Differential activation of guinea pig intrinsic cardiac neurons by the PAC1 agonists maxadilan and pituitary adenylate cyclase-activating polypeptide 27 (PACAP27)

Abstract

Pituitary adenylate cyclase-activating polypeptide (PACAP) evokes tachycardia followed by a larger cholinergic bradycardia in isolated guinea pig hearts. We used the selective PAC1 receptor agonist maxadilan and vasoactive intestinal polypeptide (VIP) to test the hypothesis that PACAP27-evoked tachycardia and bradycardia are mediated by VPAC and PAC1 receptors, respectively. Chronotropic actions of these peptides were evaluated in isolated perfused hearts. Direct neuronal actions were determined by intracellular voltage recordings from cholinergic neurons in atrial ganglion whole mounts. Administration of 1 nmol of PACAP27 to isolated hearts evoked typical biphasic rate responses, whereas 1 nmol of maxadilan caused only a minor rate decrease. Desensitization with VIP eliminated the positive chronotropic effect of PACAP27 selectively. Local application of PACAP27 to cardiac neurons frequently evoked slow depolarization and caused prolonged increase of neuronal excitability. Maxadilan rarely affected membrane potential but consistently increased excitability. VIP had no effect on excitability and evoked depolarization in only a few neurons. Because maxadilan increased neuronal excitability but did not trigger action potentials as PACAP often does, we evaluated the interaction of maxadilan with substance P (SP) in isolated hearts. SP depolarizes cardiac neurons more consistently than PACAP, often triggers neuronal action potentials, and causes bradycardia but does not increase neuronal excitability. Maxadilan had a persistent effect to augment negative chronotropic responses to SP. These findings support our hypothesis that PACAP evokes tachycardia and bradycardia through VPAC and PAC1 receptors, respectively. They also suggest that maxadilan and PACAP27 differ in activating PAC1 receptors on cardiac neurons and/or stimulating downstream signaling mechanisms.

Fig. 1.

Chronotropic responses of isolated guinea pig heart to maxadilan and PACAP27. A, recorder tracings showing changes in heart rate evoked by bolus injections of 1 nmol of maxadilan and 1 nmol of PACAP27 in separate hearts. Maxadilan caused only a minor decrease in rate, but PACAP27 produced a biphasic response (phase 1, tachycardia; phase 2, bradycardia). B, quantification of chronotropic responses. Values are means ± S.E.M. (1 nmol of maxadilan, n = 10; 3.2 nmol of maxadilan, n = 3; and 1 nmol of PACAP27, n = 9). Responses to each dose of maxadilan and PACAP27 were determined in separate hearts.

Fig. 2.

Infusion of maxadilan attenuates the negative chronotropic response to PACAP27 without affecting the positive chronotropic response. A, control heart rate response to bolus injection of 1 nmol of PACAP27. B, chronotropic response to maxadilan infusion (final concentration, 100 nM) started 15 min after PACAP27 administration. C, chronotropic responses to a second bolus of 1 nmol of PACAP27 given after 15 min of maxadilan infusion. The positive chronotropic response to PACAP27 was preserved, but its negative chronotropic effect was attenuated. Negative chronotropic responses to SP and ACh were preserved. Data are representative of two experiments.

Fig. 3.

Desensitization with VIP eliminates the positive chronotropic (phase 1) response to PACAP27. A, bolus injection of 1 nmol of VIP causes tachycardia that is accompanied by desensitization in the isolated perfused guinea pig heart. Under this condition, bolus injection of PACAP27 causes bradycardia only instead of the typical biphasic change in heart rate. B, summary data showing the loss of phase 1 response to PACAP27 after desensitization with VIP. ∗, significantly different from control response to PACAP27 shown in Fig. 1 (P = 0.003; n = 4 for VIP desensitization group).

Fig. 4.

PACAP27 causes slow depolarization of most intrinsic cardiac neurons. Maxadilan, PACAP27, or VIP was delivered by local puffer application to individual intrinsic cardiac neurons in atrial whole mount preparations while recording membrane potential (V_m). PACAP27 caused membrane depolarization in a majority of neurons, whereas maxadilan and VIP rarely affected V_m. ∗, significantly different from responses to maxadilan and VIP (ANOVA: F_2,26 = 5.03, P < 0.02).

Fig. 5.

PACAP27 and maxadilan increase neuronal excitability. Most intrinsic cardiac neurons fire only one or two action potentials during the injection of depolarizing current. Treatment with PACAP27 or maxadilan increased the number of action potentials evoked by depolarizing current, but treatment with VIP did not. Best-fit linear regression lines were calculated and compared between groups using Prism. Asterisks denote significant difference between lines. Values are the mean ± S.E.M. (PACAP, n = 10; maxadilan, n = 9; and VIP, n = 7).

Fig. 6.

Recorder tracings showing that maxadilan augments the negative chronotropic response to SP in isolated guinea pig hearts. Bolus injections of 3.2 nmol of SP were given every 20 min beginning 16 min before a single injection of 1 nmol of maxadilan. Bolus injections of 10 nmol of bethanechol were given 2 min after SP. Maxadilan had no effect on postjunctional sensitivity to the muscarinic agonist bethanechol.

Fig. 7.

A single bolus injection of 1 nmol of maxadilan caused prolonged potentiation of the negative chronotropic response to 3.2 nmol of SP. SP and maxadilan were given as described in the previous legend. ∗, significantly different from control response at −16 min (repeated measures ANOVA: F_3,18 = 28.8, P < 0.0001; n = 4).