Impact of altered gravity on aspects of cell biology.
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The paper reports the results of experiments with centrifugation and clinostating. Growth rate, cellular division and several morphofunctional characteristics of unicellular organisms of infusoria Bursaria truncatella in culture were studied under normal (1 g), elevated (hypergravity at 2 and 5 g), and compensated gravity. The data point to certain changes in the functional activity and morphology of cells consequent to long-time cultivation under these conditions. The observed regularities in the dynamics of B.truncatella growth and shifts in its physiology and morphology due to hypergravity or compensated gravity support our earlier proposed working hypothesis about the dominance of functional activity over morphological properties in sensitivity of unicellular organisms to perception and realization of the gravitational stimulus.
The effects of weightlessness on vestibular function have been studied since the beginning of manned spaceflight. The results of these studies have been highly variable and to some extent even contradictory, which makes it difficult to draw unambiguous conclusions. This variability is probably due to at least three factors: (1) individual differences in the adaptive process, (2) non-standardized experimental methods and conditions, (3) a lack of integrated experiments. For this reason, we have used a single integrated approach with a specially developed battery of tests. The results thus obtained for 21 cosmonauts on short- and long-term flights are reviewed here, and discussed in the light of the results obtained by others. Changes in the operation of the vestibular system and in all functions based on vestibular afferent input are commonly observed in spaceflight. These changes are characteristic for the process of adaptation and re-adaptation to altered gravity. They occur in a high proportion of persons exposed to such conditions, although there are individual differences with regard to severity, nature, time and duration of occurrence, and the dynamics of the process. Analysis of the observations in a large number of cosmonauts has permitted to distinguish three types of adaptation of the system to altered gravity. The first type of adaptation is characterized by a strong response to any stimulus during the initial adaptation period. The second type of adaptation is characterized by responses that are drastically decreased or even absent. The third type of adaptation is distinguished by the selective response of the sensory system to certain types of stimulation only. After long-term missions the process of re-adaptation usually takes a more severe course than the earlier process of adaptation to microgravity. Both adaptation and re-adaptation follow an undulating course, in which adaptation and re-adaptation are alternating. This is most conspicuous during long-term flights, and it suggests that in the initial stage of adaptation to weightlessness the vestibular input plays a dominant role, while at the end of the adaptation process the visual input prevails.
The effect of long-term (10 days) altered gravitational conditions upon succinate dehydrogenase (SDH) reactivity in total brain as well as in individual brain nuclei of developing cichlid fish larvae has been investigated by means of semiquantitative histochemical methods (densitometric grey value analysis). Increasing acceleration from near weightlessness (spaceflight) via 1 g controls to 3 g hypergravity (centrifuge) resulted in slightly increased total brain SDH reactivity. When focusing on distinct neuronal integration centres within the same brains in order to find the anatomical substratum of the gross histochemical data, significant effects of altered gravity on vestibulum-related brain parts were obtained. The total brain results may therefore represent the sum of such particular indirect effects but may also comprise in addition a non vestibular-related general and therefore direct influence of altered gravitational conditions, possibly on all cells.
To investigate whether changes in the gravitational field of developing neurons and myocytes affect cellular development, we rotated cultures of embryonic spinal neurons and myocytes in a horizontal clinostat. Rotation in the clinostat produces, from the cells' perspective, a "vector-free" gravity environment by continuous averaging of the vector. In this way, rotation in the clinostat simulates the microgravity of space where the gravity vector is substantially reduced. At rotation rates of 1-50 rpm, cellular and nuclear areas of myocytes were significantly enlarged and the number of presumptive nucleoli increased. In neurons, frequent and large swellings appeared along neuritic shafts. Some of these changes were reversible after cessation of rotation. Since our data are generally consistent with findings from other cell types subjected to spaceflight, we suggest that the vector-free gravity environment of the clinostat appears to simulate, at least in part, the microgravity of space. Our data further show that cellular processes are sensitive to altered gravity and suggest that cell development in the microgravity of space may be significantly altered.
Epidermal growth factor (EGF) activates a well characterized signal transduction system in human A431 epidermoid carcinoma cells, which leads to rapid and transient expression of the c-fos proto-oncogene. In order to investigate the influence of altered gravity on EGF-induced signal transduction, we have studied the EGF-induced c-fos expression under simulated hypo- and hypergravity conditions. In this report we show that EGF-induced fos expression is decreased under simulated hypogravity conditions, while hypergravity has a stimulatory effect on EGF-induced fos expression. These results show that the EGF-activated signal transduction system is influenced by gravity, and that gravity exerts its effects already in the early phases of the signal transduction cascade.
The mitochondrial enzyme, cytochrome oxidase, was localized cytochemically in the nucleus magnocellularis, a primary relay nucleus of vestibular information within the area octavolateralis in the fish brain. Cichlid fish larvae were analyzed after long-term exposure (9 days) to altered gravity situations: increased acceleration in a centrifuge (3 g) and near weightlessness during space flight. Controls (1 g) were reared under identical conditions in the centrifuge but without rotation on earth or with an acceleration resulting in gravity of 1 g in space shuttle. Quantification of highly reactive mitochondria reveals a correlation of gravity and cytochrome oxidase activity: low enzyme activity in respect to 1 g controls under near weightlessness conditions and an increased activity after hyper-g exposure in a centrifuge. This gravity effect on the energy metabolism of vestibular nuclei of developing cichild fish seems to reflect adaptational processes in response to gravity stimulation.
Glucose-6-phosphate dehydrogenase activity was studied in the brain of the cichlid fish Oreochromis mossambicus during early ontogenetic development. In general a slight but continuous decrease in enzyme activity was found (9.5 +/- 0.5 nmol substrate cleaved per mg protein and per min at developmental stage 13 [= 1 day post hatch at 28 degrees C] to a value of 7.9 +/- 0.6 in adult brain). In order to investigate the possible influence of altered gravity during early ontogenetic brain development, fish larvae were exposed to an increased acceleration of three times earth gravity (3 g) or to functional weightlessness in a fast-rotating clinostat for 7 days. A significant increase of brain G6PDH activity of approx. 15% was found after exposure to hyper gravity, whereas a significant decrease of the enzyme activity, approximately 10%, was detected following functional weightlessness in respect to the corresponding 1 g controls. Analyses concerning the regain of normal control enzyme activity of the larvae revealed dramatic fluctuations within the first 5 h after exposure to an increased acceleration of 3 g. Thereafter, between day 1 and day 3 after exposure, brain glucose-6-phosphate dehydrogenase decreased slowly. At day 3 after exposure no further differences of the hyper-g larvae compared to the controls were found. Only slight changes in total brain glucose-6-phosphate dehydrogenase activity occur during ontogenetic development of cichlid fish. This suggests that a more or less constant enzyme activity is important during brain development, but is reacting very sensitively to changes in the environmental factor gravity.
The regional metabolic activity in the otolithic sensory epithelia of the inner ear of a cichlid fish (Oreochromis mossambicus) was investigated on light- and electronmicroscopical level using the cytochemical method for detection of cytochrome oxidase activity. In adult animals a characteristic distribution of mitochondria with high enzyme activity was found in sensory and non-sensory cells of otolithic sensory epithelia, which was correlated with regions with a high energy demand. These findings were the basis for studies on the influence of long-term altered gravity conditions in developing larvae: hypogravity (10(-4) g in spaceflight), normal gravity (1 g in a centrifuge in space and 1 g on earth) and hypergravity (3 g in a laboratory centrifuge). Cytochrome oxidase activity was quantified in different parts of the sensory hair cell synapse in the vestibular sensory epithelia utricle and saccule: apical and basal cytoplasm, postsynaptic area of the afferent synapse and presynaptic region of the efferent synapse. Our results show that the energy metabolism of utricle, but not of saccule is decreased after microgravity exposure during the 2nd German Spacelab Mission D-2. However, a general effect of the spaceflight is detectable in both sensory epithelia. Long-term exposure to increased acceleration (3 g) had no effects on cytochrome oxidase activity in inner ear sensory epithelia.
Morphological and biochemical analyses of heart muscle of rats subjected to microgravity on Spacelab 3 (SL-3) flight and rats born and reared under increased gravity (1.7 G) conditions were compared with 1-G controls. Electronmicroscopic studies showed an increase in the number of lipid droplets and in areas of glycogen storage. Distribution changes of microtubules and cytoskeletal elements from both SL-3 and 1.7-G groups were observed. The high Km cyclic AMP phosphodiesterase activity was lower (P less than 0.05) in SL-3 heart muscle, and low Km activity was lower in 1.7-G males but was unaltered in females. Cyclic AMP-dependent protein kinase (cA-PK) activity was decreased in subcellular fractions of heart muscle of SL-3 animals. Recompartmentalization of cA-PK activity occurred in particulate tissue fraction of 1.7-G animals (70.3% of total for 1.7 G vs. 35.9% for controls). Phosphorylation of endogenous low-mobility proteins increased in SL-3 heart-soluble fractions. Photoaffinity labeling (18 h, 4 degrees C) decreased in type II cA-PK regulatory (R) subunits in both SL-3 and in 1.7-G male heart tissue particulate fractions. The 1.7-G female heart R subunit distribution did not differ from controls. These findings indicate that in heart muscle altered gravity conditions influenced physiological reactions similar to catecholamine-induced receptor-mediated hormonal responses.
A model was developed for evaluation of cardiovascular parameters in conscious baboons exposed to altered gravitational environments. Baboons were trained to sit quietly in a confinement chair of unique design which allowed a range of normal physical activity. They were then instrumented with high-fidelity blood pressure transducers in the aorta and left ventricle, electromagnetic flow probes encircling the proximal ascending aorta, left and right atrial fluid catheters, left ventricular sonomicrometer crystals in a 3-axis configuration, and a hydraulic occluder cuff encircling the inferior vena cava. Catheters and transducer wires were exteriorized at the midscapular region of the back. Viability of percutaneous exit sites was enhanced by use of velour cuffs on the transducer wires, providing a scaffold for wound healing. Pressure transducers and flow probes were calibrated and balanced during postoperative cardiac catheterization procedures. This instrumentation allowed measurement of beat-to-beat stroke volume and cardiac output not reliant on thermodilution techniques. Postoperative longevity was from 1 to 10 months. Instrumentation failure included endocardial trapping of ventricular pressure transducers, corrosion of ventricular sonomicrometer crystals, and catheter tip thrombosis. Acquisition of high quality data was possible with this model in several different environments of altered gravitational stress, allowing characterization of aortic flow and ventricular performance.
The mitochondrial enzyme, cytochrome oxidase, was localized cytochemically in the nucleus magnocellularis, a primary relay nucleus of vestibular information within the area octavolateralis in the fish brain. Larvae of the cichlid fish Oreochromis mossambicus were analyzed at different developmental stages (4, 10, and 35 days post-hatching) and after long-term exposure (8 days) to increased gravity (2-4 g). Quantification of highly reactive, moderately reactive, and nonreactive mitochondria reveals differences in the cytochrome oxidase activity of various cellular structures, for example, perikarya of neurons, presynaptic terminals, and myelinated and nonmyelinated cell profiles. Cytochrome oxidase activity in the mitochondria of neuronal perikarya increases during development which parallels the differentiation of the area octavolateralis. This possibly reflects the increasing energy demand during maturation and innervation of the magnocellular nucleus. Hyper-g-exposure of the larvae for 8 days (centrifuge) caused a further augmentation of cytochrome oxidase activity in the perikarya within the nucleus magnocellularis. This may reflect an increased oxidative metabolism resulting from the need for compensation of altered inputs from gravity-sensitive epithelia in the inner ear. Another possibility is that acceleration within a centrifuge causes physiological stress for the animals and, therefore, influences the cytochrome oxidase activity in neurons.
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Several studies have shown that altered gravity conditions influence mammalian cell growth and differentiation. The molecular mechanisms underlying these effects, however, remain relatively obscure. In this paper we show that microgravity reached in a sounding rocket strongly decreases epidermal growth factor (EGF)-induced expression of the proto-oncogenes c-fos and c-jun, which are both implicated in the regulation of proliferation and differentiation. Decreased activity of the serum response element (SRE), present in the c-fos promoter-enhancer region, is probably responsible for the decrease in EGF-induced c-fos expression. In addition, we show that gravity alterations differentially modulate distinctive signal transduction pathways, indicating that gravity-dependent modulations of mammalian cell proliferation are unlikely to be caused by a nonspecific stress response of the cell.
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The control of man's movements during adaptation to an altered gravity field was modelled by mathematical methods. Two types of movements were investigated: forearm displacements with various loads and repulsions from a support in zero-g and at high g's. It has been shown that movement coordination in altered gravity fields can be maintained due to a rearrangement of the control over muscle contraction which includes changes in the amplitude and pattern of joint momenta in harmony with the gravity changes.