Calcitonin stimulates lysosomal enzyme release and uptake in LLC-PK1 cells

Renal tubular targeted hormones increase urinary excretion of a lysosomal e nzyme, N-acetyl-beta-D-glucosaminidase (NAG). To elucidate the mechanism of this event, the calcitonin effect on NAG handling by LLC-PK1 cells was exa mined. Calcitonin(1 nM to 1 mu M), phorbol myristate (10 nM to 1 mu M), and ionomycin (1 to 10 mu M) promoted NAG release without any increase in lact ate dehydrogenase release or any reduction of mitochondrial dehydrogenase a ctivity. Treatment with 100 nM calphostin C or 50 mu M KN-93 partially reve rsed the calcitonin effect on NAG release. Calcitonin promoted secretion of fluorescence ceramide, a reporter of protein transport from Golgi apparatu s to cell surface. Calcitonin-stimulated NAG release was partially inhibite d by 10 mu g/ml brefeldin A, a blocker of protein transport through the Gol gi apparatus. Calcitonin accelerated cellular uptake of exogenous NAG, whic h was inhibited by low temperature, 0.1 mM monodansyl cadaverine (receptor- mediated endocytosis inhibitor), and 10 mM mannose-6-phosphate, Furthermore , calcitonin promoted progression of intracellular membranes stained by a f luorescence membrane marker, styryl pyridinium dye, from cell periphery to perinuclear regions (commonly referred to as recycling vesicles) and increa sed dye release from preloaded cells. Fluorescence release from the cells p reloaded with FITC-labeled NAG or albumin was also stimulated by calcitonin . These calcitonin effects on endocytotic and re-exocytotic pathways were i nhibited by 100 nM cytochalasin D, 100 nM nocodazole, 0.1 to 1 mu M bafilom ycin Al, or 0.1 mM monodansyl cadaverine. Increased urinary NAG excretion h as been considered to reflect renal tubular damage. However, it was demonst rated here that stimulation of secretory and recycling pathways may be an a lternative mechanism for calcitonin-induced enzymuria, which will become a new indicator of renal tubular response to this hormone.