Ca²⁺ Influx, But Not Ca²⁺ Release From Internal Stores, Is Required for the PACAP-Induced Increase in Excitability in Guinea Pig Intracardiac Neurons

Abstract

Mechanisms modulating the pituitary adenylate cyclase activating polypeptide (PACAP)-induced increase in excitability have been studied using dissociated guinea pig intrinsic cardiac neurons and intact ganglion preparations. Measurements of intracellular calcium (Ca2+) with the fluorescent Ca2+ indicator dye fluo-3 indicated that neither PACAP nor vasoactive intestinal polypeptide (VIP) at either 100 nM or 1 microM produced a discernible elevation of intracellular Ca2+ in dissociated intracardiac neurons. For neurons in ganglion whole mount preparations kept in control bath solution, local application of PACAP significantly increased excitability, as indicated by the number of action potentials generated by long depolarizing current pulses. However, in a Ca2+ -deficient solution in which external Ca2+ was replaced by Mg2+ or when cells were bathed in control solution containing 200 microM Cd2+, PACAP did not enhance action potential firing. In contrast, in a Ca2+ -deficient solution with Ca2+ replaced by strontium (Sr2+), PACAP increased excitability. PACAP increased excitability in cells treated with a combination of 20 microM ryanodine and 10 mM caffeine to interrupt release of Ca2+ from internal stores. Experiments using fluo-3 showed that ryanodine/caffeine pretreatment eliminated subsequent caffeine-induced Ca2+ release from intracellular stores, whereas exposure to the Ca2+ -deficient solution did not. In dissociated intracardiac neurons voltage clamped with the perforated patch recording technique, 100 nM PACAP decreased the voltage-dependent barium current (IBa). These results show that, in the guinea pig intracardiac neurons, the PACAP-induced increase in excitability apparently requires Ca2+ influx through Cd2+ -sensitive calcium permeable channels other than voltage-dependent Ca2+ channels, but not Ca2+ release from internal stores.

Differential Activation of Guinea Pig Intrinsic Cardiac Neurons by the PAC1 Agonists Maxadilan and Pituitary Adenylate Cyclase-Activating Polypeptide 27 (PACAP27)

“…Experiments were performed on atrial whole mount preparations containing the intrinsic cardiac ganglia as described previously (Braas et al, 1998;Tompkins et al, 2006Tompkins et al, , 2007. Atrial whole mount preparations were dissected from the heart in ice-cold, standard Krebs' solution (121 mM NaCl, 5.9 mM KCl, 2.5 mM CaCl 2 , 1.2 mM MgCl 2 , 25 mM NaHCO 3 , 1.2 mM NaH 2 PO 4 , and 8 mM glucose), with the pH maintained at 7.4 by aeration with 95% O 2 , 5% CO 2 .…”

“…For intracellular recordings, the whole mount preparations were pinned in a 2.5-ml Sylgard-lined chamber and superfused continuously (2-3 ml/min) with a modified Krebs' solution containing 10 mM HEPES buffer at 30 -32°C (Hardwick et al, 1995;Braas et al, 1998;Tompkins et al, 2006Tompkins et al, , 2007. Cardiac ganglia were visualized with an inverted microscope equipped with Hoffman optics (Modulation Optics Inc., Greenvale, NY), and individual cardiac neurons were impaled using high-impedance borosilicate microelectrodes (2 M KCl-filled; 60 -100 M⍀).…”

“…If required, hyperpolarizing current was injected through the recording electrode to electrotonically maintain the resting membrane potential between Ϫ55 and Ϫ65 mV. This ensured that action potential generation was tested at the same potential before and after peptide application (Tompkins et al, 2006(Tompkins et al, , 2007. For brief local application, PACAP27, maxadilan, or VIP (each at 50 M) was applied by pressure application (Picospritzer; General Valve, Fairfield, NJ) through ϳ5-mdiameter "puffer" pipettes positioned 50 to 100 m from the neuron.…”

Pituitary Adenylate Cyclase-Activating Polypeptide and Its Receptors: 20 Years after the Discovery

“…This effect is mediated by PAC1-R and cAMP production Tompkins and Parsons, 2008) and requires Ca 2ϩ influx (Tompkins et al, 2006). In contrast, the increased excitability of rat neonatal cardiac neurons induced by PACAP requires coactivation of PAC1-R and VPAC-R and release of Ca 2ϩ from intracellular stores (DeHaven and Cuevas, 2004).…”

Neurturin suppresses injury‐induced neuronal activating transcription factor 3 expression in cultured guinea pig cardiac ganglia

“…PACAP exhibits multiple regulatory actions on neurons (Arimura, 1998;Vaudry et al, 2000) and very potent effects on cardiac neuron excitability (Braas et al, 1998;Tompkins et al, 2006). We recently demonstrated that PACAP can be released from the preganglionic terminals during nerve stimulation (Tompkins et al, 2007).…”