Abstract
Introduction. The small intestinal undifferentiated crypt cells and differentiated villous cells serve different physiological functions in response to various stimuli. L-Arginine is the exclusive precursor for nitric oxide (NO) biosynthesis and an important nutrient for intestinal integrity during stress states. The purpose of this in vitro study was to investigate the poorly understood intracellular signaling pathways involved in the acidosis regulation of intestinal membrane arginine absorption in the undifferentiated rat intestinal crypt IEC-6 cells. Methods. IEC-6 cells were grown to subconfluence and incubated in growth media of various pHs (pH 6.6 to pH 7.4, adjusted with 1 M HCl and/or NaHCO 3), ±PKC inhibitor Chelerythrine Cl (CHEL, 0–6.6 μM), Phosphatidylinositide-3 kinase (PI-3 kinase) inhibitors wortmannin (0–10 μM), and LY 294004 (LY, 0–10 μM). 3H-arginine (5 μm) transport activity, arginine transporter MCAT1 mRNA levels, cellular protein kinase C (PKC) levels were measured. Data are means ± SD and analyzed by ANOVA with P < 0.05. Results. Desired extracellular pH (pH 6.6 to pH 7.4) was achieved in 6 h and remained stable. Chronic acidosis (pH 6.6, <48 h) resulted in a 48% decrease of arginine transport activity and a 60% increase in cellular phospho-PKC-pan levels, with significant increases in phospho-PKC-δ and phospho-PKC-μ isoforms but not the phospho-PKC-αβ, phospho-PKC-ε, or phospho-PKC-ζ isoforms. Protein kinase C inhibitor chelerythrine Cl and PI-3 kinase inhibitors wormannin and LY 294004 individually blocked the acidosis-mediated reduction in arginine transport activity and MCAT1 mRNA levels. Conclusions. Protein kinase C activation is a key intracellular regulator in the signal transduction cascade involved in this acidosis-mediated reduction in membrane arginine transport activity in the undifferentiated rat intestinal epithelial cells. TABLE—ABSTRACT 9 Control + CHEL + Wormannin + LY Control 0.12 ± 0.03 0.11 ± 0.02 0.11 ± 0.03 0.09 0.04 Acidosis 0.062 ± 0.02 ∗ 0.13 ± 0.03 0.09 ± 0.01 0.10 ± 0.01 Unit: nmole/mg/min. ∗ P < 0.05
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