Preparation and characterization of polyclonal antibodies against rat sodium pump alpha 2 subunit M1-M2 extra membrane fragment / 南方医科大学学报

<p><b>OBJECTIVE</b>To prepare polyclonal antibodies against sodium pump alpha 2 subunit M1-M2 extramembrane fragment (NKAα2 EM1) for studying the pathogenesis of hypertension.</p><p><b>METHODS</b>According to the GenBank data, the amino acid sequence of NKAα2 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 NKAα2 EM1 expression in rat aortic vascular smooth muscle cells by enzyme-linked immunosorbent assay and immunocytochemistry (ICC).</p><p><b>RESULTS</b>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 µg/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.</p><p><b>CONCLUSION</b>The anti-NKAα2 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.</p>

Effect of ouabain on intracellular Ca(2+) concentration in rat vascular smooth muscle cells in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the effect of ouabain on intracellular Ca(2+) concentration ([Ca(2+)]i) in thoracic aorta vascular smooth muscle cells (VSMCs) in vitro.</p><p><b>METHODS</b>Primary 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 Ca(2+) 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 α2 subunit was assayed in antagonizing the effect of ouabain on [Ca(2+)]i in the VSMCs.</p><p><b>RESULTS</b>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 [Ca(2+)]i in the VSMCs, which was antagonized by the application of the truncated fragment of sodium pump α2 subunit.</p><p><b>CONCLUSIONS</b>Elevations in [Ca(2+)]i in the VSMCs can be the cytological basis of high ouabain-induced hypertension. The truncated fragment of the sodium pump α2 subunit can antagonize ouabain-induced increase of [Ca(2+)]i in the VSMCs, which provides a clue for understanding the pathogenesis of and devising a therapeutic strategy for high ouabain-induced hypertension.</p>

EXPRESSION OF ENDOGENOUS OUABAIN IN MULTIPLE ADRENAL TUMORS / 药物分析学报

Objective To explore expression of endogenous ouabain(EO) in multiple adrenal tumors.Methods Thirty-one cases of adrenal tumors and 6 cases of healthy adrenal tissues were selected. The expression of EO in the adrenal tiss ue was detected with immunohistochemical streptavidin peroxadase conjugated(SP) method.Results Most of EO positive products were localized in cy toplasm of the zona reticularis of human adrenal cortex, and positive products s howed to be fine granular. There was no positive signal in the medulla. EO showe d on diffused positive in patients with pheochromocytoma accompanied high blood pressure[SBP:(165.22±7.61) mmHg, DBP:(105.52±4.26) mmHg], but there were neg ative in ones with normative blood pressure[SBP:(118.52±4.58) mmHg, DBP:(83±3.60) m mHg]. The expression of EO was positive in all adrenocortical hyperplasic, aden oma an d carcinoma, no matter its high or normative blood pressure. The degree of expre ssion of EO in adrenal tissues was related to the level of BP.Conclusion Expression of endogenous ouabain(EO) in health y adrenal tissue and adrenal tumors was a valuable morphological and pathophysio logical clue for the research on ouabain.

Preparation and molecular weight estimation of ouabain pol yclonal antibody F(ab)2 fragment / 中国生化药物杂志

Purpose　The aim is to prepare ouabain polyclonal antibody F(ab)2 fragment and to estimate its molecular weight.Methods[ KG*2 [ WTBZ]Ouabain polyclonal antibody was obtained from immunized rabbits.The antibod y was digested with pepsin.The resulting products were analyzed and the molecular weig ht of F(ab)2 fragment was estimated by HPSEC.The immune activity was detec ted by ELISA.Results　100 mg of ouabain polyclonal antibody wa s dige sted by 2 mg of pepsin for 18 hours at pH 3.0 and active ouabain polyclonal anti body F(ab)2 framgment was obtained.Its molecular weight was 107 kD.Concl usion　The active ouabain polyclonal antibody F(ab)2 fragment coul d be prepared by digesting its antibody with pepsin.

Investigation of amino acid sequence of specific ouabain conjugated peptides / 中国病理生理杂志

AIM: The purpose of the study was to investigate the gene and amino acid sequence of specific ouabain conjugated peptides (OCP) in order to get an experimental bases to block or antagonist the actions between endogenous ouabain(EO) and sodium pump in hypertension. METHODS: Screening the phage displayed 12-peptide library by biopaning for OCP. The sequence of each selected peptide was determined and the sequence was analyzed through internet. The bioactivity was determined by erythrocyte [ 86 Rb] uptaking. RESULTS: Three kinds of peptides were screened out. Peptide A (12 peptide) was occupied in 66 7%(8/12), peptide B (8 peptide) 16 7% (2/12) and peptide C (12 peptide) 8 3% (1/12). There was only one case without insertron. The analysis of protein showed that there were no homogenous between peptide A, B, C and sodium pump. The amino acid sequence of specific OCP was Leu-Leu-Ala-Asp-Thr-Thr-His-His-Arg -Pro-Trp-Thr. CONCLUSIONS: Determination of the sequence of OCP supplies an important experimental foundation for ouabain research. The results also show that phage display peptide library is an effective, simple and efficient method to select specific steroid receptors. [

Selection of specific ouabain conjugated peptides and identification of its binding activity / 中国药理学通报

AIM To select specific ouabain conjugated peptides (OCP) to decline ouabain level. METHODS Biopanning phage displayed 12 peptide library for OCP. The sequences of each selected peptide was determined and analyzed. The binding activity of synthetical OCP was identified by radioligand binding assay (RRA). RESULTS Three kinds of peptides were found out. Peptide A (12 peptide) was occupied in 0 67(8/12). The analysis of protein showed that there was no homogeneous protein in Genebank. The results of RRA showed that there were binding activity between synthetical OCP and 3H ouabain. Dissociation constant(K d) was 1 087 nmol?L -1 . Receptor density was 120 pmol?g -1 protein. IC 50 =1 46?10 -4 mol?L -1 . CONCLUSION It is important to obtain distinctive OCP, by which not only the protein sequence was analyzed, but also a valuable experimental data in detection of ouabain and therapy in hypertension also was offered.