Einfluss von Hypergravitation auf den cGMP-Efflux in Stickstoffmonoxid-stimulierten humanen Melanozyten und Melanomzellen
Gravity alteration is known to affect cell proliferation, morphology, gene expression, and me-tabolism. To our knowledge, studies concerning the gravity influence on melanocyte physiol-ogy with respect to activation and cellular responses that are important for malignant trans-formation have been rarely reported. Since melanocytes are able to sense mechanical forces and transform them into biochemical signals it was assumed that these cells likewise respond to gravity alteration. We examined whether hypergravity (up to 5g for 24 h) modulates the efflux of the second messenger cGMP under conditions of stimulated cGMP-synthesis. Due to the differential expression of functional guanylyl cyclase (GC) isoforms in normal human melanocytes (NHM) and melanoma cell lines we used direct NO donors (NONOates) which differ in their kinetics of NO release as stimulators of sGC and ANP as a GC-A modulator. Our results demonstrate that long-term application of hypergravity (5g for 24 h) in the presence of DETA/NO induced a decrease in intracellular cGMP in NHMs and non-metastatic 1F6 melanoma cells compared to cGMP levels at 1g. The corresponding extracellular cGMP levels increased suggesting that hypergravity enhances the cGMP efflux. This efflux was re-duced in the presence of trequinsin as a selective phosphodiesterase 5 inhibitor and additionally a highly selective inhibitor of multidrug resistance protein 4/5, indicating that in melanocytes the hypergravity-stimulated cGMP efflux under PDE5 inhibition is most proba-bly mediated by endogenous anion transporters and/or selective cGMP transporters like MRP 4/5. Short-term application (5g up to 6 h) was not effective. Compared to the 1g-controls there existed no differences between intra- and extracellular cGMP-levels. These results indicate that short-term centrifugation did not affect cGMP efflux in melanocytic cells. Interestingly, highly metastatic melanoma cells were apparently insensitive to a 24 hour centrifugation in the presence of ANP. These cells that do not express the NO-sensitive GC isoforms, showed no alteration in intra- and extracellular cGMP concentrations. Application of hypergravity did not alter the cAMP efflux in melanocytic cells, but the production of cAMP appeared to be stimulated by centrifugal acceleration. In summary, the presented in vitro experiments demonstrate that long-term exposure to hypergravity increases the cGMP efflux in NO-stimulated NHMs and non-metastatic melanoma cells, whereas highly metastatic melanoma cells are not sensitive. Thus an altered acceleration vector may induce cGMP-dependent signalling events in NO-stimulated human melanocytic cells.
info:eu-repo/classification/ddc/570, Biowissenschaften, Biologie, Hypergravitation, Stickstoffmonoxid, Melanozyten, cGMP
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