The short-chain fatty acid butyrate was readily taken up by Caco-2 cells. Transport exhibited saturation kinetics, was enhanced by low extracellular pH, and was Na(+) independent. Butyrate uptake was unaffected by DIDS; however, alpha-cyano-4-hydroxycinnamate and the butyrate analogs propionate and L-lactate significantly inhibited uptake. These results suggest that butyrate transport by Caco-2 cells is mediated by a transporter belonging to the monocarboxylate transporter family. We identified five isoforms of this transporter, MCT1, MCT3, MCT4, MCT5, and MCT6, in Caco-2 cells by PCR, and MCT1 was found to be the most abundant isoform by RNase protection assay. Transient transfection of MCT1, in the antisense orientation, resulted in significant inhibition of butyrate uptake. The cells fully recovered from this inhibition by 5 days after transfection. In conclusion, our data showed that the MCT1 transporter may play a major role in the transport of butyrate into Caco-2 cells.

The identity of Na+/H+ exchanger (NHE) isoforms in the human small intestine and colon and their role in vectorial Na+ absorption are not known. The present studies were undertaken to examine the regional and vertical axis distribution of NHE-1, NHE-2, and NHE-3 mRNA in the human intestine. Ribonuclease protection assays were used to quantitate the levels of mRNA of these isoforms in various regions of the human intestine. In situ hybridization technique was used to localize NHE-2 and NHE-3 mRNA in the colon. The NHE-1 isoform message was present uniformly throughout the length of the human intestine. In contrast, mRNA levels for human NHE-2 and NHE-3 isoforms demonstrated significant regional differences. The NHE-3 abundance was found in decreasing order: ileum > jejunum > proximal colon = distal colon. The NHE-2 message level in the distal colon was significantly higher than in the proximal colon but was evenly distributed in the small intestine. In addition, NHE-2 mRNA was present in surface epithelial cells as well as in cells of the crypt region, suggesting the presence of NHE-2 message throughout the vertical axis of the colonic crypts. In contrast, NHE-3 mRNA was localized to surface colonocytes in the proximal colon. On the basis of this tissue-specific localization of NHE-2 and NHE-3 mRNA, it can be speculated that the relative contribution of NHE-2 and NHE-3 isoforms in Na+ absorption in the human intestine may be region specific, and these putative apical isoforms may be differentially regulated.

The adherence of Escherichia coli to human uroepithelial cells obtained from midstream urine specimens of healthy women was studied. Bacteria labeled with [3H]uridine were used, and unattached organisms were separated from the epithelial cells by vacuum filtration with 5-,um-pore-size Nucleopore membrane

In the present report, we describe the cloning of a human colonic cDNA that describes the full-length Na+/H+exchanger (NHE) 2 coding region. The human NHE2 (hNHE2) cDNA encodes for a polypeptide of 812 amino acids with a 90% overall identity to both rabbit and rat NHE2 isoforms. In comparison with SLC9A2, recently reported as the human NHE2, the hNHE2 polypeptide is 115 amino acids longer in the NH2-terminal end and shows only an 84% DNA nucleotide sequence identity. Northern blot analysis revealed that hNHE2 message has an uneven tissue distribution, with high levels in the skeletal muscle, colon, and kidney and lower levels in the testis, prostate, ovary, and small intestine. Protein expression studies with hNHE2 clone showed that a 75-kDa protein was expressed. Stable expression of transfected full-length hNHE2 cDNA in Na+/H+exchange-deficient LAP1 cells exhibited Na+-dependent pH recovery after an acid prepulse that was inhibited by 0.1 mM amiloride. These data indicate that this cDNA is the true human NHE2 cDNA and that the encoded protein is capable of catalyzing Na+/H+exchange activity.

Regulation of cytoplasmic pH (pHi) of esophageal cells assumes importance as these cells can be exposed to mucosally absorbed acid during gastroesophageal reflux episodes. In this study, we examined whether esophageal cells possess pHi transport systems. Esophageal cells were harvested utilizing a gentle trypsin technique that yielded cells per esophagus. Cells were attached to a glass cover slip that had been pretreated with rat-tail collagen, and pHi was measured continuously in a spectrofluorometer utilizing 2',7'-bis(2-carboxyethyl)-5(-6)- carboxyfluoroscein acetoxymethyl ester as a pH-sensitive fluorescent probe. The basal pHi of cells exposed to a -containing solution averaged 7.52 ± 0.20 (n = 6). The pHi declined slightly but not significantly to 7.46 ± 0.12 with the addition of 5% and 28 mM When H2 4,4'-diisothiocyanatostilbene- 2,2'-disulfonic acid (DIDS; 0.5 mM) was added, pHi was unchanged. However, addition of M amiloride caused pHi to decrease to 7.29 ± 0.18 (P less than 0.01). When cells were acidified (pHi 6.3-7.0) using a(20 mM) pulse technique, pHi was rapidly restored toward neutrality in the presence of a -free external concentration ([]o)-containing solution (pH units/min = 0.26 ± 0.12; n = 8). Alkalinization was completely blocked with M amiloride. In the presence of M amiloride, 28 mM , and 5%, acidified cells also alkalinized, although at a slower rate (0.11 ± 0.04 pH units/min; n = 16).(ABSTRACT TRUNCATED AT 250 WORDS)