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.     

3′:5′-Cyclic AMP-dependent 3′:5′-cyclic GMP accumulation in Dictyostelium discoideum

Suspensions of 3':5'-cyclic AMP (cAMP)-sensitive cells of Dictyostelium discoideum responded to a cAMP pulse with increased 3':5'-cyclic GMP (cGMP) levels. Under the assay conditions used (2 x 10(8) cells per ml in 10 mM phosphate buffer, pH 6.0) cAMP (5 x 10(-8) M final concentration) increased cGMP levels from 1 pmol per 10(7) cells to 7 pmol per 10(7) cells in 10 sec and basal levels were recovered in 20-25 sec. cGMP accumulation did not occur when cells were in the cAMP-insensitive stage. cAMP-sensitive cells responded with increased cGMP levels when triggered by 5 x 10(-8) M 5'-CH(2)-cAMP or 10(-5) M adenosine-5'-methylmonophosphate (5'-AMPMe) but not after addition of 5 x 10(-8) M 3':5'-cyclic IMP (cIMP) or 5 x 10(-8) M 5'-AMP. As agonists of cAMP, 5'-CH(2)-cAMP and 5'-AMPMe have, respectively, more than 10% and 1% the chemotactic activity of cAMP, while cIMP has 0.01% the activity of cAMP and 5'-AMP is inactive up to a concentration of 10(-3) M. cAMP-mediated cGMP formation was dependent upon cAMP concentration, with a half-maximal cAMP concentration of about 10(-8) M. This cAMP concentration agrees closely with that necessary for half-maximal receptor occupation. cAMP-mediated cGMP formation was independent of the presence of extracellular Ca(2+); cell aggregation and chemotaxis were also independent of the presence of external Ca(2+). Therefore, cAMP action does not depend on stimulation of the Ca(2+) influx. cAMP was found to mediate desensitization of cAMP-dependent cGMP formation. Addition of 5 x 10(-8) M cAMP to sensitive cells induced a desensitization period that lasted 1-5 min. Desensitization was dependent on the cAMP concentration. Finally, we propose that the translation of a chemotactic signal from the cell surface to pseudopod formation in Dictyostelium involves changes in the levels of cGMP.     

Catecholamine-stimulated cyclic GMP accumulation in the rat pineal: apparent presynaptic site of action.

Guanosine 3':5'-cyclic monophosphate (cGMP) increased 7-fold in rat pineal glands incubated in the presence of l-norepinephrine. This response consisted of two components-one was stereospecific and inhibited by alpha-adrenergic antagonists while the other was not stereospecific and not readily inhibited by antagonists. Although l-isoproterenol was more potent than l-norepinephrine it had less intrinsic activity and its action was not stereospecifc. The increase in cGMP caused by these catecholamines, unlike that of adenosine 3':5'-cyclic monophosphate (cAMP), was dependent upon extracellular calcium. Ouabain and high levels of potassium produced a marked, calcium-dependent increase in pineal cGMP, without affecting cAMP. There was no effect of cholinergic agonists on cGMP. Surgical denervation markedly reduced the cGMP response to stimulation by l-norepinephrine, potassium, or ouabain. This was in contrast to the enhanced response of cAMP in denervated glands. The nonspecific increase in cGMP caused by l-isoproterenol, however, was not affected by denervation. These data demonstrate the existence of a calcium-dependent presynaptic mechanism for the generation of cGMP which may be mediated by an alpha-adrenergic-like receptor. In addition, the mechanisms regulating pineal cGMP appear to be physiologically distinct from those regulating cAMP.     

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.     

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.     

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.     

Mode of action of erythropoietin (Epo) in an Epo-dependent murine cell line. I. Involvement of adenosine 3',5'-cyclic monophosphate not as a second messenger but as a regulator of cell growth

A subclone of the interleukin 3 (IL-3)-dependent murine cell line DA-1 was recently established, and its growth found to be dependent on erythropoietin (Epo) as well as on IL-3. This subclone, named DA-1ER, has been used to study the mechanisms of action of these two growth factors, though most especially of Epo. In the present study, the involvement of adenosine 3',5'-cyclic monophosphate (cAMP) in the growth of DA-1ER cells was analyzed. In initial experiments, cAMP levels in cells stimulated with either recombinant Epo or recombinant IL-3 were monitored. The results showed that no significant changes in cellular cAMP levels had occurred. The cAMP-enhancing agents, N6-2'-o-dibutyryl-adenosine 3',5'-cyclic monophosphate (dbcAMP), 3-isobutyl-1-methyl-xanthine (IBMX), and cholera toxin (CT) were then applied to DA-1ER cells to see whether they could induce cell growth, but none of them was effective. The data indicated that cAMP was not the so-called second messenger for Epo or IL-3. The regulatory effects of cAMP on Epo- and IL-3-stimulated cell growth were next examined. It was found that whereas Epo-stimulated growth was markedly inhibited by cAMP-enhancing agents, IL-3-stimulated growth was relatively resistant and inhibited only by high doses of these agents. These data suggested that cAMP could play different roles in the same cells, depending on which growth factors were applied, and that it finely regulated proliferation and differentiation of multireceptor-bearing hematopoietic precursor cells.     

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.     

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.     

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.     

Hydroxyurea nitrosylates and activates soluble guanylyl cyclase in human erythroid cells

Hydroxyurea, a drug widely used for treating myeloproliferative diseases, has also been approved for the treatment of sickle cell disease by raising fetal hemoglobin (HbF). We have shown that nitric oxide (NO) and the soluble guanylyl cyclase (sGC) pathways are involved in hydroxyurea induction of HbF levels in erythroid progenitor cells (EPCs). We demonstrate now that during erythroid differentiation, endothelial NO synthase mRNA and protein levels decline steadily, as does the production of NO derivatives and cyclic adenosine monophosphate (cAMP) levels, but guanosine 3',5'-cyclic monophosphate (cGMP) levels are stable. Hydroxyurea increased intracellular cGMP levels and cAMP levels in EPCs. The NO donor, DEANONOate, induced much higher cGMP levels, but reduced cAMP levels. Hydroxyurea (1 mM) induced production of approximately 45 pM cGMP/minute/ng of purified sGC, similar to induction by 1 muM DEANONOate. We found that hydroxyurea and ProliNONOate produced iron-nitrosyl derivatives of sGC. Thus, we confirm that hydroxyurea can directly interact with the deoxy-heme of sGC, presumably by a free-radical nitroxide pathway, and activate cGMP production. These data add to an expanding appreciation of the role of hydroxyurea as an inducer of the NO/cGMP pathway in EPCs. These mechanisms may also be involved in the cytostatic effects of hydroxyurea, as well as the induction of HbF.     

Vasoactive intestinal peptide: A potent stimulator of adenosine 3′:5′-cyclic monophosphate accumulation in gut carcinoma cell lines in culture

Vasoactive intestinal peptide (VIP) is a potent and efficient stimulator of adenosine 3':5'-cyclic monophosphate (cAMP) accumulation in a human colon carcinoma cell line, HT 29. cAMP accumulation is sensitive to a concentration of VIP as low as 3x10(-12) M. Maximum VIP-induced cAMP levels were observed with 10(-9) M VIP and are about 200 times above the basal levels. Half-maximum cAMP production was obtained at 3x10(-10) M VIP. (125)I-Labeled VIP was found to bind to HT 29 cells; this binding was competitively inhibited by concentrations of unlabeled VIP between 10(-10) and 10(-7) M. Half-maximum inhibition of binding was observed with 2x10(-9) M VIP. Secretin also stimulated cAMP accumulation in HT 29 cells, but its effectiveness was 1/1000 that of VIP. The other peptides tested at 10(-7) M, such as insulin, glucagon, bovine pancreatic polypeptide, somatostatin, octapeptide of cholecystokinin, neurotensin, and substance P, did not stimulate cAMP accumulation. Prostaglandin E(1) and catecholamines stimulated cAMP production but were 1/2.3 and 1/5.5 as efficient as VIP, respectively. Another malignant cell line from the gut, the human rectal tumor cell line HRT 18, is also sensitive to VIP. In HRT 18 cells, VIP stimulated cAMP accumulation with a maximal effect at 10(-8) M; half-maximum stimulation was observed at about 10(-9) M. These results demonstrate the presence of VIP receptors in two malignant human intestinal cell lines (HT 29 and HRT 18) in culture and provide a model for studying the action of VIP on cell proliferation.     

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.     

Cyclic nucleotide metabolism in compensatory renal hypertrophy and neonatal kidney growth.

Cyclic nucleotide metabolism was investigated in growing kidneys of rats during compensatory hypertrophy and during neonatal development. After unilateral nephrectomy a mild and short-lasting decrease in cyclic 3':5" adenosine monophosphate (cAMP) was observed in the hypertrophying kidney. In contrast, cyclic 3':5' guanosine monophosphate (cGMP) showed a sharp decline to 20% of control at 15 min and a rapid rise to 200-300% above base-line at 1-72 hr. The alterations in renal tissue levels of cGMP were associated with parallel changes in the soluble, 100,000 X g supernatant guanylate cyclase activity [GTP pyrophosphate-lyase (cyclizing): EC 4.6.1.2]. No change was observed in total cGMP phosphodiesterase (3':5'-cyclic-nucleotide 5'-nucleotidohydrolase; EC 3.1.4.17). In the rapidly growing kidney of newborn rats cAMP levels were 983 +/- 65 and 833 +/- 42 pmol/g of kidney at 4 and 7 days after birth, and increased to adult levels (1518 +/- 57 pmol/g) at 21 days whereas cGMP levels were 59.8 +/- 6.8 and 92.5 +/- 13.9 pmol/g at 4 and 7 days and decreased to adult levels (36 +/- 1.5) at 21 days. The results indicate that compensatory renal hypertrophy and neonatal kidney growth are associated with changes in cAMP and cGMP metabolism.     

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.     

Comparison of cyclic AMP accumulation and morphological changes induced by beta-adrenergic stimulation of cultured vascular smooth muscle cells and fibroblasts

Cyclic 3',5'-adenosine monophosphate (cAMP) accumulation and morphological changes induced by isoproterenol (ISO) on cultured vascular smooth muscle cells (SMC) and vascular fibroblasts derived from spontaneously hypertensive rats, their stroke-prone strain and normotensive Wistar Kyoto rats were investigated. At the time points studied, ISO-induced cAMP accumulation in SMC reached a peak level at 5 min. Accumulation was dose-dependent and was maximal at a concentration of 10(-5) M ISO. Maximal cAMP levels were approximately 600-fold higher than basal levels. Maximal cAMP accumulation or half maximal stimulatory ISO concentrations were similar in SMC from the three strains. ISO had no effects on cyclic 3',5'-guanosine monophosphate (cGMP) levels in SMC. Phenylephrine had no effects on cAMP or on cGMP levels. In contrast to SMC, beta-adrenergic stimulation of vascular fibroblasts resulted in only a 4-fold increase of cAMP levels. 1.5 h after administration of ISO to SMC cultures, the morphological changes were apparent in SMC but not in fibroblasts. Morphological changes induced by ISO were reversible and morphological appearances returned to normal 16 h after exposure to ISO. 10(-3) M dibutyryl cAMP had similar effects on the morphologies of both SMC and fibroblasts. These effects were antagonized by 5 X 10(-6) M colchicine, an inhibitor of microtubule assembly. These results indicate that cultured vascular SMC possess the ability to increase markedly their cellular cAMP level in response to beta-adrenergic stimulation, while fibroblasts are less responsive to the stimulation. Furthermore, cAMP accumulation results in morphological changes of SMC and fibroblasts probably through the alteration of intracellular microtubule systems. As the morphological response to intracellular cAMP (or its lipophilic derivatives) is similar in both SMC and fibroblasts, the difference in each cell line's responsiveness to ISO may be due to a difference in: (1) the density or sensitivity of beta-adrenergic receptors on the plasma membrane of each cell type, or (2) the catalytic activity of adenylate cyclase itself.     

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.     

RIA determination and immunofluorescence localization of cyclic nucleotides in rainbow trout (Salmo gairdneri) testes

Using histological criteria, testicular development was divided into six stages (I-Va). The testicular amounts of 3',5'-cyclic adenosine- and guanosine monophosphate (cAMP, cGMP) were determined radioimmunologically, and their testicular distribution patterns were monitored by indirect immunofluorescence microscopy. During slow testicular growth (stages I-III), the nucleotide concentrations were high (0.62-1.2 pmol cAMP/mg, 0.17-0.24 pmol cGMP/mg). With the appearance of spermatozoa in stage IV, they fell to low levels which were maintained with some fluctuations until the end of the cycle (0.05-0.1 pmol cAMP/mg, 0.016-0.05 pmol cGMP/mg). The cAMP antiserum intensely stained spermatogonia, a portion of the spermatocytes, and spermatids. Spermatozoa showed almost no staining. Fluorescence labeling of somatic cells was observed in immature testes and during spermiation. Except for staining all spermatocytes, the same pattern was observed using the cGMP antiserum.     

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.     

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.