Electropharmacological actions of propofol on calcium current in guinea-pig ventricular myocytes

Propofol, a widely-used intravenous anesthetic, causes bradycardia, depression in contractility and hypotension. The cellular mechanisms responsible for these cardiac toxicity remain unclear. In this study, we examined the cellular electropharmacological actions of propofol on calcium current in guinea-pig heart. Single ventricular myocytes were freshly isolated from guinea-pig using modified enzymatic method. Whole-cell voltage-clamp technique was applied with one suction pipette. Transmembrane L-type calcium current (ICa(L)) was separated from other ionic currents by voltage-control, ionic channel blockers and ion substitution methods. Our results show that propofol decreased ICa(L) in a concentration-dependent manner (KD = 54.2 microM). Slope conductance of current-voltage relation was decreased by 56 microM propofol. Propofol did not affect the steady-state activation curve, but shifted the inactivation curve to hyperpolarizing direction. Recovery from inactivation was slowed down by propofol. Marked resting block and use-dependent block were noted. In conclusion, our results indicate that propofol inhibits cardiac L-type calcium current mainly by shifting inactivation curve and retarding the recovery from inactivation.     

Effects of hydroxyl radicals on KATP channels in guinea-pig ventricular myocytes

We studied the effects of oxygen free radicals on the ATP-sensitive potassium channel (KATP channel) of guinea-pig ventricular myocytes. Single KATP channel currents were recorded from inside-out patches in the presence of symmetrical K+ concentrations (140 mM in both bath and pipette solutions). Reaction of xanthine oxidase (0.1 U/ml) on hypoxanthine (0.5 mM) produced superoxide anions (.O2-) and hydrogen peroxide (H2O2). Exposure of the patch membrane to.O2- and H2O2 increased the opening of KATP channels, but this activation was prevented by adding 1 microM glibenclamide to the bath solution. In the presence of ferric iron (Fe3+: 0.1 mM), the same procedure produced hydroxyl radicals (.OH) via the iron-catalysed Haber-Weiss reaction.OH also activated KATP channels; however, this activation could not be prevented by, even very high concentrations of glibenclamide (10 microM). These different effects of glibenclamide suggest that the mode of action of these oxygen free radicals on KATP channels is different and that.OH is more potent than.O2-/H2O2 in activating KATP channels in the heart.     

The effects of propofol and enflurane on single calcium channel currents of guinea-pig isolated ventricular myocytes

1. The effects of the anaesthetics, propofol (100 microM) and enflurane (3%, 1.46 mM), on single L type calcium channel currents were investigated in single myocytes isolated from guinea-pig ventricles. Channel activity was recorded from membrane patches by use of the 'cell-attached' patch-clamp technique (pipette solution containing 110 mM BaCl2, 5 microM Bay K 8644, 5 microM HEPES, pH 7.4; temperature 36 degrees C). 2. Channel conductance was calculated from the slope of the relationship between single channel current and membrane potential during step depolarizations to activate the channel over a range of approximately -20 to +20 mV. Neither propofol (6 cells) nor enflurane (7 cells) caused any significant reduction in channel conductance. 3. Both propofol (7 cells) and enflurane (9 cells) decreased the probability of the channel being open during depolarizations to +10 mV (measured from histograms of the fraction of time spent by the channel at different current levels, taking areas under the Gaussian curves fitted to the open and closed components of the distributions to represent the proportion of time spent in the two states). 4. A fraction of the current traces showed no detectable channel openings in response to step depolarizations to +10 mV. Both propofol and enflurane significantly increased the fraction of silent traces. 5. Transitions across a threshold halfway between the open and closed levels were used to define periods spent in the open and closed states. Both propofol (7 cells) and enflurane (9 cells) reduced the mean open times and increased the mean closed times of the calcium channel. 6. Histograms were plotted showing the distributions of times spent by the channels in the open and closed states. Two exponentials were fitted to the open and closed time distributions. Both propofol (7 cells) and enflurane (9 cells) shortened both time constants fitted to the open times and lengthened both time constants fitted to the closed times.7. It is concluded that both propofol and enflurane appear to alter the kinetics of opening and closing of calcium channels to favour shut channels without altering channel conductance. This effect would be expected to result in a reduction of the macroscopic calcium current and thus contribute to the negative inotropic action of these anaesthetics.     

Gadolinium blocks the delayed rectifier potassium current in isolated guinea-pig ventricular myocytes

The effect of Gd3+ on the delayed rectifier potassium current (IK) in single guinea-pig ventricular myocytes was tested using whole-cell patch-clamp techniques. It was found that Gd3+ blocked 70% of the IK tail current at a concentration of 100 microM. The EC50 was 24 microM. Action potential durations were, however, reduced, consistent with a predominant effect on depolarizing L-type Ca2+ current (Ica.L). In the presence of 5 microM nifedipine Gd3+ prolonged the action potential. Using carbon fibres to stretch cells we observed that 10 microM Gd3+ was not effective in reducing a large stretch-activated increase in resting calcium. Modelling studies using the OXSOFT HEART program suggest that this lack of response is influenced by blockade of repolarizing current but is best reproduced by additional blockade of Ca2+ extrusion via the Na(+)-Ca2+ exchanger. When Gd3+ is used as a blocker of stretch-activated channels its actions upon both Ica.L and IK must therefore be accounted for.     

The effect of alterations to action potential duration on beta-adrenoceptor-mediated aftercontractions in human and guinea-pig ventricular myocytes

Aftercontractions induced by beta-adrenoceptor stimulation in human and guinea-pig cardiomyocytes may be related to changes in action potential duration (APD). We investigated the effects of altering APD during the occurrence of isoproterenol-induced aftercontractions, using the KATP channel openers cromakalim and lemakalim or the action potential voltage clamp technique, in guinea-pig and human ventricular cardiomyocytes. Contractile responses were measured at 32 degrees C using a video-based edge-detection system. In guinea-pig myocytes, action potentials, Indo-1 fluorescence and contraction were measured at 22 degrees C. Isoproterenol (< or = 12 nM) had variable effects on APD but induced aftercontractions, the majority (14/19 cells) of which occurred during the action potential. Short action potentials were produced using K+ channel openers. These compounds reduced or completely abolished the isoproterenol-induced aftercontractions. Increasing isoproterenol in the presence of K+ channel opener restored the main contraction to a level similar to or above those with isoproterenol alone, but without the reappearance of aftercontractions. When cells were stimulated to contract under action potential voltage clamp, isoproterenol-induced aftercontractions were abolished by voltage clamping with action potentials of short duration. It was possible to induce aftercontractions in some cells without application of isoproterenol if voltage clamp-imposed action potentials of very long duration were used. These aftercontractions were also abolished by shortening action potential duration. We conclude that K+ channel openers or the imposition of action potentials of short duration can dissociate positively inotropic beta-adrenoceptor stimulation from aftercontraction formation and that action potentials of long duration can be pro-arrhythmic.     

The role of oxygen free radicals in isoniazid-induced hepatotoxicity

This report describes the glycosaminoglycans, collagen and elastin--composition of leiomyosarcoma. Studies were performed on leiomyosarcoma removed during surgery. The material was taken from 7 patients. Rearrangement of extracellular matrix in leiomyosarcoma has been found. There was an increase of total collagen content in comparison to control uterus. In both tissues type I collagen was found to be the predominant one. Both tissues, normal and neoplastic contain all known glycosaminoglycans types. Among them heparan sulphate was found to be the most abundant. A slight decrease in the content of this glycosaminoglycan was observed in leiomyosarcoma. A possible role of these alterations in tumour biology is discussed.     

The voltage dependence of contraction at different stimulation rates in guinea-pig ventricular myocytes

Ventricular myocytes, isolated from the guinea-pig, were stimulated to contract by 100 ms long voltage clamp pulses from -80 to 0 mV at 0.5 and 3 Hz. An increase in frequency from 0.5 to 3 Hz led to a positive inotropic effect. Contraction-voltage relationships (CVR) were determined at each frequency. The CVR at 0.5 Hz was bell shaped and peaked between 0 and +20 mV, displaying a voltage dependence similar to the L-type Ca2+ current (ICa). At 3 Hz, contractions continued to increase at positive voltages, giving a more sigmoidal CVR. At 0.5 Hz, TTX reduced the size of steady-state contractions to 91 +/- 2% of control values, but had no effect on the shape of the CVR. At 3 Hz, TTX significantly reduced (P < 0.05) the magnitude of contractions at positive voltages (> or = +20 mV) but had no significant effect on contractions at voltages negative to 0 mV. These data illustrate that intracellular sodium activity (aNa(i)) and, in particular, Na+ entry due to the sodium current (INa) are important in determining the voltage dependence of contraction at positive voltages. Thapsigargin (2.5 microM), a blocker of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, reduced the size of steady-state contractions at 0 mV to 65 +/- 7% at 0.5 Hz. Increasing frequency to 3 Hz abolished the positive inotropy seen under control conditions. With thapsigargin present, contractions at 0.5 Hz were reduced at all potentials and the CVR was bell shaped. At 3 Hz the CVR was sigmoidal in shape. Contractions were significantly inhibited by thapsigargin at all potentials, but most significantly at more positive potentials (> or = +20 mV). These data show that, at normal body temperature, the shape of the CVR of guinea-pig ventricular myocytes changes with stimulation rate. Due to the voltage dependence of ICa, contractions evoked at positive voltages at 3 Hz must be supported by other mechanisms. The sensitivity of such contractions to TTX and thapsigargin suggests the involvement of both a Na(+)-dependent process and the SR. One possibility is that when aiNa and the Ca2+ content of the SR are raised at higher stimulation rates, enhanced Ca2+ entry via reverse Na(+)-Ca2+ exchange leads to a direct activation of the myofilaments and, to a lesser extent, the release of Ca2+ from the SR.     

Effect of oxygen free radicals on myosin in muscle fibres

The Palmaz-Schatz stent delivery system (PS 153) and "bare" PS 204 stents are relatively high-profile, rigid devices that can be difficult to deliver to lesions beyond tortuous, irregular, or rigid proximal segments. Described herein is a method of mounting and shaping a Palmaz-Schatz stent on a low-profile balloon that provides a steerable, low-profile, and secure stent delivery system. Also described is the successful use of this method in four consecutive cases where Palmaz-Schatz stents could not be delivered to the lesion site due to severely angulated, irregular, or rigid proximal vessel segments.     

The role of Gi-proteins and beta-adrenoceptors in the age-related decline of contraction in guinea-pig ventricular myocytes

A decline in contractility in myocytes from ageing guinea-pig hearts was demonstrated, which is more pronounced for maximum beta-adrenoceptor-stimulated activity than contraction in high Ca2+. In this study the role of the inhibitory G-proteins (Gi) in this process was investigated. Comparisons were made between young (Y, < 400 g, < 4 weeks), adult (A. > 600 g, > 8 weeks) and senescent guinea pigs (S, 58-65 weeks, 1136 +/- 30 g). Gi alpha activity, detected by pertussis toxin-catalysed ADP ribosylation, was significantly increased in senescent compared to young animals, but immunodetectable levels of Gi alpha were unchanged, beta-adrenoceptor number was decreased by 27% in senescent compared with young animals (P < 0.002). Pertussis toxin treatment increased the maximum response to isoproterenol in contacting myocytes so that there was no longer any significant decline with age. Maximum contraction amplitudes (sarcomere length change, micron) with isoproterenol before pertussis toxin were 0.144 +/- 0.011 (Y, n = 22 animals), 0.104 +/- 0.009 (A. 18) and 0.098 +/- 0.009 (S. 14), P < 0.01 by analysis of variance (ANOVA). Following toxin treatment amplitudes were 0.140 +/- 0.012 (Y. 12), 0.117 +/- 0.010 (A. 10) and 0.117 +/- 0.018 (S. 8), P = N.S. Pertussis toxin treatment also reversed the effects of ageing on contraction and relaxation velocity in isoproterenol. In contrast, the effect of age on contraction amplitude or velocity in maximum Ca2+ was more pronounced after toxin treatment. The EC50 value for isoproterenol increased with age: pertussis treatment decreased the EC50 in each group, but the effect was especially pronounced for senescent animals. There was no significant difference in the concentration-response curves for the negative inotropic effect of adenosine (in the presence of isoprotenerol) between the three age groups before toxin treatment. All effects of adenosine were abolished after pertussis exposure. We conclude that increased Gi alpha activity is likely to contribute to the decreased response to isoproterenol, but not to high Ca2+, in myocytes from ageing guinea-pigs.     

Biphasic effects of intrapipette cyclic guanosine monophosphate on L-type calcium current and contraction of guinea pig ventricular myocytes

The effects of intracellular cyclic guanosine monophosphate (cGMP) on L-type calcium current (lCa) and contraction of ventricular myocytes enzymatically isolated from guinea pig hearts were investigated to test the hypothesis that cGMP increases contractions along with ICa in these cells. ICa and contractions, elicited every 15 sec, were recorded simultaneously with a whole-cell voltage-clamp method and a video edge-detector, respectively. Cells were superfused with Tyrode's solution (22 degrees C); the pipette solution contained 120 mM potassium aspartate, 30 mM KCl, 4 mM ATP, 5 mM N-(2-hydroxyethyl)piperazine-N-(2-ethanesulfonic acid), 0.01 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and various concentrations of cGMP, which entered the cell interior through the patch electrode. In the presence of 3 nM isoproterenol (ISO) in the bath, ICa was increased 3.2-fold. ICa was further increased by 20% with 30 microM cGMP; cell contractions were also increased by 32%. When ICa was maximal in the presence of 30 nM ISO, cGMP no longer increased ICa or contractions, an indication that the effects of cGMP and ISO were additive. When ICa was increased maximally (4.3-fold) by 100 microM isobutylmethylxanthine, a nonselective phosphodiesterase inhibitor, application of 100 microM cGMP in the pipette decreased ICa by 53% and cell shortening by 64%. Cyclic GMP changed contraction in parallel with ICa in the presence of either ISO or isobutylmethylxanthine. 5'-GMP had no significant effect on ICa or contraction in the presence of ISO or isobutyl-methylxanthine. Cyclic GMP alone, at 30 microM, increased ICa by 25%; this effect on basal ICa was reversed by removal of cGMP from the pipette solution. We conclude that intracellular cGMP had two effects on ICa and contraction, namely, 1) an increase caused by an action on cGMP-inhibited phosphodiesterase and 2) a decrease attributed to activation of cGMP-dependent protein kinase.     

The thrombin receptor elevates intracellular calcium in adult rat ventricular myocytes

While there is evidence that thrombin receptor activation leads to contractile dysfunction and induces arrhythmias in ischemic/reperfused cardiac tissue, thrombin is variably reported to modulate intracellular calcium in cardiomyocytes. The present study demonstrates that thrombin receptor activation leads to a rise in intracellular calcium in adult ventricular myocytes and serves to reconcile previous discrepant findings. The thrombin receptor-derived agonist peptide (SFLLRN, a portion of the tethered ligand created by thrombin's proteolytic actions) increases cytosolic calcium and twitch amplitude in cardiomyocytes isolated from adult ventricles. The truncated control peptide FLLRN has no effect, establishing that the response to SFLLRN results from a specific agonist peptide-receptor interaction. However, the response to SFLLRN occurs only at high agonist peptide concentrations and thrombin itself is inactive. This result is not compatible with an action of SFLLRN at a distinct protease-activated receptor (PAR-2; which is activated by SFLLRN, but not by thrombin), since SLIGRL (a ligand which is selective for PAR-2, but not the thrombin receptor) has no effect. Rather, the enzyme-based cell isolation procedure may partially cleave the thrombin receptor and influence cell responses, since concentrations of SFLLRN which are sub-threshold in enzymatically disaggregated myocytes significantly increase the force of isometric contraction of intact rat papillary muscles. These studies provide the first evidence that thrombin receptor activation leads to a change in intracellular calcium and a positive inotropic response in adult ventricular myocardium.     

Comparison of cannabinoid binding sites in guinea-pig forebrain and small intestine

We have investigated the nature of cannabinoid receptors in guinea-pig small intestine by establishing whether this tissue contains cannabinoid receptors with similar binding properties to those of brain CB1 receptors. The cannabinoids used were the CB1-selective antagonist SR141716A, the CB2-selective antagonist SR144528, the novel cannabinoid receptor ligand, 6'-azidohex-2'-yne-delta8-tetrahydrocannabinol (O-1184), and the agonists CP55940, which binds equally well to CB1 and CB2 receptors, and WIN55212-2, which shows marginal CB2 selectivity. [3H]-CP55940 (1 nM) underwent extensive specific binding both to forebrain membranes (76.3%) and to membranes obtained by sucrose density gradient fractionation of homogenates of myenteric plexus-longitudinal muscle of guinea-pig small intestine (65.2%). Its binding capacity (Bmax) was higher in forebrain (4281 fmol mg(-1)) than in intestinal membranes (2092 fmol mg(-1)). However, the corresponding KD values were not significantly different from each other (2.29 and 1.75 nM respectively). Nor did the Ki values for its displacement by CP55940, WIN55212-2, O-1184, SR141716A and SR144528 from forebrain membranes (0.87, 4.15, 2.85, 5.32 and 371.9 respectively) differ significantly from the corresponding Ki values determined in experiments with intestinal membranes (0.99, 5.03, 3.16, 4.95 and 361.5 nM respectively). The Bmax values of [3H]-CP55940 and [3H]-SR141716A in forebrain membranes did not differ significantly from each other (4281 and 5658 fmol mg(-1)) but were both greater than the Bmax of [3H]-WIN55212-2 (2032 fmol mg(-1)). O-1184 (10 or 100 nM) produced parallel dextral shifts in the log concentration-response curves of WIN55212-2 and CP55940 for inhibition of electrically-evoked contractions of the myenteric plexus-longitudinal muscle preparation, its KD values being 0.20 nM (against WIN55212-2) and 0.89 nM (against CP55940). We conclude that cannabinoid binding sites in guinea-pig small intestine closely resemble CB1 binding sites of guinea-pig brain and that 0-1184 behaves as a cannabinoid receptor antagonist in the guinea-pig myenteric plexus-longitudinal muscle preparation.     

Purinergic facilitation of ATP-sensitive potassium current in rat ventricular myocytes

Patients who cannot be reperfused after thrombolytic therapy have a high mortality rate. Noninvasive clinical markers of reperfusion have been widely studied, yet their prognostic significance remains unclear. To assess the prognostic value of commonly used noninvasive clinical markers of early reperfusion we studied 327 patients who received intravenous thrombolytic treatment (1.5 MU streptokinase in 1 hour or 100 mg alteplase in 3 hours) within 6 hours of acute infarction. Successful clinical reperfusion (SCR) was defined as the presence of at least two of the following criteria at 2 hours after thrombolytic treatment: (1) significant relief of pain (a 5-point reduction on a 1 to 10 subjective scale), (2) > or =50% reduction of sum of ST segment elevation, and (3) abrupt initial increase of creatine kinase levels (more than twofold over the upper-normal or baseline elevated values). Clinical variables that were significantly associated by univariate analysis were tested by multivariate analysis to obtain independent predictors of 30-day mortality rate. SCR was present in 210 (64%) patients (group 1), and absent in 117 (36%) patients (group 2). The groups were similar for most baseline characteristics, although group 2 patients were slightly older (mean 60 vs 57 years, p < 0.02). Thirty-day outcomes for group 2 patients compared with group 1 patients were heart failure in 23.1% and 10.5% (p < 0.005), progression to cardiogenic shock in 12.8% and 0.5%, (p < 0.00001), and death in 16.2% and 3.8% (p < 0.0001), respectively. By multivariate analysis the Killip class at admission (p < 0.00001), the absence of SCR (p = 0.017), anterior infarct location (p = 0.021), and age (p = 0.03) were independent predictors of mortality rate, and sex (p = 0.051) had borderline significance. The absence of SCR defined a group of patients with significantly higher mortality rate (odds ratio 4.89, 95% confidence interval 2.07 to 11.57). Three simple noninvasive clinical criteria of successful reperfusion may be used to identify a group of patients with poor prognosis after thrombolytic therapy in whom alternative strategies could be applied.     

Regional differences in action potential characteristics and membrane currents of guinea-pig left ventricular myocytes

Regional differences in action potential characteristics and membrane currents were investigated in subendocardial, midmyocardial and subepicardial myocytes isolated from the left ventricular free wall of guinea-pig hearts. Action potential duration (APD) was dependent on the region of origin of the myocytes (P < 0.01, ANOVA). Mean action potential duration at 90 % repolarization (APD90) was 237 +/- 8 ms in subendocardial (n = 30 myocytes), 251 +/- 7 ms in midmyocardial (n = 30) and 204 +/- 7 ms in subepicardial myocytes (n = 36). L-type calcium current (ICa) density and background potassium current (IK1) density were similar in the three regions studied. Delayed rectifier current (IK) was measured as deactivating tail current, elicited on repolarization back to -45 mV after 2 s step depolarizations to test potentials ranging from -10 to +80 mV. Mean IK density (after a step to +80 mV) was larger in subepicardial myocytes (1.59 +/- 0.16 pA pF-1, n = 16) than in either subendocardial (1.16 +/- 0.12 pA pF-1, n = 17) or midmyocardial (1. 13 +/- 0.11 pA pF-1, n = 21) myocytes (P < 0.05, ANOVA). The La3+-insensitive current (IKs) elicited on repolarization back to -45 mV after a 250 ms step depolarization to +60 mV was similar in the three regions studied. The La3+-sensitive tail current, (IKr) was greater in subepicardial (0.50 +/- 0.04 pA pF-1, n = 11) than in subendocardial (0.25 +/- 0.05 pA pF-1, n = 9) or in midmyocardial myocytes (0.38 +/- 0.05 pA pF-1, n = 11, P < 0.05, ANOVA). The contribution of a Na+ background current to regional differences in APD was assessed by application of 0.1 microM tetrodotoxin (TTX). TTX-induced shortening of APD90 was greater in subendocardial myocytes (35.7 +/- 7.1 %, n = 11) than in midmyocardial (15.7 +/- 3. 8 %, n = 10) and subepicardial (20.2 +/- 4.3 %, n = 11) myocytes (P < 0.05, ANOVA). Regional differences in action potential characteristics between subendocardial, midmyocardial, and subepicardial myocytes isolated from guinea-pig left ventricle are attributable, at least in part, to differences in IK and Na+-dependent currents.     

Age-associated alterations in calcium current and its modulation in cardiac myocytes

Stimulatory effects of several types of adjuvants on secondary antibody response to inactivated Newcastle disease virus (iNDV) were examined in chickens. For this purpose, animals were primed with iNDV without adjuvant resulting in a low but significant antibody response, boosted with iNDV plus adjuvant 3 weeks later, and analysed for specific antibody titres in serum 3 weeks after the booster. Water-in-mineral oil emulsion (W/O) caused significant increase in antibody titres measured in an indirect enzyme-linked immunosorbent (ELISA), haemagglutination inhibition (HI), and virus neutralisation (VN) assay. The adjuvants tested included three oil-in-water emulsions (i.e. mineral oil-in-water, sulpholipo(SL)-Ficoll400/squalane-in-water and sulpholipo-cyclodextrin/squalane-in-water), three negatively-charged polymers with high molecular weight (i.e. polyacrylate, polystyrenesulphonate and sulpho(S)-Ficoll400) and two surface-active agents (i.e. dimethyldioctadecylammonium bromide (DDA) and Quil A). These adjuvants enhanced significantly the secondary immune response but none reached the titre obtained with W/O. Combinations of adjuvants with distinct physicochemical properties, i.e. polyacrylate and DDA revealed only slight, beneficial effects. We concluded that the various types of adjuvants tested can stimulate secondary immune responses in primed animals but that W/O is superior.     

Cytoplasmic sodium, calcium and free energy change of the Na+/Ca2+-exchanger in rat ventricular myocytes

The relationship between changing driving force of the Na+/Ca2+-exchanger (deltaG(exch)) and associated cytosolic calcium fluxes was studied in rat ventricular myocytes. DeltaG(exch) was abruptly reversed by the reduction of extracellular sodium ([Na+]o) with or without sustained depolarization by the elevation of potassium ([K+]o). Cytosolic sodium ([Na+]i) and calcium ([Ca2+]i) were measured with SBFI and indo-1 respectively and the time course of recovery of deltaG(exch) was calculated. Following abrupt reversal of deltaG(exch) from +4.1 to -9.2 kJ/mol [Na+]i exponentially decreased from 9.6-2.5 mmol/l (t(1/2) about 30 s) and [Ca2+]i transiently increased to a peak value after about 30 s. Negative values of deltaG(exch) were associated with an increase and positive values with a decrease of [Ca2+]i. Equilibrium (deltaG(exch) = 0) was reached after about 30 s coinciding with the time to peak [Ca2+]i. After 180 s deltaG(exch) reached a new steady state at +3.5 kJ/mol. Inhibition of SR with ryanodine or thapsigargin reduced the amplitude of the [Ca2+]i transient and shifted its peak to 80 s, but did not affect the time course of [Na+]i changes. In the presence of ryanodine or thapsigargin the time required for deltaG(exch) to recover to equilibrium was also shifted to 80 s. When we changed the deltaG(exch) to the same extent by the reduction of [Na+]o in combination with a sustained depolarization, [Na+]i decreased less and the amplitude of [Ca2+]i transient was much enhanced. This increase of [Ca2+]i was completely abolished by verapamil. DeltaG(exch) only recovered to a little above equilibrium (+1 kJ/mol). Inhibition of the Na+/K+-ATPase with ouabain entirely prevented the decrease of [Na+]i and caused a much larger increase of [Ca2+]i, which remained elevated; deltaG(exch) recovered to equilibrium and never returned to positive values. The rate of change of total cytosolic calcium was related to deltaG(exch), despite the fact that the calcium flux associated with the exchanger itself contributed only about 10%; SR related flux contributed by about 90% to the rate of change of total cytosolic calcium. In summary, reduction of [Na+]o causes reversal of the Na+/Ca2+-exchanger and its driving force deltaG(exch), a transient increase of [Ca2+]i and a decrease of [Na+]i. The influx of calcium associated with reversed deltaG(exch) triggers the release of calcium from SR. Both the decrease of [Na+]i and the increase of [Ca2+]i contribute to the recovery of deltaG(exch) to equilibrium. The time at which deltaG(exch) reaches equilibrium always coincides with the time to peak of [Ca2+]i transient. Activation of the Na+/K+-ATPase is required to reduce [Na+]i and recover deltaG(exch) to positive values in order to reduce [Ca2+]i. We conclude that deltaG(exch) is a major regulator of cytosolic calcium by interaction with SR.     

Rapid effects of cytochalasin-D on contraction and intracellular calcium in single rat ventricular myocytes

Contraction and intracellular calcium ([Ca2+]i) transients were recorded using a video edge detector and fluorescence spectrophotometry, respectively, in rat ventricular myocytes at 22-24 degreesC stimulated at a frequency of 1 Hz. Application of the F-actin disrupter cytochalasin-D (Cyt-D) caused a large reduction in the amplitude of contraction and a small increase in the [Ca2+]i transient. These responses began within a few seconds of application and were complete after 2 min of exposure. Phase-plane relationships of contraction and [Ca2+]i were consistent with cytochalasin-D causing a decrease in myofilament responsiveness to Ca2+.     

Effects of myosin light chain kinase inhibitors on carbachol-activated nonselective cationic current in guinea-pig gastric myocytes

The effects of myosin light chain kinase inhibitors on muscarinic stimulation-activated nonselective cationic current (ICCh) in guinea-pig gastric antral myocytes were studied using the whole-cell patch-clamp technique. ICCh was induced by carbachol (CCh, 50 microM) at a holding potential of -30 mV or -60 mV. ML-7, a chemical inhibitor of myosin light chain kinase (MLCK), inhibited ICCh concentration dependently in a reversible manner (53 +/- 8.6% at 1 microM, mean +/- SE, n = 11). In addition, amplitudes of ICCh were only 37 +/- 2.7% of the daily control values following the addition of a peptide inhibitor of MLCK to the pipette solution. On the other hand, ML-7 had an inhibitory effect on voltage-operated Ca2+ channel current. The peak value of Ba2+ current at 0 mV was reduced to 35 +/- 7.4% (n = 9) by 3 microM of ML-7. As ICCh is known to have an intracellular Ca2+ dependence, we tried to exclude the possibility that ML-7 inhibited ICCh indirectly via suppression of Ca2+ current and the similar inhibitory effects of ML-7 on ICCh were confirmed under the following conditions: (1) clamp of membrane potential at -60 mV; (2) clamp of intracellular [Ca2+] to 1 microM by 10 mM BAPTA; (3) pre-inhibition of Ca2+ channel by verapamil. Different from the effects on ICCh, ML-7 barely inhibited the same cationic current induced by guanosine 5'-O-(3-thiotriphosphate) (GTP[gammaS], 0.2 mM) in the pipette solution. These results suggest that a Ca2+/calmodulin-MLCK-dependent pathway can modulate the activation of ICCh in guinea-pig gastric antral myocytes.     

Fosinoprilate prolongs the action potential: reduction of iK and enhancement of the L-type calcium current in guinea pig ventricular myocytes

Changes in the gross and cellular morphology of the nucleus preopticus medianus (POMn) were measured in response to changes in photoperiod in adult male Japanese quail (Coturnix japonica). POMn volume and the soma size of a dorsolateral population of neurons within POMn decreased when birds were moved from long day housing conditions (16L,8D) to short day housing conditions (8L,16D), and then increased again when birds were moved back to long day conditions, presumably as a function of the changes in circulating testosterone that accompanied changing daylengths. Male Japanese quail exhibit sexual behavior only when housed under long day housing conditions that approximate the photoperiod of the spring/summer breeding season, and do not exhibit sexual behavior when housed under short day conditions characteristic of fall/winter. Because POMn is known to be critically involved in the expression of male copulatory behavior, these morphological changes in the adult brain likely represent key functional events associated with the seasonal regulation of sexual behavior in male Japanese quail.     

Hydrogen peroxide-induced stimulation of L-type calcium current in guinea pig ventricular myocytes and its inhibition by adenosine A1 receptor activation

Hydrogen peroxide (H2O2) produces complex cardiac effects that may involve altered calcium homeostasis. The cardiotoxic effects of H2O2 can be attenuated by adenosine A1 receptor agonists. The present study examined the effect of H2O2 on L-type Ca++ current (ICa,L) in guinea pig ventricular myocytes under two different recording conditions and the influence of adenosine receptor agonists. H2O2 (100 microM), did not have any significant effect on ICa,L, under conventional whole cell patch configuration. However, when recorded under nystatin perforated patch configuration, H2O2 caused a gradual and significant increase (84 +/- 14%) in ICa,L compared to control values. N6-cyclopentyladenosine (CPA), an adenosine A1 receptor agonist, significantly attenuated the effect of H2O2. The inhibitory effect of N6-cyclopentyladenosine was antagonized by 8cyclopentyl-1, 3-dipropylxanthine, an adenosine A1 receptor antagonist. The A2A and A3 receptor agonists, 2-p-(2-Carboxyethyl)phenethylamino-5'- N - ethylcarboxamidoadenosine (CGS-21680) and 1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-be ta-D-ribofuranuronamide, respectively, did not modulate the enhancement of ICa,L by H2O2. Moreover the effects of N6-cyclopentyladenosine were mimicked by the protein kinase C inhibitor bisindolylmaleimide. Thus, our results demonstrate a potent stimulatory effect of H2O2 on ICa,L in guinea pig ventricular myocytes. We further demonstrate that adenosine A1 receptor activation attenuates this effect. Our results suggest a potential basis for altered calcium homeostasis in response to H2O2 as well as the salutary effects of A1 receptor activation against H2O2-induced cardiotoxicity.