Angiotensin-converting enzyme-independent angiotensin formation in a human model of myocardial ischemia: modulation of norepinephrine release by angiotensin type 1 and angiotensin type 2 receptors

Angiotensin II (Ang II) promotes norepinephrine (NE) release from cardiac sympathetic nerve endings. We assessed in a human model in vitro whether locally formed Ang II contributes to NE release in myocardial ischemia. Surgical specimens of human right atrium were incubated in anoxic conditions. After 70 min of anoxia, NE release (carrier-mediated; caused by NE transporter reversal) was 8-fold greater than normoxic release. Angiotensin-converting enzyme inhibition with enalaprilat failed to reduce anoxic NE release. In contrast, prevention of chymase-dependent Ang II formation with chymostatin, Bowman-Birk inhibitor, or alpha(1)-antitrypsin significantly inhibited anoxic, but not exocytotic, NE release. Two mast-cell stabilizers, cromolyn and lodoxamide, markedly reduced NE release, implicating cardiac mast cells as a major source of chymase. Angiotensin type 1 receptor (AT(1)R) blockade with EXP3174 inhibited NE release, whereas angiotensin type 2 receptor (AT(2)R) blockade with PD123319 did not. Interestingly, PD123319 reversed the inhibitory effect of EXP3174. Furthermore, synergisms were uncovered between EXP3174 and an AT(2)R agonist, and between EXP3174 and a Na(+)/H(+) exchanger inhibitor. Thus, angiotensin-converting enzyme-independent Ang II formation via chymase is important for carrier-mediated ischemic NE release in the human heart. Locally generated Ang II promotes NE release by acting predominantly at AT(1)Rs, which are likely coupled to the Na(+)/H(+) exchanger. Effects of Ang II at AT(2)Rs, seemingly opposite to those resulting from AT(1)R activation, are uncovered when AT(1)Rs are blocked. Because NE release is associated with coronary vasoconstriction and arrhythmias, and mast-cell density and chymase content increase in the ischemic heart, the notion that chymase-generated Ang II plays a major role in carrier-mediated NE release may have important clinical implications.     

Bradykinin promotes ischemic norepinephrine release in guinea pig and human hearts

We previously reported that bradykinin (BK; 1-1000 nM) facilitates norepinephrine (NE) release from cardiac sympathetic nerves. Because BK production increases in myocardial ischemia, endogenous BK could foster NE release and associated arrhythmias. We tested this hypothesis in guinea pig and human myocardial ischemia models. BK administration (100 nM) markedly enhanced exocytotic and carrier-mediated NE overflow from guinea pig hearts subjected to 10- and 20-min ischemia/reperfusion, respectively. Ventricular fibrillation invariably occurred after 20-min global ischemia; BK prolonged its duration 3-fold. The BK B2 receptor antagonist HOE140 (30 nM) blocked the effects of BK, whereas the B1 receptor antagonist des-Arg9-Leu8-BK (1 microM; i.e., 2.5 x pA2) did not. When serine proteinase inhibitors (500 KIU/ml aprotinin and 100 microg/ml soybean trypsin inhibitor) were used to prevent the formation of endogenous BK, NE overflow and reperfusion arrhythmias were diminished. In contrast, when kininase I and II inhibitors (DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid and enalaprilat, each 1 microM) were used to prevent the degradation of endogenous BK, NE overflow and reperfusion arrhythmias were enhanced. B2 receptor blockade abolished these effects but was ineffective if kininases were not inhibited. B2 receptor stimulation, by either exogenous or endogenous BK, also markedly enhanced carrier-mediated NE release in the human myocardial ischemia model; conversely, inhibition of BK biosynthesis diminished ischemic NE release. Because atherosclerotic heart disease impairs endothelial BK production, in myocardial ischemia BK could accumulate at sympathetic nerve endings, thus augmenting exocytotic and carrier-mediated NE release and favoring coronary vasoconstriction and arrhythmias.     

LLC-PK(1) cells stably expressing the human norepinephrine transporter: A functional model of carrier-mediated norepinephrine release in protracted myocardial ischemia.

In myocardial ischemia, adrenergic terminals undergo ATP depletion, hypoxia, and intracellular pH reduction, causing the accumulation of axoplasmic norepinephrine (NE) and intracellular Na(+) [via the Na(+)-H(+) exchanger (NHE)]. This forces the reversal of the Na(+)- and Cl(-)-dependent NE transporter (NET), triggering massive carrier-mediated NE release and, thus, arrhythmias. We have now developed a cellular model of carrier-mediated NE release using an LLC-PK(1) cell line stably transfected with human NET cDNA (LLC-NET). LLC-NET cells transported [(3)H]NE and [(3)H]N-methyl-4-phenylpyridinium ([(3)H]MPP(+)) in an inward direction. This uptake was abolished by the NET inhibitors desipramine (100 nM) and mazindol (300 nM) and by extracellular Na(+) removal. Na(+)-gradient reversal induced an efflux of (3)H-substrate from preloaded LLC-NET cells. Desipramine and mazindol blocked this efflux. Because of its greater intracellular stability and higher sensitivity to Na(+)-gradient reversal, [(3)H]MPP(+) proved preferable to [(3)H]NE as an NET substrate; therefore, only [(3)H]MPP(+) was used for subsequent studies. The K(+)/H(+) ionophore nigericin (10 microM) evoked a large efflux of [(3)H]MPP(+). This efflux was potentiated by the Na(+),K(+)-ATPase inhibitor ouabain (100 microM), was sensitive to desipramine, and was blocked by the NHE inhibitor 5-(N-ethyl-N-isopropyl)-amiloride (EIPA; 10 microM). In contrast, EIPA failed to inhibit the [(3)H]MPP(+) efflux elicited by the Na(+) ionophore gramicidin (10 microM). Furthermore, [(3)H]MPP(+) efflux induced by the NHE-stimulant proprionate (25 mM) was negatively modulated by imidazoline receptor activation. Our findings suggest that LLC-NET cells are a sensitive model for studying transductional processes of carrier-mediated NE release associated with myocardial ischemia.     

Ectonucleotidase in sympathetic nerve endings modulates ATP and norepinephrine exocytosis in myocardial ischemia

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.     

Histamine 3 receptor activation reduces the expression of neuronal angiotensin II type 1 receptors in the heart

In severe myocardial ischemia, histamine 3 (H?) receptor activation affords cardioprotection by preventing excessive norepinephrine release and arrhythmias; pivotal to this action is the inhibition of neuronal Na?/H? exchanger (NHE). Conversely, angiotensin II, formed locally by mast cell-derived renin, stimulates NHE via angiotensin II type 1 (AT?) receptors, facilitating norepinephrine release and arrhythmias. Thus, ischemic dysfunction may depend on a balance between the NHE-modulating effects of H? receptors and AT? receptors. The purpose of this investigation was therefore to elucidate the H?/AT? receptor interaction in myocardial ischemia/reperfusion. We found that H? receptor blockade with clobenpropit increased norepinephrine overflow and arrhythmias in Langendorff-perfused guinea pig hearts subjected to ischemia/reperfusion. This coincided with increased neuronal AT? receptor expression. NHE inhibition with cariporide prevented both increases in norepinephrine release and AT? receptor expression. Moreover, norepinephrine release and AT? receptor expression were increased by the nitric oxide (NO) synthase inhibitor N(G)-methyl-L-arginine and the protein kinase C activator phorbol myristate acetate. H? receptor activation in differentiated sympathetic neuron-like PC12 cells permanently transfected with H? receptor cDNA caused a decrease in protein kinase C activity and AT? receptor protein abundance. Collectively, our findings suggest that neuronal H? receptor activation inhibits NHE by diminishing protein kinase C activity. Reduced NHE activity sequentially causes intracellular acidification, increased NO synthesis, and diminished AT? receptor expression. Thus, H? receptor-mediated NHE inhibition in ischemia/reperfusion not only opposes the angiotensin II-induced stimulation of NHE in cardiac sympathetic neurons, but also down-regulates AT? receptor expression. Cardioprotection ultimately results from the combined attenuation of angiotensin II and norepinephrine effects and alleviation of arrhythmias.     

Bradykinin activates a cross-signaling pathway between sensory and adrenergic nerve endings in the heart: a novel mechanism of ischemic norepinephrine release?

We had shown that bradykinin (BK) generated by cardiac sympathetic nerve endings (i.e., synaptosomes) promotes exocytotic norepinephrine (NE) release in an autocrine mode. Because the synaptosomal preparation may include sensory C-fiber endings, which BK is known to stimulate, sensory nerves could contribute to the proadrenergic effects of BK in the heart. We report that BK is a potent releaser of NE from guinea pig heart synaptosomes (EC(50) approximately 20 nM), an effect mediated by B(2) receptors, and almost completely abolished by prior C-fiber destruction or blockade of calcitonin gene-related peptide and neurokinin-1 receptors. C-fiber destruction also greatly decreased BK-induced NE release from the intact heart, whereas tyramine-induced NE release was unaffected. Furthermore, C-fiber stimulation with capsaicin and activation of calcitonin gene-related peptide and neurokinin-1 receptors initiated NE release from cardiac synaptosomes, indicating that stimulation of sensory neurons in turn activates sympathetic nerve terminals. Thus, BK is likely to release NE in the heart in part by first liberating calcitonin gene-related peptide and Substance P from sensory nerve endings; these neuropeptides then stimulate specific receptors on sympathetic terminals. This action of BK is positively modulated by cyclooxygenase products, attenuated by activation of histamine H(3) receptors, and potentiated at a lower pH. The NE-releasing action of BK is likely to be enhanced in myocardial ischemia, when protons accumulate, C fibers become activated, and the production of prostaglandins and BK increases. Because NE is a major arrhythmogenic agent, the activation of this interneuronal signaling system between sensory and adrenergic neurons may contribute to ischemic dysrhythmias and sudden cardiac death.     

Ischemia promotes renin activation and angiotensin formation in sympathetic nerve terminals isolated from the human heart: contribution to carrier-mediated norepinephrine release

We recently reported that in the ischemic human heart, locally formed angiotensin II activates angiotensin II type 1 (AT(1)) receptors on sympathetic nerve terminals, promoting reversal of the norepinephrine transporter in an outward direction (i.e., carrier-mediated norepinephrine release). The purpose of this study was to assess whether cardiac sympathetic nerve endings contribute to local angiotensin II formation, in addition to being a target of angiotensin II. To this end, we isolated sympathetic nerve endings (cardiac synaptosomes) from surgical specimens of human right atrium and incubated them in ischemic conditions (95% N(2,) sodium dithionite, and no glucose for 70 min). These synaptosomes released large amounts of endogenous norepinephrine via a carrier-mediated mechanism, as evidenced by the inhibitory effect of desipramine on this process. Norepinephrine release was further enhanced by preincubation of synaptosomes with angiotensinogen and was prevented by two renin inhibitors, pepstatin-A and BILA 2157BS, as well as by the angiotensin-converting enzyme inhibitor enalaprilat and the AT(1) receptor antagonist EXP 3174 [2-N-butyl-4-chloro-1-[2'-(1H-tetrazol-5-yl)biphenyl-4-yl] methyl]imidazole-5-carboxylic acid]. Western blot analysis revealed the presence of renin in cardiac sympathetic nerve terminals; renin abundance increased ~3-fold during ischemia. Thus, renin is rapidly activated during ischemia in cardiac sympathetic nerve terminals, and this process eventually culminates in angiotensin II formation, stimulation of AT(1) receptors, and carrier-mediated norepinephrine release. Our findings uncover a novel autocrine/paracrine mechanism whereby angiotensin II, formed at adrenergic nerve endings in myocardial ischemia, elicits carrier-mediated norepinephrine release by activating adjacent AT(1) receptors.     

Aldehyde Dehydrogenase Type 2 Activation by Adenosine and Histamine Inhibits Ischemic Norepinephrine Release in Cardiac Sympathetic Neurons: Mediation by Protein Kinase C{varepsilon}

During myocardial ischemia/reperfusion, lipid peroxidation leads to the formation of toxic aldehydes that contribute to ischemic dysfunction. Mitochondrial aldehyde dehydrogenase type 2 (ALDH2) alleviates ischemic heart damage and reperfusion arrhythmias via aldehyde detoxification. Because excessive norepinephrine release in the heart is a pivotal arrhythmogenic mechanism, we hypothesized that neuronal ALDH2 activation might diminish ischemic norepinephrine release. Incubation of cardiac sympathetic nerve endings with acetaldehyde, at concentrations achieved in myocardial ischemia, caused a concentration-dependent increase in norepinephrine release. A major increase in norepinephrine release also occurred when sympathetic nerve endings were incubated in hypoxic conditions. ALDH2 activation substantially reduced acetaldehyde- and hypoxia-induced norepinephrine release, an action prevented by inhibition of ALDH2 or protein kinase Cε (PKCε). Selective activation of G(i/o)-coupled adenosine A(1), A(3), or histamine H(3) receptors markedly inhibited both acetaldehyde- and hypoxia-induced norepinephrine release. These effects were also abolished by PKCε and/or ALDH2 inhibition. Moreover, A(1)-, A(3)-, or H(3)-receptor activation increased ALDH2 activity in a sympathetic neuron model (differentiated PC12 cells stably transfected with H(3) receptors). This action was prevented by the inhibition of PKCε and ALDH2. Our findings suggest the existence in sympathetic neurons of a protective pathway initiated by A(1)-, A(3)-, and H(3)-receptor activation by adenosine and histamine released in close proximity of these terminals. This pathway comprises the sequential activation of PKCε and ALDH2, culminating in aldehyde detoxification and inhibition of hypoxic norepinephrine release. Thus, pharmacological activation of PKCε and ALDH2 in cardiac sympathetic nerves may have significant protective effects by alleviating norepinephrine-induced life-threatening arrhythmias that characterize myocardial ischemia/reperfusion.     

The plasminogen activator system modulates sympathetic nerve function

Sympathetic neurons synthesize and release tissue plasminogen activator (t-PA). We investigated whether t-PA modulates sympathetic activity. t-PA inhibition markedly reduced contraction of the guinea pig vas deferens to electrical field stimulation (EFS) and norepinephrine (NE) exocytosis from cardiac synaptosomes. Recombinant t-PA (rt-PA) induced exocytotic and carrier-mediated NE release from cardiac synaptosomes and cultured neuroblastoma cells; this was a plasmin-independent effect but was potentiated by a fibrinogen cleavage product. Notably, hearts from t-PA-null mice released much less NE upon EFS than their wild-type (WT) controls (i.e., a 76.5% decrease; P<0.01), whereas hearts from plasminogen activator inhibitor-1 (PAI-1)-null mice released much more NE (i.e., a 275% increase; P<0.05). Furthermore, vasa deferentia from t-PA-null mice were hyporesponsive to EFS (P<0.0001) but were normalized by the addition of rt-PA. In contrast, vasa from PAI-1-null mice were much more responsive (P<0.05). Coronary NE overflow from hearts subjected to ischemia/reperfusion was much smaller in t-PA-null than in WT control mice (P<0.01). Furthermore, reperfusion arrhythmias were significantly reduced (P<0.05) in t-PA-null hearts. Thus, t-PA enhances NE release from sympathetic nerves and contributes to cardiac arrhythmias in ischemia/reperfusion. Because the risk of arrhythmias and sudden cardiac death is increased in hyperadrenergic conditions, targeting the NE-releasing effect of t-PA may have valuable therapeutic potential.     

The aminotetraline derivative (+/-)-(R,S)-5,6-dihydroxy-2-methylamino-1,2,3,4-tetrahydro-naphthalene hydrochloride (CHF-1024) displays cardioprotection in postischemic ventricular dysfunction of the rat heart

To analyze the protective effects of the aminotetraline derivative (+/-)-(R,S)-5,6-dihydroxy-2-methylamino-1,2,3,4-tetrahydro-naphthalene hydrochloride (CHF-1024), a compound endowed with DA2-dopaminergic/alpha2-adrenergic receptor agonistic activity, in myocardial ischemia/reperfusion damage. A model of isolated and perfused (15 ml/min) electrically driven (300 beats/min) rat heart subjected to global ischemia (1 ml/min for 20 min) and reperfusion (15 ml/min for 30 min) was followed. Cardiac mechanics changes were evaluated together with biochemical markers of cardiac ischemia in perfusate and tissue tumor necrosis factor-alpha (TNF-alpha). CHF-1024, perfused through the heart for 15 min before ischemia at different molar concentrations (1-100 nM), significantly improved left ventricle developed pressure during reperfusion, and normalized left ventricular end-diastolic pressure and coronary perfusion pressure. This anti-ischemic effect of CHF-1024 was associated to a decrease in creatine kinase and lactate dehydrogenase, both released during heart reperfusion. These events were concomitant with maintenance of a higher production of 6-keto-prostaglandin F1alpha The ability of CHF-1024 to improve postischemic ventricular dysfunction was correlated with a dose-dependent inhibition of the release of both norepinephrine (NE), from sympathetic nerve endings, and TNF-alpha from cardiac tissue. The effect of CHF-1024 on NE release was almost completely antagonized by specific antagonists of presynaptic inhibitory receptors domperidone and rauwolscine. The finding that this new aminotetraline derivative possesses anti-ischemic properties and limits NE release from cardiac nerve endings may bear some therapeutic potential in cardiovascular diseases.     

Histamine H3-receptor-induced attenuation of norepinephrine exocytosis: a decreased protein kinase a activity mediates a reduction in intracellular calcium

We had reported that activation of presynaptic histamine H(3)-receptors inhibits norepinephrine exocytosis from depolarized cardiac sympathetic nerve endings, an action associated with a marked decrease in intraneuronal Ca(2+) that we ascribed to a decreased Ca(2+) influx. An H(3)-receptor-mediated inhibition of cAMP-dependent phosphorylation of Ca(2+) channels could cause a sequential attenuation of Ca(2+) influx, intraneuronal Ca(2+) and norepinephrine exocytosis. We tested this hypothesis in sympathetic nerve endings (cardiac synaptosomes) expressing native H(3)-receptors and in human neuroblastoma SH-SY5Y cells transfected with H(3)-receptors. Norepinephrine exocytosis was elicited by K(+) or by stimulation of adenylyl cyclase with forskolin. H(3)-receptor activation markedly attenuated the K(+)- and forskolin-induced norepinephrine exocytosis; pretreatment with pertussis toxin prevented this effect. Similar to forskolin, 8-bromo-cAMP elicited norepinephrine exocytosis but, unlike forskolin, it was unaffected by H(3)-receptor activation, demonstrating that inhibition of adenylyl cyclase is a pivotal step in the H(3)-receptor transductional cascade. Indeed, we found that H(3)-receptor activation attenuated norepinephrine exocytosis concomitantly with a decrease in intracellular cAMP and PKA activity in SH-SY5Y-H(3) cells. Moreover, pharmacological PKA inhibition acted synergistically with H(3)-receptor activation to reduce K(+)-induced peak intracellular Ca(2+) in SH-SY5Y-H(3) cells and norepinephrine exocytosis in cardiac synaptosomes. Furthermore, H(3)-receptor activation synergized with N- and L-type Ca(2+) channel blockers to reduce norepinephrine exocytosis in cardiac synaptosomes. Our findings suggest that the H(3)-receptor-mediated inhibition of norepinephrine exocytosis from cardiac sympathetic nerves results sequentially from H(3)-receptor-G(i)/G(o) coupling, inhibition of adenylyl cyclase activity, and decreased cAMP formation, leading to diminished PKA activity, and thus, decreased Ca(2+) influx through voltage-operated Ca(2+) channels.     

Na(+)-dependent transporters mediate HCO(3)(-) salvage across the luminal membrane of the main pancreatic duct

To study the roles of Na(+)-dependent H(+) transporters, we characterized H(+) efflux mechanisms in the pancreatic duct in wild-type, NHE2(-/-), and NHE3(-/-) mice. The pancreatic duct expresses NHE1 in the basolateral membrane, and NHE2 and NHE3 in the luminal membrane, but does not contain NHE4 or NHE5. Basolateral Na(+)-dependent H(+) efflux in the microperfused duct was inhibited by 1.5 microM of the amiloride analogue HOE 694, consistent with expression of NHE1, whereas the luminal activity required 50 microM HOE 694 for effective inhibition, suggesting that the efflux might be mediated by NHE2. However, disruption of NHE2 had no effect on luminal transport, while disruption of the NHE3 gene reduced luminal Na(+)-dependent H(+) efflux by approximately 45%. Notably, the remaining luminal Na(+)-dependent H(+) efflux in ducts from NHE3(-/-) mice was inhibited by 50 microM HOE 694. Hence, approximately 55% of luminal H(+) efflux (or HCO(3)(-) influx) in the pancreatic duct is mediated by a novel, HOE 694-sensitive, Na(+)-dependent mechanism. H(+) transport by NHE3 and the novel transporter is inhibited by cAMP, albeit to different extents. We propose that multiple Na(+)-dependent mechanisms in the luminal membrane of the pancreatic duct absorb Na(+) and HCO(3)(-) to produce a pancreatic juice that is poor in HCO(3)(-) and rich in Cl(-) during basal secretion. Inhibition of the transporters during stimulated secretion aids in producing the HCO(3)(-)-rich pancreatic juice.     

Role of neuronal KATP channels and extraneuronal monoamine transporter on norepinephrine overflow in a model of myocardial low flow ischemia

Global myocardial low flow ischemia results in an uniform suppression of norepinephrine (NE) overflow from the heart. We hypothesized that opening of neuronal ATP-sensitive potassium (K(ATP)) channels as well as activation of the extraneuronal monoamine transporter (EMT) mediates attenuation of NE overflow during low flow ischemia. Isolated rat hearts were subjected to low coronary flow of 0.4 ml min(-1). Release of endogenous NE was induced by electrical field stimulation. EMT activity was measured as the transport rate of the substrate N-[methyl-3H]4-phenylpyridinium ([3H]MPP+). NE overflow decreased by 57 +/- 2% within 120 min of low flow. Five minutes of reperfusion at normal flow (8 ml min(-1)) restored NE overflow to baseline. K(ATP) channel blockade with glibenclamide as well as EMT blockade with corticosterone increased NE overflow 1.5- and 2-fold at 120 min of low flow, whereas neither drug affected NE overflow in the absence of flow reduction. At normal flow, K(ATP) channel opening with cromakalim suppressed NE overflow, both in the presence and absence of EMT blockade (14 +/- 4 and 9 +/- 1%). However, cromakalim had no effect on EMT activity as indicated by an unaffected [3H]MPP+ overflow. In conclusion, activation of both K(ATP) channels and EMT mediate suppression of NE overflow during low flow ischemia. K(ATP) channels impair NE release directly at presynaptic nerve endings, whereas EMT increases NE elimination in a manner independent of K(ATP) channels.     

Altered responsiveness to sympathetic nerve stimulation and agonists of isolated left atria of diabetic rats: no evidence for involvement of hypothyroidism

To clarify the role of hypothyroidism in diabetes-induced sympathetic neuropathy, we examined the responsiveness to sympathetic nerve stimulation and to agonists of the left atria of streptozotocin-induced diabetic rats, and compared it with those in hypothyroid, thyroxine(T4)-treated diabetic or T4-treated hypothyroid rats. Positive inotropic response of isolated left atria to transmural nerve stimulation (TNS) was examined in the presence of atropine. In diabetic rats, plasma triiodothyronine and T4 levels decreased markedly. The responses to TNS and norepinephrine (NE) also decreased. The decrease was greater in response to TNS, which may be due to the reduction of presynaptic NE release. As to the postsynaptic response, the maximal response to NE decreased without any significant change in ED50 value, and a similar decrease in the maximal response to Ca++ was also observed in spite of a slight decrease in the beta receptor density. Therefore, in diabetic rats the decreased response to TNS may result mainly from a decreased NE release from nerve endings and a decreased contractility beyond the adrenoceptor level. In hypothyroid rats, the response to TNS and NE decreased, and the decrease was again greater in response to TNS. The decrease in the NE response was due to the increased ED50 value without any change in the maximal response. Similarly, the maximal response to Ca++ did not change. Thus in the hypothyroid rats, the decreased response to TNS probably results from a decreased NE release from nerve endings as well as a decreased sensitivity to NE.(ABSTRACT TRUNCATED AT 250 WORDS)     

EctoNucleotidase in cardiac sympathetic nerve endings modulates ATP-mediated feedback of norepinephrine release.

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.     

Possible involvement of adenine nucleotides in sympathetic neuroeffector mechanisms of dog basilar artery

The transmission mechanism of sympathetic neuroeffector was studied in the isolated dog basilar artery. Electrical transmural stimulation produced an initial contractile response which was followed by a transient relaxation or a late contraction, or both relaxation and contraction. These arteries showed a marked uptake of [3H]norepinephrine ([3H]NE) or [3H]adenosine after incubation with these compounds, and electrical stimulation increased the release of [3H]NE or 3H-purine compounds. After treatment with tetrodotoxin or bretylium or in the sympathetically denervated arteries, the mechanical response to electrical stimulation and the release of 3H-compounds were attenuated; however, the mechanical response was not affected by treatment with reserpine. These results suggest that transmural stimulation releases not only [3H]NE but also 3H-purine compounds predominantly from the sympathetic nerve terminals and produces sympathetic contractile and relaxing responses. Phentolamine enhanced these sympathetic responses and augmented the release of [3H]NE and 3H-purine compounds. Exogenously applied NE produced a slowly developing contractile response and inhibited the release of 3H-purine compounds upon electrical transmural stimulation. Exogenously applied ATP produced responses which were similar in pattern to the sympathetic response and the release of [3H]NE was inhibited. Other adenine nucleotides and adenosine produced only relaxation. Theophylline attenuated the relaxing response to sympathetic nerve stimulation or to exogenously applied nucleotides. These results suggest that ATP or related nucleotides are released, concomitant with NE, from the sympathetic nerve terminals in the dog basilar artery and may act as neurotransmitters and/or modulators on presynaptic and postsynaptic membranes.     

Release of norepinephrine and dopamine-beta-hydroxylase by nerve stimulation. V. Enhanced release associated with a granular effect of a benzoquinolizine derivative with reserpine-like properties.

A reserpine-like agent, 2-hydroxy-2-ethyl-3-isobutyl-9,10-dimethoxy-1,2,3,4,6,7,-hexa-hydro-11b-H-benzo[a]quinolizine (BQZ), at concentrations that do not inhibit phosphodiesterase activity, produces a marked increase in the outflow of 3-H-dihydroxyphenyl-ethyleneglycol from the isolated, perfused cat slpeen prelabeled with 3-H-norepinephrine (3-H-NE). The increased intraneuronal levels of catechols probably account for the inhibition of the conversion of 1-minus14C-L-tyrosine to 1-minus14C-L-dopa which is observed in the presence of the drug. In addition, in the presence of 0.9 muM BQZ, there is a 2.5- to 3-fold increase in the nerve stimulation-mediated overflow of NE, 3-H-NE, total 3-H and dopamine-beta-hydroxylase activity. A highly significant positive correlation was observed between the increase in the spontaneous release of 3-H and the enhanced exocytotic release of transmitter by nerve stimulation. These results suggest that either a primary alteration of the storage granule membrane or the subsequent enhanced intraneuronal levels of NE or NE metabolites may be responsible for the enhanced exocytotic release by nerve stimulation. In the presence of 0.9 muM BQZ, addition of 3 muM cocaine produces an increase in the nerve stimulation-mediated overflow of NE and an inhibition of the formation of 3-H-dihydroxyphenylethyleneglycol. In addition, there is a 20 to 30% decrease in the overflow of 3-H and dopamine-beta-hydroxylase activity and a marked delay in the outflow of the enzyme elicited by nerve stimulation. These results suggested that, in the presence of BQZ, a large fraction of the NE released during nerve stimulation is recaptured into the nerve terminals where it is subsequently metabolized to 3-H-dihydroxyphenylethyleneglycol. The enhanced exocytotic release of NE, the extensive presynaptic metabolism of the recaptured transmitter subsequent to release by nerve stimulation, and inhibition of norepinephrine synthesis all appear to contribute to the accelerated depletion of tissue NE which is observed when the splenic nerves are stimulated in the presence of 0.9 muM BQZ. These results provide an explanation for the accelerated depletion of tissue NE in animals treated with reserpine-like compounds when the sympathetic innervation is intact.     

Ectonucleoside triphosphate diphosphohydrolase 1/CD39, localized in neurons of human and porcine heart, modulates ATP-induced norepinephrine exocytosis

Using a guinea pig heart synaptosomal preparation, we previously observed that norepinephrine (NE) exocytosis was attenuated by a blockade of P2X purinoceptors, potentiated by inhibition of ectonucleoside triphosphate diphosphohydrolase-1 (E-NTPDase1)/CD39, and reduced by soluble CD39, a recombinant form of human E-NTPDase1/CD39. This suggests that norepinephrine and ATP are coreleased upon depolarization of cardiac sympathetic nerve endings and that ATP enhances norepinephrine exocytosis by an action modulated by E-NTPDase1/CD39 activity. Whether E-NTPDase1/CD39 is localized to cardiac neurons and modulates norepinephrine exocytosis in intact heart tissue remained untested. We report that E-NTPDase1/CD39 is selectively localized in human and porcine cardiac neurons and that depolarization of porcine heart tissue elicits omega-conotoxin-inhibitable release of both norepinephrine and ATP. Inhibition of E-NTPDase1/CD39 with ARL67156 markedly potentiated ATP release, demonstrating that E-NTPDase1/CD39 is a major determinant of ATP availability at sympathetic nerve terminals. Notably, inhibition of E-NTPDase1/CD39 enhanced both ATP and NE exocytosis, whereas administration of soluble CD39 reduced both ATP and NE exocytosis. The strong correlation between ATP and norepinephrine release was abolished in the presence of the purinergic P2X receptor (P2XR) antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). We conclude that released ATP governs norepinephrine exocytosis by activating presynaptic P2XR and that this action is controlled by neuronal E-NTPDase1/CD39. Clinically, excessive norepinephrine release is a major cause of arrhythmic and coronary vascular dysfunction during myocardial ischemia. By curtailing NE release, in addition to its effects as an antithrombotic agent, soluble CD39 may constitute a novel therapeutic approach to ischemic complications in the myocardium.     

Reduction of sympathetic inotropic response after ischemia in dogs. Contributor to stunned myocardium.

Eight open chest dogs underwent 25 min of coronary occlusion to determine whether brief myocardial ischemia disrupts the normal myocardial inotropic response to sympathetic nervous stimulation. If so, this could represent a mechanism contributing to postischemic myocardial dysfunction. Myocardial segment shortening was measured using ultrasonic dimension crystals before and after coronary artery occlusion and reperfusion. Left ansa subclavia stimulation and systemic norepinephrine (NE) infusion were used to test the myocardial inotropic response to neural stimulation and direct exposure to the sympathetic mediator, respectively. Before coronary artery occlusion, base-line preischemic segment shortening (12.5 +/- 1.6%) (SEM) increased during both sympathetic stimulation (20.2 +/- 1.4%) and NE infusion (19.7 +/- 1.1%). The control segment responded similarly. After ischemia and reperfusion there was no significant change in heart rate, aortic or left ventricular pressures, nor changes in control segment shortening. In contrast, shortening in the postischemic segment was markedly reduced compared to baseline (4.1 +/- 2.4%), and no longer responded to sympathetic stimulation (2.4 +/- 2.8%), while responsiveness to systemic NE was maintained (12.9 +/- 2.0%), P less than 0.001, which suggested injury to the sympathetic-neural axis during the period of ischemia. This reduced response to neural stimulation was persistent for up to 2 h after reperfusion. Left atrial or intracoronary infusion of bretylium tosylate, which releases norepinephrine from nerve terminals, resulted in an immediate inotropic response in the postischemic segment, which indicated that total depletion of NE from nerve terminals during the ischemic period had not occurred. Disruption of sympathetic neural responsiveness is likely a component of the mechanism of postischemic myocardial dysfunction whenever there is appreciable sympathetic drive to the heart.