-HT-induced inositol phosphate production. We conclude that in airway smooth muscle contractile agonists acting on G protein-coupled receptors may activate tyrosine kinase(s), which in turn modulate calcium signaling by affecting, directly or indirectly, PLC-beta activity. It is unlikely that PLC-gamma or the mitogen-activated protein kinase pathway is involved in Ca(2+) signaling to 5-HT.     

NO(+) but not NO radical relaxes airway smooth muscle via cGMP-independent release of internal Ca(2+)

We compared the effects of two redox forms of nitric oxide, NO(+) [liberated by S-nitroso-N-acetyl-penicillamine (SNAP)] and NO. [liberated by 3-morpholinosydnonimine (SIN-1) in the presence of superoxide dismutase], on cytosolic concentration of Ca(2+) ([Ca(2+)](i); single cells) and tone (intact strips) obtained from human main stem bronchi and canine trachealis. SNAP evoked a rise in [Ca(2+)](i) that was unaffected by removing external Ca(2+) but was markedly reduced by depleting the internal Ca(2+) pool using cyclopiazonic acid (10(-5) M). Dithiothreitol (1 mM) also antagonized the Ca(2+) transient as well as the accompanying relaxation. SNAP attenuated responses to 15 and 30 mM KCl but not those to 60 mM KCl, suggesting the involvement of an electromechanical coupling mechanism rather than a direct effect on the contractile apparatus or on Ca(2+) channels. SNAP relaxations were sensitive to charybdotoxin (10(-7) M) or tetraethylammonium (30 mM) but not to 4-aminopyridine (1 mM). Neither SIN-1 nor 8-bromoguanosine 3',5'-cyclic monophosphate had any significant effect on resting [Ca(2+)](i), although both of these agents were able to completely reverse tone evoked by carbachol (10(-7) M). We conclude that NO(+) causes release of internal Ca(2+) in a cGMP-independent fashion, leading to activation of Ca(2+)-dependent K(+) channels and relaxation, whereas NO. relaxes the airways through a cGMP-dependent, Ca(2+)-independent pathway.     

Impaired Ca2+ handling in penile arteries from prediabetic Zucker rats: involvement of Rho kinase

Diabetes is associated with an increased vascular tone usually involved in the pathogenesis of diabetic cardiovascular complications such as hypertension, stroke, coronary artery disease, or erectile dysfunction (ED). Enhanced contractility of penile erectile tissue has been associated with augmented activity of the RhoA/Rho kinase (RhoK) pathway in models of diabetes-associated ED. The present study assessed whether abnormal vasoconstriction in penile arteries from prediabetic obese Zucker rats (OZRs) is due to changes in the intracellular Ca(2+) concentration ([Ca(2+)](i)) and/or in myofilament Ca(2+) sensitivity. Penile arteries from OZRs and lean Zucker rats (LZRs) were mounted on microvascular myographs for simultaneous measurements of [Ca(2+)](i) and tension. The relationships between [Ca(2+)](i) and contraction for the α(1)-adrenergic vasoconstrictor phenylephrine (PE) were left shifted and steeper in OZRs compared with LZRs, although the magnitude of the contraction was similar in both groups. In contrast, the vasoconstriction induced by the thromboxane A(2) receptor agonist U-46619 was augmented in arteries from OZRs, and this increase was associated with an increase in both the sensitivity and maximum responses to Ca(2+). The RhoK inhibitor Y-27632 (10 μM) reduced the vasoconstriction induced by PE to a greater extent in OZRs than in LZRs, without altering Ca(2+). Y-27632 inhibited with a greater potency the contraction elicited by high KCl in arteries from OZRs compared with LZRs without changing [Ca(2+)](i). RhoK-II expression was augmented in arteries from OZRs. These results suggest receptor-specific changes in the Ca(2+) handling of penile arteries under conditions of metabolic syndrome. Whereas augmented vasoconstriction upon activation of the thromboxane A(2) receptor is coupled to enhanced Ca(2+) entry, a RhoK-mediated enhancement of myofilament Ca(2+) sensitivity is coupled with the α(1)-adrenergic vasoconstriction in penile arteries from OZRs.     

Role of Cav1.2 L-type Ca2+ channels in vascular tone: effects of nifedipine and Mg2+

Ca(2+) entry via L-type voltage-gated Ca(2+) channels (LVGCs) is a key factor in generating myogenic tone (MT), as dihydropyridines (DHPs) and other LVGC blockers, including Mg(2+), markedly reduce MT. Recent reports suggest, however, that elevated external Mg(2+) concentration and DHPs may also inhibit other Ca(2+)-entry pathways. Here, we explore the contribution of LVGCs to MT in intact, pressurized mesenteric small arteries using mutant mice (DHP(R/R)) expressing functional but DHP-insensitive Ca(v)1.2 channels. In wild-type (WT), but not DHP(R/R), mouse arteries, nifedipine (0.3-1.0 microM) markedly reduced MT and vasoconstriction induced by high external K(+) concentrations ([K(+)](o)), a measure of LVGC-mediated Ca(2+) entry. Blocking MT and high [K(+)](o)-induced vasoconstriction by <1 microM nifedipine in WT but not in DHP(R/R) arteries implies that Ca(2+) entry via Ca(v)1.2 LVGCs is obligatory for MT and that nifedipine inhibits MT exclusively by blocking LVGCs. We also examined the effects of Mg(2+) on MT and LVGCs. High external Mg(2+) concentration (10 mM) blocked MT, slowed the high [K(+)](o)-induced vasoconstrictions, and decreased their amplitude in WT and DHP(R/R) arteries. To verify that these effects of Mg(2+) are due to block of LVGCs, we characterized the effects of extracellular and intracellular Mg(2+) on LVGC currents in isolated mesenteric artery myocytes. DHP-sensitive LVGC currents are inhibited by both external and internal Mg(2+). The results indicate that Mg(2+) relaxes MT by inhibiting Ca(2+) influx through LVGCs. These data provide new information about the central role of Ca(v)1.2 LVGCs in generating and maintaining MT in mouse mesenteric small arteries.     

Effect of timosaponin A-III,from Anemarrhenae asphodeloides Bunge (Liliaceae), on calcium mobilization in vascular endothelial and smooth muscle cells and on vascular tension

The effects of timosaponin A-III (TA-III), from Rhizoma Anemarrhenae, on Ca(2+) mobilization in vascular endothelial cells and smooth muscle cells and on vascular tension have been explored. TA-III increased intracellular Ca(2+) concentrations ([Ca(2+)](i)) in endothelials cells at a concentration larger than 5 microM with an EC(50) of 15 microM, and increased [Ca(2+)](i) in smooth muscle cells at a concentration larger than 1 microM with an EC(50) of 8 microM. Within 5 min, the [Ca(2+)](i) signal was composed of a gradual rise, and the speed of rising depended on the concentration of TA-III. The [Ca(2+)](i) signal was abolished by removing extracellular Ca(2+) and was recovered after reintroduction of Ca(2+). The TA-III-induced [Ca(2+)](i) increases in smooth muscle cells were partly inhibited by 10 microM nifedipine or 50 microM La(3+), but was insensitive to 10 microM verapamil and diltiazem. TA-III (10-100 microM) inhibited 0.3 microM phenylephrine-induced vascular contraction, which was abolished by pretreatment with 100 microM N(omega)-nitro-L-arginine (L-NNA) or by denuding the aorta. TA-III also increased [Ca(2+)](i) in renal tubular cells with an EC(50) of 8 microM. Collectively, the results show for the first time that TA-III causes [Ca(2+)](i) increases in the vascular system. TA-III acted by causing Ca(2+) influx without releasing intracellular Ca(2+). TA-III induced relaxation of phenylephrine-induced vascular contraction via inducing release of nitric oxide from endothelial cells.