Activation of MEK/ERK Signaling by PACAP in Guinea Pig Cardiac Neurons

Abstract

Pituitary adenylate cyclase-activating polypeptide (PACAP) signaling can increase guinea pig cardiac neuron excitability in part through extracellular signal-regulated kinase (ERK) activation. The present study examined the PACAP receptors and signaling cascades that stimulate guinea pig cardiac neuron ERK signaling using confocal microscopy to quantify PACAP-induced neuronal phosphorylated ERK (pERK) immunoreactivity. PACAP and maxadilan, but not vasoactive intestinal polypeptide (VIP), increased cardiac neuron pERK, implicating primary roles for PACAP-selective PAC1 receptor (Adcyap1r1) signaling rather than VPAC receptors (Vipr1 and Vipr2) in the generation of cardiac neuron pERK. The adenylyl cyclase (AC) activator forskolin, but not the protein kinase C (PKC) activator phorbol myristate acetate (PMA), increased pERK. Also, Bim1 did not blunt PACAP activation of pERK. Together, the results suggest PAC1 receptor signal transduction via Gs/adenylyl cyclase (AC)/cAMP rather than Gq/phospholipase C (PLC) generated neuronal pERK. Activator and inhibitor studies suggested that the PACAP-mediated pERK activation was PKA-dependent rather than an exchange protein directly activated by a cAMP (EPAC), PKA-independent mechanism. The PACAP-induced pERK was inhibited by the clathrin inhibitor Pitstop2 to block receptor internalization and endosomal signaling. We propose that the PACAP-mediated MEK/ERK activation in cardiac neurons involves both AC/cAMP/PKA signaling and PAC1 receptor internalization/activation of signaling endosomes.

Keywords: Autonomic neuron; MAPK signaling; PAC1 endocytosis; PACAP; PKA.

Figure 1

PACAP activates MEK/ERK signaling through PAC1 receptors. A: Confocal images (~1 μm optical sections) of cardiac ganglia neurons immunostained with an antiserum against pERK. A1: A control cardiac ganglia preparation. A2: A cardiac ganglia preparation exposed to 25 nM PACAP for 20 minutes. A3: A cardiac ganglia preparation exposed to PAC1 receptor agonist maxadilan (25 nM) for 20 minutes. A4: A cardiac ganglia preparation exposed to 25 nM VIP for 20 minutes. Calibration bar in A4 equals 20 μm. Basal levels of pERK immunoreactivity were evident in axonal bundles and peri-neuronal cells. Only PAC1 receptor-mediated signaling increased cardiac ganglia neuron pERK immunoreactivity. B: Averaged fold-change in fluorescence intensity/area for the treatments shown in A. Results averaged from neurons in multiple cardiac ganglia from different whole mount preparations.

Figure 2

cAMP-dependent, but EPAC-independent mechanisms increase cardiac neuron ERK phosphorylation. Cardiac ganglia whole mount preparations were treated with the adenylyl cyclase activator forskolin (5 μM) (A1), the protein kinase C activator PMA (500 nM) (A2) or the EPAC activator 8-(4-chlorophenylthio)-2′-O-methyl-cAMP (100 μM) (A3, 8-CPT) for 20 minutes at 37°C before fixation and immunocytochemical processing for pERK. Only forskolin treatments increased cardiac neuron pERK levels. B: Averaged fold change in fluorescence intensity/area for the different conditions shown in A. Data presented as averaged results from cells in multiple cardiac ganglia from different whole mount preparations.

Figure 3

Inhibition of PKA or endocytosis mechanisms diminishes PACAP-induced pERK generation in cardiac neurons. Cardiac ganglia whole mount preparations were exposed to 25 nM PACAP alone or pretreated with the clathrin-mediated endocytosis inhibitor Pitstop2 (15 μM) or the PKA inhibitor KT5902 (1 μM) for 15 minutes before addition of 25 nM PACAP + inhibitor for 20 minutes at 37°C and immunocytochemical processing. Each inhibitor blocked PACAP-induced ERK phosphorylation approximately 50%. Data presented as averaged results from cells in multiple cardiac ganglia from different whole mount preparations under each condition (PACAP, 4 whole mount preparations, Pitstop2/PACAP, 3 whole mount preparations; KT5902/PACAP, 4 whole mount preparations).

Figure 4

Schematic of PACAP/PAC1 receptor signaling to ERK activation. The PACAP/PAC1 receptor is dually coupled to Gs to activate adenylyl cyclase (AC) activity and Gq to stimulate phospholipase C (PLC) activity. From increased AC activity, the resulting increase in cAMP levels can engage PKA or EPAC (PKA-independent) pathways to ERK signaling; cardiac neurons employ the former PKA-dependent pathway. Despite potent activation of PLC signaling cascades, cardiac neuron PAC1 receptors do not appear to stimulate PKC mechanisms to ERK. However, following activation, PAC1 receptors can be associated with arrestin scaffolding molecules and internalized as signaling endosomes for long term MEK/ERK signaling. GPCR receptor signaling mechanisms to ERK is cell type specific; the pathways identified for the guinea pig cardiac neurons are boxed.