Translocation of protein kinase-C with IP3-induced calcium mobilization by heat-stable enterotoxin of Vibrio cholerae non-O1 in isolated rat enterocytes.
The activity of the calcium- and phospholipid-dependent enzyme protein kinase C (PKC) in response to heat-stable enterotoxin (NAG-ST) of Vibrio cholerae non-O1 was examined in isolated rat enterocytes. Optimal stimulation of the membrane-bound PKC activity (about 4.3-fold) was observed after 1 min of incubation of cells with 10 ng/ml toxin; and the effects were dose dependent. Following NAG-ST treatment an increase in PKC activity in the membrane fraction was found with a concomitant decrease in the cytosolic fraction suggesting the redistribution of the enzyme. The pronounced enzyme activity in presence of a classical pseudosubstrate and its complete inhibition by Gö 6976 suggested the involvement of a calcium-dependent isoform of PKC (PKC-alpha). A time course study employing an immunoblot assay provided evidence that NAG-ST led to almost complete translocation of PKC-alpha to the membrane. A 65% inhibition of enzyme activity in the membrane fraction and inhibition of its translocation to some extent by dantrolene treatment further suggested that the enzyme was translocated with the rise of intracellular calcium ([Ca2+]i). The phosphorylation of three membrane proteins by toxin-induced PKC in vitro and abolition of this phosphorylation by Gö 6976 demonstrated that phosphorylation of these membrane proteins was PKC-alpha mediated and might be involved in the alteration of membrane functions.
Hoque
Kazi Mirajul
KM
Division of Pathophysiology, National Institute of Cholera and Enteric Diseases, Beliaghata, Kolkata, India.
Pal
Amit
A
Chakrabarti
Manoj K
MK
D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D002462:Cell Membrane / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D020895:Enterocytes / Q000201:enzymology / Q000382:microbiology*; D004768:Enterotoxins / Q000378:metabolism / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D007295:Inositol Phosphates / Q000378:metabolism* / Q000494:pharmacology; D008297:Male; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects; D044163:Vibrio cholerae non-O1 / Q000378:metabolism / Q000472:pathogenicity*
D004768:Enterotoxins; D007295:Inositol Phosphates; C037420:stN protein, Vibrio cholerae; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D002118:Calcium