| ChEI has been used not only in the modern war but also in the terrific events and causes serious vocational harm. Various solutions that try to enhance antidote's antimuscarinic effect have been used, but these fail to increase the antidotal effect and are not available for clinic because of their serious side effects.Benthiactzine (benzilate thio S-2-diethylamino ethyl ester hydrochloride), our newly screened out organophosphate (OP) poisoning antidote, which has both antimuscarinic and antinicotinic effects. In the previous study, our serial in vivo and in vitro experiments demonstrate that benthiactzine possesses a less potent antimuscarinic effect than atropine, and an antinicotinic effect that atropine does not possess. Finally, we show that the antidotal effect of benthiactzine is more potent than that of atropine. We further showed that the more potent antidotal effect of antidotes does not result from its high intensity of antimuscarinic and antinicotinic properties. We further hypothesized that the ratio of antimuscarinic and antinicotinic effect of antidotes may be involved in determining their antidotal effect. The study aimed to identify the intensity and properties of benthiactzine's antimuscarinic and antinicotinic effect especially the ratio relationship.In present study, electrophysiological methods were used and the cell model was rat SCG neurons which express both mAChRs and nAChRs. To identify benthiactzine's antinicotinic effect and contrast it with mecamylamine, nicotine sensitive current was measured. To identify benthiactzine's antimuscarinic effect and contrast it with atropine, mAChRs (M1 subtype) mediated IM-inhibition and mAChRs (M2/M4 subtypes) mediated IKV- enhancement were measured.To investigate the effect on antagonism of benthiactzine and atropine against the function of muscarinic acetylcholine receptors by desensitization of nicotinic acetylcholine receptors, the whole cell recording configuration of patch-clamp technique was used and the cell model was rat SCG neurons.This study may also provide new intervention strategy for other cholinergic disorders. Main findings from this study are as follows:Part I Morphological and Functional Identification of SCG NeuronsIn present study, the cell model was primary cultured rat SCG neurons which express both nAChRs and mAChRs.1. Morphological Observation of cultured neonatal rat SCG neurons The experiment showed that the SCG neurons dissociated and cultured in our laboratory grew well and have developed a satable ACh sensitivity. The neurons had clean and accessible surface membranes, which was a requisite factor to patch-clamp techniques.2. Voltage-gated ion channels expresses on SCG neuronsThe cultured neurons expressed the voltage-gated channels, such as Na+, K+ and Ca2+ channels.Part II Research on antagonism against function of nAChRs by benthiactzine and benthiactzine action sites on nAChRs1. Dose-response relationship of nicotine-induced currents on SCG neurons nAChRs-mediated whole-cell currents were induced by the EC50 doses of nicotine(77.1μmol·L-1, 1s).2. Benthiactzine blocks nAChRs-mediated whole-cell currents The effects of benthiactzine applied at different concentrations during 1s nicotine exposure demonstrated concentration dependence of the functional block. The effect showed concentration dependent and the IC50±L95 value was 12.74±3.16μmol·L-1, b±Sb value was 2.43±0.58.The effects of mecamylamine applied at different concentrations during 1s nicotine exposure demonstrated concentration dependence of the functional block. The effect showed concentration dependent and the IC50±L95 value was 9.82±2.73μmol·L-1, b±Sb value was 2.16±1.03.3. Recovery of nAChRs-mediated whole-cell currents after the inhibition of benthiactzineThe blockade of benthiactzine and mecamylamine on nAChRs were both reversible. After washout of benthiactzine, the peak currents had recovered almost completely and rapidly, whereas after washout of 100μmol·L-1 of mecamylamine, the peak currents had recovered rapidly but not completely4. Effect of benthiactzine on desensitization of nAchRsBoth of benthiactzine and mecamylamine can accelerate the desensitization of nAChRs5. Voltage dependence of benthiactzine block of nAChRs functionThe inhibition effect of benthiactzine and mecamylamine on nAChRs had voltage-dependent6. Use dependence of benthiactzine block of nAChRs functionBoth benthiactzine and mecamylamine produces a use-dependent blockade of nAChRs.It is indicated that the effects of benthiactzine and mecamylamine show concentration-dependence of the functional block. Despite benthiactzine's IC50 value is 1.3 times larger than that of mecamylamine, the antinicotinic effect of benthiactzine and mecamylamine are equivalent. The blockade of benthiactzine and mecamylamine on nAChRs were both reversible and can accelerate the desensitization of nAChRs. Both benthiactzine and mecamylamine produce a voltage-dependent and use-dependent blockade on nAChRs. This suggested that the molecules of benthiactzine and mecamylamine may physically block the ion channels of nAChRs.PartⅢAntagonism against function of mAChRs by benthiactzine1. Antagonism against mAChRs (M1 subtype) mediated IM-inhibition by benthiactzineAfter the application of benthiactzine, the ratio of oxotremorine-induced IM-inhibition was reduced. The effect showed concentration dependent and the IC50±L95 value was 4.13±1.27μmol·L-1, b±Sb value was -2.05±0.81.Ater the application of atropine, the ratio of oxotremorine-induced IM-inhibition was reduced. The effect showed concentration dependent and the IC50±L95 value was 1.32±0.45μmol·L-1, b±Sb value was -2.67±0.59. 2. Antagonism against mAChRs (M2/M4 subtypes) mediated IKV-enhancement by benthiactzineAfter the application of benthiactzine, the ratio of oxotremorine-induced IKV-enhancement was reduced. The effect showed concentration dependent and the IC50±L95 value was 6.30±1.93μmol·L-1, b±Sb value was -2.15±0.61. After the application of atropine, the ratio of oxotremorine-induced IKV- enhancement was reduced. The effect showed concentration dependent and the IC50±L95 value was 2.56±0.87μmol·L-1, b±Sb value was -2.24±0.42.It is indicated that antagonism against function of mAChRs (M1 subtype) by benthiactzine and atropine show concentration dependent. Antagonism against mAChRs (M1 subtype) of benthiactzine is weaker than that of atropine. Benthiactzine's IC50 value is 3.13 times larger than that of atropine. Antagonism against function of mAChRs (M2/M4 subtypes) by benthiactzine and atropine show concentration dependent. Antagonism against mAChRs (M2/M4 subtypes) of benthiactzine is weaker than that of atropine. Benthiactzine's IC50 value is 2.46 times larger than that of atropine.PartⅣEffect on benthiactzine antagonism against mAChRs by desensitization of nAChRs1. Antagonism against mAChRs by benthiactzine after desensitization of nAChRs After desensitization of nAChRs, the ratio of oxotremorine-induced IM-inhibition was reduced after the application of benthiactzine. The effect showed concentration dependent and the IC50±L95 value was 52.28±9.76μmol·L-1, b±Sb value was -3.54±1.98.After desensitization of nAChRs, the ratio of oxotremorine-induced IM-inhibition was reduced after the application of atropine. The effect showed concentration dependent and the IC50±L95 value was 43.17±11.43μmol·L-1, b±Sb value was -1.17±0.15.2. Effect on benthiactzine antagonism of mAChRs by degree of desensitization of nAChRsThe antimuscarinic effect of both benthiactzine and atropine recovered gradually with the recovery of nAChRs from desensitization.3. Analysis of the ratio between benthiactzine's antimuscarinic and antinicotinic effectBenthiactzine's antimuscarinic effect is more potent than antinicotinic effect, the ratio range from 2:1 to 3.1:1.It is indicated that after desensitization of nAChRs, the antimuscarinic effect of both benthiactzine and atropine decreased significantly relative to the normal condition. The decreased amplitude of benthiactzine was weaker than that of atropine. The antimuscarinic effect of both benthiactzine and atropine recovered gradually with the recovery of nAChRs from desensitization.From above observations, it could be concluded:1. The effects of benthiactzine and mecamylamine show concentration dependence of the functional block. Despite benthiactzine's IC50 value is 1.3 times larger than that of mecamylamine, the antinicotinic effect of benthiactzine and mecamylamine are equivalent.The blockade of benthiactzine and mecamylamine on nAChRs were both reversible and can accelerate the desensitization of nAChRs. Both benthiactzine and mecamylamine produce a voltage-dependent and use-dependent blockade on nAChRs. This suggested that the molecules benthiactzine and mecamylamine may physically block the ion channels of nAChRs.2. Antagonism against function of mAChRs (M1 subtype) by benthiactzine and atropine show concentration dependent. Antagonism against mAchRs (M1 subtype) of benthiactzine is weaker than that of atropine. Benthiactzine's IC50 value is 3.67 times larger than that of atropine. Antagonism against function of mAChRs (M2/M4 subtypes) by benthiactzine and atropine show concentration dependent. Antagonism against mAChRs (M2/M4 subtypes) of benthiactzine is weaker than that of atropine. Benthiactzine's IC50 value is 2.46 times larger than that of atropine.3. After desensitization of nAChRs, the antimuscarinic effect of both benthiactzine and atropine decreased significantly relative to the normal condition. The decreased amplitude of benthiactzine was weaker than that of atropine. The antimuscarinic effect of both benthiactzine and atropine recovered gradually with the recovery of nAChRs from desensitization. Desensitization of nAChRs weakens the antagonists'antimuscarinic effect. 4. Benthiactzine's antimuscarinic effect is more potent than antinicotinic effect, the ratio range from 2:1 to 3.1:1. |
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