Endothelin-1 causes P-selectin dependent leukocyte rolling and adhesion within the rat mesenteric microvessels

April 1998 non-diabetic rats, leukocyte adhesion and emigration 30 min after reperfusion was completely abrogated by Spermine NONOate treatment. From these results we conclude that the diabetic state induces an enhanced inflammatory response to I/R that can be abrogated by treatment with NO donors. This effect is at least in part related to blockade of P-selectin upregulation during reperfusion. • G1683 PHYSICAL INTERACTION BETWEEN COLON-SPECIFIC ct AND 13 SUBUNITS OF H, K-ATPASE IN COLONIC APICAL MEMBRANE. P. Sangao, V.M. Rajeudran, M. Kashgarian and H.J Binder. Departments of Internal Medicine and Pathology, Yale University, New Haven, CT 06520. An apical membrane H,K-ATPase, a member of the P-type ATPase gene family, regulates active K absorption which is enhanced by aldosterone (aldo) and K depletion in rat distal colon. These ATPases are usually heterodimers consisting of ct and [3 subunits. Although an cc subunit (HKcc0 has been cloned and characterized, uncertainty exists whether our recently cloned [3 subunit (HKc13) is the required [3 subunit for colonic H,K-ATPase. HKc13 mRNA is expressed in several tissues, but K depletion upregulates HKc[3 mRNA only in distal colon not in proximal colon, lung or testis, tissues with highest expression. The present study sought to identify the membrane specific localization of HKc[3 protein. Western blot analysis using a polyclonal antibody produced to amino acids 87-142 of HKc13 protein identified a 42 KDa protein in both apical and basolateral membranes of distal colon. In contrast, HKccx protein is expressed exclusively in apical membrane of distal colon. NaK[31 protein was expressed solely in basolateral membrane excluding its possible role as the 13 subunit for HKcct. HKcct antibody incubated with apical membrane proteins co-immunoprecipitated the HKc13 protein that was detected by Western blot analysis using HKc13 antibody, establishing that the colonic HKcct physically interacts with HKc13 protein. The tissue-specific upregulation of HKc[3 subunit mRNA in response to K depletion, the localization of its protein in apical membrane of distal colon and its physical association with HKcot protein provide compelling evidence that HKc13 is the J3 subunit of H, K-ATPase in rat distal colon. • G1684 PERIPHERAL CORTICOTROPIN RELEASING-HORMONE INCREASES COLONIC PERMEABILITY AND IONIC SECRETION THROUGH NICOTINIC PATHWAYS AND MAST CELLS. J Santos, PR Sannders, D Yates, M Benjamin and MH Perdue. Intestinal Disease Research Program, McMaster University, Hamilton, Ontario, Canada. Clinical and experimental evidence links stress with colonic disorders present in inflammatory as well as functional bowel diseases. Corticotropin releasinghormone (CRF) is considered to he a key effector hormone of stress. Although central and peripheral effects of CRF on colonic motility are well known, the effects of CRF on epithelial physiology have not been established. Methods: Male WKY rats were exposed to restraint stress (RS) for 2 h at 8 °C followed by 2 h rest in home cage at 22 °C or injected ip with CRF (10 ~g) or saline. At the end of the test period (4h), under urethane anesthesia, distal colonic segments were excised, stripped and immediately mounted in Ussing Chambers. Short-circuit current (Isc)(ion transport), conductance (ion permeability) and mucosal-to-serosal fluxes of horseradish peroxidase (HRP) (macromolecular permeability), were measured. To test the pathways involved in CRF-induced changes, ganglionic blockade with hexamethonium (15 mg/kg), the mast-cell stabilizer, doxantrazole (20 mg/kg) or the CRF antagonist, ct-helical CRF 9-41 (50~g), were administered ip 30 min prior to CRF, in different groups of rats. Results: Both RS and peripheral CRF induced similar increases in Isc, conductance and HRP fluxes. The effects of RS and CRF on colonic tissues were blocked .by pretreatment of rats with a-helical CRF. Colonic responses to CRF were reduced or blocked in rats pretreated with hexamethonium or doxantrazole. Isc Conductance HRP flux (gA/cm2) (mS/era2) (pmol/h/cm2) Control 46.6 .+ 5.4 16.8 -+ 3.7 5.8 ,+ 1 RS 98.67 ,+ 8.9a 22.4 .+ 2.9a 16.6 .+ 3.4a CRF 87.4-+ 12.1a 26.9_+ 5.2a 14.2+ 3.3 a RS+ ~xhel CRF 61.2 _ 6.9b 19.3 _+3.2 6.3 _+3.3b CRF + tz hel CRF 54.8 _+7.7c 18.8 .+ 1.5c 3.3 _+0.5e CRF + Hex 48.8 .+ 2.6c 26 ,+ 3 5.9 -+ 1.8c CRF + Dox 60.4 _+7.9c 20.4 _+2.4c 7.5 -+4.7c n = 12-24 tissues,3-6 rats, a, (P < 0.05 vs control);b, (P < 0.05 vs RS); c, (P < 0.05 vs CRF) Conclusion: Peripheral CRF mimicks restraint stress-induced changes in colonic epithelial physiology. CRF-stimulated colonic secretion and permeability involve nicotinic pathways and mast cells. Modulation of stress responses in the gastrointestinal tract may be relevant to the management of colonic functional and inflammatory disorders.

Intestinal Disorders A413 • G1685 ENDOTHELIN-1 CAUSES P-SELECTIN DEPENDENT LEUKOCYTE ROLLING AND ADHESION WITHIN THE RAT MESENTERIC MICROVESSELS. M.J. Sanz, B. Johnston and P. Kubes, Immunology Research Group, University of Calgary, Calgary, Alberta, Canada, T2N 4N1 Endothelin-i (ET-1) is a 21-amino acid peptide with a potent vasoconstrictor activity. High plasma levels of ET-1 have been found in several pathological diseases of the intestine where increased adhesion of leukocytes to endothelium have also been observed. However, to date little is known regarding the mechanism of ET-1 induced leukocyte-endothelial cell interactions. The aim of the present study was to systematically assess the role of ET-1 on leukocyte recruitment in vivo. For this purpose intravital microscopy within the rat mesentery posteapillary venules has been used. Very brief and small amounts (10 -9 M) ET-1 superfusion (no effect on blood pressure) caused a significant increase in leukocyte rolling and adhesion. Surprisingly, the cells were restricted to the vessel wall and never emigrated. In addition, whilst arteriolar diameter was significantly reduced venular diameter remained unchanged. Previous work suggested that ET-1 may cause mast cell degranulation to induce adhesion however reduced flow was also noted. In this study, these effects were found not to be mediated by mast cell activation since ET-1 did not activate mast cells using ruthenium red a new on line mast cell activation detection system. Pretreatment of the animals with mast cell stabilizers did not result in changes in ET-l-induced effects. An anti-rat P-selectin (RMP-I) or control (RP-2) mAbs (2.5 mg/kg) dramatically reduced leukocyte rolling and adhesion (Table).

RP-2 + ET-1 RMP-1 + ET-1 * p < 0.05 Student's test

Rolling flux cells/min 63.2 ,+ 16.1 8.0 .+ 3.2*

Adhesion / 100pro length 13.8 ,+ 3.1 4.7 _+0.4*

In conclusion, this study demonstrates for the first time that ET-1 can cause leukocyte rolling and adhesion via rapid P-selectin expression but does not function as a chemoattractant (no emigration) restricting cells to the vessel wall. ET-1 may be an early inducer of leukocyte rolling and adhesion in intestinal disease associated with leukocyte recruitment. Sponsored by the Crohn's and Colitis Foundation of Canada G1686 EFFECT OF SPECIFIC PRE-INDUCTION OF HSP60 ON ACETIC ACID-INDUCED SMALL INTESTINAL LESIONS. H.Sasahara, M. Otaka, A. Okuyama, S.Itoh, MJin, S.Otani, A. Iwabuchi, M. Odashima, I. Pacheco, O. Masamune. Department of Internal Medicine-l, Akita University School of Medicine, AKITA, JAPAN. [Background] Some heat shock proteins (HSPs) are known to he involved in cytoprotection against environmental stresses mediated by their function as molecular chaperons. In the small intestinal mucosa, 72-kDa heat shock protein (HSP72) has been reported to have cytoprotective function against ischemia/reperfusion-induced mucosal damage. Previously, we have reported that the synthesis of HSP60 (chaperonin homologue) was specifically induced by water-immersion stress or thyrotropin releasing hormone (TRH) administration in rat small intestinal mucosa without histopathologic alteration. In this study, the influence of specific pre-induction of mucosal HSP60, which is known as an internal cytoprotectant in the pancreatic acinar cells, by TRH administration on acetic acid-induced small intestinal lesion was investigated. [Materials] 15-25-week old male Sprague-Dawley rats were used in this experiment. [Methods] Mucosal lesion was developed by perfusing 5% acetic acid for 15 rain into proximal small intestine (20 cm in length). The effects of preinduction of HSP60 by TRH administration on acetic acid-induced mucosal lesion were evaluated by measuring mucosal height microscopically. [Results] (1) Expression of HSP60 was specifically and dose-dependently increased after TRH administration in rat small intestinal mucosa without any pathologic alteration. (2) Pre-induction of HSP60 by TRH administration had no preventive effect against acetic acid-induced small intestinal lesion. [Conclusions] Our findings might suggest that HSP60 has no cytoprotective function against acetic acid-induced mucosal damage in the small intestine, and that the function of HSP60 might be different from that of HSP72 with respect to cytoprotection.