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Biomedical subjects

P M Conn

Publications and source records attributed to P M Conn.

At least 55 records · Page 3Linked to original sources
PubMed1995

Transient transfection of GGH3-1' cells [GH3 cells stably transfected with the gonadotropin-releasing hormone (GnRH) receptor complementary deoxyribonucleic acid] with the carboxyl-terminal of beta-adrenergic receptor kinase 1 blocks prolactin release: evidence for a role of the G protein beta gamma-subunit complex in GnRH signal transduction.

G proteins consist of heterotrimeric alpha-, beta-, and gamma-subunits. To assess the role of the beta gamma-subunit complex in GnRH receptor-mediated signal transduction, GGH3-1' cells were transfected with plasmids PRK5-beta ARK1 (495-689) containing complementary DNA (cDNA) of the carboxyl-terminal (Gly495-Leu689) of beta-adrenergic receptor kinase 1 (beta ARK1). GGH3-1' cells are GH3 cells that have been stably transfected with rat GnRH receptor cDNA. The carboxyl region of beta ARK1 (Gly495-Leu689) binds to the beta gamma complex and thereby inhibits its action. Twenty-four hours after stimulation, PRL release, cAMP release, and inositol phosphate (IP) production were measured in these cells and in control cells transfected with vector PRK5 cDNA alone. In cells expressing the beta ARK1-(495-689) sequence there was inhibition of basal PRL release (69.3%), cAMP release (61.2%), and IP production (75.5%) compared to cells containing vector only. When cells expressing the beta ARK1 fragment were stimulated with a GnRH analog (Buserelin; 10(-7) M), maximal responses were inhibited (66.1% for PRL release, 52.3% for cAMP release, and 79.1% for IP production). Scatchard analysis of GnRH analog binding was also performed in the two groups of transfected cells. No significant differences in Kd or receptor numbers were found between beta ARK1-(495-689)-transfected cells and control cells containing the vector alone. These data suggest a role for the beta gamma complex in mediation of cAMP release, IP production, and hormone release in response to agonist occupancy of the GnRH receptor.

Animals↗
PubMed1994

Gonadotropin-releasing hormone and its receptors in rat brain.

Since the chemical identification of gonadotropin-releasing hormone (GnRH) in 1971, significant progress has been made in understanding the mechanisms by which GnRH action is mediated in the anterior pituitary. In contrast, relatively little information is available which identifies the intracerebral sites and mechanisms of action of GnRH in the brain. Early immunohistochemical studies of GnRH distribution in the central nervous system, together with behavioral and electrophysiological experiments, suggested that GnRH functioned as a neurotransmitter and was, possibly, involved in the expression of reproductive behaviors. The subsequent identification and characterization of GnRH receptors in the brain further strengthened the view that GnRH caused specific effects in select regions of the brain known to be involved in the neuroendocrine regulation of the anterior pituitary and in the generation of reproductive behaviors. After the mouse pituitary GnRH receptor was cloned it became possible to identify brain neurons which contained the GnRH receptor mRNA. Comparison of the locations of the GnRH peptide, the GnRH receptor protein, and the neurons which contain the GnRH receptor mRNA suggests that the GnRH neuronal system itself can potentially provide a direct link between the neuroendocrine regulation of anterior pituitary function and the intracerebral regulation of reproductive behaviors; furthermore, it is possible that the GnRH neuronal system takes an active part in intracerebral feedback loop systems between the mediobasal hypothalamus and the septum-diagonal band which regulate GnRH release from the median eminence.

Amino Acid Sequence↗
PubMed1994

Effects of GnRH analogs on six ovarian cancer cell lines in culture.

The objective was to evaluate the possible direct anti-tumor effects of the GnRH agonists buserelin and leuprolide acetate and the GnRH antagonist antide on ovarian cancer cell lines in culture. Evidence from several hormone-responsive tumor systems suggests that GnRH analogs may have direct anti-tumor effects at the cellular level. In small clinical trials, the use of GnRH analogs in advanced refractory ovarian cancer has produced some dramatic responses. With this in mind, we evaluated the growth effects of the GnRH agonists buserelin and leuprolide and the GnRH antagonist antide over a range of concentrations on six established ovarian cancer cell lines over a period of 7 days. Cell viability was measured by an ATP-bioluminescence assay and expressed as a percentage of untreated control cultures. Analysis of variance was used to evaluate significant growth differences from control cultures. Although we were able to demonstrate statistically significant reduction in cell growth in two of six cell lines with buserelin, no dose-related response patterns were seen. While buserelin caused a maximum 16% inhibition of growth, antide had no effect on tumor growth at the same doses. Leuprolide acetate produced significant dose-dependent inhibition of growth in all six cell lines when doses were increased to supraphysiologic levels, but demonstrated no significant inhibition at doses that are clinically attainable. Furthermore, competitive binding assays showed no specific GnRH binding in any of the six ovarian cell lines tested. Although GnRH analogs have growth inhibitory effects on ovarian cancer cells in vitro, the magnitude of this effect at doses that are clinically achievable is insufficient to support direct tumor effects as the mechanism behind the clinical responses reported.

Analysis of Variance↗
PubMed1994

Gonadotropin-releasing hormone and its analogs.

GnRH and its analogues have led to exciting new avenues of therapy in virtually every subspecialty of internal medicine as well as in gynecology, pediatrics, and urology. Since their discovery in 1971, it has been demonstrated that GnRH and its analogues enable medical professionals to influence the hypothalamic-pituitary-gonadal axis in two distinct classes of therapeutic applications. The first provides natural sequence GnRH in a pulsatile fashion via portable infusion pumps to mimic the normal physiology of hypothalamic GnRH secretion and restores reproductive potential to infertile men and women with disorders of endogenous GnRH secretion. The second mode uses long-acting GnRH agonists administered in a depot delivery to produce a paradoxical desensitization of pituitary gonadotropin secretion which, in turn, results in a complete ablation of the reproductive axis. This biochemical castration induced by GnRH agonist administration is a safe, effective, complete, and reversible method of removing the overlay of gonadal steroids from a variety of diseases which they are known to exacerbate. These diseases include endometriosis and uterine fibroids in women, prostate cancer in men, and precocious puberty in both sexes. This review examines the physiologic and pharmacologic principles underlying the advances produced by these agents, the mechanism of action of GnRH and its analogues at the cellular level, and the individual therapeutic applications to which these analogues have been applied. Because virtually every subspecialty of medicine will be touched by the GnRH analogues, this review provides an overview and background of their use.

Female↗
PubMed1994

Activin modulates the intracellular signaling system activated by gonadotropin-releasing hormone: dual effect on calcium messenger system and protein kinase-C pathway.

This study was performed to assess the effects of activin on intracellular mechanisms involved in GnRH action. When rat pituitary cell cultures were pretreated with activin-A (5-80 ng/ml) for 3 days, subsequent FSH and LH release (percentage of total cellular FSH and LH released during 4 h) in response to GnRH (10(-10)-10(-6) M) was not significantly different from that in cells pretreated with medium alone. In contrast, activin pretreatment increased the potency of both A23187 (Ca2+ ionophore) and phorbol 12-myristate 13-acetate [a protein kinase-C (PKC) activator] as secretagogues for FSH and LH release. FSH or LH release in response to another Ca(2+)-mobilizing secretagogue, maitotoxin (an activator of the GnRH receptor-associated Ca2+ channel), was not increased by activin. Although PKC is capable of influencing the actions of Ca2+, which is believed to be the second messenger for GnRH action, neither GnRH- nor maitotoxin-stimulated gonadotropin release was increased by activin even when the influence of activin on PKC was eliminated by the addition of a PKC inhibitor (staurosporine; 100 nM) during the final 30 min of the 3-day pretreatment period. These results indicate that although activin does not influence GnRH action with regard to gonadotropin release, it increases the sensitivity of the system regulating gonadotropin release to increases in cytosolic Ca2+ concentrations and PKC activation. Furthermore, activin appears to exhibit an inhibitory effect(s) at some point(s) in GnRH action in a PKC-independent manner, which could be responsible for opposing the increased sensitivity of the gonadotrope to Ca2+. The differential effects of activin on gonadotropin release in response to Ca(2+)-mobilizing secretagogues (ionophore and maitotoxin) raise the possibility that the activity of the GnRH receptor-associated Ca2+ channel may be suppressed by activin.

Activins↗
PubMed1994

Stable transfection of GH3 cells with rat gonadotropin-releasing hormone receptor complementary deoxyribonucleic acid results in expression of a receptor coupled to cyclic adenosine 3',5'-monophosphate-dependent prolactin release via a G-protein.

GH3 cells, which normally release PRL in response to stimulation by TRH, have been stably transfected with rat GnRH receptor complementary DNA (GGH3-1' cells). Unlike the parent line, GGH3-1' cells express GnRH receptor, which can be measured in a radioligand assay using a metabolically stable GnRH analog. The number of receptors (11,000 +/- 2,800 receptors/cell; n = 3) and Kd (4.1 +/- 1.0 x 10(-8) M; n = 3), determined using a radioiodinated GnRH agonist, as well as binding inhibition values for GnRH agonists and antagonists and for unrelated substances suggest that this receptor is similar to those expressed in cell cultures derived from rat pituitaries, although the binding affinity is about 1 log lower in the former. Unlike GnRH-stimulated release of gonadotropins from primary pituitary cultures, which does not require protein synthesis and is not coupled to cAMP production, GnRH-stimulated PRL release from the transfected cell line is absolutely dependent on protein synthesis, and cAMP fulfills the requirements of a second messenger. The receptor appears to be coupled to adenylate cyclase-mediated PRL release through a cholera toxin-sensitive G-protein. These studies provide functional evidence to support the view that the cloned receptor is the physiological receptor for the releasing hormone, and that this receptor can differentially couple to G-proteins depending on their availability and accessibility in the target cell.

Animals↗
PubMed1994

Gonadotropin-releasing hormone (GnRH)-receptor coupling to inositol phosphate and prolactin production in GH3 cells stably transfected with rat GnRH receptor complementary deoxyribonucleic acid.

This study examines the relation between inositol phosphate (IP) production and PRL release in four GGH3 cell lines (GGH(3)1', GGH(3)2', GGH(3)6', and GGH(3)12'; lactotropic GH3 cells that have been stably transfected with rat GnRH receptor complementary DNA). Production of IPs is an early response of GGH3 cells to a GnRH agonist, measurable at 15-30 min and maximal at 60 min after treatment with Buserelin in [3H]inositol preloaded cells. In contrast, PRL release, which requires protein synthesis, is not measurable until 1-3 h and total cAMP production is not measurable until about 24 h (3). In one of the lines studied (GGH(3)2'), PRL was also released in response to TRH. Measurable expression of the PRL gene requires 1-2 days (2). All four lines produced IPs robustly after treatment with Buserelin, although the IP response to TRH is minimal in all lines, being the best in the GGH(3)2' line. Pretreatment of cells with cholera toxin (CTX) or pertussis toxin (PTX) attenuated TRH-induced IP production in GGH(3)1', GGH(3)2', or GGH(3)12' cells. No effect of CTX or PTX is measurable in GGH(3)6' cells in terms of TRH stimulation of IP production. In contrast, both toxins augment Buserelin-stimulated IP production in GGH(3)1' and GGH(3)6' cells, but have no action in the other two lines. Both CTX and PTX inhibit Buserelin-stimulated PRL production. This study suggests that IP production is the earliest measurable response of GGH3 cells to a GnRH agonist, although this event does not appear to be coupled to Buserelin-stimulated PRL release. Further, the studies with toxins suggest that Buserelin and TRH appear to regulate IP production by different mechanisms.

Animals↗
PubMed1994

Functional and morphological characterization of four cell lines derived from GH3 cells stably transfected with gonadotropin-releasing hormone receptor complementary deoxyribonucleic acid.

Four cell lines, stably transfected with rat GnRH receptor complementary DNA, have been prepared from the lactotropic GH3 cell line. All four lines (as well as the parent line and a line transfected with the vector DNA) show extensive rosettes of circular polyribosomes, characteristic of high protein synthetic activity, although secretory granules are virtually absent; the rough endoplasmic reticulum (rER) cisternae were short and straight. Instances were observed in which the ER reaches to the plasma membrane, suggesting a possible nongranular secretory route. All four lines (but not the parent or a control transfected line) expressed GnRH receptors that were down-regulated (1-5 h, depending on the cell line) after exposure to 10 nM GnRH; receptors then recovered (2-7 h). This pattern is reminiscent of the GnRH receptor in the primary gonadotrope cell cultures. All cell lines released PRL (4-96 h) in response to a GnRH agonist (D-tBuSer6-des-Gly10-Pro9-ethylamide-GnRH), an event that was inhibited by all three major classes of Ca+2 ion channel antagonists (methoxyverapamil, 1,4-dihydropyridines, and diltiazem); in contrast, GnRH-stimulated LH release from pituitary-derived primary cultures is only inhibited by methoxyverapamil. One line became refractory to GnRH analog stimulation after 24 h, although the other three released PRL vigorously up to the longest time point examined (96 h). All four lines responded substantially more robustly to 1 microgram/ml Buserelin than to 1 microgram/ml TRH. All four lines produced inositol phosphate metabolites and released immunoassayable cAMP (24 h) in response to treatment with Buserelin. These cell lines are good models for understanding the mechanisms by which the GnRH receptor is coupled to second messenger systems and for comparing these mechanisms with TRH-receptor coupling in the same cell.

Animals↗
PubMed1994

Gonadotropin-releasing hormone antagonist for postpartum contraception: outcome for the mother and male offspring in the marmoset.

GnRH antagonists have potential for use in postpartum contraception, as it is likely that they would be effective in maintaining the inhibition of ovulation associated with lactation, but possible effects on the infant by transfer of antagonist via breast milk are unknown. The aim of this study was to establish whether chronic treatment with a GnRH antagonist would prevent ovulation throughout the period of lactation using the marmoset monkey as a model and to evaluate the effects of the presence of GnRH antagonist in breast milk on the postnatal rise in plasma testosterone concentrations in male infants. Mothers who had delivered male twins were selected. Starting within 3 days postpartum, mothers (n = 5/group) were treated with either 6.0 or 0.6 mg/kg of the GnRH antagonist antide, sc, once per week for 11 weeks. Ten postpartum lactating females acted as controls. All animals were housed in family groups with fertile males. To determine possible effects on the postnatal rise in testosterone, plasma samples were collected from the male infants of the high dose antide-treated mothers at weekly intervals for 12 weeks. Plasma progesterone concentrations were monitored in lactating mothers until establishment of pregnancy in both treated and control groups. Concentrations of antide were determined by RIA in plasma from mothers in the high dose group, in their milk, and in pooled plasma samples from their infants. Ovulation followed by pregnancy occurred in all of the control animals. No ovulations occurred during treatment with antide. Antide was present throughout treatment in plasma and breast milk in the mother. In male infants feeding from mothers receiving high dose treatment, antide was not detectable in infant plasma, and plasma testosterone concentrations were within the normal range for male neonates in our colony. These results indicate that the GnRH antagonist has potential as a method of contraception postpartum without affecting the postnatal rise in testosterone in male infants.

Animals↗
PubMed1994

Evidence that signalling pathways by which thyrotropin-releasing hormone and gonadotropin-releasing hormone act are both common and distinct.

TRH and GnRH receptors are each coupled to G proteins of the Gq/11 family. Activation of each of these receptors by their respective ligands results in the stimulation of phospholipase C activity, leading to calcium mobilization and protein kinase C activation. Thus, the effects of TRH and GnRH may be mediated through the same intracellular signal transduction pathway. To compare responses to TRH and GnRH directly within one cell type, we have stably transfected the rat pituitary GH3 lactotrope cell line, which expresses the endogenous TRH receptor, with an expression vector containing rat GnRH receptor cDNA. Transfected cells specifically bound GnRH with high affinity and responded to GnRH stimulation with an increase in PRL mRNA levels, analogous to their response to TRH stimulation. Stably transfected GH3 cells, which were then transiently transfected with luciferase reporter constructs containing either the PRL or the glycoprotein hormone alpha-subunit promoter, responded to either GnRH or TRH stimulation with an increase in luciferase activity in a time- and dose-dependent fashion. The stimulatory effects of maximally effective concentrations of TRH and GnRH were additive on PRL, but not alpha-subunit, gene expression. These data, coupled with evidence of cross-desensitization of alpha-subunit, but not PRL, promoter activity stimulation by TRH and GnRH, suggest that there may be differences in the signal transduction pathways activated by TRH and GnRH receptors in the regulation of PRL and alpha-subunit gene expression.

Adenoma↗
PubMed1993

Single-dose administration of the gonadotropin-releasing hormone antagonist, Nal-Lys (antide) to healthy men.

OBJECTIVE: To evaluate the ability the Nal-Lys GnRH antagonist ([N-Ac-Nal (2)1, 4ClDPhe2, D3Pal3, Lys (Nic)5, D-Lys(Nic)6, Lys (iPr)8, D-Ala10] to suppress gonadotropins and T in humans and to assess its duration of action and its local effects. DESIGN: Placebo-controlled clinical study. SETTING: A university community. SUBJECTS: Seven normal male volunteers. INTERVENTIONS: We administered single injections of Nal-Lys (0, 10, 25, and 50 micrograms/kg body weight). Blood samples were collected before and at frequent time intervals after injection. RESULTS: Nal-Lys caused only minor local effects. At the higher doses (25 and 50 micrograms/kg), serum LH and T levels were suppressed to 50% to 70% of baseline; serum FSH levels were suppressed to 70% to 80% of baseline, and levels of all three hormones returned to basal values within 24 hours after injection. CONCLUSIONS: In humans, Nal-Lys has similar potency and duration of action to other antagonists and produces fewer local side effects. However, the utility of Nal-Lys is limited by formulation difficulties; current efforts are directed at improving the formulation in order to explore the potential clinical uses of this peptide.

Adult↗
PubMed1993

Cholera toxin and pertussis toxin provoke differential effects on luteinizing hormone release, inositol phosphate production, and gonadotropin-releasing hormone (GnRH) receptor binding in the gonadotrope: evidence for multiple guanyl nucleotide binding proteins in GnRH action.

A growing body of evidence suggests a role for guanyl nucleotide binding proteins (G proteins) in GnRH action. G protein activation provokes LH release, inositol phosphate (IP) production, decreased gonadotrope responsiveness to GnRH, increased gonadotrope responsiveness to the calcium ionophore A23187, and decreased GnRH receptor binding. The specific G proteins involved in these actions, however, are not known. This study uses pertussis toxin (PTX) and cholera toxin (CTX), which affect the activity of a number of G proteins by ADP ribosylation of a Cys or an Arg residue, respectively, of the alpha-subunit. Although not an effective LH secretagogue in itself, CTX enhanced GnRH-, NaF-, and A23187-stimulated LH release after an 18-h pretreatment period. CTX pretreatment did not affect GnRH- or NaF-stimulated IP production. Conversely, 18 h pretreatment with PTX reduced GnRH- and NaF-provoked IP production compared to control values, but did not affect LH release. In addition, pretreatment with either CTX, PTX, or Bt2cAMP provoked a decrease in GnRH receptor binding compared to control. The results of this study suggest that: 1) GnRH stimulates IP production, but not LH release, through a PTX-sensitive G protein; 2) A distinct CTX-sensitive G protein appears to provoke gonadotrope sensitization by stimulating an increase in intracellular cAMP levels; and 3) there appears to be a distinct G protein, insensitive to PTX and CTX, capable of mediating LH release independent of IP production and cAMP.

Animals↗
PubMed1993

A cholera toxin-sensitive guanyl nucleotide binding protein mediates the movement of pituitary luteinizing hormone into a releasable pool: loss of this event is associated with the onset of homologous desensitization to gonadotropin-releasing hormone.

Cholera toxin (CTX; 5 micrograms/ml), but not pertussis toxin (100 ng/ml), when preincubated with pituitary cells for 18 h, enhances the percentage of cellular LH released in response to continuous or pulsatile administration of 5 x 10(-9) M GnRH. This effect occurs without increasing total (intracellular plus extracellular) LH, indicating that it is best explained by redistribution of LH from a nonreleasable to a releasable pool. This site of action is consistent with the observation that CTX-pretreated cells are also sensitized to stimulation of LH release by the Ca2+ ionophore A23187. The observations that CTX stimulates the production of cAMP in these cells and that the sensitizing action of CTX is mimicked by (Bu)2cAMP (1 mM) are consistent with the view that a CTX-stimulated guanyl nucleotide binding protein, capable of activating adenylyl cyclase, is mediating this sensitization. We used a perifused cell system to show that the movement of LH into a releasable pool is lost with the onset of homologous desensitization due to high pulse frequency or constant administration of GnRH (5 x 10(-9) M, continuous or a pulse each 15 min). Sensitization to CTX is restored by stimulation with a high concentration of GnRH (10(-6) M) or by resetting the pulse frequency to the rate measured in vivo (a pulse each 90 min). Both of these treatments also circumvent the desensitized state, restoring LH release. These results identify a novel lesion associated with the development of desensitization in the gonadotrope and support the role of a CTX-sensitive guanyl nucleotide binding protein in regulation of pituitary responsiveness to GnRH.

Animals↗
PubMed1993

Modulation of activin A action and specificity in the rat gonadotrope by protein kinase C.

This study was undertaken to assess the role of protein kinase C (PKC) in activin action in the rat pituitary. Pretreatment with 500 nM phorbol 12-myristate 13-acetate (PMA) for 22-24 h reduced subsequent FSH and LH release (percentage of total cellular FSH and LH released) in response to 100 nM PMA. This action persisted for 2 days after the pretreatment. Pretreatment with 500 nM 4 alpha-phorbol 12,13-didecanoate (4 alpha PDD, a phorbol ester which does not activate PKC) did not affect cell responsiveness to 100 nM PMA. Both PKC-down-regulated cells and cells with a full complement of PKC responded similarly to 100 nM GnRH and 100 microM A23187 during this period. Incubation with 50 ng/ml activin A for 48 h significantly increased both FSH release and total FSH (extracellular plus intracellular) compared to corresponding basal values in PMA-pretreated cells, as well as in vehicle-or 4 alpha PDD-pretreated cells. Activin stimulation of basal FSH release and total FSH was significantly more potent in PMA-pretreated cells than in cells not pretreated with PMA. Activin did not alter basal LH release or total LH in vehicle- or 4 alpha PDD-pretreated cells but significantly increased both in PMA-pretreated cells. When PMA was present only during the initial 2 h of the 22- to 24-h pretreatment period at 50 nM, PKC was not down-regulated. In these cells, the potency of activin stimulation of basal FSH release was not affected, but stimulation of basal LH release by activin was still observed. These results suggest that PKC is not required for activin to stimulate FSH release but is involved as a modulator of potency and specificity of the activin action.

Activins↗
PubMed1993

Differential orientation of a GnRH agonist and antagonist in the pituitary GnRH receptor.

In the present study we have used a high affinity photoaffinity label (PAL) agonist (pGlu-His-Trp-Ser-125I-iodoTyr-D-Lys(para-N3-Benzoyl)-Leu-Arg-P roNHEt) and a PAL antagonist (NAcD2Nal-D4ClPhe-D3Pal-Ser-125I-iodo-NMeTyr-D-L ys(para-N3-Benzoyl)Leu-Lys(Isp) - Pro-D-Ala-NH2) to covalently label the GnRH receptor. Rat pituitary membranes were incubated 3 h with either the radioiodinated agonist or antagonist in the dark, exposed to UV light, then electrophoresed in SDS. The PAL agonist and antagonist labeled broad bands (estimated molecular weight [M(r)] 46K-60K). Labeling by either PAL agonist or antagonist was displaced by unlabeled agonist or antagonist, indicating that the agonist and antagonist bind to the same molecule. The broad band, believed to reflect differential glycosylation, was divided into six sections corresponding M(r) to 60K, 56K, 52K, 48K, 46K and 44K for the agonist and 62K, 58K, 54K, 52K, 48K and 45K for the antagonist; these were electroeluted with recovery > 80% based on radioactivity. Each could be re-electrophoresed to the same location from which it was eluted. Other eluted samples were treated with trypsin. The M(r) of the samples labeled with the agonist PAL were shifted to M(r) 48K, 42K, 40K, 37K, 35K and 33K by proteolysis. The observation that each section shifted approximately the same M(r) after trypsin treatment suggests that the backbone of the labeled proteins in each gel section is identical. Samples labeled with the antagonist PAL were shifted to M(r) < 10,000 in all cases. These data indicate that the agonist and antagonist PALs bind to different regions of the GnRH receptor and, therefore, are likely oriented differently with respect to the receptor and support the view that different strategies should be used for the design of agonists and antagonists.

Affinity Labels↗
PubMed1993

Cholera toxin and dibutyryl cyclic adenosine 3',5'-monophosphate sensitize gonadotropin-releasing hormone-stimulated inositol phosphate production to inhibition in protein kinase-C (PKC)-depleted cells: evidence for cross-talk between a cholera toxin-sensitive G-protein and PKC.

The present study assesses the relationship between G-proteins and protein kinase-C (PKC) in the gonadotrope. Cells were depleted of PKC with 1 microM phorbol 12-myristate 13-acetate for 12 h, followed by medium 199-BSA for 6 h before treatment with vehicle, pertussis toxin (PTX), cholera toxin (CTX), or (Bu)2cAMP (dBcAMP) for 18 h. PTX (10 ng/ml) significantly decreased GnRH-stimulated inositol phosphate (IP) production over a range of 10(-8)-10(-6) M GnRH. The degree of this inhibition was the same in control cells and PKC-depleted cells. Pretreatment with CTX (0.5 microgram/ml) significantly decreased GnRH-stimulated IP production over a range of 10(-9)-10(-6) M GnRH in PKC-depleted cells. This effect was mimicked by pretreatment with 3 mM dBcAMP. Although CTX and dBcAMP both decreased GnRH-stimulated IP production in control cells, this effect was enhanced in PKC-depleted cells. CTX (0.1 microgram/ml) and dBcAMP (3 mM) both enhanced GnRH-stimulated LH release, whereas PTX (100 ng/ml) had no effect. This was observed in control as well as PKC-depleted cells. Both PKA and PKC are capable of regulating IP turnover by phosphorylating phospholipase-C at distinct sites. CTX activates a G-protein that increases cAMP. cAMP can then activate PKA. In PKC-depleted cells, CTX inhibits GnRH-stimulated IP production. This effect is mimicked by dBcAMP, which suggests a role for PKA in the gonadotrope. The results of this study provide evidence for cross-talk between a CTX-sensitive G-protein and PKC.

Animals↗
PubMed1993

Assessment of the role of G proteins and inositol phosphate production in the action of gonadotropin-releasing hormone.

The first step in gonadotropin-releasing hormone (GnRH) action involves the binding of GnRH to a plasma membrane receptor. Calcium has been implicated as a second messenger molecule. More recently, it has been suggested that the products of phosphoinositide metabolism may act as a second messenger for GnRH-stimulated release of luteinizing hormone (LH). To be considered a second messenger, however, a candidate molecule must meet three requirements: in the second messenger's presence, (a) GnRH should stimulate increased production of inositol phosphate; (b) inositol phosphate production, stimulated by any means, should provoke LH release; and (c) inhibition of inositol phosphate production should block GnRH-stimulated release of LH.

Animals↗