Melatonin counteracts potentiation by homocysteine of KCL-induced vasoconstriction in human umbilical artery: relation to calcium influx

Homocysteinemia is a major and independent risk factor for vascular disease. Oxidative stress is a possible mechanism for homocysteine (HCY)-induced vascular disease. Herein, we evaluated the antioxidant property of melatonin (MLT) in relation to the vasoconstrictive effect of HCY on the human umbilical artery. Helical umbilical arterial strips without endothelium were obtained at elective Cesarean delivery near term. Changes in potassium chloride (KCl)-induced vasoconstriction were measured. Arterial strips were treated with HCY (10 or 100 microM) plus FeSO(4) (10 microM) alone or pretreated with a hydroxyl radical ((*)OH) scavenger, mannitol (20 mM), or MLT (1 or 10 microM). The effect of HCY on the response of arterial strips to external calcium (Ca(2+)) in the presence of KCl (20 mM) was determined. HCY plus FeSO(4) potentiated KCl-induced vasoconstriction in a concentration-dependent manner; pretreatment with mannitol significantly reduced this vasospastic effect. HCY (100 microM) significantly augmented the contractile response to external Ca(2+). MLT (10 microM) significantly suppressed the contractile response to external Ca(2+). These results suggest that HCY potentiates KCl-induced umbilical artery vasoconstriction, in part by increasing Ca(2+) influx in vascular smooth muscle cells via activation of Ca(2+) channels. MLT significantly suppressed the vasoconstrictive effect of HCY, probably by scavenging (*)OH arising from HCY autooxidation.     

Up4A stimulates endothelium-independent contraction of isolated rat pulmonary artery

Extracellular nucleotides, such as ATP, UDP, and UTP, regulate pulmonary vascular tone through P2X and P2Y receptors. Recently, uridine adenosine tetraphosphate (Up(4)A) was reported as a novel endothelium-derived vasoconstrictive factor. Up(4)A contains both purine and pyrimidine moieties, which potentially activate P2X and P2Y receptors. The present study examined the effect of Up(4)A on contractility of isolated rat pulmonary artery. Up(4)A at 1-100 microM stimulated contraction in a concentration-dependent manner. Up(4)A was equipotent as UTP and UDP in the endothelium-denuded artery while much more effective than UTP and UDP in endothelium-intact preparations. The vasoconstrictor effect of Up(4)A was inhibited by suramin but not IP(5)I or desensitization of P2X receptors with alpha,beta-methylene-ATP (alpha,beta-Me-ATP). Up(4)A-induced contraction was also inhibited by pretreatment with thapsigargin, nitrendipine, or EGTA but unaffected by H1152. Furthermore, unlike ATP and UTP, Up(4)A did not induce relaxation of endothelium-intact preparations precontracted with phenylephrine. These results suggest that Up(4)A is a potent vasoconstrictor, but not a vasodilator, of the rat pulmonary artery. Up(4)A likely acts through a suramin-sensitive P2Y receptor. The contractile effect of Up(4)A involves the entry of extracellular Ca(2+) and release of Ca(2+) from intracellular stores but not Ca(2+) sensitization via the RhoA/Rho kinase pathway. Up(4)A, therefore, potentially plays an important role in the regulation of pulmonary vascular tone.     

Zinc modulation of glycine receptors in acutely isolated rat CA3 neurons

Glycine and GABA are the primary inhibitory neurotransmitters in the spinal cord and brain stem, with glycine exerting its physiological roles by activating strychnine-sensitive ionotropic receptors. Glycine receptors are also expressed in the brain, including the cortex and hippocampus, but their physiological roles and pharmacological properties are largely unknown. Here, we report the pharmacological properties of functional glycine receptors in acutely isolated rat CA3 neurons using conventional whole-cell patch clamp techniques. Both glycine and taurine, which are endogenous agonists of glycine receptors, elicited Cl(-) currents in a concentration-dependent manner. The glycine-induced current (I(Gly)) was inhibited by strychnine, picrotoxin or cyclothiazide in a concentration-dependent manner. At lower concentrations (0.01-1 microM), ICS-205,930 potentiated I(Gly), but at higher concentrations (>10 microM) it inhibited I(Gly). These pharmacological properties strongly suggest that CA3 neurons express functional strychnine-sensitive glycine receptors containing alpha2 subunits. Furthermore, at lower concentrations (1-30 microM), Zn(2+) potentiated I(Gly), but at higher concentrations (>100 microM) it inhibited I(Gly). Considering that Zn(2+) is synaptically co-released with glutamate from mossy fiber terminals that make excitatory synapses onto CA3 neurons, these results suggest that endogenous Zn(2+) modulation of these glycine receptors may have an important role in the excitability of CA3 neurons.     

Mechanisms for the vasodilations induced by Ginkgo biloba extract and its main constituent,bilobalide, in rat aorta

Vasodilating actions of Ginkgo biloba extract (GBE) and bilobalide, a main constituent, were examined using rat aorta ring strips. GBE at the concentration ranges from 0.03 to 3 mg/ml had a potent concentration-dependent relaxation, reaching 70 +/- 4.5% (n = 6, P < 0.001) at 3 mg/ml. Bilobalide at 0.1 to 100 microM also caused the relaxation in a concentration-dependent manner. At 100 microM, bilobalide caused dilation by 17.6 +/- 3.9% (n = 7, P < 0.05). NG-monomethyl-L-arginine acetate (L-NMMA)(100 microM), an NO synthesis inhibitor, reduced the vasodilation of GBE (3 mg/ml) to 57.6 +/- 2.5% (n = 6, P < 0.05), and was accompanied with a decrease in the rate of relaxation. Tetraethylammonium (TEA)(100 microM), a Ca(2+)-activated K(+) channel inhibitor, also decreased the GBE (3 mg/ml)-induced relaxation to 63.1 +/- 4.6% (n = 6), but not significantly. Indomethacin tended to reduce the GBE (3 mg/ml)-induced vasorelaxation to 67.3 +/- 4.1% (n = 6). In contrast, the vasorelaxation of GBE (3 mg/ml) was strongly attenuated to 53 +/- 6.1% (n = 7, P < 0.05) in Ca(2+)-free medium. Similarly, the vasorelaxation induced by bilobalide significantly decreased both by pretreatment with NO inhibitor (L-NMMA) and in Ca(2+)-free solution. These results indicate that the relaxation induced by GBE would be due to the inhibition of Ca(2+) influx through the Ca(2+) channel and the activation of NO release, and might be in part due to the inhibitions of Ca(2+)-activated K(+) current and PGI(2) release, in the endothelium and aortic vascular muscles. Bilobalide possesses the similar mechanisms for the vasodilation.     

Cu2+ suppresses GABA(A) receptor-mediated responses in rat sacral dorsal commissural neurons

The effect of copper ions (Cu(2+)) on gamma-aminobutyric acid (GABA)-induced responses in acutely dissociated neurons from the rat sacral dorsal commissural nucleus (SDCN) was investigated using a nystatin-perforated patch recording configuration under voltage clamp conditions. The application of Cu(2+) to SDCN neurons reversibly suppressed the GABA (10 microM)-activated Cl(-) current (I(GABA)) in a concentration-dependent manner (1-1000 microM; IC(50) = 24.5 microM). In the presence of Cu(2+) (30 microM), the concentration-response curve of GABA was shifted rightward without reducing I(GABA) recorded under the maximally effective concentration of GABA, thus indicating a dependence of Cu(2+) action on GABA concentration. Inhibition of GABA (10 microM) responses by 30 microM Cu(2+) was essentially voltage independent and was not accompanied by a shift in the reversal potential of the currents. Cu(2+) antagonized the suppressive effect of Zn(2+) in a concentration-dependent manner, suggesting competition between Cu(2+) and Zn(2+) for similar binding sites. These data demonstrate that Cu(2+) is a potent inhibitor of GABA(A) receptor-mediated responses, implying a possible modulatory effect of Cu(2+) on GABAergic synaptic transmission in the mammalian SDCN.     

The anti-anginal drug fendiline elevates cytosolic Ca(2+) in rabbit corneal epithelial cells

The effect of the anti-anginal drug fendiline on intracellular free Ca(2+) levels ([Ca(2+)](i)) in a rabbit corneal epithelial cell line (SIRC) was explored using fura-2 as a fluorescent Ca(2+) indicator. At a concentration above 1 microM, fendiline increased [Ca(2+)](i) in a concentration-dependent manner with an EC(50) value of 7 microM. The [Ca(2+)](i) response consisted of an immediate rise and an elevated phase. Extracellular Ca(2+) removal decreased half of the [Ca(2+)](i )signal. Fendiline induced quench of fura-2 fluorescence by Mn(2+) (50 microM), suggesting the presence of Ca(2+) influx across the plasma membrane. This Ca(2+) influx was abolished by La(3+) (50 microM), but was insensitive to dihydropyridines, verapamil and diltiazem. Fendiline (10 microM)-induced store Ca(2+) release was largely reduced by pretreatment with thapsigargin (1 microM) (an endoplasmic reticulum Ca(2+) pump inhibitor) to deplete the endoplasmic reticulum Ca(2+). Conversely, pretreatment with 10 microM fendiline abolished thapsigargin-induced Ca(2+) release. Fendiline (10 microM)-induced Ca(2+) release was not altered by inhibiting phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). Cumulatively, this study shows that fendiline induced concentration-dependent [Ca(2+)](i )increases in corneal epithelial cells by releasing the endoplasmic reticulum Ca(2+) in a phospholipase C-independent manner, and by causing Ca(2+) influx.     

Activation of Rho/Rho kinase signaling pathway by reactive oxygen species in rat aorta

Evidence indicates that both the Rho/Rho kinase signaling pathway and reactive oxygen species (ROS) such as superoxide and H(2)O(2) are involved in the pathogenesis of hypertension. This study aimed to determine whether ROS-induced vascular contraction is mediated through activation of Rho/Rho kinase. Rat aortic rings (endothelium denuded) were isolated and placed in organ chambers for measurement of isometric force development. ROS were generated by a xanthine (X)-xanthine oxidase (XO) mixture. The antioxidants tempol (3 mM) and catalase (1,200 U/ml) or the XO inhibitor allopurinol (400 microM) significantly reduced X/XO-induced contraction. A Rho kinase inhibitor, (+)-(R)-trans-4-(1-aminoethyl-N-4-pyridil)cyclohexanecarboxamide dihydrochloride (Y-27632), decreased the contraction in a concentration-dependent manner; however, the Ca(2+)-independent protein kinase C inhibitor rottlerin did not have an effect on X/XO-induced contraction. Phosphorylation of the myosin light chain phosphatase target subunit (MYPT1) was increased by ROS, and preincubation with Y-27632 blocked this increased phosphorylation. Western blotting for cytosolic and membrane-bound fractions of Rho showed that Rho was increased in the membrane fraction by ROS, suggesting activation of Rho. These observations demonstrate that ROS-induced Ca(2+) sensitization is through activation of Rho and a subsequent increase in Rho kinase activity but not Ca(2+)-independent PKC.     

Involvement of Ca2+ channels in endothelin-1-induced MAP kinase phosphorylation, myosin light chain phosphorylation and contraction in rabbit iris sphincter smooth muscle

The purpose of the present study was to investigate the role and type of Ca2+ channels involved in the stimulatory effects of endothelin-1 (ET-1) on the Ca2+-dependent functional responses, p42/p44 MAP kinase phosphorylation, 20-kDa myosin light chain (MLC) phosphorylation and contraction, in rabbit iris sphincter, a nonvascular smooth muscle. ET-1 induced inositol phosphates production, MAP kinase phosphorylation, MLC phosphorylation (MLC20-P plus MLC20-2P) and contraction in a concentration-dependent manner with EC50 values of 71, 8, 6 and 25 nM, respectively. ET-1-induced MAP kinase phosphorylation, MLC phosphorylation and contraction were not significantly affected by nifedipine (1-60 microM), an L-type Ca2+ channel blocker, or by LOE 908 (1-100 microM), a blocker of Ca2+-permeable nonselective cation channels. However, SKF96365, a receptor-operated Ca2+ channel (ROCC) blocker, inhibited MAP kinase phosphorylation, MLC phosphorylation and contraction in a concentration-dependent manner with IC50 values of 28, 30 and 42 microM, respectively. 2-APB, a store-operated Ca2+ channel (SOCC) blocker, inhibited ET-1-induced MLC phosphorylation and contraction in a concentration-dependent manner with IC50 values of 12.7 and 19 microM, respectively, but was without effect on MAP kinase phosphorylation. The combined effects of submaximal concentrations of SKF96365 and 2-APB on ET-1-induced MLC phosphorylation and contraction were not additive, implying that their inhibitory actions could be mediated through a common Ca2+ entry channel. PD98059, a MAP kinase inhibitor, had no effect on ET-1-induced MLC phosphorylation and contraction, suggesting that these ET-1 effects in the rabbit iris muscle are MAP kinase-independent. In conclusion, the present study demonstrated for the first time that in rabbit iris sphincter (a) ET-1, through the ETA receptor, stimulates MAP kinase phosphorylation, MLC phosphorylation and contraction in a concentration-dependent manner, (b) that these Ca2+-dependent functional responses are not significantly affected by nifedipine or LOE908, and (c) that ET-1-induced MLC phosphorylation and contraction are inhibited by SKF96365 and 2-APB, suggesting that these effects are mainly due to store- and/or receptor Ca2+ entry.     

Regulation of pulmonary venous tone in response to muscarinic receptor activation

We investigated cellular mechanisms that mediate or modulate the vascular response to muscarinic receptor activation (ACh) in pulmonary veins (PV). Isometric tension was measured in isolated canine PV rings with endothelium (E+) and without endothelium (E-). Tension and intracellular Ca(2+) concentration ([Ca(2+)](i)) were measured simultaneously in fura-2-loaded E- PV strips. In the absence of preconstriction, ACh (0.01-10 microM) caused dose-dependent contraction in E+ and E- rings. ACh contraction was potentiated by removing the endothelium or by nitric oxide (NO) synthase inhibition (N-nitro-L-arginine methyl ester, P = 0.001). Cyclooxygenase inhibition (indomethacin) reduced ACh contraction in both E+ and E- PV rings (P = 0.013 and P = 0.037, respectively). ACh contraction was attenuated by inhibitors of voltage-operated Ca(2+) channels (nifedipine, P < 0.001), inositol-1,4,5-trisphosphate (IP(3))-mediated Ca(2+) release (2-aminoethoxydiphenyl borate, P = 0.001), PKC (bisindolylmaleimide I, P = 0.001), Rho-kinase (Y-27632, P = 0.002), and tyrosine kinase (TK; tyrphostin 47, P = 0.015) in E- PV rings. ACh (1 microM) caused a leftward shift in the [Ca(2+)](i)-tension relationship (P = 0.015), i.e., ACh increased myofilament Ca(2+) sensitivity. Inhibition of PKC, Rho-kinase, and TK attenuated the ACh-induced increase in myofilament Ca(2+) sensitivity (P < 0.001, P < 0.001, and P = 0.024, respectively). These findings indicate that in canine PV, ACh contraction is modulated by NO and partially mediated by metabolites of the cyclooxygenase pathway and involves Ca(2+) influx through voltage-operated Ca(2+) channels and IP(3)-mediated Ca(2+) release. In addition, ACh induces increased myofilament Ca(2+) sensitivity, which requires the PKC, Rho-kinase, and TK pathways.     

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.     

Progesterone inhibits gallbladder motility through multiple signaling pathways

Progesterone (P) has an inhibitory effect on the contractility of gastrointestinal smooth muscle, including the gallbladder. Since P levels are elevated during pregnancy, a biliary stasis may develop during pregnancy that is characterized by an increase in the fasting and residual volumes and by a decrease in emptying capacity. This study investigates the effect of P and two metabolites on contraction in guinea pig gallbladder strips. P induced a concentration-dependent relaxation in guinea pig gallbladder strips precontracted with cholecystokinin octapeptide (CCK). Pretreatment of gallbladder strips with P (50 microM) also reduced the amount of CCK-induced tension. Nifedipine (1 microM) produced a similar effect. Pretreatment of the strips with PKA inhibitor 14--22 amide myristolated (180 nM) or the PKG inhibitor KT5823 (1.2 microM) either separately or in combination significantly reduced the amount of P-induced relaxation. Rp-cAMPs (0.1mM) or H-89 (10 microM) separately or in combination significantly reduced the P-effect; however, the combination of agents produced the largest reduction. Genistein (1 microM), an inhibitor of protein tyrosine kinases, significantly (p<0.01) reduced the amount of P-induced relaxation. The use of strontium in the Kreb's solution as a substitute for Ca(2+) significantly (p<0.01) reduced the amount of CCK-induced tension. Pretreatment of the strips with 2-APB (26 microM), an inhibitor of IP(3,) induced Ca(2+) release, produced a significant (p<0.01) reduction in P-induced relaxation. We conclude that P inhibits gallbladder motility rapidly by nongenomic actions of the hormone. Several pathways that include tyrosine kinase and PKA/cAMP activity may mediate this effect.     

Selected contribution: effect of the aldehyde acrolein on acetylcholine-induced membrane current in airway smooth muscle cells.

Acrolein administered to isolated airways has been shown to alter airway responsiveness as a consequence of its effect on Ca(2+) signaling. To examine the mechanisms involved, we studied the effect of acrolein on ACh- and caffeine-induced membrane currents (patch-clamp) in myocytes freshly isolated from rat trachea. In cells clamped at -60 mV, ACh (0.1-10 microM) induced a concentration-dependent inward current, which, in approximately 50% of the cells, was followed by current oscillations in response to high concentration of ACh (10 microM). Exposure to acrolein (0.2 microM) for 10 min significantly enhanced the amplitude of the low-ACh (0.1 microM) concentration-induced initial peak of current (318.8 +/- 28.3 vs. 251.2 +/- 40.3 pA; n = 25, P < 0.05). At a high-ACh concentration (10 microM), the frequency at which subsequent peaks occurred was significantly increased (13.2 +/- 1.1 vs. 8.7 +/- 2 min(-1); n = 20, P < 0.05). ACh-induced current was identified as a Ca(2+)-activated Cl(-) current. In contrast, similar exposure to acrolein, which does not alter caffeine-induced Ca(2+) release, did not alter caffeine-induced transient membrane currents (595 +/- 45 and 640 +/- 45 pA in control cells and in cells exposed to acrolein, respectively; n = 15). It is concluded that acrolein alters ACh-induced current as a consequence of its effect on the cytosolic Ca(2+) concentration response and that the protective role of inhibitors of Cl(-) channels in air pollutant-induced airway hyperresponsiveness should be examined.     

Calcium mobilization as the endothelium-independent mechanism of action involved in the vasorelaxant response induced by the aqueous fraction of the ethanol extract of Albizia inopinata G. P. Lewis (AFL) in the rat aorta.

In a previous work, we demonstrated that, in normotensive rats, AFL induced a marked hypotension due to a decrease in total peripheral resistances (TPR), partially secondary to the release of NO by the endothelium. NO did not, however, account for the total vasodilation produced by AFL in these rats. The aim of this study was to determine the involvement of the intracellular calcium mobilization in the vasorelaxant action induced by AFL in the rat aorta. In aorta of normotensive rats AFL (10, 20, 40 and 80 microg/ml) inhibited the sustained contractions induced by KCl (80 and 30 mM) and phenylephrine (Phe, 1 microM) with similar IC50 values (54 +/- 6, 52 +/- 4 and 65 +/- 4 microg/ml, respectively). The relaxing response induced by AFL against Phe-induced contractions was modified significantly by the endothelium removal (IC50 = 132 +/- 23 and 65 +/- 4 microg/ml, endothelium removed and intact endothelium aortic rings, respectively). Nevertheless, removal of the endothelium did not significantly change IC50 values when KCl (30 and 80 mM) was used as the contractile agent. The inhibitory effect induced by AFL on high (64.5 mM) K+-induced contraction was potentiated slightly (p < 0.05) by the decrease (from 2.5 to 0.3 mM, Ca2+) and attenuated by the increase (from 2.5 to 7.5 mM Ca2+) in the external [Ca2+]. In addition, in aortas from normotensive rats, AFL antagonized transient contractions induced in Ca2+-free media induced by 1 microM noradrenaline in a concentration-dependent manner, but not those induced by 20 mM caffeine. It is suggested that the remaining vasodilator effect of AFL in normotensive rats is probably due to an inhibition of Ca2+ influx and/or inhibition of intracellular Ca2+ mobilization from the noradrenaline-sensitive stores.     

Effect of NPC-14686 (Fmoc-L-homophenylalanine) on intracellular Ca2+ levels in human hepatoma cells

The effect of NPC-14686, a potential anti-inflammatory drug, on cytosolic free Ca(2+) levels ([Ca(2+)](i)) in HA22/VGH human hepatoma cells was explored by using fura-2 as a fluorescent Ca(2+) indicator. NPC-14686 at concentrations above 10 microM increased [Ca(2+)](i) in a concentration-dependent manner with an EC(50) value of 100 microM. The Ca(2+) signal was reduced by removing extracellular Ca(2+) or by 10 microM nifedipine and was not changed by verapamil or diltiazem. Pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) to deplete the endoplasmic reticulum Ca(2+) abolished 200 microM NPC-14686-induced Ca(2+) release; and conversely pretreatment with NPC-14686 abolished thapsigargin-induced Ca(2+) release. The Ca(2+) release induced by 200 microM NPC-14686 was not changed by inhibiting phospholipase C with 2 microM U73122. Together, the results suggest that in human hepatoma cells, NPC-14686 induced a [Ca(2+)](i) increase by causing store Ca(2+) release from the endoplasmic reticulum in an phospholipase C-independent manner, and by inducing nifedipine-sensitive Ca(2+) influx.     

Differential contractile actions of reactive oxygen species on rat aorta: selective activation of ATP receptor by H2O2

This study aims to examine the effects of different reactive oxygen species (ROS) on the resting tension of endothelium-denuded rat aortic rings. In these preparations, H2O2 (30 microM) induced a fast and transient contraction, which could be abolished by pretreatment of catalase (800 U/ml), but not affected by superoxide anion scavenger, superoxide dismutase (SOD; 150 U/ml) or the hydroxyl free radical scavenger, DMSO/mannitol (each 3 mM). In contrast, pyrogallol, a putative superoxide anion donor, induced a biphasic contraction, which could be abolished by SOD, but not by catalase or DMSO/mannitol. Unlike H2O2 and pyrogallol, Vitamin C(VitC)/Fe2+ (each 100 microM), a commonly used hydroxyl radical-generating system, triggered a tonic contraction which could be prevented by DMSO/mannitol, but not by SOD or catalase. Interestingly, H2O2-induced contraction could be concentration-dependently (10-100 microM) inhibited by suramin and reactive blue-2 (RB-2), two widely used ATP receptor antagonists. On the other hand, suramin or RB-2, at concentration up to 100 microM, affected neither pyrogallol nor VitC/Fe2+-induced contraction. In conclusion, we showed for the first time that different ROS could contract rat aorta with different mechanisms of action, and H2O2 elicits a transient contraction probably as a result of the ATP receptor activation.     

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.     

Effects of tyrosine kinase inhibitors on voltage-dependent Ba(2+) currents in the guinea-pig gastric antrum.

We have investigated whether tyrosine kinases modify the activity of voltage-dependent Ba(2+) currents (I(Ba)) recorded from guinea-pig gastric myocytes by use of patch-clamp techniques. All experiments were carried on single smooth muscle cells, dispersed from the circular layer of the guinea-pig gastric antrum. Genistein ( > or = 10 microM), a specific tyrosine kinase inhibitor, reduced the peak amplitude of I(Ba) in a voltage- and concentration-dependent manner. Daidzein ( > or = 30 microM), an inactive analog of genistein, also inhibited I(Ba) in a concentration-dependent manner. Similarly, other types of tyrosine kinase inhibitors (lavendustin A and tyrphostin 23) suppressed the peak amplitude of I(Ba) in a concentration-dependent manner. These results indicate that tyrosine kinases may be essential to regulate Ca(2+) mobilization through voltage-dependent Ca(2+) channels in gastric myocytes.     

Involvement of K(+)-channel opening in endothelin-1 induced suppression of spontaneous contractions in the guinea pig taenia coli.

Effects of porcine-human endothelin-1 on mechanical as well as electrical activities and on intracellular free Ca2+ levels in the guinea pig taenia coli were compared with those of nifedipine, a voltage-dependent Ca2+ channel blocker. Endothelin-1 (0.1-100 nM) caused a concentration-dependent suppression of spontaneous contractions but did not significantly affect the sustained contraction evoked by 40 mM KCl. However, nifedipine (0.1-100 nM) inhibited both types of contractions in a concentration-dependent manner. In electrophysiological studies, endothelin-1 (30 nM) or nifedipine (30 nM) eliminated spontaneous spike discharges. Endothelin-1 produced hyperpolarization, while nifedipine did not change the resting membrane potential. The endothelin-1 induced suppression of spontaneous contractions was dose-dependently antagonized by apamin (0.01-10 nM), an inhibitor of a small conductance Ca(2+)-dependent K+ channel, and D-tubocurarine (10-100 microM), an inhibitor of Ca(2+)-dependent K+ channel, but was unaffected by 4-aminopyridine (0.01-1 mM), an inhibitor of a voltage-dependent K+ channel. In the study with fura 2 excited at 340 nm, endothelin-1 abolished, from the tissue, the fluorescence signals that were coupled with spontaneous contraction. It is suggested that the inhibitory action of endothelin-1 on spontaneous contraction may be caused by hyperpolarization of the membrane that reduces the spontaneous generation of spike discharge coupled normally to an increase in the intracellular free Ca2+ levels in the guinea pig taenia coli. The hyperpolarization may be caused by activating apamin-sensitive Ca(2+)-dependent K+ channels.     

Bidirectional modulation of P2X receptor-mediated response by divalent cations in rat dorsal motor nucleus of the vagus neurons

The modulatory effects of Zn(2+) and other divalent cations on the ATP-induced responses of preganglionic neurons acutely dissociated from the rat dorsal motor nucleus of the vagus (DMV) were examined using a nystatin-perforated patch technique under voltage-clamp. DMV neurons were identified by back-filling of DiI placed on the vagal bundle at the neck. Zn(2+) exerts a concentration-dependent effect on P2X receptor-mediated current (I(ATP)): a potentiation by low concentrations of Zn(2+) (< or = 50 microM) and an inhibition by high concentrations (> 50 microM). Inhibition of the ATP response was associated with a prolongation of the rising phase of I(ATP). Cu(2+) mimicked Zn(2+) regarding the biphasic modulation of I(ATP). On the other hand, Ni(2+) potentiated, but failed to inhibit, the ATP response even at a concentration of 3 mM. Quantitative RT-PCR revealed the similarity of P2X(2) mRNA expression between the DMV and superior cervical ganglion (SCG) but not in the dorsal root ganglion (DRG) and hypoglossal nucleus (XII). The results from the electrophysiological and molecular approaches suggest that functional P2X receptors expressed in DMV neurons are characterized mainly by the P2X(2) and P2X(2/6) subtype. DMV neurons possess similar P2X receptor characteristics to SCG neurons.