Stimulation of Cyclic GMP Efflux in Human Melanocytes by Hypergravity Generated by Centrifugal Acceleration

Abstract

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.

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

“…These pumps also function as a resistance factor for purine and pyrimidine nucleoside analogs, commonly employed in chemotherapy (Schuetz et al 1999;Wijnholds et al 2000). Interestingly, we have reported that altered gravity (e.g., hypergravity) may enhance the efflux of the second messenger cGMP in normal melanocytes and nonmetastatic melanoma cell lines, but not in metastatic MCs under conditions of limited PDE activities or induction of cGMP synthesis with NO donors partly via stimulation of MRP4-5 expression (Ivanova et al 2004(Ivanova et al , 2006. In this context, the immortalized melanocytes could be used in studies to determine the circumstances and the extent to which MRP4-5 participate in intracellular cyclic nucleotide homeostasis and to define their involvement in the (patho)physiological processes in which extruded cyclic nucleotides can be proposed as primary messengers.…”

“…Briefly, total RNA was isolated from melanocyte culture by the RNAClean System (Angewandte Gentechnologie Systeme, Heidelberg, Germany) following the manufacturer's instructions. After reverse transcription of 1 µg of total RNA by specific priming and SuperScript II RNase HReverse Transcriptase (Gibco BRL Life Technologies GmbH, Karlsruhe, Germany; ten units), the resultant single-stranded cDNA was subjected to PCR for detection using specific primers (400nM) for sGC α 1 and β 1 subunits, GC-B (Ivanova et al 2001), GC-A (Ivanova et al 2004), NOS2 (sense: 5′-TGCCTGGCAAGCC CAAGGTCTATGTTCAGGAC-3′, antisense: 5′-GGT GCTGCTTGTTAGGAGGTCAAGTAAAGGGC-3′, PCR product 500bp; Ikeguchi et al 2002), NOS3 (sense: 5′-ACCTGCAAAGCAGCAAGTCCACG-3′, antisense: 5′-CCGAACACCAAAGTCATGGGAGT-3′, PCR product 837bp; Gilchrist et al 2004), and MRP4-5 (MRP4: sense: 5′-CCATTGAAGATCTTCCTGG-3′, antisense: 5′-GGTGTTCAATCTGTGTGC-3′, PCR product 239bp; MRP5: sense: 5′-GGATAACTTCTCAGTGGG-3′, antisense: 5′-GGAATGGCAATGCTCTAAAG-3′, PCR product 381bp; Kool et al 1997) as described in literature. Briefly, after one cycle of an initial 15 min denaturation at 95°C, the PCR reaction consisted of 28 cycles (MRP4-5), 30 cycles (GC-B), 35 cycles (sGC, GC-A, and NOS2), and 40 cycles (NOS3) of denaturation (45s or 1 min at 93°C), annealing (1 min at 50°C for MRP4/5, 52°C for NOS2/3, or 57°C for GC), and elongation (1 min at 72°C), and one cycle of final extension for 10 min at 72°C.…”

Pretreatment of rat bone marrow mesenchymal stem cells with a combination of hypergravity and 5‐azacytidine enhances therapeutic efficacy for myocardial infarction

“…By adjusting the rotating speed, cells can gain different gravity values in the centrifuger. [33][34][35] Galimberti et al 36 explored the effect of hypergravity on lymphocyte motility with cell centrifuger. The centrifuger was also used in osteoblast research, 35 endothelial cells research, 37 and plant research.…”

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

“…In cells grown in space, various changes have been observed, including cytoskeleton rearrangements, altered gene expression, and changes in cell signaling [78,79]. As the GC-cGMP pathway is involved in the melanocyte response to environmental stress and has also been linked to many cellular processes, including melanoma growth and migration, we compared the regulation of the GC-cGMP signaling pathway in a panel of human melanocytes and melanoma cell lines with different metastatic potential and pigmentation in hypergravity as well as under simulated microgravity conditions [80][81][82]. We used a specific centrifuge for the hypergravity experiments (up to 10 g) [80] and a fast-rotating 2D clinostat (60 rpm) as well as a fast-rotating clinostat microscope (60 rpm) to simulate microgravity conditions [81,83].…”