Involvement of PI3′‐K, mitogen‐activated protein kinase and protein kinase B in the up‐regulation of the expression of nNOSα and nNOSβ splicing variants induced by PRL‐receptor activation in GH3 cells

It is well known that GH‐PRL secreting GH3 cells express constitutive neuronal nitric oxide synthase (nNOS) and produce nitric oxide (NO•). In addition, these cells possess plasma membrane prolactin (PRL) receptors which can be responsible for an autocrine ‘short‐loop’ feedback. The aim of the present study was to investigate whether the activation of PRL receptors modulates the expression of the different spliced forms of nNOS gene, and the transductional mechanisms involved in this action. In GH3 cells, both exon 2‐containing nNOSα and exon 2‐lacking nNOSβ were time‐dependently expressed, whereas the other two isoforms eNOS and iNOS were not. The antibodies directed against the residues 53–68 of the external domain common to both the long and short form of rat PRL receptors, and the selective D2 agonist cabergoline (1 nm) reduced both basal and exogenous PRL‐induced expressions of nNOSα and nNOSβ, but to a greater extent for the β splicing form. In line with these results, oPRL (1 and 10 μm) added to the incubation medium increased to a greater extent the expression of nNOSβ form than of the nNOSα. The receptor and non‐receptor protein tyrosine kinase (PTK) inhibitors, genistein (10 μm), the Src‐specific tyrosine kinase inhibitor PP2 (100 μm), the MAPK inhibitor PD 098059 (50 nm) and the two PI3′‐K inhibitors, wortmannin (300 nm) and LY‐294002 (25 μm) prevented both basal and exogenous PRL‐induced expression of nNOSα and nNOSβ isoforms. In addition, exogenous PRL induced a phosphorylation of protein kinase B (PKB) (Akt) that was prevented both by the two MAPK inhibitors PD 098059 and U 0126, and by the PI3′‐K inhibitors wortmannin and LY‐294002. Up‐regulation of the expression of the two splicing forms of nNOS elicited by PRL‐receptor activation was mirrored by the increased synthesis of NO•. In conclusion, PRL receptor activation up‐regulated the expression of both nNOSα and nNOSβ proteins via a PTK, PI3′‐K, MAPK and PKB signalling transduction components. This action may represent the molecular mechanism by which PRL exerts the ‘short‐loop’ feedback on its own secretion.