intracellular free calcium in HEK293 cells Melanocortin receptor-mediated mobilization of (original) (raw)
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Signaling Mechanisms Controlled by Melanocortins in Melanoma, Lacrimal, and Brain Astroglial Cells
Annals of the New York Academy of Sciences, 1993
SALOMON et af.: SIGNALING MECHANISMS 365 12-16 amino acids, respectively, and arise in various mammalian tissues along with other bioactive peptides by posttranscriptional processing from the 3 1-36-kDa POMC precursor. The melanocortin peptides contain a consensus 7 amino acid sequence flanked by peptide-specific sequences of considerable homology on both the Nand C-terminal ends.'O Other common features of these peptides relate to (1) their unique dependence on extraceflular calcium required for interaction with their respective receptor molecules2' and (2) their ability to regulate target cell function via G-p r~t e i n s~~*~~ and adenylate cyclase (AC),20724 as well as other signaling pathways as further discussed below. A requirement of extracellular Ca2+ ions for the activity of ACTH was first noted in the early 50s by Birmingham and associates,2s studying ACTH-induced steroidogenesis in rat adrenal cortical tissue. ACTH stimulation of lipolysis was also shown to be Ca2+ dependent.26 Understanding of this phenomenon increased when Birnbaumer and Rodbell demonstrated that AC in fat cells serves as the common effector enzyme for several lipolytic hormones (epinephrine, glucagon, ACTH).*' Among these hormones, only ACTH appeared to require Ca2+ ions and its stimulatory effect was reduced to basal levels by EGTA. The difficulty of obtaining biologically active radiolabeled ACTH deferred further progress in the analysis of this phenomenon until, in 1985, Cheitlin and his associates first synthesized [1251][Tyr23 Phe2 Nle41ACTH-(1-38)-a derivative that maintained full biological activity.28 Binding of this peptide to adrenal glomerulosa cells required physiological calcium concentration, Furthermore, transfer of the cells to Ca2+-free medium induced the release of the receptor-bound hormone. Studies performed in lower vertebrates on the stimulation of skin melanophores, in which MSH induces melanosome dispersion, also revealed that the response to this hormone required extracellular calcium.20~29*30 Pigment dispersion could, however, be induced in the absence of calcium by exogenous cyclic AMP (CAMP), dibutyryl cAMP or other agents, such as theophylline, that nonspecifically raise intracellular CAMP."-^^ These observations, therefore, suggested that the elevation of cAMP and, hence, the cellular response depend on extracellular calcium only when stimulated by MSH. In experiments performed with photoreactive a-MSH, De Graan et al. noted that covalent cross-linking of MSH to the MSH receptor (MSH-R), in the presence but not in the absence of calcium, resulted in persistent activation of amphibian m e l a n o p h~r e s .~~-~~ However, removal of calcium reversed the stimulation, presumably while maintaining the active covalently cross-linked receptor-MSH complex. This was inferred since replenishment of calcium in MSH-free medium restored the cellular response. These authors, therefore, suggested a dual role for Ca2+ ions in the amphibian melanocyte, at the receptor level and at sites in the MSH-responsive pathways that are distal to the MSH-R itself. In this short review, we will discuss our recent work on MSH receptors in melanoma, lacrimal, and rat brain astrocytes, tissues in which MSH or ACTH appears to control rather different cellular responses. Furthermore, the various cell-specific responses seem to be mediated by different signaling mechanisms. M2R MOUSE MELANOMA The Calcium Dependence of MSH Receptor Function in Mouse Melanoma To further understand the role of Ca2+ in MSH-R function, particularly in mammalian systems, the mouse melanoma M2R cell line was chosen as a model.
Biochemical and Biophysical Research Communications, 2002
The melanocortins are involved in the regulation of various cognitive and physiological processes such as learning, feeding, immune suppression, pigmentation, and sebum production. Five melanocortin receptors have been identified, of which the melanocortin 5 receptor (MC5R) has the most widespread distribution. This subtype is found in the brain, and at numerous peripheral sites including the skin where it is expressed in the sebaceous glands. The purpose of this study was to identify the peptide that functions as a natural ligand at the MC5R in the skin. ␣-MSH, ACTH1-39, ACTH1-17, ACTH1-10, and ACTH4-10 all increased the production of cAMP in HEK293 cells transfected with the mouse MC5R. ␣-MSH and ACTH1-17 were the most potent in this respect. In addition, all peptides stimulated a rapid and transient increase in [Ca 2؉ ] i , and, ACTH1-10 was the most potent. The increases in [Ca 2؉ ] i were of intracellular origin, but not associated with inositol phosphate production. The elevations in [Ca 2؉ ] i were reduced by ruthenium red and procaine and it is therefore possible that they were mediated via ryanodine receptors.
Neuroscience Letters, 2005
Secretion of ␣-melanophore-stimulating hormone (␣-MSH) from the neuroendocrine melanotrope cells in the intermediate lobe of the pituitary gland of the clawed frog Xenopus laevis is regulated by various inhibitory, stimulatory and autocrine factors. The neuropeptide sauvagine stimulates ␣-MSH secretion by changing the pattern of intracellular Ca 2+ oscillations and the electrical properties of the cell membrane. In the present study we investigated whether another secreto-stimulator, the extracellular Ca 2+ -sensing receptor (CaR), also affects the Ca 2+ oscillatory pattern and electrical membrane properties. Using high-speed dynamic video-imaging we show that activation of the CaR with the specific agonist l-phenylalanine (l-Phe) changes the Ca 2+ oscillatory pattern by increasing the number of Ca 2+ steps, which are the "building blocks" of the oscillations. Moreover, using patch-clamp electrophysiology it is demonstrated that l-Phe affects membrane properties by increasing frequency and duration of action currents. Compared to sauvagine, the CaR has different effects on the action current parameters, suggesting that multiple mechanisms regulate the electrical properties of the melanotrope cell membrane and, thereby, the Ca 2+ oscillation-dependent level of ␣-MSH secretion.
Melanin has a Role in Ca2+ Homeostasis in Human Melanocytes
Pigment Cell Research, 2003
We have examined whether melanin affects Ca 2+ homeostasis in cultured normal human melanocytes. Intracellular Ca 2+ concentrations ([Ca 2+ ] i), were measured in four Caucasian and in three Negroid melanocyte cultures. Under resting conditions [Ca 2+ ] i was around 100 nM in all cultures, but differences between cells within cultures were observed. All cultures responded to endothelin-1 (ET-1) with increases in [Ca 2+ ] i and there were no differences between Caucasian and Negroid cultures. However, large differences in responses between cells within cultures were observed, indicating that melanocyte cultures are very heterogeneous. The addition of 2.5 mM CaCl 2 to melanocytes kept in Ca 2+-free medium resulted in rapid and transient increases in [Ca 2+ ] i of up to 1500 nM. These increases were on average more than two times smaller in melanocyte cultures established from Negroid donors compared with Caucasian cultures. In addition, well melanized Caucasian melanocytes, cultured in the presence of 400 lM tyrosine and 10 mM NH 4 Cl, showed a reduced increase in cytoplasmic Ca 2+ concentration upon the addition of extracellular Ca 2+. The difference in maintaining Ca 2+ homeostasis between poorly and well melanized melanocytes may be the result of the clearance of cytoplasmic Ca 2+ into melanosomes and the greater capacity for this in the more pigmented melanocytes.
Intracellular signaling mechanisms of the melanocortin receptors: current state of the art
Cellular and Molecular Life Sciences, 2014
The melanocortin system is composed by the agonists adrenocorticotropic hormone and a, b and cmelanocyte-stimulating hormone, and two naturally occurring antagonists, agouti and agouti-related protein. These ligands act by interaction with a family of five melanocortin receptors (MCRs), assisted by MCRs accessory proteins (MRAPs). MCRs stimulation activates different signaling pathways that mediate a diverse array of physiological processes, including pigmentation, energy metabolism, inflammation and exocrine secretion. This review focuses on the regulatory mechanisms of MCRs signaling, highlighting the differences among the five receptors. MCRs signal through G-dependent and independent mechanisms and their functional coupling to agonists at the cell surface is regulated by interacting proteins, namely MRAPs and b-arrestins. The knowledge of the distinct modulation pattern of MCRs signaling and function may be helpful for the future design of novel drugs able to combine specificity, safety and effectiveness in the course of their therapeutic use.
Activation of protein kinase C by intracellular free calcium in the motoneuron cell line NSC-19
Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 1997
The relationship between intracellular free calcium ([Ca2+] i) and the activation of protein kinase C (PKC) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) was investigated in the NSC-19 motoneuron cell line. Increased extracellular calcium ([Ca 2+ ]o) up to 10 mM resulted in sustained elevations of [Ca 2+ ]i. Control cell cultures (1.3 mM [ Ca2+ ]o, [ Ca2+ ]i = 83 4-17 nM) contained Ca 2+-and PS/DO lipid-dependent PKC activity predominantly in the cytosol. However, elevation of [Ca2+]o up to 5 mM ([Ca2+]~ = 232 + 24 nM) resulted in almost complete loss of cytosolic PKC activity. Cells incubated in 10 mM [Ca2+]o ([Ca2+] i = 365 _+ 13 nM)
Effect of CRF and related peptides on calcium signaling in human and rodent melanoma cells
FEBS Letters, 1998
Corticotropin releasing factor (CRF) induces a rapid, within seconds, and dose-dependent increase in the intracellular Ca P+ in both human and hamster melanoma cells. This effect is inhibited by depletion of extracellular calcium using 3 mM EGTA and is attenuated by the CRF receptor antagonist, K Khelical-CRF(9-41). Other peptides of the CRF superfamily, sauvagine and urocortin, also induce increases in cytoplasmic calcium concentration but at higher concentrations than CRF. We conclude that malignant melanocytes express CRF receptors, which are coupled to activation of plasma membrane calcium channels.
Stable expression of human melanocortin 3 receptor fused to EGFP in the HEK293 cells
Biochemical and Biophysical Research Communications, 2003
Among the melanocortins a-MSH is known to be involved in feeding behavior. These hormones mediate their effects through G protein-coupled receptors by stimulating adenylate cyclase. In this study, we have developed an in vitro expression model for human melanocortin 3 receptor (hMC3R) tagged at its C terminus with EGFP. The corresponding chimeric cDNA was stably expressed in HEK293 cells. The selected clones expressing the hMC3R-EGFP exhibited cell surface fluorescence and responded to NDP-MSH stimulation by producing cAMP in a dose-dependent manner (EC 50 : 0.3 nM). Binding studies revealed a single class of binding sites with a K D of 2.24 nM. Moreover, Agouti-related protein was also demonstrated to be an antagonist of the hMC3R-EGFP. Thus, the hMC3R tagged with EGFP stably expressed in HEK293 cells, exhibiting the same characteristics than the wild-type hMC3R, is the only model of expression of this receptor allowing its direct localization inside living cells.
Melanopsin Triggers the Release of Internal Calcium Stores in Response to Light†
Photochemistry and Photobiology, 2007
Melanopsin is the photopigment that confers photosensitivity upon intrinsically photosensitive retinal ganglion cells (ipRGCs). This subset of retinal ganglion cells comprises less than 2% of all RGCs in the mammalian retina. The paucity of melanopsin-positive cells has made studies on melanopsin signaling difficult to pursue in ipRGCs. To address this issue, we have established several cell lines consisting of a transformed human embryonic kidney cell line (HEK293) stably expressing human melanopsin. With these cell lines, we have investigated the intracellular rise in calcium triggered upon light activation of melanopsin. Our human melanopsin-expressing cells exhibit an irradiance-dependent increase in intracellular calcium. Control cells expressing human melanopsin, where the Schiff-base lysine has been mutated to alanine, show no responses to light. Chelating extracellular calcium has no effect on the light-induced increase in intracellular calcium suggesting that calcium is mobilized from intracellular stores. This involvement of intracellular stores has been confirmed through their depletion by thapsigargin, which inhibits a subsequent light-induced increase in intracellular calcium. Addition of the nonselective cation channel blocker lanthanum does not alter light-induced rises in intracellular calcium, further supporting that melanopsin triggers a release of internal calcium from internal stores. HEK293 cells stably expressing melanopsin have proven to be a useful tool to study melanopsin-initiated signaling. †This paper is part of the
Melanocyte?keratinocyte interaction induces calcium signalling and melanin transfer to keratinocytes
Pigment Cell Research, 2007
Physical contact between melanocytes and keratinocytes is a prerequisite for melanosome transfer to occur, but cellular signals induced during or after contact are not fully understood. Herein, it is shown that interactions between melanocyte and keratinocyte plasma membranes induced a transient intracellular calcium signal in keratinocytes that was required for pigment transfer. This intracellular calcium signal occurred due to release of calcium from intracellular stores. Pigment transfer observed in melanocyte-keratinocyte co-cultures was inhibited when intracellular calcium in keratinocytes was chelated. We propose that a 'ligand-receptor' type interaction exists between melanocytes and keratinocytes that triggers intracellular calcium signalling in keratinocytes and mediates melanin transfer.