Regulation of nitric-oxide production in hemocytes of the ascidian Phallusia nigra

• Eight types of blood cells (hemocytes) were identified in the hemolymph of the ascidian Phallusia nigra.
• The hemocytes involved in nitric oxide (NO) production were identified.
• Zymosan A and LPS stimulated NO production in P. nigra hemocytes.
• NO is related to regulation of host defense mechanisms in ascidians.
• PKC, PKA and NFκB play an important role in NO synthase activation and subsequent NO production.

Nitric oxide (NO) production in ascidians is related to immune responses of blood cells, and also to events such as egg fertilization and notochord regression. However, the signaling pathway for NO generation has been little investigated in this animal model. The present contribution identifies the cells involved in NO production and provides new information about a pathway for NO signaling. We were able to identify eight types of blood cells in the hemolymph of the ascidian Phallusia nigra, of which signet ring cells, univacuolar refractile granulocytes, and morula cells were involved in NO production. Zymosan A and lipopolysaccharide (LPS) enhanced NO production by blood cells, and the compound N-nitro-l-arginine-methyl ester (l-NAME) reduced NO production. Finally, the application of protein kinase A (PKA) and protein kinase C (PKC) inhibitors revealed that these molecules participate, together with NFκB, in the regulation of NO production by blood cells of P. nigra. This is the first report to show that PKA and PKC are involved in a signaling pathway that leads to NO production in ascidian blood cells.

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