PACAP-Induced PAC1 Receptor Internalization and Recruitment of Endosomal Signaling Regulate Cardiac Neuron Excitability.

Pituitary adenylate cyclase-activating polypeptide (PACAP, Adcyap1) activation of PAC1 receptors (Adcyap1r1) significantly increases excitability of guinea pig cardiac neurons. This modulation of excitability is mediated in part by plasma membrane G protein-dependent activation of adenylyl cyclase and downstream signaling cascades, as well as by endosomal signaling mechanisms. PACAP/PAC1 receptor-mediated activation of plasma membrane adenylyl cyclase (AC) and the resulting increase in cellular cAMP enhances a hyperpolarization-induced nonselective cationic current Ih, which contributes to the PACAP-induced increase in cardiac neuron excitability. Further, PACAP-mediated AC/cAMP/PKA downstream signaling also appears to enhance cardiac neuron IT to facilitate the excitatory responses. PACAP activation of PAC1 receptors rapidly stimulates receptor internalization, and reducing ambient temperature or treatments with the clathrin inhibitor Pitstop2 or the dynamin I/II inhibitor dynasore to block endocytic events can suppress PACAP-enhanced neuronal excitability. Thus, endocytosis inhibitors essentially eliminate PACAP-enhanced excitability suggesting that endosomal platforms represent a primary signaling mechanism. Endosomal signaling is associated canonically with ERK activation and in accord, PACAP-enhanced cardiac neuron excitability is reduced by MEK inhibitor pretreatments. PACAP activation of MEK/ERK signaling can enhance currents through voltage-dependent Nav1.7 channels. Hence, PACAP-induced PAC1 receptor internalization/endosomal signaling, recruitment of MEK/ERK signaling, and modulation of Nav1.7 are implicated as key mechanisms contributing to the PACAP-enhanced neuronal excitability. PACAP/PAC1 receptor-mediated endosomal ERK signaling in central circuits can play key roles in development of chronic pain and anxiety-related responses; thus, PAC1 endosomal signaling likely participates in a variety of homeostatic responses within neuronal circuits in the CNS.