Cell Calcium 3: 451-465, 1982

IIWSITOL LIPIDSANDHIMBRANB FmcTIoN IN-s

David Allan

Department of Experimental Pathology, The School of Medicine, University College London, University Street, London WClE 6JJ.

Although mammalian erythrocytes are relatively simple cells and possess only a limited ability to metabolise inositol lipids, the study of these cells has generated a surprisingly large number of different hypotheses seeking to establish an involvement of inositides in membrane function. Over the years, the red cell has provided a seed-bed for the development of some of the main ideas concerning the role of inositides in cells in general, but it must be confessed that there is no wide-spread agreement as to the major function of these lipids in erythrocytes themselves. However it has been repeatedly suggested that inositol lipids may in some way be involved in the regulation of Ca++ levels within cells and that even in such a rudimentary cell as the mature erythrocyte, Ca++ may influence membrane function.

This article will attempt to survey the variety of roles which have been suggested for inositides in erythrocytes and in some cases to indicate where information gleaned from these cells may be useful in the consideration of inositide function in other cell types.

A The physical and chemical properties of inositides

The inositides are distinguished from the other common glycero- phospholipids by their possession of a carbohydrate-containing head group (inositol) which has the potential to become phosphorylated. Their relatively hydrophilic character, together with the negative charge which they carry at all except very acid pH values, seems to make them interact with proteins more than other phospholipids do, and this feature, which applies particularly to PI-P and PI-P,, generally makes them more difficult to extract from tissue samples.

Thus while most phospholipids are easily extracted by neutral chloroform/methanol mixtures, extraction of inositides and especially PI-P and PI-P, by chloroform/methanol is complete only in the presence of strong acid (1) or detergent (2). It is worth emphasising that this feature is not simply a technical nuisance but could well be an indication that inositides are involved in significant interactions with protein in membranes under physiological conditions (3).

Abbreviations:- PI, phosphatidylinositol. PI-P, phosphatidylinositol 4-phosphate. PI-P,, phosphatidylinositol 4,5_bisphosphate.

451

The other major property of inositides arising from their anionic character is their capacity to bind metal cations, particularlg+those of the alkaline earth metals. The affinity of PI for Ca++ or Mg is very much higher than the major membrane lipids but is not radically different from that of phosphatidylserine (4,5). However PI-P, to bind Ca++ considerably more strongly (6) perhaps because Ca

$yes seem can be

chelated between the multiple phosphate groups of this lipid. It may be of some significance that whereas the Na and K salts of PI-P and PI-P, readily disperse in water to give relatively small micellar aggregates, Ca salts of these lipids are insoluble in water but are readily soluble in chloroform (6). In the hydrophobic environment represented by a membrane therefore, the stability of Caf+ complexes with PI-P or PI-P, could be enhanced compared with the situat+l;on in free aqueous solution. Suggestions that PI-P/PI-P, might bind Ca than Mg++

considerably more strongly have not been confirmed (7).

Consistent with their capacity to bind metal cations, inositides also appear to interact with various organic cations. PI binds a variety of cationic amphiphilic molecules including chlorpromazine and local anaesthetics (8,9) and such molecules will compete with Ca++ for binding to the lipid (8,lO). An aminoglycoside antibiotic, neomycin, also interacts rather specifically with PI-P, and there are suggestions that this compound may be useful to probe the function of PI-P, in various tissues (11).

B The inoeitide composition of erythrocytes

Although a considerable amount of data exists regarding the inositide composition of erythrocyte membranes, it is an unfortunate fact that much of the information is subject to uncertainty. This situation arises for several reasons. Firstly, many workers have relied on treatment of red cells with neutral chloroform/methanol mixtures which are adequate for the quantitative extraction of most phospholipids (including PI) but which only extract a small and variable fraction of PI-P/PI-P,. Secondly, treatment of intact erythrocytes with acid chloroform/methanol, which will satisfactorily extract PI-P/PI-P,, also extracts haem pigments and their breakdown products which besides complicating chromatographic separations, also catalyse peroxidation of the unsaturated fatty acids that are generally prominent components of inositides (12).Acid treatment may also cause degradation of plasmalogens although these are not generally major components of the inositides. Thirdly, PI-P and PI-P2 seem to undergo a rapid turnover of their phosphomonoester groups in intact erythrocytes (13) and there is a risk that in the process of membrane isolation where resynthesis is prevented these lipids could be degraded to PI. It has been claimed that virtually all of the inositol in metabolically-replete erythrocytes is in the form of PI-P and PI-P, and that observations of significant amounts of PI are essentially artefactual in origin (13,15).

The above factors would tend to increase the apparent relative proportions of PI to PI-P/PI-P,. This is probably reflected in many of

452

the published figures for inositide content of various erythrocytes which have tended to emphasise the PI content rather than the content of PI-P and PI-P, (16). Where the most careful determinations of inositide levels have been made (in human (17) and pig cells (13-15)) PI-P and PI-P, predominate, but at this stage it cannot be asserted that this is the true situation for other species as well.

C The metabolism of inositides in erythrocytes

Mammalian erythrocytes generally have a very restricted ability to synthesise lipids compared with nucleated cells. They have a negligible capacity to synthesise lipids de novo and are limited mainly to reactions involved in the turnover of fatty acyl groups (16). Headgroup turnover involving the phosphodiester bond which links the headgroup to the glycerol backbone does not occur in general. However turnover does occur of the phosphomonoester groups present in PI-P, PI-P, and phosphatidate and these lipids (but no others) can be labelled by incubation of whole cells with 32P (13,14,17). The enzymes which catalyse turnover of PI-P, PI-PI and phosphatidate (phosphohydrolases and kinases) appear generally to be membrane bound (14,18,19) although a recent careful examination of PI-P, phosphohydrolase in human erythrocytes has shown it to be present chiefly in the cytosol (20). This enzyme will also attack inositol trisphosphate but appears to be distinct from the membrane-bound inositol trisphosphate phosphomonoesterase described by ++Downes et al (21). The inositide-utilising kinases require Mg and the concentration of Mg++ is reported to influence the proportions of PI-P and PI-P, which are produced (22).

There is one reported exception to the rule that mammalian erythrocytes cannot accomplish lipid transformations which involve the phosphodiesief bond: in human and rabbit erythrocyte membranes there is a Ca -sensitive phosphodiesterase (phospholipase C) which specifically cleaves PI-P and PI-P, (but not PI) to give 1,2-diacylglycerol and inositol bis- and trisphosphates (17,23). The 1,2-diacylglycerol which remains in the membrane provides a substrate for the endogenous 1,2-diacylglycerol kinase, leading to the production of phosphatidate (24). The net effect of this process in whole cells is thus to convert PI-P and PI-P, into phosphatidate. Similar enzymes appear to be present in rabbit, rat and guinea-pig erythrocytes but the phosphodiesterase seems to be absent from ovine, swine, bovine and avian red cells (17,25).

Originally it appeared th$$ the phosphodiesterase of human erythrocytes might be activated at Ca concentrations similar to those prevailing within normal erythrocytes (c 1pM) (26). However more recent work using ionic conditions thought to be more physiologically appropriate has suggested that the phosphodiesterase is only switched on at Ca++ concentrations in excess of 10MM and therefore should not show significant activity under normal conditions (27,28). Neomycin appears to inhibit the phosphodiesterase (23) and also the phosphomonoesterase (20,29), probably by binding to PI-P, (11).

453

It is worth noting that a number of recent reports concerning the metabolism of PI-P/PI-P, in human erythrocytes are confused regarding the route of degradation of these lipids. In particular some authors have not clearly distinguished between the phosphomonoesterase activities which degrade PI-P/PI-P, to PI and the phosphodiesterase which removes the entire inositol bis-or trisphosphate headgroup (29-31).Measurement of decreases in "P-1abelling of inositides without characterisation of the products is clearly an unsatisfactory way of determining the nature of the breakdown process.

D Proposed functions of inositides in red cells

1 Interactions of inositides with proteins (a) ATPases One of the earliest proposals for a role of lipids in membrane function was due to the Hokins (30) who made the ingenious suggestion that the turnover of the phosphate groups in phosphatidate which is catalysed by phosphatidate phosphohydrolase and diglyceride kinase and which occurs at the expense of ATP breakdown, movements of Na+ and K+

might be linked to the vectorial across cell membranes. Although this idea soon

became untenable since the rate of ATP hydrolysis by the Na+, K+ ATPase was much too high to be accounted for by turnover of phosphatidate, (12) there is still the possibility that the apparently more rapid turnover of the phosphomonoester groups in PI-P and PI-P, could contribute a significant part of the total ATPase activity in erythrocyte membranes. The reported values for the rate of incorporation of phosphate into PI-P and PI-P, in pig erythrocytes seem however to be+lo+wer by at least an order of magnitude than the values measured for Na ,K -ATPase activities (13.14).

Although the contribution of lipid phosphomonester turnover to ATPase activity thus appears to be rather small, there are indications in the literature that inositides might have some functional role in the support of ATPase activities. An increase in membrane content of PI-P and PI-Pa is apparently correlated with Ca++ in pig erythrocytes (15) and Na+,

-sensitive ATPase activity K+-ATPase activity in canine kidney

membranes is inhibited by neomycin (an antibiotic thought to interact strongly with PI-P,) and restored by addition of inositides, but not by other lipids (33).

(b) Adenylate cyclase Low and Finean (34) have produced convincing evidence that adenylate cyclase of turkey erythrocytes has a requirement for PI. These workers used a phospholipase C specific for PI and showed that the activity of adenylate cyclase in turkey erythrocyte plasma membranes was reduced as PI was degraded. They did not however demonstrate that activity could be restored by adding back PI and it is possible that some change secondary to hydrolysis of PI (eg as structural alteration induced by introduction of diacylglycerol into the membrane) was responsible for the inhibition. This possibility is rendered less likely by the observation that treatment with a non-specific phospholipase C did not cause inhibition of the adenylate cyclase.

454

(c) Anchoring of proteins in membranes In a number of different tissues, breakdown of PI in membranes by a specific phospholipase C apparently leads to the release of certain enzymes from these membranes (35-38). It is implied by these experiments that PI might be involved in anchoring some peripherally located membrane-bound enzymes. One of the best examples of this situation is the release of acetylcholinesterase from intact pig erythrocytes (and to a lesser extent, human erythrocytes) when the cells are treated with low concentrations of a purified PI-specific phospholipase C from S.aureus (36). It should particularly be noted that although more than 95% of the total PI of pig erythrocytes seems to be located on the inner leaflet of the plasma membrane since almost all of the PI of the cells is available for phosphorylation, breakdown of the 5% which is accessible to the bacterial phospholipase C is sufficient to promote the release of virtually all of the acetylcholinesterase (36).

(2) Inositides as chelators of Ca++

As noted above, inositides and especially PI-P and PI-P, will bind Ca++ and it has been speculated that these lipids could contribute to the buffering of intracellular Ca++ concentrations. An obvious objection to this idea is that physical methods have generally failed to show a marked selectivity for Ca++ binding of Mg++ (6). Mg++

binding to inositides in comparison with is usually present in cells at an ionic

concentration three to four orders of magnitude higher than the Ca++ concentration and would be expected to compete with Ca++ in binding to inositides. However, experiments with swine (15,39) and bovine (39) erythrocyte membranes have shown a clear correlation between membrane content of PI-P/PI-P, and binding of Cat+ even in the presence of physiological concentrations of Mg++. In fact Mg++ was essential not only for synthesis of PI-P/PI-P, but also for ATP-sensitive Ca++ -binding, and this together with observation that Cat+-binding increased in parallel with PI-P/PI-P, synthesis strongly suggested that these lipids were the actual sites of ATP-sensitive binding of Ca++. In contrast bov$+ne erythrocyte membranes, which did not exhibit ATP- sensitive Ca -binding showed no ability to synthesise PI-P and PI-P, (39).

There was a considerable discrepancy between the values reported by the groups referred to above regarding the actual amounts of Ca++ bound by pig erythrocyte ghosts. However in general it seems that there is sufficient PI-P/PI-P, in erythrocytes and the binding content of these lipids for Ca++ . capacity for Ca

+‘+ high enough for them t_%provide significant buffering

internal Ca++ at the upper end (~10 M) of the physiological range

of concentrations significant in buffering Ca++

(15,39,40). If these lipids are then it might be predicted that a decrease

in their concentration in intact cells would be associated with a rise in Ca++ levels. Such an effect may be observed during energy depletion, for under these conditions PI-P/PI-P, concentrations should drop and there is evidence for a concomitant rise in Cat+ levels (e.g. there is an enhancement of Ca++-sensitive Kt efflux and some release of microvesicles) (41,42). Such an effect of energy depletion could however

455

be due to a reduction of Ca++ pump activity.

It should be noted that although PI-P and PI-P, can normally be resynthesised from PI in intact cells which are energy replete, there is one situation which could lead to a permanent loss of inositides from human erythrocytes. This is when a rise of internal Ca++ activates the phosphodiesterase which attacks PI-P and PI-P, (but not apparently PI) (17,231. There is no known route by which the cells could resynthesise the PI-P/PI-P, which is lost so that if these lipids are really potent intracellular chelators of Ca+', activation of the diesterase should lead to a greatly enhanced level of free Ca++ within the cells. In principle therefore, a small rise in intracellular Ca++, sufficient to activate the diesterase, could produce a much larger rise in Ca++ levels due to loss of inositide buffering capacity for Ca++. The water-soluble products of PI-P/PI-P, breakdown (inositol bis-and trisphosphates) may still have some buffering capacity but they are rapidly degraded by a cytosolic phosphomonoesterase (21).

Although it seems unlikely that Ca++ levels in normal cells would ever rise high enough to activate the phosphodiesterase, it is posT$ble that in senescent++cells with impaired energy metabolism or Ca -pumping activity, Ca could rise sufficiently high to set in train a kind of auto-destruct mechanism which begins wi:t breakdown of PI-P/PI-P,, continues with a catastrophic rise in Ca levels, and concludes with radical alterations to cell morphology (echinocytosis and loss of microvesicles) which lead finally to the elimination of the defective cells from the bloodstream. Moreover, in certain pathological conditions involving defective erythrocytes there are definite indications of an association between raised internal Ca++ levels and alterations in inositide metabolism which may be due to activation of the phosphodiesterase (31). There is a need for a more detailed examination of the relation between inositide content and pathological status of erythrocytes.

(3) Inositides and membrane resealing

One of the most remarkable features of biological membranes is their ability to reseal spontaneously after damage to the permeability barrier. By analogy with the resealing capacities of soap films, it seems most likely that resealing of membranes is largely a property of their lipid components. However there are some indications that resealing of lysed erythrocytes is influenced by Ca++ (eg ghosts prepared in the presen+c+e of EDTA or EGTA are leaky to macromolecules and ghosts prepared in Ca contain more intracellular protein (42)). There is a tenuous suggestion that Ca++ may modulate resealing by interacting with lipid components and in particular with inositides, but firm evidence for such an hypothesis is lacking. In red cells (44) which are permeabilised by an osmotic stress there is a very marked increase in "P in inositides, which may be related to resealing of these cells. However a more trivial explanation i.e. that enhanced turnover of ATP leads to a higher specific activity of "P in inositides, has not been discounted.

456

(4) Inositides and membrane fusion

In general, fusion of membranes requires firstly that two membrane surfaces be brought close together , secondly that membrane components which normally hinder fusion (e.g. certain proteins) are removed from the site of interaction of the membranes, and (perhaps) thirdly that some critical component which positively promotes fusion is introduced into the region of the membrane where fusion occurs. Since it seems likely that as with membrane resealing the essential feature of membrane fusion is a coalescence of lipidic phases, it is natural to consider the possibility that certain lipid components of membranes may be of particular significance in the control of membrane fusion.

A variety of membrane fusion events can occur in human red cells under certain circumstances (45). Fusion of the exterior faces of the membranes of intact cells can lead to either cell-cell fusion or to endocytosis, whilst fusion of cytoplasmic faces engenders the release of exovesicles (microvesicles). Since inositides are apparently localised largely in the cytoplasmic leaflet of the red cell plasma membrane it would be expected that if they have any role at all in the mediation of membrane fusion events in red cells then their influence would be most marked in fusion directly involving the cytoplasmic leaflet. It wz interesting to note therefore that in situations where cytoplasmic Ca concentrations were elevated in human cells and PI-P/PI-P, were broken down by the endogenous phosphodiesterase, there was a concomitant echinocytosis which rapidly led to membrane fusion and release of microvesicles (46). Originally it was suggested (45) that membrane fusion might be associated with inositide degradation because (a) removal of the large highly-charged headgroup of these lipids made close apposition of membrane surfaces energetically more feasible, (b) the 1,2-diacylglycerol produced might intrinsically be a fusogenic lipid or might promote localised membrane curvature which increased the likelihood of interactions between membrane surfaces, (c) the phosphatidate produced by phosphorylation of 1,2-diacylglycerol might itself have a fusogenic action as it appeared to have in model systems (47).

Subsequent work (48) has made it less likely that (b) and (c) are factors relevant to the Ca++ -induced release of microvesicles from human red cells but (a) has still not been discounted and is further supported by the observation that Ca++ -dependent release of microvesicles does not occur in the red cells of animals which appear to lack an inositide- specific phosphodiesterase (e.g. swine and bovine cells) (25). It is also relevant to note that removal of charged headgroups from phospholipids of the outer leaflet of the red cell plasma membrane (using an exogenous phospholipase C) promotes fusion of these outer surfaces to give fused cells (49) or endovesicles (50).

(5) Erythrocyte shape and membrane fluidity

The normal discoid shape of the mammalian erythrocyte appears to depend on the presence within cells (or ghosts) of Mg++ and ATP (51). Although there may be a direct requirement for ATP to be bound without hydrolysis

457

to some specific membrane site (51), it has generally been assumed that the Mg ATP requirement indicates that phosphorylation of a membrane component is essential for the maintenance of discoid morphology. Such a component could be a protein but it has recently been suggested that phosphorylation of inositides might be a crucial factor (30). This hypothesis is based on the observations that (a) phosphorylation of inositides occurs concomitantly with the assumption of discoid morphology and (b) agents which either bind to PI-P/PI-P2 (neomycin) or promote the degradation of these lipids (C!a++) prevent ghosts from becoming discoid in the presence of Mg++ and ATP. Viscosity changes in the ghost suspension parallelled the above alterations in morphology. This work is of some interest but suffers from a lack of precision in defining the actual metabolic changes undergone by PI-P/PI-P, and also takes no account of the possibility that the effects of Ca++ could include degradation of ATP by stimulation of Ca++-dependent ATPase activity.

Recent work by Sheetz and coworkers (52) has supported the interesting suggestion that the fluidity of the lipid bilayer portion of the human red cell membrane can be influenced by PI-P,. These workers showed that addition of PI-P, to membrane appeared to increase the mobility of membrane glycoproteins. One difficulty with this type of experiment is that it is not clear how much exogenously-added PI-P, is bound to the membrane or whether it adopts a position in the membrane which is analogous to that of endogenous PI-P,. It would be interesting to know if bilayer fluidity is influenced by changes in endogenous PIP, content, making use of the kinase, phosphomonoesterase and phosphodiesterase activities (referred to above) to alter the endogenous levels of inositides.

The relevance of erythrocyte studies to other cell types

Out of the variety of suggestions that have been put forward for a role of inositides in erythrocyte membrane function, a few may be of particular significance for other kinds of cells.Perhaps the most interesting possibility stems from the indications that PI-P/PI-P2 may be able to buffer intracellular Ca+' concentrations so that alterations in the amounts of these lipids potentially could control Ca++ levels of within cells. Paradoxically, in the human erythrocyte it seems that the best-defined process in which degradation of PI-P/PI-P, occurs (i.e. by the phosphodiesterase pathway) is itself dependent on a rise in intracellular Ca++ that Ca++

concentration, but in other cell types it is possible -in+d+ependent breakdown of inositides could lead to increases in

internal Ca levels which could then trigger physiological responses. According to this hypothesis, binding of an appropriate agonist to a cell-surface receptor would stimulate breakdown of inositi+d+es in the plasma membrane thus decreasing buffering of intracellular Ca and con- sequently promoting physiological responses which depend on Ca ++ (e.g. secretion).

Such an hypothesis may be consistent with the ideas put forward by Michell (53,54) where it was suggested originally that receptor-mediated breakdown of PI might be a key event in the mechanism of stimulus-

458

response coupling in++ tissues where activation by an extracellular agonist involves Ca mobilisation. considered Ca++

Although Michell has mainly mobilisation from outside the cell there is evidence

from many tissues that Ca++ from internal as well as external sources may be utilised. Recent work (55) has also suggested that breakdown of PI-P, rather than PI may be the process most directly related to stimulus-response coupling, although if PI/PI-P/PI-P, are really as closely coupled metabolically as has been inferred (see above), then breakdown of any of these lipids will rapidly be transmitted to the others.

The data regarding the inositide composition of erythrocytes may be significant for other cell types. If other cells are comparable to erythrocytes, then their plasma membranes may contain very little PI; instead the major inositides would be PI-P/PI-P, and it would be the metabolism of these higher inositides rather than metabolism of PI directly which would seem most likely to be involved in mediation of stimulus-response coupling at the plasma membrane. Receptor-mediated inositide breakdown results in a reciprocal accumulation of phosphatidate in many tissues, due to the action of diacylglycerol kinase. Phosphatidate is of particular interest since it has been implicated as a potential ionophore for Ca++ (56,56) while PI has actually been reported to inhibit this ionophoric property of phosphatidate in a model system (56).

A diagram incorporating these ideas is shown below. According to this scheme, receptor-mediated breakdo!: of inositides in plasma membrane not only mobilises intracellular Ca , but leads to the formation of a product (phospha:$date) which may increase cell membrane permeability to extracellular Ca .

PI \

PI-P + 1,2-diacylglycerol w phosphatidate

PI-P,

Decrease in Ca++ buffering increase in membrane permeability to Ca++

Increase in intracellular [Ca++]

Largely on the basis of experiments in model systems it has also been suggested that diacylglycerol (45) and/or phosphatidate (47) might be able to promote fusion between membranes. These lipids are normally present in membranes only on tiny quantities so that a small mass increase in their amount produces a large relative increase and this could be consistent with some kind of control function for them. Moreover, the relatively hydrophobic character of these lipids which is

459

due to their small headgroups could decrease energy barriers to fusion. Conversely the presence of large hydrophobic groups as exist in the intact inositides could act as a natural barrier to fusion.These speculations have not been easy to test in cells other than erythrocytes largely because no effective inhibitor has been found for the diesterases which cause the breakdown of inositides in many cell types.

It has however been possible to test the potential role of inositides in anchoring proteins to membranes in a variety of non-erythrocytic cells (34-38). The results suggest that a number of peripherally-located membrane enzymes including alkaline phosphatase and 5' nucleotidase are attached to membranes through some kind of linkage to PI. Most studies of this kind have concerned enzymes which are located on the outer face of the plasma membrane (eg 5' nucleotidase end acetylcholinesterase) whereas most of the PI of cells resides in the cell interior, and this contrast raises the possibility that the binding of important intracellular enzymes to membranes may depend on the presence of PI in these membranes. The binding (and therefore possibly the activity) of such enzymes could even be modulated by agonist-sensitive breakdown of PI under physiological conditions. It would in particular be interesting to know whether or not the inhibition of adenylate cyclase by PI-specific phospholipase C (34,59) is related to the release of protein components from the plasma membrane.

Finally, it is worth bearing in mind the possibility that inositides in red cells have no significant function in the mature cells but instead represent a relic of some former function in erythrocytic progenitor cells (28). If this is so, the study of inositide composition and metabolism in mature cells may lead to a greater understanding of the role of inositides in these more metabolically active precursor cells.

REFERENCES

1 Dawson,R.M.C. and Eichberg,J. (1965). Diphosphoinositide triphosphoinositide in animal tissues. Biochemical Journal 634-643.

and

96,

2 Grove,R.I., Fitzpatrick,D. and Schimme1,S.D. (1981). Effects of Ca++ on triphosphoinositide extraction in fusing myoblasts. Lipids 16, 691-693.

3 Palmer,F.B. and Dawson,R,M.C. (1969) Complex formation between triphosphoinositide and experimental allergic encephalitogenic protein. Biochemical Journal 111, 637-646.

4 Abramson,M.B. Colacicco,G., Curci,R. and Rapport,M. (1968) Ionic properties of acidic lipids. Phosphatidylinositol. Biochemistry 7, 1692-1698.

5 Hauser,H., Chapman,D. and Dawson,R.M.C. (1969) Ca++ binding to monolayers of phosphatidylserine and phosphatidylinositol. Biochimica et Biophysics Acta 183 320-333.

460

6

7

8

9

10

11

12

13

14

15

16

17

18

Hendrickson,H.S. (1969) Physical properties and interactions of phosphoinositides. Annals of the New York Academy of Sciences 165, 668-676.

Henderson,H.S. and Reinertsen,J.L. (1969) Comparison of metal binding properties of 1,2 cyclohexanediol diphosphate and deacylated phosphoinositides. Biochemistry 12, 4855-4858.

Feinstein,M.B. (1964) Reaction of local anaesthetics with phospholipids. Journal of General Physiology 48, 357-374.

Allan,D, and Michel1,R.H. (1975) Enhanced synthesis de novo of phosphatidylinositol in lymphocytes treated with cationic amphiphilic drugs. Biochemical Journal 148 471-478.

Hauser,H, and Dawson,R.M.C. (1969). The displacement of Ca++ from phospholipid monolayers by phemacologically active and other organic bases. Biochemical Journal 109 909-916.

Shahid,L., Weiner,N.D. and Schacht,J. (1979) Interactions of neomycin with monomolecular films of polyphosphoinositides and other lipids. Biochimica et Biophysics Acta, 567, l-8.

Hawthorne,J.N. and White,D.A. (1975) Myoinositol lipids. Vitamins & Hormones 33, 529-573.

Peterson,S.C. and Kirschner,L.B. (1970) Di-and triphosphoinositide metabolism in intact swine erythrocytes. Biochimica et Biophysics Acta 202, 295-304.

Schneider,R.P. and Kirschner,L.B. (1970) Di-and triphosphoinositide metabolism in swine erythrocyte membranes. Biochimica et Biophysics Acta 202, 283-294.

Buckley,J.T. and Hawthorne,J.N. (1972) Erythrocyte membrane polyphosphoinositide metabolism and the regulation of calcium binding. Journal of Biological Chemistry 247, 7218-7223.

Nelson,G.J. (1972) Lipid composition and metabolism of erythrocytes In "Blood Lipids and Lipoproteins: Quantitation, Composition and Metabolism" ed. Nelson,G.J., Wiley-Interscience, New York. pp 317-385.

Allan,D. end Michel1,R.H. (1978) A Ca++ -activated polyphos- phoinositide phosphodiesterase in the plasma membrane of human and rabbit erythrocytes. Biochimica et Biophysics Acta 508, 277-286.

Hokin,L.E. and Hokin,M.R. (1963) Diglyceride kinase and other pathways for phosphatidic acid synthesis in the erythrocyte membrane. Biochimica et Biophysics Acta 67, 470-484.

461

19

20

21

22

23

24

25

26

27

28

29

30

Hokin,L.E., Hokin,M.R. and Mathison,D. (1963) Phosphatidic acid phosphatase in the erythrocyte membrane. Biochimica et Biophysics Acta 67, 485-497.

Roach,P.D. and Palmer,F.B.St.C. (1981) Human erythrocyte cytosol phosphatidylinositol bisphosphate phosphatase. Biochimica et Biophysics Acta 661, 323-333.

Downes,C.P., Mussat,M.C. and Michel1,R.H. (1982). The inositol trisphosphate phosphomonoesterase of the human erythrocyte membrane. Biochemical Journal 203, 169-177.

Marche,P., Koutouzov,S. and Meyer,P. (1982). Metabolism of phosphoinositides in the rat erythrocyte membrane. Biochimica et Biophysics Acta 710, 332-340.

Downes,C.P, end Michel1,R.H. (1981). The polyphosphoinositide phosphodiesterase of erythrocyte membranes. Biochemical Journal 198, 133-140.

Allan,D., Watts,R. and Michel1,R.H. (1976) Production of 1,2-dia- cylglycerol and phosphatidate in human erythrocytes treated with calcium ions and ionophore A23187. Biochemical Journal 156, 225-232.

Allan,D. and Michel1,R.H. (1977) Ca++-dependent diacylglycerol accumulation in erythrocytes is associated with microvesiculation but not with K+efflux. Biochemical Journal 166, 495-499.

Allan,D. and Michel1,R.H. (1976) Production of 1,2-diacylglycerol in human erythrocyte membranes exposed to low concentrations of calcium ions. Biochimica et Biophysics Acta 455, 824-830.

Allan,D. and Thomas,P. (1981) the effects of Ca++and Sr++ on Ca++ -sensitive biochemical changes in human erythrocytes and their membranes. Biochemical Journal 198, 441-445.

Downes,C.P. and Michel1,R.H. (1982) The control by Ca++ of the polyphosphoinositide phosphodiesterase and the Ca++-pump ATPase in human erythrocytes. Biochemical Journal 202, 53-58.

Lang,V., Pryhitka,G. and Buckley,J.T. (1977) Effect of neomycin and ionophore A23187 on ATP levels and turnover of polyphosphoinositides in human erythrocytes. Canadian Journal of Biochemistry 55, 1007-1012.

Quist,E.E. and Reece,K.L. (1980) The role of diphosphatidylinositol in erythrocyte membrane shape change. Biochemical and Biophysical Research Communications 95, 1023-1030.

462

31

32

33

34

35

36

37

38

39

40

41

42

43

Ponnappa,B.C., Greenquist,A.C. and Shohet,S.B. (1980). Calcium- induced changes in polyphosphoinositides and phosphatidate in normal erythrocytes, sickle cells and hereditary pyropoikilocytes. Biochimica et Biophysics Acta 598, 494-501.

Hokin,L.E. and Hokin,M.R. (1961) Diglyceride kinase and phosphatidic acid phosphatase in erythrocyte membranes. Nature (London) 189, 836-837.

Lipsky,J.J. and Lietman,P.S. (1980) Neomycin inhibition of adenosine triphosphatase: evidence for a neomycin-phospholipid interaction. Antimicrobial Agents and Chemotherapy 18, 532-535.

Low,M.G. and Finean,J.B. (1977) Effects of phosphatidylinositol- specific phospholipase C from S. aureus on plasma membrane enzymes. Biochemical Society Transactions 5, 1131-1132.

Low,M.G. and Finean,J.B. (1977) Release of alkaline phosphatase from membranes by a phosphatidylinositol-specific phospholipase C. Biochemical Journal 167, 281-284.

Low,M.G. and Finean,J.B. (1977) Non-lytic release of acetyl- cholinesterase from erythrocytes by a phosphatidylinositol-specific phospholipase C. FEBS Letters 82, 143-146.

Low,M.G. and Finean,J.B. (1973) Specific release of plasma membrane enzymes by a phosphatidylinositol-specific phospholipase C. Biochimica et Biophysics Acta 508, 565-570.

Ikezawa,H., Yamanegi,M., Taguchi,R., Miyashita,T. and 0hyabu.T (1976) Studies on phosphatidylinositol phosphodiesterase of B.cereus I. Purification, properties and phosphatase-releasing activity. Biochimica et Biophysics Acta 450, 154-164.

Kawaguchi,T and Konishi,K. (1980) Relation between phosphorylation and adenosine triphosphate-dependent Ca++ binding of swine and bovine erythrocyte membranes. Biochimica et Biophysics Acta 597, 577-586.

Harrison,P.G. and Long,C. (1968) The calcium content of human erythrocytes. Journal of Physiology (London) 199, 367-381.

Lew,V.L. & Ferreira,H.G. (1978) Calcium transport and the properties of a calcium-activated potassium channel in red cell membranes. Current Topics in Membranes and Transport 10, 217-277.

Muller,H., Schmidt,V. and Lutz,H.A. (1981) On the mechanism of vesicle release from ATP-depleted human red blood cells. Biochimica et Biophysics Acta 849, 462-470.

Bram1ey.T.A. Mg++,

and Coleman,R. (1972) Effects of inclusion of Ca++, EDTA or EGTA during the preparation of erythrocyte ghosts by

hypotonic haemolysis. Biochimica et Biophysics Acta 290, 219-228.

463

44 Redman,C.M. (1971) Phospholipid metabolism in intact and modified erythrocyte membranes. Journal of Cell Biology 49, 35-49.

45

46

47

48

49

50

51

52

53

54

55

Allan,D. and Michel1,R.H. (1979) The possible role of lipids in control of membrane fusion during secretion. Symposia of the Society for Experimental Biology 33, 323-336.

Allan,D., Billah,M.M., Finean.J.B. and Michel1,R.H. (1976) Release of diacylglycerol-enriched vesicles from erythrocytes with increased intracellular Ca++. Nature (London) 261, 58-60.

Sundler,R. and Papahadjopoulos,D. (1981) Control of membrane fusion by phospholipid head groups. Biochimica et Biophysics Acta, 849, 743-750.

Allan,D. and Thomas,P. (1981). Ca++ -induced biochemical changes in human erythrocytes and their relation to microvesiculation. Biochemical Journal 198, 443-440.

Peretz,H., Toister,Z., Laster,J. and Loyter,A. (1974) Fusion of intact human erythrocytes and erythrocyte ghosts. Journal of Cell Biology 63, l-11.

Allan,D., Low,M.G., Finean,J.B. and Michel1,R.H. (1975) Changes in lipid metabolism and cell morphology following attack by phospholipase C on red cells and lymphocytes. Biochimica et Biophysics Acta 413, 309-16.

Shukla,S.D., Billah,M.M., Coleman,R., Finean,J.B. and Michel1,R.H. (1978) Modulation of the organisation of erythrocyte membrane phospholipids by cytoplasmic ATP. Biochimica et Biophysics Acta 509, 48-57.

Sheetz,M.P., Febbroriello,P. and Koppe1,D.E. (1982) Triphospho- inositide increases glycoprotein lateral mobility in erythrocyte. membranes Nature (London) 296, 91-93.

Michel1,R.H. (1975) Inositol lipids and cell-surface receptor function. Biochimica et Biophysics Acta 415, 81-147.

Michel1,R.H. (1979) Inositol lipids in membrane fWtiOn. Trends in Biochemical Sciences 4, 128-131.

Michell,R.H., Kirk,C.J., Jones,L.M., Downes,C.P. and Creba,J.A. (1981) The stimulation of inositol lipid metabolism that accompanies calcium mobilisation in stimulated cells: defined characteristics and unanswered questions. Philosophical Transactions of The Royal Society of London B298, 123-137.

464

56 Tyson,C.A., Zande,H.V. and Green,D.E. (1976) Phospholipids as ionophores. Journal of Biological Chemistry 251, 1326-1332.

57 Putney,J.W.Jr., Weiss,S.J., van der Walle,C.M. and Haddas,R.A. (1980) Is phosphatidic acid a calcium ionophore under neurohumoral control? Nature (London) 284. 345-347.

58 Salmon,D.M. and Honeyman,T.W. (1980) Proposed mechanism of cholinergic action in smooth muscle. Nature (London) 284, 344-345.

59 Panagia,V., Michiel,D.F., Dhalla,K.S., Nijjar,M.S. and Dhalla,N.S. (1981) Role of phosphatidylinositol in basal adenyl cyclase activity of rat heart sarcolemma. Biochimica et Biophysics Acta 676, 395-400.

Received 4.10.82 Accepted 5.10.82

465