208
Brain Research, 592 (1992) 2(}8-212 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
BRES 18150
Effect of nitric oxide on mitogenesis and proliferation of cerebellar glia! cells U t t a m C. G a r g a, Lakshmi Devi b, H e r m a n T u r n d o r f a n d Mylarrao Bansinath a
a, Lewis R. G o l d f r a n k c
Departments of a Anesthesiology, b Pharmacology and c Emergency Medicine, New York Uni~'ersity Medical Center, N Y 10016 (USA)
(Accepted 12 May 1992)
Key words: Nitric oxide; Cerebellar gila; Milogenesis; Cyclic guanosine monophosphate; Cytostasis; Thymidine incorporation.
In the brain, nitric oxide (NO) has been identified as a messenger molecule and a mediator of excitatory amino acid-induced neurotoxicity. In this study, the effects of NO on serum-induced mitogenesi~ and cell proliferation of the cerebellar glial cells were assessed. NOgenerating agent, S-nitroso-N-acetylpenicillamine (SNAP) increased intracellular cyclic guanosine monophosphate (cGMP) levels. Furthermore, 2 chemically dissimilar NO.generatin~ agents, SNAP and sodium nitroprusside (SNP) inhibited serum-induced thymidine incorporation and cell proliferation. The antimitogenic effect of NO was mimicked by 8-bromo-cGMP and blocked by hemoglobin, a known inhibitor of NO. The effect of NO was not cytotoxic, since the cells were not stained with Trypan blue and did not show increased release of lactate dehydrogenase in the culture supernutants, However, NO-treated cells showed decreased conversion of tetrazolium to blue formazan suggesting that NO inhibited mitoohondrial activity in the glial cells, Those results demonstrate that NO inhibits serum-induced mitogcnesis and cell proliferation of cultured rat ¢crebeUar glial cells.
INTRODUCTION
Nitric oxide (NO), a short.lived, highly reactive molecule was initially identified as a mediator for macrophages and endothelial cells. Recently, NO has been recognized as a neuronal messenger ~. The' most striking feature of NO as a second messenger is its rapid diffusion into ceils, without involvement of any receptor, in the brain, NO is synthesized from Larginine, with stoichiometric formation of L-citrulline, by calcium calmodulin-dependent enzyme NO synthase "'ts. Using immunochemistry, this enzyme has been localized within neuronal cells in specifi,: brain regions, with highest expression in neurons of the cerebellumz22. Many physiological and pathophysiological functions, including second messenger and mediation of excitatory amino acid-induced nourotoxicity in the brain, have been attributed to NO t,T, In the cerebel. lure, excitatory amino acid agonists, particularly N-
mothyl-D-aspartate, stimulate NO release both in vivo and in vitro t4,'4. This release of NO is linked to stimulation of guanylate cyelasc and increased cGMP levels in neuronal and glial structures z4. Several studies have shown that NO is cytostatic/cytotoxic in various cell types, including cultured cortical neurons t,=°. Being highly diffusible, NO released from neurons could have cytostatie/cytotoxic effects on surrounding glial cells. Recently, it has been shown that incubation of cerebellar granular ceils with cerebellar astrocytes in the prosence of N-methyl-o-aspartate increases cGMP in the astrocytes. This increase in cGMP was due to release of NO from granular cells I~, We studied the effects of NO-generating agents S-nitroso-N-acetyl-penicillamine (SNAP) and sodium nitroprusside (SNP) on DNA synthesis and cell proliferation in cultured cerebellar glial cells. In the present communication, we report that both SNAP and SNP inhibit thymidine incorporation and cell proliferation of rat cerebellar glial cells.
Corrcspondet:ce: U.C, Garg and M. Bansinath, Department of Anesthesiology, NYU Medical Center, 550 First Avenue New York, NY 10016, USA. Fax: (i) (212) 263-7254.
209 12.
120 l
10.
• &
SNAP SNP Hemoglobin
100
u
o P
L
8
S
1
8O
e, O
.m
60
o n.
_c
4
40
E
=i
=0
I-
o
0
.eel
. . . . . . . .
.O1
SNAP, mM
Fig. 1. Effect of SNAP on intracellular cGMP. All cGMP values obtained in the presence of various concentrations of SNAP were significantly different from control (P < 0,01). Results are mean+ S.E.M. (n = 3) from a representative experiment. Similar results were obtained in a second series of the experiment; variation between the experiments was < 10%. In 'control' cGMP levels were 0.8 pmol/mg protein,
MATERIALS AND METHODS [methyl.3H]Thymidine (58 Ci/mmol) was purchased from ICN radiochemicals (lrvine, CA). SNAP was synthesized as described earlier t° by the reaction of NuNO 2 and N.acetylpenicillamine. SNP, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), 8-bromo-cGMP and fetal bovine serum were obtained from Sigma (St. Louis, Me). Dulbecco's modified essential medium (DMEM) was purchased from Mediateeh (Washington PC, MD). 2'-O-succinyl-[JZSl](iodotyrosine methyl ester)-guanosine-Y,5'-cy¢lic phosphoric acid and specific antibody against cGMP were purchased from Biomedical Technologies (Stoughton, MA). Rat cercbellar glial cells were cultured as described by Saneto and de Vellisu. Briefly, the cerebelli from 1- to 2.day-old rats (Sprague-Dawley, Taconic, Germantown, NY) were dissected and adherent meninges were removed. The tissue was dissociated mechanically and incubated at 37°C for 10-15 min in Hanks' balanced salt solution (Mediatech, Washington PC, MD) containing 0.1% trypsin. Big clumps were removed by passing the suspension through 75 ~m Nitex mesh. The cells were collected by centrifugation and suspended in DMEM supplemented with 10% fetal bovine serum. The cells were seeded at a density of ~ 1.4x 104/cm 2 for 7-10 days at 37°C in 5% CO 2. Contaminating neurons were removed by orbital shaking of the flasks overnight. To study the effect of NO on cGMP, the cells were incubated with or without ('control') varying concentrations of SNAP (Fig. 1) for 1 min. The medium was aspirated and cGMP was extracted by 0.1 N HCI. cGI'4P was measured by radioimmunoassay 5, For thymidine incorporation studies, the cells were growth arrested by incubation in serum-free medium for 24 h. The quiescent cells were cultured for 24 h in DMEM having 5% serum, containing or lacking experimental agents as shown in Figs. 2 and 3. In the last 4 h of incubation, 1 /~Ci of methyl [3H]thymidine was added to each well, to measure DNA synthesis by thymidine incorporation. The experiments were terminated by removing the media and washing the cells with phosphate-buffered saline. Acid insoluble material was precipitated with 10% trichloroacetie acid and DNA was extracted with 0.2% sodium dodecyi sulphate (SDS) in 0.5 N NaOH. Radioactivity incorporated into the DNA was measured by liquid scintillation counting,
.
. . . . . . . .
.
,1
. . . . . . . .
I
10
Concentration,mM Fig. 2. Dose-response relationship for the inhibition of sert/m-induced tbymidine incorporation by SNAP (closed squares) and SNP (triangle). The effect of hemoglobin (10 /zM) on the inhibition of serum-induced mitogenesis by SNAP (open squares) is also shown. Each value is mean + S.E.M. (n = 3-4) from a representative experiment. Similar results were obtained in other 2-3 experiments; variation between experiments was < 10%. Results are expressed as percent of 'control', defined as thymidine incorporation in the presence of 5% serum only. In 'control' there were 70x 103-80X 103 cpm/well. To determine the effect of SNAP on cell proliferation, the cells were made quiescent as described above. Subsequently, the cells were cultured for 4 days in DMEM supplemented with 5% fetal bovine serum containing various concentrations of SNAP as shown in Fig. 4. The medium was changed daily. Cells were dissociated by 0.25% trypsin-EDTA and counted by a hemocytometer. For lactate dehydrogenase assay, the cells were treated with SNAP or SNP for 24 h or 4 days and the supernatants were
120,
100~
o [: 8
o
95%) of the cells treated with SNAP or SNP were not stained with Trypan blue. Also, lactate dehydrogenase activity was not signifi-
211
cantI\;- increased in the supernatants from SNAP- or SNP-treated cells as compared with the control cells suggesting that the observed effects of NO were not due to cell death (data not shown). Tetrazolium assay is based on the cellular conversion of tetrazolium salt into a blue formazan product. This conversion is dependent on the mitochondrial activity. SNAP-treated cells showed decreased conversion of tetrazolium to blue formazan (Fig. 5). DISCUSSION NO is one of the most recently recognized messenger molecules in the brai#. It is released from the neurons by excitatory amino acid receptor activation’4*24.Since NO diffuses readily into aqueous cellular fluids14, NO could affect the cells from which it is released or it could have effects on neighboring neuronal or glial cells. Co-incubation of cerebellar granular cells and cerebellar astrocytes, in the presence of N-methyl-D-aspartate, increased cGMP in the astrocytes”. This increase in cGMP was due to release of NO from granular cells. The purpose of the present work was to investigate the effects of NO-generating agents on DNA synthesis and cell proliferation of cerebellar glial cells. Cerebellar glial cells were chosen since cerebellum is the major site for the synthesis of NO*. NO-generating agents and not gaseous NO were used in this study since gaseous NO has very short half life (l-5 s). Our data demonstrate that 2 chemically dissimilar agents, SNAP and SNP, sharing the ability to generate NO in the aqueous medium, inhibited serum-induced thymidine incorporation and cell proliferation in cultured cerebellar glial cells. Antimitogenic effect of SNAP could be reversed by hemoglobin, a known inhibitor of NO, demonstrating that the effect of SNAP was mediated by NO. The effect of NO on thymidine incorporation was mimicked by 8-bromo-cGMP, a permeable and non-hydrolyzable analog of cGMP, suggesting that the antimitogenic effect of NO may be mediated through cGMP. However, a cGMP independent effect of NO cannot be ruled out from the present studies. NO has been shown to have many effects independent of cGMP. In Balb/c 3T3 fibroblasts, NO has been shown to decrease cytosolic free-calcium by a cGMP independent mechanism’*. In addition, NO activates an endogenous ADP-ribosyltransfer&e, independent of cGMP in many tissues including brain4. Also NO-generating agents inhibit serum-induced mitogenesis and cell proliferation in Balb/c 3T3 fibroblasts by a cGMP independent mechanism13. Cyclic-GMP mimicked the antimitogenic effect of
NO in aortic smooth muscle and mesangial cellsl0J’. cGMP has been shown to cause destruction of photoreceptor cells in the retina, where NO synthase has been demonstrated by immunochemistry *v**,Furthcrmore, in cultured cerebral cortical neurons, 8-bromocGMP enhanced the cytotoxic effect of glutamate, Nmethyl-1)-aspartate and kainate’. Recently, NO has been demonstrated to mediate glutamate-induced neurotoxicity in primary cortical neuronal cultures’. Glutamate-induced neurotoxicity could be inhibited by nitroarginine and N-monomethyl+arginine, known NO synthase inhibitors. Also glutamate-induced cellular toxicity could be reversed by hemoglobin which binds to NO’. In cerebellar glial cells, the antimitogenic effect of NO was unlikely to be due to NO-induced cell death because, the cells treated with SNAP or SNP were not stained with Trypan blue, a dye known to stain dead cells. This was further supported by the finding that SNAP- or SNP-treated cells did not show increased release of lactate dehydrogenase in the supernatants. Since, SNAP-treated cells showed decreased cleavage of tetrazolium salt to blue formazan, the likely mechanism of action of NO may be to decrease the mitochondrial activity. Tetrazolium salt is cleaved in the active mitochondria*‘. Live but metabolically inactive cells such as RBC’s and resting spleen cells do not cleave tetrazolium salt*“. It has been shown that NO inhibits mitochondrial respiration by forming a complex with iron-sulfur centers of the enzymes to inactivate them’“. Another potential mechanism that could mediate the effects of NO is enhanced ADP ribosylation of specific proteins. NO induces ADP ribosylation of a 39 kDa protein in many tissues including brain4. Recently, NO has been shown to deaminate DNA which could further interfere with the DNA repli. catiorP. In summary, NO inhibits serum-induced DNA synthesis and cell proliferation of cerebellar glial cells. These findings could have clinical implications particularly in excitatoryamino acid-induced neurotoxicity. Acknowledgemenls. This work was partially supported by grants from Aaron Diamond Foundation and National Institutes of Health (NS 26880 to L-D.). Part of the results were presented in the FIDIA Research Foundation Symposium entitled ‘Excitatory amino acids 1992’ held at Yosemite, CA, February 16-21, 1992.
REFERENCES 1 Barinaga, M., Is nitric oxide the ‘retrograde messenger’7 Science, 254 (1991) 1296-1297. 2 Bredt, D.S., Hwang, P.M. and Snyder, S.H., Localization of nitric oxide synthase indicating a neural role for nitric oxide, Nature, 347 (1990) 768-770.
212 3 Bredt, D.S. and Snyder, S.H,, Nitric oxide, a novel neuronal messenger, Neuron, 8 (1992) 3-11. 4 Brune, B. and Lapetina, E.G., Activation of a cytosolic ADPribosyltransferase by nitric oxide-generating agents, J. Biol. Chem., 264 (1989) 8455-8458. 5 Brooker, G., Terasaki, W.L. and Moylan, R.D., Radioimmunoassay of cAMP and cGMP. In G. Brooker, P. Greengard and G.A. Robison (Eds.), Adt'ances in Cyclic Nucleotide Research. Vol. 10, Raven, New York, 1979, pp. 1-33. 6 Crossin, K.L., Nitric oxide (NO): a versatile second messenger in brain, Trends Biochem. Sci., 16 (1991) 81-83. 7 Daws,an, V.L., Dawson, T.M., London. E.D., Bredt. D.S. and Snyder, S.H., Nitric-oxide mediates glutamate neurotoxicity in primaw cortical cultures, Proc. Natl. Acad. Sci. USA. 88 (1991) 6368-6371. 8 Forstermann, U., Gorsky. L.D., Pollock, J.S., Ishii, K., Schmidt, H.H.H.W., Miller, M. and Murad. F., Hormone-induced biosynthesis of endothelium-derived relaxing factor/nitric oxide-like material in NIE-II5 neuroblastoma cells requires calcium and calmodulin, Mol. Pharmacol., 38 (1990) 7-13. 9 Frandsen. A.. Andersen, C.F. and Schousboe, A.. Possible role of cGMP in excitatory amino induced cytotoxicity in cultured cerebral cortical neurons, Neurochem. Res., 17 (1992) 35-43. 10 Gar'g, U.C. and Hassid. A., Inhibition of rat mesangial cell mitogenesis by nitric oxide generating vasodilators, Am. J. Phys. iol., 257 (1989) F60-F66. I I Garg, U.C. and Hassid, A., Nitric oxide-generating vasodilators and 8.bromo.cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells, J. CIin." hll'est., 83 (1989) 1774- 1777. 12 Garg, U.C. and Hassid, A., Nitric oxide decreases cytosolic freecalcium in Balb/c 3T3 fibroblasts by a cyclic GMP-independent mechanism, J. Biol. Ozem., 266 (1991) 9-12. 13 Garg, U.C. and Hassid, A., Nitric oxide.generating vasodilators inhibit mitogenesis and proliferation of Balb/c 3T3 fibrohlasts by a cyclic GMP-dependent mechanism, Biochem. Biophys. Res. Commtm., 171 (1991) 474-479, 14 Garthwaite, J., Charles, S.L. and Chess.Williams, R., Endothe. lium derived relaxing factor release on ~Lctivation ~f NMDA
receptor suggests role as intracellular messenger in the brain, Nature, 336 (1988) 385-388. 15 Hansen, M.B., Nielsen, S.E. arid Berg, K., Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill, J. lmmunol. Methods, 119 (1989) 203-210. 16 Hibbs, J.B., Taintor, R.R., Vavrin, Z. ~nd Rechlin, E.M., Nitric oxide: a cytotoxic activated macrophage effector molecule, Biochem. Biophys. Res. Commun., 157 (1988)87-94. 17 Kiedrowski, L., Costa, E. and Wroblewski, J.T., In vitro interaction between cerebellar astrocytes and granule cells: a putative role for nitric-oxide, Neurosci. Lett., 135 (1992)59-61. 18 Knowles, R.G., Palacios, M., Palmer R.M. and Monocada, S., Formation of nitric-oxide from L-arginine in the central nervous system: a transduction mechanism for stimulation of the soluble guanylate cyclase, Proc. Natl. Acad. Sci. USA, 86 (1989) 51595162. 19 Moncada, S., Palmer, R.MJ. and Higgs, E,A., Nitric oxide: pathophysiology and pharmacology. Pharmacol. Ret'., 43 (1991) 109-142.
20 Mosmann, T., Rapid ,olorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. lmmunol. Methods, 65 (1983) 55-63. 21 Saneto, R.P. and de Vellis, J., Neuronal and glial cells: cell culture of the central nervous system. In A.J. Turner and H.S. Bachelard (Eds,). Neurochemistry: A Practical Approach, IRL, Oxford, 1987, pp. 27-63. 22 Vincent, S.R. and Kimura, H., Histochemical mapping of nitric oxide synthase in the rat brain, Neuroscience, 46 (1992) 755-784. 23 Wink, D,A., Kasprzak, K.$., Ma,agos, G.M., Elespuru, R,K., Mishra, M., Dunams, T.M., Cebula, T.A., Koch, W,H,, Andrews, A.W., Alle,.i, J.S. and Keefer, L.K., DNA deaminating ability and genotoxic[ty of nitric oxide and its progenitors, Science, 254 (1991) 1001-1003. 24 Wood. P.L., Emmett, M.R,, Rao, T,S., Cler, J., Mick, S, and lyenger, $., Inhibition of nitric oxide synthase blocks N-methylD-aspartate, quisqualate, kainate, harmaline and pentylenctctraz~le.dependent increases in cerebellar cyclic GMP in vivo, J, Neurochem., 55 (I 990) 346-348.
Brain Research, 592 (1992) 2(}8-212 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
BRES 18150
Effect of nitric oxide on mitogenesis and proliferation of cerebellar glia! cells U t t a m C. G a r g a, Lakshmi Devi b, H e r m a n T u r n d o r f a n d Mylarrao Bansinath a
a, Lewis R. G o l d f r a n k c
Departments of a Anesthesiology, b Pharmacology and c Emergency Medicine, New York Uni~'ersity Medical Center, N Y 10016 (USA)
(Accepted 12 May 1992)
Key words: Nitric oxide; Cerebellar gila; Milogenesis; Cyclic guanosine monophosphate; Cytostasis; Thymidine incorporation.
In the brain, nitric oxide (NO) has been identified as a messenger molecule and a mediator of excitatory amino acid-induced neurotoxicity. In this study, the effects of NO on serum-induced mitogenesi~ and cell proliferation of the cerebellar glial cells were assessed. NOgenerating agent, S-nitroso-N-acetylpenicillamine (SNAP) increased intracellular cyclic guanosine monophosphate (cGMP) levels. Furthermore, 2 chemically dissimilar NO.generatin~ agents, SNAP and sodium nitroprusside (SNP) inhibited serum-induced thymidine incorporation and cell proliferation. The antimitogenic effect of NO was mimicked by 8-bromo-cGMP and blocked by hemoglobin, a known inhibitor of NO. The effect of NO was not cytotoxic, since the cells were not stained with Trypan blue and did not show increased release of lactate dehydrogenase in the culture supernutants, However, NO-treated cells showed decreased conversion of tetrazolium to blue formazan suggesting that NO inhibited mitoohondrial activity in the glial cells, Those results demonstrate that NO inhibits serum-induced mitogcnesis and cell proliferation of cultured rat ¢crebeUar glial cells.
INTRODUCTION
Nitric oxide (NO), a short.lived, highly reactive molecule was initially identified as a mediator for macrophages and endothelial cells. Recently, NO has been recognized as a neuronal messenger ~. The' most striking feature of NO as a second messenger is its rapid diffusion into ceils, without involvement of any receptor, in the brain, NO is synthesized from Larginine, with stoichiometric formation of L-citrulline, by calcium calmodulin-dependent enzyme NO synthase "'ts. Using immunochemistry, this enzyme has been localized within neuronal cells in specifi,: brain regions, with highest expression in neurons of the cerebellumz22. Many physiological and pathophysiological functions, including second messenger and mediation of excitatory amino acid-induced nourotoxicity in the brain, have been attributed to NO t,T, In the cerebel. lure, excitatory amino acid agonists, particularly N-
mothyl-D-aspartate, stimulate NO release both in vivo and in vitro t4,'4. This release of NO is linked to stimulation of guanylate cyelasc and increased cGMP levels in neuronal and glial structures z4. Several studies have shown that NO is cytostatic/cytotoxic in various cell types, including cultured cortical neurons t,=°. Being highly diffusible, NO released from neurons could have cytostatie/cytotoxic effects on surrounding glial cells. Recently, it has been shown that incubation of cerebellar granular ceils with cerebellar astrocytes in the prosence of N-methyl-o-aspartate increases cGMP in the astrocytes. This increase in cGMP was due to release of NO from granular cells I~, We studied the effects of NO-generating agents S-nitroso-N-acetyl-penicillamine (SNAP) and sodium nitroprusside (SNP) on DNA synthesis and cell proliferation in cultured cerebellar glial cells. In the present communication, we report that both SNAP and SNP inhibit thymidine incorporation and cell proliferation of rat cerebellar glial cells.
Corrcspondet:ce: U.C, Garg and M. Bansinath, Department of Anesthesiology, NYU Medical Center, 550 First Avenue New York, NY 10016, USA. Fax: (i) (212) 263-7254.
209 12.
120 l
10.
• &
SNAP SNP Hemoglobin
100
u
o P
L
8
S
1
8O
e, O
.m
60
o n.
_c
4
40
E
=i
=0
I-
o
0
.eel
. . . . . . . .
.O1
SNAP, mM
Fig. 1. Effect of SNAP on intracellular cGMP. All cGMP values obtained in the presence of various concentrations of SNAP were significantly different from control (P < 0,01). Results are mean+ S.E.M. (n = 3) from a representative experiment. Similar results were obtained in a second series of the experiment; variation between the experiments was < 10%. In 'control' cGMP levels were 0.8 pmol/mg protein,
MATERIALS AND METHODS [methyl.3H]Thymidine (58 Ci/mmol) was purchased from ICN radiochemicals (lrvine, CA). SNAP was synthesized as described earlier t° by the reaction of NuNO 2 and N.acetylpenicillamine. SNP, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), 8-bromo-cGMP and fetal bovine serum were obtained from Sigma (St. Louis, Me). Dulbecco's modified essential medium (DMEM) was purchased from Mediateeh (Washington PC, MD). 2'-O-succinyl-[JZSl](iodotyrosine methyl ester)-guanosine-Y,5'-cy¢lic phosphoric acid and specific antibody against cGMP were purchased from Biomedical Technologies (Stoughton, MA). Rat cercbellar glial cells were cultured as described by Saneto and de Vellisu. Briefly, the cerebelli from 1- to 2.day-old rats (Sprague-Dawley, Taconic, Germantown, NY) were dissected and adherent meninges were removed. The tissue was dissociated mechanically and incubated at 37°C for 10-15 min in Hanks' balanced salt solution (Mediatech, Washington PC, MD) containing 0.1% trypsin. Big clumps were removed by passing the suspension through 75 ~m Nitex mesh. The cells were collected by centrifugation and suspended in DMEM supplemented with 10% fetal bovine serum. The cells were seeded at a density of ~ 1.4x 104/cm 2 for 7-10 days at 37°C in 5% CO 2. Contaminating neurons were removed by orbital shaking of the flasks overnight. To study the effect of NO on cGMP, the cells were incubated with or without ('control') varying concentrations of SNAP (Fig. 1) for 1 min. The medium was aspirated and cGMP was extracted by 0.1 N HCI. cGI'4P was measured by radioimmunoassay 5, For thymidine incorporation studies, the cells were growth arrested by incubation in serum-free medium for 24 h. The quiescent cells were cultured for 24 h in DMEM having 5% serum, containing or lacking experimental agents as shown in Figs. 2 and 3. In the last 4 h of incubation, 1 /~Ci of methyl [3H]thymidine was added to each well, to measure DNA synthesis by thymidine incorporation. The experiments were terminated by removing the media and washing the cells with phosphate-buffered saline. Acid insoluble material was precipitated with 10% trichloroacetie acid and DNA was extracted with 0.2% sodium dodecyi sulphate (SDS) in 0.5 N NaOH. Radioactivity incorporated into the DNA was measured by liquid scintillation counting,
.
. . . . . . . .
.
,1
. . . . . . . .
I
10
Concentration,mM Fig. 2. Dose-response relationship for the inhibition of sert/m-induced tbymidine incorporation by SNAP (closed squares) and SNP (triangle). The effect of hemoglobin (10 /zM) on the inhibition of serum-induced mitogenesis by SNAP (open squares) is also shown. Each value is mean + S.E.M. (n = 3-4) from a representative experiment. Similar results were obtained in other 2-3 experiments; variation between experiments was < 10%. Results are expressed as percent of 'control', defined as thymidine incorporation in the presence of 5% serum only. In 'control' there were 70x 103-80X 103 cpm/well. To determine the effect of SNAP on cell proliferation, the cells were made quiescent as described above. Subsequently, the cells were cultured for 4 days in DMEM supplemented with 5% fetal bovine serum containing various concentrations of SNAP as shown in Fig. 4. The medium was changed daily. Cells were dissociated by 0.25% trypsin-EDTA and counted by a hemocytometer. For lactate dehydrogenase assay, the cells were treated with SNAP or SNP for 24 h or 4 days and the supernatants were
120,
100~
o [: 8
o
95%) of the cells treated with SNAP or SNP were not stained with Trypan blue. Also, lactate dehydrogenase activity was not signifi-
211
cantI\;- increased in the supernatants from SNAP- or SNP-treated cells as compared with the control cells suggesting that the observed effects of NO were not due to cell death (data not shown). Tetrazolium assay is based on the cellular conversion of tetrazolium salt into a blue formazan product. This conversion is dependent on the mitochondrial activity. SNAP-treated cells showed decreased conversion of tetrazolium to blue formazan (Fig. 5). DISCUSSION NO is one of the most recently recognized messenger molecules in the brai#. It is released from the neurons by excitatory amino acid receptor activation’4*24.Since NO diffuses readily into aqueous cellular fluids14, NO could affect the cells from which it is released or it could have effects on neighboring neuronal or glial cells. Co-incubation of cerebellar granular cells and cerebellar astrocytes, in the presence of N-methyl-D-aspartate, increased cGMP in the astrocytes”. This increase in cGMP was due to release of NO from granular cells. The purpose of the present work was to investigate the effects of NO-generating agents on DNA synthesis and cell proliferation of cerebellar glial cells. Cerebellar glial cells were chosen since cerebellum is the major site for the synthesis of NO*. NO-generating agents and not gaseous NO were used in this study since gaseous NO has very short half life (l-5 s). Our data demonstrate that 2 chemically dissimilar agents, SNAP and SNP, sharing the ability to generate NO in the aqueous medium, inhibited serum-induced thymidine incorporation and cell proliferation in cultured cerebellar glial cells. Antimitogenic effect of SNAP could be reversed by hemoglobin, a known inhibitor of NO, demonstrating that the effect of SNAP was mediated by NO. The effect of NO on thymidine incorporation was mimicked by 8-bromo-cGMP, a permeable and non-hydrolyzable analog of cGMP, suggesting that the antimitogenic effect of NO may be mediated through cGMP. However, a cGMP independent effect of NO cannot be ruled out from the present studies. NO has been shown to have many effects independent of cGMP. In Balb/c 3T3 fibroblasts, NO has been shown to decrease cytosolic free-calcium by a cGMP independent mechanism’*. In addition, NO activates an endogenous ADP-ribosyltransfer&e, independent of cGMP in many tissues including brain4. Also NO-generating agents inhibit serum-induced mitogenesis and cell proliferation in Balb/c 3T3 fibroblasts by a cGMP independent mechanism13. Cyclic-GMP mimicked the antimitogenic effect of
NO in aortic smooth muscle and mesangial cellsl0J’. cGMP has been shown to cause destruction of photoreceptor cells in the retina, where NO synthase has been demonstrated by immunochemistry *v**,Furthcrmore, in cultured cerebral cortical neurons, 8-bromocGMP enhanced the cytotoxic effect of glutamate, Nmethyl-1)-aspartate and kainate’. Recently, NO has been demonstrated to mediate glutamate-induced neurotoxicity in primary cortical neuronal cultures’. Glutamate-induced neurotoxicity could be inhibited by nitroarginine and N-monomethyl+arginine, known NO synthase inhibitors. Also glutamate-induced cellular toxicity could be reversed by hemoglobin which binds to NO’. In cerebellar glial cells, the antimitogenic effect of NO was unlikely to be due to NO-induced cell death because, the cells treated with SNAP or SNP were not stained with Trypan blue, a dye known to stain dead cells. This was further supported by the finding that SNAP- or SNP-treated cells did not show increased release of lactate dehydrogenase in the supernatants. Since, SNAP-treated cells showed decreased cleavage of tetrazolium salt to blue formazan, the likely mechanism of action of NO may be to decrease the mitochondrial activity. Tetrazolium salt is cleaved in the active mitochondria*‘. Live but metabolically inactive cells such as RBC’s and resting spleen cells do not cleave tetrazolium salt*“. It has been shown that NO inhibits mitochondrial respiration by forming a complex with iron-sulfur centers of the enzymes to inactivate them’“. Another potential mechanism that could mediate the effects of NO is enhanced ADP ribosylation of specific proteins. NO induces ADP ribosylation of a 39 kDa protein in many tissues including brain4. Recently, NO has been shown to deaminate DNA which could further interfere with the DNA repli. catiorP. In summary, NO inhibits serum-induced DNA synthesis and cell proliferation of cerebellar glial cells. These findings could have clinical implications particularly in excitatoryamino acid-induced neurotoxicity. Acknowledgemenls. This work was partially supported by grants from Aaron Diamond Foundation and National Institutes of Health (NS 26880 to L-D.). Part of the results were presented in the FIDIA Research Foundation Symposium entitled ‘Excitatory amino acids 1992’ held at Yosemite, CA, February 16-21, 1992.
REFERENCES 1 Barinaga, M., Is nitric oxide the ‘retrograde messenger’7 Science, 254 (1991) 1296-1297. 2 Bredt, D.S., Hwang, P.M. and Snyder, S.H., Localization of nitric oxide synthase indicating a neural role for nitric oxide, Nature, 347 (1990) 768-770.
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