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P Palade

Publications and source records attributed to P Palade.

At least 19 recordsLinked to original sources
PubMed2001

FPL-64176 modifies pore properties of L-type Ca(2+) channels.

In addition to its known effects on Ca(2+) and Ba(2+) currents, the L-type Ca(2+) channel agonist FPL-64176 was found to affect channel function in isolated rat ventricular myocytes in the absence of Ca(2+), with other ions as current carriers through the channel. FPL-64176 induced Cd(2+) current through the L-type Ca(2+) channel, suggesting that certain selectivity properties had changed, perhaps indicative of a small change in pore structure. FPL-64176 slightly but significantly decreased the effectiveness of Co(2+) as a blocker of the channel. FPL-64176 also increased conductance through single L-type Ca(2+) channels recorded in the cell-attached configuration, from 71.9 +/- 11.6 to 94.1 +/- 8.3 pS, with Na(+) carrying the current at pH 9.0. At present it is uncertain whether FPL-64176 produces small alterations of a sole open state of the channel or whether it increases the prevalence of a second, higher conductance open state. These changes, particularly the conversion of Cd(2+) from a pure blocker to a permeant ion, may be of eventual help in discriminating among different models for Ca(2+) channel selectivity.

Animals↗
PubMed2000

Sarcoplasmic reticulum calcium release is stimulated and inhibited by daunorubicin and daunorubicinol.

Cardiac effects of anthracyclines or their metabolites may include both the stimulation and inhibition of Ca(2+) release from sarcoplasmic reticulum. In this study, the ability of daunorubicin and its primary metabolite, daunorubicinol, to stimulate and inhibit Ca(2+) release from canine sarcoplasmic reticulum (SR) vesicles was investigated. It was observed that both daunorubicin and daunorubicinol were several fold more potent at inhibiting than they were at stimulating SR Ca(2+) release. Respective IC50 inhibition of daunorubicin and daunorubicinol for caffeine-induced calcium release was 1.2 and 0.6 microM, and for spontaneous Ca(2+) release was 3 and 1 microM. EC50's for daunorubicin- and daunorubicinol-induced calcium release were 30 and 15 microM, respectively. Inhibition of either spontaneous or caffeine-induced SR Ca(2+) release was inversely related to the amount of Ca(2+) loaded into the SR before exposure to daunorubicin or daunorubicinol. The free-radical scavenger dithiothreitol did not attenuate the ability of anthracyclines to inhibit SR Ca(2+) release. A nonquinone daunorubicin derivative, 5-iminodaunorubicin, was less potent than daunorubicin at inhibiting caffeine-induced Ca(2+) release. These data suggest anthracyclines and their metabolites may produce cardiotoxicity through free-radical independent, concentration-dependent effects on SR Ca(2+) release. These effects involve either inhibition or stimulation of SR Ca(2+) release and are partly dependent upon the presence of the quinone moiety.

Animals↗
PubMed2000

Kinetic effects of FPL 64176 on L-type Ca2+ channels in cardiac myocytes.

To characterize the effects of the Ca2+ channel agonist FPL 64176 on L-type Ca2+ current in isolated rat ventricular myocytes, certain of its effects were compared with those of a better known agonist, S (-) Bay K 8644. Both drugs enhance currents elicited by depolarizing pulses and enhance and slow the decay of tail currents elicited by subsequent repolarization. Both drugs shift the voltage dependence of activation and of inactivation approximately 10 mV in the negative direction, but FPL 64176 slows the rate of both activation and the decline of Ca2+ current during a depolarization, whereas Bay K 8644 accelerates the rate of current decay under the same conditions. In single channel studies in on-cell recording mode, FPL 64176 produced a great lengthening of the channel open time, produced very long openings when the channels were repolarized after a depolarizing stimulus, and had only modest effects on mean closed times and on first latency distributions. FPL 64176 and Bay K 8644 also had minimal effects on L-type channel "on" gating currents, while the "off" gating currents were slowed, particularly at positive potentials. However, the effects on gating currents were too small to account for the prolonged tails observed in FPL 64176. Once the channel is open, FPL 64176 slows transitions to closed or inactivated channel states.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1999

One calcium ion may suffice to open the tetrameric cardiac ryanodine receptor in rat ventricular myocytes.

1. The release of Ca2+ from sarcoplasmic reticulum in response to Ca2+ entering through L-type Ca2+ channels was studied in isolated voltage clamped rat ventricular myocytes at room temperature using the fluorescent Ca2+ indicators fluo-3 and Oregon Green 488 Bapta 5N. 2. Depolarizations to positive potentials elicited fluo-3 Ca2+ transients with rates of rise that were linearly related to the magnitude of the peak measured Ca2+ current in the presence of Cs+-containing pipette solutions. 3. Further experiments utilizing prepulses to preactivate a constant number of channels also revealed a linear relationship between the Ca2+ transient rate of rise and the magnitude of entering Ca2+ current at positive potentials. Under these conditions as well, the maximal rates of rise of global myoplasmic Ca2+ transients were due primarily to Ca2+ release from the sarcoplasmic reticulum as revealed by effects of ryanodine and caffeine on the Ca2+ transients. Using such prepulses, linearity between the Ca2+ transient rate of rise and the magnitude of the peak Ca2+ current was found under a variety of pulse protocols. 4. Using one such pulse protocol, linearity between the Ca2+ transient rate of rise and the magnitude of the peak Ca2+ current was also found when Ca2+ currents assessed at one potential were reduced in magnitude during the onset of block by application of Co2+. Using the same pulse protocol, linearity between the Ca2+ transient rate of rise and the magnitude of the peak Ca2+ current was also found when use of Cs+ was avoided by blocking K+ currents with extracellular TEA and 4-aminopyridine. Linearity in the relationship between the Ca2+ transient rate of rise and the magnitude of the peak Ca2+ current was also found when Ca2+ transients were measured using the low affinity Ca2+ indicator Oregon Green 488 Bapta 5N in place of fluo-3. 5. These results appear to indicate that the cardiac ryanodine receptor is capable of being activated by only one calcium ion. Alternative interpretations of the data are discussed.

Animals↗
PubMed1998

Perforated patch recording with beta-escin.

Perforated patch recording with nystatin, amphotericin B and gramicidin can be more difficult in the hands of some investigators than others. In addition, it is difficult to introduce low molecular weight substances such as dyes into the cytoplasm in such experiments. We have determined that beta-escin represents a convenient, easy-to-use alternative to less water-soluble ionophores.

Amphotericin B↗
PubMed1998

Dihydropyridine receptor isoform expression in adult rat skeletal muscle.

The expression of isoform-specific dihydropyridine receptor Ca2+ channel (DHPR) alpha1-subunit genes in rat diaphragm, soleus and extensor digitorum longus muscles was investigated using RNase protection assays. As expected, mRNA expression levels for the DHPR skeletal muscle isoform were highest in extensor digitorum longus. Unexpectedly, both diaphragm and soleus expressed mRNA for the cardiac isoform at a significant level. Moreover, immunohistochemical experiments provided evidence of the cardiac DHPR isoform at the protein level in muscle fibres. The presence of the cardiac DHPR in the soleus and diaphragm is consistent with a degree of reported cardiac-like excitation-contraction coupling in these muscles, and may be an explanation for some of the therapeutic effects of theophylline in asthmatics, but is likely to serve some other role(s) as well.

Animals↗
PubMed1998

Effects of three sarcoplasmic/endoplasmic reticulum Ca++ pump inhibitors on release channels of intracellular stores.

The three principal sarcoplasmic/endoplasmic reticulum Ca++ pump inhibitors have been compared for their effects on Ca++ fluxes across intracellular stores present in isolated skeletal muscle and brain membrane preparations. At moderate concentrations that only partially inhibited Ca++ pumping, all three inhibitors induced transient release of Ca++ from isolated sarcoplasmic reticulum membranes, and release was ruthenium red-sensitive, much faster and sustained at higher pump inhibitor concentrations. In contrast, in unidirectional 45Ca efflux assays, cyclopiazonic acid appeared to have little effect, thapsigargin decreased efflux and 2,5-di(tert-butyl)-1,4-benzohydroquinone increased efflux only slightly. These observations taken together suggest that transient releases were manifest primarily by vesicles with a high ratio of ryanodine receptors to pumps (and thus more susceptible to becoming leaky with only some pumps inhibited), and that Ca(++)-induced Ca++ release amplified releases when all pumps were blocked. These mostly indirect side effects were specific for ryanodine receptors. In similar experiments with brain cerebellar membranes, none of the three inhibitors appeared to directly reduce release induced by inositol 1,4,5-trisphosphate. These findings may affect interpretation of results of experiments involving application of these compounds to isolated membranes, cells or tissue preparations.

Animals↗
PubMed1997

Dihydropyridine receptor and ryanodine receptor gene expression in long-term denervated rat muscles.

Following disruption of the nerve supply, extensor digitorum longus (EDL) and soleus (SOL) muscles in rats are known to exhibit alterations in excitation-contraction coupling. After total RNA isolation from the denervated and the contralateral control muscles performed at 25 and 50 days following denervation, RNase protection assays were carried out with four cDNA probes specific for the skeletal and cardiac isoforms of both the DHPR alpha 1-subunit and the RyR. Longterm denervation increased the expression of the mRNA for skeletal DHPR and skeletal RyR in SOL muscle, but it also significantly increased the expression of the mRNA for the cardiac isoform of the DHPR alpha 1 subunit in EDL muscle.

Animals↗
PubMed1997

Chronic stimulation differentially modulates expression of mRNA for dihydropyridine receptor isoforms in rat fast twitch skeletal muscle.

This study examined the effects of low frequency chronic stimulation on expression of the mRNA encoding the two isoforms of the alpha1 subunit of the dihydropyridine receptor (DHPR) calcium channel, a critical component of skeletal muscle excitation-contraction coupling. RNase protection assay was used to determine alteration in isoform expression in 5-day, 9-day and 13-day chronically stimulated rat tibialis anterior muscle, and to compare it with soleus and extensor digitorum longus muscles. Low frequency chronic stimulation was associated not only with a significant decrease in the mRNA level of the skeletal isoform of the DHPR, but also with a significant increase in the mRNA level of the cardiac isoform of the DHPR, the overwhelming majority of which was the adult splice variant. Significant levels of cardiac DHPR mRNA expression were also found in normal adult slow twitch soleus muscle. These findings raise the question of a potential role for the cardiac DHPR in certain adult skeletal muscles.

Alternative Splicing↗
PubMed1997

Regulation of dihydropyridine receptor and ryanodine receptor gene expression in regenerating skeletal muscle.

One of the the major properties of mature skeletal muscle is its ability to regenerate after injury. The purpose of the present study was to determine whether the expression of genes encoding the dihydropyridine receptor calcium channel (DHPR) and the ryanodine receptor (RyR), which play a critical role in excitation-contraction coupling, is regulated by skeletal muscle regeneration. The process of regeneration was induced by bupivacaine injection in surgically exposed rat extensor digitorum longus (EDL) muscle. After total RNA isolation from the injected and the contralateral control EDL muscles performed 3, 7, 15 and 30 days following injection, Northern blot and RNase protection assays were carried out with four cDNA probes specific for the skeletal and cardiac muscle isoforms of both the DHPR alpha1-subunit and the RyR. After 3 days, an initial precipitous decrease in the expression of the genes encoding the skeletal muscle isoforms of the DHPR and RyR was observed, followed by an increase. Moreover, regenerating skeletal muscle transiently expressed mRNA for the DHPR cardiac isoform, mainly at the beginning of regeneration. No expression of mRNA for the cardiac RyR was observed. Contraction experiments, performed using EDL muscle at the same times after bupivacaine injection, showed that twitch amplitude was markedly decreased in the absence of external calcium, but only during the early stages of regeneration. Similar findings in relation to expression of skeletal and cardiac muscle DHPR message were previously reported from experiments conducted during early developmental stages using fetal skeletal muscle and muscle cell cultures [Chaudhari N, Beam KG (1993) Dev Biol 155:507-515]. These results suggest that expression of the DHPR cardiac isoform in skeletal muscle could explain certain cardiac-like aspects of excitation-contraction coupling of regenerating skeletal muscle and developing skeletal muscle as well.

Animals↗
PubMed1997

Ca2+ feedback on "quantal" Ca2+ release involving ryanodine receptors.

The influence of luminal and cytoplasmic Ca2+ on the ability of ryanodine-sensitive stores to undergo multiple partial ("quantal") releases has been assessed. Increased luminal Ca2+ levels do indeed modulate sarcoplasmic reticulum Ca2+ release by lowering the threshold agonist concentration required to elicit release, but the decrease in luminal Ca2+ that accompanies a partial release is not sufficient by itself to terminate release. Similarly, an increase in cytoplasmic Ca2+ lowers the threshold agonist concentration required to elicit release; thus, the bulk cytoplasmic Ca2+ levels attained during a release would only stimulate further release, not terminate it before it reached completion. Very high cytoplasmic Ca2+ levels (1-3 mM) also triggered release but were unable to terminate release before reaching completion. Thus, even the high local cytoplasmic Ca2+ concentration that might accompany release would also not terminate release. It is concluded that Ca2+ feedback can modulate release through ryanodine receptors but that it does not account for the properties of quantal release. The low affinity inhibitor tetracaine induces a decrease in the extent of release that cannot be explained solely by heterogeneous caffeine sensitivity of the stores. The results are interpreted in terms of a scheme that includes (i) heterogeneous sensitivity of stores, conferred in part by differences in luminal Ca2+ content and (ii) adaptive behavior on the part of individual ryanodine receptors.

Animals↗
PubMed1996

Sphingolipid actions on sodium and calcium currents of rat ventricular myocytes.

Sphingosine, an endogenous phospholipid known to produce significant decreases in myoplasmic Ca2+ transients, was shown to have a pronounced inhibitory effect on inward Na+ and L-type Ca2+ currents in rat ventricular myocytes. Sphingosine action was accompanied by a slowing of inactivation of both kinds of current. Both sphingosine and sphingosylphosphorylcholine (SPC) caused depolarizing shifts in the activation curves for the two channels. In tests on Ca2+ currents, sphingosine neither showed high affinity for inactivated states nor exhibited any use dependence. The mechanism of the blocking action of sphingosine does not appear to involve effects on bulk surface charge or, at least for Ca2+ channels, a voltage-dependent block. Instead, the results appear most consistent with an effect of sphingosine on channel gating. The shift in the voltage dependence of channel activation by sphingosine and SPC appears likely to be a feature of both the hydrocarbon chains and the net positive charge of these amphiphiles.

Animals↗
PubMed1995

Involvement of ryanodine receptors in sphingosylphosphorylcholine-induced calcium release from brain microsomes.

Sphingosylphosphorylcholine (SPC) releases Ca2+ from brain microsomes. SPC-induced CA2+ release differs from IP3-induced Ca2+ release in that it is more extensive in the cerebrum than in the cerebellum. SPC has little effect on [3H] IP3 binding but enhances [3H] ryanodine binding, as expected for an activator of ryanodine receptors. SPC-induced Ca2+ release is inhibited by ryanodine receptor blockers but not by selective blockers of IP3 receptors. We conclude that SPC releases Ca2+ from brain microsomes by activating ryanodine receptors rather than IP3 receptors. Activation of an additional SPC-sensitive pathway for releasing Ca2+ is not precluded.

Animals↗
PubMed1995

Inhibitory action of SR33557 on L-type calcium current in single ventricular myocytes of rat.

1. The effect of SR33557 on L-type Ca2+ currents in rat ventricular myocytes was investigated by use of the whole-cell patch-clamp technique. 2. SR33557 inhibited Ca2+ current (ICa) in a concentration-dependent manner without change in the current-voltage relationship. 3. The inhibitory effect of SR33557 on ICa was dependent on the holding potential (Vh). The IC50 values were estimated to be 2.2 x 10(-8) M at Vh = -50 mV and 9.0 x 10(-6) M at Vh = -80 mV. SR33557 (10(-7) M) shifted the steady state inactivation curve of ICa toward more negative potentials. Thus, the affinity of the drug for inactivated channels was considerably higher than for resting channels. 4. Blockade of ICa by SR33557 was both tonic and use-dependent. 5. The time constant of onset of block was 36.4 s at -50 mV and 41.9 +/- 11.1 s at -40 mV. 6. The time course of unblock was voltage-dependent. The time constant declined from 400.7 +/- 68.1 is at -50 mV to 5.2 +/- 1.2 s at -80 mV. 7. The rate of block of ICa was related to the number of openings per unit time and to the amount of time spent depolarized. The affinity of drug for open channels was considered to be similar to that for inactivated channels. 8. These results suggest that SR33557 inhibits L-type Ca2+ current through binding to both open and inactivated channels in rat ventricular myocytes.

Animals↗
PubMed1994

Ca(2+)-dependent negative control mechanism for Ca(2+)-induced Ca2+ release in crayfish muscle.

The mechanism of termination of Ca(2+)-induced Ca2+ release (CICR) from the sarcoplasmic reticulum has been investigated in voltage clamped cut crayfish muscle fibres loaded with rhod-2. During depolarizing steps evoking calcium current (ICa), Ca2+ release was first activated. Then the release rapidly (tau approximately 6 ms) declined, as evidenced by the rate of change of the intracellular fluorescence signal representing a Ca2+ transient. The rapid termination of release was not accounted for by inactivation of the trigger ICa or depletion of Ca2+ from the SR, since the rate at which release declined was constant under conditions where the rate of ICa inactivation and the amount of Ca2+ released varied widely. Pre-elevations of [Ca2+]i with prepulses or photolysis of caged Ca2+ caused depression of Ca2+ release during a subsequent test pulse. When the rate of ICa onset was varied by applying voltage ramps with different slopes, currents with fast onset elicited larger Ca2+ release than calcium currents with slower onset, even though the amplitude of the currents was the same. These results suggest that a Ca(2+)-dependent negative control mechanism exists which mediates the termination of CICR independently of the duration of the trigger ICa and before significant depletion of Ca2+ in the SR occurs.

Animals↗
PubMed1994

Modulation of cardiac sarcoplasmic reticulum ryanodine receptor by sphingosine.

Excitation contraction (EC) coupling in muscle cells involves the movement of calcium through the calcium release channel of the sarcoplasmic reticulum (SR) membrane known as the ryanodine receptor. We have recently shown that the novel second messenger, sphingosine, can block calcium release from skinned skeletal muscle fibers and from isolated skeletal muscle SR membranes (Sabbadini et al., J Biol Chem 267: 15475-15484, 1992). In this report, we demonstrate that sphingosine also inhibits calcium release from isolated canine cardiac SR membranes containing the ryanodine receptor when release is induced by caffeine, doxorubicin or by calcium. Sphingosine also prevents the augmentation of [3H]-ryanodine binding normally produced by caffeine and doxorubicin and exerts noncompetitive inhibition with regard to both releasing agents. Sphingosine significantly reduces in a dose-dependent manner [3H]-ryanodine binding to the high affinity site of the receptor and increases by several-fold the Kd for binding, which is consistent with a blocking action of sphingosine on the ryanodine receptor calcium channel. Sphingosine inhibits the extent of calcium-induced calcium release (CICR) and significantly shifts the threshold for CICR so that a higher level of trigger calcium is required to initiate CICR. The sphingosine inhibition of CICR is consistent with the near abolition of calcium dependent [3H]-ryanodine binding. HPLC analysis of cardiac sphingosine content indicates that sphingosine is present in the cardiac cell at moderately high levels (29.4 nmol/g wet wt for the entire cell and approximately 0.4 microM for the cytosol) which are sufficient to produce significant inhibition by sphingosine on calcium release and ryanodine binding. The data suggest that sphingosine acts on the cardiac ryanodine receptor by opposing the physiological stimulus (e.g. trigger calcium entering via the dihydropyridine receptor). We propose that sphingosine is produced by the T-tubule membranes and that sphingosine is released into the protected intracellular environment of the T-tubule/SR junction to negatively modulate calcium release. Consequently, it is possible that sphingosine is a physiologically relevant regulator of calcium levels in the heart.

Animals↗
PubMed1994

Negative control mechanism with features of adaptation controls Ca2+ release in cardiac myocytes.

The central paradox of cardiac excitation-contraction coupling is that Ca(2+)-induced Ca2+ release (CICR), an inherently self-regenerating process, is finely graded by surface membrane Ca2+ current (ICa). By using FPL64176, a novel Ca2+ channel agonist that reduces inactivation of ICa, a rapid negative control mechanism was unmasked at the Ca2+ release level in isolated rat ventricular myocytes. This mechanism terminates CICR independently of the duration of trigger ICa and before the sarcoplasmic reticulum becomes depleted of Ca2+. In its ability to be reactivated by incremental increases in trigger ICa, this mechanism differs from conventional inactivation/desensitization and is similar to the mechanism of increment detection or adaptation described for intracellular Ca2+ release channels. These results indicate that ryanodine receptor adaptation regulates Ca2+ release in cardiac muscle, accounting for or contributing to the graded nature of CICR and, additionally, permitting stores to reload at later times during Ca2+ entry.

Animals↗