Neuroscience Letters, 118 (1990) 235-237 235 Elsevier Scientific Publishers Ireland Ltd.

NSL 07224

Muscarinic receptors mediate the release of arachidonic acid from spinal cord and hippocampal neurons in primary culture

Robert Y. Kante rman 2, Alice L. Ma 1, Eileen M. Briley x, Julius Axelrod 1 and Christian C, Felder 1

1 Laboratory of Cell Biology, Section on Pharmacology, National Institute of Mental Health, Bethesda, U.S.A., 2Howard Hughes Medical Institute- National Institutes of Health Research Scholars Program, Bethesda, MD (U.S.A.)

(Received 5 March 1990; Revised version received 30 May 1990; Accepted 26 June 1990)

Key words: Phospholipase A2; Phospholipase C; Phorbol ester; Eicosanoid; Pirenzepine; Inositol phosphate

Muscarinic receptors are involved in CNS neurotransmission and have been shown to transduce their message by modulating cAMP, calcium, inositol phosphates, and more recently, by liberating arachidonic acid via phospholipase Al. We have previously shown that the aq-adrenergic and 5-HT2 serotonergic neurotransmitter receptors cause the release of arachidonic acid from spinal cord and hippocampal neurons, respectively, in primary culture. In this study, we demonstrated a muscarinic receptor-mediated release of arachidonic acid in these two neural segments which occurred independent of phosphatidylinositol-specific phospholipase C. This release of arachidonic acid was neuronal (not glial) in origin and exhi- bited M~ muscarinic receptor pharmacology.

Muscarinic receptors play an important role in neuro- transmission, particularly in the brain and spinal cord, and these receptors can activate several transmembrane signalling pathways. Among the second messengers modulated by these receptors are cAMP, calcium, and inositol phosphates [12]. The ml and m3 (muscarinic receptor subtype nomenclature based on the chronologi- cal order in which they were cloned) muscarinic recep- tors have recently been shown to liberate arachidonic acid via phospholipase A2 in fibroblast cell lines trans- fected with and stably expressing these receptors [7].

Phospholipase A2 has been proposed as the effector enzyme involved in the receptor-mediated release of ara- chidonic acid from neural cells in primary culture. 0q- adrenergic receptors mediate the release of arachidonic acid from spinal cord neurons in primary culture inde- pendent of phospholipase C and likely via phospholipase A2 [10]. The 5-HT2 serotonergic receptor has been shown to release arachidonic acid from hippocampal neurons via phospholipase A2 and independent of phospholi- pases C and D [9]. Muscarinic receptors are widely dis- tributed throughout the CNS, and the ml, m3, and m5 receptors have been shown to be expressed in the hippo- campus [15]. Therefore, we investigated whether muscar- inic receptors mediated the release of arachidonic acid from neural tissue in primary culture of both the hippo-

Correspondence: C.C. Felder, Laboratory of Cell Biology, Building 36, Room 3A-15, NIMH, Bethesda, MD 20892, U.S.A.

campus and spinal cord. The muscarinic receptor agonist, carbachol, stimu-

lated the release of arachidonic acid from both spinal cord (Fig. 1A) and hippocampal (Fig. 1B) neural/glial co-cultures in a concentration-dependent manner. How- ever, carbachol failed to stimulate the release of arachi- donic acid from spinal cord and hippocampal glia cell cultures devoid of neurons, suggesting a neuronal origin for this response. As in other examples of muscarinic receptor-mediated release of arachidonic acid [7], carba- chol also stimulated the production of inositol phos- phates (IP) from these neural cultures in a concentra- tion-dependent manner with an ECs0 comparable to that of arachidonic acid release (ECs0 AA release= 12/tM, ECs0 IP production = 15/tM).

There are multiple pathways that could give rise to the receptor-mediated release of arachidonic acid. Receptors may activate phospholipase A2, which then directly hyd- rolyzes membrane phospholipids to release arachidonic acid [1]. Alternatively, receptors may release arachidonic acid indirectly by activating phosphatidylinositol-speci- fic or phosphatidylcholine-specific phospholipase C, which generate diacylglycerol (along with inositol phos- phates and phosphocholine, respectively). Diacylgly- cerol may be further metabolized by diglyceride lipase to produce arachidonic acid [3]. Receptors may also acti- vate phospholipase D producing phosphatidic acid, an- other potential source of arachidonic acid [l 1]. The phorbol ester, phorbol 12-myristate 13-acetate (PMA),

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Fig. 1. Carbachol stimulated the release of arachidonic acid (AA) from spinal cord neurons (A) and hippocampal neurons (B), but not from their corresponding glial cells. Cells were labelled overnight in 24 well plates with 0.2/tCi per well of [3H]arachidonic acid, washed 3 times with Eagles no. 2 medium containing 1 mM CaCI2, 0.1 mM MgC12 and 0.2% BSA. Cells were incubated with carbachol at the appropriate con- centrations for 15 min and media were removed, centrifuged, and counted to measure cpm [aH]arachidonic acid released. Data represent the mean + S.E.M. for at least 3 experiments performed in triplicate.

has been previously utilized to inhibit phospholipase C [14], thus dissociating the release of arachidonic acid from the release of inositol phosphates via phospholi- pase C [1]. PMA inhibited carbachol (100 uM)-stimu- lated production of inositol phosphates in a concentra- tion-dependent manner (IC50 = 10 nM) but not the re- lease of arachidonic acid from spinal cord cultures (Fig. 2). PMA had a similar effect in hippocampal cultures (data not shown). These data suggest that the release of arachidonic acid is independent of phosphatidylinositol- specific phospholipase C, consistent with our earlier findings in neuronal cell cultures [9, 10].

Muscarinic receptor-mediated release of arachidonic acid from cells transfected with the ml and m3 (molecu- lar biology subtypes) muscarinic receptors exhibits M1 (pharmacologic subtype) muscarinic pharmacology. This is characterized by a high affinity for the muscarinic antagonist pirenzepine [5, 6]. Carbachol-stimulated re- lease of arachidonic acid in the hippocampus and spinal

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Fig. 2. Carbachol (CC)-stimulated production of inositol phosphates but not release of arachidonic acid is inhibited by the phorbol ester PMA in spinal cord cells. Ceils were incubated with PMA for 1 hour at the concentrations indicated, stimulated with 100/~M carbachol, and assayed for the release of arachidonic acid (as described in legend of Fig. 1) and production of inositol phosphates (as previously de- scribed by Berridge et al. [4] with modifications [11]). Data represents the mean + S.E.M. for at least 3 experiments performed in triplicate.

cord likewise demonstrated M1 muscarinic pharmaco- logy. In the hippocampus, atropine (10/tM) the non-se- lective muscarinic antagonist, blocked carbachol (100 /iM)-stimulated release of arachidonic acid by 93% while pirenzipine (1 pM), the M1 muscarinic antagonist, inhi- bited carbachol-stimulated release of arachidonic acid by 81%. AFDX-116, the M2 muscarinic antagonist [5], failed to block the carbachol-stimulated release of ara- chidonic acid (data not shown). Similar results were seen in the spinal cord cultures. These results indicate that the M1 muscarinic receptor mediated the release of arachi- donic acid in these neurons.

Preliminary results indicate that carbachol also stimu- lates the release of arachidonic acid from cerebral corti- cal neurons (but not cerebeltar granule cells or striatal neurons) in primary culture. The biological role of this muscarinic receptor-mediated release of arachidonic acid in the nervous system is presently unknown, but arachidonic acid and its eicosanoid metabolites are known to have a variety of effects in the CNS. Eicosa- noids are liberated in the brain during seizure, stroke, and other pathophysiological circumstances [2, 17]. The lipoxygenase metabolites of arachidonic acid have been shown to be involved in presynaptic inhibition and ion channel regulation and arachidonic acid itself has been proposed to play a role in long term potentiation and synaptic plasticity [8, 13, 16].

1 Axelrod, J., Burch, R.M. and Jelsema, C.L., Receptor-mediated activation of phospholipase A2 via GTP-binding proteins: arachi- donic acid and its metabolites as second messengers, Trends Neur- osci., 1 (1988) 117-123.

2 Bazan, N.G., Birkle, D.L., Tang, W. and Reddy, T.S., The accu- mulation of free arachidonic acid, diacylglycerols, prostaglandins,

and lipoxygenase reaction products in the brain during experimen- tal epilepsy. In A.V. Delgado-Escueta, A.A. Ward and D.M. Woodbury (Eds.), Advances in Neurology, Raven, New York, 1986, pp. 879-902.

3 Bell, R.L., Kennedy, D.A., Stanford, N. and Majerus, P.W., Digly- ceride lipasc: a pathway for arachidonate release form human pla- telets, Prec. Natl. Acad. Sei. U.S.A. 76 (1979) 3238-3241.

4 Berridge, M.J., Downes, C.P. and Hanley, M.R., Lithium amplifies agonist-dependent phosphatidytinositol responses in brain and salivary glands, Biechem. J., 206 (1982) 587-595.

5 Birdsall, N.J.M., Hulme, E.C., Keen, M., Pedder, E.K., Poyner, D., Stockton, J.M. and Wheatley, M., Soluble and membrane- bound muscarinic acetylcholine receptors, Biochem. Sec. Symp., 52 (1986) 23-32.

6 Bonner, T.I., Buckley, N.J., Young, A.C. and Brann, M.R. Identi- fication of a family of muscarinic acetylcholine receptor genes, Sci. ence, 237 (1987) 527-532.

7 Conklin, B.R., Brann, M.R., Buckley, N.J., Ma, A.L., Bonnet, T.I. and Axelrod, J., Stimulation of arachidonic acid release and inhibi- tion of mitogenesis by cloned genes for muscarinic receptor sub- types stably expressed in A9 L ceils, Prec. Natl. Acad. Sci. U.S.A., 85 (198) 8698-8702.

8 Dumuis, A., Sebben, M., Haynes, L., Pin, J.-P. and Beckaert, J., NMDA receptors activate the arachidonic acid cascade system in striatal neurons, Nature, 336 (1988) 68-70.

9 Felder, C.C., Kanterman, R.Y., Ma, A.L. and Axelrod, J., Seroto- nin stimulates phospholipase A2 and the release of arachidonic acid in hippocampal neurons via a 5-HT2 receptor, independent of

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inositolphospholipid hydrolysis, Prec. Natl. Acad. Sci. U.S.A., 87 (1990) 2187-2191.

10 Kanterman, R.Y., Felder, C.C., Brenneman, D.E., Ma, A.L., Fitz- gerald, S. and Axelrod, J., Al-adrenergic receptor mediates arachi- donic acid release in spinal cord neurons independent of inositol phospholipid turnover, J. Neurochem., 54 (1990) 1225-1232.

11 Loffelholz, K., Receptor regulation of choline phospholipid hy- drolysis, Biochem. Pharm., 38 (1989) 1543-1549.

12 Peralta, E.G., Ashkenazi, A., Winslow, J.W., Ramachandran, J. and Capon, D.J., Differential regulation of PI hydrolysis and ade- nylyl cyclase by muscarinic receptor subtypes, Nature, 334 (198) 43~437.

13 Piomelli, D., Volterra, A., Dale, N., Siegelbaum, S,A., Kandel, E.R., Schwartz, J.M. and Belardetti, F., Lipoxygenase metabolites of arachidonic acid as second messengers for pre-synaptie inhibi- tion of Aplysia sensory ceils, Nature, 328 (1987) 38-43.

14 Rhee, S.G., Suh, P.-G., Ryu, S.-H. and Lee, S.Y., Studies of inositol phospholipid-specific phospholipase C, Science, 244 (1989) 546-550.

15 Weiner, D.M. and Brann, M.R., Distribution ofml-m5 muscarinic receptor mRNAs in rat brain, Trends Pham. Sci., 10 Suppl. (1989) 115 (abstr. 72).

16 Williams, J.H., Errington, M.L., Lynch, M.A. and Bliss T.V.P., Arachidonic acid induces a long-term activity-dependent enhance- ment of synaptic transmission in the hippocampus, Nature, 341 (1989) 739-742.

17 Wolfe, L.S., Eicosanoids: prostaglandins, thromboxanes, leuko- trienes, and other derivatives of carbon-20 unsaturated fatty acids, J. Neurochem., 38 (1982) 1-14.