Calcium channels and excitation-contraction coupling in cardiac cells. I. Two components of contraction in guinea-pig papillary muscle

Biphasic contractions have been obtained in guinea-pig papillary muscle by inducing partial depolarization in K+-rich solution (17 mM) containing 0.3 microM isoproterenol; whereas in guinea-pig atria, the same conditions led to monophasic contractions corresponding to the first component of contraction in papillary muscle. The relationships between the amplitude of the two components of the biphasic contraction and the resting membrane potential were sigmoidal curves. The first component of contraction was inactivated for membrane potentials less positive than those for the second component. In Na+-low solution (25 mM), biphasic contraction became monophasic subsequent to the loss of the second component, but tetraethylammonium unmasked the second component of contraction. The relationship between the amplitude of the first component of contraction and the logarithm of extracellular Ca2+ concentration was complex, whereas for the second component it was linear. When Ca2+ ions were replaced by Sr2+ ions, only the second component of contraction was observed. It is suggested that the first component of contraction may be triggered by a Ca2+ release from sarcoplasmic reticulum, induced by the fast inward Ca2+ current and (or) by the depolarization. The second component of contraction may be due to a direct activation of contractile proteins by Ca2+ entering the cell along with the slow inward Ca2+ current and diffusing through the sarcoplasm. These results do not exclude the existence of a third "tonic" component, which could possibly be mixed with the second component of contraction.     

Rat atrial muscle responses with caffeine: dose-response, force frequency, and postrest contractions.

Caffeine has been reported to have a positive and (or) a negative inotropic effect on cardiac muscle. In this study, the force-frequency and postrest characteristics of rat atrium were studied in the presence of caffeine (1.0-10 mM) to see if the interval between beats affected the response of cardiac muscle to caffeine. When stimulation frequency was 0.5 or 2.0 Hz, there was a positive followed by a negative inotropic response with 1, 5, or 10 mM caffeine. Incomplete relaxation occurred under these circumstances, giving rise to contracture. At low frequency of stimulation (0.1 Hz) caffeine had only a negative inotropic effect, and this effect was greater with 1 mM caffeine than with 5 mM caffeine. In the absence of caffeine, when stimulation at 0.5 or 3 Hz was interrupted, a pause of 2-20 s resulted in potentiation. When caffeine was present (2.0 mM), postrest potentiation was severely attenuated, but the steady-state contraction amplitude within the range 0.5-3.0 Hz was not different. These results are consistent with the hypothesis that caffeine induces a leak of Ca2+ from the sarcoplasmic reticulum, and this Ca2+ is extruded from the cell, possibly by Na+/Ca2+ exchange. Sarcoplasmic reticular uptake of Ca2+ and the translocation to release sites appear not to be affected by caffeine within 1-5 mM concentrations.     

Voltage dependent activation of tonic contraction in cardiac myocytes.

Contractions of isolated single myocytes of guinea pig heart stimulated by rectangular depolarizing pulses consist of a phasic component and a voltage dependent tonic component. In this study we analyzed the mechanism of activation of the graded, sustained contractions elicited by slow ramp depolarization and their relation to the components of contractions elicited by rectangular depolarizing pulses. Experiments were performed at 37 degrees C in ventricular myocytes of guinea pig heart. Voltage-clamped myocytes were stimulated by the pulses from the holding potential of -40 to +5 mV or by ramp depolarization shifting voltage within this range within 6 s. [Ca2+]i was monitored as fluorescence of Indo 1-AM and contractions were recorded with the TV edge-tracking system. Myocytes responded to the ramp depolarization between -25 and -6 mV by the slow, sustained increase in [Ca2+]i and shortening, the maximal amplitude of which was in each cell similar to that of the tonic component of Ca2+ transient and contraction. The contractile responses to ramp depolarization were blocked by 200 microM ryanodine and Ca2+-free solution, but were not blocked by 20 microM nifedipine or 100-200 microM Cd2+ and potentiated by 5 mM Ni2+. The responses to ramp depolarization were with this respect similar to the tonic but not to the phasic component of contraction: both components were blocked by 200 microM ryanodine, and were not blocked by Cd2+ or Ni2+ despite complete inhibition of the phasic Ca2+ current. However, the phasic component but not the tonic component of contraction in cells superfused with Ni2+ was inhibited by nifedipine. Both components of contraction were inhibited by Ca2+-free solution superfused 15 s prior to stimulation. CONCLUSIONS: In myocytes of guinea pig heart the contractile response to ramp depolarization is equivalent to the tonic component of contraction. It is activated by Ca2+ released from the sarcoplasmic reticulum by the ryanodine receptors. Their activation and inactivation is voltage dependent and it does not depend on the Ca2+ influx by the Ca2+ channels or reverse mode Na+/Ca2+ exchange, however, it may depend on Ca2+ influx by some other, not yet defined route.     

Contribution of Na+ and membrane depolarization to contraction induced by adrenaline in the guinea pig vas deferens

The contribution of Na+ and membrane depolarization to biphasic contractions induced by adrenaline were investigated in the smooth muscle of guinea pig vas deferens. Adrenaline (5 X 10(-6) M) produced an initial small contraction (first contraction) followed by a large tonic contraction (second contraction) with subsequent rhythmic activity. The entire response to adrenaline was largely inhibited by phentolamine (5 X 10(-6) M). By adding an appropriate concentration of Mn2+ (2 X 10(-4) M) or nifedipine (3 X 10(-7) M), a Ca2+ blocker, the second contraction was strongly reduced, accompanied by abolishment of the rhythmic contraction, whereas the first contraction was virtually unaffected. However, the first contraction was markedly suppressed by a higher concentration of Mn2+. All contractions produced by adrenaline were greatly reduced in Ca2+-free solution containing 0.5 mM EGTA. By lowering external Na+ concentration, the first contraction was markedly increased without greatly affecting the second contraction. By exposure to Na+-free isotonic high K+ solution, which elicited a greater depolarization of the membrane, the first contraction produced by adrenaline was also greatly potentiated, while the second and rhythmic contractions were eliminated. These results suggest that the adrenaline-evoked first contraction may be due to an influx of membrane bound Ca2+ which is independent of membrane depolarization, while the second (rhythmic) contraction is due to an influx of extracellular Ca2+ which is dependent upon depolarization.     

Role of extracellular Ca2+ in diaphragmatic contraction: effects of ouabain, monensin, and ryanodine.

The effects of zero extracellular Ca2+ on the contractility of rat diaphragmatic strips in vitro were studied in conjunction with various pharmacological agents known to influence the intracellular Ca2+ concentration: the Na+ ionophore, monensin, and the Na(+)-K+ pump inhibitor, ouabain, which enhance [Ca2+]i, caffeine, which induces Ca2+ release from the sarcoplasmic reticulum (SR), and ryanodine, which prevents Ca2+ retention by the SR. The effect of increasing [Ca2+]i on diaphragmatic contraction was assessed by comparing contractions induced by 120 mM K+ in the small muscle strips before and after the addition of ouabain or monensin. Monensin (20 microM) and ouabain (1-100 microM) augmented contractions up to threefold. Treatment of diaphragm strips with 3 nM ryanodine increased baseline tension 360% above the original resting tension but only if the diaphragm was electrically stimulated concurrently; 100 microM ryanodine induced contracture in quiescent tissue. High K+ contractures were of greater magnitude in the presence of ryanodine compared with control, and relaxation time was prolonged by greater than 200%. Ca(2+)-free conditions ameliorated these actions of ryanodine. Ryanodine reduced contractions induced by 10 mM caffeine and nearly abolished them in Ca(2+)-free solution. The data demonstrate that extracellular Ca2+ is important in certain types of contractile responses of the diaphragm and suggest that the processes necessary to utilize extracellular Ca2+ are present in the diaphragm.     

The importance of the ion-transport systems of the sarcolemma and sarcoplasmic reticulum in changing rat cardiac contractile function under a hypersodium medium]

Following a reduced pressure in the left ventricle, elevated concentrations of sodium ions enhanced by half the contraction force of the rat isolated heart. This effect was shown to be independent of the Na-channels blockers or Na/H exchange of caffeine but quite susceptible to sodium channel blockers, caffeine, and the blocking agent for Na-Ca exchange Ni2+. A decrease in potassium concentration amplified, and elevation of K+ level attenuated the positive inotropic effect of the elevated concentration of sodium ions. The effect was preserved even after heart arrest induced by verapamil. The findings suggest that elevated concentration of sodium ions may affect the Na+/Ca2+ exchange and provoke Ca2+ release from sarcoplasmic reticulum by means of changing the sodium gradient. These data corroborate the Leblanc and Hume hypothesis of the sodium-induced calcium ions release from sarcoplasmic reticulum.     

Comparison of effects of a potassium channel opener BRL34915, a specific potassium ionophore valinomycin and calcium channel blockers on endothelin-induced vascular contraction

The effects of a potassium (K+) channel opener BRL34915 and a specific K+ ionophore valinomycin on vasoconstriction induced by endothelin (ET) were compared with those of calcium (Ca2+) channel blockers, nicardipine and verapamil, using helical strips from rat thoracic aorta. ET induced potent and persistent contraction in control solution and similar but smaller contraction in Ca2+-free solution. BRL34915 and valinomycin inhibited the ET-induced contraction dose-dependently in control solution, but not in Ca2+-free solution. The ET-induced contraction was also inhibited by nicardipine and verapamil, though less strongly. On the other hand, high K+ (35 mM)-induced vasoconstriction was strongly inhibited by nicardipine and verapamil, but not by BRL34915 or valinomycin. These results support the idea that the extracellular Ca2+-dependent component of the ET-induced contraction may be mediated by Ca2+ influx by a route other than voltage-dependent Ca2+-channels.     

The correlation between the increase in slow outward current and in contraction induced by caffeine, ryanodine, and rapid cooling in voltage-clamped frog muscle fibers.

The effects of caffeine, ryanodine, and rapid cooling were tested on the depolarization-induced contraction and the apamin-insensitive slow outward current (Iso) of voltage-clamped (double mannitol gap) single frog muscle fibers. Subthreshold caffeine concentrations (0.5-2 mM) induced a monotonic increase in contractile and Iso amplitude. Whatever the concentration, the increase in contraction was roughly twice the one in current. Similar results were obtained upon rapid cooling (20-4 degrees C) in the presence of 0.5 mM caffeine. In the absence of external Na+ (choline-substituted) 10(-5) M ryanodine induced a delayed increase (approximately 30 min) in contraction and in current, shortly before the development of a drastic and irreversible contracture. Here again, the increase in contraction was twice that in current. In the presence of 5 mM tetraethylammonium (TEA) and (or) 25 nM charybdotoxin, 2 mM caffeine still induced a strong facilitating effect on contraction but the parallel increase in current was strongly reduced. The linear relationship between the increase in current and contractile amplitude has a slope approximately 0.5 (whatever the drug used to increase contractility); it is approximately 0.1 in the presence of TEA and (or) charybdotoxin. In conclusion, provided the changes in contractile amplitude are caused by parallel changes in depolarization-induced sarcoplasmic reticulum Ca2+ release, about 50% of the apamin-insensitive Iso is controlled by internal Ca2+ release. The main part of this current corresponds to the TEA- and charybdotoxin-sensitive component of Iso.     

Cellular mechanisms involved in iso-osmotic high K+ solutions-induced contraction of the estrogen-primed rat myometrium

The aim of the present study was to investigate the mechanisms involved in the contraction evoked by iso-osmotic high K+ solutions in the estrogen-primed rat uterus. In Ca2+-containing solution, iso-osmotic addition of KCl (30, 60 or 90 mM K+) induced a rapid, phasic contraction followed by a prolonged sustained plateau (tonic component) of smaller amplitude. The KCl (60 mM)-induced contraction was unaffected by tetrodotoxin (3 microM), omega-conotoxin MVIIC (1 microM), GF 109203X (1 microM) or calphostin C (3 microM) but was markedly reduced by tissue treatment with neomycin (1 mM), mepacrine (10 microM) or U-73122 (10 microM). Nifedipine (0.01-0.1 microM) was significantly more effective as an inhibitor of the tonic component than of the phasic component. After 60 min incubation in Ca2+-free solution containing 3 mM EGTA, iso-osmotic KCl did not cause any increase in tension but potentiated contractions evoked by oxytocin (1 microM), sodium orthovanadate (160 micrM) or okadaic acid (20 microM) in these experimental conditions. In freshly dispersed myometrial cells maintained in Ca2+-containing solution and loaded with indo 1, iso-osmotic KCl (60 mM) caused a biphasic increase in the intracellular Ca2+ concentration ([Ca2+]i). In cells superfused for 60 min in Ca2+-free solution containing EGTA (1 mM), KCl did not increase [Ca2+]i. In Ca2+-containing solution, KCl (60 mM) produced a 76.0 +/- 16.2% increase in total [3H]inositol phosphates above basal levels and increased the intracellular levels of free arachidonic acid. These results suggest that, in the estrogen-primed rat uterus, iso-osmotic high K+ solutions, in addition to their well known effect on Ca2+ influx, activate other cellular processes leading to an increase in the Ca2+ sensitivity of the contractile machinery by a mechanism independent of extracellular Ca2+.     

Effects of temperature and Na0+ on the relaxation of phasic and tonic tension of guinea-pig ureter muscle

Effects of temperature and Na0+ on the relaxation of guinea-pig ureter smooth muscle were studied. Relaxation of phasic contraction was found to be highly temperature-dependent, practically independent of Na0+ and Ca02+, and resistant to vanadate. The relaxation of the tonic tension of both high-K and low-Na contracture was less temperature-dependent and affected by Na0+. The relaxation of tonic tension produced by introduction of Na0+ was about 3-5 times faster than that produced by Ca-free solution. La3+ ions were found to block the relaxation of the tonic component of the Na+-free contracture initiated by removal of Ca02+. Three systems of regulation of cell calcium are suggested to be operative in the ureter muscle: a fast one which is highly temperature-dependent and responsible for the relaxation of the phasic contraction (probably the sarcoplasmic reticulum), and two slow membrane-linked carriers, one of which is dependent on Na0+ (probably Na-Ca exchange) and another one which is independent of Na0+ and inhibited by La3+ (probably Ca-pump).     

The effects of cyanide on intracellular ionic exchange in ferret and rat ventricular myocardium

The effects of cyanide on Ca2+ exchange in isolated ventricular myocytes and on the intracellular concentrations of Ca2+, Na+ and H+ have been investigated to assess the contribution that mitochondria might play in cellular Ca2+ metabolism. Ionic levels were measured with ion-selective electrodes. KCN (2.5 mM) inhibited a component of Ca2+ exchange in myocytes that could be attributed to mitochondrial exchange, but was without effect on non-mitochondrial Ca2+ exchange. NaCN (2.5 mM) caused a transient reduction of [H+]i, [Na+]i and [Ca2+]i when applied to the superfusate bathing ventricular trabeculae or papillary muscles. The transient changes of [Na+]i were accentuated when the preparation was exposed to a solution which would be expected to increase the cellular calcium content. The reduction of [Na+]i which accompanies a reduction of the extracellular sodium concentration, [Na]o, was attenuated in the presence of NaCN, but the intracellular acidosis resulting from a reduction of [Na]o was unaffected by NaCN. A small, but significant, rise of [Ca2+]i accompanied a reduction of [Na]o but only when NaCN was present in the superfusate. It is concluded that cyanide ions have a reasonably specific action on cardiac cellular ionic metabolism. Its primary action is to prevent mitochondrial Ca2+ sequestration. It is postulated that a Na+/H+ exchange, possibly at the sarcolemma, could account for some of the changes to sarcoplasmic ionic levels observed. In a solution of low [Na]o, it is concluded that mitochondria could sequester at least 30% of the calcium accumulated by the cell even though the sarcoplasmic [Ca2+] does not exceed 0.3 microM.     

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.     

Maitotoxin-Evoked γ-Aminobutyric Acid Release Is Due Not Only to the Opening of Calcium Channels

The effects of maitotoxin (MTX) on endogenous amino acid release were tested on highly purified striatal neurons differentiated in primary culture. MTX induced a large and concentration-dependent release of gamma-aminobutyric acid (GABA). This effect was abolished when experiments were performed in the absence of external Ca2+, and restored when Ca2+ ions were added after removing the MTX-containing Ca2+-free solution. MTX-induced amino acid release was not affected by 1 microM nifedipine and only slightly inhibited by 1 mM Co2+. MTX also induced a massive accumulation of 45Ca2+ in the neurons which, in contrast to the MTX-evoked GABA release, was totally blocked in the presence of 1 mM Co2+. Whereas 500 nM tetrodotoxin was without significant effect, MTX-evoked GABA release was dependent on the presence of external Na+ and sensitive to nipecotic acid, a GABA uptake inhibitor. It is concluded that, on striatal neurons, MTX induced Na+ influx only in the presence of external Ca2+. The increase in cytoplasmic Na+ ions then triggers the release of GABA.     

Characteristics of functioning of electromechanical coupling in striated muscles of higher and lower vertebrates

A comparative pharmacological analysis of relative contributions of different signal transduction pathways in the activation of contraction (excitation-contraction coupling, ECC) in intact fast striated muscles of frog and lamprey was performed. It was found that the major mechanism responsible for the ECC in muscles of both animals is Ca2+ release from the sarcoplasmic reticulum through the ryanodine-sensitive channels. However, the ECC in lamprey muscle displays some important differences in the units of electromechanical coupling, which precede the calcium release from sarcoplasmic reticulum. The maximum contraction force in frog muscle develops during caffeine-induced contracture, which indicates that all Ca2+ stored in sarcoplasmic reticulum is released through ryanodine-sensitive channels. In contrast, in lamprey muscle, the maximum force develops not in response to high caffeine concentration, but in response to repetitive electrical stimulation. Hence, in addition to stores liberated by ryanodine-sensitive channels, some other sources of calcium ions should exist, which contribute to the contraction activation. A source of this additional Ca2+ ions can be external medium, because acetylcholine contracture is abolished in a calcium-free medium. In frog muscle, the acetylcholine contracture was abolished in a Na(+)-free solution. It was concluded that in frog muscle ECC can be triggered by changes in the transmembrane potential (depolarization-induced calcium release), while in lamprey muscle the entry of calcium ions into myoplasm as the trigger in ECC (calcium-induced calcium release). The lamprey muscle was found to be more resistant to tetrodotoxin and tetracaine, which is indicative of a role in the activation of contraction of tetrodotoxin-resistant Na+ and/or Ca2+ channels. It was concluded, that ECC mechanism in striated muscles of low vertebrates is not limited by the generally accepted scheme of depolarization-induced calcium release but can include some other schemes, which require the Ca2+ influx into the cell.     

A selective beta2-adrenergic agonist, terbutaline, improves sepsis-induced diaphragmatic dysfunction in the rat

BACKGROUND: Sepsis causes diaphragmatic dysfunction, which can lead to the development of respiratory failure. We previously reported that isoproterenol, non-selective beta-adrenergic agonist, improved contractility of the diaphragm in a septic rat model. Since beta(2)-adrenoceptor agonists are widely used in the treatment of chronic respiratory disease, we investigated the effect of terbutaline, a selective beta(2)-adrenergic agonist, on contractility of the septic rat diaphragm and the contribution of intracellular Ca(2+) to the effect of terbutaline in vitro. METHODS: Forty-eight rats were divided into a sham group (in which sham laparotomy was performed) and a CLP group (in which peritonitis was induced by cecal ligation and perforation). The left hemidiaphragm was removed at 16 h after the operation. The effect of terbutaline (10(-)(6) M) on contractility of the diaphragm was assessed by twitch characteristics (twitch tension, contraction time and contraction velocity) and force-frequency relationship. In addition, to investigate the role of calcium ions in the effect of terbutaline on contractility of the diaphragm, contractility of the diaphragm was assessed after the pre-incubation of the diaphragm with methoxy-verapamil (10(-)(5) M), Ca(2+)-free Krebs-Ringer's solution buffered with 2 mM of ethylene glycol tetra-acetic acid (EGTA), and ryanodine (10(-)(6) M). RESULTS: Terbutaline significantly improved twitch characteristics and force-frequency relationship of the diaphragm in the CLP group (P<0.01). Incubation with methoxy-verapamil or calcium-free solution with EGTA did not show any changes in the inotropic effect of terbutaline in the CLP group. However, incubation with ryanodine completely abolished the inotropic effect of terbutaline in the CLP group. CONCLUSIONS: The present study demonstrated that terbutaline increased contractility of the diaphragm in the septic rats. Since this inotropic effect was abolished by ryanodine administration, calcium release from the sarcoplasmic reticulum may contribute to the terbutaline-induced improvement in dysfunction of the septic diaphragm.     

Spontaneous Ca2+ release from the sarcoplasmic reticulum limits Ca2+- dependent twitch potentiation in individual cardiac myocytes. A mechanism for maximum inotropy in the myocardium

We hypothesized that the occurrence of spontaneous Ca2+ release from the sarcoplasmic reticulum (SR), in diastole, might be a mechanism for the saturation of twitch potentiation common to a variety of inotropic perturbations that increase the total cell Ca. We used a videomicroscopic technique in single cardiac myocytes to quantify the amplitude of electrically stimulated twitches and to monitor the occurrence of the mechanical manifestation of spontaneous SR Ca2+ release, i.e., the spontaneous contractile wave. In rat myocytes exposed to increasing bathing [Ca2+] (Cao) from 0.25 to 10 mM, the Cao at which the peak twitch amplitude occurred in a given cell was not unique but varied with the rate of stimulation or the presence of drugs: in cells stimulated at 0.2 Hz in the absence of drugs, the maximum twitch amplitude occurred in 2 mM Cao; a brief exposure to 50 nM ryanodine before stimulation at 0.2 Hz shifted the Cao of the maximum twitch amplitude to 7 mM. In cells stimulated at 1 Hz in the absence of drugs, the maximum twitch amplitude occurred in 4 mM Cao; 1 microM isoproterenol shifted the Cao of the maximum twitch amplitude to 3 mM. Regardless of the drug or the stimulation frequency, the Cao at which the twitch amplitude saturated varied linearly with the Cao at which spontaneous Ca2+ release first occurred, and this relationship conformed to a line of identity (r = 0.90, p = less than 0.001, n = 25). The average peak twitch amplitude did not differ among these groups of cells. In other experiments, (a) the extent of rest potentiation of the twitch amplitude in rat myocytes was also limited by the occurrence of spontaneous Ca2+ release, and (b) in both rat and rabbit myocytes continuously stimulated in a given Cao, the twitch amplitude after the addition of ouabain saturated when spontaneous contractile waves first appeared between stimulated twitches. A mathematical model that incorporates this interaction between action potential-mediated SR Ca2+ release and the occurrence of spontaneous Ca2+ release in individual cells predicted the shape of the Cao-twitch relationship observed in other studies in intact muscle. Thus, the occurrence of spontaneous SR Ca2+ release is a plausible mechanism for the saturation of the inotropic response to Ca2+ in the intact myocardium.     

The ATPase activity of saponin-treated rat erythrocytes: regulation by monovalent cations, calcium, ouabain, and furosemide

The ATPase activities were studied in rat erythrocytes permeabilized with saponin. The concentrations of calcium and magnesium ions were varied within the range of 0.1-60 microM and 50-370 microM, respectively, by using EGTA-citrate buffer. The maximal activity of Ca2(+)-ATPase of permeabilized erythrocytes was by one order of magnitude higher, whereas the Ca2(+)-binding affinity was 1.5-2 times higher than that in erythrocyte ghosts washed an isotonic solution containing EGTA. Addition of the hemolysate restored the kinetic parameters of ghost Ca2(+)-ATPase practically completely, whereas in the presence of exogenous calmodulin only part of Ca2(+)-ATPase activity was recovered. Neither calmodulin nor R24571, a highly potent specific inhibitor of calmodulin-dependent reactions, influenced the Ca2(+)-ATPase activity of permeabilized erythrocytes. At Ca2+ concentrations below 0.7 microM, ouabain (0.5-1 mM) activated whereas at higher Ca2+ concentrations it inhibited the Ca2(+)-ATPase activity. Taking this observation into account the Na+/K(+)-ATPase was determined as the difference of between the ATPase activities in the presence of Na+ and K+ and in the presence of K+ alone. At physiological concentration of Mg2+ (370 microM), the addition of 0.3-1 microM Ca2+ increased Na+/K(+)-ATPase activity by 1.5-3-fold. Higher concentrations of this cation inhibited the enzyme. At low Mg2+ concentration (e.g., 50 microM) only Na+/K(+)-ATPase inhibition by Ca2+ was seen. It was found that at [NaCl] less than 20 mM furosemide was increased ouabain-inhibited component of ATPase in Ca2(+)-free media. This activating effect of furosemide was enhanced with a diminution of [Na+] upto 2 mM and did not reach the saturation level unless the 2 mM of drug was used. The activating effect of furosemide on Na+/K(+)-ATPase activity confirmed by experiments in which the ouabain-inhibited component was measured by the 86Rb+ influx into intact erythrocytes.     

Rapid filtration studies of Ca2+-induced Ca2+ release from skeletal sarcoplasmic reticulum. Role of monovalent ions

We have developed a rapid filtration technique for the measurement of Ca2+ release from isolated sarcoplasmic reticulum vesicles. Using this technique, we have studied the Ca2+-induced Ca2+ release of sarcoplasmic reticulum vesicles from rabbit skeletal muscle passively loaded with 5 mM Ca2+. The effect of known effectors (adenine nucleotides and caffeine) and inhibitors (Mg2+ and ruthenium red) of this release were investigated. In a medium composed of 100 mM KCl buffered at pH 6.8 with 20 mM K/3-(N-morpholino)propanesulfonic acid the Ca2+ release rate was maximal (500 nmol of Ca2+ released.(mg of protein)-1.s-1) at 1 micron external Ca2+ and 5 mM ATP. We also observed a rapid Ca2+ release induced by micromolar Ag+ in the presence of ATP (at 1 nM Ca2+). The Ag+-induced Ca2+ release was totally inhibited by 5 micron ruthenium red. We have also investigated the effect of monovalent ions on the Ca2+ release elicited by Ca2+ or Ag+. We show that the Ca2+ release rate: 1) was dependent upon the presence of K+ or Na+ in the release medium and 2) was influenced by a K+ gradient created across the sarcoplasmic reticulum membrane. These results directly support the idea of the involvement of an influx of K+ (through K+ channels) during the Ca2+ release and allow to reconsider a possible influence of the membrane potential of the sarcoplasmic reticulum on the Ca2+ release.     

The effect of dicyclohexylcarbodiimide and cyclopiazonic acid on the difference FTIR spectra of sarcoplasmic reticulum induced by photolysis of caged-ATP and caged-Ca2+.

The photochemical release of Ca2+ from caged-Ca2+ in the absence of ATP, and the release of ATP from caged-ATP in the presence of Ca2+ induce characteristic difference FTIR spectra on rabbit sarcoplasmic reticulum that are related to the formation of Ca2-E1 and E approximately P intermediates of the Ca(2+)-ATPase, respectively. Dicyclohexylcarbodiimide (10 nmol/mg protein) abolished both the Ca(2+)-and ATP-induced difference FTIR spectra parallel with inhibition of ATPase activity. Cyclopiazonic acid (50 nmol/mg protein) inhibited the Ca(2+)-induced difference spectrum measured in the absence of ATP, but had no significant effect on the ATP-induced difference spectrum measured in the presence of 1 mM Ca2+. The dog kidney Na+,K(+)-ATPase did not give significant difference spectrum after photolysis of caged-ATP in Ca(2+)-free media containing 90 mM Na+ and 10 mM K+, with or without ouabain. We propose that both the Ca2+ and the ATP-induced difference FTIR spectra of the Ca(2+)-ATPase reflect the occupancy of the high-affinity Ca2+ transport site of the enzyme.     

Effect of cyanide and verapamil on sodium-free contractures in the frog heart

Sodium-free contractures were studied in myocardial strips from R. pipiens with extracellular sodium (Na0+) replaced by choline chloride and extracellular calcium (Ca20+) varied with EGTA buffer. At calculated Ca02+ below 2.8 X 10(-7) mol/l, no contracture occurred in most of the experiments, even in the presence of cyanide. When Ca02+ was above 2.8 X 10(-7) mol/l, relatively short tension transients of up to 80 sec duration could be avoided if the myocardial strip was previously equilibrated for 20 min in a Na+-Ca2+-free solution. Instead, contractures developed slowly within one to several hours. The maximum contracture was dependent on Ca02+ in a dose-response-like pattern. The time-course of contracture development was not affected by verapamil, but KCN significantly increased the rate of resting tension increase. In solutions with normal Na+-Ca2+ content and even in a Na+-Ca2+-free milieu, the cellular ultrastructure was normal. Development of contracture after addition of Ca2+ to the Na+-free solution was combined with ultrastructural damage of the ventricular strip. It is concluded that Na+-free contractures depend on transsarcolemmal net-Ca2+ uptake as a sum of Na-Ca-exchange-dependent Ca2+ uptake and active sequestering of intracellular free calcium Ca2+ mediated by sarcolemmal and probably intracellular Ca2+-ATPases. The negative inotropic effect of the Ca blocker verapamil seems not to be mediated by the Na-Ca exchange.