Antinicotinic action of nicotine and lobeline on frog sartorius muscle

Summary Evidence is presented to show that nicotine and lobeline cause desensitization of frog sartorius muscle cells. Nicotine (3.3 to 166 M) caused a dose-dependent depolarization. The membrane potential returned to control in 30–40 min in the presence of nicotine. The duration of the nicotine-induced depolarization was not related to the nicotine concentration. In contrast, the duration of the depolarization induced by carbachol (109–546 M) decreased as the concentration of carbachol was increased.The desensitization caused by lobeline (3–300 M) differed from that caused by nicotine and other cholinomimetic agents in that it was not preceded by a depolarization-repolarization sequence of change in the muscle cell membrane. At the time of repolarization after nicotine or during exposure to lobeline the application of a second dose of nicotine had no effect or caused a depolarization that was low in amplitude and brief in duration (<3 min).In contrast to nicotine and lobeline d-tubocurarine (d-TC: 1.4–14 M) reduced the amplitude of the nicotine depolarization but did not affect the duration 930 to 40 min). Tetraethylammonium (1–5 mM) reduced the amplitude and duration (5–15 min) of the nicotine-induced depolarization.Nicotine caused an increase in ⁴²K efflux which paralleled the time course of the nicotine-induced depolarization. Lobeline and d-TC did not stimulate ⁴²K efflux. After the muscles had been soaked for 30 to 60 min in nicotine, lobeline or d-TC, the stumulation of ⁴²K efflux caused by a second dose of nicotine was decreased. The nicotine-induced stimulation of ⁴²K efflux in desensitized muscles paralleled the nicotine-induced changes in the muscle membrane potential seen during desensitization.On the basis of the duration of the nicotine-induced depolarization it was concluded that lobeline's antinicotinic effect was due to desensitization. These data further suggest that nicotine acts as a metaphilic agonist and that lobeline acts as a metaphilic antagonist.This study was aided by grants from the American Medical Associated Education and Research Foundation awarded to Drs. Robert Volle and Edward G. Henderson.     

Chlorpyrifos and chlorpyrifos-oxon inhibit axonal growth by interfering with the morphogenic activity of acetylcholinesterase

A primary role of acetylcholinesterase (AChE) is regulation of cholinergic neurotransmission by hydrolysis of synaptic acetylcholine. In the developing nervous system, however, AChE also functions as a morphogenic factor to promote axonal growth. This raises the question of whether organophosphorus pesticides (OPs) that are known to selectively bind to and inactivate the enzymatic function of AChE also interfere with its morphogenic function to perturb axonogenesis. To test this hypothesis, we exposed primary cultures of sensory neurons derived from embryonic rat dorsal root ganglia (DRG) to chlorpyrifos (CPF) or its oxon metabolite (CPFO). Both OPs significantly decreased axonal length at concentrations that had no effect on cell viability, protein synthesis or the enzymatic activity of AChE. Comparative analyses of the effects of CPF and CPFO on axonal growth in DRG neurons cultured from AChE nullizygous (AChE -/-) versus wild type (AChE +/+) mice indicated that while these OPs inhibited axonal growth in AChE+/+ DRG neurons, they had no effect on axonal growth in AChE -/- DRG neurons. However, transfection of AChE -/- DRG neurons with cDNA encoding full-length AChE restored the wild type response to the axon inhibitory effects of OPs. These data indicate that inhibition of axonal growth by OPs requires AChE, but the mechanism involves inhibition of the morphogenic rather than enzymatic activity of AChE. These findings suggest a novel mechanism for explaining not only the functional deficits observed in children and animals following developmental exposure to OPs, but also the increased vulnerability of the developing nervous system to OPs.     

Decreased axonal density and altered expression profiles of axonal guidance genes underlying lead (Pb) neurodevelopmental toxicity at early embryonic stages in the zebrafish

Previous studies have reported that environmental lead (Pb) exposure can result in neurological alterations in children leading to reduced IQ, attention deficit hyperactivity disorder, and diminished reading and learning abilities. However, the specific alterations in neurodevelopmental morphology and the underlying genetic mechanisms of these alterations have not yet been thoroughly defined. To investigate alterations in neurologic morphology and test the hypothesis that developmental Pb neurotoxicity is partially mediated through alterations in neuronal growth and transport function of axons, the changes of specific axon tracts in the embryonic zebrafish brain were observed with anti-acetylated α-tubulin staining at several developmental time points through 36 hours post fertilization (hpf). In addition, the role of a subset of axonogenesis-related genes including shha, epha4b, netrin1b, netrin2, and noiwas investigated with real-time quantitative PCR (qPCR). Pb treatment resulted in decreased axonal density at 18, 20, and 24 hpf for specific axon tracts in the midbrain and forebrain. These observations corresponded to an observed down-regulation of shha and epha4b at 14 and 16 hpf, respectively. The axonal density in Pb exposed individuals at later stages (30 and 36 hpf) was not significantly different from controls. An overexpression of netrin2 at these two developmental stages suggests a novel role for this gene in regulating axonal density specific to Pb neurotoxicity. Although no significant differences in axonal density was observed in the two later developmental stages, further studies are needed to determine if the morphologic alterations observed at the earlier stages will have lasting functional impacts.     

Skeletal muscle degeneration induced by venom phospholipases A2: insights into the mechanisms of local and systemic myotoxicity

Local and systemic skeletal muscle degeneration is a common consequence of envenomations due to snakebites and mass bee attacks. Phospholipases A₂ (PLA₂) are important myotoxic components in these venoms, inducing a similar pattern of degenerative events in muscle cells. Myotoxic PLA₂s bind to acceptors in the plasma membrane, which might be lipids or proteins and which may differ in their affinity for the PLA₂s. Upon binding, myotoxic PLA₂s disrupt the integrity of the plasma membrane by catalytically dependent or independent mechanisms, provoking a pronounced Ca²⁺ influx which, in turn, initiates a complex series of degenerative events associated with hypercontraction, activation of calpains and cytosolic Ca²⁺-dependent PLA₂s, and mitochondrial Ca²⁺ overload. Cell culture models of cytotoxicity indicate that some myotoxic PLA₂s affect differentiated myotubes in a rather selective fashion, whereas others display a broad cytolytic effect. A model is presented to explain the difference between PLA₂s that induce predominantly local myonecrosis and those inducing both local and systemic myotoxicity. The former bind not only to muscle cells, but also to other cell types, thereby precluding a systemic distribution of these PLA₂s and their action on distant muscles. In contrast, PLA₂s that bind muscle cells in a more selective way are not sequestered by non-specific interactions with other cells and, consequently, are systemically distributed and reach muscle cells in other locations.     

Microvessel damage by B. jararacussu snake venom: pathogenesis and influence on muscle regeneration.

The loss of muscle mass consequent to poor muscle regeneration is a common sequela following the injection of Bothrops jararacussu snake venom. Since an intact microvasculature plays a central role in the success of muscle regeneration, the poor muscle regeneration seen after envenomation could be explained by damage to the local microvasculature. In this work, we investigated the pathogenesis of microvessel damage caused by B. jararacussu venom and its correlation with poor muscle regeneration. The right soleus muscle of adult mice was injected with 80 microg of venom and the mice were killed from 2 min to 3 months later. Similarly, the soleus muscle of other mice was injected with 80 microg of bothrosptoxin-I (BthTX-I), a non-vasculotoxic myotoxin. Tissue samples were prepared for analysis by electron (venom only) and light (venom and BthTX-I) microscopy. The extent of revascularization was assessed using light microscopy by examining recanalization of thrombi and calculating the individual capillary-to-fiber-ratio, the number of capillaries around a fiber and the capillary/muscle cell ratio. Microvessel damage by venom started within 5 min and, after 6 h, there was total degeneration of the capillaries with failure of the local microcirculation. The time-course of the ultrastructural lesions suggested that endothelial cells were probably damaged by a direct action of B. jararacussu venom on these cells. The revascularization of muscle damaged by venom, but not by BthTX-I, occurred later and was very poor. These results indicate a central role for vascular lesions in muscle regeneration after damage by B. jararacussu venom.     

Treatment with an anti-inflammatory drug is detrimental for muscle regeneration at Bothrops jararacussu envenoming: An experimental study

We evaluated the effects of deflazacort (DFZ) on muscle regeneration following Bothrops jararacussu envenoming. Tibialis anterior muscle from adult mice was injected with 80 μg of venom. Animals received DFZ during 6 days. Seven and 60 days after envenoming, DFZ lead to a decrease in the total number of muscle fibers and an increase in interstitial fibrosis. We conclude that DFZ treatment may aggravate the loss of muscle mass after B. jararacussu envenoming.     

Concentration- and time-dependent behavioral changes in Caenorhabditis elegans after exposure to nicotine

Nicotine induces profound behavioral responses in the model organism Caenorhabditis elegans. We tested the effect of a broad range of concentrations of nicotine (from 0.001 mM to 30 mM in nematode growth medium) on C. elegans locomotor behavior. We also followed the time-course influence on the sensitivity of C. elegans to nicotine (from 0 min to 300 min). A low concentration (0.001 mM) of this alkaloid causes a reduction of the speed of movement. By contrast, moderate concentrations (0.01 and 0.1 mM) induced acceleration of the mean speed of locomotion of C. elegans. High doses of nicotine (above 1 mM) induced slowing down of the movements and, finally, paralysis. Time-dependent analysis revealed that the stimulating effect of nicotine abolished the slowing down of C. elegans in control experiments after 30 min in the presence of 0.001, 0.1 and 10 mM nicotine. In the presence of 0.1 mM nicotine, the stimulation phase lasted up to 70 min. The evidence indicates that nicotine can have dual effects on the speed of locomotion, which is dependent on differences in its dosage and treatment time.     

The effects of combined exposure to the pyrethroids deltamethrin and S-bioallethrin on hippocampal inhibition and skeletal muscle hyperexcitability in rats

The default assumption that different pyrethroid insecticides, sharing a common mode of action, will show additivity of toxicity has not always been supported by in vitro measures, some of which have indicated antagonism. Our intention was to see whether the antagonism between pyrethroids of different classes seen in vitro could be reproduced in vivo. We therefore investigated the effects of single and combined exposures to two commonly used pyrethroids, deltamethrin (type II) and S-bioallethrin (type I) given intravenously to anaesthetised rats. We used two quantitative measures that are responsive to pyrethroids: the duration of prolongation of hippocampal dentate granule cell inhibition and the amplitude of the abnormal electromyogram discharge. At equi-toxic doses, S-bioallethrin extended the inter-stimulus interval evoking 50% inhibition in the hippocampus by 30+/-2.2 ms, and deltamethrin extended it by 199+/-21 ms. Combined administration of the same doses of deltamethrin and S-bioallethrin extended hippocampal inhibition by 164+/-14 ms, which did not differ significantly from the effect of deltamethrin alone. S-bioallethrin was without any effect on the electromyogram, and produced no significant change in the amplitude of the abnormal muscle discharges evoked by deltamethrin. The increase in arterial blood pressure evoked by the combination was significantly less than that evoked by either pyrethroid alone (p<0.001). In summary, although our electrophysiological indices provide no support for functional antagonism between these two pyrethroids, they also fail to indicate any summation of effect.     

The marine polyether gambierol enhances muscle contraction and blocks a transient K current in skeletal muscle cells

Gambierol is a complex marine toxin first isolated with ciguatoxins from cell cultures of the toxic dinoflagellate Gambierdiscus toxicus. Despite the chemical complexity of the polycyclic ether toxin, the total successful synthesis of gambierol has been achieved by different chemical strategies. In the present work the effects of synthetic gambierol on mouse and frog skeletal neuromuscular preparations and Xenopus skeletal myocytes have been studied. Gambierol (0.1-5 μM) significantly increased isometric twitch tension in neuromuscular preparations stimulated through the motor nerve. Less twitch augmentation was observed in directly stimulated muscles when comparing twitch tension-time integrals obtained by nerve stimulation. Also, gambierol induced small spontaneous muscle contraction originating from presynaptic activity that was completely inhibited by d-tubocurarine. Gambierol slowed the rate of muscle action potential repolarization, triggered spontaneous and/or repetitive action potentials, and neither affected action potential amplitude nor overshoot in skeletal muscle fibers. These results suggest that gambierol through an action on voltage-gated K⁺ channels prolongs the duration of action potentials, enhances the extent and time course of Ca²⁺ release from the sarcoplasmic reticulum, and increases twitch tension generation. Further evidence is provided that gambierol at sub-micromolar concentrations blocks a fast inactivating outward K⁺ current that is responsible for action potential prolongation in Xenopus skeletal myocytes.     

Beta3-adrenoceptor agonist stimulation of the Na+, K+ -pump in rat skeletal muscle is mediated by beta2- rather than beta3-adrenoceptors

BACKGROUND AND PURPOSE: In cardiac muscle, BRL 37344, a selective beta3-adrenoceptor agonist, activates the Na+, K+ -pump via NO signalling. This study investigated whether BRL 37344 also activates the Na+, K+ -pump via beta3-adrenoceptors in skeletal muscle. EXPERIMENTAL APPROACH: Isolated rat soleus muscles were incubated between 1 and 60 min in buffer. Intracellular Na+, K+ content and Na+, K+ -pump activity were measured using flame photometry and ouabain-suppressible 86Rb+ uptake, respectively. Additional muscles were mounted on force transducers and stimulated (60 Hz for 2 s) every 10 min. KEY RESULTS: BRL 37344 (10(-8) -10(-5) M) induced a concentration- and time-dependent reduction in intracellular Na+, and increased ouabain-suppressible 86Rb+ uptake by up to 112%. BRL 37344-induced reductions in intracellular Na+ were blocked by the beta1/beta2-adrenoceptor antagonist, nadolol (10(-7) M), and the beta2-adrenoceptor antagonist, ICI 118,551 (10(-7) -10(-5) M), but not by beta3- or beta1-adrenoceptor antagonists, SR 59230A (10(-7) M) and CGP 20712A (10(-7) -10(-5) M), respectively. Another beta3-adrenoceptor agonist, CL 316,243, did not alter intracellular Na+. BRL 37344-induced reductions in intracellular Na+ were not blocked by L-NAME, an NOS inhibitor, or ODQ, a guanylyl cyclase inhibitor. The NO donors, SNP and SNAP, did not alter intracellular Na+. BRL 37344 rapidly recovered force in muscles depressed by high [K+]o, an effect that was blocked by nadolol, but not L-NAME. CONCLUSIONS AND IMPLICATIONS: In rat soleus muscle, the beta3-adrenoceptor agonist BRL 37344 stimulated the Na+, K+ -pump via beta2-adrenoceptors. A more selective beta3-adrenoceptor agonist did not affect Na+, K+ homeostasis in skeletal muscle. NO did not seem to mediate Na+, K+ -pump stimulation in skeletal muscle.     

Blockade by lobeline of potassium exchange in skeletal muscle

Summary Lobeline (0.05 to 2.5 mM) caused depolarization of muscle fibers in frog sartorius muscle bathed in chloride-free solution. When the extent of depolarization was plotted against the log of the concentration of lobeline, the relationship was described by a straight line with a slope of 57 mV for a 10-fold change in the concentration of lobeline (0.2 to 2.0 mM). Like lobeline, butacaine (0.1 to 0.5 mM) caused muscle depolarization with a 57 mV change in membrane potential for a 10-fold change in the concentration of the local anesthetic. Lobeline (0.05 to 0.15 mM) depressed the depolarization of the muscle fibers caused by the elevation of (K⁺]₀ over a range of 2.5 to 50 mM. ⁴²K-exchange in resting muscle was also depressed by lobeline (0.05 to 0.25 mM). The blockade by lobeline of ⁴²K-efflux followed a typical dose-response relationship with 0.06 mM lobeline required to cause a 50% decrease of ⁴²K-efflux. These findings show that lobeline in concentrations not much greater than those required to block neuromuscular transmission, has a direct action on ion exchange in skeletal muscle. These actions on electrogenic mechanisms may contribute to the anti-nicotinic action of lobeline and to the desensitization of endplate acetylcholine receptors caused by lobeline.This study was supported by a grant from AMA-ERF.     

Effect of heparin treatment on the expression and activity of different ion-motive P-type ATPase isoforms from mouse extensor digitorum longus muscle during degeneration and regeneration after Bothrops jararacussu venom injection

Ca²⁺ ions are essential to myonecrosis, a serious complication of snake envenomation, and heparin seems to counteract this effect. We investigated the effect of local injection of Bothrops jararacussu venom in mouse fast-twitch extensor digitorum longus (EDL) muscle, without or with heparin, on functional/molecular alterations of two central proteins involved in intracellular Ca²⁺ homeostasis, sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) and Na⁺/K⁺-ATPase. EDL-specific SERCA1 isoform expression dropped significantly just after venom administration (up to 60% compared to control EDL values at days 1 and 3; p < 0.05) while SERCA2 and Na⁺/K⁺-ATPase α₁ isoform expression increased at the same time (3-6- and 2-3-fold, respectively; p < 0.05). Although not significant, Na⁺/K⁺-ATPase α₂ isoform followed the same trend. Except for SERCA2, all proteins reached basal levels at the 7th day. Intravenous heparin treatment did not affect these profiles. Ca²⁺-ATPase activity was also decreased during the first days after venom injection, but here heparin was effective to reinstate activity to control levels within 3 days. We also showed that B. jararacussu venom directly inhibited Ca²⁺-ATPase activity in a concentration-dependent manner. Our results indicate that EDL SERCA and Na⁺/K⁺-ATPase are importantly affected by B. jararacussu venom and heparin has protective effect on activity but not on protein expression.     

Behavioral and physiological effects of acute ketamine exposure in adult zebrafish

Ketamine is a non-competitive glutamatergic antagonist used to induce sedation and analgesia. In sub-anesthetic doses, it induces hyperlocomotion, impairs memory and evokes stereotypic circling in rodents. Zebrafish (Danio rerio) emerged as a promising new animal model to screen the effects of psychotropic compounds. Here, we investigated the effects of sub-anesthetic doses of ketamine on anxiety, locomotion, habituation and social behavior of adult zebrafish. Acute 20-min exposure to 20 and 40 mg/L (but not 2 mg/L) of ketamine reduced anxiety, impaired intra-session habituation, evoked circular swimming and disrupted zebrafish shoaling. Additionally, ketamine reduced whole-body cortisol levels and elevated brain c-fos expression in zebrafish. Our findings demonstrate the sensitivity of zebrafish to behavioral and physiological effects of sub-anesthetic doses of ketamine, further supporting the utility of this species as a model for neuropharmacological research, including testing ketamine and related drugs.     

Toxicity of cylindrospermopsin, and other apparent metabolites from Cylindrospermopsis raciborskii and Aphanizomenon ovalisporum, to the zebrafish (Danio rerio) embryo

Cyanobacteria produce a diverse array of toxic or otherwise bioactive compounds that pose growing threats to human and environmental health. We utilized the zebrafish (Danio rerio) embryo, as a model of vertebrate development, to investigate the inhibition of development pathways (i.e. developmental toxicity) by the cyanobacterial toxin, cylindrospermopsin (CYN), as well as extracts from various isolates of Cylindrospermopsis raciborskii and Aphanizomenon ovalisporum. CYN was toxic only when injected directly into embryos, but not by direct immersion at doses up to 50 μg/ml. Despite the dose dependency of toxicity observed following injection of CYN, no consistent patterns of developmental defects were observed, suggesting that toxic effects of CYN may not target specific developmental pathways. In contrast, direct immersion of embryos in all of the extracts resulted in both increased mortality and reproducible, consistent, developmental dysfunctions. Interestingly, there was no correlation of developmental toxicity observed for these extracts with the presence of CYN or with previously reported toxicity for these strains. These results suggest that CYN is lethal to zebrafish embryos, but apparently inhibits no specific developmental pathways, whereas other apparent metabolites from C. raciborskii and A. ovalisporum seem to reproducibly inhibit development in the zebrafish model. Continued investigation of these apparent, unknown metabolites is needed.     

Promotion of axonal maturation and prevention of memory loss in mice by extracts of Astragalus mongholicus

BACKGROUND AND PURPOSE: Neurons with atrophic neurites may remain alive and therefore may have the potential to regenerate even when neuronal death has occurred in some parts of the brain. This study aimed to explore effects of drugs that can facilitate the regeneration of neurites and the reconstruction of synapses even in severely damaged neurons. EXPERIMENTAL APPROACH: We investigated the effects of extracts of Astragalus mongholicus on the cognitive defect in mice caused by injection with the amyloid peptide Abeta(25-35). We also examined the effect of the extract on the regeneration of neurites and the reconstruction of synapses in cultured neurons damaged by Abeta(25-35). KEY RESULTS: A. mongholicus extract (1 g kg(-1) day(-1) for 15 days, p.o.) reversed Abeta(25-35)-induced memory loss and prevented the loss of axons and synapses in the cerebral cortex and hippocampus in mice. Treatment with Abeta(25-35) (10 microM) induced axonal atrophy and synaptic loss in cultured rat cortical neurons. Subsequent treatment with A. mongholicus extract (100 microg/ml) resulted in significant axonal regeneration, reconstruction of neuronal synapses, and prevention of Abeta(25-35)-induced neuronal death. Similar extracts of A. membranaceus had no effect on axonal atrophy, synaptic loss, or neuronal death. The major known components of the extracts (astragalosides I, II, and IV) reduced neurodegeneration, but the activity of the extracts did not correlate with their content of these three astragalosides. CONCLUSION AND IMPLICATIONS: A. mongholicus is an important candidate for the treatment of memory disorders and the main active constituents may not be the known astragalosides.     

Bisphenol A activates Maxi-K (KCa1.1) channels in coronary smooth muscle

Background and purpose:

Bisphenol A (BPA) is used to manufacture plastics, including containers for food into which it may leach. High levels of exposure to this oestrogenic endocrine disruptor are associated with diabetes and heart disease. Oestrogen and oestrogen receptor modulators increase the activity of large conductance Ca²⁺/voltage-sensitive K⁺ (Maxi-K; K_Ca1.1) channels, but the effects of BPA on Maxi-K channels are unknown. We tested the hypothesis that BPA activates Maxi-K channels through a mechanism that depends upon the regulatory β1 subunit.

Experimental approach:

Patch-clamp recordings of Maxi-K channels were made in human and canine coronary smooth muscle cells as well as in AD-293 cells expressing pore-forming α or α plus β1 subunits.

Key results:

BPA (10 µM) activated an outward current in smooth muscle cells that was inhibited by penitrem A (1 µM), a Maxi-K blocker. BPA increased Maxi-K activity in inside-out patches from coronary smooth muscle, but had no effect on single channel conductance. In AD-293 cells with Maxi-K channels composed of α subunits alone, 10 µM BPA did not affect channel activity. When channels in AD-293 cells contained β1 subunits, 10 µM BPA increased channel activity. Effects of BPA were rapid (<1 min) and reversible. A higher concentration of BPA (100 µM) increased Maxi-K current independent of the β1 subunit.

Conclusions and implications:

Our data indicate that BPA increased the activity of Maxi-K channels and may represent a basis for some potential toxicological effects.     

How mitochondrial dysfunction affects zebrafish development and cardiovascular function: an in vivo model for testing mitochondria-targeted drugs

Background and Purpose

Mitochondria are a drug target in mitochondrial dysfunction diseases and in antiparasitic chemotherapy. While zebrafish is increasingly used as a biomedical model, its potential for mitochondrial research remains relatively unexplored. Here, we perform the first systematic analysis of how mitochondrial respiratory chain inhibitors affect zebrafish development and cardiovascular function, and assess multiple quinones, including ubiquinone mimetics idebenone and decylubiquinone, and the antimalarial atovaquone.

Experimental Approach

Zebrafish (Danio rerio) embryos were chronically and acutely exposed to mitochondrial inhibitors and quinone analogues. Concentration-response curves, developmental and cardiovascular phenotyping were performed together with sequence analysis of inhibitor-binding mitochondrial subunits in zebrafish versus mouse, human and parasites. Phenotype rescuing was assessed in co-exposure assays.

Key Results

Complex I and II inhibitors induced developmental abnormalities, but their submaximal toxicity was not additive, suggesting active alternative pathways for complex III feeding. Complex III inhibitors evoked a direct normal-to-dead transition. ATP synthase inhibition arrested gastrulation. Menadione induced hypochromic anaemia when transiently present following primitive erythropoiesis. Atovaquone was over 1000-fold less lethal in zebrafish than reported for Plasmodium falciparum, and its toxicity partly rescued by the ubiquinone precursor 4-hydroxybenzoate. Idebenone and decylubiquinone delayed rotenone- but not myxothiazol- or antimycin-evoked cardiac dysfunction.

Conclusion and Implications

This study characterizes pharmacologically induced mitochondrial dysfunction phenotypes in zebrafish, laying the foundation for comparison with future studies addressing mitochondrial dysfunction in this model organism. It has relevant implications for interpreting zebrafish disease models linked to complex I/II inhibition. Further, it evidences zebrafish''s potential for in vivo efficacy or toxicity screening of ubiquinone analogues or antiparasitic mitochondria-targeted drugs.     

Characterization of kinin receptors in human cultured detrusor smooth muscle cells

BACKGROUND AND PURPOSE: Kinins have an important role in inflammatory cystitis and in animal pathophysiological models, by acting on epithelium, fibroblasts, sensory innervation and smooth muscle. The aim of this study was to characterize the receptors responsible for direct motor responses induced by kinins on human detrusor. EXPERIMENTAL APPROACH: Human detrusor cells from biopsies were isolated and maintained in culture. B(1) and B(2) kinin receptors were characterized by means of radioligand and functional experiments (PI accumulation and PGE(2) release). KEY RESULTS: [(3)H]-[desArg(9)]-Lys-BK and [(3)H]-BK saturation studies indicated receptor density (B(max)) and K (d) values of 19 or 113 fmol mg(-1), and 0.16 or 0.11 nM for the B(1) or B(2) receptors, respectively. Inhibition binding studies indicated the selectivity of the B(1) receptor antagonist [desArg(9)Leu(8)]-Lys-BK and of the B(2) receptor antagonists Icatibant and MEN16132. [DesArg(9)]-Lys-BK and BK induced PI accumulation with an EC(50) of 1.6 and 1.4 nM and different maximal responses (E(max) of [desArg(9)]-Lys-BK was 10% of BK). BK also induced prostaglandin E(2) release (EC(50) 2.3 nM), whereas no response was detected with the B(1) receptor agonist. The incubation of detrusor smooth muscle cells with interleukin 1beta (IL-1beta) or tumour necrosis factor-alpha (TNF-alpha) (10 ng ml(-1)) induced a time-dependent increase in radioligand-specific binding, which was greater for the B(1) than for the B(2) receptor. CONCLUSIONS AND IMPLICATIONS: Human detrusor smooth muscle cells in culture retain kinin receptors, and represent a suitable model to investigate the mechanisms and changes that occur under chronic inflammatory conditions.     

Purification and characterization of Taiwan cobra venom proteins with weak toxicity.

Two proteins G2a and G2b with molecular masses of approximately 24 kDa were isolated from Naja naja atra (Taiwan cobra) venom using sequential chromatography on gel filtration, ion-exchanger and reverse phase columns. The results of Edman degradation and mass analysis revealed that G2a is a cysteine-rich protein reported previously, and G2b is a novel polypeptide. CD spectra showed that the gross conformation of G2a and G2b notably differed. G2a exhibited an activity higher than that noted with G2b on inhibiting carbachol-induced muscle contraction. However, the two proteins weakly blocked muscle contraction evoked by K+. The observations that the two proteins exhibit the toxic activity in the concentration of micromolar range suggest that they are inherently weak toxins as other snake venom cysteine-rich proteins.