Differential expression of NO-sensitive guanylyl cyclase subunits during the development of rat cerebellar granule cells: regulation via N-methyl-D-aspartate receptors

In primary cultures of rat cerebellar granule cells with a functional network of glutamatergic neurons, the expression pattern of the different subunits of nitric-oxide (NO)-sensitive guanylyl cyclase changes during cell differentiation. These cells express the ₁, ₂ and ₁ subunits of NO-sensitive guanylyl cyclase and synthesize cyclic guanosine monophosphate (cGMP) in response to exogenous or endogenous nitric oxide. In this study, we determined the protein content of the ₁ and ₁ subunits and quantified ₁, ₂ and ₁ mRNA by reverse transcription coupled to a polymerase chain reaction (RT-PCR). Expression of the ₁ subunit increased with the degree of cell differentiation, although most marked changes occurred at the subunit level. In cells freshly isolated from rat pups on postnatal day 7 (P7) the most abundant subunit was ₁, while ₂ appeared as the predominant subunit of this type in cultured cells. N-methyl-D-aspartate (NMDA) receptor stimulation in 7- or 14-day-cultured cells led to the upregulation of guanylyl cyclase subunit mRNAs; ₂ mRNA levels undergoing most significant change. This enhanced subunit expression was accompanied by an increase in the amount of cGMP synthesized in response to NO. Thus, it seems that ₂ subunits are increasingly expressed as granule cells mature. The presence of this subunit in the guanylyl cyclase heterodimer facilitates its localization at synaptic membranes, where the enzyme acts as a sensor for NO formed by the postsynaptic protein 95 (PSD-95)-associated neuronal NO synthase.