F1CRhR/cyclic AMP signaling in myenteric ganglia and calbindin-D-28 intrinsic primary afferent neurons involves adenylyl cyclases I, III and IV

The aims of this study were to improve insight into cAMP signaling in myent eric neurons and glia and identify the adenylyl cyclase (AC) isoforms expre ssed in myenteric ganglia of the guinea-pig small intestine. An increase in the intracellular cAMP levels was measured indirectly by an increase in th e 520 nm/580 nm fluorescence emission ratio of the protein kinase A fluoros ensor FlCRhR. Forskolin or pituitary adenylyl cyclase activating peptide ca used an increase in cAMP levels in cell somas and neurites and elicited a s low EPSP-like response in myenteric AH/Type 2 neurons, whereas the inactive form of forskolin was without these effects. Glia displayed similar cAMP r esponses. Immunoblot analysis showed that AC I, III and IV were present in myenteric ganglia, with AC I being detected as two bands of 160 kDa and 185 kDa, AC III as two bands near 220 kDa, and AC IV as two bands of greater t han 220 kDa. Pretreatment with N-ethylmaleimide and N-glycosidase F reveale d an AC IV band at 115 kDa. Preabsorption with specific blocking peptides p revented detection of AC I or AC IV immunoreactive proteins. In ganglia whi ch expressed strong AC IV immunoreactivity, no immunoreactive bands were de tected for AC LT, AC V/VI, AC VII or AC VIII. The amount of AC isoforms exp ressed in myenteric ganglia followed the order of AC IV >> III > I. Immunof luorescent labeling studies revealed that AC I, AC III and AC TV were varia bly expressed in myenteric neurons and glia of the duodenum, jejunum and il eum. In the guinea-pig ileum, AC I, III and IV immunoreactivities were resp ectively present in 26%, 58% and 89% of calbindin-D-28-colabeled myenteric neurons. These findings suggest that (1) AC I, AC III and AC IV variably co ntribute to cAMP signaling in myenteric ganglia, (2) AC I, AC III and AC IV may be differentially expressed in distinct subsets of calbindin-D-28 neur ons which may represent intrinsic primary afferent myenteric neurons. Our s tudy also provides direct evidence for activation of cAMP-dependent protein kinase. (C) 1999 Published by Elsevier Science B.V. All rights reserved.