Calcium-induced Contractions Research Articles

SMOOTH MUSCLE RELAXANT ACTIVITY OF A DIHYDROPYRIMIDINE DERIVATIVE 5-ACYL-6-METHYL-4-PHENYL-2-S-ETHYL-1, 4-DIHYDROPYRIMIDINE (BK- VI) ON ISOLATED RAT UTERUS AND RABBIT AORTIC STRIP.

ABSTRACT Objective : To investigate the smooth muscle relaxant activity of a newly synthesized dihydropyrimidine derivative 5-acyl-6-methyl-4-phenyl-2-S-ethyl-1,4-dihydropyrimidine (BK- VI) and nifedipine on isolated rat uterus and rabbit aortic strip. Material and Methods : Effect of the test compound BK-VI on the smooth muscles of isolated rat uterus and isolated rabbit aortic strip was observed and compared with that of nifedipine. Observations were made with increasing bath concentrations of BK-VI and nifedipine.Six preparations were used for each dose of BK-VI and nifedipine.Mean effect of increasing doses of BK-VI and nifedipine on the height of calcium-induced contraction of depolarized isolated rabbit aortic strip and on K + -induced contraction of isolated rat uterus were noted and IC-50 calculated. Results : Test compound BK-VI had a significant dose-dependent relaxant effect on K + -induced contractions of isolated rat uterus. Significant relaxation was seen at bath concentration starting from 9.34x10 -4 M (IC-50=12.2x10 -4 M).Nifedipine showed significant relaxation at all bath concentrations starting from 2.8X10 -7 M(IC 50 =7.5X10 -7 M).Compound BK-VI produced potentiation of Ca 2+ -induced contractions of K+-depolarized rabbit’s aortic strip at bath concentration of 0.7×10 -5 M while significant inhibition was observed at higher bath concentrations. Nifedipine showed dose-dependent significant inhibition at all bath concentrations. Conclusion : BK-VI has a calcium channel blocking activity like nifedipine and it can inhibit the Ca 2+ dependent contractions of smooth muscles of uterus and aorta. KEY WORD: Calcium channel blockers, Dihydropyrimidines, Voltage dependent calcium channels.

Levobupivacaine-induced contraction of isolated rat aorta is calcium dependent

Levobupivacaine is a long-acting local anesthetic that intrinsically produces vasoconstriction in isolated vessels. The goals of this study were to investigate the calcium-dependent mechanism underlying levobupivacaine-induced contraction of isolated rat aorta in vitro and to elucidate the pathway responsible for the endothelium-dependent attenuation of levobupivacaine-induced contraction. Isolated rat aortic rings were suspended to record isometric tension. Cumulative levobupivacaine concentration-response curves were generated in either the presence or absence of the antagonists verapamil, nifedipine, SKF-96365, 2-aminoethoxydiphenylborate, Gd(3+), N(W)-nitro-l-arginine methyl ester (L-NAME), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), and methylene blue, either alone or in combination. Verapamil, nifedipine, SKF-96365, 2-aminoethoxydiphenylborate, low calcium concentrations, and calcium-free Krebs solution attenuated levobupivacaine-induced contraction. Gd(3+) had no effect on levobupivacaine-induced contraction. Levobupivacaine increased intracellular calcium levels in vascular smooth muscle cells. L-NAME, ODQ, and methylene blue increased levobupivacaine-induced contraction in endothelium-intact aorta. SKF-96365 attenuated calcium-induced contraction in a previously calcium-free isotonic depolarizing solution containing 100mmol/L KCl. Levobupivacaine-induced contraction of rat aortic smooth muscle is mediated primarily by calcium influx from the extracellular space mainly via voltage-operated calcium channels and, in part, by inositol 1,4,5-trisphosphate receptor-mediated release of calcium from the sarcoplasmic reticulum. The nitric oxide - cyclic guanosine monophosphate pathway is involved in the endothelium-dependent attenuation of levobupivacaine-induced contraction.

Rho-kinase inhibition attenuates calcium-induced contraction in β-escin but not Triton X-100 permeabilized rabbit femoral artery

K+-depolarization (KCl) of smooth muscle has long been known to cause Ca2+-dependent contraction, but only recently has this G protein-coupled receptor (GPCR)-independent stimulus been associated with rhoA kinase (ROCK)-dependent myosin light chain (MLC) phosphatase inhibition and Ca2+ sensitization. This study examined effects of ROCK inhibition on the concentration-response curves (CRCs) generated in femoral artery by incrementally adding increasing concentrations of KCl to intact tissues, and Ca2+ to tissues permeabilized with Triton X-100, β-escin and α-toxin. For a comparison, tissue responses were assessed also in the presence of protein kinase C (PKC) and MLC kinase inhibition. The ROCK inhibitor H-1152 induced a strong concentration-dependent inhibition of a KCl CRC. A relatively low GF-109203X concentration (1 μM) sufficient to inhibit conventional PKC isotypes also inhibited the KCl CRC but did not affect the maximum tension. ROCK inhibitors had no effect on the Ca2+ CRC induced in Triton X-100 or α-toxin permeabilized tissues, but depressed the maximum contraction induced in β-escin permeabilized tissue. GF-109203X at 1 μM depressed the maximum Ca2+-dependent contraction induced in α-toxin permeabilized tissue and had no effect on the Ca2+ CRC induced in Triton X-100 permeabilized tissue. The MLC kinase inhibitor wortmannin (1 μM) strongly depression the Ca2+ CRCs in tissues permeabilized with Triton X-100, α-toxin and β-escin. H-1152 inhibited contractions induced by a single exposure to a submaximum [Ca2+] (pCa 6) in both rabbit and mouse femoral arteries. These data indicate that β-escin permeabilized muscle preserves GPCR-independent, Ca2+- and ROCK-dependent, Ca2+ sensitization.

Characterization of a Long Acting Smooth Muscle Myosin Inhibitor, CK‐2125927, as a Novel Therapeutic Mechanism for Bronchodilation

Smooth muscle myosin is a mechanochemical enzyme that hydrolyzes ATP to generate mechanical force; ultimately all signaling pathways that modulate smooth muscle tone converge on the regulation of this motor protein. We previously showed that a novel smooth muscle myosin inhibitor, CK‐2019165, inhibited the methacholine‐induced bronchoconstriction in rat models with a short duration of action (~ 2 hours). Here we describe the discovery and characterization of a long acting smooth muscle myosin inhibitor for bronchodilation. Using high throughput screening, we identified and subsequently optimized a class of selective inhibitors of smooth muscle myosin. These compounds potently inhibit the smooth muscle myosin ATPase (IC50≤ 30 nM). An optimized member of this series, CK‐2125927,, inhibited calcium‐induced contraction of skinned rat tail artery in a concentration dependent manner with an IC50 of ~ 1 μM. CK‐2125927 induced concentration‐dependent relaxation of methacholine‐pre‐constricted rat tracheal rings with an EC50 of ~ 1 μM. Further, CK‐2125927 (10 μM) completely inhibited electrical field stimulation‐induced constriction of guinea pig tracheal rings with a 50% recovery time of > 6 hours, a duration comparable to the long‐acting beta‐adrenergic agonist salmeterol. Together these data suggest CK‐2125927 is a long‐acting smooth muscle myosin inhibitor that may provide a novel therapeutic approach for the treatment of diseases where bronchoconstriction plays a role, such as asthma and COPD.

Nongenomic uterine relaxing effect of RU 486 (mifepristone) prior to its antiprogesterone activity in the human pregnancy

The antiprogestin mifepristone (RU 486) is used for termination of pregnancy, as RU 486 blocks the quiescent action of progesterone, increases uterine contractility, sensitizes the myometrium to prostaglandins, and elicits cervical ripening. Since RU 486 represents a class of compound that is structurally related to steroid hormones, some of which possess a nongenomic uterine relaxing effect, we investigated the potential nongenomic relaxing action of RU 486 on the human pregnant myometrium. Myometrial tissues were obtained from pregnant women undergoing elective cesarean section at term and were isometrically recorded. RU 486 caused relaxation on spontaneous contractility and high potassium-induced contractions with lower relaxing efficacy than progesterone. The progesterone receptor-blocking activity of RU 486 did not antagonize the uterine relaxation of progesterone. Moreover, contractions induced by oxytocin or different prostaglandins (PGF2α, PGE2, and a prostaglandin analogue, misoprostol) were inhibited rather than increased by RU 486. RU 486 induced a rapid and reversible relaxing effect, which was unaffected by inhibitors of protein synthesis and transcription, implying that RU 486 acts through a nongenomic mechanism. This study reveals that RU 486: (i) reduced high potassium-induced contraction and prevented calcium-induced contraction in depolarized tissue; and (ii) relaxed the oxytocin- and prostaglandin-induced contractions, indicating a blockade of voltage- and receptor-operated calcium channels by RU 486. These data show that this antiprogestin may induce a rapid nongenomic antiuterotonic effect prior to its antiprogesterone action.

Excitation–Contraction Coupling of the Mouse Embryonic Cardiomyocyte

In the mammalian embryo, the primitive tubular heart starts beating during the first trimester of gestation. These early heartbeats originate from calcium-induced contractions of the developing heart muscle cells. To explain the initiation of this activity, two ideas have been presented. One hypothesis supports the role of spontaneously activated voltage-gated calcium channels, whereas the other emphasizes the role of Ca2+ release from intracellular stores initiating spontaneous intracellular calcium oscillations. We show with experiments that both of these mechanisms coexist and operate in mouse cardiomyocytes during embryonic days 9–11. Further, we characterize how inositol-3-phosphate receptors regulate the frequency of the sarcoplasmic reticulum calcium oscillations and thus the heartbeats. This study provides a novel view of the regulation of embryonic cardiomyocyte activity, explaining the functional versatility of developing cardiomyocytes and the origin and regulation of the embryonic heartbeat.

Evidence for the participation of calcium in non‐genomic relaxations induced by androgenic steroids in rat vas deferens

Androgens cause non-genomic relaxation in several smooth muscle preparations. However, such an effect has not been investigated in rat vas deferens yet. Our purpose was to study the effect of testosterone and derivatives in this tissue. The influence of androgens was tested on contraction and translocation of intracellular Ca(2+) induced by KCl in rat vas deferens in vitro. The testosterone derivative 5alpha-dihydrotestosterone produced a rapid and reversible concentration-dependent relaxation of KCl-induced contractions. Other androgens were also effective, showing the following rank order of potency: androsterone >5beta-dihydrotestosterone >androstenedione >5alpha-dihydrotestosterone >testosterone. Calcium-induced contractions were also inhibited (about 45%) by 5alpha-dihydrotestosterone (30 microM). Moreover 5alpha-dihydrotestosterone blocked the increase of intracellular Ca(2+) induced by KCl, measured by the fluorescent dye fura-2. Relaxation to 5alpha-dihydrotestosterone was resistant to the K(+) channel antagonists glibenclamide, 4-aminopyridine and charybdotoxin. It was not affected by removal of epithelium or by L-NNA (300 microM), an inhibitor of nitric oxide biosynthesis, nor by selective inhibitors of soluble guanylate cyclase, ODQ or LY 83583, indicating that nitrergic or cGMP mediated mechanisms were not involved. The androgen-induced relaxation was also not blocked by the protein synthesis inhibitor cycloheximide (300 microM) or by the classical androgen receptor flutamide (up to 100 microM), corroborating that the effect is non-genomic. Testosterone derivatives caused relaxation of the rat vas deferens, that did not involve epithelial tissue, K(+) channels, or nitric oxide-dependent mechanisms, but was related to a partial blockade of Ca(2+) influx.

Specific H +/K +-ATPase inhibitors decreased contractile responses of isolated rat vas deferens

The effect of H +/K +-ATPase inhibitors on rat vas deferens contractility was investigated in vitro. Omeprazole (100–300 μM), lansoprazole (100–300 μM) and SCH 28080 (10–100 μM) (2-methyl-8-(phenylmethoxy)-imidazo[1,2-a]pyridine-3-acetonitrile) decreased contractile responses of vas deferens to electrical field stimulation, high K + (80 mM) and phenylephrine in a reversible, reproducible and concentration-dependent manner. The inhibitory potency of lansoprazole on vas deferens contractility was increased in relatively acidic solution (pH 6.9), suggesting that the site of action may be related to H +/K +-ATPase. However, lansoprazole-induced inhibition on contractility was unaltered in K + free solution, indicating that the mechanism of action is independent from H +/K +-ATPase. Reversible nature of omeprazole and lansoprazole-induced inhibition on contractility also suggests that the effects are not due to inhibition of H +/K +-ATPase, since both compounds are irreversible inhibitors of the enzyme. Presence of ouabain (5 μM) did not decrease lansoprazole-induced inhibition on contractility but potentiated the inhibitory effect of lansoprazole, suggesting that lansoprazole-induced inhibition is not mediated by the inhibition of Na +/K +-ATPase. Calcium-induced contractions in high K +–Ca 2+ free medium were completely antagonized by lansoprazole, implying that lansoprazole inhibits Ca 2+ entry through voltage-gated channels. In conclusion, three H +/K +-ATPase inhibitors decreased contractile responses of rat vas deferens to various stimulants in vitro. They may act on a common mechanism, which plays a crucial role in regulating rat vas deferens contractility and this mechanism is probably involved in the regulation of intracellular Ca 2+.

Characterisation of the relaxant response to raloxifene in porcine coronary arteries

The present study characterises the vasorelaxant response to raloxifene in isolated rings of porcine coronary artery. Tissues precontracted either with KCl (30 mM) or prostaglandin F 2α (PGF 2α; 3 μM) were concentration-dependently relaxed by raloxifene (0.1–10 μM). Relaxation was not inhibited by the estrogen receptor antagonist 7α-[9-[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]-estra-1,3,5(10)-triene-3,17 β-diol (ICI 182,780; 1 μM). Preincubation with raloxifene (1–3 μM) caused an inhibition of the KCl or PGF 2α-induced contraction. The effects of raloxifene were independent of the endothelium. The relaxant response to raloxifene was slow in the onset and could not be reversed after repeated washings. Raloxifene did not affect Ca 2+ release from intracellular stores since it failed to inhibit a transient contraction induced by caffeine (10 mM). Raloxifene-induced relaxation was not influenced by the intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM; 10–20 μM). Calcium-induced contractions in Ca 2+-free high K + (60 mM) depolarising medium were concentration-dependently inhibited by raloxifene (0.3–3 μM). If arterial rings were incubated with the L-type Ca 2+ channel activator ( S)-(–)-1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-3-pyridine carboxylic acid methyl ester (( S)-(–)-Bay K 8644; 0.1 μM), cumulative concentration–response curves to Ca 2+ were shifted to the left. Raloxifene (0.3–3 μM) inhibited the effect of ( S)-(–)-Bay K 8644 in a concentration-dependent manner. 4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1 H-imidazole (SB 203580; 10 μM), an inhibitor of p38 mitogen-activated protein kinase (MAPK), diminished raloxifene-induced relaxation in endothelium-denuded arterial rings. Western blot analysis demonstrated that raloxifene stimulated p38 MAPK. It is concluded that raloxifene has an inhibitory effect on voltage-gated and receptor-operated L-type Ca 2+ channels in porcine coronary arteries, thus inducing vascular relaxation independent of the endothelium. p38 MAPK is, at least in part, involved in the relaxant response to raloxifene.

Calcium-induced contraction of sarcomeres changes the interaction between mitochondria and ATPases in permeabilized cardiac cells

Calcium-induced contraction of sarcomeres changes the interaction between mitochondria and ATPases in permeabilized cardiac cells

Vasodilator treatment with hydralazine increases blood flow in mdx mice resistance arteries without vascular wall remodelling or endothelium function improvement

Whereas dystrophin has a key role in striated muscle mechanotransduction, its role in vascular functions is not fully understood. In mice lacking dystrophin (mdx) both flow (shear stress)-mediated dilation (FMD) and vascular wall remodelling are decreased, suggesting a low capacity of the vasculature to adapt to metabolic needs. We assessed the capacity of a systemic vasodilator treatment to improve vascular remodelling and blood flow in mdx mice. We measured vascular structure and function in mesenteric resistance arteries (MRA) from control and mdx mice treated for 1 month with hydralazine. Although hydralazine did not significantly affect blood pressure, mesenteric blood flow was increased in control and mdx mice (150+/-25 to 194+/-30 and 110+/-26 to 143+/-18 microl/min; respectively; n=10/group). MRA (90 microm internal diameter, 75 mmHg) were isolated in vitro in arteriographs. Arterial diameter and FMD were significantly increased by hydralazine in control but not in mdx mice. Hydralazine also increased N-nitro-L-arginine methyl ester (L-NAME)-sensitive FMD in control mice, not in mdx mice. Pressure-, phenylephrine-, serotonin- and calcium-induced contraction, as well as acetylcholine- and sodium nitroprusside-induced dilation, were not affected by hydralazine whatever the strain. This study provides functional evidence that local blood flow could be improve by hydralazine in mdx mice despite a lack of vascular adaptation to flow. This study brings a new insight in the pathophysiology of dystrophin-related myopathies. Nevertheless, the consequences of an increased blood flow in non-adapted arteries remain unknown.

Calcium‐induced contraction of sarcomeres changes the regulation of mitochondrial respiration in permeabilized cardiac cells

The relationships between cardiac cell structure and the regulation of mitochondrial respiration were studied by applying fluorescent confocal microscopy and analysing the kinetics of mitochondrial ADP-stimulated respiration, during calcium-induced contraction in permeabilized cardiomyocytes and myocardial fibers, and in their 'ghost' preparations (after selective myosin extraction). Up to 3 microm free calcium, in the presence of ATP, induced strong contraction of permeabilized cardiomyocytes with intact sarcomeres, accompanied by alterations in mitochondrial arrangement and a significant decrease in the apparent K(m) for exogenous ADP and ATP in the kinetics of mitochondrial respiration. The V(max) of respiration showed a moderate (50%) increase, with an optimum at 0.4 microm free calcium and a decrease at higher calcium concentrations. At high free-calcium concentrations, the direct flux of ADP from ATPases to mitochondria was diminished compared to that at low calcium levels. All of these effects were unrelated either to mitochondrial calcium overload or to mitochondrial permeability transition and were not observed in 'ghost' preparations after the selective extraction of myosin. Our results suggest that the structural changes transmitted from contractile apparatus to mitochondria modify localized restrictions of the diffusion of adenine nucleotides and thus may actively participate in the regulation of mitochondrial function, in addition to the metabolic signalling via the creatine kinase system.

Peroxynitrite-induced relaxation in isolated rat aortic rings and mechanisms of action

The present study was designed to evaluate the effects of peroxynitrite (ONOO-), the product of superoxide and nitric oxide, on isolated segments of rat aorta. In the absence of any vasoactive agent, ONOO- (from 10(-8) to 10(-4) M) failed to alter the basal tension. In phenylephrine (PE; 5 x 10(-7) M)-precontracted rat aortic rings (RAR), ONOO- elicited concentration-dependent relaxation at concentrations of from 10(-8) to 10(-4) M. The effective concentrations producing approximately 50% of maximal relaxation (ED50) to ONOO- were 1.84 x 10(-5) M and 1.96 x 10(-5) M in intact and denuded RAR, respectively (P > 0.05). No significant differences in the relaxation responses were found between RAR with or without endothelium (P > 0.05). The presence of either 5 microM methylene blue (MB) or 5 microM 1H-[1,2,4]oxadiazolo-[4,3-alpha]quinoxalin-1-one (ODQ) significantly inhibited the relaxations induced by ONOO-. Sildenafil (10(-7) M), on the other hand, significantly potentiated the ONOO--induced relaxations. Tetraethylammonium chloride (T-2265) significantly decreased the ONOO--induced relaxations in a concentration-dependent manner. However, ONOO- had no effect on RAR precontracted by high KCL (40 mM, n = 6, P > 0.05). Addition of calyculin A also significantly decreased the ONOO--induced relaxation in a dose-dependent manner. Furthermore, ONOO- significantly inhibited calcium-induced contractions of K+-depolarized aortic rings in a concentration-related manner. Lastly, a variety of other pharmacological agents and antagonists including L-NMMA, L-arginine, indomethacin, atropine, naloxone, diphenhydramine, cimetine, glibenclamide, haloperidol, superoxide dismutase (SOD), and catalase did not influence the relaxant effects of ONOO- on RAR. Our new results suggest that ONOO--triggered relaxation on rat aortic rings is mediated by elevation of cGMP levels, membrane hyperpolarization via K+-channel activation, activation of myosin phosphatase activity, and interference with calcium movement and cellular membrane Ca2+ entry.

Spasmolytic Effect of Vitis Vinifera Leaf Hydroalcoholic Extract on Rat's Isolated Vas Deferens

Introduction: Many reports have shown that the grape (Vitis vinifera) seed extract induces antioxidant, hypotensive hypolipidemic, and vasodilatory effects. We have recently shown the relaxatory effect of grape leaf extract on rat's aorta, ileum, uterus and frog's heart rate and contractility. The objective of this study was to investigate the spasmolytic effect of Vitis Vinifera Leaf Hydroalcoholic extract (VLHE) on rat's isolated vas deferens. Materials & Methods: Vas deferens of male adult rat (Sprague Dawley) was removed, placed in an organ bath containing Tyrode’s solution (30oC, pH 7.4) and the bath bubbled with air. Then its contractions under 1 g initial tension, were recorded isometrically. The vas deferens was contracted either by KCl (80 mM) or adrenaline (2 μg/ml). Results: The results demonstrated that the VLHE at 1, 2, 4 and 8 mg/ml reduced the vas deferens contractions evoked by KCl (80 mM) significantly (P<0.0001) and dose-dependently. VLHE (1, 2 and 4 mg/ml) also reduced the adrenaline-induced vas deferens contractions (P<0.0001). In calcium free Tyrod’s solution, KCl-induced contractions were occurred only after adding normal calcium (1.77 mM) to the organ bath solution. VLHE (3mg/ml) reduced the calcium-induced contraction by KCl too. The VLHE-induced vas deferens relaxation was unaffected by the β- adrenoceptor antagonist (propranolol, 3 μM for 5 min). Nitric oxide synthase inhibitor (L-NAME, 300 μM for 10 min) was unable to modify either KCl-induced contraction or the vas deferens relaxation induced by the extract. Conclusion: These results suggested that the spasmolytic effect of Vitis vinifera leaf hydroalcoholic extract on rat's vas deferens occurred through the blockage of voltage dependent calcium channels (VDCC). Also, neither NO nor β-adrenoceptors were involved in this inhibitory effect of the extract.

VASCULAR AND ANTI‐OXIDANT ACTIONS OF FLAVONOLS AND FLAVONES

1. Flavonols and flavones are plant-derived polyphenolic compounds that are commonly consumed in the diet. Epidemiological studies indicating that high dietary intake of flavonols reduces the risk of mortality due to coronary heart disease have provoked interest in the mechanism of this cardioprotective effect. 2. We have investigated the structure-activity relationships of a range of flavonols and flavones with regard to their vascular relaxant and anti-oxidant activity. In rat isolated thoracic aorta, the synthetic flavonol 3',4'-dihydroxyflavonol (DiOHF) was found to be a significantly more potent vasorelaxant than the naturally occurring compounds chrysin, apigenin, luteolin, quercetin and fisetin. Similarly, DiOHF was significantly more potent than those compounds in the inhibition of calcium-induced contraction of the rat aorta. 3. 3',4'-Dihydroxyflavonol was also found to significantly inhibit superoxide radical generation in a cell-free system in the presence of xanthine/xanthine oxidase or by rat isolated aorta in the presence of NADPH. In the presence of oxidant stress generated by pyrogallol or xanthine/xanthine oxidase, endothelium-dependent relaxation of rat aortic rings was impaired. 3',4'-Dihydroxyflavonol was able to significantly improve endothelium-dependent relaxation in the presence of those oxygen radical generators. 4. In addition, DiOHF was found to significantly improve dilatation in the rat hindquarters vasculature after exposure to ischaemia and reperfusion. 3',4'-Dihydroxyflavonol was found to be equally effective whether applied before ischaemia or during ischaemia just before reperfusion. 5. In conclusion, DiOHF is an effective vasodilator and anti-oxidant that is able to prevent vascular reperfusion injury. We suggest that DiOHF may be useful as an adjunct to thrombolytic therapy in the management of reperfusion injury.

Peroxynitrite-induced relaxation in isolated canine cerebral arteries and mechanisms of action

The present study was undertaken to determine the vascular actions of peroxynitrite (ONOO −), the product of superoxide and nitric oxide (NO), in isolated canine cerebral arteries and to gain insight into its potential mechanisms of action. In the absence of any vasoactive agent, ONOO − (from 10 −7 to 10 −6 M) was able to reduce the basal tension. In prostaglandin F2α-precontracted canine basilar arterial rings, ONOO − elicited concentration-dependent relaxation at concentrations from 10 −8 to 10 −5 M. The effective concentrations producing approximately 50% maximal relaxation (EC 50) to ONOO − were 4.06 × 10 −6 and 4.12 × 10 −6 M in intact and denuded rings, respectively ( P > 0.05). No significant differences in relaxation responses were found in ring preparations with or without endothelium ( P > 0.05). The presence of either 5 μM methylene blue (MB) or 5 μM 1 H-[1,2,4]oxadiazolo-[4,3-α]quinoxalin-1-one (ODQ) significantly inhibited the relaxations induced by ONOO −. Tetraethylammonium chloride (T-2265) significantly decreased the ONOO −-induced relaxations in a concentration-dependent manner. However, ONOO − had no effect on rings precontracted by high KCL ( P > 0.05). Addition of low concentrations of calyculin A (50 nM) was able to abolish the ONOO −-induced relaxation. Furthermore, ONOO − significantly inhibited calcium-induced contractions of K +-depolarized canine cerebral rings in a concentration-related manner. Lastly, a variety of pharmacological agents and antagonists including L-NMMA, l-arginine, indomethacin, atropine, naloxone, diphenhydramine, cimetine, glibenclamide, haloperidol, etc., did not influence the relaxant effects of ONOO − on the rings. Our new results suggest that ONOO −-triggered relaxation, on canine cerebral arteries, is mediated by elevation of cyclic guanosine monophosphate (cGMP) levels, membrane hyperpolarization via K+ channel activation, activation of myosin light chain phosphatase activity, and interference with calcium movement and cellular membrane Ca 2+ entry.

Effects of HA-1077 and Y-27632, Two Rho-Kinase Inhibitors, in the Human Umbilical Artery

A role for the small G protein rho and rho-kinase has been shown in smooth muscle contraction regarding Ca++ sensitivity. However, there are no data in the literature assessing how this system operates in human umbilical arteries (HUA). Therefore, we evaluated the effects of HA-1077 and Y-27632, two rho-kinase inhibitors, on agonist-(5-hydroxytryptamine [5-HT]) and depolarization-induced (KCl) contractions of HUA. HA-1077 and Y-27632 inhibited 5-HT-induced contractile responses at 10-4M concentration but not at 10(-5) M. HA-1077 at 10(-4) M also significantly attenuated contractions induced by 20 mM KCl. In addition, HUA precontracted with 5-HT relaxed concentration dependently in response to HA-1077 and Y-27632. When precontracted with KCl, HUA also relaxed dose-dependently in response to HA-1077, but the maximal relaxation was significantly smaller than the response obtained when precontracted with 5-HT. To determine possible involvement of rho-kinase on agonist-induced intracellular calcium-mediated contractions, tissues were precontracted with 5-HT in Ca++-free Krebs solution before cumulative addition of HA-1077 or Y-27632 (10(-7) to 10(-4) M). Both rho-kinase inhibitors relaxed HUA completely. Maximum relaxations of HUA to HA-1077 and Y-27632 were significantly larger than the responses seen in normal Krebs solution and were obtained with lower concentrations of the drugs considered to be more specific for rho-kinase inhibition. However, preincubation of HUA with HA-1077 or Y-27632 (10(-5) M for both) did not affect the 5-HT-induced contractions in this medium. Finally, immunoblot experiments revealed the expression of rho-kinase isoform rockII protein in HUA. These results indicate that rhoA/rho-kinase pathway can contribute to agonist-induced contractions of HUA. However, this effect appears to be limited to intracellular calcium-induced contractions and may be more important in sustaining contractions rather than the initial phase of force development.

SR 33557, a novel calcium-antagonist: interaction with [3H]-(�)-nitrendipine and [3H]-(?)-desmethoxy-verapamil binding sites in cerebral membranes

We investigated the pharmacological properties of SR 33557, a novel compound with calcium-antagonist properties, in both functional tests in vitro and radioligand binding studies. SR 33557 potently antagonized calcium-induced contraction of potassium-depolarized rat aorta in vitro with an IC50 value of 5.6 +/- 0.9 nM, but was a much weaker inhibitor of noradrenaline-induced contraction of the same tissue (IC50 = 96 +/- 22 nM). SR 33557 totally inhibited [3H]-(+/-)-nitrendipine binding to rat brain membranes with a Ki value of 0.19 +/- 0.03 nM. Diltiazem, which used alone increases [3H]-(+/-)-nitrendipine binding, reversed this inhibition indicating that SR 33557 allosterically regulates [3H]-(+/-)-nitrendipine binding. SR 33557 also fully inhibited [3H]-(-)-desmethoxyverapamil binding to cerebral membranes, but inhibition curves were biphasic. IC50 value calculated for that part of the curve which reflects the high affinity binding site of SR 33557 (IC50 = 0.20 +/- 0.02 nM) was very similar to the Ki value determined for inhibition of [3H]-(+/-)-nitrendipine binding. Kinetic evidences indicate that SR 33557 binds to a site which is distinct from the 1,4-dihydropyridine or the phenylalkylamine binding sites associated with the calcium channel. To test the pharmacological specificity of these interactions, the ability of SR 33557 to interact with eight other receptors in cerebral or heart membranes was assessed by binding assays. No high-affinity interaction was observed between SR 33557 and any of the receptors investigated. We conclude that SR 33557 binds specifically and with a high affinity to a site closely associated with the voltage-operated calcium channel in cerebral membranes.

Titin determines the Frank-Starling relation in early diastole.

Titin, a giant protein spanning half the sarcomere, is responsible for passive and restoring forces in cardiac myofilaments during sarcomere elongation and compression, respectively. In addition, titin has been implicated in the length-dependent activation that occurs in the stretched sarcomere, during the transition from diastole to systole. The purpose of this study was to investigate the role of titin in the length-dependent deactivation that occurs during early diastole, when the myocyte is shortened below slack length. We developed a novel in vitro assay to assess myocyte restoring force (RF) by measuring the velocity of recoil in Triton-permeabilized, unloaded rat cardiomyocytes after rigor-induced sarcomere length (SL) contractions. We compared rigor-induced SL shortening to that following calcium-induced (pCa) contractions. The RF–SL relationship was linearly correlated, and the SL-pCa curve displayed a characteristic sigmoidal curve. The role of titin was defined by treating myocytes with a low concentration of trypsin, which we show selectively degrades titin using mass spectroscopic analysis. Trypsin treatment reduced myocyte RF as shown by a decrease in the slope of the RF-SL relationship, and this was accompanied by a downward and leftward shift of the SL-pCa curve, indicative of sensitization of the myofilaments to calcium. In addition, trypsin digestion did not alter the relationship between SL and interfilament spacing (assessed by cell width) after calcium activation. These data suggest that as the sarcomere shortens below slack length, titin-based restoring forces act to desensitize the myofilaments. Furthermore, in contrast to length-dependent activation at long SLs, length-dependent deactivation does not depend on interfilament spacing. This study demonstrates for the first time the importance of titin-based restoring force in length-dependent deactivation during the early phase of diastole.

Calcium-Antagonistic Activity of Sumatriptan in the Rat Anococcygeus Muscle

The relaxant effect of sumatriptan, a 5-HT_1B/1D receptor agonist, on the rat anococcygeus muscle was investigated. Sumatriptan induced concentration-dependent relaxations of the phenylephrine-precontracted rat anococcygeus muscle. N^G-nitro-L-arginine, methylene blue, glibenclamide, tetrodotoxin, indomethacin, GR 113808, GR 55562, methysergide, ketanserin, ICS 205930, clozapine, methiothepin, metergoline, mesulergine, and ritanserin did not inhibit the relaxations induced by sumatriptan. Sumatriptan converted calcium-induced contractions into relaxations in the preparations depolarized by a calcium-free, high-potassium solution. In membrane preparations obtained from the rat anococcygeus muscle, the basal rate of cAMP production by adenylate cyclase was 4.77 ± 0.02 pmol/mg protein/min (n = 15). The enzyme activity was increased to 104 ± 51.7 pmol/mg protein/min by forskolin (10^–4 mol/l), but did not change in the presence of sumatriptan. The mRNA expression of 5-HT_1B, 5-HT_1D, and 5-HT₇ receptors was not observed in the rat anococcygeus muscle. The results indicate that sumatriptan causes relaxation of the precontracted rat anococcygeus muscle by a calcium-antagonistic activity.