ENHANCED EXPRESSION OF CA2-FACTOR AND THE V-SRC ONCOGENE IN RAT PHEOCHROMOCYTOMA CELLS( CHANNELS BY NERVE GROWTH)

1. Rat phaeochromocytoma (PC12) cells were used to investigate the exp ression of Ca2+ channel types during neuronal differentiation. Neurona l differentiation was induced by treatment with nerve growth factor (N GF) or by activation of a temperature-sensitive tyrosine kinase (pp60v -src) in genetically modified PC12 (PC12/v-src) cells. PC12 cells diff erentiated morphologically in the presence of NGF. When grown at the p ermissive temperature of 37-degrees-C which activates the kinase activ ity of pp60v-src, PC12/v-src cells differentiated morphologically with the extension of neurites. In contrast, PC12/v-src cells grown at the non-permissive temperature of 40-degrees-C continued to divide and we re morphologically indistinguishable from control PC12 cells. 2. Whole -cell Ca2+ currents were measured in PC12 cells using Ba2+ as the char ge carrier. Ba2+ currents measured at the peak of the current-voltage curve from a holding potential of - 80 mV were - 0.28 +/- 0.04 nA (mea n +/- s.E.m.) in control PC12 cells compared to - 1.25 +/- 0.16 nA in NGF-differentiated cells. The current density increased from 9-4 +/- 0 .7 pA/pF in control PC12 cells to 22-8 +/- 2.4 pA/pF in NGF-differenti ated PC12 cells. Ba2+ currents were - 0.24 +/- 0.04 nA in undifferenti ated PC12/v-src cells grown at the non-permissive temperature of 40-de grees-C compared to -0.95 +/- 0.16 nA in differentiated PC12/v-src cel ls grown at the permissive temperature of 37-degrees-C. The current de nsity increased from 4.5 +/- 0.5 pA/pF in PC12/v-src cells grown at th e non-permissive temperature of 40-degrees-C to 13.3 +/- 2.4 pA/pF in PC12/V-src cells grown at the permissive temperature of 37-degrees-C. 3. The sensitivity of Ba2+ currents to omega-conotoxin GVIA (omega-CgT X) was determined for currents measured at the peak of the current-vol tage curve (0 mV in 10 mm Ba2+) from a holding potential of - 80 mV. I n NGF-differentiated PCl 2 cells, 10 mum omega-CgTx inhibited 68.1 +/- 3.2 % of the total Ba2+ current compared to 35.9 +/- 4.1 % in control cells. The density of the omega-CgTX-sensitive current increased from 3.3 +/- 0.4 pA/pF in control cells to 15.7 +/- 2.0 pA/pF in NGF-diffe rentiated cells. In differentiated PC12/v-src cells grown at 37-degree s-C, omega-CgTX inhibited 52.2 +/- 4.2 % of total Ba2+ current compare d to 41.1 +/- 3-8% in PC12/v-src cells grown at 40-degrees-C. The dens ity of the omega-CgTX-sensitive current increased from 1.9 +/- 0.3 to 7.4 +/- 2.0 pA/pF with v-src-mediated differentiation. 4. Ba 2+ curren ts were separated into three components based upon their inhibition by Ca2+ channel antagonists. Ba 2+ currents were measured at the peak of the current-voltage curve from a holding potential of - 60 mV. Sequen tial additions of omega-CgTX and nifedipine separated the Ba 2+ curren ts into three components: omega-CgTX sensitive, nifedipine sensitive a nd omega-CgTX and nifedipine resistant. The Ba 2+ current in control P C12 cells was 27-2 +/- 4.0 % omega-CgTX sensitive, 33.8 +/- 1.1% nifed ipine sensitive and 39.0 +/- 2-4 % resistant. In NGF-differentiated PC 12 cells, the Ba2+ current was 55.8 +/- 5.3 % omega-CgTX sensitive, 8. 4 +/- 2.5% nifedipine sensitive and 35.8 +/- 6.0 % resistant. Similarl y, differentiated PC12/v-src cells grown at 37-degrees-C had a Ba2+ cu rrent which was 50.3 +/- 4.7 % omega-CgTX sensitive, 8.0 +/- 4.6 % nif edipine sensitive and 41.7 +/- 3.6 % resistant. Control PC12/v-src cel ls grown at 40-degrees-C had a Ba2+ current which was 30.2 +/- 4.6 % o mega-CgTX sensitive, 46.1 +/- 5.8 % nifedipine sensitive and 23.7 +/- 4.8% resistant. 5. Binding of I-125-omega-CgTX increased 6-fold in PC1 2 cells treated with NGF for 6 days compared to control cells. Differe ntiated PC12/v-src cells grown at 37-degrees-C showed a 4-fold increas e in I-125-omega-CgTX binding compared to control PC12/V-src cells gro wn at 40-degrees-C. Nifedipine did not displace I-125-omega-CgTX bindi ng indicating independent binding sites. 6. Electrophysiological, phar macological and radioligand binding studies indicate an enhanced expre ssion of omega-CgTX-sensitive N-type Ca 2+ channels in both NGF-treate d and v-src differentiated PC12 cells. Additionally, a current compone nt resistant to block by both omega-CgTX and nifedipine also showed an enhanced density in both NGF-treated and v-src-differentiated PC12 ce lls. The contribution of the nifedipine-sensitive L-type current to th e total whole-cell current decreased in both NGF-treated and V-src-dif ferentiated PC12 cells.