Krassimira Ivanova

Effects of nitric oxide on the adhesion of human melanocytes to extracellular matrix components

The aim of the present study was to explore whether nitric oxide (NO) interferes with the attachment of human melanocytes to the extracellular matrix (ECM) components. Consequently, the effects have been investigated of the NO‐releasing compounds 3‐morpholino‐sydnonimine (SIN‐1) and S‐nitroso‐glutathione (GSNO) on the in vitro adhesion of human melanocytic cells to fibronectin. The NO donors induced a concentration‐dependent reduction in the adhesion of both 51CrO42−‐labelled melanocytes and melanoma cells to fibronectin. Pigmented M14 melanoma cells were more susceptible to the effect of SIN‐1 (half‐maximal inhibiting effect at about 0·5 mm) than normal human melanocytes and also than the non‐pigmented melanoma cells Mel57 (half‐maximal inhibiting effects between 0·9 and 2 mm). This effect of SIN‐1 also appeared to be related to the melanin content of normal melanocytes, whereas GSNO was significantly less active. Both flow cytometric analysis and immunocytochemical staining showed expression of neuronal NO synthase in all cell lines. The results of this study suggest that aberrant in vivo production of NO during infection and inflammation may contribute to loss of melanocytes in, for example, vitiligo, by reducing de novo attachment of melanocytes to the ECM. These findings could also be important for understanding the process of metastasis.

Soluble Guanylyl Cyclase and Platelet Functiona

Soluble guanylyl cyclase is abundantly present in platelets. This heterodimeric heme-protein is activated by nitric oxide (NO) and is involved in the inhibitory action of NO on platelet function. NO interferes with early steps of platelet activation involving calcium mobilization and inhibits both adhesion and aggregation. NO is formed endogenously from endothelium and a variety of other cell types. The nitrovasodilators, commonly used for the treatment of angina pectoris, also act through the release of NO. The release of NO from these drugs is enzyme coupled, and only a little NO is released from these drugs spontaneously. Platelets do not contain the enzyme system(s) that releases NO from organic nitrates. These drugs can therefore only weakly activate platelet-soluble guanylyl cyclase and inhibit platelet activation in vitro. However, NO is released from these drugs in the vessels and can reach the platelets close to the vessel wall in vivo. Therefore, many effects of these drugs in the primary or secondary prevention of myocardial infarction might come from their antiplatelet action rather than from their ability to dilate vessels. Knowledge of the mechanisms by which soluble guanylyl cyclase is activated has contributed considerably to our understanding of the mechanisms of regulation of platelet function and has presented us with new therapeutic possibilities.

Effects of nitric oxide-containing compounds on increases in cytosolic ionized Ca2+ and on aggregation of human platelets.

The present study was undertaken to determine the modulatory effects of nitric oxide (NO)-releasing compounds on increases in cytosolic ionized calcium ([Ca2+]i) and on aggregation of gel-filtered human platelets induced via diverse agonists. We used various sydnonimines and organic nitrates as donors of NO. Gel-filtered and fura-2-loaded platelets were stimulated with ADP (4-8 microM), collagen (2-10 micrograms/ml) or thrombin (0.02-0.05 IU/ml), respectively. Half-maximal inhibiting effects of sydnonimines on agonist-evoked increases in [Ca2+]i were observed between 30 and 1000 nM, while half-maximal inhibiting effects of the compounds on aggregation were between 3 and 500 nM. The compound C 87-3754, which is the bioactive metabolite of pirsidomine, was a much stronger inhibitor of increases in [Ca2+]i than of platelet aggregation. This was due to an enhanced NO release from this compound exposed to ultraviolet light during Ca2+ measurement. The organic nitrates isosorbide 5-mono-nitrate and nicorandil inhibited both aggregation and increase of cytosolic ionized calcium in stimulated platelets at half-maximal concentrations of approximately 200 microM. The present results suggest that some of the effects of NO on platelets are independent of cytosolic ionized calcium. The results also suggest that some of the inhibitory effects of NO-releasing compounds correspond rather to the presence of the A forms (NO-containing intermediates) than to the presence of free NO.

Amiloride increases the sensitivity of particulate guanylate cyclase to atrial natriuretic factor

The natriuretic agent amiloride induces a shift of the dose-response curve of particulate guanylate cyclase to atrial natriuretic factor (ANF) to the left. The ANF concentration for half-maximal activation of guanylate cyclase is shifted from 20 to 3 nM in the presence of 100 microM amiloride. This effect is observed with GTP*Mn2+, but not with GTP*Mg2+ as substrate. Amiloride derivatives, which inhibit a specific Na+-channel, also shift the dose-response curve to the left. These data suggest that some of the effects of amiloride may be mediated by an increased sensitivity of particulate guanylate cyclase to ANF.

Kinetic characterization of atrial natriuretic factor-sensitive particulate guanylate cyclase.

The present investigation describes kinetic characteristics of membrane-bound and Triton X-100-solubilized atrial natriuretic factor (ANF)-sensitive guanylate cyclase from bovine adrenal cortex. The kinetic analysis of both enzyme forms suggests that in the presence of manganese, ANF induces or stabilizes at least two apparent GTP*Mn2(+)- and in addition two Mn2(+)-binding sites. Addition of the natriuretic drug amiloride favors this state. ATP increases the vmax in the presence of ANF for GTP*Mg2+, but not for GTP*Mn2+ as a substrate. With GTP*Mg2+, amiloride has no effect on basal or ANF-stimulated activity, but slightly reduces the effect of ATP. Under all conditions tested, the enzyme follows regular Michaelis-Menten kinetics in the presence of Mg2+ and exhibits positive cooperativity with Mn2+. Positive cooperativity is also retained after Triton extraction. The results indicate that Triton extraction has no major influence on the kinetic properties of particulate guanylate cyclase when the extraction procedure is done carefully. The data also support the suggestion that multiple interactions of subunits might occur upon activation of the enzyme by ANF in the presence of Mn2+.

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

Abstract:  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.

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

Platelet norepinephrine (NE) and epinephrine (E) were measured as indices of long-term changes in sympathoadrenal activity. Ten normal healthy subjects were studied before and during head-down bed rest (HDBR) of 2 weeks duration, as well as during an ambulatory study period of a similar length. Platelet NE and E concentrations were studied in 5 cosmonauts, who participated in three different Soyuz missions to the International Space Station, 2 weeks before launch, within 12 hours after landing following 11 to 12 days of flight and at least 2 weeks after return to earth. Due to the long half-life of NE and E in platelets (approximately 2 days), data obtained early after landing would still reflect the microgravity state. Platelet NE decreased markedly during HDBR (p<0.001). During micro-gravity platelet NE and E increased in 4 of the 5 cosmonauts. Platelet NE and E concentrations expressed in percentage of pre-flight and pre-HDBR values, respectively, were significantly increased during microgravity as compared to HDBR (NE: 153±28% (mean±SEM) vs. 60±6%, p<0.004; E: 293±85% vs. 90±12%, p<0.01). The increase in platelet NE and E during microgravity is most likely due to an increase in sympathoadrenal activity. The reason why sympathoadrenal activity does not decrease to low levels during microgravity as one would expect remains to be elucidated. HDBR cannot be applied to simulate changes in sympathoadrenal activity during microgravity.

Melanocytes: interface of cell biology and pathobiology with a focus on nitric oxide and cGMP signaling

Human epidermal melanocytes represent a crucial protective barrier against UV irradiation and oxidative stress by generating the radical scavenging pigment melanin. Melanin is also known to act as a photosensitizer that generates active oxygen species upon UV irradiation, which may initiate hypopigmentary disorders (e.g., vitiligo) as well as UV-induced oncogene cell transformation. Finally, melanocytes in vivo are permanently targeted by environmental mechanical stimuli.

The functional importance of signal transduction pathways for the risk of cancer development in long-term spaceflight

The risk for the development of cancer in human spaceflight is still not completely understood. While many researchers focus on immediate effects of different types of space radiation on cellular damage, little is known on the effects of longterm weightlessness on cellular mechanisms that might alter the susceptibility for cancer development from a variety of reasons.