Neurally released pituitary adenylate cyclase-activating polypeptide enhances guinea pig intrinsic cardiac neurone excitability

Abstract

Intracellular recordings were made in vitro from guinea-pig cardiac ganglia to determine whether endogenous neuropeptides such as pituitary adenylate cyclase-activating polypeptide (PACAP) or substance P released during tetanic neural stimulation modulate cardiac neurone excitability and/or contribute to slow excitatory postsynaptic potentials (sEPSPs). When nicotinic and muscarinic receptors were blocked by hexamethonium and atropine, 20 Hz stimulation for 10 s initiated a sEPSP in all innervated neurones. In 40% of the cells, excitability was enhanced after termination of the sEPSP. This suggested that non-cholinergic receptor-mediated mechanisms contributed to the sEPSP and modulated neuronal excitability. Exogenous PACAP and substance P initiated a slow depolarization in the neurones whereas neuronal excitability was only increased by PACAP. When ganglia were treated with the PAC1 antagonist PACAP6-38 (500 nM), the sEPSP evoked by 20 Hz stimulation was reduced by approximately 50% and an enhanced excitability occurred in only 10% of the cells. These observations suggested that PACAP released from preganglionic nerve terminals during tetanic stimulation enhanced neuronal excitability and evoked sEPSPs. After addition of 1 nM PACAP to the bath, 7 of 9 neurones exhibited a tonic firing pattern whereas in untreated preparations, the neurons had a phasic firing pattern. PACAP6-38 (500 nM) diminished the increase in excitability caused by 1 nM PACAP so that only 4 of 13 neurones exhibited a tonic firing pattern and the other 9 cells retained a phasic firing pattern. These findings indicate that PACAP can be released by tetanic neural stimulation in vitro and increase the excitability of intrinsic cardiac neurones. We hypothesize that in vivo PACAP released during preganglionic firing may modulate neurotransmission within the intrinsic cardiac ganglia.

Figure 1. Repetitive neural stimulation can initiate a sEPSP and an increase in cardiac neurone excitability

A, HFS (20 Hz for 10 s) applied to a nerve fibre connective can elicit action potentials followed by a sEPSP in an innervated cardiac neurone. B, in the same cell as in A, neuronal excitability is enhanced following the sEPSP as evidenced from the increase in number of action potentials elicited by an identical 1 s suprathreshold depolarizing current pulse. C, during exposure to 1 mm hexamethonium, action potential generation elicited by 0.1 Hz stimulation of the nerve bundle is lost as the fast EPSP becomes progressively subthreshold. Hexamethonium was added to the bath just before the beginning of the record. D, when 1 mm hexamethonium was present in the bath solution, HFS elicited a hyperpolarization during the period of stimulation to the nerve connective, followed by a depolarization that persisted after the stimulation had ended. E, when both 1 mm hexamethonium and 1 μm atropine were present in the bath solution, only a sEPSP was elicited by HFS. The sEPSP in some cells was sufficiently large to elicit a burst of action potentials. F, in the same cell as in E, neuronal excitability was enhanced following the sEPSP as evidenced from the increase in number of action potentials elicited by an identical 1 s suprathreshold depolarizing current pulse. In A, C, D and E, the dashed line indicates the resting membrane potential.

Figure 2. Application of PACAP or substance P can initiate depolarization, but only PACAP increases cardiac neurone excitability

A, local puffer application of PACAP, indicated by arrow, could initiate a slow depolarization in the cardiac neurones. In many cells, a burst of action potentials was generated during the rising phase of the depolarization. B, in the same cell as in A, membrane excitability was enhanced following PACAP application as evidenced by the increased number of action potentials elicited by a similar 1 s suprathreshold depolarizing current pulse as given prior to peptide application. C, local puffer application of substance P, indicated by arrow consistently initiated a slow depolarization. D, in the same cell as C, there was no change in excitability (determined using a similar 1 s suprathreshold depolarizing current pulse) evident following substance P application. E, the mean amplitude of the depolarization produced in different cells by PACAP or substance P was similar. However, the range of depolarization was noticeably greater for PACAP than for substance P. F, a graph demonstrating that after PACAP application, the number of action potentials elicited by 1 s depolarizing current pulses increased with increasing stimulus strength. Data points represent mean ±s.e.m. from 10 cells following PACAP application (•). In contrast, prior to peptide application or following substance P application (n= 9 cells, ▴), the number of action potentials produced did not change markedly with increasing stimulus strength. The control curve is the average response obtained from 18 cells prior to peptide application (▾). The asterisks indicate that the number of action potentials produced was significantly greater following PACAP application than prior to peptide application.

Figure 3. An increased excitability was sustained during bath application of 1 nm PACAP

A, a recording from the same cell obtained prior to and 5 min after bath application of 1 nm PACAP. Note that the firing pattern elicited with a 1 s depolarizing pulse shifted from phasic to tonic-like during exposure to 1 nm PACAP. B, an excitability curve demonstrating that during continued exposure to 1 nm PACAP, the number of action potentials elicited by 1 s depolarizing current pulses increased with increased stimulus strength (averaged data from 9 cells that were evaluated at different times over a 5–166 min exposure to PACAP). C, the number of action potentials produced by a 0.3 nA stimulus in different cells are plotted as a function of time in 1 nm PACAP. The results show that neuronal excitability remained elevated during the sustained exposure to PACAP.