• ^
•
National Research
Council Canada
Conseil national
de recherches Canada
Canadian
Journal of
Physiology and
Pharmacology
Volume 65, Number 4, April 1987
£ Z i
-/)2bb
{ € g a ii g e o
(Janad'a
Journal
canadien de
Physiologie et
pharmacologic
Volume 65, numero 4, avril 1987
ISSN 0008-4212
CJPPA3 65(4) 4 8 9 - 7 4 5 (1987)
Canadian Journal of
Physiology and
Pharmacology
An International Journal
Journal canadien de
Physiologie et
pharmacologie
UrtlversitätsBibliothek
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1038
1
Cellular mechanisms of potassium homeostasis in the mammalian nervous system
P. G R Ä F E
Physiologisches
Institut,
Universität
München,
2
ANDK.
Pettenkofer
BALLANYI
Straße 12, D-8000 München
2, Federal
Republic
of
Germany
Received July 2 1 , 1986
G R Ä F E , P., and B A L L A N Y I , K . 1987. Cellular mechanisms o f potassium homeostasis i n the m a m m a l i a n nervous system. Can. J .
Physiol. Pharmacol. 65: 1 0 3 8 - 1 0 4 2 .
Double-barrelled ion-sensitive microelectrodes were used to measure changes in the intracellular activities o f K , N a , and
Cl~ ( a K j , tfNaj, aClj) in neurones o f rat sympathetic ganglia and in g l i a l cells o f slices f r o m guinea-pig olfactory cortex. I n
sympathetic neurones, carbachol and 7 - a m i n o b u t y r i c acid ( G A B A ) produced a reversible decrease o f aK . The decrease o f aK
during carbachol was accompanied by a rise o f aNa , whereas in the presence o f G A B A decreases o f aK and aC\[ were seen. The
reuptake o f K released d u r i n g the action o f carbachol was completely blocked by ouabain, whereas furosemide i n h i b i t e d the aK
recovery after the action o f G A B A . I n g l i a l cells, i n contrast to the observations i n the sympathetic neurones, aK and aC\\
increased, whereas aNaj decreased when neuronal activity was enhanced by repetitive stimulation o f the lateral olfactory tract. I t
was found that barium ions and ouabain strongly reduced the activity-related rise o f intraglial « K j i n slices o f guinea-pig olfactory
cortex. These data show that m a m m a l i a n neurones as w e l l as glial cells possess several K uptake mechanisms that contribute to
potassium homeostasis. O u a b a i n , furosemide, and B a
are useful pharmacological tools to separate these mechanisms.
+
+
x
x
t
t
+
t
x
+
2 +
G R Ä F E , P., et B A L L A N Y I , K . 1987. Cellular mechanisms o f potassium homeostasis in the m a m m a l i a n nervous system. Can. J .
Physiol. Pharmacol. 65 : 1 0 3 8 - 1 0 4 2 .
O n a utilise des microelectrodes ä double branche, sensibles aux ions pour determiner les variations des activites intracellulaires K , N a et C I " (aK„ aNaj, aCl\) dans des neurones des ganglions sympathiques de rat et dans des cellules gliales de
tranches de cortex o l f a t i f de cobaye. Dans les neurones sympathiques, le carbachol et 1'acide 7 - a m i n o b u t y r i q u e ( G A B A )
provoquerent une d i m i n u t i o n reversible d ' a K j . L a d i m i n u t i o n d'tfKj en presence de carbachol fut accompagnee d'une elevation
d'aNaj, alors q u ' e n presence de G A B A , on observa des diminutions d ' a K ; et d'tfClj. L a reabsorption de K libere durant Taction
du carbachol fut completement bloquee par Touabai'ne, alors que le furosemide inhiba le retablissement d'öKj apres Taction de
G A B A . Dans les cellules gliales, contrairement aux observations dans les neurones sympathiques, aK\ et aC\\, augmenterent,
alors qu'öNaj diminua lorsque Tactivite neuronale fut augmentee par la stimulation repetee du pedoncule o l f a c t i f lateral. O n a
constate que les ions b a r y u m e t Touabaine reduisaient fortement l'elevation reliee ä Tactivite de VaK intragliale dans les tranches
de cortex o l f a c t i f de cobaye. Ces resultats montrent que les neurones et les cellules gliales de mammiferent possedent plusieurs
mechanismes d'absorption de K q u i contribuent ä Thomeostasie potassique. L'ouabaine, le furosemide et le B a
sont des
elements pharmacologiques utiles pour distinguer ces mecanismes.
+
+
+
{
+
2 +
[Traduit par la revue]
been proposed to contribute to the K
Introduction
Enhanced neuronal activity i n the central nervous system is
accompanied by transient elevations i n the extracellular K
activity (aK )
+
released f r o m neurones
via voltage- or C a - d e p e n d e n t , transmitter-gated, and leaky
2+
K
channels.
+
Mammalian
mechanisms for K
Na -K
+
+
+
neurones
possess
at least t w o
reuptake. One is the ouabain-sensitive
p u m p , w h i c h can be activated, for example, by the
acetylcholine-induced rise i n the intracellular N a
+
activity and
the other one is a furosemide-sensitive, N a - d e p e n d e n t K C l
+
cotransport,
which
is activated
during
the action
measure changes i n the intracellular activities o f K , N a , and
+
+
(Somjen 1979; Nicholson 1981; Sykova 1983).
e
The rise o f aKe is the consequence o f K
of 7 -
aminobutyric acid ( G A B A ) ( B a l l a n y i et al. 1984; Ballanyi and
increase o f aK
+
during an
(literature reviewed b y W a l z and Hertz 1983).
e
However, the mechanisms o f K
uptake into g l i a l cells are not
+
yet understood i n detail. Several factors such as spatial buffering
( O r k a n d e t a l . 1966; Dietzel et al. 1982; Coles and Orkand 1983;
Gardner-Medwin 1983), N a - K
+
+
pump activity (Kukes et a l .
+
aNai, aC\ , respectively) i n g l i a l cells o f slices f r o m
Cl~ (aKj,
x
guinea-pig olfactory cortex during repetitive stimulation o f the
lateral olfactory tract ( B a l l a n y i et al. 1987). These experiments
showed that B a
2 +
and ouabain are useful pharmacological tools
to study the mechanisms underlying the K
The
+
uptake in glial cells.
present paper reviews o u r intracellular measurements
with ion-sensitive microelectrodes f r o m mammalian neurones
and glial cells. The data are summarized i n a model about
cellular mechanisms contributing to the K
+
homeostasis i n the
mammalian nervous system.
Gräfe 1985).
Glial cells, on the other hand, accumulate K
uptake process. Recently,
+
we have used double-barrelled ion-sensitive microelectrodes to
Methods
Sympathetic
neurones
Experiments were performed on neurones o f superior cervical ganglia o f rats. Ganglia were isolated, desheathed, and continuously
superfused at 30°C i n a recording chamber ( B a l l a n y i and Gräfe 1985)
with a solution containing ( i n m M ) : N a C l , 118; K C l . 4.8, N a H C O i .
2 . 5 ; K H P 0 , 1 . 2 ; M g S 0 , 1.2; C a C l , 2.5; and glucose, 10.
2
4
4
2
1976; Walz and Hertz 1 9 8 2 ; G r i s a r e t a l . 1983), K C l cotransport
(Kimelberg and Frangakis 1985; W a l z and H i n k s 1985), or the
presence o f B a - s e n s i t i v e K
2+
+
channels ( W a l z e t a l . 1984) have
'This paper was presented at the Ion-selective Microelectrodes and
Excitable Tissue S y m p o s i u m (Toronto, O n t a r i o , July 8 - 1 1 , 1986), a
Satellite Symposium o f the 30th IUPS Congress, and has undergone the
Journal's usual peer review.
A u t h o r to w h o m correspondence should be addressed.
2
Cortical glial cells
Experiments were performed o n slices o f the olfactory cortex o f
guinea pigs. After decapitation, the brain was rapidly removed and
surface slices (approximately 500 (Jim t h i c k ) were cut using a plastic
guide and a bow cutter. Slices were placed i n a perspex chamber ( v o l . 2
m L ) and superfused at 25°C w i t h a solution o f the f o l l o w i n g composition (in m M ) : N a C l , 118; K C l , 3; N a H C 0 , 2 5 ; N a H P 0 , 1.2; M g C l ,
1.0; C a C l , 1.5; and glucose, 10 (equilibrated w i t h 9 5 % 0 - 5% C 0 ;
pH 7.4). Drugs were added to the superfusion f l u i d . Cortical neurones
3
2
2
4
2
2
2
G R Ä F E AND
1039
BALLANYI
sympathetic neurone
c o r t i c a l glial eel
B
30 s
- 2 5 -|
(mV)
-40
-55
J
-90
•GABA 50;uM
•Carbachol 50
LOT 30 Hz 20 s
F I G . 1. A n overview o f changes in membrane potential ( £ ) and i n intra- and extra-cellular K activity (aK , a K , respectively) accompanying
the actions of carbachol, G A B A , and repetitive stimulation on neurones and g l i a l cells. ( A and B) Experiments w i t h isolated rat sympathetic
ganglia. (C) A n experiment w i t h a guinea-pig olfactory cortex slice. For further discussion see text. L O T , lateral olfactory tract.
+
m
were activated by electrical s t i m u l a t i o n o f the lateral olfactory tract
w i t h platinum wire electrodes.
e
aCl,
OK;
aNa,
30-
Ion-sensitive
microelectrodes
The properties and the methods used for the construction and calibration o f double-barrelled ion-sensitive microelectrodes are described in
detail elsewhere (Gräfe et al. 1985; B a l l a n y i et al. 1987). The ligands
used for the K - , C I " - , and N a - s e n s i t i v e microelectrodes were C o r n ing 477317, IE-170 (WP-Instruments), and Fluka 71176, respectively.
A l l values o f intracellular N a , K * , and C l ~ are given in activities
(Meieret a l . 1980). Intracellular impalements were achieved by means
of a piezo-driven m i c r o m a n i p u l a t o r (built by M . Frankenberger,
Munich).
+
}
mM
mM
20
J
+
-60mV
+
-70-80-90-
Results
Intracellular
activity
20
in mammalian
neurones
and glial
cells
Figure 1 summarizes observations from experiments in which
intracellular^ and extracellularly positioned K -sensitive m i croelectrodes were used simultaneously. Parts A and B are from
experiments on rat sympathetic ganglia. One can see that both
carbachol and G A B A depolarize the membrane of sympathetic
neurones. This effect is accompanied i n each case by a rise o f
a K and a decrease o f a K j . The post-carbachol K reuptake is
accompanied by an undershoot o f aK and membrane hyperpolarization. Neither phenomenon occurs after the end o f the
G A B A application. This indicates that two different mechanisms might contribute to the aK recovery after carbachol and
G A B A . This conclusion was confirmed by pharmacological
observations: ouabain blocked completely the post-carbachol
aK recoveries, whereas furosemide inhibited the K reuptake
after the action o f G A B A (Ballanyi et al. 1 9 8 4 ) .
+
+
e
e
x
+
e
In contrast to the neurones, glial cells of guinea-pig olfactory
cortex accumulate K
during a period of enhanced neuronal
activity. Figure 1C illustrates that stimulation of the lateral
olfactory tract ( 3 0 H z , 2 0 s) resulted in a glial depolarization o f
about 2 0 m V during an increase of aK o f about 4 m M (Ballanyi
et al. 1 9 8 7 ) . D u r i n g the depolarization aK increased by about
lOmM.
+
e
x
Activity-related
changes
ofintraglial
ion
activities
The base-line level o f aK± as measured in 12 cells w i t h a mean
membrane potential ( £ ) of - 8 4 . 4 ± 3.3 m V was 6 5 . 9 ± 6 . 6
m M (mean ± SD). Therefore a close correspondence between
the calculated £ o f - 8 5 . 6 m V (aK at rest = 2 . 2 m M ) and the
g l i a l £ is revealed, lntraglial aNa, had a resting level of 2 5 . 2 ±
m
K
m
e
15 s
10s
Hz.15s
1 mm
F I G . 2. Changes o f intraglial i o n activities during repetitive stimulation o f the lateral olfactory tract (20 H z for 1 0 - 1 5 s). The figure
combines recordings f r o m three different g l i a l cells in w h i c h a K - ,
N a - , or Cl"-sensitive microelectrode was used to record changes o f
these ion activities i n a glial c e l l . For a quantitative analysis o f such
recordings see text.
+
+
4.6 m M ( £ = - 83.0 ± 2.8 m V ; n = 12) and the corresponding
flClj was 6.0 ± 1.5 m M ( £ = - 8 6 . 9 ± 3.8 m V ; n = 23;
calculated under the assumption of a pure Cl~ selectivity o f the
Cl"-sensitive ligand). Figure 2 illustrates how the intracellular
activities of these ions changed when the lateral olfactory tract
was stimulated repetitively (20 Hz) for 1 0 - 1 5 s. The average
rise of aK was 16.1 ± 6.1 m M (12 glial cells), aN^ decreased
by 6.0 ± 1 . 9 m M ( n = 12), and aCl, increased by 6.0 ± 1.5 m M
(n = 23). I n conclusion, these observations demonstrate that
activity-related glial K accumulation is accompanied by a rise
of intraglial aC\ and by a fall of aNa .
m
m
{
+
{
K
+
uptake
}
mechanisms
in cortical
glial
cells
To differentiate between several possible mechanisms o f K
uptake into cortical glia, the action o f B a
was explored.
Barium ions are k n o w n to block K channels (Hille 1984) and
have been previously used as a tool to differentiate between
passive and active K uptake mechanisms in skeletal muscle
fibres (Sjodin and Ortiz 1975). These ions are also k n o w n to
interfere w i t h the K homeostasis o f cultured glial cells (Walz et
al. 1984). In our experiments, three clear effects of B a on the
glial cells were observed: (/) B a induced a membrane depolarization and led to a change f r o m a K - i n d u c e d membrane
+
2 +
+
+
+
2 +
2 +
+
C A N . J. P H Y S I O L . P H A R M A C O L . V O L . 65. 1987
1040
Ba (o.5mM,
control
1
+ 8 min)
mM
53 -
Ba *(0 S m M . * l 2 mm)
control
w a s h (+12 min
2+
70-1
mM
60-
2
Bo *(commued)
2
Ouabain 1 S^M,
.15min)
OK;
50-40-.
35
mV
-60mV
-70-
-60-
-80-80-
6.5-,
mM
L.O -
2min
LOT 20 Hz, 15s
2.2 1.5 -
F I G . 4 . Effects o f barium and ouabain on stimulus-induced increases
in intracellular K activity (aK ) and glial membrane potential ( £ ) .
The left c o l u m n shows the t y p i c a l rise o f aK during a stimulus-induced
depolarization o f the g l i a l c e l l . I n the presence o f B a
(0.5 mM) an
elevation o f the aK base-line l e v e l , smaller increase o f aK , and
stimulus-induced membrane hyperpolarization were seen. T h e B a resistant rise o f aK and the membrane hyperpolarization were c o m pletely blocked 15 m i n after ouabain (5 jxAf) was added to the B a containing solution. L O T , lateral olfactory tract.
+
LOT 10Hz,10s
t
m
t
2 +
F I G . 3. Effects o f barium on stimulus-induced increases i n intracellular K activity (aK ), extracellular K activity ( a K ) , and glial m e m brane potential ( £ ) . T h e figure shows three sections taken f r o m a
continuous recording f r o m a single glial c e l l . The left c o l u m n shows the
typical rise o f aK during a stimulus-induced increase o f aK i n normal
solution. I n the presence o f B a
(0.5 m M , central column) an even
greater rise i n aK led to a much smaller increase o f a K j and was
accompanied by a membrane hyperpolarization. Right c o l u m n shows
changes w i t h stimulation during the recovery f r o m B a . L O T , lateral
olfactory tract. (From B a l l a n y i et a l . 1987, reprinted w i t h k i n d permission o f The Physiological Society.)
+
+
t
e
m
x
c
t
x
2 +
t
2 +
2 +
e
2 +
depolarization into a K - i n d u c e d hyperpolarization, (//) Ba *"
raised the intraglial aK base-line level and partially blocked the
K uptake, and (Hi) B a
completely blocked the rise o f intraglial äClj usually seen during a stimulus-induced rise o f aK .
These effects are illustrated i n Figs. 3, 4, and 5.
Figure 3 shows three excerpts from a continuous recording
from a single cortical glial cell. The left column illustrates that in
the normal bathing solution a membrane depolarization and a
rise of aKj occur during repetitive stimulation o f the lateral
olfactory tract. The center column shows a stimulation period
with the same stimulus parameters 8 m i n after B a
(0.5 m M )
was added to the bathing solution. The glial cell is now depolarized and responds w i t h a hyperpolarization to the rise o f aK .
Now, only a small elevation o f aK is seen during the stimulation
period. The right column is from a period 12 m i n after B a was
replaced by the normal bathing solution and shows that the
effect o f B a
is reversible.
+
2-
x
+
2 +
e
-90
E
J
B o * 0.5 m M
1
1
ref
5mV]
aK
2 +
e
80-|
mM
2.2-
e
V
LOT
2 +
2 +
Figure 4 is an example o f experiments in which the mechanism o f the stimulus-related glial hyperpolarization was explored. The left column and center column show changes in the
membrane potential and aK i n the normal bathing solution and
12 m i n after B a
was added to this solution (see Fig. 3). The
membrane depolarization during the application o f B a
can be
explained by a reduced K conductance. A reduced efflux o f
K
from the glial cell in the presence o f an active N a - K
pump then results i n an increase o f aK (see also F i g . 8 in
Ballanyi et al. 1987). The right column is f r o m a period 15 m i n
after ouabain (5 \LM) was added to the B a - c o n t a i n i n g bathing
solution. Ouabain blocked both the stimulus-related remaining
rise of öKj and the membrane hyperpolarization. This indicates
that an electrogenic N a - K
pump underlies the membrane
hyperpolarization and also the B a - r e s i s t a n t K uptake. (Ouabain d i d not block the stimulus-induced rise i n aK \ on the
x
2 +
2 4
+
+
+
r
2+
+
+
2+
+
e
I
1.1-
x
20Hz.20s
2 mm
F I G . 5. Effects o f barium on intracellular Cl~ activity (aClj) and
extracellular K (aK ). A f t e r t w o control stimulation trains, B a (0.5
mM) was added to the bathing solution. The typical glial depolarization
in B a was accompanied by an increase i n aC\\ base-line. The B a induced shift o f stimulus-related depolarization into hyperpolarization
was mirrored by a shift f r o m an increase in aC\[ to a decrease. After the
washout o f B a , the membrane potential (E ) as well as #Clj recovered to base-line values. L O T , lateral olfactory tract. (From Ballanyi et
al. 1987, reprinted w i t h k i n d permission o f The Physiological Society.)
+
2 +
e
2 +
2 +
2 +
m
+
contrary, the stimulus-related rise o f aK was slightly bigger
than usual; see Fig. 6 i n Ballanyi et al. 1987.)
The behaviour o f intraglial aC\ i n the presence o f B a
indicates a high C I " conductance o f the glial membrane. I n the
experiment illustrated in F i g . 5, a Cl~-sensitive microelectrode
was used to record aC\ and E o f a glial cell (Fig. 5 A ) , whereas
a K - s e n s i t i v e microelectrode was positioned extracellularly to
measure aK (Fig. 5 B ) . T w o stimulus trains in normal solution
Q
x
x
+
e
m
2 +
1041
GRÄFE AND BALLANYI
neurone
(ACh ,Glu)
^
N
1I
a
glia
e. s.
e
IBQ'-)
+
K
( ouabain)
by a membrane hyperpolarization and an undershoot o f a K .
Examples o f such a situation are shown b y the action o f glutamate on frog spinal motoneurons (Sonnhof et al. 1976) and o f
carbachol (via stimulation o f nicotinic acetylcholine receptors)
on rat sympathetic ganglia (Ballanyi et a l . 1984). If, on the other
hand, neurones are depolarized as a consequence o f an increase
in C I " conductance (for example by the activation o f G A B A
receptors), both C I " and K are released f r o m the cell. I t was
found that in such a situation an electroneutral, Na -dependent,
and furosemide-sensitive K - C 1 ~ carrier is involved i n the
neuronal K homeostasis (Ballanyi and Gräfe 1985).
er
Ncf
A
+
+
+
(GABAA)
+
K
(furosemide)
K
+
(ouabain )
+
+
)(Na*)
[
er
+
+
t
+
N
+
+
+
+
t
+
+
+
+
F I G . 6. Summary o f K uptake mechanisms i n m a m m a l i a n neurones
and glia. After a rise o f neuronal aNa v i a the opening o f nicotinic
cholinergic ( A C h ) o r glutamate ( G l u ) receptors, the N a - K p u m p
restores the intracellular K activity. I f neurones release K together
w i t h C I " (during a depolarizing action o f G A B A ) , a furosemidesensitive, [Na] -dependent K - G ~ cotransport is found to maintain
the normal aK resting level. O u r results indicate that glial cells also
possess at least two types o f K uptake mechanisms. There is passive
K
uptake v i a B a - s e n s i t i v e K channels (together w i t h passive
uptake o f Cl~) and also the activation o f a glial N a - K pump secondary to arise o f extracullular K (see also text), e.s., extracellular space.
e
Glial cells also have at least t w o mechanisms to take up K
(Ballanyi et a l . 1987). One component is a ouabain-sensitive
N a - K pump that, i n contrast to neurones, is activated b y
excess extracellular K . The electrogenic pump current o f this
transport mechanism is normally short-circuited by the high
resting conductance o f the glial membrane. However, a ouabain-sensitive membrane hyperpolarization can be seen i n the
presence o f B a
(see Figs. 3 and 4). The N a - K pump most
probably contributes to the decrease o f intraglial aNaj w h i c h is
observed during a rise o f # K , although other factors may
contribute to this phenomenon.
2+
+
+
+
+
at the beginning o f this recording revealed the stimulus-related
rise o f aK
membrane depolarization and accompanying rise o f
tfCli usually seen i n the glial cells. B a
was added to the
bathing solution and w i t h i n a few minutes the membrane depolarized by more than 20 m V and aC\ increased to about 12
m M . Repetitive stimulation o f the lateral olfactory tract during
Ba
induced a small membrane hyperpolarization and a slight
fall in aC\\. Therefore, the increase in resting aClj, as w e l l as the
stimulus-related decrease o f aC\
clearly indicates the close
relationship between the membrane potential and aC\ . I f aC\
were to follow changes in a K , then the B a - i n d u c e d increase
of the flK base-line and the stimulus-related aK
increase
should have altered Ö C I J i n the opposite direction. Previously in
the literature pertaining to glia, results f r o m t w o kinds o f experiment led to the conclusion that g l i a l cells do not have a
significant C I " conductance. First, no change in glial membrane
potential was seen during the transition from a h i g h to a l o w
extracellular C I " m e d i u m . This fact may be explained by a lack
of C l ~ conductance. However, i f intracellular C l ~ were to leave
the cell as rapidly as the decrease o f a C l , E \ w o u l d always
remain close to £ ; there w o u l d be no potential change in spite
of a C l ~ conductance (see F i g . 4 i n B a l l a n y i et a l . 1987).
Secondly, some authors did not observe changes i n glial input
resistance i n Ci~-free extracellular m e d i u m . These experiments
should be repeated w i t h the substitution o f bigger anions, i n
view o f the discovery o f large anion-conducting channels i n
glial cells (Gray and Ritchie 1985).
ey
2 +
x
2 +
ly
{
x
2+
e
e
e
3
2 +
The other mechanism, b y w h i c h glial cells take up K , is a
B a - s e n s i t i v e K conductance. This finding, together w i t h the
observation o f a high C I " conductance o f the glial cells (see F i g .
5), indicates that the K uptake can occur v i a a mechanism
suggested by Boyle and Conway (1941) and H o d g k i n and Horowicz (1959) to explain passive uptake o f K into muscle fibres.
These authors point out that during an elevation o f <sK , the
presence o f a CI" conductance w i l l prevent E from reaching the
new E . Hence K (as w e l l as CI") can continue to flow into the
cell. Our data are compatible w i t h this view. Another mechanism by which K uptake could occur through B a - s e n s i t i v e
channels w o u l d be via spatial buffer currents (Orkand et a l .
1966; Coles and Orkand 1983; Gardner-Medwin 1983; Dietzel
et al. 1982). According to this v i e w , K w o u l d enter the glial
syncytium through K channels as a result o f a spatial potential
gradient along the membrane o f the electrically coupled glia; the
rise o f intraglial aC\ w o u l d be explained by a redistribution o f
C r ions w i t h i n the linked glial cells. Our experiments do not
exclude such a mechanism. However, the existence o f a passive
K C l uptake w o u l d allow the K to be stored i n the glial cell i n
the immediate neighbourhood o f the active neurone, where i t
might be more readily available to the neurone during recovery
from K loss (Gray and Ritchie 1985).
+
2+
+
+
+
e
m
+
K
+
Figure 6 summarizes our data obtained w i t h intracellular
ion-sensitive microelectrodes f r o m rat sympathetic neurones
and guinea-pig cortical glial cells. Neurones, o n the one hand,
exchange intracellular K
w i t h N a i f an increase i n N a
conductance leads to a membrane depolarization. A ouabainsensitive N a - K pump restores both ion concentration gradients involved, and this electrogenic reuptake is accompanied
+
+
+
+
+
2+
+
+
x
+
+
Acknowledgements
We thank Professor G . ten Bruggencate for constant encouragement and helpful discussions during the course o f this
project. Miss G . Schneider and M r s . C. Müller provided expert
technical and secretarial assistance. The w o r k was supported by
the Deutsche Forschungsgemeinschaft (SFB 220).
BALLANYI,
Discussion
+
e
C
m
+
K.,
and G R Ä F E ,
P.
1985. A n
i n t r a c e l l u l a r analysis
of
7-aminobutyric-acid-associated i o n movements i n rat sympathetic
neurones. J . Physiol. ( L o n d o n ) , 365: 4 1 - 5 8 .
B A L L A N Y I , K . , G R Ä F E , P., R E D D Y , M . M . , and T E N B R U G G E N C A T E ,
G . 1984. Different types o f potassium transport linked to carbachol
and G A B A actions in rat sympathetic neurons. Neuroscience (Oxford), 1 2 : 9 1 7 - 9 2 7 .
B A L L A N Y I , K.,
GRÄFE,
P.,
and T E N B R U G G E N C A T E , G .
1987. I o n
activities and potassium uptake mechanisms o f glial cells i n guineapig olfactory cortex slices. J . Physiol. ( L o n d o n ) . 382: 1 5 9 - 1 7 4 .
1042
B O Y L E , P.
CAN.
J.,
and
C O N W A Y , E. J .
1941.
J. P H Y S I O L . P H A R M A C O L . V O L . 65, 1987
Potassium a c c u m u l a t i o n in
muscle and associated changes. J . Physiol. ( L o n d o n ) , 100:
C O L E S , J. A.,
and
O R K A N D , R.
K.
1983.
Modification of
potassium
movement through the retina o f the drone (Apis mellifera)
uptake. J . Physiol. ( L o n d o n ) , 340: 1 5 7 - 1 7 4 .
DIETZEL, I . , HEINEMANN, U.,
H O F M E I E R , G.,
and L u x ,
1-63.
by glial
H. D .
1982.
Stimulus-induced changes i n extracellular N a and C l ~ concentration i n relation to changes i n the size o f the extracellular space. Exp.
Brain Res. 46: 7 3 - 8 4 .
G A R D N E R - M E D W I N , A . R. 1983. Analysis o f potassium dynamics in
mammalian brain tissue. J . Physiol. ( L o n d o n ) , 335: 3 9 3 - 4 2 6 .
+
G R Ä F E , P.,
B A L L A N Y I , K.,
and T E N B R U G G E N C A T E , G .
1985.
Changes
o f intracellular free ion concentrations, evoked by carbachol or
G A B A , i n sympathetic neurons. In I o n measurements i n physiology
and medicine. Edited by Kessler, M . , Harrison, D . K . , andHöper, J .
Springer, B e r l i n , Heidelberg, N e w Y o r k . pp. 1 8 4 - 1 8 8 .
G R A Y , P. T .
A.,
and R I T C H I E , J .
M.
1985.
I o n channels i n S c h w a n n
and glial cells. Trends Neurosci. 8: 4 1 1 - 4 1 5 .
GRISAR, T.,
F R A N C K , G.,
and D E L G A D O - E S C U E T A , A .
V.
1983.
Glial
contribution to seizure: K activation o f ( N a , K ) - A T P a s e in bulk
isolated glial cells and synaptosomes o f epileptogenic cortex. B r a i n
Res. 261: 7 5 - 8 4 .
H I L L E , B . 1984. Ionic channels o f excitable membranes. Sinauer
Associates I n c . , Sunderland, M A .
+
+
H O D G K I N , A . L . , and H O R O W I C Z , P.
1959.
The
+
influence o f potassium
and chloride ions on the membrane potential o f single muscle fibres.
J. Physiol. ( L o n d o n ) , 148: 1 2 7 - 1 6 0 .
KIMELBERG, H.
K.,
and
FRANGAKIS, M.
V.
1985.
Furosemide-
and
bumetanide-sensitive ion transport and volume control i n primary
astrocytes f r o m rat brain. B r a i n Res. 361: 1 2 5 - 1 3 4 .
K U K E S , G.,
E L U L , R.,
and D E V E L L I S , J .
1976.
The
ionic basis o f
membrane potential i n a rat glial cell line. B r a i n Res. 104:
M E I E R , P.
C,
AMMANN, D.,
M O R F , W.
E.,
and
the
71-92.
S I M O N , W.
1980.
Liquid-membrane ion-sensitive electrodes and their biomedical ap-
plications. In Medical and b i o l o g i c a l applications o f electrochemical
devices. Edited by K o r y t a , J . J . W i l e y & Sons L t d . , N e w Y o r k ,
pp. 1 3 - 9 1 .
N I C H O L S O N , C. 1981. Brain-cell microvenvironment as a c o m m u n i c a tion channel. In The neurosciences—Fourth study program. Edited
by Schmitt, F. O., and W o r d e n , F. G . M I T Press, Cambridge,
U . S . A . pp. 4 5 7 - 4 7 6 .
O R K A N D , R. K . ,
N I C H O L L S , J. G.,
a n d K u F F L E R , S. W .
1966.
Effect o f
nerve impulses on the membrane potential o f glial cells i n the central
nervous system o f A m p h i b i a . J . N e u r o p h y s i o l . 29: 7 8 8 - 8 0 6 .
S J O D I N , R.
A.,
and O R T I Z , O.
1975.
R e s o l u t i o n o f the potassium
ion
p u m p in muscle fibers using b a r i u m ions. J . Gen. Physiol. 66:
269-286.
S O M J E N , G . G . 1979. Extracellular potassium in the m a m m a l i a n central nervous system. A n n u . Rev. Physiol. 41: 1 5 9 - 1 7 7 .
SONNHOF, U.,
G R Ä F E , P.,
and
K R U M N I K L , J.
1976.
Interaction
of
glutamate w i t h the sodium p u m p i n spinal motoneurons o f the frog.
Pfluegers A r c h . 362: R 3 4 .
S Y K O V A , E. 1983. Extracellular K accumulation i n the central nervous system. Prog. B i o p h y s . M o l . B i o l . 42: 1 3 5 - 1 9 0 .
+
WALZ,
W.,
and
HERTZ,
L.
1982.
Ouabain-sensitive and
ouabain-
resistant net uptake o f potassium into astrocytes and neurons in
primary cultures. J . Neurochem. 39: 7 0 - 7 7 .
1983. Functional interactions between neurones and astrocytes. I I . Potassium homeostasis at the cellular level. Prog. Neurobiol.20: 133-183.
W A L Z , W.,
and H I N K S , E. C.
1985.
Carrier-mediated K C l
accumula-
tion accompanied by water movements is involved in the control o f
physiological K levels by astrocytes. B r a i n Res. 343: 4 4 - 5 1 .
+
W A L Z , W.,
SHARGOOL, M.,
and
H E R T Z , L.
1984.
Barium-induced
inhibition o f K transport mechanisms i n cortical astrocytes—its
possible contribution to the large B a
evoked extracellular K signal in brain. Neuroscience ( O x f o r d ) , 13: 9 4 5 - 9 4 9 .
+
2 +
+