Gravity-induced Changes Research Articles

Vector-averaged gravity-induced changes in cell signaling and vitamin d receptor activity in MG-63 cells are reversed by a 1,25-(OH) 2D 3 analog, EB1089

Skeletal unloading in an animal hindlimb suspension model and microgravity experienced by astronauts or as a result of prolonged bed rest causes site-specific losses in bone mineral density of 1%–2% per month. This is accompanied by reductions in circulating levels of 1,25-(OH) 2D 3, the active metabolite of vitamin D. 1,25-(OH) 2D 3, the ligand for the vitamin D receptor (VDR), is important for calcium absorption and plays a role in differentiation of osteoblasts and osteoclasts. To examine the responses of cells to activators of the VDR in a simulated microgravity environment, we used slow-turning lateral vessels (STLVs) in a rotating cell culture system. We found that, similar to cells grown in microgravity, MG-63 cells grown in the STLVs produce less osteocalcin, alkaline phosphatase, and collagen Iα1 mRNA and are less responsive to 1,25-(OH) 2D 3. In addition, expression of VDR was reduced. Moreover, growth in the STLV caused activation of the stress-activated protein kinase pathway (SAPK), a kinase that inhibits VDR activity. In contrast, the 1,25-(OH) 2D 3 analog, EB1089, was able to compensate for some of the STLV-associated responses by reducing SAPK activity, elevating VDR levels, and increasing expression of osteocalcin and alkaline phosphatase. These studies suggest that, not only does simulated microgravity reduce differentiation of MG-63 cells, but the activity of the VDR, an important regulator of bone metabolism, is reduced. Use of potent, less calcemic analogs of 1,25-(OH) 2D 3 may aid in overcoming this defect.

Mechanical culture conditions effect gene expression: gravity-induced changes on the space shuttle.

Three-dimensional suspension culture is a gravity-limited phenomenon. The balancing forces necessary to keep the aggregates in suspension increase directly with aggregate size. This leads to a self-propagating cycle of cell damage by balancing forces. Cell culture in microgravity avoids this trade-off. We determined which genes mediate three-dimensional culture of cell and tissue aggregates in the low-shear stress, low-turbulent environment of actual microgravity. Primary cultures of human renal cortical cells were flown on the space shuttle. Cells grown in microgravity and ground-based controls were grown for 6 days and fixed. RNA was extracted, and automated gene array analysis of the expression of 10, 000 genes was performed. A select group of genes were regulated in microgravity. These 1,632 genes were independent of known shear stress response element-dependent genes and heat shock proteins. Specific transcription factors underwent large changes in microgravity including the Wilms' tumor zinc finger protein, and the vitamin D receptor. A specific group of genes, under the control of defined transcription factors, mediate three-dimensional suspension culture under microgravity conditions.

Gravity-induced absorbance changes in Phycomyces: a novel method for detecting primary responses of gravitropism.

The negative gravitropism of the sporangiophores of Phycomyeces blakesleeanus Burgeff is elicited by different sensory inputs, which include flexure of the growing zone, buoyance of lipid globules and sedimentation of paracrystalline proteins, so-called octahedral crystals (C. Schimek et al., 1999a, Planta 210: 132-142). Gravity-induced absorbance changes (GIACs), which are associated with primary events of gravity sensing, were detected in the growing zones of sporangiophores. After placing sporangiophores horizontally, GIACs were detected after a latency of about 5 min, i.e. 15-25 min prior to gravitropic bending. The spectroscopic properties of the GIACs indicate that gravitropic stimulation could imply the reduction of cytochromes. The GIACs were spectrally distinct from light-induced absorbance changes (LIACs), showing that the primary responses of the light and gravity transduction chains are different. A dual stimulation with gravity and light generated GIAC-LIACs which were distinct from the absorbance changes occurring after the single stimuli and which indicate that light and gravity interact early in the respective transduction chains.

Evaluation of gravity-dependent membrane potential shift in Paramecium

It is still debated whether or not gravity can stimulate unicellular organisms. This question may be settled by revealing changes in the membrane potential in a manner depending on the gravitational forces imposed on the cell. We estimated the gravity-dependent membrane potential shift to be about 1 mV G −1 for Paramecium showing gravikinesis at 1–5 G, on the basis of measurements of gravity-induced changes in active propulsion and those of propulsive velocity in solutions, in which the membrane potential has been measured electrophysiologically. The shift in membrane potential to this extent may occur from mechanoreceptive changes in K + or Ca 2+ conductance by about 1% and might be at the limit of electrophysiological measurement using membrane potential-sensitive dyes. Our measurements of propulsive velocity vs membrane potential also suggested that the reported propulsive force of Paramecium measured in a solution of graded densities with the aid of a video centrifuge microscope at 350 G was 11 times as large as that for −29 mV, i.e., the resting membrane potential at [K +] o = 1 mM and [Ca 2+] o = 1 mM, and, by extrapolation, that Paramecium was hyperpolarized to −60 mV by gravity stimulation of 100- G equivalent, the value corrected by considering the reduction of density difference between the interior and exterior of the cell in the graded density solution. The estimated shift of the membrane potential from −29 mV to −60 mV by 100- G equivalent stimulation, i.e., 0.3 mV G −1, could reach the magnitude entirely feasible to be measured more directly.

Bioelectricity, gravity and plants.

This brief review summarizes gravity-induced changes in bioelectric parameters and evaluates their contribution to our understanding of the sensing of gravity, and the transduction and transmission of the gravity stimulus in plants. During the last few decades, information has accumulated demonstrating gravity-induced changes in surface potentials, membrane voltages, endogenous electric currents and ion fluxes. These changes point to the plasma membrane as the site of perception and transduction of the gravity signal. To date, it is reasonable to assume that gravity affects the state of ion channels (in particular, Ca2+ channels) and the activity of ion pumps (in particular, the electrogenic H(+)-ATPase) in the plasma membrane leading to intracellular and apoplasmic changes in ion activities and in membrane voltages. The flow of H+ and Ca2+ currents is probably the means by which information about gravity is amplified and transmitted from sensing to responding cells. No data are available so far about the effect of microgravity on bioelectric parameters. However, it would be interesting to learn if plants become hypersensitive to gravity during a prolonged stay in microgravity. If so, such plants might fire action potentials after return to earth, because more Ca2+ channels than usual may be activated by 1 g in microgravity-adapted plants.

Electric-field effects on gravikinesis in Paramecium

Equilibrated Paramecium caudatum cells exposed to a constant DC gradient reorient with their depolarized anterior ends toward the cathode (galvanotaxis). Voltage gradients were applied to cells swimming either horizontally or vertically. Their velocity and orientation were recorded and compared to unstimulated cells. The DC field increased the horizontal velocity (= reference) up to 175% (galvanokinesis). Swimming velocities saturated after 1 min and were unchanged during the following 4 min. The upward and downward swimming velocities of stimulated cells were below those of horizontal swimmers. The difference in vertical rates (determining gravikinesis) was independent of variations in absolute velocity. Normalization of vertical velocities to horizontal velocities (= 100%) separated DC-field dependent changes from gravity-induced changes in velocities. A weak voltage gradient (0.3 V/cm) was most effective in raising downward gravikinesis up to threefold (-202 μm/s) above the unstimulated reference (-66 μm/s) and to change sign of gravikinesis in upward swimmers (-43 μm/s →+33 μm/s). We conclude that DC-field stimulation is equivalent to a depolarizing bias on gravikinetic responses of Paramecium. The stimulation does not directly interfere with mechanoreception, but modulates somatic Ca2+ entry to induce contraction of the cell soma. This presumably affects the gating of gravisensory transduction channels.

Gravitropism: interaction of sensitivity modulation and effector redistribution.

Our increasing capabilities for quantitative hormone analysis and automated high resolution growth studies have allowed a reassessment of the classical Cholodny-Went hypothesis of gravitropism. According to this hypothesis, gravity induces redistribution of auxin toward the lower side of the organ and this causes the growth asymmetry that leads to reorientation. Arguments against the Cholodny-Went hypothesis that were based primarily on concerns over the timing and magnitude of the development of hormone asymmetry are countered by recent evidence that such asymmetry develops early and is sufficiently large to account for curvature. Thus, it appears that the Cholodny-Went hypothesis is fundamentally valid. However, recent comparative studies of the kinetics of curvature and the timing of the development of hormone asymmetry indicate that this hypothesis alone cannot account for the intricacies of the gravitropic response. It appears that time-dependent gravity-induced changes in hormone sensitivity as well as changes in sensitivity of the gravity receptor play important roles in the response.

Gravity-Induced Changes in Intracellular Potentials in Elongating Cortical Cells of Mung Bean Roots

Gravity-induced changes in intracellular potentials in primary roots of 2-day-old mung bean (Vigna mungo L. cv. black matpe) seedlings were investigated using glass microelectrodes held by 3-dimensional hydraulic micro-drives. The electrodes were inserted into outer cortical cells within the elongation zone. Intracellular potentials, angle of root orientation with respect to gravity, and position within the root of the impaled cortical cell were measured simultaneously. Gravistimulation caused intracellular potential changes in cortical cells of the elongation zone. When the roots were oriented vertically, the intracellular potentials of the outer cortical cells (2 mm behind the root apex) were approximately - 115 mV. When the roots were placed horizontally cortical cells on the upper side hyperpolarized to - 154 mV within 30 s while cortical cells on the lower side depolarized to about - 62 mV. This electrical asymmetry did not occur in cells of the maturation zone. Because attempts to insert the electrode into cells of the root cap were unsuccessful, these cells were not measured. The hyperpolarization of cortical cells on the upper side was greatly reduced upon application of N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of respiratory energy coupling. When stimulated roots were returned to the vertical, the degree of hyperpolarization of cortical cells on the previous upper side decreased within 30 s and approached that of cortical cells in non-stimulated roots. This cycle of hyperpolarization/loss of hyperpolarization was repeatable at least ten times by alternately turning the root from the vertical to the horizontal and back again. The very short (<30 s) lag period of these electrical changes indicates that they may result from stimulus-perception and transduction within the elongation zone rather than from transmission of a signal from the root cap.

HOW ROOTS PERCEIVE AND RESPOND TO GRAVITY

Graviperception by plant roots is believed to occur via the sedimentation of amyloplasts in columella cells of the root cap. This physical stimulus results in an accumulation of calcium on the lower side of the cap, which in turn induces gravicurvature. In this paper we present a model for root gravitropism integrating gravity-induced changes in electrical potential, cytochemical localization of calcium in cells of gravistimulated roots, and the interdependence of calcium and auxin movement. Key features of the model are that 1) gravity-induced redistribution of calcium is an early event in the transduction mechanism, and 2) apoplastic movement of calcium through the root-cap mucilage may be an important component of the pathway for calcium movement.

The Geoelectric Effect in Plant Shoots

The development of the geoelectric effect has been followed in Zea coleoptiles with a flowing solution electrode system, and its dependence upon auxin concentration gradients and aerobic metabolism assessed. A symmetrical source of IAA can effectively replace the coleoptile tip in allowing the geo electric potential to occur. The diffusate from coleoptile tips, when applied asymmetrically to the apex of a vertical decapitated coleoptile, generates a potential difference across the coleoptile indistinguishable from that induced by the asymmetrical application of IAA. Asymmetrical application of IAA to vertical Avena and Zea coleoptiles and Helianthus hypocotyls induces closely similar responses. Neither the geoelectric effect nor a geotropic response develops when intact Zea coleoptiles are placed horizontally after being deprived of oxygen, but they both occur when an aerobic atmosphere is restored. The lateral potential difference induced by the asymmetrical applica tion of IAA to the apex of a vertical coleoptile does not occur under anoxic conditions. With a static-drop electrode system and a decapitated Zea coleoptile, a potential difference develops immediately after reorientation of the coleoptile into the horizontal position, and attains a maximum value after about 10 min. This potential difference can be further increased by the asymmetrical application of IAA to the lower half of the apical cut surface of the coleoptile. Our data support the view that both the geoelectric potential and the geotropic response are due to the IAA concentration gradient which arises from the lateral transport of this substance from the upper to the lower half of the horizontal shoot. They also bear out our previous conclusions that the 'geoelectric potential' observed with static-drop electrodes and an intact shoot, is the resultant of two processes. The first is a physical phenomenon arising in the electrodes, or between the electrodes and the plant tissue, and the second arises in the living tissues of the shoot as the result of gravity-induced changes in auxin distribution.