Spontaneous tension oscillation in skinned bovine cardiac muscle

 Skinned fibres from bovine ventricles exhibited spontaneous tension oscillations when MgADP and inorganic phosphate (Pi) were added to the solution bathing fibres in the relaxed state (ADP-SPOC). A similar type of oscillation was observed at intermediate concentrations of free Ca²⁺ in the absence of MgADP and Pi (Ca-SPOC). To investigate the correlation between ADP-SPOC and Ca-SPOC, we constructed two-dimensional state diagrams of cardiac muscle using different concentrations of Pi (0–20 mM) and free Ca²⁺ [pCa=around 5 (+Ca²⁺), pCa=5.15–6.9 and +EGTA (–Ca²⁺)], with varying concentrations of MgADP (0–10 mM), with 2 mM MgATP and 2 mM free Mg²⁺ maintaining ionic strength at 0.15±0.01 M, pH 7.0, 25 °C. The three-dimensional (pCa-Pi-MgADP) state diagram thus obtained was divided into three regions, i.e. the contraction region in which tension oscillation was undetectable, the spontaneous tension oscillation (SPOC) region and the relaxation region. We found that the regions of ADP-SPOC and Ca-SPOC were continuously connected by a single oscillation region sandwiched between the contraction and relaxation regions. The state diagram, which encompasses physiological conditions, shows that the probability of SPOC is higher in cardiac muscle than in skeletal muscle. From these results, we suggest that, despite distinct ionic conditions, the molecular state of cross-bridges during SPOC is common to both ADP-SPOC and Ca-SPOC. Received 19 February 1996 / Received after revision: 16 July 1996 / Accepted: 14 August 1996     

Regulatory roles of MgADP and calcium in tension development of skinned cardiac muscle

We investigated the regulatory roles of MgADP and free Ca2+ in isometric tension development in skinned bovine cardiac muscle. We found that, in the relaxed state without free Ca2+, MgADP elicited a sigmoidal increase in active tension, as is the case in skeletal muscle (ADP-contraction). The critical MgADP concentration, at which the tension increment became half-maximal, increased in proportion to MgATP concentration, with a slope of approximately 1 for cardiac and 4 for skeletal muscle. Raising the free Ca2+ concentration decreased the critical MgADP concentration in proportion to the free Ca2+ concentration. In addition, the apparent Ca2+ sensitivity of tension development increased with MgADP, while decreasing with inorganic phosphate (Pi); MgADP suppressed the Ca2+- desensitizing effect of Pi in a concentration-dependent manner. These activating effects of MgADP were quantitatively assessed by means of a model based upon the kinetic scheme of actomyosin ATPase. These experimental results and model simulation suggest that the state of thin filaments is synergistically regulated by both the binding of Ca2+ to troponin and the formation of the actomyosin–ADP complex.     

Ca2+ uptake by cardiac sarcoplasmic reticulum ATPase in situ strongly depends on bound creatine kinase

The role of creatine kinase (CK) bound to sarcoplasmic reticulum (SR), in the energy supply of SR ATPase in situ, was studied in saponin-permeabilised rat ventricular fibres by loading SR at pCa 6.5 for different times and under different energy supply conditions. Release of Ca²⁺ was induced by 5 mM caffeine and the peak of relative tension (T/T _max) and the area under isometric tension curves, S _T, were measured. Taking advantage of close localisation of myofibrils and SR, free [Ca²⁺] in the fibres during the release was estimated using steady state [Ca²⁺]/tension relationship. Peak [Ca²⁺] and integral of free Ca²⁺ transients (S[Ca²⁺]_f) were then calculated. At all times, loading with 0.25 mM adenosine diphosphate, Mg²⁺ salt (MgADP) and 12 mM phosphocreatine (PCr) [when adenosine triphosphate (ATP) was generated via bound CK] was as efficient as loading with both 3.16 mM MgATP and 12 mM PCr (control conditions). However, when loading was supported by MgATP alone (3.16 mM), T/T _max was only 40% and S[Ca²⁺]_f 31% of control (P < 0.001). Under these conditions, addition of a soluble ATP-regenerating system (pyruvate kinase and phosphoenolpyruvate), did not increase loading substantially. Both S _T and S[Ca²⁺]_f were more sensitive to the loading conditions than T/T _max and peak [Ca²⁺]. The data suggest that Ca²⁺ uptake by the SR in situ depends on local ATP/ADP ratio which is effectively controlled by bound CK. Received: 23 January 1996/Received after revision: 19 April 1996/Accepted: 3 May 1996     

Role of myofibrillar creatine kinase in the relaxation of rigor tension in skinned cardiac muscle

In the absence of creatine phosphate, MgATP produced relaxation of rigor tension in chemically-skinned right papillary muscles of the rat, the half maximal effect being obtained at 1.8 mM MgATP. In the presence of 12 mM creatine phosphate and 250 M ADP, a decrease in MgATP concentration even to 10^–9 M never induced rigor tension. At a very low MgATP concentration (10^–6 M), the half maximal relaxing effect was obtained with 2 mM creatine phosphate, a value close to theK _m of isolated MM-creatine kinase for this substrate, or with 14 M MgADP, a value 5 times lower than the reportedK _m. An exogenous MgATP regenerating system (phosphoenol pyruvate + pyruvate kinase) was not able to fully relax the fibres. When MM-creatine kinase was inhibited by fluorodinitrobenzene, the dependency of rigor tension on MgATP became the same as it was without creatine phosphate. After washing out the fluorodinitrobenzene the addition of exogenous MM-creatine kinase for half an hour fully relaxed rigor tension; moreover, this effect persisted even after prolonged washout. These results show that endogenous MM-creatine kinase is able to ensure maximal efficiency of myosin ATPase by producing a localized high MgATP/MgADP ratio; they also suggest the existence of rapidly exchangeable binding sites for MM-creatine kinase in cardiac myofibrils.     

MgADP− increases maximum tension and Ca2+ sensitivity in skinned rabbit soleus fibers

Increasing concentrations of MgADP^– or MgCDP^– in the millimolar range cause an increase in the maximum Ca²⁺-activated tension that a skinned rabbit soleus muscle fiber can develop in the presence of 2 mM MgATP^2– or MgCTP² respectively. In contrast, the maximal Ca²⁺-activated ATPase activity of the fiber decreases in the presence of MgADP^–. As the nucleoside diphosphate (MgADP^– or MgCDP^–) concentration is increased, the Ca²⁺ concentration required for half-maximal activation of tension is reduced.MgADP^– has a similar effect on the Ca²⁺ concentration required to half-maximally activate the fiber ATPase. The effects on tension are due to magnesium nucleoside diphosphate and not some other form of nucleoside diphosphate since the effects occur at both low (pMg 4) and control (pMg 3) Mg²⁺ concentrations. Cooperativity, as judged by the Hill n value relating isometric tension and Ca²⁺, is less in the presence of 5 mM MgADP^– as compared to a control (no added MgADP^–) n value. Increasing concentrations of inorganic phosphate (Pi) in the millimolar range decrease maximum Ca²⁺-activated tension, and increase the concentration of Ca²⁺ required to half-maximally activate tension, effects opposite to those of MgADP. These data are consistent with the hypothesis that cooperative interactions between actin and myosin can affect the affinity of troponin for Ca²⁺.A preliminary report of this work was given at the Biophysical Society Meeting, February 1985, W. G. L. Kerrick, P. E. Hoar (1985) Effects of nucleotide diphosphate and inorganic phosphate on tension in skinned soleus and smooth muscle cells. Biophys J 47:296a     

Spontaneous tension oscillations in guinea-pig atrial trabeculae

Summary Spontaneous tension oscillations have been recorded from intact guinea-pig auricular trabeculae bathed in Na-poor and/or Ca-rich solutions.The frequency of these oscillations and that of after-contractions (oscillations following an electrically induced contraction) evoked under identical experimental conditions was the same (33°C).The amplitude of the oscillations rose when the [Ca²⁺]₀/[Na⁺]₀ ²-ratio or the intracellular Na-concentration was increased. When the increase of the [Ca²⁺]₀/[Na⁺]₀ ²-ratio was relatively small, tension oscillations only occured after a period of electrical stimulation.The oscillation-frequency increased slightly in media containing 70 instead of 5.4 mM KCl.MnCl₂ (3mM) did not affect either the amplitude or the frequency of the oscillations.Caffeine (0.5–2.5 mM) decreased the amplitude and enhanced the frequency of the oscillations. After-contractions were diminished and, at higher concentrations, abolished.It is demonstrated that the membrane potential does not participate in the process causing the tension oscillations. An increased [Ca²⁺]_i is a prerequisite for the occurrence of these oscillations. Characteristics of intracellular Ca-movement probably determine the amplitude and frequency of the spontaneous oscillations of tension.     

Sarcomere length-dependent Ca<Superscript>2+</Superscript> activation in skinned rabbit psoas muscle fibers: coordinated regulation of thin filament cooperative activation and passive force

In skeletal muscle, active force production varies as a function of sarcomere length (SL). It has been considered that this SL dependence results simply from a change in the overlap length between the thick and thin filaments. The purpose of this study was to provide a systematic understanding of the SL-dependent increase in Ca²⁺ sensitivity in skeletal muscle, by investigating how thin filament “on–off” switching and passive force are involved in the regulation. Rabbit psoas muscles were skinned, and active force measurements were taken at various Ca²⁺ concentrations with single fibers, in the short (2.0 and 2.4 μm) and long (2.4 and 2.8 μm) SL ranges. Despite the same magnitude of SL elongation, the SL-dependent increase in Ca²⁺ sensitivity was more pronounced in the long SL range. MgADP (3 mM) increased the rate of rise of active force and attenuated SL-dependent Ca²⁺ activation in both SL ranges. Conversely, inorganic phosphate (Pi, 20 mM) decreased the rate of rise of active force and enhanced SL-dependent Ca²⁺ activation in both SL ranges. Our analyses revealed that, in the absence and presence of MgADP or Pi, the magnitude of SL-dependent Ca²⁺ activation was (1) inversely correlated with the rate of rise of active force, and (2) in proportion to passive force. These findings suggest that the SL dependence of active force in skeletal muscle is regulated via thin filament “on–off” switching and titin (connectin)-based interfilament lattice spacing modulation in a coordinated fashion, in addition to the regulation via the filament overlap.     

Effects of vanadate, phosphate and 2,3-butanedione monoxime (BDM) on skinned molluscan catch muscle

The effects of orthovanadate (V_i), inorganic phosphate (P_i) and 2,3-butanedione monoxime (BDM) on tension, force transients and the catch state (passive tension maintenance) were investigated in saponin-skinned fibre bundles of the anterior byssus retractor muscle (ABRM) of the bivalve mollusc Mytilus edulis at pH 6.7. During maximal Ca²⁺ activation isometric force was depressed by V_i (0.03–10 mM), P_i (10 mM) and BDM (50 mM). Force transients following quick stretches (0.1–0.3% of fibre length) were accelerated substantially by 1 mM V_i, 10 mM P_i or 50 mM BDM. These compounds also accelerated force responses in experiments in which ATP was released rapidly from caged ATP by flash photolysis at both pCa 4.7 (force rise) and at pCa>8 (force decline). The effects on the catch state were investigated in two types of experiments: (1) Ca²⁺ removal after maximal Ca²⁺ activation and (2) rapid ATP release during high-force rigor at pCa>8. In both cases rapid relaxation was followed by slow relaxation (slower than 2% of initial force per min). This later slow relaxation (catch) was insensitive to V_i (1–10 mM), P_i (10 mM) and BDM (50 mM) but was accelerated by 0.12 mM cAMP. Complete relaxation to almost zero force was attained by changing pH from 6.7 to 7.7 (pCa>8). We conclude that catch depends on cAMP- and pH-sensitive structures linking the myofilaments and not on the force-generating actomyosin cross-bridges that are sensitive to V_i, P_i and BDM.     

Regulation of the cross-bridge cycle: the effects of MgADP,LC17 isoforms and telokin

This review summarizes the role of MgADP in force maintenance by dephosphorylated cross-bridges in smooth muscle and a potential physiological role for telokin. In tonic, compared with phasic, smooth muscles the affinity of cross-bridges is ～5 times higher for MgADP and the apparent second-order rate constant for MgATP is ～3 times lower. This gives rise to a large population of dephosphorylated cross-bridges in tonic smooth muscle. Such cross-bridges are thought to be major determinants of the different relaxation kinetics of the two types of smooth muscle and contribute to force maintenance at low levels of MLC₂₀ phosphorylation, termed ‘catch-like state’ (Somlyo & Somlyo 1967) or ‘latch’ (Dillon et al. 1981). The molecular basis of the different affinities for MgADP and MgATP between tonic and phasic smooth muscle myosin was explored by exchange of essential myosin light chain (LC₁₇) isoforms. In phasic bladder smooth muscle the exchange of LC_17b for LC_17a caused a significant decrease in the unloaded shortening velocity of non-phosphorylated, slowly cycling cross-bridges, suggesting that the LC₁₇ isoforms contribute to the nucleotide affinity of latch bridges. The role of telokin in Ca²⁺-desensitization in phasic smooth muscle is reviewed. Telokin, the independently expressed C-terminus of myosin light chain kinase, is extensively phosphorylated during forskolin- and 8-br-cGMP-induced relaxation in situ. Telokin accelerated dephosphorylation of the regulatory myosin light chain and relaxed rabbit ileum smooth muscle. The results suggest that telokin contributes to cAMP and/or cGMP kinase-mediated Ca²⁺-desensitization of phasic smooth muscles.     

Effects of MgATP on ATP utilization and force under normal and simulated ischaemic conditions in rat cardiac trabeculae

The dependency of ATP utilization and isometric force on [MgATP] was studied in skinned rat trabeculae under normal (pH 7.0) and simulated ischaemic (pH 6.2, 30 mM added Pi) conditions at 20+/-1 degrees C. At saturating [Ca2+], mean (+/-SEM) ATP utilization at 5 mM MgATP (A0) was 0.48+/-0.03 mM/s and force (F0) was 37+/-2 kN/m2. At 10 microM MgATP under normal conditions ATP utilization decreased gradually to 66+/-3% of A0, and force increased to 169+/-7% of F0. Under ischaemic conditions at 10 microM MgATP, ATP utilization decreased from 30+/-5% to 11+/-2% of A0 whereas force increased eightfold from 12+/-4% to 97+/-7% of F0. The [MgATP] at half-maximal ATP utilization (Km) under ischaemic conditions was 21+/-3 microM. At pH 7.0, Km was estimated to be less than 10 microM. These results show that tension cost decreases markedly with decreasing MgATP. Under ischaemic conditions parallel changes in Ca2+ sensitivity of force and ATP utilization were observed, corresponding to 1.3 pCa units. Reducing [MgATP] from 0.5 to 0.05 mM caused a modest reversal of this change in Ca2+ sensitivity. These changes in Ca2+ sensitivity are consistent with a marked reduction in active force and force-related ATP utilization during ischaemia but are insufficient to explain the ischaemic contracture on the basis of active force development.     

Evidence for myosin-linked regulation in guinea pig taenia coli muscle

Summary The ATPase activity of actomyosin prepared from taenia coli muscle of guinea pig was found to increase upon adding rabbit skeletal heavy meromyosin (HMM) in the absence of Ca²⁺. SDS-gel electrophoresis of muscle homogenates did not reveal the presence of troponin. Ca²⁺-regulation in taenia coli muscle thus appears to be myosin-linked.The glycerinated muscles which did not develop any tension in the presence of EGTA contracted after irrigation with rabbit skeletal myosin.Skeletal HMM could also cause tension generation in strips of glycerinated taenia coli in the presence of EGTA. The tension developed by the muscles in the presence of Ca²⁺ was increased if HMM was added. The HMM-induced tension was associated with a marked increase in ATPase activity both in the presence and in the absence of Ca²⁺. No HMM-associated tension could be detected when inactivated HMM was employed or when MgATP was substituted with Mg-pyrophosphate or Mg-AMP-PNP.The mechanical effect of HMM probably results from a mechanochemical interaction between the added HMM and muscle actin.This work was supported by a Katzir-Katchalsky fellowship (to J. B.) and by a grant from the Muscular Dystrophy Association of America (to A. O.)     

Myosin light chain phosphorylation and the cross-bridge cycle at low substrate concentration in chemically skinned guinea pigTaenia coli

Force-velocity relations, rate of ATP turnover (J_ATP), and phosphorylation of the 20,000 D myosin light chains (LC₂₀) were measured in chemically skinned guinea pigTaenia coli. Relative LC₂₀ phosphorylation at 3.2 mM MgATP was 17% in relaxed tissues at pCa 9, and increased with force at increasing [Ca²⁺] to a maximum of 67% at pCa 4.5. Force at pCa 4.5 was dependent on the MgATP concentration with a half-maximal response at about 0.1 mM. At 0.1 mM MgATP LC₂₀ phosphorylation at pCa 4.5 was 38%. Both J_ATP and the maximal shortening velocity (V _max) were reduced in 0.1 mM MgATP, to 32% and 43%, respectively, of their values at 3.2 mM MgATP. Low-MgATP thus inhibits both LC₂₀ phosphorylation and the extent and rate of cross-bridge interaction. High levels of LC₂₀ phosphorylation, independent of Ca²⁺ and MgATP concentrations, were obtained by treatment with ATP--S. Maximal force at 3.2 mM MgATP after LC₂₀ thiophosphorylation was unchanged, whereas halfmaximal force occurred at 0.065 mM MgATP after thiophosphrylation, compared to 0.13 mM after activation by Ca²⁺. The contraction in thiophosphorylated preparations at low-MgATP (0.1 mM) was associated with submaximalV _max (60%) and J_ATP (27%). The results show that LC₂₀ phosphorylation is correlated with the degree of force development in the Ca²⁺ activated contraction, both when Ca²⁺ and MgATP concentrations are varied. The reduced force and rate of crossbridge turnover in lowMgATP are however primarily mediated by an influence of MgATP on the cross-bridge cycle, which is separate from the effect on LC₂₀ phosphorylation.     

Dissociation of K+-induced tension and cellular Ca2+ retention in vascular and intestinal smooth muscle in normoxia and hypoxia

In experiments on smooth muscle preparations of rabbit aorta and guinea pig taenia coli, replacement of the external Na⁺ with K⁺ produced sustained contraction. When external K⁺ concentration was increased, cellular Ca²⁺ retention as measured by a modified lanthanum technique increased. However, when K⁺ concentration was above 80 mM, the tension decreased despite an increase in Ca²⁺ retention. Maximum amount of Ca²⁺ retained was 1280 nmol/g in aorta and 980 nmol/g in taenia coli while the control values for both tissues were approximately 430 nmol/g when the external Ca²⁺ concentration was 2.5 mM. Under hypoxia (N₂ aeration), sustained contraction was induced by 80 mM K⁺ in aorta and by 45.4 mM K⁺ (and 55 mM glucose) in taenia coli. However, no increase in the cellular Ca²⁺ retention was observed under these conditions. During the K⁺-induced sustained contraction in aorta, introduction of N₂ transiently increased, while readmission of O₂ transiently decreased the muscle tension. In taenia coli, the introduction of N₂ decreased the sustained contractile tension probably because of an ATP deficiency, while the readmission of O₂ further decreased the tension trasniently. From these results, it is concluded that, in the presence of a high concentration of K⁺, external Ca²⁺ enters the cell and activates the contractile machinery. A part of the cellular Ca²⁺ is taken up by mitochondria under normoxic but not under hypoxic conditions.     

The role of orthophosphate in crossbridge kinetics in chemically skinned rabbit psoas fibres as detected with sinusoidal and step length alterations

Summary The role of orthophosphate (P_i) ions in crossbridge kinetics was investigated in chemically skinned rabbit psoas fibres in the presence of saturating Ca²⁺. The muscle length was altered sinusoidally, and the resulting tension time courses were analysed in terms of three exponential processes (A), (B) and (C). Experiments were also performed with step length changes, and the resulting tension transients were correlated with the results of sinusoidal analysis. It was shown that addition of a low millimolar concentration of P_i increased both the rate constant and magnitude of process (B), which resulted in a dramatic increase in the oscillatory power output. The P_i effect was greater at higher oscillation amplitude and at higher MgATP concentration. At 5mm MgATP, the amplitude effect became saturated at a 6 nm length change per crossbridge, whereas the P_i effect did not become saturated in the concentration range tested (0–16mm), An introduction of MgADP to the activating saline resulted in a decrease of all rate constants, and these effects were opposite to MgATP. The effect of P_i resembled neither MgADP nor MgATP. Based on these observations, all the crossbridge reactions except for one (ADP desorption reaction) were eliminated as the possible site of action of P_i ions, supposing that P_i affects only one specific site in the crossbridge cycle. Other mechanisms, which might account for the P_i effects, are the presence of parallel hydrolysis pathways and the presence of multiple sites of action of the P_i ions.     

Modulation of large conductance calcium-activated K+ channel by membrane-delimited protein kinase and phosphatase activities

Large conductance Ca²⁺-activated K⁺ channel was identified and studied in excised inside-out membrane patches of freshly dispersed smooth muscle cells from rabbit gastric antrum. The current-voltage relationship of the single channel was linear from -80 to +80 mV of pipette voltage in which single channel conductance was 249±17.8 pS (n=19) in symmetrical concentration of K⁺ (145mM) across the patch. Activity of the channel (NPo) depended not only on cytoplasmic calcium concentration but also on membrane potential. MgATP increased NPo in a dose-dependent manner and Mg²⁺ was prerequisite for the effect. Okadaic acid (l00nM), inhibitor of protein phosphatases, increased NPo further in the presence of MgATP. Therefore, it would be concluded that activity of the calcium-activated K⁺ channel in gastric smooth muscle cells was controlled by phosphorylation state of the channel protein and the state is further modulated by membrane-delimited protein kinase and protein phosphatase activities.     

Modulation of Ca2+-activated K+ channels of human erythrocytes by endogenous cAMP-dependent protein kinase

 Single Ca²⁺-activated K⁺ channels of human erythrocytes were studied with the patch-clamp technique, to identify the mechanisms of their modulation by phosphorylation. In the cell-attached configuration, the openings of these channels were infrequent, as expected by the low cell Ca²⁺ content. After patch excision, the activity increased to levels determined by the Ca²⁺ concentration (0.5–10 μM) in the bath solution, then the channel activity ran down within a few minutes, to reach values of open probability lower than 0.10. The perfusion of the patch with MgATP increased the channel activity, with delayed and variable effects. Furthermore, the application of a mixture of cAMP (1 mM), MgATP (1 mM) and theophylline (1 mM) to the cytoplasmic side of excised patches led to dramatic enhancement of channel activity, which appeared within 20–120 s and decayed in tens of seconds after wash-out. The activation of the channel by the mixture was reversibly blocked by PKI_5–24, a peptide inhibitor specific to cAMP-dependent protein kinase (PKA). The level of activation promoted by cAMP and ATP was dependent on the Ca²⁺ concentration in the bathing solution. These results provide direct evidence that an endogenous PKA modulates the calcium sensitivity of Ca²⁺-activated K⁺ channels of human erythrocytes. Received: 19 February 1998 / Received after revision: 14 April 1998 / Accepted: 20 April 1998     

Effects of thapsigargin and cyclopiazonic acid on sarcoplasmic reticulum Ca2+ uptake,spontaneous force oscillations and myofilament Ca2+ sensitivity in skinned rat ventricular trabeculae

Thapsigargin (TG) and cyclopiazonic acid (CPA) have been reported to be potent inhibitors of the sarcoplasmic reticulum (SR) Ca²⁺ uptake in isolated SR vesicles and cells. We have examined the effect of TG and CPA on (1) the Ca²⁺ uptake by the SR in saponin-skinned rat ventricular trabeculae, using the amplitude of the caffeine-induced contraction to estimate the Ca²⁺ content loaded into the SR, (2) the spontaneous Ca²⁺ oscillations at pCa 6.6 using force oscillation as the indicator, and (3) the myofilament Ca²⁺ sensitivity in Triton X-100-treated preparations. Inhibition of Ca²⁺ loading by TG and CPA increased with time of exposure to the inhibitor over 18–24 min. TG and CPA produced half inhibition of Ca²⁺ loading at 34.9 and 35.7 μM respectively, when 18–24 min were allowed for diffusion. The spontaneous force oscillations were more sensitive to the inhibitors: 10 μM TG and 30 μM CPA both abolished the oscillations in this time. The myofilament Ca²⁺ sensitivity was not affected by 10 and 300 μM TG or CPA. The results show that the concentrations of TG and CPA necessary to inhibit the SR Ca²⁺ uptake of skinned ventricular trabeculae are much higher than the reported values for single intact myocytes. One reason for this may be slow diffusion of the inhibitors into the multicellular trabecula preparation. Received: 28 July 1995/Received after revision: 11 December 1995/Accepted: 18 December 1995     

Effects of external cadmium ions on excitation–contraction coupling in rat soleus fibres

 The actions of external Cd²⁺ on the twitch and tetanic contractions, action potentials and potassium (K⁺) contractures of rat soleus muscle fibre bundles have been investigated. Cd²⁺ at 1–1.5 mM did not significantly alter tetanic tension, but increased twitch tension and increased the duration and overshoot of action potentials. At ≥3 mM, Cd²⁺ (1) depressed tetanic contractions and initially potentiated but later depressed twitches, (2) abolished the action potential overshoot, and (3) shifted peak K⁺ contracture tension to more positive membrane potentials. Twitch and tetanic contractions, and action potentials remained depressed when Cd²⁺ was washed out of the bath. The effects of Cd²⁺ on the twitch, tetanus and action potential were mimicked by Zn²⁺, while La³⁺ and Co²⁺ at 3 mM – or Mg²⁺ and Ca²⁺ at 30 mM – depressed peak twitch and tetanic tension, but did not potentiate twitches. The results suggest that: (1) Cd²⁺ and Zn²⁺ potentiate twitch tension by prolonging action potential depolarisation; (2) Cd²⁺ depresses twitch and tetanic tension by reducing the action potential overshoot, and causing a positive shift in the voltage dependence of contraction; and (3) the irreversible depression of action potential amplitude in rat soleus muscle is a specific property of Cd²⁺ and Zn²⁺ that is not shared by Co²⁺, Mg²⁺ or Ca²⁺. Received: 14 April 1998 / Received after revision and accepted: 3 September 1998     

A nutrient-regulated cytosolic calcium oscillator in endocrine pancreatic glucagon-secreting cells

 We investigated the influence of nutrients on spontaneous cytosolic calcium oscillations in InR1-G9 glucagonoma cells, a model for pancreatic α-cells. The oscillations depended on calcium release from stores and on calcium influx, partly through voltage-dependent calcium channels. Oscillations required the presence of at least 1 mM glucose, 50 μM alanine, or 50 μM glutamine, but were terminated by higher nutrient concentrations (40 mM glucose, or above 2 mM alanine or glutamine). The effects depended on the metabolism of the nutrients. Glutamine and alanine hyperpolarized the cells. This effect was inhibited (glutamine) or attenuated (alanine) by 1 mM ouabain. Our findings suggest that [Ca²⁺]_i regulation in α-cells is dominated by slow oscillations induced by a lack of metabolic energy, resulting in decreased calcium export and storage, as well as increased calcium influx, partly due to depolarization caused by reduced sodium pump activity. These processes, leading to an elevated cytosolic calcium concentration, may mediate oscillations by calcium-induced calcium release from intracellular stores. Received: 24 June 1998 / Received after revision: 6 October 1998 / Accepted: 12 October 1998     

Addition of phosphate to active muscle fibers probes actomyosin states within the powerstroke

We have measured the effect of phosphate (P_i) on the tension and maximum shortening velocity of permeable rabbit psoas fibers. Work in a number of laboratories has established that addition of phosphate (0–25 mM) to active muscle fibers at physiological MgATP concentrations decreases isometric tension with little effect on the maximum shortening velocity. Here we extend these results to a wider range of P_i concentrations and to low MgATP concentrations. Low levels of P_i (approx. 150 M – 200 M) were obtained by using sucrose phosphorylase and sucrose to reduce contaminating P_i in the solutions used to activate the fiber, and high levels (52–73 mM) were obtained by replacing acetate with P_i as the principal anion. In an activating solution containing either 50 M or 4 mM MgATP, pH 6.2 or 7.0, isometric tension declines linearly with the logarithm of P_i concentration. Although the isometric tension decreases with increasing concentrations of H⁺ or MgATP, the slope of relative isometric tension as a function of log[P_i] is the same at the two values of pH and [MgATP]. At pH 7 and 4 mM MgATP, the velocity of contraction increased slightly as P_i increased from 0.2 to 52 mM. At 50 M MgATP the velocity decreased slightly as P_i increased from 10 to 52 mM. These results are discussed in terms of models of cross-bridge energetics. The observation that force declines linearly with the logarithm of [P_i] is compatible with models in which a major force producing state occurs subsequent to P_i release. The inhibition of shortening velocity by P_i at low concentration of MgATP can be explained by a competition between MgATP and P_i at the end of the cross-bridge powerstroke.