ILEAL MICROVILLAR PROTEIN VILLIN IS TYROSINE-PHOSPHORYLATED AND ASSOCIATES WITH PLC-GAMMA(1) - ROLE OF CYTOSKELETAL REARRANGEMENT IN THE CARBACHOL-INDUCED INHIBITION OF ILEAL NACL ABSORPTION

In ileal absorptive cells, carbachol inhibits NaCl absorption and its component brush border Na+/H+ exchanger, acting via basolateral membra ne receptors, This carbachol effect involves (i) activation of brush b order phosphatidylinositol 4,5-bisphosphate-specific phospholipase C ( PLC) activity and brush border but not basolateral membrane translocat ion of PLC-gamma(1) (Khurana, S., Kreydiyyeh, S., Aronzon, A., Hoogerw erf, W. A., Rhee, S. G., Donowitz, M., and Cohen, M. E. (1996) Biochem , J. 313, 509-518); and (ii) brush border tyrosine kinase(s) because m ucosal but not serosal addition of the tyrosine kinase inhibitor genis tein prevents the carbachol-induced inhibition of NaCl absorption and brush border Na+/H+ exchange, In the present work we identify a pool o f villin (a brush border actin-binding protein) in the microvillus mem brane fraction of rabbit ileum; this pool of villin is tyrosine-phosph orylated and associates with brush border membrane PLC-gamma(1), Villi n is present both in the Triton X-100-soluble and -insoluble fractions of the brush border, The Triton X-100-soluble pool is approximately 4 -fold smaller than the brush border pool of villin that is present in the Triton X-100- insoluble fraction, Only the villin present in the T riton X-100-soluble fraction of ileal villus brush border associates w ith PLC-gamma(1), and is tyrosine phosphorylated. Carbachol increases the tyrosine phosphorylation of villin rapidly (as early as 30 s) and transiently, Carbachol also increases the amount of tyrosine-phosphory lated villin that associates with PLC-gamma(1), These studies demonstr ate that carbachol effects on NaCl absorption are accompanied by an in crease in brush border PLC-gamma(1), association with villin and an in crease in tyrosine phosphorylation of villin, To study the role of cyt oskeletal rearrangement in carbachol-induced inhibition of NaCl absorp tion, we used the F-actin stabilizing drug jasplakinolide. Jasplakinol ide prevents the carbachol inhibition of ileal Nacl absorption, This s uggests that F-actin severing is necessary for carbachol to inhibit il eal villus NaCl absorption, Since villin is known to sever actin, thes e studies suggest a role for villin in the signaling cascade that begi ns at the basolateral membrane with carbachol binding to its receptor and ends at the apical membrane in inhibition of NaCl absorption.