A340 AGA ABSTRACTS GASTROENTEROLOGY, Vol. 108, NO. 4

E N T E R O P A T H O G E N I C E. C O L I I N F E C T I O N OF Ts4 MONOLAYERS ATTENUATES Ca2+- AND cAMP-MEDIATED CHLORIDE SECRETION. R. Yuhan A. Koutsouris, (3. Hecht. Departments of Medicine (GI) and Surgery, University of IL, Chicago, IL.

The mechanism by which enteropathogenic E. coli (EPEC) induces diarrhea is unknown. It has been hypothesized that EPEC infection may induce secretory diarrhea. This speculation is certainly plausible in view of the fact that EPEC infection significantly increases intraecllular calcium (Ca2+), a known mechanism by which CI- secretion is stimulated. To test this hypothesis, T84 monolayers were infected with EPEC strain E2348/69 and short circuit current (Isc) was measured hourly between 1- 6 hrs post- infection and after 24 hrs. No increase in baseline Isc was seen at any time point. The impact of EPEC infection on Ca2+ stimulated CI: Secretion was then investigated. T84 monolayers were infected with EPEC for 6 hrs then challenged with carbachol (10pM) and peak Isc was compared to that from control monolayers. Paradoxically, CI- secretion was attenuated by EPEC infection (58-~4 vs 33 +8 pAmp/cm2, control and EPEC; n=9, p<0.05). The response of EPEC-infected monolayers to cAMP-mediated CI- secretion was also examined by comparing the peak Ise responses to forskolin (1/zM). Interestingly, cAMP-mediated secretion was also diminished (78+4 vs 47+2 uAmp/cm2, control vs EPEC; n=14, p<0.05). Time course studies revealed that attenuation of CI- secretion occurred as early as lhr for Ca2+-mediated events but not until 6 hrs for cAMP-stimulated. To determine if a soluble factor might be responsible for the attenuation of CI- secretion, bacterial culture supernatant was filter-sterilized and incubated with T84 monolayers for 6 hrs. Stimulation with forskolin elicited identical responses from control and culture supernatant-treated monolayers (138+--6 vs 139+12 ,uAmp/cm2, respectively; n=3,p>0.05). A similar synergistic response to carbachol was also seen (285+_6 vs 290+3 ,uAmp/cm2, respectively; n=3, p>0.05). We conclude that EPEC attachment attenuates the ability of host cells to secrete CI- in response to both Ca2+ and .cAMP. We speculate that although these negative findings do not account for EPEC-induced diarrhea, they in fact represent a pathogenic factor. The flushing action of secretory diarrhea is a host defense and the turning off of Such a response would allow increased numbers of organisms to colonize the intestinal epithelium and exert additional effects which ultimately result in diarrhea. (Supported by NIDDK.)

• N- AND C-TERMINAL CHIMERAS OF Na+IH ÷ EXCHANGERS NHEI, NHE2 AND NHE3: AN EPITHELIAL N-TERMINAL DOMAIN REQUIRES AN EPITHELIAL C-TERMINAL DOMAIN FOR REGULATION BY PHORBOL MYRISTATE ACETATE (PMA) AND FIBROBLAST GROWTH FACTOR (FGF). C.H.C. Yun, C.M. Tse and M. Donowitz. GI Unit, Depts of Med and Physiol, Johns Hopkins Univ Sch of Med, Baltimore, MD.

The Na*/H ÷ exchanger (NHE) isoform, NHEI, is ubiquitously expressed whereas NHE2 and NHE3 are epithelial specific; end both are intestinal brush border NHEs. These NHEs consist of two functionally distinct domains: membrane bound N-terminus, which exchanges Na and H, and cytoplasmic C-terminus, which is required for kinase-induced regulation. Despite their similarity in structures and kinetic characteristics, the NHEs exhibit different kinetic mechanisms in response to growth factor regulation. For instance, growth factors stimulate NHE1 by a change in K'(H*); and regulate NHE2 and NHE3 by a change in V ~ . To determine 1) which domain, the transmembrane N-terminus or cytoplasmic C-terminus, plays the critical role in determining the above V,~-vs-K' effect of the Na÷/H~ isoforms, and 2) if the kinase regulation of the exchangers can be modified by swapping the cytoplasmic C-termini, we have constructed chimeric Na*/H+ exchangers by exchanging the N- and C-termini among the cloned rabbit NaVH ÷ exchangers-NHE1, NHE2 and NHE3. The cDNAs encoding each domain were amplified by PCR and chimeric NHEs were constructed by ligating the six possible pairings of the N- and C-termini. These chimeric exchangers were stably expressed in PS120 fibroblast cell line, which lacks aii endogenous Na*/H* exchange, and their activities were studied fiuorometrically using BCECF, under basal conditions and after exposure to FGF, PMA and serum. All the chimeras had functional NHE activity and showed kinetic properties similar to wild-type exchangers with non- Michaelis-Menten kinetics in response to intracellular H with a Hill coefficient of 2-3. Growth factor exposure showed that 1) the membrane-bound N- terminus is responsible for the Vma x vs K' effect of serum on the Na*/H * exchanger isoforms, and 2) PMA and FGF altered Na÷/H * exchange only in chimeras that had an epithelial N-terminal domain and an epithelial C- terminal domain. In conclusion, the kinase-induced regulation of Na÷/H * exchangers is mediated through a specific interaction between the N- and C-termini, which is restricted based on properties of both the N- and C- termini, such that an epithelial N-terminus can only communicate with an epithelial C-terminus.

TRANSCRIIrFIONAL REGULATION OF ANION EXCHANGER 2 IN CELL LINES OF DIFFERENT ORIGINS. W. Zhou and A. Chow. Emory University School of Medicine, Atlanta VA Medical Center, Atlanta, GA 30322.

]~ii.¢,kgr.0.11ad: Members of Anion Exchanger (AE) gene family encode Na+-independent chloride-bicarbonate exchangers. AE1, AE2 and AE3 mRNAs are differentially expressed in different tissues. AE2 mRNA is the major form expressed in intestinal epithelial cells. We hypothesized that tissue distribution of AE2 may be regulated at transcriptional level.

Methods: IEC-6 (rat small intestine epithelial cell line) and GH3 (a rat pituitary tumor cell line) were cultured under recommended conditions. Ribonuclease protection assays were used to compare AE2 mRNA levels. An AE2 eDNA probe corresponding to the cytoplasmic domain was used to generate the in vitro transcript for ribonuclease pruteetion assays. Relative AE2 transcription rates were determined by nuclear run-on assays. 2x 107 cpm of run-on transcription reactions from IEC-6 or GH3 was used for hybridization to linearized plasmid clones containing eDNA of 18S rRNA, AE2 and the vector itself.

Results: Ribonuclease protection assays demonstrated concentration- dependent increase of AE2 signal in IEC-6 ceils. The AE2 signal in IEC-6 ceils was at least 3 fold stronger than in GH3. Signals in nuclear run-on assays were measured by densitometry. Relative to 18S, AE2 signals were approximately 4 fold higher in IEC-6 than GH3 cells. The higher level of AE2 Wancription in IEC-6 may be due to the presence of enhancing activities or the absence of silencers.

Because earlier studies had demonstrated that thyroid hormone decreased AE2 mRNA in suckling rats, we tested if thyroid hormone can confer silencing effect on AE2 transcription in IEC-6 cells. Treatments of IEC-6 cells with 10 nM of triiodothyronine or 1 uM of L-thyroxine did not result in changes in AE2 mRNA level even though IEC-6 cells were found to display nuclear thyroxine-binding activities,

Summary: We demonstrated that the difference in AE2 mRNA levels in IEC-6 and GH3 cell lines are at least in part due to transcriptional differences. We postulate that tissue-specific factors may play a role in regulating tissue distribution of AE isoforms.