The interplay between 5-HT2C and 5-HT3A receptors in the dorsal periaqueductal gray mediates anxiety-like behavior in mice.

The monoamine neurotransmitter serotonin (5-HT) modulates anxiety by its activity on 5-HT2C receptors (5-HT2CR) expressed in the dorsal periaqueductal gray (dPAG). Here, we investigated the presence of 5-HT3A receptors (5-HT3AR) in the dPAG, and the interplay between 5-HT2CR and 5-HT3AR in the dPAG in mediating anxiety-like behavior in mice. We found that 5-HT3AR is expressed in the dPAG and the blockade of these receptors using intra-dPAG infusion of ondansetron (5-HT3AR antagonist; 3.0 nmol) induced an anxiogenic-like effect. The activation of 5-HT3ABR by the infusion of mCPBG [1-(m-Chlorophenyl)-biguanide; 5-HT3R agonist] did not alter anxiety-like behaviors. In addition, blockade of 5-HT3AR (1.0 nmol) prevented the anxiolytic-like effect induced by the infusion of the 5-HT2CR agonist mCPP (1-(3-chlorophenyl) piperazine; 0.03 nmol). None of the treatment effects on anxiety-like behaviors altered the locomotor activity levels. The present results suggest that the anxiolytic-like effect exerted by serotonin activity on 5-HT2CR in the dPAG is modulated by 5-HT3AR expressed in same region.

A biodegradable poly(amido amine) based on the antimicrobial polymer polyhexamethylene biguanide for efficient and safe gene delivery.

Inspired by the excellent membrane affinity of antimicrobial polymers, we synthesized a novel biodegradable poly(amino amine) polymer with pendent side chains that mimic the widely used biocide polyhexamethylene biguanide (PHMB) for gene delivery. Michael addition polymerization was utilized to form the polymer scaffold between N,N'-cystaminebisacrylamide (CBA) and N-Boc-1,6-diaminohexane (Boc-DAH) followed by N-Boc deprotection. Then the exposed primary amino groups were partly (about 75%) transformed into biguanide by an addition reaction with dicyandiamide to obtain the final product CBA-DAH-biguanide (CBA-DAH-BG). The polymer CBA-DAH-BG was able to condense plasmid DNA (pDNA) into nano-sized (<200 nm), positively-charged (>35 mV) polyplexes that were well resistant to heparin and DNase I. Rapid DNA release was observed in the presence of dithiothreitol (DTT), indicating that CBA-DAH-BG was equipped with biodegradability by the cleavage of disulfide bonds, which was helpful for unpacking DNA and decreasing cytotoxicity. CBA-DAH-BG/pDNA polyplexes were characterized by efficient cellular uptake efficacy, extremely low cytotoxicity, and high transfection efficiency in two cell lines (i.e., NIH/3T3 and U87 MG), compared to 25 kDa polyethyleneimine (PEI) and the intermediate product CBA-DAH that were both devoid of biguanide groups. Of note, clathrin-mediated endocytosis and lipid rafts played an important role in the internalization of the polyplexes. Taken together, this strategy described herein may represent an innovative avenue for the design of more advanced nonviral gene vectors with high transfection efficiency and biocompatibility.

Uptake of PHMB in a bacterial nanocellulose-based wound dressing: A feasible clinical procedure.

BACKGROUND: With the increase of antimicrobial resistance in recent decades, other methods of preventing and fighting infections must be considered. Burn patients, whose wound areas are often extensive, are especially prone to wound infections. The loading of bacterial nanocellulose (BNC) with antiseptics has already been successfully performed but unfortunately, the described procedure is time-consuming and thus not applicable in a clinical emergency setting. Therefore, a clinically feasible approach was established. MATERIAL AND METHODS: Sheets of BNC-based wound dressings were placed into antiseptic solutions containing PHMB (Prontosan® and LAVANID® 2) and were left to soak for up to two hours. At different time points, samples were analysed for their concentration of PHMB and antiseptic efficacy. RESULTS: Within 30min, clinically relevant concentrations of PHMB were achieved in the BNC-based wound dressing. The 30-min PHMB uptake for Prontosan® and LAVANID® 2 resulted in concentrations of 0.05% and 0.019%, respectively. Samples from the PHMB loaded dressing showed a dose dependent antiseptic efficacy for Staphylococcus aureus. CONCLUSION: This experiment showed that the loading of BNC-based wound dressings with PHMB-containing antiseptics was achieved by a simple and quick procedure. According to studies a PHMB concentration of 0.001% can already inhibits all bacterial growth, indicating that the concentrations of PHMB in the BNC-based wound dressings after 30min are higher than the minimal inhibitory concentration and the antiseptic efficacy after 120min loading analysed by an standardized bacterial disk diffusion assay was shown to be comparable to the clinically used Suprasorb® X+PHMB wound dressing.

Mapping of biguanide transporters in human fat cells and their impact on lipolysis.

AIM: To examine the cell membrane transporters involved in mediating the antilipolytic effect of biguanides in human fat cells. MATERIALS AND METHODS: Gene expression of biguanide transporters was mapped in human subcutaneous adipose tissue and in adipocytes before and after differentiation. Those expressed in mature fat cells were knocked down by RNA interference (RNAi) and the antilipolytic effects of metformin and two novel, highly potent biguanides, NT1014 and NT1044, were examined. RESULTS: Analysis of the transporter affinity of biguanides in HEK293 cells overexpressing individual transporters showed that NT1014 and NT1044 had >10 times higher affinity than metformin. Animal studies showed that NT1014 was >5 times more potent than metformin in lowering plasma glucose in mice. In human fat cells, the novel biguanides displayed higher AMP-activated protein kinase activation and antilipolytic efficacy than metformin. Five transporters, organic cation transporter (OCT)1 (SLC22A1), organic cation transporter novel type 1 (OCTN1; SLC22A4), OCT3 (SLC22A3), plasma membrane monoamine transporter (PMAT; SLC29A4) and multidrug and toxin extrusion transporter (MATE1; SLC47A1), were detectable in fat cells but only OCT3, PMAT and MATE1 increased during adipogenesis in vitro and were enriched in fat cells compared with other adipose cell types. Gene knockdown by RNAi showed that MATE1 and PMAT reduction attenuated the antilipolytic effect of metformin but only PMAT knockdown decreased the effect of all three biguanides. CONCLUSIONS: While human fat cells primarily express three biguanide transporters, our data suggest that PMAT is the primary target for development of fat cell-specific antilipolytic biguanides with high sensitivity and potency.

Heme Binding Biguanides Target Cytochrome P450-Dependent Cancer Cell Mitochondria.

The mechanisms by which cancer cell-intrinsic CYP monooxygenases promote tumor progression are largely unknown. CYP3A4 was unexpectedly associated with breast cancer mitochondria and synthesized arachidonic acid (AA)-derived epoxyeicosatrienoic acids (EETs), which promoted the electron transport chain/respiration and inhibited AMPKα. CYP3A4 knockdown activated AMPKα, promoted autophagy, and prevented mammary tumor formation. The diabetes drug metformin inhibited CYP3A4-mediated EET biosynthesis and depleted cancer cell-intrinsic EETs. Metformin bound to the active-site heme of CYP3A4 in a co-crystal structure, establishing CYP3A4 as a biguanide target. Structure-based design led to discovery of N1-hexyl-N5-benzyl-biguanide (HBB), which bound to the CYP3A4 heme with higher affinity than metformin. HBB potently and specifically inhibited CYP3A4 AA epoxygenase activity. HBB also inhibited growth of established ER+ mammary tumors and suppressed intratumoral mTOR. CYP3A4 AA epoxygenase inhibition by biguanides thus demonstrates convergence between eicosanoid activity in mitochondria and biguanide action in cancer, opening a new avenue for cancer drug discovery.

Novel biguanide-based derivatives scouted as TAAR1 agonists: Synthesis, biological evaluation, ADME prediction and molecular docking studies.

Trace amines (TAs) are endogenous neuromodulators that play a functional role in the synaptic transmission within central nervous system (CNS), targeting trace amine-associated receptors (TAARs). Starting from our previous computational studies on TAAR1 and TAAR5 interactions with the unselective ligand 3-iodothyronamine (T1AM), we investigated the functional activity at murine and human TAAR1 and murine TAAR5 receptors of twenty-seven biguanide-based derivatives, including six newly synthesized compounds. Phenyl (BIG2, BIG4, BIG8 and BIG22) or benzyl (BIG10-BIG16) biguanides were found to be selective murine and human TAAR1 agonists with potencies in nanomolar or low micromolar range, respectively. In particular, compounds BIG2 and BIG12-BIG14 were the most promising and they could be considered valuable lead compounds worthy of further investigations. In addition to the interest for developing more effective human TAAR1 ligands, the disclosed here potent murine TAAR1 agonists could offer suitable tools for studying the pharmacology of TAAR1 receptor.

L503F variant of carnitine/organic cation transporter 1 efficiently transports metformin and other biguanides.

OBJECTIVES: Carnitine/organic cation transporter 1 (OCTN1) is involved in gastrointestinal absorption and mitochondrial toxicity of biguanides in rodents, but its pharmacokinetic roles in humans are largely unknown. The purpose of this study was to clarify the transport activities of two major OCTN1 variants, L503F and I306T, for gabapentin and three biguanide drugs, metformin, buformin and phenformin. METHODS: HEK293 cells were transfected with OCTN1 gene, its variants, or vector alone, and the uptake and cytotoxicity of each drug were examined. KEY FINDINGS: Buformin was identified to be an OCTN1 substrate. Uptake of biguanides, especially metformin, mediated by OCTN1 variant L503F, which is commonly found in Caucasians, was much higher than that by the wild-type transporter (WT-OCTN1). Cytotoxicity of metformin was also greater in HEK293 cells expressing the L503F variant, compared with WT-OCTN1. Uptake of gabapentin mediated by OCTN1 variant I306T, which is commonly found in both Asians and Caucasians, was lower than that by WT-OCTN1, although uptake of the typical OCTN1 substrate ergothioneine was similar. CONCLUSION: Organic cation transporter 1 variant L503F transports biguanides, especially metformin, more efficiently than WT-OCTN1, whereas the I306T variant transports gabapentin less efficiently than WT-OCTN1, suggesting that the common OCTN1 variants may alter pharmacokinetics of these drugs.

The antimicrobial polymer PHMB enters cells and selectively condenses bacterial chromosomes.

To combat infection and antimicrobial resistance, it is helpful to elucidate drug mechanism(s) of action. Here we examined how the widely used antimicrobial polyhexamethylene biguanide (PHMB) kills bacteria selectively over host cells. Contrary to the accepted model of microbial membrane disruption by PHMB, we observed cell entry into a range of bacterial species, and treated bacteria displayed cell division arrest and chromosome condensation, suggesting DNA binding as an alternative antimicrobial mechanism. A DNA-level mechanism was confirmed by observations that PHMB formed nanoparticles when mixed with isolated bacterial chromosomal DNA and its effects on growth were suppressed by pairwise combination with the DNA binding ligand Hoechst 33258. PHMB also entered mammalian cells, but was trapped within endosomes and excluded from nuclei. Therefore, PHMB displays differential access to bacterial and mammalian cellular DNA and selectively binds and condenses bacterial chromosomes. Because acquired resistance to PHMB has not been reported, selective chromosome condensation provides an unanticipated paradigm for antimicrobial action that may not succumb to resistance.

Uptake, translocation and possible biodegradation of the antidiabetic agent metformin by hydroponically grown Typha latifolia.

The increasing load of pharmaceutical compounds has raised concerns about their potential residues in aquatic environments and ecotoxicity. Metformin (MET), a widely prescribed antidiabetic II medicine, has been detected in high concentration in sewage and in wastewater treatment effluents. An uptake and translocation study was carried out to assess the ultimate fate of MET in phytoremediation. MET was removed from media by Typha latifolia, and the removal processes followed first order kinetics. After 28 days, the removal efficiencies were in a range of 74.0±4.1-81.1±3.3%. In roots, MET concentration was increasing during the first two weeks of the experiment but thereafter decreasing. In contrast, MET concentration was continuously increasing in rhizomes and leaves. Bioaccumulation of MET in roots was much higher than in leaves and rhizomes. As degradation product of metformin in the plant, methylbiguanide (MBG) was detected whereas guanylurea was undetectable. Moreover, MBG concentration in roots was increasing with exposure time. An enzymatic degradation experiment showed the degradation rate followed the order of MET

Repurposing metformin: an old drug with new tricks in its binding pockets.

Improvements in healthcare and nutrition have generated remarkable increases in life expectancy worldwide. This is one of the greatest achievements of the modern world yet it also presents a grave challenge: as more people survive into later life, more also experience the diseases of old age, including type 2 diabetes (T2D), cardiovascular disease (CVD) and cancer. Developing new ways to improve health in the elderly is therefore a top priority for biomedical research. Although our understanding of the molecular basis of these morbidities has advanced rapidly, effective novel treatments are still lacking. Alternative drug development strategies are now being explored, such as the repurposing of existing drugs used to treat other diseases. This can save a considerable amount of time and money since the pharmacokinetics, pharmacodynamics and safety profiles of these drugs are already established, effectively enabling preclinical studies to be bypassed. Metformin is one such drug currently being investigated for novel applications. The present review provides a thorough and detailed account of our current understanding of the molecular pharmacology and signalling mechanisms underlying biguanide-protein interactions. It also focuses on the key role of the microbiota in regulating age-associated morbidities and a potential role for metformin to modulate its function. Research in this area holds the key to solving many of the mysteries of our current understanding of drug action and concerted effects to provide sustained and long-life health.

Allosteric activation of the 5-HT3AB receptor by mCPBG.

The 5-HT3AB receptor contains three A and two B subunits in an A-A-B-A-B order. However, serotonin function at the 5-HT3AB receptor has been shown to depend solely on the A-A interface present in the homomeric receptor. Using mutations at sites on both the primary (E122) and complementary (Y146) faces of the B subunit, we demonstrate that meta-chlorophenyl biguanide (mCPBG), a 5-HT3 selective agonist, is capable of binding to and activating the 5-HT3AB receptor at all five subunit interfaces of the heteromer. Further, mCPBG is capable of allosterically modulating the activity of serotonin from these sites. While these five binding sites are similar enough that they conform to a monophasic dose - response relationship, we uncover subtle differences in the heteromeric binding sites. We also find that the A-A interface appears to contribute disproportionately to the efficacy of 5-HT3AB receptor activation.

Metformin retards aging in C. elegans by altering microbial folate and methionine metabolism.

The biguanide drug metformin is widely prescribed to treat type 2 diabetes and metabolic syndrome, but its mode of action remains uncertain. Metformin also increases lifespan in Caenorhabditis elegans cocultured with Escherichia coli. This bacterium exerts complex nutritional and pathogenic effects on its nematode predator/host that impact health and aging. We report that metformin increases lifespan by altering microbial folate and methionine metabolism. Alterations in metformin-induced longevity by mutation of worm methionine synthase (metr-1) and S-adenosylmethionine synthase (sams-1) imply metformin-induced methionine restriction in the host, consistent with action of this drug as a dietary restriction mimetic. Metformin increases or decreases worm lifespan, depending on E. coli strain metformin sensitivity and glucose concentration. In mammals, the intestinal microbiome influences host metabolism, including development of metabolic disease. Thus, metformin-induced alteration of microbial metabolism could contribute to therapeutic efficacy-and also to its side effects, which include folate deficiency and gastrointestinal upset.

A coupled array of noncovalent interactions impacts the function of the 5-HT3A serotonin receptor in an agonist-specific way.

The serotonin type 3A (5-HT(3)A) receptor is a Cys-loop (pentameric) neurotransmitter-gated ion channel found in the central and peripheral nervous systems and implicated in numerous diseases. In previous studies with the endogenous agonist serotonin, we identified two interactions critical for receptor function: a cation-π interaction with W183 in loop B (TrpB) and a hydrogen bond to E129 in loop A. Here we employ mutant cycle analyses utilizing conventional and unnatural amino acid mutagenesis to demonstrate that a third residue, D124 of loop A, forms two functionally important hydrogen bonds to the backbone of loop B. We also show that these three interactions, the cation-π interaction, the backbone hydrogen bonds, and the E129 hydrogen bond, are tightly coupled to each other, suggesting they function as a single unit. We also identify key functional differences between serotonin and the competitive partial agonist m-chlorophenyl biguanide (mCPBG) at these residues. mCPBG displays no cation-π at TrpB and extreme sensitivity to the positioning of E129, on which it is reliant for initiation of channel gating.

A preservative-and-fluorescein interaction model for benign multipurpose solution-associated transient corneal hyperfluorescence.

PURPOSE: Multipurpose contact lens solution (MPS)/preservative-associated transient corneal hyperfluorescence has been suggested to represent corneal injury. To determine the validity of this assumption, the molecular-level interactions of common disinfectants in soft contact lens MPS and the corneal epithelium using an in vitro model were assessed. METHODS: A liposome-based model of the corneal epithelial surface was developed and used to assess the interactions of polyhexamethylene biguanide (PHMB), polyquaternium-1 (PQ-1), and fluorescein with membrane components and the effects of PHMB and PQ-1 on membrane integrity. The fluorescence anisotropy (a measure of interactions between molecules) was determined. Liposome integrity was assessed by measuring the liposome melting point temperature. RESULTS: Free fluorescein did not associate with the liposome (P>0.4). Both fluorescein-tagged PHMB and PQ-1 associated with liposomes (P<0.002 and P≤0.01, respectively); however, only PHMB induced free fluorescein association with membrane components. At physiological temperature, no significant shift in the melting point temperature was observed when liposomes were exposed to PHMB from 0 to 100 ppm (P>0.05). In contrast, exposure of >7 ppm PQ-1 disrupted the liposomes. CONCLUSIONS: Based on this study, PHMB-to-liposome bilayer interaction is nondestructive, even at concentrations 100 times higher than found in commercially available MPS products. In contrast, PQ-1-to-liposome bilayer interaction led to liposome disruption. This study presents molecular-level evidence to support that preservative-associated transient corneal hyperfluorescence is a benign transient phenomenon and its evaluation clinically may be an ambiguous strategy for determining biocompatibility and cell surface integrity.

Liver uptake of biguanides in rats.

Metformin is an oral antihyperglycaemic agent widely used in the management of non-insulin-dependent diabetes mellitus. The liver is the primary target, metformin being taken up into human and rat hepatocytes via an active transport mechanism. The present study was designed to compare hepatic uptake of two biguanides, metformin and phenformin, in vitro and in vivo. In in vitro experiments, performed using rat cryopreserved hepatocytes, phenformin exhibited a much higher affinity and transport than metformin, with marked differences in kinetics. The K(m) values for metformin and phenformin were 404 and 5.17µM, respectively, with CLint (V(max)/K(m)) values 1.58µl/min per 10(6) cells and 34.7µl/min per 10(6) cells. In in vivo experiments, when (14)C-metformin and (14)C-phenformin were given orally to male rats at a dose of 50mg/kg, the liver concentrations of radioactivity at 0.5 hour after dosing were 21.5µg eq./g with metformin but 147.1µg eq./g for phenformin, ratios of liver to plasma concentrations being 4.2 and 61.3, respectively. In conclusion, the results suggest that uptake of biguanides by rat hepatocytes is in line with the liver distribution found in vivo, phenformin being more efficiently taken up by liver than metformin after oral administration.

Importance of porins for biocide efficacy against Mycobacterium smegmatis.

Mycobacteria are among the microorganisms least susceptible to biocides but cause devastating diseases, such as tuberculosis, and increasingly opportunistic infections. The exceptional resistance of mycobacteria to toxic solutes is due to an unusual outer membrane, which acts as an efficient permeability barrier, in synergy with other resistance mechanisms. Porins are channel-forming proteins in the outer membrane of mycobacteria. In this study we used the alamarBlue assay to show that the deletion of Msp porins in isogenic mutants increased the resistance of Mycobacterium smegmatis to isothiazolinones (methylchloroisothiazolinone [MCI]/methylisothiazolinone [MI] and octylisothiazolinone [2-n-octyl-4-isothiazolin-3-one; OIT]), formaldehyde-releasing biocides {hexahydrotriazine [1,3,5-tris (2-hydroxyethyl)-hexahydrotriazine; HHT] and methylenbisoxazolidine [N,N'-methylene-bis-5-(methyloxazolidine); MBO]}, and the lipophilic biocides polyhexamethylene biguanide and octenidine dihydrochloride 2- to 16-fold. Furthermore, the susceptibility of the porin triple mutant against a complex disinfectant was decreased 8-fold compared to wild-type (wt) M. smegmatis. Efficacy testing in the quantitative suspension test EN 14348 revealed 100-fold improved survival of the porin mutant in the presence of this biocide. These findings underline the importance of porins for the susceptibility of M. smegmatis to biocides.

Blockade of the antinociception induced by diclofenac, but not of indomethacin, by sulfonylureas and biguanides.

There is evidence that administration of sulfonylureas, such as glibenclamide and tolbutamide, blocks diclofenac-induced antinociception, suggesting that diclofenac activates ATP-sensitive K(+) channels. However, there is no evidence for the interaction between diclofenac and other hypoglycemic drugs, such as the biguanides metformin or phenformin. Therefore, this work was undertaken to determine whether two sulfonylureas, glibenclamide and glipizide, as well as two biguanides, metformin and phenformin, have any effect on the systemic antinociception that is induced by diclofenac and indomethacin using the rat formalin test as an animal model. Systemic injections of diclofenac (10 to 30mg/kg) and indomethacin (10 to 30mg/kg) produced dose-dependent antinociception during the second phase of the test. Systemic pretreatment with glibenclamide (3 and 10mg/kg), glipizide (3 and 10mg/kg), metformin (100 and 180mg/kg) or phenformin (100 and 180mg/kg) blocked diclofenac-induced systemic antinociception in the second phase of the test (P<0.05). In contrast, pretreatment with glibenclamide, glipizide, metformin or phenformin did not block indomethacin-induced systemic antinociception (P>0.05). These data suggest that diclofenac, but not indomethacin, activated K(+) channels and metformin and phenformin-dependent mechanisms, which resulted in systemic antinociceptive effects in the rat formalin test.

The phenanthridine biguanides efficiently differentiate between dGdC, dAdT and rArU sequences by two independent, sensitive spectroscopic methods.

At submicromolar concentrations two novel phenanthridine biguanides exhibit distinctly different spectroscopic signals for dGdC and dAdT sequences, respectively, by opposite fluorimetric changes (quenching for dGdC and increase for dAdT) and especially the bis-biguanide derivative gives an opposite ICD response (negative ICD for dGC and strong positive for dAdT). This specific signalling was explained by the ability of compounds to switch the binding mode from intercalation into dGdC to minor groove binding into dAdT sequences. Both compounds bind to rArU by intercalation, yielding different fluorimetric and CD response in comparison to any of aforementioned ds-DNA. Moreover, both compounds revealed significantly higher affinity toward ds-polynucleotides in comparison to previously studied alkylamine- and urea-analogues. Furthermore, DNA/RNA binding properties of novel compounds could be controlled by pH, due to the protonation of heterocyclic nitrogen. Low in vitro cytotoxicity of both compounds against human cell lines makes them interesting spectrophotometric probes.

Persistent interactions between biguanide-based compound NB325 and CXCR4 result in prolonged inhibition of human immunodeficiency virus type 1 infection.

We previously demonstrated that the biguanide-based compound NB325 inhibits human immunodeficiency virus type 1 (HIV-1) infection by interacting with the CXCR4 viral coreceptor. This interaction also appeared to be persistent, since HIV-1 infection was inhibited even when the virus was introduced subsequent to the removal of NB325 from the cell culture medium. The present studies were conducted to determine the extent and mechanism of this prolonged antiviral activity. Persistent inhibition of HIV-1 infection by NB325 was concentration dependent and was apparent up to 8 h after removal of the compound. Flow cytometric analyses of stimulated CD4(+) T lymphocytes exposed to NB325 demonstrated concentration-dependent reductions in CXCR4 extracellular loop 2 epitope recognition that were maintained up to 24 h after removal of the compound. CXCL12-induced chemotaxis was also persistently inhibited following pre-exposure to NB325. These results demonstrate that persistent inhibition of X4 HIV-1 infection by NB325 involves extended perturbation of the viral coreceptor CXCR4.

B(2) kinin receptors mediate the Indian red scorpion venom-induced augmentation of visceral reflexes via the nitric oxide cyclic guanosine monophosphate pathway.

AIM: This study was performed to delineate the kinin (receptor)-dependent pathways in the Indian red scorpion (Mesobuthus tamulus; MBT) venom-induced pulmonary oedema as well as the augmentation of cardio-pulmonary reflexes evoked by phenyldiguanide (PDG). METHODS: In urethane-anaesthetized adult rats, the effect of venom on the PDG reflex responses (blood pressure, heart rate and respiration rate) and the pulmonary water content was ascertained using various antagonists(des- Arg, B(1) receptor antagonist; Hoe 140, B(2) receptor antagonist; N(omega)-nitro-l-arginine methyl ester (l-NAME), nitric oxide (NO) synthase inhibitor; methylene blue, soluble guanylate cyclase inhibitor; and glibenclamide, K(+)(ATP) channel blocker). The effect of phosphodiesterase V inhibitor (sildenafil citrate) on the reflex response and the pulmonary water content was also examined and compared with venom-induced responses. RESULTS: Intravenous injection of PDG (10 microg kg(-1)) evoked apnoea, bradycardia and hypotension lasting >60 s. Exposure to MBT venom (100 microg kg(-1)) for 30 min augmented the PDG reflex responses by two times and increased the pulmonary water content, significantly. Hoe 140 blocked the venom-induced responses (augmentation of PDG reflex and increased pulmonary water content) whereas des-Arg did not. l-NAME, methylene blue or glibenclamide also blocked the venom-induced responses. Furthermore, sildenafil citrate (that increases cGMP levels) produced augmentation of PDG reflex response and increased the pulmonary water content as seen with venom. CONCLUSION: The results indicate that venom-induced responses involve B(2) kinin receptors via the NO-dependent guanylate cyclase-cGMP pathway involving K(+)(ATP) channels.