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 alpha1, alpha2 and beta1 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 alpha1 and beta1 subunits and quantified alpha1, alpha2 and beta1 mRNA by reverse transcription coupled to a polymerase chain reaction (RT-PCR). Expression of the beta1 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 alpha1, while alpha2 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; alpha2 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 alpha2 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.
Jurado
Sandra
S
Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain.
Sanchez-Prieto
Jose
J
Torres
Magdalena
M
D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D002531:Cerebellum / Q000201:enzymology* / Q000254:growth & development; D006152:Cyclic GMP / Q000096:biosynthesis*; D000076122:Disks Large Homolog 4 Protein; D005260:Female; D006162:Guanylate Cyclase; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I; D021122:Protein Subunits / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000071756:Soluble Guanylyl Cyclase; D013570:Synaptic Membranes / Q000378:metabolism
D000076122:Disks Large Homolog 4 Protein; C501449:Dlg4 protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D021122:Protein Subunits; D018160:Receptors, Cytoplasmic and Nuclear; D016194:Receptors, N-Methyl-D-Aspartate; C033652:postsynaptic density proteins; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase; D006152:Cyclic GMP