Membrane depolarization induces protein kinase C translocation and voltage operated calcium channel opening in PU5-1.8 cells. Protein kinase C as a negative feedback modulator for calcium signalling.
Incubation of the murine macrophage tumour cell line PU5-1.8 in K+ (140 mM)-HEPES buffer induced depolarization of the membrane and the translocation of protein kinase C (PKC) to the subnuclear region. Membrane depolarization also induced an increase of intracellular free Ca2+ levels ([Ca2+]i) which was due to the Ca2+ influx. The amount of K(+)-mediated Ca2+ uptake was dependent on the Ca2+ concentration gradient as measured by indo-1 fluorescence and 45Ca2+ fluxes. The depolarization-mediated Ca2+ influx was suppressed by voltage sensitive Ca2+ channel blockers such as nifedipine and verapamil. Furthermore, in Na(+)-HEPES buffer, incubation of cells with a dihydropyridine agonist [3H]PN200-110 produced a dose-dependent saturable binding. On the other hand, short-term incubation of cells with phorbol 12-myristate 13-acetate (PMA) abolished the early phase of 45Ca2+ influx and the rise of indo-1 fluorescence. Depleting cells of PKC or incubating them with PKC inhibitors, H7 and sphingosine, enhanced the uptake of 45Ca2+ and the rise of indo-1 fluorescence. These observations suggest that membrane depolarization caused an activation of PKC and induced Ca2+ influx through the activation of dihydropyridine-sensitive, voltage-operated Ca2+ channels. Data also show that PKC may act as a negative modulator in controlling the Ca2+ response by closing the voltage-operated Ca2+ channel and/or by enhancing the Ca(2+)-ATPase activity during membrane depolarization in PU5-1.8 cells.
Kong
S K
SK
Department of Biochemistry, Chinese University of Hong Kong, Shatin, NT.
Choy
Y M
YM
Fung
K P
KP
Lee
C Y
CY
D000818:Animals; D002118:Calcium / Q000037:antagonists & inhibitors / Q000502:physiology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000502:physiology*; D002462:Cell Membrane / Q000502:physiology; D004789:Enzyme Activation; D005456:Fluorescent Dyes; D008264:Macrophages / Q000502:physiology; D008564:Membrane Potentials; D051379:Mice; D008807:Mice, Inbred BALB C; D011494:Protein Kinases / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D014407:Tumor Cells, Cultured
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D005456:Fluorescent Dyes; D011494:Protein Kinases; D002118:Calcium