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C H Orchard

Publications and source records attributed to C H Orchard.

At least 37 records · Page 2Linked to original sources
PubMed1997

Sarcoplasmic reticulum Ca2+ content, L-type Ca2+ current and the Ca2+ transient in rat myocytes during beta-adrenergic stimulation.

1. The effect of beta-adrenergic stimulation on the relationship between the intracellular Ca2+ transient and the amplitude of the L-type Ca2+ current (ICa) has been investigated in ventricular myocytes isolated from rat hearts. Intracellular [Ca2+] was monitored using fura-2 during field stimulation and while membrane potential was controlled using voltage clamp techniques. 2. The increase in the amplitude, and the rate of decline, of the Ca2+ transient produced by isoprenaline (1.0 mumol l-1) was not significantly different in myocytes generating action potentials and in those voltage clamped with pulses of constant duration and amplitude. 3. Under control conditions, the current-voltage (I-V) relationship for ICa was bell shaped. The amplitude of the Ca2+ transient also showed a bell-shaped voltage dependence. In the presence of isoprenaline, the amplitude of both ICa and the Ca2+ transient was greater at all test potentials and the I-V relationship maintained its bell-shaped voltage dependence. However, the size of the Ca2+ transient was no longer graded with changes in the amplitude of ICa: a small ICa could now elicit a maximal Ca2+ transient. 4. Rapid application of caffeine (10 mmol l-1) was used to elicit Ca2+ release from the sarcoplasmic reticulum (SR). Isoprenaline increased the integral of the subsequent rise in cytoplasmic [Ca2+] to 175 +/- 13% of control. 5. Abbreviation of conditioning pulse duration in the presence of isoprenaline was used to reduce the amplitude of the Ca2+ transient to control levels. Under these conditions, the amplitude of the Ca2+ transient was again graded with the amplitude of ICa in the same way as under control conditions. 6. Nifedipine (2 mumol l-1) was also used to decrease Ca2+ transient amplitude in the presence of isoprenaline. In the presence of isoprenaline and nifedipine, the amplitude of the Ca2+ transient again showed a bell-shaped voltage dependence. 7. The SR Ca(2+)-ATPase inhibitor thapsigargin (2.5 mumol l-1) reduced the effect of isoprenaline on the amplitude of the Ca2+ transient. In the presence of thapsigargin, the size of the Ca2+ transient increased as ICa increased in response to isoprenaline. 8. These data suggest that the increase in the amplitude of the Ca2+ transient produced by beta-adrenergic stimulation in cardiac muscle is due to an increase in the gain of the SR Ca2+ release process, due principally to an increase in the Ca2+ content of the SR.

Adrenergic beta-Agonists↗
PubMed1997

The effect of the venom of a Chilean tarantula, Phrixotrichus spatulatus, on isolated guinea pig ventricular myocytes.

We have investigated the effect of the venom of a Chilean tarantula, Phrixotrichus spatulatus, on cell contraction, intracellular [Ca2+] ([Ca2+]i), and the L-type Ca2+ current (ICa,L) in cells isolated from the ventricles of guinea pig hearts. Whole-cell voltage clamp techniques were used to monitor ICa,L. The action potential was recorded using whole cell current clamp. [Ca2+]i was monitored using the fluorescent indicator indo-1. The venom of P. spatulatus decreased ICa,L in a dilution-dependent manner, with half-maximal inhibition at a dilution of 1.1/10(4) (v/v). At a dilution of 1/10(4), this inhibition occurred at all potentials so that the current-voltage relationship of ICa,L was depressed. However, inhibition of ICa,L by the venom was relieved by positive potentials, the venom being less effective following pulses to positive potentials. The venom reduced the duration of the action potential (at 50% repolarization) by between 26 and 43%. The venom also decreased the amplitude of the [Ca2+]i transient and cell contraction. It is concluded that the venom of P. spatulatus is a potent, voltage-dependent inhibitor of ICa,L; this inhibition of ICa,L may account for the effects of the venom on action potential duration, [Ca2+]i, and contraction.

Action Potentials↗
PubMed1997

Acidosis alters the phosphorylation of Ser16 and Thr17 of phospholamban in rat cardiac muscle.

The effect of acidosis on the phosphorylation of Ser16 and Thr17 of phospholamban in rat cardiac muscle has been investigated using phosphorylation-site-specific antibodies to this protein. Ventricular myocytes were stimulated at 0.5 Hz for 5 min, in either control (pH 7.4) or acid (pH 6.5) physiological salt solution, in the absence or presence of isoprenaline. Site-specific phosphorylation of phospholamban was determined by Western blotting. Acidosis reduced phosphorylation of Ser16 in the absence of isoprenaline, but did not alter the isoprenaline-induced phosphorylation of Ser16. In contrast, acidosis increased Thr17 phosphorylation in the absence and presence of isoprenaline. Buffering intracellular Ca2+ ([Ca2+]i) with BAPTA inhibited the increase in Thr17 phosphorylation during acidosis but had no effect on Ser16 phosphorylation. We conclude that acidosis can alter the phosphorylation of Ser16 and Thr17 by inhibition of protein kinase A, and by an acidosis-induced increase in [Ca2+]i and the subsequent activation of a Ca2+/calmodulin-dependent protein kinase, respectively. The possible effect of these changes in phosphorylation on the activity of the Ca2+-ATPase of the cardiac sarcoplasmic reticulum during acidosis is discussed.

Acidosis↗
PubMed1997

Rate-dependent abbreviation of Ca2+ transient in rat heart is independent of phospholamban phosphorylation.

The mechanisms underlying the accelerated decline of the intracellular Ca2+ transient that occurs in cardiac muscle when stimulation rate is increased have been investigated in ventricular myocytes from rat hearts. Increasing stimulation rate from 0.1 to 0.5 and 1 Hz decreased the time taken for the Ca2+ transient to decline from its peak to 50% of its peak value in cells generating action potentials, when the duration of depolarization was held constant by voltage clamp, and when Na/Ca exchange was inhibited. The sarcoplasmic reticulum Ca2+ adenosinetriphosphatase inhibitor thapsigargin inhibited rate-dependent abbreviation of the Ca2+ transient. However, neither a chemical inhibitor of Ca(2+)-calmodulin-dependent protein kinase II (KN62) nor a peptide inhibitor of this enzyme (calmodulin-binding domain peptide) had a significant effect on rate-dependent abbreviation of the Ca2+ transient. Analysis of the phosphorylation of the regulatory sites Ser16 and Thr17 of phospholamban showed no significant change in phosphorylation with changes of stimulation rate. These data suggest that rate-dependent shortening of the Ca2+ transient is due predominantly to enhanced Ca2+ uptake by the sarcoplasmic reticulum without changes in phospholamban phosphorylation.

Action Potentials↗
PubMed1996

Changes in [Ca2+]i, [Na+]i and Ca2+ current in isolated rat ventricular myocytes following an increase in cell length.

Isolated rat ventricular myocytes were stretched using carbon fibres to investigate the mechanisms underlying the increase in contraction following stretch. 2. [Ca2+]i and [Na+]i were monitored using the fluorescent indicators fura-2 and sodium-binding benzofuran isophthalate, respectively. The L-type Ca2+ current was recorded simultaneously with contraction using the perforated patch-clamp technique. 3. Mechanical stretch caused an immediate increase in contraction, followed by a slow increase. Contraction was prolonged immediately after the stretch, but did not change during the slow phase. 4. The Ca2+ transient did not change immediately after the stretch. The slow increase in contraction was accompanied by an increase in the amplitude of the Ca2+ transient. However, diastolic [Ca2+]i did not change significantly following stretch. 5. [Na+]i did not change significantly either immediately, or during the slow increase in contraction, after the stretch. 6. The L-type Ca2+ current was not significantly altered either by mechanical loading of the cell with carbon fibres or by stretching the cell. 7. These results suggest that: (1) the rapid increase in contraction following a stretch is due to an increase in myofilament Ca2+ sensitivity rather than to changes in the L-type Ca2+ current or [Na+]i; and (2) a slow increase in the Ca2+ transient underlies the slow increase in contraction in isolated myocytes, but is not caused by either an increase in diastolic [Ca2+]i or a change in [Na+]i (and hence Ca2+ influx via Na(+)-Ca2+ exchange) or a change in myofilament Ca2+ sensitivity.

Animals↗
PubMed1996

Effects of acidosis on phosphorylation of phospholamban and troponin I in rat cardiac muscle.

Acidosis inhibits Ca2+ transport by the sarcoplasmic reticulum of cardiac muscle and decreases the Ca2+ sensitivity of the contractile proteins, although the mechanisms underlying these changes are unclear. We have investigated the hypothesis that changes in the phosphorylation of the regulatory proteins phospholamban and troponin I might play a role in the acidosis-induced changes in the function of the sarcoplasmic reticulum and the myofilaments, respectively. Langendorff-perfused rat hearts were labeled with 32P and then perfused with either control (pH 7.4) or acid (pH 6.8) physiological salt solution, in both the absence and presence of isoproterenol. The incorporation of 32P into phospholamban and troponin I was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of sarcoplasmic reticulum and myofibrillar proteins, followed by autoradiography and liquid scintillation counting. The data show that acidosis has no effect on the phosphorylation of phospholamban in the absence of isoproterenol but that, in the presence of isoproterenol, acidosis increased the phosphorylation of phospholamban. However, acidosis increased the phosphorylation of troponin I, in both the absence and the presence of isoproterenol. Acidosis did not alter the adenosine 3',5'-cyclic monophosphate content of the hearts but did inhibit type 1 phosphatase. These data show that acidosis can alter the phosphorylation of these two proteins and suggest that these changes underlie, in part the changes observed in cardiac muscle during acidosis.

Acidosis↗
PubMed1995

The effects of mechanical loading and changes of length on single guinea-pig ventricular myocytes.

1. The effects of mechanical loading and changes of length on the contraction of single guinea-pig ventricular myocytes has been investigated. 2. Cell shortening was monitored during isotonic contractions (in which the cell shortened freely) and after attaching carbon fibres of known compliance to the ends of the cell, so that the cell contracted auxotonically (the cell both shortened and developed force). 3. Mechanically loading the cells decreased the amount of shortening during a contraction and abbreviated the contraction. There were, however, no consistent changes in the action potential or the [Ca2+]i transient (measured with the fluorescent dye fura-2). 4. Increasing stimulation rate increased the size of the contraction and the [Ca2+]i transient in both isotonic and auxotonic conditions. The increase in the size of the contraction induced by an increase in stimulation rate was greater in auxotonic conditions but the increase in the size of the [Ca2+]i transient was not. 5. When cells were stretched, there was a step increase in the size of the contraction and a prolongation of its time course. However, neither the size nor the time course of the accompanying [Ca2+]i transient was significantly altered by this intervention. 6. When a stretch was maintained, a further, slow increase in the size of the contraction occurred during the following 3-11 min, in about half the cells studied. The probability of this slow response occurring was increased if the initial degree of activation of the cell was decreased. 7. These data suggest that the mechanisms underlying the responses to mechanical loading and changes of length are the same in both multicellular and single cell preparations of cardiac muscle.

Animals↗
PubMed1995

The mechanism underlying the cardiotoxic effect of the toxin from the jellyfish Chironex fleckeri.

We have investigated the mechanisms underlying the cardiac effects of the toxin from the box jellyfish Chironex fleckeri. Papillary muscles isolated from the hearts of ferrets and ventricular myocytes isolated from the hearts of ferrets and rats were used. Force, intracellular [Ca2+], and membrane potential were monitored in the papillary muscles; contraction, intracellular [Ca2+], intracellular [Na+], and membrane currents were monitored in the isolated myocytes. Application of the toxin to these preparations resulted in a large increase in intracellular [Ca2+] and the adverse symptoms of Ca2+ overload (aftercontractions, spontaneous contractions, a decrease in developed force, and an increase in resting force). The response of papillary muscles to the toxin was not inhibited by blockers of Ca2+ or Na+ channels or by inhibitors of the sarcoplasmic reticulum, Na+/K+ ATPase, or Na+/H+ exchange. The response to the toxin was, however, blocked by prior exposure to a solution which contained no Na+ and by Ni2+. In the isolated myocytes, as well as an increase in intracellular [Ca2+], the toxin also caused an increase in intracellular [Na+] and the appearance of a current which was inward at negative potentials and reversed at about -10 mV. These data can be explained by the toxin increasing Na+ influx into the cell. The increase in intracellular [Na+] will then increase intracellular [Ca2+] via the Na+/Ca2+ exchange mechanism, thus producing the observed Ca2+ overload.

Animals↗
PubMed1995

The effect of mechanical loading on the response of rat ventricular myocytes to acidosis.

The effect of mechanical loading on the negative inotropic effect of acidosis in isolated rat ventricular myocytes was investigated. The mechanical loading of the myocytes was changed by attaching carbon fibres to the ends of the cell. To monitor intracellular Ca2+ and Na+ concentrations, cells were loaded with fluorescent dyes (fura-2 for Ca2+ and SBFI for Na+) using the acetoxymethyl (AM) esters. Mechanical loading reduced cell shortening by 73.0 +/- 3.5% (mean +/- S.E.M., n = 16) and abbreviated the time course of contraction. CO2-induced acidosis caused a rapid decrease in contraction followed by a slow partial recovery. The percentage changes in contraction were not significantly different in mechanically loaded and unloaded conditions. Mechanical loading had little effect on the time course of contraction during acidosis. Changes in intracellular Ca2+ and Na+ concentrations during acidosis were unaffected by mechanical loading. The mechanical loading conditions of a region of the heart can be modified by ischaemia and the subsequent acidosis. Our results suggest that the response of cardiac muscle to acidosis is not markedly modified by such changes in mechanical loading.

Acidosis↗
PubMed1995

Effect of stretch on contraction and the Ca2+ transient in ferret ventricular muscles during hypoxia and acidosis.

The effect of stretch on cardiac muscle contraction and the Ca2+ transient was studied during hypoxia and acidosis in isolated ferret ventricular muscles. In control conditions, a maintained stretch produced an immediate increase in tension followed by a slow increase in tension and the Ca2+ transient. A stretch between contractions (diastolic stretch) caused only a slow increase in tension and the Ca2+ transient, whereas a stretch during the period of contraction (systolic stretch) produced an immediate increase in tension followed by a small slow increase in tension and the Ca2+ transient. In hypoxia, the immediate percent increase in tension was the same as in control. However, the slow increase was smaller during all three types of stretch. In acidosis, the immediate percent increase in tension was larger than in control. The slow change was the same during maintained stretch. However, the slow increase in tension was smaller during diastolic stretch and larger during systolic stretch. Thus the stretch-dependent increase in contraction is inhibited during hypoxia and modulated by acidosis.

Acidosis↗
PubMed1994

Effects of acidosis and hypoxia on the response of isolated ferret cardiac muscle to inotropic agents.

OBJECTIVE: The aim was to study the effects of acidosis and hypoxia on the response of cardiac muscle to inotropic agents which (a) act predominantly by increasing intracellular [Ca2+] (raising extracellular [Ca2+], noradrenaline, isoprenaline) and (b) act partly (phenylephrine) or predominantly (EMD 57033) by increasing myofilament calcium sensitivity. METHODS: The experiments were performed on isometrically contracting, isolated ferret papillary muscles (n = 45). For each intervention dose-response curves were performed in control solution (pH 7.35), in hypercapnic acidosis (pH 6.85), and in hypoxia (produced by replacing O2 with N2 in the superfusing solution). In some experiments, the photoprotein aequorin was microinjected into superficial cells of the preparation in order to measure intracellular [Ca2+] as well as force. RESULTS: The results were broadly similar for both classes of inotropic agent. Acidosis caused a shift of the pCa-tension curve to the right (desensitisation of the myofilaments to calcium), but had no significant effect on maximum force. A sufficient inotropic stimulus supplied by either class of inotropic agent could completely reverse the negative inotropic effects of acidosis. The main difference between the two inotropic mechanisms was that the enhanced force produced by calcium sensitisers was associated with a reduction in calcium transient amplitude, while the other inotropes increased the amplitude. The main effect of hypoxia was to decrease maximum force. All the inotropes tested were relatively ineffective in reversing the force depression due to hypoxia. CONCLUSIONS: The negative inotropic effects of acidosis can be reversed by a sufficiently large inotropic stimulus. Since calcium transient amplitude is already increased in acidosis, the results suggest that calcium sensitisers are likely to be less arrhythmogenic in this situation. The relative ineffectiveness of the inotropes in hypoxia indicates that the main mechanisms causing reduced force in this situation lie downstream of the mechanisms of action of the inotropic agents tested.

Acidosis↗
PubMed1994

Acidosis and arrhythmias in cardiac muscle.

Acidosis is a well recognised consequence of myocardial ischaemia. In this brief article we have reviewed the consequences of acidosis that might be arrhythmogenic. These include early afterdepolarisations and triggered activity, delayed afterdepolarisations, pulsus alternans, and reentry. In each case we have described the evidence that acidosis can provoke such behaviour and then discussed the possible mechanisms and consequences of each behaviour. It seems likely that changes of pH may contribute to arrhythmogenesis during ischaemia by these mechanisms.

Acidosis↗
PubMed1994

The role of the sarcoplasmic reticulum in the response of isolated ferret cardiac muscle to beta-adrenergic stimulation.

beta-Adrenergic stimulation of cardiac muscle leads to an increase in the strength of contraction and an abbreviation of its time course. We have investigated the role of the sarcoplasmic reticulum in these changes by monitoring force and cytoplasmic [Ca2+] in ferret papillary muscles, and the Ca2+ current in isolated ferret myocytes, during the application of isoprenaline in the absence and presence of the sarcoplasmic reticulum inhibitor ryanodine (10(-6) mol/l). Isoprenaline (10(-6) mol/l) led to a marked increase in the size of both the twitch and Ca2+ transient, and a decrease in their duration. In the presence of ryanodine, application of isoprenaline had no significant effect on either the size or the time course of the twitch. However, the increase in the Ca2+ current in response to isoprenaline was the same in the absence and presence of ryanodine. Increasing bathing [Ca2+] led to a prolongation of both the twitch and the Ca2+ transient. In the presence of ryanodine, increasing bathing [Ca2+] still increased the size, but decreased the duration, of the twitch. These data provide direct evidence that both the inotropic and lusitropic effects of isoprenaline are mediated via the sarcoplasmic reticulum.

Animals↗
PubMed1993

A novel thiadiazinone derivative fully reverses acidosis-induced depression of force in cardiac muscle by a calcium-sensitizing effect.

1. We examined the effects of the novel thiadiazinone derivative EMD 57033 on developed force and intracellular [Ca2+] in cardiac muscle during control conditions (pH 7.35) and in acidosis (pH 6.8). 2. In the control solution, application of EMD 57033 fully activated the muscle. Acidosis reduced developed force to 18% of maximum, but application of EMD 57033 in acid solution was able to fully reverse this effect and restore force to its previous maximum. 3. During the positive inotropic effect in acidosis, the Ca2+ transients declined to 62% of their initial amplitude, whereas force increased to 557%. 4. These observations suggest that EMD 57033 increases force by a Ca(2+)-sensitizing action in intact cardiac muscle. Since EMD 57033 is able to fully reverse the effects of acidosis on force without increasing the amplitude of the Ca2+ transients, this compound and others with similar mechanisms of action appear to hold particular promise for heart failure therapy.

Acidosis↗
PubMed1993

The effect of acidosis on beta-adrenergic receptors in ferret cardiac muscle.

Acidosis decreases the force of contraction of cardiac muscle in response to noradrenaline. The role of beta-adrenergic receptors in this response to acidosis was investigated. Radioligand techniques were used to determine beta-adrenergic receptor number and the degree of G-protein coupling, and to see whether these were altered in tissues subject to acidosis. The effect of pH on agonist and antagonist binding to these receptors was also investigated. Tissue pre-exposure to acidic conditions had no effect on numbers of beta-adrenergic receptors and no effect on the affinity of [125I]iodocyanopindolol (ICYP) for the receptors. Agonist competition experiments indicated that there was no change in the affinity of isoprenaline for these receptors, and there was no change in the relative proportions of high and low affinity binding sites. When radioligand experiments were performed under acidic conditions, however, the total number of beta-adrenergic receptors increased, and the affinity of these receptors for isoprenaline increased. This increase in agonist affinity might, therefore, minimize the shift to the right seen in the dose-response curve to noradrenaline during acidosis.

Acidosis↗
PubMed1993

The role of Na(+)-Ca2+ exchange in paired pulse potentiation of ferret ventricular muscle.

1. Stimulation of cardiac muscle with pairs of stimuli ('paired pulse stimulation') results in a large inotropic effect and experiments have been carried out on ferret ventricular muscle to investigate the underlying mechanism. 2. Aequorin was used to measure sarcoplasmic Ca2+ in papillary muscles. During paired pulse stimulation the first aequorin light transient (i.e. Ca2+ transient) and contraction of the pair increased in amplitude, whereas the second aequorin light transient and contraction were small. When the interval between the pair was decreased, the second aequorin light transient and contraction of the pair were smaller, but the increase in the first aequorin light transient and contraction was greater. 3. The relationship between contraction and the aequorin light transient was the same during paired pulse stimulation and on raising the bathing Ca2+ concentration. It is concluded that there was no change in the myofilament sensitivity to Ca2+ during paired pulse stimulation. 4. The increase in the aequorin light transient and contraction during paired pulse stimulation was prevented by ryanodine, an inhibitor of the sarcoplasmic reticulum (SR). 5. During paired pulse stimulation of ventricular myocytes there was little change in the first action potential of the pair, but the second action potential was shorter than control when the interval between the pair was short. During paired pulse stimulation of ventricular myocytes under voltage clamp control there was little change in the first Ca2+ current (iCa) of the pair, but the second iCa was smaller than control when the interval between the pair was short. Because paired pulse potentiation was greatest when the interval between the pair was short, it is concluded that paired pulse potentiation was not the result of a prolongation of the action potential or increase in iCa. 6. During paired pulse stimulation of ventricular myocytes under voltage clamp control the increase in contraction was greater, the more positive the membrane potential during the second pulse of the pair. This voltage dependence is consistent with a role for the Na(+)-Ca2+ exchanger in paired pulse potentiation. 7. During paired pulse stimulation of ventricular myocytes under voltage clamp control, changes in putative Na(+)-Ca2+ exchange current were observed consistent with a decrease of Ca2+ efflux (or increase of Ca2+ influx) via the exchanger during the second pulse of the pair. 8. A computer model of excitation-contraction coupling (Harrison, McCall & Boyett, 1992) has been used to simulate paired pulse stimulation and the results described above.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗