The role of Ni(2+)-sensitive T-type Ca(2+) channels in the regulation of spontaneous excitation in detrusor smooth muscles of the guinea-pig bladder

OBJECTIVE: To explore the role of Ni(2+)-sensitive T-type Ca(2+) channels in the generation of spontaneous excitation of detrusor smooth muscles. MATERIALS AND METHODS: In isolated detrusor smooth muscle bundles of the guinea-pig bladder, changes in the membrane potential and muscle tension were measured using intracellular microelectrodes and isometric tension recording. Changes in the intracellular Ca(2+) concentration were recorded from bundles loaded with the fluorescent dye fura-PE3. RESULTS: Detrusor smooth muscles had two types of spontaneous electrical activity, i.e. individual and bursting action potentials. Ni(2+) (30 microM), a blocker for T-type Ca(2+) channels, reduced the frequency of individual action potentials without changing their amplitude. Higher concentrations of Ni(2+) (100-300 microM) converted individual action potentials into the bursts, as did apamin (0.1 microM), a blocker of small-conductance Ca(2+)-activated K(+) channels (SK). They also increased the amplitudes of spontaneous Ca(2+) transients and corresponding contractions whilst reducing their frequencies. In preparations which generated bursting action potentials, nifedipine (1 microm) converted action potentials into spontaneous transient depolarizations (STDs), and subsequent applications of Ni(2+) (100 microm) abolished STDs. Gadolinium (100 microM) and SKF96365 (10 microM), blockers for nonselective cation channels, and niflumic acid (100 microm), a blocker for Ca(2+)-activated Cl- channels, had no effect on either the amplitude or frequency of spontaneous action potentials. CONCLUSIONS: The T-type Ca(2+) channel may have dual roles in generating spontaneous excitation in detrusor smooth muscles. First, activity of these channels may account for the preceding depolarizations that lead to action potentials. Second, Ca(2+) influx through T-type Ca(2+) channels may couple functionally to SK channels, contributing to the stability of the resting membrane potential in detrusor smooth muscle. Thus, pharmacological manipulation of T-type Ca(2+) channels in detrusor smooth muscles could be of potential value for treating the overactive bladder.     

Effects of halothane on delayed afterdepolarization and calcium transients in dog ventricular myocytes exposed to isoproterenol

The effects of halothane on isoproterenol-induced delayed after-depolarizations (DADs) were investigated in canine ventricular myocytes. In addition, the effects of halothane on the intracellular free calcium transient were determined in fura-2-loaded myocytes exposed to isoproterenol to explore the mechanisms underlying halothane effects on DADs. Isoproterenol (100 nM) induced DADs and/or undriven action potentials in myocytes stimulated electrically with the use of trains of 10 stimuli delivered at basic drive cycle lengths of 200-1,000 ms. Isoproterenol (100 nM) increased the peak ratio (350/380 nm excitation) of stimulated myocyte calcium transients; furthermore, isoproterenol induced a second spontaneous component in the calcium transients of 62% of treated myocytes (n = 72). Halothane (1.5%, 0.53 mM) significantly decreased the amplitude of isoproterenol-induced DADs (P less than 0.01). Halothane not only reduced the peak ratio of the stimulated calcium transient, but also eliminated the second spontaneous component in myocytes previously exposed to isoproterenol (n = 14). Elevated extracellular calcium concentrations (5 mM) restored the amplitudes of DADs and the second components of the calcium transients in myocytes exposed to isoproterenol and halothane. These data suggest that halothane opposes isoproterenol-induced DADs by altering intracellular calcium stores. The authors' findings do not support a role for DAD-induced triggered activity in the genesis of anesthetic-catecholamine dysrhythmias.     

Spontaneous activity of mouse detrusor smooth muscle and the effects of the urothelium

AIMS: To characterize the detrusor muscle of the mouse urinary bladder in order to understand more precisely spontaneous contractile behavior of this organ. This study examined the spontaneous electrical activity and Ca(2+) dynamics of the detrusor smooth muscle and investigated the role of the urothelium. MATERIALS AND METHODS: Detrusor smooth muscle strips were isolated from mouse bladders. The urothelium was either kept intact or removed. Changes in membrane potential were recorded using sharp electrode intracellular recording. To image Ca(2+) dynamics, tissue strips were exposed to 10 microM Oregon Green 488 BAPTA-1 AM for 70 min, and then image series were acquired with a laser-scanning confocal microscope. RESULTS: (1) Mouse detrusor smooth muscle cells (SMCs) generate nifedipine-sensitive spontaneous action potentials (sAPs) at a low frequency (1.3 +/- 0.9 min(-1), n = 11) in preparations with intact urothelium. This frequency increased when the urothelium was removed (7 +/- 8.3 min(-1), n = 17) (P < 0.05, Student's t test). (2) Frequent ATP-mediated spontaneous depolarizations were recorded in all cells. (3) The frequency of whole cell Ca(2+) flashes of detrusor smooth muscle cells was higher in preparations with the urothelium removed (median 1.2 min(-1), n = 7) than in urothelium denuded preparations (median 0.6 min(-1), n = 7) (P < 0.01, Mann-Whitney U-test). CONCLUSIONS: Spontaneous activity of the mouse detrusor smooth muscles was characterized enabling future comparative work on gene knock-out strains. Evidence suggesting release of an inhibitory factor by the urothelium was apparent.     

Effects of imatinib mesylate (Glivec) as a c-kit tyrosine kinase inhibitor in the guinea-pig urinary bladder

AIMS: In the gastrointestinal tract, slow wave activity in smooth muscle is generated by the interstitial cells of Cajal (ICC). Detrusor smooth muscle strips of most species show spontaneous contractions which are triggered by action potential bursts, however, the pacemaker mechanisms for the detrusor are still unknown. Recently, ICC-like cells have been found in guinea-pig bladder, using antibodies to the c-kit receptor. We have investigated the effects of Glivec, a c-kit tyrosine kinase inhibitor, on spontaneous action potentials in guinea-pig detrusor and intravesical pressure of isolated guinea-pig bladders. METHODS: Changes in the membrane potential were measured in guinea-pig detrusor smooth muscle using conventional microelectrode techniques. Pressure changes in the bladder were recorded using whole organ bath techniques. RESULTS: Smooth muscle cells in detrusor muscle bundles exhibited spontaneous action potentials, and spontaneous pressure rises occurred in isolated bladders. Glivec (10 microM) converted action potential bursts into continuous firing with no effects on the shape of individual action potentials. Glivec (>50 microM) reduced the amplitude of spontaneous pressure rises in the whole bladder in a dose dependent manner and abolished spontaneous action potentials in detrusor smooth muscle cells. CONCLUSIONS: The results suggest that ICC-like cells may be responsible for generating bursts of action potentials and contractions in detrusor smooth muscle. Drugs inhibiting the c-kit receptor may prove useful for treating the overactive bladder.     

The ATP—sensitive K^＋ channel and membrane potential in the pathogenesis of vascular hyporeactivity in severe hemorrhagic shock

Objective:To elucidate the mechanism of vascular hyporeactivity following severe hemorrhagic shock(HS) by studying the changes of ATP-sensitive potassium channels‘(KATP) groperties and membrane potential of mesenteric arteriolar smooth muscle cells.Methods:single channel currents were studied on cell-attached and indide-out patches of enzymatically isolated mesenteric arteriolar smooth muscle cells (ASMCs).Membrane potentials of arteriolar strips and ASMCs were recorded by intracellular membrane potential recording method and confocal microscopy,respectively.Results:KATP channels in ASMCs were activated,which induced smooth muscle hyperpolarization following vascular hyporeactivity in HS.Conclusions:Hyperpolarizing effect of KATP channel activation plays an important role in low vasoreactivity during severe hemorrhagic shock.     

Mechanisms of excitatory transmission in circular smooth muscles of the guinea pig seminal vesicle

PURPOSE: Cellular mechanisms of excitatory neuromuscular transmission in circular smooth muscles of the seminal vesicle were investigated. MATERIALS AND METHODS: Circular smooth muscles of the seminal vesicle of the guinea pig were isolated. Changes in membrane potential produced by transmural nerve stimulation were recorded using intracellular microelectrode techniques. Changes in the intracellular Ca ion concentration induced by transmural nerve stimulation were measured in preparations loaded with Ca indicator fura-PE3. Responses produced by bath applied norepinephrine and alpha,beta-methylene adenosine triphosphate (ATP) were also examined. RESULTS: Transmural nerve stimulation evoked excitatory junction potentials that triggered action potentials and also caused transient increases in [Ca2+] (Ca transients). Nifedipine abolished action potentials, leaving underlying excitatory junction potentials unchanged, and reduced the amplitude of Ca transients. Excitatory junction potentials were blocked by alpha,beta-methylene ATP or guanethidine but not by phentolamine. A train of transmural nerve stimulation evoked oscillatory changes in membrane potential and [Ca2+], which were abolished by phentolamine or inhibited by nifedipine. Nifedipine insensitive components were abolished by cyclopiazonic acid. Norepinephrine depolarized the membrane and elicited oscillatory potentials with an associated elevation in [Ca2+]. These responses were inhibited by nifedipine and abolished by additional application of cyclopiazonic acid. Transient depolarization with an associated increase in [Ca2+] was elicited by alpha,beta-methylene ATP and [Ca2+] responses but no potential changes were inhibited by nifedipine. CONCLUSIONS: Circular smooth muscles of the guinea pig seminal vesicle receive a projection of sympathetic nerves that release norepinephrine to initiate slow depolarization through the activation of alpha-adrenoceptors. These nerves also release ATP to elicit excitatory junction potentials. Neurally released norepinephrine and ATP are increased [Ca2+] by the influx of Ca2+ through L-type Ca2+ channels and also by the release of Ca2+ from internal stores.     

Potassium Channel-mediated Hyperpolarization of Mesenteric Vascular Smooth Muscle by Isoflurane

Background: A primary source of calcium (Ca2+) necessary for excitation contraction in vascular smooth muscle (VSM) is influx via voltage-dependent Ca2+ channels. Thus, force generation in VSM is coupled closely to resting transmembrane potential, which itself is primarily a function of potassium conductance. Previously, the authors reported that volatile anesthetics hyperpolarize VSM of small mesenteric resistance arteries and capacitance veins. The current study was designed to determine whether isoflurane-mediated hyperpolarization is the result of specific effects on one or more of four types of potassium channels known to exist in VSM.

Methods: Transmembrane potentials (Em) were recorded from in situ mesenteric capacitance and resistance vessels in Sprague-Dawley rats weighing 250-300 g. In separate experiments, selective inhibitors of each of four types of potassium channels known to exist in VSM were administered in the superfusate of the vessel preparations to assess their effects on isoflurane-mediated hyperpolarization.

Results: Resting VSM Em ranged from -38 to -43 mV after local sympathetic denervation. Isoflurane produced a significant hyperpolarization (2.7-4.3 mV), whereas each potassium channel inhibitor significantly depolarized (2.8-8.5 mV) the VSM. Both 100 nM iberiotoxin (inhibitor of high conductance calcium-activated potassium channels) and 1 [micro sign]M glybenclamide (inhibitor of adenosine triphosphatase-sensitive potassium channels) significantly inhibited VSM hyperpolarization induced by 1 MAC (minimum alveolar concentration) levels of inhaled isoflurane (0.1-0.9 mV E (m) change, which was not significant). In contrast, isoflurane hyperpolarized the VSM significantly despite the presence of 3 mM 4 aminopyridine (inhibitor of voltage-dependent potassium channels) or 10 [micro sign]M barium chloride (an inhibitor of inward rectifier potassium channels) (3.7-8.2 mV change in VSM Em).     

Potassium channel-mediated hyperpolarization of mesenteric vascular smooth muscle by isoflurane

BACKGROUND: A primary source of calcium (Ca2+) necessary for excitation contraction in vascular smooth muscle (VSM) is influx via voltage-dependent Ca2+ channels. Thus, force generation in VSM is coupled closely to resting transmembrane potential, which itself is primarily a function of potassium conductance. Previously, the authors reported that volatile anesthetics hyperpolarize VSM of small mesenteric resistance arteries and capacitance veins. The current study was designed to determine whether isoflurane-mediated hyperpolarization is the result of specific effects on one or more of four types of potassium channels known to exist in VSM. METHODS: Transmembrane potentials (Em) were recorded from in situ mesenteric capacitance and resistance vessels in Sprague-Dawley rats weighing 250-300 g. In separate experiments, selective inhibitors of each of four types of potassium channels known to exist in VSM were administered in the superfusate of the vessel preparations to assess their effects on isoflurane-mediated hyperpolarization. RESULTS: Resting VSM Em ranged from -38 to -43 mV after local sympathetic denervation. Isoflurane produced a significant hyperpolarization (2.7-4.3 mV), whereas each potassium channel inhibitor significantly depolarized (2.8-8.5 mV) the VSM. Both 100 nM iberiotoxin (inhibitor of high conductance calcium-activated potassium channels) and 1 microM glybenclamide (inhibitor of adenosine triphosphatase-sensitive potassium channels) significantly inhibited VSM hyperpolarization induced by 1 MAC (minimum alveolar concentration) levels of inhaled isoflurane (0.1-0.9 mV Em change, which was not significant). In contrast, isoflurane hyperpolarized the VSM significantly despite the presence of 3 mM 4 aminopyridine (inhibitor of voltage-dependent potassium channels) or 10 microM barium chloride (an inhibitor of inward rectifier potassium channels) (3.7-8.2 mV change in VSM Em). CONCLUSIONS: These results suggest that isoflurane-mediated hyperpolarization (and associated relaxation) of VSM can be attributed in part to an enhanced (or maintained) opening of calcium-activated and adenosine triphosphate-sensitive potassium channels but not voltage-dependent or inward rectifier potassium channels.     

Ca2+ regulation in detrusor smooth muscle from ovine fetal bladder after in utero bladder outflow obstruction

PURPOSE: We characterized intracellular Ca(2+) regulation in fetal bladders following outflow obstruction by examining the Ca(2+) response to agonists in smooth muscle cells. MATERIALS AND METHODS: Severe bladder outflow obstruction was induced in male fetal sheep by placing a urethral ring and urachal ligation midway through gestation at 75 days. Fetuses were examined 30 days after surgery. Intracellular Ca(2+) in single smooth muscle cells isolated from the bladder wall was measured with epifluorescence microscopy using fura-2(AM) during exposure to agonists, such as carbachol and adenosine triphosphate, and to other activators, such as caffeine and KCl. RESULTS: Detrusor smooth muscle cells from obstructed bladders had resting intracellular Ca(2+) similar to that in sham operated controls. The maximal response to carbachol was decreased following obstruction (p <0.05). Construction of dose-response curves also demonstrated higher EC(50) (p <0.05). However, these changes were not mirrored by caffeine evoked Ca(2+) release, which was not significantly different between the obstruction group and sham operated controls. Kinetic analysis of carbachol transients further revealed an attenuated maximal rate of increase in obstructed bladders (p <0.01). The magnitude of intracellular Ca(2+) to purinergic neurotransmitter adenosine triphosphate was also found to be smaller in cells from obstructed bladders (p <0.05), although transmembrane influx by high K depolarization was not significantly affected. CONCLUSIONS: Muscarinic and purinergic pathways were down-regulated in fetal detrusor muscle following outflow obstruction. These major functional receptors appeared to be more susceptible to obstruction than other Ca(2+) regulators. Their impairment may contribute to the compromised contractile function seen in in utero bladder outflow obstruction.     

Differential Effects of Sevoflurane, Isoflurane, and Halothane on Ca (2+) Release from the Sarcoplasmic Reticulum of Skeletal Muscle

Background: Although malignant hyperthermia after application of sevoflurane has been reported, little is known about its action on intracellular calcium homeostasis of skeletal muscle. The authors compared the effect of sevoflurane with that of isoflurane and halothane on Ca2+ release of mammalian sarcoplasmic reticulum and applied a novel method to quantify Ca2+ turnover in permeabilized skeletal muscle fibers.

Methods: Liquid sevoflurane, isoflurane, and halothane at 0.6 mM, 3.5 mM, and 7.6 mM were diluted either in weakly calcium buffered solutions with no added Ca2+ (to monitor Ca2+ release) or in strongly Ca2+ buffered solutions with [Ca2+] values between 3 nM and 24.9 [micro sign]M for [Ca2+]-force relations. Measurements were taken on single saponin skinned muscle fiber preparations of BALB/c mice. Individual [Ca2+]-force relations were characterized by the Ca2+ concentration at half-maximal force that indicates the sensitivity of the contractile proteins and by the steepness. Each force transient was transformed directly into a Ca (2+) transient with respect to the individual [Ca2+]-force relation of the fiber.

Results: At 0.6 mM, single force transients induced by sevoflurane were lower compared with equimolar concentrations of isoflurane and halothane (P < 0.05). Similarly, calculated peak Ca2+ transients of sevoflurane were lower than those induced by equimolar halothane (P < 0.05). The Ca2+ concentrations at half maximal force were decreased after the addition of sevoflurane, isoflurane, and halothane in a concentration-dependent manner (P < 0.05).     

Isoproterenol enhances myofilament Ca(2+) sensitivity during hypothermia in isolated guinea pig beating hearts

Isoproterenol is often required to treat acute left ventricular dysfunction during separation from cardiopulmonary bypass for cardiac surgery. We hypothesized that heart rate and intracellular Ca(2+) concentration ([Ca(2+)]i) homeostasis may be important factors when isoproterenol improves the cardiac function during hypothermia. Accordingly, we investigated the effect of isoproterenol on the cardiac functional variables, [Ca(2+)]i, and myofilament Ca(2+) sensitivity under spontaneous beating during hypothermia. Intact guinea pig hearts were perfused with a modified Krebs-Ringer solution (baseline) and Krebs-Ringer solution containing isoproterenol (1 nM) at 37 degrees C, 32 degrees C, and 27 degrees C while all cardiac variables and [Ca(2+)]i were recorded. Isoproterenol increased developed left ventricular pressure (LVP), maximum rate of increase in LVP, and coronary inflow at 27 degrees C, and it also increased heart rate and maximum rate of decrease in LVP at each temperature (P < 0.05). Isoproterenol produced a leftward shift of the curve of developed LVP as a function of available [Ca(2+)]i at 32 degrees C and 27 degrees C (P < 0.05), without changing available [Ca(2+)]i. Isoproterenol improves the cardiac function, especially systolic ventricular function, by enhancement of myofilament Ca(2+) sensitivity under spontaneous beating during hypothermia in intact guinea pig hearts. IMPLICATIONS: Enhancement of myofilament Ca(2+) sensitivity is involved in the improvement of cardiac function by isoproterenol under spontaneous beating during hypothermia.     

Changes in cyclic AMP and cyclic GMP levels in the contraction-relaxation cycle of isolated smooth muscle cells from guinea-pig taenia caeci

The present study was undertaken to investigate the role of cyclic AMP and cyclic GMP in the regulation of the contraction-relaxation cycle in isolated smooth muscle cells from guinea pig taenia caeci. The contraction of isolated smooth muscle cells induced by both caffeine (4 X 10(-3) M) and carbachol (10(-4) M) consisted of an initial activation phase and a following spontaneous relaxation phase. Isoproterenol (10(-4) M) inhibited the carbachol-induced contraction and raised intracellular cyclic AMP level of isolated smooth muscle cells. Both the inhibition of the carbachol-induced contraction and the rise in cyclic AMP level elicited by isoproterenol were blocked by propranolol (10(-4) M). Caffeine increased intracellular cyclic AMP level significantly and contracted isolated smooth muscle cells. Caffeine and carbachol contracted isolated smooth muscle cells and increased intracellular cyclic GMP level. Cyclic GMP was significantly increased in spontaneous relaxation phase. The data suggest that beta-adrenergic inhibitory effect on cholinergic excitation in the mammalian isolated smooth muscle cells is mediated by the change in intracellular cyclic AMP level and that cyclic GMP related to the relaxation in these single cells. Thus, it appears that caffeine-induced contraction might be due to the stimulation of Ca2+-induced Ca2+ release by cyclic AMP.     

Remifentanil induces l-type ca2+ channel inhibition in human mesenteric arterial smooth muscle cells

PURPOSE: Remifentanil is known to cause vasodilation at standard anesthetic concentrations. The intracellular mechanisms underlying its vasodilator action may involve the activation of ion channels. The purpose of this study was to examine whether remifentanil inhibits L-type calcium channels (Ca.(L)) and provides dose-dependent effects on L-type calcium channel Ba(2+) currents (I(Ba.L)) in human mesenteric arterial smooth muscle cells. METHODS: Using the whole-cell patch-clamp method, an in depth analysis of the mechanism of the I(Ba.L) induced by remifentanil was performed in cells which were enzymatically isolated from human mesenteric arterial smooth muscle. Ten millimolars Ba(2+) was used to replace 1.5 mM Ca(2+) to increase the amplitude of the inward current through Ca(2+)channels. L-type calcium channel Ba(2+) was elicited during 50 msec depolarizing test pulses (150 msec duration) to +80 mV (10 mV increments) from a holding potential of -60 mV. The effects of remifentanil on Ca.(L) were observed at the following concentrations: 1.21, 4.84, and 19.4 nmol.L(-1) and were compared with control. RESULTS: Remifentanil produced a concentration-dependent block of I(Ba,L) with IC(50) values of 38.90 +/- 3.96 x 10(-3) micromol.L(-1). The L-type calcium channel blocker, nifedipine, antagonized these remifentanil-induced currents. Remifentanil, at all concentrations, shifted the maximum of the current-voltage relationship in the hyperpolarizing direction of I(Ba.L). CONCLUSION: Remifentanil significantly inhibits Ca.(L) channels in a concentration-dependent manner in human mesenteric arteriolar smooth muscle cells.     

The effects of ethanol on CA(2+) sensitivity in airway smooth muscle

Halothane and other volatile anesthetics relax air-way smooth muscle (ASM) in part by decreasing the amount of force produced for a given intracellular Ca(2+) concentration (the Ca(2+) sensitivity) during muscarinic receptor stimulation. To determine whether this is a unique property of the volatile anesthetics, we tested the hypothesis that ethanol, another compound with anesthetic properties, also inhibits calcium sensitization induced by muscarinic stimulation of ASM. A beta-escin permeabilized canine tracheal smooth muscle preparation was used. Ethanol was applied to permeabilized muscles stimulated with calcium in either the absence or presence of acetylcholine. In intact ASM, ethanol produced incomplete relaxation (approximately 40%) at concentrations up to 300 mM. Ethanol significantly increased Ca(2+) sensitivity both in the presence and the absence of muscarinic receptor stimulation. Although ethanol did not affect regulatory myosin light chain (rMLC) phosphorylation during stimulation with Ca(2+) alone, it decreased rMLC phosphorylation by Ca(2+) during muscarinic receptor stimulation. Ethanol, like volatile anesthetics, inhibits increases in rMLC phosphorylation produced by muscarinic receptor stimulation at constant [Ca(2+)](i). However, despite this inhibition, the net effect of ethanol is to increase Ca(2+) sensitivity (defined as the force maintained for a given [Ca(2+)](i)) by a mechanism that is independent of changes in rMLC phosphorylation. IMPLICATIONS: In permeabilized airway smooth muscle, ethanol, like volatile anesthetics, inhibits increases in regulatory protein phosphorylation caused by stimulation of the muscle when intracellular calcium concentration is constant. However, unlike volatile anesthetics, ethanol causes a net increase in force through a process not dependent on protein phosphorylation, an action favoring bronchoconstriction.     

Characterization of the spontaneous electrical and contractile activity of smooth muscle cells in the rat upper urinary tract.

PURPOSE: We morphologically and electrophysiologically identified the cells that generate the electrical activity underlying the peristaltic contractions of the rat upper urinary tract. MATERIALS AND METHODS: Electron microscopy and tension recording techniques were used to characterize the smooth muscle cells underlying spontaneous contractions in the wall of the rat ureter, and proximal and distal renal pelvis. Intracellular microelectrodes, containing 4% neurobiotin were used to record data from the cells of the renal pelvis, which were later viewed on a confocal microscope. RESULTS: Spontaneous myogenic contractions (average 22.3 +/- 2.2 minutes(-1)) originated in the proximal renal pelvis and propagated into the distal renal pelvis and ureter in 6 preparations. Smooth muscle cells in the renal pelvis and ureter were typical in appearance with greater than 85% of their sectional area containing clumped contractile filaments. In contrast, contractile fibrils occupied only 65% of the sectional area of the smooth muscle cells within the most proximal region of the renal pelvis (pelvicaliceal junction). In strips of the renal pelvis spindle shaped cells 83 to 200 microm. long fired spontaneous action potentials (6 minutes(-1)) consisting of an initial spike, a quiescent plateau phase and abrupt hyperpolarization to a peak diastolic potential of -60 mV. Other spindle shaped cells 94 to 112 microm. long displayed small membrane transients (15 minutes(-1)) 9 to 19 mV. in amplitude, firing from a diastolic potential of -40 mV. CONCLUSIONS: It is likely that the spontaneous contractile activity of the rat upper urinary tract arises from the discharge of action potentials in typical smooth muscle cells of the proximal renal pelvis that are directly driven by the spontaneous membrane oscillations of atypical smooth muscle cells.     

Antagonistic actions of halothane and sevoflurane on spontaneous Ca2+ release in rat ventricular myocytes

BACKGROUND: Halothane has been reported to sensitize Ca(2+) release from the sarcoplasmic reticulum (SR), which is thought to contribute to its initial positive inotropic effect. However, little is known about whether isoflurane or sevoflurane affect the SR Ca(2+) release process, which may contribute to the inotropic profile of these anesthetics. METHODS: Mild Ca(2+) overload was induced in isolated rat ventricular myocytes by increase of extracellular Ca(2+) to 2 mM. The resultant Ca(2+) transients due to spontaneous Ca(2+) release from the SR were detected optically (fura-2). Cells were exposed to 0.6 mM anesthetic for a period of 4 min, and the frequency and amplitude of spontaneous Ca(2+) transients were measured. RESULTS: Halothane caused a temporary threefold increase in frequency and decreased the amplitude (to 54% of control) of spontaneous Ca(2+) transients. Removal of halothane inhibited spontaneous Ca release before it returned to control. In contrast, sevoflurane initially inhibited frequency of Ca(2+) release (to 10% of control), whereas its removal induced a burst of spontaneous Ca(2+) release. Isoflurane had no significant effect on either frequency or amplitude of spontaneous Ca(2+) release on application or removal. Sevoflurane was able to ameliorate the effects of halothane on the frequency and amplitude of spontaneous Ca(2+) release both on application and wash-off. CONCLUSIONS: Application of halothane and removal of sevoflurane sensitize the SR Ca(2+) release process (and vice versa on removal). Sevoflurane reversed the effects of halothane, suggesting they may act at the same subcellular target on the SR.     

Remifentanil-induced mechanical responses and membrane potential changes in human umbilical arteries

BACKGROUND: The aim of this study was to evaluate the characteristic features of the mechanical responses and membrane potential changes induced by remifentanil in human umbilical arteries (HUAs). The ionic mechanisms underlying the electrophysiological responses were pharmacologically assessed using two K(+) channel blockers. METHODS: Thirty-eight HUAs were obtained. Contraction-relaxation, membrane potential changes and electrical responses of the HUAs were recorded. RESULTS: Remifentanil produced concentration-dependent relaxation in both endothelium-intact and endothelium-denuded HUA rings. Remifentanil produced a significantly greater relaxation response in intact than in denuded HUA rings. In endothelium-intact rings, pre-treatment with L-nitroarginine [N(w)-NITRO-(L)-ARGININE (L-NO-ARG)] or indomethacin decreased the degree of remifentanil-induced relaxation. Remifentanil (10(-9)-10(-6) mol/l) produced a transient concentration-dependent membrane hyperpolarization, which was not decreased by pre-treatment with L-NO-ARG or indomethacin. It also produced a small concentration-dependent hyperpolarization in the presence of charybdotoxin or tetraethylammonium. CONCLUSION: In both endothelium-intact and endothelium-denuded HUAs, remifentanil induces concentration-dependent vasorelaxation and simultaneously releases nitric oxide, prostaglandins and possibly an endothelium-derived hyperpolarizing factor. In addition, it produces hyperpolarization in a dose-dependent manner. Hyperpolarization induced by remifentanil involves the activation of Ca(2+)-dependent and Ca(2+)-independent potassium channels regulated by intracellular Ca(2+).     

Simultaneous recording of mechanical and intracellular electrical activity in human urinary bladder smooth muscle

OBJECTIVE: To elucidate the role of the membrane potential in human detrusor smooth muscle contraction, by simultaneously recording mechanical and intracellular electrical activity in muscle strips. Materials and methods The agonists acetylcholine and carbachol were applied to induce a contraction on muscarinic receptor stimulation; to block the response, atropine was added to the bath. The Ca2+ necessary for activating the contractile machinery can be recruited via two pathways: release from intracellular stores or influx from the extracellular matrix. High potassium was applied to induce Ca2+ influx through voltage-sensitive Ca2+ channels. RESULTS: There were significant changes in the force when agonist, antagonist and high potassium was administered. However, there were significant changes in membrane potential only when KCl was applied to the bath and not with muscarinic agonist or antagonist application. Activity in the form of spike potentials did not change significantly on applying any of the test substances. CONCLUSION: The present results indicate that the Ca2+ mobilized on M3 receptor stimulation originates primarily from intracellular stores, with no systematic changes in membrane potential. Atropine only caused a relaxation in muscle previously contracted by M3-receptor agonist stimulation; it had no effect on relaxed muscle strips.     

Differential effects of sevoflurane, isoflurane, and halothane on Ca2+ release from the sarcoplasmic reticulum of skeletal muscle.

BACKGROUND: Although malignant hyperthermia after application of sevoflurane has been reported, little is known about its action on intracellular calcium homeostasis of skeletal muscle. The authors compared the effect of sevoflurane with that of isoflurane and halothane on Ca2+ release of mammalian sarcoplasmic reticulum and applied a novel method to quantify Ca2+ turnover in permeabilized skeletal muscle fibers. METHODS: Liquid sevoflurane, isoflurane, and halothane at 0.6 mM, 3.5 mM, and 7.6 mm were diluted either in weakly calcium buffered solutions with no added Ca2+ (to monitor Ca2+ release) or in strongly Ca2+ buffered solutions with [Ca2+] values between 3 nM and 24.9 microm for [Ca+]-force relations. Measurements were taken on single saponin skinned muscle fiber preparations of BALB/c mice. Individual [Ca2+]force relations were characterized by the Ca2+ concentration at half-maximal force that indicates the sensitivity of the contractile proteins and by the steepness. Each force transient was transformed directly into a Ca2+ transient with respect to the individual [Ca2+]-force relation of the fiber. RESULTS: At 0.6 mM, single force transients induced by sevoflurane were lower compared with equimolar concentrations of isoflurane and halothane (P < 0.05). Similarly, calculated peak Ca2+ transients of sevoflurane were lower than those induced by equimolar halothane (P < 0.05). The Ca2+ concentrations at half maximal force were decreased after the addition of sevoflurane, isoflurane, and halothane in a concentration-dependent manner (P < 0.05). CONCLUSION: Whereas sevoflurane, isoflurane, and halothane similarly increase the Ca2+ sensitivity of the contractile apparatus in skeletal muscle fibers, 0.6 mM sevoflurane induces smaller Ca2+ releases from the sarcoplasmic reticulum than does equimolar halothane.     

Effects of protamine on vascular smooth muscle of rabbit mesenteric artery

Systemic hypotension is commonly observed in association with protamine administration after cardiopulmonary bypass. However, little information is available concerning the action of protamine on vascular smooth muscle. Thus, we investigated the action of protamine on vascular tissues using tension recording and microelectrode methods. Protamine (5-500 micrograms/ml) inhibited contractions induced by norepinephrine (NE)- or elevated K+ in a concentration-dependent manner in both endothelium-intact and -denuded strips. Protamine inhibition of NE contractions was less profound after endothelial denudation, whereas protamine inhibition of K(+)-induced contractions was less affected by prior denudation. In endothelium-intact strips, the protamine-induced inhibition was significantly reduced by inhibitors of the endothelium-derived relaxing factor pathway, including oxyhemoglobin, methylene blue, or NG-nitro-L-arginine, whereas the contractile inhibition was enhanced by superoxide dismutase. In endothelium-denuded strips, protamine inhibited Ca(2+)-induced contraction evoked in Ca(2+)-free solution containing 100 mM K+ and inhibited the NE-induced contraction under the following conditions: 1) in Ca(2+)-free solution; 2) after nifedipine treatment; and 3) after depletion of stored Ca2+ by A23187 or ryanodine. In membrane-permeabilized strips, protamine did not modify Ca(2+)-induced contraction. Protamine (50-500 micrograms/ml) did not modify the membrane potential of either endothelium-intact or -denuded strips. Furthermore, protamine irreversibly impaired acetylcholine-induced endothelium-dependent relaxant response, implying a toxic effect of protamine on the endothelium. We conclude that protamine exerts its inhibition on vascular smooth muscles in both an endothelium-dependent and -independent manner; i.e., the endothelium-dependent component is mediated probably by endothelium-derived relaxing factor, and direct smooth muscle effects are due to the inhibition of both Ca(2+)-influx and the NE-induced Ca2+ release from intracellular stores.