Search PubMed⌕ Search

Biomedical subjects

P M Conn

Publications and source records attributed to P M Conn.

At least 37 records · Page 2Linked to original sources
PubMed1998

The third intracellular loop of the rat gonadotropin-releasing hormone receptor couples the receptor to Gs- and G(q/11)-mediated signal transduction pathways: evidence from loop fragment transfection in GGH3 cells.

The GnRH receptor (GnRH-R) belongs to the rhodopsin/beta-adrenergic family of G protein-coupled receptors. The intracellular domains of these receptors, particularly the regions closest to the plasma membrane in intracellular loops 2 (2i) and 3 (3i) as well as some regions located in the membrane-proximal end of the COOH-terminus, are frequently important sites for G protein coupling and specificity determination. Although studies in mouse and human GnRH-R have identified loop 2i as a critical determinant for coupling the receptor to the G(q/11)-mediated signal transduction pathway, given the functional similarity among the members of this particular G protein-coupled receptor subfamily and the fact that the GnRH-R lacks the typical intracellular COOH-terminal domain of its superfamily (a potential site for G protein coupling), we investigated the possibility that loop 3i of this receptor also participates in GnRH-R coupling to G proteins. GGH(3)1' cells, a pituitary-derived cell line that expresses a functional rat GnRH-R coupled to both Gs and G(q/11) proteins, were transiently transfected with a plasmid DNA containing a complementary DNA (cDNA) coding for the entire loop 3i of the GnRH-R as well as with other expression plasmids containing cDNAs encoding loop 3i of other Gs-, G(i/o)-, or G(q/11)-coupled receptors. The effects of coexpression of these loops with the wild-type GnRH-R on inositol phosphate (IP) production, cAMP accumulation, and PRL release were then examined. Transfection of GGH(3)1' cells with the cDNA for loop 3i of the rat GnRH-R (efficiency, 35-45%) maximally inhibited buserelin-stimulated IP turnover by 20% as well as cAMP accumulation and PRL secretion by 30%. This attenuation in cellular responses to a GnRH agonist was statistically significant (P < 0.05) compared with the responses exhibited by GGH(3)1' cells transfected with a control plasmid and stimulated with the same GnRH agonist. Transfection of minigenes coding for loop 3i of the M1Ach-muscarinic and the alpha1B-adrenergic (G(q/11)-coupled) receptors resulted in 25-55% inhibition of maximal GnRH-evoked IP turnover. Paradoxically, loop 3i from the M1Ach-muscarinic receptor also maximally inhibited GnRH agonist-stimulated cAMP accumulation and PRL release by 40% (both effects mediated through activation of the Gs protein). Transfection of loop 3i from the D1A -dopamine receptor (coupled to the Gs protein) produced a selective attenuation (40%) in Gs-mediated cellular responses. In contrast, receptor/G protein coupling appeared unaffected by expression of loop 3i domains derived from two receptors coupled to G(i/o) proteins (M2Ach-muscarinic and alpha2A-adrenergic receptors). These data indicate that the third intracellular loop of the rat GnRH-R is involved in receptor G(q/11) protein coupling and/or selectivity, and in the GGH(3)1' cell line, this loop is also involved in signal transduction mediated through the Gs protein pathway.

Amino Acid Sequence↗
PubMed1998

Gonadotropin and gonadal steroid release in response to a gonadotropin-releasing hormone agonist in Gqalpha and G11alpha knockout mice.

In this study, we used mice lacking the G11alpha [G11 knockout (KO)] or Gqalpha gene (Gq KO) to examine LH release in response to a metabolically stable GnRH agonist (Buserelin). Mice homozygous for the absence of G11alpha and Gqalpha appear to breed normally. Treatment of (5 wk old) female KO mice with the GnRH agonist Buserelin (2 microg/100 microl, sc) resulted in a rapid increase of serum LH levels (reaching 328 +/- 58 pg/25 microl for G11 KO; 739 +/- 95 pg/25 microl for Gq KO) at 75 min. Similar treatment of the control strain, 129SvEvTacfBr for G11 KO or the heterozygous mice for Gq KO, resulted in an increase in serum LH levels (428 +/- 57 pg/25 microl for G11 KO; 884 +/- 31 pg/25 microl for Gq KO) at 75 min. Both G11 KO and Gq KO male mice released LH in response to Buserelin (2 microg/100 microl of vehicle; 363 +/- 53 pg/25 microl and 749 +/- 50 pg/25 microl 1 h after treatment, respectively). These values were not significantly different from the control strain. In a long-term experiment, Buserelin was administered every 12 h, and LH release was assayed 1 h later. In female G11 KO mice and control strain, serum LH levels reached approximately 500 pg/25 microl within the first hour, then subsided to a steady level (approximately 100 pg/25 microl) for 109 h. In male G11 KO mice and in control strain, elevated LH release lasted for 13 h; however, LH levels in the G11 KO male mice did not reach control levels for approximately 49 h. In a similar experimental protocol, the Gq KO male mice released less LH (531 +/- 95 pg/25 microl) after 13 h from the start of treatment than the heterozygous male mice (865 +/- 57 pg/25 microl), but the female KO mice released more LH (634 +/- 56 pg/25 microl) after 1 h from the start of treatment than the heterozygous female mice (346 +/- 63 pg/25 microl). However, after the initial LH flare, the LH levels in the heterozygous mice never reached the basal levels achieved by the KO mice. G11 KO mice were less sensitive to low doses (5 ng/per animal) of Buserelin than the respective control mice. Male G11 KO mice produced more testosterone than the control mice after 1 h of stimulation by 2 microg of Buserelin, whereas there was no significant difference in Buserelin stimulated testosterone levels between Gq KO and heterozygous control mice. There was no significant difference in Buserelin stimulated estradiol production in the female Gq KO mice compared with control groups of mice. However, female G11 KO mice produced less estradiol in response to Buserelin (2 microg) compared with control strain. Although there were differences in the dynamics of LH release and steroid production in response to Buserelin treatment compared with control groups of mice, the lack of complete abolition of these processes, such as stimulated LH release, and steroid production, suggests that these G proteins are either not absolutely required or are able to functionally compensate for each other.

Animals↗
PubMed1998

Transcriptional activation of gonadotropin-releasing hormone (GnRH) receptor gene by GnRH and cyclic adenosine monophosphate.

GnRH appears to regulate messenger RNA levels and synthesis of its own receptor (GnRHR). In this study, we examined the regulation of GnRHR gene transcription by GnRH and cAMP in the GGH3 cell line (GH3 cells stably expressing GnRHR). Transient transfection of GGH3 cells with luciferase reporter gene vector (GnRHR-pXP2) containing a 1226-bp promoter fragment (-1164 to +62, relative to the major transcription start site) of mouse GnRHR gene resulted in an increase in reporter gene (GnRHR-Luc) activity (11- to 22-fold) compared with the promoterless vector. GnRH or a GnRH agonist (Buserelin) significantly stimulated the GnRHR-Luc activity in a dose-dependent manner. Time-course studies using 10(-7) M Buserelin revealed that GnRHR-Luc activity increased progressively from 1.5-6 h, with a peak at 6 h. The increase in GnRHR-Luc activity was lower at 12 and 24 h. Both cholera toxin and dBcAMP significantly stimulated GnRHR-Luc activity. Pretreatment with dBcAMP also enhanced the extent of stimulation of GnRHR-Luc activity in response to Buserelin. Pertussis toxin did not induce basal or Buserelin-stimulated GnRHR-Luc activity. Treatment of GGH3 cells with 10(-9) or 10(-7) M Buserelin for 6 h was sufficient to stimulate a significant increase in cAMP release. An adenylate cyclase inhibitor SQ 22536 did not affect the basal GnRHR-Luc activity but significantly reduced Buserelin-activated GnRHR-Luc activity. These results suggest that GnRH and cAMP activate transcriptional activity of the GnRHR gene and that GnRH activates GnRHR transcriptional activity, in part, through the cAMP pathway. Progressive 5'-deletion analysis revealed that basal and Buserelin- or dBcAMP-stimulated GnRHR-Luc activity were consistently retained after 5'-deletion at position -456, -381, or -331 relative to the major transcription start site but were significantly decreased after subsequent truncation of the promoter from -331 to -255 relative to the major transcription start site. However, the -255 construct still retained responsiveness to Buserelin and dBcAMP, and the relative activity remained similar under both stimulation conditions. These results suggest that elements located between -331 and -255 necessary for transcriptional activity of the GnRHR gene in GGH3 cells, and that the response elements on the mouse GnRHR gene for both GnRH and cAMP reside at two different sites: between -331 and -255 and between -255 and +62.

Animals↗
PubMed1998

Addition of catfish gonadotropin-releasing hormone (GnRH) receptor intracellular carboxyl-terminal tail to rat GnRH receptor alters receptor expression and regulation.

Mammalian GnRH receptor (GnRHR) is unique among G protein-coupled seven-transmembrane segment receptors due to the absence of an intracellular C-terminal tail frequently important for internalization and/or desensitization of other G protein-coupled receptors. The recent cloning of nonmammalian (i.e. catfish, goldfish, frog, and chicken) GnRHRs shows that these contain an intracellular C terminus. Addition of the 51-amino acid intracellular C terminus from catfish GnRHR (cfGnRHR) to rat GnRHR (rGnRHR) did not affect rGnRHR binding affinity but elevated receptor expression by about 5-fold. Truncation of the added C terminus impaired the elevated receptor-binding sites by 3- to 8-fold, depending on the truncation site. In addition, introducing the C terminus to rGnRHR altered the pattern of receptor regulation from biphasic down-regulation and recovery to monophasic down-regulation. The extent of down-regulation was also enhanced. The alteration in receptor regulation due to the addition of a C terminus was reversed by truncation of the added C terminus. Furthermore, addition of the cfGnRHR C terminus to rGnRHR significantly augmented the inositol phospholipid (IP) response of transfected cells to Buserelin, but this did not result from the elevation of receptor-binding sites. Addition of the C terminus did not affect Buserelin-stimulated cAMP and PRL release. GH3 cells transfected with wild-type cfGnRHR did not show measurable Buserelin binding or significant stimulation of IP, cAMP, or PRL in response to Buserelin (10[-13]-10[-9] M). GH3 cells transfected with C terminus-truncated cfGnRHR showed no IP response to Buserelin (10[-13]-10[-7] M). These results suggest that addition of the cfGnRHR intracellular C terminus to rGnRHR has a significant impact on rGnRHR expression and regulation and efficiency of differential receptor coupling to G proteins.

Amino Acid Sequence↗
PubMed1997

Regulation of G(q/11)alpha by the gonadotropin-releasing hormone receptor.

Evidence from use of pertussis and cholera toxins and from NaF suggested the involvement of G proteins in GnRH regulation of gonadotrope function. We have used three different methods to assess GnRH receptor regulation of G(q/11)alpha subunits (G(q/11)alpha). First, we used GnRH-stimulated palmitoylation of G(q/11)alpha to identify their involvement in GnRH receptor-mediated signal transduction. Dispersed rat pituitary cell cultures were labeled with [9,10-(3)H(N)]-palmitic acid and immunoprecipitated with rabbit polyclonal antiserum made against the C-terminal sequence of G(q/11)alpha. The immunoprecipitates were resolved by 10% SDS-PAGE and quantified. Treatment with GnRH resulted in time-dependent (0-120 min) labeling of G(q/11)alpha. GnRH (10(-12), 10(-10), 10(-8), or 10(-6) g/ml) for 40 min resulted in dose-dependent labeling of G(q/11)alpha compared with controls. Cholera toxin (5 microg/ml; activator of G(i)alpha), pertussis toxin (100 ng/ml; inhibitor of G(i)alpha actions) and Antide (50 nM; GnRH antagonist) did not stimulate palmitoylation of G(q/11)alpha above basal levels. However, phorbol myristic acid (100 ng/ml; protein kinase C activator) stimulated the palmitoylation of G(q/11)alpha above basal levels, but not to the same extent as 10(-6) g/ml GnRH. Second, we used the ability of the third intracellular loop (3i) of other seven-transmembrane segment receptors that couple to specific G proteins to antagonize GnRH receptor-stimulated signal transduction and therefore act as an intracellular inhibitor. Because the third intracellular loop of alpha1B-adrenergic receptor (alpha1B 3i) couples to G(q/11)alpha, it can inhibit G(q/11)alpha-mediated stimulation of inositol phosphate (IP) turnover by interfering with receptor coupling to G(q/11)alpha. Transfection (efficiency 5-7%) with alpha1B 3i cDNA, but not the third intracellular loop of M1-acetylcholine receptor (which also couples to G(q/11)alpha), resulted in 10-12% inhibition of maximal GnRH-evoked IP turnover, as compared with vector-transfected GnRH-stimulated IP turnover. The third intracellular loop of alpha2A adrenergic receptor, M2-acetylcholine receptor (both couple to G(i)alpha), and D1A-receptor (couples to G(s)alpha) did not inhibit IP turnover significantly compared with control values. GnRH-stimulated LH release was not affected by the expression of these peptides. Third, we assessed GnRH receptor regulation of G(q/11)alpha in a PRL-secreting adenoma cell line (GGH(3)1') expressing the GnRH receptor. Stimulation of GGH(3)1' cells with 0.1 microg/ml Buserelin (a metabolically stable GnRH agonist) resulted in a 15-20% decrease in total G(q/11)alpha at 24 h following agonist treatment compared with control levels; this action of the agonist was blocked by GnRH antagonist, Antide (10(-6) g/ml). Neither Antide (10(-6) g/ml, 24 h) alone nor phorbol myristic acid (0.33-100 ng/ml, 24 h) mimicked the action of GnRH agonist on the loss of G(q/11)alpha immunoreactivity. The loss of G(q/11)alpha immunoreactivity was not due to an effect of Buserelin on cell-doubling times. These studies provide the first direct evidence for regulation of G(q/11)alpha by the GnRH receptor in primary pituitary cultures and in GGH3 cells.

Animals↗
PubMed1997

Brain gonadotropin releasing hormone receptors: localization and regulation.

In this review, the current information about the location of GnRH receptor protein and GnRH receptor mRNA in the rat central nervous system is summarized as well as the changes that occur in the GnRH receptor mRNA levels during different endocrine conditions of the animals. The results of these studies show that GnRH receptor protein and mRNA levels change in parallel in the hippocampus, suggesting that pretranscriptional factors control the synthesis of the receptor. In the arcuate and ventromedial nuclei of the hypothalamus, GnRH receptor mRNA levels are highest during the early morning of proestrus and during the morning of an estrogen-progesterone-induced LH surge. The timing of the changes in GnRH receptor mRNA levels indicates that increasing levels of estradiol are responsible for the increase in GnRH receptor synthesis. Binding of GnRH agonist to the brain GnRH receptor causes a dose-dependent increase in inositol phosphates as well as changes in intracellular Ca++ levels of the target neurons. Together, it is suggested that GnRH functions in the brain as a neurotransmitter and/or modulator linking the peripheral endocrine effects of GnRH to actions of the peptide inside the central nervous system where it can facilitate, for example, reproductive behaviors.

Animals↗
PubMed1996

Contour of the GnRH pulse independently modulates gonadotropin secretion in the human male.

GnRH pulse frequency, amplitude, and interpulse interval have all been demonstrated to regulate gonadotropin secretion individually. We tested the hypothesis that the contour of the GnRH pulse also modulates gonadotropin output in 10 men with isolated GnRH deficiency in whom a fixed GnRH dose was administered at a constant physiologic frequency by either instantaneous bolus or by 1-, 5-, or 30-min infusions. LH, FSH and free alpha subunit (FAS) responses were also compared to spontaneous gonadotropin secretion in normal adult men. While the LH and FAS pulses following the instantaneous bolus and 1-min infusion of GnRH were indistinguishable, further increases in the duration of gonadotrope stimulation by GnRH were associated with progressive decreases in all parameters of gonadotropin secretion (mean levels, amplitude, peak levels, AUC). FSH secretion was also decreased following variations in the contour of the GnRH pulse, although overall changes were less dramatic than for LH and FAS. The LH pulses following the bolus GnRH stimulation were indistinguishable from spontaneous LH pulses occurring in normal men whereas those stimulated by the 1-, 5-, and 30-min infusions of GnRH became progressively blunted with the lowest levels of secretion occurring after the longest infusion. In sharp contrast, FAS pulse parameters in the GnRH-deficient subjects greatly exceeded those of normal men regardless of the contour of the GnRH stimulus, whereas mean FSH levels were all modestly (although significantly) higher than those of normal adult men. These results demonstrate that the pituitary is sensitive to subtle changes in the contour of the GnRH stimulus, with a more prolonged duration of GnRH stimulation resulting in a diminished pituitary response. Alterations of the contour of endogenous GnRH secretion may represent an additional mechanism for altering gonadotrope function and provide additional evidence for the differential regulation of LH, FAS, and FSH by GnRH. However, the previously reported elevated levels of FAS secretion in GnRH-deficient men undergoing long-term GnRH replacement are not explained by abnormalities of GnRH contour.

Adult↗
PubMed1996

Effects of Asn318 and Asp87Asn318 mutations on signal transduction by the gonadotropin-releasing hormone receptor and receptor regulation.

GnRH receptor (GnRH-R) contains Asn87 and Asp318 instead of the more frequently observed Asp87 and Asn318 found in other G protein-coupled receptors. In the present study, site-directed mutagenesis was used to introduce Asn318 and Asp87Asn318 into GnRH-R. The effect on coupling and regulation of GnRH-R was studied by stable expression of wild and mutant mouse GnRH-R in the lactotropic GH3 cells; these normally release PRL in response to TRH stimulation. The responses to Buserelin (a metabolically stable GnRH analog) in three different cell lines, M1, N8, and ND1 (expressing wild-type, Asn318 mutant, and Asp87Asn318 mutant mouse GnRH-R, respectively) were compared with that observed in the previously characterized GGH3-1' cells, which stably express rat GnRH-R. The Asn318 and Asp87Asn318 mutations had no measurable effect on ligand binding, but abolished the initial down-regulation of receptor that was observed in M1 and GGH3-1' cells, suggesting that the normal location of Asn87 and Asp318 in GnRH-R is involved in the regulation of GnRH-R. In N8 and ND1 cells, Buserelin-stimulated inositol phosphate (IP) production was attenuated, but the release of both cAMP and PRL was stimulated in a dose- and time-dependent manner. These mutations apparently impaired the coupling between GnRH-R and G proteins involved in IP production, but not those involved in cAMP release. In M1 cells, Buserelin stimulation produced a significant increase in IP production, but neither cAMP nor PRL release was significantly stimulated. These findings are consistent with the previous suggestion that GnRH-stimulated PRL release is mediated by a cAMP second messenger system in transfected GGH3 cells.

Amino Acid Sequence↗
PubMed1996

Biphasic action of cyclic adenosine 3',5'- monophosphate in gonadotropin-releasing hormone (GnRH) analog-stimulated hormone release from GH3 cells stably transfected with GnRH receptor complementary deoxyribonucleic acid.

GH3 cells are a PRL-secreting adenoma cell line derived from pituitary lactotropes. These cells have been stably transfected with rat GnRH receptor complementary DNA to produce four cell lines: GGH(3)1', GGH(3)2', GGH(3)6', and GGH(3)12'. In response to either GnRH or Buserelin (a metabolically stable GnRH agonist), these cell lines synthesize PRL in a cAMP-dependent manner. Only GGH(3)6' cells desensitize in response to persistent treatment with 10(-7) g/ml Buserelin. GGH(3)1', GGH(3)2', and GGH(3)12' cells, however, can be made refractory to Buserelin stimulation by raising cAMP levels either by the addition of (Bu)2cAMP to the medium or by treatment with cholera toxin. In GGH(3) cells, low levels of cAMP fulfill the requirements for a second messenger, whereas higher levels appear to mediate the development of desensitization. The observation that in GGH(3)6' cells, cAMP production persists after the onset of desensitization is consistent with the view that the mechanism responsible for desensitization is distal to the production of cAMP. Moreover, the absence of any significant difference in the amount of cAMP produced per cell in GGH(3)2', GGH(3)6', or GGH(3)12' cells suggests that elevated cAMP production per cell does not explain the development of desensitization in GGH(3)6' cells. We suggest that Buserelin-stimulated PRL synthesis in GGH(3)6' cells is mediated by a different cAMP-dependent protein kinase pool(s) than that in nondesensitizing GGH(3) cells. Such a protein kinase A pool(s) may be more susceptible to degradation via cAMP-mediated mechanisms than the protein kinase pools mediating the Buserelin response in nondesensitizing GGH(3) cells. A similar mechanism has been reported in other systems.

Animals↗
PubMed1996

Progesterone diminishes the sensitivity of gonadotropin-releasing hormone-stimulated luteinizing hormone (LH) release and protects an LH pool from desensitization: actions opposed by cholera toxin.

In the present study we used characterized perifusion model for the development of homologous desensitization to GnRH in the pituitary gonadotrope. This system relies on immobilized primary pituitary cultures that are perifused with various pulse patterns and concentrations of GnRH; these patterns and concentrations are selected to maintain, inhibit, restore, circumvent the desensitization process. The data indicate that progesterone (P) inhibits LH release in response to pulsatile administration of GnRH; this effect is blocked by the P antagonist, RU486, and is opposed by cholera toxin, an agent that provokes the formation of cAMP. In multiple and rapid pulse administration of 5 nM GnRH, which is usually associated with desensitization, the area under the LH release curve is progressively diminished when cells are preincubated with medium only or with medium containing cholera toxin. In contrast, cells pretreated with P are less responsiveness to GnRH, but maintain a relatively constant level of LH release (area under the curve). These data suggest a modulatory role for P in the development of the desensitized state and indicate that P can functionally protect a pool of LH from the onset of desensitization.

Animals↗
PubMed1996

Gonadotropin releasing hormone agonist provokes homologous receptor microaggregation: an early event in seven-transmembrane receptor mediated signaling.

Gonadotropin releasing hormone (GnRH) mediates interactions between the neural and the endocrine systems. The GnRH receptor is a member of the ubiquitous seven-transmembrane segment G-protein-coupled receptor class and is the target of drug development for treatment of breast and prostate cancer, regulation of fertility, endometriosis and a range of other medical and veterinary uses. This study shows that occupancy of the receptor by an agonist (but not an antagonist) promotes receptor-receptor interactions which appears to be an early event in hormone action.

Animals↗
PubMed1996

Leukemia inhibitory factor (LIF) induces acute adrenocorticotrophic hormone (ACTH) secretion in fetal rhesus macaque primates: a novel dynamic test of pituitary function.

Leukemia Inhibitory Factor (LIF), a pituitary cytokine, and LIF receptors are expressed in human fetal and adult adenohypophyseal cells. LIF induces adrenocorticotropin hormone (ACTH) secretion in vitro and potently synergizes with both corticotropin-releasing hormone (CRH) and cAMP-induced pro-opiomelanocortin (POMC) transcription. We therefore investigated the effects of intra-carotid administration of recombinant human LIF to chronically catheterized fetal non-human primates. LIF induced fetal monkey ACTH secretion in a time- and dose dependent manner. Maximal ACTH induction (12-fold) was achieved with 100 micrograms/kg after 60 minutes (p < 0.01). CRH (10 micrograms/kg) also induced ACTH secretion 4.8-fold at 60 minutes. Co-injection of LIF (50 micrograms/kg) and CRH (10 micrograms/kg) synergistically induced ACTH levels in a time-dependent manner up to 23-fold after 60 minutes. Thus, LIF alone induces ACTH secretion and LIF acts in synergy with CRH in vivo. As LIF is expressed early in human fetal pituitary development, and potentiates corticotroph function both in vitro and in vivo, this immuno-regulatory cytokine may be useful for clinical testing of the hypothalamic-pituitary-adrenal axis.

Adrenocorticotropic Hormone↗
PubMed1995

A mechanism for the differential regulation of gonadotropin subunit gene expression by gonadotropin-releasing hormone.

The hypothalamic hormone gonadotropin-releasing hormone (GnRH) is released in a pulsatile fashion, with its frequency varying throughout the reproductive cycle. Varying pulse frequencies and amplitudes differentially regulate the biosynthesis and secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by pituitary gonadotropes. The mechanism by which this occurs remains a major question in reproductive physiology. Previous studies have been limited by lack of available cell lines that express the LH and FSH subunit genes and respond to GnRH. We have overcome this limitation by transfecting the rat pituitary GH3 cell line with rat GnRH receptor (GnRHR) cDNA driven by a heterologous promoter. These cells, when cotransfected with regulatory regions of the common alpha, LH beta, or FSH beta subunit gene fused to a luciferase reporter gene, respond to GnRH with an increase in luciferase activity. Using this model, we demonstrate that different cell surface densities of the GnRHR result in the differential regulation of LH and FSH subunit gene expression by GnRH. This suggests that the differential regulation of gonadotropin subunit gene expression by GnRH observed in vivo in rats may, in turn, be mediated by varying gonadotrope cell surface GnRHR concentrations. This provides a physiologic mechanism by which a single ligand can act through a single receptor to regulate differentially the production of two hormones in the same cell.

Animals↗
PubMed1995

Regulation of hippocampal gonadotropin releasing hormone (GnRH) receptor mRNA and GnRH-stimulated inositol phosphate production by gonadal steroid hormones.

The purpose of the present study was to determine if gonadal steroids can alter the amounts of GnRH receptor mRNA in the pyramidal and granule neurons of the hippocampus of female and male rats and if GnRH causes a change in the production of inositol phosphates in hippocampal slices in vitro. The results show that in the ovariectomized rat the amount of GnRH receptor mRNA is increased to 137% in area CA1 and to 147% in area CA3 and in the dentate gyrus when compared to the ovariectomized, estradiol-progesterone treated animal. Similarly, in the orchidectomized male rat the amount of GnRH receptor mRNA is increased to 155% in area CA1, to 146% in area CA3 and to 145% in the dentate gyrus when compared to the intact male rat. There was no significant difference in the relative amounts of GnRH receptor mRNA when gonadectomized male and female rats were compared. Addition of GnRH (100 pM-1 microM) to hippocampal slices in vitro caused a dose-dependent increase in the production of [3H]inositol phosphate which was abolished by co-administration of a GnRH antagonist. The increase in inositol phosphate production was significantly higher at low doses of GnRH (100 pM-1 nM) in estradiol-progesterone treated female and in intact male rats when compared to gonadectomized rats. The results suggest that the amount of GnRH receptor mRNA in the hippocampus is at least in part regulated by gonadal steroids and that the steroid hormones can sensitize the GnRH target neurons to respond more robustly to a GnRH stimulus.

Animals↗
PubMed1995

GH3 cells transfected with gonadotropin-releasing hormone (GnRH) receptor complementary deoxyribonucleic acid release secretogranin-II through a constitutive pathway after GnRH analog-regulated synthesis: evidence that secretory proteins do not contain a sequence that obligates processing through a secretory granule or by regulated secretion.

GGH3 cells (lactotropic GH3 cells transfected with rat GnRH receptor complementary DNA) respond to GnRH agonists with time- and dose-dependent release of secretogranin-II (SII), a molecule believed to be a marker of the regulated pathway of release and a component of secretory granules. Release requires both protein and RNA synthesis and is also stimulated by analogs of the cyclic nucleotides. Release, as such, appears to be determined by the availability of SII, because this process is constitutive once the molecule is synthesized, and intracellular levels are at the same steady state whether the receptor is occupied by an agonist or an antagonist or is unoccupied. This synthetic requirement is consistent with the lag in time from stimulation to release and the inhibitory actions of either cycloheximide or actinomycin-D on secretion as well as the morphologic observation that storage granules (i.e. secretory granules) are absent in these cells. SII, accordingly, can be released constitutively and does not require processing through the so-called regulated secretory pathway. These observations suggest, in addition, that proteins associated with release through regulated pathways do not have "sorting" domains that preclude release via constitutive routes or require processing through secretory granules.

Animals↗