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Heat shock proteins (Hsps) are highly conserved proteins that play a role in cytoprotection and maintaining intestinal homeostasis. Glutamine is essential for the optimal induction of intestinal epithelial Hsp expression, but its mechanisms of action are incompletely understood. Glutamine is a substrate for polyamine synthesis and stimulates the activity of ornithine decarboxylase (ODC), a key enzyme for polyamine synthesis, in intestinal epithelial cells. Thus we investigated whether polyamines (putrescine, spermidine, or spermine) and their precursor ornithine mediate the induction of Hsp expression in IEC-18 rat intestinal epithelial cells. As previously observed, glutamine was required for heat stress induction of Hsp70 and Hsp25, although it had little effect under basal conditions. Under conditions of glutamine depletion, supplementation of ornithine or polyamines restored the heat-induced expression of Hsp70 and Hsp25. When ODC was inhibited by α-difluoromethylornithine (DFMO), an irreversible ODC inhibitor, the heat stress induction of Hsp70 and Hsp25 was decreased significantly, even in the presence of glutamine. Ornithine, polyamines, and DFMO did not modify the nuclear localization of heat shock transcription factor 1 (HSF-1). However, DFMO dramatically reduced glutamine-dependent HSF-1 binding to an oligonucleotide with heat shock elements (HSE), which was increased by glutamine. In addition, exogenous polyamines recovered the DNA-binding activity. These results indicate that polyamines play a critical role in the glutamine-dependent induction of the intestinal epithelial heat shock response through facilitation of HSF-1 binding to HSE. [ABSTRACT FROM AUTHOR]

During cell swelling, cells release organic osmolytes via a volume-activated channel as part of the regulatory volume decrease. The erythrocyte membrane protein AE1 (band 3), has been shown to be involved in regulatory volume responses of fish erythrocytes. Previous studies showed that the expression of trout AE1 in Xenopus laevis oocytes induces band 3 anion exchange activity and organic osmolyte channel activity. However, an endogenous swelling-activated anion channel, IClswell, is present in Xenopus oocyte membranes. Therefore, it is not yet known whether a new organic osmolyte channel is formed or whether the endogenous channel, IClswell, is activated when trout AE1 is expressed in the oocytes. The purpose of this study was to determine whether the expression of trout AE1 in Xenopus oocytes leads to the formation and membrane insertion of a new organic osmolyte channel or activates IClswell. To differentiate between the two possibilities, we compared the time courses, pH profiles and inhibitor sensitivities of both trout AE1 and IClswell. The results of taurine-uptake experiments show that the time courses and pH levels for optimum expression of trout AE1 and IClswell differ significantly. The inhibitor sensitivities of the organic osmolyte channel mediated by trout AE1 and IClswell are also significantly different, strongly suggesting that the expression of trout AE1 in Xenopus oocytes does not activate IClswell, but rather forms a new organic osmolyte channel. [ABSTRACT FROM AUTHOR]

Na-K-Cl cotransport stoichiometry and affinities for Na, K and Cl were determined in flounder intestine. Measurement of simultaneous NaCl and RbCl influxes resulted in ratios of 2.2 for Cl/Na and 1.8 for Cl/Rb. The effect of Na and Rb on Rb influx showed first order kinetics with K1/2 values of 5 and 4.5 mM and Hill coefficients of 0.9 and 1.2, respectively. The effect of Cl on rubidium influx showed a sigmoidal relationship with K1/2 of 20 mM and a Hill coefficient of 2.0. The effects of variations in Na and Cl concentration on short-circuit current (Isc) were also determined. The K1/2 for Na was 7 mM with a Hill coefficient of 0.9 and the K1/2 for Cl was 46 mM with a Hill coefficient of 1.9. Based on the simultaneous influx measurements, a cotransport stoichiometry of 1Na:1K:2Cl is concluded. The Hill coefficients for Cl suggest a high degree of cooperativity between Cl binding sites. Measurements of the ratio of net Na and Cl transepithelial fluxes under short-circuit conditions (using a low Na Ringer solution to minimize the passive Na flux) indicate that the Cl/Na flux ratio is approximately 2:1. Therefore, Na recycling from serosa to mucosa does not significantly contribute to the Isc. Addition of serosal ouabain (100 microM) inhibited Rb influx, indicating that Na-K-Cl cotransport is inhibited by ouabain. This finding suggests that a feedback mechanism exists between the Na-K-ATPase on the basolateral membrane and the apical Na-K-2Cl cotransporter. [ABSTRACT FROM AUTHOR]

This article was originally published with the wrong title. [ABSTRACT FROM AUTHOR]