beta-Adrenergic receptor-sensitive adenosine cyclic 3',5'-monophosphate accumulation in homogenates of the rat corpus striatum. A comparison with the dopamine receptor-coupled adenylate cyclase.

The conversion of newly formed [³H]adenosine triphosphate (ATP) to [³H]adenosine cyclic 3′,5′-monophosphate (cAMP) was studied in osmotically shocked, crude synaptosomal fractions of the rat corpus striatum. Of the β-hydroxylated catecholamines tested, the potency of isoproterenol (Ec₅₀ about 0.01 μM) was greater than that of norepinephrine (Ec₅₀ about 1.0 μM). Stereoselectivity was displayed with the (?)isomer of isoproterenol being more potent that its (+)isomer. Of the non-β-hydroxylated catecholamines studied, N-isopropyldopamine demonstrated greater potency than dopamine, whereas apomorphine was inactive. No “additive stimulatory effect” was observed when dopamine was combined with a maximum effective concentration of isoproterenol. The β-adrenergic antagonist propranolol, completely blocked both the dopamine- and norepinephrine-induced increases in cAMP formation. Properties characteristic of a β-type system were, likewise, exhibited by homogenates of both the cerebral cortex and hindbrain. Furthermore, the conversion to [³H]cAMP was increased only slightly (about 16 per cent) by exposure to sodium fluoride, and the stimulatory response to isoproterenol was not altered significantly by high concentrations of ATP but was lost upon sonication of the tissues. In contrast, by assaying adenylate cyclase activity with a high saturating concentration of exogenous [³H]ATP, striatal and cerebral cortical homogenates exhibited responses characteristic of a specific dopamine receptor-coupled adenylate cyclase. Sonication of tissues did not alter the stimulatory effect of dopamine, and sodium fluoride produced about a 2-fold stimulation of the adenylate cyclase activity. Thus, findings in osmotically shocked, crude synaptosomal fractions of the corpus striatum suggest that the paniculate component of adenylate cyclase, which utilizes exogenous ATP as substrate, exhibits properties characteristic of a dopamine receptor-coupled adenylate cyclase. In contrast, the membrane-enclosed, particulate component of adenylate cyclase, which utilizes endogenously synthesized ATP as substrate, conforms to criteria identified with a β-adrenoreceptor-linked adenylate cyclase.     

Effects of phenothiazines on the membrane-bound guanylate and adenylate cyclase in tetrahymena pyriformis

The particulate-bound guanylate cyclase activity of Tetrahymena pyriformis was shown previously to be Ca²⁺-dependent and to be activated by an endogenous calmodulin-like protein (Tetrahymena Ca²⁺-binding protein, TCBP) [S. Nagao, Y. Suzuki, Y. Watanabe and Y. Nozawa, Biochem. biophys. Res. Commum.90, 261 (1979)]. Phenothiazine derivatives, such as chlorpromazine and trifluoperazine, that interact with calmodulin were found to inhibit the Ca²⁺-dependent guanylate cyclase activity and the TCBP-induced activation of the guanylate cyclase activity. Ethylene glycol-bis (β-aminoethyl ether)-N, N'-tetraacetic acid (EGTA), a Ca²⁺ chelator, also inhibited the activation of guanylate cyclase. However, the mechanisms by which EGTA and trifluoperazine act were different. The EGTA-induced inhibition could not be overcome by increasing the concentration of TCBP, whereas the trifluoperazine-induced inhibition could be overcome by increasing the concentration of TCBP, but not by increasing the concentration of Ca²⁺. These findings suggest that the mechanism by which trifluoperazine inhibits the activation of guanylate cyclase involves competition with TCBP.     

Effects of local anesthetics on calmodulin-dependent guanylate cyclase in the plasma membrane of Tetrahymena pyriformis

A highly purified preparation of Tetrahymena calmodulin activated a membrane-bound guanylate cyclase by more than 40-fold. This activation of guanylate cyclase by calmodulin was inhibited completely by local anesthetics such as dibucaine, tetracaine, lidocaine and procaine at concentrations that had no appreciable effect on the activities of basal guanylate cyclase (without calmodulin) and adenylate cyclase. The inhibition by dibucaine of calmodulin-mediated activation of the enzyme activity was not reversed by calcium but was partially overcome by increasing the concentration of calmodulin. Kinetic analysis of local anesthetic-induced inhibition of activation of guanylate cyclase demonstrated a mixed type of antagonism. These results suggest the possibility that the inhibition of calmodulin-dependent guanylate cyclase resulted, in part, from interaction of the drugs with calmodulin.     

Positive dromotropic effect of dibutyryl cyclic adenosine 3',5'-monophosphate on the atrioventricular node.

The effect of atrioventricular (A-V) conduction of N6-2'-0-dibutyryl cyclic 3',5'-adenosine monophosphate (dibutyryl cyclic AMP) was investigated in comparison with those of norepinephrine, cyclic 3',5'-adenosine monophosphate (cyclic AMP), adenosine-5'-monophosphate (5'-AMP), and adenosine by the use of the isolated, blood-perfused A-V node preparation of the dog. Single injections of dibutyryl cyclic AMP (3-300 micronmol) and norepinephrine (0.1-1 nmol) into the posterior septal artery of the preparation (the upper part of the A-V node is mainly perfused through this artery) produced a dose-dependent decrease in the A-V conduction time. The time to the peak effect and the duration of the effect of dibutyryl cyclic AMP were much longer than with norepinephrine. The positive dromotropic effect of dibutyryl cyclic AMP was resistant to the beta-adrenoceptor blocking action of propranolol. Unlike dibutyryl cyclic AMP, cyclic AMP (above 30 nmol), 5'-AMP and adenosine (above 1 nmol) injected into the posterior septal artery prolonged the A-V conduction time in a dose-dependent manner. The results indicate that dibutyryl cyclic AMP has a mode of action on A-V nodal cells which differs distinctly from that of either norepinephrine or cyclic AMP, 5'-AMP, and adenosine.     

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.     

Reduced adenosine 3',5'cyclic monophosphate levels in patients with reversible obstructive airways disease

1. Patients were grouped into categories of ‘no airways disease’, ‘obstructive airways disease without response to bronchodilator’ and ‘obstructive airways disease with bronchodilator responsiveness’. 2. Cyclic nucleotides were assayed in specimens of lung tissue that were excised during surgery. 3. Reduced levels of adenosine 3′,5′-cyclic monophosphate (cyclic AMP) were found in pulmonary tissue obtained from patients with reversible obstructive airways disease, lending support to the β-adrenergic theory of asthma.     

Determination of 8-chloroadenosine 3',5'-monophosphate in dog plasma by capillary electrophoresis

A method for the quantitative determination of 8-chloroadenosine 3′,5′-monophosphate (8-Cl-cAMP) in dog plasma by capillary zone electrophoresis (CE) has been developed and validated. Samples of plasma (with 2′-O-monobutyryladenosine-3′,5′-monophosphate as internal standard) were deproteinized with two volumes of acetonitrile. The supernatant was evaporated and reconstituted in water. A BioFocus 2000 system (Bio-Rad, Hercules, CA, USA) was used. The UV detector was set at 261 nm. The samples were loaded into uncoated fused silica capillary (40 cm×50 μm) by pressure injection. A running electrolyte contained 30 mM SDS, 100 mM Tris, pH 7.55, with 20% of methanol added. The typical analytical conditions were: voltage, 18 kV; injection, 12 psi×s; capillary and carousel temperature were 20°C. The linear relationship was observed between 0.063–2.00 μM using the time-corrected peak area ratio of 8-Cl-cAMP to internal standard with correlation coefficient greater than 0.99. The intra-day and inter-day coefficients of variation (CV's) were less than 12%. The developed method was used for the analysis of plasma samples from beagle dogs (n=12) to examine the toxicity of the anticancer drug, 8-Cl-cAMP, following two, 5-day cycles of continuous intravenous infusion at various doses of 8-Cl-cAMP as the sodium salt.     

Potentiation by homocysteine of adenosine-stimulated elevation of cellular adnosine 3',5'-monophosphate.

The novel ability of l-homocysteine (Hcy) to potentiate the cellular elevation of adenosine 3′,5′-monophosphate (cAMP) caused by adenosine (Ado) is described. This effect of Hcy is highly selective in that it is not mimicked by l-cysteine, and Hcy does not potentiate the elevation of cellular cAMP caused by either 2-chloroadenosine or prostaglandin E₁. Hcy also augments the Ado-stimulated increase in 2-fluoroadenosine 3′,5′-monophosphate in cells preloaded with nucleotides of 2-fluoroadenosine. Addition of Hcy to cells during their incubation with radioactive Ado results in a decrease in the cellular content of radioactive Ado and a concomitant buildup of S-adenosylhomocysteine. The enhancive effect of Hcy on the Ado-stimulated elevation of cAMP may be due to this associated reduction in the intracellular pool of Ado (due to condensation of Ado with Hcy via S-adenosylhomocysteinase) and to a resultant reduction in inhibition of adenylate cyclase by intracellular Ado, thereby allowing greater net stimulation of the cyclase by extracellular Ado.     

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.     

Inhibitory effects of calmodulin antagonists on plasma membrane cyclases in Tetrahymena: calmodulin-dependent guanylate cyclase and calmodulin-independent adenylate cyclase

Trifluoperazine was shown previously to inhibit the activation of Tetrahymena guanylate cyclase activity by calmodulin [S. Nagao, S. Kudo and Y Nozawa, Biochem. Pharmac. 19, 2709 (1981)]. The present paper reports that N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), another representative calmodulin inhibitor, inhibited the calmodulin-induced activation of the guanylate cyclase, and that trifluoperazine and W-7 also inhibited Tetrahymena adenylate cyclase. The adenylate cyclase activity was found to be present in a membrane-bound form and not to be influenced by calmodulin. The inhibitions of the adenylate cyclase activity by these agents were dose-dependent and not Ca2+-dependent. These findings suggest that the inhibitory actions of these drugs may not necessarily be specific for calmodulin-dependent enzymes in T. pyriformis.     

Effect of modification of the 1-, 2-, and 6-positions of 9-beta-D-ribofuranosylpurine cyclic 3',5'-phosphate on the cyclic nucleotide specificity of adenosine cyclic 3',5'-phosphate- and guanosine cyclic 3',5'-phosphate-dependent protein kinases

A group of analogs of adenosine cyclic 3′,5′-phosphate (cAMP) and guanosine cyclic 3′,5′-phosphate (cGMP) with modifications in the 1-, 2- (or N²), and 6 (or N⁶)-positions of the purine ring were compared as activators of a cAMP-dependent protein kinase [PK(cAMP)] from bovine brain and of cGMP-dependent protein kinase [PK(cGMP)] from lobster tail muscle. The results suggest that the 6-amino group of cAMP is not required for the activation of PK(cAMP) by cAMP and that the 6-oxygen and 2-amino moieties of cGMP are required for the activation of PK(cAMP) by cAMP aIn the case of PK(cGMP) activation by cGMP, the 6-oxygen apparently accepts a proton from the enzyme and the 2-amino group apparently donates a proton to the enzyme.     

Behavioral effects of norepinephrine and dibutyryl 3',5'AMP in centrally sympathectomized rats

Male Wistar rats were injected intraventricularly with two doses of 250 μg of 6-hydroxydopamine (6-OHDA) at a 48 hr interval. Seven days after the second injection, 50 μg of norepinephrine (NE) or 100 μg of dibutyryl cyclic AMP (DCAMP) were injected intraventricularly. There were no differences in gross behavior but an increase in irritability was observed in rats treated with 6-OHDA compared with controls. NE increased locomotor activity and irritability of animals. Chemical sympathectomy intensified locomotor excitation and irratibility caused by NE. DCAMO caused an increase of locomotor activity, irritability and convulsions 30 min after injection. These behavioral phenomena were intensified in animals treated with 6-OHDA. It is suggested that 6-OHDA sensitizes the central nervous system to action of NE and DCAMP.     

Cyclic adenosine 3',5'-monophosphate concentration in rabbit parotid slices following stimulation by secretagotues

The effects of sympathomimetic and parasympathomimetic drugs on cyclic AMP concentration in rabbit parotid slices were investigated. Catecholamines caused a large increase of cyclic AMP which was inhibited by the β-adrenoreceptor blocking agent propranolol; the α-adrenoreceptor blocking agent phentolamine was ineffective. Parasympathomimetic drugs, potent inducers of secretion in vivo and in vitro, did not change the cyclic AMP concentration. These data suggest that the secretory process mediated by cholinergic innervation of the parotid gland is independent of cyclic AMP.     

Studies on beta-adrenoceptors mediating changes in mechanical events and adenosine 3',5'-monophosphate levels. Guinea-pig trachea.

In quinea-pig tracheal smooth muscle, (?)-isoproterenol and (?)-soterenol increase cyclic AMP levels, the latter being classified as a partial agonist. The ED₅₀ values were 1.74 × 10^?7 M and 2.48 × 10^?7M for isoproteronol and soteronol, respectively. Isoproterenol and soterenol were also examined as competitive antagonists of isoproterenol-induced increase in cyclic AMP levels. The apparent dissociation constants calculated from dose ratios for antogonism were 2.19 × 10^?8 M for isoproterenol and 08.38 × 10^?8 M for soterenol. These values are the same as apparent dissociation constants obtained in previous studies on relaxation produced by these agonsits. Carbachol, the functional antagonist use to estimate agonist dissociation constants for production of relaxation, antagonized isoproterenol- and soterenol-induced increases in cyclic AMP only in the central region of their dose-response curves without blocking effects of small or large doses of either agonist. This antagonism was demonstrated to be calcium-dependent. The results are consistent with the view that β-receptors mediating relaxation and diseases in cyclic AMP levels in guinea-pig trahea are of the same type and that differences in concentrations required to elicit the two responses is a result of the receptor reserve. A larger receptor reserves for β-agonist in tracheal smooth muscle vs. heart is considered to be a major determinant in quantitative responses produced in the two tissues.     

The effect of intracerebroventricularly administered dibutyryl adenosine 3',5'-cyclic monophosphate on cerebrospinal fluid and serum dopamine-beta-hydroxylase in rabbits

Intracerebrospinal injected or infused dibutyryl adenosine 3′,5′-cyclic monophosphate provoked (a) an initial small decrease of cerebrospinal fluid dopamine-β-hydroxylase activity which lasted for 2 hours, followed by a significant rise of a 4–6 hours duration, together with a rise in total cerebrospinal fluid protein concentration, (b) a significant increase in serum dopamine-β-hydroxylase activity. Pretreatment of rabbits with two intracisternal injections of 6-hydroxydopamine completely abolished the dibutyryl adenosine 3′,5′-cyclic monophosphate induced release of dopamine-β-hydroxylase in the cerebrospinal fluid but only partially the release of dopamine-β-hydroxylase in the serum.     

Studies on beta-adrenoceptors mediating changes in mechanical events and adenosine 3',5'-monophosphate levels. Rat atria.

Increasing concentrations of the functional antagonist carbachol resulted in a shift to the right of the dose-response curves for (--)-isoproterenol-induced positive chronotropic and inotropic responses and a reduction of the maximum degree of response that could be produced relative to that produced by theophylline. Carbachol only reduced the maximum responses to the partial agonist (--)-soterenol. The ED50 value for isoproterenol-induced increases in adenosine 3',5'-monophosphate (cyclic AMP) levels was more than 10-fold larger than those for production of mechanical events and soterenol did not produce a measurable increase in cyclic AMP above baseline values. Soterenol was analyzed as a competitive antagonist of isoproterenol-induced responses and the apparent dissociation constants (KA) for soterenol calculated to be 1.21 X 10(-7) M for receptors mediating positive chronotropic responses, 3.56 X 10(-7) M for receptors mediating positive inotropic responses, and 2.96 X 10(-7) M for receptors mediating increases in cyclic AMP levels. By two additional theoretical models of drug-receptor interactions, the KA values for soterenol were between 1.5 and 4.5 X 10(-7) M. These KA values were essentially the same as the ED50 values for soterenol-induced mechanical effects. By one of the models, the KA for isoproterenol was calculated to be about 2.14 X 10(-8) M, a value close to the ED50 value for isoproterenol-induced increases in cyclic AMP (5.45 X 10(-8) M). These results suggest that the beta-adrenoceptors mediating the three responses in rat atria are of the same type and that differences in concentrations required to produce mechanical vs. cyclic AMP changes results with agonists possessing high efficacy for which there is a receptor reserve.     

Elevation of adenosine 3',5'-monophosphate in the perfusate of rat kidney after addition of dopamine.

Dopamine (10-4 M) and vasopressin (1 mU/ml) were found to increase the level of cyclic AMP in the perfusate of rat kidney. There were some differences in the mode of action of these two drugs. Firstly, the effect of dopamine, but not of vasopressin, was completely antagonized by spiroperidol. Secondly, the maximal response was attained within 1 min after dopamine perfusion, but 8 min after vasopressin perfusion. These results suggest that a specific dopamine receptor which acts to increase the concentration of cyclic AMP is located in the vascular tissue of rat kidney.     

1-Ethyl-4-(isopropylidenehydrazino)-1H-pyrazolo-(3,4-b)-pyridine-5-carboxylic acid, ethyl ester, hydrochloride (SQ 20009)--a potent new inhibitor of cyclic 3',5'-nucleotide phosphodiesterases

The properties of SQ 20009 [1-ethyl-4-(isopropylidenehydrazino)-1H-pyrazolo-(3,4-b)-pyridine-5-carboxylic acid, ethyl ester, HCl] as a cyclic nucleotide phosphodiesterase inhibitor have been investigated. The phosphodiesterase preparations used in this study were ammonium sulfate-fractionated supernatants of homogenates of rat brain, rabbit brain, rat adrenal, rat lipocyte and cat heart; commercially available beef heart phosphodiesterase was also studied. The concentrations of SQ 200009 required to inhibit these phosphodiesterase activities 50 per cent were 2·0, 4·8, 20, 21, 27 and 60 μM, respectively, using 1·6 × 10⁻⁷ M cyclic AMP as substrate. SQ 20006 (the parent of SQ 20009 lacking the 4-isopropylidene moiety), theophylline and caffeine were also tested against all six enzyme preparations. Whereas SQ 20009 was more potent than SQ 20006 using the phosphodiesterase prepared from rat adrenal, the potencies were reversed when the lipocyte enzyme was used. SQ 20009 was approximately 60 and 75 times as potent an inhibitor of rat brain cyclic AMP phosphodiesterase as were theophylline and caffeine respectively. The kinetic properties of the phosphodiesterases prepared from rat brain, cat heart and beef heart were also investigated. Using the rat brain enzyme, two K_m values for cyclic AMP, 4·0 × 10⁻⁶ and 1·2× 10⁻⁴ M and a single K_m, 2·0 × 10⁻⁵ M, for cyclic GMP were confirmed. The K_i of SQ 20009 against the low K_m cyclic AMP phosphodiesterase was 2·0 × 10⁻⁶ M and that for cyclic GMP hydrolysis was 2·4 × 10⁻⁵ M. The inhibition by SQ 20009 of the hydrolysis of both cyclic nucleotides by both the rat brain and beef heart phosphodiesterases was competitive. The cat heart cyclic nucleotide phosphodiesterase was inhibited non-competitively by SQ 20009; the K_i for cyclic AMP hydrolysis was 6·4 × 10⁻⁵ M, and the K_i for cyclic GMP hydrolysis was 3·0 × 10⁻⁵ M. The inhibition by SQ 20009 of cyclic AMP hydrolysis by both the rat brain and cat heart preparations was reversible.