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Biomedical subjects

S Rosberg

Publications and source records attributed to S Rosberg.

At least 73 records · Page 4Linked to original sources
PubMed1985

Effects of FSH and LH on adenylate cyclase activity in rat granulosa cell membranes during follicular maturation.

Adenylate cyclase activity was measured in membranes prepared of granulosa cells isolated from PMSG-treated immature rats and the effects of ovine, highly purified human and rat gonadotrophins were compared. Furthermore, a comparison of the effects of the human preparations (hLH, hFSH) on the adenylate cyclase activity in membranes prepared from granulosa cells isolated at different stages of follicular maturation, was performed. The adenylate cyclase in membranes of immature granulosa cells was stimulable with FSH but not with LH, while in pre-ovulatory granulosa cell membranes, both gonadotrophins were stimulatory with FSH generally being more effective than LH. Surprisingly, the dose-response curve for ovine LH (oLH) was biphasic with a plateau at a level of adenylate cyclase activity corresponding to the maximal stimulatory effect of hCG. With increasing oLH concentrations the response resumed and the maximal stimulation corresponded to that of oFSH. With highly purified rat gonadotrophins the FSH response was significantly higher than the response to LH at all concentrations tested. Using highly purified human gonadotrophins the maximal FSH response was 50% higher than the maximal LH response and by adding increasing concentrations of hFSH to a maximally stimulatory concentration of hLH it was possible to mimic the biphasic dose-response curve for oLH. When the membranes were prepared from granulosa cells isolated after the pro-oestrus LH/FSH surge there was clear increase in the sensitivity of the adenylate cyclase to stimulation with LH although the maximal response was unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1984

Stimulation and inhibition of rat oocyte meiosis by forskolin.

The adenylate cyclase activator forskolin was used to study the role of cAMP for oocyte meiosis and follicular steroid secretion. Follicular and cumulus cAMP production was stimulated dose-dependently by forskolin, as was the follicular secretion of progesterone, testosterone and estradiol. Forskolin induced meiosis in follicle-enclosed oocytes with a maximal effect at 1 microM, with lower and higher concentrations being less effective. The spontaneous resumption of meiosis in isolated cumulus-enclosed oocytes was dose-dependently retarded by forskolin. Meiosis of cumulus-free oocytes was also retarded but only slightly. These data support the earlier hypothesis that a limited increase in follicular cAMP levels triggers meiosis, whereas sustained levels of cAMP in the oocyte itself prevent meiosis.

Animals↗
PubMed1984

Beta-adrenergic receptor concentration in corpora lutea of different ages obtained from pregnant mare serum gonadotropin-treated rats.

We have measured the beta-adrenergic receptor content in 1- to 10-day-old corpora lutea. Corpora lutea of defined ages were obtained by sc injection of 8 IU PMSG to 26-day-old rats, leading to ovulation and formation of corpora lutea in the early morning of day 29. Membrane fractions of isolated corpora lutea were incubated with various concentrations of the beta-adrenergic antagonist [125I]iodohydroxybenzylpindolol [125I]iodo-HYP, 12.5-2500 pM) for 60 min at 22 C. Alprenolol (10(-5) M) was used to determine nonspecific binding. Bound and free [125I]iodo-HYP was separated by filtration and washing on Whatman GF/C filters under vacuum. There was a 3-fold increase in beta-adrenergic receptor concentration during the first 2-3 days of corpus luteum formation, followed by a decline in the beta-adrenergic receptor content with luteal age. The rat luteal beta-adrenergic receptor seems to be of the beta 2-subtype as determined from adenylate cyclase stimulation as well as from displacement of [125I]iodo-HYP binding by various beta-adrenergic agonists.

5-alpha-Dihydroprogesterone↗
PubMed1984

The antilipolytic, insulin-like effect of growth hormone is caused by a net decrease of hormone-sensitive lipase phosphorylation.

The mechanism of the antilipolytic effect of GH and the cause of refractoriness to its own action was studied in isolated rat adipocytes. Human GH rapidly inhibited catecholamine-stimulated lipolysis rate, with a time course similar to that of insulin, but only in cells which had been preincubated in the absence of GH for 2-3 h. Half-maximal inhibition was obtained with a GH concentration of 100 ng/ml. Parallel determinations of the lipolysis rate (with a pH-stat titration technique) and the extent of phosphorylation of hormone-sensitive lipase, the rate-controlling enzyme in adipose tissue lipolysis, were made. The extent of lipase phosphorylation, 1.7-fold enhanced by previous noradrenaline stimulation, was rapidly reversed by addition of GH, and the decrease was followed by a parallel decrease in the lipolysis rate. The time course and magnitude of these effects was similar to those obtained with exposure of the cells to insulin, indicating that the antilipolytic effect of both hormones was exerted through the same mechanism: a net dephosphorylation of the hormone-sensitive lipase. To study the refractoriness of the fat cells to the action of GH (which was not found with insulin) adipocytes were prepared from hypophysectomized rats 24 h after surgery. With such cells no preincubation was required to obtain the effects of GH on the lipolysis rate and extent of hormone-sensitive lipase phosphorylation. The effects of GH on both of the parameters studied were similar to those obtained in nonhypophysectomized rats. These results suggest that the refractoriness of the fat cells to GH may be explained by a functional inhibition at a site(s) in the series of metabolic events initiated by GH action, which precedes the activation of the hormone-sensitive lipase.

Adipose Tissue↗
PubMed1983

Prostaglandin F2 alpha inhibition of epinephrine stimulated cyclic AMP and progesterone production by rat corpora lutea of various ages.

Epinephrine can mimic the stimulatory effects of LH in vitro on cyclic AMP (cAMP) and progesterone production by isolated rat corpora lutea. The aim of the present study was to test whether the effects of epinephrine in vitro on the rat corpus luteum, as with LH, can be inhibited by prostaglandin F2 alpha (PGF2 alpha). The stimulatory effect of epinephrine on tissue levels of cAMP in 1-day-old corpora lutea was not inhibited by PGF2 alpha. A dose-dependent inhibition by PGF2 alpha (0.5-50 microM) was seen for 3-day-old corpora lutea and this inhibition could not be overcome by higher concentrations of epinephrine (0.165-165 microM). The stimulation by epinephrine on progesterone production was inhibited by PGF2 alpha (5 microM) in 3- and 5-day-old, but not in 1-day-old corpora lutea. Thus, PGF2 alpha can inhibit the stimulatory effect of epinephrine in 3- and 5-day-old corpora lutea, but not in the newly formed corpora lutea (1-day-old) and PGF2 alpha shows in this respect the same age dependent inhibitory pattern as in relation to LH stimulation.

Aging↗
PubMed1983

Forskolin effects on the cAMP system and steroidogenesis in the immature rat ovary.

The diterpene forskolin was found to activate the adenylate cyclase system in intact tissue and membrane preparations of the immature rat ovary. The cyclic AMP (cAMP) response reached a maximal level after 5 min and no decline was observed even after 4 h of incubation. Forskolin stimulated production of both progesterone and testosterone in a pattern similar to that produced by luteinizing hormone (LH) or dibutyryl-cAMP (dbcAMP). In combination with LH, follicle-stimulating hormone (FSH) or prostaglandin E2 (PGE2), forskolin potentiated the hormone effects on adenylate cyclase activity in membrane preparations. Pretreatment with LH or PGE2 desensitized the cells to further hormone stimulation, while the forskolin response was unaffected. Pre-exposure to forskolin did not desensitize the cells to a subsequent stimulation by LH or PGE2. The presence of 8-bromo-cAMP (brcAMP) in the preincubation medium reduced the subsequent hormone response. These results demonstrate a rapid and sustained activation of the adenylate cyclase system by forskolin in the rat ovary. The steroidogenic response was similar to that of known stimulators of ovarian cells (LH, dbcAMP). The inability of forskolin to induce desensitization of the adenylate cyclase system demonstrates, however, important differences between hormone and non-hormone activation. Consequently, forskolin can be a useful tool for investigation of the mechanisms involved in the desensitization process.

Adenylyl Cyclases↗
PubMed1982

Inhibition of adenylate cyclase activity in muscles by growth hormone.

The acute effects of GH have a characteristic lag period of 10-30 min before a short lasting stimulation is seen, suggesting that some metabolic events precede the observable stimulation. There is evidence that cyclic nucleotides are involved in the mechanism of action of GH, which this work attempts to evaluate further. The effect of GH (rat, bovine, and human) on adenylate cyclase activity was measured in diaphragms and hearts of young normal and hypophysectomized rats. Measurements were made both after short term in vivo treatment and after addition directly to isolated muscle membranes. The muscles were homogenized and centrifuges, and the adenylate cyclase activity in the pellet was assayed. The injection of 4.5-450 pmol GH, iv, induced, within 3 min, inhibition of basal as well as epinephrine-stimulated adenylate cyclase activities, in both the absence and presence of a GTP analog, in the tissues studied. When added directly to the muscle membranes in the adenylate cyclase assay, 0.45-45 nM GH caused a dose-dependent inhibition of the adenylate cyclase activity, but only in the presence of a GTP analog. These findings clearly demonstrate that a physiological dose of GH has a rapid and direct inhibitory effect on muscle membrane adenylate cyclase, well within the lag period of the anabolic effect of the hormone.

Adenylyl Cyclase Inhibitors↗
PubMed1980

Desensitization of the cyclic AMP system in rat corpora lutea. Comparison between the effects of hCG and LH.

The changes in hormonal responsiveness of corpora lutea produced by a single injection of hCG or ovine LH were studied in PMSG-treated immature rats with well-characterized corpora lutea of different ages. The hormonal response was measured as LH-stimulated accumulation of cyclic AMP in isolated, whole corpora lutea and, in some experiments, as LH stimulated luteal adenylate cyclase activity. Injection of hCG produced a complete desensitization (refractoriness) of very long duration (4-5 days) as has earlier been found in rats with heavily luteinized ovaries, while the injection of LH in equivalent doses, produced only a partial desensitization of much shorter duration (12-48 h). Morphological observations, after injection of trypan blue to a small number of the rats, showed that follicular growth, ovulation and formation of new corpora lutea could occur after both hCG and LH. The possibility that the corpora lutea which started to respond to LH stimulation 5-7 days after the hCG injection were not the ones originally desensitized by hCG but new corpora lutea, is discussed. The very rapid recovery of the LH sensitivity after desensitization with LH, can, however, not be explained in the same way. It represents a true reversal to the 'normal' hormonal sensitivity of the cells originally desensitized. The marked difference between hCG and LH in these desensitizing experiments is discussed in relation to earlier known differences between these two gonadotrophins, e.g. in binding properties to ovarian receptors and in rate of metabolism in the body.

Adenylyl Cyclases↗
PubMed1979

Acute suppression by PGF2 alpha on LH, epinephrine and fluoride stimulation of adenylate cyclase in rat luteal tissue.

Injection of PGF2 alpha (250 microgram/rat) 15 min prior to isolation of corpora lutea (CL) from PMSG treated immature rats significantly reduced the LH stimulation of adenylate cyclase in CL membranes. The LH stimulation did not return to normal even 24 h after PGF2 alpha injection. A transient decrease in epinephrine and fluoride stimulation of AC was also observed, the response returning to normal 6-12 h after PGF2 alpha treatment. In vitro incubation of whole isolated CL with 0.005 micrometer or higher concentrations of PGF2 alpha markedly reduced the LH and fluoride stimulation of AC in the CL membranes. Exposure of CL to PGF2 alpha for 15 min in vitro reduced the LH and fluoride response. The results are discussed in relation to the suppressive action of PGF2 alpha on LH receptors in CL, and a mechanism is proposed to explain the discrepancy in time relation between our results and the LH receptor studies. The proposed mechanism might also explain the transient decrease in epinephrine and fluoride stimulation of AC.

20-alpha-Dihydroprogesterone↗
PubMed1978

Acute influence of LH and FSH on cyclic AMP formation in isolated granulosa cells of the rat.

A technique for the mechanical isolation of granulosa cells from the rat ovary is described. Cyclic AMP formation by the isolated granulosa cells of the follicles in various stages of development was studied in response to the administration in vitro of gonadotrophins. In granulosa cells from small to medium-sized follicles FSH but no LH stimulated cAMP formation, while in cells from pre-ovulatory follicles both gonadotrophins had a stimulatory effect. The effects of both gonadotrophins were transient with a maximal response after 15 to 60 min of incubation. In the presence of the phosphodiesterase inhibitor, 3-isobutyl-methylxanthine, the action of FSH was potentiated and prolonged while the response to LH was unaffected. These data indicate that both gonadotrophins activate the adenylate cyclase system of the isolated granulosa cells while FSH in addition stimulates the phosphodiesterase activity. Consecutive determinations of cAMP during and after the pre-ovulatory LH-FSH surge, demonstrated a rise of cAMP levels in granulosa cells from the pre-ovulatory follicles following endogenous gonadotrophin release. cAMP levels remained high or increased until the time of ovulation.

Animals↗
PubMed1977

Effect of insulin and adrenaline on cyclic AMP in the diaphragm of normal and diabetic rats.

The effects of insulin and adrenaline on cyclic AMP (cAMP) levels in diaphragms of normal, streptozotocin-diabetic and insulin-treated diabetic rats were studied. Adrenaline caused a biphasic rise in cAMP with peak values of cAMP within the first few minutes. Diaphragms of diabetic rats showed an increased responsiveness to adrenaline. Injection of insulin to diabetic rats normalized the rise in cAMP after addition of adrenaline. There was no difference in basal levels of cAMP between diaphragms of normal, diabetic or insulin-treated diabetic rats. Insulin in vitro did not affect basal cAMP-levels or the release of cAMP from the tissue but significantly decreased adrenaline-induced peak levels of cAMP. This effect of insulin was abolished by theophylline. The results of the present study suggest that experimental diabetes is associated with changes of the adenylate cyclase and/or phosphodiesterase enzyme activities in skeletal muscle resulting in an increased responsiveness to adrenaline. Since insulin in vitro depressed the adrenaline-induced elevation of cAMP the increased responsiveness in diaphragms of diabetic rats might be attributed to the specific lack of insulin.

Animals↗
PubMed1976

Effect of adrenaline on the release of cyclic AMP by the rat diaphragm.

An investigation was made of the effect of adrenaline on the tissue level and release of cyclic AMP (cAMP) into the incubation medium by the isolated rat diaphragm preparation. Adrenaline, added in vitro, caused a dose-dependent biphasic increase in the tissue level of the nucleotide. A significant elevation of the level of cAMP in the tissue was observed throughout a 60 min incubation period in the presence of 0.01, 0.1 and 10 mug/ml of adrenaline. The hormone also produced a small but significant increase in the release of cAMP into the incubation medium. The accumulation of cAMP in the incubation medium ceased after 10 min of incubation in the presence of adrenaline, although the tissu e level of the nucleotide remained considerably elevated throughout the entire incubation period. Theophylline potentiated the effect of adrenaline on both the accumulation of cAMP in the tissue and in the incubation medium. Theophylline by itself also increased the tissue level of cAMP slightly. It is concluded from the present study that the elimination of cAMP from the rat diaphragm muscle by release of the nucleotide from the cells is small.

Animals↗
PubMed1976

Differences in action of LH and FSH on the formation of cyclic AMP in the prepubertal rat ovary.

The actions of LH (NIH-LH-B8) and FSH (NIH-FSH-S9) on the cyclic AMP (cAMP) system in ovaries of 23-24 day old rats have been analyzed. An intravenous injection of LH increased ovarian cAMP levels in vivo after only 20 seconds. Maximal cAMP levels were seen after 15 min. Addition of LH or FSH in vitro to the isolated ovaries produced dose dependent increases of cAMP in the tissue as well as in the incubation medium. Low concentrations of LH caused a release of cAMP into the incubation medium without any detectable change in the tissue levels. The levels of cAMP in the incubation media for all concentrations of FSH were lower than the tissue levels, whereas for LH the opposite was found. In time-course experiments where the concentrations of LH (10 mug/ml) and FSH (100 mug/ml) were chosen to give similar tissue levels of cAMP, the release of the cyclic nucleotide into the incubation medium was approximately 2-3 times greater for LH than for FSH at the time periods studied (5-240 min). When LH and FSH were tested together in high concentrations, their effects were additive. When the ovaries were first incubated with FSH for 120 min followed by an incubation with LH, the stimulatory effect of LH was considerably reduced. When the order of the incubations was reversed, however, LH did not change the response to FSH. The results show that both LH and FSH have intrinsic effects on the cAMP system in the prepubertal rat ovary, but that the effects of the two gonadotrophins are not identical.

Animals↗
PubMed1976

Stimulatory effect of FSH in vitro on the extracellularly active cyclic AMP phosphodiesterase in the prepubertal rat ovary.

Intact prepubertal rat ovaries were incubated with radioactively labelled adenosine 3',5'-cyclic monophosphate (cAMP) in Krebs bicarbonate buffer containing glucose. The rate of degradation of cAMP was determined by measuring the radioactivity in the medium after precipitation with Ba(OH)2 and ZnSO4. The fate of the nucleotide was followed by measuring the products in the incubation medium. Paper chromatography was used for the separation and identification of these products. It was found that cAMP was degraded to AMP, which in turn was degraded to inorganic phosphate (Pi) and adenosine. An uptake of labelled products was also observed. NIH-FSH-S9 (10 and 100 mug/ml), but not NIH-LH-B8 (0.1-100 mug/ml), increased the degradation of cAMP. Concomitantly, an increased accumulation of labelled adenosine and Pi as well as an increased uptake of labelled products were seen. Kinetic studies with low concentrations of cAMP (0.125-0.025 mumol/l) revealed an apparent Km value of 0.12 mumol/l for the phosphodiesterase (PDE) activity. FSH significantly changed the slope of the curve in the Lineweaver-Burk plot by increasing the PDE activity. The increased PDE activity in the presence of FSH is discussed in relation to earlier findings of differences in action betweeh LH and FSH on the cAMP system in the prepubertal rat ovary.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1975

Characterization of the metabolism of exogenous cyclic AMP by perfused rat heart and incubated prepubertal rat ovary.

In order to study the metabolism of extracellular 3',5'-adenosine monophosphate (cAMP), rat hearts were perfused and prepubertal rat ovaries incubated with 3H- and 32P-labelled cAMP (0.025-1 muM). The rate of disappearance of cAMP from the medium was determined by "Ba-Zn-precipitation" and degradation products of 3H- and 32P-CAMP by paper chromatography. Both tissues degraded cAMP to 5'-adenosine monophosphate (AMP), but the enzyme kinetic for this phosphodiesterase activity was different (apparent Km value for the heart 3.95 muM and for the ovary 0.2 muM). AMP was further degraded, since also other labelled substances were found in the medium. An uptake of both 3H- and 32P-labelled substance(s) into the heart and the ovary was noticed. Tissue extracts contained several labelled purines, but the amounts of labelled cAMP did not exceed expected amounts in the extracellular space. In the ovary the uptake of cAMP and AMP seemed to be low, since the uptake of labelled substances was inhibited by high concentrations of unlabelled AMP or adenosine. The degradation of 32P-cAMP was unchanged when AMP was present, strongly suggesting that the phosphodiesterase enzyme was acting extracellularly. In the heart added AMP was very rapidly degraded making it impossible to elucidate whether cAMP was degraded extracellularly or not. It is concluded that elimination of extracellular cAMP under physiological conditions can be due to degradation of cAMP by various tissues. At least for the ovary this phosphodiesterase enzyme is extracellularly active.

Adenine↗