©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Hypotonically Induced Calcium Release from Intracellular Calcium Stores (*)

(Received for publication, November 27, 1995; and in revised form, January 4, 1996)

Ludwig Missiaen (§) Humbert De Smedt Jan B. Parys (¶) Ilse Sienaert (**) Sara Vanlingen Guy Droogmans Bernd Nilius Rik Casteels

From the Laboratorium voor Fysiologie, K. U. Leuven Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

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.


MATERIALS AND METHODS

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(2) 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^2) at a density of approximately 10^4 cells cm and for the intracellular [Ca] measurements in Coverglass Chambers (Nunc Inc., Naperville, IL) at a density of 5times10^4 cells cm.

Ca fluxes on monolayers of saponin-permeabilized A7r5 cells (3 times 10^5 cells/4-cm^2 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(2), 5 mM ATP, 0.44 mM EGTA, 10 mM NaN(3), 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(2)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 times 10^5 cells/4-cm^2 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(2), 11.6 mM Hepes (pH 7.3), 11.5 mM glucose, and 1.5 mM CaCl(2) (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(2), 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 times (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.


RESULTS AND DISCUSSION

Permeabilized A7r5 cells loaded to equilibrium with Ca slowly lost their Ca during incubation in an isoosmotic (300 mosm/kg H(2)O) Ca-free medium. A long-lasting reduction in medium osmolality to 180 mosm/kg H(2)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(2)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(2)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(3)) (^1)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 (circle), 40 mM (), or 0 mM (bullet) 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(3)-sensitive store, since the InsP(3)-insensitive compartment in permeabilized A7r5 cells contains only about 5% of the stored Ca(14) . Activation of the InsP(3) receptor by endogenous InsP(3) 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(3)-induced Ca release from partially depleted stores(14, 19, 20, 21) . A second argument against the involvement of the InsP(3) receptor is that 10 µM thimerosal, a pharmacological activator of InsP(3) receptors(22, 23, 24, 25, 26) , did not stimulate the hypotonically induced Ca release (Fig. 3B). A third argument indicating that the InsP(3) 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(3) receptor, which was found in control experiments to completely inhibit the Ca release in response to up to 3.2 µM InsP(3) (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(3)-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(3) 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 (bullet) or after 20 min (circle). B illustrates the effect of removing the mannitol in the absence (bullet) and in the presence of 10 µM thimerosal (box) or 10 µM ruthenium red (circle), added from time 0 onward. The inset shows the effect of removing the mannitol in the absence (bullet) and in the presence of 50 µg ml heparin (circle), 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(2) 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(3) 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(3)-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(2) (), MgCl(2) (circle), MnCl(2) (), CoCl(2) (), and NiCl(2) (box) 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 (bullet) 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 (circle) and absence (bullet) of 2 mM MgCl(2). 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(2)]. The increase in [NiCl(2)] 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(3) receptors and ryanodine receptors, it is possible that, in the intact cell, released Ca subsequently activates phospholipase C or A(2), thereby generating InsP(3) 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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 32-16-345-720; Fax: 32-16-345-991; ludwig.missiaen{at}med.kuleuven.ac.be.

Senior Research Assistant of the Belgian National Foundation for Scientific Research (N.F.W.O.).

**
Research Assistant of the Belgian National Foundation for Scientific Research (N.F.W.O.).

(^1)
The abbreviation used is: InsP(3), inositol 1,4,5-trisphosphate.


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©1996 by The American Society for Biochemistry and Molecular Biology, Inc.