Effect of an anti-substance P serum on prolactin and gonadotropins in hyperprolactinemic rats

The effects of the acute injection of a rabbit anti-substance P serum (ASPS) were studied in normal rats and rats with hyperprolactinemia induced by 5-hydroxytryptophan and estradiol given as a short or chronic treatment. The anti-substance P serum decreased the release of prolactin induced by 5-hydroxytryptophan when this serotonin precursor was injected 24 h, but not 1 h, after the administration of the antiserum. ASPS reduced the hyperprolactinemia induced by short and chronic treatment with estradiol in castrated rats. This effect was observed 24 h after the injection of the antiserum. On the other hand, the injection of ASPS induced a significant decrease in LH levels in serum of intact male rats injected with 5-hydroxytryptophan 24 h after ASPS, and in castrated rats treated with short-term and chronic administration of estradiol, 24 h after the injection of the antiserum. These results suggest that substance P may have a role in the control of prolactin secretion and could play a part in the hyperprolactinemic effects of estradiol. On the other hand, substance P, under certain circumstances, may stimulate LH release.     

Effects of short photoperiod on the ability of golden hamster pituitaries to secrete prolactin and gonadotropins in vitro

Transfer of male golden (Syrian) hamsters from a 14L:10D (light:dark) to a 5L:19D photoperiod induced significant changes in pituitary function tested in vitro. Within 27 days after transfer to a 5L:19D photoperiod, basal prolactin (Prl) release was significantly depressed and response to dopamine (DA) was significantly enhanced as compared to Prl release by pituitaries from 14L: 10D hamsters. Follicle-stimulating hormone (FSH) release tended to be depressed after 9 or 27 days of 5L:19D exposure, but the effect was not significant. After 77 days of 5L:19D exposure, Prl release was further suppressed, while FSH release surpassed that seen in 14L:10D pituitaries. In vitro FSH response to luteinizing hormone releasing hormone (LHRH) was also enhanced at this time. After 15 weeks of exposure to a short photoperiod, FSH secretion was still elevated above control levels, but Prl release and Prl response to DA were no longer different from that of 14L: 10D controls. Secretion of luteinizing hormone (LH) in vitro, either basal or LHRH stimulated, was not affected by photoperiod at any time tested. From these results, we conclude that short photoperiod exposure does not reduce the pituitary's ability to secrete LH or FSH, although secretion of Prl is severely attenuated.     

Depletion and release of prolactin from rat pituitaries and pituitary tumors in vitro

Depletion of pituitary prolactin (PRL) and PRL release into culture medium were simultaneously examined over a 3.5- to 4.0-hr incubation period from anterior pituitary fragments obtained from Fischer-344 or Wistar-Furth female rats treated with estrogen for 5 days, in pituitary tumors induced by 8 weeks of diethylstilbestrol (DES) treatment in Fischer-344 rats and in MtTW15 pituitary tumors transplanted subcutaneously in Wistar-Furth rats for 4 weeks. Our objective was to determine if the event known as transformation, which we define as a loss in the tissue PRL content without a corresponding and equivalent increase in the medium PRL content, occurs in rat pituitary tumors. Our results indicated that transformation did not occur in vitro in rat anterior pituitary tumors induced in Fischer-344 rats by DES treatment but was present in pituitaries from Fischer-344 rats treated for 5 days with estrogen, which served as controls. We also observed in vitro transformation in the anterior pituitary of Wistar-Furth rats treated with estrogen for 5 days (controls) and in the pituitaries of Wistar-Furth rats inoculated with the MtTW15 tumor for 4 weeks, but not in the MtTW15 tumor itself. Although transformation was present in both Fischer-344 and Wistar-Furth rats treated acutely with estrogen the timing of the transformation was delayed 1-2 hr in the Fischer-344 rats compared with Wistar-Furth females. We concluded that transformation does not precede release of prolactin in rat pituitary tumors and that in normal pituitaries the mechanisms of transformation are induced differently between the strains of rats examined.     

In vivo and in vitro effects of cholecystokinin octapeptide on the release of prolactin in rats

The effect of cholecystokinin octapeptide (CCK-8) on the release of prolactin (PRL) in rats was studied in vivo and in vitro. Intravenous injection of 5 micrograms/100 g BW of CCK-8 resulted in significant increase in the plasma PRL level after 10 and 20 min. CCK-8 at concentrations of 10(-11) M to 10(-7) M also caused dose-dependent stimulation of PRL release from dispersed cells of rat anterior pituitary. On the other hand, dopamine inhibited PRL release from dispersed cells of rat anterior pituitary in a dose-related manner at concentrations of 10(-8) M to 10(-6) M. Release of PRL from the cells was increased by addition of K+ at high concentration (53 mM) in a Ca++-dependent manner. Addition of 10(-3) M verapamil to the incubation medium inhibited CCK-8-induced PRL release from the cells. Addition of dopamine (10(-7) M) to the incubation medium inhibited PRL release from the cells induced by CCK-8 or high K+ (53 mM). These results indicate that CCK-8 acts directly on the anterior pituitary cells to stimulate PRL release and that calcium ion is involved in the mechanism of this effect.     

Concentration of native prolactin and prolactin binding sites in hepatic subcellular fractions from hyperprolactinemic rats

Lactogen binding and prolactin content were measured in hepatic subcellular fractions from tumor-bearing rats (TBR; MtT/F4, MtT/W5, MtT/W10) with elevated prolactin and growth hormone levels and from control animals. Specific binding of 125I-oPRL to Golgi fractions from tumor-bearing animals was 2.5 to 7 fold greater than that from controls. Binding to plasmalemma was 6-fold greater in tumor-bearing rats. The specific binding of 125I-labelled bGH and insulin showed less marked differences between TBR and controls. Subcellular fractions were extracted with HCl to determine hormonal content. The content of prolactin and growth hormone in Golgi fractions from TBR was at least 20-fold that in fractions from controls. Rat prolactin extracted from Golgi heavy elements was 50% as effective as native material in binding to lactogen receptors as judged by radioreceptor assay. These studies demonstrate that the chronic elevation of prolactin was associated with an increase of receptors not only in the intracellular compartment but on the cell surface as well. Furthermore, they demonstrate that native prolactin is internalized and accumulated in rat liver Golgi fractions.     

Effect of treatment with cortisol in vivo on secretion of gonadotropins in vitro

To investigate the site of action of glucocorticoids in modulating secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from pituitaries of male rats, we implanted intact male rats with 250-mg pellets of cortisol (F) or cholesterol (C). Four days later, we collected and enzymatically dispersed the pituitaries. After the dispersed pituitaries had been in culture for 2 days, we treated the cells with gonadotropin-releasing hormone (GnRH) (0-150 nM) and determined the concentrations of LH and FSH in the medium after 6 h of incubation. Cells from donor animals pretreated with F secreted 30-60% more LH approximately 75% more FSH than cells from donor animals pretreated with C. This increase occurred regardless of the presence of F or C in the incubation medium in vitro. The slopes and ED50s of the GnRH dose-response curves were not altered. These data show that glucocorticoids have stimulatory effects on both LH and FSH. The inhibitory effects observed in vivo must be exerted by some mechanism that is not carried over to the in vitro model, and perhaps involve sites of action in addition to the pituitary.     

Blockade of the preovulatory release of gonadotropins and prolactin by spinal transections in female rats

The effect of spinal transections on the preovulatory release of gonadotropins and PRL was investigated in female rats. A preovulatory rise in serum LH, FSH and PRL and subsequent ovulation were prevented by complete spinal transections (CST) at high thoracic levels (T3-T7), but not at low thoracic and lumbar levels (T8-L5), performed at 1000-1230 h on proestrus. Norepinephrine (NE) concentrations in the preoptic-anterior hypothalamic area at 1700-1800 h on proestrus were also significantly reduced by CST at high thoracic levels, but not at lumbar levels. Either electrochemical stimulation of the suprachiasmatic part of the preoptic area or NE injection into the third ventricle at 1400-1500 h on proestrus restored ovulation in animals with CST at high thoracic levels. Animals with CST at lumbar levels exhibited relatively regular 4-day cycles, but those with CST at high thoracic levels showed prolonged periods of diestrous (8-20 days) before they resumed cyclicity. In the case of partial transections, bilateral transections of the lateral columns, but not transections of the dorsal or medial columns, of the spinal cord at T4-T5 significantly blocked the preovulatory gonadotropin release and the occurrence of ovulation. Unilateral transections of the lateral columns of the spinal cord or unilateral electrolytic lesions of the ventrolateral part of the medulla oblongata (VLMO) failed to block ovulation. When combinations of them were performed ipsilaterally, ovulation occurred, but when they were performed contralaterally, the incidence of ovulation was significantly decreased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of in vivo and in vitro treatment with L-carnitine on isolated hearts from chronically diabetic rats

The effects of in vivo and in vitro L-carnitine administration on cardiac function were studied in isolated perfused working hearts from control and diabetic rats. Injection of L-carnitine (3 g.kg-1.day-1, i.p.) for 2 weeks into rats previously diabetic for 6 weeks partially reversed the adverse effects of chronic diabetes on heart function. In a second experiment, a lower dose of L-carnitine (0.5 g.kg-1.day-1, i.p.) injected for 6 weeks prevented the onset of heart dysfunction in chronically diabetic rats. The protective action of L-carnitine in the myocardium appeared to be independent of any direct pharmacological effects. In both studies, L-carnitine was a potent lipid-lowering agent. The data suggests that L-carnitine administration at either dose had a protective effect against myocardial damage seen during diabetes. The mechanism(s) underlying these effects remains to be elucidated but are discussed.     

Effects of estradiol on the prolactin surges and the feedback mechanisms of gonadotropins in pseudopregnant rats

The influences of estradiol on the prolactin (PRL) surges and on the secretion of gonadotropins (LH and FSH) were investigated in the pseudopregnancy (PSP) of acutely ovariectomized rats. The four following experimental groups were prepared: 1) intact PSP as a control, 2) ovariectomy was performed on day 0 of PSP (OVX), 3) a Silastic tube containing estradiol was implanted for day 1-4 into the OVX rats (OVX-E 1-4), and 4) the Silastic tube was implanted for day 5-8 by the same manner into the OVX rats (OVX-E 5-8). In the OVX group nocturnal (N) PRL surges were observed at 0500 h on days 4, 8 and 12 examined, and increased secretions of LH and FSH were noted. In the OVX-E 1-4 group, the N PRL surge was suppressed on day 4, and the suppressed N PRL surge did not occur on day 8, after the removal of the implanted tubes. Diurnal (D) PRL surges with LH surges were observed at 1700 h on day 4 in these rats. Similarly, more remarkable results were obtained on days 8 and 12 in the OVX-E 5-8 group than in the OVX-E 1-4.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of arginine vasotocin on levels of plasma gonadotropins and prolactin in ovariectomized conscious rats

Arginine vasotocin was injected into the third ventricle or intravenously in conscious, ovariectomized rats and its effect on gonadotropin and prolactin release evaluated. The peptide lowered plasma levels of both LH and prolactin in doses of 40 or 100 ng given intraventricularly. The higher dose was slightly more effective than the lower dose. Intravenous injection of a 1-microgram dose of vasotocin failed to alter plasma LH in the ovariectomized animals; however, a 5-micrograms dose induced a slight depression apparent at only 60 min following injection. Intravenous injection of 1 microgram produced a significant lowering of plasma prolactin, whereas a dramatic lowering followed the injection of the higher dose. Plasma FSH was unaffected in these experiments. Incubation of dispersed anterior pituitary cells from ovariectomized rats with various doses of vasotocin revealed no effect of the peptide on the release of FSH, LH, or prolactin. It also did not alter the response to LHRH, but it partially blocked the action of dopamine to inhibit prolactin release. The data indicate that quite low doses of arginine vasotocin act within the brain to inhibit LH and prolactin secretion in ovariectomized, conscious animals.     

Effects of polyamines on the release of gonadotropin-releasing hormone and gonadotropins in developing female rats

Polyamines, putrescine (PUT), spermidine (SPD), spermine (SPM), and agmatine (AGM), are polycationic amines related to multiple cell functions found in high concentrations during the development of hypothalamus and pituitary. In previous works, we demonstrated that alpha-difluoromethylornithine (DFMO), an inhibitor of polyamines biosynthesis, induced a delay in puberty of female rats, accompanied by high, sustained follicle-stimulating hormone (FSH) levels during the infantile period. Also, DFMO treatment induced changes in polyamine concentration both in hypothalamus and pituitary of rats, mainly a decrease of PUT and SPD, an increase in SPM, and no change in AGM. In the present work, we investigated the direct effects of polyamines on the secretion of hypothalamic GnRH and pituitary gonadotropins in 6- and 15-day-old female rats. In 6-day-old animals, in vitro incubations with PUT, SPD, and AGM of hypothalami or anterior pituitaries were able to inhibit GnRH, FSH, and leutinizing hormone (LH) secretion, respectively. SPM showed a nonspecific transient inhibitory effect on FSH. When challenged with either high K(+) (hypothami) or GnRH (pituitaries), the tissues incubated in the presence of polyamines showed no differences when compared with their controls. No effects of polyamines in 15-day-old rats in either tissue were observed. Pituitary cell cultures of 6-day-old animals incubated with DFMO for 4 days showed a significant increase in FSH, but not in LH. We conclude that high PUT, SPD, and AGM levels during the first 10 days of life are important for the development of the hypothalamic-hypophyseal unit, probably related to an inhibitory effect on GnRH and gonadotropins. Therefore, polyamine participation, especially PUT and SPD, is of importance in the regulation of GnRH and gonadotropin secretion in the neonatal and infantile periods, critical stages in the establishment of sexual differentiation.     

An in vitro study of LH release, synthesis and heterogeneity in pituitaries from proestrous and short-term ovariectomized rats

It is known that acute ovariectomy (OVX) greatly attenuates the pituitary luteinizing hormone (LH) response to gonadotropin-releasing hormone (GnRH) in vitro. The present study evaluated possible quantitative and/or qualitative differences in the biosynthesis and secretion of LH in pituitaries from proestrous and acutely (72 h) OVX rats. Paired anterior pituitary glands were incubated for 4 h in a medium containing +/- 10 nM GnRH. Pituitary and secreted LH were measured by radioimmunoassay with differences in total LH (tissue plus medium) +/- GnRH being indicative of GnRH-stimulated LH synthesis. Qualitative changes in LH were evaluated by isoelectrofocusing (IEF). The results show that the major form of LH stored in and released from the pituitaries consisted of LH molecules with an isoelectric point (pI) in the alkaline pH range (alkaline LH), and a lesser amount (approximately 30%) of LH molecules in the acidic pH range (acidic LH). The ratio of alkaline/acidic LH observed in the pituitary and medium was similar in the proestrous and OVX groups, although the amount of alkaline and acidic LH release in response to GnRH was 2-3 times greater in the proestrous group. In both groups, the alkaline/acidic LH ratio of secreted LH was higher in the presence of GnRH than in its absence. Alkaline LH synthesis was increased by GnRH in both groups, with the response being greater in the proestrous than in the OVX group; GnRH-stimulated acidic LH synthesis was observed only in the proestrous group. In both groups, the amount of LH synthesized was about 60% of the amount released, which suggests that LH synthesis does not fully account for differences in GnRH-stimulated LH release. Treatment of pituitary extracts with neuraminidase decreased acidic LH, and proportionately increased alkaline LH. These results suggest that the quality of LH stored in and secreted from pituitaries of proestrous and OVX rats is similar, and that there is a preferential release of the major alkaline LH isoform in response to GnRH. The ovarian steroid environment, presumably estradiol, proportionately increases the amount of alkaline and acidic LH released, and differentially affects the amounts of the various isoforms synthesized in response to GnRH. The charge heterogeneity of alkaline and acidic LH may be related to the sialic acid content of the LH molecule.     

Ultrastructural studies on prolactin and growth hormone cells in Anguilla pituitaries in vitro

Summary Eel hemi-pituitaries were cultured in vitro on high or low sodium media which are known to affect differentially prolactin and growth hormone release. Ultrastructural examination of the prolactin cells after 24 h culture showed the Golgi bodies were markedly more abundant and widely distributed in hemi-pituitaries from the low sodium medium. Secretory granule release profiles and dense bodies were also more frequent, but the percentage of the cytoplasmic volume occupied by secretory granules was lower than on the high sodium medium. RER was only slightly modified. Significant differences were noted in the shape and processes of the non-granulated (stellate) cells of the RPD, but there were only slight differences in the ultrastructure of the somatotropes.     

Quantification of the cellular proliferation on freshly dispersed cells from rat anterior pituitaries after in vivo and in vitro labelling with bromodeoxyuridine.

Summary The labelling index i.e., the proportion of cells in S phase of the cell cycle, has been calculated in cytospin preparations of rat anterior pituitary cells after labelling eitherin vivo orin vitro with the thymidine analogue bromodeoxyuridine (BrdU). The aims of this work were (1) to check whether enzymatic digestion interferes with the incorporation of BrdU into S phase cells and/or whether it has any deletereous effect on the immunohistochemical detection of cells that have already incorporated BrdU, and (2) to check the viability of simultaneous staining for BrdU and markers for the different types of pituitary cells in the cytospins. No statistical difference was found between the labelling index afterin vivo orin vitro labelling with BrdU. Identification of doubly-immunostained cells was straightforward and up to 40% of BrdU-labelled cells were immunopositive for pituitary hormones. It is suggested that cytospin preparations from biopsy samples may be used to study cellular proliferation without exposing the patient to the hazardous effects of BrdU infusion and without the interference of cell culture methods.     

Effect of castration and steroid treatment on the release of gonadotropins by the rat pituitary-hypothalamus complex in vitro

Comparative in vitro studies on the release of LH and FSH by pituitary-hypothalamus complex (PHC) with intact portal plexus and whole pituitary (PI) from adult male rats showed that PHC released LH at a greater rate and in larger amounts than PI. PHC and PI released FSH in comparable amounts and rates. Attempts were made to correlate serum gonadotropin levels to that released by PHC and PI at 1, 3, 7, 14, 21 and 46 days of post-castration (PC). Sham operated animals served as controls. Castration increased serum LH and FSH levels but in different profiles. CPHC and CPI (PHC and PI from castrated rats) released less LH than NPHC and NPI (PHC and PI from sham operated controls) till day 14 PC after which CPHC and CPI released more LH than NPHC and NPI respectively. Castration abolished the intrinsic capacity of PHC to secrete more LH than PI. CPHC and CPI secreted significantly less FSH than NPHC and NPI at 1, 3 and 7 days PC. At days 14 and 21 of post-castration PCNCP or CPI and NPHC or NPI released similar amounts of FSH. Administration of 5 alpha-dihydrotestosterone (DHT, 1 mg/rat/day) or estradiol valerate (EV, 1 microgram/rat/day) immediately following castration prevented the rise in serum LH and FSH but increased the amounts of LH and FSH released by CPHC and CPI. The treatment caused a marked stimulation of FSH released by CPI.(ABSTRACT TRUNCATED AT 250 WORDS)