CALCIUM AND PROTEIN-KINASE-C PLAY AN IMPORTANT ROLE IN CAMPYLOBACTER JEJUNI-INDUCED CHANGES IN NA- TRANSPORT IN RAT ILEUM IN-VITRO( AND CL)

The pathophysiological mechanism of Campylobacter jejuni (enterotoxige nic) induced secretory diarrhoea remains least understood. To investig ate the mechanism(s) involved, the unidirectional fluxes of Na+ and Cl - were measured across the C. jejuni live culture infected and control (non infected) rat ileum (unstriped), in vitro by Ussing technique un der short circuit conditions, in the presence or absence of: Ca2+ iono phore A23187 (5 mu M), 1-verapami1 (100 mu M), calmodulin (CaM) antago nist W-7 (100 mu M), dantrolene (25 mu M), protein kinase C (PKC) acti vator PMA (100 ng/ml) and H-7 (60 mu M), selective inhibitor of PKC. T here was net absorption of Na+ and enhanced Cl- secretion in infected animals while in control animals there was net absorption of Na+ and m arginal secretion Cl-. Ca2+ ionophore A23187 mimicked the effects of C . jejuni infection whereas 1-verapamil had significant antisecretory e ffect on Na+ and Cl- secretion in infected animals. In vitro measureme nt of unidirectional Ca-45 fluxes in Ussing chamber experiments reveal ed net absorption of Ca2+ in infected rat ileum as compared to net sec retion of Ca2+ in control rat ileum, These observations clearly indica te that there is increased stimulation of Ca2+ uptake from extracellul ar milieu to the enterocytes during C. jejuni-induced diarrhoea. The i ntracellular isolated from C. jejuni infected ileum as compared to the enterocytes from control ileum. The observed increase in [Ca2+](i) in enterocytes isolated from C. jejuni live culture supernatant treated rat ileum further shows the involvement of enterotoxin in diarrhoeal p rocess. Dantrolene decreased significantly C. jejuni-induced net Na+ a nd Cl- secretion but it could not reverse it to absorption suggesting the partial involvement of Ca2+ mobilised from intracellular stores in mediating secretion. W-7 failed to inhibit the C. jejuni-induced net Na+ and Cl- secretion. In addition the CaM activity estimated in intes tinal microvillar core remained same in both the control and C. jejuni infected animals. This indicates that C. jejuni-induced diarrhoea is not mediated through the activation of Ca2+-CaM complex pathway of the Ca2+ messenger system. The PKC activator PMA, induced net secretion o f Na+ and Cl- in the control animals but it could not enhance further the C. jejuni-induced Na+ and Cl- secretion, suggesting that there is overlapping effect of PMA and C. jejuni live culture infection. H-7 si gnificantly inhibited (P < 0.001) net Na+ and Cl- secretion in the inf ected animals but had no effect on basal rat ileal Na+ and Cl- transpo rt in control animals. This again suggest the activation of PKC signal in C. jejuni-induced ileal Na+ and Cl- secretion. Although intracellu lar cAMP levels were found to be significantly raised in the enterocyt es isolated from C. jejuni live culture infected and C. jejuni culture supernatant treated rat ileum, the mechanism of interaction between c AMP and Ca2+ messenger system in infection remains undefined. Taken to gether, the present results therefore, indicate that enterotoxigenic C . jejuni live culture-induced Na+ and Cl- secretion is a calcium-depen dent process where both increased Ca2+ entry from extracellular milieu and Ca2+ mobilization from intracellular stores result in increased e nterocyte (Ca2+](i). The activation of PKC further appears to be the i mportant intracellular mediator pathway involved in Ca2+-dependent sti mulation of C. jejuni-induced diarrhoea.