Introduction

Top Abstract Introduction Experimental Procedures Results Discussion References

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 Ca²⁺-independent 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 LPS-induced 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 serine-threonine 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 possibility that LPS administration activates the Ca²⁺-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

Top Abstract Introduction Experimental Procedures Results Discussion References

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 min¹ to remove dead cells. The distended colonic sacs were then suspended in 25 ml of RPMI 1640 medium (1 mg ml¹) containing 1 mM EDTA and 1 mg ml¹ 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 experiments.

Treatments

In some experiments, the rats were treated with bacterial LPS from Escherichia coli (serotype 0111:B4; Sigma; 3 mg kg¹ i.v.) Animals were killed and the colons removed for cell harvest at intervals of 1 to 6 h after LPS treatment. In further experiments, groups of rats were treated with the protein kinase C activator phorbol-12-myristate 13-acetate (PMA; 3 mg kg¹ intracolonically; Biomol, Plymouth Meeting, PA). The PMA was instilled in a volume of 0.5 ml or less. In these experiments, animals were killed and the colons excised for cell isolation 1 to 6 h after instillation of the PMA.

Animals in these groups of studies were also treated with the following agents: the highly selective PKC antagonist GF 109203X (Toullec et al., 1991) administered in vivo at a concentration of 25 ng kg¹ i.v., and the specific inhibitor of iNOS 1400W (N-(3-aminomethyl)benzyl) acetamidine; 5 mg kg¹ s.c.; Alexis Biochemicals, San Diego, CA) (Garvey et al., 1997). These agents used at the doses described above have been shown to effectively inhibit PKC and iNOS activities, respectively, in vivo (Laszlo and Whittle, 1997; Brown et al., 1999). Both agents were given 15 min before either LPS or PMA treatments and animals killed 2 h after LPS administration and 4 h after PMA treatment. Furthermore, in the LPS studies, groups of animals were pretreated with anti-neutrophil serum (ANS; Accurate Chemical and Scientific Corporation, Westbury, NY; 10 µl of antiserum i.p. 2 h before LPS), which at this dose and with the same treatment regime has previously been shown to reduce the number of circulating neutrophils to less than 5% of control numbers (Brown et al., 1998). In these experiments rats were killed, the colons excised, and cells harvested 2 h after LPS treatment as described above. In a final group of studies, rats were treated with ANS as described above and 2 h later the animals were killed, the colons excised, and cells harvested. Control animals were treated with a similar volume of normal rabbit serum. The resultant cellular suspensions were then treated in vitro with the PKC activator PMA in the concentration range 0.1 to 10 µM. Cells were incubated with PMA for 20 min at 37°C after which time cells were examined for viability, PKC activity, and iNOS activation.

Trypan Blue Dye Uptake

In all experiments an aliquot of cells was examined for viability as determined by Trypan blue dye exclusion (0.5% Trypan blue in phosphate-buffered saline), which has previously been shown to be a reliable index of gastrointestinal epithelial cell injury (Tepperman et al., 1991). Cells were counted in a randomized manner by a naïve observer using a hemocytometer and the number of nonviable cells was determined by light microscopy (200× magnification) by counting those cells that failed to exclude the dye.

Measurement of Myeloperoxidase (MPO) Activity

MPO levels were measured to provide an index of polymorphonuclear leukocyte infiltration. MPO activity was determined as described by Wallace (1987). Briefly, samples of cells (4 × 10⁶ cells) were resuspended in 50 mM phosphate buffer containing 0.5% hexadecyltrimethylammonium bromide (1 ml; pH 6.0). Samples were frozen in liquid nitrogen and thawed. This procedure was repeated twice more. Samples were then centrifuged at 40,000g for 15 min at 4°C. MPO activity in the supernatant was determined by adding 100 µl of the supernatant to 2.9 ml of 50 mM phosphate buffer containing 0.167 mg ml¹ o-dianisidine hydrochloride (Sigma) and 0.0005% w/v hydrogen peroxide. The change in absorbance at 460 nm over a 3-min period was measured. MPO activity is presented as moles of hydrogen peroxide converted to water per 4 × 10⁶ cells.

Measurement of Protein Kinase C Activity

Cells were centrifuged at 2000g for 10 min (4°C) and then were resuspended in 50 mM Tris-HCl buffer (pH 7.4) containing EDTA (5 mM), EGTA (10 mM), phenylmethylsulfonyl fluoride (50 µg ml¹), benzamide (10 mM), soybean trypsin inhibitor (10 µg ml¹), leupeptin (10 µg ml¹), aprotinin (10 µg ml¹), -mercaptoethanol (0.3% w/v), and okadaic acid (10 nM). The cells were lysed by sonication (10 s). A 25-µl aliquot of the sonicate was removed for determination of PKC activity using a commercially available kit (Amersham, Burlington, Ontario, Canada), which measures the transfer of [-³²P]ATP to a peptide specific for PKC. Results are expressed as picomoles per minute per 10⁶ cells.

Measurement of Protein Kinase C Content

Materials. Affinity-purified rabbit polyclonal antibodies to the -, -, -, and -isoforms of protein kinase C were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The secondary antibody was a goat anti-rabbit antibody conjugated to horseradish peroxidase purchased from Amersham (Arlington Heights, IL). Rainbow electrophoresis molecular weight marker, the enhanced chemiluminescence (ECL) kit, Hybond ECL nitrocellulose membrand, and Hyperfilm ECL were also purchased from Amersham.

Preparation of Cytosolic and Particulate Fractions. Some cell samples were resuspended in buffer and sonicated for 15 s on ice. The buffer consisted of 50 mM Tris-HCl (pH 7.5); 0.25 M sucrose; 2 mM EDTA; 1 mM EGTA; 25 µg ml¹ each of aprotonin, leupeptin, and pepstatin; 1 µg ml¹ soybean trypsin inhibitor; 50 µg ml¹ phenylmethylsulfonyl fluoride; and 10 mM -mercaptoethanol. The samples were centrifuged at 100,000g for 60 min. The supernatant was taken as the cytosolic fraction. The pellet was resuspended in the buffer described above to which was added 10% Triton X-100 and extracted at 4°C for 1 h before centrifugation again under the same conditions. The whole cellular sample was extracted by the same homogenization buffer containing Triton X-100 and centrifuged at 25,000g for 20 min. The protein concentration of each sample was subsequently determined.

Immunoblotting of Cellular Samples. Each sample of 10 to 15 µg of protein was boiled for 10 min in an equal volume of sample buffer (125 mM Tris-HCl, pH 6.8; 20% glycerol; and 10% mercaptoethanol) before subjecting to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrophoresis, the gel was soaked for 30 min in transfer buffer and electroblotted onto nitrocellulose membranse using Mini-Trans blot. A corresponding gel with the same loading samples and broad-range standard protein markers (Bio-Rad, Hercules, CA) were stained with Coomassie Brilliant blue R-250. The bands appearing on the whole gel were scanned to demonstrate standards of equal loading. Furthermore, after electrophoresis, the gel was cut according to the position of the standard protein marker. The region around the PKC protein molecular weight was cut for further blotting analysis. The lower portions were stained to demonstrate the bands on the gel as a marker of equal loading.

Measurement of NOS Activity

Cellular NOS activity was assessed by determining the formation of radiolabeled citrulline from the substrate [¹⁴C]arginine as described previously for colonic mucosal cells (Tepperman et al., 1994). Briefly, enzyme activity was released from cells into the buffer by freezing in liquid nitrogen and then thawing at 37°C three times. The broken cell fractions were then centrifuged at 10,000g for 20 min at 4°C. Conversion of radiolabeled arginine to the NO coproduct citrulline was determined in an assay system (70-µl total volume, pH 7.2) containing 20 µl of broken cell supernatant and the following components (final concentrations): 30 mM potassium phosphate, 150 µM CaCl₂, 0.7 mM MgCl₂, 15 µM L-[¹⁴C]arginine (700,000 disintegrations min¹ ml¹; Amersham) and 0.1 mM NADPH, as well as 10 mM L-valine to inhibit any arginase.

Incubation normally lasted for 10 min at 37°C, after which time 1 ml of a 1:1 suspension of Dowex (AG 50 W-8; Sigma) in water was added to bind arginine. The resin was allowed to settle and the supernatant was removed for estimation of the radiolabeled products by scintillation counting. Product formation that was inhibited by the in vitro incubation with the NO synthase inhibitor N^G-monomethyl-L-arginine (300 µM) but not with EGTA (1 mM) was used as an index of inducible Ca²⁺-independent iNOS activity.

Statistics

Data are shown as means ± S.E.M. of six to eight experiments each done in duplicate. Statistical significance was assessed by the t test for paired data or one-way analysis of variance and Duncan's multiple range test where P < .05 was taken as significant.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Effect of Lipopolysaccharide. Cells isolated from the rat colonic mucosa by the techniques used here were identified by light microscopy as being 90 to 95% epithelial cells. Lipopolysaccharide treatment resulted in an increase in the extent of cell injury as assessed by Trypan blue dye uptake (Fig. 1A). A significant increase in the extent of cell damage was not observed until 2 h after LPS administration. The peak extent of cell damage was seen 4 h after LPS.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Effect of E. coli LPS (3 mg kg1 i.v.) on the extent of cellular damage as assessed by Trypan blue dye uptake (A), PKC activity (B), and Ca2+-independent inducible NOS activity (C) in cells harvested from the rat colon 1 to 6 h after administration of LPS. Values are displayed as means ± S.E.M. of six to seven independent experiments. Asterisks (*) indicate significant increases over cells harvested from untreated animals (0 h) as determined by Duncan's multiple range test.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Effect of pretreatment (15 min) with either the selective PKC inhibitor GF 109203X (25 ng kg1 i.v.) or the selective iNOS inhibitor 1400W (5 mg kg1 s.c.) on the extent of cellular injury as assessed by Trypan blue dye uptake (A), PKC activity (B), and Ca2+-independent NOS activity (C) in response to LPS administration. Animals were killed 2 h after LPS administration. Values are displayed as means ± S.E.M. of six to eight independent experiments. Asterisks (*) indicate significant reductions from the LPS alone data as determined by Duncan's multiple range test.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Effect of pretreatment with anti-neutrophil serum (10 µl of antiserum i.p.) on LPS-mediated responses (3 mg kg1 i.v.) in rat colonic epithelial cells. The measured responses include cell viability as assessed by Trypan blue dye uptake (A), neutrophil content as assessed by MPO activity (B), PKC activity (C), and Ca2+-independent NOS activity (D). All parameters were assessed 2 h after LPS administration. The anti-neutrophil serum was given 1 h before LPS injection. Values are displayed as means ± S.E.M. of seven to eight independent experiments. Asterisks (*) indicated significant increases over control (CNTL), whereas crosses (+) indicate significant decreases from the responses observed to LPS alone as determined by Duncan's multiple range test.

View larger version (52K): [in this window] [in a new window]   Fig. 4.   Effect of LPS treatment (3 mg kg1 i.v.) on PKC isoforms in lysates of colonic epithelial cells. Representative Western immunoblots for PKC-, PKC-, PKC-, and PKC- from control and LPS-treated cells are displayed. Cells were harvested from rats 1 to 6 h after LPS treatment. The immunoblot patterns displayed were confirmed in at least three separate experiments. The bottom portion of the figure displays the gel loaded with the same sample as was used for detection of PKC isozyme protein between standard proteins at positions 60 kDa and 30 kDa, which was cut and stained with Coomassie Brilliant blue to show equal loading protein amounts. M1 is the Rainbow electrophoresis marker and M2 is the polypeptide standard molecular weight marker.

Effect of Phorbol Myristate Acetate. Phorbol myristate acetate treatment of cells in vitro resulted in a dose-dependent increase in the extent of cell damage (Fig. 5A) as well as an increase in PKC activity in cells harvested from control rats (Fig. 5B). PMA treatment resulted in a significant increase in iNOS activity in response to 10 µM PMA (Fig. 5C). Repetition of this experiment in cells harvested from animals made neutropenic by ANS treatment resulted in significant reductions in the extent of cell injury, and PKC and iNOS activities in response to PMA treatment (Fig. 5) although, especially at the highest concentration of phorbol ester used, the values were significantly greater that those observed for the respective control samples.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Effect of neutropenia on the response of isolated colonic mucosal cells to in vitro treatment with PMA (0.1-10 µM). Control cells were harvested from animals treated with normal rabbit serum. Cells were treated as described under Experimental Procedures and examined for the effects of PMA on cellular integrity as estimated by Trypan blue dye uptake (A), PKC activity (B), and iNOS activity (C). Results are the means ± S.E.M. of six to eight experiments each from a separate rat. Asterisks (*) indicate significant increases in response to PMA compared with the respective group of untreated cells, whereas crosses (+) indicate significant reductions in the ANS group at any one concentration of PMA when compared with the responses of cells harvested from control animals. Significance was determined by Duncan's multiple range test.

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Effect of intracolonic instillation of PMA (3 mg kg1) on cell viability as estimated by Trypan blue dye uptake (A), PKC activity (B), and Ca2+-independent NOS activity (C) in cells harvested from the rat colon. Cells were isolated 1 to 6 h after PMA instillation. Cell were also taken from animals treated (0.5 ml) with the vehicle for PMA (20% ethanol). Values are displayed as means ± S.E.M. of six independent experiments. Asterisks (*) indicate significant differences between PMA and ethanol control animals as determined by the t test for paired data.

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Effect of PMA (3 mg kg1) on cell viability as assessed by Trypan blue dye uptake (A) and PKC activity in epithelial cells isolated from rat colon (B). Cells were harvested 4 h after PMA administration. In some experiments animals were pretreated 15 min before PMA administration with either the iNOS inhibitor 1400W or the selective PKC inhibitor GF 109203X (GFX). Values are displayed as means ± S.E.M. of seven experiments. Asterisks (*) indicate a significant increase over responses evident in cells harvested from animals treated with the vehicle for PMA (20% ethanol, control). Crosses (+) indicate significant decreases over the responses observed with PMA alone. Significance was determined by Duncan's multiple range test.

View larger version (40K): [in this window] [in a new window]   Fig. 8.   Effect of PMA treatment (3 mg kg1 intracolonically) on PKC isoforms in lysates of colonic epithelial cells. Cells were harvested 2 h after PMA treatment. A, displays representative Western immunoblots of samples from vehicle (20% ethanol)-treated control or PMA-treated rats. The whole Western blot, demonstrating equal loading is displayed in B. (M1 is the Rainbow electrophoresis marker and M2 is the polypeptide standard molecular weight marker; C1 and C2 represent control samples, and P1 and P2 represent samples taken from PMA-treated rats). C, displays the mean ± S.E.M. protein band intensity for each isoform determined in the cytosolic and membrane fraction of the cellular lysate from six independent experiments. Asterisks (*) indicate significant differences from the respective control as determined by the t test for paired data.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

The results of the present study indicate that administration of bacterial endotoxin LPS to experimental animals resulted in an increase in the extent of cell injury as well as an increase in inducible NOS activity in epithelial cells harvested from the colonic mucosa. This confirms previous findings that LPS treatment would activate iNOS and the resultant NO thus liberated could account for the increase in colonic cellular damage (Tepperman et al., 1994). Furthermore, the present data indicate that LPS treatment results in an increase in the activity of PKC in the cells isolated from the colonic mucosa. The increase in PKC occurs within the 1st h after LPS treatment, whereas the increase in iNOS activity was not observed until 4 h after LPS injection, a time that corresponded to the increase in the extent of cell injury. This temporal relationship suggests that the increase in PKC may mediate the cellular injury via activation of iNOS. This suggestion is further supported by the finding that the effect of LPS on iNOS activation could be inhibited by pretreating animals with the selective PKC inhibitor GF 109203X. On the other hand, the selective iNOS inhibitor 1400W did not affect cellular PKC activity. These findings support previous studies, which have demonstrated that LPS administration would increase iNOS activity via PKC activation in a variety of cell types, including cardiac cells (McKenna et al., 1995), aortic smooth muscle cells (Paul et al., 1997), macrophages (Shapira et al., 1997), and microglial cells (Fiebich et al., 1998).

The present study also demonstrated that this increase in PKC activity in vivo as well as the increase in cell injury in response to LPS was dependent, at least in part, upon neutrophil infiltration. These data could suggest that the neutrophil is the source of the PKC activity determined in this study, and its reduction after anti-neutrophil serum treatment accounts for the reduction in both iNOS activity and the extent of cell injury in response to LPS. These data do not exclude the possibility that the neutrophil may also contribute directly to the increase in iNOS activity seen after LPS or PMA activation. Alternatively, these data may also indicate that the infiltrating cells that secrete a wide variety of inflammatory mediators such as tumor necrosis factor- and interleukin-1 (Nathan, 1987) may stimulate the colonic epithelial cells to increase PKC activity and subsequently, iNOS activity. Previous studies have demonstrated that such cytokines can increase PKC activity in some epithelial cells (Wyatt et al., 1997; Prasanna et al., 1998; Fischer et al., 1999). This possibility is supported to some extent by our in vitro findings in which elimination of neutrophils by ANS treatment resulted in a reduction in the extent of cell injury as well as PKC and iNOS activities in response to direct stimulation of the cells with the PKC activator PMA. The remaining epithelial cells were able to respond to the phorbol ester but only at the highest concentration used here. Therefore, in the present study it is likely that activation of PKC and iNOS occur predominantly within the neutrophils

We have also examined the effects of direct activation of PKC via the intraluminal administration of the phorbol ester PMA. In the present study, we have determined that administration of PMA by this route resulted in a rapid increase in the extent of cellular injury as well as an increase in both PKC and iNOS activities. The increase in PKC activity preceded that observed for iNOS. The ability of intraluminal PMA to decrease cellular integrity confirms and extends findings from in vivo experiments in which PKC activation via phorbol ester treatment was shown to mediate colonic mucosal and cellular injury (Fretland et al., 1990; Berin and Buell, 1995). Furthermore, we have recently demonstrated a direct cytotoxic action of PKC activators toward isolated colonic epithelial cells (Tepperman et al., 2000).

PKC activation via PMA treatment appeared to mediate the increase in iNOS activity. This is suggested by the temporal relationship between the activities of the two enzyme systems. Furthermore, inhibition of PMA-mediated increases in PKC activity with GF109203X reduced the extent of cell injury. In contrast, the iNOS inhibitor 1400W reduced the extent of cell damage but did not affect PKC activity. This confirms previous studies, which have demonstrated that PMA administration could activate iNOS via a PKC-dependent mechanism (Hortelano et al., 1993; Fujihara et al., 1994; Zauli et al., 1996; Paul et al., 1997). Furthermore, the induction of NOS appears to play a role in the degree of cellular integrity because treatment with 1400W reduced the extent of cell injury in response to intraluminal PMA treatment.

The present study revealed the presence of various PKC isoforms in cells harvested from the unstimulated as well as the LPS- and PMA-activated colon. The presence of multiple PKC isozymes has previously been demonstrated in the colonic epithelium of the rat (Jiang et al., 1995). PKC isoforms have been divided on the basis of the dependence of their enzyme activity on Ca²⁺ and their susceptibility to treatment with phorbol esters. The conventional PKCs, including PKC-, are Ca²⁺-dependent and respond to phorbol esters; the novel PKCs (PKC-, PKC-) are Ca²⁺-independent but respond to phorbol esters; and the atypical PKCs such as PKC- are independent of both Ca²⁺ and phorbol esters (Dekker and Parker, 1994). In the present study, LPS treatment resulted in activation of all isoforms examined, whereas PMA administration was associated with selective activation of the novel isoforms PKC- and PKC-. Previous studies in other tissues and cells have demonstrated that these isoforms are up-regulated in response to LPS treatment (Fujihara et al., 1994; Shapira et al., 1997). Studies directed at examining the isoforms activated in response to PMA have suggested that depending upon the tissue or cell type under investigation, the profile of PKC isoform activation can vary. However, many of these studies have suggested that PMA-mediated activation of PKC- may play an important role in the mediation of NOS induction and subsequent cellular injury (Fujihara et al., 1994; Keenan et al., 1997; Shapira et al., 1997). The functional role of these isoforms in the regulation of colonic cellular integrity is currently under investigation in this laboratory.

In conclusion, the present study has shown that LPS and PMA treatment in the rat can result in activation of colonic cellular PKC activity with subsequent induction of the Ca²⁺-independent NOS. The increase in iNOS is further associated with damage to the cells harvested from the colonic epithelium. Although a number of PKC isoforms may be involved in these responses, PMA treatment increased the translocation of PKC- and PKC- from cytosol to membrane, suggesting that these isoforms may play central roles in this process.