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 conjuncti on with whole-cell current recordings to examine the involvement of th e immediate early genes, c-fos and c-jun, in the expression of calcium channel currents. 2. Phaeochromocytoma cells (PC12 clone) were expose d to nerve growth factor (NGF) and to depolarizing concentrations of K Cl for 60 min every day. Cells challenged with NGF developed extensive networks of neurites within 3 days. Cells depolarized periodically re tained their undifferentiated morphology even after 5 days of treatmen t. 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-Conoto xin (5-10 mu M) inhibited 24.6 +/- 8.5% of I-Ca in undifferentiated ce lls 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. Nai ve 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 t he 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-ju n, 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 wit h 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 c oncentrations of omega-conotoxin blocked 19.2 +/- 5.4% of I-Ca in cont rol cells, the current increase induced by c-fos and c-jun was support ed by up to 11-fold enhancement of the omega-conotoxin-sensitive compo nent of 7. The transfection experiments suggest that c-fos and c-jun, can regulate membrane calcium conductances in a time frame of days. Re newed induction of both c-fos and c-jun may be required for sustained enhancement of I-Ca.