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At least 19 records
All sourcesNASA NTRSDOE OSTI
NASA NTRS2003

Fluid shear stress inhibits TNF-alpha-induced apoptosis in osteoblasts: a role for fluid shear stress-induced activation of PI3-kinase and inhibition of caspase-3

In bone, a large proportion of osteoblasts, the cells responsible for deposition of new bone, normally undergo programmed cell death (apoptosis). Because mechanical loading of bone increases the rate of new bone formation, we hypothesized that mechanical stimulation of osteoblasts might increase their survival. To test this hypothesis, we investigated the effects of fluid shear stress (FSS) on osteoblast apoptosis using three osteoblast cell types: primary rat calvarial osteoblasts (RCOB), MC3T3-E1 osteoblastic cells, and UMR106 osteosarcoma cells. Cells were treated with TNF-alpha in the presence of cyclohexamide (CHX) to rapidly induce apoptosis. Osteoblasts showed significant signs of apoptosis within 4-6 h of exposure to TNF-alpha and CHX, and application of FSS (12 dyne/cm(2)) significantly attenuated this TNF-alpha-induced apoptosis. FSS activated PI3-kinase signaling, induced phosphorylation of Akt, and inhibited TNF-alpha-induced activation of caspase-3. Inhibition of PI3-kinase, using LY294002, blocked the ability of FSS to rescue osteoblasts from TNF-alpha-induced apoptosis and blocked FSS-induced inhibition of caspase-3 activation in osteoblasts treated with TNF-alpha. LY294002 did not, however, prevent FSS-induced phosphorylation of Akt suggesting that activation of Akt alone is not sufficient to rescue cells from apoptosis. This result also suggests that FSS can activate Akt via a PI3-kinase-independent pathway. These studies demonstrate for the first time that application of FSS to osteoblasts in vitro results in inhibition of TNF-alpha-induced apoptosis through a mechanism involving activation of PI3-kinase signaling and inhibition of caspases. FSS-induced activation of PI3-kinase may promote cell survival through a mechanism that is distinct from the Akt-mediated survival pathway. Copyright 2002 Wiley-Liss, Inc.

Non-NASA Center↗
NASA NTRS2020

Let Dependent Low Dose and Synergistic Inhibition of Human Angiogenesis by Charged Particles: Validation of Micrornas That Drive Inhibition

The microvasculature permeates all tissues at the microscopic level making the whole body a target for Space Radiation. Depending on the trajectory a single heavy ion particle would traverse many micro-vessels as it passes through the body causing a widespread response. Studies on the effects of charged particles on the development of human 3D micro-vessel models show that angiogenesis is inhibited by all ions detectible at a dose of 1.25c Gy. However, there are two distinct mechanisms of inhibition depending on the linear energy transfer (LET). We show here that the inhibition of angiogenesis is demarcated into two groups. The blockage of the early motile stage of micro-vessel development occurs after exposure to light ions (LET 3 KeV/n or lower) whereas the prevention of the later stages of development when the microvessels widen into tubes occurs after exposure to heavy ions (LET 8 KeV/n or higher). Strikingly, the combined effect of each ion has a synergistic effect detectible as low as 0.6 cGy. The low fluence of these doses indicates a bystander effect where the response is transmitted to other cells by the secretion of molecules by targeted cells. Prime candidates are microRNAs (miRNAs) and we have previously identified three angiogenesis-associated miRNA regulators from serum, cardiac tissue, and micro-vessels exposed to a simplified Galactic Cosmic Radiation (GCR) simulation – a mixture of heavy and light ions found in Space. Synthetic molecules (Antagomirs) designed to inhibit the predicted miRNAs were successfully used to reverse the inhibition of angiogenesis by Space Radiation. With the simple application of the antagomirs as a countermeasure we have for the first time shown an amelioration of the complex response(s) of angiogenesis to Space Radiation. This opens a new avenue of possible mitigation strategies based on the regulation and understanding of master regulators. These results not only have impact with Space Radiation research, but also provide clues of possible therapeutics for terrestrial diseases in the clinical setting.

Yen-Ruh Wuu↗
NASA NTRS1998

Inhibition of auxin movement from the shoot into the root inhibits lateral root development in Arabidopsis

In roots two distinct polar movements of auxin have been reported that may control different developmental and growth events. To test the hypothesis that auxin derived from the shoot and transported toward the root controls lateral root development, the two polarities of auxin transport were uncoupled in Arabidopsis. Local application of the auxin-transport inhibitor naphthylphthalamic acid (NPA) at the root-shoot junction decreased the number and density of lateral roots and reduced the free indoleacetic acid (IAA) levels in the root and [3H]IAA transport into the root. Application of NPA to the basal half of or at several positions along the root only reduced lateral root density in regions that were in contact with NPA or in regions apical to the site of application. Lateral root development was restored by application of IAA apical to NPA application. Lateral root development in Arabidopsis roots was also inhibited by excision of the shoot or dark growth and this inhibition was reversible by IAA. Together, these results are consistent with auxin transport from the shoot into the root controlling lateral root development.

NASA Discipline Plant Biology↗
NASA NTRS2003

Extracellular ATP inhibits root gravitropism at concentrations that inhibit polar auxin transport

Raising the level of extracellular ATP to mM concentrations similar to those found inside cells can block gravitropism of Arabidopsis roots. When plants are grown in Murashige and Skoog medium supplied with 1 mM ATP, their roots grow horizontally instead of growing straight down. Medium with 2 mM ATP induces root curling, and 3 mM ATP stimulates lateral root growth. When plants are transferred to medium containing exogenous ATP, the gravity response is reduced or in some cases completely blocked by ATP. Equivalent concentrations of ADP or inorganic phosphate have slight but usually statistically insignificant effects, suggesting the specificity of ATP in these responses. The ATP effects may be attributable to the disturbance of auxin distribution in roots by exogenously applied ATP, because extracellular ATP can alter the pattern of auxin-induced gene expression in DR5-beta-glucuronidase transgenic plants and increase the response sensitivity of plant roots to exogenously added auxin. The presence of extracellular ATP also decreases basipetal auxin transport in a dose-dependent fashion in both maize (Zea mays) and Arabidopsis roots and increases the retention of [(3)H]indole-3-acetic acid in root tips of maize. Taken together, these results suggest that the inhibitory effects of extracellular ATP on auxin distribution may happen at the level of auxin export. The potential role of the trans-plasma membrane ATP gradient in auxin export and plant root gravitropism is discussed.

Non-NASA Center↗
NASA NTRS2014

GPM Timeline Inhibits For IT Processing

The Safety Inhibit Timeline Tool was created as one approach to capturing and understanding inhibits and controls from IT through launch. Global Precipitation Measurement (GPM) Mission, which launched from Japan in March 2014, was a joint mission under a partnership between the National Aeronautics and Space Administration (NASA) and the Japan Aerospace Exploration Agency (JAXA). GPM was one of the first NASA Goddard in-house programs that extensively used software controls. Using this tool during the GPM buildup allowed a thorough review of inhibit and safety critical software design for hazardous subsystems such as the high gain antenna boom, solar array, and instrument deployments, transmitter turn-on, propulsion system release, and instrument radar turn-on. The GPM safety team developed a methodology to document software safety as part of the standard hazard report. As a result of this process, a new tool safety inhibit timeline was created for management of inhibits and their controls during spacecraft buildup and testing during IT at GSFC and at the launch range in Japan. The Safety Inhibit Timeline Tool was a pathfinder approach for reviewing software that controls the electrical inhibits. The Safety Inhibit Timeline Tool strengthens the Safety Analysts understanding of the removal of inhibits during the IT process with safety critical software. With this tool, the Safety Analyst can confirm proper safe configuration of a spacecraft during each IT test, track inhibit and software configuration changes, and assess software criticality. In addition to understanding inhibits and controls during IT, the tool allows the Safety Analyst to better communicate to engineers and management the changes in inhibit states with each phase of hardware and software testing and the impact of safety risks. Lessons learned from participating in the GPM campaign at NASA and JAXA will be discussed during this session.

Inhibits↗
NASA NTRS1999

Angiotensin-converting enzyme and matrix metalloproteinase inhibition with developing heart failure: comparative effects on left ventricular function and geometry

The progression of congestive heart failure (CHF) is left ventricular (LV) myocardial remodeling. The matrix metalloproteinases (MMPs) contribute to tissue remodeling and therefore MMP inhibition may serve as a useful therapeutic target in CHF. Angiotensin converting enzyme (ACE) inhibition favorably affects LV myocardial remodeling in CHF. This study examined the effects of specific MMP inhibition, ACE inhibition, and combined treatment on LV systolic and diastolic function in a model of CHF. Pigs were randomly assigned to five groups: 1) rapid atrial pacing (240 beats/min) for 3 weeks (n = 8); 2) ACE inhibition (fosinopril, 2.5 mg/kg b.i.d. orally) and rapid pacing (n = 8); 3) MMP inhibition (PD166793 2 mg/kg/day p.o.) and rapid pacing (n = 8); 4) combined ACE and MMP inhibition (2.5 mg/kg b.i.d. and 2 mg/kg/day, respectively) and rapid pacing (n = 8); and 5) controls (n = 9). LV peak wall stress increased by 2-fold with rapid pacing and was reduced in all treatment groups. LV fractional shortening fell by nearly 2-fold with rapid pacing and increased in all treatment groups. The circumferential fiber shortening-systolic stress relation was reduced with rapid pacing and increased in the ACE inhibition and combination groups. LV myocardial stiffness constant was unchanged in the rapid pacing group, increased nearly 2-fold in the MMP inhibition group, and was normalized in the ACE inhibition and combination treatment groups. Increased MMP activation contributes to the LV dilation and increased wall stress with pacing CHF and a contributory downstream mechanism of ACE inhibition is an effect on MMP activity.

Non-NASA Center↗
NASA NTRS1994

Inhibition of the Vacuolar-like ATPase from Halobacterium saccharovorum by Thiol Reagents: Evidence for Different Functional Thiols

N-Ethylmaleimide (NEM) inhibited the vacuolar-like ATPase from Halobacterium saccharovorum (K(sub i) approximately 1 mM) by modifying one or more of the thiols located on the largest of the subunit. ATP protected against inhibition and coincidentally prevented NEM binding which suggested that NEM acts at or near the catalytic site. p-Chloromercuriphenylsulfonate (PCMS) also inhibited this ATPase (K(sub i) approximately 90 microM). ATP did not protect against PCMS inhibition. Dithiothreitol (DTT) partially reversed PCMS inhibition and restored approximately half of the initial activity of 90% inhibited enzyme. DTT did not restore activity of the NEM-inhibited enzyme or the PCMS-inhibited enzyme when it was subsequently incubated with NEM. The failure of ATP to protect against PCMS inhibition and the inability of DTT to restore activity of enzyme incubated in the presence of PCMS and NEM suggests these reagents react with different thiols and that the PCMS-sensitive thiol may have a structural role.

Hochstein, L. I.↗
NASA NTRS1998

Two genetically separable phases of growth inhibition induced by blue light in Arabidopsis seedlings

High fluence-rate blue light (BL) rapidly inhibits hypocotyl growth in Arabidopsis, as in other species, after a lag time of 30 s. This growth inhibition is always preceded by the activation of anion channels. The membrane depolarization that results from the activation of anion channels by BL was only 30% of the wild-type magnitude in hy4, a mutant lacking the HY4 BL receptor. High-resolution measurements of growth made with a computer-linked displacement transducer or digitized images revealed that BL caused a rapid inhibition of growth in wild-type and hy4 seedlings. This inhibition persisted in wild-type seedlings during more than 40 h of continuous BL. By contrast, hy4 escaped from the initial inhibition after approximately 1 h of BL and grew faster than wild type for approximately 30 h. Wild-type seedlings treated with 5-nitro-2-(3-phenylpropylamino)-benzoic acid, a potent blocker of the BL-activated anion channel, displayed rapid growth inhibition, but, similar to hy4, these seedlings escaped from inhibition after approximately 1 h of BL and phenocopied the mutant for at least 2.5 h. The effects of 5-nitro-2-(3-phenylpropylamino)-benzoic acid and the HY4 mutation were not additive. Taken together, the results indicate that BL acts through HY4 to activate anion channels at the plasma membrane, causing growth inhibition that begins after approximately 1 h. Neither HY4 nor anion channels appear to participate greatly in the initial phase of inhibition.

Non-NASA Center↗
NASA NTRS2000

Nitric oxide inhibits calpain-mediated proteolysis of talin in skeletal muscle cells

We tested the hypothesis that nitric oxide can inhibit cytoskeletal breakdown in skeletal muscle cells by inhibiting calpain cleavage of talin. The nitric oxide donor sodium nitroprusside prevented many of the effects of calcium ionophore on C(2)C(12) muscle cells, including preventing talin proteolysis and release into the cytosol and reducing loss of vinculin, cell detachment, and loss of cellular protein. These results indicate that nitric oxide inhibition of calpain protected the cells from ionophore-induced proteolysis. Calpain inhibitor I and a cell-permeable calpastatin peptide also protected the cells from proteolysis, confirming that ionophore-induced proteolysis was primarily calpain mediated. The activity of m-calpain in a casein zymogram was inhibited by sodium nitroprusside, and this inhibition was reversed by dithiothreitol. Previous incubation with the active site-targeted calpain inhibitor I prevented most of the sodium nitroprusside-induced inhibition of m-calpain activity. These data suggest that nitric oxide inhibited m-calpain activity via S-nitrosylation of the active site cysteine. The results of this study indicate that nitric oxide produced endogenously by skeletal muscle and other cell types has the potential to inhibit m-calpain activity and cytoskeletal proteolysis.

NASA Discipline Musculoskeletal↗
NASA NTRS1995

Role of calcium in growth inhibition induced by a novel cell surface sialoglycopeptide

Our laboratory has purified an 18 kDa cell surface sialoglycopeptide growth inhibitor (CeReS-18) from intact bovine cerebral cortex cells. Evidence presented here demonstrates that sensitivity to CeReS-18-induced growth inhibition in BALB-c 3T3 cells is influenced by calcium, such that a decrease in the calcium concentration in the growth medium results in an increase in sensitivity to CeReS-18. Calcium did not alter CeReS-18 binding to its cell surface receptor and CeReS-18 does not bind calcium directly. Addition of calcium, but not magnesium, to CeReS-18-inhibited 3T3 cells results in reentry into the cell cycle. A greater than 3-hour exposure to increased calcium is required for escape from CeReS-18-induced growth inhibition. The calcium ionophore ionomycin could partially mimic the effect of increasing extracellular calcium, but thapsigargin was ineffective in inducing escape from growth inhibition. Increasing extracellular calcium 10-fold resulted in an approximately 7-fold increase in total cell-associated 45Ca+2, while free intracellular calcium only increased approximately 30%. However, addition of CeReS-18 did not affect total cell-associated calcium or the increase in total cell-associated calcium observed with an increase in extracellular calcium. Serum addition induced mobilization of intracellular calcium and influx across the plasma membrane in 3T3 cells, and pretreatment of 3T3 cells with CeReS-18 appeared to inhibit these calcium mobilization events. These results suggest that a calcium-sensitive step exists in the recovery from CeReS-18-induced growth inhibition. CeReS-18 may inhibit cell proliferation through a novel mechanism involving altering the intracellular calcium mobilization/regulation necessary for cell cycle progression.

Non-NASA Center↗
NASA NTRS2012

Global Precipitation Measurement (GPM) Safety Inhibit Timeline Tool

The Global Precipitation Measurement (GPM) Observatory is a joint mission under the partnership by National Aeronautics and Space Administration (NASA) and the Japan Aerospace Exploration Agency (JAXA), Japan. The NASA Goddard Space Flight Center (GSFC) has the lead management responsibility for NASA on GPM. The GPM program will measure precipitation on a global basis with sufficient quality, Earth coverage, and sampling to improve prediction of the Earth's climate, weather, and specific components of the global water cycle. As part of the development process, NASA built the spacecraft (built in-house at GSFC) and provided one instrument (GPM Microwave Imager (GMI) developed by Ball Aerospace) JAXA provided the launch vehicle (H2-A by MHI) and provided one instrument (Dual-Frequency Precipitation Radar (DPR) developed by NTSpace). Each instrument developer provided a safety assessment which was incorporated into the NASA GPM Safety Hazard Assessment. Inhibit design was reviewed for hazardous subsystems which included the High Gain Antenna System (HGAS) deployment, solar array deployment, transmitter turn on, propulsion system release, GMI deployment, and DPR radar turn on. The safety inhibits for these listed hazards are controlled by software. GPM developed a "pathfinder" approach for reviewing software that controls the electrical inhibits. This is one of the first GSFC in-house programs that extensively used software controls. The GPM safety team developed a methodology to document software safety as part of the standard hazard report. As part of this process a new tool "safety inhibit time line" was created for management of inhibits and their controls during spacecraft buildup and testing during 1& Tat GSFC and at the Range in Japan. In addition to understanding inhibits and controls during 1& T the tool allows the safety analyst to better communicate with others the changes in inhibit states with each phase of hardware and software testing. The tool was very useful for communicating compliance with safety requirements especially when working with a foreign partner.

Dion, Shirley↗
NASA NTRS1993

S-Ketoprofen Inhibits Tenotomy-Induced Bone Loss and Dynamics in Weanling Rats

The objects of this study were to determine whether S-ketoprofen, a non-steroidal anti-inflammatory drug (NSAID), can prevent immobilization (tenotomy)-induced bone loss in weanling rats. Forty five 4 week-old Sprague-Dawley female rats were either sham-operated or subjected to knee tenotomy and treated simultaneously with 0, 0.02, 0.1, 0.5 or 2.5 mg of S-ketoprofen/kg per day for 21 days. We then studied double-fluorescent labeled proximal tibial longitudinal sections and tibial shaft cross sections using static and dynamic histomorphometry. Less cancellous bone mass in proximal tibial metaphyses was found in tenotomized controls than in basal (36%) and sham-operated (54%) controls. This was due to the inhibition of age-related bone gain and induced bone loss due to increased bone resorption and decreased bone formation. S-ketoprofen prevented both the inhibition of age-related bone gain and the stimulation of bone loss at the 2.5 mg/kg per day dose level, while it only prevented bone loss at the 0.5 mg/kg dose levels. In cancellous bone, dynamic histomorphometry showed that S-ketoprofen prevented the tenotomy induced decrease in bone formation and increase in bone resorption. In the tibial shaft, tenotomy inhibited the enlargement of total tissue area by depressing periosteal bone formation, and thus inhibited age-related cortical bone gain. S-ketoprofen treatment did not prevent this change at all dose levels, but reduced marrow cavity area to increase cortical bone area at the 0.1, 0.5 and 2.5 mg/kg per dose levels compared to tenotomy controls. However, the cortical bone area in the 0.1 and 0.5 mg dose-treated treated tenotomy rats was still lower than in the age-related controls. S-ketoprofen also prevented the increase in endocortical eroded perimeter induced by tenotomy. In summary, tenotomy inhibited age-related bone gain and stimulated bone loss in cancellous bone sites, and only inhibited age-related bone gain in cortical bone sites. S-ketoprofen treatment at the highest dose levels prevented the changes in cancellous bone, and reduced marrow area to increase cortical bone in the tibial shafts.

Zeng, Q. Q.↗
NASA NTRS2002

Increased GABA(A) inhibition of the RVLM after hindlimb unloading in rats

Attenuated baroreflex-mediated increases in renal sympathetic nerve activity (RSNA) in hindlimb unloaded (HU) rats apparently are due to changes within the central nervous system. We hypothesized that GABA(A) receptor-mediated inhibition of the rostral ventrolateral medulla (RVLM) is increased after hindlimb unloading. Responses to bilateral microinjection of the GABA(A) antagonist (-)-bicuculline methiodide (BIC) into the RVLM were examined before and during caudal ventrolateral medulla (CVLM) inhibition in Inactin-anesthetized control and HU rats. Increases in mean arterial pressure (MAP), heart rate (HR), and RSNA in response to BIC in the RVLM were significantly enhanced in HU rats. Responses to bilateral CVLM blockade were not different. When remaining GABA(A) inhibition in the RVLM was blocked by BIC during CVLM inhibition, the additional increases in MAP and RSNA were significantly greater in HU rats. These data indicate that GABA(A) receptor-mediated inhibition of RVLM neurons is augmented after hindlimb unloading. Effects of input from the CVLM were unaltered. Thus, after cardiovascular deconditioning in rodents, the attenuated increase in sympathetic nerve activity in response to hypotension is associated with greater GABA(A) receptor-mediated inhibition of RVLM neurons originating at least in part from sources other than the CVLM.

Non-NASA Center↗
NASA NTRS1998

1,25-Dihydroxyvitamin D3 inhibition of col1a1 promoter expression in calvariae from neonatal transgenic mice

We studied the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on organ cultures of transgenic mouse calvariae containing segments of the Col1a1 promoter extending to -3518, -2297, -1997, -1794, -1763, and -1719 bp upstream of the transcription start site fused to the chloramphenicol acetyltransferase (CAT) reporter gene. 1,25(OH)2D3 had a dose-dependent inhibitory effect on the expression of the -3518 bp promoter construct (ColCAT3.6), with maximal inhibition of about 50% at 10 nM. This level of inhibition was consistent with the previously observed effect on the endogenous Col1a1 gene in bone cell models. All of the shorter constructs were also inhibited by 10 nM 1,25(OH)2D3, suggesting that the sequences required for 1, 25(OH)2D3 inhibition are downstream of -1719 bp. The inhibitory effect of 1,25(OH)2D3 on transgene mRNA was maintained in the presence of the protein synthesis inhibitor cycloheximide, suggesting that the inhibitory effect on Col1a1 gene transcription does not require de novo protein synthesis. We also examined the in vivo effect of 1,25(OH)2D3 treatment of transgenic mice on ColCAT activity, and found that 48 h treatment caused a dose-dependent inhibition of CAT activity in calvariae comparable to that observed in organ cultures. In conclusion, we demonstrated that 1,25(OH)2D3 inhibits Col1A1 promoter activity in transgenic mouse calvariae, both in vivo and in vitro. The results indicate that there is a 1, 25(OH)2D3 responsive element downstream of -1719 bp. The inhibitory effect does not require new protein synthesis.

NASA Discipline Cell Biology↗
NASA NTRS2000

Generational Analysis Reveals that TGF-Beta1 Inhibits the Rate of Angiogenesis in Vivo by Selective Decrease in the Number of New Vessels

Quantitative analysis of vascular generational branching demonstrated that transforming growth factor-beta1 (TGF-beta1), a multifunctional cytokine and angiogenic regulator, strongly inhibited angiogenesis in the arterial tree of the developing quail chorioallantoic membrane (CAM) by inhibition of the normal increase in the number of new, small vessels. The cytokine was applied uniformly in solution at embryonic day 7 (E7) to the CAMs of quail embryos cultured in petri dishes. After 24 h the rate of arterial growth was inhibited by as much as 105% as a function of increasing TGF-beta1 concentration. Inhibition of the rate of angiogenesis in the arterial tree by TGF-beta1 relative to controls was measured in digital images by three well-correlated, computerized methods. The first computerized method, direct measurement by the computer code VESGEN of vascular morphological parameters according to branching generations G(sub 1) through G(sub greater than or equal to 5), revealed that TGF-beta1 selectively inhibited the increase in the number density of small vessels, N(sub v greater than or equal to 5), (382 plus or minus 85 per square centimeter) for specimens treated with 1 microgram TGF-beta1/CAM for 24 h, compared to 583 plus or minus 99 per square centimeter for controls), but did not significantly affect other parameters such as average vessel length or vessel diameter. The second and third methods, the fractal dimension (D(sub f)) and grid intersection (rho (sub v)), are statistical descriptors of spatial pattern and density. According to D(sub f) and rho(sub v), arterial density increased in control specimens from 1.382 plus or minus 0.007 and 662 plus or minus 52 per square centimeters at E7 (0 h) to 1.439 plus or minus 0.013 and 884 plus or minus 55 per square centimeters at E8 (24 h), compared to 1.379 plus or minus 0.039 and 650 plus or minus 111 per square centimeter for specimens treated with 1 microgram TGF-beta1/CAM for 24 h. TGF-beta1 therefore regulates vascular pattern and the rate of angiogenesis in a unique fingerprint manner, as do other major angiogenic regulators that include VEGF, FGF-2 (bFGF), and angiostatin. TGF-beta1 did not stimulate angiogenesis significantly at low cytokine concentrations, which suggests that this quail CAM model of angiogenesis is not associated with an inflammatory response.

Parsons-Wingerter, Patricia↗
NASA NTRS2002

Inhibition of the active lymph pump by flow in rat mesenteric lymphatics and thoracic duct

There are only a few reports of the influence of imposed flow on an active lymph pump under conditions of controlled intraluminal pressure. Thus, the mechanisms are not clearly defined. Rat mesenteric lymphatics and thoracic ducts were isolated, cannulated and pressurized. Input and output pressures were adjusted to impose various flows. Lymphatic systolic and diastolic diameters were measured and used to determine contraction frequency and pump flow indices. Imposed flow inhibited the active lymph pump in both mesenteric lymphatics and in the thoracic duct. The active pump of the thoracic duct appeared more sensitive to flow than did the active pump of the mesenteric lymphatics. Imposed flow reduced the frequency and amplitude of the contractions and accordingly the active pump flow. Flow-induced inhibition of the active lymph pump followed two temporal patterns. The first pattern was a rapidly developing inhibition of contraction frequency. Upon imposition of flow, the contraction frequency immediately fell and then partially recovered over time during continued flow. This effect was dependent on the magnitude of imposed flow, but did not depend on the direction of flow. The effect also depended upon the rate of change in the direction of flow. The second pattern was a slowly developing reduction of the amplitude of the lymphatic contractions, which increased over time during continued flow. The inhibition of contraction amplitude was dependent on the direction of the imposed flow, but independent of the magnitude of flow. Nitric oxide was partly but not completely responsible for the influence of flow on the mesenteric lymph pump. Exposure to NO mimicked the effects of flow, and inhibition of the NO synthase by N (G)-monomethyl-L-arginine attenuated but did not completely abolish the effects of flow.

NASA Discipline Musculoskeletal↗
NASA NTRS2002

Oxidative stress inhibits caveolin-1 palmitoylation and trafficking in endothelial cells

During normal and pathological conditions, endothelial cells (ECs) are subjected to locally generated reactive oxygen species, produced by themselves or by other vessel wall cells. In excess these molecules cause oxidative injury to the cell but at moderate levels they might modulate intracellular signalling pathways. We have investigated the effect of oxidative stress on the palmitoylation and trafficking of caveolin-1 in bovine aortic ECs. Exogenous H2O2 did not alter the intracellular localization of caveolin-1 in ECs. However, metabolic labelling experiments showed that H2O2 inhibited the trafficking of newly synthesized caveolin-1 to membrane raft domains. Several mechanisms potentially responsible for this inhibition were examined. Impairment of caveolin-1 synthesis by H2O2 was not responsible for diminished trafficking. Similarly, the inhibition was independent of H2O2-induced caveolin-1 phosphorylation as shown by the markedly different concentration dependences. We tested the effect of H2O2 on palmitoylation of caveolin-1 by the incorporation of [3H]palmitic acid. Exposure of ECs to H2O2 markedly inhibited the palmitoylation of caveolin-1. Comparable inhibition was observed after treatment of cells with H2O2 delivered either as a bolus or by continuous delivery with glucose and glucose oxidase. Kinetic studies showed that H2O2 did not alter the rate of caveolin-1 depalmitoylation but instead decreased the 'on-rate' of palmitoylation. Together these results show for the first time the modulation of protein palmitoylation by oxidative stress, and suggest a cellular mechanism by which stress might influence caveolin-1-dependent cell activities such as the concentration of signalling proteins and cholesterol trafficking.

NASA Discipline Cell Biology↗
NASA NTRS1985

In vivo inhibition of polyamine biosynthesis and growth in tobacco ovary tissues

Post fertilization growth of tobacco ovary tissues treated with inhibitors of polyamine (PA) biosynthesis was examined in relation to endogenous PA titers and the activities of arginine decarboxylase (ADC, EC 4.1.1.19) and ornithine decarboxylase (ODC, EC 4.1.1.17). DL-alpha-Difluoromethylornithine (DFMO) and DL-alpha-difluoromethylarginine (DFMA), specific, irreversible ("suicide") inhibitors of ODC and ADC in vitro, were used to modulate PA biosynthesis in excised flowers. ODC represented >99% of the total decarboxylase activity in tobacco ovaries. In vivo inhibition of ODC with DFMO resulted in a significant decrease in PA titers, ovary fresh weight and protein content. Simultaneous inhibition of both decarboxylases by DFMO and DFMA produced only a marginally greater depression in growth and PA titers, indicating that ODC activity is rate-limiting for PA biosynthesis in these tissues. Paradoxically, DFMA alone inhibited PA biosynthesis, not as a result of a specific inhibition of ADC, but primarily through the inactivation of ODC. In vivo inhibition of ODC by DFMA appears to result from arginase-mediated hydrolysis of this inhibitor to urea and DFMO, the suicide substrate for ODC. Putrescine conjugates in tobacco appear to function as a storage form of this amine which, upon hydrolysis, may contribute to Put homeostasis during growth.

NASA Program Space Biology↗