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Nitric oxide synthase activity in the hippocampus, frontal cerebral cortex, and cerebellum of the guinea pig: Ontogeny and in vitro ethanol exposure
Alcohol,Vol. 12, No. 4, pp. 329-333, 1995 Copyright©1995ElsevierScienceLtd Printedin the USA.All rightsreserved 0741-8329/95 $9.50 + .00
Pergamon 0741-8329(95)00006-2
Nitric Oxide Synthase Activity in the Hippocampus, Frontal Cerebral Cortex, and Cerebellum of the Guinea Pig: Ontogeny and In Vitro Ethanol Exposure J A M E S F. B R I E N , 1 J A M E S D. R E Y N O L D S , M I C H A E L A. C U N N I N G H A M , A N N M. P A R R , S A R A H W A D D O C K A N D B E T T I N A E. K A L I S C H
Department o f Pharmacology and Toxicology, Faculty o f Medicine, Queen's University, Kingston, Ontario, Canada K7L 3N6 Received 11 N o v e m b e r 1994; Accepted 7 D e c e m b e r 1994 BRIEN, J. F., J. D. REYNOLDS, M. A. CUNNINGHAM, A. M. PARR, S. WADDOCK AND B. E. KALISCH.
Nitric oxide synthase activity in the hippocampus,frontal cerebralcortex, and cerebellum of the guineapig: Ontogeny and in vitro ethanol exposure. ALCOHOL 12(4) 329-333, 1995.--Decreased nitric oxide (NO) formation, resulting from inhibition of NO synthase (NOS), may be important in the pathogenesis of ethanol central nervous system teratogenesis. The objectives of this study were to determine the ontogeny of NOS activity in the hippocampus, frontal cerebral cortex, and cerebellum of the developing guinea pig, and to test the hypothesis that direct exposure to ethanol inhibits NOS activity in these brain regions at selected developmental ages. NOS activity was quantitated by an optimized radiometric assay. The ontogeny study demonstrated that NOS activity in the hippocampus and frontal cortex was not fully developed prenatally, and apparently increased during postnatal life to attain adult level of activity at postnatal day > 60. In the cerebellum, NOS activity increased during prenatal life to an apparent maximum in the mature near-term fetus at gestational day 63 (term, about 68 days), and then apparently declined during postnatal life to attain adult level of activity. In vitro ethanol exposure (25-100 raM) did not affect NOS activity in the hippocampus, frontal cortex, or cerebellum at any developmental age studied. These data indicate that, although the ontogeny of NOS activity varies between brain regions, ethanol does not directly affect NOS activity in the developing guinea pig. The effects of acute and chronic in utero ethanol exposure on NOS activity in these brain regions are currently being investigated. Nitric oxide synthase Ontogeny Ethanol
Hippocampus
Frontal cerebral cortex
Cerebellum
Guinea pig
L-arginine to L-citrulline, catalyzed by NO synthase (NOS) (16). In the brain, NO is predominantly formed by the constitutive isoform of NOS, which is a Ca2+/calmodulin-dependent enzyme (3) localized in the neuron (13). NO has been proposed to play a neur,otrophic role in brain development (10,11). Glu also plays an important role in CNS development by regulating neuronal survival and synaptogenesis (15). As Glu activation of the NMDA receptor can result in NO formation, it is conceivable that NO acts as a second messenger to mediate some of the neurotrophic actions of Glu. Recent data from our laboratory have shown that, in the fetal guinea pig hippocampus, in vitro ethanol exposure
THE hippocampus, frontal cerebral cortex, and cerebellum have been identified as target sites for ethanol central nervous system (CNS) teratogenesis (20). L-Glutamate (Glu) appears to be the major excitatory neurotransmitter in these brain regions. The postsynaptic actions of Glu are mediated by several different excitatory amino acid receptor subtypes, including the N-methyl-D-aspartate (NMDA) receptor (18). The binding of Glu to the NMDA receptor leads to ion channel opening and the influx of Ca 2+. This increase in intracellular Ca 2+ concentration can result in the formation of nitric oxide (NO), a novel gaseous second messenger with a short biological halflife (17). NO is produced during the enzymatic oxidation of
To whom requests for reprints should be addressed. 329
330
BRIEN ET AL.
decreases stimulated and spontaneous release of Glu (19). This depressant action of ethanol could result in reduced NMDA receptor activation leading to decreases in NOS activity and consequent NO formation. A decrease in NO formation could account, at least in part, for the disruption of neuronal development produced by prenatal ethanol exposure. This proposal is supported by the observation that injection of a NOS inhibitor into the amniotic fluid of the cultured whole rat conceptus at midgestation produces dysmorphogenic effects (12). In vitro ethanol exposure inhibits Glu release (19), but it does not appear to affect the density or affinity of the NMDA receptor (Abdollah and Brien, submitted for publication). To continue our investigation of the effects of ethanol on the developing Glu neurotransmitter-NMDA receptor system, it was important to determine whether in vitro ethanol exposure had a direct effect on NOS activity. The first objective of this study was to determine the ontogeny of NOS activity in the guinea pig hippocampus, frontal cerebral cortex, and cerebellum at selected developmental ages, as there apparently are no ontogeny data reported in the literature. The second objective was to test the hypothesis that in vitro exposure to ethanol directly inhibits NOS activity in these brain regions. Our laboratory has used the guinea pig to investigate the neuroteratogenie effects of prenatal ethanol exposure because of this animal's extensive prenatal brain development, including the brain growth spurt, which is more similar to the human than is the case for the rat (7). METHOD
Breeding Procedure for Experimental Animals Male and female Dunkin-Hartley strain guinea pigs (Charles River Canada Inc., St. Constant, Que.) were bred using an established procedure (5,8). Day zero of pregnancy was established as the last day of full vaginal-membrane opening [term, about gestational day (GD) 68]. Pregnant and nonpregnant animals were housed in groups of up to four in stainless steel wire cages at 23°C with a 12-h light/12-h dark cycle. Food (Purina Guinea Pig Chow 5025 ®) and water were provided ad lib. Pregnancy status of the breeding female animals and the general health of all the guinea pigs were monitored every other day. The animals were cared for in accordance with the principles and guidelines of the Canadian Council on Animal Care. The experimental protocol was approved by the Queen's University Animal Care Committee.
Chemicals and Solutions Dowex 50W/50X8-400, N-[2-hydroxyethyl]-piperazine-N'[2-ethanesulfonic acid] (HEPES), ethylenediaminetetraacetic acid disodium salt (EDTA), dithiothreitol (DTT), and leupeptin were obtained from Sigma Chemical Co. (St. Louis, MO). L-[~+C]Arginine (331.2 mCi/mmol, 99% radiochemical purity) was supplied by Dupont-New England Nuclear (Lachine, Que.). The dye reagent used for the protein assay (2) was purchased from Bio-Rad Laboratories (Mississauga, Ont.). All other chemicals were at least reagent grade quality and were obtained from a variety of suppliers. Deionlzed water was used to prepare the aqueous chemical solutions. Dowex 50W/50X8-400 was converted to the sodium salt form by treating an aqueous mixture containing the resin with 1 M NaOH to raise the pH above 9.0, followed by rinsing the resin with deionized water until the pH of the wash was 7.0.
Tissue Isolation and Preparation of Subcellular Fraction Containing NOS Fetal guinea pigs at mean GD51 (range, GD50-52) and mean GD63 (range, GD62-64), and adult male and female guinea pigs at postnatal day (PD) > 60 were used in this study. Guinea pigs were killed by decapitation (9), and the fetuses were removed by cesarean section. Each fetus was decapitated; the brain was excised from the skull, and the hippocampi, frontal cerebral cortex, and cerebellum were dissected. For each brain region, pooled tissue from all fetuses in the litter was homogenized in ice-cold solution containing 50 mM HEPES, 1 mM EDTA, 1 mM DTT, 10 #g/ml leupeptin (pH 7.4) to produce an 80-90% (w/v) homogenate. The homogenate was centrifuged at 4°C for 30 min at 25,000 x g. The supernatant was removed and stored at - 7 0 ° C . In a similar manner, the hippocampi, frontal cortex, and cerebellum of individual adult guinea pigs were isolated, and the supernatant samples were prepared and stored at - 70°C. NOS activity of the supernatant was analyzed within 2 weeks of preparation of the subcellular fraction.
NOS Assay The radiometric method used for the quantitation of NOS activity was a modification of the procedure of Bredt and Snyder (3). The method was optimized for the hippocampus, frontal cortex, and cerebellum of the developing guinea pig. A 25-#1 volume of supernatant sample (150-300/~g protein for the fetus at GD51, 260-420/~g protein for the fetus at GD63, and 200-450/zg protein for the adult at PD > 60, depending on the brain region) was added to a reaction tube. A 100-/~1 volume of reaction buffer (50 mM HEPES, 1 mM EDTA, 1 mM DTT, 1.25 mM CaCI2, 2 mM NADPH, pH 7.4) and 25/~I of L-[14C]arginine in an aqueous solution of 180 #M nonradiolabelled L-arginine (radioactivity, about 2.0 x 104 dpm/sampie) were added to each tube, and the contents were incubated at 37°C for 15 min. The reaction was stopped by the addition of 2.0 ml of ice-cold buffer containing 20 mM HEPES and 2 mM EDTA (pH 5.5). Experimental blanks were prepared by adding the stop buffer and reaction buffer to the sample prior to the addition of L-[mC]arginlne/180 /~M nonradiolabelled L-arginine and then incubating at 37°C for 15 rain. Each sample was subjected to chromatographic separation by loading the 2.15-ml volume of reaction solution onto a Dowex 50W/50X8-400 (sodium form) column (4 cm high, 0.4 cm internal diameter) with a silanized wool plug. The column was washed with 2.0 ml deionized water to elute the [l+C]citrulline. The eluate was collected and mixed with 10 ml of Scintiverse ® scintillation fluid (Fisher Scientific, Unionville, Ont.). [~+C]Citrulline radioactivity was quantitated using a Beckman LS 3800 liquid scintillation counter, in which each sample was counted for a 2-rain period. The protein content of each sample was measured using the protein-dye-binding method of Bradford (2) with bovine serum albumin as the standard.
In Vitro Ethanol Exposure and NOS Activity A 25-/zl aliquot of the supernatant sample of each brain region was preincubated with 25, 50, 75, or 100 mM ethanol (final concentration) for 15 rain at 37°C in a sealed tube that had been aerated with 95% 02/5% CO2. Only 10/~1 of preincubation buffer (50 mM HEPES, 1 mM EDTA, pH 7.4) containing the appropriate volume of ethanol to give the desired
E T H A N O L A N D BRAIN NOS ACTIVITY final ethanol concentration were added to each tube to minimize dilution o f the sample. Then, a 10-#1 aliquot o f reaction buffer containing ethanol to produce a final concentration of 25, 50, 75, or 100 mM in 1:he reaction mixture, 100 #1 of normal reaction buffer, and 25 /~1 o f L-["C]arginine in an aqueous solution of 180 ~M nonradiolabelled L-arginine (radioactivity, about 2.0 × 104 dpm/sample) were added to each sample, followed by incubation at 37°C for 15 min. The reaction was terminated by the addition of 2.0 ml o f stop buffer after the 15-rain incubation period. ["C]Citrulline formation was quantitated as described above.
Data Analysis NOS activity in each tissue sample was expressed as pmol [m4C]citrulline formed • 15 miin -t • #g protein -l. The data are presented as group means + SD. To determine if the level of NOS activity in the hippocaznpus, frontal cortex, or cerebellum changed during development, statistical analysis o f the data was conducted by using one-way randomized-design ANOVA. Similarly, one-way randomized-design A N O V A was used to determine if the level o f NOS activity varied among the three brain regions at each selected developmental age. To determine if in vitro ethanol exposure affected the level of NOS activity in each brain region, the data at each developmental age were anaiyzed b y repeated-measures ANOVA. Heterogeneity of variance was assessed by Cochran's test prior to conducting the ANOVA, and the data were transformed as necessary (21). Tukey-Kramer post hoc test was conducted if a significant F statistic ( p < 0.05) was obtained in the ANOVA. Two groups o f data were considered to be statistically different when p < 0.05.
331 mined at GD51 (immature fetus), GD63 (mature fetus), and PD > 60 (adult), and the data are presented in F i g . 1. In the hippocampus and frontal cortex, NOS activity was similar for the immature and mature fetus, and was less at each fetal age compared with the adult ( p < 0.05). In the cerebellum, the level of enzyme activity was higher in the mature fetus compared with the immature fetus and the adult ( p < 0.05). The data demonstrate that, in the hippocampus and frontal cortex, NOS activity is not fully developed during prenatal life and apparently increases during postnatal life to attain adult level by PD > 60. In the cerebellum, NOS activity increases during prenatal life to apparent maximal activity in the mature fetal guinea pig, and then apparently decreases during postnatal life to the adult level. In contrast to our findings in the guinea pig, rat cytosolic cerebellar NOS activity is low in the neonate, and then increases progressively to adult activity (14). These data and the results of our investigation demonstrate that there are Species differences in the ontogeny of constitutive NOS activity in these selected regions of the developing brain. In this regard, it is important to note that most brain development occurs prenatally in the guinea pig and postnatally in the rat (7). There were also regional differences in the level of NOS activity during prenatal development. NOS activity was lower in the hippocampus and frontal cortex compared with the cerebellum for both the immature and mature fetus ( p <
4
I-HPPOCAMPUS
RESULTS AND DISCUSSION
Assay for NOS Activity NOS activity in the hippocampus, frontal cerebral cortex, and cerebellum of the developing guinea pig was quantitated by a modified radiometric as:my used to determine NOS activity in rat brain (3). The assay, using L-[~4C]arginine, was optimized for the determination of Ca2+-dependent constitutive NOS activity in the guinea piF brain with respect to incubation time, protein concentration, L-arginine and N A D P H concentrations, and temperature. ]'he optimized conditions were identical for the hippocampus, frontal cerebral cortex, and cerebellum of the fetal and adult guinea pig. An assay blank (supernatant sample with reaction buffer and stop buffer, containing 2 mM EDTA, added prior to incubation) was run with each set of experimental samples in an attempt to control for any Ca2+-independent production of [t4C]citrulline from L-[liC]arginine. To ensure that NOS activity was being quantitated, a series of experiments~ was conducted using N-nitro-Larginine methyl ester (L-NAlVIE), a selective inhibitor of NOS. Incubation of supernatant samples from the adult guinea pig hippocampus, frontal cortex, and cerebellum with increasing concentrations of L-NAME produced a concentration-dependent decrease in the production o f [t4C]citrulline (data not shown). The highest concentration of L-NAME (1 mM) completely inhibited ["C]citrulline formation in these brain regions (data not shown). These data indicate that virtually all the [14C]citrulline quantitated in our assay results from the oxidation of L-[~4C]arginine, catalyzed by NOS.
Ontogeny of NOS Activity The developmental profiles of NOS activity in the guinea pig hippocampus, frontal Cortex, and cerebellum were deter-
,T
*
CEREBRAL CORTEX
m~
, T ~
J_
• ,
4
GD 51
GD 63 AGE
PD>60
( days )
FIG. 1. Ontogeny of NOS activity in the hippocampus, frontal cerebral cortex, and cerebellum of the guinea pig at GD51 (n = 5), GD63 (n = 5), and PD > 60 (n = 6). The data are presented as group means + SD. *Denotes statistical difference for the: h i p p o c a m p u s , F(2, 15) = 57.25, p < 0.0001, frontal cortex, F(2, 15) = 15.41, p = 0.0004, and cerebellum, F(2, 15) = 19.02, p = 0.0001, compared with the same brain region at PD > 60. T(GD51) and ~(GD63) denote statistical difference compared with the cerebellum at the same developmental age: GD51, F(2, 14) = 65.14,p < 0.0001, and GD63, F(2, 11) = 51.56,p < 0.0001.
332
BRIEN ET AL.
1.0
In Vitro Ethanol Exposure and NOS Activity
GD51
0.5 0.0 1.O.
r.) <_-
r~
0.5 .]
~.~ o ~ • N
0.0 '
0.5 0.0
0
25
50
75
100
[ETHANOL] (raM) FIG. 2. Effect of in vitro ethanol exposure (0-100 mM) on NOS activity in the hippocampus of the guinea pig at GD51 (n = 3), GD63 (n = 4), and PD > 60 (n = 6). The data are presented as group means + SD. 0.05), but was similar in these three brain regions for the adult guinea pig. There was no gender difference in NOS activity for the adult guinea pig (data not presented). This differential ontogeny of NOS activity in the guinea pig brain results in greater enzyme activity in the cerebellum compared with the hippocampus and frontal cortex during prenatal life, but not during adulthood. This is the first reported characterization of the ontogeny of NOS activity in the hippocampus and frontal cerebral cortex of a rodent species and in these two brain regions, along with the cerebellum, of the developing guinea pig.
The hippocampus, frontal cortex, and cerebellum were studied because these brain regions are target sites of ethanol neurobehavioural teratogenesis (20). To assess the effect of direct ethanol exposure on NOS activity, the subcellular fraction containing NOS was incubated with ethanol (25-100 raM). The 15-min incubation time (for a total ethanol exposure time of 30 min including the 15-min enzymatic reaction time) was selected in view of the pharmacokinetics of ethanol in the maternal-fetal unit, including the fetal brain, of the pregnant guinea pig (6). This ensured complete distribution of ethanol throughout the supernatant sample containing NOS activity, and mimicked the time duration that brain tissue would be exposed to a particular ethanol concentration. The data for hippocampai NOS activity in the immature fetus (GD51), mature fetus (GD63), and adult (PD > 60) are presented in Fig. 2. In vitro ethanol exposure did not alter NOS activity in the hippocampus. Similarly, in vitro ethanol exposure did not affect NOS activity in the frontal cortex or cerebellum of the fetal or adult guinea pig (data not shown). These data indicate that the direct exposure of hippocampal, frontal cerebral cortical, or cerebellar NOS to ethanol does not affect enzymatic formation of NO, when the substrate and cofactor are not rate limiting. The next step in our investigation of the effects of ethanol on the developing Glu neuronal system is to determine whether ethanol can act indirectly to alter NOS activity via fetal, placental, and/or maternal effects that ultimately impact on the fetal hippocampus, frontal cortex, and/ or cerebellum. We plan to examine NOS activity in these brain regions of the guinea pig following chronic prenatal exposure to an ethanol regimen that, when administered daily throughout gestation, produces CNS teratogenesis (1,4). ACKNOWLEDGEMENTS This research was supported by an operating grant (MT-8073) from the Medical Research Council of Canada (MRC). M.A.C. and A.M.P. were the recipients Of summer student research scholarships from the Pharmaceutical Manufacturers Association of CanadaHealth Research Foundation/MRC program. The authors wish to thank Mr. Bruce Connop for his excellent technical assistance in setting up the NOS assay.
REFERENCES I. AbdoUah, S.; Catlin, M. C.; Brien, J. F. Ethanol neurobehavioural teratogenesis in the guinea pig: Behavioural dysfunction and hippocampal morphologic change. Can. J. Physiol. Pharmacol. 71:776-782; 1993. 2. Bradford, M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254; 1976. 3. Bredt, D. S.; Snyder, S. H. Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme. Proc. Natl. Acad. Sci. USA 87: 682-685; 1990. 4. Catlin, M. C.; Abdollah, S.; Brien, J. F. Dose-dependent effects of prenatal ethanol exposure in the guinea pig. Alcohol 10:109115; 1993. 5. Clarke, D. W.; Steenaart, N. A. E.; Breedon, T. H.; Brien, J. F. Differential pharmacokinetics for oral and imraperitoneal administration of ethanol to the pregnant guinea pig. Can. J. Physiol. Pharmacol. 63:169-172; 1985. 6. Clarke, D. W.; Steenaart, N. A. E.; Slack, C. J.; Brien, J. F. Pharmacokinetics of ethanol and its metabolite, acetaldehyde,
7. 8. 9. 10. 11. 12.
and fetolethality in the third-trimester pregnant guinea pig for oral administration of acute, multiple-doseethanol. Can. J. Physiol. Pharmacol. 64:1060-1067; 1986. Dobbing, J.; Sands, J. Comparative aspects of the brain growth spun. Early Hum. Dev. 3:79-83; 1979. Elvidge, H. Production of dated pregnant guinea pigs without postpartum matings. J. Inst. Anita. Tech. 23:111-117; 1972. Holson, R. R. Euthanasia by decapitation: Evidence that this technique produces prompt painless unconsciousness in laboratory rodents. Neurotoxicol. Teratol. 14:253-257; 1992. Kandel, E. R.; O'Dell, T. J. Are adult learning mechanisms also used for development? Science258:243-245; 1992. Lancaster, F. E. Alcohol, nitric oxide, and neurotoxicity: Is there a connection?-a review. Alcohol. Clin. Exp. Res. 16:539-541; 1992. Lee, Q. P.; Juchau, M. R. Dysmorphogeniceffects of nitric oxide (NO) and NO-synthase inhibition: Studies with intra-amniotic injections of sodium nitroprusside and N~-monomethyl-L-arginine. Teratology 49:452-464; 1994.
E T H A N O L A N D B R A I N NOS A C T I V I T Y 13. Marietta, M. A. Nitric oxide synthase structure and mechanism. J. Biol. Chem. 268:12231-12234; 1993. 14. Matsumoto, T.; Pollock, J, S.; Nakane, M.; Forstermann, U. Developmental changes in cytosolic and particulate nitric oxide synthase in rat brain. Dev. Brain Res. 73:199-203; 1993. 15. McDonald, J. W.; Johnston, M. V. Physiological and pathophysiological roles of excitatoD' amino acids during central nervous system development. Brain ires. Rev. 15:41-70; 1990. 16. Moncada, S.; Palmer, R. M. J.; Higgs, E. A. Biosynthesis of nitric oxide from L-arginine: A pathway for the regulation of cell function and communication. Biochem. Pharmacol. 38:17091715; 1989. 17. Moncada, S.; Palmer, R. M. J.; Higgs, E. A. Nitric oxide: Physi-
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18. 19. 20. 21.
ology, pathophysiology, and pharmacology. Pharmacol. Rev. 43: 109-142; 1991. Nakanishi, S. Molecular diversity of glutamate receptors and implications for brain function. Science 258:597-603; 1992. Reynolds, J. D.; Brien, J. F. Effects of acute ethanol exposure on glutamate release in the hippocampus of. the fetal and adult guinea pig. Alcohol I 1:259-267; 1994. West, J. R.; Pierce, D. R. Perinatal alcohol exposure and neuronal damage. In: West, J. R., ed. Alcohol and brain development. New York: Oxford University Press; 1986:120-157. Zivin, J. A.; Bartko, J. J. Statistics for disinterested scientists. Life Sci. 18:15-26; 1976.
Pergamon 0741-8329(95)00006-2
Nitric Oxide Synthase Activity in the Hippocampus, Frontal Cerebral Cortex, and Cerebellum of the Guinea Pig: Ontogeny and In Vitro Ethanol Exposure J A M E S F. B R I E N , 1 J A M E S D. R E Y N O L D S , M I C H A E L A. C U N N I N G H A M , A N N M. P A R R , S A R A H W A D D O C K A N D B E T T I N A E. K A L I S C H
Department o f Pharmacology and Toxicology, Faculty o f Medicine, Queen's University, Kingston, Ontario, Canada K7L 3N6 Received 11 N o v e m b e r 1994; Accepted 7 D e c e m b e r 1994 BRIEN, J. F., J. D. REYNOLDS, M. A. CUNNINGHAM, A. M. PARR, S. WADDOCK AND B. E. KALISCH.
Nitric oxide synthase activity in the hippocampus,frontal cerebralcortex, and cerebellum of the guineapig: Ontogeny and in vitro ethanol exposure. ALCOHOL 12(4) 329-333, 1995.--Decreased nitric oxide (NO) formation, resulting from inhibition of NO synthase (NOS), may be important in the pathogenesis of ethanol central nervous system teratogenesis. The objectives of this study were to determine the ontogeny of NOS activity in the hippocampus, frontal cerebral cortex, and cerebellum of the developing guinea pig, and to test the hypothesis that direct exposure to ethanol inhibits NOS activity in these brain regions at selected developmental ages. NOS activity was quantitated by an optimized radiometric assay. The ontogeny study demonstrated that NOS activity in the hippocampus and frontal cortex was not fully developed prenatally, and apparently increased during postnatal life to attain adult level of activity at postnatal day > 60. In the cerebellum, NOS activity increased during prenatal life to an apparent maximum in the mature near-term fetus at gestational day 63 (term, about 68 days), and then apparently declined during postnatal life to attain adult level of activity. In vitro ethanol exposure (25-100 raM) did not affect NOS activity in the hippocampus, frontal cortex, or cerebellum at any developmental age studied. These data indicate that, although the ontogeny of NOS activity varies between brain regions, ethanol does not directly affect NOS activity in the developing guinea pig. The effects of acute and chronic in utero ethanol exposure on NOS activity in these brain regions are currently being investigated. Nitric oxide synthase Ontogeny Ethanol
Hippocampus
Frontal cerebral cortex
Cerebellum
Guinea pig
L-arginine to L-citrulline, catalyzed by NO synthase (NOS) (16). In the brain, NO is predominantly formed by the constitutive isoform of NOS, which is a Ca2+/calmodulin-dependent enzyme (3) localized in the neuron (13). NO has been proposed to play a neur,otrophic role in brain development (10,11). Glu also plays an important role in CNS development by regulating neuronal survival and synaptogenesis (15). As Glu activation of the NMDA receptor can result in NO formation, it is conceivable that NO acts as a second messenger to mediate some of the neurotrophic actions of Glu. Recent data from our laboratory have shown that, in the fetal guinea pig hippocampus, in vitro ethanol exposure
THE hippocampus, frontal cerebral cortex, and cerebellum have been identified as target sites for ethanol central nervous system (CNS) teratogenesis (20). L-Glutamate (Glu) appears to be the major excitatory neurotransmitter in these brain regions. The postsynaptic actions of Glu are mediated by several different excitatory amino acid receptor subtypes, including the N-methyl-D-aspartate (NMDA) receptor (18). The binding of Glu to the NMDA receptor leads to ion channel opening and the influx of Ca 2+. This increase in intracellular Ca 2+ concentration can result in the formation of nitric oxide (NO), a novel gaseous second messenger with a short biological halflife (17). NO is produced during the enzymatic oxidation of
To whom requests for reprints should be addressed. 329
330
BRIEN ET AL.
decreases stimulated and spontaneous release of Glu (19). This depressant action of ethanol could result in reduced NMDA receptor activation leading to decreases in NOS activity and consequent NO formation. A decrease in NO formation could account, at least in part, for the disruption of neuronal development produced by prenatal ethanol exposure. This proposal is supported by the observation that injection of a NOS inhibitor into the amniotic fluid of the cultured whole rat conceptus at midgestation produces dysmorphogenic effects (12). In vitro ethanol exposure inhibits Glu release (19), but it does not appear to affect the density or affinity of the NMDA receptor (Abdollah and Brien, submitted for publication). To continue our investigation of the effects of ethanol on the developing Glu neurotransmitter-NMDA receptor system, it was important to determine whether in vitro ethanol exposure had a direct effect on NOS activity. The first objective of this study was to determine the ontogeny of NOS activity in the guinea pig hippocampus, frontal cerebral cortex, and cerebellum at selected developmental ages, as there apparently are no ontogeny data reported in the literature. The second objective was to test the hypothesis that in vitro exposure to ethanol directly inhibits NOS activity in these brain regions. Our laboratory has used the guinea pig to investigate the neuroteratogenie effects of prenatal ethanol exposure because of this animal's extensive prenatal brain development, including the brain growth spurt, which is more similar to the human than is the case for the rat (7). METHOD
Breeding Procedure for Experimental Animals Male and female Dunkin-Hartley strain guinea pigs (Charles River Canada Inc., St. Constant, Que.) were bred using an established procedure (5,8). Day zero of pregnancy was established as the last day of full vaginal-membrane opening [term, about gestational day (GD) 68]. Pregnant and nonpregnant animals were housed in groups of up to four in stainless steel wire cages at 23°C with a 12-h light/12-h dark cycle. Food (Purina Guinea Pig Chow 5025 ®) and water were provided ad lib. Pregnancy status of the breeding female animals and the general health of all the guinea pigs were monitored every other day. The animals were cared for in accordance with the principles and guidelines of the Canadian Council on Animal Care. The experimental protocol was approved by the Queen's University Animal Care Committee.
Chemicals and Solutions Dowex 50W/50X8-400, N-[2-hydroxyethyl]-piperazine-N'[2-ethanesulfonic acid] (HEPES), ethylenediaminetetraacetic acid disodium salt (EDTA), dithiothreitol (DTT), and leupeptin were obtained from Sigma Chemical Co. (St. Louis, MO). L-[~+C]Arginine (331.2 mCi/mmol, 99% radiochemical purity) was supplied by Dupont-New England Nuclear (Lachine, Que.). The dye reagent used for the protein assay (2) was purchased from Bio-Rad Laboratories (Mississauga, Ont.). All other chemicals were at least reagent grade quality and were obtained from a variety of suppliers. Deionlzed water was used to prepare the aqueous chemical solutions. Dowex 50W/50X8-400 was converted to the sodium salt form by treating an aqueous mixture containing the resin with 1 M NaOH to raise the pH above 9.0, followed by rinsing the resin with deionized water until the pH of the wash was 7.0.
Tissue Isolation and Preparation of Subcellular Fraction Containing NOS Fetal guinea pigs at mean GD51 (range, GD50-52) and mean GD63 (range, GD62-64), and adult male and female guinea pigs at postnatal day (PD) > 60 were used in this study. Guinea pigs were killed by decapitation (9), and the fetuses were removed by cesarean section. Each fetus was decapitated; the brain was excised from the skull, and the hippocampi, frontal cerebral cortex, and cerebellum were dissected. For each brain region, pooled tissue from all fetuses in the litter was homogenized in ice-cold solution containing 50 mM HEPES, 1 mM EDTA, 1 mM DTT, 10 #g/ml leupeptin (pH 7.4) to produce an 80-90% (w/v) homogenate. The homogenate was centrifuged at 4°C for 30 min at 25,000 x g. The supernatant was removed and stored at - 7 0 ° C . In a similar manner, the hippocampi, frontal cortex, and cerebellum of individual adult guinea pigs were isolated, and the supernatant samples were prepared and stored at - 70°C. NOS activity of the supernatant was analyzed within 2 weeks of preparation of the subcellular fraction.
NOS Assay The radiometric method used for the quantitation of NOS activity was a modification of the procedure of Bredt and Snyder (3). The method was optimized for the hippocampus, frontal cortex, and cerebellum of the developing guinea pig. A 25-#1 volume of supernatant sample (150-300/~g protein for the fetus at GD51, 260-420/~g protein for the fetus at GD63, and 200-450/zg protein for the adult at PD > 60, depending on the brain region) was added to a reaction tube. A 100-/~1 volume of reaction buffer (50 mM HEPES, 1 mM EDTA, 1 mM DTT, 1.25 mM CaCI2, 2 mM NADPH, pH 7.4) and 25/~I of L-[14C]arginine in an aqueous solution of 180 #M nonradiolabelled L-arginine (radioactivity, about 2.0 x 104 dpm/sampie) were added to each tube, and the contents were incubated at 37°C for 15 min. The reaction was stopped by the addition of 2.0 ml of ice-cold buffer containing 20 mM HEPES and 2 mM EDTA (pH 5.5). Experimental blanks were prepared by adding the stop buffer and reaction buffer to the sample prior to the addition of L-[mC]arginlne/180 /~M nonradiolabelled L-arginine and then incubating at 37°C for 15 rain. Each sample was subjected to chromatographic separation by loading the 2.15-ml volume of reaction solution onto a Dowex 50W/50X8-400 (sodium form) column (4 cm high, 0.4 cm internal diameter) with a silanized wool plug. The column was washed with 2.0 ml deionized water to elute the [l+C]citrulline. The eluate was collected and mixed with 10 ml of Scintiverse ® scintillation fluid (Fisher Scientific, Unionville, Ont.). [~+C]Citrulline radioactivity was quantitated using a Beckman LS 3800 liquid scintillation counter, in which each sample was counted for a 2-rain period. The protein content of each sample was measured using the protein-dye-binding method of Bradford (2) with bovine serum albumin as the standard.
In Vitro Ethanol Exposure and NOS Activity A 25-/zl aliquot of the supernatant sample of each brain region was preincubated with 25, 50, 75, or 100 mM ethanol (final concentration) for 15 rain at 37°C in a sealed tube that had been aerated with 95% 02/5% CO2. Only 10/~1 of preincubation buffer (50 mM HEPES, 1 mM EDTA, pH 7.4) containing the appropriate volume of ethanol to give the desired
E T H A N O L A N D BRAIN NOS ACTIVITY final ethanol concentration were added to each tube to minimize dilution o f the sample. Then, a 10-#1 aliquot o f reaction buffer containing ethanol to produce a final concentration of 25, 50, 75, or 100 mM in 1:he reaction mixture, 100 #1 of normal reaction buffer, and 25 /~1 o f L-["C]arginine in an aqueous solution of 180 ~M nonradiolabelled L-arginine (radioactivity, about 2.0 × 104 dpm/sample) were added to each sample, followed by incubation at 37°C for 15 min. The reaction was terminated by the addition of 2.0 ml o f stop buffer after the 15-rain incubation period. ["C]Citrulline formation was quantitated as described above.
Data Analysis NOS activity in each tissue sample was expressed as pmol [m4C]citrulline formed • 15 miin -t • #g protein -l. The data are presented as group means + SD. To determine if the level of NOS activity in the hippocaznpus, frontal cortex, or cerebellum changed during development, statistical analysis o f the data was conducted by using one-way randomized-design ANOVA. Similarly, one-way randomized-design A N O V A was used to determine if the level o f NOS activity varied among the three brain regions at each selected developmental age. To determine if in vitro ethanol exposure affected the level of NOS activity in each brain region, the data at each developmental age were anaiyzed b y repeated-measures ANOVA. Heterogeneity of variance was assessed by Cochran's test prior to conducting the ANOVA, and the data were transformed as necessary (21). Tukey-Kramer post hoc test was conducted if a significant F statistic ( p < 0.05) was obtained in the ANOVA. Two groups o f data were considered to be statistically different when p < 0.05.
331 mined at GD51 (immature fetus), GD63 (mature fetus), and PD > 60 (adult), and the data are presented in F i g . 1. In the hippocampus and frontal cortex, NOS activity was similar for the immature and mature fetus, and was less at each fetal age compared with the adult ( p < 0.05). In the cerebellum, the level of enzyme activity was higher in the mature fetus compared with the immature fetus and the adult ( p < 0.05). The data demonstrate that, in the hippocampus and frontal cortex, NOS activity is not fully developed during prenatal life and apparently increases during postnatal life to attain adult level by PD > 60. In the cerebellum, NOS activity increases during prenatal life to apparent maximal activity in the mature fetal guinea pig, and then apparently decreases during postnatal life to the adult level. In contrast to our findings in the guinea pig, rat cytosolic cerebellar NOS activity is low in the neonate, and then increases progressively to adult activity (14). These data and the results of our investigation demonstrate that there are Species differences in the ontogeny of constitutive NOS activity in these selected regions of the developing brain. In this regard, it is important to note that most brain development occurs prenatally in the guinea pig and postnatally in the rat (7). There were also regional differences in the level of NOS activity during prenatal development. NOS activity was lower in the hippocampus and frontal cortex compared with the cerebellum for both the immature and mature fetus ( p <
4
I-HPPOCAMPUS
RESULTS AND DISCUSSION
Assay for NOS Activity NOS activity in the hippocampus, frontal cerebral cortex, and cerebellum of the developing guinea pig was quantitated by a modified radiometric as:my used to determine NOS activity in rat brain (3). The assay, using L-[~4C]arginine, was optimized for the determination of Ca2+-dependent constitutive NOS activity in the guinea piF brain with respect to incubation time, protein concentration, L-arginine and N A D P H concentrations, and temperature. ]'he optimized conditions were identical for the hippocampus, frontal cerebral cortex, and cerebellum of the fetal and adult guinea pig. An assay blank (supernatant sample with reaction buffer and stop buffer, containing 2 mM EDTA, added prior to incubation) was run with each set of experimental samples in an attempt to control for any Ca2+-independent production of [t4C]citrulline from L-[liC]arginine. To ensure that NOS activity was being quantitated, a series of experiments~ was conducted using N-nitro-Larginine methyl ester (L-NAlVIE), a selective inhibitor of NOS. Incubation of supernatant samples from the adult guinea pig hippocampus, frontal cortex, and cerebellum with increasing concentrations of L-NAME produced a concentration-dependent decrease in the production o f [t4C]citrulline (data not shown). The highest concentration of L-NAME (1 mM) completely inhibited ["C]citrulline formation in these brain regions (data not shown). These data indicate that virtually all the [14C]citrulline quantitated in our assay results from the oxidation of L-[~4C]arginine, catalyzed by NOS.
Ontogeny of NOS Activity The developmental profiles of NOS activity in the guinea pig hippocampus, frontal Cortex, and cerebellum were deter-
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*
CEREBRAL CORTEX
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, T ~
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GD 63 AGE
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FIG. 1. Ontogeny of NOS activity in the hippocampus, frontal cerebral cortex, and cerebellum of the guinea pig at GD51 (n = 5), GD63 (n = 5), and PD > 60 (n = 6). The data are presented as group means + SD. *Denotes statistical difference for the: h i p p o c a m p u s , F(2, 15) = 57.25, p < 0.0001, frontal cortex, F(2, 15) = 15.41, p = 0.0004, and cerebellum, F(2, 15) = 19.02, p = 0.0001, compared with the same brain region at PD > 60. T(GD51) and ~(GD63) denote statistical difference compared with the cerebellum at the same developmental age: GD51, F(2, 14) = 65.14,p < 0.0001, and GD63, F(2, 11) = 51.56,p < 0.0001.
332
BRIEN ET AL.
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In Vitro Ethanol Exposure and NOS Activity
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[ETHANOL] (raM) FIG. 2. Effect of in vitro ethanol exposure (0-100 mM) on NOS activity in the hippocampus of the guinea pig at GD51 (n = 3), GD63 (n = 4), and PD > 60 (n = 6). The data are presented as group means + SD. 0.05), but was similar in these three brain regions for the adult guinea pig. There was no gender difference in NOS activity for the adult guinea pig (data not presented). This differential ontogeny of NOS activity in the guinea pig brain results in greater enzyme activity in the cerebellum compared with the hippocampus and frontal cortex during prenatal life, but not during adulthood. This is the first reported characterization of the ontogeny of NOS activity in the hippocampus and frontal cerebral cortex of a rodent species and in these two brain regions, along with the cerebellum, of the developing guinea pig.
The hippocampus, frontal cortex, and cerebellum were studied because these brain regions are target sites of ethanol neurobehavioural teratogenesis (20). To assess the effect of direct ethanol exposure on NOS activity, the subcellular fraction containing NOS was incubated with ethanol (25-100 raM). The 15-min incubation time (for a total ethanol exposure time of 30 min including the 15-min enzymatic reaction time) was selected in view of the pharmacokinetics of ethanol in the maternal-fetal unit, including the fetal brain, of the pregnant guinea pig (6). This ensured complete distribution of ethanol throughout the supernatant sample containing NOS activity, and mimicked the time duration that brain tissue would be exposed to a particular ethanol concentration. The data for hippocampai NOS activity in the immature fetus (GD51), mature fetus (GD63), and adult (PD > 60) are presented in Fig. 2. In vitro ethanol exposure did not alter NOS activity in the hippocampus. Similarly, in vitro ethanol exposure did not affect NOS activity in the frontal cortex or cerebellum of the fetal or adult guinea pig (data not shown). These data indicate that the direct exposure of hippocampal, frontal cerebral cortical, or cerebellar NOS to ethanol does not affect enzymatic formation of NO, when the substrate and cofactor are not rate limiting. The next step in our investigation of the effects of ethanol on the developing Glu neuronal system is to determine whether ethanol can act indirectly to alter NOS activity via fetal, placental, and/or maternal effects that ultimately impact on the fetal hippocampus, frontal cortex, and/ or cerebellum. We plan to examine NOS activity in these brain regions of the guinea pig following chronic prenatal exposure to an ethanol regimen that, when administered daily throughout gestation, produces CNS teratogenesis (1,4). ACKNOWLEDGEMENTS This research was supported by an operating grant (MT-8073) from the Medical Research Council of Canada (MRC). M.A.C. and A.M.P. were the recipients Of summer student research scholarships from the Pharmaceutical Manufacturers Association of CanadaHealth Research Foundation/MRC program. The authors wish to thank Mr. Bruce Connop for his excellent technical assistance in setting up the NOS assay.
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