Affinity, Subtype And Tissue Selectivity Of Benthiactzine To Muscarinic Acetylcholine Receptors

AChE inhibitor (ChEI) poisoning, in which circumstances AChE is highly inhibited, is a major global public health problem, causing hundreds of thousands of deaths each year. Medical management of ChEI poisoning is difficult, with case fatality often over 20%. Current therapeutic strategies to decrease acetylcholinesterase inhibitor (ChEI) poisoning include atropine to reduce the muscarinic syndrome, oxime to reactivate OP-inhibite AChE. Atropine which is used to antagonize the syndrome caused by excess Ach includes: miosis, profuse secretions, bradycardia, bronchoconstriction, hypotension, and diarrhea, is considered the drug of choice for ChEI poisoning since the late 1940s.The tissue and subtype selectivity of atropine is not distinguished. Although atropine alone attenuates the muscarinic syndrome resulting from the exposure of guinea pigs to the Ops, it does not afford significant protection against their lethality. Our previous reviews revealed that desensitized nicotinic receptor can still mediate ChEI toxicity by facilitating mAChRs functions.The Non-neuronal Acetylcholine System has been identified in numerous non-neuronal cells and tissues,including keratinocytes,cancer cells, immune cells and reproductive organs. NNAs is not the same as neuronal acetylcholine system, and is not the same in different cells. Our previous study demonstrated the gene sequences and the pharmacological feature of NNMRs subtypes 1-5 expressed by primary cerebral cortex astrocyte cells were quite different from them with NMRs subtypes 1-5 expressed by neurons.In our previous studies, benthiactzine showed both antimuscarinic and antinicotinic effects and immediate and potent protection against ChEI poisoning in animal models, possesses more potent effect than atropine in counteracting ChEI poisoning, could relieve the functional injury of gastrointestinal mucosa barrier induced by endotoxin in the blood and antagonize circulation failures induced by cholinesterase inhibitor effectively and immediately. To test our hypothesis: we further compared the antimuscarinic and antinicotinic effect between benthiactzine and atropine in vitro.1. The affinity and selectivity of benthiactzine to five subtypes muscarinic acetylcholine receptors.To evaluate the antimuscarinic effect of benthiactzine and atropine to different subtypes, specific ³H-QNB binding experiment was conducted. Benthiactzine was less potent than atropine to 5 different subtypes of human mAChRs (hM1-hM5). It was also noted that benthiactzine and atropine possessed different selectivity of mAChRs subtypes. Benthiactzine bonded to hM1 > hM3 > hM5 > hM2≈hM4, while atropine's affinity to hM3≈hM1 > hM4≈hM2≈hM5.2. The affinity and selectivity of benthiactzine to muscarinic acetylcholine receptors expressed in different tissues.To evaluate the antimuscarinic effect of benthiactzine and atropine to different tissues, specific ³H-QNB binding experiment was conducted. It was noted that benthiactzine was less potent than atropine in binding to mAChRs expressed in nine different peripheral tissues (guinea pig's submaxillary gland, ventricular myocardium, lung, gastric smooth muscle, gastric mucosa-submucosa, small intestinal smooth muscle, small intestine mucosa-submucosa, colonic smooth muscle and colonic mucosa-submucosa) and five central tissues (guinea pig's cerebral cortex, spinal cord, forebrain, cerebellum, myelencephalon). ³H-QNB bound to muscarinic acetylcholine receptors expressed in cerebral cortex with high affinity (Kd=0.3476nmol/L) and maximal bounding (Bmax=1181fmol/mg). Ki value for benthiactzine (27.18nmol/L) in cerebral cortex is larger than atropine (Ki= 8.40nmol/L, P<0.01). It was noted that, the affinity of benthiactzine differed largely when binding to mAChRs expressed in different tissues. The effect of benthiactzine on mAChRs was most potent in submaxillary gland (IC₅₀ = 21.48±3.05nM), and was least potent in alimentary tract mucosa-submucosas (IC₅₀ all inμM level), especially in small intestine mucosa-submucosa, where it had no binding even at the extremely high concentration of 16μM. In contrast, the difference between affinities of atropine to mAChRs expressed in different tissues was much smaller, The effect of atropine on mAChRs was most potent in gastric mucosa-submucosa (IC₅₀= 8.66±3.41nM), and was least potent in myelencephalon (IC₅₀=46.90±5.87nmol/L).3. The affinity of benthiactzine to non-neuronal muscarinic acetylcholine receptors expressed in astrocytes.Benthiactzine and atropine inhibited specific ³H-QNB binding to muscarinic acetylcholine receptors expressed in astrocytes in a concentration-depended manner in vitro. Interestingly, benthiactzine and atropine inhibited specific ³H-QNB binding to muscarinic acetylcholine receptors expressed in astrocytes, with the IC₅₀ values 2591.55nmol/L and 14.73nmol/L, respectively. Thus, the IC₅₀ value for benthiactzine was 174 times larger than that for atopine. This difference was statistically significant (P<0.01). These results suggested that benthiactzine has low affinity to non-neuronal muscarinic acetylcholine receptors expressed in astrocytes which was different from atropine.4. The targets of benthiactzine to nicotinic acetylcholine receptors expressed in Wistar rats'cerebral cortex and SH-EP1 cells.³H-nicotine bound to nicotinic acetylcholine receptors expressed in Wistar rats'cerebral cortex and SH-EP1 cells. It is significant that benthiactzine has no bounding even at the extremely high concentration of 0.01mmol/L and 0.1mmol/L (P>0.05,n=4).This result suggested the binding sites of benthiactzine to nicotinic acetylcholine receptors were the same with acetylcholine. It is conformed with the results of patch clamp.Therefore, we come to the conclusions: 1. The affinity of benthiactzine to muscarinic receptors is lower than atropine. 2. Benthiactzine and atropine possessed different selectivity of mAChRs subtypes. Benthiactzine bonded to hM1≈hM3 > hM5 > hM2≈hM4。Benthiactzine was less potent than atropine in binding to mAChRs expressed in nine different peripheral tissues and five central tissues. The affinity of benthiactzine differed largely when binding to mAChRs expressed in different tissues. The effect of benthiactzine on mAChRs was most potent in submaxillary gland, and was least potent in alimentary tract mucosa-submucosas, especially in small intestine mucosa-submucosa. In contrast, the difference between affinities of atropine to mAChRs expressed in different tissues was much smaller.3. Benthiactzine has no block effect on ³H-nicotine bound to nicotinic acetylcholine receptors expressed in Wistar rats'cerebral cortex and SH-EP1 cells. This result suggested the binding sites of benthiactzine to nicotinic acetylcholine receptors were the same with acetylcholine. It is conformed with the results of patch clamp.