Pituitary Adenylate Cyclase 1 Receptor Internalization and Endosomal Signaling Mediate the Pituitary Adenylate Cyclase Activating Polypeptide-Induced Increase in Guinea Pig Cardiac Neuron Excitability

Abstract

Following G protein-coupled receptor activation and signaling at the plasma membrane, the receptor complex often is rapidly internalized via endocytic vesicles for trafficking into various intracellular compartments and pathways. Formation of signaling endosomes is recognized to be a mechanism to produce sustained intracellular signals, which may be distinct from those generated at the cell surface, for cellular responses including growth, differentiation and survival. Pituitary adenylate cyclase activating polypeptide (PACAP; Adcyap1) is a potent neurotransmitter/neurotrophic peptide and mediates its diverse cellular functions in part through internalization of its cognate G protein-coupled PAC1 receptor (Adcyap1r1). In the current studies, we examined whether PAC1 receptor endocytosis participates in regulation of neuronal excitability. While PACAP increased excitability in 90% of guinea pig cardiac neurons, pretreatment with Pitstop 2 or dynasore to inhibit clathrin and dynaminI/II, respectively, suppressed the PACAP effect. Subsequent addition of inhibitor, after the PACAP- induced increase in excitability developed, gradually attenuated excitability with no changes in action potential properties. Likewise, the PACAP-induced increase in excitability was markedly decreased at ambient temperature. Receptor trafficking studies with GFP-PAC1 cell lines demonstrated the efficacy of Pitstop 2 and dynasore and low temperature to suppress PAC1 receptor endocytosis. In contrast, brefeldin A pretreatments to disrupt Golgi vesicle trafficking did not blunt the PACAP effect, and PACAP/PAC1 receptor signaling still increased neuronal cAMP production even with endocytic blockade. In aggregate, these studies demonstrate that PACAP/PAC1 receptor complex endocytosis is a key step for the PACAP modulation of cardiac neuron excitability.

PACAP-induced ERK activation in HEK cells expressing PAC1 receptors involves both receptor internalization and PKC signaling

“…Our previous studies demonstrated that treatment with the inhibitors Pitstop 2 and dynasore or temperature reduction to blunt clathrin-and dynamin-dependent endocytosis did not suppress PACAP-induced cAMP generation in sympathetic neuronal cultures, suggesting that the second messenger was generated primarily at the plasma membrane of primary neurons (31). Thus we evaluated whether the same mechanisms could be observed in the PAC1 receptor-expressing cell lines.…”

“…However, even at the higher concentration, dynasore did not significantly blunt the PACAP-induced ERK activation after 120 min (data not shown). Our earlier studies demonstrated that reducing treatment temperature from 37°C to ambient conditions (22-24°C) very effectively blunted the PACAP-induced PAC1 receptor internalization (31). Consequently, the temperature dependence of PACAP-induced ERK activation was examined to complement the endocytosis inhibitor studies.…”

“…PAC1Hop1 receptor-expressing cells were seeded onto 24-well culture plates and maintained in Dulbecco's modified Eagle's medium/Ham's F-12 containing 8% fetal bovine serum and 50 g/ml G418. Assays for PACAP-or forskolin-induced cAMP production were performed as described previously (4,31). After PACAP treatments in medium containing the phosphodiesterase inhibitor RO20-1724 (50 M), the cultures were extracted in absolute ethanol containing 100 M RO20-1724 and processed for cAMP immunoassay using the Biotrak nonacetylation protocol (Amersham GE Healthcare, Piscataway, NJ).…”

“…PAC1 receptor activation of MEK/ERK has been shown to be one of the principal mediators of many trophic responses, including fiber outgrowth during development and neuroplasticity during stressrelated challenges. Recently, our laboratories showed that internalization of PACAP/PAC1 to form a signaling endosome supports many of the diverse effects of PACAP, ranging from neuroprotection during proapoptotic challenges to regulation of membrane excitability (28,31). In the current work, using a stable PAC1 receptor-expressing cell line, we have evaluated mechanisms of PACAP/PAC1 receptor-mediated MEK/ERK activation and determined whether the signaling cascade was affected by paradigms that attenuate PAC1 receptor internalization.…”

A New Recombinant PACAP-Derived Peptide Efficiently Promotes Corneal Wound Repairing and Lacrimal Secretion

“…7,44 But there is yet no report on the signal transduction mechanism of trigeminal ganglion cell injury repair. This work showed that the genes of MAPK signal pathway molecules were significantly differentially expressed in MPAPO-treated injured mouse trigeminal ganglion cells.…”