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We recently reported that ATP, coreleased with norepinephrine (NE) from cardiac sympathetic nerves, increases NE exocytosis via a positive feedback mechanism. A neuronal ectonucleotidase (E-NTPDase) metabolizes the released ATP, decreasing NE exocytosis. Excessive NE release in myocardial ischemia exacerbates cardiac dysfunction. Thus, we studied whether the ATP-mediated autocrine amplification of NE release is operative in ischemia and, if so, whether it can be modulated by E-NTPDase and its recombinant equivalent, solCD39. Isolated, guinea pig hearts underwent 10- or 20-min ischemic episodes, wherein NE was released by exocytosis and reversal of the NE transporter, respectively. Furthermore, to restrict the role of E-NTPDase to transmitter ATP, sympathetic nerve endings were isolated (cardiac synaptosomes) and subjected to increasing periods of ischemia. Availability of released ATP at the nerve terminals was either increased via E-NTPDase inhibition or diminished by enhancing ATP hydrolysis with solCD39. P2X receptor blockade with PPADS was used to attenuate the effects of released ATP. We found that, in short-term ischemia (but, as anticipated, not in protracted ischemia, where NE release is carrier-mediated), ATP exocytosis was linearly correlated with that of NE. This indicates that by limiting the availability of ATP at sympathetic terminals, E-NTPDase effectively attenuates NE exocytosis in myocardial ischemia. Our findings suggest a key role for neuronal E-NTPDase in the control of adrenergic function in the ischemic heart. Because excessive NE release is an established cause of dysfunction in ischemic heart disease, solCD39 may offer a novel therapeutic approach to myocardial ischemia and its consequences.

Platelets are responsible for maintaining vascular integrity. In thrombocytopenic states, vascular permeability and fragility increase, presumably due to the absence of this platelet function. Chemical or physical injury to a blood vessel induces platelet activation and platelet recruitment. This is beneficial for the arrest of bleeding (hemostasis), but when an atherosclerotic plaque is ulcerated or fissured, it becomes an agonist for vascular occlusion (thrombosis). Experiments in the late 1980s cumulatively indicated that endothelial cell CD39-an ecto-ADPase-reduced platelet reactivity to most agonists, even in the absence of prostacyclin or nitric oxide. As discussed herein, CD39 rapidly and preferentially metabolizes ATP and ADP released from activated platelets to AMP, thereby drastically reducing or even abolishing platelet aggregation and recruitment. Since ADP is the final common agonist for platelet recruitment and thrombus formation, this finding highlights the significance of CD39. A recombinant, soluble form of human CD39, solCD39, has enzymatic and biological properties identical to the full-length form of the molecule and strongly inhibits human platelet aggregation induced by ADP, collagen, arachidonate, or TRAP (thrombin receptor agonist peptide). In sympathetic nerve endings isolated from guinea pig hearts, where neuronal ATP enhances norepinephrine exocytosis, solCD39 markedly attenuated norepinephrine release. This suggests that NTPDase (nucleoside triphosphate diphosphohydrolase) could exert a cardioprotective action by reducing ATP-mediated norepinephrine release, thereby offering a novel therapeutic approach to myocardial ischemia and its consequences. In a murine model of stroke, driven by excessive platelet recruitment, solCD39 reduced the sequelae of stroke, without an increase in intracerebral hemorrhage. CD39 null mice, generated by deletion of apyrase-conserved regions 2 to 4, exhibited a decrease in postischemic perfusion and an increase in cerebral infarct volume when compared with controls. "Reconstitution" of CD39 null mice with solCD39 reversed these changes. We hypothesize that solCD39 has potential as a novel therapeutic agent for thrombotic diatheses.

ATP and norepinephrine (NE) are cotransmitters released from many postganglionic sympathetic nerves. In this article, we review the evidence for ATP and NE cotransmission in the rodent vas deferens with special attention to the mechanisms involved in removing the cotransmitters from the neuroeffector junction. Although the clearance of NE is well understood (e.g., the primary mechanism being reuptake into the nerves), the clearance of ATP is just beginning to be explained. The general belief has been that ATP is metabolized by cell-fixed ecto-nucleotidases. It now seems, however, that when ATP is released from nerves as a transmitter there is a concomitant release of nucleotidases that rapidly degrade ATP sequentially to ADP, AMP, and adenosine, thereby terminating the action of ATP. In the guinea pig vas deferens, there appear to be at least two enzymes, one that converts ATP to ADP and ADP to AMP (an ATPDase) and a second enzyme that converts AMP to adenosine (an AMPase). An important feature of this process is that the transmitter-metabolizing nucleotidases are released into the synaptic space as opposed to being fixed to cell membranes. A preliminary characterization of these enzymes suggests that the releasable ATPDase exhibits some similarities to known ectonucleoside triphosphate/diphosphohydrolases, whereas the releasable AMPase exhibits some similarities to ecto-5'-nucleotidases.

Previously, we have demonstrated that stimulation of the sympathetic nerves of the guinea pig vas deferens evokes release not only of the cotransmitters ATP and norepinephrine but also of soluble nucleotidases that break down extracellular ATP, ADP, and AMP into adenosine. In this study we show that the apparent K(m) values of the releasable enzyme activity vary depending on which of these adenine nucleotides is used as initial substrate. The K(m) value for ATP was 33.6 +/- 2.3 microM, 21.0 +/- 2.3 microM for ADP, and 10.0 +/- 1.1 microM for AMP. The ratios of the V(max) values for each enzyme reaction were 4:2:3. We have also found a different sensitivity of the metabolism of ATP and AMP by releasable nucleotidases to known nucleotidase inhibitors. Suramin inhibited the breakdown of ATP by releasable nucleotidases in a noncompetitive manner and with a K(i) value of 53 microM, but had no effect on the breakdown of AMP. The 5'-nucleotidase inhibitor alpha,beta-methylene ADP inhibited the breakdown of AMP but not that of ATP. Concanavalin A inhibited the breakdown of AMP but had neither inhibitory nor facilitatory effects on the breakdown of ATP. 6-N,N-Diethyl-beta,gamma-dibromomethylene-D-ATP (ARL67156), an ecto-ATPase inhibitor, suppressed ATPase and AMPase activities, whereas NaN(3) (10 mM) affected neither reaction, but inhibited the ADP metabolism. Phosphatase- and phosphodiesterase inhibitors did not affect the activity of the releasable nucleotidases. This evidence suggests that the soluble nucleotidases released during neurogenic stimulation of the guinea pig vas deferens combine an ecto-5'-nucleotidase-like and an ecto-nucleoside triphosphate diphosphohydrolase-like activity.

ATP, coreleased with norepinephrine, affects adrenergic transmission by acting on purinoceptors at sympathetic nerve endings. Ectonucleotidases terminate the actions of ATP. Previously, we had preliminary evidence for ectonucleotidase activity in cardiac sympathetic nerve terminals. Therefore, we investigated whether this ectonucleotidase might influence norepinephrine release in the heart. Sympathetic nerve endings isolated from guinea pig heart (cardiac synaptosomes) were rich in Ca(2+)-dependent ectonucleotidase activity, as measured by metabolism of exogenously added radiolabeled ATP or ADP. By its inhibitor profile, ectonucleotidase resembled ectonucleoside triphosphate diphosphohydrolase 1 (E-NTPDase1). Exogenous ATP elicited concentration-dependent norepinephrine release from cardiac synaptosomes (EC(50) 0.96 microM). This release was antagonized by the P2X receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (10 microM) and potentiated by the P2Y receptor antagonist 2'-deoxy-N(6)-methyladenosine-3',5'-diphosphate (MRS 2179) (30 nM). Norepinephrine release promoted by ATP was also potentiated by the nucleotidase inhibitor 6-N,N-diethyl-beta-gamma-dibromomethylene-D-adenosine-5'-triphosphate (ARL67156) (30 microM) and blocked by a recombinant, soluble form of human E-NTPDase1 (solCD39). In contrast, ARL67156 had no effect on norepinephrine release induced by the nonhydrolyzable analog, alpha, beta-methyleneadenosine-5'-triphosphate (alpha,beta-MeATP). Depolarization of cardiac synaptosomes with K(+) elicited release of endogenous norepinephrine. This was attenuated by PPADS and solCD39 and potentiated by MRS 2179 and ARL67156. Importantly, our results demonstrate that facilitation of ATP-induced norepinephrine release from cardiac sympathetic nerves is a composite of two autocrine components: positive, mediated by P2X receptors, and negative, mediated by P2Y receptors. Modulation of norepinephrine release by coreleased ATP is terminated by endogenous as well as exogenous ectonucleotidase. We propose that ectonucleotidase control of norepinephrine release should provide cardiac protection in hyperadrenergic states such as myocardial ischemia.

Subtotal destruction of central noradrenergic neurons with the neurotoxin, 6-hydroxydopamine, is known to increase tyrosine hydroxylase (TH) activity within surviving nerve terminals. The present report demonstrates a similar, dose-related elevation of TH activity in the adrenal medulla and sympathetic nerves after systemic 6-hydroxydopamine treatment. Two temporally distinct processes were observed in the sympathetic nerves: a rapid activation of TH, present during the first few days after the lesion, and a more gradual increase in maximal TH activity, most probably due to enzyme induction. In this regard, the stimulatory effect on catecholamine biosynthesis of cyclic AMP-dependent phosphorylation was attenuated substantially within cardiac sympathetic nerves 3 days after the lesion, but by 3 weeks the efficacy of cyclic AMP-dependent phosphorylation was restored completely. Similarly, the normal activation of cardiac TH activity elicited by insulin-induced hypoglycemia and cold stress was absent soon after 6-hydroxydopamine treatment, but returned 3 weeks later. These latter findings suggest that the adaptations within the sympathoadrenal system after subtotal sympathectomy can preclude further adaptations to stress.

A procedure is described for measuring the activity of tyrosine hydroxylase in the intact adrenergic neurons of the mouse vas deferens. In this procedure, the L-dopa-1-14C which is formed from L-tyrosine-1-14C by the action of tyrosine hydroxylase is selectively and quantitatively decarboxylated by endogenous aromatic-L-amino acid decarboxylase. Although considerable tyrosine hydroxylase activity can be demonstrated with the intact mouse vas deferens in the absence of exogenous tetrahydropterin cofactor, the addition of 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine results in increased activity. Exogenous norepinephrine inhibits the activity of tyrosine hydroxylase in the intact mouse vas deferens and this inhibitory effect is competitively antagonized by 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine. When the vesicular catecholamine uptake mechanism is blocked by treatment of mice with 5 mg/kg of reserpine 24 hours prior to sacrifice, the activity of tyrosine hydroxylase in intact vas deferens is reduced and the inhibitory effect of exogenous norepinephrine is enhanced. Inhibition of monoamine oxidase with 0.15 mM pargyline does not affect the activity of tyrosine hydroxylase in the intact mouse vas deferens when the vesicular catecholamine uptake mechanism is intact but has a pronounced inhibitory effect following reserpine treatment. These observations lend further support to the conclusion that the activity of tyrosine hydroxylase in the intact adrenergic neuron is inversely related to the catecholamine concentration within an extravesicular pool. They also suggest that the catecholamines tend to accumulate within this extravesicular pool and thus become accessible to the action of monoamine oxidase when the vesicular uptake mechanism is inactivated or when the vesicular stores are filled to capacity.