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

Abstract

In antral mucous cells, acetylcholine (ACh, 1 M) activates Ca 2ϩ -regulated exocytosis, consisting of an initial peak that declines rapidly (initial transient phase) followed by a second slower decline (late phase) lasting during ACh stimulation. The addition of 8-bromo-cGMP (8-BrcGMP) enhanced the initial phase, which was inhibited by the protein kinase G (PKG) inhibitor guanosine 3=,5=-cyclic monophosphorothoiate, ␤-phenyl-1,N 2 -etheno-8-bromo, Rp-isomer, sodium salt (Rp-8-BrPETcGMPS, 100 nM). However, Rp-8-BrPETcGMPS produced a delayed, but transient, increase in the exocytotic frequency during the late phase that was abolished by a protein kinase A (PKA) inhibitor (PKI-amide), suggesting that Rp-8-BrPETcGMPS accumulates cAMP. The cGMP-dependent phosphodiesterase 2 (PDE2), which degrades cAMP, may exist in antral mucous cells. The PDE2 inhibitor BAY-60-7550 (250 nM) mimicked the effect of Rp-8-BrPETcGMPS on ACh-stimulated exocytosis. Measurement of the cGMP and cAMP contents in antral mucosae revealed that ACh stimulates the accumulation of cGMP and that BAY-60-7550 accumulates cAMP similarly to Rp-8-BrPETcGMPS during ACh stimulation. Analyses of Western blot and immunohistochemistry demonstrated that PDE2A exists in antral mucous cells. In conclusion, Rp-8-BrPETcGMPS accumulates cAMP by inhibiting PDE2 in AChstimulated antral mucous cells, leading to the delayed, but transient, increase in the frequency of Ca 2ϩ -regulated exocytosis. PDE2 may prevent antral mucous cells from excessive mucin secretion caused by the cAMP accumulation.acetylcholine; guanosine 3=,5=-cyclic monophosphate; adenosine 3=,5=-cyclic monophosphate; gastric; mucin secretion GASTRIC MUCINS, WHICH ARE high-molecular-weight glycoproteins that protect the gastric mucosa from acid-peptic injury, are stored in intracellular granules and secreted to the lumen by Ca 2ϩ -regulated exocytosis. Ca 2ϩ -regulated exocytosis in antral mucous cells consists of three biochemically distinct steps: docking, priming, and fusion. The priming step is regulated by ATP, and the fusion step is regulated by Ca 2ϩ (12,20,23). 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).In antral mucous cells, Ca 2ϩ -regulated exocytosis is activated by ACh and is modulated by many substances, such as cAMP, cGMP, arachidonic acid (AA), and intracellular Cl Ϫ (3, 4, 10, 11, 14 -18). An accumulation of cAMP, which accelerates the priming step and the fusion step, also significantly increases the frequency of the initial phase in Ca 2ϩ -regulat...

<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>

“…The Ca 2+ -regulated exocytotic events is characterized by an initial peak in exocytotic events that declined rapidly (initial phase) followed by a second slower decline (late phase) lasting during ACh stimulation (8,9,14,19,(21)(22)(23)(24). In a previous study (28), an increase in NO/cGMP stimulated by PPARα enhanced the initial phase of Ca 2+ -regulated exocytotic events in antral mucous cells and a NOS1 inhibitor (N-PLA) or a PKG inhibitor (Rp8BrPETcGMPS) abolished the enhancement of the initial phase and produced the delayed, but transient, increase in the late phase (27,28). The similar experiments were carried out using GW7647, GW6471, and ACh in this study.…”

“…The frequencies of initial phase and late produced a delayed, but transient, increase in the late phase stimulated by GW7647 plus ACh (4.5 ± 0.7 events/cell/30s, n = 11). A previous study demonstrated that antral mucous cells have cGMP-dependent PDE2 and the PDE2 inhibited by BAY60-7550 (a selective inhibitor of PDE2) produces the delayed, but transient, increase in the late phase (27). BAY60-7550 induced cAMP accumulation and the delayed, but transient, increase in the late phase was abolished by PKI-amide (1 μM, a PKA inhibitor).…”

“…The procedures for protein extraction and Western blotting have already been described in details (27,28). The antibodies used were an anti-NOS1 antibody (LS-B3272; LifeSpan BioSciences, WA, USA), an anti-PI3K antibody (No.…”

“…This indicates that the inhibition of NO synthesis inhibits cGMPdependent PDE2 (27,28). We examined the effects of wortmannin (50 nM) and AKT-inh (100 nM) on the exocytotic events stimulated by GW7647 plus ACh in antral mucous cells.…”

“…In the course of experiments, we found that the inhibitors of PI3K and Akt abolished the enhancement of Ca 2+ -regulated exocytotic events stimulated by GW7647 plus ACh (Tanaka unpublished observation). Our previous report have shown that the inhibitors of NOS1 and PKG also inhibited the enhancement of Ca 2+ -regulated exocytotic events stimulated by GW7647 plus ACh (27,28). Thus, PPARα agonist may phosphorylate NOS1 mediated via PI3K/Akt to produce NO in antral mucous cells.…”