beta-Adrenergic catecholamine-dependent properties of rat myometrium primary cultures

We have used cultured trypsin-collagenase-dispersed cells from uteri of 21-day-old rats to investigate the mechanism of control of uterine motility by the beta-adrenergic receptor. After 5 to 7 days in RPMI 1640 the cells started to assume some of the morphological characteristics of smooth muscle cells. When cultures were incubated with 45Ca2+ for 3 h then washed free of isotope and incubated in medium with unlabeled Ca2+, efflux from the prelabeled intracellular pools was linear for up to 60 min. The potent beta-adrenergic agonist isoproterenol had a rapid effect on the rate of efflux and increased it almost sevenfold. Isoproterenol's effect was blocked by propranolol and could be duplicated by the addition of 8-bromo-adenosine 3',5'-cyclic monophosphate or cholera toxin. The cultured myometrial cells had adenylate cyclase properties similar to those of intact muscle strips when these were determined by the conversion of radioactive substrate (alpha-32P-ATP) to 32P-cAMP using a broken-cell preparation. Adenylate cyclase was sensitive to stimulation by GTP and by isoproterenol in the presence but not in the absence of GTP. Adenylate cyclase was also sensitive to stimulation by Ca2+ in the absence of GTP. We conclude that the primary cultures had the properties expected of smooth muscle cells including beta-adrenergic receptors that were coupled to a physiologically important function, Ca2+ flux. The beta-adrenergic receptor's effect on Ca2+ flux was cAMP mediated, and the divalent cation may also regulate its rate of flux by an effect on Ca2+-sensitive cAMP production.