The Cl⁻ channel blocker niflumic acid releases Ca²⁺ from an intracellular store in rat pulmonary artery smooth muscle cells

Abstract

1 The effect of the Cl À channel blockers niflumic acid (NFA), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), 4,4 0 -diisothiocyanatostilbene-2,2 0 -disulfonic acid (DIDS), and anthracene-9-carboxylic acid (A-9-C), on Ca 2 þ signalling in rat pulmonary artery smooth muscle cells was examined. Intracellular Ca 2 þ concentration ([Ca 2 þ ] i ) was monitored with either fura-2 or fluo-4, and caffeine was used to activate the ryanodine receptor, thereby releasing Ca 2 þ from the sarcoplasmic reticulum (SR). 2 NFA and NPPB significantly increased basal [Ca 2 þ ] i and attenuated the caffeine-induced increase in [Ca 2 þ ] i . These Cl À channel blockers also increased the half-time (t 1/2 ) to peak for the caffeineinduced [Ca 2 þ ] i transient, and slowed the removal of Ca 2 þ 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 Ca 2 þ in studies involving these Cl À channel blockers. Both DIDS and A-9-C increased basal fluo-4 fluorescence, indicating an increase in intracellular Ca 2 þ , and while DIDS had no significant effect on the t 1/2 to peak for the caffeine-induced Ca 2 þ transient, it was significantly increased by A-9-C. 3 In the absence of extracellular Ca 2 þ , NFA significantly increased basal [Ca 2 þ ] i , suggesting that the release of Ca 2 þ from an intracellular store was responsible for the observed effect. 4 Depleting the SR with the combination of caffeine and cyclopiazonic acid prevented the increase in basal [Ca 2 þ ] i induced by NFA. Additionally, incubating the cells with ryanodine also prevented the increase in basal [Ca 2 þ ] i induced by NFA. 5 These data show that Cl À channel blockers have marked effects on Ca 2 þ signalling in pulmonary artery smooth muscle cells. Furthermore, examination of the NFA-induced increase in [Ca 2 þ ] i indicates that it is likely due to Ca 2 þ release from an intracellular store, most probably the SR.

Potassium-induced intermittent vasomotion in rat isolated pulmonary artery

“…In rat pulmonary artery smooth muscle cells, niflumic acid has been noted to cause Ca 2+ release from intracellular stores (Cruickshank et al, 2003). However, the stimulatory effect seen here may not be due to such a mechanism since the concentration of niflumic acid utilized by these authors was of the order of 50 M, a much higher concentration than the one employed here.…”

Dynamics of Ca2+-Dependent Cl- Channel Modulation by Niflumic Acid in Rabbit Coronary Arterial Myocytes

“…This diminished potency of NFA to inhibit I Cl(Ca) was also noticed by Piper et al (2002) in rabbit pulmonary myocytes dialyzed with elevated [Ca 2ϩ ] i (250 nM to 1 M). Whether the higher efficacy of NFA for blocking STICs relative to I Cl(Ca) elicited by a sustained elevated intracellular Ca 2ϩ level is caused by the much higher Ca 2ϩ concentrations (tens of micromolar) generated by Ca 2ϩ sparks (Jaggar et al, 2000) or the ability of NFA to interfere with Ca 2ϩ release from internal stores (Cruickshank et al, 2003) remains to be investigated. An alternative possibility is Fig.…”

“…Flufenamic acid and tolfenamic acids relaxed guinea pig tracheal contractions through a direct inhibition of voltagegated Ca 2ϩ channels (Li et al, 1998). Niflumic acid stimulated Ca 2ϩ release from internal stores in rat pulmonary artery smooth muscle cells (Cruickshank et al, 2003) and was shown, along with 5-nitro-2-(3-phenylpropylamino)-benzoic acid and 4,4-diisothiocyanato-stilbene-2,2-disulfonic acid, to inhibit endothelin-1-induced contractions of rat pulmonary arterial myocytes by a mechanism independent of block of Cl Ca channels (Kato et al, 1999). Moreover, the IC 50 for NFA block of spontaneously occurring I Cl(Ca) is significantly lower than that required to block I Cl(Ca) evoked by caffeine or an agonist such as norepinephrine (Large and Wang, 1996).…”

CaMKII inactivation by extracellular Ca2+ depletion in dorsal root ganglion neurons

“…6). However, there have been reports of the related compound niflumic acid releasing Ca 2+ from ryanodine-sensitive stores that might act to reverse the affects on Ca 2+ -influx [17]. We also tested several di-and trivalent cations; neither Gd 3+ nor La 3+ at 300 μM blocked activation of CaMKII.…”