Search PubMed⌕ Search

Biomedical subjects

D M Bers

Publications and source records attributed to D M Bers.

At least 91 records · Page 5Linked to original sources
PubMed1995

Protein kinase inhibitor H-89 reverses forskolin stimulation of cardiac L-type calcium current.

Calcium currents (ICa) and barium currents (IBa) were measured in freshly isolated single ferret ventricular myocytes, using the whole cell patch-clamp and perforated patch-clamp techniques with Na and K currents blocked by tetraethylammonium and Cs. The membrane potential (Em) dependence of activation and steady-state inactivation curves were determined using a Boltzmann relation, where E0.5 is the Em at half-maximal conductance. Forskolin (1 microM) increased the rate of ICa inactivation, especially in perforated patch, but slowed IBa inactivation. The acceleration is likely to be due to greater Ca-dependent inactivation of ICa, where the slowing of IBa inactivation may be due to protein kinase A-dependent slowing of Em-dependent inactivation. Forskolin (1-10 microM) also increased ICa amplitude by two- to threefold and shifted the E0.5 for both activation and inactivation to more negative potentials by 7-8 mV. The effect of forskolin on the amplitude of ICa could be reversed by an inhibitor of adenosine 3',5'-cyclic monophosphate-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89; 1-10 microM). However, H-89 did not reverse the shift of E0.5 induced by forskolin. H-89 application by itself does not decrease basal ICa but does shift the E0.5 of both activation and inactivation to more negative values of Em. It is possible that H-89 reverses the shift induced by regulatory phosphorylation (due to forskolin) but induces a coincidental negative shift itself.

Animals↗
PubMed1995

Fractional SR Ca release is regulated by trigger Ca and SR Ca content in cardiac myocytes.

The release of sarcoplasmic reticulum (SR) Ca in cardiac muscle during excitation-contraction coupling is known to be graded by the amount of activating Ca outside the SR (i.e., Ca-induced Ca release). However, little is known about how intra-SR Ca affects the release process. In this study we assessed how the fractional SR Ca release as described by Bassani et al. [Am. J. Physiol. 265 (Cell Physiol. 34): C533-C540, 1993] is affected by alteration of trigger Ca and of SR Ca content. Experiments were done with isolated ferret ventricular myocytes using indo 1 to measure Ca concentration, perforated patch to measure Ca current (ICa), caffeine application to release SR Ca, and thapsigargin to completely block SR Ca uptake. For what we consider a Normal SR Ca load and trigger Ca [action potential at 0.5 Hz with 2 mM extracellular Ca concentration ([Ca]o)], 35 +/- 3% of the SR Ca content was released at a twitch. Changing trigger Ca by altering [Ca]o (to 0.5 and 8 mM) at a test twitch changed this fractional SR Ca release to 10 +/- 2 and 59 +/- 6%, with the same SR Ca load (and peak ICa changed in a parallel manner in separate voltage-clamp experiments). Three different levels of SR Ca load were studied (Low, Normal, and High; by action potential stimulation at different frequencies from 0.05 to 0.8 Hz) using the same standard test trigger Ca (2 mM). Surprisingly, the High-load condition only increased SR Ca content by approximately 4% but appeared to be very close to the limiting SR Ca capacity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

CaMKII is responsible for activity-dependent acceleration of relaxation in rat ventricular myocytes.

We investigated the role of Ca/calmodulin-dependent protein kinase (CaMKII) in relaxation and cytosolic free [Ca] ([Ca]i) decline during steady-state (SS) and postrest (PR) twitches in intact rat ventricular myocytes. Half-time of mechanical relaxation and time constant of [Ca]i decline (tau) were twofold greater during PR than with SS at 1 Hz. This difference was 1) abolished by inhibition of sarcoplasmic reticulum (SR) Ca accumulation by thapsigargin or caffeine; 2) greater at higher stimulation frequency and extracellular [Ca], which affected only SS tau; 3) abolished by the protein phosphatase inhibitor okadaic acid (10 microM, which selectively accelerated [Ca]i decline during PR); 4) still present during stimulation or inhibition of adenosine 3',5'-cyclic monophosphate-dependent protein kinase (PKA) by 10 microM forskolin or 1 microM H-89, respectively (SS and PR tau values were abbreviated and prolonged, respectively); and 5) suppressed by 10 microM KN-62, a selective inhibitor of CaMKII, which selectively prolonged [Ca]i decline during SS twitches. Both protein kinase inhibitors were also shown to decrease the SR Ca-uptake rate in digitonin-permeabilized rat myocytes. We conclude that CaMKII plays a major role in modulation of relaxation in rat ventricular myocytes, enhancing SR Ca uptake in a activity-dependent fashion. Our results are also compatible with a background, activity-independent stimulation of SR Ca uptake by PKA in intact rat myocytes.

Animals↗
PubMed1994

Relaxation in rabbit and rat cardiac cells: species-dependent differences in cellular mechanisms.

The roles of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase and Na(+)-Ca2+ exchange in Ca2+ removal from cytosol were compared in isolated rabbit and rat ventricular myocytes during caffeine contractures and electrically stimulated twitches. Cell shortening and intracellular calcium concentration ([Ca2+]i) were measured in indo-1-loaded cells. Na(+)-Ca2+ exchange was inhibited by replacement of external Na+ by Li+. To avoid net changes in cell or SR Ca2+ load during a twitch in 0 Na+ solution, intracellular Na+ (Na+i) was depleted using a long pre-perfusion with 0 Na+, 0 Ca2+ solution. SR Ca2+ accumulation was inhibited by caffeine or thapsigargin (TG). Relaxation of steady-state twitches was 2-fold faster in rat than in rabbit (before and after Na+i depletion). In contrast, caffeine contractures (where SR Ca2+ accumulation is inhibited), relaxed faster in rabbit cells. Removal of external Na+ increased the half-time for relaxation of caffeine contractures 15- and 5-fold in rabbit and rat myocytes respectively (and increased contracture amplitude in rabbit cells only). The time course of relaxation in 0 Na+, 0 Ca2+ solution was similar in the two species. Inhibition of the Na(+)-Ca2+ exchange during a twitch increased the [Ca2+]i transient amplitude (delta[Ca2+]i) by 50% and the time constant of [Ca2+]i decline (tau) by 45% in rabbit myocytes. A smaller increase in tau (20%) and no change in delta[Ca2+]i were observed in rat cells in 0 Na+ solution. [Ca2+]i transients remained more rapid in rat cells. Inhibition of the SR Ca(2+)-ATPase during a twitch enhanced delta[Ca2+]i by 25% in both species. The increase in tau after TG exposure was greater in rat (9-fold) than in rabbit myocytes (2-fold), which caused [Ca2+]i decline to be 70% slower in rat compared with rabbit cells. The time course of [Ca2+]i decline during twitch in TG-treated cells was similar to that during caffeine application in control cells. Combined inhibition of these Ca2+ transport systems markedly slowed the time course of [Ca2+]i decline, so that tau was virtually the same in both species and comparable to that during caffeine application in 0 Na+, 0 Ca2+ solution. Thus, the combined participation of slow Ca2+ transport mechanisms (mitochondrial Ca2+ uptake and sarcolemmal Ca(2+)-ATPase) is similar in these species. We conclude that during the decline of the [Ca2+]i transient, the Na(+)-Ca2+ exchange is about 2- to 3-fold faster in rabbit than in rat, whereas the SR Ca(2+)-ATPase is 2- to 3-fold faster in the rat.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1994

Relaxation in ferret ventricular myocytes: unusual interplay among calcium transport systems.

Transport systems responsible for removing Ca2+ from the myoplasm during relaxation in isolated ferret ventricular myocytes were studied using caffeine-induced contractures. Internal calcium concentration ([Ca2+]i) was measured with the fluorescent calcium indicator indo-1, and the results were compared with our recent detailed characterizations in rabbit and rat myocytes. Relaxation and [Ca2+]i decline during a twitch in ferret myocytes were fast and similar to that in rat myocytes (i.e. half-time, t 1/2 approximately 100-160 ms). During a caffeine-induced contracture (SR Ca2+ accumulation prevented), relaxation was still relatively fast (t 1/2 = 0.57 s) and similar to relaxation in rabbit supported mainly by a strong Na(+)-Ca2+ exchange. When both the SR Ca2+ uptake and Na(+)-Ca2+ exchange are blocked (by caffeine and 0 Na+, 0 Ca2+ solution) relaxation in the ferret myocyte is remarkably fast (approximately 5-fold) compared with rabbit and rat myocytes. The decline of the Cai2+ transient was also fast under these conditions. These values were similar to those in rat under conditions where relaxation is due primarily to Na(+)-Ca2+ exchange. Additional inhibition of either the sarcolemmal Ca(2+)-ATPase or mitochondrial Ca2+ uptake caused only modest slowing of the relaxation of caffeine-induced contracture in 0 Na+, 0 Ca2+ (t 1/2 increased to approximately 3 s). In rabbit myocytes the relaxation t 1/2 is slowed to 20-30 s by these procedures. Even when the systems responsible for slow relaxation in rabbit ventricular myocytes are inhibited (i.e. sarcolemmal Ca(2+)-ATPase and mitochondrial Ca2+ uptake) along with the SR Ca(2+)-ATPase and Na(+)-Ca2+ exchange, relaxation and [Ca2+]i decline in ferret myocytes remain rapid compared with rabbit myocytes. Ca2+ taken up by mitochondria in rabbit myocytes during a caffeine contracture in 0 Na+, 0 Ca2+ solution gradually returns to the SR after caffeine removal, but this component appears to be much smaller in ferret myocytes under the same conditions. We tested for possible residual Ca2+ transport by each of the four systems which suffice to explain Ca2+ removal from the cytoplasm in rabbit (SR Ca(2+)-ATPase, Na(+)-Ca2+ exchange, sarcolemmal Ca(2+)-ATPase and mitochondrial Ca2+ uptake). We conclude that there is an additional calcium transport system at work in ferret myocytes. For this additional system, our results are most compatible with a trans-sarcolemmal Ca2+ transport, but neither a cation exchanger nor a Ca(2+)-ATPase with characteristics like that in other cardiac cells. This additional system appears able to transport Ca2+ nearly as fast as the Na(+)-Ca2+ exchange in rat ventricular myocytes.

Animals↗
PubMed1994

Regulation of the cloned L-type cardiac calcium channel by cyclic-AMP-dependent protein kinase.

Hormones can regulate cardiac L-type Ca2+ channels via cAMP-dependent protein kinase (PKA) phosphorylation. However, regulation of the cloned L-type Ca2+ channel has been difficult to demonstrate conclusively. We stably transfected a human embryonic kidney (HEK-293) cell with the cardiac alpha 1 and beta 2 subunits, then examined PKA modulation of the Ca2+ current. Although forskolin did not increase basal Ca2+ current, the PKA inhibitors, H-89 and Rp-cAMPS, could inhibit basal current. We reversed H-89 inhibition with either forskolin or okadaic acid. We conclude that the channel was phosphorylated under basal conditions, and that inhibition of PKA allowed dephosphorylation. These studies demonstrate that reversible PKA regulation of cloned Ca2+ channels can be studied in HEK-293 cells.

Animals↗
PubMed1994

Na-Ca exchange is required for rest-decay but not for rest-potentiation of twitches in rabbit and rat ventricular myocytes.

The influence of the Na-Ca exchange (NaCaX) on the effects of rest (30-300 s) on twitch amplitude and SR Ca content (assessed by caffeine contractures) was studied in ventricular myocytes isolated from rat and rabbit. In control conditions, rabbit cells showed monotonic rest-decay of the amplitudes of both twitch and caffeine contractures, while rat myocytes developed rest-potentiation of twitches without change in SR Ca content. Inhibition of the Na-Ca exchange during rest by perfusion with 0Na,0Ca solution did not affect the responses in rat cells but abolished rest-dependent SR Ca loss in rabbit cells. Indeed, when NaCaX was blocked during rest, then rabbit cells, like rat, displayed rest-potentiation of twitches. Stimulation of net Ca extrusion via NaCaX during rest by perfusion with 0Ca solution induced rest-decay of twitches and caffeine contractures in rat cells similar to that observed in rabbit cells. This maneuver also accelerated decline in SR Ca during rest and amplitude of the first post-rest twitch in rabbit myocytes. These effects were only slightly enhanced by preperfusion with 0Na,0Ca solution to deplete Nai. We were thus able to interconvert the contractile responses to rest between these cell types solely by modifying the driving force for Ca transport by the exchange. Our results indicate that SR Ca is lost during quiescence in both species, but only if the NaCaX is able to promote diastolic Ca extrusion will net decline of SR Ca (and twitch amplitude) occur. On the other hand, post-rest twitch potentiation in both rat and rabbit cells can occur without a change in SR Ca content. This effects might be attributable, at least in part, to a slow phase of recovery of excitation-contraction coupling.

Action Potentials↗
PubMed1994

Intrinsic cytosolic calcium buffering properties of single rat cardiac myocytes.

Intracellular passive Ca2+, buffering was measured in voltage-clamped rat ventricular myocytes. Cells were loaded with indo-1 (K+ salt) to an estimated cytosolic concentration of 44 +/- 5 microM (Mean +/- SEM, n = 5), and accessible cell volume was estimated to be 24.5 +/- 3.6 pl. Ca2+ transport by the sarcoplasmic reticulum (SR) Ca-ATPase and sarcolemmal Na-Ca exchange was inhibited by treatment with thapsigargin and Na-free solutions, respectively. Extracellular [Ca2+] was maintained at 10 mM and, in some experiments, the mitochondrial uncoupler "1799" was used to assess the degree of mitochondrial Ca2+ uptake. To perform single cell titrations, intracellular Ca2+ ([Ca2+]i) was increased progressively by a train of depolarizing voltage clamp pulses from -40 to +10 mV. The total Ca2+ gain with each pulse was calculated by integration of the Ca current and then analyzed as a function of the rapid change in [Ca2+]i during the pulse. In the range of [Ca2+]i from 0.1 to 2 microM, overall cell buffering was well described as a single lumped Michaelis-Menten type species with an apparent dissociation constant, KD, of of 0.63 +/- 0.07 microM (n = 5) and a binding capacity, Bmax, of 162 +/- 15 mumol/l cell H2O. Correction for buffering attributable to cytosolic indo-1 gives intrinsic cytosolic Ca2+ buffering parameters of KD = 0.96 +/- 0.18 microM and Bmax = 123 +/- 18 mumol/l cell H2O. The fast Ca2+ buffering measured in this manner agrees reasonably with the characteristics of known rapid Ca buffers (e.g., troponin C, calmodulin, and SR Ca-ATPase), but is only about half of the total Ca2+ buffering measured at equilibrium. Inclusion of slow Ca buffers such as the Ca/Mg sites on troponin C and myosin can account for the differences between fast Ca2+ buffering in phase with the Ca current measured in the present experiments and equilibrium Ca2+ buffering. The present data indicate that a rapid rise of [Ca2+]i from 0.1 to 1 microM during a contraction requires approximately 50 microM Ca2+ to be added to the cytosol.

Animals↗
PubMed1994

Protein kinase inhibitors reduce SR Ca transport in permeabilized cardiac myocytes.

Phosphorylation of the sarcoplasmic reticulum (SR) protein phospholamban by adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) and Ca-calmodulin-dependent protein kinase (CaM-KII) stimulates Ca-adenosinetriphosphatase (ATPase) activity and SR Ca transport, but the role of CaM-KII-dependent phosphorylation is not well defined. We studied the PKA- and CaM-KII-dependent regulation of SR Ca transport in digitonin-permeabilized rabbit ventricular myocytes. SR Ca uptake and free Ca concentration were measured on line with indo 1 and Ca electrodes in the presence of 20 microM ruthenium red and 10 mM oxalate. neither N5,2'-w-dibutyryl-cAMP (up to 500 microM) nor the nonhydrolyzable cAMP agonist adenosine 3'5'-cyclic monophosphorothioate sodium salt (Sp-cAMP[S]; up to 275 microM) affected the maximum uptake rate (Vmax) or the dissociation constant (Kd) for Ca uptake. However, the PKA inhibitor H-89 significantly increased Kd (e.g., from 307 +/- 67 to 826 +/- 62 nM Ca at 40-65 microM H-89) without significantly affecting Vmax. Both CaM-KII inhibitors, KN-62 (60 microM) and a CaM-KII inhibitory peptide (10 microM), significantly decreased Vmax from 11.95 +/- 0.5 to 9.48 +/- 0.6 nmol.mg-1.min-1 and from 10.95 +/- 1.72 to 7.37 +/- 0.94 nmol.mg-1.min-1, respectively, without consistently changing Kd. The effects of H-89 on Kd and of KN-62 on Vmax were prevented by a monoclonal antibody to phospholamban 2D12 (consistent with the antibody removing the inhibitory effect of phospholamban on the SR Ca-ATPase).(ABSTRACT TRUNCATED AT 250 WORDS)

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1994

Ca-dependent facilitation of cardiac Ca current is due to Ca-calmodulin-dependent protein kinase.

Repetitive membrane potential (Em) depolarization from -90 to 0 mV in rabbit and ferret ventricular myocytes induces a facilitation or "staircase" of Ca current (ICa), which is Ca (not Em) dependent and takes several seconds to accumulate and dissipate. That is, ICa at the tenth pulse at 1-2 Hz exceeds that at the first pulse (I10 > I1). The ICa staircase was completely abolished by dialysis with either of two inhibitory peptides of Ca-calmodulin-dependent protein kinase (CaMKII) CaMKII(290-309) and CaMKII(273-302)], implicating this kinase. Inclusion of ATP gamma S in the patch pipette gradually increased ICa but also abolished the staircase implicating phosphorylation. KN-62, a nonpeptide CaMKII inhibitor, reversed the ICa staircase (I1 > I10). However, this effect of KN-62 was largely attributed to a slower recovery from inactivation and a gating shift to more negative Em (not seen with CaMKII peptides). Similar results were obtained with H-89 and staurosporine (inhibitors of adenosine 3',5'-cyclic monophosphate and phospholipid-/Ca-dependent protein kinase, respectively). The reversal of the ICa staircase with H-89 and KN-62 could be prevented by more negative interpulse Em or elevation of extracellular [Ca] (which could counteract changes in channel gating due to a reduction in internal negative surface potential). That is, these kinase inhibitors might decrease phosphorylation at the inner membrane surface. In approximately 30% of the cells studied with H-89 and staurosporine the characteristic kinetic difference in ICa inactivation (faster at I1 than I10) was also diminished. This might be due to a relatively nonspecific inhibition of the same protein kinase inhibited by the CaMKII peptides. We conclude that the Ca-dependent ICa facilitation is due to activation of CaMKII and phosphorylation of a site on or near the Ca channel. KN-62, H-89, and staurosporine shifted ICa gating to more negative potentials and slowed recovery from inactivation, effects that could be due to reduction in phosphorylation at the inner membrane surface. Thus the reversal of the ICa staircase by KN-62, H-89, and staurosporine may not be Ca channel specific.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1994

Contractile arrest increases sarcoplasmic reticulum calcium uptake and SERCA2 gene expression in cultured neonatal rat heart cells.

We developed protocols with intact cultured neonatal rat myocytes to directly evaluate the function of the sarcoplasmic reticulum (SR) Ca-ATPase (or SERCA2), Na-Ca exchange (Na-CaX), and slow Ca transport systems (mitochondria and sarcolemmal Ca-ATPase). Spontaneously beating control cells were compared with cells cultured for 2 days in the presence of verapamil (verapamil-arrested cells, VA). Intracellular calcium (Cai) transients were measured by use of indo-1 during (1) spontaneous twitches, (2) contractures induced by rapid application of caffeine (CafC, with and without Nao), and (3) twitches induced by brief depolarizations with high [K]o solution (K-twitches). We also measured mRNA levels for the SR Ca-ATPase and Na-CaX in the same experimental preparations. The t1/2 for [Ca]i decline when both the SR Ca uptake and Na-CaX were prevented was the same for control and VA cells (approximately 20 seconds), indicating unaltered slow Ca transport systems. Similarly, there was no significant difference in the t1/2 of CafC when Na-CaX was the main mechanism responsible for [Ca]i decline (t1/2 approximately 1.5 seconds), indicating unaltered Na-CaX. Conversely, we found nearly a twofold increase in the rate of [Ca]i decline during K-twitches (control t1/2, 0.84 +/- 0.05 seconds; VA t1/2, 0.48 +/- 0.06 second; P < .001), indicating an increase in SR Ca-pumping activity in VA cells. This was also reflected by a 56% increase in the peak [Ca]i reached during CafC used to assess maximal SR Ca content (427 +/- 49 nmol/L in control versus 665 +/- 75 nmol/L in VA cells).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Paradoxical twitch potentiation after rest in cardiac muscle: increased fractional release of SR calcium.

Rest interval dependent changes in contractile force (rest decay and rest potentiation) were studied in rabbit, rat and ferret ventricular muscle and myocytes. The SR Ca content was assessed by rapid cooling contractures or caffeine induced contractures. Intracellular Ca transients, action potentials and Ca current were also recorded. Rest decay of twitches in rabbit ventricle are roughly paralleled by a decline in SR Ca content. Rat ventricle exhibits primarily rest potentiation, which is not necessarily paralleled by an increased SR Ca content. Ferret ventricle exhibits both rest potentiation and rest decay. However, the SR Ca content in ferret appears to decline monotonically throughout the rest. It is demonstrated that the rest potentiation is not due to an increase in Ca current or in action potential duration. We conclude that there is an increase in the fraction of SR Ca content which is released during the time that rest potentiation develops. The differences in post-rest contractile function among different cardiac preparations can be described by a simple unifying mechanistic model. In this model there is an exponential time dependent recovery of the ability of the SR to release Ca (e.g. recovery from inactivation) which can be considered to increase the fraction of SR Ca release in response to activation. This fractional SR Ca release is multiplied by the SR Ca content (which may decline exponentially) to provide a measure of the Ca available for activation of contractile force.

Animals↗
PubMed1993

Competition and redistribution among calcium transport systems in rabbit cardiac myocytes.

Normally the sarcoplasmic reticular calcium pump and sarcolemmal Na/Ca exchange compete and are the primary mechanisms responsible for reducing [Ca]i during cardiac relaxation. The sarcolemmal calcium pump and mitochondrial calcium uptake are much slower, but may also participate in [Ca]i regulation. The aim of this study was to provide a clearer understanding of the interaction of these mechanisms. Myocyte shortening and [Ca]i transients (using indo-1 fluorescence) were measured in rabbit ventricular myocytes, with similar results. The t1/2 of twitch relaxation was 0.17(SEM 0.03) s. Contractures induced by 10 mM caffeine (caffeine contraction) relaxed more slowly [t1/2 = 0.54(0.07) s] due to prevention of sarcoplasmic reticular calcium uptake. When the Na/Ca exchange was also blocked by perfusion with 0Na,0Ca solution, the peak of the caffeine contraction was increased (by 44%) and relaxation was slowed about 16-fold [t1/2 = 8.8(0.8) s]. Blocking mitochondrial calcium uptake by including 1 microM FCCP + 1 microM oligomycin in the 0Na,0Ca solution slowed the relaxation of the caffeine contraction further [t1/2 = 19.7(3.2) s]. Inhibition of the sarcolemmal calcium pump by perfusion with 0Na, 10 mM Ca during caffeine contraction also increased the relaxation t1/2 to 27.5(6.9) s. Inhibition of all four calcium transport systems almost abolished relaxation. It is also shown that calcium which was taken up by the mitochondria during relaxation of the caffeine contraction in 0Na,0Ca gradually redistributed (with tau = 41 s) back to the sarcoplasmic reticulum after caffeine was removed. A second caffeine contraction could then be elicited.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Ca2+ cycling between sarcoplasmic reticulum and mitochondria in rabbit cardiac myocytes.

1. Shortening and intracellular Ca2+ (Ca2+i) transients were measured in isolated rabbit ventricular myocytes during paired contractures induced by rapid application of 10 mM caffeine. 2. Caffeine-induced contractures relax despite maintained presence of caffeine. In control solution, a second phasic caffeine contracture failed to appear, unless the sarcoplasmic reticulum (SR) was refilled by a series of electrically stimulated twitches during the interval between caffeine exposures. 3. The relaxation of caffeine-induced contractures in 0 Na(+)-0 Ca2+ solution has previously been shown to rely on mitochondrial Ca2+ uptake and sarcolemmal Ca2(+)-ATPase. Thus, a second caffeine contracture (T2) while still in 0 Na(+)-0 Ca2+ was greatly reduced compared to the first one (T1). However, the amplitude of T2 increased exponentially with the time interval, attaining a maximum of approximately 50% of T1 for an interval of 180-300 s, with a time constant (tau) of 41.2 s. Similar results were found for Ca2+i transients (tau = 45 s). 4. Inhibition of the mitochondrial Ca2+ uptake by the oxidative phosphorylation uncoupler, FCCP during T1 dramatically depressed T2. On the other hand, inhibition of the sarcolemmal Ca2(+)-ATPase (by increasing extracellular Ca2+ concentration, [Ca2+]o) resulted in increase of T2. Spermine inclusion during T1 also increased T2, possibly by an increase of mitochondrial Ca2+ uptake. 5. We conclude that Ca2+ taken up by mitochondria during the decline of T1 moves back to the SR after caffeine is removed, with a tau approximately 40 s. 6. Partial intracellular Na+ depletion by prolonged (3 min) perfusion with 0 Na(+)-0 Ca2+ solution before T1 (a) accelerated relaxation and [Ca2+]i decline during T1, and (b) slowed, but did not abolish, the recovery of T2 as the interval was increased. This effect was particularly pronounced when choline was used instead of Li+ as the Na+ substitute. 7. We further conclude that the mitochondrial Na(+)-Ca2+ antiporter influences the rate of net Ca2+ uptake by mitochondria and is also important in Ca2+ efflux from mitochondria during rest.

Animals↗
PubMed1993

Passive Ca buffering and SR Ca uptake in permeabilized rabbit ventricular myocytes.

Passive Ca binding was measured with a Ca-selective minielectrode in suspensions of permeabilized rabbit ventricular myocytes equilibrated with 5 microM thapsigargin and 30 microM ruthenium red to prevent sarcoplasmic reticulum (SR) or mitochondrial Ca uptake. Passive Ca binding was obtained by titration of the myocytes with Ca and subtraction of Ca binding in a blank titration without myocytes. Passive Ca binding could be described by a Michaelis binding curve with two sites: K1 = 0.42 microM n1 = 1.27 nmol/mg cell protein and K2 = 79 microM, n2 = 4.13 nmol/mg cell protein. The passive Ca buffering over the physiological Ca concentration was approximately twice the value expected from the values compiled by Fabiato [A. Fabiato. Am. J. Physiol. 245 (Cell Physiol. 14): C1-C14, 1983]. The maximal SR Ca uptake in the presence of 30 microM ruthenium red was fit by an uptake curve with a maximal uptake of 5.16 nmol/mg cell protein and a K 1/2 of 1.0 microM. In the presence of 5 microM thapsigargin and no ruthenium red, a significant Ca uptake attributed to mitochondria was measured between 10 and 100 microM free Ca. Rapid changes in free Ca concentration ([Ca]) measured with a Ca electrode were slower than simultaneous measurements of free [Ca] with indo-1 in permeabilized myocytes. However, oxalate, which buffers Ca and maximizes SR Ca uptake, increased the uptake rate and eliminated the difference in free [Ca] measured with Ca electrode and indo-1. This suggests that spatial gradients of [Ca] exist in permeabilized myocytes without Ca buffering. The new estimates of the buffering of intracellular Ca in cardiac myocytes should be valuable in developing quantitative insights into cardiac Ca regulation.

Animals↗
PubMed1993

Ratio of ryanodine to dihydropyridine receptors in cardiac and skeletal muscle and implications for E-C coupling.

We measured dihydropyridine receptor (DHPR) and ryanodine receptor (RYR) density in isolated ventricular myocytes from rabbits, rats, ferrets, and guinea pigs and also from rabbit ventricular homogenate, skeletal muscle homogenate, and isolated triads. In skeletal muscle homogenate and triads the RYR/DHPR ratio was 0.7 and 0.52, respectively. This stoichiometry is reasonably consistent with excitation-contraction (E-C) coupling models in skeletal muscle where the DHPR molecule itself may transmit the signal for Ca release to the sarcoplasmic reticulum (SR) and with the molecular arrangement proposed for toadfish swimbladder from ultrastructural studies by B. A. Block, T. Imagawa, K. P. Campbell, and C. Franzini-Armstrong. (J. Cell Biol. 107: 2587-2600, 1988). That is, there could be approximately two RYR for each four DHPR or two RYR feet per DHPR tetrad in an organized array (assuming 1 high-affinity RYR/foot and 4 DHPR/tetrad). The fraction of rabbit ventricular protein that is cardiac myocyte protein was also estimated (< or = 55-62%), assuming that RYR and DHPR are useful but not exclusive markers for myocytes in the ventricle. In cardiac myocytes the RYR/DHPR was much higher than in skeletal muscle and varied among different mammalian myocytes. The RYR/DHPR ratios were 3.7 in rabbit, 4.3 in guinea pig, 7.3 in rat, and 10.2 in ferret myocytes. In contrast to skeletal muscle, these results indicate that there are many more RYR feet per DHPR in cardiac muscle, and this ratio depends on species (i.e., 4-10 times and would be 4 times higher still per putative DHPR tetrad if that structure exists in mammalian heart).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗