Voltage-sensitive calcium channels regulate guanosine 3'',5''-cyclic monophosphate levels in neuroblastoma cells.

Veratridine or high potassium concentration increased guanosine 3',5'-cyclic monophosphate (cGMP) levels in neuroblastoma cells of clone N1E-115 without affecting levels of adenosine 3',5'-cyclic monophosphate (cAMP). The increases in cGMP appear to be a direct result of the depolarizing action of these agents and not due to the action of substances released from the cells upon depolarization. The increase in cGMP produced by depolarization was dependent upon extracellular calcium and could be prevented by the calcium channel blockers D600 and cobalt. Carbachol, acting on muscarinic acetylcholine receptors, also caused a calcium-dependent increase in cGMP in these cells. The carbachol and potassium effects were additive from 5 to 100 mM potassium and from 1 to 3 mM calcium. The carbachol response was nearly as sensitive as the potassium response to inhibition by D600 but was much less sensitive to inhibition by cobalt. The results suggest that depolarization increases cGMP levels in these cells by opening voltage-sensitive calcium channels and that activation of muscarinic receptors opens separate, voltage-insensitive calcium channels.     

Hormone and neurotransmitter receptors in an established vascular endothelial cell line.

A cell line from the intima of the rabbit aorta has been established. This cell line exhibits strict contact inhibition, and morphologically resembles intimal endothelial cells. B-type blood group antigens and the presence of fibrinolytic activity also distinguish these cells from smooth muscle cells and from fibroblasts of the aortic wall. Endothelial cells were assayed for changes in levels of adenosine 3':5'-cyclic monophosphate (cAMP) and guanosine 3':5'-cyclic monophosphate (cGMP) in response to a series of vasoactive drugs. Control levels for cAMP and cGMP are 7.01 +/- 0.82 and 1.50 +/- 0.06, respectively (mean +/- SEM). Norepinephrine, acetylcholine, 5-hydroxytryptamine, and phenylephrine increased the levels of both nucleotides significantly. Propranolol (10-5 M) and phentolamine (10-5M) inhibited, respectively, the cAMP and cGMP response to norepinephrine. Angiotensin II and histamine significantly increased cGMP levels but not cAMP levels of the endothelial cells. The cGMP increases with acetylcholine were inhibited by atropine. These results indicate that the established cell line is endothelial in nature and contains cellular receptors to a variety of vasoactive agents.     

Epinephrine-induced elevation of guanosine 3′:5′-cyclic monophosphate in isolated fat cells of rat

The effects of epinephrine (as low as 0.1 muM) on guanosine 3':5'-cyclic monophosphate (cGMP) and adenosine 3':5'-cyclic monophosphate (cAMP) in isolated fat cells were examined. Epinephrine increased both cGMP and cAMP levels, with the elevation of cAMP preceding the rise of cGMP. Maximal elevation was obtained with 1 muM epinephrine for each nucleotide. The increase in content of cGMP and cAMP due to epinephrine was completely blocked by a beta-adrenergic antagonist (5 muM propranolol). Phentolamine (10-100 muM), an alpha-adrenergic antagonist, enhanced the response to epinephrine resulting in elevation of cAMP levels, whereas a high concentration (100 muM) of phentolamine suppressed the elevation of cGMP. The ability of epinephrine to increase cGMP and cAMP levels was markedly diminished by "feedback regulator" partially purified from the incubation mixtures of isolated fat cells exposed to epinephrine [Ho, R.J. & Sutherland, E. W. (1971) J. Biol. Chem. 246, 6822-6827], whereas an increase in cGMP, but not cAMP, levels was observed in isolated fat cells incubated with "feedback regulator" alone (without epinephrine). These observations suggest the possibility that the epinephrine-induced elevation of cGMP levels in isolated fat cells might be mediated by an increase in formation of intracellular "feedback regulator" due to an elevation of cAMP by epinephrine.     

Cyclic GMP may serve as a second messenger in peptide-induced muscle degeneration in an insect.

At the end of metamorphosis, the intersegmental muscles of the moth Antheraea polyphemus undergo rapid degeneration in response to the peptide eclosion hormone (EH). Muscle death was preceded by a 22-fold increase in muscle guanosine-3',5'-cyclic monophosphate (cGMP) titers, which peaked 60 min after peptide exposure; adenosine-3'5'-cyclic monophosphate (cAMP) titers remained unchanged. EH induced a dose-dependent increase in muscle cGMP content with a threshold dose similar to that needed to induce cell death. Exogenous cGMP, but not cAMP, mimicked the action of EH. Sodium nitroprusside, a potent stimulator of guanylate cyclase, and methylated xanthines, a class of 3',5'-cyclic-nucleotide phosphodiesterase inhibitors, also induced the selective death of these muscles. It is concluded that an elevation of cGMP level is involved in EH-induced muscle degeneration. The intersegmental muscles become sensitive to EH at the end of adult development in response to the declining titers of the steroid molting hormones, the ecdysteroids. At earlier times, treatment with EH, exogenous cGMP, sodium nitroprusside, or methylated xanthines was ineffective in causing cell death. Nevertheless, treatment with EH at this time resulted in a marked increase in intersegmental-muscle cGMP. Thus, the onset of physiological responsiveness to the peptide hormone presumably results from biochemical changes distal to the EH receptors and guanylate cyclase.     

Atypical synergistic alpha 1- and beta-adrenergic regulation of adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate in rat pinealocytes

The adrenergic control of cAMP and 3',5'-cyclic GMP (cGMP) in dispersed adult rat pinealocytes was investigated. Norepinephrine treatment increased cAMP and cGMP content 60- and 400-fold, respectively; both alpha- and beta-adrenoceptors had to be activated for these responses to occur. Beta-Adrenergic stimulation alone produced only about 6- and 2-fold increase in cAMP and cGMP content, respectively. Alpha-Adrenergic stimulation, which alone had no effect on either cyclic nucleotide concentration, markedly amplified the beta-adrenergic stimulation of both cAMP and cGMP. The relative potency of alpha-adrenergic agonists and antagonists indicates the alpha 1-subclass of adrenoceptors is involved. A role of alpha 1-adrenoceptors in the control of pineal cAMP is consistent with published evidence of the presence of alpha 1-adrenoceptors on pinealocytes and their role in the regulation of N-acetyltransferase activity and melatonin production.     

Regulation of hepatic nuclear guanylate cyclase.

In immunohistochemical studies of rat liver tissue slices and purified nuclei, adenosine 3':5'-cyclic monophosphate (cAMP) and guanosine 3':5'-cyclic monophosphate (cGMP) immunofluorescence on the nuclear membrane are sequentially increased after glucagon administration. An explanation for the increased cGMP immunofluorescence was sought in experiments in which guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2]activity of hepatic subcellular fractions was determined. The results showed that a nuclear guanylate cyclase exists which can be distinguished from the soluble and crude particulate guanylate cyclases. The activity of the nuclear enzyme was increased by 35% in nuclei isolated from rats 30 min after glucagon injection, the time at which maximal nuclear membrane cGMP immunofluorescence is observed. Because glucagon altered both cAMP location and levels prior to the observed changes in nuclear cGMP metabolism, the hypothesis that cAMP acted as the second messenger was tested. In vitro incubation of nuclei isolated from control rats with 10(-5) M cAMP produced a 25% increase in nuclear guanylate cyclase activity. With nuclei isolated from glucagon-treated rats, no significant increase in enzyme activity was observed; this indicates that maximal stimulation of nuclear guanylate cyclase by cAMP occurred at levels that are obtained in vivo after glucagon administration. These findings suggest that hepatic nuclear cGMP content may be regulated by a specific organelle guanylate cyclase and that cAMP may be one of the determinants of this enzyme's activity.     

Role of cyclic nucleotides in rapid platelet adhesion to collagen

Adhesion of human platelets to type I collagen under arterial flow conditions is extremely fast, being mediated primarily by the alpha 2 beta 1 integrin (glycoprotein Ia/IIa). We have investigated the involvement of cyclic nucleotides in platelet adhesion to soluble native collagen immobilized on Sepharose beads using a new microadhesion assay under arterial flow conditions. To prevent platelet stimulation by thromboxanes and adenosine diphosphate (ADP), experiments were performed with aspirin-treated platelets in the presence of ADP-removing enzyme systems such as creatine phosphate/creatine phosphokinase or apyrase. Rapid reciprocal changes in platelet adenosine 3'5'-cyclic monophosphate (cAMP) and guanosine 3'5'-cyclic monophosphate (cGMP) occurred during adhesion. cAMP levels in adherent platelets were 2.4-fold lower than in effluent platelets or in static controls, whereas cGMP levels were increased 2.4-fold. These results suggest that contact between platelets and collagen stimulates guanylate cyclase and inhibits adenylate cyclase. This occurs in the absence of the platelet release reaction. We also studied short-term effects of agents that regulate cyclic nucleotide synthesis, prostaglandin E1 (PGE1) and sodium nitroprusside (SNP). After only 3.8 seconds at 10 to 30 dyne/cm2, PGE1 (10 mumol/L) increased cAMP 16.4- fold, whereas SNP (50 mumol/L) increased cGMP ninefold and caused a 3.2- fold increase in cAMP. Both PGE1 and SNP rapidly (< 5 seconds) inhibited platelet adhesion in a dose-dependent manner that was correlated with the increase in cyclic nucleotides. Our data suggest that cAMP and cGMP play a regulatory role in the initial phases of platelet adhesion to collagen mediated by the alpha 2 beta 1 integrin receptor.     

Immunohistochemical localization of 3′:5′-cyclic AMP and 3′:5′-cyclic GMP in rat renal cortex: Effect of parathyroid hormone

Adenosine 3':5'-cyclic monophosphate (cAMP) and guanosine 3':5'-cyclic monophosphate (cGMP) were localized in cells of rat kidney cortex by an immunocytochemical technique before and after perfusion with parathyroid hormone (PTH). In control tissues the cAMP antiserum detected approximately the same intensity of fluorescence in cytoplasmic epithelial cell elements of cortical tubules and glomeruli (cells of Bowman's capsule and podocytes). PTH increased fluorescence in these glomerular cells and increased cAMP fluorescence in cytoplasmic granules in proximal tubular cells. These granules, whose structure has not been identified, were located predominantly on the luminal side of the tubular cells. In control rats, the renal cortical fluorescence detected with the cGMP antiserum was more pronounced in glomeruli (predominantly in the mesangial areas) and lesser amounts of fluorescence were observed in tubules. After PTH treatment, cGMP fluorescence increased in glomeruli and in renal tubular cells. A bright linear pattern of fluorescence was found in the area of the tubular luminal membrane. Perfusion with PTH caused relatively small increases in total tissue cAMP and no consistent increases in total tissue cGMP. Our observations suggest that both cAMP and cGMP are involved in the glomerular and tubular responses to PTH and point out the added dimension that this immunocytochemical technique brings to studies of cyclic nucleotide dynamics in heterogeneous tissues.     

cAMP and forskolin decrease gamma-aminobutyric acid-gated chloride flux in rat brain synaptoneurosomes.

The effects of the cyclic nucleotide cAMP on gamma-aminobutyric acid-gated chloride channel function were investigated. The membrane-permeant cAMP analog N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate inhibited muscimol-induced 36Cl- uptake into rat cerebral cortical synaptoneurosomes in a concentration-dependent manner (IC50 = 1.3 mM). The inhibition was due to a decrease in the maximal effect of muscimol, with no change in potency. Similar effects were observed with 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate, 8-bromoadenosine 3',5'-cyclic monophosphate, and the phosphodiesterase inhibitor isobutylmethylxanthine. The effect of endogenous cAMP accumulation on the gamma-aminobutyric acid-gated Cl- channel was studied with forskolin, an activator of adenylate cyclase. Under identical conditions, in the intact synaptoneurosomes, forskolin inhibited muscimol-induced 36Cl- uptake and generated cAMP with similar potencies (IC50 = 14.3 microM; EC50 = 6.2 microM, respectively). Surprisingly, 1,9-dideoxyforskolin, which does not activate adenylate cyclase, also inhibited the muscimol response, suggesting that forskolin and its lipophilic derivatives may interact with the Cl- channel directly. Indeed, forskolin inhibition of muscimol-induced 36Cl- uptake was extremely rapid (within 5 sec), preceding the accumulation of sufficient levels of cAMP. After 5 min, a slower phase of inhibition was seen, similar to the time course for cAMP accumulation. The data suggest that gamma-aminobutyric acid (GABAA) receptor function in brain can be regulated by cAMP-dependent phosphorylation.     

Evidence for alpha-adrenergic receptors acting through the guanylate cyclase system in human cultured thyroid cells

In order to investigate the presence of alpha-adrenergic receptors in human thyroid, we have studied the effect of alpha-adrenergic agonists and antagonists on cGMP cellular content of human thyroid cells in primary culture. Epinephrine as well as TSH were not able to modify the cGMP cellular levels, while norepinephrine significantly increased cGMP accumulation already at 10 nM, a dose inactive on cAMP accumulation. A non selective alpha-adrenergic antagonist, phentolamine, significantly inhibited cGMP accumulation induced by norepinephrine. Norepinephrine-induced cGMP accumulation was unaffected by prazosin, an alpha 1-adrenergic antagonist, but was abolished by yohimbine, an alpha 2-adrenergic antagonist. Phenylephrine, an alpha-adrenergic agonist, produced an increase of cellular cGMP levels without modifying cAMP content. In the presence of TSH, the cGMP response to norepinephrine was not modified; however, the increase of cAMP levels was inhibited by norepinephrine at doses inactive on cAMP accumulation, but active on cGMP levels. The present results demonstrate the existence in human thyroid cells of alpha 2-adrenergic receptors, regulating the guanylate cyclase system. It may be postulated that the counter-regulation exerted by alpha-adrenergic agonists on the response to TSH operates on the TSH-dependent adenylate cyclase.     

Insulin influences the nitric oxide cyclic nucleotide pathway in cultured human smooth muscle cells from corpus cavernosum by rapidly activating a constitutive nitric oxide synthase

AIMS: We have evaluated, in cultured human cavernosal smooth muscle cells, the expression and activity of calcium-dependent constitutive nitric oxide synthase (cNOS) and the ability of insulin to induce nitric oxide (NO) production and to increase intracellular cyclic nucleotides guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate (cAMP). METHODS: cNOS mRNA was detected by RT-PCR amplification, cNOS protein by immunofluorescence, cNOS activity as l-[3H]-citrulline production from l-[3H]-arginine and cyclic nucleotides by radioimmunoassay. RESULTS: cNOS mRNA and cNOS protein were found in cultured cells; cNOS activity was increased by 5-min exposure to 1 micro mol/l calcium ionophore ionomycin (from 0.1094+/-0.0229 to 0.2685+/-0.0560 pmol/min per mg cell protein, P=0.011) and to 2 nmol/l insulin (from 0.1214+/-0.0149 to 0.2045+/-0.0290 pmol/min per mg cell protein, P=0.041). Insulin increased both cGMP and cAMP in a dose- and time-dependent manner (i.e. with 2 nmol/l insulin, cGMP rose from 2.71+/-0.10 to 6.80+/-0.40 pmol/10(6) cells at 30 min, P=0.0001; cAMP from 1.26+/-0.06 to 3.02+/-0.30 pmol/10(6) cells at 60 min, P=0.0001). NOS inhibitor N(G)-monomethyl-l-arginine and phosphatidylinositol 3-kinase (PI 3-kinase) inhibitors wortmannin and LY 294002 blunted these effects of insulin. The action of insulin on cyclic nucleotides persisted in the presence of phosphodiesterase inhibition, guanylate cyclase activation by NO donors and adenylate cyclase activation by Iloprost or forskolin. CONCLUSION: Human cavernosal smooth muscle cells, by expressing cNOS activity, are a source of NO and not only its target; in these cells, insulin rapidly activates cNOS through a PI 3-kinase pathway, with a consequent increase of both cyclic nucleotides, thus directly influencing the mechanisms involved in penile vascular tone and interplaying with classical haemodynamic mediators.     

Immunocytochemical localization of cyclic GMP: Light and electron microscope evidence for involvement of neuroglia

Guanosine 3',5'-cyclic monophosphate (cGMP) immunoreactivity in the rat's cerebellum was studied with light and electron microscopy by the indirect fluorescence method and the peroxidase-antiperoxidase method. Labeled cells included neuroglial cells in the cerebellar cortex, white matter, and deep nuclei; some stellate and basket cells in the cortex; and some large neurons in the deep nuclei. No evidence was found for sagittal microzonation in the cGMP distribution. In the labeled cells, cGMP immunoreactive sites were localized to surface membranes, organelles, and the cytoplasmic matrix. Specificity was indicated by the same pattern of labeling after treatment with cGMP immunoglobulin that had been adsorbed with adenosine 3',5'-cyclic monophosphate (cAMP) and by the failure to label after treatment with normal rabbit sera or with cGMP immunoglobulin that had been adsorbed with 1 mM cGMP. Cerebella treated with cAMP antisera, however, showed immunoreactivity in Purkinje cells, granule cells, and Golgi cells in addition to neuroglia in cortex and deep nuclei. Sequential norepinephrine and glutamate superfusions generally intensified cGMP immunoreactivity, not only in neuroglial cells but also in the background. Under these conditions some Purkinje cells and some granule cells were also labeled. Increased cGMP immunoreactivity was also obtained by treatment with harmaline, gamma-aminobutyric acid and aminooxyacetic acid, muscimol, gamma-aminobutyric acid, or apomorphine in order of decreasing effectiveness. Serotonin and colchicine produced no detectable increase of cGMP immunoreactivity above normal, and diazepam and sodium pentobarbital decreased it. In these experiments, diethyl ether was preferable to sodium pentobarbital for anesthesia on account of the depressive action of the latter on cGMP immunoreactivity. Thus, drugs that increase cerebellar activity enhance cGMP levels, whereas those that decrease cerebellar activity decrease cGMP levels. However, it is not clear whether these fluctuations in cGMP levels are a direct consequence of neurotransmitter function or are sequelae to other related events. The present study suggests that some neurons and many neuroglial cells are the major sites of cGMP in the cerebellum.     

Nitric oxide/cGMP signaling inhibits TSH-stimulated iodide uptake and expression of thyroid peroxidase and thyroglobulin mRNA in FRTL-5 thyroid cells.

OBJECTIVE: Nitric oxide (NO) induces morphological and functional alterations in primary cultured thyroid cells. The aim of this paper was to analyze the direct influence of a long-term exposition to NO on parameters of thyroid hormone biosynthesis in FRTL-5 cells. DESIGN: Cells were treated with the NO donor sodium nitroprusside (SNP) for 24-72 h. MAIN OUTCOME: SNP (50-500 micromol/L) reduced iodide uptake in a concentration-dependent manner. The inhibition of iodide uptake increased progressively with time and matched nitrite accumulation. SNP inhibited thyroperoxidase (TPO) and thyroglobulin (TG) mRNA expression in a concentration-dependent manner. SNP enhanced 3',5'-cyclic guanosine monophosphate (cGMP) production. 3',5'-cyclic adenosine phosphate (cAMP) generation was reduced by a high SNP concentration after 48 h. 8-Bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP), a cGMP analog, inhibited iodide uptake as well as TPO and TG mRNA expression. The cGMP-dependent protein kinase (cGK) inhibitor KT-5823 reversed SNP or 8-Br-cGMP-inhibited iodide uptake. Thyroid-stimulating hormone pretreatment for 24-48 h prevented SNP-reduced iodide uptake although nitrite levels remained unaffected. CONCLUSION: These findings favor a long-term inhibitory role of the NO/cGMP pathway on parameters of thyroid hormone biosynthesis. A novel property of NO to inhibit TPO and TG mRNA expression is supported. The NO action on iodide uptake could involve cGK mediation. The long-term inhibition of steps of thyroid hormonogenesis by NO could be of interest in thyroid pathophysiology.     

Cyclic AMP mediation of the hormonal stimulation of 14C-acetate incorporation by Heliothis armigera pheromone glands in vitro

Evidence is presented for hormone-controlled adenosine 3',5'-cyclic monophosphate (cAMP)-mediated incorporation of 14C-acetate by in vitro pheromone gland preparations of Heliothis armigera. Basal intracellular cAMP concentrations of in vitro pheromone glands increase 28-fold in the presence of methanolic brain-complex extracts after 5 min of incubation. This intracellular cAMP increase shows a dose-dependent response. Studies on isolated pheromone gland preparations show that 8-bromo-cAMP added to the incubation media stimulates acetate incorporation thereby mimicking the hormonal response. This effect was duplicated by forskolin and isobutyl-methylxanthine. In addition, forskolin exhibits a dose-dependent stimulatory effect on the incorporation of 14C-acetate by such isolated in vitro pheromone glands. High-pressure liquid chromatographic analysis of the radioactive products, extracted by hexane from pheromone glands and media, revealed a stimulation by forskolin of a product exhibiting the same retention time as (Z)-11-hexadecenal, the main pheromone component of H. armigera. In addition, thin-layer chromatographic analysis revealed co-elution with (Z)-11-hexadecenal on stimulation by 8-bromo-cAMP.     

Effects of norepinephrine on cyclic nucleotide levels in dog thyroid slices.

Addition of dibutyryl adenosine 3'-5'-cyclic monophosphate (DBcAMP) or dibutyryl guanosine 3',5'-cyclic monophosphate (DBcGMP; 1--6mM) to to enzymatically dispersed, overnight-cultured rat anterior pituitary cell preparations stimulated the release of gonadotropins (LH and FSH) from the cells into the incubation medium. Stimulation of gonadotropin release by DBcGMP was observed after only 10 min of incubation, whereas that caused by DBcAMP appeared at 180 min. Synthetic LHRH (2.5 X 10(-9) M) induced a small, transient increase in intracellular cAMP content (+16%, P less than 0.05, after 5 min coincubation), while levels of cGMP in the same cells were rapidly and markedly low for 2 h. The decrease in cGMP content was accompanied by a discharge of gonadotropins lasting for 2 h, which was detectable after 5 min in the case of LH and after 10 min in the case of FSH. These results strongly suggest that cGMP might be an intracellular mediator in the process of LHRH-stimulated release of gonadotropins.     

Influence of insulin and of insulin resistance on platelet and vascular smooth muscle cell function

In this short review, we present the main results obtained in our laboratory in the last 15 years concerning the influence exerted by insulin on platelets and human vascular smooth muscle cells (VSMCs). In particular, we discuss: (i) the insulin ability to rapidly activate a constitutive nitric oxide synthase (NOS) in both cell types, with a consequent increase of the two nucleotides guanosine-3',5'-cyclic monophosphate (cGMP) and adenosine-3',5'-cyclic monophosphate (cAMP), well-known mediators of antiaggregation and vasodilation; (ii) the interplay of insulin with substances able to activate adenylate cyclase in both cell types; (iii) the impairment of the antiaggregating insulin effects in insulin-resistant subjects; (iv) the insulin-induced increase on endothelin in the VSMCs; (v) the insulin ability to slightly stimulate VSMC proliferation.     

Adenosine effects on the rat pineal gland in vitro: Cyclic adenosine monophosphate levels, N-acetyltransferase, and thyroxine type II 5''-deiodinase activities and melatonin production

The presence of adenosine and its receptors in the pineal gland suggests that they could have a regulatory influence on pineal physiology. Rat pineal glands cultured in the presence of the adenosine analog 5'-N-ethylcarboxamido adenosine (NECA) promoted a significant increase in pineal adenosine 3'5'-monophosphate (cAMP) content, similar to that caused by the beta-adrenergic receptor agonist isoproterenol (ISO). A potentiation of the cAMP response occurred when the glands were simultaneously stimulated with both NECA and ISO. Whereas high cAMP levels induced by ISO were associated with an increased N-acetyltransferase (NAT) activity and rise in the melatonin production and release, the NECA-induced rise in cAMP concentration failed to promote an increase in the activity of either NAT or another pineal enzyme thyroxine 5'-deiodinase type II; likewise, pineal melatonin levels did not rise.     

Calcium-dependent regulation of guanosine 3',5'-monophosphate in renal cortex: effects of ionophore A23187 and tetracaine and evidence for independent control of adenosine 3',5'-monophosphate.

The effects of carbamylcholine (Cch), the divalent cation ionophore A23187 and Ca2+ on the cyclic 3',5'-guanosine monophosphate (cGMP) and cyclic 3',5'-adenosine monophosphate (cAMP) content of rat renal cortical slices were examined. In both the presence and absence of 10 mM theophylline, Cch detectably increased cGMP within 15 sec, with peak responses noted by 2 min. The maximal cGMP response to Cch alone (0.05 mM) was an increase of two- to three-fold over control. Theophylline, which was routinely present in the incubations and which alone increased cGMP of the slices two-fold over basal during 20 min incubations, potentiated the response to Cch (maximal increase, five- to sixfold over theophylline alone). The action of Cch to increase renal cortical cGMP was blocked by prior addition of atropine and was dependent upon the presence of Ca2+ in the incubation media. Exclusion of Ca2+ lowered basal cGMP and abolished increases mediated by Cch, while exclusion of Mg2+ was without detectable effect on cGMP. In slices incubated initially without Ca2+, reexposure to Ca2+ for 1min partially restored the cGMP response to Cch, and reexposure for 3 min completely restored this response. Since prior incubation of tissue in Ca2+-free buffer for only 2 min was sufficient to block the cGMP responses to Cch, depletion of tissue Ca2+ did not appear to be involved. A23187 also increased renal cortical cGMP fivefold in the presence of Ca2+. Its effects were not additive with those of Cch and were not additive with those of Cch and were not expressed by Mg2+ in Ca2+-free media. By contrast, tetracaine, which blocks Ca2+ transport across or binding to biologic membranes, reduced basal cGMP and inhibited the actions of Cch and A23187 to increase cGMP in cortical slices incubated with Ca2+. The action of 1 mM tetracaine to block Cch-mediated increases in cGMP was partially reversed by increasing media Ca2+ from 1.5 to 5 mM, but not by increasing media Mg2+ to 5 mM. In contrast to their effects on cGMP, Cch, A23187, Ca2+ exclusion, and tetracaine did not detectably alter basal renal cortical cAMP or cAMP responses to parathyroid hormone (PTH). Conversely, concentrations of PTH, glucagon, and isoproterenol which maximally increased renal cortical cAMP did not alter cGMP. Furthermore, prior incubation of slices with Cch did not alter their subsequent cAMP response to PTH at a time when cGMP levels were still elevated, while prior incubation with PTH did not affect the subsequent cGMP response to Cch at a time when cAMP was increased. These studies demonstrate modulation of renal cortical cGMP by cholinergic stimuli and Ca2+. They also indicate that cGMP and cAMP in renal cortex can be regulated independently.     

Prostaglandin F2 alpha-induced functional luteolysis: interactions of LH, prostaglandin F2 alpha and forskolin in cyclic AMP and progesterone synthesis in isolated rat luteal cells

The acute antigonadotrophic action of prostaglandin F2 alpha (PGF2 alpha) was examined in dispersed luteal cell preparations from immature superluteinized rat ovaries. Cell suspensions prepared by collagenase digestion and purification over a Percoll density gradient were incubated for 1 h in Eagle's minimum essential medium in the presence and/or absence of LH, PGF2 alpha, N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate (dbcAMP) and forskolin. Medium was assayed for total progesterone and adenosine 3',5'-cyclic monophosphate (cAMP). Luteal cell preparations showed typical steroidogenic (progesterone) responses to LH, mimicked by both dbcAMP and forskolin. Whilst the threshold LH dose to increase cAMP synthesis was greater than that for progesterone (100 micrograms/l compared with 1 microgram/l), 24 mumol forskolin/l was the threshold dose for both cAMP and progesterone responses. Furthermore, combined doses of LH and forskolin synergistically raised cAMP yet produced less than additive increases in progesterone. Similarly, combinations of dbcAMP plus forskolin produced less than additive progesterone increases. These data suggest that forskolin may not act as a simple mimic of LH. Prostaglandin F2 alpha dose-dependently inhibited forskolin-induced cAMP and progesterone synthesis and also inhibited progesterone synthesis induced by dbcAMP. These data suggest that the antigonadotrophic effect of PGF2 alpha has more than one locus of action, i.e. it both inhibits an adenylate cyclase event associated with cAMP generation and blunts the cellular response to cAMP. The present uncertainty over the exact locus of forskolin's action within the adenylate cyclase complex limits further delineation of the inhibitory action of PGF2 alpha on LH-responsive adenylate cyclase.     

Role of Calcium and Adenosine-3′:5′-Cyclic Monophosphate in Controlling Fly Salivary Gland Secretion

The action of 5-hydroxytryptamine (5-HT) on an insect salivary gland was associated with a rise in adenosine-3':5'-cyclic monophosphate (cAMP) concentration and an increase in calcium uptake. An increase in secretion induced either by 5-HT or exogenous cAMP required extracellular calcium. Both 5-HT and exogenous cAMP increased (45)Ca efflux from previously labeled glands, but only 5-HT caused an increase in calcium uptake. The transepithelial potential in this tissue became more negative after addition of 5-HT, but more positive after addition of cAMP. 5-HT and cAMP induced a more negative potential when calcium was removed from the medium. It was concluded that both calcium and cAMP serve as intracellular messengers when 5-HT acts upon fly salivary gland.