Dual Action ofFRC8653, Novel Derivative, Ba2` Recorded …circres.ahajournals.org/content/circresaha/67/4/993.full.pdf · 994 Circulation Research Vol67, No4, October 1990 ... evoked

993

Dual Action of FRC8653, a NovelDihydropyridine Derivative, on the Ba2`

Current Recorded From the RabbitBasilar Artery

Masahiro Oike, Yoshihito Inoue, Kenji Kitamura, and Hirosi Kuriyama

Actions ofFRC8653 on the macroscopic and unitary Ba2` currents were studied using the rabbitbasilar artery. Application of (+)-FRC8653 (<1 ,uM) increased the amplitude of the inwardcurrent when depolarization pulses more negative than -10 mV were applied but inhibited itwhen depolarization was more positive than 0 mV (in each case from a holding potential of -80mV). At a holding potential of -40 mV, (-)-FRC8653 (>0.1 nM) consistently inhibited theinward current. (-)-FRC8653 (>1 nM) inhibited the amplitude of the inward current evokedby a depolarizing pulse more positive than -10 mV (the holding potential being -80 mV). Atthe holding potential of -80 mV, but not at -40 mV, (+)-FRC8653 (1 ,uM) enhanced thecurrent amplitude evoked by a depolarizing pulse more negative than -10 mV but inhibited thecurrent evoked by a pulse more positive than 0 mV. (-+-)-FRC8653 shifted the voltage-dependentinhibition curves to the left, and the slope of the curve became steeper (test pulse of + 10 mV).Two types of single Ca2' channel currents (12 and 23 pS) were recorded from the basilar arteryby the cell-attached patch-clamp method. Opening of the 12-pS channel occurred with a

depolarizing pulse (-20 mV) from a holding potential of -80 mV, but not from one of -60 mV.(+)-FRC8653 activated, and (-)-FRC8653 inhibited, the 23-pS channel. These results indicatethat (+)-FRC8653 had dual actions (excitatory and inhibitory) on the Ca21 channels in therabbit basilar artery. Excitatory actions of (+)-FRC8653 result mainly from the actions of(+)-FRC8653 on the 23-pS channel, but this was reversed to an inhibitory action at a holdingpotential of -40 mV. Therefore, FRC8653 dominantly acts as a Ca21 channel blocker in therabbit basilar artery, since this tissue has a resting membrane potential of -50 to -60 mV.(Circulation Research 1990;67:993-1006)

R ecently, two types of voltage-dependent Ca2'channels have been revealed in smooth mus-cle cells.'-6 In smooth muscle cells, dihydro-

pyridine (DHP) derivatives such as nifedipine andnitrendipine are known to inhibit the high-thresholdCa2' channel or L-type channel without effect on thelow-threshold Ca2' channel, the T-type.3-57 More-over, it has been reported that DHP derivatives inhib-ited other ion channels such as the voltage-dependentK' channels and Na+ channels in cardiac and smoothmuscle cells, although the required concentration was200-1,000 times higher.8'9 These results indicate thatDHP derivatives have selective inhibitory actions onthe high-threshold-type (L-type) Ca2' channel in

From the Department of Pharmacology, Faculty of Medicine,Kyushu University, Fukuoka, Japan.Address for correspondence: Dr. M. Oike, Department of

Pharmacology, Faculty of Medicine, Kyushu University, Fukuoka812, Japan.

Received January 5, 1990; accepted June 12, 1990.

smooth muscle cells. However, the low-threshold Ca2+channel (T-type) recorded from the rat aorta could beinhibited by nifedipine, albeit with higher concentra-tions than those needed to block the high-thresholdCa2' channels.l1

In contrast, some DHP derivatives, namely, Bay K8644 and 202-791, are reported to enhance thehigh-threshold Ca21 channels but not the low-threshold one.11'2 Because such augmentation of theCa2+ current by DHP derivatives was reported usingnifedipine as well as nitrendipine, this action may bea common property of DHP derivatives and may playa role in their apparent inhibitory actions.'3 There-fore, it seems that the potency of the inhibitoryactions of DHP derivatives observed in the variousvascular tissues may depend on the types of Ca21channels distributed in the smooth muscle cells ofparticular vascular tissue and the relative potency ofthe inhibitory actions of each DHP derivative com-pared with its excitatory actions.

by guest on July 1, 2018http://circres.ahajournals.org/

Dow

nloaded from

http://circres.ahajournals.org/

994 Circulation Research Vol 67, No 4, October 1990

In guinea pig basilar artery, Fujiwara andKuriyama14 reported that nicardipine, a DHP deriv-ative, inhibited the action potential and the high-K+-induced contraction but not the 5-hydroxy-tryptamine-induced contraction, and they postulatedthat nicardipine selectively inhibited Ca + perme-ation through the voltage-dependent Ca2' channel inthe membrane. However, no patch-clamp investiga-tion has been performed using basilar artery.

(± )-FRC8653 [2-methoxyethyl (E)-3 -phenyl-2-propen- 1-yl( ± )-1,4 -dihydro-2,6 -dimethyl- 4 -(3-nitrophenyl)pyridine-3,5-dicarboxylate] is a newlysynthesized DHP derivative and produces vasodila-tion, especially in the basilar and coronaryarteries.15The present experiments were performed to

investigate in detail the actions of (+t)-FRC8653and its enantiomers on smooth muscle cells of therabbit basilar artery.

Materials and MethodsMale albino rabbits (1.7-2.0 kg) were anesthe-

tized with sodium pentobarbital (40 mg/kg i.v.;Pitman-Moore Inc., Washington Cross, N.J.) andexsanguinated. The basilar artery was dissectedalong with brain and brain stem and isolated inphysiological salt solution (PSS). Tunica adventitiaand surrounding connective tissue were carefullyremoved, as far as possible, with fine scissors andforceps under a binocular microscope. Endotheliumwas also removed by gentle rubbing with a smallcotton ball. The procedure for cell dispersion wassimilar to that described by Momose and Gomi16and Terada et al.17 In brief, small segments of thetissue were rinsed with Ca2+-free PSS, then incu-bated in warmed Ca2+-free PSS containing 0.3%collagenase (Wako Pure Chemicals, Osaka, Japan),0.1% bovine serum albumin (essentially fatty acidfree; Sigma Chemical Co., St. Louis), and 0.1%trypsin inhibitor (type II-S, Sigma) at 360 C for 70minutes. After collagenase treatment, tissues weretransferred to fresh Ca2+-free PSS, and single cellswere dispersed by gentle agitation with a glasspipette. Dispersed cells were collected by mildcentrifugation (600 rpm, 1 minute) after removal ofpieces of undigested tissue with fine nylon mesh(10Ox100 gm). Dispersed cells were resuspendedin fresh 0.5 mM Ca2+-0.5 mM Mg2+ solution andstored in ice-cold water. Experiments were per-formed at room temperature (20-250 C).

Recording of the Membrane CurrentsRecordings of the macroscopic and single-

channel currents were made in ways similar to thosedescribed by Hamill et al.18 One drop of the cellsuspension was added to a small chamber (0.2-mlvolume) placed on the stage of a differential inter-ference inverted microscope (TMD-Diaphoto,Nikon Co., Tokyo). Patch electrodes (o.d., 2.0-3.5,ttm; 3-5 Mfl) were prepared by means of anelectrode puller and heat polisher (models PP-83and MF-83, Narishige Scientific Instruments Labo-

ratory, Tokyo) and were manipulated by three-dimensional oil-driven or electric manipulators(model MO-102, Narishige; Manipulator-E, Leitz-Wetzler GmbH., Wetzlar, FRG). A high-resistanceseal (>10 GQ) was obtained by application ofnegative pressure to the patch electrode (10-30 cmH20). For single-channel current recording, thepipette was filled with 100 mM Ba`+ solution withhigh-K' solution being superfused in the bath. Formacroscopic current recording, the pipette wasfilled with high-Cs+ solution (for recording the Ca21or Ba2+ current) or high-K' solution (for recordingthe K' current) with 10 mM Ca2' or Ba`+ solution(for the Ca2+ or Ba2` current) or PSS (for the K+current) superfused in the bath.Membrane currents were monitored on a high-gain

digital oscilloscope and a conventional thermo-writing pen recorder (models VC-10 and RJG-4124,Nihon Kohden, Tokyo) through a patch-clamp ampli-fier (model EPC-5 or EPC-7, List Medical Electron-ics, Darmstadt, FRG) and stored on magnetic tapeby an FM tape recorder (model A-65, Sony-Magnescale Inc., Tokyo) or videocasette recorder viaa PCM data recording system (model NV-21,National Co., Tokyo; model PCM-501E, Sony Co.,Tokyo).

Capacitative and leak currents were subtractedusing the P/n (n =2-4) method described by Almersand Palade19 for measurement of macroscopic cur-rents on a digital oscilloscope (model 4094B, Nico-let Instrument Corp., Madison, Wis.), and a hardcopy was obtained with an X-Y plotter (model7440A, Hewlett-Packard Co., San Diego). Forsingle-channel current recording, capacitative andleak currents were averaged from 30 to 50 tracingswith no appearance of the single-channel current("blank" tracings); then the capacitative and leakcurrents in the traces with the appearance of thesingle-channel current ("nonblank" tracings) weresubtracted from the averaged capacitative and leakcurrents on a digital storage oscilloscope (model4094B, Nicolet).

Solutions and DrugsIonic millimolar composition of PSS was NaCl

134, KCl 6, CaCl2 2.5, and glucose 12. The Ca2+-freesolution was made by replacement of CaCl2 with anequimolar amount of NaCl. The 0.5 mM Ca2+-0.5mM Mg2+ solution was made by adding 0.5 mMCaCl2 and 0.5 mM MgCl2 to the Ca2+-free solution.The 10 mM Ba21 or Ca2+ solution contained BaCl210 mM or CaCl2 10 mM with tetraethylammoniumchloride 135 mM and glucose 10 mM. Ionic milli-molar composition of the 100 mM Ba2+ solution wasBaCl2 100 and glucose 12. The high-K+ solution(bath solution for single-channel recording) had thefollowing ionic millimolar composition: KCl 145,MgCl2 5, and EGTA 4.The high-K' solution (pipette solution for mac-

roscopic current recording) was made by adding 5mM Na2ATP to the high-K+ solution used in the


Dow

nloaded from


Oike et al Actions of a Ca2' Antagonist on the Basilar Artery 995

No NO2

H

CH3OCH2CH200C COOCH2H

H3C, N CH3H

FIGURE 1. Chemical structure of (+)-FRC8653 [2-methoxyethyl (E)-3-phenyl-2-propen-1-yl(±)-1,4-dihydro-2,6-dimethyl- 4- (3-nitro-phenyl)pyridine-3,5-dicarboxylate].

bath. The high-Cs+ solution was made by replace-ment of KCl in the high-K' solution (the pipettesolution) with an equimolar amount of CsCl. ThepH of the solutions was adjusted to 7.25-7.30 by 10mM HEPES titrated with Tris. The solution in thechamber was perfused at a constant rate (1 ml/min)throughout the experiments. Exchange of the solu-tion was accomplished within 1 minute.Drugs used in the present experiments were

FRC8653 and its enantiomers (Fuji-Rebio Inc.,Tokyo). The chemical structure of (±)-FRC8653(MW 492.53) is shown in Figure 1. FRC8653 (100mM) was dissolved in 50% polyethylene glycol(PEG)-50% ethanol solution and diluted with etha-nol to keep the final concentration of ethanol con-stant (0.1%). Although the maximum concentrationsof PEG (0.05%) and ethanol (0.1%) used in thepresent experiments did not produce any effect onthe membrane currents, the same concentrations of

PEG (0.05%) and ethanol (0.1%) were added in thecontrol solutions.

ResultsUnder control conditions, the Ba2` inward current

amplitude remained the same for over 30 minutes;therefore, all experiments were performed within 30minutes. Figure 2 shows the time course of inhibitionof the Ba21 inward current evoked by a depolarizingpulse of + 10 mV from the holding potential of -80mV. When depolarizing pulses were applied every 20seconds, (±)-FRC8653 (100 nM) gradually inhibitedthe amplitude of the Ba21 inward current, and max-imum inhibition was reached after 4 minutes (Figure2A, open circles). The Ba2+ inward current amplitudewas not completely restored after removal of (+)-FRC8653 even after 30 minutes.

Effects of (±)-FRC8653 on Macroscopic Ca2+ (Ba2+)Currents in the Rabbit Basilar Artery

In 10 mM Ca2+ solution, application of a depolariz-ing pulse (more positive than -40 mV) evoked aninward current, and the maximum amplitude wasevoked by depolarizing pulses of +10 to +20 mV(Figure 3Aa, open circles). Application of (+)-FRC8653 (100 nM) inhibited the inward current ampli-tude evoked by depolarizing pulses more positive than0 mV, but slightly enhanced the current amplitudeevoked by depolarizing pulses more negative than -10mV (Figure 3Aa, closed circles). Under the influence ofthe drug, the maximum inward current amplitude wasobtained by depolarizing pulses of 0 to + 10 mV. Figure

+lOmV

-80mV -F--L 0.05Hz

0

b c

b c d

aa

FRC8653 1OOnM

B0

A Control00 FRC8653

d

a 5OpA

25msec0' 3' 6'l 9'l 12',TTM"n

FIGURE 2. Changes in ampli-tude ofthe Ba21 inward current inthe presence or absence of (+)-FRC8653 (100 nM). Depolareingpulses (100 msec in duration) to+10 mVfrom the holdingpoten-tial of -80 mVwere applied every20 seconds. Panel A: Effect of(+)-FRC8653 when the stimula-tion was unbroken (o) or inter-rupted for the first 3.5 minutesafter application of drug (a) andstimulation in the absence ofdrug(A). The amplitude of the Ba2+inward current recorded 3 minutesbefore application of (_)-FRC8653 was normalized as 1.0.Values of control are shown asmean +SD (n=3). Panel B:Tracings of the Ba2+ inward cur-rent recorded before (a) and dur-ing (b and c) application and afterremoval (d) of 100 nM (+)-FRC8653. Tracings (a-d) wereobtained at the time indicated byeach letter in panel A.

A

1.0.

0Q

0ao. 0.5.

,2g)

00C6 by guest on July 1, 2018

http://circres.ahajournals.org/D

ownloaded from



Aa b

-20mV-

iOmV+10mY~~~~~~~~~~~~~~~~

+1ornV -

+3OmV

-20inV - A

0

+lOmV 40o

+30mV

FIGURE 3. Current-voltage relations in theabsence orpresence of (±)-FRC8653. Various

L .60pA amplitudes of depolarizing pulses were applied5mc from holding potentials (Vh) of -80 mV (pan-

els A and B) or -40 mV (panel C). In panelA, excess Ca2' solution (10 mM Ca2') wassuperfused in the bath. In panels B and C,excess Ba2` solution (10 mM Ba2`) was super-fused in the bath. In panels A and B, 100 nM(±)-FRC8653 was applied in the bath, and inpanel C, 10 nM was applied. In panels Aa, Ba,and Ca, amplitudes of the inward current atpeak (circles) and at the end (triangles) of thedepolarization pulse are plotted against theamplitude of the depolarizing pulse, and inpanels Ab, Bb, and Cb, tracings of the Ba2ìnward current in the presence (closed symbols)and absence (open symbols) of (±)-FRC8653are shown. The amplitude of the depolarizing

1O' pulse (100 msec in duration) is indicated at thescc left side of each tracing.

-tOmV 0

+lOrnV

+3OmV *

3Ab shows typical inward current tracings recorded inthe absence or presence of (±)-FRC8653 (100 nM).(± )-FRC8653 consistently inhibited the inward cur-

rent amplitude measured at peak or at the end ofthe depolarizing pulse (100 msec in duration andmore positive than 0 mV). However, (±)-FRC8653more profoundly inhibited the current amplitudemeasured at the end of depolarizing pulse than itdid that measured at the peak amplitude. As shownin the top tracing in Figure 3Ab, the augmentationinduced by (±+)-FRC8653 is seen only in the earlyphase of the inward current evoked by a depolariz-ing pulse of -20 mV.To enable further study of the actions of (±)-

FRC8653, 10 mM Ba2+ solution was superfused in

the bath instead of the 10 mM Ca2' solution,because the Ca2' current has been reported to beinactivated by an increase in concentration of intra-cellular Ca2', as well as by depolarization of themembrane.20-22 In the 10 mM Ba21 solution, theBa2+ inward current amplitude was larger and thedecay was slower than their equivalents in the Ca21inward current experiments. However, the shape ofthe current-voltage relation in the absence of (±+)-FRC8653 was almost the same as that observed inthe presence of 10 mM Ca2+, also without (+)-FRC8653 (open circles, Figure 3Ba versus Figure3Aa). In 10 mM Ba2+ solution, (±)-FRC8653 (100nM) reduced the inward current amplitude evokedby depolarizing pulses more positive than 0 mV but

Vh -8omV

0o Contol* A FRC8E53

10iOM

Ba

-50

pAb

bC

Vh -80mV

0O Control* A FRC8653

100nM

pAa

Vh -40MV

0 6 Control* A FRC8653

lOnMJ50PA

50meCC


Dow

nloaded from



B+lOmV

0 -8OmVJL+lOmV

A -40mV-FL

2.01

a-

0

0 1.5.

0

i 1.0

4

0.1 1 10 100nM 1

[FRC6531

-20mV

-80mV

{

10 104iM 0.1 1 1o 1bnM i

[FRC86531

{-109hj

10 10CtiM

FIGURE 4. Relation betweenamplitude of the Ba2` inwardcurrent and concentration of(±)-FRC8653. Panel A: TheBa2` inward current was evokedby depolarizing pulses to +10mVfrom the holdingpotential of-80 mV (o, n=5-7) or -40mV (A, n=3). Panel B: TheBa2` inward current was evokedby depolarizing pulses to -20mVfrom the holdingpotential of-80 mV (n=5-7). The ampli-tude of the Ba2+ inward currentwas measured at peak and thatrecorded before application of(±)-FRC8653 was normalizedas 1.0.

increased the amplitude evoked by depolarizationpulses more negative than - 10 mV (holding poten-tial, -80 mV; Figure 3Ba, closed circles). Enhance-ment of the inward current induced by (±)-

FRC8653 (100 nM) was seen only during the earlyphase of the inward current (Figure 3Bb, top trac-ing), and decay of the inward current evoked by anygiven depolarizing pulse was steeper than thatobserved in the control (Figure 3Bb).At a holding potential of -40 mV, the Ba2`

inward current amplitude evoked by depolarizingpulses more positive than -20 mV was reducedwith no detectable change in the shape of the Ba21inward current (compare open symbols of Figures3C and 3B). (+)-FRC8653 (10 nM) reduced theamplitude but not the decay of the Ba21 inwardcurrent evoked by any given depolarizing pulse(Figures 3Ca and Cb).

Figure 4 shows the relation between the concentra-tion of (±)-FRC8653 and the relative amplitude of theBa21 inward current evoked by depolarizing pulses of+10 mV or -20 mV from holding potentials of either-80 mV or -40 mV. Figure 4A shows that at theholding potential of -80 mV, (±)-FRC8653 (>0.1 nM)inhibited the Ba2+ inward current evoked by a depolar-izing pulse of + 10 mV, but that even application of 20,M (±)-FRC8653 did not completely inhibit the Ba2+inward current. The 50% inhibitory concentration(IC50) of (±)-FRC8653 on the Ba2+ inward currentevoked by the depolarizing pulse of + 10 mV was 1 ,uMat the holding potential of -80 mV. On the other hand,at the holding potential of -40 mV, (±)-FRC8653potently inhibited the Ba2+ inward current; applicationof 1 ,uM (±)-FRC8653 completely inhibited the Ba21inward current, and the ICO value was 1 nM. Incontrast, an enhancement of the Ba2+ inward currentamplitude occurred in a concentration-dependentmanner (1 nM to 1 ,uM), when the effects of (±)-

FRC8653 were explored using a depolarizing pulse of-20 mV from the holding potential of -80 mV (Figure4B). A higher concentration of (±)-FRC8653 (20 ,tM)

reduced the Ba2` inward current amplitude to less thanthe control value.

Effects of (+)- and (-)-FRC8653 on the Ba2Ìnward Current in the Rabbit Basilar Artery

Application of 100 nM (-)-FRC8653 reduced theamplitude of the Ba2` inward current evoked by a

depolarizing pulse more positive than + 10 mV.However, augmentation of the Ba2+ inward currentwas not clearly observed when a depolarizing pulsemore negative than -20 mV was applied (Figure5A). On the other hand, 100 nM (+)-FRC8653 didenhance the Ba2` inward current amplitude evokedby any depolarizing pulses more negative than - 10mV but inhibited the current evoked by pulses morepositive than 0 mV (Figure 5B). The peak of thecurrent-voltage curve was shifted in a more negativedirection (20 mV) by (+)-FRC8653; however, thethreshold potential of the inward current remainedthe same (-40 mV) whether or not (+)-FRC8653was present. Both (+)- and (-)-FRC8653 acceler-ated the decay of the inward current. When themembrane potential was held at -40 mV, (+)-FRC8653 produced no enhancement of the Ba2+inward current amplitude (Figure SC).

Figure 6 shows the relation between the relativeamplitude of the Ba2+ inward current evoked by twodifferent depolarizing pulses (+10 mV and -20mV) and the concentration of (+)- or (-)-FRC8653. The inhibitory action of (-)-FRC8653on the Ba2+ inward current was more potent thanthat of the (+) enantiomer [IC50 of (-)-FRC8653,60 nM; IC50 of (+)-FRC8653, 1.5 ,uM; Figure 6A].When a depolarizing pulse of -20 mV from theholding potential of -80 mV was applied, (-)-FRC8653 (<10 uM) inhibited, but the (+) enan-

tiomer (< 100 nM) augmented, the Ba2+ inwardcurrent amplitude in a concentration-dependentmanner. A higher concentration of (+)-FRC8653(10 uM) caused no increase above control in theBa2+ current amplitude (Figure 6B).

A

1.0.

0U0

I 0.5

0.2

cc

4

4


Dow

nloaded from



Aa

-50

bpA

-2OmV A

* A

-20mv t:

OmV

A

+10mV A

+3OmV

-3OmV

-20mV

OmV

+20mV

FIGURE 5. Current-voltage relations in the50pA absence or presence of (+)- or (-)-FRC8653.

5omsec Panel A: 100 nM (-)-FRC8653 was applied inthe bath. The holding potential (Vh) was -80mV Panel B: 100 nM (+)-FRC8653 wasapplied in the bath. The Vh was -80 mVPanel C: 10 nM (+)-FRC8653 was applied inthe bath. The Va was -40 mV In panels Aa,Ba, and Ca, amplitudes of the inward currentat the peak (circles) and at the end (triangles)of the depolarizingpulse are plotted against theamplitude of depolarizing pulse, and in panelsAb, Bb, and Cb, tracings of the Ba2` inwardcurrent in the absence (open symbols) andpresence (closed symbols) are shown. Theamplitudes of the depolarizing pulse are indi-cated at the left side of the tracings.

J 10OpA

50msec

-lOmV A

4-lOmV

+30mV-

These results indicate that the augmentation of theBa2` inward current amplitude observed on applica-tion of (±+ )-FRC8653 is closely related to the presenceof the (+) enantiomer and is seen only when depolar-izing pulses of less than -10 mV are applied from alow holding potential level of -80 mV.

Effects of (±)-FRC8653 on Voltage-DependentInactivation in Smooth Muscle Cells of the RabbitBasilar ArteryWhen various amplitudes of long conditioning

pulses (5 seconds) were applied just before appli-cation of the test pulse (depolarization to + 10 mV,20-msec interval, 100 msec in duration), the Ba2+inward current amplitude evoked by the test pulse

was decreased depending on the amplitude of theconditioning pulse (Figure 7A). However, even withapplication of a conditioning pulse of +20 mV,about 15% of the Ba2+ inward current was notinactivated (noninactivating component13,23). Whena Boltzmann's distribution was adopted for thepresent voltage-dependent inactivation curve, thecurve did not fit with a single slope factor, as shownin Figure 7A (continuous line with open circles).(±+)-FRC8653 (100 nM) inhibited the noninactivat-ing component completely and shifted the voltage-dependent inactivation curve to the left. Whensimilar experiments were performed using a testpulse of -20 mV (Figure 7B), (±)-FRC8653 (100nM) augmented the Ba2+ inward current evoked by

Vh -8omV

0 A Control0 A (-)FRC8653

10OnM

B

a-50

b+50 mV

Vh -80mV

0 A Control* A (+)FRC8653

100nM

Ca

-50

b

Vh -4OmVCA Control* A (+)FRC8653

lOnM J 5OpA50msec


Dow

nloaded from


Oike et al Actions of a Ca2+ Antagonist on the Basilar Artery 999

+l0mV

-80mVA.F--B-20~-2mVB -8OmVX

5.0.

* (+)FR08653O (-)FRC8653 4.J.

0

0

o 3.0'

I2.0.

0

?--1.0.a1cc,

4

0.1 i ib 1OOnM 1 1OpM[FRC86531

I9

0.1 1 io10 OOnM 1[FRC86531

FIGURE 6. Relation between relativeamplitude of the Ba2` inward currentand concentration of (+)- or (-)-FRC8653. Panel A: The Ba2` inward

* (+)FRC8653 current was evoked by depolarizingo (_)FR653 pulses to +10 mV from the holding

potential of -80 mV. 0, (+)-FRC8653; o, (-)-FRC8653 (n=3).Panel B: The Ba2` inward current wasevoked by depolarizing pulses to -20mVfrom the holding potential of -80mV (n=3). The amplitude of the Ba2`

4 current before application ofeither drugi10OpM was normalized as 1.0.

conditioning pulses more negative than - 60 mV butinhibited the current evoked by conditioning pulsesmore positive than -50 mV.

Effects of (±)-FRC8653 on the K' Outward Currentin Smooth Muscle Cell Membranes of the RabbitBasilar ArteryTo investigate the effects of (±)-FRC8653 on the

K' outward current, various concentrations of (+)-FRC8653 were applied to the cells in a PSS bath. Asshown in Figure 8A, (±)-FRC8653 (3 and 10 gM)inhibited the amplitude of the outward currentevoked by any given depolarizing pulse (-30 to +40mV; holding potential, -60 mV). The inhibition ofthe outward current was completely reversible afterremoval of the drug. Applications of (±)-FRC8653(> 1 ,uM) inhibited the outward current in aconcentration-dependent manner (Figure 8B). TheIC50 value of (+)-FRC8653 for the K' outwardcurrent was about 3 ,uM; this value was three timeshigher than that for the Ba2` inward current mea-sured at the holding potential of -80 mV, but 3,000times higher than that measured at the holdingpotential of -40 mV (see Figure 4).

Recording of the Single Ba2` Channel Current FromSmooth Muscle Cells of the Rabbit Basilar Artery

Figure 9A shows the current-voltage relations ofthe two different amplitudes of the single Ba2` chan-nel current. The values of the large and small singleBa2` channel conductance were 23 and 12 pS, respec-tively. When the holding potential was elevated to-60 mV, the 12-pS channel was not evoked. Figure9B shows the amplitudes of the single-channel cur-rents evoked by various intensities of depolarizingpulses (-30 to + 10 mV) from the holding potentialof -80 mV in 100 mM Ba2` solution. When themembrane was depolarized to -30 to -10 mV, twodifferent amplitudes of single-channel currents wererecorded in the same membrane patch. On the otherhand, when depolarizing pulses more positive than 0mV were applied in the same membrane patch, the

small amplitude inward current ceased. In Figure 9C,summated single-channel currents of the 12- and 23-pS channels are shown. The amplitude of the sum-mated current of the 12-pS channel declined duringthe depolarization (150 msec in duration), while thatof the 23 -pS channel was sustained at the same level.

Effects of (+)- or (-)-FRC8653 on the Single Ba2+Channel Current in Smooth Muscle Cells of theRabbit Basilar Artery

Figure 10 shows the effects of (+)-FRC8653 on the12-pS channel current evoked by repetitively applieddepolarizing pulses to -20 mV from the holdingpotential of -80 mV (20-second interval, 150 msec induration). In the absence of (+)-FRC8653, 43 of 72depolarization pulses evoked channel opening (non-blank tracings), but the other 29 pulses did not (blanktracings). Essentially, the same result was obtained inthe presence of 100 nM (+)-FRC8653 (42 nonblankand 31 blank tracings from 73 depolarizing pulses).In Figure 10, the lower tracings show summatedcurrents of the 12-pS channel in the absence orpresence of 100 nM (+)-FRC8653. There is nosignificant difference in the amplitude or the shapebefore or after application of (+)-FRC8653. Table 1shows the proportion of nonblank tracings, meanopen and closed times, open probability, and aver-aged number of events (channel opening) per tracingrecorded from two different membrane patches. (+)-FRC8653 (100 nM) did not modify the above param-eters. This means that (+)-FRC8653 does not modifythe activity of the 12-pS channel currents in smoothmuscle cells of the rabbit basilar artery.

Figure 11A shows the effects of (+)-FRC8653 (100nM) on the 23-pS channel current evoked by adepolarizing pulse of -20 mV. In this membranepatch, both 12- and 23-pS channels were simultane-ously opened, and in 56 of 72 depolarizing pulses, the23-pS channel opened. In the presence of 100 nM(+)-FRC8653, the number of depolarizing pulsesneeded to record 50 nonblank tracings was reducedto 58. As shown in the bottom tracings, the amplitudeof the summated currents was increased by 100 nM

A

1.0

c000

0.5.

0


Dow

nloaded from



A +lOmV

-lOOmV F L5sec 100msec

1.0 0 Control 0* FRC8653

lOOnM

E

J0 1OOpA-100 -00 0 +20 mV

Conditioning potential

B 2mV30msec

0~ ~ ~ ~ ~~2m

-100mV -5 L5sec 100msec

1.5 O Control 01.5 ~~~~~0FRC865310OnM

01i.00

0.5-

0 _ _ _ _ _ _ _3OpA-100 -50 0mV

Conditioning potential 30msec

FIGURE 7. Voltage-dependent inactivation curves recorded from smooth muscle cells of the rabbit basilar artery. Panel A: Theamplitude of the conditioning pulse (5 seconds in duration) is plotted on the horizontal axis, and relative amplitude of the Ba2ìnward current evoked by the test pulse (+10 mV, 100 msec in duration) on the vertical axis. An interval of 20 msec was allowedbetween the conditioning and test pulses to eliminate possible contamination of the capacitative current in the current measurement.O, Control (mean +SD, n=4); 0, presence of 100 nM (+)-FRC8653 (mean -+-SD, n=4); solid lines, absence or presence of(+)-FRC8653, the Ba2+ inward current amplitudes recorded after application of conditioning potential (-100 mV) beingnormalized as 1.0; dashed line, Ba2+ inward current amplitudes recorded after application of the drug expressed relative to thoserecorded in the absence of drug. The lines were drawn theoretically using the following equation: I=(Imn,-C)/{1+exp[(V-V0.5)lk]}+C, where I, Im,, V, Vo5, k, and C are the relative amplitudes of the Ba21 inward currents observed at various amplitudes ofthe conditioningpulse (I) and observed with application of the conditioningpulse of -100 mV(Imn,>), amplitude of the conditioningpulse (V), and that where the amplitude of the Ba2+ inward current was reduced to half (V05), slope factor (k) and fraction of thenoninactivating component of the Ba2+ inward current (C). The curves in the absence or presence of (±)-FRC8653 were drawnusing the following values: (control) 'ma=1.0, V05= -22 mV, k=9 mV, and C=0.15; [(±)-FRC8653] I,",=0.61, VO5= -43 mV,k=9 mV, and C=0.02. Tracings ofthe Ba2` inward current recorded with various amplitudes ofthe conditioningpulse (-100, -40,-20, and +20 mV) are shown on the right. Panel B: Amplitude of the Ba2+ inward current evoked by the test pulse depolarizationto -20 mV (100 msec in duration) was plotted against the amplitude of the conditioningpulse (5 seconds in duration). o, Absenceof (±)-FRC8653; *, presence of 100 nM (±)-FRC8653 (mean+SD, n=4). The amplitude of the Ba2` inward current wasmeasured at peak. The Ba2+ inward current amplitude evoked by the conditioning pulse of -100 mV in the absence of(±)-FRC8653 was normalized as a relative amplitude of 1.0. Curves were drawn by eye. Tracings recorded with conditioningpulsesof -100, -50, -30, and -10 mV, are shown on the right.

(+)-FRC8653; however, no decay of the current was of channel openings per tracing. On the other hand,seen during the membrane depolarization (150 msec 100 nM (+)-FRC8653 reduced the fast component ofin duration). (+)-FRC8653 did not modify the ampli- the closed time but not the slow component (Tabletude of the 23-pS channel current, but it did increase 2). Figure llB shows the effects of (-)-FRC8653 onthe proportion of nonblank tracings, the mean open the 23-pS channel current evoked by a depolarizingtime, the averaged open probability, and the number pulse to +10 mV from the holding potential of -80


Dow

nloaded from



AO Control

FRC8653* luM

A 3iM* 10pM

Vh -60mV

-50

B

100

m

pA

200

0o +50 mV

OmV

-60mV fi

1.0.-

c0

0

0.5.

0

o 00 IK

0.1 1 10

tFRC8653l (UM)

Control

J50PA

100msec

FIGURE 8. Effects of (+)-FRC8653 on the current-voltagerelation ofthepeak amplitude ofK' outward current recordedfrom smooth muscle cells ofthe rabbit basilar artery. Bath was

superfused with physiological salt solution, and the pipette wasfilled with high-K' (4 mMEGTA) solution. Panel A: Variousconcentrations of (±)-FRC8653 were applied to the same cell.Each concentration ofdrug was applied after the amplitude ofthe outward current was restored completely by removal ofprevious application of drug. Holding potential (Vh) was keptat -60 mV Panel B: The outward current was evoked by a

depolarizing pulse to 0 mVfrom the Vh of -60 mV, and theamplitude before application of the drug was normalized as

1.0. Tracings of the K' outward current (IK) in the absence or

presence of (±)-FRC8653 are shown on the right.

mV. In the control, 50 of 61 depolarization pulsesevoked single-channel currents of the 23-pS channel,and the summated current of the 50 nonblank trac-ings showed no reduction in the current amplitudeduring the depolarizations. On the other hand, in thepresence of (-)-FRC8653, to obtain 50 nonblanktracings, 108 depolarization pulses were required,and the summated current showed a rapid decay inamplitude. This means that the channel openingoccurs during the early phase of depolarization.When two summated inward currents of 50 nonblanktracings were compared in the absence or presence of100 nM (-)-FRC8653, the peak amplitudes of thesummated currents were the same.

Table 2 also shows the values of the mean openand closed times of the 23-pS channels in the

absence or presence of (-)-FRC8653 recorded onapplication of depolarizing pulses to +10 mV fromthe holding potential of -80 mV. The inhibitoryaction of (-)-FRC8653 observed at +10 mV wasmainly due 1) to an increase in the number of blanktracings [proportion of nonblank tracings: control,79.1%, but in the presence of (-)-FRC8653, 47.5%]and 2) to a reduction in the number of events pertracing [control, 8.5+ 1.0, n=3; (-)-FRC8653,4.2±0.8, n=3]. However, the mean open and closedtimes were not modified (control, t.=2.1±0.3 msec,tc,fast=4.3±0.4 msec, t,,0w =14.1±0.8 msec, n=3; (-)-FRC8653, to=2.0±0.3 msec, tc,fast=4.0±0.7 msec,tc,slow=15.1±0.9 msec, n=3). As a consequence, meanopen probability was reduced to 0.03±0.01 from0.09±0.02 in the presence of 100 nM (-)-FRC8653(n=3). (-)-FRC8653 prolonged the time betweenthe final closing of the 23-pS channel and the end ofthe depolarizing pulse (control, 20.5±5.0 msec, n =3;(-)-FRC8653, 57.5±4.6 msec, n=3).

DiscussionIn dispersed smooth muscle cells of the rabbit

basilar artery, two types of voltage-dependent Ca21channels were present with single-channel conduc-tances of 12 or 23 pS (in the 100 mM BaCl2 solution).The summated current of the 12-pS channel wasinactivated within 100 msec, but that of the 23-pSchannel was long lasting. Both 12- and 23-pS channelcurrents were activated by depolarizing pulses above-30 mV from the holding potential of -80 mV;however, the 12-pS channel was not evoked bydepolarizing pulses from the holding potential of-60 mV. These characteristics are consistent withthe properties reported as belonging to the T- andL-types of voltage-dependent Ca21 channels in vari-ous excitable cells.3'7'24 In the whole-cell voltage-clamp experiments, however, no clear evidence forthe existence of T- or L-type voltage-dependent Ca21channels was obtained. For example, no additionalpeak on the low membrane potential level wasobserved in the current-voltage relation, and a largerapidly inactivating component was not observed onthe decay phase of the inward current at the holdingpotential of -80 mV. The resting membrane poten-tial of the rabbit basilar artery has been reported tobe -50 to -60 mV25,26; therefore, almost all T-typeCa21 channels might be inactivated at the restingmembrane potential level. It is not yet clear what therole of the T-type Ca21 channel is, but whatever it is,modification of the T-type Ca>2 channel may not beinvolved in the vasodilating actions of (±)-FRC8653.The characteristics of (±)-FRC8653, a novel DHP

derivative, as determined in the present experimentsmay be summarized as follows. 1) (±)-FRC8653 hada dual action, producing both excitatory and inhibi-tory effects, on the Ba inward current. The (+)enantiomer mainly had excitatory, and the (-) enan-tiomer inhibitory, actions. The excitatory action ofthe (+) enantiomer was easily reversed to an inhib-itory action by elevation of the membrane potential


Dow

nloaded from



OmV _ _~f*4

lOmV

10mV --e -~

FIGURE 9. Two types ofunitaryBa2` channel current observed insmooth muscle cells of the rabbitbasilar artery. The pipette wasfilled with 100 mM BaCI2 solu-tion, and the bath was superfusedwith high-K' solution containing4 mM EGTA. The membranewas maintained at -80 mV, anda depolarizing pulse for patchmembrane was applied. Panel A:Relation between amplitude oftheunitary Ba21 current (I) and thepatch membrane potential (V).Panel B: Sample tracings of theunitary Ba21 current of two dif-ferent amplitudes. Panel C: Sum-mated unitary Ba2` current with50 nonblank tracings evoked byrepetitive depolarizing pulses to-20 mV (a) and to +10 mV (b)(150 msec in duration, 15-secondinterval).

(to -40 mV). 2) (+)-FRC8653 did not act on the12-pS channel, which was classified as a T-type Ca2+channel,7 and thus both the excitatory and inhibitoryactions induced by (+)-FRC8653 result from actionson the 23-pS channel in the rabbit basilar artery.Although the T- and L-type Ca2 channels are

present in the smooth muscle cell membrane of therabbit basilar artery, both the excitatory and inhibitoryactions of (±t)-FRC8653 were caused by actions of thedrug on the 23-pS channel (L-type Ca2+ channel). It iswell known that many DHP derivatives, such as Bay K8644, PN200-110, nitrendipine, and nifedipine, andnewly synthesized derivatives, such as CV-4093 or

benidipine, have both agonistic and antagonisticactions on the L-type Ca2 channels in various musclecells. Furthermore, one of the enantiomers has apredominantly agonistic action.13,17,27-29 In thisrespect, (±)-FRC8653 appears to be a typical DHPderivative; that is, (+)-FRC8653 has a predominantlyagonistic action, but that of the (-) enantiomer ismarginal.One of the interesting properties of (±+)-FRC8653

is that this drug can inhibit the voltage-dependentCa2+ channel over a very wide range of concentra-tions (<0.1 nM to >20 ,uM) (Figure 4, at a holdingpotential of -80 mV). When the Ba2+ current ampli-tude was measured at peak with a depolarizing pulseof +10 mV from the holding potential of -80 mV,(±+)-FRC8653 did not completely inhibit the inwardcurrent, presumably because of activation of theT-type Ca>2 channel and partly because of participa-tion of the agonistic action of the (+) enantiomer.However, the contribution of the former may besmall, because (-)-FRC8653 (10 gM) produced anearly complete block of the Ba21 current. On the

other hand, a step observed between 10 and 100 nMin the concentration-inhibition curve (as shown inFigure 4A at a holding potential of -80 mV) mayreflect a contamination of the agonistic action of thedrug, because such a step was not observed in theconcentration-inhibition curve observed at the hold-ing potential of -40 mV (Figure 4A). Similarly, theturning down of the augmentation curve of the Ba2`current (depolarizing pulse, -20 mV; holding poten-tial, -80 mV) observed after application of (±)-

FRC8653 and of the (+) enantiomer (>10 ,M) alsomay be due to contamination of antagonistic actions.Therefore, the overall actions of (±)-FRC8653 canbe expressed as the sum of the agonistic and antag-onistic actions of the (+) and (-) enantiomers.The voltage-dependent inactivation curves showed

that 15% of the inward current was not inactivatedabove +10 mV of conditioning depolarization andthat (±)-FRC8653 (100 nM) inhibited this compo-nent, as reported in other smooth muscle cells.13,23(±+)-FRC8653 shifted the voltage-dependent inacti-vation curve to the left (17 mV), suggesting that thisdrug binds at the inactivated state with highaffinity.30'31 However, in the present experiments (seeFigure 7), the voltage-dependent inactivation curvein the absence of (±)-FRC8653 did not fit withBoltzmann's equation with a single slope factor; thatis, the upper part of the curve fitted with a slopefactor of 16 but the lower part with a factor of 9.These results may be due to contamination of theactivation of the T-type Ca21 channel at lower con-ditioning potentials (<-40 mV, upper part of thecurve). Various Ca2+ antagonists, such as diltiazem,verapamil, flunarizine, and many DHP derivatives,are known to shift the voltage-dependent inactivation

Ab

-8OmB1(pA)

±-- l*f-30mV

_gv -20mV

23.2pSI 2pA

5omsec

bCa

-2OmV

5 Mgem -iomv - --- .

- ---T


Dow

nloaded from


Oike et al Actions of a Ca2' Antagonist on the Basilar Artery

Control-20mV ~ I

-80mV

__ e -1

rvrm

Ir

43/72

(+)FRC 10OnM-20mV I

-80mV

20

_ r~~W

FIGURE 10. Effects of 100 nM(+)-FRC8653 on the 12-pS chan-nel current. Seventy-two repetitivedepolarizing pulses (150 msec induration, 20-second interval) to-20 mV in the absence of (+)-FRC8653 were applied; then 100nM (+)-FRC8653 was superfused.Ten minutes after application ofdrug, 73 pulses were applied. Trac-ings shown were obtained frompulses 33 to 43 in control and from20 to 30 in the presence of (+)-FRC8653. Bottom tracings are twosummated currents superimposed inthe absence or presence of (+)-FRC8653. Fraction numbers in thebottom tracings indicate (number ofnonblank tracings)I(total number oftracings).

42/73

- ----- 30

2pA

50msec

trolJ 4pA

50msec

TABLE 1. Effects of 100 nM (+)-FRC8653 on the Single-Channel Activities of the 12-pS Channel

No. ofMean closed time Mean open events/

time probability tracingProportion of N Mean open time Fast Slow

Preparation (NiT) N (N) (N) (N) (T) (N) (T)1 (Control) 58.3% (42/72) 3.4 msec 2.8 msec 19.5 msec 0.09 0.06 3.4 2.01 (FRC) 58.9% (43/73) 2.8 msec 2.8 msec 18.9 msec 0.09 0.05 3.9 2.32 (Control) 60.7% (34/56) 2.5 msec 2.9 msec 23.7 msec 0.04 0.03 4.0 2.42 (FRC) 61.1% (33/55) 2.2 msec 2.7 msec 22.5 msec 0.05 0.03 3.8 2.31 (FRC/control) 1.01 0.82 1.00 0.97 1.00 0.83 1.15 1.152 (FRC/control) 1.01 0.88 0.93 0.94 1.25 1.00 0.95 0.96

N, nonblank tracings; T, total tracings; FRC, (+)-FRC8653.

1003

~_*. 43


Dow

nloaded from



BControl (+) FRC 10OnMmYr F ThL

nVt _ 1

1 Lg.#¶+ *p±±rrr

-vw

Control"WOm L

-janmV

(-) FRC 10OnM

1 53

,M[ k- ±---- .--

ru1 1-.

rw

fTW--t

-1-8- I-L-25--mow

±T

50/58 50/

m -

h

1w,t

1_f T r---1---

1 LWTFr

4qqqq--- i.--

It 1 50/108

20 64 2pA

50msec

X4pA50msec

FIGURE 11. Effects of (+)- and (-)-FRC8653 on the 23-pS channel current evoked by repetitive depolarizing pulses to -20 mV(panel A) and +10 mV (panel B). Panel A: Sample tracings of the unitary Ba2+ channel currents evoked by depolarizing pulsesto -20 mV (150 msec in duration, 20-second interval). In the absence orpresence of (+)-FRC8653, both 12- and 23-pS channelsopened. Tracings shown were obtained from pulses 9 to 18 in control and from 16 to 25 in the presence of (+)-FRC8653. PanelB: Tracings of the unitary Ba2+ channel current evoked by the depolarizing pulse to +10 mV (150 msec in duration, 20-secondinterval). (-)-FRC8653 (100 nM) was applied in the bath. Recording of tracings in the presence of drug was started 10 minutesafter application. Tracings shown were obtainedfrom pulses 11 to 20 in control andfrom 53 to 64 in thepresence of (-)-FRC8653.In panels A and B, bottom tracings show the summated unitary current of all nonblank tracings in the absence or presence ofFRC8653 (100 nM). Fraction numbers above the bottom tracings indicate (number of nonblank tracings)I(total number oftracings).

curve to the left in a parallel manner.32 In the presentexperiments, (±+ )-FRC8653 shifted the voltage-dependent inactivation curve and also changed theslope of the upper part of the curve. This may suggestthat (±)-FRC8653 has a more potent agonistic actionthan other Ca2' antagonists.Enhancement of the Ba2` inward current ampli-

tude was reversed to an inhibition by elevation of theholding potential (-40 mV). When the voltage-dependent inactivation curves (conditioning pulse, 5seconds in duration) were produced using a short testpulse of -20 mV [at which (±)-FRC8653 producedthe largest augmentation of the Ba21 inward current;see Figure 3Ba], these curves in the presence orabsence of (+)-FRC8653 crossed at near -50 mV(Figure 7B). With the microelectrode technique, themean resting membrane potential of the rabbit basi-lar artery has been reported to be -50 to -60mV.25,26 Although the conditions for these measure-

ments of the resting membrane potential and theBa` inward current were quite different, augmenta-tion of the Ba` current may not play an importantrole in the action of this drug under physiologicalconditions. Furthermore, augmentation of the Ca'inward current by this drug was smaller than thatobserved in the same concentration of Ba`; this mayreinforce the above postulation.

Inoue et a133 studied the inhibitory action of nifed-ipine on the single Ba> channel current in rabbitintestinal smooth muscle cells and reported thatnifedipine both increases the proportion of blanktracings and reduces the mean open time; however,the former action is more important than the latter inthe overall inhibitory actions of nifedipine. They alsoreported that nifedipine has a voltage-dependentinhibitory action on the single Ba> channel current,since an increase in the slope of the current decay isthought to be due to the occurrence of a long shut in

A-20r-80n

T 1 .1.-1 ..

-T

56/72

-1 - -1- TRUT

09 J-F ---1


Dow

nloaded from


Oike et al Actions of a Ca2+ Antagonist on the Basilar Artery 1005

TABLE 2. Effects of (+)- or (-)-FRC8653 on the Single-Channel Activities of the 23-pS Channel

Number ofMean closed time Mean open probability events/tracing

Proportion of N Mean open time Fast SlowPreparation (N/T) (N) (N) (N) (N) (T) (N) (T)

100 nM (+)-FRC86531 (Control) 73.6% (53/72) 2.1 msec 6.4 msec 16.3 msec 0.04 0.03 3.1 2.3

1 (FRC) 82.7% (48/58) 4.0 msec 3.0 msec 18.5 msec 0.12 0.09 4.6 3.8

2 (Control) 49.0% (50/102) 1.3 msec 6.1 msec 14.1 msec 0.05 0.02 2.0 1.0




1 (FRC/control) 1.12 1.90 0.47 1.13 3.00 3.00 1.48 1.65

2 (FRC/control) 1.30 2.08 0.39 1.17 2.60 4.00 1.65 2.10

3 (FRC/control) 1.24 1.40 0.59 1.21 2.00 2.33 1.52 1.92

Mean 1.22+0.09 1.79±0.35 0.48±0.1 1.17±0.04 2.53±0.50 3.11±0.84 1.55±0.09 1.89±0.23

100 nM (-)-FRC86531 (Control) 82.0% (50/61) 1.8 msec 4.8 msec 15.1 msec 0.13 0.10 9.2 7.5






1 (FRC/control) 0.63 0.89 0.96 0.97 0.56 0.4 0.58 0.37

2 (FRC/control) 0.57 1.05 0.82 1.09 0.54 0.27 0.37 0.21

3 (FRC/control) 0.61 0.96 1.05 1.16 0.67 0.43 0.55 0.40

Mean 0.60+0.03 0.97±0.08 0.94±0.12 1.07±0.10 0.59±0.07 0.37±0.09 0.50±0.11 0.33±0.10

N, nonblank tracings; T, total tracings; FRC, (+)- or (-)-FRC8653.

the early phase of the depolarizing pulse. In thepresent experiments, (- )-FRC8653 had similaractions on the 23-pS channel current evoked by adepolarizing pulse of + 10 mV, except for changes inthe mean open and closed times, which may be due toa contribution from the agonistic action of (-)-FRC8653. The present experiments showed thatapplication of 100 nM (-)-FRC8653 reduces themean open probability to 0.3-0.4 times the control,because of an increase in the number of blanktracings and a decrease in the number of events(channel opening) per tracing. On the other hand,the increase in the mean open probability thatoccurred after application of (+)-FRC8653 may becaused by an increase in the proportion of nonblanktracings, mean open time, and number of events pertracing.

In conclusion, (+)-FRC8653 shares the commonproperties of DHP derivatives, including the dif-ferent actions of the two enantiomers. Because (+)-FRC8653 had a moderate agonistic action, theactions of the racemic form of this drug, (+)-FRC8653, would be modified by the resting mem-

brane potential of various smooth muscle cells.Therefore, this drug may selectively relax certainvascular tissues in which the cells have a relativelyless negative resting membrane potential.

AcknowledgmentsWe thank Dr. R.J. Timms for kindly reading the

manuscript. FRC8653 was kindly provided by Fuji-Rebio Inc. and Ajinomoto Inc., Tokyo, Japan.

References1. Aalonson PI, Benham CD, Bolton TB, Hess P, Lang RJ, TsienRW: Two types of single-channel and whole-cell calcium orbarium currents in single smooth muscle cells of rabbit earartery and the effects of noradrenaline (abstract). J Physiol(Lond) 1986;377:36p

2. Worley JF III, Deitmer JW, Nelson MT: Single nisoldipine-sensitive calcium channels in smooth muscle cells isolatedfrom rabbit mesenteric artery. Proc Natl Acad Sci USA 1986;83:5746-5750

3. Benham CD, Hess P, Tsien RW: Two types of calcium channelsin single smooth muscle cells from rabbit ear artery studied withwhole-cell and single-channel recordings. Circ Res 1987;61(suppll):1-10-I-16


Dow

nloaded from



4. Yatani A, Seidel CL, Allen J, Brown AM: Whole-cell andsingle-channel calcium currents of isolated smooth musclecells from saphenous vein. Circ Res 1987;60:523-533

5. Yoshino M, Someya T, Nishio A, Yabu H: Whole-cell andunitary Ca channel currents in mammalian intestinal smoothmuscle cells: Evidence for the existence of two types of Cachannels. Pflugers Arch 1988;411:229-231

6. Inoue Y, Oike M, Nakao K, Kitamura K, Kuriyama H:Endothelin augments unitary Ca channel currents on thesmooth muscle cell membrane of guinea-pig portal vein. JPhysiol (Lond) 1990;423:171-191

7. Nilius B, Hess P, Lansman JB, Tsien RW: A novel type ofcardiac calcium channel in ventricular cells. Nature 1985;316:443-446

8. Hume JR: Comparative interactions of organic Ca' channelantagonists with myocardial Ca++ and K' channels. JPharma-col Exp Ther 1985;234:134-140

9. Terada K, Kitamura K, Kuriyama H: Different inhibitions ofthe voltage-dependent K' current by Ca'+ antagonists insmooth muscle cell membrane of rabbit small intestine.Pfiugers Arch 1987;408:558-564

10. Akaike N, Kanaide H, Kuga T, Nakamura M, Sadoshima J,Tomoike H: Low-voltage-activated calcium current in rataorta smooth muscle cells in primary culture. J Physiol (Lond)1989;416:141-160

11. Kokubun S, Prod'hom B, Becker C, Prozig H, Reuter H:Studies on Ca channels in intact cells: Voltage-dependenteffects and cooperative interactions of dihydropyridine enan-

tiomers. Mol Pharmacol 1987;30:571-58412. Sanguinetti MC, Krafte DS, Kass RS: Voltage-dependent

modulation of Ca channel current in heart cells by Bay K8644.J Gen Physiol 1986;88:369-392

13. Aalonson PI, Bolton TB, Lang RJ, MacKenzie I: Calciumcurrents in single isolated smooth muscle cells from the rabbitear artery in normal-calcium and high-barium solutions. JPhysiol (Lond) 1988;405:57-75

14. Fujiwara S, Kuriyama H: Effects of agents that modulatepotassium permeability on smooth muscle cells of the guinea-pig basilar artery. Br J Pharmacol 1983;79:23-35

15. lida H, Fujiyoshi T, Ikeda K, Hosono M, Yamamura T, KaseN, Sekine A, Uematsu T: Antihypertensive and cardiovasculareffects of a new dihydropyridine derivative, 2-methoxyethyl(E)-3-phenyl-2-propen-1-yl-1,4-dihydro-2,6-dimethyl-4-(3 -

nitrophenyl)pyridine-3,5-dicarboxylate (FRC-8653)(abstract). Jpn J Pharmacol 1987;43(suppl):296p

16. Momose K, Gomi Y: Studies on isolated smooth muscle cells:IV. Dispersion procedures for acetylcholine-sensitive smoothmuscle cells of guinea-pig (abstract in English). Jpn J SmoothMuscle Res 1980;16:29-36

17. Terada K, Nakao K, Okabe K, Kitamura K, Kuriyama H:Action of the 1,4-dihydropyridine derivative, KW-3049, on thesmooth muscle cell membrane of the rabbit mesenteric artery.Br J Pharmacol 1987;92:615-625

18. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth F:Improved patch-clamp techniques for high-resolution currentrecording from cells and cell-free membrane patches. PflugersArch 1981;391:85-100

19. Almers W, Palade PT: Slow calcium and potassium currentsacross frog muscle membrane: Measurements with a Vaseline-gap technique. J Physiol (Lond) 1981;312:159-176

20. Ohya Y, Terada K, Kitamura K, Kuriyama H: Membranecurrents recorded from a fragment of rabbit intestinal smoothmuscle cell. Am J Physiol 1986;251:C335-C346

21. Ohya Y, Kitamura K, Kuriyama H: Regulation of calciumcurrent by intracellular calcium in smooth muscle cells fromthe rabbit portal vein. Circ Res 1988;62:375-383

22. Jmari K, Mironneau C, Mironneau J: Inactivation of calciumchannel current in rat uterine smooth muscle: Evidence forcalcium- and voltage-mediated mechanism. J Physiol (Lond)1986;380:11 1-126

23. Imaizumi Y, Muraki K, Watanabe M: Ionic currents in singlesmooth muscle cells from the ureter of the guinea-pig. JPhysiol(Lond) 1989;411:131-159

24. Nowycky MC, Fox AP, Tsien RW: Three types of neuronalcalcium channel with different calcium agonist sensitivity.Nature 1985;316:440-443

25. Nagao T, Suzuki H, Kuriyama H: Effects of flunarizine onelectrical and mechanical responses of smooth muscle cells inbasilar and ear arteries of the rabbit. Pflugers Arch 1986;333:431-438

26. Surprenant A, Neild TO, Holman ME: Membrane propertiesof rabbit basilar arteries and their responses to transmuralstimulation. Pflugers Arch 1987;410:92-101

27. Hess P, Lansman JB, Tsien RW: Different modes of Cachannel behavior favoured by dihydropyridine Ca agonists andantagonists. Nature 1984;311:538-544

28. Brown AM, Kunze DL, Yatani A: Dual effects of dihydropy-ridines on whole cell and unitary calcium currents in singleventricular cells of guinea-pig. J Physiol (Lond) 1986;379:495-514

29. Okabe K, Terada K, Kitamura K, Kuriyama H: Selective andlong lasting inhibitory actions of the dihydropyridine deriva-tive, CV-4093, on calcium currents in smooth muscle cells ofthe rabbit pulmonary artery. J Pharmacol Exp Ther 1987;243:703-710

30. Sanguinetti MC, Kass RS: Voltage-dependent block of cal-cium channel current in the calf cardiac Purkinje fiber bydihydropyridine calcium channel antagonists. Circ Res 1984;55:336-348

31. Bean BP: Nitrendipine block of cardiac calcium channels:High-affinity binding to the inactivated state. Proc Natl AcadSci USA 1984;81:6388-6392

32. Terada K, Kitamura K, Kuriyama H: Blocking actions of Ca2'antagonists on the Ca2' channels in the smooth muscle cellmembrane of rabbit small intestine. Pflugers Arch 1987;408:552-557

33. Inoue Y, Xiong ZL, Kitamura K, Kuriyama H: Modulationproduced by nifedipine of the unitary Ba current of dispersedsmooth muscle cells of the rabbit ileum. Pflugers Arch 1989;414:534-542

KEY WORDS * Ca2l antagonist * dihydropyridine derivativeCa2' channel * basilar artery * Ca2+ agonist


Dow

nloaded from


M Oike, Y Inoue, K Kitamura and H Kuriyamarecorded from the rabbit basilar artery.

Dual action of FRC8653, a novel dihydropyridine derivative, on the Ba2+ current

Print ISSN: 0009-7330. Online ISSN: 1524-4571 Copyright © 1990 American Heart Association, Inc. All rights reserved.is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation Research

doi: 10.1161/01.RES.67.4.9931990;67:993-1006Circ Res.

http://circres.ahajournals.org/content/67/4/993World Wide Web at:

The online version of this article, along with updated information and services, is located on the

http://circres.ahajournals.org//subscriptions/

is online at: Circulation Research Information about subscribing to Subscriptions:

http://www.lww.com/reprints Information about reprints can be found online at: Reprints:

document. Permissions and Rights Question and Answer about this process is available in the

located, click Request Permissions in the middle column of the Web page under Services. Further informationEditorial Office. Once the online version of the published article for which permission is being requested is

can be obtained via RightsLink, a service of the Copyright Clearance Center, not theCirculation Researchin Requests for permissions to reproduce figures, tables, or portions of articles originally publishedPermissions:


Dow

nloaded from

http://circres.ahajournals.org/content/67/4/993

http://www.ahajournals.org/site/rights/

http://www.lww.com/reprints

http://circres.ahajournals.org//subscriptions/


Documents

Dual Action ofFRC8653, Novel Derivative, Ba2` Recorded …circres.ahajournals.org/content/circresaha/67/4/993.full.pdf · 994 Circulation Research Vol67, No4, October 1990 ... evoked