Regulation of cyclic AMP levels by phorbol esters in bovine adrenal medullary cells

Cyclic AMP responses to phorbol esters were studied in cultured bovine adrenal medullary cells. Phorbol esters that activate protein kinase C (PKC: phorbol 12,13-dibutyrate, phorbol 12,13-didecanoate) increased cellular cyclic AMP levels by up to 100% over 5 min, and this was maintained for up to 3 h. The effect was mimicked by 1,2-dioctanoyl-sn-glycerol but not by inactive phorbol esters. The effect of active phorbol esters was concentration dependent over the range 50–500 nM, and was abolished by the PKC inhibitor, Ro 31–8220 (10μM). The response was enhanced by 3-isobutyl-1-methylxanthine (1 mM) and by forskolin (0.3 μM), was enhanced following pertussis toxin pretreatment (100 ng/ml, 7.5 h) and was unaffected by removing extracellular Ca²⁺. The phorbol ester cyclic AMP response was additive with that to K⁺ depolarisation, and synergised with those to prostaglandin E₁ and dimaprit. The results indicate PKC activation increases cyclic AMP formation in bovine adrenal medullary cells, probably by a direct action on adenylate cyclase or G_s.     

Ethanol Dependent Interaction between Prostaglandins and Lipoxygenase Products in Human Peripheral Lymphocytes

The effect of lipoxygenase products, 12-L-HETE and 15-L-HPETE, on cyclic AMP levels in human peripheral lymphocytes was examined in the absence and in the presence of a prostaglandin of the E-type (0.6–3.0 μM) or isoprenaline (33 μM). The studies were performed either in the absence or in the presence of 6 per cent ethanol. For comparison the effect of arachidonic acid and linolenic acid were studied. In the absence of ethanol 12-L-HETE and 15-L-HPETE had no significant effect on cyclic AMP accumulation. However, in the presence of ethanol 12-L-HETE (above 1 μM) inhibited prostaglandin E₁ but not isoprenaline induced cyclic AMP accumulation. 15-L-HPETE had a biphasic effect on prostaglandin E₂ induced cyclic AMP accumulation. Concentrations below 1 μM stimulated, those above inhibited. Virtually complete inhibition was seen at 15 μM. The two other fatty acids inhibited both prostaglandin E₂ and isoprenaline induced cyclic AMP accumulation in the presence, but not in the absence of ethanol. The results show that lipoxygenase products have little or no effect on cyclic AMP accumulation in human peripheral lymphocytes unless ethanol is present. In the presence of ethanol both 12-L-HETE and 15-L-HPETE appeared to selectively affect the cyclic AMP accumulation stimulated by PGE.     

Inhibition of cyclic AMP phosphodiesterase by 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylic acid 3-[2-(N-benzyl-N-methylamino)] ethyl ester 5-methyl ester hydrochloride (YC-93), a potent vasodilator.

A new, potent vasodilator (YC-93), 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylic acid 3-[2-(N-benzyl-N-methyl amino)] ethyl ester 5-methyl ester hydrochloride, competitively inhibited cyclic adenosine 3′,5′-monophosphate (cyclic AMP) phosphodiesterase in the 105,000 g supernatant solutions from canine basilar, carotid, coronary and femoral arteries. The inhibition constant (K_i) of YC-93 for these enzyme preparations was in the range of 2.0–4.3 μM at substrate concentrations near the low K_m (about 1 μM for each enzyme preparation), and was 4.0–12 μM at substrate concentrations near the high K_m (50–70 μM). The potency of YC-93 for inhibition of coronary phosphodiesterase at 1 μM cyclic AMP and 50 μM cyclic AMP was much greater than that of papaverine and 3-isobutyl-l-methyl xanthine (IBMX). Commercially available cyclic AMP phosphodiesterase purified from beef heart was also strongly inhibited by YC-93 in a competitive manner and its K_i value was 2.0 μM in the wide range of substrate concentrations tested. Studies on the structure-activity relationship using low K_m phosphodiesterase from canine coronary artery and high K_m phosphodiesterase from beef heart, demonstrated that 3,5-diethoxycarbonyl-1,4-dihydro-2, 4,6-collidine, the simplest 1,4-dihydropyridine derivative (tested in the present studies) resulted in slightly less inhibition than papaverine, and the inhibitory potency of the former compound was greatly increased mainly by two structural modifications. Firstly, addition of a nitrophenyl group at position 4 of the 1,4-dihydropyridine ring, secondly, the replacement of ethylester at position 3 of the 1,4-dihydro-pyridine ring by N-benzyl-N-methylaminoethyl ester. A few dihydropyridine derivatives together with YC-93 were the most potent inhibitors of cyclic AMP phosphodiesterase among the compounds tested. The finding that the level of cyclic AMP in canine arterial strips was increased by 64 per cent (P < 0.01) even after 1 min exposure to 1 μM YC-93 supports the possibility of at least a partial involvement of phosphodiesterase inhibition in vasodilation by the drug.     

Effect of histamine on adenosine 3',5'-cyclic monophosphate levels in granulation tissue

The effect of histamine on cyclic AMP levels in rat granulation tissue was examined in vivo and in vitro. Granulomas formed by subcutaneous implantation of formalin-soaked filter paper disks were used as granulation tissue. Histamine injection (5 mg/kg, i.p.) produced a significant increase in the cyclic AMP levels for 10–30 min after injection. Administration of either burimamide or mepyramine at 1 mg/kg, s.c., 30 min before histamine administration blocked the cyclic AMP increase induced by 5 mg/kg histamine. Burimamide was more effective than mepyramine. Neither of these histamine antagonists at 1 mg/kg blocked the cyclic AMP increase induced by adrenaline (1 mg/kg, i.p.). Propranolol at 1 mg/kg, s.c., reversed the cyclic AMP increase due to adrenaline but partially blocked a similar effect of histamine. Histamine (1 mM and less), added alone to the incubation medium in which the chopped granulation tissue was immersed, had little or no effect on cyclic AMP levels in the granulation tissue. However, when histamine (1 and 10 mM) was added concomitantly with 3-isobutyl-1-methylxanthine (IBMX) (1 mM), a significant increase in cyclic AMP occured, compared to the addition of IBMX alone. IBMX (1 mM) alone caused a significant increase in cyclic AMP compared to controls in which the drug was omitted. 2-Methylhistamine (5 mM) induced a cyclic AMP increase in the presence of IBMX (1 mM). Metiamide (0.01 mM) partially blocked the cyclic AMP increase caused by histamine (1 mM) in the presence of IBMX (1 mM). Mepyramine (0.01 mM) completely blocked this histamine effect. Propranolol (0.01 mM) markedly inhibited the effect of histamine(1 mM). These data suggest that histamine causes the cyclic AMP increase partly by the activation of H₂-receptors on the cells of granulation tissue and partly through the β-receptor action of catecholamines released, although the involvement of H₁-receptors cannot be excluded.     

Role of calcium and cyclic adenosine 3′,5′-monophosphate in the antilipolytic effect of tolbutamide and glibenclamide

Summary Tolbutamide (1 mM) and glibenclamide (0.1 mM) reduced lipolysis due to adrenaline, noradrenaline, isoprenaline, glucagon, and ACTH, whereas theophylline-induced glycerol production was only marginally affected. Furthermore, the effect of dibutyryl cyclic AMP on lipolysis was markedly decreased by low concentrations of both drugs. The antilipolytic action of glibenclamide and, to a smaller degree, of tolbutamide was highly dependent on the calcium concentration in the incubation medium used after preparation of fat cells in the absence of calcium. In the presence of isoprenaline as a lipolytic agent the antilipolytic effect of glibenclamide was progressively enhanced by raising the calcium concentration up to 1.2 mM. Howver, further elevation of calcium to 2.4 mM resulted in a loss of antilipolytic effect. Basal cyclic AMP levels of isolated fat cells were increased twofold by glibenclamide or tolbutamide. In contrast to the effect of glibenclamide, cyclic AMP levels in the presence of isoprenaline were further increased by tolbutamide. Low K _m cyclic AMP phosphodiesterase of fat cells was inhibited by tolbutamide as well as by glibenclamide. The results indicate that the antilipolytic action of sulfonylurea drugs is closely related to changes in calcium metabolism but independent of cyclic AMP levels.     

Phorbol esters impair endothelium-dependent and independent relaxation in rat aortic rings

• 1.1. This study examined the ability of various nitro-vasodilators, 8-bromo cyclic guanosine 3′:5′ monophosphate (8-BrcGMP) and forskolin to relax rings of rat thoracic aorta pre-contracted with either noradrenaline (0.1 μM) or the protein kinase C activators, phorbol 12,13-dibutyrate (PDB, 0.1 μM) or phorbol 12-myristate 13-acetate (PMA, 0.5 μM).
• 2.2. In noradrenaline pre-contracted rings, acetylcholine (10 nM−10 μM), sodium nitroprusside (1 nM−0.5 μM), the calcium ionophore A23187 (10 nM−10 μM) and 8-BrcGMP (10 mM) totally reversed the smooth muscle contraction. In PDB-contracted aortic rings acetylcholine, sodium nitroprusside and 8-BrcGMP-induced relaxation was reduced compared to that in noradrenaline-contracted aortic rings, but A23187 and forskolin-induced relaxations were unaffected. Both acetylcholine and A23187-induced relaxations in PDB-contracted rings were abolished in the presence of the nitric oxide synthesis inhibitor Nω-nitro-l-arginine (NOLA, 100 μM).
• 3.3. Acetylcholine and sodium nitroprusside were even less potent in their ability to relax PMA-contracted aortic rings compared with noradrenaline and PDB-contracted rings. A23187-induced relaxation was also inhibited in PMA-contracted rings.
• 4.4. These results show that protein kinase C activation reduces the ability of agents which liberate nitric oxide to induce smooth muscle relaxation, and also inhibits the biochemical pathways which are subsequently activated by nitric oxide and lead to vascular smooth muscle relaxation.

     

Studies on the mechanism of the positive inotropic action evoked by epinine on the rabbit isolated papillary muscle at different rates of beating

1. Effects of epinine on cyclic AMP and contractility were investigated in rabbit papillary muscles driven at a rate of 0.5 or 2.0 Hz. 2. When the frequency of stimulation was increased from 0.5 to 2.0 Hz, the log dose-response curve for the positive inotropic effect of epinine was displaced to the left, whereas the maximum of the developed tension was not changed. 3. At both frequencies phentolamine (1 μmol/1) shifted the lower part of the log dose-response curve for epinine to the right, whereas pindolol (30 nmol/1) affected mainly the upper part. In the presence of both a- and β-adrenoceptor antagonists, the whole curve was shifted to the right in a parallel manner. However, cocaine (30 μmol/1) did not significantly influence the log dose-response curve of epinine. 4. At 0.5 Hz a submaximal effective concentration of epinine (100 μmol/1) led to an approximately 100% increase of the cyclic AMP level after 60 s; the same increase of the cyclic AMP level was induced at 2.0 Hz by one-third the concentration of epinine (30 μmol/1). 5. Phentolamine (1 μmol/1) did not affect the increase of the cyclic AMP level evoked by epinine, whereas pindolol (30 μmol/1) completely depressed it. 6. The present results indicate that epinine produces its positive inotropic effect through direct stimulation of myocardial α-adrenoceptors as well as β-adrenoceptors, depending upon the concentration: in lower concentrations it acts mainly on α-adrenoceptors, whereas in higher concentrations it acts predominantly on β-adrenoceptors. The positive inotropic effect through α-adrenoceptor stimulation is mediated by cyclic AMP, while that through a-adrenoceptors is not.     

Identification of a beta 2-adrenergic receptor in mammalian epidermis

The presence of a beta₂-adrenergic receptor in the epidermis has been demonstrated, based on the following pieces of information: (1) the addition of salbutamol, a beta₂-agonist, to slices of epidermal tissue increased the levels of cyclic AMP in the tissue above control levels in a dose-dependent manner with a maximum response after 5 min of incubation in 5 × 10^?5 M salbutamol, (2) the addition of butoxamine, a beta₂-antagonist, in conjunction with isoproterenol or salbutamol reduced the epidermal cyclic AMP levels when compared to levels obtained with agonist alone, (3) practolol, a beta₁-antagonist. had little effect on the salbutamol-induced increases in the cyclic AMP levels and further elevated the levels of cyclic AMP obtained by the addition of isoproterenol, (4) the addition of propranolol to the tissue in conjunction with isoproterenol or salbutamol reduced the levels of cyclic AMP to near control values, and (5) Ro 20-1724, a cyclic nucleotide phosphodiesterase inhibitor, maintained the salbutamolelevated cyclic AMP levels for a longer period of time.     

Stimulation of adenosine 3′,5′-monophosphate formation in guinea-pig cerebral cortical slices in a calcium free medium

Summary In guinea-pig cerebral cortical slices cyclic AMP concentrations increase during incubation with histamine+noradrenaline. After 10 min of incubation the levels of cyclic AMP start to decline. When calcium ions are omitted from the incubation medium, cyclic AMP levels do increase to a greater extent under the same conditions and do not drop during 30 min incubation. In the presence of calcium ions cyclic AMP synthesis can not be elicited by noradrenaline alone. In calcium-free Krebs-Ringer solution a pronounced effect of noradrenaline on cyclic AMP levels is observed. This effect of noradrenaline is shown to be mediated by a classical -type receptor. 5-Hydroxytryptamine, prostaglandin E ₁ and dopamine do not significantly enhance cyclic AMP formation in guinea-pig brain slices in either the presence in, or the absence of calcium ions from the incubation medium. Under depolarizing conditions of incubation the stimulatory effect of ouabain or 125 mM K⁺ is blocked in a calcium-free medium, while with the depolarizing agent veratridine no significant reduction of cyclic AMP formed during incubation in a calcium-free medium is obtained.     

Augmentation by external Mg ions of beta-adrenoceptor-mediated actions in the longitudinal muscle of rat uterus.

1. The longitudinal muscle isolated from the uterus of oestrogen-treated rats was not spontaneously active in Locke solution, and electrical stimulation evoked phasic contraction. Isoprenaline (3 x 10(-11) - 10(-8) M) and dibutyryl cyclic AMP (db cyclic AMP, 0.1-0.8 mM) depressed the phasic contraction; the depression was enhanced in the presence of 0.6 mM Mg. 2. The contracture generated by 40 mM K was partially relaxed by isoprenaline (10(-11) - 10(-8) M) and db cyclic AMP (0.1-0.8 mM). Mg (0.6 mM) enhanced the isoprenaline-induced relaxation, but not that induced by db cyclic AMP. 3. The membrane potential of the muscle was -61 mV, and electrical stimulation induced an action potential which consisted of spike and plateau components. Application of isoprenaline and db cyclic AMP mainly reduced the duration of the plateau potential. The effect was potentiated by 0.6 mM Mg. 4. The membrane was hyperpolarized, accompanied by a decrease in membrane resistance, when 10(-8) M isoprenaline or 0.8 mM db cyclic AMP was applied. The effects of isoprenaline were prominently augmented in the presence of 1.2 mM Mg, while those of db cyclic AMP were slightly potentiated. 5. Forskolin (0.1 microM) or papaverine (10 microM) inhibited the phasic contraction and the K-contracture. The effect on the phasic contraction was potentiated by 0.6 mM Mg, while that on the K-contracture was not affected. 6. Forskolin shortened the action potential at 0.3 microM, and hyperpolarized the membrane with a decrease in membrane resistance at 3.0 microM. The membrane effects were augmented by 0.6 and 1.2 mM Mg, respectively. 7. It was hypothesized that external Mg ions could affect at least two processes involved in actions at beta-adrenoceptors on rat myometrium; receptor-agonist interaction and cyclic AMP-mediated inhibition of membrane excitability.     

Effect of cyclic nucleotides on activity of cyclic 3',5'-adenosine monophosphate phosphodiesterase

A series of cyclic 2′,3′-nucleotides, cyclic 3′,5′-nucleotides and derivatives of cyclic 3′,5′-adenosine monophosphate (cyclic AMP) with a substituent at the C-8 position were investigated as inhibitors of partially purified cyclic AMP phosphodiesterases (PDE) of cat heart and rat brain. The assays were carried out at a substrate concentration (0.06 μM) where the contribution to the total enzyme activity by phosphodiesterases with K_m values for cyclic AMP above 100 μM was insignificant; consequently the activity measured was that of low K_m enzymes.Cyclic 3′,5′-guanosine monophosphate (cyclic GMP) and cyclic 3′,5′-inosine monophosphate (cyclic IMP) were shown to be the most potent inhibitors of PDE of cat heart (I₅₀ = 1 and 2 μM, respectively). Of the cyclic AMP derivatives tested that have a substituent at the C-8 position, 8-bromo cyclic AMP was the most potent inhibitor; next most potent were the derivatives with a sulfur atom, whereas derivatives with oxygen- or nitrogen-containing substituents were the least potent inhibitors of PDE of cat heart or rat brain. Most of the cyclic nucleotides that were tested were more potent inhibitors of the PDE of cat heart than that of PDE of rat brain.The kinetic properties of PDE of cat heart were also investigated in the presence of cyclic GMP, cyclic IMP and 8-bromocyclic AMP. All three compounds were found to be competitive inhibitors, with apparent K_i values of 0.51, 2.3 and 20 μM respectively. The possible pharmacologic role of cyclic nucleotides is discussed.     

The alpha-adrenergic control of rabbit liver glycogenolysis.

We have confirmed that the electrical stimulation of the splanchnic nerve in the rabbit causes glycogenolysis m a cyclic AMP-independent way as found by Shimazu and Amakawa [1]; glycogen phosphorylase (1,4-α-d-Glucan: orthophosphate α-glucosyltransferase, EC 2.4.1.1) was activated, but phosphorylase b kinase (ATP: phosphorylase b phosphotransferase, EC 2.7.1.38) was not. We could, however, not confirm the observation of a decrease in phosphorylase phosphatase (phosphorylase a phosphohydrolase, EC 3.1.3.17) activity. Pretreatment of the rabbits with the α-adrenergic blocking agent phentolamine prevented the splanchnic nerve stimulation from activating glycogen phosphorylase.The addition of norepinephrine (10^?7 M) to isolated rabbit hepatocytes activated glycogen phosphorylase without an activation of phosphorylase b kinase. At 10^?6 M, norepinephrine activated both enzymes. Phentolamine blocked the activation of glycogen phosphorylase by norepinephrine at 10^?7M but not at 10^?6M. Absence of Ca²⁺ from the incubation medium prevented norepinephrine (10^?7 M) from activating glycogen phosphorylase. The ionophore A 23187 also caused an activation of phosphorylase (but not of phosphorylase b kinase) provided that Ca²⁺ was present in the incubation medium. These data indicate that sympathetic nervous control of liver glycogenolysis is achieved, via α-adrenergic receptors, by an increased concentration of cytosolic Ca²⁺ ions which stimulate rather than activate phosphorylase b kinase. The neurotransmitter involved is most probably norepinephrine.     

Cyclic nucleotides and contractility in human and sheep umbilical arteries

Strips of human umbilical artery (HUA) and sheep umbilical artery (SUA) were freeze-clamped at selected times during drug-induced contraction or relaxation. Tissue concentrations of cyclic AMP and cyclic GMP were measured. 5-Hydrpxytryptamine (10 μM) contracted HUA and SUA, but had no detectable effect on cyclic GMP; cyclic AMP rose in SUA (but not in HUA) after contractions had begun. Histamine (4 gmM) elevated cyclic GMP in HUA with a time course that lagged behind contraction. Prostaglandin E₁ (PGE₁), PGE₂ and PGF_2α (2.8 μM with 0.1 % v/v ethanol) contracted HUA and decreased cyclic GMP. Nitroglycerin (3 μM) relaxed KCl-contracted HUA; this relaxation was preceded by an initial 29-fold elevation of cyclic GMP. Our data shows no consistent correlation of cyclic nucleotide changes with contraction of unbilical arteries and suggest that such changes are not essential for contraction of vascular smooth muscle. In contrast, the temporal relationship between cyclic GMP elevation and relaxation induced by nitroglycerin suggests a possible role for cyclic GMP.     

Cyclic AMP independent inhibition by papaverine of histamine release induced by compound 48/80.

Compound 48/80-induced histamine release from isolated rat peritoneal mast cells was inhibited in a dose-dependent manner by papaverine (ic₅₀ approx 20 μM). This effect of papaverine was not influenced by PGE₁ (14–140 μM), even though PGE₁ markedly increased must cell cAMP levels. Papaverine (0.5 mM) completely inhibited histamine release without causing any change in cAMP levels. Theophylline (0.1 and 0.5 mM) potentiated histamine release induced by submaximal concentrations of compound 48/80, while cAMP levels were increased. IBM X was as potent as papaverine in causing inhibition of mast cell phosphodiesterase. IBM X (0.14–0.7 mM) had no effect on histamine release but caused a 6–20 fold increase in mast cell cyclic AMP. Papaverine inhibition of histamine release was gradual at the onset and was parallelled by a depletion of mast cell ATP content. The inhibition of 48/80-induced histamine release and depletion of mast cell ATP levels was reversed by glucose. It is concluded that papaverine induced inhibition of 48/80-induced histamine release is independent of cAMP, is unrelated to phosphodiesterase inhibition but is dependent upon inhibition of energy production.     

Effect of isoproterenol and acetylcholine treatment in vitro on cyclic nucleotides in mouse sex accessory organs.

The effect of treatment in vitro with either isoproterenol or acetylcholine on the levels of cyclic AMP and cyclic GMP in male mouse prostate gland and seminal vesicle was studied. In each experiment, tissues were preincubated with aminophylline (5 mM) for 10 min prior to exposure to the autonomic agonist. Treatment with 1 × 10^?5 M isoproterenol for 5 min resulted in significant increases in endogenous levels of cyclic AMP in both prostate glands and seminal vesicles obtained from intact mature male mice. This effect of isoproterenol on cyclic AMP could be blocked by pretreatment for 10 min with an equimolar (1 × 10^?5 M) concentration of propranolol. On the other hand, isoproterenol (1 × 10^?5 M for 5 min) had no effect on sex accessory organ levels of cyclic GMP. If sex accessory organs obtained from intact mature male mice were subjected to acetylcholine (1 × 10^?5 M for 5 min), significant increases (P < 0.05) in cyclic GMP were noted in the seminal vesicles; this effect could be blocked by atropine pretreatment (1 × 10^?5 M for 10 min). Just as isopro erenol had no effect on sex accessory cyclic GMP levels, acetylcholine produced no changes in cyclic AMP in these tissues. Although treatment with neurotransmitters resulted in striking increases in cyclic nucleotides in sex accessory tissues obtained from normal mice. this treatment had little or no effect on cyclic nucleotide levels in tissues obtained from 7-day castrate mice.     

Effect of adenosine on cyclic AMP accumulation in ventricular myocardium.

Adenosine stimulated cyclic AMP accumulation in guinea pig ventricular slice preparations. The response to adenosine was dose-dependent over the range 0.1 to 100 μM; half-maximal stimulation occurred at 25 μM. The response to the nucleoside was rapid; maximum levels of cyclic AMP were obtained in 3 min. Examination of a variety of adenosine analogues revealed that the 2'-, 3'-, and 5'-hydroxyl groups of the ribose moiety were important for activity. Agonist activity also required an amino group in the 6-position. Substitution of one hydrogen atom on the primary amino nitrogen did not alter activity, but substitution of both hydrogens abolished activity. Replacement of the N in position 7 of the purine ring with a C atom, or substitution of the hydrogen atom on position 8 with either an amino group or bromine atom abolished activity. Examination of the effect of several agents, papaverine, 5'-deoxyadenosine, 6-chloropurine riboside and $\text{6-N[(p-}\text{nitrobenzyl}\text{)}\text{thio}\text{]-9-β-}\text{d}\text{-}\text{ribofuranosyl}$ purine, which inhibited adenosine uptake into cardiac cells, provided evidence which suggested that the action of the nucleoside was mediated via interaction with a receptor on the external cell surface. Several phosphodiesterase inhibitors (papaverine, SQ 20009, RO-20-1724 and 3-isobutyl-1-methylxanthine) potentiated the effect of adenosine; theophylline, on the other hand, antagonized adenosine stimulation of cyclic AMP levels. Hexobencline potentiated the stimulatory action of low (10 μM) concentrations of adenosine, and seemed to do so by preventing adenosine uptake. Lidoflazine potentiated the action of both low (10 μM) and high (100 μM) concentrations of adenosine and appeared to act primarily as a phosphodiesterase inhibitor. Dipyridamole potentiated the actions of both low and high concentrations of adenosine probably by blocking adenosine uptake and by inhibiting phosphodiesterase.     

Adenosine antagonism by purines,pteridines and benzopteridines in human fibroblasts

Theophylline (K_i 5 μM) is a competitive inhibitor of the increase in cyclic AMP caused by adenosine in the VA13 fibroblast line. More than 100 purine bases and structurally related heterocycles were tested as adenosine antagonists. Three families of adenosine antagonists were found: xanthines, benzo[g]pteridines and 9-substituted adenines. For the xanthines, the optimal group at the 1-position was butyl (5-fold improvement versus methyl), at the 7-position was 2-chloroethyl (5-fold improvement versus hydrogen) and at the 8-position was p-bromophenyl (100-fold improvement versus hydrogen). The receptors appeared to have butyl- and phenyl-sized “pockets” at the 1- and 8-positions, respectively, since compounds with larger groups had greatly reduced activity.     

Lack of effect of cholinergic agents or endogenous guanosine 3':5'-monophosphate on renin release by slices of rat renal cortex in vitro

Cyclic GMP, in concentrations exceeding 50 μM, and cyclic AMP, in concentrations exceeding 1 μM, significantly increased the release of renin by slices of rat renal cortex. Stimulants of cyclic AMP accumulation, such as epinephrine or norepinephrine, 1 μM, produced a significant increase of renin release but did not alter cyclic GMP levels. In contrast, acetylcholine, carbamylcholine, bethanachol, methachol and NaN₃ increased cyclic GMP accumulation without modifying renin release. These results suggest that exogenous cyclic GMP acts as a weak agonist of cyclic AMP on renin release in renal cortex. The effects observed with exogenous cyclic GMP, however, may not represent a physiological action since various stimulants of endogenous cyclic GMP accumulation did not alter renin secretion.     

Effects of adenosine analogs and adenine nucleotides on adenosine 5'-diphosphate-induced rat platelet aggregation

Various adenosine analogs and adenine nucleotides have been tested as inhibitors of ADP-induced aggregation of rat platelets. The potent inhibitors of human platelet aggregation, adenosine, 2-fluoroadenosine, 2-chloroadenosine, carbocyclic adenosine and N⁶-phenyl adenosine, had little effect on rat platelet aggregation (0–30 per cent inhibition). The effects of adenosine or its analogs on ADP-induced aggregation of cross-species platelet-rich plasmas (PRPs) (human platelets suspended in rat plasma or rat platelets in human plasma) were similar to those with the native PRPs, indicating that these species differences were due to intrinsic factors in the platelets and not in the plasma. When these analogs were tested in the presence of the cyclic AMP phosphodiesterase inhibitor papaverine, strong inhibiton of rat platelet ADP-induced aggregation was seen. 2′-Deoxyadenosine and 3′-deoxyadenosine were not inhibitory to ADP-induced aggregation of rat PRP even in the presence of papaverine. Adenosine 5′-tetraphosphate strongly inhibited both human and rat platelet aggregation. AMP, like adenosine, did not inhibit rat platelet aggregation but became strongly inhibitory in the presence of papaverine. This inhibitory effect was abolished by preincubating rat PRP with an adenylate cyclase inhibitor, 2′, 5′-dideoxyadenosine or adenosine deaminase. In the later case, however, if the adenosine deaminase inhibitor 2′-deoxycoformycin was included in the incubation mixture, the inhibition by AMP plus papaverine was similar to adenosine plus papaverine. About 50 per cent of [¹⁴C]AMP was converted to [¹⁴ C]adenosine in rat platelet-free plasma or PRP after a 10-min incubation. α,β-Methylene-ADP and β,γ-methylene-ATP (200 μM) inhibited rat platelet aggregation by 50 and 64 per cent, respectively. Cyclic AMP phosphodiesterase of rat and human platelets gave comparable K_m, and V_max values (K_m 0.53 and 0.21μM and V_max 6.0 and 6.7 pmoles/min/10⁷ platelets, respectively).     

Effects of compound 48/80 on mast cells,histamine and cyclic AMP in isolated superior cervical ganglia

Summary Superior cervical ganglia of the rat contain mast cells which are sensitive to degranulation by compound 48/80. The granulation process is shown to the independent of the ATP content of the ganglion. Compound 48/80 released histamine into the incubation medium, thereby decreasing the histamine content of the ganglia. Moreover, the release of ³H-noradrenaline was accelerated by the compound. Histamine and adrenaline induced a rapid accumulation of cyclic AMP in the ganglia. This effect of the amines was specifically blocked by diphenhydramine or propranolol with an ID₅₀ of 1.5×10^–9 M and 2.2×10^–7 M, respectively.In contrast to other findings with isolated mast cell preparations, compound 48/80 induced a rapid and marked accumulation of cyclic AMP in intact ganglia and an enhanced release of cyclic AMP into the incubation fluid. Diphenhydramine prevented the accumulation in the tissue but only partly inhibited the enhanced appearance of cyclic AMP in the medium. The accumulation of the cyclic nucleotide in the tissue was partly blocked by propranolol, suggesting an additional action of compound 48/80 on cyclic AMP through catecholamines.The cyclic nucleotide phosphodiesterase activity in homogenates of superior cervical ganglia was completely inhibited by compound 48/80 at 7 g/ml when low cyclic AMP concentrations were used.In addition to cyclic AMP release, rapid and marked efflux of ATP into the medium was observed during incubations with compound 48/80. The lactate dehydrogenase activity in the incubation medium was significantly enhanced with incubation periods of 40 to 60 min indicating rather slowly occurring toxic damage to cell membranes by compound 48/80.This work was supported by the Deutsche Forschungsgemeinschaft (SFB 70).