Local Regulatory Mechanisms In Simulated Microgravity-induced Differential Changes Of Arterial Vessels--L-RAS And Contractile Proteins

Studies on the mechanisms of postflight cardiovascular deconditioning and its countermeasures have important theoretical and practical implications to fully unravel the cardiovascular effects of microgravity and to realize the manned-spaceflight project in the new millennium. Recent observations during spaceflight or by ground simulations have both suggested that adaptive alterations in the arterial vasculatures may play a pivotal role in the occurrence of postflight orthostatic intolerance. Previous findings from our laboratory have demonstrated that simulated microgravity may result in atrophic changes with depressed vasoconstrictor responsiveness in hindquarter vessels, and hypertrophic changes with enhanced vasoconstrictor responsiveness in cerebral arteries of rats. However, the mechanisms of this differential adaptation are still not well understood. We hypothesized that local renin-angiotensin system (L-RAS) might function asan local regulatory mechanism. In the first part of the present work, the expression changes of angiotensinogen (AGT) and angiotensin-converting enzyme (ACE) as well as its time course characteristics were investigated by RT-PCR, in situ hybridization and Western blotting. We also prepared the polyclonal antiserum against AGT for the following work. Our recent work also suggest that simulated microgravity may induce the contractile proteins in the arterial vessles to be changed differentially. Therefore, in the second part of the present work the alterations of a-SM-actin and calponin were observed by immunohistochemistry and Western blotting.The major findings of the present work are as follows:1. mRNA expression of the key elements of L-RASAGT and ACE mRNA were expressed in myocardium, basilar arterial, carotid arterial, femoral arterial and abdominal aortic tissues as shown by RT-PCR. In situ hybridization also confirmed the existence of AGT mRNA in these tissues. The AGT mRNA hybridization signals were localized dispersively in the myocytes of left ventricle. While in the arterial wall, the intense signals were not restricted to the media. They were also found in adventitia and even in perivascular fat cells. Previous studies failed to reach a solid conclusion about the expression of L-RAS in ventricular myocardium and observations of L-RAS in vessles were mainly limited to aorta. Our present finding provides a clear evidence that cardiovascular tissues have the ability to produce L-RAS independently.2. Effects of 4-wk simulated microgravity on the mRNA expression of AGT and ACE in arterial tissues of different body regions of rats4-wk simulated microgravity induced a significant increase of AGT mRNA expression in basilar arterial tissues (P < 0.05, n = 5), and a decreasing tendency in femoral arterial tissues (P > 0.05, n = 5). No significant changes were observed in carotid arterial and abdominal aortic tissues. ACE mRNAexpression showed no changes as compared with control rats. Our finding gives the first proof that L-RAS is differentially changed by simulated microgravity.3. Preparation of polyclonal antiserum against angiotensinogenIn order to study AGT in protein level and also for other continuing work, the C-teminus of rat angiotensinogen was expressed in E. coll. Rabbits were immunized by this expressed 6His-AGTc protein and serum from different rabbits were raised. A high titre (1:25600) of the prepared polyclonal antiserum was shown by ELISA. Immunohistochemistry and Western blotting further confirmed that the prepared antiserum could recognize not only the prokaryotic expressed 6His-AGTc, but also the endogenous AGT protein in many tissues of rat and human. Compared with the known antibodies, the antiserum we prepared is highly specific and rules out the possibility of cross-reacting with angiotensin fragments.4. Time course characteristics of AGT mRNA and protein expressions in different arterial tissues induced by simulated microgravityIn basilar arterial tissue, AGT mRNA expression increased with the extension of simulated micr...