H. Sipma et al., THE EFFECT OF THE PKC INHIBITOR GF109203X ON THE RELEASE OF CA2-2 CELLS( FROM INTERNAL STORES AND CA2+ ENTRY IN DDT1 MF), British Journal of Pharmacology, 119(4), 1996, pp. 730-736
1 The effect of the specific protein kinase C (PKC) inhibitor, GF10920
3X, were measured on the cytoplasmic Ca2+ concentration ([Ca2+](i)), a
nd on histamine H-1 receptor- and thapsigargin-mediated increases in [
Ca2+](i) in DDT1 MF-2 smooth muscle cells. 2 After pretreatment of cel
ls with GF109203X (5 mu M, 45 min), the histamine (100 mu M)-induced i
nitial rise in [Ca2+](i), representing Ca2+ mobilization from internal
stores, was inhibited (by 59 +/- 7%). The slowly declining phase of t
he histamine induced Ca2+ response, reflecting Ca2+ entry, was enhance
d (83 +/- 26%) in the presence of the PMC inhibitor. 3 The histamine i
nduced release of Ca2+ from internal stores, measured after blocking C
a2+ entry with LaCl3 was inhibited by GF109203X in a concentration-dep
endent manner (IC50:3.1 +/- 1.1 mu M). 4 Histamine-induced formation o
f 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 mu M), strongly reduced histamine-induced
ins(1,4,5)P-3 formation (58 +/- 16%). This effect was reversed by GF1
09203X (5 mu M). Furthermore, PMA diminished histamine evoked Ca2+ rel
ease (50 +/- 6%) and blocked Ca2+ entry completely. 6 The rise in [Ca2
+](i) caused by blocking endoplasmic reticulum Ca2+-ATPase with thapsi
gargin (1 mu M), was strongly reduced (57 +/- 3%) after pretreatment o
f cells with GF109203X. Downregulation of PKC by long-term pretreatmen
t of cells with PMA (1 mu M, 48 h) did not abolish this effect of GF10
9203X (48 +/- 3% inhibition). 7 In permeabilized DDT1 MF-2 cells prelo
aded with Ca-45(2+) in the presence of GF109203X, the amount of Ca-45(
2+) released by Ins(1,4,5)P-3 (10 mu M) was markedly reduced (42 +/- 9
%). GF109203X did not release Ca2+ itself and did not impair Ins(1,4,5
)P-3 receptor function. 8 Uptake of Ca-45(2+) by intact cells, represe
nting Ca2+ entry, was enhanced by GF109203X (65 +/- 11%), by histamine
(24 +/- 6%) and also by thapsigargin (121 +/- 10%). The GF109203X- an
d the thapsigargin-induced uptake of Ca-45(2+) were not additive. 9 Th
ese data suggest that GF109203X reduces the filling-state of intracell
ular Ins(1,4,5)P-3 sensitive Ca2+ stores by inhibiting the Ca2+ uptake
into these stores, thereby promoting store-dependent (capacitive) Ca2
+ entry.