Ca²⁺-activated Cl^– channels (Cl_Ca) play an important role in the regulation of resting membrane potential of vascular smooth muscle cells during agonist-mediated tone. Niflumic acid (NFA) is recognized as the most potent inhibitor of Cl_Ca. However, when Cl_Ca in rabbit pulmonary artery myocytes was evoked by a sustained level of intracellular calcium, NFA both inhibits and stimulates the current (Piper et al. 2002). We examined this phenomenon further by studying the effect of NFA on I_Cl(Ca) evoked in coronary artery myocytes by dialysis with K⁺-free pipette solutions containing 500 nM Ca²⁺ using the whole-cell patch-clamp technique. Coronary myocytes were freshly isolated from New Zeland White rabbits that were killed by an anaesthetic overdose (pentobarbital 1 mg kg^-1) via the ear vein. Cells were prepared as described previously (Greenwood et al. 2001). All pooled data are expressed as means ± S.E.M. Application of 100 mM NFA inhibited I_Cl(Ca), which was followed by a marked increase in current level above control values after washout (mean increase of instantaneous, late and tail currents was 47 ± 13, 80 ± 28 and 50 ± 16 %, respectively, n = 4). The activation kinetics of enhanced I_Cl(Ca) during a depolarizing step increased (at +90 mV, t = 0.4 ± 0.1 and 0.2 ± 0.02 s before and after washout of NFA, respectively; P < 0.05, Student’s paired t test, n = 4) while the mono-exponential decay of the tail current at -80 mV became biexponential upon washout of the drug (t = 44 ± 4 ms in control, t_fast = 19 ± 2 ms and t_slow = 176 ± 29 ms after washout of NFA; P < 0.05, ANOVA, n = 4). We next sought to determine the concentration-dependence of enhanced I_Cl(Ca) after washout of NFA. Cells were stepped to +60 mV for 30 s (HP = -50 mV) and exposed to a given concentration of NFA with a fast flow perfusion system for 2 s. Whilst the relative increase of I_Cl(Ca) following washout of NFA was independent of drug concentration, the time to achieve 50 % of the maximal increase of current was concentration dependent (almost instantaneous, 641 ± 169 ms and 1421 ± 400 ms following a 2 s exposure to 10 mM, 100 mM or 1 mM NFA, respectively; P < 0.05, Student’s paired t test, n = 4). Moreover, increased I_Cl(Ca) could be blocked as effectively and as quickly as the control current by a second exposure to 100 mM NFa after a 500 ms washout. This indicates that both control and stimulated I_Cl(Ca) currents can be inhibited by NFA. We propose the existence of at least two binding sites on smooth muscle Cl_Ca: a high-affinity inhibitory site, which is hiding a low-affinity site revealed upon washout of the drug.

All procedures accord with current local guidelines.