The Effect of Lithium on Unstimulated and Glucagon-stimulated Urinary Cyclic AMP Excretion in Rat and Man

Abstract The purpose of this study was to determine whether lithium also inhibits hormone-stimulated adenylate cyclase in vivo. The effect of long-term lithium treatment on unstimulated and glucagon-stimulated cyclic AMP excretion was studied in the rat and in man. The influence of lithium on plasma glucagon degradation was also investigated. It was found that in the rat lithium doubled unstimulated and glucagon-stimulated urinary cyclic AMP excretion. In lithium treated rats plasma glucagon concentrations thirty minutes after intraperitoneal injection were twice that of the control rats. In man, lithium affected neither cyclic AMP excretion nor glucagon degradation. These results offer no support for the hypothesis that in vivo lithium in general inhibits hormone-stimulated adenylate cyclase. However, in the intact organism lithium may have additional pharmacological actions, and complex regulatory mechanisms which may modify the cyclic AMP metabolism. Therefore it may be premature to conclude that lithium per se does not have an inhibitory action on glucagon-sensitive adenylate cyclase in vivo.     

Effect of ethanol on cyclic AMP levels in intact PC12 cells

Two subclones of the rat pheochromocytoma cell line, PC12, were used to compare the effects of ethanol on adenylate cyclase activity in isolated membranes with its effects on cyclic AMP accumulation in intact cells. Consistent with previous reports, ethanol increased basal and 2-chloroadenosine-stimulated adenylate cyclase activity in isolated membrane preparations from both subclones. However, ethanol had opposite effects on agonist-stimulated cyclic AMP accumulation in intact cells of the two subclones, enhancing accumulation in one subclone, and inhibiting it in the other. The inhibition of cyclic AMP accumulation did not result from stimulation of phosphodiesterase activity, activation of the inhibitory guanyl nucleotide regulatory protein, Gi, or stimulation of protein kinase C. The results indicate that extrapolation of the effects of ethanol from one cell type to another, or from in vitro to in vivo systems, may be complicated by the interaction of ethanol with regulatory processes that influence second messenger systems, and can differ in various types of intact cells.     

Forskolin as an activator of cyclic AMP accumulation and lipolysis in rat adipocytes

Forskolin increased cyclic AMP accumulation in isolated adipocytes and markedly potentiated the elevation of cyclic AMP due to isoproterenol. In adipocyte membranes, forskolin stimulated adenylate cyclase activity at concentrations of 0.1 microM or greater. Forskolin did not affect the EC50 for activation of adenylate cyclase but did increase the maximal effect of isoproterenol. Neither the soluble nor particulate low-Km cyclic AMP phosphodiesterase activity was affected by forskolin. Low concentrations of forskolin (0.1-1.0 microM), which significantly elevated cyclic AMP levels, did not increase lipolysis, whereas similar increases in cyclic AMP levels due to isoproterenol elevated lipolysis. Forskolin did not inhibit the activation of triacylglycerol lipase by cyclic AMP-dependent protein kinase or the subsequent hydrolysis of triacylglycerol. Higher concentrations of forskolin (10-100 microM) did increase lipolysis. Both the increased cyclic AMP production and lipolysis due to forskolin were inhibited by the antilipolytic agents insulin and N6-(phenylisopropyl)adenosine. Hypothyroidism reduced the ability of forskolin to stimulate cyclic AMP production and lipolysis. These results indicate that forskolin increases cyclic AMP production in adipocytes through an activation of adenylate cyclase. Lipolysis is activated by forskolin but at higher concentrations of total cyclic AMP than for catecholamines.     

Effects of ethanol on the cyclic AMP system of the dog gastric mucosa.

The effect of ethanol on the cyclic AMP system of the dog fundic mucosa was studied in vitro. The gastric mucosal content of cyclic AMP was increased by 2.5% ethanol, whereas 10 and 20% ethanol decreased the mucosal content of cyclic AMP. The activity of adenylate cyclase was increased by 2.5 and 5% ethanol, whereas 10% ethanol did not significantly affect it. The activity of cyclic AMP phosphodiesterase was inhibited by ethanol in a competitive manner. The increase in the gastric mucosal content of cyclic AMP, induced by low concentrations of ethanol, is apparently due to the stimulation of adenylate cyclase and inhibition of phosphodiesterase. Changes in the phosphodiesterase or adenylate cyclase activites do not explain the decrease of the mucosal content of cyclic AMP by higher concentrations of ethanol. The mechanism of the decrease is discussed.     

Involvement of a plasma membrane phosphodiesterase in the negative control of cyclic AMP levels by vasopressin in rat hepatocytes

Vasopressin has been shown previously to lower the glucagon-induced increase of cyclic AMP levels in isolated rat hepatocytes by way of an enhanced phosphodiesterase (EC 3.1.4.17) activity. Five phosphodiesterase inhibitors were tested for their ability to prevent vasopressin from lowering cyclic AMP levels in intact hepatocytes and for their inhibitory effect in vitro on soluble and particulate phosphodiesterase activities partially purified from hepatocytes. Three soluble activities have been separated by DEAE-cellulose chromatography: a phosphodiesterase hydrolyzing both cyclic AMP and cyclic GMP, a form stimulated by cyclic GMP and a cyclic AMP-specific activity. The absence of any statistically significant correlation between the in vivo (in intact cells) and the in vitro (on isolated phosphodiesterases) potencies of the inhibitors does not support a role for the cytosolic phosphodiesterases in mediating the vasopressin-induced decrease in cyclic AMP levels. No statistically significant correlation was observed between the inhibition of the vasopressin effect on cyclic AMP accumulation and the inhibition of phosphodiesterase activity either associated with the native plasma membranes or solubilized from these membranes with 0.4 M NaCl. In contrast, a statistically significant correlation was observed between the degree of inhibition of the vasopressin effect in the intact cells and the degree of inhibition of the intrinsic phosphodiesterase still associated with the plasma membranes after high-salt treatment. These data indicate that a phosphodiesterase activity integral to the plasma membrane is very likely involved in the negative control of cyclic AMP levels by vasopressin.     

Role of cyclic AMP in cardiac beta-adrenoceptor desensitization: studies using prenalterol and inhibitors of phosphodiesterase

We examined the effects of prolonged exposure of cardiac cells in primary culture to the partial beta-adrenoceptor agonist prenalterol and inhibitors of phosphodiesterase on their subsequent ability to increase intracellular cyclic AMP during a 5-min exposure to 50 microM isoprenaline (receptor responsiveness). Although prenalterol possesses only 7% of the agonist activity of isoprenaline on adenylate cyclase, it induces extensive beta-adrenoceptor desensitization. Three hours after exposing the cells to 1 microM prenalterol, beta-adrenoceptor responsiveness was reduced by 40% (p less than 0.05), whereas after 12 h the reduction averaged 55%. Prolonging the incubation time to 48 h had no further effect on the magnitude of receptor desensitization. The magnitude of the desensitization was concentration dependent. On exposure of cells to 10(-8) M prenalterol for 16 h, receptor responsiveness was reduced by 19%, and at concentrations of 1 microM and higher responsiveness was reduced by 60% (p less than 0.01). Receptor desensitization appeared to be due to an inability of receptors to activate adenylate cyclase as well as to receptor loss. To investigate if beta-adrenoceptor desensitization as well as receptor loss could be mediated by cyclic AMP, the cells were exposed for 16 h to inhibitors of phosphodiesterase. Exposure of cells to the phosphodiesterase inhibitor isobutylmethylxanthine (0.1 mM) (which increased intracellular cyclic AMP by between 50 and 150%) also induced receptor desensitization. The reduction in receptor responsiveness averaged 62% (p less than 0.01). The loss in responsiveness could be accounted for by an inability of receptors to activate adenylate cyclase as well as by receptor loss.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship between prostacyclin biosynthesis and cyclic AMP in cultured rabbit mesothelial cells

Calcium ionophore A23187 (10 microM) as well as thrombin (10 U/ml) stimulated the biosynthesis of prostacyclin in cultured rabbit mesothelial cells; in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (Mix, 1 mM) the cyclic AMP (cAMP) content was also elevated. Both effects were inhibited by indomethacin (28 microM). Exogenous prostacyclin elicited by itself a clear enhancement of intracellular cAMP. An increased cAMP content was also obtained with isoproterenol (10 microM), whose activity was antagonized by propranolol (10 microM). These two products however, had no effect on the prostacyclin release. In all these experiments, inhibition of phosphodiesterase with Mix, was necessary to obtain detectable cAMP levels. In the presence of Mix, the stimulation of prostacyclin production by A23187 and thrombin was significantly lower as compared to the stimulation in the absence of Mix. Our results suggest that increased prostacyclin biosynthesis results in adenylate cyclase stimulation. This rise in intracellular cAMP in the presence of Mix, is accompanied by a downward regulation of further prostacyclin production.     

Effect of ethanol on the cyclic AMP system in rat brain.

The effects of ethanol on adenylate cyclase and phosphodiesterase activity in vitro, and on cyclic AMP, ATP and adenosine levels in vivo were studied. Ethanol appeared to affect the cyclic AMP system indirectly, probably through its effect on neurotransmitter release or on adenosine formation.     

Increases in rat liver cyclic AMP and glycogen phosphorylase activity caused by the herbicide atrazine.

The in vivo effects of the triazine herbicide atrazine on rat liver glycogen metabolism was investigated. After administration of atrazine, liver c-AMP was elevated up to threefold, the activity of glycogen phosphorylase increased, the content of glycogen in the liver decreased and the level of blood glucose increased. Cyanuric acid, although not a herbicide but a simple triazine, did alter neither liver c-AMP nor phosphorylase activity. In vitro adenylate cyclase was not activated or inhibited by triazine. However, phosphodiesterase was inhibited non-competitively by the herbicide (0.1–0.4 mM), resembling theophylline in this respect. When atrazine was added to isolated hepatocytes the effect of glucagon-stimulated c-AMP accumulation was potentiated. Thus, atrazine can inhibit phophodiesterase in vitro and in the intact cell. This phophodiesterase inhibition may also occur in vivo and this may lead to the observed effects on glycogen metabolism. However, additional hormonal and/or nervous alterations caused in vivo by the herbicide can not be excluded.No changes in the activities of adenylate cyclase and of phosphodiesterase could be detected in response to in vivo administration of atrazine. However, these negative results may be the consequence of dissociation of the enzyme-atrazine complex during preparation of the liver fractions.     

Protein kinase C activators sensitize cyclic AMP accumulation by intact 1321N1 human astrocytoma cells.

Pretreatment of 1321N1 human astrocytoma cells with phorbol 12-myristate-13-acetate or other activators of protein kinase C led to 2.5- to 5-fold increases (sensitization) in subsequent stimulation by forskolin of intracellular cyclic AMP accumulation. These compounds caused much smaller or no increases in receptor-mediated stimulation of cyclic AMP accumulation induced by isoproterenol and by prostaglandin E1. Carbachol and histamine, agonists acting at receptors coupled to polyphosphoinositide turnover in these cells, induced less sensitization of subsequent stimulation by forskolin but greater sensitization of stimulation by isoproterenol and by prostaglandin E1. The specificities of various analogs of phorbol 12-myristate-13-acetate, for induction of sensitization of forskolin stimulation were consistent with involvement of protein kinase C. The effects of protein kinase inhibitors and of down-regulation of protein kinase C activity also indicated involvement of protein kinase C in sensitization of forskolin stimulation, although additional mechanisms are likely to be involved in sensitization of isoproterenol stimulation. Neither pertussis toxin pretreatment nor inclusion of isobutylmethylxanthine during assays of cyclic AMP accumulation were able to prevent or mimic these sensitization phenomena, suggesting that the primary site of modification responsible for sensitization is neither the inhibitory guanine nucleotide-binding protein nor cyclic AMP phosphodiesterase. Sensitization was only observed in assays with intact cells. These results, together with those from our previous study describing protein kinase C-mediated desensitization of broken cell adenylate cyclase activity, indicate that activation of protein kinase C leads to multiple changes in the receptor-stimulated adenylate cyclase signal transduction pathway of these cells.     

Cardiac adenylate cyclase, cyclic nucleotide phosphodiesterase and lactate dehydrogenase in normotensive and spontaneously hypertensive rats.

To understand altered physiological responses of hypertrophied spontaneously hypertensive rat (SHR) myocardium to beta-adrenergic receptor stimulation in vivo, myocardial tissues from SHR, normotensive Wistar (NR) and normotensive Wistar-Kyoto (WKY) rats were analyzed for adenylate cyclase, phosphodiesterase and lactic dehydrogenase. While WKY left ventricular adenylate cyclase activity exceeded that of SHR at low (1 and 5 μM) norepinephrine concentrations, there were no further differences. Norepinephrine stimulation of NR and SHR left ventricular adenylate cyclase was the same. In all rat strains similar responses of adenylate cyclase to glucagon were observed. Cyclic AMP phosphodiesterase activity of whole left ventricular homogenates were not significantly different in NR, WKY or SHR when assayed at either 1 mM or μM cyclic AMP. While it was not possible to entirely explain previous hemodynamic findings in vivo on the basis of abnormal cAMP-related enzyme activities, certain other interstrain enzymatic differences were observed. The hypertrophied SHR left ventricle contained higher levels of lactate dehydrogenase (LDH) and an altered isozymic composition as compared to both normotensive strains. These changes may indicate a shift in glycolytic enzyme needs as also seen with artificially produced cardiac hypertrophy. In all rat strains the right ventricular wall has less norepinephrine-stimulatable adenylate cyclase activity and more cyclic AMP phosphodiesterase activity than the left ventricle. These results demonstrate differences in cyclic AMP-related activities between the ventricles and increased LDH activity in the hypertrophied SHR left ventricle.     

Molecular basis of the synergistic inhibition of platelet function by nitrovasodilators and activators of adenylate cyclase: inhibition of cyclic AMP breakdown by cyclic GMP

We investigated the roles of cyclic GMP and cyclic AMP in the inhibition of rabbit platelet aggregation and degranulation by two nitrovasodilators, sodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1; the active metabolite of molsidomine), with particular reference to the synergistic interaction of these drugs with prostaglandin E1 (PGE1). Changes in platelet cyclic [3H]GMP and cyclic [3H]AMP were measured by rapid and sensitive prelabeling techniques, the validity of which were confirmed by radioimmunoassays. Incubation of the platelets with 0.1 to 10 microM SNP alone for 0.5 min caused progressively greater inhibitions of platelet function associated with large dose-dependent increases in cyclic [3H]GMP and 1.4- to 3.0-fold increases in cyclic [3H]AMP. However, addition of SNP with the adenylate cyclase activator, PGE1, at a concentration of the latter that had little effect alone, caused much larger increases in cyclic [3H]AMP and greatly enhanced the inhibition of platelet aggregation. SIN-1 had effects similar to those of SNP, although it was less active. The adenylate cyclase inhibitor 2',5'-dideoxyadenosine (DDA) diminished the increases in cyclic [3H]AMP caused by SNP or SIN-1 in both the presence and absence of PGE1 but reduced the inhibition of platelet function caused by the nitrovasodilators only in the presence of PGE1. These results suggest that, although cyclic GMP may mediate the inhibition of rabbit platelet function by high concentrations of nitrovasodilators added alone, the synergistic interaction of lower concentrations with PGE1 depends on an enhanced accumulation of cyclic AMP. Synergistic effects on cyclic [3H]AMP accumulation were also observed on incubation of platelets with SNP and adenosine, another activator of adenylate cyclase. Hemoglobin, which binds nitric oxide, blocked or reversed the increases in both cyclic [3H]GMP and cyclic [3H]AMP in platelets caused by the nitrovasodilators added either alone or with PGE1. Cilostamide, a selective inhibitor of platelet low Km cyclic AMP phosphodiesterase, had effects on platelet cyclic [3H]AMP accumulation identical to those of SNP, suggesting that the action of the latter depends on inhibition of the same enzyme. M&B 22,948, a selective inhibitor of cyclic GMP phosphodiesterase, potentiated the increases in both cyclic [3H]GMP and cyclic [3H]AMP caused by SNP. A hyperbolic relationship was found between the increases in cyclic [3H]GMP and cyclic [3H]AMP caused by different concentrations of SNP; this relationship was not affected by addition of M&B 22,948. The results strongly suggest that the increases in platelet cyclic [3H]AMP caused by nitrovasodilators in the presence or absence of activators of adenylate cyclase are mediated by the inhibition by cyclic GMP of cyclic AMP breakdown.(ABSTRACT TRUNCATED AT 400 WORDS)     

The absence of stimulation of lipolysis by papaverine, a strong inhibitor of phosphodiesterase

Quantitative studies of the action of theophylline and papaverine were performed in rat epididymal fat pads, both on the lipolytic effect and on the activity of phosphodiesterase, adenylate cyclase and protein kinase. Papaverine, a stronger inhibitor of phosphodiesterase than theophylline, did not produce lipolysis. The maximum lipolytic effect (glycerol release) of theophylline was much higher than that of epinephrine and nearly approached the effect exerted by dibutyryl cyclic AMP. While theophylline potentiated or was without any effect on lipolysis produced by epinephrine and dibutyryl cyclic AMP, papaverine at concentration 10⁻³M reduced the effect of both drugs as well as of theophylline by 90%. These concentrations of papaverine also strongly inhibited the activity of adenylate cyclase. Neither papaverine nor theophylline prevented the activation of protein kinase by cyclic AMP.

The data suggest that the lack of a lipolytic effect of papaverine might be caused by a combination of its inhibitory effect on adenylate cyclase and direct inhibition of activation of triglyceride lipase     

On the possible role of biliverdin stimulation of cyclic AMP levels as a trigger for liver regeneration in the rat

Contrary to a recent report by Okazaki et al. (Biochem. biophys. Res. Commun., 1978, 81, 512-520) we show that high concentrations of biliverdin (100 mg/kg, i.p.) slightly reduce the mitotic rate in rat liver. Adenylate cyclase and both the "high Km" and "low Km" phosphodiesterase of rat liver plasma membranes are inhibited by high concentrations of biliverdin. Biliverdin has no effect on cyclic AMP production in isolated hepatocytes. An intracellular binding protein (glutathione-S-transferase B) is shown to bind biliverdin providing a mechanism for maintaining a low free intracellular concentration of the tetrapyrrole analogous to that of albumin in plasma. In conclusion, these results are not consistent with a role for biliverdin in stimulating liver regeneration in the rat via a mechanism involving elevated cyclic AMP levels.     

Morphine-induced changes in cyclic AMP metabolism and protein kinase activity in the brain.

The effects of consecutive oral administration of morphine on the cyclic AMP synthesizing system and cyclic AMP dependent protein kinase activity in the cerebral cortex of mice were examined. The administration of morphine (2--4 weeks) induced an increase of the cyclic AMP formation by activating adenylate cyclase, whereas responses of the cyclic AMP synthesizing system to biogenic amines (norepinephrine, dopamine and histamine) added in vitro was found to be significantly attenuated in these animals. Cyclic AMP dependent protein kinase activity in the cerebral cortex was also increased following a consecutive oral administration of morphine. These changes in the activities of adenylate cyclase and protein kinase were found mainly in crude mitochondrial and/or synaptosomal fractions. Morphine induced decrease in the response of the cyclic AMP synthesizing system to biogenic amines was rapidly reversed, and a significant increase of the cyclic AMP formation in the presence of added norepinephrine compared with that found in morphinized animals was observed following the administration of levallorphan, a narcotic antagonist. On the other hand, the changes in adenylate cyclase and cyclic AMP dependent protein kinase activities were not affected significantly by levallorphan administration. These results suggest that alterations in activities of cyclic AMP synthesizing system and of cyclic AMP dependent protein kinase may be involved in processes of the formation of morphine dependence. Possible involvement of abrupt increments in the sensitivity of "norepinephrine receptor-adenylate cyclase" system and a subsequent increase in cerebral cyclic AMP is also suggested as a cause of morphine withdrawal syndrome.     

Cyclic AMP and Ca-binding in microsomal fractions isolated from rabbit colon smooth muscle.

From a homogenate of rabbit colon muscle two ATP dependent Ca-accumulating microsomal fractions were isolated by differential centrifugation on a sucrose density grandient at 35% and 35-45% sucrose. Adenylate cyclase and phosphodiesterase activities were found in the fractions. The Ca-accumulation and the ATPase activity of these fractions were stimulated by cyclic AMP (10(-5)M) at an ATP concentration of 0.35 mM ATP. In the presence of higher concentrations of ATP (5 mM) cyclic AMP had no effect on the Ca-binding. The higher concentration of ATP markedly increased the cyclic AMP formation in relation to the activity found at the lower concentration of ATP. Isoprenaline (2 X 10(-6)M) stimulated the Ca-accumulation in the 35-45% fraction and increased the hydrolysis of ATP. These effects were absent in the fraction isolated at 35% sucrose. In the former fraction isoprenaline also stimulated the adenylate cyclase activity at 0.35 mM but not at 5 mM ATP. Both the effect of isoprenaline on the Ca-binding and the adenylate cyclase activity were inhibited by the adrenergic beta-receptor blocking agent sotalol. In the 35-45% fraction papaverine (1 X 10(-3)M) stimulated the Ca-accumulation and inhibited the phosphodiesterase activity. It is suggested that cyclic AMP and agents which influence the cyclic AMP metabolism in the microsomes may have a regulatory role on the Ca-binding of the microsomes.     

Forskolin and ethanol both perturb the structure of liver plasma membranes and activate adenylate cyclase activity

Both forskolin and ethanol elicit the activation of basal and ligand-stimulated adenylate cyclase activities in rat liver plasma membranes. Ethanol is most potent at activating the fluoride- and glucagon-stimulated activities whilst having little effect on basal activity. In contrast forskolin exerts its greatest effect on basal activity. Over the concentration range that ethanol activates adenylate cyclase, it also increases bilayer fluidity as indicated by a decrease in the values of the order parameters for an incorporated fatty acid spin probe. At high concentrations forskolin does increase bilayer fluidity. However, it only begins to do so at concentrations above those where forskolin has already exerted its maximal effect in activating adenylate cyclase. Forskolin can still activate, albeit to a reduced extent, detergent-solubilized adenylate cyclase whereas ethanol cannot. Forskolin elicits a pronounced rise in hepatocyte intracellular cyclic AMP concentrations, whereas ethanol does not. Both forskolin and ethanol reduce the temperature of onset of the lipid phase separation occurring in rat liver plasma membranes. This is detected in Arrhenius plots of both glucagon-stimulated adenylate cyclase activity and order parameters of an incorporated fatty acid spin probe, where we find that forskolin is particularly potent in decreasing the temperature at which this lipid phase separation occurs. Our results are consistent with the notion that forskolin exerts its effect on adenylate cyclase primarily by a direct action on the catalytic unit of the enzyme. However, as forskolin is a potent perturber of the organisation of the lipid bilayer it is possible that this could modulate its effect on adenylate cyclase and might be expected to affect the activity of other membrane enzymes.     

Further studies of the action of cyclic AMP on the electrical and mechanical activities of intestinal smooth muscle.

Effects of externally applied cyclic AMP and other adrenergic stimulants on the electrical and mechanical activities of the cat small intestine were observed by using pressure electrodes. The electrical and mechanical activities were suppressed by cyclic AMP and beta-stimulants. Those inhibitory actions of cyclic AMP and beta-stimulants were potentiated under the treatment with caffeine, theophylline and papaverine which inhibits the phosphodiesterase activity. On the other hand, the inhibitory action of cyclic AMP and beta-stimulants was decreased in imidazole, an agent that increases phosphodiesterase activity. Exogenous applied concanavalin A, an agent that inhibits the adenyl cyclase activity, showed no observable changes in both activities but the effects of beta-stimulants were decreased after treatment with concanavalin A. No obvious changes on both activities were obtained in cyclic GMP and dibutyryl cyclic GMP. These findings tentatively support the hypothesis that cyclic AMP is a second messenger in the inhibitory responses to beta-stimulants on the intestinal smooth muscle. However, it is also concluded that the inhibition of mechanical activity caused by cyclic AMP is partially due to suppression of the membrane activity.     

Effect of chlorpropamide and phenformin on rat liver: the effect on plasma membrane-bound enzymes and cyclic AMP content of hepatocytes in vitro.

Chlorpropamide and phenformin inhibited (Na+ - K+)-ATPase and stimulated a high affinity cyclic AMP-phosphodiesterase of isolated liver plasma membrane when tested in vitro. In addition, the two drugs decreased the intracellular cyclic AMP content of isolated hepatocytes without being effective on plasma membrane-bound adenylate cyclase. The results suggest that the plasma membrane plays an important role in the mechanism of action of the two hypoglycemic drugs, but do not exclude the presence of intracellular targets.