Summary
Primary mycolardial cell cultures and freshly isolated cardiac cells in suspension resprensent two isolated, whole cell models for investigating cellular transsarcolemmal45Ca++ exchange in response to a receptor-coupled stimulus. Studies were performed to characterize beta-adrenergic receptor binding, beta-adrenergic receptor mediated cellular calcium (45Ca++) exchange, and viability in purified primary myocardial cell cultures and freshly isolated cardiac cells in suspension obtained from 3-to 3-d-old Sprague-Dawley rats. In addition, beta-adrenergic receptor binding was characterized in whole-heart crude membrane preparations. All three preparations had saturable beta-adrenergic binding sites with the antagonist [125I]iodopindolol ([125I]IPIN). The suspensions had a significantly lower B max (42±6 fmol/mg protein) than the membranes and cultures (77±8 and 95±10 fmol/mg protein, respectively). The K D of the cultures (218±2.0 pM) was significantly higher than that for the suspensions (107 ±1.3 pM) and membranes (93±1.3 pM). Viability was significantly lower in the suspensions (57%) when compared to 94% viability in myocardial cell cultures after 3 h of incubation in Kreb's Henseleit buffer. Incubation of the cultures with 5.0×10−7 M isoproterenol resulted in a significant increase in45Ca++ exchange as early as 15 s. In contrast,45Ca++ exchange into the suspensions was not increased. Although both primary cell cultures and cardiac cells in suspension possess saturable beta-adrenergic receptors, only the monolayer cultures exhibited functional beta-adrenergic receptor-mediated45Ca++ exchange. Of the two intact cell models investigated, these data suggest that primary myocardial cell cultures are more suitable than cell suspensions for investigating beta-adrenergic receptor binding and functions in the postnatal rat heart.
Similar content being viewed by others
References
Acosta, D.; Sorensen, E. M. B.; Anuforo, D. C., et al. An in vitro approach to the study of target organ toxicity of drugs and chemicals. In Vitro 21: 495–504; 1985.
Baily, J. L. Techniques in protein chemistry. New York: Elsevier; 1967: 341–346.
Barovsky, K.; Brooker, G. (−)-[125I]-Iodopindolol, a new highly selective radioiodinated beta-adrenergic receptor antagonist: measurment of beta-receptors on intact rat astrocytoma cells. J. Cyclic Nucleotide Res. 6: 297–307; 1980.
Bhalla, R. C.; Sharma, R. V.; Ramanathan, S. Ontogenetic development of isoproterenol subsensititivity of myocardial adenylate cyclase and beta-adrenergic receptors in spontaneously hypertensive rats. Biochim. Biophys. Acta. 632: 497–506; 1980.
Butler, A. A. W.; Smith, M. A.; Farrar, R. P., et al. The effects of ethanol on cellular calcium content in primary myocardial cell cultures from offspring of sedentary and swim-trained rats. Biochem. Biophys. Res. Commun. 142: 496–500; 1987.
Danpure, C. J. Lactate dehydrogenase and cell injury. Cell Biochem. Funct. 2: 144–148; 1984.
Dowell, R. T. Cardiac adaptations to exercise. Exercise Sport Sci. Rev. 11: 99–117; 1983.
Hoyer, D.; Reynolds, E. E.; Molinoff, P. B. Agonist-induced changes in the properties of beta-adrenergic receptors on intact S49 lymphoma cells. Mol. Pharmacol. 25: 209–218; 1984.
Karliner, J. S.; Simpson, P. C.; Hondo, N., et al. Mechanisms and time course of beta1-adrenoceptor desensitization in mammalian cardiac myocytes. Cardiovasc. Res. 20: 221–228; 1986.
Katz, A. M. In: Physiology of the heart. New York: Raven Press; 1977.
Lau, Y. H.; Robinson, R. B.; Rosen, M. R., et al. Sub-classification of beta-adrenergic receptors in cultured rat cardiac myoblasts and fibroblasts. Circ. Res. 47: 41–48; 1980.
Leslie, S. W.; Barr, E.; Chandler, L. J. Comparison of voltage-dependent45Ca2+ uptake rates by synaptosomes isolated from rat brain regions. J. Neurochem. 41: 1602–1605; 1983.
Lundgren, E.; Terracio, L.; Allen, D. O., et al. Modulation of beta-receptors as adults and neonatal cardiac myocytes progress into culture. In Vitro 24: 28–34; 1988.
Marsh, J. D.; Barry, W. H.; Smith, T.W. Desensitization to the inotropic effect of isoproterenol in cultured ventricular cells. J. Pharmacol. Exp. Ther. 223: 60–67; 1982.
Marsh, J. D.; Smith, T. W. Receptors for beta-adrenergic agonists in cultured chick ventricular cells. Mol. Pharmacol. 27: 10–18; 1985.
Michell, D. B.; Santone, K. S.; Acosta, D. Evaluation of cytotoxicity in cultured cells by enzyme leakage. J. Tissue Cult. Methods 6: 113–116; 1980.
Moore, R. L.; Riedy, M.; Gollnick, P. D. Effect of training on beta-adrenergic receptor number in rat heart. J. Appl. Physiol. 52: 1133–1137; 1982.
Opie, L. H. In: The heart: physiology, metabolism, pharmacology, and therapy. London: Grune and Stratton; 1984.
Ramos, K.; Combs, A. B.; Acosta, D. Role of calcium in isoproterenol cytotoxicity to cultured myocardial cells. Biochem. Pharmacol. 33: 1989–1992; 1984.
Scatchard, G. The attractions of proteins for small molecules and ions. Ann. NY Acad. Sci. 51: 660–672; 1949.
Stiles, G. L.; Lefkowitz, R. J. Cardiac adrenergic receptors. Ann. Rev. Med. 35: 149–164; 1984.
Tipton, C. M.; Scheuer, J. Symposium on experimental preparations to study the effects of training on the cardiovascular system. Med. Sci. Sports Exercise 9: 219–267; 1977.
Tsien, R. W.; Bean, B. P.; Hess, P., et al. Mechanisms of calcium channel modulation by beta-adrenergic agents and dihydropyridine calcium agonists. J. Mol. Cell. Cardiol. 18: 691–710; 1986.
Wenzel, D. G.; Wheatley, J. W.; Byrd, G. D. Effects of nicotine on cultured rat heart cells. Toxicol. Appl. Pharmacol. 17: 774–785; 1970.
Whitsett, J. A.; Pollinger, J.; Matz, S. Beta-adrenergic receptors and catecholamine sensitive adenylate cyclase in developing rat ventricular myocardium: effect of thyroid status. Pediatr. Res. 16: 463–469; 1982.
Wolfe, B. B.; Harden, T. K. Guanine nucleotides modulate the affinity of antagonists at beta-adrenergic receptors. J. Cyclic Nucleotide Res. 303–312; 1981.
Wollenberger, A. Isolated heart cells as a model of the myocardium. Basic Res. Cardiol. Suppl. 80: 9–13; 1985.
Author information
Authors and Affiliations
Additional information
This research was supported by The University of Texas Research Institute, a grant from the Texas Advanced Research Technology Program awarded to S. W. Leslie and R. E. Wilcox, and contract 223-86-2109 from the Food and Drug Administration.
Rights and permissions
About this article
Cite this article
Welder, A.A., Machu, T., Leslie, S.W. et al. Beta-adrenergic receptor characteristics of postnatal rat myocardial cell preparations. In Vitro Cell Dev Biol 24, 771–777 (1988). https://doi.org/10.1007/BF02623647
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02623647