L. S. Cheah

Autonomic effects of some scorpion venoms and toxins.

1. The autonomic effects of venoms and toxins from several species of scorpions, including the Indian red scorpion Mesobuthus tamulus, the Chinese scorpion Buthus martensi Karsch and the Israeli scorpion Leiurus quinquestriatus quinquestriatus, all belonging to Buthidae, and the Asian black scorpions Heterometrus longimanus and Heterometrus spinifer, belonging to Scorpionidae, are reviewed.

Neuromuscular effects of candoxin, a novel toxin from the venom of the Malayan krait (Bungarus candidus)

Candoxin (MW 7334.6), a novel toxin isolated from the venom of the Malayan krait Bungarus candidus, belongs to the poorly characterized subfamily of nonconventional three‐finger toxins present in Elapid venoms. The current study details the pharmacological effects of candoxin at the neuromuscular junction. Candoxin produces a novel pattern of neuromuscular blockade in isolated nerve‐muscle preparations and the tibialis anterior muscle of anaesthetized rats. In contrast to the virtually irreversible postsynaptic neuromuscular blockade produced by curaremimetic α‐neurotoxins, the neuromuscular blockade produced by candoxin was rapidly and completely reversed by washing or by the addition of the anticholinesterase neostigmine. Candoxin also produced significant train‐of‐four fade during the onset of and recovery from neuromuscular blockade, both, in vitro and in vivo. The fade phenomenon has been attributed to a blockade of putative presynaptic nicotinic acetylcholine receptors (nAChRs) that mediate a positive feedback mechanism and maintain adequate transmitter release during rapid repetitive stimulation. In this respect, candoxin closely resembles the neuromuscular blocking effects of d‐tubocurarine, and differs markedly from curaremimetic α‐neurotoxins that produce little or no fade. Electrophysiological experiments confirmed that candoxin produced a readily reversible blockade (IC50∼10 nM) of oocyte‐expressed muscle (αβγδ) nAChRs. Like α‐conotoxin MI, well known for its preferential binding to the α/δ interface of the muscle (αβγδ) nAChR, candoxin also demonstrated a biphasic concentration–response inhibition curve with a high‐ (IC50∼2.2 nM) and a low‐ (IC50∼98 nM) affinity component, suggesting that it may exhibit differential affinities for the two binding sites on the muscle (αβγδ) receptor. In contrast, curaremimetic α‐neurotoxins have been reported to antagonize both binding sites with equal affinity.

Evidence For The Existence Of A Constitutive Nitric Oxide Synthase In Vascular Smooth Muscle

1. We have identified a neuronal nitric oxide synthase (NOS)‐like constitutive form of NOS in vascular smooth muscle (VSM) using a functional contractility approach as well as immunohistochemical methods.

The black scorpion Heterometrus longimanus: Pharmacological and biochemical investigation of the venom

Documentation on the biological activity (including the lethality) of the venom (BSV) from the black scorpion Heterometrus longimanus is lacking. We have investigated the effects of BSV on adrenergic transmission using the rat isolated anococcygeus muscle (Acm), since the venom from several species of scorpions causes peripheral sympathetic nerve stimulation with enhanced adrenergic responses. The catecholamine content in BSV was also measured by HPLC. The effects of phentolamine (5 microM), guanethidine (5 microM), desipramine (1.5 microM), tetrodotoxin (2 microM) and reserpine pretreatment in vivo (5 mg/kg s.c. x 24 hr and 5 mg/kg i.p. x 3 hr) on contractile responses of the rat Acm to field stimulation, crude BSV (2-10 microliters in 6 ml bath), noradrenaline (3 microM), tyramine (10-15 microM), carbachol (2-3 microM) and potassium chloride (50-75 mM) were investigated. BSV mimicked the agonist actions of noradrenaline (NA) by acting directly on postjunctional alpha-adrenoceptors in the anococcygeus muscle. The LD50 of crude BSV injected i.v. into mice was 0.13 ml per kg mouse. Sequential ultrafiltration of the crude BSV revealed the presence of a substance of low mol. wt which mediates the postjunctional alpha-agonist actions of BSV. HPLC measurements confirmed the presence of noradrenaline (NA; mean concentration of 1.8 +/- 0.3 mM) in BSV; the dopamine concentration (mean of 31 +/- 4 microM) was 60-fold lower than that of NA, whereas adrenaline was not detected in all the 15 samples investigated. Thus, the presence of NA in BSV can account for the postjunctional alpha-agonist actions of the venom in the Acm.

Biochemical and pharmacological characterization of the venom of the black scorpion Heterometrus spinifer.

The sting of the black scorpion Heterometrus spinifer, which can cause intense localized pain, has not been reported to produce lethal cardiovascular complications, which are well known to result from scorpion envenomation as a consequence of a massive release of catecholamines. Therefore, we have undertaken a biochemical and pharmacological characterization of the venom of H. spinifer. Pharmacologically, the venom (0.125 microL/mL) produced a marked, reversible contracture in the chick biventer cervicis muscle that was blocked by d-tubocurarine (2 microM) but not by tetrodotoxin (5 microM) and omega-conotoxin GVIA (3 microM). The anticholinesterase neostigmine (1 microM) potentiated the contracture by 5.3-fold. An ultra-filtrate fraction of MW < 3000 (F3K) of the venom produced a similar contracture in the biventer muscle, whereas the retentate of MW > 3000 did not. In the rat anococcygeus muscle, the venom produced a contractile response that was partially (37.4 +/- 1.6%) blocked by atropine (5 microM); phentolamine (5 microM) blocked the remaining response. Tetrodotoxin (5 microM) did not block the contractile response of the venom on the anococcygeus muscle. Electrospray ionization-mass spectrometry/mass spectrometry confirmed the presence of high concentrations of acetylcholine (79.8 +/- 1.7 microM) and norepinephrine (146.7 +/- 19.8 microM) in H. spinifer venom, which can fully account for the observed cholinergic and adrenergic effects. In contrast to scorpion venoms that selectively target neuronal ion channels in mediating transmitter release, our data show that H. spinifer venom does not possess such activity, which likely explains the apparent lack of lethality of black scorpion envenomation.

Involvement of the L-arginine-nitric oxide synthase pathway in the relaxant responses of the rat isolated anococcygeus muscle to a scorpion (Leiurus quinquestriatus quinquestriatus) venom

Venom from several species of scorpions can cause generalized depolarization of peripheral nerves with enhancement of neurotransmitter release. The effects of the venom (LQV) from the scorpion Leiurus quinquestriatus quinquestriatus were investigated using the rat isolated carbachol (CCh) precontracted anococcygeus muscle (Acm) mounted in Krebs solution containing phentolamine (5 microM). LQV (0.2 microgram/ml and 1.5 micrograms/ml) markedly relaxed the tone of the CCh precontracted (non-stimulated) Acm by 31.5 +/- 5.1% and 35.5 +/- 4.7%, respectively; the onset was immediate after the high dose, followed by a slow and gradual return of the muscle tone to 88.7 +/- 1.8% of the initial peak tension in 50.2 +/- 4 min. Subsequent doses of LQV produced essentially no appreciable changes in muscle tone, but the addition of L-arginine (250 microM) or, especially sodium nitroprusside (SNP; 1 microM) produced marked and rapid relaxations of the Acm. Similar results were obtained with LQV during electrical field stimulation (EFS) of the precotracted Acm; however, during the gradual return of the muscle tone from the relaxed state induced by LQV, the EFS-induced relaxant (NANC) responses were also progressively inhibited by 84.9 +/- 3.4% and 64.3 +/- 4.5 by LQV 0.2 microgram/ml and 1.5 micrograms/ml, respectively. Tetrodotoxin (TTx; 2 microM) or NG-nitro-L-arginine methylester (L-NAME; 50 microM) markedly inhibited the relaxant responses of the Acm to EFS as well as to LQV but not the responses to SNP: L-arginine (250 microM) partially restored the relaxant (NANC) responses of the Acm to EFS. Thus, the L-arginie-nitric oxide synthase-nitric oxide pathway is involved in mediating the marked relaxant responses of the CCh precontracted Acm to LQO.

ANTICHOLINESTERASE ACTIVITY OF AND POSSIBLE ION-CHANNEL BLOCK BY CIMETIDINE, RANITIDINE AND OXMETIDINE IN THE TOAD ISOLATED RECTUS ABDOMINIS MUSCLE

1. Responses of the toad isolated rectus abdominis muscle to cumulative doses of acetylcholine were recorded in the absence or presence of varying concentrations of cimetidine, ranitidine or oxmetidine. The corresponding cumulative log concentration‐response curves for acetylcholine were then plotted for each antagonist studied.

Pharmacological characterization of mikatoxin, an α-neurotoxin isolated from the venom of the New-Guinean small-eyed snake Micropechis ikaheka

Symptoms of envenomation by the New-Guinean small-eyed snake Micropechis ikaheka (Elapidae) include peripheral neurotoxicity and myotoxicity. We have now purified to homogeneity a long-chain neurotoxin, mikatoxin, from M. ikaheka venom by successive gel filtration and reverse-phase chromatography. Electrospray ionization mass spectrometry showed mikatoxin to be a homogenous peptide of MW 7775.6. Mikatoxin was devoid of any phospholipase A(2) activity associated with the crude venom and did not exhibit any intrinsic anticholinesterase activity. In the chick biventer cervicis muscle, it produced an irreversible, concentration-dependent block of responses to exogenously applied acetylcholine and carbachol as well as twitches evoked by nerve, but not by direct muscle stimulation. Moreover, mikatoxin, like alpha-bungarotoxin and erabutoxin-b, did not show significant fade response to train-of-four stimulation of the mouse phrenic nerve-hemi diaphragm muscle. It also failed to block ganglionic transmission in the guinea pig ileum and muscarinic responses in the rat anococcygeus muscle. Our study provides strong evidence for the presence of a neurotoxin (mikatoxin) in M. ikaheka venom that produces neuromuscular blockade in skeletal muscle attributable to selective and irreversible antagonism of postsynaptic nicotinic acetylcholine receptors of the neuromuscular junction and likely contributes to the peripheral neurotoxicity observed in M. ikaheka envenomation.

Prejunctional action of the venom from the Indian red scorpion Mesobuthus tamulus on adrenergic transmission in vitro

Venom (RSV) from the Indian red scorpion, Mesobuthus tamulus (Buthus tamulus), can cause increased peripheral sympathetic activity with consequent enhancement of adrenergic responses. We have therefore investigated the effects of RSV on adrenergic transmission in the rat isolated anococcygeus muscle. The effects of phentolamine (5 microM), tetrodotoxin (2 microM), guanethidine (5 microM), desipramine (1 microM) and reserpine pretreatment in vivo (5 mg/kg s.c. x 24 hr and 5 mg/kg i.p. x 3 hr) on the contractile responses of the rat anococcygeus muscle to RSV (1.5 microgram/ml), field stimulation, noradrenaline (NA, 1 microM or 3 microM) and tyramine (15 microM) were compared. The contractile responses to RSV and to field stimulation were completely blocked by phentolamine, tetrodotoxin, guanethidine and reserpine pretreatment, but the responses were potentiated by desipramine. The contractile responses to tyramine were completely blocked by phentolamine, reserpine pretreatment as well as desipramine. The responses to NA were completely blocked by phentolamine, but were potentiated by guanethidine, desipramine and reserpine. Relatively low concentrations (0.1 microgram/ml x 4) of RSV which did not produce any observable increase in tone of the anococcygeus muscle, potentiated the contractile response of the anococcygeus muscle to field stimulation, but not the responses to exogenous NA; 4-aminopyridine (25 microM x 2) also potentiated the muscle responses to field stimulation. HPLC measurements revealed only very low concentrations (0.10 +/- 0.03 mumol/g venom) of NA in RSV. Thus, the adrenergic agonist action of RSV in the rat isolated anococcygeus muscle can be attributed to the involvement of some prejunctional mechanism(s) of action that stimulates the release of neurotransmitter which differs from the indirect action mediated by tyramine.

TRAIN‐OF‐FOUR FADE DURING NEUROMUSCULAR BLOCKADE INDUCED BY TUBOCURARINE, SUCCINYLCHOLINE OR α‐BUNGAROTOXIN IN THE RAT ISOLATED HEMIDIAPHRAGM

1. Nerve‐evoked maximal twitches (T1, T2, T3, T4) of the rat isolated hemidiaphragm to train‐of‐four (TOF) stimulation (2 Hz2 s) were recorded continuously in the absence or presence of tubocurarine (1.5 μmol/1), succinylcholine (40 μmol/1) or α‐bungarotoxin (1 μmol/1). The T1 and T4 response‐time profiles for the three drugs were analysed with respect to amplitude depression and the TOF ratio (T4/T1) during the development of and recovery from neuromuscular blockade.