CHANGES IN LEPTIN, BLOOD PRESSURE AND RR INTERVAL DURING PASSIVE TILTING

F. Strollo, A. Mambro, M.A. Masini*, M. Morè, G. Strollo, A. Scano and B. Uva*
Endocrine Unit, IRCCS-INRCA – University "La Sapienza", Via Cassia 1167, I-00189 Rome, Italy * Experimental and Environmental Biology Department, University, Via Benedetto XV 5, I-16132 Genoa, Italy

Leptin (L) is a newly identified polypeptide hormone signalling to the hypothalamus the amount of fat tissue energy stores. It enhances adrenergic activity with cardiovascular effects, including increased heart rate (shortened RR interval in electrocardiographic records) and elevated systolic and diastolic blood pressure (SBP and DBP): this makes L a typical neuroendocrine signal. Studies dealing with real microgravity pointed to increased adrenergic activity, while long term simulated microgravity experiments using head down tilt (HDT) at –6° showed decreased rest energy expenditure and increased L levels. The aim of this study was to verify whether passive short term –6° HDT and / or +70° HUT (head up tilt) modify L, RR, SBP and DBP.
Eight healthy female volunteers aged 18 to 45, with a body mas index (BMI) £ 30Kg/m² underwent a tilt test consisting of two randomly assigned sequences (n=4 each):HUT and HDT. The former of 30 min in the supine position followed by 10 min at +70° (tilting speed: 5 sec). This was stopped at 5 min in three cases, because of orthostatic intolerance. HDT had a similar 30 min clinostatic adaptation phase followed by 30 more min a –6°. The following parameters were evaluated: means of three consecutive standard sphigmomanometric SBP and DBP measurements; means of 30 consecutive RR intervals as recorded on the ECG D2 lead at 50 mm/sec; L levels as measured in a single session by a highly sensitive and accurate coated tube IRMA method on serum samples stored at -20°C until assayed (DSL, TX, USA, sensitivity: 0.15 ng/ml, intra-assay c.v. of 3.6%; reference range of 2 to 15 ng/ml in our lab). Sampling times were: –10’, -5’, 0’, +5’, +10’ for HUT and –10’, -5’, 0’, +30’for HDT. The statistical analysis was based upon the paired U Wilcoxon test and Repeated Measures ANOVA as needed.
RESULTS. Our results may be summarized as follows: during HUT decreased RR interval at 10’ (0.93± 0.03 à 0.73± 0.04 msec), increased L at 10’ (15.6± 0.9 à 17.3± 0.9) and DBP at 5’ (78± 4.9 à 84.8± 6.2 mmHg) were observed; during HDT only a slight increase in DBP (72.6± 2.7 à 75.5± 2.7 mmHg) was found, with a trend of L to decrease and of RR to increase.
DISCUSSION AND CONCLUSIONS. During HUT, signs of enhanced sympathetic activity, namely RR decrease and DBP increase, were accompanied by increased L levels L (p<0.05). Conversely, during HDT only an opposite trend was noticed and, what complicates the interpretation of our results, DBP slightly increased, as it happened during HUT. An explanation might come from the fact that the adaptation phase in the supine position might have attenuated the short-term response to HDT, so that it seems more useful to compare HDT to orthostatic position, as recently suggested by experiments led in the fields of kidney and cardio-pulmonary physiology. Moreover, with respect to HDT, the duration of the test was too short as compared to expected hemodynamic responses to allow significant changes to become apparent. When referring to HUT, instead, the system looks solicited enough and the strong relation between L and RR (sympathetic activity index) is confirmed.
To date our group explored only women in terms of physiological response to posture changes in such experimental conditions. We therefore are now ready to go on with the study in both sexes with some protocol changes in terms of tilting angle and test duration.


LEPTIN AND SEX HORMONES IN MEN AND WOMEN DURING SHORT TERM HDT

F. Strollo, F. Celotti*, P. Magni*, A. Mambro, M. Morè, G. Strollo and G. Riondino
Endocrine Unit, IRCCS-INRCA – University "La Sapienza", Rome, Italy * Endocrinology Institute, University, Milan, Italy

Leptin (L) is a newly identified polypeptide hormone produced by the adipose tissue as a function of fat energy stores representing a neuroendocrine signal enhancing adrenergic activity and thus increasing heart rate (HR) together with systolic and diastolic blood pressure arteriosa (SBP and DBP) and inhibiting 17,20-lyase activity within the testis, thus decreasing testosterone [T] synthesis in men at high dosage. Physiological regulation of many systems looks different in the two genders: women, for instance, have almost double L levels as compared to men and much more often suffer from orthostatic intolerance, probably due to hyperadrenergic tone with consequent faster response exhaustion. Moreover, our group already showed that T response to gravity follows the so-called "principle of continuity", by decreasing at 0xg in man and increasing at 2xg in primates. Conversely, men under prolonged head down tilt (HDT) at - 6° (a 0xg simulation method) show a dramatic increase in L levels, while rats have an opposite response under 2xg, thus behaving again according to the "principle of continuity". The aim of this study was to verify the gender differences in the response to HDT.
In particular, men were analyzed in terms of Androstenedione (A) and T, women in terms of oestrone (E₁) and oestradiol (E₂) and both in terms of HR, SBP and DBP and L. 18 healthy volunteers (M:F=9:9) matched for age (F = 36-55 years, M = 33-55 years) and body mass index (BMI) (F = 21.7-41.5 Kg/m²; M = 22.4-45.3 Kg/m²) underwent 5 hour HDT after overnight fast. Immediately before and after the test, the following parameters were monitored: mean of 3 consecutive SBP and DBP values measured by a standard Riva Rocci sphygmomanometer; mean of 30 consecutive HR levels measured from ECG standard lead recordings at a speed of 50 mm/sec; serum levels of L (IRMA), T, A, E₂ and E₁ (RIA) by coated tube methods with high sensitivity and specificity. Statistical analysis was based upon the paired U Wilcoxon test and 1-way ANOVA.
RESULTS: (A) Between genders: women had lower 0h SBP (115±2.8
vs 126.7±2.0 mmHg), 0h DBP (68.9±1.4 vs. 83.9±4.9 mmHg) and higher L concentrations at 0h (35.4± 5.5 vs 16.0±2.9 ng/ml) and at 5h (34.7± 5.1
vs. 16.3± 2.9 ng/ml) than men (p<0.01). (B) Within genders: (b.1) women showed no significant changes between 0h and 5h.; (b.2) men showed a significant decrease (p<0.05) in HR (from 69.3±2.2 to 66.7±2.1 b/min), SBP (from 126.7±2.0 to 116.1±5.2 mmHg), DBP (from 83.9±4.9 to 72.2±3.0 mmHg) and
A (from 2.2±0.3 to 1.8±0.2 ng/ml).
DISCUSSION AND CONCLUSIONS: The lack of variations in L and T levels was in contrast to what we had hypothesized to occur on the basis of current knowledge in gravitational physiology and might depend on a different behaviour than normal with respect to overweight subjects, or on the inability of the test to elicit hemodynamic responses during five hours or, again, on the fact that HDT should be compared more to the orthostatic (representing ordinary 1xg conditions) than to the supine position. To investigate upon possible leptin transport or receptor--binding abnormalities we set up and validated a FPLC (Fast Protein Liquid Chromatography) method for gel-chromatographic separation of free from bound L and RT-PCR (reverse transcription-polymerase chain reaction) analysis for the study of L receptor isoform expression in peripheral lymphocytes. We will use such methods in our future studies including a larger number of subjects of both genders examined at closer intervals than those chosen this time and for a different duration test.

NUCLEAR ALTERATIONS IN CULTURED GLIAL CELLS SUBMITTED TO SIMULATED MICROGRAVITY

B.M. Uva, M.A. Masini, G. Tagliafierro and F. Strollo*
Dipartimento di Biologia Sperimentale, Università di Genova, I-16132 Genova, Italy *U.O. Endocrinologia e Malattie del Ricambio, IRCCS-INRCA, Università "La Sapienza", Roma, Italy

Glial cells are necessary to the proper functioning of the nervous system; they are fundamental for nutrition, neurogenesis, neural regeneration and signal transmission. Cytoskeletal disorganisation, by causing damages to glial cells and to glial-neuron interaction, eventually leads to CNS dysfunctions.
In our previous ground-based studies on cultured glial cells submitted to simulated microgravity, we investigated upon the possibility that the neurophysiological impairment, experienced by astronauts during space-flights, might be traced back to cytomorphology. In simulated weightlessness, we observed, several alterations in the glial cell cytoskeleton: the organisation of the microfilaments, intermediate filaments and microtubules were severely altered after 30’ of simulated microgravity (Uva et al., 2000). The shape of the nucleus and its position inside the cytoplasm is secured by microfilaments and intermediate filaments that anchor the nucleus to the cell outer membrane. The microtubules organise cell division. Any alterations of these three components of the cytoskeleton may lead to dislocation of the nucleus, impairment of cell division and eventually cell death.
Aim of the present research was to investigate on the nuclear shape, the nuclear position in the cytoplasm and on cell divisions in cultured glial cells submitted to simulated microgravity. For this purpose we used C6 glioma cell line in monolayer cultures kept in a Fokker three dimensional clinostat under continuous rotation for 15’, 30’, 1h, 20h and 32h (simulated microgravity 0g). Control cultures (1g) were positioned on the support of the rotating frames of the clinostat in order to submit the cells to the same vibrations.
After fixation with 4% paraformaldehyde, immunohistochemistry standard techniques were applied to the samples, using antibodies to apoptosis-related peptides and to the proliferating cell nuclear antigen (PCNA). Nuclei were stained with propidium iodide or DAPI. The stained cells were then observed through a conventional or confocal laser microscope.
After 15’, 30’, 1h, 20h and 32h, at 1g the nuclei showed a normal shape and were centrally positioned in the pyramidal glial cells.
Conversely, the nuclei of the cells kept at 0g from 15’ to 20h lost their original shape they were often fragmented and showed blebs, with chromatin condensed into discrete patches. Moreover, the number of the cells observed in the 0g samples was severely reduced.
After 32h at 0g the remaining cells were no more distinguishable from 1g cells; in fact 0g cells had acquired a normal shape again, their nuclei had a normal aspect and were centrally positioned. Clusters of undifferentiated cells were numerous; the cells showed immuno-positivity for the proliferating cell nuclear antigen, in fact mitotic figures were often present. Therefore, luckily enough, the cell population might be restored showing the potential plasticity of the glial cells. Nevertheless, the dramatic decrease observed in the number of cells undergoing simulated microgravity might depend on apoptosis or necrosis and deserves further investigation.

Effect of weightlessness on cytoskeleton architecture and proliferation of human Breast cancer cell line mcf-7

J. Vassy1, S. Portet1, M. Beil2, G. Millot3, F. Fauvel-Lafève4, A. Karniguian4, G. Gasset5, T. Irinopoulou6, F. Calvo3, JP. Rigaut1, D. Schoevart1
(1) AIPC Lab., Université Paris 7, IUH, Hôpital Saint Louis, 1 avenue Claude Vellefaux, F-75475 Paris cedex 10, France, (2) Dept. Of Internal Medicine I, University Hospital, Ulm; Germany, (3) Pharmacologie Lab., IUH, Hôpital Saint Louis, 1 avenue Claude Vellefaux, F-75475 Paris cedex 10, France, (4) U553 INSERM, IUH, Hôpital Saint Louis, 1, avenue Claude Vellefaux, 75475 Paris cedex 10, (5) GSBMS, Université Paul Sabatier, Toulouse, France, (6) U430 INSERM, Hôpital Broussais, Paris, France.

Because cells are sensitive to mechanical forces, weightlessness might act on stress-dependent cell changes. We hypothesized that the integration of environmental factors might induce specific cytoskeletal architecture patterns, characterized by quantitative image analysis.
Human breast cancer cells MCF-7, flown in space in a Photon capsule, were fixed after 1.5, 22 and 48 h in orbit. Cells subjected to weightlessness were compared to 1g in-flight and ground controls. Post-flight, fluorescent labellings were performed to visualize cell proliferation (Ki-67), signal transduction (phosphotyrosine), three cytoskeleton components (microtubules, microfilaments and intermediate filaments) and the chromatin structure. Confocal microscopy and image analysis were used to quantify cycling cells and mitosis, modifications of the cytokeratin network and the chromatin structure.
In weightlessness, phosphotyrosine signal transduction was lower, more cells were cycling and mitosis was prolonged. Finally, cell proliferation was reduced as a consequence of a cell-cycle blockade. Microtubules were altered in many cells. The perinuclear cytokeratin network was more loosely "woven" and the chromatin structure was modified.
The prolongation of mitosis can be explained by an alteration of the microtubule self-organization in weightlessness, involving reaction-diffusion processes. The loosening of the perinuclear cytokeratin network and the modification of the chromatin distribution are in agreement with basic predictions of cellular tensegrity.

Microgravity induced lesions in lymphocyte signal transduction: impairment in locomotion involves protein kinase C.

A. Sundaresan, D. Risin and N.R. Pellis
Cellular Biotechnology, Wyle Laboratories, and Cellular Biotechnology, NASA Johnson Space Center, Houston, Texas, USA.

In space travel and long-term space residence, crew, animals and cells are exposed to microgravity, radiation and other space stressors. In turn these are potential hazards to the immune system. Much of active immunity is dependent on lymphocyte function. T cell activation occurs through a series of events inclusive of signal transduction. Receptor mediated signaling is inhibited both in microgravity and modeled microgravity (MMG). Lymphocyte locomotion was also shown to be inhibited in microgravity and MMG (Pellis et al, 1997). However activation of lymphocytes using CD3 and IL-2 prior to MMG exposure resulted in normal lymphocyte movement. Thus the locomotion loss in MMG may be due to a defect in signal transduction.
Direct targeting of Protein Kinase C (PKC) bypassing cellular membrane events using the phorbol ester PMA, lead to a rescue effect on MMG inhibited lymphocyte locomotion. The calcium ionophore ionomycin had no rescue effect. These observations may suggest that calcium independent PKC isoforms are involved in MMG induced locomotion inhibition and rescue. Both calcium dependent PKC isoforms and calcium independent isoforms were investigated to assess their role in locomotion in 1g and MMG cultured lymphocytes. Human lymphocytes were cultured and harvested at 24, 48, 72 and 96 hours. Samples were then subjected to the locomotion assay using type I collagen, protein and molecular analysis.
Transcriptional and translational status of PKC alpha, delta and epsilon were assessed by RT-PCR, flow cytometry and Immunoblotting. Upstream events leading to PKC functionality and activation such as phosphorylation of Phospholipase C gamma (PLC-gamma), in MMG were investigated. Results indicated that PKC isoforms delta and epsilon were down regulated by more than 50% at the transcriptional and translational levels in MMG cultured lymphocytes compared to 1g controls. Levels of activated PLC-gamma protein were also down regulated by more than 60% in MMG grown lymphocytes.
PKC pathways are of paramount importance in lymphocyte function. The role of PKC in the rescue effect of lymphocyte locomotion in MMG would contribute to elucidation of microgravity’s multiple effects on the immune system. A detailed understanding of microgravity induced lymphocyte defects serves as a basis for the formulation of countermeasure strategies necessary for long duration occupation of low gravity environments.

SIMULATED MICROGRAVITY PROMOTES THE DIFFERENTIATION OF FLG 29.1 CELLS ON THE OSTEOCLASTIC PATHWAY

M. Monici(1), G. Agati(2), F. Fusi(3), A. Cogoli(4), M. Paglierani (5) and P. A. Bernabei(6)
(1) Centro di Eccellenza Optronica, Florence, Italy; (2) Istituto di Elettronica Quantistica, CNR, Florence, Italy; (3) Dip. di Fisiopatol. Clinica, Univ. of Florence, Italy; (4) Space Biology Group, ETH-Technopark, Zurich, Switzerland; Dip. di Patol. Umana ed Oncol., Policlin. Careggi, Florence, Italy; (6) Div. Ematologia, Policlin. Careggi, Florence, Italy

The importance of gravity in modulating some biological processes, like plant gravitropism and the adaptation of the skeleton related to its load bearing function, is known for a long time. When life science studies in space started, the observation of not expected and, mostly, unexplained effects in weightlessness conditions highlighted the need for a deeper understanding of the importance of gravity in biological processes.
Regarding cell biology, our knowledge about the influence of gravity on cell proliferation, cell growth and differentiation, cell activation, cell-cell interaction, cell-surface interaction is not enough. Actually we have the opportunity to enrich our knowledge performing controlled experiments on model systems in microgravity, gravity and hypergravity conditions and comparing the behaviour of the models in the different environments.
The research here deals with the effect of simulated microgravity conditions on the capability to differentiate of FLG 29.1 cells. This stabilized leukemic cell line was characterized as an osteoclastic precursor model. Osteoclasts are monoblastic cells of haemopoietic origin that, following a differentiation process, assume a peculiar morphological aspect of large, multinucleated cells and the capability to resorb bone. Physiological remodelling of bone mostly depends on osteoclast activity. Enhanced bone resorption is thought to be the key of osteoporotic diseases, those observed in astronauts included. We don’t know why weightlessness fosters osteoporosis and if enhanced bone resorption is due to an increased osteoclast activity or to an increased number of cells or both. The cell line FLG 29.1, chosen as experimental model, is particularly suitable to investigate microgravity effect on osteoclastic differentiation and the possible consequences on bone resorption activity. Cell samples exposed to microgravity conditions (60h), simulated by a Random Positioning Machine, and controls at 1g were analysed and compared by standardized histochemical and immunohistochemical techniques. Moreover, Autofluorescence Micro- spectroscopy and Multispectral Imaging Autofluorescence Microscopy techniques, allowing direct analysis on single living cell, were used in order to study the cell morphofunctional state. Bone resorption activity was evaluated by a new method, based on surface profilometry and applied to the surface analysis of suitably finished bone slices.
The results obtained demonstrate that FLG 29.1 cells under simulated microgravity conditions assume characteristics of more mature elements, in comparison with control cells, and consistent with a differentiation process on the osteoclastic pathway. On this basis an experimental model can be developed, useful for studies on osteoporotic diseases and their pharmacological treatments.
This research is part of a wide program of studies on the modulation of cell differentiation by physical factors. It has been reported that mechanical stimuli, i.e. changes of the strains on cell membrane, can induce a succession of events, from the cytoskeleton rearrangement to the modulation of gene expression. A deeper knowledge of cell differentiating mechanisms, from the signals inducing the process to the reaching of cell morpho-functional maturity, can be considered among the main objectives of basic research. It is a common opinion that a therapy based on the induction of cell differentiation could be an alternative strategy in the treatment of several diseases, as in t hyper-proliferative disorders especially.

GRAVITY DEPENDENCE OF MICROTUBULE SELF-ORGANISATION

James Tabony*, Nicolas Glade*$, Jacques Demongeot$
* Commissariat à l'Energie Atomique, Département de Biologie Moléculaire et Structurale, Laboratoire de Résonance Magnétique en Biologie Métabolique, D.S.V, C.E.A. Grenoble, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France. $ Institut d'Informatique et Mathématique Appliquées de Grenoble, Laboratoire de Technique de l'Imagerie, de la Modélisation et de la Cognition, Faculté de Médecine, Domaine de la Merci, 38706 La Tronche Cedex, France.

The molecular processes by which gravity affects biological systems are poorly, if at all, understood. Under equilibrium conditions, chemical and biochemical reactions do not depend upon gravity. It has been proposed that biological systems might depend on gravity by way of the bifurcation properties of certain types of non-linear chemical reactions that are far-from-equilibrium. In such reactions, the initially homogenous solution spontaneously self organises by way of a combination of reaction and diffusion. Theoreticians have predicted that the presence or absence of an external field, such as gravity, at a critical moment early in the self-organising process may determine the morphology that subsequently develops. We have found that the formation in-vitro of microtubules, a major element of the cellular skeleton, shows this type of behaviour. The microtubule preparations spontaneously self-organise by way of reaction and diffusion, and the morphology of the state that forms depend upon gravity at a critical bifurcation time early in the process. Space experiments show that the presence of gravity at the bifurcation time triggers the self-organising process. Self-organisation can also be triggered by other weak effects such as magnetic fields and shearing. We have developed a numerical reaction-diffusion scheme, based on the chemical dynamics of a population of microtubules, that simulates the experimental self-organisation. These simulations illustrate how self-organisation occurs. Microtubules are chemically anisotropic, growing and shrinking along the direction of their long axis. This leads to the formation of chemical trails, comprised of regions of high and low local tubulin concentration from their shrinking and growing ends respectively. These concentration trails are oriented along the direction of the microtubule. Neighbouring microtubules will preferentially grow into regions where the local concentration of tubulin is highest. When microtubules first form from the tubulin solution they are still in a growing phase and have an isotropic arrangement. However, this isotropic arrangement becomes unstable once significant disassembly from the shrinking end occurs. At this point, if a few microtubules start to take up a preferred orientation then neighbouring microtubules will also grow into the same orientation. Once started, the process mutually reinforces itself with time and leads to self-organisation. When the isotropic arrangement is unstable at the bifurcation time, any effect that leads to a slight directional bias, such as somewhat different rates of molecular transport in the up-down and left-right directions, will trigger self-organisation. Gravity acts by way of its directional interaction with the macroscopic density fluctuations present in the solution arising from microtubule disassembly at the instability.