Hypotonicity Stimulates Phosphatidylcholine Hydrolysis and Generates Diacylglycerol in Erythrocytes”

Exposure of skate erythrocytes to hypotonic medium stimulates a rapid increase in levels of 1,2-diacylglycerol. Other treatments which produce cell swelling such as replacement of a portion of medium NaCl with the permeant solutes ethylene glycol or ammonium chloride also stimulate increases in diacylglycerol. Whereas the reduction of medium osmolarity to 460 mosm (from 940) stimulated a persistent diacylglycerol increase, the increase after reduction to 660 mosm was transient, peaking at 2.5 min and then slowly declining. This decline could be prevented by preincubation with the diacylglycerol kinase inhibitor R59022. To investigate the source of the increased diacylglycerol, the rate of incorporation of [32P]PO4 into each major phospholipid was measured. Reduction of osmolarity to 660 mosm stimulated the incorporation of phosphate into phosphatidylcholine markedly, with a smaller increase observed into phosphatidylinositol. To demonstrate phosphatidylcholine hydrolysis, erythrocytes were prelabeled with [32P]PO4. Subsequent exposure to hypotonic (660 mosm) medium stimulated a decrease in radioactivity in phosphatidylcholine and a large increase in radioactivity in phosphatidic acid. When stimulated in the presence of ethanol, 32PO4-labeled phosphatidylethanol was formed, suggesting activation of phospholipase D. In addition, the initial formation of 32PO4-labeled phosphatidic acid was not sensitive to inhibition of diacylglycerol kinase, supporting the role of direct activation of phospholipase D. These results indicate that hypotonicity and the accompanying cell swelling induce cell membrane phospholipid turnover, predominantly phosphatidylcholine, and production of the protein kinase C activator, diacylglycerol, which appears to occur via activation of phospholipase D.

D. In addition, the initial formation of 32P04-labeled phosphatidic acid was not sensitive to inhibition of diacylglycerol kinase, supporting the role of direct activation of phospholipase D. These results indicate that hypotonicity and the accompanying cell swelling induce cell membrane phospholipid turnover, predominantly phosphatidylcholine, and production of the protein kinase C activator, diacylglycerol, which appears to occur via activation of phospholipase D.
Exposure of little skate (Ruj, erinacea) erythrocytes to hypotonic medium stimulates influx of water, increasing cell volume followed by a regulatory volume decrease (l-4). The regulatory volume decrease is due to efflux of cellular osmolytes, including the potassium and the p-amino acid taurine, accompanied by cell water. If a portion of medium NaCl is replaced by the permeant solute ethylene glycol, a rapid and  (3). When a portion of medium NaCl is replaced by NH&l, cell volume increases, but the increase is not rapid and is of smaller magnitude than with either ethylene glycol-containing medium or hypotonic shock (3). The stimulation of taurine efflux by hypotonicity can be mimicked by the addition of either phorbol ester or the divalent ionophore A23187 in standard (940 mosm) medium (1). Reduction of osmolarity from 940 to 660 mosm stimulates both the generation of inositol l-phosphate and the rate of incorporation of exogenous arachidonic acid into 1,2-diacylglycerol, suggesting generation of diacylglycerol (2). Thus a possible mechanism responsible for the stimulation of potassium and taurine efflux may be DAG' generation from membrane phospholipids and activation of protein kinase C.
Increased phospholipid metabolism has been demonstrated as the signal transduction mechanism of many neurohumoral agents (5-7) and as a response to hypotonic exposure (8). Not only can membrane phosphatidylinositols be hydrolyzed by stimuli (8-11 as limited examples), but also phosphatidylethanolamine (12) and phosphatidylcholine (13-23). In addition, although not neurohumoral, calcium ionophores and the protein kinase C activators phorbol esters are known to stimulate phosphatidylcholine metabolism in a number of cell systems (19,20,22,23). Both synthetic and degradative processes for phosphatidylcholine can be stimulated (18). Relevant to the generation of diacylglycerol, it has recently been demonstrated that phosphatidylcholine-"specific" phospholipase D activity followed by a phosphatidic acid hydrolase, rather than direct activation of phospholipase C activity, appears to be the major pathway for diacylglycerol formation in dimethyl sulfoxide-differentiated HL-60 granulocytes (17, 20) as well as human neutrophils (19,21,23) stimulated with chemotactic peptide.
The following study was undertaken to determine whether conditions that induce swelling of skate erythrocytes might stimulate phospholipid turnover and diacylglycerol production. Using a sensitive mass quantitative technique to measure cell DAG with recombinant DAG kinase (24), we determined whether the increased incorporation of ['%]arachidonic acid into DAG found previously (2) is accompanied by an increase in concentration of DAG. As a measure of phospholipid turnover, rates of incorporation of phosphate into membrane phospholipid were assayed to determine the phospholipid source of the stimulated levels of DAG. To directly determine whether hydrolysis occurred, cells were prelabeled with [3'P] PO, and decreases in radioactivity in phospholipids deter- The addition of HCl in the extraction procedure did not cause significant acyl migration over the period used (15 min). Samples were always extracted as rapidly as possible and analvsis of DAG nerformed on the same dav. Omitting HCl from the extraction procedure and including NaCl had no effect-on dioleyl-DAG standards measured by the assay (data not shown).
To determine that drying the samples in a vacuum oven did not affect the recovery of DAG due to oxidation, 100 ng of dioleyl-DAG and sn-lstearoyl-2-arachidonyl-DAG were extracted and DAG measured concurrently.
In  (Fig. LA). DAG elevation occurs in medium reduced to either one-half (460 mosm) or twothirds (660 mosm). Stimulation of DAG levels is rapid (significant at 40 s) and persistent, lasting over 1 h after stimulation. The maximal elevations stimulated by either change in osmolarity are not statistically different; however, the rise stimulated by reducing osmolarity to 660 mosm does not persist as long as the elevation stimulated by the further reduction of osmolarity to 460 mosm. To determine whether endogenous DAG kinase activity may be responsible for this decline, cells were preincubated with a DAG kinase inhibitor R59022 (26) and then exposed to reduced (660 mosm tiated (to a small degree) the elevation of cell DAG and also prevented subsequent metabolism back to basal levels ( Fig.   1B).
To determine whether other media alterations which also cause cell swelling stimulate DAG formation, erythrocytes were transferred to media where a portion of the NaCl was replaced with either ethylene glycol or NH&l. Both of these agents have been shown to stimulate cell swelling in skate erythrocytes, but to differing degrees and with different kinetics (3). Ethylene glycol penetrates the cell rapidly and stimulates a rapid, large increase in cell volume which maintained for at least 1 h. NH&l does not stimulate cell swelling until 5-15 min after exposure to the salt, and swelling occurs more slowly. Exposure of skate erythrocytes to medium with ethylene glycol stimulates a rapid and persistent increase of DAG, whereas the stimulation of DAG by NH&l is slower in onset and smaller in magnitude (Fig. 1C). For the alterations above it appears that the time course of elevation of DAG correlates with the time of onset and speed of cell swelling.

Phospholipid
Source of DAG-To determine whether phospholipid turnover may be responsible as a source for the DAG, the rate of incorporation of [32P]P04 was measured into all phospholipid classes following transfer of erythrocytes to 660 mosm EIM. As shown in Fig. 2 phosphatidylinositol. Stimulation of skate erythrocytes stimulates the formation of inositol 1-monophosphate without the generation of inositol 1,4-bisphosphate or inositol 1,4,5trisphosphate (2), a finding which is supported by lack of stimulation in the rate of 32P04 incorporation into phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5bisphosphate. The turnover of phospholipid suggests that phosphatidylcholine and to some extent phosphatidylinositol may be sources of the DAG formed after osmotic stress and cell swelling in these cells.
Hydrolysis of Phosphatidylcholine and Generation of Phosphutidic Acid and Phosphutidylethanol-To directly demonstrate that hydrolysis of phosphatidylcholine occurred, we initially attempted to label cells with [3H]choline. Incorporation of the label was minimal over 4 h; therefore, the erythrocytes were prelabeled with [32P]P04. After labeling for 4 h at 20% hematocrit in 940 mosm EIM, cells were diluted lofold into either 940 or 660 mosm EIM. Samples were removed at varying times, lipids extracted and analyzed for the major phospholipids as well as phosphatidic acid as described under "Experimental Procedures." Exposure to hypotonicity stimulated a rapid decrease in label in phosphatidycholine (Fig.  3A). The decrease in phosphatidylcholine was paralleled by an increase in the label in phosphatidic acid (Fig. 3B). The increase in phosphatidic acid accounted for a majority (>90%) of the early decrease of phosphatidylcholine but did not account for the decrease at later times (>5 min), perhaps due to metabolism of the phosphatidic acid.
To determine if phospholipase D was involved in the hydrolysis of phosphatidylcholine, ethanol was included in the incubation reactions. It has been demonstrated by a number of investigators that in the phospholipase D-mediated hydrolysis of phospholipids in the presence of ethanol transphosphatidylation can occur, resulting in the formation of phosphatidylethanol (17,19,22). Formation of phosphatidylethanol can only occur via this pathway (22) and not via phospholipase C followed by modification of the resulting diacylglycerol. When 32P04 -prelabeled erythrocytes were exposed to hypotonic medium in the presence of 0.5% ethanol, both 32P04-labeled phosphatidic acid and phosphatidylethanol were formed (Fig. 4). Thus it appears likely that the diacylglycerol formed results from the actions of phospholipase D followed by a phosphohydrolase (21). As further evidence that the labeled phosphatidic acid formed was generated by phospholipase D-mediated hydrolysis, cells were exposed to hypotonicity in the presence of the diacylglycerol kinase inhibitor R59022. If a phosphoiipase C were involved followed by phosphorylation, then R59022 should greatly decrease the amount of labeled phosphatidic acid formed. Incubation with 10 pM R59022, however, did not significantly alter the amount of labeled phosphatidic acid formed (Fig. 5), suggesting that a phospholipase C/diacylglycerol kinase pathway was not involved. DISCUSSION A number of changes in the environment of the erythrocyte may stimulate cell swelling and subsequent responses to return back to a preselected, set volume, the regulatory volume FIG decrease (l-4). Skate erythrocytes take up water and swell rapidly after exposure to hypotonicity or to permeant solutes. They react to this change by increased efflux of two major cell osmolytes, potassium and the p-amino acid taurine (1,4). The mechanism(s) which mediate this stimulation of osmolyte efflux are not completely understood. Within 1 min after exposure to hypotonicity, the formation of inositol l-mono-phosphate, but not 1,4-bisphosphate or 1,4,5-trisphosphate, is stimulated along with the rate of incorporation of arachidonic acid into DAG (2) as well as an increase in concentration of DAG (Fig. 1). The generation of DAG, an activator of protein kinase C, suggests that protien kinase C may be involved in the alteration of cell transport to combat increased volume. Indeed, stimulation of skate erythrocytes in isoosmotic medium (940 mosm) with phorbol ester mimics the effect of hypotonic exposure (1). This effect can also be duplicated by the divalent ionophore A23187, suggesting that alterations in intracellular calcium may also be involved (1). A number of neurohumoral agents are known to stimulate phospholipid turnover and to be pivotal for cellular activation (for a review, Refs. 6 and 7). Early emphasis focused on the well characterized phosphatidylinositol cycle by phospholipase C-mediated breakdown of inositol containing membrane phospholipids, directly generating DAG and inositol phosphates (5, 6). One of the inositol phosphates, inositol 1,4,5trisphosphate, was shown to release calcium from an intracellular calcium store. Initially it was thought to be only a portion of the endoplasmic reticulum (27); however, multiple organelles such as the calciosome now appear to be involved in calcium regulation (28). In skate erythrocytes, no stimulated increase of inositol 1,4,5-trisphosphate was observed (2) nor elevation of cytosolic calcium (29), which might be expected if inositol 1,4,5-trisphosphate were generated. Thus phospholipase C activity on phosphatidyl inositol(s) could not explain the intracellular signals involved in responding to hypotonicity.
When stimulated by hypotonicity, the predominant phospholipid which showed increased incorporation of [32P]P04 was phosphatidylcholine (Fig. 2). This suggests that phosphatidylcholine turnover might mediate the effects of hypotonic exposure in these cells. To demonstrate that phosphatidylcholine breakdown occurred, cells prelabeled with [32P]P04 were exposed to hypotonic conditions and a decrease in the radioactivity in phosphatidylcholine was in fact observed (Fig.  3). Although not shown, phosphatidylinositol showed a small ((0.5%) decrease in radioactivity and no changes were observed in phosphatidylethanolamine or phosphatidylserine. Thus, phosphatidylcholine appears to be the major source of the DAG generated after hypotonic shock.
The generation of DAG from phosphatidylcholine could occur by the action of phospholipase C directly or by the action of phospholipase D followed by a phosphohydrolase. The latter pathway appears to be the major source of DAG generated in human neutrophils after stimulation with the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (21, 23). Phospholipase D activities have been shown to be stimulated by a variety of neurohumoral and other agents (16,(18)(19)(20). The rapid generation of phosphatidic acid in [32P]P04 prelabeled cells (maximal at 1 min) appears to precede the changes in DAG (maximal at 2 min). To demonstrate that phospholipase D is involved, we used the specific phospholipase D-mediated transphosphatidylation reaction. Phospholipase D can mediate the generation of phosphatidylethanol. This reaction has been best characterized in the phospholipase D-mediated breakdown of phosphatidylcholine in neutrophils and HL-60 granulocytes (17,(20)(21)(22). Phosphatidylethanol can only be formed by this reaction (23). Thus phosphatidylethanol formation is strong evidence that phospholipase D mediates the hydrolysis of a membrane phospholipid. A rapid increase in phosphatidylethanol was observed in erythrocytes exposed to hypotonicity in the presence of ethanol. Thus it appears that the phosphatidic acid formed is generated directly from phosphatidylcholine by the action of phospholipase D. Subsequently, a phosphohydrolase would convert the phosphatidic acid to diacylglycerol. We also have demonstrated that the early rise of phosphatidic acid formed does not result from the action of a diacylglycerol kinase (Fig.  5). Using the diacylglycerol kinase inhibitor R59022 we did not observe significant decrease in the phosphatidic acid formed. This result suggests that phospholipase C activity followed by diacylglycerol kinase does not account for the phosphatidic acid formed. Along with the formation of phosphatidylethanol, the data strongly indicate a role for phospholipase D in DAG formation.
In summary, the present results suggest that cell swelling stimulates DAG formation, probably via phospholipase D which prefers phosphatidylcholine as its substrate. The generation of DAG is required to activate protein kinase C. The activated kinase may have phosphoprotein targets which are the transporters responsible for stimulated efflux of potassium and taurine. Although not fully characterized, these transporters may be: 1) a KC1 cotransporter (for K') which is well characterized to mediate the volume decrease in erythrocytes of many species (4,30,31) or possibly Na'/H+ or K'/ H+ exchangers which are believed to mediate the regulatory volume decrease in Amphiuma red blood cells (32); and 2) the anion transporter band III which appears to be responsible for the stimulated efflux of taurine (33). Whether these transport activities are regulated by protein kinase C in skate erythrocytes is currently unknown.