Journal of Applied Physiology Journal of Neurophysiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

J Appl Physiol 88: 1607-1613, 2000;
8750-7587/00 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (25)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Steiner, A. A.
Right arrow Articles by Branco, L. G. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Steiner, A. A.
Right arrow Articles by Branco, L. G. S.
Vol. 88, Issue 5, 1607-1613, May 2000

Central CO-heme oxygenase pathway raises body temperature by a prostaglandin-independent way

Alexandre A. Steiner and Luiz G. S. Branco

Faculdade de Odontologia and de Medicina de Ribeirão Preto, Universidade de São Paulo, 14040-904 Ribeirão Preto, São Paulo, Brazil


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Recently, the carbon monoxide (CO)-heme oxygenase pathway has been shown to play an important role in fever generation by acting on the central nervous system, but the mechanisms involved have not been assessed. Thus the present study was designed to determine whether prostagandins participate in the rise in body temperature (Tb) observed after induction of the CO-heme oxygenase pathway in the central nervous system. Intracerebroventricular (ICV) injection of heme-lysinate (152 nmol/4 µl), which is known to induce the CO-heme oxygenase pathway, caused an increase in Tb [thermal index (TI) = 5.3 ± 0.5°C · h], which was attenuated by ICV administration of the heme oxygenase inhibitor ZnDPBG (200 nmol/4 µl; TI = 2.5 ± 1.7°C · h; P < 0.05). No change in Tb was observed after intraperitoneal injection of the cyclooxygenase inhibitor indomethacin (5 mg/kg), whereas indomethacin at the same dose attenuated the fever induced by ICV administration of lipopolysaccharide (LPS) (10 ng/2 µl) (vehicle/LPS: TI = 4.5 ± 0.5°C · h; indomethacin/LPS: TI = 1.7 ± 1.0°C · h; P < 0.05). Interestingly, indomethacin did not affect the rise in Tb induced by heme-lysinate (152 nmol/4 µl) ICV injection (vehicle/heme: TI = 4.5 ± 1.4°C · h; indomethacin/heme: TI = 4.2 ± 1.0°C · h). Finally, PGE2 (200 ng/2 µl) injected ICV evoked a rise in Tb that lasted 1.5 h. The heme oxygenase inhibitor ZnDPBG (200 nmol/4 µl) failed to alter PGE2-induced fever. Taken together, these results indicate that the central CO-heme oxygenase pathway increases Tb independently of prostaglandins.

temperature; fever; lipopolysaccharide; cyclooxygenase; prostaglandin H synthase; indomethacin; central nervous system


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

WHEN THE NATURAL BARRIERS of the body are invaded by an infectious microorganism, an array of systemic reactions promptly develops to protect the host from the deleterious effects of the invading agent and, ultimately, to restore health. Among this set of early responses of the host, called "acute-phase reaction," the febrile response is the most outstanding (cf. Ref. 2). By definition, fever is a regulated increase in body temperature (Tb) characterized by a raised thermoregulatory set point (for a review, see Ref. 14).

A series of studies has shown that the increase in Tb by a few degrees in response to infection is protective, because it improves the efficiency of macrophages in killing invading agents and impairs the replication of many microorganisms, thus giving the immune system an adaptive advantage (14, 28). Accordingly, the importance of fever is emphasized by reports that show that moderate fever has a beneficial effect on the outcome of infections (for a review, see Ref. 14). Although fever is considered to be beneficial, evidence has demonstrated that it is critical to the host that Tb does not get too close to lethal limits, because such high temperatures could elicit injury of neurons, seizures, and even death (cf. Ref. 14).

Several studies have been conducted to identify the mechanisms underlying fever, and it is generally believed that fever generation involves the induction of cytokines, such as interleukin (IL)-1beta and IL-6, interferons, and tumor necrosis factor (13, 14), and subsequent stimulation of the generation of PGs, namely PGE2, in the central nervous system (20). Classically, PGE2 is thought to act as a final mediator of fever by acting in the preoptic area of the anterior hypothalamus (POAH) (for a review, see Ref. 20), which is considered to be the thermoregulatory site of the central nervous system (19, 20). Evidence in favor of the role of PGE2 as a final mediator of fever can be summarized as follows: 1) the levels of PGE2 rise in the POAH in conjunction with the generation of fever (31); 2) PGE2 causes a rise in Tb when administered into the POAH (24, 30), which has been attributed to a rise in the thermoregulatory set point (3); 3) pretreatment of humans and experimental animals with inhibitors of cyclooxygenase (COX), the enzyme that synthesize PGs, attenuates fever (20, 39, 40); and 4) COX inhibitors do not affect PGE-induced fever (21).

Besides the notion that PGE2 is the final mediator of fever, some endogenous pyrogens have been reported to evoke PG-independent fever, i.e., a fever that is not blocked by a COX inhibitor (4, 6, 27, 40). To our knowledge, the occurrence of a PG-independent fever was first reported in 1989 by Davatelis et al. (4). In that study, it was observed that the macrophage inflammatory protein-1-induced fever was not altered by the COX inhibitor ibuprofen. Later on, other pyrogens, such as IL-8 (40), endothelin-1 (ET-1) (6), and corticotropin-releasing factor (CRF) (27), have also been shown to produce PG-independent fever. Taken together, these studies suggest that another pathway, besides that dependent on PGs, plays a role in fever generation.

Recently, the central carbon monoxide-heme oxygenase pathway has been shown to be involved in a series of physiological and pathophysiological processes (for a review, see Refs. 5, 10). Heme oxygenase is the enzyme responsible for carbon monoxide synthesis in vivo and catalyzes the metabolism of heme to biliverdin, free iron, and carbon monoxide (1, 18, 23). Three distinct heme oxygenase isoforms have been identified, among which heme oxygenase-1 (inducible) and heme oxygenase-2 (constitutive) have been the most extensively studied (17). The mode of action of carbon monoxide arising from the metabolism of heme is exquisitely paracrine, because it acts only at a short distance from its sites of generation and in most cases the gas produces an elevation in cGMP levels (for a review, see Ref. 5). Moreover, lipopolysaccharide (LPS) (11, 12) and cytokines (26) have been reported to induce heme oxygenase-1 in vivo and in vitro. Finally, a recent study (32) provided evidence that the gas carbon monoxide plays an important role in fever generation by acting on the central nervous system in the rat. However, no information exists about the mechanisms involved.

In view of these considerations, the aim of the present study was to assess the role of PGs in the rise in Tb observed after induction of the carbon monoxide-heme oxygenase pathway in the central nervous system. We tested the effect of the COX inhibitor indomethacin on the increase in Tb produced by the intracerebroventricular (ICV) injection of heme, which has been shown to induce the carbon monoxide-heme oxygenase pathway (10, 32). To further investigate the interaction between carbon monoxide and PGE2, we also tested the hypothesis that the carbon monoxide-heme oxygenase pathway is downstream from PGE2 in the fever cascade by assessing the effect of the heme oxygenase inhibitor zinc deuteroporphyrin 2,4-bis glycol (ZnDPBG) on the rise in Tb induced by ICV injection of PGE2.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Experiments were performed in adult male Wistar rats weighing 250-300 g, housed at controlled temperature (25.1 ± 1.3°C), and exposed to a daily 12:12-h light-dark cycle with lights on at 6:00 AM. The animals were allowed free access to water and food. Experiments were performed between 9:00 AM and 4:00 PM.

Drugs. The heme oxygenase inhibitor ZnDPBG was obtained from Porphyrin Products. ZnDPBG was dissolved in 50 mmol/l Na2CO3 (50 µmol/ml) and stored in the dark. LPS (from Escherichia coli, serotype 0111:B4) was obtained from Sigma Chemical and dissolved in pyrogen-free sterile saline. Heme-L-lysinate (38 mmol/l) was prepared as previously described (15, 33). Heme-free preparations were used as amino acid (L-lysine) vehicle control solutions. Indomethacin and PGE2 (gifts from Dr. Fernando Q. Cunha, Department of Pharmacology, Medical School of Ribeirão Preto, University of São Paulo, Brazil) were dissolved in Tris buffer (pH 8) and pyrogen-free sterile saline, respectively, at the time of injection.

Surgery. Animals were anesthetized with 2,2,2-tribromoethanol (Aldrich, Milwaukee, WI) and fixed in a stereotaxic frame. A stainless steel guide cannula (0.7 mm OD) was introduced into the right lateral cerebral ventricle (coordinates: anterior, -1.0 mm; lateral, -1.6 mm; dorsal, 3.2-3.7 mm) (25). The displacement of the meniscus in a water manometer ensured correct positioning of the cannula in the lateral ventricle. The cannula was attached to the bone with stainless steel screws and acrylic cement. A tight-fitting stylet was kept inside the guide cannula to prevent occlusion. Subsequently, each animal was removed from the stereotaxic frame and submitted to a paramedian laparotomy for the insertion of a biotelemetry probe capsule (model VM-FH, Mini-Mitter, Sunriver, OR) into the peritoneal cavity. The wound was then closed with skin sutures, and the implanted capsule was used for measurements of Tb. The surgical procedures were performed over a period of 30-40 min. After surgery, animals were treated with 100,000 U of benzyl-penicillin and allowed to recover for 1 wk.

Tb measurement. Tb was measured by biotelemetry (Mini-Mitter). On the day preceding the experiment, the fully conscious rats previously implanted with the telemetry probes were conditioned in the experimental cages. On the day of the experiment, Tb was measured by moving each cage gently on a Mini-Mitter receiver each time Tb was to be measured; the receiver was connected to a frequency counter. The frequency of each probe had a corresponding Tb value in a calibration table. The measurements were performed at 30-min intervals to minimize possible stress due to movement of the cages. This method is validated by our observations of stable baseline Tb values in animals that received no treatment.

Determination of the central effect of the heme-lysinate preparation on Tb. Rats previously cannulated in the lateral cerebral ventricle were left undisturbed for at least 24 h before the experiment, after which initial Tb (Tbi) was determined by three measurements made at 30-min intervals. Rats were then treated ICV with heme-lysinate (152 nmol in 4 µl) or the same volume of the L-lysine vehicle mixture, and Tb was measured every 30 min for 4.5 h after the injections. This dose of heme-lysinate was chosen on the basis of a previous study (32). A 10-µl Hamilton syringe and a dental injection needle (Missy, 200 µm OD) were used for all the ICV injections. Injection was performed over a period of 2 min, and 1 min was allowed to elapse before the injection needle was removed from the guide cannula to avoid reflux.

In another set of experiments, Tbi was measured, and animals were injected ICV with ZnDPBG (200 nmol/4 µl) or its vehicle. Fifteen minutes later, heme-lysinate (152 nmol/4 µl) was injected into the lateral ventricle of both groups, and Tb was measured every 30 min for a period of 4.5 h.

Thermal indexes (TIs) were used for data analysis. They were calculated as areas under the temperature curves (°C · h) for a total period of 4.5 h.

Determination of the effect of intraperitoneal injection of the COX inhibitor indomethacin on Tb. Rats were housed in a plastic chamber (5 liters) for at least 24 h before Tbi was measured. The animals were then treated intraperitoneally with indomethacin (5 mg/kg body wt), and Tb was measured every 30 min for 4.5 h. The volume of each injection was 0.5 ml. Control animals received intraperitoneal injections of Tris buffer (0.5 ml). This dose of indomethacin was chosen on the basis of previous studies (38) and because, when preliminary doses were tested, the thermoregulatory responses to the dose of 5 mg/kg was the most consistent and repeatable.

Determination of the effect of indomethacin on the heme-induced rise in Tb. Rats previously cannulated in the lateral ventricle were housed in a plastic chamber (5 liters) for at least 24 h before the experiment. After Tbi was measured, rats were injected intraperitoneally with indomethacin (5 mg/kg) or Tris buffer in a final volume of 0.5 ml. Thirty minutes later, heme-lysinate (152 nmol/4 µl) was administered ICV, and Tb was measured every 30 min for 4.5 h.

TIs were calculated as areas under the temperature curves (°C · h) for a total period of 4.5 h.

Determination of the effect of indomethacin on LPS-induced fever. After Tbi was determined, rats were treated with indomethacin (5 mg/kg) or Tris buffer by intraperitoneal injection. Thirty minutes later, rats were injected ICV with LPS (10 ng/2 µl), and Tb was measured every 30 min for 4.5 h. Another group of animals received only injections of Tris intraperitoneally and ICV saline as control.

TIs were calculated as areas under the temperature curves (°C · h) for a total period of 4.5 h.

Determination of the effect of the heme oxygenase inhibitor ZnDPBG on PGE2-induced fever. Previously cannulated rats were housed in their cages for 24 h before the experiment. After Tbi was measured, animals were injected ICV with ZnDPBG (200 nmol/4 µl) or the same volume of vehicle. Fifteen minutes later, PGE2 (200 ng/2 µl) was injected into the lateral ventricle of both groups, and Tb was measured every 30 min for a total period of 1.5 h. The dose of PGE2 and the time after injection along which Tb was determined were chosen on the basis of previous studies (20, 35).

Statistical analysis. All values in this study are reported as means ± SE. The values of Tb are the changes from the basal values (Tbi). Tbi for each group was determined as the Tb averaged over the last three 30-min-interval measurements preceding all the injections. Differences among changes in Tb were evaluated by ANOVA for repeated measurements. The difference between means was assessed by the Tukey-Kramer multiple-comparisons test. Ordinary ANOVA or Student's t-test was used to assess differences between TIs. Values of P < 0.05 were considered to be significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In all experimental protocols, Tb ranged from 36.6 to 37.9°C during the control period. Although this is a relatively large variation, there is no difference among the mean Tbi values, indicating that this variation was homogeneous among the different groups. These values are shown in the legends of Figs. 1-5. During the experiments, room temperature was 26.2 ± 0.6°C.

Temporal effect of ICV injection of heme-lysinate on Tb. Figure 1 shows the effect of heme-lysinate injected into the cerebral ventricle on Tb. Heme-lysinate (152 nmol/4 µl) evoked an elevation in Tb (TI = 5.3 ± 0.5°C · h), whereas the L-lysine-vehicle mixture caused no significant change in Tb (TI = 0.2 ± 0.1°C · h). ZnDPBG (200 nmol/4 µl) administration attenuated the increase in Tb elicited by the heme preparation (TI = 2.5 ± 1.7°C · h), but ICV treatment with the ZnDPBG vehicle did not alter the pyretic effect of heme-lysinate (TI = 5.9 ± 0.8°C · h).


View larger version (26K):
[in this window]
[in a new window]
  		Fig. 1.   Top: effect of heme-lysinate (Heme-lys) alone or combined with zinc deuteroporphyrin 2,4-bis glycol (ZnDPBG) injected intracerebroventricularly (ICV) on body temperature (Tb) in rats. ZnDPBG or its vehicle (Veh) was given 15 min before heme-lysinate injection. Bottom: 4.5-h thermal indexes (TIs) between 0 and 4.5 h after injections of rats shown in top. Initial Tb (Tbi) values are as follows: vehicle = 37.3 ± 0.1, heme-lysinate = 37.0 ± 0.1, Na2CO3/heme-lysinate = 37.4 ± 0.1, and ZnDPBG/heme-lysinate = 37.3 ± 0.1°C. * Significant difference between TI compared with control group (vehicle of heme-lysinate) (P < 0.05). Values are means ± SE; n = 5 rats/group.

Temporal effect of intraperitoneal injection of indomethacin on Tb. No significant change in Tb was observed after intraperitoneal injection of indomethacin (5 mg/kg) or its vehicle. These data are shown in Fig. 2.


View larger version (14K):
[in this window]
[in a new window]
  		Fig. 2.   Change in Tb over time in rats injected intraperitoneally with indomethacin (Indo) at 5 mg/kg dose or its vehicle (Tris buffer) as control. None of the treatments significantly altered Tb. Tbi values are as follows: Tris = 37.0 ± 0.2 and indomethacin = 36.9 ± 0.1°C. Values are means ± SE; n = 5 rats/group.

Effect of the intraperitoneal injection of indomethacin on the rise in Tb produced by the ICV injection of heme-lysinate. Figure 3 shows the effect of indomethacin on ICV injected heme-induced hyperthermia. Neither the intraperitoneal injection of Tris (0.5 ml; TI = 4.5 ± 1.4°C · h) nor indomethacin (5 mg/kg; TI = 4.2 ± 1.0°C · h) altered the rise in Tb observed after the ICV administration of heme-lysinate (152 nmol/4 µl).


View larger version (24K):
[in this window]
[in a new window]
  		Fig. 3.   Effect of the cyclooxygenase inhibitor indomethacin (5 mg/kg ip) or its vehicle (Tris ip) on the rise in Tb observed after heme-lysinate (152 nmol) ICV injection. Top: change in Tb over time in rats injected intraperitoneally with indomethacin or its vehicle 30 min before ICV injection of heme-lysinate. Bottom: 4.5-h TI between 0 and 4.5 h after injections of rats shown in top. There is no difference between TIs. Tbi values are as follows: Tris-heme = 37.1 ± 0.2 and indomethacin-heme = 37.1 ± 0.3°C. Values are means ± SE; n = 6 rats/group.

Effect of the intraperitoneal injection of indomethacin on LPS-induced fever. LPS (10 ng/2 µl) administered ICV evoked a monophasic fever that started to increase significantly 1.5 h after the injections, whereas vehicles (TI = 0.1 ± 0.1°C · h) caused no significant change in Tb. Rats treated with indomethacin (5 mg/kg) 30 min before LPS developed an attenuated fever (TI = 1.7 ± 1.0°C · h) compared with the group treated with Tris (TI = 4.5 ± 0.5°C · h). These data are plotted in Fig. 4.


View larger version (25K):
[in this window]
[in a new window]
  		Fig. 4.   Effect of the cyclooxygenase inhibitor indomethacin (5 mg/kg ip) or its vehicle (Tris) on lipopolysaccharide (LPS; 10 ng ICV)-induced fever. Top: change in Tb over time in rats injected intraperitoneally with indomethacin or its vehicle 30 min before ICV injection of LPS. Sal, saline. Bottom: 4.5-h TI between 0 and 4.5 h after injections of rats shown in top. Tbi values are as follows: Tris/saline = 37.2 ± 0.2, Tris/LPS = 37.4 ± 0.2, and indomethacin/LPS = 37.4 ± 0.2°C. * Significant difference between TI compared with control group (Tris/saline) (P < 0.05). Values are means ± SE; n = 6 rats/group.

Effect of the heme oxygenase inhibitor ZnDPBG on PGE2-induced fever. A rapid increase in Tb that lasted 1.5 h was observed after the PGE2 (200 ng/2 µl) ICV injection. No difference in the response to PGE2 was observed between the groups pretreated ICV with Na2CO3 (50 mM; TI = 1.6 ± 0.4°C · h) or ZnDPBG (200 nmol/4 µl; TI = 1.7 ± 0.7°C · h). These data are plotted in Fig. 5.


View larger version (21K):
[in this window]
[in a new window]
  		Fig. 5.   Effect of the heme oxygenase inhibitor ZnDPBG (200 nmol ICV) or its vehicle (Na2CO3 50 mM ICV) on PGE2 (200 ng ICV)-induced fever. Top: change in Tb over time in rats injected ICV with ZnDPBG or its vehicle 15 min before ICV injection of PGE2. Bottom: 1.5-h TI between 0 and 1.5 h after injections of rats shown in top. There is no difference between TIs. Tbi values are as follows: vehicle-PGE2 = 37.0 ± 0.2 and ZnDPBG-PGE2 = 37.3 ± 0.2°C. Values are means ± SE; n = 5 rats/group.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study provides evidence that a heme oxygenase product acting in the central nervous system raises Tb independently of PGs, which have been extensively referred to as the final mediators of fever (20). This product is likely to be carbon monoxide, because a previous study from our laboratory (32) has reported that ICV injection of carbon monoxide-saturated saline produced an increase in Tb in euthermic rats and partially reversed the antipyretic effect of the heme oxygenase inhibitor ZnDPBG. In that study (32), the increase in Tb observed after ICV injection of carbon monoxide-saturated saline was smaller and less consistent than the effect of heme-lysinate (substrate) ICV injection, which is known to induce the carbon monoxide-heme oxygenase pathway. These differences might be due to 1) a lower amount of carbon monoxide delivered to the brain or 2) a rapid diffusion of the gas carbon monoxide out of the brain because this gas has a high affinity for hemoglobin. Thus we have chosen to use heme-lysinate ICV injection because it produces a more pronounced and consistent response. Furthermore, the heme oxygenase inhibitor ZnDPBG did not affect PGE2-induced fever, indicating that carbon monoxide is not located downstream from PGE2 in the fever cascade.

To our knowledge, Milton and Wendlandt in 1970 (22) reported for the first time that PGs could play a thermoregulatory role in the central nervous system. In that study, the authors observed that ICV injection of PGE1 in microgram amounts produced a marked rise in Tb that lasted ~1.5 h. This observation was also made in the present study (Fig. 5). A few months later, Vane (34) showed that nonsteroidal anti-inflammatory drugs inhibited the enzyme COX, which is responsible for the synthesis of PGs. Furthermore, it was observed that PGE1 elicits hyperthermia by acting in the POAH (20, 24, 30), which is considered to be the thermoregulatory site in the central nervous system (19, 20). As to endogenously produced PGs, it has been reported that PGE2 and not PGE1 levels rise in the POAH, cerebrospinal fluid, and in plasma in conjunction with the production of fever (20, 31), a fact that is abolished by treatment with a COX inhibitor (8). PGE2 has been shown to be equipotent to PGE1 in elevating Tb (22). Additionally, it has been reported that, when PGs are administered ICV, the thermoregulatory strategy to increase Tb varies at different ambient temperatures and is not due to changes in a single effector mechanism (3), indicating that PGs cause an increase in the thermoregulatory set point, i.e., fever. Finally, PGE2-induced fever has been shown to be resistant to COX inhibitors (21). Altogether, this evidence strongly indicates that PGs, namely PGE2, are the final mediators of fever, acting directly in the POAH to cause an increase in the thermoregulatory set point. Besides the POAH, Matsuda et al. (19) have shown that PGE2 also alters the activity of thermosensitive neurons in the organum vasculosum laminae terminalis, which has been suggested to be one of the main sites through which cytokines signal the brain to produce fever (2).

Besides this conventional viewpoint, some studies have demonstrated that some pyrogens produce fever by a PG-independent pathway. In this context, the rise in Tb observed after injection of macrophage inflammatory protein-1 (4), IL-8 (in rats but not in rabbits) (39, 40), ET-1 (6), and CRF (27), which are all produced and released during fever, has been shown to be unaltered by pretreatment with COX inhibitors such as ibuprofen and indomethacin. As to the position of these substances in the fever cascade, it is likely that the pyretic effect of IL-8 is mediated by CRF, because the CRF antagonist alpha -helical CRF9-41 has been shown to antagonize the effects of IL-8 (27). Moreover, the pyretic effect of CRF has been shown to be attenuated by the ET-1 antagonist BQ-788 in rats (7).

Recently, a new biologically active molecule has been described, i.e., the gaseous compound carbon monoxide (for a review, see Ref. 5). Heme oxygenase is the enzyme responsible for carbon monoxide synthesis in vivo and seems to be extensively distributed throughout the body, including the central nervous system (16, 17). This enzyme catalyzes the oxidation of the heme molecule in concert with NADPH-cytochrome P-450 reductase, with resulting specific cleavage of the heme molecule into biliverdin, free iron, and carbon monoxide (11). The physiological importance of the carbon monoxide-heme oxygenase pathway can be demonstrated by the inhibition of the enzyme heme oxygenase by using metalloporphyrins such as ZnDPBG or inducing the pathway by using heme preparations (10, 32).

We have demonstrated that carbon monoxide arising from the heme metabolism by heme oxygenase may perform a thermoregulatory action (30), but this pathway is unlikely to account for the thermoregulatory action of carbon monoxide exposition (9), because inspired carbon monoxide widely binds to hemoglobin, thus eliciting hypoxia, which by itself has been shown to cause hypothermia (9). Actually, we have demonstrated (32) that the ICV administration of the nonselective heme oxygenase inhibitor ZnDPBG, which acts on both the constitutive and inducible isoforms of the enzyme (10), attenuates LPS-induced fever in rats without affecting Tb in euthermic animals. This effect of the drug can be reversed by ICV injection of heme-lysinate (substrate) or carbon monoxide-saturated saline, indicating that a heme oxygenase product, probably carbon monoxide, plays a role in fever generation by acting on the central nervous system. Accordingly, heme-lysinate injected ICV has been shown to elicit an elevation in Tb in euthermic rats, which has been attributed to an induction of the carbon monoxide-heme oxygenase pathway, probably by an increase in the production of carbon monoxide, because ICV injection of carbon monoxide-saturated saline also elicits an increase in Tb in euthermic rats (32). However, the mechanisms involved have not been assessed.

In the present study, heme-lysinate also led to an increase in Tb, which was attenuated by the heme oxygenase inhibitor ZnDPBG (Fig. 1). It should be pointed out that we now monitored Tb for 4.5 h and not 2 h as we have done previously (32), a fact that allowed us to evaluate the plateau phase of the increase in Tb induced by heme-lysinate injection. Moreover, it is important to emphasize that heme-lysinate has been shown to exert its effects when administered systemically at the dose of 45 µmol/kg (cf. Ref. 10), indicating that the rise in Tb observed after ICV injection of the 300-fold smaller amount (152 nmol) is centrally mediated and not due to a systemic action of the drug. To our knowledge, no study has assessed so far whether the effect of heme ICV injection is fever (a regulated rise in Tb) or just hyperthermia. Consequently, in this study we refer to the rise in Tb after ICV administration of heme-lysinate as hyperthermia.

The nonselective COX inhibitor indomethacin administered intraperitonealy caused no change in Tb in euthermic rats (Fig. 2), whereas it attenuated LPS-induced fever (Fig. 4), a fact consistent with the notion that PGs have no tonic role in Tb control but are essential for fever generation (20). It should be pointed out that COX inhibitors have been reported to attenuate the night rise in Tb (29), indicating that PGE2 may play a tonic role in controlling Tb during the night. However, this is not the case in the present study, which monitored Tb in the animals during the morning and afternoon when COX inhibitors have been shown to cause no change in Tb. Intraperitoneal injection of indomethacin has been shown to inhibit peripherally and centrally both isoforms of the enzyme COX, i.e., the inducible (COX-2) and the constitutive (COX-1) isoform (cf. Refs. 6, 39, 40). In our experiments, we were unable to inject indomethacin ICV because this would require more concentrated solutions that are above the solubility of the drug. However, because PGs are known to act in the central sites to produce fever and LPS was injected ICV at a dose that does not cause fever when injected peripherally (cf. Ref. 36), we can infer that the antipyretic effect of indomethacin on LPS-induced fever observed is due to a reduction in PG levels in the central nervous system. Interestingly, indomethacin did not alter the hyperthermia induced by the ICV injection of heme-lysinate (Fig. 3), indicating that the carbon monoxide-heme oxygenase pathway in the central nervous system elicits hyperthermia by a PG-independent pathway.

It should be pointed out that, besides the action of indomethacin blocking COX, a few studies have reported that an increase in the antipyretic activity of vasopressin might contribute to the antipyretic effect of indomethacin (37). However, this does not complicate the interpretation of our results because indomethacin does inhibit COX (cf. Refs. 6, 39, 40). Thus the fact that indomethacin did not affect heme-induced hyperthermia emphasizes that the COX pathway does not mediate the rise in Tb produced by ICV injection of heme-lysinate.

Previous studies have reported that carbon monoxide may also exert its physiological actions by increasing the synthesis of PGs (18). However, this is not the case for the role of carbon monoxide in fever, because we observed that the rise in Tb produced by ICV injection of heme-lysinate is not blocked by indomethacin.

Because heme-induced hyperthermia remains unchanged after indomethacin administration, one could suggest that this effect result from the fact that the carbon monoxide-heme oxygenase pathway is downstream from PGE2. Actually, we observed that, in the case of PGE2-induced fever, the heme oxygenase inhibitor ZnDPBG did not alter the thermoregulatory response to ICV injection of PGE2 (Fig. 5), showing that the central carbon monoxide-heme oxygenase pathway is not located downstream from PGE2.

In summary, our data indicate that the carbon monoxide-heme oxygenase pathway in the central nervous system causes hyperthermia, a fact that is not altered by treatment with indomethacin. Moreover, we also observed that ICV PGE2-induced fever is unaltered by the heme oxygenase inhibitor ZnDPBG. Taken together, these results indicate that the carbon monoxide-heme oxygenase pathway may belong to the list of pyrogens that increase Tb independently of PGs.


    ACKNOWLEDGEMENTS

We thank Mauro F. Silva for excellent technical assistance and Dr. F. Q. Cunha for the kind gift of indomethacin and PGE2.


    FOOTNOTES

This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico. A. A. Steiner was the recipient of a FAPESP fellowship.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: L. G. S. Branco, Faculdade de Odontologia de Ribeirão Preto/USP, 14040-904 Ribeirão Preto, São Paulo, Brazil (E-mail: branco{at}forp.usp.br).

Received 17 June 1999; accepted in final form 10 January 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abraham, NG, Lin JHC, Schwartzman ML, Levere RD, and Shibahara S. The physiological significance of heme oxygenase. Int J Biochem 20: 543-558, 1988[Web of Science][Medline].

2.   Blatteis, CM, and Sehic E. Fever: how may circulating pyrogens signal the brain? News Physiol Sci 12: 1-9, 1997[Abstract/Free Full Text].

3.   Bligh, J, and Milton AS. The thermoregulatory effects of prostaglandin E1 when infused into a lateral cerebral ventricle of the Welsh Mountain sheep at different ambient temperatures. J Physiol (Lond) 229: 30P-31P, 1973.

4.   Davatelis, G, Wolpe SD, Sherry B, Dayer J-M, Chicheportiche R, and Cerami A. Macrophage inflammatory protein-1: a prostaglandin-independent endogenous pyrogen. Science 243: 1066-1068, 1989[Abstract/Free Full Text].

5.   Dawson, TM, and Snyder SH. Gases as biological messengers: nitric oxide and carbon monoxide in the brain. J Neurosci 14: 5147-5159, 1994[Abstract].

6.   Fabricio, ASC, Silva CAA, Rae GA, D'orleans-Juste P, and Souza GEP Essential role for endothelin ETB receptors in fever induced by LPS (E. coli) in rats. Br J Pharmacol 125: 542-548, 1998[Web of Science][Medline].

7.   Fabricio, ASC, Vercesi JA, D'orleans-Juste P, Rae GA, and Souza GEP Endothelins (Ets) mediation of the pyretic effect of CRH in rats (Abstract). Bull Chim Farm 138: CCLII, 1999.

8.   Feldberg, W, Grupta KP, Milton AS, and Wendlandt S. Effect of pyrogen and antipyretics on prostaglandin activity in cisterna csf of anaesthetized cats. J Physiol (Lond) 234: 279-303, 1973[Abstract/Free Full Text].

9.   Gautier, H, Murariu C, and Bonora M. Ventilatory and metabolic responses to ambient hypoxia or hypercapnia in rats exposed to CO hypoxia. J Appl Physiol 83: 253-261, 1997[Abstract/Free Full Text].

10.   Johnson, RA, Kozma F, and Colombari E. Carbon monoxide: from an old toxin to endogenous modulator of cardiovascular functions. Braz J Med Biol Res 32: 1-14, 1999[Web of Science][Medline].

11.   Kitamura, Y, Furukawa M, Matsuoka Y, Tooyama I, Kimura H, Nomura Y, and Taniguchi T. In vitro and in vivo induction of heme oxygenase-1 in rat glial cells: possible involvement of nitric oxide from inducible nitric oxide synthase. Glia 22: 138-148, 1998[Web of Science][Medline].

12.   Kitamura, Y, Matsuoka Y, Nomura Y, and Taniguchi T. Induction of inducible nitric oxide synthase and heme oxygenase-1 in rat glial cells. Life Sci 62: 1717-1721, 1998[Web of Science][Medline].

13.   Klir, JJ, McClellan JL, and Kluger MJ. Interleukin-1beta causes an increase in anterior hypothalamic interleukin-6 during LPS-induced fever in rats. Am J Physiol Regulatory Integrative Comp Physiol 266: R1845-R1848, 1994[Abstract/Free Full Text].

14.   Kluger, MJ. Fever: role of pyrogens and cryogens. Physiol Rev 71: 93-127, 1991[Abstract].

15.   Lindén, I-B, Tokola O, Karlsson M, and Tenhunen R. Fate of haem after parenteral administration of haem arginate to rabbits. J Pharm Pharmacol 39: 96-102, 1987[Web of Science][Medline].

16.   Maines, MD. Heme oxygenase: function, multiplicity, regulatory mechanisms, and clinical applications. FASEB J 2: 2557-2568, 1988[Abstract].

17.   Maines, MD. The heme oxygenase system: a regulator of second messenger gases. Annu Rev Pharmacol Toxicol 37: 517-554, 1997[Web of Science][Medline].

18.   Mancuso, C, Tringali G, Grossman A, Preziosi P, and Navarra P. The generation of nitric oxide and carbon monoxide produces opposite effects on the release of immunoreactive interleukin-1beta from the rat hypothalamus in vitro: evidence for the involvement of different signaling pathways. Endocrinology 139: 1031-1037, 1998[Abstract/Free Full Text].

19.   Matsuda, T, Hori T, and Nakashima T. Thermal and PGE2 sensitivity of the organum vasculosum laminae terminalis region and preoptic area in rat brain slices. J Physiol (Lond) 454: 197-212, 1992[Abstract/Free Full Text].

20.   Milton, AS. Thermoregulatory actions of eicosanoids in the central nervous system with particular regard to the pathogenesis of fever. Ann NY Acad Sci 559: 392-410, 1989[Web of Science][Medline].

21.   Milton, AS, and Wendlandt S. A possible role for prostaglandin E1 as a modulator for temperature regulation in the central nervous system of the cat. J Physiol (Lond) 207: 76P-77P, 1970.

22.   Milton, AS, and Wendlandt S. Effects on body temperature of prostaglandins of the A, E and F series on injection into the third ventricle of unanaesthetized cats and rabbits. J Physiol (Lond) 218: 325-336, 1971[Abstract/Free Full Text].

23.   Morita, T, Perrella MA, Lee ME, and Kourembanas S. Smooth muscle cell-derived carbon monoxide is a regulator of cGMP. Proc Natl Acad Sci USA 92: 1475-1479, 1995[Abstract/Free Full Text].

24.   Oka, K, Oka T, and Hori T. Prostaglandin E2 may induce hyperthermia through EP1 receptor in the anterior wall of the third ventricle and neighboring preoptic regions. Brain Res 767: 92-99, 1997[Web of Science][Medline].

25.   Paxinos, G, and Watson C. The Rat Brain in Stereotaxic Coordinates (2nd ed.). San Diego, CA: Academic, 1986, p. plate 20, fig. 20.

26.   Rizzardini, M, Terao M, Falciani F, and Cantoni L. Cytokine induction of haem oxygenase mRNA in mouse liver. Biochem J 290: 343-347, 1993.

27.   Rothwell, NJ. Mechanisms of the pyrogenic actions of cytokines. Eur Cytokine Netw 1: 211-213, 1990[Medline].

28.   Saper, CB, and Breder CD. The neurologic basis of fever (seminars in medicine of the Beth Israel hospital, Boston). N Engl J Med 330: 1880-1886, 1994[Free Full Text].

29.   Scales, WE, and Kluger MJ. Effect of antipyretic drugs in circadian rhythm in body temperature of rats. Am J Physiol Regulatory Integrative Comp Physiol 253: R306-R313, 1987[Abstract/Free Full Text].

30.   Scammell, TE, Elmquist JK, Griffin JD, and Saper CB. Ventromedial preoptic prostaglandin E2 activates fever-producing autonomic pathways. J Neurosci 16: 6246-6254, 1996[Abstract/Free Full Text].

31.   Sehic, E, Ungar AL, and Blatteis CM. Interaction between norepinephrine and prostaglandin E2 in the preoptic area of guinea pigs. Am J Physiol Regulatory Integrative Comp Physiol 271: R528-R536, 1996[Abstract/Free Full Text].

32.   Steiner, AA, Colombari E, and Branco LGS Carbon monoxide as a novel mediator of the febrile response in the central nervous system. Am J Physiol Regulatory Integrative Comp Physiol 277: R499-R507, 1999[Abstract/Free Full Text].

33.   Tenhumen, R, Tokola O, and Lindén I-B. Haem arginate: a new stable haem compound. J Pharm Pharmacol 39: 780-786, 1987[Web of Science][Medline].

34.   Vane, JR. Inhibition of prostaglandins synthesis as a mechanism of action for aspirin-like drugs. Nat New Biol 231: 232-235, 1971[Web of Science][Medline].

35.   Watanabe, T, Saiki Y, and Sakata Y. The effect of central angiotensin II receptor blockade on interleukin-1beta - and prostaglandin E-induced fevers in rats: possible involvement of brain angiotensin II receptor in fever induction. J Pharmacol Exp Ther 282: 873-881, 1997[Abstract/Free Full Text].

36.   Wilkinson, MF, and Kasting NW. The antipyretic effects of centrally administered vasopressin at different ambient temperatures. Brain Res 415: 275-280, 1987[Web of Science][Medline].

37.   Wilkinson, MF, and Kasting NW. Vasopressin release within the ventral septal area of the rat brain during drug-induced antipyresis. Am J Physiol Regulatory Integrative Comp Physiol 264: R1133-R1138, 1993[Abstract/Free Full Text].

38.   Xu, RH, Kaleckman Y, Albeck M, and Srendni B. The cytoprotective effect of the immunomodulator AS101 against hydrochloride induced gastric lesions. Res Commun Mol Pathol Pharmacol 87: 4-20, 1995[Web of Science][Medline].

39.   Zampronio, AR, Silva CAA, Cunha FQ, Ferreira SH, Pelá IR, and Souza GEP Indomethacin blocks the febrile response induced by interleukin-8 in rabbits. Am J Physiol Regulatory Integrative Comp Physiol 269: R1469-R1474, 1995[Abstract/Free Full Text].

40.   Zampronio, AR, Souza GEP, Silva CAA, Cunha FQ, and Ferreira SH. Interleukin-8 induces fever by a prostaglandin-independent mechanism. Am J Physiol Regulatory Integrative Comp Physiol 266: R1671-R1674, 1994.

J APPL PHYSIOL 88(5):1607-1613
8570-7587/00 $5.00 Copyright © 2000 the American Physiological Society


This article has been cited by other articles:


Home page
 J. Leukoc. Biol.Home page
E. Tavares, F. J. Minano, R. Maldonado, and M. J. Dascombe
Endotoxin fever in granulocytopenic rats: evidence that brain cyclooxygenase-2 is more important than circulating prostaglandin E2
J. Leukoc. Biol., December 1, 2006; 80(6): 1375 - 1387.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
K. C. Bicego, A. A. Steiner, J. Antunes-Rodrigues, and L. G. S. Branco
Indomethacin impairs LPS-induced behavioral fever in toads
J Appl Physiol, August 1, 2002; 93(2): 512 - 516.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
A. A. Steiner and L. G. S. Branco
Carbon monoxide is the heme oxygenase product with a pyretic action: evidence for a cGMP signaling pathway
Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2001; 280(2): R448 - R457.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
F. C. Baker, H. S. Driver, J. Paiker, G. G. Rogers, and D. Mitchell
Acetaminophen does not affect 24-h body temperature or sleep in the luteal phase of the menstrual cycle
J Appl Physiol, April 1, 2002; 92(4): 1684 - 1691.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (25)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Steiner, A. A.
Right arrow Articles by Branco, L. G. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Steiner, A. A.
Right arrow Articles by Branco, L. G. S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online