Molecular Pharmacological Mechanism Of Nimodipine On The Inhibition Of TMEM16A/CaCC Chloride Channels

As an alkaloid calcium channel blocker,nimodipine is often used in the treatment of cardiovascular and cerebrovascular diseases such as cerebral vasospasm and subarachnoid hemorrhage.The main physiological activity of nimodipine is to regulate intracellular calcium levels and selectively dilate brain blood vessels to protect brain cells from promoting memory.Nimodipine has a slight expansion effect on peripheral blood vessels,thereby achieving a blood pressure lowering effect.Since nimodipine can inhibit Ca²⁺influx and reduce intracellular Ca²⁺concentration,the cell TMEM16A chloride channel activity is closely related to the cell Ca²⁺concentration,and the decrease of Ca²⁺concentration leads to the decrease of TMEM16A chloride channel activity.In recent years,it has been found that the TMEM16A chloride channel participates in many important physiological functions in the body,such as fluid transport,smooth muscle contraction,and olfactory conduction.Therefore,we speculate that the antihypertensive effect of nimodipine may be related to its inhibition of TMEM16A chloride channel activity.In this study,we utilized a functional assay fluorescence model?FRT?established by the Fischer rat thyroid epithelial?FRT?cell line stably cotransfected with human TMEM16A andgreenfluorescentproteinsmutantYFP-H148Q/I152Lsensitivetohalide?FRT-TMEM16A/YFP-H148Q/I152L?and short-circuit current experiments,analyzing the effect of nimodipine on the activity of TMEM16A chloride channel;The effects of nimodipine on vascular smooth muscle contraction were studied using a rat vascular ring experiment.The effect of nimodipine on the activity of CaCC chloride channel with unknown molecular identity on TMEM16A,CFTR and intestinal epithelium was studied using mouse smooth muscle experiment.The expression of TMEM16A in rodent,thoracic and abdominal aorta and intestinal smooth muscle was analyzed by immunohistochemistry.Experimental results:1.Functional analysis performed on FRT/TMEM16A/YFP-H148Q/I152L showed that nimodipine inhibited TMEM16A/CaCC chloride channel activity in a dose-dependent manner with an IC₅₀0 value of approximately 5.91?M.Time dynamics and reversibility analysis showed that nimodipine has a rapid and reversible pharmacological effect on the inhibition of TMEM16A channel activity.2.The short-circuit current measurement on FRT/TMEM16A/YFP-H148Q/I152L showed that nimodipine can inhibit the TMEM16A chloride ion current induced by the selective activator E_actct in a dose-dependent manner,and achieve the maximum inhibition rate at 100?M?>90%?,IC₅₀0 value is approximately 53.2?M.3.mouse ileal short-circuit current measurement results show that nimodipine inhibits the calcium-activated chloride channel in the mucosal side of mice,the maximum inhibition rate is 51%,in the absence of extracellular calcium ions,nimodipine The inhibitory effect of carbachol-mediated transient activation is weak.4.Short-circuit current results on FRT/CFTR/YFP-H148Q/I152L cells showed that nimodipine could activate CFTR-mediated chloride current,but showed significant current on FSK-induced currents on T84 cells.Inhibition,the maximum inhibition rate was 56%,IC₅₀value was 23.12?M;nimodipine significantly inhibited calcium-activated chloride currents mediated by T84 and HT-29 cells,with maximum inhibition rates of 98%and 92.1%,respectively.The IC₅₀0 values are 90.2?M and 5.64?M,respectively.5.The intracellular calcium concentration measurement showed that nimodipine?10?M?inhibited the increase of intracellular calcium concentration induced by ATP,CCh and ionomycin,and the inhibition rates were about 88%and 81.7%,respectively.87%.6.Nimodipine?100?M?had no significant effect on the Na⁺-K⁺-ATPase activity on the serosal side,and showed a significant inhibitory effect on the serosal K⁺channel,with an inhibition rate of 22.7%.7.Immunohistochemical analysis showed that nimodipine had higher expression in TMEM16A in rat thoracic aorta,abdominal aorta,ileum and mouse ileum.8.The results of rat vascular ring tension test showed that both nimodipine and T16A_inh-A01 can significantly inhibit vasoconstriction induced by phenylephrine,and the maximum inhibition efficiency is 60%.The above two compounds can completely inhibit TMEM16A specific activator.E_act-mediated vasoconstriction.Conclusion:This study found that nimodipine inhibits the activity of TMEM16A/CaCC chloride channel by reducing intracellular Ca²⁺concentration,and its inhibitory activity has rapid and reversible pharmacodynamic characteristics.The inhibition was measured on the short-circuit current mainly by inhibiting the extracellular calcium ion influx,in part by directly inhibiting TMEM16A.Smooth muscle experiments confirmed that nimodipine can relax the contraction caused by smooth muscle and inhibit CaCCs and TMEM16A.The effect of nimodipine on relaxation of vascular smooth muscle can be achieved by acting on the TMEM16A/CaCC chloride channel as well as on the calcium channel.The inhibitory activity of nimodipine on the TMEM16A/CaCC chloride channel may be one of the molecular mechanisms of its antihypertensive effect.