Initial Transient Contraction Research Articles

Monitoring of Caffeine Consumption Effect on Skin Blood Properties by Diffuse Reflectance Spectroscopy.

Caffeine is the most widely consumed psychoactive substance worldwide, affecting numerous tissues and organs, with notable impacts on the central nervous system, heart, and blood vessels. The effect of caffeine on vascular smooth muscle cells is an initial transient contraction followed by significant vasodilatation. In this study we investigate the use of diffuse reflectance spectroscopy (DRS) for monitoring of vascular changes in human skin induced by caffeine consumption. DRS spectra were recorded on volar sides of the forearms of eight healthy volunteers at time intervals of 0, 30, 60, 120, and 180 min after consumption of caffeine, while one subject served as a negative control. Analytical diffusion approximation solutions for diffuse reflectance from three-layer structures were used to assess skin composition (e.g. dermal blood volume fraction and oxygen saturation) by fitting these solutions to experimental data. The results demonstrate that cutaneous vasodynamics induced by caffeine consumption can be monitored by DRS, while changes in the control subject not consuming caffeine were insignificant.

Temperature Effect on Nerve Conduction Block Induced by High-Frequency (kHz) Biphasic Stimulation

ObjectivesThis study aims to determine temperature effect on nerve conduction block induced by high-frequency (kHz) biphasic stimulation (HFBS). Materials and MethodsFrog sciatic nerve-muscle preparation was immersed in Ringer’s solution at a temperature of 15 or 20 °C. To induce muscle contractions, a bipolar cuff electrode delivered low-frequency (0.25 Hz) stimulation to the nerve. To induce nerve block, a tripolar cuff electrode was placed distal to the bipolar cuff electrode to deliver HFBS (2 or 10 kHz). A bipolar hook electrode distal to the blocking electrode was used to confirm that the nerve block occurred locally at the site of HFBS. A thread tied onto the foot was attached to a force transducer to measure the muscle contraction force. ResultsAt 15 °C, both 2- and 10-kHz HFBSs elicited an initial transient muscle contraction and then produced nerve block during the stimulation (ie, acute block), with the 10 kHz having a significantly (p < 0.001) higher acute block threshold (5.9 ± 0.8 mA peak amplitude) than the 2 kHz (1.9 ± 0.3 mA). When the temperature was increased to 20 °C, the acute block threshold for the 10-kHz HFBS was significantly (p < 0.0001) decreased from 5.2 ± 0.3 to 4.4 ± 0.2 mA, whereas the 2-kHz HFBS induced a tonic muscle contraction during the stimulation but elicited nerve block after terminating the 2-kHz HFBS (ie, poststimulation block) with an increased block duration at a higher stimulation intensity. ConclusionTemperature has an important influence on HFBS-induced nerve block. The blocking mechanisms underlying acute and poststimulation nerve blocks are likely to be very different.

Role of endothelial nitric oxide in pulmonary and systemic arteries during hypoxia

Our aim was to investigate the role played by endothelial nitric oxide (NO) during acute vascular response to hypoxia, as a modulator of both vascular tone (through guanylate cyclase (sGC) activation) and mitochondrial O2 consumption (through competitive inhibition of cytochrome-c-oxydase (CcO)). Organ bath experiments were performed and O2 consumption (Clark electrode) was determined in isolated aorta, mesenteric and pulmonary arteries of rats and eNOS-knockout mice. All pre-contracted vessels exhibited a triphasic hypoxic response consisting of an initial transient contraction (not observed in vessels from eNOS-knockout mice) followed by relaxation and subsequent sustained contraction. Removal of the endothelium, inhibition of eNOS (by L-NNA) and inhibition of sGC (by ODQ) abolished the initial contraction without altering the other two phases. The initial hypoxic contraction was observed in the presence of L-NNA+NO-donors. L-NNA and ODQ increases O2 consumption in hypoxic vessels and increases the arterial tone in normoxia but not in hypoxia. When L-NNA+mitochondrial inhibitors (cyanide, rotenone or myxothiazol) were added, the increase in tone was similar in normoxic and hypoxic vessels, which suggests that inhibition of the binding of NO to reduced CcO restored the action of NO on sGC. ConclusionA complex equilibrium is established between NO, sGC and CcO in vessels in function of the concentration of O2: as O2 falls, NO inhibition of mitochondrial O2 consumption increases and activation of sGC decreases, thus promoting a rapid increase in tone in both pulmonary and systemic vessels, which is followed by the triggering of NO-independent vasodilator/vasoconstrictor mechanisms.

Carbon dioxide-induced inhibition of mechanical activity in gastrointestinal smooth muscle preparations isolated from the guinea-pig

Mechanical responses of smooth muscle elicited by application of CO2-gas bubbled physiological salt solution (CO2-gas solution) were investigated in isolated stomach antrum and colon preparations of the guinea-pig. Circular smooth muscle preparations of both colon and stomach were spontaneously active with periodic generation of phasic contractions. In colonic preparations, the CO2-gas solution produced a biphasic response, with an initial small transient contraction followed by a sustained inhibition of phasic contractions. Removal of the CO2-gas solution allowed a slow recovery of the spontaneous contractions over a period of about 40 min. The recovery developed with a similar time course irrespective of the length of time exposed to CO2-gas solution. The inhibitory responses elicited by CO2-gas solution were not modulated by atropine, Nω-nitro-L-arginine or neostigmine. Atropine-sensitive excitatory responses of smooth muscle elicited by transmural nerve stimulation or exogenously applied acetylcholine were attenuated or abolished in the presence of CO2-gas solution. In stomach preparations, the CO2-gas solution elicited a tri-phasic response, with an initial transient relaxation followed by a transient contraction and then a sustained inhibition of the rhythmic contractions. The peak amplitude of the transient contraction was about 2.5 times larger than the spontaneous phasic contractions. The pH of the CO2-gas solution was reduced to about 6. Application of pH 6 solution again produced a tri-phasic response, as was the case for the CO2-gas solution, however the amplitude of the transient contraction was only about 0.4 times that of the spontaneous contractions. The re-appearance of the abolished phasic contraction was quicker with the pH 6 solution (about 1.8 min) than it was for the CO2-gas solution (about 6 min). The inhibitory responses elicited by the CO2-gas solution could be simulated only partly by the acidified solution, and a possible involvement of additional factors in the inhibition elicited by CO2-gas solution was considered.

Relaxation Patterns of Human Gastric Corporal Smooth Muscle by Cyclic Nucleotides Producing Agents

To elucidate the mechanism of cyclic nucleotides, such as adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5' -cyclic monophosphate (cGMP), in the regulation of human gastric motility, we examined the effects of forskolin (FSK), isoproterenol (ISO) and sodium nitroprusside (SNP) on the spontaneous, high K(+) and acetylcholine (ACh)-induced contractions of corporal circular smooth muscle in human stomach. Gastric circular smooth muscle showed regular spontaneous contraction, and FSK, ISO and SNP inhibited its phasic contraction and basal tone in a concentration-dependent manner. High K(+) (50 mM) produced sustained tonic contraction, and ACh (10 microM) produced initial transient contraction followed by later sustained tonic contraction with superimposed phasic contractions. FSK, ISO and SNP inhibited high K(+)-induced tonic contraction and also ACh-induced phasic and tonic contraction in a reversible manner. Nifedipine (1 microM), inhibitor of voltage-dependent L-type calcium current (VDCC(L)), almost abolished ACh-induced phasic contractions. These findings suggest that FSK, ISO and SNP, which are known cyclic nucleotide stimulators, inhibit smooth muscle contraction in human stomach partly via inhibition of VDCC(L).

Purinergic and adrenergic Ca2+ transients during neurogenic contractions of rat mesenteric small arteries.

Contraction of small arteries is regulated by the sympathetic nervous system, but the Ca2+ transients during neurally stimulated contraction of intact small arteries have not yet been recorded. We loaded rat mesenteric small arteries with the fluorescent Ca2+ indicator fluo-4 and mounted them in a myograph that permitted simultaneous (i) high-speed confocal imaging of fluorescence from individual smooth muscle cells, (ii) electrical stimulation of perivascular nerves, and (iii) recording of isometric tension. Sympathetic neuromuscular transmission was achieved by electrical field stimulation (EFS) (frequency, 10 Hz; pulse voltage, 40 V; pulse duration, 0.2 ms) in the presence of capsaicin and scopolamine (to inhibit 'sensory' and cholinergic nerves, respectively). During the first 20 s of EFS, force rose to a small peak and then declined. During this time, junctional Ca2+ transients (jCaTs) were present at relatively high frequency. We have previously attributed jCaTs to influx of Ca2+ through post-junctional P2X receptors activated by ATP. Propagating asynchronous Ca2+ waves, previously associated with bath-applied alpha1-adrenoceptor agonists, were not initially present. During the next 2.5 min of EFS, force rose slowly, and asynchronous propagating Ca2+ waves appeared. The selective alpha1-adrenoceptor antagonist prazosin abolished both the slowly developing contraction and the Ca2+ waves, but reduced the initial transient contraction by only ~25 %. During 3 min of EFS in prazosin, the frequency of jCaTs declined markedly; at sites at which at least one jCaT occurred, the average probability of a jCaT was 0.008 +/- 0.002 pulse-1 in the first 20 s and 0.0007 +/- 0.0002 pulse-1 in the last 20 s. We suggest that (i) ATP released from sympathetic varicosities activates the initial, transient, contraction and the activator Ca2+ is derived largely from jCaTs, and (ii) sympathetically released noradrenaline (NA) activates the later, major contraction through mechanisms involving alpha1-adrenoceptors and which are associated with propagating Ca2+ waves.

Acute Effects of Serotonin on Rat Bladder Contractility

Introduction: The purpose of this study is to investigate in vitro the effects of serotonin on the rat detrusor. In particular, it examines which drugs inhibit the serotonin-induced detrusor contractions. Materials and Methods: Isometric tension changes of isolated rat bladder muscle strips were recorded in an organ bath using a force transducer. Acute effects of serotonin (0.0001–0.01 mM) on resting tension were assessed. Electrical field stimulation (EFS); bethanechol (0.0001–0.01 mM); ATP (1–3 mM)- or KCl (63.5–254 mM)-induced contractions using an application in an organ bath were compared with serotonin-induced contractions. In order to examine the action mechanism of serotonin-induced stimulation, EFS-, bethanechol-, ATP- or KCl-induced contraction on serotonin treatment (0.001 mM) was assessed and serotonin (0.001–0.1 mM) was cumulatively added to the organ bath following preincubation with propranolol, ketanserin, tropisetron, propiverine, sodium nitroprusside or doxazosin. Results: The serotonin-induced response has two phases: an initial transient contraction and a prolonged tonic phase. Serotonin produced a reversible and dose-dependent contraction of the detrusor strips. Responses to bethanechol significantly increased with a concentration of 0.001 mM serotonin (p < 0.05). There was no effect on the responses to ATP, KCl, or EFS on 0.001 mM serotonin. The 5-HT₂ receptor is mainly responsible for serotonin-induced contractions of the detrusor (p < 0.05), while the 5-HT₁ receptor is partially responsible. Doxazosin and propiverine each significantly suppressed the response to serotonin, while sodium nitroprusside and tropisetron each had no effect (p < 0.05). Conclusions: Because the 5-HT₂ antagonist blocked the effect of serotonin-induced bladder contractions and the stimulation of the adrenoreceptors, the 5-HT₂ antagonist seems to improve lower urinary tract symptoms.

Thiol oxidation activates a novel redox-regulated coronary vasodilator mechanism involving inhibition of Ca2+ influx.

This study examines the mechanism of relaxation of isolated endothelium-removed bovine coronary arteries (BCAs) to the thiol oxidant diamide. BCAs precontracted with KCl or the thromboxane A(2) receptor agonist U46619 showed a concentration-dependent reversible relaxation on exposure to 10 micromol/L to 1 mmol/L diamide. This relaxation was enhanced by an inhibitor of glutathione reductase, and it was not altered by severe hypoxia, the presence of inhibitors of soluble guanylate cyclase, K(+) channels, tyrosine kinases, or probes that modulate levels of superoxide. The relaxation was almost eliminated when BCAs were precontracted with a phorbol ester that causes a contraction that is largely independent of extracellular Ca(2+). The initial transient contraction elicited by 5-hydroxytryptamine in Ca(2+)-free solution was not altered by the presence of 1 mmol/L diamide; however, a subsequent tonic contraction on addition of CaCl(2) was inhibited by diamide. Diamide also inhibited contractions caused by the addition of CaCl(2) to Ca(2+)-free Krebs' buffer containing Bay K8644 (an L-type Ca(2+) channel opener) or KCl. Relaxation to diamide was attenuated by L-type Ca(2+) channel blockers (nifedipine and diltiazem). Thus, thiol oxidation elicited by diamide appears to activate a novel redox-regulated vasodilator mechanism that seems to inhibit extracellular Ca(2+) influx.

Investigation of endogenous neurotransmitters of guinea pig gallbladder using nicotinic agonist stimulation.

Gallbladder motility is modulated by intrinsic nerves, the identities of which are not well established. The aim of this study was to determine the effect of nicotinic receptor stimulation of intrinsic nerves on gallbladder muscle contractility. Guinea pig gallbladder muscle strips were studied in vitro. Histamine 1 microM was used to increase baseline tone. The nicotinic receptor agonist, 1,1-dimethyl-4-phenylpiperazinium (DMPP), produced a biphasic response characterized by an initial transient contraction followed by a sustained relaxation. The initial contraction was inhibited by the neural blocker tetrodotoxin, the nicotinic antagonist hexamethonium, and the muscarinic antagonist atropine, but not by a substance P receptor antagonist or a bombesin receptor antagonist. The relaxation response to DMPP was not affected by tetrodotoxin, but was reduced by hexamethonium and omega-conotoxin GVIA, an inhibitor of neurotransmitter release. The relaxation response was reduced by the nitric oxide synthase inhibitor L-NAME, but not by a vasoactive intestinal peptide antagonist or propranolol. DMPP produces a biphasic response in the guinea pig gallbladder. The initial contractile response is mediated by nicotinic receptors on the cell body or axon of cholinergic nerves. The relaxation response appears to result, in part, from activation of nicotinic receptors on nerve terminals of nitric oxide-releasing nerves. These results suggest nicotinic receptors have heterogeneity in location depending on excitatory or inhibitory neuronal function.

Activation of tyrosine kinases in H2O2-induced contraction in pulmonary artery.

Hydrogen peroxide (H2O2) is an important reactive oxygen species implicated in lung vascular constriction and injury. The purpose of this study was to investigate the role of tyrosine kinases in H2O2-induced vascular contraction and dysfunction. In our study, H2O2 (200 microM) caused an initial transient contraction followed by a strong, sustained contraction in isolated rat pulmonary arteries. Genistein, a tyrosine kinase inhibitor, attenuated both the initial and the sustained contractions. Aminogenistein and tyrphostin 51, specific inhibitors of tyrosine kinases, had the same effects as genistein. Exposure of pulmonary arteries to H2O2 for 1 h caused a significant reduction in the contractile response to KCl or phenylephrine and in the vasodilatory response to acetylcholine (smooth muscle dysfunction). Although tyrosine kinase inhibitors significantly blocked contractions induced by H2O2, pretreatment of pulmonary arteries with these inhibitors before H2O2 exposure did not prevent the decreases in responses to KCl, phenylephrine, or acetylcholine. Removal of extracellular Ca2+ and depletion of intracellular Ca2+ pools by ryanodine or thapsigargin did not inhibit the initial and sustained contractions in response to H2O2. W-7, a calmodulin antagonist, or ML-9, a myosin light chain kinase inhibitor, significantly inhibited the sustained contractions but did not prevent smooth muscle dysfunction induced by H2O2. These data show that 1) exposure to H2O2 causes smooth muscle contractions and dysfunction in isolated pulmonary arteries and 2) activation of tyrosine kinases mediates H2O2-induced contractions; however, tyrosine kinases do not appear to be involved in H2O2-induced inhibition of arterial responses to vasoactive substances. These data suggest that different signaling pathways and mechanisms are involved in H2O2-induced smooth muscle contraction and dysfunction.

Involvement of protein kinase C in muscarinic agonist-induced contractions of guinea pig real longitudinal muscle

1. 1. Carbachol (10 −5M) caused an initial transient contraction (phasic contraction) and a subsequent late contraction (tonic contraction) in the guinea pig ileum. The phasic contraction was markedly suppressed by calmodulin antagonist W-7, but not by protein kinase C inhibitor H-7. 2. 2. The tonic contraction was suppressed by H-7, but not by W-7. 3. 3. The carbachol-induced phasic contraction in the absence of extracellular Ca 2+ was suppressed by both W-7 and H-7. 4. 4. Carbachol (10 −5M) stimulated the formation of inositol phosphates (IPs) in the guinea pig ileum. The carbacholinduced IPs formation was dependent on the extracellular Ca 2+ concentration.

Effects of PKC downregulation on norepinephrine- and prostaglandin F2 alpha-induced contraction in rat aorta

The purpose of this study was to investigate, through phorbol ester-induced protein kinase C (PKC) downregulation, the role of PKC in the regulation of alpha-adrenergic agonist- and prostaglandin (PG) F2 alpha-induced contraction in vascular smooth muscle. In rat aorta, long-term phorbol ester exposure (10 microM phorbol dibutyrate for 17 h), a procedure that decreased PKC activity by > 95%, and maximal phorbol myristate acetate (PMA)-induced contraction by approximately 75%, decreased tissue sensitivity to norepinephrine (NE) and PGF2 alpha 2.8- and 4.6-fold, respectively, while maximal contraction was not significantly decreased. In contrast, long-term phorbol ester exposure did not alter tissue sensitivity to KCl, while maximal KCl contraction was decreased by 40%. Long-term phorbol ester exposure, as well as short-term phorbol ester exposure (1 microM PMA for 1 h), abolished the initial transient NE contraction elicited in Ca(2+)-free solution. In contrast, long-term phorbol ester exposure did not alter the plateau NE or PGF 2 alpha contraction elicited in Ca(2+)-free solution. Short-term phorbol ester exposure of PKC-downregulated tissues potentiated the plateau PGF2 alpha contraction elicited in Ca(2+)-free solution, although the magnitude of potentiation was less than that observed in non-PKC downregulated tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Critical role of endothelium in sustained arterial contraction during prolonged hypoxia

Acute anoxia or severe hypoxia causes an initial transient contraction followed by marked relaxation of vascular tissues. We observed a spontaneous gradual sustained contraction of rat aortic rings following relaxation when hypoxia was prolonged. Deendothelialization as well as treatment of the endothelium-intact rings with nitric oxide synthase inhibitors or oxyhemoglobin abolished the late hypoxic contraction despite prolonged hypoxia. The prolonged hypoxia-induced sustained contraction was not affected by adenosine receptor blockade, cyclooxygenase inhibition, free radical scavengers, or the endothelin receptor antagonists. The ATP-sensitive K+ channel blocker glibenclamide abbreviated the duration of hypoxic relaxation and potentiated the magnitude of late hypoxic contraction. These data suggest that the late-sustained hypoxic contraction of arterial tissues is dependent on the presence of intact functional endothelium. Activation of ATP-sensitive K+ channels may participate in the genesis of hypoxic relaxation. However, cyclooxygenase products, free oxygen radicals, adenosine, and endothelin are not involved in the regulation of hypoxia-mediated events in rat aortic rings.

Mechanisms of hypoxic vasoconstriction in the canine isolated pulmonary artery: role of endothelium and sodium pump

Contraction of canine pulmonary artery to hypoxia in vitro is both endothelium dependent and independent. The mechanisms which underlie this phenomenon were studied. Rings of canine pulmonary artery were suspended for isometric force recording in tissue baths containing modified Krebs-Ringer bicarbonate solution. Tissues were first contracted with norepinephrine [effective dose at 35% (ED35) concentration]. Subsequent exposure to hypoxia induced a triphasic response: an initial phasic transient contraction (phase 1), a transient reduction in force (phase 2), followed by a sustained tonic contraction (phase 3). In the absence of endothelium, all phases of the hypoxic response were reduced, and phase 2 was reversed from a contraction to a relaxation (with endothelium: 0.68 +/- 0.2 g; without endothelium: -0.34 +/- 0.1 g). Similar data were obtained in the presence of nitro-L-arginine (3 x 10(-5) M). In the absence of endothelium, indomethacin (10(-5) M) abolished the phase 2 relaxation and converted phase 3 from a contraction to a relaxation (control: 0.99 +/- 0.2 g; indomethacin: -0.44 +/- 0.1 g); and ONO-3708 (thromboxane A2/prostaglandin H2 receptor antagonist) diminished phase 3 (control: 0.99 +/- 0.2 g; ONO-3708: 0.3 +/- 0.04 g). In the absence of endothelium, but in the presence of indomethacin (10(-5) M), K(+)-free solution diminished phase 1 (contraction) and converted phase 2 (relaxation) to a contraction (control: -0.74 +/- 0.1 g; K(+)-free solution: 0.1 +/- 0.06 g). Similar results were obtained with ouabain (4 x 10(-7) M), and cooling of the bathing medium (20 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS)

Photodynamic action of aluminium phthalocyanine tetrasulphonate (A1PcS4) on smooth muscle: effects of thiols and a cyclic GMP analogue.

1. The smooth muscle system of the guinea-pig taenia caeci has been used in vitro to characterize the photodynamic action of aluminium phthalocyanine tetrasulphonate (A1PcS4) in the presence or absence of the thiol reductants L-cysteine (Cys), N-acetyl-L-cysteine (NAC), DL-dithiothreitol (DTT) or reduced glutathione (GSH). 2. In all photodynamic experiments the muscle was exposed to A1PcS4 (10(-5) M) for 30 min, followed by a 30 min washout period before photon irradiation at 32,000 lux (lambda > 570 nm) for 30 min. Photodynamic contractions were measured relative to the contractile response to carbachol (5 x 10(-5) M) and relaxation responses were determined in muscle precontracted with either carbachol 5 x 10(-5) M or KCl 23.5 mM. 3. Photon-activation of A1PcS4-sensitized smooth muscle evoked a triphasic response: an initial transient contraction and subsequent relaxation followed by a secondary sustained contraction. Cys 10 mM, NAC 10 mM and DTT 5 mM had no effect on the initial photodynamic contraction but significantly decreased the magnitude of the sustained contraction from mean values of 98% to 18%, 95% to 72% and 93% to 6% of the standard carbachol contraction (5 x 10(-5) M), respectively; GSH 10 mM was without significant effect on either the initial or sustained contraction. 4. In the absence of extracellular calcium the A1PcS4-sensitized smooth muscle did not respond to photon activation but re-introduction of calcium after cessation of illumination produced a sustained contraction which was markedly inhibited by Cys 10 mM. 5. In precontracted AlPcS4-treated muscle preparations photon activation produced a triphasic relaxation response, i.e. a rapid relaxation followed by a transient contraction and a secondary more sustained relaxation. The sustained phase of photodynamic relaxation was potentiated significantly by Cys 10 mM,NAC 10 mM, DTT 5 mM and GSH 10 mM, the relaxation being approximately doubled in magnitude from mean values of 34% to 68%, 30% to 73%, 34% to 68%, and 48% to 77%, respectively, relative to the standard carbachol (5 x l0-5 M) response.6. The cyclic GMP analogue, 8-(4-chlorophenylthio)-guanosine-3':5'-cyclic monophosphate (8-PCPTcGMP)(2 x 10-4 M) alone caused a triphasic relaxation response similar to that produced by photon activation of an AIPcS4-sensitized precontracted preparation in the presence of thiol reductants. The pattern of 8-PCPT-cGMP-induced relaxation was similar in muscle precontracted with carbachol 5 x 10-5M or KCI 23.5 mM.7. It is concluded that the rapid generation of reactive intermediates by photon-activation of boundAlPcS4 leads to membrane permeabilization, calcium entry and muscle contraction. These effects may be opposed by a direct stimulatory action of singlet oxygen on guanylate cyclase which is enhanced by the action of thiol reagents and mimicked by the cyclic GMP analogue, 8-PCPT-cGMP.

Specific G proteins mediate endothelin induced contraction

Endothelin is a potent vasoconstrictor peptide which has recently been localized in the gastrointestinal tract. We have investigated the transmembrane signaling properties of endothelin in isolated smooth muscle cells of the rabbit rectosigmoid. Endothelin induced a dose dependent contraction of smooth muscle cells in a range of 10 −10 to 10 −6M. In normal buffer, contraction peaked at 30 sec and was sustained for up to 8 min. Incubation in 0Ca/2mM EGTA abolished the sustained contraction induced by endothelin, but had no effect on the initial transient contraction. Preincubation of saponin treated cells with G protein antisera had no effect on control cell length. Preincubation of saponin treated isolated smooth muscle cells with specific G protein antisera (rabbit antisera) for Goα or Gs for 60 minutes did not inhibit contraction induced by endothelin. Preincubation with an antiserum to Gi 3α inhibited the initial transient contraction induced by endothelin and preincubation with an antiserum to Gi 1−2α inhibited the sustained phase of the endothelin induced contraction. Our data indicate that: 1) Endothelin induces a direct sustained contraction of smooth cells from the rectosigmoid; 2) The transmembrane signalling of endothelin is through two specific GTP binding components that are Giα, one for the initial transient contraction, and the other for the sustained phase of the contraction.

Phorbol esters induce oscillatory contractions of intestinal smooth muscles

The actions of tumor-promoting phorbol esters in smooth muscle excitation-contraction coupling were studied in isolated guinea pig ileum in the presence of various contractile agents. Muscarinic agonists, histramine and bradykinin elicited an initial transient phasic contraction and a subsequent sustained tonic contraction in guinea pig ileum. The Ca 2+ channel antagonist nifedipinc selectively inhibited the tonic contraction. Phorbol esters, protein kinase C activators, induced immediate muscle relaxation followed by oscillatory contractions when added during the tonic phase of contraction. Phorbol esters, when added in adBance, slightly altered the ligand-induced phasic contraction but converted tonic contractions into oscillatory spikes. The amplitude, frequency and shape of the oscillation induced by phorbol esters were dependent upon the dose of phorbol ester: amplitude was increased and frequency was decreased by increasing the doses of phorbol ester. In contrast, the phorbol ester potentiated the tonic contraction induced by high potassium chloride with little effect on the phasic component. It also sensitized the muscles to Bay K 8644. Bay K 8644, which was ineffective in stimulating muscle contraction at 1 pM, became a very effective stimulator in the presence of the phorbol ester. All of these phorbol ester-induced potentiations and oscillations were sensitive to inhibition by staurosporinc or nifedipine. These data suggest that in guinea pig ileum, protein kinase C plays a positive regulatory role in Ca 2+ channel activation and promotes a complex regulatory effect on Ca 2+ -mobilizing ligand-stimulated Ca 2+ channel activity, which results in oscillatory contractile responses to carbachol, methacholine, histamine and bradykinin.

An examination of the sources of calcium for contractions mediated by postjunctional α1‐ and α2‐adrenoceptors in several blood vessels isolated from the rabbit

1. The roles of intracellular and extracellular-derived Ca2+ in alpha-adrenoceptor-mediated contractions to noradrenaline (NA) have been investigated in several isolated blood vessels from the rabbit by examining responses in the presence of a modified Krebs-Henseleit saline with 2.5 mM Ca2+ and a Ca2(+)-buffered saline with 0.1 microM free Ca2+. 2. NA was tested in preparations of the abdominal aorta, distal saphenous artery, renal vein, lateral saphenous vein, plantaris vein and ear vein exposed to a Ca2(+)-buffered saline with 0.1 microM [Ca2+]. A concentration of NA which was maximally effective in modified Krebs-Henseleit saline, produced an initial transient contraction (ITC) followed by a relaxation towards baseline. This is evidence that alpha-adrenoceptor-mediated responses in all these blood vessels depend upon calcium from both sources. 3. The ITC was particularly pronounced in the arteries and was associated more closely with the alpha 1-receptor subtype. In the abdominal aorta, distal saphenous artery and renal vein the ITC can almost exclusively be attributed to an alpha 1-adrenoceptor (prazosin-sensitive, rauwolscine-resistant). In the ear vein, and to a lesser extent the plantaris vein, the ITC was mediated in part by an alpha 2-adrenoceptor (prazosin-resistant, rauwolscine-sensitive). 4. alpha 2-Adrenoceptors in the lateral saphenous vein largely account for the response to NA in modified Krebs-Henseleit saline, but alpha 1-adrenoceptors mediate the ITC in Ca2(+)-buffered saline. After selective inactivation of alpha 1-adrenoceptors with a combination of phenoxybenzamine and rauwolscine, responses to NA in modified Krebs-Henseleit saline are slow in onset and there is no ITC in Ca2(+)-buffered saline. 5. The possible significance of the coupling of postjunctional alpha 2-adrenoceptors to dual sources of Ca2 + is discussed in relation to the interaction between alpha-adrenoceptor subtypes and the ease of demonstrating functional alpha 2-adrenoceptors in isolated blood vessels.

Two phases of the prostaglandin F2?-induced contraction in guinea-pig taenia coli involve different Ca2+ channels

Properties of the contraction produced by PGF2 alpha in the guinea-pig taenia coli were compared to those produced by ACh. Prostaglandin (PG) F2 alpha (3 x 10(-7) M) and acetylcholine (ACh, 10(-5)M) induced an initial transient contraction (phasic contraction) and a subsequent late contraction (tonic contraction). Both phasic and tonic contractions produced by PGF2 alpha or ACh were abolished in Ca2+ -free Krebs solution containing 0.5 mM EGTA. The tonic contractions caused by PGF2 alpha and ACh were markedly suppressed by alpha-[3-[[2-(3,4-dimethoxy-phenyl)-ethyl]-methylamino]-propyl]- 3,4,5-trimethoxy-alpha-(1-methylethyl)benzeneacetonitrile hydrochloride (D600, greater than 10(-7)M) as well as nifedipine (5 x 10(-9)M), a Ca2+-antagonist. However, the phasic contraction produced by PGF2 alpha, but not by ACh, was greatly inhibited by Mn2+ (greater than 10(-4)M). Furthermore, the phasic contraction caused by PGF2 alpha was abolished in 18 mM K+ Krebs solution with D600 (2 x 10(-7)M), whereas that induced by ACh and the tonic contractions produced by PGF2 alpha as well as by ACh were unaffected in this high K+ solution without D600. Membrane potentials of the tissue in normal (K+, 5.9 mM) and 18 mM K+ Krebs solution containing D600 were about -55 mV and -43 mV, respectively. In a fluorescence study which used Fura-2 an intracellular free Ca2+ indicator in the presence of D600, PGF2 alpha and ACh increased fluorescence intensity in the tissue, which coupled with the magnitude of contractions.(ABSTRACT TRUNCATED AT 250 WORDS)

Verapamil-sensitive and less sensitive contractions in the intestinal smooth muscle of the guinea-pig taenia caeci.

Effect of verapamil, a Ca2+ channel blocker, on the contractions in the intestinal smooth muscle of taenia isolated from guinea-pig caecum was examined. In normal medium, 10 mM-80 mM KCl or 10(-7) M-10(-5) M carbachol induced a transient contraction followed by a sustained one. When the muscle strips were treated with a Ca2+-free solution for 2 min, these stimulants failed to induce contraction except an initial transient contraction induced by 10(-6) M-10(-5) M carbachol. Cumulative application of verapamil during the sustained contraction induced muscle relaxation. Higher concentration of verapamil was needed to inhibit the sustained contraction induced by 10 mM or 20 mM K+ than that induced by 40 mM or 80 mM K+. Similarly, the sensitivity to verapamil of the sustained contraction induced by 10(-7) M carbachol was lower than that induced by 10(-6) M or 10(-5) M carbachol. In the presence of verapamil, addition of either high K+ or carbachol induced an initial transient contraction, although the sustained contraction was strongly inhibited. These results suggest that high K+ and carbachol activate two types of Ca2+ channels; lower concentrations of the stimulants open the channel which is less sensitive to verapamil, and higher concentrations open the channel which is more sensitive to verapamil.