Guinea Pig Antral Mucous Cells Research Articles

PPARα autocrine regulation of Ca²⁺-regulated exocytosis in guinea pig antral mucous cells: NO and cGMP accumulation.

In antral mucous cells, acetylcholine (ACh, 1 μM) activates Ca(2+)-regulated exocytosis, consisting of a peak in exocytotic events that declines rapidly (initial phase) followed by a second slower decline (late phase) lasting during ACh stimulation. GW7647 [a peroxisome proliferation activation receptor α (PPARα) agonist] enhanced the ACh-stimulated initial phase, and GW6471 (a PPARα antagonist) abolished the GW7647-induced enhancement. However, GW6471 produced the delayed, but transient, increase in the ACh-stimulated late phase, and it also decreased the initial phase and produced the delayed increase in the late phase during stimulation with ACh alone. A similar delayed increase in the ACh-stimulated late phase is induced by an inhibitor of the PKG, Rp8BrPETcGMPS, suggesting that GW6471 inhibits cGMP accumulation. An inhibitor of nitric oxide synthase 1 (NOS1), N(5)-[imino(propylamino)methyl]-L-ornithine hydrochloride (N-PLA), also abolished the GW7647-induced-enhancement of ACh-stimulated initial phase but produced the delayed increase in the late phase. However, in the presence of N-PLA, an NO donor or 8BrcGMP enhanced the ACh-stimulated initial phase and abolished the delayed increase in the late phase. Moreover, GW7647 and ACh stimulated NO production and cGMP accumulation in antral mucosae, which was inhibited by GW6471 or N-PLA. Western blotting and immunohistochemistry revealed that NOS1 and PPARα colocalize in antral mucous cells. In conclusion, during ACh stimulation, a PPARα autocrine mechanism, which accumulates NO via NOS1 leading to cGMP accumulation, modulates the 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

In antral mucous cells, acetylcholine (ACh, 1 μM) activates Ca(2+)-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, β-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 ACh-stimulated antral mucous cells, leading to the delayed, but transient, increase in the frequency of Ca(2+)-regulated exocytosis. PDE2 may prevent antral mucous cells from excessive mucin secretion caused by the cAMP accumulation.

Peroxisome proliferation activation receptor α modulation of Ca2+‐regulated exocytosis via arachidonic acid in guinea‐pig antral mucous cells

Indomethacin (IDM, 10 microm), not aspirin (ASA; 10 microm), enhanced the Ca(2+)-regulated exocytosis stimulated by 1 microm acetylcholine (ACh) in guinea-pig antral mucous cells. Indomethacin inhibits prostaglandin G/H (PGG/H) and 15R-hydroperoxy-eicosatetraenoic acid (15R-HPETE) production from arachidonic acid (AA), while ASA inhibits PGG/H production but accelerates 15R-HPETE production. This suggests that IDM accumulates AA. Arachidonic acid (2 microm) enhanced Ca(2+)-regulated exocytosis in antral mucous cells to a similar extent to IDM. Moreover, a stable analogue of AA, arachidonyltrifluoromethyl ketone (AACOCF(3)), also enhanced Ca(2+)-regulated exocytosis, indicating that AA, not products from AA, enhances Ca(2+)-regulated exocytosis. We hypothesized that AA activates peroxisome proliferation activation receptor alpha (PPARalpha), because AA is a natural ligand for PPARalpha. A PPARalpha agonist (WY14643; 1 microm) enhanced Ca(2+)-regulated exocytosis, and a PPARalpha blocker (MK886; 50 microm) abolished the enhancement of Ca(2+)-regulated exocytosis induced by AA, IDM, AACOCF(3) and WY14643. Western blotting and immunohistochemical examinations demonstrated that PPARalpha exists in antral mucous cells. Moreover, MK886 decreased the frequency of Ca(2+)-regulated exocytosis activated by 1 microm ACh or 2 microm thapsigargin alone by 25-30%. Thus, ACh stimulates AA accumulation via an [Ca(2+)](i) increase, which activates PPARalpha, leading to enhancement of Ca(2+)-regulated exocytosis in antral mucous cells. A novel autocrine mechanism mediated via PPARalpha enhances Ca(2+)-regulated exocytosis in guinea-pig antral mucous cells.

Arachidonic Acid/ppara Enhancement of Ca2+-Regulated Exocytosis in Antral Mucous Cells of Guinea Pig

Indomethacin (IDM, 10 μM) enhanced the Ca2+-regulated exocytosis stimulated by 1 μM ACh in guinea-pig antral mucous cells, but not aspirin (ASA, 10 μM). The differences in pharmacological actions between IDM and ASA suggest that IDM accumulates arachidonic acid (AA), which enhances Ca2+-regulated exocytosis. AA (2 μM) enhanced Ca2+-regulated exocytosis in antral mucous cells similarly to IDM, moreover, an analogue of AA, AACOCF3 (Arachidonyltrifluoromethyl ketone, a PLA2 blocker) also enhanced it. These indicate that the Ca2+-regulated exocytosis is directly enhanced by AA, not by the products of the AA cascade, such as PGs, LXs and LTs. We examined the effects of MK886 (an inhibitor of peroxisome proliferation activation receptor α, PPARα) on the AA-induced enhancement of Ca2+-regulated exocytosis, because AA is a natural ligand for PPARα. MK-886 (40 μM) abolished the enhancement of Ca2+-regulated exocytosis induced by AA, IDM and AACOCF3. Moreover, WY14643 (a PPARα agonist) enhanced the Ca2+-regulated exocytosis, similarly to AA. MK-886 decreased the frequency of the Ca2+-regulated exocytosis activated by 1 μM ACh or thapsigargin by 25-30 %. Western blotting and immunohistochemical examinations demonstrated that PPARα exists in antral mucous cells. Thus, ACh stimulates AA accumulation via increases in [Ca2+]i, and then, AA activates PPARα, which enhances Ca2+-regulated exocytosis in antral mucous cells. A novel autocrine mechanism mediated via PPARα maintains Ca2+-regulated exocytosis of the antral mucous cells of guinea pig.

Ca2+‐regulated exocytosis modulated by [Cl−]i in ACh‐stimulated antral mucous cells of guinea pig

The effects of [Cl−]i on ACh‐stimulated exocytosis were studied in guinea pig antral mucous cells. ACh activated Ca2+‐regulated exocytosis (initial phase followed by a sustained phase). Bumetanide or an NO3− solution enhanced it, in contract, NPPB decreased it and eliminated the enhancement. ATP depletion studies demonstrated that exposure of NO3− solution prior to ATP depletion induced an enhanced initial phase followed by a sustained phase, whereas exposure of NO3− solution after ATP depletion induced only a sustained phase. Measurements of [Cl−]i revealed that ACh decreases [Cl−]i. and that bumetanide and NO3− solution decreased [Cl−]i and enhanced the ACh‐evoked [Cl−]i decrease; in contrast, NPPB increased [Cl−]i and inhibited the [Cl−]i decrease induced by ACh, bumetanide or NO3− solution. [Ca2+]i measurements showed that a decrease in [Cl−]i enhanced the ACh‐stimulated [Ca2+]i increase. In conclusion, a decrease in [Cl−]i accelerates ATP‐dependent priming and [Ca2+]i increase, which enhance Ca2+‐regulated exocytosis in ACh‐stimulated antral mucous cells.

Cl−]imodulation of Ca2+-regulated exocytosis in ACh-stimulated antral mucous cells of guinea pig

The effects of intracellular Cl- concentration ([Cl-]i) on acetylcholine (ACh)-stimulated exocytosis were studied in guinea pig antral mucous cells by video microscopy. ACh activated Ca2+-regulated exocytosis (an initial phase followed by a sustained phase). Bumetanide (20 microM) or a Cl- -free (NO3-) solution enhanced it; in contrast, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, a Cl- channel blocker) decreased it and eliminated the enhancement induced by bumetanide or NO3- solution. ACh and Ca2+ dose-response studies demonstrated that NO3- solution does not shift their dose-response curves, and ATP depletion studies by dinitrophenol or anoxia demonstrated that exposure of NO3- solution prior to ATP depletion induced an enhanced initial phase followed by a sustained phase, whereas exposure of NO3- solution after ATP depletion induced only a sustained phase. Intracellular Ca2+ concentration ([Ca2+]i) measurements showed that bumetanide and NO3- solution enhanced the ACh-stimulated [Ca2+]i increase. Measurements of [Cl-]i revealed that ACh decreases [Cl-]i and that bumetanide and NO3- solution decreased [Cl-]i and enhanced the ACh-evoked [Cl-]i decrease; in contrast, NPPB increased [Cl-]i and inhibited the [Cl-]i decrease induced by ACh, bumetanide, or NO3- solution. These suggest that [Cl-]i modulates [Ca2+]i increase and ATP-dependent priming. In conclusion, a decrease in [Cl-]i accelerates ATP-dependent priming and [Ca2+]i increase, which enhance Ca2+-regulated exocytosis in ACh-stimulated antral mucous cells.

ACh‐stimulated exocytosis enhanced by arachidonic acid in guinea pig antral mucous cells: cGMP accumulation via PPARα

Arachidonic acid (AA, 10 nM – 2 μM) enhanced Ca2+-regulated exocytosis in acetylcholine (ACh) stimulated antral mucous cells of guinea pig. The enhancement in ACh-stimulated exocytosis induced by AA was not mediated via PGE2 accumulation. The AA actions were inhibited by MK886 (50 μM, an inhibitor of by peroxisome proliferation activation receptor ? (PPAR?), and were mimicked by PPAR? agonists (Eicosatetraynoic acid (ETYA) and WY14643). The enhancement of ACh-stimulated exocytosis induced by AA, ETYA and WQY14643 was eliminated by a PKG inhibitor (Rp8BrPET-cGMPS) and was mimicked by 8Br-cGMP. Nitro-L-arginine methyl ester (L-NAME) also inhibited the enhancement induced by AA, ETYA and WY14643, and 1-Hydroxy-2-oxo-3-(N-ethyl-2-aminoethyl)-3-ethyl-1-triazene (NOC-12) enhanced ACh-stimulated exocytosis similar to AA, ETYA and WY14643, which was inhibited by Rp8BrPET-cGMPS. AA, ETYA or WY14643 did not increase intracellular Ca2+ concentration ([Ca2+]i) and did not enhance ACh-stimulated increases in [Ca2+]i. Measurements of cGMP contents in antral mucosa demonstrated that stimulation with ACh, AA and PPAR? agonists stimulate cGMP accumulation, which is inhibited by MK886 and L-NAME. In conclusion, in ACh-stimulated antral mucous cells, AA accumulated stimulates PPAR?, which activates the NO/cGMP cascade, and PKG activated by cGMP enhances Ca2+-regulated exocytosis in antral mucous cells by increasing number of the primed granule.μ μ μ μ μ

CGMP modulation of ACh-stimulated exocytosis in guinea pig antral mucous cells

In guinea pig antral mucous cells, ACh stimulates the Ca(2+)-regulated exocytosis, which has a characteristics feature: an initial transient phase followed by a sustained phase. The effects of cGMP on ACh-stimulated exocytosis were studied in guinea pig antral mucous cells using video microscopy. cGMP enhanced the frequency of ACh-stimulated exocytotic events, whereas cGMP alone did not induce any exocytotic events under the ACh-unstimulated condition. cGMP did not stimulate either Ca(2+) mobilization or cAMP accumulation. The Ca(2+) dose-response studies demonstrated that cGMP shifted the dose-response curve upward with no shift to the lower concentration. This indicates that cGMP increased maximal responsiveness of the Ca(2+)-regulated exocytosis, but not the Ca(2+) sensitivity. Moreover, under a condition of ATP depletion by dinitrophenol (DNP) or anoxia (N(2) bubbling), ACh evoked only a sustained phase in exocytotic events with no initial transient phase. However, ACh evoked an initial transient phase followed by a sustained phase with addition of cGMP before ATP depletion, whereas only a sustained phase was evoked in a case of cGMP addition after ATP depletion. Thus cGMP-induced enhancement in ACh-stimulated exocytotic events requires ATP, suggesting that cGMP modulates ATP-dependent priming of Ca(2+)-regulated exocytosis in antral mucous cells. In conclusion, cGMP increases the number of primed granules via acceleration of the ATP-dependent priming, which enhances the Ca(2+)-regulated exocytosis stimulated by ACh.

Enhancement of Ca2+-regulated exocytosis by indomethacin-induced arachidonic acid accumulation in guinea pig antral mucous cells

Enhancement of Ca2+-regulated exocytosis by indomethacin-induced arachidonic acid accumulation in guinea pig antral mucous cells

Cyclic GMP modulates ACh-stimulated exocytosis in guinea pig antral mucous cells

Cyclic GMP modulates ACh-stimulated exocytosis in guinea pig antral mucous cells

ACh-stimulated exocytosis modulated by indomethacin-induced accumulation of arachidonic acid in guinea pig antral mucous cells

ACh-stimulated exocytosis modulated by indomethacin-induced accumulation of arachidonic acid in guinea pig antral mucous cells

Inhibition of ACh-stimulated exocytosis by NSAIDs in guinea pig antral mucous cells: autocrine regulation of mucin secretion by PGE2.

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.

Acetylcholne-stimulated exocytosis inhibited by indomethacin in guinea pig antral mucous cells

Acetylcholne-stimulated exocytosis inhibited by indomethacin in guinea pig antral mucous cells

EP1 and EP4 receptors mediate exocytosis evoked by prostaglandin E(2) in guinea-pig antral mucous cells.

Effects of prostaglandin E(2) (PGE(2)) on exocytosis of mucin were studied in mucous cells isolated from guinea-pig antrum using video-microscopy. Stimulation with PGE(2) elicited a sustained increase in the frequency of exocytotic events in a dose-dependent manner, which was under regulation by both Ca(2+) and cAMP. Stimulation with a selective prostanoid EP4 receptor agonist (ONO-AEI-329, 10 microM), which activates cAMP signals, elicited a sustained increase in the frequency of exocytotic events (30 % of that evoked by 1 microM PGE(2)). Stimulation with an EP1 agonist (17-P-T-PGE(2), 1 microM), which activates Ca(2+) signals, increased the frequency of exocytotic events to a lesser extent (5 % of that evoked by 1 microM PGE(2)), while addition of an EP1 antagonist (ONO-8713, 10 microM) decreased the frequency of exocytotic events (approximately 40 % of that evoked by 1 microM PGE(2)). 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 Ca(2+) signal activated via the EP1 receptor potentiates the cAMP-regulated exocytotic events activated via the EP4 receptor during PGE(2) stimulation, by increasing the sensitivity of the exocytotic response to cAMP. In conclusion, exocytotic events in PGE(2)-stimulated antral mucous cells were regulated by interactions between EP1 and EP4 receptors. Experimental Physiology (2001) 86.4, 451-460.

Na+]i, [K+]i and [Cl-]i regulation of exocytosis in guinea-pig antral mucous cells.

Effects of intracellular Na+, K+ and Cl- on Ca(2+)-regulated exocytosis activated by 10 microM acetylcholine (ACh) were studied in guinea-pig antral mucous cells which are permeabilized by nystatin treatment. Ca(2+)-regulated exocytotic events were modulated by [Na+]i, [K+]i and [Cl-]i via mediation of PTX-sensitive G proteins. Increases in [Na+]i and PTX inhibit G protein (G(Na)), which suppressed the exocytosis. Increases in [K+]i caused the exchange of G proteins (from G(Na) to G(K)) to increase, and GK evoked activation of the exocytosis and was inhibited by PTX. Increases in [Cl-]i and PTX inhibit G protein (G(Cl)), which stimulates exocytotic events. Based on these observations, the exocytosis in antral mucous cells were modulated by intracellular ions, concentration of which were increased or decreased by cell volume changes caused by Ach.

Inhibition of Ca(2+) entry caused by depolarization in acetylcholine-stimulated antral mucous cells of guinea pig: G protein regulation of Ca(2+) permeable channels.

The effects of depolarizing conditions resulting from increasing extracellular K(+) concentration or nystatin treatment on intracellular Ca(2+) concentration ([Ca(2+)](i)) were studied in guinea pig antral mucous cells following acetylcholine (ACh) stimulation. ACh stimulation evoked a biphasic increase in [Ca(2+)](i), that is, an initial transient increase followed by a plateau. Depolarizing conditions reduced the [Ca(2+)](i) in the plateau phase during ACh stimulation. However, pertussis toxin (PTX, a G protein inhibitor) treatment caused [Ca(2+)](i) in the ACh-evoked plateau phase to increase under depolarizing conditions, while it had no effect on [Ca(2+)](i) under hyperpolarized conditions. Based on these observations, Ca(2+) permeable channels are regulated by a G protein which is activated by depolarized conditions and inhibited by hyperpolarized conditions and PTX; activation of the G protein (depolarization) causes Ca(2+) permeable channels to inhibit, and in turn, inhibition of the G protein (hyperpolarization) causes them to activate.