Response of intestinal epithelial cells to Trichuris suis excretory-secretory products and the influence on Campylobacter jejuni invasion under in vitro conditions.

We previously developed a swine animal model in which natural host resistance to Campylobacter jejuni is altered by experimental infection with low numbers of the nematode Trichuris suis. Pigs naturally colonized with C. jejuni experience colitis because of the invasion of the bacterium approximately 21 days after exposure to T. suis. To better understand the mechanism of T. suis-dependent C. jejuni colitis, we evaluated the effects of T. suis excretory-secretory products (ESPs) on intestinal epithelial cells (IECs) and the influence of ESP on C. jejuni invasion in IECs under in vitro conditions. Viability assays revealed a dose-dependent cytotoxic response in ESP-treated IECs, particularly IPEC-1 and INT407 cells. Transepithelial electrical resistance dropped significantly in IPEC-1 cells treated on apical and basolateral surfaces, but not in those treated only on apical surfaces. Using the gentamicin-killing assay, reduced numbers of intracellular C. jejuni were recovered from IECs treated with ESP at 1 mg protein/ml concentration. This observation can be at least partially explained by a novel antibacterial activity in ESP. Contrary to our hypothesis, ESP at subtoxic concentrations did not enhance invasion. In addition to mechanical damage from worms, these results suggest that soluble products released by T. suis contribute to IEC damage at the site of worm attachment.

NHE3-dependent cytoplasmic alkalinization is triggered by Na(+)-glucose cotransport in intestinal epithelia.

Cytoplasmic pH (pH(i)) was evaluated during Na(+)-glucose cotransport in Caco-2 intestinal epithelial cell monolayers. The pH(i) increased by 0.069 +/- 0.002 within 150 s after initiation of Na(+)-glucose cotransport. This increase occurred in parallel with glucose uptake and required expression of the intestinal Na(+)-glucose cotransporter SGLT1. S-3226, a preferential inhibitor of Na(+)/H(+) exchanger (NHE) isoform 3 (NHE3), prevented cytoplasmic alkalinization after initiation of Na(+)-glucose cotransport with an ED(50) of 0.35 microM, consistent with inhibition of NHE3, but not NHE1 or NHE2. In contrast, HOE-694, a poor NHE3 inhibitor, failed to significantly inhibit pH(i) increases at <500 microM. Na(+)-glucose cotransport was also associated with activation of p38 mitogen-activated protein (MAP) kinase, and the p38 MAP kinase inhibitors PD-169316 and SB-202190 prevented pH(i) increases by 100 +/- 0.1 and 86 +/- 0.1%, respectively. Conversely, activation of p38 MAP kinase with anisomycin induced NHE3-dependent cytoplasmic alkalinization in the absence of Na(+)-glucose cotransport. These data show that NHE3-dependent cytoplasmic alkalinization occurs after initiation of SGLT1-mediated Na(+)-glucose cotransport and that the mechanism of this NHE3 activation requires p38 MAP kinase activity. This coordinated regulation of glucose (SGLT1) and Na(+) (NHE3) absorptive processes may represent a functional activation of absorptive enterocytes by luminal nutrients.

Cloning of a novel EGFR-related peptide: a putative negative regulator of EGFR.

Although epidermal growth factor receptor (EGFR) plays a key role in regulating cell proliferation, differentiation, and transformation in many tissues, little is known about the factor(s) that may modulate its function. We have isolated a cDNA clone from the rat gastroduodenal mucosa whose full length revealed 1,958 bp that contained 227 bp of 5'-untranslated region (UTR) and an open-reading frame encoding 479 amino acids, followed by 290 bp of 3'-UTR. It showed ~85% nucleotide homology to the external domain of the rat EGFR. We refer to the product of the newly isolated cDNA as EGFR-related protein (ERRP). In Northern blot analysis with poly(A)(+) RNA from different rat tissues, ERRP cDNA hybridized to several mRNA transcripts with the strongest reaction noted with a transcript of approximately 2 kb. Maximal expression of the 2-kb mRNA transcript was observed in the small intestine, followed by colon, liver, gastric mucosa, and other tissues. Transfection of ERRP cDNA into a colon cancer cell line, HCT116, resulted in a marked reduction in proliferation in monolayer and colony formation in soft agar compared with the vector-transfected controls. In another colon cancer cell line, Caco-2, with a tetracycline-regulated promoter system, induction of ERRP expression in the absence of doxycycline was associated with a marked reduction in EGFR activation and proliferation. We conclude that the ERRP cDNA may represent a new member of the EGFR gene family and that ERRP plays a role in regulating cell proliferation by modulating the function of EGFR.

Transepithelial resistance can be regulated by the intestinal brush-border Na(+)/H(+) exchanger NHE3.

Initiation of intestinal Na(+)-glucose cotransport results in transient cell swelling and sustained increases in tight junction permeability. Since Na(+)/H(+) exchange has been implicated in volume regulation after physiological cell swelling, we hypothesized that Na(+)/H(+) exchange might also be required for Na(+)-glucose cotransport-dependent tight junction regulation. In Caco-2 monolayers with active Na(+)-glucose cotransport, inhibition of Na(+)/H(+) exchange with 200 microM 5-(N,N-dimethyl)- amiloride induced 36 +/- 2% increases in transepithelial resistance (TER). Evaluation using multiple Na(+)/H(+) exchange inhibitors showed that inhibition of the Na(+)/H(+) exchanger 3 (NHE3) isoform was most closely related to TER increases. TER increases due to NHE3 inhibition were related to cytoplasmic acidification because cytoplasmic alkalinization with 5 mM NH(4)Cl prevented both cytoplasmic acidification and TER increases. However, NHE3 inhibition did not affect TER when Na(+)-glucose cotransport was inhibited. Myosin II regulatory light chain (MLC) phosphorylation decreased up to 43 +/- 5% after inhibition of Na(+)/H(+) exchange, similar to previous studies that associate decreased MLC phosphorylation with increased TER after inhibition of Na(+)-glucose cotransport. However, NHE3 inhibitors did not diminish Na(+)-glucose cotransport. These data demonstrate that inhibition of NHE3 results in decreased MLC phosphorylation and increased TER and suggest that NHE3 may participate in the signaling pathway of Na(+)-glucose cotransport-dependent tight junction regulation.

PKC-dependent regulation of transepithelial resistance: roles of MLC and MLC kinase.

The mechanisms by which protein kinase C (PKC) activation results in increased transepithelial resistance (TER) are unknown [G. Hecht, B. Robinson, and A. Koutsouris. Am. J. Physiol. 266 (Gastrointest. Liver Physiol. 29): G214-G221, 1994]. We have previously shown that phosphorylation of the regulatory light chain of myosin II (MLC) is associated with decreases in TER and have suggested that contraction of the perijunctional actomyosin ring (PAMR) increases tight junction (TJ) permeability [J. R. Turner, B. K. Rill, S. L. Carlson, D. Carnes, R. Kerner, R. J. Mrsny, and J. L. Madara. Am. J. Physiol. 273 (Cell Physiol. 42): C1378-C1385, 1997]. We therefore hypothesized that PKC activation alters TER via relaxation of the PAMR. Activation of PKC by the phorbol ester phorbol 12-myristate 13-acetate (PMA) resulted in a progressive dose-dependent increase in TER that was apparent within 15 min (111% of controls) and maximal within 2 h (142% of controls). Similar increases were induced by a diacylglycerol analog, and the effects of both PMA and the diacylglycerol analog were prevented by the PKC inhibitor bisindolylmaleimide I. PMA treatment caused progressive decreases in MLC phosphorylation, by 12% at 15 min and 41% at 2 h. Phosphorylation of MLC kinase (MLCK) increased by 64% within 15 min of PMA treatment and was stable over 2 h (51% greater than that of controls). Thus increases in MLCK phosphorylation preceded decreases in MLC phosphorylation. These data suggest that PKC regulates TER via decreased phosphorylation of MLC, possibly due to inhibitory phosphorylation of MLCK. The decreased phosphorylation of MLC likely reduces PAMR tension, leading to decreased TJ permeability.