Regulation of force and shortening velocity by calcium and myosin phosphorylation in chemically skinned smooth muscle

The phosphatase inhibitor okadaic acid (OA) was used to study the relationship between [Ca2+], rates of phosphorylation/dephosphorylation and the mechanical properties of smooth muscle fibres. Force/velocity relationships were determined with the isotonic quick release technique in chemically skinned guinea-pig taenia coli muscles at 22 degrees C. In the maximally thiophosphorylated muscle neither OA (10 microM) nor Ca2+ (increase from pCa 9.0 to pCa 4.5) influenced the force-velocity relationship. When the degree of activation was altered by varying [Ca2+] in the presence of 0.5 microM calmodulin, both force and the maximal shortening velocity (Vmax) were altered. At pCa 5.75, at which force was about 35% of the maximal at pCa 4.5, Vmax was 55% of the maximal value. When OA was introduced into fibres at pCa 6.0, force was increased from less than 5% to 100% of the maximal force obtained in pCa 4.5. The relationship between the degree of myosin light chain phosphorylation and force was similar in the two types of activation; varied [OA] at constant [Ca2+] and at varied [Ca2+]. The relation between force and Vmax when the degree of activation was altered with OA was almost identical to that obtained with varied [Ca2+]. The results show that Ca2+ and OA do not influence force or Vmax in the maximally phosphorylated state and suggest that the level of myosin light chain phosphorylation is the major factor determining Vmax. The finding that the relationship between force and Vmax was similar when activation was altered with OA and Ca2+ suggests, however, that alterations in the absolute rates of phosphorylation and dephosphorylation at a constant phosphorylation level do not influence the mechanical properties of the skinned smooth muscle fibres.     

Protein kinase A increases the tension cost and unloaded shortening velocity in skinned rat cardiac muscle

To address controversies concerning the effect of beta-adrenergic stimulation on the rate of cross-bridge cycling in cardiac muscle, we measured ca(2+)-induced isometric tension development, unloaded shortening velocity (Vmax) and ATPase activity of demembranated (Triton X-100 skinned) rat right ventricular trabeculae before and after treatment with the catalytic subunit of protein kinase A (PKA), which is known to mimic the action of beta-adrenergic agonists in demembranated preparations. PKA treatment (1 U/microliter, 40 min) shifted the pCa-tension relation to the right from 5.41 to 5.26 at pCa50 (the [Ca2+] required for half maximal steady state tension) without changing the steepness of the pCa-tension relation and the maximum Ca(2+)-activated tension; Vmax, as determined by the slack test, was increased for a given pCa value, despite the reduced level of isometric tension. PKA treatment also shifted the pCa-ATPase activity to the right slightly from 5.47 to 5.40 at pCa50 (the [Ca2+] required for half maximal ATPase activity), but increased the ATPase activity during a given level of steady isometric tension generation, resulting in a 33% increase of the tension cost (ATPase activity/tension). All the results obtained strongly suggest that, in rat right ventricular trabeculae, beta-adrenergic stimulation may increase the rate of cross-bridge cycling by increasing the rate of cross-bridge detachment from actin through a PKA-mediated mechanism, although PKA reduces the Ca(2+)-sensitivity of the contractile system.     

Effects of Mg2+ on Ca2+ handling by the sarcoplasmic reticulum in skinned skeletal and cardiac muscle fibres

The influence of myoplasmic Mg2+ (0.05-10 mM) on Ca2+ accumulation (net Ca2+ flux) and Ca2+ uptake (pump-driven Ca2+ influx) by the intact sarcoplasmic reticulum (SR) was studied in skinned fibres from the toad iliofibularis muscle (twitch portion), rat extensor digitorum longus (EDL) muscle (fast twitch), rat soleus muscle (slow twitch) and rat cardiac trabeculae. Ca2+ accumulation was optimal between 1 and 3 mM Mg2+ in toad fibres and reached a plateau between 1 and 10 mM Mg2+ in the rat EDL fibres and between 3 and 10 mM Mg2+ in the rat cardiac fibres. In soleus fibres, optimal Ca2+ accumulation occurred at 10 mM Mg2+. The same trend was obtained with all preparations at 0.3 and 1 microM Ca2+. Experiments with 2,5-di-(tert-butyl)-1,4-benzohydroquinone, a specific inhibitor of the Ca2+ pump, revealed a marked Ca2+ efflux from the SR of toad iliofibularis fibres in the presence of 0.2 microM Ca2+ and 1 mM Mg2+. Further experiments indicated that the SR Ca2+ leak could be blocked by 10 microM ruthenium red without affecting the SR Ca2+ pump and this allowed separation between SR Ca2+ uptake and SR Ca2+ accumulation. At 0.3 microM Ca2+, Ca2+ uptake was optimal with 1 mM Mg2+ in the toad iliofibularis and rat EDL fibres and between 1 and 10 mM Mg2+ in the rat soleus and trabeculae preparations. At higher [Ca2+] (1 microM), Ca2+ uptake was optimal with 1 mM Mg2+ in the iliofibularis fibres and between 1 and 3 mM Mg2+ in the EDL fibres.(ABSTRACT TRUNCATED AT 250 WORDS)     

Force regulation by Ca2+ in skinned single cardiac myocytes of frog

Atrial and ventricular myocytes 200 to 300 microm long containing one to five myofibrils are isolated from frog hearts. After a cell is caught and held between two suction micropipettes the surface membrane is destroyed by briefly jetting relaxing solution containing 0.05% Triton X-100 on it from a third micropipette. Jetting buffered Ca2+ from other pipettes produces sustained contractions that relax completely on cessation. The pCa/force relationship is determined at 20 degrees C by perfusing a closely spaced sequence of pCa concentrations (pCa = -log[Ca2+]) past the skinned myocyte. At each step in the pCa series quick release of the myocyte length defines the tension baseline and quick restretch allows the kinetics of the return to steady tension to be observed. The pCa/force data fit to the Hill equation for atrial and ventricular myocytes yield, respectively, a pK (curve midpoint) of 5.86 +/- 0.03 (mean +/- SE.; n = 7) and 5.87 +/- 0.02 (n = 18) and an nH (slope) of 4.3 +/- 0.34 and 5.1 +/- 0.35. These slopes are about double those reported previously, suggesting that the cooperativity of Ca2+ activation in frog cardiac myofibrils is as strong as in fast skeletal muscle. The shape of the pCa/force relationship differs from that usually reported for skeletal muscle in that it closely follows the ideal fitted Hill plot with a single slope while that of skeletal muscle appears steeper in the lower than in the upper half. The rate of tension redevelopment following release restretch protocol increases with Ca2+ >10-fold and continues to rise after Ca2+ activated tension saturates. This finding provides support for a strong kinetic mechanism of force regulation by Ca2+ in frog cardiac muscle, at variance with previous reports on mammalian heart muscle. The maximum rate of tension redevelopment following restretch is approximately twofold faster for atrial than for ventricular myocytes, in accord with the idea that the intrinsic speed of the contractile proteins is faster in atrial than in ventricular myocardium.     

A novel phasic contraction induced by dithiothreitol in frog skeletal muscle

1. Dithiothreitol (DTT), at 50-100 mM, induced a phasic reversible contraction of frog skeletal muscle. 2. Exposure of single fibers to nifedipine (20 microM), an L-type Ca2+ antagonist, blocked the twitch and tetanus tensions but never affected the DTT-induced contraction. 3. DTT also produced a phasic contraction in fibers where voltage sensors were inactivated in the presence of high K+ concentration (190 mM). 4. A fiber was mechanically skinned after observation of DTT-induced contraction. The skinned fiber contracted in response to a DTT concentration similar to that required to produce contraction in intact fibers before skinning. 5. In skinned fibers, DTT, at 100 or 200 mM, inhibited the accumulation of Ca2+ by SR, but not Ca2+ ATPase activity. 6. These results suggest that a high concentration of DTT triggers Ca2+ efflux from the SR through action on the Ca2+ release channel and/or closely associated proteins, such as triadin and FK-506 binding protein.     

Expression analysis of the mixed function oxidase system in rat brain by the polymerase chain reaction

To characterize the calcium (Ca2+)-releasing effects of histamine and GTP gamma S, the drug-induced tension developments were measured in beta-escin-treated skinned longitudinal smooth muscle of guinea pig ileum. Intracellular Ca2+ stores were loaded with Ca2+ by incubating the muscle for 10 min in a Ca(2+)-containing solution. Histamine (10-100 microM), applied after Ca(2+)-loading, produced a transient rise in tension. The effect of histamine was not preserved after treatment with 20 mM caffeine, a Ca(2+)-store releaser. The effect of histamine was potentiated by GTP; inhibited by GDP beta S, an antagonist of GTP for binding to G-proteins; or heparin, an antagonist of inositol 1,4,5-trisphosphate (IP3) for binding to its receptor; and mimicked by IP3. When GTP gamma S (20 microM) was applied and continued to be present for 15 min, a transient rise in tension followed by a small, sustained rise in tension was elicited. The effect of GTP gamma S was completely inhibited by GDP beta S. The initial, transient component of the biphasic GTP gamma S response was abolished or markedly inhibited after treatment with caffeine, heparin or the calcium ionophore A23187. The present results suggest that histamine and GTP gamma S cause a release of Ca2+ from caffeine-sensitive stores which is mediated by IP3 formed through a G-protein-coupled mechanism. The GTP gamma S-induced Ca2+ release is not considered to involve such an IP3-independent process as described in chemically-skinned arterial muscle.     

Calcium modulates the influence of length changes on the myofibrillar adenosine triphosphatase activity in rat skinned cardiac trabeculae

The relationship between adenosine triphosphate (ATP) turnover and muscle performance was investigated in skinned cardiac trabeculae of the rat at different [Ca2+] and two different sarcomere lengths (1.8 microns and 2.2 microns) at 20 degrees C. ATP turnover was measured photometrically by enzymatic coupling of the regeneration of ATP to the oxidation of reduced nicotinamide adenine dinucleotide. The trabeculae were studied under isometric conditions and when the length was altered repetitively at a frequency of 23 Hz, with a square wave, by 5% of the initial length. The isometric ATPase activity amounted to 0.48 mM/s. Isometric ATP turnover and force were proportional at different [Ca2+]. During length changes at maximal activation (pCa 4.27) and 2.2 microns sarcomere length, ATPase activity increased to up to 162% whereas at low [Ca2+], ATPase activity decreased with respect to the isometric value at that pCa. At pCa 5.5, ATPase activity was reduced to 33%. These results indicate that during the length changes the apparent cross-bridge detachment rate is increased and the apparent attachment rate is decreased. The findings suggest that the Fenn effect, i.e. the increase in energy turnover above the isometric value during shortening, is present in cardiac trabeculae at high levels of activation, but is absent or reversed at lower levels of activity.     

MgADP promotes a catch-like state developed through force-calcium hysteresis in tonic smooth muscle

Tonic rabbit femoral artery and phasic rabbit ileum smooth muscles permeabilized with Triton X-100 were activated either by increasing [Ca2+] from pCa > 8.0 to pCa 6.0 (calcium-ascending protocol) or contracted at pCa 6.0 before lowering [Ca2+] (calcium-descending protocol). The effects of, respectively, high [MgATP]/low [MgADP] [10 mM MgATP + creatine phosphate (CP) + creatine kinase (CK)] or low [MgATP]/[MgADP] (2 mM MgATP, 0 CP, 0 CK) on the "force-[Ca]" relationships were determined. In femoral artery at low, but not at high, [MgATP]/[MgADP] the force and the ratio of stiffness/force at pCa 7.2 were significantly higher under the calcium-descending than calcium-ascending protocols (54% vs. 3% of Po, the force at pCa 6.0) (force hysteresis); the levels of regulatory myosin light chain (MLC20) phosphorylation (9 +/- 2% vs. 10 +/- 2%) and the velocities of unloaded shortening V0 (0.02 +/- 0.004 l/s with both protocols) were not significantly different. No significant force hysteresis was detected in rabbit ileum under either of these experimental conditions. [MgADP], measured in extracts of permeabilized femoral artery strips by two methods, was 130-140 microM during maintained force under the calcium-descending protocol. Exogenous CP (10 mM) applied during the descending protocol reduced endogenous [MgADP] to 46 +/- 10 microM and abolished force hysteresis: residual force at low [Ca2+] was 17 +/- 5% of maximal force. We conclude that the proportion of force-generating nonphosphorylated (AMdp) relative to phosphorylated cross-bridges is higher on the Ca2+-descending than on the Ca2+-ascending force curve in tonic smooth muscle, that this population of positively strained dephosphorylated cross-bridges has a high affinity for MgADP, and that the dephosphorylated AMdp . MgADP state makes a significant contribution to force maintenance at low levels of MLC20 phosphorylation.     

Effects of pH and temperature on myocardial calcium-activation in Pterygoplichthys (catfish)

Fish are chronically exposed to a wide range of temperatures and acidic environments. Fish hearts have to therefore adapt to these changes in order to maintain contractility. Myofibrillar responsiveness to Ca2+ is exquisitely sensitive to both temperature and pH in mammalian myocardium. To evaluate myofilament calcium-activation, we chemically skinned ventricular myocardium from catfish (Pterygoplichthys). A decrease in pH from 7.5 to 6.8, irrespective of temperature change, shifted the calcium-force curve towards higher calcium concentrations without affecting maximal Ca(2+)-activated force. The contractile elements are therefore sensitive to changes in pH. In intact muscle preparations the active twitch force was decreased with increasing temperature (10-22 degrees C). However, the sensitivity of the myofilaments to Ca2+ was independent of temperature. These data suggest a possible role of the sarcoplasmic reticulum (SR) in mediating the effects of temperature. The response of intact muscle preparations to changes in temperature is therefore not likely due to temperature-dependent changes in myofilament calcium responsiveness.     

Cross-bridge movement in rat slow skeletal muscle as a function of calcium concentration

A single fibre bundle from rat soleus muscle was chemically skinned with saponin and the transfer of myosin heads from the thick filaments to the thin filaments at a sarcomere length of 2.4 microm was measured as a function of Ca2+ concentration using an x-ray diffraction method at 4-7 degrees C. In the relaxed state, the 1,0 spacing was 42.08 nm. The spacing showed no significant decrease when the Ca2+ concentration was below the threshold (-log10 [Ca2+] or pCa 5.8). No significant transfer of the myosin heads occurred when the Ca2+concentration was below the threshold (pCa 5.8). When the muscle was maximally activated at pCa 4.4, the spacing decreased to 40.35 nm. During the maximum isometric contraction at pCa 4.4, 54. 9 +/- 6.5% (+/-SE of the mean) of the myosin heads were transferred to the thin filaments. The transfer of the myosin heads was approximately proportional to relative tension. These results suggest that myosin heads of both fast-twitch and slow-twitch skeletal muscles transferred on the common movement as a function of Ca2+ concentration.     

Molecular mechanisms in the control of limb regeneration: the role of homeobox genes

The Ca2+ sensitivity of cardiac myofibrillar force production can be decreased by acidosis or inorganic phosphate (P(i)) and increased by caffeine. To investigate whether the source of tissue influences the potency of these agents, we compared the actions of acidosis (change of pH from 7.0 to 6.2), P(i) and caffeine (both 20 mM) on force production of skinned cardiac muscles from adult ventricle, adult atrium and neonate ventricle of the rat. Maximum Ca(2+)-activated force was reduced by all three interventions and the responses of the different muscle types to a given intervention were similar. Acidosis reduced myofibrillar Ca2+ sensitivity by 1.09 and 1.04 pCa units in adult ventricle and atrium, respectively, and P(i) reduced it by 0.19 and 0.22 pCa units. However, each effect was only one-third as great in the neonate ventricle, which showed falls of 0.33 pCa units for acidosis and 0.06 for P(i). In contrast, caffeine raised the Ca2+ sensitivity by the same amount (approximately 0.4 pCa units) in all three muscle types. The differential effect between adult and neonate seen with both acidosis and P(i) suggests some similarity in the mechanisms by which these factors decrease Ca2+ sensitivity. In contrast, the equal effects of caffeine on neonate and adult suggests that caffeine acts by a completely different mechanism. The lower pH- and P(i)-sensitivity of the neonatal ventricle can help to explain why neonatal and adult myocardium exhibit differential force responses to ischaemia (or hypoxia alone).     

Relationship between the ability of sunscreens containing 2-ethylhexyl-4''-methoxycinnamate to protect against UVR-induced inflammation, depletion of epidermal Langerhans (Ia+) cells and suppression of alloactivating capacity of murine skin in vivo

Phalloidin was shown to increase the ATPase activity and Ca2+ sensitivity of both bovine cardiac and rabbit psoas myofibrils when assayed in a solution containing 50 mM KCl, 100 mM MOPS (pH 7.0), 2 mM MgCl2, 1 mM ATP, 2 mM EGTA, and varying concentrations of Ca2+ (temperature 21-22 degrees C). The phalloidin effect in cardiac myofibrils developed over a time course of several minutes in the presence of 50 microM phalloidin. Relative increase of ATPase activity was maximal at pCa 8 and decreased with decrease in pCa. In cardiac myofibrils the increase was about 70% at pCa 8 and 20% at pCa 4 following 20-30 min pre-incubation with 2 microM or 50 microM phalloidin. The effect persisted after excess phalloidin was washed out. The increase in Ca2+ sensitivity was approximately 0.15 pCa units. For skeletal myofibrils treated with 2 microM phalloidin all changes were considerably less than those seen with cardiac myofibrils and the changes were even less when the myofibrils were exposed to 50 microM phalloidin. These results show that when specifically bound to actin, phalloidin can change the kinetic parameters of the cross-bridge cycle and may also alter the Ca2+ sensitivity of the contractile system. The effects of phalloidin seem to vary with muscle type.     

Mitochondrial calcium in relaxed and tetanized myocardium

The elemental composition of rat cardiac muscle was determined with electron probe x-ray microanalysis (EPMA) of rapidly frozen papillary muscles and trabeculae incubated with ryanodine (1 microM) in either 1.2 or 10 mM [Ca2+]o-containing solutions, paced at 0.6 Hz or tetanized at 10 Hz. Total mitochondrial calcium increased significantly, by 4.2 mmol/kg dry weight during a 7 s tetanus, only in muscles tetanized in the presence of 10 mM [Ca2+]o when cytoplasmic Ca2+ is 1-4 microM (Backx, P. H., W.-D. Gao, M. D. Azan-Backx, and E. Marban. 1995. The relationship between contractile force and intracellular [Ca2+] in intact rat trabeculae. J. Gen. Physiol. 105:1-19). Comparison of total mitochondrial with free mitochondrial Ca2+ reported in the literature indicates that the total/free ratio is approximately 6000 at physiological or near-physiological levels of total mitochondrial calcium. Increases in free mitochondrial [Ca2+] consistent with regulation of mitochondrial enzymes should be associated with increases in total mitochondrial calcium detectable with EPMA. However, such increases in mitochondrial calcium occur only as the result of prolonged, unphysiological elevations of cytosolic [Ca2+].     

Alterations in the force-frequency relationship by tert-butylbenzohydroquinone, a putative SR Ca2+ pump inhibitor, in rabbit and rat ventricular muscle

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.     

Isometric and dynamic contractile properties of porcine skinned cardiac myocytes after stunning

The purpose of this study was to investigate myofibrillar mechanisms of depressed contractile function associated with myocardial stunning. We first tested whether the degree of stunning was directly related to changes in myofilament Ca2+ sensitivity. Variable degrees and durations of low-flow ischemia were followed by 30 minutes of reperfusion in an open-chest porcine model of regional myocardial stunning (n = 27). Ca2+ sensitivity of isometric tension was measured in skinned myocytes obtained from endocardial biopsies taken during control aerobic flow and after 30 minutes of reperfusion. The degree of stunning, as assessed by percent systolic wall thickening, ranged from -3% to 75% of control but did not correlate (r = .11) with changes in pCa50, ie, pCa for half-maximal tension. Only in the group (n = 10) with the most severe level of ischemia was there a significant decrease in pCa50 (from 5.97 +/- 0.06 in the control condition to 5.86 +/- 0.07 after ischemia, P < .05). Less severe levels of ischemia (n = 17) resulted in significant stunning (percent systolic wall thickening, 38 +/- 4% of control) but no change in pCa50. To investigate the possibility that alterations in myofibrillar cross-bridge kinetics contribute to depressed function in stunning, maximum velocity of shortening (Vo) was measured in postischemic myocytes. Vo in postischemic myocytes was reduced to 56 +/- 4% of Vo in control myocytes and was independent both of the degree of stunning (r = .26) and changes in Ca2+ sensitivity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Uridine triphosphate-sensitive pathway of Ca2+ release from the sarcoplasmic reticulum of rat skeletal muscle fibers

The pyrimidine nucleotide, uridine triphosphate (UTP), was tested with skinned skeletal muscle fibers in order to investigate the UTP-sensitive pathway of Ca2+ release from the sarcoplasmic reticulum. The presence of ryanodine (200 microM), ruthenium red (10 microM) or heparin (2.5 mg/ml) did not affect the tension elicited in the presence of UTP, demonstrating that the UTP-induced Ca2+ release involved neither ryanodine nor inositol triphosphate-sensitive channels. Drugs such as compound 48/80 or cyclopiazonic acid used to inhibit Ca2+-ATPase in its reverse function appeared to be, respectively, non-specific or without any inhibitory effect on the tension induced by UTP. Finally, the UTP-induced tension as well as the trifluoperazine-induced tension were abolished in the presence of spermidine (50 mM), supporting the hypothesis that the UTP-sensitive pathway of the SR Ca2+ release might occur through the uncoupled calcium ATPase.     

Steady state relation between cytoplasmic free Ca2+ concentration and force in intact frog skeletal muscle fibers

The steady state relation between cytoplasmic Ca2+ concentration ([Ca2+]i) and force was studied in intact skeletal muscle fibers of frogs. Intact twitch fibers were injected with the dextran-conjugated Ca2+ indicator, fura dextran, and the fluorescence signals of fura dextran were converted to [Ca2+]i using calibration parameters previously estimated in permeabilized muscle fibers (Konishi and Watanabe. 1995. J. Gen. Physiol. 106:1123-1150). In the first series of experiments, [Ca2+]i and isometric force were simultaneously measured during high K+ depolarization. Slow changes in [Ca2+]i and force induced by 15-30 mM K+ appeared to be in equilibrium, as instantaneous [Ca2+]i versus force plot tracked the common path in the rising and relaxation phases of K+ contractures. In the second series of experiments, 2,5-di-tert-butylhydroquinone (TBQ), an inhibitor of the sarcoplasmic reticulum Ca2+ pump, was used to decrease the rate of decline of [Ca2+]i after tetanic stimulation. The decay time courses of both [Ca2+]i and force were dose-dependently slowed by TBQ up to 5 micro M; the instantaneous [Ca2+]i- force relations were nearly identical at >/=1 micro M TBQ, suggesting that the change in [Ca2+]i was slow enough to reach equilibrium with force. The [Ca2+]i-force data obtained from the two types of experiments were consistent with the Hill curve using a Hill coefficient of 3.2-3.9 and [Ca2+]i for half activation (Ca50) of 1.5-1.7 micro M. However, if fura dextran reacts with Ca2+ with a 2.5-fold greater Kd as previously estimated from the kinetic fitting (Konishi and Watanabe. 1995. J. Gen. Physiol. 106:1123-1150), Ca50 would be 3.7-4.2 micro M. We also studied the [Ca2+]-force relation in skinned fibers under similar experimental conditions. The average Hill coefficient and Ca50 were estimated to be 3.3 and 1.8 microM, respectively. Although uncertainties remain about the precise levels of [Ca2+]i, we conclude that the steady state force is a 3rd to 4th power function of [Ca2+]i, and Ca50 is in the low micromolar range in intact frog muscle fibers, which is in reasonable agreement with results obtained from skinned fibers.     

Mechanisms of action of isoflurane on contraction of rabbit conduit artery

Isoflurane may cause differential effects on different vascular beds of the same animal species. The mechanisms of this action have not been elucidated. Accordingly, we compared in rabbit aorta and femoral artery the effects of isoflurane (1-3.3%) in isolated rings (endothelium denuded) activated by norepinephrine, and isoflurane effects on Ca2+ fluxes from the sarcoplasmic reticulum in skinned strips. When < 30 nM norepinephrine was used to cause ring contraction, isoflurane increased the force of contraction in aortic rings, but decreased force in femoral arterial rings. At 30 nM norepinephrine stimulation, 3.3% isoflurane decreased the force and, in the presence of verapamil, isoflurane actually increased the force in both arterial types. In skinned strips of both arterial types, isoflurane present during Ca2+ uptake decreased the caffeine-induced tension transients, whereas isoflurane present during Ca2+ release enhanced the transients. Isoflurane potentiated the depression of the tension transients by ryanodine. Isoflurane directly caused contracture even in the absence of caffeine. Thus, isoflurane has similar cellular mechanisms of action in the aortic and femoral arterial smooth muscle: inhibiting Ca2+ influx through the sarcolemma, decreasing Ca2+ uptake by the sarcoplasmic reticulum, and enhancing caffeine-induced Ca2+ release from the sarcoplasmic reticulum.     

High selectivity for inhibition of phosphodiesterase III and positive inotropic effects of MCI-154 in guinea pig myocardium

MCI-154 (0.3-100 microM) exerted a concentration-dependent positive inotropic effect in isolated guinea pig papillary muscles (EC50 0.8 microM). The efficacy of MCI-154 (253% of predrug value) was 1.7-fold higher than that of saterinone but comparable to that of milrinone. Carbachol markedly reduced the increase in force of contraction (FOC) of MCI-154. In intact contracting papillary muscles, the positive inotropic effect was accompanied by an increase in cyclic AMP content to 0.78 +/- 0.09 pmol/mg wet weight (n = 10), corresponding to 150% of the basal value (0.51 +/- 0.05 pmol/mg wet weight, n = 21) in the presence of submaximal cyclic AMP phosphodiesterase (PDE) isoenzyme III inhibiting concentrations of MCI-154 (30 microM). MCI-154 (1-1,000 microM) concentration-dependently inhibited the activity of PDE III from homogenates of guinea pig myocardium. The IC50 was 3.8 microM. PDE I, II, and IV were not significantly affected up to 100 microM (PDE I and IV) and up to 1,000 microM (PDE II). In comparison, milrinone and saterinone were PDE III/IV-selective PDE inhibitors. Rolipram inhibited PDE IV only. IBMX and theophylline were nonselective PDE inhibitors. MCI-154 had only a marginal positive chronotropic effect. The frequency of spontaneously beating right auricles from guinea pig heart was increased by 8.7% at most (n = 5). MCI-154 increased Ca2+ sensitivity in chemically skinned porcine ventricular muscle fibers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium handling by the sarcoplasmic reticulum during oscillatory contractions of skinned skeletal muscle fibres

Isometric ATP consumption and force were investigated in mechanically skinned fibres from iliofibularis muscle of Xenopus laevis. Measurements were performed at different [Ca2+], in the presence and absence of caffeine (5 nM). In weakly Ca2+-buffered solutions without caffeine, spontaneous oscillations in force and ATPase activity occurred. The repetition frequency was [Ca2+]-and temperature-dependent. The Ca2+ threshold (+/- SEM) for the oscillations corresponded to a pCa of 6.5 +/- 0.1. The maximum ATP consumption associated with calcium uptake by the sarcoplasmic reticulum (SR) reached during the oscillations was similar to the activity under steady-state conditions at saturating calcium concentrations in the presence of caffeine. Maximum activity was reached when the force relaxation was almost complete. The calculated amount of Ca2+ taken up by the SR during a complete cycle corresponded to 5.4 +/ 0.4 mmol per litre cell volume. In strongly Ca2+-buffered solutions, caffeine enhanced the calcium sensitivity of the contractile apparatus and, at low calcium concentrations, SR Ca uptake. These results suggest that when the SR is heavily loaded by net Ca uptake, there is a massive calcium-induced calcium release. Subsequent net Ca uptake by the SR then gives rise to the periodic nature of the calcium transient.