W. A. Large

Action of niflumic acid on evoked and spontaneous calcium‐activated chloride and potassium currents in smooth muscle cells from rabbit portal vein

1 The action of niflumic acid was studied on spontaneous and evoked calcium‐activated chloride (ICl(Ca)) and potassium (IK(Ca)) currents in rabbit isolated portal vein cells. 2 With the nystatin perforated patch technique in potassium‐containing solutions at a holding potential of – 77 mV (the potassium equilibrium potential), niflumic acid produced a concentration‐dependent inhibition of spontaneous transient inward current (STIC, calcium‐activated chloride current) amplitude. The concentration to reduce the STIC amplitude by 50% (IC50) was 3.6 × 10−6 m. 3 At – 77 mV holding potential, niflumic acid converted the STIC decay from a single exponential to 2 exponential components. In niflumic acid the fast component of decay was faster, and the slow component was slower than the control decay time constant. Increasing the concentration of niflumic acid enhanced the decay rate of the fast component and reduced the decay rate of the slow component. 4 The effect of niflumic acid on STIC amplitude was voltage‐dependent and at – 50 and + 50 mV the IC50 values were 2.3 × 10−6 m and 1.1 × 10−6 m respectively (cf. 3.6 × 10−6 m at −77 mV). 5 In K‐free solutions at potentials of – 50 mV and + 50 mV, niflumic acid did not induce a dual exponential STIC decay but just increased the decay time constant at both potentials in a concentration‐dependent manner. 6 Niflumic acid, in concentrations up to 5 × 10−5 m, had no effect on spontaneous calcium‐activated potassium currents. 7 Niflumic acid inhibited noradrenaline‐ and caffeine‐evoked ICl(Ca) with an IC50 of 6.6 × 10−6 m, i.e. was less potent against evoked currents compared to spontaneous currents. In contrast niflumic acid (2 × 10−6 m–5 × 10−5 m) increased noradrenaline‐ and caffeine‐induced IK(Ca). 8 The results are discussed with respect to the mechanism of block of ICl(Ca) by niflumic acid and its suitability as a pharmacological tool for assessing the role of ICl(Ca) in physiological mechanisms.

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).

Activation of store‐operated channels by noradrenaline via protein kinase C in rabbit portal vein myocytes

In the present study we have investigated the role of diacylglycerol (DAG) and protein kinase C (PKC) in mediating activation of Ca2+‐permeable store‐operated channels (SOCs) by noradrenaline in rabbit portal vein smooth muscle cells. With cell‐attached recording, bath application of noradrenaline, 1‐oleoyl‐acetyl‐sn‐glycerol (OAG) and phorbol 12,13‐dibutyrate (PDBu) evoked single channel currents. The biophysical properties of these channel currents were similar to those of the channel currents activated by depletion of internal Ca2+ stores with cyclopiazonic acid (CPA). The activation of SOCs in cell‐attached recording by noradrenaline, OAG, PDBu, CPA and the acetoxymethyl ester form of BAPTA (BAPTA‐AM) was markedly inhibited by the PKC inhibitors chelerythrine and RÖ‐31‐8220. In isolated outside‐out patches CPA did not evoke SOCs but noradrenaline stimulated SOC activity, which was reduced by about 90 % by PKC inhibitors. The addition of the serine/threonine phosphatase inhibitors calyculin A and microcystin also stimulated SOCs in isolated outside‐out patches. It is concluded that in rabbit portal vein myocytes, noradrenaline activates SOCs via DAG and PKC, possibly by a store‐independent mechanism. In addition in this cell type it appears that PKC and phosphorylation may play an important role in stimulating SOC activity in response to depletion of internal Ca2+ stores by CPA and BAPTA‐AM.

Receptor-Operated Ca2+-Permeable Nonselective Cation Channels in Vascular Smooth Muscle: A Physiologic Perspective

Nonselective Cation Channels in Vascular Myocytes. This article summarizes the literature on receptor‐operated Ca2+‐permeable nonselective cation channels in vascular smooth muscle cells. One of these conductances, the P2X1 receptor, is a classic ligand‐gated channel, but others are likely to be mediated via G‐protein‐coupled receptors. The most studied receptor‐operated channel in vascular myocytes is the norepinephrine‐evoked nonselective cation channel in rabbit portal vein myocytes. The data regarding the transduction mechanisms and biophysical properties of whole‐cell and single‐channel currents in this preparation are described. The channels have a conductance of 20 to 25 pS and complex kinetic behavior with at least two open and two closed states. These channels are activated by norepinephrine and acetylcholine via G‐protein‐coupled receptors linked to phospholipase C and by diacylglycerol (DAG). The action of DAG occurs by a mechanism independent of protein kinase C, but other kinases may mediate the responses to norepinephrine and DAG. In addition, activation of tyrosine kinases leads to opening of this channel. Other vasoconstrictors, such as endothelin, vasopressin, serotonin, and angiotensin II, open Ca2+‐permeable nonselective cation channels, but there may be differences between these conductances and the norepinephrine‐evoked channels. A homologue of the transient receptor potential protein (TRPC6) is an essential component of the norepinephrine‐activated channel in rabbit portal vein, and it is likely that this family of proteins plays an important role in mediating Ca2+ influx in vascular smooth muscle.

Alpha 1‐adrenoceptor activation of a non‐selective cation current in rabbit portal vein by 1,2‐diacyl‐sn‐glycerol.

1. The transduction mechanisms involved in the activation and modulation of the noradrenaline‐activated cation current (Icat) were investigated with whole‐cell patch clamp techniques in rabbit portal vein smooth muscle cells. 2. Intracellular application of guanosine 5‐O‐(3‐thiotriphosphate) (GTP gamma S, 500 microM) evoked a ‘noisy’ inward current at ‐50 mV with a similar current‐voltage relationship and reversal potential to the current evoked by bath application of noradrenaline (100 microM). Guanosine 5‐O‐(2‐thiodiphosphate) (GDP beta S, 1 mM) markedly inhibited noradrenaline‐activated Icat. 3. The phospholipase C (PLC) inhibitor U73122 inhibited the amplitude of the noradrenaline‐activated Icat in a concentration‐ and time‐dependent manner and the IC50 was about 180 nM. U73122 had similar effects on the cation current evoked by GTP gamma S. 4. Intracellular application of myo‐inositol 1,4,5‐trisphosphate (IP3, 100 microM) from the patch pipette did not activate any membrane current in cells where intracellular calcium concentration ([Ca2+]i) was buffered to 14 nM, but subsequent addition of noradrenaline evoked Icat. 5. Bath application of the 1,2‐diacyl‐sn‐glycerol (DAG) analogue 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG, 10 microM) activated Icat, whereas the phorbol ester phorbol 12,13‐dibutyrate (PDBu, 0.1‐5 microM) failed to activate Icat, in every cell examined. Icat activated by OAG after bath application of PDBu was not significantly different from OAG‐activated Icat in the absence of PDBu. The DAG lipase inhibitor RHC80267 (10 microM) activated Icat in some cells, whereas the DAG kinase inhibitor R59949 (10 microM) never activated Icat. 6. Bath application of the protein kinase C inhibitor chelerythrine (1‐10 microM) had no effect on either OAG‐or noradrenaline‐activated Icat. 7. It is concluded that noradrenaline activates Icat via a G‐protein coupled to PLC and that the resulting DAG product plays a central role in the activation of cation channels via a protein kinase C‐independent mechanism.

A Ca2+-permeable non-selective cation channel activated by depletion of internal Ca2+ stores in single rabbit portal vein myocytes

In vascular smooth muscle cells many agonists cause the release of Ca2+ ions from internal stores. An important problem concerns the mechanism by which the intracellular stores are refilled subsequent to depletion. In the present study, we describe the properties of a Ca2+‐permeable non‐selective cation channel current that is activated in rabbit portal vein myocytes by depletion of internal Ca2+ stores. Application of cyclopiazonic acid (CPA), which depletes internal Ca2+ stores, activated whole‐cell currents that had a reversal potential (Er) of about +50 mV in 1.5 mm external Ca2+ (Ca2o+). In 0 mm Ca2o+, the currents were larger and Er was ∼0 mV. Application of CPA and caffeine during cell‐attached recording activated single inward channel currents at negative potentials, which had a slope conductance of 2–3 pS and an Er of +20 mV. The slope conductance in 0 and 110 mm Ca2o+ was 7 and 1.5 pS, respectively, and Er values indicated that these non‐selective cation channels are highly permeable to Ca2+ ions. Bath application of the cell‐permeant Ca2+ chelator, BAPTA‐AM, also activated similar currents, indicating that these channels are not activated by Ca2+. Spontaneous channel currents with similar properties to store‐operated channels were observed in some patches. Application of W‐7, an inhibitor of the Ca2+‐binding protein calmodulin, also activated similar Ca2+‐permeable channel currents. In conclusion, it is demonstrated that agents that deplete Ca2+ stores and inhibit calmodulin binding activate Ca2+‐permeable non‐selective cation channel currents in rabbit portal vein myocytes. These channels may have an important role in vascular smooth muscle in providing an influx of Ca2+ to refill depleted internal Ca2+ stores and appear to possess different characteristics to store‐operated channels described in other vascular smooth muscle preparations.

Effects of Cl channel blockers on Ca-activated chloride and potassium currents in smooth muscle cells from rabbit portal vein

1 The effects of some chloride channel antagonists were studied on the calcium‐activated chloride current (ICl(ca)) in smooth muscle cells from the rabbit portal vein with the perforated patch technique. 2 4‐Acetamido‐4′‐isothiocyanatostilbene‐2,2′‐disulphonic acid (SITS) and 4,4′‐diisothiocyanato‐stilbene‐2,2′‐disulphonic acid (DIDS) reduced the amplitude of spontaneous transient inward currents (STICs, calcium‐activated chloride currents) in a concentration‐dependent manner. The concentrations required to reduce the amplitude by 50% (IC50) of STICs were 2.1 × 10−4m and 6.4 × 10−4m for DIDS and SITS, respectively. This effect was not voltage‐dependent. 3 The time constant of decay of STICs (τ), which is voltage‐dependent, was increased by about 30% by SITS and decreased by about 20% by DIDS. The effect of DIDS and SITS on τ was similar at holding potentials of − 50 and + 50 mV. 4 These compounds did not modify the characteristics of spontaneous transient outward currents (STOCs, calcium‐activated potassium currents). 5 DIDS and SITS decreased the amplitude of ICl(Ca) evoked by noradrenaline and caffeine less potently than STICs with IC50 values of 7.5 × 10−4m and 1.8 × 10−3m, respectively. 6 DIDS and SITS increased the calcium‐activated potassium current (IK(Ca)) evoked by noradrenaline and caffeine by 3–6 fold. 7 Anthracene‐9‐carboxylic acid (A‐9‐C) inhibited STICs in a voltage‐dependent fashion and was about 3 fold more active at + 50 mV than at − 50 mV. A‐9‐C increased STIC τ and this effect was enhanced by depolarization. 8 A‐9‐C also inhibited caffeine‐evoked ICl(ca) but less potently than STICs and also increased the evoked IK(ca) without altering spontaneous IK(ca). 9 The results from the present work are compared with the pharmacology of other chloride conductances and the mechanism of action of the chloride channel antagonists in vascular smooth muscle is discussed.

Synergism between inositol phosphates and diacylglycerol on native TRPC6-like channels in rabbit portal vein myocytes.

In rabbit portal vein myocytes noradrenaline activates a non‐selective cation current (Icat) which involves a transient receptor potential protein (TRPC6). Previously we have shown that the diaylglycerol (DAG) analogue 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG) stimulates Icat via a protein kinase C (PKC)‐independent mechanism, and in the present study we have investigated the interaction between inositol phosphates (InsPs) and OAG on Icat. With whole‐cell recording of Icat from freshly isolated rabbit portal vein myocytes the amplitude and rate of activation of noradrenaline‐evoked Icat were much greater than those of OAG‐induced Icat. Inclusion of inositol 1,4,5‐trisphosphate (Ins(1,4,5)P3) in the pipette solution did not evoke Icat but greatly potentiated the amplitude and rate of activation of OAG‐induced Icat. With isolated outside‐out patches Ins(1,4,5)P3 markedly increased the rate of activation and the open probability of OAG‐evoked channel activity, with no change in unitary conductance, channel mean open times or burst durations. The effects of Ins(1,4,5)P3 were mimicked by Ins(2,4,5)P3, 3‐F‐Ins(1,4,5)P3 and Ins(1,4)P2 but not by Ins(1,3,4,5)P4 and the potentiating effects of InsPs were not inhibited by heparin. Therefore it is concluded that both DAG and InsPs are necessary for full activation of Icat by noradrenaline and the effect of InsPs is via a heparin‐insensitive mechanism and represents a novel action of InsPs.

Time course of spontaneous calcium-activated chloride currents in smooth muscle cells from the rabbit portal vein.

1. The time course of spontaneous calcium‐activated chloride currents was studied with the perforated patch technique in freshly dispersed smooth muscle cells from the rabbit portal vein. 2. In potassium‐containing solutions the spontaneous transient outward current (STOC, a calcium‐activated potassium current) was more commonly recorded than spontaneous transient inward currents (STICs, a calcium‐activated chloride current). In addition the duration of STOCs was much briefer (about 100 ms) than the duration of STICs (about 400 ms). 3. The decay of STICs could be described by a single exponential but the STOC decay appeared to be more complex. The decay time constant of STICs was not determined significantly by amplitude. 4. The time constant of decay of STICs (tau) was 86 ms at ‐50 mV and was increased by depolarization. Between ‐90 and +50 mV the relationship between tau and membrane potential was exponential and tau changed e‐fold for a change of membrane potential of 120 mV. 5. The I‐V relationship of STIC amplitude was linear between ‐10 and +50 mV but at more negative potentials the chord conductance was reduced and the I‐V relationship exhibited negative slope conductance between ‐50 and ‐90 mV. 6. There was good agreement between the STIC tau values and the exponential relaxations to voltage steps evoked during caffeine‐induced calcium‐activated chloride currents. 7. In the presence of the chloride channel blocking agent anthracene‐9‐carboxylic acid the STIC amplitude was reduced and tau was increased. This effect was voltage dependent with a much greater effect at positive potentials. 8. The evidence suggests that the decay of STICs represents closure of chloride channels and tau approximates to the channel mean open time.

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.