Effect of TRH and dopamine on cyclic AMP levels in enriched mammotroph and thyrotroph cells

160

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 E₁, 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.

Section: Discussionsupporting

confidence: 81%

Section: Discussionmentioning

confidence: 99%

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

Thorner¹,

Hackett²,

Murad³

et al. 1980

160

“…Inhibition of adenylate cyclase activity has been impli cated in the mediation of the inhibitory action of DA on PRL release [2,7,15,16,46]. DA inhibits the activity of ade nylate cyclase in homogenates of pituitary glands and tu mors [7,15,16] and decreases cAMP concentrations in en riched populations of lactotrophs [2,46].…”

Section: Discussionmentioning

confidence: 99%

“…There is evidence that DA may inhibit PRL release by in hibiting the activation of both Ca:+/protein kinase C [12,24,25,29,41,44,45,47,48] and cAMP [2,7,15,16,46] systems and that the combination of a cAM P analog, a Ca2* ionophore and a protein kinase-C activator, is required to fully overcome the inhibitory action of DA [9]. The present studies were designed to determine whether, under conti nual DA infusion, the brief administration of either the cAMP analog 8-Br-cAMP, the Ca2* ionophore A23I87, or the protein kinase-C activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA) would enhance the release of PRL in re sponse to a subsequent challenge with TRH.…”

mentioning

confidence: 99%

Mechanism(s) by which the Transient Removal of Dopamine Regulation Potentiates the Prolactin-Releasing Action of Thyrotropin-Releasing Hormone

Escalera¹,

Weiner²

1988

The transient removal of dopamine (DA) selectively potentiated the prolactin (PRL) releasing action of thyrotropin-releasing hormone (TRH) but not vasoactive intestinal peptide (VIP). Consistent with these findings, the PRL-stimulating actions of agents which activated the Ca²⁺/protein kinase C second messenger pathway but not the adenylate cyclase system were also potentiated. In the current study we have extended these findings to determine the second messenger system mediating the potentiating action of the removal of DA. Dispersed anterior pituitary cells from E₂-treated Sprague-Dawley rats were cultured on plastic coverslips. Cells tonically superfused with DA (500 nM were challenged with TRH (100 nM) 20 min after no additional treatment or a 10-min treatment with 8-Br-cyclic adenosine monophosphate (8-Br-cAMP), the Ca²⁺ ionophore A23187,12–0-tetradecanoyl-phorbol-13-acetate (TPA), TRH, or VIP. The potentiation of the TRH response was compared to the 4- to 5-fold potentiation observed following the removal of DA for 10 min. 8-Br-cAMP at the concentration used (500 µM) was unable to alter the basal rate of PRL release, but, as VIP (500 nM), potentiated 2- to 3-fold the PRL-releasing action of TRH. A prior administration of TRH (100 nM) did not affect the responsiveness of the cells to a second challenge with TRH 20 min later. Both A23187 (20 µM) and TPA (5 or 50 nM) induced a sustained rise in the rate of PRL release. TPA-treated cells showed an increased responsiveness to TRH, whereas A23187-treated cells did not. These results suggest that increasing cAMP levels either by VIP or 8-Br-cAMP in part mimics the potentiation caused by the transient removal of DA. Although TPA potentiated the PRL-releasing action of TRH, stimulating the influx of Ca^{2 +} with the ionophore A23187 or the presumed activation of Ca²⁺/protein kinase C pathway by a priming treatment with TRH did not. Therefore, the mechanism by which TPA is potentiating the response to TRH is unclear. The finding that prior removal of DA or exposure to VIP can potentiate the action of TRH supports the concept that a secretory response can represent the integration of the action of multiple regulatory neurohormones delivered in a specific sequence, a so-called ‘pleotropic regulation’.

“…Indomethacin, a specific inhibitor of the cycloxygenase pathway, had no significant effect on basal and TRH-stimulated prolactin release. The results suggest that arachidonic acid metabolism is involved in basal and TRH-stimulated pro lactin secretion and that lipoxygenase pathway products are at least partially responsible for these effects.The biochemical mechanisms controlling prolactin se cretion from the anterior pituitary gland are not clearly de lineated; only recently have certain implicated processes, i.e., cyclic nucleotide involvement [3,23, 38] and the Ca2+-calmodulin system [16,30], been at least partially elucidat ed. Recent evidence suggests that phosphatidylinositol hy drolysis is involved in basal and stimulated prolactin re lease [5,27, 37], Phospholipids, including phosphatidylinositol, are cel lular sources of arachidonic acid.…”

mentioning

confidence: 99%

Arachidonic Acid Metabolism and Prolactin Secretion in vitro: A Possible Role for the Lipoxygenase Products

Canonico¹,

Schettini²,

Valdenegro³

et al. 1983

This study was designed to investigate basal and thyrotropin-releasing hormone (TRH)-stimulated prolactin release in the presence of agents that influence arachidonic acid metabolism. Agents that decrease its production by blocking phospholipase A2 activity, i.e., quinacrine and 4-bromophenacylbromide, significantly decreased prolactin secretion from anterior pituitary glands in vitro and from dispersed pituitary cells in a perifusion column. Phospholipase A2 and phorbol myristate acetate, substances that increase intracellular concentrations of arachidonic acid, markedly stimulated prolactin release by dispersed pituitary cells and by anterior pituitary glands incubated in vitro. The involvement in prolactin secretion of arachidonic acid metabolic products produced via the lipoxygenase pathway was investigated indirectly using nordihydroguaiaretic acid (NDGA), a specific inhibitor of this enzyme. NDGA progressively (dose-related) inhibited the release of prolactin in vitro and blocked the stimulating effect of 50 nM TRH on prolactin release from hemipituitary glands. Indomethacin, a specific inhibitor of the cycloxygenase pathway, had no significant effect on basal and TRH-stimulated prolactin release. The results suggest that arachidonic acid metabolism is involved in basal and TRH-stimulated prolactin secretion and that lipoxygenase pathway products are at least partially responsible for these effects.