Renal effects of cyclic AMP in normal and congenital diabetes insipidus rats

The administration of dibutyryl cyclic AMP to normal rats undergoing water diuresis and to rats with congenital diabetes insipidus resulted in a rise in the excretion of Na⁺ and K⁺. A reduction in free water clearance was also observed in the normal rat, but this could not be entirely attributed to the effect of the nucleotide alone. Infusion of cyclic AMP to Brattleboro rats led to a modest rise in urine osmolality and a fall in urine flow, free water clearance and solute excretion, all of which could be explained on the basis of a fall in GFR. From the present experiments, it may be concluded that at the doses used neither cyclic AMP nor its dibutyryl derivative mimic the effects of ADH on water reabsorption by the kidney in vivo.     

Similar and opposite effects of dibutyryl cyclic AMP and insulin on glucose metabolism in adipose tissue

The effects of N⁶-2′-O-dibutyryl cyclic AMP on glucose metabolism and lipolysis in fragments of rat epididymal adipose tissue were studied. Measurements were made of glucose uptake, conversion of glucose carbon to CO₂ and tissue fatty acids and glyceride-glycerol, lactate production, and glycerol release. Low concentrations of dibutyryl cyclic AMP (0.1–0.5 mM) increased all parameters of glucose metabolism and inhibited glycerol release in tissue from both normally fed and fasted rats. Higher concentrations of dibutyryl cyclic AMP (3–5 mM) diminished glucose utilization and greatly accelerated lipolysis. Insulin, 50 μunits/ml, accelerated glucose metabolism in the presence of either low or high concentrations of dibutyryl cyclic AMP though the effect of insulin was greatly reduced by 3 mM dibutyryl cyclic AMP. Tissue exposed to concentrations of dibutyryl cyclic AMP which inhibited glucose metabolism (5 mM), then rinsed and reincubated without dibutyryl cyclic AMP, displayed increased glucose utilization. The results of these experiments emphasize the need for caution in interpretation of the effects of dibutyryl cyclic AMP on adipose tissue metabolism and the need for further research to elucidate the role of cyclic AMP in the regulation of glucose metabolism.     

STIMULATION OF BRAIN SEROTONIN SYNTHESIS BY DIBUTYRYL-CYCLIC AMP IN RATS

Cyclic AMP and dibutyryl-cyclic AMP, a derivative of cyclic AMP resistant to phosphodiesterase inactivation, were injected into the lateral ventricles of rats. These nucleotides did not change the level of brain 5-HT but increased the brain level of its principal metabolite, 5-hydroxyindoleacetic acid. Cyclic AMP was less potent than dibutyryl-cyclic AMP. Butyrate and 5′-AMP were inactive. The effect of dibutyryl cyclic AMP on 5-HT metabolism was studied both in vivo and in vitro. The rate of synthesis of 5-HT was measured by the rate of accumulation of 5-hydroxyindoleacetic acid after the transport of this acid out of the brain was blocked with probenecid. The rate of synthesis of brain 5-HT increased from 0-38 μg/g/h in control rats to 0-65 μg/g/h after dibutyryl-cyclic AMP. In addition cyclic AMP and dibutyryl-cyclic AMP markedly increased brain tryptophan, while AMP was inactive. Since brain tryptophan hydroxylase has a K_m for its substrate that is much higher than the concentrations of tryptophan normally present in the brain, it is likely that the increase in the rate of synthesis of brain 5-HT is secondary to the cyclic AMP induced increase in the levels of brain tryptophan. In vitro studies revealed that dibutyryl-cyclic AMP increased the uptake of radioactive labelled tryptophan into slices of rat brain stem and the formation of 5-HT and 5-hydroxyindoleacetic acid.     

Cyclic AMP-induced tyrosinase synthesis in Neurospora crassa

Cyclic AMP induces the synthesis of tyrosinase in $\text{Neurospora crassa}$. Adenine, adenosine, 3′-AMP, 5′-AMP, and 2′,3′-cyclic AMP have no inductive effect while 8-bromocyclic AMP and dibutyryl cyclic AMP are good inducers. Caffeine and theophylline, inhibitors of cyclic AMP phosphodiesterase, also induce tyrosinase. A possible relationship between cyclic AMP induction and previously reported induction by cycloheximide is suggested.     

Mechanism of allylisopropylacetamide-induced increase of delta-aminolevulinate synthetase in liver mitochondria. Effects of administration of glucose, cyclic AMP, and some hormones related to glucose metabolism

The allylisopropylacetamide-induced increase of δ-aminolevulinate synthetase in the rat liver was significantly reduced when any one of glucose, ATP, cyclic 3′,5′-AMP, dibutyryl cyclic 3′,5′-AMP, theophylline, insulin, or glucagon was given to rats simultaneously with the administration of allylisopropylacetamide. Administration of these substances to the rats not given allylisopropylacetamide resulted in decrease in enzyme activity in the liver. However, when these substances were given to rats after an intensive induction had commenced, the level og δ-aminolevulinate synthetase in the liver cytosol increased greatly, while the enzyme level in the mitochondria decreased markedly, so that the increase in the total activity of δ-aminolevulinate synthetase in the liver was not appreciably reduced except that the total activity in the glucose-treated rats was considerably lower than that in the control rats. Moreover, the half-life of the δ-aminolevulinate synthetase in cytosol was much longer when rats were given dibutyryl cyclic AMP. These findings are quite similar to those observed after the administration of hemin to rats treated or untreated with allylisopropylacetamide and suggest that these substances, as well as hemin, inhibit in some way both the induction of δ-aminolevulinate synthetase and the conversion of the cytosol δ-aminolevulinate synthetase to the mitochondrial δ-aminolevulinate synthetase. Dibutyryl cyclic AMP and glucagon were effective even in alloxan-diabetic rats, suggesting that the effects of cyclic AMP and glucagon may not be mediated by insulin.     

Interralation of prostaglandin endoperoxide (prostaglandin G2) and cyclic 3′,5′-adenosine monophosphate in human blood platelets

The prostaglandin endoperoxide, prostaglandin G₂, in platelet-rich plasma may produce reversible platelet aggregation without secretion, irreversible aggregation with secretion of platelet constituents inhibited by indomethacin, or the latter effects despite indomethacin, depending on the concentration of the endoperoxide. Irreversible aggregation and platelet secretion induced by prostaglandin G₂ apparently result from the action of ADP, since these responses are inhibited by 2-n-amylthio-5′-AMP (an inhibitor of the actions of ADP on platelets) and they do not occur in heparinized platelet-rich plasma. Prostaglandin G₂ lowers the platelet level of cyclic 3′,5′-AMP. Its actions are inhibited by elevation of cyclic AMP levels by prostaglandin E₁ or dibutyryl cyclic AMP or adenosine. Like malondialdehyde production induced by thrombin, ADP, or arachidonic acid, prostaglandin G₂-induced malondialdehyde production is reduced by dibutyryl cyclic AMP and prosraglandin E₁. Platelet activation by prostaglandin G₂ is enhanced by the adenylate cyclase inhibitor, 9-(tetrahydro-2-furyl)-adenine.The action of prostaglandin G₂ on platelets is more complex then previously reported.     

Apparent opposing effects of cyclic AMP and dibutyryl-cyclic GMP on the neuronal firing of the blowfly chemoreceptors

Cyclic AMP and its dibutyryl derivative inhibit neuronal firing of the labellar sugar sensitive receptor of the blowfly when applied in conjunction with the stimulant sucrose. Furthermore, simultaneous application of aminophylline (phosphodiesterase inhibitor) and sucrose or in combination with cyclic AMP caused a similar depression of the sugar receptors response. In contrast, dibutyryl cyclic GMP elicited an increase in sugar receptor firing when applied with sucrose to the sugar receptor. Either 5′-AMP or 5′-GMP in combination with sucrose had no discernable effect on the sugar receptors response. Different ratio combinations of cyclic AMP and dibutyryl cyclic GMP showed the striking inhibitory effect of cyclic AMP upon the dibutyryl cyclic GMP elicited increases in receptor firing frequency. Therefore, it is suggested that these two nucleotides may be mediating different but complimentary aspects of sugar receptor function in a push-pull manner.     

N6,O2′-dibutyryl adenosine 3′,5′-monophosphate-stimulated release of proteoglycans from cultured immature rabbit ear cartilage

The stimulatory effects of N⁶,O^2′-dibutyryl adenosine 3′,5′-monophosphate on proteoglycans released from immature rabbit ear cartilage were studied in vitro. Cartilage incubated in medium containing dibutyryl cyclic AMP resulted in a significant increase of proteoglycans released in concentrations above 0.5 mM. Theophylline (1 mM) which did not significantly stimulate proteoglycans released alone, was found to potentiate the action of this nucleotide. ATP, 5′-AMP and butyric acid in the presence of theophylline, did not stimulate proteoglycans released. The addition of protein or RNA synthesis inhibitors depressed proteoglycans released by dibutyryl cyclic AMP and theophylline.Gel chromatographic and chemical investigations of the proteoglycans released into the culture media in the presence of dibutyryl cyclic AMP indicated a reduction in the proportion of protein associated with these complexes. This result, together with enzyme inhibitor studies, leads us to speculate that the observed action of dibutyryl cyclic AMP on rabbit ear cartilages may be mediated by the neural proteases.     

The effect of prostaglandin synthetase inhibitors on renal function: Studies in normal rats during sodium or water diuresis and in rats with chronic renal insufficiency

The effect of acute infusion of the prostaglandin synthetase inhibitors — meclofenamate or indomethacin — was examined in awake rats. Studies were performed in normal rats undergoing either sodium or water diuresis and in salt-replete rats with chronic renal insufficiency. Prostaglandin synthetase inhibitors had no effect on renal plasma flow, glomerular filtration rate or fractional excretion of sodium in any of the groups. Absolute urinary excretion rates for sodium and potassium decreased only in the normal, salt-replete rats. In contrast, prostaglandin synthetase inhibitors consistently decreased urinary flow and osmolar clearance under all experimental conditions studied. In the normal, salt-replete rats the fall in urine flow was preceded by an increase in urinary excretion of cyclic AMP. These results show that inhibitors of prostaglandin synthesis enhance the ability of the kidney to reabsorb water. This effect may be secondary to increased cyclic AMP generation and to increased urea recirculation resulting in higher urea accumulation in the renal medulla.     

Renal effect of acute hypobaric pressure breathing in normal and diabetes insipidus rats

The role of blood volume regulatory mechanisms located in the low pressure system in the control of urinary excretion was studied using hypobaric pressure breathing in normal and diabetes insipidus (Brattleboro strain with a congenital lack of vasopressin) rats. Rats were placed in an altitude simulator chamber for 4 h. A pump maintained pressure reduced to 701, 577 and 472 mbar simulating respectively altitude of 3,000, 4,500 and 6,000 m. In normal rats, hypobaric breathing induced an increase in urine flow, urinary urea and K+ excretion and urinary pH but did not significantly modify creatinine and Na+ excretion. In diabetes insipidus rats, hypobaric breathing produced oliguria and an decrease in urea, creatinine, Na+, K+, Cl- urinary excretions. Since acute hypobaric pressure breathing induced opposed effects in normal and Brattleboro rats, it is suggested that this kind of experimental procedure which increases intrathoracic blood volume elicits a diuretic response through an inhibition of vasopressin release. These experiments confirm the main role of vasopressin in the control of central blood volume.     

The effect of prostaglandin synthetase inhibitors on renal function: studies in normal rats during sodium or water diuresis and in rats with chronic renal insufficiency

The effect of acute infusion of the prostaglandin synthetase inhibitors - meclofenamate or indomethacin - was examined in awake rats. Studies were performed in normal rats undergoing either sodium or water diuresis and in salt-replete rats with chronic renal insufficiency. Prostaglandin synthetase inhibitors had no effect on renal plasma flow, glomerular filtration rate or fractional excretion of sodium in any of the groups. Absolute urinary excretion rates for sodium and potassium decreased only in the normal, salt-replete rats. In contrast, prostaglandin synthetase inhibitors consistently decreased urinary flow and osmolar clearance under all experimental conditions studied. In the normal, salt-replete rats the fall in urine flow was preceded by an increase in urinary excretion of cyclic AMP. These results show that inhibitors of prostaglandin synthesis enhance the ability of the kidney to reabsorb water. This effect may be secondary to increased cyclic AMP generation and to increased urea recirculation resulting in higher urea accumulation in the renal medulla.     

Nicotinic acid reverses fasting ketosis by lowering the level of cyclic AMP.

When nicotinic acid was administered intraperitoneally to fasted rats, it reversed ketosis, decreased concentrations of cyclic AMP in adipose tissue and liver, and partially suppressed lipolysis. Administration of dibutyryl cyclic AMP reinduced ketosis in fasted rats previously treated with nicotinic acid. The results that nicotinic acid reverses ketosis by lowering tissue levels of cyclic AMP with a consequent suppression of lipolysis and ketogenesis.     

Cyclic AMP-binding proteins in human blood platelets detected by photoaffinity labelling

Cyclic AMP inhibits platelet aggregation induced by physiological agents. 8 Azido [³²P]cyclic AMP (N₃ cyclic AMP) has been utilized as a photoaffinity probe to define the cyclic AMP-binding proteins present in unperturbed human platelets and their subcellular fractions. Specificity of cyclic AMP binding was determined by contrasting binding in the presence and absence of excess unlabelled cyclic AMP, cyclic GMP and 5′-AMP. Binding was unaffected by 5′-AMP and obliterated by cyclic AMP. Four major species of binding proteins, 49 000, 42 000, 39 000, 37 000, were obtained in all platelet fractions (crude homeogenate, cytosol, membranes and granules). Two-dimensional gel electrophoresis of platelet cytosol resolved the major molecular weight species into 15 specific cyclic AMP binding proteins of four molecular weight classes differing by charge density. These studies suggest that platelets contain an array of specific cyclic AMP-binding proteins which may function in hemostatic regulation.     

Glutamine metabolism of isolated rat hepatocytes. Evidence for catecholamine activation of alpha-ketoglutarate dehydrogenase

Effects of norepinephrine on gluconeogenesis and ureogenesis from glutamine by hepatocytes from fasted rats were assessed. Comparisons were made to asparagine metabolism and to the effects of NH4Cl and dibutyryl cyclic AMP. With asparagine as substrate, aspartate content was very high but norepinephrine, dibutyryl cyclic AMP, or NH4Cl had little effect on gluconeogenesis or ureogenesis. Metabolism of asparagine could be greatly enhanced by the combination of oleate, ornithine, and NH4Cl. However, even under these conditions, asparatate content remained high, and norepinephrine and dibutyryl cyclic AMP had little influence on glucose or urea synthesis. With glutamine as substrate, aspartate content was much lower, but was greatly elevated by norepinephrine, dibutyryl cyclic AMP, or NH4Cl. Each of these effectors strongly stimulated glucose and urea formation from glutamine. NH4Cl stimulation was accompanied by an increased glutamate and decreased alpha-ketoglutarate content. This suggests the mechanism for NH4Cl stimulation is a near-equilibrium adjustment to ammonia by glutamate dehydrogenase and aspartate aminotransferase rather than a principal involvement of glutaminase. Although both norepinephrine and dibutyryl cyclic AMP lowered alpha-ketoglutarate to the same extent, norepinephrine more rapidly increased aspartate content and led to a smaller accumulation of glutamate than did dibutyryl cyclic AMP. Moreover, only norepinephrine led to a rapid increase in succinyl-CoA concentration. The catecholamine effect could not be explained by specific changes in cytosolic or mitochondrial redox states. The results suggest that alpha-ketoglutarate dehydrogenase is a site of catecholamine action in rat liver. Since purified alpha-ketoglutarate dehydrogenase is known to be Ca2+ stimulated and Ca2+ flux is involved in catecholamine action, these findings also suggest that mitochondrial Ca2+ is elevated by catecholamines.     

Inhibition of "spontaneous," notochord-induced, and collagen-induced in vitro somite chondrogenesis by cyclic AMP derivatives and theophylline.

The present study represents a first step in investigating the possible involvement of cyclic AMP in the stimulation of somite chondrogenesis elicited by extracellular matrix components produced by the embryonic notochord. Dibutyryl cyclic AMP (db-cAMP) at 1.0 mM severely impairs “spontaneous” somite chondrogenesis, i.e., inhibits the formation of the small amount of cartilaginous matrix normally formed by embryonic somites in vitro in the absence of inducing tissues. This inhibition of cartilaginous matrix formation is reflected in a 30–40% reduction in sulfated glycosaminoglycan (GAG) accumulation. 8-Bromo-cyclic AMP also severely inhibits cartilage formation and sulfated GAG accumulation by somite explants. This impairment is limited to cyclic AMP derivatives; dibutyryl cyclic GMP, 5′-AMP, and 2′,3′-AMP have no effect. The inhibitory effect of cyclic AMP derivatives is mimicked by the cyclic AMP-phosphodiesterase inhibitor, theophylline, and potentiated by the addition of both db-cAMP and theophylline. Dibutyryl cyclic AMP and/or theophylline also inhibit the stimulation of cartilaginous matrix formation and sulfated GAG accumulation normally elicited by the embryonic notochord, reducing accumulation to a level similar to that found in somite explants without notochord. The inhibition of chondrogenesis by cyclic AMP in notochord-somite explants appears to result from an inability of somites to respond and not from an effect on the inductive capacity of the notochord, since db-cAMP has no detectable effect on the synthesis of molecules (sulfated GAG and collagen) by the notochord that have been implicated in its inductive activity. Finally, db-cAMP and/or theophylline inhibit the stimulation of somite chondrogenesis normally elicited by purified Type I collagen substrates. Dibutyryl cyclic AMP and theophylline reduce sulfated GAG accumulation by somites cultured on collagen to a level even below that accumulated by somites cultured in the absence of collagen, i.e., on Millipore filters.     

Measurements of brain amobarbital concentrations in rats anesthetized and overdosed by amobarbital and treated centrally with dibutyryl cyclic AMP.

Three doses of amobarbital, an anesthetic dose (80 mg/kg), a minimum lethal dose (130 mg/kg), and a high lethal dose (180 mg/ kg) were administered intraperitoneally to groups of rats. All rats were injected intracerebroventricularly with 0.9% saline or dibutyryl cyclic AMP, 200 μg/rat. While those rats treated with saline solution were still sleeping at decapitation and those treated with dibutyryl cyclic AMP were awake, there were no significant differences in their brain concentrations of amobarbital.     

Possible influence of tachykinins on body fluid homeostasis in the rat

The effect on water intake, urine flow and vasopressin release of intracranial injections of substance P, physalaemin and eledoisin was studied in Wistar and Brattleboro, homozygous and heterozygous, rats. The tachykinins strongly inhibited water intake both in Wistar and in Brattleboro, homozygous and heterozygous, rats. Physalaemin and eledoisin reduced urine flow in Wistar and heterozygous, but not in homozygous, Brattleboro rats. Substance P never affected urine elimination. Physalaemin and eledoisin produced a dose-dependent, long lasting release of vasopressin in Wistar rats. Substance P did not affect the release of vasopressin. The results suggest that both substance P and physalaemin could influence brain mechanisms which control water intake, acting as thirst inhibitors, and that physalaemin could also participate in body fluid control by conserving water through vasopressin release.     

1,N6-etheno-2-aza-adenosine 3',5'-monophosphate: a new fluorescent substrate for cycle nucleotide phosphodiesterase

1,N⁶-etheno-2-aza-adenosine 3′,5′-monophosphate (cyclic 2-aza-?-AMP) has been shown to be a sensitive and an efficient substrate for the assay of cyclic-nucleotide phosphodiesterase. The relative activity is 75% compared to cyclic AMP. Two K_m values of 503 and 15 μm were observed with the beef heart enzyme.     

Cyclic AMP and platelet prostaglandin synthesis

The present study has investigated the influence of agents which elevate intracellular levels of endogenous platelet adenosine 3′5′-cyclic monophosphate (cyclic AMP), and the effect of the exogenous cyclic AMP analog, dibutyryl cyclic AMP, on the conversion of ¹⁴C-arachidonic acid by washed platelets. Prostaglandin E₁ (PGE₁), PGE₁ with theophylline, or dibutyryl cyclic AMP incubated with washed platelets prevented arachidonic acid induced platelet aggregation, but had no effect on the conversion of arachidonic acid to 12L-hydroxy-5,8,10, 14-eicosatetraenoic acid (HETE), 12L-hydroxy-5,8,10 heptadecatrienoic acid (HHT), or thromboxane B₂. Ultrastructural studies of the platelet response revealed that agents acting directly or indirectly to increase the level of cyclic AMP inhibited the action of arachidonic acid on washed platelets and prevented internal platelet contraction as well as aggregation. The influence of PGE₁ with theophylline, and dibutyryl cyclic AMP on the thrombin induced release of ¹⁴C-arachidonic acid from platelet membrane phospholipids was also investigated. These agents were found to be potent inhibitors of the thrombin stimulated release of arachidonic acid from platelet phospholipids, due most likely to an inhibition of platelet phospholipase A activity. The results show that dibutyryl cyclic AMP and agents which elevate intracellular cyclic AMP levels act to inhibit platelet activation at two steps 1) internal contraction and 2) release of arachidonic acid from platelet phospholipids.