PRL release was studied in ovariectomized (OVX) rats pretreated with estradiol benzoate (EB), progesterone (P), or a combination of both steroids using a protocol that was selected to mimic ovarian steroid changes that have been observed during the female rat 4-day estrous cycle and early pregnancy. On the morning of the experiment, the animals received injections of either the dopamine (DA) antagonist domperidone (0.01 mg/rat iv) or vehicle (acetic acid in saline). Five minutes later, all animals received injections of the DA agonist 2-bromo-alpha-ergocryptine (CB-154; 0.5 mg/rat, iv) followed 60 min later by the administration of TRH (1.0 microgram/rat, iv). Plasma obtained from blood samples taken during the experiment was assayed for PRL by RIA. In OVX or P-treated OVX rats, a transient blockade of DA by domperidone did not alter the sensitivity of the pituitary to TRH administration, as measured by an increase in plasma PRL. However, such an effect of DA blockade was induced by 2 days of EB treatment and was maintained and amplified by P administration after EB injections. We conclude that enhancement of the PRL-releasing effect of TRH by DA antagonism, a mechanism we previously observed in female rats during midlactation, proestrus, estrus, and metestrus using the present drug protocol, can be induced by estrogen and maintained by P. Further, our data suggest that the previously observed loss of this secretory mechanism on the morning of diestrus may be due to the decrease in plasma P that takes place between metestrus and diestrus.

The effect of transient dopamine (DA) antagonism on the sensitivity of pituitary lactotrophs to the PRL-releasing effect of TRH was investigated in rats on days 3, 9, 15, and 21 of pregnancy. Each animal, bearing an indwelling intraatrial catheter, received injections of either the DA antagonist domperidone (0.01 mg/rat, iv) or saline at 0930 h on the day of the experiment. Five minutes later, all animals were given the DA agonist 2-bromo-alpha-ergocryptine maleate (CB-154; 0.5 mg/rat, iv), followed 60 min later by the administration of TRH (1.0 microgram/rat iv). Plasma samples obtained during the experiment were assayed by RIA for PRL and progesterone (P). The results showed that transient DA antagonism increased the sensitivity to TRH as a PRL-releasing stimulus on the morning of day 3 of pregnancy, but not on days 9 and 15. However, the response was present on day 9 in animals that were hysterectomized (HS) on day 6 of pregnancy. The increase in sensitivity of lactotrophs to TRH after DA blockade was observed on day 21 of pregnancy. Plasma levels of P were high on days 3, 9, and 15, but decreased markedly by day 21. In a second experiment, the anterior pituitary (AP) PRL content was determined on days 3, 9, 15, and 21 of pregnancy. The results demonstrated that AP PRL significantly decreased between days 3 and 9 of pregnancy in both intact and HS animals. However, AP PRL concentrations in animals killed on days 15 and 21 were significantly greater than that on day 9 but were not different from that observed on day 3 of pregnancy. We conclude that the ability to transform AP PRL to a TRH-releasable pool by the transient blockade of DA is present in early and late pregnancy, but is absent in midpregnancy. Since this secretory mechanism is retained on day 9 after hysterectomy on day 6 of pregnancy, it appears that the secretory products of the uterine-placental unit are inhibitory to transformation. Further, this inhibitory effect at midpregnancy cannot simply be the result of decreased AP PRL content or changes in plasma P. Finally, the return of the transformation mechanism on the day before parturition (day 21) may be due to the increase in estrogen secretion that occurs in late pregnancy, since we have previously shown that estrogen can induce this AP secretory mechanism.

The effectiveness of TRH in releasing PRL after transient dopamine (DA) blockade was investigated in female rats between days 3 and 11 of pseudopregnancy (PSP). At 0930 h on the morning of the experiment, each animal was injected with the DA antagonist domperidone (0.01 mg/rat, iv) or vehicle (acetic acid in saline); 5 min later, the DA agonist 2-bromo-alpha-ergocryptine maleate (CB-154; 0.5 mg/rat, iv) was administered. Sixty minutes later, TRH (1.0 micrograms/rat, iv) was administered. Blood samples were withdrawn via indwelling catheters before, 5, 20, 40, and 70 min after domperidone or vehicle administration, and 5 and 10 min after TRH administration. On day 3 of PSP, TRH-induced PRL release was significantly enhanced by the domperidone-CB154 treatment compared to that in vehicle-treated control rats. By day 9 of PSP, the effectiveness of TRH in stimulating PRL release after domperidone treatment was decreased by 50% compared to that on day 3 of PSP. This reduction in PRL response to TRH was not due to decreased progesterone levels, as no difference was observed in plasma progesterone between days 3 and 9. Rats that were given domperidone on day 11 of PSP did not exhibit a significant increase in sensitivity to TRH; however, the effectiveness of TRH was enhanced by domperidone on day 11 of PSP in animals that were hysterectomized on day 2 of PSP. Since DA receptor blockage increased the sensitivity to a putative PRL-releasing factor (TRH) and this mechanism was eliminated around the time that the PRL surges of PSP disappear, we suggest that this pituitary mechanism is a critical component of the PRL release mechanism during the surges of PSP. Further, the observed loss of the mechanism between days 9 and 11 of PSP may be due to the direct influence at the anterior pituitary of a uterine PRL inhibitory factor which has been recently described.

These experiments were conducted to test the hypothesis that the effectiveness of VIP in releasing prolactin is, like TRH, enhanced when preceded by a short period of dopamine receptor antagonism. Chronically catheterized, ovariectomized rats pretreated with estradiol benzoate and progesterone to mimic early pregnancy were used throughout these studies. In the first experiment, animals were injected either with the dopamine (DA) antagonist domperidone (DOM, 0.01 mg/rat, iv) or with vehicle (acetic acid in saline). Five minutes later, all animals were treated with the DA agonist 2-Br-a-ergocryptine maleate (CB-154,0.5 mg/rat, iv) followed 60 min later by the administration of thyrotropin-releasing hormone (TRH, 1.0 pg/rat iv) or vasoactive intestinal peptide (VIP, 25 pg/rat, iv). The injection of TRH following DOM treatment increased mean plasma PRL levels 100 ng/ml above levels found in vehicle-injected rats. VIP administration, however, increased PRL levels in the blood in DOM-treated rats only 6 ng/ml above the levels in vehicle-injected animals. The same treatment protocol was used in the second experiment except that the DA antagonist, sulpiride (0.01 mg/rat, iv) was administered instead of DOM, and CB-154 was not given. In this experiment both TRH and VIP released PRL. The response to TRH, but not to VIP, was significantly greater following sulpiride than in animals treated with sulpiride vehicle. In the third experiment animals were treated with DOM, VIP, DOM plus VIP, or vehicle. Five minutes later all rats received CB-154 injections, followed 60 min later by TRH administration. The final experiment was a replicate of the third except that sulpiride was substituted for domperidone and no CB-154 was given. The resulting data revealed that ( 1) dopamine antagonism enhanced the effectiveness of TRH but not VIP and (2) that VIP augmented the effectiveness of DA blockade on PRL release and was additive with domperidone (but not sulpiride) on increasing the responsivenss to TRH. However, VIP administration without concurrent administration of domperidone or sulpiride did not increase the effectiveness of TRH compared to vehicle-injected animals. From these data we concluded that VIP is a PRL-releasing hormone the effect of which is not affected by interruption in dopamine tone as is observed for TRH. Second, VIP can potentiate the stimulatory actions of at least one DA receptor antagonist and TRH on PRL release. This later finding suggests that VIP may play a modulatory role in the neuroendocrine regulation of PRL secretion in the female rat. 0 1988 Society for Experimental Biology and Medicine.34 1

Previous studies have shown that stimulation of cultured beating cardiac myocytes with calcitonin gene-related peptide (CGRP) produces increased beating frequency, increased cellular cAMP concentration, and a homologous desensitization of the cAMP-elevating action of CGRP. In the present study, the characteristics and regulation of [125I]CGRP binding sites in cultured cardiac myocytes were investigated. Binding of [125I] CGRP to membranes prepared from these cells was selective, saturable, and of high affinity. Scatchard transformation of the saturation isotherm generated a linear plot suggesting the existence of a homogeneous population of binding sites with an equilibrium binding constant of 41 +/- 7 pM and maximum binding capacity of 31 +/- 5 fmol/mg protein. Binding of [125I]CGRP to membranes was inhibited completely by guanosine 5'-(3-O-thio)triphosphate (250 microM), suggesting association of the binding sites with a G protein. Consistent with the saturation binding data, association kinetic studies indicated that [125I]CGRP associated with a single population of binding sites. Dissociation kinetic data, in contrast, indicated that [125I]CGRP dissociated from two affinity component sites on membranes, suggesting the existence of multiple affinity states of the G protein-coupled forms of the CGRP receptor. Nonequilibrium dissociation kinetic experiments revealed a time-dependent conversion of [125I] CGRP binding sites from a fast- to a slow-dissociating state. Desensitization of cells to CGRP by prior exposure to CGRP (10 nM) for 5 min reduced the maximal cAMP response of cells to further CGRP challenge and the number of [125I]CGRP binding sites in membranes prepared from these cells approximately 90% and 80%, respectively. These results demonstrate the existence of high affinity CGRP receptors in cardiac myocytes which appear coupled to G proteins and which undergo ligand-induced affinity alterations and desensitization-induced loss of receptor activity. The present findings also suggest the existence of multiple affinity states of the CGRP:receptor:G protein ternary complex.