The effects of the cyclosporin A, a P-glycoprotein inhibitor, on the pharmacokinetics of baicalein in the rat: a microdialysis study.

1. Baicalein is a bioactive flavonoid isolated from the root of Scutellaria baicalensis Georgi, a medicinal herb that has been used since ancient times to treat bacterial infections. As little is known concerning its pharmacokinetics, this study focussed on its pharmacokinetics as well as the possible roles of the multidrug transporter P-glycoprotein on its distribution and disposition. 2. Three microdialysis probes were simultaneously inserted into the jugular vein, the hippocampus and the bile duct of male Sprague-Dawley rats for sampling in biological fluids following the administration of baicalein (10, 30 and 60 mg kg(-1)) through the femoral vein. The P-glycoprotein inhibitor cyclosporin A was used to help delineate its roles. 3. The study design consisted of two groups of six rats in parallel: control rats which received baicalein alone and the cyclosporin A treated-group in which the rats were injected cyclosporin A, a P-glycoprotein inhibitor, 10 min prior to baicalein administration. 4. Cyclosporin A treatment resulted in a significant increase in elimination half-life, mean residence time and area under the concentration versus time curve (AUC) of unbound baicalein in the brain. However, AUC in the bile was decreased. 5. The decline of baicalein in the hippocampus, blood and bile suggested that there was rapid exchange and equilibration between the peripheral compartment and the central nervous system. In addition, the results indicated that baicalein was able to penetrate the blood-brain barrier as well as undergoing hepatobiliary excretion. 6. Although no direct transport studies were undertaken and multiple factors may affect BBB penetration and hepatobiliary excretion, strong association of the involvement of P-glycoprotein in these processes is indicated.

Calcium channel blockade in vascular smooth muscle cells: major hypotensive mechanism of S-petasin, a hypotensive sesquiterpene from Petasites formosanus.

In vivo and in vitro studies were carried out to examine the putative hypotensive actions of S-petasin, a sesquiterpene extracted from the medicinal plant Petasites formosanus. Intravenous S-petasin (0.1-1.5 mg/kg) in anesthetized rats produced a dose-dependent hypotensive effect. In isolated aortic ring, isometric contraction elicited by KCl or the L-type Ca2+ channel agonist Bay K 8644 was reduced by S-petasin (0.1-100 microM), an action not affected by the cyclooxygenase inhibitor indomethacin, nitric-oxide synthase inhibitor N(omega)-nitro-L-arginine, guanylyl cyclase inhibitor methylene blue, or removal of vascular endothelium. Pretreatment with S-petasin for 10 min shifted the concentration-response curve for KCl (15-90 mM)-induced contraction to the right and reduced the maximal response. In Ca2+-depleted and high K+-depolarized aortic rings preincubation with S-petasin attenuated the Ca2+-induced contraction in a concentration-dependent manner, suggesting that S-petasin reduced Ca2+ influx into vascular smooth muscle cells (VSMCs). Moreover, in cultured VSMCs, whole-cell patch-clamp recording indicated that S-petasin (1-50 microM) inhibited the L-type voltage-dependent Ca2+ channel (VDCC) activities. Intracellular Ca2+ concentration ([Ca2+[(i)) estimation using the fluorescent probe 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2'-amino-5'-methylphenoxy)-ethane-N,N,N,N-tetraacetic acid pentaacetoxymethyl ester indicated that S-petasin (10, 100 microM) suppressed the KCl-stimulated increase in ([Ca2+[(i)). Taken together, the results suggested that a direct Ca2+ antagonism of L-type VDCC in vascular smooth muscle may account, at least in part, for the hypotensive action of S-petasin.

Production of macrophage inflammatory protein-2 following hypoxia/reoxygenation in glial cells.

Polymorphonuclear neutrophils (PMNs) are known to mediate brain inflammation following hypoxia/reoxygenation (H/R), but the precise mechanisms leading to PMN recruitment are undefined. The alpha-chemokine macrophage inflammatory protein-2 (MIP-2) has specificity for the recruitment of PMNs. In this study, we found that 8 or 12 h of hypoxia followed by 24-h reoxygenation (H8/R24 or H12/R24) induced MIP-2 secretion in cultures of enriched microglia or mixed glia, respectively. Microglia, however, could not survive longer duration (>12 h) of hypoxia. Astrocytes did not produce any significant amount of MIP-2 even though astrocytes maintained 98-99% viability following H12/R24. We also found that microglia survived the H/R treatment better (following H24) in the presence of astrocytes (mixed glial culture) than in microglia-enriched culture. Reoxygenation for prolonged periods (3 and 5 days) following H24 resulted in progressively larger increases in MIP-2 production (20- and 60-fold, respectively) in mixed glial cultures. Immunocytochemical staining revealed that the cells expressing MIP-2 in response to H/R were microglia rather than astrocytes in mixed glial cultures. Examination of MIP-2 mRNA expression showed that H/R upregulated MIP-2 gene expression. Taken together, our data suggest that microglial cells are an important source of MIP-2 production and suggest a potential injury mechanism involving brain-derived production of MIP-2 in H/R.

Simultaneous blood and brain sampling of cephalexin in the rat by microdialysis and microbore liquid chromatography: application to pharmacokinetics studies.

To circumvent the need for laborious sample clean-up and multiple blood sampling, a system was developed consisting of on-line microdialysis coupled to microbore liquid chromatography and ultraviolet detection. The system was designed for the simultaneous and continuous monitoring of unbound blood and brain cephalexin in the rat following single bolus intravenous administrations (10 mg/kg, n = 6). Microdialysis probes were inserted into the jugular vein and brain striatum, respectively, for blood and brain sampling. Chromatographic conditions consisted of a mobile phase of methanol-100 mM monosodium phosphoric acid (20:80, v/v, pH 5.0) pumped through a microbore reversed-phase column at a flow-rate of 0.05 ml/min. Detection wavelength was set at 260 nm. The method was validated for response linearity as well as intra- and inter-day variabilities. Rapid appearance of cephalexin in the striatal dialysate suggested good blood-brain barrier penetration. This study provided pharmacokinetics information for cephalexin as well as demonstrated the applicability of this continuous sampling method for pharmacokinetics studies.

Concurrent quantification and pharmacokinetic analysis of cefotaxime in rat blood and brain by microdialysis and microbore liquid chromatography.

A simple but effective coupling of microdialysis and microbore liquid chromatograph with UV detection technique was applied to the simultaneous studying of the pharmacokinetics of cefotaxime in both the peripheral compartment and central nervous system. The mobile phase consisted of methanol-100 mM monosodium phosphoric acid (25:75, v/v, pH 5.5) pumped through a C18 microbore column at a flow-rate of 0.05 ml/min. Detection of cefotaxime was set at a UV wavelength of 254 nm. Microdialysis probes were inserted into the jugular vein and striatum of the rat. Following stabilization of microdialysate levels, rats received cefotaxime (20 mg/kg, i.v., n=6) via the femoral vein, and complete concentration versus time profiles for blood and striatum were constructed. The results indicated that cefotaxime rapidly (within 10 min) entered the extracellular fluid of brain striatum following intravenous administration. Noncompartmental pharmacokinetics analysis indicated that the area under the concentration versus time ratio of cefotaxime in rat brain and blood was 6.9%, suggesting appreciable blood-brain barrier penetration. The method was relatively simple, imposed minimal physiological perturbance as it involved no body fluid consumption and sampled in particular protein-unbound drugs, generally believed to be the active fraction.

Determination of chlorogenic acid in rat blood by microdialysis coupled with microbore liquid chromatography and its application to pharmacokinetic studies.

To investigate the pharmacokinetics of unbound chlorogenic acid, a sensitive microbore liquid chromatographic method for the determination of chlorogenic acid in rat blood by microdialysis has been developed. A microdialysis probe was inserted into the jugular vein of male Sprague-Dawley rats, to which chlorogenic acid (20, 40, 60 or 80 mg/kg, i.v.) had been administered. On-line microdialysate was directly injected into a microbore column using a methanol-100 mM sodium dihydrogenphosphate (30:70, v/v, pH 2.5 adjusted with orthophosphoric acid) as the mobile phase and ultraviolet detection at 325 nm. The method is rapid, easily reproduced, selective and sensitive. The limit of detection for chlorogenic acid was 0.01 microg/ml and the limit of quantification was 0.05 microg/ml. The in vivo recovery of the chlorogenic acid of the microdialysis probe, based on a 5 microg/ml standard, was approximately 49-65% (n=6). The disposition of chlorogenic acid at each dose was best fitted to a two-compartment pharmacokinetic model. The area under the concentration curve increased greater than in direct proportion with the dose and terminal disposition become much slower as the dose was increased. The results indicated that the pharmacokinetics of unbound chlorogenic acid in rat blood is non-linear.

Enterohepatic circulation of chloramphenicol and its glucuronide in the rat by microdialysis using a hepato-duodenal shunt.

A system consisting of a hepato-duodenal shunt in which the bile of a drug-treated donor rat was diverted to the duodenum of an untreated recipient rat via a bile cannula was used to assess the role of hepatic metabolism and enterohepatic circulation in the pharmacokinetics of chloramphenicol. Blood concentrations of unbound chloramphenicol and its glucuronide were measured by on-line microdialysis coupled to a microbore liquid chromatographic system. Results indicated that chloramphenicol and its glucuronide were detected in the blood of both donor and recipient rats following an intravenous 100 mg/kg dose of chloramphenicol succinate to the donor rat. Our finding suggests that although enterohepatic circulation contributed only to a minor extent (approximately 1.8%) was involved in the disposition of unbound chloramphenicol in the rat on-line microdialysis techniques were applicable for such studies.

Simultaneous blood and biliary sampling of esculetin by microdialysis in the rat.

Biliary excretion and intestinal reabsorption in enterohepatic circulation play major dispositional roles for some drugs. To circumvent multiple blood sampling and interruption of enterohepatic circulation in conventional biliary cannulation, the present study utilized the minimally invasive sampling technique of microdialysis in pharmacokinetics and biliary excretion studies. Microdialysis probes were inserted into the jugular vein and bile duct in the anesthetized rat for simultaneous and continuous sampling following intravenous administration of esculetin, a bioactive coumarin derivative. Placements of the microdialysis probes were designed to minimize obstruction to normal flows of the body fluids. Separation and quantitation of esculetin in the dialysates were achieved using high performance liquid chromatography (HPLC) coupled to UV detection. The results indicated higher drug concentrations in the bile than in the blood, suggesting active biliary excretion. The study also provided an example of successful application of in vivo microdialysis as an interesting and feasible alternative for pharmacokinetics and biliary drug excretion studies.

Inhibition of aldosterone production by testosterone in male rats.

In vivo and in vitro experiments were designed to assess the effect of testosterone on aldosterone secretion in male rats. Orchidectomized rats were injected subcutaneously with oil or testosterone propionate ([TP] 2 mg/kg) for 7 days. Intact rats were injected with oil only. The results indicate that the plasma aldosterone level was higher in orchidectomized versus intact and TP-replaced rats. In the in vitro study, testosterone caused a marked decrease of aldosterone secretion by zona glomerulosa (ZG) cells, but failed to alter the accumulation of intracellular adenosine 3',5'-cyclic monophosphate (cAMP). Testosterone significantly decreased the corticotropin (ACTH)-stimulated production of aldosterone and accumulation of cAMP in rat ZG cells. The conversion of corticosterone to aldosterone and of 25-OH-cholesterol to pregnenolone, as well as angiotensin II (ANG II)-stimulated production of aldosterone, were decreased by testosterone. These results suggest that testosterone inhibits the basal and ANG II- and ACTH-stimulated release of aldosterone, via inhibition of aldosterone synthase activity and cytochrome P-450 side-chain cleavage (P450scc) activity, and ACTH-stimulated cAMP accumulation in rat ZG cells.

Ethanol modulates induction of nitric oxide synthase in glial cells by endotoxin.

Although ethanol has long been recognized as an immunosuppressant, the effects of ethanol on immune functions in the central nervous system (CNS) have not been well characterized. Glial cells function as immune effector cells within the CNS. Nitric oxide (NO), generated by inducible NO synthase (iNOS) of activated glial cells, appears to participate in the immune defense and the pathogenesis of brain injury and several neurologic diseases. The goal of the present study was to examine the effects of ethanol on NO production and mRNA expression of iNOS following its induction by bacterial endotoxin lipopolysaccharide (LPS) in cultured glial cells. After incubation of mixed glia with LPS for 24 hr, the levels of nitrite in the culture medium were assayed by Griess reaction. We found that LPS (10-500 ng/ml) induced a concentration-dependent increase in the production of NO which was abolished by the selective iNOS inhibitor aminoguanidine. While ethanol treatment (25 to 400 mM, 24 hr exposure) had no direct effect on basal NO production, it significantly suppressed the LPS-induced increase of nitrite levels in a concentration-dependent manner. Using a semiquantitative reverse transcriptase polymerase chain reaction, we found that while ethanol by itself was unable to induce iNOS mRNA, it nevertheless suppressed LPS-induced iNOS mRNA expression. Our results that ethanol had no direct effect on NO production but inhibited LPS-induced NO, indicated an immunomodulatory role by ethanol. These findings suggest that ethanol may ameliorate the consequences of overwhelming NO generation through iNOS induction in glial cells following infection, inflammation or CNS injuries.

Inhibitory effect of dehydroevodiamine and evodiamine on nitric oxide production in cultured murine macrophages.

Possible antiinflammatory effects of dehydroevodiamine (1) and evodiamine (2) were examined by assessing their effects on NO production in the murine macrophage-like cell line RAW 264.7. The results indicated that both 1 and 2 inhibited the IFN-gamma/LPS-stimulated NO production in a concentration-dependent manner. However, 1 appeared to inhibit NO production by interfering not only with the priming signal initiated by IFN-gamma but also with iNOS protein synthesis, while 2 affected the former only.

Studies of the cellular mechanisms underlying the vasorelaxant effects of rutaecarpine, a bioactive component extracted from an herbal drug.

We conducted studies to investigate the nature and underlying mechanisms of the vascular effects of rutaecarpine (Rut), an alkaloid isolated from the Chinese herbal drug Evodia rutaecarpa. By using largely the effects on phenylephrine (PE)-induced contraction in the isolated rat aorta as the experimental index and by comparison with several known vascular muscle relaxants such as acetylcholine (ACh), histamine, and A23187, Rut relaxed PE-precontracted aorta in concentration-(10(-7)-10(-4) M) and endothelium-dependent manners. Studies with appropriate antagonists indicated that this was coupled to nitric oxide (NO) and guanylyl cyclase. Extracellular Ca2+ removal and treatment with the intracellular Ca2+ antagonist, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), suggested that influx of extracellular Ca2+ was the major factor contributing to the action of Rut. Pertussis toxin suppressed the relaxation potency of histamine but had no effects on the actions of Rut. NaF, the G proteins activator, attenuated the actions of ACh, but only minimally affected Na-NP, A23187, and Rut. 1-[6-{[17 beta-3-methoxyestra-1,2,3(10)-trien-17-yl]amino} hexyl]-1H-pyrrole-2,5-dione (U73122), the phospholipase C inhibitor, again suppressed the actions of ACh but had few effects on A23187 and Rut. Taken together, these results suggest that these vasorelaxants had different cellular mechanisms and that neither pertussis toxin-sensitive Gi protein, other G proteins, nor phospholipase C activation was involved in the cellular response to rutaecarpine.

Central serotonergic lesions increase voluntary alcohol consumption in Sprague Dawley rats: moderation by long-term ethanol administration.

The relationship between central serotonergic activities and voluntary alcohol consumption was studied in Sprague-Dawley rats, which normally have low alcohol preference. After initial screening for an evenly matched baseline alcohol preference, selective central serotonergic lesioning was induced by intracisternal injection of the serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT). Control rats received injections of vehicle only. Both 5,7-DHT and vehicle-treated rats were further divided into two subgroups, which either had continued free access to ethanol (alcohol-drinking) or were deprived of it (alcohol-free). All rats were then tested again for alcohol preference. All rats were then killed, and the levels of monoamines in the brains were determined by high performance liquid chromatography with electrochemical detection. Behavioral results indicated that all 5,7-DHT-treated rats had significantly higher alcohol preference and consumption than the corresponding sham controls. Except in the cerebellum, the 5,7-DHT-treated rats had significantly lower levels of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in most brain regions compared with those in the corresponding sham controls. Treatment with 5,7-DHT also resulted in a decrease in serotonin turnover in all brain regions in the alcohol-free rats, except in the cerebellum. In alcohol-drinking rats, however, 5,7-DHT treatment only reduced serotonin turnover in the pons. The levels of norepinephrine and dopamine in several brain regions were not significantly different. Thus, it appeared that in the Sprague Dawley rats, 5,7-DHT treatment depleted 5-HT and 5-HIAA levels in most brain regions while increasing alcohol consumption. Chronic alcohol-drinking attenuated the increase in alcohol consumption associated with serotonergic lesions. Voluntary alcohol consumption seemed more associated with 5-HT turnover than with tissue 5-HT levels. Our data also suggested that tolerance to alcohol-induced hypothermia was primarily attributable to long-term alcohol drinking rather than serotonergic lesioning.

Mechanisms of vasorelaxant effect of dehydroevodiamine: a bioactive isoquinazolinocarboline alkaloid of plant origin.

We examined the mechanisms underlying the vasorelaxant effect of dehydroevodiamine (DeHE), one of the bioactive components of the Chinese herbal drug Evodia rutaecarpa that has been shown to produce vasorelaxant and hypotension. DeHE (10(-7)-10(-4) M) concentration-dependently relaxed isolated rat mesenteric arteries precontracted with phenylephrine (PE). This vasorelaxant potency was diminished by 15% by endothelial removal, L-NG-nitro arginine, or methylene blue (MB), but not indomethacin treatment, indicating that the vasorelaxant effect of DeHE was partially endothelium dependent and mediated by nitric oxide (NO) and the cyclic GMP pathway. In endothelium-denuded preparations, DeHE caused a rightward shift of the contractile concentration-response curve (CRC) to PE in a dose-dependent manner with a pA2 value of 6.15. Maximal response was unaffected. Receptor binding assay indicated that DeHE competed with alpha 1-adrenoceptor ligand prazosin with a Ki value of 3.57 microM. Potassium channel activity-attenuating conditions such as increased level of extracellular K+ (20 mM) and treatment with the antagonist tetraethylammonium (TEA) significantly inhibited DeHE's effect, suggesting a mode of action similar to that of a potassium channel activator. In addition, high concentrations of DeHE (3 x 10(-5) and 10(-4) M) relaxed high K+ (80 mM)-evoked contraction, indicating that DeHE might possess K+ channel blocking properties. Multiple-action mechanisms, including endothelium dependence, alpha 1-adrenoceptor blockade, K+ channel activation, and Ca2+ channel blockade were probably involved in the vasorelaxant effects of DeHE.

Role of plasma catecholamines, autonomic, and left ventricular function in normotensive and hypotension prone dialysis patients.

The current study looked at plasma catecholamines, clinical autonomic function tests, and hemodynamic parameters in 10 ESRD patients (five men and five woman, aged 56.4 +/- 3.6) with dialysis hypotension and 10 patients (five men and five women, aged 58.6 +/- 4.2) without dialysis hypotension. Catecholamines were measured using high performance liquid chromatography--electrochemical detection (HPLC-ECD). Dialysis led to a significant decrease in mean arterial pressure (MAP) in the hypotensive group as compared with the normotensive group. Significantly higher basal (predialysis) plasma norepinephrine (NE) and dopamine levels (DA) were found in the hypotensive uremic group as compared with the normotensive group. Levels of plasma epinephrine (EP) were not significantly different between the normotensive and hypotensive groups. In response to postural stimulation, blood pressure fell in both groups, but the fall in the hypotensive group was significantly greater. Percentage increments of plasma catecholamines in response to postural stimulation in both groups were similar, however. Among the measured hemodynamic parameters, including total peripheral vascular resistance and left ventricular function (cardiac index and fractional shortening), only the cardiac index showed significantly lower values in the hypotensive group after dialysis, as compared with the normotensive group. Results of four tests of autonomic function indicated that although both groups responded similarly to hand-grip and cold-pressor tests, impaired responses to orthostasis and Valsalva maneuver after dialysis were observed in the hypotensive group. The MAP changes in dialysis in the hypotension prone group correlated inversely with predialysis plasma NE, but not with EP and DA.(ABSTRACT TRUNCATED AT 250 WORDS)

High-performance liquid chromatography of the quinazolinocarboline alkaloid dehydroevodiamine.

A high-performance liquid chromatographic method has been developed for the determination of dehydroevodiamine (DeHE), an active principle from Evodia fruit. Plasma was denatured with acetonitrile and centrifuged, the supernatant was separated and blown dry, and the residue was redissolved in water. Bile was acidified with perchloric acid and centrifuged to yield the supernatant. Aliquots were used for analysis. Elution was isocratic on a reversed-phase column with acetonitrile-water-phosphoric acid (64:35:0.8, v/v) adjusted to pH 3.5 as the mobile phase. Ultraviolet detection was at a wavelength of 367 nm. The detection limits were 2 ng/ml for plasma and 10 ng/ml for bile. The intra-day and inter-day variations were mostly below 10%.

Pharmacokinetics of glycyrrhizin after intravenous administration to rats.

A simple high-performance liquid chromatographic method was developed to study the pharmacokinetics of glycyrrhizin in the rat after bolus intravenous administration at a dose of 20, 50, or 100 mg/kg. The decline in the concentration of glycyrrhizin in plasma was generally biexponential at each dose, but the terminal disposition became much slower as the dose was increased. A greater-than-proportional increase in the glycyrrhizin concentration in plasma was observed with an increase in the dose, a result suggesting a dose-dependent glycyrrhizin disposition. The disposition of drug in plasma at each dose fitted well to a two-compartment pharmacokinetic model. The apparent total body clearance decreased significantly with increases in the dose. On the other hand, the apparent distribution volume after intravenous administration was unaffected by the dose. The results indicate that the pharmacokinetics of glycyrrhizin is nonlinear.

Increase of plasma neuropeptide Y-like immunoreactivity following chronic hypoxia in the rat.

In an attempt to understand the changes of circulating neuropeptide Y (NPY) during hypoxia, the plasma level of NPY was investigated by radioimmunoassay. Exposure of rats to hypobaric hypoxia at an altitude of 18,000 ft for 4 weeks causes an increase of pulmonary pressure and an elevation of plasma NPY-like immunoreactivity (NPY-LI). However, the systemic blood pressure was not elevated by this chronic hypoxia. Also, plasma noradrenaline (NA) estimated by chromatographic analysis (HPLC-ECD) was not markedly raised. Failure of bretylium and guanethidine, sympathetic neuron blockers, in reducing the plasma NPY-LI level of these rats ruled out the participation of adrenergic nervous terminals. Adrenal medulla seems responsible for this elevation of plasma NPY-LI because this magnitude disappeared in adrenalectomized rats. These data suggest that chronic hypoxia induced an elevation of circulating NPY from the adrenal gland of rats.

The vasorelaxant effect of evodiamine in rat isolated mesenteric arteries: mode of action.

The roles of the endothelium, Ca2+ and K+ fluxes in the evodiamine-induced attenuation of vascular contractile responses to vasoactive agents were examined. The results showed that: (1) in rat mesenteric artery rings, evodiamine elicited a concentration-dependent attenuation in the contractile response generated by phenylephrine. The inhibitory potency was greater for intact than for endothelium-denuded preparations. Thus, the vasodilator action of evodiamine appeared to be partially endothelium-interactive (dependent). (2) Evodiamine pretreatment had a greater inhibitory effect on the phenylephrine-induced tonic contraction (via Ca2+ influx) than on the phasic contraction (via Ca2+ release). In addition, evodiamine was more potent to inhibit the restoration by CaCl2 of contractile responses to phenylephrine than a potassium depolarizing solution in media that had been kept calcium-free. These results suggest that block of the Ca2+ influx through receptor-mediated Ca2+ channels may be the major mechanism underlying the vasodilator effect of evodiamine. (3) A K+ channel blocker, tetraethylammonium, almost completely abolished the vasodilatation induced by minoxidil (a known K+ channel opener) but not evodiamine. The possible involvement of K+ channel activation of the vasodilator effect produced by evodiamine was therefore excluded.

The role of interscapular brown adipose tissue in cold acclimation in the rat.

The role of the interscapular brown adipose tissue (IBAT) in the defense against cold exposure and subsequent acclimation to it was studied in the rat, a homeotherm. When placed at 4 degrees C, the rat was able to maintain a constant core temperature even though skin and even more so tail temperature dropped drastically. Metabolic rate became significantly higher than control animals kept at 23 degrees C after 24 hrs and continued to rise throughout the 6 weeks exposure period. Starting at week 1, there was hypertrophy of the IBAT with concomitant proliferation of mitochondria. Bilateral surgical removal of IBAT led to significant reduction in metabolic rate only in the fully cold-acclimated rat. However, the survival rate was not affected. It is thus concluded that IBAT played an important but not absolutely essential role in cold acclimation.