In both sexes, orthostatic responses are impaired by spaceflight or head-down bed-rest (HDBR), with a greater impact in women. We hypothesized that cardiovascular differences arise as soon as 4 hours after HDBR, and that responses will be poorer in women. We measured heart rate (HR), mean arterial blood pressure (MAP; Finometer), cardiac output (Q; Doppler ultrasound), stroke volume (SV; Doppler ultrasound), central venous pressure (CVP), and total peripheral resistance (TPR) in response to lower body negative pressure (LBNP; 0 to -40mmHg) in men and women before and after 4-hr HDBR or seated control (SEAT). We studied women on day 8-11 after menstruation (all subjects on oral birth control). In both men and women, LBNP resulted in increased HR, decreased CVP, decreased SV, increased TPR and decreased Q (HDBR and SEAT). In women, 4-hr SEAT tended to result in lower resting CVP (P=0.1), lower SV at -40mmHg (P=0.095), and higher TPR at -40mmHg (P=0.081). After 4-hr HDBR: 1) men exhibit lower MAP (P=0.051) with no MAP differences in women (P=0.913), 2) both men and women exhibit a higher HR response to -40mmHg (Men: P=0.024; Women: P=0.038), 3) men and women showed lower resting CVP (Men: P=0.054; Women: P=0.009) which was not different than pre-HDBR at -40mmHg (Men: P=0.661; Women: P=0.126), 4) women show higher resting SV (P=0.052) and Q (P=0.101) which was not different than pre-HDBR at -40mmHg (SV: P=0.449; Q: P=0.396), 5) no differences in SV or Q were observed in men, 6) baseline TPR in women was unchanged (P=0.346); however at -40mmHg TPR was significantly lower than pre-HDBR (P=0.040), and 7) no differences in TPR were observed in men. Our results indicate that the inactivity and/or circadian rhythm of bed-rest (as shown by the seated position) may be an important factor to consider when studying women. We have also shown that after 4-hr HDBR, women have poorer increases in TPR and greater drops in SV which could partially account for greater orthostatic hypotension. Future analysis of this project will determine changes in venous compliance and vasoactive hormones. We will also be studying the same women on day 18-24 after menstruation, and we will be comparing these women to those not on oral birth control. Funding provided by the CSA (Space Science Enhancement Program).

We investigated fluid shifts and regulatory responses to variations of posture, exercise, Gz level and radius of rotation in subjects riding NASA Ames' 20G centrifuge. Results are from 4 protocols that address radius and exercise effects only.

Protocol A: After 10 min supine control, 12 healthy men (35 ± 9 yr, 82.8 ± 7.9 kg) were exposed to rotational 1 Gz (2.5 m radius) for 2 min followed by 20 min alternating between 1 and 1.25 Gz. Blood samples were taken pre and post spin.

Protocol B: Same as A, but lower limb exercise (70% V02max) preceded ramps to 1.25 Gz.

Protocol C: Same as A but radius of rotation 8.3 m.

Protocol D: Same as B but at 8.3 m.

RESULTS: The 8 subjects who completed all protocols, increased heart rate (HR) from control, on average, by: A: 5, B: 39, C: 11, D: 44 bpm. For thoracic fluid volume, (bioimpedance), the 8 subjects changed from control, on average: A: -394, B: -548, C: -537, D: -708 mL. For thigh fluid volume, changes from control, on average, were: A: -137, B: 129, C: -75, D: 159 mL. Hematocrit changes from control were: A: 2.3, B: 3.5, C: 2.3, D: 4.3 %.

SUMMARY: Radius effects were mild and included greater loss of fluid from the thorax, less fluid loss from the thigh and increased heart rate at the longer radius. Pre-acceleration exercise effects were more dramatic and included additional loss of fluid from the chest, increased fluid volume of the thigh, increased hematocrit and greater heart rate increases. We propose that short bouts of intense exercise can be used to magnify the cardiovascular stress delivered by artificial gravity (AG) training and the combination of AG with exercise training may be fine-tuned to preserve orthostatic tolerance of astronauts during spaceflight. Supported by NASA EPSCoR WKU52611 and Ames Res. Center.

Human spaceflight is associated with a loss of body protein most likely caused by muscle degradation. Additionally astronauts tend towards a high dietary intake of sodium chloride (NaCl), which has recently been shown to induce low grade metabolic acidosis (Frings-Meuthen et al. JBMR, Epub 2007). In several patterns, e.g. chronical renal failure, metabolic acidosis is associated with protein catabolism. We therefore hypothesized that high dietary intake of NaCl enforces protein losses in HDBR, a model for physiological changes in microgravity (µG). Eight healthy male subjects (mean age 26.25 ± 3.5; mean body weight: 78.5 ± 4.1 kg) participated in a 14-day bed rest study in the metabolic ward of the DLR - Institute of Aerospace Medicine, Cologne, Germany. The study was carried out in a cross over design, consisting of two phases, each lasting 22 days (5 days adaptation, 14 days 6° HDBR and 3 days recovery). Both study phases were identical with respect to environmental conditions and study protocol. Subjects received an individually tailored, weight-maintaining diet containing 1.3 g protein/kg/day. The diet was identical in both study phases with the exception of NaCl-intake: Every subject received a low NaCl diet (0.7 mmol/kg/day) in one phase and a high NaCl diet (7.7 mmol/kg/day) in another one. Blood gas for analysis of acid-base balance was implemented at days 4 and 5 of adaptation, days 2, 5, 7, 10, 12, 14 of HDBR and days 2, 3 of recovery. Continuous urine collection started on the first day in the metabolic ward to analyze nitrogen excretion. Nitrogen balance was calculated from the difference between protein intake and urinary nitrogen excretion, determined by use of chemiluminescence. Plasma pH did not change significantly (p=0.285), but plasma bicarbonate and base excess decreased with high NaCl intake in HDBR compared to the low NaCl diet (bicarbonate high salt 25,2 ± 0,16 mmol/l, low salt 26,4 ± 0,39 mmol/l (p=0.0175); base excess high salt 0,88 ± 0,20, low salt 2,0 ± 0,44 mmol/l (p=0.0093)). Nitrogen balance in HDBR was negative, as expected in immobilization with low NaCl diet ( 0.34 ± 1.2 g/d). However, high NaCl intake in HDBR exacerbated the negative nitrogen balance to 1.34 ± 1.0 g/d (p<0.001) compared to low NaCl. We conclude that high dietary NaCl intake induces low grade metabolic acidosis during HDBR. Low grade metabolic acidosis may be a reason for an increased protein turnover reflected by an exaggerated negative nitrogen balance in HDBR. Accordingly, a high dietary NaCl intake may exacerbate loss of body protein in µG via low grade metabolic acidosis.

Cytoskeletal alterations occur in several cell types including lymphocytes, glial cells, and osteoblasts, during spaceflight and under simulated microgravity (SMG). One potential mechanism for cytoskeletal gravisensitivity of cell is disruption of extracellular matrix (ECM) and integrin interactions. Focal adhesions are specialized sites of cell-matrix interaction composed of integrins and the diversity of focal adhesion-associated cytoplasmic proteins including vinculin, talin, á-actinin, and actin filaments. Integrins are heterodimeric transmembrane receptors produce signals essential for proper cellular function, survival and differentiation. Binding of integrins to the ECM may facilitate the transfer of mechanical signals from the external environment across the membrane to activate intracellular signaling cascades. Therefore, we investigated the effects of SMG on F-actin cytoskeleton structure, vinculin focal adhesions, expression of some integrin subtypes and cellular adhesion molecules (CAMs) in mesenchymal stem cells derived from human bone marrow (hMSCs). Simulated microgravity was produced by 3D-clinistat (RPM, manufactured by Dutch Space, The Netherlands). Culture flasks with MSCs were settled on the inner platform of RPM for 30 minutes, 6, 24, 48 and 120 hours. Structure changes of F-actin (TRITC-phalloidin staining) and vinculin (anti-vinculin antibodies) were studied with fluorescent microscopy (AxioVert 25, Zeiss, Germany). The expression of integrin á2 (CD49b), á4 (CD49d) and â1 (CD29); ICAM-1 (CD54) and VCAM-1 (CD106) were evaluated by means of flow cytometry (Epics XL, Beckman Coulter, USA) after 120 hours of SMG. The results were compared with static (1g) and dynamic (shaker) controls.

Staining of actin fibers with TRITC-phalloidin showed reorganization even after 30 minutes of simulated microgravity. Randomization of gravity vector altered dimensional structure of stress fibers and resulted in redistribution of total actin inside the cells. After 6 hours of clinorotation number of cells with disrupted F-actin cytoskeleton increased. The mean value of F-actin fluorescence intensity on the same length of exposure decreased after 24 hours in RPM. In addition, we observed vinculin redistribution inside the cells after 6 hours and subsequent terms of rotation. This process was accompanied with increasing of fluorescence intensity of clinorotated cells relative to both controls. The expression of integrin á2 increased 1,5-3-fold in clinorotated hMSCs. Interestingly integrin á2 forms receptors to collagen and laminin in heterodimeric complexes with integrin â1 and has been reported to interact directly with F-actin. Also we observed decrease in number of VCAM-1-positive cells and changes in expression of ICAM-1. Taken together, our findings indicate that SMG leads to microfilament and adhesion alterations of hMSCs most probably associated with activation of certain integrin subtypes.

Multidrug resistance proteins (MRPs) are transmembrane proteins with the potential to export a wide range of substances into the extracellular space preventing cells from toxification. MRP4, 5, and 8, on which this investigation is focussed, particularly transport cyclic nucleotides, e.g., guanosine 3', 5'-cyclic monophosphate (cGMP) and other nucleoside analogues. cGMP plays an important signaling role in melanocytic physiology. In previous studies we could show that long-term exposure to hypergravity leads to an increased cGMP efflux in non-metastatic melanoma cell lines, whereas the highly metastatic phenotype appeared to be insensitive.

Biochemical analyses let assume that overexpressed MRP4, 5 and 8 could be responsible for these effects. Based on these results the aim of the present study was to investigate the effects of hypergravity (5xg for 24 h) on mRNA and protein levels of MRP4, 5, and/or 8 using a modified STATEX centrifuge from the spacelab mission D2. The mRNA analyses were carried out by a relative calibrator-normalized and efficiency corrected quantitative polymerase chain reaction (LightCycler^®, Roche). Protein levels were detected by flow cytometry with the FACScan (Becton Dickinson). Our data show that the mRNA as well as the protein levels of MRP4 and MRP5 were about 1.4-fold higher in non-metastatic melanoma cells exposed to hypergravity in comparison to 1xg controls. In contrast, the expressions of MRP4 and 5 in highly metastatic cells remained unaffected. For MRP8 we basically measured low mRNA and protein expression levels in all investigated cell lines independent of gravity alterations. Our studies indicate that the previously found elevated cGMP export in hypergravity could be a consequence of an upregulated expression of MRP4 and MRP5 in non-metastatic melanoma cells, whereas a MRP8-driven export can be excluded. Since enhanced MRP expression is responsible for a decreased drug uptake in cells, a gravity-modified MRP expression on long-term space flights can lead to an altered drug tolerance and efficiency in astronauts. Regarding this aspect as a critical factor for medical applications in space, investigations on MRP expression in simulated microgravity are currently under investigation.