碩士 / 國防醫學院 / 藥理學研究所 / 96 / Abstract
Background：
Changing intracellular pH (pHi) influences many cellular functions, such as enzyme activities, permeability of channels, and cell volume. Vascular smooth muscles (VSMs) typically display significant pHi signaling associated with physiologically relevant regulatory stimuli, and a variety of correlations between pHi and VSM function have been reported. To date , the pHi regulators include, at least, Na+/H+ exchanger (NHE), Na+/HCO3- symporter (NHS), Cl-/OH- exchanger (CHE) and Cl-/HCO3- exchanger (AE) in the heart, neuron and many cells. Many epidemiological and clinical studies have shown that heavy alcohol (Alc) consumption is associated with increased risk of coronary heart disease (CHD) and total mortality. Human urotensin II (U-II), the most potent endogenous vasoconstrictor peptide identified to date, but the role of U-II in cardiovascular physiology and diseases remains largely uncertain. Therefore the aims of the present study were to (Ⅰ) examine the possible pHi regulators in the human aorta smooth muscle cells ; (Ⅱ) investigate the effect of alcohol on pHi and further explore the possible underlying mechanism. ; (Ⅲ) investigate the effect of U-II on pHi and further explore the possible underlying mechanism.
Materials and Methods:
Human aorta smooth muscle cells (HASMCs) was cultured by Explant Method and cell identification was checked by immunocytochemistry. We used solid phase peptide synthesis method to produce U-II peptides. In the whole study, pHi change was measured by the technique of microspectrofluorimetry with the H+-sensitive fluoroprobe, 2’,7’-Bis-(2-carboxyethyl)-5(6)- carboxy- fluorescein (BCECF).
Results:
1. We have successfully cultured single cells from tissue of human aorta with identification of antibody of HASMCs, as well as specificity of morphology.
2. In HEPES-buffered Tyrode solution, either removal of extracellular Na+ or adding HOE 694 (a specific NHE inhibitor) totally inhibited the pHi recovery from NH4Cl-induced intracellular acidosis, which proves the existence of NHE.
3. In bicarbonate-buffered Tyrode solution, adding either HOE 694 or DIDS (a specific NHS inhibitor) partially inhibited the pHi recovery from NH4Cl-induced intracellular acidosis, while removal of Na+ or adding together HOE 694 and DIDS can totally inhibited the recovery. These results demonstrate the existence of NHS.
4. In bicarbonate-buffered Tyrode solution, adding DBDS (a specific CHE inhibitor) or DIDS (a specific AE inhibitor) or adding together DBDS and DIDS only slowed the pHi recovery from Na+ acetate-induced intracellular alkalosis, while removal extracellular Cl- totally inhibited the recovery. These results demonstrate the existence of CHE, AE and other Cl--dependent acid loaders.
5. In HEPES-buffered Tyrode solution, alcohol affected pHi biphasic, increasing at lower concentration (10 ~ 100 mM) while decreasing at higher concentration (300 mM). In bicarbonate-buffered Tyrode solution, pHi was increased by alcohol (10 ~ 300 mM) .
6. Alcohol affected the activity of NHE, NHS, CHE and AE in a way of biphasic, increasing at lower concentration (10 mM ~100 mM ), while decreasing at higher concentration (300 mM).
7. In HEPES-buffered Tyrode solution, U-II (30~ 100 nM) increased pHi and NHE activity.
Conclusions
1. We demonstrated for the first time that, two acid-extruders (NHE and NHS) and two acid-loaders (CHE and AE), are functionally existed in the HASMCs.
2. The alcohol-induced pHi changes are caused by the combination effects of inhibition or activation of NHE, NHS, CHE and AE in the HASMCs.
3. U-II ( 30~300 nM ) increases NHE activity and pHi in HASMCs.
| Identifer | oai:union.ndltd.org:TW/096NDMC0550012 |
| Date | January 2008 |
| Creators | Chan-Jung, Tsao, 曹展榮 |
| Contributors | Shih-Hurng Loh, 羅時鴻 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 100 |
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