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W J Lederer

Publications and source records attributed to W J Lederer.

At least 109 records · Page 6Linked to original sources
PubMed1991

Restoring forces in cardiac myocytes. Insight from relaxations induced by photolysis of caged ATP.

Concentration jumps of intracellular ATP were produced by photolysis of P3-1-(2-nitrophenyl)ethyl (NPE)-caged ATP and were used to investigate the passive relengthening properties in unloaded cardiac myocytes. Patch-clamp pipettes in the whole-cell mode were used to voltage-clamp the myocytes and to load the cells with caged ATP while optical methods were applied to record sarcomere length or cell length simultaneously. Cell length was varied using energy deprivation contractures while intracellular Ca2+ was controlled with EGTA. At sarcomere lengths between 1.8 and 1.4 microns cellular relengthening after photolysis of caged ATP was rapid (t1/2 approximately 100 ms) and could be well described by a simple mechanical model. However, ATP jumps made at sarcomere lengths approximately 1.1 microns led to slow relengthening (t1/2 approximately seconds), comparable to the slow reextensions observed in skinned myocytes after bulk solution changes. We attribute the slow and incomplete relengthening of intact and skinned myocytes after severe rigor shortening to deformation and alteration of structural elements inside the cell. Relengthening from intermediate sarcomere lengths in intact cells is elastic and provides information about the underlying relengthening forces inside the cell. The data do not support the presence of a significant discontinuity in elastic modulus at a sarcomere length of approximately 1.6 microns expected from ultrastructural features of the sarcomeres and from observations in skinned myocytes. Our results suggest that the cell length measurements usually performed in this preparation provide an adequate description of the force produced by the unloaded cell in the steady state. The results also provide a way to estimate the error arising from viscous forces during rapid shortening.

Adenosine Triphosphate↗
PubMed1991

Properties of L-type calcium channel gating current in isolated guinea pig ventricular myocytes.

Nonlinear capacitative current (charge movement) was compared to the Ca current (ICa) in single guinea pig ventricular myocytes. It was concluded that the charge movement seen with depolarizing test steps from -50 mV is dominated by L-type Ca channel gating current, because of the following observations. (a) Ca channel inactivation and the immobilization of the gating current had similar voltage and time dependencies. The degree of channel inactivation was directly proportional to the amount of charge immobilization, unlike what has been reported for Na channels. (b) The degree of Ca channel activation was closely correlated with the amount of charge moved at all test potentials between -40 and +60 mV. (c) D600 was found to reduce the gating current in a voltage- and use-dependent manner. D600 was also found to induce "extra" charge movement at negative potentials. (d) Nitrendipine reduced the gating current in a voltage-dependent manner (KD = 200 nM at -40 mV). However, nitrendipine did not increase charge movement at negative test potentials. Although contamination of the Ca channel gating current from other sources cannot be fully excluded, it was not evident in the data and would appear to be small. However, it was noted that the amount of Ca channel gating charge was quite large compared with the magnitude of the Ca current. Indeed, the gating current was found to be a significant contaminant (19 +/- 7%) of the Ca tail currents in these cells. In addition, it was found that Ca channel rundown did not diminish the gating current. These results suggest that Ca channels can be "inactivated" by means that do not affect the voltage sensor.

Animals↗
PubMed1991

Ca2+ and voltage inactivate Ca2+ channels in guinea-pig ventricular myocytes through independent mechanisms.

1. L-type Ca2+ currents and Ca2+ channel gating currents were studied in isolated guinea-pig ventricular heart cells using the whole-cell patch-clamp technique, in order to investigate the mechanism of Ca(2+)-dependent inactivation. The effect of altering the intracellular Ca2+ concentration ([Ca2+]i) on these currents was studied through photorelease of intracellular Ca2+ ions using the photolabile Ca2+ chelators DM-nitrophen and nitr-5. 2. We found that step increases in [Ca2+]i produced by photorelease could either increase or decrease the L-type Ca2+ current. Specifically, Ca2+ photorelease from DM-nitrophen almost exclusively caused inactivation of the Ca2+ current. In contrast, Ca2+ photorelease from nitr-5 had a biphasic effect: a small, rapid inactivation of the Ca2+ current was followed by a slow potentiation. These two Ca(2+)-dependent processes seemed to differ in their Ca2+ dependence, as small Ca2+ photoreleases elicited potentiation without a preceding inactivation, whereas larger photoreleases elicited both inactivation and potentiation. 3. The mechanism of the Ca(2+)-dependent inactivation of Ca2+ channels was explored by comparing the effects of voltage and photoreleased Ca2+ on the Ca2+ current and the Ca2+ channel gating current. Voltage was found to reduce both the Ca2+ current and the gating current proportionally. However, Ca2+ photorelease from intracellular DM-nitrophen inactivated the Ca2+ current without having any effect on the gating current. 4. The dephosphorylation hypothesis for Ca(2+)-dependent inactivation was tested by applying isoprenaline to the cells before eliciting a maximal rise of [Ca2+]i (maximal flash intensity, zero external [Na+]i). Isoprenaline could completely prevent Ca(2+)-dependent inactivation under these conditions, even when [Ca2+]i rose so high as to cause an irreversible contracture of the cell. 5. We concluded from these experiments that voltage and Ca2+ ions inactivate the L-type Ca2+ channel through separate, independent mechanisms. In addition, we found that Ca(2+)-dependent inactivation does not result in the immobilization of gating charge, and apparently closes the Ca2+ permeation pathway through a mechanism that does not involve the voltage-sensing region of the channel. Furthermore, we found that Ca(2+)-dependent inactivation is entirely sensitive to beta-adrenergic stimulation. These facts suggest that either Ca(2+)-dependent inactivation results from Ca(2+)-dependent dephosphorylation of the Ca2+ channel, or that Ca(2+)-dependent inactivation is modulated by protein kinase A.

Acetates↗
PubMed1991

Adenosine triphosphate-sensitive potassium channels in the cardiovascular system.

ATP is normally available in cells at millimolar concentrations and is "buffered" by intracellular pools of other high-energy phosphates, such as creatine phosphate. Thus intracellular [ATP] [( ATP]i) may seem an unlikely candidate for a regulatory signal inside cells. Recent evidence suggests, however, that [ATP]i regulates the behavior of a class of potassium (KATP) channels that are found throughout the cardiovascular system. KATP channels are present in cardiac, skeletal, and vascular smooth muscle. The channels are inhibited by micromolar [ATP]i, and this inhibition is relieved by micromolar [ADP]i. We present evidence in support of the idea that variations of [ATP]i and [ADP]i, even within normal concentration ranges, may influence cellular function in the heart and vascular system via a direct action on the KATP channel. Furthermore, very specific modulators of KATP channel activity are available. We discuss the mechanism of action of these agents and their interaction with endogenous modulators and consider their potential roles in cardiovascular therapy.

Adenosine Triphosphate↗
PubMed1991

ATP-sensitive potassium channel modulation of the guinea pig ventricular action potential and contraction.

The role of ATP-sensitive potassium (KATP) channels in modulating the action potential and contraction of guinea pig ventricular myocytes was investigated. Under voltage clamp, the maximum whole-cell KATP channel conductance was estimated (195 +/- 10 nS, n = 6) by exposing the cells to complete metabolic blockade (2 mM cyanide in the presence of 10 mM 2-deoxy-glucose). In isolated inside-out membrane patches, the ATP dependence of KATP channel activity under relevant conditions was measured (half-maximal inhibition at 114 microM). Under current clamp (with intracellular ATP concentration = 5 mM), the effect of graded KATP channel activation on the action potential and the twitch was estimated by injection of a current (proportional to voltage) that simulated the KATP conductance. As this "conductance" was increased, the action potential was shortened, and contractile amplitude declined, as expected. From the results of these experiments, the quantitative dependence of the action potential duration on intracellular ATP concentration was estimated, without relying on a mathematical model of the cell membrane. The results imply that KATP-dependent action potential shortening is likely to occur if ATP concentration falls below normal levels (approximately 5 mM), as may happen regionally, or globally, during myocardial ischemia.

Action Potentials↗
PubMed1991

Angiotensin-induced desensitization of the phosphoinositide pathway in cardiac cells occurs at the level of the receptor.

Previous studies show that angiotensin II (Ang II) increases phosphoinositide turnover in cultured neonatal heart cells. Ang II has also been shown to transiently increase spontaneous beating behavior in these cells. In this study we seek to identify the molecular mechanism underlying this rapid (3-5-minute) desensitization. Time-course studies on the accumulation of [3H]inositol phosphates indicate that the loss in functional responsiveness correlates with reduced efficacy of Ang II to activate the phosphoinositide path. Binding studies with 125I-Ang II revealed that there was no change in surface receptor binding capacity during the time in which desensitization developed. Normal phosphoinositide and functional responses are observed when desensitized cells are treated with probes that activate the cardiac phosphoinositide pathway at discrete steps. These studies reveal that the functional status of the major components of the phosphoinositide signaling pathway, including G proteins, phospholipase C, and protein kinase C (PKC), are normal during maintained Ang II desensitization. To study the potential role of PKC in Ang II desensitization, the cells are treated with TPA for 24 hours, which downregulates PKC activity. PKC-depleted cells rapidly desensitize after Ang II application. We conclude that the selective Ang II-evoked biochemical/functional desensitization involves inhibition at the level of the receptor, rather than at a component downstream in the path, and that this process is independent of PKC and loss of surface binding capacity.

Angiotensin II↗
PubMed1991

Molecular and cellular actions of platelet-activating factor in rat heart cells.

Platelet-activating factor (PAF) is a phospholipid with cardiovascular actions at low concentrations (1-100 nM) but with uncertain direct myocardial actions. We investigated the cellular and molecular effects of PAF on heart cells using isolated adult and neonatal rat myocytes. Addition of PAF, in the superfusion solution, decreased twitch amplitude and contractile velocity in both systems. Concentrations of PAF below 1 nM stimulated reproducible responses with maximal effects seen at 100 nM. These functional actions of PAF could be blocked by the known PAF antagonist, BN 50739, in a dose-dependent manner. Parallel biochemical studies showed that nanomolar PAF rapidly stimulated the phosphoinositide pathway in cultured myocytes, evidenced by the accumulation of [3H]inositol phosphates in prelabeled cultured myocytes. The potency and specificity of PAF, as well as the time course, for the response were nearly identical in the biochemical and functional assays. PAF produced no functional changes in protein kinase C-depleted myocytes, but it did stimulate inositol trisphosphate accumulation in such cells. We conclude that: (a) PAF exerts a direct negative inotropic effect on myocardial tissue; (b) the effects of PAF are mediated by a specific, high affinity cardiac receptor; (c) an underlying biochemical mechanism for the action of PAF includes the activation of the phospholipase C/phosphatidylinositol intracellular signaling pathway, which leads to activation of protein kinase C.

Animals↗
PubMed1990

Voltage-independent calcium release in heart muscle.

The Ca2+ that activates contraction in heart muscle is regulated as in skeletal muscle by processes that depend on voltage and intracellular Ca2+ and involve a positive feedback system. How the initial electrical signal is amplified in heart muscle has remained controversial, however. Analogous protein structures from skeletal muscle and heart muscle have been identified physiologically and sequenced; these include the Ca2+ channel of the sarcolemma and the Ca2+ release channel of the sarcoplasmic reticulum. Although the parallels found in cardiac and skeletal muscles have provoked valuable experiments in both tissues, separation of the effects of voltage and intracellular Ca2+ on sarcoplasmic reticulum Ca2+ release in heart muscle has been imperfect. With the use of caged Ca2+ and flash photolysis in voltage-clamped heart myocytes, effects of membrane potential in heart muscle cells on Ca2+ release from intracellular stores have been studied. Unlike the response in skeletal muscle, voltage across the sarcolemma of heart muscle does not affect the release of Ca2+ from the sarcoplasmic reticulum, suggesting that other regulatory processes are needed to control Ca2(+)-induced Ca2+ release.

Acetates↗
PubMed1990

Modulation of ATP-sensitive potassium channel activity by flash-photolysis of 'caged-ATP' in rat heart cells.

We have used 'caged-ATP' to investigate the kinetic behavior of KATP channels in ventricular cells from rat heart. In whole cells, loaded with 'caged-ATP', an increase of intracellular [ATP] following a UV light flash produced a decrease of KATP channel current that was too slow (tau approximately 300 ms) to be explained by the expected time-course of ATP release (tau approximately 3 ms) and the time-course of channel blockade by ATP (tau approximately 20 ms). In isolated membrane patches, caged-ATP itself caused partial blockade of KATP) channels. Under these conditions, photorelease of ATP caused channel activity to decline further. The results suggest that caged-ATP can bind to the KATP) channel but, on binding, decreases the open probability to a lesser extent than does ATP. Additionally, the observations indicate that for photolytically-generated ATP to bind to the channel, caged-ATP must first unbind (slowly) from the channel. We conclude that 'caged-ATP' is not fully caged with respect to its allosteric action on the KATP channel.

Adenosine Triphosphate↗
PubMed1990

Measurement of intracellular Ca2+ in BC3H-1 muscle cells with Fura-2: relationship to acetylcholine receptor synthesis.

Synthesis of acetylcholine receptors (AChR) can be affected by calcium, but the role played by this cation is controversial. The effect of changes in extracellular calcium, [Ca2+]o, on AChR synthesis was examined in a cultured mouse muscle cell line, BC3H-1. Reduction of [Ca2+]o for long periods (approximately 22 h) leads to a decrease in total surface AChR levels, a finding that is consistent with inhibition of AChR synthesis. A half-maximal reduction in surface AChR levels is observed when [Ca2+]o is decreased from 1.8 to approximately 5o microM. Under these conditions, however, total protein synthesis is also largely inhibited, suggesting that the effect of [Ca2+]o on AChR synthesis may be relatively non-specific. Increasing [Ca2+]i by adding the Ca2+ ionophore, A23187 (in the presence of 1.8 mM [Ca2+]o) also gives similar and significant reductions of both AChR and protein synthesis. Since the time course of changes in intracellular calcium [( Ca2+]i) produced by these manoeuvres is unknown, we examined the effects of briefer (1-6 h) reductions in [Ca2+]o and achieved a more specific reduction in AChR synthesis. A direct measurement of the changes in [Ca2+]i resulting from changes in [Ca2+]o was made using the fluorescent indicator Fura-2 and video fluorescence microscopy. Our results show that in BC3H-1 muscle cells the resting intracellular calcium decreases reversibly over 20 min when [Ca2+]o is decreased. We suggest that a reduction of [Ca2+]i produced by the lower [Ca2+]o underlies the reduction in AChR synthesis observed in these experiments.

Animals↗
PubMed1990

Excitation-contraction coupling in heart cells. Roles of the sodium-calcium exchange, the calcium current, and the sarcoplasmic reticulum.

Experimental data do not support the idea that excitation-contraction coupling in heart muscle can be explained by a simple calcium-induced calcium release mechanism alone or a simple voltage-dependent calcium release mechanism alone. Our data on excitation-contraction coupling combined with the results of others suggest the need to either develop more complex and more sophisticated single-mechanism models, or to establish dual-control models.

Animals↗
PubMed1990

The regulation of ATP-sensitive K+ channel activity in intact and permeabilized rat ventricular myocytes.

1. In isolated rat heart ventricular myocytes exposed to 2 mM-cyanide in the presence of 10 mM-2-deoxyglucose (complete metabolic blockade), there is a time-dependent increase in ATP-sensitive potassium (KATP) channel activity. The increase in KATP channel activity accompanies the decline of twitch amplitude. Channel activation and decline of the twitch amplitude precede the development of a 'rigor' contracture. 2. We measured KATP channel activity in permeabilized cells using the open-cell attached (O-C-A) patch configuration (by establishing a cell-attached patch and then permeabilizing the cell by exposure to saponin). The apparent ATP dependence of KATP channel activity could be described by a sigmoid curve with ki, ATP (ATP concentration required for half-maximum inhibition of channel activity) = 122 microM and H (Hill coefficient) = 1.225. 3. In the O-C-A patch configuration, 10 mM-creatine phosphate (CrP) decreased the apparent ki, ATP from 122 microM to about 10 microM, and the maximal activity (in zero ATP) was decreased to about 30% of the maximal activity in the absence of CrP. 4. In isolated inside-out (I-O) patches, ATP inhibited KATP channel activity at much lower [ATP] than in the O-C-A patch configuration (ki, ATP = 25 microM, H = 2). CrP was without effect on I-O patches. 5. These results are consistent with the hypothesis that the difference in the ATP dependence of KATP channel activity in the O-C-A and I-O patch configurations arises because of ATP consumption in the O-C-A patch configuration. The results suggest that hydrolysis of ATP to ADP by endogenous ATPases leads to the development of gradients of [ATP] and [ADP] between the bath and the 'inside' of the open cell. By re-phosphorylating ADP, CrP is able to dissipate these gradients, revealing the 'true' ATP dependence of channel activity, which is the same as that in the I-O patch configuration. 6. In order to estimate the contribution of KATP channel activity to the rat cardiac action potential at different [ATP] we have made the following measurements. Using electrodes of resistance 2-8 M omega the density of KATP channels was 10.3 +/- 0.1 channels per patch (n = 162).(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1990

The role of ATP in energy-deprivation contractures in unloaded rat ventricular myocytes.

Energy-deprivation contractures were investigated in unloaded rat ventricular myocytes. Application of 2 mM cyanide in the presence of 10 mM 2-deoxyglucose (metabolic blockade) led to a rapid shortening "contracture" (maximum speed 1.5 +/- 0.2% control cell length/s). Cells shortened to a constant length of 69 +/- 1.6% of the control length. Removal of cyanide caused cells to shorten further ("recontracture"), before relaxing towards the control length. Cells shortened to 57 +/- 2.0% during the recontracture. Similar behaviour was observed in zero extracellular [Ca2+]. Cells permeabilized with saponin (0.1% w/v) responded to the removal of ATP from the bathing solution, and to readdition of ATP, as intact cells did to complete metabolic blockade and its removal. In these permeabilized cells, the extent and speed of contracture shortening were similar at pCa = 7 and pCa greater than 9. When the bath concentration of ATP ([ ATP]b) was lowered to zero, shortening stopped at about 70% of the control length. However, when [ATP]b was lowered to an intermediate level (4-20 microM), cells contracted to lengths as short as 30% of the control length. Similarly, when [ATP]b was restored from zero to an intermediate concentration (4-20 microM), recontracture shortening continued without relaxation. The peak speed of this Ca2(+)-independent shortening showed a sigmoidal dependence on pMgATP (pMgATP0.5 = 4.0). Phosphocreatine (10 mM) shifted the ATP dependence of Ca2(+)-independent shortening to lower [ATP]b (pMgATP0.5 = 5.0), suggesting that gradients of [ATP] could exist between the bath and the myofilaments. Ca2(+)-independent shortening was inhibited by the chemical phosphatase 2,3-butanedione monoxime (BDM), although BDM did not relax cells from the shortened state during energy deprivation. Using a simple model, we show that the results can be explained by cross-bridge cycling occurring independently of Ca2+ over a "window" range of [MgATP] (0.1-100 microM). Therefore, when [MgATP] falls, cross-bridge cycling occurs and the cell shortens. As [MgATP] falls to very low levels ([ MgATP] less than 1 microM), shortening ceases as the rate of cross-bridge cycling declines. Recontracture occurs on restoring ATP production, because stiffness falls and Ca2(+)-independent cross-bridge cycling initially increases. As [MgATP] rises above 100 microM, Ca2(+)-independent cross-bridge cycling ceases and the cell relaxes towards the control length. We conclude that energy-deprivation contractures, and recontractures, can result from changes in [MgATP] and do not necessarily require changes in [Ca2+]i.

Adenosine Triphosphate↗
PubMed1990

Intracellular Ca transients in rat cardiac myocytes: role of Na-Ca exchange in excitation-contraction coupling.

Membrane current and intracellular Ca concentration ([Ca]i) transients were recorded from isolated rat ventricular myocytes under voltage-clamp control. The cells were dialyzed by the patch pipette solution, which contained the fluorescent Ca indicator indo-1 and 0.5 mM Na. Under these experimental conditions, Ca entry via Na-Ca exchange did not appear to be appreciable even in the absence of extracellular Na. Increasing the duration of voltage-clamp pulses from 5 to 80 ms produced [Ca]i transients of increasing amplitude, while the peak Ca current was not changed. This duration dependence of the [Ca]i transient was most demonstrable at more negative test potentials (e.g., -20 to -30 mV) and was not qualitatively modified by Na-free solutions. This latter result indicates that Ca extrusion by Na-Ca exchange is not responsible for the smaller [Ca]i transients observed when the membrane is repolarized after very brief depolarizations. Although the peak Ca current was not changed by increasing pulse duration, the integrated Ca current was increased. These observations are consistent with a Ca-release mechanism in cardiac excitation-contraction coupling in which 1) the Ca-release process can be modulated by membrane potential or 2) the Ca entering the cell via Ca channels has a preferential access [compared with Ca from the sarcoplasmic reticulum (SR)] to the site(s) that control SR Ca release. The role of Na-Ca exchange in the decline of [Ca]i during relaxation was also explored. Removal of extracellular Na (Nao) resulted in 20% slowing of the decline in [Ca]i during relaxation. From this, we conclude that the Na-Ca exchange competes with SR to remove Ca from the cytoplasm and that under our control conditions the exchanger may account for 20% of this decline. The Nao dependence of relaxation was reduced at more positive membrane potentials and increased by SR Ca loading.

Animals↗