Involvement of protein kinase C and Na+/K+-ATPase in the contractile response induced by myricetin in rat isolated aorta

The role of PKC and Na+/K+-ATPase in the vascular smooth muscle responses induced by the bioflavonoid myricetin was investigated. KCl induced a concentration-dependent relaxation in arteries exposed to K+-free solution that was mainly mediated by an activation of Na+/K+-ATPase. Myricetin (50 microM) partially inhibited this vasorelaxant effect induced by KCl in intact rings, being unaffected in the endothelium-denuded rings. This inhibitory effect induced by myricetin was suppressed by the PGH2-TXA2 receptor antagonist, SQ 29,548, and the PKC inhibitor, staurosporine. Myricetin also induced an endothelium-dependent contractile response which was increased in the presence of PMA and reduced by staurosporine. In conclusion, myricetin both modulates Na+/K+-ATPase-induced vasodilatation acting as a functional inhibitor of Na+/K+-ATPase activity and activates protein kinases, including PKC, to induce contraction. These effects appear to be related to the activation of PGH2-TXA2 receptors on vascular smooth muscle by the TXA2 released from endothelium.NA:noradrenalineNA+/K+-ATPase pump:sodium-potassium-activated ATPasePKC:protein kinase CPMA:phorbol 12-myristate 13-acetateTXA2:thromboxane A2The role of PKC and Na+/K+-ATPase in the vascular smooth muscle responses induced by the bioflavonoid myricetin was investigated. KCl induced a concentration-dependent relaxation in arteries exposed to K+-free solution that was mainly mediated by an activation of Na+/K+-ATPase. Myricetin (50 microM) partially inhibited this vasorelaxant effect induced by KCl in intact rings, being unaffected in the endothelium-denuded rings. This inhibitory effect induced by myricetin was suppressed by the PGH2-TXA2 receptor antagonist, SQ 29,548, and the PKC inhibitor, staurosporine. Myricetin also induced an endothelium-dependent contractile response which was increased in the presence of PMA and reduced by staurosporine. In conclusion, myricetin both modulates Na+/K+-ATPase-induced vasodilatation acting as a functional inhibitor of Na+/K+-ATPase activity and activates protein kinases, including PKC, to induce contraction. These effects appear to be related to the activation of PGH2-TXA2 receptors on vascular smooth muscle by the TXA2 released from endothelium.     

Mechanisms of hydroxyl radical-induced contraction of rat aorta

The present study was designed to investigate the effects of hydroxyl radicals (*OH), generated via the Fe2+-mediated Fenton reaction, on isolated rat aortic rings with and without endothelium. In the absence of any vasoactive agent, generation of *OH alone elicited an endothelium-independent contraction in rat aortic rings in a concentration-dependent manner. Hydroxyl radical-induced contractions of denuded rat aortic rings appeared, however, to be slightly stronger than those on intact rat aortic rings. The contractile responses to *OH were neither reversible nor reproducible in the same ring; even small concentrations of *OH radicals resulted in tachyphylaxis. Removal of extracellular calcium ions (Ca2+) or buffering intracellular Ca2+ with 10 microM acetyl methyl ester of bis(o-aminophenoxy) ethane-N,N,N',N',-tetraacetic acid (BAPTA-AM) significantly attenuated the contractile actions of *OH radicals. The presence of 1 microM staurosporine, 1 microM bisindolylmaleimide I, 1 microM G?6976 [inhibitor of protein kinase C (PKC)], 2 microM PD-980592 (inhibitor of ERK), 10 microM genistein, and 1 microM wortmannin significantly inhibited the contractions induced by *OH. Proadifen (10 microM), on the other hand, significantly potentiated the hydroxyl radical-induced contractions. Exposure of primary cultured aortic smooth muscle cells to *OH produced significant, rapid rises of intracellular free Ca2+ ([Ca2+]i). Several, specific antagonists of possible endogenously formed vasoconstrictors did not inhibit or attenuate either hydroxyl radical-induced contractions or the elevation of [Ca2+]i. Our new results suggest that hydroxyl radical-triggered contractions on rat aortic rings are Ca2+-dependent. Several intracellular signal transduction systems seem to play some role in hydroxyl radical-induced vasoconstriction of rat aortic rings.     

Release of prostaglandins during the contraction of rat aorta induced by ouabain and K+-free solution

Contractile effects of ouabain and K+-free solution on rat aortic strips were investigated. In the aorta without endothelium, application of ouabain or K+-free solution produced, after a latency period, a slow contraction reaching the maximum after 80-100 min. Pretreatment of the muscle with either indomethacin (20 microM) or verapamil (1 microM) decreased the maximum level of these contractions, whereas verapamil, but not indomethacin, prolonged the latency period. Simultaneous application of these inhibitors showed additive inhibitory effects. In the presence of endothelium, the latency period slightly increased without changing the maximum contractile tension. Methylene blue (5 microM) shortened the latency period only in the aorta with endothelium. These results suggest that the contractions of rat aorta induced by ouabain and K+-free solution are due not only to membrane depolarization and Na+-Ca2+ exchange but also to the release of prostaglandins. Endothelium-derived relaxing factor seems to inhibit a part of these contractions.     

Effects of high calcium diet on arterial smooth muscle function and electrolyte balance in mineralocorticoid-salt hypertensive rats.

1. The effects of a high calcium diet (2.5%) on blood pressure, electrolyte balance, plasma and tissue atrial natriuretic peptide (ANP), cytosolic free Ca2+ concentration ([Ca2+]i), and arterial smooth muscle responses were studied in one-kidney deoxycorticosterone (DOC)-NaCl hypertensive Wistar rats. 2. Calcium supplementation for 8 weeks markedly attenuated the development of DOC-NaCl hypertension and the associated cardiac hypertrophy, and prevented the DOC-NaCl-induced sodium-volume retention as judged by reduced plasma Na+, and decreased plasma and ventricular ANP concentrations in high calcium-fed DOC-NaCl rats. However, calcium supplementation did not affect the DOC-NaCl-induced rise in platelet [Ca2+]i. 3. Smooth muscle contractions of isolated mesenteric arterial rings in response to depolarization by K+ (20-30 mM) were enhanced in DOC-NaCl-treated rats, this enhancement being abolished by concurrent oral calcium loading. The Ca2+ entry blocker nifedipine (10 nM) inhibited the contractions induced by K+ (30-125 mM) more effectively in DOC-NaCl rats than in controls, while the inhibition in calcium-loaded DOC-NaCl rats was significantly greater than in controls only with 30 mM K+. 4. The contractions of mesenteric arterial rings induced by omission of K+ from the organ baths were used to evaluate cell membrane permeability to ions. In chemically denervated rings the onset of the gradual rise in contractile force in K(+)-free medium occurred earlier, and the rate of the contraction was faster in DOC-NaCl-treated rats than in controls and high calcium-fed DOC-NaCl rats. Smooth muscle relaxation induced by 0.5 mM K+ upon K(+)-free contractions was clearly slower in DOC-NaCl rats than in controls and calcium-supplemented DOC-NaCl rats. 5. The functions of arterial smooth muscle Na+, Ca2+ exchange and Ca(2+)-ATPase were evaluated by the aortic contractions elicited by low Na+ medium, and the subsequent relaxation responses induced by Ca(2+)-free solution (in the presence of 5 mM caffeine, 1 microM nifedipine and 10 microM phentolamine). The rate of aortic low Na+ contractions (evaluating Ca2+ influx via Na+, Ca2+ exchange), as well as that of subsequent relaxations was slower in DOC-NaCl-treated rats than in controls, whether the relaxation was induced in normal (144.0 mM) or low (1.2 mM) organ bath Na+ concentration (reflecting Ca2+ extrusion by both Ca(2+)-ATPase and Na+, Ca2+ exchange, and by Ca(2+)-ATPase alone, respectively). However, in calcium-supplemented DOC-NaCl rats the aortic responses did not differ from control.(ABSTRACT TRUNCATED AT 400 WORDS)     

Lead induces endothelium- and Ca2+-independent contraction in rat aortic rings

The contractile effect of lead on rat aortic rings was examined. Lead (0.1-3.1 mM) elicited concentration-dependent but endothelium-independent contractions, which were unaffected by prazosin (1 microM). The contractile effects of lead were similar when the aortic rings were bathed either in the absence or presence of external Ca2+. Lanthanum (1 mM) but not verapamil (I pM) inhibited the lead contractions; hence non-L-calcium channels are involved in such effect. In addition, lead induced contractions on aortic rings incubated in Ca2+-free EGTA-containing solution for 70 min., an experimental condition in which intracellular Ca2+-stores are depleted. Finally, the contractile effect of lead was not modified by calphostin C (an inhibitor of protein kinase C). In conclusion, the present results suggest that in rat aorta, the lead-induced contraction is independent of extra- and intracellular calcium stores. In addition, the effect of lead is independent of either catecholamines or protein kinase C. It is likely that in rat aorta, lead enters into the smooth muscle cells through non-L-calcium channels, and when acting like calcium on the contractile machinery it produces contraction. The differences observed between our results and those obtained by other authors may indicate that the mechanism of the contractile effect of lead varies among the different blood vessels.     

Endothelial nitric oxide attenuates Na+/Ca2+ exchanger-mediated vasoconstriction in rat aorta

BACKGROUND AND PURPOSE: The Na+/Ca2+ exchanger (NCX) may be an important modulator of Ca2+ entry and exit. The present study investigated whether NCX was affected by prostacyclin and nitric oxide (NO) released from the vascular endothelium, as NCX contains phosphorylation sites for PKA and PKG. EXPERIMENTAL APPROACH: Rat aortic rings were set up in organ baths. Tension was measured across the ring with a force transducer. KEY RESULTS: Lowering extracellular [Na+] ([Na+]o) to 1.18 mM induced vasoconstriction in rat endothelium-denuded aortic rings. This effect was blocked by the NCX inhibitor KB-R7943 (2-2-[4-(4-nitrobenzyloxy)phenyl] ethyl isothiourea methanesulphonate; 1 microM). In endothelium-intact aortic rings, decreasing [Na+]o did not constrict the aortic rings significantly, but after treatment with the guanylate cyclase inhibitor ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; 1 microM) or the NOS inhibitor L-NAME (N(omega)-nitro-L-arginine methyl ester; 50 microM), a vasoconstriction that was similar in size to that in endothelium-denuded preparations was evident. The vasorelaxation induced by the NO donor sodium nitroprusside sodium nitroprusside dihydrate (30 nM) was the same in the endothelium-denuded aortic rings preconstricted with either low Na+ (1.18 mM), the thromboxane A2 agonist U46619 (9,11-dideoxy-9alpha, 11alpha-methanoepoxy prostaglandin F(2alpha); 0.1 microM) or high K+ (80 mM). CONCLUSIONS AND IMPLICATIONS: The results suggest that the endothelium inhibits NCX operation via guanylate cyclase/NO. This is stronger than for other constrictors such as phenylephrine and may relate to concomitant NCX-stimulated NO release from the endothelium. This finding may be important where NCX operates in reverse mode, such as during ischaemia, and highlights a new mechanism whereby the endothelium modulates Ca2+ homoeostasis in vascular smooth muscle.     

Dual regulation of cerebrovascular tone by UTP: P2U receptor-mediated contraction and endothelium-dependent relaxation.

1. The mechanisms of vascular tone regulation by extracellular uridine 5'-triphosphate (UTP) were investigated in bovine middle cerebral arterial strips. Changes in cytosolic Ca2+ concentration ([Ca2+]i) and force were simultaneously monitored by use of front-surface fluorometry of fura-2. 2. In the arterial strips without endothelium, UTP (0.1 microM-1 mM) induced contraction in a concentration-dependent manner. However, when the endothelium was kept intact, cumulative application of UTP (0.1-100 microM) (and only at 1 mM) induced a modest phasic contraction in arterial strips. This endothelium-dependent reduction of the UTP-induced contraction was abolished by 100 microM N omega-nitro-L-arginine (L-NOARG) but not by 10 microM indomethacin. In the presence of intact endothelium, UTP (30 microM) induced a transient relaxation of the strips precontracted with 30 nM U-46619 (a stable analogue of thromboxane A2), which was completely inhibited by pretreatment with L-NOARG but not with indomethacin. 3. In the endothelium-denuded strips, the contractile response to UTP was abolished by desensitization to either ATP gamma S or ATP (P2U receptor agonists), but not by desensitization to alpha, beta-methylene-ATP (P2x receptor agonist) or to 2-methylthio-ATP (P2Y receptor agonist). Desensitization to UTP abolished the contractile response to ATP. 4. In the endothelium-denuded artery, a single dose application of UTP induced an initial transient, and subsequently lower but sustained increase in [Ca2+]i and force. In the absence of extracellular Ca2+, UTP induced only the initial transient increases in [Ca2+]i and force, while the sustained increases in [Ca2+]i and force were abolished. UTP (1 mM) had no effect on the basic [Ca2+]i-force relationship obtained on cumulative application of extracellular Ca2+ at steady state of 118 mM K(+)-depolarization-induced contraction. 5. We conclude that in the presence of an intact endothelium, UTP-induced relaxation of preconstricted middle cerebral artery is mainly mediated indirectly, by the production of an endothelium-derived relaxing factor, but at high doses of UTP, vascular smooth muscle contraction is mediated directly via activation of P2U purinoceptor and [Ca2+]i elevation without Ca(2+)-sensitization of the contractile apparatus. UTP may thus exert a dual regulatory effect upon cerebrovascular tone, but in cases where the endothelium is impaired, it may also act as a significant vasoconstrictor.     

Magnesium modulates contractile responses of rat aorta to thiocyanate: A possible relationship to smoking-induced atherosclerosis.

Thiocyanate anions (SCN-) as the end products of tobacco smoke and found in the blood of cigarette smokers have been implicated in atherogenesis and heart diseases. Magnesium deficiency has also been implicated in the etiology of atherogenesis. The contractile responses of rat aorta to SCN- and the modulation of extracellular magnesium ions ([Mg2+]o) on the effect of SCN- were, therefore, studied in isolated rat aortic rings. SCN- exposure at a range of concentrations (from 10(-5) to 5 x 10(-2) M) induces contractile responses of isolated rat aortic rings with and without endothelium in a concentration-dependent manner. Significant differences in responsiveness to SCN- were found in rat aortic ring segments with and without endothelial cells. Preincubation of these vessels with low [Mg2+]o markedly shifted the contractile concentration-effect curves to the left, and the contractile effects of SCN- in rat aortic rings were potentiated. In contrast to lowering [Mg2+]o, increasing [Mg2+]o to 2.4 mM was found to dramatically attenuate the contractile responses to SCN-. In the absence of extracellular Ca2+ ([Ca2+]o), SCN--induced contractions were, however, almost abolished after exposure to Mg2+-free medium. In order to investigate the mechanisms of [Mg2+]o modulation of SCN--induced contractile response of rat aorta, changes in intracellular Ca2+ ([Ca2+]i) were measured in cultured primary smooth muscle cells isolated from rat aorta. The resting level of [Ca2+]i in the rat aortic smooth muscle cells was 80.6 +/- 6.6 nM. Exposure of these cells to SCN- (5 x 10(-5) to 5 x 10(-3) M) produced rises in [Ca2+]i in a concentration-dependent manner. Preincubation of these cells with low [Mg2+]o (0 or 0.3 mM, the lowest physiological range) for 24 h significantly potentiated increments in [Ca2+]i induced by SCN-. These rises in [Ca2+]i induced by SCN- were completely inhibited by pretreating the cells with 2.4 mM [Mg2+]o for 24 h. These results support a hypothesis whereby cigarette smoking or exposure to smoking can induce cardiovascular diseases, at least partly, probably by causing spasm and thickening of arterial blood vessels as a consequence of large rises in [Ca2+]i in vascular smooth muscle cells. The chronic presence of or exposure to both thiocyanate and low Mg2+ in the blood of smokers can result in rapid flux of Ca2+ into vascular smooth muscle cells, thus accelerating or initiating atherosclerotic processes in smokers.     

In the presence of L-NAME SERCA blockade induces endothelium-dependent contraction of mouse aorta through activation of smooth muscle prostaglandin H2/thromboxane A2 receptors

1. The mechanism of transient contractions induced by the sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA) blocker cyclopiazonic acid (CPA) in the presence of L-NAME was investigated in mouse aorta. 2. The contractions elicited by 10 micro M CPA required an intact endothelium, were dependent upon external Ca(2+) and were prevented by 10 micro M indomethacin, the inhibitor of prostaglandin synthesis, or 1 micro M SQ29548, the specific prostaglandin H2/thromboxane A2 (PGH2/TXA2) receptor blocker. 3. A blocker of receptor/store operated Ca(2+) channels and voltage gated calcium channels (VGCC), SK&F 96365 (10 micro M), completely abolished the contractions, while a specific blocker of VGCC nifedipine (1 micro M) inhibited them by one third. 4. Dichlorobenzamyl hydrochloride, a blocker of Na(+)/Ca(2+) exchange effectively prevented return of tension to baseline value. 5. At higher concentrations (30-100 micro M) CPA induced indomethacin-resistant tonic contractions of mouse aorta. The CPA dose response curve for tonic contractions is shifted to the right compared to the transient contractions suggesting that smooth muscle is less sensitive to CPA than endothelium. 6. PGH2/TXA2 receptors in mouse aorta are highly sensitive to the thromboxane analogue U46619 (EC(50) : 1.93 nM). This compound stimulates contractions even in the absence of external Ca(2+), which are abolished by the Rho-kinase inhibitor HA-1077. 7. The results suggest that 10 micro M CPA induced capacitive Ca(2+) entry in endothelial cells stimulating the release of PGH2/TXA2, which subsequently caused smooth muscle contraction dependent on Ca(2+) influx and myofilament sensitization by Rho-kinase. Higher concentrations of CPA (30-100 micro M) directly induced contraction of mouse aortic smooth muscle.     

Endothelin-1 and platelet activating factor stimulate thromboxane A2 biosynthesis in rat vascular smooth muscle cells

The effect of endothelin-1 (ET-1) on the release of thromboxane A2 (TXA2) was examined in cultured rat vascular smooth muscle cells (VSMC). ET-1 (10(-11) to 10(-6) M) significantly stimulated the release of thromboxane B2 (TXB2), a stable metabolite of TXA2. These effects of ET-1 were blocked by a cyclooxygenase inhibitor (indomethacin), a TXA2 synthetase inhibitor (CV-1451) and a specific platelet activating factor (PAF) antagonist (CV-6209). Additionally, PAF (10(-11) to 10(-6) M) stimulated the TXB2 release. Pretreatment with the phospholipase A2 inhibitor dexamethasone potently inhibited both ET-1 and PAF-induced elevation of cytosolic free Ca2+ concentrations [( Ca2+]i) in fura-2-loaded VSMC. These results clearly demonstrate that both ET-1 and PAF stimulate TXA2 biosynthesis in cultured rat VSMC, and TXA2 may contribute to the elevation of [Ca2+]i induced by ET-1 or PAF in VSMC. Furthermore, the stimulation of TXA2 biosynthesis may be a result of PLA2 activation by not only ET-1 but also PAF.     

Contractile responses to calcium chloride in rat aortic rings bathed in K+-free solution are resistant to organic calcium antagonists.

1. In rat aortic rings, devoid of functional endothelium, suspended in a modified Krebs solution (KCl: 0 mM; CaCl2: 0.63 mM), addition of CaCl2 (0.89-10 mM) produced concentration-related increases in tension (Emax = 2.38 +/- 0.10 g, EC50 = 2.31 +/- 0.15 mM, n = 36). 2. The Ca2+ evoked contractile responses were not modified by cinnarizine (10 microM), diltiazem (1 microM), ryanodine (10 microM), verapamil (1 microM), or the dihydropyridines, nitrendipine (1 microM) and (-)-Bay K 8644 (0.003-0.3 microM). 3. Cobalt chloride (0.1-1 mM) competitively antagonized the Ca2+ concentration-response curve; the Schild plot (slope 1.08 +/- 0.04), gave a pA2 value of 3.3 +/- 0.01 (n = 27). Nickel chloride (0.5-1 mM) displaced the Ca2+ concentration-response curve to the right, without an effect on the maximum response. Cadmium chloride (3-30 microM) depressed the maxima of concentration-response curves to Ca2+ with an IC50 of 15.5 +/- 1.1 microM (n = 6). 4. Monochlorobenzamil (100 microM), a Na+-Ca2+ exchange inhibitor, failed to modify the Ca2+-induced contractions. 5. In conclusion, Ca2+ evoked concentration-related contractile responses of rat aortic rings bathed in a K+-free medium; these effects were attenuated by the divalent cations cobalt, nickel and cadmium, but not modified by several organic calcium antagonists. The lack of effect of diltiazem verapamil and the dihydropyridines would suggest that, under these experimental conditions, extracellular Ca2+ enters the cytosol via pathways which are distinct from the slow (L-type) calcium channels.     

Influence of extracellular Ca2+ on the modulation of alpha-adrenergic-induced contractions in rat aorta.

1. Rat thoracic aortic rings with and without endothelium incubated in a Ca2(+)-free solution showed a significant reduction of the maximal contraction (E max) induced by norepinephrine (NE) and phenylephrine (Ph), while the contraction induced by a 70 mM KCl depolarizing solution was completely abolished. 2. After Ca2+ removal, pD2 values (-log ED50) for NE and Ph were significantly reduced only in vessels without endothelium. 3. Under control conditions, clonidine (C) induced a contraction only in vessels without endothelium; this response was completely abolished by Ca2+ removal and by nifedipine (10(-8) M). 4. Pre-incubation with nifedipine (10(-8) M) produced an effect similar, although less pronounced, than the removal of Ca2+ in vessels with and without endothelium. 5. Nevertheless, aortic rings with intact endothelium showed a greater reduction of E max than rings without endothelium, suggesting that the endothelial layer may act in synergism with calcium channel blockers to inhibit the contractions induced by alpha-adrenergic agonists. 6. The modulation exerted by the endothelium on alpha 1- and alpha 2-adrenergic-induced contractions in rat aortic rings seems to depend both on extracellular Ca2+ concentration and on the release and action of endothelial relaxing factor(s).     

Relaxation of rat thoracic aorta induced by the Ca(2+)-ATPase inhibitor, cyclopiazonic acid, possibly through nitric oxide formation.

1 The effect of the Ca(2+)-ATPase inhibitor, cyclopiazonic acid (CPA), was studied on rat thoracic aortic ring preparations. 2 At concentrations above 0.3 microM, CPA induced relaxation in the arteries precontracted with phenylephrine. Removal of the endothelium abolished CPA-induced relaxation. 3 The nitric oxide (NO) synthase inhibitor NG-nitro L-arginine (3-300 microM), the free radical scavenger haemoglobin (0.1-3 microM), the soluble guanylate cyclase inhibitor, LY83583 (0.1-10 microM), each inhibited the endothelium-dependent relaxation to CPA. The potassium channel blocker, glibenclamide (10 microM) and cyclo-oxygenase inhibitor, indomethacin (100 microM for 60 min and then washed out) did not alter the action of CPA. 4 The calmodulin inhibitors calmidazolium (3-10 microM) and W-7 (100 microM) also abolished CPA-induced relaxation. 5 CPA (10 microM) increased guanosine 3':5'-cyclic monophosphate (cyclic GMP) levels in arteries with an intact endothelium, without affecting adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels. 6 The inhibitors of NO synthesis and actions, the calmodulin inhibitor and removal of the endothelium abolished the CPA-stimulated increase in the levels of cyclic GMP. 7 In Ca(2+)-free solution, CPA failed to induce relaxation or to stimulate cyclic GMP production. Relaxation to nitroprusside was not affected under these conditions. 8 These results suggest that CPA can stimulate NO synthesis, possibly by inhibiting a Ca(2+)-ATPase, which replenishes Ca2+ in the intracellular storage sites in endothelial cells. Depletion of the Ca2+ store in the endothelium may then trigger influx of extracellular Ca2+, contributing to an increase in free Ca2+ in the endothelial cells, which activates NO synthase and NO formation.     

Activation of rabbit platelets by Ca2+ influx and thromboxane A2 release in an external Ca(2+)-dependent manner by zooxanthellatoxin-A, a novel polyol.

1. Zooxanthellatoxin-A (ZT-A), a novel polyhydroxylated long chain compound, isolated from a symbiotic marine alga Simbiodinium sp., caused aggregation in rabbit washed platelets in a concentration-dependent manner (1-4 microM), accompanied by an increase in cytosolic Ca2+ concentration ([Ca2+]i). 2. ZT-A did not cause platelet aggregation or increase [Ca2+]i in a Ca(2+)-free solution, and Cd2+ (0.1-1 mM), Co2+ (1-10 mM) and Mn2+ (1-10 mM) inhibited ZT-A-induced aggregation. SK&F96365 (1-100 microM), a receptor operated Ca2+ channel antagonist, and mefenamic acid (0.1-10 microM), a non-specific divalent cation channel antagonist, inhibited platelet aggregation and the increase in [Ca2+]i induced by ZT-A. 3. Indomethacin (0.1-10 microM), a cyclo-oxygenase inhibitor, and SQ-29548 (0.1-10 microM), a thromboxane A2 (TXA2) receptor antagonist, inhibited platelet aggregation and the increase in [Ca2+]i induced by ZT-A. 4. Methysergide (0.01-1 microM), a 5-HT2 receptor antagonist, inhibited ZT-A-induced platelet aggregation but did not affect the increase in [Ca2+]i induced by ZT-A. 5. Tetrodotoxin (1 microM), a Na+ channel blocker and chlorpheniramine (1 microM), a H1-histamine receptor antagonist, neither affected ZT-A-induced platelet aggregation nor the increase in [Ca2+]i induced by ZT-A. 6. Genistein (1-100 microM), a protein tyrosine kinase inhibitor, and staurosporine (0.01-1 microM), a protein kinase C inhibitor, also inhibited ZT-A-induced platelet aggregation. 7. The present results suggest that ZT-A elicits Ca(2+)-influx from platelet plasma membranes. The resulting increase in [Ca2+]i subsequently stimulates the secondary release of TXA2 from platelets.(ABSTRACT TRUNCATED AT 250 WORDS)     

Contractions induced by potassium-free solution and potassium relaxation in vascular smooth muscle of hypertensive and normotensive rats.

1. Vascular contractions induced by K(+)-free solution and relaxation responses following the return of K+ to the organ bath were studied in mesenteric arterial rings from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) with particular focus on the role of vascular adrenergic nerve-endings and endothelium. 2. In endothelium-denuded rings the omission of K+ from the incubation medium resulted in gradual contractions, the rate of which was slower in SHR than WKY. Nifedipine (1 microM) inhibited the contractions more effectively in SHR than WKY. 3. Adrenergic denervation in vitro with 6-hydroxydopamine reduced the contractions induced by the K(+)-free medium in endothelium-denuded rings. The remaining contractions after denervation were markedly greater in SHR than WKY. 4. The presence of intact vascular endothelium attenuated the K(+)-free contractions in both strains, the attenuation being smaller in SHR than WKY. NG-nitro-L-arginine methyl ester (L-NAME, 0.1 mM) and methylene blue (10 microM), but not indomethacin (10 microM), abolished the attenuating effect of endothelium on the K(+)-free contractions. L-Arginine (1 mM) reversed the effect of L-NAME in WKY but not in SHR. 5. The re-addition of K+ after full K(+)-free contractions dose-dependently relaxed the rings. The rate of this K(+)-induced relaxation was significantly slower in SHR than WKY at all K+ concentrations (0.1-5.9 mM) studied, whether the endothelium or functioning adrenergic nerve-endings were present or not. Ouabain (1 mM) totally inhibited the K+ relaxation in SHR but only partially in WKY.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxygen-derived free radical-induced vasoconstriction by thromboxane A2 in aorta of the spontaneously hypertensive rat

This study was performed to clarify the mechanism of vasoconstriction induced by oxygen-derived free radicals in spontaneously hypertensive rats. The isometric tension of aortic rings from spontaneously hypertensive rats and Wistar-Kyoto rats was measured in Krebs-Henseleit solution. Oxygen-derived free radicals were generated by mixing xanthine and xanthine oxidase. The removal of endothelium enhanced the contractions induced by oxygen-derived free radicals. The inhibition of nitric oxide production with NG-nitro-L-arginine methyl ester (10(-4) M) enhanced the contractions. Treatment with the thromboxane A2 (TXA2) synthetase inhibitor OKY-046 (10(-4) M) or RS-5186 (10(-4) M) markedly reduced the contractions. Treatment with the cyclooxygenase inhibitor indomethacin (10(-5) M) and a TXA2/prostaglandin H2 (PGH2) receptor antagonist, ONO-3708 (10(-6) M), completely abolished the oxygen-derived free radical-induced contractions. In contrast, treatment with the PGI2 synthetase inhibitor tranylcypromine (10(-4) M) did not attenuate the oxygen-derived free radical-induced contractions. Whether endothelium was present or not, the release of TXB2, PGE2, and 6-keto-PGF1alpha, but not PGF2alpha, was increased by the production of oxygen-derived free radicals. Catalase and the hydroxyl radical scavenger deferoxamine plus mannitol markedly inhibited the oxygen-derived free radical-induced contractions. These results suggest that oxygen-derived free radical-induced vasoconstriction in spontaneously hypertensive rat aorta is caused by TXA2 and PGH2 released in smooth muscle.     

Study of the mechanisms involved in the vasorelaxation induced by (-)-epigallocatechin-3-gallate in rat aorta

This study investigated several mechanisms involved in the vasorelaxant effects of (-)-epigallocatechin-3-gallate (EGCG). EGCG (1 microM-1 mM) concentration dependently relaxed, after a transient increase in tension, contractions induced by noradrenaline (NA, 1 microM), high extracellular KCl (60 mM), or phorbol 12-myristate 13-acetate (PMA, 1 microM) in intact rat aortic rings. In a Ca2+ -free solution, EGCG (1 microM-1 mM) relaxed 1 microM PMA-induced contractions, without previous transient contraction. However, EGCG (1 microM-1 mM) did not affect the 1 microM okadaic acid-induced contractions. Removal of endothelium and/or pretreatment with glibenclamide (10 microM), tetraethylammonium (2 mM) or charybdotoxin (100 nM) plus apamin (500 nM) did not modify the vasorelaxant effects of EGCG. In addition, EGCG noncompetitively antagonized the contractions induced by NA (in 1.5 mM Ca2+ -containing solution) and Ca2+ (in depolarizing Ca2+ -free high KCl 60 mM solution). In rat aortic smooth muscle cells (RASMC), EGCG (100 microM) reduced increases in cytosolic free Ca2+ concentration ([Ca2+]i) induced by angiotensin II (ANG II, 100 nM) and KCl (60 mM) in 1.5 mM CaCl2 -containing solution and by ANG II (100 nM) in the absence of extracellular Ca2+. In RASMC, EGCG (100 microM) did not modify basal generation of cAMP or cGMP, but significantly reversed the inhibitory effects of NA (1 microM) and high KCl (60 mM) on cAMP and cGMP production. EGCG inhibited the enzymatic activity of all the cyclic nucleotide PDE isoenzymes present in vascular tissue, being more effective on PDE2 (IC50 approximately 17) and on PDE1 (IC50 approximately 25). Our results suggest that the vasorelaxant effects of EGCG in rat aorta are mediated, at least in part, by an inhibition of PDE activity, and the subsequent increase in cyclic nucleotide levels in RASMC, which, in turn, can reduce agonist- or high KCl concentration-induced increases in [Ca2+]i.     

Complex effects of Gillichthys urotensin II on rat aortic strips.

The aim of this study was to determine whether the fish neuropeptide, Gillichthys urotensin II (GUII), possesses significant biological activity on rat aortic strips. On intact strips, pre-contracted by noradrenaline (100 nM), low concentrations (0.1-0.5 nM) of GUII produced relaxations, while higher concentrations (1-10 nM) caused further contraction. On strips rubbed to remove endothelial cells, relaxations were absent but contractile responses to higher concentrations of GUII remained. GUII (0.02-10 nM) produced dose-related contractions of quiescent, intact aortic strips. These contractions consisted of two components, tonic and phasic, and were potentiated in rubbed strips and in the presence of the antioxidant drug hydroquinone (10 microM). Mepacrine (40 microM) and p-bromophenacyl bromide (50 microM) completely abolished contractions to GUII, but indomethacin (10 microM) and nordihydro-guaiaretic acid (10 microM) were without effect. The phasic, but not the tonic, component of the contractile response was inhibited by nitrendipine (200 nM), and was absent in bathing medium from which Ca2+ had been omitted. Addition of EGTA (2 mM) to Ca2+-free bathing medium abolished the residual tonic component. GUII-induced contractions were completely abolished by the calmodulin antagonists trifluoperazine (50 microM) and W-7 (30 microM). It is concluded that GUII, previously considered devoid of significant activity on mammalian tissues, produces potent endothelium-dependent relaxations and endothelium-independent contractions of rat aorta, and possible mechanisms underlying each response are discussed.     

Vasodilatation produced by orientin and its mechanism study

In this paper we investigated the vascular activity and possible mechanism of Orientin, from bamboo leaves (Phyllostachys nigra), in isolated thoracic aortic rings from New Zealand rabbit. Among the four compounds, studied, only Orientin relaxed phenylephrine-induced contractions with an IC50 value of 2.28 microM in the endothelium intact and with an IC50 value around 7.27 microM in the endothelium removed aortic rings. The vasorelaxant effect of Orientin on endothelium-intact thoracic aortic rings was attenuated by the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine methyl ester, but not by indomethacin (a cyclooxygenase inhibitor), tetraethylammonium chloride (K+ channels inhibitor) or propranolol (beta-receptor inhibitor). Furthermore, Orientin inhibited norepinephrine (NE), CaCl2 and KCl-induced vasoconstriction concentration dependently in a non-competitive manner, and also reduced both the initial fast release and the sustained phases of phenylephrine-induced contractions. Orientin can stimulate NO production from endothelial cells. Orientin also increased cyclic guanosine 3,5-cyclic monophosphate (cGMP) levels without changes in adenosine-3',5'-cyclic phosphoric acid (cAMP) in rabbit aorta. The results showed that Orientin relaxed thoracic aortic rings by the nitric oxide-cGMP pathway, and in the vascular smooth muscle inhibited the contraction induced by the activation of receptor-operating and voltage-dependent Ca2+ channels. Cyclooxygenase pathway, potassium channels, beta-receptors and cAMP pathway, on the other hand, had no apparent roles. The inhibition of both intracellular Ca2+ release and extracellular Ca2+ influx may be one of the main vasorelaxant mechanisms of Orientin.     

Endothelium-dependent and -independent relaxation induced by pinocembrin in rat aortic rings

The aim of present study was to evaluate the vasorelaxant effects of the flavonone pinocembrin and its possible mechanisms in isolated rat aortic rings. Pinocembrin (5 approximately 100 microM) induced relaxation in aortic rings pre-contracted with norepinephrine (NE, 1 microM) or KCl (60 mM), with pEC(50) value 4.37+/-0.02 and 4.52+/-0.04. Pretreatment with pinocembrin (30 or 50 microM) also inhibited contractile responses to NE and KCl. The vasorelaxant effect of pinocembrin relied on intact endothelium partially, and incubation with n(omega)-nitro-l-arginine methyl ester (l-NAME, 100 microM) or methylene blue (10 microM) significantly inhibited the effect, however indomethacin (5 microM) had no influence on the action. In endothelium-denuded rings, the vasorelaxant effect of pinocembrin was reduced by glibenclamide (10 microM), tetraethylammonium (5 mM) and 4-aminopyridine (100 microM). Pinocembrin also reduced NE-induced transient contraction in Ca(2+)-free solution and inhibited contraction induced by increasing external calcium in Ca(2+)-free medium plus 60 mM KCl. Our results suggest that pinocembrin induces relaxation in rat aortic rings through an endothelium-dependent pathway, involving NO-cGMP, and also through an endothelium-independent pathway, opening K(+) channels and blockade of Ca(2+) channels.