Effects of aminoglycosides on proximal tubule brush border membrane phosphatidylinositol-specific phospholipase C

Rat kidney proximal tubule brush border membrane (BBM)-associated phosphatidylinositol-specific phospholipase C (PI-PLC) has been characterized previously in our laboratory. Here we report the effect of aminoglycosides on this enzyme. Enzyme activity is determined at 37° C by increases in diacylglycerol or decreases in PI in the presence of Ca⁺⁺, deoxycholate and [³H] arachidonate-labeled phospholipids in Tris buffer. Whereas activity of PI-PLC is inhibited 90% by gentamicin (1.5 mM) at pH 6 to 7, inhibition decreases to 72% at pH 7.4 and to zero at pH 7.8. As pH is raised from 7.8 to 9.5, gentamicin elicits a pH-dependent stimulation of activity. Alterations in Ca⁺⁺ concentration (1-7.5 mM) have no effect on inhibition of PI-PLC by gentamicin, although the enzyme itself is critically dependent on divalent cations. Double reciprocal plots of activity vs. substrate concentration in the presence of 0, 1.0 and 1.5 mM gentamicin demonstrate uncompetitive interaction between enzyme and drug. V(max) of PI-PLC in the absence of gentamicin is 0.73 μmol/h/mg of protein and the K(m) is 7.05 μM (PI). V(max) and apparent K(m) decrease with increasing drug concentration. Comparative inhibition of PI-PLC by other aminoglycosides (at 1-2 mM concentrations) approximates their known nephrotoxic potential: streptomycin ≤ kanamycin ≤ amikacin < tobramycin < gentamicin < neomycin. Kidney cortex cytosolic PI-PLC activity is also inhibited by gentamicin. However, BBM PI-PLC specific activity is 15 to 20-fold greater than for the cytosolic enzyme. Because marked gentamicin binding to BBM and damage/loss of BBM occurs early after administration of the drug, gentamicin-induced modulation of BBM PI-PLC may be an important factor in ensuing nephrotoxicity.