European Journal of Pharmacolog}, - Molecular Pharmacology Section, 188 ( 19~q~ I 399-401 399 Elsevier

EJPMOL 80020

Sh,~rl communication

Hydrolysis of polyphosphoinosifides in asiroeytes by p|atelet-activating factor

Scan Murphy and Greg WeN

Department of Phurmaeolo9"o; College c:.: Medicine. Unt~,er,:ily o] Iowa, Iowa Cio; IA 52242. L\S.A.

Received 10 Jmnuary I990, accepted t5 February t990

In primary astroeyte cultures, picomolar concemrations of p!atelet-activating factor (1-O-alky]-2-acetyl-sn-giycero- 3-phosphoeholine, PAF) evoked th,z formation of inositol phosphates (InsP), including inositot tnsphospttate. This effect was not observed with the biologically inert lyso-PAF, nor in cells pretreated with phorbol mynsta~e acetate to downregulate receptors. PAF at concentrations >~ 10 -9 M did not elevate lnsP, suggesting some form of unco~pling of the receptor from phospholipase C. The responsiveness of astrocytes to PAF is further evidence for !he role of the~e cells in the central nervous system response to trauma.

Platelet-activating factor (PAF); Astrocyte: Ca2+: Phesphoinc sitides; (Trauma); (Receptor)

1. Introduction

Platelet-activating factor (1-O-alkyl-2-acetyl- sn-glycero-3-phosphocholine, PAF) is a potent lipid autacoid with inflammatory and hypotensive properties and is produced by endothelial cells from diverse vascular beds (Braquet et al., 1987). Recently, PAF has been reported present in the avascular chick retina, where its origin must be in neuronal and /or g!ial cells (Bussolino et al., 1988). In hepatic Kupffer cells (Fisher et at., 1989) and neutrophils (Naccache et al., 1985), PAF hydro- lyzes phosphoinositides (PI) and elevates intracell- ular Ca 2+, PAF increases Ca :+ influx into neuro- nal cells (Kornecki and Ehrlich, 1988), an event which may follow injury and precede cell death, and indeed PAF antagonists have been shown to reduce the accumulation of free fatty acids associ. ated with injury (Birkle et a l , 1988). In ~h, central nervous system (CNS) the effects of PAC are as yet ill-defined. As astroglial cells are closely asso-

Correspondence ~o: S. Murphy, Department of Pharmacology, BSB, University of Iowa, Iowa City, IA 52242. U.S.A.

ciated via spec!alized end-feet with the cerebral vasculamre, and are highly responsive to trauma. their responsiveness to PAF has been investigated.

2, MateriMs and methods

As.:rocyte-enfich,:d cultures were prepared from newbon? rat cerebral cortex and grown to con- fluence (18 days in vitro). Cultures were pre- labelled for 18 h in physiological saline solution (PSS) conta;ning t ,u.Ci/ml myo-[2-3H]inositol (20 Ci/mmot. Amersham), Cultures ,,ere washed in PSS, preincubated for t5 mix with 5 mM LiCI. and PAF (bound to 0.25% bovine serum albumin) added for 30 mix. The cells were aa~,ested, and the [3Hlinositol phosphate ([3HllnsP; predonfi- nantly inosito! monophosphate. InsP~) e:dracted and separated by ion exchange chromatography (Fearce et al., 1986). In some experiments tt*,e indi~ddual [3H]InsP formed were determined. In- cubations were carried out for 1 win in the ab- sence of Li +. The labelled lnsP 1, it~osttoJ bis- phosphate (InsP2) and iaositol trisphosphate (lnsF~) were separated by ion-exchange chro-

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400

170

160

150

140

~ 130 m

° 120

110

100 f

9 o / , .... I i I t I ! I i I -13 -12 -11 -10 -9 - 8

log CM] PAF

Fig. t. Dose-dependent effect of PAF on [3H]IBsP accumula- tion in astrocyte cultures. Cells were prelabelled with [3H]itt- ositol and exposed to PAF for 30 mitt in the presence of LiCI. Results (expressed as % basal) represent the means_+S.E, of at

least three separate experiments.

matography and eluted with formate solutions of increasing strength.

3. Results

PAF at concentrations as low as 10-11 M caused a small (50~g above basal) increase in the accumu- lation of InsP after 30 rain (see fig. 1). Lyso-PAF, the biologically inactive form of PAF, did not promote the formation of InsP (data not shown). Interestingly, at concentrations of PAF 10 -9 M and above there was no elevation in InsP accumu- lation. Short incubations (1 min) revealed that polyphosphoinositides (PPI) were being hydro- lyzed by 10 - t° M PAF, as small increases (46% above basal) in the accumulation of InsP 2 and InsP 3 were detected (see table 1). In two experi- ments, cells were pretreated with phorbol myri- state acetate (PMA) for 15 min before the ad- dition of PAF, and this abolished the accumula- tion of the polyphosphates.

4. Discuss ion

PAF at physiologically relevant concentrations (10 -12 to 10 -9 M) evokes the hydrolysis of PPI in ast~ocytes and causes the accumulation of InsP including InsP3. The maximal response in astro- cytes is small, but in Kupffer cells PAF-induced InsP formation was approximately 50% above basal at 10 -1° M, though the maximal effect achieved reached seven-fold (Fisher et al., 1989). That PAF is acting specifically via a receptor mechanism is suggested by the observations that lyso-PAF is inactive and that the accumulation of InsP is inhibited by PMA pretreatment. Phorbol esters, through protein kinase C activation, uncou- ple receptors from phospholipase C in astrocytes (Pearce et al., I98g), and PAF receptors have been shown to be sensitive to this treatment (Fisher et al., 1989).

The fact that concentrations of PAF >i 10 -9 M caused little turnover of PI may have one of several explanations. Microsome fractions from the CNS have been reported (Marcheselli et al., 1989) to express two PAF binding sites (K a of 22 pM and 25 riM), Activation of the high-affinity site could therefore be associated with the hydrol- ysis of inositol phospholipids. The lack of effect on InsP accumulation of PAF at concentrations 10 -9 M and above could represent activation of the second receptor which in some way inhibits or uncouples the high-affinity receptor from phos- pholipase C. Alternatively, the higher concentra- tions of PAF could lead to rapid desensitization and /o r permeabilization of the cells,

Thus, PAF released in vivo either from vascular cells, from neurons or indeed from astrocytes

TABLE l

The effects of PMA pretreatment on the formation of individ- ual IttsP evoked by PAF. Astrocyte cultures prelabelled with [3Hlittositol were exposed to 10 -!° M PAF for 1 rain (in the absence of LiCI) and the [3H]lnsP separated by ton exchange chromatography. Values (expressed as • basal) are meatts:E S.EM. (tt = 6). PMA pretreatmettt was for 15 rain and the results of two experimems are shown.

IttsP t IttsP 2 InsP~

No pretreatment 122.6 + 13.9 145.9 + 5.26 146.7 +_ 7.8 PMA (1 izM) 126; 122 97; 97 g0:87

401

themselves induces PPI turnover and presumably

mobilizes intracellular Ca 2÷ in astrocytes. The

functional significance of this response to PAF is

not yet clear. Astrocytes release araehidonate and

its metabolites in response to some CaZ+-mobiiiz-

ing agonists (Murphy et al., !988) and so these

cells in response to PAF could contribute to the

changes in lipid metabolism associated with

t rauma (Birkle et al., 1988).

Acknowledgements

We should like to thank Dr. Rory Fisher for providing PAF and lyso-PAF. This work was supported by NIH Grant NS- 24621.

References

Birkle. D.L., P. Kudan, P. Braquet and N.G. Bazan. 1988, Platelet-activating factor antagonist BN52021 decreases ac- cumulation of free polyunsaturated fatty acid in mouse brain during ischemia and electroconvulsive shock, J. Neu- rochem. 51, 1900.

Braquet, P., L. Touqui, T,Y. Shen and B.B. Vargaftig. 1987, Perspectives in platdet activating factor research, Pharma- col. Rev. 39, 97.

Bussotino. F. G, Pescarmona. G. Camussi. and F. Gremo. 1988, Acetylchol/ne and dopamine promote the production of platelet activating factor in immature cells of ch~ck embryonic retina. J. Neurochem. 51. 1755.

Fisher~ R.A., R.V. Sharma and R.C. Bhalla, 1989, Platetet* activating factor increases inositol phosphate production and eytosolic free calcium in cultured rat Kupffer cells. FEBS Lett. 251.22.

Kornecki, E. and Y.H. Ehrlich, 1988, Neuroregulatory and neuropathological actions of the ether-phospholipid plate- let-activating factor, Science 240. 1792.

Marcheselli. V.L., M. Rossowska. P. Braqaet and N.G. Bazan, 1989. Platelet activating factor binding sites in microsomes of rat brain cortex, Soc. Neurochem. Abstr. 2~), 135.

Murphy, S.. B. Pearce, J. Jeremy and P. Dandona, 1988, Astrocytes as eicosanoid-produc~,g cells, Glia 1, 241.

Naccache, P.H., M.M. Molski, M, Volpk E.L. Beeker and RA. Sha'afi, 1985, Unique inhibitory profile of plateIet activat- ing factor induced calcium mobilization, polyphos- phoinositide turnover and granule er,,7_yme secretion in rab- bit neutrophils towards pertussis toxin and phorbol ester, Biochim. Biophys. Res. Commun. 130. 677.

Pearce, B.. C. Morrow and S. Murphy, lC86, Receptor-media- ted inositol phospholipid hydrolysis in CNS astroc).teg. European J. Pharmacol. 12l, 23L

Pearce, B.. C. Morrow and S. Murphy, 1988, Characteristics of phorbol ester- and agonist-indt~ced dowxtregulation of ~- trocyte receptors coupled to inos~.tol phospholipid metabo- lism, J. Neurochem. 50. 936.