TRPC6 channels stimulated by angiotensin II are inhibited by TRPC1/C5 channel activity through a Ca2+- and PKC-dependent mechanism in native vascular myocytes.

The present work investigated interactions between TRPC1/C5 and TRPC6 cation channel activities evoked by angiotensin II (Ang II) in native rabbit mesenteric artery vascular smooth muscle cells (VSMCs). In low intracellular Ca(2+) buffering conditions (0.1 mm BAPTA), 1 nm and 10 nm Ang II activated both 2 pS TRPC1/C5 channels and 15-45 pS TRPC6 channels in the same outside-out patches. However, increasing Ang II to 100 nm abolished TRPC6 activity but further increased TRPC1/C5 channel activity. Comparison of individual patches revealed an inverse relationship between TRPC1/C5 and TRPC6 channel activity suggesting that TRPC1/C5 inhibits TRPC6 channel activity. Inclusion of anti-TRPC1 and anti-TRPC5 antibodies, raised against intracellular epitopes, in the patch pipette solution blocked TRPC1/C5 channel currents but potentiated by about six-fold TRPC6 channel activity evoked by 1-100 nm Ang II in outside-out patches. Bath application of T1E3, an anti-TRPC1 antibody raised against an extracellular epitope, also increased Ang II-evoked TRPC6 channel activity. With high intracellular Ca(2+) buffering conditions (10 mm BAPTA), 10 nm Ang II-induced TRPC6 channel activity was increased by about five-fold compared to channel activity with low Ca(2+) buffering. In addition, increasing intracellular Ca(2+) levels ([Ca(2+)](i)) at the cytosolic surface inhibited 10 nm Ang II-evoked TRPC6 channel activity in inside-out patches. Moreover, in zero external Ca(2+) (0 [Ca(2+)](o)) 100 nm Ang II induced TRPC6 channel activity in outside-out patches. Pre-treatment with the PKC inhibitor, chelerythrine, markedly increased TRPC6 channel activity evoked by 1-100 nm Ang II and blocked the inhibitory action of [Ca(2+)](i) on TRPC6 channel activity. Co-immunoprecipitation studies shows that Ang II increased phosphorylation of TRPC6 proteins which was inhibited by chelerythrine, 0 [Ca(2+)](o) and the anti-TRPC1 antibody T1E3. These results show that TRPC6 channels evoked by Ang II are inhibited by TRPC1/C5-mediated Ca(2+) influx and stimulation of PKC, which phosphorylates TRPC6 subunits. These conclusions represent a novel interaction between two distinct vasoconstrictor-activated TRPC channels expressed in the same native VSMCs.

Store-operated Ca2+-permeable non-selective cation channels in smooth muscle cells.

Over twenty years ago it was shown that depletion of the intracellular Ca2+ store in smooth muscle triggered a Ca2+ influx mechanism. The purpose of this review it to describe recent electrophysiological data which indicate that Ca2+ influx occurs through discrete ion channels in the plasmalemma of smooth muscle cells. The effect of external Ca2+ on the amplitude and reversal potential of whole-cell and single channel currents suggests that there are at least two, and probably more, distinct store-operated channels (SOCs) which have markedly different permeabilities to Ca2+ ions. Two activation mechanisms have been identified which involve Ca2+ influx factor and protein kinase C (PKC) activation via diacylglycerol. In addition, in rabbit portal vein cells there is evidence that stimulation of alpha-adrenoceptors can stimulate SOC opening via PKC in a store-independent manner. There is at present little knowledge on the molecular identity of SOCs but it has been proposed that TRPC1 may be a component of the functional channel. We also summarise the data showing that SOCs may be involved in contraction and cell proliferation of smooth muscle. Finally, we highlight the similarities and differences of SOCs and receptor-operated cation channels that are present in native rabbit portal vein myocytes.

Membrane potential responses to ionophoretically applied alpha-adrenoceptor agonists in the mouse anococcygeus muscle.

1 Noradrenaline phenylephrine, naphazoline and oxymetazoline were applied by ionophoresis to the mouse anococcygeus muscle and the membrane potential was recorded with an intracellular microelectrode. 2 The ionophoretic application of noradrenaline and phenylephrine produced dose-related depolarizations in 96% of the cells tested; in contrast, naphazoline and oxymetazoline depolarized only 62% of the cells although contraction was always seen. 3 The depolarizations produced by all four drugs had similar characteristics in shape and time course except that the latency of responses induced by the imidazoline-related drugs was significantly longer than the value obtained with the phenylethanolamines. This discrepancy was not due to the difference in susceptibility to neuronal uptake of the two groups of drugs. 4 The time to peak depolarization for naphazoline and oxymetazoline was longer than that for noradrenaline and phenylephrine but was not sufficient to account for the considerably slower contraction produced by the former drugs. 5 At room temperature the sensitivity of the mouse anococcygeus to ionophoretically applied naphazoline and oxymetazoline was significantly lower than that to noradrenaline and phenylephrine but at 35 degrees C the sensitivity was similar for all drugs. 6 These results suggest that there might be two subclasses of alpha 1-adrenoceptor in the mouse anococcygeus; stimulation of one type leads to depolarization and contraction and activation of the other class produces contraction with no change in membrane potential.

Effect of tetramethylenedisulphotetramine on the membrane conductance increase produced by gamma-amino-butyric acid at the crab neuromuscular junction.

The effect of tetramethylenedisulphotetramine (TETS) on the increase in membrane conductance produced by gamma-aminobutyric acid (GABA) at the hermit crab neuromuscular junction was investigated. TETS produced a dose-dependent antagonism of GABA, which was characterized by a non-parallel shift of the log dose-conductance curve for GABA with a reduced maximal conductance change.

The action of noradrenaline on single smooth muscle cells freshly dispersed from the guinea-pig pulmonary artery.

Responses to ionophoretically-applied noradrenaline were investigated with micro-electrodes in whole tissue preparations and with patch pipettes in isolated cells dispersed from the guinea-pig pulmonary artery. In whole tissue and dispersed cells noradrenaline evoked monophasic depolarizations which had a similar time course. In dispersed cells the amplitude of electronic potentials was reduced during the noradrenaline-evoked depolarization. Under voltage clamp noradrenaline elicited an inward current, which persisted in 18 mM external potassium with the membrane potential set at the potassium equilibrium potential. In voltage clamp experiments the amplitude of current steps to hyperpolarizing voltage jumps was increased during the noradrenaline-induced inward current. These data suggest that the depolarization to noradrenaline in the guinea-pig pulmonary artery is mediated by an increase in membrane conductance.

Comparison of the biphasic excitatory junction potential with membrane responses to adenosine triphosphate and noradrenaline in the rat anococcygeus muscle.

The effects of field stimulation and ionophoretic application of adenosine triphosphate (ATP) and noradrenaline were studied in the rat anococcygeus by means of an intracellular microelectrode. Field stimulation at room temperature produced three types of electrical membrane response: (a) a 'fast' excitatory junction potential (e.j.p.) which had a latency of less than 100 ms and a time to peak of 300 ms; (b) a 'slow' e.j.p. which had a latency of several hundred ms and a time to peak of 1-2 s, and (c) an inhibitory junction potential (i.j.p.) which had a time to peak of about 1.5 s. All three responses were blocked by tetrodotoxin. The ionophoretic application of ATP produced both monophasic and biphasic depolarizations; these responses had a latency of less than 30 ms and a time to peak of 150-300 ms. In contrast, ionophoretically-applied noradrenaline produced a depolarization which had a mean latency of 471 ms and a time to peak of 861 ms. The 'slow' e.j.p. and the noradrenaline-induced depolarization were blocked by prazosin whereas the 'fast' e.j.p. and the ATP responses were resistant to this antagonist and also to atropine. These results are further evidence that the 'fast' e.j.p. in some smooth muscle tissues is mediated by ATP.

Mechanism of action of alpha-adrenoceptor activation in single cells freshly dissociated from the rabbit portal vein.

1. The action of noradrenaline was studied in freshly dispersed cells of the rabbit portal vein using microelectrode techniques. 2. In normal physiological salt solution, the ionophoretic application of noradrenaline evoked an alpha-adrenoceptor-mediated depolarization and sometimes a beta-adrenoceptor-mediated hyperpolarization. Experiments were carried out in the presence of propranolol to study the membrane mechanism associated with alpha-adrenoceptor activation. 3. In the current clamp mode of recording, the equilibrium potential of the noradrenaline-evoked depolarization was -1.9 mV. The depolarization was brought about by an increase in membrane conductance. 4. Under voltage clamp conditions, noradrenaline produced an inward current with a reversal potential of -7 +/- 3 mV (mean +/- s.e. mean). 5. The relationship between the noradrenaline-induced inward current and clamp potential was non-linear. Depolarization enhanced the conductance elicited by noradrenaline. 6. Evidence is presented which suggests that an additional conductance mechanism (probably an increase in potassium conductance) is also evoked by alpha-adrenoceptor stimulation in dispersed cells of rabbit portal vein.

Membrane mechanism associated with muscarinic receptor activation in single cells freshly dispersed from the rat anococcygeus muscle.

1 The mechanism of action of carbachol was studied on freshly dispersed cells of the rat anococcygeus using microelectrodes and patch pipettes. 2 Micro-ionophoretic application of carbachol evoked reproducible depolarizations which were reduced or blocked by atropine (10(-7)-10(-6) M). The time courses of the responses to noradrenaline and carbachol were similar. 3 The reversal potential of the carbachol-induced response was -3.8 mV and similar to the value (-6.2 mV) found for noradrenaline. 4 During the response to carbachol the membrane conductance was increased. At depolarized membrane potentials carbachol evoked biphasic membrane responses suggesting an increase in two separate ionic conductances. 5 With patch pipettes in the whole-cell configuration under voltage-clamp, carbachol produced an inward current at a holding potential of -50 mV. The inward current was associated with an increase in membrane conductance with an equilibrium potential of about 0 mV. 6 It is suggested that muscarinic receptors and adrenoceptors in the rat anococcygeus may activate similar membrane conductances. The most prominent mechanism is an increase in chloride ion conductance.

A comparison between mechanisms of action of different nicotinic blocking agents on rat submandibular ganglia.

The blocking properties of tubocurarine, decamethonium, hexamethonium and trimetaphan on nicotinic agonists applied by repetitive ionophoretic pulses were examined in rat submandibular ganglion cells using a two-microelectrode voltage-clamp technique at 30 degrees C. Hexamethonium, a proposed slowly dissociating, open-channel blocker at concentrations of 2-20 microM did not produce a 'use-dependent' run-down of responses, but its antagonism was clearly dependent on membrane potential. The voltage-dependent reduction of agonist response by hexamethonium was not dependent on the nature of agonist used. Similar results were obtained with acetylcholine (ACh) and carbamylcholine (CCh) ionophoresis. (+)-Tubocurarine (5 microM) and decamethonium (10 microM) produced 'use-dependent' run-down of agonist responses which became more pronounced at higher frequency and as the cell was hyperpolarized, consistent with open-channel blockade. In contrast, trimetaphan (2.5 microM), a receptor antagonist did not cause 'use-dependent' run-down of responses. Hence, the antagonism produced by hexamethonium, unlike tubocurarine and decamethonium, could not be accounted for in terms of open-channel blockade but requires an alternative mechanism, the nature of which is discussed.

Evidence for two mechanisms of depolarization associated with alpha 1-adrenoceptor activation in the rat anococcygeus muscle.

Membrane potential responses in the rat isolated anococcygeus to bath-applied noradrenaline and field stimulation have been investigated by use of intracellular microelectrode and combined extracellular electrical and mechanical (sucrose gap) recording techniques. Intracellular recordings were made usually from tissues immobilized with hypertonic Krebs solution. Bath-application of noradrenaline produced depolarizations which consisted of two components; an initial 'fast' phase which peaked within 1-2 s and which was followed by a 'slow' sustained depolarization. Both components were concentration-dependent. Noradrenaline could also evoke oscillations in membrane potential which, unlike the 'fast' component of depolarization, were prevented by conditioning hyperpolarization of the membrane and were evoked by direct membrane depolarization with externally applied current pulses. Thus, the oscillations are voltage-dependent phenomena. Replacement of the external NaCl of the Krebs solution with an equimolar amount of Na benzenesulphonate abolished the noradrenaline-evoked 'fast' depolarization while the 'slow' phase was unaffected. This suggests that two mechanisms of depolarization are activated in this muscle by the bath-application of noradrenaline. The adrenergic excitatory junction potential was also abolished in Na benzenesulphonate. Prazosin reduced both the 'fast' and 'slow' components of depolarization produced by noradrenaline indicating their mediation by alpha 1-adrenoceptors. The membrane potential (-29 mV) at the maximum amplitude of the 'fast' depolarization was similar to the equilibrium potential (-27 mV) for the depolarization evoked by ionophoretically applied noradrenaline and which was obtained by extrapolation from the relationship between amplitude of the ionophoretic response and membrane potential displacement in the partition chamber. These results suggest that the 'fast' depolarization and the ionophoretic response are due to an increased membrane conductance, possibly to chloride.

Electrophysiological analysis of neurogenic vasodilatation in the isolated lingual artery of the rabbit.

The nature of neurogenic vasodilatation was investigated in isolated segments of rabbit lingual artery. In separate experiments membrane responses to nerve stimulation were studied by use of microelectrodes. In the presence of guanethidine to block constrictor responses and noradrenaline to induce tone, field stimulation with trains of pulses (8 Hz for 0.5 to 4 s) produced vasodilatation. Atropine (10(-6) M) reduced the relaxations to about 50% of the control values while the induced vasodilatations were potentiated by physostigmine. Tetrodotoxin (TTX, 10(-7) M) blocked all nerve-evoked responses. These data suggest that there is a cholinergic and a non-cholinergic component of the vasodilatation produced by nerve stimulation in the rabbit lingual artery. Single stimuli did not evoke electrophysiological responses. With parameters similar to those used in the mechanical studies, periarterial stimulation in the presence of guanethidine evoked membrane hyperpolarizations which achieved amplitudes of up to 11 mV. The ionophoretic application of acetylcholine (ACh) produced hyperpolarization. The inhibitory junction potentials (i.j.ps) but not the ionophoretic-induced responses were blocked by TTX. The nerve-evoked and the ACh-induced hyperpolarizations were potentiated by physostigmine (5 X 10(-7) M) and totally blocked by atropine (10(-7) M). I.j.ps and hyperpolarization to ionophoresis of ACh were recorded from arteries in which the endothelium had been removed by mechanical rubbing. Mechanical relaxation to field stimulation and ACh was observed in preparations without endothelium. These data suggest that the cholinergic component of the neurogenic vasodilatation in the rabbit lingual artery is accompanied by hyperpolarization. The non-cholinergic component does not appear to possess an electrophysiological correlate. In addition, it seems that the action of nerve-released ACh is mediated by muscarinic receptors which are situated directly on the vascular smooth muscle cells.

The effect of alpha, beta-methylene ATP on the depolarization evoked by noradrenaline (gamma-adrenoceptor response) and ATP in the immature rat basilar artery.

Depolarizations evoked by noradrenaline that were resistant to alpha- and beta-adrenoceptor antagonists were recorded in the rat basilar artery. These gamma-adrenoceptor-mediated responses and the depolarizations to adenosine triphosphate (ATP) were blocked by pretreatment of the tissue with alpha, beta-methylene ATP. These data are discussed with respect to the selectivity of alpha, beta-methylene ATP.

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, ICl(Ca)) in a concentration-dependent manner. The potency sequence was niflumic > flufenamic > mefenamic acid. 3. At -77 mV 1 x 10(-5) M flufenamic acid increased the STIC exponential decay time constant (tau). At higher concentrations the STIC decay was described by 2 exponentials with an initial decay (tau f) faster than the control tau value and a second exponential (tau s) which had a time constant slower than the control tau value. Low concentrations of mefenamic acid had no effect or decreased the tau 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 > or = mefenamic acid. 6. All these compounds in concentrations greater than 5 x 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 x 10(-4) M flufenamic acid and 5 x 10(-4) M niflumic acid was not affected by 1 x 10(-5) M glibenclamide but was markedly inhibited by 1 x 10(-3) M 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 x 10(-4) M) and niflumic acid (5 x 10(-4) M) both evoked large outward currents when recordings were made with either 1 x 10(-4) M or 1 x 10(-2) M 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 ICl(Ca) by a mechanism similar to niflumic acid, possibly open channel blockade. Furthermore, at concentrations greater than 5 x 10(-5) M all three fenamates inhibited STOC activity and evoked directly an outward current which resembled IK(Ca).

Effect of atropine on the decay of miniature end-plate currents at the frog neuromuscular junction.

1 The effect of atropine on the time constant of decay (tau) of miniature end-plate currents was investigated. 2. The atropine-induced shortening of tau was accompanied by a reduction in the voltage-sensitivity of tau. 3. This effect of atropine was reversible and dose-dependent, the voltage sensitivity being abolished with high concentrations of atropine (2.5 X 10(-5)M).

Effect of penicillin on the increase in membrane conductance induced by gamma-aminobutyric acid at the crab neuromuscular junction.

The effects of penicillin and picrotoxin on the increase in membrane conductance produced by gamma-aminobutyric acid (GABA) at the hermit crab neuromuscular junction were investigated. Penicillin failed to block the effects of GABA, while picrotoxin proved to be a potent antagonist.

Microelectrode study on the ionic mechanisms which contribute to the noradrenaline-induced depolarization in isolated cells of the rabbit portal vein.

1. Experiments were carried out to determine the identity of the ionic mechanisms which contribute to the noradrenaline-evoked depolarization recorded with microelectrodes in freshly dispersed rabbit portal vein cells. 2. In normal physiological salt solution with microelectrodes containing 1 M NaCl the reversal potential (Er) of the noradrenaline-induced response was -7.6 +/- 2.9 mV. When the external NaCl was replaced by equipmolar concentrations of NaI, NaBr and NaNO3, Er was -33 +/- 3.5 mV, -29.1 +/- 5.2 mV and -18.4 +/- 1.1 mV, respectively. 3. In physiological salt solution Er of noradrenaline-evoked responses recorded with electrodes filled with 1 M NaI or 1 M NaNO3 was +16.3 +/- 3.9 mV and +10.0 +/- 7.6 mV, respectively. These results suggest that an increase in anion conductance contributes to the depolarization to noradrenaline. 4. Data from experiments with organic anions indicated that glutamate behaves as a less permeant anion but that benzenesulphonate blocks the anion conductance to unmask another conductance mechanism activated by noradrenaline. 5. When external NaCl was substituted by choline Cl and Tris Cl Er was -21.3 +/- 3.7 mV and -20.5 +/- 2.8 mV, respectively. These results suggest that noradrenaline also activates a cation conductance mechanism in freshly dispersed rabbit portal vein cells. It is concluded that the depolarization to noradrenaline recorded with a microelectrode is produced by the simultaneous activation of an anion channel and a separate cation channel.

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 x 10(-4) M and 6.4 x 10(-4) M for DIDS and SITS, respectively. This effect was not voltage-dependent. 3. The time constant of decay of STICs (tau), 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 tau 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 x 10(-4) M and 1.8 x 10(-3) M, 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 tau and this effect was enhanced by depolarization. 8. A-9-C also inhibited caffeine-evoked IC1(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.

Some characteristics of pre- and post-synaptic inhibitory receptors at the hermit crab neuromuscular junction.

1 The effects of gamma-aminobutyric acid (GABA), beta-guanidinopropionic acid (betaGP) and picrotoxin on the pre- and post-synaptic receptors of the hermit crab neuromuscular junction were studied quantitatively usine electrophysiological techniques. Reductions in excitatory junction potential (e.j.p.) amplitude and membrane resistance were measured simultaneously from the same cells. 2 The pre- and post-synaptic receptors were activated by the same order of concentration of GABA, whereas betaGP stimulated the pre-synaptic receptors at a concentration ten times lower than was required to affect the post-synaptic membrane. 3 Picrotoxin appeared to antagonize the pre-synaptic action of betaGP in a competitive manner. The affinity constants (+/- s.e. mean) for picrotoxin 5 times 10(-6)M and 2 times 10(-4)M were 6.80(+/-0.46) times 10(5)M-1 and 6.42(+/-1.8) times 10(5)M-1 respectively. 4 The effect of GABA on e.j.p. amplitude also appeared to be antagonized competitively by picrotoxin whereas the post-synaptic effect was antagonized in a non-competitive manner. 5 Possible differences in the nature of the pre- and post-synaptic receptors are discussed.

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 x 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 x 10(-6) M and 1.1 x 10(-6) M respectively (cf. 3.6 x 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 x 10(-5) M, had no effect on spontaneous calcium-activated potassium currents. 7. Niflumic acid inhibited noradrenaline- and caffeine-evoked IO(Ca) with an ICM50 of 6.6 x 10-6 M, i.e.was less potent against evoked currents compared to spontaneous currents. In contrast niflumic acid(2 x 10-6 M-5 x 105 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 Ic(ca) in physiological mechanisms.

Activation of potassium currents by inhibitors of calcium-activated chloride conductance in rabbit portal vein smooth muscle cells.

1. The conventional whole-cell recording technique was used to study the effects of the chloride channel inhibitors ethacrynic acid, anthracene-9-carboxylic acid (A-9-C) and indanyloxyacetic acid (IAA) on membrane currents in rabbit portal vein smooth muscle cells at a holding potential of 0 mV. 2. Using a pipette solution that contained 1 x 10(-4) M 1,2-bis (2-aminophenoxy)-ethane-N,N,N,N,-tetraacetic acid (BAPTA) and a normal bathing solution the addition of ethacrynic acid (2 x 10(-4) M to 1 x 10(-3) M) inhibited spontaneous transient outward currents (STOCs) and evoked a concentration-dependent current at a holding potential of 0 mV. A similar current was activated by IAA (5 x 10(-4) M to 1 x 10(-3) M) but not by A-9-C (1-5 x 10(-3) M) at a holding potential of 0 mV. 3. The amplitude of the current evoked by ethacrynic acid and IAA was linearly related to potential between -30 and 0 mV and displayed outward rectification at positive potentials. The current induced by A-9-C was evident only at potentials positive to +20 mV. 4. Glibenclamide (1 x 10(-5) M) abolished the current evoked by ethacrynic acid and IAA at potentials negative to +10 mV and partially inhibited the current positive to +10 mV. The glibenclamide-insensitive current at positive potentials was completely inhibited by 1 x 10(-3) M TEA. The A-9-C-evoked current was insensitive to glibenclamide and abolished by 1 x 10(-3) M TEA. 5. The glibenclamide-sensitive current activated by ethacrynic acid was not sustained and declined to control levels in the continued presence of ethacrynic acid. However, the outwardly rectifying current recorded at +50 mV was well maintained over the same period. 6. Outwardly rectifying currents evoked by ethacrynic acid and A-9-C were observed with a pipette solution containing 1 x 10(-2) M BAPTA in cells bathed in Ca-free extracellular solution containing 5 x 10(-4) M BAPTA and 1 x 10(-5) M cyclopiazonic acid. 7. It is concluded that all three chloride-channel blockers activated an outwardly rectifying, TEA-sensitive current. Moreover, ethacrynic acid and IAA evoked an additional glibenclamide-sensitive current which was present at all potentials between -30 and +50 mV.