The effects of vasopressin and catecholamines on cyclic AMP in homogenates of rat brain

Vasopressin is known to mediate its action on the kidney through increasing the concentrations of cyclic AMP. As vasopressin is widely distributed in many extra hypothalamic areas of the brain and can be shown to act centrally, we have investigated the effect of vasopressin on cyclic AMP levels in homogenates of the striatal and locus coeruleus areas. In contrast with the effect obtained on the kidney, vasopressin did not stimulate adenyl cyclase activity in rat brain homogenates in a dose-related manner. The stimulation of cyclic AMP observed with dopamine or noradrenaline in these brain areas and the hippocampus was not affected by the presence of vasopressin. These observations suggest that the action of vasopressin on the brain is not mediated through cyclic AMP.     

Differential long-term effects of tannic acid on adenyl cyclase activity and lipolysis in rat adipocytes

Plants elaborate a variety of secondary metabolites such as hydrolysable tannins which are relatively abundant in fruits, vegetables and beverages in the human diet. We have studied the in vivo long-term effect consumption of tannic acid-supplemented drinking water (0.05%, w/v) on the rat adipocyte adenyl cyclase system and on lipolysis. We found that 14-day tannic acid supplementation did not significantly affect either body growth or food consumption, while fat pads weight was higher than that of the control, although the difference was not significant. On the other hand, tannic acid supplementation decreased both basal and isoproterenol-stimulated lipolysis significantly whereas cyclic AMP production as well as adenyl cyclase activity increased significantly. These results are at a first glance contradictory as cyclic AMP accumulation and lipolysis are positively correlated in rat adipocytes. They suggest at least that the tannic acid diet led to an inhibition of cyclic AMP-dependent protein kinase activity followed by a decrease in lipolysis in rat adipocytes, and to an increased activity of the type VI adenyl cyclase subunit of rat fat cells. This subunit is known to be negatively regulated under phosphorylation by cyclic AMP-dependent protein kinase. More in-depth studies are required to examine whether tannic acid could at least modify the expression of the catalytic subunit of adenyl cyclase, G-proteins and cyclic AMP-dependent protein kinase and/or alter their activities.     

Effects of adrenergic blocking agents on lipolysis and adenyl cyclase activity induced by vasoactive intestinal polypeptide (VIP).

VIP stimulates lipolysis and adenyl cyclase activity in the rat adipose tissue. VIP-induced lipolysis and adenyl cyclase activity are not affected by phenoxybenzamine. VIP-induced lipolysis is inhibited by propranolol but VIP-induced adenyl cyclase activity is not.     

Vasopressin affects adenylate cyclase activity in rat brain: a possible neuromodulator

The effect of vasopressin on adenylate cyclase activity was measured in the homogenates of selected rat brain regions. Adenylate cyclase activity in homogenate of the caudate nucleus did not change significantly with various concentrations of vasopressin. Furthermore, vasopressin did not reliably alter adenylate cyclase activity in various brain regions. Vasopressin in low concentrations significantly enhanced the activation of caudate adenylate cyclase activity by dopamine. This effect of vasopressin was dose dependent. Maximal enhancement by vasopressin occurred at 100 microM vasopressin. These results indicate that vasopressin may not have a direct effect on brain adenylate cyclase activity but appears to modulate the action of dopamine on brain adenylate cyclase.     

ONTOGENETIC DEVELOPMENT OF ADENYL CYCLASE AND PHOSPHODIESTERASE IN RAT BRAIN

Abstract— The activities of adenyl cyclase and phosphodiesterase were determined in homogenates of cerebrum, cerebellum and brain stem of rats of 1 day to 9 weeks of postnatal age. The activity of cerebral and brain stem adenyl cyclase measured either in the absence or presence of sodium fluoride increased rapidly for 2 weeks, reached at 20 days a maximum about three times (brain stem) or six times (cerebrum) that seen on day 1 and then declined slightly during the next several weeks. In contrast, activity of cerebrellar adenyl cyclase increased more slowly and reached a maximum at about 32 days. Activity of phosphodiesterase in cerebrum and brain stem increased several-fold during brain maturation; however, enzymic activity in the cerebellum decreased during the entire 9 week period.
In the pineal gland, adenyl cyclase activity measured in the absence of norepinephrine or sodium fluoride did not change significantly with age. However, enzymic activity measured in the presence of these agents increased with the age of the rat. Bilateral removal of the superior cervical ganglia at 1 day of age is known to arrest the sympathetic innervation of the pineal gland but did not prevent the development of this adenyl cyclase system activated by catecholamines or fluoride.     

Actions of vasopressin and isoprenaline on the ionic transport across the isolated frog skin in the presence and the absence of adenyl cyclase inhibitors MDL12330A and SQ22536.

1. The effects of both adenyl cyclase inhibitors (MDL12330A and SQ22536) have been studied on the ionic transport induced by vasopressin and isoprenaline across the frog skin. 2. MDL12330A inhibits the vasopressin action on the short-circuit current (SCC), confirming that this effect is cAMP-mediated. 3. On the other hand, isoprenaline action on the SCC is unaffected by MDL12330A. However, this lack of effect is not a sufficient argument against the role of cAMP in this action; in fact, as MDL12330A is also an inhibitor of cAMP phosphodiesterase, this action could mask the inhibitory effect of the drug on adenyl cyclase. 4. By using the other adenyl cyclase inhibitor (SQ22536), probably deprived of effect on the cAMP phosphodiesterase, we obtained a strong inhibition of isoprenaline action on the SCC. Thus we conclude that the actions of isoprenaline on the ionic transport across the frog skin are also cAMP-mediated.     

Effects of starvation, refeeding, and fat feeding on adipocyte ghost adenyl cyclase activity

Basal adenyl cyclase activity and its response to epinephrine and glucagon were studied in isolated adipocyte ghosts obtained from fed, starved, refed, and fat-diet-adapted rats. Epinephrine stimulation of adenyl cyclase was significantly increased in fasted rats, but the glucagon response did not change. Rats fasted for 48 hr and refed a high carbohydrate, low fat diet for 48 or 96 hr showed no differences from chow-fed animals in either basal or hormone-stimulated adenyl cyclase activity. Rats adapted to a high fat, low carbohydrate diet showed an initial and transitory increase in basal activity but a progressive loss of epinephrine- and glucagon-stimulated enzyme activities. The loss in hormone responsiveness correlated well with a decrease in hormone-stimulated lipolysis of fat pads and was associated with a significant increase in fat cell diameter.     

Adenyl cyclase activities in rat erythrocytes during stress erythropoiesis: localization of the enzyme in the reticulocytes

NaF- and isoproterenol-stimulated adenyl cyclase activity found in preparations from rat erythrocytes is obviously localized mainly in the reticulocytes. After treatment with acetylphenyl hydrazine, both reticulocyte counts and adenyl cyclase activity increase dose-dependently. Evidence is given that adenyl cyclase activity in the red blood cell is lost during the maturation process.     

Direct anesthetic-like effects of forskolin on the nicotinic acetylcholine receptors of PC12 cells

Forskolin is thought to be a highly specific activator of adenyl cyclase. However, when applied to rat pheochromocytoma (PC12) cells at concentrations of 1 microM or higher it caused an immediate, concentration-dependent inhibition of carbachol-stimulated uptake of 86Rb+ through the nicotinic receptors, which did not appear to be related to activation of adenyl cyclase. The inhibition of receptor activation occurred instantaneously whereas cellular cAMP content did not increase for a measureable period of time. Normal receptor function was recovered rapidly upon removal of forskolin. Additional evidence that this effect of forskolin was not related to cAMP was obtained when 1,9-dideoxyforskolin (an analog of forskolin which does not activate adenyl cyclase) also caused a rapid, concentration-dependent, rapidly reversible inhibition of receptor-mediated influx of 86Rb+ into the cells. An examination of the effect of forskolin on 86Rb+ uptake at various concentrations of carbachol showed that forskolin was not acting by competing with carbachol for the receptor activation site. Given the lipophilic nature of forskolin, it probably acts like a general anesthetic to perturb the plasma membrane lipid structure and alter the function of the nicotinic acetylcholine receptors, possibly by increasing the rate of closure of open channels.     

Effect of butaclamol and its enantiomers upon striatal homovanillic acid and adenyl cyclase of olfactory tubercle in rats.

Butaclamol, a new neuroleptic agent, and its (+)- enantiomer caused a pronounced dose-related elevation of rat striatal homovanillic acid concentration $\text{in}$$\text{vivo}$. In addition, each blocked the dopamine-induced increase in adenyl cyclase activity of homogenates of the olfactory tubercle, a limbic area in the brain. The (-)-enantiomer of butaclamol did not exhibit these activities indicating a stereochemical specificity for dopamine receptor-blockade activity. The (+)-enantiomer was 2–3 times more potent than butaclamol, exhibiting activities similar to those of fluphenazine. The present findings are consistent with the existence of relationships between changes in dopamine turnover in the striatum and the production of extrapyramidal side effects and between changes in adenyl cyclase activity of olfactory tubercle and antipsychotic activity.     

Adenyl Cyclase and the Differentiation of β-Adrenoreceptors

WE have studied the enzyme adenyl cyclase to extend the evidence that β-adrenoreceptors consist of at least two types, β-1 and β-2 (refs. 1-4.) Cyclic 3′,5′-AMP has been shown to be the intracellular mediator of the action of various hormones, including the catecholamines and it has been suggested that adenyl cyclase is an integral part of the β-adrenoreceptor^5,6. Thus adenyl cyclase preparations from certain tissues should differ in their responsiveness to β-adrenoreceptor agonists and antagonists in a manner analogous to the intact tissues. We have therefore examined the differential effects of some sympathomimetic amines and their antagonists on the activity of adenyl cyclase preparations from rat heart and lung.     

Ultracytochemical localisation and comparison of adenyl cyclase activities in pineal bodies of Wistar rats and mongolian gerbils

Summary In the pineal bodies of Wistar rats and mongolian gerbils (Meriones unguiculati) the main concentration of adenyl cyclase was found in capillary endothelial cells, their basal laminae, pericapillary amorphous material and in nerve endings. In the intercellular clefts between the rat pineal cells there was no adenyl cyclase activity, but a considerable amount of this enzyme was detected in the gerbils. The adenyl cyclase lacks completely in the pinealocyte cytoplasms of both species. It was concluded that the gerbil pineal cells may be more reactive to various hormonal influence than those of the rat.With the technical assistance of Miss Ch. Thommen and Mr. P.-A. MilliquetDedicated to Professor Dr. med. O. Bucher on the occasion of his 65th birthday     

Studies on the assay and activities of guanyl and adenyl cyclase of rat liver

In applying recently developed methods for measuring adenyl and guanyl cyclase activities, we found that some modifications produced much better cyclic nucleotide recovery, lower assay backgrounds, and greater reliability than previously reported. The reliability and specificity of the assay methods were confirmed by substrate and product analysis. Kinetic analysis of rat liver guanyl and adenyl cyclase was subsequently performed to investigate regulatory properties of both enzymes. The Michaelis-Menton constant of guanyl cyclase activity of a 30,000g supernatant fraction of rat liver for guanosine 5′-triphosphate (GTP) was 0.04 mm. This enzyme was competitively inhibited by adenosine 5′-triphosphate (ATP) (K_i = 0.011 mM). Guanyl cyclase was activated in vitro by secretin but unaffected by carbamylcholine, hist-amine, methoxamirie, serotonin, glucagon, and pancreozymin. Liver homogenate adenyl cyclase had a Michaelis-Menten constant for ATP of 0.2 mm. This enzyme was activated by secretin, pancreozymin, glucagon, sodium fluoride, and isoproterenol. GTP (0.005 mm) enhanced the activation by both isoproterenol and glucagon. Methoxamine had no effect on adenyl cyclase activity in the presence or absence of GTP. These results suggest that both guanyl cyclase and adenyl cyclase may be mediators of hormone action in the liver.     

Effect of guanyl nucleotides on the stimulation of adenyl cyclase activity in human thyroid plasma membranes by thyroid-stimulating hormone and prostaglandin E2

Thyroid homogenates and thyroid plasma membranes were prepared from human thyroid and the effects of thyroid-stimulating hormone (thyrotropin), NaF, and prostaglandins E₁ and E₂ on adenyl cyclase activity in these preparations were studied. The basal level of adenyl cyclase activity in plasma membranes was 5–8 times greater than that of the original homogenates. Adenyl cyclase activity in plasma membranes was stimulated 4.7-fold by 100 munits/ml of thyrotropin and 5-fold by 10 mM of NaF, but the activity in the homogenates was only stimulated 2-fold by either thyrotropin or NaF. Prostaglandin E₁ (10^?6?10^?3 M) and prostaglandin E₂ (10^?7?10^?4 M) failed to stimulate adenyl cyclase activity in plasma membranes, but they did stimulate adenyl cyclase activity in the homogenates. A marked stimulatory effect of prostaglandin E₂ (10^?5 M) on adenyl cyclase activity in plasma membranes resumed in the presence of GTP (10^?7?10^?4 M), although GTP itself only slightly stimulated enzyme activity. GDP and GMP were also effective in this respect, although their potencies varied from compound to compound. GTP potentiated slightly the action of thyrotropin on adenyl cyclase in plasma membranes, but it significantly depressed an increase of enzyme activity produced by NaF. Since GTP did not affect the ATP-regenerating system, it seems that GTP, GDP or GMP was required for the manifestation of prostaglandin E₂ action on adenyl cyclases of human thyroid plasma membranes.     

The cytochemical localization of adenyl cyclase activity in rat sublingual gland.

Adenyl cyclase activity in mucous acinar cells and serous demilune cells of the rat sublingual gland was localized cytochemically. After incubation with adenylyl-imidodiphosphate (AMP-PNP) as substrate, deposits of reaction product are found along the cell membranes bordering the secretory surfaces of serous demilune cells. These are the membranes which participate directly in secretion by fusing with the granule membranes. The granule membranes of the demilune cells do not reveal reaction product, but the membranes of the granules which are fused with and become part of the cell membrane do show deposits. Thus, it appears that the cell membranes which fuse with granule membranes during secretion are associated with a high level of adenyl cyclase activity. In support of this, the luminal membranes of the mucous acinar cells which do not fuse with granule membranes during secretion are not associated with detectable amounts of adenyl cyclase activity.     

Metabolism of Adenosine 3′,5′-Cyclic Monophosphate and Induction of Fruiting Bodies in Coprinus macrorhizus

The adenyl cyclase and phosphodiesterase metabolizing adenosine 3',5'-cyclic monophosphate (cyclic AMP) were detected in mycelia of strains of Coprinus macrorhizus which form fruiting bodies, but not in those of strains which do not form fruiting bodies. The adenyl cyclase synthesized cyclic AMP from adenosine triphosphate. The phosphodiesterase degr[UNK]ded cyclic AMP to adenosine-5'-monophosphate and was inhibited by adenosine-3'-monophosphate, theophylline, and caffeine. The strains which form fruiting bodies incorporated and metabolized cyclic AMP, but strains which do not form fruiting bodies did not. The possible participation of cyclic AMP in the induction of fruiting bodies is discussed.     

Distribution of enzymes of glycogen metabolism and calcium uptake in skeletal muscle

Homogenates of rat skeletal muscle yield on centrifugation a particulate fraction containing relatively large vesicles capable of calcium uptake and a second fraction of smaller vesicles devoid of this capacity. Presumably the vesicles are derived from the sarcoplasmic reticulum. The glycogen synthase, adenyl cyclase, and phospho-diesterase specific activities are similar in both fractions, while in fractions of muscle from diabetic rats there are increased adenyl cyclase specific activities compared to muscle from normal rats. These results are in accord with the hypothesis that insulin acts upon the sarcotubular system of muscle.     

Desensitization of immature rat testicular ornithine decarboxylase to arginine vasopressin

Prior exposure of immature rat testis to arginine vasopressin caused the testis refractory at 24 h in terms of ornithine decarboxylase activity. Arginine vasopressin caused desensitization both in Leydig cells and seminiferous tubules. Arginine vasopressin induced desensitization was found to be both time and dose-dependent. Arginine vasopressin desensitized testis was refractory to luteinizing hormone, follicle stimulating hormone, norepinephrine, dibutyryl cAMP, phorbol-myristate acetate and cholera toxin at 24 h. Arginine vasopressin desensitized testis showed recovery of response to norepinephrine at 48 h after the first injection. On the contrary arginine vasopressin could stimulate ornithine decarboxylase in luteinizing hormone desensitized testis. These results indicate that in arginine vasopressin desensitized testis the block is at post cAMP step which is common to both cAMP dependent and protein kinase C-diacylglycerol system in stimulating testicular ornithine decarboxylase.     

Glucagon antagonists. Synthesis and inhibitory properties of Asp3-containing glucagon analogs

In an effort to find analogs of glucagon that would bind to the glucagon receptor of the rat liver membrane but would not activate membrane-bound adenyl cyclase, several hybrid molecules were synthesized which contained sequences from both glucagon and secretin. [Asp3, Glu9]Glucagon and [Asp3, Glu9, Arg12]glucagon were inactive in the adenyl cyclase assay even at high concentrations but retained some binding affinity for the receptor. They were able to displace 125I-glucagon completely from its receptor and could completely inhibit the activation of adenyl cyclase by natural or synthetic glucagon. The inhibition index [I/A]50 was approximately 110 for both analogs. [Asp3]Glucagon, [Glu3]glucagon and [Asp3, Lys17, 18, Glu21]glucagon were weak partial agonists, while [Asp3, Glu21]glucagon was inactive and a poor inhibitor. The peptides were synthesized by solid-phase methods and purified to homogeneity by reverse-phase high-performance liquid chromatography on C18 silica columns. These are the first fully synthetic competitive glucagon antagonists to be reported.     

Regulation of diamine oxidase expression by beta 2-adrenoceptors in normal and hypertrophic rat kidney

The administration of preferential adrenergic receptor antagonists to uninephrectomized rats revealed the beta 2-adrenergic mediation in diamine oxidase activity increase that occurs in the remaining kidney undergoing compensatory hypertrophy. In fact, beta 1, beta 2- or beta 2, but not alpha 1-, alpha 2-, or beta 1-receptor-blocking agents prevented this enzyme enhancement. Further studies with adrenoceptor agonists, such as epinephrine (alpha 1, alpha 2, beta 1, beta 2), isoproterenol (beta 1, beta 2) or terbutaline (beta 2) showed that also in normal rat kidney diamine oxidase activity is under the control of catecholamine-beta 2-receptors through a mechanism that involves new synthesis of mRNA and protein. Theophylline, an inhibitor of phosphodiesterase, or forskolin, an activator of adenyl cyclase, increased diamine oxidase activity as does epinephrine or nephrectomy. Thus, catecholamine-triggered beta 2-receptors coupled to adenyl cyclase are involved in the regulation of diamine oxidase activity in normal and hypertrophic rat kidney.