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First published online 10 June 2003
doi: 10.1242/jcs.00620

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Journal of Cell Science 116, 3165-3175 (2003)
doi: 10.1242/jcs.00620

Research Article

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

Sandra Jurado, José Sánchez-Prieto and Magdalena Torres*

Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain

* Author for correspondence (e-mail: mitorres{at}vet.ucm.es)

Accepted 15 April 2003

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 {alpha}1, {alpha}2 and ß1 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 {alpha}1 and ß1 subunits and quantified {alpha}1, {alpha}2 and ß1 mRNA by reverse transcription coupled to a polymerase chain reaction (RT-PCR). Expression of the ß1 subunit increased with the degree of cell differentiation, although most marked changes occurred at the {alpha} subunit level. In cells freshly isolated from rat pups on postnatal day 7 (P7) the most abundant {alpha} subunit was {alpha}1, while {alpha}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; {alpha}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 {alpha}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.

Key words: Cerebellum, Granule cells, NO-sensitive guanylyl cyclase, N-methyl-D-aspartate receptors




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