Investigation of calcium antagonist—L-type calcium channel interactions by a vascular smooth muscle cell membrane chromatography method

Hui Du; Jianyu He; Sicen Wang; Langchong He
July 2010
Analytical & Bioanalytical Chemistry;Jul2010, Vol. 397 Issue 5, p1947
Academic Journal
The dissociation equilibrium constant ( KD) is an important affinity parameter for studying drug–receptor interactions. A vascular smooth muscle (VSM) cell membrane chromatography (CMC) method was developed for determination of the KD values for calcium antagonist–L-type calcium channel (L-CC) interactions. VSM cells, by means of primary culture with rat thoracic aortas, were used for preparation of the cell membrane stationary phase in the VSM/CMC model. All measurements were performed with spectrophotometric detection (237 nm) at 37 °C. The KD values obtained using frontal analysis were 3.36 × 10−6 M for nifedipine, 1.34 × 10−6 M for nimodipine, 6.83 × 10−7 M for nitrendipine, 1.23 × 10−7 M for nicardipine, 1.09 × 10−7 M for amlodipine, and 8.51 × 10−8 M for verapamil. This affinity rank order obtained from the VSM/CMC method had a strong positive correlation with that obtained from radioligand binding assay. The location of the binding region was examined by displacement experiments using nitrendipine as a mobile-phase additive. It was found that verapamil occupied a class of binding sites on L-CCs different from those occupied by nitrendipine. In addition, nicardipine, amlodipine, and nitrendipine had direct competition at a single common binding site. The studies showed that CMC can be applied to the investigation of drug–receptor interactions. [Figure not available: see fulltext.]


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