A Chloride Ion Channel in Halocynthia roretzi Hemocytes is Associated with PO Activity but Not Pigmentation During the Contact Reaction

Abstract

When hemocytes of two different individuals of the solitary ascidian Halocynthia roretzi come into contact (allogeneic recognition), they devacuolate in several seconds following contact, release phenoloxidase (PO) into the supernatant, and form coagulates. These coagulates show brown pigmentation. This reaction is referred to as the contact reaction (CR). In this study, the CR-inhibitory monoclonal antibody ku-4-96, which inhibits devacuolation, increase in PO activity, coagulation, and pigmentation, was constructed. This antibody is thought to exert its inhibitory action at an early stage in the CR. A differential display analysis was conducted by using ku-4-96 to search exhaustively for differentially expressed genes involved in the CR. One of the genes cloned was downregulated in the presence of ku-4-96 and upregulated during the CR. This gene showed very high similarity to the Cl(-) channel gene ClC-2 and was named HrClC-2. We examined the effects of Cl(-) channel inhibitors on the CR to examine whether the Cl(-) channel was involved in the CR signal cascade. Devacuolation, coagulation, and pigmentation were not affected by different concentrations of these inhibitors, which inhibited PO activity. This suggests that the PO activity is independent of these other phenomena occurring during the CR.