Inhibition by cyclic AMP of lactose production in lactating guinea pig mammary gland slices.

Addition of the cyclic AMP phosphodiesterase inhibitors theophylline (10- minus 2 M) or papaverine (10- minus 4 M) leads to a complete inhibition of lactose synthesis in incubated guinea pig mammary gland slices. Addition of 10- minus 5 M cyclic AMP or dibutyryl cyclic AMP results in 1 30-40% inhibition of the synthesis, which effect is not increased by applying higher concentrations of these compounds. A 30-40% inhibition can also be obtained with epinephrine (5 - 10- minus 5 M), or isoproterenol (10- minus 4 M), but the polypeptide hormones glucagon (10- minus 7 M), insulin (1 munit/ml) and relaxin (10 mug/ml) do not significantly affect lactose synthesis. Cytochalasin B (5 mug/ml) inhibits lactose production by 58and colchicine (10- minus 5 M) by 25%. These experiments suggest that an increase in the intracellular level of cyclic AMP either through its addition, through hormonal stimulation of its synthesis, or through inhibition of its intracellular breakdown, leads to an inhibition of lactose production in lactating mammary gland. This effect of cyclic AMP is similar to that of progesterone, which is known to inhibit lactation in vivo and the withdrawal of which at parturition has been postulated to initiate lactogenesis.     

Calmodulin content of rat mammary tissue and isolated cells during pregnancy and lactation.

The calmodulin content of heat-treated extracts of rat mammary tissue and isolated cells was measured by using stimulation of cyclic nucleotide phosphodiesterase (PDE) activity and radioimmunoassay (r.i.a.) procedures. The calmodulin content of mammary tissue increased 2.5-fold near the time of parturition, remained at the elevated level during lactation, then, after the onset of involution, decreased to values similar to those measured from mammary tissue of pregnant rats. When tissue from 15 animals in different stages of pregnancy, lactation and involution were compared, the r.i.a. gave 2.6-fold higher results than the PDE assay. To investigate further the increase in calmodulin content of mammary tissue, secretory and myoepithelial cells were enzymically dissociated from rat mammary tissue during different stages of pregnancy, lactation and involution. Protein, DNA, lactose, glucose-6-phosphate dehydrogenase and alkaline phosphatase were assayed to characterize the cell fractions. By using r.i.a., the calmodulin content per mg of protein in isolated secretory-cell fractions was high near parturition, then decreased and remained relatively constant during lactation. The amount of calmodulin expressed per mg of DNA in secretory cells did not show a marked change near parturition, suggesting a constant amount of calmodulin per cell. The calmodulin content of myoepithelial cells dissociated from mammary tissue and measured by using r.i.a. was 6-fold lower than in secretory cells and remained relatively constant during the course of lactation. The changing levels of calmodulin in rat mammary tissue during development are suggested to be related to proliferation and destruction of secretory epithelial cells, events that occur near parturition and involution respectively.     

Evidence for a calcium/calmodulin involvement in density-dependent melanogenesis in murine B16 melanoma cells.

Previous work from our laboratory has shown that both cyclic AMP and calcium/calmodulin appear to be involved in the regulation of melanogenesis in murine B16 melanoma cells. In these cells as in murine Cloudman S91 cells, melanogenic responsiveness to melanocyte-stimulating hormone (MSH) varies with cell density in culture. Our objective in this study was to learn more about the intracellular systems involved in the control of melanogenesis, particularly the role played by calcium. The melanogenic response to alpha MSH was compared to the response to drugs affecting intracellular free calcium and calmodulin over a range of cell densities in B16F1 cells. alpha MSH-stimulated melanin production was extremely density-dependent but alpha MSH-stimulated cyclic AMP production was independent of cell density. The melanogenic response to agents that increased intracellular calcium (A23187) or inhibited intracellular calmodulin varied with cell density. A drug (TMB8) that lowered intracellular free calcium, however, increased melanogenesis independently of cell density. At high cell density it was found that an elevation in calcium decreased melanogenesis, whereas agents that reduced calcium or inhibited calmodulin activity increased melanogenesis. At low cell density, however, the inhibitory response to A23187 was lost and in some experiments even stimulated melanogenesis. These data suggest that the calcium/calmodulin signalling system has an inhibitory influence on melanogenesis, and its expression, which depends upon cell density, may also modulate the response to stimulatory agents such as alpha MSH.     

Evidence for a Calcium/Calmodulin Involvement in Density-Dependent Melanogenesis in Murine B16 Melanoma Cells

Previous work from our laboratory has shown that both cyclic AMP and calcium/calmodulin appear to be involved in the regulation of melanogenesis in murine B16 melanoma cells. In these cells as in murine Cloudman S91 cells, melanogenic responsiveness to melanocyte-stimulating hormone (MSH) varies with cell density in culture. Our objective in this study was to learn more about the intracellular systems involved in the control of melanogenesis, particularly the role played by calcium. The melanogenic response to αMSH was compared to the response to drugs affecting intracellular free calcium and calmodulin over a range of cell densities in B16F1 cells. αMSH-stimulated melanin production was extremely density-dependent but αMSH-stimulated cyclic AMP production was independent of cell density. The melanogenic response to agents that increased intracellular calcium (A23187) or inhibited intracellular calmodulin varied with cell density. A drug (TMB8) that lowered intracellular free calcium, however, increased melanogenesis independently of cell density. At high cell density it was found that an elevation in calcium decreased melanogenesis, whereas agents that reduced calcium or inhibited calmodulin activity increased melanogenesis. At low cell density, however, the inhibitory response to A23187 was lost and in some experiments even stimulated melanogenesis. These data suggest that the calcium/calmodulin signalling system has an inhibitory influence on melanogenesis, and its expression, which depends upon cell density, may also modulate the response to stimulatory agents such as αMSH.     

Calmodulin activation of cyclic AMP phosphodiesterase in the B16 mouse melanoma

Mouse B16 melanoma extracts of both cultured cells and tumour tissue contain cyclic AMP phosphodiesterase activity, with 95% present in the soluble fraction. Although activation of the enzyme by added calmodulin did not occur, it was found that endogenous calmodulin was present at a level sufficient to activate fully the enzyme. The ability of Ca-calmodulin to stimulate cyclic AMP phosphodiesterase in this tissue was shown by the inhibitory effect of N-(6-aminohexyl)-5-chloronaphthalenesulphonamide (W7), a known calmodulin antagonist; by the activation of the enzyme with exogenous calmodulin observed in supernatants depleted of endogenous calmodulin by passage over fluphenazine-Sepharose 6B in the presence of Ca2+; by the Ca-dependent binding of the enzyme to calmodulin-agarose and its activation by Ca-calmodulin after elution from the column with EGTA-containing buffer. It was calculated that about 50% of the total cyclic AMP phosphodiesterase activity was calmodulin-activated in this tissue.     

Fructose 2,6-bisphosphate, sugar phosphates and adenine nucleotides in the regulation of glucose metabolism in the lactating rat mammary gland

Fructose 2,6-bisphosphate is present in the rat mammary gland, rising from a value of 1.4 nmol/g in pregnancy to 4.3 nmol/g tissue at 14 days lactation; the equivalent values calculated/ml intracellular water are 5.2 and 11.6 nmol, respectively. The tissue content of fructose 6-phosphate, fructose 1,6-bisphosphate, ATP and phosphoenolpyruvate remain relatively constant in the transition from pregnancy to the height of lactation. The changes in AMP, cyclic AMP, and citrate content of the mammary gland during lactation are such as to promote an increase in fructose 2,6-bisphosphate formation and flux through phosphofructokinase.     

Inhibitors of intracellular cyclic AMP accumulation affect differentiation of sporogenous mutants of Dictyostelium discoideum

Abstract Sporogenous mutants of Dictyostelium discoideum strain V12M2 were used to determine whether the intracellular levels of cyclic AMP or other second messengers regulate differentiation. Increasing external concentrations of cyclic AMP promoted spore formation. Caffeine and progesterone, which lower intracellular cyclic AMP levels by different mechanisms, blocked spore formation and favored stalk cell formation. In contrast, differentiation of both spore and stalk cells occurred normally in the presence of agents that disrupt calcium/calmodulin or protein kinase C-based second messenger systems. The data are in accord with the view that (1) intracellular cyclic AMP is essential for terminal differentiation of both cell types, and (2) higher levels are required for formation of spores than for stalk cells.     

Induction of cellulase in trichoderma reesei grown on lactose

Intracellular concentrations of ATP, cyclic AMP and glucose-6-phosphate were monitored during growth of partially catabolically derepressed strain of Trichoderma reesei CC II in medium containing lactose as the sole carbon source. The induction of cellulase synthesis occured when the concentration of lactose in the medium decreased below 7 mg/ml. The onset of cellulase synthesis was preceded by a transient peak of intracellular concentration of ATP and by the increase of the cyclic AMP contents in the mycelium whereby the intracellular level of glucose-6-phosphate was at its minimum. By keeping the lactose concentration in the medium at 2 mg/ml, it was possible to support the continuation of cellulase synthesis over the prolonged periods without appreciable growth of biomass.     

Effect of calcium on cyclic nucleotide phosphodiesterase in parathyroid tissue

The hydrolysis of cyclic AMP and cyclic GMP by homogenates of normal bovine parathyroid gland and human parathyroid adenomas was decreased by EGTA. When supernatants were chromatographed on DEAE-cellulose it was found that sheep brain calmodulin in the presence of calcium stimulated cyclic AMP and cyclic GMP phosphodiesterase activity. The response to calmodulin in two human parathyroid adenomas was less than that in normal bovine parathyroid. Calmodulin was detected in heat-treated supernatants of 11 parathyroid adenomas by its ability to activate calmodulin-free sheep brain phosphodiesterase. The results suggest a role for calcium in the hydrolysis of cyclic nucleotides in parathyroid tissue.     

Steroidogenesis in isolated adrenocortical cells. Correlation with receptor-bound adenosine 3′:5′-cyclic monophosphate

Because several groups have recently questioned a mediating role for cyclic AMP in adrenocortical steroidogenesis, we analysed the problem in more detail by measuring three different cyclic AMP pools in cells isolated from decapsulated rat adrenals. Extra-cellular, total intracellular and bound intracellular cyclic AMP were determined by radioimmunoassay in comparison with corticosterone production induced by low corticotropin concentrations. The increase in extracellular and total intracellular cyclic AMP with low corticotropin concentrations was dependent on the presence of a phosphodiesterase inhibitor and short incubation times. Bound intracellular cyclic AMP was less dependent on these two parameters. In unstimulated cells cyclic AMP bound to its receptor represents only a small fraction of the total intracellular cyclic AMP. After stimulation by a concentration of corticotropin around the threshold for corticosterone production, an increase in bound cyclic AMP was observed which correlated very well with steroidogenesis both temporally and with respect to corticotropin concentration. This finding was complemented by measuring a concomitant decrease in free receptor sites. Full occupancy of the receptors was not necessary for maximal steroidogenesis. Binding kinetics of cyclic [(3)H]AMP in concentrations equivalent to the intracellular cyclic AMP concentration suggest the presence of at least three different intracellular cyclic AMP pools. These observations are in agreement with a possible role for cyclic AMP as a mediator of acute steroidogenesis induced by low corticotropin concentrations.     

Human relaxin increases cyclic AMP levels in cultured anterior pituitary cells

Although relaxin acts at several abdominal sites and mammary tissue associated with pregnancy and parturition, the scope of target tissues and the signals conveying the relaxin message into the cell are poorly defined. We found that human relaxin rapidly elevates the cyclic AMP content of cultured rat anterior pituitary cells. This is a graded response (EC50 0.3 nM relaxin) that can be blocked by anti-relaxin antibodies or the hormones somatostatin and dopamine. Furthermore, other hormones with some sequence homology to relaxin, such as insulin and insulin-like growth factor-I, have no such action. We conclude that the anterior pituitary may be a target tissue for relaxin and that cyclic AMP may act as an intracellular messenger for relaxin in these cells.     

Calmodulin modulates prolactin secretion in vitro: studies with calmodulin containing liposomes

The control of prolactin secretion by Ca calmodulin and cyclic AMP was studied. Ca++ ionophore A23187 stimulated both cyclic AMP accumulation and prolactin release by primary culture of anterior pituitary cells in vitro. The increase of cyclic AMP formation by A23187 preceded that of prolactin release. To test the calmodulin involvement in these processes we used either selective calmodulin antagonist, the naphthalene sulphonamide derivative W7, or calmodulin containing liposomes. W7 dose dependently inhibited both basal or A23187 stimulated cyclic AMP accumulation and prolactin secretion. Insertion of Ca calmodulin within the cells stimulated prolactin secretion without modifying cyclic AMP accumulation. W7 inhibited the Ca calmodulin containing liposomes stimulation of prolactin release. These results suggest that calmodulin participates to the process of prolactin release.     

Possible involvement of Ca2+-calmodulin system in cyclic AMP action in cholesterol ester hydrolytic response to ACTH in bovine adrenocortical cells

In order to elucidate the relationship between cyclic AMP and the Ca2+-calmodulin system in the steroidogenic response to adrenocorticotropic hormone (ACTH), the effects of calmodulin inhibitors (trifluoperazine and W-7) on cortisol production and cellular cholesterol ester hydrolysis induced by ACTH or dibutyryl cyclic AMP in bovine adrenocortical cells were examined in the absence of extracellular Ca2+. These calmodulin inhibitors inhibited not only the cortisol production and the cholesterol ester hydrolysis induced by ACTH in the absence of extracellular Ca2+, but also inhibited the dibutyryl cyclic AMP-induced cortisol production and the cholesterol ester hydrolysis in the absence of extracellular Ca2+. These results suggested the possibility that cyclic AMP action was mediated by the Ca2+-calmodulin system in the activation process of cellular cholesterol ester hydrolysis in the steroidogenic response to ACTH.     

Photoreactive (caged) cyclic AMP analogs induce DNA synthesis in mammary epithelial cells

Dimethoxy-2-nitrobenzyl adenosine-3′,5′ cyclic monophosphate (DMNB) is a metabolically active, photolabile cyclic AMP analog that yields free cyclic AMP upon UV hydrolysis. The analog is useful in that it permits short term, transient elevations of intracellular cyclic AMP. Addition of DMNB (1-10 μM) to mouse mammary epithelial cells, followed by UV irradiation of cells, caused a significant increase in DNA synthesis over that observed with controls, UV irradiation alone or DMNB alone. In subsequent studies, DMNB exhibited a modest, but statistically significant, interaction with epidermal growth factor in promoting DNA synthesis. Effects of DMNB were observed if DNA synthesis was measured as either ³H-thymidine incorporation into DNA or as percent S-phase cells. These results indicate that previously observed effects of agents such as cholera toxin and phosphodiesterase resistant cyclic AMP analogs on mammary epithelial proliferation can be mimicked, at least in part, by a short term pulse of cyclic AMP.     

Acute change in the cyclic AMP content of rat mammary acini in vitro. Influence of physiological and pharmacological agents.

The cyclic AMP content of acini, freshly prepared from mammary tissue of lactating rats, was measured during incubation in vitro. Neither adrenergic agonists nor cyclic AMP phosphodiesterase inhibitors alone caused a change of more than 2-fold in the basal cyclic AMP content of acini. Together, however, these agents provoked increases of around 20-fold in acini cyclic AMP content. Forskolin caused similar effects. The relative potency of adrenergic agonists in increasing cyclic AMP in acini, together with the ability of selective antagonists to oppose such rises, indicated that beta 2-adrenergic receptors were involved in mediating the effects. Receptor-binding experiments using [3H]dihydroalprenolol and selective beta-antagonists confirmed the predominant presence of beta 2-adrenergic receptors on acini membranes and on membranes prepared from purified mammary secretory epithelial cells. These results elucidate some previous findings [Robson, Clegg & Zammit (1984) Biochem. J. 217, 743-749; Williamson, Munday, Jones, Roberts & Ramsey (1983) Adv. Enzyme Regul. 21, 135-145], questioning the role of cyclic AMP in the regulation of lipogenesis in mammary acini.     

Regulation of the proliferation rate of cultured,androgen-responsive cells does not involve changes in cyclic AMP levels

The proliferation rate of cultured cells from the mouse mammary carcinoma Shionogi 115 is regulated both by local cell population density and by androgens. Measurement of intracellular levels of cyclic AMP has shown that these levels are constant over a wide range of proliferation rates (mean doubling times varied from 23 hr to more than 200 hr). Addition of dibutyryl cyclic AMP or theophylline to the culture medium resulted in inhibition of growth—even in the presence of androgen. This inhibition of growth and the relationship between cyclic AMP levels and cell proliferation is discussed.     

The regulation of membrane fusion during sexual development in Dictyostelium discoideum

During the first 24 h of sexual development in Dictyostelium discoideum, three sequential events of membrane fusion occur: gamete fusion, pronuclear fusion, and phagocytosis. The early events of sexual development are regulated by a diverse group of endogenous molecules: (i) a volatile sexual pheromone, (ii) a protein cell fusion inducing factor (CFIF), (iii) a low molecular weight autoinhibitor, (iv) and cyclic AMP. CFIC enhances cell fusion while the autoinhibitor and cyclic AMP both inhibit the event. Both extracellular and intracellular calcium ions are essential for cell and pronuclear fusion. Pharmacological analyses show that the intracellular functions of the divalent cation in these processes are mediated by calmodulin. The autoinhibitor appears to function by inhibiting calmodulin activity. Glucose, mannose, and N-acetylglucosamine containing glycoproteins have been shown to function in both cell fusion and phagocytosis. The interplay between all of these diverse molecules is examined and a review of all of the recent literature is presented.     

The role of calcium ions in the mechanism of ACTH stimulation of cortisol synthesis

Removal of free calcium ions from the incubation medium of isolated bovine adrenocortical cells with EGTA reduced basal cortisol synthesis and blocked the effects of ACTH; additional calcium restored normal steroid synthesis. Calcium channel blockers, verapamil and nitrendipine and the calmodulin antagonist, trifluoperazine inhibited ACTH-stimulated cortisol synthesis in a dose-dependent manner (IC50s of 6.2, 10 and 5.2 microM, respectively). Steroidogenic effects of dibutyryl cyclic AMP were prevented with 50 microM verapamil or trifluoperazine. Calcium ionophore A23187 at 1 microM increased cortisol synthesis 2-3 fold which was less than the normal response to ACTH. Stimulatory effects of ionophore and cyclic AMP or ACTH were not additive. ACTH-stimulation of cortisol synthesis appears to involve cyclic AMP-dependent uptake of extracellular calcium ions, possibly by a mechanism requiring calmodulin. Increases in intracellular calcium ions cannot wholly mimic ACTH actions.     

Modulation and role of Ca2+ in LH and LHRH agonist action in rat Leydig cells

The results of our recent studies on purified rat Leydig cells indicate that there are no major qualitative differences in the stimulating effects of LH and LHRH agonists on steroidogenesis via mechanisms that are dependent on calcium. This was demonstrated by using inhibitors of calmodulin and the lipoxygenase pathways of arachidonic acid metabolism. Using the fluorescent indicator quin-2, it was shown that LH and LHRH agonist increase intracellular calcium levels; LH was more potent than LHRH agonist (max increase in concentrations obtained were 500 nM and 60 nM respectively). This difference was probably the result of a direct effect of cyclic AMP (whose production is stimulated by LH but not by LHRH) because cyclic AMP analogues were as potent as LH in increasing calcium levels. These studies indicate a major role for calcium in the control of steroidogenesis in testis Leydig cells.