Protein Kinase C Mediates Lipopolysaccharide-and Phorbol-Induced Nitric-Oxide Synthase Activity and Cellular Injury in the Rat Colon 1

The role of protein kinase C (PKC) in lipopolysaccharide (LPS)and phorbol ester-induced changes in rat colonic cellular integrity and Ca-independent inducible nitric-oxide synthase (iNOS) activity was investigated. LPS treatment (3 mg kg i.p.) increased colonic cellular PKC activity within 1 h after administration. The percentage of nonviable cells and iNOS activity in response to LPS were reduced by pretreatment with the selective PKC antagonist GF 109203X (25 ng kg i.v.). Pretreatment with the selective iNOS inhibitor 1400W (5 mg kg s.c.) reduced the extent of cellular injury and iNOS activity but did not affect the increase in LPS-mediated PKC activation. Reduction of circulating neutrophils with anti-neutrophil serum reduced cell damage as well as the increases in PKC and iNOS activities in response to LPS. Intracolonic administration of the phorbol ester phorbol-12-myristate-13-acetate (PMA; 3 mg kg) increased colonic cellular PKC activity within 2 h after instillation. Cellular iNOS activity did not increase until 6 h after PMA administration. The colonic responses to PMA were eliminated by GF 109203X. The selective iNOS inhibitor 1400W reduced the increase in cell injury but did not affect the PKC activation in response to PMA. LPS treatment also increased in the proteins for PKC-a, PKC-d, PKC-e, and PKC-z. PMA treatment resulted in PKC-d and PKC-e translocation from cytosol to membrane. These data suggest that PKC mediates iNOS activation and subsequent colonic cell injury in response to LPS administration. The dand e-isozymes appear to be most closely associated with these responses. Administration of bacterial endotoxin via lipopolysaccharide (LPS) treatment to rats has been associated with changes in intestinal vascular pemeability and with cytotoxic actions on small and large intestinal epithelial cells (Boughton-Smith et al., 1993; Tepperman et al., 1994). Furthermore, LPS administration has also been shown to induce Caindependent nitric-oxide synthase (NOS) activity. The large amounts of nitric oxide (NO) elaborated as a result of this enhanced enzyme activity have been shown to mediate the injury associated with LPS treatment and inhibition of this induction can ameliorate the extent of cellular damage (Tepperman et al., 1994). The mechanism whereby the LPS signal is transduced into the enhanced inducible NOS (iNOS) activity in intestinal epithelial cells is unknown. However, it has been shown that protein kinase C (PKC) is an important mediator for LPSinduced NOS activity as well as dysfunctional changes in the contractility of rat vascular tissue (McKenna et al., 1994). Similarly, it has been shown in rat macrophages that direct activation of PKC can induce iNOS activity (Hortelano et al., 1993). Protein kinase C consists of a family of at least 12 serinethreonine protein kinases that have been implicated in many cellular signaling pathways (Nishizuka, 1992). In addition to its signal transduction role, PKC has also been associated with tissue injury. PKC activation is associated with inflammation of a number of tissues, including the colon (Gupta et al., 1988; Sakanoue et al., 1992). Furthermore, direct activation of PKC via intraluminal instillation of phorbol ester has been shown to induce ileal and colonic inflammation in experimental animals (Fretland et al., 1990; Buell and Berin, 1994; Berin and Buell, 1995; Overdahl et al., 1995). Recently, this laboratory has demonstrated that trinitobenze sulfonic acid-induced colonic mucosal injury is mediated by increases in PKC activity (Brown et al., 1999). Furthermore, activation of PKC has also been shown to compromise the viability of a variety of cell types in vitro and PKC activity has been shown to be elevated in cells in response to a number of inflammatory challenges (Kuruvilla et al., 1993; Koong et al., 1994; Jan et al., 1997; Jones et al., 1997). Therefore, in the present study we have examined the Received for publication April 13, 2000. 1 This study was supported by Grant MT 6426 from the Medical Research Council of Canada. ABBREVIATIONS: LPS, lipopolysaccharide; NOS, nitric-oxide synthase; NO, nitric oxide; iNOS, inducible nitric-oxide synthase; PKC, protein kinase C; PMA, phorbol-12-myristate 13-acetate; ANS, anti-neutrophil serum; MPO, myeloperoxidase; ECL, enhanced chemiluminescence. 0022-3565/00/2953-1249$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 295, No. 3 Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics 2813/864751 JPET 295:1249–1257, 2000 Printed in U.S.A. 1249 at A PE T Jornals on July 0, 2017 jpet.asjournals. org D ow nladed from possibility that LPS administration activates the Ca-independent iNOS in colonic epithelial cells via an increase in PKC activity and by this route mediates the observed decreases in cellular integrity. We have also examined the effect of direct activation of PKC in colonic epithelium via intraluminal administration of phorbol ester on promoting cellular iNOS as well as changes in integrity. Experimental Procedures Isolation of Colonic Epithelial Cells Nonfasted male Sprague-Dawley rats (250–300 g) were sacrificed by cervical dislocation, and colonic epithelial cells were isolated from the colonic mucosa. Briefly, the colon was excised, everted, rinsed in ice-cold saline, and distended with Dulbecco’s phosphate buffer (pH 7.2). The colonic segment was incubated for 15 min at 37°C in 50 ml of Dulbecco’s in a water bath that shook at 50 oscillations min to remove dead cells. The distended colonic sacs were then suspended in 25 ml of RPMI 1640 medium (1 mg ml) containing 1 mM EDTA and 1 mg ml hyaluronidase (Sigma, St. Louis, MO) for 30 min at 37°C. Cells were removed by vigorous shaking and the cells collected were centrifuged at 2000g for 2 min. The cells were washed twice in RPMI 1640 buffer and filtered once more before their use in the