Blue Fluorescent cGMP Sensor for Multiparameter Fluorescence Imaging

Cyclic GMP (cGMP) regulates many physiological processes by cooperating with the other signaling molecules such as cyclic AMP (cAMP) and Ca²⁺. Genetically encoded sensors for cGMP have been developed based on fluorescence resonance energy transfer (FRET) between fluorescent proteins. However, to analyze the dynamic relationship among these second messengers, combined use of existing sensors in a single cell is inadequate because of the significant spectral overlaps. A single wavelength indicator is an effective alternative to avoid this problem, but color variants of a single fluorescent protein-based biosensor are limited. In this study, to construct a new color fluorescent sensor, we converted the FRET-based sensor into a single wavelength indicator using a dark FRET acceptor. We developed a blue fluorescent cGMP biosensor, which is spectrally compatible with a FRET-based cAMP sensor using cyan and yellow fluorescent proteins (CFP/YFP). We cotransfected them and loaded a red fluorescent probe for Ca²⁺ into cells, and accomplished triple-parameter fluorescence imaging of these cyclic nucleotides and Ca²⁺, confirming the applicability of this combination to individually monitor their dynamics in a single cell. This blue fluorescent sensor and the approach using this FRET pair would be useful for multiparameter fluorescence imaging to understand complex signal transduction networks.     

Current good manufacturing practice in plant automation of biological production processes

The production of biologicals is subject to strict governmental regulations. These are drawn up in current good manufacturing practices (cGMP), a.o. by the U.S. Food and Drug Administration. To implement cGMP in a production facility, plant automation becomes an essential tool. For this purpose Manufacturing Execution Systems (MES) have been developed that control all operations inside a production facility. The introduction of these recipe-driven control systems that follow ISA S88 standards for batch processes has made it possible to implement cGMP regulations in the control strategy of biological production processes. Next to this, an MES offers additional features such as stock management, planning and routing tools, process-dependent control, implementation of software sensors and predictive models, application of historical data and on-line statistical techniques for trend analysis and detection of instrumentation failures. This paper focuses on the development of new production strategies in which cGMP guidelines are an essential part.     

cGMP modulation of ACh-stimulated exocytosis in guinea pig antral mucous cells

In guinea pig antral mucous cells, ACh stimulates the Ca(2+)-regulated exocytosis, which has a characteristics feature: an initial transient phase followed by a sustained phase. The effects of cGMP on ACh-stimulated exocytosis were studied in guinea pig antral mucous cells using video microscopy. cGMP enhanced the frequency of ACh-stimulated exocytotic events, whereas cGMP alone did not induce any exocytotic events under the ACh-unstimulated condition. cGMP did not stimulate either Ca(2+) mobilization or cAMP accumulation. The Ca(2+) dose-response studies demonstrated that cGMP shifted the dose-response curve upward with no shift to the lower concentration. This indicates that cGMP increased maximal responsiveness of the Ca(2+)-regulated exocytosis, but not the Ca(2+) sensitivity. Moreover, under a condition of ATP depletion by dinitrophenol (DNP) or anoxia (N(2) bubbling), ACh evoked only a sustained phase in exocytotic events with no initial transient phase. However, ACh evoked an initial transient phase followed by a sustained phase with addition of cGMP before ATP depletion, whereas only a sustained phase was evoked in a case of cGMP addition after ATP depletion. Thus cGMP-induced enhancement in ACh-stimulated exocytotic events requires ATP, suggesting that cGMP modulates ATP-dependent priming of Ca(2+)-regulated exocytosis in antral mucous cells. In conclusion, cGMP increases the number of primed granules via acceleration of the ATP-dependent priming, which enhances the Ca(2+)-regulated exocytosis stimulated by ACh.     

Cyclic GMP diffusion coefficient in rod photoreceptor outer segments.

Cyclic GMP (cGMP) is the intracellular messenger that mediates phototransduction in retinal rods. As photoisomerizations of rhodopsin molecules are local events, the longitudinal diffusion of cGMP in the rod outer segment should be a contributing factor to the response of the cell to light. We have employed the truncated rod outer segment preparation from bullfrog (Rana catesbeiana) and tiger salamander (Ambystoma tigrinum) to measure the cGMP diffusion coefficient. In this preparation, the distal portion of a rod outer segment was drawn into a suction pipette for measuring membrane current, and the rest of the cell was then sheared off with a glass probe, allowing bath cGMP to diffuse into the outer segment and activate the cGMP-gated channels on the surface membrane. Addition and removal of bath cGMP were fast enough to produce effectively step changes in cGMP concentration at the open end of the outer segment. When cGMP hydrolysis is inhibited by isobutylmethylxanthine (IBMX), the equation for the diffusion of cGMP inside the truncated rod outer segment has a simple analytical solution, which we have used to analyze the rise and decay kinetics of the cGMP-elicited currents. From these measurements we have obtained a cGMP diffusion coefficient of approximately 70 x 10(-8) cm2 s-1 for bullfrog rods and approximately 60 x 10(-8) cm2 s-1 for tiger salamander rods. These values are six to seven times lower than the expected value in aqueous solution. The estimated diffusion coefficient is the same at high (20-1000 microM) and low (5-10 microM) concentrations of cGMP, suggesting no significant effect from buffering over these concentration ranges.     

NO induces a cGMP-independent release of cytochrome c from mitochondria which precedes caspase 3 activation in insulin producing RINm5F cells.

Exposure of RINm5F cells to interleukin-1beta and to several chemical NO donors such as sodium nitroprusside (SNP), SIN-1 and SNAP induce apoptotic events such as the release of cytochrome c from mitochondria, caspase 3 activation, Bcl-2 downregulation and DNA fragmentation. SNP exposure led to transient activation of soluble guanylate cyclase (sGC) and prolonged protein kinase G (PKG) activation but apoptotic events were not attenuated by inhibition of the sGC/PKG pathway. Prolonged activation of the cGMP pathway by exposing cells to the dibutyryl analogue of cGMP for 12 h induced both apoptosis and necrosis, a response that was abolished by the PKG inhibitor KT5823. These results suggest that NO-induced apoptosis in the pancreatic beta-cell line is independent of acute activation of the cGMP pathway.     

cGMP-dependent ABA-induced stomatal closure in the ABA-insensitive Arabidopsis mutant abi1-1

? The drought hormone abscisic acid (ABA) is widely known to produce reductions in stomatal aperture in guard cells. The second messenger cyclic guanosine 3', 5'-monophosphate (cGMP) is thought to form part of the signalling pathway by which ABA induces stomatal closure. ? We have examined the signalling events during cGMP-dependent ABA-induced stomatal closure in wild-type Arabidopsis plants and plants of the ABA-insensitive Arabidopsis mutant abi1-1. ? We show that cGMP acts downstream of hydrogen peroxide (H(2) O(2) ) and nitric oxide (NO) in the signalling pathway by which ABA induces stomatal closure. H(2) O(2) - and NO-induced increases in the cytosolic free calcium concentration ([Ca(2+) ](cyt) ) were cGMP-dependent, positioning cGMP upstream of [Ca(2+) ](cyt) , and involved the action of the type 2C protein phosphatase ABI1. Increases in cGMP were mediated through the stimulation of guanylyl cyclase by H(2) O(2) and NO. We identify nucleoside diphosphate kinase as a new cGMP target protein in Arabidopsis. ? This study positions cGMP downstream of ABA-induced changes in H(2) O(2) and NO, and upstream of increases in [Ca(2+) ](cyt) in the signalling pathway leading to stomatal closure.     

The implementation of tissue banking experiences for setting up a cGMP cell manufacturing facility

Cell manufacturing for clinical applications is a unique form of biologics manufacturing that relies on maintenance of stringent work practices designed to ensure product consistency and prevent contamination by microorganisms or by another patient's cells. More extensive, prolonged laboratory processes involve greater risk of complications and possibly adverse events for the recipient, and so the need for control is correspondingly greater. To minimize the associate risks of cell manufacturing adhering to international quality standards is critical. Current good tissue practice (cGTP) and current good manufacturing practice (cGMP) are examples of general standards that draw a baseline for cell manufacturing facilities. In recent years, stem cell researches have found great public interest in Iran and different cell therapy projects have been started in country. In this review we described the role of our tissue banking experiences in establishing a new cGMP cell manufacturing facility. The authors concluded that, tissue banks and tissue banking experts can broaden their roles from preparing tissue grafts to manufacturing cell and tissue engineered products for translational researches and phase I clinical trials. Also they can collaborate with cell processing laboratories to develop SOPs, implement quality management system, and design cGMP facilities.     

Predicting sporulation events in a bioreactor using an electronic nose

An electronic nose (EN) based on a non- specific multi-sensor array was used to accurately estimate sporulation events and the spore concentration of Bacillus subtilis cultures. The array included 6 metal oxide sensors (MOS), 10 metal oxide semiconductor field effect transistors (MOSFET), one CO(2) infrared sensor and one humidity sensor. The EN was used to monitor the gas emissions from B. subtilis bioreactions during both batch and fed-batch operation. The signal pattern produced by the sensors was evaluated by principal component analysis (PCA) and training cultivations were used to build a model. The arc length of the PCA trajectories was successfully correlated to the off-line spore count; a strong linear correlation (R(2) = 0.992) between the numerical integration of the curves and the measured spore concentration was established. The fast responses of the sensors in combination with the robust correlation with the off-line determination of spore concentration establish this EN device as a convenient tool for monitoring sporulation events in bioprocesses.     

Elevated glutamate and NMDA disrupt production of the second messenger cyclic GMP in the early postnatal mouse cortex

The second messenger cyclic guanosine monophosphate (cGMP) plays many roles during nervous system development. Consequently, cGMP production shows complex patterns of regulation throughout early development. Elevated glutamate levels are known to increase cGMP levels in the mature nervous system. A number of clinical conditions including ischemia and perinatal asphyxia can result in elevated glutamate levels in the developing brain. To investigate the effects of elevated glutamate levels on cGMP in the developing cortex we exposed mouse brain slices to glutamate or N‐methyl D ‐aspartate (NMDA). We find that at early postnatal stages when the endogenous production of cGMP is high, glutamate or NMDA exposure results in a significant lowering of the overall production of cGMP in the cortex, unlike the situation in the mature brain. However, this response pattern is complex with regional and cell‐type specific exceptions to the overall lowered cGMP production. These data emphasize that the response of the developing brain to physiological disturbances can be different from that of the mature brain, and must be considered in the context of the developmental events occurring at the time of disturbance. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

Optogenetic manipulation of cyclic guanosine monophosphate to probe phosphodiesterase activities in megakaryocytes

Cyclic guanosine monophosphate (cGMP) signalling plays a fundamental role in many cell types, including platelets. cGMP has been implicated in platelet formation, but mechanistic detail about its spatio-temporal regulation in megakaryocytes (MKs) is lacking. Optogenetics is a technique which allows spatio-temporal manipulation of molecular events in living cells or organisms. We took advantage of this method and expressed a photo-activated guanylyl cyclase, Blastocladiella emersonii Cyclase opsin (BeCyclop), after viral-mediated gene transfer in bone marrow (BM)-derived MKs to precisely light-modulate cGMP levels. BeCyclop-MKs showed a significantly increased cGMP concentration after illumination, which was strongly dependent on phosphodiesterase (PDE) 5 activity. This finding was corroborated by real-time imaging of cGMP signals which revealed that pharmacological PDE5 inhibition also potentiated nitric oxide-triggered cGMP generation in BM MKs. In summary, we established for the first-time optogenetics in primary MKs and show that PDE5 is the predominant PDE regulating cGMP levels in MKs. These findings also demonstrate that optogenetics allows for the precise manipulation of MK biology.     

Acidic pH increases cGMP accumulation through the OGR1/phospholipase C/Ca2 +/neuronal NOS pathway in N1E-115 neuronal cells

Neuronal NO synthase (nNOS)-mediated cGMP accumulation has been shown to affect a variety of neuronal cell activities, regardless of whether they are detrimental or beneficial, depending on the amount of their levels, under the physiological and pathological situations. In the present study, we examined the role of proton-sensing G protein-coupled receptors (GPCRs), which have been identified as new pH sensors, in the acidic pH-induced nNOS/cGMP activity in N1E-115 neuronal cells. In this cell line, ovarian cancer G protein-coupled receptor 1 (OGR1) and G protein-coupled receptor 4 (GPR4) mRNAs are expressed. An extracellular acidic pH increased cGMP accumulation, which was inhibited by nNOS-specific inhibitors. Acidic pH also activated phospholipase C/Ca^2 + pathways and Akt-induced phosphorylation of nNOS at S1412, both of which have been shown to be critical regulatory mechanisms for nNOS activation. The acidic pH-induced activation of the phospholipase C/Ca^2 + pathway, but not Akt/nNOS phosphorylation, was inhibited by small interfering RNA specific to OGR1 and YM-254890, an inhibitor of G_q/11 proteins, in association with the inhibition of cGMP accumulation. Moreover cGMP accumulation was inhibited by 2-aminoethoxydiphenyl borate, an inhibitor of inositol 1,4,5-trisphosphate channel; however, it was not by wortmannin, a phosphatidylinositol 3-kinase inhibitor, which inhibited Akt/nNOS phosphorylation. In conclusion, acidic pH stimulates cGMP accumulation preferentially through the OGR1/G_q/11 proteins/phospholipase C/Ca^2 +/nNOS in N1E-115 neuronal cells. Akt-mediated phosphorylation of nNOS, however, does not appreciably contribute to the acidification-induced accumulation of cGMP.     

Regulation of cyclic GMP elevation in the developing antennal lobe of the Sphinx moth, Manduca sexta.

In the moth, Manduca sexta, 3',5'-guanosine monophosphate (cGMP) is transiently elevated during adult development in about 100 neurons of the antennal lobe. We demonstrate that nearly all of these neurons are local interneurons of the lateral cluster I, that their capacity to show a strong cGMP response during development is regulated by the steroid hormone 20-hydroxyecdysone, and that in a subpopulation of these neurons cGMP elevation seems to be controlled directly by the gaseous messenger molecule nitric oxide (NO). Treatment with the acetylcholine esterase inhibitor eserine, antennal nerve transection, and electrical stimulation of the antennae suggest that NO/cGMP signaling during development is an activity-dependent process. Besides input from the antennae, input from the central brain and the ventral ganglia is involved in upregulating cGMP in the antennal-lobe neurons. Possible sources are centrifugal aminergic neurons, since application of serotonin and histamine enhances the GMP signal in local interneurons. Comparing the time course of cGMP elevation with events occurring during development leads us to the hypothesis that the NO/cGMP signaling pathway might be involved in synapse formation of a subset of antennal-lobe neurons.     

Real-time monitoring of phosphodiesterase inhibition in intact cells

Phosphodiesterases (PDEs) are hydrolytic enzymes, which convert cyclic AMP (cAMP) and cyclic GMP (cGMP) into their corresponding monophosphates. PDE-dependent hydrolysis shape gradients of these second messengers in cells, which may form the basis of their compartmentation and play a key role in a vast number of physiological and pathological processes. Here, we present a novel approach for real-time monitoring of local cAMP and cGMP levels associated with particular PDEs. We used HEK 293 cells expressing genetic constructs encoding a PDE of interest (PDE3A, PDE4A1 or PDE5A) fused to cAMP and cGMP sensors, which allow to directly visualize changes in cyclic nucleotide concentrations in the vicinity of PDE molecules by fluorescence resonance energy transfer (FRET). FRET was detected by imaging of single cells on 96-well plates and demonstrated specific effects of PDE inhibitors on local cyclic nucleotide levels. In addition, this approach reported physiological regulation of PDE3A activity, its activation by PKA-dependent phosphorylation and inhibition by cGMP. In conclusion, our assay provides a unique and highly sensitive method to analyze PDE activity in living cells. It allows to sense cAMP gradients around particular PDE molecules and to study the pharmacological effects of selective inhibitors on localized cAMP signalling.     

Biochemical evidence for a cGMP-regulated protein kinase in Pharbitis nil

It is known that the level of cGMP is modulated in response to a number of stimuli in plant cells but intracellular events distal to cGMP metabolism are not clear. Cyclic GMP-dependent protein kinase (Pk-G) is a major effector of cGMP action in animals and yeasts. We wanted to determine whether such kinase is present in plant cells. A soluble protein kinase was isolated from seedlings of Pharbitis nil and purified following purification methods including anion-exchange and affinity-chromatography. The enzyme consists of a single polypeptide of M(r) 70 kDa as determined by SDS-PAGE. From conventional modulators only cyclic GMP, when applied in low concentration, was able to accelerate the enzyme activity in the presence of histones. The enzyme autophosphorylated on serine and threonine residues and phosphorylated some substrates only on serine residues. Mixture of histones and histones H2B, H3 were the best phosphate acceptors. The process of autophosphorylation was accelerated by a low concentration of cGMP and reduced by high concentration of this second messenger. Antibodies raised against catalytic domain of animals Pk-G I alpha and beta cross-reacted with protein kinase from Pharbitis nil tissue. These data, taken together, demonstrate the presence of functional enzyme, which activity is regulated by cGMP and allow to classify this protein kinase as a member of the second messenger regulated group of enzymes.     

Disruption of a Plasmodium falciparum cyclic nucleotide phosphodiesterase gene causes aberrant gametogenesis

Phosphodiesterase (PDE) and guanylyl cyclase (GC) enzymes are key components of the cGMP signalling pathway and are encoded in the genome of Plasmodium falciparum . Here we investigate the role of specific GC and PDE isoforms in gamete formation – a process that is essential for malaria transmission and occurs in the Anopheles mosquito midgut following feeding on an infected individual. Details of the intracellular signalling events controlling development of the male and female gametes from their precursors (gametocytes) remain sparse in P. falciparum . Previous work involving the addition of pharmacological agents to gametocytes implicated cGMP in exflagellation – the emergence of highly motile, flagellated male gametes from the host red blood cell. In this study we show that decreased GC activity in parasites having undergone disruption of the PfGCβ gene had no significant effect on gametogenesis. By contrast, decreased cGMP-PDE activity during gametocyte development owing to disruption of the PfPDEδ gene, led to a severely reduced ability to undergo gametogenesis. This suggests that the concentration of cGMP must be maintained below a threshold in the developing gametocyte to allow subsequent differentiation to proceed normally. The data indicate that PfPDEδ plays a crucial role in regulating cGMP levels during sexual development.     

Regulation of cyclic GMP elevation in the developing antennal lobe of the sphinx moth,Manduca sexta

In the moth, Manduca sexta, 3′,5′‐guanosine monophosphate (cGMP) is transiently elevated during adult development in about 100 neurons of the antennal lobe. We demonstrate that nearly all of these neurons are local interneurons of the lateral cluster I, that their capacity to show a strong cGMP response during development is regulated by the steroid hormone 20‐hydroxyecdysone, and that in a subpopulation of these neurons cGMP elevation seems to be controlled directly by the gaseous messenger molecule nitric oxide (NO). Treatment with the acetylcholine esterase inhibitor eserine, antennal nerve transection, and electrical stimulation of the antennae suggest that NO/cGMP signaling during development is an activity‐dependent process. Besides input from the antennae, input from the central brain and the ventral ganglia is involved in upregulating cGMP in the antennal‐lobe neurons. Possible sources are centrifugal aminergic neurons, since application of serotonin and histamine enhances the GMP signal in local interneurons. Comparing the time course of cGMP elevation with events occurring during development leads us to the hypothesis that the NO/cGMP signaling pathway might be involved in synapse formation of a subset of antennal‐lobe neurons. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 359–375, 1999     

Concurrent Bursty Behavior of Social Sensors in Sporting Events

The advent of social media expands our ability to transmit information and connect with others instantly, which enables us to behave as “social sensors.” Here, we studied concurrent bursty behavior of Twitter users during major sporting events to determine their function as social sensors. We show that the degree of concurrent bursts in tweets (posts) and retweets (re-posts) works as a strong indicator of winning or losing a game. More specifically, our simple tweet analysis of Japanese professional baseball games in 2013 revealed that social sensors can immediately react to positive and negative events through bursts of tweets, but that positive events are more likely to induce a subsequent burst of retweets. We confirm that these findings also hold true for tweets related to Major League Baseball games in 2015. Furthermore, we demonstrate active interactions among social sensors by constructing retweet networks during a baseball game. The resulting networks commonly exhibited user clusters depending on the baseball team, with a scale-free connectedness that is indicative of a substantial difference in user popularity as an information source. While previous studies have mainly focused on bursts of tweets as a simple indicator of a real-world event, the temporal correlation between tweets and retweets implies unique aspects of social sensors, offering new insights into human behavior in a highly connected world.     

Correlation of cyclic GMP and cyclic AMP immunofluorescence with cytochemical patterns during dormancy release and development from gemmules in Spongilla lacustris L. (Porifera: Spongillidae)

The spongillid freshwater sponges asexually produce an encapsulated dormant stage, the gemmule. With release from dormancy, internal, yolk-laden, binucleate thesocytes differentiate into histoblasts or archeocytes. The histoblasts emerging first from the gemmule form the initial pinacoderm of the hatching sponge. Immunohistochemistry was employed to examine the distribution of cyclic GMP (cGMP) and cyclic AMP (cAMP) following dormancy release and during gemmule germination and hatching in the freshwater sponge, Spongilla lacustris L. Cyclic nucleotide fluorescence patterns were analyzed in relation to the distribution of cytochemically demonstrable macromolecular constituents and intracellular organelles. Twenty-four hours following temperature-activated release from dormancy, cGMP fluorescence levels are elevated in thesocytes at the gemmule periphery prior to histoblast formation. The cAMP fluorescence in the gemmule also occurs first in those thesocytes differentiating into histoblasts. Cytochemical patterns in germinating gemmules are comparable with those described by Ruthmann ('65) and Tessenow ('69). However, cytochemically demonstrable events of cytodifferentiation follow the earlier appearance of cGMP and cAMP in the histoblast precursors by approximately 12 hours. In addition, cGMP appears to be associated with the membranes of cytoplasmic organelles, possibly lysosomes or lipid inclusions, in the region of vitelline platelets and with symbiotic algae. cAMP is located primarily on the membranes of the vitelline platelets and on membranes of vacuoles involved in forming the spicular skeleton These observations suggest that cGMP and cAMP are involved in the mobilization of nutrient reserves and in ion transport during dormancy release and development from gemmules in freshwater sponges.     

Structure, function and evolution of microbial adenylyl and guanylyl cyclases

Cells respond to signals of both environmental and biological origin. Responses are often receptor mediated and result in the synthesis of so-called second messengers that then provide a link between extracellular signals and downstream events, including changes in gene expression. Cyclic nucleotides (cAMP and cGMP) are among the most widely studied of this class of molecule. Research on their function and mode of action has been a paradigm for signal transduction systems and has shaped our understanding of this important area of biology. Cyclic nucleotides have diverse regulatory roles in both unicellular and multicellular organisms, highlighting the utility and success of this system of molecular communication. This review will examine the structural diversity of microbial adenylyl and guanylyl cyclases, the enzymes that synthesize cAMP and cGMP respectively. We will address the relationship of structure to biological function and speculate on the complex origin of these crucial regulatory molecules. A review is timely because the explosion of data from the various genome projects is providing new and exciting insights into protein function and evolution.     

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.