Iain A. Greenwood

Comparison of the effects of fenamates on Ca‐activated chloride and potassium currents in rabbit portal vein smooth muscle cells

1 . The perforated patch and conventional whole‐cell recording techniques were used to study the action of flufenamic, mefenamic and niflumic acid on calcium‐activated chloride and potassium currents in rabbit portal vein smooth muscle cells. 2 . In K‐conditions at a holding potential of —77 mV flufenamic acid and mefenamic acid decreased the amplitude of spontaneous transient inward currents (STICs, calcium‐activated chloride currents, ICI(ca)) in a concentration‐dependent manner. The potency sequence was niflumic > flufenamic > mefenamic acid. 3 . At —77 mV 1 × 10−5 m flufenamic acid increased the STIC exponential decay time constant (τ). At higher concentrations the STIC decay was described by 2 exponentials with an initial decay (τf) faster than the control τ value and a second exponential (τs) which had a time constant slower than the control τ value. Low concentrations of mefenamic acid had no effect or decreased the τ value whereas in higher concentrations biphasic currents were recorded. 4 . In K‐free conditions the inhibitory effect of both flufenamic and mefenamic acid on STIC amplitude was greater at + 50 mV compared to − 50 mV, showing that the effect of these agents was voltage‐dependent. 5 . In cells held at 0 mV in K‐containing conditions the fenamates reduced both the frequency and amplitude of spontaneous transient outward currents (STOCs, calcium‐activated potassium currents, IK(ca)). The concentration range to produce these effects was higher than that to decrease STIC amplitude and the potency sequence was flufenamic> niflumic ≥mefenamic acid. 6 . All these compounds in concentrations greater than 5 × 10 −5 m evoked a ‘noisy’ potassium current at 0 mV which reached a maximum after approximately 3 min. This current was readily reversible on washout of the drug and could be elicited several times in the same cell. The current‐voltage relationship of the fenamate‐evoked current exhibited pronounced outward rectification characteristic of IK(ca). 7 . The current evoked by 2 × 10−4 m flufenamic acid and 5 × 10−4 m niflumic acid was not affected by 1 × 10−5 m glibenclamide but was markedly inhibited by 1 × 10−3m tetraethylammonium. Furthermore, large currents were activated by flufenamic and niflumic acid in the presence of caffeine and cyclopiazonic acid (an inhibitor of the sarcoplasmic reticulum Ca‐ATPase) to deplete intracellular Ca‐stores. 8 . Conventional whole‐cell recording was performed with pipette solutions in which the ability to buffer changes in intracellular calcium was varied by altering the concentration of the calcium chelator (2‐aminophenoxy)‐ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA). Flufenamic acid (2 × 10−4 m) and niflumic acid (5 × 10−4 m) both evoked large outward currents when recordings were made with either 1 × 10−4 m or 1times10−2m BAPTA. Furthermore, bathing the cells in nominally calcium‐free extracellular solution did not reduce the amplitude of the evoked currents. 9 . It is concluded that both flufenamic and mefenamic acid inhibit ICI(Ca) by a mechanism similar to niflumic acid, possibly open channel blockade. Furthermore, at concentrations greater than 5 × 10−5 m all three fenamates inhibited STOC activity and evoked directly an outward current which resembled IK(Ca).

Differential regulation of Ca2+-activated Cl− currents in rabbit arterial and portal vein smooth muscle cells by Ca2+-calmodulin-dependent kinase

1 Ca2+‐activated chloride currents (ICl(Ca)) were recorded from smooth muscle cells isolated from rabbit pulmonary (PA) and coronary artery (CA) as well as rabbit portal vein (PV). The characteristics and regulation by Ca2+‐calmodulin‐dependent kinase II (CaMKII) were compared between the three cell types. 2 In PA and CA myocytes dialysed and superfused with K+‐free media, pipette solutions containing fixed levels of free Ca2+ in the range of 250 nm to 1 μm evoked well sustained, outwardly rectifying ICl(Ca) currents in about 90 % of cells. The CaMKII inhibitor KN‐93 (5 μm) increased the amplitude of ICl(Ca) in PA and CA myocytes. However, the threshold intracellular Ca2+ concentration for detecting this effect was different in the two arterial cell types. KN‐93 also enhanced the rate of activation of the time‐dependent current during depolarising steps, slowed the kinetics of the tail current following repolarisation, and induced a negative shift of the steady‐state activation curve. 3 In PA myocytes, the effects of KN‐93 were not mirrored by its inactive analogue KN‐92 but were reproduced by the inclusion of autocamtide‐2‐related CaMKII inhibitory peptide (ARIP) in the pipette solution. Cell dialysis with constitutively active CaMKII (30 nm) significantly reduced ICl(Ca) evoked by 500 nm Ca2+. 4 In PV myocytes, ICl(Ca) was evoked by pipette solutions containing up to 1 μm free Ca2+ in less than 40 % of cells. Application of KN‐93 to cells where ICl(Ca) was sustained produced a small inhibition (≈25 %) of the current in 70 % of the cells. 5 The present study shows that regulation of Ca2+‐dependent Cl− channels by CaMKII differs between arterial and portal vein myocytes.

Effect of niflumic acid on noradrenaline‐induced contractions of the rat aorta

1 The effects of niflumic acid, an inhibitor of calcium‐activated chloride channels, were compared with the actions of the calcium channel antagonist nifedipine on noradrenaline‐evoked contractions in isolated preparations of the rat aorta. 2 The cumulative concentration‐effect curve to noradrenaline (NA) was depressed by both nifedipine and niflumic acid in a reversible and concentration‐dependent manner. The degree of inhibition of the maximal contractile response to NA (1 μm) produced by 10 μm niflumic acid (38%) was similar to the effect of 1 μm nifedipine (39%). 3 Contractions to brief applications (30 s) of 1 μm NA were inhibited by 55% and 62% respectively by 10 μm niflumic acid and 1 μm nifedipine. 4 In the presence of 0.1 μm nifedipine, niflumic acid (10 μm) produced no further inhibition of the NA‐evoked contractions. Thus, the actions of niflumic acid and nifedipine were not additive. 5 In Ca‐free conditions the transient contraction induced by 1 μm NA was not inhibited by niflumic acid (10 μm) and therefore this agent does not reduce the amount of calcium released from the intracellular store or reduce the sensitivity of the contractile apparatus to calcium. 6 Niflumic acid 10 μm did not inhibit the contractions produced by KCl (up to 120 mM) which were totally blocked by nifedipine. Contractions induced by 25 mM KCl were completely inhibited by 1 μm levcromakalim but were unaffected by niflumic acid. 7 It was concluded that niflumic acid produces selective inhibition of a component of NA‐evoked contraction which is probably mediated by voltage‐gated calcium channels. These data are consistent with a model in which NA stimulates a calcium‐activated chloride conductance which leads to the opening of voltage‐gated calcium channels to produce contraction.

Inhibitory action of niflumic acid on noradrenaline- and 5-hydroxytryptamine-induced pressor responses in the isolated mesenteric vascular bed of the rat

The effects of niflumic acid, an inhibitor of calcium‐activated chloride currents, were compared with the actions of the calcium channel blocker nifedipine on noradrenaline‐ and 5‐hydroxytryptamine (5‐HT)‐induced pressor responses of the rat perfused isolated mesenteric vascular bed. Bolus injections of noradrenaline (1 and 10 nmol) increased the perfusion pressure in a dose‐dependent manner. Nifedipine (1 μm) inhibited the increase in pressure produced by 1 nmol noradrenaline by 31±5%. Niflumic acid (10 and 30 μm) also inhibited the noradrenaline‐induced increase in perfusion pressure and 30 μm niflumic acid reduced the pressor response to 1 nmol noradrenaline by 34±6%. The increases in perfusion elicited by 5‐HT (0.3 and 3 nmol) were reduced by niflumic acid (10 and 30 μm) in a concentration‐dependent manner and 30 μm niflumic acid inhibited responses to 0.3 and 3 nmol 5‐HT by, respectively, 49±8% and 50±7%. Nifedipine (1 μm) decreased the pressor response to 3 nmol 5‐HT by 44±9%. In the presence of a combination of 30 μm niflumic acid and 1 μm nifedipine the inhibition of the pressor effects of noradrenaline (10 nmol) and 5‐HT (3 nmol) was not significantly greater than with niflumic acid (30 μm) alone. Thus the effects of niflumic acid and nifedipine were not additive. In Ca‐free conditions the transient contractions induced by 5‐HT (3 nmol) were not reduced by 30 μm niflumic acid, suggesting that this agent does not inhibit calcium release from the intracellular store or the binding of 5‐HT to its receptor. Niflumic acid 30 μm did not inhibit the pressor responses induced by KCl (20 and 60 μmol) which were markedly reduced by 1 μm nifedipine. In addition, 1 μm levcromakalim decreased pressor responses produced by 20 μmol KCl. These data suggest that niflumic acid does not block directly calcium channels or activate potassium channels. It is concluded that niflumic acid selectively reduces a component of noradrenaline‐ and 5‐HT‐induced pressor responses by inhibiting a mechanism which leads to the opening of voltage‐gated calcium channels. Our data suggest that the Ca2+‐activated chloride conductance may play a pivotal role in the activation of voltage‐gated calcium channels in agonist‐induced constriction of resistance blood vessels.

Dual effect of blocking agents on Ca2+-activated Cl- currents in rabbit pulmonary artery smooth muscle cells

The effects of the Cl− channel antagonists, niflumic acid (NFA), dichloro‐diphenylamine 2‐carboxylic acid (DCDPC) and diisothiocyanato‐stilbene‐2,2′‐disulphonic acid (DIDS) on Ca2+‐activated Cl− current (ICl(Ca)) evoked by adding fixed intracellular calcium concentrations ([Ca2+]i) to the pipette solution were studied in rabbit pulmonary artery myocytes. With 250 and 500 nm[Ca2+]i bath application of NFA (100 μm) increased inward current at negative potentials, but inhibited outward current at positive potentials. On wash out of NFA, ICl(Ca) was greatly enhanced at all potentials. When external Na+ ions were replaced by N‐methyl‐d‐glucamine (NMDG+) NFA still enhanced ICl(Ca) at negative potentials but the increase of ICl(Ca) on wash out was blocked. When the mean reversal potential (Er) of ICl(Ca) was shifted to negative potentials by replacing external Cl− with SCN−, NFA increased inward current but blocked outward current suggesting that the effect of NFA is dependent on current flow. Inclusion of NFA in the pipette solution had no effect on ICl(Ca). Voltage jump experiments indicated that ICl(Ca) displayed characteristic outward current relaxations at +70 mV and inward current relaxations at −80 mV that were abolished by NFA. DCDPC (100 μm) produced similar effects to NFA but 1 mm DIDS produced inhibition of ICl(Ca) at both positive and negative potentials and there was no increase in current on wash out of DIDS. These results suggest that NFA and DCDPC, but not DIDS, simultaneously enhance and block ICl(Ca) by binding to an external site, probably close to the mouth of the chloride channel.

Modulation of Ca2+‐activated Cl− currents in rabbit portal vein smooth muscle by an inhibitor of mitochondrial Ca2+ uptake

1 The effects of carbonyl cyanide m‐chlorophenyl hydrazone (CCCP), an inhibitor of mitochondrial Ca2+ uptake, was investigated on the properties of Ca2+‐activated chloride currents (ICl(Ca)) in rabbit portal vein smooth muscle cells using the perforated patch whole‐cell voltage‐clamp technique to ascertain whether this Ca2+ uptake process influences the time course of the subsarcolemmal Ca2+ signal that activates ICl(Ca). 2 In cells bathed in either physiological calcium (2 mm Ca2+o) or high calcium (10 mm Ca2+o) external solutions, application of CCCP (1–2 μm) evoked an inward current and prolonged the exponential decay time constant (τ) of Ca2+‐activated Cl−‘tail’ currents (Itall) evoked by Ca2+ influx through voltage‐dependent calcium channels (VDCCs). The effect of CCCP on τ was greater in cells where the amplitude of Itall was relatively large and, in different cells, the effect of CCCP on T was positively correlated with the amplitude of Itall. 3 CCCP abolished spontaneously occurring transient Ca2+‐activated Cl− currents (STICs), but did not alter their time course before complete block. 4 Thapsigargin and cyclopiazonic acid (inhibitors of the sarcoplasmic Ca2+‐ATPase) inhibited STICs, but did not affect the decay of Itall or STICs. 5 In conclusion, when Ca2+ enters the cell through VDCCs, the time course of the consequent Ca2+ signal in the subsarcolemmal domain containing Ca2+‐activated chloride channels appears to be regulated by Ca2+ uptake into mitochondria. In contrast, inhibition of Ca2+ uptake by the sarcoplasmic reticulum ATPase does not seem to influence the time course of ICl(Ca).

Comparison of the properties of CLCA1 generated currents and ICl(Ca) in murine portal vein smooth muscle cells

Calcium‐activated chloride currents (ICl(Ca)) have been recorded in various smooth muscle cells but, to date, there has been no information as to the molecular nature of the channel underlying this conductance. We have characterised native ICl(Ca) in freshly dispersed smooth muscle cells isolated from murine portal vein using whole‐cell voltage clamp. ICl(Ca) exhibited time‐dependent activation at depolarised potentials and rapid deactivation upon repolarisation. The reversal potential of ICl(Ca) was close to the theoretical equilibrium potential (ECl) and was shifted by replacement of external Cl− by SCN− or isethionate. Dithiothreitol (DTT, 1 mm), a blocker of CLCA1, had no effect on the ICl(Ca) current in myocytes. RT‐PCR demonstrated the expression of mCLCA1 transcripts, but not mCLCA3 transcripts, in various murine smooth muscle cells including portal vein, as well as cardiomyocytes, and the levels of mCLCA1 transcriptional expression were quantified by real time quantitative RT‐PCR. Stable transfection of HEK293 cells with the cDNA encoding mCLCA1 cloned from murine portal vein smooth muscle yielded a current with notable differences in Ca2+ sensitivity, channel kinetics and modulation by DTT from the native ICl(Ca). However, there was some similarity in the pore properties and these data suggest that mCLCA1 alone does not comprise the Cl− channel in portal vein smooth muscle cells.

Modulation of Ca2+-dependent Cl− channels by calcineurin in rabbit coronary arterial myocytes

The role of the Ca2+‐dependent phosphatase calcineurin (CaN) in the modulation of Ca2+‐dependent Cl‐ channels (ClCa) was studied in freshly isolated rabbit coronary arterial myocytes. Immunocytochemical experiments showed that calmodulin‐dependent protein kinase II (CaMKII) and CaN were distributed evenly throughout the cytoplasm of coronary myocytes at rest and translocated to the plasmalemma when intracellular Ca2+ was increased. ClCa currents (ICl(Ca)) elicited by cell dialysis with fixed intracellular Ca2+ levels up to 500 nm were inhibited by 10 μm cyclosporin A (CsA), a specific inhibitor of CaN, in a voltage‐dependent manner, whereas currents evoked by 1 μm Ca2+ were not affected. Inhibition of CaN with CsA also led to a significant reduction in Ca2+ sensitivity of the channel at +50 mV; half‐maximal activation increased from 363 ± 16 nm Ca2+ in control to 515 ± 40 nm Ca2+ in the presence of CsA. Similar effects were observed on ICl(Ca) when a specific peptide fragment inhibitor of CaN (CaN‐AF, 5 μm) was dialysed into the cell via the pipette (500 nm Ca2+). Application of KN‐93 (10 μm), a specific inhibitor of CaMKII, enhanced ICl(Ca) in myocytes dialysed with 1 μm Ca2+ but produced no significant effect on this current when the cells were dialysed with 350 or 500 nm Ca2+. These results are consistent with the notion that in coronary arterial cells, the activity of ClCa is enhanced by dephosphorylation of the channel or a closely associated regulatory protein. Moreover the balance of CaN and CaMKII regulating ICl(Ca) is dependent on the level of Ca2+ used to activate ICl(Ca).

Modulation of volume-sensitive chloride current by noradrenaline in rabbit portal vein myocytes

The effect of noradrenaline on the volume‐sensitive chloride current (ICl(swell)) was studied with conventional whole‐cell recording techniques in freshly dispersed isolated smooth muscle cells of the rabbit portal vein. In the absence of receptor antagonists, noradrenaline produced an increase in the amplitude of ICl(swell) in some cells and a decrease in others. In the presence of the β‐adrenoceptor antagonist propranolol, noradrenaline increased ICl(swell) and in the presence of the α1‐adrenoceptor antagonist prazosin, noradrenaline reduced ICl(swell). The phospholipase C (PLC) inhibitor U73122 reduced the amplitude of ICl(swell) whereas the inactive analogue U73343 had no effect. The phorbol esters phorbol‐12‐myristate‐13‐acetate (PMA) and phorbol‐12,13‐dibutyrate (PDBu) increased the amplitude of ICl(swell) by approximately 60 and 100 %, respectively, in a voltage‐independent fashion. Inhibitors of protein kinase C (PKC) chelerythrine and calphostin‐C decreased the amplitude of ICl(swell) in a concentration‐dependent but voltage‐independent manner. Bath application of 8‐Br‐cAMP decreased ICl(swell) by about 60 % whereas the inhibitor of protein kinase A (PKA) KT5720 increased the amplitude of ICl(swell) by approximately 80–90 %. In the presence of propranolol, chelerythrine prevented the increase of ICl(swell) by noradrenaline; in the presence of prazosin, KT5720 blocked the inhibitory action of noradrenaline. The results show that in rabbit portal vein myocytes noradrenaline enhances ICl(swell) by acting on α1‐adrenoceptors and reduces ICl(swell) by stimulating β‐adrenoceptors. The data suggest that the potentiating and inhibitory effects of noradrenaline are mediated, respectively, by PKC and PKA.

Modulation of the decay of Ca2+-activated Cl− currents in rabbit portal vein smooth muscle cells by external anions

1 The effects of external anions on the decay kinetics of Ca2+‐activated Cl− currents (ICl(Ca)) were studied in smooth muscle cells isolated from rabbit portal vein using the perforated patch whole‐cell voltage clamp technique. 2 In normal NaCl‐containing external solution the decay of spontaneous Ca2+‐activated Cl− currents (STICs) and Ca2+‐activated Cl−‘tail’ currents (Itail) was described by a single exponential with a time constant (τ) that was prolonged by external anions which are more permeable than Cl− (Br−, I− and SCN−) and accelerated by less permeant anions. However, intracellular I− did not affect the τ of STICs and Itail. 3 There was a positive correlation between the ability of an external anion to affect the decay τ of ICl(Ca) and its permeability relative to Cl−. 4 The voltage dependence of STIC and Itail decay was not affected by external or internal anions. 5 External permeating anions were not obligatory for activation of ICl(Ca) and STIC τ was not altered in Cl−‐free external solution. 6 Modulation of τ by mole fractions of SCN− and Cl− ions was fitted by a logistic curve, suggesting competition between SCN− and Cl− ions for a binding site. 7 In conclusion, external anions affect the decay of ICl(Ca) by a mechanism compatible with an interaction with a binding site which modulates Cl− channel kinetics.