Quantification of Ca2+ binding to melanin supports the hypothesis that melanosomes serve a functional role in regulating calcium homeostasis

Calcium regulation in melanocytes affects numerous biological pathways including protecting the redox balance in the cell and regulating the supply of substrate, l ‐tyrosine, for melanogenesis. The pigment contained in the melanocytes, melanin, has been implicated in maintaining calcium homeostasis in the cell and is known to be involved with calcium ion regulation in the inner ear. Herein, the association constant for Ca²⁺ binding to Sepia melanin is determined by isothermal titration calorimetry to be 3.3 (±0.2) × 10³/M. This value is comparable with other well‐established intracellular calcium‐binding proteins that serve to buffer calcium concentrations, lending further support to the hypothesis that melanosomes serve as intracellular mediators of calcium homeostasis in melanocytes. Using this binding constant and the data from a fluorescent Ca²⁺ displacement assay, the pK_a of the carboxyl group coordinated to Ca²⁺ is determined to be 3.1 ± 0.1.     

Melanin protects melanocytes and keratinocytes against H2O2-induced DNA strand breaks through its ability to bind Ca2+

Reactive oxygen species (ROS) such as hydrogen peroxide (H(2)O(2)) are produced in the skin under the influence of UV radiation. These compounds are highly reactive and can induce DNA lesions in epidermal cells. Melanin is considered to protect human skin against DNA damage by absorbing UV radiation. We have investigated whether melanin can, in addition, offer protection against the effects of H(2)O(2) in human melanocytes and HaCaT keratinocytes. In the present study, it was shown that 40 and 100 microM H(2)O(2) increased the number of DNA strand breaks as measured using the comet assay, in melanocytes of Caucasian origin. In melanocytes of the same origin in which melanin levels were increased by culturing in presence of 10 mM NH(4)Cl and elevated l-tyrosine, H(2)O(2)-induced DNA damage was reduced compared to that in control melanocytes. Similarly, HaCaT cells that were loaded with melanin were better protected against H(2)O(2)-induced DNA strand breaks than control HaCaT cells. These protective effects of melanin were mimicked by the intracellular Ca(2+)-chelator BAPTA. Thus, BAPTA reduced the level of H(2)O(2)-induced DNA strand breaks in melanocytes. Like BAPTA, melanin is known to be a potent chelator of Ca(2+) and this was confirmed in the present study. It was shown that melanin levels in melanocytic cells correlated directly with intracellular Ca(2+) binding capacity and, in addition, correlated inversely with H(2)O(2)-induced increases in intracellular Ca(2+). Our results show that melanin may have an important role in regulating intracellular Ca(2+) homeostasis and it is suggested that melanin protects against H(2)O(2)-induced DNA strand breaks in both melanocytes and keratinocytes and through its ability to bind Ca(2+).     

Physiology and pathophysiology of inner ear melanin

The presence of melanin in the inner ear was established more than a century ago, but the exact biological function of the pigment in the labyrinth has yet to be determined. In this brief review, the correlation of pigmentation and inner ear function, as well as the presumed role of melanocytes in hereditary diseases are discussed. Special attention was drawn to the composition of melanin and its presumed function as a biological reservoir for divalent ions and as an ion exchanger, as well as an intracellular buffering system for calcium. It is pointed out that melanin is capable of binding ototoxic drugs. Finally, morphological responses of melanocytes to local disturbance of Ca++ homeostasis in the inner ear are described as 1) intracellular movement and intraepithelial deposition of melanosomes; 2) cell motility; 3) neomelanogenesis; and 4) enhanced exocytotoxic/endocytotic activity. The possible consequences of this malfunction of the melanocytes on the inner ear function are discussed.     

Ca2+ binding to EF hands 1 and 3 is essential for the interaction of apoptosis-linked gene-2 with Alix/AIP1 in ocular melanoma

Apoptosis-linked gene-2 (ALG-2) encodes a 22 kDa Ca(2+)-binding protein of the penta EF-hand family that is required for programmed cell death in response to various apoptotic agents. Here, we demonstrate that ALG-2 mRNA and protein are down-regulated in human uveal melanoma cells compared to their progenitor cells, normal melanocytes. The down regulation of ALG-2 may provide melanoma cells with a selective advantage. ALG-2 and its putative target molecule, Alix/AIP1, are localized primarily in the cytoplasm of melanocytes and melanoma cells independent of the intracellular Ca(2+) concentration or the activation of apoptosis. Cross-linking and analytical centrifugation studies support a single-species dimer conformation of ALG-2, also independent of Ca(2+) concentration. However, binding of Ca(2+) to both EF-1 and EF-3 is necessary for ALG-2 interaction with Alix/AIP1 as demonstrated using surface plasmon resonance spectroscopy. Mutations in EF-5 result in reduced target interaction without alteration in Ca(2+) affinity. The addition of N-terminal ALG-2 peptides, residues 1-22 or residues 7-17, does not alter the interaction of ALG-2 or an N-terminal deletion mutant of ALG-2 with Alix/AIP1, as might be expected from a model derived from the crystal structure of ALG-2. Fluorescence studies of ALG-2 demonstrate that an increase in surface hydrophobicity is primarily due to Ca(2+) binding to EF-3, while Ca(2+) binding to EF-1 has little effect on surface exposure of hydrophobic residues. Together, these data indicate that gross surface hydrophobicity changes are insufficient for target recognition.     

Altered calcium homeostasis in the pathogenesis of myocardial ischemic and hypoxic injury

Pathological calcification, observed in infarcted myocardium under certain conditions, is the most severe manifestation of abnormal calcium (Ca2+) homeostasis induced by ischemia and related forms of myocardial injury. Specialized techniques for measurement of intracellular electrolytes, i.e., electron probe X-ray microanalysis, and intracellular free Ca2+, i.e. carboxylate indicators including fura-2, are providing new insights into regulation of intracellular Ca2+ and the role of altered Ca2+ homeostasis in the pathogenesis of myocardial cell injury. Several lines of investigation indicate that increased intracellular Ca2+ develops in association with other electrolyte alterations, altered cell volume regulation, and altered membrane phospholipid composition during the progression of myocardial cell injury.     

HCO(3)(-) ions modify the role of PKC isoforms in the modulation of rat mast cell functions

PKC and the intracellular calcium signal are two well-known intracellular signaling pathways implicated in the induction of mast cell exocytosis. Both signals are modified by the presence or absence of HCO(3)(-) ions in the external medium. In this work, we studied the regulation of the exocytotic process by PKC isozymes and its relationship with HCO(3)(-) ions and PKC modulation of the calcium entry. The calcium entry, induced by thapsigargin and further addition of calcium, was inhibited by PMA, a PKC activator, and enhanced by 500 nM GF109203X, which inhibits Ca(2+)-independent PKC isoforms. PMA inhibition of the Ca(2+) entry was reverted by 500 and 50 nM GF109203X, which inhibit Ca(2+)-independent and Ca(2+)-dependent isoforms, respectively, and G?6976, a specific inhibitor of Ca(2+)-dependent PKCs. Thus, activation of Ca(2+)-dependent and Ca(2+)-independent PKC isoforms inhibit Ca(2+) entry in rat mast cells, either in a HCO(3)(-)-buffered or a HCO(3)(-)-free medium. PMA, GF109203X, G?6976 and rottlerin, a specific inhibitor of PKC delta, were also used to study the role of PKC isoforms in the regulation of exocytosis induced by thapsigargin, ionophore A23187 and PMA. The results demonstrate that Ca(2+)-dependent PKC isoforms inhibit exocytosis in a HCO(3)(-)-dependent way. Moreover, Ca(2+)-independent PKC delta was the main isoform implicated in promotion of Ca(2+)-dependent mast cell exocytosis in the presence or absence of HCO(3)(-). The role of PKC isoforms in the regulation of mast cell exocytosis depends on the stimulus and on the presence or absence of HCO(3)(-) ions in the medium, but it is independent of PKC modulation of the Ca(2+) entry.     

Regulation of acetylcholinesterase expression by calcium signaling during calcium ionophore A23187- and thapsigargin-induced apoptosis

We have recently reported that acetylcholinesterase expression was induced during apoptosis in various cell types. In the current study we provide evidence to suggest that the induction of acetylcholinesterase expression during apoptosis is regulated by the mobilization of intracellular Ca(2+). During apoptosis, treatment of HeLa and MDA-MB-435s cells with the calcium ionophore A23187 resulted in a significant increase in acetylcholinesterase mRNA and protein levels. Chelation of intracellular Ca(2+) by BAPTA-AM (1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester), an intracellular Ca(2+) chelator, inhibited acetylcholinesterase expression. A23187 also enhanced the stability of acetylcholinesterase mRNA and increased the activity of acetylcholinesterase promoter, effects that were blocked by BAPTA-AM. Perturbations of cellular Ca(2+) homeostasis by thapsigargin resulted in the increase of acetylcholinesterase expression as well as acetylcholinesterase promoter activity during thapsigargin induced apoptosis in HeLa and MDA-MB-435s cells, effects that were also inhibited by BAPTA-AM. We further demonstrated that the transactivation of the human acetylcholinesterase promoter by A23187 and thapsigargin was partially mediated by a CCAAT motif within the -1270 to -1248 fragment of the human acetylcholinesterase promoter. This motif was able to bind to CCAAT binding factor (CBF/NF-Y). These results strongly suggest that cytosolic Ca(2+) plays a key role in acetylcholinesterase regulation during apoptosis induced by A23187 and thapsigargin.     

Emanuel Strehler's work on calcium pumps and calcium signaling

Cells are equipped with mechanisms to control tightly the influx, efflux and resting level of free calcium (Ca 2+ ). Inappropriate Ca 2+ signaling and abnormal Ca 2+ levels are involved in many clinical disorders including heart disease, Alzheimer’s disease and stroke. Ca 2+ also plays a major role in cell growth, differentiation and motility; disturbances in these processes underlie cell transformation and the progression of cancer. Accordingly, research in the Strehler laboratory is focused on a better understanding of the molecular "toolkit" needed to ensure proper Ca 2+ homeostasis in the cell, as well as on the mechanisms of localized Ca 2+ signaling. A longterm focus has been on the plasma membrane calcium pumps (PMCAs), which are linked to multiple disorders including hearing loss, neurodegeneration, and heart disease. Our work over the past 20 years or more has revealed a surprising complexity of PMCA isoforms with different functional characteristics, regulation, and cellular localization. Emerging evidence shows how specific PMCAs contribute not only to setting basal intracellular Ca 2+ levels, but also to local Ca 2+ signaling and vectorial Ca 2+ transport. A second major research arearevolves around the calcium sensor protein calmodulin and an enigmatic calmodulin-like protein (CALML3) that is linked to epithelial differentiation. One of the cellular targets of CALML3 is the unconventional motor protein myosin-10, which raises new questions about the role of CALML3 and myosin-10 in cell adhesion and migration in normal cell differentiation and cancer.     

Drosophila bestrophin-1 chloride current is dually regulated by calcium and cell volume

Mutations in the human bestrophin-1 (hBest1) gene are responsible for Best vitelliform macular dystrophy, however the mechanisms leading to retinal degeneration have not yet been determined because the function of the bestrophin protein is not fully understood. Bestrophins have been proposed to comprise a new family of Cl(-) channels that are activated by Ca(2+). While the regulation of bestrophin currents has focused on intracellular Ca(2+), little is known about other pathways/mechanisms that may also regulate bestrophin currents. Here we show that Cl(-) currents in Drosophila S2 cells, that we have previously shown are mediated by bestrophins, are dually regulated by Ca(2+) and cell volume. The bestrophin Cl(-) currents were activated in a dose-dependent manner by osmotic pressure differences between the internal and external solutions. The increase in the current was accompanied by cell swelling. The volume-regulated Cl(-) current was abolished by treating cells with each of four different RNAi constructs that reduced dBest1 expression. The volume-regulated current was rescued by transfecting with dBest1. Furthermore, cells not expressing dBest1 were severely depressed in their ability to regulate their cell volume. Volume regulation and Ca(2+) regulation can occur independently of one another: the volume-regulated current was activated in the complete absence of Ca(2+) and the Ca(2+)-activated current was activated independently of alterations in cell volume. These two pathways of bestrophin channel activation can interact; intracellular Ca(2+) potentiates the magnitude of the current activated by changes in cell volume. We conclude that in addition to being regulated by intracellular Ca(2+), Drosophila bestrophins are also novel members of the volume-regulated anion channel (VRAC) family that are necessary for cell volume homeostasis.     

Changes in calcium dynamics following the reversal of the sodium-calcium exchanger have a key role in AMPA receptor-mediated neurodegeneration via calpain activation in hippocampal neurons

Proteolytic cleavage of the Na(+)/Ca(2+) exchanger (NCX) by calpains impairs calcium homeostasis, leading to a delayed calcium overload and excitotoxic cell death. However, it is not known whether reversal of the exchanger contributes to activate calpains and trigger neuronal death. We investigated the role of the reversal of the NCX in Ca(2+) dynamics, calpain activation and cell viability, in alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor-stimulated hippocampal neurons. Selective overactivation of AMPA receptors caused the reversal of the NCX, which accounted for approximately 30% of the rise in intracellular free calcium concentration ([Ca(2+)](i)). The NCX reverse-mode inhibitor, 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea (KB-R7943), partially inhibited the initial increase in [Ca(2+)](i), and prevented a delayed increase in [Ca(2+)](i). In parallel, overactivation of AMPA receptors strongly activated calpains and led to the proteolysis of NCX3. KB-R7943 prevented calpain activation, cleavage of NCX3 and was neuroprotective. Silencing of NCX3 reduced Ca(2+) uptake, calpain activation and was neuroprotective. Our data show for the first time that NCX reversal is an early event following AMPA receptor stimulation and is linked to the activation of calpains. Since calpain activation subsequently inactivates NCX, causing a secondary Ca(2+) entry, NCX may be viewed as a new suicide substrate operating in a Ca(2+)-dependent loop that triggers cell death and as a target for neuroprotection.     

Functional alteration of dihydropyridine-sensitive Ca(2+) channels in the adrenal glomerulosa of pregnant rats

Our previous work on aldosterone secretion suggested that dihydropyridine-sensitive calcium channels, one type of voltage-dependent calcium channels (VDCC), are functionally impaired in adrenal capsule preparations from the pregnant rat. The aim of this study was to determine whether, during pregnancy, the density and/or activity of these channels is altered in the adrenal zona glomerulosa. These VDCC measured with [(3)H]nitrendipine binding were not different between membrane preparations of nonpregnant and pregnant rats. Western blots were performed using two different antibodies, a polyclonal (PcAb) directed against the alpha(1)-subunit of VDCC and a monoclonal (McAb) that recognizes an intracellular domain of that protein. McAb immunoreactivity showed a significant decrease in preparations from pregnant rats, whereas no difference was observed with PcAb. VDCC activity was estimated by (45)Ca(2+) uptake in isolated adrenal cortex and by intracellular calcium concentration ([Ca(2+)](i)) in adrenal glomerulosa cells with the Ca(2+) probe fura PE3. These measurements revealed that KCl stimulation produced greater Ca(2+) influx in nonpregnant than in pregnant rats. Nifedipine (a blocker of VDCC) inhibited this stimulation only in nonpregnant rats, whereas BAY K 8644 (an activator of VDCC) increased Ca(2+) influx in pregnant rats only. These data suggest that, during pregnancy, the altered regulation of calcium homeostasis in adrenal glomerulosa is linked to a conformational alteration of VDCC.     

Glucocorticoids prolong Ca(2+) transients in hippocampal-derived H19-7 neurons by repressing the plasma membrane Ca(2+)-ATPase-1

Calcium ions (Ca(2+)) play an important role in mediating an array of structural and functional responses in cells. In hippocampal neurons, elevated glucocorticoid (GC) levels, as seen during stress, perturb calcium homeostasis and result in altered neuronal excitability and viability. Ligand- and voltage-gated calcium channels have been the presumed targets of hormonal regulation; however, circumstantial evidence has suggested the possibility that calcium extrusion might be an important target of GC regulation. Here we demonstrate that GC-induced repression of the plasma membrane Ca(2+)-ATPase-1 (PMCA1) is an essential determinant of intracellular Ca(2+) levels ([Ca(2+)](i)) in cultured hippocampal H19-7 cells. In particular, GC treatment caused a prolongation of agonist-evoked elevation of [Ca(2+)](i) that was prevented by the expression of exogenous PMCA1. Furthermore, selective inhibition of PMCA1 using the RNA interference technique caused prolongation of Ca(2+) transients in the absence of GC treatment. Taken together, these observations suggest that GC-mediated repression of PMCA1 is both necessary and sufficient to increase agonist-evoked Ca(2+) transients by down-regulating Ca(2+) extrusion mechanisms in the absence of effects on calcium channels. Prolonged exposure to GCs, resulting in concomitant accumulation of [Ca(2+)](i), is likely to compromise neuronal function and viability.     

Calmodulin X (Ca2+)4 is the active calmodulin-calcium species activating the calcium-, calmodulin-dependent protein kinase of cardiac sarcoplasmic reticulum in the regulation of the calcium pump

Calcium-, calmodulin-dependent phosphorylation of cardiac sarcoplasmic reticulum increases the rate of calcium transport. The complex dependence of calmodulin-dependent phosphoester formation on free calcium and total calmodulin concentrations can be satisfactorily explained by assuming that CaM X (Ca2+)4 is the sole calmodulin-calcium species which activates the calcium-, calmodulin-dependent, membrane-bound protein kinase. The apparent dissociation constant of the E X CaM X (Ca2+)4 complex determined from the calcium dependence of calmodulin-dependent phosphoester formation over a 100-fold range of total calmodulin concentrations (0.01-1 microM) was 0.9 nM; the respective apparent dissociation constant at 0.8 mM free calcium, 1 mM free magnesium with low calmodulin concentrations (0.1-50 nM) was 2.60 nM. These results are in good agreement with the apparent dissociation constant of 2.54 nM of high affinity calmodulin binding determined by 125I-labelled calmodulin binding to sarcoplasmic reticulum fractions at 1 mM free calcium, 1 mM free magnesium and total calmodulin concentration ranging from 0.1 to 150 nM, i.e. conditions where approximately 98% of the total calmodulin is present as CaM X (Ca2+)4. The apparent dissociation constant of the calcium-free calmodulin-enzyme complex (E X CaM) is at least 100-fold greater than the apparent dissociation constant of the E X CaM X (Ca2+)4 complex, as judged from non-saturation 125I-labelled calmodulin binding at total calmodulin concentrations of up to 150 nM, in the absence of calcium.     

Calcium binds to leptospiral immunoglobulin-like protein, LigB, and modulates fibronectin binding

Pathogenic Leptospira spp. express immunoglobulin-like proteins, LigA and LigB, which serve as adhesins to bind to extracellular matrices and mediate their attachment on host cells. However, nothing is known about the mechanism by which these proteins are involved in pathogenesis. We demonstrate that LigBCen2 binds Ca(2+), as evidenced by inductively coupled plasma optical emission spectrometry, energy dispersive spectrometry, (45)Ca overlay, and mass spectrometry, although there is no known motif for Ca(2+) binding. LigBCen2 binds four Ca(2+) as determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The dissociation constant, K(D), for Ca(2+) binding is 7 mum, as measured by isothermal titration calorimetry and calcium competition experiments. The nature of the Ca(2+)-binding site in LigB is possibly similar to that seen in the betagamma-crystallin superfamily, since structurally, both families of proteins possess the Greek key type fold. The conformation of LigBCen2 was significantly influenced by Ca(2+) binding as shown by far- and near-UV CD and by fluorescence spectroscopy. In the apo form, the protein appears to be partially unfolded, as seen in the far-UV CD spectrum, and upon Ca(2+) binding, the protein acquires significant beta-sheet conformation. Ca(2+) binding stabilizes the protein as monitored by thermal unfolding by CD (50.7-54.8 degrees C) and by differential scanning calorimetry (50.0-55.7 degrees C). Ca(2+) significantly assists the binding of LigBCen2 to the N-terminal domain of fibronectin and perturbs the secondary structure, suggesting the involvement of Ca(2+) in adhesion. We demonstrate that LigB is a novel bacterial Ca(2+)-binding protein and suggest that Ca(2+) binding plays a pivotal role in the pathogenesis of leptospirosis.     

Effects of endothelins on signal transduction and proliferation in human melanocytes

We demonstrate here that human melanocytes could be regulated by endothelin (ET) derivatives, potent vasoconstrictive peptides synthesized by endothelial cells, to stimulate their proliferation and melanization via a receptor-mediated signal transduction pathway. Receptor-binding assay using [125I]ET indicated that unlabeled ET-1 or ET-2 competitively inhibited each binding of labeled ETs to melanocytes with a concentration for half-maximal inhibition (IC50) of 0.7 or 0.9 nM, respectively. The dissociation constant (Kd) and the number of sites of the specific bindings of ET-1 and those of ET-2 were almost the same (Kd: 1.81 nM, binding sites: 7.0-8.0 x 10(4) per cell). Upon incubation with cultured cells, the mass contents of inositol 1,4,5-trisphosphate and intracellular calcium level were substantially increased by 10 nM ET-1, ET-2, and ET-3, but not by big-ET with maximal response at 80-130-s postincubation. The addition of ET-1 and ET-2 at 1-50 nM concentrations caused human melanocytes to significantly stimulate DNA [( 3H]thymidine incorporation) and melanin synthesis (3H2O release and [14C] thiouracil incorporation). Furthermore, ETs exhibited an additive stimulatory effect on basic fibroblast growth factor-stimulated DNA synthesis. In a long-term serum-free culture system, the strongest stimulation of growth by 10 nM ET-1 or ET-2 was observed in the presence of 10 nM cholera toxin and 0.2% bovine pituitary extract, resulting in a 4.5-fold increase in cell number for 12 culture days. These findings strongly suggest involvement of ET in the mechanism regulating proliferation and melanization of human melanocytes.     

Internal calcium-binding proteins

Ca(2+)-ions play a key role in the regulation of many cellular processes and impairment of calcium homeostasis has been implicated in several diseases. Intracellularly the Ca(2+)-signal is transmitted by two families of proteins, the 'EF-hand'- and the Ca(2+)-dependent and phospholipid binding proteins. Their protein and gene structures as well as possible functional roles are summarized.     

Distinct Regulation of Cytoplasmic Calcium Signals and Cell Death Pathways by Different Plasma Membrane Calcium ATPase Isoforms in MDA-MB-231 Breast Cancer Cells

Plasma membrane calcium ATPases (PMCAs) actively extrude Ca(2+) from the cell and are essential components in maintaining intracellular Ca(2+) homeostasis. There are four PMCA isoforms (PMCA1-4), and alternative splicing of the PMCA genes creates a suite of calcium efflux pumps. The role of these different PMCA isoforms in the control of calcium-regulated cell death pathways and the significance of the expression of multiple isoforms of PMCA in the same cell type are not well understood. In these studies, we assessed the impact of PMCA1 and PMCA4 silencing on cytoplasmic free Ca(2+) signals and cell viability in MDA-MB-231 breast cancer cells. The PMCA1 isoform was the predominant regulator of global Ca(2+) signals in MDA-MB-231 cells. PMCA4 played only a minor role in the regulation of bulk cytosolic Ca(2+), which was more evident at higher Ca(2+) loads. Although PMCA1 or PMCA4 knockdown alone had no effect on MDA-MB-231 cell viability, silencing of these isoforms had distinct consequences on caspase-independent (ionomycin) and -dependent (ABT-263) cell death. PMCA1 knockdown augmented necrosis mediated by the Ca(2+) ionophore ionomycin, whereas apoptosis mediated by the Bcl-2 inhibitor ABT-263 was enhanced by PMCA4 silencing. PMCA4 silencing was also associated with an inhibition of NFκB nuclear translocation, and an NFκB inhibitor phenocopied the effects of PMCA4 silencing in promoting ABT-263-induced cell death. This study demonstrates distinct roles for PMCA1 and PMCA4 in the regulation of calcium signaling and cell death pathways despite the widespread distribution of these two isoforms. The targeting of some PMCA isoforms may enhance the effectiveness of therapies that act through the promotion of cell death pathways in cancer cells.     

Diabetes alters intracellular calcium transients in cardiac endothelial cells

Diabetic cardiomyopathy (DCM) is a diabetic complication, which results in myocardial dysfunction independent of other etiological factors. Abnormal intracellular calcium ([Ca(2+)](i)) homeostasis has been implicated in DCM and may precede clinical manifestation. Studies in cardiomyocytes have shown that diabetes results in impaired [Ca(2+)](i) homeostasis due to altered sarcoplasmic reticulum Ca(2+) ATPase (SERCA) and sodium-calcium exchanger (NCX) activity. Importantly, altered calcium homeostasis may also be involved in diabetes-associated endothelial dysfunction, including impaired endothelium-dependent relaxation and a diminished capacity to generate nitric oxide (NO), elevated cell adhesion molecules, and decreased angiogenic growth factors. However, the effect of diabetes on Ca(2+) regulatory mechanisms in cardiac endothelial cells (CECs) remains unknown. The objective of this study was to determine the effect of diabetes on [Ca(2+)](i) homeostasis in CECs in the rat model (streptozotocin-induced) of DCM. DCM-associated cardiac fibrosis was confirmed using picrosirius red staining of the myocardium. CECs isolated from the myocardium of diabetic and wild-type rats were loaded with Fura-2, and UTP-evoked [Ca(2+)](i) transients were compared under various combinations of SERCA, sarcoplasmic reticulum Ca(2+) ATPase (PMCA) and NCX inhibitors. Diabetes resulted in significant alterations in SERCA and NCX activities in CECs during [Ca(2+)](i) sequestration and efflux, respectively, while no difference in PMCA activity between diabetic and wild-type cells was observed. These results improve our understanding of how diabetes affects calcium regulation in CECs, and may contribute to the development of new therapies for DCM treatment.