Heterogeneous increases of cytoplasmic calcium: distinct effects on down-regulation of cell surface sodium channels and sodium channel subunit mRNA levels

Abstract

1. Long-term (> or = 12 h) treatment of cultured bovine adrenal chromaffin cells with A23187 (a Ca(2+) ionophore) or thapsigargin (TG) [an inhibitor of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)] caused a time- and concentration-dependent reduction of cell surface [(3)H]-saxitoxin (STX) binding capacity, but did not change the K:(D:) value. In A23187- or TG-treated cells, veratridine-induced (22)Na(+) influx was reduced (with no change in veratridine EC(50) value) while it was enhanced by alpha-scorpion venom, beta-scorpion venom, or Ptychodiscus brevis toxin-3, like in nontreated cells. 2. The A23187- or TG-induced decrease of [(3)H]-STX binding was diminished by BAPTA-AM. EGTA also inhibited the decreasing effect of A23187. A23187 caused a rapid, monophasic and persistent increase in intracellular concentration of Ca(2+) ([Ca(2+)](i)) to a greater extent than that observed with TG. 2,5-Di-(t-butyl)-1,4-benzohydroquinone (DBHQ) (an inhibitor of SERCA) produced only a rapid monophasic increase in [Ca(2+)](i), without any effect on [(3)H]-STX binding. 3. Reduction in [(3)H]-STX binding capacity induced by A23187 or TG was attenuated by Gö6976 (an inhibitor of conventional protein kinase C) or calpastatin peptide (an inhibitor of calpain). When the internalization rate of cell surface Na(+) channels was measured in the presence of brefeldin A (an inhibitor of vesicular exit from the trans-Golgi network), A23187 or TG accelerated the reduction of [(3)H]-STX binding capacity. 4. Six hours treatment with A23187 lowered Na(+) channel alpha- and beta(1)-subunit mRNA levels, whereas TG had no effect. 5. These results suggest that elevation of [Ca(2+)](i) caused by A23187, TG or DBHQ exerted differential effects on down-regulation of cell surface functional Na(+) channels and Na(+) channel subunit mRNA levels.

Figure 1

Effects of treatment with A23187, TG, and DBHQ on [³H]-STX binding capacity in adrenal chromaffin cells. (A) Cells were treated for up to 96 h with DMSO, 1 μM A23187, 100 nM TG, 10 μM or 100 μM DBHQ, then washed with KRP buffer, and tested for [³H]-STX binding assay. In a parallel experiment, cells exposed for 48 h to 1 μM A23187 or 100 nM TG were washed with culture medium (indicated by arrow), then incubated without A23187 or TG, and subjected to [³H]-STX binding assay at 72 and 96 h. Mean±s.e.mean (n=5). ^*P<0.05, significant decrease by A23187 or TG compared to DMSO-treated cells. (B) Cells were treated for 48 h with DMSO, A23187 or TG at indicated concentrations, and tested for [³H]-STX binding ability. Mean±s.e.mean (n=5). ^*P<0.05, significant decrease by A23187 or TG. (C) Scatchard plot of [³H]-STX binding to cells treated with DMSO, 1 μM A23187 or 100 nM TG for 48 h. Data are representative of one experiment from three independent experiments with similar results.

Figure 2

Treatment of adrenal chromaffin cells with A23187 and TG: effects on veratridine-induced ²²Na⁺ influx in the absence and presence of ouabain. Cells were treated for 48 h with DMSO, (A) 1 μM A23187 or (B) 100 nM TG, and washed. To measure ²²Na⁺ influx, cells were incubated with 2 μCi ²²NaCl at 37°C for 5 min in the absence or presence of 1-560 μM veratridine and 100 μM ouabain. Basal ²²Na⁺ influx values (nmol 4×10⁶ cells⁻¹ 5 min⁻¹; n=5) were similar among nontreated (19.6±2.0), A23187 (18.6±1.8)- and TG (18.2±2.4)-treated cells, and these values were subtracted from the data. ²²Na⁺ influx values obtained with ouabain alone were similar among nontreated (70.8±3.2), A23187 (70.4±2.8)- and TG (70.4±2.8)-treated cells. Mean±s.e.mean (n=5). ^*P<0.05, significant decrease by A23187 or TG; #P<0.05, significant enhancement by ouabain of veratridine's effect within each cell group.

Figure 3

Enhancement of veratridine-induced ²²Na⁺ influx by α-scorpion venom, β-scorpion venom and PbTx-3 in nontreated, A23187- and TG-treated adrenal chromaffin cells. Cells were treated for 48 h with DMSO, 1 μM A23187 or 100 nM TG, washed, and incubated with 2 μCi ²²NaCl at 37°C for 5 min in the absence (−) or presence (+) of 0.5 μg ml⁻¹ α-scorpion venom, 5 μg ml⁻¹ β-scorpion venom, 1 μM PbTx-3 or 30 μM veratridine. Basal ²²Na⁺ influx values at 37°C were subtracted for each value. Mean±s.e.mean (n=3). ^*P<0.05, combined with DMSO-treated cells; #P<0.05, significant enhancement by venom or toxin of veratridine's effect. @P<0.05; significant enhancement by scorpion venom compared to value obtained in cells exposed to veratridine and PbTx-3.

Figure 4

Ca²⁺-dependent decrease of [³H]-STX binding capacity in A23187- and TG-treated adrenal chromaffin cells. (A) Cells were treated for 24 h with DMSO, 1 μM A23187 or 100 nM TG in the absence (none) or presence of 5 mM EGTA or 50 μM BAPTA-AM, washed, and subjected to [³H]-STX binding assay. Mean±s.e.mean (n=5). ^*P<0.05, significant decrease by A23187 or TG within each cell group. (B) Cells were exposed to DMSO, 1 μM A23187 or 100 nM TG for the first 24 h, then treated with or without 50 μM BAPTA-AM in the continuous presence of DMSO, A23187 or TG for up to 48 h, and used for [³H]-STX binding assay. Mean±s.e.mean (n=3). ^*P<0.05, compared with A23187 or TG alone.

Figure 5

Time-course of [Ca²⁺]_i increase in adrenal chromaffin cells: effects of A23187, TG and DBHQ treatment in the presence or absence of extracellular Ca²⁺. Cells preloaded with fura-2 were treated with 1 μM A23187, 100 nM TG or 100 μM DBHQ (as indicated by the horizontal bars under recordings) in (A) Ca²⁺-containing HEPES-buffered solution (HBS) for 60 min and (B) Ca²⁺-free HBS containing EGTA for 30 min. Each recording shown here was obtained in a single cell and is typical of independent multiple experiments with similar results. (C) Increment of [Ca²⁺]_i over the basal value (Δ [Ca²⁺]_i). Cells were treated with or without A23187, TG or DBHQ in the presence (Ca²⁺ (+)) or absence (Ca²⁺ (−)) of extracellular Ca²⁺. Peak, maximum increase of Δ [Ca²⁺]_i; 30 min, increase of Δ [Ca²⁺]_i measured at 30 min after the exposure to A23187, TG or DBHQ. Basal values of [Ca²⁺]_i were 109.4±6.8 nM (n=68) in Ca²⁺-containing medium, and 100.4±5.2 nM (n=114) in Ca²⁺-free medium. Parenthesis (number of cells examined). (D) Culture dishes were divided into nontreated, A23187- or TG-treated cell group; cells were treated in cultured medium with DMSO, 1 μM A23187 or 100 nM TG for 24, 48 and 96 h, and then subjected to [Ca²⁺]_i measurement. Mean±s.e.mean. ^*P<0.05, significant difference; n.s., no significant difference.

Figure 6

Decrease of [³H]-STX binding capacity in A23187- and TG-treated adrenal chromaffin cells: prevention by Gö6976 and calpain inhibitor. Cells were treated for 24 h with DMSO, 1 μM A23187 or 100 nM TG in the absence (None) or presence of 1 μM Gö6976, or/and 1 μM calpastatin peptide or 1 μM biologically inactive calpastatin analogue [calpastatin (−)], and used for [³H]-STX binding assay. Mean±s.e.mean (n=5). ^*P<0.05, significant decrease by A23187 or TG; #P<0.05, significant prevention by Gö6976 or/and calpastatin.

Figure 7

Enhancement by BFA of A23187-, and TG-induced decrease of [³H]-STX binding capacity in adrenal chromaffin cells. Cells were treated with DMSO, 1 μM A23187 or 100 nM TG for 6 or 12 h in the absence (None) or presence of 10 μg ml⁻¹ BFA, and subjected to [³H]-STX binding assay. Mean±s.e.mean (n=5). ^*P<0.05, significant decrease by BFA, A23187 or TG; #P<0.05, compared with BFA alone.

Figure 8

Northern blot analysis: distinct effects of A23187 and TG treatment on Na⁺ channel α- and β₁-subunit mRNA levels in adrenal chromaffin cells. (A) Cells were treated with DMSO (−), 1 μM A23187 (+) or 100 nM TG (+) for up to 48 h; poly(A)⁺ RNA was extracted, separated by electrophoresis on agarose gel, and transferred to membrane. The membrane was sequentially hybridized to each ³²P-labelled cDNA probe for hNE-Na (top), rat brain β₁-subunit (middle), and GAPDH (bottom) after removal of the former probe. Data shown are typical one from three independent analyses with similar results. (B) Levels of α- and β₁-subunit mRNAs and GAPDH mRNA in (A) were quantified by a Bioimage analyser, and the relative amount of α- or β₁-subunit mRNA GAPDH mRNA⁻¹ in A23187- or TG-treated cells is shown. A value of 100% represents the relative level obtained in DMSO-treated cells at the left lane of each incubation time. Mean±s.e.mean (n=3). ^*P<0.05, compared with DMSO-treated cells.