Schistosoma mansoni: Possible involvement of protein kinase C in linoleic acid-induced proteolytic enzyme release from cercariae

Abstract

The possible involvement of protein kinase C and Ca 2+ metabolism in the proteolytic enzyme release from schistosome cercariae was studied. Cercariae were placed in dechlorinated tap water containing 0.37 m M calcium in the small glass petri dish and exposed to the stimuli-(linoleic acid, phorbol esters, and Ca 2+ ionophore) with or without inhibitors of protein kinase C or Ca 2+ metabolism. The proteolytic activity of incubation medium of cercariae thus treated was measured by the azocoll assay. The penetration response of cercariae induced by linoleic acid, a physiological stimulus, was mimicked by phorbol esters. When exposed to phorbol esters, 0.02 to 2 μM of 12- O-tetradecanoylphorbol-13-acetate (TPA) and 0.2 to 2 μM of phorbol-12,13-dibutyrate (PDBu), cercariae ceased the swimming movement, began a rhythmic thrusting of the anterior tip of the parasite, and released the proteolytic enzyme, but they did not shed the tails. Lowering Ca 2+ in water by addition of 5 m M ethylene glycol-bis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid (EGTA), phorbol ester-induced release of enzyme was completely inhibited. Phorbol ester-induced release of enzyme was partially inhibited by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), an inhibitor of protein kinase C, at a concentration of 100 μ M. H-7 alone, at a concentration of 100 μ M, did not affect the swimming movement of cercariae. The cercariae were stimulated to release the enzyme by high concentrations (10 and 100 μ M) of the Ca 2+ ionophore, A23187, but enzyme was not released by low concentrations (0.5 and 1 μ M) of this drug. Cercariae exposed to A23187 behaved differently from those exposed to phorbol esters. They ceased swimming, showed strong muscle contraction, and shed their tail. A23187 stimulated cercariae to release the enzyme in the water containing 5 m M EGTA. A23187-induced enzyme release was not inhibited by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin antagonist, trifluoperazine (TFP), a better calmodulin antagonist on schistosome, or by verapamil, a Ca 2+ channel blocker. Linoleic acid-induced release of enzyme was partially inhibited by 0.5 and 5 m M of EGTA and by 1 to 100 μ M of H-7. While it was not inhibited by N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8) and N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004), inhibitors of cyclic nucleotide-dependent protein kinase which were used as negative controls of H-7, W-7, TFP, 8-( N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8), an intracellular Ca 2+ antagonist, and verapamil. These results strongly suggest the involvement of protein kinase C in the proteolytic enzyme release from cercariae which come into contact with skin lipid. The mechanisms by which phorbol esters and linoleic acid stimulate cercariae to release the proteolytic enzyme and the necessity of Ca 2+ in release of proteolytic enzyme were discussed.