Biomedical subjects

D M Bers

Publications and source records attributed to D M Bers.

At least 127 records · Page 7Linked to original sources

PubMed1990

Ca current facilitation during postrest recovery depends on Ca entry.

Whole cell Ca current (ICa) recovery after periods of rest was examined in voltage-clamped rabbit ventricular myocytes with Na and K currents suppressed. To evaluate rest-dependent changes in ICa independent of the effects of sarcoplasmic reticular (SR) Ca release, the intracellular Ca ([Ca]i) transients were usually buffered by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (10 mM) in the patch pipette. When voltage-clamp pulses were resumed (at 0.5 Hz) after a period of rest, several pulses were required to reattain steady-state peak ICa levels. From depolarized holding potentials between -40 and -50 mV, peak ICa of the first pulse was large and gradually decayed to steady-state levels (negative ICa staircase). This potentiation of postrest ICa was mediated by increased recovery from inactivation of Ca channels during the rest period. In contrast, with more negative holding potentials (-70 to -90 mV), the initial postrest ICa was relatively small (rest depression) and facilitation of ICa was then observed for subsequent pulses (positive ICa staircase). This ICa facilitation was mediated by a progressive decrease in the ICa inactivation rate. Depression of the initial postrest ICa required 10-15 s of rest to fully develop and became relatively constant for longer rest intervals (30-300 s). Postrest ICa depression (i.e., subsequent ICa facilitation) was abolished by replacement of extracellular Ca ([Ca]o) with either Ba or Sr. Thus ICa facilitation depends on Ca entry. Increasing [Ca]o increased postrest ICa facilitation and reducing [Ca]o had an opposite effect. When ICa was altered by changing step potential, maximal ICa facilitation occurred when ICa was maximal. Thus ICa facilitation can be graded by the amount of Ca entry. As ICa facilitation was not altered by ryanodine, this response is not likely to be due to SR Ca release. However, increasing [Ca]i buffering by using 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid in the pipette abolished the ICa staircase. Our results indicate that Ca entry can facilitate subsequent ICa, presumably through actions occurring near the sarcolemma. These local changes in [Ca]i lead to a progressive slowing in the rate of ICa inactivation.

Animals↗

PubMed1989

Influence of temperature on the calcium sensitivity of the myofilaments of skinned ventricular muscle from the rabbit.

The steady-state myofilament Ca sensitivity was determined in skinned cardiac trabeculae from the rabbit right ventricle (diameter, 0.13-0.34 mm) at 36, 29, 22, 15, 8, and 1 degree C. Muscles were stimulated to 0.5 Hz and stretched to a length at which maximum twitch tension was generated. The preparation was then skinned with 1% vol/vol Triton X-100 in a relaxing medium (10 mM EGTA, pCa 9.0). Each preparation was exposed to a series of Ca-containing solutions (pCa 6.3-4.0) at two of the six temperatures studied (temperature was regulated to +/- 0.1 degree C). The pCa values (mean +/- SD, n = 6) corresponding to half maximal tension at 36, 29, 22, 15, 8, and 1 degree C were 5.47 +/- 0.07, 5.49 +/- 0.07, 5.34 +/- 0.05, 5.26 +/- 0.09, 4.93 +/- 0.06, and 4.73 +/- 0.04, respectively. Mean (+/- SD) maximum tension (Cmax) developed by the preparation as a percentage of that at 22 degrees C was 118 +/- 10, 108 +/- 5, 74 +/- 6, 57 +/- 7, and 29 +/- 5% at 36, 29, 15, 8, and 1 degree C, respectively. As cooling led to a shift of Ca sensitivity towards higher [Ca2+] and a reduction of Cmax, the Ca sensitivity curves over this range of temperatures do not cross over as has been described for canine Purkinje fibers (Fabiato 1985). Since tension is decreased by cooling at all levels of [Ca2+] it is unlikely that changes in myofilament Ca sensitivity play a role in the large hypothermic inotropy seen in rabbit ventricular muscle. The increase in sensitivity of the myofilaments to Ca on warming from 1 to 29 degrees C might be related to the increase in force seen on rewarming from a rapid cooling contracture in intact rabbit ventricular muscle.

Actin Cytoskeleton↗

PubMed1989

Intracellular Ca2+ transients during rapid cooling contractures in guinea-pig ventricular myocytes.

1. We measured intracellular Ca2+ transients during rapid cooling contractures (RCCs) in guinea-pig ventricular myocytes using the fluorescent Ca2+ indicator, Indo-1. 2. Rapid cooling of myocytes from 22 to 0-1 degrees C induced a rapid increase in [Ca2+]i which preceded the peak of the contraction and was sometimes large enough to saturate Indo-1. This indicates that [Ca2+]i may reach greater than 10 microM during an RCC. 3. The [Ca2+]i during the RCC slowly declined from its peak value and most of this decline in [Ca2+]i can be attributed to slow reaccumulation of Ca2+ by the sarcoplasmic reticulum (SR) in the cold. RCCs induced in the absence of Cao2+, were not different from control, supporting previous conclusions that RCCs depend exclusively on intracellular Ca2+ stores. 4. RCCs are depressed by long rest periods (rest decay) or by exposure to ryanodine or caffeine, which supports conclusions that RCCs are due to Ca2+ release from the SR. The rest decay of RCCs can be almost completely prevented by applying Nao(+)-free solution during the rest period. This implies that the loss of SR Ca2+ during rest depends on the sarcolemmal Na(+)-Ca2+ exchange (and not the sarcolemmal Ca2(+)-ATPase pump). 5. Rapid rewarming during an RCC normally leads to an additional transient contraction (or rewarming spike), without any increase in [Ca2+]i. Thus, the rewarming spike might be attributable to an increase in myofilament Ca2+ sensitivity induced by rewarming. 6. A second RCC is used to assess the fraction of Ca2+ which is re-sequestered by the SR during relaxation from the first RCC. In control solution progressive RCCs decline in amplitude, but in Na(+)-free, Ca2(+)-free solution they are of constant amplitude. We conclude that the SR Ca2+ pump and Na(+)-Ca2+ exchange are responsible for relaxation and that the latter may account for 20-50% of relaxation. 7. These results support the use of RCCs as a useful means of assessing SR Ca2+ content in intact cardiac muscle cells.

Animals↗

PubMed1989

SR Ca loading in cardiac muscle preparations based on rapid-cooling contractures.

The influence of rest periods on twitches and rapid-cooling contractures (RCCs) was examined in trabeculae from rabbit, rat, guinea pig, and frog ventricle and rabbit atrium. RCCs were used as a relative index of sarcoplasmic reticulum (SR) Ca content. After increasing rest duration, rabbit and guinea pig ventricles exhibit a decline of both twitch force and RCC force (rest decay). When stimulation is resumed, both twitches and RCCs recover to steady-state levels. The SR (and cells) in these tissues may lose Ca during quiescence and become reloaded with progressive stimulation. Rat ventricle and rabbit atrium exhibited an increase in both twitch and RCC tension as a function of rest duration (rest potentiation). Resumption of stimulation resulted in parallel declines of both twitch and RCC tension approaching steady state. Thus stimulation in rat ventricle and rabbit atrium may lead to a net Ca loss from the SR (and the cell) and quiescence may lead to replenishment of cellular Ca. This major difference in Ca metabolism in mammalian cardiac muscles might be due to a fundamental difference in SR properties or, alternatively, different sarcolemmal transport properties (e.g., action potential configuration, Na-pump). After long rest intervals in rabbit and guinea pig ventricle, RCCs return toward their steady-state value in considerably fewer beats than does twitch tension. This implies that something other than SR refilling is responsible for the slow phase of twitch recovery after rest. In rabbit ventricle increasing frequency or extracellular Ca concentration ([Ca]o) generally increases both twitch and RCC tension. However, decreasing [Ca]o (to 0.2 mM) does not decrease RCCs much despite a dramatic decline in twitch tension (suggesting low twitch tension despite a loaded SR). Rapid rewarming during an RCC usually results in a transient rise in tension (or rewarming "spike"), which is due to a warming-induced increase in myofilament Ca sensitivity. Differences in rewarming spikes among the tissues studied suggest differences in temperature effects on myofilament Ca sensitivity.

Animals↗

PubMed1989

Rat vs. rabbit ventricle: Ca flux and intracellular Na assessed by ion-selective microelectrodes.

Trans sarcolemmal Ca movements in rabbit and rat ventricular muscle were compared using extracellular double-barreled Ca-selective microelectrodes. In rabbit ventricle, steady-state twitches were associated with transient extracellular Ca (Cao) depletions, indicative of Ca uptake during the twitch. In contrast, steady-state twitches in rat ventricle were associated with net cellular Ca extrusion. Rest periods in rabbit ventricle lead to a net loss of cell Ca and resumption of stimulation induces a net uptake of Ca by the cells. Conversely, in rat ventricle rest periods lead to cellular Ca gain and resumption of stimulation induces a net Ca loss from the cells. Thus stimulation is associated with net Ca gain in rabbit ventricle and net Ca loss in rat ventricle. These observations provide an explanation for some of the functional differences between rat and rabbit ventricle (e.g., negative force-frequency staircase and rest potentiation in rat vs. positive staircase and rest decay in rabbit). Resting intracellular Na activity (alpha iNa) was 12.7 +/- 0.6 mM in rat and 7.2 +/- 0.5 mM in rabbit ventricle. This alpha iNa in rat ventricle is sufficiently high that Ca entry via Na-Ca exchange is thermodynamically favored at the resting membrane potential. This may explain why rest potentiation is observed in rat ventricle. In contrast, the lower alpha iNa in rabbit ventricle would favor Ca extrusion via Na-Ca exchange at rest (and consequent rest decay). In rat ventricle, the increase of intracellular [Ca] ([Ca]i) associated with contraction, coupled with the short action potential duration, strongly favor Ca extrusion via Na-Ca exchange and explain the observed Cao accumulation observed during twitches in rat. The high plateau of the rabbit ventricular action potential tends to prevent Ca extrusion via Na-Ca exchange during the contraction and explains the Cao depletions observed in rabbit. It is concluded that the higher alpha iNa and shorter action potential duration in rat vs. rabbit ventricle can explain many of the functional differences observed in these tissues.

Animals↗

PubMed1989

Correction of proton and Ca association constants of EGTA for temperature and ionic strength.

The association constants of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) for protons and Ca can be used to calculate the apparent association constant of EGTA for Ca (K'CaEGTA). This value is often used in calculating the free [Ca2+] of complex solutions such as those used to determine the Ca sensitivity of skinned muscle fibers. As association constants are usually measured at 0.1 M ionic strength and between 20 and 25 degrees C, these constants must first be adjusted for conditions different from those at which they were measured, before calculation of K'CaEGTA. The proton and Ca association constants (and their delta H values) from A. E. Martell and R. M. Smith (Critical Stability Constants, New York: Plenum, vol. 1, 1974) adjusted for ionic strength and temperature using a semiempirical Debye-Hückel formalism and Van't Hoff isochore, respectively, closely describe the influence of both ionic strength and temperature on K'CaEGTA. Errors in the adjustment or calculation of association constants can dramatically affect the calculated value of K'CaEGTA and hence the calculated free [Ca2+] of complex solutions, such as those used to mimic the intracellular environment.

Calcium↗

PubMed1989

Rapid cooling contractures as an index of sarcoplasmic reticulum calcium content in rabbit ventricular myocytes.

Rapid cooling contractures (RCCs) were used to assess changes in sarcoplasmic reticulum (SR) Ca content in both isolated rabbit ventricular myocytes and multicellular preparations. The main difference observed between these preparations was the magnitude of RCCs relative to twitches, apparently due to differences in measured parameters, i.e., unloaded shortening vs. isometric tension. When multicellular preparations were unloaded, RCC shortening was similar to that observed in myocytes. RCC magnitude decreased as the time between the last electrical stimulation and the RCC was increased (rest decay). RCC rest decay closely paralleled that of postrest twitches, suggesting that SR Ca loss is responsible for this process. Paired RCC experiments were used to investigate RCC relaxation and rest decay. When a second RCC (RCC2) was induced immediately after the first (RCC1), a large contracture was still observed (RCC2/RCC1 x 100 = 77.8 +/- 7.3%, mean +/- SD), indicating that the SR resequestered the majority of Ca on rewarming. This fraction was increased (to 92.9 +/- 5.5%) if Na and Ca-free solution was used during RCCs and rewarming, indicating that Na-Ca exchange also contributes to RCC relaxation. Increasing the interval between paired RCCs led to a decrease in RCC2, analogous to rest decay. This rest decay was abolished by inhibiting Na-Ca exchange, indicating that SR Ca loss during rest is mediated primarily by this process. RCCs were abolished by 10 mM caffeine. Ryanodine (1 microM) greatly accelerated RCC rest decay but had less effect on RCCs generated immediately after a train of stimulation. This accelerated rest decay was also dependent on Na-Ca exchange.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Relaxation of rabbit ventricular muscle by Na-Ca exchange and sarcoplasmic reticulum calcium pump. Ryanodine and voltage sensitivity.

We studied relaxation during rapid rewarming of rabbit ventricular muscles that had been activated by rapid cooling. Rewarming from 1 degree to 30 degrees C (in less than 0.5 second) activates mechanisms that contribute to the reduction of intracellular calcium concentration and thus relaxation (e.g., sarcoplasmic reticulum [SR] calcium pump and sarcolemmal Na-Ca exchange and calcium pump). Rapid rewarming in normal Tyrode's solution induces relaxation with a half-time (t1/2) of 217 +/- 14 msec (mean +/- SEM). During cold exposure, changing the superfusate to a sodium-free, calcium-free medium with 2 mM CoCl2 (to eliminate Na-Ca exchange) slightly slows relaxation upon rewarming in the same medium (t1/2 = 279 +/- 44 msec). Addition of 10 mM caffeine (which prevents SR calcium sequestration) to normal Tyrode's solution during cold superfusion slows relaxation somewhat more (t1/2 = 376 +/- 31 msec) than sodium-free, calcium-free solution. However, if both interventions are combined (sodium-free + caffeine) during the cold exposure and rewarming, the relaxation is greatly slowed (t1/2 = 2,580 +/- 810 msec). These results suggest that either the SR calcium pump or, to a lesser extent, sarcolemmal Na-Ca exchange can produce rapid relaxation, but if both systems are blocked, relaxation is very slow. If muscles are equilibrated with 500 nM ryanodine before cooling, relaxation upon rewarming is not greatly slowed (t1/2 = 266 +/- 37 msec) even if sodium-free, calcium-free solution is applied during the cold and rewarming phases (t1/2 = 305 +/- 66 msec). This result suggests that ryanodine does not prevent the SR from accumulating calcium to induce relaxation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Electrodiffusion of ions approaching the mouth of a conducting membrane channel.

The movement of ions in the aqueous medium as they approach the mouth (radius a) of a conducting membrane channel is analyzed. Starting with the Nernst-Planck and Poisson equations, we derive a nonlinear integrodifferential equation for the electric potential, phi(r), a less than or equal to r less than infinity. The formulation allows deviations from charge neutrality and dependence of phi(r) on ion flux. A numerical solution is obtained by converting the equation to an integral equation that is solved by an iterative method for an assumed mouth potential, combined with a shooting method to adjust the mouth potential until the numerical solution agrees with an asymptotic expansion of the potential at r-a much greater than lambda (lambda = Debye length). Approximate analytic solutions are obtained by assuming charge neutrality (Läuger, 1976) and by linearizing. The linear approximation agrees with the exact solution under most physiological conditions, but the charge-neutrality solution is only valid for r much greater than lambda and thus cannot be used unless a much greater than lambda. Families of curves of ion flux vs. potential drop across the electrolyte, phi(infinity)-phi (a), and of permeant ion density at the channel mouth, n1(a), vs. flux are obtained for different values of a/lambda and S = a d phi/dr(a). If a much greater than lambda and S = O, the maximum flux (which is approached when n1(a)----0) is reduced by 50% compared to the value predicted by the charge-neutrality solution. Access resistance is shown to be a factor a/[2 (a + lambda)] times the published formula (Hille, 1968), which was derived without including deviations from charge neutrality and ion density gradients and hence does not apply when there is no counter-ion current. The results are applied to an idealized diffusion-limited channel with symmetric electrolytes. For S = O, the current/voltage curves saturate at a value dependent on a/lambda; for S greater than O, they increase linearly for large voltage.

Diffusion↗

PubMed1988

Differences in action potential configuration in ventricular trabeculae correlate with differences in density of transverse tubule-sarcoplasmic reticulum couplings.

The goal of the present study was to identify structural correlates to an observed variability in the response of ventricular muscle action potential configuration to ryanodine. In a previous study, rabbit ventricular trabeculae were shown to display either a shortened action potential (Group I) or lengthened action potential (Group II) in response to the negative inotropic drug ryanodine. The configuration of control action potentials were also different in these two groups. Action potentials in Group I trabeculae exhibited a prominent early repolarization and low plateau, while those in Group II exhibited a small early repolarization and high plateau. Electron microscope morphometric data showed that there is a significant, positive linear correlation between the number of transverse tubules displaying internal couplings with sarcoplasmic reticulum and the change in action potential duration in the presence of ryanodine. No other differences in internal coupling structure were observed. Although variability in the prominence of M-lines was also apparent between myocytes in both Group I and Group II, there is no correlation between the M-line prominence and ryanodine-induced change in electrophysiological behavior. No other structural variability was noted between Group I and Group II trabeculae. It is suggested that the difference in electrophysiological characteristics in the two groups might be due to a difference in T-tubular density. This hypothesis seems most plausible if there is a differential distribution of ion channels between the surface membrane and T-tubular membrane.

Action Potentials↗

PubMed1988

Can Ca entry via Na-Ca exchange directly activate cardiac muscle contraction?

Developed twitch tension and action potentials were recorded in rabbit ventricular muscle in physiological saline at 30 degrees C stimulated at 0.5 Hz. Addition of 5 microM nifedipine to block Ca entry via Ca channels almost abolished twitches (to 2.5 +/- 0.7%, S.E.M., n = 10 of control). This suggests that under normal conditions Ca entry via Na-Ca exchange is insufficient to activate contractions. However, when muscles are first exposed to 4 microM acetylstrophanthidin to elevate [Na]i the same exposure to nifedipine only partially suppresses twitches (to 59 +/- 12% of the original control). This suggests that when [Na]i is elevated, Ca entry via the Na-Ca exchange may be adequate to partially activate contraction. From this result it is not clear whether Ca entry via Na-Ca exchange is sufficient to activate contraction directly or whether sarcoplasmic reticulum (SR) Ca release is required. When these experiments were carried out in the presence of 5 to 10 mM caffeine or 100 nM ryanodine similar results were obtained. That is, nifedipine still abolished contractions in the presence of caffeine or ryanodine (to 3.8 +/- 0.3% and 1.3 +/- 0.4%, respectively), but only partially inhibited contractions in the presence of caffeine + acetylstrophanthidin (to 21 +/- 5%) or ryanodine + acetylstrophanthidin (10 +/- 2%). Thus, it appears that even in the absence of a functional SR and with Ca current blocked, Na-Ca exchange might bring sufficient Ca into the cell to activate appreciable contractions, but only when [Na]i is elevated. Action potential duration is decreased by nifedipine and acetylstrophanthidin and is further decreased when nifedipine is added on top of acetylstrophanthidin. If this Ca entry is by an electrogenic 3 Na: 1 Ca exchange, Ca entry will be favored at more positive membrane potentials. If the action potential were not so abbreviated with these drugs, Na-Ca exchange might bring in more Ca and activate additional tension.

Action Potentials↗

PubMed1988

Effect of acetylstrophanthidin on twitches, microscopic tension fluctuations and cooling contractures in rabbit ventricle.

1. We have measured the effect of the aglycone acetylstrophanthidin (ACS) on twitches, cooling contractures and microscopic tension fluctuations in rabbit ventricular muscle. 2. Both developed twitches and cooling contractures are strengthened by applications of ACS in the range 1-4 microM. This positive inotropy averages 150-160% of control (zero ACS) in both twitches and cooling contractures. Cooling contracture magnitude is assumed to reflect the availability of sarcoplasmic reticulum (SR) Ca2+ for contraction (Bridge, 1986). We infer that ACS increases the availability of SR Ca2+ by enlarging SR Ca2+ stores and this may contribute to the positive inotropy. 3. However, twitches appear to increase at lower concentrations of ACS than those required to increase cooling contractures. This observation suggests that the initial ACS inotropy may be achieved without an increase in SR Ca2+. Furthermore, low doses of ACS produce positive inotropy in the presence of 10.0 mM-caffeine where cooling contractures are abolished. This also suggests that positive inotropy occurs in the absence of SR Ca2+ accumulation. 4. Rest decay of both cooling contractures and twitches is significantly slowed in 4 and 8 microM-ACS. We infer that ACS slows the rate of decline of SR Ca2+ available for contraction by slowing the rate at which Ca2+ is lost from the cell during rest. This suggests that ACS produces a net slowing of Ca2+ efflux during activity which in the absence of altered Ca2+ influx will result in net Ca2+ gain and presumably enlarged SR Ca2+ stores. 5. Increasing the concentration of ACS (6-10 microM) results in a decline in developed twitch tension, total tension and an increase in rest tension. Measurement of microscopic tension fluctuations indicates that as developed twitches decline, the root mean square (r.m.s.) of the tension fluctuations increases in a reciprocal manner. This supports the suggestion of others that the decline in developed twitch tension and the appearance of tension fluctuations are causally related. 6. Although ACS (6-10 microM) causes a decline in twitch tension, rapid cooling contractures remain elevated. We suggest that in the presence of Ca2+ oscillations the magnitude of cooling contractures reflects the sum of cytosolic Ca2+ and Ca2+ that is available for release. If microscopic tension fluctuations do represent Ca2+ moving between the SR and cytosol the sum of SR and cytosolic Ca2+ and hence cooling contracture might not decline.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1987

The effect of temperature and ionic strength on the apparent Ca-affinity of EGTA and the analogous Ca-chelators BAPTA and dibromo-BAPTA.

The apparent calcium association constants (K'Ca) of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and 1,2-bis 2-bis(o-amino-5-bromophenoxy)ethane-N,N,N',N'-tetraacetic acid (dibromo-BAPTA) were measured using the method described by Bers (Am. J. Physiol. 242 (1982) C404-408). The purity of the three ligands determined from the chi-intercept of Scatchard plots were 96.3%, 79.0% and 97.3% for EGTA, BAPTA and dibromo-BAPTA, respectively. The impurity of BAPTA was found to be water by drying several samples to constant weight. Increasing temperature from 1 to 36 degrees C led to an increase in K'Ca which was of similar magnitude for the three ligands. Increasing ionic strength from 0.104 to 0.304 M led to a reduction of K'Ca in all cases, though EGTA was affected much less than BAPTA or dibromo-BAPTA. Experimental results were compared with values of K'Ca calculated from the individual association constants of the ligands for calcium and protons which were modified for the experimental conditions using the Debye-Hückel limiting law and the Van't Hoff Isochore to correct for ionic strength and temperature, respectively. The experimental values of K'Ca of EGTA agree well with those in the literature and with the calculated values. Good agreement was also found between the experimental and calculated values of K'Ca for the temperature and ionic strength dependence of BAPTA and dibromo-BAPTA.

Calcium↗

PubMed1987

Two different electrophysiological responses to ryanodine: evidence for two populations of muscles isolated from the rabbit right ventricle.

We have observed that small trabeculae isolated from the rabbit right ventricle can be grouped into two populations on the basis of action potential configuration and electrophysiological response to ryanodine. These two populations have been designated Groups I and II. Action potentials in Group I muscles were characterized by a long action potential, a fast early phase of repolarization and shortened when exposed to 0.1 microM ryanodine (from 210 +/- 22 (6) ms (Mean +/- S.E.M. (n)) to 176 +/- 20 (6). Group II muscles showed a shorter action potential with a higher plateau phase and was prolonged by 0.1 microM ryanodine (from 175 +/- 7 (9) ms to 236 +/- 18 (9) ms). No significant difference in the mechanical response to ryanodine was observed between the two groups. Two populations of muscles, isolated from the rabbit right ventricle, have been previously described and distinguished on the basis of the presence or absence of transverse (T) tubules. We have attempted to correlate the electrophysiological observations with the presence or absence of (T) tubules. Electron microscopic examination, however, revealed that T-tubules were clearly present in both groups of trabeculae and therefore the two populations cannot be distinguished simply on the basis of the presence or absence of T-tubules. It was considered possible that the difference in the two populations may be related to the existence, and/or predominance, of two transient outward currents (one activated by SR Calcium release and the other that is 4-aminopyridine (4-AP) sensitive). The relative importance of these two currents to repolarization may be different in the two groups. This was investigated by adding ryanodine in the presence of 4-AP. The differential response to ryanodine seen in the two groups was, however, still observed in the presence of 4-AP. In these experiments, three out of seven muscles showed a decrease in action potential duration and four out of seven showed an increase. Other possible explanations for the differences in the two groups of muscles, and their response to ryanodine, are discussed.

Action Potentials↗

PubMed1987

Mechanisms contributing to the cardiac inotropic effect of Na pump inhibition and reduction of extracellular Na.

Reduction of the transsarcolemmal [Na] gradient in rabbit cardiac muscle leads to an increase in the force of contraction. This has frequently been attributed to alteration of Ca movements via the sarcolemmal Na/Ca exchange system. However, the specific mechanisms that mediate the increased force at individual contractions have not been clearly established. In the present study, the [Na] gradient was decreased by reduction of extracellular [Na] or inhibition of the Na pump by either the cardioactive steroid acetylstrophanthidin or by reduction of extracellular [K]. Contractile performance and changes in extracellular Ca (sensed by double-barreled Ca-selective microelectrodes) were studied in order to elucidate the underlying basis for the increase in force. In the presence of agents that inhibit sarcoplasmic reticulum (SR) function (10 mM caffeine, 100-500 nM ryanodine), reduction of the [Na] gradient produced increases in contractile force similar to that observed in the absence of caffeine or ryanodine. It is concluded that an intact, functioning SR is not required for the inotropic effect of [Na] gradient reduction (at least in rabbit ventricle). However, this does not exclude a possible contribution of enhanced SR Ca release in the inotropic response to [Na] gradient reduction in the absence of caffeine or ryanodine. Acetylstrophanthidin (3-5 microM) usually leads to an increase in the magnitude of extracellular Ca depletions associated with individual contractions. However, acetylstrophanthidin can also increase extracellular Ca accumulation during the contraction, especially at potentiated contractions. This extracellular Ca accumulation can be suppressed by ryanodine and it is suggested that this apparent enhancement of Ca efflux is secondary to an enhanced release of Ca from the SR. Under conditions where Ca efflux during contractions is minimized (after a rest interval in the presence of ryanodine), acetylstrophanthidin increased both the rate and the extent of extracellular Ca depletions. Thus, acetylstrophanthidin can increase both Ca influx and Ca efflux during the cardiac muscle contraction. These results can be explained by a simple model where the direction of net Ca flux via Na/Ca exchange during the action potential is determined by the changes in reversal potential of the Na/Ca exchange. Reduction of the [Na] gradient may well lead to net cellular Ca uptake (via Na/Ca exchange) and may also elevate the resting intracellular [Ca].(ABSTRACT TRUNCATED AT 400 WORDS)

Alkaloids↗

PubMed1987

The mechanism of ryanodine action in rabbit ventricular muscle evaluated with Ca-selective microelectrodes and rapid cooling contractures.

Cellular Ca uptake and efflux in rabbit ventricular muscle was measured using double-barreled Ca microelectrodes in the extracellular space. When repetitive stimulation was stopped there was a slow loss of cellular Ca. Upon resumption of stimulation Ca was taken up by the cells. These Ca movements are thought to represent the loss of Ca from the sarcoplasmic reticulum and the cell during rest and the refilling of the sarcoplasmic reticulum during stimulation. Ryanodine (100 nM) greatly enhanced both the efflux of Ca during rest and the uptake of Ca induced by stimulation. These results are consistent with the conclusions drawn below, but they are dependent upon the interpretation that these extracellular Ca depletions are indicative of sarcoplasmic reticulum Ca movements. To examine further this process, contractures induced by rapid cooling to 0 degrees C were used as an independent assay of sarcoplasmic reticulum Ca content. These rapid cooling contractures were smaller after longer rest intervals (declining with a half time of 1.5 min). In the presence of ryanodine, the rapid cooling contracture immediately after a contraction was greater than that seen under control conditions. However, in the presence of ryanodine these rapid cooling contractures decline as a function of rest duration with a half time of about 1 s. These results suggest that in the presence of ryanodine the sarcoplasmic reticulum can still take up Ca, but that it also loses this Ca very rapidly at the onset of rest.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaloids↗

PubMed1987

Effects of rest duration and ryanodine on changes of extracellular [Ca] in cardiac muscle from rabbits.

Cumulative depletions of extracellular Ca were measured using double-barreled Ca-sensitive microelectrodes in the extracellular space of rabbit ventricular muscle. Depletions were produced by 1-Hz stimulation after rest intervals of 10 s to 10 min. With longer rest intervals, depletion size increased while the first postrest contraction decreased in a reciprocal manner. The depletions may represent refilling of sarcoplasmic reticulum (SR) Ca stores that have become partially depleted of Ca during the rest. Within this interpretive framework, the longer the rest interval the lower the SR Ca content, so the SR is then capable of taking up larger amounts of Ca. This may be related to the rest decay of tension of the first postrest beat, since this is thought to be SR dependent. Ryanodine (1 microM) increased the size of the depletions after short rest intervals (less than 2 min) but not after longer (greater than or equal to 2 min) intervals. Ryanodine also increased the rate of Ca loss from the cell on cessation of stimulation. This increased rate of Ca loss with ryanodine may deplete the SR of Ca such that more Ca can be taken up during subsequent stimulation than in untreated muscles. Thus cumulative depletions after short rest intervals are enhanced by ryanodine. When a Ca load was produced during 1) quiescence [by removal of extracellular Na (Nao)] or 2) continuous stimulation (in the presence of 3 microM acetylstrophanthidin), addition of ryanodine (5-10 microM) did not produce any apparent Ca loss. Caffeine (10 mM), added after ryanodine, induced contractures accompanied by Ca efflux, implying there was Ca in the SR after ryanodine exposure. The results of other investigators have suggested that ryanodine may inhibit cardiac SR Ca release. The present study suggests that ryanodine also enhances the loss of cellular (and probably SR) Ca on cessation of stimulation but not when applied during continuous stimulation or quiescence.

Alkaloids↗

PubMed1987

Ryanodine and the calcium content of cardiac SR assessed by caffeine and rapid cooling contractures.

The ability of rabbit ventricular muscle sarcoplasmic reticulum (SR) to accumulate and retain Ca during Na-free perfusion was assessed using caffeine contractures and rapid cooling contractures (RCC). Muscles were exposed to a Na-free medium for 15 min, during which time a transient contracture developed and relaxed back to the resting tension level. Muscles were then either exposed to 20 mM caffeine or rapidly cooled to less than 5 degrees C, both of which produced large contractures. The magnitudes of those contractures are believed to be a relative index of SR Ca content. Reduction of extracellular [Ca] from 2 to 0.2 mM did not significantly alter the magnitude of either Na-free caffeine contractures or RCC. These are not the maximum contractures that can be obtained. The possibility that low extracellular [Ca] ([Ca]o) may increase passive Ca permeability is suggested in explanation of this effect. After equilibration with 100 nM ryanodine, both Na-free caffeine contractures and RCC are virtually eliminated. This suggests that even if the SR could accumulate Ca during the initial Na-free exposure in the presence of ryanodine, it could not retain that Ca in Na-free medium. It is proposed that the sarcolemmal Ca pump can extrude Ca from the cells at a rate sufficient to deplete the ryanodine-treated SR. When removal of Na was accompanied by increase of [K]o (to 12, 20, or 30 mM), the Na-free RCC were enhanced. Increasing [K]o during Na-free superfusion in the presence of ryanodine resulted in demonstrable RCC that were graded with [K] and completely suppressed by nifedipine.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaloids↗