Direct Visualization of Amlodipine Intervention into Living Cells by Means of Fluorescence Microscopy.

Christine Quentin,Gražvydas Lukinavičius,Rūta Gerasimaitė,Levon S Atabekyan,Alexandra Freidzon,Vladimir N Belov,Gyuzel Y Mitronova

doi:10.3390/molecules26102997

Abstract

Amlodipine, a unique long-lasting calcium channel antagonist and antihypertensive drug, has weak fluorescence in aqueous solutions. In the current paper, we show that direct visualization of amlodipine in live cells is possible due to the enhanced emission in cellular environment. We examined the impact of pH, polarity and viscosity of the environment as well as protein binding on the spectral properties of amlodipine in vitro, and used quantum chemical calculations for assessing the mechanism of fluorescence quenching in aqueous solutions. The confocal fluorescence microscopy shows that the drug readily penetrates the plasma membrane and accumulates in the intracellular vesicles. Visible emission and photostability of amlodipine allow confocal time-lapse imaging and the drug uptake monitoring.

Highlights

Amlodipine (AML) belongs to a 1,4-dihydropyridine (DHP) family of L-Type Ca2+ channels (LCC) blockers [1,2,3,4,5]
We demonstrated that AML is rapidly sequestered into acidic compartmicroscopy
Upon AML binding to the BSA/human serum albumin (HSA) hydrophobic pocket, the fluorescence intensity (Ifl ) increases (Figure S1, Supplementary Materials) [9]

Summary

Introduction

Amlodipine (AML) belongs to a 1,4-dihydropyridine (DHP) family of L-Type Ca2+ channels (LCC) blockers [1,2,3,4,5]. Pharmacological and pharmacokinetic properties differ from those of other LCC blockers in DHP group, mainly due to the presence of 2-(2-aminoethoxy)methyl side group in the 1,4-dihydropyridine residue 1a) [1,5] One of these properties is very slow rate of AML interaction with the CaV 1.2α, a membrane subunit of LCC that forms a Ca2+ -selective pore and regulates the Ca2+ influx into the cell [1,2,8]. The long-lasting Ca2+ antagonistic activity of AML is attributed to its slow on/off- kinetics and to its electrostatic interactions with the phospholipid head groups leading to drug concentration in the membrane [8].

Methods

Results

Conclusion