George I. Drummond

Effect of adenosine on cyclic AMP accumulation in ventricular myocardium

Abstract 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 6-N[(p- nitrobenzyl ) thio ]-9-β- d - 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 metabolism in microvessels from heart and brain.

Activities of several adenosine metabolizing enzymes were examined in capillary preparations isolated from rabbit ventricle. Vmax and Km values for 5-nucleotidase were 2.3 nmol/min/mg and 10 microM, respectively. For adenosine deaminase the corresponding values were 7.8 nmol/min/mg and 32 microM. S-adenosyl-homocysteine hydrolase, which forms adenosine by the hydrolysis of S-adenosylhomo-cysteine, was also present (Vmax, 0.07 nmol/min/mg; Km, 0.81 microM), as were adenosine kinase (Vmax, 0.2 nmol/min/mg; Km, 0.52 microM) and purine nucleoside phosphorylase (Vmax, 13.8 nmol/min/mg; Km, 96 microM). These enzymes were also present in microvessels (capillaries and arterioles) purified from rabbit brain. Activities of several enzymes, especially 5-nucleotidase and adenosine deaminase, were much lower in myocytes isolated from rabbit ventricle. The study provides evidence that endothelial cells of the microvasculature from heart and brain are capable of activity forming and degrading adenosine. It is possible that adenosine formed by these cells may contribute to the local regulation of blood flow.

(Ca2+ + Mg2+)-ATPase in enriched sarcolemma from dog heart

An enriched fraction of plasma membranes was prepared from canine ventricle by a process which involved thorough disruption of membranes by vigorous homogenization in dilute suspension, sedimentation of contractile proteins and mitochondria at 3000 X g followed by sedimentation of a microsomal fraction at 200 000 X g. The microsomal suspension was then fractionated on a discontinuous sucrose gradient. Particles migrating in the density range 1.0591--1.1083 were characterized by (Na+ + K+)-ATPase activity and [3H]ouabain binding as being enriched in sarcolemma and were comprised of nonaggregated vesicles of diameter approx. 0.1 micron. These fractions contained (Ca2+ + Mg2+)-ATPase which appreared endogenous to the sarcolemma. The enzyme was solubilized using Triton X-100 and 1 M KCl and partially purified. Optimal Ca2+ concentration for enzyme activity was 5--10 microM. Both Na+ and K+ stimulated enzyme activity. It is suggested that the enzyme may be involved in the outward pumping of Ca2+ from the cardiac cell.

Activation and stabilization of cardiac adenylate cyclase by GTP analog and fluoride.

Abstract The rate of cyclic AMP formation by rabbit heart membrane particles decreased at assay temperatures greater than 30 °C. Adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity (assayed at 24 °C) decreased exponentially with time of preincubation at 30 or 37 °C, providing evidence for the instability of this enzyme. The half-life, t1/2, of the enzyme at 37 °C was 9.9 min in the absence and 4.4 min in the presence of MgCl2. The activity was most labile in the presence of 50 m m Mg2+ and 1 m m ATP, having t1/2 = 1.3min. Prior incubation of membranes with the GTP analog, guanyl-5′-yl imidodiphosphate [Gpp(NH)p], 0.1 m m , for 30 min at 37 °C produced maximal activation of adenylate cyclase; the rate of activation was temperature dependent and was increased in the presence of isoproterenol. The Gpp(NH)p-activated enzyme had increased thermal stability, t1/2 = 170 min, and was also markedly more stable in the presence of Mg-ATP, t1/2 = 72min, than nonactivated enzyme. Preactivation with F− (30 min at 24 °C) also stabilized the activity; t1/2 > 70 min in the absence or presence of Mg-ATP. The Mg2+ concentration required for maximal activity was reduced from approximately 60 m m for nonactivated enzyme to 10 m m for the Gpp(NH)p- and F−activated enzyme.

Inactivation of the β-adrenergic receptor in skeletal muscle by dithiols

Abstract The effect of sulfhydryl compounds on binding of the β-adrenergic antagonist (−)-[ 3 H]dihydroalprenolol [(−)-[ p3 H][DHA] to a microsomal fraction from rabbit skeletal muscle was examined. Inhibition of binding by a variety of adrenergic agonists and antagonist and the effects of these agents on adenylate cyclase were consistent with the β-adrenergic receptor in this tissue being of the β 2 -subtype. Binding of (−)-[ 3 H]DHA was reduced by incubating the membranes with dithiols such as dithiothreitol (DTT), 1,3-dimercapto-2-propanol and 1,4-dimercaptobutane; monothiols were much less potent. DTT-induced decline in (−) [ 3 H]DHA binding resulted from a decrease in receptor number. Inactivation was partially reversed by the oxidant H 2 O 2 . Binding sites could be locked in the inactivated state by incubating DTT-treated membranes with the alkylating agent iodoacetamide. Both β-adrenergic agonists and antagonists protected against inactivation. Adenylate cyclase activity in the membranes was increased by DTT. The enzyme was rapidly inactivated by H 2 O 2 , and this could be partially reversed by DTT. It is concluded that the β-adrenergic receptor of skeletal muscle contains an essential disulfide moiety which can be inactivated by reducing dithiols. Adenylate cyclase, on the other hand, contains at least one essential sulfhydryl which is preserved by dithiols.

Biochemical properties of adenylate cyclase in the gill of Aplysia californica

1. n1. Several biochemical properties of adenylate cyclase were examined in particulate and detergent-solubilized preparations of gill tissue in Aplysia californica. n n2. n2. Enzyme activity in a 38,000 g particulate fraction was stimulated 250-fold by NaF and 23-fold by guanyl-5′-yl-imidodiphosphate. Stimulation of activity by dopamine and 5-hydroxytryptamine displayed an absolute requirement for guanine nucleotide. n n3. n3. Enzyme activity was supported by both Mg2+ and Mn2+, activity being 8-fold greater in the presence of saturating concentrations of the latter cation. n n4. n4. Solubilization of the enzyme with nonionic detergents caused a loss in sensitivity to guanyl-5′-yl-imidodiphosphate while NaF stimulation was enhanced. n n5. n5. In the presence of calmodulin, Ca2+ augmented basal as well as dopamine- and serotonin-stimulated activity at concentrations below 10 μM; concentrations above this were inhibitory. n n6. n6. The diterpene forskolin had no stimulatory action on either the membrane-bound or detergent-solubilized enzyme. n n7. n7. The data show that neurohormone-sensitive adenylate cyclase in this invertebrate tissue resembles, in many respects, the enzyme from mammalian tissues.

Properties of detergent-dispersed adenylate cyclase from cerebral cortex. Presence of an inhibitor protein.

Abstract: Adenylate cyclase was solubilized from washed paniculate fraction of rabbit cerebral cortex with the nonionic detergent Lubrol 12A9 and subjected to either gel filtration on Ultrogel AcA 34 or chromatography on DEAE Bio‐Gel A. By both procedures the enzyme was resolved into two components, one insensitive to guanyl 5′‐yl imidodiphosphate [Gpp(NH)p] and NaF but stimulated by Ca2+ and calmodulin, and another that was sensitive to Gpp(NH)p and NaF but relatively insensitive to Ca2+ and calmodulin. The data support the possibility that two independent forms of adenylate cyclase exist in cerebral cortex, one regulated by guanine nucleotide regulatory protein and another by Ca2+‐calmodulin. Fractions containing the guanylnucleotide‐sensitive activity were found to contain a factor that inhibited basal and Ca2+‐stimulated adenylate cyclase in the Ca2+‐sensitive fraction. The inhibitor was inactivated by heating at 60°C and by incubation with trypsin. Inhibition was not time‐dependent, and it was not due to destruction of cAMP by phosphodiesterase or of ATP by ATPase. Inhibitory action was not reversed by calmodulin and therefore it does not appear to be a calmodulin binding protein. Sucrose density gradient sedimentation indicated a sedimentation coefficient of 4S for the inhibitor; by this technique it co‐sedimented with the adenylate cyclase sensitive to Gpp(NH)p and NaF.

Inactivation of cardiac adenylate cyclase by oxidation, trivalent arsenicals, and N-ethylmaleimide☆

Abstract Cardiac adenylate cyclase whether membrane-bound or soluble underwent rapid oxidation in the absence of thiol; loss of activity was reversed by dithiothreitol and several monothiols. The enzyme was also inactivated by hydrogen peroxide; in this case activity could not be fully recovered by dithiothreitol. In both instances stimulation by NaF and Gpp(NH)p declined in parallel with basal activity suggesting that the catalytic unit was the primary site of attack. Enzyme converted to the activated form by prior incubation with either NaF or Gpp(NH)p was relatively resistant to oxidation. The nonactivated enzyme was rapidly inactivated by p -aminophenyl arsenoxide and p -aminophenyl dichloroarsine. Activity in the presence of NaF and Gpp(NH)p declined with greater facility than basal suggesting an attack on the regulatory protein as well as the catalytic unit. Inhibition was reversed by dithiothreitol and 2,3-dimercaptopropanol; monothiols were much less effective. The F − - and Gpp(NH)p-activated enzyme was much less sensitive to these reagents than the native form. N -Ethylmaleimide rapidly inactivated both the particulate and the soluble enzyme. Action of this monosulfhydryl reagent on the F − and Gpp(NH)p-activated enzyme was complex; 60% of the activity disappeared rapidly while the remainder declined much less rapidly. It is suggested that the catalytic unit of adenylate cyclase contains vicinal thiols which are crucial for catalysis and which are in sufficiently close proximity to allow cyclic dithioarsenite formation. When the enzyme is associated with the regulatory component that confers F − and Gpp(NH)p stimulatability the sulfhydryls must be covered or spatially distorted. It is suggested that sulfhydryls may be crucial not only for catalysis but for association of the regulatory component with the catalytic unit of adenylate cyclase.

Stimulation of adenylate cyclase in the heart of Aplysia californica by biogenic amines

The effects of serotonin (5-HT), dopamine (DA), several peptides including FMRFamide and arginine vasotocin, the diterpene forskolin and Ca2+ were examined on adenylate cyclase in a particulate fraction from hearts of Aplysia californica. Enzyme activity was stimulated 6-7-fold by 5-HT (EC50, 1 microM) in the presence of GTP. Several 5-HT analogs particularly 5-methoxytryptamine and 5-methoxy-N-N-dimethyltryptamine were also active. The stimulatory action of 5-HT was antagonized by the 5-HT receptor blockers methergoline and metitepine and by the DA receptor blocker chlorpromazine. Dopamine had weak stimulatory action (EC50, 10 microM) and an efficacy relative to that of 5-HT of 0.3. The action of DA was antagonized by chloropromazine and metitepine. Several peptides including FMRFamide and arginine vasotocin had no effect on adenylate cyclase when tested over the concentration range 0.1-100 microM. The enzyme was stimulated 6-fold by the diterpene forskolin (EC50, 2 microM). 5-HT-stimulated activity was strongly inhibited by Ca2+. Calmodulin had no action on the enzyme in the presence of Ca2+.

Heart microvessels: presence of adenylate cyclase stimulated by catecholamines, prostaglandins, and adenosine.

Microvessels (capillaries) were isolated in pure form from rabbit ventricle by a process which involved fine mincing of the tissue, repeated incubation with collagenase to effect cell dispersal, passage of the suspended cells over a column of glass beads, and finally concentration of the capillary fraction on a step-wise sucrose gradient. Adenylate cyclase in the capillary preparation was stimulated by beta adrenergic agonists in a potency order which suggested coupling to a beta 2 subtype adrenergic receptor. Catecholamine-stimulated activity was antagonized by methoxamine, but this did not seem to be mediated through alpha adrenergic receptor activation, since it was not reversed by the alpha adrenergic antagonist phentolamine. Adenylate cyclase was stimulated by adenosine and several adenosine analogs in a potency order which suggested enzyme coupling to a stimulatory A2 receptor. Prostaglandins were also effective stimulators of enzyme activity, those of the E and A series being more potent than members of the F series. It is possible that these agents may exert their physiological actions on the microvasculature via cyclic AMP formed in response to activation of adenylate cyclase.