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

Abstract

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 α1,α 2 and β1 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 α1 and β1 subunits and quantified α1, α2 and β1mRNA by reverse transcription coupled to a polymerase chain reaction (RT-PCR). Expression of the β1 subunit increased with the degree of cell differentiation, although most marked changes occurred at the α subunit level. In cells freshly isolated from rat pups on postnatal day 7 (P7) the most abundant α subunit was α1, whileα 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; α2 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 thatα 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.

Suppression of guanylyl cyclase (β1 subunit) expression impairs neurite outgrowth and synapse maturation in cultured cerebellar granule cells

“…Although this experimental approach does not accurately define the location of sGC, these results indicate that active sGC is present in these presynaptic boutons, consistent with the presence of this enzyme in presynaptic terminals and close to the active zone. 11,12,30 The presence of sGC in neuronal processes and in synaptic boutons would also explain its increased expression during development 5 and its importance in regulating different steps of neuronal development.…”

“…33 In addition, GAP-43 mRNA expression is controlled by glutamatergic NMDA receptors, 34 and the formation of NO and cGMP is one consequence of NMDA receptor stimulation. 5,35 We previously demonstrated that NMDA receptor stimulation regulates the stability of different mRNAs via the NO/cGMP pathway, altering the levels of the AUF1 proteins that bind to and destabilize them. 23 Such a process might be active in this paradigm as GAP-43 mRNA has a long 3 0 UTR bearing specific determinant pyrimidinerich sequences that AUF1 can bind to, thereby destabilizing this mRNA.…”

“…4 The expression of the different guanylyl cyclase subunits (sGC), the main physiological receptor for nitric oxide (NO), augments throughout development in different areas of the rat brain and in in vitro models of neuronal maturation. [5][6][7] Accordingly, these proteins may be important for the formation and growth of neuronal processes, as well as for the establishment of intercellular contacts and the refinement of functional neuronal connections within the peripheral and central nervous system. 1,8,9 There are two known isoforms of sGC, the ubiquitous a 1 b 1 and a 2 b 1 , which has a more limited distribution.…”

“…20 The expression of both the nNOS and sGC (subunits-b1 and -a2) augments during the development of rat cerebellar granule cells in vitro, as well as in the cerebellum. 5,21 Moreover, NMDA receptor stimulation, which is essential for the survival and differentiation of developing cerebellar granule cells, 22 upregulates the expression of these proteins in vitro. 5,23 Together, these data reflect the importance of such proteins for the correct development of cerebellar granule cells.…”

“…5,21 Moreover, NMDA receptor stimulation, which is essential for the survival and differentiation of developing cerebellar granule cells, 22 upregulates the expression of these proteins in vitro. 5,23 Together, these data reflect the importance of such proteins for the correct development of cerebellar granule cells. Given that the b 1 subunit of sGC is the obligatory dimerization partner for both a-subunits to render a functional enzyme, we analyzed the effects of knocking down sGCb1 in granule cells.…”