Pituitary adenylate cyclase activating polypeptide (PACAP) decreases neuronal somatostatin immunoreactivity in cultured guinea-pig parasympathetic cardiac ganglia

Abstract

Postganglionic parasympathetic neurons in guinea-pig cardiac ganglia exhibit choline acetyltransferase (ChAT)-immunoreactivity, and a large fraction (60%) of the ChAT-positive cardiac neurons co-express somatostatin-immunoreactivity. This co-expression remained when the cardiac ganglia explants were maintained in culture for 72 h (40% somatostatin-immunoreactive). The guinea-pig cardiac ganglia neurons express the high affinity pituitary adenylate cyclase activating polypeptide (PACAP)-selective PAC1 receptor, and treatment of the ganglia explants with 20 nM PACAP27 for 72 h to evaluate PACAP regulation of somatostatin expression revealed a dramatic 85% decrease in the number of somatostatin-IR neurons (6% somatostatin-IR neurons) compared with untreated control explant preparations. The decrease in percentage of somatostatin-IR neurons by PACAP27 was time- and concentration-dependent, and selective for PACAP27; PACAP38 and vasoactive intestinal polypeptide were less effective. PACAP6-38, a PACAP antagonist, eliminated the PACAP27-induced change in somatostatin positive neurons. The PACAP-mediated decrease in somatostatin-IR neurons was eliminated in calcium-deficient solutions and by the addition of nifedipine, indicating a requirement for calcium influx through L-type calcium channels. The addition of either the calmodulin inhibitor N-(4-aminobutyl)-1-naphthalenesulfonamide or the MEK inhibitor PD98059, also eliminated the PACAP27-induced decrease in somatostatin-IR cells. The PACAP27-mediated effect on somatostatin expression was not affected by inhibitors of protein kinase A or phospholipase C, but was reduced by the adenylyl cyclase inhibitor SQ22356, suggesting cAMP involvement. Semiquantitative and quantitative reverse transcription PCR prosomatostatin transcript measurements showed that cardiac ganglia prosomatostatin mRNA levels were not diminished by chronic PACAP27 exposure despite the dramatic decrement in somatostatin-expressing neurons. Neuronal peptide-IR content represents a balance between production and secretion. These results suggested that one of the primary effects of PACAP exposure may be enhanced levels of neuropeptide release that exceeded production levels, resulting in somatostatin depletion and a decrement in the number of identifiable somatostatin-expressing cardiac neurons.