The Stimulation of Luteinizing Hormone Secretion from Anterior Pituitary Cells in Culture by Substance P: A Dissertation
Faculty AdvisorH. Maurice Goodman
Academic ProgramCell Biology
UMass Chan AffiliationsMicrobiology and Physiological Systems
Document TypeDoctoral Dissertation
Amino Acids, Peptides, and Proteins
Animal Experimentation and Research
Hormones, Hormone Substitutes, and Hormone Antagonists
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AbstractThe observations that substance P (SP) is localized in the anterior pituitary gland (AP) and is regulated by the hormonal status of the animal, as well as the demonstration of SP binding sites in the AP, have led to the idea that SP may participate in the regulation of AP function. Numerous and sometimes contradictory reports of SP effects on AP hormone secretion, particularly on luteinizing hormone (LH), left the question of whether SP acts directly at the level of the AP to regulate LH secretion still unanswered. To investigate a possible physiological function of SP in the AP, the effects of exogenous SP on LH secretion from AP cells from adult and prepubertal male and female rats in short term culture were studied. It was found that SP (100nM-1μM) significantly stimulates LH release in cultured AP cells and that this effect varies as a function of age and sex. SP has no significant effect on LH release from AP cells of male and female prepubertal rats. After day 30 a sharp increase in the response to SP occurs in both sexes. This level of responsiveness continues through adulthood in AP cells from the female rat. In contrast, AP cells from male rats failed to respond during adulthood (over 50 days of age) but were highly responsive during the peripubertal period (30-35 days). The possibility that the responsiveness to SP is influenced by the endocrine status of the animal was investigated by exposing AP cells from responding animals to androgens in vivo and in vitro. It was found that AP cells from female rats treated with androgen were less responsive to 100nM SP but did respond at higher doses of SP. SP effects on AP function were further analyzed in experiments using radioligand binding assays to assess possible changes in SP receptor number or affinity as related to age and sex. In AP membranes from female rats, maximum binding is 8-fold higher (Bmax=4.2 pmo1/mg membrane protein) than in AP membranes from male rats (Bmax=560fmo1/ mg membrane protein). These studies suggest a role for SP as a secondary regulator of LH secretion with possible physiological significance for reproductive function.
Permanent Link to this Itemhttp://hdl.handle.net/20.500.14038/31424
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CIS/SOCS Proteins in Growth Hormone Action: A DissertationDu, Ling (2000-10-01)CIS/SOCS (cytokine-inducible SH2 protein/suppressor of cytokine signaling) are a family of proteins that are thought to act as negative regulators of signaling by erythropoetin, interleukin-6 and other cytokines whose receptors are related to the growth hormone receptor (GHR), and like growth hormone (GH), signal through the JAK/STAT pathway. We examined the possibility that CIS/SOCS proteins may also be involved in GH signaling, in particular, in termination of the transient insulin-like effects of GH. mRNAs for CIS, SOCS3, and to a lesser extent SOCS1 were detectable by Northern blot analysis of rat adipocyte total RNA, and the expression of CIS and SOCS3 was markedly increased 30 min after incubation with 500 ng/ml hGH. Both CIS and SOCS3 were detected in adipocyte extracts by immunoprecipitation and immunoblotting with their corresponding antisera. GH stimulated the tyrosine phosphorylation of a 120 kDa protein (p120) that was co-precipitated from adipocyte extracts along with αCIS and detected in Western blots with phospho-tyrosine antibodies. However, no tyrosine phosphorylated proteins in these cell extracts were immunoprecipitated with antibodies to CIS3/SOCS3. p120 was later identified as the GHR based on the observations that two GHR antibodies recognized p120 in scale-up experiments and that p120 and the GHR share several characteristics, including their molecular weights, tyrosine phosphorylation upon GH stimulation, interaction with CIS, similar extent of glycosylation as judged by electrophoretic mobility shift after Endo F digestion, comparable mobility shifts upon thrombin digestion, and N-terminal histidine-tagging. The findings, however, do not rule out the possibility that there might be other tyrosine phosphorylated 120 kDa protein(s) that interact with CIS and contribute to the p120 signal, as well as the GHR. Further studies of the association of CIS with the GHR revealed that CIS might selectively interact with multiply tyrosine phosphorylated forms of the GHR, and these tyrosines are likely located near the carboxyl end of the GHR. Overexpression of CIS partially inhibited GH-induced STAT5 phosphorylation in CHO cells. Studies in freshly isolated and GH-deprived (sensitive) adipocytes revealed that the abundance of CIS does not correlate with the termination of the insulin-like effects of GH or the emergence of refractoriness. Neither the association of CIS with the GHR nor the tyrosine phosphorylation status of the GHR, JAK2 and STAT5 appear responsible for refractoriness in adipocytes. These data imply that some negative regulators other than CIS might contribute to the termination of GH-induced insulin-like effects in adipocytes.
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