April 1995 Intestinal Disorders A271

• THE ROLE OF SOMATOSTATIN IN HUMAN COLONIC ION TRANSPORT M. Am, I. Ross, R. Lfibeke, M. Grigor, G. Warhurst & G.O. Barbezat. Departments of Medicine & Biochemistry, University of Otago Medical School, Dunedin, New Zealand and Department of Medicine, University of Manchester, UK. Aims: In cultured human neoplastic colonic crypt cells (HT29) and in colon prepa- rations of rodents somatostatin (SST), and its long acting analogue octreotide (OCT), have been shown to inhibit secretion. To date no data are available about the effect on normal human tissue in vitro. We established an in vitro model of secretory diarrhoea using the cAMP agodist prostaglandin E 2 (PGE2) to measure the effect of SST and OCT on normal human colon. Methods: Secretion was assessed by changes in short-circuit current (Also across human or rat colonic tissue prepara- tions, mounted in modified Ussing chambers. Human tissues (n=109 from 19 indi- viduals) were obtained from patients undergoing colon surgery for benign or malig- nant pathologies. Tissues were bathed in mammalian Ringer's plus indomethacin, stimulated by human EC50 PGE 2 (3x10"7mol/l) prior to adding SST or OCT. Bu- mete'de (10"Stool/l) served as phammcological probe to help c ~ r i z e chloride (C1) secretion. Results: A sustained increase of lsc (pAmp cm "z) above baseline current was achieved with 60 PGE 2 (Also [mean~SEM]: human: 83,2--~:3,0; rat: 40 35,7±2,7; equivalent to 100% secretion) serving as a model for defined C1 seem- ~ 2o tion. Addition of bumetanide ~" 0 abolished Also by 65% in _~- both human and rat. In rats ~ -20 (n=36 preparations from 5 animals) SST (~10"7mol/l) < -40 and OCT (>~10"~mol/1) in- hibited secretion by 100% -60 (EC50 20nmol/l and 2nmogl

mean ± SEM human n = 12

n = 4-6

1,0OE- eo 1,00E-09 1,00E-tIS 1,00E-07 1,00E46

respectively). In contrast, i n SST/Oet Imolal humans SST (>/10-6mol/l) and OCT (>/10-6reel/l) increased Alse by a maximum of 23% (ECs0: 10nmol/l for both). Conclusions: In humans and rats Alsc induced by PGE 2 represents active Cl--seeretion. Different sensitivity of Also to SST and OCT in rats and hunmns indicate activation of different SST receptors (SSTR's) possibly linked to different effeetor pathways with different effects on secretion. Our in vitro model does not provide evidence for an antiseeretory effect of SST or OCT in hu- man PGE 2 stimulated colonic tissue and does not support the use of SST or OCT as anti-secretagogue in the human colon. Characterization of the SSTR's, the coupling to second messenger (G proteins) and the activation of secretory pathways (cAMP or Ca 2+) is under investigation.

• INCREASED SMALL BOWEL EPITHELIAL APOPTOSIS REFLECTS CELIAC SPRUE ACTIVITY AL ARia, SF Moss, JRF Waiters, S Wang, PR Holt. St. Luke's- Roosevelt Hospital Center /Columbia University, New York, NY & Gastro Unit, Royal Postgraduate Medical School, London, UK.

Celiac Sprue (CS) is characterized by small intestinal mucosal damage and rapid epithelial cell turnover which improves with a gluten-free diet (GFD). Apoptosis, programmed cell death, is a feature of normal enterocyte turnover. We measured apoptosis in 35 small bowel biopsies from 27 adult patients with CS, 16 with active disease and 11 after at least 3 months on GFD, including 5 patients with biopsies both before and after GFD. Methods: Apoptotic cells were identified in situ by staining fragmented DNA, using terminal deoxynucleotidyl transferase and digoxigenin- labeled dUTP. Specimens were compared with H&E stained adjacent sections. Enterocyte apoptosis was graded on a scale of 1 + ( < 5 % positive cells), 2 + (between 5-20%), and 3 + (greater than 20%) per crypt-villus unit. Results: Apoptotic cells were not seen with H&E but were readily visible by in situ end-labelling histochemistry. In active CS, apoptotic cells were located diffusely throughout the crypt and blunted villi whereas in treated CS, 47% of apoptotic enterocytes were concentrated at the villus tip, and 21% in the basal half of the crypt - this pattern was similar to controls, even in those treated CS cases which were not histologically normal. The median apoptotic grade in active CS was 3 + ( > 20%) compared with 1 + (0.5 %) in the patients on GFD (p < 0.001). In the patients with paired biopsies, the median apoptotic grade fell from 3 + to 1+ on GFD (p< 0.05). Conclusion: Enterocyte apoptosis is greatly increased in untreated celiac sprue and falls to normal with GFD, prior to complete histological improvement. Increased programmed cell death may protect the intestine from genetic damage induced by the hyperproliferati0n and/or immunologic or toxic injury in sprue.

ROLE OF NITRIC OXIDE IN RADIATION-INDUCED ALTERATIONS IN ELECTROLYTE TRANSPORT IN THE RAT COLON IN VITRO, A.R. Aurora and W.K. MaeNanghton, Defense Research Establishment Ottawa and Digestive Diseases Research Group, Department of Physiology, University of Ottawa, Ottawa, Canada

Abdominopelvie radiation often predisposes patients to acute diarrhea which may occur due to stimulation of an inflammatory response in the gut and/or alterations in mechanisms regulating intestinal electrolyte transport. Nitric oxide is thought to be one of the potential mediators of bot h intestinal inflammation and electrolyte transpor t. In this study male Sprague-Dawley rats Were injected with either the non-specific NOS inhibitor, L-NAME (100 mg/kg s.e.) or with phy~siologieal saline (1 ml/kg s.c.), one hour prior to whole body, 10 Gy "/-radiation froin s ~37Cs source. Shams were placed in the irradiator but Were not exposed to the source. At 2, 24 and 48 h post-irradiation, segments of colon were removed, sthpped of the external muscle layers and placed in standard Ussing flux chambers. The short circuit current (Ise) was measured as the indicator of active ion transport. After a stabilization period of 15 rain., ~ thg~ Ise responses to electrical field stimulation (EFS) of 1, 2.5, 5, 10 and 25 Hz or to 0.1pM PGE 2 were recorded. Irradiation resulted in a'signifieant decrease in the Ise response to EFS at 24 and 48 h post-irradiation in saline pretreated rats. However, the response to PGE 2 was only significantly decreased at 24 h post-irradiation in saline pretreated rats. L- NAME pretreatment decreased the response to EFS in shams when compared to saline pretreated rats although this decrease on13, reached significance at the EFS frequency of l0 Hz. Furthermore, L-NAME pretreatment appeared to accelerate the effects of radiation by significantly decreasing the lse response to EFS as early as 2 h post-irradiation i n L-NAME pretreated rats. L-NAME pretreatment did not affect the radiation-induced depression in EFS-evoked transport at 24 and 48 h. In L-NAME pretreated rats, the response to PGE~ was significantly decreased at 48 h. The ~C-L-arginine to ~4C-L-eitrulline conversion assay confirmed that L- NAME pretreatment virtually abolished colonic NOS activity at 2 and 24 h but returned to basal levels by 48 h. Because mast cell-nerve interactions regulate electrolyte transport, segments of eohin from all groups were fixed and stained for determination of mast cells. Mast cells decreased 24 and 48 h post-irradiation, but this was not affected by L-NAME pretreatment. These results imply that NO plays an important role in modulating colonic transport function in normal tissue, but that this relationship is uncoupled by 24 or 48 h post-irradiation. The effects of NOS inhibition on neurally--evoked electrolyte transport are unrelated to changes in mast cell numbers.

HISTOPATHOLOGICAL FEATURES OF SMALL BOWEL MUCOSA IN CHOLERA-LIKE DISEASE IN BANGLADESH CAUSED BY VIBRIO CHOLERAE O139. P.K. Bardhan, M.M. Islam, M.J. Albert, M. Mathan, U. Dhar, D. Mahalanabis, R.B. Sack. International Center for Diarrhoeal Disease Research, Dhaka, Bangladesh; Christian Medical College, Vellore, India.

Until lately K cholerae (VC) non-O1 has been associated with only sporadic cases of diarrhoea. Recently, a new strain of VC non- O1, named VC O139, caused large epidemics of cholera-like disease in many countries including Bangladesh. A light microscopic (LM) and electronmicroscopic (EM) study of proximal small intestinal biopsy specimens obtained through endoscopes from 12 patients with VC O139 disease was carried out. For LM, all specimens were placed in neutral buffered formalin, dehydrated and embedded in paraffin. Hematoxylin and eosin stain was used, followed by periodic acid-Schiff stain. For EM, specimens were fixed in chilled 2.5% glutaraldehyde.

Biopsy specimens from all the patients demonstrated moderate to marked infiltration of mononuclear cells, consisting of lymphocytes and plasma cells, and mild to moderate infiltration of polymorpho- nuclear cells. Blunting of villi were noted in 40% of patients. No denudation, ulceration or erosion was noted. However, these features wei'e also found in biopsies from 14 healthy controls. The changes which were noted only among the patients and not in the controls were: edema of the lamina propria, dilatation of the lumens of the crypts, dilatation of the villous capillaries, discharging goblet cells in the crypts, swelling of the mitochondria, increase in number of vesicles of the Golgi apparatus, and widening of the interepithelial spaces. These alterations are thought to be functional changes that are not indicative of structural damage.

It is concluded that VC O139 infection is not associated with any morphologic damage to the proximal small intestinal mucosa.