Neuropeptide Y reduces acetylcholine release and vagal bradycardia via a Y2 receptor-mediated, protein kinase C-dependent pathway

Abstract

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 ³H-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 μM, n = 4). NPY also significantly attenuated the release of ³H-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 μM, n = 5) and chelerythrine chloride (25 μM, n = 6) but not the PKA inhibitor H89 (0.5 μM, n = 6). Conversely, the PKC activator Phorbol-12-myristate-13-acetate (0.5 μM, n = 7) mimicked the effect of NPY and significantly reduced ³H-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.

Introduction

High cardiac sympathetic tone and reduced vagal activity is the characteristic autonomic phenotype associated with myocardial infarction and congestive heart failure and is an independent predictor of mortality (e.g. [1], [2], [3], [4]). Prolonged sympathetic activation in vivo is also associated with a reduced chronotropic response to peripheral stimulation of the vagus nerve, an effect that is thought to be due to crosstalk of slowly diffusing sympathetic co-transmitters [5]. Recent evidence suggests that the co-transmitter neuropeptide Y (NPY) is a key player in this process as the NPY Y2 receptor antagonist BIIE 0246 prevents reduced vagal bradycardia post-sympathetic stimulation in vivo (in the mouse [6] and dog [7]). Interestingly, plasma NPY concentrations are significantly elevated in many pathophysiological states such as myocardial ischaemia and infarction [8] and congestive heart failure [9], [10].

Exogenously applied NPY can mimic prolonged sympathetic nerve activation and reduce the heart rate response to vagal nerve stimulation both in vitro (in the guinea pig [11], [12]) and in vivo (in the guinea pig [13], dog [14], mouse [6] and rat [13], [15]), an effect that is also abolished by the Y2 receptor antagonist BIIE 0246 (in the mouse [6] and rat [16]) or genetic knockout of this receptor [17]. However, the chronotropic response to muscarinic agonists in vivo is not affected by NPY [10], [13] and similar observations are found in in vitro organ bath experiments which are not complicated by possible changes in circulating factors [11], [12], [18]. NPY appears therefore to act via a pre-synaptic mechanism to reduce vagal neurotransmission. This may occur by inhibiting cardiac acetylcholine release or its synthesis, or by increasing its re-uptake.

The Y2 receptor is coupled to inhibitory G proteins that can reduce adenylate cyclase activity and cAMP/protein kinase A (PKA)-dependent phosphorylation. These G proteins can also be coupled to the activation of the phospholipase C/IP3/diacylglycerol system to increase protein kinase C (PKC)-dependent phosphorylation [19], [20]. We have shown previously that cAMP/PKA-dependent phosphorylation appears to tonically activate cardiac vagal N-type calcium channels and both neuronal nitric oxide and natriuretic peptides can increase acetylcholine release and bradycardia via stimulating PKA activity [21], [22], [23]. An inhibitory effect on adenylate cyclase activity via the Y2 receptor could therefore lead to a reduction in the calcium-induced exocytosis of acetylcholine during nerve stimulation. PKC activation by NPY can also inhibit other neuronal calcium channels (e.g. L- and P-type) in neuroendocrine cells [24] and NPY can cause a PKC-dependent increase in the delayed rectifier potassium current (IK) via myocyte Y2 receptors [25] both mechanisms that could reduce the release of acetylcholine if present in cardiac vagal neurons. We therefore investigated whether NPY reduces acetylcholine release and vagal bradycardia via a pre-synaptic Y2 receptor-mediated pathway that is dependent on PKA or PKC.