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PubMed1999

The transduction of very small hydrostatic pressures.

This paper reviews experiments in which cells, subjected to hydrostatic pressures of 20 kPa or less, (micro-pressures), demonstrate a perturbation in growth and or metabolism. Similarly, the behavioural responses of aquatic animals (lacking an obvious compressible gas phase) to comparable pressures are reviewed. It may be shown that in both cases the effect of such very low hydrostatic pressures cannot be mediated through the thermodynamic mechanisms which are invoked for the effects of high hydrostatic pressure. The general conclusion is that cells probably respond to micro-pressures through a mechanical process. Differential compression of cellular structures is likely to cause shear and strain, leading to changes in enzyme and/or ion channel activity. If this conclusion is true then it raises a novel question about the involvement of 'micro-mechanical' effects in cells subjected to high hydrostatic pressure. The responses of aquatic animals to micro-pressures may be accounted for, using the model case of the crab, by the mechanical, bulk, compression of hair cells in the statocysts, the organ of balance. If this is true, it raises the interesting question of why the putative cellular mechanisms of micro-pressure transduction appear to have been superseded by the statocyst.

Adaptation, Physiological↗
PubMed1980

The effects of increased hydrostatic pressure upon normal and regenerated rabbit corneal endothelium.

The effect of increased hydrostatic pressure upon the ability of normal and regenerated endothelium to deturgesce preswollen, de-epithelialized rabbit corneas was studied. Stromal deturgescence occurred as a biphasic response when hydrostatic pressure at the endothelial surface was increased above baseline values. Initially there was a rapid phase of stromal thinning which was dependent upon hydrostatic pressure nad endothelial function. This was followed by a slower phase of corneal thinning which was independent of hydrostatic pressure at the endothelial surface for pressures between 15 and 50 mm Hg. The slow phase of thinning represents the steady-state ability of the endothelium to deturgesce the stroma. Regenerated rabbit endothelium functioned similarly to normal endothelium in deturgescing the stroma. In addition, short-term increases in hydrostatic pressure at the endothelial surface did not produce ultrastructural changes in normal or regenerated corneal endothelial cells.

Animals↗
PubMed2005

Stabilization of partially folded states in protein folding/misfolding transitions by hydrostatic pressure.

In the last few years, hydrostatic pressure has been extensively used in the study of both protein folding and misfolding/aggregation. Compared to other chemical or physical denaturing agents, a unique feature of pressure is its ability to induce subtle changes in protein conformation, which allow the stabilization of partially folded intermediate states that are usually not significantly populated under more drastic conditions (e.g., in the presence of chemical denaturants or at high temperatures). Much of the recent research in the field of protein folding has focused on the characterization of folding intermediates since these species appear to be involved in a variety of disease-causing protein misfolding and aggregation events. The exact mechanisms of these biological phenomena, however, are still poorly understood. Here, we review recent examples of the use of hydrostatic pressure as a tool to obtain insight into the forces and energetics governing the productive folding or the misfolding and aggregation of proteins.

Amyloidosis↗
PubMed2003

High hydrostatic pressure treatment of finfish to inactivate Anisakis simplex.

High hydrostatic pressure has been demonstrated to be a useful technique for treating food to reduce the number of pathogenic organisms and to extend shelf life. Most research in this area has focused on bacteria. However, a concern in the sashimi (raw fish) industry is that nematode worms such as Anisakis simplex occur naturally in cold-water marine fish. The objectives of this research were to perform a pilot study to determine the effect of high hydrostatic pressure on the viability of Anisakis simplex larvae, commonly found in king salmon and arrowtooth flounder, and to evaluate the effects of high hydrostatic pressure on the color and texture of the fish fillets. Pieces of fish (ca. 100 g per bag) containing 13 to 118 larvae were exposed to pressures of up to 80,000 lb/in2 (552 MPa) for up to 180 s. The times and pressures required to kill 100% of the larvae were as follows: 30 to 60 s at 60,000 lb/in2 (414 MPa), 90 to 180 s at 40,000 lb/in2 (276 MPa), and 180 s at 30,000 lb/in2 (207 MPa). For all salmon treatments that killed 100% of the larvae, a significant increase in the whiteness of the flesh was observed. Although high hydrostatic pressure was effective in killing A. simplex larvae in raw fish fillets, its significant effect on the color and overall appearance of the fillet may limit its application to the processing of fish for raw-fish markets.

Animals↗
PubMed1994

Proteins under pressure. The influence of high hydrostatic pressure on structure, function and assembly of proteins and protein complexes.

Oceans not only cover the major part of the earth's surface but also reach into depths exceeding the height of the Mt Everest. They are populated down to the deepest levels (approximately 11,800 m), which means that a significant proportion of the global biosphere is exposed to pressures of up to 120 MPa. Although this fact has been known for more than a century, the ecology of the 'abyss' is still in its infancy. Only recently, barophilic adaptation, i.e. the requirement of elevated pressure for viability, has been firmly established. In non-adapted organisms, increased pressure leads to morphological anomalies or growth inhibition, and ultimately to cell death. The detailed molecular mechanism of the underlying 'metabolic dislocation' is unresolved. Effects of pressure as a variable in microbiology, biochemistry and biotechnology allow the structure/function relationship of proteins conjugates to be analyzed. In this context, stabilization by cofactors or accessory proteins has been observed. High-pressure equipment available today allows the comprehensive characterization of the behaviour of proteins under pressure. Single-chain proteins undergo pressure-induced denaturation in the 100-MPa range, which, in the case of oligomeric proteins or protein assemblies, is preceded by dissociation at lower pressure. The effects may be ascribed to the positive reaction volumes connected with the formation of hydrophobic and ionic interactions. In addition, the possibility of conformational effects exerted by moderate, non-denaturing pressures, and related to the intrinsic compressibility of proteins, is discussed. Crystallization may serve as a model reaction of protein self-organization. Kinetic aspects of its pressure-induced inhibition can be described by a model based on the Oosawa theory of molecular association. Barosensitivity is known to be correlated with the pressure-induced inhibition of protein biosynthesis. Attempts to track down the ultimate cause in the dissociation of ribosomes have revealed remarkable stabilization of functional complexes under pseudo-physiological conditions, with the post-translational complex as the most pressure-sensitive species. Apart from the key issue of barosensitivity and barophilic adaptation, high-pressure biochemistry may provide means to develop new approaches to nonthermic industrial processes, especially in the field of food technology.

Bacterial Proteins↗
PubMed1999

Modulation of Na+ x H+ exchange by hydrostatic pressure in isolated bovine articular chondrocytes.

The effects of increased hydrostatic pressure on Na+ x H+ exchange activity in bovine articular chondrocytes have been characterized. Chondrocytes were isolated from the cartilage matrix and the cells were loaded with the pH-sensitive fluorophore BCECF. Cells were acidified by ammonium rebound and the rate of recovery of pHi back to control levels was determined using cuvette fluorimetry. The application of hydrostatic pressure (1-300 atm) to cells within the fluorimeter was found to stimulate the rate of recovery from acidification, recorded as proton fluxes, in MOPS buffered media. This increase was dependent on the presence of extracellular Na+ ions and was inhibited by the Na+ x H+ exchange inhibitor EIPA. The pressure-stimulated increase in H+ flux is therefore mediated completely by Na+ x H+ exchange. In addition, the stimulation could be abolished by the kinase inhibitor staurosporine, was not additive with the stimulation of Na+ x H+ exchange elicited by the addition of serum and was unaffected by low concentrations of the myosin light chain kinase inhibitor ML-7. We therefore conclude that hydrostatic pressure activates Na+ x H+ exchange in this cell type by a pathway which involves direct phosphorylation of the transporter protein itself. This is the first demonstration of the activation of Na+ x H+ exchange by hydrostatic pressure and the relevance of this finding to the biology of cartilage tissue is discussed.

Acid-Base Equilibrium↗
PubMed2005

Effect of high hydrostatic pressure on the bacterial mechanosensitive channel MscS.

We have investigated the effect of high hydrostatic pressure on MscS, the bacterial mechanosensitive channel of small conductance. Pressure affected channel kinetics but not conductance. At negative pipette voltages (corresponding to membrane depolarization in the inside-out patch configuration used in our experiments) the channel exhibited a reversible reduction in activity with increasing hydrostatic pressure between 0 and 900 atm (90 MPa) at 23 degrees C. The reduced activity was characterized by a significant reduction in the channel opening probability resulting from a shortening of the channel openings with increasing pressure. Thus high hydrostatic pressure generally favoured channel closing. Cooling the patch by approximately 10 degrees C, intended to order the bilayer component of the patch by an amount similar to that caused by 50 MPa at 23 degrees C, had relatively little effect. This implies that pressure does not affect channel kinetics via bilayer order. Accordingly we postulate that lateral compression of the bilayer, under high hydrostatic pressure, is responsible. These observations also have implications for our understanding of the adaptation of mechanosensitive channels in deep-sea bacteria.

Biophysics↗
PubMedDate not supplied

A heat-shock-like response with cytoskeletal disruption occurs following hydrostatic pressure in MG-63 osteosarcoma cells.

Human osteosarcoma cells, MG-63, were exposed to a hydrostatic pressure shock of 4.0 MPa for 20 min. Changes in subcellular distribution of the cytoskeletal elements and heat shock protein 70 (hsp70) were followed by indirect immunofluorescence and by avidin-biotin-peroxidase protocols. During recovery, total cellular RNA was determined and actin and aldolase mRNA content was followed using reverse transcription-polymerase chain reaction techniques. Hydrostatic pressure caused cell rounding (but not cell death), disruption of microtubules, collapse of intermediate filaments to a perinuclear location, collapse of actin stress fibers into globular aggregates in the cytoplasm, and the formation of several large elongated intranuclear actin inclusions. During recovery, the cells flattened, reorganized microtubules, and redistributed intermediate filaments prior to the reappearance of actin stress fibers. At 20 and 60 min following the initiation of hydrostatic pressure, there was increased anti-hsp 70 staining at the nuclear membrane and concentration of hsp70 in four to six granules in the nucleus. At 120 min following the hydrostatic pressure, hsp70 showed intense staining in the cytoplasm and hsp70-containing granules in the nucleus disappeared. Cellular RNA decreased during the first 120-min posthydrostatic pressure shock and then recovered to near prehydrostatic pressure treatment levels by 240 min. Actin mRNA abundance, in relation to aldolase mRNA abundance, showed the same temporal pattern of initial decrease, followed by increase as did total RNA. Review of the literature indicated that eukaryotic cells respond to heat shock and to hydrostatic pressure by disruption of the cytoskeletal elements and by similar modifications in genetic expression. In this study, the observed responses of MG-63 cells to a 4-MPa hydrostatic pressure shock was like the reported response of mammalian cells to a 43 degrees C heat shock.

Actins↗
PubMed2006

Gravitational valves: relevant differences with different technical solutions to counteract hydrostatic pressure.

Two different technical principles of gravitational valves (G-valves) have been presented: counterbalancer and switcher G-valves. The objective of our prospective study was to look for clinically relevant differences between both. A total of 54 patients with normal-pressure hydrocephalus (NPH) were treated; 30 patients received an Aesculap-Miethke GA-Valve (GAV; counterbalancer), and in 24 patients an Aesculap-Miethke Dualswitch-Valve (DSV; switcher) was implanted. The opening pressure of the posture-independent valve was 5 cm H2O in both devices. The outcome was clearly better with the usage of the GAV than with the DSV. The frequency and severity of complications was pronounced in the DSV group. We recommend the Aesculap-Miethke-GAV valve with a low opening pressure in a posture-independent valve for patients with NPH.

Adult↗
PubMed2006

Age of donor alters the effect of cyclic hydrostatic pressure on production by human macrophages and osteoblasts of sRANKL, OPG and RANK.

BACKGROUND: Cyclic hydrostatic pressure within bone has been proposed both as a stimulus of aseptic implant loosening and associated bone resorption and of bone formation. We showed previously that cyclical hydrostatic pressure influenced macrophage synthesis of several factors linked to osteoclastogenesis. The osteoprotegerin/soluble receptor activator of NF-kappa beta ligand /receptor activator of NF-kappa beta (OPG/ RANKL/ RANK) triumvirate has been implicated in control of bone resorption under various circumstances. We studied whether cyclical pressure might affect bone turnover via effects on OPG/ sRANKL/ RANK. METHODS: In this study, cultures of human osteoblasts or macrophages (supplemented with osteoclastogenic factors) or co-cultures of macrophages and osteoblasts (from the same donor), were subjected to cyclic hydrostatic pressure. Secretion of OPG and sRANKL was assayed in the culture media and the cells were stained for RANK and osteoclast markers. Data were analysed by nonparametric statistics. RESULTS: In co-cultures of macrophages and osteoblasts, pressure modulated secretion of sRANKL or OPG in a variable manner. Examination of the OPG:sRANKL ratio in co cultures without pressurisation showed that the ratio was greater in donors <70 years at the time of operation (p < 0.05 Mann Whitney U) than it was in patients >70 years. However, with pressure the difference in the OPG:sRANKL ratios between young and old donors was not significant. It was striking that in some patients the OPG:sRANKL ratio increased with pressure whereas in some it decreased. The tendency was for the ratio to decrease with pressure in patients younger than 70 years, and increase in patients > or = 70 years (Fishers exact p < 0.01). Cultures of osteoblasts alone showed a significant increase in both sRANKL and OPG with pressure, and again there was a decrease in the ratio of OPG:RANKL. Secretion of sRANKL by cultures of macrophages alone was not modulated by pressure. Only sRANKL was assayed in this study, but transmembrane RANKL may also be important in this system. Macrophages subjected to pressure (both alone and in co-culture) stained more strongly for RANK on immunohistochemstry than non-pressurized controls and 1,25-dihydroxyvitamin D3 (1,25 D3) further increased this. Immunocytochemical staining also demonstrated that more cells in pressurized co-cultures exhibited osteoclast markers (tartrate-resistant acid phosphatase, vitronectin receptor and multinuclearity) than did unpressurized controls. CONCLUSION: These data show that in co-cultures of osteoblasts and macrophages the ratio of OPG : sRANKL was decreased by pressure in younger patients but increased in older patients. As falls in this ratio promote bone resorption, this finding may be important in explaining the relatively high incidence of osteolysis around orthopaedic implants in young patients. The finding that secretion of OPG and sRANKL by osteoblasts in monoculture was sensitive to hydrostatic pressure, and that hydrostatic pressure stimulated the differentiation of macrophages into cells exhibiting osteoclast markers indicates that both osteoblasts and preosteoclasts are sensitive to cyclic pressure. However, the effects of pressure on cocultures were not simply additive and coculture appears useful to examine the interaction of these cell types. These findings have implications for future therapies for aseptic loosening and for the development of tests to predict the development of this condition.

Acid Phosphatase↗
PubMed2000

Responses of different cell lines from ocular tissues to elevated hydrostatic pressure.

BACKGROUND/AIMS: Mechanical forces are thought to induce cellular responses through activation of signalling pathways. Cells within the intraocular environment are exposed to constant changes in the levels of intraocular pressure. In this study, an attempt was made to determine the acute effects of elevated hydrostatic pressure on different intraocular cells grown in culture. METHODS: Different cell lines derived from ocular tissues including non-pigmented and pigmented ciliary epithelium, trabecular meshwork, retina, and lamina cribrosa were incubated in a pressurised chamber at 50 mm Hg in a culture incubator at 37 degrees C for up to 6 hours. Control cells were incubated at atmospheric pressure. The viability of the cells was examined using their intracellular esterase activity. The morphology and cytoskeleton of the cells were investigated using microscopy and phalloidin staining. Adenylyl cyclase activity was assessed by measuring the conversion of [(3)H]-cAMP from [(3)H]-ATP in response to elevated hydrostatic pressure for 1-6 hours. In addition, at the end of incubation period under elevated hydrostatic pressure the recovery of adenylyl cyclase activity to control levels was examined. RESULTS: Cell viability did not change following exposure to elevated hydrostatic pressure for 6 hours. Cells subjected to elevated hydrostatic pressure demonstrated morphological differences characterised by a more rounded shape and a redistribution of actin stress fibres that was most prominent in lamina cribrosa astrocytes. A time dependent increase in basal adenylyl cyclase activity, and a decrease in maximum forskolin stimulated activity were observed in all cell lines following exposure to elevated hydrostatic pressure. CONCLUSION: These observations demonstrate that cell lines from different ocular tissues are sensitive to changes in external pressure in vitro. They exhibit morphological and cytoskeletal changes as well as significant alterations of intracellular adenylyl cyclase activity following exposure to acute and sustained levels of elevated hydrostatic pressure of up to 6 hours' duration.

Actins↗
PubMed1993

Morphological and proliferative responses of endothelial cells to hydrostatic pressure: role of fibroblast growth factor.

Subconfluent bovine pulmonary artery endothelial cells on rigid substrates were exposed to 1.5-15 cm H2O sustained hydrostatic pressure for up to 7 days and exhibited elongation, cytoskeletal rearrangement, increased cell proliferation, and bilayering. The role of basic fibroblast growth factor (bFGF) in the mechanism(s) of these endothelial cell responses to sustained hydrostatic pressure was investigated. Evidence that bFGF was released from endothelial cells exposed to sustained hydrostatic pressure or compression was provided by the following experimental results: 1) Cells exposed to control (3 mm H2O) pressure displayed intense nuclear and cytoplasmic bFGF staining by immunocytochemical techniques; this staining was absent in cells exposed to 10 cm H2O for 7 days. 2) Conditioned medium from endothelial cells exposed to 10 cm H2O for 7 days contained a transferable, growth-promoting activity exhibiting heparin-Sepharose affinity, lability to both heat and freeze/thawing, and neutralization by anti-bovine bFGF. 3) Suramin (0.1 mM), a growth-factor receptor inhibitor, abrogated the proliferative and morphological responses of endothelial cells exposed to sustained hydrostatic pressure. Endothelial cells exposed to elevated hydrostatic pressure demonstrated no detectable decrement in cell viability as assessed by Trypan blue exclusion. The results of the present study indicate that hydrostatic pressure or compression can induce bFGF release from endothelial cells independent of cell injury or death; bFGF is subsequently responsible for the morphological, proliferative, and bilayering responses of endothelial cells to hydrostatic pressure.

Analysis of Variance↗
PubMed1997

Effects of hydrostatic pressure on matrix synthesis and matrix metalloproteinase production in the human lumbar intervertebral disc.

STUDY DESIGN: This study is a unique in vitro study on the effects of hydrostatic pressure on human intervertebral disc metabolism. OBJECTIVE: To investigate the effects of hydrostatic pressure on matrix synthesis and matrix metalloproteinase production in the human lumbar intervertebral disc. SUMMARY OF BACKGROUND DATA: Mechanical stress and hydrostatic pressures influence proteoglycan and protein synthesis rates in bovine articular cartilage and coccygeal discs. However, the mechanism of matrix synthesis regulation of the intervertebral disc under mechanical stress has not been elucidated. METHODS: Twenty-eight human lumbar intervertebral discs obtained from surgery and from cadavers at autopsy were used. Each tissue fraction was charged with medium in a plastic syringe and placed in a water-filled hydrostatic pressure-control vessel. The hydrostatic pressures applied were 1 (control), 3, and 30 atm (atm = atmospheres) for 2 hours. The proteoglycan and protein synthesis rates were determined by radioisotope incorporation. The production of matrix metalloproteinase-3 and tissue inhibitor of metalloproteinases-1 were measured by a one-step enzyme immunoassay method using monoclonal antibodies. RESULTS: Three atm pressure stimulated proteoglycan synthesis rates in the nucleus pulposus and inner anulus (n = 14 in each tissue). Compared with the control group, 30 atm pressure significantly inhibited proteoglycan synthesis in the inner anulus (P = 0.011). In the nucleus pulposus, matrix metalloproteinase-3 production was stimulated at a pressure of 30 atm relative to 3 atm (P = 0.014, n = 16 in each tissue). The highest tissue inhibitor of metalloproteinases-1 production showed highest values at 3 atm pressure in the inner anulus (n = 16 in each tissue). CONCLUSION: The results suggest that hydrostatic pressure influences intervertebral disc cell metabolism. A physiologic level of hydrostatic pressure (3 atm) may act as an anabolic factor for stimulation of proteoglycan synthesis and tissue inhibitor of metalloproteinases-1 production. This may be essential for maintaining the matrix of the disc. If the pressure was 30 atm or more or 1 atm or less, a catabolic effect will be predominant, with reduction of proteoglycan synthesis rate and increase of matrix metalloproteinase-3 production. Abnormal hydrostatic pressure, therefore, may accelerate disc degeneration.

Cadaver↗
PubMed1988

Hydrostatic pressure enhances baroreceptor reflexes in the rat.

The effects of increased hydrostatic pressure on baroreceptor reflexes in the decerebrate rat have been investigated. Baroreceptor 'sensitivity' was assessed from the relationship between cardiac interval and systolic blood pressure following infusion of phenylephrine to animals exposed to increases in hydrostatic pressure (1-30 bar) with helium. Resting heart rate and systolic blood pressure were reduced and the 'sensitivity' of the baroreflex heart rate response was significantly increased by exposure to pressure. This enhancement of cardiovascular reflex function, elicited by moderate increases in hydrostatic pressure may, in part, be centrally mediated.

Animals↗
PubMed2003

Upregulation of aggrecan and type II collagen mRNA expression in bovine chondrocytes by the application of hydrostatic pressure.

The aim of this study was to investigate the effect of hydrostatic pressure on the expression of messenger ribonucleic acid (mRNA) for specific extracellular matrix proteins in chondrocytes. Chondrocytes obtained from bovine metatarsophalangeal joints were embedded in cylindrical 2% agarose gels. A novel experimental system was used to apply 5 MPa of static hydrostatic pressure to these chondrocytes for 4 hours. The application of hydrostatic pressure caused a significant increase in the level of aggrecan mRNA by almost four fold (p<0.01) as well as a 50% increase in the level of type II collagen mRNA (p<0.05). However, there was no significant change in the level of TIMP-1 mRNA. It was suggested that the application of hydrostatic pressure, in the absence of cell deformation, can bring about changes in the matrix components which may play an important role in the homeostasis and mechanical properties of articular cartilage.

Aggrecans↗