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J. Smooth Muscle Res.(2002) 38 (1, 2):39-50 39

Effects of Flufenamic Acid on Smooth Muscle of the

Carotid Artery Isolated from Spontaneously

Hypertensive Rats

Keiichi SHIMAMuRAi, Ming ZHOui, Yasuko IToi, Shinichi kMuRAi, L.-B. Zou2, Fumiko SEKIGucHI2, Kenji KITAMuRA3, and Satoru SuNAN02

'Dopartment of Ctinical Pharmacolag),, Flacutty of IVtarmaceutical Sciences, Heatth

Sciences Ubeiversity ofHokkaido, 17Zi7 Klina2awa, lshihanL71obets", Hohkaido 061- 0293, lbPan

allaculty ofPltamaaeeutical Sciences. Kinki Uhiversity, 3q-1 Kbwakae, Higashi-Osaka 57rs5a2, foPan 3DqPartment ofJlharmacotqgy, Eetleuoha Dental Collage, 7lamant, Saware-ku, Fukuoha 8140103, foPan

Abstract

Endothelium-removed carotid artery strips from stroke-prone spontaneously

hypertensive rats spontaneously developed a tonic rnyogenic contraction. Flufenamic

'I>rrode's acid reduced the resting tone observed during superfusion with solution, in a

concentration-dependent manner. Flufenamic acid also inhibited contractions produced

by high-K' solutions in a concentration-dependent manner. The resting membrane

potential of smooth muscle cells in the artery was around -32 mV, with occasional oscillatory potentials. Flufenamic acid hyperpolarizecl the membrane in a concentration-

dependent mannen The voltage-dependent outward currents recorded in isolated cells -60 with micropipettes fi11ed with high-K' solution (holding potential, mV) were enhanced by flufenamic acid and inhibited by tetraethylammoniurn. When the recording

micropipette was fi11ed with high Cs' to inhibit the K'-current, clepolarizing step pulses

evoked nifedipine-sensitive inward currents. Flufenamic acid inhibited the inward

currents. IIIiese results indicate that flufenamic acid inhibits the spontaneous active tone

of the carotid artery by inhibiting L-type Ca2'-channels and possibly by membrane

hyperpolarization through activation of the voltage-dependent K'-channels.

Key words: Flufenamic acid, Carotid artery, K' current, Ca2' current, SHR

Introduction

Large arteries of hypertensive rats have been shown to develop spontaneous tonic myogenic contractions (Noon et al., 1978; Sada et al,, 1990), We have reported that the endothelium-

Correspondence to: K. Shimamura, Department of Clinical Pharmacology, Faculty of Pharrnaceutical Sciences, Health Science University of Hokkaido, Kanazawa 1757, Ishlkari-Tobetsu, Hokkaide 061-0293, JapanPhone: +81-1332-3-1211 ext,3160 Fax: +81-1332-3-1669 e-mail: [email protected]

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40 K, SHIMAMURA et at,

removed carotid artery of stroke-prone spontaneously hypertensive rats (SHRSP) contracts spontaneously with an increase in intracellular free Ca2' concentration (Sekiguchi et al., 1996). This preparation was thought to be suitable for the study of the cellular mechanisms involved in the action of chemicals that may cause relaxation of arteries during hypertension. Flufenamic acid, a diphenylamine carboxylate derivative, is a cyclooxygenase inhibitor (Flower, 1974), and has been shown to modulate several types of ion channels in different smooth muscle tissues. This chemical potentiates the potassium cttrrent (K'-current) in the canine and human jejunum (Farrugia et al,, 1993a; 1993b), the rabbit portal vein (Greenwood and Large, 1995), the canine coronary artery (Xu et al., 1994) and the pig coronary artery (Ottolia and Toro, 1994). Flufenamic acid inhibits the L-type Ca2'-current in the rat basilar artery (Doughty et al., 1998), the Ca2'-dependent Cl--current in the rabbit portal vein (Greenwood and Large, 1995) and the non-selective cationic current in the guinea pig ileum (Chen et al,, 1993). Although these effects of flufenamic acid on ion channels can inhibit contraction of smooth muscle, no report is available on the effects of this chemical on the membrane potential or ionic currents of arterial smooth muscle obtained from hypertensive rats. In the present study, we investigated the effects of flufenamic acid on the spontaneous tonic contractions and on the membrane potennial and membrane currents of smooth muscle isolated from the carotid artery of SHRSP. A part of this study was reported as a preliminary report (Shimamura et al., 1999).

Materials and Metltods

Animals andProparations Sixteen week old male SHRSP (Okamoto et al,, 1974), with a systolic blood pressure of 220-

260 mmHg, were anesthetized with ethyl ether and exsanguinated. The left or right common

carotid artery was dissected from the animals and the endothelial cells were removed by

rubbing the lumenal surface. Helically cut preparations were prepared from the artery segments. The hepatic portal vein was also dissected, opened by a longitudinal incision and the

lumenal surface rubbed to remove the endothelial cells.

Mechanical recerdings

Both ends of the tissue segments were tied with a tungsten wire (30 "m diameter) and mounted venically in a recording organ bath fi11ed with modified TYrode's soiution (warmed to 370C), One of the wires was connected to a force-displacement transducer (UL-10, Minebea, Karuizawa, Japan) to record contractile forces isometrically, A stretch of 1,3 tirnes the original length was applied to each preparation and the preparation was subjected to high K'-induced

contraction and was then allowed to incubate until development of spontaneous tonic

. contractlons.

'IYrode's The composition of the modified solution was as follow (in mM); NaCl 137, KCI 5.4, CaC12 2.0, MgC12, 1.0, NaH2P04 O.e4, glucose 5.6. The solution was equilibrated with a gas mixture of 95% 02 and 5% C02, and the pH of the solution maintained at 7.3. High-K' solution

([K']. - 80 mM) was prepared by replacing equimolar amounts of NaCl with KCI. Nominally

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Mufenamic acid on SHR carotid artery 41

Ca2' free Tyrode's solution (Ca2'-free solution) was prepared by omitting CaC12 from the 'IYrode's solution.

Permeabilized preparations of circular muscle of the rat portal vein were made, as described previously (Shimamura et al,, 1998). Both ends of a thin strip (50 um thick, 100 "m wide, 2 mm long) of the circular muscle were tied to tungsten wires with silk thread. The preparations were

'IYrode's set horizontally in a recording chamber (volume, 1 ml) and fi11ed with solution at 250C.

One of the wires was connected to an arm of the micromanipulator, while the other was

connected to a lever of the isometric force-displacement transducer (UL-10 Minebea). After 'IYrode's equilibration in the solution for 1 hr, the tissue was stimulated with high-K solution to elicit contraction. The tissues were then permeabilized with I3-escin, using the methods reported by Gonzalez et al. (1995). Briefiy, the muscle strip was incubated in a relaxing solution containing 10 "M ffescin for 30 min. Permeabilizing treatment was considered successful when

the amplitude of contraction induced by a solution containing high-Ca (pCa = 6.0) exceeded that of the high K-solution Uensen, 1994). After removal of Pescin from the incubating solution, responses of muscle strips to solutions containing different concentrations of Ca2' were tested

using Ca2'-buffered solutions, according to the methods reported by Kerrick and Hoar (1994).

Free Ca2' concentrations in the solution were calculated with the association constants in

accordance with the temperature and pH of the solution (Fabiato and Fabiato, 1979). The composition of the incubating solution was as follows (in mM); NaCl 130, KCI 5.6, glucose 11, Tris 20, MgC12 1, and CaC12 2,5. High-K solution ([K']. ; 80 mM) was prepared by an equimolar replacement of NaCl with KCI. Composition of the relaxing solution was as follows (in mM): K propionate 130, Tris-malate 20, EGTA 10, MgC12 1, ATPMg 3, Creatine phosphate Na2 10 and Creatine phosphokinase 10 unitlml (pH - 6.8).

Mbmbrane Potential recording The rnembrane potential was recorded intracellularly using a glass capillary microelectrode (tip resistance, 40-80 M9) fi11ed with 3M KCI, as previously described (Sekiguchi et al,, 1996). The reference electrode was an Ag-AgCl plate. The potential differences between these two electrodes were measured using a microelectrode amplifier (MEZ-8201, Nihon Kohden, Tokyo), and were displayed on a dual beam cathode-ray oscilloscope (VC-10, Nihon Kohden, Tokyo) and also on an ink-writing pen oscillograph (8561 ME-IH Sanei, Tokyo) and a tape recorder (RMG- 5304, Nihon Kohden). Arterial strips were mounted on a silicon rubber plate, with the lumenal side up, in a recording organ bath (volume, 1 ml). The strips were superfused continuously with 'IYrode's modified solution (370C) at a flow rate of 4 mllmin, Successful impalements of the

recording electrode into cells were characterized by a sudden negative shift in the voltage

followed by a stable negative potential for at least 5 min.

Cell dispersion and whole cett recording using the Patch clamP method Preparations were incubated in nominally Ca2tfree physiological salt solution (PSS) containing 2 mglml collagenase (type I, Wako pure Chemical, Osaka Japan), 1 mglml papain (Wako), 1 mglml soybean trypsin inhibitor (type II, Wako), O.5 mglml dithiothreitol and 1,5 mglml bovine serum albumin (Fraction V, Sigma) at 370C for 60 min. The digested tissue was

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42 K. SHIMArviuRA et al,

cut into pieces and triturated in Ca2'-free PSS. Cells were stored at 4eC until used. Standard whole-cell recording was performed as described by Hamill et al. (1981). Currents were recorded with a patch clamp amplifier (CEZ-2200, Nihon Kohden) and an A/D, DIA converter (Labmaster DMA, Scientific Solutions, INC, Solon, USA) with pClamp v.5.5 programs (Axon

Instuments, Foster City USA). The capacitance of the cell membrane was measured using small ramp pulses (10 mV depolarization in 10 msec) and the current amplitudes obtained by the step pulses normalized by the capacitance. All experiments were carried out at room temperature. In the patch clamp experiments, the bathing solution (- PSS) contained the following ienic concentrations (mM): NaCl 140, KCI 5.4, glucose 12, MgC12 1.2, CaC12 2, HEPES 10 (titrated to pH 7,35-7.45 by NaOH). The nominally Ca2'-free PSS was made by omitting CaC12 from the PSS. The composition of the high-K' micropipette solutions was as fo11ows (in mM): KCI 135, glucose 12, MgC12 5, ATP Na2 5, EGTA 5 and HEPES 10 (pH adjusted to 7.3 by KOH). The CsCl micropipette solution contained the following ions (in mM): CsCl 135, glucose 12, MgC12 5, ATP2Na 5, EGTA 5 and HEPES 10 (pH adjusted to 7.3 by CsOH). Flufenamic acid (Wako pure Chemical, Osaka, Japan) was dissolved in a mixture of 10% dimethylsulfoxide (DMSO, Sigma, USA) and O.1 N NaOH, The maximum final concentration of DMSO in the bath solution was O,Ol%, Nifedipine, pinacidil, papaverine, and tetraethylammonium chleride were supplied by Sigma (USA).

Statistics Data were expressed as the mean ± S.E,M., with the number of animals in parentheses. Statistical analysis was performed using the Student's t-test, and probabilities of less than 5% (P

Results

orzaracteristicsofactivetone in arteries .fivm SHRSP

In TYrode's solution, endothelium-removed preparations of the carotid artery isolated from

SHRSP developed spontaneous tonic contraction or active tone. The mean amplitude of this

active tone was 74,9 ± 5,6% (n-14) of the contraction produced by high-K' solution. The

nominally Ca2'-free Tyrode's solution caused a complete inhibition of the active tone. Re-

'IYrode's administration of solution containing CaCl2 2 mM restored the tone. Papaverine 10" M

did not produce any further relaxation during the relaxation induced by Ca2'-free solution. Application of 10-6 M of nifedipine, a L-type Ca2'-channel antagonist, or 10T6 M pinacidil, an ATP- sensitive K'-channel opener, also inhibited the active tone (data not shown), Flufenamic acid (10-6-10-` M) inhibited the active tone of the carotid artery strips in a concentration-dependent manner (Fig. 1). Decreases of the active tone by flufenamic acid at 10-5

and were ± and ± M 10" M 36.7 8.1% (n-17) 97.6 O.9%(n=15), lrespectively.Indomethacin10"5M did not alter either the active tone or the inhibition of active tone by flufenamic acid 10-5 M (35,6

± 10.1%, n-6, P>O.05).

Flufenamic aeid also inhibited the contraction induced by high-K' in the carotid artery strips in a concentration-dependent manner (Fig. 1). At 10-` M, fiufenamic acid decreased the

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Flufenamic acid on SHR carotid artery 43

%

=ot-co=1OO

¢ vo.2re5"

50

o -7 -6 -5-log[Flufi-4 -3

Fig. 1'Mie relationship between the concentration of fiufenamic acid and its inhibitien of both spontaneously active tone (open circles) and the contractile responses induced by high- K' (fi11ed circles) in endothelium-removed carotid artery strips isolated from stroke- prone spontaneously hypertensive rats (SHRSP). The amplitude of the tension in the presence of flufenamic acid was shown relative to that before application of flufenamic acid (%), Number of animals: 10-17 for data ef spontaneously active tone and 9-14 for data of high-K induced contractions.

contractions induced by high-K' by 65.0 ± 7.3% (n=15). Pinacidil 10-6 or 10'5 M did not inhibit

the contractions induced by high-K'.

The effects of fiufenamic acid on contractile proteins were examined in skinned circular muscle of the hepatic portal vein. In the circular muscle of the intact hepatic portal vein,

flufenamic acid 10" M relaxed preparations which had been contracted with high-K' solution (Fig. 2A). However, in the frescin permeabilized preparations, flufenamic acid did not modulate the contraction produced by solutions containing either pCa 6 or pCa5 (Fig. 2B). The amplitude of contractions elicited by 10" M of flufenamic acid was 101.4 ± 2.5% (n=8) of the pCa 6induced contraction and 98.8 ± 11.1% (n-4) of the pCa 5induced contraction (P>O,05 for both).

ELfi2ict offltdenamic acid on the membrane Potential The resting membrane potential of smooth muscle cells in preparations of the endothelium- -32 removed carotid artery of SHRSP was ± 2.3 mV (n=18). In some preparations (6 out of 23), the membrane potential showed spontaneous oscillations with an amplitude of a few mV.

Flufenamic acid 10-3 M hyperpolarized the membrane and inhibited this spontaneous oscillation

of the membrane (Fig. 3A). Figure 3B shows the changes in membrane potential in the presence of different concentrations of fiufenamic acid. Flufenamic acid hyperpolarized the

membrane in a concentration-dependent mannen The EDse of the hyperpolarization induced by

fiufenamic acid was approximately 10" M. Nifedipine (10'5 M) did not alter the resting membrane potential of smooth muscle cells in

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44 K. SHIMAMURA et aL

A

'tK80 tFIUf B

50 mN

. 40 min tpCa 6tFtuf tRlx

Fig. 2Effects of flufenamic acid on contractions of intact (A) and skinned smooth muscle preparation (B) of the rat hepatic portal vein. A: A typical trace showing the effect of fiufenamic acid 10'` M (FluD on the high-K (K80) induced tonic contraction of intact preparations. B: A typical trace showing pCa 6-induced tonic contraction of permeabilized preparations of circular muscle of the rat hepatic pertal vein and the effects of flufenamic acid 10" M (FluD, Rlx: relaxing solution,

-36 preparations of the endothelium-removed carotid artery (control, ± 1,2 mV, n=7; in

nifedipine, -37.1 ± 2,O mV, n=7; P>O.05).

EL62ect offldenamic acid on membtune currents

In enzymatically isolated smooth muscle cells of the carotid artery, held in whole-cell patch

clamp mode in PSS solution, voltage-dependent outward currents were recorded. The

membrane capacitance ranged between 20 pF and 60 pF, Using high-K" solution in the micropipette, depolarizing step pulses with duration of 500 msec evoked outward currents in a

voltage-dependent way (Fig. 4A). The outward current was abolished by tetraethylammonium

20 mM (data not shown), Flufenamic acid enhanced the outward current elicited by -120 depolarization (Fig. 4A). Ramp pulses of from mV to +80 mV (300 msec in pulse duration, -60 with mV holding potential) evoked a voltage-dependent current with a reversal potential of -70 -60 approximately to mV (data net shown), The relationship between the amplitude of the

outward current and the membrane potentials obtained from 6 preparations is summarized in Fig. 4B. Flufenamic acid (10" and 10-A M) markedly increased the outward current, In some preparations, when currents were recorded with high-K' solution in the micropipette, a small transient inward current preceded the outward current in response to the depolarizing pulse. When outward potassium currents were inhibited by adding cesium to the micropipette, inward currents were evoked with depolarizing step pulses of 300 msec duration at -60 the holding potential (Vh) of mV. The current amplitude peaked within 10 msec of the commencement of the depolarization steps and then decayed exponentially. The threshold

depolarization for the inward current was around -30 mV, and the current amplitude peaked at around +10 mV. These inward currents were abolished in the presence of 10'6 M nifedipine

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Mufenamic acid on SHR carotid artery 45 A

-36 mV

tomv.

20 sec

B

-20 Eii'

-30 2gk:5Ee I

40

6 - -5 4-logrelufi -3 control

Fig. 3A: A typical trace of the mernbrane potential recorded from a smeoth muscle cell in a preparation from the endothelium-rernoved carotid artery iselated from stroke prone spontaneously hypertensive rats. Flufenamic acid 10-3 M was applied at the arrow, B: Membrane potentials measured in smooth muscle cells in the endothelium-removed carotid artery of stroke prone spontaneously hypertensive rats in the presence of different concentrations of fiufenamic acid (10'5-1tr3 M). Control, n-21; in the presence of fiufenamic acid, n= 4-9.

(data not shown), indicating that they were carried through voltage-gated Iftype Ca2'-channels. The Flufenamic acid (3 × 10" M) inhibited the voltage-gated Ca2'-current (Fig. 5, A and B). inhibitory actions of flufenamic acid on the Ca2'-currents occurred in a concentration-dependent

manner (Fig. 5C).

Discussion

Endothelium-removed preparations of large arteries from spontaneously hypertensive have been shown to develop spontaneous active tone (Noon et al,, 1978; Sada et aL, 1990).ratsThe

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46 K, SHIMAMuRA et al. A +10mV n60V

Ftufenamlcacid 3xlo'4M

2oopA

.

2oo ms

M

M

806040-20 o 20 4e 60 80 mV

Fig. 4Effects of flufenamic acid on outward currents elicited by depolarization of the membrane in isolated single smooth muscle cells of the carotid artery of stroke-prone spontaneously hypertensive rats. A: Outward currents were recorded by applying step pulses of 300 msec duration, from the holding potential of -60 mV to +10 mV, in the absenee (control) and presence of 3 × 10" M flufenamic acid. B: The I-V relationships of outward currents evoked by depolarizing step pulses (Vh=-60 mV) in isolated smoeth muscle cells of carotid artery strips from stroke-prone spontaneously hypertensive rats, in the absence (control) and presence of fiufenamic acid 10" M and 10'3 M. Symbols with error bars indicate the mean ± S.E.M. (n=6). AJnplitudes of the outward current were expressed with current density, which was calculated by dividing current amplitudes by the cell capacitance,

present experiments indicated that the tone is sensitive to either extracellular Ca2' or to CaZ'

antagonists. These results suggest that the opening of voltage-gated L-typeCa2' channels

contributes to the generation of active tone. In the present experiments with carotid artery

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Flufenamic acid on SHR carotid artery 47

A pAtpF mv -50 so -

Fluf -O.2

-4 control

o.

+20 mV 6V

Ftutenamic acld 3xlo-4 M

1OO pA Control

200 rnsec c-*vlegggegor

E

6 -5 4 .3

-log[Hutbnamicaclq

Fig, 5Effects of flufenamic acid on inward currents measurecl in enzymatically isolated

smooth muscle cells of carotid artery strips from stroke-prone spontaneously

hypertensive rats, A: I-V relationship of the inward currents measured ln the absence (control) and presence of flufenamic acid 3 x 10-" M (FluD. The currents were evoked -60 by depolarizing step pulses of 300 msec duration from the holding potential of mV to +20 mV, and the peak amplitudes were measured, Error bars indicate the mean ± S.E.M. (n=22 for control, n=4 for fiur). B: Inward current traces evoked by clepolarizing -60 pulses from mV to +20 mV, in the absence (control) and presence of fiufenamic acid 3 x 10" M. The recording micropipette contained high Cs. C: The concentration- response relationship of the effects of flufenamic acid en the peak amplitude of the -60 inward currents evoked by clepolarizing pulses from mV to + 10 mV or +20 mV. Symbols with error bars indicate the mean ± S.E.M. (n=4-8).

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48 K, SHIMAMuRA et al.

preparations from SHRSP, the resting membrane potential of smooth muscle cells was approximately -30 mV, which is less negative than that of arterial smooth muscle cells in other

mammals (Kuriyama et al., 1995). Although our patch clamp experiments did not detect inward currents at the membrane potential of -30 mV, pharmacological evidence provided by the use of

a Ca2' antagonist and a ATP-sensitive K' channel opener, showed that Ca2" may flow into cells

through voltage-gated Iv-type Ca2' channels. Thus, it is reasonable to consider that the active

tone is produced by a spontaneous increase in Ca2' infiux through voltagegated channels. Studies of the response of arterial smooth muscle to chemicals that inhibit spontaneous active tone may help to elucidate the mechanisms involved in its development. They would also

facilitate the development of a new therapeutic strategy for hypertension. Nonsteroidal anti-

inflammatory drugs, that are derivatives of diphenylamine-2-carboxylic acid, have been shown to block a variety of ion channels, such as nonselective cation channels (Chen et al., 1993; Hescheler and Schultz, 1993) and calcium-activated chloride channels (Greenwood and Large, 1995). These chemicals activate potassium currents in jejunal smooth muscle cells (Farrugia et in mouse mandibular cell lines al. , 1993a;1993b)and release Ca2 from internalstores (Poronnik et al., 1992). It has been reported that flufenamic acid potentiates large conductance Ca2'- activated potassium channels (Ottolia and Toro, 1994). The potassium currents recorded in the present study were similar to those reported previously in hypertensive rats (Liu et al,, 1995). Although we did not have enough evidence to determine the channel type involved in the outward currents, Ca2'-activated potassium channels present in blood vessels of SHRSP may be

the site of action of flufenamic acid.

Flufenamic acid has been reported to inhibit I-type Ca2'-channel in both cardiomyocytes (Conforti et aL, 1994) and the rat basilar artery (Dougherty et al., 1998). In the present study on carotid artery, high-K" induced contractions were inhibited by flufenamic acid, The present

experiments also indicated that the nifedipine-sensitive inward Ca2' currents were recorded

when the outward current was inhibited by cesium in the micropipette. The current amplitude

achieved its peak within 10 msec of the depolarization step and decayed exponentially as reported in other arterial smooth muscle cells in hypertensive rats (Ohya et al,, 1993).

Flufenamic acid inhibited the Irtype Ca2' currents in a concentration-dependent manner. The

effects of flufenamic acid on contractile proteins were unlikely to have contributed to the

inhibition of contractions produced by the high-K solution, because flufenamic acid did not change the contraction of permeabilized circular smooth muscle cells of the hepatic portal vein. Comparison of the inhibitory actions of flufenamic acid indicated that the effects on active tone appeared at lower concentrations than those on high-K' induced contraction (Fig, 1). This suggests that, at the resting membrane potential level, unidentified mechanisms may be

contributing to the inhibition of active tone by flufenamic acid, in addition to the inhibition of

voltage-gated Ltype Ca2' channels. Although we did not examine the effects of fiufenamic acid

on the membrane potential in the presence of K'-channel blockers, it is speculated that, in the presence of flufenamic acid, the increased activity of K'-channel which induces

hyperpolarization of the smooth muscle membrane may close the voltage-gated Ca2' channels

and inhibit Ca2' influx. This will finally diminish the spontaneous active tension. The reversal -60 potential for the current evoked by ramp pulse was distributed at approximately mV. The

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Flufenamic acid on SHR carotid artery 49

present results indicate that the Ca2' channels were active at a potential close to the resting mernbrane potential of approximately -30 mV. Abnormalities in the membrane mechanisms during arterial hypertension have been reviewed (Sunano et al., 2000). In pulmonary hypertension of the rat, active tone in the pulmonary artery and elevated chloride channel dctivity have been reported in smooth muscle cells (Nakazawa et at., 2001). Flufenamic acid has been shown to inhibit Ca2'-activated chloride current (Greenwood and Large, 1995) and non-selective cationic current (Chen et al., 1993; Hescheler and Schultz, 1993), The possible contribution of an effect of flufenamic acid on membrane hyperpolarization needs to be further examined.

In conclusion, flufenamic acid inhibits the active tone of smooth muscle in the carotid artery of SHRSP by inhibiting Ca2' influx, by direct inhibition of the voltage-gated Ca2'-channels and indirectly through hyperpolarization of the membrane due to activation of K'-channels.

Acknowledgement

'Iliis The authors are gratefu1 to T. Tsukahara for assistance in computer system. work was supported in part by a Grant-in-Aid fbr High Technology Research Programs from the Ministry

of Education, Science, Sports and Culture of Japan.

References

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(Received April 7, 2002: Accepted April 14, 2002)

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