Jacques Noireaud

Abstract: We evaluated celiprolol-induced vasodilatation in aorta taken from 12-week-old spontaneously hypertensive rats (SHR) and the effect of AT(1) angiotensin II receptor antagonism on the vasodilatory action of celiprolol in Wistar Kyoto (WKY) rats and SHR. In WKY rats, the celiprolol-induced relaxation was greatly decreased in denuded aorta, and completely abolished in intact aorta by N(omega)-nitro-l-arginine methyl ester (l-NAME, 100 microM). In SHR, celiprolol-induced relaxation was reduced compared to WKY rats (E(max) (value obtained for the highest concentration, 300 microM)=39.1+ or - 3.78%, n=21 vs. 80.4 + or - 3% in WKY rats, n=10; P<0.0001). Endothelium removal or pre-treatment with l-NAME did not alter celiprolol-induced relaxation in SHR. In both strains, relaxation to celiprolol was decreased in the presence of nadolol (a beta(1)/beta(2)-adrenoceptor antagonist, 10 microM). N-[[3-[(2S)-2-hydroxy-3-[[2-[4-[(phenylsulfonyl)amino] phenyl]ethyl]amino] propoxy]phenyl]methyl]-acetamide (L748337, a beta(3)-adrenoceptor antagonist, 7 microM) had no effect. A 12-day treatment with candesartan cilexetil (an AT(1) receptor antagonist, 0.37 or 1mg/kg/day) reduced systolic blood pressure in both strains, but only improved relaxation to celiprolol in SHR, and only at the highest dose (E(max)=64.2+/-3.9%, n=10, P<0.0001 vs. SHR control). In both strains, local aortic AT(1) receptor antagonism with candesartan CV11974 (100 microM) had no effect. The endothelial beta(1)/beta(2) relaxation induced by celiprolol was therefore impaired in SHR aorta and AT(1) receptor antagonism improved the response to celiprolol, in conjunction with a reduction in blood pressure. This work highlights the need to analyse the potential benefit of a combination of celiprolol/AT(1) receptor antagonist in the treatment of hypertension.

Abstract: In experimental pharmacology, drug effect studies currently establish and analyse cumulative concentration-response curves (CCRC) under repeated measurements designs. Usually the CCRC parameters are estimated using the Hill's function in a nonlinear regression for independent data. The two-way analysis of variance is generally used to identify a statistical difference between the responses for two treatments but that analysis does not take into account the nonlinearity of the model and the heteroscedasticity (uneven distribution) of the data. We presently tested the possibility of finding a statistical solution for the nonlinear response in repeated measurements data using the nonlinear mixed effects (nlme) models.

Abstract: To investigate the role of superoxide anions in the lipopolysaccharide (LPS)-induced impairment of beta-adrenoceptor-mediated equine digital vein (EDV) vasodilation.

Abstract: The beta(3)-adrenoceptors (beta(3)-ARs) have been identified and characterized in the human heart. Specific beta(3)-AR stimulation, unlike beta(1)-AR or beta(2)-AR stimulation, decreases cardiac contractility, partly via the G(i)-NO pathway. However, the precise role of cardiac beta(3)-ARs is not yet completely understood. Indeed, under normal conditions, the beta(3)-AR response is present only to a very low degree in rats and mice. Therefore, we evaluated whether beta(3)-ARs were present and functional in rabbit ventricular cardiomyocytes, and whether the rabbit could serve as a relevant model for the study of cardiac beta(3)-ARs. We used RT-PCR and Western blot to measure the beta(3)-AR transcripts and protein levels in rabbit ventricular cardiomyocytes. We also analysed the effect of beta(3)-AR stimulation using isoproterenol in combination with nadolol or SR 58611A on cardiomyocyte shortening, Ca(2+) transient, L-type Ca(2+) current (I(Ca,L)), delayed rectifier potassium current (I(Ks)) and action potential duration (APD). For the first time, we show that beta(3)-ARs are expressed in rabbit ventricular cardiomyocytes. The mRNA and protein sequences present a high homology to those of rat and human beta(3)-ARs. Furthermore, beta(3)-AR stimulation decreases cardiomyocyte shortening, Ca(2+) transient and I(Ca,L) amplitudes, via a G(i)-NO pathway. Importantly, beta(3)-AR stimulation enhances I(Ks) amplitude and shortens the APD. Taken together, our results indicate that the rabbit provides a relevant model, easily used in laboratories, to study the roles of cardiac beta(3)-ARs in physiological conditions.

Abstract: CGP12177 is a non-conventional partial agonist, known to have cardiostimulating and vasorelaxant properties related to its agonist action on the low affinity state of the beta(1)-adrenoceptor (beta(1LA)-adrenoceptor). In normotensive Wistar Kyoto (WKY) rats and spontaneously hypertensive rats (SHR), CGP12177-induced vasorelaxant effects were analysed in hindquarter vessels to assess modifications in hind limb vascular resistance, and in femoral artery rings. The global haemodynamic effects induced by CGP12177 were also investigated using telemetry in conscious animals. In hindquarters vasculature precontracted with 5-hydroxytryptamine, CGP12177 (0.16 to 475 microg) produced a similar dose-dependent decrease in hindquarters perfusion pressure in both strains. Vasorelaxation was not modified by nadolol, a beta(1) and beta(2)-adrenoreceptor antagonist, nor by L748337, a beta(3)-adrenoceptor antagonist, but was concentration dependently inhibited by bupranolol, a beta(1LA)-adrenoceptor antagonist at high concentrations. In femoral artery rings from WKY rats and SHR, CGP12177 produced a concentration-dependent relaxation, which was unaffected by nitric oxide synthases inhibition but was significantly reduced in the presence of bupranolol. With double cardiac autonomic blockade (atropine plus atenolol) in conscious WKY rats and SHR, CGP12177 greatly increased heart rate with minor changes in mean arterial pressure in both strains. Conversely, in the absence of double cardiac autonomic blockade, the amplitude of CGP12177-induced heart rate increase was less pronounced and had an hypotensive effect. The reduction in tachycardia and the hypotension were significantly greater in SHR compared to WKY rats. In conclusion, in both strains, CGP12177 produced vasodilating effects in hindquarter vessels and femoral arteries that can be attributed to a beta(1LA)-adrenoceptor stimulation. In conscious WKY rats and SHR, CGP12177-induced cardiostimulation and hypotension were not significantly different after baroreflex blockade, but were decreased and increased respectively, in the presence of baroreflex activity.

Abstract: Nebivolol, a selective beta-adrenoceptor (beta1-AR) antagonist, induces vasodilatation by an endothelium- and NO-cGMP-dependent pathway. However, the mechanisms involved in the vascular effect of nebivolol have not been established. Thus, we evaluated the role of alpha1 and beta3-ARs in nebivolol-induced vasodilatation. The responses to nebivolol were investigated in vitro in thoracic aortic rings isolated from male Sprague-Dawley rats. Nebivolol (0.1-10 microM) significantly shifted the concentration-response curve to phenylephrine, an alpha1-AR agonist, to the right in a concentration-dependent manner (pA2 = 6.5). Conversely, the concentration-response curve to endothelin 1 (ET1) was unaffected by nebivolol. In ET1-precontracted rings, nebivolol induced a concentration-dependent relaxation, which was unaffected by nadolol (a beta1/beta2-AR antagonist) but was significantly reduced by L-748,337 (a beta3-AR antagonist), endothelium removal or pretreatment with L-NMMA (an NOS inhibitor). Similar results were obtained with a beta3-AR agonist, SR 58611A. It was concluded that, in rat aorta, nebivolol-induced relaxation results from both inhibition of alpha1-ARs and activation of beta3-ARs. In addition, we confirmed that the endothelium and the NO pathway are involved in the vascular effect of nebivolol. The identification of these vascular targets of nebivolol indicate that it has therapeutic potential for the treatment of pathological conditions associated with an elevation of sympathetic tone, such as heart failure and hypertension.

Abstract: The voltage-sensitive Na(+) channel Na(v)1.5 plays a crucial role in generating and propagating the cardiac action potential and its dysfunction promotes cardiac arrhythmias. The channel takes part into a large molecular complex containing regulatory proteins. Thus, factors that modulate its biosynthesis, localization, activity, and/or degradation are of great interest from both a physiological and pathological standpoint. Using a yeast 2-hybrid screen, we unveiled a novel partner, 14-3-3eta, interacting with the Na(v)1.5 cytoplasmic I interdomain. The interaction was confirmed by coimmunoprecipitation of 14-3-3 and full-length Na(v)1.5 both in COS-7 cells expressing recombinant Na(v)1.5 and in mouse cardiac myocytes. Using immunocytochemistry, we also found that 14-3-3 and Na(v)1.5 colocalized at the intercalated discs. We tested the functional link between Na(v)1.5 and 14-3-3eta using the whole-cell patch-clamp configuration. Coexpressing Na(v)1.5, the beta1 subunit and 14-3-3eta induced a negative shift in the inactivation curve of the Na(+) current, a delayed recovery from inactivation, but no changes in the activation curve or in the current density. The negative shift was reversed, and the recovery from inactivation was normalized by overexpressing the Na(v)1.5 cytoplasmic I interdomain interacting with 14-3-3eta. Reversal was also obtained with the dominant negative R56,60A 14-3-3eta mutant, suggesting that dimerization of 14-3-3 is needed for current regulation. Computer simulations suggest that the absence of 14-3-3 could exert proarrhythmic effects on cardiac electrical restitution properties. Based on these findings, we propose that the 14-3-3 protein is a novel component of the cardiac Na(+) channel acting as a cofactor for the regulation of the cardiac Na(+) current.

Abstract: The aim of the present study was to analyze whether beta3-adrenoceptors (beta3-ARs) were effectively present and functional in the human internal mammary artery (IMA).

Abstract: Knowledge of the sympathetic system is a basic element in the understanding of numerous physiological and physiopathological phenomena. In the two last decades, new pharmacological, biochemical and molecular tools have changed our approach to the roles of beta-adrenoceptors in the cardiovascular system. In the heart, the positive inotropic effect of predominant beta 1-adrenoceptor stimulation is classically recognised. Several studies reveal a significant physiological relevance of the beta 2-adrenoceptor which could activate different signalling pathways in addition to that of cAMP. Moreover, the detection of a third beta-adrenoceptor subtype, beta 3, in human heart, responsible for a negative inotropic effect through a NO signalling pathway, has changed the classically admitted paradigm on the regulation of heart function by the beta-adrenergic system. The identification of atypical beta-adrenoceptors, based on pharmacological tools, led to the discovery of "putative" beta 4-adrenoceptors, which constituted a low affinity state of the beta 1-adrenoceptors. In vessels, all beta-adrenoceptors subtypes, beta 1, beta 2 and beta 3, mediated a vasodilation, but the signalling pathway involved in this effect was variable according to their localization (endothelial or smooth muscle cells), the species and the vascular bed. beta-adrenoceptors are involved in several cardiovascular disease and could constitute a determinant therapeutic target. The efficiency of some beta-blockers used in the treatment of heart failure could result from action on beta 3-adrenoceptors. Moreover, a mutation of the beta-adrenoceptor subtype suggested a role in hypertension and diabetes mellitus.

Abstract: Protein kinase C (PKC) is thought to be involved in the regulation of the mammalian cardiac excitation-contraction coupling process by vasoactive peptides like endothelin-1 (ET-1). However, the demonstration of a causal link between activation of specific PKC isoforms and the increase in contractility mediated by ET-1 is still inferential.

Abstract: Investigations of the coupling between contraction and relaxation (contraction-relaxation [CRC] process) in isometric conditions are essential in determining whether pharmacologic interventions or cardiac diseases specifically modify isometric relaxation (intrinsic lusitropic effect) or change it in proportion with the accompanying changes in contractility (or inotropy). For this purpose, the CRC process is quantified by various indexes, derived from differentiation and/or curve fitting the whole or relaxation phase of the isometric twitch, one of the most used being tau, the time constant of the final iso(volu)metric phase of relaxation. Nevertheless, the possible redundancy and validity of such indexes have not been thoroughly investigated. Accordingly, we performed a pharmacologic evaluation of such indexes in isolated rabbit ventricular muscles isometrically contracting in vitro, using modifiers of either intracellular Ca(2)+ handling (nifedipine, ryanodine, 2,5-di-tert-butyl-benzohydroquinone, all negative inotropic compounds, and BAY K 8644, a positive inotropic drug), or myofibrillar Ca(2)+ sensitivity (CGP 48506, a Ca(2)+ sensitizer, and butanedione monoxime, a Ca(2)+ desensitizer, respectively positive and negative inotropic compounds). The isometric twitch in control conditions and in the presence of increasing concentration of each compound was analyzed to determine the classically used CRC and/or lusitropic indexes, derived either from single parameters such as the maximal rate or contraction and relaxation (+dT(max) and -dT(max), respectively), or from curve fitting of the whole, or part, of the twitch. As the rate of isometric relaxation is dependent on myofilament properties, we expected that compounds modifying myofibrillar Ca(2)+ sensitivity in an opposite direction (CGP 48506 vs butanedione monoxime) would be the only drugs exerting an intrinsic lusitropic and opposite effect on a validated CRC index. Results showed that (1) none of the tested compounds affected the slope of the linear relationship between peak twitch tension and dT(max), a previously assumed CRC index, sensitive only to myofibrillar Ca(2)+ sensitivity modifiers; (2) the lusitropic parameter B, derived from mathematical curve fitting of the whole isometric twitch, and the ratio +dT(max)/dT(max), exhibited similar drug- and dose-dependency, but no opposite sensitivity to CGP 48506 and BDM for either index; and (3) negative inotropic compounds dose-dependently slowed relaxation (and conversely for positive inotropes), whether the latter was quantified by the rate constant beta, derived from double exponential curve fitting of the whole relaxation phase, or by the time constants tau(L) and tau(E), derived from the curve fitting (logistic and monoexponential, respectively) of the final phase of relaxation. Nevertheless, the pharmacologicly induced changes in beta were statistically significant at lower concentrations and exhibited less individual variability, compared with the time constants. We demonstrate that intrinsic lusitropic changes can be quantified by the value of the slope of the relationship relating beta to peak isometric tension: the slope value was unchanged by Ca(2)+ handling modifiers, decreased by CGP 48506, and reversed by BDM (indicating number, negative, and positive intrinsic lusitropic effects respectively). Based on these data, we propose that the linear relationship between beta and peak isometric tension could be used a new method to assess whether pharmacologic interventions or cardiac diseases exert intrinsic effects on isometric relaxation.

Abstract: During postnatal development of extensor digitorum longus (EDL) muscle, sodium withdrawal contractures were observed during the first 6 days after birth, and not after this time. In regenerating EDL muscles, zero-Na contractures were demonstrated: (1) 7 days after bupivacaine injection, but not 14 or 90 days after this injection; (2) 7, 14, and 90 days after autotransplantation; and (3) 7, 14, and 90 days after the intervention in sliced muscles. The present findings emphasize the role of the denervation in the development of zero-Na contractures in the regenerating muscles and suggest that a calcium-sodium exchange across the sarcolemma may appear in these muscles.

Abstract: In the present study we have investigated the involvement of sensitized mice immunoglobulins and some electrophysiological alterations that participate to the antigenic sensitization-induced hyperreactivity of isolated mouse vas deferens. Active sensitization was performed by subcutaneous injection of egg albumen. Contractile responses to noradrenaline were isometrically recorded in the isolated vas deferens. Low external Na(+)-induced contractions and rapid cooling contractures were evaluated. Resting membrane potential (Er) and intracellular Na activity were measured in control and actively sensitized vas deferens by using conventional KCl-filled and Na(+)-sensitive microelectrodes respectively. Active sensitization-induced hyperreactivity to noradrenaline was reproduced by in vitro passive sensitization of control vas deferens with sensitized mice immunoglobulins. The inhibition of the nitric oxide synthesis by N-nitro-L-arginine methyl ester (L-NAME) did not change control vas deferens reactivity in vitro to noradrenaline and acetylcholine. Rapid cooling contractures, performed after lowering external Na+ concentration, were not altered by active sensitization. However, sensitization increased significantly the strength of the low external Na+-induced contractions. In control vas deferens Er was a mean of -49.2+/-0.3 mV (mean+/-SEM). Sensitization resulted in reduction of Er by 14 mV. In sensitized preparations, relative insensitivity of Er to ouabain, external K+ removal and cooling were observed. The intracellular Na+ activity was increased by about 40% in sensitized vas deferens. It is concluded that sensitization-induced hyperreactivity is mediated by immunoglobulins and produced smooth muscle cells depolarisation. The low Er of sensitized muscle may be partly the result of an increase in membrane permeability to Na+ which could interfere with intracellular Ca2+ homeostasis.

Abstract: Sarcoplasmic reticulum (SR) calcium handling in diaphragm was compared between mdx mice (7-8 weeks old) and age-matched controls. The total SR Ca2+ load was released from the SR by rapidly cooling muscle bundles from 22 to -1 degree C. The plateau amplitude of the rapid cooling contracture (RCC) was considered as an index of the SR Ca2+ content. The steady-state RCC amplitude was significantly lower by 50% in mdx bundles mainly because of a decreased capacity of the dystrophic diaphragm to generate maximal tension. There was no significant difference between either RCC time to peak or the time to half-relaxation of the transient, spike-like, contractile response induced by muscle rewarming. The recovery process of RCC was studied by using a paired RCC protocol. In both groups, at the shortest interval (10 s) between two RCCs, the amplitude of the second RCC was decreased by 25% compared with the first RCC. Increasing the time interval led to progressive monoexponential recovery of the second RCC with similar time constants in control and mdx diaphragm. These results indicate that the dystrophic process does not significantly alter SR Ca2+ uptake nor Ca2+ redistribution within the muscular cell.

Abstract: Smooth muscles hyperresponsiveness is a common feature in anaphylaxis and allergic diseases. The aim of the present work was to investigate whether the enhanced reactivity of sensitized guinea-pig vas deferens was associated with changes in the resting membrane potential (Er) of the smooth muscle cells. Active sensitization was performed by subcutaneous injection of egg albumen. Er was measured in vitro in isolated vas deferens with conventional KCl-filled microelectrodes. Quantification of [3H]ouabain binding sites, measurements of 86Rb efflux, and measurements of Na and K contents were also performed. In normal physiological solution, at 35 degrees C, Er was a mean of -54.1+/-0.3 mV (mean +/- SEM) in control vas deferens. Sensitization resulted in depolarizing Er by about 7 mV. In control and sensitized preparations, the 3H-ouabain binding site concentration, the efflux of 86Rb, and the K content were similar. In guinea-pig vas deferens, active sensitization induced a partial depolarization of the resting membrane potential of the smooth muscle cells, which did not result from a downregulation of Na+ -K+ pump sites.

Abstract: One of the the major properties of mature skeletal muscle is its ability to regenerate after injury. The purpose of the present study was to determine whether the expression of genes encoding the dihydropyridine receptor calcium channel (DHPR) and the ryanodine receptor (RyR), which play a critical role in excitation-contraction coupling, is regulated by skeletal muscle regeneration. The process of regeneration was induced by bupivacaine injection in surgically exposed rat extensor digitorum longus (EDL) muscle. After total RNA isolation from the injected and the contralateral control EDL muscles performed 3, 7, 15 and 30 days following injection, Northern blot and RNase protection assays were carried out with four cDNA probes specific for the skeletal and cardiac muscle isoforms of both the DHPR alpha1-subunit and the RyR. After 3 days, an initial precipitous decrease in the expression of the genes encoding the skeletal muscle isoforms of the DHPR and RyR was observed, followed by an increase. Moreover, regenerating skeletal muscle transiently expressed mRNA for the DHPR cardiac isoform, mainly at the beginning of regeneration. No expression of mRNA for the cardiac RyR was observed. Contraction experiments, performed using EDL muscle at the same times after bupivacaine injection, showed that twitch amplitude was markedly decreased in the absence of external calcium, but only during the early stages of regeneration. Similar findings in relation to expression of skeletal and cardiac muscle DHPR message were previously reported from experiments conducted during early developmental stages using fetal skeletal muscle and muscle cell cultures [Chaudhari N, Beam KG (1993) Dev Biol 155:507-515]. These results suggest that expression of the DHPR cardiac isoform in skeletal muscle could explain certain cardiac-like aspects of excitation-contraction coupling of regenerating skeletal muscle and developing skeletal muscle as well.

Abstract: Contractile responses were studied in autotransplanted and sliced extensor digitorum longus (EDL) muscles at different times after the surgical intervention (3 and 12 months). The contraction of autotransplanted and sliced EDL muscles remained strongly dependent upon the extracellular Ca2+ concentration ([Ca]o) 3 and even 12 months after the intervention. It could be suggested that in autotransplanted and sliced EDL muscles, neural control could be involved in the persistent dependence of contractility toward [Ca]o.

Abstract: Using an isolated ferret heart preparation (Langendorff perfusion, perfusion pressure 90 mmHg), energy metabolism has been characterized in right and left ventricles from control and hypertrophied hearts. Hypertrophy was induced by pulmonary artery clipping for 30-45 days (right ventricle wall weight/body weight ratio increased by 70%). Myocardial contents of high energy phosphate compounds, glycogen and lactate, and the activities of some enzymes were biochemically measured in perfused hearts and also after ischemic arrest (30 min global ischemia). In hypertrophied right ventricles, PCr (-46%), Cr (-34%) levels, creatine kinase activity (-18%) were significantly decreased compared with control. ATP and Pi levels were not affected by hypertrophy. The adenylate energy charges were similar (0.85-0.86) in both types of heart. The activities of hexokinase (+26%), aldolase (+212%), pyruvate kinase (+14%) and glucose 6-phosphate dehydrogenase (+107%) were increased by hypertrophy. The LDH isozyme pattern was significantly changed such that LDH3 was decreased by 11%, and LDH4 and LDH5 were increased by a factor 1.4 and 2.9 respectively in hypertrophy. After 30 min of global ischemia, PCr level was decreased by 89 and 79% in control and hypertrophied ventricles respectively. ATP level was depressed by 41 in control and only by 21% in hypertrophied muscles. Altogether, the present data suggested that, in the adult ferret heart, the capacity for the ATP synthesis could be maintained during hypertrophy by the enhancement of the glycolytic pathway. The smaller decline of ATP after ischemia in hypertrophied tissue could be explained by a lower consumption of ATP in the hypertrophied compared to the control heart during the earliest period of ischemia.

Abstract: Following disruption of the nerve supply, extensor digitorum longus (EDL) and soleus (SOL) muscles in rats are known to exhibit alterations in excitation-contraction coupling. After total RNA isolation from the denervated and the contralateral control muscles performed at 25 and 50 days following denervation, RNase protection assays were carried out with four cDNA probes specific for the skeletal and cardiac isoforms of both the DHPR alpha 1-subunit and the RyR. Longterm denervation increased the expression of the mRNA for skeletal DHPR and skeletal RyR in SOL muscle, but it also significantly increased the expression of the mRNA for the cardiac isoform of the DHPR alpha 1 subunit in EDL muscle.

Abstract: The expression of isoform-specific dihydropyrine receptor-calcium channel (DHPR) alpha 1-subunit genes was investigated in mdx and control mouse diaphragm (DIA) and tibialis anterior (TA). RNase protection assays were carried out with a rat DHPR cDNA probe specific for skeletal muscle and a mouse DHPR cDNA probe specific for cardiac muscle. The level of expression of the gene encoding the cardiac DHPR was very weak in TA muscle from both control and mdx mice. Compared to TA, DIA expressed mRNA for the cardiac isoform at significantly higher levels, but mdx and control mouse DIA levels were similar to one another. In contrast, mRNA expression levels for the DHPR skeletal muscle isoform were lower in control DIA than TA. However, there was a dramatic increase in the expression for the DHPR skeletal muscle isoform in mdx DIA compared with control DIA, reaching the TA expression level, whereas dystrophy did not affect TA expression. [3H]-PN200-110 binding was used to further assess DIA DHPR expression at the protein level. The density of binding sites for the probe was not significantly affected in DIA muscles of mdx vs. control mice, but it was reduced in older mdx and control mice. The increase in DHPR mRNA levels without a consequent increase in DHPR protein expression could be secondary to possible enhanced protein degradation which occurs in mdx DIA. The altered DHPR expression levels found here do not appear to be responsible for the severe deficits in contractile function of the mdx DIA.

Abstract: The effects of perchlorate (1-20 mM) on myofibrillar calcium responsiveness have been tested in Triton X-100-skinned fibre bundles from rat soleus (slow-twitch) and extensor digitorum longus (fast-twitch) skeletal muscles. In extensor digitorum longus and soleus, perchlorate dose-dependently shifted the pCa (-log[Ca2+])/tension relationship towards lower free calcium concentration (sensitizing effect) and maximal tension was unchanged. The degree of sensitization was greater in extensor digitorum longus than in soleus bundles. Reversibility after exposure to 12 mM perchlorate was complete in soleus but not in extensor digitorum longus muscles. In fact, the 'return' pCa/tension relationship in extensor digitorum longus was shifted to higher free calcium concentration (desensitizing effect) compared with control. Perchlorate (12 mM) also enhanced myofibrillar calcium responsiveness of frog semitendinosus skinned skeletal fibres. Assuming a passive distribution of perchlorate across the sarcolemma, this sensitizing effect is probably not involved in perchlorate-induced potentiation of contractile responses of intact muscles and thereby supports the specificity of perchlorate as an agonist of the excitation/calcium release sequence in skeletal muscle fibres.

Abstract: To answer the questions: (a) What is the effect of hypertrophy on the intracellular pH (pHi) and buffering power of cardiac muscle, and (b) How does hypertrophy affect the ability of cardiac muscle to recover from intracellular acidosis induced by hypoxia.

Abstract: 1. The effects of 2,5 di-(tert-butyl)-1,4-benzohydroquinone (TBQ), a putative inhibitor of the sarcoplasmic reticulum Ca2+ pump, on mechanical relaxation and contraction-relaxation coupling have been studied at different frequencies (0.5-3 Hz) in isometrically contracting isolated right ventricular preparations of rabbit and rat at 37 degrees C. Two types of mechanical responses have been studied: the twitch tension and the force transient (rewarming spike, RSp) following a rapid cooling contracture (RCC, an index of sarcoplasmic reticulum Ca2+ content) on return to 37 degrees C. 2. The coupling between contraction and relaxation was assessed by two methods: (a) by evaluation of the variation of the slope relating the maximal rate of tension fall to twitch peak tension; (b) by modelling the twitch according to the following equation: TwT (t) = C x (t/A)B x exp(1-(t/AB) where TwT(t) is the time course of isometric tension, t is time, C and A are an inotropic and a chronotropic index respectively and B, a contraction-relaxation coupling index (Nwasokwa, 1993). 3. In the rabbit ventricle, 30 microM TBQ did not prevent the frequency-induced shortening of the twitch time to half-relaxation (t1/2) and of the time constant (tau) describing the final part of the RSp relaxation (tau decreased from 140 ms (0.5 Hz) to 133 ms (3 Hz) in control and from 253 ms (0.5 Hz) to 197 ms (3 Hz) after exposure to TBQ). By contrast, at a given frequency, the prolongation of relaxation induced by TBQ was proportional to its inotropic effect (unchanged slopes and B values) but TBQ did not prevent the acceleration of relaxation observed at high frequencies: B increased from 2.02 (0.5 Hz) to a peak value of 2.18 (1 Hz) in control and from 1.88 (0.5 Hz) to a maximum of 2.48 (2 Hz) after TBQ exposure. TBQ significantly attenuated the decay of RCCs elicited after increasingly longer periods of muscle quiescence as normally observed in control conditions. 4. In the rat ventricle, TBQ depressed relaxation more than expected on the basis of its negative inotropic effects (B decreased from 2.16 to 1.84 at 0.5 Hz and from 2.15 to 1.66 at 3 Hz). TBQ also slowed the rate of RSp relaxation (tau increased from 95 ms to 168 ms at 0.5 Hz, and from 109 ms to 149 ms at 3 Hz) and increased twitch t1/2. By contrast with the results obtained in the rabbit ventricle, B, tau and t1/2 were frequency-insensitive whether or not TBQ was present. 5. TBQ exerts negative inotropic effects consistent with inhibition of the SR Ca2+ pump. In the rabbit ventricle, the TBQ-induced potentiation of relaxation acceleration at high pacing frequencies suggests the involvement of counteracting Ca(2+)-mediated mechanisms probably via Ca(2+)-calmodulin-activated kinases. In the rat ventricle, TBQ did not have any differential effect on relaxation depending on the frequency, probably because the extent of the negative staircase was small in the present experimental conditions.

Abstract: 1. The effects of 2,5 di-(tert-butyl)-1,4-benzohydroquinone (TBQ), a putative inhibitor of the sarcoplasmic reticulum (SR) Ca2+ pump, on twitch tension, time course and SR Ca2+ content have been studied at different stimulation frequencies (0.5-3 Hz) in isolated preparations from the rabbit and rat right ventricle, at 37 degrees C. 2. At 0.5Hz, 30 microM TBQ induced a marked negative inotropic effect in both species (-57% in the rabbit and -68% in the rat) and decreased the rate of rise and fall of twitch tension. In parallel, SR Ca2+ content (assessed by rapid cooling contractures) was depressed in the rabbit by 42%. The force-frequency relationship (positive for the rabbit and negative for the rat) was significantly attenuated. In the rabbit, this alteration was shown to rely on insufficient SR Ca2+ reloading with increasing frequencies. 3. Exposure of TBQ-treated preparations to 8 mM extracellular Ca2+ or 5 microM isoprenaline were effective in reloading the SR with Ca2+ whereas 20 mM caffeine emptied this compartment. 4. In the rabbit ventricle, increase in stimulation frequency shortened control twitch time course by decreasing both the time to peak tension (TTP) and the time to half relaxation (t1/2). TBQ did not differentially affect the pattern for t1/2 but significantly attenuated the frequency-induced decrease of TTP. 5. In rabbit ventricular muscle, the action potential duration increased between 0.5 and 3 Hz whether or not TBQ was present. However, TBQ induced a small but significant additional action potential shortening. 6. TBQ decreased twitch tension in the rat ventricle between 0.5 and 3 Hz but the negative staircase was not differentially affected by the SR Ca2+ pump inhibitor. In control conditions and in the presence of 30 microM TBQ, t1/2 was frequency-independent but TBQ consistently increased this parameter (by approximately 29%). 7. These data argue in favour of a specific and partial inhibition of the SR Ca2+ pump by 30 microM TBQ in the rabbit and rat ventricle and emphasise the importance of SR Ca2+ uptake in the force-frequency phenomenon.

Abstract: The effect of the intramuscular injection of bupivacaine hydrochloride on selected morphological characteristics and contractile properties of adult rat extensor digitorum longus muscle was studied. Recovery of normal fiber size was already present 30 days after bupivacaine injection and at 90 days after injection, values of the normalized twitch tension (mN/mg of tissue) and of the fatigue index approached those measured in control muscle, whereas the normalized tetanic tension remained 57% of control. At 7-30 days postinjection, twitch force was decreased by reducing [Ca2+]zero (substituted by Mg2+) or adding Co2+ (5 mmol/L-1). By contrast potentiation of the twitch was recorded in the presence of Cd2+ (2 mmol/L-1). Glycerol treatment only reduced, but did not eliminate twitches developed by muscles 7 days after injection. Present results emphasize the importance of the recovery process in the loss of the susceptibility of the contractile responses to extracellular calcium in bupivacaine-injected muscles. These data may be of interest in the evaluation of functional aspects of muscles in which injections of viral vector or autologous myoblasts have been performed.

Abstract: The effects of perchlorate (6-40 mM) on contractile properties and depolarization-contraction coupling of diaphragm have been tested in muscle fibre bundles from 8 to 12-week-old mdx and control mice. Twitch contractile properties were affected by perchlorate to a similar extent in mdx and control muscles. In the absence of perchlorate, voltage-dependent parameters of both activation and inactivation were similar in mdx and control muscles. In both mdx and control muscles, perchlorate induced a shift of activation and inactivation curves toward more negative potentials. However, mdx diaphragm fibres were less sensitive to perchlorate in the low range of concentrations (6-12 mM). These results are unlikely to be due to a possible effect of the lack of dystrophin on the excitation-contraction coupling voltage sensor or ryanodine receptor. However, necrosis and regeneration occur in mdx mouse diaphragm and it is postulated that the presence of a high proportion of not fully mature fibres could contribute to the reduced sensitivity of mdx diaphragm fibres to perchlorate.

Abstract: Two types of nerve lesions were performed at birth in rat extensor digitorum longus muscle: sciatic nerve transection (group A) and sciatic nerve crush allowing further reinnervation (group B). Contractile responses were then studied at different times after the denervation (7, 14, 30, and 60 days) and compared with control. Sixty days after the intervention, twitch and tetanic tensions remained dependent upon the extracellular Ca2+ concentration ([Ca]o) both in groups A and B. However, the depression of tensions following Ca2+ withdrawal was more important in group A. Sixty days after birth, in the presence of a Ca2+ channel blocker, Cd2+ (2 mmol L-1), a depression of the twitch tension was observed in group A (similarly to control 1-7 days postnatal muscles), whereas Cd2+ potentiated twitch tension in group B (similarly to control 14-60 days postnatal muscles). After glycerol treatment (detubulating procedure) performed in 60-day-old muscles, twitch tension was abolished in group B and control, whereas twitch tension was potentiated in group A. Thus, in developing muscles, neural control could be involved in the dependence of contractility toward [Ca]o. These results may be relevant for the understanding of the contractile properties of neuromuscular disorders with early onset.

Abstract: In newborn rat skeletal extensor digitorum longus (EDL) muscle, it has been found that an influx of calcium from the extracellular medium is necessary for contraction, in contrast to the situation observed in adult EDL muscle. The aim of the present study was to determine the influence of the extracellular calcium concentration ([Ca]o) upon the contractile responses elicited in developing as well as in regenerating (notexin-injected) soleus (SOL) muscle. A morphological study was performed to follow the steps of postnatal development and regeneration in SOL muscle. In nominally calcium-free solution, the amplitudes of the twitch and tetanic tensions were greatly reduced in 1-14-day-old developing SOL muscles, as well as in notexin-injected SOL muscles. With longer times after birth, twitch and tetanic tensions of SOL muscle were less affected by the absence of calcium. This contrasts with notexin-injected SOL muscle in which the amplitudes of the contractions remained strongly dependent on [Ca]o. The present finding suggests that some functional characteristics are different in regenerating muscle fibers and may be of interest in the evaluation of the contractile properties of muscles in which injections of genetically engineered or not autologous myoblasts or viral vector have been performed.

Abstract: The effects of preventing oxidative phosphorylation on pHi were compared in papillary muscles from right ventricles of normal and pressure-overloaded ferret hearts. Hypertrophy was induced by pulmonary artery clipping for 30-45 days. pHi was recorded with pH-sensitive microelectrodes. Resting pHi and the relationship between intracellular buffering power and pHi were not modified by the hypertrophy. At 22 degrees C, the initial intracellular alkalosis following exposure to oxygen-free Tyrode solution (containing the reducing agent sodium dithionite, 1 mM), as well as the transient acidosis on return to oxygenated solution, were reduced in hypertrophied papillary muscles. During hypoxia, exposure to alpha-cyano-4-hydroxycinnamate (5 mM) induced a larger intracellular acidification in hypertrophied than in control muscle. The initial alkalosis during hypoxia and the extra acidification on recovery from hypoxia were also significantly reduced in hypertrophied muscles at 35 degrees C. Moreover, the acidification during hypoxia was markedly accentuated in hypertrophied preparations at this temperature. [Mg2+]i and [Ca2+]i were also measured during metabolic inhibition, using mag-fura-2 and fura-2 respectively, in isolated cells from control and hypertrophied right ventricles. Hypertrophy increased the resting level of [Ca2+]i and of [Mg2+]i by a factor of 2.5 (P < 0.001) and 1.3 (P < 0.05) respectively. Upon application of 15 mM 2-deoxyglucose, [Mg2+]i was increased to a similar extent in control and hypertrophied cells. It is concluded that right ventricular hypertrophy could modify creatine phosphate metabolism and the capacity to recruit anaerobic glycolysis.

Abstract: The regenerative capacity of mdx Extensor Digitorum Longus (EDL) muscle after iterative muscle crush injuries was examined and compared with that of age-matched control C57BL/10 mice. Muscle crush injuries were performed at 8 weeks and repeated at 12 and 16 weeks. Contralateral non-crushed EDLs from mdx and C57BL/10 mice were used as internal controls for histopathology, histoenzymology, morphometry and for the study of the contractile properties. Morphological examinations were performed at 12, 16 and 20 weeks, respectively one month after a single, a second or a third crush. Contractile properties were studied at 12 to 20 weeks. By 20 weeks, no difference in the number of fibres with internal nuclei could be observed between crushed EDL from both strains, and non-crushed mdx EDL; the area and the diameter of crushed EDL from mdx mice were, respectively, 1.5- and 1.2-fold higher than the ones from crushed EDL from C57BL/10 strain. By 20 weeks, diameter distribution of crushed EDL muscles from C57BL/10 mice were shifted towards smaller fibre diameter, whereas in mdx mice, diameter distribution of crushed EDL muscles paralleled that of non-crushed EDL muscles. By 20 weeks, crushed mdx and C57BL/10 EDL muscles produced 77 and 47% of normalized tetanus tension respectively of non-crushed mdx and C57BL/10 EDL muscles. Following crush injury, both 12- and 20-week mdx and C57BL/10 EDL exhibited a slowed time to peak (TTP) and half-relaxation time (H1/2R) of twitch. There was no difference in posttetanic potentiation between the different groups. Crushed EDL of both strains showed an increased resistance to fatigue, compared to the non-crushed controls. The present study provides morphological and functional evidence for the greater recovery of mdx muscle compared to C57BL/10 muscle following iterative crush injury; however, the recovery does not completely prevent the appearance of necrosis/regeneration features.

Abstract: Some contractile and electrophysiological properties of muscle fibers isolated from the slow-twitch soleus (SOL) and fast-twitch extensor digitorum longus (EDL) muscles of rats were compared with those measured in SOL muscles from suspended rats. In suspended SOL (21 days of tail-suspension) membrane potential (Em), intracellular sodium activity (aiNa) and the slope of the relationship between Em and log [K]o were typical of fast-twitch muscles. The relation between the maximal amplitude of K-contractures vs Em was steeper for control SOL than for EDL and suspended SOL muscles. After suspension, in SOL muscles the contractile threshold and the inactivation curves for K-contractures were shifted to more positive Em. Repriming of K-contractures was unaffected by suspension. The exposure of isolated fibers to perchlorate (ClO4-)-containing (6-40 mM) solutions resulted in a similar concentration-dependent shift to more negative Em of activation curves for EDL and suspended SOL muscles. On exposure to a Na-free TEA solution, SOL from control and suspended rats, in contrast to EDL muscles, generated slow contractile responses. Suspended SOL showed a reduced sensitivity to the contracture-producing effect of caffeine compared to control muscles. These results suggested that the modifications observed due to suspension could be encounted by changes in the characteristics of muscle fibers from slow to fast-twitch type.

Abstract: Some contractile properties of soleus muscle (SOL) fibers isolated from tail-suspended (21 days) rats were compared with those determined in the slow-twitch SOL and the fast-twitch extensor digitorum longus muscle (EDL) of control rats. In SOL from suspended rats, the resting membrane potential and the intracellular Na+ activity were typical of fast-twitch muscles. The relationship between the amplitude of K+ contractures and the membrane potential was steeper for control SOL than for control EDL and suspended SOL. The inactivation curve was also shifted to more positive potentials after suspension. In the presence of perchlorate anions, the tension activation curves of control EDL and suspended SOL were similarly shifted to more negative potentials. Thus, in the present study, modifications induced by suspension in SOL, mainly related to changes in the voltage-sensing process involved in the excitation-contraction coupling mechanism, were that the SOL assumed some of the characteristics of fast-twitch muscles.

Abstract: Some contractile properties of small bundles (100-200 microns diameter) of muscle fibres isolated from the extensor digitorum longus muscle of rats at different times of development were compared. An increase of resting potential was observed in these muscles from -26.9 mV at 1 day of age to -72.6 mV at 3 months. Twitch tension and duration of postnatal muscles 1-7 days were diminished by reducing [Ca]o (substituted by Mg2+) or adding inorganic cations (Ni2+, Cd2+, La3+), unlike in the oldest animals (14 days-3 months postnatal) where twitch responses were unaffected. In the latter, potentiation of the twitch tension was even recorded in the presence of Ni2+ (0.5-1 mmol.1-1) and Cd2+ (0.5-2 mmol.1-1). Properties of activation and inactivation of the developed tension following elevation of [K]o to 15-200 mmol.1-1 were analysed at the same stages of postnatal development. In contrast to the tension-membrane potential curves for activation, which presented an average negative shift of -17.6 mV between 1 day postnatal and 3 months of age, a voltage dependence of inactivation similar to that encountered in adult extensor digitorum longus muscles, was already reached at 7 days of age. These results suggest an asynchronism in the maturation of the potential-dependent characteristics of the depolarization-contraction coupling mechanism. Furthermore, during the first week postnatal, in relation with poorly developed membrane systems and low [Ca]i-recycling capability, [Ca]o plays a fundamental role in maintaining contraction by replenishing the intracellular calcium pool.

Abstract:

Abstract: Some contractile, histochemical, morphological and electrophysiological properties of ferret, Mustela putorius furo, cremaster muscle have been estimated. Histochemical fibre typing revealed the presence of two types of fibres (type I 66.2%, type II 33.8%). Morphometry performed on ATPase-stained transverse sections showed that type I was composed of a large amount (40%) of small (less than 1400 microns2) cells. In mammalian Ringer two groups of fibres could be recognized on the basis of the values of resting potential (-69.7 mV and -59.1 mV) intracellular sodium activity (8.3 mmol.l-1 and 14.1 mmol.l-1, respectively). In experiments on fibre bundles, the elevation of extracellular potassium concentration to 15-200 mmol.l-1 produced contractures that consisted of a well-defined transient or phasic tension followed by a sustained or tonic tension. Properties of activation and inactivation of the tension analysed in small bundles of cut fibres (lengths 0.5-1.0 cm) were of fast- and slow-twitch type for phasic and tonic phase, respectively. In contrast to the phasic component of K contractures, the tonic phase was abolished by Ca2+ withdrawal and inhibited by Ni2+, Cd2+, Co2+, Gd3+ and gallopamil (D600). In Ca(2+)-free medium the sustained tension was restored by adding Sr2+. It is concluded that in ferret cremaster muscle the presence of slow-twitch fibres would give rise to the tonic component of the K contracture in which an extracellular source of activator Ca2+ is involved. The ability of these fibres to contract with a maintained tension for prolonged periods of time might participate in the temperature regulation of the testes.

Abstract: The characteristics of caffeine (1.25-80 mM) transient contractures have been examined in small atrial trabeculae (diameters 50-250 microns) isolated from young (1-1.5 months) ferret hearts. In the control medium, the half-saturation constant and the maximum contracture strength (at infinite caffeine concentration) were 37.8 +/- 10.2 mM and 0.9 +/- 0.2 kN.kg-1 (n = 11), respectively. The contractile response to caffeine was markedly enhanced following reduction of external sodium (70-0 mM). The perfusion of young ferret trabeculae with the sodium-free medium (up to 3 min) decreased the half-saturation constant by a factor of three (12.4 +/- 1.6 mM, n = 8) with an increase in maximum contracture strength (1.09 +/- 0.3 kN.kg-1, n = 8). The effects of various divalent and trivalent cations have been tested on the 10 mM caffeine contracture in trabeculae perfused with Na-containing (140 mM) solution. The order of cation effectiveness is Gd3+ (half effect 0.04-0.07 mM) greater than Cd2+ (0.15-0.25 mM) greater than Ni2+ (2-2.5 mM) greater than Co2+ (7-7.5 mM) much greater than Mn2+. In conclusion, the present work has shown that in atrial trabeculae isolated from young ferret hearts, the strength of the caffeine contracture was markedly affected by the activity of the sarcolemmal Na-Ca exchange.

Abstract:

Abstract: The inotropic effects of external calcium concentration ([Ca2+]o] and rest periods have been compared in papillary muscles isolated from control (n = 4) and pressure-overloaded right (n = 5) ventricles of adult ferrets. Hypertrophy was induced by pulmonary artery clipping for 30-45 days. Under control conditions (3 mM [Ca2+]o, 0.1 Hz), the isometric twitch force of hypertrophied muscles was decreased by 75%, time to peak was increased by 30% and time to half-relaxation was increased by 50% compared with non-hypertrophied preparations. The sensitivity of contraction to [Ca2+]o was decreased in hypertrophied muscles compared with control ([Ca2+] required for half-maximal contraction: 4.1 mM vs 1.7 mM) and the maximal contraction reached at high [Ca2+]o was smaller in pressure-overloaded muscles compared with control (8.3 +/- 2.0 mN mm-2 vs 19.0 +/- 2.1 mN mm-2 respectively). In both groups, rest periods longer than the steady-state interval were initially accompanied by a potentiation of the first post-rest contraction compared with steady-state. Peak potentiation occurred after a rest of 120 s in hypertrophied muscles and after a rest of 60 s in control. The maximal relative potentiation, i.e. compared with the steady-state twitch, was higher in hypertrophied muscles (+75%) than in control (+20%). After peak potentiation, the amplitude of the first post-rest contraction progressively decreased with increasing periods of rest, although at a slower rate in hypertrophy compared with control. The time constants of post-rest decay were 1203 +/- 99 s and 528 +/- 24 s respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: The existence of possible differences of calcium (Ca2+) fluxes through the sarcolemmal sodium-calcium (Na+/Ca2+) exchanger during hypertrophy has been tested by comparing the characteristics of the contracture--as an indicator of the intracellular Ca2+ concentration--induced by partial or total withdrawal of external sodium (Na+), in the absence of external potassium, in the right ventricular trabeculae of adult ferret hearts. Pressure-overload was induced by pulmonary artery clipping and led to an increase of the right ventricular weight of 60%. At an external Ca2+ concentration ([Ca2+]o) of 3 mM, the dependence of the contractures on extracellular sodium concentration ([Na+]o), the rate of tension development, the time course of spontaneous relaxation and the time course for the repriming of the contracture were unchanged by hypertrophy. However, the relationship between [Ca2+]o and contracture amplitude at various [Na+]o showed that the apparent affinity of the contracture for [Ca2+]o was decreased in hypertrophied preparations. Thus, in 0 mM [Na+]o, half-maximal contracture was induced at a [Ca2+]o of 0.012 +/- 0.016 mM and 0.171 +/- 0.021 mM in control (n = 11) and hypertrophy (n = 12) respectively (P less than 0.001). Although these data may be indicative of a decreased Ca2+ influx through the Na+/Ca2+ exchanger, it cannot be excluded that intracellular buffering mechanism may also be involved in this differential response to [Na+]o withdrawal.

Abstract: Because of the role of intracellular Na on cardiac contractility and of the depressed isometric contractile response of the hypertrophied myocardium, the effects of pressure overload on the intracellular Na activity (aiNa) have been investigated in papillary muscles isolated from the ferret right ventricle. In animals subjected to pulmonary artery clipped for 1-2 months, right ventricle-to-body weight ratio was increased by about 39% in comparison with the control group. aiNa was measured in quiescent papillary muscles, by means of Na-sensitive micro-electrodes, at room temperature (19-22 degrees C). aiNa values were, in the control ventricular cells, 7.8 +/- 1.1 mM (mean +/- SD; n = 20) and in the hypertrophied ones, 8.0 +/- 1.2 mM (n = 49). During superfusion by medium with a reduced extracellular Na concentration ([Na]0), aiNa declined in control and pressure-overloaded muscles to similar steady-state levels at a given [Na]0. aiNa fall was mono-exponential and was characterized by a smaller time constant in the hypertrophied group upon total withdrawal of Na0 (control 209 +/- 19 s, n = 4; hypertrophied 128 +/- 42 s, n = 6). In the absence of external K, aiNa increased to levels that were not significantly different between both groups. It was concluded that, in quiescent preparations, steady-state aiNa was not modified by the hypertrophic process. However, pressure overload induced a modification of aiNa regulation by a possible alteration of the sarcolemmal Na/Ca exchange, although other mechanisms, such as mitochondrial Ca transport, could be involved in the differential response to Na0 removal.

Abstract: Isometric tension of Na+-withdrawal contractures, membrane potential and intracellular Na+ activity (aiNa) have been measured in vitro under conditions which modify the activity of the Na+-Ca2+ exchanger of dystrophic limb muscles of (dy2J/dy2J) C57 BL/6J mice. In dystrophic soleus (Sol) and extensor digitorum longus (EDL) muscles exposed to Na+-free medium, there was a decrease in aiNa associated with a hyperpolarization of the membrane and the generation of a transient contracture. By contrast, exposing normal or denervated EDL muscles to Na+-free solution induced no change in aiNa nor in the resting tension. This study shows that Na+-Ca2+ exchange is still present in dystrophic Sol muscle. However from thermodynamic considerations, it could be suggested that the higher resting aiNa compared with that in normal muscle, may decrease the efficacy of the exchange.

Abstract: Ionic currents and contraction were recorded under voltage clamp conditions in single fibres isolated from rat soleus muscles denervated for more than 20 days. The effects of amphiphiles on depolarization-contraction (d.c.) coupling in Na-free TEA-containing solutions were analyzed. An anionic amphiphile, sodium dodecyl sulfate (1-10 microM), caused a dose-dependent reduction of the contractile response at all amplitudes of depolarization while a cationic amphiphile, dodecyltrimethylamine (1-10 microM), increased the maximum developed tension with a shift in the contractile threshold. A neutral amphiphile, lauryl acetate (20 microM), induced no significant variation. The effects of charged amphiphiles were found to be strongly dependent on the external calcium concentration and on membrane potential. The effect of sodium dodecyl sulfate to decrease tension was reduced or changed to positive inotropy following hyperpolarization of the membrane by, respectively, +10 and +20 mV. In hyperpolarized (+20 mV) cells, dodecyltrimethylamine reduced the amplitude of the contraction. The results demonstrated that changes in Ca-binding properties of surface membrane modified d.c. coupling in denervated slow twitch skeletal muscle.

Abstract: The effects of the glycoside saponin on ferret ventricular muscle have been investigated. Saponin produced a positive inotropic effect, the extent of which was determined by the bathing calcium concentration. If the bathing calcium concentration is reduced to levels equivalent to those found intracellularly then similar saponin concentrations are able to "skin" the cardiac cells. The effects were further investigated in sheep heart Purkinje fibres. In the presence of normal extracellular calcium concentrations (to prevent skinning), saponin produced increases in sodium activity, decreases in potassium activity but little change in intracellular pH. The decreases of potassium activity were compared to the effects of strophanthidin. The changes in intracellular ion levels were accompanied by the development of a contracture. The effects of saponin could be explained by its interaction with cholesterol in the cell membrane resulting in an increase in membrane permeability to sodium which may be part of a nonspecific increase in membrane permeability.

Abstract: Isometric tension of K+ contractures, membrane potential, internal Na+ activity (alpha iNa) and intracellular pH (pHi) have been measured in vitro under conditions which modify sarcolemma-bound calcium and the activity of the Na+-Ca2+ exchange of normal mammalian soleus muscle. In the absence of external Na+ and for a given high external K+, the maximum amplitude of the contracture was increased (Na+ replaced by TEA+) or decreased (Na+ replaced by Li+ or Cs+) compared with that obtained in the presence of Na+. Replacement of external Na+ by another monovalent cation (TEA+) did not induce any change of pHi. However the concomitant decrease of alpha iNa was related to a Na+-Ca2+ exchange across the sarcolemmal membrane. The data suggest that in soleus muscle, a sarcolemmal calcium pool is involved during the development of K+ contractures.

Abstract: In the double mannitol gap arrangement the contraction was estimated in single fibres isolated from rat extensor digitorum longus (e.d.l.) muscles that had been denervated for 2-48 days. Denervation induced large changes in the characteristics of the action potential and of the twitch. Up to 15-20 days after denervation the contraction-depolarization curve was sigmoidal and the maximum amplitude of the contraction was not modified by variation of [Ca]o or [Na]o. After 15-20 days of denervation a bell-shaped curve described the relation between contraction and potential. The maximum amplitude was dependent upon the [Ca]o. In Ca-free solution no contractile response was obtained. In Na-free, Ca-containing solution the relationship between contraction and potential was not modified by the addition of divalent cations Co2+, Cd2+, Mn2+, Mg2+, Ni2+, or Ba2+. The contraction, which appeared in Ca-free solution, was restored by adding Sr2+. D600, verapamil and bepridil failed to change the amplitude of the contraction while a marked reduction was found with dihydropyridines. The reduction was overcome by increasing [Ca]o. The present results suggest that the slow calcium current is not involved in the generation of the contractile responses developed by denervated muscles in Na-free (TEA) solution.

Abstract: 1. The changes of intracellular pH (pHi) of papillary muscles from ferret and Purkinje fibres from sheep heart during hypoxia and recovery from hypoxia were recorded with pH-sensitive micro-electrodes filled with neutral H+ carrier. 2. Hypoxia was produced by replacement of O2 with N2 in the superfusing solutions. When oxidative phosphorylation was prevented, developed tension fell within 20 min to about 16 and 21% of its control value for papillary muscle and Purkinje fibres respectively. On restoration of O2, recovery of developed tension in ferret papillary muscle is preceded by a transient additional decrease. 3. In ferret papillary muscle, the pHi first increased by a mean value of 0.11 pH units after 3 min hypoxia, then decreased by about 0.24 pH units after 20 min. In sheep Purkinje fibres, the initial alkalosis was small or absent, and after 5-9 min, the pHi started to fall reaching 0.17 pH units after 20 min of hypoxia. On return to oxygenated solution, a transient additional intracellular acidification occurred. This acidification reached its peak of 0.31 pH units in papillary muscle and of 0.13 pH units in Purkinje fibres. In both preparations hypoxia was accompanied by a depolarization of a few millivolts. 4. The presence of cyanide (1-2 mM) or fluorodinitrobenzene (20-40 microM) prevented the additional intracellular acidification occurring on return to oxygenated solution. Removal of cyanide itself produced a transient but smaller and slower acidification. 5. On both preparations, exposure to a Tyrode solution containing 10 mM-L-lactate produced a transient intracellular acidification. After recovery from this acidification the acidification produced by hypoxia was increased without affecting the extra acidification on reintroduction of O2. 6. After reduction of the rate of glycolysis by removal of glucose and application of 2-deoxyglucose, the transient intracellular acidification, occurring on return to oxygenated solution after hypoxia, was inhibited in both preparations. In ferret papillary muscle, insulin (100 mU/ml) potentiated the changes of pHi occurring during hypoxia. 7. Using Na+-sensitive glass micro-electrodes it was found that the intracellular Na+ activity rose slightly during the later stages of hypoxia and rose transiently on readdition of O2. These results are consistent with a Na+-H+ exchange being stimulated by acidosis. 8. The origins of the pH changes during and after hypoxia are discussed as are the differences between the responses of sheep Purkinje fibres and ferret papillary muscle.

Abstract: Under voltage clamp conditions contractile responses and ionic currents of single fibres isolated from rat soleus, denervated for more than 20 days, were recorded in Na-free TEA containing solutions. The relationship between membrane potential and contraction has been analysed under various conditions. The addition of trivalent cations (La3+, Gd3+) resulted in a dose dependent reduction of the contractile response and similar effects were produced by polymyxin B (0.05-0.5 mM). By contrast in the presence of phospholipase D (1-5 U/ml) contractions were significantly increased for all values of depolarization. The time course of the change of tension amplitude after the application of Ca-free medium, was dependent on the amplitude, the duration and the frequency of the depolarization. Upon depolarization glycerol-treated fibres generated contractile responses which were similar to those recorded in normal muscle and were also dependent on [Ca]o. It is proposed that in denervated soleus muscle the negatively charged phospholipids at the outside of the membrane were involved in the depolarization-contraction coupling by means of their Ca binding properties. The quantity of Ca binding sites would be dependent on [Ca]o and membrane potential and their binding properties modified during and/or following variation in membrane potential.

Abstract: The strength of contractures, produced by 15 to 146 mM [K]0 (as L-glutamate), was measured in isolated small bundles of muscle fibers from the fast-twitch extensor digitorum longus and from the slow-twitch soleus of normal and dystrophic (C57 BL/6J dy2J/dy2J) mice. The analysis of the relation between the maximal amplitude of the contracture vs the membrane potential and the time constant of relaxation of the K-contractures has shown that dystrophy induced an attenuation of the differences between fast- and slow-twitch muscles. The repriming of K-contractures was more affected by changes in [Ca]0 in normal soleus than in normal extensor digitorum longus and this difference was unaffected by dystrophy. For both types of muscles, the ability of caffeine to produce contractures was reduced in dystrophic muscle and this modification was not related to a change in the fiber typing.

Abstract: Small bundles of muscle fibres were isolated from diaphragm, extensor digitorum longus (EDL) and soleus (SOL) muscles of normal and dystrophic (C57 BL/6J dy2J/dy2J) mice, and their isometric tension developed in response to acetylcholine (ACh) was recorded. For each type of muscle the relationship between the maximum amplitude of the ACh-contracture and log [ACh] was similar in normal and dystrophic animals. However, this relationship was steeper for normal and dystrophic SOL than for EDL and diaphragm muscles. Dystrophy did not induce changes in the time course of the ACh-contractures, except a significant 'speeding' of dystrophic SOL that appeared in the time to peak of the contractile response. The amplitude of ACh-contractures of both normal and dystrophic diaphragm preparations increased by about 50% after perfusion for 80-90 min in physiological solution containing phospholipase C 5 mU/ml. ACh-sensitivity was measured in normal and dystrophic diaphragm preparations by iontophoretic application of ACh from high resistance pipettes. ACh-potentials were similar in time course in the two types of muscle fibres, and there was also no significant difference in the length of sensitive fibre segments and maximum sensitivity values. Extrajunctional ACh-sensitivity was absent in normal as well as in dystrophic fibres. It is concluded that the absence in dystrophic muscles of stronger ACh-contractures and of extrajunctional sensitivity can be considered as evidence against a primary neuronal involvement in murine dystrophy of the dy2J strain.

Abstract: Potassium and caffeine contractures of isolated small bundles (100 to 200 micron diameter) of muscle fibers isolated from the diaphragm of normal and dystrophic (C57BL/6J dy2J/dy2J) mice were compared. In diaphragms of pathologic mice (3 to 5 months old) the resting potential, the characteristics of the twitch, and some histological examinations were typical of dystrophic muscles. The slopes of the relationships between the steady membrane potential and log [K]0 were similar for the two types of cells. In 110 mM and 146 mM K there were no significant differences in the time course of the contractures and reduction in [Ca]0 decreased the time to peak and the time constant of relaxation to the same extent; the relative efficiency of [Mg]0 compared with [Ca]0 was equivalent. Repriming of K contractures at different external calcium concentrations indicated that the normal diaphragm did not have any special advantage. The exposure of isolated strips to a solution containing caffeine resulted in a similar increase of the strength of the regularly evoked twitch responses. However, the contractures elicited by 1.25 to 20 mM caffeine showed a subsensitivity of the dystrophic diaphragm (KmDys = 9.3 KmN) and the rate of relaxation was significantly slower than in normal muscle (in 20 mM caffeine, 50% decay time for normal muscle was 25.2 +/- 7.6 s and for dystrophic muscle 54.8 +/- 11.2 s. These results suggest an absence of major alterations in the mechanism of excitation-contraction coupling associated with dystrophy, except for a change in the specific element of the sarcoplasmic reticulum where caffeine acts.

Abstract: The distribution of acetylcholine (ACh)-sensitive membrane areas was determined in 11-75 days denervated slow muscle fibres of Rana temporaria by iontophoretic application of acetylcholine. The fibres were also stimulated directly, and their electrical activity was recorded with an intra- and an extracellular electrode. During the first two weeks following denervation the size of the ACh-sensitive fibre surface was similar to that of normal slow fibres, but a spreading out of ACh-sensitivity occurred between the 13th and 20th day. The slow fibre membrane did not become homogeneously ACh-sensitive; even after long periods of denervation large local sensitivity gradients could be observed. Throughout the denervation period maximum values of ACh-sensitivity were in the same range as in normal slow fibres. Action potentials were fully developed when ACh-sensitivity started to spread out. Extracellularly recorded inward currents varied in amplitude along the fibre surface, and either one or two peaks were observed in individual fibres. The spatial relationship between inward current peaks and peaks of ACh-sensitivity was investigated in 12 fibres. Fifteen inward current peaks were located at distances of 30-640 microns from points of maximum ACh-sensitivity; only once did the centers of ACh-sensitivity and excitability coincide. It is concluded that Na channels are incorporated into nonjunctional membrane areas of denervated slow fibres; this process precedes the incorporation of ACh receptors by approximately one week.

Abstract: Physostigmine-induced contractions of isolated small bundles (100 micron diameter) of muscle fibres isolated from the diaphragm of C57 BL mice were studied under various conditions. At different external pH the changes in the maximal amplitude of the contraction were related to the external activity of the permeant neutral form of the drug. However changes in the internal pH showed that the amplitude of the contractile response depended directly on the internal activity of the protonated form of the drug. The amplitude of the contraction was enhanced by caffeine treatment and depended on the external calcium concentrations. In mammalian skeletal muscle, physostigmine appeared to produce contractions due to its anticholinesterase property. The release of calcium seemed to occur from an intracellular store different from that involved in the action of caffeine.

Abstract: The distribution of acetylcholine-sensitive membrane areas in slow muscle fibres of pyriformis muscles of Rana temporaria was examined by iontophoretic application of acetylcholine from high resistance pipettes. ACh-sensitivity varied considerably along individual slow fibres and from fibre to fibre. In some fibres the sensitivity was restricted to segments of less than 100 microns, in others it was continuous over several millimeter. Segments of variable length, but up to several millimeter, were completely insensitive to acetylcholine. Highly sensitive spots (greater than 1,000 mV/nC) were found occasionally, their diameter being of the order of 10-20 microns only. The occurrence at rather regular intervals of ACh-sensitive areas was a rare observation; no evidence was found for a generalized ACh-sensitivity. There were marked differences in the lengths of ACh-sensitive segments between surface fibres and fibres located in deeper layers of the muscles. It is concluded that the ACh-sensitive membrane areas correspond to individual nerve muscle contacts of the small motor system whose spatial distribution is extremely variable. In superficial slow fibres the synaptic contacts seem to be located predominantly on the internal circumference of the fibres.