A288 AGA ABSTRACTS GASTROENTEROLOGY, Vol. 108, No. 4

• P O S T - T R A N S C R I P T I O N A L U P - R E G U L A T I O N OF O R N I T H I N E DECARBOXYLASE DURING REGULATORY VOLUME DECREASE IN CACO-2 CELLS. A.G. Haliine, M. Hankewych, M. Esantsi. Dept. of Medicine, University of Illinois at Chicago and VA Westside Medical Center, Chicago, IL

Many cells exposed to hypotonic conditions undergo a rapid but transient increase in cell size followed by a return to normal size secondary to extrusion of intracellular potassium and chloride during the process of regulatory volume decrease (RVD). In a variety of cell types, hypotonic stress is also a potent activator of ormihine decarboxylase (ODC), the rate-limiting enzyme of polyamine synthesis. The mechanism of ODC stimulation following hypotonic stress, the relationship of RVD to this process, and the role of hypotonically-induced ODC stimulation on cell proliferation was explored using the Caco-2 human colon cancer cell line.

METHODS: Caco-2 cells grown in DMEM containing 20% fetal calf serum were exposed to either isotonic Earle's balanced salt solution (control) or to hypotonic solutions (150, 200, and 250 mOsm/L) for various lengths of time and harvested for measurement of ODC activity and mRNA level. The effect of hypotonic stimulation on proliferation was assessed by serial cell counts during the pre- and post-confluent phase of cell growth. The effect of blockade of K+ channels (1 mM Ba÷), C1- channels (0.5 mM diphenyl-2-carboxylate, DPC) and the C1-/HCO3- exchanger (1 mM dlisothiocyanosdibene-2,2'-disulfonic acid, DIDS) on hypotonically stimulated ODC was also examined.

RESULTS: In cells exposed to mild hypotonic media (250 mOsm/L) for 4 hours, there was no change in ODC activity compared to cells maintained under isotonic conditions. At 200 mOsm/L, however, there was a large increase in ODC activity and cells maintained in this media remained viable for extended periods of time and showed no evidence oflysis as determined by the LDH release assay. At 150 mOsm/L, however, there was a marked decrease in ODC activity and in cell viability. ODC is significantly increased in cells stimulated with hypotonic media (200 mOsm/L) after only 30 minutes of incubation and by 4 hours there is an approximate 50-fold increase in ODC activity. Despite this marked'increase in enzyme activity, there was no change on ODC mRNA levels as assessed by Northern blot analysis. There was no difference in cell numbers between controls and hypotonically treated cells during the log phase of cell growth but hypotonic treatment resulted in a 30-40% increase in cell numbers during the post-confluent period. Pre-treatment with Ba + and DPC caused a significant iniaibition in stimulated ODC activity following exposure to hypotonic solution, but DIDS had no effect.

CONCLUSION: Exposure of Caco-2 cells to moderate hypotonicity results in marked ODC up-regulation which appears to be secondary to post- transcriptional mechanisms. Furthermore, these results suggest that ODC activation is intimately associated with the process of RVD since ion channel blockers which are known to specifically inhibit this regulatory decrease in cell size were potent blockers of ODC stimulation. ODC activation in response to hypotonic stress may be an important factor in the regulation of cellular proliferation.

AGE AND SE(~4ENTAL DIFFERENCES IN 5-HYDROXYTRYPTA-

MINE-INDUCED HYPERSECRETION IN PIG SMALL INTESTINE. M.B. Hansen, M.L. Gr~ndahl, J.E. Thorb~ll, E. Skadhauge. Department of Anatomy and Physiology, The Royal Veterinary and Agricultural University of Copenhagen, Denmark.

Background. The incidence and severity of enterotoxigenlc infections decrease with age and in the aboral direction of the intestine. 5-Hydroxy- tryptamine (5-HT) is a mediator of E. coli and cholera-induced hypersecretion. This study elucidates the hypothesis, that the age- and segmental-related decrease In secreEory response to E. coli and cholera is partly due Eo a lesser secretory sensitivity co 5-HT. Methods. 5-HT- induced fluid hypersecretion (FHS) and short- circuit curren~ (SCC) were measured using tied-off loops and mucosal sheets mounted in the Ussing chamber. Results. Luminal 5-HT elicited dose-depen- dent increases in FHS, and serosal 5-HT induced conc.-dependent increases in SCC, which is partly a direct measure of stimulated CI- secretion and inhibited Na" absorption in pig je3unum. In the proximal intestinal part of piglets [2 weeks of age), the EM~x of FHS was 4.8 ~ I.I mg fluid x dry loop -I x 45 rain -~ with an EDsc of 9.2 ± 7.0 mM, while

the Ewx of SCC was 66.7 ± 4.8 ~A x cm -2 with an ECs0

of 1.2 z 0.5 p_M. Compared ~o piglets, the E~ of FHS for the young (8 weeks of age) and old (14 weeks of age) pigs, was 23 % and 62 % less, with EDs0 values being 179 % and 238 % higher, respec- tively. EMAx of SCC was 61 % less, with an ECs0 67 % higher, for the old pigs. In young pigs, the E~x of FHS in the mid and distal parts, compared to proximal, was decreased with 32 % and 67 %, with increased EDs0 values of 104 % and 102 %. Conclu- sions. Results support the hypothesis, since the secretory response to 5-HT decreases with increas- ing age and in the aboral direction.

• ROLE OF SELECTINS AND HYDROSTATIC PRESSURE IN PAF-INDUCED INCREASES IN FLUID FILTRATION FROM MESENTERIC CAPILLARIES. N.R. Harris and D.N. Granger. Dept. of Physiology, LSUMC, Shreveport, LA.

Fluid filtration rate (J/S)from rat mesenteric capillaries was measured prior to and following superfusion of platelet- activating factor (PAF), a lipid that has been implicated as a mediator of endothelial dysfunction associated with ischemia- reperfusion and septic shock. J~'S was calculated using a modified Landis technique whereby the decreasing distance (and volume) between two red blood cells in a selected capillary was measured following occlusion of the vessel with a micropipette. FOllowing PAF supeffusion, J~/S increased approximately 4-fold compared to baseline values. Microvascular hydrostatic pressure (measured with a servo- nulling pressure system) increased by 5-10 mm Hg following PAF superfusion, an increase which may be responsible for a 1.2 to 1.4-fold increase in J/S. However, the larger portion of the increase in J~JS following PAF superfusion can be attributed to increased capillary permeability. The PAF- induced increase in J/S was reduced by i.v. administration of a soluble form of sialyI-Lewis" (SLC), an oligosaccharide ligand for selectins, molecules involved in leukocyte- endothelial cell adhesion (LECA) observed in postcapillary venules. However, a nonbinding fucose-deficient SLC provided no such protection. These results suggest that prevention of selectin-mediated LECA inhibits PAF-induced increases in capillary permeability. (Supported by HL26441).

ENTERIC SENSORY NERVES MEDIATE A RISE IN SHORT-CIRCUIT CURRENT IN RESPONSE TO DISTENSION OF HUMAN JEJUNUM. R.P.Harris,J.M.Kellum,Deptof Surgery, Medical College Virginia, Richmond VA

Small bowel obstruction is associated with mucosal electrolyte secretion from the distended bowel proximal to the obstruction. We used an in-vitro model ofjejnnal distension to examine the change in short-circuit current (Isc), an index of net electrolyte fluxes across human jejunal muensa. Segments of human jejunum were stripped of muscularis and mounted as flat sheets in Ussing chambers under short- circuit conditions. After equilibration, successive volumes of buffer were removed from the submucosal chamber producing a pressure gradient and distension. The change in Is~ (AI~o) in response to repeat challenges with 9 cm H~O pressure gradients was recorded in the absence (AI~ A) and presence (AI,~ B) of the autonomic sensory neural blocker, capsaicin (100~tM), the 5-HT3/5-HT4. antagonist, ICS 205-930 (10~tM), atropine ( 1 gM), hexamethnninm ( 100gM),the basolateral Na + / K + / CI" transporter blocker, bumetanide (100p, M), tetrodotoxin (lgM), or buffer alone.The AI,: value is considered as a fraction of the baseline I,c (Mean = 43.5 + 9 }xAmps/cm2). RESULTS: Pressure (cm. H~O) : 3 6 9 AI,c/Baseline I~: 0.12 + 0.04* 0.18 _+ 0.04* 0.30 _+ 0.06* *p < 0.05. vs Baseline.Paired t-test. (u=4). (Mean + SEM). Additive: Control Capsaicin Bumetanide AI~ A/Baseline I,~ 0.30 + 0.03 0.39 + 0.07 0.29 + 0.08 AIooB/Baseline Ioo 0.29 + 0.04 0.16 + 0.04* 0.18 -+ 0.05, *p < 0.01 vs pretreatment (n=8), paired t test. A repeat pressure clmllenge in controls resulted in a AI~ response not statistically different than the initial challenge but different than that in the capsalcin-treated group (1o<0.05 t test n=8). The tetrodotoxin, ICS 205-930, atropine and hexamethoninm groups were not significantly different than the control group.The capsaicin-treated group remained responsive to 5-HT (100gM) stimulation ; AI~o= 16.2 vs. 15.7p.A/cm 2 CTL (p=0.91, t-test), indicating retained cell integrity. Bumctanide significantly reduced the baseline I~, AI~ and the AlsdBaseline I~¢ ratio (*p<0.05 paired t-test, n=6). CONCLUSION: Mucosal distension produces a significant dose-dependent clmnge in I~¢ which can be inhibited by enteric sensory neural blockade. This action appears specific, unlike that of bumetanide, which reduces the baseline and stimulated fluxes similarly.