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E K Asem

Publications and source records attributed to E K Asem.

69 records · Page 4Linked to original sources
PubMed1987

Effects of calcium channel activators BAY-K8644 and CGP-28392 on steroidogenesis in granulosa cells of the domestic hen.

The effects of calcium agonists BAY-K8644 and CGP-28392 on steroidogenesis was examined in chicken granulosa cells in short term incubation. BAY-K8644 (5-500 nM) and low doses of CGP-28392 (1-10 microM) failed to appreciably affect basal and LH-stimulated progesterone production whether tested in calcium free, low (0.05 mM) or high (3 mM) calcium containing medium. However, higher concentrations of CGP-28392 (50-250 microM) inhibited significantly (P less than 0.01) both basal and LH-stimulated steroidogenesis in a dose-related manner independently of extracellular calcium availability. The suppressive effect of CGP-28392 was manifest with submaximally and maximally stimulating LH doses. In additional experiments with non-hormonal agonists such as forskolin, dibutyryl cyclic AMP and kaurenol, BAY-K8644 and low CGP-28392 concentrations were again without effect on steroidogenesis. By comparison, higher CGP-28392 doses suppressed the stimulatory effects of all three agonists dose-dependently. These results demonstrate that, the calcium channel agonists are incapable of inducing a steroidogenic response in chicken granulosa cells. Since BAY-K8644 and CGP-28392 (low dose, 1-10 microM) failed to influence steroidogenesis in the dose range that induced maximal physiologic responses and calcium influx in a variety of cells, it is concluded that chicken granulosa cells lack the type(s) of channels specific for them. Hence the usefulness of BAY-K8644 and CGP-28392 as Ca2+ probes may be tissue-specific. The inhibitory effects of CGP-28392 appear to be non-specific.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1987

In vitro effect of prostaglandins on the accumulation of cyclic AMP in the avian oviduct.

The effects of prostaglandins E1, E2, and F2 alpha (PGE1, PGE2, and PGF2 alpha, respectively) on cyclic AMP production in tissue samples from the uterus (U), uterovaginal sphincter (UVS), and vagina (V) of regularly laying hens as well as the effects of PGE2 and forskolin on U and V of Japanese quail were studied. Both PGE1 and PGE2 enhanced cyclic AMP production in the hen oviductal segments, particularly in the V in a dose-related manner, PGE1 being the more effective agonist. PGF2 alpha had no significant effect. Quail U appeared to be less responsive to PGE2 which caused a 40% increase in cyclic AMP levels at the highest concentration tested (28 microM), whereas forskolin at 10 microM increased cyclic AMP production in quail U and V 7- and 20-fold, respectively. It is suggested that the primary function of PGE2 and PGE1 is to relax the terminal portion of the oviduct, allowing the expulsion of the egg under the contractile influence of PGF2 alpha and other oxytocic factors.

Animals↗
PubMed1987

Trifluoperazine inhibits ovarian mitochondrial side-chain cleavage enzyme activity in the absence of calcium.

In a previous report we described the inhibitory effect of trifluoperazine (TFP) on steroidogenesis in avian granulosa cells. To clarify the possible site of action of TFP we measured the cholesterol side-chain cleavage enzyme (CSCC) activity in a mitochondrial preparation of granulosa cells isolated from mature and developing ovarian follicles. Using a calcium free medium, TFP inhibited CSCC in a dose related manner with an IC50 of 50 microM. Kinetic parameters (apparent Km and Vmax) obtained in the presence of TFP are indicative of uncompetitive inhibition of CSCC. Moreover, enzyme activity increased during follicular maturation while the efficacy of TFP was similar in both young and mature follicles. Because the inhibitory effects of TFP were manifest in medium from which calcium was omitted, it is suggested that the drug acts independently of the calcium-calmodulin system to suppress CSCC activity.

Animals↗
PubMed1987

Differential effects of prostaglandin F2 alpha and of prostaglandins E1 and E2 on cyclic 3',5'-monophosphate production and intracellular calcium mobilization in avian uterine smooth muscle cells.

The effects of prostaglandins (PGs) E1 (PGE1), E2 (PGE2) and F2 alpha (PGF2 alpha) on cyclic 3',5'-adenosine monophosphate (cAMP) production and intracellular Ca mobilization were examined in smooth muscle cells of chicken uterus grown in primary culture. At subnanomolar concentrations, both PGE1 and PGE2 significantly suppressed cAMP levels. However, at higher concentrations (0.1-100 microM), both agonists caused a dose-related increase in cAMP production. PGF2 alpha, on the other hand, had no effect on cAMP production. Forskolin (1-100 microM), which also stimulated cAMP production in a dose-dependent fashion, potentiated the effects of both PGE1 and PGE2. In digitonin-permeabilized uterine cells preloaded with 45Ca2+, the addition of PGF2 alpha caused a biphasic 45Ca2+ efflux. There was a small but significant 45Ca2+ release (10.0 +/- 1.5%) within 30 s (rapid phase), followed by a larger one (32.0 +/- 2.0%) within 5 min (slow phase). PGE2, at doses above 1 nM (which significantly increased cAMP accumulation), promoted 45Ca2+ sequestration. This action of PGE2 was observed as early as 1 min and was complete by 5 min. In addition, 0.001 nM PGE2 (a dose that was ineffective on 45Ca2+ mobilization) enhanced PGF2 alpha-induced 45Ca2+ mobilization from 22.5 +/- 5% to 57.0 +/- 3.5%. These results show that PGs of the E series have distinctly different effects on cAMP production and intracellular Ca mobilization. PGF2 alpha action may be linked directly to intracellular Ca mobilization, whereas the effects of PGE may be exerted at multiple sites depending on its local concentration. At low concentrations, its action may be mediated by the suppression of cAMP levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Alprostadil↗
PubMed1987

Luteinizing hormone-induced intracellular calcium mobilization in granulosa cells: comparison with forskolin and 8-bromo-adenosine 3',5'-monophosphate.

Digitonin-permeabilized avian granulosa cells were used as a model to study the effects of LH, 8-bromo-cAMP (8-Br-cAMP), and forskolin on intracellular calcium mobilization. LH caused a biphasic calcium efflux. There was a small but significant (P less than 0.05) calcium release within 1-2 min (rapid phase) with an ED50 of 35 ng/ml, followed by a larger one after 5 min (slow phase) with an ED50 of 10 ng/ml. The intracellular calcium antagonist TMB-8 inhibited the action of LH in both phases. Forskolin had no effect on calcium efflux during the rapid phase. However, it stimulated intracellular calcium efflux in a dose-dependent manner in the slow phase with an ED50 of 30 microM. Low doses of 8-Br-cAMP caused a small but significant increase in calcium uptake corresponding to the rapid phase, although high concentrations of 8-Br-cAMP caused efflux in the slow phase (ED50, 0.7 mM). Both LH and 8-Br-cAMP mobilized calcium from a nonmitochondrial pool, whereas forskolin induced calcium efflux from a nonendoplasmic reticular site. Apparently receptor-mediated (LH) and nonreceptor-mediated (forskolin and 8-Br-cAMP) intracellular calcium mobilization are qualitatively different, suggesting that rapid intracellular calcium mobilization may represent one of the initial steps in the mechanism of action of LH.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1987

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.

Animals↗
PubMed1986

Trifluoperazine inhibits progesterone and cyclic AMP production in granulosa cells of the hen (Gallus domesticus).

Unstimulated (basal) as well as luteinizing hormone (LH)-promoted progesterone production in collagenase-dispersed hen granulosa cells was inhibited in a dose-related manner by two phenothiazines, trifluoperazine (TFP) and chlorpromazine (CP), both of which are known calmodulin antagonists. Using TFP, the more potent antagonist of the two, it was found that LH-stimulated cyclic AMP production was also suppressed. Moreover, TFP attenuated the steroidogenic effects of both 8-bromo-cyclic AMP and isobutylmethylxanthine but had no effect on the conversion of pregnenolone to progesterone. The inhibitory effects of TFP on steroidogenesis were reversible. It is concluded that phenothiazines inhibit steroidogenesis in ovarian granulosa cells by acting at multiple sites both proximal and distal to cyclic AMP generation without influencing the enzyme complex responsible for the conversion of pregnenolone to progesterone. The results are discussed in relation to calmodulin- and non-calmodulin-mediated actions of phenothiazines.

Animals↗
PubMed1986

Role of calcium in luteinizing hormone-induced progesterone and cyclic AMP production in granulosa cells of the hen (Gallus domesticus).

The effects of calcium on steroidogenesis and cyclic AMP production in chicken granulosa cells were examined. For the expression of full steroidogenic effects by LH, forskolin, and 8-bromo cyclic AMP the presence of calcium in the incubation medium was essential, the optimal concentration being 1 mM. Moreover, calcium antagonists (verapamil, TMB-8) significantly suppressed steroidogenesis in response to all three agonists. The metabolism of 25-hydroxycholesterol and the conversion of pregnenolone to progesterone, however, were not affected by the lack of calcium or by coincubation with calcium antagonists. LH-stimulated cyclic AMP production was also suppressed in calcium-deficient medium and in the presence of the putative calcium channel blocker, verapamil. However, TMB-8 did not affect LH-induced cyclic AMP production. Moreover, neither forskolin- nor IBMX-induced cyclic AMP accumulation was significantly affected by lack of calcium or verapamil. These results are interpreted to indicate that the continuous presence of extracellular calcium is essential for hormonal regulation of steroidogenesis and is important for events both proximal and distal to cyclic AMP generation up to pregnenolone synthesis.

1-Methyl-3-isobutylxanthine↗
PubMed1986

Steroidogenesis and cAMP production in isolated avian granulosa cells during follicular maturation: lack of positive correlation.

Luteinizing hormone- and forskolin-induced acute steroidogenesis and cAMP production were studied in dispersed chicken granulosa cells in relation to follicular maturation. Cells isolated from the 6 largest preovulatory follicles (F1-F6) were used in this study. Basal steroidogenic activity increased with progressive maturation of the follicle without a corresponding rise in basal cAMP production. Both LH- and forskolin-promoted progesterone production was directly correlated with the maturational stage of the follicle and the dose of the agonists. On the other hand, LH caused the greatest increase in cAMP production in the third and fourth largest preovulatory follicles (F3-F4), whereas forskolin-stimulated cAMP accumulation was maximal in the least mature granulosa cells (F5-F6). Thereafter it decreased in proportion with follicular development. It is concluded that LH-induced acute steroidogenesis in chicken granulosa cells is not positively correlated with cAMP production during the last few days of follicular maturation. Therefore the steroidogenic responsiveness of granulosa cells of the largest follicle to LH cannot be ascribed solely to receptor-coupled adenylate cyclase activity. As shown by the forskolin experiment, the intrinsic activity of the adenylate cyclase-cAMP system is greatest in the small F5, F6 follicles which respond only minimally in terms of steroidogenesis to either a receptor dependent agonist (LH) or a nonreceptor agonist (forskolin).

Animals↗
PubMed1986

Clomiphene and tamoxifen inhibit the cholesterol side-chain cleavage enzyme activity in hen granulosa cells.

A radiochemical assay was utilized to study the inhibitory effects of clomiphene and tamoxifen on the cholesterol side-chain cleavage enzyme activity in a mitochondrial preparation of granulosa cells isolated from mature ovarian follicles of laying hens. At saturating substrate concentrations, both clomiphene and tamoxifen were able to suppress enzyme activity in a dose-related manner (IC50 1.8 X 10(-5) M). Double reciprocal plots of kinetic data show that the inhibition is mixed, exhibiting competitive kinetics at low concentrations, whereas at high concentrations, the inhibition is of a non-competitive nature. The competitive inhibition constants as determined from Dixon plots are 2 X 10(-5) M for clomiphene and 2.3 X 10(-5) M for tamoxifen. It is concluded that, in granulosa cells, clomiphene and tamoxifen directly inhibit the mitochondrial cholesterol side-chain cleavage activity. This inhibition may represent an important aspect of the mode of action of clomiphene and tamoxifen.

Animals↗
PubMed1985

Influence of follicular maturation on luteinizing hormone-, cyclic 3',5'-adenosine monophosphate-, forskolin- and cholesterol-stimulated progesterone production in hen granulosa cells.

The influence of follicular maturation on progesterone production by collagenase-dispersed hen granulosa cells was measured in short-term incubations. Granulosa cells of the largest follicle (F1) produced up to ten times more progesterone than cells from smaller follicles (F3-F5), not only in response to luteinizing hormone (LH), but also when stimulated by exogenous cyclic AMP or forskolin, both of which raise intracellular cyclic AMP levels by nonreceptor-mediated mechanisms. Moreover, when granulosa cell progesterone synthesis was stimulated by incorporating 25-hydroxy-cholesterol into the incubation medium, an identical pattern was obtained. This could be attributed to a corresponding increase in the specific activity of the mitochondrial cholesterol side-chain cleavage enzyme (20,22 desmolase). An increase in the apparent Vmax was observed without a change in the apparent Km values. Pregnenolone substrate at concentrations which raised progesterone production to levels similar to those observed in response to LH stimulation was utilized equally by granulosa cells of mature and developing follicles. However, at high pregnenolone concentrations, granulosa cells of mature follicles converted significantly more of the precursor to progesterone. Assay of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) showed that the enzyme has two Kms: a low Km present in cells of both mature and developing follicles, and a high Km found only in granulosa cells of more mature follicles. It is concluded that LH-promoted progesterone synthesis in granulosa cells of developing chicken follicles is restricted not so much by the availability of receptors and the competence of the adenylate cyclase/cyclic AMP system, but by the activity of key enzymes, principally the cholesterol-20,22 desmolase.

Animals↗
PubMed1984

Steroidogenesis in granulosa cells during follicular maturation: evidence for desensitization-resensitization during the ovulation cycle.

Output of progesterone, the end product of the steroidogenic pathway, was studied in isolated chicken granulosa cells in relation to follicular maturation and during the ovulation cycle with particular reference to the period between the LH peak and ovulation. The evidence gathered from a series of experiments conducted during the past 2-3 years in the authors' laboratory indicate that the steroidogenic capacity of granulosa cells during follicular maturation is regulated not so much by receptor-coupled adenylate cyclase activity as has been proposed by other investigators, but by the activity of key steroidogenic enzymes, particularly the cholesterol 20,22 desmolase. Furthermore, granulosa cells undergo cyclic sensitization following the endogenous LH surge reaching maximal responsiveness about 1 hr before oviposition. This is followed by a rapid desensitization shortly before ovulation. This desensitization extends to the second and subsequent developing follicles probably in proportion to the evolving LH receptors. It is suggested that granulosa cells remain in such a desensitized state for several hours postovulation, during which time progesterone responses to LH are attenuated and consequently ovulation does not occur prematurely. It is proposed that this intraovarian mechanism is an important component of the physiological events that regulate the ovulation cycle in the domestic fowl.

Adenylyl Cyclases↗
PubMed1983

Comparison of turkey luteinizing hormone (LH)- and ovine LH-induced progesterone production in granulosa cells of the turkey (Meleagris gallopavo) and of the domestic fowl (Gallus domesticus).

Progesterone production was compared in short-term cultures of turkey and fowl granulosa cells isolated from preovulatory follicles of different maturities. In both species cells from the most mature follicle produced the greatest amount of progesterone under stimulated conditions and in response to ovine or turkey LH, the latter being significantly more potent. Whereas basal production of progesterone was higher in turkey cells, the response to LH or cAMP stimulation was significantly greater in chicken granulosa cells both in absolute terms and particularly in relation to unstimulated levels. These species differences may reflect difference in the intensity of reproductive activity of the turkey and the domestic fowl selected for egg production.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1983

Effects of forskolin on progesterone and cyclic adenosine monophosphate production in avian granulosa cells.

Forskolin, a novel activator of adenylate cyclase, promoted progesterone production by isolated chicken granulosa cells. At submaximal levels its steroidogenic action was greatly potentiated by isobutyl methylxanthine or the addition of submaximally stimulating concentrations of ovine luteinizing hormone (oLH). When all three agonists were combined, the oLH dose-response curve was left shifted by about two orders of magnitude (from ED50 of 9 ng/ml to 0.1 ng/ml). However, forskolin alone could not stimulate progesterone production to the same extent as oLH alone did, even though cyclic AMP levels in response to forskolin were higher than those observed in response to oLH. Moreover, whereas one to two orders to magnitude discrepancy exists between the steroidogenic and adenylate cyclase activating effects of LH, the efficacy of forskolin to provoke steroidogenesis and to increase cyclic AMP production in granulosa cells was found to be similar. Therefore, forskolin-promoted steroidogenesis appears to be a direct consequence of the activation of the adenylate cyclase/cyclic AMP system, whereas oLH-induced steroidogenesis, a receptor-mediated event, is probably more complex and may involve cellular events in addition to those regulated by cyclic AMP.

1-Methyl-3-isobutylxanthine↗