Adaptation of cAMP signaling system in SH-SY5Y neuroblastoma cells following expression of a constitutively active stimulatory G protein alpha, Q227L Gsalpha

Heterotrimeric GTP-binding proteins (G protein) are known to participate in the transduction of signals from ligand activated receptors to effector molecules to elicit cellular responses. Sustained activation of cAMP-G protein signaling system by agonist results in desensitization of the pathway at receptor levels, however it is not clear whether such receptor responses induce other changes in post-receptor signaling path that are associated with maintenance of AMP levels, i.e. cAMP-forming adenylate cyclase (AC), cAMP-degrading cyclic nucleotide phosphodiesterase (PDE) and cAMP-dependent protein kinase (PKA). Experiments were performed to determine the expression of AC, PDE, and PKA isoforms in SH-SY5Y neuroblastoma cells, in which cAMP system was activated by expressing a constitutively activated mutant of stimulatory G protein (Q227L Gsalpha). Expression of ACI mRNA was increased, but levels of ACVIII and ACIX mRNA were decreased. All of the 4 expressed isoforms of PDE (PDE1C, PDE2, PDE 4A, and PDE4B) were increased in mRNA expression; the levels of PKA RIalpha, RIbeta, and RIIbeta were increased moderately, however, those of RIIalpha and Calpha were increased remarkably. The activities of AC, PDE and PKA were also increased in the SH-SY5Y cells expressing Q227L Gsalpha. The similar changes in expression and activity of AC, PDE and PKA were observed in the SH-SY5Y cells treated with dbcAMP for 6 days. Consequently, it is concluded that the cAMP system adapts at the post-receptor level to a sustained activation of the system by differential expression of the isoforms of AC, PDE, and PKA in SH-SY5Y neuroblastoma. We also showed that an increase in cellular cAMP concentration might mediate the observed changes in the cAMP system.

Multi-facet expressions of adenylate cyclase isoforms in B16-F10 melanoma cells differentiated by forskolin treatment

The terminal differentiation of malignant melanoma cells is known to be induced by activating cAMP signaling pathway with alpha-MSH or cAMP analogues. However, sustained activation of cAMP signaling system that induces the differentiation of melanoma cells, also induces the desensitization of the pathway at the receptor level. Nevertheless, the adaptation of adenylate cyclase (AC) expression by sustained activation of cAMP signaling system has not been clearly understood. This study was performed to examine whether the sustained activation of cAMP system induce changes in the expression AC isoforms as an adaptation mechanism. Treatment of B16/F10 murine melanoma cells with 100 mM forskolin for 6 days resulted in differentiation, melanin accumulation and increased expression of tyrosine hydroxylase mRNA. In the forskolin-treated melanoma cells, change in expression of various AC isoform at the transcription level was detected by reverse-transcription polymerase chain reaction (RT-PCR). Expression of AC isoform mRNA: ACI, III, VI, VII, and IX increased to the level of 196-392% of the control whereas the level of ACII was decreased by 30%. The cAMP concentration was increased both in basal and alpha-MSH stimulated cells, but the AC activity was decreased in the forskolin treated cells. Thus, these results suggest that sustained activation of cAMP system induces differential expression of AC isoforms, which results in increase of cAMP accumulation.