TRPV1 antagonists that cause hypothermia, instead of hyperthermia, in rodents: Compounds' pharmacological profiles, in vivo targets, thermoeffectors recruited and implications for drug development.

AIM: Thermoregulatory side effects hinder the development of transient receptor potential vanilloid-1 (TRPV1) antagonists as new painkillers. While many antagonists cause hyperthermia, a well-studied effect, some cause hypothermia. The mechanisms of this hypothermia are unknown and were studied herein. METHODS: Two hypothermia-inducing TRPV1 antagonists, the newly synthesized A-1165901 and the known AMG7905, were used in physiological experiments in rats and mice. Their pharmacological profiles against rat TRPV1 were studied in vitro. RESULTS: Administered peripherally, A-1165901 caused hypothermia in rats by either triggering tail-skin vasodilation (at thermoneutrality) or inhibiting thermogenesis (in the cold). A-1165901-induced hypothermia did not occur in rats with desensitized (by an intraperitoneal dose of the TRPV1 agonist resiniferatoxin) sensory abdominal nerves. The hypothermic responses to A-1165901 and AMG7905 (administered intragastrically or intraperitoneally) were absent in Trpv1-/- mice, even though both compounds evoked pronounced hypothermia in Trpv1+/+ mice. In vitro, both A-1165901 and AMG7905 potently potentiated TRPV1 activation by protons, while potently blocking channel activation by capsaicin. CONCLUSION: TRPV1 antagonists cause hypothermia by an on-target action: on TRPV1 channels on abdominal sensory nerves. These channels are tonically activated by protons and drive the reflectory inhibition of thermogenesis and tail-skin vasoconstriction. Those TRPV1 antagonists that cause hypothermia further inhibit these cold defences, thus decreasing body temperature. SIGNIFICANCE: TRPV1 antagonists (of capsaicin activation) are highly unusual in that they can cause both hyper- and hypothermia by modulating the same mechanism. For drug development, this means that both side effects can be dealt with simultaneously, by minimizing these compounds' interference with TRPV1 activation by protons.

Skin temperature: its role in thermoregulation.

This review analyses whether skin temperature represents ambient temperature and serves as a feedforward signal for the thermoregulation system, or whether it is one of the body's temperatures and provides feedback. The body is covered mostly by hairy (non-glabrous) skin, which is typically insulated from the environment (with clothes in humans and with fur in non-human mammals). Thermal signals from hairy skin represent a temperature of the insulated superficial layer of the body and provide feedback to the thermoregulation system. It is explained that this feedback is auxiliary, both negative and positive, and that it reduces the system's response time and load error. Non-hairy (glabrous) skin covers specialized heat-exchange organs (e.g. the hand), which are also used to explore the environment. In thermoregulation, these organs are primarily effectors. Their main thermosensory-related role is to assess local temperatures of objects explored; these local temperatures are feedforward signals for various behaviours. Non-hairy skin also contributes to the feedback for thermoregulation, but this contribution is limited. Autonomic (physiological) thermoregulation does not use feedforward signals. Thermoregulatory behaviours use both feedback and feedforward signals. Implications of these principles to thermopharmacology, a new approach to achieving biological effects by blocking temperature signals with drugs, are discussed.

Does obesity affect febrile responsiveness?

BACKGROUND AND OBJECTIVE: A decreased resistance to infection and impairments of immunity are common in obese humans and in rodents with hereditary obesity. Since brown fat thermogenesis is also suppressed in obese rodents, we hypothesized that obesity leads to a decreased febrile responsiveness. METHODS: We compared the fever responses to intravenous E. coli lipopolysaccharide (10 microg/kg) between Zucker fa/fa (obese due to a defective leptin receptor) and Fa/? (lean) rats and between Otsuka Long-Evans Tokushima Fatty (OLETF; obese due to the lacking cholecystokinin-A receptor) and Long-Evans Tokushima Otsuka (lean) rats. Obesity of Zucker fa/fa and OLETF rats was verified by increased body mass and fat content, hypertriglyceridemia and hypercholesterolemia. RESULTS: Neither fa/fa nor OLETF animals exhibited a decreased febrile responsiveness; if anything, their fevers tended to be higher than those in their lean counterparts. CONCLUSION: Obesity per se does not lead to antipyresis.

Neural route of pyrogen signaling to the brain.

In the pathogenesis of systemic inflammation and fever, peripheral inflammatory and pyrogenic signals gain access to the brain via humoral and neural routes. One of the neural routes is represented by chemosensitive afferent fibers of the abdominal vagus. We summarize our recent studies of the role of the abdominal vagus in fever. We conclude that capsaicin-sensitive fibers traveling within the hepatic vagal branch constitute a necessary component of the afferent mechanism of the febrile response to low, but not high, doses of circulating pyrogens. We speculate that this mechanism is triggered by blood-borne prostaglandins of the E series.

Heat defense control in an experimental heat disorder.

Both whole-body heat exposure and intraperitoneal heating (IPH) result in a body temperature (T(b)) fall that occurs once heating is abated ("hyperthermia-induced hypothermia"). This phenomenon involves a decrease in the threshold T(b) (T(b-thresh)) for activation of metabolic heat production (cold defense). Whether the T(b-thresh) for ear skin vasodilation (heat defense) also changes during hyperthermia-induced hypothermia remains unknown. In experiment 1, we applied IPH to guinea pigs by perfusing water through a preimplanted intraperitoneal thermode and delivered the total heat load of either approximately 1.5 kJ ("short" IPH; perfusion duration: 14 min) or approximately 3.0 kJ ("long" IPH; 40 min). Short IPH caused skin vasodilation and a 1.1 degrees C rise in T(b); no hypothermia occurred when IPH ceased. Long IPH caused vasodilation and hyperthermia of a comparable magnitude (1.4 degrees C) that were followed by a T(b) fall to 1.9 degrees C below the preheating value. In experiment 2, the Tb-thresh for skin vasodilation was measured twice: at the beginning of long IPH and at the nadir of the post-IPH hypothermia. The two T(b-thresh) values were 39.0 (SEM 0.1)degrees C and 39.2 (SEM 0.2)degrees C respectively. In the controls, the T(b-thresh) was measured at the beginning and after short IPH; both control values were 39.0 (SEM 0.2)degrees C. We conclude that the hyperthermia-induced hypothermia, although previously shown to be coupled with a decrease in the T(b-thresh) for cold defense, occurs without any substantial change in the T(b-thresh) for heat defense. We speculate that postheating thermoregulatory disorders are associated with threshold dissociation, thus representing the poikilothermic (wide dead-band) type of T(b) control.

Epidemic diphtheria in Belarus, 1992-1997.

In 1990, epidemic diphtheria reemerged in Russia and spread to Belarus in 1992, when 66 cases were reported. Diphtheria cases doubled each year in 1993 and 1994 and peaked in 1995, when 322 cases were reported. Intensified routine immunization of young children and mass vaccination of older children and selected groups of adults were conducted in 1995 and were followed by mass vaccination campaigns targeting all adults in 1996. By the end of 1996, full immunization of >95% of children and coverage of>87% of adults with >/=1 dose resulted in a rapid decline in diphtheria cases. In 1998, only 36 cases of diphtheria were reported. More than 70% of the 965 cases and 26 fatalities reported during 1990-1998 occurred among persons >14 years of age. High levels of immunity among the entire population are needed for rapid control of diphtheria epidemics in the vaccine era.

Does the formation of lipopolysaccharide tolerance require intact vagal innervation of the liver?

The study was designed to test whether intact vagal innervation of the liver is required for the formation of tolerance to lipopolysaccharide (LPS). Wistar rats were subjected to either denervation of the liver (transection of the hepatic and both celiac branches of the abdominal vagus) or sham surgery. Two weeks later, each rat had an osmotic pump implanted subcutaneously. The pump was filled with either a suspension of Escherichia coli LPS (18 mg/ml) in saline or saline alone. Via a catheter, the pump delivered its content into the right jugular vein at a rate of approximately 0.72 microl/kg/h (approximately 13 microg/kg/h of LPS) over 28 d. On day 25 of the infusion, each animal had another catheter implanted into the left jugular vein. Three days later, each rat was injected with a lethal bolus dose of LPS (15 mg/kg) and had its colonic temperature recorded. The saline-infused sham-operated rats responded to the bolus injection of LPS with hypothermia followed by a fever (mean response magnitude 1.0+/-0.2 degrees C); 91% of the animals died within 24 h. The LPS-primed shams developed marked tolerance: When challenged with a lethal dose of LPS, they exhibited a significantly smaller thermal response (magnitude 0.5 +/- 0.2 degrees C) and none died. No group of the vagotomized animals, whether LPS- or saline-primed, became tolerant: Both groups exhibited similar hypothermic responses to the bolus LPS injection and a substantial mortality rate (40 and 100%, respectively). The study shows that prolonged infusion of low doses of LPS leads to the formation of tolerance and that vagal denervation of the liver by hepato-celiac vagotomy suppresses this process. The mechanisms of vagal control of the formation of LPS tolerance remain speculative.

Lipopolysaccharide transport from the peritoneal cavity to the blood: is it controlled by the vagus nerve?

Vagotomy suppresses fever and hyperalgesia caused by intraperitoneal lipopolysaccharide (LPS) but has little effect on the febrile response to intravenous or intramuscular LPS. This suggests that some vagus-mediated mechanisms are recruited only when LPS is administered via the intraperitoneal route. We hypothesized that such mechanisms are associated with LPS transport from the peritoneal cavity to the circulation. Adult Wistar rats underwent total subdiaphragmatic, bilateral selective celiac, or sham vagotomy. On day 28-32 after surgery, they were injected IP with Escherichia coli LPS (5, 20, or 100 microg/kg) or saline and decapitated 90 min thereafter. Their plasma levels of LPS and their plasma interleukin-6, adrenocorticotropin, and corticosterone responses to LPS were measured. Success of intraperitoneal administration of LPS was verified by increased interleukin-1beta and interleukin-6 concentrations in the peritoneal lavage fluid. Effectiveness of vagotomies was confirmed by increased stomach mass (food retention) and pancreas mass (hypertrophy). In the shams, LPS caused a dose-dependent endotoxemia and increased plasma levels of interleukin-6, adrenocorticotropin, and corticosterone. Neither celiac nor total vagotomy affected any of these responses. LPS escapes from the peritoneal cavity by two primary routes, viz., the hematogenous (via the portal vein) and lymphogenous (via the lymphatic system). The design of the present study did not allow for evaluating the rapid, hematogenous transport. The results obtained suggest that the abdominal vagus does not control the slow. lymphogenous escape of LPS from the peritoneal cavity.

Thermoregulatory manifestations of systemic inflammation: lessons from vagotomy.

The multiple effects of vagotomy on the thermoregulatory response to systemic inflammation are reviewed (primarily, for the model of intravenous lipopolysaccharide administration in the rat). The following conclusions are drawn. (1) Vagotomy-associated thermoeffector insufficiency is likely to account for the attenuation of the fever response observed in some--but not all--studies; such an insufficiency is, however, preventable by postoperative care, including the use of a liquid diet. (2) The febrile response to low doses of lipopolysaccharide (monophasic fever) is mediated by the hepatic (but not gastric or celiac) vagal fibers, presumably afferent; the same fibers are likely to be involved in the development of tolerance to low doses of circulating endotoxins. (3) Phase 1 of the polyphasic febrile response to moderate doses of lipopolysaccharide involves capsaicin-sensitive afferents (either nonvagal only or both nonvagal and vagal), does not involve cholecystokinin A-receptors, and may involve peripheral prostaglandins. (4) Febrile phase 2 does not require the integrity of abdominal nerve fibers, either vagal or nonvagal, at least in the rat. (5) Phase 3 of the febrile response to intravenous lipopolysaccharide (and perhaps the response to intraperitoneal lipopolysaccharide) involves capsaicin-insensitive vagal fibers, presumably efferent; the involvement of these fibers in febrigenic mechanisms is strongly modulated by an unknown factor. (6) A hepatoceliac vagal, presumably efferent, mechanism ('an anti-inflammatory pathway') counteracts the development of lipopolysaccharide-induced hypothermia and shock.

Multiple neural mechanisms of fever.

In rats, fevers induced by moderate-to-high doses of intravenous lipopolysaccharide consist of three phases (phases 1, 2 and 3) with body temperature peaks at approximately 1, 2, and 5 h postinjection, respectively. In this study, the effects of bilateral truncal subdiaphragmatic vagotomy and intraperitoneal capsaicin desensitization on febrile phases 1-3 were assessed in adult Wistar rats. Surgical vagotomy was performed approximately 30 d before the experiment; this procedure interrupts both afferent and efferent vagal fibers. Capsaicin was administered intraperitoneally in two consecutive injections (2 and 3 mg/kg, 3 h apart) 1 week prior to the experiment; this procedure desensitizes afferent fibers, primarily within the abdominal cavity, and does not lead to the known thermal effects of systemic capsaicin desensitization. At a neutral ambient temperature, the rats were given Escherichia coli lipopolysaccharide (10 microg/kg) through a preimplanted jugular catheter, and their colonic temperature wes measured by thermocouples for 7 h. The control rats exhibited the typical triphasic febrile responses. Confirming our earlier studies, subdiaphragmatic vagotomy did not affect phases 1 and 2; it did, however, result in a 2.5-fold reduction of phase 3. Capsaicin desensitization modified the febrile response differently: phases 2 and 3 were unaffected, but phase 1 disappeared. We suggest that neural afferent fibers (nonvagal but perhaps vagal as well) play an important role in the early febrile response (phase 1) by transducing peripheral pyrogenic signals to the brain. We also suggest that vagal efferent fibers are likely to participate in the later febrile response (phase 3) via an unknown mechanism.

Vagotomy does not affect thermal responsiveness to intrabrain prostaglandin E2 and cholecystokinin octapeptide.

Subdiaphragmatic vagotomy has been repeatedly shown to attenuate the febrile response to peripherally injected pyrogens. In the present study, we investigated whether vagotomy-induced attenuation of febrile responsiveness reflects a decreased sensitivity of the brain to central fever mediators, prostaglandin E2 (PGE2) and cholecystokinin octapeptide (CCK-8). Male rats were subjected to subdiaphragmatic vagotomy (or sham surgery) on day 0 and had a cannula implanted into the lateral cerebral ventricle on day 24. On day 30-36, the thermal responsiveness of the rats to PGE2 or CCK-8 was tested. Each animal was injected in the ventricle with either PGE2 (0, 10, 100, or 500 ng) in pyrogen-free saline with 0.5% ethanol (5 microl) or CCK-8 (0 or 1.6 microg) in artificial cerebro-spinal fluid (5 microl). While the 0-dose of either PGE2 or CCK-8 (vehicle alone) induced no thermal response, all the higher doses of either agent caused a body temperature rise preceded by tail skin vasoconstriction. The vagotomized rats did not respond differently than their sham-operated counterparts to any dose of either drug. It is concluded that subdiaphragmatic vagotomy does not change the rat's thermal responsiveness to intrabrain PGE(2) and CCK-8.

Blood-borne, albumin-bound prostaglandin E2 may be involved in fever.

Although the involvement of blood-borne PGE2 in fever has been hypothesized by several authors and has substantial experimental support, the current literature often rejects this hypothesis because several attempts to induce fever by a peripheral PGE2 failed. However, it is usually ignored that the amphipathic molecules of PGE2 are readily self-associating and that such an aggregation could have prevented the peripherally administered PGE2 (free form) from expressing its pyrogenic activity, thus leading to false negative results. To ensure disaggregation of PGE2, we prepared its complex within a carrier protein, human serum albumin (HSA). HSA was purified with activated charcoal and polymixin B-polyacrylamide gel and incubated with PGE2 for 1 h at 37 degrees C. Adult Chinchilla rabbits were injected intravenously with PGE2-HSA complex in either the higher (75 micrograms/kg PGE2:30 mg/kg HSA) or the lower (15 micrograms/kg:6 mg/kg) dose, and the rectal temperature (Tr) was measured. In the controls, either the ligand alone or the carrier alone was administered. At the higher dose, neither free PGE2 nor albumin alone was pyrogenic, whereas the PGE2-HSA complex produced a fever characterized by a short latency (<10 min) and a maximal Tr rise of 0.7 +/- 0.2 degrees C. At the lower dose, none of the substances affected the Tr. This study demonstrates a marked pyrogenic activity of the intravenous PGE2-HSA, but not of the free PGE2. Administration of a preformed complex may be more physiologically relevant than administration of the free ligand because of the ligand's disaggregation, protection from enzymatic degradation, and facilitated delivery to targets. Our study supports the hypothesis that peripheral PGE2 is involved in fever genesis.

Signaling the brain in systemic inflammation: which vagal branch is involved in fever genesis?

Recent evidence has suggested a role of abdominal vagal afferents in the pathogenesis of the febrile response. The abdominal vagus consists of five main branches (viz., the anterior and posterior celiac branches, anterior and posterior gastric branches, and hepatic branch). The branch responsible for transducing a pyrogenic signal from the periphery to the brain has not as yet been identified. In the present study, we address this issue by testing the febrile responsiveness of male Wistar rats subjected to one of four selective vagotomies: celiac (CBV), gastric (GBV), hepatic (HBV), or sham (SV). In the case of CBV, GBV, and HBV, only the particular vagal branch(es) was cut; for SV, all branches were left intact. After the postsurgical recovery (26-29 days), the rats had a catheter implanted into the jugular vein. On days 29-32, their colonic temperature (Tc) responses to a low dose (1 microg/kg) of Escherichia coli lipopolysaccharide (LPS) were studied. Three days later, the animals were subjected to a 24-h food and water deprivation, and the effectiveness of the four vagotomies to induce gastric food retention, pancreatic hypertrophy, and impairment of the portorenal osmotic reflex was assessed by weighing the stomach and pancreas and measuring the specific gravity of bladder urine, respectively. Stomach mass, pancreas mass, and urine density successfully separated the four experimental groups into four distinct clusters, thus confirming that each type of vagotomy had a different effect on the indexes measured. The Tc responses of SV, CBV, and GBV rats to LPS did not differ and were characterized by a latency of approximately 40 min and a maximal rise of 0.7 +/- 0.1, 0.6 +/- 0.1, and 0.9 +/- 0.2 degrees C, respectively. The fever response of the HBV rats was different; practically no Tc rise occurred (0.1 +/- 0.2 degrees C). The HBV appeared to be the only selective abdominal vagotomy affecting the febrile responsiveness. We conclude, therefore, that the hepatic vagus plays an important role in the transduction of a pyrogenic signal from the periphery to the brain.

"Biphasic" fevers often consist of more than two phases.

This paper disproves the common belief that all doses of lipopolysaccharide (LPS) that are commonly referred to as biphasic fever inducing (>/=2 microg/kg) cause truly biphasic responses. A catheter was implanted into the right jugular vein of several strains of adult male rats, and the animals were habituated to the experimental conditions. At an ambient temperature of 30.0 degrees C, loosely restrained animals were injected with a 10 microg/kg dose of LPS (various preparations), and their colonic (Tc) and tail skin temperatures were monitored (from >/=1 h before to >/=7 h after the injection). The results are presented as time graphs and phase-plane plots; in the latter case the rate of change of Tc is plotted against Tc. In experiment 1 the intravenous injection of LPS (from Escherichia coli 0111:B4, phenol extract) into the rats (Bkl:Wistar) induced a triphasic febrile response, as is obvious from time graphs of Tc (3 peaks), time graphs of effector activity (3 waves of tail skin vasoconstriction), and phase-plane plots (3 complete loops); the injection of saline (control) induced no Tc changes. We analyzed whether the triphasic pattern was due to some peculiarities of the experimental design, i.e., the pyrogen preparation used (experiment 2) or the rat strain tested (experiment 3) or whether this pattern reflects a more general law. In experiment 2 we used the same (phenol) preparation of different LPS (from Shigella flexneri 1A and Salmonella typhosa) and a different preparation (TCA extract) of the same LPS (E. coli). Regardless of the LPS used, rats of the Bkl:Wistar strain responded to the 10 microg/kg dose with the triphasic fever. In experiment 3, rats of other strains [Bkl:Sprague-Dawley and Sim:(LE)fBR(Black-hooded)] were tested. Again, all animals responded to the 10 microg/kg dose of E. coli LPS (phenol extract) with the triphasic fever. Because all fevers caused by four different LPS preparations in three rat strains were triphasic, the triphasic pattern is likely to constitute an intrinsic characteristic of the febrile response.

Methodology of fever research: why are polyphasic fevers often thought to be biphasic?

This study explains why the recently described triphasic lipopolysaccharide (LPS) fevers have been repeatedly mistaken for biphasic fevers. Experiments were performed in loosely restrained male Wistar rats with a catheter implanted into the right jugular vein. Each animal was injected with Escherichia coli LPS, and its colonic (Tc) and tail skin temperatures were monitored. The results are presented as time graphs and phase-plane plots; in the latter case the rate of change of Tc is plotted against Tc. At an ambient temperature (Ta) of 30.0 degrees C, the response to the 10 microg/kg dose of LPS was triphasic, as is obvious from time graphs of Tc (3 peaks), time graphs of effector activity (3 waves of tail skin vasoconstriction), and phase-plane plots (3 complete loops). When the Ta was below neutral (22.0 degrees C) or the LPS dose was higher (100 or 1,000 microg/kg), the time graph of Tc did not allow for the reliable detection of all three febrile phases, but the phase-plane plot and time graph of effector activity clearly revealed the triphasic pattern. In a separate experiment, LPS (10 microg/kg) or saline was injected via one of two different procedures: in the first group the injection was performed through the jugular catheter, from outside the experimental chamber; in the second group the same nonstressing injection was combined with opening the chamber and pricking the animal in its lower abdomen with a needle. In the first group the febrile response was obviously triphasic, and none of the phases was due to the procedure of injection per se (injection of saline did not affect Tc). In the second group the fever similarly consisted of three Tc rises, but it might have been readily mistaken for biphasic because the first rise was indistinguishable from stress hyperthermia occurring in the saline-injected (and needle-pricked) controls. We conclude that several methodological factors (dose of LPS, procedure of its injection, and Ta) have contributed, although each in a different way, to the common misbelief that there are only two febrile phases.

Fever and hypothermia: two adaptive thermoregulatory responses to systemic inflammation.

Entering both the old dispute (whether fever is adaptive or maladaptive) and its more recent modification (whether hypothermia is protective or detrimental in systemic inflammation), we suggest a new solution. We hypothesize that fever and hypothermia represent two different strategies of fighting systemic inflammation, each developed as an adaptive response to certain conditions, and each beneficial under these conditions. The antimicrobial and immunostimulating benefits of a high body temperature could be easily offset by its high energy cost. Fever, therefore, is protective only when there is no immediate threat of a substantial energy deficit. Hypothermia, on the other hand, constitutes a response aimed at energy conservation and, as such, is beneficial exactly under the conditions of a substantial energy deficit. The two thermoregulatory responses represent two complementary strategies of survival in systemic inflammation: fever ensures the active attack against the pathogen; hypothermia secures the defense of the host's vital systems. The importance of each response's contribution to the whole campaign depends on the severity of the pathogenic insult, premorbid pathology, and current conditions (stress, nutrition, ambient temperature, etc.).