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Anesth Analg 2001;93:1217-1221
© 2001 International Anesthesia Research Society

ANESTHETIC PHARMACOLOGY

The Effects of Nitric Oxide Synthase Inhibitors on the Sedative Effect of Clonidine

Roberto Soares de Moura, MD PhD*, Anna Amélia S. Rios, MD{dagger}, Luiz F. de Oliveira, MD PhD*, Ângela C. Resende, PhD*, Miguel de Lemos Neto, MD PhD*, Edmar J. A. Santos, MD*, Marcelo L. G. Correia, MD*, and Tania Tano, PhD*

*Department of Pharmacology, State University of Rio de Janeiro, Rio de Janeiro; and {dagger}Department of Pharmacology, Fluminense Federal University, Niteroi, Rio de Janeiro, Brazil

Address Correspondence and reprint requests to Roberto Soares de Moura, Departamento de Farmacologia, Instituto de Biologia, Centro Biomédico, Universidade do Estado do Rio de Janeiro, Av. 28 de Setembro, 87, Rio de Janeiro, RJ, Brasil, CEP 20551-030. Address e-mail to demoura{at}uerj.br


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The mechanism underlying the Niteroi, Rio de Janeiro sedative effect of clonidine, an {alpha}2-adrenoceptor agonist, remains uncertain. Because activation of {alpha}2-adrenoceptors induces release of nitric oxide (NO), we tested the hypothesis that the sedative effect of clonidine depends on NO-related mechanisms. The effect of 7-nitro indazole on the sleeping time induced by clonidine was studied in Wistar rats. In addition, we examined the effect of clonidine, {alpha}-methyldopa, and midazolam on the thiopental-induced sleeping time in rats pretreated with NG-nitro-L-arginine-methyl-esther (L-NAME). The sleeping time induced by clonidine was significantly decreased by 7-nitro indazole. Thiopental sleeping time was increased by clonidine, {alpha}-methyldopa, and midazolam. L-NAME reduced the prolongation effect of clonidine and {alpha}-methyldopa, but did not alter the effect of midazolam on the thiopental-induced sleeping time. The inhibitory effect of L-NAME on clonidine-dependent prolongation of thiopental-induced sleeping time was reversed by L-arginine. These results suggest that NO-dependent mechanisms are involved in the sedative effect of clonidine. In addition, this effect seems to be specific for the sedative action of {alpha}2-adrenoceptors agonists.

IMPLICATIONS: Clonidine, an antihypertensive drug, is also a sedative. This sedative effect, although an adverse event in the treatment of hypertensive patients, can be helpful for sedation of surgical patients. The mechanism of this effect, however, is unknown. In this study, we show that the sedative effect of clonidine is mediated by nitric oxide, because it could be prevented by pretreatment with nitric oxide synthase inhibitors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Activation of {alpha}2-adrenoceptors located in the central nervous system (1,2) by clonidine causes sympathoinhibition and decreases arterial pressure. However, clonidine also produces sedation (1–3) that limits its use in hypertension treatment. Additionally, {alpha}2-adrenoceptor agonists exhibit an antinociceptive effect (4–6). The antihypertensive (7), sedative (7–9), and antinociceptive (4,5,8) actions of clonidine are reduced by {alpha}2-adrenergic antagonists, suggesting that these effects may share common mechanisms. We have previously shown that the hypotensive effect of clonidine is reduced by inhibition of nitric oxide (NO) synthase (10,11). Therefore, in this study, we tested the hypothesis that NO synthase inhibition reduces the sedative effect of clonidine. To address this issue, we examined the effect of 7-nitro indazole (7-NI), a specific neuronal NO synthase antagonist (12), and of NG-nitro-L-arginine-methyl-esther (L-NAME), a nonspecific inhibitor of NO synthase (13), on the sedation produced by clonidine in Wistar rats. In addition, we studied the effect of L-NAME on the action of {alpha}-methyldopa and midazolam on thiopental-induced sleeping time.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Male Wistar rats (weighing 250–350 g) were housed in the Animal Care Facility under 27°C and maintained in a 12-h light/dark cycle (lights on at 7 AM). Rats were allowed free access to tap water and were fed regular rodent chow. Animal handling and experimental procedures were approved by the Ethical Committee for Use of Experimental Animals of the State University of Rio de Janeiro. Sedation was assessed by the sleeping time caused by IV injections of clonidine and by prolongation of thiopental-induced sleeping time after intraperitoneal (IP) injections of clonidine, {alpha}-methyldopa, or midazolam. The sleeping time was defined as the elapsed time between the disappearance and recovery of the righting reflex measured with a chronometer and expressed in minutes. For sleeping time measurements, rats were placed within an acrylic 40 x 30 cm box maintained in a quiet and illuminated room. For IV administration of clonidine, a heparinized catheter (500 IU/mL) was inserted into the jugular vein under ether anesthesia, 24 h before the experiments. For barbiturate-induced sleep, the animals received a single IP injection of thiopental sodium (30 mg/kg). Active drugs or vehicle were given in a volume of 1 mL/kg. All drugs, except 7-NI, were dissolved in saline and administrated 30 min before clonidine or thiopental injections. 7-NI was diluted in almond oil and administered IP 60 min before clonidine.

The following five experimental protocols were studied:

1. The sedative effect of clonidine: The sleeping time induced by IV administration of clonidine IV 80 (n = 6), 100 (n = 6), and 150 µg/kg (n = 7) was obtained after IP injection of saline.
2. The action of 7-NI on clonidine-induced sleeping time: The sleeping time produced by IV injection of clonidine, 100 and 150 µg/kg, was obtained in rats pretreated with 7-NI (15 mg/kg; n = 8) or vehicle (n = 6). The effect of 7-NI IP on the rat behavior during 2 h was studied in six animals.
3. The effect of clonidine on thiopental-induced sleeping time: The sleeping time induced by IP administration of thiopental (30 mg/kg) was obtained in rats pretreated with IP injection of saline or clonidine 10, 30, and 60 µg/kg (n = 6 for all groups).
4. The action of L-NAME on the potentiating effect of clonidine on the thiopental-induced sleeping time: The sleeping time induced by IP administration of thiopental (30 mg/kg) was obtained in rats pretreated with saline (n = 10), clonidine (60 µg/kg; n = 9) or clonidine (60 µg/kg) plus L-NAME (10 mg/kg; n = 12). The effect of L-NAME IP on the rat behavior during 2 h was studied in six animals. In this group, we also examined the effect of L-arginine on the L-NAME-induced response by treating rats with clonidine (60 µg/kg) plus L-NAME (10 mg/kg) plus L-arginine (200 mg/kg; n = 11).
5. The action of L-NAME on the potentiating effect of {alpha}-methyldopa and midazolam on the thiopental-induced sleeping time: The sleeping time induced by IP administration of thiopental (30 mg/kg) was obtained in rats pretreated with IP injection of saline (n = 6), {alpha}-methyldopa (50 mg/kg; n = 8), or {alpha}-methyldopa (50 mg/kg) plus L-NAME (10 mg/kg; n = 8). The sleeping time induced by thiopental was also obtained in rats pretreated with saline (n = 6), midazolam (5 mg/kg; n = 6), or midazolam (5 mg/kg) plus L-NAME (10 mg/kg; n = 5).

The results were reported as the mean ± SEM. One-way analysis of variance was used to compare the responses with different doses of drug tested. Significant differences between two groups were determined by Student’s t-test for unpaired samples. Statistical significance was set at P < 0.05.

Clonidine hydrochloride was a gift from Boehringer de Angeli Quimica e Farmaceutica, São Paulo, Brazil; thiopental sodium was purchased from Abbott Laboratorios do Brasil, São Paulo, Brazil; 7-NI, L-NAME, and L-arginine were purchased from Sigma, St. Louis, MO.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The IV administration of clonidine induced a dose-dependent sedative effect, as measured by the sleeping time (Fig. 1). Pretreatment with 7-NI did not induce loss of the righting reflex, but substantially reduced the sleep duration induced by clonidine 100 and 150 µ/kg (Fig. 2). In addition, clonidine 30 and 60 µg/kg significantly (P < 0.001) increased thiopental-induced sleeping time as compared with saline-treated rats (Figs. 3 and 4). Pretreatment with L-NAME alone did not induce loss of righting reflex, but the thiopental sleep duration in rats pretreated with clonidine plus L-NAME was significantly shorter than that of rats treated with clonidine alone (Fig. 4). Furthermore, the inhibitory effect of L-NAME on the potentiation effect of clonidine on thiopental-induced sleeping time was reversed by L-arginine treatment (Fig. 4).



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  		Figure 1. Sleeping time induced by IV injections of clonidine. P < 0.01 (analysis of variance).

 


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  		Figure 2. The effect of 7-nitro indazole (7-NI) (15 mg/kg IP) on the sleeping time induced by IV injections of clonidine (CLO). *P < 0.001 versus CLO alone.

 


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  		Figure 3. The effect of saline (S) and clonidine intraperitoneally (IP) on the sleeping-time duration induced by 30 mg/kg thiopental IP *P < 0.001 versus saline; P < 0.01 for clonidine treatment (analysis of variance).

 


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  		Figure 4. The effect of NG-nitro-L-arginine-methyl-esther (L-NAME) (10 mg/kg) on the potentiation induced by clonidine (CLO) (60 µ/kg) on thiopental sleeping time and reversal of the inhibitory effect of L-NAME by L-arginine (L-ARG) (200 mg/kg). *P < 0.001 versus saline (S); **P < 0.001 versus clonidine alone; {phi}P < 0.001 versus clonidine plus L-NAME.

 
Also, the sleep duration of thiopental in rats pretreated with saline was significantly increased by {alpha}-methyldopa (Fig. 5). The potentiation effect of {alpha}-methyldopa on thiopental-induced sleeping time was significantly (P < 0.001) inhibited as compared with rats pretreated with {alpha}-methyldopa plus L-NAME (Fig. 5).



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  		Figure 5. The Effect of NG-nitro-L-arginine-methyl-esther (L-NAME) (10 mg/kg) on the potentiation induced by {alpha}-methyldopa ({alpha}-MDOPA) (50 mg/kg) and midazolam (5 mg/kg) on thiopental sleeping time. *P < 0.001 versus saline (S); **P < 0.001 versus {alpha}-MDOPA alone.

 
Finally, midazolam (5 mg/kg) prolonged the thiopental sleeping time as compared with saline pretreated rats (P < 0.001) (Fig. 5). However, L-NAME pretreatment did not inhibit midazolam-dependent potentiation of thiopental sleeping time (midazolam alone versus midazolam plus L-NAME; P = 0.1) (Fig. 5).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results confirm the observations of Mason and Angel (5) that {alpha}2-adrenoceptor activation prolongs barbiturate-induced sleeping time. In addition, we have shown that the IV administration of clonidine produces dose-dependent sedation. Most importantly, we have demonstrated that both the sleep duration after IV injections of clonidine and the clonidine-dependent prolongation of thiopental-induced sleeping time were substantially reduced by NO synthase inhibitors. In contrast, midazolam-dependent prolongation of the sleep induced by thiopental was not altered by NO synthase inhibition.

NO may be a modulator of the sedative effect of clonidine, because it is an important modulator of neuronal function (14). In addition, it may be a neurotransmitter involved in consciousness, analgesia, and anesthesia (15). The mechanisms by which clonidine modulates the NO-dependent pathway in the central nervous system are not well established but probably involve activation of {alpha}2-adrenoceptors.

The sedative effect of clonidine seems to be dependent on the activation of {alpha}2-adrenoceptors in the locus ceruleus where it reduces sympathetic outflow (2,3). Because clonidine releases NO by activation of endothelial {alpha}2-adrenoceptors (16), and also induces NO release in the spinal cord (17), we hypothesized that NO may modulate the sedative effect of clonidine in the central nervous system. This hypothesis is corroborated by the findings of Lothe et al. (18) who showed that the analgesic effect of intrathecal administration of clonidine was reduced by NO synthase inhibition.

Our present results add experimental evidence that NO may be a modulator of the sedative effect of {alpha}2-adrenoceptor agonists, because we showed for the first time that the sedative effect of clonidine and {alpha}-methyldopa was significantly decreased by 7-NI and L-NAME. The involvement of NO is further supported because the inhibitory effect of L-NAME was reversed by L-arginine. The systemic vasoconstrictor effect of L-NAME could alter the pharmacokinetics of {alpha}2-adrenoceptor agonists and, consequently, their sedative effect. However, it seems unlikely that the vascular effect of L-NAME could alter {alpha}2-adrenoceptor agonists’ sedative responses because the administration of 7-NI that presumably lacks vascular actions (12), elicited similar inhibitory responses on clonidine-dependent sedation as L-NAME treatment. Most importantly, L-NAME did not change the prolongation effect of midazolam on thiopental-induced sleeping time.

The participation of NO-cyclic guanosine 3',5'-monophosphate (cGMP) systems on the mechanism of the sedative effect of clonidine has to be considered, because {alpha}2-adrenoceptor agonists reduce cGMP concentration on mice cerebellum, cerebral cortex, hippocampus, caudate nucleus (19), and in the rat locus ceruleus (20). Contrasting with clonidine, L-NAME did not induce sedation despite a reduced cGMP in locus ceruleus (20). Therefore, the reduction of cGMP may not play an important role in the sedative effect of clonidine. This conclusion is further supported by our results showing that L-NAME and 7-NI did not induce loss of righting reflex despite a presumed reduction in central nervous system cGMP.

The inhibitory action of the NO synthase antagonists on the sedative effect of clonidine does not seem to be a direct effect of L-NAME or 7-NI, because these two compounds did not change the normal behavior of the rat. L-NAME could also act nonspecifically to inhibit the sedative effect of clonidine. However, this possibility seems improbable because L-NAME did not alter the potentiating effect of midazolam on the thiopental sleeping time. This result also suggests that clonidine and {alpha}-methyldopa prolongation of barbiturate-induced sleep is specific for {alpha}2-adrenoceptor activators.

In conclusion, the present results suggest that the sedative effect of {alpha}2-adrenoceptor agonists is probably caused by activation of a nitrergic mechanism because the loss of righting reflex induced by IV injections of clonidine is inhibited by 7-NI and the {alpha}2-adrenoceptor agonists-dependent potentiation of barbiturate-induced sleeping time is reduced by L-NAME. The involvement of NO in the sedative effect of {alpha}2-adrenoceptor agonists is further supported by the findings that L-NAME does not change the potentiation effect of midazolam on the thiopental-induced sleeping time and that L-arginine prevents the inhibitory effect of L-NAME on the clonidine potentiation of the barbiturate-dependent sleeping time.


   Acknowledgments
 
Supported in part by CNPq and FAPERJ.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Schmitt H. The pharmacology of clonidine and related products. In: Gross F, ed. Antihypertensive agents. Berlin: Springer-Verlag, 1977: 299.
  2. Kobinger W. Central alpha-adrenergic systems as targets for hypotensive drugs. Rev Physiol Biochem Pharmacol 1978; 81: 39–100.[ISI][Medline]
  3. Drew GM, Gower AJ, Marriott AS. Alpha 2-adrenoceptors mediate clonidine-induced sedation in the rat. Br J Pharmacol 1979; 67: 133–41.[ISI][Medline]
  4. Yaksh TL, Reddy SV. Studies in the primate on the analgesic effects associated with intrathecal actions of opiates, alpha-adrenergic agonists and baclofen. Anesthesiology 1981; 54: 451–67.[ISI][Medline]
  5. Mason ST, Angel A. Anaesthesia: the role of adrenergic mechanisms. Eur J Pharmacol 1983; 91: 29–39.[ISI][Medline]
  6. van Giersbergen PL, van Duinkerken E, Sweep CG, et al. {alpha}-Methyldopa induces a naltrexone-insensitive antinociception and hypomotility in rats. Br J Pharmacol 1990; 99: 467–72.[ISI][Medline]
  7. Timmermans PB, Schoop AM, Kwa HY, Van Zwieten PA. Characterization of alpha-adrenoceptors participating in the central hypotensive and sedative effects of clonidine using yohimbine, rauwolscine and corynanthine. Eur J Pharmacol 1981; 70: 7–15.[ISI][Medline]
  8. Maze M, Tranquilli W. Alpha-2 adrenoceptor agonists: defining the role in clinical anesthesia. Anesthesiology 1991; 74: 581–605.[ISI][Medline]
  9. Delbarre B, Schmitt H. Sedative effects of alpha-sympathomimetic drugs and their antagonism by adrenergic and cholinergic blocking drugs. Eur J Pharmacol 1971; 13: 356–63.[ISI][Medline]
  10. Soares de Moura R, Cardoso Leão M. Involvement of endothelium on the anti-hypertensive effect of clonidine. J Vasc Res 1997; 34: 38.
  11. Soares de Moura R, Leão CM, Castro Resende A, et al. Actions of L-NAME and methylene blue on the hypotensive effects of clonidine and rilmenidine in the anesthetized rat. J Cardiovasc Pharmacol 2000; 35: 791–5.[ISI][Medline]
  12. Moore PK, Wallance P, Gaffen Z, et al. Characterization of the novel nitric oxide synthase inhibitor 7-nitro indazole and related indazoles: antinociceptive and cardiovascular effects. Br J Pharmacol 1993; 110: 219–24.[ISI][Medline]
  13. Traystman RJ, Moore LE, Helfaer MA, et al. Nitro-L-arginine analogues: dose- and time-related nitric oxide synthase inhibition in brain. Stroke 1995; 26: 864–9.[Abstract/Free Full Text]
  14. Garthwhaite J, Boulton CL. Nitric oxide signaling in the central nervous system. Annu Rev Physiol 1995; 57: 683–706.[ISI][Medline]
  15. Tobin JR, Martin LD, Breslow MJ, Traystmans RJ. Selective anesthetic inhibition of brain nitric oxide synthase. Anesthesiology 1994; 81: 1264–9.[ISI][Medline]
  16. Miller VM, Vanhoutte PM. Endothelial {alpha}2-adrenoceptor in canine pulmonary and systemic blood vessels. Eur J Pharmacol 1985; 118: 123–9.[ISI][Medline]
  17. Xu Z, Chen SR, Eisenach C, et al. Role of spinal muscarinic and nicotinic receptors in clonidine-induced nitric oxide release in a rat model of neuropathic pain. Brain Res 2000; 861: 390–8.[ISI][Medline]
  18. Lothe A, Li P, Tong C, et al. Spinal cholinergic {alpha}2-adrenergic interactions in analgesia and hemodynamic control: role of muscarinic receptors subtypes and nitric oxide. J Pharmacol Exp Ther 1994; 270: 1301–6.[Abstract/Free Full Text]
  19. Vulliemoz Y, Shen H, Virag L. Alpha-2 adrenoceptor agonist decreased cyclic guanosine 3'5'-monophosphate in the mouse brain. Anesthesiology 1996; 85: 544–50.[ISI][Medline]
  20. Vulliemoz Y, Whittington RA, Virag L. The nitric oxide-cGMP system of the locus coeruleus and the hypnotic action of alpha-2 adrenergic agonists. Brain Res 1999; 849: 169–74.[ISI][Medline]
Accepted for publication June 12, 2001.





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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press