Selecting for a sustainable workforce to meet the future healthcare needs of rural communities in Australia.

An undersupply of generalists doctors in rural communities globally led to widening participation (WP) initiatives to increase the proportion of rural origin medical students. In 2002 the Australian Government mandated that 25% of commencing Australian medical students be of rural origin. Meeting this target has largely been achieved through reduced standards of entry for rural relative to urban applicants. This initiative is based on the assumption that rural origin students will succeed during training, and return to practice in rural locations. One aim of this study was to determine the relationships between student geographical origin (rural or urban), selection scores, and future practice intentions of medical students at course entry and course exit. Two multicentre databases containing selection and future practice preferences (location and specialisation) were combined (5862), representing 54% of undergraduate medical students commencing from 2006 to 2013 across nine Australian medical schools. A second aim was to determine course performance of rural origin students selected on lower scores than their urban peers. Selection and course performance data for rural (461) and urban (1431) origin students commencing 2006-2014 from one medical school was used. For Aim 1, a third (33.7%) of rural origin students indicated a preference for future rural practice at course exit, and even fewer (6.7%) urban origin students made this preference. Results from logistic regression analyses showed significant independent predictors were rural origin (OR 4.0), lower Australian Tertiary Admissions Rank (ATAR) (OR 2.1), or lower Undergraduate Medical and Health Sciences Admissions Test Section 3 (non-verbal reasoning) (OR 1.3). Less than a fifth (17.6%) of rural origin students indicated a preference for future generalist practice at course exit. Significant predictors were female gender (OR 1.7) or lower ATAR (OR 1.2), but not rural origin. Fewer (10.5%) urban origin students indicated a preference for generalist practice at course exit. For Aim 2, results of Mann-Whitney U tests confirmed that slightly reducing selection scores does not result in increased failure, or meaningfully impaired performance during training relative to urban origin students. Our multicentre analysis supports success of the rural origin WP pathway to increase rural student participation in medical training. However, our findings confirm that current selection initiatives are insufficient to address the continuing problem of doctor maldistribution in Australia. We argue for further reform to current medical student selection, which remains largely determined by academic meritocracy. Our findings have relevance to the selection of students into health professions globally.

Pharmacokinetics and pharmacodynamics of the myotoxic venom of Pseudechis australis (mulga snake) in the anesthetised rat.

CONTEXT: Myotoxicity is a common clinical effect of snake envenoming and results from either local or systemic myotoxins in snake venoms. Although numerous myotoxins have been isolated from snake venoms, there has been limited study on the relationship between the time course of venom concentrations (pharmacokinetics) and the time course of muscle injury measured as a rise in creatine kinase (CK) (pharmacodynamics). OBJECTIVE: The aim of this study was to develop an in vivo model of myotoxicity to investigate the time course of myotoxicity and the effect of antivenom. MATERIALS AND METHODS: Anesthetised rats were administered Pseudechis australis (mulga snake) venom either through i.v., i.m. or s.d. route, including a range of doses (5-100 µg/kg). Serial blood samples were collected for measurement of venom using enzyme immunoassay and measurement of CK and creatinine. Antivenom was administered before, 1 and 6 h after venom administration to investigate its effect on muscle injury. Plots of venom and CK versus time were made and the area under the curve (AUC) was calculated. RESULTS: There was a significant dose-dependent increase in CK concentration after administration of P. australis venom, which was greatest for i.v. administration. Timed measurement of venom concentrations showed a rapid absorption through s.d. and i.m. routes and a delayed rise in CK concentrations following any route. Antivenom prevented myotoxicity shown by a decrease in the CK AUC, which was most effective if given earliest. There was a rise in creatinine following i.v. venom administration. CONCLUSION: The study shows the delayed relationship between venom absorption and the rise in CK, consistent with the delayed onset of myotoxicity in human envenoming. Antivenom prevented myotoxicity more effectively if given earlier.

Development of a sensitive enzyme immunoassay for measuring taipan venom in serum.

The detection and measurement of snake venom in blood is important for confirming snake identification, determining when sufficient antivenom has been given, detecting recurrence of envenoming, and in forensic investigation. Venom enzyme immunoassays (EIA) have had persistent problems with poor sensitivity and high background absorbance leading to false positive results. This is particularly problematic with Australasian snakes where small amounts of highly potent venom are injected, resulting in low concentrations being associated with severe clinical effects. We aimed to develop a venom EIA with a limit of detection (LoD) sufficient to accurately distinguish mild envenoming from background absorbance at picogram concentrations of venom in blood. Serum samples were obtained from patients with taipan bites (Oxyuranus spp.) before and after antivenom, and from rats given known venom doses. A sandwich EIA was developed using biotinylated rabbit anti-snake venom antibodies for detection. For low venom concentrations (i.e. <1 ng/mL) the assay was done before and after addition of antivenom to the sample (antivenom difference method). The LoD was 0.15 ng/mL for the standard assay and 0.1 ng/mL for the antivenom difference method. In 11 pre-antivenom samples the median venom concentration was 10 ng/mL (Range: 0.3-3212 ng/mL). In four patients with incomplete venom-induced consumption coagulopathy the median venom concentration was 2.4 ng/mL compared to 30 ng/mL in seven patients with complete venom-induced consumption coagulopathy. No venom was detected in any post-antivenom sample and the median antivenom dose prior to this first post-antivenom sample was 1.5 vials (1-3 vials), including 7 patients administered only 1 vial. In rats the assay distinguished a 3-fold difference in venom dose administered and there was small inter-individual variability. There was small but measurable cross-reactivity with black snake (Pseudechis), tiger snake (Notechis) and rough-scale snake (Tropidechis carinatus) venoms with the assay for low venom concentrations (<1 ng/mL). The use of biotinylation and the antivenom difference method in venom EIA produces a highly sensitive assay that will be useful for determining antivenom dose, forensic and clinical diagnosis.

An in vivo examination of the stability of venom from the Australian box jellyfish Chironex fleckeri.

We have previously characterised the pharmacological activity of a number of jellyfish venoms with a particular emphasis on the profound cardiovascular effects. It has been suggested that jellyfish venoms are difficult to work with and are sensitive to pH, temperature and chemical changes. The current study aimed to examine the working parameters of the venom of the Australian box jellyfish Chironex fleckeri to enable fractionation and isolation of the toxins with cardiovascular activity. C. fleckeri venom was made up fresh each day and subjected to a number of different environments (i.e. a pH range of 5-9 and a temperature range of 4-30 degrees C). In addition, the effect of freeze drying and reconstituting the venom was investigated. Venom (50 microg/kg, i.v.) produced a transient hypertensive response followed by cardiovascular collapse in anaesthetised rats. This biphasic response was not significantly effected by preparation of the venom at a pH of 5, 7 or 9. Similarly, venom (50 microg/kg, i.v.) did not display a loss of activity when exposed to temperatures of 4, 20 or 30 degrees C for 1.5h. However, the cardiovascular activity was abolished by boiling the venom. Freeze drying, and then reconstituting, the venom did not significantly affect its cardiovascular activity. However, repeated freeze drying and reconstituting of extracted venom resulted in a significantly loss of activity. This study provides a more detailed knowledge of the parameters in which C. fleckeri venom can be used and, while supporting some previous studies, contradicts some of the perceived problems of working with the venom.

Presynaptic neuromuscular activity of venom from the brown-headed snake (Glyphodon tristis).

The brown-headed snake (Glyphodon tristis) inhabits the forest regions of Papua New Guinea, Torres Strait Islands, and far northern Queensland, Australia. Although bites by Glyphodon dunmalli have been reported, G. tristis was regarded as innocuous until 1989 when a healthy 20 year old man was bitten (Sutherland, S.K., Tibballs, J., 2001. Australian Animal Toxins, the Creatures, their Toxins and Care of the Poisoned Patient. University Press, Oxford). Treatment of envenomation by this species is empirical with no specific antivenom available. While no published studies on the venom of G. tristis are available, unpublished studies suggest neurotoxicity as being the main symptom of envenomation. In this study, the in vitro effects of G. tristis venom were examined using the chick biventer cervicis nerve muscle (CBCNM) preparation. Venom (10 microg/ml) inhibited indirect (0.2 ms, 0.1 Hz, supramaximal V) twitches of the CBCNM. This inhibition appeared to be presynaptic in origin as evidenced by the lack of effect of venom on responses to exogenous acetylcholine (1 mM), carbachol (20 microM) and KCl (40 mM) in the non-stimulated CBCNM. Prior addition (10 min) of polyvalent snake antivenom (5 U/ml; CSL Ltd) attenuated twitch inhibition. The venom (10-50 microg/ml) also appears to be myotoxic as indicated by a slowly developing contracture and inhibition of direct (2 ms, 0.1 Hz, supramaximal V, in the presence of tubocurarine 10 microM) twitches. Myotoxicity was confirmed by subsequent histological examination of tissues. This myotoxicity was prevented by the prior addition of polyvalent snake antivenom (30 U/ml). The phospholipase A inhibitor 4-BPB (1.8 mM) significantly attenuated the inhibition of indirect and direct twitches of the CBCNM preparation, indicating the involvement of a PLA2 component in the toxic action of the venom.

The in vitro and in vivo pharmacological activity of Boiga dendrophila (mangrove catsnake) venom.

The great taxonomic and prey base diversity of colubrids (non-front-fanged snakes) suggests that their venoms may represent a 'literal gold mine' for scientists eager to find novel pharmacological probes. While pharmacological characterization is lacking for most of these venoms, this is even more so with regard to activity of colubrid venoms on the mammalian autonomic nervous system. This study characterizes the activity of venom from the colubrid, Boiga dendrophila using in vitro smooth muscle preparations and the anaesthetized rat. In the prostatic segment of the rat vas deferens, cumulative additions of venom (1-150 microg ml(-1)) induced concentration-dependent inhibition of electrically evoked (0.2 Hz, 0.3 ms, 70-100 V) twitches. The inhibitory effect of venom (100 microg ml(-1)) was attenuated by 8-phenyltheophylline (8-PT) (20 microM) and 8-cyclopentyl-1, 3-dipropylxanthine (20 microM) but not idazoxan (1 microM), or a combination of ranitidine (0.2 microM) and thioperamide (10 microM). The inhibitory effect of venom (100 microg ml(-1)) was augmented by dipyridamole (10 microM) but abolished by pretreatment with adenosine deaminase (7.5 units/100 microl) suggesting that it contains components with adenosine A(1) receptor activity, most likely adenosine. In isolated segments of guinea-pig ileum, venom (10-100 microg ml(-1)) caused concentration-dependent contractions which were inhibited by the muscarinic receptor antagonist atropine (0.1 microM) but not by the histamine receptor antagonist mepyramine (0.5 microM). In the anaesthetized rat, venom (5-7.5 mg kg(-1), i.v.) caused a hypotensive effect. Our data suggest that the venom contains components with purinergic and muscarinic receptor activity.

Cardiovascular, haematological and neurological effects of the venom of the Papua New Guinean small-eyed snake (Micropechis ikaheka) and their neutralisation with CSL polyvalent and black snake antivenoms.

Cardiovascular and haematological effects of venom of the small-eyed Snake (Micropechis ikaheka) were examined in ventilated anaesthetised piglets. Neurotoxic effects were examined in chick biventer cervicis nerve-muscle preparations. Immunoreactivity of venom was tested against the monovalent antivenom components in a CSL Ltd Venom Detection Kit. Neutralisation was tested in vivo and in vitro with CSL Ltd polyvalent snake and Black Snake (Pseudechis australis) antivenoms. Venom in 0.1% bovine serum albumin in saline was infused into piglets in doses 1-2000 microg/kg. Pulmonary hypertension (P= 0.0007) and depression of cardiac output (P= 0.002) were observed up to 3 h after 150-160 microg/kg. The concentration of plasma free-haemoglobin increased more than 50-fold, indicating haemolysis. Neither coagulopathy nor thrombocytopenia occurred. Creatine phosphokinase and serum potassium levels did not increase suggesting absence of acute rhabdomyolysis. The venom caused post-synaptic neurotoxicty. Immunoreactivity of venom with Black Snake antivenom was observed at very high venom concentrations. Cardiovascular effects were absent and haemolysis was less after venom was pre-incubated at 37 degrees C for 30 min with polyvalent antivenom. Neutralisation by Black Snake antivenom was less effective. The neurotoxicity was neutralised by polyvalent or Black Snake antivenoms. Human envenomation may be treated with CSL Ltd polyvalent snake antivenom.

Species and regional variations in the effectiveness of antivenom against the in vitro neurotoxicity of death adder (Acanthophis) venoms.

Although viperlike in appearance and habit, death adders belong to the Elapidae family of snakes. Systemic envenomation represents a serious medical problem with antivenom, which is raised against Acanthophis antarcticus venom, representing the primary treatment. This study focused on the major Acanthophis variants from Australia and islands in the Indo-Pacific region. Venoms were profiled using liquid chromatography-mass spectrometry, and analyzed for in vitro neurotoxicity (0.3-10 microg/ml), as well as the effectiveness of antivenom (1-5 units/ml; 10 min prior to the addition of 10 microg/ml venom). The following death adder venoms were examined: A. antarcticus (from separate populations in New South Wales, Queensland, South Australia, and Western Australia), A. hawkei, A. praelongus, A. pyrrhus, A. rugosus, A. wellsi, and venom from an unnamed species from the Indonesian island of Seram. All venoms abolished indirect twitches of the chick isolated biventer cervicis nerve-muscle preparation in a dose-dependent manner. In addition, all venoms blocked responses to exogenous acetylcholine (1 mM) and carbachol (20 microM), but not KCl (40 mM), suggesting postsynaptic neurotoxicity. Death adder antivenom (1 unit/ml) prevented the neurotoxic effects of A. pyrrhus, A. praelongus, and A. hawkei venoms, although it was markedly less effective against venoms from A. antarcticus (NSW, SA, WA), A. rugosus, A. wellsi, and A. sp. Seram. However, at 5 units/ml, antivenom was effective against all venoms tested. Death adder venoms, including those from A. antarcticus geographic variants, differed not only in their venom composition but also in their neurotoxic activity and susceptibility to antivenom. For the first time toxicological aspects of A. hawkei, A. wellsi, A. rugosus, and A. sp. Seram venoms were studied.

A pharmacological examination of venoms from three species of death adder (Acanthophis antarcticus, Acanthophis praelongus and Acanthophis pyrrhus).

The common (A. antarcticus), northern (A. praelongus) and desert (A. pyrrhus) death adders are species belonging to the Acanthophis genus. The present study compared some pharmacological aspects of the venoms of these species and examined the in vitro efficacy of death adder antivenom. Neurotoxicity was determined by the time to produce 90% inhibition (t(90)) of indirect (0.1 Hz, 0.2 ms, supramaximal voltage) twitches in the chick biventer cervicis nerve-muscle (3-10 microg/ml) and mouse phrenic nerve-diaphragm (10 microg/ml) preparations. A. praelongus venom was significantly less neurotoxic than A. antarcticus venom but was not significantly different from A. pyrrhus venom. In the biventer muscle, all three venoms (3-10 microg/ml) abolished responses to exogenous ACh (1 mM) and carbachol (20 microM), but not KCl (40 mM), indicating activity at post-synaptic nicotinic receptors. All venoms (30 microg/ml) failed to produce significant inhibition of direct twitches (0.1 Hz, 2.0 ms, supramaximal voltage) in the chick biventer cervicis nerve-muscle preparation. However, A. praelongus (30 microg/ml) venom initiated a significant direct contracture of muscle, indicative of some myotoxic activity. The prior (10 min) administration of death adder antivenom (1 unit/ml), which is raised against A. antarcticus venom, markedly attenuated the twitch blockade produced by all venoms (10 microg/ml). Administration of antivenom (1.5 units/ml) at t(90) markedly reversed, over a period of 4 h, the inhibition of twitches produced by A. praelongus (3 microg/ml, 72+/-6% recovery) and A. pyrrhus (3 microg/ml, 51+/-9% recovery) but was less effective against A. antarcticus venom (3 microg/ml, 22+/-7% recovery). These results suggest that all three venoms contain postsynaptic neurotoxins. Death adder antivenom displayed differing efficacy against the in vitro neurotoxicity of the three venoms.

Stonefish (Synanceia spp.) antivenom neutralises the in vitro and in vivo cardiovascular activity of soldierfish (Gymnapistes marmoratus) venom.

The soldierfish (Gymnapistes marmoratus), which is related to the stonefish (Synanceia spp.), inhabits the western, southern and lower eastern coastlines of Australia. We have previously found that G. marmoratus venom possesses pharmacological activity similar to Synanceia trachynis venom (Hopkins, B.J., Hodgson, W.C., 1998. Cardiovascular studies on venom from the soldierfish (Gymnapistes marmoratus). Toxicon 36, 973-872; Church, J.E., Hodgson, W.C., 2000. Dose-dependent cardiovascular and neuromuscular effects of stonefish (Synanceja trachynis) venom. Toxicon 38, 391-407), namely an action at muscarinic receptors and adrenoceptors. The aim of this study was to determine the effectiveness of Synanceia antivenom in neutralising the in vitro and in vivo cardiovascular activity of G. marmoratus venom. Venom extract (0.4-12 microg protein/ml) caused dose- and endothelium-dependent relaxation in porcine U46619-precontracted coronary arteries. This relaxation was abolished by 10 min prior exposure of the tissue to Synanceia antivenom (3 units/ml). In rat paced (5 ms, 2 Hz, 7-12 V) left atria, G. marmoratus venom extract (40 microg protein/ml) produced a transient negative, followed by a sustained positive inotropic response. In spontaneously beating right atria, venom extract (40 microg protein/ml) produced similar changes in rate. Prior incubation of venom extract with Synanceia antivenom (1 unit/4 microg venom extract protein, 10 min) significantly attenuated both components of the inotropic response, and abolished the positive chronotropic response. In the anaesthetised rat, venom extract (400 microg protein/kg, i.v.) produced a transient depressor response, followed by a more sustained pressor response. Prior incubation of venom extract with Synanceia antivenom (1 unit/4 microg venom extract protein, 10 min) significantly attenuated both components of the in vivo response. As Synanceia antivenom neutralised the cardiovascular activity of G. marmoratus venom both in vitro and in vivo, we suggest that the venoms of the two species may share a similar component(s).

Neurotoxic activity of venom from the Australian eastern mouse spider (Missulena bradleyi) involves modulation of sodium channel gating.

Mouse spiders represent a potential cause of serious envenomation in humans. This study examined the activity of Missulena bradleyi venom in several in vitro preparations. Whilst female M. bradleyi venom at doses up to 0.05 microl ml(-1) failed to alter twitch or resting tension in all preparations used, male venom (0.02 and 0.05 microl ml(-1)) produced potent effects on transmitter release in both smooth and skeletal neuromuscular preparations. In the mouse phrenic nerve diaphragm preparation, male M. bradleyi venom (0.02 microl ml(-1)) caused rapid fasciculations and an increase in indirectly evoked twitches. Male venom (0.02 and 0.05 microl ml(-1)) also caused a large contracture and rapid decrease in indirectly evoked twitches in the chick biventer cervicis muscle, however had no effect on responses to exogenous ACh (1 mM) or potassium chloride (40 mM). In the chick preparation, contractile responses to male M. bradleyi venom (0.05 microl ml(-1)) were attenuated by (+)-tubocurarine (100 microM) and by tetrodotoxin (TTX, 1 microM). Both actions of male M. bradleyi venom were blocked by Atrax robustus antivenom (2 units ml(-1)). In the unstimulated rat vas deferens, male venom (0.05 microl ml(-1)) caused contractions which were inhibited by a combination of prazosin (0.3 microM) and P(2X)-receptor desensitization (with alpha,beta-methylene ATP 10 microM). In the rat stimulated vas deferens, male venom (0.05 microl ml(-1)) augmented indirectly evoked twitches. Male venom (0.1 microl ml(-1)) causes a slowing of inactivation of TTX-sensitive sodium currents in acutely dissociated rat dorsal root ganglion neurons. These results suggest that venom from male M. bradleyi contains a potent neurotoxin which facilitates neurotransmitter release by modifying TTX-sensitive sodium channel gating. This action is similar to that of the delta-atracotoxins from Australian funnel-web spiders.

Sex differences in the pharmacological activity of venom from the white-tailed spider (Lampona cylindrata).

This study compared the pharmacological activity of venom from male and female white-tailed spiders (L. cylindrata). In guinea-pig ileum, male L. cylindrata venom (1-10 microg/ml) caused dose-dependent contractions. The response to venom (5 microg/ml) was significantly inhibited by mepyramine (0.5 microM). Venom (5-50 microg/ml) from female L. cylindrata had no contractile activity in this tissue. However, female L. cylindrata venom (50 microg/ml) inhibited electrically-evoked twitches of guinea-pig ileum. This inhibitory effect was attenuated by 8-phenyltheophylline (10 microM) or by prior exposure of venom to adenosine deaminase. In the rat vas deferens, male (5 microg/ml) and female (50 microg/ml) L. cylindrata venom inhibited electrically-evoked twitches. 8-Phenyltheophylline (20 microM) significantly attenuated the response to female L. cylindrata venom, while the histamine H(2)- and H(3)-receptor antagonists ranitidine (10 microM) and thioperamide (0.2 microM) significantly attenuated the response to male L. cylindrata venom. Male L. cylindrata venom (5-20 microg/ml) caused dose-dependent contractions in the epididymal segment of the rat vas deferens. The response to male L. cylindrata venom (10 microg/ml) was significantly inhibited by prazosin (0.3 microM) but was unaffected by depleting monoamine stores with reserpine. Male L. cylindrata venom (5-15 microg/ml) caused dose-dependent increases in rate and force of rat atria which were significantly inhibited by propranolol (5 microM) but not by reserpine. Female L. cylindrata venom (50 microg/ml) had no effect in atria. In the anaesthetised (pentobarbitone, 100 mg/kg, i.p.) rat, male L. cylindrata venom (10-300 microg/kg, i.v.) caused dose-dependent depressor responses while venom (up to 1 mg/kg, i.v.) from female L. cylindrata had no effect on arterial pressure. A histamine content of 5 and 0.01% (dry weight) was detected in venom from male and female L. cylindrata, respectively. Venom from male L. cylindrata was found to contain 56 pg noradrenaline/microg whereas venom from the female contained negligible noradrenaline. The results of this study show the presence of histamine and noradrenaline in venom from male L. cylindrata. Although devoid of significant quantities of these amines, female L. cylindrata venom has activity at adenosine receptors.

Dose-dependent cardiovascular and neuromuscular effects of stonefish (Synanceja trachynis) venom.

There has been recent debate regarding the labile nature of stonefish venoms and the pharmacology of their breakdown products. The present study examined the cardiovascular and neuromuscular effects of lyophilised venom, and conducted a preliminary investigation of freshly milked venom. Lyophilised venom (20 microg/ml) caused endothelium-dependent relaxation in rat aortae that was abolished by atropine (0.1 microM). In contrast, an endothelium-independent contractile response occurred in porcine coronary arteries. However, in the presence of atropine (10 nM), this became a relaxation response which was attenuated by the B2 antagonist FR-173657 (0.1 microM) or by a combination of idazoxan (1 microM) and propranolol (1 microM). In rat isolated atria, lyophilised venom (4 microg/ml) caused a biphasic inotropic response consisting of an initial decrease, and then increase, in force which were attenuated by atropine (0.5 microM) and propranolol (5 microM), respectively. The increase in force produced by venom was unaffected by reserpine pre-treatment suggesting a direct action at adrenoceptors. In the anaesthetised rat, lyophilised venom (1-300 microg/kg, i.v.), caused a dose-dependent depressor response, with a subsequent pressor response at higher concentrations (30-300 microg/kg, i.v.). In the presence of atropine (1 mg/kg, i.v.), the depressor response to venom was abolished, a transient pressor response unmasked and the secondary pressor response augmented. In the additional presence of prazosin (50 microg/kg, i.v.), the transient pressor response was abolished and the secondary pressor response attenuated. Lyophilised venom had no significant effect on nerve-evoked (10 microg/ml) or directly-evoked (100 microg/ml) twitches of the chick biventer cervicis muscle preparation. Milked venom (1 microl/ml) caused a biphasic response (i.e., an initial relaxation followed by contraction) in rat aortae, a contraction in porcine coronary arteries, complete cessation of rat isolated atrial activity and markedly inhibited both nerve-evoked and directly-evoked twitches of the chick biventer cervicis muscle preparation. In the anaesthetised rat, milked venom (15 microl/kg, i.v.) caused immediate cardiovascular collapse. It appears that the cardiovascular effects of stonefish venom are mediated by a dose-dependent action at muscarinic receptors and adrenoceptors.

A pharmacological examination of venom from the Papuan taipan (Oxyuranus scutellatus canni).

The Papuan taipan (Oxyuranus scutellatus canni) is the third most venomous terrestrial snake in the world, however, little is know about the pharmacology of the venom. In the chick biventer cervicis muscle, venom (10 microg/ml) abolished nerve-mediated twitches (time to 90% inhibition (t90) 44+/-5 min, n = 9). This inhibition was unaffected by prior incubation of the venom with the phospholipase A inhibitor 4-bromophenacyl bromide (4-BPB; 0.72 mM) (t90 48+/-7 min, n = 8). The mouse phrenic nerve diaphragm preparation displayed greater sensitivity to venom (10 microg/ml) (t90 25+/-1 min, n = 6). In the chick biventer muscle, venom (10 microg/ml) significantly inhibited responses to acetylcholine (1 mM) and carbachol (20 microM), but not KCI (40 mM), indicating activity at post-synaptic nicotinic receptors. Venom (10 microg/ ml) did not affect direct muscle stimulation. Venom (3-30 microg/ml) produced dose-dependant contractions of the guinea-pig ileum. Contractile responses were significantly inhibited by indomethacin (1 microM) or prior incubation of the venom with 4-BPB (0.72 mM) indicating involvement of a PLA component. In rat phenylephrine (0.3 microM) precontracted aortae, venom (3-100 microg/ml) produced endothelium-independent relaxation which was unaffected by prior incubation of venom (30 microg/ml) with 4-BPB (0.72 mM). In anaesthetised rats, 10 microg/kg (i.v.) venom produced rapid respiratory and cardiovascular collapse while 5 microg/kg (i.v.) venom produced only a small transient decrease in mean arterial blood pressure. Prior administration of 5 microg/kg (i.v.) venom enabled subsequent administration of 10 and 100 microg/kg (i.v.) venom without respiratory or cardiovascular collapse. Further work is required to identify specific toxins with the above pharmacological activity.

The effects of antivenom on the in vitro neurotoxicity of venoms from the taipans Oxyuranus scutellatus, Oxyuranus microlepidotus and Oxyuranus scutellatus canni.

The venoms of the inland (Oxyuranus microlepidotus), coastal (O. scutellatus) and Papuan (O. s. canni) taipans are among the most potent in the world. The present study compared the in vitro neurotoxic effects of these venoms and the protective effects of taipan antivenom. Venom (10 microg/ml) from all three snakes abolished nerve-mediated twitches of the chick biventer cervicis muscle preparation with the following rank order of potency (based on the time taken to inhibit 90% of the twitch response; t90): O. microlepidotus (27+/-3 min) > O. scutellatus (42+/-3 min) = O. S. canni (48+/-5 min). This inhibitory effect of all three venoms was primarily postsynaptic in origin as evidenced by the inhibition of responses to exogenous acetylcholine (ACh; 1 mM) and carbachol (CCh; 20 microM), but not potassium chloride (40 mM). In contrast, the presynaptic neurotoxins taipoxin (3 microg/ml) and paradoxin (3 microg/ml) abolished nerve-mediated twitches without producing a significant effect on contractile responses to exogenous agonists. Prior incubation of the tissue with taipan antivenom (1 unit/ml for 10 min) markedly attenuated the inhibitory effects of taipoxin (3 microg/ml) and paradoxin (3 microg/ml), as well as O. scutellatus (10 microg/ml) and O. s. canni (10 microg/ml) venom. However, in the presence of antivenom, O. microlepidotus venom (10 microg/ml) still abolished nerve-mediated twitches and responses to ACh and CCh. The results of the current study indicate that taipan antivenom, raised against O. scutellatus venom, is effective, in vitro, against the neurotoxic effects of venom from the Papuan and coastal taipans, as well as the presynaptic effects of venom from the inland taipan. However, the antivenom appears less effective against the postsynaptic effects of the latter. It is possible that inland taipan venom contains a component not neutralised by the antivenom which may contribute to the extreme potency of this venom.

The smooth muscle relaxant effects of venom from the inland taipan (Oxyuranus microlepidotus).

Venom (10 microg/ml) relaxed phenylephrine-precontracted aortae. This relaxation was unaffected by removal of the endothelium or a combination of N(G)-nitro-L-arginine (L-NOARG; 0.1 mM), oxyhaemoglobin (10 microM) and indomethacin (10 microM). 4-BPB (0.78 mM), propranolol (1 microM), or a combination of apamin (0.1 microM), charybdotoxin (0.1 microM) and glibenclamide (10 microM) did not effect endothelium-independent relaxation, suggesting a lack of PLA2 activity or an effect at beta-adrenoceptors or K+ channels. Venom (10 microg/ml) reversed Bay K 8644 (0.1 microM)-induced contraction indicating the venom may have an effect on L-type Ca2+ channels.

Cardiovascular studies on venom from the soldierfish (Gymnapistes marmoratus).

This study examined some of the effects of soldierfish (Gymnapistes marmoratus) venom on the cardiovascular system of rats. Venom (20 microg/ml) produced a biphasic response on rat isolated spontaneously beating atria. This was characterised by a negative, followed by a positive, inotropic and chronotropic action. The increase in force and rate was significantly reduced by propranolol (5 microM) or pretreatment of the rats with reserpine. The decrease in force was significantly inhibited by atropine (0.5 microM). Venom (20-60 microg/ml) produced dose-dependent relaxation in rat isolated endothelium-intact aortae but no response in endothelium-denuded, aortae. Relaxation to venom (30 microg/ml) was significantly inhibited by the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine (NOLA; 0.1 mM) but was unaffected by atropine (0.5 microM). Venom (200 microg/kg, i.v.) produced a biphasic response in anaesthetized rats, consisting of an initial decrease (phase 1) followed by a prolonged increase (phase 2) in mean arterial pressure. Indomethacin (5 mg/kg, i.v.) significantly inhibited phase 1 of the response to venom and significantly potentiated phase 2. NOLA (30 mg/kg, i.v.) significantly inhibited phase 1 of the response to venom and had no significant effect on phase 2. Propranolol (0.5 mg/kg, i.v.) had no significant effect on phase 1 of the response to venom but significantly potentiated phase 2. Neither phase of the response to venom was significantly affected by atropine (2 mg/kg, i.v.), methysergide (2 mg/kg, i.v.) or prazosin (50 microg/kg, i.v.). These results suggest that soldierfish venom acts indirectly at beta-adrenoceptors to produce a positive inotropic and chronotropic effect in atria, and acts at muscarinic receptors to produce a negative inotropic effect. In addition, beta-adrenoceptors mediate a delayed depressor component in vivo that is absent throughout the initial depressor response to the venom and present during, but masked by, the pressor response. Soldierfish venom also appears to stimulate the release of nitric oxide from endothelial cells to produce relaxation of vascular smooth muscle and contribute to the depressor response produced by the venom in anaesthetized rats. The depressor response also appears to be partially mediated by vasodilator prostanoids.

Enzyme and biochemical studies of stonefish (Synanceja trachynis) and soldierfish (Gymnapistes marmoratus) venoms.

Venoms from the scorpaeniformes Synanceja trachynis and Gymnapistes marmoratus were quantitatively analyzed for enzymic activity. S. trachynis venom displayed significantly higher hyaluronidase activity than G. marmoratus venom, and G. marmoratus venom displayed significantly higher levels of esterase, acid phosphatase, alkaline phosphatase and phosphodiesterase activity. No detectable quantities of phospholipase A2 activity were found in G. marmoratus venom. SDS-polyacrylamide gel electrophoresis of S. trachynis venom indicated the presence of 6 protein bands (20 kDa-295 kDa). G. marmoratus venom displayed 8 protein bands (11 kDa-109 kDa).

Evidence that histamine is the principal pharmacological component of venom from an Australian wolf spider (Lycosa godeffroyi).

Wolf spiders are common throughout Australia and have been known to cause severe reactions in both animals and humans. However, little work has been done on the pharmacological activity of Australian lycosids. The purpose of this study was to obtain a preliminary pharmacological profile of the venom from an Australian wolf spider (Lycosa godeffroyi). The venom caused dose-dependent contractions of guinea-pig isolated ileum (1-4 microg/ml), endothelium-dependent relaxation in rat isolated aortae (10 microg/ml), a decrease in mean arterial blood pressure in the anaesthetised rat (100 microg/kg, i.v.) and an increase in insufflation pressure in the anaesthetised guinea-pig (50 microg/kg, i.v.). All of these responses were significantly inhibited by the H1-receptor antagonist mepyramine at concentrations that selectively inhibited responses to histamine. Venom (5 microg/ml) caused a decrease in twitch height of the rat stimulated (0.3 msec, 0.2 Hz, 100 V) vas deferens (prostatic segment). A fluorometric assay for histamine detected a concentration of 44.5 ng/microg venom protein. It appears that the in vitro and in vivo activity of L. godeffroyi venom observed in the present study is due to the presence of histamine.

Some pharmacological studies of venom from the inland taipan (Oxyuranus microlepidotus).

The present study was designed to obtain a basic pharmacological profile of venom from the inland taipan (Oxyuranus microlepidotus). Venom (0.05-50 micrograms/ml) produced dose-dependent contractions in guinea-pig ileum, which could not be reproduced upon second administration. The cyclooxygenase inhibitor indomethacin (1 microM), a preceding anaphylactic response induced by egg albumin and inactivation of phospholipase A2 (PLA2) by incubation with 4-bromophenacyl bromide (1.8 mM) all significantly inhibited responses to venom (0.5 micrograms/ml). Venom (0.5 micrograms/ml) caused inhibition of stimulation-induced contractions in the prostatic segment of rat vas deferens which was not significantly affected by the alpha 2-adrenoceptor antagonist idazoxan (0.3 microM). Venom (10 micrograms/ml) caused time-dependent inhibition of the rat electrically stimulated phrenic nerve-diaphragm preparation, positive inotropic and chronotropic responses in rat isolated atria and relaxation in rat endothelium-denuded and -intact isolated aortae. In endothelium-intact aortae, the nitric oxide synthase inhibitor N-nitro-L-arginine (NOLA, 0.1 mM) significantly inhibited the response to venom (10 micrograms/ml). Venom (50 micrograms/kg, i.v.) caused an immediate drop in blood pressure followed by cardiovascular collapse in anaesthetised rats. Venom (10 micrograms/kg, i.v.) caused a gradual fall in blood pressure which was sometimes accompanied by a temporary cessation of respiration. A PLA2 assay detected the presence of PLA2 in the venom. These results suggest that the venom contains a component capable of causing the synthesis of arachidonic acid metabolites and a component capable of relaxing vascular smooth muscle. The inhibitory effect on the phrenic nerve-diaphragm is probably due to the previously identified neurotoxin (paradoxin).