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Biomedical subjects

G A Langer

Publications and source records attributed to G A Langer.

At least 37 records · Page 2Linked to original sources
PubMed1995

Localization of the Na/Ca exchange-dependent Ca compartment in cultured neonatal rat heart cells.

It has been previously established, in both adult and cultured neonatal cardiac cells, that there is a discrete Na/Ca exchange-dependent Ca compartment. It has been proposed that a component of junctional sarcoplasmic reticulum (JSR) Ca and Ca bound to the apposed inner sarcolemmal leaflet represent together the subcellular locus of the compartment. The present study examines this proposal. The amount of Ca in the total compartment is measured isotopically in intact functional cells (using the on-line "scintillation disk" technique) under a variety of perfusion conditions. Under identical labeling conditions, sarcolemmal membranes are rapidly (within a few hundred milliseconds) isolated from another set of intact cells by "gas dissection," and the amount of Ca bound to the membranes is measured. Probes that specifically decrease SR Ca content (thapsigargin, caffeine, low-dose ryanodine) decrease total cell content and sarcolemmal binding proportionally. High-dose ryanodine (producing closure of SR channels) markedly reduces sarcolemmal binding relative to total content of the compartment. The sarcolemmal sites saturate between 1 and 2 mM extracellular Ca ([Ca]o), whereas the total compartment saturates between 4 and 6 mM [Ca]o. Below 1 mM [Ca]o, sarcolemmal binding is maintained relative to total compartment content. Finally, the total compartment increases after reversal of the intracellular Na to extracellular Na ([Na]i/[Na]o) gradient with sarcolemmal content-to-total content ratio dependent on the method used to reverse the [Na]i/[Na]o ratio. The results are consistent with localization of the Na/Ca exchange-dependent compartment to the subsarcolemmal region ("cleft") where JSR Ca is in equilibrium with anionic inner sarcolemmal leaflet Ca binding sites.

Animals↗
PubMed1994

The pathogenesis of free radical-induced calcium leak in cultured rat cardiomyocytes.

Elevated levels of intracellular calcium are generally accepted to be of critical importance in determining the outcome of cardiac myocytes exposed to ischemia and reperfusion. The mechanisms involved are obscure, but a favorite candidate is the Na-Ca exchanger operating in reversed mode, permitting calcium influx. Using exogenous free radicals and a unique, on-line, isotopic technique to measure calcium fluxes in cultured neonatal rat cardiomyocytes, we show that Na-Ca exchange is not the primary mechanism of calcium overload in this cell type during free radical exposure. We also demonstrate that neither L-type calcium channels nor general sarcolemmal defects are responsible. A specific calcium leak is present at a time when the sarcolemma remains intact with respect to its potassium permeability. The leak shares many properties with specific calcium leak channels demonstrated electrophysiologically in other tissues. It can be modulated by alterations in surface charge such that cationic amphiphiles inhibit the leak and anionic amphiphiles augment it. It is concluded that the calcium leak, in this cell type, is specific to calcium and that leak channels may be responsible.

Animals↗
PubMed1993

Role of calcium current and sarcoplasmic reticulum calcium release in control of myocardial contraction in rat and rabbit myocytes.

The relative importance of calcium current and sarcoplasmic reticulum in supplying contraction-related calcium was investigated in isolated rat and rabbit cardiac ventricular myocytes using patch-clamp and video techniques. Calcium current and contractile response were manipulated via variation of [Ca2+]o between 0.25 and 8.0 mM. Sarcolemmal calcium influx was derived from integration of the calcium current. An increase in [Ca2+]o from 0.25 to 8 mM augmented the amplitude of contractile shortening from 7 to 375% of control (amplitude at 1.0 mM) in rat but only from 36.5 to 177.6% in rabbit. Upon variation in [Ca2+]o from 0.5 to 8.0 mM calcium current increased from 67 to 175% of control in rat and from 65 to 150% in rabbit. Sarcolemmal calcium influx in rat was, however, only about half of that in rabbit for the entire range of [Ca2+]o and saturated at [Ca2+]o over 2.0 mM in both species. Application of 1 microM ryanodine increased calcium current and slowed its inactivation time course in the rat, but in the rabbit a slight decrease in current was observed and the inactivation time course was not obviously affected. The sarcoplasmic reticulum calcium release:transsarcolemmal calcium flux ratio is at least four-fold greater in the rat than in the rabbit. The results from this study further explain the well-known marked differences in contractile control in the two species.

Animals↗
PubMed1993

A discrete Na+/Ca2+ exchange dependent, Ca2+ compartment in cultured neonatal rat heart cells. Characteristics, localization and possible physiological function.

A pool of calcium has been identified in cultured neonatal rat heart cells whose exchange is dependent on extracellular sodium and calcium. In the absence of extracellular sodium and calcium this pool retains at least 760 mumol calcium/kg dry weight, which is released upon the introduction of sodium and calcium, with a t1/2 of 4.4 s. Video microscopy experiments of non-stimulated cells showed that the cells, despite the retention of 760 mumoles calcium/kg dry weight, do not develop contracture, indicating that the majority of the pool is not retained in the general cytoplasm. Neither is the pool displaced by lanthanum. Several probes for the sarcoplasmic reticulum were used. Ryanodine (1 microM) had no significant effect on the size of the pool. Caffeine reduced the size of the pool by 60% and thapsigargin, an inhibitor of the sarcoplasmic reticulum Ca-ATPase reduced the content of the pool by 70%. High concentrations of ryanodine (1 mM) reduced the pool even further. The experimental data, in association with recently developed concepts of the diadic region [1], indicate that the pool may reside in the sub-sarcolemmal space between the sarcoplasmic reticulum and the sub-sarcolemma. The physiological significance of this pool is discussed.

Animals↗
PubMed1993

Modulation of voltage-dependent sodium and potassium currents by charged amphiphiles in cardiac ventricular myocytes. Effects via modification of surface potential.

Modulation of voltage-dependent sodium and potassium currents by charged amphiphiles was investigated in cardiac ventricular myocytes using the patch-clamp technique. Negatively charged sodium dodecylsulfate (SDS) increased amplitude of INa, whereas positively charged dodecyltrimethylammonium (DDTMA) decreased INa. Furthermore, SDS shifted the steady-state activation and inactivation of INa in the negative direction, whereas DDTMA shifted the curves in the opposite direction. These shifts provided an explanation for the changes in current amplitude. Activation and inactivation kinetics of INa were accelerated by SDS but slowed by DDTMA. These changes in both steady-state gating and kinetics of INa are consistent with a decrease of the intramembrane field by SDS and an increase of the field by DDTMA due to an alteration of surface potential after their insertion into the outer monolayer of the sarcolemma. The effect of SDS on the steady-state inactivation of INa was concentration dependent and partially reversed by screening surface charges with increased extracellular [Ca2+]. These amphiphiles also altered the activation of the delayed rectifier K+ current (IK,del), producing a shift in the negative direction by SDS but in the positive direction by DDTMA. These results suggest that the insertion of charged amphiphiles into the cell membrane alters the behavior of voltage-dependent INa and IK,del by changing the surface charge density, and consequently the surface potential and implies, although indirectly, that the lipid surface charges are important to the voltage-dependent gating of these channels.

Animals↗
PubMed1993

Further characterization of the Na-Ca exchange-dependent Ca compartment in rat ventricular cells.

A discrete subcellular Ca compartment, absolutely dependent upon Na-Ca exchange for its exchange, has been previously identified in ventricular cells from both adult rat and from neonatal rat cultures. The present study further characterizes this compartment in the adult ventricular myocyte. The compartment's exchange is significantly decreased by 20 microM 3,4-dichlorobenzamil. Its content responds significantly to variation of the extracellular Ca concentration ([Ca]o) and to reversal of the Na-Ca exchanger by reduction of extracellular Na concentration to zero. Its content decreases to 155 +/- 24 mumol/kg dry cell wt in 0.2 mM [Ca]o and increases to 623 +/- 48 mumol/kg at 6.5 mM [Ca]o, where it appears to be near saturation. The compartment is significantly reduced by 45Ca labeling in the presence of 10 mM caffeine (23%) and 1 mM ryanodine (22%). This compartment is not significantly affected by 1 microM thapsigargin. 45Ca activity is retained in the compartment when 45Ca washout is conducted without Na or Ca in the washout solution, i.e., Na-Ca exchange is not operative. Under these conditions, addition of 1 mM Cd to the washout produces a significant release of 45Ca from the compartment. The results are interpreted to indicate that the compartment in the adult cell has two components that are in equilibrium: a relatively small sarcoplasmic reticulum (SR) component and a larger "non-SR" component. This is in contrast to the compartment in the cultured cell in which the compartment is 2.6 times larger and has a larger SR component. The possibility that the non-SR component represents Ca bound to the cytoplasmic leaflet of the sarcolemma at SR junction regions is discussed.

Animals↗
PubMed1993

Sarcolemmal phospholipid asymmetry and Ca fluxes on metabolic inhibition of neonatal rat heart cells.

The present study examines the hypothesis that during depletion of high-energy phosphates a change will occur in the phospholipid topology and in Ca fluxes in cultured neonatal cells and that these two events may be causally related. A combination of 2-deoxyglucose and iodoacetic acid was used to produce graded changes in the adenine nucleotides in the cells. An on-line technique for 45Ca measurement was used to follow Ca uptake and compartmentation by the cells, and chemical and enzymatic probes were used to study sarcolemmal phospholipid topology. After 15 min of metabolic inhibition (ATP = 10% of control) an increase in cellular Ca occurs, which progresses with time. Over 70% of this Ca accumulates in the mitochondria. After 30 min of metabolic inhibition (ATP < 10% of control) a change in the phospholipid topology is observed, and an increased amount (two times control) of sarcolemmal phosphatidylethanolamine is present in the outer monolayer of the sarcolemma. This change in phospholipid topology was independent of the extracellular Ca concentration. The sequence of altered Ca fluxes and distribution followed by the altered phospholipid topology is discussed in terms of its possible role in the pathogenesis of sarcolemmal disruption.

Animals↗
PubMed1993

Cationic amphiphiles prevent calcium leak induced by ATP depletion in myocardial cells.

Excessive calcium influx is important in the irreversible injury of cardiac myocytes and other cell types. The mechanism is unknown, but possibilities include L-type channels, Na(+)-Ca2+ exchange, sarcolemmal (SL) defects, and calcium leak channels. In this study, metabolic inhibition was used to induce ATP depletion and augmented calcium influx in cultured cardiac myocytes. Inhibition of the L-type calcium channel and Na(+)-Ca2+ exchanger had no significant effect on the calcium leak. There was no significant lactate dehydrogenase release, indicating that the leak did not occur through major SL defects. No alterations in the asymmetric distribution of SL phospholipids were demonstrated. Phospholipid rearrangements were therefore not responsible. The leak was unaffected by 0.5 mM cadmium and 1 microM nifedipine but was augmented by 50 microM nifedipine, characteristics in common with calcium leak channels. Insertion of the cationic amphiphiles dodecyltrimethylammonium bromide or polymyxin B sulfate into the SL had a profound inhibitory effect on the calcium leak. The anionic amphiphile sodium dodecyl sulfate had the opposite effect, and the neutral amphiphile lauryl acetate had no effect. These results suggest that an alteration in the SL surface charge affects calcium leak. It is proposed that the augmented calcium influx occurs via calcium leak channels and that these can be modulated by charged amphiphiles.

Adenosine Triphosphate↗
PubMed1993

Protamine and other polycationic drugs inhibit calcium leak in cardiac cells during metabolic inhibition and free radical exposure.

We studied the effect of the polycationic compounds protamine, polybrene and gentamicin on the calcium leak induced by metabolic inhibition and free radical exposure in cultured neonatal rat cardiac cells. All of the drugs tested inhibited the calcium leak. The potency of the compound in this respect was related to the magnitude of the positive charge borne by the molecule. Protamine was the most potent drug tested. None of the drugs tested had more than a small effect on lactate dehydrogenase release. The physicochemical properties of these compounds lead us to predict that they are acting at negatively charged sites on the outer surface of the sarcolemma. We conclude that polycationic compounds, exemplified by protamine, are able to inhibit calcium overload. It is anticipated that this group of compounds may be effective as cardioprotective agents.

Animals↗
PubMed1992

Calcium and the heart: exchange at the tissue, cell, and organelle levels.

Cellular calcium (Ca) exchange in arterially perfused whole heart is markedly perfusion-limited. Therefore, a large fraction of exchangeable Ca recycles within the cell before exchanging with extracellular Ca. Ca enters the cell via sarcolemmal (SL) transient (T) and long-lasting (L) channels activated at more negative and less negative membrane potential, respectively. The larger L currents are controlled via phosphorylation and G protein interaction to provide Ca to induce Ca release from sarcoplasmic reticulum (SR) and for direct myofilament activation. Troponin C (TNC) binds 3 mol of Ca/mol. Only the low affinity site is responsible for activation of force and regulation of myofilament ATPase rate. This occurs through a shift in troponin I (TNI) with respect to actin induced by the TNC Ca binding. Relaxation depends on reduction of cytosolic (Ca) and occurs via 1) Ca pumping into the longitudinal SR modulated by phospholamban; 2) the recently cloned high-capacity electrogenic SL Na-Ca exchanger; and 3) the SL Ca pump under complex regulation including calmodulin control. Mitochondria transport Ca, but this transport is directed primarily to the regulation of various Ca-sensitive dehydrogenases so that oxidative metabolism can be adjusted to changes in energy demand. The regulation of Ca movements consumes about 25% of the cell's total energy output. Mitochondrial Ca exchanges with extracellular Ca most slowly (t 1/2 = 3.6 min), SR Ca quite rapidly (t 1/2's = 3 and 19 s), and an Na-Ca exchange-dependent compartment very rapidly (t 1/2 = 500 ms). After further description of Ca handling by the individual organelles, Ca movement is followed through the cell during the course of contraction, and the contribution of each organelle or compartment to overall cellular Ca exchange is defined.

Actin Cytoskeleton↗
PubMed1992

Sarcolemmal calcium binding sites in heart: I. Molecular origin in "gas-dissected" sarcolemma.

Calcium in the myocardial cell is highly compartmentalized and a fast, an intermediate, a slow and a nonexchangeable calcium pool have been described. The fast pool contains 66% of the total cell exchangeable calcium in cultured neonatal rat heart cells with a t1/2 of less than 1.5 sec. Though the cellular origin of this fast pool is unknown, its rapidity and its displacement by La3+ most likely places it at the sarcolemma or at least in rapid equilibrium with the sarcolemma. We isolated the sarcolemma of cultured neonatal rat heart cells using the gas-dissection technique, which yields a pure sarcolemmal preparation in less than a second, thereby precluding membrane changes which might occur during conventional plasma membrane isolation. We determined the calcium binding characteristics of these membranes, using an on-line technique to monitor 45Ca, which allows measurement of 45Ca binding characteristics in the presence of unbound 45Ca. Two classes of calcium binding sites were determined: (i) Kd of 13 microM, capacity 7 nmol/mg and (ii) Kd of 1.1 mM, capacity of 84 nmol/mg. To assess the molecular origin of the sarcolemmal calcium binding we treated the membranes with a variety of enzymes. Protease or neuraminidase treatment did not cause large changes in these parameters. Simultaneous treatment with two different phospholipases C or the extraction of the lipids with isopropanol resulted in a dramatic loss of the low-affinity binding sites. These results, in association with previously defined sarcolemmal phospholipid distribution, places the low-affinity binding sites at the cytoplasmic leaflet. The physiological implication of this localization as it pertains to cellular calcium exchange is discussed.

1-Propanol↗
PubMed1992

Sarcolemmal calcium binding sites in heart: II. Mathematical model for diffusion of calcium released from the sarcoplasmic reticulum into the diadic region.

We present a model for predicting the temporal and spatial dependence of [Ca] in the cardiac subsarcolemmal diadic region (cleft), following Ca release from the "feet" of the sarcoplasmic reticulum. This region is modeled as a disc 10 nm thick, 430 nm in radius, with or without Ca binding sites and open at its periphery to the cytosol. [Ca] is computed for three diffusion coefficients (100, 20 and 4% of aqueous diffusion), following release of a 20-msec square pulse sufficient to produce 50% maximal contractile force, or repetitive release (400/min) of such pulses. Numerical solutions are obtained for the general diffusion/binding problem and analytic solutions for the case of no binding sites. For the middle value of diffusion coefficient, and in the absence of binding sites, [Ca] rises to approximately 1.5 mM in 20-msec and then falls to approximately 0.1 microM in less than 3 msec. Adding binding sites reduces peak [Ca] to approximately 0.6 mM but prolongs its decline, requiring approximately 200 msec to reach 20 microM. For repetitive release [Ca] is greater than 100 microM for roughly half of each cycle. Two major implications of the predicted [Ca] are: (i) The effect of Ca binding sites on [Ca] will cause Ca efflux from the cleft via the Na-Ca exchanger (Km(Ca) approximately 20 microM) to continue at a significant level for greater than 200 msec. (ii) The time constant for inactivation of release from the "feet" must be much greater than for activation if Ca-induced Ca release is to continue for greater than 1-2 msec.

Animals↗
PubMed1992

Cellular origin of the rapidly exchangeable calcium pool in the cultured neonatal rat heart cell.

Calcium in the myocardial cell is highly compartmentalized and in the cultured neonatal rat heart cells over 66% of the exchangeable calcium exchanges extremely fast (t1/2 < 1 s). The goal of the present study was to investigate, in the intact cell, the locus of this pool. By comparing myoblasts and fibroblasts and their respective plasma membranes, it is concluded that in the intact myocyte a significant fraction of the large lanthanum displaceable calcium pool is attributable to intracellular components, not present in the fibroblast. At least 30% of the lanthanum displaceable pool resides intracellularly, as is shown with the use of the drugs nifedipine, ryanodine and thapsigargin. It is proposed that the diadic subsarcolemmal junctional region represents a significant locus for the pool.

Animals↗
PubMed1992

A discrete Na-Ca exchange-dependent Ca compartment in rat ventricular cells: exchange and localization.

Application of a new rapid perfusion (up to 4.8 ml/s) technique to 45Ca-labeled ventricular cells from adult rat heart has defined a discrete intracellular calcium (Ca) compartment with the following characteristics: 1) its exchange is absolutely dependent on operation of the Na-Ca exchanger, i.e., its isotopic content remains constant during washout in the absence of Na and Ca and is released only upon addition of Na and Ca to the perfusate. 2) At an extracellular Ca concentration of 1.0 mM it contains 350 mumol/kg dry wt cells and exchanges with half time of 650 ms. Ca flux from the compartment is 385 mumol.kg dry wt-1.s-1 or 20% of the total nonperfusion limited flux from the cells. 3) Its content is decreased 19% by 10 mM caffeine but not diminished by exposure of the cells to 10(-6) M ryanodine and not accessible to lanthanum (La) displacement. 4) Only limited exchange occurs when only Na or Ca is present and exchange is virtually eliminated by substitution of extracellular Li for extracellular Na. 5) Replacement of Na and Ca to the perfusate after various periods of removal produces no contraction (despite immediate Ca release from the cell). The results define a discrete intracellular Ca compartment which exchanges only via the Na-Ca exchanger. It is not La accessible, not in the ryanodine- or caffeine-sensitive portions of the sarcoplasmic reticulum, not in the mitochondria nor at the myofilaments, but may reside at inner sarcolemmal leaflet binding sites.

Animals↗
PubMed1991

Effects of neuraminidase on cellular calcium and contraction in cultured cardiac myocytes.

Mammalian plasma membranes, including the myocardial sarcolemma, are abundantly glycosylated. Sialic acid is a ubiquitous anionic sugar found at the periphery of sarcolemmal glycoconjugates. The physiological role of this sugar is not clear, but neuraminidase, which specifically hydrolyzes sialic acid from the sarcolemma, has been found to increase calcium exchange, cause electrophysiological abnormalities, and enhance the transient (T) calcium current in cardiac myocytes. The purpose of this study was to better characterize the effect of neuraminidase on cellular calcium (Ca) and contractile function. Neuraminidase removed up to 57% of total sialic acid from the cells. 45Ca exchange was measured and neuraminidase was found to increase cell calcium proportional to the amount of sialic acid removed (18.6 +/- 0.8 mmol/kg dry w, maximally). Over 80% of the increment in calcium remained rapidly exchangeable (t1/2 less than 15 s) under non-perfusion limited conditions and was inhibited by cations (La greater than Cd greater than Mn greater than Mg) and nifedipine. Using a video-monitoring system, neuraminidase was observed to transiently increase cell shortening during contraction (30 +/- 9%), with progression to arrhythmias followed by cessation of contraction. These results indicate that neuraminidase, probably by removing sarcolemmal sialic acid residues, greatly augments cellular calcium in cultured cardiac myocytes. Most of the increment in Ca induced by neuraminidase was very rapidly exchangeable and most likely mediated by a Ca specific mechanism. Additionally, neuraminidase treatment altered contractile function in a manner consistent with elevated cellular Ca. Despite the many-fold increase in cellular Ca induced by sialic acid removal, cells recovered and demonstrated rhythmic contractions upon return to control incubation conditions.

Animals↗
PubMed1991

Effects of charged amphiphiles on cardiac cell contractility are mediated via effects on Ca2+ current.

Exposure of isolated adult rabbit myocytes to the negatively charged amphiphile dodecylsulfate (DDS; 10 microM) increased the contraction amplitude to 185% of control. The positively charged amphiphile dodecyltrimethylammonium (DDTMA; 10 microM) decreased the amplitude to 58%. DDS increased Ca2+ uptake by the same cells, but this uptake was partially prevented by nifedipine. DDTMA had no effect on Ca2+ uptake. Ca2+ binding to isolated sarcolemma of neonatal heart cells was increased by 10 microM DDS and, at higher concentrations, reduced by DDTMA. Single-cell voltage-clamp studies, using isolated rabbit myocytes, showed that DDS enhanced L-type Ca2+ currents (ICa,L), whereas DDTMA depressed ICa,L. DDS shifted current-voltage (I-V) and isochronal inactivation curves of ICa,L in the negative direction, whereas DDTMA shifted them in positive direction. Furthermore, DDS depressed T-type Ca2+ currents (ICa,T), and DDTMA enhanced ICa,T. The inotropic effects of the amphiphiles are therefore mediated to a significant degree by ICa,L. The shifts in the I-V and inactivation curves of ICa,L and the effect on ICa,T can be explained by changes in the actual membrane potential (Em), induced by the insertion of the amphiphiles in the outer monolayer of the sarcolemma. However, the changes in the Em do not explain the effect on the maximal current, indicating effects on the channel per se, possibly by an alteration of the lipid environment.

Action Potentials↗