Showing papers in "Endocrinology in 1981"

Frequency and Amplitude of Gonadotropin-Releasing Hormone Stimulation and Gonadotropin Secretion in the Rhesus Monkey

Abstract: In adult ovariectomized rhesus monkeys bearing hypothalamic lesions which reduced circulating LH and FSH to undetectable levels, sustained elevated gonadotropin concentrations were reestablished by the intermittent administration of gonadotropin-releasing hormone (GnRH) at the rate of 1 microgram/min for 6 min once every hour. The effects of varying either the frequency or the amplitude of these GnRH pulses on gonadotropin secretion were examined in such animals. Increasing the frequency of GnRH administration from the physiological one pulse per h to two, three, or five pulses h while maintaining a constant infusion rate and pulse duration resulted in gradual declines in plasma gonadotropin concentrations. These declines were most profound at the highest frequencies and the consequence of reduced pituitary responses to individual GnRH pulses. Decreasing the frequency of GnRH pulses from one per h to one every 3 h led to variable declines in plasma LH levels, but circulating FSH invariably rose. Reducing the GnRH infusion rate from 1 to 0.1 mg/min while maintaining constant frequency and pulse duration resulted in abrupt declines in plasma LH and FSH to immeasurable levels, although pulsatile increments in circulating GnRH concentrations without a concomitant reduction in plasma LH concentrations, which remained unchanged. An infusion rate of 0.5 microgram/min resulted in unstable plasma LH and FSH levels. These results demonstrate that changes in the frequency or amplitude of hypophysiotropic stimulation have profound effects on plasma gonadotropin levels as well as on FSH to LH ratios in the circulation. The physiological implications of these observations are discussed.

Characterization of Several Clonal Lines of Cultured Ley dig Tumor Cells: Gonadotropin Receptors and Steroidogenic Responses*

Abstract: Several clonal lines of cultured Leydig tumor cells have been established and characterized in terms of gonadotropin receptors and steroid production. Although freshly isolated cells derived from the M5480P tumor have functional hCG receptors, only two of the five clonal lines established were shown to bind significant quantities of hCG. In these clones, steroid production can be stimulated to the same extent by hCG, cholera toxin, and 8-Br-cAMP. The other three clones bind a small amount of hCG and respond to the hormone with a marginal increase in steroidogenesis. Steroid production, however, is significantly stimulated by cholera toxin or 8-Br-cAMP. A comparison of the steroids produced by freshly isolated cells and two of the clones revealed some changes in the steroidogenic pathway. The most obvious change is an increase in the ability of the cultured cells to synthesize 20α-dihydroprogesterone (20α-hydroxypregn-4-en-3-one). These clonal lines may provide a suitable model system for the study of gona...

1,25-dihydroxyvitamin D3 and malignant melanoma: the presence of receptors and inhibition of cell growth in culture.

Abstract: In this study we demonstrate the presence of specific, high-affinity receptors for 1,25-dihydroxyvitamin D3 in malignant melanoma. Receptors are present both in cultured melanoma cells and in melanoma tumor tissue produced by inoculation of cells into athymic rats. The receptor sediments at 3.25 on sucrose density gradients, possesses a preferential affinity for 1,25-(OH)2D3 and has an apparent Kd of 0.18 nM by Scatchard analysis. We also demonstrate that human melanoma cells are responsive to 1,25-(OH)2D3 in vitro. Inclusion of 1,25-(OH)2D3 in the culture medium produced a marked increase in cell doubling time. This inhibitory effect of the hormone on melanoma cell proliferation was dose-related and represents the first demonstration of a 1,25-(OH)2D3 mediated action on tumor cells.

Direct inhibitory effect of glucocorticoids upon testicular luteinizing hormone receptor and steroidogenesis in vivo and in vitro.

Abstract: The direct effects of glucocorticoids on testicular LH receptor content and steroidogenesis were studied in vivo and in vitro. Immature hypophysectomized rats were treated with varying doses of dexamethasone, corticosterone, or a synthetic progestin, 17,21-dimethyl-19-nor-pregna-4,9-diene-3,20-dione (R5020). Some animals were also treated concomitantly with FSH to prevent the hypophysectomy-induced decrease in testis functions. At the end of 5 days of treatment, testicular LH/hCG receptor content was measured by [125I]hCG binding assay while steroidogenic responsiveness was measured by in vitro incubation of testes. Dexamethasone decreased testicular LH receptor in control and FSH-treated hypophysectomized rats in doses as low as 10 microgram/day, whereas corticosterone (10 microgram/day) decreased testicular LH receptor in the FSH-treated rats but had no effect in rats not treated with FSH. In contrast, R5020 had no effect on testicular LH receptor content. In vivo treatment of hypophysectomized rats with FSH increased both basal and hCG-stimulated production of androstanediol in vitro. In contrast, concomitant treatment with dexamethasone, but not R5020, decreased both basal and hCG-stimulated testicular androstanediol production. The direct effect of glucocorticoids on testicular steroidogenic potentials was also studied in primary culture of testicular cells obtained from adult hypophysectomized rats. Treatment of cultured testicular cells wtih hCG increased testosterone production. The addition of various natural and synthetic glucocorticoids, but not R5020, to hCG-treated cells decreased testosterone production in a dose- and time-related manner (triamcinolone greater than or equal to dexamethasone greater than cortisol greater than or equal to corticosterone). A 40% decrease in testosterone production was apparent at 6 h after addition of 10(-7) M dexamethasone to hCG-treated cells. These results demonstrate the direct inhibitory effect of glucocorticoids on testicular LH receptor content and steroidogenesis, suggesting the adrenal glucocorticoids may regulate testis functions.

Insulin enhancement of luteinizing hormone and follicle-stimulating hormone release by cultured pituitary cells.

Abstract: The role of insulin in the regulation of basal and gonadotropin-releasing hormone (GnRH)-stimulated release of LH and FSH was investigated in vitro using primary cultures of rat anterior pituitary cells from adult ovariectomized rats. Anterior pituitary cells were incubated for 2 days in the presence or absence of insulin in a serum-free medium. At the end of the insulin treatment, the cells were washed and reincubated in the presence or absence of GnRH, and the LH and FSH released into the medium were measured by RIA. Treatment with insulin (1.0 microgram/ml) for 2 days resulted in significant increases in both the basal and the maximal release of LH and FSH, as well as a 3.2- and 6.3-fold decrease in the ED50 values for GnRH in terms of LH and FSH release, respectively. Treatment with increasing concentrations (0.1-10,000 ng/ml) of insulin, led to a dose-dependent increase in the GnRH (3 X 10(-10) M)-stimulated release of both LH and FSH. This effect of insulin was significant (P less than 0.05) at a physiological concentration of 1 ng/ml (24 microU/ml) with an ED50 value of 40 ng/ml. Increasing duration of exposure to insulin resulted in time-dependent increases in the GnRH (3 X 10(-10) M)-stimulated release of LH, becoming significant at 24 h with maximal enhancement observed by 48 h. The effect of insulin was specific; epidermal or fibroblast growth factor did not enhance LH release. The augmenting effect of insulin was not associated with cellular proliferation or an overall change in protein or LH synthesis. Furthermore, the effect of insulin was independent of the ambient glucose concentration. Insulin was, however, without effect on gonadotrophs cultured in a serum-supplemented medium. Our findings suggest that the gonadotroph constitutes a target cell of insulin and that insulin may act directly on the anterior pituitary in the regulation of gonadotropin release.

Gonadotropin-Binding Sites in the Rhesus Monkey Ovary: Role of the Vasculature in the Selective Distribution of Human Chorionic Gonadotropin to the Preovulatory Follicle

Abstract: These experiments were initiated to determine if differences exist in the vasculature of individual follicles in the rhesus monkey ovary during the late follicular phase of the menstrual cycle and to determine whether differences in vascularity result in differential exposure of certain follicles to gonadotropic hormones. The density of blood vessels within the thecal layer of the dominant follicle and other antral follicles was determined in ovaries from four animals removed on day 9 or 10 of the menstrual cycle. Blood vessels were identified using a histochemical stain for hemoglobin. Morphometric analysis indicated that the percentage of the thecal layer occupied by blood vessels in the dominant follicles (48%) was significantly greater (P less than 0.005) than that of other smaller antral follicles either within the same ovary as the dominant follicle (24%) or in the contralateral ovary (26%). To determine if differences in vascularity result in a differential supply of gonadotropins to the dominant follicle, we studied, by autoradiography, the in vivo and in vitro binding of [125I]hCG in four rhesus monkeys on day 9 of the menstrual cycle. Results of in vitro binding studies indicated that the thecal layer of virtually every antral follicle possessed hCG-binding sites. However, when [125I]hcg was injected iv into animals and allowed to distribute via the vasculature, the dominant follicle was heavily labeled while other smaller antral follicles accumulated little, if any, radioiodinated hCG. These observations indicate that increased vascularization of individual follicles results in preferential delivery of gonadotropins, and suggest that blood flow to individual follicles may play an instrumental role in the selective maturation of the preovulatory follicle in the rhesus monkey.

Functional properties of hormonally responsive cultured normal and malignant rat osteoblastic cells.

Abstract: Certain metabolic properties of hormonally responsive osteogenic sarcoma cells derived from a transplantable rat tumor have been compared with those of related normal rat bone cells. All studies were carried out on cells grown in monolayer culture. Normal rat bone cells derived by repeated collagenase/trypsin digestion of newborn rat calvaria. Bone cells selected for comparison were thought to be osteoblast-like, as judged by enrichment of alkaline phosphatase and adenylate cyclase responsiveness to parathyroid hormone and prostaglandin E2. The adenylate cyclases of the two cell strains were similarly stimulated by a range of prostanoids and their metabolites and analogs. Morphology showed the two cell strains to be similar; the only obvious difference was a multilayering of cells in the sarcoma cultures, while the normal cultures showed abundant extracellular fibril formation which was not seen in the tumor cells. Investigation of the cAMP-dependent protein kinase isoenzymes showed the presence of two forms in both cell types, one eluting at a low salt concentration and the other at a high salt concentration. There was approximately twice the amount of the first isoenzyme compared to the second isoenzyme. The results indicate the usefulness of the two cell strains to elucidate further the molecular mechanisms of action of parathyroid hormone and prostaglandins.

Somatostatin in rat tissues is depleted by cysteamine administration.

Abstract: Administration of cysteamine (mercaptoethylamine) induces in rats severe perforating duodenal ulcers. Because the ulcerogenic properties of cysteamine are markedly reduced by treatment with somatostatin, we considered the possibility that cysteamine-induced duodenal ulcer might be mediated by depletion of tissue somatostatin, and thereby of its paracrine influences on gastrin and gastric acid secretion. To test this hypothesis, we measured the concentration of immunoreactive somatostatin (IR-somatostatin) in stomach and duodenal mucosa at intervals after administration of a single ulcerogenic dose (30 mg/kg by stomach tube). IR-somatostatin in these tissues fell rapidly to reach a minimum at 4 h (stomach 31%, duodenum 60% of control respectively). IR-somatostatin in hypothalamus and pancreas decreased gradually to a minimum at 7 h. Another duodenal ulcerogen, propionitrile (10 mg/100 g bw, s.c.) which is more toxic than cysteamine, and several stressful procedures including ether anesthesia, restraint and s.c. formalin did not lower stomach or duodenal IR-somatostatin. Gut, pancreas and hypothalamic VIP levels were not influenced by cysteamine. These findings suggest that cysteamine is a relatively specific depletor of tissue somatostatin. Because blood levels of somatostatin fell, and only trivial amounts of the peptide were found in the stomach lumen after cysteamine administration, it appears likely that this agent acts at the cellular level to cause breakdown of preformed somatostatin and/or to acutely reduce its synthesis.

A Comparison of Estrogen Effects on Uterine and Pituitary Growth and Prolactin Synthesis in F344 and Holtzman Rats

Abstract: The pituitary response to chronic estrogen treatment was studied in two strains of rats, the Fischer 344 (F344) and the Holtzman. Silastic tubing implants containing diethylstilbestrol (DES) were placed in weanling animals. Uterine and pituitary growth and protein synthesis were monitored over an 8-week period. The incorporation of [3H]leucine into PRL and GH was measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both F344 and Holtzman strains respond to chronic DES with an increase in PRL synthesis. PRL synthesis is 7% of the total protein synthesis in ovariectomized female controls of both strains and increases to 40–50% in 8-week DEStreated F344 rats and to 35–45% in Holtzman rats. The uterine growth responses were similar in both strains and reached a plateau after approximately 2 weeks. Despite their similarity in PRL synthesis and uterine growth, these two strains differ dramatically in their pituitary growth response to DES. In the F344 strain, pituitaries from treated animals a...

Age-associated reduction in nocturnal pineal melatonin levels in female rats.

Abstract: Pineal N-acetyltransferase (NAT) activity and radioimmunoassayable levels of melatonin were compared in 2-month-old (young), 12-month-old (middle-aged), and 29-month-old (old) female rats killed at 1600 h (during the light) and at 2300 h (4 h after darkness onset) and 0100 h (6 h after darkness onset). During the light period, NAT levels were equivalent in pineals from each age group. With the onset of darkness NAT levels rose sharply and were again equivalent in all groups at 2300 h. At 0100 h pineal NAT values in the old rats were lower than in the other two groups. Melatonin values were low in pineal glands of all animals killed at 1600 h in light. By 4 h after darkness onset pineal melatonin content in the young rats had increased 12-fold compared to a 6-fold rise in the old animals. Melatonin levels in the middle aged rats were intermediate between the other two groups. Similar relationships were observed in the rats killed at 0100 h. By this time the young rats had melatonin levels 17 times higher than during the day while the increase in the old rats was only 7-fold; 12-month-old rats again had intermediate levels. The finding show a marked reduction in pineal melatonin with aging in female rats.

Factors Controlling Proliferation and Progesterone Production by Bovine Granulosa Cells in Serum-Free Medium*

Abstract: Bovine granulosa cells seeded in the presence of serum on extracellular matrix-coated dishes proliferate actively when exposed to serum-free medium supplemented with insulin (2 microgram/ml), fibroblast growth factor (FGF, 100 ng/ml), and high density lipoprotein (HDL, 30 microgram protein/ml). The final density of the cultures is 80-120% that of cultures grown in the presence of medium supplemented with optimal concentration (10%) of calf serum. Insulin has the greatest effect on cell proliferation when added alone to serum-free medium, since it induced an increase in cell number that was 35-60% that observed with optimal serum concentration. Somatomedin C can replace insulin when added alone. FGF, epidermal growth factor, or HDL had no significant effect on cell proliferation by themselves. When these factors were added together with insulin, they acted synergistically in stimulating cell proliferation. When cultures were seeded in the total absence of serum, the addition of transferrin (10 microgram/ml) to serum-free medium was required in order for insulin and FGF to be mitogenic. Cultures maintained on extracellular matrix and exposed to serum-free medium alone have a lifespan in culture of 4 generations. Addition of insulin, FGF, and HDL increases the lifespan of the cultures to 12 generations. Bovine granulosa cells, which proliferate in a defined medium, respond to dibutyryl cAMP by releasing progesterone into the medium. Addition of FSH to the defined medium resulted in a 30% decrease in cell proliferation and in a 2.1-fold increase in the amount of progesterone released into the medium in response to dibutyryl cAMP. This release of progesterone reached a level similar to that observed with cultures grown in medium supplemented with optimal concentration of serum and exposed or not to FSH during their growth phase and at confluence. These results demonstrate that bovine granulosa cells can actively proliferate in a serum-free medium and maintain their differentiated function, as indicated by their ability to produce progesterone.

Design, Synthesis, and Biological Activity of Peptides which Release Growth Hormone in Vitro

Abstract: Experimental observations showed that the analogs [D-Trp2]- and [D-Phe2]methionine enkephalin amide were weakly active in releasing GH from rat pituitary in vitro. These observations were used to design more active GH-releasing factors. Conformational energy calculations were carried out, and energetically favored conformations of these polypeptides were found. Structural similarities as well as structural differences between active and inactive analogs were examined, and new sequences were predicted. Progressively more active analogs were designed, then synthesized, and tested. This cycle of steps was repeated, each time using structural and chemical concepts as design guides, until a series of very active analogs resulted. The most active analog to date, Tyr-D-Trp-Ala-Trp-D-Phe-NH2, was shown to release GH in vitro at 10-30 ng/ml medium, which is approximately 10(3) times more active than the two starting enkephalin-based analogs. From the structure-activity data, a mechanism for binding at the receptors is formulated, and a comparison is made between the structural relationships of the GH-releasing peptide analogs and the GH inhibitor, somatostatin.

Adrenal sensitivity to adrenocorticotropin varies diurnally.

Abstract: To further characterize diurnal changes in the rhythm in adrenal responsiveness to ACTH, we have measured ACTH distribution volume, MCR, and t 1/2. These do not change between morning and evening in groups of untreated, dexamethasone-pretreated, or hypophysectomized female rats. To characterize the nature of the change in adrenal responsiveness to ACTH, dexamethasone-pretreated rats were infused for 2 h with a variety of doses of ACTH in the morning and evening. The adrenal response to an infusion rate of ACTH that maximally stimulated the adrenals (200 pg/100 g BW.min) was the same in the morning and evening, showing that adrenal capacity does not change. However, infusion of ACTH at lower rates (50-100 pg/100 g BW.min) revealed that the slope of the steroid response curve increased between morning and evening, demonstrating a diurnal change in adrenal sensitivity to ACTH. These results together with previous data showing that the magnitude and time course of the adrenal cAMP response to ACTH changes diurnally strongly suggest that ACTH receptor affinity or coupling with adenylate cyclase changes diurnally. In other experiments, plasma ACTH and corticosterone levels were determined in groups of young and adult male and adult female untreated rats killed at 4-h intervals around the clock. Peak sensitivity to ACTH was found at lights-out, and trough sensitivity was found at lights-on, suggesting that the experimentally demonstrated rhythm occurs normally.

Effects of Estradiol and Progesterone on Catecholamine Turnover Rates in Discrete Hypothalamic Regions in Ovariectomized Rats

Abstract: Norepinephrine (NE) and dopamine (DA) activities were measured in several discrete brain areas (medial preoptic nucleus, suprachiasmatic nucleus, median eminence, and arcuate nucleus) in ovariectomized (OVX) and OVX steroidtreated rats We wished to determine whether catecholamine (CA) mechanisms are involved in estradiol (E2)-induced LH surges and if amplification of such surges by progesterone (P)affects these CA systems Animals were OVX, and 1 week later (day 0), groups were treated with Silastic capsules containing oilor an E2 solution (150 μg/ml in sesame oil) At 0900 h on day 2, E2-treated animals received Silastic capsules containing oil or P (50 mg/ml in oil) This steroid treatment results in physiologicalserum concentrations of both steroids CA concentrations were determined by radioenzymatic assay in animals killed at 1000 or 1500 h on day 2 and in animals which were sacrificed 2 h after receiving a-methyl paratyrosine (400 mg/kg) at 1000 or 1500 h Serum LH, FSH, PRL, E2, and P concentratio

Correction of Abnormal Bone and Mineral Metabolism in Chronic Streptozotocin-Induced Diabetes Mellitus in the Rat by Insulin Therapy*

Abstract: Mineral homestasis and skeletal morphology were studied in freely fed control, streptozotocin-induced diabetic, and insulin-treated diabetic rats 7 weeks after the induction of diabetes. The untreated diabetic animals were characterized by modest hypercalcemia, hyperphosphatemia, and striking hypercalciuria and phosphaturia. Insulin treatment corrected the hypercalcemia and markedly reduced the calciuria (P < 0.001), but had no significant effect on the urinary phosphate levels. Circulating immunoreactive parathyroid hormone (iPTH) was detectable in only 6% of untreated diabetic animals compared to 30% of controls. Furthermore, in untreated diabetic animals, the circulating levels of iPTH, when detectable, approximated the lower limits of the assay. The urinary cAMP levels of untreated diabetic animals were markedly decreased. Both iPTH and urinary cAMP approximated control levels in insulin-treated rats. Conversely, plasma immunoreactive calcitonin was increased in the diabetic rats compared to control a...

Growth hormone secretory dynamics in streptozotocin diabetes: evidence of a role for endogenous circulating somatostatin.

Abstract: The present studies examined the effects of streptozotocin-induced diabetes on the dynamics of GH, insulin (IRI), and glucose secretion in 68 chronically cannulated male rats. Animals were rendered diabetic by a single iv injection of streptozotocin (65 mg/kg) and sampled every 15 min for periods of 6h both before and at various times after treatment. The typical ultradian rhythm of GH secretion was evident in normal rats, with most peak GH values greater than 500 ng/ml (mean 6-h GH level, 125.3 ± 17.2 ng/ml). After streptozotocin administration, a significant depression in the amplitude of GH pulses was observed as early as 18 h post treatment (mean 6-h GH level, 54.4 ± 9.4 ng/ml; P < 0.01) concomitant with a marked elevation in plasma glucose levels and severe depression of plasma IRI levels. The amplitude and duration of the GH secretory episodes continued to decline over a 4-week period in the presence of persisting hyperglycemia and mostly undetectable plasma IRI levels. The pituitary GH concentratio...

Regulation of Glucocorticoid Receptors by Glucocorticoids in Cultured HeLa S3 Cells

Abstract: HeLa S3 cells contain high affinity, saturable receptors specific for glucocorticoids (congruent to 20,000 per cell). Growth of HeLa S3 cells in media containing dexamethasone (10(-6) M) results in a pronounced (congruent to 70%) reduction in the total cellular level of nuclear or cytoplasmic dexamethasone receptor number without any alteration in steroid-receptor dissociation constant (Kd congruent to 1 X 10(-9) M). This reduction in receptor number is not the result of simple receptor occupation, since this effect also occurs when receptor number is modulated by the direct addition of [3H]dexamethasone. Both control and dexamethasone-treated cells attain equilibrium states of receptor binding by 120 min at 0 C and by 60 min at 37 C, enabling estimation of receptor number and affinity by Scatchard analysis of saturation curves. Down-regulation of glucocorticoid receptor occurs at steroid concentrations as low as 10(-9) M (40% reduction). This alteration in receptor occurs after 24 h of hormone but not after either 2 or 6 h. Only the active glucocorticoids, (dexamethasone, cortisol) down-regulate glucocorticoid receptors. Cortexolone, estradiol, and 5 alpha-dihydrotestosterone have no apparent effect on this process, whereas progesterone (10(-7) M) is partially effective. Subcellular distribution studies indicate that glucocorticoids affect only that population of receptors capable of nuclear translocation at 37 C. We conclude that glucocorticoids can regulate the metabolism of their own receptors, via undefined mechanisms.

Multiple Forms of Immunoreactive Somatostatin: Comparison of Distribution in Neural and Nonneural Tissues and Portal Plasma of the Rat*

Abstract: The present study was undertaken to partly characterize and compare the relative amounts of the different molecular forms of somatostatin in the major somatostatin-containing tissues and portal plasma of the rat. Immunoreactive somatostatin (IRS) was extracted from hypothalamus, cerebral cortex, neural retina, sciatic nerve, pancreas, stomach, duodenum, jejunum, ileum, colon, and portal venous plasma and fractionated on Sephadex G-50 columns equilibrated with 6 M urea. Three peaks with somatostatin-like immunoreactivity (SLI) were identified. Peak I had an apparent molecular weight of 14,000 daltons, (14K SLI), peak II coeluted with synthetic somatostatin-28 (3K SLI), and peak III coeluted with SRIF (1.6K SLI). The relative proportions of the three molecular forms were unchanged after treatment with 8 M guanidine hydrochloride and thioglycol and varied considerably from tissue to tissue. In the hypothalamus and cerebral cortex, approximately 70% of the IRS was present as 1.6K SLI, 25% as 3K SLI, and 5% as...

Inhibition of ovarian and testicular steroiodogenesis by epidermal growth factor

Abstract: Epidermal growth factor (EGF), but not fibroblast growth factor, inhibits the gonadotropin stimulation of estrogen and testosterone production by primary cultures of ovarian granulosa and testicular Leydig cells, respectively. EGF also inhibits gonadal steroidogenesis induced by cholera toxin and (Bu)2cAMP, and the inhibitory effect of EGF is not associated with cell proliferation. EGF may play an important endocrine role in the regulation of steroidogenic functions of gonadal cells.

Prolactin Regulation of Milk Secretion and Biochemical Differentiation of Mammary Epithelial Cells in Periparturient Cows

Abstract: PRL involvement in periparturient mammary development and onset of milk secretion was studied in 17 multiparous, monotocous Holstein cows. Mammary glands were obtained 10 days prepartum from untreated cows and 10 days postpartum from untreated cows and cows treated with CB154 (2-Br-α-ergokryptin) or CB154 plus PRL. CB154 was administered for 12 days before expected parturition through 10 days postpartum. PRL was infused continuously for 6 days immediately before parturition in cows receiving CB154 plus PRL. Treatment with CB154 reduced basal serum PRL concentrations approximately 80% and blocked the normal surges in serum PRL concentrations at parturition and during milking. Average milk production in cows given CB154 alone was 11.4 kg/day lower than in controls. Periparturient infusion of PRL in cows treated with CB154 (CB154 plus PRL) prevented reductions in milk production. Treatment with CB154 had no effect on feed intake or periparturient serum concentrations of GH, progesterone, and glucocorticoids....

Catecholamine turnover rates in discrete hypothalamic areas and associated changes in median eminence luteinizing hormone-releasing hormone and serum gonadotropins on proestrus and diestrous day 1.

Abstract: We have correlated catecholamine [CA; i.e. norepinephrine (NE), dopamine (DA), and epinephrine (E)] turnover rates in discrete hypothalamic nuclei and in the median eminence (ME), with concentration changes in ME LHRH and serum LH, FSH, PRL, estradiol, and progesterone levels at various times during proestrus and diestrous day 1 in 4-day cyclic rats. CA concentrations were measured with a radioenzymatic assay at 0, 60, and 120 min after ip injection of 400 mg/kg alpha-methyl-p-tyrosine, and rate constants and turnover rates were calculated. In a separate assay NE, DA, and E were separated by two-dimensional thin layer chromatography, and concentrations and turnover rates of CAs were calculated. The microdissected hypothalamic nuclei examined for NE turnover rates included the medial preoptic nucleus (MPN), suprachiasmatic nucleus (SCN), arcuate nucleus (AN), and ME. DA turnover rates also were measured in the MPN, ME, and AN. ME LHRH and serum hormone concentrations were measured by RIA. Between 0900--1200 h, proestrous serum estradiol was elevated, but other serum hormones were basal, and CA turnover rates in the brain were low. However, ME LHRH concentrations increased significantly between 0900--1200 h on proestrus. Between 1200--1500 h, serum LH, FSH, PRL, and progesterone levels increased and ME LHRH levels declined significantly; during this time interval (1200--1400 h), a significant rise in ME NE and DA turnover rates occurred. Between 1500--1700 h on proestrus, while serum gonadotropins were still rising toward peak concentrations, increased ME NE turnover rates were maintained, but increased NE turnover rates also were evident in MPN, SCN, and AN. During this same time interval (1500--1700 h), a marked decline in ME and AN DA turnover rates occurred, although such rates remained unchanged within the MPN. There were no corresponding changes in MPN E turnover rates at any of the time intervals studied. The increased turnover rates of ME NE coupled with the concomitant decline in ME LHRH levels and the rise in plasma LH and FSH levels suggest that increased NE release may be important in initiating preovulatory LH and FSH surges. These changes in brain neurotransmitters and serum hormones are not the result of a diurnal rhythm, since corresponding changes in CA turnover rates or serum gonadotropins did not occur between 0900--1100 h and 1500--1700 h diestrous day 1.

Evidence for Noradrenergic Mediation of Opioid Effects on Luteinizing Hormone Secretion

Abstract: Ovariectomized rats received estradiol benzoate (EB; 7.5 μg/rat, on day 0 at 1000 h) and progesterone (P; 5 mg/rat, on day 2 at 1000 h). On day 2, these EB- and P-treated (EBP) rats received either saline (control; at 1215 and 1230 h), morphine sulfate (M; 45 μg/kg, at 1230 h), or naloxone (NAL; 2 μg/kg, at 1215 h), followed by M (at 1230 h), and were killed between 1445–1515 h. M treatment alone blocked the P-induced afternoon increases in serum LH and the medial basal hypothalamic LHRH levels, whereas NAL pretreatment counteracted the M-induced LH suppression. The involvement of central adrenergic systems in the M- and NAL-induced serum LH responses in EBP-treated and young male rats was next evaluated. In these rats, NAL injection elicited rapid increases in serum LH levels, which were blocked by M pretreatment. The dopamine agonist, apomorphine, or antagonist, pimozide, failed to inhibit LH release induced by NAL in EBP rats. When norepinephrine (NE) neurotransmission was selectively suppressed by an ...

Intraventricularly injected growth hormone stimulates somatostatin release into rat hypophysial portal blood.

Abstract: The effects of GH on the release of somatostatin from the hypothalamus were assessed by measuring the concentrations of immunoreactive somatostatin (IRS) in hypophysial portal blood of urethane-anesthetized male rats. A significant and dose-related increase of IRS in hypophysial portal blood was observed during 20–80 min after a single injection of rat GH (5 and 25 μg) into the third ventricle. An intraventricular injection of ovine LH or vehicle alone did not affect IRS values in hypophysial portal blood. When rat GH was repeatedly injected into the cerebral ventricle at 75-min intervals, IRS in hypophysial portal blood rose following each injection in a similar pattern with a latency of 30–45 min. These findings suggest that the release of somatostatin from the hypothalamus is regulated, at least in part, by GH. Furthermore, in view of the inhibitory effect of somatostatin on GH secretion, stimulation by GH of somatostatin release into hypophysial portal blood may be involved in the mechanism by which G...

Desensitization to gonadotropin-releasing hormone observed in superfused pituitary cells on Cytodex beads.

Abstract: Chronic exposure to gonadotropin-releasing hormone (GnRH) in vivo eventually results in a failure to maintain maximal gonadotropin secretion, suggesting a loss of pituitary responsiveness. To examine changes in pituitary sensitivity in vitro, we have developed a superfusion technique in which dissociated rat anterior pituitary cells are attached to Cytodex beads. In this system, continuous administration of 30 nM GnRH caused an initial 20-fold increase in LH and FSH secretion rates. However, hormone secretion declined rapidly, reaching basal levels within 12 h. Both the calcium ionophore A23187 and the cocarcinogen phorbol myristate acetate were effective in releasing additional LH and FSH from cells made refractory to 30 nM GnRH, whereas higher doses of GnRH and 8-bromo-cAMP were minimally active. A rapid loss of response was also seen with a superactive GnRH agonist but was not observed with a GnRH antagonist. Desensitization to prolonged superfusion with phorbol myristate acetate was also observed, alt...

Effects of Growth Hormone Excess and Deficiency on Hypothalamic Somatostatin Content and Release and on Tissue Somatostatin Distribution

Abstract: Considerable indirect evidence now exists to suggest that hypothalamic somatostatin (SRIF) is the physiological inhibitory regulator of pituitary GH release. To support this relationship further, we studied the effect of in vivo modifications of GH homeostasis on hypothalamic SRIF content and in vitro release in an attempt to document a feedback relationship between the two peptides. GH administration to normal rats resulted in increased hypothalamic SRIF concentration and release. GH deficiency, in contrast, resulted in decreased hypothalamic SRIF concentration and release. This effect appears to be, at least in part, a direct action of GH, since a dose-related stimulation of hypothalamic SRIF release was demonstrated in the presence of GH concentrations ranging from 10(-9)-10(-5) M. The lowest dose causing stimulation (10(-9) M) is well within the normal concentration range of plasma GH in the rat, suggesting that the effect may be physiological. Specificity of the effect is suggested by a much greater sensitivity of the medial basal hypothalamus than the septum and preoptic area to the effects of GH. The perturbations of GH homeostasis studied had no effect on extrahypothalamic neural or gastrointestinal SRIF concentrations, suggesting a different regulatory mechanism in these areas.

Calcium: its role in the mechanism of action of angiotensin II and potassium in aldosterone production.

Abstract: The role of calcium in the angiotensin II- or potassium-mediated increase in aldosterone production was analyzed in isolated glomerulosa cells prepared from bovine adrenal glands. The response to potassium was highly dependent on the extracellular calcium concentration, and a maximal response was observed at 0.5 mM calcium. The response to angiotensin II was also a function of the calcium concentration between 0 and 0.5 mM Ca but was independent of calcium concentration above this value. The divalent ionophore A23187 also increased aldosterone production in a calcium-dependent manner. Methoxyverapamil blocked the stimulation of steroidogenesis due to angiotensin II and potassium. Calcium fluxes were studied during angiotensin II and potassium stimulation of aldosterone production. Incubation of zona glomerulosa cells with either angiotensin II or potassium at a concentration for maximal stimulation in the presence of radioactive calcium showed a significant increase in calcium uptake. Angiotensin II at a ...

Continuous infusion of growth hormone feminizes hepatic steroid metabolism in the rat.

Abstract: The metabolism of 4-[4-14C]androstene-3,17- dione was studied in the microsomal fraction of rat livers after continuous administration of human GH (hGH) in Alzet osmotic minipumps under varying conditions. hGH caused a complete feminization of hepatic steroid metabolism (i.e. increased the 5α- reductase and decreased the 6β- and 16α-hydroxylase activities) in normal male rats when infused at a rate of 5 μg/h for 7 days. Hypophysectomy and castration or adrenalectomy and thyroidectomy of male rats did not reduce the feminizing capacity of hGH, indicating that the adrenals and the thyroid gland are not involved in the mediation of the feminizing effect of hGH. The same dose (5 μg/h for 7 days) of hGH was also able to refeminize the liver steroid metabolism in hypophysectomized-ovariectomized female rats. The effect of the homologous hormones, rat PRL and rat GH on hepatic steroid metabolism was also investigated. Either hormone was infused at a rate of 10 μg/h for 7 days, a dose which was sufficient to incr...

Calcitonin receptors in the central nervous system of the rat.

Abstract: Specific binding sites for calcitonin (CT) were sought in preparations of rat brain in vitro Whole rat brains or discrete regions of the brain were homogenized, and the 10,000 × g fraction was tested for binding of 125I-labeled salmon CT ([125I]sCT) The highest total and specific binding occurred in the hypothalamus, and the least occurred in the cortex and cerebellum, relative to whole brain Intermediate levels of binding of the radioligand were found in the pons-midbrain and medulla More detailed characterization of the hypothalamusradioligand interaction revealed that the binding was saturable and linear with increasing concentrations of tissue protein from 25–800 μg/ml Furthermore, it was both time and temperature dependent, reaching equilibrium by 1 h at 4, 23, and 37 C After 1 h of incubation at 4 C, half-maximal inhibition of binding of the radioligand occurred at a sCT concentration of 1 × 10-9 M Both porcine and human CT were less effective than sCT in inhibiting the binding of [125I]sCT t

Evidence that granulosa cell aromatase induction/activation by follicle-stimulating hormone is an androgen receptor-regulated process in-vitro.

Abstract: The role of androgen in aromatase induction/activation by follicle-stimulating hormone (FSH) was studied in cultured granulosa cells from estrogen-pretreated, immature rat ovaries. Aromatase activity was measured in washed cell monolayers after a 48-h culture in medium containing hFSH and/or various sex steroids or their analogues. Culture with hFSH (100 ng/ml) plus 10(-7) M testosterone (T) stimulated aromatase activity to a level similar to that of granulosa cells from preovulatory follicles in the cyclic adult on the morning of proestrus. But if T was omitted, or replaced by estrogen (DES) or progesterone (P), the response to hFSH was at least 90% lower. The abilities of T, androstenedione, five nonaromatizable 5 alpha-reduced androgens, an aromatase reaction intermediate (19-hydroxyandrostenedione), and a pharmacological competitive aromatase inhibitor (delta 1-testoloalactone) to stimulate the aromatase response to hFSH were proportionate to their stimulatory effects on P production during the culture. By both criteria T was the most potent androgen while 19-hydroxyandrostenedione and delta 1-testololactone were completely inactive. The stimulatory effect of 10(-7) M T on the aromatase response to FSH was inhibited by the nonsteroidal antiandrogen SCH 16423 (ID50 = 3.6 x 10(-6) M). These results indicate that granulosa cell aromatase induction/activation by hFSH is an androgen receptor-regulated process in vitro.

Regulation of Pituitary Gonadotropin-Releasing Hormone Receptors by Gonadal Hormones

Abstract: The regulatory role of pituitary gonadotropinreleasing hormone (GnRH) receptors in the control of gonadotropin secretion was investigated in male and female rats after castration and sex steroid hormone replacement. GnRH receptors were measured in homogenates of individual pituitaries by equilibration with 125I-labeled [D-Ser(tBu)6]des-Gly10-GnRH Nethylamide, and compared with serum and pituitary LH concentrations. The equilibrium association constants (Ka) were 6.1 and 5.1 × 109 M-1 for intact and castrate male rat pituitaries, respectively. After orchidectomy, pituitary GnRH receptor concentration increased by 75% at 24 h, from 150 fmol to 250 fmol/gland, while serum LH levels increased 10-fold (30 to 300 ng/ml). There was a further slight increase in the GnRH receptor concentration (to 370 fmol/gland) and serum LH (to 500 ng/ml) over the ensuing 10 days, and at 15 and 20 days GnRH receptors were 304 and 306 fmol/gland, respectively. There was a highly significant (P < 0.001) positive correlation betwee...