Erica K. Potter

Effects of a selective neuropeptide Y Y2 receptor antagonist, BIIE0246, on Y2 receptors at peripheral neuroeffector junctions

This study investigated the effects of BIIE0246, a novel neuropeptide Y (NPY) Y2 receptor antagonist, on the inhibition of cholinergic neuroeffector transmission in rat heart and guinea‐pig trachea and purinergic neuroeffector transmission in guinea‐pig vas deferens produced by the NPY Y2 receptor agonist, N‐acetyl [Leu28,31] NPY 24‐36. In pentobarbitone anaesthetized rats, supramaximal stimulation every 30u2003s, of the vagus nerve innervating the heart, increased pulse interval by approximately 100u2003ms. This response was attenuated by intravenous administration of N‐acetyl [Leu28,31] NPY 24‐36 (10u2003nmolu2003kg−1). Transmural stimulation of segments of guinea‐pig trachea at 1u2003min intervals with 5u2003s trains of stimuli at 0.5, 5, 10, 20 and 40u2003Hz evoked contractions which were reduced in force by N‐acetyl [Leu28,31] NPY 24‐36 (2u2003μM). In guinea‐pig vasa deferentia, the amplitude of excitatory junction potentials evoked by trains of 20 stimuli at 1u2003Hz was reduced in the presence of N‐acetyl [Leu28,31] NPY 24‐36 (1u2003μM). In all preparations BIIE0246 attenuated the inhibitory effect of N‐acetyl [Leu28,31] NPY 24‐36 but had no effect when applied alone. The findings support the view that the nerve terminals of postganglionic parasympathetic and sympathetic neurones possess neuropeptide Y Y2 receptors which, when activated, reduce neurotransmitter release.

A novel neuropeptide Y analog, N-acetyl [Leu28,Leu31]neuropeptide Y-(24-36), with functional specificity for the presynaptic (Y2) receptor.

We have carried out functional and in vitro studies on a novel analog of neuropeptide Y which shows selectivity for the prejunctional or neuropeptide Y Y2 receptor. In anaesthetised rats N-acetyl [Leu28,Leu31]neuropeptide Y-(24-36) attenuates cardiac vagal action (a prejunctional or neuropeptide Y Y2 action) and has no significant pressor effects (postjunctional or neuropeptide Y Y1 action). In the human neuroblastoma cell line (SMS-KAN) which expresses and endogenous Y2-like neuropeptide Y receptor, N-acetyl [Leu28,Leu31]neuropeptide Y-(24-36) competes with peptide YY for binding sites with an IC50 of 0.5 +/- 0.1 nM. In contrast in a fibroblast Chinese hamster ovary cell line which expresses the cloned human neuropeptide Y Y1 receptor and is used to study changes in cytosolic calcium evoked by (a neuropeptide Y Y1 effect), N-acetyl [Leu28,Leu31]neuropeptide Y-(24-36) showed no activity even at high concentrations. The steric structure for this novel compound has been determined using proton nuclear magnetic resonance (NMR) spectroscopy and it is consistent with the C-terminal end of published structures of neuropeptide Y. We suggest acetylation and amino acid substitutions stabilise the molecule and allow it to bind only to the neuropeptide Y Y2 receptor.

Role of neuropeptide Y Y2 receptors in modulation of cardiac parasympathetic neurotransmission

The aim of the study was to clarify the role of the Y(2) receptor in regulation of vagal control of the heart, using Y(2)((-/-)) receptor-knockout mice. Adult Y(2)((+/+),(-/-)) mice (50% C57BL/6-50% 129/SvJ background) were anaesthetised and artificially ventilated. Arterial blood pressure and pulse interval was recorded and both vagus nerves were cut. The cardiac end of the right vagus nerve was stimulated supra-maximally every 30 s (7 V, 2-2.5 Hz, 5 s). Neuropeptide Y (NPY) and a Y(2) receptor agonist, N-acetyl [Leu(28, 31)]NPY 24-36, were injected intravenously in both groups of mice. N-acetyl [Leu(28, 31)] NPY 24-36 was also administered to control mice in the presence of a Y(2) receptor antagonist, BIIE0246. Stimulation of the vagus nerve increased pulse interval (PI) by approximately 100 ms. NPY and N-acetyl [Leu(28, 31)] NPY 24-36 attenuated the increase in PI evoked by vagal stimulation in control mice only. The attenuation was reduced in the presence of BIIE0246. The results presented here show in Y(2)((-/-)) receptor-knockout mice that NPY and N-acetyl [Leu(28, 31)] NPY 24-36 have no effect on PI evoked by vagal stimulation. These findings demonstrate that NPY attenuates parasympathetic activity to the heart via the Y(2) receptor.

Modulation by neuropeptide Y of parasympathetic nerve‐evoked nasal vasodilatation via Y2 prejunctional receptor

1 In pentobarbitone anaesthetized dogs, preganglionic stimulation of the superior cervical sympathetic nerve (15 V, 1 ms, 10 Hz) induced marked reduction of nasal arterial blood flow, whereas parasympathetic nerve stimulation (5 V, 1ms, 10–30 Hz) evoked frequency‐dependent vasodilatation. 2 Sympathetic nerve stimulation for 3min at 10 Hz evoked significant (P < 0.05) and prolonged attenuation of the vasodilator response to subsequent parasympathetic stimulation. Pretreatment with phentolamine (0.5 mg kg−1 h−1), propranolol (1 mg kg−1) and atropine (0.5 mg kg−1) reduced the vasoconstrictor effect of sympathetic stimulation by 35 ± 4% whereas the parasympathetic nerve‐evoked vasodilatation was not significantly modified. Atropine‐resistant parasympathetic vasodilatation remained significantly attenuated for more than 30 min after non‐adrenergic sympathetic nerve‐evoked vasoconstriction. 3 Vasodilator effects of exogenous vasoactive intestinal polypeptide and peptide histidine isoleucine and vasoconstrictor effects of exogenous neuropeptide Y (NPY) and the NPY analogue [Leu31, Pro34] NPY (Y1‐receptor agonist, 8 nmol kg−1), were not altered by adrenoceptor antagonists and atropine whereas the effects of exogenous noradrenaline and acetylcholine were virtually abolished. Attenuation of parasympathetic‐evoked vasodilatation could be mimicked by exogenous NPY (8 nmol kg−1) and the NPY analogue, N‐acetyl [Leu28, Leu31] NPY 24–36 (Y2‐receptor agonist, 20 nmol kg−1) but not by exogenous Y1receptor agonist. The Y2‐receptor agonist did not show significant vasoconstrictor action. 4 It is concluded that sympathetic nerve stimulation attenuates parasympathetic vasodilatation via NPY release acting on prejunctional Y2 receptors.

Effects of pituitary adenylate cyclase-activating polypeptide on cardiovascular and respiratory responses in anaesthetised dogs

This study examines some of the cardiovascular and respiratory effects of pituitary adenylate cyclase-activating polypeptide (PACAP) in anaesthetised dogs. Intravenous injection of PACAP 27 caused an increase in arterial blood pressure and an increase in heart rate. The blood pressure response was significantly reduced by adrenoceptor blockade suggesting a mechanism of action mediated in part via catecholamines. The heart rate increase was unaltered by adrenoceptor blockade suggesting a direct effect of PACAP 27. PACAP 27 also caused potentiation of cardiac slowing caused by stimulation of the vagus nerve. In addition, PACAP 27 powerfully stimulated breathing. This was probably evoked by stimulation of arterial chemoreceptors, because bilateral section of the carotid sinus nerves abolished this effect. PACAP 27 had no effect on the ability of the cardiac sympathetic nerve to increase heart rate, nor on the interaction between the sympathetic and parasympathetic systems in the heart.

Galanin and neuropeptide Y reduce cholinergic transmission in the heart of the anaesthetised mouse

This study investigated the effects of galanin (GAL) on inhibition of cholinergic (vagal) activity in the mouse heart using control galanin knockout (GAL‐KO) and GAL‐1R receptor knockout (GAL‐1R‐KO) mice. In pentobarbitone anaesthetised mice, supramaximal stimulation every 30 s of the vagus nerve innervating the heart, increased pulse interval (PI) by approximately 50 ms or decreased heart rate by approximately 100 beats min−1. This response was attenuated by intravenous administration of GAL (dose ranged from 0.8 to 13 nmol kg−1) in a dose‐dependent manner. In GAL‐KO mice, the magnitude of inhibition of the increase in PI (ΔPI) following a bolus dose of GAL was not different from the ΔPI in control mice, and neuropeptide Y (NPY), previously shown to attenuate vagal inhibitory activity in mice, evoked a comparative inhibition of ΔPI in GAL‐KO mice. In GAL‐1R‐KO mice, an intravenous, bolus injection of GAL had no inhibitory effect on vagal activity. In control mice, stimulation of the sympathetic nerve at 25 V, 10 Hz for 2 min in the presence of propranolol evoked a long‐lasting attenuation of ΔPI. The inhibitory effect on ΔPI was reduced in the presence of the NPY Y2 antagonist, BIIE0246. In GAL‐1R‐KO mice, stimulation of the sympathetic nerve in the presence of propranolol evoked an attenuation of ΔPI not significantly different from the response in control mice in the presence of BIIE0246. Following administration of BIIE0246 in GAL‐1R‐KO mice, the inhibition of ΔPI that followed stimulation of the sympathetic nerve was abolished. These findings support the view that the nerve terminals of parasympathetic neurons in the mouse heart possess both GAL‐1R and NPY Y2 receptors which, when activated, reduce acetylcholine release.

[Leu31, Pro34] NPY, a selective functional postjunctional agonist at neuropeptide-Y receptors in anaesthetised rats

Neuropeptide Y (NPY), a sympathetic cotransmitter, has both prejunctional and postjunctional actions in the cardiovascular system. In the bioassay system used here NPY attenuates cardiac vagal action (an indicator of prejunctional or Y2 action) and increases blood pressure (an indicator of postjunctional or Y1 action). [Leu31,Pro34]NPY has little or no prejunctional activity i.e. does not attenuate cardiac vagal action, but increases blood pressure as effectively as NPY. [Leu31,Pro34]NPY can therefore be used in functional experiments to distinguish between the two types of NPY receptor.

Sympathetic and parasympathetic interaction in vascular control of the nasal mucosa in anaesthetized cats.

1. In cats anaesthetized with pentobarbitone sodium (45 mg kg‐1), electrical stimulation of the parasympathetic nerve fibres to the nasal mucosa evoked frequency‐dependent increases in nasal arterial blood flow whereas stimulation of the superior cervical sympathetic nerve induced marked vasoconstriction. 2. Sympathetic nerve stimulation for 3 min at 10 Hz evoked significant (P < 0.05) and prolonged (> 30 min) attenuation of the vasodilatory response to subsequent parasympathetic stimulation. 3. Combined pretreatment with adrenergic and cholinergic blockers reduced the vasoconstrictory effect of sympathetic stimulation by 28 +/‐ 4% (mean +/‐ S.E.M.) and the parasympathetically evoked vasodilatation by 20 +/‐ 5%. 4. The vasodilatory effects of exogenous vasoactive intestinal polypeptide, peptide histidine isoleucine and galanin, and the vasoconstrictory effects of exogenous neuropeptide Y (NPY) and alpha,beta‐methylene adenosine 5‐triphosphate were not altered by adrenoceptor antagonists and atropine whereas the effects of exogenous noradrenaline and acetylcholine were virtually abolished. 5. The atropine‐resistant parasympathetic vasodilatation remained significantly attenuated for more than 30 min after the non‐adrenergic sympathetically evoked vasoconstriction. 6. Exogenous NPY (25 x 10(‐9) mol) mimicked the effect of sympathetic stimulation in attenuating subsequent parasympathetically evoked vasodilatation.

Cardiac function in neuropeptide Y Y4 receptor-knockout mice

Autonomic control of cardiovascular function in neuropeptide Y (NPY) Y4 receptor-knockout mice was investigated using pancreatic polypeptide (PP), NPY and specific agonists and antagonists for other NPY receptors well characterised in cardiovascular function. Y4 receptor-knockout mice, anaesthetised with sodium pentobarbitone, displayed slower heart rate, indicated by a higher pulse interval and lower blood pressure compared to control mice. After vagus nerves were cut heart rate increased but was still significantly slower than in control mice. PP had no effect on blood pressure or cardiac vagal activity in either group of mice, which was consistent with earlier studies in other species. Injection of NPY evoked an increase in blood pressure but the response was significantly reduced in Y4 receptor-knockout mice compared to the controls. The reduction in pressor activity was not Y1 mediated as the selective Y1 antagonist, BIBP 3226, was effective in blocking NPY pressor activity in knockout mice. In addition, cardiac vagal inhibitory activity evoked by low doses of NPY was also reduced when compared to control responses. As N-acetyl [Leu(28, 31)] NPY 24-36 inhibited vagal activity dose dependently in both groups of mice with no difference in response at any dose, it is unlikely that this effect also is receptor mediated. We propose that the reduced vasoconstrictor and vagal inhibitory activity evoked by NPY in Y4 receptor-knockout mice is due to a lack of adrenergic tone bought about by a proposed reduction in sympathetic activity, possibly resulting from altered NPY activity secondarily affecting adrenergic transmission. We conclude that Y4 receptor deletion disrupts autonomic balance within the cardiovascular system.

Sympathetic-parasympathetic interactions at the heart in the anaesthetised rat.

This study observed the effects of stimulation of the cardiac sympathetic nerve on vagal slowing of the heart in rats, and compared these with any actions of exogenous neuropeptide Y (NPY) and galanin (GAL). In rats anaesthetised with pentobarbitone, stimulation of the cardiac sympathetic nerve for 2 min at 20 Hz in the rat evoked an attenuation of subsequent cardiac vagal action, which could be mimicked by exogenous NPY, but not GAL. The galanin antagonist, GAL1-13/NPY24-36, known to block the inhibitory action of galanin on the cardiac vagus in cats, did not alter the effect of sympathetic stimulation on cardiac vagal activity. We suggest on the basis of results here that in the rat, NPY released during stimulation of the cardiac sympathetic nerve, causes inhibition of acetylcholine release from the vagus nerve.