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Regulation of intracellular free calcium and protein kinase C in the motoneuron cell line NSC-19

University of British Columbia

Changes in the concentrations of intracellular free calcium ([Ca²⁺][sub i]) were determined in response to the metabolic inhibitors, Amytal and carbonyl cyanide /w-chlorophenylhydrazone (CCCP), and the relationship between [Ca²⁺][sub i] and protein kinase C (PKC) activation was investigated in the motoneuron cell line NSC-19. Amytal (5 mM) produced transient elevations of [Ca²⁺][sub i] of approximately 80 nM. CCCP (10 µM) produced sustained elevations of [Ca²⁺][sub i] of about 280 nM. These responses were reduced by 32% and 47%, respectively, when cells were studied in Ca²⁺-free solutions. In spite of the sustained elevation of [Ca²⁺][sub i] induced by CCCP, no reduction in cell viability was seen after 48 hours when compared to controls. These results indicate that exposure of NSC-19 cells to Amytal and CCCP produce Ca²⁺ increments by release from internal stores, as well as by transmembrane influx, and demonstrate that small increments in [Ca²⁺][sub i] due to metabolic inhibitors or other toxins may not be associated with immediate cell death. In Ca²⁺-containing solutions, inhibition of Na⁺Ca²⁺ exchange led to increased [Ca²⁺][sub i], as did blockade of Ca²⁺-ATPase, suggesting that these membrane transporters are functional in NSC-19. [Ca²⁺][sub i] in these cells was modified by changes in extracellular Ca²⁺ concentrations ([Ca²⁺][sub o]). Exposure of cells to increased [Ca²⁺][sub o] of up to 10 mM resulted in sustained elevations of [Ca²⁺][sub i], which maintained steady state levels for at least 10 min. PKC activity in cytosolic and membrane extracts from cells exposed to varying [Ca²⁺][sub o] was assessed by the ability of the enzyme to phosphorylate histone HI in the presence or absence of Ca²⁺, diolein, and phosphatidylserine. Extracts from cultures incubated in 1.3 mM [Ca²⁺][sub o] (control, [Ca²⁺][sub i] was 83 ± 17 nM) contained PKC activity predominantly in the cytosol fractions. A 10 min exposure of the cells to 2.5 mM [Ca²⁺][sub o] ([Ca²⁺][sub i] was 140 ± 8 nM) resulted in the partial translocation of cytosolic PKC activity to the membrane, and exposure to 5 mM [Ca²⁺][sub o] ([Ca²⁺][sub i] was 232 ± 24 nM) resulted in almost complete translocation. Cells exposed to 10 mM [Ca²⁺][sub o] ([Ca²⁺][sub i] was 365 ± 13 nM) showed a two-fold increase in cytosolic PKC activity and an eleven-fold increase in membrane-associated PKC activity, suggesting that the increased activation was translocation-independent. Total PKC activity decreased in extracts from cells exposed to 25 mM and 50 mM [Ca²⁺][sub o]. [Ca²⁺][sub i] rose transiently to over 600 nM and 900 nM, respectively, and then returned to steady state values of 202 ± 1 4 nM and 122 ± 6 nM. Under these conditions, cytosolic and membrane-bound protein kinase M (PKM) activity rose approximately eight-fold and ten-fold, respectively, for both groups. Thus, three modes of PKC activation by increased [Ca²⁺][sub i] were observed: (i) translocation of PKC activity from the cytosol to the membrane at [Ca²⁺][sub i] between 140 nM and 230 nM; (ii) apparent translocation-independent activation of PKC at [Ca²⁺][sub i] values around 365 nM; and (iii) proteolytic cleavage of PKC to P KM following a Ca²⁺ transient of greater than 600 nM. These three distinct modes of PKC activation may have unique physiological consequences that depend on the amplitude and duration of the initiating [Ca²⁺][sub i] signal.

Thesis/Dissertation

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2009-02-10

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http://hdl.handle.net/2429/4371

Experimental Medicine

University of British Columbia

UBCV

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