Free calcium rise and mitogenesis in glial cells caused by endothelin

The peptide endothelin causes a biphasic rise in intracellular free calcium levels in cultured Type-1 astrocytes and C6 glioma cells, suggesting that glial cells may be the physiological target of endothelin in the brain. Endothelin also causes a calcium-dependent increase [3H]thymidine incorporation in primary cultures of rat cerebellum, indicating that, among other possible roles, this peptide may mediate mitogenesis in brain.     

Astrocytes Are Target Cells for Endothelins and Sarafotoxin

Endothelin-1, endothelin-3, and the snake venom toxin sarafotoxin S6b stimulate the hydrolysis of phosphatidylinositol by phospholipase C with similar potencies in primary cultures of astrocytes prepared from rat brain cortex. In indo 1-loaded cells, endothelin-1, endothelin-2, endothelin-3, and sarafotoxin induce the rapid mobilization of intracellular Ca2+ stores and promote a more slowly developing influx of Ca2+. These responses were insensitive to pertussis toxin and to inhibitors of cyclooxygenase and lipoxygenase. Similar actions of endothelins and sarafotoxin were observed using astrocytes from the cerebellum and glioma cells from the C6 and NN cell lines. The endothelin receptor of astrocytes differs from the receptor previously characterized in endothelial cells from brain microvessels in that it has a high affinity for endothelin-3. Thus, brain endothelin-1 and endothelin-3 have different target cells in the brain and may have different functions.     

Heterogeneity of cell surface endothelin receptors

Two distinct cell surface endothelin receptors were identified, namely a 73-kDa protein referred to as ET-R1 and a 60-kDa protein named ET-R2. ET-R1 was expressed as the sole endothelin receptor on rat A10 vascular smooth muscle cells and C6 glial cells. Binding of 125I-ET-1 to these cells was inhibited by 50-200 pM endothelin-1 and -2, whereas endothelin-3 did not compete for this receptor subtype. Binding of 125I-ET-1 to intact A10 and C6 cells was reversible, indicating that ET-R1 is located on the cell surface. Affinity labelling of a single 73-kDa band on sodium dodecyl sulfate-polyacrylamide gels by 125I-ET-1 in A10 and C6 cells was inhibited by endothelin-1 but not by endothelin-3. In A10 cells, endothelin-1 but not endothelin-3 elicited a concentration-dependent increase in intracellular inositol trisphosphate levels. ET-R1 was also expressed in cultured rat glomerular mesangial cells based on findings of a subset of receptors with an apparent molecular mass of 73 kDa that bound 125I-ET-1 displacable by endothelin-1 and endothelin-2 but not by endothelin-3. These cells also expressed the ET-R2 receptor subtype, based on findings of a 60-kDa binding site that could be labeled by both 125I-ET-1 and 125I-ET-3. Labeling of ET-R2 by the radioactive endothelins-1 and -3 was inhibited competitively by endothelins-1, -2, and -3. Furthermore, ET-R2 was shown to be a functional receptor, as endothelin-3 caused inositol trisphosphate levels to rise in mesangial cells. An endothelin binding site with high affinity for endothelin-3 was also identified on rat PC12 pheochromocytoma cells, although the apparent molecular mass of this receptor could not be verified by cross-linking studies. Since endothelin-1 or -3 failed to augment inositol trisphosphate levels in these cells, this binding site could represent a third endothelin receptor subtype. Thus, two distinct functional receptors for endothelins were identified on rat cells, namely the 73-kDa ET-R1 which has an exceedingly low affinity for endothelin-3 and the 60-kDa ET-R2 which binds endothelin-3 with high affinity. Whether an additional endothelin receptor subtype exists in PC12 cells remains to be shown with certainty.     

The endothelin receptor antagonist, BQ-123, inhibits angiotensin II-induced contractions in rabbit aorta.

The purpose of this study was to examine the specificity of the cyclic pentapeptide ET(A) receptor antagonist BQ-123. BQ-123 competitively antagonized endothelin-1-induced contractions in rabbit aorta, increases in inositol phosphates in cultured rat vascular smooth muscle A10 cells, and binding of [125I]endothelin-1 to the cloned ETA receptor cDNA expressed in Cos 7 cells. In contrast, BQ-123 was a weak antagonist of [125I]endothelin-3 binding to rat cerebellar membranes and to membranes from Cos 7 cells transfected with the cloned ETB receptor cDNA. BQ-123 shifted concentration-response curves in isolated rabbit aorta elicited by angiotensin II, but did not bind to angiotensin II receptors nor affect angiotensin II-induced increases in inositol phosphates. BQ-123 also did not affect contractions induced by KCl or norepinephrine. These data suggest that endothelin may play a role in angiotensin II-induced contractions of rabbit aorta.     

Beta-adrenoceptor-mediated cyclic AMP signal in different types of cultured nerve cells in normoxic and hypoxic conditions

β-adrenergic neurotransmission is an important factor regulating brain activity such as neuronal and glial survival, plasticity, membrane transport or cellular metabolism. Intracellular β-adrenergic signaling, via a stimulatory G protein (G_s), activates two major down-stream effectors, i.e., adenylyl cyclase (AC) and cAMP-dependent protein kinase A (PKA). The aim of this work was to study the ability of endogenous (adrenaline and noradrenaline) and exogenous (isoprenaline) β-adrenergic receptor agonists to increase cAMP in different types of nerve cells. Moreover, we wanted to precisely identify the receptor isoform involved in the observed phenomenon using selective β₁-, β₂- β₃-adrenoceptor blockers. In an additional study, the negative influence of hypoxia on the AC/cAMP intracellular signaling system was tested. The study was conducted in parallel on rat primary glial (astrocytes) cultures, primary neuronal cultures, C6 glioma cells and human T98G glioma cells. The formation of [³H] cAMP by agonists and antagonists was measured in [³H] adenine prelabeled cells under normoxic and hypoxic conditions. The obtained results revealed that adrenaline, noradrenaline and isoprenaline strongly stimulated cAMP production in all tested cell types (with highest potency in C6 glioma cells). In glial and neuronal cells the adrenaline-evoked cAMP effect was mediated mainly by the β₁-adrenoceptor, whereas in tumor cells the effect was probably mediated by all three β-subtype specific drugs. The AC/cAMP intracellular signaling system is affected by hypoxic conditions. Considering both physiological and therapeutic importance of β-family receptors the present work characterized the β-adrenoceptor-mediated cAMP signal transduction pathway in different nerve cells in normoxic and hypoxic conditions. The proposed in vitro model of hypoxic conditions may serve as a good model system to study the biological effects of endogenous catecholamines as well as potential therapeutics targeting adrenergic receptors, which are impaired during ischemia in vivo.     

ET-1 induces cortical spreading depression via activation of the ETA receptor/phospholipase C pathway in vivo

Recently, it has been shown that brain topical superfusion of endothelin (ET)-1 at concentrations around 100 nM induces repetitive cortical spreading depressions (CSDs) in vivo. It has remained unclear whether this effect of ET-1 is related to a primary neuronal/astroglial effect, such as an increase in neuronal excitability or induction of interastroglial calcium waves, or a penumbra-like condition after vasoconstriction. In vitro, ET-1 regulates interastroglial communication via combined activation of ET(A) and ET(B) receptors, whereas it induces vasoconstriction via single activation of ET(A) receptors. We have determined the ET receptor profile and intracellular signaling pathway of ET-1-induced CSDs in vivo. In contrast to the ET(B) receptor antagonist BQ-788 and concentration dependently, the ET(A) receptor antagonist BQ-123 completely blocked the occurrence of ET-1-induced CSDs. The ET(B) receptor antagonist did not increase the efficacy of the ET(A) receptor antagonist. Direct stimulation of ET(B) receptors with the selective ET(B) agonist BQ-3020 did not trigger CSDs. The phospholipase C (PLC) antagonist U-73122 inhibited CSD occurrence in contrast to the protein kinase C inhibitor G?-6983. Our findings indicate that ET-1 induces CSDs through ET(A) receptor and PLC activation. We conclude that the induction of interastroglial calcium waves is unlikely the primary cause of ET-1-induced CSDs. On the basis of the receptor profile, likely primary targets of ET-1 mediating CSD are either neurons or vascular smooth muscle cells.     

Sarcoplasmic-endoplasmic reticulum Ca2+-ATPase inhibition prevents endothelin A receptor antagonism in rat aorta

This study tested whether sarcoplasmic-endoplasmic reticulum Ca(2+)-ATPase regulates the ability of endothelin receptor antagonist to inhibit the endothelin-1 constriction. The endothelin A receptor antagonist BQ-123 (1 microM) completely relaxed constriction to 10 nM endothelin-1 in endothelium-denuded rat aorta. Challenge with cyclopiazonic acid (10 microM), a sarcoplasmic-endoplasmic reticulum Ca(2+)-ATPase inhibitor, during the plateau of endothelin-1 constriction enhanced the constriction by approximately 30%. BQ-123 relaxed the endothelin-1 plus cyclopiazonic acid constriction by only approximately 10%. In contrast, prazosin (1 microM), an alpha-adrenergic receptor antagonist, still completely relaxed the 0.3 muM phenylephrine constriction in the presence of cyclopiazonic acid. Verapamil relaxed the endothelin-1 plus cyclopiazonic acid constriction by approximately 30%, whereas Ni(2+) and 2-aminoethoxydiphenyl borate, nonselective cation channel and store-operated channel blockers, respectively, completely relaxed the constriction. These results suggest that lowered sarcoplasmic-endoplasmic reticulum Ca(2+)-ATPase activity selectively decreases the ability of endothelin receptor antagonist to inhibit the endothelin A receptor. The decreased antagonism may be related to the opening of store-operated channels and subsequent greater internalization of endothelin A receptor.     

Endothelin mobilizes Ca2+ from a caffeine- and ryanodine-insensitive intracellular pool in rat atrial cells

Endothelin-1 is a powerful inotropic peptide for the rat atrium. Its action can develop in the absence of L-type Ca2+ channel activity provided that the external Ca2(+)-concentration has been raised to supraphysiological concentrations. Endothelin stimulates phosphatidylinositol hydrolysis in new born rat atrial cells via a mechanism that is insensitive to pertussis toxin. The diacylglycerol/protein kinase C signaling pathway cannot account for the contractile action of endothelin but its activation by phorbol esters induces a partial desensitization of phospholipase C activity. Endothelin-1 and the related peptides, endothelin-2, endothelin-3, and sarafotoxin S6b, raise intracellular Ca2+ levels in rat atrial cells. The actions of endothelin-1, endothelin-2, and sarafotoxin on [Ca2+]i are mutually exclusive, suggesting that they act at the same receptor site. The rise in [Ca2+]i induced by endothelins results both from the mobilization of intracellular stores and from Ca2+ entry through the sarcolemma via a pathway that is not voltage-dependent L-type Ca2+ channels. The Ca2+ store that is mobilized in response to endothelin retains its Ca2+ content when cells were incubated for long periods of time in a 50 nM Ca2+ solution. It is insensitive to caffeine and ryanodine. These two properties distinguish it from the sarcoplasmic reticulum. Contraction experiments in which the pacing rate has been altered to favor Ca2+ accumulation into terminal cisternae of the sarcoplasmic reticulum also suggest that the Ca2+ load of the sarcoplasmic reticulum is increased in endothelin treated rat atria.     

Selective autoregulation of endothelins in primary astrocyte cultures: endothelin receptor-mediated potentiation of endothelin-1 secretion.

Observations that primary rat astrocytes express high-affinity binding sites for endothelins and, in addition, are capable of producing not only endothelin-3 but also endothelin-1 prompted the investigation of a possible relation between endothelin peptides and receptors in these cells. Sarafotoxin S6b, an endothelin receptor agonist, was used as a tool to study endothelin receptor-mediated changes in the secretion of endothelin-1 and -3. The effects of sarafotoxin S6b and endothelin-1 in stimulating inositolphospholipid turnover as well as in inducing AP1 in primary astrocyte cultures were found to be similar. A low cross-reactivity of sarafotoxin S6b with endothelin-1 and -3 in the endothelin radioimmunoassays used here, along with a distinctly different elution position in high-performance liquid chromatography, allowed a clear discrimination between sarafotoxin and endothelins in the culture media. Stimulation of primary rat astrocytes with 10(-7) M sarafotoxin S6b for 1 hour resulted in a substantial increase in endothelin-1 immunoreactivity in the medium. This immunoreactivity reached a peak at 3 hours and showed no further increase after 8 and 24 hours. Treatment of our cultures with phorbol myristate acetate, lipopolysaccharide, tumor necrosis factor alpha, and norepinephrine for 24 hours led to only a moderate elevation of endothelin-1 immunoreactivity. Immunoreactive endothelin-3 was not affected by any of the treatments tested. Thus, our data suggest that endothelins in primary rat astrocytes are subject to selective autoregulation, as demonstrated by the potentiation of endothelin-1 secretion after activation of glial endothelin receptors.     

Histamine H1 and Endothelin ETB Receptors Mediate Phospholipase D Stimulation in Rat Brain Hippocampal Slices

Abstract: Different neurotransmitter receptor agonists [carbachol, serotonin, noradrenaline, histamine, endothelin-1, and trans -(1 S ,3 R )-aminocyclopentyl-1,3-dicarboxylic acid ( trans -ACPD)], known as stimuli of phospholipase C in brain tissue, were tested for phospholipase D stimulation in [³²P]P_i-prelabeled rat brain cortical and hippocampal slices. The accumulation of [³²P]phosphatidylethanol was measured as an index of phospholipase D-catalyzed transphosphatidylation in the presence of ethanol. Among the six neurotransmitter receptor agonists tested, only noradrenaline, histamine, endothelin-1, and trans -ACPD stimulated phospholipase D in hippocampus and cortex, an effect that was strictly dependent of the presence of millimolar extracellular calcium concentrations. The effect of histamine (EC₅₀ 18 µ M ) was inhibited by the H₁ receptor antagonist mepyramine with a K _i constant of 0.7 n M and was resistant to H₂ and H₃ receptor antagonists (ranitidine and tioperamide, respectively). Endothelin-1-stimulated phospholipase D (EC₅₀ 44 n M ) was not blocked by BQ-123, a specific antagonist of the ET_A receptor. Endothelin-3 and the specific ET_B receptor agonist safarotoxin 6c were also able to stimulate phospholipase D with efficacies similar to that of endothelin-1, and EC₅₀ values of 16 and 3 n M , respectively. These results show that histamine and endothelin-1 stimulate phospholipase D in rat brain through H₁ and ET_B receptors, respectively.     

Proliferation arrest and induction of CDK inhibitors p21 and p27 by depleting the calcium store in cultured C6 glioma cells

C6 glioma - Ca2+ depletion - proliferation arrest morphology change - CDK inhibitor In this study, we investigated the role of the intracellular calcium store in modulating the cellular proliferation and the expression of cell cycle regulatory proteins in cultured C6 glioma cells. By means of microspectrofluorimetry and Ca(2+)-sensitive indicator fura-2, we found that the intracellular Ca2+ pump inhibitors, thapsigargin (TG) irreversibly and 2,5-ditert-butyl-hydroquinone (DBHQ) reversibly depleted the Ca(2+)-store accompanied with the induction of G0/G1 arrest, an increase in glial fibrillary acidic protein (GFAP) expression and morphological changes from a round flat shape to a differentiated spindle-shaped cell. The machinery underlying these changes induced by Ca(2+)-store depletion was investigated. The results indicated that Ca(2+)-store depletion caused an increased expression of p21 and p27 proteins (cyclin-dependent kinase inhibitors), with unchanged mutant p53 protein of C6 cells but reduced amounts of the cell cycle regulators: cyclin-dependent kinase 2 (CDK2), cdc2, cyclin C, cyclin D1, cyclin D3 and proliferating cell nuclear antigen (PCNA) in a time-dependent manner. These findings indicate a new function of the endoplasmic reticulum (ER) Ca2+ store in regulating cellular proliferation rate through altering the expression of p21 and p27 proteins. Moreover, cellular differentiation as revealed by spindle-shaped morphology and induced GFAP expression were also modulated by the ER Ca2+ store. The implication of this finding is that the abnormal growth of cancer cells such as C6 glioma cells may be derived from a signalling of the ER which can be manipulated by depleting the Ca2+ store.     

Calcium mobilization by muscarinic receptors in human astrocytoma cells: measurements with quin 2

Activation of muscarinic cholinergic receptors on 1321N1 human astrocytoma cells leads to Ca2+ mobilization as measured by quin 2 fluorescence. Acetylcholine and methacholine were full and potent agonists, while carbachol and muscarine, were fully efficacious but 6- and 10-fold less potent than acetylcholine. The carbachol-induced Ca2+ response was also observed in absence of extracellular Ca2+ and was blocked by muscarinic receptor antagonists but not by organic Ca2+ channel blockers, tetrodotoxin (TTX), tetraethylammonium (TEA) or metal cations, suggesting that Ca2+ is mobilized from intracellular storage sites rather than through plasma membrane ion channels. Muscarinic receptor-mediated Ca2+ release was also detected in kidney epithelial cells but not in rat fibroblasts, glial cells or differentiated neuroblastoma x glioma hybrid cells.     

Endothelin-1 stimulates c-fos mRNA expression in C6 glioma cells via MAP kinase pathway.

Exposure of C6 glioma cells to endothelin-1 (ET-1) caused dose-dependent (10(-11) M to 10(-7) M) increments in intracellular calcium concentration ([Ca2+]i) and c-fos mRNA expression (4.5-fold) that were abolished by the endothelinA receptor antagonist, BQ610, and by inhibition of phospholipase C with U73122. ET-1 stimulated c-fos mRNA expression was also inhibited by protein kinase C inhibition (chelerythrine) and by the MAP kinase kinase inhibitor PD98059, but not by inhibitors of tyrosine kinases, protein kinase A type I or II, calmodulin kinase II, or calcium channel blockade. C6 cells treated with ET-1 demonstrated a significant increase in MAP kinase activity as evidenced by Western blotting. These results indicate a mechanism of long-term signaling by ET-1 involving an ET(A) receptor-mediated, phospholipase C(beta)-linked pathway that is dependent on protein kinase C and MAP kinase activation.     

Expression of Endothelin-Converting Enzyme in Both Neuroblastoma and Glial Cell Lines and Its Localization in Rat Hippocampus

Abstract: Endothelin-converting enzyme is a phosphoramidon-sensitive metalloprotease that cleaves big endothelin to the potent vasoconstrictor peptide, endothelin. The converting enzyme is expressed in endothelial cells in a variety of tissues and in some secretory cells. In the present study, phosphoramidon-sensitive endothelin-converting enzyme activity has been demonstrated by radioimmunoassay in the neuroblastoma cell line, SH-SY5Y, and in Bu17 and C6 glioma lines. The identity of the activity was confirmed by immunoblotting, revealing a polypeptide of ∼120 kDa in each of these lines, in D384 glioma cells, and in primary astrocytes. Immunofluorescence revealed the cell-surface location of endothelin-converting enzyme in the neuronal and glial cell lines and in primary astrocytes. Pretreatment of SH-SY5Y and Bu17 cells with phosphoramidon resulted in an apparent concentration of the enzyme protein in an intracellular compartment. Immunoperoxidase-staining of rat brain sections located this metalloprotease to the pyramidal cells of the hippocampus. Endothelin-converting enzyme-1 was revealed by in situ hybridisation in the neuronal and glial cell lines.     

Interactive modulation of renal myogenic autoregulation by nitric oxide and endothelin acting through ET-B receptors

In rats, nitric oxide modulates renal autoregulation in steady-state experiments and the myogenic mechanism in dynamic studies. Interactive modulation of autoregulation by nitric oxide and endothelin-1, predominantly involving endothelin B receptors, has been reported although it remains unclear whether the interaction is synergistic or obligatory or whether it affects the myogenic component of autoregulation. Nonselective inhibition of nitric oxide synthase (L(omega)-nitro-l-arginine methyl-ester; l-NAME) with endothelin A and B selective receptor antagonists BQ-123 and BQ-788, all infused into the renal artery, plus time series analysis were used to test the interactive actions of nitric oxide and endothelin on renal vascular conductance and on autoregulation. Nonselective endothelin receptor antagonism blunted the constrictor response to subsequent l-NAME but had no effect on previously established l-NAME-induced vasoconstriction. BQ-123 did not affect conductance and caused only minor reduction in myogenic autoregulatory efficiency. Responses to BQ-123 and l-NAME were additive and not interactive. BQ-788 and l-NAME each caused strong vasoconstriction alone and in the presence of the other, indicating that coupling between nitric oxide- and endothelin B-mediated events is not obligatory. l-NAME augmented myogenic autoregulation, and subsequent BQ-788 did not alter this response. However, BQ-788 infused alone also enhanced myogenic autoregulation but resulted in significant impairment of myogenic autoregulation by subsequent l-NAME. Thus the interaction between nitric oxide and endothelin is clearly nonadditive and, because it is asymmetrical, cannot be explained simply by convergence on a common signal pathway. Instead one must postulate some degree of hierarchical organization and that nitric oxide acts downstream to endothelin B activation.     

Responses to neither exogenous nor endogenous endothelin-1 are altered in patients with hypercholesterolemia

There is some controversy regarding whether vascular responses to endothelin are altered in hypercholesterolemia. Studies performed to date have been compromised by the use of endothelin antagonists at inappropriate concentrations. In the current study, we examine the role of endothelin-1 in hypercholesterolemic patients using lower, more selective doses of specific endothelin antagonists. Twenty-two patients with hypercholesterolemia (total plasma cholesterol > 6.0 mmol/l) and 17 healthy controls were recruited. Forearm vascular responses to endothelin-1 (5 pmol/min), the endothelin A antagonist BQ-123 (10 nmol/min), and the endothelin B antagonist BQ-788 (1 nmol/min) were obtained. Endothelin-1 caused a significant vasoconstriction in both hypercholesterolemic and control subjects, an effect that was not significantly different between the two groups (P = 0.784). BQ-123 caused a significant vasodilatation that was not significantly different between the two groups (P = 0.899). Similarly, responses to BQ-788 (P = 0.774) and mean plasma endothelin-1 levels were not different (control vs. hypercholesterolemia, 1.16 +/- 0.18 vs. 1.06 +/- 0.15 fmol/ml; P = 0.64). Responses to neither exogenous nor endogenous endothelin are influenced by plasma cholesterol levels in humans. It is thus unlikely that the endothelin system contributes to early vascular disease pathology in patients with hypercholesterolemia.     

Molecular modelling of the structures of endothelin antagonists. Identification of a possible structural determinant for ET-A receptor binding.

Computer-aided molecular modelling of the endothelin (ET-A) receptor antagonists, BQ-123 and BE-18257B, shows that they have very similar 3D structures. Parts of their 3D structures are also shown to match closely with that reported for residues 6-8 in endothelin-1. On the basis of these similarities (and with supporting evidence from literature data on endothelin structure-activity relationships) a structural determinant is proposed for ET-A receptor binding, and novel designs of peptide are suggested for providing more potent and selective ET-A receptor antagonists.     

Activities of glycolytic enzymes in rapidly proliferating and differentiated C6 glioma cells

Comparisons of glycolytic enzymes between rapidly proliferating and Bt2 cAMP-induced differentiated C6 glioma cells have been made. Rapidly proliferating cells had higher concentrations of glucose-6-phosphate, fructose-6-phosphate and fructose-1,6-bisphosphate compared to morphologically differentiated cells. Under maximally activating conditions, the specific activity and Vmax of hexokinase and phosphofructokinase enzymes were reduced by approximately 3- and 28-fold, respectively, in differentiated cells, without any change in Km values. These results suggest that hexokinase and phosphofructokinase occupy special control positions and the rate of glycolysis is correlated with cellular proliferation of C6 glioma cells.     

Cholinergic stimulation of arachidonic acid and phosphatidic acid metabolism in C62B glioma cells

Glioma C62B cells were incubated for 18 h with [1-14C]arachidonic acid. Most (80%) of the added [1-14C] arachidonic acid was taken into the intracellular pool; less than 1% of the intracellular [1-14C]arachidonic acid remained unesterified; the rest was present in glycerophospholipids. Acetylcholine stimulation of the prelabeled cells resulted in the rapid accumulation of free [1-14C]arachidonic acid, presumably liberated by hydrolysis from phospholipids. Labeled unesterified [1-14C]arachidonic acid peaked by 90 s and returned to basal levels by 5 min. Paralleling the transient increase of unesterified [1-14C]arachidonic acid were increases in level of radioactivity in an unidentified lipoxygenase metabolite of arachidonic acid and of radioactive phosphatidic acid. The release of arachidonic acid induced by acetylcholine or carbachol was blocked by muscarinic but not nicotinic receptor antagonists; adrenergic or histaminergic receptor agonists were ineffective at stimulating arachidonic acid liberation. In contrast to the transient effects of stimulation with cholinergic agonists, stimulation with the divalent cation ionophore A23187 resulted in a linear increase in the accumulation of liberated arachidonic acid for at least 1 h. Furthermore, the pattern of metabolites synthesized from arachidonic acid in response to ionophore stimulation was more complex than that observed following cholinergic stimulation and included also several metabolites derived from cyclooxygenase activity. We conclude that muscarinic receptor agonists rapidly induce specific changes in arachidonic acid and phosphatidic acid metabolism in a glioma cell line and suggest that similar responses may occur in glial cells and play a physiologically significant role in neural metabolism.