Increase In Baseline Tension Research Articles

In Vitro Toxicity of Chinese Russell's Viper (Daboia siamensis) Venom and Neutralisation by Antivenoms.

Daboia siamensis (Russell’s viper) is a highly venomous and medically important snake in China, as well as much of Asia. There is minimal information on the pharmacological activity of the venom of the Chinese species, and currently no commercially available specific antivenom in China. This has led to the use of non-specific antivenoms to treat D. siamensis envenomation. In this study, the in vitro neurotoxicity and myotoxicity of D. siamensis venom was examined and the efficacy of four antivenoms was investigated, including the recently developed Chinese D. siamensis monovalent antivenom (C-DsMAV) and three commercially available antivenoms (Thai D. siamensis (Thai-DsMAV) monovalent antivenom, Deinagkistrodon acutus monovalent antivenom (DaAV), and Gloydius brevicaudus monovalent antivenom (GbAV). D. siamensis venom (10–30 µg/mL) caused the concentration-dependent inhibition of indirect twitches in the chick biventer cervicis nerve muscle preparation, without abolishing contractile responses to exogenous agonists ACh or CCh, indicating pre-synaptic neurotoxicity. Myotoxicity was also evident at these concentrations with inhibition of direct twitches, an increase in baseline tension, and the partial inhibition of ACh, CCh, and KCl responses. The prior addition of C-DsMAV or Thai-DsMAV prevented the neurotoxic and myotoxic activity of D. siamensis venom (10 µg/mL). The addition of non-specific antivenoms (GbAV and DaAV) partially prevented the neurotoxic activity of venom (10 µg/mL) but failed to neutralize the myotoxic effects. We have shown that D. siamensis venom exhibits in vitro weak presynaptic neurotoxicity and myotoxicity, which can be prevented by the pre-addition of the Chinese and Thai Russell’s viper antivenoms. Non-specific antivenoms were poorly efficacious. There should be further development of a monospecific antivenom against D. siamensis envenomation in China.

The mast cell activator Compound 48/80 causes phasic urinary bladder smooth muscle contractions independent of histamine release

Inflammatory mediators released from mast cells cause both transient and phasic contractions of urinary bladder smooth muscle (UBSM). These contractions are viewed as initial disruptors of normal bladder function and can lead to profound bladder pathologies. However, the mechanisms responsible for each of these contractions has not been thoroughly investigated. Previously, we discovered that histamine-mediated contractions rapidly desensitize, whereas the mast cell activator compound 48/80 (10 µg/ml) caused both a long-lasting increase in the amplitude and frequency of phasic contractions in UBSM strips as well as a transient increase in baseline tension. Thus, we hypothesized that phasic contractions caused by compound 48/80 depended on the release of histamine from mast cells. Isometric contractility was performed with urothelium-intact UBSM strips from C57BL/6 mice in the presence or absence of the following drugs: the cholinergic antagonist atropine (2 µM); the 5HT2A receptor antagonist MDL 11939 (1 µM); the histamine H1 receptor antagonist fexofenadine (10 µM), the histamine H2 receptor antagonist roxatidine (25 µM), the H2 antagonist cimetidine (10 µM); or the mast cell stabilizer cromolyn (100 µM). None of the compounds tested significantly reduced the amplitude and duration of the spontaneous phasic contractions caused by compound 48/80. However, both MDL 11939 and cromolyn significantly reduced the baseline contraction. Our data suggest that responses to the mast cell activator compound 48/80 are a superimposition of 2 components: (1) a slowly desensitizing increase in baseline tension caused by release of 5-HT from mast cells, and (2) large-amplitude phasic contractions that are histamine- and 5HT-independent. However, the mechanism responsible for increases in the amplitude of phasic contractions remains unclear.

An Examination of the Neutralization of In Vitro Toxicity of Chinese Cobra (Naja atra) Venom by Different Antivenoms.

The Chinese Cobra (Naja atra) is an elapid snake of major medical importance in southern China. We describe the in vitro neurotoxic, myotoxic, and cytotoxic effects of N. atra venom, as well as examining the efficacy of three Chinese monovalent antivenoms (N. atra antivenom, Gloydius brevicaudus antivenom and Deinagkistrodon acutus antivenom) and an Australian polyvalent snake antivenom. In the chick biventer cervicis nerve-muscle preparation, N. atra venom (1–10 µg/mL) abolished indirect twitches in a concentration-dependent manner, as well as abolishing contractile responses to exogenous acetylcholine chloride (ACh) and carbamylcholine chloride (CCh), indicative of post-synaptic neurotoxicity. Contractile responses to potassium chloride (KCl) were also significantly inhibited by venom indicating myotoxicity. The prior addition of Chinese N. atra antivenom (0.75 U/mL) or Australian polyvalent snake antivenom (3 U/mL), markedly attenuated the neurotoxic actions of venom (3 µg/mL) and prevented the inhibition of contractile responses to ACh, CCh, and KCl. The addition of Chinese antivenom (0.75 U/mL) or Australian polyvalent antivenom (3 U/mL) at the t90 time point after the addition of venom (3 µg/mL), partially reversed the inhibition of twitches and significantly reversed the venom-induced inhibition of responses to ACh and CCh, but had no significant effect on the response to KCl. Venom (30 µg/mL) also abolished direct twitches in the chick biventer cervicis nerve-muscle preparation and caused a significant increase in baseline tension, further indicative of myotoxicity. N. atra antivenom (4 U/mL) prevented the myotoxic effects of venom (30 µg/mL). However, G. brevicaudus antivenom (24 U/mL), D. acutus antivenom (8 U/mL) and Australian polyvalent snake antivenom (33 U/mL) were unable to prevent venom (30 µg/mL) induced myotoxicity. In the L6 rat skeletal muscle myoblast cell line, N. atra venom caused concentration-dependent inhibition of cell viability, with a half maximal inhibitory concentration (IC50) of 2.8 ± 0.48 μg/mL. N. atra antivenom significantly attenuated the cytotoxic effect of the venom, whereas Australian polyvalent snake antivenom was less effective but still attenuated the cytotoxic effects at lower venom concentrations. Neither G. brevicaudus antivenom or D. acutus antivenom were able to prevent the cytotoxicity. This study indicates that Chinese N. atra monovalent antivenom is efficacious against the neurotoxic, myotoxic and cytotoxic effects of N. atra venom but the clinical effectiveness of the antivenom is likely to be diminished, even if given early after envenoming. The use of Chinese viper antivenoms (i.e., G. brevicaudus and D. acutus antivenoms) in cases of envenoming by the Chinese cobra is not supported by the results of the current study.

Alterations in histamine responses between juvenile and adult urinary bladder urothelium, lamina propria and detrusor tissues

Inflammatory mediators may have a role in various lower urinary tract disorders. Histamine is known to induce significant increases in both the tension and frequency of spontaneous phasic contractions in both urothelium with lamina propria (U&LP) and detrusor muscle via the activation of H1 receptor in juvenile animal models. However, it is unclear whether age affects these contractile responses to histamine. This study assessed the histamine receptor subtypes mediating contraction in juvenile and adult porcine bladders and compared the urothelium with lamina propria and detrusor responses to histamine. Isolated tissue bath studies were conducted using strips of porcine U&LP and detrusor obtained from juvenile (6 months) and adult (3 years) animals exposed to histamine receptor agonists and antagonists. Treatment with histamine (100 µM) in U&LP of juvenile animals caused increases in baseline tension by 47.84 ± 6.52 mN/g (p < 0.001, n = 51) and by 50.76 ± 4.10 mN/g (p < 0.001, n = 55) in adult animals. Furthermore, the frequency of spontaneous phasic contractions was significantly enhanced in response to histamine in U&LP of both juvenile and adult tissues (p < 0.001 for both age groups). Treatment with an H2 agonist in U&LP of juvenile animals decreased baseline tension by 13.97 ± 3.45 mN/g (n = 12, p < 0.05), but had no effect in adult animals. Inhibition of H1 receptors resulted in significantly reduced contractile responses of U&LP and detrusor to histamine in both juvenile and adult animals (p < 0.05). Treatment with an H2 receptor antagonist significantly enhanced contractions in juvenile preparations (n = 10, p < 0.05) but had no effect in adult preparations (n = 8). In detrusor, treatment with histamine (100 µM) in juvenile tissues showed a significantly higher increase in baseline tension of 19.10 ± 4.92 mN/g (n = 51) when compared to adult tissues exhibiting increases of 8.21 ± 0.89 mN/g (n = 56, p < 0.05). The increases in the baseline tension were significantly inhibited by the presence of H1 receptor antagonists in both juvenile and adult detrusor preparations. Treatment with either the H2 receptor antagonist or agonist in detrusor had no effect on both juvenile and adult tissues. Therefore, the histamine receptor system may play an essential role in the maintenance of bladder function or in bladder dysfunction observed in some lower urinary tract disorders.

6 weeks consumption of pure fresh coconut milk caused up-regulation of eNOS and CSE protein expression in middle-aged male rats

Coconut milk (CCM) has been an important cooking ingredient in the Asia-Pacific region since ancient time. Due to its high content of saturated fatty acids, it has been considered atherogenic. We have tested if chronic consumption of fresh coconut milk by middle-aged male rat affects vascular function, plasma glucose and lipid profiles. Compared to control, CCM caused lower maximal contraction to phenylephrine of thoracic aortic rings and increased relaxation to acetylcholine that was abolished by N G-nitro-L-arginine (L-NA) or disruption of the endothelium. DL-propargylglycine caused slight increase in baseline tension of L-NA treated aortic rings of CCM-treated rats and produced higher contractile response of the aortic rings to low concentrations of phenylephrine. The aortic eNOS- and cystathionine-γ-lyase(CSE) proteins expression of the CCM-treated rats were also higher than in controls. Except for lower fasting plasma glucose there were no changes in blood chemistry for the CCM treated rats. CCM consumption caused up-regulation of eNOS and CSE protein expression which resulted in increased production of NO and H2S from the blood vessels with attenuation of vasocontraction to phenylephrine and increased relaxation to acetylcholine. These novel benefits may be expected to reduce the development of cardiovascular risk factors in the aging rat.

In vitro toxic effects of puff adder (Bitis arietans) venom, and their neutralization by antivenom.

This study investigated the in vitro toxic effects of Bitis arietans venom and the ability of antivenom produced by the South African Institute of Medical Research (SAIMR) to neutralize these effects. The venom (50 µg/mL) reduced nerve-mediated twitches of the chick biventer muscle to 19% ± 2% of initial magnitude (n = 4) within 2 h. This inhibitory effect of the venom was significantly attenuated by prior incubation of tissues with SAIMR antivenom (0.864 µg/µL; 67% ± 4%; P < 0.05; n = 3–5, unpaired t-test). Addition of antivenom at t50 failed to prevent further inhibition or reverse the inhibition of twitches and responses to agonists. The myotoxic action of the venom (50 µg/mL) was evidenced by a decrease in direct twitches (30% ± 6% of the initial twitch magnitude) and increase in baseline tension (by 0.7 ± 0.3 g within 3 h) of the chick biventer. Antivenom failed to block these effects. Antivenom however prevented the venom induced cytotoxic effects on L6 skeletal muscle cells. Venom induced a marginal but significant reduction in plasma clotting times at concentrations above 7.8 µg/100 µL of plasma, indicating poor procoagulant effects. In addition, the results of western immunoblotting indicate strong immunoreactivity with venom proteins, thus warranting further detailed studies on the neutralization of the effects of individual venom toxins by antivenom.

Differential Myotoxic and Cytotoxic Activities of Pre‐synaptic Neurotoxins from Papuan Taipan (Oxyuranus scutellatus) and Irian Jayan Death Adder (Acanthophis rugosus) Venoms

Pre-synaptic PLA(2) neurotoxins are important components of many Australasian elapid snake venoms. These toxins disrupt neurotransmitter release. Taipoxin, a pre-synaptic neurotoxin isolated from the venom of the coastal taipan (Oxyuranus scutellatus), causes necrosis and muscle degeneration. The present study examined the myotoxic and cytotoxic activities of venoms from the Papuan taipan (O. scutellatus) and Irian Jayan death adder (Acanthophis rugosus), and also tested their pre-synaptic neurotoxins: cannitoxin and P-EPTX-Ar1a. Based on size-exclusion chromatography analysis, cannitoxin represents 16% of O. scutellatus venom, while P-EPTX-Ar1a represents 6% of A. rugosus venom. In the chick biventer cervicis nerve-muscle preparation, A. rugosus venom displayed significantly higher myotoxic activity than O. scutellatus venom as indicated by inhibition of direct twitches, and an increase in baseline tension. Both cannitoxin and P-EPTX-Ar1a displayed marked myotoxic activity. A. rugosus venom (50-300 μg/ml) produced concentration-dependent inhibition of cell proliferation in a rat skeletal muscle cell line (L6), while 300 μg/ml of O. scutellatus venom was required to inhibit cell proliferation, following 24-hr incubation. P-EPTX-Ar1a had greater cytotoxicity than cannitoxin, inhibiting cell proliferation after 24-hr incubation in L6 cells. Lactate dehydrogenase levels were increased after 1-hr incubation with A. rugosus venom (100-250 μg/ml), O. scutellatus venom (200-250 μg/ml) and P-EPTX-Ar1a (1-2 μM), but not cannitoxin (1-2 μM), suggesting venoms/toxin generated cell necrosis. Thus, A. rugosus and O. scutellatus venoms possess different myotoxic and cytotoxic activities. The proportion of pre-synaptic neurotoxin in the venoms and PLA(2) activity of the whole venoms are unlikely to be responsible for these activities.

Evidence for the role of mast cells in colon–bladder cross organ sensitization

This study examined the contribution of mast cells to colon-bladder cross organ sensitization induced by colon irritation with trinitrobenzene sulfonic acid (TNBS-CI). In urethane anesthetized rats 12 days after TNBS-CI, the voiding interval was reduced from 357 s to 201 s and urothelial permeability, measured indirectly by absorption of sodium fluorescein from the bladder lumen, increased six-fold. These effects were blocked by oral administration of ketotifen (10 mg/kg, for 5 days), a mast cell stabilizing agent. TNBS-CI in wild type mice produced a similar decrease in voiding interval (from 319 s to 209 s) and a 10-fold increase in urothelial permeability; however this did not occur in KitªWª/KitªW-vª mast cell deficient mice. Contractile responses of bladder strips elicited by Compound 48/80 (50 μg/ml), a mast cell activating agent, were significantly larger in strips from rats with TNBS-CI (145% increase in baseline tension) than in control rats (55% increase). The contractions of strips from rats with TNBS-CI were reduced 80-90% by pretreatment of strips with ketotifen (20 μM), whereas contractions of strips from control animals were not significantly changed. Bladder strips were pretreated with SLIGRL-NH2 (100 μM) to desensitize PAR-2, the receptor for mast cell tryptase. SLIGRL-NH2 pretreatment reduced by 60-80% the 48/80 induced contractions in strips from rats with TNBS-CI but did not alter the contractions in strips from control rats. These data indicate that bladder mast cells contribute to the bladder dysfunction following colon-bladder cross-sensitization.

Dynamic Skeletal Muscle Exercise and Fatigue Development in Post Infarction Heart Failure Rats and Effect of Warm-Up

Skeletal muscle fatigue develops during physical exercise. Most reports on skeletal muscle fatigue use isometricfatigue protocols with a fall in force production as a measure of fatigue. However, most muscles shorten during normal use and we propose that the fatigue mechanism will be different during dynamic contractions as compared with isometric contractions. Methods: Fatigue was induced in rat soleus muscles from post infarction heart failure (HF) rats and controls (SHAM) by in situ isotonic shortening contractions (1s at 30Hz every 2s for 15min). The muscles were allowed to shorten isotonically against a load (AL) corresponding to 1/3 of maximal isometric force. After 15min recovery period the muscles were stimulated with a similar protocol. MLC2s phosphorylation and metabolites were measured at different points of time. Results: During 1st protocol, fatigue development was confirmed by a significant fall in shortening in both SHAM and HF. After 100s of stimulation soleus from SHAM showed an increase in baseline tension (Tbl) between stimulation trains, which was much less pronounced in HF (p<0.01). At this time there was a significant higher lactate concentration in SHAM muscle compared with HF. The increase in Tbl at 100s was correlated to both isotonic and isometric relaxation rate. At 100s in 2nd protocol, the isotonic and isometric relaxation rates were nearly recovered leading to almost no rise in Tbl in both SHAM and HF. There was a linear correlation (p=0.99) between maximal shortening and MLC2s phosphorylation in protocols with variable AL. We propose that MLC2s phosphorylation participates in the regulation of shortening, while metabolites (especially lactate) may contribute to the reduced relaxation rates leading to increased Tbl.

Effects of carbon monoxide on trout and lamprey vessels

Carbon monoxide (CO) is endogenously produced by heme oxygenase (HO) and is involved in vascular, neural, and inflammatory responses in mammals. However, the biological activities of CO in nonmammalian vertebrates is unknown. To this extent, we used smooth muscle myography to investigate the effects of exogenously applied CO (delivered via a water-soluble CO-releasing molecule, CORM-3) on isolated lamprey (Petromyzon marinus) dorsal aortas and examined its mechanisms of action on trout (Oncorhynchus mykiss) efferent branchial (EBA) and celiacomesenteric (CMA) arteries. CORM-3 dose-dependently relaxed all vessels examined. Trout EBA were twofold more sensitive to CORM-3 when precontracted with norepinephrine (NE) than KCl and CORM-3 relaxed five-fold more of the NE- than KCl-induced tension. Glybenclamide (10 microM), an ATP-sensitive potassium channel inhibitor, inhibited NE-induced contraction, but did not affect CORM-3-induced relaxation. NS-2028 (10 microM), a soluble guanylyl cyclase inhibitor, had no effect on a NE-contraction, but inhibited a subsequent CORM-3-induced relaxation. Zinc protopophyrin-IX (ZnPP-IX, 0.3-30 microM), a HO inhibitor, elicited a small, yet dose-dependent and significant, increase in baseline tension but did not have any effect on subsequent NE-induced contractions or a nitric oxide-induced relaxation (via sodium nitroprusside). [ZnPP-IX] greater than 3 microM, however, significantly reduced the predominant vasodilatory response of trout EBA to hydrogen sulfide. These results implicate an active HO/CO pathway in trout vessels having an impact on resting vessel tone and CO-induced vasoactivity that is at least partially mediated by soluble guanylyl cyclase.

Effects of carbon monoxide on sea lamprey and rainbow trout systemic arteries

Carbon monoxide (CO) is an endogenously produced gas involved in vascular, neural, and inflammatory responses in mammals. CO activity in non‐mammalian vertebrates, however, is unknown. To this extent, we utilized smooth muscle myography to investigate the effects of exogenously applied CO (via CORM‐3) on isolated lamprey (Petromyzon marinus) dorsal aortas and trout (Oncorhynchus mykiss) efferent branchial (EBA) and celiacomesenteric (CMA) arteries. CORM dose‐dependently relaxed norepinephrine‐ (NE) and U46619‐, thromboxane‐A2 mimetic, ‐induced contractions of lamprey dorsal aortas. CORM relaxed both NE (10μM)‐and KCl (80mM)‐ prestimulated trout EBA and CMA dose dependently. EBA were 2‐fold more sensitive to CORM when prestimulated with NE than KCl and CORM relaxed 5‐fold more of the NE‐ than KCl‐induced tension. Glybenclamide (lOμM) inhibited a NE‐induced contraction, but did not effect a CORM induced relaxation. NS‐2028 (10μM), a solubule guanylyl cyclase inhibitor, had no effect on a NE‐contraction, but inhibited a subsequent CORM‐induced relaxation. Zinc protopophyrin‐IX (30μM), a heme‐oxygenase (HO) inhibitor, elicited a significant increase in baseline tension and eliminated the tri‐phasic EBA response to Na2S. These results implicate an active HO/CO pathway in trout vessels on resting vessel tonus and H2S‐induced vasoactivity that is partially mediated by soluble guanylyl cyclase.

Isolation and characterisation of acanmyotoxin-2 and acanmyotoxin-3, myotoxins from the venom of the death adder Acanthophis sp. Seram

Death adder (genus Acanthophis) venoms display neurotoxic activity but were thought to be devoid of myotoxic components. Studies from our laboratory have shown that some species (i.e. Acanthophis rugosus and Acanthophis sp. Seram) posses venom with myotoxic activity [Wickramaratna JC, Fry BG, Aguilar M, Kini RM, Hodgson WC. Isolation and pharmacological characterisation of a phospholipase A 2 myotoxin from the venom of the Irian Jayan death adder ( A. rugosus). Br J Pharmacol 2003;138:333–342; Wickramaratna JC, Fry BG, Hodgson WC. Species-dependent variations in the in vitro myotoxicity of death adder ( Acanthophis) venoms. Toxicol Sci 2003;74:352–360]. The present study describes the isolation and characterisation of two myotoxins (acanmyotoxin-2 and acanmyotoxin-3) from A. sp. Seram venom. Venom was fractionated into approximately 12 major peaks using reverse phase high performance liquid chromatography. Two components caused concentration (0.1–1 μM) dependent inhibition of direct (2 ms, 0.1 Hz, supramaximal V) twitches and an increase in baseline tension in the chick biventer cervicis nerve-muscle. Histological examination of the muscle confirmed damage. PLA 2 activity was detected in both acanmyotoxin-2 (390.2 ± 19.7 μmol/(min mg); n = 4) and acanmyotoxin-3 (14.2 ± 7.7 μmol/(min mg); n = 4). In comparison, A. sp. Seram whole venom had a specific activity of 461.3 ± 90.4 μmol/(min mg) ( n = 3). Mass spectrometry analysis indicated acanmyotoxin-2 had a mass of 13,082 Da and acanmyotoxin-2 13,896 Da. Acanmyotoxin-2 and acanmyotoxin-3 accounted for approximately 7 and 4% of total venom composition, respectively. N-terminal sequencing of the first 30 amino acids of each toxin indicated they shared some sequence homology with known myotoxins. In conclusion, clinicians should be aware that symptoms of envenoming by some species of death adder may include signs of myotoxicity as well as neurotoxicity. Future studies will investigate the efficacy of the current antivenom treatment against the myotoxic components of A. sp. Seram venom.

Isolation and pharmacological characterization of a phospholipase A2 myotoxin from the venom of the Irian Jayan death adder (Acanthophis rugosus).

1. It has long been thought that death adder venoms are devoid of myotoxic activity based on studies done on Acanthophis antarcticus (Common death adder) venom. However, a recent clinical study reported rhabdomyolysis in patients following death adder envenomations, in Papua New Guinea, by a species thought to be different to A. antarcticus. Consequently, the present study examined A. rugosus (Irian Jayan death adder) venom for myotoxicity, and isolated the first myotoxin (acanmyotoxin-1) from a death adder venom. 2. A. rugosus (10-50 micro g ml(-1)) and acanmyotoxin-1 (MW 13811; 0.1-1 micro M) were screened for myotoxicity using the chick directly (0.1 Hz, 2 ms, supramaximal V) stimulated biventer cervicis nerve-muscle (CBCNM) preparation. A significant contracture of skeletal muscle and/or inhibition of direct twitches were considered signs of myotoxicity. This was confirmed by histological examination. 3. High phospholipase A(2) (PLA(2)) activity was detected in both A. rugosus venom (140.2+/-10.4 micro mol min(-1) mg(-1); n=6) and acanmyotoxin-1 (153.4+/-11 micro mol min(-1) mg(-1); n=6). Both A. rugosus venom (10-50 micro g ml(-1)) and acanmyotoxin-1 (0.1-1 micro M) caused dose-dependent inhibition of direct twitches and increase in baseline tension (n=4-6). In addition, dose-dependent morphological changes in skeletal muscle were observed. 4. Prior incubation (10 min) of CSL death adder antivenom (5 units ml(-1); n=4) or inactivation of PLA(2) activity with 4-bromophenacyl bromide (1.8 mM; n=4) prevented the myotoxicity caused by acanmyotoxin-1 (1 micro M). 5. Acanmyotoxin-1 (0.1 micro M; n=4) displayed no significant neurotoxicity when it was examined using the indirectly (0.1 Hz, 0.2 ms, supramaximal V) stimulated CBCNM preparation. 6. In conclusion, clinicians may need to be mindful of possible myotoxicity following death adder envenomation in Irian Jaya.

Xenon does not induce contracture in human malignant hyperthermia muscle

Xenon has many characteristics of an ideal anaesthetic agent. It is not known whether xenon is a safe alternative to the potent inhalational anaesthetics in patients susceptible to malignant hyperthermia (MH). We investigated the effect of xenon, halothane and caffeine on muscle specimens of 31 individuals, referred to the MH Unit of the University of Ulm, and performed genetic epidemiology. Thirteen individuals were classified as MH susceptible and 18 as MH negative. Xenon 70% did not cause an increase in baseline tension of any MH-susceptible muscle specimen in contrast to halothane and caffeine. The evoked twitch response increased transiently in MH-susceptible and normal specimens indicating a mechanism independent of MH susceptibility. These results suggest that xenon, in concentrations up to 70% may be a safe anaesthetic for MH-susceptible patients.

Neural modulation of bovine mesenteric lymph node contraction.

Spontaneous isometric contractions and field-evoked responses were recorded in vitro from strips of bovine mesenteric lymph node capsule. Field stimulation (0.3 ms pulses, 60 V nominal, 2 min trains) at frequencies > or = 2 Hz evoked a frequency-dependent increase in baseline tension which was maximum at frequencies > or = 16 Hz. Evoked contractions were significantly reduced (P = 0.02) by the neurotoxin tetrodotoxin (10(-6) M) although they were unaffected by the alpha-adrenoceptor antagonists phentolamine, rauwolscine and prazosin (3 x 10(-6) M). Similarly, responses were unaffected by the beta-adrenoceptor antagonist propranolol (10(-6) M), the cholinergic antagonist atropine (10(-6) M) or the uptake blocker cocaine (10(-6) M). Field-evoked contraction was also unaffected by a 30 min exposure to alpha,beta-methylene ATP (10(-6) M). The results suggest that bovine lymph node capsular smooth muscle is innervated by excitatory nerves which are non-adrenergic, non-cholinergic and non-purinergic in nature.

The effects of field stimulation on bovine mesenteric lymph node contractility

In these studies isometric tension was recorded from isolated strips of bovine mesenteric lymph node capsule. The possible influence of intrinsic nerves on their contractile activity was examined using field stimulation and the pharmacology of the response was investigated. Over 75% of the tissues studied demonstrated regular spontaneous activity at a frequency of 3.9 +/- 0.2 contractions/5 min under control conditions. Field stimulation at 2.8 and 32 Hz (pulse width = 0.3 ms, nominal voltage 60 V) produced frequency-dependent increases in the rate of this spontaneous activity associated with a raised baseline tension. These responses were not diminished on repeat stimulation under control conditions. The contraction frequency response to stimulation at 8 Hz was blocked completely in the presence of 1 microM tetrodotoxin, while the increase in baseline tension was reduced by over 80%, suggesting that field stimulation was activating intrinsic nerves. The responses to stimulation (8 Hz) were also greatly reduced by 10 microM phentolamine but were unaffected by 10 microM propranolol. These experiments suggest that intrinsic nerves can modulate the contractile activity of lymph node capsule acting, at least in part, through excitatory alpha-adrenoceptors. The possible functional significance of this is discussed.

Muscarinic receptor interaction with full and partial beta-adrenoceptor agonists in the rat colon strip.

beta-Adrenoceptor agonists inhibit contractile activity in isolated colon strips. In order to demonstrate that beta-adrenoceptors are located at different functional levels within the colon wall, increasing concentrations of muscarinic agonists were used to interact functionally with the beta-adrenoceptor-mediated inhibition of spontaneous colon activity. The effects of the full agonists isoprenaline and terbutaline and of the partial agonist prenalterol were functionally antagonized by carbachol (0.03 and 0.3 mumol/l) and bethanechol (1.3 and 30 mumol/l). This functional antagonism was parallelled by an increase in baseline tension and spontaneous contractile activity of the isolated colon strip. At lower concentrations of carbachol (0.003 and 0.01 mumol/l) or bethanechol (0.03 and 0.3 mumol/l) no effect on the contractile status of the smooth muscle or on the pD2-values of the full agonists was seen. However, at these lower concentrations of muscarinic agonists a marked decrease in the maximal inhibitory response to the partial beta-adrenoceptor agonist prenalterol was demonstrated. The inhibitory response to prenalterol showed a biphasic concentration-response curve. The muscarinic antagonist atropine produced an increase in the maximal response of the high potency component of the concentration-response curve for prenalterol and an increase in the sensitivity to isoprenaline. These results demonstrate the presence of a high cholinergic tone in the colon preparation of a magnitude that clearly reduces the sensitivity to beta-adrenoceptor agonists. The different responses to full and partial beta-adrenoceptor agonists in the presence of increasing concentrations of muscarinic agonists may indicate that beta-adrenoceptors are located on two different functional units within the colon wall.

Electrophysiological studies of some semisynthetic cardiac glycoside derivatives in isolated papillary muscle of the guinea-pig.

The effects of digitoxin, 3 alpha-methyl-digitoxigenin-3 beta-monoglucoside (3 alpha-MDM), 3 alpha-methyl-digitoxigenin (3 alpha-MD), proscillaridin, 4, 5-methylene-procillaridin (4, 5-MP), and 3 beta-hydroxy-4, 5-methylene-A, B-trans-scillarenin (3 beta-HMTS) on force of contraction and on the transmembrane action potentials were examined in isolated papillary muscles of guinea-pigs. All derivatives exhibited the typical cardiac glycoside effects: i.e. they increased the force of contraction and shortened the action potential duration at 20% (plateau phase) and 90% of repolarization. With digitoxin, 3 beta-HMTS and 4, 5-MP a transient prolongation in action potential duration was observed at the lower concentrations. The action potential amplitude and the resting membrane potential were reduced consistently only with the higher concentrations used. The onset of the positive inotropic effects of 3 alpha-MDM, 3 alpha-MD and 3 beta-HMTS was more rapid than that of digitoxin and proscillaridin. The increment in contractile force reached a maximum well before the full shortening effect on the action potential duration had developed. The shortening of the action potential is thought to be responsible for the biphasic nature of the positive inotropic effect. With 3 alpha-MD and 3 alpha-MDM even toxic effects, e.g. increase in baseline tension, were completely reversible after washing in drug-free solution. The dose-response curves for the positive inotropism can only be compared reliably once the equilibrium of drug action has been established. This steady state is probably reflected by the development of the full shortening in action potential duration.

Alteration by vanadate of contractility in vascular and intestinal smooth muscle preparations.

Intestinal muscle was more sensitive than vascular muscle to vanadate. Vanadate caused a biphasic response in intestinal muscle; inhibition of spontaneous contractile activity was followed by a return of phasic contractions and a sustained increase in baseline tension. Then inhibitory response appeared to be due to vanadate-induced release of an inhibitory transmitter from intramural nerve endings. The excitatory response appeared to be due to the action of vanadate at an intracellular site, possibly by inhibition of a Ca-ATPase that controls intracellular Ca2+ levels. Vanadate did not alter intracellular Na+ and K+ levels in isolated longitudinal muscle. Thus, inhibition of intestinal muscle NaK-ATPase cannot account for the alterations in contractility.