Increase In Cyclic AMP Production Research Articles

Acquisition of sensitivity to LH in relation to foetal development stimulation of cyclic AMP and testosterone production in the rat testis

The ability of LH to stimulate, in vitro, adenylate cyclase activity and testosterone secretion was studied in foetal rat testes after prelabelling with [ 14C]adenine. As little as 0.1 ng/ml LH produced significant synthesis of cyclic AMP and testosterone secretion. Increase of cyclic AMP production was observed as early as 1 min after addition of LH (100 ng/ml), preceding the rise in testosterone synthesis and secretion. FSH and prolactin were not effective. LH-stimulation of cyclic AMP and testosterone production appeared concomitantly in rat foetal testes on day 15 of gestation and reached a maximum on day 18. Immature testes (14 days) developed functional receptors when cultured in a medium devoid of hormone. The results of the present study suggest that cyclic AMP mediates the effect of LH on steroidogenesis in foetal testes and that the differentiation of functional receptors occurs at the same time as the capacity for testosterone synthesis.

Hormonal control of phenylalanine hydroxylase activity in isolated rat hepatocytes

Glucagon addition to isolated rat hepatocytes increases the level of Cyclic AMP inside the cells and the activity of the enzyme phenylalanine hydroxylase. These effects of glucagon are time and dose dependent and are detectable at hormone concentration as low as 0.02nM. The glucagon concentrations causing half-maximal increases in Cyclic AMP production and phenylalanine hydroxylase activity are 0.2nM and 0.1 nM respectively. When hepatocytes are incubated with norepinephrine or the ionophore A23187, at concentrations between 1 nM and 10 μM, a slight increase in enzyme activity is seen only at the highest dose of either drug. The effect of norepinephrine can be completely antagonized by 20 μM propranolol but not by 20 μM ergocryptine. These results suggest that the activity of phenylalanine hydroxylase can be hormonally regulated, in vivo , through a phosphorylation mechanism catalyzed by a Cyclic AMP-dependent protein kinase.

Bone remodelling induced by physical stress is prostaglandin E 2 mediated

In vitro cultured bone cells were found to be responsive to hormones and physical forces. A simple device has been developed which enables the direct application of physical forces to tissue culture dishes to which cells are firmly attached. The physical forces created a deformation of the dish. It was found that prostaglandin E 2 synthesis underwent a rapid increase, reaching a maximum after 20 min and then declined. Concurrent with the increase in prostaglandin E 2 was an increase in cyclic AMP production, having a maximum around 15 min. The increase in cyclic AMP was blocked by indomethacin, the prostaglandin E 2 synthesis inhibitor, indicating the dependence of cyclic AMP production on the de novo synthesis of prostaglandin E 2. Prostaglandin E 2 added to cells mimicked the effect of physical forces on the production of cyclic AMP. The increase in cyclic AMP resulted from an early rise in adenyl cyclase activity (within 5 min) and a later (10 min) increase in phosphodiesterase activity. The same physical forces also stimulatedthe incorporation of thymidine into DNA after 24 h. On addition of prostaglandin E 2 the increase in DNA synthesis was also mimicked. Pretreatment of the cells with indomethacin abolished the effect of physical forces on DNA synthesis. The results suggest a stimulus receptor mechanism for physical forces which, like hormonal effectors, are mediated by prostaglandins and stimulate cyclic AMP and DNA synthesis. We believe that physical forces stimulate bone remodelling through such a stimulus receptor system, mediated by prostaglandins.

Characterization of a vasoactive intestinal peptide-sensitive adenylate cyclase in rat intestinal epithelial cell membranes

A vasoactive intestinal peptide-sensitive adenylate cyclase in intestinal epithelial cell membranes was characterized. Stimulation of adenylate cyclase activity was a function of vasoactive intestinal peptide concentration over a range of 1 · 10 −10−1 · 10 −7 M and was increased six-times by a maximally stimulating concentration of vasoactive intestinal peptide. Half-maximal stimulation was observed with 4.1 ± 0.7 nM vasoactive intestinal peptide. Fluoride ion stimulated adenylate cyclase activity to a higher extent than did vasoactive intestinal peptide. Under standard assay conditions, basal, vasoactive inteetinal peptide- and fluoride-stimulated adenylate cyclase activities were proportional to time of incubation up to 15 min and to membrane concentration up to 60 μg protein per assay. The vasoactive intestinal peptide-sensitive enzyme required 5–10 mM Mg 2+ and was inhibited by 1 · 10 −5 M Ca 2+. At sufficiently high concentrations, both ATP (3 mM) and Mg 2+ (40 mM) inhibited the enzyme. Secretin also stimulated the adenylate cyclase activity from intestinal epithelial cell membranes but its effectiveness was 1/1000 that of vasoactive intestinal peptide. Prostaglandins E 1 and E 2 at 1 · 10 −5 M induced a two-fold increase of cyclic AMP production. Vasoactive intestinal peptide was the most potent stimulator of adenylate cyclase activity, suggesting an important physiological role of this peptide in the cyclic AMP-dependent regulation of the intestinal epithelial cell function.

Stimulatory Action of Gonadotropins and Prostaglandins on Adenosine-3′,5′-Monophosphate Production by Isolated Rat Granulosa Cells1

The effect of purified preparations of follicle-stimulating hormone (FSH) and luteinizing hormone (LH), and of prostaglandins, on adenosine- 3′,5′-monophosphate (cyclic AMP) production by granulosa cells isolated from ovaries of intact immature estrogen-treated rats has been studied. Incubation of the cells with FSH resulted in a 60-fold increase in cyclic AMP production in 10 min. Both NIH-LH and purified LH (LH-HP) were able to elicit only slight increases in cyclic AMP production. Prostaglandins E1 and E2, but not prostaglandin F20, were also able to stimulate cyclic AMP production, although not to as great an extent as FSH. The stimulatory action of FSH was not antagonized in the presence of inhibitors of prostaglandin synthesis, suggesting that prostaglandin biosynthesis does not mediate the action of FSH on cyclic AMP production. The ability of both FSH and LH to stimulate cyclic AMP production in granulosa cells from estrogen- treated rats was slightly decreased when compared to granulosa cells fro...

Studies on sex-organ development. The hormonal regulation of steroidogenesis and adenosine 3′:5′-cyclic monophosphate in embryonic-chick ovary

1. We investigated the production of steroid hormones by the ovaries of the developing embryonic chick under conditions of organ culture. Radioimmunoassay techniques were used to measure the amount of steroid hormone released into the culture medium. Stimulation of the production of steroid hormones by choriogonadotropin from the urine of pregnant human was dose-dependent. Oestradio and testosterone production was optimal when 20 i.u. of gonadotropic hormone was present in the culture medium 2. During development, both left and right ovaries responded to gonadotropic hormone stimulation with a 2.5-3-fold increase in oestrogen production. However, the right ovary was twice as efficient in testosterone production as the left one. The presence of dibutyryl cyclic AMP in the culture medium of the embryonic ovaries mimicked the effect of the gonadotropic hormone. 3. The human choriogonadotropic hormone stimulated cyclic AMP production in the embryonic ovarian tissue. Thyrotropin, growth hormone and insulin had no stimulating effect. 3-Isobutyl-1-methylxanthine potentiated the gonadotropic hormone effect by increasing the concentration of cyclic AMP in the ovarian tissue. 4. The amount of cyclic AMP synthesized in the embryonic ovary was gradually increased (from 1.2 to 6.5 pmol/mg of tissue) when incubated with increasing doses of human choriogonadotropic hormone in vitro. The newly synthesized cyclic AMP reached the maximum concnentration after 30 min of incubation, then decreased at 2 h of incubation. A portion of the newly synthesized cyclic AMP was released into the culture medium. 5. At various developmental stages, both left and right embryonic-chick ovaries responded to stimulation by gonadotropic hormone with an increase in cyclic AMP production. The cyclic AMP concentration in the right ovary was 80% higher than that in the corresponding left ovary.

The Effect of Calcium on Steroidogenesis in Isolated Human Adrenal Cells

The present experiment was planned to verify the effect of calcium on adenyl cyclase in isolated human adrenal cells. Normal adrenal glands were obtained surgically from patients with primary aldosteronism and advanced breast cancer. Isolated adrenal cells were prepared by the modified Haning's method. They were incubated at 37C under a gas mixture of 95 percent O2: 5 percent CO2 in calcium-free Krebs-Ringer bicarbonate buffer solution containing 0.2 percent glucose and 0.5 percent fatty acid-free bovine serum albumin, to which various doses of CaCl2 or ACTH were added. Thirty minutes later, cyclic-AMP was measured by cyclic-AMP assay kit (The Radio-chemical Center, Amersham). 11-OHCS was estimated fluorometrically by the modified Silber's method after incubation for 2 hours. In the calcium-free incubation medium, productions of 11-OHCS and cyclic-AMP were negligible. In the concentration of 2.54 mM/L of calcium, 11-OHCS production increased with significant difference statistically, while the increase of cyclic-AMP production was not significant. In the concentration of 12.70 mM/L of calcium, however, cyclic-AMP production increased remarkably. When ACTH was added to the incubation medium containing 2.54 mM/L of calcium, productions of 11-OHCS and cyclic-AMP also increased remarkably. These results indicate that adenyl cyclase of human adrenocortical cells is directly stimulated by calcium and suggest that calcium acts as the second messenger of ACTH.

Calcium Modulation of Acth and Cholera Toxin Stimulated Adrenal Steroid and Cyclic-AMP Biosynthesis

Monolayer cultures of mouse adrenal cortex tumors have been used to investigate the effects of Ca2+ on the binding of 125I-labelled s1-24-ACTH peptide to the cell, ACTH-stimulated cyclic-AMP and steroid biosynthesis. Stimulation with ACTH results in increased intracellular cyclic-AMP levels and release of cyclic-AMP into the medium. The specific activities of intracellular and extracellular cyclic-AMP produced by cells incubated with 3H-adenine are the same, suggesting that they are products of a common pool of ATP. ACTH-stimulated cyclic-AMP release continues after intracellular cyclic-AMP levels have decreased and reflects continuing cyclic-AMP biosynthesis. In incubations of two hours or less with 5.0 mM Ca2+, maximum steroidogenesis occurs with 0.1 milliunit/ml ACTH. Further increases in cyclic-AMP production are elicited with higher concentrations of ACTH. In the absence of Ca2+, both cyclic-AMP and steroid syntheses are attenuated, maximum steroidogenesis requiring > 10 milliunits/ml ACTH.The propor...