Biomedical subjects

W J Lederer

Publications and source records attributed to W J Lederer.

At least 91 records · Page 5Linked to original sources

PubMed1994

A confocal laser scanning microscope designed for indicators with ultraviolet excitation wavelengths.

In this paper we describe the modifications necessary to upgrade, at affordable cost, a commercially available confocal laser scanning microscope for use with ultraviolet (UV) excitation. The optical problems associated with these modifications are described in detail, and easy solutions to solve them are suggested. The optical resolution of the instrument was tested with fluorescent beads and was found to be close to diffraction limited. The light losses due to lateral chromatic aberration were assessed in a thick fluorescent specimen and were found to be comparable to those usually observed with visible light. For a more visual example of the resolution of this instrument, isolated ventricular heart muscle cells were loaded with the fluorescent Ca2+ indicator indo 1. This allowed us to visualize subcellular structural detail and to illustrate the optical sectioning capability of the UV confocal microscope when recording indo 1 emission. Dual-emission line scans were used to perform ratiometric time-resolved detection of Ca2+ transients in voltage-clamped heart muscle cells loaded with the salt form of indo 1. The system presented in this paper should significantly broaden the range of fluorescent indicators that can be used in confocal microscopy.

Animals↗

PubMed1993

Calcium sparks: elementary events underlying excitation-contraction coupling in heart muscle.

Spontaneous local increases in the concentration of intracellular calcium, called "calcium sparks," were detected in quiescent rat heart cells with a laser scanning confocal microscope and the fluorescent calcium indicator fluo-3. Estimates of calcium flux associated with the sparks suggest that calcium sparks result from spontaneous openings of single sarcoplasmic reticulum (SR) calcium-release channels, a finding supported by ryanodine-dependent changes of spark kinetics. At resting intracellular calcium concentrations, these SR calcium-release channels had a low rate of opening (approximately 0.0001 per second). An increase in the calcium content of the SR, however, was associated with a fourfold increase in opening rate and resulted in some sparks triggering propagating waves of increased intracellular calcium concentration. The calcium spark is the consequence of elementary events underlying excitation-contraction coupling and provides an explanation for both spontaneous and triggered changes in the intracellular calcium concentration in the mammalian heart.

Aniline Compounds↗

PubMed1993

Cloning and expression of an inwardly rectifying ATP-regulated potassium channel.

A complementary DNA encoding an ATP-regulated potassium channel has been isolated by expression cloning from rat kidney. The predicted 45K protein, which features two potential membrane-spanning helices and a proposed ATP-binding domain, represents a major departure from the basic structural design characteristic of voltage-gated and second messenger-gated ion channels. But the presence of an H5 region, which is likely to form the ion conduction pathway, indicates that the protein may share a common origin with voltage-gated potassium channel proteins.

Adenosine Triphosphate↗

PubMed1993

Activation of Na-Ca exchange current by photolysis of "caged calcium".

Intracellular photorelease of Ca2+ from "caged calcium" (DM-nitrophen) was used to investigate the Ca(2+)-activated currents in ventricular myocytes isolated from guinea pig hearts. The patch-clamp technique was applied in the whole-cell configuration to measure membrane current and to dialyze the cytosol with a pipette solution containing the caged compound. In the presence of inhibitors for Ca2+, K+, and Na+ channels, concentration jumps of [Ca2+]i induced a rapidly activating inward Na-Ca exchange current which then decayed slowly (tau approximately 500 ms). The initial peak of the inward current and the time-course of current decay were voltage-dependent, and no reversal of the current direction was found between -100 and +100 mV. The observed shallow voltage dependence can be described in terms of the movement of an apparently fractional elementary charge (+0.44e-) across an energy barrier located symmetrically in the electrical field of the membrane. The currents were dependent on extracellular Na+ with a half-maximal activation at 73 mM and a Hill coefficient of 2.8. No change of membrane conductance was activated by the Ca2+ concentration jump when extracellular Na+ was completely replaced by Li+ or N-methyl-D-glucamine (NMG) or when the Na-Ca exchange was inhibited by extracellular Ni2+, La3+, or dichlorobenzamil (DCB). The velocity of relengthening after a twitch induced by photorelease of Ca2+ was only reduced drastically when both the sarcoplasmic reticulum and the Na-Ca exchange were inhibited suggesting that all other Ca2+ removing mechanisms have a low transport capacity under these conditions. In conclusion, we have used a novel approach to study Na-Ca exchange activity with photolysis of "caged" calcium. We found that in guinea pig heart muscle cells the Na-Ca exchange is a potent mechanism for Ca2+ extrusion, is weakly voltage-dependent (118 mV for e-fold change) and can be studied without contamination with other Ca(2+)-activated currents.

Acetates↗

PubMed1993

Does the use of DM-nitrophen, nitr-5, or diazo-2 interfere with the measurement of indo-1 fluorescence?

Emission spectra of the photolabile Ca2+ chelators DM-nitrophen, nitr-5, and diazo-2 were studied alone, and in the presence of indo-1, to investigate potential interactions that would make the simultaneous manipulation and ratiometric measurement of the intracellular Ca2+ concentration difficult. Neither diazo-2 nor its photoproduct were found to be significantly fluorescent, and consequently concentrations of diazo-2 up to 20 times that of indo-1 did not distort the emission spectra of indo-1. DM-nitrophen was scarcely fluorescent, but its fluorescence did increase upon photolysis. In contrast to diazo-2 and DM-nitrophen, nitr-5 itself was found to be quite fluorescent, and this fluorescence was significantly increased upon photolysis. Thus, combined use of nitr-5 and indo-1 poses the most difficulty. The emission spectra of all the investigated compounds were used to define experimental conditions and calibration procedures that make possible simultaneous measurement and manipulation of the intracellular Ca2+ concentration.

Acetates↗

PubMed1993

Sodium/calcium exchanger in heart muscle: molecular biology, cellular function, and its special role in excitation-contraction coupling.

The Na/Ca exchanger has been examined with respect to its molecular biology, its cellular function, and its role in excitation-contraction coupling. The Na/Ca exchanger plays a central part in excitation-contraction coupling, setting the level of sarcoplasmic reticular calcium and contributing to the triggering of sarcoplasmic reticular calcium release. Functional biophysical studies with isolated single cells and caged calcium provide evidence that the Na/Ca exchanger works as a two step sequential transporter. In the heart there are about 250 exchangers.mu-2, operating at a turnover rate of up to about 2500.s-1, with the exchanger carrying -2.56 charges under normal conditions. The Na/Ca exchanger has been recently cloned from diverse mammalian species and several tissues and is largely conserved. It is clear, however, that the function of the Na/Ca exchanger is different in the different tissues. Thus work is in progress in several laboratories, including ours, to determine how the Na/Ca exchanger achieves its tissue specific function. Several modulatory motifs have been seen in studies of the exchanger that may explain some of the tissue specific differences. Interestingly the modulation of the Na/Ca exchanger (for example, by protons, sodium, calcium, ATP, calmodulin) seems to arise from interactions with the intracellular loop.

Animals↗

PubMed1993

On the mechanism of inhibition of KATP channels by glibenclamide in rat ventricular myocytes.

INTRODUCTION: The mechanism by which glibenclamide inhibits KATP channel activity has been examined in membrane patches from isolated rat ventricular cells. METHODS AND RESULTS: Inside-out patches were exposed to zero, or low, [ATP] to activate KATP channels. Glibenclamide did not affect single channel conductance, but reversibly reduced channel open probability from either side of the membrane. Internal (cytoplasmic) glibenclamide inhibited with half-maximal inhibitory [glibenclamide] = 6 microM, Hill coefficient = 0.35. Complete channel inhibition was not observed, even at 300 microM [glibenclamide]. The response to step increases of internal [glibenclamide] could be resolved into two phases of channel inhibition (t1/2,fast < 1 sec, t1/2, slow = 10.5 +/- 0.9 sec, n = 8). Step decrease of [glibenclamide] caused a single resolvable phase of reactivation (t1/2 = 20.4 +/- 0.7 sec, n = 16). Channel inhibition by internal glibenclamide could be relieved by ADP, but only in the presence of Mg2+. CONCLUSION: Glibenclamide can inhibit KATP channels from either side of the membrane, with block from one side being competitive with block from the other. Internal MgADP antagonizes the blocking action of glibenclamide. Glibenclamide inhibition of cardiac KATP channels differs quantitatively and qualitatively from the inhibition of pancreatic KATP channels.

Adenosine Diphosphate↗

PubMed1993

Calcium current in single human cardiac myocytes.

INTRODUCTION: Significant species-, tissue-, and age-dependent differences have been described for the L-type calcium current (ICa). Therefore, extrapolation of data obtained from the many animal models to human cardiac physiology is difficult. In this study, we have characterized the voltage-dependent properties of ICa from pediatric and adult, atrial and ventricular human heart tissue. METHODS AND RESULTS: ICa was measured in single human heart muscle cells using the "whole cell," voltage clamp method. Single myocytes were isolated from myocardial specimens obtained intraoperatively from both pediatric and adult patients (ages 3 months to 75 years) undergoing cardiac surgery. Cells obtained for these experiments appeared to be healthy; the resting potential was between -80 and -85 mV. The action potential shape and duration and current-voltage relationship for ICa were similar to that reported by others for human heart cells. The steady-state activation variable, d infinity, was found to be similar in both pediatric atrial and ventricular cells but shifted approximately 5 mV negative in the adult atrial and ventricular cells. ICa of all cells displayed biexponential inactivation and steady-state inactivation was incomplete at positive potentials (steady-state inactivation curves turned up at positive potentials) consistent with inactivation arising from voltage-dependent and calcium-dependent processes as reported in heart cells from many species. The potential of maximal inactivation was more negative for adult cells (around -10 mV) than pediatric cells (around 0 mV). Estimates of the calcium "window" current, using a modified Hodgkin-Huxley model, could explain measured differences in action potential shape and duration. CONCLUSION: Human cardiac ICa can be investigated using whole cell, voltage clamp methods and a modified Hodgkin-Huxley model. Quantitative characterization of many of the properties of ICa in human heart tissue suggests that important species differences do exist and that further investigations are required to characterize the dependence of inactivation on [Ca2+]i in human heart cells. Since the array of characteristics of ICa in different species varies, the study of human myocardial cells per se continues to be important when examining human cardiac physiology.

Action Potentials↗

PubMed1993

Na/Ca exchanger isoforms expressed in kidney.

The cardiac (versus retinal rod) Na/Ca exchanger gene has been cloned, sequenced and shown by RNA analysis to be present in diverse tissues. Analysis of published sequences shows that a single isoform is found in heart tissue from many species (NACA1 isoform). We provide evidence here by ribonuclease (RNase) protection assays and by reverse transcriptase-polymerase chain reaction (PCR) amplification with sequence analysis that a new isoform encoding the Na/Ca exchanger is present in renal tissue. This isoform (NACA3) reveals a 7-amino acid deletion in the tested region compared with the NACA2 isoform described by Reilly and Shugrue [Am. J. Physiol. 262 (Renal Fluid Electrolyte Physiol. 31): F1105-F1109, 1992] and is the dominant exchanger transcript in kidney. Analysis of the sequence of all isoforms indicates that the differences in the isoforms reside in the large intracellular loop region of the protein. Alternative splicing of a single Na/Ca exchanger message may be responsible for these tissue-specific transcripts.

Animals↗

PubMed1993

Mapping of the human cardiac Na+/Ca2+ exchanger gene (NCX1) by fluorescent in situ hybridization to chromosome region 2p22-->p23.

The cDNA that encodes the human Na+/Ca2+ exchanger (NCX1) involved in regulation of intracellular calcium levels has been isolated from a cardiac cDNA library. Using fluorescent in situ hybridization, the human cDNA was mapped to chromosome region 2p23-->p22 by co-hybridization with fluorescinated alu517-PCR amplified total human DNA to obtain an R-banding pattern.

Carrier Proteins↗

PubMed1993

The mechanism by which cytoplasmic protons inhibit the sodium-calcium exchanger in guinea-pig heart cells.

1. We recorded cardiac sodium-calcium exchange current (INa-Ca) in giant excised membrane patches obtained from cardiac myocytes of the adult guinea-pig. 2. Rapid changes in ion concentrations on the cytoplasmic side of the excised membrane patch were produced using a modified oil-gate bath. 3. Sodium-calcium exchange current was activated by step increases in sodium concentration on the cytoplasmic side of the membrane ([Na+]i), which led to an increase in outward INa-Ca to a new steady-state level. The [Na+]i required to half-maximally activate the sodium-calcium exchange current (K1/2) was 21 mM. 4. Step increases in cytoplasmic calcium concentration ([Ca2+]i) stimulated the [Na+]i-activated INa-Ca up to 1 microM [Ca2+]i, then inhibited the exchange current at very high [Ca2+]i (1 mM). 5. A step decrease in cytoplasmic pH from 7.2 to 6.4 (increase in [H+]i) produced a biphasic but monotonic decrease in INa-Ca. Alkalinization of cytoplasmic pH from 7.2 to 8.0 caused a large, biphasic increase in INa-Ca. 6. When INa-Ca was activated by a step increase in [Na+]i and [H+]i was simultaneously increased, the outward current rose to a peak and then declined to a low steady level. The peak current seen was always less than the maximum current produced by an identical elevation of [Na+]i at constant pHi. This reduction in peak outward current reflected a rapid 'primary' inhibition of the sodium-calcium exchange by protons. The decay of the sodium-calcium exchange current following the peak was slow and corresponded to the time course of the onset of a 'secondary' proton block. 7. Rapid primary inhibition of the sodium-calcium exchanger could also be produced by cytoplasmic acidification in the absence of cytoplasmic sodium. The primary blockade was revealed when a subsequent increase in [Na+]i activated INa-Ca and a smaller peak outward current was observed. Secondary inhibition of the sodium-calcium exchanger was not, however, produced by cytoplasmic acidification in the absence of cytoplasmic sodium. Regardless of the duration of exposure to elevated [H+]i, the 'secondary' block by protons was still seen on activation of INa-Ca by increased [Na+]i as a gradual reduction of outward current amplitude. 8. Treatment of the sodium-calcium exchanger with the proteolytic enzyme alpha-chymotrypsin largely removed its sensitivity to protons. 9. We conclude that the action of alpha-chymotrypsin on the monomeric sodium-calcium exchange protein is in part to remove a proton-sensitive regulatory component(s) or render the regulation ineffective.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

Scorpion toxins targeted against the sarcoplasmic reticulum Ca(2+)-release channel of skeletal and cardiac muscle.

We report the purification of two peptides, called "imperatoxin inhibitor" and "imperatoxin activator," from the venom of the scorpion Pandinus imperator targeted against ryanodine receptor Ca(2+)-release channels. Imperatoxin inhibitor has a M(r) of approximately 10,500, inhibits [3H]ryanodine binding to skeletal and cardiac sarcoplasmic reticulum with an ED50 of approximately 10 nM, and blocks openings of skeletal and cardiac Ca(2+)-release channels incorporated into planar bilayers. In whole-cell recordings of cardiac myocytes, imperatoxin inhibitor decreased twitch amplitude and intracellular Ca2+ transients, suggesting a selective blockade of Ca2+ release from the sarcoplasmic reticulum. Imperatoxin activator has a M(r) of approximately 8700, stimulates [3H]ryanodine binding in skeletal but not cardiac sarcoplasmic reticulum with an ED50 of approximately 6 nM, and activates skeletal but not cardiac Ca(2+)-release channels. These ligands may serve to selectively "turn on" or "turn off" ryanodine receptors in fragmented systems and whole cells.

Animals↗

PubMed1992

Thapsigargin inhibits contraction and Ca2+ transient in cardiac cells by specific inhibition of the sarcoplasmic reticulum Ca2+ pump.

Regulation of the level of ionized calcium, [Ca2+]i, is critical for its use as an important intracellular signal. In cardiac and skeletal muscle the control of fluctuations of [Ca2+]i depend on sarcolemmal and sarcoplasmic reticulum ion channels and transporters. We have investigated the sesquiterpine lactone, thapsigargin (TG), because of its reported action to alter cellular calcium regulation in diverse cell types, including striated muscle cells. We have combined biochemical and physiological methods at the cellular level to determine the site of action of this agent, its specificity, and its cellular effects. Using a patch-clamp method in whole cell configuration while measuring [Ca2+]i with Indo-1 salt, we find that TG (100 nM) largely blocks the contraction and the [Ca2+]i transient in rat ventricular myocytes. Analysis of these data indicate that no sarcolemmal current or transport system is directly altered by TG, although indirect [Ca2+]i-dependent processes are affected. In permeabilized myocytes, TG blocked oxalate-stimulated calcium uptake (half-maximal effect at 10 nM) into the SR. However, TG (100 microM) had no effect on Ca(2+)-induced Ca(2+)-release in purified muscle (ryanodine-receptor enriched) vesicles while clearly blocking Ca(2+)-ATPase activity in purified (longitudinal SR) vesicles. We conclude that in striated muscle TG markedly alters calcium metabolism and thus alters contractile function only by its direct action on the Ca(2+)-ATPase.

Animals↗

PubMed1992

Immunofluorescence localization of the Na-Ca exchanger in heart cells.

We investigated the localization of the Na-Ca exchanger in fixed, isolated heart cells from rat and guinea pig using immunocytochemical methods with epifluorescence and confocal microscopy. We found that the Na-Ca exchanger is distributed throughout all membranes in contact with the extracellular space, including the sarcolemma, the transverse tubules (T-tubules), and the intercalated disks. Microscopic nonuniformities in the fluorescent labeling appear to reflect varying views of the membranes containing Na-Ca exchanger protein. Confocal thin-section imaging reveals a regular grid of discrete foci of fluorescence, which represent Na-Ca exchanger in T-tubules viewed en face. These foci are 1.80 +/- 0.01 microns apart from sarcomere to sarcomere and are aligned with the Z-line. Along each Z-line, these foci are spaced at 1.22 +/- 0.11-microns intervals. Longitudinal sections of the sarcolemma-T-tubule junction show a comblike appearance, with T-tubules extending inward from the heavily labeled sarcolemma. Our finding that the Na-Ca exchanger is widely distributed over the cell surface may provide further insight into the role of Na-Ca exchange in the heart.

Animals↗

PubMed1992

Expression of the Na-Ca exchanger in diverse tissues: a study using the cloned human cardiac Na-Ca exchanger.

In many cells including cardiac myocytes, cytoplasmic Ca is importantly controlled by the plasmalemmal Na-Ca exchanger (3, 8). The tissue diversity and differences in cellular environment raise the question whether the same exchanger is found in all tissues. Recent experiments using rod cells have demonstrated that at least two forms of Na-dependent Ca transport exist. We have examined this issue in various rat and human tissues using the cloned human cardiac Na-Ca exchanger cDNA. Northern blot analysis in these two species show that the major transcript of the Na-Ca exchanger is 7.2 kilobases in heart, brain, kidney, liver, pancreas, skeletal muscle, placenta, and lung. Furthermore, ribonuclease protection analysis in rats shows conservation of the 348-base pair segment tested in heart, brain, kidney, skeletal muscle, and liver. Additionally, Southern blot analysis suggests that a single gene encodes this Na-Ca exchanger. Finally, we show that the clone used to generate our probes encodes a completely functional Na-Ca exchanger. With the use of COS cells and 293 cells transfected with the cloned human cardiac Na-Ca exchanger, we tested the Ca transport properties of the Na-Ca exchanger, the voltage dependence of the Na-Ca exchanger, as well as the Na dependence of the transport function of the Na-Ca exchanger. We conclude that the cardiac form of the Na-Ca exchanger is completely functional when the cDNA is expressed in mammalian cell lines, and, furthermore, this "cardiac" form of the Na-Ca exchanger is naturally expressed in all human and rat tissues tested (but at varying levels).

Amino Acid Sequence↗

PubMed1992

Comparison of the effects of BAY K 8644 on cardiac Ca2+ current and Ca2+ channel gating current.

Effects of (-)-BAY K 8644 on Ca2+ channel function were studied in guinea pig ventricular myocytes. It was found that the compound has both voltage-dependent stimulatory and inhibitory effects on the Ca2+ current (ICa), in agreement with prior studies. The basis for these effects was studied by evaluating the effects of (-)-BAY K 8644 on the Ca2+ channel gating current. It was found that the voltage-dependent inhibitory effects of the drug on ICa could be well explained by similar reductions in the amount of gating charge moved. However, the stimulatory effect of (-)-BAY K 8644 on ICa could not be simply correlated with changes in the amount of gating charge moved. Although the drug produced a shift of the charge-voltage relationship to more negative potentials, the drug actually reduced the total amount of movable gating charge. Thus it could be demonstrated that there are membrane potentials where (-)-BAY K 8644 reduced the Ca2+ channel gating current while enhancing ICa. In addition, the drug was found to slow the decay of the gating current during repolarization. It seems likely that (-)-BAY K 8644 has a dual effect on Ca2+ channels: affecting both the voltage dependence of gating charge and the relationship between open probability and charge movement.

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

PubMed1991

Molecular operations of the sodium-calcium exchanger revealed by conformation currents.

The sodium-calcium exchanger is critical in the normal functioning of many cells. In heart muscle, it is the principal way by which the cells keep the concentration of intracellular calcium low, pumping out the Ca2+ that enters the cytosol through L-type Ca2+ channels. The exchanger may also contribute to the triggering of Ca2+ release during voltage-activated excitation-contraction coupling in heart. Time resolved examination of the conformational changes of macromolecules in living cells has so far been largely restricted to ion-channel proteins whose gating is voltage-dependent. We have now directly measured electrical currents arising from the molecular rearrangements of the sarcolemmal Na-Ca exchanger. Changes in the conformation of the exchanger protein were activated by a rapid increase in the intracellular calcium concentration produced by flash photolysis of caged calcium in voltage-clamped heart cells. Two components of membrane current were produced, reflecting a calcium-dependent conformational change of the transporter proteins and net transport of ions by the exchanger. The properties of these components provide evidence that the Na-Ca exchanger protein undergoes two consecutive membrane-crossing molecular transitions that each move charge, and that there are at least 250 exchangers per micron 2 turning over up to 2,500 times per second.

Amiloride↗

PubMed1991

ATP dependence of KATP channel kinetics in isolated membrane patches from rat ventricle.

The dependence of KATP channel activity on [ATP] has been examined in isolated membrane patches from rat ventricular myocytes. The steady-state [ATP] dependence of channel open probability could be described by a sigmoidal relationship with the ki ([ATP] causing half-maximal inhibition of open probability) = 25 microM and Hill coefficient of 2. Description of channel open- and closed-time distributions required at least 2, and 3, time constants, respectively. Long open-channel lifetimes decreased with [ATP]; unconditional mean channel closed-times increased with [ATP]. Step decrease (jump) in bathing [ATP] resulted in a delay (of up to hundreds of milliseconds) followed by a pseudo-exponential rise of current (with a time constant of up to hundreds of milliseconds). The time course of channel current after changes of [ATP] (or the ATP-analogue AMP-PNP) was shown to be predominantly determined by the time course of diffusion into the tip of the electrode and to the membrane. This time course of diffusion of ATP into the pipette tip had to be taken into account when analyzing the current response to [ATP] steps. Several possible kinetic models of the ATP-dependent regulation of channel activity were considered. Adequate explanation of the data required a model with sequential ATP-binding sites. The model can account for the time course of channel opening after steps of [ATP], as well as for the steady-state dependence of P0 on [ATP]. The model predicts [ATP]-dependent closed and open lifetimes as were observed experimentally.

Adenosine Triphosphate↗