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.

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.

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.

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.

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.

On the purification of notexin. Isolation of a single amino acid variant from the venom of Notechis scutatus scutatus.

Venom of the Australian tiger snake, Notechis scutatus scutatus was fractionated by conventional ion-exchange chromatography. The fraction containing notexin, a well-known single-chain toxic phospholipase A2, was further purified by reverse-phase high-performance liquid chromatography. Two main components were isolated and the major one corresponded to notexin. The other component, designated as notechis Ns, was an isoform of notexin. Notechis Ns and notexin possessed similar in vitro esterase activity, in vitro neuromuscular activity and in vivo lethality. Amino acid composition and sequence of the Staphylococcus aureus V8-protease peptides demonstrated that primary structures of notechis Ns and notexin differed from each other by a single substitution amongst 119 amino acids: Lys----Arg at position 16.

On the site by which alpha-dendrotoxin binds to voltage-dependent potassium channels: site-directed mutagenesis reveals that the lysine triplet 28-30 is not essential for binding.

We constructed a synthetic gene encoding the published amino acid sequence of DTx from Dendroaspis angusticeps, a ligand of voltage-dependent postassium channels that facilitates neurotransmitter release. We expressed it in Escherichia coli as a fusion protein secreted in the culture medium. The recombinant DTx was generated in vitro by chemical treatment and recovered as two isoforms. One of them (rDTx), like the venom toxin, has an N-terminal pyroglutamate whereas the other (rQDTx) has a free N-terminal glutamine. Chromatographic differences between rDTx and natural DTx led us to re-examine the amino acid sequence of natural DTx. In contrast to what was previously published, position 12 was an Asp and not Asn. Despite this difference, rDTx and DTx had similar toxicity in mice and binding affinity to synaptosomes, suggesting that residue 12 is not important for DTx function. Nor is the N-terminal residue implicated in DTx function since rDTx and rQDTx also had similar biological activities. We also synthesized and expressed a mutant of the DTx gene in which the lysine triplet 28-30 was changed into Ala-Ala-Gly. The two resulting recombinant isoforms exhibited only small decreases in biological activity, excluding the possibility that the positively charged lysine triplet 28-30 of DTx is directly involved in the toxin functional site.

Modification of ionic currents underlying action potentials in mouse nerve terminals by the thiol-oxidizing agent diamide.

The effect of diamide, a thiol-oxidizing agent, was tested using electrophysiological techniques to determine whether its ability to alter neuromuscular transmission in vitro could be attributed to alterations of ion channels controlling neuronal excitability and/or acetylcholine release. In mouse triangularis sterni preparations, diamide transiently increased the evoked release of acetylcholine and then blocked release. Extracellular recording of perineural waveforms associated with neuronal action potentials at motor nerve terminals showed that diamide reduced the waveforms associated with the delayed rectifier K+ current, a Ca2+ current and a Ca(2+)-activated K+ current (IK,Ca). Inhibition of quantal transmitter release was not associated with failure of action potentials to invade nerve terminals. Thus, diamide modifies the ionic currents underlying the nerve terminal action potential, some of these changes probably account for the complex effects of diamide on quantal transmission.

Polyamine FTX-3.3 and polyamine amide sFTX-3.3 inhibit presynaptic calcium currents and acetylcholine release at mouse motor nerve terminals.

FTX-3.3 is the proposed structure of a calcium-channel blocking toxin that has been isolated from the funnel web spider (Agelenopsis aperta). The effects of FTX-3.3 and one of its analogues, sFTX-3.3, on acetylcholine release, on presynaptic currents at mouse motor nerve terminals and on whole-cell sodium currents in SK.N.SH cells (a human neuroblastoma cell line) have been studied. FTX-3.3 (10-30 microM) and sFTX-3.3 (100-300 microM) reversibly reduced release of acetylcholine by approximately 70-90% and 40-60%, respectively. FTX-3.3 (10 microM) blocked the fast component of presynaptic calcium currents by approximately 60%. sFTX-3.3 (100 microM) reduced the duration of the slow component of presynaptic calcium currents by about 50% of the control and also reduced presynaptic sodium current by approximately 20% of the control. sFTX-3.3 (100 microM) reduced whole-cell sodium current recorded from SK.N.SH cells by approximately 15%, whereas FTX-3.3, even at 200 microM, did not affect this current. Since the only difference in chemical structures of these toxins is that sFTX-3.3 has an amide function which is absent in FTX-3.3, the amide function may be responsible for the reduced potency and selectivity of sFTX-3.3. This study also provides further support for the existence of P-type calcium channels at mouse motor nerve terminals.

Potassium channel blocking actions of beta-bungarotoxin and related toxins on mouse and frog motor nerve terminals.

1. beta-Bungarotoxin and other snake toxins with phospholipase activity augment acetylcholine release evoked from mouse motor nerve terminals before they produce blockade. This action of the toxins is independent of their phospholipase A2 activity, but the underlying mechanism for the facilitation of release is unclear. To determine whether the toxins affect ionic currents at motor nerve terminals, extracellular recordings were made from perineural sheaths of motor nerves innervating mouse triangularis sterni muscles. 2. Perineural waveforms had a characteristic shape, with two major negative deflections, the first being associated with nodal Na+ currents and the second with terminal K+ currents. Block of the K+ currents revealed a Ca2+-dependent component. 3. During the facilitatory phase of its action, beta-bungarotoxin (150 nM) reduced the second negative component of the perineural waveform by 30-50%. 4. The reduction could be a consequence of a decreased K+ ion contribution or of an increase in the current carried by Ca2+. As beta-bungarotoxin had similar effects in solutions which contained no added Ca2+, it is unlikely to be acting on the Ca2+ current. Also, it is unlikely to be blocking the Ca2+-activated K+ current, which is suppressed in zero Ca2+ conditions. 5. Other prejunctionally active snake toxins (taipoxin, notexin and crotoxin) had similar effects to those of beta-bungarotoxin, but a similar basic phospholipase of low toxicity from cobra venom had no effect. 6. Thus, beta-bungarotoxin and related toxins block a fraction of the K+ current in the motor nerve terminals of mouse preparations. Such an effect could explain the facilitation of acetylcholine release caused by these toxins before the onset of presynaptic blockade. 7. In frog cutaneous pectoris preparations, f-bungarotoxin reduced endplate potential amplitude but had little effect on perineural waveforms. Therefore, the consequences of toxin binding must be different in frog terminals.

Reversal by cysteine of the cadmium-induced block of skeletal neuromuscular transmission in vitro.

1. Neuromuscular transmission in isolated nerve-muscle preparations was blocked by exposure to Cd2+ for less than 30 min or more than 2 h. The abilities of cysteine, Ca2+ or 3,4-diaminopyridine (3,4-DAP) to reverse the blockade induced by Cd2+ were studied. 2. On the mouse hemidiaphragm preparation, exposure to Cd2+ (10 microM) for 10 to 20 min induced a blockade which was easily reversed by increasing the extracellular Ca2+ concentration (5-10 mM) or by 3,4-DAP (100 microM). Exposure to Cd2+ (3-10 microM) for over 2 h led to a blockade which was not reversed by Ca2+ (5-15 mM) or 3,4-DAP (100 microM). Cysteine (1 mM) was able to reverse completely the blockade induced by both brief and prolonged exposures to Cd2+. 3. In chick biventer cervicis preparations, Cd2+ (100 microM) decreased the twitch height of indirectly stimulated preparations without affecting responses to exogenously applied acetylcholine, carbachol or KCl. Cysteine (1-3 mM) had no appreciable effect on twitch responses to indirect stimulation or to exogenously applied agonists but fully reversed the blockade induced by Cd2+ (100 microM). 4. In mouse triangularis sterni preparations, Cd2+ (1-30 microM) depressed the evoked quantal release of acetylcholine. Concentrations of Cd2+ which completely blocked endplate potentials (e.p.ps) were without significant effect on miniature endplate potential (m.e.p.p.) amplitude and frequency or time constant of decay. Cysteine (1-10 mM) alone had no effect on e.p.ps or m.e.p.ps, but completely reversed the blockade induced by Cd2+.6. In addition to the competitive blocking action of Cd2+ at the prejunctional Ca2+ channels, long exposure to Cd2+ leads to a blockade that is not competitive. This probably involves binding of Cd2+" at an extracellular thiol site on, or close to, voltage-operated Ca2+' channels.

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.

Characterization of a P2X-purinoceptor in cultured neurones of the rat dorsal root ganglia.

1. The electrophysiological actions of the P2-purinoceptor agonists, adenosine 5'-triphosphate (ATP), 2-methylthio ATP (2-meSATP) and alpha, beta-methyleneATP (alpha, beta-meATP) and of uridine 5'-triphosphate (UTP) were studied under concentration and voltage-clamp conditions in dissociated neurones of 1-6 day old rat dorsal root ganglia. 2. ATP (10 nM-100 microM) applied rapidly via a U-tube perfusion system (equilibration time < 10 ms) activated concentration-dependent inward currents with a latency to onset of a few ms, an EC50 of 719 nM and a Hill slope of 1.47. 3. 2-meSATP (10 nM- 100 microM) and alpha, beta-meATP (100 nM - 100 microM) also evoked transient inward currents. The EC50 and Hill slopes were 450 nM and 1.58 for 2-meSATP and 1.95 microM and 1.53 for alpha, beta-meATP respectively. There was no significant difference between the maximum currents evoked by the three agonists. 4. As the concentration of ATP increased so the rate of rise and decay of the currents also increased. At 100 and 300 nM ATP the decay of the current was best fitted by a single exponential, but at 1 microM and above two exponentials were required. Log-log plots of the rise time or time constants of decay versus concentration were linear. Currents evoked by 2-meSATP and alpha, beta-meATP showed a similar concentration-dependence in their kinetics. 5. Inward currents evoked by ATP, 2-meSATP and alpha, beta-meATP (300 nM) were abolished by the P2-purinoceptor antagonist, suramin (100 microM). 6. UTP (10 microM) evoked similar transient inward currents, which were sensitive to suramin (100 microM). ATP (10 microM), applied 2 min beforehand, reduced the response to UTP (10 microM) by 80 +/- 10%. 7. This study shows that ATP, 2-meSATP and alpha, beta-meATP act via a suramin-sensitive P2x-purinoceptor to evoke rapid, transient inward currents in dissociated neurones of rat dorsal root ganglia. The pyrimidine nucleotide, UTP, was also active. It is likely that the agonists were acting at the P2x3-subtype to produce these effects.

Effects of tacrine, velnacrine (HP029), suronacrine (HP128), and 3,4-diaminopyridine on skeletal neuromuscular transmission in vitro.

1. The effects of tacrine (9-amino-1,2,3,4-tetrahydroacridine), velnacrine (HP029, 9-amino-1,2,3,4-tetrahydroacridin-1-ol maleate), suronacrine (HP128, 9-benzylamino-1,2,3,4-tetrahydroacridin-1-ol maleate), and 3,4-diaminopyridine on neuromuscular transmission were compared on isolated nerve-muscle preparations. 2. Tacrine, HP029, and 3,4-diaminopyridine augmented responses of chick biventer cervicis preparations to nerve stimulation, with tacrine and HP029 increasing responses to exogenously applied acetylcholine. HP128 blocked responses to nerve stimulation and to carbachol, but increased responses to acetylcholine. 3. In mouse diaphragm preparations that were partially paralysed by tubocurarine or low calcium solutions, tacrine, HP029, and 3,4-diaminopyridine reversed the twitch block. HP128 deepened the block. 4. In mouse triangularis sterni preparations, tacrine and HP029 prolonged the decay phase of endplate potentials and miniature endplate potentials, but had no effect on quantal content at 36 degrees C; above 10 microM, they reduced endplate potential amplitude. 3,4-Diaminopyridine increased quantal content without affecting the time course of the endplate potentials. HP128 (1-10 microM) had no effect on amplitude or time course of endplate potentials, but reduced their amplitude at higher concentrations. 5. Extracellular recording of nerve terminal currents from triangularis sterni preparations revealed that 3,4-diaminopyridine and HP128 had a selective blocking action on the waveform associated with K+ currents, tacrine reduced and prolonged the K(+)-related waveform, and HP029 had nonselective blocking actions only seen at high concentrations. 6. Tacrine and HP029 behave predominantly as anticholinesterase agents, while HP128 has weaker anticholinesterase actions that are masked by cholinoceptor blockade. Tacrine and HP128, but not HP029, have some blocking actions on K+ currents of mouse motor nerve terminals.

On the blockade of acetylcholine release at mouse motor nerve terminals by beta-bungarotoxin and crotoxin.

1. beta-Bungarotoxin and crotoxin are phospholipose A2 neurotoxins, which block irreversibly the evoked release of acetylcholine from motor nerve terminals of mouse triangularis sterni preparations. 2. Extracellular recording of nerve terminal action potentials reveal that inhibition of transmitter release is not associated with failure of the action potential to invade nerve terminals. 3. When evoked transmitter release (measured as intracellularly recorded endplate potentials) was blocked by beta-bungarotoxin, spontaneous acetylcholine release was stimulated as in control experiments by K(+)-induced depolarization and by the Ca2(+)-ionophore A23187. 4. The site of action of the toxins remains to be elucidated but would appear to be associated with the coupling of action potential induced-depolarization to the release mechanism, rather than with the release mechanism itself.

Apparent block of K+ currents in mouse motor nerve terminals by tetrodotoxin, mu-conotoxin and reduced external sodium.

1. In mouse triangularis sterni nerve-muscle preparations, reduced extracellular Na+ concentrations and low concentrations of the Na+ channel blocking toxins tetrodotoxin (TTX, 18-36 nM) and mu-conotoxin GIIIB (0.4-2.0 microM) selectively decreased the amplitude of the component of perineural waveforms associated with nerve terminal K+ currents, without affecting the main Na+ spike. 2. Intracellular recording of endplate potentials (e.p.ps) and miniature endplate potentials (m.e.p.ps) from triangularis sterni preparations revealed that TTX and mu-conotoxin GIIIB depressed the evoked quantal release of acetylcholine without significant effects on m.e.p.p. amplitude, frequency or time constant of decay. 3. The apparent block of K+ current by low concentrations of TTX and mu-conotoxin is probably not a direct effect on K+ channels but results from a decrease in the passive depolarization of nerve terminals following blockade of a small proportion of axonal Na+ channels.

Effects of charybdotoxin, a blocker of Ca2+-activated K+ channels, on motor nerve terminals.

1. The contribution of Ca2+-activated K+ currents (IK,Ca) to the control of electrical excitability of motor nerve terminals and the control of acetylcholine release was assessed by studying the effects of the specific K(Ca) channel blocking toxins charybdotoxin and apamin. Electrical activity of the terminal regions of motor nerves was assessed by extracellular recording from an electrode placed in the perineural sheaths of nerves in the mouse triangularis sterni and frog cutaneous pectoris preparations. Acetylcholine release was monitored by intracellular recording of endplate potentials (e.p.ps). 2. Charybdotoxin (20-300 nM), but not apamin (10 nM-2.5 microM), selectively reduced the amplitude of an IK,Ca unmasked by prior blockade of the delayed rectifier K+ current with 3,4-diaminopyridine (3,4-DAP). 3. In the combined presence of 3,4-DAP and charybdotoxin, large Ca2+-dependent plateau responses developed, but only moderate and transient increases in acetylcholine release occurred. 4. In the absence of 3,4-DAP, charybdotoxin did not alter the electrical activity of, or the transmitter release from motor nerve terminals. 5. A possible role of the charybdotoxin-sensitive IK,Ca in the control of transmitter release is discussed.

The prejunctional inhibitory effect of suramin on neuromuscular transmission in vitro.

The P2 purinoceptor antagonist suramin reverses skeletal muscle paralysis evoked by non-depolarizing neuromuscular blocking agents in vitro and in vivo. To further study the action of suramin on neuromuscular transmission, (miniature) endplate potentials ((m.)e.p.ps), motor nerve terminal currents and the release of radiolabeled acetylcholine was measured in isolated nerve-muscle preparations. In preparations paralysed by low Ca2+/high Mg2+ conditions, suramin (10 microM-1 mM) induced a concentration-dependent decrease in quantal content of the e.p.ps without affecting m.e.p.ps. Suramin reversed neuromuscular block by d-tubocurarine in these preparations. In erabutoxin paralysed preparations, suramin (40 microM-1 mM) inhibited the motor nerve terminal currents related to Ca2+ influx concentration-dependently, but did not affect Na+ currents. Suramin-induced inhibition of Ca2+ currents was not antagonized by ATP gamma S. Suramin (300 microM) reduced [14C]acetylcholine outflow in non-paralysed rat phrenic nerve-hemidiaphragm preparations by 32%. As suramin did not chelate Ca2+, these results indicate that suramin inhibits neuromuscular transmission by blocking prejunctional Ca2+ channels, thereby decreasing acetylcholine release upon nerve stimulation.

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.

Neuromuscular effects of nigexine, a basic phospholipase A2 from Naja nigricollis venom.

Nigexine is a basic phospholipase A2 from the venom of the spitting cobra Naja nigricollis. In addition to its anticoagulant and cytolytic properties, nigexine also affects neuromuscular transmission in vitro. On chick biventer cervicis preparations, 1.5 microM nigexine caused a slowly developing block of responses to nerve stimulation, and a progressive loss of postjunctional sensitivity. Nigexine was at least 10 times less potent than notexin. On frog cutaneous pectoris preparations, nigexine caused a transient facilitation of evoked acetylcholine release, followed by a block. Spontaneous release was not abolished, and nigexine induced the release of abnormally large packets of transmitter. Nigexine also caused contracture of muscle fibres, accompanied by depolarization and degeneration. Nigexine appears to be able to cause prejunctional blockade and direct muscle damage to isolated skeletal muscle preparations.