Possible role of adenosine cyclic 3':5'-monophosphate phosphodiesterase in the morphological transformation of Chinese hamster ovary cells mediated by N6,O2-dibutyryl adenosine cyclic 3':5"-monophosphate.

We have demonstrated that in Chinese hamster ovary (CHO) cells, N6,O2'-dibutyryl adenosine cyclic 3':5'-monophosphate (dibutyryl cyclic AMP) has a remarkable morphogenetic effect in converting cells of a compact, epithelial-like morphology into a spindle-shaped, fibroblast-like form. Homogenates of CHO cells were found to contain two adenosine cyclic 3':5'-monophosphate (cyclic AMP) phosphodiesterase (EC 3.1.4.c) activities, which differ in apparent Km with respect to their substrate, cyclic AMP. These were designated cyclic AMP phosphodiesterase I, with a low Km of 2 to 5 muM and cyclic AMP phosphodiesterase II, with a high Km of 1 to 3 mM. Cyclic AMP phosphodiesterase I was competitively inhibited by N6-monobutyryl and dibutyryl cyclic AMP, with apparent Ki values of 40 to 60 muM and 0.25 to 0.35 mM, respectively. Experimental evidence demonstrates that the effect of exogenous dibutyryl cyclic AMP on cell morphology is a result of an increase in the endogenous level of cyclic AMP. This increase appears to be due largely to the inhibitory action of intracellular N6-monobutyryl cyclic AMP on cyclic AMP phosphodiesterase I, which results in a decreased rate of degradation of intracellular cyclic AMP.     

Influence of glucagon and cyclic adenosine 3':5'-monophosphate on the phosphorylation of rat liver ribosomal protein S6.

The administration of glucagon, adenosine 3':5'-monophosphate, or N6,O2'-dibutyryl adenosine 3':5'-monophosphate caused an increase in the phosphorylation of rat liver ribosomes. The increase (approximately 3-fold) was in the protein of the small ribosomal subunit. The proteins were separated by two-dimensional polyacrylamide gel electrophoresis and radioautographs were made of the gels. The effect of the hormone and of the nucleotides was entirely due to an increase in the phosphorylation of the 40 S ribosomal subunit protein S6.     

Hyperkalemia, hyperglycemia, and plasma levels of cyclic AMP and cyclic GMP induced by portal vein injection of cyclic nucleotides and their butyryl derivatives into dogs

The levels of serum potassium, blood glucose, and plasma adenosine cyclic 3':5'-monophosphate (cAMP) and guanosine cyclic 3':5'-monophosphate (cGMP) were studied after the portal vein injection of cyclic nucleotides and their derivatives, (cAMP, cGMP, N6, O2'-dibutyryl adenosine 3':5'-monophosphate (DBcAMP), N6-monobutyryl adenosine cyclic 3':5'-monophosphate (NMBcAMP), and O2'-monobutyryl adenosine cyclic 3':5'-monophosphate (OMBcAMP), into dogs. Dose-related hyperglycemic responses were observed after the injection of DBcAMP (1-8 mg/kg). Transient and prominent hyperkalemia and hyperglycemia were caused by the injection of DBcAMP, NMBcAMP, and OMBcAMP (4 mg/kg). The hyperkalemic response was highest with NMBcAMP (1.22 mequiv./L), followed by OMBcAMP (0.64), DBcAMP (0.54), cGMP (0.47), and cAMP (0.41), whereas the hyperglycemic response was highest with NMBcAMP (146 mg/100 mL), followed by DBcAMP (93.6), OMBcAMP (77.1), and cAMP (56.0), and there was only a slight change with cGMP (28.4) compared with the control. The plasma level of cAMP was maximal with DBcAMP (1.92 nmol/mL), followed by NMBcAMP (1.28) and OMBcAMP (0.76), whereas the plasma levels of cGMP showed no evident change, except that caused by DBcAMP (0.27). Of the cyclic nucleotides tested, NMBcAMP was found to be most potent in causing both hyperkalemia and hyperglycemia. Based on these results, possible correlations between hyperkalemia, hyperglycemia, and plasma levels of cAMP and cGMP are discussed.     

Stimulation of active uptake of nucleosides and amino acids by cyclic adenosine 3' :5'-monophosphate in the yeast Schizosaccharomyces pombe.

In conditions of glucose starvation, the maximum velocity of the mediated transport of nonmetabolized and metabolized amino acids, uridine, adenosine, and sucrose across the plasma membrane is stimulated by a factor of two by the addition of 1 mM adenosine 3':5'-monophosphate to Schizosaccharomyces pombe 972h- wild strain, to the glucose-super-repressed and derepressed mutants COB5 and COB6, and to Saccharomyces cerevisiae strain IL 216-IA. The mediated uptake of 2-D-deoxyglucose and the apparently nonmediated uptake of guanosine are not stimulated by the cyclic nucleotide. N6,O2'-Dibutyryl adenosine 3':5'-monophosphate is also efficient, whereas theophylline, guanosine 3':5'-monophosphate, 5'-AMP, ATP, and adenosine are ineffective. The cellular ATP content of glycerol-grown S. pombe COB5 is about 10 nmol per mg of protein and is not decreased by further incubation in the starvation medium. The addition of 100 mM glucose markedly enhances transport without any increase of the cellular ATP content. The addition of antimycin A or Dio-9 decreases markedly both cellular ATP content and transport. The addition of 2.5 mM glucose to antimycin A-containing medium restores both transport is not necessarily of mitochondrial origin. The uptake of 2-D-deoxyglucose is unaffected by the respiratory inhibitors. Stimulation of uptake by cyclic adenosine 3':5'-monophosphate occurs only in glucose-deprived cells. The addition of 10 mM glucose elicits the disappearance of the stimulation and prevents the 30% decrease of the cellular adenosine 3':5'-monophosphate content produced by glucose starvation. Adenosine 3':5'-'monophosphate does not enhance the steady state ATP level but requires cellular ATP produced either by endogenous respiration or, in the absence of respiration blocked by antimycin A, by further addition of 2.5 mM glucose. Stimulation of active uptake by adenosine 3':5'-monophosphate does not require protein synthesis because the addition of cycloheximide or anisomycin does not prevent the stimulation of L-leucine uptake. In the absence of respiration, Dio-9, and ATPase inhibitor, suppresses instantaneously the cellular ejection of protons as well as the uptake of uridine and amino acids. It abolishes also the adenosine 3':5'-monophosphate-stimulated transport. In the presence of antimycin A, specific mitochondrial ATPase inhibitors such as venruricidin A do not inhibit metabolite uptakes and their stimulation by adenosine 3':5'-monophosphate. These results suggest that in these conditions, the target of Dio-9 is not the mitochondrial ATPase but a plasma membrane proton-translocating function generating an electrochemical gradient required for active transport. That adenosine 3':5'-monophosphate enhances the Dio-9-sensitive proton extrusion supports the view that the cyclic nucleotide might modulate the plasma membrane ATPase.     

Cyclic adenosine 3':5'-monophosphate and the induction of deoxyribonucleic acid synthesis in liver.

A mixture containing glucagon and thyroid hormone was previously devised that enhances markedly nuclear DNA replication and mitosis in the parenchymal liver cells of the unoperated rat. It is now shown that the glucagon of the stimulatory solution can be completely replaced by a mixture of a butyryl derivative of cyclic adenosine 3':5'-monophosphate and theophylline. Cyclic guanosine 3':5'-monophosphate and its butyryl derivatives and insulin and high levels of glucose are inactive. The inactivity of N2-monobutyryl cyclic guanosine 3':5'-monophosphate cannot be ascribed to rapid breakdown in the animal or to the impenetrability of the liver cell since the coumpound elevates the rate of hepatic amino acid transport and the activity of ornithine decarboxylase. The observation of others (MacManus, J.P., Franks, D.J., Youdale, T. & Braceland, B.M. (1972) Biochem. Biophys. Res. Commun. 49, 1201-1207) that the level of cylcic adenosine 3':5'-monophosphate is raised during most of the prereplicative period after 70% hepatectomy is confirmed. The evidence supports a positive role for adenosine 3':5-monophosphate in regulating DNA synthesis in the liver.     

A slowly dissociating form of the cell surface cyclic adenosine 3':5'-monophosphate receptor of Dictyostelium discoideum.

Experiments using a phosphodiesterase-minus mutant of Dictyostelium discoideum indicate that ligand-induced loss of cell surface cyclic adenosine 3':5'-monophosphate binding sites (down regulation) can be evoked with concentrations of cyclic adenosine 3':5'-monophosphate as low as 10(-8) M. The loss of receptor sites is observed after 5 min of cell preincubation with cyclic adenosine 3':5'-monophosphate and can be as extensive as 75 to 80%. This decrease in binding sites is correlated with the appearance of a slowly dissociating cyclic adenosine 3':5'-monophosphate binding component. Radioactive cyclic adenosine 3':5'-monophosphate bound to this form of receptor cannot be competed for by nonradioactive cyclic adenosine 3':5'-monophosphate or adenosine 5'-monophosphate and is not accessible to hydrolysis by cyclic adenosine 3':5'-monophosphate phosphodiesterase. The extent of appearance of this binding component is dependent upon the concentration of cyclic adenosine 3':5'-monophosphate used to elicit the down regulation response and the temperature of the incubation medium.     

Regulation of cyclic nucleotide phosphodiesterases in cultured hepatoma cells by dexamethasone and N6,O2'-dibutyryl adenosine 3':k'-monophosphate.

DEAE-Bio-Gel chromatography of 100,000 X g supernatant from cultured HTC hepatoma cells separated cyclic nucleotide phosphodiesterase into three forms, numbered E I, E II, and E III in order of elution from the column, E I had a low Km for cyclic guanosine 3':5'-monophosphate (cGMP) and a high Km for cyclic adenosine 3':5'-monophosphate (cAMP), E II exhibited anomalous kinetics. At low substrate concentrations (0.5 muM) cGMP was hydrolyzed more rapidly than cAMP and hydrolysis of 0.5 muM cAMP was stimulated by 1 muM cGMP. E III had a low Km for cAMP. Incubation of cells with 1 muM dexamethasone for 72 h decreased the activity of E I and E II. In cells incubated with N6,O2'-dibutyryl cAMP plus 3-isobutyl-1-methylxanthine for 14 h the activity of E III was increased approximately 100%. Similar activities of calcium-dependent, heat stable phosphodiesterase activator were recovered from supernatants from all cells. These studies have established the presence, in a homogeneous population of hepatoma cells, of at least three forms of cyclic nucleotide phosphodiesterase, the activities of which can be independently regulated.     

Induction of phosphodiesterase by cyclic adenosine 3':5'-monophosphate in differentiating Dictyostelium discoideum amoebae.

Cyclic adenosine 3':5'-monophosphate added to the starvation media of Dictyostelium discoideum amoebae induces both intracellular and extracellular phosphodiesterase activities of these cells. The induced enzyme activity appears several hours earlier than that in starved cells which have not been induced with cyclic nucleotide. In both cases, the appearance of enzyme is inhibited by cycloheximide, and actinomycin D, and daunomycin. The KmS for the extracellular enzyme(s) of nucleotide-induced and uninduced control cells are identical. The induction of enzyme activity seems specific for cyclic adenosine 3':5'-monophosphate since cyclic guanosine 3':5'-monophosphate, as well as other nucleotides, have no effect. No differences in the activity or excretion of either N-acetylglucosaminidase or the inhibitory of the extracellular phosphodiesterase are observed between cyclic adenosine 3':5'-monophosphate-induced and control cells. A direct activation of phosphodiesterase by cyclic adenosine 3':5'-monophosphate can be excluded, since the addition of this nucleotide to cell lysates has no effect on the enzyme activity.     

Effects of estrogen, glucocorticoid, glucagon, and adenosine 3':5'-monophosphate on catalytic activity, amount, and rate of de novo synthesis of hepatic histidase.

The mechanisms by which estrogen, glucocorticoid, glucagon, and adenosine 3':5'-monophosphate (cAMP), regulators which participate in the postnatal development of rat liver histidase, elevate the catalytic activity of this enzyme have been explored. A monospecific antibody against homogeneously purified preparations of rat liver histidase has been elaborated in the goat. Employing this antibody in immunotitration experiments, it has been demonstrated that the elevations of hepatic histidase activity elicited by administration in vivo of estradiol-17beta, cortisol acetate, glucagon, and N6,O2'-dibutyryl adenosine 3':5'-monophosphate (dibutyryl cAMP) are paralleled, in each instance, by equivalent increments in immunoprecipitable histidase protein. Following administration of each of the three hormones and dibutyryl cAMP, rates of [14C]leucine incorporation in vivo into rat liver histidase, isolated by immunoprecipitation, relative to incorporation rates into total soluble hepatic protein, increase in magnitudes which are comparable to increases in enzyme amount and catalytic activity. It is thus inferred that estrogen, glucocorticoids, and glucagon, via cAMP, each regulate rat liver histidase development at specific postnatal stages by inducing increases in histidase biosynthetic rates.     

Regulation of aryl hydrocarbon (benzo-(A)-pyrene) hydroxylase activity in mammalian cells. Induction of hydroxylase activity by N6,O2'-dibutyryl8 adenosine 3':5'-monophosphate and aminophylline.

Treatment of hamster BHK cells with N6,O2'-dibutyryl adenosine 3':5'-monophosphate (Bt2cAMP), aminophylline, theophylline, or papaverine increased the level of aryl hydrocarbon (benzo(a)pyrene) hydrolxylase activity. The highese increase, 100-fold, was obtained with Bt2cAMP plus aminophylline or theophylline. N2,O2-Dibutyryl guanosine 3':5'-monophosphate gave a lower induction than Bt2cAMP. The level of hydroxylase activity started to decrease 6 hours after treatment with the inducer and was reduced to almost the uninduced level after 24 hours. Repeated addition of Bt2cAMP and aminophylline did not prevent this decrease. The hydroxylase can also be induced by treating cells with benz(a)anthracene, and the level of this induced activity was maintained for 24 hours. Aminophylline gave a 2- to 8-fold stimulation of the induction by benz(a)anthracene. The enzyme activity induced by Bt2cAMP, aminophylline, and benz(a)anthracene converted benzo(a)pyrene to similar alkali-extractable metabolities with a fluorescence spectra similar to that of 3-hydroxybenzo(a)pyrene. These induced enzyme activities also showed a similar heat stability. Induction by Bt2cAMP and aminophylline, like induction by benz(a)anthracene, required continued protein synthesis and only an initial period of RNA synthesis. Compared to the benz(a)anthracene-induced hydroxylase with a Km of 4.3 muM, the hydroxylase induced by Bt2cAMP and aminophylline showed a Km of 0.14 muM, and was 100-fold more sensitive to inhibition by 7,8-benzoflavone. Increasing the serum concentration in the culture medium stimulated the induction by aminophylline but did not stimulate induction by benz(a)anthracene. The results indicate that aryl hydrocaarbon (benzo(a)pyrene) hydroxylase can be induced by compounds that increase the level of adenosine 3':5'-monophosphate and that this induction and induced enzyme activity differs from that caused by benz(a)anthracene.     

Thyrotropin effects on thyroid cells in culture. Effects of trypsin on the thyrotropin receptor and on thyrotropin-mediated cyclic 3':5'-AMP changes.

Dog, human, and bovine thyroid cells in culture have been shown to develop follicle-like structures when cells are cultured in conditions of confluency and when cells are incubated in the presence of bovine thyrotropin or N6,O2'-dibutyryl cyclic adenosine 3':5'-monophosphate during the first 24 to 48 hours after trypsinization. If thyrotropin is added 48 hours after trypsinization, these cells do not form follicle-like structures but remain as a monolayer culture. Although thyroid cells which grow as a monolayer have a thyrotropin receptor on their plasma membranes with the same in vitro binding properties as the thyrotropin receptor on the plasma membranes of the follicle-forming thyroid cells, there is a 1- to 2-fold greater number of receptors per mg of membrane protein when follicle-forming and monolayer cultures are compared...     

Induction of rat liver phosphoenolpyruvate carbonxykinase (GTP) by cyclic AMP during starvation. The permissive action of glucocorticoids.

The effect of starvation on the activity of hepatic phosphoenolpyruvate carboxykinase (GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32), and on the response of the enzyme to N6-O2'dibutyryl adenosine 3', 5'-monophosphate was investigated in intact and glucocorticoid-deprived rats. In the liver of intact animals, starvation produced a rapid increase in the concentration of cyclic AMP and a corresponding increase in the activity of phosphoenolpyruvate carboxykinase. The kinetics of both changes were not affected by adrenalectomy. Injection of N6-O2'-dibutyryl adenosine 3', 5'-monophosphate into intact starved rats resulted in an immediate, dramatic increase in phosphoenolpyruvate carboxykinase activity above the starvation level. Adrenalectomy completely blocked the response of the enzyme to the cyclic nucleotide. Restoration of hydrocortisone to the adrenalectomized animals restored the full N6-I2'dibutyryl adenosine 3', 5'-monophosphate effect after a lag period of 2 h. The physiological significance of these findings is considered. The data are interpreted with regard to the current hypothesis that glucocorticoids promote an increase in translatable phosphoenolpyruvate carboxykinase mRNA, while cyclic AMP enhances the translation of preexisting specific mRNA templates.     

Effect of insulin on metabolism of cyclic adenosine 3',5'-monophosphate by plasma cell tumor (MPC-11).

Addition of insulin to cultured mouse plasma tumor cells (MPC-11) increases the entry of tritiated cyclic adenosine 3',5'-monophosphate (3H-cAMP). No increase of entry of N6-O2-dibutyryl adenosine 3',5' cyclic monophosphate (DBcAMP), 5' adenosine monophosphate (5' AMP) or adenosine was noted in the presence of insulin. The stimulation of cAMP transport by insulin was concentration dependent and inactivated insulin had no effect on nucleotide transport. Intracellular radioactivity after transport of cAMP was largely 5'AMP, while most of the extracellular radioactivity remained as cAMP after incubation.     

Activity of protein kinase dependent on adenosine 3':5'-monophosphate and of its thermostable protein inhibitor in rat hepatoma (HTC) cells. Unlikely role in the permissive action of glucocorticoids.

Normal expression of a variety of hormonal effects which depend on cyclic AMP (adenosine 3':5'-monophosphate) requires the presence of glucocorticoids. Our hypothesis was that glucocorticoids control directly or indirectly the activity of cyclic-AMP-dependent protein kinase. This has been investigated in cultured hepatoma (HTC) cells in which N6,O2'-dibutyryladenosine 3':5'-monophosphate increases the activity of tyrosine transaminase only after glucocorticoid treatment. In these cells, we have determined the concentration and half-life of protein kinase, the sensitivity of this enzyme in vitro to cyclic AMP and to its thermostable protein inhibitor, the state of dissociation of protein kinase holoenzyme in vivo and its sensitivity, in the intact cell, to dibutyryladenosine 3':5'-monophosphate and to the inhibitor diamide, and we have also determined the concentration of endogenous thermostable protein inhibitor of protein kinase. None of these parameters were influenced by glucocorticoids under conditions where these hormones stimulate the activity of tyrosine transaminase and restore sensitivity to dibutyryladenosine 3':5'-monophosphate. It is concluded that the permissive action of glucocorticoids probably results from a control of cyclic-AMP-dependent processes exerted at a level beyond the protein kinase system.     

Cyclic adenosine 3',5'-monophosphate levels in Pseudomonas putida and Pseudomonas aeruginosa during induction and carbon catabolite repression of histidase synthesis.

Inducibility of histidase (histidine ammonia-lyase, EC 4.3.1.3) in Pseudomonas putida and Pseudomonas aeruginosa was observed to be strongly affected by succinate-provoked catabolite repression, but this did not occur as a consequence of reduced intracellular cyclic adenosine 3',5'-monophosphate levels, and repression could not be alleviated by exogenously added cyclic adenosine 3,'5'-monophosphate. Milder repression of histidase by lactate was also not reversed by the addition of cyclic adenosine 3',5'-monophosphate. These results, along with data showing intracellular cyclic adenosine 3',5'-monophosphate levels remained essentially constant during growth on such diverse carbon sources as histidine, acetamide, glucose, and succinate, indicated that catabolite repression of histidase synthesis by efficient carbon sources was not mediated through variations in internal cyclic adenosine 3,'5'-monophosphate.     

Phospholipid metabolism and lysosomal enzyme secretion by leukocytes. Effects of dibutyryl cyclic adenosine 3':5'-monophosphate and ATP.

The effects of dibutyryl cyclic adenosine 3':5'-monophosphate and ATP on isotope incorporation into phospholipids and the release of beta-glucuronidase into the extracellular medium were studied in polymorphonuclear leukocytes from guinea pig peritoneal exudates. Exogenous dibutyryl cyclic adenosine 3':5'-monophosphate (0.1--1.0 mM) reduced beta-glucoronidase release induced by cytochalasin B in the absence of inert particles. It selectively inhibited 32Pi incorporation into phosphatidic acid and the phosphoinositides and the incorporation of myo-[2-3H]inositol into the phosphoinositides. Added ATP (0.1--1.0 MM), but not other nucleotides, was found to potentiate beta-glucuronidase release provoked by cytochasin B, but it impaired the labeling of the phosphoinositides by myo-[2-3H]inositol. The mechanism of the inhibition the isotope incarparation into these acidic phospholipids by the two mucleotides has not been defined. Dibutyryl cyclic adenosine 3':5'-monophosphate at 2--4 mM concentration was not found to appreciably alter the incorporation of [gamma-32P]ATP into phosphatidic acid, phosphatidylinositol, diphosphoinositide, and triphosphoinositide.     

Demonstration that cyclic adenosine 3',5'-monophosphate mediates the lipolytic action of parathyroid hormone.

Studies were carried out with rat epididymal fat pads first to compare the effects of the synthetic N-terminal 1-34 peptide of bovine parathyroid hormone and of the native hormone to determine whether this portion of the molecule is responsible for the lipolytic action of the hormone and second to determine whether this biologic action of parathyroid hormone is mediated by cyclic adenosine 3',5'-monophosphate. The N-terminal polypeptide was as effective as the native hormone in stimulating lipolysis in the concentration range between 10(-8) M and 10(-6) M. Parathyroid hormone stimulated lipolysis by isolated fat cells. The concentration of cyclic adenosine 3',5'-monophosphate in the fat pads was significantly increased by the hormone (10(-6)M). Lipolytic stimulation by parathyroid hormone (10(-6)M) was diminished by insulin (100 muU/ml) and prostaglandin E1 (1 mug/ml), both of which are known inhibitors of lipolysis. The findings indicate that the amino-terminal 1-34 peptide portion of parathyroid hormone is responsible for the lipolytic action and that this effect is mediated through cyclic adenosine 3',5'-monophosphate.     

Induction of hepatic tyrosine aminotransferase in vivo by derivatives of cyclic adenosine 3':5'-monophosphate.

A number of 8- and N6-SUBSTITUTED DERIVATIVES OF CYCLIC ADENOSINE 3':5'-MONOPHOSPHATE-DEPENDENT PROTEIN KINASE, AND AS SUBSTRATES FOR RAT LIVER CYCLIC NUCLEOTIDE PHOSPHODIESTERASE. All of the analogs tested were able to induce the transaminase. The induction by the analogs was shown to be the result of an actual increase in the amount of enzyme, and the mechanism of induction was an increase in the rate of synthesis of the transaminase. The induced enzyme appeared to be immunologically similar to the non-induced enzyme. A good correlation was found to exist between the dose that produced 50% of maximal induction and a combination of the activation constant for cyclic adenosine 3':5'-monophosphate-dependent protein kinase by the analog and its susceptibility to hydrolysis by cyclic nucleotide phosphodiesterase. These data suggest that the phosphorylation of some site is involved in the mechanism by which cyclic adenosine 3':5'-monophosphate affects the rate of synthesis of tyrosine aminotransferase.     

Elevation of guanosine 3',5'-monophosphate level by adenosine in cerebellar slices of guinea pig.

Effect of adenosine on the level of guanosine 3',5'-monophosphate in guinea pig cerebellar slices was investigated. Adenosine increased the concentration of guanosine 3',5'-monophosphate in the slices 3--4 fold. Upon removal of adenosine from the medium, the concentration of guanosine 3',5'-monophosphate returned to the initial level. AMP, ADP or ATP also increased the guanosine 3',5'-monophosphate level to the same extent as adenosine, while adenine or other nucleosides were not effective. In the absence of Ca2+ in the incubation medium, adenosine did not increase the concentration of guanosine 3',5'-monophosphate in cerebellar slices although level of adenosine 3',5'-monophosphate was elevated by adenosine. Anticholinergic agents, adrenergic blocking agents or antihistaminics did not prevent the increase of guanosine 3',5'-monophosphate by adenosine indicating that the effect of adenosine was not mediated by the release of neurotransmitters. The combination of adenosine with depolarizing agents showed an additive effect on the level of guanosine 3',5'-monophosphate indicating that adenosine increased the level of guanosine 3',5'-monophosphate by a different mechanism from the depolarization.