cAMP modulation of Ca²⁺-regulated exocytosis in ACh-stimulated antral mucous cells of guinea pig

Abstract

Effects of cAMP accumulation on ATP-dependent priming and Ca(2+)-dependent fusion in Ca(2+)-regulated exocytosis were examined in antral mucous cells of guinea pigs by using video-enhanced contrast microscopy. The Ca(2+)-regulated exocytosis activated by 1 microM ACh consisted of two phases, an initial transient phase followed by a sustained phase, which were potentiated by cAMP accumulation. Depletion of ATP by 100 microM dinitrophenol (uncoupler of oxidative phosphorylation) or anoxia induced the sustained phase without the initial transient phase in Ca(2+)-regulated exocytosis. However, accumulation of cAMP before depletion of ATP induced and potentiated an initial transient phase followed by a sustained phase in Ca(2+)-regulated exocytosis. This suggests that the initial transient phase of Ca(2+)-regulated exocytosis is induced by fusion of all primed granules maintained by ATP and that accumulation of cAMP accelerates ATP-dependent priming of the exocytotic cycle. Moreover, ACh and Ca(2+) dose-response studies showed that accumulation of cAMP shifted the dose-response curves to the low concentration side, suggesting that it increases Ca(2+) sensitivity in the fusion of the exocytotic cycle. In conclusion, cAMP accumulation increases the number of primed granules and Ca(2+) sensitivity of the fusion, which potentiates Ca(2+)-regulated exocytosis in antral mucous cells.

A PKG inhibitor increases Ca2+-regulated exocytosis in guinea pig antral mucous cells: cAMP accumulation via PDE2A inhibition

“…After the stimulations, the antral mucosa was immediately frozen in liquid nitrogen and lyophilized at Ϫ80°C for 24 h. The lyophilized, antral mucosae were weighed, and their cAMP or cGMP contents were measured using a cAMP EIA kit (Cayman Chemical) or a cGMP EIA kit (Amersham International, Little Chalfont, UK). The cGMP and cAMP content was expressed as picomoles per gram of dry tissue weight (10,11).…”

“…In antral mucous cells, Ca 2ϩ -regulated exocytosis has characteristic features of frequency: an initial peak that declines rapidly (initial phase or initial transient phase) followed by a second slower decline (late phase) lasting during acetylcholine (ACh) stimulation. The initial phase, which is transient and has a high frequency of exocytotic events, is caused by an immediate fusion of the primed granules (the pool of releasable granules), and the late phase, which has a low frequency of exocytotic events, is caused by a fusion of granules that are in the process of priming (10).…”

<b>PPARα induced NOS1 phosphorylation via PI3K/Akt in guinea pig antral mucous cells: NO-enhancement in Ca</b>^<b>2+</b><b>-regulated </b><b>exocytosis </b>

“…In antral mucous cells, the Ca 2+ -regulated exocytosis which is activated by ACh (acetylcholine) is the main mechanism for mucin release (8,14,15,19) and is modulated by many substances, such as cAMP, NO, cGMP, intracellular Cl − , and arachidonic acid (AA) (8,14,19,(21)(22)(23)(24). NO is synthesized by nitric oxide synthase 1 (NOS1) and enhances the Ca 2+ -regulated exocytosis mediated via cGMP accumulation in antral mucous cells (28).…”

“…Guinea pigs were sacrificed by intraperitoneal injection of pentobarbital sodium (100 mg/kg), and then, the stomach was removed from the animal. The procedures for the cell preparation have been previously described (8,9,14,15,19,(22)(23)(24). Briefly, the gastric antrum was excised from the stomach, and the mucosal layer was stripped from the muscle layer in cold phosphate buffered saline (4°C) using glass slides.…”

“…The isolated antral tubular shaped glands were mounted on a coverslip pre-coated with neutralized Cell-Tak (Becton Dickinson Labware, Bedford, MA, USA) for firm attachment of the cells. The coverslip with the cells was set in a perfusion chamber mounted on the stage of a differential interference contrast microscope (BX50Wi; Olympus, Tokyo, Japan), which was connected to a videoenhanced contrast system (ARGUS-20; Hamamatsu Photonics, Hamamatsu, Japan) (8,9,14,15,19,(22)(23)(24). Images were recorded continuously using a video recorder.…”