RESUMO
The inducible nitric oxide synthase (iNOS) plays a central role in the inflammatory reactions that follow infection or tissue damage. Induction of nitric oxide (NO) synthesis by bacterial lipopolysaccharide (LPS) depends on activation of G protein-coupled receptors in mammals. Thus, it was our intention to evaluate whether similar mechanisms are involved in iNOS activation in fish leukocytes. Therefore, the participation of membrane-bound receptors which activate effectors via G proteins has been confirmed using the G protein inhibitor suramin. Furthermore, the NO produced by iNOS performs both beneficial and detrimental actions. It is thus conceivable that regulatory mechanisms exist which control the timing and intensity of NO production by iNOS in order to outweigh protective effects against detrimental ones. The second messenger cAMP produced by adenylyl cyclases (ACs) plays a key role in the regulation of many cellular functions. Since cAMP signaling inhibits numerous immunological reactions, studies have been carried out to determine whether cAMP-dependent pathways could inhibit NO production by carp leukocytes as well. To measure cellular responses such as NO production by carp leukocytes derived from head and trunk kidneys treatments were performed with the cAMP elevating agents forskolin and dibutyryl-cAMP (db-cAMP) prior to stimulation with Aeromonas hydrophila. Pharmacological studies in stimulated kidney leukocytes showed that increased intracellular cAMP levels lead to reduced NO formation. This reduction of NO production was not due to decreased cell numbers, since a tetrazolium dye-based assay revealed no reduction of cell viability by cyclic nucleotide elevating agents. Thus, our data provide evidence that the AC/cAMP signaling pathway is well established in carp leukocytes. Cyclic AMP leads to type II immune response. We provide evidence that the predominant AC in fish leukocytes is a particulate enzyme due to its sensitivity to forskolin. Treatment of leukocytes with agents increasing intracellular cAMP gave clear evidence for participation of this cyclic nucleotide in immune signaling.
