Aldehyde dehydrogenase type 2 activation by adenosine and histamine inhibits ischemic norepinephrine release in cardiac sympathetic neurons: mediation by protein kinase Cε

Abstract

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.

Fig. 1.

Release of NE from cardiac synaptosomes during 30-min hypoxia. Bars (means ± S.E.M.) represent the hypoxia-induced increase in NE release above the normoxic basal level of 273 ± 11.5 pmol/mg protein (n = 49). A, activation of ALDH2 with Alda-1 (20 μM; 12 min) reduces hypoxia-induced NE release. Desensitization of ALDH2 with GTN (2 μM; 30 min) prevents the effect of Alda-1 (n = 7–12). *, P < 0.05 versus hypoxia. #, P < 0.05 versus Alda-1 by unpaired t test. B, PKCε activation with ΨεRACK (PKCε activator; 500 nM; 12 min) reduces hypoxia-induced NE release. ALDH2 desensitization with GTN (2 μM; 30 min) prevents the effect of ΨεRACK (n = 7–8). *, P < 0.05 versus hypoxia. #, P < 0.05 versus ΨεRACK by unpaired t test. C, activation of adenosine A₁ receptor with 2′-MeCCPA (A₁R agonist; 10 nM; 12 min) diminishes hypoxia-induced NE release. ALDH2 desensitization with GTN (2 μM; 30 min) and PKCε blockade with εV_1–2 (PKCε inhibitor; 1 μM; 20 min) each prevents the effect of 2′-MeCCPA (n = 7–10). **, P < 0.005 versus hypoxia. #, P < 0.05 versus 2′-MeCCPA by unpaired t test. D, blockade of adenosine A₁-receptor with DPCPX (A₁R antagonist; 300 nM; 12 min) enhances hypoxia-induced NE release (n = 15–17). *, P < 0.05 versus hypoxia by unpaired t test. E, selective activation of adenosine A₃ receptor with IB-MECA (A₃R agonist; 50 nM; 12 min) reduces hypoxia-induced NE release. ALDH2 desensitization with GTN (2 μM; 30 min) and PKCε blockade with εV_1–2 (1 μM; 20 min) each prevents the effect of IB-MECA (n = 7–11). **, P < 0.01 versus hypoxia. ##, P < 0.005 versus IB-MECA. ###, P < 0.001 versus IB-MECA by unpaired t test. F, blockade of adenosine A₃ receptor with MRS1523 (A₃R antagonist; 100 nM; 12 min) enhances hypoxia-induced NE (n = 7–9). *, P < 0.05 versus hypoxia by unpaired t test.

Fig. 2.

Incubation of isolated cardiac synaptosomes with acetaldehyde elicits a concentration-dependent increase in NE release, which is inhibited by the activation of either ALDH2 or PKCε. Points (means ± S.E.M.; n = 4–46 and 12–24 in A and B, respectively) represent percentage increases in NE release above a mean basal control level of 205 ± 7.4 pmol/mg protein (n = 92). A, ALDH2 activation with Alda-1 (20 μM; 10 min) attenuates the acetaldehyde-induced release of NE. ALDH2 desensitization with GTN (2 μM; 30 min) prevents the effect of Alda-1. B, PKCε activation with ΨεRACK (500 nM, 10 min) attenuates the release of NE. ALDH2 desensitization with GTN (2 μM; 30 min) prevents the effect of ΨεRACK. ***, P < 0.0001 from control and GTN + Alda-1 in A and from control and GTN + ΨεRACK in B, by unpaired t test.

Fig. 3.

Activation of adenosine A₁ and A₃ receptors inhibits the acetaldehyde-induced NE release from isolated cardiac synaptosomes: prevention by either PKCε inhibition or ALDH2 desensitization. Points (means ± S.E.M.; n = 8–42) represent percentage increases in NE release above a mean basal control level of 267 ± 6.6 pmol/mg protein (n = 124). A, selective activation of A₁ receptors with 2′-MeCCPA (10 nM; 10 min) attenuates NE release by acetaldehyde, an action that is prevented by A₁-receptor blockade with DPCPX (300 nM; 10 min). B and C, the A₁ receptor-induced attenuation of NE release is prevented either by PKCε inhibition with εV_1–2 (1 μM; 10 min) (B) or ALDH2 desensitization with GTN (2 μM; 30 min) (C). D, selective activation of A₃ receptors with IB-MECA (50 nM; 10 min) attenuates NE release by acetaldehyde, an action that is prevented by A₃-receptor blockade with MRS1523 (100 nM; 10 min). E and F, the A₃ receptor-induced attenuation of NE release is prevented either by PKCε inhibition with εV_1–2 (1 μM; 10 min) (E) or ALDH2 desensitization with GTN (2 μM; 30 min) (F). **, P < 0.01 and ***, P < 0.001 from control and A₁- and A₃-receptor agonists in combination with respective antagonists or in combination with εV_1–2 or GTN, by unpaired t test.

Fig. 4.

ALDH2 activation inhibits the positive chronotropic effect of acetaldehyde and associated increase in NE overflow in guinea pig hearts ex vivo. Top, time course of the increase in spontaneous heart rate during perfusion with acetaldehyde (500 μM). Pretreatment with the ALDH2 activator Alda-1 (20 μM; 10 min) inhibits the acetaldehyde-induced tachycardia and attenuates the associated increase in NE overflow. Points are heart rates recorded at the corresponding times on the abscissa (means ± S.E.M.; n = 5 and 6 for control and Alda-1, respectively). †, P < 0.05 and ††, P <0.01 from control by one-way ANOVA + Bonferroni's test. **, P < 0.01 and ***, P < 0.001 from acetaldehyde, by two-way ANOVA + Bonferroni's test. Bottom, time course of the increase in NE overflow (measured in 5-min intervals) in the same hearts as in Top. Bars are means (means ± S.E.M.; n = 5 and 6 for control and Alda-1, respectively). †, P < 0.05 and ††, P < 0.01 from control by one-way ANOVA + Bonferroni's test. **, P < 0.01 from acetaldehyde, by two-way ANOVA + Bonferroni's test.

Fig. 5.

Incubation of isolated cardiac synaptosomes with acetaldehyde elicits a concentration-dependent increase in NE release that is potentiated by the NE transporter inhibitor DMI (300 nM; 10 min). A selective N-type Ca²⁺ channel blocker, ω-CTX (100 nM; 10 min), markedly reduces the acetaldehyde-induced NE release. Points (means ± S.E.M.; n = 8–32) represent percentage increases in NE release above a mean basal control level of 246 ± 6.80 pmol/mg protein (n = 32). ***, P < 0.001 from acetaldehyde control by unpaired t test.

Fig. 6.

Release of NE from cardiac synaptosomes during 30-min hypoxia. Bars (means ± S.E.M.) represent the hypoxia-induced increase in NE release above the normoxic basal level of 291 ± 24 pmol/mg protein (n = 14). Treatment with the selective histamine H₃-receptor agonist methimepip (1 nM; 12 min) reduces hypoxia-induced NE release, and this effect is blocked by a previous addition of the selective histamine H₃-receptor antagonist JNJ5207852 (30 nM; 12 min). PKCε blockade with εV_1–2 (1 μM; 20 min) and ALDH2 desensitization with GTN (2 μM; 30 min) each prevents the effect of methimepip (n = 5–16). ***, P < 0.001 versus hypoxia. ##, P < 0.005 versus methimepip. ###, P < 0.0005 versus methimepip by unpaired t test.

Fig. 7.

Activation of histamine H₃ receptors inhibits the acetaldehyde-induced NE release from isolated cardiac synaptosomes: prevention by either PKCε inhibition or ALDH2 desensitization. Points (means ± S.E.M.; n = 12–28) represent percentage increases in NE release above a mean basal control level of 268 ± 9.2 pmol/mg protein (n = 96). A, selective activation of H₃ receptors with methimepip (1 nM; 10 min) attenuates NE release by acetaldehyde, an action that is prevented by H₃-receptor blockade with JNJ5207852 (30 nM; 10 min). B and C, the H₃-receptor-induced attenuation of NE release is prevented either by PKCε inhibition with εV_1–2 (1 μM; 10 min) (B) or ALDH2 desensitization with GTN (2 μM; 30 min) (C). ***, P < 0.0001 from control and H₃-receptor agonists with respective antagonists or εV_1–2 and GTN, by unpaired t test.

Fig. 8.

Activation of adenosine A₁ receptors, A₃ receptors, or histamine H₃ receptors increases ALDH2 activity in PC12-H₃ cells. Incubation of PC12-H₃ cells with the selective ALDH2 activator Alda-1 (100 μM; 20 min) increases ALDH2 activity (measured by the rate of NADH production at 340 nm). A, incubation of PC12-H₃ cells with the A₁-receptor agonist 2′-MeCCPA (10 nM; 20 min) increases ALDH2 activity. Selective desensitization of ALDH2 with GTN (2 μM; 30 min) prevents the effects of Alda-1 and A₁-receptor agonist. Pretreatment of PC12-H₃ cells with the selective A₁-receptor antagonist DPCPX (300 nM; 30 min) or the selective PKCε inhibitor εV_1–2 (1 μM; 30 min) prevents the effects of A₁-receptor activation. B, incubation of PC12-H₃ cells with the A₃-receptor agonist IB-MECA (50 nM; 20 min) increases ALDH2 activity. Selective desensitization of ALDH2 with GTN (2 μM; 30 min) prevents the effects of Alda-1 and IB-MECA. Pretreatment of PC12-H₃ cells with the selective A₃-receptor antagonist MRS1523 (100 nM; 30 min) or the selective PKCε inhibitor εV_1–2 (1 μM; 30 min) prevents the effects of A₃-receptor activation. C, incubation of PC12-H₃ cells with the H₃-receptor agonist methimepip (1 nM; 20 min) increases ALDH2 activity. Selective desensitization of ALDH2 with GTN (2 μM; 30 min) prevents the effects of Alda-1 and methimepip. Pretreatment of PC12-H₃ cells with the selective H₃-receptor antagonist JNJ5207852 (30 nM; 30 min) or the selective PKCε inhibitor εV_1–2 (1 μM; 30 min) prevents the effects of H₃-receptor activation. Bars are mean percentage increases from control (± S.E.M.; n = 4–16). Basal NADH production was 7.47 ± 0.13 μmol/min/mg protein. ***, P < 0.0001 from control. †††, P < 0.0001 from Alda-1. ♦♦♦, P < 0.0001 from A₁R agonist. ‡‡‡, P < 0.001 from A₃R agonist. ###, P < 0.0001 from methimepip by unpaired t test.

Fig. 9.

Proposed pathway for the inhibition of NE release from cardiac sympathetic neurons upon activation of mitochondrial ALDH2. This involves the pivotal phosphorylation/activation of mitochondrial ALDH2 by PKCε, which is translocated/activated when G_i/o-coupled receptors, such as A₁, A₃, and H₃ receptors, are activated by adenosine and histamine released in ischemic conditions in close proximity to sympathetic nerve endings.