Spontaneous Ca2+ transients in rat pulmonary vein cardiomyocytes are increased in frequency and become more synchronous following electrical stimulation.

The pulmonary veins have an external sleeve of cardiomyocytes that are a widely recognised source of ectopic electrical activity that can lead to atrial fibrillation. Although the mechanisms behind this activity are currently unknown, changes in intracellular calcium (Ca2+) signalling are purported to play a role. Therefore, the intracellular Ca2+ concentration was monitored in the pulmonary vein using fluo-4 and epifluorescence microscopy. Electrical field stimulation evoked a synchronous rise in Ca2+ in neighbouring cardiomyocytes; asynchronous spontaneous Ca2+ transients between electrical stimuli were also present. Immediately following termination of electrical field stimulation at 3 Hz or greater, the frequency of the spontaneous Ca2+ transients was increased from 0.45 ± 0.06 Hz under basal conditions to between 0.59 ± 0.05 and 0.65 ± 0.06 Hz (P < 0.001). Increasing the extracellular Ca2+ concentration enhanced this effect, with the frequency of spontaneous Ca2+ transients increasing from 0.45 ± 0.05 Hz to between 0.75 ± 0.06 and 0.94 ± 0.09 Hz after electrical stimulation at 3 to 9 Hz (P < 0.001), and this was accompanied by a significant increase in the velocity of Ca2+ transients that manifested as waves. Moreover, in the presence of high extracellular Ca2+, the spontaneous Ca2+ transients occurred more synchronously in the initial few seconds following electrical stimulation. The ryanodine receptors, which are the source of spontaneous Ca2+ transients in pulmonary vein cardiomyocytes, were found to be arranged in a striated pattern in the cell interior, as well as along the periphery of cell. Furthermore, labelling the sarcolemma with di-4-ANEPPS showed that over 90% of pulmonary vein cardiomyocytes possessed T-tubules. These findings demonstrate that the frequency of spontaneous Ca2+ transients in the rat pulmonary vein are increased following higher rates of electrical stimulation and increasing the extracellular Ca2+ concentration.

Dataset on the kinetics of the inhibition of PTP1B by the flavonoids and pheophytin A from Allophylus cominia.

The data presented in this article are related to the research article under the title "in vitro anti-diabetic activity of flavonoids and pheophytins from Allophylus cominia Sw. on PTP1B, DPPIV, alpha-glucosidase and alpha-amylase enzymes" (Semaan et al., 2017) [3]. This article defines the kinetics of inhibition of flavonoids and pheophytin A extracts from A. cominia which showed an inhibition of the PTP1B enzyme activity. The main reason to make these results public is to confirm that this study was followed up and no more experiments are needed, also to confirm that these compounds can be reported as PTP1B inhibitors.

In vitro anti-diabetic effect of flavonoids and pheophytins from Allophylus cominia Sw. on the glucose uptake assays by HepG2, L6, 3T3-L1 and fat accumulation in 3T3-L1 adipocytes.

BACKGROUND AND PURPOSE: Based on ethno-botanical information collected from diabetic patients in Cuba and firstly reported inhibition of PTP1B and DPPIV enzymes activities, Allophylus cominia (A. cominia) was identified as possible source of new drugs that could be used for the treatment of type 2 diabetes mellitus (T2-DM). EXPERIMENTAL APPROACH: in this study, the activity of the characterised extracts from A. cominia was tested on the glucose uptake using HepG2 and L6 cells, 3T3-L1 fibroblasts and adipocytes as well as their effect on the fat accumulation using 3T3-L1 adipocytes. KEY RESULTS: on 2-NBDG glucose uptake assay using HepG2 and L6 cells, extracts from A. cominia enhanced insulin activity by increasing glucose uptake. On HepG2 cells Insulin EC50 of 93 ± 21nM decreased to 13 ± 2nM in the presence of the flavonoids mixture from A.cominia. In L6 cells, insulin also produced a concentration-dependent increase with an EC50 of 28.6 ± 0.7nM; EC50 decreased to 0.08 ± 0.02nM and 5 ± 0.9nM in the presence of 100µg/ml of flavonoids and pheophytins mixtures, respectively. In 3T3-L1 fibroblasts, insulin had an EC50 of >1000nM that decreased to 38 ± 4nM in the presence of the flavonoids extract. However, in adipocytes, insulin produced a significant concentration-dependent increase and an EC50 of 30 ± 8nM was a further confirmation of the insulin responsiveness of the adipocytes to the insulin. At 100µg/ml, flavonoids and pheophytins extracts decreased fat accumulation in 3T3-L1 adipocytes by two folds in comparison to the control differentiated cells (p < 0.05). The crude extract of A. cominia did not show any enhancement of 2-NBDG uptake by 3T3-L1 adipocytes in the presence or absence of 100nM insulin. In addition, in fully differentiated adipocytes, both extracts produced significant decrease in lipid droplets in the cells and no lipid accumulation were seen after withdrawal of the extracts from the cell growth medium. However, there was no effect of both extracts on total protein concentration in cells as well as on Glut-4 transporters. CONCLUSIONS AND IMPLICATIONS: the pharmacological effects of the extracts from A. cominia observed in experimental diabetic models were shown in this study. A. cominia is potentially a new candidate for the treatment and management of T2-DM.

In vitro anti-diabetic activity of flavonoids and pheophytins from Allophylus cominia Sw . on PTP1B, DPPIV, alpha-glucosidase and alpha-amylase enzymes.

BACKGROUND: Ethno-botanical information from diabetic patients in Cuba led to the identification of Allophylus cominia as a possible source of new drugs for the treatment of type 2 diabetes mellitus (T2-DM). EXPERIMENTAL: Chemical characterization of the extracts from A. cominia was carried out using chromatographic and spectroscopic methods. The extracts were tested for their activity on PTP1B, DPPIV, α-glucosidase enzymes and α-amylase. RESULTS: The flavonoid rich fractions from A. cominia inhibited DPPIV enzyme (75.3±2.33%) at 30µg/ml and produced a concentration-dependent inhibition against DPPIV with a Ki value of 2.6µg/ml. At 30µg/ml, flavonoids and pheophytins extracts significantly inhibited PTP1B enzyme (100±2.6% and 68±1% respectively). The flavonoids, pheophytin A and pheophytin B fractions showed significant concentration-dependent inhibition against PTP1B with Ki values of 3µg/ml, 0.64µg/ml and 0.88µg/ml respectively. At 30µg/ml, the flavonoid fraction significantly inhibited α-glucosidase enzyme (86±0.3%) in a concentration-dependent pattern with a Ki value of 2µg/ml. None of the fractions showed significant effects on α-amylase. Fatty acids, tannins, pheophytins A and B, and a mixture of flavonoids were detected in the methanolic extract from A. cominia. The identified flavonoids were mearnsitrin, quercitrin, quercetin-3-alloside, and naringenin-7-glucoside. CONCLUSION: The pharmacological effects of the extracts from A. cominia earlier observed in experimental diabetic models was confirmed in this study. Thus a new drug or formulation for the treatment of T2-DM could be developed from A. cominia.

Snake venom phospholipases A(2): a novel tool against bacterial diseases.

The majority of snake venom phospholipases A(2) (svPLA(2)s) are toxic and induce a wide spectrum of biological effects. They are cysteine-rich proteins that contain 119-134 amino acids and share similar structures and functions. About 50% of the residues are incorporated into α-helices, whereas only 10% are in ß-sheets. Fourteen conserved cysteines form a network of seven disulfide bridges that stabilize the tertiary structure. They show a high degree of sequence and structural similarity, and are believed to have a common calcium- dependent catalytic mechanism. Additionally, svPLA(2)s display an array of biological actions that are either dependent or independent of catalysis. The PLA(2)s of mammalian origin also exert potent bactericidal activity by binding to anionic surfaces and enzymatic degradation of phospholipids in the target membranes, preferentially of Gram-positive species. The bactericidal activity against Gram-negatives by svPLA(2) requires a synergistic action with bactericidal/permeability-increasing protein (BPI), but is equally dependent on enzymatic- based membrane degradation. Several hypotheses account for the bactericidal properties of svPLA(2)s, which include "fatal depolarization" of the bacterial membrane, creation of physical holes in the membrane, scrambling of normal distribution of lipids between the bilayer leaflets, and damage of critical intracellular targets after internalization of the peptide. The present review discusses several svPLA(2)s and derived peptides that exhibit strong bactericidal activity. The reports demonstrate that svPLA(2)-derived peptides have the potential to counteract microbial infections. In fact, the C-terminal cationic/hydrophobic segment (residues 115-129) of svPLA(2)s is bactericidal. Thus identification of the bactericidal sites in svPLA(2)s has potential for developing novel antimicrobials.

A review of epidemiological, clinical and in vitro physiological studies of envenomation by the scorpion Hemiscorpius lepturus (Hemiscorpiidae) in Iran.

Many of the published accounts of envenomation by Hemiscorpius lepturus (H. lepturus) are in a form that makes it difficult for non Farsi readers to access. The purpose of this review is to summarise the Iranian literature on the toxinological effects of the venom of H. lepturus using both in vivo and in vitro data and where appropriate, details of methods and ethics statements will be reported.

The effect of the venom of the yellow Iranian scorpion Odontobuthus doriae on skeletal muscle preparations in vitro.

The yellow Iranian scorpion Odontobuthus doriae can cause fatal envenoming, but its mechanism of action is unclear. One of the reported manifestations of envenoming is moderate to severe involuntary tremor of skeletal muscle. In order to understand better the mechanism of action of this venom on skeletal muscle function, we examined the effects of the venom in vitro on chick biventer cervicis (CBC) and mouse hemidiaphragm (MHD) nerve muscle preparations. O. doriae venom (0.3-10mug/ml) initially increased and then decreased twitch height. The venom also caused contracture in both preparations. In mouse triangularis sterni preparations, used for all intracellular recording techniques, the venom enhanced the release of acetylcholine and induced repetitive firing of nerve action potentials and endplate potentials in response to single-shock stimulation. With extracellular recording techniques, scorpion venom (1mug/ml) was found to cause changes to the perineural waveform associated with nerve terminal action potentials consistent with effects on Na(+) and K(+) currents. The main facilitatory effects of O. doriae venom are likely to be due to toxins that affect Na(+) channels in nerve-muscle preparations similar to most Old World scorpion venoms, but blocking effects on K(+) channels are also possible. Such effects could lead to initial enhancement of transmitter release that could underlie the muscle tremors seen in victims. Toxins acting on Na(+) and K+ currents have been isolated from the venom [Jalali, A., Bosmans, F., Amininasab, M., Clynen, E., Cuypers, E., Zaremirakabadi, A., Sarbolouki, M.N., Schoofs, L., Vatanpour, H., Tytgat, J., 2005. OD1, the first toxin isolated from the venom of the scorpion Odontobuthus doriae active on voltage-gated Na(+) channels. FEBS Lett. 579, 4181-4186; Abdel-Mottaleb, Y., Clynen, E., Jalali, A., Bosmans, F., Vatanpour, H., Schoofs, L., Tytgat, J., 2006. The first potassium channel toxin from the venom of the Iranian scorpion Odontobuthus doriae. FEBS Lett. 580, 6254-6258]; however, the muscle paralysis seen at higher concentrations of venom may be due to additional, as yet uncharacterised, components of the venom.

Crossing the pain barrier: P2 receptors as targets for novel analgesics.

In 1995 the P2X3 receptor was found to be expressed at high levels in nociceptive sensory neurones, consistent with earlier reports that ATP induced pain in humans and animals. At first it was thought that ATP was most likely to play a role in acute pain, following its release from damaged or stressed cells and since then a wide variety of experimental techniques and approaches have been used to study this possibility. Whilst it is clear that exogenous and endogenous ATP can indeed acutely stimulate sensory neurones, more recent reports using gene knockout and antisense oligonucleotide technologies, and a novel, selective P2X3 antagonist, A-317491, all indicate that ATP and P2X3 receptors are more likely to be involved in chronic pain conditions, particularly chronic inflammatory and neuropathic pain. These reports indicate that P2X3 receptors on sensory nerves may be tonically activated by ATP released from nearby damaged or stressed cells, or perhaps from the sensory nerves themselves. This signal, when transmitted to the CNS, will be perceived consciously as chronic pain. In addition, it is now clear that several subtypes of P2Y receptor are also expressed in sensory neurones. Although their distribution and functions have not been as widely studied as P2X receptors, the effects that they mediate indicate that they might also be considered as therapeutic targets in the treatment of pain. Although our ability to treat persistent painful conditions, such as chronic inflammatory and neuropathic pain, has improved in recent years, these conditions are often resistant to currently available therapies, such as opioids or non-steroidal anti-inflammatory drugs. This reflects a limited understanding of the underlying pathophysiology. It is now clear that the development and maintenance of chronic pain are mediated by multiple factors, but many of these factors, and the receptors and mechanisms through which they act, remain to be identified. Chronic pain is debilitating and can greatly decrease quality of life, not just due to the pain per se, but also because of the depression that can often ensue. Thus a greater understanding of the mechanisms that underlie chronic pain will help identify new targets for novel analgesics, which will be of great therapeutic benefit to many people.

An electrophysiological study on the effects of Pa-1G (a phospholipase A(2)) from the venom of king brown snake, Pseudechis australis, on neuromuscular function.

The effects of Pa-1G, a phospholipase A(2) (PLA(2)) from the venom of the Australian king brown snake (Pseudechis australis) were determined on the release of acetylcholine, muscle resting membrane potential and motor nerve terminal action potential at mouse neuromuscular junction. Intracellular recording from endplate regions of mouse triangularis sterni nerve-muscle preparations revealed that Pa-1G (800 nM) significantly reduced the amplitude of endplate potentials within 10 min exposure. The quantal content of endplate potentials was decreased to 58+/-6% of control after 30 min exposure to 800 nM Pa-1G. The toxin also caused a partial depolarisation of mouse muscle fibres within 60 min exposure. Extracellular recording of action potentials at motor nerve terminals showed that Pa-1G reduced the waveforms associated with both sodium and potassium conductances. To investigate whether this was a direct or indirect effect of the toxin on these ionic currents, whole cell patch clamp experiments were performed using human neuroblastoma (SK-N-SH) cells and B82 mouse fibroblasts stably transfected with rKv1.2. Patch clamp recording experiments confirmed that potassium currents sensitive to alpha-dendrotoxin recorded from B82 cells and sodium currents in SK-N-SH cells were not affected by the toxin. Since neither facilitation of acetylcholine release at mouse neuromuscular junction nor depression of potassium currents in B82 cells has been observed, the apparent blockade of potassium currents at mouse motor nerve endings induced by the toxin is unlikely to be due to a selective block of potassium channels.

The facilitatory actions of snake venom phospholipase A(2) neurotoxins at the neuromuscular junction are not mediated through voltage-gated K(+) channels.

Electrophysiological investigations have previously suggested that phospholipase A(2) (PLA(2)) neurotoxins from snake venoms increase the release of acetylcholine (Ach) at the neuromuscular junction by blocking voltage-gated K(+) channels in motor nerve terminals. We have tested some of the most potent presynaptically-acting neurotoxins from snake venoms, namely beta-bungarotoxin (BuTx), taipoxin, notexin, crotoxin, ammodytoxin C and A (Amotx C & A), for effects on several types of cloned voltage-gated K(+) channels (mKv1.1, rKv1.2, mKv1.3, hKv1.5 and mKv3.1) stably expressed in mammalian cell lines. By use of the whole-cell configuration of the patch clamp recording technique and concentrations of toxins greater than those required to affect acetylcholine release, these neurotoxins have been shown not to block any of these voltage-gated K(+) channels. In addition, internal perfusion of the neurotoxins (100 microg/ml) into mouse B82 fibroblast cells that expressed rKv1.2 channels also did not substantially depress K(+) currents. The results of this study suggest that the mechanism by which these neurotoxins increase the release of acetylcholine at the neuromuscular junction is not related to the direct blockage of voltage-activated Kv1.1, Kv1.2, Kv1.3, Kv1.5 and Kv3.1 K(+) channels.

Anti-GQ1b ganglioside antibodies mediate complement-dependent destruction of the motor nerve terminal.

Miller-Fisher syndrome is an autoimmune neuropathy characterized by ataxia, areflexia and ophthalmoplegia, and in the majority of cases the presence of high titres of anti-GQ1b ganglioside antibodies. In an ex vivo model, human and mouse anti-GQ1b antibodies have been shown previously to induce a complement-dependent alpha-latrotoxin-like effect on the murine motor endplate, i.e. they bring about massive quantal release of acetylcholine and eventually block neuromuscular transmission. Using immunofluorescence microscopy with image analysis, we show here that the late stages of this electrophysiological effect temporally coincide with the loss of heavy neurofilament (200 kDa) and type III beta-tubulin immunostaining and structural breakdown of the nerve terminal, as demonstrated by electron microscopy. Ultrastructurally, axon terminals were disorganized, depleted of vesicles, and subdivided by the infiltrating processes of capping Schwann cells. These findings provide clear pathological evidence to support a role for anti-ganglioside antibodies in mediating nerve terminal injury and further advance the view that this site may be of importance as a target in some human neuropathies.

What does beta-bungarotoxin do at the neuromuscular junction?

beta-Bungarotoxin from the Taiwan banded krait, Bungarus multicinctus is a basic protein (pI=9.5), with a molecular weight of 21,800 consisting of two different polypeptide subunits. A phospholipase A(2) subunit named the A-chain and a non-phospholipase A(2) subunit named the B-chain, which is homologous to Kunitz protease inhibitors. The A-chain and the B-chain are covalently linked by one disulphide bridge. On mouse hemi-diaphragm nerve-muscle preparations, partially paralysed by lowering the external Ca(2+) concentration, beta-bungarotoxin classically produces triphasic changes in the contraction responses to indirect nerve stimulation. The initial transient inhibition of twitches (phase 1) is followed by a prolonged facilitatory phase (phase 2) and finally a blocking phase (phase 3). These changes in twitch tension are mimicked, to some extent, by similar changes to end plate potential amplitude and miniature end plate potential frequency. The first and second phases are phospholipase-independent and are thought to be due to the B-chain (a dendrotoxin mimetic) binding to or near to voltage-dependent potassium channels. The last phase (phase 3) is phospholipase dependent and is probably due to phospholipase A(2)-mediated destruction of membrane phospholipids in motor nerve terminals.

Effects of brucine, a plant alkaloid, on M(1) muscarinic receptors and alpha(1)-adrenoceptors in the rabbit vas deferens preparation.

The plant alkaloid brucine is an analogue of strychnine and is known to be an allosteric modulator at cloned M(1) muscarinic receptors. The functional effects of brucine were examined on the M(1) muscarinic receptors in the rabbit isolated vas deferens preparation. Brucine (10-100 microM) enhanced the effects of the muscarinic agonist McN-A-343 at presynaptic M(1) muscarinic receptors in the rabbit isolated vas deferens preparation, but only when brucine was added prior to McN-A-343. This effect is indicative of a positive allosteric action. It was poorly reversed on washing. Brucine did not affect the responses to the mamba venom muscarinic toxins MT2 and MT4, which are also allosteric activators in this preparation. Brucine (10-100 microM) caused a significant decrease in the twitch response to electrical stimulation in the rabbit vas deferens preparation, which was not antagonised by 100 nM pirenzepine (an M(1) muscarinic antagonist). Brucine and MT4, but not MT2, caused significant decreases (p<0.05) in the responses to noradrenaline in the rabbit vas deferens preparation. Responses to ATP and KCl were not affected. In radioligand binding assays, brucine displaced the alpha(1)-adrenoceptor ligand prazosin from its specific binding sites in membranes made from rat cerebral cortex and rat vas deferens. The apparent K(i) values were 150 and 3.4 microM in the cortical and vas deferens membranes, respectively. The positive allosterism found with brucine at cloned M(1) receptors seems to be mirrored at native M(1) receptors. However, the unexpected blocking effects at alpha(1)-adrenoceptors indicates that more selective ligands than brucine are required as starting points for the design of specific enhancers of the activity of M(1) receptors with therapeutic potential.

The effects of lignocaine on actions of the venom from the yellow scorpion "Leiurus quinquestriatus" in vivo and in vitro.

Many toxins from scorpion venoms activate sodium channels, thereby enhancing neurotransmitter release. The aim of the present work was to determine if the in vivo and in vitro effects of Leiurus quinquestriatus venom (LQQ) could be ameliorated by lignocaine, a sodium channel blocker. In urethane anaesthetised rabbits, LQQ venom (0.5 mg kg(-1), i.v.) caused initial hypotension and bradycardia followed by hypertension, pulmonary oedema, electrocardiographic changes indicating conduction defects, ischaemia, infarction, and then hypotension and death. Lignocaine (1 mg kg(-1) i.v. bolus initially, followed by i.v. infusion of 50 microg kg(-1) min(-1)) significantly attenuated the majority of the venom-evoked effects and reduced mortality. Addition of LQQ venom (1, 3 and 10 microg ml(-1)) to chick biventer cervicis, guinea pig ileum, and rat vas deferens preparations, increased the height of electrically-induced twitches, elevated resting tension, and caused autorhythmic oscillations. Lignocaine (3 x 10(-4)-1.2 x 10(-3) M) greatly attenuated these venom-evoked actions in the three preparations. Antagonists of appropriate neurotransmitters were also tested to determine the contribution of released transmitters to LQQ effects. Atropine significantly decreased the venom-elicited effects on guinea pig ileum preparations, while prazosin and guanethidine significantly reduced the venom's actions on rat vas deferens. In chick biventer cervicis preparations, tubocurarine and hexamethonium significantly attenuated the venom-induced effects. This study supports the hypothesis that many effects of LQQ venom involve the release of neurotransmitters and may be ameliorated by treatment with lignocaine.

Mapping the functional anatomy of BgK on Kv1.1, Kv1.2, and Kv1.3. Clues to design analogs with enhanced selectivity.

BgK is a peptide from the sea anemone Bunodosoma granulifera, which blocks Kv1.1, Kv1.2, and Kv1.3 potassium channels. Using 25 analogs substituted at a single position by an alanine residue, we performed the complete mapping of the BgK binding sites for the three Kv1 channels. These binding sites included three common residues (Ser-23, Lys-25, and Tyr-26) and a variable set of additional residues depending on the particular channel. Shortening the side chain of Lys-25 by taking out the four methylene groups dramatically decreased the BgK affinity to all Kv1 channels tested. However, the analog K25Orn displayed increased potency on Kv1.2, which makes this peptide a selective blocker for Kv1.2 (K(D) 50- and 300-fold lower than for Kv1.1 and Kv1.3, respectively). BgK analogs with enhanced selectivity could also be made by substituting residues that are differentially involved in the binding to some of the three Kv1 channels. For example, the analog F6A was found to be >500-fold more potent for Kv1.1 than for Kv1.2 and Kv1.3. These results provide new information about the mechanisms by which a channel blocker distinguishes individual channels among closely related isoforms and give clues for designing analogs with enhanced selectivity.

Anti-ganglioside antibodies can bind peripheral nerve nodes of Ranvier and activate the complement cascade without inducing acute conduction block in vitro.

The neurophysiological effects of nine neuropathy-associated human anti-ganglioside antisera, three monoclonal antibodies to ganglioside GM1 (GM1) and of the cholera toxin B subunit (a GM1 ligand) were studied on mouse sciatic nerve in vitro. GM1 antisera and monoclonal antibodies from patients with chronic motor neuropathies and Guillain-Barre syndrome, and GQ1b/ disialosyl antisera and monoclonal antibodies from patients with chronic ataxic neuropathies and Miller Fisher syndrome were studied. In vitro recording, for up to 6 h, of compound nerve action potentials, latencies, rise times and stimulus thresholds from isolated desheathed sciatic nerve was performed in the presence of antiganglioside antibodies and fresh human serum as an additional source of complement. No changes were observed over this time course, with 4-6 h values for all electrophysiological parameters being within 15% of the starting values for both normal and antibody containing sera and for the cholera toxin B subunit. Parallel experiments on identically prepared desheathed nerves performed with 0.5 nM saxitoxin led to complete conduction block within 10 min of application. Under identical conditions to those used for electrophysiological recordings, quantitative immunohistological evaluation revealed a significant increase in IgM (immunoglobulin M) deposition at nodes of Ranvier from 5.3+/-3.1% to 28.7+/-8.4% (mean+/-SEM) of desheathed nerves exposed to three normal and three antibody containing sera, respectively (P < 0.03). Complement activation was seen at 100% of normal and 79% of disease-associated IgM positive nodes of Ranvier. These data indicate that anti-ganglioside antibodies can diffuse into a desheathed nerve, bind to nodes of Ranvier and fix complement in vitro without resulting in any overt physiological deterioration of the nerve over 4-6 h. This suggests that the node of Ranvier is relatively resistant to acute antiganglioside antibody mediated injury over this time scale and that anti-ganglioside antibodies and the cholera toxin B subunit are unlikely to have major direct pharmacological effects on nodal function, at least in comparison with the effect of saxitoxin. This in vitro sciatic nerve model appears of limited use for analysing electrophysiologically the effects of anti-ganglioside antibodies on nerve function, possibly because its short-term viability and isolation from circulating systemic factors do not permit the evolution of an inflammatory lesion of sufficient magnitude to induce overt electrophysiological abnormalities. In vivo models may be more suitable for identifying the effects of these antibodies on nerve conduction.

What can toxins tell us for drug discovery?

Toxins are of interest in drug design because the toxins provide three-dimensional templates for creating small molecular mimics with interesting pharmacological properties. Toxins are also useful in drug discovery because they can be used as pharmacological tools to uncover potential therapeutic targets. With their high potency and selectivity, toxins are often more useful in functional experiments than standard pharmacological agents. We have used two groups of neurotoxins, the dendrotoxins and the muscarinic toxins (MTs), to explore the involvement of subtypes of potassium ion channels and muscarinic receptors, respectively, in processes involved in cognition and the changes in neuronal properties with aging. From our current work, quantitative autoradiographic studies with radiolabelled dendrotoxins reveal widespread distribution of binding sites throughout rat brain sections, but few differences exist between young adult and aged rats. However, displacement studies with toxin K, which preferentially binds to the Kv1.1 subtype of cloned potassium channel, show the selective loss of such sites in regions of the hippocampus and septohippocampal pathway with aging. MTs have been tested for effects on performance of rats in memory paradigms. MT2, which activates m1 receptors, improves performance of rats in a step-down inhibitory avoidance test, whereas MT3, which blocks m4 receptors, decreases performance when given into the hippocampus. This is the first clear demonstration of a role for m4 muscarinic receptors in cognition.

Delineation of the functional site of alpha-dendrotoxin. The functional topographies of dendrotoxins are different but share a conserved core with those of other Kv1 potassium channel-blocking toxins.

We identified the residues that are important for the binding of alpha-dendrotoxin (alphaDTX) to Kv1 potassium channels on rat brain synaptosomal membranes, using a mutational approach based on site-directed mutagenesis and chemical synthesis. Twenty-six of its 59 residues were individually substituted by alanine. Substitutions of Lys5 and Leu9 decreased affinity more than 1000-fold, and substitutions of Arg3, Arg4, Leu6, and Ile8 by 5-30-fold. Substitution of Lys5 by norleucine or ornithine also greatly altered the binding properties of alphaDTX. All of these analogs displayed similar circular dichroism spectra as compared with the wild-type alphaDTX, indicating that none of these substitutions affect the overall conformation of the toxin. Substitutions of Ser38 and Arg46 also reduced the affinity of the toxin but, in addition, modified its dichroic properties, suggesting that these two residues play a structural role. The other residues were excluded from the recognition site because their substitutions caused no significant affinity change. Thus, the functional site of alphaDTX includes six major binding residues, all located in its N-terminal region, with Lys5 and Leu9 being the most important. Comparison of the functional site of alphaDTX with that of DTX-K, another dendrotoxin (Smith, L. A., Reid, P. F., Wang, F. C., Parcej, D. N., Schmidt, J. J., Olson, M. A., and Dolly, J. O. (1997) Biochemistry 36, 7690-7696), reveals that they only share the predominant lysine and probably a leucine residue; the additional functional residues differ from one toxin to the other. Comparison of the functional site of alphaDTX with those of structurally unrelated potassium channel-blocking toxins from venomous invertebrates revealed the common presence of a protruding key lysine with a close important hydrophobic residue (Leu, Tyr, or Phe) and few additional residues. Therefore, irrespective of their phylogenetic origin, all of these toxins may have undergone a functional convergence. The functional site of alphaDTX is topographically unrelated to the "antiprotease site" of the structurally analogous bovine pancreatic trypsin inhibitor.

Pharmacological properties of P2X3-receptors present in neurones of the rat dorsal root ganglia.

1. The electrophysiological actions of several agonists which may differentiate between P2X1- and P2X3-receptors were studied under concentration and voltage-clamp conditions in dissociated neurones of 1-4 day old rat dorsal root ganglia. 2. Beta,gamma-Methylene-D-ATP (beta,gamma-me-D-ATP) (1-300 microM), diadenosine 5',5'''-P1,P5-pentaphosphate (AP5A) (100 nM - 300 microM), diadenosine 5',5'''-P1,P4-tetraphosphate (AP4A) (300 nM - 300 microM) and uridine 5'-triphosphate (UTP) (1 microM - 1 mM) all activated concentration-dependent inward currents with a latency to onset of a few ms. 3. The concentration-response curves for beta,gamma-me-D-ATP and AP5A and ATP had similar maximum values, while that for AP4A had a lower maximum. The concentration-response curve to UTP was shallow and did not reach a maximum. Beta,gamma-Methylene-L-ATP was virtually inactive. The rank order of agonist potency was ATP > AP5A approximately AP4A > beta,gamma-me-D-ATP > UTP > > beta,gamma-methylene-L-ATP. 4. The inward currents were inhibited by the P2-receptor antagonists suramin (100 microM) and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (10 microM). PPADS also inhibited responses to ATP (800 nM) and alpha,beta-methylene ATP (2 microM) in a concentration-dependent manner. 5. This study shows that beta,gamma-me-D-ATP, AP5A, AP4A and UTP all act via a suramin- and PPADS-sensitive P2X-receptor to evoke rapid, transient inward currents in dissociated neurones of rat dorsal root ganglia. The very low activity of beta,gamma-methylene-L-ATP suggests that the agonists were acting at the P2X3-subtype to produce these effects.