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R A Altschuld

Publications and source records attributed to R A Altschuld.

At least 37 records · Page 2Linked to original sources
PubMed1994

Evidence against norepinephrine-stimulated efflux of mitochondrial Mg2+ from intact cardiac myocytes.

We investigated the hypotheses that norepinephrine stimulates Mg2+ efflux from intact isolated adult rat ventricular cardiomyocytes and that adenosine 3',5'-cyclic monophosphate stimulates Mg2+ efflux from permeabilized myocytes and isolated mitochondria. Norepinephrine stimulation of Mg2+ release from cardiac myocytes was observed only when cells at approximately 20 mg protein/ml in Mg(2+)-containing buffer were diluted 50- to 60-fold into an Mg(2+)-free medium. Under these conditions, > 30% of total cellular lactic acid dehydrogenase activity was also released, indicating that a significant portion of the cells had died. In other protocols, where Mg2+ efflux from myocytes was not observed, extracellular Mg2+ removal and administration of 10 microM norepinephrine increased 45Ca2+ accumulation by cells in suspension. In single myocytes, Mg2+ removal and norepinephrine administration increased intracellular free [Ca2+] as measured by fura-2 fluorescence microscopy, and this was accompanied by vigorous spontaneous contractile activity followed by Ca2+ overload hypercontracture. With permeabilized myocytes and isolated mitochondria from a variety of sources, adenosine 3',5'-cyclic monophosphate did not stimulate Mg2+ efflux. These results suggest that recent evidence for direct hormonal regulation of myocardial Mg2+ homeostasis may need to be reevaluated.

Animals↗
PubMed1993

Cardiac nucleotide levels and mitochondrial respiration in copper-deficient rats.

1. Male weanling Long-Evans rats were randomly assigned to either copper-adequate (6.0 mg Cu/kg feed) or copper-deficient (< 1.0 mg Cu/kg feed) diets for a 5 week period. 2. Cardiac ATP levels and energy charges did not differ between the two groups. Levels of NAD and phosphocreatine as well as the sum of pyridine nucleotides were greater in copper-deficient rats compared with copper-adequate rats using HPLC analysis. 3. Mitochondrial respiratory control ratios and oxygen consumption rates from copper-deficient rat hearts were depressed, although ADP:O ratios were similar to copper-adequate rat hearts.

Adenine Nucleotides↗
PubMed1993

Effects of obesity and hypertension on ventricular myocytes: comparison of cells from adult SHHF/Mcc-cp and JCR:LA-cp rats.

OBJECTIVE: The aim was to compare beta adrenergic receptors, cAMP production, and Ca2+ accumulation by the sarcoplasmic reticulum in ventricular cardiomyocytes from female SHHF/Mcc-cp and JCR:LA-cp rats. Whereas rats from both strains exhibit gross obesity when the animals are homozygous for the recessive "corpulent" gene, the SHHF rats, which are hypertensive, all develop heart failure during their second year of life. The normotensive JCR:LA-cp animals do not. METHODS: beta Adrenergic receptor number, ligand affinity, isoprenaline and forskolin stimulated cyclic AMP production, and ATP dependent, phosphate supported 45Ca2+ uptake by the sarcoplasmic reticulum were compared in ventricular cardiomyocytes isolated from 6 months old obese female SHHF/Mcc-cp and obese and lean female JCR:LA-cp rats. RESULTS: Bmax and Kd for (-)-[125iodo]-cyanopindolol (125ICYP) binding were each approximately 50% lower in SHHF/Mcc-cp v JCR:LA-cp myocytes. Cyclic AMP production in response to isoprenaline, isoprenaline plus isobutylmethylxanthine (IBMX), and forskolin plus IBMX was also significantly depressed in the SHHF/Mcc-cp cells. In addition, sarcoplasmic reticular 45Ca2+ uptake by SHHF/Mcc-cp cells was 35% lower than in lean or obese JCR:LA-cp myocytes. Isoprenaline stimulated cAMP production and sarcoplasmic reticular Ca2+ uptake by the lean JCR:LA-cp cells were comparable to that described previously for myocytes from normal Sprague-Dawley rats. By contrast, Bmax and Kd for 125ICYP binding by the JCR myocytes differed substantially from previously described results for normal Sprague-Dawley rats, whereas values for the SHHF cells did not. CONCLUSIONS: Declines in Ca sequestration by the sarcoplasmic reticulum of ventricular cardiomyocytes from obese, hypertensive SHHF rats are not related to their obesity. However, obesity may contribute to the decline in cAMP production. This may account, in part, for the exacerbation by obesity of cardiac dysfunction in essential hypertension.

Animals↗
PubMed1993

Protection of ROC-1 hybrid glial cells by polyethylene glycol following ATP depletion.

Oligodendroglia-glioma hybrid cells (ROC-1) subjected to inhibition of glycolytic and oxidative ATP synthesis undergo a sequence of changes, including ATP depletion, parallel processes of cell swelling and blebbing, and finally plasma membrane disruption and cell death. The morphological and biochemical changes that follow ATP depletion were studied in the presence and absence of polyethylene glycol (M(r) 8,000), a nonpermeant oncotic agent. Polyethylene glycol prevented cell swelling and membrane blebbing. It significantly delayed, but did not prevent, the release of lactate dehydrogenase into the medium; it did not affect the fall in [ATP]. These results suggest that osmotic cell swelling may be a contributing factor in the loss of cell viability when ROC-1 cells are depleted of ATP.

Adenosine Triphosphate↗
PubMed1993

Adenosine stimulation of AMP deaminase activity in adult rat cardiac myocytes.

Using an in situ assay for analyzing AMP deaminase activity in isolated adult rat ventricular myocytes, we have shown that IMP production is stimulated approximately twofold in cardiac cells incubated with 10 microM adenosine. This effect of adenosine was not blocked by the adenosine A1-receptor antagonist 8-cyclophenyl-1,3-dipropylaxanthine (0.01-1 microM) except at a concentration (100 microM) that may inhibit adenosine transport. Similarly, in situ AMP deaminase activity was not enhanced by treatment with the specific adenosine A1-receptor agonists N6-phenylisopropyl adenosine or cyclopentyladenosine, nor was it sensitive to prior treatment of cells with pertussis toxin. The nucleoside transport blockers S-4-nitrobenzyl-6-thioinosine, dipyridamole, and papaverine inhibited adenosine-induced increases in IMP production by 75-85%, suggesting an intracellular site of action. Modulation of enzyme activity via the transmethylation pathway could not be implicated since incubation of cardiac cells under conditions known to elevate intracellular S-adenosyl-L-homocysteine had no demonstrable effect on AMP deaminase. Furthermore, a direct allosteric effect of adenosine on the partially purified rat cardiac enzyme was not observed. The results indicate that intracellular adenosine modulates rat cardiac AMP deaminase by an unknown mechanism.

AMP Deaminase↗
PubMed1992

Cell swelling, blebbing, and death are dependent on ATP depletion and independent of calcium during chemical hypoxia in a glial cell line (ROC-1).

The morphological and biochemical changes that occur during chemical hypoxic injury in a neural cell line were studied in the presence and absence of calcium. Oligodendroglial-glioma hybrid cells (ROC-1) were subjected to inhibitors of glycolytic and oxidative ATP synthesis (chemical hypoxia). Complete respiratory inhibition depleted [ATP] to less than 5% of control by 4 min. Blebs appeared on the cell surfaces and cells began to swell within a few minutes of ATP depletion. A 200% increase in cell volume and bleb coalescence preceded irreversible cell injury (lactate dehydrogenase release) which began at approximately 20 min with 50% cell death by 40 min. In energized cells an equivalent degree of osmotic swelling induced by ouabain inhibition of the Na+, K(+)-ATPase pump did not produce blebbing or cell death. Partial inhibition of respiration decreased [ATP] to approximately 10% of control by 40 min. Blebbing and swelling began at 40 min and bleb coalescence preceded plasma membrane disruption which began at approximately 55 min. ATP depletion, blebbing, swelling, and death followed similar time courses in the presence or absence of extracellular calcium ([Ca2+]e). Intracellular calcium ([Ca2+]i) was measured using fura-2. In calcium-containing medium metabolic inhibition caused a transient increase in resting [Ca2+]i (100 +/- 17 nM) followed by a low steady-state level preceding plasma membrane disruption. Following deenergization in calcium-free medium, [Ca2+]i remained below 60 nM throughout injury and death. These data suggest that decreased ATP initiates a sequence of events including bleb formation and cell swelling that lead to irreversible cell injury in the absence of large increases in [Ca2+]i.

Adenosine Triphosphate↗
PubMed1992

Cyclosporin inhibits mitochondrial calcium efflux in isolated adult rat ventricular cardiomyocytes.

Exchangeable intracellular Ca2+ as measured by 45Ca2+ uptake more than doubled when isolated adult rat ventricular cardiomyocytes were incubated 30 min with 8 microM cyclosporin; nevertheless the cells retained a normal rod-shaped morphology. High concentrations of ouabain caused a similar increase in 45Ca2+ uptake, but in this case the Ca2+ overload caused nearly all cells to hypercontract into a round disorganized form. The response to cyclosporin was concentration dependent with an apparent half-maximal effective concentration of 0.5 microM for enhancement of net 45Ca2+ accumulation. Verapamil (1 microM) could not inhibit this cyclosporin effect, but it was abolished by a 5-min preincubation with 12 microM crude ruthenium red. Cyclosporin also decreased the rate of 45Ca2+ efflux from prelabeled myocytes into Ca(2+)-containing and Ca(2+)-free media. These data are consistent with inhibition of mitochondrial 45Ca2+ efflux through the cyclosporin-sensitive mitochondrial inner membrane pore. It would appear that periodic transient increases in mitochondrial inner membrane permeability provide a pathway for mitochondrial Ca2+ extrusion under relatively normal conditions in isolated adult rat heart cells.

Animals↗
PubMed1992

Effects of simulated ischemia and reperfusion on the sarcoplasmic reticulum of digitonin-lysed cardiomyocytes.

ATP-dependent, inorganic phosphate-supported 45Ca2+ uptake by digitonin-lysed adult rat ventricular cardiomyocytes was used to evaluate the effects of simulated ischemia and reperfusion on the physically intact sarcoplasmic reticulum. Mitochondrial reactions were inhibited with rotenone and oligomycin. 45Ca2+ accumulation in the presence of the calcium efflux inhibitors, procaine (10 mM) and ruthenium red (30 microM), was used to characterize unidirectional uptake kinetics. A decrease in pH from 7.2 to 6.6 increased the [Ca2+] K0.5 from 0.5 to 2.0 microM and reduced the apparent Vmax by 28%. In the absence of procaine and ruthenium red, at a free [Mg2+] of 0.5 mM, maximum net uptake occurred at pCa 6.2 when pH was 7.2 and at pCa 6.0 when pH was 6.6. At lower pCa, net Ca2+ accumulation declined. Increasing free [Mg2+] from 0.5 to 1 mM at pH 6.6 or to 2.5 mM at pH 7.2 increased net 45Ca2+ accumulation in the absence of procaine and ruthenium and shifted maximum uptake to pCa 5.6 and 6.0, respectively. Increases in cytosolic free [Mg2+] thought to occur during myocardial ischemia are therefore capable of inhibiting calcium efflux from the sarcoplasmic reticulum. Reducing [ATP] from 10 to 1 mM reduced maximum net 45Ca2+ uptake by 30% both in the presence and absence of efflux inhibitors. Preincubation of intact myocytes under conditions designed to simulate ischemia and reperfusion decreased 45Ca2+ uptake greater than or equal to 50%. The data indicate that myocardial ischemia and reperfusion can alter both Ca2+ accumulation and calcium release by the sarcoplasmic reticulum.

Animals↗
PubMed1991

Phorbol esters and cyclic AMP activate AMP deaminase in adult rat cardiac myocytes.

Using rapid deenergization as a probe for adenylate deaminase activity in intact adult rat cardiac myocytes, we have previously established that IMP formation is enhanced by alpha-adrenergic agonists. In the present study, the effect of adrenergic agents on adenylate deaminase was further characterized. Phenylephrine (PE)3 increased IMP production in a dose-dependent fashion with an EC50 of 8 x 10(-7) M. The response to PE was reversed within 10 min by the alpha 1-antagonist, prazosin. Likewise, adenylate deaminase was also activated in ventricular myocytes challenged with phorbol 12-myristate 13-acetate (PMA, EC50 = 5 nM); cardiac cells presented with 100 nM PMA increased IMP production from 4.4 +/- 0.5 (control) to 15.7 +/- 0.9 nmol/mg protein when subsequently deenergized. The effects of PMA and PE were attenuated 85 +/- 5% and 96 +/- 4%, respectively, by pretreatment of cells with 150 nM staurosporine, an inhibitor of protein kinase C. Furthermore, incubation of cardiac cells with 1 microM PMA for 24 h blunted the response to both PMA and phenylephrine 85-90%. Elevating cyclic AMP (cAMP) content to greater than 15 pmol/mg by treatment with forskolin or isoproterenol plus isobutylmethylxanthine also resulted in enhanced adenylate deaminase activity, but this stimulatory effect was not abolished by 24 h incubation with 5 microM PMA. Forskolin and PMA-induced increases in IMP production appeared to be additive. However, 0.5 microM isoproterenol inhibited the cellular response to phenylephrine by about 30% but did not affect PMA-stimulated adenylate deaminase activity. We conclude that both cAMP and protein kinase C stimulate adenylate deaminase, perhaps through selective activation of different isoforms. However, cAMP also exerts partial inhibition on alpha-adrenoreceptor-mediated increases in IMP production.

AMP Deaminase↗
PubMed1991

Energy depletion in culture. Adenine nucleotides are altered as in vivo.

Modeling of ischemic phenomena in vitro has been hindered by the inability to create specific alterations in the variables of interest over a defined time-frame. In particular, changes in the adenine nucleotide pool have been quite difficult to mimic because of the putative low metabolic rate in culture and the long times necessary to achieve even partial chemical energy depletion. Here we present evidence for a rapid method of producing a profound chemical energy depletion with the combination of a NADH dehydrogenase inhibitor (amytal) and a mitochondrial proton ionophore (CCCP). Treatment with our protocol in enriched spinal cultures results in a 40% decrease in ATP within 2 min and a fall to one-third of control values by 15 min. The overall pool size of the total adenine nucleotides is decreased 46% by 15 min and does not completely recover after 5 min of reenergization. The ATP/ADP ratio declines to one-third of control values during deenergization and returns to control values after 5 min in control buffer. Such a loss of the total adenylate pool closely mimics that seen in vivo during ischemia and provides an in vitro model system in which the effects of the combination of this means of cellular injury with others (e.g., excitotoxins) may be examined.

Adenine Nucleotides↗
PubMed1991

Estimation of myocardial ischemic injury during ventricular fibrillation with total circulatory arrest using high-energy phosphates and lactate as metabolic markers.

STUDY OBJECTIVE: To define the time course of myocardial ischemic injury using high-energy phosphate (HEP) depletion and the cessation of lactate production as metabolic markers. SETTING: Data were collected in a laboratory animal model. TYPE OF PARTICIPANTS: Ten immature mixed breed swine weighing 23.2 +/- 3.5 kg. DESIGN: After thoracotomy, transmural myocardial biopsies were taken in vivo during normal sinus rhythm and at designated times during ventricular fibrillation with total circulatory arrest (VF-TCA). MEASUREMENTS AND MAIN RESULTS: Frozen tissue samples were analyzed for adenine nucleotides, by high-performance liquid chromatography, and lactate by enzymatic assay. At five minutes of VF-TCA, myocardial adenosine triphosphate averaged 50% of control. At 15 minutes of VF-TCA, 89% of animals had myocardial adenosine triphosphate levels above 20% of control and adenylate charge ratio above 0.60. With more than 30 minutes of VF-TCA, all animals had adenosine triphosphate levels below 10% of control and adenylate charge ratio below 0.30. In addition, myocardial lactate levels plateaued after 30 minutes of VF-TCA, indicating the cessation of lactate production. CONCLUSION: These results suggest that the myocardium can tolerate VF-TCA for as long as 15 minutes without irreversible injury; however, post-ischemic myocardial dysfunction may occur after as little as five minutes of VF-TCA. With more than 30 minutes of VF-TCA, myocardial injury is likely to be irreversible.

Adenosine Diphosphate↗
PubMed1991

Response of isolated adult canine cardiac myocytes to prolonged hypoxia and reoxygenation.

Isolated adult canine ventricular myocytes incubated in the absence of glucose with the respiratory inhibitor rotenone retained 67% of ATP (control, 26.0 +/- 0.9 nmol/mg protein) during 3-h incubation, yet phosphocreatine fell to 23% of initial content. Lactate production proceeded at a constant rate of 5 nmol.mg-1.min-1 in rotenone-treated glucose-free myocytes. A 36% decline in rod-shaped cells and an increase in percent 22Na permeation from 37% in aerobic cells (approximately 13 mM intracellular sodium) to 68% in rotenone-treated glucose-free myocytes paralleled the loss of ATP. Total exchangeable calcium was maintained at control aerobic levels. Exposure of canine cells to 3-h hypoxia in the absence of glucose followed by 5-min reoxygenation resulted in a 73% decrease in ATP, a rise in calcium from 3.3 +/- 0.2 to 6.6 +/- 1.6 nmol/mg, and an increase in 22Na permeation to 111%. Under these conditions the number of rod-shaped myocytes declined by 77%, with corresponding increases in viable contracted and hypercontracted myocytes. The response of canine myocytes to severe hypoxia and reaeration contrasts greatly to earlier studies using adult rat cardiac myocytes [see Hohl et al. Am. J. Physiol. 242 (Heart Circ. Physiol. 11): H1022-H1030, 1982]. Species differences with respect to basal metabolism, rates of ATP production and degradation, and regulation of cation movements are most likely responsible for the observed differences.

Adenosine Diphosphate↗
PubMed1990

Calcium accumulation and release by the sarcoplasmic reticulum of digitonin-lysed adult mammalian ventricular cardiomyocytes.

We have developed a model for characterizing calcium handling by the intact cardiac sarcoplasmic reticulum (SR) that yields data consistent with both mathematical simulations of in situ SR Ca2+ uptake and deduced behavior of the Ca2(+)-induced Ca2+ efflux channels in mechanically skinned single cardiac cells. In Na(+)-based media (37 degrees C, pH 7.2, 50 mM Pi, 10 mM MgATP, pMg 3.3, 10 mM phosphocreatine), SR 45Ca2+ uptake by digitonin-lysed rat myocytes as a function of free [Ca2+] peaked at pCa 6.2, declined until pCa 5.6 and increased again at lower pCa. When Ca2(+)-induced Ca2+ efflux was inhibited with 30 microM ruthenium red and 10 mM procaine, uptake was saturable with a Vmax of 160 +/- 5 nmol.min-1.mg-1, K0.5 of 500 nM free [Ca2+] and slope factor of 1.6. In K(+)-based media, maximum Pi- and oxalate-supported uptake increased to 220 and 260 nmol.min-1.mg-1, respectively. Without phosphocreatine, 45Ca2+ uptake declined under all conditions; this was correlated with a decrease in ATP/ADP. Vmax for 45Ca2+ uptake was increased 20% in hyperthyroid myocytes but depressed 30% in myocytes from heart failure-prone rats. In canine myocytes, Vmax was the same as in normal rat cells, but K0.5 was 830 nM. Without efflux inhibitors, ryanodine caused a concentration-dependent decline in net Pi-supported 45Ca2+ uptake at pCa 6.3 (K0.5 = 1 microM), while 10 microM ryanodine depressed uptake at all pCa between 7.2 and 5.6. Ruthenium red/procaine fully reversed this effect.

Adenosine Triphosphate↗
PubMed1990

Cellular glutathione and the response of adult rat heart myocytes to oxidant stress.

Freshly isolated adult rat heart myocytes contain total glutathione and reduced glutathione (GSH) at levels quite comparable to those in intact rat heart. Total glutathione can be depleted from 11 to 1 nmol/mg protein or less by treatment with cyclohex-2-ene-1-one without effect on either cellular ATP, rod-cell morphology or the integrity of the sarcolemma. Glutathione levels and redox state are not altered significantly when the Ca-tolerant, quiescent cells are subjected to a period of anoxia followed by reoxygenation. This oxygen paradox protocol results in irreversible hypercontracture of the contractile elements into an amorphous mass in the bulk of the cells, but little loss of sarcolemmal integrity. When the myocytes are subjected to an externally applied oxidant stress by the addition of either diamide or t-butylhydroperoxide, GSH is rapidly depleted with accumulation of oxidized glutathione (GSSG. On continued aerobic incubation both of these reagents promote a slower depletion of cellular ATP and a parallel hypercontracture. Cells treated with t-butylhydroperoxide, but not those with diamide, also generate increasing amounts of thiobarbituric acid reactive species as an indication of lipid peroxidation and show a parallel loss of sarcolemmal integrity. It is concluded that respiring myocytes and those subjected to the oxygen paradox do not produce oxygen radicals in sufficient amounts to displace the GSH/GSSG redox poise and depletion of myocyte glutathione per se is not detrimental to the short term survival of the cells. In addition, aerobic myocytes subjected to external oxidant stress can be damaged irreversibly by two pathways, a hypercontracture that correlates with depletion of ATP and a loss of sarcolemmal integrity that correlates with lipid peroxidation.

Animals↗
PubMed1990

Effects of calcium depletion and loading on injury during metabolic inhibition of isolated adult rat myocytes.

The hypothesis that calcium influxes from the extracellular space play an important role in the pathogenesis of irreversible anoxic injury was tested using isolated adult rat myocytes. Myocytes treated with 6 mM amytal and 3 mM iodoacetate and subsequently incubated in either calcium-containing (1.12 mM) or calcium-free media (with or without 1 mM EGTA) developed rigor contracture (cell squaring) and cell death (trypan blue permeability) at the same rate. The rates of cell death in both calcium-containing and calcium-free media were increased by incubation in hypotonic media even though the rates of contracture development remained unaltered. Cells developed osmotic fragility prior to membrane permeability increases. The calcium ionophore, A23187 (10 microM), induced rapid rounding of rod-shaped cells subjected only to mitochondrial inhibition in calcium containing media, confirming its ability to cause an increase in cellular permeability to calcium. However, A23187 did not alter the rates of cell death of totally metabolically inhibited myocytes in either calcium-containing or calcium-free media with EGTA. The results indicate that influxes of calcium are not necessary for the development of irreversible injury in metabolically inhibited, isolated myocytes.

Animals↗