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Involvement of neuronal nitric oxide synthase in restraint stress-induced fever in rats
Physiology & Behavior 75 (2002) 261 – 266
Involvement of neuronal nitric oxide synthase in restraint stress-induced fever in rats Daniela B. Sanchesa, Alexandre A. Steinerb, Luiz G.S. Brancoa,* a
Department of Morphology, Estomatology, and Physiology, Dental School of Ribeira˜o Preto, University of Sa˜o Paulo, 14040-904 Ribeira˜o Preto, SP, Brazil b Department of Physiology, Faculty of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo, 14040-904 Ribeira˜o Preto, SP, Brazil Received 30 August 2001; received in revised form 15 October 2001; accepted 9 November 2001
Abstract Nitric oxide (NO) has been shown to be an important modulator of the febrile response to pyrogens and to psychological stress. In the present study, we aimed to identify the nitric oxide synthase (NOS) isoform (neuronal or inducible, nNOS and iNOS, respectively) involved in restraint stress fever. Colonic temperature (Tc) was measured in unanesthetized rats before and after treatment with the more selective nNOS inhibitor 7-nitroindazole or with the selective iNOS inhibitor aminoguanidine (AG) under unrestrained or restrained conditions. Intraperitoneal injection of AG (25 or 50 mg/kg) did not affect restraint fever, indicating that iNOS is unlikely to be involved in restraint fever. On the other hand, intraperitoneal injection of 7-nitroindazole (25 mg/kg) significantly attenuated the rise in the Tc caused by restraint stress, whereas it caused no change in Tc of euthermic animals. These data show that NO produced by nNOS plays an important role in the genesis of restraint stress-induced fever. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Body temperature; Nitric oxide; Aminoguanidine; 7-Nitroindazole; Thermoregulation; Psychological stress; Hyperthermia; Fever
1. Introduction It is now widely accepted that the febrile rise in body core temperature (Tc) in response to infection is the result of an elevated thermoregulatory set point. This response seems not to be mediated directly by exogenous pyrogens, such as endotoxin, viruses, and Gram-positive bacteria, but rather by endogenously produced protein mediators (endogenous pyrogens) such as interleukin (IL)-1b, IL-6, interferons, and tumor necrosis factor [18], and by mediators of lipid origin such as prostaglandins [3,26]. Among these mediators, prostaglandin E2 is thought to be the most proximal mediator of fever, acting directly on the preoptic region to raise the thermoregulatory set point [3,26]. Several reports have shown that acute exposure to psychological stress, including handling [5,6], cage switching [32], exposure to an open field [20,22], and restraint [10,23,47], increases Tc in a wide variety of * Corresponding author. Faculdade de Odontologia de Ribeira˜o Preto, Universidade de Sa˜o Paulo, 14040-904 Ribeira˜o Preto, SP, Brazil. Tel.: +55-16-602-4501; fax: +55-16-633-0999. E-mail address: [email protected] (L.G.S. Branco).
animals. For many years, this rise was thought to be simply hyperthermia, but recent evidence has suggested that it may be a true fever, i.e., a regulated rise in Tc, sharing similar mechanisms with endotoxin fever. In support, it has been observed that: (1) the vasomotor responses correlate with the rise in Tc during stress, which maintains the same magnitude independently of ambient temperature, suggesting a shift in the thermoregulatory set point to a higher level [5]; (2) lizards, whose thermoregulation is primarily behavioral and therefore is related to changes in the thermoregulatory set point, choose a higher preferred Tc in a thermal gradient after handling [6]; (3) the levels of IL-6 and IL-1b, which are putative mediators of fever [18], rise during psychological stress in an open field [17,20,27]; (4) injections of anti-tumor necrosis factor-a antiserum, which enhances LPS fever [21], also magnifies stress fever [22]; (5) pretreatment of animals with nonsteroidal antipyretic drugs, which inhibit the enzyme cyclooxygenase and therefore prevents a rise in prostaglandin E2 levels, attenuates stress fever, at least in rats [19,32,43]; and (6) intracerebroventricular administration of arginine– vasopressin, which has been recognized as an important
0031-9384/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 3 1 - 9 3 8 4 ( 0 1 ) 0 0 6 5 7 - 6
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antipyretic molecule during LPS fever, also attenuates restraint stress fever [47]. Nitric oxide (NO), a diffusible free radical gas, has been shown to have signaling properties [9,29], many of which are the result of activation of soluble guanylate cyclase and the consequent increase in intracellular cyclic GMP levels [33]. Endogenous NO is produced by the conversion of L-arginine yielding L-citrulline and NO, a reaction catalyzed by the enzyme nitric oxide synthase (NOS). Three NOS isoforms encoded by different genes have been found to date. Among them, neuronal NOS (nNOS) and endothelial NOS (eNOS) are constitutively expressed, whereas inducible NOS (iNOS) is usually not expressed under basal condition but may be overexpressed in response to a series of stimuli, including immune challenge [9,29]. Although NO was first reported as a messenger molecule in the cardiovascular system [12,14,35], it has also been shown to be involved in other physiological and pathophysiological processes, including thermoregulation and fever (for a review, see Ref. [44]). In this regard, systemic administration of nonselective NOS inhibitors has been reported to reduce fever induced by endotoxin in rats and guinea pigs [16,38,41], by muramyl dipeptide in guinea pigs [16], by yeast in rats [2], and by IL-1 in rats [37,39]. Interestingly, both endotoxin and IL-1 fever seem to be dependent on constitutive NOS since systemic treatment with selective iNOS blockers does not affect IL-1 fever in rats [37] or endotoxin fever in guinea pigs [40]. Conversely, muramyl dipeptide fever has been shown to be impaired by systemic aminoguanidine (AG), a selective iNOS inhibitor [16]. As to psychological stress-induced fever, a recent study from our laboratory has demonstrated that intravenous administration of the nonselective NOS inhibitor L-NAME significantly impairs restraint fever in rats [10]. In agreement, Soszynski [46] reported that systemically administered L-NAME also abrogates the rise in Tc evoked by exposure to an open field. Taken together, these data indicate that, as observed in experimental models of infectious fever, the NO pathway plays a major role in psychological stress-induced fever. However, the NOS isoform involved remains unknown. Therefore, the aim of the present study was to identify the NOS isoform involved in stress-induced fever. To this end, we used the more selective nNOS inhibitor 7-nitroindazole [30,31] and the selective iNOS inhibitor AG [8,28].
2. Materials and methods 2.1. Animals Experiments were performed on adult male Wistar rats weighing 200 – 250 g, housed at regulated temperature (25.0 ± 1.0 C) on a 12:12-h light/dark cycle, with lights on at 6:00 a.m. The animals had free access to food and drinking water. In order to obviate possible changes in Tc
due to circadian variations, all experiments were started at 9:00 a.m. 2.2. Drugs The iNOS selective inhibitor AG was obtained from Sigma (St. Louis, MO, USA) and dissolved in pyrogen-free sterile saline. The nNOS selective inhibitor 7-nitroindazole was obtained from Sigma and dissolved in a vehicle consisting of sesame oil/dimethyl sulfoxide (9:1). 2.3. Measurement of Tc Rats were housed in a plastic chamber for at least 24 h before control Tc was measured by inserting a thermocouple (Cole Palmer, model 8502-10, Chicago, IL, USA) 40 mm into the colon each time Tc was to be measured. For all protocols, 2 days before the experiment, the animals were trained for Tc measurements that were performed quickly to avoid any handling-induced elevation in Tc. Rats were trained about 10 times to the measurement procedure each day. In the first day of training, the rats presented stressassociated rises in Tc after insertion of the thermoprobe, but this response disappeared after training. The rats were housed singly in their cages in the 2 days preceding the experiments and during the experiments. 2.3.1. Protocol 1: Determination of the effect of AG on Tc Rats received an intraperitoneal injection of AG (25 or 50 mg/kg) or pyrogen-free saline and Tc was measured before and at 15, 30, 40, 50, 60, 70, 85, 100, and 115 min after the injection. The injected volume was 0.25 ml. This dose of AG was chosen on the basis of a previous study [16]. 2.3.2. Protocol 2: Determination of the effect of AG on restraint stress-induced fever After basal Tc was measured, pyrogen-free saline or AG (25 and 50 mg/kg) was injected intraperitoneally and 30 min later the animals were subjected to restraint. The animals were stressed by being subjected to 40 min of restraint in a standard Plexiglas tube (160 mm in length and 55 mm in diameter) with perforated sides to allow dissipation of body heat. During the period the animals were inside the restraint cage, the thermoprobe was kept inside the colon and fixed to the tail with adhesive tape. This procedure was chosen because it was impossible to insert the thermoprobe intermittently into the colon while the animals were kept into the restrain tube. Immediately after, the animals were returned to their cages and Tc was measured every 15 min for a total period of 45 min. This experimental protocol was based on a previous study [10]. 2.3.3. Protocol 3: Determination of the effect of 7-nitroindazole on Tc Rats received 7-nitroindazole (25 mg/kg) or its vehicle intraperitoneally and Tc was measured as in Protocol 1. The
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volume of all injections was 0.4 ml. This dose of 7-nitroindazole was chosen on the basis of a previous study [45]. 2.3.4. Protocol 4: Determination of the effect of 7-nitroindazole on restraint stress-induced fever After basal Tc was measured, rats received an intraperitoneal injection of 7-nitroindazole (25 mg/kg) or its vehicle. Thirty minutes later, the animals were subjected to restraint and Tc was determined as in Protocol 2. 2.4. Statistical analysis All values are reported as means ± S.E. The values of Tc are the changes from the initial values (initial Tc, Tci). The Tci for each group was determined as the mean of three Tc recorded at 15-min intervals preceding all treat-
Fig. 2. Effects of 7-nitroindazole (7-NI, 25 mg/kg ip) or its vehicle (sesame oil/dimethyl sulfoxide 9:1, ip) on Tc of unrestrained (A) or restrained (B) rats. The data are expressed as changes in Tc (DTc) relative to their initial levels (Tci) (see text for details). The arrowhead indicates the time of injections and the horizontal bar indicates the period when the animals were restrained. The values are means ± S.E.; n = number of animals. * P < .05 compared to basal Tc (time zero); + P < .05 compared to the data obtained by treatment with vehicle.
ments. Two-way ANOVA followed by the Tukey– Kramer multiple comparisons test or the Student t test was used for data analysis. Values of P < .05 were considered to be significantly different.
3. Results In all experimental protocols, Tc ranged from 36.5 to 37.3 C during the control period and the baseline values did not differ significantly from the vehicle group in any experiment. Room temperature was kept at 25.0 ± 1.0 C during the experiments. Fig. 1. Effects of AG (AG, 25 or 50 mg/kg ip) or its vehicle (intraperitoneally administered pyrogen-free saline) on Tc of unrestrained (A) or restrained (B) rats. The data are expressed as changes in Tc (DTc) relative to their initial levels (Tci) (see text for details). The arrowhead indicates the time of injections and the horizontal bar indicates the period when the animals were restrained. The values are means ± S.E.; n = number of animals. * P < .05 compared to basal Tc (time zero) and applies for all the curves.
3.1. Experiment 1: Effect of intraperitoneal AG on Tc When vehicle or AG at 25 and 50 mg/kg was injected intraperitoneally, no significant change in Tc was observed. These data are plotted in Fig. 1A.
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3.2. Experiment 2: Effect of intraperitoneal AG on restraint stress-induced fever All restrained animals presented a significant ( P < .05) increase in Tc, which was already evident 10 min after the beginning of stress and remained elevated until the animals were released from restraint. Immediately after the animals were released from restraint, Tc started to return towards baseline control values. Neither intraperitoneal saline nor AG at both doses (25 and 50 mg/kg) affected restraint-induced fever. These data are plotted in Fig. 1B. 3.3. Experiment 3: Effect of intraperitoneal 7-nitroindazole on Tc No significant change in Tc was observed after intraperitoneal injection of 7-nitroindazole (25 mg/kg) or its vehicle. These data are depicted in Fig. 2A. 3.4. Experiment 4: Effect of intraperitoneal 7-nitroindazole on restraint stress-induced fever When animals pretreated intraperitoneally with the 7-nitroindazole vehicle were restrained (Fig. 4), their Tc rose similarly to that of noninjected rats subjected to restraint (Fig. 1B). However, intraperitoneal treatment with 7-nitroindazole at dose of 25 mg/kg significantly attenuated ( P < .005) the rise in Tc evoked by restraint compared to the group treated with vehicle. This attenuation was already evident 10 min after the animals were placed in the restraint cages. Fig. 2B illustrates these results.
4. Discussion The results of the present study show that nNOS plays a major role in restraint stress-induced fever since intraperitoneal administration of 7-nitroindazole, a more selective nNOS inhibitor, significantly attenuated the rise in Tc caused by restraint stress (Fig. 2). On the other hand, the selective iNOS inhibitor AG did not affect the Tc of euthermic animals or restraint-induced fever (Fig. 1). The NOS enzymes are widely distributed throughout the body [9,29]. Since NOS is encountered in various tissues involved in the regulation of Tc [44,45] and of the immune system [42], it seems probable that this gas influences thermoregulation and fever. To date, most of the data on the role of NO in fever were obtained in experimental models of infectious fever, such as endotoxin, muramyl dipeptide, and yeast (for a review, see Ref. [44]). In this context, systemic administration of NOS inhibitors has been reported to impair fever produced by endotoxin in rats and guinea pigs [16,38,41], by muramyl dipeptide in guinea pigs [16], and by yeast [2] and IL-1 [37,39] in rats. On the other hand, intracerebroventricular treatment
with NOS inhibitors has been shown to enhance endotoxin fever in rats [1] and rabbits [13], indicating that the feverinhibiting effect of systemically administered NOS inhibitors is due to a peripheral action of these drugs. Taken together, these data support the notion that NO plays differential thermoregulatory effects by acting at the periphery and in the central nervous system, with peripheral NO being a pyretic molecule and central NO an antipyretic molecule. Accordingly, recent studies reported that systemically administered L-NAME (a nonselective NOS inhibitor) also impairs restraint- and open field-induced fever [10,46], whereas intracerebroventricular L-NAME enhances the rise in Tc evoked by restraint [10]. Since in the present experiments we used systemic injections of selective NOS inhibitors in order to identify the NOS isoform involved in psychological stress fever, it is important to point out that the results obtained are related to the peripheral pyretic action of NO. AG, a molecule containing the guanidino group of L-arginine linked to hydrazine, is at least as equipotent as L-NMMA (a nonselective NOS inhibitor) to inhibit iNOS [8,28], but lacks activity against nNOS and eNOS. Thus, we used AG in order to verify if iNOS is involved in restraint fever in rats. It was observed that intraperitoneal AG at the doses of 25 and 50 mg/kg did not affect the Tc of euthermic animals (Fig. 1), a result that is in agreement with previous studies [40]. Moreover, intraperitoneal AG at 50 mg/kg caused no change in the course of restraint-induced fever (Fig. 1B). Considering that the overexpression of iNOS mRNA requires 3 –6 h to be induced [34] and that restraint fever was already evident 10 min after the beginning of the stimulus, this result is not surprising. In agreement, AG has been reported to have no effect on endotoxin fever in guinea pigs [40] and to have little effect on IL-1 induced fever in rats [37], fever that have onset latencies no longer than 1 h under the experimental conditions used. In contrast, AG seems to impair muramyl dipeptide-induced fever [16], although this fever takes about 1 h to start. Since our data indicate that iNOS is not the NOS isoform involved in restraint fever in rats, we hypothesised that nNOS is involved in restraint stress-induced fever. We then used 7-nitroindazole, which has been shown to be a more selective inhibitor of nNOS [24,30,31]. In agreement, we [45] and others [30,31] have reported that intraperitoneal 7-nitroindazole does not affect arterial blood pressure, a response that has been attributed to a reduction in the activity of eNOS [11,29]. In previous studies that investigated the thermoregulatory effects of nNOS, it was observed that 7-nitroindazole at the dose of 30 mg/kg causes a significant drop in the Tc of euthermic animals, probably by reducing thermogenesis [36,45]. This response certainly represents a limitation to the study of the participation of nNOS in fever. Thus, it would be necessary to use a dose of 7-nitroindazole without effect on basal Tc. Since we [45] have demonstrated that 7-nitroindazole at
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the lower dose of 25 mg/kg does not affect the Tc of euthermic rats but is able to attenuate the drop in Tc evoked by hypoxia, we have chosen to use this dose to study the role of nNOS in restraint fever. In agreement, Fig. 2A shows that under the present experimental conditions, intraperitoneal 7-nitroindazole at 25 mg/kg caused no change in the Tc of euthermic rats. Interestingly, intraperitoneal 7-nitroindazole at 25 mg/kg significantly attenuated the rise in Tc elicited by restraint stress (Fig. 2B), indicating that the nNOS isoform plays an important role in the development of restraint stressinduced fever. Similarly, 7-nitroindazole also seems to reduce the rise in Tc induced by peripherally administered endotoxin [36], supporting the notion that the fever evoked by psychological stress and immune challenge share similar mechanisms. It should be emphasised that the effect of intraperitoneal 7-nitroindazole on restraint fever reported in the present study was very similar to that observed for intravenous L-NAME in a previous study from our laboratory [10]. Therefore, it is tempting to suggest that nNOS may be the only NOS isoform that accounts for the pyretic effect of NO in psychological stress-induced fever. However, a possible participation of eNOS cannot be excluded. To our knowledge, no eNOS selective inhibitor is commercially available to date. The use of NOS knockout animals could be a useful tool to solve this issue. Although evidence from studies using NOS inhibitors supports the notion that peripheral NO seems to be a pyretic molecule during infectious and psychological stress fever in rats [44], the mechanism by which NO acts on stressinduced fever is still unknown. It seems that systemic administration of L-NAME impairs endotoxin fever without affecting the cytokine (tumor necrosis factor-a and IL-6) network in guinea pigs [38]. Whether the same is true for stress fever remains to be determined. Since NO acts as a neuromodulator [9], it could affect the discharge of vagal afferents, which have been shown to play a major role in endotoxin fever [4]. But this is unlikely to evoke stress fever since a recent study has demonstrated that the vagus nerve is not involved in this response [7]. Moreover, systemic administration of L-NAME might also attenuate stress fever by increasing the levels of ACTH and corticosterone, which have been reported to be important antipyretic molecules. In fact, L-NAME seems to enhance the rise in ACTH induced by the inflammatory agent turpentine [48], but not by IL-1b [37]. Furthermore, a recent study has demonstrated that the antipyretic effect of systemically administered L-NAME is related to impairment in metabolic rate [25], a fact that seems to be associated with reduced thermogenesis [15]. However, whether this phenomenon is mediated directly by the action of L-NAME on thermogenic tissues or is allied to an attenuation of the actions and release of febrile mediators by L-NAME remains to be established. In summary, the present data indicate that NO produced by nNOS mediates restraint stress-induced fever in rats. Similarly, peripheral NO also seems to be a pyretic molecule
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in endotoxin fever, being produced by the constitutive NOS isoforms, probably nNOS (for a review, see Ref. [44]).
Acknowledgments This work was supported by the Fundac˛a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq). A.A. Steiner and D.B. Sanches were recipients of FAPESP fellowships.
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Involvement of neuronal nitric oxide synthase in restraint stress-induced fever in rats Daniela B. Sanchesa, Alexandre A. Steinerb, Luiz G.S. Brancoa,* a
Department of Morphology, Estomatology, and Physiology, Dental School of Ribeira˜o Preto, University of Sa˜o Paulo, 14040-904 Ribeira˜o Preto, SP, Brazil b Department of Physiology, Faculty of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo, 14040-904 Ribeira˜o Preto, SP, Brazil Received 30 August 2001; received in revised form 15 October 2001; accepted 9 November 2001
Abstract Nitric oxide (NO) has been shown to be an important modulator of the febrile response to pyrogens and to psychological stress. In the present study, we aimed to identify the nitric oxide synthase (NOS) isoform (neuronal or inducible, nNOS and iNOS, respectively) involved in restraint stress fever. Colonic temperature (Tc) was measured in unanesthetized rats before and after treatment with the more selective nNOS inhibitor 7-nitroindazole or with the selective iNOS inhibitor aminoguanidine (AG) under unrestrained or restrained conditions. Intraperitoneal injection of AG (25 or 50 mg/kg) did not affect restraint fever, indicating that iNOS is unlikely to be involved in restraint fever. On the other hand, intraperitoneal injection of 7-nitroindazole (25 mg/kg) significantly attenuated the rise in the Tc caused by restraint stress, whereas it caused no change in Tc of euthermic animals. These data show that NO produced by nNOS plays an important role in the genesis of restraint stress-induced fever. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Body temperature; Nitric oxide; Aminoguanidine; 7-Nitroindazole; Thermoregulation; Psychological stress; Hyperthermia; Fever
1. Introduction It is now widely accepted that the febrile rise in body core temperature (Tc) in response to infection is the result of an elevated thermoregulatory set point. This response seems not to be mediated directly by exogenous pyrogens, such as endotoxin, viruses, and Gram-positive bacteria, but rather by endogenously produced protein mediators (endogenous pyrogens) such as interleukin (IL)-1b, IL-6, interferons, and tumor necrosis factor [18], and by mediators of lipid origin such as prostaglandins [3,26]. Among these mediators, prostaglandin E2 is thought to be the most proximal mediator of fever, acting directly on the preoptic region to raise the thermoregulatory set point [3,26]. Several reports have shown that acute exposure to psychological stress, including handling [5,6], cage switching [32], exposure to an open field [20,22], and restraint [10,23,47], increases Tc in a wide variety of * Corresponding author. Faculdade de Odontologia de Ribeira˜o Preto, Universidade de Sa˜o Paulo, 14040-904 Ribeira˜o Preto, SP, Brazil. Tel.: +55-16-602-4501; fax: +55-16-633-0999. E-mail address: [email protected] (L.G.S. Branco).
animals. For many years, this rise was thought to be simply hyperthermia, but recent evidence has suggested that it may be a true fever, i.e., a regulated rise in Tc, sharing similar mechanisms with endotoxin fever. In support, it has been observed that: (1) the vasomotor responses correlate with the rise in Tc during stress, which maintains the same magnitude independently of ambient temperature, suggesting a shift in the thermoregulatory set point to a higher level [5]; (2) lizards, whose thermoregulation is primarily behavioral and therefore is related to changes in the thermoregulatory set point, choose a higher preferred Tc in a thermal gradient after handling [6]; (3) the levels of IL-6 and IL-1b, which are putative mediators of fever [18], rise during psychological stress in an open field [17,20,27]; (4) injections of anti-tumor necrosis factor-a antiserum, which enhances LPS fever [21], also magnifies stress fever [22]; (5) pretreatment of animals with nonsteroidal antipyretic drugs, which inhibit the enzyme cyclooxygenase and therefore prevents a rise in prostaglandin E2 levels, attenuates stress fever, at least in rats [19,32,43]; and (6) intracerebroventricular administration of arginine– vasopressin, which has been recognized as an important
0031-9384/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 3 1 - 9 3 8 4 ( 0 1 ) 0 0 6 5 7 - 6
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antipyretic molecule during LPS fever, also attenuates restraint stress fever [47]. Nitric oxide (NO), a diffusible free radical gas, has been shown to have signaling properties [9,29], many of which are the result of activation of soluble guanylate cyclase and the consequent increase in intracellular cyclic GMP levels [33]. Endogenous NO is produced by the conversion of L-arginine yielding L-citrulline and NO, a reaction catalyzed by the enzyme nitric oxide synthase (NOS). Three NOS isoforms encoded by different genes have been found to date. Among them, neuronal NOS (nNOS) and endothelial NOS (eNOS) are constitutively expressed, whereas inducible NOS (iNOS) is usually not expressed under basal condition but may be overexpressed in response to a series of stimuli, including immune challenge [9,29]. Although NO was first reported as a messenger molecule in the cardiovascular system [12,14,35], it has also been shown to be involved in other physiological and pathophysiological processes, including thermoregulation and fever (for a review, see Ref. [44]). In this regard, systemic administration of nonselective NOS inhibitors has been reported to reduce fever induced by endotoxin in rats and guinea pigs [16,38,41], by muramyl dipeptide in guinea pigs [16], by yeast in rats [2], and by IL-1 in rats [37,39]. Interestingly, both endotoxin and IL-1 fever seem to be dependent on constitutive NOS since systemic treatment with selective iNOS blockers does not affect IL-1 fever in rats [37] or endotoxin fever in guinea pigs [40]. Conversely, muramyl dipeptide fever has been shown to be impaired by systemic aminoguanidine (AG), a selective iNOS inhibitor [16]. As to psychological stress-induced fever, a recent study from our laboratory has demonstrated that intravenous administration of the nonselective NOS inhibitor L-NAME significantly impairs restraint fever in rats [10]. In agreement, Soszynski [46] reported that systemically administered L-NAME also abrogates the rise in Tc evoked by exposure to an open field. Taken together, these data indicate that, as observed in experimental models of infectious fever, the NO pathway plays a major role in psychological stress-induced fever. However, the NOS isoform involved remains unknown. Therefore, the aim of the present study was to identify the NOS isoform involved in stress-induced fever. To this end, we used the more selective nNOS inhibitor 7-nitroindazole [30,31] and the selective iNOS inhibitor AG [8,28].
2. Materials and methods 2.1. Animals Experiments were performed on adult male Wistar rats weighing 200 – 250 g, housed at regulated temperature (25.0 ± 1.0 C) on a 12:12-h light/dark cycle, with lights on at 6:00 a.m. The animals had free access to food and drinking water. In order to obviate possible changes in Tc
due to circadian variations, all experiments were started at 9:00 a.m. 2.2. Drugs The iNOS selective inhibitor AG was obtained from Sigma (St. Louis, MO, USA) and dissolved in pyrogen-free sterile saline. The nNOS selective inhibitor 7-nitroindazole was obtained from Sigma and dissolved in a vehicle consisting of sesame oil/dimethyl sulfoxide (9:1). 2.3. Measurement of Tc Rats were housed in a plastic chamber for at least 24 h before control Tc was measured by inserting a thermocouple (Cole Palmer, model 8502-10, Chicago, IL, USA) 40 mm into the colon each time Tc was to be measured. For all protocols, 2 days before the experiment, the animals were trained for Tc measurements that were performed quickly to avoid any handling-induced elevation in Tc. Rats were trained about 10 times to the measurement procedure each day. In the first day of training, the rats presented stressassociated rises in Tc after insertion of the thermoprobe, but this response disappeared after training. The rats were housed singly in their cages in the 2 days preceding the experiments and during the experiments. 2.3.1. Protocol 1: Determination of the effect of AG on Tc Rats received an intraperitoneal injection of AG (25 or 50 mg/kg) or pyrogen-free saline and Tc was measured before and at 15, 30, 40, 50, 60, 70, 85, 100, and 115 min after the injection. The injected volume was 0.25 ml. This dose of AG was chosen on the basis of a previous study [16]. 2.3.2. Protocol 2: Determination of the effect of AG on restraint stress-induced fever After basal Tc was measured, pyrogen-free saline or AG (25 and 50 mg/kg) was injected intraperitoneally and 30 min later the animals were subjected to restraint. The animals were stressed by being subjected to 40 min of restraint in a standard Plexiglas tube (160 mm in length and 55 mm in diameter) with perforated sides to allow dissipation of body heat. During the period the animals were inside the restraint cage, the thermoprobe was kept inside the colon and fixed to the tail with adhesive tape. This procedure was chosen because it was impossible to insert the thermoprobe intermittently into the colon while the animals were kept into the restrain tube. Immediately after, the animals were returned to their cages and Tc was measured every 15 min for a total period of 45 min. This experimental protocol was based on a previous study [10]. 2.3.3. Protocol 3: Determination of the effect of 7-nitroindazole on Tc Rats received 7-nitroindazole (25 mg/kg) or its vehicle intraperitoneally and Tc was measured as in Protocol 1. The
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volume of all injections was 0.4 ml. This dose of 7-nitroindazole was chosen on the basis of a previous study [45]. 2.3.4. Protocol 4: Determination of the effect of 7-nitroindazole on restraint stress-induced fever After basal Tc was measured, rats received an intraperitoneal injection of 7-nitroindazole (25 mg/kg) or its vehicle. Thirty minutes later, the animals were subjected to restraint and Tc was determined as in Protocol 2. 2.4. Statistical analysis All values are reported as means ± S.E. The values of Tc are the changes from the initial values (initial Tc, Tci). The Tci for each group was determined as the mean of three Tc recorded at 15-min intervals preceding all treat-
Fig. 2. Effects of 7-nitroindazole (7-NI, 25 mg/kg ip) or its vehicle (sesame oil/dimethyl sulfoxide 9:1, ip) on Tc of unrestrained (A) or restrained (B) rats. The data are expressed as changes in Tc (DTc) relative to their initial levels (Tci) (see text for details). The arrowhead indicates the time of injections and the horizontal bar indicates the period when the animals were restrained. The values are means ± S.E.; n = number of animals. * P < .05 compared to basal Tc (time zero); + P < .05 compared to the data obtained by treatment with vehicle.
ments. Two-way ANOVA followed by the Tukey– Kramer multiple comparisons test or the Student t test was used for data analysis. Values of P < .05 were considered to be significantly different.
3. Results In all experimental protocols, Tc ranged from 36.5 to 37.3 C during the control period and the baseline values did not differ significantly from the vehicle group in any experiment. Room temperature was kept at 25.0 ± 1.0 C during the experiments. Fig. 1. Effects of AG (AG, 25 or 50 mg/kg ip) or its vehicle (intraperitoneally administered pyrogen-free saline) on Tc of unrestrained (A) or restrained (B) rats. The data are expressed as changes in Tc (DTc) relative to their initial levels (Tci) (see text for details). The arrowhead indicates the time of injections and the horizontal bar indicates the period when the animals were restrained. The values are means ± S.E.; n = number of animals. * P < .05 compared to basal Tc (time zero) and applies for all the curves.
3.1. Experiment 1: Effect of intraperitoneal AG on Tc When vehicle or AG at 25 and 50 mg/kg was injected intraperitoneally, no significant change in Tc was observed. These data are plotted in Fig. 1A.
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3.2. Experiment 2: Effect of intraperitoneal AG on restraint stress-induced fever All restrained animals presented a significant ( P < .05) increase in Tc, which was already evident 10 min after the beginning of stress and remained elevated until the animals were released from restraint. Immediately after the animals were released from restraint, Tc started to return towards baseline control values. Neither intraperitoneal saline nor AG at both doses (25 and 50 mg/kg) affected restraint-induced fever. These data are plotted in Fig. 1B. 3.3. Experiment 3: Effect of intraperitoneal 7-nitroindazole on Tc No significant change in Tc was observed after intraperitoneal injection of 7-nitroindazole (25 mg/kg) or its vehicle. These data are depicted in Fig. 2A. 3.4. Experiment 4: Effect of intraperitoneal 7-nitroindazole on restraint stress-induced fever When animals pretreated intraperitoneally with the 7-nitroindazole vehicle were restrained (Fig. 4), their Tc rose similarly to that of noninjected rats subjected to restraint (Fig. 1B). However, intraperitoneal treatment with 7-nitroindazole at dose of 25 mg/kg significantly attenuated ( P < .005) the rise in Tc evoked by restraint compared to the group treated with vehicle. This attenuation was already evident 10 min after the animals were placed in the restraint cages. Fig. 2B illustrates these results.
4. Discussion The results of the present study show that nNOS plays a major role in restraint stress-induced fever since intraperitoneal administration of 7-nitroindazole, a more selective nNOS inhibitor, significantly attenuated the rise in Tc caused by restraint stress (Fig. 2). On the other hand, the selective iNOS inhibitor AG did not affect the Tc of euthermic animals or restraint-induced fever (Fig. 1). The NOS enzymes are widely distributed throughout the body [9,29]. Since NOS is encountered in various tissues involved in the regulation of Tc [44,45] and of the immune system [42], it seems probable that this gas influences thermoregulation and fever. To date, most of the data on the role of NO in fever were obtained in experimental models of infectious fever, such as endotoxin, muramyl dipeptide, and yeast (for a review, see Ref. [44]). In this context, systemic administration of NOS inhibitors has been reported to impair fever produced by endotoxin in rats and guinea pigs [16,38,41], by muramyl dipeptide in guinea pigs [16], and by yeast [2] and IL-1 [37,39] in rats. On the other hand, intracerebroventricular treatment
with NOS inhibitors has been shown to enhance endotoxin fever in rats [1] and rabbits [13], indicating that the feverinhibiting effect of systemically administered NOS inhibitors is due to a peripheral action of these drugs. Taken together, these data support the notion that NO plays differential thermoregulatory effects by acting at the periphery and in the central nervous system, with peripheral NO being a pyretic molecule and central NO an antipyretic molecule. Accordingly, recent studies reported that systemically administered L-NAME (a nonselective NOS inhibitor) also impairs restraint- and open field-induced fever [10,46], whereas intracerebroventricular L-NAME enhances the rise in Tc evoked by restraint [10]. Since in the present experiments we used systemic injections of selective NOS inhibitors in order to identify the NOS isoform involved in psychological stress fever, it is important to point out that the results obtained are related to the peripheral pyretic action of NO. AG, a molecule containing the guanidino group of L-arginine linked to hydrazine, is at least as equipotent as L-NMMA (a nonselective NOS inhibitor) to inhibit iNOS [8,28], but lacks activity against nNOS and eNOS. Thus, we used AG in order to verify if iNOS is involved in restraint fever in rats. It was observed that intraperitoneal AG at the doses of 25 and 50 mg/kg did not affect the Tc of euthermic animals (Fig. 1), a result that is in agreement with previous studies [40]. Moreover, intraperitoneal AG at 50 mg/kg caused no change in the course of restraint-induced fever (Fig. 1B). Considering that the overexpression of iNOS mRNA requires 3 –6 h to be induced [34] and that restraint fever was already evident 10 min after the beginning of the stimulus, this result is not surprising. In agreement, AG has been reported to have no effect on endotoxin fever in guinea pigs [40] and to have little effect on IL-1 induced fever in rats [37], fever that have onset latencies no longer than 1 h under the experimental conditions used. In contrast, AG seems to impair muramyl dipeptide-induced fever [16], although this fever takes about 1 h to start. Since our data indicate that iNOS is not the NOS isoform involved in restraint fever in rats, we hypothesised that nNOS is involved in restraint stress-induced fever. We then used 7-nitroindazole, which has been shown to be a more selective inhibitor of nNOS [24,30,31]. In agreement, we [45] and others [30,31] have reported that intraperitoneal 7-nitroindazole does not affect arterial blood pressure, a response that has been attributed to a reduction in the activity of eNOS [11,29]. In previous studies that investigated the thermoregulatory effects of nNOS, it was observed that 7-nitroindazole at the dose of 30 mg/kg causes a significant drop in the Tc of euthermic animals, probably by reducing thermogenesis [36,45]. This response certainly represents a limitation to the study of the participation of nNOS in fever. Thus, it would be necessary to use a dose of 7-nitroindazole without effect on basal Tc. Since we [45] have demonstrated that 7-nitroindazole at
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the lower dose of 25 mg/kg does not affect the Tc of euthermic rats but is able to attenuate the drop in Tc evoked by hypoxia, we have chosen to use this dose to study the role of nNOS in restraint fever. In agreement, Fig. 2A shows that under the present experimental conditions, intraperitoneal 7-nitroindazole at 25 mg/kg caused no change in the Tc of euthermic rats. Interestingly, intraperitoneal 7-nitroindazole at 25 mg/kg significantly attenuated the rise in Tc elicited by restraint stress (Fig. 2B), indicating that the nNOS isoform plays an important role in the development of restraint stressinduced fever. Similarly, 7-nitroindazole also seems to reduce the rise in Tc induced by peripherally administered endotoxin [36], supporting the notion that the fever evoked by psychological stress and immune challenge share similar mechanisms. It should be emphasised that the effect of intraperitoneal 7-nitroindazole on restraint fever reported in the present study was very similar to that observed for intravenous L-NAME in a previous study from our laboratory [10]. Therefore, it is tempting to suggest that nNOS may be the only NOS isoform that accounts for the pyretic effect of NO in psychological stress-induced fever. However, a possible participation of eNOS cannot be excluded. To our knowledge, no eNOS selective inhibitor is commercially available to date. The use of NOS knockout animals could be a useful tool to solve this issue. Although evidence from studies using NOS inhibitors supports the notion that peripheral NO seems to be a pyretic molecule during infectious and psychological stress fever in rats [44], the mechanism by which NO acts on stressinduced fever is still unknown. It seems that systemic administration of L-NAME impairs endotoxin fever without affecting the cytokine (tumor necrosis factor-a and IL-6) network in guinea pigs [38]. Whether the same is true for stress fever remains to be determined. Since NO acts as a neuromodulator [9], it could affect the discharge of vagal afferents, which have been shown to play a major role in endotoxin fever [4]. But this is unlikely to evoke stress fever since a recent study has demonstrated that the vagus nerve is not involved in this response [7]. Moreover, systemic administration of L-NAME might also attenuate stress fever by increasing the levels of ACTH and corticosterone, which have been reported to be important antipyretic molecules. In fact, L-NAME seems to enhance the rise in ACTH induced by the inflammatory agent turpentine [48], but not by IL-1b [37]. Furthermore, a recent study has demonstrated that the antipyretic effect of systemically administered L-NAME is related to impairment in metabolic rate [25], a fact that seems to be associated with reduced thermogenesis [15]. However, whether this phenomenon is mediated directly by the action of L-NAME on thermogenic tissues or is allied to an attenuation of the actions and release of febrile mediators by L-NAME remains to be established. In summary, the present data indicate that NO produced by nNOS mediates restraint stress-induced fever in rats. Similarly, peripheral NO also seems to be a pyretic molecule
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in endotoxin fever, being produced by the constitutive NOS isoforms, probably nNOS (for a review, see Ref. [44]).
Acknowledgments This work was supported by the Fundac˛a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq). A.A. Steiner and D.B. Sanches were recipients of FAPESP fellowships.
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