INDUCTION OF CA2+ CALMODULIN-STIMULATED CYCLIC-AMP PHOSPHODIESTERASE (PDE1) ACTIVITY IN CHINESE-HAMSTER OVARY CELLS (CHO) BY PHORBOL 12-MYRISTATE 13-ACETATE AND BY THE SELECTIVE OVEREXPRESSION OF PROTEIN-KINASE-C ISOFORMS/

The cAMP phosphodiesterase (PDE) activity of CHO cells was unaffected by the addition of Ca2+ + calmodulin (CaM), indicating the absence of any PDE1 (Ca2+/CaM-stimulated PDE) activity. Treatment with the tumour promoting phorbol ester phorbol 12-myristate 13-acetate (PMA) led to the rapid transient induction of PDE1 activity which attained a maximu m value after about 13 h before slowly decreasing. Such induction was attenuated by actinomycin D. PCR primers were designed to hybridize wi th two regions identified as being characteristic of PDE1 forms found in various species and predicted to amplify a 601 bp fragment. RT-PCR using degenerate primers allowed an approx. 600 bp fragment to be ampl ified from RNA preparations of rat brain but not from CHO cells unless they had been treated with PMA. CHO cells transfected to overexpress protein kinase C (PKC)-alpha and PKC-epsilon, but not those transfecte d to overexpress PKC-beta 1 or PKC-gamma, exhibited a twofold higher P DE activity. They also expressed a PDE1 activity, with Ca2+/CaM effect ing a 1:8-2.8-fold increase in total PDE activity. RT-PCR, with PDE1-s pecific primers, identified an approx. 600 bp product in CHO cells tra nsfected to overexpress PKC-alpha and PKC-epsilon, but not in those ov erexpressing PKC-beta I or PKC-gamma. Treatment of PKC-alpha transfect ed cells with PMA caused a rapid, albeit transient, increase in PDE1 a ctivity, which reached a maximum some Ih after PMA challenge, before r eturning to resting levels some 2 h later. The residual isobutylmethyl xanthine (IBMX)-insensitive PDE activity was dramatically reduced (app rox. 4-fold) in the PKC-gamma transfectants, suggesting that the activ ity of the cyclic AMP-specific IBMX-insensitive PDE7 activity was sele ctively reduced by overexpression of this particular PKC isoform. Thes e data identify a novel point of 'cross-talk' between the lipid and cy clic AMP signalling systems where the action of specific PKC isoforms is shown to cause the induction of Ca2+/CaM-stimulated PDE (PDE1) acti vity. It is suggested that this protein kinase C-mediated process migh t involve regulation of PDE1 gene expression by the AP-1 (fos/jun) sys tem.