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R A Altschuld

Publications and source records attributed to R A Altschuld.

At least 55 records · Page 3Linked to original sources
PubMed1989

Energy depletion-repletion and calcium transients in single cardiomyocytes.

Rapid fluctuations of intracellular free calcium in single adult rat heart myocytes were monitored by time-resolved fura-2 fluorescence microscopy. Under controlled aerobic conditions (35 degrees C, pH 7.3), electrical stimulation at 0.5 Hz produced a concave negative staircase of calcium transients. When the myocytes were challenged with 3 mM amobarbital (Amytal) and 2 microM carbonyl cyanide m-chlorophenylhydrazone (CCCP) to deplete ATP, the cells became unresponsive to electrical stimulation within 1 min but responded to 10 mM caffeine with a large increase in free calcium. After the development of rigor contracture, the cellular response to caffeine was blunted. Free calcium increased at a variable rate in individual cells, reaching values of 300-1,000 nM after 15 min. When the inhibitors were removed, calcium declined toward control values, and spontaneous contractile activity and calcium transients were invariably observed. During subsequent electrical stimulation, there was a decrease in the half-widths of the calcium transients and an attenuation of the negative staircase. Parallel experiments with cells in suspension indicated that Amytal and CCCP caused ATP to fall from 27.6 +/- 1.6 to 0.7 +/- 0.2 nmol/mg protein, and the percent rod-shaped cells to fall from 70 to 0% in 5 min. Removal of the inhibitors after 15 min caused a rebound in ATP to 5.3 +/- 1.5 nmol/mg within 2 min and 6.6 +/- 1.3 nmol/mg after 10 min.

Adenosine Triphosphate↗
PubMed1989

Hyperthyroid adult rat cardiomyocytes. I. Nucleotide content, beta- and alpha-adrenoreceptors, and cAMP production.

Ventricular myocytes isolated from the hypertrophied hearts of thyrotoxic adult rats have an increase in mean protein content per myocyte (6.3 +/- 0.2 vs. 4.4 +/- 0.2 ng) compared with euthyroid cells. Viability and adenine nucleotide profiles are similar in both populations, but NAD content of the hyperthyroid myocytes is depressed (4.9 +/- 0.2 vs. 5.5 +/- 0.2 nmol/mg for controls) and UTP is higher (1.2 +/- 0.09 vs. 0.9 +/- 0.04 nmol/mg). Binding of (-)-[125I]iodocyanopindolol to intact hyperthyroid myocytes is increased by 42% compared with controls, with no change in the dissociation constant (Kd). This elevation in beta-receptor number is correlated to enhanced beta-agonist-induced adenosine 3',5'-cyclic monophosphate (cAMP) production. The half-maximal effective concentration (EC50) for the euthyroid isoproterenol dose-response curve is 2.14 x 10(-7) M but is decreased to 2.51 x 10(-8) M in hyperthyroid cardiac cells. Basal adenylate cyclase activity is apparently not affected by thyroid hormones, since basal cAMP levels for both groups are identical (5 pmol/mg) and both rise roughly twofold in the presence of a phosphodiesterase inhibitor. Forskolin-induced cAMP production and cAMP-specific phosphodiesterase activity are similar as well. In contrast to beta-adrenergic response, there are no significant differences in alpha 1-antagonist [3H]prazosin binding parameters between hyperthyroid and euthyroid cardiomyocytes.

1-Methyl-3-isobutylxanthine↗
PubMed1989

Hyperthyroid adult rat cardiomyocytes. II. Single cell electrophysiology and free calcium transients.

The effects of hyperthyroidism on electrophysiological properties and intracellular free calcium transients in single adult rat cardiomyocytes were studied using conventional microelectrodes and time-resolved single cell fura-2 fluorescence microscopy. Under control conditions, resting membrane potentials and triggered action potentials were not different in euthyroid and hyperthyroid myocytes. Calcium transients produced by electrical stimulation, however, were markedly abbreviated in hyperthyroid myocytes. During a train of stimuli, the duration of the calcium transients at half peak amplitude (half time) was 124 +/- 14 ms at the fifth beat in hyperthyroid cells vs. 287 +/- 35 ms in euthyroid cells. Isoproterenol (1 microM) prolonged time to 50% repolarization (APD50) of the action potentials and increased the peak calcium transients in both euthyroid and hyperthyroid myocytes. It also shortened the half time of the calcium transients in euthyroid myocytes but had little effect on the half time in hyperthyroid cells. These data are consistent with the electrophysiology and mechanical performance in intact euthyroid and hyperthyroid cardiac tissues, and the intrinsic changes in hyperthyroid tissues can therefore be illustrated in single ventricular myocytes. Furthermore, the results suggest that alterations in intracellular calcium handling by sarcoplasmic reticulum may account for contractile changes of the heart induced by hyperthyroidism.

Action Potentials↗
PubMed1989

Magnesium content of serum, circulating mononuclear cells, skeletal muscle, and myocardium in congestive heart failure.

Deranged magnesium concentrations in serum and cardiovascular structures have been implicated in the pathophysiology of hypertension, ischemic heart disease, arrhythmias, and sudden death. This study was conducted to determine the status and interrelation of serum and tissue concentrations of magnesium in patients with congestive heart failure, a clinical setting purportedly predisposed to the development of depleted levels of this cation. Magnesium concentrations of serum, circulating mononuclear cells, skeletal muscle, and myocardium were measured in 23 patients with heart failure on standard therapy. Two patients were hypomagnesemic (less than 1.6 meq/l). Poor or no correlations were found between serum and tissue magnesium concentrations and among the magnesium concentrations of the three tissues studied. Strong direct correlations were, however, noted between magnesium and potassium concentrations of the tissues examined. The prevalence of hypomagnesemia in this typical ambulatory heart failure population is relatively low (9%) and serum, circulating mononuclear cell, skeletal muscle, and myocardial magnesium concentrations correlate poorly with each other. Serum, circulating mononuclear cell, and skeletal muscle magnesium concentrations are thus of little predictive value in assessing the status of myocardial magnesium in humans with heart failure.

Adult↗
PubMed1989

IMP production by ATP-depleted adult rat heart cells. Effects of glycolysis and alpha 1-adrenergic stimulation.

A rapid deenergization procedure was used to probe the regulation of in situ adenylate deaminase and 5'-nucleotidase in isolated adult rat heart cells. In cells depleted of ATP, the rate of ionosine monophosphate (IMP) production was fourfold greater in cells that had been respiring prior to deenergization than in cells that had been maintaining ATP stores through anaerobic glycolysis. This effect of respiratory inhibition was fully reversed by reaeration. When phenylephrine was present during preincubation, IMP production during a subsequent 5-minute rapid deenergization was increased by 70% in respiring cells and by 88% in those that had not been respiring. These effects of phenylephrine were abolished by prazosin. Adenosine production by cells without ATP was inversely related to that of IMP, whereas it was positively correlated with the amount of AMP remaining in cells after 5 minutes. We conclude from these data that rat heart adenylate deaminase is regulated by a product(s) of anaerobic glycolysis and by alpha 1-adrenergic stimulation. The production of intracellular adenosine in cells without ATP, on the other hand, is governed primarily by the concentration of AMP and appears to be catalyzed by the cytosolic type I 5'-nucleotidase.

AMP Deaminase↗
PubMed1988

Extraction and measurement of myocardial nucleotides, nucleosides, and purine bases by high-performance liquid chromatography.

Nucleotides, nucleosides, and purine bases were extracted from human endomyocardial biopsies, freeze-clamped rat hearts, and porcine coronary sinus plasma. Perchloric acid extracts were neutralized with Freon-trioctylamine and analyzed at 250 nm by reverse-phase ion-pairing high-performance liquid chromatography. To achieve the sensitivity necessary for analyzing small (1-3 mg wet wt) tissue samples, a small-bore, 2.1-mm-internal-diameter, C18, 5-micron reverse-phase column and a flow rate of 0.2 ml/min were used. All of the myocardial nucleotides and AMP degradation products were resolved in a total separation time of 27 min with 30 mM KH2PO4, 7.5 mM tetrabutylammonium phosphate buffers, and binary pH and acetonitrile gradients.

Animals↗
PubMed1988

Myocyte deenergization and intracellular free calcium dynamics.

Intracellular free calcium in adult rat heart ventricular myocytes was monitored by single cell fura-2 fluorescence microscopy. The average resting free calcium in rod-shaped quiescent cells was 125 nM (range 70-200 nM). When cells were deenergized with an inhibitor (amytal) and an uncoupler (carbonyl-cyanide m-chlorophenylhydrazone) of oxidative phosphorylation, there was a small but significant increase (125-380 nM) in intracellular free calcium during the transition to a highly contracted (square) rigor form. After the onset of contracture, which occurred 5-15 min after addition of the above compounds, the increase in free calcium was slow for the first 20 min, reaching a value of only 750 nM. Thereafter, the rate of increase accelerated and 50 min after contracture, free calcium was approximately 3 microM. The increase in free calcium was absolutely dependent on extracellular calcium but was not inhibited by high concentrations of verapamil (2-7 microM), suggesting influx via the Na+-Ca2+ exchange transporter as the cause of calcium increase. However, in calcium repletion protocols the rate of increase in sodium-loaded myocytes was greatly accelerated if cells were not depleted of ATP, confirming suggestions that ATP loss partially inhibits Na+-Ca2+ exchange.

Adenosine Triphosphate↗
PubMed1987

Estimation of intramitochondrial pCa and pH by fura-2 and 2,7 biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) fluorescence.

Isolated heart mitochondria hydrolyze the acetoxymethyl esters of the Ca2+-sensitive fluorescent probe fura-2 and the fluorescent pH indicator biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). The free acid forms of both probes are retained in the matrix and their fluorescence can be used to monitor the pCa and pH, respectively, of this compartment. When fura-2 loaded rat heart myocytes are lysed with digitonin, a portion of the dye is retained in the mitochondrial fraction and its fluorescence reports the uptake and release of Ca2+ by the mitochondria. It is concluded that fura-2 and BCECF may report mitochondrial as well as cytosol parameters when the probes are used in intact cells.

Animals↗
PubMed1987

Degradation and resynthesis of adenine nucleotides in adult rat heart myocytes.

The degradation and short-term resynthesis of adenine nucleotides have been examined in a preparation of isolated rat heart myocytes. These myocyte preparations are essentially free of vascular and endothelial cells, contain levels of adenine nucleotides quite comparable to those of intact heart tissue, and retain these components remarkably well for up to 2 h of aerobic incubation in the presence of 1 mM Ca2+. When the cells are rapidly and synchronously de-energized by addition of uncoupler, an inhibitor of respiration and iodoacetate, cellular ATP is degraded almost quantitatively to AMP. The AMP is then converted to either intracellular adenosine, which accumulates to high concentrations before release to the cell exterior, or to IMP. The relative contribution of these two pathways depends on the metabolic state of the cells just prior to de-energization, with IMP production favored when respiring cells are de-energized and adenosine formation predominant when glycolyzing myocytes are subjected to this treatment. Cells de-energized by anaerobiosis in the absence of glucose lose ATP and adenine nucleotides with the production of IMP and adenosine. Upon reoxygenation, these cells restore a high adenylate energy charge and about 60% of control levels of GTP. There is a net resynthesis of 5-7 nmol of adenine nucleotides.mg-1 protein with a corresponding decline in IMP. Added [14C]adenosine labels the adenine nucleotide pool, but little net resynthesis of adenine nucleotides via adenosine kinase can be detected. It therefore appears that a rapid regeneration of adenine nucleotides can occur via the enzymes of the purine nucleotide cycle in heart myocytes and is limited by the size of the IMP pool retained.

Adenine Nucleotides↗
PubMed1987

Quantitation of intracellular free calcium in single adult cardiomyocytes by fura-2 fluorescence microscopy: calibration of fura-2 ratios.

Isolated rat myocytes incubated with the acetoxy methyl ester of fura 2 contained partially hydrolyzed esters, necessitating in vivo calibration of the signals obtained by fluorescence microscopy for calculation of pCa. Ionophores did not produce reliable R'max and R'min values in respiring myocytes, and elevated free calcium caused individual cells to hypercontract and burst. These difficulties were overcome by superfusion with a glucose-free buffer containing an inhibitor and an uncoupler of oxidative phosphorylation. R'max and R'min values obtained by ionophore treatment of deenergized myocytes were normalized to an in vitro calibration curve. Resting pCa derived from the individual curves averaged 6.9 for calcium-tolerant rod-shaped myocytes.

Adenosine Triphosphate↗
PubMed1986

Alpha and beta forms of cytochrome c oxidase observed in rat heart myocytes by low temperature Fourier transform infrared spectroscopy.

Carbon monoxide bound to myoglobin and cytochrome c oxidase in separated adult rat heart myocytes has been observed with Fourier transform IR spectroscopy at low temperatures. CO complexes of these two proteins can be spectrally separated through temperature manipulation of the relaxation of the photolyzed systems. Photolyzed carboxymyoglobin relaxes very rapidly above 80 K, whereas the CO photolyzed from cytochrome a3 associates with CuB and relaxes very slowly below 140 K. Cytochrome c oxidase is found to be present in two major molecular forms which we designate alpha and beta. Each form contains an a3Fe and its associated CuB which we observe by their CO complexes. The predominant FeCO band, the alpha form of cytochrome oxidase, is similar to that previously seen in beef heart mitochondria, but with a slightly larger activation enthalpy, delta H = 46 kJ/mol. At least one of the beta forms is similar, but two have not been observed in beef heart mitochondria. Upon photolysis of alpha-FeCO, the alpha-CuCO species is formed. This band splits into two at low temperature. Up to half of the FeCO band area of the intact myocytes is distributed among three or more minor species (beta forms). The beta-FeCO bands all appear to be associated with only one beta-CuCO band which does not split at low temperature. After photo-dissociation of CO, the beta forms relax considerably faster than the alpha form, achieving 50% recombination in 10% of the time required for the alpha form. In a tissue slice from an opossum heart exposed to CO, we observed alpha and beta forms of cytochrome oxidase very similar to those in the rat heart myocytes. The cause of the differences between the alpha and beta forms of the enzyme is unknown, but their possible role in the control of respiration is discussed. Carboxymyoglobin contained within intact rat heart myocytes was very similar to sperm whale carboxymyoglobin, but with a much smaller amount of the lower frequency minor component.

Animals↗
PubMed1986

Intracellular free Ca2+ and the hypercontracture of adult rat heart myocytes.

The Ca2+ sensitivity of a population of isolated adult rat heart myocytes has been related to the Na+ content of the cells prior to Ca2+ exposure, and the intracellular free Ca2+ as reported by quin2 fluorescence when the cells are challenged with millimolar external Ca2+. Myocytes exposed to Ca2+ during quin2 loading show a resting intracellular free Ca2+ of 150 +/- 30 nM and retain the rod cell morphology of heart cells in situ. The myocytes take up Na+ and lose K+ when incubated in the cold in the absence of Ca2+. Large numbers of these rod-shaped, Na+-loaded myocytes hypercontract into grossly distorted round cell forms when exposed to physiological levels of Ca2+. The number of cells that hypercontract is proportional to the Na+ content of the cells prior to Ca2+ addition and can be directly related to the intracellular free Ca2+ concentration attained following Ca2+ addition. Fifty percent of the cells in a myocyte population hypercontract when the internal free Ca2+ concentration reported by quin2 reaches 400 nM and virtually all of the cells hypercontract when this value reaches 1 microM. The entry of Ca2+ into Na+-loaded myocytes is biphasic with one phase inhibited by Ca2+ channel blockade. This suggests that Ca2+ enters Na+-loaded myocytes by the Ca2+ channel as well as by Na+/Ca2+ exchange.

Aminoquinolines↗
PubMed1986

Magnesium in cardiac energy metabolism.

Free intracellular magnesium ion, which influences many metabolic processes, is the subject of ongoing research. Its concentration has been difficult to measure because the available methods, including dye injection, microelectrodes, and nuclear magnetic resonance measurements, are invasive or indirect. Concentrations ranging from 0.1 mM in frog muscle to 6 mM in barnacle muscle have been reported. We describe recent experimental evidence regarding the concentration of free intracellular magnesium and consider the limitations of these methods. A substantial body of evidence, including our models of cardiac energy metabolism and its magnesium-related processes, indicates that intracellular concentrations of free magnesium are low (ca. 0.4 mM) and vary with time and conditions.

Animals↗
PubMed1986

Modification of caffeine-induced injury in Ca2+-free perfused rat hearts. Relationship to the calcium paradox.

The pathogenesis of the calcium paradox has not been established. In calcium-free perfused hearts, caffeine, which releases calcium from the sarcoplasmic reticulum, causes severe myocardial injury, with creatine kinase (CK) release and contraction band necrosis similar in many respects to the calcium paradox. It has been postulated that contracture, initiated by a small rise in intracellular calcium, may cause sarcolemmal injury in both the calcium paradox and caffeine-induced myocardial injury. The present study was initiated to determine whether interventions which modulate caffeine-induced contracture will also correspondingly alter cellular injury. The effects of caffeine dose, procaine, extended calcium-free perfusion, elevated potassium, temperature, and increasing intracellular sodium on caffeine-induced contracture were examined in Langendorff-perfused adult rat hearts. Caffeine-induced contracture at 22 C increased over a dose range of 5-40 mM caffeine. Procaine, which inhibits caffeine-induced calcium release at doses between 5 and 20 mM, progressively reduced contracture caused by addition of 20 mM caffeine at 22 C. Hearts perfused with calcium-free solution containing 16 mM K+ showed a reduction in caffeine-induced contracture. Extended calcium-free perfusion (20 minutes) at temperatures from 18 to 37 C resulted in a progressive reduction of caffeine-induced contracture. Each of these interventions was also found to inhibit caffeine-induced injury at 37 C. Low temperature was found to have complex effects. Hypothermia enhanced caffeine contractures but also protected hearts from cell separations and CK release. Increasing intracellular sodium was found to enhance caffeine-induced contracture at 37 C. There was a direct correlation between measured intracellular sodium levels and the magnitude and duration of caffeine-induced contracture. These results demonstrate a direct correlation between the magnitude of contracture and myocardial injury in calcium-free hearts. It is proposed that contracture is the primary mediator of sarcolemmal membrane injury in hearts with intercalated disks weakened by prior calcium-free perfusion.

Animals↗
PubMed1986

Energy dependence of contraction band formation in perfused hearts and isolated adult myocytes.

Aggregation of sarcomeres into contraction bands is a prominent feature of the oxygen paradox, the calcium paradox, and caffeine injury to calcium-free perfused hearts. For investigation of the mechanism of contraction banding, it was necessary to devise a method of evaluating the degree of sarcomere contraction and to define objectively a contraction band. Hearts with mechanical detachment of cells caused by hypocalcemic perfusion and isolated myocytes both allow unrestrained contracture of cells and permit direct optical measurements to quantitate the degree of cell contracture. With the use of the calcium paradox as a model of contraction band necrosis, it was found that cells with lengths of less than 37.3 mu could be considered as containing contraction bands. It was found that the mitochondrial inhibitors cyanide and amytal, as well as the uncoupler 2,4-dinitrophenol, allowed cell contracture but inhibited hypercontracture of sarcomeres into contraction bands during both the calcium paradox and caffeine injury to perfused hearts. However, when 2mM adenosine triphosphate (ATP) was included in the perfusion media, contraction band formation occurred despite the continued presence of cyanide or amytal. In isolated myocyte preparations the addition of the glycolytic inhibitor iodoacetate (IAA, 5 mM) and the mitochondrial inhibitor amytal (3 mM) caused relaxed rod-shaped cells (length/width ratio greater than 3:1) to contract into a stable population of square-shaped forms (length/width ratio less than 3:1), indicating an abrupt and severe decline in cellular ATP levels. Removal of amytal from the incubation medium in the presence of IAA produced a significant conversion of square-shaped cells into round-shaped cells containing contraction bands. Either IAA alone or amytal alone resulted in a mixed population of square and round cells. The results indicate that ATP is required for the formation of contraction bands in intact hearts and for the rounding of isolated myocytes. Formation of contraction bands appears to be an energy-dependent process requiring ATP.

2,4-Dinitrophenol↗
PubMed1985

Structural and functional properties of adult rat heart myocytes lysed with digitonin.

Low concentrations of digitonin disrupt the sarcolemma of adult rat heart myocytes selectively and completely. When the digitonin lysis is carried out in the presence of 10 mM Mg-ATP, the permeabilized cells retain the rod-cell morphology typical of heart cells in situ and show spontaneous phasic contractions. The rate of contraction is a function of the free Ca2+ concentration from a pCa of 7.2 to 5.2. Higher levels of free Ca2+ result in hypercontracture of the myocytes into round cells with characteristically distorted morphology. The sarcoplasmic reticulum of digitonin-lysed myocytes takes up Ca2+ in an ATP-dependent reaction that is inhibited and reversed by caffeine and strongly enhanced by procaine or ruthenium red. The Ca2+ accumulation has a Km of 0.6 microM Ca2+, depends on Pi (Km of 13 mM), and is strongly inhibited by bicarbonate ion. The hypercontracture of digitonin-lysed myocytes is a function of both the pCa and the Mg-ATP concentration of the suspending medium. Hypercontracture requires ATP. Hypercontracture due to Ca2+ overload occurs at lower Ca2+ concentrations when Mg-ATP is decreased from 10 to 1 mM. However, at low concentrations of Mg-ATP (in the range from 1 to 10 microM), hypercontracture also occurs and is essentially Ca2+-independent. Since hypercontracture of heart myocytes appears analogous to the formation of contraction bands in situ, these observations may be relevant to the phenomena of oxygen paradox and of Ca2+ paradox in intact myocardial tissue.

Adenosine Triphosphate↗
PubMed1985

Mitochondrial enzyme retention by irreversibly damaged rectangular isolated adult rat heart myocytes.

A one hour hypoxic incubation causes the release of a small but significant amount of cytosolic lactic dehydrogenase from glucose-deprived isolated adult rat heart myocytes. However, enzymes associated with the mitochondria are not liberated, and there is no increase in the number of hypercontracted cells. These observations led Piper et al. (Life Sciences 35, 127-134 [1984]) to conclude that reversibly injured myocytes can release cytosolic proteins. This conclusion was based on the supposition that irreversibly hypoxic injury must cause mitochondrial enzyme efflux and hypercontracture. The present study establishes that this supposition is invalid.

Adenosine Triphosphate↗