The gastric mucosa is covered with a viscoelastic and lubricant mucous layer. The chief determinants of the physiological role of the mucous layer are mucins, which are high molecular weight glycoproteins. Gastric mucins are produced in and secreted from specialized differentiated mucous cells located in the epithelium lining the gastric pits, and play an important role in the protection of gastric mucosa from acid-peptic injury. The production and release of mucins are regulated by neurotransmitters, hormones and biologically active peptides. Mucins are synthesized in the Golgi apparatus, and stored in intracellular granules which are transported to the luminal surface of the cell (Forstner & Forstner, 1994). These mucin granules finally discharge their contents through holes in the plasma membrane. This process is generally known as exocytosis. The first event in exocytosis is the fusion of a granule with the plasma membrane at the fusion pore, and is mediated by the exocytosis-related proteins, which are activated by intracellular Ca¥, protein kinase A (PKA) and protein kinase C (PKC) (Forstner & Forstner, 1994). Intracellular Ca¥, in particular, is widely accepted as playing a key role in exocytosis in epithelial cells, endocrine cells and nerve endings. In the gastric mucosa, elevation of intracellular Ca¥ concentration, [Ca¥]é, also increases mucin release (Seidler & Sewing, 1989), and cholinergic stimulation is well known to increase [Ca¥]é. The present experiments were designed to investigate the effects of acetylcholine (ACh) on the frequency of exocytotic events.

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.

The effects of indomethacin (IDM) and aspirin (ASA) on ACh (10 microM) -stimulated exocytotic events were studied in guinea pig antral mucous cells by using video optical microscopy. IDM or ASA, which inhibits cyclooxygenase (COX), decreased the frequency of ACh-stimulated exocytotic events by 30% or 60%, respectively. The extent of inhibition induced by ASA (60%) decreased by 30% when IDM or arachidonic acid (AA, the substrate of COX) was added. IDM, unlike ASA, appears to induce the accumulation of AA, which enhances the frequency of ACh-stimulated exocytotic events in ASA-treated cells. ONO-8713 (100 microM; an inhibitor of the EP1-EP4 prostaglandin receptors) and N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, HCl (H-89, 20 microM; an inhibitor of PKA) also decreased the frequency of ACh-stimulated exocytotic events by 60%. However, the supplementation of PGE(2) (1 microM) prevented the IDM-induced decrease in the frequency of ACh-stimulated exocytotic events. SC-560 (an inhibitor of COX-1) decreased the frequency of ACh-stimulated exocytotic events by 30%, but NS-398 (an inhibitor of COX-2) did not. Moreover, IDM decreased the frequency of exocytotic events stimulated by ionomycin, suggesting that COX-1 activity is stimulated by an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)). ACh and ionomycin increased PGE(2) release in antral mucosal cells. In conclusion, in ACh-stimulated antral mucous cells, an increase in [Ca(2+)](i) activates Ca(2+)-regulated exocytotic events and PGE(2) release mediated by COX-1. The released PGE(2) induces the accumulation of cAMP, which enhances the Ca(2+)-regulated exocytosis. The autocrine mechanism mediated by PGE(2) maintains the high-level mucin release from antral mucous cells during ACh stimulation.

This study demonstrated that PDE1 (phosphodiesterase 1) existing in the ciliary beat frequency (CBF)-regulating metabolon regulates CBF in procaterol-stimulated lung airway ciliary cells of mouse. Procaterol (an β-agonist) increased the ciliary bend angle (CBA) and CBF via cAMP accumulation in the ciliary cells of mice: interestingly, the time course of CBF increase was slower than that of CBA increase. However, IBMX (3-isobutyl-1-methylxanthine, an inhibitor of PDE) increased CBA and CBF in an identical time course. Lowering an intracellular Ca concentration ([Ca]) caused by switching to an EGTA-containing Ca-free solution from normal one elevated the procaterol-induced increasing rate of CBF. These observations suggest that Ca-dependent PDE1 controls cAMP-stimulated CBF increase. Either application of 8MmIBMX (8-methoxymethyl-IBMX, a selective PDE1 inhibitor), BAPTA-AM (an intracellular Ca chelator), or calmidazolium (an inhibitior of calmodulin) alone increased CBA and CBF in the lung airway ciliary cells and increased cAMP contents in the isolated lung cells, and like IBMX, each application of the compound made the time courses of CBA and CBF increase stimulated by procaterol identical. The immunoelectron microscopic examinations revealed that PDE1A exists in the space between the nine doublet tubules ring and plasma membrane in the lung airway cilium, where the outer dynein arm (a molecular motor regulating CBF) functions. In conclusion, PDE1A is a key factor slowing the time course of the procaterol-induced increase in CBF via degradation of cAMP in the CBF-regulating metabolon of the mouse lung airway cilia.

Effects of prostaglandin E2 (PGE2) on exocytosis of mucin were studied in mucous cells isolated from guinea‐pig antrum using video‐microscopy. Stimulation with PGE2 elicited a sustained increase in the frequency of exocytotic events in a dose‐dependent manner, which was under regulation by both Ca2+ and cAMP. Stimulation with a selective prostanoid EP4 receptor agonist (ONO‐AEI‐329, 10 μM), which activates cAMP signals, elicited a sustained increase in the frequency of exocytotic events (30% of that evoked by 1 μM PGE2). Stimulation with an EP1 agonist (17‐P‐T‐PGE2, 1 μM), which activates Ca2+ signals, increased the frequency of exocytotic events to a lesser extent (5% of that evoked by 1 μM PGE2), while addition of an EP1 antagonist (ONO‐8713, 10 μM) decreased the frequency of exocytotic events (approximately 40% of that evoked by 1 μM PGE2). However, addition of the EP1 agonist potentiated the frequency of exocytotic events evoked by the EP4 agonist or forskolin (which elevates cAMP levels) and increased the sensitivity of the exocytotic events to forskolin. These results suggest that the Ca2+ signal activated via the EP1 receptor potentiates the cAMP‐regulated exocytotic events activated via the EP4 receptor during PGE2 stimulation, by increasing the sensitivity of the exocytotic response to cAMP. In conclusion, exocytotic events in PGE2‐stimulated antral mucous cells were regulated by interactions between EP1 and EP4 receptors.