Paeciloxanthone, a new cytotoxic xanthone from the marine mangrove fungus Paecilomyces sp. (Tree1-7)

The metatrophic fungus Paecilomyces sp. (Tree1-7) was isolated from an estuarine mangrove from the Taiwan Strait. The methanol extract of the fungal mycelium exhibited cytotoxicity against hepG2. Paeciloxanthone (1), a new xanthone, and the known compounds emodin (2) and chrysophanol (3), were isolated from the extract. Their structures were elucidated by spectroscopic experiments. Paeciloxanthone (1) exhibited in vitro cytotoxicity against hepG2 (IC₅₀ = 1.08 μg/mL), acetylcholineesterase (AChE) inhibitory (IC₅₀ = 2.25 μg/mL) and antimicrobial activities.     

Inhibition of P-type ATPases by [(dihydroindenyl)oxy]acetic acid (DIOA), a K+ -Cl- cotransporter inhibitor

[(Dihydroindenyl)oxy]acetic acid (DIOA) has been used as a potent inhibitor of K⁺–Cl⁻ cotransporter (IC₅₀ = 10 μM). Here we found that DIOA inhibited activities of P-type ATPases such as dog kidney Na⁺,K⁺-ATPase (IC₅₀ = 53 μM), hog gastric H⁺,K⁺-ATPase (IC₅₀ = 97 μM) and rabbit muscle Ca²⁺-ATPase (IC₅₀ = 127 μM). In the membrane preparation of the LLC-PK1 cells stably expressing rabbit gastric H⁺,K⁺-ATPase, DIOA inhibited activities of the endogenous Na⁺,K⁺-ATPase (IC₅₀ = 95 μM) and the exogenous H⁺,K⁺-ATPase (IC₅₀ = 75 μM). 5-Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), a Cl⁻ channel blocker, had no effects on the DIOA-elicited inhibition of the P-type ATPases. These findings suggest that lower concentration of DIOA (< 20–30 μM) should be used for evaluation of the activity of K⁺–Cl⁻ cotransporter without affecting the activities of coexisting Na⁺,K⁺-ATPase and/or H⁺,K⁺-ATPase in cells.     

Inhibition of the myeloperoxidase chlorinating activity by non-steroidal anti-inflammatory drugs: flufenamic acid and its 5-chloro-derivative directly interact with a recombinant human myeloperoxidase to inhibit the synthesis of hypochlorous acid

The present in vitro study was designed to assess the inhibition of the myeloperoxidase (MPO)/H₂O₂/Cl⁻ system by several non steroidal anti-inflammatory drugs (NSAIDs) of the oxicam family and of nimesulide and to compare their effect with flufenamic acid in order to investigate their influence on the chlorinating activity of MPO as a protective mechanism during chronic inflammatory syndromes. The inhibition of the system was assessed by measurement of the taurine chlorination while the accumulation of compound II was used to investigate the mechanism of inhibition. The oxidation products of NSAIDs by the MPO/H₂O₂/Cl⁻ system were identified and flufenamic acid and derivatives were also assessed in the inhibition of LDL oxidation in two models. Flufenamic acid (IC₅₀ = 1.1 ± 0.3 μM) is the most efficient inhibitor of the MPO/H₂O₂/Cl⁻ system and nimesulide (IC₅₀ = 2.1 ± 0.3 μM) is more active than the other NSAIDs of the oxicam family (IC₅₀ = 8–12 μM). The accumulation of compound II revealed that flufenamic acid acts as an electron donor while the other NSAIDs are antagonists of chloride anions. The identification of the oxidation products confirms that flufenamic behaves like an electron donor and is directly oxidized in the 5-hydroxy-derivative but gives also the 5-chloro-derivative which similarly inhibits the MPO/H₂O₂/Cl⁻ system. Flufenamic acid has the best inhibiting activity towards the MPO/H₂O₂/Cl⁻ system. However, in models that assess the LDL oxidation, flufenamic acid and its derivatives were unable to properly inhibit MPO activity as the enzyme is adsorbed on macrostructures such as LDL molecules.     

Importance of acyl-coenzyme A:cholesterol acyltransferase 1/2 dual inhibition for anti-atherosclerotic potency of pactimibe

Pactimibe sulfate, [7-(2,2-dimethylpropanamido)-4,6-dimethyl-1-octylindolin-5-yl]acetic acid hemisulfate, a novel Acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibitor, was investigated in vitro and in vivo to characterize its potential. Pactimibe exhibited dual inhibition for ACAT1 and ACAT2 (concentrations inhibiting 50% [IC₅₀s] at micromolar levels) more potently than avasimibe. Kinetic analysis revealed pactimibe is a noncompetitive inhibitor of oleoyl-CoA (K_i value: 5.6 μM). Furthermore, pactimibe markedly inhibited cholesteryl ester formation (IC₅₀: 6.7 μM) in human monocyte-derived macrophages, and inhibited copper-induced oxidation of low density lipoprotein more potently than probucol. Pactimibe exerted potent lipid-lowering and anti-atherosclerotic effects in atherogenic diet-fed hamsters. At doses of 3 and 10 mg/kg for 90 days, pactimibe decreased serum total cholesterol by 70% and 72%, and aortic fatty streak area by 79% and 95%, respectively. Despite similar cholesterol lowering, fatty streak area reduction was greater by 10 mg/kg. These results suggest that ACAT1/2 dual inhibitor pactimibe has anti-atherosclerotic potential beyond its plasma cholesterol-lowering activity.     

β-Eudesmol suppresses tumour growth through inhibition of tumour neovascularisation and tumour cell proliferation

In the present study, we investigated the potential anti-angiogenic mechanism and anti-tumour activity of β-eudesmol using in vitro and in vivo experimental models. Proliferation of human umbilical vein endothelial cells (HUVEC) stimulated with vascular endothelial growth factor (VEGF, 30 ng/ml) and basic fibroblast growth factor (bFGF, 30 ng/ml) was significantly inhibited by β-eudesmol (50-100 μM). β-Eudesmol (100 μM) also blocked the phosphorylation of cAMP response element binding protein (CREB) induced by VEGF (30 ng/ml) in HUVEC. β-Eudesmol (10-100 μM) inhibited proliferation of HeLa, SGC-7901, and BEL-7402 tumour cells in a time- and dose-dependent manner. Moreover, β-eudesmol treatment (2.5-5 mg/kg) significantly inhibited growth of H₂₂ and S₁₈₀ mouse tumour in vivo. These results indicated that β-eudesmol inhibited angiogenesis by suppressing CREB activation in growth factor signalling pathway. This is the first study to demonstrate that β-eudesmol is an inhibitor of tumour growth.     

Thromboxane A(2)-mediated Cl(-) secretion induced by platelet-activating factor in isolated rat colon

Thromboxane A₂ is a novel endogenous secretagogue of Cl⁻ secretion in the distal colon. Here, we examined if the Cl⁻ secretion caused by platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is mediated by thromboxane A₂ production using isolated mucosae of the rat colon. Furosemide (100 μM) and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB; 300 μM) completely inhibited PAF (10 μM)-induced increase in short-circuit current (Isc) across the mucosa, indicating that PAF caused a Cl⁻ secretion in the rat colon. A selective thromboxane A₂ receptor antagonist (sodium(E)-11-[2-(5,6-dimethyl-1-benzimidazolyl)-ethylidene]-6,11-dihydrobenz[b,e]oxepine-2-carboxylate monohydrate; KW-3635), and a selective thromboxane synthase inhibitor (sodium 4-[-hydroxy-5-(1-imidazolyl)-2-methylbenzyl]-3,5-dimethylbenzoate dihydrate; Y-20811) inhibited the PAF-induced Cl⁻ current in a concentration-dependent manner. The IC₅₀ values of KW-3635 and Y-20811 were 2.1 and 0.5 μM, respectively. 30 μM KW-3635 and 1 μM Y-20811 inhibited the PAF response by 92% and 83%, respectively. These inhibitors did not affect the prostaglandin E₂-induced increase in Isc. A 5-lipoxygenase-activating protein inhibitor (3-[1-(p-chlorobenzyl)-5-(isopropyl)-3-t-butylthioindol-2-yl]-2,2-dimethyl-propanoic acid sodium; MK-886) (5 μM) did not affect the PAF-induced Cl⁻ current. The present study suggests that the PAF-induced Cl⁻ secretion in the rat colonic mucosa is mainly mediated by a release of thromboxane A₂.     

Leucocyte recruitment induced by type II phospholipases A(2) into the rat pleural cavity.

Bothropstoxin-I (BthTX-I) and bothropstoxin-II (BthTX-II) are Lys-49 and Asp-49 phospholipases A₂ (PLA₂s), respectively, isolated from Bothrops jararacussu venom. Piratoxin-I (PrTX-I) is a Lys-49 PLA₂ isolated from Bothrops pirajai venom. In this study, the ability of BthTX-I, BthTX-II and PrTX-I to recruit leucocytes into the rat pleural cavity and potential mechanisms underlying this effect were investigated. Intrapleural injection of either BthTX-I or PrTX-I (10–100 μg/cavity each) caused a significant leucocyte infiltration at 12 h after injection. The maximal cell migration was observed with the dose of 30 μg/cavity (14.9±15.5 and 17.6±1.6×10⁶ cells/cavity, respectively). Leucocyte counts consisted mainly of mononuclear cells, but significant amounts of neutrophils and eosinophils were also observed. Intrapleural injection of BthTX-II (10–100 μg/cavity) caused a marked leucocyte infiltration at 6 and 12 h after injection. The maximal response was observed with the dose of 100 μg/cavity (57.3±3.4×10⁶ cells/cavity, 6 h). The leucocyte counts were mainly composed of neutrophils and mononuclear cells. The treatment of either BthTX-I (30 μg/cavity, 12 h) or BthTX-II (30 μg/cavity, 6 h) with the PLA₂ inhibitor p-bromophenacyl bromide (p-BPB) had no effect on the total and differential leucocyte counts induced by these proteins. The same treatment partially reduced the PrTX-I-induced pleural leucocyte infiltration. In rats depleted of the histamine and 5-hydroxytryptamine (5-HT) stores by chronic treatment with compound 48/80, the total leucocyte counts in response to BthTX-I, BthTX-II and PrTX-I was not significantly affected compared to control animals. In addition, BthTX-I, BthTX-II and PrTX-I (100 μg/ml each) significantly degranulated pleural mast cells in vitro leading to the release of [¹⁴C]5-hydroxytryptamine ([¹⁴C]5-HT). p-BPB and heparin (50 IU/ml) significantly reduced the [¹⁴C]5-HT release induced by these PLA₂s. Our results demonstrate that BthTX-I, BthTX-II and PrTX-I recruit leucocyte into the pleural cavity of the rat by mechanisms unrelated to enzymatic activity and pleural mast cell degranulation.     

Involvement of mitogen-activated protein kinases and protein kinase C in cadmium-induced prostaglandin E2 production in primary mouse osteoblastic cells

We previously reported that cadmium (Cd) induced prostaglandin E₂ (PGE₂) biosynthesis through the activation of cytosolic phospholipase A₂ (cPLA₂) and induction of cyclooxygenase 2 (COX-2) in primary mouse osteoblastic cells. In the present study, we further investigated the mechanism of PGE₂ production by Cd focusing on the main mitogen-activated protein kinase (MAPK) subfamilies that mediate prostaglandin synthesis, extracellular signal-regulated kinase (ERK1/2 MAPK), c-jun-amino-terminal kinase (JNK MAPK) and p38 MAPK, and protein kinase C (PKC) which is activated by Cd in several kinds of cells. Cd at 2 μM and above stimulated PGE₂ production in osteoblastic cells and its production was inhibited by the kinase-specific inhibitors PD98059, SB203580, curcumin, and calphostin C. Calphostin C also inhibited the production of PGE₂ by phorbol 12-myristate 13-acetate (PMA), which is a potent activator of PKC. PD98059 inhibited PGE₂ production stimulated by PMA as well as Cd, indicating that activation of PKC by ERK1/2 MAPK was necessary for Cd-stimulated PGE₂ production. Moreover, Cd stimulated the phosphorylation of these three MAPKs, and inhibition of the phosphorylation of ERK1/2 MAPK by calphostin C was also observed. On the other hand, Cd was found to phosphorylate cPLA₂ and the phosphorylation was inhibited by PD98059, indicating that cPLA₂ was activated by Cd through ERK1/2 MAPK and released arachidonic acid (AA), a substrate of COX-2, from membranous phospholipids. From these results, it was suggested that activation of each of the ERK1/2, p38, and JNK MAPK cascades in addition to that of PKC and cPLA₂ played an important role in the Cd-stimulated biosynthesis of PGE₂ in mouse osteoblastic cells.     

Studies on anti-tumour activities of pseudolaric acid-B (PLAB) and its mechanism of action

Pseudolaric acid-B (PLAB), a diterpene acid, was isolated from the root and trunk barks of Pseudolarix kaempferi. It showed antifungal and anti-fertility effects as well as cytotoxic activities in previous studies. The present study investigates cytotoxic activity on cultured human cancer cells, inhibition on the growth of transplantable tumours in mice and the mechanism of these actions. The experimental results showed that PLAB had potent cytotoxic effects on cancer cells derived from different tissues. MTT assay showed that its IC₅₀ towards these tumour cells was 0.17 to 5.20 μmol/L, and towards one normal human kidney proximal tubular epithelial cell (HKC) was 5.77 μmol/L. Furthermore, the results of cell growth curve and colony formation of cancer cells matched the above results. The results in vivo demonstrated that PLAB significantly inhibited the growth of transplantable tumours, such as Lewis lung cancer and hepatocarcinoma 22 (H22) in mice. The inhibitory rate to H22 was 14.4% and 40.1%, and to Lewis lung cancer reached 39.1% and 47.0%, when PLAB was given by intraperitoneal injection (i.p.) at a dose of 30 mg/kg/day and 60 mg/kg/day for 10 days, respectively. It is suggested that PLAB also showed obvious anticancer activity in vivo. Inducing apoptosis by PLAB in HeLa cells was assessed by various morphological and biochemical characteristics, including cell shrinkage, chromatin condensation, membrane blebbing, formation of apoptotic bodies, and internucleosomal DNA fragmentation. A typical 'sub-G₁ peak' was also checked through flow cytometry. These results were accompanied by up-regulating P53, down-regulating Bcl-2 and activating Caspase-3, which was revealed by Western blotting. PLAB also caused cell cycle arrest to G₂/M phase in a dose-dependent manner. The experiments suggest that PLAB is a new potent anti-tumour agent.     

In vitro and in vivo effects of endothelin-1 and YM598, a selective endothelin ET A receptor antagonist, on the lower urinary tract

We investigated the contractile response of the lower urinary tract to endothelin-1 in vitro (rabbits) and in vivo (dogs). We also assessed the effects of a selective endothelin ET_A receptor antagonist, (E)-N-[6-methoxy-5-(2-methoxyphenoxy)[2, 2′-bipyrimidin]-4-yl]-2-phenylethenesulfonamide monopotassium salt (YM598), on endothelin-1-induced contractile responses. In the in vitro study, endothelin-1 induced contractile responses in isolated rabbit bladder base, urethra, and prostate tissues. YM598 (10^− 7–10^− 5 M) antagonized these endothelin-1-induced contractile responses without affecting the maximal responses. In the in vivo study, endothelin-1 induced the elevation of non-prostatic urethral pressure as well as prostatic urethral pressure even in the presence of tamsulosin (10 μg/kg, i.v.) in anesthetized male dogs. YM598 (0.1–3 mg/kg, i.v.) inhibited these endothelin-1-induced contractile responses in a dose-dependent fashion. These results suggest that endothelin ET_A receptors play an important role in the lower urinary tract contraction, and that the selective endothelin ET_A receptor antagonist YM598 has ameliorating effects on various urinary dysfunctions, including benign prostatic hyperplasia.     

Evaluation of the cytotoxicity of 10 chemicals in human and rat hepatocytes and in cell lines: Correlation between in vitro data and human lethal concentration

The cytotoxicity of 10 chemicals from the Multicentre Evaluation of In vitro Cytotoxicity (MEIC) list (nos 21–30) was evaluated in human and rat cultured hepatocytes and in two established cell lines (HepG2 and 3T3) according to the MEIC programme organized by the Scandinavian Society of Cell Toxicology. The MTT test was used as the endpoint of cytotoxicity after 24hr of exposure to the chemicals. Theophylline, phenobarbital and paraquat were the least cytotoxic compounds in the cellular systems (IC₅₀ = 450-17,000 μm) except for the 3T3 cells. The seven remaining chemicals (dextropropoxyphene, propranolol, arsenic trioxide, cupric sulfate, mercuric chloride, thioridazine and thallium sulfate) showed a similar relative cytotoxic ranking in the four in vitro systems in the lower range of concentrations (IC₅₀ = 2–350 μm). The data suggest that these 10 chemicals have a basal cytotoxic effect common to the four in vitro systems, and probably none of these compounds could be considered either hepatotoxic or species specific. The correlation between in vitro data and human lethal blood concentrations showed that the predictability of the in vitro systems was similar to that of in vivo rodent tests (LD₅₀) only when low cytotoxic concentrations (IC₁₀) were used for correlation.     

Growth inhibition and induction of early apoptosis by arenicolsterol A, a novel cytotoxic enolic sulphated sterol from the marine annelid, Arenicola cristata

Arenicolsterol A (ASA), a novel cytotoxic enolic sulphated sterol, was isolated from the marine annelid, Arenicola cristata (AC). Growth inhibition of this compound on cancer cell lines was determined by MTT assay and suppression of tumour stem cells colony formation. The results showed that ASA was selectively cytotoxic on HeLa cell line (IC₅₀ = 6.00 ± 1.16 μmol L^- 1 on HeLa cell line, IC₅₀ = 10.85 ± 0.97 μmol L^- 1 on 929 cell line and 14.72 ± 1.55 μmol L^- 1 on NCI-h6 cell line). In addition, the apoptosis induced by ASA was verified from monitoring the stainability with Annexin V and propidium iodine by a fluorescence-activated cell sorter. The experimental data confirmed that ASA could induce apoptosis in HeLa cells by arresting early stage in apoptosis. Meanwhile, the apoptosis was found to be correlative with the inhibition of the protein tyrosine phosphatases (cdc25A, cdc25B, JSP1, etc). Therefore, ASA might be a novel promising precursor of anticancer medicines.     

O-methyl nakafuran-8 lactone, a new sesquiterpenoid from a hainan marine sponge Dysidea sp

A new sesquiterpenoid, O-methyl nakafuran-8 lactone (1) has been isolated from a Hainan sponge Dysidea sp. and the structure of the new compound proposed by spectral data, was confirmed by X-ray diffraction analysis. The complete ¹H- and ¹³C-NMR assignments were made on the basis of detailed 2D NMR spectral analysis.

Compound 1 showed strong inhibitory bioactivity against PTP1B with IC₅₀ value of 1.58 μM.     

5-HT3 receptors augment neuronal, cholinergic contractions in guinea pig trachea European Journal of Pharmacology, 234 (1993) 109–112

Serotonin (5-HT) and the selective 5-HT₃ receptor agonist, 2-methyl-5-hydroxytryptamine enhanced electrical field stimulated contractions of the isolated guinea pig trachea. 5-HT (EC₅₀ = 3.5 μM) was twice as potent as 2-methyl-5-hydroxytryptamine (EC₅₀ = 7.4 μM). The effects of 5-HT and 2-methyl-5-hydroxytryptamine were antagonized by the selective 5-HT₃ receptor antagonist, zacopride (apparent pA₂ = 7.60 against 2-methyl-5-hydroxytryptamine). 2-Methyl-5-hydroxytryptamine (10 μM) had no effect on contractile responses to exogenous acetylcholine. Furthermore, the increase in electrical field stimulated contraction by 2-methyl-5-hydroxytryptamine was unchanged by hexamethonium (100 μM) but contractions were blocked by atropine (1 μM). These results suggest that excitatory 5-HT₃ receptors exist on postganglionic cholinergic nerves in the isolated guinea pig trachea.     

Signal peptide peptidase dependent cleavage of type II transmembrane substrates releases intracellular and extracellular signals

The intramembrane-cleaving proteases (I-CLiPs) presenilin-1 and -2 (PS1 and PS2), signal peptide peptidase (SPP) and the Site-2 protease (S2P) catalyze critical steps in cell signaling and are implicated in diseases such as Alzheimer's disease, hepatitis C virus (HCV) infection and cholesterol homeostasis. Here we describe the development of a cellular assay based on cleavage of the transmembrane sequence of the HCV core protein precursor, releasing intra- and extra-cellular signals that represent sequential signal peptidase and SPP cleavage, respectively. We find that the SPP inhibitor (Z-LL)2-ketone (IC₅₀ = 1.33 μM) and the γ-secretase potent inhibitors NVP-AHW700-NX (IC₅₀ = 51 nM) and LY411575 (IC₅₀ = 61 nM) but not DAPT dose dependently inhibited SPP but not signal peptidase cleavage. Our data confirm that type II orientated substrates, like the HCV transmembrane sequence, are sequentially cleaved by signal peptidase then SPP. This dual assay provides a powerful tool to pharmacologically analyze sequential cleavage events of signal peptidase and SPP and their regulation.     

Heterogeneity of functional GABA(A) receptors in rat dentate gyrus neurons revealed by a change in response to drugs during the whole-cell current time-course

We examined if the drug sensitivity of GABA_A receptors in dentate gyrus granule neurons changed during the whole-cell current time-course. Effects of drugs on currents evoked immediately (the peak current) upon drug application and currents remaining about two seconds later (semi-plateau current) were compared. The apparent affinity for GABA (EC₅₀) of the peak and the semi-plateau current were 14 and 4 μM, respectively. Bicuculline inhibited 50% of the peak and the semi-plateau current (IC₅₀) at 7 and 36 μM, respectively, while 100 μM was required for full inhibition of the 100 μM GABA-evoked current. Zinc inhibited about 50% of the peak current with an IC₅₀ value of 94 μM whereas biphasic, but complete inhibition of the semi-plateau current was recorded with IC₅₀ values of 3 and 558 μM. The decay phase of the 100 μM GABA-evoked current was fitted by a fast (τ₁, 100–300 ms) and a slow (τ₂, 1–2 s) time-constants in all cells. The relative current amplitude associated with the fast (A1) and the slow (A2) component varied. The A1 current amplitude appeared more sensitive to bicuculline than the A2 current while the opposite was true for zinc. The results are consistent with heterogenous population of functional GABA_A receptors in the dentate gyrus granule neurons.     

Inhibition of mouse osteoblast proliferation and prostaglandin E2 synthesis by Ulmus davidiana Planch (Ulmaceae)

Ulmus davidiana Planch (Ulmaceae) (UD) is a widely used Korean herbal medicine that has been used historically in anti-inflammatory and anticancer therapy. Since UD has been known to have anti-inflammatory and protective effects on damaged tissue, inflammation and bone among other functions, this study was undertaken to address whether the water extract of the bark of UD could modulate proliferation of mouse osteoblasts in vitro and to investigate its effect on cyclooxygenase-2 (COX-2), which converts arachidonic acid to prostaglandin E2 (PGE2). Mouse osteoblasts were tested in vitro for growth inhibition, proliferating cell nuclear antigen (PCNA) expression, and COX-2 activity and expression after treatment with UD extract. Its effects were compared with those of indomethacin (a nonselective COX inhibitor) and celecoxib (a selective COX-2 inhibitor). UD demonstrated a strong growth inhibition in tested mouse osteoblasts. The IC50s were 10 μg/ml for UD, 6 μM for celecoxib and 42 μM for indomethacin. UD, as well as celecoxib and indomethacin, suppressed PCNA expression and PGE2 synthesis in osteoblasts. UD inhibited COX-2 expression, whereas celecoxib inhibited COX-2 activity directly. UD selectively and effectively inhibits osteoblasts cell growth in vitro. Inhibition of PGE2 synthesis via suppression of COX-2 expression may be responsible for its anti-inflammatory activity.     

Indian red scorpion (Buthus tamulus) venom-induced augmentation of cardiac reflexes is mediated through the involvement of peripheral 5-HT3 and central 5-HT1A receptor subtypes

The present study was undertaken to identify 5-HT receptor subtypes involved in Buthus tamulus (BT) venom-induced augmentation of cardiac reflexes elicited by phenyldiguanide (PDG). Intravenous injection of PDG (10 μg/kg) produced parallel decrease in mean arterial pressure (MAP) and heart rate (HR) in urethane anaesthetized rats (r=0.82; p<0.001). Injection of PDG (1–40 μg/kg, i.v.) produced concentration-dependent decrease in time-response area of the HR. After BT venom (20 μg/kg) the concentration-response curve was shifted to the left. Further, fall of MAP and HR in response to submaximal concentration of PDG (10 μg/kg) were augmented significantly. Pretreatment with 5-HT₃ receptor antagonist (ondansetron; 10 μg/kg) intravenously, blocked the BT venom-induced augmentation of PDG reflex but spiperone (100 μg/kg; 5-HT_1A/5-HT₂ antagonist) or ketanserin (300 μg/kg; 5-HT₂ antagonist) failed to do so. Afferent discharges elicited by PDG (10 μg/kg) in vagus nerve were doubled after exposure to BT venom. Ondansetron (100 μg/kg, i.v.) totally abolished the discharges after exposure to BT venom but not by spiperone or ketanserin. Intracerebroventricular injection of spiperone (100 μg/kg) but not ketanserin or ondansetron, blocked the BT venom-induced augmentation of PDG reflex. Results show that the BT venom-induced augmentation of reflex elicited by PDG is mediated through the involvement of 5-HT₃ receptors peripherally and 5-HT_1A type of receptors centrally.