h-89 and Bradycardia

The co-transmitter neuropeptide Y (NPY), released during prolonged cardiac sympathetic nerve stimulation, can attenuate vagal-induced bradycardia. We tested the hypothesis that NPY reduces acetylcholine release, at similar concentrations to which it attenuates vagal bradycardia, via pre-synaptic Y2 receptors modulating a pathway that is dependent on protein kinase A (PKA) or protein kinase C (PKC). The Y2 receptor was immunofluorescently colocalized with choline acetyl-transferase containing neurons at the guinea pig sinoatrial node. The effect of NPY in the presence of various enzyme inhibitors was then tested on the heart rate response to vagal nerve stimulation in isolated guinea pig sinoatrial node/right vagal nerve preparations and also on (3)H-acetylcholine release from right atria during field stimulation. NPY reduced the heart rate response to vagal stimulation at 1, 3 and 5 Hz (significant at 100 nM and reaching a plateau at 250 nM NPY, p<0.05, n=6) but not to the stable analogue of acetylcholine, carbamylcholine (30, 60 or 90 nM, n=6) which produced similar degrees of bradycardia. The reduced vagal response was abolished by the Y2 receptor antagonist BIIE 0246 (1 microM, n=4). NPY also significantly attenuated the release of (3)H-acetylcholine during field stimulation (250 nM, n=6). The effect of NPY (250 nM) on vagal bradycardia was abolished by the PKC inhibitors calphostin C (0.1 microM, n=5) and chelerythrine chloride (25 microM, n=6) but not the PKA inhibitor H89 (0.5 microM, n=6). Conversely, the PKC activator Phorbol-12-myristate-13-acetate (0.5 microM, n=7) mimicked the effect of NPY and significantly reduced (3)H-acetylcholine release during field stimulation. These results show that NPY attenuates vagal bradycardia via a pre-synaptic decrease in acetylcholine release that appears to be mediated by a Y2 receptor pathway involving modulation of PKC.

Topics: Acetylcholine; Animals; Arginine; Benzazepines; Bradycardia; Carbachol; Choline O-Acetyltransferase; Cyclic AMP-Dependent Protein Kinases; Female; Guinea Pigs; Heart Rate; Immunohistochemistry; Isoquinolines; Neuropeptide Y; Protein Kinase C; Receptors, Neuropeptide Y; Signal Transduction; Sinoatrial Node; Sulfonamides; Vagus Nerve

1. We tested the hypothesis that nitric oxide (NO) augments vagal neurotransmission and bradycardia via phosphorylation of presynaptic calcium channels to increase vesicular release of acetylcholine. 2. The effects of enzyme inhibitors and calcium channel blockers on the actions of the NO donor sodium nitroprusside (SNP) were evaluated in isolated guinea-pig atrial-right vagal nerve preparations. 3. SNP (10 microM) augmented the heart rate response to vagal nerve stimulation but not to the acetylcholine analogue carbamylcholine (100 nM). SNP also increased the release of [3H]acetylcholine in response to field stimulation. No effect of SNP was observed on either the release of [3H] acetylcholine or the HR response to vagal nerve stimulation in the presence of the guanylyl cyclase inhibitor 1H-(1,2,4)-oxadiazolo-(4,3-a)-quinoxalin-1-one (ODQ, 10 microM). 4. The phosphodiesterase 3 (PDE 3) inhibitor milrinone (1 microM) increased the release of [3H] acetylcholine and the vagal bradycardia and prevented any further increase by SNP. SNP was still able to augment the vagal bradycardia in the presence of the protein kinase G inhibitor KT5823 (1 microM) but not after protein kinase A (PKA) inhibition with H-89 (0.5 microM) or KT5720 (1 microM) had reduced the HR response to vagal nerve stimulation. Neither milrinone nor H-89 changed the HR response to carbamylcholine. 5. SNP had no effect on the magnitude of the vagal bradycardia after inhibition of N-type calcium channels with omega-conotoxin GVIA (100 nM). 6. These results suggests that NO acts presynaptically to facilitate vagal neurotransmission via a cGMP-PDE 3-dependent pathway leading to an increase in cAMP-PKA-dependent phosphorylation of presynaptic N-type calcium channels. This pathway may augment the HR response to vagal nerve stimulation by increasing presynaptic calcium influx and vesicular release of acetylcholine.

Topics: Acetylcholine; Alkaloids; Animals; Bradycardia; Calcium Channel Blockers; Calcium Channels; Carbachol; Carbazoles; Cardiotonic Agents; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Electric Stimulation; Enzyme Inhibitors; Female; Guanylate Cyclase; Guinea Pigs; Heart Rate; In Vitro Techniques; Indoles; Isoquinolines; Milrinone; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; omega-Conotoxin GVIA; Oxadiazoles; Phosphorylation; Presynaptic Terminals; Quinoxalines; Sinoatrial Node; Sulfonamides; Synaptic Transmission; Tritium; Vagus Nerve

We tested the hypothesis that natriuretic peptide receptors (NPRs) that are coupled to cGMP production act in a similar way to nitric oxide (NO) by enhancing acetylcholine release and vagal-induced bradycardia. The effects of enzyme inhibitors and channel blockers on the action of atrial natriuretic peptide (ANP), brain-derived natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) were evaluated in isolated guinea pig atrial-right vagal nerve preparations. RT-PCR confirmed the presence NPR B and A receptor mRNA in guinea pig sinoatrial node tissue. BNP and CNP significantly (P < 0.05) enhanced the heart rate (HR) response to vagal nerve stimulation. CNP had no effect on the HR response to carbamylcholine and facilitated the release of [(3)H]acetylcholine during atrial field stimulation. The particulate guanylyl cyclase-coupled receptor antagonist HS-142-1, the phosphodiesterase 3 inhibitor milrinone, the protein kinase A inhibitor H89, and the N-type calcium channel blocker omega-conotoxin all blocked the effect of CNP on vagal-induced bradycardia. Like NO, BNP and CNP facilitate vagal neurotransmission and bradycardia. This may occur via a cGMP-PDE3-dependent pathway increasing cAMP-PKA-dependent phosphorylation of presynaptic N-type calcium channels.

Topics: Acetylcholine; Animals; Atrial Natriuretic Factor; Bradycardia; Calcium Channel Blockers; Carbachol; Cardiotonic Agents; Cyclic GMP; Enzyme Inhibitors; Female; Gene Expression; Guanylate Cyclase; Guinea Pigs; Heart Rate; Isoquinolines; Milrinone; Natriuretic Peptide, Brain; Natriuretic Peptide, C-Type; Nitric Oxide; omega-Conotoxins; Phosphodiesterase Inhibitors; Polysaccharides; Receptors, Atrial Natriuretic Factor; RNA, Messenger; Sulfonamides; Synaptic Transmission; Tritium; Vagus Nerve