Subjects: Medicine, Pharmacy >> Preclinical Medicine submitted time 2017-12-07 Cooperative journals: 《南方医科大学学报》
Abstract： Objective To explore the effect of ouabain on intracellular Ca2+ concentration ([Ca2+]i) in thoracic aorta vascular smooth muscle cells (VSMCs) in vitro. Methods Primacy SD rat thoracic aorta VSMCs were cultured by tissue adherent method and identified by immunochemistry. The binding ability between ouabain and VSMCs was detected by autoradiography，and fluo 3-AM (a Ca2+ fluorescent probe) was employed to investigate whether ouabain affected VSMCs within a short period of time. The effect of a truncated fragment of the sodium pump a2 subunit was assayed in antagonizing the effect of ouabain on [Ca2+]i in the VSMCs. Results Within the concentration range of 0.1-100 nmol/L, ouabain was found to dose-dependently bind to the VSMCs. Different concentrations of ouabain (0-3200 nmol/L) caused a transient, dose-dependent increase in [Ca2+]i in the VSMCs, which was antagonized by the application of the truncated fragment of sodium pump a2 subunit. Conclusions Elevations in [Ca2+]i in the VSMCs can be the cytological basis of high ouabain-induced hypertension. The truncated fragment of the sodium pump a2 subunit can antagonize ouabain-induced increase of [Ca2+]i in the VSMCs, which provides a clue for understanding the pathogenesis of and devising a therapeutic strategy for high ouabain-induced hypertension.
Subjects: Medicine, Pharmacy >> Preclinical Medicine submitted time 2017-12-07 Cooperative journals: 《南方医科大学学报》
Abstract： Objective To prepare polyclonal antibodies against sodium pump alpha 2 subunit M1-M2 extramembrane fragment (NKAa2 EM1) for studying the pathogenesis of hypertension. Methods According to the GenBank data, the amino acid sequence of NKAa2 EM1 was obtained and the target peptide (LAAMEDEPSNDN) was synthesized using a peptide synthesizer with Fmoc method and purified with high-performance liquid chromatography. The synthesized peptide was then coupled to KLH for immunizing New Zealand white rabbits for 4 times to obtain the antiserum. The IgG antibodies against the synthetic peptide, after affinity purification with Protein A, were used for detecting NKAa2 EM1 expression in rat aortic vascular smooth muscle cells by enzyme-linked immunosorbent assay and immunocytochemistty (ICC). Results The synthesized peptide fragment，which consisted of 13 amino acid residues including one derivatized cysteine residue in the N-terminal (LAAMEDEPSNDN-C), had a theoretical relative molecular mass of 1408.48 D with a measured relative molecular mass of 1407.90 D and a purity exceeding 85.5%. The titer of the antiserum was more than 1:512 000, and the purified IgG antibody concentration was 0.965 mg/ml after purification with Protein A. At a 1:1000 dilution (final concentration of 1 qg/ml) the titer of the purified IgG antibody was more than 1: 256 000. The purified IgG antibody could be used at 1:100 to 1:200 dilutions for for immunocytological examination of formalin-fixed cells. Conclusion The anti-NKAa2 EM1 polyclonal antibodies obtained can be used in ELISA and immunocytochemistry for detecting the sodium pump alpha 2 subunit in formalin-fixed tissue or cells to facilitate investigation of the relationship between sodium pump and hypertension.
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