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Biomedical subjects

A W Trafford

Publications and source records attributed to A W Trafford.

At least 19 recordsLinked to original sources
PubMed2001

Coordinated control of cell Ca(2+) loading and triggered release from the sarcoplasmic reticulum underlies the rapid inotropic response to increased L-type Ca(2+) current.

The aim of this study was to investigate how sarcoplasmic reticulum (SR) Ca(2+) content and systolic Ca(2+) are controlled when Ca(2+) entry into the cell is varied. Experiments were performed on voltage-clamped rat and ferret ventricular myocytes loaded with fluo-3 to measure intracellular Ca(2+) concentration ([Ca(2+)](i)). Increasing external Ca(2+) concentration ([Ca(2+)](o)) from 1 to 2 mmol/L increased the amplitude of the systolic Ca(2+) transient with no effect on SR Ca(2+) content. This constancy of SR content is shown to result because the larger Ca(2+) transient activates a larger Ca(2+) efflux from the cell that balances the increased influx. Decreasing [Ca(2+)](o) to 0.2 mmol/L decreased systolic Ca(2+) but produced a small increase of SR Ca(2+) content. This increase of SR Ca(2+) content is due to a decreased release of Ca(2+) from the SR resulting in decreased loss of Ca(2+) from the cell. An increase of [Ca(2+)](o) has two effects: (1) increasing the fraction of SR Ca(2+) content, which is released on depolarization and (2) increasing Ca(2+) entry into the cell. The results of this study show that the combination of these effects results in rapid changes in the amplitude of the systolic Ca(2+) transient. In support of this, the changes of amplitude of the transient occur more quickly following changes of [Ca(2+)](o) than following refilling of the SR after depletion with caffeine. We conclude that the coordinated control of increased Ca(2+) entry and greater fractional release of Ca(2+) is an important factor in regulating excitation-contraction coupling.

4-Aminopyridine↗
PubMed2001

The role of intracellular Ca buffers in determining the shape of the systolic Ca transient in cardiac ventricular myocytes.

We have studied the kinetics of decay of cardiac Ca transients elicited by either caffeine or electrical stimulation. The decay of the caffeine-evoked increase of intracellular Ca concentration ([Ca2+]i) could not be fit by a single exponential. A two exponential fit showed an initial rapid component. The rate of decay of total Ca was calculated from measured free Ca and the buffering properties of the cell. There was no initial rapid phase of decay of total Ca. We conclude that the rapid phase of decay of free Ca is due to a decrease of Ca buffering power at elevated [Ca2+]i. In contrast, the decay of the Ca transient, produced by voltage-clamp depolarization or field stimulation was fit by a single exponential. We suggest that these apparently simple kinetics arise because a tendency to saturation at elevated [Ca2+]i of the sarcoplasmic reticulum Ca-ATPase offsets the decrease of Ca buffering power. These data show the importance of Ca buffers as well as transporters in determining the kinetics of changes of [Ca2+]i.

Aniline Compounds↗
PubMed2001

The effects of exogenous calcium buffers on the systolic calcium transient in rat ventricular myocytes.

The aim of this work was to characterize the effects that two commonly used "caged" calcium buffers (NP-EGTA and nitr-5) have on the amplitude and time course of decay of the calcium transient. We made quantitative measurements of both free and total calcium using the measured buffering properties of the cell. Intracellular calcium concentration ([Ca(2+)](i)) was measured with fluo-3 in rat ventricular myocytes. Incorporation of the buffer NP-EGTA decreased both the amplitude and rate of decay of the caffeine response. The slowing could be quantitatively accounted for by the measured increased buffering. These effects were removed by photolysis of NP-EGTA. Similar results were obtained with nitr-5 except that the effects were not completely removed by photolysis. This was shown to be due to the persistence of a component of the increased buffering after photolysis. Both buffers decreased the amplitude of the systolic calcium transient. However, although nitr-5 produced a simple slowing of the decay, NP-EGTA resulted in an initial rapid phase of decay. This rapid phase of decay is attributed to calcium binding to NP-EGTA. This work represents the first quantitative analysis of the effects that extra buffering by a fast and a slow calcium chelator may have on the calcium transient.

Aniline Compounds↗
PubMed2000

The effect of acidosis on systolic Ca2+ and sarcoplasmic reticulum calcium content in isolated rat ventricular myocytes.

We have investigated the mechanisms responsible for the changes of systolic Ca2+ that occur in voltage-clamped rat ventricular myocytes during acidosis produced by application of the weak acid butyrate (30 mM). Intracellular pH regulation was inhibited with dimethylamiloride (bicarbonate-free solution). The application of butyrate produced an intracellular acidification of 0.33 pH units. This was accompanied by a decrease in systolic Ca2+ to about 50% of control. However, within 2 min, systolic Ca2+ returned to control levels. The decrease in systolic Ca2+ was accompanied by a decrease in the Na+-Ca2+ exchange current observed on repolarisation so that the calculated Ca2+ efflux on Na+-Ca2+ exchange was less than the entry on the L-type Ca2+ current. The magnitude of the Na+-Ca2+ exchange current recovered along with systolic Ca2+ until it equalled the Ca2+ entry on the L-type Ca2+ current. From the measurement of Ca2+ fluxes, it was calculated that, during acidosis, the cell gains 121.6+/-16.2 micromol l(-1) of Ca2+. This is equal to the measured increase of sarcoplasmic reticulum (SR) calcium content obtained by applying caffeine (20 mM) and integrating the resulting Na+-Ca2+ exchange current. We conclude that the recovery of the amplitude of the systolic Ca2+ transient is due to decreased SR calcium release, resulting in reduced Ca2+ efflux from the cell leading to increased SR calcium content.

Acidosis↗
PubMed2000

Integrative analysis of calcium cycling in cardiac muscle.

The control of intracellular calcium is central to regulation of contractile force in cardiac muscle. This review illustrates how analysis of the control of calcium requires an integrated approach in which several systems are considered. Thus, the calcium content of the sarcoplasmic reticulum (SR) is a major determinant of the amount of Ca(2+) released from the SR and the amplitude of the Ca(2+) transient. The amplitude of the transient, in turn, controls Ca(2+) fluxes across the sarcolemma and thence SR content. This control of SR content influences the response to maneuvers that modify, for example, the properties of the SR Ca(2+) release channel or ryanodine receptor. Specifically, modulation of the open probability of the ryanodine receptor produces only transient effects on the Ca(2+) transient as a result of changes of SR content. These interactions between various Ca(2+) fluxes are modified by the Ca(2+) buffering properties of the cell. Finally, we predict that, under some conditions, the above interactions can result in instability (such as alternans) rather than ordered control of contractility.

Biological Transport↗
PubMed2000

Modulation of CICR has no maintained effect on systolic Ca2+: simultaneous measurements of sarcoplasmic reticulum and sarcolemmal Ca2+ fluxes in rat ventricular myocytes.

1. The effects of modulating Ca2+-induced Ca2+ release (CICR) in single cardiac myocytes were investigated using low concentrations of caffeine (< 500 microM) in reduced external Ca2+ (0.5 mM). Caffeine produced a transient potentiation of systolic [Ca2+]i (to 800 % of control) which decayed back to control levels. 2. Caffeine decreased the steady-state sarcoplasmic reticulum (SR) Ca2+ content. As the concentration of caffeine was increased, both the potentiation of the systolic Ca2+ transient and the decrease in SR Ca2+ content were increased. At higher concentrations, the potentiating effect decayed more rapidly but the rate of recovery on removal of caffeine was unaffected. 3. A simple model in which caffeine produces a fixed increase in the fraction of SR Ca2+ which is released could account qualitatively but not quantitatively for the above results. 4. The changes in total [Ca2+] during systole were obtained using measurements of the intracellular Ca2+ buffering power. Caffeine initially increased the fractional release of SR Ca2+. This was followed by a decrease to a level greater than that under control conditions. The fraction of systolic Ca2+ which was pumped out of the cell increased abruptly upon caffeine application but then recovered back to control levels. The increase in fractional loss is due to the fact that, as the cytoplasmic buffers become saturated, a given increase in systolic total [Ca2+] produces a larger increase in free [Ca2+] and thence of Ca2+ efflux. 5. These results confirm that modulation of the ryanodine receptor has no maintained effect on systolic Ca2+ and show the interdependence of SR Ca2+ content, cytoplasmic Ca2+ buffering and sarcolemmal Ca2+ fluxes. Such analysis is important for understanding the cellular basis of inotropic interventions in cardiac muscle.

Algorithms↗
PubMed2000

Measurement of calcium entry and exit in quiescent rat ventricular myocytes.

The aim of this work was to obtain the first quantitative measurements of Ca2+ influx and efflux in quiescent cardiac cells. The relationship between free and total Ca2+ was obtained during a caffeine application. This buffering curve was then used to calculate changes of total Ca2+ from measurements of free cytosolic [Ca2+] ([Ca2+]i) made with Indo-1. The rate of Ca2+ removal from the cytoplasm was calculated by differentiating total Ca2+ with respect to time. The dependence of d(total Ca2+)/dt on [Ca2+]i was hyperbolic. Inhibition of either Na+-Ca2+ exchange (by addition of 10 mmol l(-1) NiCl2 or removal of external Na+) or the sarcolemmal Ca2+-activated adenosine triphosphatase (Ca2+-ATPase) (with carboxyeosin) decreased the calculated efflux. In both cases, the main effect was on the apparent maximum rate (Vmax) with little effect on the Michaelis-Menten constant (Km). These results suggest that the Na+-Ca2+ exchange and Ca2+-ATPase have very similar affinities for [Ca2+]i and that their fractional contributions do not change over the systolic range of [Ca2+]i. Ca2+ influx was quantified in two ways. The first method was to extrapolate the curve relating Ca2+ efflux to [Ca2+]i to zero [Ca2+]i. This gave a value of 4.49+/-0.54 micromol l(-1) s(-1) which was reduced to zero by either removal of external Ca2+ or addition of Ni2+. In other experiments external Ca2+ was removed and the maximum rate of fall of total Ca2+ calculated as 2.53+/-0.93 micromol l(-1) s(-1). This approach can be used to provide a quantitative analysis of the control of resting [Ca2+]i.

Animals↗
PubMed2000

The effects of low concentrations of caffeine on spontaneous Ca release in isolated rat ventricular myocytes.

We have investigated the effects on spontaneous SR Ca release of modulating the sarcoplasmic reticulum ryanodine receptor (RyR) with low (<0.5 mM) concentrations of caffeine. Experiments were performed on isolated rat ventricular myocytes. Intracellular Ca concentration was measured with Indo-1 or Fluo-3 in voltage-clamped cells. Spontaneous Ca release was produced by elevating external Ca to 5 mM. Caffeine application increased the frequency of spontaneous release. Both the magnitude of the spontaneous Ca transients and the integral of the resulting Na-Ca exchange current were decreased by caffeine. The combination of increased frequency of spontaneous release and decreased Ca efflux per event meant that the Ca efflux per unit time was unaffected by low concentrations of caffeine. The SR Ca content was reduced by caffeine. The extra Ca efflux calculated from the Na-Ca exchange current integrals occurring during the initial burst of spontaneous activity on application of caffeine accounted for this reduction of SR Ca content. In contrast to these maintained effects on spontaneous release, caffeine had only transient effects on stimulated Ca release produced by depolarizing pulses. We conclude that stimulation of the RyR results in spontaneous release at SR Ca contents lower than those at which release would normally occur. Therefore, the balance between normal and spontaneous Ca release can be shifted by modulation of the RyR. This will have important consequences for arrhythmogenesis due to spontaneous Ca release.

Animals↗
PubMed1999

A novel, rapid and reversible method to measure Ca buffering and time-course of total sarcoplasmic reticulum Ca content in cardiac ventricular myocytes.

This paper outlines a simple method of estimating both the Ca-buffering properties of the cytoplasm and the time-course of changes of sarcoplasmic reticulum (s.r.) Ca concentration during systole. The experiments were performed on voltage-clamped ferret single ventricular myocytes loaded with the free acid of fluo-3 through a patch pipette. The application of caffeine (10 mM) resulted in a Na-Ca exchange current and a transient increase of the free intracellular Ca concentration ([Ca2+]i). The time-course of change of total Ca in the cell was obtained by integrating the current and this was compared with the measurements of [Ca2+]i to obtain a buffering curve. This could be fit with a maximum capacity for the intrinsic buffers of 114+/-18 micromol l-1 and Kd of 0.59+/-0.17 microM (n=8). During the systolic rise of [Ca2+]i, the measured changes of [Ca2+]i and the buffering curve were used to calculate the magnitude and time-course of the change of total cytoplasmic Ca and thence of both s.r. Ca content and Ca release flux. This method provides a simple and reversible mechanism to measure Ca buffering and the time-course of both total cytoplasmic and s.r. Ca.

Animals↗
PubMed1998

Ca-activated chloride current and Na-Ca exchange have different timecourses during sarcoplasmic reticulum Ca release in ferret ventricular myocytes.

The aim of this work was to measure membrane currents activated by Ca release from the cardiac sarcoplasmic reticulum (s.r.). Intracellular Ca concentration ([Ca2+]i) was measured using fluo-3 in patch clamped cells. Calcium release from the s.r. (whether occurring spontaneously or evoked by caffeine) produced changes of membrane current which could be separated into a Ca-activated Cl current which was inhibited by DIDS or Cl removal and a Na-Ca exchange current. Both these currents had different time courses from the measured [Ca2+]i. Furthermore the Ca-activated Cl current decayed more quickly than did Na-Ca exchange. Possible explanations for the different kinetics of these two Ca-sensitive currents are discussed.

Animals↗
PubMed1998

2,3-Butanedione monoxime (BDM) decreases sarcoplasmic reticulum Ca content by stimulating Ca release in isolated rat ventricular myocytes.

The effects of 2,3-butanedione monoxime (BDM) were examined using rat ventricular myocytes loaded with Indo-1 to measure the intracellular Ca concentration ([Ca2+]i). BDM (10 mM) produced a transient increase of the systolic Ca transient with no steady-state effect on its magnitude. This transient increase was more marked when BDM was applied after having decreased the external Ca concentration from 1 to 0.1 mM. There was a transient increase of resting [Ca2+]i in both quiescent and electrically stimulated cells. Prior application of BDM decreased the rise of [Ca2+]i produced by caffeine. In voltage-clamped cells the rise of [Ca2+]i produced by BDM was accompanied by a transient inward current attributed to the electrogenic Na-Ca exchange. The amount of Ca lost from the cell upon application of 10 mM BDM could be estimated either from the integral of the BDM-evoked current or from the reduction of the integral of a caffeine-evoked current and corresponded to about 50% of the sarcoplasmic reticulum (s.r.) Ca content. The decrease of s.r. Ca content and the transient potentiation of the systolic Ca transient suggest that BDM acts by stimulating Ca-induced Ca release. These effects must be allowed for when using BDM.

Animals↗
PubMed1998

Stimulation of Ca-induced Ca release only transiently increases the systolic Ca transient: measurements of Ca fluxes and sarcoplasmic reticulum Ca.

OBJECTIVE: To investigate the effects of stimulating calcium induced Ca release with low concentrations (100-200 microM) of caffeine and, in particular, to study the cellular mechanisms responsible for the transient responses found previously. METHODS: Experiments were performed on isolated rat ventricular myocytes. Intracellular calcium concentration ([Ca2+]i) was measured with Indo-1, the cells were voltage-clamped with the perforated patch technique and sarcoplasmic reticulum (s.r.) Ca content was estimated from the integral of the caffeine-evoked current. RESULTS: The systolic Ca transient produced by the first depolarization in the presence of caffeine was larger than the control. Over the next few pulses the magnitude of the Ca transient returned to control levels despite the maintained presence of caffeine. The s.r. Ca content was decreased by 9% after one pulse in caffeine and by 21% after several pulses in caffeine. The first pulse in the low concentration of caffeine was followed by an enhanced inward (Na-Ca exchange) current tail indicating increased efflux of calcium from the cell. The extra loss of calcium calculated from the tail current agreed quantitatively with that from the change of s.r. Ca content. CONCLUSIONS: These results show that stimulating calcium induced calcium release produces only a transient increase of the systolic Ca transient. This is due to the larger Ca transient decreasing the s.r. Ca content. It is concluded that any agent whose sole mode of action is stimulation of calcium-induced calcium release will not produce a maintained inotropic effect. The consequences of this for the effects of other modulators of calcium induced calcium release are discussed.

Animals↗
PubMed1998

The control of Ca release from the cardiac sarcoplasmic reticulum: regulation versus autoregulation.

This review discusses the mechanism and regulation of Ca release from the cardiac sarcoplasmic reticulum. Ca is released through the Ca release channel or ryanodine receptor (RyR) by the process of calcium-induced Ca release (CICR). The trigger for this release is the L-type Ca current with a small contribution from Ca entry on the Na-Ca exchange. Recent work has shown that CICR is controlled at the level of small, local domains consisting of one or a small number of L-type Ca channels and associated RyRs. Ca efflux from the s.r. in one such unit is seen as a 'spark' and the properties of these sparks produce controlled Ca release from the s.r. A major factor controlling the amount of Ca released from the s.r. and therefore the magnitude of the systolic Ca transient is its Ca content. The Ca content depends on both the properties of the s.r. and the cytoplasmic Ca concentration. Changes of s.r. Ca content and the Ca released affect the sarcolemmal Ca and Na-Ca exchange currents and this acts to control cell Ca loading and the s.r. Ca content. The opening probability of the RyR can be regulated by various physiological mediators as well as pharmacological compounds. However, it is shown that, due to compensatory changes of s.r. Ca, modifiers of the RyR only produce transient effects on systolic Ca. We conclude that, although the RyR can be regulated, of much greater importance to the control of Ca efflux from the s.r. are effects due to changes of s.r. Ca content.

Animals↗
PubMed1997

Measurement of sarcoplasmic reticulum Ca2+ content and sarcolemmal Ca2+ fluxes in isolated rat ventricular myocytes during spontaneous Ca2+ release.

1. Intracellular calcium concentration ([Ca2+]i) and Na(+)-Ca2+ exchange currents were measured in calcium-overloaded voltage-clamped rat ventricular myocytes loaded with the Ca(2+)-sensitive fluorescent indicator indo-1. Sarcoplasmic reticulum (SR) Ca2+ content was measured from the integral of the caffeine-evoked current. In cells that had spontaneous SR Ca2+ release in 1 mM external Ca2+ concentration ([Ca2+]o)i raising [Ca2+]o increased the frequency of release with no effect on SR Ca2+ content. In quiescent cells, increased [Ca2+]o produced spontaneous Ca2+ release associated with increased SR Ca2+ content. Further increase of [Ca2+]o had no effect on SR Ca2+ content. The amount of Ca2+ leaving the cell during each release was constant over a wide range of frequencies and [Ca2+]o values. It appears there is a maximum level of SR Ca2+ content, perhaps because spontaneous Ca2+ release results when the content reaches a threshold. 2. From the relationship between [Ca2+]i and Na(+)-Ca2+ exchange current during a caffeine response, it is possible to estimate the changes in Na(+)-Ca2+ exchange current expected from a change of [Ca2+]i. The data show that the calcium oscillations contribute a significant fraction of the total extra Ca2+ efflux induced by increasing [Ca2+]o. Raising [Ca2+]o decreased the rate of calcium removal from the cell as measured from the rate of decay of the caffeine response, suggesting that both inhibition of Ca2+ efflux and increased Ca2+ entry account for the Ca2+ overload at elevated [Ca2+]o. 3. Inhibiting spontaneous SR Ca2+ release increases resting [Ca2+]i. The Ca2+ efflux is identical to that in the presence of release. It is concluded that spontaneous release of calcium, although potentially arrhythmogenic, is an effective way to activate Ca2+ efflux in overloaded conditions and minimizes any increase of diastolic tension.

Animals↗