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J L Sutko

Publications and source records attributed to J L Sutko.

At least 55 records · Page 3Linked to original sources
PubMed1987

Alterations by saponins of passive Ca2+ permeability and Na+-Ca2+ exchange activity of canine cardiac sarcolemmal vesicles.

Saponins can both permeabilize cell plasma membranes and cause positive inotropic effects in isolated cardiac muscles. Different saponins vary in their relative abilities to cause each effect suggesting that different mechanisms of action may be involved. To investigate this possibility, we have compared the effects of seven different saponins on the passive Ca2+ permeability and Na+-Ca2+ exchange activity of isolated canine cardiac sarcolemmal membranes. Saponins having hemolytic activity reversibly increased the passive efflux of Ca2+ from sarcolemmal vesicles preloaded with 45Ca2+ with the following order of potency: echinoside-A greater than echinoside-B greater than holothurin-A greater than holothurin-B greater than sakuraso-saponin. Ginsenoside-Rd and desacyl-jego-saponin, which lack hemolytic activity, had no significant effect on this variable. The saponins also stimulated Na+-Ca2+ exchange activity measured as Na+-dependent Ca2+ uptake by sarcolemmal vesicles. Ginsenoside-Rd and desacyl-jego-seponin, which did not affect passive Ca2+ permeability, stimulated the uptake, while in contrast, echinoside-A and -B only slightly increased or decreased this latter variable. Thus, the abilities of these compounds to enhance Na+-Ca2+ exchange activity seem to be inversely related to their abilities to increase the Ca2+ permeability. Effects by the echinosides on Na+-Ca2+ exchange may be masked by the loss of Ca2+ from the vesicles due to the increased permeability. These results suggest that the saponins interact with membrane constituent(s) that can influence the passive Ca2+ permeability and the Na+-Ca2+ exchange activity of cardiac sarcolemmal membranes.

Animals↗
PubMed1987

The effects of ryanodine on passive calcium fluxes across sarcoplasmic reticulum membranes.

Ryanodine at concentrations of 0.01-10 microM increased, while greater concentrations of 10-300 microM decreased the calcium permeability of both rabbit fast twitch skeletal muscle junctional and canine cardiac sarcoplasmic reticulum membranes. Ryanodine did not alter calcium binding by either sarcoplasmic reticulum membranes or the calcium binding protein, calsequestrin. Therefore, the effects by this agent appear to involve only changes in membrane permeability, and the characteristics of the calcium permeability pathway affected by ryanodine were those of the calcium release channel. Consistent with this, the actions by ryanodine were localized to junctional sarcoplasmic reticulum membranes and were not observed with either longitudinal sarcoplasmic reticulum or transverse tubular membranes. In addition, passage of the junctional sarcoplasmic reticulum membranes through a French press did not diminish the effects of ryanodine indicating that intact triads were not required. Under the conditions used for the permeability studies, the binding of [3H]ryanodine to skeletal junctional sarcoplasmic reticulum membranes was specific and saturable, and Scatchard analyses indicated the presence of a single binding site with a Kd of 150-200 nM and a maximum capacity of 10.1-18.9 pmol/mg protein. [3H]ryanodine binding to this site and the increase in membrane calcium permeability caused by low concentrations of ryanodine had similar characteristics suggesting that actions at this site produce this effect. Depending on the assay conditions used, ryanodine (100-300 microM) could either increase or decrease ATP-dependent calcium accumulation by skeletal muscle junctional sarcoplasmic reticulum membranes indicating that the alterations of sarcoplasmic reticulum membrane calcium permeability caused by this agent can be determined in part by the experimental environment.

Action Potentials↗
PubMed1987

Calcium- and voltage-activated plateau currents of cardiac Purkinje fibers.

We have used the two-microelectrode voltage-clamp technique to investigate the components of membrane current that contribute to the formation of the early part of the plateau phase of the action potential of calf cardiac Purkinje fibers. 3,4-Diaminopyridine (50 microM) reduced the net transient outward current elicited by depolarizations to potentials positive to -30 mV but had no consistent effect on contraction. We attribute this effect to the blockade of a voltage-activated transient potassium current component. Ryanodine (1 microM), an inhibitor of sarcoplasmic reticulum calcium release and intracellular calcium oscillations in Purkinje fibers (Sutko, J.L., and J.L. Kenyon. 1983. Journal of General Physiology. 82:385-404), had complex effects on membrane currents as it abolished phasic contractions. At early times during a depolarization (5-30 ms), ryanodine reduced the net outward current. We attribute this effect to the loss of a component of calcium-activated potassium current caused by the inhibition of sarcoplasmic reticulum calcium release and the intracellular calcium transient. At later times during a depolarization (50-200 ms), ryanodine increased the net outward current. This effect was not seen in low-sodium solutions and we could not observe a reversal potential over a voltage range of -100 to +75 mV. These data suggest that the effect of ryanodine on the late membrane current is attributable to the loss of sodium-calcium exchange current caused by the inhibition of sarcoplasmic reticulum calcium release and the intracellular calcium transient. Neither effect of ryanodine was dependent on chloride ions, which suggests that chloride ions do not carry the ryanodine-sensitive current components. Strontium (2.7 mM replacing calcium) and caffeine (10 mM), two other treatments that interfere with sarcoplasmic reticulum function, had effects in common with ryanodine. This supports the hypothesis that the effects of ryanodine may be attributed to the inhibition of sarcoplasmic reticulum calcium release.

Action Potentials↗
PubMed1987

Effects of ryanodine and caffeine on contractility, membrane voltage, and calcium exchange in cultured heart cells.

To investigate the mechanisms of action of ryanodine and caffeine, changes in mechanical and electrical activity caused by these agents were correlated with alterations in 45Ca fluxes and cell Ca contents in chick embryo ventricular cell monolayer cultures. Ryanodine (10(-10)-10(-5) M) irreversibly decreased contraction amplitude by 10-70% relative to control in a concentration-dependent manner with minimal effects on electrical activity. Ryanodine caused a slight decrease in rapid 45Ca uptake, but no change in total exchangeable calcium content or rapid 45Ca efflux. Caffeine (1-20 mM) caused a transient (less than 10 seconds) 5-12% increase in contraction amplitude followed by a sustained 9-76% decrease in contraction amplitude and a 10 mV decrease in diastolic membrane voltage. Caffeine caused a decrease in rapid 45Ca uptake, a decrease in total exchangeable calcium content, and an increase in rapid 45Ca efflux. These results suggest that caffeine produces a decrease in sarcoplasmic reticulum (SR) Ca2+ uptake, and/or an increase in SR Ca2+ release that eventually depletes the SR of Ca2+, presumably accounting for the negative inotropic effect. The ryanodine effects on contraction are more difficult to account for solely in terms of alterations of transsarcolemmal Ca2+ fluxes and Ca2+ contents. Our data indicate an important role for the SR in excitation-contraction coupling in cultured chick embryo ventricular cells and suggest that SR Ca2+ is part of the rapidly exchanging Ca2+ compartment noted in 45Ca flux studies.

Alkaloids↗
PubMed1986

Postrest inotropy in rabbit ventricle: Na+-Ca2+ exchange determines sarcoplasmic reticulum Ca2+ content.

To determine whether Na+-Ca2+ exchange is a physiologically significant Ca2+ efflux mechanism in rabbit ventricle, we investigated the effects exerted on postrest contractions by interventions that alter the transmembrane distribution of Na+ or Ca2+ so as to retard Ca2+ efflux via this system. Contractions elicited after rest periods of 0.25-10 min in duration were studied. The following interventions increased postrest contractions much more than those elicited by rhythmic stimulation: 1) Na+ pump inhibition by cardiac glycosides or by a reduction in extracellular K+, 2) reduction of extracellular Na+ (maintaining a constant [Ca2+]-to-[Na+]2 ratio), and 3) elevation of extracellular Ca2+. In contrast, isoproterenol, norepinephrine, and histamine produced comparable increases in both rhythmically stimulated and postrest contractions, suggesting that the postrest contractile potentiation was not just the result of a general increase in inotropic state. Ryanodine, which appears to antagonize sarcoplasmic reticulum (SR) Ca2+ release in cardiac muscle, markedly reduced the amplitude of the postrest contractions, but only modestly decreased rhythmically stimulated responses. Results suggest 1) that Ca2+ released from SR is involved in postrest response, 2) that Na+-Ca2+ exchange serves as a Ca2+ efflux pathway in normally polarized resting rabbit ventricle, and 3) that this activity in part determines the amount of Ca2+ available for release from SR.

Animals↗
PubMed1986

Comparison of effects of ryanodine and caffeine on rat ventricular myocardium.

Ryanodine and caffeine have the ability to diminish sarcoplasmic reticulum (SR) calcium release in cardiac muscle cells. To determine whether these agents also share a common mechanism of action, we compared their effects on rat papillary muscles using two different experimental approaches. First, using the protocol of Jundt et al. (19), in which quiescent rat papillary muscles were exposed to sodium-free solutions, we found that 1 microM ryanodine decreased resting force, phosphorylase alpha activity, and the scattered light intensity fluctuations (SLIF) due to calcium-dependent myofilament interactions. In contrast, 10-20 mM caffeine increased both resting force and phosphorylase alpha levels and initially increased then decreased SLIF to below detectable levels. In a second series of experiments, contractures were elicited by exposing rat papillary muscles to 125 mM KCl. Pretreatment with ryanodine (1 microM) or caffeine (10 mM) abolished the initial phasic component of this response, while increasing the subsequent tonic component. These effects were different from those of isoproterenol, which decreased tonic contracture force. The depression of twitch force produced by ryanodine developed more rapidly in the presence of 125 mM KCl than in normal buffer, suggesting that the negative inotropic effects of this agent may, in part, depend on membrane depolarization. The results of these experiments suggest that ryanodine and caffeine affect SR calcium release through different mechanisms of action. Ryanodine appears to decrease, while caffeine initially increases, cytoplasmic calcium. Once these effects have occurred, the alterations of SR function produced by both agents can similarly alter other inotropic responses.

Alkaloids↗
PubMed1986

Ryanodine inhibits the release of calcium from intracellular stores in guinea pig aortic smooth muscle.

We have examined the effects of ryanodine, an inhibitor of the release of sarcoplasmic reticulum calcium in cardiac muscle, on contractile tension and calcium-45 movement in aortic smooth muscle of guinea pigs to learn whether this agent also modifies the release of stored calcium in vascular smooth muscle. Ryanodine (3-100 microM) suppressed the phasic contractions induced by caffeine and norepinephrine in calcium-free medium and prevented the stimulation of calcium-45 efflux by these agonists. Ryanodine did not significantly alter either the contractile response or the increased cellular influx of calcium-45 caused by high potassium in more than 1 mM calcium, suggesting that this agent does not affect depolarization-induced calcium entry into the cells. In a calcium-free, high potassium solution, the addition of calcium at concentrations of 1 mM and less resulted in a contraction which appeared to depend largely on the release of calcium from intracellular stores. This contraction was blocked by ryanodine. These data are consistent with the hypothesis that ryanodine causes a diminished release of calcium from the intracellular store in vascular smooth muscle, as it does in cardiac muscle. Moreover, our results indicate that a calcium-induced calcium release may exist in smooth muscle, and that this release is antagonized by ryanodine.

Alkaloids↗
PubMed1986

Effects of amrinone and milrinone on calcium influx into the myocardium.

We have examined the effects of milrinone on calcium influx into cardiac cells by testing its effects on three mechanisms of calcium entry: the slow inward calcium current, sodium-calcium exchange diffusion, and the passive calcium permeability of sarcolemmal membranes. Milrinone increased the magnitude of the slow inward calcium current in voltage-clamped calf cardiac Purkinje fiber. High concentrations of this agent also caused a small, inconsistent inhibition of calcium uptake mediated by sodium-calcium exchange in bovine cardiac sarcolemma membranes, but did not alter the time course of passive efflux of calcium from these vesicles. In conclusion, milrinone and its analogue amrinone appear to increase the entry of calcium into myocardial cells, primarily by increasing the influx of this cation during the slow inward calcium current. A small and sodium-calcium exchange may also influence calcium influx, particularly at high drug concentrations. The relationship existing between these effects and the inotropic responses produced by amrinone and milrinone is discussed.

Aminopyridines↗
PubMed1985

Phenothiazine suppression of transient depolarizations in rabbit ventricular cells.

We have examined the effects of trifluperazine and fluphenazine on action potentials and transient depolarizations of rabbit ventricular cells. Isolated myocytes were prepared by perfusing rabbit hearts with low calcium enzyme-containing solutions, and action potentials were stimulated at 1 Hz and recorded using patch-type pipettes. In normal saline, 10 microM trifluperazine or fluphenazine shifted the action potential plateau to more negative potentials and increased the rate of phase 2 repolarization. Transient diastolic depolarizations appeared in solutions containing 50 nM isoproterenol plus 1 microM strophanthidin. These transient depolarizations were abolished by the addition of 10 microM trifluperazine or fluphenazine. In addition, spontaneous transient depolarizations were occasionally observed, and these too were abolished by the phenothiazines. Because these compounds are potent inhibitors of calmodulin, these data raise the possibility that calcium-calmodulin-regulated processes are important in the generation of arrhythmogenic transient depolarizations.

Action Potentials↗
PubMed1985

Ryanodine: a modifier of sarcoplasmic reticulum calcium release in striated muscle.

We have proposed that the naturally occurring alkaloid ryanodine reduces the release of calcium from the sarcoplasmic reticulum (SR) in cardiac muscle cells. We summarize the data that support this hypothesis and discuss possible mechanisms for 1) the differences in sensitivity to ryanodine displayed by intact skeletal and cardiac muscle preparations vs. that of skinned cardiac cells and isolated SR membranes, 2) the ability of ryanodine to cause either an increase or a decrease in calcium accumulation by isolated skeletal muscle SR vesicles depending on experimental conditions, and 3) the positive inotropic effects produced by ryanodine in cardiac muscle preparations under certain experimental circumstances. In addition, we also show how ryanodine can be used to evaluate the contributions made by SR calcium release to cellular events in striated muscle.

Alkaloids↗
PubMed1983

Competitive interactions of sodium and calcium with the sodium-calcium exchange system of cardiac sarcolemmal vesicles.

The initial velocity of Ca2+ uptake via the Na-Ca exchange system in cardiac sarcolemmal vesicles is competitively inhibited by extravesicular Na+. The Hill plots for multisite competitive inhibition are nonlinear, exhibiting a limiting slope of 1 at low Na+ concentrations (less than 20 mM) and 1.6-2.0 at higher concentrations. The Ki for Na+ is approximately 16 mM. Thus, the Ca2+ binding site of the Na-Ca exchange carrier interacts with either 1 or 2 Na+ ions and the binding of a single Na+ is sufficient to block Ca2+ binding. These results, in conjunction with the observation that at least 3 Na+ ions are required at the trans membrane surface to bring about Ca2+ movements by Na-Ca exchange, suggest that the exchange carrier contains two classes of cation binding sites: a divalent site which can bind either 1 Ca2+ or 1-2 Na+ ions and a second site which binds at least one additional Na+. The classical observation that cardiac contractility depends upon the [Ca2+]/[Na+]2 ratio of the extracellular medium can be readily explained in terms of the competitive interactions of Na+ and Ca2+ at the divalent site.

Animals↗
PubMed1983

Equilibrium calcium-calcium exchange in cardiac sarcolemmal vesicles.

Ca-Ca isotopic exchange activity in bovine cardiac sarcolemmal vesicles was measured under equilibrium conditions, i.e. where intra- and extravesicular media have identical compositions. The results demonstrate that the monovalent cations K+, Li+, and Rb+ stimulate the process of Ca-Ca exchange. The presence of monovalent cations increases the Vmax for this process but has little effect on the Km for Ca2+. Maximal stimulation of exchange activity occurs when the monovalent cations are present on both sides of the vesicle membrane; a lesser degree of stimulation is observed when the cations are present at only one membrane surface. Na+ also stimulates Ca-Ca exchange at low concentrations (0.5-8 mM) but at high concentrations causes a marked inhibition. The latter effect is due to a competition between Na+ and Ca2+ for a site on the exchange carrier. The presence of monovalent cations alters the pH profile for Ca-Ca exchange. In the absence of stimulating cations, Ca-Ca exchange activity exhibits a maximum at pH 7.0 and then drops to approximately half the peak value at pH 7.5 or higher. In contrast, the rate of Ca-Ca exchange in the presence of monovalent cations continues to increase as the pH is raised from pH 6 to pH 9. Measurements of 86Rb+ fluxes demonstrate that Rb-stimulated Ca-Ca exchange does not involve the co-transport of Rb+. Monovalent cations produce no stimulation of Na-dependent Ca2+ fluxes (Na-Ca exchange) when present on the same side of the membrane as Ca2+. The results are discussed in terms of an extension of the model for the Na-Ca exchanger that was described in the accompanying paper (Reeves, J.P., and Sutko, J.L. (1983) J. Biol. Chem. 258, 3178-3182).

Animals↗
PubMed1983

Ryanodine modification of cardiac muscle responses to potassium-free solutions. Evidence for inhibition of sarcoplasmic reticulum calcium release.

To test whether ryanodine blocks the release of calcium from the sarcoplasmic reticulum in cardiac muscle, we examined its effects on the aftercontractions and transient depolarizations or transient inward currents developed by guinea pig papillary muscles and voltage-clamped calf cardiac Purkinje fibers in potassium-free solutions. Ryanodine (0.1-1.0 microM) abolished or prevented aftercontractions and transient depolarizations by the papillary muscles without affecting any of the other sequelae of potassium removal. In the presence of 4.7 mM potassium and at a stimulation rate of 1 Hz, ryanodine had only a small variable effect on papillary muscle force development and action potential characteristics. In calf Purkinje fibers, ryanodine (1 nM-1 microM) completely blocked the aftercontractions and transient inward currents without altering the steady state current-voltage relationship. Ryanodine also abolished the twitch in potassium-free solutions, but it enhanced the tonic force during depolarizing voltage-clamp steps. This latter effect was dependent on the combination of ryanodine and potassium-free solutions. The slow inward current was not blocked by 1 microM ryanodine, but ryanodine did appear to abolish an outward current that remained in the presence of 0.5 mM 4-aminopyridine. Our observations are consistent with the hypothesis that ryanodine, by inhibiting the release of calcium from the sarcoplasmic reticulum, prevents the oscillations in intracellular calcium that activate the transient inward currents and aftercontractions associated with calcium overload states.

Action Potentials↗
PubMed1982

Amrinone activates K+-depolarized atrial and ventricular myocardium of guinea pigs.

Amrinone is a new synthetic drug that increases contractile strength of mammalian heart muscle; however, its mechanism of positive inotropic action has not been determined. We now report that amrinone (0.053-5.3 mM) consistently restores typical slow response electromechanical activity fo K+-depolarized atrial and ventricular myocardial preparations from guniea pigs. This action was blocked in both tissues by D-600 (1 microM), but it was not significantly inhibited by either tetrodotoxin (23.5 microM), d,l-propranolol (1 microM), or phentolamine (10 microM). Cimetidine (3 microM) or metiamide (10 microM) slightly inhibited amrinone's effect only in the ventricle, whereas pyrilamine (10 microM) slightly inhibited amrinone's response only in the atrium. These data indicate that amrinone's positive inotropic action may involve augmented Ca++ influx via the slow inward Ca++ current, and that although this action is independent of adrenoceptor mechanisms, it seems to include a small histaminergic component.

Aminopyridines↗
PubMed1981

Cardiac sarcolemmal and sarcoplasmic reticulum membrane vesicles exhibit distinctive (Ca-Mg)-ATPase substrate specificities.

The nucleoside 5'-triphosphate (NTP) substrate specificities for Ca-stimulated ATPase and ATP-dependent Ca2+ uptake activities have been examined in cardiac sarcolemma (SL) and sarcoplasmic (SR) membrane vesicles. The results indicate that SL membrane vesicles exhibit a much narrower range of NTP substrate specificities than SR membranes. In SR membrane vesicles, the Ca-stimulated Mg-dependent hydrolysis of ATP and dATP occurred at nearly equivalent rates, whereas the rates of hydrolysis of GTP, ITP, CTP, and UTP ranged from 16-33% of that for ATP. All of the above nucleotides also supported Ca2+ transport into SR vesicles; dATP was somewhat more effective than ATP while GTP, ITP, CTP, and UTP ranged from 28-30% of the activity for ATP. In the presence of oxalate, the initial rate of Ca accumulation with dATP was 4-fold higher than for ATP, whereas the activity for GTP, ITP, CTP, and UTP ranged from 35-45% of that for ATP. For the SL membranes, Ca-activated dATP hydrolysis occurred at 60% of the rate for ATP; GTP, ITP, CTP, and UTP were hydrolyzed by the SL preparations at only 7-9% of the rate for ATP. NTP-dependent Ca2+ uptake in SL membranes was supported only by ATP and dATP, with dATP 60% as effective as ATP. GTP, ITP, CTP, and UTP did not support the transport of Ca2+ by SL vesicles. The results indicate that the SL and SR membranes contain distinctly different ATP-dependent Ca2+ transport systems.

Animals↗
PubMed1980

Sodium-calcium exchange activity generates a current in cardiac membrane vesicles.

Sarcolemmal membrane vesicles isolated from canine ventricular tissue accumulate calcium through the sodium-calcium exchange system when an outwardly directed sodium gradient is generated across the vesicle membrane. Moreover, calcium uptake under these conditions is accompanied by the transient accumulation of the lipophilic cation tetraphenylphosphonium. Since the distribution of tetraphenylphosphonium across biological membranes reflects the magnitude and direction of transmembrane potential differences and the characteristics of the transient accumulation of this cation closely resemble those of sodium-calcium exchange activity, it is concluded that a membrane potential, interior negative, is produced during calcium accumulation through the exchange system. Thus, the operation of the sodium-calcium exchange system generates a current in cardiac membrane vesicles, suggesting that three or more sodium ions exchange for each calcium ion.

Biological Transport, Active↗