Regulation of magnesium-inhibited cation current by actin cytoskeleton rearrangement

Magnesium-inhibited, non-selective cation current (I(MIC)) is activated by depletion of intracellular Mg(2+) and ATP. I(MIC) transports various divalent cations including Mg(2+) and Ca(2+), and is involved in cell viability. We investigated the effect of actin dynamics on I(MIC). Formation of a stable cortical actin network by calyculin A inhibited the activation of I(MIC), while the actin depolymerizing reagent, cytochalasin D, reversed the inhibition. Induction of a dense cortical actin layer by transfecting the constitutively active form of RhoA also inhibited the activation of I(MIC). These results suggest that the activation of I(MIC) may be dynamically regulated by actin cytoskeleton rearrangement.     

Local Ca2+ rise near store operated Ca2+ channels inhibits cell coupling during capacitative Ca2+ influx

Using a new fluorescence imaging technique, LAMP, we recently reported that Ca(2+) influx through store operated Ca(2+) channels (SOCs) strongly inhibits cell coupling in primary human fibroblasts (HF) expressing Cx43. To understand the mechanism of inhibition, we studied the involvement of cytosolic pH (pH(i)) and Ca(2+)([Ca(2+)](i)) in the process by using fluorescence imaging and ion clamping techniques. During the capacitative Ca(2+) influx, there was a modest decline of pH(i) measured by BCECF. Decreasing pH(i) below neutral using thioacetate had little effect by itself on cell coupling, and concomitant pH(i) drop with thioacetate and bulk [Ca(2+)(i) rise with ionomycin was much less effective in inhibiting cell coupling than Ca(2+) influx. Moreover, clamping pH(i) with a weak acid and a weak base during Ca(2+) influx largely suppressed bulk pH(i) drop, yet the inhibition of cell coupling was not affected. In contrast, buffering [Ca(2+)(i) with BAPTA, but not EGTA, efficiently prevented cell uncoupling by Ca(2+) influx. We concluded that local Ca(2+) elevation subjacent to the plasma membrane is the primary cause for closing Cx43 channels during capacitative Ca(2+) influx. To assess how Ca(2+) influx affects junctional coupling mediated by other types of connexins, we applied the LAMP assay to Hela cells expressing Cx26. Capacitative Ca(2+) influx also caused a strong reduction of cell coupling, suggesting that the inhibitory effect by Ca(2+) influx may be a more general phenomenon.     

Ca(2+)-dependent inactivation of cardiac L-type Ca2+ channels does not affect their voltage sensor

Inactivation of currents carried by Ba2+ and Ca2+, as well as intramembrane charge movement from L-type Ca2+ channels were studied in guinea pig ventricular myocytes using the whole-cell patch clamp technique. Prolonged (2 s) conditioning depolarization caused substantial reduction of charge movement between -70 and 10 mV (charge 1, or charge from noninactivated channels). In parallel, the charge mobile between -70 and -150 mV (charge 2, or charge from inactivated channels) was increased. The availability of charge 2 depended on the conditioning pulse voltage as the sum of two Boltzmann components. One component had a central voltage of -75 mV and a magnitude of 1.7 nC/microF. It presumably is the charge movement (charge 2) from Na+ channels. The other component, with a central voltage of approximately - 30 mV and a magnitude of 3.5 nC/microF, is the charge 2 of L-type Ca2+ channels. The sum of charge 1 and charge 2 was conserved after different conditioning pulses. The difference between the voltage dependence of the activation of L-type Ca2+ channels (half-activation voltage, V, of approximately -20 mV) and that of charge 2 (V of -100 mV) made it possible to record the ionic currents through Ca2+ channels and charge 2 in the same solution. In an external solution with Ba2+ as sole metal the maximum available charge 2 of L-type Ca2+ channels was 10-15% greater than that in a Ca(2+)-containing solution. External Cd2+ caused 20-30% reduction of charge 2 both from Na+ and L-type Ca2+ channels. Voltage- and Ca(2+)-dependent inactivation phenomena were compared with a double pulse protocol in cells perfused with an internal solution of low calcium buffering capacity. As the conditioning pulse voltage increased, inactivation monitored with the second pulse went through a minimum at about 0 mV, the voltage at which conditioning current had its maximum. Charge 2, recorded in parallel, did not show any increase associated with calcium entry. Two alternative interpretations of these observations are: (a) that Ca(2+)- dependent inactivation does not alter the voltage sensor, and (b) that inactivation affects the voltage sensor, but only in the small fraction of channels that open, and the effect goes undetected. A model of channel gating that assumes the first possibility is shown to account fully for the experimental results. Thus, extracellular divalent cations modulate voltage-dependent inactivation of the Ca2+ channel. Intracellular Ca2+ instead, appears to cause inactivation of the channel without affecting its voltage sensor.     

Effects of altered gravity on the actin and microtubule cytoskeleton of human SH-SY5Y neuroblastoma cells

Summary. Human SH-SY5Y neuroblastoma cells were used to study the effects of altered gravity on the actin and microtubule cytoskeleton dynamics. A cholinergic stimulation of the cells during a 6 min period of changing gravity (3 parabolas) resulted in an enhanced actin-driven protrusion of evoked lamellipodia. Likewise, the spontaneous protrusive activity of nonactivated cells was promoted during exposure to changing gravity (6 up to 31 parabolas). Ground-based experiments revealed a similar enhancement of the spontaneous and evoked lamellar protrusive activity when the cells were kept at 2 g hypergravity for at least 6 min. This gravity response was independent of the direction of the acceleration vector in respect to the cells. Exposure of the cells to “simulated weightlessness” (clinorotation) had no obvious influence on this type of lamellar actin cytoskeleton dynamics. A 20 min exposure of the cells to simulated weightlessness or to changing gravity (6 to 31 parabolas) – but not to 2 g (hypergravity, centrifugation) – resulted in an altered arrangement of microtubules indicated by bending, turning, and loop formation. A similar altered arrangement was shown by microtubules which had polymerized into lamellipodia after release from a taxol block at simulated weightlessness (clinorotation) or during changing gravity (5 parabolas). Our data suggest that in human SH-SY5Y neuroblastoma cells, microgravity affects the dynamics and spatial arrangement of microtubules but has no influence on the Rac-controlled lamellar actin cytoskeleton dynamics and cell spreading. The latter, however, seems to be promoted at hypergravity. Correspondence and reprints: Cell and Developmental Neurobiology, Institute of Zoology, University of Hohenheim, Garbenstrasse 30, 70593 Stuttgart, Federal Republic of Germany.     

A labile,Ca2+-dependent cytoskeleton in rhabdomeral microvilli of blowflies

Summary Rhabdomeral microvilli of photoreceptors of the blowfly Lucilia are shown to contain a cytoskeleton. An axial filament ( 6–11 nm) in each microvillus is inserted into a terminal cap distally, and into a plug filling the narrow neck of the microvillus proximally. In some states, the axial filament projects beyond the neck; within the microvillus it is surrounded by amorphous material. Together, they form an axial complex, which supports side-arms linking it to the plasma membrane. Conventional fixation for examination with the electron microscope destroys the cytoskeleton. To preserve it, retinae are pre-treated with a Ringer's solution buffered with 20 mM imidazole and containing, minimally, the following components: (i) a protease inhibitor, usually phenylmethylsulphonyl-fluoride (PMSF); (ii) either the Ca²⁺-chelator EGTA, or the calmodulin-blocking agent trifluoperazine (TFP); and (iii) a source of divalent cations to preserve the side-arms. When EGTA is used, Mg²⁺, Sr²⁺, Ba²⁺, Mn²⁺ and Co²⁺ are effective, Ba²⁺ giving the most satisfactory contrast, and Mg²⁺ and Co²⁺ the best preservation. It is inferred that the cytoskeletal complex includes at least one Ca²⁺-activated protease, and possibly calmodulin. Microvilli are bonded together by intermicrovillar bridges with a periodicity of 11–17nm. The cytoskeleton is destroyed by pretreatment with 1 mM dithiothreitol (DTT), possibly by the activation of a thiol protease. It does not survive osmication unless treated with low molecular weight tannic acid (LMWT). The evidence does not discriminate between actin and intermediate filaments as the basis of the cytoskeleton. Attention is drawn to similarities and differences between the rhabdomeral cytoskeleton and that of vertebrate intestinal brush-borders. The extreme lability of the rhabdomeral cytoskeleton to conventional methods of fixation is attributed in part to the Ca²⁺ fluxes experienced by invertebrate photoreceptors, and in part to the effects of osmication.The authors thank Dr. Lindsay Barton-Brown and Tom Van Gerwen for supplying flies from CSIRO cultures: Smith Kline and French Laboratories Ltd., French's Forest, N.S.W. for a generous gift of trifluoperazine, and Mallinckrodt, Inc., St. Louis, Missouri, USA, for a gift of low molecular weight tannic acid. Many colleagues, especially Richard Payne, Steve Shaw and Gert De Couet helped by discussing the results. George Weston and the staff of the Electron Microscope Unit provided support and advice. Sandy Smith prepared Table 1     

The formation of cortical actin arrays in human trabecular meshwork cells in response to cytoskeletal disruption

The cytoskeleton of human trabecular meshwork (HTM) cells is known to be altered in glaucoma and has been hypothesized to reduce outflow facility through contracting the HTM tissue. Latrunculin B (Lat-B) and Rho-associated protein kinase (ROCK) inhibitors disrupt the actin cytoskeleton and are in clinical trials as glaucoma therapeutics. We have previously reported a transient increase in HTM cell stiffness peaking at 90 min after Lat-B treatment with a return to pretreatment values after 270 min. We hypothesize that changes in actin morphology correlate with alterations in cell stiffness induced by Lat-B but this is not a general consequence of other cytoskeletal disrupting agents such as Rho kinase inhibitors. We treated HTM cells with 2 µM Lat-B or 100 µM Y-27632 and allowed the cells to recover for 30–270 min. While examining actin morphology in Lat-B treated cells, we observed striking cortical actin arrays (CAAs). The percentage of CAA positive cells (CPCs) was time dependent and exceeded 30% at 90 min and decreased after 270 min. Y-27632 treated cells exhibited few CAAs and no changes in cell stiffness. Together, these data suggest that the increase in cell stiffness after Lat-B treatment is correlated with CAAs.     

Interplay between TRP channels and the cytoskeleton in health and disease

Transient receptor potential (TRP) channels are a family of cation channels that play a key role in ion homeostasis and cell volume regulation. In addition, TRP channels are considered universal integrators of sensory information required for taste, vision, hearing, touch, temperature, and the detection of mechanical force. Seminal investigations exploring the molecular mechanisms of phototransduction in Drosophila have demonstrated that TRP channels operate within macromolecular complexes closely associated with the cytoskeleton. More recent evidence shows that mammalian TRP channels similarly connect to the cytoskeleton to affect cytoskeletal organization and cell adhesion via ion-transport-dependent and -independent mechanisms. In this review, we discuss new insights into the interplay between TRP channels and the cytoskeleton and provide recent examples of such interactions in different physiological systems.     

Binding of Par-4 to the actin cytoskeleton is essential for Par-4/Dlk-mediated apoptosis

Prostate apoptosis response-4 (Par-4) is a 38-kDa protein originally identified as a gene product upregulated in prostate cancer cells undergoing apoptosis. Cell death mediated by Par-4 and its interaction partner DAP like kinase (Dlk) is characterized by dramatic changes of the cytoskeleton. To uncover the role of the cytoskeleton in Par-4/Dlk-mediated apoptosis, we analyzed Par-4 for a direct association with cytoskeletal structures. Confocal fluorescence microscopy revealed that endogenous Par-4 is specifically associated with stress fibers in rat fibroblasts. In vitro cosedimentation analyses and in vivo FRET analyses showed that Par-4 directly binds to F-actin. Actin binding is mediated by the N-terminal 266 amino acids, but does not require the C-terminal region of Par-4 containing the leucine zipper and the death domain. Furthermore, the interaction of Par-4 with actin filaments leads to the formation of actin bundles in vitro and in vivo. In rat fibroblasts, this microfilament association is essential for the pro-apoptotic function of Par-4, since both disruption of the actin cytoskeleton by cytochalasin D treatment and overexpression of Par-4 constructs impaired in actin binding result in a significant decrease of apoptosis induction by Par-4 and Dlk. We propose a model, in which Par-4 recruits Dlk to stress fibers, leading to enhanced phosphorylation of the regulatory light chain of myosin II (MLC) and to the induction of apoptosis.     

Voltage- and Ca2+-activated potassium channels in Ca2+ store control Ca2+ release

Ca2+ release from Ca2+ stores is a 'quantal' process; it terminates after a rapid release of stored Ca2+. To explain the quantal nature, it has been supposed that a decrease in luminal Ca2+ acts as a 'brake' on store release. However, the mechanism for the attenuation of Ca2+ efflux remains unknown. We show that Ca2+ release is controlled by voltage- and Ca2+-activated potassium channels in the Ca2+ store. The potassium channel was identified as the big or maxi-K (BK)-type, and was activated by positive shifts in luminal potential and luminal Ca2+ increases, as revealed by patch-clamp recordings from an exposed nuclear envelope. The blockage or closure of the store BK channel due to Ca2+ efflux developed lumen-negative potentials, as revealed with an organelle-specific voltage-sensitive dye [DiOC5(3); 3,3'-dipentyloxacarbocyanine iodide], and suppressed Ca2+ release. The store BK channels are reactivated by Ca2+ uptake by Ca2+ pumps regeneratively with K+ entry to allow repetitive Ca2+ release. Indeed, the luminal potential oscillated bistably by approximately 45 mV in amplitude. Our study suggests that Ca2+ efflux-induced store BK channel closures attenuate Ca2+ release with decreases in counter-influx of K+.     

Acanthamoeba castellanii: identification and distribution of actin cytoskeleton

The presence of the cytoskeleton of Acanthamoeba castellanii was observed by means of cryo-electronmicroscopy and immunofluorescence techniques. This structure is formed largely by fibers and networks of actin located mainly in cytoplasmic locomotion structures as lamellipodia and as well as in various endocytic structures. In addition, the comparision between total actin content in whole extracts among different amoebae was made. The molecular weight of actin in A. castellanii was 44 kDa, and 45 kDa for Naegleria fowleri and Entamoeba histolytica.     

Arabidopsis vacuolar H+-ATPase (V-ATPase) B subunits are involved in actin cytoskeleton remodeling via binding to, bundling, and stabilizing F-actin

Vacuolar H(+)-ATPase (V-ATPase) is a membrane-bound multisubunit enzyme complex composed of at least 14 different subunits. The complex regulates the physiological processes of a cell by controlling the acidic environment, which is necessary for certain activities and the interaction with the actin cytoskeleton through its B and C subunits in both humans and yeast. Arabidopsis V-ATPase has three B subunits (AtVAB1, AtVAB2, and AtVAB3), which share 97.27% sequence identity and have two potential actin-binding sites, indicating that these AtVABs may have crucial functions in actin cytoskeleton remodeling and plant cell development. However, their biochemical functions are poorly understood. In this study, we demonstrated that AtVABs bind to and co-localize with F-actin, bundle F-actin to form higher order structures, and stabilize actin filaments in vitro. In addition, the AtVABs also show different degrees of activities in capping the barbed ends but no nucleating activities, and these activities were not regulated by calcium. The functional similarity and differences of the AtVABs implied that they may play cooperative and distinct roles in Arabidopsis cells.     

Mythical origins of the actin cytoskeleton

The origin of the eukaryotic cell is one of the greatest mysteries in modern biology. Eukaryotic-wide specific biological processes arose in the lost ancestors of eukaryotes. These distinctive features, such as the actin cytoskeleton, define what it is to be a eukaryote. Recent sequencing, characterization, and isolation of Asgard archaea have opened an intriguing window into the pre-eukaryotic cell. Firstly, sequencing of anaerobic sediments identified a group of uncultured organisms, Asgard archaea, which contain genes with homology to eukaryotic signature genes. Secondly, characterization of the products of these genes at the protein level demonstrated that Asgard archaea have related biological processes to eukaryotes. Finally, the isolation of an Asgard archaeon has produced a model organism in which the morphological consequences of the eukaryotic-like processes can be studied. Here, we consider the consequences for the Asgard actin cytoskeleton and for the evolution of a regulated actin system in the archaea-to-eukaryotic transition.     

Intracellular motility and the evolution of the actin cytoskeleton during development of the male gametophyte of wheat (Triticum aestivum L.)

The uniaperturate pollen of wheat is dispersed in a partially hydrated condition. Amyloplasts are concentrated in the apertural hemisphere where they surround the two sperms, while vigorously moving polysaccharide-containing wall precursor bodies (P-particles) together with the vegetative nucleus occupy the other. This disposition is the product of a post-meiotic developmental sequence apparently peculiar to the grasses. During vacuolation of the spore after release from the tetrad, the nucleus is displaced to the pole of the cell opposite the site of the germination aperture, already defined in the tetrad. Following pollen mitosis, the vegetative nucleus migrates along the wall of the vegetative cell towards the aperture, leaving the generative cell at the opposite pole isolated by a callose wall. As the vacuole is resorbed, the generative cell rounds up, loses its wall and follows the vegetative nucleus, passing along the wall of the vegetative cell towards the aperture where it eventually divides to produce the two sperms. Throughout this period of nucleus and cell manoeuvrings, minor inclusions of the vegetative cell cytoplasm, including mitochondria, lipid globuli and developing amyloplasts, move randomly. Coordinated vectorial movement begins after the main period of starch accumulation, when the amyloplasts migrate individually into the apertural hemisphere of the grain, a final redistribution betokening the attainment of germinability. In the present paper we correlate aspects of the evolution of the actin cytoskeleton with these events in the developing grain, and relate the observations to published evidence from another monocotyledonous species concerning the timing of the expression of actin genes during male gametophyte development, as revealed in the synthesis of actin mRNA.     

Interaction of the Coxsackie and adenovirus receptor (CAR) with the cytoskeleton: binding to actin

The Coxsackie and adenovirus receptor (CAR) is a cell adhesion molecule that is highly expressed in the developing brain. CAR is enriched in growth cone particles (GCP) after subcellular fractionation. In GCP, we identified actin as an interaction partner of the cytoplasmic domain of CAR. In vivo, actin and CAR co-immunoprecipitate and co-localize. In vitro, the binding is direct, with a K(d) of approximately 2.6 microM, and leads to actin bundling. We previously demonstrated that CAR interacts with microtubules. These data suggest a role for CAR in processes requiring dynamic reorganization of the cytoskeleton such as neurite outgrowth and cell migration.     

Inhibition of vascular ATP-sensitive K+ channels does not affect reactive hyperemia in human forearm

The extent to which ATP-sensitive K(+) channels contribute to reactive hyperemia in humans is unresolved. We examined the role of ATP-sensitive K(+) channels in regulating reactive hyperemia induced by 5 min of forearm ischemia. Thirty-one healthy subjects had forearm blood flow measured with venous occlusion plethysmography. Reactive hyperemia could be reproducibly induced (n = 9). The contribution of vascular ATP-sensitive K(+) channels to reactive hyperemia was determined by measuring forearm blood flow before and during brachial artery infusion of glibenclamide, an ATP-sensitive K(+) channel inhibitor (n = 12). To document ATP-sensitive K(+) channel inhibition with glibenclamide, coinfusion with diazoxide, an ATP-sensitive K(+) channel opener, was undertaken (n = 10). Glibenclamide did not significantly alter resting forearm blood flow or the initial and sustained phases of reactive hyperemia. However, glibenclamide attenuated the hyperemic response induced by diazoxide. These data suggest that ATP-sensitive K(+) channels do not play an important role in controlling forearm reactive hyperemia and that other mechanisms are active in this adaptive response.     

Alteration of the cortical actin cytoskeleton deregulates Ca2+ signaling, monospermic fertilization, and sperm entry

Background

When preparing for fertilization, oocytes undergo meiotic maturation during which structural changes occur in the endoplasmic reticulum (ER) that lead to a more efficient calcium response. During meiotic maturation and subsequent fertilization, the actin cytoskeleton also undergoes dramatic restructuring. We have recently observed that rearrangements of the actin cytoskeleton induced by actin-depolymerizing agents, or by actin-binding proteins, strongly modulate intracellular calcium (Ca²⁺) signals during the maturation process. However, the significance of the dynamic changes in F-actin within the fertilized egg has been largely unclear.

Methodology/Principal Findings

We have measured changes in intracellular Ca²⁺ signals and F-actin structures during fertilization. We also report the unexpected observation that the conventional antagonist of the InsP₃ receptor, heparin, hyperpolymerizes the cortical actin cytoskeleton in postmeiotic eggs. Using heparin and other pharmacological agents that either hypo- or hyperpolymerize the cortical actin, we demonstrate that nearly all aspects of the fertilization process are profoundly affected by the dynamic restructuring of the egg cortical actin cytoskeleton.

Conclusions/Significance

Our findings identify important roles for subplasmalemmal actin fibers in the process of sperm-egg interaction and in the subsequent events related to fertilization: the generation of Ca²⁺ signals, sperm penetration, cortical granule exocytosis, and the block to polyspermy.     

Interference of heavy metal cations with fluorescent Ca2+ probes does not affect Ca2+ measurements in living cells

In studies about the effects of heavy metals on intracellular Ca2+, the use of fluorescent probes is debated, as metal cations are known to affect the probe signal. In this study, spectrofluorimetric experiments in free solution, using Fluo-3 and Fura-2, showed that Zn2+ and Cd2+ enhanced the probe signal, Cu2+ quenched it, and Hg2+ had no effect. Addition of GSH prevented most of these effects, suggesting the occurrence of a similar protective role in living cells. Digital imaging of living mussel haemocytes loaded with Fura-2/AM or Fluo-3/AM showed that Hg2+, Cu2+ and Cd2+ induced a rise in probe fluorescence, whereas up to 200 microM Zn2+ had no effect. In particular, Cd2+ produced the strongest probe signal rise in free solution, but the lowest fluorescence increase in cells. Probe calibration yielded [Ca2+]i values characteristic of resting levels in control and Zn2+-exposed cells, and, as expected, indicated Ca2+ homeostasis impairment in cells exposed to Cd2+, Cu2+ and Hg2+. Our results show that Ca2+ probe responses to heavy metals in living cells are completely different from those obtained in free solution, indicating that fluorescent probes can be a suitable tool to record the effects of heavy metals on [Ca2+]i.     

Pannexin1 drives multicellular aggregate compaction via a signaling cascade that remodels the actin cytoskeleton

Pannexin 1 (Panx1) is a novel gap junction protein shown to have tumor-suppressive properties. To model its in vivo role in the intratumor biomechanical environment, we investigated whether Panx1 channels modulate the dynamic assembly of multicellular C6 glioma aggregates. Treatment with carbenoxolone and probenecid, which directly and specifically block Panx1 channels, respectively, showed that Panx1 is involved in accelerating aggregate assembly. Experiments further showed that exogenous ATP can reverse the inhibitive effects of carbenoxolone and that aggregate compaction is sensitive to the purinergic antagonist suramin. With a close examination of the F-actin microfilament network, these findings show that Panx1 channels act as conduits for ATP release that stimulate the P(2)X(7) purinergic receptor pathway, in turn up-regulating actomyosin function. Using a unique three-dimensional scaffold-free method to quantify multicellular interactions, this study shows that Panx1 is intimately involved in regulating intercellular biomechanical interactions pivotal in the progression of cancer.     

Methylglyoxal evokes acute Ca2+ transients in distinct cell types and increases agonist-evoked Ca2+ entry in endothelial cells via CRAC channels

Methylglyoxal (MG) is a by-product of glucose metabolism and its accumulation has been linked to the development of diabetic complications such as retinopathy and nephropathy by affecting multiple signalling pathways. However, its influence on the intracellular Ca²⁺ homeostasis and particularly Ca²⁺ entry, which has been reported to be mediated via TRPA1 channels in DRG neurons, has not been studied in much detail in other cell types. In this study, we report the consequences of acute and long-term MG application on intracellular Ca²⁺ levels in endothelial cells. We showed that acute MG application doesn’t evoke any instantaneous changes in the intracellular Ca²⁺ concentration in immortalized mouse cardiac endothelial cells (MCECs) and murine microvascular endothelial cells (muMECs). In contrast, an MG-induced rise in intracellular Ca²⁺ level was observed in primary mouse mesangial cells within 30 s, indicating that the modulation of Ca²⁺ homeostasis by MG is strictly cell type specific. The formation of the MG-derived advanced glycation end product (AGE) MG-H1 was found to be time and concentration-dependent in MCECs. Likewise, MG pre-incubation for 6 h increased the angiotensin II-evoked Ca²⁺ entry in MCECs and muMECs which was abrogated by inhibition of Calcium release activated calcium (CRAC) channels with GSK-7975A, but unaffected by an inhibitor specific to TRPA1 channels. Quantitative PCR analysis revealed that MG pre-treatment did not affect expression of the genes encoding the angiotensin receptors AT1R (Agtr 1a & Agtr 1b), Trpa1 nor Orai1, Orai2, Orai3, Stim1, Stim2 and Saraf which operate as constituents or regulators of CRAC channels and store-operated Ca²⁺ entry (SOCE) in other cell types. Together, our results show that long-term MG stimulation leads to the formation of glycation end products, which facilitates the agonist-evoked Ca²⁺ entry in endothelial cells, and this could be a new pathway that might lead to MG-evoked vasoregression observed in diabetic vasculopathies.