The Cl(-) channel blocker niflumic acid releases Ca(2+) from an intracellular store in rat pulmonary artery smooth muscle cells

Abstract

The effect of the Cl- channel blockers niflumic acid (NFA), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and anthracene-9-carboxylic acid (A-9-C), on Ca2+ signalling in rat pulmonary artery smooth muscle cells was examined. Intracellular Ca2+ concentration ([Ca2+]i) was monitored with either fura-2 or fluo-4, and caffeine was used to activate the ryanodine receptor, thereby releasing Ca2+ from the sarcoplasmic reticulum (SR). NFA and NPPB significantly increased basal [Ca2+]i and attenuated the caffeine-induced increase in [Ca2+]i. These Cl- channel blockers also increased the half-time (t1/2) to peak for the caffeine-induced [Ca2+]i transient, and slowed the removal of Ca2+ from the cytosol following application of caffeine. Since DIDS and A-9-C were found to adversely affect fura-2 fluorescence, fluo-4 was used to monitor intracellular Ca2+ in studies involving these Cl- channel blockers. Both DIDS and A-9-C increased basal fluo-4 fluorescence, indicating an increase in intracellular Ca2+, and while DIDS had no significant effect on the t1/2 to peak for the caffeine-induced Ca2+ transient, it was significantly increased by A-9-C. In the absence of extracellular Ca2+, NFA significantly increased basal [Ca2+]i, suggesting that the release of Ca2+ from an intracellular store was responsible for the observed effect. Depleting the SR with the combination of caffeine and cyclopiazonic acid prevented the increase in basal [Ca2+]i induced by NFA. Additionally, incubating the cells with ryanodine also prevented the increase in basal [Ca2+]i induced by NFA. These data show that Cl- channel blockers have marked effects on Ca2+ signalling in pulmonary artery smooth muscle cells. Furthermore, examination of the NFA-induced increase in [Ca2+]i indicates that it is likely due to Ca2+ release from an intracellular store, most probably the SR.

Figure 1

Caffeine increases [Ca²⁺]_i via a ryanodine-sensitive Ca²⁺ store. Caffeine (20 mM) was applied to single PASMCs via a pressure-ejection pipette positioned approximately 100 μm from the cell. Reproducible [Ca²⁺]_i transients were obtained when a 5–6 min recovery period was allowed between caffeine applications (a). The break between transients represents the recovery period during which time recordings were not made. Cells were incubated with ryanodine (50 μM) for 5 min and caffeine re-applied. Subsequent caffeine-induced [Ca²⁺]_i transients were abolished (b). Data are shown as mean values±s.e.m. (n=6).

Figure 2

Effect of the Cl⁻ channel blockers NFA, NPPB, DIDS, and A-9-C on fura-2 excitation spectra. Excitation spectra for 1 μM fura-2 in Ca²⁺-free and Ca²⁺-containing solution in the absence or presence of NFA (50 μM) (a). Excitation spectra for 1 μM fura-2 in Ca²⁺-free and Ca²⁺-containing solution in the absence or presence of NPPB (10 μM) (b). Excitation spectra for 1 μM fura-2 in Ca²⁺-free and Ca²⁺-containing solution in the absence or presence of DIDS (100 μM) (c). Excitation spectra for 1 μM fura-2 in Ca²⁺-free and Ca²⁺-containing solution in the absence or presence of A-9-C (50 μM) (d). Emission was measured at 510 nm with a 10 nm bandpass filter.

Figure 3

Effect of NFA on caffeine-induced [Ca²⁺]_i transients in rat PASMCs. Averaged caffeine-induced [Ca²⁺]_i transients from six cells, before and after the addition of 50 μM NFA (a). [Ca²⁺]_i was not recorded during the preincubation period (2 min) with NFA. Summary data showing: the effect of NFA on basal [Ca²⁺]_i (b); the change in [Ca²⁺]_i produced by caffeine ([Ca²⁺]_i) (c); the half-time (t_1/2) to peak for the caffeine-induced [Ca²⁺]_i transient (d); and the half-time (t_1/2) for Ca²⁺ removal following caffeine application (e). Data are shown as mean values±s.e.m. (n=6) and ^*P<0.05.

Figure 4

Effect of NPPB on caffeine-induced [Ca²⁺]_i transients in rat PASMCs. Averaged caffeine-induced [Ca²⁺]_i transients from five cells, before and after the addition of 10 μM NPPB (a). [Ca²⁺]_i was not recorded during the preincubation period (2 min) with NPPB. Summary data showing: the effect of NPPB on basal [Ca²⁺]_i (b); the change in [Ca²⁺]_i produced by caffeine ([Ca²⁺]_i) (c); the half-time (t_1/2) to peak for the caffeine-induced [Ca²⁺]_i transient (d); and the half-time (t_1/2) for Ca²⁺ removal following caffeine application (e). Data are shown as mean values±s.e.m. (n=5) and ^*P<0.05.

Figure 5

Effect of DIDS on caffeine-induced changes in fluo-4 fluorescence in rat PASMCs. Averaged caffeine-induced changes in fluo-4 fluorescence, recorded from six cells, before and after the addition of 100 μM DIDS (a). Fluorescence was not recorded during the preincubation period (2 min) with DIDS. Summary data showing the effect of DIDS on basal fluo-4 fluorescence (b); the change in fluorescence produced by caffeine (Δfluo-4) (c); the half-time (t_1/2) to peak for the caffeine-induced fluorescence transient (d); and the half-time (t_1/2) for Ca²⁺ removal following caffeine application (e). Data are shown as mean values±s.e.m. (n=6) and ^*P<0.05.

Figure 6

Effect of A-9-C on caffeine-induced changes in fluo-4 fluorescence in rat PASMCs. Averaged caffeine-induced changes in fluo-4 fluorescence, recorded from seven cells, before and after the addition of 500 μM A-9-C (a). Fluorescence was not recorded during the preincubation period (2 min) with A-9-C. Summary data showing the effect of A-9-C on basal fluo-4 fluorescence (b); the change in fluorescence produced by caffeine (Δfluo-4) (c); the half-time (t_1/2) to peak for the caffeine-induced fluorescence transient (d); and the half-time (t_1/2) for Ca²⁺ removal following caffeine application (e). Data are shown as mean values±s.e.m. (n=7) and ^*P<0.05.

Figure 7

Effect of NFA on [Ca²⁺]_i in PASMCs. Concentration–response curve showing the magnitude of the increase in [Ca²⁺]_i when NFA was applied to single PASMCs using a pressure-ejection pipette. Data are shown as mean values±s.e.m., and each point is representative of 5–7 different cells (a). PASMCs were incubated in Ca²⁺-free extracellular medium (containing 1 mM EGTA) for 1 min prior to application of NFA (50 μM). NFA was applied to a single PASMC, using a pressure-ejection pipette positioned approximately 100 μm from the cell. Data are shown as mean values±s.e.m. (n=5).

Figure 8

Effect of depleting the SR Ca²⁺ stores on NFA-induced increases in [Ca²⁺]_i. Cells were incubated with the SR Ca²⁺ ATPase inhibitor CPA (10 μM) for 1 min prior to repeated caffeine (20 mM) application. Thereafter, NFA (50 μM) was applied. Data are shown as mean values±s.e.m. (n=5).

Figure 9

Effect of ryanodine on the NFA-induced increase in [Ca²⁺]_i. Repeated application of NFA (50 μM) from a pressure-ejection pipette increased [Ca²⁺]_i. Cells were then incubated with ryanodine (50 μM) and caffeine (20mM) was applied to activate the ryanodine receptor (not shown). Thereafter, NFA was applied. Data are shown as mean values±s.e.m. (n=3).