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.

Inactivation of the beta-adrenergic receptor in cardiac muscle by dithiols.

The effect of sulfhydryl reagents on binding of the beta-adrenergic antagonist (-)-[3H]dihydroalprenolol hydrochloride [-)-[3H]DHA) to a microsomal fraction of rabbit ventricular muscle was studied. Incubation with the disulfide reducing agents dithiothreitol (DTT), 2-mercaptoethanol, and reduced glutathione resulted in loss of (-)-[3H]DHA binding. At 500 microM DTT, less than 50% of specific binding activity remained; at 100 mM, binding was completely eliminated. 2-Mercaptoethanol and reduced glutathione were less effective than DTT at inhibiting binding activity. The total binding capacity (Bmax) decreased from 155.4 fmol mg-1 of protein, in the absence of DTT, to 92.4 and 77.5 fmol mg-1 at 0.25 and 0.7 mM DTT, respectively. The equilibrium dissociation constant (KD) increased from 7.6 nM, in the absence of DTT, to 10.3 nM at 0.25 mM DTT and to 20.8 nM at 0.7 mM DTT. Thus, DTT-induced decline in (-)-[3H]DHA binding results from a decrease in both the number and affinity of membrane binding sites for the tracer. Receptors could be protected from DTT inactivation by preincubation with beta-adrenergic ligands. Oxidants could not reverse inactivation, with the exception of o-iodosobenzoate which was only partially effective. Thus, the beta-adrenergic receptor of rabbit ventricular muscle contains essential disulfide moietie(s) which can be inactivated by reducing thiols.

The effects of small cardioactive peptide B on the isolated heart and gill of Aplysia californica.

Effects of small cardioactive peptide B on the physiology of the isolated heart and gill preparations from the mollusc Aplysia californica were examined. In addition, the effects of small cardioactive peptide B and FMRFamide (Phe-Met-Arg-Phe-NH2) on adenylate cyclase activity were compared in particulate fractions of heart and gill tissues, respectively. Small cardioactive peptide B was found to exert dose-dependent, reversible changes in cardiac activity when perfused through the isolated heart. The EC50 values effecting changes in heart rate and force of contraction were 3 X 10(-11) and 3 X 10(-10) M, respectively; minimum concentrations found to effect changes in heart rate and force of contraction were normally 10(-15) and 10(-12) M, respectively. However, some winter hearts demonstrated threshold sensitivity to small cardioactive peptide B at concentrations as low as 10(-17) M. When perfused through the isolated gill, small cardioactive peptide B was found to suppress the gill withdrawal response amplitude with a threshold concentration of 10(-14) M and an EC50 value of 3 X 10(-11) M. Suppression of the gill withdrawal response amplitude by small cardioactive peptide B was found to be dose dependent and reversible up to a concentration of 10(-9) M. At higher concentrations, the suppression tended to persist irreversibly. Small cardioactive peptide B stimulated adenylate cyclase activity in particulate fractions of both heart and gill tissues with an EC50 of 0.1 and 1.0 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Resolution and properties of the catalytic unit of cardiac adenylate cyclase.

The catalytic unit (C) of cardiac adenylate cyclase was resolved from the guanine nucleotide-binding regulatory protein (G/F) by fractionation of Lubrol 12A9 extracts with 33% saturated (NH4)2SO4 and by gel filtration in the presence of 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS). Catalytic activity in both preparations was supported by Mg2+ and Mn2+, Vmax values being 12 fold higher in the presence of the latter cation. NaF-activated preparations were also subjected to gel filtration in the presence of CHAPS. In this case, both the catalytic activity and G/F emerged in the activated state. G/F could be deactivated by dialysis of the preparation in the absence of NaF; the catalyst however remained activated following dialysis. NaF sensitivity was reconstituted in C by nonactivated and NaF-activated preparations of G/F isolated by gel filtration. Reconstitution was dependent upon the amount of both G/F and C in the assay. Nonactivated G/F also reconstituted guanine nucleotide sensitivity in C. Catalytic activity was thermally labile, but was stabilized at 25 degrees C by substrate (Mn2+ ATP). C was stimulated up to 25-fold by forskolin. The NaF-activated catalyst resolved by gel filtration was relatively insensitive to this agent. Forskolin, however, augmented NaF-sensitivity by both non-activated and NaF-activated G/F provided it was added to the assay before G/F. Similarly, forskolin augmented guanine nucleotide sensitivity of nonactivated G/F in the presence of GTP gamma S. The P site agent 2',5'-dideoxyadenosine (DDA) was a weak inhibitor of nonactivated C, but a powerful inhibitor of C stimulated by forskolin or activated by G/F in the presence of NaF. In all respects C precipitated by (NH4)2SO4 appeared to be identical with that resolved by gel filtration. Ammonium sulfate precipitation, because of its simplicity, speed, relatively good yield, and adaptability for large scale operation, may be the preferred method for preparing cardiac C for purification studies

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+.

Effect of forskolin on phosphorylation of a 25,000 Mr protein in perfused guinea pig hearts.

The effects of forskolin on phosphorylation of proteins of a 100,000 X g fraction was examined in isolated beating guinea pig hearts. Hearts were perfused with [32P] inorganic phosphate to label intracellular adenine nucleotides. Forskolin was injected into the coronary circulation and after freeze-clamping, phosphorylated proteins in a fraction were separated by sodium dodecyl sulfate-polyacrylamide gel electro-phoresis. Forskolin increased the incorporation into a 25,000 Mr protein approximately 15 fold over control. Incorporation of label was time and dose dependent and was temporally coincident with increases in developed tension. A sarcolemmal fraction prepared from perfused hearts contained a similar 25,000 Mr protein. The data provides evidence that forskolin induced inotropy is accompanied by cAMP-dependent protein kinase mediated phosphorylation. The phosphorylation may be of the same protein whose phosphorylation is associated with epinephrine-induced increase in contractility.

Properties of the resolved catalytic unit of skeletal muscle adenylate cyclase.

Attempts were made to resolve the catalytic unit (C) of adenylate cyclase from the guanine nucleotide-binding regulatory protein (G/F) in detergent extracts of pigeon breast muscle. When preparations solubilized in 1% Lubrol 12A9 were fractionated at 33% saturated (NH4)2SO4, catalytic activity precipitated which was insensitive to NaF and guanine nucleotide. G/F was concentrated in a fraction precipitating between 33 and 47% saturated (NH4)2SO4, but was not obtained free of C. C was also resolved from G/F by gel filtration in the presence of 13 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps), but recoveries of C were lower than those obtained by salt fractionation. NaF-Activated preparations were also subjected to gel filtration in the presence of Chaps, in which case both the catalytic activity and G/F emerged in the activated state. G/F from such columns was deactivated by removal of NaF by dialysis. NaF and guanine nucleotide sensitivity could be reconstituted in nonactivated preparations of C by G/F in the presence of NaF or guanylylimidodiphosphate. Reconstitution was dependent upon both the amount of C and G/F in the assay. C in both preparations was strongly stimulated by the diterpene, forskolin, whereas the NaF-activated enzyme resolved by gel filtration was only marginally stimulated by this agent. C was only weakly inhibited by the P-site agent 2',5'-dideoxyadenosine. However, when C was stimulated by forskolin, dideoxyadenosine was a potent inhibitor. The NaF-activated catalytic unit was also strongly inhibited by this agent. Preparations of C obtained by (NH4)2SO4 precipitation may be suitable starting material for attempted purification of this component of adenylate cyclase.

Evidence for FMRF-amide as a neurotransmitter in the gill of Aplysia californica.

In Aplysia californica, multiple regulatory mechanisms are involved in the actions of neurotransmitters on the gill. Neurotransmitter receptors and adenylate cyclase were examined in a particulate fraction of gill homogenates. The neuropeptide FMRF-amide stimulated enzyme activity 7- to 8-fold (EC50, 1 microM) via receptors that were pharmacologically distinct from those for dopamine and serotonin. FMRF-amide augmented cyclic AMP levels in slices of gill tissue with a time course similar to that for adenylate cyclase activation. Increases in cyclic AMP levels produced by the neuropeptide were potentiated by the phosphodiesterase inhibitor theophylline. Physiological responses to neuropeptides and cyclic AMP analogues were examined in a perfused, isolated gill preparation. Phasic contractions evoked by FMRF-amide (EC50, 0.1 microM) were mimicked by membrane-permeable analogues of cyclic AMP. Comparison of FMRF-amide effects on adenylate cyclase and gill behavior suggests an association between cyclic AMP and phasic contractions. In addition, FMRF-amide-like immunoreactivity, detected by antisera raised against the neuropeptide, was found in nerve fibers innervating the gill. These findings indicate that in Aplysia, FMRF-amide or a closely related peptide neurotransmitter may be involved in the physiological regulation of gill behavior.

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.

Inactivation of the beta-adrenergic receptor in skeletal muscle by dithiols.

The effect of sulfhydryl compounds on binding of the beta-adrenergic antagonist (-)-[3H]dihydroalprenolol [(-)-[3H]DHA] to a microsomal fraction from rabbit skeletal muscle was examined. Inhibition of binding by a variety of adrenergic agonists and antagonists and the effects of these agents on adenylate cyclase were consistent with the beta-adrenergic receptor in this tissue being of the beta 2-subtype. Binding of (-)-[3H]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 (-)-[3H]DHA binding resulted primarily from a decrease in receptor number. Inactivation was partially reversed by the oxidant H2O2. Binding sites could be locked in the inactivated state by incubating DTT-treated membranes with the alkylating agent iodoacetamide. Both beta-adrenergic agonists and antagonists protected against inactivation. Adenylate cyclase activity in the membranes was increased by DTT. The enzyme was rapidly inactivated by H2O2, and this could be partially reversed by DTT. It is concluded that the beta-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.

Mg2+ and Mn2+ effects on membrane-bound and detergent-solubilized adenylate cyclase.

Mg2+ and Mn2+ stimulation of basal, F-, and guanyl-5'-yl imidodiphosphate (GPP(NH)P) stimulated adenylate cyclase in particulate and detergent-solubilized preparations of skeletal muscle, cardiac muscle, and erythrocytes was examined. Solubilization decreased the concentrations of Mg2+ required for half-maximal velocity and for saturation. Concentrations of Mn2+ required for saturation and for half-maximal velocity of particulate preparations was much lower than for Mg2+ and these values were not markedly reduced by solubilization. Particulate and soluble preparations were similarly stimulated by NaF and GPP(NH)P. Activation of the heart and skeletal muscle enzyme by NaF and GPP(NH)P greatly reduced the Mg2+ requirement; this was seen with both particulate and solubilized preparations. It is suggested that solubilization removes Mg2+ action at a regulatory site; MgATP and MnATP are both effective at the catalytic site, the latter producing higher Vmax.

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

Adenylate cyclase was solubilized from washed particulate 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 degrees 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.

Proteolysis of skeletal muscle adenylate cyclase. Destruction and reconstitution of fluoride and guanylnucleotide sensitivity.

Adenylate cyclase was measured in skeletal muscle plasma membranes incubated with subtilisin. Under specific conditions the protease preferentially inactivated fluoride and guanylnucleotide sensitivity. Following protease treatment, membranes were solubilized with Lubrol 12A9 and subjected to ion-exchange chromatography. Adenylate cyclase was eluted with 200 mM NaCl; the enzyme recovered was completely unresponsive to either NaF or guanylyl imidodiphosphate. Responsiveness to the two ligands was restored by adding a heart fraction in which basal activity had been destroyed by heating at 40 degrees C or by adding a soluble skeletal muscle fraction in which basal activity had been largely destroyed by N-ethylmaleimide. The solubilized subtilisin-treated skeletal muscle preparation may serve as a source of catalytic activity for the study and purification of regulatory factors for adenylate cyclase.

(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.

Catecholamine and guanine nucleotide activation of skeletal muscle adenylate cyclase.

Activation of adenylate cyclase by guanine nucleotide and catecholamines was examined in plasma membranes prepared from rabbit skeletal muscle. The GTP analog, 5'-guanylyl imidodiphosphate caused a time and temperature-dependent activation of the enzyme which was persistent, the Ka was 0.05 microM. 5'-Guanylyl imidodiphosphate binding to the membranes was time and temperature dependent, KD 0.07 microM. Beta adrenergic amines accelerated the rate of 5'-guanylyl imidodiphosphate activation of the enzyme with an order of potency isoproterenol approximately soterenol approximately salbutamol greater than epinephrine greater than norephrine. Catecholamine activation was antagonized by propranolol and the beta2 antagonist butoxamine; the beta1 antagonist practolol was inactive. [3H]Dihydroalprenolol bound to the membranes and binding was antagonized by beta adrenergic agonists with an order of potency similar to the activation of adenylate cyclase and was antagonized by butoxamine but not by practolol. The data are consistent with the idea that adenylate cyclase in skeletal muscle plasma membranes is coupled to adrenergic receptors of the beta2 type.