Force development at elevated [Mg2+]o and [K+]o in myocardium from the freshwater turtle (Trachemys scripta) and influence of factors associated with hibernation

The effects of high [Mg(2+)](o) on force development were examined for heart muscle of freshwater turtle. Plasma [Mg(2+)] during hibernation may increase drastically and like plasma [K(+)] approach values as high as 10 mM. Each experiment performed at either 20 or 5 degrees C involved four ventricular preparations of which one pair was exposed to 10, and one to 1 mMMg(2+). One preparation of each pair was furthermore exposed to 10 mM K(+), whereas the other was maintained at 2.5 mM K(+). During oxygenation, high relative to low [Mg(2+)](o) displayed a weak tendency to reduce twitch force; a tendency that was not reduced by elevations of [Ca(2+)](o). Severe hypoxia accentuated the negative effect of high [Mg(2+)](o). This effect disappeared, however, when hypoxia was combined with acidosis obtained by 24 mM lactic acid. In comparison to [Mg(2+)](o), high [K(+)](o) strongly depressed force development under both oxygenation and hypoxia, but no consistent interplay between the two ions was revealed. The negative inotropic effects of both high [Mg(2+)](o) and high [K(+)](o) were reduced or eliminated by 10 muM adrenaline. In conclusion the cardiac effects of elevations in [Mg(2+)](o) appear to be small during hibernation, in particular when considering the concomitant adrenergic stimulation and acidosis.     

Oxygen consumption and force development in turtle and trout cardiac muscle during acidosis and high extracellular potassium

Relative to species such as rainbow trout, freshwater turtle shows a high tolerance to challenges involving acidosis and increases in extracellular K+. Therefore, the effects of acidosis or high K+ on twitch force and oxygen consumption were examined in ventricular ring preparations from these two species. The oxygen consumption associated with force development was estimated by net oxygen consumption (oxygen consumption during twitch force development minus that during rest). For turtle, elevation of CO2 from 2% (pH 7.7) to 12% (pH 6.9) in the gas equilibrating the muscle bath decreased twitch force by 20% without any effects on oxygen consumption. Decreasing pH from 7.7 to 6.9 with 22 mM lactic acid had similar effects. For trout, CO2-induced acidosis decreased twitch force by approximately 60%. Furthermore, force development became energetically less efficient as it fell disproportionately more than net oxygen consumption. This was not observed for lactic acidosis. For trout but not for turtle, acidosis resulted in an increase in oxygen consumption during rest. An increase in extracellular K+ from 2.5 mM to 10 mM depressed force and oxygen consumption proportionately for both species. Adrenaline (10 microM) increased twitch force for both species and oxygen consumption for trout; it attenuated the effects of high extracellular K+. Neither adrenaline nor high K+ influenced the ratio of force to net oxygen consumption. As opposed to high extracellular K+, acidosis appears to increase the energetic cost of contractility, particularly for the trout heart.     

Contribution of sarcolemmal sodium-calcium exchange and intracellular calcium release to force development in isolated canine ventricular muscle

The aim of this work was to determine the relationship between peak twitch amplitude and sarcoplasmic reticulum (SR) Ca2+ content during changes of stimulation frequency in isolated canine ventricle, and to estimate the extent to which these changes were dependent upon sarcolemmal Na(+)-Ca2+ exchange. In physiological [Na+]o, increased stimulation frequency in the 0.2-2-Hz range resulted in a positive inotropic effect characterized by an increase in peak twitch amplitude and a decrease in the duration of contraction, measured as changes in isometric force development or unloaded cell shortening in intact muscle and isolated single cells, respectively. Action potentials recorded from single cells indicated that the inotropic effect was associated with a progressive decrease of action potential duration and a marked reduction in average time spent by the cell near the resting potential during the stimulus train. The frequency-dependent increase of peak twitch force was correlated with an increase of Ca2+ uptake into and release from the SR. This was estimated indirectly using the phasic contractile response to rapid (less than 1 s) lowering of perfusate temperature from 37 degrees C to 0-2 degrees C and changes of twitch amplitude resulting from perturbations in the pattern of electrical stimulation. Lowering [Na+]o from 140 to 70 mM resulted in an increase of contractile strength, which was accompanied by a similar increase of apparent SR Ca2+ content, both of which could be abolished by exposure to ryanodine (1 x 10(-8) M), caffeine (3 x 10(-3) M), or nifedipine (2 x 10(-6) M). Increased stimulation frequency in 70 mM [Na+]o resulted in a negative contractile staircase, characterized by a graded decrease of peak isometric force development or unloaded cell shortening. SR Ca2+ content estimated under identical conditions remained unaltered. Rate constants derived from mechanical restitution studies implied that the depressant effect of increased stimulation frequency in 70 mM [Na+]o was not a consequence of a decreased rate of refilling of a releasable pool of Ca2+ within the cell. These results demonstrate that frequency-dependent changes of contractile strength and intracellular Ca2+ loading in 140 mM [Na+]o require the presence of a functional sarcolemmal Na(+)-Ca2+ exchange process. The possibility that the negative staircase in 70 mM [Na+]o is related to inhibition of Ca(2+)-induced release of Ca2+ from the SR by various cellular mechanisms is discussed.     

The effects of different types of acidosis and extracellular calcium on the mechanical activity of turtle atria

Summary The effects of acidosis and extracellular calcium were examined at 20°C in the isolated spontaneously contracting atria of the freshwater turtle (Chrysemys picta bellii). The atria were subjected to treatments of lactic acidosis, hypercapnic acidosis or chloride acidosis in the presence of both normal (2.0 mM) and high (10.0 mM) calcium, which simulated levels of acidosis and calcium observed in vivo. In all cases of acidosis, pH was reduced to 6.80 from a control pH of 7.80.All three forms of acidosis significantly depressed the force of atrial contraction. During lactic and chloride acidosis a progressive decrease in contractile force was seen, while during hypercapnic acidosis a spontaneous partial recovery was observed following an initial sharp drop in tension. Hypercapnic acidosis had the most rapid effect on contractility, while chloride had the slowest effect.Elevated levels of calcium during lactic and hypercapnic acidoses significantly moderated the negative inotropic effects of acidosis, although contractile force was still below pre-acid values. During chloride acidosis with increased [Ca], no decline in contractile force was observed compared to the control values. Each of the three types of acidoses caused a significant decrease in the frequency of the spontaneous atrial contractions but this effect was not significantly improved with acidosis plus increased [Ca].Based on the present findings and on related observations of acidosis, it appears that the fresh-water turtle is able to compensate for the negative inotropic effects on the heart of both lactic and hypercapnic acidosis, and these compensations may contribute to its remarkable tolerance to anoxia.     

Inotropic effects of adrenaline on the anoxic or hypercapnic myocardium of rainbow trout and eel

Summary The effects of adrenaline on the development of force under anoxia and hypercapnic acidosis (13% CO₂ in 30 mM HCO ₃ ^– ) were examined in isolated, electrically stimulated cardiac ventricle strips of rainbow trout and eel.During anoxia or acidosis applied 15 min in advance, the adrenaline concentration of the bathing solution was increased in 5 steps from 0 to 10^–4 M with 5 min at each step. Before levelling off the contractile tension increased by 145±42% (mean±SE) in the anoxic, 80±14% in the acidotic and 152±41% in the control trout cardiac strips. Except for the acidotic strips the corresponding values tended to be lower for the eel strips being 46±9%, 57±17% and 57±9%, respectively. The inotropic affinity for adrenaline was lower in the trout than in the eel myocardium. For the trout myocardium it remained unchanged, while it decreased somewhat for the eel myocardium under anoxia or acidosis.Adding to the muscle bath 10^–5 M adrenaline resulted in an increase in force development by about 90% for the trout myocardium and 50% for the eel myocardium. 5 min later anoxia or hypercapnic acidosis was applied for 30 min followed by 30 min at control conditions. Relative to the force values recorded just before anoxia or acidosis was applied, the changes in contractile force during these periods were the same with and without adrenaline. Thus adrenaline appears to have a persistent positive inotropic effect in the fish myocardium during and after oxygen lack or acidosis.     

Different effects of simple anoxic lactic acidosis and simulated in vivo anoxic acidosis on turtle heart.

We compared responses of turtle heart at 20 degrees C to an anoxic lactic acidosis solution (LA) containing 35 mM lactic acid in an otherwise normal turtle Ringers equilibrated with 3% CO2/97% N2 at pH 7.0) to a solution simulating in vivo anoxic acidosis (VA), with elevated concentrations of lactate, Ca2+, Mg2+, and K+, and decreased Cl-, equilibrated with 10.8% CO2/89.2% N2 at pH 7.0. We examined mechanical properties on cardiac muscle strips and determined intracellular pH (pHi) and high energy phosphates on perfused hearts using 31P-NMR. Maximum active force (Fmax) and the maximum rate of force development (dF/dtmax) of muscle strips were significantly higher during VA than during LA superfusion. An elevation of Ca2+ alone (to 6 mM) in LA significantly increased both Fmax and dF/dtmax but the effects diminished toward the end of the exposure; however, hypercapnic anoxic lactic acidosis (addition of 20 mM HCO3- to LA, equilibrated with 10.8% CO2/89.2% N2, pH 7.0) did not significantly affect Fmax or dF/dtmax. During VA perfusion, pHi (6.73 +/- 0.01) was significantly higher than that during LA perfusion (pHi 6.69 +/- 0.013), but the difference is probably too small to have physiological significance. ATP, creatine phosphate, and inorganic phosphate were not significantly different in the two anoxic solutions. We conclude that the reduction of cardiac mechanical function in vivo is minimized by the integrated effects of changes of ionic concentrations, but the observed changes in Ca2+ and pHi cannot fully explain the effect.     

Lactic acid restores skeletal muscle force in an in vitro fatigue model: are voltage-gated chloride channels involved?

High interstitial K(+) concentration ([K(+)]) has been reported to impede normal propagation of electrical impulses along the muscle cell membrane (sarcolemma) and then also into the transverse tubule system; this is one considered underlying mechanism associated with the development of muscle fatigue. Interestingly, the extracellular buildup of lactic acid, once considered an additional cause for muscle fatigue, was recently shown to have force-restoring effects in such conditions. Specifically, it was proposed that elevated lactic acid (and intracellular acidosis) may lead to inhibition of voltage-gated chloride channels, thereby reestablishing better excitability of the muscle cell sarcolemma. In the present study, using an in vitro muscle contractile experimental setup to study functionally viable rectus abdominis muscle preparations obtained from normal swine, we examined the effects of 20 mM lactic acid and 512 μM 9-anthracenecarboxylic acid (9-AC; a voltage-gated chloride channel blocker) on the force recovery of K(+)-depressed (10 mM K(+)) twitch forces. We observed a similar muscle contractile restoration after both treatments. Interestingly, at elevated [K(+)], myotonia (i.e., hyperexcitability or afterdepolarizations), usually present in skeletal muscle with inherent or induced chloride channel dysfunctions, was not observed in the presence of either lactic acid or 9-AC. In part, these data confirm previous studies showing a force-restoring effect of lactic acid in high-[K(+)] conditions. In addition, we observed similar restorative effects of lactic acid and 9-AC, implicating a beneficial mechanism via voltage-gated chloride channel modulation.     

Extracellular determinants of cardiac contractility in the cold anoxic turtle

Painted turtles (Chrysemys picta) survive months of anoxic submergence, which is associated with large changes in the extracellular milieu where pH falls by 1, while extracellular K+, Ca++, and adrenaline levels all increase massively. While the effect of each of these changes in the extracellular environment on the heart has been previously characterized in isolation, little is known about their interactions and combined effects. Here we examine the isolated and combined effects of hyperkalemia, acidosis, hypercalcemia, high adrenergic stimulation, and anoxia on twitch force during isometric contractions in isolated ventricular strip preparations from turtles. Experiments were performed on turtles that had been previously acclimated to warm (25 degrees C), cold (5 degrees C), or cold anoxia (submerged in anoxic water at 5 degrees C). The differences between acclimation groups suggest that cold acclimation, but not anoxic acclimation per se, results in a downregulation of processes in the excitation-contraction coupling. Hyperkalemia (10 mmol L(-1) K+) exerted a strong negative inotropic effect and caused irregular contractions; the effect was most pronounced at low temperature (57%-97% reductions in twitch force). Anoxia reduced twitch force at both temperatures (14%-38%), while acidosis reduced force only at 5 degrees C (15%-50%). Adrenergic stimulation (10 micromol L(-1)) increased twitch force by 5%-19%, but increasing extracellular [Ca++] from 2 to 6 mmol L(-1) had only small effects. When all treatments were combined with anoxia, twitch force was higher at 5 degrees C than at 25 degrees C, whereas in normoxia twitch force was higher at 25 degrees C. We propose that hyperkalemia may account for a large part of the depressed cardiac contractility during long-term anoxic submergence.     

Measurement of sarcoplasmic reticulum Ca2+ content in intact amphibian skeletal muscle fibres with 4-chloro-m-cresol.

Single skeletal muscle fibres were isolated from the toad (Bufo marinus) and isometric force and myoplasmic free calcium concentration ([Ca2+]i) were measured. Brief applications of 4-chloro- m-cresol (4-CmC, 0.2-5 mM) elevated [Ca2+]i reversibly in a dose-dependent manner. The lowest concentration of 4-CmC which reliably gave maximal [Ca2+]i was 2 mM and it was, therefore, used for measurement of sarcoplasmic reticulum (SR) Ca2+ content. Tetanic stimulations (100 Hz) increased [Ca2+]i from a resting level of 105 +/- 47 nM (n = 10) to 1370 +/- 220 nM (n = 6). Application of 2 mM 4-CmC produced a contracture that was 54 +/- 16% (n = 6) of the tetanic force and elevated [Ca2+]i to a peak of 3520 +/- 540 nM (n = 8). Both force and [Ca2+]i levels (resting and tetanic) were restored after 10 min of washout of 4-CmC. In skinned muscle fibres, the myofibrillar Ca(2+)-sensitivity was not changed by 4-CmC, but maximal force was reduced to 74 +/- 10% (n = 4). The magnitude of the peak of the 4-CmC-induced Ca2+ transient was not significantly changed by removal of extracellular Ca2+ nor by inhibiting the SR Ca2+ pump with 2,5-di-tert-butylhydroquinone. Treatment of intact fibres with 30 mM caffeine produced a peak Ca2+ level that was indistinguishable from 2 mM 4-CmC. These results indicate that it is possible to measure the SR Ca2+ content in the same fibre with 4-CmC without loss of normal muscle function.     

Zero extracellular K+ and prostaglandin E release in the guinea-pig taenia coli

Prostaglandin E release rates from isolated strips of guinea-pig taenia coli increased during exposure to zero K+ bathing fluid, from control values of 0.78 +/- 0.11 ng/g per min to levels as high as 29.2 ng/per min. Release rates increased for 40-50 min and then remained constant or fell despite progressive increases in intracellular sodium [Nai+] or fall in intracellular potassium [Ki+]. Readmittance of K+ to the bathing solution resulted in rapid reversal of elevated prostaglandin E release rates. [Nai+] and [Ki+] were markedly more abnormal in strips exposed to zero K+ for 70-201 min compared to 30-min exposures. Upon the readdition of K+ after long zero K+ exposure, the rate of prostaglandin E release fell long before [Nai+] and [Ki+] returned to control levels. After K+ was readded to the bathing solution, the ion concentration of tissues exposed to zero K+ for 30 min returned to normal much more quickly than did those of tissues exposed for the longer time periods, yet the exponential rate constants for fall of prostaglandin E release rate after K+ was added were not significantly different after short or long zero K+ exposure. Thus there was a dissociation between the return of [Nai+] and [Ki+] and the fall of prostaglandin E release rate to control levels. Ouabain augmented prostaglandin E release under conditions where [Ki+] could not fall. Addition of known neurotransmitters present in this tissue to the bathing fluid did not augment prostaglandin E release. Guinea-pig taenia coli strips that had been incubated with [3H]arachidonic acid, constantly released [3H]arachidonic acid and [3H]prostaglandin E and a prostaglandin which cochromatographed with prostaglandin E but could not be converted to prostaglandin B by alkali and was shown to be 6-ketoprostaglandin F1 alpha. Release of [3H]arachidonic acid and [3H]prostaglandin E plus 6-[3H]ketoprostaglandin F1 alpha was increased when strips were exposed to zero K+. Data obtained in this study suggest the augmented prostaglandin E release seen during zero K+ or ouabain is related to increased availability of unbound arachidonic acid at the site of cyclooxygenase in the cell. Augmented prostaglandin E release is apparently not related to alterations in intracellular electrolyte concentrations or release of known neurotransmitters.     

Acidosis facilitates spontaneous sarcoplasmic reticulum Ca2+ release in rat myocardium

Previous studies have shown that acidosis increases myoplasmic [Ca2+] (Cai). We have investigated whether this facilitates spontaneous sarcoplasmic reticulum (SR) Ca2+ release and its functional sequelae. In unstimulated rat papillary muscles, exposure to an acid solution (produced by increasing the [CO2] of the perfusate from 5 to 20%) caused a rapid increase in the mean tissue Cai, as measured by the photoprotein aequorin. This was paralleled by an increase in spontaneous microscopic tissue motion caused by localized Ca2+ myofilament interactions, as monitored in fluctuations in the intensity of laser light scattered by the muscle. In regularly stimulated muscles, acidosis increased the size of the Ca2+ transient associated with each contraction and caused the appearance of Cai oscillations in the diastolic period. In unstimulated single myocytes, acidosis depolarized the resting membrane potential by approximately 5 mV and enhanced the frequency of spontaneous contractile waves. The small sarcolemmal depolarization associated with each contractile wave increased and occasionally initiated spontaneous action potentials. In regularly stimulated myocytes, acidosis caused de novo spontaneous contractile waves between twitches; these waves were associated with a decrease in the amplitude of the subsequent stimulated twitch. Ryanodine (2 microM) abolished all evidence of spontaneous Ca2+ release during acidosis, markedly reduced the acidosis-induced increase in aequorin light, and reduced resting tension. We conclude that acidosis increases the likelihood for the occurrence of spontaneous SR Ca2+ release, which can cause spontaneous action potentials, increase resting tension, and negatively affect twitch tension.     

Isometric contractile properties and instantaneous stiffness of amphibian skeletal muscle in the temperature range from 0 to 20 degrees C

The isometric contractile properties of frog (Rana pipiens) and toad (Bufo bufo) sartorii have been studied over the temperature range from 0 to 20 degrees C. The isometric twitch tension was found to vary considerably between these two species and between muscles in the same species. Between 0 and 4 degrees C there was very little change in maximum isometric twitch tension. Between 4 and 12 degrees C several muscles from frog or toad showed a potentiation of twitch tension whereas others showed a decline. Over this temperature range the toad sartorii consistently demonstrated a greater potentiation. By 12 degrees C a steady decline in twitch tension in both muscles was seen as the temperature range the toad sartorii consistently demonstrated a greater potentiation. By 12 degrees C a steady decline in twitch tension in both muscles was seen as the temperature approached 20 degrees C. The maximum isometric tetanic tension recorded between 18 and 20 degrees C increased fractionally to an average of 1.504 +/- 0.029 (n = 4) for frog sartorii and to 1.377 +/- 0.008 (n = 5) for toad sartorii. The time to peak twitch tension and the half-relaxation time decreased markedly with an increase in temperature. Moreover, the half-relaxation time was reduced by a greater proportion than the time to peak twitch tension. Measurements of instantaneous stiffness by controlled velocity releases from the plateau of isometric tetani revealed that the large increase in isometric tetanus tension as the muscle was warmed was not accompanied by a corresponding increase in the total number of active cross-bridges. The possibility that a decreased availability of intracellular Ca2+ ions at the contractile sites contributing to the fall of isometric twitch tension at elevated temperatures is discussed. The possibility exists that at elevated temperatures a change inthe intrinsic contractile ability of the muscle occurs which produces an increased tension per cross-bridge.     

Excitation-contraction coupling in cardiac muscle of lobster (Homarus americanus); the role of the sarcolemma and sarcoplasmic reticulum

The T-tubules and sarcoplasmic reticulum (SR) serving excitation-contraction (EC) coupling in lobster (Homarus americanus) cardiac muscle are similar to those in mammalian myocardium. Tetanic contraction is elicited by a burst of action potentials from the cardiac ganglion. In this study we evaluated the roles of the sarcolemma and SR in EC coupling of the ostial valve muscle (orbicularis ostii m. or OOM) of lobster heart. The OOM was mounted in a bath with saline on a microscope stage; force was measured by strain gauge. [Ca2+]i was measured using iontophoretically micro-injected fura-2 salt. Peak [Ca+]i, peak tetanic force and time to peak [Ca2+]i increased with that of stimulus train duration (TD), to a maximum at a TD of 500 ms. Force increased with [Ca2+]. Cd2+ reduced force by 90%; ryanodine and caffeine reduced tetanic [Ca2+]i transients by 80% and 70%, and force by 90% and 80%, respectively. Ryanodine, caffeine and cyclopiazonic acid slowed the decline of [Ca2+]i and force during relaxation. Relaxation required [Na+]o. The rate of decline of [Ca2+]i appeared to be a sigmoidal function of the [Ca2+]i and increased for any [Ca2+]i with TD. Inactivity slowed relaxation of force; stimulation accelerated relaxation. These data suggest important contributions of Ca2+ transport both across the sarcolemma and across the SR membrane during EC-coupling of lobster cardiac muscle, while average cytosolic [Ca2+]i regulates the rate of [Ca2+]i elimination during relaxation.     

Aging augments interstitial K+ concentrations in active muscle of rats.

Skeletal muscle performance declines with advancing age, and the underlying mechanism is not completely understood. A large body of convincing evidence has demonstrated a crucial role for interstitial K+ concentration ([K+]o) in modulating contractile function of skeletal muscle. The present study tested the hypothesis that during muscle contraction there is a greater accumulation of [K+]o in aged compared with adult skeletal muscle. Twitch muscle contraction was induced by electrical stimulation of the sciatic nerves of 8- and 32-mo-old Fischer 344 x Brown Norway rats. Levels of [K+]o were measured continuously by a microdialysis technique with the probes inserted into the gastrocnemius muscle. Stimulation at 1, 3, and 5 Hz elevated muscle [K+]o by 52, 64, and 88% in adult rats, and by 78, 98, and 104% in aged rats, respectively, and the increase was significantly higher in aged than in adult rats. Recovery for [K+]o, as measured by the time for [K+]o to recover by 20 and 50% from peak response after stimulation, was slower in aged rats. Ouabain (5 mM), a specific inhibitor of the Na+-K+ pump, was added in the perfusate to inhibit the reuptake of K+ into the cells to assess the role of the pump in the overall K+ balance. Ouabain elevated muscle [K+]o at rest, and the effect was significantly attenuated in aged animals. The present data demonstrated an augmented [K+]o in aged skeletal muscle compared with adult skeletal muscle, and the data suggested that an alteration in the function of the Na+-K+ pump may contribute, in part, to the deficiency in K+ balance in skeletal muscle of aged rats.     

Reaccumulation of [K+]o in the toad retina during maintained illumination

Using K+-selective microelectrodes, [K+]o was measured in the subretinal space of the isolated retina of the toad, Bufo marinus. During maintained illumination, [K+]o fell to a minimum and then recovered to a steady level that was approximately 0.1 mM below its dark level. Spatial buffering of [K+]o by Müller (glial) cells could contribute to this reaccumulation of K+. However, superfusion with substances that might be expected to block glial transport of K+ had no significant effect upon the reaccumulation of K+. These substances included blockers of gK (TEA+, Cs+, Rb+, 4-AP) and a gliotoxin (alpha AAA). Progressive slowing of the rods' Na+/K+ pump (perhaps caused by a light-evoked decrease in [Na+]i) also could contribute to this reaccumulation of K+ by reducing the uptake of K+ from the subretinal space. As evidence for a major contribution by this mechanism, treatments designed to prevent such slowing of the pump reversibly blocked reaccumulation. These treatments included superfusion with 2 microM ouabain, or lowering [K+]o, PO2, or temperature. It is likely that such treatments inhibit the pump, increase [Na+]i, and attenuate any light-evoked decrease in [Na+]i. The results are consistent with the following hypothesis. At light onset, the decrease in rod gNa will reduce the Na+ influx and the resulting rod hyperpolarization will reduce the K+ efflux. In combination with these reduced passive fluxes, the continuing active fluxes will lower both [K+]o and [Na+]i, which in turn will inhibit the pump. In support of this hypothesis, the solutions to a pair of coupled differential equations that model changes in both [K+]o and [Na+]i match quantitatively the time course of the observed changes in [K+]o during and after maintained illumination for all stimuli examined.     

Effects of the rod receptor potential upon retinal extracellular potassium concentration

It has been hypothesized that the light-evoked rod hyperpolarization (the receptor potential) initiates the light-evoked decrease in extracellular potassium ion concentration, [K+]o, in the distal retina. The hypothesis was tested using the isolated, superfused retina of the toad, Bufo marinus; the receptor potential was recorded intracellularly from red rods, and [K+]o was measured in the photoreceptor layer with K+-specific microelectrodes. In support of the hypothesis, variations in stimulus irradiance or duration, or in retinal temperature, produced qualitatively similar effects on both the receptor potential and the decrease in [K+]o. A mechanism for the relationship between the receptor potential and the decrease in [K+]o was suggested by Matsuura et al. (1978. Vision Res. 18:767-775). In the dark, the passive efflux of K+ out of the rod is balanced by an equal influx of K+ fromthe Na+/K+ pump. The light-evoked rod hyperpolarization is assumed to reduce the passive efflux, with little effect on the pump. Thus, the influx will exceed the efflux, and [K+]o will decrease. Consistent with this mechanism, the largest and most rapid decrease in [K+]o was measured adjacent to the rod inner segments, where the Na+/K+ pump is most likely located; in addition, inhibition of the pump with ouabain abolished the decrease in [K]o more rapidly than the rod hyperpolarization. Based upon this mechanism, Matsuura et al. (1978) developed a mathematical model: over a wide range of stimulus irradiance, this model successfully predicts the time-course of the decrease in [K+]o, given only the time-course of the rod hyperpolarization.     

Opposite effects of cooling on twitch contractions of skeletal muscle isolated from tropical toads (Leptodactylidae) and northern frogs (Ranidae)

Cooling increases the twitch force of frog skeletal muscle (Rana temporaria; Rana pipiens), but decreases the twitch force of tropical toad muscle (Leptodactylus insularis). Action potentials and intramembranous charge movement in frog and toad fibers were slowed identically by cooling. Cooling increased the integral of twitch Ca²⁺ detected by aequorin in frog fibers (1.4-fold), while also decreasing the peak and slowing the rate of decay. Conversely, cooling decreased the integral (0.6-fold) and the peak of twitch Ca²⁺ in toad fibers, without affecting the rate of decay. The difference in entire Ca²⁺ transients may account for cold-induced twitch potentiation in frogs and twitch paralysis in toads. In sustained contractions of toad fibers, cooling markedly decreased maximum force caused by: (i) tetanic stimulation, (ii) two-microelectrode voltage clamp steps, (iii) high [K⁺], or (iv) caffeine. Maximum force in sustained contractions was decreased moderately by cooling frog fibers. Rapid rewarming and simultaneous removal of high [K⁺] or caffeine during a sustained contraction, caused toad muscle force to rise towards the value corresponding to the warm temperature. This did not occur after removing high [K⁺] or caffeine from toad fibers kept in the cold. Transmission electron micrographs showed no relevant structural differences. Parvalbumins are thought to promote relaxation of frog muscle in the cold. The unique parvalbumin isoforms in toad muscle apparently lack this property. Accepted: 27 August 1998     

Changes in force and calcium sensitivity in the developing avian heart.

The aim of this study was to characterize the development of the contractile properties of intact and chemically skinned muscle from chicken heart and to compare these characteristics with those of developing mammalian heart reported by others. Small trabeculae were dissected from left ventricles of Arbor Acre chickens between embryonic day 7 and young adulthood (7 weeks post-hatching). At all ages, increasing extracellular calcium (0.45-3.6 mM) progressively increased twitch force of electrically stimulated trabeculae. Twitch force at 1.8 mM extracellular calcium, normalized to cross-sectional area, increased to a maximum at 1 day post-hatching, remained constant through 3 weeks post-hatching, but then decreased at 7 weeks post-hatching. The maximal calcium-activated force of trabeculae chemically skinned with Triton X-100 detergent increased to a maximum 2 days before the time of hatching and was not significantly changed up to 7 weeks post-hatching. Over the ages studied, average twitch force in 1.8 mM calcium was between 26 and 66% of maximal calcium-activated force after skinning, suggesting that the contractile apparatus is not fully activated during the twitch in normal Ringer. In skinned trabeculae, the calcium sensitivity of the contractile apparatus was higher in the embryo than in the young adult. These age-dependent changes in calcium sensitivity are correlated with isoform switching in troponin T. A decrease in pH from 7.0 to 6.5 decreased the calcium sensitivity of the contractile apparatus to a greater degree in skinned trabeculae from young adult hearts than in those from embryonic hearts. This change in susceptibility to acidosis is temporally associated with isoform switching in troponin I.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased excitability of acidified skeletal muscle: role of chloride conductance

Generation of the action potentials (AP) necessary to activate skeletal muscle fibers requires that inward membrane currents exceed outward currents and thereby depolarize the fibers to the voltage threshold for AP generation. Excitability therefore depends on both excitatory Na+ currents and inhibitory K+ and Cl- currents. During intensive exercise, active muscle loses K+ and extracellular K+ ([K+]o) increases. Since high [K+]o leads to depolarization and ensuing inactivation of voltage-gated Na+ channels and loss of excitability in isolated muscles, exercise-induced loss of K+ is likely to reduce muscle excitability and thereby contribute to muscle fatigue in vivo. Intensive exercise, however, also leads to muscle acidification, which recently was shown to recover excitability in isolated K(+)-depressed muscles of the rat. Here we show that in rat soleus muscles at 11 mM K+, the almost complete recovery of compound action potentials and force with muscle acidification (CO2 changed from 5 to 24%) was associated with reduced chloride conductance (1731 +/- 151 to 938 +/- 64 microS/cm2, P < 0.01) but not with changes in potassium conductance (405 +/- 20 to 455 +/- 30 microS/cm2, P < 0.16). Furthermore, acidification reduced the rheobase current by 26% at 4 mM K+ and increased the number of excitable fibers at elevated [K+]o. At 11 mM K+ and normal pH, a recovery of excitability and force similar to the observations with muscle acidification could be induced by reducing extracellular Cl- or by blocking the major muscle Cl- channel, ClC-1, with 30 microM 9-AC. It is concluded that recovery of excitability in K(+)-depressed muscles induced by muscle acidification is related to reduction in the inhibitory Cl- currents, possibly through inhibition of ClC-1 channels, and acidosis thereby reduces the Na+ current needed to generate and propagate an AP. Thus short term regulation of Cl- channels is important for maintenance of excitability in working muscle.     

Differences between mouse and rat myocardial contractile responsiveness to calcium

Genetically altered mice have become an increasingly important tool for the study of mechanisms of cardiac function, and therefore it is vital to characterize the basic contractile properties of the mouse heart. As a first approach to this goal, we first optimized perfusion conditions and characterized the effect of incremental left ventricular balloon inflation on end-diastolic, systolic and developed pressures in the isovolumically-contracting mouse heart. Under constant loading conditions, we determined developed pressure in response to changing perfusate calcium (1.25, 2.5, 3.75 and 5.0 mM) and perfusate temperature (30 and 37 degrees C). We then compared the intrinsic inotropic responsiveness to changes in extracellular calcium of left ventricular myocardium from mouse to that from the rat. In the baseline state (1.25 mM extracellular calcium; [Ca2+]o), both isometric contraction duration and normalized active force at the peak of the active force-length relationship (Lmax) were less in mouse than in rat myocardium. Under isotonic conditions, temporal parameters of shortening and the relative shortening were less in mouse vs rat myocardium. Increasing [Ca2+]o from 1.25 to 2.5 mM markedly increased active isometric force and rate of force development (+dF/dt) in the mouse. However, rat myocardium responded to a lesser extent. Under isotonic conditions, peak shortening and the rate of shortening also increased to a greater extent in mouse relative to rat myocardium. Increasing the bath calcium concentration to 5.0 mM increased isometric force and +dF/dt further in the rat but not the mouse, suggesting that two species operate at different points on the force vs [Ca2+]o relationship. We conclude that mouse myocardium exhibits increased sensitivity to changes in [Ca2+]o within the physiologic range in comparison to rat. These differences do not appear to be due to differences in loading conditions. The data suggest that differences in inotropic responsiveness to calcium may reflect intrinsic differences in myocardial calcium sensitivity between species.