Evidence for a possible prostaglandin link in ACTH-induced steroidogenesis.

The steroidogenic response to ACTH prostaglandin E2 (PGE2) was studied in cat adrenocortical cells dispersed with trypsin. The dose-response curves of both agents were qualitatively and quantitatively similar. Exposure to PGE2 or ACTH in the presence of labeled steroid precursor (acetate) resulted in the accumulation of comparable levels of steroid intermediates and end-product. Submaximal or maximal concentrations of ACTH and PGE-2 given simultaneously elicited a response which was no greater than that obtained with either stimulant alone. Although calcium was required for optimal PGE-2 stimulation of steroid production, this requirement with ACTH as the stimulant, but greater than with butyryl cyclic AMP. PGE-2-induced increase in the adrenal cyclic AMP was not statistically significant and was small in relation to that found with equipotent steroidogenic ACTH concentrations. The possible relationship between prostaglandins, cyclic AMP, and calcium in the action of ACTH is discussed.     

Prostacyclin: A potent stimulator of adrenal steroidogenesis

The relative potencies of various prostaglandins were investigated in trypsin-dispersed cat adrenocortical cells. Prostacyclin proved to be the most potent steroidogenic prostaglandin, being 100–1000 times more potent than PGE₂. This stimulant effect of prostacyclin was only partially dependent upon the presence of extracellular calcium and was associated with increased levels of cyclic AMP. These data suggest a possible role for prostacyclin in corticosteroidogenesis.     

Comparison of the Ca ++ requirement for the steroidogenic effect of ACTH acid dibutyryl cyclic AMP in rat adrenal cell suspension

Calcium requirement for ACTH and Dibutyryl cyclic AMP (DBCAMP) stimulation of steroidogenesis was compared in rat adrenal cell suspensions. In the absence of added calcium ACTH at low concentrations (< 1 mU/ml) was ineffective; however, the calcium requirement decreased when higher concentrations of ACTH were used. This was not the case with DBCAMP. At all levels of the nucleotide tested, the Ca⁺⁺ requirement was about the same. When the cells were preincubated with EGTA, the Ca⁺⁺ requirement became more pronounced for ACTH than for DBCAMP. The results indicate that the events before the formation of cyclic AMP show a greater dependence on Ca⁺⁺ than the events following its formation.     

On the mechanism of action of cholera toxin on isolated rat adrenocortical cells Comparison with the effects of adrenocorticotropin on steroidogenesis and cyclic AMP output

The effects of cholera toxin on isolated rat adrenocortical cells have been investigated. Both steroid and cyclic AMP output from adrenal cells were increased by the toxin in a dose dependent fashion. The concentration of toxin for half maximal stimulation for both of these responses was about 40 ng/ml. Maximal steroidogenesis and cyclic AMP output was obtained with similar concentrations of the toxin. A correlation was observed between the low amounts of cyclic AMP produced in response to all doses of cholera toxin and to physiologically significant concentrations of adrenocorticotropin (ACTH) (< 0.1 munit/ml; i.e. submaximal for steroidogenesis in this system). This was in direct contrast to the much higher levels of cyclic AMP generated by concentrations of ACTH greater than 1 munits/ml. Time course studies demonstrated a time-lag between toxin addition and steroid response of at least 40 min. Binding of cholera toxin to adrenal cells was rapid and was 90% complete within 15 min at both 37 and 0°C. These data indicate that most of the delay in response to cholera toxin is due to processes subsequent to the initial binding interaction. Following the initial delay the subsequent maximal rate of steroidogenesis brought about by cholera toxin was very similar to that obtained with a concentration of ACTH that was maximal for steroidogenesis. Significant increases in cyclic AMP levels were detected about 20 min before increased steroidogenesis was apparent. Possible explanations for this result are considered. The results presented indicate great potential use for cholera toxin in the study of adrenal steroidogenic control mechanisms, particularly at the level of receptor mechanisms and the role of cyclic AMP.     

A Dual Pathway for Acth Steroidogenic Action in Purified Adrenocortical Cells

Abstract

Isolated adrenal fasciculata cells were purified by centrifugation through a 0-50% hyperbolic gradient of Percoll^R. The dose-dependence and kinetics of both intracellular cyclic AMP accumulation and steroido-genesis in response to ACTH_1-39 and ACTH_5-24 (corticotropin-(1-39) and corticotropin-(5-24)-peptides) were determined using purified cells. The rate of intracellular cyclic AMP formation was maximal during the first five minutes after hormone addition and remained constant or fell thereafter. Therefore intracellular cyclic AMP accumulation, assessed after 5 min., was compared with steroid output after 20 min. Maximal steroidogenesis was elicited by ACTH^5-24 without discerning a significant stimulation of intracellular cyclic AMP accumulation. ACTH_6-24 (corticotropin-(6-24)-peptide) could completely inhibit the intracellular cyclic AMP accumulation elicited by ACTH_1-39 or by ACTH_5-24 at concentrations that only partially inhibited steroidogenesis.

It is possible that there are two pathways for the steroidogenic action of ACTH, one of which is obligatorily mediated by intracellular cyclic AMP, and another which involves a different mediator.     

Effects of prostaglandins and other drugs on the cyclic AMP content of cultured bone cells

Prostaglandins of the E-series (PGE₁ and PGE₂) may be involved in disease-related, localized loss of bone. E-prostaglandins increase the cyclic AMP content of many cells; and, to determine if their effects on bone are mediated by cyclic AMP, we examined the effects of E-prostaglandins and of other agents on the cyclic AMP content of cultured bone cells. PGE₂ produced a rapid, marked and dose-related increase in the cyclic AMP content of confluent monolayers of bone cells isolated from newborn rat calvaria. At 2.8 × 10⁻⁶ M, PGE₁ and PGE₂ had approximately the same effect, while the effect of PGF_2α was much less pronounced. In the presence of theophylline, PGE₂ had a more marked effect than parathyroid hormone (PTH) and the combination of PGE₂ and PTH had a synergistic effect. The divalent, cationic, ionophore, A23187, produced an increase in cellular cyclic AMP and had an additive effect in combination with PGE₂. Synthetic salmon calcitonin (CT), which inhibits the bone resorptive effect of PGE₂, increased cellular cyclic AMP and had an additive effect in combination with PGE₂. A prostaglandin antagonist, SC-19220, partially inhibited the resorptive effect of PGE₂ and reduced its effect on cellular cyclic AMP. The calcium antagonist, D600, inhibited the bone resorptive effects of PGE₂ but had no effect on increased cellular cyclic AMP produced by PGE₂.The marked effect of PGE₂ on bone cell cyclic AMP suggests that this action is involved in the mechanism of PGE₂-related bone loss. The fact that agents with different effects on PGE₂-induced increases in cellular cyclic AMP can inhibit its resorptive actions, suggests that PGE₂-induced changes in cyclic AMP may be related less to its resorptive actions than to its inhibitory effect on bone formation.     

Inhibition of ACTH-stimulated steroidogenesis in isolated rat adrenal cells treated with neuraminidase

The possible role of membrane sialic acid in the action of ACTH was investigated in rat adrenal cells. After treatment with neuraminidase, the cells showed a diminished steroidogenic response to ACTH while the response to cyclic AMP and dibutyryl cyclic AMP was unaffected. 11β-hydroxylation of deoxycorticosterone (DOC) was also not impaired. Dose response curves for three ACTH peptides (ACTH_1–39′, ACTH_1–24 and ACTH_1–10) with neuraminidase treated cells suggest that sialic acid residues on the glycoproteins of the plasma membrane may either impart affinity to the plasma membrane for ACTH molecule or facilitate transmission of the signal arising from ACTH-receptor interaction to the catalytic site of adenyl cyclase.     

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.     

Loss of the PGE1 requirement for MDCK cell growth associated with a defect in cyclic AMP phosphodiesterase

Prostaglandin E₁(PGE₁), one of the components in the hormone-supplemented, serum-free medium for Madin Darby Canine Kidney (MDCK) cells (Medium K-1), is required for both long-term growth and for dome formation. Variant cells have been isolated from MDCK populations, which lack the PGE₁, requirement for long-term growth in Medium K-1. These variants will be useful in identifying the molecular events initiated by PGE₁ which are necessary for the growth response to be observed. The growth and functional properties of five independently isolated PGE₁ independent clones have been examined. Normal MDCK cells grew at an equivalent rate in Medium K-1 and in serum-supplemented medium; the growth rate was lower in Medium K-1 lacking PGE₁. In contrast, PGE₁ independent clone 1 grew at an equivalent rate in Medium K-1 minus PGE₁, and in serum-supplemented medium. When PGE₁ was added to K-1 minus PGE₁, less growth of PGE₁ independent clone 1 was observed. A similar observation was made with one other PGE₁ independent clone which was studied. A hormone deletion study indicated that PGE₁ independent clone 1 still retained growth responses to the other four supplements in Medium K-1 (insulin, transferrin, T₃, and hydrocortisone). The molecular alterations associated with loss of the PGE₁ requirement for long-term growth were examined. At confluency, all of the PGE₁ independent clones studied had higher intracellular cyclic AMP levels following PGE₁ treatment, as compared with normal MDCK cells. The increased cyclic AMP levels in the variant cells could result from a number of different types of defects, including reduced cyclic adenylic acid (cyclic AMP) efflux, an increased affinity of PGE₂ for the PGE₁ receptor, or a defect in cyclic AMP metabolism. However, in all of the variant clones studied a decreased rate of cyclic AMP degradation by cyclic AMP phosphodiesterase was observed. Thus, the increased cyclic AMP levels in the PGE₁ independent variants may result from alterations which affect cyclic AMP metabolism. The effect of PGE₁ on dome formation by the variant cells was also examined. The frequency of dome formation by PGE₁ independent clone 1 was enhanced in a dosage-dependent manner, like normal MDCK cells. This observation suggests that PGE₁ affects MDCK cell growth and dome formation by different mechanisms.     

An investigation of the involvement of adenosine 3'':5''-cyclic monophosphate in steroidogenesis by using isolated adrenal cell column perfusion.

The involvement of cyclic AMP in corticosteroidogenesis was investigated by using isolated adrenal cell column perfusion. Steroids were produced in response to 0.5, 1.0 and 5.0 mg of cyclic AMP/ml. Analysis of the shape of the response curves indicated an inverse relationship between rate of onset of steroid production and dose. A further increase in steroid production during the washout period after the 5 mg/ml dose was considered to indicate an intracellular inhibitory effect of cyclic AMP. Release of cyclic AMP into the perfusate only occurred in response to supramaximal steroidogenic doses of ACTH (adrenocorticotrophin). A connexion between dose and response was demonstrated over a narrow concentration range. Variation in the time-lag before cyclic AMP production and in the duration of the response was marked; further, no reproducible ratio of steroid output to cyclic AMP output was shown at any level of stimulation. These results are discussed together with those of other recent investigations. It is considered that these findings do not support an obligatory role for cyclic AMP as mediator of ACTH action in the adrenal.     

Role of cyclic AMP and protein kinase on the steroidogenic action of ACTH, prostaglandin E1 and dibutyryl cyclic AMP in normal adrenal cells and adrenal tumor cells from humans.

The role of the cyclic AMP-protein kinase system in mediating the steroidogenic effect of ACTH, prostaglandin E1 and dibutyryl cyclic AMP, induced similar stimulations of protein kinase activity, cyclic AMP was studied using human adrenal cells isolated from normal and adrenocortical secreting tumors. At high concentrations of ACTH, complete activation of protein kinase of normal adrenal cells was observed within 3 min, at the time when cyclic AMP production was slightly increased and there was still no stimulation of steroidogenesis. At supramaximal concentrations, ACTH, PGE1 and dibutyryl cyclic AMP and cortisol productions in adrenal cells isolated from normal and from one adrenocortical tumor. In one tumor in which the adenylate cyclase activity was insensitive to ACTH, the hormone was unable to stimulate protein kinase or steroidogenesis, but the cells responded to both PGE1 and dibutyryl cyclic AMP. In another tumor in which the adenylate cyclase was insensitive to PGE1, this compound also did not increase protein kinase activity or steroidogenesis, but both parameters were stimulated by ACTH and dibutyryl cyclic AMP. After incubation of normal adrenal cells with increasing concentrations of ACTH (0.01-100 nM) marked differences were found between cyclic AMP formation and cortisol production. However at the lowest concentrations of ACTH exerting an effect on steroid production a close linked correlation was found between protein kinase activation and cortisol production, but half-maximal and maximal cortisol production occurs at lower concentration of ACTH than was necessary to induce the same stimulation of protein kinase. Similar findings were found after incubating the adrenal cells with dibutyryl cyclic AMP (0.01-10 mM). The results implicate an important role of the cyclic AMP-protein kinase system during activation of adrenal cell steroidogenesis by low concentrations of steroidogenic compounds.     

The in vivo effect of indomethacin and prostaglandin E2 on ACTH and DBCAMP-induced steroidogenesis in hypophysectomized rats

The level of plasma corticosterone attained in hypophysectomized rats stimulated with ACTH was significantly reduced by pretreatment with indomethacin, an inhibitor of prostaglandin synthesis. This effect was not seen in animals stimulated with dibutyryl cyclic AMP. Intraperitoneal injection of prostaglandin E₂ to indomethacin treated rats restored the normal response to ACTH stimulation. However, PGE₂ itself did not have any significant effect on plasma corticosterone levels. These findings suggest that prostaglandins are involved, perhaps in an allosteric fashion, in the mechanism of action of ACTH.     

Prostaglandin I2 stimulation of granulosa cell cyclic AMP production

The ability of prostaglandin I₂ (PGI₂) to stimulate cyclic AMP production by granulosa cells, isolated from intact immature rats, has been demonstrated in vitro. The minimal effective dose was 15 ng/ml, which was comparable to the minimal effective dose for PGE₂. However, a concentration of 15 μg/ml PGI₂ was required to stimulate cyclic AMP production maximally, compared to a concentration of 1 μg/ml PGE₂, which produced the maximum response. It therefore appears that PGI₂ is not more effective than PGE₂ in stimulating cyclic AMP production in granulosa cells, and is possibly less effective. Submaximal concentrations of PGI₂ appeared to be able to modify the stimulation of cyclic AMP production by follicle- stimulating hormone (FSH), but whether or not PGI₂ plays any role in follicular function remains to be established.     

Measurement of the dynamics of stimulation and inhibition of steroidogenesis in isolated rat adrenal cells by using column perfusion

Isolated adrenal cells were perfused in a small column by using Bio-Gel polyacrylamide beads as an inert supporting matrix, and the time-course of the response to various stimuli was observed by measuring fluorogenic 11-hydroxycorticosteroids in the effluent. A small but significant response was observed 1 min after stimulation with physiological concentrations of ACTH (adrenocorticotrophin), but the response did not start to build up rapidly for 3-4min and eventually reached a plateau after 9-10min. A similar pattern of events was observed for the decay of the steroid output on removal of ACTH. ACTH analogues, including one with a long duration of action in vivo, were found to produce responses with similar kinetics. However, cyclic AMP caused a more rapid increase in steroidogenesis and its effects were more short-lived after withdrawal. If, as present evidence suggests, cyclic AMP is produced rapidly after ACTH stimulation the delayed build-up of the steroidogenic response to ACTH would indicate that cyclic AMP may not be the intracellular mediator. When inhibitors were applied during ACTH stimulation, aminoglutethimide, which blocks mitochondrial conversion of cholesterol into pregnenolone (3beta-hydroxypregn-5-en-20-one), caused a rapid fall in steroid output (1 min), whereas cycloheximide took longer to achieve its full effect. Nevertheless, the response had fallen by 50% in 2 min, indicating a much shorter half-life than that previously reported for the labile protein implicated in steroidogenesis. In addition the rapid response to cyclic AMP makes it unlikely that steroid production is induced as a result of initiation of protein synthesis. This suggests that the labile protein plays an obligatory but permissive role in the development of the response. Column perfusion has proved to be a simple technique which can readily yield accurate data on responses of cells to stimulants and inhibitors.     

Cyclic AMP response to prostaglandin E1 in mononuclear cells from peripheral blood and synovial fluid of patients with rheumatoid arthritis

The cyclic AMP response to prostaglandin E₁ (PGE₁) was studied in peripheral blood (PB) and synovial fluid (SF) mononuclear cells from patients with rheumatoid arthritis (RA). The PGE₁ induced accumulation of cyclic AMP was consistently (7 of 8 patients) less in cell suspensions derived from SF than in suspensions of equivalent numbers of mononuclear cells obtained simultaneously from PB. The high PB/SF cyclic AMP ratio was seen most clearly at the lowest concentration (10⁻⁶M) of PGE₁ tested. There was no correlation between the patients' therapy and cyclic AMP response to PGE₁. The high PB/SF cyclic AMP ratio was not accounted for by the presence of platelets in PB cell suspensions.     

Stimulatory effects of prostaglandin E1 on rat anterior pituitary cyclic amp and luteinizing hormone release

The effect of prostaglandin E₁ (PGE₁) on rat anterior pituitary cyclic AMP accumulation and luteinizing hormone (LH) release was studied both in vivo and in vitro. Addition of PGE₁ to incubation medium over a concentration range of 10^-6 to 10^-4 M produced a graded increase in pituitary cyclic AMP. At the lowest concentration (10^-6 M) there was no significant increase in LH release, but proportional increments in LH release were seen with increasing concentrations of PGE₁.Ten minutes after intravenous administration of 5 μg of PGE₁ to adult male rats, pituitary cyclic AMP was substantially increased while serum LH levels were not changed. Administration of a higher dose of PGE₁ (20 μg) produced a greater increase in pituitary cyclic AMP; and, at this dose serum LH was significantly increased. These results suggest that the PGE₁ effect on LH release is mediated by the adenyl cyclase — cyclic AMP system.     

Prostaglandin biosynthesis and stimulation of cyclic AMP in primary monolayer cultures of epithelial cells from mouse mammary gland

Cyclic AMP levels in primary monolayer cultures of epithelial cells prepared from mid-pregnant mice are stimulated by prostaglandin E₁ and E₂. Prostaglandin F_1α and F_2α have only a slight effect upon cyclic AMP levels. In the absence of phosphodiesterase inhibitors the rise in cyclic AMP produced by PGE₁ is only transient and the levels return to normal within 30 minutes. High concentrations (16 mM) of theophylline are needed to prevent this decline, suggesting that the phosphodiesterase activity of epithelial cells in culture is high. However, theophylline alone produced only a small increase in basal cyclic AMP levels even over a 2-hour period indicating that basal cyclic AMP is turned over more slowly than cyclic AMP produced in response to stimulation with PGE₁.Both PGE and PGF synthesis were monitored using radioimmunoassay procedures previously reported. The observed levels were found to decrease as cell density increased and were sensitive to the addition of agents such as collagen and naproxen.     

Relationship Between the Actions of Calcium Ions, Opioids and Prostaglandin E1 on the Level of Cyclic AMP in Neuroblastoma × Glioma Hybrid Cells

Abstract: Neuroblastoma × glioma hybrid cells increase their intracellular concentration of cyclic AMP in response to prostaglandin E₁ (PGE₁). This effect is inhibited by opioids. The response to PGE₁ is positively correlated with the concentration of Ca²⁺ in the incubation medium. The Ca²⁺ antagonists Co²⁺ and La³⁺, the Ca²⁺ chelator EGTA and a blocker of Ca²⁺ influx into cells, Segontin, inhibit the response to PGE₁. At low external concentrations of Ca²⁺ the response to PGE₁ is enhanced by the Ca²⁺ ionophore A23187. The effects of A23187 and Segontin point to a cytosolic site of Ca²⁺ action. Lack of Ca²⁺ reduces the level of cyclic AMP even in the absence of PGE₁ and the presence of an inhibitor of cyclic AMP phosphodiesterase. Ca²⁺ is required even for an increase in the level of cyclic AMP in cells pretreated with cholera toxin. The increases in level of cyclic AMP evoked by PGE, in a neuroblastoma and by PGE₁ or noradrenaline in a glioma cell line do not depend on Ca²⁺. The response of the hybrid cells to the opioid leucine-enkephalin appears not to rely on the presence of Ca²⁺. Even changing the intracellular concentration of Ca²⁺ by the ionophore A23187 does not alter the effect of the opioid. The analogy between opioids and lack of Ca²⁺ in the short-term (minutes) experiments mentioned holds also for long-term (hours) experiments. Cells chronically exposed to opioids or to low concentrations of Ca²⁺ display an enhanced maximal response to PGE₁.     

Calcium-mediated effects of calcitonin on cyclic AMP formation and lymphoblast proliferation in thymocyte populations exposed to prostaglandin E 1

The calcitonin (SCT) from salmon ultimobranchial bodies which (like mammalian calcitonins) lowers the plasma calcium concentration in mammals can also affect cyclic AMP (cyclic adenosine 3′,5′-monophosphate) metabolism and proliferation of lymphoblasts in normal and prostaglandin E₁ (PGE₁)-treated rat thymocyte populations in three different ways. In the first case, low concentrations (0.5–5.0 ng per milliliter) of SCT lower (by a calcium-mediated process) the ability of PGE₁ to transiently increase cyclic AMP synthesis, but the reduced surge of cyclic AMP production is still ample to stimulate lymphoblasts in the cell population to initiate deoxyribonucleic acid (DNA) synthesis. Secondly, these low SCT concentrations affect the eventual progression of the PGE₁-stimulated, DNA-synthesizing lymphoblasts into mitosis by a calcium-mediated process. Depending on the extracellular calcium concentration and the magnitude of the initial increment in the intracellular cyclic AMP content, SCT can either promote or inhibit the progression of the stimulated cells into mitosis. SCT's third action is a rapid (within 5 minutes), calcium-independent elevation of the cellular cyclic AMP content in otherwise untreated thymic lymphocyte populations exposed to a very high concentration (100 ng per milliliter) of the hormone. This early, transient rise in the cyclic AMP level is followed by a calcium-dependent increase in lymphoblast proliferation. An attempt is made to interrelate and explain the different actions of SCT on cyclic AMP metabolism and mitogenesis.