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Biomedical subjects

G A Langer

Publications and source records attributed to G A Langer.

At least 91 records · Page 5Linked to original sources
PubMed1983

Calcium compartmentalization in cultured and adult myocardium: activation of a caffeine-sensitive component.

Calcium (Ca) exchange was studied under various perfusion conditions in monolayer myocardial culture and in the interventricular septum of the rabbit. In cultured cells perfused in HEPES buffered medium or in 10 mM phosphate (Pi), pH less than 7.2, 10 mM caffeine produced no change in 45Ca uptake rate. By contrast, increase of pH to 7.35 in the presence of 10 mM Pi caused 45Ca uptake rate to increase by more than two-fold when caffeine was added. Control 45Ca uptake (prior to caffeine) was markedly increased in 10 mM Pi, pH = 7.35 as compared to the two other perfusion conditions in the cultured cells. The same sequence of response of 47Ca uptake rate to caffeine was found in the rabbit septum, i.e. no increased uptake under HEPES or Pi, pH less than 7.2 perfusion, but significant increase under 10 mM Pi, pH 7.35 with development of progressive contracture only in the last case. Two other conditions produced sensitivity (both in 47Ca uptake and contracture) to caffeine in the septum. Preperfusion with ouabain in HEPES buffer increased caffeine sensitivity proportional to ouabain concentration (5 X 10(-7) to 10(-5) M) as did preperfusion with vanadate at low concentration (1 to 3 X 10(-6) M). The results suggest that activation of Ca uptake by the sarcoplasmic reticulum (SR) is dependent upon a threshold of cellular Ca and that a stable contractile state is possible in the absence of SR activation in both cultured cells and adult ventricular tissue.

Animals↗
PubMed1983

Control of calcium movement in the myocardium.

A model for calcium (Ca) movement in the course of excitation-contraction of mammalian myocardium is presented. At physiological levels of [Ca]o between 500-700 mumol Ca/kg wet tissue is bound within the sarcolemmal-glycocalyx--most at anionic phospholipid sites. This Ca feeds two systems responsible for influx--the channel under control of protein phosphorylation and the Na-Ca exchanger regulated by the level of [Na]i. Respiring mitochondria are activated for Ca exchange in the presence of a proton donor (e.g. phosphate, bicarbonate). The sarcoplasmic reticulum (SR) may not be activated at relatively low levels of Ca influx but is activated as the 'Ca load' within the cell rises. Basic control of resting membrane Ca permeability resides at two sites--within the glycocalyx where sialic acid plays a role and within the lipid bilayer where phospholipid disruption leads to increased permeability.

Animals↗
PubMed1983

Effects of cations, phospholipases, and neuraminidase on calcium binding to "gas-dissected" membranes from cultured cardiac cells.

Sarcolemmal membranes prepared by "gas dissection" from monolayers of cultured neonatal rat heart cells were studied with respect to their ability to bind calcium. Lanthanum displacement of calcium was 168 +/- 7 nmol/mg sarcolammel protein. This represents 3.21 mmol Ca/kg dry weight original cells on the basis of the measured membrane protein: dry cell weight ratio of 19.1 g/kg. Lanthanum-displaceable calcium from whole cells was essentially equal (3.32 mmol/kg dry weight), which indicates that all calcium displaceable from whole cells by lanthanum is localized to sarcolemmal sites. The potency of a series of divalent cations for calcium displacement from the sarcolemma was according to similarity of their crystal radii to that of calcium (cadmium greater than manganese greater than magnesium). This order was the same for the cations' ability to displace calcium from whole cells and for their ability to uncouple excitation from contraction in neonatal papillary muscle. The membranes were treated with four enzymes: phospholipase A2, phospholipase C, phopholipase D, and neuraminidase. Phospholipase A2 and phospholipase D produced significantly increased calcium-binding. The increased binding secondary to phospholipase A2 treatment was eliminated by an albumin wash which was indicative of binding to the fatty acid product of hydrolysis. The increase after phospholipase D treatment can be attributed to an increase in phosphatidate, with attendant increase in net anionic charge on the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Influence of polymyxin B, a probe for anionic phospholipids, on calcium binding and calcium and potassium fluxes of cultured cardiac cells.

Polymyxin B, an amphiphilic, cationic peptidolipid, which is thought to bind to anionic phospholipids in cell membranes, is shown to interact with the cellular calcium of cultured neonatal rat myocardial cells in a dose-dependent, partially reversible manner. At concentrations of less than or equal to 0.1 mM, it has two distinct effects. First, it results in displacement of 1.4 +/- 0.3 mmol Ca/kg dry weight, which is equivalent to 18.1 +/- 3.4% of the total exchangeable cellular calcium. Total calcium displaced by polymyxin B and a nonspecific cationic probe, lanthanum, at its maximal displacing concentration (1 mM), was 5.9 +/- 1.3 mmol/kg dry weight. Thus, the total displaceable calcium represented 76.3 +/- 2.5% of the total exchangeable calcium. Second, polymyxin B (less than or equal to 0.1 mM) causes a reduction in net uptake of calcium, and slows the efflux of both calcium and potassium. Concentrations of polymyxin B higher than 0.1 mM result in an initial displacement of calcium, followed by an irreversible and sustained period of enhanced net calcium uptake. Efflux of calcium is slowed at the higher polymyxin B concentrations, whereas efflux of potassium is enhanced. Cellular contractile activity and electrical activity are irreversibly altered only by the higher concentrations. The results suggest that polymyxin B is a useful probe for the role of membrane phospholipids in control of ion binding and fluxes.

Animals↗
PubMed1983

Activation-dependent cumulative depletions of extracellular free calcium in guinea pig atrium measured with antipyrylazo III and tetramethylmurexide.

We have used a spectrophotometric method to monitor mean free extracellular calcium concentrations in isolated left atria of guinea pigs via extracellular application of the calcium-sensitive absorption dyes, antipyrylazo III and tetramethylmurexide. Exchange of extracellular free calcium with the bathing medium takes several minutes and closely parallels contractile response in this preparation. Under conditions favoring a rapid positive force staircase response during repetitive stimulation after a long rest period (2-10 minutes), cumulative depletions of extracellular calcium can clearly be differentiated from motion artifact due to muscle movement by multiple-wavelength spectrophotometry. Responses of similar magnitude and characteristics are obtained with both dyes employed. In the presence of 10(-7) M isoproterenol, the mean extracellular calcium concentration falls by at least 5% (0.25-0.8 mM total calcium concentration) in four beats at 0.5 Hz; extracellular calcium replenishes during rest with an apparent t1/2 of 25-60 seconds. A 10-minute pretreatment with 10(-8) M ryanodine greatly reduces the contraction force and motion artifact of the first beat after a rest period, whereby the magnitude of depletion response to one post-rest stimulation is increased 2- to 3-fold. With further ryanodine treatment, the magnitude of depletion responses remains stable, and the rate of calcium replenishment during rest increases many-fold. After ryanodine treatment and 10(-7) M isoproterenol, at least 10% of total dye accessible calcium (0.25-1.0 mM) is lost during two to five rapid stimulations, and returns to the extracellular space within 20 seconds of rest. Cumulative extracellular calcium depletion responses are strongly suppressed by 10(-6) M nifedipine. Cumulative depletion responses are also inhibited by 10 mM caffeine, whereby contraction and corresponding motion artifacts are increased at post-rest stimulation.

Animals↗
PubMed1982

Binding and movement of calcium in the myocardial cell.

This report presents a summary of work done at UCLA over the past 10 years on calcium (Ca) exchange in the myocardium. Four major aspects are considered: binding, permeability, compartmentalization, and transport. Calcium bound within the sarcolemmal-glycocalyx complex plays a significant role in the control of contractile force either in supplying Ca directly to the myofilaments or by serving as a trigger to modulate release from the sarcotubular system. Approximately 80% of bound Ca is associated with sarcolemmal phospholipid. Calcium permeability is controlled at a minimum of two sites--at the glycocalyx and at the lipid bilayer. Removal of sialic acid in cultured cells markedly increases permeability without evidence for disruption of bilayer structure. If, however, the phosphoryl group is cleaved from bilayer phospholipid with phospholipase C, the permeability is also increased. Again, in cultured cells, there is evidence of marked Ca compartmentalization. Surface-bound Ca can participate in excitation-contraction coupling without, in effect, mixing or exchanging with intracellular organelles. If, however, inorganic phosphate (Pi) is added to the perfusion solution, intracellular Ca2+ exchange is markedly augmented. A component of the intracellular Ca2+ is directed to the mitochondria and can be blocked by inhibitors of mitochondrial respiration. Finally, it is clear that Na+ -Ca2+ exchange plays a role in transsarcolemmal Ca2+ transport, with the level of intracellular sodium [Na]i being a most important factor in control of carrier movement under physiological conditions. Elevations of [Na]i will operate to depress Ca2+ efflux and augment Ca2+ influx. Therefore, interventions such as increased frequency or digitalis administration that lead to elevation of [Na]i will be associated with augmented Ca2+ uptake by the myocardium.

Animals↗
PubMed1982

Effects of inorganic phosphate on ion exchange, energy state, and contraction in mammalian heart.

Effects of inorganic phosphate (Pi) on contractile function, ionic exchange, and cellular energetic state were investigated in isolated, arterially perfused, interventricular rabbit septa. The addition of 10 or 20 mM Pi to a N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffered perfusate showed 1) a decrease in developed tension due to a decrease in free Ca concentration; 2) an increase in diastolic tension; 3) a decrease in 42K uptake at the same time that 42K efflux was depressed; 4) an increase of 24Na activity; 5) an increase in tissue 47Ca activity; and 6) a decrease in tissue adenosine 5'-triphosphate (ATP) levels. If a 10-mM Pi intervention was applied without changing the free Ca concentration of the perfusate, the mechanical record showed a transient positive inotropic effect without changes in K efflux. These results are consistent with Pi-induced stimulated mitochondrial Ca uptake and Na-K pump inhibition that combine to increase cellular Ca uptake. The increase in cellular Ca and the decrease in cellular ATP would both tend to increase diastolic tension. In the presence of antimycin A, respiration-dependent Ca uptake is inhibited, and mitochondrial Ca uptake is then maintained at the expense of cellular ATP. This process of mitochondrial Ca uptake could account for the deficiency of ATP and the rigor type contracture that occurs with antimycin A. This energy-deficient state is modified if mitochondrial ATP consumption is blocked by oligomycin, and such treatment prevents the ATP-deficient rigor. Under these conditions, although mitochondrial Ca uptake is blocked by the presence of antimycin A and oligomycin, Ca uptake remains augmented secondary to Na-K pump inhibition by Pi.

Adenosine Triphosphate↗
PubMed1982

On-line calorimetry in the arterially perfused rabbit interventricular septum.

A method has been developed to measure myocardial heat production simultaneously with mechanical (developed tension, rate of contraction and relaxation) and metabolic parameters in the arterially perfused interventricular septum of the rabbit. The system allows control of rest tension, frequency of contraction, temperature, and composition of the perfusate. The technique is based on the differential measurement of the heat flux from the muscle to the calorimetric bath. This technique is able to resolve changes in heat production as small as 0.06 mW. The resting heat production measured with the present calorimeter (1.62 +/- 0.1 mW/g wet tissue) agrees with that obtained with thermopiles and with that calculated from measurements of oxygen consumption. The heat per contraction (6.5 +/- 1.4 mJ/g wet tissue) also agreed with that measured with thermopiles in rabbit papillary muscles. The heat production measured at 22 degrees C under severe hypoxia can be fully explained by the addition of the expected change of enthalpy due to the reaction 0.5 glucose-lactate, calculated on the basis of the lactate measured in the perfusate, and the expected change of enthalpy of oxygen consumption [assuming that all remaining O2 in perfusate (0.09 vol%) is used for combustion of glucose]. These results clearly demonstrated the feasibility of this method for the correlation of changes in energy turnover, through the measurement of the heat production, with mechanical and metabolic processes on-line in arterially perfused septum.

Animals↗
PubMed1982

Calcium depletion in rabbit myocardium. Calcium paradox protection by hypothermia and cation substitution.

The purpose of this study was to define further the basis of control of myocardial membrane permeability by further examination of the "calcium paradox." To this end, the protective effect of hypothermia and addition of micromolar amounts of divalent cations during the Ca-free perfusion period were studied. Damage during Ca++ repletion to the isolated arterially perfused, interventricular rabbit septum was assessed by contracture development, loss of developed tension, and loss of 42K and creatine kinase. Progressive hypothermia prolongs the time of Ca-free perfusion needed to cause similar 42K, creatine kinase and developed tension losses upon Ca++ repletion. Complete protection against the Ca-paradox after 30-60 minutes Ca-free perfusion is seen at 18 degree C. The inclusion of 50 microM Ca++ during 30 minutes "Ca-free" perfusion also provides complete protection during Ca++ repletion i.e., there was full mechanical recovery with no 42K or creatine kinase loss. Other divalent cations perfused in 50 microM concentrations during the Ca-free period exhibited variable ability to protect when Ca++ was reperfused. The order of effectiveness (Ca++ greater than Cd++ greater than Mn++ greater than Co++ greater than Mg++) was related to the crystal ionic radius, with those cations whose radii are closest to that of Ca++ (0.99 A) exerting the greatest protective effect. The cation sequence for effectiveness in Ca-paradox protection is the same sequence for potency of excitation-contraction uncoupling. The mechanism of hypothermic protection is likely a phase transition in the membrane lipids (from a more liquid to a less liquid state) which stabilizes membrane structure and preserves Ca++ permeability characteristics during the Ca-free period. The mechanism of protection via cation addition is perhaps a cation's ability to substitute for Ca++ (dependent on unhydrated crystal ionic radius) at critical sarcolemmal binding sites to preserve control of Ca++ permability during the Ca-free period.

Animals↗
PubMed1982

Ca++ distribution after Na+ pump inhibition in cultured neonatal rat myocardial cells.

The influence of inhibition of the Na+ pump, with secondary stimulation of Na+-Ca++ exchange, on cellular Ca++ distribution is examined using the on-line scintillation disk technique and cultured neonatal rat myocardial cells. Under control conditions, La+++ displaced 78.1 +/- 1.13% (SEM) of the total cell associated 45Ca. Application of 1 mm ouabain or reduction of [K+]o to 0.5 mM resulted in a net increase of 10.6 +/- 1.3% and 13.8 +/- 2%, respectively, in total cell-associated Ca++. Of this added 45Ca, 75.9 +/- 2.7% and 78.4 +/- 2.1%, respectively remained La+++-displaceable. The 45Ca-binding characteristics of isolated sarcolemma, prepared from the cultured neonatal rat myocardial cells using the gas dissection technique, were examined. When treated with either ouabain or low [K+]o solutions, sarcolemmal 45Ca binding did not change. This result indicates that functional, intact tissue is necessary to observe the Ca++ increase. Treatment of the cells with verapamil before and during ouabain exposure failed to inhibit the ouabain-induced increase in cell-associated 45Ca. The evidence indicates that inhibition of the Na+-pump, and secondary stimulation of Na+-Ca++ exchange, result in a net increase of 11-15% in cell-associated Ca++, 78% of which remains La+++-displaceable and is, therefore, localized to the sarcolemma-glycocalyx complex.

Adenosine Triphosphatases↗
PubMed1981

Sodium efflux in rabbit myocardium: relationship to sodium-calcium exchange.

1. The washout of (22)Na, (45)Ca and (58)CoEDTA(-) has been observed in arterially perfused rabbit interventricular septa both in the presence and absence of intoxicating doses of the aglycone, 3 acetylstrophanthidin (ACS).2. The washout of (22)Na from paced septa (21.0/min) was rapid with approximately 99% of the exchange occurring in 30 min.3. Septa labelled with (22)Na and washed out in the presence of 10(-5)m-ACS were quiescent, exhibited marked contracture and a slowed (22)Na exchange with 99% of the initial (22)Na leaving the tissue in approximately 200 min.4. Comparison of the washout of the extracellular marker (58)CoEDTA(-) from paced and intoxicated septa revealed no differences, suggesting that regions of the extracellular space had not become inexchangeable as a result of the contracture that these septa exhibited.5. Simultaneous measurements of the distribution of (22)Na and (58)CoEDTA(-) in the presence of 10(-5)m-ACS revealed an apparent cellular Na concentration of 54.8+/-10.4 (s.e.). This was considerably in excess of the maximum amount of Na contributing to the slowest component of Na efflux from intoxicated septa. Origins of this discrepancy are considered.6. The slowest component of (22)Na efflux from intoxicated septa could be markedly stimulated by increasing the external Ca concentration from a nominal 1.5 to 16 mm. The magnitude of stimulation was 44.3+/-7.8% (s.e.). Increases in the concentration of Mg from 1.0 to 16.0 mm were without effect on the (22)Na efflux.7. Application of 10(-5)m-ACS produced marked depolarization of the membrane to new stable levels that lay between -10 and -20 mV.8. Washout of ACS after several hours of intoxication resulted in a partial decline in contracture tension, an acceleration of Ca efflux, little change in (22)Na efflux and a repolarization of the cell membrane.9. Some of the Na and Ca movements reported in this paper have been interpreted by supposing that there is, in this preparation, a coupled exchange of three Na ions for a single Ca ion.

Animals↗
PubMed1981

Cardiac contractility and sarcolemmal calcium binding in several cardiac muscle preparations.

Striking correlations are found between cardiac contractility and Ca2+ binding to isolated cardiac sarcolemma in rabbit, neonatal rat, and frog ventricular tissue. Deviations from this correlation are seen in the adult rat ventricle and rabbit atrium. The observation of this correlation in the three former tissues and under various ionic conditions suggests that this correlation is not coincidental and that Ca2+ bound to the cardiac sarcolemma is of major importance in the control of myocardial contractility. The data are consistent with a functional Ca2+-induced Ca2+ release system in the sarcoplasmic reticulum (SR) of all the tissues (which is controlled by Ca2+ entry from sarcolemmal sites), with the adult rat ventricular and rabbit atrial SR Ca2+ release being much more sensitive to CA2+. It is suggested that the frog, neonatal rat, and rabbit ventricles depend more directly on the entry of Ca2+ from sarcolemmal sites for the control of tension development, whereas the adult rat ventricle and rabbit atrium depend to a greater extent on CA2+ released from the SR.

Age Factors↗
PubMed1981

Correlation of alterations in cation exchange and sarcolemmal ultrastructure produced by neuraminidase and phospholipases in cardiac cell tissue culture.

Myoblasts and fibroblasts in cultured derived from neonatal rat hearts were exposed to neuraminidase and phospholipases C (PLC) and A2 (PLA2). Calcium (Ca) and potassium (K) exchange of the cells was measured before and after enzymatic exposure. The exchange characteristics of control and treated cells were correlated with cellular ultrastructure including assessment of intramembrane particle (IMP) density and aggregation by freeze-fracture. Neuraminidase exposure (removal of sialic acid) known to produce marked increase in calcium permeability without change in potassium permeability in myoblasts produced no change in IMP configuration of these cells. PLC, however, produced marked increase in both Ca and K permeabilities, permitted entry of La, and was associated with IMP aggregation in myoblastic cells. PLA2 produced no change in ionic permeability and no alteration in intramembrane particle configuration in myoblasts. Exposure of fibroblasts to PLC caused no change in either Ca or K permeability and no change in IMP distribution. These results, coupled with those of previous studies of permeability changes induced by sialic acid removal, indicate that control of cellular Ca permeability resides in at least two separate sites at the cellular surface: (1) the glycocalyx and (2) the lipid bilayer. By contrast, K permeability control is based within the bilayer. Ultrastructural correlations suggest that IMP aggregation may be associated with changes in bilayer permeability.

Animals↗