The potassium channel opener cromakalim (BRL 34915) activates ATP-dependent K+ channels in isolated cardiac myocytes

Vol.

154,

No.

July

29,

1988

2, 1988

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

THE POTASSIUM CHANNEL OPENER CROM+KALIM (BRL ACTIVATES ATP-DEPENDENT K+ CHANNELS IN ISOLATED CARDIAC MYOCYTES Denis

Escande*,

Dominique

Thuringer, Icilio

Laboratory

Sylvain

620-625

34915)

Leguern

and

Cavero

of Cellular Electrophysiology, BP158, 92231 Gennevilliers

Phone-Poulenc Cedex, France

Sante,

Received June 9, 1988 SUMMARY. In cardiac myocytes, cromakalim (BRL 34915), a potassium channel opener, activates a time-independent K+ current exhibiting poor voltage-sensitivity. This effect of cromakalim is antagonized by low concentrations of glibenclamide, a specific blocker of ATP-dependent K+ channels in cardiac cells. Direct recording of the activity of K+ channels in inside-out membrane patches, confirmed that cromakalim is a potent activator of ATP-dependent 191988Academic Press,Inc. K+ channels in cardiac myocytes.

Potassium

INTRODUCTION.

pinacidil

are

a novel

and antihypertensive mediated

via

present

unknown

selectivity channel

for openers

markedly paper

the

deals

cells

by

using

both

with

one

of

class

of

the the

of

also

in

action

patch-clamp

technique

*To whom all

correspondence

0 1988 by Academic Press. Inc. of reproduction in any form reserved.

whose identity a certain cells

striated

be

is

at

degree

(2),

myocytes

to

of

potassium since

they

The present

of the K+ channel

activated

in cardiac

channel inside-out

in

isolated

should

be sent.

0006-291X188 $1.50 Copyright All rights

vasodilatory

had been proposed

muscle

or

(5-7).

and (8)

cromakalim

potential

potassium

whole-cell

that

as

produce

exhibiting

active

nature

which

smooth

cardiac

such

a K+ channel

Although

a variety

the

drugs

(1,2)

activation (l-4).

openers

of

effects

are

shorten

channel

620

openers,

cromakalim.

configurations cardiac

myocytes,

of

By the we

Vol.

154,

No.

2, 1988

demonstrate

BIOCHEMICAL

that

ATP-dependent

cromakalim

K+ channels

AND

BIOPHYSICAL

is

a

first

RESEARCH

potent

described

COMMUNICATIONS

activator in

the

heart

of

the

by

Noma

(9). MATERIALS AND METHODS. Cell isolation. The technique used for isolating guinea-pig ventricular myocytes was a derivative of that described by Mitra and Morad (lo), using a Langendorff column at 37'C for coronary perfusion and both collagenase (type I; Sigma Chemical, St. Louis, MO, USA; 2 mg/ml) and protease (type XIV; Sigma; 0.28 mg/ml) for enzymatic dispersion. Isolated cells were stored until used at room temperature in a high Kf low Cl- storage medium (11) (composition in mM: taurine 10, glutamic acid 70, KC1 25, KH2P04 10, glucose 22, EGTA 0.5, pH 7.4 with KOH). Whole-cell experiments. In the whole-cell configuration (8), membrane currents were recorded by means of a L/M-EPC7 List amplifier. Isolated cells were continuously perfused with an extracellular solution prewarmed at 33-35'C (composition in mM: NaCl 135, KC1 5.4, MgC12 1.0, CaC12 1.8, NaH2P04 0.33, HEPES buffer 10, pH adjusted with NaOH to 7.3). L-type Ca+' channels 10, glucose and Nat channels were blocked by adding 3 /.lM nitrendipine (Bayer) or 3 mM CoCl (Sigma) and 50 PM tetrodotoxin (Sigma) to the extracellular medium. Patch pipettes (2-4 Mfi) were filled with an intracellular medium (composition in mM: K-aspartate 85, KC1 50, Na-pyruvate 5, MgClz 1, EGTA 10, HEPES buffer 10, Mg-ATP 3, D-glucose 11, pH 7.3 with KOH) . Cromakalim (synthetized by RhGne-Poulenc Ltd., Dagenham, UK) and glibenclamide (Sigma) were dissolved as stock solutions in ethanol or in dimethylsulfoxyde. Control experiments were conducted out to ensure that the vehicle recorded in guinea-pig myocytes. did not affect the K+ currents Drugs were applied in close vicinity of the chosen cell by means of a micr'opressure ejection system (Medical System). On-line data acquisition was performed using an IBM-AT computer expanded with a TECMAR TM 40 Labmaster interface. Further analysis was achieved using pCLAMP software from Axon Instrument. Single channel recordings. Single channel current recordings were conducted at room temperature in the inside-out configuration The bath solution (intracellular solution) had the following (8). composition (mM): KC1 127, HEPES 10, KOH 13, EGTA 5, glucose 11, pH 7.2, whereas the pipette medium (extracellular solution) contained (mM): KC1 140, CaC12 2, MgC12 1, HEPES 10, glucose 11, pH 7.2 with NaOH. Data were displayed on a Nicolet 3091 digital oscilloscope. They were stored on a digital video-recording system and analyzed off-line through a 8-pole Bessel low pass (Sony) filter (Frequency Devices Inc. 902LPF) at 100-500 Hz.

RESULTS of

AND

DISCUSSION.

croma-kalim

current ramps current voltage

(3-300

measured (4.7

either

mV/s)

measured steps

from at

the

elicited

Whole-cell PM) as

experiments.

were

determined

the

-80

to

end

of

from

current +60

on

response

mV

(Fig.

40 621

mV

the to

(Fig.lB).

or

effects

background slow

1A & 1B)

1 s depolarizing -

The

voltage or

as

hyperpolarizing At

potentials

the

Vol.

154,

No.

BIOCHEMICAL

2, 1988

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

1. A: effects of cromakalim (300 FM) on the background current (upper trace) induced by 30 s voltage ramps from -80 to +60 mV (lower trace). Cromakalim was applied during the indicated period. B: current-voltage relationship of the steady-state current recorded in control and in the presence of cromakalim (300 values measured at the end of 1 s jN . Symbols are current voltage steps elicited from -40 mV to the indicated voltage. Curves are current responses to 140 mV depolarizing ramps from -80 mV. Same cell as in A.

Figure

positive

to

outward

shift

about

of

Osterrieder another

are

30 or

sensitivity

conditions).

This

was mainly

carried

sulfonylurea,

is

pancreatic pancreatic state

under

cells

they

and

8.8

z!z 1.9

been

observed At

with

- 78.5

It

current

by

pinacidil current

2.2

& 0.8

exhibited

& 0.6

mV under

the

reported

+ 60 mV, the

(n=7).

and

(Fig.lB).

was respectively nA

at

relationship

already

opener.

that

poor

mV (theoretical

our

experimental

induced

by cromakalim

by Kt ions. been

shown

a potent (13) K+-ATP

glibenclamide,

in

glycolysis

an antidiabetic

of ATP-dependent

and

in

cardiac

channels

are

conditions

normally

would

that

blocker

physiological

which

a dose-related

rectification

those

-85.3

confirms

8 cells,

are

have

to

channel

or

I3 cells

especially

substance

which

potential:

has

going

and re-versed

K+ equilibrium

It

inward

300 PM cromakalim

n=6)

(mean & SEM;

induced

current-voltage

comparable

potassium

by

voltage

the

cromakalim

(6),

activated nA

steady-state

abolished

These effects

cromakalim

80 mv,

in the

reversibly

(121,

-

a closed are

specifically 622

myocytes

thought (151,

to

unless

impaired

(16). Kt-ATP

(14).

be in

whereas

state

affect

K+ channels

in

in In

an open cardiac

ATP production Therefore, channels

a is

Vol.

154,

No.

expected

2, 1988

to

myocytes.

have In

specific background

current were

to

avoid

proceed dialysis

with

the

delayed

nor

rectifier

iK

of

K+-ATP

al.

(7)

the

normal

bases

illustrated elicited

by

guinea-pig 0.3 at

current

was

10 different

the

support

a specific

inhibitor

suggested

by Mestre does

papillary

by

not

et

modify

muscles.

As

reduced

PM. A complete

produced

the

delayed

further

PM glibenclamide 300

on the

neither of

glibenclamide

slow

the

blockade

of

3 PM glibenclamide

2B).

Single

channel

K+-ATP

At

3A).

recordings.

channels

conductance

recorded

in

be

inner

side

In

easily

and their

a holding

potential

as upward

deflections

single Fig.

ATP-free

could

were

(9,18)

outward

the

already

may

due to

modified

is

in

that

no effect

observations

that

cromakalim

cromakalim-induced

shown

as

& 2B,

pipette

In

curve

the

These

current

17).

be

on

the

exerted

glibenclamide

of

2A

Fig.

ref.

FM)

These

results

in

K+-ATP

activation

heart,

effects K+ current.

glibenclamide

potential

in

current

of

action

the

cardiac would

its

rectifying

(see

(0.03-3

the

of

ATP depletion

medium

that

COMMUNICATIONS

glibenclamide

intracellular

evidence

on the

current

of

(6 experiments).

in

background

3 mM Mg-ATP

steady-state

channels

RESEARCH

explored

activation

Moreover,

the

available

(Fig.

with

pipette

current.

amplitude

in

and on the

glibenclamide

background

to

we

conducted

BIOPHYSICAL

whether

channels,

a progressive

experiments,

the

check

possible

from

AND

on the

to

Kt-ATP

experiments

the

no effect

order

for

order

BIOCHEMICAL

channel

3B,

at

medium detected

10 different

of the

identified high of

membrane

membrane

the

(Fig.3A

11

of

presence

300

Comparable

patches. 623

& 3B).

channel

elementary

3 mM Mg-ATP

were

corresponding In

were

the

example

recorded

opening

PM cromakalim results

(Fig.

channels

trace

channels

additive

patches,

large

Ki-ATP

current

2 distinct

patch.

their to

+ 50 mV,

of

whereas the

by

membrane

sensitivity

currents

least

in

inside-out

were

in levels

at

the

obtained

Vol.

154,

No.

2, 1988

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

7 - (nA)

B

2

A

cmk

cmk gbl 0.3

cmk

Cl

-60

pM

-60

1

-40

-20

-11

0

20

40 " Cm")

Figure 2. A: inhibition by glibenclamide (gbl: 0.3 FM) of the outward current induced by cromakalim (cmk: 300 FM). Upper traces are current responses to 140 mV depolarizing voltage ramps. Lower trace is the voltage. Holding potential was - 80 mV. B: current voltage relationships of the steady-state current recorded in control conditions in the presence of cromakalim (300 NM) (11, of cromakalim (300 PM) plus 0.3 PM (3) or (2), or in the presence 3 J1M qlibenclamide (4). Voltage ramps (30 s in duration) were elicited from - 80 mV. Same cell throughout.

Our

channels (i) the

show that

results

in

in rat

cardiac aortic

relaxation

stimulated

cromakalim cells.

rings induced

by

cromakalim

It

is

a potent

has been

or portal

veins,

by cromakalim (19,20)

activator

reported

elsewhere

glibenclamide

and inhibits

and that;

of

the

(i i)

in

K+-ATP that:

antagonizes 86Rb+ efflux anesthetized

2

3 ::

Mg-ATP

cromakalim

Figure 3. A: inhibition by ATP (3 mM) of the ATP-modulated K+ channels recorded in an inside-out membrane patch. C indicates the closed state of the channel whereas 1 and 2 indicate numbers of simultaneous channel openings. Membrane potential was + 50 mV. B: activation by cromakalim (300 PM) of at least 11 K+-ATP channels in the same membrane patch as in A. Holding potential + 50 mV. Traces were filtered at 500 Hz. 624

60

Vol.

154,

No.

2, 1988

normotensive

rats,

hypotension vascular

smooth

as

assessed.

muscle

cells acts

demonstrated

herein the

blocked

activated

by

pharmacological

ATP-sensitive

the

specifically

are

channels common

However,

However,

cromakalim

AND

glibenclamide

(21).

cromakalim cells

BIOCHEMICAL

the

existence

of

never

at

K+-ATP

in

RESEARCH

blocks

has

fact by

BIOPHYSICAL

been

that

the

in

properties

K+-ATP

channels

demonstrated. in

myocytes

suggests smooth with

smooth

muscle to

effects that

muscle the

in

Whether

remains

vascular

glibenclamide

cromakalim

cromakalim-induced

channels

cardiac

COMMUNICATIONS

be of

the

shares

K+ some

myocardial

K+ channels.

ACKNOWLEDGEMENTS. We are grateful to D. Girdlestone for help with the manuscript. The expert technical assistance Courteix and M. Laville is also acknowledged.

her

kind of J.

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