Salman Azhar

LH biosynthesis and secretion in rat anterior pituitary cell cultures: Stimulation of LH glycosylation and secretion by GNRH and an agonistic analogue and blockade by an antagonistic analogue

Abstract In rat anterior pituitary cell cultures GnRH (1nM) stimulated a progressive increase in LH release into the medium from 1 to 8 h of incubation, while cellular LH showed a corresponding decrease. GnRH (1nM) neither modified the uptake nor the incorporation of [3H]-glucosamine and [3H]-proline into total protein. The incorporation of [3H]-proline into cellular LH also was unaffected by GnRH. In contrast, GnRH stimulated a 3 to 4-fold increase in [3H]-glucosamine incorporation into cellular LH. The agonistic analogue, [des GlyNH210]-LHRH ethylamide, mimicked the GnRH effects and was 5 to 6 times more potent than GnRH. The antagonistic analogue, [D-Phe2, D-Phe6]-LHRH blocked the GnRH-stimulated effects. These results suggest that GnRH and agonistic analogues may preferentially regulate turnover or synthesis of the carbohydrate moiety of LH.

Receptor-mediated gonadotropin action in the ovary. Demonstration of acute dependence of rat luteal cells on exogenously supplied steroid precursor (sterols) for gonadotropin-induced steroidogenesis.

Incubation of luteal cells with human, horse and rat sera, but not bovine sera resulted in enhanced basal and hCG-stimulated progesterone accumulation. The stimulatory effect of human or rat sera on basal, hCG- or 8 Br-cyclic AMP-induced progesterone synthesis in luteal cells was evident within 15-30 min after incubation, reaching a maximum after 3-4 h. The stimulatory effects of hCG and/or sera were blocked by inhibitors of RNA and protein synthesis. Similarly, lysosomotropic agents, chloroquine (100 microM) and ammonium chloride (10 mM), partly blocked the steroidogenic response of luteal cells to hCG and/or human or rat sera. Incubation of cells in the presence of 2-deoxyglucose, sodium azide and phenylmethylsulfonyl fluoride resulted in partial inhibition of progesterone secretion in response to hCG or sera. Fractionation of human or rat sera into various lipoprotein fractions demonstrated that LDL and HDL most effectively supported and potentiated the steroidogenic response to hCG. Lipoprotein-deficient serum, however, did not alter gonadotropin-induced steroid production. Incubation of luteal cells with increasing concentrations of h-LDL and h-HDL enhanced both basal and hCG-mediated steroidogenesis in a dose-related manner, although very high concentrations of these lipoproteins were inhibitory. Further, [3H]cholesterol from [3H]cholesteryl linoleate-LDL was incorporated into luteal cell progesterone and the extent of this incorporation was enhanced by hCG. Addition of excess unlabeled h-LDL, h-HDL, as well as r-HDL, drastically reduced the incorporation of radioactive label into progesterone. These studies suggest that (a) serum potentiation of steroidogenesis was due to presence of lipoproteins, mainly LDL and HDL, and (b) the lipoprotein-bound cholesterol is delivered into the luteal cells and utilized for steroidogenesis.

Evidence that danazol inhibits gonadotropin-induced ovarian steroidogenesis at a point distal to gonadotropin-receptor interaction and adenosine 3′,5′ cyclic monophosphate formation

The action of danazol on 125I-human chorionic gonadotropin (hCG) binding, gonadotropin-stimulated adenosine 3,5 cyclic monophosphate (cAMP) accumulation and progesterone production has been investigated in luteinized rat ovaries. Preincubation of luteal cells for short periods of time with increasing concentrations of danazol caused a significant inhibition of gonadotropin-stimulated steroidogenesis. The inhibitory effect of danazol was both concentration and time dependent. Danazol also reduced progesterone production in response to cholera enterotoxin and 8 bromo-adenosine-cAMP, but it had no effect on hCG, luteinizing hormone, and cholera enterotoxin stimulated cAMP formation. Similarly danazol did not affect 125I-hCG binding as assessed by the equilibrium dissociation constant (Kd) and number of hormone-binding sites on the luteal cell surface. These results suggest that in intact luteal cells danazol inhibits steroidogenesis at a point distal to hormone-receptor interaction and cAMP formation.

Receptor mediated gonadotropin action in gonadal tissues: Relationship between blood cholesterol levels and gonadotropin stimulated steroidogenesis in isolated rat leydig and luteal cells

The present studies were performed to evaluate the role of steroid precursors and plasma lipoproteins in gonadal tissue steroidogenesis. Leydig cell suspension isolated from rat testes responded to hCG. Bt2cAMP, 8 Br-cAMP and cholera toxin with an increase in testosterone response. Administration of 4-aminopyrazolo[3,4-d]pyrimidine (4-APP) reduced the plasma cholesterol and testosterone levels in a time and dose dependent manner. This treatment also reduced the steroidogenic capacity of isolated Leydig cells both under basal conditions and in response to trophic hormone. Different doses of 4-APP up to 25 mg/kg BW and up to 4 days of treatment, however, did not modulate cholesterol and cholesterol ester contents of isolated Leydig cells. 4-APP treatment also had no effect on testis weight, phospholipid content, protein synthesis and energy metabolism in isolated Leydig cells. Similarly, administration of 4-APP (12.5 mg/kg) to PMSG-hCG primed rats beginning on day 3, post hCG, drastically reduced the circulating cholesterol and progesterone levels. Injection of the drug also produced an inhibition in vitro luteal cell steroidogenesis and a reduction in cellular cholesterol esters and free cholesterol contents. Addition of LDL or HDL to incubation medium reversed the inhibitory effect of 4-APP on luteal cell steroidogenesis while this inhibition persisted in Leydig cells. Injection of rats with Triton-WR-1339 (mg/kg BW) resulted in a 10-fold increase in plasma cholesterol and a contrasting decrease in testosterone levels. This treatment, however, produced no effect on in vitro Leydig cell steroidogenesis or cellular content of cholesterol esters and free cholesterol. It appears that the Leydig and luteal cells process and utilize lipoprotein-delivered cholesterol for steroidogenesis through different mechanism(s). These studies thus demonstrate differential actions and an acute regulatory role of lipoproteins in gonadotropin modulated steroidogenesis in two different gonadal tissue.

Adenosine 3′,5′-monophosphate dependent phosphorylation of ribosomes and ribosomal subunits from bovine corpus luteum

Abstract In a previous publication the purification and properties of two protein kinases (KI and KII) from a soluble fraction of bovine corpus luteum and the stimulation of the latter fraction by cyclic AMP and luteinizing hormone was reported (Menon, K.M.J. (1973) J. Biol. Chem. 248, 494–501). We have now studied the effects of cyclic AMP and luteinizing hormone on ribosomal protein phosphorylation of corpus luteum by protein kinase II. Protein kinase II catalyzed the phosphorylation of ribosomes by transfer of terminal phosphate of ATP to ribosomal proteins. Extraction with hot trichloroacetic acid and non-aqueous solvent revealed that about 80% of total radioactivity incorporated remain associated with the protein residue. Radioactivity was identified in the phosphoserine and phosphothreonine residues of polypeptides by high voltage paper electrophoresis. The extent of phosphorylation was stimulated by cyclic AMP but not by luteinizing hormone. At least 9 proteins of 80-S ribosomes and 12 proteins of the 60-S ribosomal subunit were phosphorylated in the presence of cyclic AMP as resolved by urea polyacrylamide gel electrophoresis. However, only one major and four minor bands were phosphorylated in the case of 40-S ribosomal subunit under the influence of cyclic AMP. The ribosomal protein phosphorylation catalyzed by protein kinase II is regulated by cyclic AMP whereas luteinizing hormone has no effect on ribosome phosphorylation.

ROLE OF GANGLIOSIDES IN GONADOTROPIN AND CHOLERA ENTEROTOXIN STIMULATED STEROIDOGENESIS IN ISOLATED RAT OVARIAN CELLS

fifty-fold increase in progesterone production. Both cholera enterotoxin and hCG-stimulated progesterone response was accompanied by a lag period. The duration of the lag period in the production of the progesterone depended on the concentration ot gonadotropin or cholera enterotoxin, and with maximally stimulating dose it was 20-30.minutes. Addition of highly purified mixed gangliosides to the incubation medium abolished the stimulatory effect of cholera enterotoxin on progesterone response. In contrast, under iaentical experimental conditions, ganglioside addition produced no effect on progesterone response elicited by hCG or LH. Similarly mixed gangliosides did not prevent the specific binding of [ 1251]hCG to the ovarian cells or to the membranes isolated from the ovary. In addition preincubation of [12!jI]hCG with ganglioside did not alter the subsequent binding of the hormone to the ovarian cell surface receptor. These findings suggest that gangliosides are not involved in the hormone receptor interactions and subsequent receptor mediated physiological response.

Differential actions of gangliosides on gonadotropin and cholera enterotoxin stimulated adenosine 3′:5′ cyclic monophosphate dependent protein kinase in isolated rat ovarian cells

SUMMARY: Rat ovarian cells were exposed to cholera enterotoxin, and the effect on progesterone synthesis as well as on protein kinase stimulation was examined. Cholera enterotoxin stimulated ovarian steroidogenesis in a dose dependent manner similar to that of hCG. The stimulation of protein kinase by cholera toxin was followed by a lag period, whereas hCG effect was imnediate. Mixed gangliosides, when added to the incubation medium, blocked the cholera enterotoxin-stimulated protein kinase activity and abolished the decrease in exogenous [sH] cyclic AMP receptor activity brought about by the toxin. In contrast, under similar experimental conditions ganglioside addition elicited no effect on protein kinase activation produced by hCG or LH. The data suggest that gangliosides do not appear to be directly involved in gonadotropin binding to ovarian cell membrane and subsequent mediation of physiological response.

Sedimentation behavior of solubilized gonadotropin receptor from plasma membranes of bovine corpus luteum

The gonadotropin receptors associated with plasma membrane fractions were solubilized by detergents, including Triton X-100, Lubrol WX, Lubrol PX and sodium deoxycholate before and after equilibration with 125I-labelled human chorionic gonadotropin. The binding activity remained in solution even after centrifugation at 300 000 X g for 3 h. The solubilized gonadotropin receptor or gonadotropin receptor complex was characterized by gel filtration and sucrose density gradient centrifugation. Sucrose density gradient centrifugation of solubilized gonadotropin-receptor complex in the presence of Triton X-100 had a sedimentation coefficient of 6.5 S whereas the solubilized uncomplexed receptor had a sedimentation coefficient of 5.1 S. In the absence of the detergent, solubilized hormone receptor complex from plasma membrane fractions I and II sedimented with an apparent sedimentation coefficient of 6.6 S and 7.4 S, respectively. Similarly, the free receptor also showed higher sedimentation profile with an apparent sedimentation coefficient of 6.7 S for fraction I and 7.2 S for fraction II. Treatment of plasma membranes with phospholipase A and C inhibited the binding of 125I-labelled human chorionic gonadotropin in a dose dependent manner, whereas phospholipase D was without any effect. Doses of 1.4 mI. U. of phospholipase A or 0.6 mI.U. of phospholipase C were required to produce 50% inhibition of the binding activity. These phospholipases had no effect on the preformed 125I-labelled human chorionic gonadotropin-receptor complex nor on the sedimentation profile of solubilized gonadotropin receptor complex.

Cyclic adenosine 3', 5' -- monophosphate and luteinizing hormone stimulated protein kinase from bovine corpus luteum: Evidence for activation through separate mechanisms

Protein kinases catalyze the transfer of the terminal phosphate of ATP to a variety of acceptor proteins [ 11. It has been demonstrated that cyclic adenosine 3’, 5’-monophosphate (cyclic AMP) activates protein kinase (holoenzyme, RC) by forming a complex with the regulatory subunit (R) to release the catalytic subunit (C) in the active state [2,3]. In a previous communication from this laboratory the purification and properties of two protein kinases (KI and KII) from bovine corpus luteum has been described [4]. In addition to stimulation by cyclic AMP, KII was also stimulated directly by luteinizing hormone (LH). From these studies it was inferred that LH may have a direct control on the activity of KII and that this effect is independent of cyclic AMP. The present investigation was undertaken to compare the effect of LH with that of cyclic AMP on the stimulation of KII from bovine corpus luteum. Evidence is presented to suggest that cyclic AMP stimulates the activity of KII by binding to the regulatory subunit thereby releasing the activated catalytic subunit whereas LH acts without dissociating the regulatory-catalytic subunit complex.

Gonadotropin Induced Stimulation and Desensitization of Cyclic 3′,5′-Adenosine Monophosphate Dependent Protein Kinase(S) in the Rat Ovary

Incubation of rat ovarian slices with choriogonadotropin resulted in stimulation of cyclic AMP dependent protein kinase activity in a time and dose dependent manner. However, prior exposure of animals to choriogonadotropin in vivo resulted in a decreased sensitivity to subsequent in vitro stimulation by the same hormone. This decreased sensitivity was due to a lesion in the hormone receptor adenylate cyclase system rather than a change in the property of protein kinase with respect to its stimulation by cyclic AMP. Unchanged levels of cyclic AMP formation in these ovaries were evidenced by the lack of inhibition of [3H] cyclic AMP binding. In the untreated controls, however, choriogonadotropin in vitro resulted in inhibition [3H] cyclic AMP binding activity suggesting endogenous occupation parallel to protein kinase activation. These results are indicative of the linear coupling of hormone receptor-adenylate cyclase-protein kinase system and point out the in vivo role of protein kinase activation in choriogonadotropin mediated regulation of ovarian function.