Ileal microvillar protein villin is tyrosine-phosphorylated and associates with PLC-γ₁. 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 membrane receptors. This carbachol effect involves (i) activation of brush border phosphatidylinositol 4,5-bisphosphate-specific phospholipase C (PLC) activity and brush border but not basolateral membrane translocation of PLC-γ₁ (Khurana, S., Kreydiyyeh, S., Aronzon, A., Hoogerwerf, 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 mucosal but not serosal addition of the tyrosine kinase inhibitor genistein prevents the carbachol-induced inhibition of NaCl absorption and brush border Na⁺/H⁺ exchange. In the present work we identify a pool of villin (a brush border actin-binding protein) in the microvillus membrane fraction of rabbit ileum; this pool of villin is tyrosine-phosphorylated and associates with brush border membrane PLC-γ₁. Villin 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 Triton X- 100-soluble fraction of ileal villus brush border associates with PLC-γ₁ and is tyrosine-phosphoryiated. Carbachol increases the tyrosine phosphorylation of villin rapidly (as early as 30 s) and transiently. Carbachol also increases the amount of tyrosine-phosphorylated villin that associates with PLC-γ₁. These studies demonstrate that carbacbol effects on NaCl absorption are accompanied by an increase in brush border PLC-γ₁ association with villin and an increase in tyrosine phosphorylation of villin. To study the role of cytoskeletal rearrangement in carbachol-induced inhibition of NaCl absorption, we used the F-actin stabilizing drug jasplakinolide. Jasplakinolide prevents the carbachol inhibition of ileal NaCl absorption. This suggests that F-actin severing is necessary for carbachol to inhibit ileal villus NaCl absorption. Since villin is known to sever actin, these studies suggest a role for villin in the signaling cascade that begins at the basolateral membrane with carbachol binding to its receptor and ends at the apical membrane in inhibition of NaCl absorption.