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The role of nitric oxide in the regulation of cerebral blood flow
248
Brain Research, 610 (1993) 248-255 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
BRES 18779
The role of nitric oxide in the regulation of cerebral blood flow Joel E. Buchanan
and John W. Phillis
Department of Physiology, Wayne State Unit,ersity School of Medicine, Detroit, MI 48201 (USA) (Accepted 1 December 1992)
Key words." Nitric oxide; Endothelium-derived relaxing factor; N-Nitro-e-arginine methyl ester; Cerebral blood flow; Hypoxia; Hypercapnia; Hypotension
The role of nitric oxide in the cerebral circulation under basal conditions and when exposed to hypoxic, hypercapnic and hypotensive stimuli, was studied in mechanically ventilated rats using a venous outflow technique, by examining the effects of inhibition of nitric oxide synthase with N-nitro-e-arginine methyl ester (e-NAME). e-NAME (10 or 30 mg/kg injected intravenously) raised mean arterial blood pressure by 14% and 24%, and increased cerebrovaseular resistance (CVR) by 20% and 24%, respectively. Cerebral blood flow (CBF) was unaltered, as were blood gases and pH. The increases in MABP and CVR were attenuated by L-arginine (300 mg/kg). Following the administration of L-NAME, the increases in CBF elicited by ventilation with 8% oxygen for 25 s were unaltered, in comparison to control responses. L-NAME attenuated the increases in CBF and reduced the time for recovery to basal flow rates evoked by ventilation with 10% carbon dioxide. These effects were reversed by L-, but not by D-, arginine. Autoregulation by CBF during hypotensive episodes, as measured by comparisons of CVR values, was unaffected by L-NAME. The results suggest that endogenous nitric oxide is involved in the responses of the cerebral vasculature to elevated levels of CO 2 in the arterial blood. Nitric oxide does not appear to play a major role in autoregulation to increases or decreases in MABP, or in hypoxia-evoked vasodilation.
INTRODUCTION It is now recognized that the vascular endothelium can play a key role in regulating vascular tone by releasing vasoactive factors and thus modulating vascular responses to various stimuli 4. Experimental evidence suggests that nitric oxide, or a closely related compound, synthesized from L-arginine, is an endothelium-derived relaxing factor ~1'~8 which relaxes vascular smooth muscle, including that of cerebral arteries 1 by stimulation of guanylate cyclase t°'15. The synthesis of endothelial nitric oxide can be inhibited by several analogues of L-arginine, and this inhibition induces an increase in the resistance of several vascular beds, including that of the cerebrovascular system 6'27. Contraction of pial arteries was observed when the arginine analogue N-monomethyl-L-arginine ( L - N M M A ) w a s superfused over the brain s u r f a c e 24, and of the basilar artery when L-NMMA was applied topically 2. Thus a basal release of nitric oxide appears to be responsible
for maintaining a vasodilator tone in the cerebrovascular, as well as other regional vascular, systems 6'27. The present experiments were performed to study the role of nitric oxide in setting basal vascular tone and in the vasodilation of cerebral blood vessels during hypoxia, hypercapnia and hypotension in anesthetized, mechanically ventilated rats. Cerebral blood flow was measured by a venous outflow technique 22'25 and the effects of a potent inhibitor of nitric oxide synthesis, N-nitro-L-arginine methyl ester (L-NAME) a'23 were investigated both on basal cerebral blood flow and on the responses elicited during brief episodes of hypoxia, hypercapnia and hypotension. MATERIALS AND METHODS Male Sprague-Dawley rats (Charles River: 350-450 g) were used to study CBF, using a venous outflow technique involving cannulation of the retroglenoid vein (RGV), first described by Nilsson and Siesj6 iv. The original method was refined by the inclusion of an extracorporeal circulation pump which permits continuous recording
Correspondence." J.W. Phillis, Department of Physiology, Wayne State University School of Medicine, 540 E. Canfield, Detroit, MI 48201, USA. Fax: (1) (313) 577-5494.
249 of cerebral blood flow for several hours 16'22'2s, conferring the advantage of continuous monitoring of CBF before, during and after various tests are performed on the same animal. This method was chosen because of previous successful use of this technique in our laboratory during other CBF studies, The general procedure for the experiments was as follows. T h e animals were anesthetized with 3% halothane in air to allow tracheotomy. During and after surgery, the animals were maintained on a mixture of methoxyflurane; 30% oxygen (O 2) and 70% nitrogen (N2). The animals were placed on a heating pad actuated by a rectal probe, to maintain body temperature at 37°C. Surgery was performed to expose the right and left RGV. Initial incisions were m a d e just in front of the external auditory meatus, The temporal muscle was then lifted and the blood vessels were separated and freed from the surrounding tissues to expose the R G V at their exits through the retroglenoid foramen. 5.0 surgical silk threads were placed around the veins, T h e left femoral artery was exposed, tied off and cannulated. The right femoral artery and vein were then exposed, tied off and cannulated. T h e right artery cannula was attached to a Grass Polygraph to continuously record arterial blood pressure. The venous cannula was attached to the extracorporeal p u m p and served to return blood to the animal. The left femoral artery cannula was used in the hypotensive portion of the experiment for withdrawing and reinjecting blood while maintaining a continuous blood pressure recording. It was also used to obtain small (0.35 ml) samples of arterial blood for pH and blood gas analysis. Following femoral cannulation the animal was heparinized (1 U / g ) . The left R G V was tied off distally with a long 5.0 thread. A small weight (alligator clamp) was attached to the end of this thread and allowed to pull on the RGV. This weight facilitated cannulation and blood flow through the cannula after its insertion. The left R G V was cannulated with an angiocatheter (Deseret, 22GA, 1 inch), which was advanced until its tip was flush with the retroglenoid foramen opening. T h r o u g h this opening the transverse sinus drains blood which
DROPS
A
originates in the cerebrum, the cerebellum and possibly the brainstem into the RGV. The angiocatheter was tied in place with 5.0 surgical silk thread. The extracorporeal venous flow system was then connected. The thread on the right R G V was tied off to prevent the possibili~ of entry of extracranial blood into the cerebral venous system. Blood in the left R G V flowed through the angiocatheter and an attached tube and was then allowed to drip past an electric eye into a collecting chamber. The outlet of the tube was positioned at the level of the retroglenoid foramen. Each time a drop passed the electric eye, a mark was recorded on the Grass Polygraph. Blood was then transferred from the collecting chamber back into the animal with an extracorporeal p u m p connected to the femoral venous cannula. To prevent blood loss in the subject animal, the blood volume requirement in the extracorporeal return system (4-5 ml) was filled by an additional animal killed by decapitation to donate blood to this system. Blood in the extracorporeal circuit was passed between the animal and the heating pad for warming before entering the femoral venous cannula. The subject animal was connected to a respiratory p u m p and respired at a frequency of 60-80 s t r o k e s / m i n on a gas mixture of m e t h o x y f l u r a n e / 3 0 % O 2 / 7 0 % N 2. The subject was then administered pancuronium bromide (Pavulon, 1 m g / k g ) and the concentration of methoxyflurane was decreased to 0.5-1% and adjusted such that blood pressure remained constant when pressure was applied to a forepaw. A period of 10-20 min was allowed for the animal to stabilize. Blood gases were measured in a blood sample and respiration was adjusted so that pH was approximately 7.38-7.42 and p C O 2 was approximately 32-36 mmHg. Additional samples were withdrawn during the course of the experiment and, if necessary, p u m p ventilation was adjusted to maintain blood gases and pH at their original levels. Additional injections of pancuronium bromide were administered if the animal evinced any tendency to make spontaneous respiratory movements. Following stabilization, the animals were given a series of challenges. Brief (25 and 36 s, respectively) challenges were given
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Fig. l. Responses of cerebral blood flow and arterial blood pressure to hypoxic (A,C,E) and hypercapnic (B,D,F) episodes. Cerebral blood flow, as represented by outflow from the retroglenoid sinus, is recorded as drops. Hypoxia was induced by mechanically ventilating the rat with 8% oxygen for 25 s; hypercapnia by vcntilating with ]0% CO 2 for 36 s. C and D were recorded after intravenous administration of L-NAME (30 mg/kg). E and F were rccorded after subsequent intravcnous administration of L-arginine (300 mg/kg).
250 TABLE I
istration of a gas mixture of 10% CO z / 3 0 % 0 2 / 6 0 % N 2 were given
Effects of L-NAME on MABP and cerebrot,ascular resistance (CVR)
every 10-12 rain. Hypotensive challenges were administered by first changing the inspiratory gas from 30% 0 2 / 7 0 % N z to 40% 0 2 / 6 0 % N z. The animal was allowed to respire this mixture for 10-12 min before the hypotensive challenge was administered as described above. To reduce dead space in the delivery system, two identical anesthetic vaporizers were used; one for the standard oxygen-enriched respiratory gas, the other for the hypoxic or hypercapnic challenge gases. For the hypoxic and hypercapnic challenges, observations and recordings were made for basal CBF rate, peak CBF rate and time to recovery. For the bypotensive challenge, CBF rate was recorded for the basal level, during the challenge and after recovery. In addition to CBF, MABP was recorded for the 2-rain hypotensive period. A brief delay occurred before animals began to increase CBF in response to hypercapnic or hypoxic challenges. The increased CBF rate continued for up to 2 rain after the basal gas was reconnected. Peak flow rate was defined as the greatest number of drops recorded in any 10-s interval. This number was then compared to the basal flow rate, which was defined as the number of drops recorded in a continuous l-rain interval immediately preceding the challenge period. Peak flow rates were adjusted (multiplied by 6) in order to present the data in a drops per minute format. Subjects were very stable during the basal period with constant flow (drop) rates and MABP (Fig. 1). To avoid interference with the MABP and CBF recordings, no blood samples were taken during the test challenges. Since the challenge durations were rigorously controlled, it is assumed that comparable blood gas and pH changes would have been present in each challenge. The percentage peak increases in CBF presented in Table II were calculated from the formula: % increase = (peak flow rate/basal flow rate × i 0 0 ) - 100. Recovery times represent time from the end of the challenge until flow rates returned to a new stable baseline. Cerebrovascular resistance (CVR) was calculated according to the formula CVR = MABP/CBF. CBF ( m l / m i n ) was determined from the d r o p s / m i n data using a conversion factor obtained from data presented in Phillis et al. 22. In evaluating the effects of L-NAME, k-arginine and i>arginine, the control responses preceding drug administration were compared with those after drug injection. Corn-
Values are expressed as the means-+S.E.M.
MABP
Basal flow (drops/min)
CVR (mmHg.ml-1 .min-1)
114.0-+3.8
21.2_+1.4
228.7_+15.6
130.4_+3.3 **
19.7+ 1.1
273.1 -+ 14.0 *
126.5+ 4.7
22.6 + 2.7
232.0 + 20.7
115.7+4.4
19.6-+2.2
231.2+24.7
143.5_+5.6 **
21.4_+ 2.2
295.6_+24.5 *
136.3_+5.2
19.7_+ 2.5
296.8_+34.7
Group I (n = 19) Control t,-NAME (10 m g / k g ) L,-Arginine (300 m g / k g ) Group II (n = 12) Control L-NAME (30 m g / k g ) L-Arginine (300 m g / k g )
• P < 0.05 compared to control, • * P < 0.01 compared to control,
tbr the bypoxic and hypercapnic episodes. A hypotensive challenge consisted of a 1-min period of blood withdrawal to reduce the MABP to 30-40 mmHg, along with an observation period of 2 rain where MABP was kept at 30-40 mmHg, followed by a 1-min reinjection of the blood originally withdrawn to re-establish the pre-hypotension MABP. Hypoxic and hypercapnic challenges were frequently administered alternatively to the same animal, with at least two control challenges of each type being recorded prior to drug administration. The responses to each type of challenge in a given animal were averaged before being combined with the responses of other rats. Challenges were administered as follows: hypoxic challenges consisting of 8% oxygen in nitrogen for 25-s duration were administered 10-12 min apart. Hypercapnic challenges consisting of a 36-s admin-
TABLE 1I
Effects of L-NAME and L-arginine on responses to hypoxia and hypercapnia Values are expressed as the means_+ S.E.M.
Condition
No. of rats
Group I Control hypoxia L-NAME (30 m g / k g ) t,-Arginine (300 m g / k g )
7
Group II Control hypercapnia ~,-NAME (10 m g / k g ) L-Arginine (300 m g / k g )
11
Group III Control hypercapnia t,-NAME (30 m g / k g ) L-Arginine (300 m g / k g )
11
Group 1V Control hypercapnia L-NAME (10 m g / k g ) D-Arginine (300 m g / k g )
9
Basal flow rate (drops/min)
% Increase in peak flow
Time to recot,ery (s)
23.4_+ 3.1 20.4 +_2.6 20.9-+ 1.3
49.4 + 6.4 49.1 + 6.7 53.7 + 7.8
69.9 + 8.1 51.4 _+23.2 64.4 + 19.9
21.4 _+ 1.7 20.4_+ 1.3 23.6 -+ 2.5
75.8 _+ 10.6 74.7+ 10.8 72.6 + 10.4
93.5 +_ 10.8 * 64.6_+ 7.3 105.9 _+20.3 *
20.4+_2.9 21.2 + 2.4 18.7 _+2.6
81.1 +_ 12.9 * 47.4 + 10.1 77.4 -+ 13.8
93.4_+ 12.9 * * 38.8_+ 5.8 81.8 -+ 15.8 *
22.1 + 1.9 21.9_+ 1.7 22.7 + 2.3
85.4 -+ 11.1 70.6_+ 13.7 58.4 -+ 11.0
79.5 _+ 9.4 60.3+ 7.6 50.0 _+ 11.9 +
• P < 0.05 in comparison to value following L-NAME administration. • * P < 0.01 in comparison to value following L-NAME administration. P < 0.05 in comparison to control.
251 parisons were performed between control data and post-drug responses using an analysis of variance and a t-test. Results are presented as means + S.E.M. Drugs used were: N-nitro-L-arginine methyl ester (L-NAME, Sigma Co.; 10 or 30 mg/kg), L-arginine (Sigma Co.; 300 mg/kg) and D-arginine (Sigma Co.; 300 mg/kg) administered intravenously in isotonic saline. Administration of isotonic saline alone at the volumes employed in the present study had no systemic or cerebral blood flow effects. RESULTS
Effects of N-nitro-L-arginine methyl ester Mean values for mean systemic arterial blood pressure, basal cerebral venous outflow and a calculated C V R before and 10 min after administration of LN A M E are presented in Table I. Intravenous administration of L-NAME increased m e a n arterial blood pressure by about 14 and 24% at the doses of 10 and 30 m g / k g , respectively. Venous outflow was unaltered and C V R increased by 19% and 28%. The increase in C V R was significant at both doses. Effects became apparent within 60 s of the onset of L-NAME administration and had stabilized by the 10-min period of measurement. Blood gases and p H following L-NAME administration were not significantly different from those obtained under control conditions. The hemodynamic effects of L-NAME were still present at least 60 min after administration of the low dose, and continued for the duration of the experiment, up to 2 h, after the higher dose. Administration of L-arginine failed to reverse the effects of L-NAME (30 m g / k g ) , but did attenuate the increase in C V R and M A B P elicited by the lower dose
of L-NAME, although neither reduction was statistically significant.
Responsesto hypoxic episodes The hypoxic challenges consisted of repeated brief (25 S) periods of inhalation of 8% oxygen in nitrogen. Hypoxic episodes of this duration elicited a fall in paO2 values from a resting level of 98.6 m m H g to 41.6 m m H g during control challenges. By using a challenge of this brevity, it was possible to avoid the reductions in blood pressure usually associated with hypoxia in anesthetized animals. Prechallenge MABPs of the 7 rats in this series were 102.6 + 6.4 m m H g , with an average M A B P during control hypoxic challenges of 100.6 +_ 5.7 mmHg. L-NAME (30 m g / k g ) did not alter the basal drop rate in these animals, nor did it affect the % increase peak flow rate or recovery time following hypoxic challenges (Table II, Group 1). L-Arginine (300 m g / k g ) administered after L-NAME, was also without effect on the responses to hypoxia (Table II). Examples of the lack of effect of L-NAME and L-arginine on the vascular responses to hypoxia are illustrated in Fig. 1A,C,E. L-NAME (30 m g / k g ) failed to reduce the increase in venous outflow evoked by 8% oxygen inhalation and following L-arginine (300 m g / k g ) administration; the hyperemia was still evident even though basal flow rates had declined at this time.
Responsesduring hypercapnia Hypercapnic challenges of the same duration as those used in these experiments (10% CO z in 30% 0 2
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Fig. 2. Responses of arterial blood pressure and cerebral blood flow to hypercapnic challenges (36 s) before (A) and following sequential doses of L-NAME (10 mg/kg) (B); D-arginine (300 mg/kg) (C) and L-arginine (300 mg/kg) (D).
252 for 36 s) elicited a significant rise in p~CO 2 of + 9.6 +_ 0.66 mmHg from a prechallenge value of 32.2 -+ 0.7 mmHg to an end of challenge value of 41.8 + 0.7 mmHg in control animals. The duration of the hypercapnic challenge was selected so as to have minimal effects on MABP. The pre-challenge MABP for 23 rats was 115.6 + 3.8 mmHg, and that during the hypercapnic challenge was 117.7 + 3.7 mmHg. The drop rate increased dramatically in response to the hypercapnic challenge, and remained elevated for a brief period (80-93 s) after the animal had been returned to its normal (30% 0 2) respiratory gas (Table II, Figs. 1 and 2). Peak increases in blood flow were in the range of 75-85%. Following the administration of L-NAME (10 or 30 mg/kg) MABP pressures rose, but there were no significant alterations in MABP during the hypercapnic challenges (135.4 + 4.3 vs. 134.2 + 3.9 for the L-NAME (10 m g / k g ) animals; 145 + 5.8 vs. 141.4_+ 4.8 for the L-NAME (30 mg/kg) rats). L-NAME (10 mg/kg) was without effect on the peak rate of flow during the hypercapnic challenges but did significantly reduce the time to recovery (Table II). At 30 mg/kg, L-NAME significantly reduced both the peak rate of flow and the time to recovery (Table II). The effects of L-NAME on both peak flow rate and time to recovery were reversed by the subsequent administration of L-arginine (300 mg/kg), but not by D-arginine (300 mg/kg), The effects of L-NAME and L- and D-arginine are illustrated in Figs. 1 and 2. The responses in Fig. 1B,D,F were recorded from the same rat as the responses to 8% oxygen inhalation. Fig. 1B illustrates a control response to 10% CO 2 inhalation for 36 s. Following L-NAME (30 mg/kg) administration, CO 2 evoked a markedly attenuated hyperemia (Fig. 1D), but after administration of L-arginine (300 mg/kg) the hyperemic response was restored, Fig. 2 A - D illustrates a succession of responses to 10% CO 2 inhalation, t - N A M E (10 mg/kg) administration greatly attenuated the hyperemic response to CO 2 (Fig. 2B), and its effects were not blocked by D-arginine
(300 mg/kg). A subsequent administration of L-arginine (300 mg/kg) resulted in a clear reversal of the effect of L-NAME.
Responsesduring hypotension Ten minutes prior to the start of a hypotensive challenge, the animals were ventilated with 40% oxygen to minimize the severity of cerebral hypoxia as a result of the reduction in blood flow. The results from 6 rats are presented in Table III. L-NAME (10 mg/kg) had no effect on either flow rate or calculated cerebrovascular resistance either prior to, during or following a hypotensive episode. L-Arginine (300 mg/kg) administered after L-NAME was also without effect. The results from one experiment are illustrated in Fig. 3. The drop rate fell from 20.0/min to 15.5/min (a 22% decline; CVR during hypotension = 88) during the 2 rain of hypotension. Following L-NAME (10 mg/kg) administration, the drop rate declined from 21.0/min to l l . 5 / m i n (a 45% decline) but as MABP during this episode of hypotension was actually lower than in the control (36 vs. 29 mmHg), the CVR was unchanged at 88. A pronounced reactive hyperemia followed the reinfusion of withdrawn blood. A subsequent injection of L-arginine did not affect the response during hypotension and partially reversed the increase in magnitude of the reactive hyperemia. Similar increases in the magnitude of the reactive hyperemias following hypotensive episodes after L-NAME administration were observed in all six of these experiments. Reversal by L-arginine was never complete. DISCUSSION The present results demonstrate that intravenous administration of I,-NAME, a potent inhibitor of nitric oxide synthase 8'23 produced moderate increases in mean arterial blood pressure, in the absence of any alteration of cerebral venous outflow as a result of an increase in cerebrovascular resistance. This finding is consistent with previous reports that nitric oxide syn-
TABLE III Effects of L-NAME and l_-arginine on cerebrovascular responses during hypotension Values are expressed as the means+ S.E.M. Data recorded during the 2-min period following blood reinfusion. + MABP was calculated by averaging values observed at 15-s intervals during the 2 rain of hypotension. CVR = mmHg. ml 1.min 1. Condition Control(n=6) L-NAME(10mg/kg) L-Arginine(300mg/kg)
Pre-hypotension
Hypotension
Drop rate
113.7_+1.6 123.7+-4.3" 122.6-+5.7
20.7_+2.0 227.2_+15.6 34.3+_1.9 13.6+_1.(I 92.5_+7.9 111.0_+3.6 21.7_+2.2 180.6+_39.3 19.1+-1.6 262.5_+20.2 32.5_+0.8 10.8±1.2 105.5+_10.0 119.7_+2.9 19.9+-1.9 251.7+-18.7 21.9-+3.9 249.7_+34.1 39.3_+3.0 12.6_+2.6 124.5+22.6 117.5+_6.3 22.7_+3.4 227.0_+28.9
* P < 0.05 compared to control.
CVR
MABP
Post-hypotension
MABP +
Drop rate CVR
MABP
Drop rate
CVR
253 A
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BLOODPRESSURE 150r ~ ~ ~ I L
an effect of L-NAME on resting CBF may, therefore, have been a result of these low P a C O 2 values. This possibility is supported by the observation 3° that if PaCO2 values were reduced, there was no further reduction of CBF after inhibition of nitric oxide synthe-
2 rain HYPOTENSION~ _---AL ¥ , ~ r
100["
~-"
5;[mmHgntB
15o[-~lmlL_,
,0o~-~ 1 ~ 5OI-
Ol"mmHg
--- ....
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15o[100[" 5o[ 0 - mmHg Fig. 3. Responses of cerebral blood flow and arterial blood pressure to the withdrawal and reinfusion of arterial blood. Blood was withdrawn over a 1-min period; the hypotension was maintained for 2 min; and then blood was reinfused over a 1-min period. After recording the control response (A), L-NAME (10 mg/kg) was administered and 10 rain later the hypotensive challenge was repeated (B). After injection of L-arginine (300 mg/kg), another response to the hypotensive challenge (C) was obtained.
thesis inhibitors increase resistance in the cerebral and other vascular b e d s 6'14'23'27, and that a direct contraction of cerebral arteries is observed in the presence of such inhibitors 2'24. Our data would be consistent with the concept that cerebral arterioles, share, with resistance vessels all over the body, an endothelium-dependent mechanism that can oppose the development of vascular tone. The reversal of the effects of the lower dose of L-NAME on cerebrovascular resistance by Larginine is consistent with the identification of the relaxant factor as nitric oxide, Our observation of an increase in CVR, in the absence of any change in cerebral blood flow following nitric oxide synthase inhibition, stands in contrast to the findings of other investigators in the r a t 14'21'27'29'3°. Some studies on other animal species have, however, failed to reveal any effects of nitric oxide synthesis inhibition on resting CBF (rabbit3; dog26). It may be significant that the P a C O 2 levels in the majority of studies in which a reduction in CBF was observed were in the range of 37-40 mmHg, whereas they were in the range of 31-34 mmHg in the present study. The lack of
sis.
The fact that, in this present study, the CVR increased in the resting state in proportion to the increase in MABP, whilst CBF remained constant, suggests that autoregulation was preserved intact following nitric oxide synthesis inhibition. L-NAME administration failed to alter the peak increase in CBF during a brief (8% 0 2 for 25 s) hypoxic episode. The time for a return of CBF to a stable baseline rate was reduced by 26.5%, but this Change was not significant. L-Arginine administration reversed the decrease in the time to recovery. Our results therefore suggest that nitric oxide is unlikely to play more than a minor role in eliciting the hyperemia associated with transient hypoxic episodes. These findings are generally consistent with those of Kozniewska et al. 14, who observed similar increases in CBF and decreases in CVR during hypoxia in control and LNMMA-treated rats, and concluded that nitric oxide w a s n o t the main factor responsible for the increase in CBF during hypoxia. In c o n t r a s t t o both our results and those of Kozniewska e t al. 14, Pelligrino et al. 2° observed a n increase in hypoxia-evoked CBF in rats following LN A M E administration in comparison to the control responses. These authors have interpreted the enhanced hypoxic response in the face of an inability to release nitric oxide as an indication that, under normal circumstances, hypoxia is associated with a diminished NO synthesis, which may act to limit the CBF increase during hypoxia. Pearce et al. 19 have previously suggested that an endothelially derived relaxing factor may be involved in hypoxia-evoked dilation of cerebral vessels, as scraping of the endothelium, or application of Methylene blue, prevented the dilation. However, our results appear to confirm the conclusion of Kozniewska et al. 14 that, at least for brief hypoxic episodes, nitric oxide is not the main factor eliciting cerebrovascular dilation. Iadecola 9, Pelligrino et al. 2° and Wang et al. 3° have previously demonstrated that nitric oxide is a critical participant in the increase in CBF elicited by hypercapnia, in that the administration of nitric oxide synthase inhibitors attenuated the CO2-induced hyperemia by 60-90%. Similar results were obtained in the present experiments. L-NAME (10 mg/kg) significantly reduced the time to recovery to baseline blood flow following a hyperemic episode (10% CO 2 for 36 s), but
254 did not reduce the peak increase in CBF. Its effects
conditions. T h e evidence suggests that while nitric ox-
were reversed by a s u b s e q u e n t a d m i n i s t r a t i o n of L-, but not D-, arginine. L - N A M E (30 m g / k g ) significantly
ide is an i m p o r t a n t factor in the vasodilation associated with hypercapnia, it is not a critical c o m p o n e n t of the
r e d u c e d the p e a k increase in C B F (by 42%, P < 0.05) and the time to recovery (by 89%, P < 0.01) associated
m e c h a n i s m s involved in cerebrovascular responses to hypoxia or in a u t o r e g u l a t i o n .
with hypercapnic episodes, a n d the effects were a t t e n u ated by L-arginine. T h e s e findings confirm that argin i n e - d e r i v e d nitric oxide plays a n i m p o r t a n t role in the vasodilation associated with increases in paCO2. D-Arginine (300 m g / k g ) a p p e a r e d to e n h a n c e the inhibitory effects of E - N A M E (10 m g / k g ) in the responses to h y p e r c a p n i a (Table II). Whilst D-arginine does n o t inhibit nitric oxide synthase ~3, it is an effective a n t a g o n i s t of L-arginine t r a n s p o r t 31'32 with a K i of a b o u t 1 mM. At the dose level e m p l o y e d (300 m g / k g ) , it can be a n t i c i p a t e d that the D-arginine c o n c e n t r a t i o n s in the arterial blood would be a d e q u a t e to inhibit L-arginine u p t a k e into vascular cells, thus r e d u c i n g intracellular levels of L-arginine a n d p o t e n t i a t i n g the inhibitory effects of L - N A M E on h y p e r c a p n i a - e v o k e d increases in CBF. T h e precise site of nitric oxide f o r m a t i o n d u r i n g h y p e r c a p n i a r e m a i n s unresolved. In isolated dog cerebral arteries, removal of the e n d o t h e l i u m did not affect the relaxation elicited by h y p e r c a p n i a 28, raising the possibility that the e n d o t h e l i u m may not be the site (or only site) of nitric oxide p r o d u c t i o n . T h e m o l e c u l a r m e c h a n i s m s by which CO 2 a n d / o r H + might affect nitric oxide p r o d u c t i o n also r e m a i n u n c e r t a i n , L - N A M E failed to alter cerebrovascular resistance d u r i n g 2-min periods of h y p o t e n s i o n in c o m p a r i s o n to the p r e - L - N A M E responses (Table III), suggesting that nitric oxide is not involved in the a u t o r e g u l a t o r y responses to m a r k e d decreases in M A B P . This conclusion is consistent with a previous report of preserved C B F a u t o r e g u l a t i o n over a wide range of arterial blood pressures 29. T h e increase in the m a g n i t u d e of the hyperemic response occurring after 2 min of severe h y p o t e n s i o n caused by L - N A M E is of some interest. A similar p h e n o m e n o n has b e e n observed by Garcia et al. 5 in a study on the role of nitric oxide in the coronary circulation of a n e s t h e t i z e d goats. In the p r e s e n c e of L-NAME, the hyperemic responses following a brief coronary occlusion were increased in c o m p a r i s o n to control, p r e - L - N A M E , responses. A possible e x p l a n a t i o n for this p h e n o m e n o n may be the d e v e l o p m e n t of a supersensitivity of the vascular system to factors released by the e n d o t h e l i u m or s u r r o u n d i n g tissues w h e n nitric oxide p r o d u c t i o n is reduced, In conclusion, the results p r e s e n t e d h e r e i n support the idea that e n d o g e n o u s nitric oxide may be involved in the r e g u l a t i o n of the cerebral circulation u n d e r basal
Acknowledgements. Supported by a grant from the Wayne State
University Neuroscience Program. J.E. Buchanan was a Student ResearchFellow of the American Heart Association of Michigan. REFERENCES
1 Alonso, M.J., Salaices, M., Sanchez-Ferrer, C.F. and Marin, J., Predominant role for nitric oxide in the relaxation induced by acetylcholine in cat cerebral arteries, J. Pharmacol. Exp. Ther., 261 (1992) 12-20. 2 Faraci, F.M., Role of nitric oxide in regulation of basilar artery tone in vivo, Am. J. Physiol., 259 (1990) H1216-H1221. 3 Faraci, F. and Heistad, D.D., Does basal production of nitric oxide contribute to regulation of brain-fluid balance, Am. J. Physiol., 262(1992)H340-H344. 4 Furchgott, R.E. and VanHoutte, P.M., Endothelium-derived relaxing and contracting factors, FASEB J., 3 (1989) 2007-2018. 5 Garcia, J.L., Fernandez, N., Garcia-Villalon, A.L., Monge, L., Gomez, B. and Dieguez, G., Effects of nitric oxide synthesis inhibition on the goat coronary circulation under basal conditions and after vasodilator stimulation, Br. J. Pharmacol.. 106 (1992) 563-567. 6 Gardiner, S.M., Compton, A.M., Bennett, T., Palmer, R.M.J. and Moncada, S., Control of regional blood flow by endothelium-derived nitric oxide, Hypertension, 15 (1991) 486-492. 7 Harder, D.R., Pressure-induced myogenic activation of cat cerebral arteries is dependent on intact endothelium, Circ. Res., 60 (1987) 102-107. 8 Hobbs, A.J. and Gibson, A., L-N-nitro-arginine and its methyl ester are potent inhibitors of non-adrenergic, non-cholinergic transmission in the rat anococcygeus, Br. J. Pharmacol., 100 (1990) 749-752. 9 ladecola, C., Does nitric oxide mediate the increases in cerebral blood flow elicited by hypercapnia?. Proc. Natl. Acad. Sci. USA, 89 (1992) 3913-3916. 10 Ignarro, L.J., Endothelium-derived nitric oxide: actions and properties, FASEBJ., 3 (1989)31-36. 11 Ignarro, L.J., Buga, G.M., Wood, K.S., Byrns, R.E. and Chaudhurl, G., Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide, Proc. Natl. Acad. Sci. usA, 84 (1987) 9265-9269. 12 Katusic, Z.S., Shepherd, J.T. and VanHoune, P.M., Endothelium-dependent contraction to stretch in canine basilar arteries, Ant. J. Physiol., 252 (1987) H671-H673. 13 Knowles, R.G., Palacios, M., Palmer, R.M.J. and Moncada, S., Formation of nitric oxide from ~-arginine in the central nervous system: a transduction mechanism for stimulation of the soluble guanylate cyclase, Proc. Natl. Acad. Sci. USA, 86 (1989) 5159 5162. 14 Kozniewska, E., Oseka, M. and Stys, T., Effects of endotheliumderived nitric oxide in cerebral circulation during normoxia and hypoxia in the rat, J. Cereb. Blood Flow Metab., 12 (1992) 311-317. 15 Moncada, S., Palmer, R.M.J. and Higgs, E.A., Nitric oxide: physiology, pathophysiology, and pharmacology, Pharmacol. Rec., 43 (1991) 109-142. 16 Morii, S., Ngai, A.C., Ko, K.R. and Winn, H.R., A venous outflow method for continuously monitoring cerebral blood flow in the rat, Am. J. Physiol., 250 (1986) H304-H312. 17 Nilsson, B. and Siesj6, B.K., A method for determining blood flow and oxygen consumption in the rat brain, Acta Physiol. Scand., 96 (1976)72-82. 18 Palmer, R.M.J., Ashton, D.S. and Moncada, S., Vascular endothelial cells synthesize nitric oxide from L-arginine, Nature. 333 (1988) 664-666.
255 19 Pearce, W.J., Ashwal, S. and Cuevas, J., Direct effects of graded hypoxia on intact and denuded rabbit cranial arteries, Am. J. Physiol., 257 (1989) H824-H833. 20 Pelligrino, D.A., Koenig, H. and Sharp, A., Nitric oxide (NO) synthesis and cerebral vasodilatory responses in the rat, FASEB J., 6 (1992) A1462. 21 Pelligrino, D.A., Miletich, D.J. and Albrecht, R.F., Diminished muscarinic receptor-mediated cerebral blood flow response in streptozotocin-treated rats, Am. J. Physiol., 262 (1992) E447E454. 22 Phillis, J.W., Preston, G. and DeLong, R.E., Effects of anoxia on cerebral blood flow in the rat brain: evidence for a role of adenosine in autoregulation, J. Cereb. Blood Flow Metab., 4 (1984) 586-592. 23 Rees, D.D., Palmer, R.M.J., Schulz, R., Hodson, H.R. and Moncada, S., Characterization of three inhibitors of endothelial nitric oxide synthase in vitro and in vivo, Br. J. Pharmacol., 101 (1990) 746-752. 24 Rosenblum, W.I., Nishimura, H. and Nelson, G.H., Endothelium-dependent L-Arg- and L-NMMA-sensitive mechanisms regulate tone of brain microvessels, Am. J. Physiol., 259 (1990) H1396-H1401. 25 Simpson, R.E., Phillis, J.W. and Buchanan, J.E., A comparison of cerebral blood flow during basal, hypotensive, hypoxic and hypercapnic conditions between normal and streptozotocin diabetic rats, Brain Res., 531 (1990) 136-142.
26 Sonntag, M., Deussen, A. and Schrader, J., Role of nitric oxide in local blood flow control in the anaesthetized dog, Pflugers Arch., 420 (1992) 194-199. 27 Tanaka, K., Gotoh, F., Gomi, S., Takashima, S., Mihara, B., Shirai, T., Nogawa, S. and Nagata, E., Inhibition of nitric oxide synthesis induces a significant reduction in local cerebral blood flow in the rat, Neurosci. Lett., 127 (1991) 129-132. 28 Toda, W., Hatano, Y. and Mori, K., Mechanisms underlying responses to hypercapnia and bicarbonate of isolated dog cerebral arteries, Am. J. Physiol., 257 (1989) H141-H146. 29 Wang, Q., Paulson, O.B. and Lassen, N.A., Is autoregulation of cerebral blood flow in rats influenced by nitro-L-arginine, a blocker of the synthesis of nitric oxide?, Acta Physiol. Scand., 145 (1992) 297-298, 1992. 30 Wang, Q., Paulson, O.B. and Lassen, N.A., Effect of nitric oxide blockade by N-nitro-L-arginine on cerebral blood flow response to changes in carbon dioxide tension, Z Cereb. Blood Flow Metab., 12 (1992) 947-953. 31 White, M.F., Gazzola, G.C. and Christensen, H.N., Cationic amino acid transport into cultured animal cells. I. Influx into cultured human fibroblasts, J. Biol. Chem., 257 (1982) 4443-4449. 32 White, M.F. and Christensen, H.N., Cationic amino acid transport into cultured animal cells. II. Transport system barely perceptible in ordinary hepatocytes, but active in hepatoma cell lines, J. Biol. Chem., 257 (1982) 4450-4457.
Brain Research, 610 (1993) 248-255 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
BRES 18779
The role of nitric oxide in the regulation of cerebral blood flow Joel E. Buchanan
and John W. Phillis
Department of Physiology, Wayne State Unit,ersity School of Medicine, Detroit, MI 48201 (USA) (Accepted 1 December 1992)
Key words." Nitric oxide; Endothelium-derived relaxing factor; N-Nitro-e-arginine methyl ester; Cerebral blood flow; Hypoxia; Hypercapnia; Hypotension
The role of nitric oxide in the cerebral circulation under basal conditions and when exposed to hypoxic, hypercapnic and hypotensive stimuli, was studied in mechanically ventilated rats using a venous outflow technique, by examining the effects of inhibition of nitric oxide synthase with N-nitro-e-arginine methyl ester (e-NAME). e-NAME (10 or 30 mg/kg injected intravenously) raised mean arterial blood pressure by 14% and 24%, and increased cerebrovaseular resistance (CVR) by 20% and 24%, respectively. Cerebral blood flow (CBF) was unaltered, as were blood gases and pH. The increases in MABP and CVR were attenuated by L-arginine (300 mg/kg). Following the administration of L-NAME, the increases in CBF elicited by ventilation with 8% oxygen for 25 s were unaltered, in comparison to control responses. L-NAME attenuated the increases in CBF and reduced the time for recovery to basal flow rates evoked by ventilation with 10% carbon dioxide. These effects were reversed by L-, but not by D-, arginine. Autoregulation by CBF during hypotensive episodes, as measured by comparisons of CVR values, was unaffected by L-NAME. The results suggest that endogenous nitric oxide is involved in the responses of the cerebral vasculature to elevated levels of CO 2 in the arterial blood. Nitric oxide does not appear to play a major role in autoregulation to increases or decreases in MABP, or in hypoxia-evoked vasodilation.
INTRODUCTION It is now recognized that the vascular endothelium can play a key role in regulating vascular tone by releasing vasoactive factors and thus modulating vascular responses to various stimuli 4. Experimental evidence suggests that nitric oxide, or a closely related compound, synthesized from L-arginine, is an endothelium-derived relaxing factor ~1'~8 which relaxes vascular smooth muscle, including that of cerebral arteries 1 by stimulation of guanylate cyclase t°'15. The synthesis of endothelial nitric oxide can be inhibited by several analogues of L-arginine, and this inhibition induces an increase in the resistance of several vascular beds, including that of the cerebrovascular system 6'27. Contraction of pial arteries was observed when the arginine analogue N-monomethyl-L-arginine ( L - N M M A ) w a s superfused over the brain s u r f a c e 24, and of the basilar artery when L-NMMA was applied topically 2. Thus a basal release of nitric oxide appears to be responsible
for maintaining a vasodilator tone in the cerebrovascular, as well as other regional vascular, systems 6'27. The present experiments were performed to study the role of nitric oxide in setting basal vascular tone and in the vasodilation of cerebral blood vessels during hypoxia, hypercapnia and hypotension in anesthetized, mechanically ventilated rats. Cerebral blood flow was measured by a venous outflow technique 22'25 and the effects of a potent inhibitor of nitric oxide synthesis, N-nitro-L-arginine methyl ester (L-NAME) a'23 were investigated both on basal cerebral blood flow and on the responses elicited during brief episodes of hypoxia, hypercapnia and hypotension. MATERIALS AND METHODS Male Sprague-Dawley rats (Charles River: 350-450 g) were used to study CBF, using a venous outflow technique involving cannulation of the retroglenoid vein (RGV), first described by Nilsson and Siesj6 iv. The original method was refined by the inclusion of an extracorporeal circulation pump which permits continuous recording
Correspondence." J.W. Phillis, Department of Physiology, Wayne State University School of Medicine, 540 E. Canfield, Detroit, MI 48201, USA. Fax: (1) (313) 577-5494.
249 of cerebral blood flow for several hours 16'22'2s, conferring the advantage of continuous monitoring of CBF before, during and after various tests are performed on the same animal. This method was chosen because of previous successful use of this technique in our laboratory during other CBF studies, The general procedure for the experiments was as follows. T h e animals were anesthetized with 3% halothane in air to allow tracheotomy. During and after surgery, the animals were maintained on a mixture of methoxyflurane; 30% oxygen (O 2) and 70% nitrogen (N2). The animals were placed on a heating pad actuated by a rectal probe, to maintain body temperature at 37°C. Surgery was performed to expose the right and left RGV. Initial incisions were m a d e just in front of the external auditory meatus, The temporal muscle was then lifted and the blood vessels were separated and freed from the surrounding tissues to expose the R G V at their exits through the retroglenoid foramen. 5.0 surgical silk threads were placed around the veins, T h e left femoral artery was exposed, tied off and cannulated. The right femoral artery and vein were then exposed, tied off and cannulated. T h e right artery cannula was attached to a Grass Polygraph to continuously record arterial blood pressure. The venous cannula was attached to the extracorporeal p u m p and served to return blood to the animal. The left femoral artery cannula was used in the hypotensive portion of the experiment for withdrawing and reinjecting blood while maintaining a continuous blood pressure recording. It was also used to obtain small (0.35 ml) samples of arterial blood for pH and blood gas analysis. Following femoral cannulation the animal was heparinized (1 U / g ) . The left R G V was tied off distally with a long 5.0 thread. A small weight (alligator clamp) was attached to the end of this thread and allowed to pull on the RGV. This weight facilitated cannulation and blood flow through the cannula after its insertion. The left R G V was cannulated with an angiocatheter (Deseret, 22GA, 1 inch), which was advanced until its tip was flush with the retroglenoid foramen opening. T h r o u g h this opening the transverse sinus drains blood which
DROPS
A
originates in the cerebrum, the cerebellum and possibly the brainstem into the RGV. The angiocatheter was tied in place with 5.0 surgical silk thread. The extracorporeal venous flow system was then connected. The thread on the right R G V was tied off to prevent the possibili~ of entry of extracranial blood into the cerebral venous system. Blood in the left R G V flowed through the angiocatheter and an attached tube and was then allowed to drip past an electric eye into a collecting chamber. The outlet of the tube was positioned at the level of the retroglenoid foramen. Each time a drop passed the electric eye, a mark was recorded on the Grass Polygraph. Blood was then transferred from the collecting chamber back into the animal with an extracorporeal p u m p connected to the femoral venous cannula. To prevent blood loss in the subject animal, the blood volume requirement in the extracorporeal return system (4-5 ml) was filled by an additional animal killed by decapitation to donate blood to this system. Blood in the extracorporeal circuit was passed between the animal and the heating pad for warming before entering the femoral venous cannula. The subject animal was connected to a respiratory p u m p and respired at a frequency of 60-80 s t r o k e s / m i n on a gas mixture of m e t h o x y f l u r a n e / 3 0 % O 2 / 7 0 % N 2. The subject was then administered pancuronium bromide (Pavulon, 1 m g / k g ) and the concentration of methoxyflurane was decreased to 0.5-1% and adjusted such that blood pressure remained constant when pressure was applied to a forepaw. A period of 10-20 min was allowed for the animal to stabilize. Blood gases were measured in a blood sample and respiration was adjusted so that pH was approximately 7.38-7.42 and p C O 2 was approximately 32-36 mmHg. Additional samples were withdrawn during the course of the experiment and, if necessary, p u m p ventilation was adjusted to maintain blood gases and pH at their original levels. Additional injections of pancuronium bromide were administered if the animal evinced any tendency to make spontaneous respiratory movements. Following stabilization, the animals were given a series of challenges. Brief (25 and 36 s, respectively) challenges were given
B
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Fig. l. Responses of cerebral blood flow and arterial blood pressure to hypoxic (A,C,E) and hypercapnic (B,D,F) episodes. Cerebral blood flow, as represented by outflow from the retroglenoid sinus, is recorded as drops. Hypoxia was induced by mechanically ventilating the rat with 8% oxygen for 25 s; hypercapnia by vcntilating with ]0% CO 2 for 36 s. C and D were recorded after intravenous administration of L-NAME (30 mg/kg). E and F were rccorded after subsequent intravcnous administration of L-arginine (300 mg/kg).
250 TABLE I
istration of a gas mixture of 10% CO z / 3 0 % 0 2 / 6 0 % N 2 were given
Effects of L-NAME on MABP and cerebrot,ascular resistance (CVR)
every 10-12 rain. Hypotensive challenges were administered by first changing the inspiratory gas from 30% 0 2 / 7 0 % N z to 40% 0 2 / 6 0 % N z. The animal was allowed to respire this mixture for 10-12 min before the hypotensive challenge was administered as described above. To reduce dead space in the delivery system, two identical anesthetic vaporizers were used; one for the standard oxygen-enriched respiratory gas, the other for the hypoxic or hypercapnic challenge gases. For the hypoxic and hypercapnic challenges, observations and recordings were made for basal CBF rate, peak CBF rate and time to recovery. For the bypotensive challenge, CBF rate was recorded for the basal level, during the challenge and after recovery. In addition to CBF, MABP was recorded for the 2-rain hypotensive period. A brief delay occurred before animals began to increase CBF in response to hypercapnic or hypoxic challenges. The increased CBF rate continued for up to 2 rain after the basal gas was reconnected. Peak flow rate was defined as the greatest number of drops recorded in any 10-s interval. This number was then compared to the basal flow rate, which was defined as the number of drops recorded in a continuous l-rain interval immediately preceding the challenge period. Peak flow rates were adjusted (multiplied by 6) in order to present the data in a drops per minute format. Subjects were very stable during the basal period with constant flow (drop) rates and MABP (Fig. 1). To avoid interference with the MABP and CBF recordings, no blood samples were taken during the test challenges. Since the challenge durations were rigorously controlled, it is assumed that comparable blood gas and pH changes would have been present in each challenge. The percentage peak increases in CBF presented in Table II were calculated from the formula: % increase = (peak flow rate/basal flow rate × i 0 0 ) - 100. Recovery times represent time from the end of the challenge until flow rates returned to a new stable baseline. Cerebrovascular resistance (CVR) was calculated according to the formula CVR = MABP/CBF. CBF ( m l / m i n ) was determined from the d r o p s / m i n data using a conversion factor obtained from data presented in Phillis et al. 22. In evaluating the effects of L-NAME, k-arginine and i>arginine, the control responses preceding drug administration were compared with those after drug injection. Corn-
Values are expressed as the means-+S.E.M.
MABP
Basal flow (drops/min)
CVR (mmHg.ml-1 .min-1)
114.0-+3.8
21.2_+1.4
228.7_+15.6
130.4_+3.3 **
19.7+ 1.1
273.1 -+ 14.0 *
126.5+ 4.7
22.6 + 2.7
232.0 + 20.7
115.7+4.4
19.6-+2.2
231.2+24.7
143.5_+5.6 **
21.4_+ 2.2
295.6_+24.5 *
136.3_+5.2
19.7_+ 2.5
296.8_+34.7
Group I (n = 19) Control t,-NAME (10 m g / k g ) L,-Arginine (300 m g / k g ) Group II (n = 12) Control L-NAME (30 m g / k g ) L-Arginine (300 m g / k g )
• P < 0.05 compared to control, • * P < 0.01 compared to control,
tbr the bypoxic and hypercapnic episodes. A hypotensive challenge consisted of a 1-min period of blood withdrawal to reduce the MABP to 30-40 mmHg, along with an observation period of 2 rain where MABP was kept at 30-40 mmHg, followed by a 1-min reinjection of the blood originally withdrawn to re-establish the pre-hypotension MABP. Hypoxic and hypercapnic challenges were frequently administered alternatively to the same animal, with at least two control challenges of each type being recorded prior to drug administration. The responses to each type of challenge in a given animal were averaged before being combined with the responses of other rats. Challenges were administered as follows: hypoxic challenges consisting of 8% oxygen in nitrogen for 25-s duration were administered 10-12 min apart. Hypercapnic challenges consisting of a 36-s admin-
TABLE 1I
Effects of L-NAME and L-arginine on responses to hypoxia and hypercapnia Values are expressed as the means_+ S.E.M.
Condition
No. of rats
Group I Control hypoxia L-NAME (30 m g / k g ) t,-Arginine (300 m g / k g )
7
Group II Control hypercapnia ~,-NAME (10 m g / k g ) L-Arginine (300 m g / k g )
11
Group III Control hypercapnia t,-NAME (30 m g / k g ) L-Arginine (300 m g / k g )
11
Group 1V Control hypercapnia L-NAME (10 m g / k g ) D-Arginine (300 m g / k g )
9
Basal flow rate (drops/min)
% Increase in peak flow
Time to recot,ery (s)
23.4_+ 3.1 20.4 +_2.6 20.9-+ 1.3
49.4 + 6.4 49.1 + 6.7 53.7 + 7.8
69.9 + 8.1 51.4 _+23.2 64.4 + 19.9
21.4 _+ 1.7 20.4_+ 1.3 23.6 -+ 2.5
75.8 _+ 10.6 74.7+ 10.8 72.6 + 10.4
93.5 +_ 10.8 * 64.6_+ 7.3 105.9 _+20.3 *
20.4+_2.9 21.2 + 2.4 18.7 _+2.6
81.1 +_ 12.9 * 47.4 + 10.1 77.4 -+ 13.8
93.4_+ 12.9 * * 38.8_+ 5.8 81.8 -+ 15.8 *
22.1 + 1.9 21.9_+ 1.7 22.7 + 2.3
85.4 -+ 11.1 70.6_+ 13.7 58.4 -+ 11.0
79.5 _+ 9.4 60.3+ 7.6 50.0 _+ 11.9 +
• P < 0.05 in comparison to value following L-NAME administration. • * P < 0.01 in comparison to value following L-NAME administration. P < 0.05 in comparison to control.
251 parisons were performed between control data and post-drug responses using an analysis of variance and a t-test. Results are presented as means + S.E.M. Drugs used were: N-nitro-L-arginine methyl ester (L-NAME, Sigma Co.; 10 or 30 mg/kg), L-arginine (Sigma Co.; 300 mg/kg) and D-arginine (Sigma Co.; 300 mg/kg) administered intravenously in isotonic saline. Administration of isotonic saline alone at the volumes employed in the present study had no systemic or cerebral blood flow effects. RESULTS
Effects of N-nitro-L-arginine methyl ester Mean values for mean systemic arterial blood pressure, basal cerebral venous outflow and a calculated C V R before and 10 min after administration of LN A M E are presented in Table I. Intravenous administration of L-NAME increased m e a n arterial blood pressure by about 14 and 24% at the doses of 10 and 30 m g / k g , respectively. Venous outflow was unaltered and C V R increased by 19% and 28%. The increase in C V R was significant at both doses. Effects became apparent within 60 s of the onset of L-NAME administration and had stabilized by the 10-min period of measurement. Blood gases and p H following L-NAME administration were not significantly different from those obtained under control conditions. The hemodynamic effects of L-NAME were still present at least 60 min after administration of the low dose, and continued for the duration of the experiment, up to 2 h, after the higher dose. Administration of L-arginine failed to reverse the effects of L-NAME (30 m g / k g ) , but did attenuate the increase in C V R and M A B P elicited by the lower dose
of L-NAME, although neither reduction was statistically significant.
Responsesto hypoxic episodes The hypoxic challenges consisted of repeated brief (25 S) periods of inhalation of 8% oxygen in nitrogen. Hypoxic episodes of this duration elicited a fall in paO2 values from a resting level of 98.6 m m H g to 41.6 m m H g during control challenges. By using a challenge of this brevity, it was possible to avoid the reductions in blood pressure usually associated with hypoxia in anesthetized animals. Prechallenge MABPs of the 7 rats in this series were 102.6 + 6.4 m m H g , with an average M A B P during control hypoxic challenges of 100.6 +_ 5.7 mmHg. L-NAME (30 m g / k g ) did not alter the basal drop rate in these animals, nor did it affect the % increase peak flow rate or recovery time following hypoxic challenges (Table II, Group 1). L-Arginine (300 m g / k g ) administered after L-NAME, was also without effect on the responses to hypoxia (Table II). Examples of the lack of effect of L-NAME and L-arginine on the vascular responses to hypoxia are illustrated in Fig. 1A,C,E. L-NAME (30 m g / k g ) failed to reduce the increase in venous outflow evoked by 8% oxygen inhalation and following L-arginine (300 m g / k g ) administration; the hyperemia was still evident even though basal flow rates had declined at this time.
Responsesduring hypercapnia Hypercapnic challenges of the same duration as those used in these experiments (10% CO z in 30% 0 2
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30 sec
Fig. 2. Responses of arterial blood pressure and cerebral blood flow to hypercapnic challenges (36 s) before (A) and following sequential doses of L-NAME (10 mg/kg) (B); D-arginine (300 mg/kg) (C) and L-arginine (300 mg/kg) (D).
252 for 36 s) elicited a significant rise in p~CO 2 of + 9.6 +_ 0.66 mmHg from a prechallenge value of 32.2 -+ 0.7 mmHg to an end of challenge value of 41.8 + 0.7 mmHg in control animals. The duration of the hypercapnic challenge was selected so as to have minimal effects on MABP. The pre-challenge MABP for 23 rats was 115.6 + 3.8 mmHg, and that during the hypercapnic challenge was 117.7 + 3.7 mmHg. The drop rate increased dramatically in response to the hypercapnic challenge, and remained elevated for a brief period (80-93 s) after the animal had been returned to its normal (30% 0 2) respiratory gas (Table II, Figs. 1 and 2). Peak increases in blood flow were in the range of 75-85%. Following the administration of L-NAME (10 or 30 mg/kg) MABP pressures rose, but there were no significant alterations in MABP during the hypercapnic challenges (135.4 + 4.3 vs. 134.2 + 3.9 for the L-NAME (10 m g / k g ) animals; 145 + 5.8 vs. 141.4_+ 4.8 for the L-NAME (30 mg/kg) rats). L-NAME (10 mg/kg) was without effect on the peak rate of flow during the hypercapnic challenges but did significantly reduce the time to recovery (Table II). At 30 mg/kg, L-NAME significantly reduced both the peak rate of flow and the time to recovery (Table II). The effects of L-NAME on both peak flow rate and time to recovery were reversed by the subsequent administration of L-arginine (300 mg/kg), but not by D-arginine (300 mg/kg), The effects of L-NAME and L- and D-arginine are illustrated in Figs. 1 and 2. The responses in Fig. 1B,D,F were recorded from the same rat as the responses to 8% oxygen inhalation. Fig. 1B illustrates a control response to 10% CO 2 inhalation for 36 s. Following L-NAME (30 mg/kg) administration, CO 2 evoked a markedly attenuated hyperemia (Fig. 1D), but after administration of L-arginine (300 mg/kg) the hyperemic response was restored, Fig. 2 A - D illustrates a succession of responses to 10% CO 2 inhalation, t - N A M E (10 mg/kg) administration greatly attenuated the hyperemic response to CO 2 (Fig. 2B), and its effects were not blocked by D-arginine
(300 mg/kg). A subsequent administration of L-arginine (300 mg/kg) resulted in a clear reversal of the effect of L-NAME.
Responsesduring hypotension Ten minutes prior to the start of a hypotensive challenge, the animals were ventilated with 40% oxygen to minimize the severity of cerebral hypoxia as a result of the reduction in blood flow. The results from 6 rats are presented in Table III. L-NAME (10 mg/kg) had no effect on either flow rate or calculated cerebrovascular resistance either prior to, during or following a hypotensive episode. L-Arginine (300 mg/kg) administered after L-NAME was also without effect. The results from one experiment are illustrated in Fig. 3. The drop rate fell from 20.0/min to 15.5/min (a 22% decline; CVR during hypotension = 88) during the 2 rain of hypotension. Following L-NAME (10 mg/kg) administration, the drop rate declined from 21.0/min to l l . 5 / m i n (a 45% decline) but as MABP during this episode of hypotension was actually lower than in the control (36 vs. 29 mmHg), the CVR was unchanged at 88. A pronounced reactive hyperemia followed the reinfusion of withdrawn blood. A subsequent injection of L-arginine did not affect the response during hypotension and partially reversed the increase in magnitude of the reactive hyperemia. Similar increases in the magnitude of the reactive hyperemias following hypotensive episodes after L-NAME administration were observed in all six of these experiments. Reversal by L-arginine was never complete. DISCUSSION The present results demonstrate that intravenous administration of I,-NAME, a potent inhibitor of nitric oxide synthase 8'23 produced moderate increases in mean arterial blood pressure, in the absence of any alteration of cerebral venous outflow as a result of an increase in cerebrovascular resistance. This finding is consistent with previous reports that nitric oxide syn-
TABLE III Effects of L-NAME and l_-arginine on cerebrovascular responses during hypotension Values are expressed as the means+ S.E.M. Data recorded during the 2-min period following blood reinfusion. + MABP was calculated by averaging values observed at 15-s intervals during the 2 rain of hypotension. CVR = mmHg. ml 1.min 1. Condition Control(n=6) L-NAME(10mg/kg) L-Arginine(300mg/kg)
Pre-hypotension
Hypotension
Drop rate
113.7_+1.6 123.7+-4.3" 122.6-+5.7
20.7_+2.0 227.2_+15.6 34.3+_1.9 13.6+_1.(I 92.5_+7.9 111.0_+3.6 21.7_+2.2 180.6+_39.3 19.1+-1.6 262.5_+20.2 32.5_+0.8 10.8±1.2 105.5+_10.0 119.7_+2.9 19.9+-1.9 251.7+-18.7 21.9-+3.9 249.7_+34.1 39.3_+3.0 12.6_+2.6 124.5+22.6 117.5+_6.3 22.7_+3.4 227.0_+28.9
* P < 0.05 compared to control.
CVR
MABP
Post-hypotension
MABP +
Drop rate CVR
MABP
Drop rate
CVR
253 A
DROPS
BLOODPRESSURE 150r ~ ~ ~ I L
an effect of L-NAME on resting CBF may, therefore, have been a result of these low P a C O 2 values. This possibility is supported by the observation 3° that if PaCO2 values were reduced, there was no further reduction of CBF after inhibition of nitric oxide synthe-
2 rain HYPOTENSION~ _---AL ¥ , ~ r
100["
~-"
5;[mmHgntB
15o[-~lmlL_,
,0o~-~ 1 ~ 5OI-
Ol"mmHg
--- ....
C
15o[100[" 5o[ 0 - mmHg Fig. 3. Responses of cerebral blood flow and arterial blood pressure to the withdrawal and reinfusion of arterial blood. Blood was withdrawn over a 1-min period; the hypotension was maintained for 2 min; and then blood was reinfused over a 1-min period. After recording the control response (A), L-NAME (10 mg/kg) was administered and 10 rain later the hypotensive challenge was repeated (B). After injection of L-arginine (300 mg/kg), another response to the hypotensive challenge (C) was obtained.
thesis inhibitors increase resistance in the cerebral and other vascular b e d s 6'14'23'27, and that a direct contraction of cerebral arteries is observed in the presence of such inhibitors 2'24. Our data would be consistent with the concept that cerebral arterioles, share, with resistance vessels all over the body, an endothelium-dependent mechanism that can oppose the development of vascular tone. The reversal of the effects of the lower dose of L-NAME on cerebrovascular resistance by Larginine is consistent with the identification of the relaxant factor as nitric oxide, Our observation of an increase in CVR, in the absence of any change in cerebral blood flow following nitric oxide synthase inhibition, stands in contrast to the findings of other investigators in the r a t 14'21'27'29'3°. Some studies on other animal species have, however, failed to reveal any effects of nitric oxide synthesis inhibition on resting CBF (rabbit3; dog26). It may be significant that the P a C O 2 levels in the majority of studies in which a reduction in CBF was observed were in the range of 37-40 mmHg, whereas they were in the range of 31-34 mmHg in the present study. The lack of
sis.
The fact that, in this present study, the CVR increased in the resting state in proportion to the increase in MABP, whilst CBF remained constant, suggests that autoregulation was preserved intact following nitric oxide synthesis inhibition. L-NAME administration failed to alter the peak increase in CBF during a brief (8% 0 2 for 25 s) hypoxic episode. The time for a return of CBF to a stable baseline rate was reduced by 26.5%, but this Change was not significant. L-Arginine administration reversed the decrease in the time to recovery. Our results therefore suggest that nitric oxide is unlikely to play more than a minor role in eliciting the hyperemia associated with transient hypoxic episodes. These findings are generally consistent with those of Kozniewska et al. 14, who observed similar increases in CBF and decreases in CVR during hypoxia in control and LNMMA-treated rats, and concluded that nitric oxide w a s n o t the main factor responsible for the increase in CBF during hypoxia. In c o n t r a s t t o both our results and those of Kozniewska e t al. 14, Pelligrino et al. 2° observed a n increase in hypoxia-evoked CBF in rats following LN A M E administration in comparison to the control responses. These authors have interpreted the enhanced hypoxic response in the face of an inability to release nitric oxide as an indication that, under normal circumstances, hypoxia is associated with a diminished NO synthesis, which may act to limit the CBF increase during hypoxia. Pearce et al. 19 have previously suggested that an endothelially derived relaxing factor may be involved in hypoxia-evoked dilation of cerebral vessels, as scraping of the endothelium, or application of Methylene blue, prevented the dilation. However, our results appear to confirm the conclusion of Kozniewska et al. 14 that, at least for brief hypoxic episodes, nitric oxide is not the main factor eliciting cerebrovascular dilation. Iadecola 9, Pelligrino et al. 2° and Wang et al. 3° have previously demonstrated that nitric oxide is a critical participant in the increase in CBF elicited by hypercapnia, in that the administration of nitric oxide synthase inhibitors attenuated the CO2-induced hyperemia by 60-90%. Similar results were obtained in the present experiments. L-NAME (10 mg/kg) significantly reduced the time to recovery to baseline blood flow following a hyperemic episode (10% CO 2 for 36 s), but
254 did not reduce the peak increase in CBF. Its effects
conditions. T h e evidence suggests that while nitric ox-
were reversed by a s u b s e q u e n t a d m i n i s t r a t i o n of L-, but not D-, arginine. L - N A M E (30 m g / k g ) significantly
ide is an i m p o r t a n t factor in the vasodilation associated with hypercapnia, it is not a critical c o m p o n e n t of the
r e d u c e d the p e a k increase in C B F (by 42%, P < 0.05) and the time to recovery (by 89%, P < 0.01) associated
m e c h a n i s m s involved in cerebrovascular responses to hypoxia or in a u t o r e g u l a t i o n .
with hypercapnic episodes, a n d the effects were a t t e n u ated by L-arginine. T h e s e findings confirm that argin i n e - d e r i v e d nitric oxide plays a n i m p o r t a n t role in the vasodilation associated with increases in paCO2. D-Arginine (300 m g / k g ) a p p e a r e d to e n h a n c e the inhibitory effects of E - N A M E (10 m g / k g ) in the responses to h y p e r c a p n i a (Table II). Whilst D-arginine does n o t inhibit nitric oxide synthase ~3, it is an effective a n t a g o n i s t of L-arginine t r a n s p o r t 31'32 with a K i of a b o u t 1 mM. At the dose level e m p l o y e d (300 m g / k g ) , it can be a n t i c i p a t e d that the D-arginine c o n c e n t r a t i o n s in the arterial blood would be a d e q u a t e to inhibit L-arginine u p t a k e into vascular cells, thus r e d u c i n g intracellular levels of L-arginine a n d p o t e n t i a t i n g the inhibitory effects of L - N A M E on h y p e r c a p n i a - e v o k e d increases in CBF. T h e precise site of nitric oxide f o r m a t i o n d u r i n g h y p e r c a p n i a r e m a i n s unresolved. In isolated dog cerebral arteries, removal of the e n d o t h e l i u m did not affect the relaxation elicited by h y p e r c a p n i a 28, raising the possibility that the e n d o t h e l i u m may not be the site (or only site) of nitric oxide p r o d u c t i o n . T h e m o l e c u l a r m e c h a n i s m s by which CO 2 a n d / o r H + might affect nitric oxide p r o d u c t i o n also r e m a i n u n c e r t a i n , L - N A M E failed to alter cerebrovascular resistance d u r i n g 2-min periods of h y p o t e n s i o n in c o m p a r i s o n to the p r e - L - N A M E responses (Table III), suggesting that nitric oxide is not involved in the a u t o r e g u l a t o r y responses to m a r k e d decreases in M A B P . This conclusion is consistent with a previous report of preserved C B F a u t o r e g u l a t i o n over a wide range of arterial blood pressures 29. T h e increase in the m a g n i t u d e of the hyperemic response occurring after 2 min of severe h y p o t e n s i o n caused by L - N A M E is of some interest. A similar p h e n o m e n o n has b e e n observed by Garcia et al. 5 in a study on the role of nitric oxide in the coronary circulation of a n e s t h e t i z e d goats. In the p r e s e n c e of L-NAME, the hyperemic responses following a brief coronary occlusion were increased in c o m p a r i s o n to control, p r e - L - N A M E , responses. A possible e x p l a n a t i o n for this p h e n o m e n o n may be the d e v e l o p m e n t of a supersensitivity of the vascular system to factors released by the e n d o t h e l i u m or s u r r o u n d i n g tissues w h e n nitric oxide p r o d u c t i o n is reduced, In conclusion, the results p r e s e n t e d h e r e i n support the idea that e n d o g e n o u s nitric oxide may be involved in the r e g u l a t i o n of the cerebral circulation u n d e r basal
Acknowledgements. Supported by a grant from the Wayne State
University Neuroscience Program. J.E. Buchanan was a Student ResearchFellow of the American Heart Association of Michigan. REFERENCES
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