The cardiac sympathetic co-transmitter galanin reduces acetylcholine release and vagal bradycardia: implications for neural control of cardiac excitability

Abstract

The autonomic phenotype of congestive cardiac failure is characterised by high sympathetic drive and impaired vagal tone, which are independent predictors of mortality. We hypothesize that impaired bradycardia to peripheral vagal stimulation following high-level sympathetic drive is due to sympatho-vagal crosstalk by the adrenergic co-transmitters galanin and neuropeptide-Y (NPY). Moreover we hypothesize that galanin acts similarly to NPY by reducing vagal acetylcholine release via a receptor mediated, protein kinase-dependent pathway. Prolonged right stellate ganglion stimulation (10 Hz, 2 min, in the presence of 10 μM metoprolol) in an isolated guinea pig atrial preparation with dual autonomic innervation leads to a significant (p<0.05) reduction in the magnitude of vagal bradycardia (5 Hz) maintained over the subsequent 20 min (n=6). Immunohistochemistry demonstrated the presence of galanin in a small number of tyrosine hydroxylase positive neurons from freshly dissected stellate ganglion tissue sections. Following 3 days of tissue culture however, most stellate neurons expressed galanin. Stellate stimulation caused the release of low levels of galanin and significantly higher levels of NPY into the surrounding perfusate (n=6, using ELISA). The reduction in vagal bradycardia post sympathetic stimulation was partially reversed by the galanin receptor antagonist M40 after 10 min (1 μM, n=5), and completely reversed with the NPY Y(2) receptor antagonist BIIE 0246 at all time points (1 μM, n=6). Exogenous galanin (n=6, 50-500 nM) also reduced the heart rate response to vagal stimulation but had no effect on the response to carbamylcholine that produced similar degrees of bradycardia (n=6). Galanin (500 nM) also significantly attenuated the release of (3)H-acetylcholine from isolated atria during field stimulation (5 Hz, n=5). The effect of galanin on vagal bradycardia could be abolished by the galanin receptor antagonist M40 (n=5). Importantly the GalR(1) receptor was immunofluorescently co-localised with choline acetyl-transferase containing neurons at the sinoatrial node. The protein kinase C inhibitor calphostin (100 nM, n=6) abolished the effect of galanin on vagal bradycardia whilst the protein kinase A inhibitor H89 (500 nM, n=6) had no effect. These results demonstrate that prolonged sympathetic activation releases the slowly diffusing adrenergic co-transmitter galanin in addition to NPY, and that this contributes to the attenuation in vagal bradycardia via a reduction in acetylcholine release. This effect is mediated by GalR(1) receptors on vagal neurons coupled to protein kinase C dependent signalling pathways. The role of galanin may become more important following an acute injury response where galanin expression is increased.

Copyright © 2011 Elsevier Ltd. All rights reserved.

Figures

Fig. 1

Expression of galanin in stellate sympathetic neurons and GalR1 on cardiac cholinergic neurons. (A) Reverse transcription-polymerase chain reaction (RT-PCR) products viewed on an ethidium bromide gel after amplification of freshly dissected stellate ganglia and brain mRNA. NTC, no template control, is a negative control assessing the absence of contamination. Galanin mRNA is present in both right and left stellate ganglia. (B) All stellate neurons from freshly dissected left and right stellate ganglia exhibit immunoreactivity for tyrosine hydroxylase (TH, left panel, 20 μm sections) but only occasionally do TH immunoreactive neurons also exhibit galanin immunoreactivity (< 5%, right panel, calibration bar = 20 μm). (C) Dissociated stellate neurons cultured for 3-days exhibit immunoreactivity for TH (left hand panel) and galanin (right hand panel, calibration bar = 20 μm). (D) RT-PCR bands for the galanin receptors GalR1 (253 base pairs) and GalR3 (234 base pairs) from mRNA isolated from cardiac ganglia. Guinea pig brain was used as a positive control, and reaction mix without template was the negative control (NTC). (E) Immunohistochemistry of right atrial slices showing expression of GalR1 receptor protein in neuronal structures only (see inset picture at higher magnification, calibration bar = 10 μm). GalR1, GalR2 and GalR3 expression was demonstrated in guinea pig midbrain as a positive control (calibration bar = 20 μm). (F) Immunohistochemistry showing choline acetyltransferase (ChAT) staining with Texas Red in a neuron in right atrial slices also staining positive for the galanin GalR1 receptor in green (fluorescein). Co-localization was demonstrated by overlap of staining in yellow (calibration bar = 20 μm).

Fig. 2

Release of co-transmitters after high frequency sympathetic stimulation and the corresponding reduction in vagal bradycardia. Representative raw data traces (A and D) and group mean data (B and E) showing the heart rate response to vagal nerve stimulation (5 Hz, 30 s) before and during the 20 minute period after high frequency stimulation of the right stellate ganglion (SNS, 10 Hz, 2 min, in the presence of 10 μM metoprolol). A control experiment is shown in black (baseline heart rate 187 ± 9 bpm, n = 6), whilst in a separate set of experiments shown in blue (A and B), this was repeated in the presence of the neuropeptide-Y receptor antagonist BIIE 0246 (1 μM, baseline heart rate 180 ± 9 bpm, n = 6) and in a further set of experiments shown in red (D and E) this was repeated in the presence of the galanin GalR1 receptor antagonist M40 (1 μM, baseline heart rate 183 ± 13 bpm, n = 5). The heart rate response to vagal nerve stimulation using norepineprhine (10 μM in the presence of 10 μM metoprolol, n = 5) rather than sympathetic pre-stimulation was not significantly different to that using sympathetic pre-stimulation in the presence of BIIE 0246 (data not shown). C and F show the organ bath concentrations of neuropeptide-Y (n = 6, blue) and galanin (n = 6, red) before and during the 20 min after high frequency sympathetic nerve stimulation (10 Hz, 2 min in the presence of metoprolol 10 μM) measured in 50 μl organ bath samples analysed using ELISA. (*p < 0.05 vs control).

Fig. 3

Exogenous galanin reduces vagal bradycardia. Representative raw data traces (A at 5 Hz) and group mean data (B) showing the reduction in the magnitude of the bradycardia (beats per minute, bpm, baseline heart rate 192 ± 14 bpm, n = 6) to stimulation of the right vagus nerve (5, 3 or 1 Hz, for 30 s) in the presence of increasing concentrations of galanin (5–500 nM). (*p 

Fig. 4

Galanin acts via the GalR1 receptor but not via the neuropeptide-Y Y2 receptor. Representative raw data traces (A and C) and group mean data (B and D) showing the effect of galanin (100 and 250 nM) on the heart rate response (beats per minute, bpm) to vagal nerve stimulation (5 Hz, 30 s) in the presence of antagonists of the GalR1 receptor (M40 1 μM, baseline heart rate 200 ± 6 bpm, n = 5, A and B) or the neuropeptide-Y Y2 receptor (BIIE 0246 1 μM, baseline heart rate 169 ± 12 bpm, n = 5, C and D). (*p < 0.05 vs control and BIIE 0246).

Fig. 5

A pre-synaptic action of galanin reduces vagal bradycardia and acetylcholine release. Galanin (500 nM) significantly reduced (*p 3H-acetylcholine to field stimulation (5 Hz, S1 vs S2, n = 5) as demonstrated by the representative raw data trace (C, left panel) and group mean data (C, right panel).

Fig. 6

Inhibition of protein kinase C, but not protein kinase A prevents the reduction in vagal bradycardia to galanin. Inhibition of protein kinase A (H89 500 nM, baseline heart rate 204 ± 9 bpm, n = 6) significantly reduced (+ p 

Fig. 7

Cardiac sympatho-vagal crosstalk by adrenergic co-transmitters. A diagram hypothesizing the mechanism by which the adrenergic co-transmitters neuropeptide-Y (NPY, red square) and galanin (Gal, red square) are released during high frequency sympathetic stimulation in the presence of beta blockade (norepinephrine, NE, black circle, and metoprolol, yellow cross). Neuropeptide-Y and galanin act via the Y2 and GalR1 receptors respectively to reduce acetylcholine (ACh, black triangle) release and subsequent vagal bradycardia at the sinoatrial node. Both GalR1 and Y2 receptor signalling directly or indirectly involves protein kinase C (PKC).