Stimulation by epinephrine of adenyl cyclase activity, cyclic AMP formation, DNA synthesis and cell proliferation in populations of rat thymic lymphocytes

During the first ten minutes after the beginning of a continuous exposure of rat thymocyte populations (maintained in vitro) to epinephrine, there is an increase in the cellular concentration of cyclic AMP. The hormone also increases the activity of a crude preparation of the thymocyte's cyclic AMP-forming enzyme, adenyl cyclase. Between 30 and 45 minutes after the beginning of exposure to epinephrine, an additional part of the cell population begins to synthesize deoxyribonucleic acid (DNA). These changes are finally followed two to four hours later by an increase of the flow of cells into mitosis. Since cyclic AMP itself is known to stimulate both the initiation of DNA synthesis and thymocyte proliferation, and since the mitogenic action of epinephrine is shown to be potentiated by caffeine and inhibited by imidazole, it is concluded that the mitogenic action of this hormone is mediated by the cyclic nucleotide.     

The influence of sex steroids on calcium- and magnesium-induced mitogenesis in isolated rat thymic lymphocytes

Elevated calcium and magnesium concentrations promoted mitotic activity in rat thymic lymphocyte cultures. Oestradiol inhibited calcium- but not magnesium-induced mitogenesis. One prerequisite for the mitogenic action of calcium is a raised intracellular concentration of cyclic adenosine 3′5′ monophosphate (cyclic AMP) but cyclic AMP-induced mitogenesis was insensitive to oestradiol. This suggests that the steroid blocks the mitogenic process at a stage preceding the endogenous cyclic AMP elevation. Furthermore the mitogenic actions of adrenaline, which stimulates adenylate cyclase (the enzyme responsible for cyclic AMP biosynthesis), and caffeine, which inhibits phosphodiesterase (the enzyme which degrades cyclic AMP) were also insensitive to oestradiol inhibition. This precludes a direct effect of the steroid on these enzymes. However, oestradiol did inhibit the mitogenic action of parathyroid hormone (PTH). Since the mitogenic action of PTH probably involves increased calcium entry to the cell, oestradiol may block this ion influx. The inhibition of calcium- and PTH-induced mitogenesis must be attributable to some structurally specific action of oestradiol. The steroids cholesterol, progesterone and testosterone all failed to reduce calcium-induced mitogenesis, whereas both α and β oestradiol were effective. In addition to its insensitivity to oestradiol inhibition, magnesium-stimulated mitosis was unaffected by both imidazole and calcitonin at concentrations which significantly reduced calcium-stimulated proliferation. These findings are compatible with the thesis that magnesium-induced mitogenesis does not involve the elevation of cyclic AMP concentrations.     

The possible mediation by cyclic AMP of the stimulation of thymocyte proliferation by vasopressin and the inhibition of this mitogenic action by thyrocalcitonin

Lysine vasopressin (antidiuretic hormone), like cyclic adenosine 3',5'-monophosphate (cyclic AMP), rapidly (in less than 1 hour) stimulates the initiation of deoxyribonucleic acid synthesis and thereby increases the flow of cells into mitosis in rat thymic lymphocyte populations in vitro. This mitogenic action of vasopressin, again like that of cyclic AMP, is potentiated by caffeine, an inhibitor of the intracellular phosphodiesterase which catalyzes the degradation of cyclic AMP. On the other hand, vasopressin's mitogenic action (also like that of cyclic AMP) is blocked by imidazole, an activator of cyclic nucleotide phosphodiesterase activity. The hormone, thyrocalcitonin (calcitonin) which is known to block the cyclic AMP-mediated mitogenic effect of parathyroid hormone by interfering with cyclic AMP action, also blocks the mitogenic action of vasopressin. The inhibitory effects of imidazole and thyrocalcitonin on vasopressin's mitogenic action are both overcome by the phosphodiesterase inhibitor, caffeine. It is concluded from these observations that the mitogenic action of vasopressin is mediated by cyclic AMP.     

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.     

The possible mediation by cyclic AMP of parathyroid hormone-induced stimulation of mitotic activity and deoxyribonucleic acid synthesis in rat thymic lymphocytes

Purified parathyroid hormone (PTH) strongly stimulates the initiation of deoxyribonucleic acid (DNA) synthesis and thereby raises the flow of cells into mitosis in rat thymic lymphocyte populations maintained in vitro. These actions of PTH are potentiated by caffeine and inhibited by imidazole which indicates that the hormonal action is mediated by cyclic adenosine 3′,5′,-monophosphate (cyclic AMP). The feasibility of cyclic AMP being the mediator of PTH action is established by the observation that a low concentration (10^?7 M) of dibutyryl cyclic AMP precisely mimics the stimulatory action of the hormone on DNA synthesis and cell proliferation.     

Restoration by cyclic AMP of the differentiated phenotype of chondrocytes from de-differentiated cells pretreated with retinoids

Summary Parathyroid hormone (PTH) increases the cyclic AMP level in rabbit costal chondrocytes in culture. PTH, dibutyryl cyclic AMP (DBcAMP), and 8-bromo cyclic AMP (8-Br cAMP) induce ornithine decarboxylase (ODC) and expression of the differentiated phenotype of chondrocytes in this cell system. On the other hand, retinoids inhibit expression of the differentiated phenotype of chondrocytes. In the present study, the effects of PTH, DBcAMP, and 8-Br cAMP on rabbit costal chondrocytes pretreated with retinoids were examined.PTH did not increase the cellular cyclic AMP level in de-differentiated cells that had been pretreated with retinyl acetate or retinoic acid for three days, but it did increase the cyclic AMP level four days after removal of retinoids. PTH did not stimulate ODC activity or expression of the differentiated phenotype of chondrocytes in the de-differentiated state. On the other hand, DBcAMP or 8-Br cAMP stimulated expression of the differentiated phenotype of chondrocytes even in de-differentiated cells, as judged by morphological and bistological changes of the cells and increase in glycosaminoglycan synthesis. Cyclic AMP analogues also induced ODC in these cells.     

Increase of adenylate cyclase catalytic-unit activity by dexamethasone in rat osteoblast-like cells.

Glucocorticoids are known to increase the cyclic AMP response to parathyroid hormone (PTH) in cultured bone organs or bone cells. Using the osteoblast-like cell line ROS 17/2.8, which possesses receptors for both PTH and glucocorticoids, we investigated which component of the complex hormone receptor-guanine nucleotide regulatory unit--adenylate cyclase was affected by dexamethasone treatment. In response to PTH, isoproterenol or forskolin, a compound that is supposed to act directly on the catalytic unit, cyclic AMP production by intact cells and adenylate cyclase activity in purified plasma membrane were markedly increased by dexamethasone. Whereas NaF, guanosine 5'-[beta gamma-imido]triphosphate and Mn/ stimulated adenylate cyclase activity were similarly enhanced in membranes isolated from glucocorticoid-treated cells, the activity of the stimulatory guanine nucleotide regulatory unit, as assessed by reconstitution into membranes from the CYC- clone, which is genetically devoid of this component, was not altered. Thus in osteoblast-like cells dexamethasone appears to increase cyclic AMP synthesis by influencing the catalytic unit. Moreover, since it has been reported that glucocorticoids may produce changes in cell calcium metabolism, we evaluated cytoplasmic free Ca2+ concentration ([Ca2+]i) and intracellular Ca2+ stores mobilizable by the bivalent-cationophore ionomycin, by using the intracellular fluorescent indicator Quin-2. The results indicated that dexamethasone treatment did not influence [Ca2+]i but markedly decreased ionomycin-releasable Ca2+ stores.     

Activation of renal adenylate cyclase by forskolin: assessment of enzymatic activity in animal models of the secondary hyperparathyroid state

The effects of forskolin on kidney slice cyclic AMP content and membrane adenylate cyclase activity were studied in order to determine whether or not activation of the enzyme by forskolin was affected in experimental animal models of the secondary hyperparathyroid state. Forskolin was found to be a potent activator of renal adenylate cyclase in rats and chicks, and the diterpene produced a marked potentiation of the cyclic AMP response to parathyroid hormone (PTH). The diterpene had no effect on the binding of PTH to renal receptors. Activity of adenylate cyclase in the presence of forskolin was similar in renal membranes from either vitamin D-deficient rats or chicks compared to control. Forskolin did not restore full responsiveness to PTH in renal slices from chicks raised on diets that were deficient in either vitamin D or calcium although the diterpene was capable of potentiating the cyclic AMP response to PTH in these tissues. Forskolin also augmented the activation of membrane adenylate cyclase by PTH although this effect of the diterpene was much less prominent in membrane preparations than that observed in renal slices. This study provided additional evidence that the downregulation of renal PTH-dependent adenylate cyclase in experimental models of secondary hyperparathyroidism is due to a specific reduction in receptor-mediated regulation of cyclic AMP formation. Adenylate cyclase activity as assessed by forskolin-stimulated enzyme activity was fully maintained in kidney membranes from these animal models. Thus, forskolin appears to be a useful drug for measuring total enzymatic activity in situations where altered responsiveness of adenylate cyclase to hormones has been demonstrated to be mediated by changes in hormone receptors.     

The influence of calcium on the cyclic AMP-mediated stimulation of DNA synthesis and cell proliferation by prostaglandin E 1

Prostaglandin type E₁ (PGE₁) rapidly stimulates cyclic AMP formation and the initiation of deoxyribonucleic acid (DNA) synthesis in rat thymic lymphocytes suspended in vitro by reactions which are not affected by wide variations in the extracellular calcium concentration. On the other hand, the operation of the associated reaction(s) responsible for the subsequent progression of the stimulated cells into mitosis is profoundly affected by the extracellular calcium level. If the maximum intracellular cyclic AMP concentration is in the lower range of stimulatory values (e.g., 150 × 10^?8 picomoles per cell as produced by an exposure to 0.5 μg of PGE₁ per milliliter of medium), an extracellular calcium concentration of 0.5 to 1.0 mM is needed to obtain maximum cell proliferation, but not the maximum stimulation of DNA synthesis. Contrariwise, if the cellular cyclic AMP content is raised to a much higher level (260 × 10^?8 picomoles per cell) by exposure to a greater PGE₁ concentration (5.0 μg per millilter), cell proliferation is maximally stimulated in calcium-free medium and increasing the extracellular calcium concntration above 0.2 mM actually prevents the stimulation of cell proliferation (but does not affect the stimulation of DNA synthesis). Thus, the ultimate translation of PGE₁'s early cyclic AMP-mediated reactions into increased cell proliferation is determined by both the intracellular cyclic AMP level and the extracellular calcium concentration.     

Parathyroid hormone and prostaglandin E2 stimulate both inositol phosphates and cyclic AMP accumulation in mouse osteoblast cultures.

Parathyroid hormone (PTH) and prostaglandin E2 (PGE2) are physiological agonists which stimulate bone cells to resorb bone, a process by which the mineralized extracellular bone matrix is dissolved. Bone resorption has a key role in the maintenance of plasma calcium levels. It has been established that both PTH and PGE2 activate adenylate cyclase in osteoblasts, but it is apparent that (1) the two agents have qualitatively different effects on osteoblasts, and (2) the generation of cyclic AMP cannot account for all the effects of PTH on bone cell metabolism. Others have demonstrated that PTH and PGE2 may also elevate intracellular calcium levels, but the mechanism by which this is achieved has not been fully defined. Here we have investigated the effects of PTH on neonatal mouse osteoblasts in culture and shown that physiological concentrations of the hormone (50 nM) caused a small increase (22%) in total inositol phosphates accumulation, with a larger increase (40%) in inositol trisphosphate. We found that this activation occurred at lower concentration than was necessary to activate adenylate cyclase. PGE2 was a more effective activator of inositol phosphates accumulation than PTH, causing up to 300% increase in the total inositol phosphates after 30 min. Both PTH and PGE2 stimulated cyclic AMP accumulation, but the activation of adenylate cyclase by forskolin did not enhance inositol phosphates production. We conclude that both PTH and PGE2 stimulate phosphoinositide turnover in mouse osteoblasts and suggest that this mechanism may contribute to their elevation of intracellular calcium in bone cells.     

The ability of calcium to change cyclic AMP from a stimulator to an inhibitor to thymic lymphoblast proliferation

Calcium is a major regulator of thymic lymphoblast proliferation in vivo and in vitro. The proliferative activity of the lymphoblasts in thymic lymphocyte (thymocyte) populations in vitro is both constant and low in the presence of calcium concentrations between 0 and 1.0 mM, but higher concentrations increase proliferation by an endogenous cyclic AMP-mediated promotion of the initiation of DNA synthesis. Lower concentrations (10^?7 to 10^?5 M) of exogenous cyclic AMP (but not 5′-AMP) stimulate lymphoblast proliferation in a low-calcium (0.5 mM) medium, but higher concentrations do not. However, all exogenous cyclic AMP concentrations between 10^?7 and 10^?3 M (but again not 5′-AMP) block the stimulation of lymphoblast proliferation in a high-calcium (1.5 mM) medium. Exogenous cyclic AMP does not prevent calcium from “activating” lymphoblasts, but it reversibly blocks the reaction responsible for the initiation of DNA synthesis in these stimulated cells. Finally, cyclic AMP's inhibitory action, in contrast to its stimulatory action in low-calcium medium, is not specific for the cyclic nucleotide since a low, non-mitogenic concentration of cyclic GMP also prevents calcium from stimulating DNA synthesis and cell proliferation.     

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 X 10(-6) M, PGE1 and PGE2 had approximately the same effect, while the effect of PGF2alpha 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.     

Second Messengers and the Action of Prothoracicotropic Hormone in Manduca sexta

SYNOPSIS. The large (26 kDa) prothoracicotropic hormone of Manducasexta stimulates ecdysteroid secretion by the prothoracic glandsthrough the action of cyclic AMP (cAMP). Adenylate cyclase inthe prothoracic glands is sensitive to calcium/calmodulin, andenhancement of intracellular calcium levels may be the meansby which PTTH stimulates cAMP synthesis. The cyclic nucleotidein turn activates cAMP-dependent protein kinase and proteinphosphorylation, most notably of a 34 kDa membraneassociatedprotein. It does not appear that protein kinase C plays a rolein the acute action of PTTH, nor has the hormone been foundto stimulate formation of inositol trisphosphate undercurrentassay conditions. PTTH rapidly increases protein synthesis bythe prothoracic glands, and translation inhibitors block PTTH-stimulatedecdysteroid secretion. Connections between protein phosphorylation,protein synthesis, and ecdysone secretion remain to be clarified.     

Bioactivity of amino terminal fragments of parathyroid hormone and parathyroid hormone related peptide in rat kidney slices: no effect of dietary phosphate deprivation

Humoral hypercalcemia of malignancy has been associated with the production of a recently cloned peptide human parathyroid hormone related protein (hPTHRP). One of the markers of this disease is an increased urinary excretion of cyclic AMP. The postreceptor mechanism of action and physiological role of hPTHRP remain obscure. To study the activity of hPTHRP 1-34 compared to rat and human parathyroid hormone (PTH) 1-34 we incubated these peptides with rat kidney slices and measured the cyclic AMP generated in the supernatant. hPTHRP 1-34 was equipotent with human PTH 1-34 but both were 5 times less active than rat PTH 1-34. Previous studies have suggested that a low dietary phosphate intake results in renal resistance to the phosphaturic action of PTH perhaps mediated by reduced adenylate cyclase activation by PTH. To determine whether, during dietary phosphate restriction, hPTHRP 1-34 has actions different from hPTH 1-34 we studied their effects following dietary phosphate deprivation. Dietary phosphate restriction had no significant effect on the cyclic AMP generating activity of any of the peptides. We conclude that hPTHRP 1-34 may be operating through similar mechanisms as human PTH 1-34 and that the previously observed effects of dietary phosphate deprivation on PTH mediated cyclic AMP generation in a broken cell preparation do not occur in intact cell preparations.     

Activation of murine lymphocytes by cyclic guanosine 3′,5′-monophosphate: Specificity and role in mitogen action

Cyclic guanosine 3′,5′-monophosphate (cyclic GMP) stimulates nucleic acid synthesis in lymphocytes, and has been implicated as the intracellular effector of the actions of mitogenic agents on these cells. In the present study, we examined the specificity of the mitogenic activity of cyclic GMP and of its 8-bromo (Br) derivatives, and the effects of the T cell mitogens, concanavalin A, phytohemagglutinin, and staphylococcal entertoxin B (SEB) on the cyclic GMP content and guanylate cyclase activity of mouse splenic lymphocytes. Cyclic GMP and guanosine modestly increased the incorporation of [³H]thymidine into DNA by cultured lymphocytes, but were far less effective than their 8-Br-derivatives. However, on a molar basis the mitogenic activity of both 8-Br-guanosine and 8-Br-5′-GMP exceeded that of 8-Br-cyclic GMP, when tested in the presence and absence of serum in the culture media. Combined addition of maximal doses of these nucleotides did not give additive stimulatory effects, suggesting an action on a common subpopulation of cells, and possibly a common mechanism. By contrast, cyclic AMP, 8-Br-cyclic AMP, 8-Br-adenosine, cholera toxin and prostaglandin E₁ suppressed both basal [³]thymidine incorporation and stimulation of this parameter by T-cell line mitogens and the guanosine nucleotides. Rapid effects of concanavalin A, phytohemagglutinin, SEB, guanosine, 5′-GMP, 8-Br-guanosine, and 8-Br-5′-GMP on the cyclic GMP content of murine lymphocytes could not be demonstrated. Similarly, concanalin A, phytohemagglutinin and SEB failed to alter guanylate cyclase activity when added directly to cellular homogenates or pre-incubated with intact cels. Conversely, carbamylcholine rapidly increased lymphocyte cyclic GMP but was not mitogenic.These results are consistent with the hypothesis that cyclic GMP and cyclic AMP are antagonistic in their influence on lymphocyte mitogenesis. However, they also demonstrate that related nucleotides are more potent mitogens than cyclic GMP and suggest that activation of murine lymphocytes by concanavalin A, phytohemagglutinin and SEB may not be mediated by rapid increases in cellular cyclic GMP content. Since high concentrations of exogenous cyclic GMP and related nucleotides must be used to influence DNA synthesis, the biologic significance of this effect remains uncertain.     

Multiple mechanisms of growth inhibition by cyclic AMP derivatives in rat GH1 pituitary cells: isolation of an adenylate cyclase-deficient variant

GH pituitary cells have been widely utilized for studies of hormone response mechanisms. Studies reported here were motivated by the desirability of isolating characterized GH clones defective in cyclic AMP synthesis or action. Spontaneously occurring GH1 cell variants resistant to the growth-inhibitory effects of cyclic AMP analogs were isolated. Characterization of four variants showed that these were deficient in adenosine kinase and had acquired resistance to the cytotoxic effects of purine nucleoside derivatives formed in the culture medium. A second-stage selection was undertaken with mutagenized adenosine kinase-deficient cells. One 8 Br cAMP-resistant variant was found to have normal cyclic AMP-dependent protein kinase activity but exhibited altered adenylate cyclase activity. Activation of cyclase activity by fluoride, guanyl nucleotides, cholera toxin, and hormone (VIP) was subnormal in the variant. Mn-dependent cyclase activity was also subnormal, suggesting that the 8 Br cAMP-resistant variant may have a deficiency in the catalytic moiety of adenylate cyclase. Surprisingly, adenosine 3':5'-monophosphate and 5'-monophosphate derivatives were found to be equally potent in growth-inhibiting adenosine kinase-deficient cells. Cross-resistance to 8 Br AMP was observed in the 8 Br cAMP-resistant variant. We conclude that cyclic AMP derivatives inhibit growth of GH cells by an unanticipated mechanism that is, nonetheless, related to endogenous cyclic AMP synthesis.     

Dissociation by cooling of hormone and cholera toxin activation of adenylate cyclase in intact cells

Cholera toxin, through adenylate cyclase activation reproduced cyclic AMP-mediated effects of thyroid-stimulating hormone (TSH) in dog thyroid slices, i.e protein iodination, [1-¹⁴C]glucose-oxidation and hormone secretion. Iodide and carbamylcholine decreased the cyclic AMP accumulation induced by cholera toxin as well as by TSH, which supports the hypothesis of an action of these agents beyond the steps of hormone-receptor and receptor-adenylate cyclase interaction. Cooling to 20°C did not impair the TSH induced cyclic AMP accumulation in thyroid slices, but completely suppressed the cholera toxin effect.This observation has been extended to other hormones and target tissues, such as the parathyroid hormone (PTH) (kidney cortex), adrenocorticotropic hormone (ACTH) (adrenal cortex)_and luteinizing hormone (LH) (ovary systems). As in thyroid, cooling dissociated the cholera toxin and hormonal effects on cyclic AMP accumulation. In homogenate, cooling decreased cyclic AMP generation in the presence of cholera toxin but at 20°C and 16°C a cholera toxin stimulation was still observed. These results bear strongly against the hypothesis that the glycoprotein hormones TSH and LH activate adenylate cyclase by a mechanism identical to cholera toxin.     

Forskolin Stimulates Adenylate Cyclase Activity, Cyclic AMP Accumulation, and Adrenocorticotropin Secretion from Mouse Anterior Pituitary Tumor Cells

Abstract: The effects of forskolin, an adenylate cyclase activator, were investigated on adrenocorticotropin (ACTH) secretion from AtT-20/D16-16 mouse pituitary tumor cells. Forskolin increased adenylate cyclase activity in these cells in the absence of added guanyl nu-cleotide, an effect blocked by somatostatin. Cyclic AMP synthesis and ACTH secretion increased in a concentration-dependent manner, not only in the clonal cells, but in primary cultures of rat anterior pituitary as well. Somatostatin inhibited cyclic AMP synthesis and ACTH secretion in response to forskolin. When forskolin was coapplied with corticotropin releasing factor, cyclic AMP synthesis was potentiated and ACTH secretion additive. The calcium channel blocker, nifedipine, inhibited forskolin, and 8-bromocyclic AMP stimulated ACTH secretion. These data suggest that ACTH secretion may be regulated at the molecular level by changes in cyclic AMP formation, which in turn regulate a calcium gating mechanism.     

Dissociation by cooling of hormone and cholera toxin activation of adenylate cyclase in intact cells.

Cholera toxin, through adenylate cyclase activation reproduced cyclic AMP-mediated effects of thyroid-stimulating hormone (TSH) in dog thyroid slices, i.e. protein iodination, [1-14C]glucose-oxidation and hormone secretion. Iodide and carbamylcholine decreased the cyclic AMP accumulation induced by cholera toxin as well as by TSH, which supports the hypothesis of an action of these agents beyond the steps of hormone-receptor and receptor-adenylate cyclase interaction. Cooling to 20 degrees C did not impair the TSH induced cyclic AMP accumulation in thyroid slices, but completely suppressed the cholera toxin effect. This observation has been extended to other hormones and target tissues, such as the parathyroid hormone (PTH) (kidney cortex), adrenocorticotropic hormone (ACTH) (adrenal cortex) and luteinizing hormone (LH) (ovary systems). As in thyroid, cooling dissociated the cholera toxin and hormonal effects on cyclic AMP accumulation. In homogenate, cooling decreased cyclic AMP generation in the presence of cholera toxin but at 20 degrees C and 16 degrees C a cholera toxin stimulation was still observed. These results bear strongly against the hypothesis that the glycoprotein hormones TSH and LH acetivate adenylate cyclase by a mechanism identical to cholera toxin.     

Independence of cyclic AMP and relA gene stimulation of glycogen synthesis in intact Escherichia coli cells.

Previous studies from our laboratory established that in Escherichia coli, glycogen synthesis is regulated by both the relA gene, which mediates the stringent response, and by cyclic AMP. However, those studies raised the question of whether this dual regulatory system functions in an independent or a dependent manner. We show here that this regulation is independent, i.e., each regulatory process can express its action in the absence of the other. Triggering the stringent response by amino acid starvation increased glycogen synthesis even in mutants lacking the ability to synthesize cyclic AMP or lacking cyclic AMP receptor protein; and cyclic AMP addition stimulated glycogen synthesis in relA mutant strains. We also show that physiological concentrations of GTP inhibit ADP-glucose synthetase (glucose-1-phosphate adenylyltransferase, EC 2.7.7.27), the rate-limiting enzyme of bacterial glycogen synthesis, in vitro. Because the stringent response is known to cause an abrupt decrease in the cellular level of GTP, modulation of ADP-glucose synthetase activity by this nucleotide could account for a substantial portion of the step-up in the cellular rate of glycogen synthesis observed when the stringent response is triggered.