Differential activation of potassium channels in cerebral and hindquarter arteries of rats during simulated microgravity

The purpose of this study was to test the hypothesis that differential autoregulation of cerebral and hindquarter arteries during simulated microgravity is mediated or modulated by differential activation of K(+) channels in vascular smooth muscle cells (VSMCs) of arteries in different anatomic regions. Sprague-Dawley rats were subjected to 1- and 4-wk tail suspension to simulate the cardiovascular deconditioning effect due to short- and medium-term microgravity. K(+) channel function of VSMCs was studied by pharmacological methods and patch-clamp techniques. Large-conductance Ca(2+)-activated K(+) (BK(Ca)) and voltage-gated K(+) (K(v)) currents were determined by subtracting the current recorded after applications of 1 mM tetraethylammonium (TEA) and 1 mM TEA + 3 mM 4-aminopyridine (4-AP), respectively, from that of before. For cerebral vessels, the normalized contractility of basilar arterial rings to TEA, a BK(Ca) blocker, and 4-AP, a K(v) blocker, was significantly decreased after 1- and 4-wk simulated microgravity, respectively. VSMCs isolated from the middle cerebral artery branches of suspended rats had a more depolarized membrane potential (E(m)) and a smaller K(+) current density compared with those of control rats. Furthermore, the reduced total current density was due to smaller BK(Ca) and smaller K(v) current density in cerebral VSMCs after 1- and 4-wk tail suspension, respectively. For hindquarter vessels, VSMCs isolated from second- to sixth-order small mesenteric arteries of both 1- and 4-wk suspended rats had a more negative E(m) and larger K(+) current densities for total, BK(Ca), and K(v) currents. These results indicate that differential activation of K(+) channels occur in cerebral and hindquarter VSMCs during short- and medium-term simulated microgravity. It is further suggested that different profiles of channel remodeling might occur in VSMCs as one of the important underlying cellular mechanisms to mediate and modulate differential vascular adaptation during microgravity.     

Daily short-period gravitation can prevent functional and structural changes in arteries of simulated microgravity rats.

This study was designed to clarify whether simulated microgravity-induced differential adaptational changes in cerebral and hindlimb arteries could be prevented by daily short-period restoration of the normal distribution of transmural pressure across arterial vasculature by either dorsoventral or footward gravitational loading. Tail suspension (Sus) for 28 days was used to simulate cardiovascular deconditioning due to microgravity. Daily standing (STD) for 1, 2, or 4 h, or +45 degrees head-up tilt (HUT) for 2 or 4 h was used to provide short-period dorsoventral or footward gravitational loading as countermeasure. Functional studies showed that Sus alone induced an enhancement and depression in vasoconstrictor responsiveness of basilar and femoral arterial rings, respectively, as previously reported. These differential functional alterations can be prevented by either of the two kinds of daily gravitational loading treatments. Surprisingly, daily STD for as short as 1 h was sufficient to prevent the differential functional changes that might occur due to Sus alone. In morphological studies, the effectiveness of daily 4-h HUT or 1-h STD in preventing the differential remodeling changes in the structure of basilar and anterior tibial arteries induced by Sus alone was examined by histomorphometry. The results showed that both the hypertrophic and atrophic changes that might occur, respectively, in cerebral and hindlimb arteries due to Sus alone were prevented not only by daily HUT for 4 h but also by daily STD even for 1 h. These data indicate that daily gravitational loading by STD for as short as 1 h is sufficient to prevent differential adaptational changes in function and structure of vessels in different anatomic regions induced by a medium-term simulated microgravity.     

Effects of simulated microgravity on arterial nitric oxide synthase and nitrate and nitrite content.

The aim of the present work was to investigate the alterations in nitric oxide synthase (NOS) expression and nitrate and nitrite (NOx) content of different arteries from simulated microgravity rats. Male Wistar rats were randomly assigned to either a control group or simulated microgravity group. For simulating microgravity, animals were subjected to hindlimb unweighting (HU) for 20 days. Different arterial tissues were removed for determination of NOS expression and NOx. Western blotting was used to measure endothelial NOS (eNOS) and inducible NOS (iNOS) protein content. Total concentrations of NOx, stable metabolites of nitric oxide, were determined by the chemiluminescence method. Compared with controls, isolated vessels from simulated microgravity rats showed a significant increase in both eNOS and iNOS expression in carotid arteries and thoracic aorta and a significant decrease in eNOS and iNOS expression of mesenteric arteries. The eNOS and iNOS content of cerebral arteries, as well as that of femoral arteries, showed no differences between the two groups. Concerning NOx, vessels from HU rats showed an increase in cerebral arteries, a decrease in mesenteric arteries, and no change in carotid artery, femoral artery and thoracic aorta. These data indicated that there were differential alterations in NOS expression and NOx of different arteries after hindlimb unweighting. We suggest that these changes might represent both localized adaptations to differential body fluid redistribution and other factors independent of hemodynamic shifts during simulated microgravity.     

Short-term simulated weightlessness enhances response of L-type calcium channel to angiotensin II in cerebral vascular smooth muscle cells in rats

Some studies suggest that the calcium channels and rennin-angiotensin system (RAS) play pivotal roles in the region-specific vascular adaptation due to simulated weightlessness. This study was designed to clarify if angiotensin II (Ang II) was involved in the adaptational change of the L-type calcium channel (Ca(L)) in the cerebral arterial vascular smooth muscle cells (VSMCs) under simulated weightlessness. Tail suspension (SUS) for 3 d was used to simulate immediate early cardiovascular changes to weightlessness. Then VSMCs in cerebral basilar artery were enzymatically isolated using papain, and Ca(L) current (barium instead of calcium as current carrier) in VSMCs was measured by whole-cell patch-clamp techniques. The results showed that 3-day simulated weightlessness significantly increased current density of Ca(L). However, I-V relationships of normalized peak current densities and steady-state activation curves of Ca(L) were not affected by simulated weightlessness. Although Ang II significantly increased current densities of Ca(L) in both SUS and control rats, the increase of Ca(L) current density in SUS rats was much more than that in control rats. These results suggest that 3-day simulated weightlessness induces the adaptational change of Ca(L) in cerebral VSMCs including increased response to Ang II, indicating that Ang II may play an important role in the adaptational change of cerebral arteries under microgravity.     

Mitochondrial Regulation of NADPH Oxidase in Hindlimb Unweighting Rat Cerebral Arteries

Exposure to microgravity results in post-flight cardiovascular deconditioning and orthostatic intolerance in astronauts. Vascular oxidative stress injury and mitochondrial dysfunction have been indicated in this process. To elucidate the mechanism for this condition, we investigated whether mitochondria regulated NADPH oxidase in hindlimb unweighting (HU) rat cerebral and mesenteric arteries. Four-week HU was used to simulate microgravity in rats. Vascular superoxide generation, protein and mRNA levels of Nox2/Nox4, and the activity of NADPH oxidase were examined in the present study. Compared with control rats, the levels of superoxide increased in cerebral (P<0.001) but not in mesenteric vascular smooth muscle cells. The protein and mRNA levels of Nox2 and Nox4 were upregulated significantly (P<0.001 and P<0.001 for Nox2, respectively; P<0.001 and P<0.001 for Nox4, respectively) in HU rat cerebral arteries but not in mesenteric arteries. NADPH oxidases were activated significantly by HU (P<0.001) in cerebral arteries but not in mesenteric arteries. Chronic treatment with mitochondria-targeted antioxidant mitoTEMPO attenuated superoxide levels (P<0.001), decreased the protein and mRNA expression levels of Nox2/Nox4 (P<0.01 and P<0.05 for Nox2, respectively; P<0.001 and P<0.001 for Nox4, respectively) and the activity of NADPH oxidase (P<0.001) in HU rat cerebral arteries, but exerted no effects on HU rat mesenteric arteries. Therefore, mitochondria regulated the expression and activity of NADPH oxidases during simulated microgravity. Both mitochondria and NADPH oxidase participated in vascular redox status regulation.     

Regulation of the cerebrovascular smooth muscle cell phenotype by mitochondrial oxidative injury and endoplasmic reticulum stress in simulated microgravity rats via the PERK-eIF2α-ATF4-CHOP pathway

Microgravity exposure results in vascular remodeling and cardiovascular dysfunction. Here, the effects of mitochondrial oxidative stress on vascular smooth muscle cells (VSMCs) in rat cerebral arteries under microgravity simulated by hindlimb unweighting (HU) was studied. Endoplasmic reticulum (ER)-resident transmembrane sensor proteins and phenotypic markers of rat cerebral VSMCs were examined. In HU rats, CHOP expression was increased gradually, and the upregulation of the PERK-eIF2α-ATF4 pathway was the most pronounced in cerebral arteries. Furthermore, PERK/p-PERK signaling, CHOP, GRP78 and reactive oxygen species were augmented by PERK overexpression but attenuated by the mitochondria-targeting antioxidant MitoTEMPO. Meanwhile, p-PI3K, p-Akt and p-mTOR protein levels in VSMCs were increased in HU rat cerebral arteries. Compared with the control, HU rats exhibited lower α-SMA, calponin, SM-MHC and caldesmon protein levels but higher OPN and elastin levels in cerebral VSMCs. The cerebral VSMC phenotype transition from a contractile to synthetic phenotype in HU rats was augmented by PERK overexpression and 740Y-P but reversed by MitoTEMPO and the ER stress inhibitors tauroursodeoxycholic acid (TUDCA) and 4-phenylbutyric acid (4-PBA). In summary, mitochondrial oxidative stress and ER stress induced by simulated microgravity contribute to phenotype transition of cerebral VSMCs through the PERK-eIF2a-ATF4-CHOP pathway in a rat model.     

四周模拟失重大鼠后身动脉平滑肌细胞钾电流的改变

本文采用全细胞膜片钳方法观察4周尾部悬吊大鼠（tail-suspended rats,SUS)隐动脉及肠系膜的动脉第2-6级动脉分支血管平滑肌细胞（vascular smooth muscle cells,VSMCs)钾电流密度的变化,结果表明：SUS大鼠后身动脉VSMCs的静息电位（RP）较对照大鼠（CON）后身动脉VSMCs的RP更负,SUS组隐动脉和肠系膜小鼠后身动脉VSMCs的静息电位（RP）较对照大鼠（CON）后身动脉VSMCs的RP更负,SUS组隐动脉和肠系膜小动脉VSMCs的全细胞钾电流密度较CON组显著增加,其中,SUS组的隐动脉和肠系膜小动脉VSMCs的大电导钙激活钙离子通道（BKca)和电压激活钾离子通道（Kv)电流密度较CON组的BKca和Kv电流密度均显著增加,以上结果提示,VSMCs的超极化及进一步引起的通过电压依赖性钙离子通道的钙内流减少可能是模拟失重引起后身动脉反应性降低的电生理机制之一。     

模拟失重增强大鼠脑动脉血管平滑肌细胞大电导钙激活钾通道功能

本工作旨在探讨短期模拟失重大鼠脑动脉血管平滑肌细胞（vascular smooth muscle cells,VSMCs）大电导钙激活钾通道（large conductance calcium-activated potassium channels,BKCa channels）功能的改变。以尾部悬吊大鼠模型模拟失重对脑血管的影响。应用激光扫描共聚焦显微镜测定VSMCs胞内游离钙浓度（[Ca^2＋]i）;采用细胞贴附模式,记录BKCa通道的单通道活动。结果表明,模拟失重1周后,大鼠脑动脉VSMCs的[Ca^2＋]i比对照组显著升高（P〈0．05）：BKCa通道的开放概率（Po）与平均开放时间（To）显著增加（P〈0．05）,而单通道电导与平均关闭时间（Tc）则无显著变化。总之,1周模拟失重可引起脑动脉VSMCs的BKCa通道功能显著增强,且与细胞[Ca^2＋]i的升高同步出现。结果提示,脑动脉VSMCs的离子通道机制可能参与介导模拟失重引起的脑血管适应性变化。     

Simulated microgravity alters rat mesenteric artery vasoconstrictor dynamics through an intracellular Ca(2+) release mechanism

Previous work has shown that orthostatic hypotension associated with cardiovascular deconditioning results from inadequate peripheral vasoconstriction. We used the hindlimb-unloaded (HU) rat in this study as a model to induce cardiovascular deconditioning. The purpose of this study was to test the hypothesis that 14 days of HU diminishes vasoconstrictor responsiveness of mesenteric resistance arteries. Mesenteric resistance arteries from control (n = 43) and HU (n = 44) rats were isolated, cannulated, and pressurized to 108 cm H(2)O for in vitro experimentation. Myogenic (intralumenal pressure ranging from 30 to 180 cm H(2)O), KCl (2-100 mM), norepinephrine (NE, 10(-9)-10(-4) M) and caffeine (1-20 mM) induced vasoconstriction, as well as the temporal dynamics of vasoconstriction to NE, were determined. The active myogenic and passive pressure responses were unaltered by HU when pressures remained within physiological range. However, vasoconstrictor responses to KCl, NE, and caffeine were diminished by HU, as well as the rate of constriction to NE (C, 14.8 +/- 3.6 microm/s vs. HU 7.6 +/- 1.8 microm/s). Expression of sarcoplasmic reticulum Ca(2+)ATPase 2 and ryanodine 3 receptor mRNA was unaffected by HU, while ryanodine 2 receptor mRNA and protein expression were diminished in mesenteric arteries from HU rats. These data suggest that HU-induced and microgravity-associated orthostatic intolerance may be due, in part, to an attenuated vasoconstrictor responsiveness of mesenteric resistance arteries resulting from a diminished ryanodine 2 receptor Ca(2+) release mechanism.     

Differential regulation and recovery of intracellular Ca2+ in cerebral and small mesenteric arterial smooth muscle cells of simulated microgravity rat

Background

The differential adaptations of cerebrovasculature and small mesenteric arteries could be one of critical factors in postspaceflight orthostatic intolerance, but the cellular mechanisms remain unknown. We hypothesize that there is a differential regulation of intracellular Ca²⁺ determined by the alterations in the functions of plasma membrane Ca_L channels and ryanodine-sensitive Ca²⁺ releases from sarcoplasmic reticulum (SR) in cerebral and small mesenteric vascular smooth muscle cells (VSMCs) of simulated microgravity rats, respectively.

Methodology/Principal Findings

Sprague-Dawley rats were subjected to 28-day hindlimb unweighting to simulate microgravity. In addition, tail-suspended rats were submitted to a recovery period of 3 or 7 days after removal of suspension. The function of Ca_L channels was evaluated by patch clamp and Western blotting. The function of ryanodine-sensitive Ca²⁺ releases in response to caffeine were assessed by a laser confocal microscope. Our results indicated that simulated microgravity increased the functions of Ca_L channels and ryanodine-sensitive Ca²⁺ releases in cerebral VSMCs, whereas, simulated microgravity decreased the functions of Ca_L channels and ryanodine-sensitive Ca²⁺ releases in small mesenteric VSMCs. In addition, 3- or 7-day recovery after removal of suspension could restore the functions of Ca_L channels and ryanodine-sensitive Ca²⁺ releases to their control levels in cerebral and small mesenteric VSMCs, respectively.

Conclusions

The differential regulation of Ca_L channels and ryanodine-sensitive Ca²⁺ releases in cerebral and small mesenteric VSMCs may be responsible for the differential regulation of intracellular Ca²⁺, which leads to the altered autoregulation of cerebral vasculature and the inability to adequately elevate peripheral vascular resistance in postspaceflight orthostatic intolerance.     

Functional alterations in cerebrovascular K(+) and Ca(2+) channels are comparable between simulated microgravity rat and SHR

Exposure to microgravity leads to a sustained elevation in transmural pressure across the cerebral vasculature due to removal of hydrostatic pressure gradients. We hypothesized that ion channel remodeling in cerebral vascular smooth muscle cells (VSMCs) similar to that associated with hypertension may occur and play a role in upward autoregulation of cerebral vessels during microgravity. Sprague-Dawley rats were subjected to 4-wk tail suspension (Sus) to simulate the cardiovascular effect of microgravity. Large-conductance Ca(2+)-activated K(+) (BK(Ca)), voltage-gated K(+) (K(V)), and L-type voltage-dependent Ca(2+) (Ca(L)) currents of Sus and control (Con) rat cerebral VSMCs were investigated with a whole cell voltage-clamp technique. Under the same experimental conditions, K(V), BK(Ca), and Ca(L) currents of cerebral VSMCs from adult spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) were also investigated. K(V) current density decreased in Sus rats vs. Con rats [1.07 +/- 0.14 (n = 22) vs. 1.31 +/- 0.28 (n = 16) pA/pF at +20 mV (P < 0.05)] and BK(Ca) and Ca(L) current densities increased [BK(Ca): 1.70 +/- 0.37 (n = 23) vs. 0.88 +/- 0.22 (n = 19) pA/pF at +20 mV (P < 0.05); Ca(L): -2.17 +/- 0.21 (n = 35) vs. -1.31 +/- 0.10 (n = 26) pA/pF at +10 mV (P < 0.05)]. Similar changes were also observed in SHR vs. WKY cerebral VSMCs: K(V) current density decreased [1.03 +/- 0.33 (n = 9) vs. 1.62 +/- 0.64 (n = 9) pA/pF at +20 mV (P < 0.05)] and BK(Ca) and Ca(L) current densities increased [BK(Ca): 2.54 +/- 0.47 (n = 11) vs. 1.12 +/- 0.33 (n = 12) pA/pF at +20 mV (P < 0.05); Ca(L): -3.99 +/- 0.53 (n = 12) vs. -2.28 +/- 0.20 (n = 10) pA/pF at +20 mV (P < 0.05)]. These findings support our hypothesis, and their impact on space cardiovascular research is discussed.     

模拟失重大鼠肠系膜小动脉平滑肌细胞电压依赖性钙离子通道电流的改变

本工作旨在探讨短、中期模拟失重下人鼠肠系膜小动脉血管平滑肌细胞(vascular smooth muscle cells,VSMCs)电压依赖性钙离子通道(voltage-dependent calcium channels,VDC)功能的改变。以尾部悬吊大鼠模型模拟失重对不同部位血管的影响。采用全细胞膜片钳实验技术,以Ba^2 作为载流子,测定1周及4周模拟失重人鼠肠系膜小动脉VSMCs的VDC电流密度、稳态激活与失活曲线及有关参数,并与对照组结果进行比较。研究表明,本实验所记录到的内向电流主要为钡离子通过长时程VDC(L-VDC)所形成的电流。与对照组相比,1周模拟失重大鼠肠系膜小动脉VSMCs的L-VDc电流密度仪呈降低趋势;但4周模拟失重人鼠肠系膜小动脉VSMCs的L-VDC电流密度则已显著降低。此外,与对照组相比,1、4周模拟失重大鼠肠系膜小动脉VSMCs的膜电容、翻转电位与L-VDC的一些动力学特征值,如通道的开放与关闭速率,通道电流稳态激活与火活曲线及其特征拟合参数V0.5与K的值,均末见有显著改变。结果提示：模拟失重下后身小动脉VSMCs的VDC功能降低可能是模拟失重引起人鼠后身动脉收缩反应性降低及适应性萎缩变化的电生理机制之一。     

Effectiveness of intermittent -Gx gravitation in preventing deconditioning due to simulated microgravity.

This study was designed to compare the effectiveness of daily short-duration -Gx gravity exposure in preventing adverse changes in skeletal and cardiac muscles and bone due to simulated microgravity. Tail suspension for 28 days was used to simulate microgravity-induced deconditioning effects. Daily standing (STD) at 1 G for 1, 2, or 4 h/day or centrifugation (CEN) at 1.5 or 2.6 G for 1 h/day was used to provide -Gx gravitation as a countermeasure. The results indicate that the minimum gravity exposure requirements vary greatly in different systems. Cardiac muscle is most responsive to such treatment: 1 h/day of -Gx gravitation by STD was sufficient to prevent adverse changes in myocardial contractility; bone is most resistant: 4 h/day of -Gx gravitation only partially alleviated the adverse changes in physical and mechanical properties of the femur. The responsiveness of skeletal muscle is moderate: 4 h/day of -Gx gravitation prevented mass reduction and histomorphometric changes in the soleus muscle during a 28-day simulation period. Increasing gravitational intensity to 2.6 G showed less benefit or no additional benefit in preventing adverse changes in muscle and bone. The present work suggests that system specificity in responsiveness to intermittent gravity exposure should be considered one of the prerequisites in proposing intermittent artificial gravity as a potential countermeasure.     

Changes of angiotensinogen expression in arteries of tail-suspended rats

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. Local renin-angiotensin system (L-RAS) has been found to be actively involved in the remodeling of arteries. We hypothesized that L-RAS may function as a local regulatory mechanism in the microgravity-induced differential changes of arterial vessles. Angiotensinogen (AGT) is the only and indispensable substrate of local renin-angiotensin system (L-RAS). In the present work, the expression changes of AGT mRNA and protein level as well as its time course characteristics were examined.     

Exercise within lower body negative pressure partially counteracts lumbar spine deconditioning associated with 28-day bed rest.

Astronauts experience spine deconditioning during exposure to microgravity due to the lack of axial loads on the spine. Treadmill exercise in a lower body negative pressure (LBNP) chamber provides axial loads on the lumbar spine. We hypothesize that daily supine LBNP exercise helps counteract lumbar spine deconditioning during 28 days of microgravity simulated by bed rest. Twelve sets of healthy, identical twins underwent 6 degrees head-down-tilt bed rest for 28 days. One subject from each set of twins was randomly assigned to the exercise (Ex) group, whereas their sibling served as a nonexercise control (Con). The Ex group exercised in supine posture within a LBNP chamber for 45 min/day, 6 days/wk. All subjects underwent magnetic resonance imaging of their lumbar spine before and at the end of bed rest. Lumbar spinal length increased 3.7 +/- 0.5 mm in the Con group over 28-day bed rest, whereas, in the Ex group, lumbar spinal length increased significantly less (2.3 +/- 0.4 mm, P = 0.01). All lumbar intervertebral disk heights (L5-S1, L4-5, L3-4, L2-3, and L1-2) in the Con group increased significantly over the 28-day bed rest (P < 0.05). In the Ex group, there were no significant increases in L5-S1 and L4-5 disk heights. Lumbar lordosis decreased significantly by 3.3 +/- 1.2 degrees during bed rest in the Con group (P = 0.02), but it did not decrease significantly in the Ex group. Our results suggest that supine LBNP treadmill exercise partially counteracts lumbar spine lengthening and deconditioning associated with simulated microgravity.     

NAD(P)H oxidase inhibiting with apocynin improved vascular reactivity in tail-suspended hindlimb unweighting rat

Recent studies suggested that reactive oxygen species derived from nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase is of functional importance in modulating vascular tone, and we have previously detected excessive superoxide production in tail-suspended hindlimb unweighting (HU) rat cerebral and carotid arteries. HU rat was a widely used model to simulate physiological effects on the vasculature. The present study tended to investigate whether NAD(P)H oxidase inhibition with apocynin influences vasoconstriction, endothelium-dependent relaxation, and nitrite/nitrate (NOx) content in HU rat cerebral and carotid arteries. Vascular contractile and dilate responses were assessed in a myograph organ bath. NOx content in cerebral and carotid arteries was measured. We found enhanced maximal contractile response and impaired endothelium-dependent relaxation in HU rat basilar (P < 0.01) and common carotid artery (P < 0.05); however, chronic treatment of apocynin (50 mg/kg/day) partially reversed abnormal vascular response. Furthermore, 21-day HU increased arterial NOx content (P < 0.01) in cerebral and carotid arteries compared with control rats; however, apocynin treatment restored it toward near-normal values. These data demonstrated that NAD(P)H oxidase-derived oxidative stress mediated abnormal vasoreactivity though nitric oxide mechanism in the settings of simulated microgravity.     

Biomechanical and phenotypic changes in the vasospastic canine basilar artery after subarachnoid hemorrhage.

Because it has been argued that active myogenic tone prolongs cerebral vasospasm for >2 wk after subarachnoid hemorrhage (SAH), we attempted to identify the mechanism that plays the main role in sustaining the prolonged cerebral vasospasm. We especially focused on the roles of biomechanical and phenotypic changes in the cerebral arteries in the mechanisms of prolonged vasospasm after SAH. We used the basilar arteries from a "two-hemorrhage" canine model to make serial measurements of maximal contraction capacity and arterial stiffness (papaverine-insensitive tone) until day 28. We also examined hematoxylin-eosin-stained vasospastic canine basilar arteries for histological changes and immunohistochemically examined them for expression of myosin heavy chain isoforms (SMemb, SM1, and SM2), which are markers of smooth muscle phenotypic changes. Changes in collagen concentration in canine basilar arteries were also measured. Angiographic cerebral vasospasm persisted until day 14 and then gradually diminished; artery diameter returned to the control diameters on day 28. Maximal contraction capacity decreased until day 21 and showed some recovery by day 28. Arterial stiffness, on the other hand, progressed until day 28. Histological examination revealed medial thickening and increased connective tissue until day 21 and a return to control findings by day 28. The increased connective tissue was not accompanied by changes in collagen concentration, suggesting a role of some other protein in the increase in connective tissue. Immunohistochemical studies with anti-SMemb, anti-SM1, and anti-SM2 antibodies showed enhanced expression of SMemb from day 7 to day 21 and disappearance of SM1 and SM2 on days 14 and 21. The changes in myosin heavy chain isoform expression returned to normal on day 28. The above results indicate that biomechanical and phenotypic changes may play a pivotal role in sustaining cerebral vasospasm for >2 wk after SAH, with minimal changes in active myogenic arterial tone.     

Role of iPLA2 and store-operated channels in agonist-induced Ca2+ influx and constriction in cerebral, mesenteric, and carotid arteries

Store-operated channels (SOC) and store-operated Ca2+ entry are known to play a major role in agonist-induced constriction of smooth muscle cells (SMC) in conduit vessels. In microvessels the role of SOC remains uncertain, in as much as voltage-gated L-type Ca2+ (Ca2+L) channels are thought to be fully responsible for agonist-induced Ca2+ influx and vasoconstriction. We present evidence that SOC and their activation via a Ca2+-independent phospholipase A2 (iPLA2)-mediated pathway play a crucial role in agonist-induced constriction of cerebral, mesenteric, and carotid arteries. Intracellular Ca2+ in SMC and intraluminal diameter were measured simultaneously in intact pressurized vessels in vitro. We demonstrated that 1) Ca2+ and contractile responses to phenylephrine (PE) in cerebral and carotid arteries were equally abolished by nimodipine (a Ca2+L) inhibitor) and 2-aminoethyl diphenylborinate (an inhibitor of SOC), suggesting that SOC and Ca2+L channels may be involved in agonist-induced constriction of cerebral arteries, and 2) functional inhibition of iPLA2beta totally inhibited PE-induced Ca2+ influx and constriction in cerebral, mesenteric, and carotid arteries, whereas K+-induced Ca2+ influx and vasoconstriction mediated by Ca2+L channels were not affected. Thus iPLA2-dependent activation of SOC is crucial for agonist-induced Ca2+ influx and vasoconstriction in cerebral, mesenteric, and carotid arteries. We propose that, on PE-induced depletion of Ca2+ stores, nonselective SOC are activated via an iPLA2-dependent pathway and may produce a depolarization of SMC, which could trigger a secondary activation of Ca2+L channels and lead to Ca2+ entry and vasoconstriction.     

In-vivo and ex-vivo studies on region-specific remodeling of large elastic arteries due to simulated weightlessness and its prevention by gravity-based countermeasure

The present study was designed to test the hypothesis that a medium-term simulated microgravity can induce region-specific remodeling in large elastic arteries with their innermost smooth muscle (SM) layers being most profoundly affected. The second purpose was to examine whether these changes can be prevented by a simulated intermittent artificial gravity (IAG). The third purpose was to elucidate whether vascular local renin-angiotensin system (L-RAS) plays an important role in the regional vascular remodeling and its prevention by the gravity-based countermeasure. This study consisted of two interconnected series of in-vivo and ex-vivo experiments. In the in-vivo experiments, the tail-suspended, hindlimb unloaded rat model was used to simulate microgravity-induced cardiovascular deconditioning for 28 days (SUS group); and during the simulation period, another group was subjected to daily 1-hour dorso-ventral (-G(x)) gravitation provided by restoring to normal standing posture (S + D group). The activity of vascular L-RAS was evaluated by examining the gene and protein expression of angiotensinogen (Ao) and angiotensin II receptor type 1 (AT1R) in the arterial wall tissue. The results showed that SUS induced an increase in the media thickness of the common carotid artery due to hypertrophy of the four SM layers and a decrease in the total cross-sectional area of the nine SM layers of the abdominal aorta without significant change in its media thickness. And for both arteries, the most prominent changes were in the innermost SM layers. Immunohistochemistry and in situ hybridization revealed that SUS induced an up- and down-regulation of Ao and AT1R expression in the vessel wall of common carotid artery and abdominal aorta, respectively, which was further confirmed by Western blot analysis and real time PCR analysis. Daily 1-hour restoring to normal standing posture over 28 days fully prevented these remodeling and L-RAS changes in the large elastic arteries that might occur due to SUS alone. In the ex-vivo experiments, to elucidate the important role of transmural pressure in vascular regional remodeling and differential regulation of L-RAS activity, we established an organ culture system in which rat common carotid artery, held at in-vivo length, can be perfused and pressurized at varied flow and pressure for 7 days. In arteries perfused at a flow rate of 7.9 mL/min and pressurized at 150 mmHg, but not at 0 or 80 mmHg, for 3 days led to an augmentation of c-fibronectin (c-FN) expression, which was also more markedly expressed in the innermost SM layers, and an increase in Ang II production detected in the perfusion fluid. However, the enhanced c-FN expression and increased Ang II production that might occur due to a sustained high perfusion pressure alone were fully prevented by daily restoration to 0 or 80 mmHg for a short duration. These findings from in-vivo and ex-vivo experiments have provided evidence supporting our hypothesis that redistribution of transmural pressures might be the primary factor that initiates region-specific remodeling of arteries during microgravity and the mechanism of IAG is associated with an intermittent restoration of the transmural pressures to their normal distribution. And they also provide support to the hypothesis that L-RAS plays an important role in vascular adaptation to microgravity and its prevention by the IAG countermeasure.     

Daily 4-h head-up tilt is effective in preventing muscle but not bone atrophy due to simulated microgravity

To assess the potential value of intermittent artificial gravity as an efficient countermeasure, our previous studies have showed that daily 4-h standing (STD) is sufficient in counteracting muscle atrophy but not bone atrophy induced by simulated microgravity. The aim of the present study was to determine whether intermittent gravitational loading by daily 2-h or 4-h, +45 degrees head-up tilt (HUT) is more effective than STD in counteracting muscle and, particularly, bone atrophy due to simulated microgravity. Sprague-Dawley male rats weighing 290-300 g were subjected to a 28-d tail-suspension to simulate microgravity deconditioning. Daily HUT for 2, or 4 h was used to provide intermittent gravitational loading in foot-ward and tail-ward directions. The results showed that 4 h/d HUT was sufficient, and 2 h/d was less effective, in preventing adverse changes in muscle weights, fiber types, and cross-sectional areas (CSA) of muscles due to a 28-d simulated microgravity. The % protections by 4 h/d HUT in maintaining the CSAs of type I fibers in soleus, medial and lateral gastrocnemius and extensor digitorum longus muscles were 103%, 82%, 102%, and 83%, respectively. However, according to changes in physical and mechanical properties of femur, daily 4-h HUT was ineffective in attenuating the adverse changes in bone due to a 28-d simulated microgravity. Reductions in wet, dry, and ash weights and decreases in mechanical strength of femur did not show significant improvement by daily 2-h or 4-h HUT. Taken together, the findings indicate that the countermeasure effectiveness of daily 2-h or 4-h HUT for muscles is comparable with that by daily STD with the same durations. Daily 4-h HUT, as 4-h STD, is also ineffective in attenuating adverse changes in bone mass, but seems partially effective in preventing declines in mechanical properties due to simulated microgravity.