BIOCHEMICAL

Vol. 182, No. 3, 1992

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1429-1434

February 14, 1992

CALCIUM-ACTIVATED

POTASSIUM

CHANNELS

IN

CHONDROCYTES M.Grandolfo*, *Din

P. D’Andrea*.

Biochimica, OIst.

Received

di

M. Martina*,

Biofisica

e Chimica

Fisiologia,

December

Via

29,

F. Ruzzier’,

delle

A.Fleming Trieste,

and

Macromolecole,

22, Universitl Italy

F. Vittur*

Via di

A.Valerio

Trieste,

22,

34127

1991

The presence of calcium-activated potassium channels in chondrocytes of growResults obtained with furaon cultured resting ing cartilage was tested. chondrocytes indicate that he cells respond to an elevation of extracellular calcium concentration ( [Ca t+ 1 1 f om 0.1 to 2 mM increasing the intracellu1 f rom 117 to 187 nM. This lar concentration of the ion Y [Cah+I. increment may be blocked by 3pM La3+. P tch clan& experiments in cell-attached configuration showed that, when [Ca 1+ Ii rises, the open probability (PO) of the K+ channels increases. Increments in both PO and channels can be obtained after applying 2.5pM A23187 with 2mM the results demonstrate that, in chondrocytes,a channels is present and their activity is related to an B 1992

The

Academic

Press,

membrane

cytes

Inc.

potential

of growth

plate

approaching

the

tion

membrane

of the

other

pump,

the

tive

types

K+ channels.

channels

that

are

frequently

Ca2+ -activated free

voltage

was

sensitive

modulated Kt-channels

Ca2+ concentration

it

that

system

(3)

by calcium in and the

chondrocytes. K+-channel

a key

least

in

interest

the

regula-

to study

(>

200

activity

kinds

and the that pS) Since

aim of this The

include

three

demonstrated

the

chondro-

differentiation

role

systems

activated

and TEA.

(21,

cell

in

correlation

the

Na*-Kt

of

conduc-

volume

sensitive

chondrocytes

from

voltage

dependent

channels

of this

work

was to between

K+ kind

look

for

cytosolic

was investigated. 0006-291x/92

1429

the

membrane.

and at

conductance

to quinidine

of

of

these

the Ca2*

activity

stage

therefore

plasma

et al.

high

the

Kt plays

is

found

sated,

Grandolfo have

are

(1).

and

it

potassium

with

of chondrocyte

(2)

Recently

cartilage

surface

co-transport

the

resting

change

potential

Nat-K+-2Cl-

channels:

intracellular

cartilage

systems

cell

the

mineralizing

K+ transporting In

and

$1.50

Copyright 0 1992 by Academic Press, Inc. All righfs of reproduction in any form reserved.

Vol.

182,

No.

Lsolation

3, 1992

md

BIOCHEMICAL

culture

ti

AND

PreoS8eOUg

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

chondrocvtes

Scapulas from 40 to 70 kg pigs were excised from animals imnediately after death. The scapulas were freed of perichondrium and the cartilagineous portion was cleaved off. The resting region, furthest from the mineralizing surface, was selected and dissected as already described (4). The enzymatic disaggregation of the tissue and the chondrocyte plating and culture conditions have been previously reported (5). Furameasurements & jCa’+li These measurements were carried out as described by D’Andrea et al. (5). Briefly, 48 hrs serum starved confluent cultures were treated by trypsin (0.05X)-EDTA (0.02%) solution. The cell suspension in DMEM was incubated with 5 pM fura-2-acetoxy methylester (Molecular Probes, USA) for 1 h at 37’C. The cells where then rinsed with buffer (in mM: 140 NaCl, 5 KCl. 1.5 M&12, 5 glucose, 10 Hepes (Sigma), 0.1% bovine serum albumin, fraction V (Sigma); pH 7.4 with Tris), and resuspended at a concentration of IO6 cells/ml. When neces ary, cells where washed twice in the above buffer solution to which 0.1 IIS! CaS+ was added. To obtain 2 r&l or higher calcium concentrations, a few microliters of stock CaCl2 solution were added. Fluorescence was measured under continuous stirring in a Jasco spectrofluorometer. Signal calibration was performed as described by Meldolesi et al. (6). Patch-clrecordinConfluent cells were employed for the electrophysiological experiments which were performed as described by Grandolfo et al. (3). The cells were rinsed and then maintained for 1 h in the following solution (in mM): 130 NaCl, 3 KCl, 0.1 CaC12, 10 Na-Hepes: pH 7.2. Pipettes were filled with the same solution as above. Single channel currents were recorded in the cell-attached and the outward current, i.e. current flowing from the cytoconfiguration, plasmic to the external side of the membrane, are shown as positive deflections. The electrophysiological experiments were done at room temperature (20-25 ‘C), whereas the fluorescence was measured at 37 OC.

RESULTS To evaluate maintained

the in

by using

role

of

bathing

solutions

fluorescence

Fura-

records

Fig.

[Ca’+li

in response

to

logical

saline of

at

low

content

Ca 2+ concent

resting

[Ca2+Io.

The cells,

further

( widely (Pig. in

1 B). the

. The

used Ca 2* channel Moreover, fluorescence

when

entry blockers

the

signal

were

studied

regulate

their

is

of not

cells

are

are

observed

1430

from [Ca2+Io

blocked

) but

is

in

mM) respond

[Ca”Ii

increments

Ca2+

rat ion

maintained

= 0.1

[Ca2+lo up to 1.8 - 2mM, by increasing

[Ca’+li

chondrocytes

chondrocytes

([Ca2+Io

Additional

K+-channels,

of

methods.

that

calcium

alter

no changes

different

of

not

La3+

of

Change83

to 187 f 23 nM (n = 37).

3vM

the

1 A show

20)

verapamil

activity

on

and electrophysiological

in

crease

ICa’+Ii

(data

to

an

in-

117 f 17 nM (n = up to 15 a&l does by nifedipine

completely

depolarized

a physio-

by not

inhibited

and by

KC1 (40 mM), shown).

These

Vol. 182, No. 3, 1992

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

200 z c

100 I

.-

1.8mM

[Ca2’lo

200

7

N

%iI

100 I

Pig. 1 Influence

of tCa**l on ICa**l. and blockage A) The cells we eOloaded witch fur-a-z, and [Ca*+l The [Ca I+ 1 was then raised to the was esffmated from the fluorescence ion (B) The cells maintained at O.ldl [Ca calcium entry was observed when

findings

suggest

at

Ca 24 channels

dependent The activity studied

that,

of

using

ty occurs

A 0.1

is

(Pig.

mM

are

K+ channels the

When [Ca2+lo

least

patch

raised 2A).

under

our

of

calcium

entry

by

La3’.

initially exposed (A) to O.lmM indicated values and the [Ca2’li (see Materials and methods). were treated by 3 pM La3*: no

experimental

conditions,

voltage-

undetectable. in

resting

clamp from

B

I

--

technique

0.1

Likewise,

chondrocytes

to 2mM,

in

tCa2+lo,

different

cell-attached

increment

an

at higher

the

at

open

was

configuration.

of the the

[Ca2*lo

K* channel

activi-

probability,

PO, as

B

[Ca2*],

2 mM[Ca2+lo

10pA Vpipz

Vpip

-3OmV 20

hnV)

msec

pis.s

Single cytes.

channel

currents

across

voltage-activated

K*

channels

in

chondro-

(A) Representative currents recorded in cell-attached configuration at a -3OmV pipette pqt+ential (Vpip) when exposing the cell to 0.1 mM (upper trace) or 2mM tCa lo (lower trace). (B) Voltage dependence of channel activity expressed as open probability (PO) at (0-e) O.lmM or (A-A) 21d4 tCa2+lo. 1431

Vol.

182, No. 3, 1992

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

600,

[Ca2fo=

2 ti~M

A

400,

[Ca2+]

i

nM

d+

200,

A23187

1 min.

I

a

u Cytosolic treated

with

[Ca 2+ 1 chanses A23187.

and

single-channel

K+

currents

in

chondrocytes

Chondrocytes were exposed to 2mM [Ca2’Io and A23187 wa added to a concentration of 2.5nM (see arrow). (A) A typical trace of [Ca82+Ii variation by furaafter ionophore addition and as a function of time, and determined (Bl Single-channel K+ currents measured at a Vpip of -40mV at the basal (a), transient fbl and final steady-state (cl levels of [Ca”It.

a function resting

of

Vpip

membrane

mM [Ca2+lo

(pipette

potential

(Po=O)

when

extracellular of the

increase

of

reached

the

K+ channels creased an increase

of

A23187

terms

3 8).

In

the

unitary

in

the

presence

600 nM (Fig.

3 AI.

fact,

PO and

unitary

at Vpip=O current

of

pS,

of

of about

1432

in the

15%. The

the

at

0.1

at

0.1

2 mM

or

respectively).

2 mM [Ca2*lo, 350

from

At

mean slope

of

nM,

amplitude

the PO changes

observed

presence

n=4.

2B).

observed

As a consequence, current

about

is

observed

in the

112*17

a plateau

of

are

and

180 nM to

is

activity

incubated

n=4,

(Figure

activity

evident

are

pS,

from

of almost

( in (Pip.

but

differences

chondrocytes (96?35

increases

= 0) no channel

a modest

ionophore

[Ca2+li

a peak

voltage)

No significant

Ca 2*

addition

(Vpip

while

[Ca*+ IO (Po=O.OOlI. conductance

applied

The

induces after

an

having

the

activity

1 is

highly

in-

to 0.02

with

is

evi-

0.001

phenomenon

2 mM

more

of

Vol.

182, No. 3, 1992

dent

BIOCHEMICAL

at a Vpip=-4OmV

current

increases

when the

AND BIOPHYSICAL

PO changes

from

RESEARCH COMMUNICATIONS

0.85

to

0.3

and

the

unitary

of 80%.

QISCUSSION The

aim of

channel

experiments

activity.

amount Ca2+

our

Two facts

of extracellular Kt

The

variation

physiological of Ca ”

zation

from

[Ca*+ Ii

the

um channels of

is

in

experimental

the

rise,

cells

rapidly

of K+- channels allows

and

(see

Fig.

were

obtained

fura-

in

iB1,

depletion

lar

space.

and the

to

as

already

when

using

shows of

A clear

different

that

clamp

time

are

[Ca’+Ii

for

other

ionophore

A23187

evokes

stores was observed levels

instead

(see

of

a marked and to

between Fig. 1433

2B).

not

presence

of

(8.9)

or

influx

the

activity

cells,

of

these

of

Similar

of from of

chan-

the

level

the

may

activity

which

the

changing

the

observed

on the

for

and

a [Ca2+lo

the

(11-131.

transient

voltage cells

excitable

to

types

blocker

were

conductance

of calci-

active”

channels”

cell

of the

following

effect

related

within

through

these

“tonically

The

strictly

in

in

of

elevation

are

However,

has a significant

recordings.

[Ca2+Io

changes

found

of

changes

( an aspecific

of Ca 2+,

as “maxi-K+

shown an

(10)

the

a class

sustained

ICa’+Ii

the

that

of Ca*+ mobili-

channels

verapamil.

K*-

as a consequence

detectable

The entry

example

[Ca’+ Ii

intracellular link

no

channels”

in

patch

open

fact,

and

be classified

the

response

the

our

them

probability

“fast rise

not

of

seems to occur

calcium

is

by KCl.

as for

by

but

entry

channel

by nifedipine

modulated

as a result

a little

these

In

that

by 3pM La3+

Apparently,

inactivating

and not

Only

second,

only

on the

first,

increase

occur

inhibited

depolarized

The

to

space

of

are an

[Ca’+Ii

results:

and,

and

The calcium

of Ca 2+ -channels,

be hypothesized.

nels

(7).

type

blocked

[Ca’+Ii

appears

of

present

following

conditions.

were

was not

some types the

as this

by the

stores.

(7)).

(WCs)

these

[Ca’+Iit

completely

channels

an influence

present

extracellular

observed

operated

when

of

the

as it

calcium

are

intracellular

has been

proved

(O.l-2mM).

from

prove

modifies

channels

limits

entry

are

calcium

activated

ICaZ+lis

was to

open

[Ca’+Ii results

[Ca2+Io.

[Ca2+Ii, the the

The due to

extracelluK+channels

Vol.

182,

All

No.

these in

channels is

are

It

growth

is

the

clearly

present

that

(14).

Although

&Q

membrane

and tissue

not

only

(D.l-2mM),

potential

that

total

these

calcium

as a function these

situation,

cart i lage , may influence cyte

BIOPHYSICAL

RESEARCH

that but

the

also

COMMUNICATIONS

lCa’*li

that

depends

CaZt-activated

on Kt

in chondrocytes.

varies

b

range

to hypothesize known

AND

demonstrate

physiological

plate,

surface to

the

tempting

tion.

BIOCHEMICAL

results

[Ca2+lo

It

3, 1992

the the

(11,

the

on cell

in

from

can only

existing

conductances

with

in calcifica-

different the

in part

gradient,

differentiation,

a role the

distance

studies

calcium membrane

may play

concentration, of

in vitro

changes

zones

mineralization be extrapolated

Jo w an

matrix

of

effect vesicle

in on

growth chondro-

formation

calcification.

ACKNOWLEDGMENTS This work Miss M.T. Fondo per ter, and scapulas.

was supported by research grants from CNR and MURST, Italy. We thank Nicodemo for her valid technical assistance. We also acknowledge the lo Studio delle Malattie de1 Fegato for use of the spectrofluoromethe Uanetto Delicatessen Factory for the generous supply of pip

REFERENCES 1.

2. 3. 4. 5. 6. 7. a. 9. ::: 12. 13. 14.

Edelman. A., Thil, C.L., Garabedian, M., Plachot, J.J., Guillozo, H., Fritsch, J., Thomas, S.R., Balsan, S. (1985) Min. Electr. Metab. 11,97-105. Grinstein, S. and Dixon, S.J. (1989) Physiol. Rev. 69.417-481. Crandolfo, M.. Martina, M., Ruzzier, F., Vittur, F. (1990). Calcif. Tissue Tnt. 47, 302-307. Vittur, F., Pugliarello, M.C., de Bernard, B. (1971) Experientia 27,126-127. D’Andrea, P., Grandolfo, M.. de Bernard, 6. Vittur, F. (1990) Exp. Cell Res. 191, 22-26. Meldolesi I J., Huttner, W.B., Tsien, R.Y., Pozzan, T. (1984) Proc . Nat. Acad. Sci. USA 81,620-624. Nemeth, E.F. (1990) Cell Calcium 11.323-327. Benham, C.D. and Tsien, R.W. (1987) in Cell calcium and the control of membrane transport (L. J. Mandel, D.C. Eaton, Eds), pp 45-64. The Rockefeller University Press, New York. Rosenberg, R.L., Hess, P., Tsien. R.W. (1988) J. Gen. Physiol. 92.27-54. Meldolesi, J. and Pozzan, T. (1987) Exp. Cell Res. 171, 271-283. Latorre, R.. Vergara M.C., Hidalso, C. (1982) Proc. Natl. Acad.Sci. USA 79.805-809. Schwarz, W.and Passow, H. (1983) Ann. Rev. Physiol. 45.359-374. Latorre. R., Oberhauser, A.. Labarca, P.. Alvarez, 0. (1989) Ann. Rev. Physiol. 51.385-399. Howell, D.S., Delchamps, E., Riemer. W., Kiem, I. (1960) J. Clin. Invest. 39.919-929. 1434