Phorbol ester inhibits luteinizing hormone-, forskolin-, and dibutyryl cyclic AMP-induced progesterone production in chicken granulosa cells

The possible role of protein kinase C in avian granulosa cell steroidogenesis was studied in vitro by examining the effect of tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) on progesterone synthesis in chicken granulosa cells in short-term (3h) incubations. TPA (1-100 nM) caused a marginal but nonsignificant increase in progesterone production in granulosa cells isolated from the largest preovulatory follicle. When incubated in combination with luteinizing hormone (5-100 ng/mL), TPA suppressed the stimulatory effects of submaximally and maximally effective doses of the gonadotropin in a concentration-related manner. Similarly, the phorbol ester inhibited the steroidogenic responses to forskolin and dibutyryl cyclic AMP. By comparison, TPA had no appreciable effect on the metabolism of exogenous pregnenolone substrate to progesterone. Our data indicate that the tumor-promoting phorbol ester influences steroidogenic steps distal to cyclic AMP generation but at or before pregnenolone formation, and that protein kinase C may be a negative regulator of steroid biosynthesis in chicken granulosa cells.     

Arachidonic acid inhibits luteinizing hormone-stimulated progesterone production in hen granulosa cells

Arachidonic acid has been proposed to function as a hormone-induced second messenger in a variety of mammalian endocrine tissues. The present studies were conducted to evaluate whether arachidonic acid, either added exogenously or released endogenously following treatment with physiologic (phospholipase A2) or pharmacologic (melittin) agents, influences basal and/or luteinizing hormone (LH)-induced cyclic adenosine 3',5'-monophosphate (cAMP) and progesterone production in granulosa cells from domestic hens. Phospholipase A2 (PLA2) and melittin treatments failed to alter basal concentrations of progesterone, whereas arachidonic acid had a slight stimulatory effect (only at the 50-microM dose) on progesterone levels, and no effect on cAMP. By contrast, arachidonic acid, PLA2, and melittin each inhibited LH-promoted progesterone production in a dose-dependent fashion. The inhibitory effects of arachidonic acid on the progesterone response were determined to occur both prior and subsequent to cAMP formation since cAMP levels in arachidonic acid-treated cells were attenuated after treatment with 10 ng LH or 100 microM forskolin (at 10- to 100-microM doses of arachidonic acid), and progesterone production was decreased in the presence of 1 mM 8-bromo-cAMP (with 50 and 100 microM arachidonic acid). The post-cAMP mechanism of action is characterized by the inability of cells to convert 25-hydroxy-cholesterol, but not pregnenolone, to progesterone. The effects of arachidonic acid are probably direct, since pharmacologic inhibitors of the lipoxygenase (nordihydroguaiaretic acid) and cyclooxygenase (indomethacin) pathways of arachidonic acid metabolism failed to alter the suppression of     

Influence of follicular maturation on 3-hydroxy-methylglutaryl coenzyme A reductase activity in hen granulosa cells

The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase: EC 1.1.1.34) was measured in a microsomal preparation of the granulosa of rapidly growing ovarian follicles of laying hens in the late preovulatory period (2-3 h before expected ovulation). The specific activity of the enzyme was measured in the five largest (F1-F5) preovulatory follicles, F1 being the follicle destined to ovulate first. Enzyme activity increased concomitantly with follicle size. The apparent Km of the enzyme decreased 60-80% from the smallest to the largest preovulatory follicle. There was no significant change in the Vmax during follicle development. Although our results have demonstrated the presence of HMG/CoA reductase in chicken granulosa cells and the progressive increase of its activity with follicular maturation, the quantitative significance of de-novo synthesized cholesterol as steroid hormone precursor remains to be ascertained.     

Influence of follicular maturation on luteinizing hormone and guanosine 5'-O-thiotriphosphate-promoted breakdown of phosphoinositides and calcium mobilization in chicken granulosa cells

Previous studies in this laboratory have shown a remarkable increase in the capacity of avian granulosa cells to produce progesterone during the last 2-3 days of folliculogenesis, with concomitant increases in the activities of key steroidogenic enzymes. In view of the proposed involvement of inositol phospholipids and their hydrolytic products in signal transduction in steroidogenic cells, we investigated in this study the influence of follicular maturation on phosphoinositide-specific phospholipase C (PLC) and intracellular Ca2+ release in chicken granulosa cells. Resting concentrations of intracellular calcium ([Ca2+]i) as measured in Fura 2/AM-loaded cells increased significantly during the last 48 h of follicular maturation, from 185 +/- 9 nM in the third largest follicle (F3) to 355 +/- 26 in the cells of the largest (F1) follicle. Luteinizing hormone (LH) caused a dose-related rise in [Ca2+]i, but the dose response was left-shifted by more than one order of magnitude in F1 cells compared to F2 cells. In granulosa cells of less developed follicles, LH failed to raise [Ca2+]i. To assess PLC activity, granulosa cells from F1, F2, and F3 follicles were labeled with [3H]inositol for 2 h and then stimulated with LH (0.1 microgram/ml). Time course studies showed that within 30 s, phosphatidylinositol-4,5 bisphosphate (PIP2) decreased by 33%, 13%, and 11% in F1, F2, and F3 cells, respectively. Similar responses were obtained when permeabilized cells were exposed to guanosine 5'-O-thiotriphosphate, which also caused a corresponding increase in 45Ca efflux from F1 and F2 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of cyclic 3',5'-adenosine monophosphate on central neurons

In experiments on unanesthetized non-immobilized rabbits, unit responses in the cortex and thalamus to the cyclic 3',5'-adenosine monophosphate (cAMPh) were studied by means of microionophoresis. It was shown that cAMPh changes the pattern of background unit activity, increasing or decreasing the discharge frequency. cAMPh changes unit responses both to acetylcholine and noradrenaline. These data permit, to assume that cAMPh participates both in adrenergic and in acetylocholinergic mechanisms of excitation processing in brain neurones.     

Influence of cyclic 3',5'-adenosine monophosphate on uracil uptake by rifampicin treated Escherichia coli cells.

Incubation of cells from a wild type strain of E. coli with 0.3 mg/ml rifampicin for 15 minutes lead to a complete inhibition of RNA synthesis measured as the uracil incorporation into the trichloroacetic acid insoluble fraction. In these rifampicin-treated cells [14C]uracil incorporation tended to decrease during a further incubation at 37 degrees. Addition of cyclic AMP increased the inactivation of the system responsible for [14C]uracil uptake. The cyclic nucleotide effect seems to be specific since ATP or 5'AMP did not increase such inactivation.     

Biphasic effect of calcium on luteinizing hormone-stimulated cyclic adenosine 3',5'-monophosphate production in granulosa cells of the fowl (Gallus domesticus).

Both cAMP and Ca2+ play important roles in the steroidogenic action of LH in hen granulosa cells. However, the interaction of these intracellular messengers is not fully understood. In the present study we used two calcium ionophores (ionomycin and A23187), as well as trifluoperazine (TFP), an inhibitor of calmodulin, to investigate LH- and forskolin-induced cAMP production in granulosa cells isolated from the largest (F1) preovulatory follicle of White Leghorn laying hens. Between 0.1 and 1.0 microM, both ionophores significantly potentiated cAMP responses to LH in the presence of 0.1 mM extracellular Ca2+. When calcium was omitted from the medium, ionophores had no effect. When either calcium was raised above 1 mM, or the concentration of ionophores was increased above 1 microM, LH-induced cAMP production was drastically inhibited. In the presence of 0.5-2.0 mM calcium, A23187 inhibited forskolin-promoted cAMP synthesis. TFP, while having no effect on basal cAMP, suppressed LH-induced responses and the potentiating effect of ionomycin. It is concluded that for full activation of the adenylate cyclase/cAMP system by LH, Ca-calmodulin is required at a site upstream from the catalytic component of the enzyme. However, high intracellular Ca2+ and/or other effects of ionophores (such as uncoupling of oxidative phosphorylation) inhibit LH-induced cAMP production.     

Deglycosylated human follitropin: characterization and effects on adenosine cyclic 3',5'-phosphate production in porcine granulosa cells

Chemical deglycosylation of gonadotropins with hydrogen fluoride (HF) has facilitated the investigation of the structure-function relationship of the individual peptide and oligosaccharide moieties in the mechanism of hormone action. These studies have dealt almost exclusively with lutropin or human choriogonadotropin. We report here the chemical characterization and biological properties of deglycosylated human follitropin (degly hFSH). Results indicate that deglycosylation of hFSH by HF removes 89% of the total carbohydrate without disruption of the peptide chain or significant loss of amino acid residues. However, a change in the conformation of the molecule was observed by measurement of the far-ultraviolet circular dichroic spectrum. The degly hFSH showed a 44% reduction in binding when tested in a FSH radioimmunoassay utilizing a polyclonal antibody. Binding of the degly hFSH to FSH-responsive tissues showed that the altered hormone bound with equal or better avidity than the intact hormone while the association constants were approximately the same for both preparations. The degly hFSH alone did not stimulate the FSH-stimulatable adenylyl cyclase (AC) activity of cellular homogenates of small follicle porcine granulosa cells. Furthermore, degly hFSH was a potent antagonist of hFSH-stimulatable AC activity when coincubated with intact hFSH. In intact granulosa cells, both the hFSH and the degly hFSH stimulated cAMP production and release by these cells. However, the degly hFSH was one-tenth as effective as the intact hormone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of interleukin-8 on estradiol and progesterone production by bovine granulosa cells from large follicles and progesterone production by luteinizing granulosa cells in culture

Interleukin 8 (IL-8) is a chemoattractant involved in the recruitment and activation of neutrophils and is associated with the ovulate process. We examined the possible role of IL-8 in steroid production by bovine granulosa cells before and after ovulation. The concentration of IL-8 in the follicular fluid of estrogen-active dominant (EAD) and pre-ovulatory follicles (POF) was higher than that of small follicles (SF). CXCR1 mRNA expression was higher in the granulosa cells of EAD and POF than that of SF. In contrast, CXCR2 mRNA expression was lower in granulosa cells of EAD and POF than in SF. IL-8 inhibited estradiol (E2) production in follicle-stimulating hormone (FSH)-treated granulosa cells at 48 h of culture. IL-8 also suppressed CYP19A1 mRNA expression in FSH-treated granulosa cells. IL-8 stimulated progesterone (P4) production in luteinizing hormone (LH)-treated granulosa cells at 48 h of culture. Although IL-8 did not alter the expression of genes associated with P4 production, it induced StAR protein expression in LH-treated granulosa cells. The expression of CXCR1 mRNA in corpus luteum (CL) did not change during the luteal phase. In contrast, the expression of CXCR2 mRNA in middle CL was significantly higher than in early and regression CL during the luteal phase. In luteinizing granulosa cells, an in vitro model of granulosa cell luteinization, CXCR2 mRNA expression was downregulated, whereas CXCR1 mRNA expression was unchanged. IL-8 also stimulated P4 production in luteinizing granulosa cells. These data provide evidence that IL-8 functions not only as a chemokine, but also act as a regulator of steroid synthesis in granulosa cells to promote luteinization after ovulation.     

Effect of cyclic 3',5'-adenosine monophosphate on the sporulation of Saccharomyces cerevisiae

Cyclic 3',5'-adenosine monophosphate and sodium dibutyryl cyclic3',5'-adenosine monophosphate had no effect on sporulation ofSaccharomyces cerevisiae, when added to a sporulation mediumnot enriched with glucose. They did, however, reverse the repressionof sporulation by glucose, when added to the sporulation mediumtogether with glucose. 5'-AMP, 5'-ADP and 5'-ATP did not reversethe repression of sporulation by glucose. (Received February 24, 1972; )     

Photoaffinity labeling of three renal cyclic 3',5'-adenosine monophosphate-binding proteins.

Evidence is presented for the presence of multiple cyclic AMP binding components in the plasma membrane and cytosol fractions of porcine renal cortex and medulla. N6-(Ethyl-2-diazomalonyl)-3',5'-adenosine monophosphate, a photoaffinity label for cyclic AMP binding sites, exhibits non-covalent binding characteristics similar to cyclic AMP in membrane and soluble fractions. Binding data for either compound to the plasma membrane fraction yields biphasic Scatchard plots while triphasic plots are obtained with the dialyzed cytosol. When covalently labeled fractions are separated on SDS-polyacrylamide gel electrophoresis, the cyclic AMP photoaffinity label is found on 49 000 and 130 000 dalton components in each kidney fraction. DEAE-cellulose and gel filtration chromatography of the labeled cortical cytosol fraction establishes that the three components suggested by the binding data correspond to two 49 000 dalton species and a 130 000 component. The 49 000 species have higher affinities for cyclic AMP than the 130 000 component (Ka(1) = 2.0 . 10(9), Ka(2) = 1.7 . 10(8), Ka(3) = 1.0 . 10(7)). The 49 000 components are associated with protein kinase activity while the 130 000 component does not exhibit protein kinase, adenosine deaminase, or cyclic nucleotide phosphodiesterase activity. Immunologic results and effects of phosphorylation and cyclic GMP on cyclic AMP binding further suggest that the 49 000 components are regulatory subunits of cyclic AMP-dependent protein kinases. Cyclic AMP binding to the 130 000 component is markedly inhibited by adenosine and adenine nucleotides, but not cyclic GMP. Thus, this component may reflect an aspect of adenosine control or metabolism which may or may not be a cyclic AMP-related cellular function.