Interaction of neuronal nitric-oxide synthase with alpha 1-syntrophin in rat brain

Neuronal nitric-oxide synthase (nNOS) has a PSD-95/ Dlg/ZO-1 (PDZ) domain t hat can interact with multiple proteins. nNOS has been known to interact wi th PSD-95 and a related protein, PSD-93, in brain and with alpha 1-syntroph in in skeletal muscle in mammals. In this study, we have purified an nNOS-i nteracting protein from bovine brain using an affinity column made of Sepha rose conjugated with glutathione S-transferase-rat nNOS fusion protein and identified it as alpha 1-syntrophin by microsequencing. Immunostaining of p rimary cultures of rat embryonic brain neuronal cells with antibodies again st these proteins showed that nNOS and alpha 1-syntrophin were colocalized in neuronal cell bodies and neurites. Immunohistochemical analysis indicate d that the nNOS- and alpha 1-syntrophin-like immunoreactive substances were highly expressed in the rat hypothalamic suprachiasmatic nucleus (SCN) and paraventricular nucleus. In the SCN, nNOS- and alpha 1-syntrophin-like imm unoreactive substances were colocalized in the same neurons as detected by confocal microscopy. These results indicate that nNOS in brain interacts wi th alpha 1-syntrophin in specific neurons of the SCN and paraventricular nu cleus and that this interaction might play a physiological role in function s of these neurons.