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Microiontophoresis of dopamine, apomorphine, cyclic adenosine monophosphate (cyclic AMP) and monobutyryl cyclic AMP depresses tile spontaneous or drug-evoked discharge of the majority of neurons in the rat caudate nucleus. Responses to dopamine are enhanced only slightly by the phosphodiesterase (PDE) inhibitors aminophylline or papaverine, both of which also directly slow caudate neurons. However, iontophoresis of the PDE inhibitor methyl isobutyl xanthine (IBMX), or combination of IBMX and papaverine produced marked potentiations of dopamine-induced inhibitions. IBMX alone has little or no direct actions. Chlorpromazine, but not tile beta adrenergic biocker MJ-1999, antagonized dopamine, but not cyclic AMP responses. After destruction of tile nigral striatal dopamine bundle by focal injection of 6-hydroxydopamine, caudate neurons show supersensitivity to dopamine and apomorphine. These results provide electrophysiological support for the hypothesis that the dopamine receptor in caudate may be functionally related to tile adenyl cyclase system, as suggested by recent biochemical findings.

1. Dibutyryl cyclic AMP, injected towards the superior cervical ganglion of the cat, produced no consistent responses. 2. Imidazole, injected towards the ganglion, regularly produced facilitation, both during intermittent and continuous preganglionic stimulation. This effect was dose-dependent and lasted 2-10 minutes. 3. Aminophylline, injected towards the ganglion, regularly produced depression of ganglionic transmission, both during intermittent and continuous preganglionic stimulation. This effect was also dose-dependent and lasted 1-4 minutes. Papaverine produced the same type of response as aminophylline. 4. Imidazole potentiated the ganglionic response to 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), while aminophylline depressed it. 5. The results of the present experiments are consistent with the view that cyclic AMP may have a mediating role in the process of ganglionic transmission.

The relationship between cellular levels of cyclic adenosine monophosphate (AMP) and slow inward calcium ion (Ca 2+ ) current was studied in isolated perfused guinea pig hearts in which fast sodium ion (Na + ) channels had been inactivated by depolarization with potassium ions (K + ) or blockade with tetrodotoxin. Perfusion with 22 mM K + depolarized cardiac tissue to approximately -40 mv and rendered hearts inexcitable. Tetrodotoxin (3 x 10 -5 M) blocked excitability without altering resting membrane potential. Excitability and contractions could be restored to these hearts with a variety of inotropic agents that also raised the measured tissue levels of cyclic AMP or with high concentrations of Ca 2+ . The magnitude of steady-state tension developed by restored hearts was directly related to the external Ca 2+ concentration as well as to the concentration of the restoring agent used. The tension of restored hearts was markedly reduced by Ca 2+ -channel antagonists. Elevation of cyclic AMP levels preceded restoration of excitability to inactivated hearts. A highly significant positive correlation was found between the magnitude of Ca 2+ -dependent tension developed by restored hearts and the level of cyclic AMP in those hearts. Glucagon and ouabain, inotropic drugs that do not elevate myocardial levels of cyclic AMP, failed to restore depolarized or tetrodotoxin-blocked hearts. Therefore, cyclic AMP appears to modulate slow Ca 2+ influx channels in myocardial cells.

Summary Experiments were designed to study the effect of cAMP1) on stomatal opening in isolated epidermal peels of Vicia faba L. leaves. In presence of KCl, cAMP greatly stimulated stomatal opening, but the role of GA3 in the process was less clear. ABA, which induces stomatal closure, counteracted the action of cAMP as well as GA3. The results indicate that the action of cAMP can be modulated by endogenous inhibitors.

The effects of two different invasive strains (TML and SL 1027) of Salmonella tryphimurium on Na and Cl transport in the absence of an electrochemical gradient were measured across rabbit ileal mucosa in vitro. Only strain TML elicits ileal fluid secretion in vivo. Net Na transport is negligible and net Cl secretion occurs across mucosae invaded by TML. In contrast, mucosae invaded by SL 1027 show absorption of both Na and Cl, as occurs in normal ileum. The luminal addition of glucose stimulates Na absorption without affecting Cl transport across ileum invaded by either strain. Theophylline stimulates Na transport across TML-invaded mucosa, causing net secretion, and enhances Cl secretion. Across SL 1027-invaded tissues, theophylline reverses spontaneous ion absorption, causing secretion of both Na and Cl. The data suggest that stimulation of active Cl secretion combined with inhibition of spontaneous Na absorption may account for the diarrhea of salmonella enteritis. However, invasion of the mucosa alone by S. typhimurium is not necessarily associated with major alterations in ion transport, suggesting that a factor, in addition to invasion, is necessary for fluid secretion. The effects of theophylline are compatible with strain TML exerting submaximal stimulation of cyclic adenosine monophosphate activity or involving another metabolic pathway which also inhibits spontaneous Na absorption and stimulates Cl secretion.

The interaction in vitro of cyclic [3H]AMP and a partially purified cyclic [3H] AMP-binding protein preparation from calf ovary cytosol with nuclei and chromatin of calf ovaries was investigated. Cyclic [3H]AMP, complexed with a calf ovary cytosol protein (the cyclic AMP-binding protein), binds to ovary nuclei and chromatin, whereas free cyclic [3H]AMP does not bind. Concomitantly with the nuclear association of cyclic [3H]AMP-binding protein, cyclic AMP-independent protein kinase activity in nuclei and chromatin incubated with a calf ovary cyclic [3H]AMP-binding protein preparation is significantly enhanced. The increase of nuclear protein kinase activity is dependent upon the presence of cyclic AMP complexed to ovary cytosol protein, and neither free cyclic AMP nor cyclic AMP-free binding protein stimulate endogenous nuclear protein kinase activity in ovary nuclei or chromatin. The association in vitro of cyclic [3H]AMP-binding protein with ovary chromatin and nuclei as well as the concomitant increase ...

Application of adenosine 3′: 5′-cyclic monophosphate (CAMP), N 6 , O 2′ -dibutyryl cyclic AMP (DBCAMP) or N 6 -monobutyryl cyclic AMP (N6-MBCAMP), from concentrations of 10 −7 to 10 −2 M had no effect on fertilization or cleavage rates in sea urchin eggs up to early gastrula stages. In order to study the permeability of CAMP and DBCAMP in sea urchin eggs, they were incubated with the nucleotides. CAMP was readily taken up by the eggs. There was no significant breakdown of the nucleotide in the extracellular medium, indicating that CAMP entered the cell as such and was not resynthesized from a metabolic breakdown product. When eggs were incubated with DBCAMP, no dibutyryl derivative could be recovered from egg-extracts, the compound having been converted to N 6 MBCAMP. Homogenates of the eggs similarly were able to deacylate DBCAMP, indicating the presence of a potent esterase activity. The intracellular levels of CAMP and N 6 -MBCAMP reached concentrations greater than 10 −5 M, at least two orders of magnitude higher than the CAMP level normally found in sea urchin eggs. These results indicate that CAMP does not participate in regulation of mitosis in early embryogenesis in sea urchin eggs.

The effects of isoproterenol (a β-adrenergic stimulating agent) and propranolol (a β-adrenergic blocking agent) were studied on immediate hypersensitivity skin test reactions in 15 atopic subjects. Forearm skin of these subjects was pretreated with these agents by local iontophoretic application and compared to areas locally pretreated with saline or diphenhydramine hydrochloride. Following pretreatment of skin, scratch or intradermal tests were performed with the most reactive antigen for each patient. A significant increase of skin reactivity occurred in areas of skin pretreated with propranolol. A significant decrease in reactivity followed pretreatment with isoproterenol. Thus the iontophoresis of β-adrenergic agents can alter the immediate hypersensitivity skin test reactions of atopic patients. This alteration is consistent with a modification of the local synthesis of cyclic adenosine monophosphate (CAMP) induced by these pharmacologic agents. It is not clear whether this presumed alteration in CAMP is exerting its effect on mediator release from dermal mast cells or directly on the dermal blood vessels or both.

We investigated the chemotactic responses of individual human polymorphonuclear leukocytes in defined fields of adenosine and guanosine 5′monophosphate, dibutyryl cyclic adenosine monophosphate, cyclic guanosine 3′,5′monophosphate, Kaolin and uric acid. We observed positive chemotaxis towards dibutyryl cyclic AMP, positive and negative chemotaxis towards cyclic GMP and no response towards the other chemicals. These results are discussed and compared with the known response to cyclic AMP.

The frequency of miniature endplate potentials (mepps) in rat diaphragms was markedly increased by epinephrine and norepinephrine in preparations exposed to 15 mM K+. The effect was rapid in onset but gradually declined during continued exposure to the catecholamines. N6, O2'-dibutyryl adenosine 3',5'-monophosphate (dibutyryl-cAMP) also caused transient frequency increases resembling in time-course those observed with catecholamines. Contrary to previous reports, catecholamines and dibutyryl-cAMP had little effect on mepp frequency in preparations not treated with K+. Sustained increases with theophylline and decreases with adenosine were found in both K+-treated and untreated preparations. Analysis of the data obtained with catecholamines showed the intensity of the response to be a function of nerve terminal polarization. The inability of catecholamines and dibutyryl-cAMP to affect mepp frequency of untreated preparations argues against an obligatory role for cAMP in the neurosecretory mechanism. The findings are consistent with an action of catecholamines and cAMP in the regulation of transmitter release at fatigued preparations.

The strong repression of inducible synthesis of the enzymes of fatty acid degradation by glucose can be partially relieved by the addition of cyclic adenosine 3′,5′ monophosphate (cyclic AMP) to the growth medium. This reversal of the glucose effect by cyclic AMP is not observed in a mutant (K29) that is unable to grow on fatty acids as sole carbon source and that was found to synthesize low levels of several enzymes specified by the fad regulon. In a revertant selected for the ability to grow on oleate these effects are concomitantly relieved. By both genetic (co-transduction of the mutation with the strA locus) and biochemical experiments (an extract of the mutant strain does not show the cyclic AMP-dependent stimulation of the deoxyribonucleic acid-directed in vitro synthesis of the enzymes of the gal operon), it is demonstrated that the mutant lacks functional cyclic AMP receptor protein (CR protein). It is concluded that, like many other inducible enzyme systems, expression of the enzymes of the fad system requires cyclic AMP and the CR protein.

In order to assess the effects of time requirements of different tissue inactivation methods, concentrations of cyclic adenosine monophosphate in rat brain were determined. Fixation of tissues was obtained by the following methods: decapitation with removal of brain and freezing in liquid nitrogen; decapitation into liquid nitrogen; whole animal immersion in liquid nitrogen; 1.5 kW maximal field strength microwave irradiation for 8 seconds; and, 5 kW maximal field strength microwave irradiation for 2 seconds. Results of these studies indicate that as the time is reduced for inactivation of brain adenyl cyclase and phosphodiesterase, levels of cyclic adenosine monophosphate become progressively lower. This same correlation is also evident in studies of regional brain concentrations of cyclic adenosine monophosphate after 1.5 kW and 5 kW microwave inactivation. It is concluded that 5 kW maximal field strength microwave exposure is the most rapid means of enzyme inactivation permitting a more accurate estimation of endogenous cyclic adenosine monophosphate concentrations. Its use offers rapid inactivation with minimization of trauma and facilities the study of regional metabolites through ease of dissection.

Some properties were examined of the isolated membranes of zymogen granules from the rat pancreas. It was possible to detect an endogenous protein kinase activity capable of phosphorylating 8 to 10 membrane protein constituents as well as added histones. Cyclic adenosine monophosphate (AMP), and cyclic guanosine monophosphate (GMP), to a lesser extent, exerted moderate stimulatory effects. These effects of cyclic nucleotides on membrane phosphorylation became more apparent upon addition of a partially purified soluble protein kinase also extracted from the rat pancreas., SCOPUS: ar.j, info:eu-repo/semantics/published

1. 1. The activity of 2′,3′-cyclic adenosine monophosphate 3′-phosphoesterhydrolase was determined in the brains of several elasmobranchs and teleosts by a new fluorometric method using a fluorescent analogue of 2′,3′-cAMP, 1,N 6 -ethenoadenosine-2′,3′-cyclic monophosphate, as substrate. 2. 2. Enzymatic activity of lemon shark brain was optimal at a pH of 6·5 in acetate buffer and at a temperature of about 30°C. 3. 3. Brain enzyme activity in lemon, nurse and brown sharks was similar and was 0·17–0·25 m-moles/g wet wt. tissue per hr. Brain homogenates of sting rays ( Dasyatis spp.) had an activity of 0·32 and 0·34, respectively, and the brain homogenates from two teleost fishes assayed as 0·34 and 0·39, respectively. 4. 4. Assay of enzyme activity in various portions of elasmobranch brain showed that activity in the telencephalon was consistently higher than that in the whole brain while that in the medulla was lower; the optic lobes and cerebellum exhibited intermediate activity.

Human malignant trophoblast cells in continuous culture were employed to investigate the causal relationship between N6,O2′-dibutyryl cyclic adenosine 3′,5′-monophosphoric acid. (dbcAMP) stimulation of human chorionic gonadotropin (hCG) secretion and dbcAMP stimulation of estrone and 17β-estradiol (E) secretion. The cells secreted up to 25 times more hCG when propagated in medium containing 10% serum, compared to growth in the absence of serum. By contrast, serum had little effect on E secretion. In the presence of serum, dbcAMP resulted in marked stimulation of hCG and E secretion; both processes were potentiated by theophylline. When cells were incubated with various concentrations of dbcAMP or theophylline, stimulation of hCG secretion was disproportionately greater than the stimulation of E secretion. Furthermore, the relative stimulation of secretion of the two hormones was not constant. The rate of hCG, but not of E, secretion was elevated for at least 2 days following a 7-hr pulse with dbc...

1 The efflux of (45)Ca from rat pancreas was investigated in relation to the secretion of alpha-amylase.2 The (45)Ca space in the pancreas was increased 1.6 times when pancreatic tissue was incubated in media in which the extracellular calcium concentration was reduced from 1.0 to 0.05 mM.3 Carbachol increased the rate of (45)Ca efflux from the tissue and this effect was associated with an increase in the release of alpha-amylase. Dibutyryl cyclic adenosine monophosphate (AMP) also evoked a secretory response, but did not alter the rate of (45)Ca efflux. In combination with carbachol, the dibutyryl analogue of cyclic AMP reduced the carbachol-stimulated increase in (45)Ca efflux while enhancing the release of secretory protein.4 The stimulatory effect of carbachol on (45)Ca efflux was observed when pancreatic tissues were incubated in media containing a high concentration of ethyleneglycolbis (beta-aminoethyl)-N,N'-tetraacetic acid (EGTA).5 Atropine blocked the effects of carbachol on both (45)Ca efflux and secretion of alpha-amylase.6 It was concluded that intracellular calcium can and may sustain stimulus-secretion coupling in the rat exocrine pancreas.

Cyclic adenosine 3':5'-monophosphate (cyclic AMP), when added in a concentration of 5 mm to incubations of rat liver slices with [1-14C]acetate or [1-14C]octanoate, was found to depress markedly both cholesterol and fatty acid biosynthesis but not CO2 or ketone body production. The inhibitory effects of the nucleotide were lost when its concentration was reduced to 0.5 mm. These findings, therefore, confirm and extend those made by others. However, it also was observed that perfusion of rat livers with the above mentioned substrates in the presence of sufficient glucagon to raise the tissue cyclic AMP level by at least 50-fold was totally without effect on rates of cholesterol or fatty acid synthesis. In addition, such treatment of the livers failed to reduce the activity of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase in subsequently isolated microsomes. It is concluded that in the intact liver cholesterol and fatty acid synthesis are independent of acutely induced changes in the intracellular cyclic AMP concentration over a very wide physiological range.

Some properties of cyclic 3', 5'-nucleotide phosphodiesterase (PDE) in the rat cerebral cortex and heart muscle and inhibitory effect of 3, 7-dimethyl-1-(5-oxo-hexyl)-xanthine (BL 191) on PDE have been investigated by comparison with theophylline and caffeine as PDE inhibitor. 1) Cyclic adenosine monophosphate (AMP) hydrolysis in the supernate of the cerebral cortex and heart muscle is linear with respect to time (up to 30 min) and protein concentration (up to 150 μg and 400 μg in the incubation medium on the brain and heart respectively). 2) PDE activity obtained in the absence of Mg2+ could be increased up to 70 per cent by adding Mg2+ with the supernate of the cerebral cortex as the enzyme. 3) Substantial activity was present in all of the fraction although most was contained in the supernate. 4) From typical Lineweaver-Burk plots, Km values for the supernate of the cerebral cortex and heart muscle were found to be 130 μM and 74 μM respectively. 5) Inhibitory effect of BL 191 on PDE from the rat cerebral cortex supernate was similar to the action of theophylline and was slightly more potent than that of caffeine. The inhibition of BL 191, theophylline and caffeine was noncompetitive ; the Ki was calculated to be 3.0 mM, 3.1 mM and 5.1 mM respectively. The Ki values of 1.65 mM, 1.80 mM and 1.55 mM were found in the heart muscle supernate treated with BL 191, theophylline and caffeine respectively : the type of inhibition was competitive.

An E. coli mutant with a temperature sensitive enzyme I of the phosphoenolpyruvate dependent transferase system (PTS) is described. Its phenotype at 41° C is Mtl− Glu± Fru− Man− Lac− Glp− Mal− Ara+ Gal+; at 28°C, wild type. The half-life of the enzyme in extracts is 1.5–2.0 minutes at 41°C. The cellular content of 3′,5′ cyclic adenosine monophosphate (cAMP) in this mutant was measured and was the same at both temperatures. It is concluded that the effect of loss of enzyme I on the utilization of lactose1, maltose, and glycerol is not mediated through cellular cAMP levels. The mutant was used to study carbohydrate uptake and it was found that destruction of enzyme I by heating affected the uptake of those carbohydrates phosphorylated by the PTS but did not significantly affect the uptake of carbohydrates not phosphorylated by the PTS.

The effect of a number of agents on the in vitro migration of guinea pig peritoneal macrophages and on the activity of macrophage migration inhibitory factor was studied. It was found that: 1. Dibutyryl cyclic adenosine monophosphate and theophylline cause pronounced inhibition of migration. Migration was not modified by cyclic guanosine monophosphate, noncyclic nucleotides or by prostaglandins E1, E2, and F2α. Theophylline (10−4M), paradoxically, abolishes the migration inhibitory effect of migration inhibitory factor. 2. Migration was not influenced by subtoxic doses of actinomycin D, puromycin and cycloheximide, but macrophages escaped inhibition partially, when exposed to migration inhibitory factor in the presence of puromycin (1–2 μg/ml) and of cycloheximide (0.1 μg/ml). 3. The polyanions dextran sulfate and heparin promoted macrophage migration and dextran sulfate (40 μg/ml) partially abolished migration inhibitory factor action. 4. Treatment of macrophages with the proteolytic enzymes trypsin and pronase resulted in enhanced migration while exposure to neuraminidase and wheat germ lipase was without effect. 5. Macrophage motility was dependent upon the presence of serum and increased in proportion with raising serum concentrations. Migration inhibitory factor was inactive at high serum concentrations. 6. Macrophage migration was enhanced and migration inhibitory factor activity abolished in the absence of calcium and magnesium.

The phosphorylation of specific membrane proteins by an endogenous protein kinase has been studied in purified membrane fractions from rat adipocytes using trichloroacetic acid precipitation and sodium dodecyl sulfate polyacrylamide disc gel electrophoresis. The endogenous phosphorylation of two specific membrane proteins is completely dependent on the presence of cyclic adenosine 3':5'-monophosphate (cyclic AMP) and magnesium ions. This phosphorylation occurs very rapidly at 24°, reaching maximal levels at 1 min. The number of sites specifically phosphorylated in the presence of cyclic AMP probably does not exceed 50,000 per fat cell, requiring the use of very high specific activity (10 to 50 Ci per mmole) [γ-32P]ATP for these studies. The minimal molecular weights of the two specifically phosphorylated proteins are about 22,000 and 16,000 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. The same two proteins are phosphorylated when intact fat cells are exposed briefly to low concentrations of exogenous ATP, a process which results in the suppression of the insulin-stimulated rates of d-glucose transport. At least 95 % of the cyclic AMP-dependent 32P incorporated into trichloroacetic acid-precipitable protein is in the form of protein-bound phosphoserine. The concentration of cyclic AMP required for maximal stimulation of phosphorylation is about 1.5 µm. Cyclic guanosine 3':5'-monophosphate has no significant effect unless its concentration is increased to 10-4 m. Phosphorylation is inhibited by calcium ions. The cyclic AMP-stimulated phosphorylation is inhibited by phloretin and by 5'-adenylyl-β, γ-methylene triphosphonate. The specific cyclic AMP-dependent membrane phosphorylation is also found in membranes from cells of obese rats, an insulin-resistant animal. This phosphorylation system, however, cannot be detected in membranes from guinea pig fat cells. The lack of phosphorylation of specific membrane proteins in guinea pig fat cell membranes is correlated with the insulin insensitivity of this tissue to glucose transport (but not lipolysis) and with the inability of ATP to inhibit the slight stimulation which insulin exerts on glucose transport. The possibility is considered that this specific cyclic AMP-dependent phosphorylating system is involved in the hormonal regulation of glucose transport and the modulation of insulin sensitivity.

A comparison has been made between the capacity to hydrolyse 2′,3′-cyclic adenosine monophosphate and 3′,5′-cyclic adenosine monophosphate in subcellular fractions of normal and neoplastic (lymphosarcoma) spleen of C 57 BL mice. The effect of X-irradiation on these activities was tested. Subcellular fractionation of normal and lymphosarcoma spleen points to a different overall localization of the enzymes. The 2′,3′-cyclic nucleotide phosphodiesterase (2′,3′-cAMPase) has its highest specific activity in the particulate fractions of the cell, while the data on 3′,5′-cyclic nucleotide phosphodiesterase (3′,5′-cAMPase) show the highest activity in the soluble fraction. The 2′,3′-cAMPase activity is higher in the tumor as compared to the normal tissue, while the opposite holds for 3′,5′-cAMPase. Total body irradiation of normal mice with a dose of 600 rads of X-rays, results in a clear drop in 2′,3′-cAMPase 48 hours after the exposure. The 3′,5′-cAMPase is hardly affected at this time. Neither imidazol nor Mg ++ has any influence on the 2′,3′-cAMPase. The pH optimum for 3′,5′-cAMPase and 2′,3′-cAMPase appears to be 7.7 and 6.2 respectively. This report suggests a no-identity of the two enzymes in mouse spleen, a situation different from that found in certain plants.

Summary The release of chemical mediators from sensitized cells is inhibited by drugs which increase intra-cellular cyclic adenosine monophosphate. Theophylline. which increases cyclic AMP through inhibition of phosphodiesterase, has been shown to inhibit mediator release in several in vitro systems. This study was undertaken to determine whether theophylline. as used for asthma therapy, would have this effect in vivo in humans. In three of six atopic subjects, inhibition of mediator release (as studied with the leucocyte hislamine release system) could be demonstrated after theophylline given orally in dosage commonly employed in asthma, This suggests that, in some asthmatic patients, theophylline may have beneficial effects other than bronchodilatalion.

The interactions of parathyroid hormone (PTH), calcitonin (CT) and vitamin D in mineral and skeletal homeostasis are discussed. Evidence is presented that not only does PTH control the renal synthesis of 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2 D 3 ), the biologically active form of vitamin D 3 , but also that 1,25-(OH) 2 D 3 acts as a feedback inhibitor of PTH secretion. Thus, the plasma concentrations of both 1,25-(OH) 2 D 3 and calcium are controlled by the actions of PTH, and both of these metabolites operate as feedback inhibitors of PTH secretion. At the level of bone cell function, present evidence indicates that both PTH and CT influence the size of the osteoclast and osteoblast cell pools and the respective activities of the cells in these pools: PTH increases the size of the osteoclast pool and the activity of the cells in this pool; CT has the opposite effects; and 1,25-(OH) 2 D 3 seems to increase the activity of these cells as well as those in the osteoblast pool without altering the size of these pools. These different effects are interpreted in terms of the actions of these three hormonal agents on the concentration of three second messengers, cyclic adenosine monophosphate (AMP), calcium ion and monohydrogen phosphate, within the cell.

✓ Topical dibutyryl cyclic adenosine monophosphate (AMP) was used to reverse experimental cerebral vasospasm of the basilar artery in the cat. The combination of dibutyryl cyclic AMP and theophylline caused prolonged dilatation of the basilar artery. Dibutyryl cyclic AMP seems to be specific as a topical vasodilator, which may be useful in the postoperative management of subarachnoid hemorrhage.

A ouabain-sensitive, Mg 2+ -dependent, Na + , K + -stimulated adenosinetriphosphatase (ATPase) isolated from canine mesenteric arteries was activated by the following vasodilators: hydralazine, diazoxide, PGE 1 , PGE 2 , PGA 2 , and minoxidil. Epinephrine, norepinephrine, and isoproterenol also stimulated the ATPase, but PGF 2α was ineffective. Since these vasodilators activate the adenylate cyclase of vascular smooth muscle, the effects of cyclic adenosine monophosphate (AMP) and theophylline on the Na + , K + -ATPase were studied; both substances caused a concentration-dependent increase in enzymatic activity. Propranolol blocked the catecholamine-induced stimulation of Na + , K + -ATPase, and polyphloretin phosphate antagonized the effects of the prostaglandins. It is concluded that vasodilatation in response to these substances is associated with the stimulation of the Na + , K + -ATPase of vascular smooth muscle probably mediated through cyclic AMP.

Incubation of the salivary glands of the larvae ofDrosophila hydei in a control medium containing 2×10−3 m cyclic adenosine monophosphate (cAMP) induces a considerable increase in passive electrical cel communication. This is caused by a decrease in permeabilty of the nonjunctional membrane part, together with an increase of the permeability of the low-resistance junctions. Similar changes in intercellular communication in the salivary gland ofDrosophila hydei were seen in a majority of experiments in which 10−3 m dibutyryl cycli adenosine monophosphate (dBcAMP), 5×10−3 m theophylline or ecdysterone (100 μg/ml) were added to the control medium. Hyperpolarization of the gland cells can be observed concomitant with the increase in communication. A hypothesis is discussed for a possible molecular regulation of passive electrical cell communication in which the intracellular cAMP level plays a significant part.

Calcium stimulates leucine incorporation into protein during incubations of sections and cell-free preparations of the rat adrenal. Like adrenocorticotrophic hormone (ACTH) action, calcium enhances the transfer of amino acid from transfer RNA to protein. Stimulation of leucine incorporation by ACTH and cyclic adenosine monophosphate is best observed when sections are incubated in limiting calcium concentrations.

A protein kinase which is intimately associated with equine herpesvirus (equine abortion virus) was found by using adenosine triphosphate-γ- 32 P as a phosphate donor and virus protein as an acceptor. Consistent demonstration of the activity requires prior removal of phosphohydrolase. The kinase activity requires Mg 2+ , is not stimulated by cyclic adenosine monophosphate, but is enhanced by added protamine or arginine-rich histone. The labeled product is resistant to ribonuclease, deoxyribonuclease, and chloroform-methanol but is sensitive to Pronase. Other tests suggest that serine and threonine residues are the acceptor sites. In the in vitro reaction, the incorporation represents an average of approximately 4,500 phosphate residues per virion, and all 17 virus protein bands resolved by polyacrylamide gel electrophoresis appear to be labeled.

Adenosine and 2'-deoxyadenosine at concentrations in the range of 5 to 50 µm inhibited the activation of adenylate cyclase activity in fat cell "ghosts" by norepinephrine. 2',5'-Dideoxyadenosine was more potent than adenosine while arabinosyl adenosine, 3'-deoxyadenosine, and 2-fluoroadenosine were equi-potent to adenosine as inhibitors of adenylate cyclase. Adenine was inactive as an inhibitor of adenylate cyclase along with nucleosides such as phenylisopropyl adenosine, inosine, 2'-deoxyinosine, guanosine, and 2'-deoxyguanosine. Adenosine, 2'-deoxyadenosine, and 2-fluoroadenosine were also inhibitors of the low Km phosphodiesterase found in the supernatant fraction obtained after centrifugation of fat cell homogenates. The large accumulation of cyclic adenosine 3',5'-monophosphate (cyclic AMP) seen in fat cells incubated for 5 min with catecholamines plus theophylline was markedly inhibited by 2',5'-dideoxyadenosine, adenosine, and 2-fluoroadenosine. The small increase in cyclic AMP due to norepinephrine or theophylline alone was also blocked by adenosine and 2',5'-dideoxyadenosine. However, there was no correlation between the effects of the nucleosides on cyclic AMP accumulation and lipolysis since 2',5'-dideoxyadenosine did not inhibit lipolysis. Adenosine inhibited the lipolytic action of theophylline or low concentrations of norepinephrine but did not inhibit the lipolytic action of higher concentrations of norepinephrine under conditions in which it virtually abolished the increase in cyclic AMP accumulation due to norepinephrine. These data indicate that marked inhibition by nucleosides of the increase in total cyclic AMP accumulation seen 5 min after the addition of theophylline, norepinephrine, or norepinephrine plus theophylline is not necessarily associated with any decrease in lipolysis. Apparently most of the increased cyclic AMP accumulation seen in the presence of lipolytic agents is unrelated to the regulation of lipolysis. The effect of adenosine and related nucleosides on cyclic AMP accumulation suggest that they may play a physiological role as feedback regulators of fat cell adenylate cyclase.

We evaluated the role of cyclic adenosine monophosphate (cyclic AMP) in the energetics of gastric secretion in the canine stomach in three ways: (1) by inhibiting the enzyme, cyclic nucleotide phosphodiesterase, which degrades cyclic AMP, (2) by determining the effect of a secretagogue, histamine, on activity of the cyclic AMP-forming enzyme, adenyl cyclase, and (3) by administering to the stomach a derivative of cyclic AMP which often elicits physiological responses more readily in intact tissue than cyclic AMP. Adenyl cyclase was assayed by the method of Krishna et al. (J Pharmacol Exp Ther 163:379-385, 1968), and phosphodiesterase W as measured by modification of the methods of Butcher and Sutherland (J Biol Chem 237: 1244-1250, 1962) and of Buell et al. (J Biol Chem 232:979-987, 1958). The findings are as follows: (1) theophylline (12.5 ռա) inhibited gastric mucosal phosphodiesterase 50% and papaverine (1.25 mM) inhibited phosphodiesterase 90% in vitro but neither agent initiated gastric secretion in the anesthetized dog stomach. Phosphodiesterase activity was not significantly altered by in vivo administration of either of the drugs; (2) histamine, in all concentrations studied, did not increase gastric mucosal adenyl cyclase activity in vitro, and histamine in a dose (30 μg per kg-hr) which stimulated acid secretion from the anesthetized dog stomach did not increase mucosal adenyl cyclase activity in vivo; and, (3) dibutyryl cyclic AMP (0.1 and 0.2 mg per kg-min) infused intraarterially into a gastric artery for 60 min increased blood flow but did not initiate gastric secretion. Our results suggest that initiation of secretion in the canine stomach does not depend upon accumulation of cyclic AMP.

This article by Rall, Sutherland, and Berthet on hormone action in cell-free homogenates announced the discovery of the biological activity of cyclic adenosine monophosphate (cAMP). The article influenced much of the subsequent research on hormone action, including the Rodbell's work.

Data obtained in the dog during thyroid perfusion support the hypothesis that glucagon and epinephrine stimulate thyrocalcitonin release via adenyl cyclase activation and consequently by the intracellular level of cyclic adenosine monophosphate. The studies also suggest that agonists acting at alpha adrenergic loci may lead to a reduction in cyclic adenosine monophosphate in the parafollicular thyroid cells. (Endocrinology 88: 1337, 1971)

Heme oxygenase (HO), the enzyme system catalyzing the conversion of heme to bilirubin, was studied in the liver and spleen of fed, fasted, and refed rats. Fasting up to 72 hr resulted in a threefold increase in hepatic HO activity, while starvation beyond this period led to a gradual decline in enzyme activity. Refeeding of rats fasted for 48 hr depressed hepatic HO activity to basal values within 24 hr. Splenic HO was unaffected by fasting and refeeding. Hypoglycemia induced by injections of insulin or mannose was a powerful stimulator of hepatic HO. Glucose given together with the insulin abolished the stimulatory effect of the latter. Parenteral treatment with glucagon led to a twofold, and with epinephrine to a fivefold, increase of hepatic HO activity; arginine, which releases endogenous glucagon, stimulated the enzyme fivefold. These stimulatory effects of glucagon and epinephrine could be duplicated by administration of cyclic adenosine monophosphate (AMP), while thyroxine and hydroxortisone were ineffective. Nicotinic acid, which inhibits lipolysis, failed to modify the stimulatory effect of epinephrine. None of these hormones altered HO activity in the spleen. These findings demonstrate that the enzymatic mechanism involved in the formation of bilirubin from heme in the liver is stimulated by fasting, hypoglycemia, epinephrine, glucagon, and cyclic AMP. They further suggest that the enzyme stimulation produced by fasting may be mediated by glucagon released in response to hypoglycemia. The possibility is considered that the enhanced HO activity in the liver may increase hepatic heme turnover and hence, bilirubin production, which may explain the rise of unconjugated serum bilirubin observed in fasting or hypoglycemic individuals.

Cardiac microsomes, which represent an enriched but not pure preparation of the heart's sarcoplasmic reticulum, can remove calcium from solution by 2 kinetically dissimilar mechanisms. In the presence of adenosine triphosphate (ATP), Ca ++ is taken up by cardiac microsomes by a process designated Ca-binding , which exhibits saturation kinetics. The rate and extent of Ca-binding, and the high affinity of the Ca-binding sites could allow this process to cause the intact cell to relax. When anions that permit Ca ++ to be precipitated within the mlcrosomal vesicles are included along with ATP, much larger amounts of Ca ++ are taken up by cardiac microsomes. This second process, designated Ca-uptake , does not follow saturation kinetics. Instead, the rate of Ca-uptake increases linearly with increasing Ca ++ concentration until Ca-uptake becomes inhibited at higher Ca ++ concentrations. The finding of 2 kinetically distinct Ca ++ transport processes in cardiac microsomes, both of which are highly active in the micromolar range of Ca ++ concentration, suggests that Ca ++ movements in the intact myocardial cell may be controlled by 2 mechanisms. It is suggested that one of these, possibly manifest in vitro as Ca-binding, represents an intracellular release site that initiates systole by delivering Ca ++ to the contractile proteins. The second process, possibly manifest in vitro as Ca-uptake, is suggested to represent the uptake of Ca ++ into an intracellular storage site whose Ca ++ content indirectly determines the amount of Ca ++ that is delivered to the contractile proteins. These 2 intracellular Ca ++ pools can be tentatively related to Ca ++ movements into and out of the myocardial cell, permitting the formulation of a model by which a number of inotropic interventions might modulate myocardial contractility. Cardiac glycosides had no detectible effect on either cardiac microsomal Ca-binding or Ca-uptake. Cyclic adenosine monophosphate (cAMP), which by itself was without effects on cardiac microsomes, more than doubled the rate of Ca-uptake in the presence of a cyclic AMP-dependent protein kinase. The resulting increase in rate of Ca-uptake could explain the actions of epinephrine to enhance contractility at the same time that systole is abbreviated.

The effect of electrical stimulation of autonomic nerves on c-AMP levels in the tibial muscle of rats was studied after pretreatment with a phosphodiesterase inhibitor and a muscle paralyzant. Cyclic-AMP levels in the skeletal muscle increase significantly. This increase is not the result of changes in muscle blood flow which might have resulted from autonomic nerve stimulation. These studies indicate that the adenyl cyclase-cyclic AMP system in skeletal muscle may be controlled by the autonomic nervous system.

1. Cyclic adenosine 3',5'-monophosphate and N6-2'-O-dibutyryl cyclic adenosine 3',5'-monophosphate stimulate the active cellular entry and cumulative uptake of several amino acids in rat kidney cortex slices. 2. Parathyroid hormone, known to augment cellular production of cyclic adenosine 3',5'-monophosphate by stimulating renal cortical adenyl cyclase, increases the renal cortical uptake of amino acids. Antidiuretic hormone and insulin, hormones which do not stimulate renal cortical adenyl cyclase, have no effect on the renal cortical uptake of amino acids. 3. Characteristics of the N6-2'-O-dibutyryl cyclic adenosine 3',5'-monophosphate stimulation of amino acid transport include a delay in stimulation of transport, blockade of stimulation by inhibitors of protein synthesis, and inhibition of the expression of the effect by either sodium-free incubation or ouabain exposure.

1. Significant inhibition of CO2 production and lipogenesis from acetate-1-14C and pyruvate-2-14C occurred in the presence of 1.6 µm vasopressin, in rat liver slices and in homogenates. 2. This effect cannot be reproduced by cyclic adenosine monophosphate, nor can it be accounted for by lipolysis or by glycogenolysis-induced dilution of a precursor pool. 3. Vasopressin has no significant effect on lipogenesis from citrate-1,5-14C and stimulates the formation of fatty acids from acetyl-1-14C-coenzyme A, in rat liver homogenates. 4. The failure of vasopressin to inhibit acetylhydroxamate formation as well as the stimulation of fatty acid synthesis from acetate-1-14C in a mitochondria-free homogenate, focuses attention on the mitochondria as a possible site of action of vasopressin in inhibiting lipogenesis. 5. Vasopressin noncompetitively inhibits purified citrate-condensing enzyme. The inhibitory effects of the hormone on lipogenesis are explainable by this observation.

Colchicine, vinblastine, podophyllotoxin, and cytochalasin B inhibit the action of vasopressin and cyclic adenosine monophosphate on osmotic water movement across the toad bladder. The findings suggest that microtubules, and possibly microfilaments, play a role in the action of vasopressin, perhaps through involvement in the mechanism of release of secretory material from the bladder epithelial cells.

In an effort to better define the role of betaadrenergic blockade in human bronchial asthma, peripheral blood leukocytes and lymphocytes from individuals with this condition were studied for possible alterations in cyclic AMP metabolism. Using a previously described radioimmunoassay to measure cyclic AMP, cells from asthmatic subjects were shown to have a highly significant decrease in their cyclic AMP response to beta-adrenergic agents (isoproterenol, norepinephrine, and epinephrine) by comparison with normal control cells. The alteration in responsiveness was most marked at the time of severe active asthma and returned toward normal during periods of clinical remission. Evidence was presented to indicate that the reduced response in cells from asthmatic individuals was not due to marked alterations in the proportion of T and B lymphocytes. Five normal volunteers were treated with an oral bronchodilator preparation containing theophylline and ephedrine over a 2 wk period without a significant change in the lymphocyte cyclic AMP response. These results provide unambiguous evidence for altered adrenergic responsiveness in bronchial asthma and indicate that purified peripheral blood lymphocytes should be a suitable in vitro system for further elucidation of the abnormality. Despite the reduction in catecholamine responsiveness in the asthmatic population as a whole, major alterations were largely restricted to individuals with severe, chronic asthma. Conclusive evidence for beta-adrenergic blockade in individuals who have not had recent asthmatic symptoms was not obtained, casting some doubt on the theory that bronchial asthma is due to a congenital derangement of cyclic AMP metabolism. Moreover, transient episodes of bronchospasm were often accompanied by a normal cyclic AMP response indicating that episodes of asthma frequently occur in the absence of easily demonstrable adrenergic blockade.

In the isolated abdominal ganglion of Aplysia, previously incubated in adenine-3H, the amount of 3H-labeled adenosine-3',5' monophosphate (cAMP) doubled after electrical stimulation of nerves at a physiological rate (1/sec). No change was detected after 4 min of stimulation. An increase in cAMP was first seen after 15 min; lengthening the period of stimulation to 1 hr did not increase the extent of the effect. ATP contained 50% of the total radioactivity taken up from adenine-3H, cAMP about 0.1%. During stimulation both the total amount and the specific radioactivity of adenosine triphosphate (ATP) did not change. Thus, the increased amount of radioactivity found in cAMP after stimulation represented an increase in its rate of synthesis. During stimulation formation of cAMP-3H was not altered in nerves or in the cell body of an identified neuron (R2). In addition, no changes were detected in the total amounts of cAMP in the ganglion and in the cell body of R2. It seems likely that the increase was initiated by synaptic activity rather than by action potentials. It was blocked by elevating the concentration of Mg, which also blocks synaptic activity without impairing conduction of impulses. Moreover, impulse activity induced by ouabain and glutamate did not result in increased formation of cAMP.

1. 1. Accumulation of intracellular radioactivity was measured during incubation of isolated bovine thyroid cells with cyclic [ 32 P]AMP, cyclic [8- 3 H]AMP and dibutyryl cyclic [8- 3 H]AMP. With cyclic [ 32 P]AMP, 32 P cell/medium ratios ranged from 0 to to 0.04 compared to a maximum 3 H cell/medium ratio of 0.29 with cyclic [ 3 H]AMP and 0.16 with dibutyryl cyclic [ 3 H]AMP. The excess of intracellular cyclic [ 3 H] over cyclic [ 32 P]AMP radioactivity was due to extracellular formation of more penetrable dephosphorylated cyclic AMP metabolites which probably served as precursor of intra-cellular cyclic AMP. 2. 2. Less than 10% of intracellular cyclic [ 32 P] of cyclic [ 3 H]AMP survived enzymatic degradation whereas only one-half of intracellular dibutyryl cyclic [ 3 H]AMP was degraded during a 60-min incubation. Nonetheless, these data indicate that incubation of cells with 3 mM cyclic AMP would result in intracellular cyclic AMP levels considerably higher than those measured in thyrotrophin-stimulated cells. 3. 3. Conclusions: (a) the ability of dibutyryl cyclic AMP to simulate TSH effects on thyroid in vitro , and the inability of cyclic AMP to do so do not appear to be due to the greater accumulation of the substituted nucleotide inside the cell, (b) although susceptibility of cyclic AMP to intracellular enzymic hydrolysis may account for its biological ineffectiveness, intracellular cyclic AMP levels achieved in the presence of exogenous cyclic AMP are of sufficient magnitude to warrant other considerations, and, (c) effects of dibutyryl cyclic AMP on thyroid in vitro may be due predominantly to the substituted nucleotide itself or to N 6 -monobutyryl cyclic AMP rather than to cyclic AMP formed by deacylation.