AJP - Regu Watch the video to learn how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Regul Integr Comp Physiol 288: R872-R878, 2005. First published December 2, 2004; doi:10.1152/ajpregu.00514.2004
0363-6119/05 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
288/4/R872    most recent
00514.2004v1
Right arrow Alert me when this article is cited
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 (8)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ivanov, A. I.
Right arrow Articles by Romanovsky, A. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ivanov, A. I.
Right arrow Articles by Romanovsky, A. A.

CALL FOR PAPERS
Physiology and Pharmacology of Temperature Regulation

Albumin is not an irreplaceable carrier for amphipathic mediators of thermoregulatory responses to LPS: compensatory role of {alpha}1-acid glycoprotein

Andrei I. Ivanov, Alexandre A. Steiner, Shreya Patel, Alla Y. Rudaya, and Andrej A. Romanovsky

Systemic Inflammation Laboratory, Trauma Research, St. Joseph's Hospital and Medical Center, Phoenix, Arizona

Submitted 30 July 2004 ; accepted in final form 29 November 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In view of the potential involvement of peripherally synthesized, circulating amphipathic mediators [such as platelet-activating factor (PAF) and prostaglandin E2] in the systemic inflammatory response to lipopolysaccharide (LPS), we hypothesized that transport of amphipaths by albumin is essential for conveying peripheral inflammatory signals to the brain. Our first specific aim was to test this hypothesis by studying LPS-induced fever and hypothermia in Nagase analbuminemic rats (NAR). NAR from two different colonies and normalbuminemic Sprague-Dawley rats were preimplanted with jugular catheters, and their febrile responses to a mild dose of LPS (10 µg/kg iv) at thermoneutrality and hypothermic responses to a high dose of LPS (500 µg/kg iv) in the cold were studied. NAR of both colonies developed normal febrile and hypothermic responses, thus suggesting that transport of amphipathic mediators by albumin is not indispensable for LPS signaling. Although alternative carrier proteins [such as {alpha}1-acid glycoprotein (AGP)] are known to assume transport functions of albumin in NAR, it is unknown whether inflammatory mediators are capable of inducing their actions when bound to alternative carriers. To test whether PAF, the most potent amphipathic pyrogen, causes fever when administered in an AGP-bound form was our second aim. Sprague-Dawley rats were preimplanted with jugular catheters, and their thermal responses to infusion of a 1:1 [PAF-AGP] complex (40 nmol/kg iv), AGP (40 nmol/kg iv), or various doses of free (aggregated) PAF were studied. The complex, but neither free PAF nor AGP, caused a high (~1.5°C) fever with a short (<10 min) latency. This is the first demonstration of a pyrogenic activity of AGP-bound PAF. We conclude that, in the absence of albumin, AGP and possibly other carriers participate in immune-to-brain signaling by binding and transporting amphipathic inflammatory mediators.

systemic inflammation; fever; hypothermia; body temperature; platelet-activating factor; Nagase analbuminemic rats

BACTERIAL LIPOPOLYSACCHARIDE (LPS) is widely used to induce systemic inflammation and its thermoregulatory manifestations, that is, fever and hypothermia, in laboratory animals. In the rat, an intravenous dose of LPS administered at a neutral or supraneutral ambient temperature (Ta) typically causes fever (51, 52, 55, 63). When Ta is subneutral, intravenous LPS evokes fever at low doses, mild hypothermia followed by fever at intermediary doses, and pronounced hypothermia at high, shock-inducing doses (52, 53, 55, 63). Both the febrile and hypothermic responses to LPS are mediated by bioactive lipids, such as platelet-activating factor (PAF), prostaglandins (PG) E2 and D2, and leukotrienes (22, 24, 46, 68). Peripherally synthesized, blood-borne pools of these lipids are thought to play a role in conveying febrigenic and hypothermic signals to the brain; such a role is best documented by the mediation of LPS fever by circulating PGE2 (for review, see Ref. 24). First, the earliest (first) phase of LPS fever in rats is accompanied by massive expression of PGE2-synthesizing enzymes in the liver and in lungs but not in the brain (23). Second, PGE2 concentration in the venous blood of rabbits and sheep (37, 57, 61) and in the carotid blood of sheep (61) increases simultaneously with the onset of fever. Third, peripherally administered inhibitors of PG synthesis (38, 57), inhibitors of the release of a PG precursor (see Ref. 35), and antibodies to PGE2 (Steiner AA, Ivanov AI, Rudaya AY, Dragic AS, and Romanovsky AA, unpublished observations) block the febrile response even if they do not cross the blood-brain barrier. Fourth, intravenous and intracarotid PGE2 have been repeatedly shown to enter the brain (2, 6, 8, 27) and produce dose-dependent fevers (4, 8, 45, 49, 60, 61) in several animal species.

In the blood, amphipathic lipid mediators, including PGE2 and PAF, exist predominantly as complexes with their principal carrier protein, albumin (31, 48, 69). In the free form, these lipids are poorly soluble in water and readily self-aggregate (7, 28). Binding to albumin typically enhances biological activity of an amphipathic mediator by shifting the equilibrium from the free, aggregated (less biologically active) form to the bound, monomeric (most active) form; it also protects the mediator from rapid degradation by extracellular enzymes (13, 47). In the case of PGE2 or PAF, intravenous infusion of an albumin complex with either mediator produces a more pronounced febrile response at a neutral Ta than infusion of the same mediator in its free (aggregated) form (22, 49). Similarly, intravenous PAF produces a more pronounced hypothermic response at a subneutral Ta when administered as a [PAF-albumin] complex than when infused in the free form (22). In view of the potential involvement of peripherally synthesized, circulating amphipaths in the systemic inflammatory response to LPS, we hypothesized that binding of amphipathic mediators to and their transport by albumin are essential for conveying peripheral inflammatory signals to the brain and, therefore, for normal development of the inflammatory response. The first specific aim of the present study was to test this hypothesis by studying LPS-induced fever and hypothermia in Nagase analbuminemic rats (NAR; see Refs. 40, 41).

These Sprague-Dawley (SD)-derived rats produce virtually no albumin due to a mutation in the albumin gene; this mutation results in transcription of defective mRNA (10, 59). The lack of albumin in NAR readily reveals itself by altered pharmacokinetics (increased free fraction, accelerated clearance) of some exogenous amphipathic drugs (15, 16, 19, 39, 42). However, the functionally verifiable lack of albumin in NAR causes relatively few and relatively mild side effects. Due to multiple compensatory mechanisms, these rats have essentially normal content of total protein in the plasma, colloid osmotic pressure, blood pressure, and hepatic and renal functions (17, 25, 26, 36, 39). This makes NAR a unique, reasonably pathology-free model to study the role of albumin. However, the other side of the issue is that the same compensatory mechanisms may mask the effect of analbuminemia on the transport of the mediator studied, even if this mediator is normally carried by albumin, whether exclusively or predominantly. Hence, negative results obtained in NAR are difficult to explain, and the potential compensatory mechanisms should be considered.

An important compensatory role in analbuminemia is played by {alpha}1-acid-glycoprotein (AGP), a 44-kDa glycoprotein, considered the next important carrier for circulating amphipaths after albumin (20). Because AGP binds (although with a lower affinity) many amphipathic mediators that are normally bound to albumin, it "automatically" becomes a principal carrier of these mediators in the absence of albumin (29, 47), even if the concentration of AGP does not increase. For those cases of analbuminemia when the concentration of AGP increases (for review, see Ref. 17), binding to AGP becomes an even more powerful compensatory mechanism. Importantly, AGP is known to bind PAF, the most proximal mediator of LPS action (30) and the most potent amphipathic pyrogen (22). In an aqueous solution containing a physiological concentration of AGP (25 µM) and no albumin, ~99% of PAF is AGP bound (32, 33). Although AGP is known to assume transport functions of albumin in NAR (20), it is unknown whether inflammatory mediators are capable of inducing their actions when bound to this alternative carrier. We hypothesized that binding to AGP preserves the pyrogenic activity of PAF, and to test this hypothesis was the second specific aim of the present study. This aim was met by studying the febrile responses of normalbuminemic SD rats to free and AGP-bound PAF.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals

Male SD-derived NAR were obtained from two different colonies: from the Utrecht University (Utrecht, The Netherlands; NAR/SaU) and from SLC (Hamamatsu, Japan; NAR/Slc). Wild-type SD rats were purchased from Harlan (Indianapolis, IN). All rats weighed 260–320 g at the time of the experiments. Initially, the rats were housed three per standard "shoe box"; after surgery, they were caged individually. The cages were kept in a rack equipped with a Smart Bio-Pack ventilation system (model SB4100) and Thermo-Pak temperature control system (model TP2000; Allentown Caging Equipment, Allentown, NJ); the temperature of the incoming air was maintained at 28°C. Standard rat chow (Teklad Rodent Diet "W" 8604; Harlan Teklad, Madison, WI) and tap water were available ad libitum. The room was on a 12:12-h light-dark cycle (lights on at 7:00 AM). The cage space was enriched with artificial "rat holes" (cylindrical confiners made of stainless steel wire). In addition to spending time in the confiners voluntarily, the rats were systematically habituated to them (7 daily training sessions, 4 h each). The same confiners were used later in the experiments. Rodents are readily adaptable to restraint to an extent that they respond to it with neither stress fever (54) nor other signs of stress (1, 12, 34, 62). Each rat was used in an experiment once. The rats were euthanized with pentobarbital sodium (20 mg/kg iv). The protocols were approved by the St. Joseph's Hospital Animal Care and Use Committee.

Surgery

On day 0, each rat was subjected to jugular catheterization under ketamine-xylazine-acepromazine (55.6, 5.5, and 1.1 mg/kg, respectively, ip) anesthesia and antibiotic (1.2 mg/kg sc enrofloxacin) protection. The animal was placed on an operating board, and a 1-cm longitudinal incision was made on the ventral surface of the neck, 1 cm left of the trachea. The muscles were retracted, and the left jugular vein was exposed, freed from its surrounding connective tissue, and ligated. A silicon catheter (0.5 mm ID, 0.9 mm OD) filled with heparinized (50 USP units/ml) pyrogen-free saline was passed into the superior vena cava through the jugular vein and secured in place with ligatures. The free end of the catheter was knotted, tunneled under the skin, and exteriorized at the nape. The surgical wounds were sutured. On days 1 and 3 postsurgery, the catheter was flushed with heparinized (50 USP units/ml) saline.

Instrumentation

The experiments were performed on day 5. The animals were instrumented between 8:00 and 9:00 AM. For experiment 1, each rat was placed in a confiner and equipped with a copper-constantan thermocouple (Omega Engineering, Stamford, CT) to measure colonic temperature (Tc). This colonic thermocouple was inserted 9–10 cm beyond the anal sphincter. For experiment 2, each rat was additionally instrumented with a second copper-constantan thermocouple used to record tail skin temperature (Tsk). This was done to increase the chance of revealing thermoregulatory responses in this experiment. Indeed, changes in Tsk are often easier to detect than changes in Tc (52) because Tsk has a much wider operational range (50). The skin thermocouple was positioned on the lateral surface of the tail (at the boundary of the proximal and middle thirds) and fixed in place with adhesive tape. The thermocouples were plugged to a data logger (model AI-24; Dianachart, Rockaway, NJ) connected to a personal computer. The rat was transferred to a climatic chamber (Forma Scientific, Marietta, OH) set to either a subneutral (20.0°C) or neutral (30.0°C) Ta (50). The jugular catheter was extended with a length of PE-50 tubing filled with saline, and the extension was passed through a wall port and connected to a syringe filled with the drug of interest. The syringe was kept outside the chamber. This setup permitted injection of the drug without disturbing the rat. The instrumented rats were allowed to rest for at least 2 h before drug administration.

Drugs

All drugs and reagents were purchased from Sigma-Aldrich (St. Louis, MO). A stock suspension of E. coli 0111:B4 LPS (2.5 mg/ml) in saline was stored at –20°C. At the time of the experiment, the stock was diluted to a final concentration of either 10 µg/ml to induce fever or 500 µg/ml to cause hypothermia (experiment 1). To prepare a [PAF-AGP] complex for experiment 2, an ethanol solution of PAF ({beta}-acetyl-{gamma}-O-alkyl-L-{alpha}-phosphatidylcholine, from bovine heart lecithin; 2 µmol/ml) and a saline solution of bovine serum AGP (1 µmol/ml) were used as stocks (stored at –80°C and –20°C, respectively). On the day of the experiment, an aliquot (20 µl) of the ethanol stock of PAF (40 nmol) was dried in a SpeedVac centrifuge (model SVC-200H; Thermal Savant, Holbrook, NY). The dried PAF was suspended in saline (0.4 ml), and the AGP stock and saline were added to the suspension to achieve final concentrations of PAF and AGP of 40 nmol/ml each. The same molar concentrations of PAF and AGP were used because PAF binds to a single major site in the AGP molecule (32, 33). The PAF-AGP-saline system was sonicated for 3 min and incubated for 1 h at 37°C. The incubation conditions were identical or similar to those used by others and by us in the past to prepare a [PAF-albumin] complex (22) and AGP complexes with various amphipaths (44, 70). In addition to the [PAF-AGP] complex, a solution of AGP alone (40 nmol/ml) and a suspension of free (aggregated) PAF (40, 80, or 160 nmol/ml) were used in experiment 2. The solution of AGP and the suspension of free PAF were sonicated and incubated exactly as the solution of the [PAF-AGP] complex. The higher concentrations of free PAF in the suspension were used because the loss of PAF during the incubation and intravenous infusion due to the adherence to plastic and glass is up to four times higher for a suspension of free PAF than for a solution of PAF in a protein-bound form (22).

Experimental Protocols

Experiment 1: LPS-induced fever and hypothermia in NAR. The febrile and hypothermic responses to LPS of NAR/SaU, NAR/Slc, and wild-type SD rats were studied. To induce fever, LPS was injected intravenously at a moderate dose of 10 µg/kg at a neutral Ta (30°C). Hypothermia was induced by a high dose (500 µg/kg iv) of LPS at a subneutral Ta (20°C). At either Ta, the controls were injected with saline (1 ml/kg iv). Six rats from each colony were allowed to recover from the experiment for 1 wk and used for verification of albumin content in their blood plasma (see below).

Experiment 2: thermal effect of a [PAF-AGP] complex. At Ta of 30°C, the SD rats were infused with the [PAF-AGP] complex (40 nmol/kg iv), AGP alone (40 nmol/kg iv), or free (aggregated) PAF (40, 80, or 160 nmol/kg iv). All infusions were performed at a rate of 1 ml·kg–1·h–1 over 1 h.

Verification of Analbuminemia of NAR

The absence of albumin in the plasma of the NAR/SaU and NAR/Slc and its presence in the plasma of SD rats were confirmed by electrophoresis. The rats were anesthetized with a small dose of ketamine-xylazine-acepromazine cocktail (5.56, 0.55, and 0.11 mg/kg, respectively, iv), and the arterial blood (3 ml) was collected by cardiac puncture with the use of a sterile syringe. The blood was immediately transferred to a Vacutainer tube (Becton Dickinson, Franklin Lakes, NJ) containing 100 USP units of sodium heparin. The plasma was separated by centrifugation (3,000 g, 10 min) at 4°C and stored at –80°C. The albumin level in the plasma was determined by electrophoresis in agarose gel. The separated proteins were visualized by Amido Black 10B staining.

Data Processing and Analysis

The absolute value of Tc, rather than the change in Tc, was used to evaluate deep body temperature responses in experiments 1 and 2 (for justification, see Ref. 56). The heat loss index (HLI) was used to evaluate thermoeffector responses of tail skin vasculature in experiment 2. As justified elsewhere (50), the HLI was calculated according to the formula: HLI = (Tsk – Ta)/(Tc – Ta). The theoretical limits of the HLI are 0 (maximal skin vasoconstriction) and 1 (maximal vasodilation). In practice, however, the upper limit depends on the position of the tail skin thermocouple. When Tsk is measured at the boundary of the proximal and middle thirds of the tail, as in the present study, the HLI rarely exceeds 0.6. The Tc and HLI responses were compared across treatments and time points by a two-way ANOVA for repeated measures. Tukey's (honest significant difference) post hoc test was used to determine whether there was a significant change in Tc or HLI over time ("effect of time") and whether there was a significant intertreatment difference in a Tc or HLI response ("effect of treatment"). The analysis was performed with Statistica AX'99 (StatSoft, Tulsa, OK). The effects were considered significant when P < 0.05. The data are reported as means ± SE.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Experiment 1: LPS-induced Fever and Hypothermia in NAR

At a neutral Ta of 30°C, the SD rats responded to a moderate dose of LPS (10 µg/kg iv) with a typical polyphasic fever (P < 0.0001, effect of time). As reported in previous studies (e.g., Ref. 54), three consecutive febrile phases peaked at ~60, 150, and 320 min postinjection (Fig. 1A). Febrile responses of similar duration and magnitude were observed in both colonies of analbuminemic rats, NAR/SaU (Fig. 1B) and NAR/Slc (Fig. 1C). A peculiar characteristic of the febrile responses of both NAR/SaU and NAR/Slc was the absence of a clear transition between the second and third febrile phases. In all rats studied, injection of saline was thermally ineffective (Fig. 1).



View larger version (27K):
[in this window]
[in a new window]
  		Fig. 1. Effects of intravenous injection (arrowhead) of lipopolysaccharide (LPS; 10 µg/kg) or saline (1 ml/kg) on the colonic temperature of normalbuminemic Sprague-Dawley (SD) rats (A), Nagase analbuminemic rats from Utrecht University (NAR/SaU; B), and Nagase analbuminemic rats from SLC (NAR/Slc; C). Experiments were conducted at a neutral ambient temperature of 30°C.

 
At a subneutral Ta of 20°C, the SD rats responded to a high dose of LPS (500 µg/kg iv) with a marked hypothermia (P < 0.0001, effect of time). As seen in previous studies (e.g., Ref. 55), this response had a latency of ~40 min and reached its nadir ~110 min postinjection (Fig. 2A). The hypothermic responses of either colony of NAR [NAR/SaU (Fig. 2B) and NAR/Slc (Fig. 2C)] did not differ from the response of the SD rats. Treatment with saline produced no thermal effect (Fig. 2).



View larger version (21K):
[in this window]
[in a new window]
  		Fig. 2. Effects of intravenous injection (arrowhead) of LPS (500 µg/kg) or saline (1 ml/kg) on the colonic temperature of SD rats (A), NAR/SaU (B), and NAR/Slc (C). Experiments were conducted at a subneutral ambient temperature of 20°C.

 
The absence of albumin in NAR/SaU and NAR/Slc was confirmed by comparing the electrophoretic profiles of their plasma proteins with the profile of the SD rats (Fig. 3). Three main protein fractions corresponding to albumin, {alpha}-globulins, and {beta}-globulins (40, 41) were robust in the SD rats. In the NAR of either colony, albumin was missing, but both {alpha}- and {beta}-globulins were overexpressed. The overexpression was marked in the case of {alpha}-globulins and marginal in the case of {beta}-globulins.



View larger version (64K):
[in this window]
[in a new window]
  		Fig. 3. Representative electrophoretograms (2 for each colony) of plasma proteins of SD rats (top), NAR/SaU (middle), and NAR/Slc (bottom). Fractions of interest are marked.

 
Experiment 2: Thermal Effect of a [PAF-AGP] Complex

At thermoneutrality, intravenous infusion of the [PAF-AGP] complex (40 nmol/kg) in the SD rats caused a fever characterized by a short (<10 min) latency and high (~1.5°C) magnitude (P < 0.0002, effect of time; Fig. 4A). A significant (P < 0.004, effect of time) decrease in the HLI (indicates tail skin vasoconstriction) paralleled the febrile rise in Tc (Fig. 4A). AGP alone tended (P < 0.2) to cause a slow (latency, ~40 min), small (magnitude, ~0.5°C) rise in Tc and produced weak vasoconstriction (P < 0.014, effect of time; Fig. 4B), possibly due to the immunogenicity of bovine AGP in the rat or a contamination of the 99% purity AGP preparation used with pyrogenic substances. When infused at a dose of 40 nmol/kg, free PAF was thermally ineffective (Fig. 4C) and so was saline (data not shown). However, to accurately compare effects of the [PAF-AGP] complex with those of free PAF, one should account for the fact that free PAF more readily binds to plastic and glass surfaces than protein-bound PAF. In the experimental paradigm used, the amount of PAF actually received by the animal is up to four times higher in the case of the protein-bound form than the free form (22). Hence, two and four times higher doses of free PAF (that is, 80 and 160 nmol/kg) were also used in the present work. At 80 nmol/kg, free PAF was still less effective than the 40-nmol/kg dose of the [PAF-AGP] complex (P < 0.017, effect of treatment): free PAF caused a very slow (latency of ~90 min), small (magnitude of ~0.5°C) Tc rise accompanied by vasoconstriction (Fig. 4D). Infusion of the highest dose of free PAF (160 nmol/kg) caused death of all the rats at ~50 min (Fig. 4E), thus indicating that the amount of PAF delivered was higher than the amount delivered by infusing the [PAF-AGP] complex (which induced no mortality). However, the fever response to even such a high, lethal dose of free PAF was still substantially lower (maximal rise ~0.5°C; P < 0.0005, effect of treatment) than that to the [PAF-AGP] complex.



View larger version (18K):
[in this window]
[in a new window]
  		Fig. 4. Effects of intravenous infusion (solid bar) of the following substances on the colonic temperature (left ordinate axis) and heat loss index (HLI, right ordinate axis) of SD rats: [PAF-AGP], a 1:1 preformed complex of platelet-activating factor (PAF) and {alpha}1-acid glycoprotein (AGP) at a dose of 40 nmol/kg (A); AGP, 40 nmol/kg (B); free (aggregated) PAF, 40 nmol/kg (C); free PAF, 80 nmol/kg (D); and free PAF, 160 nmol/kg (E). At the highest dose, free PAF caused death ({dagger}) of all rats at ~50 min after the beginning of infusion. The experiments were conducted at a neutral ambient temperature of 30°C.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The first aim of this study was to test whether transport of amphipathic inflammatory mediators by albumin is essential for development of the systemic inflammatory response to LPS. We addressed this aim by studying LPS-induced fever and hypothermia in NAR, a conventional animal model of albumin depletion (40, 41). We have found that both the febrile and hypothermic responses to LPS are normal in NAR, thus indicating that transport of peripherally synthesized, amphipathic mediators by albumin is not essential for LPS-induced thermoregulatory responses. These results have a twofold explanation. The first explanation is that peripherally synthesized, amphipathic mediators of LPS action normally carried by albumin are unimportant for the thermoregulatory responses to LPS. However, this possibility is highly improbable because it contradicts the large body of experimental evidence, suggesting a crucial importance of the circulating pool of at least one of these mediators, PGE2, in at least one of the thermoregulatory responses, fever (see the Introduction). The second, more likely, explanation is that peripheral amphipathic mediators of LPS action are important, but that they are bound to and transported by alternative carriers in NAR.

Albumin binds the majority of ligands with a moderate to low affinity characterized by dissociation constants in a micromolar to millimolar range (47). Because such binding is less specific than a typical ligand-receptor interaction, other carrier proteins can often replace albumin in transporting its customary ligands (14, 21). In the absence of albumin (as happens in NAR), a customary ligand binds to the protein characterized by the most favorable combination of the binding constant and plasma concentration. Such compensation can be strictly functional, i.e., an alternative carrier can pick up the albumin's ligand without being overexpressed. Compensation can also involve overexpression of the alternative carrier; the large list of proteins overexpressed in the absence of albumin (see Refs. 3, 11, 17) includes {alpha}- and {beta}-globulins, as seen in the present study (Fig. 3).

Functions that normally depend on transport of an amphipathic substance by albumin are compensated in NAR both functionally and expressionally. For example, pharmacokinetics of tryptophan (normally transported by albumin) are unaltered in NAR because tryptophan binds to and is carried by {alpha}2-macroglobulin in these rats (9). The level of {alpha}2-macroglobulin is increased in NAR (11); this is an example of expressional compensation. Plasma binding of bilirubin and its delivery to the liver are also normal in NAR (18, 72), possibly due to the bilirubin-binding property of high-density lipoproteins (65). However, this is a purely functional mechanism, as the level of high-density lipoproteins is not increased (and is even decreased) in NAR (see Ref. 17). For other examples of transport of amphipathic ligands by alternative carriers in hypoalbuminemia, see Refs. 9, 14, 21, 65, and 67.

AGP is considered the next important carrier for circulating amphipaths after albumin (20). Although the binding profiles of albumin and AGP are not identical (14, 21), they overlap substantially, and under conditions of analbuminemia AGP carries many amphipathic mediators normally carried by albumin (29, 47). Because the plasma concentration of AGP either remains unchanged (11) or increases (17) in analbuminemia, AGP is likely involved in both functional and expressional compensation. That amphipathic inflammatory mediators (e.g., PAF) bind to AGP in the absence of albumin is known (32, 33). It is unknown, however, whether binding to AGP enhances the ability of the amphipathic mediator to induce a thermoregulatory response, as does its binding to albumin (22, 49). To answer this question for the case of the pyrogenic activity of the AGP-bound form of PAF, a highly potent mediator of LPS action, was the second aim of the present study.

We have found that the intravenous [PAF-AGP] complex is much more pyrogenic than free PAF. The [PAF-AGP] complex, but not the free form, produced a marked fever with a very short latency. Similarly to fevers induced at thermoneutrality by LPS (66), PGE2 (5, 64), or a [PAF-albumin] complex (22), the fever evoked by the [PAF-AGP] complex was brought about, at least partially, by tail skin vasoconstriction (as shown by the associated decrease in the HLI). This is the first demonstration of the ability of a ligand to exhibit a pyrogenic activity when bound to AGP. It is also the first demonstration of an enhanced pyrogenic activity of a ligand in the AGP-bound form. We conclude that circulating AGP may participate in immune-to-brain signaling by transporting PAF and possibly other amphipathic inflammatory mediators in genetic analbuminemia.

Perspectives

The proposed role of AGP can be extended to other cases of hypoalbuminemia. For example, systemic inflammation and sepsis are accompanied by a moderate (up to 2-fold) decrease in the plasma level of albumin (21, 58) and strong (~15-fold) increase in the concentration of AGP (43), which unavoidably results in redistribution of ligands among carrier proteins (21, 71). Under such conditions, the importance of AGP-mediated transport can be expected to increase drastically.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The study was funded by National Institute of Neurological Disorders and Stroke Grant R01 NS-41233 (A. A. Romanovsky).


   ACKNOWLEDGMENTS
 
We thank Dr. Jaap A. Joles (Utrecht University, Utrecht, The Netherlands) for donating the NAR/SaU; Patricia R. Hills, Robert K. Velander, Kathy B. Ulrich, and Marcia K. Schulmerich (Legacy Central Laboratory, Portland, OR) for performing the plasma protein electrophoresis; and Dr. Victoria F. Turek (Oregon Health and Science University, Portland, OR) for reading the manuscript and providing important feedback.

Present address of A. I. Ivanov: Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA 30322. Permanent address of A. Y. Rudaya: Institute of Physiology, National Academy of Sciences, Minsk, Belarus 220072.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. A. Romanovsky, Trauma Research, St. Joseph's Hospital, 350 W. Thomas Rd., Phoenix, AZ 85013 (E-mail: aromano{at}chw.edu)

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. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Abercrombie ED and Jacobs BL. Single-unit response of noradrenergic neurons in the locus coeruleus of freely moving cats. II. Adaptation to chronically presented stressful stimuli. J Neurosci 7: 2844–2848, 1987.[Abstract]
  2. Abul HT, Davidson J, Milton AS, and Rotondo D. Prostaglandin E2 enters the brain following stimulation of the acute phase immune response. Ann NY Acad Sci 813: 287–295, 1997.[Medline]
  3. Baldo-Enzi G, Baiocchi MR, Vigna G, Andrian C, Mosconi C, and Fellin R. Analbuminaemia: a natural model of metabolic compensatory systems. J Inherit Metab Dis 10: 317–329, 1987.[Medline]
  4. Coceani F, Bishai I, Engelberts D, House RV, and Adamson SL. Response of newborn and adult sheep to pyrogens: relation between fever and brain eicosanoid changes. Brain Res 700: 191–204, 1995.[CrossRef][Web of Science][Medline]
  5. Crawshaw LI and Stitt JT. Behavioural and autonomic induction of prostaglandin E1 fever in squirrel monkeys. J Physiol 244: 197–206, 1975.[Abstract/Free Full Text]
  6. Dascombe MJ and Milton AS. Study on the possible entry of bacterial endotoxin and prostaglandin E2 into the central nervous system from the blood. Br J Pharmacol 66: 565–572, 1979.[Web of Science][Medline]
  7. Edmonds JW and Duax WL. Molecular conformation of prostaglandin E2. Prostaglandins 5: 275–281, 1974.[CrossRef][Web of Science][Medline]
  8. Eguchi N, Hayashi H, Urade Y, Ito S, and Hayashi O. Central action of prostaglandin E2 and its methyl ester in the induction of hyperthermia after their systemic administration in urethane-anesthetized rats. J Pharmacol Exp Ther 247: 671–679, 1988.[Abstract/Free Full Text]
  9. Emori T, Sugiyama K, and Nagase S. Tryptophan metabolism in analbuminemic rats. J Biochem (Tokyo) 94: 623–632, 1983.[Free Full Text]
  10. Esumi H, Takahashi Y, Sato S, Nagase S, and Sugimura T. A seven-base-pair deletion in an intron of the albumin gene of analbuminemic rats. Proc Natl Acad Sci USA 80: 95–99, 1983.[Abstract/Free Full Text]
  11. Goto K, Saito A, Nagase S, and Sinohara H. Acute phase response of plasma proteins in analbuminemic rats. J Biochem (Tokyo) 104: 952–955, 1988.[Abstract/Free Full Text]
  12. Hashimoto K, Suemaru S, Takao T, Sugawara M, Makino S, and Ota Z. Corticotropin-releasing hormone and pituitary-adrenocortical responses in chronically stressed rats. Regul Pept 23: 117–126, 1988.[CrossRef][Web of Science][Medline]
  13. Heirwegh KP. Effects of binding of amphipathic compounds to carrier proteins on their enzymatic biotransformation by membrane-bound enzymes. Biochem Soc Trans 12: 11–13, 1984.[Web of Science][Medline]
  14. Herve F, Urien S, Albengres E, Duche JC, and Tillement JP. Drug binding in plasma. A summary of recent trends in the study of drug and hormone binding. Clin Phamacokinet 26: 44–58, 1994.
  15. Hirate J, Horikoshi I, and Nagase S. Disposition of phenytoin in analbuminemic rats. Biopharm Drug Dispos 11: 773–783, 1990.[CrossRef][Web of Science][Medline]
  16. Hirate J, Kato Y, Horikoshi I, Nagase S, and Ueda CT. Further observations on the disposition characteristics of salicylic acid in analbuminemic rats. Biopharm Drug Dispos 10: 299–309, 1989.[CrossRef][Web of Science][Medline]
  17. Inoue M. Metabolism and transport of amphipathic molecules in analbuminemic rats and human subjects. Hepatology 5: 892–898, 1985.[Web of Science][Medline]
  18. Inoue M, Hirata E, Morino Y, Nagase S, Chowdhury JR, Chowdhury NR, and Arias IM. The role of albumin in the hepatic transport of bilirubin: studies in mutant analbuminemic rats. J Biochem (Tokyo) 97: 737–743, 1985.[Abstract/Free Full Text]
  19. Inoue M, Koyama H, Nagase S, and Morino Y. Hepato-renal transport of phenolsulfonphthalein in analbuminemic rats: albumin is essential for hepatic compensatory elimination of a nephrophilic ligand in animals with renal dysfunction. J Biochem (Tokyo) 100: 201–206, 1986.[Abstract/Free Full Text]
  20. Israili ZH and Dayton PG. Human alpha-1-glycoprotein and its interactions with drugs. Drug Metab Rev 33: 161–235, 2001.[CrossRef][Web of Science][Medline]
  21. Ivanov AI, Gavrilov VB, Furmanchuk DA, Aleinikova OV, Konev SV, and Kaler GV. Fluorescent probing of the ligand-binding ability of blood plasma in the acute-phase response. Clin Exp Med 2: 147–155, 2002.[CrossRef][Web of Science][Medline]
  22. Ivanov AI, Patel S, Kulchitsky VA, and Romanovsky AA. Platelet-activating factor: a previously unrecognized mediator of fever. J Physiol 553: 221–228, 2003.[Abstract/Free Full Text]
  23. Ivanov AI, Pero RS, Sheck AC, and Romanovsky AA. Prostaglandin E2-synthesizing enzymes in fever: differential transcriptional regulation. Am J Physiol Regul Integr Comp Physiol 283: R1104–R1117, 2002.[Abstract/Free Full Text]
  24. Ivanov AI and Romanovsky AA. Prostaglandin E2 as a mediator of fever: synthesis and catabolism. Front Biosci 9: 1977–1993, 2004.[Web of Science][Medline]
  25. Joles JA, Willekes-Koolschijn N, Braam B, Kortlandt W, Koomans HA, and Dorhout Mees EJ. Colloid osmotic pressure in young analbuminemic rats. Am J Physiol Renal Fluid Electrolyte Physiol 257: F23–F28, 1989.[Abstract/Free Full Text]
  26. Joles JA, Willekes-Koolschijn N, van Tol A, Geelhoed-Mieras MM, Danse LH, van Garderen E, Kortlandt W, Erkelens DW, and Koomans HA. Hyperlipoproteinemia in one-year-old analbuminemic rats. Atherosclerosis 88: 35–47, 1991.[CrossRef][Web of Science][Medline]
  27. Jones SA, Adamson SL, Engelberts D, Bishai I, Norton J, and Coceani F. PGE2 in the perinatal brain: local synthesis and transfer across the blood brain barrier. J Lipid Mediators 6: 487–492, 1993.[Web of Science][Medline]
  28. Kramp W, Pieroni G, Pinckard RN, and Hanahan DJ. Observations on the critical micellar concentration of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine and a series of its homologs and analogs. Chem Phys Lipids 35: 49–62, 1984.[CrossRef][Web of Science][Medline]
  29. Kremer JMH, Wilting J, and Janssen LMH. Drug binding to human {alpha}-1-glycoprotein in health and disease. Pharmacol Rev 40: 1–48, 1988.[Web of Science][Medline]
  30. Kuipers B, van Der Poll T, Levi M, van Deventer SJ, ten Cate H, Imai Y, Hack CE, and ten Cate JW. Platelet-activating factor antagonist TCV-309 attenuates the induction of the cytokine network in experimental endotoxemia in chimpanzees. J Immunol 152: 2438–2446, 1994.[Abstract]
  31. Kulikov VI and Bergel'son LD. Binding of 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine (thrombocyte-activating factor) by plasma components. Exchange of the thrombocyte-activating factor between lipoproteins and thrombocytes. Biokhimiia 49: 1310–1315, 1984 (in Russian).[Medline]
  32. McNamara PJ, Brouwer KR, and Gillespie MN. Autacoid binding to serum proteins. Interaction of platelet activating factor (PAF) with human serum alpha-1-acid glycoprotein (AGP). Biochem Pharmacol 35: 621–624, 1986.[CrossRef][Web of Science][Medline]
  33. McNamara PJ, Jewell RC, and Gillespie MN. The interaction of alpha-1-acid glycoprotein with endogenous autacoids, in particular, platelet activating factor (PAF). Prog Clin Biol Res 300: 307–319, 1989.[Medline]
  34. Melia KR, Ryabinin AE, Schroeder R, Bloom FE, and Wilson MC. Induction and habituation of immediate early gene expression in rat brain by acute and repeated restraint stress. J Neurosci 14: 5929–5938, 1994.[Abstract]
  35. Milton AS. Prostaglandins and fever. Prog Brain Res 115: 129–139, 1998.[Web of Science][Medline]
  36. Minamiyama Y, Takemura S, and Inoue M. Albumin is an important vascular tonus regulator as a reservoir of nitric oxide. Biochem Biophys Res Commun 225: 112–115, 1996.[CrossRef][Web of Science][Medline]
  37. Morimoto A, Morimoto K, Watanabe T, Sakata Y, and Murakami N. Does an increase in prostaglandin E2 in the blood circulation contribute to a febrile response in rabbits? Brain Res Bull 29: 189–192, 1992.[CrossRef][Web of Science][Medline]
  38. Morimoto A, Murakami N, Nakamori T, and Watanabe T. Evidence for separate mechanisms of induction of biphasic fever inside and outside the blood-brain barrier in rabbits. J Physiol 383: 629–637, 1987.[Abstract/Free Full Text]
  39. Nagase S. Studies on analbuminemic rats. Prog Clin Biol Res 229: 27–34, 1987.[Medline]
  40. Nagase S, Shimamune K, and Shumiya S. Albumin-deficient rat mutant. Science 205: 590–591, 1979.[Abstract/Free Full Text]
  41. Nagase S, Shimamune K, and Shumiya S. Albumin-deficient rat mutant: an animal model for analbuminemia. Jikken Dobutsu 29: 33–38, 1980.[Medline]
  42. Okajima K, Inoue M, Itoh K, Nagase S, and Morino Y. Role of plasma albumin in renal elimination of a mercapturic acid. Analyses in normal and mutant analbuminemic rats. Eur J Biochem 150: 195–199, 1985.[Web of Science][Medline]
  43. Ostberg JR, Taylor SL, Baumann H, and Repasky EA. Regulatory effects of fever-range whole-body hyperthermia on the LPS-induced acute inflammatory response. J Leukoc Biol 68: 815–820, 2000.[Abstract/Free Full Text]
  44. Otagiri M, Maruyama T, Imai T, Suenaga A, and Imamura Y. A comparative study of the interaction of warfarin with human {alpha}1-acid glycoprotein and human albumin. J Pharm Pharmacol 39: 416–420, 1987.[Web of Science][Medline]
  45. Parrott RF, Vellucci SV, Forsling ML, and Goode JA. Hyperthermic and endocrine effects of intravenous prostaglandin administration in the pig. Domest Anim Endocrinol 12: 197–205, 1995.[CrossRef][Web of Science][Medline]
  46. Paul L, Fraifeld V, and Kaplanski J. Evidence supporting involvement of leukotrienes in LPS-induced hypothermia in mice. Am J Physiol Regul Integr Comp Physiol 276: R52–R58, 1999.[Abstract/Free Full Text]
  47. Peters T Jr. All About Albumin: Biochemistry, Genetics and Medical Applications. San Diego, CA: Academic, 1996.
  48. Raz A. Interaction of prostaglandins with blood plasma proteins. I. Binding of prostaglandin E2 to human plasma proteins and its effect on the physiological activity of prostaglandin E2 in vitro and in vivo. Biochim Biophys Acta 280: 602–613, 1972.[Medline]
  49. Romanovsky AA, Ivanov AI, and Karman EK. Blood-borne, albumin-bound prostaglandin E2 may be involved in fever. Am J Physiol Regul Integr Comp Physiol 276: R1840–R1844, 1999.[Abstract/Free Full Text]
  50. Romanovsky AA, Ivanov AI, and Schimansky YP. Ambient temperature for experiments in rats: a new method for determining the zone of thermal neutrality. J Appl Physiol 92: 2667–2679, 2002.[Abstract/Free Full Text]
  51. Romanovsky AA, Kulchitsky VA, Akulich NV, Koulchitsky SV, Simons CT, Sessler DI, and Gourine VN. First and second phases of biphasic fever: two sequential stages of the sickness syndrome? Am J Physiol Regul Integr Comp Physiol 271: R244–R253, 1996.[Abstract/Free Full Text]
  52. Romanovsky AA, Kulchitsky VA, Simons CT, and Sugimoto N. Methodology of fever research: why are polyphasic fevers often thought to be biphasic? Am J Physiol Regul Integr Comp Physiol 275: R332–R338, 1998.[Abstract/Free Full Text]
  53. Romanovsky AA, Shido O, Sakurada S, Sugimoto N, and Nagasaka T. Endotoxin shock: thermoregulatory mechanisms. Am J Physiol Regul Integr Comp Physiol 270: R693–R703, 1996.[Abstract/Free Full Text]
  54. Romanovsky AA, Simons CT, and Kulchitsky VA. "Biphasic" fevers often consist of more than two phases. Am J Physiol Regul Integr Comp Physiol 275: R323–R331, 1998.[Abstract/Free Full Text]
  55. Romanovsky AA, Simons CT, Székely M, and Kulchitsky VA. The vagus nerve in the thermoregulatory response to systemic inflammation. Am J Physiol Regul Integr Comp Physiol 273: R407–R413, 1997.[Abstract/Free Full Text]
  56. Romanovsky AA, Sugimoto N, Simons CT, and Hunter WS. The organum vasculosum laminae terminalis (OVLT) in immune-to-brain febrigenic signaling: a reappraisal of lesion experiments. Am J Physiol Regul Integr Comp Physiol 285: R420–R428, 2003.[Abstract/Free Full Text]
  57. Rotondo D, Abul HT, Milton AS, and Davidson J. Pyrogenic immunomodulators increase the level of prostaglandin E2 in the blood simultaneously with the onset of fever. Eur J Pharmacol 154: 145–152, 1988.[CrossRef][Web of Science][Medline]
  58. Ruot B, Papet I, Béchereau F, Denis P, Buffière C, Gimonet J, Glomot F, Elyousfi M, Breuillé D, and Obled C. Increased albumin plasma efflux contributes to hypoalbuminemia only during early phase of sepsis in rats. Am J Physiol Regul Integr Comp Physiol 284: R707–R713, 2003.[Abstract/Free Full Text]
  59. Shalaby F and Shafritz DA. Exon skipping during splicing of albumin mRNA precursors in Nagase analbuminemic rats. Proc Natl Acad Sci USA 87: 2652–2656, 1990.[Abstract/Free Full Text]
  60. Siren AL. Central cardiovascular and thermal effects of prostaglandin E2 in rats. Acta Physiol Scand 116: 229–234, 1982.[Web of Science][Medline]
  61. Skarnes RC, Brown SK, Hull SS, and McCracken. Role of prostaglandin E in the biphasic fever response to endotoxin. J Exp Med 154: 1212–1224, 1981.[Abstract/Free Full Text]
  62. Stamp JA and Herbert J. Multiple immediate-early gene expression during physiological and endocrine adaptation to repeated stress. Neuroscience 94: 1313–1322, 1999.[CrossRef][Web of Science][Medline]
  63. Steiner AA, Dogan MD, Ivanov AI, Patel S, Rudaya AY, Jennings DH, Orchinik M, Pace TWW, O'Connor KA, Watkins LR, and Romanovsky AA. A new function of the leptin receptor: mediation of the recovery from lipopolysaccharide-induced hypothermia. FASEB J 18: 1949–1951, 2004.[Abstract/Free Full Text]
  64. Stitt JT. Prostaglandin E1 fever induced in rabbits. J Physiol 232: 163–179, 1973.[Abstract/Free Full Text]
  65. Suzuki N, Yamaguchi T, and Nakajima H. Role of high-density lipoprotein in transport of circulating bilirubin in rats. J Biol Chem 263: 5037–5043, 1988.[Abstract/Free Full Text]
  66. Székely M and Szelényi Z. Endotoxin fever in the rat. Acta Physiol Acad Sci Hung 53: 265–277, 1979.[Web of Science][Medline]
  67. Tsao SC, Sugiyama Y, Shinmura K, Sawada Y, Nagase S, Iga T, and Hanano M. Protein-mediated hepatic uptake of rose bengal in analbuminemic mutant rats (NAR). Albumin is not indispensable to the protein-mediated transport of rose bengal. Drug Metab Dispos 16: 482–489, 1988.[Abstract]
  68. Ueno R, Narumiya S, Ogorochi T, Nakayama T, Ishikawa Y, and Hayaishi O. Role of prostaglandin D2 in the hypothermia of rats caused by bacterial lipopolysaccharide. Proc Natl Acad Sci USA 79: 6093–6097, 1982.[Abstract/Free Full Text]
  69. Unger WG. Binding of prostaglandin to human serum albumin. J Pharm Pharmacol 24: 470–477, 1972.[Web of Science][Medline]
  70. Urien S, Giroud Y, Tsai RS, Carrupt PA, Brée F, Testa B, and Tillement JP. Mechanism of ligand binding to {alpha}1-acid glycoprotein (orosomucoid): correlated thermodynamic factors and molecular parameters of polarity. Biochem J 306: 545–549, 1995.[Medline]
  71. Wasan KM and Cassidy SM. Role of plasma lipoproteins in modifying the biological activity of hydrophobic drugs. J Pharm Sci 87: 411–427, 1998.[CrossRef][Web of Science][Medline]
  72. Yamashita M, Adachi Y, Kambe A, Nanno T, Nagase S, and Yamamoto T. Serum binding and biliary excretion of bilirubin after bilirubin loading in Nagase analbuminemic rats and heterozygous (Jj) Gunn rats. J Lab Clin Med 112: 443–449, 1988.[Web of Science][Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
Y. Ootsuka, W. W. Blessing, A. A. Steiner, and A. A. Romanovsky
Fever response to intravenous prostaglandin E2 is mediated by the brain but does not require afferent vagal signaling
Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2008; 294(4): R1294 - R1303.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
A. A. Romanovsky
Thermoregulation: some concepts have changed. Functional architecture of the thermoregulatory system
Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2007; 292(1): R37 - R46.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
A. A. Romanovsky
Vioxx, Celebrex, Bextra....Do we have a new target for anti-inflammatory and antipyretic therapy?
Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2005; 288(5): R1098 - R1099.
[Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
S. Saha, L. Engstrom, L. Mackerlova, P.-J. Jakobsson, and A. Blomqvist
Impaired febrile responses to immune challenge in mice deficient in microsomal prostaglandin E synthase-1
Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2005; 288(5): R1100 - R1107.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
288/4/R872    most recent
00514.2004v1
Right arrow Alert me when this article is cited
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 (8)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ivanov, A. I.
Right arrow Articles by Romanovsky, A. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ivanov, A. I.
Right arrow Articles by Romanovsky, A. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.