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Gunnar Selstam and Sten Rosberg

ABSTRACT

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 μg/ml), but not NIH-LH-B8 (0.1–100 μg/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 μmol/l) revealed an apparent Km value of 0.12 μmol/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 between LH and FSH on the cAMP system in the prepubertal rat ovary.

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Kerstin Albertsson-Wikland and Sten Rosberg

The rate of GH secretion and the pattern of GH peaks were compared in a group of nine prepubertal children during their prepubertal period in repeated 24-h GH profiles. At investigation, the children were 6–1 3 years old (at first profile 6–11 years old) and of normal height (±2 sd). Two profiles were obtained per child, (with a mean time interval of 1.5 years, range 0.7 to 3.5 years. The calculated GH secretions of the first and second profiles were compared. As a group, no significant differences in secreted amount of GH, when expressed as data from the second profile as a percentage of data from the first profile (93±8%), number of peaks (98±7%) or mean peak amplitudes (92±11%), were obtained. Between the repeated curves of an individual child, maximal difference in secretion, number of peaks and mean peak amplitudes ranged around±30%, with a mean intraindividual cv of 12%. The reproducibility in the peak distribution for all profiles was also analysed. The relative frequencies is a percentage of the GH peak amplitudes and peak widths were virtually identical in the repeated profiles. Reproducibility of the temporal pattern of profiles was analysed using time-series analysis (Fourier analysis) and showed no difference in rhythmicity between the different occasions. In conclusion, a high reproducibility of both GH secretion and GH pattern was found for the whole group of prepubertal children. The high degree of reproducibility of the 24-h GH profiles of the whole group of children indicated that the information from these curves, in terms of both pattern and total secretion, can be used for clinical as well as for physiological purposes. The intraindividual reproducibility was less pronounced, however, leading to a sound scepticism when relating biological phenomena to a single profile of an individual child.

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Lars Nilsson, Sten Rosberg and Kurt Ahrén

ABSTRACT

Isolated pre-ovulatory follicles from PMSG-injected immature rats, described in a previous publication, have been used for the investigation of the pattern of cyclic 3′,5′-AMP synthesis after in vitro addition of gonadotrophins and prostaglandins. The follicles were incubated in a modified Krebs bicarbonate buffer for periods up to 4 h and the cyclic 3′,5′-AMP content in tissue and medium was analyzed with the protein binding assay of Gilman (1970).

Addition of LH, FSH or prostaglandin E2 (PGE2) increased the cyclic 3′,5′-AMP level in follicular tissue dramatically. HCG and TSH, but not STH or prolactin, also induced a similar increase. The effect of FSH was blocked by a highly specific antiserum to LH. The effect of LH had a maximum after approximately 1 h of incubation, whereas the maximal effect of PGE2 was seen between 5 and 15 min of incubation. When LH was present in the incubation medium a significant release of cyclic 3′,5′-AMP into the medium occurred. Theophylline potentiated the effects of LH and PGE2.

The different time-courses of the effects of LH and PGE2 on the cyclic 3′,5′-AMP production by follicular tissue are discussed in relation to the hypothesis of prostaglandins as obligatory mediators of all LH effects on the ovary (Kuehl et al. 1970).

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Jan Åman, Sten Rosberg and Kerstin Albertsson-Wikland

Aman J. Rosberg S, Albertsson-Wikland K. Effect of growth hormone treatment on insulin secretion and glucose metabolism in prepubertal boys with short stature. Eur Endocrinol 1994;131:246–50. ISSN 0804–4643

The purpose of this study was to evaluate the effect on insulin secretion and glucose metabolism of daily growth hormone (GH) treatment, 0.1 U/kg. for up to 3 years in 42 short prepubertal boys without GH deficiency. Their median height standard deviation (sd) score increased from −2.7 to −1.7, whereas their weight for height sd score was unchanged after 3 years of treatment. Fasting plasma glucose concentrations were unchanged, but median fasting insulin concentrations increased from 6.0 mU/l before treatment to 7.8 mU/l (p < 0.05) after the first year. No further increase was seen during the second or third years. The median insulin area under the curve 10–60 min after an intravenous glucose tolerance test increased from 480 mU·1−1·min−1 before treatment to 799 mU·1−1 · min−1 (p < 0.05) after 1 year. The median glucose disposal rate (K value) before GH treatment, 2.2%/min, was unchanged after 1 year of treatment. A significant positive correlation was found between the change in the height sd score and the change in fasting insulin concentration during the first (r = 0.45; p < 0.01) and second (r = 0.56; p < 0.05) years of GH treatment. It was concluded that GH treatment in prepubertal children without GH deficiency caused a moderate increase in fasting and stimulated insulin concentrations during the first year of treatment. There was no further change during the following years of treatment, and there were no negative effects on fasting plasma glucose concentrations or glucose disposal rates. The increase in insulin concentration was related positively to the growth response.

Jan Åman, Department of Pediatrics, Örebro Medical Centre Hospital, S-701 85 Örebro, Sweden

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Staffan Edén, Kerstin Albertsson-Wikland, Sten Rosberg and Olle Isaksson

ABSTRACT

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.

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Sten Rosberg, Ensio Norjavaara, Monica Sender Baum and Iqbal Khan

Abstract. Adenylate cyclase activity was studied in membranes from isolated corpora lutea of defined ages obtained from pregnant mare's serum gonadotropin treated rats and the effects of luteinizing hormone (LH), isoproterenol, guanylylimidodiphosphate (Gpp (NH)p), fluoride and forskolin were compared. The effect of LH on adenylate cyclase activity increased with the luteal age up to nine days of age, while the effect of isoproterenol increased dramatically during the first days, reaching a maximum at 2–3 days of age and then declined. Forskolin potentiated the effects of both LH and isoproterenol without affecting the patterns of age-dependency. The effect of forskolin itself was fairly constant during the luteal phase, indicating a relatively constant amount of the catalytic unit in the corpus luteum. The effects of fluoride and Gpp(NH)p on the other hand increased markedly during the first days and then remained constant for the rest of the period studied. These results suggest that the regulatory Ns-protein develops during the first days of luteal life. It is speculated that the close correlation between the development of β-adrenergic response and the development of Ns-protein are causally related.

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M. Iqbal Khan, Sten Rosberg, Christina Bergh and Kurt Ahrén

Abstract.

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.

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Hans Herlitz, Lars Hamberger, Sten Rosberg and Kurt Ahrén

ABSTRACT

Corpora lutea were isolated from 34–39 day-old rats injected with 10 IU PMSG when 30 days old leading to ovulation early in the morning of day 33.

Three day-old corpora lutea were incubated in Krebs bicarbonate buffer containing 5.5 mmol/l glucose. Addition of LH to the incubation medium increased accumulation of cyclic 3′, 5′-AMP (cAMP) in a dose-dependent way in the isolated tissue and caused a release of cAMP to the incubation medium. cAMP was determined by the protein binding technique of Gilman (1970). There was a continuous rise in the levels of cAMP in both tissue and medium with increasing incubation time in presence of LH. Neither prolactin nor FSH could influence the cAMP levels in the corpus luteum tissue in vitro. Prostaglandin E2 (PGE2) caused a stimulation with respect to cAMP formation. The time-course of the PGE2 effect was quite different from that of LH: PGE2 gave maximal tissue cAMP content within 5–15 min with decreasing levels thereafter, compared to the continuous rise with LH stimulation for at least 1 h.

In experiments performed on corpora lutea of different ages (1–7 day-old), tissue levels of cAMP were determined after incubations with LH or PGE2. There was a dramatic increase of tissue cAMP in the very young corpus luteum and a marked decrease in sensitivity to LH with increasing age of the corpus luteum with a very small but still significant effect on the old corpora lutea. The stimulatory effect of PGE2 on cAMP levels on the other hand showed only a minimal change.

A few experiments on the first generation of corpora lutea from mature cycling rats are also reported for comparison.

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Lars Hamberger, Knut Nordenström, Sten Rosberg and Anita Sjögren

ABSTRACT

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 not 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.

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Gunnar Selstam, Sten Rosberg, Jan Liljekvist, Lena Grönquist, Torsten Perklev and Kurt Ahrén

ABSTRACT

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 μg/ml) and FSH (100 μg/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.