Exogenous Acetylcholine Research Articles

Cholinergic properties of soy

Objectives Soybeans have been shown to have numerous health benefits, but the underlying mechanisms are poorly understood. The aim of this study was to characterize some pharmacologic properties of active substances in aqueous soy extract. Methods The pharmacologic actions of the extract were tested by measuring mechanical activity of isolated guinea-pig ileum in an organ bath. Results The ileum contracted in response to soy extract in a concentration-dependent manner. This response was unaffected by the nerve blocker tetrodotoxin (10 −6 M) but was completely inhibited by atropine (10 −9 M), indicating an action via muscarinic receptors on the muscle. In the presence of the M 3 muscarinic antagonist 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide and to a lesser extent in the presence of the M 2 muscarinic antagonist 11-([2-[(diethylamino)methyl]-1-piperidinyl]acetyl)-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one, the response was decreased. When acetylcholine (ACh) esterase inhibitors were added to the medium before the addition of soy extract, the response to the extract was potentiated. Preincubation of the extract with exogenous ACh esterase reduced its activity. The response to choline, ACh, and phosphorylcholine was also tested, and none of these substances accurately replicated the response to soy extract. However, some qualitative similarities were observed between the effect of choline and ACh to that of the extract. Conclusion These results indicate the presence of an ACh-like substance in soy. Due to the abundance and importance of muscarinic receptors, the presence of a cholinergic substance in soy could have numerous implications. The role of this substance in the beneficial effect of soy on various body systems merits further investigation.

Abnormal modulation of cholinergic neurotransmission by endomorphin 1 and endomorphin 2 in isolated bronchus of type 1 diabetic rats

To assess whether diabetes alters the regulatory effects of μ-opioid receptor (MOR) agonists on the cholinergic bronchoconstriction, we investigated the inhibitory effects of endomorphins (EMs) on the electrical field stimulation (EFS)-induced cholinergic bronchoconstriction in type 1 diabetic rats. At 4 weeks after the onset of diabetes, both the EFS- and exogenous acetylcholine (ACh)-induced bronchoconstriction in diabetes in vitro were greater than those in non-diabetes rats. Furthermore, endomorphin 1 (EM1) and endomorphin 2 (EM2) inhibited the response to EFS in diabetic rat isolated bronchus in a concentration- and frequency-dependent manner, which is in agreement with that in non-diabetes. However, the inhibitory effects of EMs on the EFS-induced bronchoconstriction in diabetes were significantly weaker than those in non-diabetes. Both EM1 and EM2 (1 μM) had no effect on the contractile response to exogenous ACh, indicating a prejunctional effect. Furthermore, the inhibitory effect on the EFS-induced bronchoconstriction was blocked by naloxone (10 μM). Eight weeks after the induction of diabetes, both the EFS- and exogenous ACh-induced bronchoconstrictions in diabetes were further enhanced compared to those in short-time (4 weeks) diabetic rats. Moreover, the inhibitory effects of EMs on the EFS-induced bronchoconstriction were further attenuated. These results suggest that dysfunction of presynaptic inhibitory modulation through opioid receptor by EMs may take place in the bronchus of diabetic rats.

Effect of Calcium on Spontaneous Quantal Transmitter Secretion from ACh-Loaded Myocytes

Spontaneous secretions occur in both neurons and non-neuronal cells, and calcium is important for these secretion processes. However, the detailed roles of calcium on the secretions have not yet been identified. In the present study, cultured Xenopus myocytes loaded with exogenous acetylcholine (ACh) into the cytoplasm in the absence of extracellular Ca 2+ undergo spontaneous quantal ACh secretion as detected by the appearance of pulsatile miniature endplate currents. Analysis of the frequencies, amplitudes, and time courses of these currents suggests that similar cellular mechanisms are involved in the secretions of ACh in normal medium and Ca 2+-free solution. Various doses of ryanodine were used to regulate the intracellular Ca 2+ to different levels. The spontaneous ACh secretion from myocytes in Ca 2+-free medium was decreased by reducing intracellular Ca 2+ levels and enhanced by increasing cytosolic Ca 2+ levels. These observations demonstrate that the spontaneous secretion from isolated myocytes and the effect of ryanodine on ACh-loaded cells are both independent of extracellular Ca 2+ while Ca 2+ in the sarcoplasmic reticulum plays a crucial role in the secretions.

Isolation and pharmacological characterisation of hostoxin-1, a postsynaptic neurotoxin from the venom of the Stephen's banded snake ( Hoplocephalus stephensi)

Envenoming by the Stephen's banded snake ( Hoplocephalus stephensi) is not usually characterised by neurotoxicity. The present study describes the pharmacological characterisation of hostoxin-1 (MW 6660 Da), the first neurotoxin to be isolated from the venom of the Stephen's banded snake. Hostoxin-1 (0.3–1.0 μM) caused concentration-dependent inhibition of indirect twitches of the chick biventer cervicis nerve–muscle preparation. The neurotoxic activity of hostoxin-1 (0.3 μM) was irreversible by washing, but significantly reversed by the addition of CSL tiger snake antivenom (5 units/ml) added at t 90 (i.e. time at which twitches were inhibited by 90%). In addition, hostoxin-1 (0.3 μM) inhibited responses to exogenous acetylcholine and carbachol, but not KCl, indicating a postsynaptic mode of action. Hostoxin-1 (5–30 nM) displayed pseudo-irreversible antagonism at the skeletal muscle nicotinic receptor with a pA 2 value of 8.45 ± 0.32 (i.e. approximately 100-fold more potent than tubocurarine). H. stephensi venom displayed a high level of PLA 2 activity (specific activity 100.1 ± 4.4 μmol/min/mg). However, the activity of hostoxin-1 was negligible. Partial N-terminal sequencing of hostoxin-1 indicates that it has high sequence homology with other elapid short-chain neurotoxins.

Multiple Motor Effects of ATP and their Inhibition by P2 Purinoceptor Antagonist, Pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic Acid in the Small Intestine of the Guinea‐Pig

Adenosine 5'-triphosphate (ATP) may be an important neurotransmitter in the gastrointestinal tract. The present study examined the motor effects of exogenous ATP on longitudinally-oriented preparations of the guinea-pig isolated ileum and the influence of drugs on the ATP-induced responses. High micromolar concentrations of ATP caused two types of contraction, a phasic, cholinergic response and a tonic, tetrodotoxin-resistant contraction. The phasic contraction was reduced by hexamethonium (5x10(-5) M), but left uninfluenced by capsaicin tachyphylaxis or tachyphylaxis to alpha,beta-methylene ATP. The tonic response was resistant to atropine, hexamethonium, capsaicin, omega-conotoxin GVIA, or pretreatment with alpha,beta-methylene ATP. Both types of ATP-induced contraction were diminished or abolished by the P2 purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 3x10(-6) and 3x10(-5) M, respectively). In the precontracted, atropine-treated ileum ATP (10(-6)-10(-4) M) caused guanethidine-resistant relaxation. This response was not influenced by tetrodotoxin, omega-conotoxin GVIA, or NG-nitro-L-arginine, but was abolished by apamin (10(-7) M), and inhibited by PPADS (3x10(-5) M) or reactive blue 2 (10(-5) M), in a surmountable manner. A high degree of tachyphylaxis was observed with the relaxant effect of ATP (10(-5)-10(-4) M). A high concentration (3x10(-4) M) of PPADS failed to influence ileum contractions to exogenous acetylcholine or histamine. It is concluded that, in addition to its direct contractile action in the guinea-pig ileum, ATP can activate (partly preganglionic) cholinergic neurones, an effect whose mechanism is largely different from that of alpha,beta-methylene ATP. ATP also causes relaxation by a direct, probably P2Y-receptor-mediated effect on the smooth muscle. All motor effects of ATP are inhibited by the antagonist PPADS.

Altered neurogenic and mechanical responses to acetylcholine, ATP and substance P in detrusor from rat with outlet obstruction

The well-known side effects of anticholinergic compounds used to treat urinary incontinence caused by detrusor overactivity have addressed the interest on other pharmacological intervention. The purpose of the present work was to investigate the possible changes in purinergic and cholinergic components of parasympathetic neurotransmission in obstructed rat bladders with detrusor overactivity, and to examine the effect of the association of suramin, atropine and indomethacin on nerve-mediated responses to electrical field stimulation (EFS). Mechanical responses to exogenous acetylcholine, ATP and substance P were also evaluated. Altered sensitivities to acetylcholine and to the sensory neurotransmitter substance P, but unchanged sensitivity to the stable ATP analogue α,β-methyleneATP were observed in bladders from obstructed rats. Suramin and atropine inhibited purinergic and cholinergic components of the neurogenic responses evoked by EFS in detrusor strips from control and obstructed rats. Interestingly, suramin enhanced the antagonistic effect of atropine on neurogenic responses of detrusor strips at all frequencies of stimulation tested. Our results suggest that the association between an antimuscarinic drug and an antagonist of P2X purinoceptors such as suramin might be helpful to reduce the therapeutic dosage of the antimuscarinic drug, along with its side effects. This approach may be of interest in the therapy of patients with bladder incontinence caused by detrusor overactivity, which do not even respond to a maximal dosage of antimuscarinic drug.

Endomorphin1 and endomorphin2, endogenous potent inhibitors of electrical field stimulation (EFS)-induced cholinergic contractions of rat isolated bronchus

In the present study, we determined whether endomorphin1 (EM1) and endomorphin2 (EM2), selective endogenous μ-opioid receptor (MOR) agonists, inhibited the response to EFS in rat isolated bronchus in a concentration- and frequency-dependent manner. EM1 (1 μM) produced significant inhibition at relatively low frequencies (<5 Hz) (74.02 ± 5.53%, 56.16 ± 10.24% and 37.64 ± 5.92% inhibition at 1, 2 and 4 Hz, respectively, p < 0.05 versus control), but no significant inhibition at 8, 16, 32 and 64 Hz (17.15 ± 9.4%, 14.51 ± 4.23%, 9.11 ± 2.38% and 5.93 ± 3.5%, respectively, p > 0.05 versus control). Similar modulations were observed in response to EM2 (1 μM). It is therefore considered that the inhibition effects of EM1 and EM2 may take place at frequencies under physiological conditions. Furthermore, EM1 and EM2 (0.01–10 μM) induced inhibition of cholinergic constriction in a dose-dependent manner at 1, 2 and 4 Hz. The inhibitory effect on EFS was blocked by the opioid receptor antagonist naloxone (10 μM), indicating that opioid receptors were involved. Neither EM1 nor EM2 (1 μM) had an effect on the contractile response to exogenous acetylcholine, indicating a prejunctional effect. All the results indicate that EM1 and EM2 are potent inhibitors of EFS-induced cholinergic bronchoconstriction. These also imply that EM1 and EM2 may modulate cholinergic bronchoconstriction under physiological conditions and that these tetrapeptides could have therapeutic potential in the treatment of airway diseases.

The hallucinogenic herb Salvia divinorum and its active ingredient salvinorin A inhibit enteric cholinergic transmission in the guinea‐pig ileum

Salvia divinorum is a widespread hallucinogenic herb traditionally employed for divination, as well as a medicament for several disorders including disturbances of gastrointestinal motility. In the present study we evaluated the effect of a standardized extract from the leaves of S. divinorum (SDE) on enteric cholinergic transmission in the guinea-pig ileum. SDE reduced electrically evoked contractions without modifying the contractions elicited by exogenous acetylcholine, thus suggesting a prejunctional site of action. The inhibitory effect of SDE on twitch response was abolished by the opioid receptor antagonist naloxone and by the kappa-opioid antagonist nor-binaltorphimine, but not by naltrindole (a delta-opioid receptor antagonist), CTOP (a mu-opioid receptor antagonist), thioperamide (a H(3) receptor antagonist), yohimbine (an alpha(2)-receptor antagonist), methysergide (a 5-hydroxytryptamine receptor antagonist), N(G)-nitro-L-arginine methyl ester (an inhibitor of NO synthase) or apamin (a blocker of Ca(2+)-activated K(+) channels). Salvinorin A, the main active ingredient of S. divinorum, inhibited in a nor-binaltorphimine- and naloxone-sensitive manner electrically induced contractions. It is concluded that SDE depressed enteric cholinergic transmission likely through activation of kappa-opioid receptors and this may provide the pharmacological basis underlying its traditional antidiarrhoeal use. Salvinorin A might be the chemical ingredient responsible for this activity.

Neurotoxic and myotoxic actions from Lachesis muta muta (surucucu) whole venom on the mouse and chick nerve–muscle preparations

Lachesis genus is one of the less studied among others from Viperidae's genera, mainly due to difficulties in obtaining the venom. Accidents by Lachesis snakes cause severe envenoming syndrome, eventually leading victims to shock. This work is part of a comprehensive study aimed at studying the venom and its effects. Herein the neurotoxicity and myotoxicity of L. muta muta venom were investigated on mouse phrenic nerve-diaphragm (PNDp) and chick biventer cervicis (BCp) preparations. For both preparations the time required to venom produces 50% neuromuscular blockade was indirectly concentration-dependent, being for PNDp: 117.6±6.5 min (20 μg/ml), 70.1±8.6 min (50 μg/ml) and 43.6±3.8 min (100 μg/ml), and for BCp: 28±1.8 min (50 μg/ml), 30.4±2.3 min (10 μg/ml), 50.4±4.3 min (5 μg/ml) and 75.2±0.7 min (2 μg/ml), ( n=5/dose). In BCp, a venom dose of 50 μg/ml significantly reduced contractures elicited by exogenous acetylcholine (55 μM) and KCl (20 mM), as well as increased the release of creatine kinase (442.7±39.8 IU/l in controls vs 4322.6±395.2 IU/l, after 120 min of venom incubation ( P<0.05). Quantification of myonecrosis in BCp indicated the doses 50 and 10 μg/ml as significantly myotoxic affecting 59.7±6.2%, and 20.8±1.2% of fibers, respectively, whereas 5 and 2 μg/ml that affected 13.5±0.8% and 5.4±0.6% of fibers, were considered weakly- and non-myotoxic, respectively. We concluded that there are neurotoxins present in the venom, the concentration of which governs its pre- (if low) or postsynaptic (if high) activity. Since myotoxicity in the avian preparation is negligible at lower venom doses, but not neurotoxicity, we suggest that this effect may contribute minimum to the venom neurotoxic effect. The BCp is more sensible than PNDp to Lachesis m. muta venom.

Abstract no.: 5 Endothelium‐dependent relaxation and impact of a Western type diet in the aorta of apolipoprotein E‐deficient mice

Previously we reported that even in old (18 months) apolipoprotein E‐deficient mice (apoE‐/‐) on regular chow (RC), all nitric oxide (NO)‐mediated responses are preserved in plaque‐free vessels. Only when plaques develop, localised vasomotor dysfunction occurs. The impact of increased oxidative stress on relaxations was evaluated by feeding apoE‐/‐ mice either a Western type diet (WD; 0.15% cholesterol, 41% fat) or RC (no cholesterol, 4% fat) for 8, 16 and 24 weeks. The aorta was systematically sectioned and segments of the aortic root (1 mm) and the thoracic aorta (5 × 2 mm, covering whole vessel, varying degrees of atherosclerosis) were studied in organ baths followed by morphometry. The WD enhanced plasma levels of total cholesterol (1382 ± 109 vs. 425 ± 21 mg/dl) and soluble ICAM‐1 (μg/ml; 8 weeks 43 ± 3 vs. 28 ± 1; 16 weeks 37 ± 2 vs. 25 ± 1; 24 weeks 41 ± 4 vs. 29 ± 1). In RC mice, relaxations to exogenous NO and acetylcholine (ACh) were preserved in all segments up to 24 weeks. When fed the WD however, maximum relaxation (Emax) to exogenous NO (acidified NaNO2) was impaired in the aortic root at 8 (83 ± 3 vs. 92 ± 1%), 16 (71 ± 4 vs. 87 ± 3%) and 24 weeks (59 ± 7 vs. 83 ± 1%); shifts in the pD2 values pointed to decreased sensitivity. ACh‐relaxation was unaltered at 8 weeks, but deteriorated in the aortic root at 16 (74 ± 6 vs. 99 ± 1%) and 24 weeks (51 ± 6% vs. 95 ± 3%). Similar changes occurred in the most distal thoracic aorta segment of WD mice. However, in all those segments plaques were detectable, and average lesion size was increased 6‐fold by the WD. Furthermore, responses to ACh (Emax rs = −0.54; pD2 rs = −0.36, P &lt; 0.001) and exogenous NO (Emax rs = −0.59; pD2 rs = −0.30, P &lt; 0.01) inversely correlated to plaque size in WD mice, as previously reported for RC apoE‐/‐ mice. The dysfunction was apparent only after plaque development. Indeed, segments of the central thoracic aorta were largely free of atherosclerosis and showed unaltered relaxation, even after the onset of atherogenesis at 24 weeks WD. In conclusion, the elevation of soluble ICAM‐1 points to enhanced oxidative stress in WD apoE‐/‐ mice. Yet, endothelium‐dependent relaxation is still maintained until plaques develop. As opposed to the human situation, endothelial dysfunction in hypercholesterolemic apoE‐/‐ mice is not a systemic but a local process, strictly associated with lesion formation. Finally, the preservation of vasomotor responses in plaque‐free segments, and the correlation between lesion size and dysfunction, further emphasise that it is crucial to document plaque dimensions in vasomotor studies in atherosclerosis research.

Acetylcholine-induced vasodilation is mediated by nitric oxide and prostaglandins in human skin

Acetylcholine (ACh) can effect vasodilation by several mechanisms, including activation of endothelial nitric oxide (NO) synthase and prostaglandin (PG) production. In human skin, exogenous ACh increases both skin blood flow (SkBF) and bioavailable NO levels, but the relative increase is much greater in SkBF than NO. This led us to speculate ACh may dilate cutaneous blood vessels through PGs, as well as NO. To test this hypothesis, we performed a study in 11 healthy people. We measured SkBF by laser-Doppler flowmetry (LDF) at four skin sites instrumented for intradermal microdialysis. One site was treated with ketorolac (Keto), a nonselective cyclooxygenase antagonist. A second site was treated with NG-nitro-L-arginine methyl ester (L-NAME) to inhibit NO synthase. A third site was treated with a combination of Keto and L-NAME. The fourth site was an untreated control site. After the three treated sites received the different inhibiting agents, ACh was administered to all four sites by intradermal microdialysis. Finally, sodium nitroprusside (SNP) was administered to all four sites. Mean arterial pressure (MAP) was monitored by Finapres, and cutaneous vascular conductance (CVC) was calculated (CVC = LDF/MAP). For data analysis, CVC values for each site were normalized to their respective maxima as effected by SNP. The results showed that both Keto and L-NAME each attenuated the vasodilation induced by exogenous ACh (ACh control = 79 +/- 4% maximal CVC, Keto = 55 +/- 7% maximal CVC, L-NAME = 46 +/- 6% maximal CVC; P < 0.05, ACh vs. Keto or L-NAME). The combination of the two agents produced an even greater attenuation of ACh-induced vasodilation (31 +/- 5% maximal CVC; P < 0.05 vs. all other sites). We conclude that a portion of the vasodilation effected by exogenous ACh in skin is due to NO; however, a significant portion is also mediated by PGs.

Pharmacological characterisation of a neurotoxin from the venom of Boiga dendrophila (Mangrove catsnake)

In this study, we have pharmacologically characterised boigatoxin-A, a three finger toxin isolated from the venom of the colubrid, Boiga dendrophila (Mangrove catsnake). In the chick biventer cervicis nerve-muscle preparation boigatoxin-A (1 μM) displayed poorly reversible postsynaptic blockade as evidenced by the inhibition of indirect (0.1 Hz, 0.2 ms, supramaximal V) twitches and responses to exogenous acetylcholine (1 mM) and carbachol (20 μM). Boigatoxin-A (0.3–0.5 μM) caused a concentration-dependent depression of the maximum response of cumulative concentration response curves to CCh (0.6–80 μM). Boigatoxin-A (1 μM) induced readily reversible inhibition of electrically evoked (0.2 Hz, 0.3 ms, 70–100 V) twitches of the prostatic segment of the rat vas deferens. This inhibition was not significantly attenuated by 8-phenyltheophylline (20 μM) or idazoxan (1 μM). Boigatoxin-A (1 μM) did not affect α,β-mATP (10 μM) or noradrenaline (25 μM) responses in unstimulated epididymal segments of the rat vas deferens. Our data suggests that this toxin has weak postsynaptic neurotoxicity in skeletal muscle and also prejunctional neurotoxic activity in the smooth muscle of the rat vas deferens to inhibit the release of neurotransmitter(s), but not via prejunctional purinergic or adrenergic receptors. This is the first report of such activity for a toxin isolated from snake venom and reinforces the largely untapped potential of colubrid venoms.

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 μg/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 μM) 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 μg/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 μM) 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 PLA 2 component in the toxic action of the venom.

Neurotoxic effects of venoms from seven species of australasian black snakes (Pseudechis): Efficacy of black and tiger snake antivenoms

1. Pseudechis species (black snakes) are among the most widespread venomous snakes in Australia. Despite this, very little is known about the potency of their venoms or the efficacy of the antivenoms used to treat systemic envenomation by these snakes. The present study investigated the in vitro neurotoxicity of venoms from seven Australasian Pseudechis species and determined the efficacy of black and tiger snake antivenoms against this activity. 2. All venoms (10 microg/mL) significantly inhibited indirect twitches of the chick biventer cervicis nerve-muscle preparation and responses to exogenous acetylcholine (ACh; 1 mmol/L), but not to KCl (40 mmol/L), indicating activity at post-synaptic nicotinic receptors on the skeletal muscle. 3. Prior administration of either black or tiger snake antivenom (5 U/mL) prevented the inhibitory effects of all Pseudechis venoms. 4. Black snake antivenom (5 U/mL) added at t90 (i.e. the time-point at which the original twitch height was reduced by 90%) significantly reversed the effects of P. butleri (28+/-5%), P. guttatus (25+/-8%) and P. porphyriacus (28+/-10%) venoms. Tiger snake antivenom (5 U/mL) added at the t90 time-point significantly reversed the neurotoxic effects of P. guttatus (51+/-4%), P. papuanus (47+/-5%) and P. porphyriacus (20+/-7%) venoms. 5. We show, for the first time, the presence of neurotoxins in the venom of these related snake species and that this activity is differentially affected by either black snake or tiger snake antivenoms.

A biochemical and pharmacological examination of Rhamphiophis oxyrhynchus (Rufous beaked snake) venom

The venom of R. oxyrhynchus, a member of the psammophiine subfamily of the colubrid assemblage, was examined for biological activity using biochemical and pharmacological techniques. Venom displayed a high protein content, a complex electrophorectic profile and PLA 2 activity but no detectable proteolytic or haematological activities. In the chick biventer cervicis nerve muscle preparation, venom (1–10 μg/ml) displayed postsynaptic neurotoxic activity as evidenced by inhibition of indirect (0.1 Hz, 0.2 ms, supramaximal V) twitches and responses to exogenous acetylcholine (1 mM) and carbachol (20 μM). This inhibitory effect was poorly reversible by washing. Venom (30–50 μg/ml) caused a rapid and readily reversible inhibition of direct (0.1 Hz, 2 ms, supramaximal V) twitches of the chick biventer cervicis nerve muscle preparation without morphological changes to the muscle fibers. Venom (30–100 μg/ml) inhibited electrically-evoked (0.2 Hz, 0.3 ms, 70–100 V) twitches of the prostatic segment of the rat vas deferens. This inhibitory effect was not significantly attenuated by 8-phenyltheophylline (8-PT; 20 μM), idazoxan (1 μM), a combination of ranitidine (0.2 μM) and thioperamide (10 μM) or capsazepine (10 μM). Venom (5 mg/kg) induced hypotension with subsequent cardiovascular collapse in the anaesthetised rat. The cardiovascular collapse was prevented by artificial respiration of the animals prior to venom administration. The biological activities demonstrated by R. oxyrhynchus venom may aid in prey envenomation strategies such as prey immobilisation. This study provides further evidence that colubrid venoms are comprised of multiple components which can display a variety of actions, some of which may be novel, therefore reinforcing the largely untapped potential of colubrid venoms.

A “Synaptoplasmic Cistern” Mediates Rapid Inhibition of Cochlear Hair Cells

Cochlear hair cells are inhibited by cholinergic efferent neurons. The acetylcholine (ACh) receptor of the hair cell is a ligand-gated cation channel through which calcium enters to activate potassium channels and hyperpolarize the cell. It has been proposed that calcium-induced calcium release (CICR) from a near-membrane postsynaptic store supplements this process. Here, we demonstrate expression of type I ryanodine receptors in outer hair cells in the apical turn of the rat cochlea. Consistent with this finding, ryanodine and other store-active compounds alter the amplitude of transient currents produced by synaptic release of ACh, as well as the response of the hair cell to exogenous ACh. Like the sarcoplasmic reticulum of muscle, the "synaptoplasmic" cistern of the hair cell efficiently couples synaptic input to CICR.

The stimulant cathartic, emodin, contracts the rat isolated ileum by triggering release of endogenous acetylcholine

Anthraquinone stimulant cathartics, such as emodin, are believed to increase the rate of contraction of ileum tissue in vitro via multiple mechanisms. The aim of this study was to probe the effects of emodin on acetylcholine (ACh)-induced contraction of the rat isolated ileum preparation. 2 Ileal sections were incubated in Tyrode's solution and responses to methacholine, ACh and emodin obtained in the absence and presence of the muscarinic antagonist atropine and the choline uptake inhibitor hemicholinium (HC-3). Depletion of endogenous ACh in the presence of HC-3 was achieved by construction of an ACh dose-response curve, using exogenous ACh, prior to re-testing the effects of emodin in the presence of HC-3. 3 Emodin caused dose-dependent tissue contraction that was abolished by inclusion of atropine (1 microM) in the buffer. Atropine (1 microM) antagonized the response caused by methacholine. Incubation of tissues with HC-3 (1 and 10 microM) reduced the maximum response caused by emodin by 45% and 71% respectively, but had no effect on ACh-induced tissue contraction. These data suggest that, emodin causes contraction of the ileum by triggering the release of endogenous ACh which acts on muscarinic receptors to cause contraction of the rat isolated ileum preparation.

A secoisopimarane diterpenoid from Salvia cinnabarina inhibits rat urinary bladder contractility in vitro.

We have evaluated the effect of 3,4-secoisopimar-4(18),7,15-triene-3-oic acid (compound 1), isolated from the aerial parts of Salvia cinnabarina, on the contractile response elicited by electrical field stimulation (EFS) in the rat isolated urinary bladder. Compound 1 (10 ( - 7) - 10 ( - 4) M) produced a concentration-dependent inhibition of the EFS contractile response without modifying the contractions produced by exogenous acetylcholine (10 ( - 6) M). A number of antagonists/inhibitors including a combination of atropine (10 ( - 6) M), phentolamine (10 ( - 6) M), propranolol (10 ( - 6) M) and hexamethonium (10 ( - 4) M), the NK (1) receptor antagonist SR140333 (10 ( - 7) M) plus the NK (2) receptor antagonist SR48968 (10 ( - 6) M), naloxone (10 ( - 6) M), verapamil (10 ( - 7) M), capsazepine (10 ( - 5) M) and the CB (1) receptor antagonist SR141716A (10 ( - 6) M) did not modify the inhibitory effect of compound 1. However, the nitric oxide (NO) synthase inhibitor L-NAME (3 x 10 ( - 4) M), significantly reduced the inhibitory effect of compound 1. It is concluded that compound 1 inhibits rat bladder contractility with a mechanism involving, at least in part, NO production.

Acetylcholine and choline amperometric enzyme sensors characterized in vitro and in vivo.

Acetylcholine (ACh) and choline (Ch) are important neuroactive molecules, yet detection of these substances in vivo presents significant analytical challenges. New multienzyme amperometric biosensors are presented here with measurement of physiologically relevant levels of ACh and Ch in vivo. Poly(m-(1,3)-phenylenediamine) (pmPD) electropolymerized on a platinum iridium wire (Pt) served as a template for immobilization of enzymes. A multienzyme layer containing choline oxidase (ChOx) and ascorbic acid oxidase (AAO) for a Ch sensor or ChOx, acetylcholinesterase (AChE), and AAO for a ACh/Ch sensor was immobilized with bovine serum albumin by cross-linking with glutaraldeyhyde. The pmPD enzyme sensors displayed enhanced sensitivity, stability, and selectivity compared to the same multienzyme systems immobilized to solvent cast Nafion and cellulose acetate-modified Pt. Sensor response was linear up to 100 microM ACh or Ch. Detection limits were 0.66 +/- 0.46 microM ACh and 0.33 +/- 0.09 microM Ch, and response times were <1 s. Selectivity for Ch and ACh relative to potential interferences and pharmacological agents commonly used to examine cholinergic physiology was demonstrated. Temperature and pH dependence and the effect of storage conditions on sensor sensitivity and selectivity were determined. Exogenous and endogenous Ch and ACh were measured in the rat brain in vivo.

Angiotensin AT 1 receptor antagonism normalizes retinal blood flow and acetylcholine-induced vasodiliation in normotensive diabetic rats

The renin angiotensin system is emerging as a potential therapeutic target for diabetic retinopathy. This study examines the effects of angiotensin-converting-enzyme inhibition by captopril and angiotensin AT(1) receptor antagonism using candesartan-cilexetil on retinal blood flow and acetylcholine-stimulated vasodilatation in normotensive diabetic rats. Non-diabetic or streptozotocin-induced diabetic rats were treated for 2 weeks with captopril (100 mg/kg/day) or candesartan cilexetil (2 mg/kg/day). Retinal haemodynamics were measured using video fluorescein angiography. Effects of exogenous acetylcholine on retinal haemodynamics were examined following intravitreal injection. Total retinal diacylglycerol was labelled using diacylglycerol kinase, separated by thin-layer chromatography, and quantified using autoradiography. Diabetic rats had prolonged retinal mean circulation time and decreased retinal blood flow compared with non-diabetic rats. Treatment of diabetic rats with either captopril or candesartan blocked the development of these blood flow abnormalities. Intravitreal injection of acetylcholine (10(-5) mol/l) in non-diabetic rats increased retinal blood flow by 53.9+/-22.0% relative to baseline whereas this response to acetylcholine was blunted in diabetic rats (4.4+/-19.6%, p<0.001). Candesartan treatment of diabetic rats restored the acetylcholine-stimulated retinal blood flow response to 60.0+/-18.7% compared with a 56.2+20.1% response in candesartan-treated non-diabetic rats. Total retinal diacylglycerol levels were increased in diabetic rats (3.75+/-0.98 nmol/mg, p<0.05) compared with non-diabetic rats (2.13+/-0.25 nmol/mg) and candesartan-treatment of diabetic rats normalized diacylglycerol levels (2.10+/-0.25 nmol/mg, p<0.05). This report provides evidence that angiotensin-converting enzyme inhibition and AT(1) receptor antagonism ameliorates retinal haemodynamic dysfunctions in normotensive diabetic rats.