title: 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: After G-protein-coupled receptor activation and signaling at the plasma membrane, the receptor complex is often rapidly internalized via endocytic vesicles for trafficking into various intracellular compartments and pathways. The formation of signaling endosomes is recognized as a mechanism that produces sustained intracellular signals that 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 (PAC1R; Adcyap1r1). In the present study, we examined whether PAC1R endocytosis participates in the regulation of neuronal excitability. Although PACAP increased excitability in 90% of guinea pig cardiac neurons, pretreatment with Pitstop 2 or dynasore to inhibit clathrin and dynamin I/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, dynasore, and low temperatures at suppressing PAC1R endocytosis. In contrast, brefeldin A pretreatments to disrupt Golgi vesicle trafficking did not blunt the PACAP effect, and PACAP/PAC1R signaling still increased neuronal cAMP production even with endocytic blockade. Our results demonstrate that PACAP/PAC1R complex endocytosis is a key step for the PACAP modulation of cardiac neuron excitability.
authors: Department of Neurological Sciences, College of Medicine, University of Vermont, Burlington, Vermont 05405, USA.
authors: Laura A
authors: LA
authors: Merriam
authors: Caitlin N
authors: CN
authors: Baran
authors: Beatrice M
authors: BM
authors: Girard
authors: Jean C
authors: JC
authors: Hardwick
authors: Victor
authors: V
authors: May
authors: Rodney L
authors: RL
authors: Parsons
mesh_terms: D000200:Action Potentials / Q000187:drug effects* / Q000235:genetics; D000818:Animals; D000831:Animals, Newborn; D017609:Barium Compounds / Q000494:pharmacology; D001703:Biophysics; D020126:Brefeldin A / Q000494:pharmacology; D002478:Cells, Cultured; D002712:Chlorides / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D004334:Drug Administration Schedule; D004558:Electric Stimulation; D011992:Endosomes / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006168:Guinea Pigs; D006801:Humans; D006835:Hydrazones / Q000494:pharmacology; D066298:In Vitro Techniques; D008297:Male; D009206:Myocardium / Q000166:cytology*; D009474:Neurons / Q000187:drug effects* / Q000502:physiology; D018408:Patch-Clamp Techniques; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide / Q000494:pharmacology*; D051381:Rats; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology; D013449:Sulfonamides / Q000494:pharmacology; D017783:Superior Cervical Ganglion / Q000166:cytology; D013696:Temperature; D053778:Thiazolidines / Q000494:pharmacology; D014162:Transfection
chemical_list: D017609:Barium Compounds; D002712:Chlorides; D004791:Enzyme Inhibitors; D006835:Hydrazones; C511794:N'-(3,4-dihydroxybenzylidene)-3-hydroxy-2-naphthahydrazide; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I; D013449:Sulfonamides; D053778:Thiazolidines; C578512:pitstop 2; C024986:barium chloride; D049452:Green Fluorescent Proteins; D020126:Brefeldin A; D000242:Cyclic AMP