Activity of blood cells, erythrocytes, leucocytes, and platelets, in microcirculation was observed by using an intravital microscope and confocal laser scanning microscope connected with an image processing system including fluorescence and phosphorescence emission methods. Dynamic functions of the blood flow were mainly observed in mesentery, brain, and liver tissues of rats. The results are summarized as follows: Deformability of diabetic erythrocytes was significantly lower than that of healthy controls, particularly at high shear rate. The spring constant and Young’s modulus of diabetic erythrocytes obviously stiffened, making them hard to deform in the capillary. During hemorrhagic shock and thrombosis, flow velocity and oxygen partial pressure of blood decreased in the brain and liver tissues that can be visualized by using FITC stained erythrocytes and Pd-porphyrin derivative as a pO2 probe. Platelet adhesion and thrombus formation in the micro-vessels accelerated under the photodynamic reaction; diabetic platelets showed augmented adhesion and aggregation on the vessel wall which was followed by acute thromboembolism. Active oxygen radicals take part in thrombus formation, accompanied with adhesion of the activated leucocytes. Fluorescent dye probes, rhodamine G and acridine orange, are quite useful for visualization of the flow behavior of platelets and leucocytes, respectively.
Since voltage-sensitive dyes (VSDs) change their optical properties in response to membrane potential changes, non-invasive optical measurements of membrane excitation using VSDs have been applied to various types of excitable tissue. This article deals with the authors’ recent work using this technique in addition to basic and technical information about VSD imaging. By applying this technology to hippocampal brain slices, we have found that astrocytes play indispensable roles in sequestering glutamate, maintenance of synaptic transmission, and neuronal survival. Finally, the potential of VSD imaging for drug development was discussed.
Recent developments in optoelectronics permit real-time Ca2+ imaging of thin planes within cells utilizing laser scanning confocal microscopy (LSCM). However, a major complication associated with this imaging system involves increased phototoxicity with improved spatiotemporal resolution. Two-photon excitation microscopy (TPEM) helps to minimize phototoxicity due to the restriction of this technique to the volume proximal to the geometric focus of the light. In this study, the capability of Ca2+ imaging was investigated employing recently developed real-time TPEM, RTS2000MP (Bio-Rad, Tokyo) with a mode-locked Ti-sapphire laser. Z-axis resolution of RTS2000MP with high NA objectives defined as full-width at half maximum (FWHM) with a 0.5-μm fluorescent bead provided values nearly identical to those obtained with LSCM at a small pinhole (0.2 mm) (approximately 0.6 μm). When serial sectioning of 21 sequential images at 0.3-μm intervals in cultured endothelial cells loaded with calcein and tetramethyl-rhodamine methylester were performed with TPEM, the z-axis resolution was higher than that observed with LSCM; moreover, the photobleaching rate was significantly lower than that obtained with LSCM. Maximum fluorescence intensities were detected at 780 nm in excitation spectra of fluo-3 and fluo-4 Ca2+-sensitive probes with TPEM. Fluorescence images in mouse arterial endothelial cells loaded with fluo-4 could be clearly visualized by TPEM in situ. Application of acetylcholine caused oscillatory increase in [Ca2+]i of endothelial cells; subsequently, relaxation along the major axis of smooth muscle cells was evident. Furthermore, consecutive long-lasting experiments could be repeated with identical response in the same microscopic field. In conclusion, fluorescence imaging employing TPEM is useful for Ca2+ imaging in blood vessels in situ.
Rapid-scanning cofocal microscopy has been applied to the analysis of early phase Ca2+ transients in ventricular and atrial cardiomyocytes. On electrical stimulation of ventricular myocytes, Ca2+ concentration begins to rise earliest at the Z-line level and becames uniform throughout the cytoplasm within about 10 ms after the onset of the action potential; transsarcolemmal Ca2+ influx triggers Ca2+ release from release sites on the junctional sarcoplasmic reticulum (SR) coupled to T-tubules at the Z-line throughout the cytoplasm. In atrial myocytes lacking the T-tubular network, transsarcolemmal Ca2+ influx during an action potential triggers SR Ca2+ release only at subsarcolemmal region. SR Ca2+ release then spreads towards the central region of the cell thourgh a propagated Ca2+-induced-Ca2+ release mechanism. The atrio-ventricular difference in excitation-contraction coupling mechanisms underlies some of the atrio-ventricular difference in response to physiological and pharmacological stimuli.
Single-molecule imaging is an ideal technology to study molecular mechanisms of biological reactions in vitro. Recently, this technology has been extended to real-time observation of fluorescent dye-labeled molecules in living cells. Total internal reflection fluorescence microscopy is the major technique for this purpose. Using this technique, we have studied the process of early signal transduction of epidermal growth factor (EGF) in single molecules: binding of EGF to its receptor (EGFR) on the cell surface, dimerization of EGFR induced by binding of EGF, fluctuation of the structure of EGFR clusters, activation of EGFR through tyrosine phosphorylations on its cytoplasmic domain, and recognition of activated EGFR by a cytoplasmic adaptor protein, Grb2. EGF induces intracellular calcium response, sometimes caused by less than one hundred EGF molecules. Single-molecule studies suggested that this highly sensitive response to EGF was due to the amplification of the EGFR signal using dynamic clustering, reorganization of the dimers, and lateral mobility of EGFR on the cell surface. Through these studies, single-molecule analysis has proven to be a powerful technology to analyze intracellular protein systems.
The effects of imipramine on 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT), the 5-hydroxytryptamine (5-HT)1A-receptor full agonist, -induced hypothermia was examined in rats. Single administration of imipramine (30 mg/kg, i.p.) attenuated 8-OH-DPAT-induced hypothermia. This effect of imipramine was blocked by the 5-HT2A-receptor antagonist ketanserin. 8-OH-DPAT-induced hypothermia was not altered 24 h after repeated administration of imipramine (1 – 10 mg/kg per day) for 14 days. However, 8-OH-DPAT-induced hypothermia was significantly enhanced in repeated imipramine (10 mg/kg per day)-treated rats that received 8-OH-DPAT plus imipramine 24 h after the final imipramine administration for 14 days. The 5-HT2A-receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane ((±)-DOI) attenuated the 8-OH-DPAT-induced hypothermia in drug naive rats. The inhibitory effect of (±)-DOI (0.3 mg/kg, s.c.) on 8-OH-DPAT-induced hypothermia was attenuated by repeated administration of imipramine (10 mg/kg per day) for 14 days. These findings suggest that enhancement of the 5-HT1A receptors by repeated administration of imipramine may be due to reduction of the inhibitory effects from the 5-HT2A receptors to the 5-HT1A receptors.
Tetrahydrobiopterin (BH4) acts as an essential cofactor for the enzymatic activity of nitric oxide (NO) synthases. Biosynthesis of the cofactor BH4 starts from GTP and requires 3 enzymatic steps, which include GTP cyclohydrolase I (GCH I) catalysis of the first and rate-limiting step. In this study we examined the effects of cGMP on GCH I activity in human umbilical vein endothelial cells under inflammatory conditions. Exogenous application of the cGMP analogue 8-bromo-cGMP markedly inhibited GCH I activity in the short term, whereas an cAMP analogue had no effect on GCH I activity under the same condition. NO donors, NOR3 and sodium nitroprusside, elevated the intracellular cGMP level and reduced GCH I activity in the short term. This inhibition of GCH I activity was obliterated in the presence of an NO trapper carboxy-PTIO. NO donors had no effect on GCH I mRNA expression in the short term. Moreover, cycloheximide did not alter the inhibition by NO donors of GCH I activity. These findings suggest that stimulation of the cGMP signaling cascade down-regulates GCH I activity through post translational modification of the GCH I enzyme.
To explore cellular signaling molecules that respond to amyloid β protein (Aβ), we investigated the effect of Aβ on tyrosine phosphorylation of cellular proteins in cultured rat cortical astrocytes. Western blotting with the phosphotyrosine-specific monoclonal antibody 4G10 demonstrated that exposure of cultured rat cortical astrocytes to 20 μM Aβ1 – 40 or Aβ25 – 35 for 24 h resulted in a prominent increase in the phosphotyrosine content of 44-kDa protein. The Aβ-induced increase in tyrosine phosphorylation of 44-kDa protein was blocked by U0126, a specific inhibitor of the extracellular signal-regulated kinase (ERK) kinase MEK. Western blotting with anti-phospho-ERK1/2 antibody and anti-ERK1/2 antibody demonstrated that Aβ1 – 40 or Aβ25 – 35 induced an increase in the dually (tyrosine and threonine) phosphorylated form of ERK1 and ERK2, with no change in total ERK1/2 level. In addition, immunofluorescent staining with anti-ERK1/2 antibody revealed that Aβ induced a significant increase in the number of cells expressing ERK1/2 mainly in the nucleus. These results suggest that Aβ specifically promotes tyrosine phosphorylation and nuclear translocation of ERK in astrocytes.
We analyzed the effect of HMG-CoA reductase inhibitors on Ca2+ release from the sarcoplasmic reticulum (SR) using chemically skinned skeletal muscle fibers from the mouse and the rat. Cerivastatin (>20 μM) released Ca2+ from the SR, while pravastatin showed only a little effect. The rates of Ca2+ release were increased by cerivastatin at all Ca2+ concentrations tested. Cerivastatin-induced Ca2+ release in the presence of Ca2+ was affected by adenosine monophosphate, Mg2+, and procaine in essentially the same way as for caffeine-induced Ca2+ release. The Ca2+-uptake capacity of the SR was reduced after co-treatment with ryanodine and cerivastatin at pCa 6.0 to a much greater extent than with ryanodine alone. Thus, cerivastatin-induced Ca2+ release in the presence of Ca2+ must be a result of the activation of the Ca2+-induced Ca2+ release (CICR) mechanism of the ryanodine receptor. However, even when CICR was maximally inhibited by Mg2+ and procaine, or in the practical absence of Ca2+ (pCa >8), cerivastatin still caused Ca2+ release. These results indicate that cerivastatin causes Ca2+ release also by activating some other mechanism(s) in addition to the activation of CICR. Either or both of these effects might be related to its adverse effect, rhabdomyolysis.
Effects of KRN4884 (5-amino-N-[2-(2-chlorophenyl)ethyl]-N'-cyano-3-pyridinecarboxamidine), a novel K+ channel opener, on ionic currents were examined in rabbit femoral arterial myocytes (RFAMs). Under whole-cell clamp conditions where cells were superfused with 5.9 mM K+ bathing solution, KRN4884 elicited an outward current at −30 mV. KRN4884-induced current had a reversal potential of −78 mV and was abolished by application of glibenclamide (glib). KRN4884 was approximately 43 times more potent than levcromakalim in activating an ATP-sensitive K+ current (IK-ATP). On the other hand, KRN4884 affected neither voltage-dependent Ca2+ nor delayed rectifier K+ channel currents. In the inside-out patch clamp configuration where cells were superfused with the symmetrical 140 mM K+ solution, KRN4884 activated 47 pS K+ channels in the presence of adenosine diphosphate. Similar 47 pS K+ channels, which were reversibly inhibited by glib, were recorded under outside-out patch conditions. Using RT-PCR analysis, we found that inward rectifier K channel 6.1 (Kir6.1) and sulfonylurea 2B (SUR2B) transcripts were predominantly expressed in rabbit femoral artery. These results indicate that KRN4884 potently activates IK-ATP in RFAMs. The KRN4884-sensitive 47 pS K+ channel activity underlying IK-ATP is a vascular type KATP channel consisting of Kir6.1 and SUR2B and has similar characteristics to those of ATP-sensitive K+ channels activated by K+ channel openers in other types of smooth muscles.
The involvement of spinopetal noradrenergic and serotonergic systems in antinociception induced by endomorphin-1 (EM-1) and endomorphin-2 (EM-2) given supraspinally or spinally were investigated in male CD-1 mice. Groups of mice were pretreated intrathecally (i.t.) with 6-hydroxydopamine (6-OHDA, 20 μg) or 5,7-dihydroxytryptamine (5,7-DHT, 50 μg) for 3 days before intracerebroventricular (i.c.v.) or i.t. injection of different doses of EM-1 or EM-2, and the tail-flick response was measured for antinociceptive effects. I.t. pretreatment with 6-OHDA for 3 days, which markedly depleted noradrenaline (NA) contents by more than 90%, but not serotonin (5-HT) in the spinal cord, completely abolished the antinociception induced by i.c.v.-administered EM-1 or EM-2. Intrathecal pretreatment with 5,7-DHT for 3 days, which markedly reduced 5-HT contents by more than 92%, but only reduced NA by 14 – 25% in the spinal cord, also markedly attenuated the antinociception induced by i.c.v.-administered EM-1 or EM-2. However, the antinociception induced by i.t.-administered EM-1 or EM-2 was not affected in either 6-OHDA or 5,7-DHT pretreated mice. It is concluded that NA and 5-HT in the spinal cord are involved in the antinociception induced by supraspinally, but not spinally administered EM-1 and EM-2.
Propolis is a bee product, which has long been used in folk medicine for the management of different diseases. In this study we evaluated the analgesic and anti-inflammatory effects of a standard ethanolic extract of Bulgarian propolis (Et-Blg) in mice and its in vitro effect on airway smooth muscle. Et-Blg inhibited acetic acid-induced abdominal contortions with an ID50 = 7.4 ± 0.7 mg · kg−1. In the formalin test, the extract caused a significant reduction in pain in mice treated with 100 mg · kg−1 Et-Blg during the neurogenic phase and for the inflammatory phase with all doses of the extract, with an ID50 = 2.5 ± 0.4 mg · kg−1. Et-Blg inhibited also the capsaicin-induced ear edema in mice; however, this extract was ineffective when assessed in the tail-flick and hot-plate thermal assays. The analgesic effect of Et-Blg was associated with the inhibition of inflammatory responses and not to a simple irritation of nervous terminals. In vitro, this extract inhibited the contraction of trachea smooth muscle induced by histamine (IC50 = 50 ± 5 μg · mL−1), capsaicin (IC50 = 26.8 ± 3 μg · mL−1), 80 mM KCl (IC50 = 27.8 ± 3 μg · mL−1), and carbachol (IC50 = 54 ± 2 μg · mL−1).
The inhibitory effect of an anti-ulcer drug irsogladine [2,4-diamino-6-(2,5-dichlorophenyl)-s-triazine maleate] on monochloramine (NH2Cl)-induced gastric mucosal lesions and its mechanisms of action were clarified in rats. Irsogladine dose-dependently prevented the formation of gastric mucosal lesions induced by 60 mM NH2Cl. The mucosal protective effect of irsogladine was not influenced by capsaicin-sensitive sensory defunctionalization. On the other hand, its protective effect was diminished by the inhibitor of nitric oxide synthase NG-nitro-L-arginine methylester (L-NAME), but not by the inducible nitric oxide synthase selective inhibitor aminoguanidine. Irsogladine restored the NH2Cl-induced decrease in the gastric cGMP formation as an index of nitric oxide synthesis, while it alone had no influence on the cGMP formation in intact tissues. Pretreatment with L-NAME abolished the recovery of cGMP by irsogladine. Furthermore, irsogladine ameliorated the NH2Cl-induced decrease in gastric mucosal blood flow, which was also reversed by pretreatment with L-NAME. These findings suggest that the improvement of the decrease in mucosal blood flow subsequent to the disturbance of gastric nitric oxide synthesis is involved in the protective effect of irsogladine on gastric mucosal lesions caused by NH2Cl.
Cimetidine is known to suppress the growth of several tumors, including gastrointestinal cancer, in humans and animals. Nonetheless, whether other histamine H2-receptor antagonists exert such tumor suppressive effects remains unclear. The effect of roxatidine acetate hydrochloride (roxatidine), an H2-receptor antagonist, on the growth of colon cancer implanted in mice was examined and compared with that of cimetidine. Drugs were orally delivered for 26 – 29 days beginning before or after implantation of syngeneic colon cancer (Colon 38) in C57BL/6 mice. Tumor volume was determined throughout and histochemical analysis was also performed. Tumor tissue and serum vascular endothelial growth factor (VEGF) levels were measured. In vitro cell growth was assessed by the MTT assay. Both roxatidine and cimetidine significantly suppressed the growth of Colon 38 tumor implants. Histologic analysis revealed that such antagonists markedly increased necrotic areas and decreased the density of microvessels in tumor tissue. Both H2-receptor antagonists suppressed VEGF levels in tumor tissue and significantly decreased serum VEGF levels in Colon 38-bearing mice. Such drugs, however, failed to suppress in vitro growth of the cell line. In conclusion, both roxatidine and cimetidine were found to exert suppressive effects on the growth of colon cancer implants in mice by inhibiting angiogenesis via reducing VEGF expression.
When kakkalide, which was isolated from Flos Puerariae, was incubated with human fecal bacteria, kakkalide was metabolized to irisolidone via kakkalidone. When kakkalide (250 mg/kg) was orally administered to rats, irisolidone, but not kakkalide, was detected in the blood. The mortality associated with ethanol treatment was slightly reduced when the mice were intraperitoneally treated with kakkalide. Intraperitoneally administered kakkalide and kakkalidone did not reduce alcohol toxicity. However, orally administered kakkalide and intraperitoneally administered irisolidone significantly reduced the mortality. Orally administered kakkalide and intraperitoneally injected irisolidone greatly reduced serum alanine aminotransferase and aspartate aminotransferase activities in ethanol-intoxified mice. Orally administered kakkalide and intraperitoneally administered irisolidone significantly lowered the level of blood ethanol. The results indicate that kakkalide is a prodrug of irisolidone in protecting against ethanol-induced lethality and hepatic injury.
The protective effect of teprenone, an anti-ulcer drug, against acute gastric mucosal lesions was examined in rats with a single intraperitoneal injection of compound 48/80 (0.75 mg/kg). Teprenone (50, 100, or 200 mg/kg) was orally administered 0.5 h before compound 48/80 treatment. Administered teprenone prevented gastric mucosal lesion development found at 3 h after compound 48/80 treatment dose-dependently, although no dose of teprenone affected the decreased gastric mucosal blood flow and increased serum serotonin and histamine concentrations found at 3 h after the tresatment. Increases in the activities of myeloperoxdiase (an index of neutrophil infiltration) and xanthine oxidase and the content of thiobarbituric acid reactive substances (an index of lipid peroxidation) and decreases in the contents of hexosamine (a marker of gastric mucus) and adherent mucus occurred in gastric mucosal tissues at 3 h after compound 48/80 treatment. Administered teprenone dose-dependently attenuated all these changes found at 3 h after compound 48/80 treatment. These results indicate that orally administered teprenone protects against compound 48/80-induced acute gastric mucosal lesions in rats possibly through its stimulatory action on gastric mucus synthesis and secretion and its inhibitory action on neutrophil infiltration and enhanced lipid peroxidation in the gastric mucosal tissue.
The nuclear receptor PPAR (peroxisome proliferator-activated receptor) has three subtypes named α, δ(β), and γ that may act as receptors for a range of compounds including antihyperglycaemic drugs, insulin sensitizers, and non-steroidal anti-inflammatory drugs (NSAIDs). Although profiling of the subtype selectivity of the compounds for PPAR is indispensable to elucidate their pharmacological action, the absence of an appropriate transactivation assay for PPARδ led us to develop a sensitive and reproducible method. We found that co-expression of PPARδ, retinoid X receptor (RXR) α, and coactivators such as CBP and SRC-1 enhanced basal and agonist-dependent activation of PPAR responsive element (PPRE)-driven transcription by PPARδ, rendering a PPRE-driven reporter assay reliable and sensitive. Utilizing this assay for PPARδ, we re-evaluated the subtype selectivity of a variety of anti-inflammatory drugs for human PPAR. The PPAR agonists tested included two leukotriene (LT) D4 antagonist, seven NSAIDs, and two anti-rheumatoid drugs. We found that a novel LTD4 antagonist, FK011 ([2-(((2-(4-tert-butyl-1,3-thiazol-2-yl)-1-benzofuran-5-yl)oxy)methyl)phenyl]acetic acid), showed marked agonistic activity for PPARγ. NSAIDs were classified into the following three groups: those showing no activity for all subtypes, those that were selective for PPAR γ such as indomethacin and diclofenac, and those showing agonistic activity for the δ and γ subtypes such as ibuprofen. These results will be important to studies on the molecular mechanisms of pharmacological actions of LTD4 antagonists and NSAIDs.
Adenosine receptors are expressed in various mammalian tissues where they mediate the effects of adenosine on cellular functions through a number of signalling mechanisms. 18F-NECA is the positron-emitting derivative of the A2-receptor agonist NECA (5'-n-ethyl-carboxamidoadenosine) and is a radioligand for PET imaging of adenosine receptors. Contractility and relaxation studies were performed on guinea pig atrial myocardium, pulmonary artery, and thoracic aorta to compare the pharmacological effects of NECA and F-NECA (a non-emitting derivative) on tissues. Furthermore, the effect of NECA and F-NECA on the potassium conductance was investigated in DDT1 MF-2 smooth muscle cells with the patch-clamp technique. Both NECA and F-NECA reduced the contractile force in atrial myocardium and evoked phasic contraction in pulmonary artery (A1 adenosine-receptor-mediated actions) in a dose dependent manner; however, the apparent affinity was lower for F-NECA. No difference was found in relaxation induced by these compounds in 1 μM noradrenaline-precontracted aorta and pulmonary artery (in the presence of DPCPX, an A1 adenosine receptor antagonist, tissue containing A2B adenosine receptors). NECA (5 μM) and F-NECA (5 μM) also decreased the peak current and accelerated activation and inactivation properties of the potassium channels, but F-NECA was less effective. These results suggest that while NECA and F-NECA are equivalent agonists of vascular A2B receptors, they mediate different changes of some parameters. When evaluating the data obtained by the use of radiolabelled ligands, one has to take into consideration the possible physiological effects of the ligands besides its binding properties to tissues.
The effect of Y-931 (8-fluoro-12-(4-methylpiperazin-1-yl)-6H-benzothieno[2,3-b][1,5]benzodiazepine maleate), a novel potential atypical antipsychotic candidate, in producing dystonia in Cebus monkeys was investigated. Y-931 induced relatively weak dystonia in several observation periods at doses greater than 0.1 mg/kg, i.m. Although Y-931 significantly increased total dystonia scores (the sum of 15 to 360 min after injection) at doses greater than 0.5 mg/kg, i.m., the scores did not exceed 20, up to a dose of 1.0 mg/kg, i.m. and lacked a dose-response relationship. The present result suggests that Y-931 is predicted to have a low risk of extrapyramidal side effects.
Harpagophytum procumbens (Pedaliaceae) has been used for the treatment of pain and arthritis. The effect of Harpagophytum procumbens against lipopolysaccharide-induced inflammation was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, reverse transcription-polymerase chain reaction, prostaglandin E2 (PGE2) immunoassay, and nitric oxide detection on mouse fibroblast cell line L929. The aqueous extract of Harpagophytum procumbens was shown to suppress PGE2 synthesis and nitric oxide production by inhibiting lipopolysaccharide-stimulated enhancement of the cyclooxygenase-2 and inducible nitric oxide synthase (iNOS) mRNAs expressions in L929 cells. These results suggest that Harpagophytum procumbens exerts anti-inflammatory and analgesic effects probably by suppressing cyclooxygenase-2 and iNOS expressions.
κ-Opioid receptor agonists decrease the levels of extracellular dopamine in vivo and in vitro. However, the mechanism(s) underlying these actions are unclear. The objective of this study was to distinguish between an effect of the selective κ-opioid receptor agonist U-50,488H ((trans-(±)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]cyclohexyl)benzeneacetamide methanesulfonate) on secretion and reuptake of dopamine by PC12 cells. The data show that U-50,488H has both a modest effect to increase dopamine release and a more pronounced effect to inhibit dopamine uptake. Neither effect was sensitive to nor-binaltorphimine or naloxone, suggesting that they are not mediated through an opioid receptor.
Intravenous injection of the supernatant fluids from human peripheral blood mononuclear cells (PBMC) incubated with lipopolysaccharide (LPS) caused fever in rabbits. The fever was in parallel with the levels of either interleukin-1β (IL-1β), IL-6, or tumor necrosis factor-α (TNF-α) in supernatant fluids. When incubating the platonin with the LPS-human PBMC, both the levels of IL-1β, IL-6, or TNF-α in supernatant fluids and the pyrogenicity of supernatant fluids were significantly suppressed. The febrile response to supernatant fluids from the LPS-stimulated PBMC was attenuated almost completely by adding anti-IL-1β, but not anti-IL-6 or anti-TNF-α, monoclonal antibody to supernatant fluids. In addition, both the fever and the increased levels of either IL-1β, IL-6, or TNF-α in rabbit serum following an intravenous administration of LPS were significantly attenuated by pretreatment with an intravenous dose of platonin. Furthermore, the fever induced by intravenous injection of IL-1β was reduced by pretreatment of rabbits with intravenous injection of platonin. The data indicate that platonin inhibits production of pyrogenic cytokines (in particular, IL-1β) from PBMC and results in antipyresis.