Protein kinase C is involved in mediating the effects of elevated Ca2+ in ileal villus Na+ absorbing cells to inhibit NaCI absorption. The present studies were undertaken to understand the mechanism by which this occurs. The effects of carbachol and the calcium ionophore A23187, agents which elevate intracellular Ca2" and inhibit NaCl absorption in ileal villus cells, were studied. Carbachol treatment of villus cells caused a rapid decrease in protein kinase C activity in cytosol, with an accompanying increase in microvillus membrane C kinase. Exposure of the villus cells to calcium ionophore also caused a quantitatively similar decrease in cytosol C kinase and increase in C kinase activity in the microvillus membrane. This increase caused by carbachol and Ca2+ ionophore was specific for the microvillus membrane. In fact, 30 s and 10 min after exposure of the cells to carbachol, basolateral membrane protein kinase C decreased, in a time-dependent manner, whereas 10 min of Ca2" ionophore exposure did not alter basolateral C kinase. Exposure of villus cells to Ca2" ionophore or carbachol caused similar increases in microvillus membrane diacylglycerol content. As judged by the ability to inhibit Na+/H+ exchange measured in ileal villus cell brush border membrane vesicles, the protein kinase C which translocated to the microvillus membrane was functionally significant. Inhibition of Na+/H+ exchange required ATP and was reversed by the protein kinase C antagonist H-7. In conclusion, the effect of carbachol and Ca2" ionophore in regulation of ileal NaCl absorption is associated with an increase in microvillus membrane diacylglycerol content and functionally active protein kinase C. The effects of both carbachol and Ca2" ionophore are different on brush border and basolateral membrane distribution of protein kinase C. (J. Clin. Invest. 1991. 88:855-863.)

Beal brush border membranes contain an endogenous Ca2+/cahnodulin (CaM)-dependent protein kinase activity that modulates the activity of the apical membrane Na+/H+ exchanger. To further characterize this kinase, synapsin I, a substrate for Ca2+/CaM-dependent protein kinases, was added to preparations of ileal brush border membranes. In the presence of Ca2+/CaM, synapsin I was phosphorylated.Phosphopeptide mapping demonstrated that the addition of Ca2+/CaM to brush border membranes stimulated the phosphorylation of sites in synapsin I specific for Ca2W/CaM-dependent protein kinase II. Immunoblots containing brush border and microvillus membrane proteins were probed with an antibody that recognizes the 50-kDa subunit of rat brain Ca2+/CaM-dependent protein kinase II. This antibody labeled major and minor species of 50 and 53 kDa, respectively, with more labeling of the brush border than the microvillus membranes. Right-side-out ileal villus cell brush border vesicles were prepared containing CaM, ATP, and 350 nM free Ca2 . Na+/H+ exchange was inhibited by the presence of Ca2+/ CaM/ATP within the vesicles. A 21-mino acid peptide inhibitor of CaM kinase II was enclosed within some vesicle preparations by freeze-thaw. The effect on Na+/H+ exchange of Ca2 /CaM/ATP was partially reversed by the inhibitor peptide. These studies demonstrate the presence of Ca2+/CaMdependent protein kinase II in rabbit ileal villus cell brush border membranes. Based on the effect of a specific inhibitor peptide of Ca2+/CaM kinase II, it is concluded that this kinase inhibits brush border Na+/H+ exchange, which participates in

Patients with AIDS-associated refractory watery diarrhea, especially those without identifiable pathogens, may respond favorably to subcutaneously administered octreotide. This drug deserves further study in a randomized, placebo-controlled trial.