(Received for publication, November 27, 1995; and in revised form, January 4, 1996)
From the
Osmotic cell swelling induced by hypotonic stress is associated
with a rise in intracellular Ca concentration, which
is at least partly due to a release of Ca
from
internal stores. Since osmotic influx of water dilutes the cytoplasmic
milieu, we have investigated how nonmitochondrial Ca
stores in permeabilized A7r5 cells respond to a reduction in
cytoplasmic tonicity. We now present experimental evidence for a direct
Ca
release from the stores when exposed to a
hypotonic medium. The release is graded, but does not occur through the
inositol trisphosphate or the ryanodine receptor. Ca
seems to be released through the passive leak pathway, and this
phenomenon can be partially inhibited by divalent cations in the
following order of potency: Ni
=
Co
> Mn
> Mg
> Ba
. This release also occurs in intact
A7r5 cells. This novel mechanism of hypotonically induced
Ca
release is therefore an inherent property of the
stores, which can occur in the absence of second messengers.
Intracellular stores can therefore act as osmosensors.
Most cells exposed to anisosmotic solutions activate volume
regulatory processes to prevent damage by cell swelling or shrinkage (1, 2, 3) . Osmotic cell swelling in response
to hypotonic stress is associated with a rise in intracellular
[Ca], which is at least partly due to a
release of Ca
from internal
stores(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) .
The link between changes in external osmotic concentration and internal
Ca
release is not known: inositol trisphosphate (3) and arachidonic acid (5) may be involved, but it is
also possible that no messenger is needed(2) . Since the influx
of water dilutes the cytoplasm, we have investigated how the
Ca
stores in permeabilized A7r5 cells respond to a
reduction in cytoplasmic tonicity. We now present experimental evidence
for a direct Ca
release from the nonmitochondrial
stores when exposed to a hypotonic medium. This hypotonically induced
Ca
release through the passive leak pathway is an
inherent property of the stores, which can occur in the absence of
second messengers.
A7r5 cells, an established cell line derived from embryonic
rat aorta, were used between the 7th and the 18th passage after receipt
from the American Type Culture Collection (Bethesda, MD) and
subcultured weekly by trypsinization. The cells were cultured at 37
°C in a 9% CO incubator in Dulbecco's modified
Eagle's medium supplemented with 10% fetal calf serum, 3.8 mML-glutamine, 0.9% (v/v) nonessential amino acids, 85 IU
ml
penicillin, and 85 µg ml
streptomycin. The cells were seeded for the
Ca
fluxes in 12-well dishes (Costar, 4
cm
) at a density of approximately 10
cells
cm
and for the intracellular
[Ca
] measurements in Coverglass Chambers
(Nunc Inc., Naperville, IL) at a density of 5
10
cells cm
.
Ca
fluxes on monolayers of saponin-permeabilized A7r5 cells (3
10
cells/4-cm
well) at 25 °C were
done as described(14) . The stores were loaded for 40 min in
120 mM KCl, 30 mM imidazole (pH 6.8), 5 mM MgCl
, 5 mM ATP, 0.44 mM EGTA, 10
mM NaN
, and 150 nM free Ca
(50 µCi ml
). The wells were then washed
twice in an isoosmotic efflux medium containing 60 mM KCl, 120
mM mannitol, 30 mM imidazole (pH 6.8), 1 mM EGTA, and 2 µM thapsigargin (measured osmolality of
300 mosm/kg H
O). 1 ml of medium was then added at time 0
and replaced every 6 s or every 2 min. Osmolality changes were induced
by changing the mannitol concentration to prevent changes in ionic
concentration and ionic strength.
For the Ca
fluxes on monolayers of intact A7r5
cells (3
10
cells/4-cm
well), the cells
were loaded for 60 min in a modified Krebs solution containing 135
mM NaCl, 5.9 mM KCl, 1.2 mM MgCl
, 11.6 mM Hepes (pH 7.3), 11.5 mM glucose, and 1.5 mM CaCl
(50 µCi
ml
) at 25 °C. The efflux was performed in a
medium containing 65 mM NaCl, 120 mM mannitol, 5.9
mM KCl, 1.2 mM MgCl
, 11.6 mM Hepes (pH 7.3), 11.5 mM glucose, and 2 mM EGTA.
The hypotonic shock was induced by removing the mannitol.
Single-cell intracellular [Ca]
measurements were performed using a laser-scanning MRC-1000 system
(Bio-Rad, Hertfordshire, UK) attached to an inverted Nikon Diaphot 300
epifluorescence microscope with a CF Fluor 40
(numeric aperture
= 1.3) oil immersion objective. The cells were incubated for 30
min with 5 µM Indo-1/AM dissolved in the modified Krebs
solution and then further incubated for 1 to 2 h in the absence of
Indo-1. During the experiment at 25 °C, the cells were continuously
superfused from a pipette placed on top of the cell. The solutions were
the same as for the
Ca
fluxes in intact
cells.
Permeabilized A7r5 cells loaded to equilibrium with Ca
slowly lost their
Ca
during incubation in an isoosmotic
(300 mosm/kg H
O) Ca
-free medium. A
long-lasting reduction in medium osmolality to 180 mosm/kg
H
O transiently increased the rate of Ca
release (Fig. 1A). This effect occurred despite
the reduced concentration gradient for Ca
across the
store membrane as a result of the decreased luminal
[Ca
] by osmotic H
O influx. This
40% reduction in tonicity is of the same order as used experimentally
in intact cells (range 30-50%, (4, 5, 6, 7, 8, 9, 10, 11, 12) ).
The release could be elicited again after reloading the stores with
Ca
(data not shown). Increasing the
osmolality to 420 mosm/kg H
O had no effect but the
returning to isoosmotic solution again resulted in a transient,
although less pronounced, Ca
release (Fig. 1B).
Figure 1:
Effect
of anisosmotic solutions on the rate of Ca release
from permeabilized A7r5 cells. A illustrates the effect of
lowering the medium osmolality by leaving out 120 mM mannitol (filled bar). B shows the effect of a short-lasting
increase in mannitol concentration from 120 to 240 mM (hatched bar). Typical for 4
experiments.
Ca release through the
inositol trisphosphate (InsP
) (
)receptor (15) and ryanodine receptor (16, 17) is
graded, i.e. continuous submaximal stimulation is unable to
completely empty the entire Ca
pool. The
hypotonically induced Ca
release is also graded: a
long-lasting moderate decrease in tonicity released less Ca
than a more pronounced decrease (Fig. 2A). Graded
responses allow increment detection(18) . Increment detection
for the hypotonically induced Ca
release is shown in Fig. 2B: decreasing the osmolality stepwise resulted in
concomitant phases of Ca
release.
Figure 2:
Hypotonically induced Ca
release is a graded process. The permeabilized cells were first
incubated in efflux medium containing 120 mM mannitol, and the
Ca
content of the stores was followed as a function
of time. In A, the mannitol concentration was reduced to 80
mM (
), 40 mM (
), or 0 mM (
)
after 14 min. In B, the mannitol concentration was reduced to
80 mM after 14 min and to 0 mM after 28 min. The dashed lines are the control efflux curves in isoosmotic
medium. Typical for 4 experiments.
The
hypotonically induced Ca release (up to 60% of the
total store Ca
content in Fig. 2) originated
from the InsP
-sensitive store, since the
InsP
-insensitive compartment in permeabilized A7r5 cells
contains only about 5% of the stored Ca
(14) .
Activation of the InsP
receptor by endogenous InsP
formation was, however, not involved. First, a hypotonic
challenge after 20 min (less filled stores) released more
Ca
and therefore resulted in a lower Ca
content at 30 min than a challenge at 0 min (full stores, Fig. 3A). This larger Ca
release from
less filled stores is in contrast with the less complete
InsP
-induced Ca
release from partially
depleted stores(14, 19, 20, 21) . A
second argument against the involvement of the InsP
receptor is that 10 µM thimerosal, a pharmacological
activator of InsP
receptors(22, 23, 24, 25, 26) ,
did not stimulate the hypotonically induced Ca
release (Fig. 3B). A third argument indicating
that the InsP
receptor was not involved is that the release
was unaffected by the presence of 50 µg ml
heparin (Fig. 3B, inset). Heparin is a
competitive inhibitor of the InsP
receptor, which was found
in control experiments to completely inhibit the Ca
release in response to up to 3.2 µM InsP
(data not shown). Finally, the release was unaffected when the
cytosolic free [Ca
] was varied over a
concentration range (10 nM to 10 µM) that would
profoundly affect the InsP
-induced Ca
release (27, 28, 29) (data not shown).
Figure 3:
Hypotonically induced Ca
release in permeabilized A7r5 cells does not occur through the
InsP
or ryanodine receptor. A shows how the
Ca
content decreased during incubation in isotonic
efflux medium and how this Ca
content was affected by
removing all mannitol for 2 min, either at time 0 (
) or after 20
min (
). B illustrates the effect of removing the mannitol
in the absence (
) and in the presence of 10 µM thimerosal (
) or 10 µM ruthenium red (
),
added from time 0 onward. The inset shows the effect of
removing the mannitol in the absence (
) and in the presence of 50
µg ml
heparin (
), added from time 0 onward.
Typical for 4 experiments.
Ryanodine receptors were not involved in the release because 10
µM ruthenium red was without effect (Fig. 3B) and because less Ca was
released from filled stores (Fig. 3A), while ryanodine
receptors are stimulated by luminal Ca
(30) .
Ca
was not released via Ca
pumps,
because the presence or absence of 2 µM thapsigargin, a
blocker of pump-mediated Ca
release(31) , did
not affect the hypotonically induced Ca
release.
Phospholipase A
and C blockers (10 µM 4-bromophenacyl bromide, 10 µM manoalide) had no
effect (data not shown), indicating that endogenous production of
arachidonic acid or InsP
was not involved. Also, modulators
of microfilaments or microtubules (10 µM phalloidin, 50
µM cytochalasin B, 50 µM taxol, 10 µM demecolcine) had no effect (data not shown).
Divalent cations
(2 mM) inhibited the hypotonically induced Ca release with the following order of potency: Ni
= Co
> Mn
>
Mg
> Ba
(Fig. 4A). The inhibition by 2 mM Mg
(Fig. 4B) and the other
cations (data not shown) was more effective at moderate decreases in
tonicity. These ions also decreased the passive
InsP
-independent Ca
leak with the same
order of potency (Fig. 4A, inset). We
therefore propose that Ca
was released through this
passive leak pathway. The overall inhibition by these cations was
relatively small and actually became even smaller when the
[Ni
] was increased from 2 to 10 mM (Fig. 4B, inset).
Figure 4:
Effect of divalent cations on the
hypotonically induced Ca release in permeabilized
A7r5 cells. A illustrates the effect of 2 mM BaCl
(
), MgCl
(
), MnCl
(
), CoCl
(
), and NiCl
(
) on the Ca
release induced by
removing mannitol. The addition of these ions is indicated by the horizontal line. KCl (3 mM) was added to the control
(
) for the same time period as the divalent ions to maintain
constant tonicity. The inset in A shows how the
passive leak was affected when the divalent ions were added from time 0
onward. B shows the effect of gradually lowering the mannitol
concentration in 40 steps of 3 mM reduction each, both in the
presence (
) and absence (
) of 2 mM MgCl
. The decrease of tonicity is schematically
presented by the thickness of the bar. The inset in B shows the hypotonically induced Ca
release
(mean ± S.E., n = 4) as a function of the
[NiCl
]. The increase in
[NiCl
] was balanced by a decrease in
[KCl].
The hypotonically
induced Ca release also occurred in intact A7r5 cells
incubated in Ca
-free medium. A 40% reduction in
extracellular osmolality induced a transient increase in intracellular
[Ca
] in 60% of the cells investigated (closed symbols in Fig. 5A). This
Ca
did not come from outside since the external
medium contained no Ca
. To discriminate whether this
[Ca
] increase represented a Ca
release from internal stores or an inhibited Ca
extrusion, we have investigated the effect of hypotonic stress on
the rate of
Ca
release from intact A7r5
cells. Fig. 5B shows an enhanced rate of
Ca
extrusion during the hypotonic shock,
indicating that Ca
release from intracellular stores
and not inhibition of the extrusion caused the
[Ca
] increase in the intact cell. 40% of
the cells showed no rise in intracellular
[Ca
] in response to the hypotonic challenge (open circles in Fig. 5A), although a
subsequent vasopressin stimulation (10 µM) could release
internal Ca
.
Figure 5:
Hypotonically induced Ca
release from intracellular stores in intact A7r5 cells during
incubation in Ca
-free medium. A illustrates
how the intracellular [Ca
] in 2 intact A7r5
cells was affected by leaving out 120 mM mannitol from the
Ca
-free modified Krebs solution (filled bar)
and by a subsequent application of 10 µM vasopressin (open bar). The closed circles are typical for 60% of
the cells, and the open circles for 40% of the cells
investigated (n = 90). B shows how removing
the 120 mM mannitol from the Ca
-free
modified Krebs solution affected the rate of
Ca
release from a monolayer of intact
A7r5 cells (n = 3).
We conclude that hypotonically
induced Ca release through the passive leak pathway
is an inherent property of the intracellular stores. Although this
phenomenon is not mediated by classical Ca
channels
such as InsP
receptors and ryanodine receptors, it is
possible that, in the intact cell, released Ca
subsequently activates phospholipase C or A
, thereby
generating InsP
or arachidonic acid and its metabolites.
These second messengers may provide a positive feedback loop for the
internal Ca
release or alternatively activate the
necessary mechanisms for volume recovery.