Role of protein kinase C in intestinal ischemic preconditioning.

INTRODUCTION: Tissue protection by ischemic preconditioning (IPC) has been previously characterized in organs such as the heart and involves at least in part PKC activation. It is not yet clear whether such preconditioning against ischemia/reperfusion (I/R) injury operates in the intestine, and, if so, whether IPC involves protein kinase C (PKC). MATERIALS AND METHODS: IPC of the small intestine in male Sprague Dawley rats was induced by 10-min superior mesenteric artery (SMA) clamp followed by 120-min reperfusion. Sham-operated control or IPC rats were then rechallenged with 20-min SMA clamp. Histological injury to jejunal mucosa was assessed by microscopic examination and Parks' injury score (Grade 0-4; 0 = no damage). PKC activity was determined by immunoprecipitation of specific isoforms followed by in vitro kinase assay using mucosal scrapings of the harvested jejunum. Data were expressed as mean +/- SEM and analyzed by one-way ANOVA with multiple comparison tests. RESULTS: Ten-minute SMA clamp led to epithelial damage that was fully reversed by 120-min reperfusion. Activity of several PKC isoforms (PKCalpha, -delta, -epsilon) increased after 10-min ischemia. Epithelial injury associated with 20-min SMA clamp was attenuated by prior IPC. The protective effect of IPC on intestinal mucosa was prevented when animals were pretreated with the conventional (c) and novel (n) PKC inhibitor Go6850, but not with Go6976 (selective cPKC inhibitor), rottlerin (selective PKCdelta inhibitor), or saline control. CONCLUSIONS: Brief mesenteric ischemia induces a reversible epithelial injury in rats associated with activation of several PKC isoforms. Injury induced by mesenteric ischemia is reduced by brief ischemic preconditioning, an effect that is abolished by nonselective PKC inhibition but not by a selective inhibitor of cPKC or PKCdelta. The results suggest that activation of nPKC isoform(s), especially PKCepsilon during and following ischemic insults (IPC), may play an important role in protection against I/R injury in the intestine.

Bryostatin-1 enhances barrier function in T84 epithelia through PKC-dependent regulation of tight junction proteins.

Protein kinase C (PKC) is known to regulate epithelial barrier function. However, the effect of specific PKC isozymes, and their mechanism of action, are largely unknown. We determined that the nonphorbol ester PKC agonist bryostatin-1 increased transepithelial electrical resistance (TER), a marker of barrier function, in confluent T84 epithelia. Bryostatin-1, which has been shown to selectively activate PKC-alpha, -epsilon, and -delta (34), was associated with a shift in the subcellular distribution of the tight junction proteins claudin-1 and ZO-2 from a detergent-soluble fraction into a detergent-insoluble fraction. Bryostatin-1 also led to the appearance of a higher-molecular-weight form of occludin previously shown to correspond to protein phosphorylation. These changes were attenuated by the conventional and novel PKC inhibitor Gö-6850 but not the conventional PKC inhibitor Gö-6976 or the PKC-delta inhibitor röttlerin, implicating a novel isozyme, likely PKC-epsilon. The results suggest that enhanced epithelial barrier function induced by bryostatin-1 involves a PKC-epsilon-dependent signaling pathway leading to recruitment of claudin-1 and ZO-2, and phosphorylation of occludin, into the tight junctional complex.

Bryostatin-1 attenuates TNF-induced epithelial barrier dysfunction: role of novel PKC isozymes.

Tumor necrosis factor (TNF) increases epithelial permeability in many model systems. Protein kinase C (PKC) isozymes regulate epithelial barrier function and alter ligand-receptor interactions. We sought to define the impact of PKC on TNF-induced barrier dysfunction in T84 intestinal epithelia. TNF induced a dose- and time-dependent fall in transepithelial electrical resistance (TER) and an increase in [(3)H]mannitol flux. The TNF-induced fall in TER was not PKC mediated but was prevented by pretreatment with bryostatin-1, a PKC agonist. As demonstrated by a pattern of sensitivity to pharmacological inhibitors of PKC, this epithelial barrier preservation was mediated by novel PKC isozymes. Bryostatin-1 reduced TNF receptor (TNF-R1) surface availability, as demonstrated by radiolabeled TNF binding and cell surface biotinylation assays, and increased TNF-R1 receptor shedding. The pattern of sensitivity to isozyme-selective PKC inhibitors suggested that these effects were mediated by activation of PKC-epsilon. In addition, after bryostatin-1 treatment, PKC-delta and TNF-R1 became associated, as determined by mutual coimmunoprecipitation assay, which has been shown to lead to receptor desensitization in neutrophils. TNF-induced barrier dysfunction occurs independently of PKC, but selective modulation of novel PKC isozymes may regulate TNF-R1 signaling.

Electrogenic ion transport in mammalian colon involves an ammonia-sensitive apical membrane K+ conductance.

It is remarkable that high ammonia concentrations can be present within the colonic lumen without compromising normal epithelial function. We investigated the impact of luminal ammonia on Cl- secretion in native tissue. Stripped human colonic mucosa and unstripped rat distal colon were used. Paired samples were mounted in modified Ussing chambers for electrophysiological studies. In rat distal colon, apical ammonia dose-dependently blocked forskolin-activated short-circuit current with an IC50 to approximately 5 mM. Basolateral NH4Cl was less effective. Luminal methylamine (50 mM), chromanol 293B (10-50 microM), and Ba2+ (5 mM) blocked cAMP-activated short-circuit current but apical clotrimazole (100 microM) was without effect. In stripped human colonic mucosa, luminal but not basolateral NH4Cl (10 mM) and luminal Ba2+ (5 mM) suppressed forskolin-activated short-circuit current. Ammonia may be an endogenous regulator of colonic water and salt secretion. Apical K+ channels may be involved in the regulation of cAMP-stimulated Cl- secretion in mammalian colon.