The effect of the PKC inhibitor GF109203X on the release of Ca2+ from internal stores and Ca2+ entry in DDT1 MF-2 cells.

1. The effects of the specific protein kinase C (PKC) inhibitor, GF109203X, were measured on the cytoplasmic Ca2+ concentration ([Ca2+]i), and on histamine H1 receptor- and thapsigargin-mediated increases in [Ca2+]i in DDT1 MF-2 smooth muscle cells. 2. After pretreatment of cells with GF109203X (5 microM, 45 min), the histamine (100 microM)-induced initial rise in [Ca2+]i, representing Ca2+ mobilization from internal stores, was inhibited (by 59 +/- 7%). The slowly declining phase of the histamine induced Ca2+ response, reflecting Ca2+ entry, was enhanced (83 +/- 26%) in the presence of the PKC inhibitor. 3. The histamine induced release of Ca2+ from internal stores, measured after blocking Ca2+ entry with LaCl3 was inhibited by GF109203X in a concentration-dependent manner (IC50: 3.1 +/- 1.1 microM). 4. Histamine-induced formation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) was not changed in the presence of GF109203X. 5. The PKC activating phorbol ester, phorbol 12-myristate 13-acetate (PMA, 1 microM), strongly reduced histamine-induced Ins(1,4,5)P3 formation (58 +/- 16%). This effect was reversed by GF109203X (5 microM). Furthermore, PMA diminished histamine evoked Ca2+ release (50 +/- 6%) and blocked Ca2+ entry completely. 6. The rise in [Ca2+]i caused by blocking endoplasmic reticulum Ca2(+)-ATPase with thapsigargin (1 microM), was strongly reduced (57 +/- 3%) after pretreatment of cells with GF109203X. Downregulation of PKC by long-term pretreatment of cells with PMA (1 microM, 48 h) did not abolish this effect of GF109203X (48 +/- 3% inhibition). 7. In permeabilized DDT, MF-2 cells preloaded with 45Ca2+ in the presence of GF109203X, the amount of 45Ca2+ released by Ins(1,4,5)P3 (10 microM) was markedly reduced (42 +/- 9%). GF109203X did not release Ca2+ itself and did not impair Ins(1,4,5)P3 receptor function. 8. Uptake of 45Ca2+ by intact cells, representing Ca2+ entry, was enhanced by GF109203X (65 +/- 11%), by histamine (24 +/- 6%) and also by thapsigargin (121 +/- 10%). The GF109203X- and the thapsigargin-induced uptake of 45Ca2+ were not additive. 9. These data suggest that GF109203X reduces the filling-state of intracellular Ins(1,4,5)P3 sensitive Ca2+ stores by inhibiting the Ca2+ uptake into these stores, thereby promoting store-dependent (capacitive) Ca2+ entry.