Steroidogenic refractoriness of bovine adrenocortical cells to dibutyryl cyclic AMP

The long-term effects of dibutyryl cyclic AMP [(Bu)₂-cAMP] on steroidogenesis in bovine adrenocortical cells maintained in primary culture were investigated. During the first 36 h, total steroid production by cells incubated with (Bu)₂-cAMP increased progressively. Thereafter, however, there was a marked fall in steroid output. During the first 36 h adrenocortical cells incubated in the presence of (Bu)₂-cAMP produced substantially more C₁₉-steroids and 17α-hydroxylated C₂₁ -steroids than did cells incubated in the absence of (Bu)₂-cAMP. By 48 h, however, such steroid secretion by cells incubated in the continued presence of (Bu)₂-cAMP declined toward control levels. By contrast, the secretion of corticosterone and 11-deoxycorti-costerone was consistently less by cells maintained in the presence of (Bu)₂-cAMP than by cells maintained in its absence. These results suggest that refractoriness results, at least in part, from events which occur distal to the formation of cAMP. The action of ACTH and (Bu)₂cAMP to promote the secretion of 17α-hydroxylated C₂₁-steroids and C₁₉-steroids, on the other hand, appears to reflect an increase in the rate of cholesterol side-chain cleavage, as well as an increase in 17α-hydroxylase and possibly also 17,20-lyase activities.     

Inhibition of dibutyryl cyclic AMP induced steroidogenesis in rat adrenocortical cells by the putative calcium antagonist TMB-8

A significant proportion of the steroidogenic response of isolated rat adrenocortical cells to dibutyryl cyclic AMP does not require extracellular calcium, and this component is profoundly depressed by low concentrations of the putative calcium antagonist, TMB-8. The inhibition is reversed by either the readdition of calcium or the calcium ionophore A23187. The steroidogenic response to pregnenolone, whose mode of action does not require calcium, was not depressed by TMB-8. Corticotropin (ACTH)-induced steroidogenesis, which requires extracellular calcium, was markedly depressed by TMB-8, although enhanced cyclic AMP formation is only slightly depressed by this drug. Adrenal cortical microsomes possess an ATP-dependent 45calcium (45Ca2+) uptake system which responded to EGTA with a rapid efflux of 45Ca2+; EGTA-induced calcium efflux from this microsomal fraction was markedly reduced by a concentration of TMB-8 that blocked dibutyryl cyclic AMP-evoked steroidogenesis. TMB-8 produced a smaller but significant reduction of EGTA-facilitated 45Ca2+ efflux from a mitochondrial-enriched fraction. We interpret these results to mean that TMB-8 blocks the steroidogenic effect of dibutyryl cyclic AMP by interfering with the mobilization of a cellular pool of calcium that is probably localized to the endoplasmic reticulum. The physiological implications of these findings in relation to the complex interactions between calcium and cyclic AMP in adrenal steroidogenesis are discussed.     

Effect of lutropin and cycloheximide on lutropin receptors and cyclic AMP production in Leydig tumour cells in vitro.

A system to study lutropin-induced desensitization of tumour Leydig cells in vitro has been investigated. Tumour Leydig cells were purified on a Percoll gradient and then incubated for 30 min with lutropin (0-1000ng/ml). The cells were then washed and incubated in suspension media at 32 degrees C. 125I-labelled human choriogonadotropin binding and basal and lutropin-stimulated cyclic AMP production were determined at various times. Initially the cells showed a dose-dependent decrease in human choriogonadotropin binding (1.18 and 0.13fmol/10(6) cells respectively) followed by an increase at 1 h (2.32 and 0.87fmol/10(6) cells respectively). Human choriogonadotropin binding remained elevated in the cells pre-incubated without lutropin, whereas the cells pre-incubated with lutropin showed a dose-dependent decrease over the next 10 h (2.20-0.18fmol/10(6) cells respectively). Basal production of cyclic AMP initially reflected the pre-incubation conditions (1.17-21.19ng/10(6) cells per h for 0-1000ng of lutropin/ml respectively). However, by 1 h there was a marked rise in basal cyclic AMP production which returned to the initial lower values by 4 h. At all time intervals studied, lutropin-induced cyclic AMP production showed a decrease that was proportional to lutropin concentration in the pre-incubated media. The decreases in human choriogonadotropin binding produced by pre-incubations with lutropin (100ng/ml) was partially inhibited by the presence of cycloheximide in the pre-incubation media and totally prevented by the continuous presence of cycloheximide. These results demonstrate that desensitization of tumour Leydig cells occurs after exposure to lutropin in vitro. This desensitization involves both a loss of plasma membrane receptors for lutropin and lutropin-stimulated adenylate cyclase. These events can be prevented by cycloheximide and are therefore probably dependent on protein synthesis.     

Transformation-dependent effect of dibutyryl cyclic AMP on surface properties of rat cultured cells

The possible differential effect of dibutyryl cyclic AMP on the surface properties of ts-NT3-KR rat cells that express a normal phenotype at 37 degrees C and a transformed morphology and behavior at 33 degrees C has been studied. Electrophoretic examination of glycosylated macromolecules revealed a 350,000 dalton glycoconjugate in phenotypically normal cells but not in the corresponding samples from phenotypically transformed cells or in phenotypically "normal" cells rounded by exposure to the cyclic nucleotide. A decreased exposure of a major 100,000 dalton surface component characteristic of cells that expressed a transformed phenotype, was observed when the corresponding cultures were exposed to dibutyryl cyclic AMP. No change in the 230,000 dalton fibronectinlike molecule of phenotypically normal cells was apparent even in the corresponding cultures exposed to the cyclic nucleotide.     

Control of steroidogenesis in small and large bovine luteal cells

Evidence was cited to show that: (1) prostacyclin (PGI2) plays a luteotrophic role in the bovine corpus luteum and that products of the lipoxygenase pathway of arachidonic acid metabolism, especially 5-hydroxyeicosatetraenoic acid play luteolytic roles; (2) oxytocin of luteal cell origin plays a role in development, and possibly in regression, of the bovine corpus luteum; and (3) luteal cells arise from two sources; the characteristic small luteal cells at all stages of the oestrous cycle and pregnancy are of theca cell origin; the large cells are of granulosa cell origin early in the cycle, but a population of theca-derived large cells appears later in the cycle. Results of in vitro studies with total dispersed cells and essentially pure preparations of large and small luteal cells indicate that: (1) the recently described Ca2+-polyphosphoinositol-protein kinase C second messenger system is involved in progesterone synthesis in the bovine corpus luteum; (2) activation of protein kinase C is stimulatory to progesterone synthesis in the small luteal cells; (3) activation of protein kinase C has no effect on progesterone synthesis in the large luteal cells; and (4) protein kinase C exerts its luteotrophic effect in total cell preparations, in part at least, by stimulating the production of prostacyclin. The protein kinase C system may cause down regulation of LH receptors in the large cells.     

Effects of phorbol esters on steroidogenesis in small bovine luteal cells

The possible influence of an activator of protein kinase C, the tumor-promoting phorbol ester, PMA (phorbol-12-myristate-13-acetate), upon small bovine luteal cell steroidogenesis was investigated in vitro, PMA had no significant effect on basal and dibutyryl cyclic AMP (dbcAMP)-stimulated progesterone production but markedly modulated the LH-stimulated progesterone and cAMP productions. PMA potentiated the LH-stimulated cAMP accumulation whatever the dose of LH used. It also potentiated the LH-induced progesterone production in the presence of low doses of LH. Paradoxically, in the presence of maximal or submaximal effective doses of LH, PMA exerted a time- and dose-dependent inhibition of progesterone synthesis. Diacylglycerol was able to mimic the effects of PMA on LH-induced steroidogenesis. These observations suggest that the Ca2+- and phospholipid-dependent protein kinase C can modulate the regulation by LH of small bovine luteal cell steroidogenesis at a step before the synthesis of cAMP. They also suggest that the interaction between LH and its receptor is able to trigger a negative regulatory signal which would be only expressed for high doses of LH and in the presence of an activator of PKC.     

Plasma membrane vesicles from isolated hepatocytes retain the increase of amino acid transport induced by dibutyryl cyclic AMP in intact cells

We have investigated the effect of cyclic AMP on hepatic amino acid transport by measuring the uptake of L-alanine in plasma membrane vesicles purified from hepatocytes incubated without or with dibutyryl cyclic AMP. The application of an Na+ gradient to vesicles from hepatocytes exposed to dibutyryl cyclic AMP, compared to control, resulted in a greater transient accumulation of L-alanine, whereas in the presence of a K+ gradient a similar slow equilibration of L-alanine was observed. Kinetic analysis of L-alanine influx revealed that the increased uptake resulted from an increased capacity (Vmax) with no change in the affinity (Km) of the carriers for L-alanine. These results strongly suggest that dibutyryl cyclic AMP induces stable changes at the membrane level probably by increasing the number of amino acid carrier molecules.     

Coexpression of mutant and wild type protein kinase in lymphoma cells resistant to dibutyryl cyclic AMP.

A mutant clone resistant to dibutyryl cyclic AMP was isolated from S49 mouse lymphoma cells. The mutant expressed a form of cyclic AMP-dependent protein kinase distinguishable from wild type kinase by its decreased sensitivity to activation by cyclic AMP and its increased thermal lability. Hybrids formed between mutant and wild type cells were resistant to dibutyryl cyclic AMP and expressed both mutant and wild type activities in about equal amount. The parent mutant cells also appeared to express wild type kinase activity, but at a lower level. We conclude that wild type S49 cells have and express two identical alleles for the regulatory subunit of protein kinase, one of which has undergone mutation in the mutant cells.     

Maturation-stimulation effect of brain extract and dibutyryl cyclic AMP on dissociated embryonic brain cells in culture

Extracts from adult rat brains contain a macromolecular factor capable of transforming dissociated embryonic rat brain cells in a monolayer culture. The factor simulates the activity of dibutyryl cyclic AMP but is not identical with either cyclic AMP or prostaglandin.     

Effects of dibutyryl cyclic AMP and cytochalasin B on cultured human glioma cells.

Cultured human glioma cells (138 MG) exposed to dibutyryl cyclic AMP (dbc-AMP; 0.1--5 mM) attained an arborized shape with thin processes extending from a rounded cell body. Cytochalasin B (CB; 1--1 muM) induced similar morphological changes. The processes in both dbc-AMP and CB treated cells were formed by retraction of the cell margin. Colchicine (1muM) completely and liver treated phalloidin (0.1 mg/ml) partially inhibited the morphological alterations induced by dbc-AMP and CB. Dbc-AMP was found to arrest cell movement, cell division and uptake of 2-deoxy-D-glucose. CB has the same effects but was more potent. The effects of dbc-AMP and CB could be due to interference with a common cellular structure, e.g. microfilaments.     

Effect of norepinephrine and dibutyryl cyclic AMP on S-100 protein level in C6 glioma cells

S-100 Protein level was determined in C6 glioma cells after treatments by norepinephrine. In growing cells norepinephrine induces an important increase of S-100 protein level continuing during the stationary phase to reach a level higher than in untreated quiescent cells. In quiescent, low density, thymidine blocked cells, S-100 protein level is also enhanced by norepinephrine. In high density, contact inhibited cells, S-100 protein level is not modified although cAMP level is much more stimulated by norepinephrine than is low density cells. Exogenous addition of dibutyryl cyclic AMP mimics the effects of norepinephrine.Our results suggest that cyclic AMP level can mudulate S-100 protein level in C6 cells but that in density inhibited cells, a subsequent step involved in the regulation is no more operative.     

Endogenous cyclic AMP in thyroid cell culture. Effect of thyroid-stimulating hormone and dibutyryl cyclic AMP.

We have studied the variations of endogenous cyclic AMP levels in thyroid cells cultured over a period of 7 days in several conditions: in the presence of thyroid-stimulating hormone or dibutyryl cyclic AMP which both promote the aggregation of isolated cells into follicles, and in their absence when cells develop as a typical monolayer. In follicle-forming cells, the cyclic AMP level was found to rise during the first day of culture, then to fall rapidly. In monolayer-forming cells, the cyclic AMP content slightly increases attaining the same level as found in other cells at the fourth day, which remains stable till the seventh day. We have investigated the response of these cells cultured in the presence of dibutyryl cyclic AMP retain the capability of increasing their cyclic AMP concentration whereas monolayer-forming cells do not preserve this quality of thyroid cells.     

Estradiol-17beta stimulates the renewal of spermatogonial stem cells in males.

In this work, we examined the functions of the female hormone "estrogen" on spermatogenesis of the Japanese eel (Anguilla japonica). Estradiol-17beta (E(2)), a natural estrogen in vertebrates, was present in the serum and its receptor was expressed in the testis during the whole process of spermatogenesis. Spermatogonial stem cell renewal was promoted by E(2) implantation but was suppressed by tamoxifen (an antagonist of estrogen). In vitro, 10 pg/ml of E(2) was sufficient to induce spermatogonial stem cell division in cultured testicular tissue, therefore confirming the in vivo observations. These results clearly show that estrogen is an indispensable "male hormone" in the early spermatogenetic cycle.     

Glucose synthesis by isolated guinea-pig hepatocytes. Effect of cyclic AMP and dibutyryl cyclic AMP.

In isolated guinea-pig hepatocytes, dibutyryl cyclic AMP stimulated gluconeogenesis from 2 mM galactose by 25 and 40% respectively. In the presence of 0.5 mM theophylline, cyclic AMP (0.1 mM) increased glucose synthesis from lactate and galactose by 26 and 34% respectively.