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 post fertilization (dpf) with 10 µM forskolin or 1 µM 8-bromo cAMP significantly increased Na+ uptake by 3- and 2-fold, respectively. The cAMP-dependent stimulation of Na+ uptake was probably unrelated to protein trafficking via microtubules because pre-treatment 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 2-fold elevation in whole body cAMP levels and attenuated by inhibiting protein kinase A (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- co-transporter (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 trans-membrane adenylyl cyclase within multiple subtypes of ionocytes expressing beta-adrenergic receptors. Taken together, results of the present study demonstrate that cAMP-mediated intracellular signalling may regulate multiple Na+ transporters, and plays an important role in regulating Na+ uptake in zebrafish larvae during acute exposure to an acidic environment.