The interaction of dopaminergic and serotonergic drugs on plasma prolactin in ovariectomized, estrogen-treated rats.

Pharmacologic agents that either block or stimulate dopaminergic or block serotonergic receptors were administered intraarterially alone or in combination to ovariectomized, estrogen-treated rats. Prolactin was estimated by radioimmunoassay of plasma collected by serial blood sampling from chronic aortic catheters. Apomorphine (10 mg/kg) depressed, whereas Pimozide (50 and 250 mug/kg) and Methiothepin (50, 500 and 5000 mug/kg) markedly increased prolactin levels. Apomorphine completely blocked the increases induced by the lowest does of Pimozide and Methiothepin, as well as the initial increases at the higher doses. However, at later time intervals after large doses of Pimozide and Methiothepin in apomorphine-pretreated rats there was a marked rebound of prolactin which was greater than the level induced by each drug alone. The serotonin antagonists SQ 10,631 and methysergide reduced the Pimozide-induced elevation in prolactin levels, suggesting that part of the increase in prolactin following dopaminergic blockage may result from the action of a tonically stimulatory serotonin system that is normally masked by the intense tonic inhibition by the dopaminergic system.

The influence of cholinergic, adrenergic, and serotonergic drugs on the afternoon surge of plasma prolactin in ovariectomized, estrogen-treated rats.

The effects of systemic administration of cholinergic, adrenergic, and serotonergic drugs on the afternoon surge of plasma prolactin was investigated using ovariectomized, polyestradiol phosphate (PEP)-injected rats bearing aortic catheters. Basal prolactin levels were elevated and similar after PEP administration for a period of 5 weeks, and an afternoon surge in plasma prolactin persisted for a period of 3 weeks before the magnitude of the surge diminished. The plasma estradiol levels were significantly higher for the 1100 and 1300 h samples than for the 1500 and 1700 h samples. Cornified vaginal epithelia were predominant in the vaginal smears of all animals throughout the 49-day experimental period. The cholinergic agonists arecoline, nicotine, and carbachol significantly inhibited the afternoon surge of prolactin. The muscarinic antagonist atropine resulted in a partial inhibition of the surge while the nicotinic antagonist mecamylamine did not have any inhibitory effect. The alpha-adrenergic blockers phenoxybenzamine and phentolamine and the beta-blocker propranolol inhibited the prolactin surge, with phenoxybenzamine being most effective. The administration of the serotonergic antagonist methysergide resulted in only a partial blockade of the afternoon prolactin surge. The data suggest that both the adrenergic and serotonergic systems may have a positive input in the afternoon surge of plasma prolactin. It appears that the cholinergic system does not play a significant role in the afternoon surge.

The influence of raphe lesions, p-chlorophenylalanine, and ketanserin on the estrogen-induced afternoon prolactin surge.

Female Sprague-Dawley rats were ovariectomized and 2 weeks later injected sc with 100 micrograms polyestradiol phosphate, a long-acting estrogen, to induce the afternoon PRL surge. In one series of experiments, the dorsal raphe (DRN), median raphe (MRN), and median raphe-pontine (MRN-RPn) regions were lesioned using a radiofrequency lesion maker set at 56 degrees C for 1 min. Lesions were induced, and atrial catheters were implanted on the day of estrogen injection. Six days later, blood samples were obtained every 2 h from 1100-2100 h to monitor the afternoon PRL surge. The animals were killed, and the hypothalami were quickly frozen on dry ice and stored at -60 degrees C for determination of amine content using HPLC with electrochemical detection. The rest of the brain was fixed and sectioned to verify the location of brain lesions. Complete lesions of the DRN markedly attenuated the PRL surge (P less than 0.001) compared to the effect of sham or incomplete lesions. MRN lesions did not significantly alter the PRL surge; however, MRN lesions that included the RPn significantly (P less than 0.001) attenuated the afternoon PRL surge. Significant decreases in serotonin (5-HT) and 5-hydroxyindoleacetic acid concentrations were observed in the arcuate, ventromedial, suprachiasmatic, and medial preoptic nuclei of the hypothalamus, but not in the median eminence of the lesioned rats. DRN and MRN-RPn lesions decreased the 5-HT concentration in the ARC and MPN compared to sham lesion and DRN incomplete lesion values. Other hypothalamic areas did not show a similar effect of the lesions. The concentrations of norepinephrine, dopamine, or dihydroxyphenylacetic acid were not altered by the lesions. In a second series of experiments one group of animals was injected ip with 10 mg/kg ketanserin, a serotonergic antagonist, at 1200 h on the sampling day. A second group was given 250 mg/kg p-chlorophenylalanine (PCPA) at 1700 h for 2 days before blood sampling was initiated on the third day. Both ketanserin and PCPA completely blocked the PRL surge. In the PCPA-injected animals, the 1100 and 1300 h PRL values were significantly higher (P less than 0.01) than those in vehicle-injected controls. Animals injected with ketanserin at 1200 h had a 1300 h PRL value significantly higher (P less than 0.01) than was observed in vehicle-injected animals.(ABSTRACT TRUNCATED AT 400 WORDS)

Central nervous system regions involved in the estrogen-induced afternoon prolactin surge. II. Implantation studies.

In the intact cycling female rat, there is a surge of plasma PRL during the afternoon of proestrus. Ovariectomy on diestrous day 1 eliminated the PRL surge completely, and injection of 100 micrograms polyestradiol phosphate, a long-acting estrogen, not only maintained the surge, but amplified and prolonged it. Bilateral implantation of an estradiol (E2)-containing cannula [diluted 1:4 with cholesterol (C)] in the medial preoptic area (MPO), but not in the cerebral cortex (CC), also maintained the surge. In long term ovariectomized rats, bilateral implantation of E2-containing cannulae (1:5, 1:10, 1:20, and 1:200) in the corticomedial amygdala (CMA) or ventromedial nucleus of the hypothalamus or a singular implantation in the third ventricle failed to induce a PRL surge 12 days later. Similar singular implantation in the anterior pituitary (AP) increased the basal levels of plasma PRL compared to those in C-implanted controls, but no surge was evident. Only bilateral implantation of E2 in the MPO induced a small but significant rise of plasma PRL at 1700 and 1900 h. Using higher concentration implants with a higher E2 concentration (1:4) in the MPO and sampling at shorter intervals, significant afternoon PRL surges were induced on days 2-4. However, systemic effects of E2, i.e. vaginal cornification and uterine weight enlargement, were also evident. Similar implants in other brain regions had the same results. Further increasing the E2 to C dilution from 1:10 to 1:200 eliminated the systemic effect of E2 implants, while the PRL surge-inducing ability persisted. It appears that the highest diluted E2 implants (1:150 and 1:200) gave the highest PRL response and persisted for the greatest number of days. Using the highly diluted E2 implants (1:100 and 1:200) in various brain regions, the MPO and the ventromedial hypothalamus were the most sensitive areas in inducing the PRL surge; the other areas studied, including suprachiasmatic nuclei, CMA, AP, and CC were ineffective. In conclusion, highly diluted E2 implants in the brain appear to be effective in obtaining a specific effect on the afternoon PRL surge; the CMA, suprachiasmatic nuclei, AP, and CC are not estrogen feedback sites for the induction of PRL release, and the MPO was the most sensitive area of the estrogen action in the brain regions examined for the induction of the afternoon PRL surge.

Central nervous system regions involved in the estrogen-induced afternoon prolactin surge. I. Lesion studies.

Central nervous system regions were examined in long term ovariectomized rats to determine if they are involved in the estrogen-induced afternoon surge in plasma PRL. Adult female rats were ovariectomized 2-3 weeks before bilateral radiofrequency or electrolytic lesions of the brain were placed on day 0. In short term lesion studies, catheterizations and sc injections of polyestradiol phosphate (PEP) were done after the lesion was made; blood sampling was performed on day 2, 3, 4, or 6. In long term lesion studies, the catheterization and PEP injection were done on day 21; blood was collected on day 28. In short term experiments, extensive lesions in the medial preoptic area/suprachiasmatic nuclei (MPO/SCN) completely blocked the PEP-induced afternoon PRL surges sampled on days 2, 3, 4, and 6, while bilateral lesions in the corticomedial amygdala (CMA) had no effect. Discrete bilateral lesions of either MPO or SCN eliminated the afternoon PRL surge on day 6. Discrete, yet complete, lesions of the ventromedial nuclei of the hypothalamus also blocked the PRL surge; however, lesions in the dorsomedial nuclei of the hypothalamus increased, the magnitude of the afternoon PRL surge. In long term studies, lesions of the CMA delayed and attenuated the PEP-induced PRL surge, and lesions of the stria terminalis for 4 weeks had a similar effect. As in the short term lesion studies, long term lesions of the MPO/SCN eliminated the daily rhythm of PRL secretion, although small sporadic rises in plasma PRL levels could be observed throughout the sampling period. It can be concluded that structural integrity of the MPO/SCN and ventromedial hypothalamic nuclei is essential for the estrogen-induced afternoon PRL surge; destruction of the dorsomedial hypothalamic nuclei can increase the magnitude of the afternoon PRL surge; and the CMA is not essential for induction of the PRL surge even though removing its neural input to the hypothalamus for an extended period can delay the onset of and suppress the magnitude of hormone release.

The influence of adrenergic, dopaminergic, cholinergic and serotoninergic drugs on plasma prolactin levels in ovariectomized, estrogen-treated rats.

Levels of plasma prolactin were estimated in ovariectomized, estrogen-treated rats following the systemic administration of several neural blocking and stimulating drugs. Phenoxybenzamine, an alpha-adrenergic blocker, at high doses, increased plasma prolactin. Phenotlamine, another alpha-adrenergic blocker, and propranolol, a beta-adrenergic blocker, also increased prolactin but the responses were small and transient. Clonidine, an alpha-adrenergic stimulating drug, elevated prolactin levels whereas the beta-adrenergic stimulator isoproterenol had no effect. Dopaminergic blockade by pimozide increased levels of prolactin while stimulation of dopamine receptors by apomorphine decreased prolactin release. Atropine (a muscarinic chilinergic blocker), arecoline (a muscarinic stimulator) and nicotine (a nicotinic cholinergic stimulating drug) did not affect basal prolactin levels. Mecamylamine (a nicotinic blocker) produced a small transient elevation in plasma prolactin. Methiothepin, an alleged serotoninergic blocker, markedly increased prolactin secretion, as did serotonin. The data suggested the involvement of several neurotransmitters in the control of basal secretion of prolactin.

Identification by analytical flow cytometry of prolactin receptors on immunocompetent cell populations in the mouse.

Five monoclonal antibodies (T1, T6, U5, U6, and E21) made to the external portion of the rat PRL receptor (PRL-R) were conjugated to fluorescein isothyrocynate and used to examine the presence of PRL-R on mouse lymphocytes and macrophages using analytical flow cytometry. The monoclonals were initially evaluated using Nb2 cells, a cloned line from a rat lymphoma, and NOG-8 cells, a cloned line from normal mouse mammary gland tissue, which are known to have PRL-R. All monoclonal antibodies bound to these cells, but the U5 monoclonal gave the best separation from unstained cells. CTLL-2 cells, a mouse lymphoma cell line containing interleukin-2 receptors, did not bind to any of the monoclonals. Isolated thioglycolate-induced peritoneal macrophages contained PRL-R, and the PRL-R monoclonal U5 gave the best separation from unstained cells. Eighty-five percent of macrophages constitutively had PRL-R using this monoclonal. In vivo stimulation of the popliteal lymph node by the injection of Concanavalin-A (Con-A) into the right foot pad of intact and ovariectomized (OVX) BALB/c mice induced, at the end of 10-12 h, a marked increase in interleukin-2 receptor (IL-2R) expression on CD4, CD8, and B-cells compared to the unstimulated left popliteal lymph node. The number of CD4 and CD8 cells from OVX animals with IL-2R was twice that from intact animals, whereas no difference in the percentage of B-cells with IL-2R was evident. PRL-R were constitutively expressed on 5% of the CD4 cells and 20% of the CD8 cells and were increased in the Con-A-stimulated lymph node when examined with the U5 PRL-R monoclonal. A higher percentage of CD4 and CD8 cells from OVX animals constitutively expressed PRL-R, and when stimulated with Con-A, a further increase was observed compared to the level in intact animals. Using the U5 monoclonal, over 80% of the B220 cells constitutively expressed PRL-R; however, when T1, T6, and U6 monoclonals were examined, the percentage was considerably below (20%) than that observed for U5. Con-A stimulation did not alter the percentage of B220 cells expressing PRL-R. These results show the importance of identifying lymphocyte subsets and examining a number of PRL-R monoclonals in determining lymphocyte PRL-R expression on the surface of the cell.

The influence of blinding, olfactory bulbectomy, and pinealectomy on twenty four-hour plasma prolactin levels in normal and neonatally androgenized female rats.

The influence of blinding (BLD), olfactory bulbectomy (ANOS), and pinealectomy (PX) on 24-h plasma PRL levels was examined in normal and neonatally androgenized (NA) female rats. NA pups were treated at 3 days of age with 1.25 mg testosterone propionate. Surgery was performed on both NA and normal animals at 22-25 days of age. At 10 weeks of age, animals were vaginally smeared, and at 15 weeks, they were ovariectomized, injected with 0.5 mg polyestradiol phosphate, and fitted with atrial catheters for blood sampling. At 16 weeks of age, blood samples were taken over a 24-h period. In normal animals, BLD plus ANOS resulted in half of the animals exhibiting a diestrous vaginal smear, while the other half exhibited at least one vaginal estrus during the 7-day period when vaginal smears were obtained. Ovarian, oviductal, and uterine weights for blinded (BLD) plus olfactory bulbectomized (ANOS) animals exhibiting diestrous smears were significantly less than those of BLD plus ANOS animals exhibiting periodic vaginal estrus. Plasma PRL levels were lower in both animal groups compared with those in sham controls, but BLD plus ANOS animals with small ovaries had significantly lower plasma PRL and BLD plus ANOS animals with large ovaries. PX of BLD plus ANOS animals resulted in endocrine organ weights comparable to those in controls and elevated the plasma PRL levels to those observed in BLD plus ANOS animals with large ovaries. The PRL levels in BLD, ANOS, and pinealectomized animals, however, were still below those in sham control animals. Sensory deprived animals had peak PRL values at various times of day, and regrouping of the data from the peak PRL time for each animal suggested the presence of a free-running plasma PRL rhythm. In NA animals, a decrease was also observed in endocrine organ weights and plasma PRL for BLD plus ANOS animals similar to that observed for normal BLD plus ANOS animals. However, all animals in the NA BLD plus ANOS group responded with a constant diestrous smear, small ovaries, and low plasma PRL values. PX prevented the decrease in endocrine organ weights and plasma PRL observed in BLD plus ANOS animals. As in normal animals, NA animals also exhibited a free-running PRL rhythm when sensory deprived.(ABSTRACT TRUNCATED AT 400 WORDS)

The effect of transient dopamine antagonism on thyrotropin-releasing hormone-induced prolactin release in pregnant rats.

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 effects of transient dopamine antagonism on thyrotropin-releasing hormone-induced prolactin release in pseudopregnant rats.

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.

The effect of transient dopamine antagonism on thyrotropin-releasing hormone-induced prolactin release in ovariectomized rats treated with estradiol and/or progesterone.

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.

Pituitary gonadotropin-releasing hormone (GnRH) receptor responses to GnRH in hypothalamus-lesioned rats: inhibition of responses by hyperprolactinemia and evidence that testosterone and estradiol modulate gonadotropin secretion at postreceptor sites.

Increased hypothalamic GnRH secretion appears to influence positively the number of pituitary GnRH receptors (GnRH-R). GnRH-R increase after castration in male rats, and this rise can be prevented by testosterone (T), anti-GnRH sera, or hypothalamic lesions. GnRH also increases serum LH and GnRH-R in hypothalamus-lesioned rats, and these animals injected with exogenous GnRH are, therefore, a good model in which to study the site of steroid feedback at the pituitary level. Adult male and female rats were gonadectomized, and radiofrequency lesions were placed in the hypothalamus. Males received T implants, and females received estradiol implants at the time of surgery. Empty capsules were placed in the control animals. Beginning 3-5 days later, animals in each group were injected every 8 h with vehicle (BSA) or GnRH (0.002-200 micrograms/day) for 2 days. After these GnRH injections, all rats received 6.6 micrograms GnRH, sc, 1 h before decapitation to determine acute LH and FSH responses. GnRH-R were determined by saturation analysis using 125I-D-Ala6-GnRH ethylamide as ligand. In males, GnRH injections increased GnRH-R. T inhibited acute LH and FSH responses to GnRH in all groups, but had little effect on GnRH-R, indicating that T inhibits gonadotropin secretion at a post-GnRH receptor site. In females, the GnRH-R response to GnRH was less marked, and only the 200 micrograms/day dose of GnRH increased GnRH-R, indicating that the positive feedback effects of estradiol at the pituitary level are also exerted at a site distal to the GnRH receptor. There was no positive correlation between the number of GnRH-R and GnRH-stimulated gonadotropin release in males or females. Female rats with hypothalamic lesions had markedly elevated serum PRL levels (greater than 300 ng/ml). Suppression of PRL secretion by bromocryptine resulted in augmented GnRH-R responses to GnRH, and GnRH-R concentrations rose to the same values induced in males. This suggests that hyperprolactinemia inhibits GnRH-R responses to GnRH in females by a direct action on the pituitary gonadotroph.

Influence of the pineal gland, olfactory bulbs and photoperiod on surges of plasma prolactin in the female rat.

The role of the pineal gland, olfactory bulbs and photoperiod in the regulation of the two daily surges of plasma prolactin in the pseudopregnant rat has been investigated. Pinealectomy had no effect on the surges of prolactin in pseudopregnant rats maintained on either a long (14 h light : 10 h darkness; 14L : 10D) or a short (2L : 22D) photoperiod, but olfactory bulbectomy decreased the nocturnal surge in animals maintained on 14L : 10D. This effect of bulbectomy was eliminated if the rats maintained on 14L : 10D were also pinealectomized. After cervical stimulation, bulbectomized rats maintained on a 2L : 22D photoperiod had nocturnal-type prolactin surges similar to those of intact rats maintained on the same photoperiod. These results indicate that the pineal gland andlength of photoperiod are not involved in the regulation of the surges of plasma prolactin in pseudopregnant rats but that the olfactory bulbs may enhance the nocturnal surge.

Influence of restraint on plasma prolactin and corticosterone in female rats.

The effect of restraint on plasma prolactin and corticosterone concentrations was investigated in chronically catheterized, ovariectomized (OVX) or ovariectomized, oestrogen-treated (OVX-PEP) rats. Restraint was induced by tying the hind legs together. In OVX rats, prolactin levels were unchanged following restraint, either during the morning (10.00 h) or afternoon (14.00 h). Prolactin levels increased in OVX-PEP animals when restraint was initiated in the morning; when restraint was initiated in the afternoon the prolactin response depended upon the level of prolactin before restraint. If levels were low (pre-surge) the response to restraint was similar to that observed in the morning; if prolactin levels were high (surge) the response to restraint was reversed and the prolactin level declined. The morning prolactin response to restraint was significantly inhibited and the afternoon surge was retarded in adrenalectomized OVX-PEP (OVX-PEP-ADX) rats; however, in OVX-PEP animals maintained on 0-9% NaCl drinking solution, the morning prolactin response to restraint was also blunted, although the afternoon surge was normal. In OVX-PEP-ADX animals injected with either vehicle alone, or 2 or 4 mg corticosterone for 4 days, and sampled on the morning of day 5, the prolactin response to restraint was absent. Furthermore, when OVX-PEP animals were injected daily with either vehicle or 4 mg corticosterone/day, they showed no increase in prolactin in response to restraint when values were compared with those of uninjected animals. Corticosterone levels after restraint were higher than initial values in all of the above experimental conditions.

Influence of anaesthetics on basal, perphenazine-induced and thyrotrophin releasing hormone-induced prolactin secretion in ovariectomized, oestrogen-treated rats.

Plasma levels of prolactin were determined, by radioimmunoassay, in ovariectomized, oestrogen-treated rats after administration of ether, sodium pentobarbitone, urethane, chloral hydrate or ketamine. These anaesthetics, when administered alone, induced sustained increases in the plasma level of prolactin (continuous ether inhalation), no change in prolactin secretion (urethane), or depressions in the level of prolactin (sodium pentobarbitone, chloral hydrate and ketamine). These same anaesthetics when given before perphenazine failed to alter the stimulatory effect of this phenothiazide on prolactin secretion. Sodium pentobarbitone did not alter the normal increase in prolactin concentration after intra-arterial administration of synthetic thyrotrophin releasing hormone (TRH). These results indicated that anaesthetics do not affect the response of either the central nervous system or the anterior pituitary to perphenazine or TRH although they affect prolactin secretion when administered alone. The site of action of anaesthetics must, therefore, be different from that of perphenazine or perphenazine must be capable of overcoming their influence by direct action on the pituitary.

The occurrence of pituitary prolactin depletion-transformation in lactating rats: dependence on strain of rats, homogenization conditions and method of assay.

The objectives of this study were to determine whether pre-release transformation (depletion) of pituitary prolactin occurs as the result of suckling to the same extent in several strains of lactating rats, the molecular nature of the transformed hormone, whether the quantity of transformed (depleted) prolactin recovered is dependent upon the type of homogenization buffer used and whether the method of assay influences the extent to which transformed prolactin is detected. During the course of these experiments other factors such as the methods of handling and storing pituitaries and homogenates were also found to influence the amount of prolactin recovered. The results indicated that transformation of prolactin is a very labile event which is affected by many factors. Strain and supplier of rats was critical to the observation of suckling-induced depletion of prolactin, with Spartan- and Holtzman-derived Sprague-Dawley strains exhibiting the most consistent responses. When transformation was observed, it mattered little which buffer was used for homogenization; however, alkaline or acidic buffers extracted more prolactin than did neutral buffers. Triton X-100 also significantly enhanced the efficiency of extraction by neutral buffers. Maintaining pituitaries on dry ice immediately upon removal from the animal increased the amount of prolactin recovered, as did freezing the homogenate for 1-5 weeks before assay. The addition of the protease inhibitor, benzamidine hydrochloride, did not affect the pituitary content of prolactin. Assay of prolactin by polyacrylamide electrophoresis and densitometry yielded more prolactin than either radioimmunoassay or the Nb2 lymphoma bioassay. The molecular nature of pituitary prolactin, extracted at neutral pH, as judged by gel filtration was altered slightly but consistently by suckling, such that large molecular forms increased at the expense of the smallest molecular form. We conclude from these studies that great care must be exercised when attempting to characterize dynamic changes in pituitary prolactin content in lactating rats. Strain and supplier of rats, methods of handling and storing pituitaries, types of buffers used for homogenization and methods of assay all influence the amount of prolactin recovered and can influence the extent to which rapid changes in pituitary prolactin are detected.

Prolactin modulates the incidence of diabetes in male and female NOD mice.

The nonobese diabetic (NOD) mouse develops diabetes spontaneously due to autoimmune destruction of the pancreatic islets with a higher incidence in the female than the male. Prolactin (PRL), a hormone whose role has been previously focused on reproduction and lactation has been demonstrated to influence immune responses. In this study, we investigated the effect of hypoprolactinemia and hyperprolactinemia on the incidence of diabetes in male and female NOD mice. Our hypoprolactinemia model was induced from the time of weaning (21 days of age) to 112 days of age by daily injections of 200 micrograms of bromocriptine (CB-154). A hyperprolactinemic model was induced by a syngeneic anterior pituitary transplant (APT) to the kidney capsule at 35 days of age and maintained until 112 days of age. Additional experimental groups were also investigated. A group of males received pituitary transplants combined with daily subcutaneous injections of CB-154. A group of females treated with CB-154 was also given daily subcutaneous injections of 30 micrograms of oPRL. An ovariectomized (OVX-Control) group of females was also established to serve as a second control for the OVX-APT group. Bromocriptine administration did not significantly decrease plasma PRL levels compared to controls (CTRL) while APT animals had plasma PRL levels that were significantly higher (P < 0.01) than those of CTRL and CB-154 animals. These differences were observed in animals of both sexes. Bromocriptine treatment of APT groups significantly lowered plasma PRL levels from their respective controls. Plasma PRL from the OVX-Control group was markedly lower than the intact female control. The incidence of diabetes was significantly lower in female mice receiving CB-154 injections compared to the intact female CTRL group at 84, 98 and 112 days of age.(ABSTRACT TRUNCATED AT 250 WORDS)

The lymphocyte and macrophage profile in the pancreas and spleen of NOD mice: percentage of interleukin-2 and prolactin receptors on immunocompetent cell subsets.

The NOD mouse is a model for autoimmune diabetes that develops symptoms similar to Type I diabetes. The incidence of diabetes is greater in females but the degree of insulitis is comparable in both sexes. The purpose of this study was to assess the populations of lymphocytes and macrophages in the pancreas and spleen of NOD mice. Comparisons were made between male and female; young (32-40 days old) and old (197-297 days old); diabetic and non-diabetic mice. Using analytical fluorescent cell cytometry we quantitated the percentages of CD4, and CD8 T-cells, B-cells and macrophages and the percentages of these subsets expressing interleukin-2 (IL-2R), prolactin (PRLR) and Hi-intensity PRL (Hi-PRLR) receptors. Evaluation of T-splenocytes indicated a 2:1 ratio of CD4 to CD8 T-lymphocytes in the spleen. The pancreas had higher percentages of all of the subsets in the old male and female groups compared to their young counterparts. Pancreatic immunocompetent cell subsets expressed lower percentages of IL-2R, PRLR and Hi-PRLR compared to splenocytes. The results did not demonstrate any dramatic differences in the immunocompetent cell populations of the spleen or pancreas between male and female animals, however we were able to establish the percentage of immunocompetent cells with IL-2R, PRLR and Hi-PRLR as a reference for future studies.

Temporal and anatomical considerations involved in the effects of amygdaloid lesions on prolactin secretion in ovariectomized estrogenized rats.

Plasma prolactin (PRL) levels were investigated in chronically catheterized control, sham-lesioned, and amygdala (AMYG)-lesioned rats under a variety of conditions. Ovariectomized (OVX) rats injected with 0.5 mg of polyestradiol phosphate (PEP) exhibited a circadian rhythm of PRL secretion characteristic of proestrus rats. Small bilateral lesions of the central AMYG area had no effect on this pattern of PRL secretion in animals bled over a 24-h period, 1 week following lesioning. A second group of rats bearing lesions or sham lesions of the corticomedial AMYG nuclei were studied at 1, 2 and 3 weeks following surgery and PEP treatment. PEP produced a significant afternoon surge of PRL in controls at weeks 1 and 2 but not week 3. Lesions of the corticomedial AMYG nuclei had no effect on basal PRL, but significantly blunted the afternoon surge occurring at weeks 1 and 2. The effect of a larger lesion encompassing a greater proportion of the corticomedial AMYG was examined in another group of rats at 6 weeks post-lesion and 2 weeeks after PEP injection. This lesion significantly decreased PRL levels compared to controls at 5 of 6 time periods examined over a 24-h period. The effect of this lesion on PRL secretion in non-estrogenized OVX rats was also examined. There was no difference in the low, non-fluctuating 24-h PRL levels of control and lesioned rats bled at 3 or 4 weeks post-lesion.