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Abstract
Studies on the mechanisms which govern the release of prolactin were undertaken using
two 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 perifused with Earle’s basic
salt solution. The eluates containing prolactin were then collected at short intervals.
Test substances were added to the perifusion medium and their effect on prolactin
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 prolactin. Perifusion of dispersed pituitary
cells with dopamine produced a marked inhibition of prolactin release within 3 min,
and maximal suppression was noted 11 min after initiating the perifusion. Upon withdrawal
of dopamine, prolactin 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 calcium
also produced a marked reduction in prolactin release which was restored after reexposure
of the cells to calcium. The addition of manganese and D-600, agents which block calcium
channels, also caused reversible inhibition of prolactin release. The effects of the
ionophores A23187 and X537A on prolactin release were studied. The presence of calcium
ionophore A23187 did not effect prolactin release but it reversed the dopamine-mediated
inhibition of prolactin release. In the absence of calcium, both ionophores stimulated
release of prolactin. Tetrodotoxin, a blocker of sodium channels had no effect on
prolactin release. Agents such as prostaglandin E<sub>1</sub>, and cholera toxin increased
cyclic AMP levels, but no positive correlation was obtained on prolactin release patterns.
Gpp(NH)p-stimulated adenylate cyclase activity in homogenates of anterior pituitary
tissue was unaffected by dopamine. In contrast, addition of dibutyryl cyclic AMP to
perifused pituitary glands stimulated prolactin release and theophylline added to
hemipituitary gland completely reversed the inhibitory effect of dopamine on prolactin
release and caused a concomitant increase in cyclic AMP levels. It is suggested that
the tonic high level of prolactin release is maintained by influx of extracellular
calcium and that dopamine inhibits this process. The role of intracellular cyclic
AMP is undefined; however, the effects of dibutyryl cyclic AMP and theophylline may
be due to mobilization of intracellular calcium and thereby stimulate prolactin release
by this mechanism rather than through cyclic AMP. In summary, we present evidence
that regulation of prolactin secretion by normal lactotropes is a calcium-mediated
process.