Effects of gentamicin on sphingomyelinase activity in cultured human renal proximal tubular cells

We have previously shown that cultured human proximal tubular cells (PT) incubated with gentamicin contain numerous 'myeloid bodies'. This morphological change was accompanied by the storage of phosphatidylcholine and sphingomyelin. In order to delineate the biochemical mechanisms responsible for the accumulation of sphingomyelin in cells incubated with gentamicin, we pursued detailed studies on the activity of sphingomyelinase. Characterization studies on sphingomyelinase revealed that this enzyme has a bimodal pH optima in PT cells. Optimum activity was observed at pH 5.6 (designated as acid sphingomyelinase, A-SMase) and at pH 7.4 (designated as neutral sphingomyelinase, N-SMase). The activity of both the enzymes increased proportionately in control cells as a function of days of incubation. The activity of A-SMase was 16% lower in cells incubated with gentamicin as compared to control. The most striking observation was a gradual decline in the activity of N-SMase in cells incubated with gentamicin. Thus, following 21 days of incubation of cells with 0.3 mM gentamicin, the N-SMase was 2.7-fold lower than control cells. Mg²⁺ stimulated and Triton X-100 inhibited the activity of N-SMase. Whereas Mg²⁺ had no effects, Triton X-100 stimulated the activity of the A-SMase in PT cells. Moreover, A-SMase was relatively more heat-resistant than the N-SMase. The K(m) values for sphingomyelin using A-SMase in control cells and cells incubated with gentamicin were 0.07 x and 0.016 x 10^-7 M, respectively, whereas the K(m) values for sphingomyelin using N-SMase in control cells and cells incubated with gentamicin were 1.8 x and 1.5 x 10^-7 M, respectively. These findings suggest that gentamicin exerts a competitive inhibition of the A-SMase in PT cells. In contrast, gentamicin exerts a noncompetitive inhibition of the N-SMase in PT cells. Subcellular fractionation studies revealed that A-SMase was exclusively localized in the 'lysosome-rich' fraction, whereas most, if not all, the N-SMase was localized in the microsomal fraction and 'plasma-membrane'-rich fraction in cultured PT cells. Cells incubated with gentamicin for 21 days contained 25% lower activity of A-SMase associated with the lysosomal fraction as compared to control. In contrast, N-SMase activity in the microsomal and plasma membrane fraction was one-half as compared to control. We conclude that gentamicin-mediated decrease in sphingomyelinase activity may be responsible for the storage of sphingomyelin in cultured human PT cells. In this regard, we particularly emphasize the role played by the N-SMase in gentamicin-induced nephrotoxicity in cultured human PT cells, which is more than has been previously realized.