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• BASOLATERAL MEMBRANE LOCALIZATION OF THE AE2 ISOFORM OF THE ANION EXCHANGER FAMILY IN BOTH STOMACH AND ILEUM. H. Rossmann. M Nader, U. Seidler, M. Classen, S. Alper$. I1. Med. Klinik der TU Mfinchen and $Molecular Medicine and Renal Units, Beth Israel Hospital, Boston.

Baekround: AE2 cDNA and cDNA fragments have been cloned from rabbit ileum and rabbit parietal cells, and Northern blot and PCR analysis have demonstrated the expression of AE2 mRNA in ileal enterooytes and parietal cells (Chow et al, Am.J. Physiol 263:G345-352, 1992, and Rossmann et al, Pfl/Jgers Archives, 420:71, 1993). Isotope flux studies in rabbit parietal cell basolataral (BLM) membranes (Lamprecht et al, Am J. Physiol. 265:G903- 910,1993) and a specific antibody against AE2 in rat stomach have localized tl-t~ anion exchanger to the basolateral membrane (Stuart-Titley et al, Am. J. Physiol. 266:C559-568, 1994). In the ileum, a well-characterized anion exchanger is, located on the brush border membrane (BBM) and is suggested to represent the AE2 gene product (Chow et al, Am: J. Physiol 263:G345-352, 1992). However, we have recently found that effect of pH i and osmolarity on maximal anion exchange rates is completely different between the parietal cell basolateral and ileal brush border anion exchanger (Nader et al, J. Physiol. 481:605-615). Aim: Using an antibody directed against the AE2 C-terminus, ~ we studied the membrane localization of AE2 in rabbit ileal and gastric BBM and BLM membranes. We then performed isotope flux studies to verify the presence of anion exchange activity in the respective membrane vesicles. Methods and Results: Western blot analysis revealed a strong 163-170 kD band and a second broad band at 130-150 kD (deglycosylated forms) in rat and rabbit whole stomach lysate and muc h weaker bands in the same location in rat and rabbit ileum. The same bands were detected in BLM vesicles from rabbit stomach and ileum, where they appeared strongly enriched compared the whole organ lysate. A very weak band of the respective size was detected in stomach apical membranes (cross-contamination from BLM) and no band n that size range was detected in ileal BBM membranes, DIDS-sensitive 36CI-

/CI- exchange was present in gastric BLM and ileal BBM and also in ileal BLM vesicles, and absent in gastric apical membrane vesicles. The 36CI-/CI- up- take into ileal BLM vesicles had a faster time course than that into ileal BBM membrane vesicles but similar to that in gastric BLM vesicles and is unlikely to represent cross-contamination. Conclusions: AE2 is very strongly expressed in parietal cells, is localized on the basolateral membrane, and likely repre- sents the well-characterized parietal cell anion exchanger. On the other hand, this isoform is only weakly expressed in the ileum, and appears to be also lo- cated on the basolateral membrane. This suggests that the well-characterized ileal brush border anion exchanger may be an as yet uncloned isoform.

• BUTYRATE-INDUCED REGULATION OF INTRACELLULAR pH AND VOLUME IN COLONOCYTES IS NOT ALTERED BY CHRONIC EXPOSURE TO BUTYRATE. William A. Rowe. Stephen M. Bobar, Rohini Polavarapu. Department of Medicine, The Pennsylvania State University College of Medicine / The Milton S. Hershey Medical Center, Hershey, PA.

The short chain fatty acids are bacterial waste products found throughout the colon. They have been found to impact on colonocytes as a nutrient source and appear to be involved in transcellular transport, likely through a mechanism involving intracellular pH regulation. Upon exposure to the short chain fatty acids (including butyrate), colonocytes acidify and swell, then recover intracellular pH and volume back toward normal, with the initial component of pH recovery mediated by Na+/H + exchange. A human colonocyte cell line (HT29-18-cl) was grown in the presence artd absence of butyrate to determine whether chronic exposure to low and moderate levels of butyrate altered pH and volume regulation upon exposure to high concentrations of butryate.

Methods: Cells were cultured in Dulbecco's Modified Eagle Medium with transferrin and 10% fetal bovine serum. Medium was then changed with increasing concentrations of butyrate (up to 5 raM), or allowed to grow to near confluence in DMEM, with medium change to DME base with 25 rnM butyrate substituting for glucose. Cell protein synthesis was measured by 3H-leucine incorporation. Isolated cells were washed with an isosmotic Ringer medium at 37°C and pH 7.4, then cell volume was measured (Coulter Counter) and intracellular pH was measured in cells loaded with 2',7'-bis-(2-carboxycthyl)-5- (and-6)carboxyflourescein [BCECF] by ratio of dual wavelength excitation.

Results: The presence of butyrate (or propionate) in the growth media had no effect on protein synthesis when compared with controls. Cells maintained in 25 mM butyrate-containing growth medium showed a mean cell volume of 1270 -+ 33 femtuliters (compared with 1281 + 16 fi for controls); butyrate-induced swelling was similar for both groups (146 _+38 v. 138 _+ 18 fl/min), as was peak volume (1400 _+ 25 v. 1397 _+ 23 fi) and the rate of regulatory volume decrease after butyrate exposure (36 + 7 v. 33 _+ 6 fi/min)[all btuyrate-pretreated v. control, p=NS]. In addition, studies of intracellular pH revealed similar resting pH values (7.00 _+ .05 v. 7.03 _+ .02), similar pH minima after butyrate exposure (6.10 _+. 18 v. 6.16 _+ .03) and similar initial rates of intracellular pH recovery after butyrate exposure (1.43 -+ .09 v. 1.89 _+ .88 x 1016 protons/sec)[all butyrare-pretreated v. control, p=NS].

Conclusions: I) Chronic exposure to moderate doses of butyrate does not appear to alter butyrate-induced intracellular pH or volume regulation in HT29- 18-cl colonocytes.

Support." NIH DK02069;Glaxo Institute for Digestive Health Basic Research Award.

• INTRACELLULAR pH OF COLONOCYTES IS ALTERED IN PATIENTS WITH CROHN'S DISEASE. William A. Rowe, Stephen M. Bobar. Department of Medicine, The Pennsylvania State University College of Medicine / The Milton S. Hershey Medical Center, Hershey, PA.

The short chain fatty acids are bacterial waste products found throughout the cofun~ They have been found to impact on colonocytes as a nutrient source and appear to be involved in transcellular transport, likely through a mechanism involving intracellular pH regulation. Upon exposure to the short chain fatty acids (including butyrate), coionocytes acidify, then recover intracellular pH back toward normal, with the initial component of pH recovery mediated by Na+/H + exchange. Butyrare enemas appear to be effective in the treatment of ulcerative colitis, but not Crohn's colitis, so patients with Crohn's disease were studied to see if the intracellular pH response to butyrate exposure was altered.

Methods: Colonic tissue was obtained from Crohn's disease patients undergoing colectomy. Patients with distant malignancy and constipation were used as controls. Colonocytes were isolated from normal colon, colon with inactive Crohn's disease, and colon with active Crohn's disease by treatment with Dispase and col!agenase type IV. Isolated cells were washed with an isosmotic Ringer medium at 37°C and pH 7.4, then intracellular pH was measured in cells loaded with 2',7'-bis-(2-carboxyethyl)-5-(and- 6)carboxyflourescein [BCECF] by ratio of dual wavelength excitation.

Results: There were marked differences between the baseline intracellular pH from patients with Crohn's disease cbmpared with the intracellular pH from patients with normal colonic mucosa. The baseline pH of patients with Crohn's disease was significantly lower (intracellular pH 6.68 + 0.06) compared with normals (intracellular pH 7.05 _+ 0.03, p=0.013). This difference was present whether the samples were obtained from areas with active Crohn's colitis or inactive colonic disease. The initial rate of intracellular pH recovery after exposure to 130 mM sodium butyrate was determined. The cells reached similar pH minima after exposure (intracellular pH 5.90 -+ .23 [Crohn's] v. pH 5.91 + .14 [control], p=NS). Similarly, the initial rates ofpH recovery were similar between the two groups (2.79 + 1.77 [Crohn's] v. 4.65 + 2.39 [control] xl016protons extruded/second, p=NS). There was no significant difference in either the pH minima or the pH recovery rate between the active and inactive Crohn's disease groups.

Conclusions: 1) Resting colonocyte intracetlular pH is markedly decreased in patients with Crohn's disease. Inflammation does not appear to play a role in this decrease as patients with both active and inactive Crohn's disease had similar baseline acidification. 2) The response to butyrate exposure in patients with Crohn's disease is not altered; the same pH minima is reached and the same initial rate of pH recovery is seen when compared with controls, indicating that some level of Na+/H + exchange remains intact. 3) Alterations in basal intracellular pH regulation may play a role in the etiology of Crohn's disease.

Support: Glaxo Inst. for Digestive Health Basic Res. Award; NIH DK02069.

ANALYSIS OF ENDOTHELIAL DYSFUNCTION IN MESENTERIC AND ILIAC ARTERIES AFTER PROLONGED HYPERCHOLESTEROLEMIA IN VIVO IN RABBITS. Z. Rozsa J. Pararicza, J. Nemeth*, J. Gy. Papp. Albert Szent-Gyorgyi Medical University, Department of Pharmacology and First Department of Medicine*, Szeged, Hungary

Endothelial dysfunction and abnormal vascular responsiveness appears to con- tribute to the pathophysiology of atherosclerosis. Objective: The effects of hy- percholesterolemia on the endothelium-dependent and independent vascular reac- tivity of mesenteric (SMA) and lilac (RCIA) arteries were examined in anes- thetized rabbits. We assessed the contribution of endothelin to the abnormal vas- cular reponsiveness in hypercholesterolemia. Methods: Rabbits were fed either standard or a cholesterol-enriched diet for 24 wks. Plasma lipids and the changes of the plasma and vascular tissue contents of endothelth 1/2 were detected. The functional severity of atherosclerosis was confirmed by examining vascular re- sponses in the isolated thoracic aorta and from morphological changes in the en- dothelial cells analysed by scanning electron microscope. The SMA and RCIA blood flow (BF) were measured by transit-time flowmetry and drugs were in- jected through an intra-abdominal aortic catheter . Results: The morphologic le- sions of endothelium in the thoracic aorta correlated positively with the total cholesterol exposure, in contrast, no major abnormalities were found in the en- dothelium of the SMA. Acetylcholine (ACh), (5, 10, 20 #g.kg -t) elicited dose- dependent vasodilation in both vascular beds but in hypercholesterolemic animals the response to ACh was completely diminished and a high dose produced para- doxical vasoconstriction. The plasma and the vascular tissue endothelin 1/2 levels were significantly elevated in hypemholesterolemic animals. Cromakalim at a dose Of 3/~g.kg -1, which was found to be potent relaxant of the SMA and RCIA in control animals, exerted similar vasodilation in these blood vessels in hypercholesterolemic animals. Conclusions: 1) the endothelium-dependent re- sponses of SMA and RCIA to ACh are functionally impaired by hypercholes- terolemia 2) this altered vascular reactivity correlates with the elevation of the circulating and of the vascular tissues endothelin levels, thereby endothelin may contribute to the impaired endothelium-dependent relaxation of these blood ves- sels. 3) in hypercholesterolemia the dilation of these arteries in response to K + channel opener cromakalim is entirely preserved, suggesting that KAT p channel opener may be of potential interest for a new therapeutic approach to peripheral atherosclerosis.