Pharmacological Characterization of Muscarinic Receptor-Activated Cation Channels in Guinea-Pig Ileum Shan Chen, 'Ryuji Inoue & Yushi Ito

Home , Nicardipine, Tetraethylammonium

'." Macmillan Press Ltd, 1993 Br. J. Pharmacol. (I(1993),993), 109, 793 801 Macmillan Press Ltd, 1993 Pharmacological characterization of muscarinic receptor-activated cation channels in guinea-pig ileum Shan Chen, 'Ryuji Inoue & Yushi Ito

Department of Pharmacology, Faculty of Medicine, Kyushu University, Fukuoka 812, Japan 1 The pharmacological properties of cationic currents activated by acetylcholine (ACh) (Icat) in guinea-pig ileal smooth muscle cells were investigated, with conventional single patch electrode or nystatin-perforated whole-cell recording. Cs-aspartate was used as the internal solution to allow selective measurement of Icat 2 Well-known K channel blockers, tetraethylammonium (TEA), 4-aminopyridine (4-AP), procaine and quinine as well as a Ca releasing agent, caffeine, all produced concentration-dependent inhibition of Ica, with rapid onset (time constant - 100 ms), when applied externally. The recovery from the inhibition on washout also occurred rapidly in the order of 100 ms except in the case of quinine. Approximate values of the half inhibitory concentrations (IC50) were 10 mM for TEA and caffeine, 1-5 mM for 4-AP and procaine, and 1 tiM for quinine. The mode of inhibition was voltage-dependent, i.e., depolarization relieved the inhibition with no change in reversal potential. 3 Externally applied diphenylamine-2-carboxylate (DPC) derivatives, DCDPC and flufenamic acid, produced potent inhibition of Ica, at micromolar concentrations (IC50s were <30 t4M for DCDPC and 32 fIM for flufenamic acid). The onset of and recovery from inhibition occurred slowly and the degree of inhibition depended on the membrane potential only weakly, without any discernible change in the reversal potential. 4 All of the above-tested drugs exhibited comparable inhibitory actions on the voltage-dependent Ca current in the concentration ranges effective at inhibiting Ica,. However, amongst them, quinine and flufenamic acid seemed to have several-fold better selectivity for the Icat channel than for the voltage- dependent Ca channel. 5 Internally dialysed GTPyS (100 IM) induced inward cationic currents. The effects of drugs on these currents were similar to their effects on the Icat current. 6 These results clearly indicate that many drugs used as pharmacological tools in smooth muscle research exert considerable nonspecific effects on various types of channels. The mechanism of inhibition and the relevance to use of these drugs as blockers for the Icat channel are discussed. Keywords: Acetylcholine (ACh); nonselective cation channels; blockers; smooth muscle

Introduction In mammalian intestinal smooth muscles muscarinic receptor increased as the membrane is depolarized (Sturgess et al., activation leads to at least two major consequences, produc- 1987; Ehara et al., 1988), as is observed in the case of Icat tion of inositol 1,4,5 trisphosphate and membrane depolariz- channel (Inoue & Isenberg, 1990b), and the hcat channel is ation due to increased Na+ permeability. The latter can be also sensitive to changes in the intracellular Ca2+ concentra- ascribed to the opening of cation (Icat), channels, which serve tion (Inoue & Isenberg, 1990c). These observations imply the as the main route for Na+ as well as Ca2" influx. Although presence of considerable structural homology between the some details of the biophysical nature of the Icat channel have Ca2"-activated cation channels and the hcat channel. It is already been published and the involvement of a pertussis therefore of great interest to examine how the above-men- toxin-sensitive guanosine 5'-triphosphate (GTP)-binding pro- tioned blockers for the Ca2"-activated cation channels act on tein in its activation has been suggested (e.g., Inoue, 1991b), the 'cat channel. very little effort has been made to characterize the phar- In the present study, we have investigated the effects of macological properties of this channel, and thus information DPC-derivatives as well as well-known K channel blockers about selective blockers is lacking. and some other agents which are frequently used as phar- Recently, it was reported that diphenylamine-2-carboxylate macological tools in electrophysiological studies on excitable (DPC) derivatives such as 3',5-dichloro-DPC (DCDPC) and membranes. As a result of this work, we found that quinine flufenamic acid are very potent and relatively selective block- and DPC derivatives such as DCDPC and flufenamic acid ers for Ca2 +-activated cation channels in colonic epithelial are very potent inhibitors of Icat, and unfavourable non- cells (Gogelein & Capek, 1990). Although the Ca2'-activated specific actions on other types of channels are also evident. cation channels appear to be very different from the Icat channels in mode of activation and voltage-dependence, some essential properties are similar. For example, single channel Methods conductances of both types of channels range between 20-30 pS and the kinetics are slow in both cases (Inoue et al., 1987; Cell dispersion and electrical recordings Swandulla & Partridge, 1990; Vogalis et al., 1990) as compared with voltage-dependent Na,K and Ca channels. In The procedures used for dispersion of single ileal smooth addition, some of the Ca2'-activated cation channels are muscle cells, the system for recording the membrane currents reported to be voltage-sensitive and the open probability is and the method of rapid application of drugs were the same as described previously (Inoue, 1991a; Inoue & Kuriyama, 1993). Briefly, guinea-pigs of either sex weighing 600-1000 g ' Author for correspondence. were stunned and exanguinated. Thin sheets of longitudinal 794 S. CHEN et al. smooth muscle (1-2 mm x 5-IO mm) were gently stripped dipped in a nystatin-free internal solution and then backfilled off by hand from the terminal part of ileum, then incubated with one complemented with nystatin. The timing of the in Ca-free solution containing 0.5-1.0 mg ml' actinase (Ka- dipping was a critical step to determine the success of form- ken, Tokyo, Japan) and 1 mg ml' collagenase (type I, Sig- ing a 'giga-seal' and subsequent chemical perforation. The ma) at 35°C for 15-20 min. The digested sheets of ileal other procedures of whole-cell recording were the same as muscle were cut into pieces and triturated in Ca free solution described above. until yielding a sufficient number of cells. Currents were recorded with a commercial patch clamp amplifier (Axopatch Solutions 1D, Axon Instruments, Burlingame, CA, U.S.A.) in conjunc- tion with an A/D, D/A converter (Labmaster TL-1 DMA, The bathing and pipette solutions were the following (mM): Axon Instruments), which was run under the control of an modified Krebs solution: Na+ 140, K+ 5, Mg2+ 1.2, Ca2+ 2, IBM compatible personal computer (AMSC-ATC, Alps Elec- Cl- 151.4, HEPES/Tris 10 (pH 7.35-7.4); Cs aspartate inter- tronic, Japan) with 'pClamp v.5.5' programmes (Axon nal solution: Cs+ 140, Mg2+ 2, Cl- 24, aspartate- 120, Instruments). All experiments were carried out at room EGTA10/4 - 5Ca2+ and HEPES 10 (titrated to 7.2-7.25 temperature. with Tris base). The Ca-free solution was made by simply omitting Ca2+. Nystatin-perforated whole-cell recording Chemicals Patch pipettes (3-5 Mohm) were made from 1.5 mm pyrex glass capillaries. Fresh nystatin stock solution was made Flufenamic acid and niflumic acid were purchased from every day by dissolving the powder (Sigma) in dimethylsul- Wako (Osaka, Japan). DPC and DCDPC were generous gifts phoxide (DMSO) to give a concentration of 50 mg ml1'. This from Dr R. Greger (University of Freiburg). DPC derivates was ultrasonicated for about 15 min. The stock solution was were freshly dissolved into DMSO and then diluted 1000- further diluted 200-500 times with the internal solution (Cs- 10000 times by the bathing solution so as to give a final aspartate complemented with 1 mM EGTA) and ultrasoni- DMSO concentration of no more than 0.1% for any experi- cated again for 15 min just before use. Whole-cell recording ment; 0.1% DMSO did not show any effects on its own. The was achieved with a pipette which was first momentarily other chemicals used in the present study were purchased

.-: b

jJ25 pA 0mM s10 :-:'M

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ACh ACh *l 10. ---S!--- i ___:: *------1 -* ------'------

A-iCh v

Quinine 10 M 10pm Figure 1 Rapid reduction in the acetylcholine (ACh)-evoked inward currents by K channel blockers and caffeine. Recordings were made with conventional patch electrode whole-cell recording, in which the cell membrane was disrupted by negative pressure after a 'giga-seal' was formed. Throughout the following figures this recording configuration was used, unless otherwise stated (i.e. Figures 4 and 5). The bath and pipette contained modified Krebs solution and Cs-aspartate internal solution, respectively. In the continued presence of 10 gM ACh (indicated by long bars), clamped cells were swiftly moved from the control bathing solution to one containing either tetraethylammonium (TEA) 10 mm (a), caffeine 10 mm (b), procaine 5 mM (c), 4-aminopyridine (4-AP) 1 mM (d) or quinine 10 j.M (e) (indicated by short bars). The onset and offset of solution changes are approximate. The speed and efficacy of the rapid solution change are demonstrated in Figure 2. The membrane was held at - 50 mV. Vertical deflections represent slow ramps as shown in Figure 7. BLOCKERS ON CATION CHANNELS 795 from the following: TEA, indomethacin and nystatin (Sig- included in the internal solution to clamp the [Ca2+]i to ma), 4-AP (Nacalai, Kyoto, Japan), procaine and caffeine 100-200 nM (see Methods) to minimize any secondary chan- (Wako, Osaka, Japan), quinine and quinidine (Tokyo Kasei, ges in the current amplitude and kinetics due to the altered Tokyo). Changes in pH due to tested drugs were minimized [Ca2+], (Inoue & Isenberg, 1990c). This strategy appeared to by adjusting the pH of stock solutions with addition of Tris work satisfactorily since the potentiating effect of voltage- base or HEPES as appropriate before use. dependent Ca2+ influx on the Icat (Inoue & Isenberg, 1990c) was only brief and marginal. Furthermore, membrane depolarizations to - 30 mV-O mV, which generate a long-lasting Results Ca inward current and subsequent contraction of smooth muscle cells when [Ca2+], is buffered weakly, did not contract The effect of K channel blockers on Ica, cells examined over 1 min. Under these conditions, however, the inward currents evoked by 10 tLM ACh showed a decay to As I,,a, is very sensitive to changes in the intracellular Ca2+ a varying extent, with a time constant of 30-60 s, and under- concentration ([Ca2+]i), a mixture of EGTA and Ca2" was went a strong rundown on repeated application. Therefore, we first assessed the effects of blockers on Icat with a fast 0- I~~~~~~~~~~~~~ solution exchange device based not on sequential but on a single administration of ACh as performed previously (Inoue, -50- 1991a). Figure 1 demonstrates the time course of several well- established blockers on ,,a,. K channel blockers such as TEA, -100- 4-AP and procaine all caused clear inhibition. The inhibitions Na removal seemed to occur rapidly within about 50-100 ms, the speed -150- T=40ms of which compares to that of solution exchange as demonstrated by Na removal in Figure 2. The inhibitory action of -200-t these agents was reversible because the degree of inhibition evaluated at onset and recovery from blockade was in close l5O I agreement. For example, with TEA 1O mM, the percentage 100 200 300 400 500 inhibition was 37% and 35% respectively at onset and _30b recovery (Figure la). Caffeine, a Ca2+ releaser from the sarcoplasmic reticulum (SR), also produced inhibition of Icat < -40- at a concentration of 10 mM, the nature of which appeared 0. similar to that produced by the K channel blockers (also see V0D -50- below). Figure 3a summarizes the results pooled from many cells with various concentrations of these blockers. The half a E -60- inhibitory concentrations (IC5os) were between 2 and 5 mM TEA10mM for 4-AP, 1 and 5 mM for procaine, and about 1O mM for C -70 T = 41 ms TEA and caffeine. It should be noted that all the IC50 values t-E;A. L) -80 \'

-HU e I 1 100 200 300 400 500 1.0- . ., -100- O. . -200- -300- -400-

XfFlufenamic acid 100 ._ -500- FLM Tlv = 207 ms -600- T T2 = 2153ms 2 5 10 P i5 10 -700 4-AP " Procaine TEA Caffeine co -800_#W% 1 0 2000 4000 6000 8000 i.doo 104 Z 00.64 2.8 I Time (ms) -W Figure 2 Efficiency of the rapid solution changing system evaluated a: - by Na removal and the onset of tetraethylammonium (TEA) or f.I" flufenamic acid induced inhibition of the acetylcholine (ACh)-evoked inward current. (a) At a holding potential of - 50 mV, Na+ was removed from the bathing solution, by switching from normal Krebs solution to Na+-free Krebs solution (Tris replacement) in the con- 0.2 tinued presence of 10 fM ACh. The time constant of decrease in the Icat amplitude is 40 ms in this experiment (on average, 39 ± 9 ms, 0.0 EWE n = 4). On reintroduction of Na+, the current recovered with the 1 10 60 600 (5M) time constant of 42 ± 5 ms (n = 4). The slower time constant of the Quinine solution change as compared to a similar method applied to rat and Figure 3 Concentration-dependence of the inhibition of the acetyl- bullfrog sensory neurons (ca. 10ms; Bean, 1990) may be due to the choline (ACh)-evoked inward current by K channel blockers and spindle shape of ileal smooth muscle cells. (b and c) The time course caffeine. The results obtained from experiments similar to those of inhibition of the ACh-evoked current by 10 mm TEA (b) or shown in Figure 1 are summarized. Each column represents mean 1I00 1M flufenamic acid (c). Note that the time constant for TEA- (and s.e.) calculated from 5-10 experiments by measuring the frac- induced inhibition (41 ms) compares to that for Na removal, while tion of the ACh (10ILM)-evoked current which remained in the that for flufenamic acid is considerably slower (fast and slow time presence of the tested drug. In the case of quinine (b), the best fit constants are 207 and 2153 ms, respectively). The fast time constant using the equation in Figure 5 gave values of 0.84 llM and 1.94 for K for flufenamic acid was not always observed. and n, respectively. 796 S. CHEN et al.

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Figure 4 Inhibition of the acetylcholine (ACh)-evoked inward current by diphenylamine-2-carboxylate (DPC) derivatives. (a) Currents were recorded with conventional whole-cell recording. ACh and DPC derivatives were applied at bars with a fast solution change. Conditions used here are the same as in Figure 1. (b) Currents recorded with the nystatin-perforated whole-cell recording before (b(i)), after 10 min pretreatment with (b(ii)) and 10 min after washout of (b(iii)) 50 gAM flufenamic acid. Flufenamic acid was added in the bath with ordinary bath superfusion, while 10 M ACh was rapidly applied at bars with a fast solution exchange device described in Figure I in the continued presence of flufenamic acid. The holding potential was - 70 mV. ACh 10 JAM was added during the bars. obtained here fall in the concentration-range effective for reversal potential of the inward current evoked by 10 JAM blocking certain types of K channels (Cook & Quast, 1990) ACh did not shift significantly on halving the extracellular or causing Ca2+ release from the SR. Cl- concentration (8.0 ± 5.4 mV for normal Krebs (n = 11) Quinine is different from the above K channel blockers. It vs. 8.6 ± 2.9 mV for 75 mM Cl Krebs (n = 4)). Under these is very potent at inhibiting Icat, but the recovery after conditions, time-dependent reduction in the Ica, amplitude washout was quite slow or in some cases only partial over was no more than 20% over 30 min on repeated applications 30 s (Figure le). The concentration-inhibition relationship of 1O JM ACh with an interval of 5-O min (also see Figure reveals that the half inhibition occurs at about 1 JAM (see 1 in Inoue & Kuriyama, 1992), otherwise the data were legend); the value is extremely low as compared to those for discarded. This greatly reduced errors of overestimating the the other K channel blockers (Figure 3b). effects of blockers, which may arise from rundown. As shown in Figure 4b (ii), administration of 50 JAM flufenamic acid for DPC derivatives potently block Ical 10 min resulted in reduction in the ,a, current by 70%. This inhibitory effect persisted over a few minutes after the Figure 4a shows actual traces of inhibition of Icat by two washout of flufenamic acid, and was evident when the drug DPC derivatives, flufenamic acid and DCDPC, which are was administered for several minutes and then washed out reported to be potent blockers of Ca2"-activated cation chan- just before the application of ACh. The latter observation nels (Gogelein & Capek, 1990). The time course of inhibition excludes the involvement of open channel blockade was slow and could not be clearly distinguished from those mechanism reported for e.g., MK801 which is known as a of desensitization. It seemed that the time constant was one potent and selective blocker of NMDA-type cation channels order of magnitude slower than in the cases of K channel (Huetter & Bean, 1988). Figure 5 summarizes the concen- blockers such as those shown in Figure 2. Recovery after tration-dependent inhibition caused by various DPC deriva- washing out the drugs was incomplete, since the degree of tives. The IC50s are smaller than 30 JAM for DCDPC but inhibition evaluated at onset and recovery did not seem to larger than 100JAM for DPC and niflumic acid (Figure 5a). coincide. For this reason, we decided to evaluate the inhi- The concentration-inhibition curve for flufenamic acid gives bitory potency of the DPC derivatives on repeated applica- an IC50 of 32 JM and a cooperativity number of one (open tion of ACh employing the nystatin-perforated whole-cell circles in Figure Sb). recording method (Horn & Marty, 1988; also see Methods). Figure 4b shows actual current traces before, during and Effect of blockers of the Ical channel on the after the application of 50 JAM flufenamic acid with nystatin- voltage-dependent Ca channel perforated whole-cell recording. At a holding potential as negative as - 70 mV with Cs' in the pipete solution, con- The selectivity of these agents was investigated in a study of tamination of I,ht with another possible inward current, e.g. a their effects on the voltage-dependent Ca channels. Figure 6a Ca2'-activated Cl- current, seemed negligible because the illustrates the current-voltage relationship in the presence and BLOCKERS ON CATION CHANNELS 797

1.0 30a 0 Amomwd= I-:L -30-

&- 90 0 - 120 Quinine -150 10 AM I -180 I I i I -60 -40 -20 0 iO 40 60 0.0 Membrane potential (mV) b 1.2, 4- 1.0- 0.2 C 0.8- a- 4 c:Q0 0.6 0.4 tT 052 .5'5 0 0.2 0.0-L ., .,,. 3 mM 10 mM ¶ 5 10 FM Procaine TEA Quinine 021 1.2- -5.5,5 5 4.5 4 3353 0 1.0- lo.g (PhFke.oemt, 0.8 C.) i 0.6 Figure 5 Concentration-dependence of the inhibition of the acetyl- 0 choline (ACh)-evoked inward currents by diphenylamine-2-carboxy- 0.4- S0 late (DPC) derivatives. The results of nystatin-perforated whole-cell .5 recording seen in Figure 4b are summarized. The degree of inhibition 0.2 was calculated as the ratio of It current amplitude in the presence of 0.0 a drug tested versus that in control response. Control responses were 30 100({M) obtained before and after application of DPC derivatives, in order to DCDPC Flufenamic Indomethacin check the rundown of currents (see text), and the first response was acid used as the reference. (a) Each column represents mean ( + s.d.) calculated from 3-6 cells, respectively. (b) Concentration-inhibition Figure 6 Inhibition of voltage-dependent Ca channels by various curve of the ACh (10tiM)-evoked inward current for flufenamic acid drugs. The bath contained modified Krebs solution and the pipette (0). The curve was fitted by an empirical Hill type equation (1/ Cs-aspartate solution in which only 10 mM EGTA, instead of (1 + ([flufenamic acid]/K)1) where [flufenamic acid], K and n denote EGTA/Ca mixture, and 2mm ATP were added. (a) An 800ms the concentration of flufenamic acid, the concentration of flufenamic voltage ramp (- 120 to 80 mV) was applied at a rate of 0.25 V ' to acid to produce 50% inhibition and the cooperatively number. The activate voltage-dependent Ca currents. Leak currents due to depol- best fit of the data points (0) gave K = 32 jaM and n = 1; (0) arization were obtained in the presence of 5 gim nicardipine and represent the fraction of voltage-dependent Ca currents remaining in subtracted. The currents were plotted against membrane potential as the presence of flufenamic acid. The currents were measured either shown in Figure 7. (b and c) The fraction of Ca current remaining in with voltage-ramps (see Figure 6a) or by depolarizing pulses. Data the presence of the drugs named in the figure. points were obtained from 3-7 cells. Influence of blockers on the ionic selectivity and absence of 10 JlM quinine, evaluated by a 800 ms ramp pulse. voltage-dependence of the Icat current The presence of quinine resulted in reduction of the voltage- dependent Ca current, the extent of which was virtually The mechanisms underlying the inhibitory actions of the voltage-independent. As summarized in Figure 6b, quinine above-mentioned agents on Iat may be complicated, e.g., the reduced the voltage-dependent Ca current by no more than onset and recovery time course seem to be different among 20% at a concentration of 1O tLM, which inhibited Ihat by different agents as shown in Figure 2. In order to get further more than 90% (Figure 3b). In contrast, 3 mM procaine insight, we compared the voltage-dependence and ionic selec- produced inhibition of the voltage-dependent Ca current and tivity of the Ih,t current before and after the applications of 4cat to a similar extent. blockers with ramp command voltages. This approach is With DPC derivatives, 30 j1M DCDPC, which is the most practical because only one ramp instead of a set of step potent at inhibiting Icat, also caused a comparable inhibition pulses allows not only measurement of the reversal potential of the voltage-dependent Ca current, whereas 1OO1M flufe- but a more complete delineation of the voltage-dependence namic acid, which produces ca. 80% reduction in I:at, affected of the Icat current. As illustrated in Figure 7a (the uppermost the voltage-dependent Ca current only slightly (Figure 6c and trace), the membrane was first held at - 120 mV for 250 ms, filled circles in Figure Sb). to set the Acat current in a steady state (time constant of These results indicate that quinine and flufenamic acid are relaxation at this potential is 40-50 ms, Inoue & Isenberg, the preferred blockers among the above-tested drugs against 1990b), and then depolarized by a ramp at a rate of h, channel with regard to selectivity on the voltage-depen- 0.1 V s l. The net current corresponding to this voltage dent Ca channels. The ratio of the inhibitory effect on Icat change in the absence of ACh was nearly ohmic (the middle versus that on the voltage-dependent Ca current is 4.5 and pale trace), but changed to an inverted bell-shaped curve in 7.9 for 10 AM quinine and 10011M flufenamic acid respec- the presence of ACh (the bottom thick trace). The current tively, while this value is near to 1.0 in the case of 30 JAM component sensitive to ACh was then defined as a difference DCDPC (1.47). of net currents in the presence and absence of ACh (i.e., the 798 S. CHEN et al.

+80 mV only a few mV for flufenamic acid but amounted to 12 mV for TEA (Figure 8a(ii) and a(i)). Furthermore, the degree of inhibition at a given membrane potential is reduced as the membrane is depolarized in the case of TEA, while only a small relief is observed for flufenamic acid (Figure 8b(i) and b(ii)). The ratio of the degree of inhibition at + 80 mV over that at - 80 mV (180/1L80) is 1.9 ± 0.4 (n = 4) for TEA 10 mM and 1.2 ± 0.2 (n = 3) for flufenamic acid 50 tLM, clearly 4) indicating the more pronounced voltage-dependence of the z TEA effect. Table 1 tabulates the results obtained for other drugs. Voltage-dependence was also clearly observed for 4-AP, Time (ms) caffeine and quinine. In one available experiment with 600b DCDPC, only weak voltage-dependence was observed as in the case of flufenamic acid. 400' GTPyS-induced inward currents also show a similar -9 200- pharmacological profile TEA 10 mM i-xL ileal smooth CDL - 00-O It was previously reported that in guinea-pig u 114 muscle intracellularly applied GTPyS induces an inward -200- cationic current, the properties of which resemble those of ~-Control the ICat current (Inoue & Isenberg, 1990a; Komori & Bolton, A This curent is thought to be induced through persis- - 4UU v 1 . . I . . . . 1990). -120 -80 -40 0 40 80 tent activation of a pertussis toxin-sensitive G-protein that Membrane potential (mV) might be coupled to the I< channel rather directly, where the association-dissociation process of ACh to the muscarinic 600& receptor can be excluded. We therefore tested whether or not / the sties of actions of the above-mentioned drugs are located 400- downstream of the receptor, by observing their effects on the GTPyS-induced inward current. After the start of internal 200- Flufenamic acid dialysis with 100 GTPyS, noisy inward currents were -CL jtM 50 FM - - a1) observed and reached a level ranging from 10 to 75 pA (I I (- 35 ± 21 pA, n = 10) within 10-20 min. Usually, these currents were maintained for 30-60 min and declined slowly. As -200- shown in Figure 9, addition of 5 mM procaine, 10 jAM quinine - Control ",-.%., -, or 30 l4M DCDPC in the bath suppressed the noisy inward nn_ I _- ...... a I I . . . -4UU1 .- current almost completely, while 10 mM TEA and 10 mM -120 -80 -40 0 40 80 caffeine (data not shown) reduced the current to about the Membrane potential (mV) half of the original amplitude. After washout of the drugs, recovery from the inhibition occurred in several minutes with Figure 7 Changes in the current-voltage relationships of the acetyl- quinine and DCDPC. Quinidine (10 jtM), which is a stereo- choline (ACh)-evoked inward current by addition of tetraethylam- isomer of and equipotent with quinine, also markedly dim- monium (TEA) or flufenamic acid. The bath and pipette conditions inished the GTPyS-induced inward current. Other classes of were the same as in Figure 1. (a) The voltage ramp protocol em- K channel blockers such as charybdotoxin and glibenclamide ployed. Three traces represent a voltage change (uppermost bold showed no discernible effects on the GTPyS-induced currents. line), corresponding currents in the absence (middle pale) and the The inhibitory potencies and time courses of the drugs presence (bottom thick) of 10 tM ACh, respectively. In order to tested are consistent with those described for the I,at current. block the voltage-dependent Ca current, 5 pM nicardipine was pres- act somewhere ent. (b and c) The difference currents (bottom trace - middle trace This supports the hypothesis that the drugs in a) were converted into current-voltage curves. downstream of the receptor, most likely at the level of the I, channel protein. bottom trace - the middle trace). Figure 7b and c plot the ACh-sensitive currents obtained in this way against membrane potential. The validity of this method of assessing the Discussion voltage dependency was verified by the close agreement of Boltzmann parameters obtained by the two different voltage The main findings of the present study are: (1) many agents protocols under the same ionic conditions: the half maximum used as pharmacological tools in smooth muscle research activation potential (Vh) and the slope factor (k) were including TEA, 4-AP, procaine, quinine and caffeine are all - 47.9 ± 4.9 mV and - 18.0 3.7 mV for the above ramp able to inhibit the Icat channel at concentrations in normal protocol (n = 10) and - 45.9 4.6 mV and - 21.4 ± 2.1 mV use, but micromolar quinine might be useful to block for the multiple step pulse protocol, respectively (see Figure 8 selectively the Icat channel. (2) Potent blockers for the Ca2+- of this paper and Table 1 in Inoue, 1991a). activated cation channel, e.g. DPC derivatives, are also effect- Both TEA 10 mM and flufenamic acid 50 tLM flattened the ive at inhibiting the Icat channel at micromolar concen- current-voltage relationship for Icat but the reversal potential trations. The observed effects are likely to be actions on the was unaffected (Figure 7b and c). As summarized in Table 1, I.at current itself, since any indirect effects of these agents no significant shift of reversal potential was observed in the were minimized with internal dialysis with Cs aspartate and presence of any blocker listed. This suggests that the blockers clamping [Ca2+]i with a Ca2+/EGTA mixture. The mode of do not change the ionic selectivity of the Icat channel. inhibition of the various agents appears to differ in time The steady state activation curves calculated from the course and voltage-dependence and will be discussed below. current-voltage relationships were shifted to the right in the Such differences apparently divide the agents into two presence of TEA, while little shift was observed in the groups, fast (K channel blockers and caffeine) and slow presence of flufenamic acid: the shift of Vh value (AVh) was blockers (DPC derivatives). BLOCKERS ON CATION CHANNELS 799

b (i) 1-1 0)c CE 0.8- TEA 10 mM a) J L.1 L : 0.6- = 0)" 0) 'V'#I, ln = 0.4- .q. (.10) I; O-01' 'A'V <, 0.2- . v

A V lTX X X X X -120 -100 -80 -60 -40 -20 -100 -60 -20 20 60 Membrane potential (mV) Membrane potential (mV)

a (ii) b (ii) 1-, 1-, 0) C 0.8- C 0.8- Flufenamic acid Flufenamic acid / 50 FIM 50 FiM ' 0.6- Vh = -46.8 mV < 0.6-I C:. ` 0) - 'L . x cr L.C P."# 1% /% 0.4- Control S.' 0.4- WV, I 0. Vh = -43.3 mV C) r- . > 0 0.2- X- 0.2-

0 l 1 1 1 --- a X X X -120 100 -80 -60 -40 -20 0 -100 -60 -20 20 60 Membrane potential (mV) Membrane potential (mV)

Figure 8 Steady state activation curve and voltage-dependence of the acetylcholine (ACh)-evoked inward current in the presence of either tetraethylammonium (TEA) or flufenamic acid. (a) Examples of the steady state activation curve of I,,. evaluated from Figure 7b and c. The chord conductance, the ratio of the Ica, amplitude over the driving force (the membrane potential - the reversal potential), was calculated at a given membrane potential and then normalized to its maximum. At membrane potentials positive to 0 mV, which are near to the reversal potential, serious scatter in calculated values were observed due to the small current amplitudes and driving forces, thus such data were not included in the evaluation. Smooth sigmoid curves represent theoretical ones drawn from using parameters of the best fit according to the Boltzmann equation 1/(1 + exp((Vm-Vh)/k)), where Vm, Vh and k denote the membrane potential, the half-maximum activation potential and the slope factor, respectively. (b) Changes in the degree of inhibition by TEA 10 mM (i) or flufenamic acid 50 gM (ii) as a function of the membrane potential.

a b d Procaine_5 mm TEA 10 mM Quinidine1lCt0FM 10 min -

m;mII 94 PA 1 min -j 40 pA 1 min VH: -150mV I Quinine 10 pM 0 DCDPC 30 FM ~~~~~~~~~~~~_ - ffi - - -1 if - i7 min _J 40 pA I mi

Figure 9 Effects of various drugs on the GTPyS-induced inward current. The same conditions as in Figure 1 were used except that 100 jlM GTPyS was included in the pipette. At a holding potential of - 50 mV, various drugs were superfused into the bath at a rate of 0.07-0.08 ml s' (the chamber volume is 0.2 ml). 800 S. CHEN et al.

Table 1 Comparison of the reversal potential, voltage-dependence of the inhibitory effect (180/L-80) and shift in the half-maximum activation potential (AVh) in the presence of various drugs Erev (mV) I80/I-80 A Vh (mV) Control 8.0 ± 5.4 (1 1) NA NA TEA (10 mM) 6.8 ± 1.6 (4) 1.9 ± 0.4 (4) 14.6 ± 2.7 (3) Caffeine (10 mM) 5.2 ± 1.7 (3) 1.9 ± 0.5 (3) 20.7 4-AP (1 mM) 10.9 ± 1.6 (4) 3.1 ±0.7 (4) 13.9± 2.2 (3) Quinine (1 pM) 9.8 ± 1.4 (2) 2.2 ± 0.3 (3) 10.7± 2.9 (2) Flufenamic acid (50 pM) 6.1 ± 5.6 (3) 1.2 ± 0.2 (3) 4.0 ± 2.3 (3) DCDPC (30 JAM) 5.7 1.1 0.5 The numbers in parentheses represent the number of experiments. For the definition of 180/L_80 see the text. Control indicates the modified Krebs solution without drugs. NA indicates no available data. For abbreviations, see text.

K channel blockers and caffeine as nonspecific blockers activated cation channels by use of single channel recordings (Gogelein & Pfannmiiller, 1989; G6gelein et al., 1990; Siemer As generally documented in the literature, TEA in millimolar & Gogelein, 1992). The sequence of inhibitory efficacy is concentration is able to block several distinct classes of K DCDPC > flufenamic acid >> DPC > niflumic acid and the channels including Ca2"-dependent K channels, the delayed IC50 for DCDPC or flufenamic acid is about 10 JAM, although rectifier K channel, and the ATP-sensitive K channel (Cook the tested drugs were administered from the outer side of the & Quast, 1990). Amongst them, a large conductance K chan- cell membrane whilst most of data for the Ca2"-activated nel (designated as the maxi-K channel) is the most suscepti- cation channels were obtained in the inside-out patch ble to TEA applied both externally and internally, the IC,0 configuration. In rat exocrine pancreatic cells, DPC for the external TEA being as low as 0.3 mM in various derivatives block Cl conductances with comparable efficacy smooth muscles. Millimolar concentrations of 4-AP are also to Ca2'-activated cation channels (Gogelein & Pfannmiiller, reported to block the delayed rectifier (e.g. Beech & Bolton, 1989). However, conflicting evidence has been provided on 1990) as well as fast inactivating voltage-dependent K cur- the inability of 10 JAM DCDPC to affect Cl channels in distal rents (A-like currents; Okabe et al., 1987; Beech & Bolton, colonic epithelial cells of the same species (Gogelein & 1989; Imaizumi et al., 1990; Smirnov & Aaronson, 1992). Capek, 1990). In our experiments, we did not observe the Procaine (1-10 mM), together with other local anaesthetics, effects of these drugs on the Cl conductance in the guinea-pig blocks not only a broad range of voltage-dependent channels ileum, since such a conductance could hardly be detected (Na and K channels, Cook & Quast, 1990; Ca channels, the under the experimental conditions employed. However, the present work) but also the Ca2+ release process from the voltage-dependent Ca current showed almost the same sensi- internal stores of smooth muscles (Itoh et al., 1981). Caffeine tivity to 30 JM DCDPC as did the Icat current. Furthermore, (5-20 JM) is frequently used as an experiment tool to 100 JM flufenamic, DPC and DCDPC exerted inhibitory mobilize Ca2+ into the cytosol from the SR (lino, 1989), but effects on the transient outward currents during depolarizing also inhibited the Icat channel (IC50 is c.a. 10 mM) not due to pulses, the properties of which are similar to those described its Ca2+ releasing action since the [Ca2+]i was clamped in the e.g. by Benham & Bolton (1986) and Ohya et al. (1987) range of 100-200 nM in the present experiments. These (Chen, Inoue & Ito, unpublished data). These observations observations clearly indicate that the effective concentration suggest that internal Ca stores are also a major target of ranges of K channel blockers and caffeine considerably over- these drugs, since Poronik et al. (1992) have recently pro- lap those which inhibit the Icat channel, and thus emphasize vided evidence that several synthesized DPC derivatives in- the need of special care for designing and interpreting cluding flufenamic acid and DPC possess the capability of experiments with the aid of such pharmacological agents. provoking calcium transients in the absence of external cal- Quinine seems to be one of the most desirable blockers of cium. In addition, we noticed in our preliminary microelec- the Ica, channel from the present results, but the literature trode experiments that 3O0AM-100 1M flufenamic acid and reveals a variety of its nonspecific actions (Cook & Quast, DCDPC abolish slow membrane oscillations (the so-called 1990). For example, it is reported that quinine is a fairly slow waves, Bolton, 1979), resulting in membrane hyper- selective blocker of the Ca2+-dependent K channels (IC50 is polarizations of 10-20 mV and subsequent cessation of spike > 10 jAM) but in submillimolar concentrations it also sup- discharge. These observations obviously restrict the use of presses voltage-dependent Na and Ca channels. Further, in these drugs as selective blockers for the I,at current. angiotensin II-induced contractions, a component remaining after blockade of voltage-dependent Ca or K channels is Possible mechanisms involved in the action of blockers found to be further inhibited by quinine (Cook & Quast, for the Icat current 1990). These observations seem to exclude largely the pos- sibilty of using quinine as a selective Icat channel blocker. From the results of some mechanistic approaches undertaken However, it deserves attention that the reported IC50 values in the present study, at least two important different features against other currents are not lower than 51M, most fre- have become evident between caffeine and K channel block- quently 1O iM-1 mM, and are several fold higher than the ers, and DPC derivatives. First, inhibition and recovery of IC50 obtained for the Ica, current (1 JlM). This raises the the I,a, current by the former drug group occurred in the possibility that quinine can serve as a relatively selective order of 100 ms, while those by the latter were considerably blocker for the cation channels in guinea-pig ileal muscle slower (see Figure 2). Secondly, the degree of inhibition was with limited concentration ranges (ca. 5-10IOtM). In fact, in strongly attenuated by depolarizing the membrane in the case preliminary experiments with the intracellular recording tech- of K channel blockers and caffeine but was relieved only nique, 10 gAM quinine abolished depolarizations evoked by weakly in the cases of DPC derivatives. In addition, the site 1 ILLM ACh with only modest changes in spike discharge. of action of blockers is not on the muscarinic receptor but probably on the Ia, channel itself from the observations on The effects of DPC derivatives on the Icat channel GTPyS-induced inward currents. One possible explanation may be as follows. In cation-permeable channels, large The inhibitory potencies of DPC derivatives on the Iha, chan- positively charged molecules such as TEA, in the extracel- nel accord approximately with those observed against Ca2+- lular space, are believed to be driven into the orifice or BLOCKERS ON CATION CHANNELS 801 attracted near to a negatively charged binding site located the Ihat currents persisted for several minutes even after midway down the channel pore along the transmembrane washout of the drugs, and their efficacies on the I, channel potential gradient, and to interfere with the permeation of are similar to those on the Ca2"-activated cation channels, cations (e.g. Hille, 1984). Such interference could occur where the drugs are applied at the intracellular side of the rapidly due to their easy access and be relieved by plasma membrane. In addition, this view is consistent with depolarization of the membrane due to reduction of the the finding that DPC derivatives decrease the open pro- potential gradient which might slow the binding process of bability of the Ca2"-activated cation channels but such an the drug molecules. On the other hand, DPC derivatives effect is not associated with the appearance of flickering possess high lipophilicity and are highly membrane- within long opening intervals, which is usually observed for permeable, although most of the molecules are negatively open channel blockade (Gogelein & Capek, 1990). charged at a physiological pH (Gogelein & Phannmuller, 1989). This might enable the drugs to pass through the We are grateful to Dr A.F. Brading (University Department of plasma membrane to an internal site. This mechanism seems Pharmacology, Oxford) for her critical reading of our manuscript, consistent with slow onset and recovery of their inhibitions. and Dr R. Greger (University of Freiburg) for his kind gifts of DPC Indeed, the inhibitory effect of flufenamic acid or DCDPC on derivatives.

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