Isolation and characterization of adult rat heart cells

SUMMARY The contractile and biochemical effects of the divalent cation ionophore A23187 were studied in isolated perfused guinea pig and rat ventricles. A23187 increased both cyclic adenosine 3':5'-monophosphate (cyclic AMP) and cyclic guanosine 3':5'-monophosphate (cyclic GMP) concentrations in guinea pig ventricles. Associated with these changes in nucleotide content were positive inotropic effects and activation of giycogen phosphorylase. Pretreatment of «nirrmln with reserpine to deplete endogenous catecholamine stores or pretreatment of hearts with propranolol failed to alter these ionophore-induced change*. In contrast, blockade of histamine (Hi) receptors with metiamide completely abolished the positive inotropic effects and activation of giycogen phosphorylase induced by A23187. In these metiamide-treated hearts, the ionophore-induced increase in cyclic AMP concentration was significantly attenuated, whereas the increase in cyclic GMP content persisted. A23187 produced a negative inotropic effect in hearts taken from rats treated with reserpine. In these rat hearts, cyclic AMP levels and giycogen phosphorylase activity were unchanged, whereas cyclic GMP concentrations were markedly increased. These results indicate that A23187 produces both indirect and direct effects on guinea pig ventricles. The indirect effects are mediated primarily by histamine, presumably released from tissue mast cells by the ionophore. Histamine in turn increases cyclic AMP levels, which leads to activation of phosphorylase and positive inotropic effects. The direct biochemical effect of A23187 is an increase in cyclic GMP concentrations. Ore Rea 44: [473][474][475][476][477][478][479][480][481][482] 1979

Section: Biochemical Assaysmentioning

confidence: 99%

Indirect and direct effects of the divalent cation ionophore A23187 on guinea pig and rat ventricular myocardium.

Lindemann¹,

Besch²,

Watanabe³

1979

“…Our method 21 for isolating cells is a modification of that described by Berry et al 22 Briefly, this involves Langendorff perfusion of rat hearts at 37°C with a well oxygenated, nominally Ca 2+ -free and Mg 2+ -free buffer solution containing (ITIM): NaCl, 134; KC1, 5; sodium phosphate (pH 7.4), 6; and glucose, 10. After about 5 minutes, collagenase (80 U/ml) and hyaluronidase (82 U/ml) were added to the perfusion fluid and the perfusion was continued at 37°C for an additional 20 minutes.…”

Section: Isolation Of Cellsmentioning

confidence: 99%

“…The cells were collected from the filtrate by centifugation at a force of 37 g, washed twice by resuspension and recentrifugation in an incubation medium that contained (ITIM): NaCl, 120; KCl,5;MgCl 2 , 1.2; sodium phosphate (pH 7.4), 10; and glucose, 5. Further details of the method are described by Farmer et al 21 Cell viability was determined by the criterion of trypan blue exclusion: 23 the percentage of cells in three high power fields which were able to exclude 0.3% trypan blue was determined under a microscope using a hemocytometer counting chamber and a hand tally counter.…”

Section: Isolation Of Cellsmentioning

confidence: 99%

“…21 Only those features of the cells which are directly relevant to this study are considered in this paper. Assessed within 5 minutes after preparing the cells, the percent of the total number of cells which were viable, as judged by their ability to exclude 0.3% trypan blue, was 72 ± 1 % (mean ± SEM) for eight separate preparations.…”

Section: Viability Of Isolated Cellsmentioning

confidence: 99%

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alpha-Adrenergic reduction of cyclic adenosine monophosphate concentrations in rat myocardium.

Watanabe

Hathaway

Besch

et al. 1977

SUMMARY We determined the effect of a-adrenergic receptor stimulation on cyclic adenosine monophosphate (cyclic AMP) concentrations in isolated myocytes derived from adult rat hearts and in isolated perfused rat hearts. Activation of a-adrenergic receptors with either phenylephrine (10~8 M to 10~6 M) or epinephrine (10" M to 10~6 M) plus propranolol (10 ' ! M) resulted in a reduction in cyclic AMP levels in isolated myocytes. The action of phenylephrine was antagonized by phentolamine (10~6 M). Phenylephrine (10 s M) attenuated cyclic AMP generation in response to isoproterenol (10" s M and 10 s M). However, this effect of phenylephrine was not antagonized by phentolamine. Elevation of cyclic AMP concentrations produced by glucagon and by theophylline in isolated myocytes was attenuated by phenylephrine and by epinephrine plus propranolol and the attenuation was antagonized by phentolamine. In isolated perfused rat hearts epinephrine (10 (i M), when given with propranolol, diminished the rate of development of tension and also reduced tissue levels of cyclic AMP. Epinephrine alone, as well as isoproterenol, increased contractility and myocardial cyclic AMP concentrations as expected. These results indicate that catecholamines may increase or decrease cyclic AMP levels in rat myocardium, depending on the intensity of stimulation of receptor types. Increases are mediated by /3-adrenergic receptors, whereas decreases appear to be mediated by a-adrenergic receptors. IN 1967, Robison et al. 1 hypothesized that activation of either a-adrenergic or /3-adrenergic receptors may modify the activity of adenylate cyclase and result in changes in the rate of conversion of ATP to cyclic adenosine monophosphate (cyclic AMP). They further predicted that asympathomimetic agents could induce a decrease in the activity of the enzyme in some tissues. This original hypothesis has since been corroborated by studies which indicate that some a-adrenergic effects are mediated by a reduction in the tissue level of cyclic AMP. 2 " 5 Although there is an abundance of evidence that activation of a-adrenergic receptors can lead to alterations in the electrophysiological and mechanical functions of the heart, 6 " 18 no intracellular mediator or biochemical basis for these effects has been identified. It has been fairly convincingly shown that these a-adrenergic effects are not mediated by an increase in myocardial cyclic AMP concentrations. 1920 The question remains, however, whether any of the a-adrenergic effects on the heart might be mediated by a decrease in tissue cyclic AMP levels. The purpose of the present study was to attempt to demonstrate whether stimulation of myocardial a-adrenergic receptors is associated with a fall in cyclic AMP concentrations. The studies were carried out using isolated adult rat heart cells. To extend the findings to the intact heart, we also performed a limited parallel study of a-adrenergic effects on contractility and cyclic AMP levels of isolated, perfused rat hearts. Methods ISOLATION OF CELLSOur me...

“…For example, the abnormal hypersensitivity to Ca 2+ seen in many heart cell preparations (cf. Nag et al, 1977;Farmer et al, 1977;Altschuld et al, 1980) suggests that significant alterations may have been introduced by the process of enzymatic disaggregation of the cells. However, we have recently reported that preparations of Ca 2+ -hypersensitive cells in which the sarcolemma is intact can be made Ca 2+ -tolerant when physiological Na + /K + ratios are restored by a preincubation step in the absence of Ca 2+ (Altschuld et al, 1981).…”

mentioning

confidence: 99%

Response of isolated rat heart cells to hypoxia, re-oxygenation, and acidosis.

Altschuld¹,

Hostetler²,

Brierley³

1981

SUMMARY Responses of isolated adult rat heart cells to conditions that emphasize various aspects of ischemia have been evaluated. Cells maintained in hypoxic media with limited gubstrate deteriorate mort rapidly than aerobic controls supplemented with glucose. Two distinct irreversible pathways for cell alteration can be distinguished as follows: (1) continued anaerobic aging in the absence of glucose results in the production of large numbers of cells which retain the rod-shaped morphology of heart cells in situ, but which have lost saxcolemmal integrity, and (2) after a period of anaerobic aging, reaeration of the cells produces large numbers of rounded cells in irreversible contracture. These cells maintain an intact sarcolemma and are indistinguishable from those produced by addition of 1 mM Ca 1+ to Na + -loaded, aerobic cells. Contracture of isolated cells on re-aeration is at least superficially analogous to the oxygen paradox in situ, but since the isolated cells maintain an intact sarcolemma, there is no loss of creatine phosphokinase or other components of the cytosol. Incubation of isolated heart cells at acid pH (pH 8.8 to 6.2) largely prevents both Ca I+ -dependent contracture and a Ca 1+ -dependent loss of respiratory capacity. The acidic conditions virtually eliminate the net influx of "Ca 1 * into isolated cells that occurs at neutral pH, and the inhibition appears to be localized at the sarcolemma.