Differential regulation of NHE3 expression in Caco2 cells by PKCα

GASTROENTEROLOGY Vol. 114, No. 4

A348 AGA ABSTRACTS • G1418 5 - H T ACTIVATES AN ENTERIC NEURAL REFLEX M O D U L A T I N G T R A N S P O R T IN RESPONSE TO MUCOSAL STROKING IN MAN.

F.C. Albuquerque. Jr., M.C. Stoner, J.M. Kellum. Dept. of Surgery, Medical College of Virginia/Virginia Commonwealth University, Richmond, VA. A recent report (Sidhu and Cooke, tLIP 1995) documented an enteric neural reflex which mediates the CI- secretory response to mucosal stimulation in guinea pig colon. Having previously reported 5-HT release and C1- secretion from human jejunal mucosa in response to stroking, we postulated the presence of an enteric neural reflex, modulating chloride secretion and activated by release of 5-HT from human jejunal mucosa, in response to stroking. METHODS: Segments of proximal jejunum were harvested from gastric bypass patients, stripped of the muscularis and mounted in modified Ussing chambers equipped with a brush under short-circuit conditions. Initially, change in short circuit current (A_I) was measured after different mucosal stroking stimuli without any drugs. After determining the ideal number of strokes, the mucosa was stroked with a soft brush at l/s for 5s, first in the absence and then in the presence of various 5-HT antagonists and neural blockers. RESULTS: Mucosal stroking caused a stroke-dependent increase in ~,: which was significantly inhibited by 100 pM capsaicin (n=16), 100 pM hexamethonium (n=10) and 1 pM tetrodotoxin (n=9) but not by atropine. The A/~ was also significantly inhibited by 10 laM tropisetron and 10 laM SDZ 205-557 (5-HT~.4 and 5-HT4 receptor antagonists, n=6 and n=8, respectively). Receptor antagonists preferential for 5-HTjp, 5-HT2^ and 5-HT3 receptors did not inhibit the Io response to mucosal stroking. Short Circuit Current After Stroking (mean ± SEM) Control Cap Hex TTX SDZ Tropis 205557 45.4±2.5 49.4±4.9 46.7±5.4 46.8±3.1 37.8±3.3 46.0±2.9

Before drags After 44.4±3.7 28.5±3.8"t 38.3+_3.2* 32.8±5.5* 32.2±3.2:~ 37.2±3.0* drugs t p < 0.001, * p < 0.02, * p < 0.05, Student's t-test for paired data CONCLUSION: These results suggest that mucosal stroking of human jejunum causes release of 5-HT from EC cells which evokes chloride secretion by activating 5-HT4 receptors on afferent enteric neurons, and reflex pathways, containing nicotinic cholinergic receptors. This research was supported, in part, by NIH grant, DK43899. • G1419

CRYPT CELL APOPTOSIS AND INCREASED MUCOSAL PERMEABILITY INDUCED BY ACTIVATED T CELLS. Z Alna~im. R Koehler, Z Kayali, P Efthimiou, S Hurst, S Mandrea, E Chang, D Green*, T Lin* and TA Barrett. VA Lakeside Hosp and Northwestern Univ Med School, Chicago IL. 60611. *La Jolla Inst for Allergy/Immunology, San Diego, CA.. Increased levels of crypt cell apoptosis are observed in inflammatory bowel disease (IBD), graft versus host disease (GVHD), AIDS colitis and small bowel (SB) allograft rejection. To address mechanisms of T cell-induced crypt cell apoptosis in vivo we utilized an ovalbumin (OVA) antigen (Ag)specific DOll.10 T cell receptor transgenic (Tg) murine model. Results of TUNEL staining revealed that 24 hr after T cell activation with i.p. OVA peptide injection, nearly all crypt epithelial cells in the terminal ileum undergo apoptosis with lower, yet significant levels in the duodenum and colon. Interestingly, inhibition of endogenous T cell activation with CTLA41g led to reduced crypt cell apoptosis in control (unstimulated) as well as Agstimulated mice suggesting that T cell activation plays a role in naturallyoccurring crypt cell apoptosi s. Furthermore, T cell activation increased mucosal permeability to small sugars (sucrose, lactulose and mannitol) and relatively large probes (14C-labeled inulin). Pretreatment of mice with antitumor necrosis factor-ct (TNF-tx) or anti-interferon-T (IFN-'/) mAbs reduced effects of T cell activation on permeability and crypt cell apoptosis by 40 and 70% respectively. Crypt cell apoptosis was also induced in nontransgenic mice by T cell activation with systemic anti-CD3 (2C11) mAb administration. Levels of anti-CD3-induced crypt cell apoptosis were reduced (-70%) in CD95 (lpr/lpr) and CD95L (gld/gld)-deficient mice compared to controls and completely abrogated with anti-TNF-ct mAb. In conclusion, T cell activation

induced crypt cell apoptosis through a combination of CD95 and TNFR-1mediated pathways and increased mucosal permeability through IFN-7 and TNF-ct-mediated pathways. We suspect that mucosal permeability is increased, in part, due to crypt cell apoptosis. The induction of crypt cell apoptosis and mucosal permeability via IFN-), and TNF-ct-mediated pathways in this model of T cell activation may be relevant to mechanisms which operate in Crohn's disease as well as GVHD, SB graft rejection and AIDS enteropathy. • G1420 DIFFERENTIAL REGULATION OF NHE3 EXPRESSION IN Caco2 CELLS BY PKC~. W.A. Alrefai, B. Scaglione-Sewell*, S. Tyagi, L. Wartman, T.A. Brasitus*, K. Ramaswamy and P.K. Dudeja. Departments of Medicine, University of Illinois and VAMC Chicago (Westside), *University of Chicago, Chicago, IL Na+-H+ exchange (NHE) isoform activities have been shown to be regulated by many protein kinases in a variety of cells and tissues. The human colon

cancer cell line Caco2 has been extensively used to characterize electrolyte transport processes of the small intestine and colon, and has recently been used as an experimental model to study expression of PKC isoforms. Aim of our current studies with Caco2 cells was to determine if protein kinase C isoforms could regulate the expression and/or function of various human NHE isoforms. Caco2 ceils over-expressing or under-expressing PKCct were prepared by stable transfection of sense and antisense cDNAs for PKCct, respectively. Message levels of the human NHE1, 2 and 3 were measured by a semi-quantitative RT-PCR technique. Initially, message levels of the NHE isoforrns in wild type Caco2 cells were assessed in response to PKC down regulation by long term exposure t o PMA (1 laM for 24 h). PKC down regulation significantly stimulated the NHE3 transcript levels by more than 60%, with no changes in NHE1 or NHE2. To elucidate the role of PKCc~, one of the isoforms down regulated by PMA, the relative abundance Of NHE mRNAs in Caco2 cells over-expressing or under-expressing PKCct was assessed. From our results shown below, it can be concluded that the level of only NHE3 but not NHE1 or NHE2 was regulated by PKCct isoform. To further examine the functional relevance of the alteration in NHE3 mRNA, Na+-H÷ exchange activity in the ceils over-expressing and under-expressing PKCct was evaluated. Consistent with mRNA results, our data showed that amiloride-sensitive 22Na uptake (nmol/mg/5min) was significantly lower in cells over-expressing PKCct (8.4 +_0.4), and higher in ceils under-expressing PKCct (12.8 +_0.4) compared to control (11.6 +_0.5). N H E message levels normalized to Histone 3.3 or G 3 P D H a

Isoform Wild type NHE3 1.41+_0.09 NHE2 0.62 +_0.09 NHE1 0.95+_0.08 a. Mean _+SEM of 3-6 preps;

Empty vector Sense Antisense 1.31+_0.32 0.59+_0.08* 2.10+_0.11" 0.84 +_0.35 0.78 +_0.06 0.84 +_0.11 1.03+_0.10 0.92+0.12 0.88+_0.08 *P < 0.05 compared to empty vector

Conclusion. These data demonstrate differential regulation of NHE3 but not

NHE2 or NHE1 expression in Caco2 cells by PKCtt isoform. Supported by NIDDK and the Dept. of Veterans Affairs. G1421

KINASE C-MEDIATED PHOSPHORYLATION OF MYOSIN LIGHT CHAIN KINASE (MLCK) INHIBITS MYOSIN LIGHT CHAIN (MLC) PHOSPHORYLATION AND RESULTS IN INCREASED TRANSEPITHELIAL RESISTANCE (TER). J.M. Angle 1, J.L. Joyal 2, E.D. Black 1, H. Alli 1, D.B. Sacks 2,/.L. Madara 3, and J.R. Turner 1. Departments of Pathology, 1Wayne State University and Harper Hospital, Detroit, MI, 2Brigham and Women's Hospital, Boston, MA, and 3Emory University, Atlanta, GA. PROTEIN

Activation of PKC with phorbol esters causes a rapid transient increase in TER (Hecht et al., 1994. Amer. J. Physiol.). The mechanism of this effect is unknown. We have previously shown that phosphorylation of MLC by MLCK is associated with decreases in TER, perhaps by increasing contraction of the perijunctional actomyosin ring (Turner et al., 1997. Amer. J. Physiol.). Studies in non-epithelial systems have shown that PKC can alter mYosin function by phosphorylation of MLCK, resulting in decreased MLCK activity, or by direct phosphorylation of MLC at sites which decrease actomyosin contraction (i.e. sites distinct from those phosphorylated by MLCK). The aim of this study was to determine if the PKC-dependent TER increase is associated with phosphorylation of MLCK or MLC. M E T H O D S : Caco-2 cell monolayers (clone 5E6L) were grown on semipermeable supports. Electrophysiology and 32p radiolabelling were assessed by standard methods. PKC activity was measured using partially purified PKC, a synthetic substrate, and 3/p-ATP incorporation. RESULTS: Activation of PKC by phorbol ester (PMA, 0.17-1.6uM) resulted in a dose-dependent increase in TER to as much as 142% +_ 13% of control monolayers. A TER increase of 22% +_6% was reduced to an increase of 4% +_3% by the specific PKC inhibitor bisindolylmaleimide I (5uM), supporting the conclusion that the increases in TER are due to PKC activation. PMA treatment resulted in a 79% +_2% increase in membraneassociated PKC activity and a 31% +_3% decrease in cytosolic PKC activity. After stimulation of monolayers with PMA, MLC phosphorylation decreased by 39%. Peptide mapping of purified MLC did not detect additional phosphorylation sites in PMA-treated monolayers, and phosphoamino acid analysis showed only phosphoserine residues on purified MLC. In contrast to MLC, phosphorylation of MLC kinase (MLCK) increased by 24% after PMA stimulation. CONCLUSIONS: These data demonstrate that (1) activation of PKC results in increased TER of intestinal epithelial monolayers, (2) that this increase in TER may be prevented by PKC inhibitors, and (3) that activation of PKC is accompanied by decreased MLC phosphorylation. The decreased MLC phosphorylation is likely due to inactivation of MLCK by PKC-mediated phosphorylation. Thus, the mechanism by which PKC increases TER may be to inhibit MLCK, leading to reduced contraction of the perijunctional actomyosin ring and diminished tension on the epithelial tight junction. Supported by the NIH/NIDDK DK-K08-02503 (J.R.T.) and DK-R37-35932 (J.L.M.) and the F.M.R.E. of Wayne State University.