Cardiac output is regulated by the coordinate interactions of stimulatory sympathetic and inhibitory parasympathetic signals. Intracardiac parasympathetic ganglia are integrative centers of cardiac regulation, and modulation of the parasympathetic drive on the heart is accomplished by altering intrinsic cardiac ganglion neuron excitability. The pituitary adenylate cyclase-activating polypeptide (PACAP)/vasoactive intestinal peptide (VIP) family of peptides modulates cardiac function, and in guinea pig heart, PACAP appears to act directly on intrinsic parasympathetic cardiac ganglia neurons through PACAP-selective receptors. A multidisciplinary project tested whether cardiac PACAP peptides act through PACAP-selective receptors as excitatory neuromodulators amplifying the parasympathetic inhibition from guinea pig cardiac ganglia. The in vivo sources of regulatory PACAP peptides were localized immunocytochemically to neuronal fibers and a subpopulation of intrinsic postganglionic cardiac neurons. RT-PCR confirmed that cardiac ganglia expressed proPACAP transcripts and have PACAP peptide biosynthetic capabilities. Messenger RNA encoding PACAP-selective PAC1 receptor isoforms were also present in cardiac ganglia. Alternative splicing of PAC1 receptor transcripts produced predominant expression of the very short variant with neither HIP nor HOP cassettes; lower levels of the PAC1HOP2 receptor mRNA were present. Almost all of the parasympathetic neurons expressed membrane-associated PAC1 receptor proteins, localized immunocytochemically, which correlated with the population of cells that responded physiologically to PACAP peptides. PACAP depolarized cardiac ganglia neurons and increased neuronal membrane excitability. The rank order of peptide potency on membrane excitability in response to depolarizing currents was PACAP27>PACAP38>VIP. The PACAP-induced increase in excitability was not a function of membrane depolarization nor was it caused by alterations in action potential configuration. These results support roles for PACAP peptides as integrative modulators amplifying, through PACAP-selective receptors, the parasympathetic cardiac ganglia inhibition of cardiac output.

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Many regulated events guide neuropeptide biosynthesis, processing, and secretion. For PACAP peptides, these events have not been well examined. In our studies of PACAP expression in sympathetic neurons, we discovered that neuronal depolarization not only increased the levels of the 2.2 kb form of proPACAP mRNA identified in neuronal tissues, but also induced a novel 0.9 kb PACAP transcript, which appeared similar in size to a form present in testes. Using reverse-transcription PCR and 3' RACE studies, we demonstrated that the 0.9 kb PACAP mRNA in depolarized SCG neurons was not identical to the testicular PACAP mRNA, but represented shortened, more stable, forms of the 2.2 kb transcript resulting from alternative upstream polyadenylation site usage. These results demonstrate that post-transcriptional mechanisms play important roles in determining cellular PACAP levels and provide several important insights. For example, alternative upstream polyadenylation can elicit a major influence on the amount of bioactive peptide that can by synthesized, since short 3' UTR transcripts are usually more stable due to elimination of destabilizing elements present in the longer messages. In cells such as testicular germ cells, which have restricted transcriptional periods, stable mRNAs allow longer translational events and extended periods of peptide production. The neuronal PACAP system adopts a similar post-transcriptional strategy following neuronal depolarization, and although the roles of PACAP remain unclear, this suggests important roles for PACAP peptides during increased neuronal activity. Additionally, unlike alternative polyadenylation described for many genes, alternative site usage in the proPACAP transcript does not result from alternative splicing. The mechanism of alternative site usage may be related to changes in the expression and binding of polyadenylation factors to the short and long 3' UTR proPACAP sites leading to production of more stable transcripts and increased PACAP precursor biosynthesis. The implications of increased PACAP production following altered neurophysiological states and the mechanisms underlying alternative polyadenylation site choice are important considerations for future inquiries.

Peptide alpha-amidation, an essential posttranslational modification that confers bioactivity to many neuroendocrine peptides, is catalyzed by peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3). To complement our previous studies on the distribution of PAM in neuroendocrine organs, we have examined expression of the PAM gene in several endocrine tissues by in situ hybridization and immunocytochemistry. In all instances, the autoradiographic densities for PAM mRNA correlated with staining patterns for PAM immunoreactivity. Very high levels of PAM mRNA were found in all heart atrial cardiomyocytes, while much lower levels were present in ventricular cells. In the sublingual gland, PAM was expressed diffusely in both acinar and tubule cells. In contrast, expression of PAM was confined to granular convoluted tubule cells in the submaxillary gland. PAM was expressed at high levels in a subset of adrenal medullary chromaffin cells, and low levels of PAM mRNA and immunoreactivity were also detected in the adrenal cortex. PAM was found predominantly in the calcitonin-producing parafollicular C-cells in the thyroid gland and in the glucagon-containing A-cells in the endocrine pancreas. Collecting and distal tubule cells of the kidney expressed both PAM mRNA and immunoreactivity. The basal cells in testicular seminiferous tubules containing PAM may represent developing germ and Sertoli cells. The cellular localization of PAM within the thyroid gland, adrenal gland, testis, and pancreas correlated with known peptidergic systems, and some of the observed cellular heterogeneity in PAM mRNA expression and immunoreactivity may reflect differences in the levels of amidated peptide production. The expression of PAM in cells not known to produce high levels of alpha-amidated peptides may indicate the production of yet unidentified alpha-amidated bioactive peptides or alternative functions of the PAM protein.