Graduation Year

2005

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Pharmacology and Therapeutics

Major Professor

Javier Cuevas, Ph.D.

Committee Member

David Morgan, Ph.D.

Committee Member

Paul E. Gottschall, Ph.D.

Committee Member

Eric S. Bennett, Ph.D.

Committee Member

Keith R. Pennypacker, Ph.D.

Keywords

Intracardiac ganglia, Pacap, Vip, Vpac2, Pac1, Calcium

Abstract

Autonomic control of cardiac function depends on the coordinated activity generated by neurons within the intracardiac ganglia, and intrinsic feedback loops within the ganglia provide precise control of cardiac function. Both pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are important regulators of cell-to-cell signaling within the intracardiac ganglia, and PACAP and VIP action on these ganglia, mediated through associated receptors, play an important role in the regulation of coronary blood flow, cardiac contraction, relaxation, and heart rate. Results reported here using PACAP and VIP provide direct evidence of some of the complex signaling which occurs in neurons of the rat intracardiac ganglia.

The expression of PACAP and VIP receptors was investigated using single-cell RT-PCR. Individual neurons were shown to express multiple isoforms of the PACAP-selective receptor, PAC1, including the -short, -HOP1 and -HOP2 variants. These splice variants affect ligand binding, G-protein coupling and selectivity. Intracardiac neurons also express the non-selective receptors, VPAC1 and VPAC2, with VPAC2 being found in greater proportion of cells. These results demonstrate heterogeneity of PAC1 and VPAC receptors expressed in intracardiac neurons, which has significant implications on the effects of PACAP and VIP on cellular function.

Calcium imaging and electrophysiology were used to examine the physiological effects of PACAP and VIP on isolated intracardiac neurons. Both neuropeptides, through the activation of VPAC2 receptors, evoked rapid increases in cytosolic calcium concentrations ([Ca2+]i) that exhibited both transient and sustained components. The transient increases in [Ca2+]i were mediated through the activation of ryanodine receptors, whereas the sustained [Ca2+]i elevations were dependent on extracellular Ca2+ and pharmacologically resembled canonical transient receptor potential (TRPC) channels.

PACAP and VIP also depolarized intracardiac neurons, and PACAP was further shown to augment action potential firing in these cells. The depolarization was dependent on activation of VPAC2 receptors and the concomitant increases in [Ca2+]i, while PAC1 receptor stimulation potentiated the VPAC2 receptorinduced depolarizations. Pharmacological evidence suggest TRPC channels mediate this link between neuropeptide evoked changes in membrane properties and [Ca2+]i. Thus, these results give insight into the complex PACAP and VIP signaling which occurs in the intracardiac ganglia.

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