Katsumi Irikura, Kenneth I. Maynard, and Michael A. Moskowitz. Stroke Research ..... Sokoloff L (1993) Effect of nitric oxide synthase inhibitors on increase in ...
Journal of Cerebral Blood Flow and Metabolism 14:45-48 © 1994 The International Society of Cerebral Blood Flow and Metabolism Published by Raven Press, Ltd., New York
Importance of Nitric Oxide Synthase Inhibition to the Attenuated Vascular Responses Induced by Topical L- Nitroarginine
During Vibrissal Stimulation
Katsumi Irikura, Kenneth I. Maynard, and Michael A. Moskowitz Stroke Research Laboratory, Neurosurgery and Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, U.S.A.
Summary: We assessed the regional cerebral blood flow (rCBF) response to vibrissal stimulation before and after nitric oxide synthase (NOS) inhibitors were topically ap plied through a closed cranial window placed over the cortical barrel fields in anesthetized Sprague-Dawley rats. In the presence of L-nitroarginine (l mM), both the maximum and total responses became reduced, but only in those animals demonstrating >50% inhibition of NOS activity as determined by the conversion of [3H]arginine to eHlcitrulline within homogenates taken from cortical gray matter under the cranial window. The degree of en-
zyme inhibition depended in part, upon duration after topical application of NOS inhibitor. When >50%, en zyme inhibition correlated with the decrease in maximum and total rCBF response (p < 0.01). These findings em phasize the merits of assessing enzyme activity after ad ministering NOS inhibitors, and suggest that NO gener ated from parenchymal NOS activity plays an important role in the cerebrovascular response to physiologic so matosensory stimulation under the stated conditions. Key Words: Nitric oxide-Nitric oxide s y n t h a s e-L Nitroarginine-Cortical barrel fields-rCBF.
The mechanism which couples blood flow and metabolism within the brain is not well understood. Recently, nitric oxide was suggested as a potential mediator because it is formed and released from neurons (Garthwaite et aI., 1988; Knowles et aI., 1989; Bredt et aI., 1990), diffuses readily (e.g., 0.5 fJ.m theoretical distance) (Knowles and Moncada, 1992), crosses cellular membranes freely, and is a potent vasodilator (Palmer et al., 1987). Although certain publications note that nitric oxide synthase (NOS) inhibitors attenuate the blood flow response elicited by somatosensory stimulation (Northington et aI., 1992; Dirnagl et aI., 1993), others have not observed this relation (Adachi et aI., 1993; Ngai et aI., 1993). Studies were undertaken to establish a possible
role for NO as a coupling mediator for regional CBF (rCBF) and metabolism, and to extend our own pre liminary negative findings examining the effects of topical NOS inhibitors on the rCBF response to vibrissal stimulation 30 min after topical application (Irikura et aI., 1993). Accordingly, NOS activity was determined in vitro based on the conversion of eH]arginine to [3H]citrulline within homogenates taken from subjacent cerebral cortex. Our results support an important role for NO under the condi tions of this assay, and for the need to assess NOS activity in vitro in the presence of L-arginine ana logue inhibitors. MATERIALS AND METHODS General preparation Male Sprague-Dawley rats were housed under diurnal lighting conditions and allowed food and water ad libitum. Rats were anesthetized initially with 1.5% halothane, and mechanically ventilated in the unparalyzed state after tra cheostomy. The femoral artery and vein were cannulated for continuous monitoring of arterial blood pressure and the intravenous administration of drugs, respectively. Halothane was subsequently withdrawn and a-chloralose (50 mg/kg) and urethane (500 mg/kg) were injected intra peritoneally (i.p.). For maintenance of anesthesia, sup plemental doses of a-chloralose (10 mg/kg, i.p.) and ure thane (l00 mg/kg, i.p.) were given. In some animals, an-
Received June 18 , 1993; final revision received July 6, 1993; accepted July 6, 1993. Address correspondence and reprint requests to Dr. Michael A. Moskowitz, Massachusetts General Hospital, 32 Fruit Street, Boston, MA 02114, U.S.A. Abbreviations used: CSF, cerebrospinal fluid; EDTA, ethyl enediaminetetra-acetic acid; HEPES, N-2-hydroxyethylpipera zine-N' -2-ethanesulfonic acid; L-NA, NG-nitro-L-arginine; L-NAME, �-nitro-L-arginine methylester; �-NADPH, �-nico tinamide adenine dinucleotide phosphate; NOS, nitric oxide syn thase; rCBF, regional CBF.
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46
esthesia was maintained by a continuous infusion of u-chloralose (40 mg kg-I/h-I, i.v.). End-tidal COz was continuously monitored (Model 126017000 Novametrix Medical Systems, Wallingford, CT, U.S.A.) and arterial blood gases and pH were mea sured periodically (Corning 178 blood gas/pH analyzer, Ciba Corning Diag., Medford, MA, U.S.A.). Rectal tem perature was kept at 36.5-37SC using a homeothermic blanket (Harvard Bioscience, Natick, MA, U.S.A.).
Closed cranial window preparation The head was fixed in a stereotaxic frame after which the left parietotemporal bone was exposed by a longitu dinal midline skin incision and the temporal muscle re moved. Dental acrylic was applied to the intact calvarium surrounding the region of the left barrel cortex (Welker, 1971). Two polyethylene tubes were embedded in the dental acrylic for superfusion. A craniectomy (6-mm di ameter) was created in the center of the acrylic doughnut using a power drill while cooling with saline. After re flecting the dura mater along with the closely attached arachnoidal membrane, the exposed pial surface was con tinuously superfused with artificial cerebrospinal fluid (CSF) and sealed immediately with a cover glass 12 mm in diameter using cyanoacrylate as described by Morii et al. (1986). The volume under the window was �0.8 ml. In tracranial pressure was maintained at 5-8 mm Hg by ad justing the outlet tube to an appropriate height. rCBF was measured (Laser-Flo model BPM403A, TSI, St. Paul, MN, U.S.A.) by a laser-Doppler probe (0.8 mm diameter) placed over the closed cranial window and above the whisker barrel field. rCBF and systemic arterial pressure were recorded continuously using a Mac Lab data acqui sition and analysis system (Mac Lab NSW 2154, Austra lia). Artificial CSF consisted of Na+ 156.5 mEq/L, K+ 2.95 z z mEq/L, Ca + 2.5 mEq/L, Mg + 1.33 mEq/L, Cl- 138.7 mEq/L, HC0 - 24.6 mEq/L, dextrose 66.5 mg/dl, and 3 urea 40.2 mg/dl. NOS inhibitors (�-nitro-L-arginine: L-NA, �-nitro-L-arginine methyl ester: L-NAME) were obtained from Sigma Chemical Co. (St. Louis, MO,
U.S.A.) and dissolved in artificial CSF. Artificial CSF was equilibrated with 5% COz, 10% Oz, the balance Nz at 37°C. Paz, Pcoz and pH were maintained at 80-90 mm Hg, 30-40 mm Hg, and 7.35-7.43, respectively.
Experimental protocol After stabilizing the blood pressure and ensuring that the corneal reflex and tail pinch responses were absent, rCBF was recorded during manual deflection (2-3 Hz) of the contralateral whiskers. Mechanical stimulation was continued for 1 min and repeated two or three times at 3-min intervals to obtain mean values for maximum rCBF increase and total response (Fig. 1). Artificial CSF con taining either L-NAME (10 j.LM or 1 mM) or L-NA (1 mM) was superfused by manual injection (0.5 mllmin) for 20 min, after which it remained under the window for an additional 10 or 40 min. rCBF measurements were then repeated during vibrissal stimulation. In randomly selected animals, rCBF responses to pa paverine superfusion (1 mM or 10 j.Lm) were compared before and after L-NA. Percentage of inhibition was determined by comparing the responses to whisker stimulation (maximum rCBF change; total rCBF response) before and after topical ap plication of NOS inhibitors.
Assay for NOS activity Immediately after the experiment, the brain was re moved and rapidly frozen. The cortex just beneath the cranial window (100 mg) was used for NOS assay. NOS activity was measured in the L-NA-treated group only, and was determined by the conversion of eH]arginine (58.4 Cilmmol, NENlDupont) to [3H]citrulline according to the method described by Bredt et al. (1989). After weighing, the samples were homogenized in 500 j.LI of 50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) and 1 mM EDTA (pH 7.4). Homogenates were centrifuged at 2,500 rpm for 5 min and the superna tant (4°C) retained. The reaction mixture consisted of 100 j.LI of HEPES (50 mM) ethylenediaminetetra-acetic acid (EDTA) (1 mM), l3-nicotinamide adenine dinucleotide phosphate, reduced form (I3-NADPH, 1 mM) and Ca2+ (1 ,
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FIG. 1. reBF response recorded over the cortical barrel fields to vibrissal stimulation (2-3 Hz, manual deflection, 1 min duration) by laser-Doppler flowmetry in anesthetized Sprague-Dawley rats (see Methods). A: Before L·NA. B: After L-NA (1 mM, 60 min). Maximum increase and the total response (response integral; shaded area) were recorded and computed by a Mac Lab data acquisition system. Blood pressure was recorded from the femoral artery.
J Cereb Blood Flow Metab, Vol. 14, No.1, 1994
L-NA AND ReBF DURING VIBRISSAL STIMULATION mM [3H]arginine (1 mCilm!), and 25 f11 of cortical super natant. Samples were assayed in duplicate at room tem perature. After 10 min, the reaction was stopped with HEPES (20 mM) and EDTA (2 mM) The mixture was then applied to columns containing Dowex AG50WX-8 (Na + form) (Bio-Rad, Richmond, CA, U.S.A.), and eluted with 2 ml of distilled water. eH]citrulline was quantified in the eluate by a liquid scintillation spectrom eter. NOS activity was expressed as cpm mg-I 10 min -I. Enzyme activity was determined in comparable samples from sham-operated animals (control tissues) run in par allel within the same experiment. Percentage of inhibition was calculated by comparing enzyme activity within tis sues exposed to topical inhibitors and controls. .
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Data analysis Data are expressed as mean ± SD. Paired Student's t test was used to compare the rCBF response (maximum or total) before and after superfusion. Differences in NOS activity among groups was determined by one-way anal ysis of variance (ANOV A) followed by unpaired Stu dent's t test. Least-squares regression analysis was used to determine the correlation between the rCBF response and NOS inhibition. p < 0.05 was considered significant.
Baseline rCBF was affected by topical L-NA. Af ter 1 mM, rCBF decreased by 15 ± 5 (n 8) and 24 ± 7% (n 6) at 30 and 60 min, respectively. Top ical L-NAME (1 mM; n 4) reduced baseline by 18 ± 9% at 30 min. rCBF oscillations were observed in some animals after administration of the inhibitors. After the start of whisker stimulation, rCBF in creased within a few seconds,reached its maximum within 5 s and then decreased toward baseline (Fig. 1). After discontinuing the stimulation, rCBF fell below baseline for 30--45 s but returned to baseline within 1-2 min. Responses of similar magnitude and duration were obtained with repetitive stimulation. In sham-operated animals (n 6), the rCBF re sponse (maximum increase; response integral or to tal response) did not change 60 min after superfu sion with artificial CSF (Table 1). Superfusion with L-NAME did not change the maximum increase at 30 min [1 mM, 22 ± 3 vs. 24 ± 3% (n 5 ); 10 fLM, 28 ± 11 vs. 30 ± 10% (n 3)]. L-NA (1 mM) su perfused for 30 min (n 12), however, attenuated the total response (5 24 ± 173 vs. 408 ± 138 % s, p < 0.01) but not the peak response (16 ± 5 vs. 16 ± 5%). By contrast, L-NA (1 mM) applied for 60 min =
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TABLE 1. rCBP responses to whisker stimulation in sham group rCBF response Max. response (%)
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FIG. 2. Inhibition of rCBF responses correlates with inhibi tion of NOS activity (L-NA, 1 mM) as measured within ho mogenates of cortical gray matter taken from below the closed cranial window. Closed and open circles denote L-NA topical application for 30 and 60 min, respectively. Both the maximum response (A) (r2 = 0.63, p < 0.01) and the total response (8) (r2 = 0.40, p < 0.01) are correlated with NOS inhibition when >50% was achieved (p < 0.01).
(n 8) attenuated both the maximum and the total response (18 ± 7 vs. 11 ± 4%, p < 0.01 and 636 ± 324 vs. 339 ± 206 %/s, p < 0.01, respectively). rCBF response to topical papaverine did not differ statistically before and after L-NA (1 mM) [papav erine 10 fLM (n 3),14 ± 6% before vs. 25 ± 11% after L-NA; 1 mM (n 5 ) 49 ± 13 vs. 5 9 ± 10% after L-NA). Cortical NOS activity was 160 ± 5 8 (n 9),62 ± 20 (n 8),29 ± 23 (n 12) cpm mg-110 min -I for the sham,L-NA [30 min (p < 0.05 ) and 60 min (p < 0.05 ) after superfusion] groups,respectively. A sig nificant correlation (p < 0.01) was found between the extent of physiologic blockade and the degree of NOS inhibition after >50% inhibition of enzyme ac tivity was achieved (Fig. 2). =
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DISCUSSION
Our results demonstrate the importance of as sessing brain NOS activity following the adminis-
J Cereb Blood Flow Metab. Vol.14. No.1. 1994
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K. IRIKURA ET AL.
tration of NOS inhibitors, and the need for this in formation when interpreting the relevance of NO to a particular physiologic response. The importance of NO may be underestimated if results are not evaluated in this context. For any given system, the reduction in enzyme activity depends in part upon the delivery of inhib itor and its clearance. Steady-state concentrations may vary depending upon the choice and dosage of NOS inhibitors as well as routes, times, and dura tion of administration, as well as conditions unique to each experimental preparation. We suspect that differences in superfusion technique may affect the rate of tissue penetration and thereby the degree of enzyme inhibition to explain discrepant findings in the literature. In our preparation, superfusion was done manually and maintained for only 20 min (0.5 ml/min), after which the superfusate remained in place for an additional 10 or 40 min before physio logic testing. In other preparations, NOS inhibitors were superfused via a pump (rates vary between laboratories) and maintained for either 10 min (Ngai et aI., 1993) or 60 min (Dirnagl et al., 1993; Iadecola, 1992) before testing, or were infused for 20 min by intracisternal administration (Adachi et aI., 1993). In our hands, inhibition of the rCBF response was most convincing >30 min after topical application whereas others noted significant physiologic inhibi tion at :;;;;30 min (U. Dirnagl; C. Iadecola, personal communications). It seems likely that L-NA-induced inhibition of NOS activity was underestimated within cortical tissue directly under the flow probe. First, the en zyme inhibitor is diluted at least fivefold during tis sue homogenization and diluted again within the re action mixture. Assuming that inhibition is revers ible [which may (Moore et aI., 1990) or may not be the case for L-NA (Dwyer et al., 1991)], dilution would affect apparent enzyme activity in vitro. Sec ondly, the highest L-N A tissue concentrations should be in closest proximity to the superfusate. Hence, enzyme inhibition should occur sooner and be more complete under the probe (1 mm3), a point that was possibly obscured by the relatively large amounts of cortical gray matter (100 mg) used for assay. We infer from the above that very high levels of inhibition are required to block the rCBF response to whisker stimulation. However, the long "incu bation period" required to block the physiologic re sponse may reflect some other fundamental change in the tissue such as expression of a new enzyme or enhanced activity of existing enzymes within corti cal gray matter. Of note, there was no breakdown of the blood-brain barrier after 60 min following topiJ Cereb Blood Flow Metab. Vol.14, No. I, 1994
cal L-NA (1 mM) as determined by the Evans blue technique (Irikura and Moskowitz, unpublished). We urge caution for those offering simple expla nations to account for the effects of NOS inhibition on brain blood flow, and await additional experi mental evidence directed at this problem. Never theless, the findings reported herein suggest the ad vantages of measuring NOS activity when adminis tering inhibitors of this enzyme. Acknowledgment: These studies were supported by NIH grant NS 10828 (Interdepartmental Stroke Program Project).
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