J oirrnal qf Ncirrochctnisirj~ Raven Press, Ltd.. New York 0 1992 International Society for Neurochemistry
Induction of Nitric Oxide Synthase in Glial Cells Martha L. Simmons and Sean Murphy Departtncwt of Pharmacology.. Universiry of IOMU Collqy cf Mdicine. I ~ MCily, Y I Iow~a,U.S.A
~~
Abstract: Primary astrocyte cultures, C6 glioma cells, and N I8 neuroblastoma cells were assayed for nitric oxide synthase (NOS) activity with a bioassay of cyclic GMP production in RFL-6 fibroblasts. Treatment of astrocyte cultures for 16-18 h with lipopolysaccharide (LPS) induced NOSlike activity that was L-arginine and NADPH dependent, Ca2+independent, and potentiated by superoxide dismutase. Induction was evident after 4 h, was dependent on the dose of LPS, and required protein synthesis. Treatment of astrocyte cultures with leucine methyl ester reduced microglial cell contamination from 7 to 1%. with a loss of44% of
NOS-like activity. C6 cells treated with LPS also showed Ca2+-independent and L-arginine-dependent NOS-like activity. N 18 cells demonstrated constitutive Ca2+-dependent NOS-like activity that was not enhanced by LPS induction. These data indicate that NOS-like activity can be induced in microglia, astrocytes. and a related glioma cell line as it can in numerous other cell types, but not in neuron-like N18 cells. Key Words: Astrocyte-Nitric oxide-Microglia-LArginine-Endotoxin-Neuroblastoma. Simmons M. L. and Murphy S. Induction of nitric oxide synthase in glial cells. J. Ncirrochcin. 59, 897-905 (1 992).
One of the endothelium-derived relaxing factors (EDRFs) is nitric oxide (NO) or another nitrosyl compound, which is released from endothelial cells and causes vasorelaxation by stimulation of soluble guanylate cyclase in adjacent vascular smooth muscle (for review, see Moncada et al., 1991). Similar NO release has been described in cerebellar neurons in response to glutamate (Bredt and Snyder, 1989; Garthwaite et al., 1989a,b) and more recently from other neuronal types in response to various stimuli (Bult et al., 1990; Bauer et al., 1991). Neuronal NO release stimulates cyclic GMP production (Knowles et al., 1989)and has been implicated in cell-cell signaling (Snyder and Bredt, 1991) and in some forms of excitotoxic injury (Dawson et al., 199 1). The NO synthase (NOS) responsible for neuronal production of NO has been purified from rat cerebellum, characterized, cloned, and sequenced (Bredt and Snyder, 1990; Bredt et al., 1991; Schmidt et al., 1991 ). It is a constitutive, calcium/calmodulin (CaM)-dependent enzyme (Bredt and Snyder, 1990)that requires NADPH and FAD as cofactors (Giovanelli et al., 1991 ; Mayer et al., 1991) and has sequence homology to cy-
tochrome P-450 reductase (Bredt et al., 1991). The substrate for NOS is L-arginine, with equimolar amounts of L-citrulline and NO being produced (Bredt and Snyder, 1990). Using an antiserum, NOS has been localized to endothelial cells and neuronal elements in the CNS (Bredt et al., 1990). Release of NO from macrophages is implicated in cell killing, and this NOS has been purified and characterized (Stuehr and Nathan, 1989; Stuehr et al., 199 1; Yui et al., 1991). Although the macrophage enzyme appears to catalyze the same reaction as the neuronal form and requires NADPH and FAD, it is calcium independent and must be induced by agents such as bacterial lipopolysaccharide (LPS: endotoxin) or cytokines. Smooth muscle cells and endothelial cells have also been shown to contain an inducible enzyme with properties similar to those of the macrophage enzyme (Kilbourn and Belloni, 1990; Fleming et al., 1991;Gross et al., 1991;Schini et al., 199 1 ). The finding that endothelial cells contain both constitutive and inducible forms of NOS suggests that other cells may also express multiple isoforms of the enzyme. Cloning of NOS indicates that there may be
Received December 9, I99 I : revised manuscript received February 17. 1992: accepted February 24. 1992. Addrcss correspondence and reprint requests to Dr. S. Murphy at Department of Pharmacology. University of Iowa College of Medicine. Iowa City. I A 52247. U.S.A. :lhl,~c,~irr/io/l.r ~ r . s t dBME. basal medium Eagle: CaM, calmodulin: Dil-Ac-LDL. acetylated low-density lipoprotein labeled with 1. I ‘-dioctadccyl- 1-3.3.3’.3’-tetramethylindocarbocyanine perchlo-
rate: DIV. days in vitro: EBSS. Earle’s balanced salt solution: EDRF. endothelium-derived relaxing factor: EMEM. Eagle’s minimal essential medium: FCS. fetal calf serum: IBMX. 3-isobutyl- I -methylxanthine: LME. leucine methyl ester: LPS. lipopolysaccharide: NAME. Kw-nitro-L-arginine methyl ester: NMA. NG-monomethyI-L-arginine: NO, nitric oxide: NOS. nitric oxide synthase: SOD, supcroxide dismutase.
897
M . L. SIMMONS AND S. MURPHY
898
several gene products in the NOS family (Bredt et al., 1991), but it remains to be seen which specific cell types express each. Recently, we (Murphy et al., 1990, 1991) and others (Mollace et al., 1990; Agullo and Garcia, I99 1, 1992) have described a nitrosyl factor released from astrocytes that is capable of stimulating soluble guanylate cyclase and eliciting vasorelaxation. Here we show that astrocytes, microglia, and C6 glioma cells express an LPS-inducible NOS that is calcium independent.
MATERIALS AND METHODS Cell culture Primary astrocyte cultures were prepared from neonatal rat cortices as previously described (Murphy, 1990) and grown in TI50 flasks for at least 14 days in vitro (DIV) in Eagle’s minimal essential medium (EMEM) supplemented with 33 mM glucose, 2 mA4 glutamine, 50 pg/ml of gentamicin, and 10% (vol/vol) fetal calf serum (FCS). All cells were kept at 37°C in 5% C0,/95% air. These primary cultures have been characterized by immunocytochemistry and routinely consist of 90-9596 glial acidic fibrillary protein-positive cells (astrocytes) with contaminating microglia (3-7%), fibroblasts ( 1-3%), oligodendrocytes, and vascular cells (
Induction of Nitric Oxide Synthase in Glial Cells Martha L. Simmons and Sean Murphy Departtncwt of Pharmacology.. Universiry of IOMU Collqy cf Mdicine. I ~ MCily, Y I Iow~a,U.S.A
~~
Abstract: Primary astrocyte cultures, C6 glioma cells, and N I8 neuroblastoma cells were assayed for nitric oxide synthase (NOS) activity with a bioassay of cyclic GMP production in RFL-6 fibroblasts. Treatment of astrocyte cultures for 16-18 h with lipopolysaccharide (LPS) induced NOSlike activity that was L-arginine and NADPH dependent, Ca2+independent, and potentiated by superoxide dismutase. Induction was evident after 4 h, was dependent on the dose of LPS, and required protein synthesis. Treatment of astrocyte cultures with leucine methyl ester reduced microglial cell contamination from 7 to 1%. with a loss of44% of
NOS-like activity. C6 cells treated with LPS also showed Ca2+-independent and L-arginine-dependent NOS-like activity. N 18 cells demonstrated constitutive Ca2+-dependent NOS-like activity that was not enhanced by LPS induction. These data indicate that NOS-like activity can be induced in microglia, astrocytes. and a related glioma cell line as it can in numerous other cell types, but not in neuron-like N18 cells. Key Words: Astrocyte-Nitric oxide-Microglia-LArginine-Endotoxin-Neuroblastoma. Simmons M. L. and Murphy S. Induction of nitric oxide synthase in glial cells. J. Ncirrochcin. 59, 897-905 (1 992).
One of the endothelium-derived relaxing factors (EDRFs) is nitric oxide (NO) or another nitrosyl compound, which is released from endothelial cells and causes vasorelaxation by stimulation of soluble guanylate cyclase in adjacent vascular smooth muscle (for review, see Moncada et al., 1991). Similar NO release has been described in cerebellar neurons in response to glutamate (Bredt and Snyder, 1989; Garthwaite et al., 1989a,b) and more recently from other neuronal types in response to various stimuli (Bult et al., 1990; Bauer et al., 1991). Neuronal NO release stimulates cyclic GMP production (Knowles et al., 1989)and has been implicated in cell-cell signaling (Snyder and Bredt, 1991) and in some forms of excitotoxic injury (Dawson et al., 199 1). The NO synthase (NOS) responsible for neuronal production of NO has been purified from rat cerebellum, characterized, cloned, and sequenced (Bredt and Snyder, 1990; Bredt et al., 1991; Schmidt et al., 1991 ). It is a constitutive, calcium/calmodulin (CaM)-dependent enzyme (Bredt and Snyder, 1990)that requires NADPH and FAD as cofactors (Giovanelli et al., 1991 ; Mayer et al., 1991) and has sequence homology to cy-
tochrome P-450 reductase (Bredt et al., 1991). The substrate for NOS is L-arginine, with equimolar amounts of L-citrulline and NO being produced (Bredt and Snyder, 1990). Using an antiserum, NOS has been localized to endothelial cells and neuronal elements in the CNS (Bredt et al., 1990). Release of NO from macrophages is implicated in cell killing, and this NOS has been purified and characterized (Stuehr and Nathan, 1989; Stuehr et al., 199 1; Yui et al., 1991). Although the macrophage enzyme appears to catalyze the same reaction as the neuronal form and requires NADPH and FAD, it is calcium independent and must be induced by agents such as bacterial lipopolysaccharide (LPS: endotoxin) or cytokines. Smooth muscle cells and endothelial cells have also been shown to contain an inducible enzyme with properties similar to those of the macrophage enzyme (Kilbourn and Belloni, 1990; Fleming et al., 1991;Gross et al., 1991;Schini et al., 199 1 ). The finding that endothelial cells contain both constitutive and inducible forms of NOS suggests that other cells may also express multiple isoforms of the enzyme. Cloning of NOS indicates that there may be
Received December 9, I99 I : revised manuscript received February 17. 1992: accepted February 24. 1992. Addrcss correspondence and reprint requests to Dr. S. Murphy at Department of Pharmacology. University of Iowa College of Medicine. Iowa City. I A 52247. U.S.A. :lhl,~c,~irr/io/l.r ~ r . s t dBME. basal medium Eagle: CaM, calmodulin: Dil-Ac-LDL. acetylated low-density lipoprotein labeled with 1. I ‘-dioctadccyl- 1-3.3.3’.3’-tetramethylindocarbocyanine perchlo-
rate: DIV. days in vitro: EBSS. Earle’s balanced salt solution: EDRF. endothelium-derived relaxing factor: EMEM. Eagle’s minimal essential medium: FCS. fetal calf serum: IBMX. 3-isobutyl- I -methylxanthine: LME. leucine methyl ester: LPS. lipopolysaccharide: NAME. Kw-nitro-L-arginine methyl ester: NMA. NG-monomethyI-L-arginine: NO, nitric oxide: NOS. nitric oxide synthase: SOD, supcroxide dismutase.
897
M . L. SIMMONS AND S. MURPHY
898
several gene products in the NOS family (Bredt et al., 1991), but it remains to be seen which specific cell types express each. Recently, we (Murphy et al., 1990, 1991) and others (Mollace et al., 1990; Agullo and Garcia, I99 1, 1992) have described a nitrosyl factor released from astrocytes that is capable of stimulating soluble guanylate cyclase and eliciting vasorelaxation. Here we show that astrocytes, microglia, and C6 glioma cells express an LPS-inducible NOS that is calcium independent.
MATERIALS AND METHODS Cell culture Primary astrocyte cultures were prepared from neonatal rat cortices as previously described (Murphy, 1990) and grown in TI50 flasks for at least 14 days in vitro (DIV) in Eagle’s minimal essential medium (EMEM) supplemented with 33 mM glucose, 2 mA4 glutamine, 50 pg/ml of gentamicin, and 10% (vol/vol) fetal calf serum (FCS). All cells were kept at 37°C in 5% C0,/95% air. These primary cultures have been characterized by immunocytochemistry and routinely consist of 90-9596 glial acidic fibrillary protein-positive cells (astrocytes) with contaminating microglia (3-7%), fibroblasts ( 1-3%), oligodendrocytes, and vascular cells (
