Yukinori Sawabe

Abnormal expression of ENaC and SGK1 mRNA induced by dietary sodium in Dahl salt-sensitively hypertensive rats

Epithelial sodium channel (ENaC) plays a crucial role in controlling sodium reabsorption in the kidney keeping the normal blood pressure. We previously reported that the expression of ENaC mRNA in the kidney of Dahl salt‐sensitive (DS) rats was abnormally regulated by aldosterone, however it is unknown if dietary sodium affects the expression of ENaC and serum and glucocorticoid‐regulated kinase 1 (SGK1), which plays an important role in ENaC activation, in DS rats. In the present study, we investigated whether dietary sodium abnormally affects the expression of ENaC and SGK1 mRNA in DS rats. DS and Dahl salt‐resistant (DR) rats (8 weeks old) were divided into three different groups, respectively: (1) low sodium diet (0.005% NaCl), (2) normal sodium diet (0.3% NaCl), and (3) high sodium diet (8% NaCl). The high sodium diet for 4 weeks in DS rats elevated the systolic blood pressure, but did not in any other groups. The expression of α‐ENaC mRNA in DS rats was abnormally increased by high sodium diet in contrast to DR rats, while it was normally increased by low sodium diet in DS rats similar to DR rats. The expression of β‐ and γ‐ENaC mRNA in DS rats was also abnormally increased by high sodium diet unlike DR rats. The expression of SGK1 mRNA was elevated by high sodium diet in DS rats, but it was decreased in DR rats. These observations indicate that the expression of ENaC and SGK1 mRNA is abnormally regulated by dietary sodium in salt‐sensitively hypertensive rats, and that this abnormal expression would be one of the factors causing salt‐sensitive hypertension.

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

Indomethacin (IDM, 10 μm), not aspirin (ASA; 10 μm), enhanced the Ca2+‐regulated exocytosis stimulated by 1 μm 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 μm) enhanced Ca2+‐regulated exocytosis in antral mucous cells to a similar extent to IDM. Moreover, a stable analogue of AA, arachidonyltrifluoromethyl ketone (AACOCF3), also enhanced Ca2+‐regulated exocytosis, indicating that AA, not products from AA, enhances Ca2+‐regulated exocytosis. We hypothesized that AA activates peroxisome proliferation activation receptor α (PPARα), because AA is a natural ligand for PPARα. A PPARα agonist (WY14643; 1 μm) enhanced Ca2+‐regulated exocytosis, and a PPARα blocker (MK886; 50 μm) abolished the enhancement of Ca2+‐regulated exocytosis induced by AA, IDM, AACOCF3 and WY14643. Western blotting and immunohistochemical examinations demonstrated that PPARα exists in antral mucous cells. Moreover, MK886 decreased the frequency of Ca2+‐regulated exocytosis activated by 1 μm ACh or 2 μm thapsigargin alone by 25–30%. Thus, ACh stimulates AA accumulation via an [Ca2+]i increase, which activates PPARα, leading to enhancement of Ca2+‐regulated exocytosis in antral mucous cells. A novel autocrine mechanism mediated via PPARα enhances Ca2+‐regulated exocytosis in guinea‐pig antral mucous cells.

PPARα induced NOS1 phosphorylation via PI3K/Akt in guinea pig antral mucous cells: NO-enhancement in Ca(2+)-regulated exocytosis.

A PPARα (peroxisome proliferation activation receptor α) agonist (GW7647) activates nitric oxide synthase 1 (NOS1) to produce NO leading to cGMP accumulation in antral mucous cells. In this study, we examined how PPARα activates NOS1. The NO production stimulated by GW7647 was suppressed by inhibitors of PI3K (wortmannin) and Akt (AKT 1/2 Kinase Inhibitor, AKT-inh), although it was also suppressed by the inhibitors of PPARα (GW6471) and NOS1 (N-PLA). GW7647 enhanced the ACh (acetylcholine)-stimulated exocytosis (Ca(2+)-regulated exocytosis) mediated via NO, which was abolished by GW6471, N-PLA, wortmannin, and AKT-inh. The Western blotting revealed that GW7647 phosphorylates NOS1 via phosphorylation of PI3K/Akt in antral mucous cells. The immunofluorescence examinations demonstrated that PPARα existing with NOS1 co-localizes with PI3K and Akt in the cytoplasm of antral mucous cells. ACh alone and AACOCF3, an analogue of arachidonic acid (AA), induced the NOS1 phosphorylation via PI3K/Akt to produce NO, which was inhibited by GW6471. Since AA is a natural ligand for PPARα, ACh stimulates PPARα probably via AA. In conclusion, PPARα activates NOS1 via PI3K/Akt phosphorylation to produce NO in antral mucous cells during ACh stimulation.

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.