The role of cAMP-mediated intracellular signaling in regulating Na+ uptake in zebrafish larvae.

In the current study, the role of cAMP in stimulating Na(+) uptake in larval zebrafish was investigated. Treating larvae at 4 days postfertilization (dpf) with 10 μM forskolin or 1 μM 8-bromo cAMP significantly increased Na(+) uptake by three-fold and twofold, respectively. The cAMP-dependent stimulation of Na(+) uptake was probably unrelated to protein trafficking via microtubules because pretreatment with 200 μM colchicine or 30 μM nocodazole did not attenuate the magnitude of the response. Na(+) uptake was stimulated markedly following acute (2 h) exposure to acidic water. The acid-induced increase in Na(+) uptake was accompanied by a twofold elevation in whole body cAMP levels and attenuated by inhibiting PKA with 10 μM H-89. Knockdown of Na(+)-H(+) exchanger 3b (NHE3b) attenuated, but did not abolish, the stimulation of Na(+) uptake during forskolin treatment. In glial cell missing 2 morphants, in which the role of NHE3b in Na(+) uptake is diminished and the Na(+)-Cl(-) cotransporter (NCC) becomes the predominant route of Na(+) entry, forskolin treatment continued to increase Na(+) uptake. These data suggest that at least NHE3b and NCC are targeted by cAMP in zebrafish larvae. Staining of larvae with fluorescent forskolin and propranolol revealed the presence of transmembrane adenylyl cyclase within multiple subtypes of ionocytes expressing β-adrenergic receptors. Taken together, results of the present study demonstrate that cAMP-mediated intracellular signaling may regulate multiple Na(+) transporters and plays an important role in regulating Na(+) uptake in zebrafish larvae during acute exposure to an acidic environment.