Calcium Rather Than Cyclic AMP as the Physiological Intracellular Regulator of Prolactin Release

Abstract

Studies on the mechanisms which govern prolactin [PRL] release were undertaken using 2 in vitro techniques. A dispersed preparation of rat anterior pituitary cells was made by mechanical means in the presence of trypsin. These washed cells were drawn up into a small column together with a Bio-Gel matrix and perfused with Earle''s basic salt solution. The eluates containing PRL were then collected at short intervals. Test substances were added to the perfusion medium and their effect on PRL release was measured. The results of these studies were compared with those obtained by incubating hemipituitary glands in Medium 199 to measure the effect of test substances on the release of radioimmunoassayable PRL. Perifusion of dispersed pituitary cells with dopamine [DA] produced a marked inhibition of PRL release within 3 min, and maximal suppression 11 min after initiating the perifusion. Upon withdrawal of DA, PRL release began to recover within 1 min and continued to rise to 80% of baseline at 6.5 min. Perifusion of pituitary cells in medium free of Ca also produced a marked reduction in PRL release which was restored after reexposure of the cells to Ca. The addition of Mn and D-600 [.alpha.-isopropyl-.alpha.-[N-methyl-N-homoveratryl-.gamma.-aminopropyl]-3,4,5-trimethoxyphenylacetonitrile], agents which block Ca channels, also caused reversible inhibition of PRL release. The effects of the ionophores A23187 and X537A on PRL release were studied. The presence of Ca ionophore A23187 did not effect prolactin release but it reversed the DA-mediated inhibition of PRL release. In the absence of Ca, both ionophores stimulated PRL. Tetrodotoxin, a Na channel blocker, had no effect on PRL release. Agents such as prostaglandin E1 and cholera toxin increased cAMP levels, but no positive correlation was obtained on PRL release patterns. Gpp(NH)p[5''-guanylyl.beta.-.gamma.-imidodiphosphate]-stimulated adenylate cyclase activity in homogenates of anterior pituitary tissue was unaffected by DA. Addition of dibutyryl cAMP to perifused pituitary glands stimulated PRL release and theophylline added to hemipituitary gland completely reversed the inhibitory effect of DA on PRL release, and caused a concomitant increase in cAMP levels. The tonic high level of PRL release may be maintained by influx of extracellular Ca, and DA inhibits this process. The role of intracellular cAMP is undefined; the effects of dibutyryl cAMP and theophylline may be due to mobilization of intracellular Ca and thereby stimulate PRL release by this mechanism rather than through cAMP. Regulation of PRL secretion by normal lactotropes is evidently a Ca-mediated process.