Serotonin (5-HT) stimulated the release of arachidonic acid in hippocampal neurons cocultured with glial cells but not in glial cultures alone. Similar results were observed for the 5-HT-stimulated release of inositol phosphates. These results suggest a neural but not glial orign of both responses.Pharmacological studies suggested that release of arachidonic acid and inositol phosphates was mediated by a type 2 5-HT (5-HT2) receptor. 5-HT-stimulated release of arachidonic acid was also detected in cortical neurons, which contain high levels of 5-HT2 receptors, but not striatum, spinal cord, or cerebellar granule cells, which have very low levels or are devoid of 5-HT2 receptors. The phorbol ester phorbol 12-myristate 13-acetate augmented the 5-HT-stimulated release of arachidonic acid but inhibited the 5-HT-stimulated release of inositol phosphates. 5-HT-stimulated release of arachidonic acid, but not inositol phosphates, was dependent on extracellular calcium. 5-HT stimulated the release of [3U1lysophosphatidylcholine from [3UHcholine-labeled cells with no increase in the release of [3HUcholine or phospho[3Ulcholine. These data suggest that 5-UT stimulated the release of arachidonic acid in hippocampal neurons through the activation of phospholipase A2, independent of the activation of phospholipase C.The excitable membranes of the central nervous system are enriched in 20-carbon unsaturated fatty acids, particularly arachidonic acid (1). Arachidonic acid serves as a precursor to a number of biologically active acid lipids, including prostaglandins, leukotrienes, and thromboxanes. Arachidonic acid and its eicosanoid metabolites are released after activation ofcertain neurotransmitter receptors and may play a role in neurotransmission. Recent studies have demonstrated (2-4) receptor-mediated release of eicosanoids in brain cells in primary culture. N-Methyl-D-aspartate stimulated the release of arachidonic acid, 11-HETE, and 12-HETE (where HETE is hydroxyeicosatetraenoic acid) in striatal cells (2) as well as arachidonic acid in cerebellar granule cells (3). Bradykinin stimulated arachidonic acid mobilization from dorsal root ganglion neurons (4). It is not clear from these studies whether the release of arachidonic acid is independent of inositolphospholipid turnover and whether the response is neural or glial in origin. Recent studies demonstrated a-adrenergic receptor stimulation caused arachidonic acid release from spinal cord, hippocampal, and cortical neurons, but not from glia cells, grown in primary culture that was independent of inositolphospholipid turnover (5).Central serotonin (5-HT) receptors are involved in sleep, depression, hallucinations, anxiety, sexual behavior, memory, appetite control, and thermoregulation (6). 5-HT receptors exist as several subtypes and transduce their signals by way of multiple intracellular messengers (7). Types 1A, 1B, and 1D 5-HT receptors (5-HT1A, 5-HT1B, and 5-HTlD) modulate adenylate cyclase activity and types 1C and 2 5-HT (5-HT1c and 5-HT2) receptors incr...

A family of genes encoding four distinct muscarinic receptors (designated ml-m4) has been cloned and stably expressed in A9 L cells. When the ml and m3 receptors were stimulated with carbachol, there was a rapid rise of liberated arachidonic acid, inositol phosphates, and cAMP, while m2 and m4 receptor stimulation had no detectable stimulation of these second messengers. Pretreatment with phorbol 12-myristate 13-acetate (PMA) caused a marked acceleration and amplification of ml and m3 receptor-mediated arachidonic acid release. In contrast, ml-and m3-mediated inositol phosphate formation was inhibited by the same PMA pretreatment. Arachidonic acid release was unaffected by manipulations of cAMP levels. Arachidonic acid production was inhibited by calcium-free medium and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8; an inhibitor of cytosolic calcium mobilization) yet was unaffected by verapamil, a calcium-channel blocker. These experiments show that arachidonic acid release induced by the ml and m3 receptors is regulated independently of phospholipase C and cAMP accumulation. Carbachol stimulation of the ml and m3 receptors also markedly decreased mitogenesis as measured by thymidine incorporation. The ml receptor-mediated inhibition of mitogenesis could be partially blocked by indomethacin, a cyclooxygenase inhibitor. The inhibition of mitogenesis could be mimicked by cAMP elevation.The muscarinic acetylcholine receptor has been pharmacologically classified into two subtypes based on pirenzepinebinding studies (1, 2). The classification of the muscarinic receptor subtypes has been extended recently by the cloning of a family of genes encoding muscarinic receptors named ml-m4 (3), based on the chronological order in which their primary structure was determined (4-6). When the cloned muscarinic receptor genes are expressed in COS-7 (3) and A9 L cells (7), the m2 receptor has low-affinity binding for pirenzepine and the ml, m3, and m4 receptors have higher affinity binding for pirenzepine. These muscarinic receptors are members of a family of receptors that have seven proposed membrane-spanning regions and are thought to be coupled to guanine nucleotide-binding proteins (G proteins).The genes for the four muscarinic receptors have been transfected and stably expressed in the murine A9 L cell line (7). In other studies, we have shown that activation of the ml and m3 receptors with a muscarinic agonist causes increased levels of cAMP, inositol phosphates, and arachidonic acid (7) and the opening of calcium-dependent potassium channels (8-10), while an agonist causes none of these effects on m2 or m4 receptors.Muscarinic receptor-stimulated arachidonic acid release has been observed in a variety of tissues and cell lines (11). These reports have proposed that the release of arachidonic acid is secondary to the activation of phospholipase C (12, 13). Receptor-mediated mobilization of arachidonic acid can occur through the stimulation of either phospholipase A2 or phospholipase C. Recent evide...

Several members of the family of guanine nucleotide-binding protein (G protein)-coupled receptors have recently been shown to induce agonist-dependent foci development in NIH 3T3 cells and tumors in nude mice. We selected the five subtypes of the muscarinic acetylcholine receptor family to investigate their role in tumor suppression. When transfected and expressed in CHO-Kl Chinese hamster ovary cells, ml, m3, and m5 muscarinic acetylcholine receptor activation resulted in a morphology change. Receptor activation did not slow or inhibit monolayer growth of CHOm5 cells in culture but markedly inhibited density-independent growth in soft agar and suppressed tumor formation in nude mice. Receptor-mediated tumor suppression was found to be agonistdependent and reversible and was blocked with a muscarinic receptor antagonist. Of the five signaling pathways associated with the ml, m3, and m5 receptors, only receptor-operated, and inositol trisphosphate-independent, calcium influx was found to correlate with inhibition of tumorigenicity. These data suggest a pivotal role for inositol trisphosphate-independent receptor-regulated calcium homeostasis in CHO-Kl tumor suppression.

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The alpha 1-adrenergic receptor has been shown to mediate the release of arachidonic acid in FRTL5 thyroid cells and MDCK kidney cells. In primary cultures of spinal cord cells, norepinephrine stimulated release of arachidonic acid (from neurons only) and turnover of inositol phospholipids (from neurons and glia) via alpha 1-adrenergic receptors. These two responses were dissociated by treatment with phorbol ester and pertussis toxin, which inhibited production of inositol phosphates with no appreciable effect on release of arachidonic acid. Extracellular calcium was required for release of arachidonic acid, but not for production of inositol phosphates. The calcium channel blockers nifedipine and verapamil inhibited release of arachidonic acid only. However, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), a compound that blocks intracellular calcium release, diminished production of inositol phosphates, but had little effect on release of arachidonic acid. These results suggest that alpha 1-adrenergic receptors couple to release of arachidonic acid in primary cultures of spinal cord cells by a mechanism independent of activation of phospholipase C, possibly via the activation of phospholipase A2.