PROTOPORPHYRINS MODULATE VOLTAGE-GATED CA CURRENT IN ATT-20 PITUITARY-CELLS

1. Zinc-protoporphyrin-IX (ZnPP-IX) is an inhibitor of the enzyme heme-oxygenase-2 (HO-2) and consequently has been used to examine the role of carbon monoxide production in neural tissues. We have measured voltage-gated Ca current in AtT-20 pituitary cells using the whole-cell patch-clamp technique and have assessed the effects of extracellularly applied ZnPP-IX and related compounds. 2. Ca currents evoked by depolarizing steps from a holding potential of -90 mV were of the high-threshold, slowly inactivating type. Fifty-six percent of this current was blocked by 10 muM nifedipine and 16% by 3 muM omega-conotoxin with the remainder resistant to both drugs in combination, suggesting that the total Ca current was a mixture of L, N, and possibly P-type conductances. 3. Bath application of ZnPP-IX resulted in an irreversible, dose-dependent attenuation of Ca current. Five micromolar ZnPP-IX produced a 62% reduction of peak current amplitude with no shift in the current-voltage relation, 0.5 muM produced a 19% reduction, and 0.05 muM produced a variable response, either a small transient attenuation or potentiation. 4. The attenuation of Ca current by 5 muM ZnPP-IX could be nearly completely blocked by co-application of superoxide dismutase in the bath (90 U/ml) but not by addition of an inhibitor of cGMP-dependent protein kinase to the internal saline (KT5823, 1 muM). 5. Other inhibitors of heme-oxygenase with similar potency such as tin-protoporphyrin-IX (Sn-PP-IX) and Zn-deuteroporphyrin-bis-glycol (ZnBG) did not attenuate Ca current when applied at 5 muM. Rather, these compounds produced a small, transient and variable potentiation. A similar effect was also seen with 5 muM copper-protoporphyrin-IX (CuPP-IX), a compound which may not inhibit heme oxygenase. KT-5823 did not block the small, transient potentiation produced by 5 muM CuPP-IX. 6. Protoporphyrin-IX (PP-IX) is known to activate soluble guanylyl cyclase. Application of 5 muM PP-IX produced a large (55%) transient potentiation of Ca current with no shift in the current-voltage relation. This potentiation was nearly completely blocked by KT-5823. 7. These results indicate that ZnPP-IX attenuates voltage-gated Ca current, and suggest that it does so through a mechanism other than inhibition of HO-2 or soluble guanylyl cyclase, possibly one related to the production of free radicals. Furthermore, PP-IX potentiates voltage-gated Ca current through a mechanism that is likely to involve activation of soluble guanylyl cyclase and the consequent activation of cyclic guanosine 3,5'-monophosphate (cGMP)-dependent protein kinase.