Immortalization of human melanocytes does not alter the de novo properties of nitric oxide to induce cell detachment from extracellular matrix components via cGMP.

Nitric oxide (NO) is an important mediator in many (patho)physiological processes including inflammation and skin cancer. A key transducer in NO signaling is the soluble guanylyl cyclase (sGC) that catalyzes the formation of guanosine 3',5'-cyclic monophosphate (cGMP). The basic mechanism of NO-cGMP signaling in melanocytic cells is, however, not well elucidated. A setback for such studies is the limited availability of patient-derived melanocytes. Here, we report that immortalized human normal and vitiliginous cell lines generated via cell transfection with human papilloma virus 16 genes E6 and E7 express NO synthase and guanylyl cyclase isoforms and the multidrug resistance-associated proteins 4 and 5 as selective cGMP exporters. Donors of NO (e.g., the NONOate (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2-diolate (PAPA-NO) and reactive nitrogen oxygen species (RNOS) like 3-morpholino-sydnonimine (SIN-1) as a donor of peroxynitrite as well as YC-1 as a NO-independent sGC stimulator increased intracellular cGMP levels in immortalized melanocytes (up to eightfold over controls), indicating the expression of functional sGC in these cells. PAPA-NO and SIN-1 also reduced the attachment of immortalized melanocytes to extracellular matrix (ECM) components like fibronectin which was dependent on cellular melanin content and cGMP. Such effects on melanoma cells were positively related to metastatic potential and were cGMP independent. Intriguingly, nonpigmented metastatic melanoma cells were more sensitive to exogenous sources of RNOS than of NO. Thus, immortalized melanocytes can be used as a tool for further research on differences in cell signaling between the different melanocytic lineages in particular towards impairment of cell-ECM adhesion by NO or RNOS, which may be important in metastasis and vitiligo pathogenesis.

Towards human exploration of space: the THESEUS review series on muscle and bone research priorities.

Without effective countermeasures, the musculoskeletal system is altered by the microgravity environment of long-duration spaceflight, resulting in atrophy of bone and muscle tissue, as well as in deficits in the function of cartilage, tendons, and vertebral disks. While inflight countermeasures implemented on the International Space Station have evidenced reduction of bone and muscle loss on low-Earth orbit missions of several months in length, important knowledge gaps must be addressed in order to develop effective strategies for managing human musculoskeletal health on exploration class missions well beyond Earth orbit. Analog environments, such as bed rest and/or isolation environments, may be employed in conjunction with large sample sizes to understand sex differences in countermeasure effectiveness, as well as interaction of exercise with pharmacologic, nutritional, immune system, sleep and psychological countermeasures. Studies of musculoskeletal biomechanics, involving both human subject and computer simulation studies, are essential to developing strategies to avoid bone fractures or other injuries to connective tissue during exercise and extravehicular activities. Animal models may be employed to understand effects of the space environment that cannot be modeled using human analog studies. These include studies of radiation effects on bone and muscle, unraveling the effects of genetics on bone and muscle loss, and characterizing the process of fracture healing in the mechanically unloaded and immuno-compromised spaceflight environment. In addition to setting the stage for evidence-based management of musculoskeletal health in long-duration space missions, the body of knowledge acquired in the process of addressing this array of scientific problems will lend insight into the understanding of terrestrial health conditions such as age-related osteoporosis and sarcopenia.

L-arginine, the natural precursor of NO, is not effective for preventing bone loss in postmenopausal women.

UNLABELLED: NO is an important regulator of bone turnover. L-Arginine, the natural precursor of NO, can enhance NO production. However, no effect of L-arginine hydrochloride supplementation was found on bone metabolism or on BMD, bone mass, or bone structure of healthy postmenopausal women. INTRODUCTION: Recent studies indicate that NO exerts an anabolic effect on bone cell activity. The NO level of the human body can be elevated by administering pharmacological NO donors. Animal studies and the first human trial showed that NO donor administration had a positive effect on bone formation and a negative effect on bone resorption. L-arginine, the natural precursor of NO, can enhance NO production. This study was conducted to examine the effect of an oral L-arginine supplement on bone metabolism of healthy postmenopausal women. MATERIALS AND METHODS: The participants in this study were 30 healthy, age-matched postmenopausal women, divided into two groups. For 6 months, one group (54.5 +/- 4.1 years; 66.3 +/- 10.5 kg) received a daily oral supplement with 18 g L-arginine hydrochloride (14.8 g free L-arginine). The other 15 volunteers (55.3 +/- 4.4 years; 64.2 +/- 9.1 kg) received 18 g dextrose as a placebo. To verify compliance, 24-h urinary excretion of nitrogen was analyzed for 2 consecutive days at baseline and after 2, 4, and 6 months. At baseline and after 2, 4, and 6 months of supplementation, blood was drawn for analysis of insulin-like growth factor-I (IGF-I) and biomarkers of bone metabolism. At baseline, after 6 months, and after 1 year, pQCT measurements were performed at trabecular and cortical sites of the radius and tibia. The two groups of subjects were compared by repeated measures ANOVA. RESULTS: As expected, in the group with L-arginine hydrochloride supplementation, nitrogen excretion rose, and in the placebo group, it remained constant. Only bone formation marker, procollagen type I propeptides (PICP), increased significantly (p < 0.05) after 6 months of L-arginine supplementation. The results from pQCT showed no significant changes at any site in either group. No significant change in IGF-I concentration, which might have been caused by the L-arginine hydrochloride supplementation, was evident. CONCLUSIONS: We conclude from these results that supplementation with L-arginine hydrochloride is not effective for improving bone mass in humans.

Immobilization induces a very rapid increase in osteoclast activity.

We studied in a randomized, strictly controlled cross-over design, the effects of 6 days 6 degrees head-down tilt bed rest (HDT) in eight male healthy subjects in our metabolic ward. The study consisted of two periods (phases) of 11 days each in order to allow for the test subjects being their own controls. Both study phases were identical with respect to environmental conditions, study protocol and diet. Two days before arriving in the metabolic ward the subjects started with a diet. The diet was continued in the metabolic ward. The metabolic ward period (1l days) was divided into three parts: 4 ambulatory days, 6 days either HDT or control and 1 recovery day. Continuous urine collection started on the first day in the metabolic ward to analyze calcium excretion and bone resorption markers. On the 2nd ambulatory day in the metabolic ward and on the 5th day in HDT or control blood was drawn to analyze serum calcium, parathyroid hormone, and bone formation markers. Urinary calcium excretion was, as early as the first day in immobilization, increased (p<0.01). CTX- and NTX-excretion stayed unchanged in the first 24 h in HDT compared to the control. But already on the 2nd day of immobilization, both bone resorption markers significantly increased. We conclude from these results--pronounced rise of bone resorption markers--that already 24 h of immobilization induce a significant rise in osteoclast activity in healthy subjects. Thus, it appears possible to use short-term bed rest studies as a first step for the development of countermeasures to immobilization.

Bone resorption is induced on the second day of bed rest: results of a controlled crossover trial.

The aim of the study was to analyze the kinetics of short-term changes in bone turnover. We studied in a randomized crossover design the effects of 6 days of bed rest on eight healthy male subjects (mean body wt: 70.1 +/- 5.7 kg; mean age: 25.5 +/- 2.9 yr). The metabolic ward period was divided into three parts: 4 ambulatory days, 6 days of either bed rest or non-bed rest periods, and 1 recovery day. The diet was identical in both bed rest and non-bed rest phases. Continuous urine collection started on the first day in the metabolic ward to analyze excretion of bone resorption markers, namely C-telopeptide (CTX) and N-telopeptide (NTX), creatinine, urea, and 3-methylhistidine. On the second ambulatory day and on the fifth day of bed rest or during the non-bed rest phase, blood was drawn to analyze bone formation markers and amino acid concentrations. Urinary calcium excretion was increased as early as the first day of bed rest (P < 0.01). CTX and NTX excretion stayed unchanged during the first 24 h of bed rest compared with the non-bed rest period. However, already on the second day, both resorption markers had increased significantly. NTX excretion increased by 28.7 +/- 14.0% (P < 0.01), whereas CTX excretion rose by 17.8 +/- 8.3% (P < 0.001). Creatinine, urea, and 3-methylhistidine excretion did not change. We conclude that 24 h of bed rest are sufficient to induce a significant rise in osteoclast activity in healthy subjects.

Chronobiotic effects of the melatonin agonist LY 156735 following a simulated 9h time shift: results of a placebo-controlled trial.

INTRODUCTION: The melatonin agonist LY 156735 (LY) is a new investigational drug under development to treat circadian rhythm disorders. The present study assessed the efficacy of LY to alleviate the symptoms of shift lag and to enhance readaptation of desynchronized circadian rhythms to a new time zone. SUBJECTS AND METHODS: Eight healthy male volunteers of age 25-35 yr participated in three identical trials of 13d duration in a temporal isolation unit separated by washout intervals. A high dose (HD) of 5 mg and a low dose (LD) of 0.5 mg of LY and placebo (PL) were administered double-blinded in a three-period cross-over design. Each trial consisted of an adaptation period, a pre-shift period for baseline measurements, a simulated 9h phase-advance shift, and a post-shift period for follow-up. The time shift was performed at 23:00h of day 6 by advancing the laboratory time to 08:00h of day 7. Double-blind study medication was administered at 14:30h on day 6, and at 22:30h on days 7-10. Subjective ratings of jet lag, alertness, tenseness, and daytime fatigue were assessed using visual analog scales (VAS) and standardized questionnaires. The objective markers of readaptation included core body temperature, wrist actigraphy, cortisol and electrolyte excretion, and a battery of computerized performance tests. RESULTS: HD but not LD enhanced the readaptation speed of all physiological rhythms investigated, as demonstrated by a significantly faster movement of acrophases towards the post-shift target time. HD (p = 0.05) significantly blunted the post-shift deterioration of performance in those tests that were sensitive to shift lag. Parameters of subjective well-being were not significantly affected by either dose. CONCLUSION: This pilot study demonstrates the chronobiotic efficacy of LY when taken at a dose of 5 mg/d.

Influence of microgravity on plasma levels of gastroenteropancreatic peptides: a case study.

BACKGROUND: No data are available about the short- or long-term influences of microgravity in space on the release of gastroenteropancreatic peptides, although these peptides are involved in the regulation of gastrointestinal functions. METHODS: Fasting plasma samples were gained during the EUROMIR-94 mission from a European Space Agency (ESA) astronaut who experienced no signs of space motion sickness in orbit. Plasma concentrations of nine gastroenteropancreatic peptides were measured with sensitive and specific radioimmunoassays. RESULTS: Fasting plasma levels of motilin, pancreatic polypeptide (PP), vasoactive intestinal peptide (VIP), and secretin were increased and plasma level of cholecystokinin (CCK) was decreased by acute exposure of the astronaut to microgravity. Chronic (4 wk) exposure caused an enhancement of plasma CCK, motilin, neurotensin, VIP, and insulin whereas plasma concentrations of PP, secretin, gastrin, and somatostatin showed no changes. During the 25-d stay on MIR station plasma levels of CCK, motilin, and neurotensin increased. Short-time body rotations caused an elevation of plasma levels of PP but decreased plasma motilin. CONCLUSIONS: As the influence of microgravity on the peptide levels was not uniform, an effect due to other factors (e.g., change in fluid balance or body weight) is unlikely. Moreover, adaptive changes of some peptides occurred during the stay in orbit. The release of PP and motilin seems to be very sensitive to rotation forces. These results have to be confirmed in more subjects in space to be able to link changes of gastroenteropancreatic peptide release to alterations of gastrointestinal functions.

Positive pressure breathing by total body negative pressure: a new simulation model for fluid balance in weightlessness?

A systems study is proposed to explore correctly the effects of the combination of HDT (Head Down Tilt) and TBNP (Total Body Negative Pressure).

Salt-dependent chemotaxis of macrophages.

Besides their role in immune system host defense, there is growing evidence that macrophages may also be important regulators of salt homeostasis and blood pressure by a TonEBP-VEGF-C dependent buffering mechanism. As macrophages are known to accumulate in the skin of rats fed under high salt diet conditions and are pivotal for removal of high salt storage, the question arose how macrophages sense sites of high sodium storage. Interestingly, we observed that macrophage-like RAW264.7 cells, murine bone marrow-derived macrophages and peritoneal macrophages recognize NaCl hypertonicity as a chemotactic stimulus and migrate in the direction of excess salt concentration by using an in vitro transwell migration assay. While RAW264.7 cells migrated toward NaCl in a dose-dependent fashion, no migratory response toward isotonic or hypotonic media controls, or other osmo-active agents, e.g. urea or mannitol, could be detected. Interestingly, we could not establish a specific role of the osmoprotective transcription factor TonEBP in regulating salt-dependent chemotaxis, since the specific migration of bone marrow-derived macrophages following RNAi of TonEBP toward NaCl was not altered. Although the underlying mechanism remains unidentified, these data point to a thus far unappreciated role for NaCl-dependent chemotaxis of macrophages in the clearance of excess salt, and suggest the existence of novel NaCl sensor/effector circuits, which are independent of the TonEBP system.

Long-term space flight simulation reveals infradian rhythmicity in human Na(+) balance.

The steady-state concept of Na(+) homeostasis, based on short-term investigations of responses to high salt intake, maintains that dietary Na(+) is rapidly eliminated into urine, thereby achieving constant total-body Na(+) and water content. We introduced the reverse experimental approach by fixing salt intake of men participating in space flight simulations at 12 g, 9 g, and 6 g/day for months and tested for the predicted constancy in urinary excretion and total-body Na(+) content. At constant salt intake, daily Na(+) excretion exhibited aldosterone-dependent, weekly (circaseptan) rhythms, resulting in periodic Na(+) storage. Changes in total-body Na(+) (±200-400 mmol) exhibited longer infradian rhythm periods (about monthly and longer period lengths) without parallel changes in body weight and extracellular water and were directly related to urinary aldosterone excretion and inversely to urinary cortisol, suggesting rhythmic hormonal control. Our findings define rhythmic Na(+) excretory and retention patterns independent of blood pressure or body water, which occur independent of salt intake.

ESSC-ESF position paper--science-driven scenario for space exploration: report from the European Space Sciences Committee (ESSC).

In 2005 the then ESA Directorate for Human Spaceflight, Microgravity and Exploration (D-HME) commissioned a study from the European Science Foundation's (ESF) European Space Sciences Committee (ESSC) to examine the science aspects of the Aurora Programme in preparation for the December 2005 Ministerial Conference of ESA Member States, held in Berlin. A first interim report was presented to ESA at the second stakeholders meeting on 30 and 31 May 2005. A second draft report was made available at the time of the final science stakeholders meeting on 16 September 2005 in order for ESA to use its recommendations to prepare the Executive proposal to the Ministerial Conference. The final ESSC report on that activity came a few months after the Ministerial Conference (June 2006) and attempted to capture some elements of the new situation after Berlin, and in the context of the reduction in NASA's budget that was taking place at that time; e.g., the postponement sine die of the Mars Sample Return mission. At the time of this study, ESSC made it clear to ESA that the timeline imposed prior to the Berlin Conference had not allowed for a proper consultation of the relevant science community and that this should be corrected in the near future. In response to that recommendation, ESSC was asked again in the summer of 2006 to initiate a broad consultation to define a science-driven scenario for the Aurora Programme. This exercise ran between October 2006 and May 2007. ESA provided the funding for staff support, publication costs, and costs related to meetings of a Steering Group, two meetings of a larger ad hoc group (7 and 8 December 2006 and 8 February 2007), and a final scientific workshop on 15 and 16 May 2007 in Athens. As a result of these meetings a draft report was produced and examined by the Ad Hoc Group. Following their endorsement of the report and its approval by the plenary meeting of the ESSC, the draft report was externally refereed, as is now normal practice with all ESSC-ESF reports, and amended accordingly. The Ad Hoc Group defined overarching scientific goals for Europe's exploration programme, dubbed "Emergence and co-evolution of life with its planetary environments," focusing on those targets that can ultimately be reached by humans, i.e., Mars, the Moon, and Near Earth Objects. Mars was further recognized as the focus of that programme, with Mars sample return as the recognized primary goal; furthermore the report clearly states that Europe should position itself as a major actor in defining and leading Mars sample return missions. The report is reproduced in this article. On 26 November 2008 the Ministers of ESA Member States decided to give a high strategic priority to the robotic exploration programme of Mars by funding the enhanced ExoMars mission component, in line therefore with the recommendations from this ESSC-ESF report.

Non-linear function model of voice pitch dependency on physical and mental load.

The present work describes that under increasing physical load the voice fundamental frequency (voice pitch) remains on a given level as long as the physical load is well tolerated by the subject, whereas heart rate and blood pressure continuously increase during increasing physical load. This voice pitch level was compared to voice pitch levels under mental load. Using a word recognition system, 11 well trained, young male subjects had to solve 2 moderate mental load tasks. Before, during and after each task, there were structured relaxation phases. The physical load protocol was a standard bicycle stress test. In each protocol phase the subjects had to count from 1 to 10 in order to provide a standardized speech sample. Heart rate and blood pressure were recorded in all phases. Voice frequency was at average 106 +/- 5.2 Hz in the relaxation phases ('rest level') and was increased under mental load (115.9 +/- 5.7 Hz, Pillais-P = 0.037). During physical stress testing, voice pitch remained unchanged ('tolerated load level') between 100 and 200 W (117.4 +/- 4.8 Hz) and increased shortly before physical exhaustion ('exhaustion level', 275-350 W, 142.9 +/- 5.6 Hz, Pillais-P = 0.007). In contrast, heart rate and blood pressure increased continuously with the physical load. Three voice pitch levels could be verified also individually for each subject. For the practical monitoring of emotional stress the individual anchor frequencies for these levels must be assessed. These data indicate that the relationship between both types of load and voice pitch is non-linear with multiple plateaus and transition functions between them.

Non-lesional vitiliginous melanocytes are not characterized by an increased proneness to nitric oxide-induced apoptosis.

Nitric oxide (NO) is a reactive endogenous molecule with multiple functions including inflammation and immunity. NO stimulates melanogenesis by activating soluble guanylyl cyclase (sGC) resulting in increases in intracellular guanosine 3',5'-cyclic monophosphate (cGMP). In vitro experiments showed that NO could inhibit the de novo attachment of melanocytes to extracellular matrix (ECM) suggesting that NO-induced aberrant perturbation of melanocyte-ECM interaction could be a reason for melanocyte loss in vitiliginous lesions. Here, we examined whether there might be differences between normal melanocytes and vitiliginous melanocytes (VMs) with respect to NO-induced detachment from ECM and whether cGMP is involved. We used the direct NO donor (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2-diolate and the peroxynitrite donor 3-morpholino-sydnonimine for the present studies. These donors induced detachment of both normal melanocytes and non-lesional VMs in a time- and concentration-dependent manner with comparable susceptibility and similar expression profile of sGC. Treatment of melanocytes with caspase inhibitors reduced cell detachment, indicating that a major part of the detachment is due to apoptosis. The NO-induced detachment but not apoptosis was partly inhibited in the presence of sGC and cGMP-dependent protein kinase inhibitors. In addition, the membrane-permeable cGMP analog 8-(4-chlorophenyethio/guanosine-3',5'-cyclic monophosphate (PCPT) cGMP was not able to induce apoptosis in melanocytes, suggesting that NO-induced detachment of melanocytes via apoptosis is cGMP-independent. The present results also indicate that there are no apparent differences between NO-induced detachment of non-lesional vitiliginous and normal melanocytes from ECM.

Effects of L-arginine supplementation on bone metabolism.

Recent experimental studies indicate that nitric oxide (NO) is an important regulator of bone turnover in humans by exerting an anabolic effect on bone cell activity. NO is synthesised from the nonessential amino acid L-arginine. Therefore, a supplementation with oral L-arginine might be highly potent to affect bone cell activity via NO synthesis from L-arginine. In our study we examined the effect of a six months oral supplementation with L-arginine-hydrochloride (18 g) on bone metabolism in 15 healthy postmenopausal women. We analysed nitrogen excretion, markers of bone turnover and calcium concentration. The results show neither a change in serum calcium concentration nor in bone turnover as shown by bone markers. In conclusion, L-arginine-hydrochloride supplementation at that concentration seems to have no effect on bone cell activity in healthy postmenopausal women.

Stimulation of cyclic GMP efflux in human melanocytes by hypergravity generated by centrifugal acceleration.

Gravity alteration (micro- and hypergravity) is known to influence cell functions. As guanosine 3',5'-cyclic monophosphate (cGMP) plays an important role in human melanocyte functions and different guanylyl cyclase isoforms are responsible for cGMP synthesis in human non-metastatic and metastatic melanoma cells, we investigated the effects of hypergravity on the regulation of cGMP levels in cultured human melanocytes and in melanoma cell lines with different metastatic potentials. Hypergravity was produced by horizontal centrifugal acceleration. Here we report that long-term application of hypergravity (up to 5 g for 24 h) stimulated cGMP efflux in cultured melanocytes and in non-metastatic melanoma cells in the presence of 0.1 mM 3-isobutyl-1-methylxanthine (IBMX), a non-selective phosphodiesterase (PDE) inhibitor. Under these conditions, cAMP synthesis and melanin production were up-regulated in pigmented melanocytes and non-metastatic melanoma cells. Hypergravity also stimulated cGMP transport in the presence of 1 microM trequinsin, an inhibitor of cGMP-binding PDE (PDE5) and of transport by multidrug resistance proteins MRP4/5, whereas 50 microM trequinsin partially inhibited cGMP transport. Transport was further inhibited by probenecid, an inhibitor of endogenous non-selective transporters as well as of MRP4/5 and by cycloheximide as an inhibitor of de novo protein synthesis. In contrast, hypergravity did not affect cGMP efflux in metastatic melanoma cells, which might be related to an up-regulated cGMP efflux at 1 g. The results of the present study indicate that hypergravity may stimulate cGMP efflux in melanocytes and in non-metastatic melanoma cells most probably by an enhanced expression of endogenous transporters and/or MRP4/5. Thus, an altered acceleration vector may induce signaling events in melanocytic cells.

Gravity and cyclic GMP levels in melanocytic cells.

Guanosine 3',5'-cyclic monophosphate (cyclic GMP) is a major second messenger molecule, that is believed to play a role in various physiological and pathophysiological processes. Here we report that hypergravity induces differential effects on cyclic GMP turnover in melanocytic cells. Nonmetastatic melanoma cells responded to long-time exposure (24 h) of hypergravity (up to 5 x g) with decrease in intracellular cyclic GMP accumulation in the presence of an universal inhibitor of phosphodiesterases (IBMX), whereas the extracellular cyclic GMP increase. In contrast, there were no changes in cyclic GMP turnover in metastatic melanocytes. The expression of the guanylyl cyclases appeared to be not affected. These results suggest that cyclic GMP signaling may be involved in adaptation of human melanocytes to altered gravity conditions.