Guanylyl Cyclase-cGMP Signaling Pathway in Melanocytes: Differential Effects of Altered Gravity in Non-Metastatic and Metastatic Cells.

Human epidermal melanocytes as melanin producing skin cells represent a crucial barrier against UV-radiation and oxidative stress. It was shown that the intracellular signaling molecule cyclic guanosine-3',5'-monophosphate (cGMP), generated by the guanylyl cyclases (GCs), e.g., the nitric oxide (NO)-sensitive soluble GC (sGC) and the natriuretic peptide-activated particulate GC (GC-A/GC-B), plays a role in the melanocyte response to environmental stress. Importantly, cGMP is involved in NO-induced perturbation of melanocyte-extracellular matrix interactions and in addition, increased NO production during inflammation may lead to loss of melanocytes and support melanoma metastasis. Further, the NO-sensitive sGC is expressed predominantly in human melanocytes and non-metastatic melanoma cells, whereas absence of functional sGC but up-regulated expression of GC-A/GC-B and inducible NO synthase (iNOS) are detected in metastatic cells. Thus, suppression of sGC expression as well as up-regulated expression of GC-A/GC-B/iNOS appears to correlate with tumor aggressiveness. As the cGMP pathway plays important roles in melanocyte (patho)physiology, we present an overview on the differential effects of altered gravity (hypergravity/simulated microgravity) on the cGMP signaling pathway in melanocytes and melanoma cells with different metastatic potential. We believe that future experiments in real microgravity may benefit from considering cGMP signaling as a possible factor for melanocyte transformation and in medication.

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.

Sympathetic nervous activity decreases during head-down bed rest but not during microgravity.

We tested the hypothesis that sympathoadrenal activity in humans is low during spaceflight and that this effect can be simulated by head-down bed rest (HDBR). Platelet norepinephrine and epinephrine were measured as indexes of long-term changes in sympathoadrenal activity. Ten normal healthy subjects were studied before and during HDBR of 2-wk duration, as well as during an ambulatory study period of a similar length. Platelet norepinephrine concentrations (half-life = 2 days) were studied in five cosmonauts, 2 wk before launch, within 12 h after landing after 11-12 days of flight, and at least 2 wk after return to Earth. Because of the long half-life of platelet norepinephrine, data obtained early after landing would still reflect the microgravity state. Platelet norepinephrine decreased markedly during HDBR (P < 0.001), whereas there were no significant changes when subjects were ambulatory. Platelet epinephrine did not change during HDBR. During microgravity, platelet norepinephrine and epinephrine increased in four of the five cosmonauts. Platelet norepinephrine concentrations expressed in percentage of preflight and pre-HDBR values, respectively, were significantly different during microgravity compared with HDBR [153 +/- 28% (mean +/- SE) vs. 60 +/- 6%, P < 0.004]. Corresponding values for platelet epinephrine were also significant (293 +/- 85 vs. 90 +/- 12%, P < 0.01). The mechanism of the platelet norepinephrine and epinephrine response during spaceflight flight is most likely related to the concomitant decrease in plasma volume. HDBR cannot be applied to simulate changes in sympathoadrenal activity during microgravity.

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.

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.

Variable acceleration influences cyclic AMP levels in Paramecium biaurelia.

Paramecium is used as a model system to analyse the gravity signal transduction pathway, that leads to gravitaxis and gravikinesis. In order to prove whether gravistimulation is coupled with second messenger production (cyclic AMP: hyperpolarization, cyclic GMP: depolarization) Paramecium was fixated under variable accelerations (1 x g, 9 x g and 10(-4) x g) on a centrifuge and during a sounding rocket flight (TEXUS 39). The analysis of cAMP and cGMP levels revealed an acceleration-dependent change in cAMP, while cGMP-levels showed gravity-independent variations. Hypergravity did not only induce an amplification of gravitaxis and gravikinesis, but also an increase in cAMP compared to the 1 x g-data. We conclude that the increased pressure of the cytoplasm on the lower membrane of upward swimming cells enhance the number of open K+(-)channels, thus causing hyperpolarization and change in cAMP concentration. Consequently, transition to microgravity declines gravitaxis and gravikinesis, and decreases cAMP concentration due to the loss of pressure on the cell membrane.

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.