CONSTITUTIVE UP-REGULATION OF CALCIUM-CHANNEL CURRENTS IN RAT PHEOCHROMOCYTOMA, CELLS - ROLE OF C-FOS AND C-JUN

1. Northern blot analysis and cell transfection were used in conjunction with whole-cell current recordings to examine the involvement of the immediate early genes, c-fos and c-jun, in the expression of calcium channel currents. 2. Phaeochromocytoma cells (PC12 clone) were exposed to nerve growth factor (NGF) and to depolarizing concentrations of KCl for 60 min every day. Cells challenged with NGF developed extensive networks of neurites within 3 days. Cells depolarized periodically retained their undifferentiated morphology even after 5 days of treatment. 3. The maximal amplitude of high-voltage-activated calcium currents (I-Ca) increased from the control level of 117.8 +/- 48.3 (mean +/- S.D.) to 387.2 +/- 90.1 pA within 3 days of NGF treatment. omega-Conotoxin (5-10 mu M) inhibited 24.6 +/- 8.5% of I-Ca in undifferentiated cells and 57.8 +/- 6.9% in NGF-treated cells. 4. The levels of c-fos and c-jun mRNAs increased transiently during each daily exposure to NGF. The level of c-fos mRNA also increased transiently during repeated KCl-induced depolarizations but c-jun mRNA remained low or absent. 5. Naive PC12 cells were transiently co-transfected with expression plasmids that contained the full length of c-fos and c-jun cDNA. After 2 days following transfection, the PC12 cells could be grouped according to the size of I-Ca. In 58% of cells, I-Ca was similar to control currents (106.1 +/- 37.4 pA). In the remaining 44% of cells, I-Ca showed a 2.2-fold enhancement with respect to control cells. Transfection of only c-fos had no effect on I-Ca but, in 24% of cells transfected with c-jun, I-Ca was 176.6 +/- 124.6 pA. Since periodic membrane depolarization induced c-fos but not c-jun mRNA, c-jun transfection was combined with a high-K+ treatment over 3 days. In 18% of treated cells, I-Ca, was 3.7 times larger than control currents. Morphological differentiation was not observed in transfected cells. 6, In PC12 cells co-transfected with c-fos and c-jun or treated with high K+ after transfection of c-jun, omega-conotoxin (5-10 mu M) inhibited 68.7 +/- 11.9% of I-Ca when the current amplitude was in the range of 200-600 pA. Since similar concentrations of omega-conotoxin blocked 19.2 +/- 5.4% of I-Ca in control cells, the current increase induced by c-fos and c-jun was supported by up to 11-fold enhancement of the omega-conotoxin-sensitive component of 7. The transfection experiments suggest that c-fos and c-jun, can regulate membrane calcium conductances in a time frame of days. Renewed induction of both c-fos and c-jun may be required for sustained enhancement of I-Ca.