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M Morad

Publications and source records attributed to M Morad.

At least 19 recordsLinked to original sources
PubMed1992

Gating of the cardiac Ca2+ release channel: the role of Na+ current and Na(+)-Ca2+ exchange.

In cardiac myocytes, calcium influx through the calcium channel is the primary pathway for triggering calcium release. Recently it has been suggested that the calcium-induced calcium release mechanism can also be activated indirectly by the sodium current, which elevates the sodium concentration under the cell membrane, thereby favoring the entry of "trigger" calcium via the sodium-calcium exchanger. To test this hypothesis, sodium current was suppressed by reducing the external sodium concentration or applying tetrodotoxin. At potentials positive to -30 millivolts, calcium release was unaffected. A small calcium release at more negative potentials could be attributed to partial activation of calcium channels, because it was unaltered by replacement of sodium with lithium and was blocked by cadmium. Thus, sodium influx or its accumulation does not initiate calcium release. In addition, sodium-calcium exchange-related calcium release at potentials positive to +80 millivolts has slower kinetics than calcium channel-induced release. Therefore, only the calcium channel gates the fast release of calcium from the sarcoplasmic reticulum in the range of the action potential.

Animals↗
PubMed1992

Modulation of contraction by intracellular Na+ via Na(+)-Ca2+ exchange in single shark (Squalus acanthias) ventricular myocytes.

1. The effect of direct alteration of intracellular Na+ concentration on contractile properties of whole-cell clamped shark ventricular myocytes was studied using an array of 256 photodiodes to monitor the length of the isolated myocytes. 2. In myocytes dialysed with Na(+)-free solution, the voltage dependence of Ca2+ current (ICa) and contraction were similar and bell shaped. Contractions activated at all voltages were completely suppressed by nifedipine (5 microM), and failed to show significant tonic components, suggesting dependence of the contraction on Ca2+ influx through the L-type Ca2+ channel. 3. In myocytes dialysed with 60 mM Na+, a ICa-dependent and a ICa-independent component of contraction could be identified. The Ca2+ current-dependent component was prominent in voltages between -30 to +10 mV. The ICa-independent contractions were maintained for the duration of depolarization, increased with increasing depolarization between +10 to +100 mV, and were insensitive to nifedipine. 4. In such myocytes, repolarization produced slowly decaying inward tail currents closely related to the time course of relaxation and the degree of shortening prior to repolarization. 5. With 60 mM Na+ in the pipette solution, positive clamp potentials activated decaying outward currents which correlated to the size of contraction. These outward currents appeared to be generated by the Na(+)-Ca(2+)-exchanger since they depended on the presence of intracellular Na+, and were neither suppressed by nifedipine nor by K+ channel blockers. 6. The results suggest that in shark (Squalus acanthias) ventricular myocytes, which lack functionally relevant Ca2+ release pools, both Ca2+ channel and the Na(+)-Ca2+ exchanger deliver sufficient Ca2+ to activate contraction, though the effectiveness of the latter mechanism was highly dependent on the [Na+]i.

Animals↗
PubMed1992

Permeation of divalent cations through the Ca2+ channel of rabbit portal vein myocytes.

The divalent selectivity of the Ca2+ channel in the rabbit portal vein myocyte was examined by the whole cell clamp method. A concentration-dependent selectivity of divalent ion permeation was found such that when Ca2+ was replaced by Ba2+ or Sr2+, the order of maximum current was Ca2+ = Ba2+ greater than Sr2+ at 2 mM and Ba2+ greater than Sr2+ greater than or equal to Ca2+ at 5-10 mM. The possibility of block of the Ca2+ channel by micromolar concentrations of "contaminant" Ca2+ as a determinant of change in the order of selectivity of divalents was examined. Ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (500 microM) significantly increased maximum Ba2+ current (IBa) or ISr in solution containing 5 mM Ba2+ or Sr2+. Furthermore, at 5 mM extracellular Ba2+ concentration, addition of 10, 20, 50, and 100 microM Ca2+ caused a 6, 14, 22, and 33% decrease in IBa, respectively. These results suggest that the portal vein Ca2+ channel has three orders of magnitude higher selectivity for Ca2+ over Ba2+ and Sr2+ such that micromolar Ca2+ may block permeation of other divalents through the channel.

Animals↗
PubMed1992

Characteristics of calcium currents in rabbit portal vein myocytes.

The properties of voltage-dependent Ca2+ channels were studied in isolated portal vein myocytes using the whole cell voltage-clamp method. Ca2+ currents (ICa) were identified based on their activation and inactivation potential, their dependence on external Ca2+ ([Ca2+]o), their suppression by organic or inorganic Ca2+ channel blockers, their augmentation by BAY K 8644, and their insensitivity to tetrodotoxin or alterations in external Na+ ([Na+]o). Changing the holding potential from -90 to -40 mV decreased ICa from 4.6 +/- 0.6 to 2.0 +/- 0.3 pA/pF at 0 mV but did not shift its voltage dependence significantly. The voltage dependence of steady-state inactivation and activation was represented by Boltzmann distributions with the following parameters: inactivation, half-maximal voltage (V0.5) = -32 +/- 7 mV and slope factor (k) = 6.1 +/- 0.2 mV; activation, V0.5 = -15 +/- 4 mV and k = 5.6 +/- 0.6 mV. Doubling the [Ca2+]o increased ICa and shifted the voltage dependence of its activation and inactivation by approximately 10 mV toward more positive potentials without altering the window currents. Substituting Na+, Ba2+, or Sr2+ for Ca2+ as the charge carrier through the Ca2+ channel slowed the rate of its inactivation and shifted its voltage dependence toward more negative potentials. Divalent selectivity of the Ca2+ channel showed an apparent concentration dependence: at 2 mMISr less than IBa = ICa, while at 10 mM ICa less than ISr = IBa. Because 50-100 microM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid abolished the apparent concentration dependence of the divalent ion selectivity, this phenomenon was attributed to a high Ca2+ selectivity of the channel. Our data support the presence of only one type of Ca2+ channel in rabbit portal vein myocytes with characteristics similar to the L-type Ca2+ channel described in other cells, but with somewhat different divalent selectivity, holding potential, and [Na+]o dependence.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1991

Modulation of cardiac sodium channels by cAMP receptors on the myocyte surface.

The phosphorylation of the cardiac sodium channel by adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase A leads to its inactivation. It was shown that extracellular cAMP can also modulate the sodium channel of rat, guinea pig, and frog ventricular myocytes in a rapid (less than 50 milliseconds), reversible, and dose-dependent manner. The decrease in the sodium current was accompanied by a 10- to 15-millivolt shift in the steady-state availability of the sodium channel toward more negative potentials and was inhibited by guanosine-5'-O-(2-thiodiphosphate) or pertussis toxin, suggesting that the extracellular modulation of the sodium channel by cAMP is mediated by a membrane-delimited mechanism that includes a pertussis toxin-sensitive G protein.

Animals↗
PubMed1991

Permeance of Cs+ and Rb+ through the inwardly rectifying K+ channel in guinea pig ventricular myocytes.

Inward currents carried by external Cs, Rb, NH4 and K through the IK1 channel were studied using a whole-cell voltage clamp technique. Cs, NH4, and Rb currents could be recorded negative to -40 mV following depolarizing prepulses (greater than or equal to 0 mV and 200-1000 msec in duration). The current activation displayed an instantaneous component followed by a monoexponential increase (tau a) to a peak amplitude. Subsequent inactivation was fit by a single exponential, tau ia. With hyperpolarization, tau a and tau ia decreased e-fold per 36 and 25 mV, respectively. In Ca-free external solutions (pipette [Mg] approximately 0.3 mM), inactivation was absent, consistent with the hypothesis that inactivation represents time- and voltage-dependent block of Cs, NH4, and Rb currents by external Ca. The inactivation and degree of steady-state block was greatest when Cs was the charge carrier, followed by NH4, and then Rb. K currents, however, did not inactivate in the presence of Ca. Na and Li did not carry any significant current within the resolution of our recordings. Comparison of peak inward current ratios (Ix/IK) as an index of permeability revealed a higher permeance of Cs (0.15), NH4 (0.30), and Rb (0.51) relative to K (1.0) than that obtained by comparing the steady-state current ratios (Cs:NH4:Rb:K approximately 0.01:0.06:0.21:1.0). At any given potential, tau a was smaller the more permeant the cation. In the absence of depolarizing prepulses, the amplitude of tau a was reduced. Divalent-free solutions did not significantly affect activation in the presence of 0.3 mM pipette [Mg]. When pipette [Mg] was buffered to approximately 50 microM, however, removal of external Ca and Mg lead to a four- to fivefold increase in Cs currents and loss of both time-dependent activation and inactivation (reversible upon repletion of external Ca). These results suggest that (i) permeability ratios for IK1 should account for differences in the degree to which monovalent currents are blocked by extracellular Ca and (ii) extracellular or intracellular divalent cations contribute to the slow phase of activation which may represent either (a) the actual rate of Mg or Ca extrusion from the channel into the cell, a process which may be enhanced by repulsive interaction with the incoming permeant monovalent cation or (b) an intrinsic gating process that is strongly modulated by the permeant monovalent ion and divalent cations.

Ammonia↗
PubMed1991

Role of Ca2+ channel in cardiac excitation-contraction coupling in the rat: evidence from Ca2+ transients and contraction.

1. Optical methods were used to measure simultaneously unloaded cell shortening and intracellular Ca2+ transients in whole-cell voltage clamped rat ventricular myocytes. Red light (greater than 670 nm) was used to measure cell shortening with a linear photodiode array. The dyes Fura-2 (Kd = 140 nM) and Mag-Fura-2 (Kd = 44 microM) were used as Ca2+ indicators with fluorescence excitation at 340 and 410 nm and emission at 510 nm. 2. Repeated measurements at 6 s intervals as 0.4 mM-Fura-2 diffused into the cell from the tip of the voltage clamp pipette showed no decrease in the rate of rise and peak value of the intracellular Ca2+ transient and only a small suppression of cell shortening, suggesting that the molecular mechanisms regulating the Ca2+ release were not significantly altered by the buffering capacity of the Fura-2. 3. Experiments in which the sarcoplasmic reticulum (SR) was depleted of Ca2+ either by exposure to caffeine or by repeated brief (20 ms) voltage clamp depolarizations confirm that the SR is the major source of activator Ca2+. 4. Mag-Fura-2 (1 or 5 mM) was used to register the initial rapid development of the [Ca2+]i transient but the later time course of the Ca2+ transients measured with this dye was obscured by motion artifacts resulting from cell shortening. 5. Both Fura-2 and Mag-Fura-2 showed that depolarization to 0 mV from a holding potential of -80 mV resulted in a [Ca2+]i transient which developed with a delay of 3-9 ms and approached its peak value in an additional 8-19 ms. Both Ca2+ indicators also showed that the Ca2+ transient approached its peak value more slowly as the clamped membrane potential was made increasingly more positive. 6. The voltage dependencies of the Ca2+ signal (Fura-2) and cell shortening were both bell-shaped and were qualitatively similar to the voltage dependence of Ca2+ current simultaneously measured. This was observed with holding potentials of both -40 and -80 mV. 7. Comparison of the temporal relation of the Ca2+ current, ICa, and intracellular Ca2+ transient (Fura-2) and cell shortening at different membrane potentials showed that Ca2+ transient measured 25 ms into the depolarization correlated closely to the integral of the Ca2+ current measured prior to this time. Cell shortening, on the other hand, peaked about 100 ms later and correlated with measurements of the Ca2+ activity at the later time.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

The transient K+ current in rat ventricular myocytes: evaluation of its Ca2+ and Na+ dependence.

1. The transient outward K+ current (Ito) was studied in enzymatically isolated rat ventricular myocytes using the whole-cell patch clamp technique. 2. At holding potentials between -100 and -60 mV, depolarizing pulses activated outward current which was composed of transient and maintained components. These components differed from each other in their activation voltage range as well as in their kinetics of inactivation. 3. The transient component, in turn, appeared to be composed of rapidly and slowly inactivating components. Subtraction of ICa from the total current, or nifedipine pre-treatment, eliminated the slowly inactivating component of Ito indicating that the time course of inactivation of Ito may be contaminated by ICa. 4. Reduction of the holding potential from -100 mV to less negative holding potentials reduced all components of Ito, such that at holding potentials of -40 mV, very little or no Ito could be measured. 5. Elevation of [Ca2+]o activated Ito at holding potentials of -40 mV, and substitution of external Ca2+ by Sr2+ suppressed Ito, consistent with findings from other preparations and in support of a Ca(2+)-activated component of Ito. 6. Elevations of [Ca2+]o, however, also shifted the steady-state activation and inactivation parameters of the transient K+ current, such that a greater proportion of Ito channels were activated at the less negative holding potentials. 7. The shifts in the activation and inactivation parameters of the transient outward current were not mimicked by equivalent changes in external Mg2+. 8. Modulators of Ca2+ release from the sarcoplasmic reticulum (SR) such as caffeine and ryanodine suppressed Ito regardless of whether the myocytes were dialysed with low or high concentrations of Ca2+ buffers (EGTA or BAPTA, 0.5-14 mM) or whether nifedipine was used to block ICa. 9. 4-Aminopyridine (4-AP) blocked Ito in a dose-dependent manner, completely suppressing it at 10 mM. Similarly, tedisamil, a new K+ channel blocker, completely and reversibly blocked Ito at 5-20 microM concentrations. 10. TTX (10 microM) or removal of external Na+ decreased Ito, consistent with the idea that a component of Ito was Na+ activated. Both interventions, however, also shifted the voltage dependence of the activation and inactivation of Ito to more negative potentials, such that at -100 mV neither intervention had a significant effect on Ito. Alterations in [Na+]i had no effect on Ito.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Regulation of the secretory response in bovine chromaffin cells.

The nicotine-induced current and the Ca2+ current were studied in cultured bovine chromaffin cells using the whole cell patch-clamp technique. The dose-response curve for the nicotinic current gave a dissociation constant of 53 microM and a Hill coefficient of 1.3. Desensitization of the nicotinic current was rapid, with time constants of 22 and 155 ms at 10 microM nicotine. At higher concentrations of nicotine, both time constants decreased somewhat, but the most prominent effect was on the ratio of the two components. Recovery from desensitization was fitted by a single exponential with a time constant of approximately 6 s. Ca2+ current and catecholamine secretion were highly sensitive to changes in extracellular H+ concentration ([H+]o), such that small increases in [H+]o markedly decreased both. The Ca2+ current measured in a chromaffin cell located within a cluster of cells, but not in a single isolated cell, was markedly suppressed when KCl or nicotine was used to induce secretion, suggesting possible local feedback of secretory agents. Among agents secreted by chromaffin cells, ATP, enkephalins, epinephrine, and protons, only protons significantly suppressed the Ca2+ current. Our findings suggest that the secretory response of chromaffin cells may be modulated by rapid desensitization of the nicotinic receptor and a secretion-dependent suppression of the Ca2+ current.

Adrenal Medulla↗
PubMed1991

Divalent cations modulate the transient outward current in rat ventricular myocytes.

The modulation of the transient outward K+ current (Ito) by divalent cations was studied in enzymatically isolated rat ventricular myocytes with the whole cell patch-clamp technique. At holding potentials negative to -70 mV, 1 mM Cd2+ suppressed Ito, whereas, at potentials positive to -50 mV, the current was augmented. These effects were caused by shifts in the voltage dependence of both activation and inactivation of Ito toward more positive potentials. Cd2+ also slowed the activation kinetics of Ito by shifting the voltage dependence of its rate of activation, but the rate of inactivation was unaffected. Other divalent cations produced similar shifts but at markedly different concentrations. Thus, in the millimolar range, a rightward shift of approximately 20 mV was produced by 3 Co2+, 5 Ni2+, and 10 Ca2+, whereas 10 microM concentrations of Cu2+ and Zn2+ produced equivalent shifts. Similar effects were seen in hippocampal neurons with micromolar concentrations of Zn2+. Thus divalent cations have marked and specific effects on the kinetics and voltage dependence of Ito and may serve as a regulatory mechanism in its activation, particularly in cells with resting potentials positive to -60 mV.

Animals↗
PubMed1991

Phospholamban mediates the beta-adrenergic-enhanced Ca2+ uptake in mammalian ventricular myocytes.

To probe the molecular mechanism responsible for the relaxant effect of catecholamines in heart muscle, we studied the effect of a monoclonal antibody (2D12) against phospholamban in intact whole cell clamped guinea pig ventricular myocytes, in which intracellular Ca2+ transient and Ca2+ current were simultaneously measured. The antibody stimulated Ca2+ uptake in guinea pig ventricular sarcoplasmic reticular vesicles, shifting the apparent dissociation constant for activation by Ca2+ from 200 to 60 nM. The stimulatory effect of the antibody could be mimicked by the catalytic subunit of adenosine 3',5'-cyclic monophosphate-dependent kinase and could be blocked by phospholamban peptide 2-25. Dialysis of ventricular myocytes with the antibody enhanced the rate of uptake of Ca2+ and significantly suppressed the ability of isoproterenol to enhance the rate of uptake and release of Ca2+ by depolarizing pulses. These data suggest that not only is phosphorylation of phospholamban crucial in sequestration of Ca2+ by the sarcoplasmic reticulum, but that this process may account for the catecholamine-enhanced rate of Ca2+ uptake release in heart muscle.

Adrenergic beta-Agonists↗
PubMed1990

Atrionatriuretic peptide transforms cardiac sodium channels into calcium-conducting channels.

The atrionatriuretic peptide (ANP) is released from atrial cells in response to increased extracellular fluid volume and reduces sodium absorption by the kidney, thus reducing the blood volume. In this report, ANP suppressed the calcium and sodium currents in rat and guinea pig ventricular myocytes. The suppression of sodium current was caused by enhanced permeability of the sodium channel to calcium without significant changes in the kinetics or the tetrodotoxin sensitivity of the channel. Thus, ANP may regulate the sodium channel by altering its cationic selectivity site to calcium, thereby repressing the sodium current. The suppression of sodium and calcium channels and the resultant depressed excitability of the atrial cells may help to regulate ANP secretion.

Animals↗
PubMed1990

Modulation of the N-methyl-D-aspartate channel by extracellular H+.

The influence of external [H+] on whole-cell and single-channel currents activated by glutamate agonists was studied in rat hippocampal neurons. In the pH range between 6.6 and 8.0, changes in external [H+] had negligible influence on the amplitude and kinetics of the monovalent ion-carrying currents activated by the agonists quisqualate and kainate. The divalent ion-carrying N-methyl-D-aspartate (NMDA)-activated current, on the other hand, was strongly modulated by extracellular [H+]. Increased external [H+] suppressed, whereas decreased external [H+] enhanced, the NMDA-activated current. Changes in internal [H+] had little or no effect on the NMDA-activated current. Modulation of the NMDA-activated current resulted primarily from changes in the number of channel openings. Neither the unitary conductance nor the individual open dwell-times were significantly affected. These results suggest that the protonation site is on the external aspect of the channel and is far removed from the channel permeation pathway. Because interactions between H+, NMDA, and glycine in activating the current were predominantly noncompetitive, our results suggest that the modulatory effect of H+ was not associated with changes in receptor-agonist affinities. These results suggest that modulation of the NMDA-receptor channel by [H+] may be an intrinsic protective mechanism by which calcium influx into neurons is regulated, particularly in hypoxic/ischemic conditions.

Animals↗
PubMed1990

Ca2(+)-induced Ca2+ release as examined by photolysis of caged Ca2+ in single ventricular myocytes.

In cardiac muscle, entry of Ca2+ through the voltage-gated Ca2+ channel and its interaction with an intracellular site are thought to trigger the release of the intracellular Ca2+ pools and to activate contraction. The availability of a novel "caged calcium" compound, and its effective use in neuronal and heart cells to modulate Ca2+ channel and contraction, made it possible to examine directly the Ca2(+)-induced Ca2+ release hypothesis in intact mammalian cardiac myocytes. We used the caged Ca2+ compound DM-nitrophen, which on photolysis, rapidly (less than 200 microseconds) changes its Ca2(+)-binding affinity from 3 X 10(-9) to 2 X 10(-3) M at pH 7.0. In isolated whole cell clamped guinea pig ventricular myocytes dialyzed with unphotolyzed DM-nitrophen (Ca2+ buffered to values less than 10(-7) M), we found that a 160-microseconds light pulse photoreleased sufficient Ca2+ to activate contraction. Photorelease of Ca2+ failed to activate significant contraction in myocytes pretreated with caffeine, supporting the idea that the release of Ca2+ from intracellular pools was necessary to generate tension. However, photorelease of Ca2+ after the depolarization-induced Ca2+ release failed to suppress contraction, as predicted from the Ca2(+)-induced inactivation hypothesis. The failure to suppress contraction was not sufficient to definitively reject the Ca2(+)-induced inactivation hypothesis, since the intracellular Ca2+ concentration may not have risen sufficiently to inactivate the release channel.

Acetates↗
PubMed1990

Tedisamil blocks the transient and delayed rectifier K+ currents in mammalian cardiac and glial cells.

The potassium currents in rat and guinea pig ventricular myocytes and mouse astrocytes were studied using tedisamil, a novel antiarrhythmic agent. A 1 to 20 microM dosage of tedisamil caused marked prolongation of the action potential in isolated rat ventricular myocytes, mimicking its reported effects on multicellular rat heart preparations. Under voltage clamp conditions, tedisamil caused a dose-dependent increase in the speed of inactivation of the transient outward K+ current (Ito), the predominant outward current in rat ventricular myocytes. In cardiac myocytes, the tedisamil block was neither use- nor voltage-dependent. The slow reversibility of drug action when applied from the outside, and its effectiveness when applied intracellularly, suggested an internal site of drug action. In guinea pig ventricular myocytes, tedisamil blocked the slowly developing time-dependent delayed rectifier K+ current (IK) over the same concentration range as that found for Ito in the rat myocytes. Tedisamil reduced this current without changing the characteristics of its slow (tau approximately 1 sec) activation. The effects of tedisamil on Ito and IK were independent of the phosphorylation state of the channel, as assessed by the equal effectiveness of the drug in the presence or absence of isoproterenol. Tedisamil also blocked the transient K+ current and the delayed rectifier current (IK) in mouse astrocytes over the same concentration range as that found in the cardiac myocytes and by a process that accelerated (transient K+ current) or mimicked (IK) inactivation. At concentrations of up to 50 microM, tedisamil had little effect on the time-dependent inward rectifier K+ current, or inward calcium current in rat or guinea pig ventricular myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗
PubMed1989

Regulation of calcium release is gated by calcium current, not gating charge, in cardiac myocytes.

In skeletal muscle, intramembrane charge movement initiates the processes that lead to the release of calcium from the sarcoplasmic reticulum. In cardiac muscle, in contrast, the similarity of the voltage dependence of developed tension and intracellular calcium transients to that of calcium current suggests that the calcium current may gate the release of calcium. Nevertheless, a mechanism similar to that of skeletal muscle continues to be postulated for cardiac muscle. By using rapid exchange (20 to 50 milliseconds) of the extracellular solutions in rat ventricular myocytes in which the intracellular calcium transients or cell shortening were measured, it has now been shown that the influx of calcium through the calcium channel is a mandatory link in the processes that couple membrane depolarization to the release of calcium. Thus, intramembrane charge movement does not contribute to the release of calcium in heart muscle.

Animals↗
PubMed1989

Quisqualate activates a rapidly inactivating high conductance ionic channel in hippocampal neurons.

Glutamate activates a number of different receptor-channel complexes, each of which may contribute to generation of excitatory postsynaptic potentials in the mammalian central nervous system. The rapid application of the selective glutamate agonist, quisqualate, activates a large rapidly inactivating current (3 to 8 milliseconds), which is mediated by a neuronal ionic channel with high unitary conductance (35 picosiemens). The current through this channel shows pharmacologic characteristics similar to those observed for the fast excitatory postsynaptic current (EPSC); it reverses near 0 millivolts and shows no significant voltage dependence. The amplitude of the current through this channel is many times larger than that through the other non-NMDA (N-methyl-D-aspartate) channels. These results suggest that this high-conductance quisqualate-activated channel may mediate the fast EPSC in the mammalian central nervous system.

Animals↗