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The effect of the PKC inhibitor GF109203X on the release of Ca 2+ from internal stores and Ca 2+ entry in DDT 1 MF‐2 cells
Author(s) -
Sipma Henk,
Zee Lucie,
Akker Jan,
Hertog Adriaan,
Nelemans Adriaan
Publication year - 1996
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.1996.tb15733.x
Subject(s) - protein kinase c , thapsigargin , histamine , phorbol , endocrinology , medicine , chemistry , endoplasmic reticulum , biology , signal transduction , biochemistry
1 The effects of the specific protein kinase C (PKC) inhibitor, GF109203X, were measured on the cytoplasmic Ca 2+ concentration ([Ca 2+ ] i ), and on histamine H 1 receptor‐ and thapsigargin‐mediated increases in [Ca 2+ ] i in DDT 1 MF‐2 smooth muscle cells. 2 After pretreatment of cells with GF109203X (5 μ m , 45 min), the histamine (100 μ m )‐induced initial rise in [Ca 2+ ] i , representing Ca 2+ mobilization from internal stores, was inhibited (by 59 ± 7%). The slowly declining phase of the histamine induced Ca 2+ response, reflecting Ca 2+ entry, was enhanced (83 ± 26%) in the presence of the PKC inhibitor. 3 The histamine induced release of Ca 2+ from internal stores, measured after blocking Ca 2+ entry with LaCl 3 was inhibited by GF109203X in a concentration‐dependent manner (IC 50 :3.1 ± 1.1 μ m ). 4 Histamine‐induced formation of inositol 1,4,5‐trisphosphate (Ins(1,4,5)P 3 ) was not changed in the presence of GF109203X. 5 The PKC activating phorbol ester, phorbol 12‐myristate 13‐acetate (PMA, 1 μ m ), strongly reduced histamine‐induced Ins(1,4,5)P 3 formation (58 ± 16%). This effect was reversed by GF109203X (5 μ m ). Furthermore, PMA diminished histamine evoked Ca 2+ release (50 ± 6%) and blocked Ca 2+ entry completely. 6 The rise in [Ca 2+ ] i caused by blocking endoplasmic reticulum Ca 2+ ‐ATPase with thapsigargin (1 μ m ), was strongly reduced (57 ± 3%) after pretreatment of cells with GF109203X. Downregulation of PKC by long‐term pretreatment of cells with PMA (1 μ m , 48 h) did not abolish this effect of GF109203X (48 ± 3% inhibition). 7 In permeabilized DDT 1 MF‐2 cells preloaded with 45 Ca 2+ in the presence of GF109203X, the amount of 45 Ca 2+ released by Ins(1,4,5)P 3 (10 μ m ) was markedly reduced (42 ± 9%). GF109203X did not release Ca 2+ itself and did not impair Ins(1,4,5)P 3 receptor function. 8 Uptake of 45 Ca 2+ by intact cells, representing Ca 2+ 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 45 Ca 2+ were not additive. 9 These data suggest that GF109203X reduces the filling‐state of intracellular Ins(1,4,5)P 3 sensitive Ca 2+ stores by inhibiting the Ca 2+ uptake into these stores, thereby promoting store‐dependent (capacitive) Ca 2+ entry.