Induction of cyclic AMP and cyclic GMP 3′:5′‐cyclic nucleotide phosphodiesterase activities in neuroblastoma lines under differentiating conditions

Abstract

It is now widely accepted that cyclic nucleotide phosphodiesterases (PDEs) play fundamental roles in signal transduction pathways; they show a remarkable molecular complexity, different tissue distribution and complex regulatory mechanisms. Here we report PDE isoforms expression in two dibutyryl cyclic AMP differentiated murine cell lines: the hybrid neuroblastoma-glioma 108CC15 and the parental neuroblastoma N18TG2. They differ in the ability to establish functional synapses, a feature present only in the former. Ionic exchange chromatography elution profiles of N18TG2 and 108CC15 undifferentiated cell extracts show two main peaks of activity. The first one hydrolyzes cyclic GMP and is specifically inhibited by Zaprinast, thus representing a member of the PDE5 family. The second peak hydrolyzes cyclic AMP and is significantly inhibited by rolipram, as all the PDE4 family members. The induction of differentiation by dibutyryl cyclic AMP in both clonal lines results in an increase of PDE activities only after 3 hr of treatment, suggesting that protein neosynthesis is involved. Interestingly in both clones, besides the increase in cyclic AMP hydrolyzing specific activity (3.1-fold in 108CC15 and 2.5-fold in N18TG2), we also observed an increase in cyclic GMP hydrolyzing activity (1.7-fold in 108CC15 and 4.3-fold in N18TG2). While the induction of PDE4, previously reported also in other cellular systems, could be considered as a feedback response to the higher cyclic AMP levels, this is not true for the isoform that hydrolyzes cyclic GMP. These data suggest that the induction of PDE isoforms in neuroblastoma cells could be related to the activation of neuronal differentiative pathway.

cAMP‐dependent induction of PDE5 expression in murine neuroblastoma cell differentiation

“…A similar mechanism may be operating in N18TG2 cells, where no increase in PDE5 mRNA level has been observed. However, the timing of enzyme induction in N18TG2 cells, which is not rapidly attained [18], is not consistent with a post‐translational regulation of the enzyme. It may be therefore proposed that in these cells cAMP‐induced activation of PDE5 requires the transcriptional regulation of an unknown regulatory element, possibly of the type described in airway smooth cells [36].…”

“…In both cell lines their activity is higher in the presence of dbcAMP [17, 18]. The time course of PDE4 and PDE5 activity induction suggested a possible involvement of transcription in the mechanisms of enzyme regulation activated by cAMP [18]. We have focused our attention on the induction of PDE5 since only post‐translational regulation by cAMP has so far been reported for this PDE isoform [35, 36], while cAMP activation of PDE4 transcription is known [33, 34].…”

“…Both mouse and bovine cDNA probes were then used to establish whether the differentiating agent dbcAMP elicited a higher transcription of the PDE5 gene, as suggested by the timing of enzyme activity induction [18].…”

“…Extraction of the PDE activity was performed as previously described [18]. Cells were homogenized at 4°C on 20 mM Tris‐HCl buffer pH 7.2, containing 0.2 mM EGTA, 5 mM MgCl 2 , 1 mM phenylmethylsulfonylfluoride (PMSF), 5 mM β‐mercaptoethanol, 10 μg/ml leupeptin, 5 μg/ml bestatin, 10 μg/ml pepstatin A, 0.1% Triton X‐100, in a glass‐teflon homogenizer with 20 strokes.…”

“…Among them, little is known on PDE5 (cGMP‐binding cGMP‐specific) expression in the nervous system. It has been described in neuronal cell lines [17, 18], in rat superior cervical ganglion [19]and in cerebellum Purkinje cells [20].…”