31Hiroshima J . Med. Sci.
Vo1.45, No.1, 31 - 35, March, 1996
HIJM 45-5

Effect of N-Feruloyl Tyramine (an Analogue of Tyramine)on Inwardly Rectifying Potassium Channel In
Frog Ventricular Myocytes
Makoto MUNEMORIll, Kaoru YAMAOKAll*, Irawan YUSUFll** , Kotaro SUMIIlI,
Hideaki OTSUKA 21 and Kazuo YAMASAKI2 l1) Department of Physiology, Hiroshima University School of Medicinc
2) Institute of Pharmaceutical Science, Hiroshima Uniuersity School of Medicinc. Hiroshima . Japan

ABSTRACTElectrophysiological effects of N-feruloyl tyramine (NFT), an analogue of tyramine, on potassium currents in frog ventricular myocytes were examined using single-chan ne l recording andwhole-cell voltage clamp technique. Extracellular application of NFT induc d a concentrationdependent decrease of macroscopic inward rectifier potassium current (iKl) with ID50 of 19,11M, while tyramine (100 JiM) was ineffective in producing an inhibitory effect on iK1 . NFT
reduced the mean open time of iK1 to 1.3 ms from 3.1 ms in control without affecting theamplitude of single-channel conductance. It is indicated that boi containing NFT produces aprolongation of the plateau phase caused by the suppression of inwardly rectifying K channel.
Thus, this prolongation may induce an increase in the inflow of Ca ions, which in turn leads
to a positive inotropic effect.
Key words: NFT, ~Kl, Suppression
It has been reported that a Chinese folklore
medicine, boi , containing extracts from Sinomenium acutwn, suppresses the maximum rate ofrise of action potential and prolongs its durationin the frog heart, and that consequently the contraction force is increased by 50% of the contro1 4 ).
We previously showed that the main extracts ofboi, N-feruloyl tyramine (NFT) and tyramine, exert inhibitory action on sodium (Na) channels
without affecting calcium (Ca) channels by
suppressing the maximum conductance and also
shifting the steady state inactivation curve in thehyperpolarizing direction 10). Since no electrophysiological findings obtained so far explain the positive inotropic effect of boi 41, we have examinedthe effect of NFT and tyramine on the potassi um(K) channels in single ventricular frog myocytes.

MATERIALS AND METHODS
Cell preparationThe method for the isolation of single ventricular cells from the frog (Rana catesbeiana) was essentially the same as that in a previous
experiment (Seyama & Yamaoka, 1988). Briefly, aheart was mounted on a Langendorff apparatus
and retrogradely perfused via the aorta with aCa 2+ free solution containing collagenase (0.1 mg/ml; Yakult, Tokyo, Japan) and trypsin (0.06 mg/ml; type I, Sigma Chemical, St. Louis, MO, USA)for 20 min at 32°C. Then the isolated ventricle
was cut into pieces and dispersed by pipetteagitation. The single cells were kept in a solution
containing a low Ca 2+ concentration (200 JiM) for
30 min and then centrifuged for 1 min at 93 x
g and stored in Leibovitz's L-15 medium (GIBCO,Grand Island, NY, USA) for experimental use .

Solutions and chemicals
The compositions of the solutions are given In
Table 1. Whole-cell currents other than K currents were eliminated by the use of an externalsolution containing 1 JiM tetrodotoxin (TTX)(Sankyo Co. , Ltd. Tokyo, Japan) and 3.0 mM
MgS04 without adding CaC12 (external-I).
Single-channel currents were recorded with external-2 and internal-2 soluti ons.
Tyramine (4-hydroxyphenylethylamin) was purchased from Sigma Chemical Co. NFT was syn"'All correspondence should be add res cd to Dr. K. Y&\1.AOKA.
Mailing address: Dr. K. YAMAOKA
Department of Physiology, Hiroshima University School of Medicine. Hiros hima 734 Japan''''" Present addre s: Dr. Irawan YUSUF
Hasanuddin University, Faculty of Medicine, Department of Phy iology Kampu. Tamalanrea ,JL . Perintis Kemerdekaan jung Pandang-Indonesia

32M. Munemori et al

Table 1.

NaCI

external-1

internal-1

ll3.5

10.0

K:lATP

external-2internal-2 *MJW

5.0

MgATPTa2-creati ne phospha tc
KCIK-aspal'tate

30.0

110.0

113.5

l.0
3.0

glucos('5.05.0

HEPES

10.0

10.0

10.0

10.0

7.0

7.2

7.0

"' Free Ca:l+ conccntrations ofinternal-2 were calculated to be l.0 x 10-8 M, usin g Schoenmakers' program 7)

th sized from tyramine and feruloic acid. Briefly,
O-acetylfcruloic acid was condensed with tyramine to give O-acetylferuloyl tyramine. NFT wasobtained by a lkalin e hydrolysis of O-acetylferuloyl tyramine and purified by crystallization. Thech mical ~tructurcs of the reagent~ are shown inFig. 1. tock so lution of tyramine (100 mM ) andNFT (] 00 mM) were prepared in dimehtylsulfoxide (DMSOl. All the test solution were freshly
prepared before each experim ent. All experiments
were conduct d at room temperature (23-26°C)
unl ss otherwise stated.

Electrophysiological recording and analysis
The experim nts were carried out in whole-celland in s idp-oul configurations u ing the conventional patch-clamp lechnique al. The records ob-'[) l'<1ll1in('~C;II 'III JV

Fig. 1. Structure of 1 -feruloyl tyramine and tyramine ,~ 0.5

Sb(8)

~0 .0
10 8

10.0

7.2(10)e50 ms

10.0pH•(14)

5.0

5.00.001

(4)0]

0.62

EGTATTX• NTF 100 ~ M

•

I-2

100.0

C'aC1 2MgSO,

J

3.0
5.4

o control• NTF 100 ~ M

InAI

3.0

~----------InA)

contro,Single-channel current experiment

1.0B

A

Composition of the solutions (in mM)
Whole-cell experiment33

NFT and Inwardly Rectifying Potassium Channel

tained by a patch-clamp amplifier (Axopatch
200A, Axon Instruments, Inc" Foster City, CA,USA) were stored on tapes using a DAT tape-recorder (DTC-1000 ES, SONY, Tokyo, Japan), Data
were filtered through a low-pass filter ofAxopatch 200A, having a -3 dB cut-off frequency of5 kHz, Data were recorded by DAT sampled at 5
kHz and were filtered with 1 kHz for analysis.
Data were expressed as mean ± standard error(S,E.). The statistical significance between groups
was determined by the Mann-Whitney test,
p<0.05 being considered signifcant.

RESULTS
Suppression of macroscopic inward-rectifierpotassium current (iKl) by external application of NFT
K channels in frog ventricular myocytes consist
mainly of inwardly rectifying K channels andATP-sensitive K (KATP) channels, Since weemployed an ATP-rich internal solution for thewhole-cell current recording, K current observedin this configuration should be mainly iKl' Onapplying 175 ms pulses of variou amplitudes
from a holding potential of -65 m V, the resultant
current-voltage (IN) curve showed a characteristic N-shaped configuration indicating that thecurrents recorded in this condition were mainly
carried by K ion through inwardly rectifying K
channels 6 ,g l (Fig. 2), NFT (100 JIM) in the external olution significantly suppressed iKl at membrane potential negative to 0 m V However, 100,liM tyramine applied externally did not suppress
iKl in any of the three cell tested.

Fig. 2. A. Record s of iKJ in the pre enc and absence of NFT (100 11M!.
IKIS were evoked by hyperpolarizing test pulses to-110, - 40 and 0 mV from a holding potential of -65
mV. B. Current-voltage (IN) relationship for iKl ·Amplitudes of th e steady state iKl current at theend of 175 ms test pulses are plotted against themembrane potentia1.

NFT (IJM)

010~-S::<:100

washout

10 7

10 6

10 5-,-T

10 4

10 3

NFT (M)

Fig. 4. Do_ -responsc curve for NFT.
All data in NFT w re refclTcd to those in controland we)' plotted aga in st t h e con entl'ation of FT
The lin c was fitted by th cquation of' lI( 1+([NFT[/ID 5 0)1l), wh 1'e lD5() incli at s half inhibitiondose of NFT [01' the channcl and 11, Hill's cocf'ficienL. Fitting thc equation to thc data gave [I)S() ofl.98 x 10 M and n of 0.26. Vertical bars indicatc
mean ± standard error (S,b,). Numb'r 0(' cells 'xamined is shown in parentheses.

(nA)

or-0.5'0C

pronounced at 100 ,11M NFT. The dose-response
curve is shown in Fig. 4. 1D50 wa estimated tobe 198 ,uM with a Hill' coefficient of 0,26.

-1~:::l0'0~- 1.0a~

E-1.5

I05

1015202530Time (min)Fig. 3. Time course for the effect of TFT on steadystate iKl 'NFT (1, 10 and 100 I"M) was consecutively appliedexternally. Test pulses to -llO mV from a holding
potential of -65 m V were applied every 30 s. Amplitudes of iKl at the end of 175 ms pulse (measuredas a difference b tween t h e holding current andiKIJ were plotted again t time.

Dose-response relationship for NFT action
The dose-response relationship wa studied bymeasuring the macroscopic iKl during a 175 ms
test pulse to -110 m V from a holding potential of-65 m V The test pulses were applied every 30 s,
As shown in Fig, 3, iKl was slightly decreased at10 ,aM NFT and the suppression became more

Effect of NFT on the single-channel activities
of inwardly rectifying K channeZ,c;.
To determine the mechanism underlying thesuppression of iKl, we recorded si ngle-channel
currents for inward ly rectifying K channels infrog ventricular myocytes. Since frog card iac myocytes have been reported to contain 2- 3 mM
ATPll, KATP channels are thought to remain inert
in a physiological condition, Therefore, the singlechannel currents obtained in the following experiments were considered to be iKl' The
extrapolated IN curves for iKl with and without
NFT were made from the collected data of s veral
individual patches (Fig, 5), Since thline
adopted to data obtain d with 110 mM IK+ 10 and
113.5 mM IK+ I; cross the abscissa at 0 m V, regardless of the presence of NFT, the inwardly recti(ying K channel is highly selective to K ion andits ionic selectivity is not affected by NFT The
conductance was e timated to be 3 .9 pS and38.5 pS with and without NFT, reo pectively, Combined open time histograms were made fromthree patches, The bin width was et to 0.4 m .
The mean open time were shortened from 3.1 ms
to 1.3 ms by application of NFT (Fig. 6 ). Amplitude histograms were compiled from the record

..

34- .. . . _-.. _.

,. . ... . . ,..

NFT and Inwardly Rectifyi.ng Pota ium Ch annel

M. Munemori et al2Control

100iJM NFT

Control100(p A)20mVa 20100 80 60(mV)~ IM... ' -'"CQJ>
wQ;.0~z-60mV-3~VI~

60'0-40mV )Yr~ ~.,...

-280-_40
20~3PA
300ms-4

NFT lOOI1M:100Fig. 5. The IN relation for single-channel curre ntof i foe}.
A. Dc La in the absenc ( ) and presence (e l ofF'1' gave almost id entical linear conductances(38.9 pS and :38.5 pS wiLh and without NFT, respecLively) when fiLLed by straight lin es. DaLa were obLained from inside-ouL patch . of three cell s. Vertica l bars ind1caLe m an ± S.E. B. These figures indicaLe representa Live records for each condition atdifferent membrane potential s.

250250~c- ~~~~~ 2PA200Q)

>

W150

<lOms

"6200150

2 100100

E::JZ

50

50

t= 1.3ms

00010Time (ms)15205101580Q)

>

w

60

'0Q;
.0

E

40

::J

Z

200-2-1o23(pA)

Fig. 7. Amplitude histograms for iKl with andwithout FT.
Amplitude hi stogr am s were constructed from theoriginal record sh own in the inset of Fig. 6 withthe bin width set at 0.1 pA. Gaussian curves werefitted to these histograms and unitary conductanceswere determi ned to be l.51 pA and 1.45 pA withand without NFT, r espectively. Membrane potentials were -40 mY.

NFT 100l1M

Control'"C20

Time (ms)Fig. 6. Dw'lI tim histogram and si.ngle-channel
records fil!' il<l in th presence and abse nce of NFT.
The paLch membrane potential wa - 40 mY. A sampi ' of Lh· single-channel currents is shown in Lhein set. Open time hi sLograms were fitted by a sin gle
exponential viith time constanLs of 3.1 m (control)and 1.:3 ms ( FT), respectively.

hown in Fig. 6 (Fig. 7). The single-ch annel amplitud was 1. 4 ± 0.24 pA (n = 3) in the control
oluti on and 1.72 ± 0.2 pA (n = 3) in the pre nc of NFT, i.. , th conductance wa not affected b, NFT.

DISCUSSION
The prc cnt stud, ha unveiled everal charact ri. tics of thc acb n of NFT on inwardly rectifying K channel in frog ventricular myocyte .
First, NFT uppr s d inwardly rectifying K
channels at membrane potential n gative to 0
mV when applied ' tra ellularl,. econd, NFT

shortened the mean open time for inwardly rectifying K channels from 3.1 ms in control to 1.3 ms
in the presence of 100 pM NFT without affecting
the single-channel conductance. Third, ID50 was
estimated to be 198 ,uM. Fourth, 100 .uM tyramine had no effect on iKl'Hirose et al 4 ) have reported that the main
pharmacological actions of boi containing NFT
are a decrease in the maximum rate of rise ofaction potential, an increase in the duration ofaction potential, and positive inotropic action.
Since the generation of action potential mainly
depends on the excess i.nflow of Na ions to depolarize the membrane to the equilibrium potential
for Na ion 2 ,5), partial suppression of the inflow of
Na ion by boi does not affect the amplitude ofaction potential but reduces the maximum rate ofrise of action potential. It is well known that theformation of the plateau phase in cardiac cells isdue to the inflow of Ca ions through Ca channels
and the limited outflow of K ions through inwardly rectifying K channels in the low value ofthe membrane conductance 2 ). Since K current
pa ~ing through inwardly rectifying K channels
i respon ible for the repolarization of action potential, the uppression of this K channel by NFT

should prolong the plateau phase of action potential and, consequently, the influx of Ca ion may
increase, resulting in the enhancement of contraction; the positive inotropic effect. The findingin our previous studylO ) that main pharmacological action of tyramine and NFT is the suppression of Na channels without affecting calcium
channels accords well with the notion discussedabove.
Since tyramine contains two hydrophilic groups
per eight carbon atoms and NFT three per seventeen (see Fig. 1), it is reasonable to assume thattyramine is more hydrophilic than NFT. In
comparing their inhibitory action on N a channel ,NFT has been reported to be more potent thantyramine lO ). When the results obtained in thisstudy are reconciled with those on sodium channel, a hydrophobic character seems to be criticalto pharmacological action on channels. Thus, one
can assume that the binding site in K channels
accessible from the external surface may be in amore hydrophobic environment. Another noticeable finding is that Hill's coefficient = 0.26. It issuggested that receptors for NFT in a hydrophobic environment retain a strong negative cooperativity: several receptors around the binding site
being subjected to a suppressive influence.

ACKNOWLEDGMENTS
We would like to express our sincere thanks toProf. 1. Seyama for his guidance and for reviewing the manuscript. This work was supported bya Grant-in-Aid for Scientific Research on Priority
Areas from the Japanese Ministry of Education,
Science and Culture (Grant number 02257101 ,03253101 and 04248101 ), and the Uehara Memorial Foundation.
(Received J anuary 12, 1996)(Accepted February 1, 1996)

35

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