Identification of intracellular signaling cascades mediating the PACAP-induced increase in guinea pig cardiac neuron excitability.
The pituitary adenylate cyclase-activating polypeptide (PACAP) increases excitability of guinea pig cardiac neurons, an effect mediated through activation of PAC1 receptors. The signaling cascades that couple activation of the PAC1 receptor to alterations in membrane ionic conductances responsible for the PACAP effect are unknown. Intracellular recordings were made from neurons in kinase inhibitor-treated cardiac ganglia preparations to determine which of the intracellular cascades activated by PAC1 receptor stimulation mediate the PACAP effect. In control cells, long depolarizing-current steps elicited one to three action potentials. In contrast, during the application of 10 nM PACAP, depolarizing-current pulses elicited multiple action potential firing (greater than or equal to five action potentials) in 79% of the neurons. Pretreatment with an adenylyl cyclase inhibitor, SQ 22536 (100 microM), suppressed the PACAP-induced increase in excitability, whereas the presence of U-73122 (10 microM), a potent phospholipase C (PLC) inhibitor, had no effect. Thus, the activation of adenylyl cyclase, but not PLC, was a critical step mediating the PACAP effect. Pretreatment with H-89 (1 microM), a protein kinase A inhibitor, and PD 98059 (50 microM), a MEK kinase inhibitor, also significantly blunted the PACAP-induced increase in excitability. Furthermore, treatment with forskolin (5 microM), an activator of adenylyl cyclase, or exposure to the cell-permeable cyclic adenosine monophosphate (cAMP) analogue, 8-bromo-cAMP (1 mM), partially recapitulated the effect of PACAP on excitability. We conclude that the activation of signaling cascades downstream of cAMP mediate the PACAP-induced increase in cardiac neuron excitability.
Tompkins
John D
JD
Department of Anatomy and Neurobiology, College of Medicine, University of Vermont, Burlington, VT 05405, USA. john.tompkins@uvm.edu
Parsons
Rodney L
RL
D000225:Adenine / Q000031:analogs & derivatives / Q000378:metabolism; D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D004963:Estrenes / Q000378:metabolism; D005419:Flavonoids / Q000378:metabolism; D006168:Guinea Pigs; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009206:Myocardium / Q000166:cytology* / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D017777:Parasympathetic Fibers, Postganglionic / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide / Q000494:pharmacology*; D011760:Pyrrolidinones / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology
D000067956:Adenylyl Cyclase Inhibitors; D004791:Enzyme Inhibitors; D004963:Estrenes; D005419:Flavonoids; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D011760:Pyrrolidinones; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C017759:9-(tetrahydro-2-furyl)-adenine; D017868:Cyclic AMP-Dependent Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D000262:Adenylyl Cyclases; D000225:Adenine; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one