The present study was undertaken to develop an improved and stabilized method for isolating cardiomyocytes from perfused rat heart. Different lots of the commercial collagenases used for isolating cardiomyocytes give variable results both with respect to the total cell yield and the percentage of elongated cells obtained. When trypsin was present both before and during collagenase treatment of the tissue, the performance of the collagenases was improved and stabilized, and a high and stable cell yield (7.5 x 10(6) cells per heart), and a high percentage of elongated cells (about 70%) was regularly obtained. The cells possessed alpha 1-adrenergic binding sites with binding properties (Bmax = 43.5 fmol/mg protein and Kd = 125.5 pmol/l) in agreement with values previously reported. The cells were able to respond functionally, as the cellular uptake of 86Rb+ increased by 18% after alpha 1-adrenoceptor stimulation with phenylephrine. These criteria indicate that the cells were well preserved during the isolation procedure.

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The aim of the present investigation was to study the effect of alpha(1)- and beta-adrenoceptor stimulation, alone and in combination, on potassium uptake in isolated ventricular cardiomyocytes from adult rat heart, using the potassium analogue 86Rb+. The reliability of 86Rb+ as a potassium analogue was also investigated. Alpha(1)-, beta-, And combined adrenoceptor stimulation was achieved by using noradrenaline in the presence and absence of appropriate adrenoceptor antagonists. The uptake of 86Rb+ was found to increase linearly with time up to 20 min., both during basal and receptor-stimulated conditions. The basal uptake rate was about 0.18 ml/g protein x min. At 15 min. both alpha(1)-, beta- and combined adrenoceptor stimulation dose-dependently increased the 86Rb(+)-uptake with a -logEC50 of 7.05, 6.68 and 6.73, respectively. The maximal increase in these series achieved by 5 x 10(-5) mol/l noradrenaline was 29%, 24% and 41% above basal level, respectively. Comparison of the maximal effects in the same cell preparations, with the observed value for combined adrenoceptor stimulation in each experiment as 100%, gave a relative maximal increase in 86Rb(+)-uptake after separate alpha(1)-adrenoceptor stimulation of 67 +/- 8%, and of 68 +/- 6% after separate beta-adrenoceptor stimulation. The theoretically calculated value for combined adrenoceptor stimulation, if additivity, was 135 +/- 11%, which was significantly higher than the observed value (100%) (P = 0.026). The effect of noradrenaline was not limited by the maximal 86Rb(+)-uptake capacity, as 10(-5) mol/l forskolin increased the 86Rb(+)-uptake more than noradrenaline. Examining the reliability of 86Rb+ as potassium-analogue by combining 42K+ and 86Rb+ in the same experiments, showed that combined adrenoceptor stimulation dose-dependently increased both the 42K(+)- and 86Rb(+)-uptake with the same potency and to the same extent. Thus 86Rb+ is a reliable potassium-analogue for these effects. In conclusion both alpha(1)- and beta-adrenoceptor stimulation dose-dependently increased the cellular 86Rb(+)-uptake to the same extent and with the same potency. The observed maximal 86Rb(+)-uptake after combined adrenoceptor stimulation was significantly higher than the maximal effect after either form of separate receptor stimulation, but significantly lower than expected if the effects were purely additive. The results thus show inhibitory interaction between the two receptor systems.

Abstract:The aim of the present study was to investigate the accumulation of inositol-l,4,5-trisphosphate (IP,) in isolated adult rat ventricular cardiomyocytes after a,-and P-adrenoceptor stimulation, separate and in combination, in order to elucidate a possible influence of concomitant P-adrenoceptor stirnulation on the al-adrenoceptor stimulated response. IP3 was measured by a radioligand binding assay based on an (1,4,5)IP3-specific binding protein from bovine adrenal cortex. The basal IP, content was 4.06?0.31 pmol/mg protein (N=56). a,-Adrenoceptor stimulation resulted in a rapid increase in the IP3 level, which reached a plateau, 50-80% above basal level, at 10-30 sec. The plateau lasted at least up to 120 sec., while at 300 sec. there was no significant difference between control values and values after a,-adrenoceptor stimulation. Li+ did not affect either the basal IP3 level, or the magnitude or time course of al-adrenoceptor-stimulated IP, accumulation. Combined adrenoceptor stimulation gave a similar response as separate al-adrenoceptor stimulation, whereas there was no significant change in the IP, level after P-adrenoceptor stimulation. No inhibitory influence of simultaneous P-adrenoceptor stimulation on the al-adrenoceptor-stimulated increase of IP3 mass was revealed.The existence of both a l -and P-adrenoceptors in rat myocardium is well established (Osnes et al. 1985;Terzic et al. 1993). al-Adrenoceptor stimulation activates phospholipase C, resulting in breakdown of phosphoinositide 4,5-bisphosphate to inositol-1,4,5-trisphosphate (IP3) and diacylglycer-01. IP, releases Ca2+ from internal stores in most cell types, and diacylglycerol activates protein kinase C (Terzic et al. 1993). Whereas protein kinase C has been implicated in the al-adrenergic inotropic effect (Terzic et al. 1993), and in the regulation of cardiac growth (Sugden & Bogoyevitch 1995), the physiological role of IP3 in the heart remains unclear, since a clearcut effect on Ca*+-release has not been found (Terzic et al. 1993).When al-adrenoceptors are stimulated under physiological conditions, P-adrenoceptors are activated simultaneously. An inhibitory influence of concomitant P-adrenoceptor stimulation has been reported for several q-adrenoceptor-mediated responses in the myocardium. At the functional level, the al-adrenoceptor-stimulated inotropic response was attenuated by 0-adrenoceptor stimulation (Skomedal et al. 1988; Osnes et af. 1989). The a,-adrenoceptor-stimulated increase in potassium uptake rate of isolated ventricular cardiomyocytes was also attenuated by concomitant P-adrenoceptor stimulation (Viko et al. 1996). However, when myocardial phospholipase C activity was determined after labelling of cells with radioactive inositol (Berridge & Irvine 1989), simultaneous P-adrenoceptor stimulation either inhibited (Guse et al. 1991) Changes in labelled inositol phosphates may not reflect regulation of the IP3 mass, and thus a possible mediator function of IP3. Generation of inositol phosphates may occur independently of...

Various cells and tissues contain high basal levels of inositol 1,4,5-trisphosphate, raising questions about the functional significance of inositol 1,4,5-trisphosphate in some tissues such as the heart. We used intact tissue and isolated cells from heart and liver of adult rats to examine if different fixation procedures might artificially elevate the level of inositol 1,4,5-trisphosphate. The basal level of inositol 1,4,5-trisphosphate in intact, freeze-clamped cardiac tissue from adult rats was 10 times higher than in isolated, non-frozen cardiomyocytes, while freeze-clamped liver contained approximately 4 times higher inositol 1,4,5-trisphosphate levels than isolated, non-frozen hepatocytes. Stimulation with norepinephrine induced a significant increase in the inositol 1,4,5-trisphosphate level in isolated cardiomyocytes, whereas no significant increase was observed in freeze-clamped cardiac tissue. Freezing of isolated cardiomyocytes or hepatocytes before extraction increased basal inositol 1,4,5-trisphosphate levels 3 times. In cellular homogenates prepared in the presence of EGTA and stored at 4 ae, readdition of calcium resulted in a time-dependent increase in inositol 1,4,5-trisphosphate mass and a decrease in the mass of phosphatidylinositol 4,5-bisphosphate (PIP 2 ). The reaction was essentially complete within 30 sec. in homogenates from cardiomyocytes, while PIP 2 hydrolysis was slower in hepatocyte homogenates. Perfusion of intact rat hearts with EGTA present during the last 2 min. of perfusion, followed by freeze-clamping, resulted in basal inositol 1,4,5-trisphosphate levels comparable to those in isolated cardiomyocytes, and norepinephrine stimulation increased inositol 1,4,5-trisphosphate mass by approximately 80%. The presence of EGTA did not significantly affect PIP 2 levels in perfused hearts. The results suggest that freezing or homogenization of intact tissue and isolated cells may result in Ca 2π -dependent activation of phospholipase C, leading to high basal inositol 1,4,5-trisphosphate levels that may mask agonist-induced changes.In order to understand the functional role of inositol 1,4,5-trisphosphate, determination of the tissue content is of interest. Various cells and tissues contain high basal levels of inositol 1,4,5-trisphosphate that may be well above the level required for release of intracellular Ca 2π , raising questions about the functional role of inositol 1,4,5-trisphosphate in some tissues (Horstman et al.