Assessment of Mitochondrial Ca2+ Uptake.

Mitochondrial Ca2+ uptake regulates mitochondrial function and contributes to cell signaling. Accordingly, quantifying mitochondrial Ca2+ signals and elaborating the mechanisms that accomplish mitochondrial Ca2+ uptake are essential to gain our understanding of cell biology. Here, we describe the benefits and drawbacks of various established old and new techniques to assess dynamic changes of mitochondrial Ca2+ concentration ([Ca2+]mito) in a wide range of applications.

Postural hemodynamic parameters in older persons have a seasonal dependency : A pilot study.

AIMS: It is known that blood pressure regulation differs seasonally. It is unknown, however, how the cardiovascular system in patients with a stroke reacts to postural changes in different seasons. The aim was therefore to investigate how different temperatures in cold and warm seasons influence the reactions of haemodynamic mechanisms as well as heart rate variability during a sit-to-stand test in patients with stroke and a control group. METHODS: Hemodynamic responses were assessed in both groups during a sit-to-stand test (5 min sitting followed by 5 min standing) beat to beat within two different seasons. Systolic blood pressure (SBP), diastolic blood pressure (DBP), mean blood pressure (MBP), heart rate (HR), stroke index (SI), cardiac index (CI) and heart rate variability (HRV) were continuously monitored. RESULTS: During the sitting baseline period delta values of DBP (+15.1 [Standard error (SE) 3.75] mmHg, p < 0.05) and MBP (+14.35 [SE 4.18] mmHg, p < 0.05) were significantly higher in colder months compared to warmer months whereas SI (-3.86 [SE 1.43] ml/beat/m2, p < 0.05) and CI (-0.4 [SE 0.11] l/min/m2, p < 0.05) were lower in colder months compared to warmer months in non-stroke participants. In patients with stroke during sitting, baseline period delta values of DBP (+19.92 [SE 8.03] mmHg, p < 0.05) and MBP (+19.29 [SE 8.6] mmHg, p < 0.05) were significantly higher in colder months compared to warmer months but SI (-5.43 [SE 1.96] ml/beat/m2, p < 0.05) was significantly lower in colder months compared to warmer months. After standing, there was a significant decrease in SBP in warmer months (-16.84 [SE 4.38] mmHg, p < 0.05) and a decrease in DBP in warmer months (-7.8 [SE 2.3] mmHg, p < 0.05) and colder months (-6.73 [SE 1.5] mmHg, p < 0.05) in non-stroke participants and a decrease in MBP in warmer months (-12.5 [SE 2.8] mmHg, p < 0.05) and colder months (-8.93 [SE 1.8] mmHg, p < 0.05) in non-stroke participants and in warmer months (-14.54 [SE 4.1] mmHg, p < 0.05) in patients with stroke. CONCLUSION: Elderly with and without stroke respond to orthostatic stress with a greater drop in blood pressure in the warmer seasons.

Cannabinoids and Cardiovascular System.

Cannabinoids influence cardiovascular variables in health and disease via multiple mechanisms. The chapter covers the impact of cannabinoids on cardiovascular function in physiology and pathology and presents a critical analysis of the proposed signalling pathways governing regulation of cardiovascular function by endogenously produced and exogenous cannabinoids. We know that endocannabinoid system is overactivated under pathological conditions and plays both a protective compensatory role, such as in some forms of hypertension, atherosclerosis and other inflammatory conditions, and a pathophysiological role, such as in disease states associated with excessive hypotension. This chapter focuses on the mechanisms affecting hemodynamics and vasomotor effects of cannabinoids in health and disease states, highlighting mismatches between some studies. The chapter will first review the effects of marijuana smoking on cardiovascular system and then describe the impact of exogenous cannabinoids on cardiovascular parameters in humans and experimental animals. This will be followed by analysis of the impact of cannabinoids on reactivity of isolated vessels. The article critically reviews current knowledge on cannabinoid induction of vascular relaxation by cannabinoid receptor-dependent and -independent mechanisms and dysregulation of vascular endocannabinoid signaling in disease states.

Ca2+-dependent potassium channels and cannabinoid signaling in the endothelium of apolipoprotein E knockout mice before plaque formation.

Endothelial Ca2+-dependent K+ channels (KCa) regulate endothelial function. We also know that stimulation of type 2 cannabinoid (CB2) receptors ameliorates atherosclerosis. However, whether atherosclerosis is accompanied by altered endothelial KCa- and CB2 receptor-dependent signaling is unknown. By utilizing an in situ patch-clamp approach, we directly evaluated the KCa channel function and the CB2 receptor-dependent electrical responses in the endothelium of aortic strips from young ApoE-/- and C57Bl/6 mice. In the ApoE-/- group, the resting membrane potential (-30.1±1.1mV) was less negative (p<0.05) compared to WT (-38.9±1.4mV) and voltage ramps generated an overall KCa current of reduced amplitude. The peak hyperpolarization to 2µM Ach was not different between the groups. However, the sustained component was significantly reduced in ApoE-/- strips. In contrast, the peak hyperpolarization to 0.2µM Ach was increased in the ApoE-/- group, and SKA-31, a direct IKCa/SKCa channel opener, produced a hyperpolarization and whole-cell current of greater amplitude. The BKCa opener NS1619 produced hyperpolarization that was enhanced in ApoE-/- group. N-arachidonoyl glycine, a BKCa opener, produced a hyperpolarization of enhanced amplitude in ApoE-/- arteries. Selective CB2 receptor agonist AM1241 (5µM) had no effect on endothelial membrane potential in WT group; however, in ApoE-/- group, it elicited hyperpolarization that was inhibited by a selective CB2 receptor antagonist AM630. Conclusively, our data point to functional down-regulation of basal IKCa activity in unstimulated endothelium of ApoE-/- mice. Direct and indirect IKCa stimulation resulted in increased recruitment of the channels. In addition, our data point to up-regulation of endothelial BKCa channels and CB2 receptors in ApoE-/- arteries.

The quest for endothelial atypical cannabinoid receptor: BKCa channels act as cellular sensors for cannabinoids in in vitro and in situ endothelial cells.

Endothelium-dependent component of cannabinoid-induced vasodilation has been postulated to require G-protein-coupled non-CB1/CB2 endothelial cannabinoid (eCB) receptor. GPR18 was proposed as a candidate for eCBR. To address the hypothesis that the effects attributed to eCBR are mediated by G-protein-coupled receptor (GPCR)-independent targets, we studied the electrical responses in endothelial cells, focusing on BKCa channels. In patches excised from endothelial-derived EA.hy926 cells, N-arachidonoyl glycine (NAGly) and abnormal cannabidiol (abn-cbd), prototypical agonists for eCB receptor, stimulate single BKCa activity in a concentration- and Ca2+-dependent manner. The postulated eCB receptor inhibitors rimonabant and AM251 were found to inhibit basal and stimulated by NAGly- and abn-cbd BKCa activity in cell-free patches. In isolated mice aortas, abn-cbd and NAGly produced endothelial cell hyperpolarization that was sensitive to paxilline, a selective BKCa inhibitor, but not to GPR18 antibody, and mimicked by NS1619, a direct BKCa opener. In excised patches from mice aortic endothelium, single channel activity with characteristics similar to BKCa was established by the addition of abn-cbd and NAGly. We conclude that the two cannabinoids abn-cbd and NAGly initiate a GPR18-independent activation of BKCa channels in mice aortic endothelial cells that might contribute to vasodilation to cannabinoids.

Big conductance calcium-activated potassium channel openers control spasticity without sedation.

BACKGROUND AND PURPOSE: Our initial aim was to generate cannabinoid agents that control spasticity, occurring as a consequence of multiple sclerosis (MS), whilst avoiding the sedative side effects associated with cannabis. VSN16R was synthesized as an anandamide (endocannabinoid) analogue in an anti-metabolite approach to identify drugs that target spasticity. EXPERIMENTAL APPROACH: Following the initial chemistry, a variety of biochemical, pharmacological and electrophysiological approaches, using isolated cells, tissue-based assays and in vivo animal models, were used to demonstrate the activity, efficacy, pharmacokinetics and mechanism of action of VSN16R. Toxicological and safety studies were performed in animals and humans. KEY RESULTS: VSN16R had nanomolar activity in tissue-based, functional assays and dose-dependently inhibited spasticity in a mouse experimental encephalomyelitis model of MS. This effect occurred with over 1000-fold therapeutic window, without affecting normal muscle tone. Efficacy was achieved at plasma levels that are feasible and safe in humans. VSN16R did not bind to known CB1 /CB2 /GPPR55 cannabinoid-related receptors in receptor-based assays but acted on a vascular cannabinoid target. This was identified as the major neuronal form of the big conductance, calcium-activated potassium (BKCa ) channel. Drug-induced opening of neuronal BKCa channels induced membrane hyperpolarization, limiting excessive neural-excitability and controlling spasticity. CONCLUSIONS AND IMPLICATIONS: We identified the neuronal form of the BKCa channel as the target for VSN16R and demonstrated that its activation alleviates neuronal excitability and spasticity in an experimental model of MS, revealing a novel mechanism to control spasticity. VSN16R is a potential, safe and selective ligand for controlling neural hyper-excitability in spasticity.

Direct activation of Ca2+ and voltage-gated potassium channels of large conductance by anandamide in endothelial cells does not support the presence of endothelial atypical cannabinoid receptor.

Endocannabinoid anandamide induces endothelium-dependent relaxation commonly attributed to stimulation of the G-protein coupled endothelial anandamide receptor. The study addressed the receptor-independent effect of anandamide on large conductance Ca2+-dependent K+ channels expressed in endothelial cell line EA.hy926. Under resting conditions, 10µM anandamide did not significantly influence the resting membrane potential. In a Ca2+-free solution the cells were depolarized by ~10mV. Further administration of 10µM anandamide hyperpolarized the cells by ~8mV. In voltage-clamp mode, anandamide elicited the outwardly rectifying whole-cell current sensitive to paxilline but insensitive to GDPßS, a G-protein inhibitor. Administration of 70µM Mn2+, an agent used to promote integrin clustering, reversibly stimulated whole-cell current, but failed to further facilitate the anandamide-stimulated current. In an inside-out configuration, anandamide (0.1-30µM) facilitated single BKCa channel activity in a concentration-dependent manner within a physiological Ca2+ range and a wide range of voltages, mainly by reducing mean closed time. The effect is essentially eliminated following chelation of Ca2+ from the cytosolic face and pre-exposure to cholesterol-reducing agent methyl-ß-cyclodextrin. O-1918 (3µM), a cannabidiol analog used as a selective antagonist of endothelial anandamide receptor, reduced BKCa channel activity in inside-out patches. These results do not support the existence of endothelial cannabinoid receptor and indicate that anandamide acts as a direct BKCa opener. The action does not require cell integrity or integrins and is caused by direct modification of BKCa channel activity.

GPR55 agonist lysophosphatidylinositol and lysophosphatidylcholine inhibit endothelial cell hyperpolarization via GPR-independent suppression of Na+-Ca2+ exchanger and endoplasmic reticulum Ca2+ refilling.

Lysophosphatidylinositol (LPI) and lysophosphatidylcholine (LPC) are lipid signaling molecules that induce endothelium-dependent vasodilation. In addition, LPC suppresses acetylcholine (Ach)-induced responses. We aimed to determine the influence of LPC and LPI on hyperpolarizing responses in vitro and in situ endothelial cells (EC) and identify the underlying mechanisms. Using patch-clamp method, we show that LPI and LPC inhibit EC hyperpolarization to histamine and suppress Na+/Ca2+ exchanged (NCX) currents in a concentration-dependent manner. The inhibition is non-mode-specific and unaffected by intracellular GDPßS infusion and tempol, a superoxide dismutase mimetic. In excised mouse aorta, LPI strongly inhibits the sustained and the peak endothelial hyperpolarization induced by Ach, but not by SKA-31, an opener of Ca2+-dependent K+ channels of intermediate and small conductance. The hyperpolarizing responses to consecutive histamine applications are strongly reduced by NCX inhibition. In a Ca2+-re-addition protocol, bepridil, a NCX inhibitor, and KB-R7943, a blocker of reversed NCX, inhibit the hyperpolarizing responses to Ca2+-re-addition following Ca2+ stores depletion. These finding indicate that LPC and LPI inhibit endothelial hyperpolarization to Ach and histamine independently of G-protein coupled receptors and superoxide anions. Reversed NCX is critical for ER Ca2+ refilling in EC. The inhibition of NCX by LPI and LPC underlies diminished endothelium-dependent responses and endothelial dysfunction accompanied by increased levels of these lipids in the blood.

PRMT1-mediated methylation of MICU1 determines the UCP2/3 dependency of mitochondrial Ca(2+) uptake in immortalized cells.

Recent studies revealed that mitochondrial Ca(2+) channels, which control energy flow, cell signalling and death, are macromolecular complexes that basically consist of the pore-forming mitochondrial Ca(2+) uniporter (MCU) protein, the essential MCU regulator (EMRE), and the mitochondrial Ca(2+) uptake 1 (MICU1). MICU1 is a regulatory subunit that shields mitochondria from Ca(2+) overload. Before the identification of these core elements, the novel uncoupling proteins 2 and 3 (UCP2/3) have been shown to be fundamental for mitochondrial Ca(2+) uptake. Here we clarify the molecular mechanism that determines the UCP2/3 dependency of mitochondrial Ca(2+) uptake. Our data demonstrate that mitochondrial Ca(2+) uptake is controlled by protein arginine methyl transferase 1 (PRMT1) that asymmetrically methylates MICU1, resulting in decreased Ca(2+) sensitivity. UCP2/3 normalize Ca(2+) sensitivity of methylated MICU1 and, thus, re-establish mitochondrial Ca(2+) uptake activity. These data provide novel insights in the complex regulation of the mitochondrial Ca(2+) uniporter by PRMT1 and UCP2/3.

Treatment with the GPR55 antagonist CID16020046 increases neutrophil activation in mouse atherogenesis.

Endocannabinoids modulate atherogenesis by triggering different receptors. Recently, orphan G protein-coupled receptors (GPRs) were suggested to be activated by endocannabinoids, possibly regulating vasorelaxation. Here, we investigated whether GPR55 antagonism with CID16020046 would impact on atherosclerotic size and inflammation in two mouse models of early and more advanced atherogenesis. Eleven-week old ApoE-/- mice were fed either a normal diet ([ND] for 16 weeks) or a high-cholesterol diet ([HD] for 11 weeks), resulting in different degrees of hypercholesterolaemia and size of atherosclerosis. CID16020046 (0.5 mg/kg) or vehicle were intraperitoneally administrated five times per week in the last three weeks before euthanasia. Treatment with CID1602004 was well-tolerated, but failed to affect atherosclerotic plaque and necrotic core size, fibrous cap thickness, macrophage and smooth muscle cell content as well as Th cell polarisation. In ND mice, treatment with CID1602004 was associated with increased chemokine production, neutrophil and MMP-9 intraplaque content as well as reduced collagen as compared to vehicle-treated animals. In HD mice, CID1602004 increased intraplaque MMP-9 and abrogated collagen content without affecting neutrophils. In vitro, serum from CID1602004-treated ND mice increased mouse neutrophil chemotaxis towards CXCL2 as compared to serum from vehicle-treated animals. CID1602004 dose-dependently induced neutrophil degranulation that was reverted by co-incubation with the GPR55 agonist Abn-CBD. In supernatants from degranulation experiments, increased levels of the endocannabinoid and putative GPR55 ligand anandamide (AEA) were found, suggesting its possible autocrine control of neutrophil activity. These results indicate that GPR55 is critical for the negative control of neutrophil activation in different phases of atherogenesis.

UCP2 modulates single-channel properties of a MCU-dependent Ca(2+) inward current in mitochondria.

The mitochondrial Ca(2+) uniporter is a highly Ca(2+)-selective protein complex that consists of the pore-forming mitochondrial Ca(2+) uniporter protein (MCU), the scaffolding essential MCU regulator (EMRE), and mitochondrial calcium uptake 1 and 2 (MICU1/2), which negatively regulate mitochondrial Ca(2+) uptake. We have previously reported that uncoupling proteins 2 and 3 (UCP2/3) are also engaged in the activity of mitochondrial Ca(2+) uptake under certain conditions, while the mechanism by which UCP2/3 facilitates mitochondrial Ca(2+) uniport remains elusive. This work was designed to investigate the impact of UCP2 on the three distinct mitochondrial Ca(2+) currents found in mitoplasts isolated from HeLa cells, the intermediate- (i-), burst- (b-) and extra-large (xl-) mitochondrial/mitoplast Ca(2+) currents (MCC). Using the patch clamp technique on mitoplasts from cells with reduced MCU and EMRE unveiled a very high affinity of MCU for xl-MCC that succeeds that for i-MCC, indicating the coexistence of at least two MCU/EMRE-dependent Ca(2+) currents. The manipulation of the expression level of UCP2 by either siRNA-mediated knockdown or overexpression changed exclusively the open probability (NPo) of xl-MCC by approx. 38% decrease or nearly a 3-fold increase, respectively. These findings confirm a regulatory role of UCP2 in mitochondrial Ca(2+) uptake and identify UCP2 as a selective modulator of just one distinct MCU/EMRE-dependent mitochondrial Ca(2+) inward current.

Assessment of mitochondrial Ca²âº uptake.

Mitochondrial Ca(2+) uptake regulates mitochondrial function and contributes to cell signaling. Accordingly, quantifying mitochondrial Ca(2+) signals and elaborating the mechanisms that accomplish mitochondrial Ca(2+) uptake are essential to gain our understanding of cell biology. Here, we describe the benefits and drawbacks of various established old and new techniques to assess dynamic changes of mitochondrial Ca(2+) concentration ([Ca(2+)]mito) in a wide range of applications.

Endothelial atypical cannabinoid receptor: do we have enough evidence?

Cannabinoids and their synthetic analogues affect a broad range of physiological functions, including cardiovascular variables. Although direct evidence is still missing, the relaxation of a vast range of vascular beds induced by cannabinoids is believed to involve a still unidentified non-CB1 , non-CB2 Gi/o protein-coupled receptor located on endothelial cells, the so called endothelial cannabinoid receptor (eCB receptor). Evidence for the presence of an eCB receptor comes mainly from vascular relaxation studies, which commonly employ pertussis toxin as an indicator for GPCR-mediated signalling. In addition, a pharmacological approach is widely used to attribute the relaxation to eCB receptors. Recent findings have indicated a number of GPCR-independent targets for both agonists and antagonists of the presumed eCB receptor, warranting further investigations and cautious interpretation of the vascular relaxation studies. This review will provide a brief historical overview on the proposed novel eCB receptor, drawing attention to the discrepancies between the studies on the pharmacological profile of the eCB receptor and highlighting the Gi/o protein-independent actions of the eCB receptor inhibitors widely used as selective compounds. As the eCB receptor represents an attractive pharmacological target for a number of cardiovascular abnormalities, defining its molecular identity and the extent of its regulation of vascular function will have important implications for drug discovery. This review highlights the need to re-evaluate this subject in a thoughtful and rigorous fashion. More studies are needed to differentiate Gi/o protein-dependent endothelial cannabinoid signalling from that involving the classical CB1 and CB2 receptors as well as its relevance for pathophysiological conditions.

Mitochondrial Ca(2+) uniporter (MCU)-dependent and MCU-independent Ca(2+) channels coexist in the inner mitochondrial membrane.

A protein referred to as CCDC109A and then renamed to mitochondrial calcium uniporter (MCU) has recently been shown to accomplish mitochondrial Ca(2+) uptake in different cell types. In this study, we investigated whole-mitoplast inward cation currents and single Ca(2+) channel activities in mitoplasts prepared from stable MCU knockdown HeLa cells using the patch-clamp technique. In whole-mitoplast configuration, diminution of MCU considerably reduced inward Ca(2+) and Na(+) currents. This was accompanied by a decrease in occurrence of single channel activity of the intermediate conductance mitochondrial Ca(2+) current (i-MCC). However, ablation of MCU yielded a compensatory 2.3-fold elevation in the occurrence of the extra large conductance mitochondrial Ca(2+) current (xl-MCC), while the occurrence of bursting currents (b-MCC) remained unaltered. These data reveal i-MCC as MCU-dependent current while xl-MCC and b-MCC seem to be rather MCU-independent, thus, pointing to the engagement of at least two molecularly distinct mitochondrial Ca(2+) channels.

Effect of orthostasis on endothelial function: a gender comparative study.

As the vascular endothelium has multiple functions, including regulation of vascular tone, it may play a role in the pathophysiology of orthostatic intolerance. We investigated the effect of orthostasis on endothelial function using EndoPAT®, a non-invasive and user-independent method, and across gender. As sex steroid hormones are known to affect endothelial function, this study examined the potential effect of these hormones on the endothelial response to orthostasis by including females at different phases of the menstrual cycle (follicular and luteal-where the hormone balance differs), and females taking an oral contraceptive. A total of 31 subjects took part in this study (11 males, 11 females having normal menstrual cycles and 9 females taking oral contraceptive). Each subject made two visits for testing; in the case of females having normal menstrual cycles the first session was conducted either 1-7 (follicular) or 14-21 days (luteal) after the start of menstruation, and the second session two weeks later, i.e., during the other phase, respectively. Endothelial function was assessed at baseline and following a 20-min orthostatic challenge (active standing). The EndoPAT® index increased from 1.71 ± 0.09 (mean ± SEM) at baseline to 2.07 ± 0.09 following orthostasis in females (p<0.001). In males, the index increased from 1.60 ± 0.08 to 1.94 ± 0.13 following orthostasis (p<0.001). There were no significant differences, however, in the endothelial response to orthostasis between females and males, menstrual cycle phases and the usage of oral contraceptive. Our results suggest an increased vasodilatatory endothelial response following orthostasis in both females and males. The effect of gender and sex hormones on the endothelial response to orthostasis appears limited. Further studies are needed to determine the potential role of this post orthostasis endothelial response in the pathophysiology of orthostatic intolerance.

N-Arachidonoyl glycine suppresses Na⁺/Ca²âº exchanger-mediated Ca²âº entry into endothelial cells and activates BK(Ca) channels independently of GPCRs.

BACKGROUND AND PURPOSE: N-Arachidonoyl glycine (NAGly) is a lipoamino acid with vasorelaxant properties. We aimed to explore the mechanisms of NAGly's action on unstimulated and agonist-stimulated endothelial cells. EXPERIMENTAL APPROACH: The effects of NAGly on endothelial electrical signalling were studied in combination with vascular reactivity. KEY RESULTS: In EA.hy926 cells, the sustained hyperpolarization to histamine was inhibited by the non-selective Na⁺/Ca²âº exchanger (NCX) inhibitor bepridil and by an inhibitor of reversed mode NCX, KB-R7943. In cells dialysed with Cs⁺-based Na⁺-containing solution, the outwardly rectifying current with typical characteristics of NCX was augmented following histamine exposure, further increased upon external Na⁺ withdrawal and inhibited by bepridil. NAGly (0.3-30 µM) suppressed NCX currents in a URB597- and guanosine 5'-O-(2-thiodiphosphate) (GDPßS)-insensitive manner, [Ca²âº]i elevation evoked by Na⁺ removal and the hyperpolarization to histamine. In rat aorta, NAGly opposed the endothelial hyperpolarization and relaxation response to ACh. In unstimulated EA.hy926 cells, NAGly potentiated the whole-cell current attributable to large-conductance Ca²âº-activated K⁺ (BK(Ca)) channels in a GDPßS-insensitive, paxilline-sensitive manner and produced a sustained hyperpolarization. In cell-free inside-out patches, NAGly stimulated single BK(Ca) channel activity. CONCLUSION AND IMPLICATIONS: Our data showed that NCX is a Ca²âº entry pathway in endothelial cells and that NAGly is a potent G-protein-independent modulator of endothelial electrical signalling and has a dual effect on endothelial electrical responses. In agonist pre-stimulated cells, NAGly opposes hyperpolarization and relaxation via inhibition of NCX-mediated Ca²âº entry, while in unstimulated cells, it promotes hyperpolarization via receptor-independent activation of BK(Ca) channels.

Characterization of distinct single-channel properties of Ca²âº inward currents in mitochondria.

Previous studies have demonstrated several molecularly distinct players involved in mitochondrial Ca(2+) uptake. In the present study, electrophysiological recordings on mitoplasts that were isolated from HeLa cells were performed in order to biophysically and pharmacologically characterize Ca(2+) currents across the inner mitochondrial membrane. In mitoplast-attached configuration with 105 mM Ca(2+) as a charge carrier, three distinct channel conductances of 11, 23, and 80 pS were observed. All types of mitochondrial currents were voltage-dependent and essentially depended on the presence of Ca(2+) in the pipette. The 23 pS channel exhibited burst kinetics. Though all channels were sensitive to ruthenium red, their sensitivity was different. The 11 and 23 pS channels exhibited a lower sensitivity to ruthenium red than the 80 pS channel. The activities of all channels persisted in the presence of cylosporin A, CGP 37187, various K(+)-channel inhibitors, and Cl(-) channel blockers disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate and niflumic acid. Collectively, our data identified multiple conductances of Ca(2+) currents in mitoplasts isolated from HeLa cells, thus challenging the dogma of only one unique mitochondrial Ca(2+) uniporter.

The endocannabinoid N-arachidonoyl glycine (NAGly) inhibits store-operated Ca2+ entry by preventing STIM1-Orai1 interaction.

The endocannabiniod anandamide (AEA) and its derivate N-arachidonoyl glycine (NAGly) have a broad spectrum of physiological effects, which are induced by both binding to receptors and receptor-independent modulations of ion channels and transporters. The impact of AEA and NAGly on store-operated Ca(2+) entry (SOCE), a ubiquitous Ca(2+) entry pathway regulating many cellular functions, is unknown. Here we show that NAGly, but not AEA reversibly hinders SOCE in a time- and concentration-dependent manner. The inhibitory effect of NAGly on SOCE was found in the human endothelial cell line EA.hy926, the rat pancreatic ß-cell line INS-1 832/13, and the rat basophilic leukemia cell line RBL-2H3. NAGly diminished SOCE independently from the mode of Ca(2+) depletion of the endoplasmic reticulum, whereas it had no effect on Ca(2+) entry through L-type voltage-gated Ca(2+) channels. Enhanced Ca(2+) entry was effectively hampered by NAGly in cells overexpressing the key molecular constituents of SOCE, stromal interacting molecule 1 (STIM1) and the pore-forming subunit of SOCE channels, Orai1. Fluorescence microscopy revealed that NAGly did not affect STIM1 oligomerization, STIM1 clustering, or the colocalization of STIM1 with Orai1, which were induced by Ca(2+) depletion of the endoplasmic reticulum. In contrast, independently from its slow depolarizing effect on mitochondria, NAGly instantly and strongly diminished the interaction of STIM1 with Orai1, indicating that NAGly inhibits SOCE primarily by uncoupling STIM1 from Orai1. In summary, our findings revealed the STIM1-Orai1-mediated SOCE machinery as a molecular target of NAGly, which might have many implications in cell physiology.

Mitochondrial Ca2+ uptake 1 (MICU1) and mitochondrial ca2+ uniporter (MCU) contribute to metabolism-secretion coupling in clonal pancreatic ß-cells.

In pancreatic ß-cells, uptake of Ca(2+) into mitochondria facilitates metabolism-secretion coupling by activation of various matrix enzymes, thus facilitating ATP generation by oxidative phosphorylation and, in turn, augmenting insulin release. We employed an siRNA-based approach to evaluate the individual contribution of four proteins that were recently described to be engaged in mitochondrial Ca(2+) sequestration in clonal INS-1 832/13 pancreatic ß-cells: the mitochondrial Ca(2+) uptake 1 (MICU1), mitochondrial Ca(2+) uniporter (MCU), uncoupling protein 2 (UCP2), and leucine zipper EF-hand-containing transmembrane protein 1 (LETM1). Using a FRET-based genetically encoded Ca(2+) sensor targeted to mitochondria, we show that a transient knockdown of MICU1 or MCU diminished mitochondrial Ca(2+) uptake upon both intracellular Ca(2+) release and Ca(2+) entry via L-type channels. In contrast, knockdown of UCP2 and LETM1 exclusively reduced mitochondrial Ca(2+) uptake in response to either intracellular Ca(2+) release or Ca(2+) entry, respectively. Therefore, we further investigated the role of MICU1 and MCU in metabolism-secretion coupling. Diminution of MICU1 or MCU reduced mitochondrial Ca(2+) uptake in response to d-glucose, whereas d-glucose-triggered cytosolic Ca(2+) oscillations remained unaffected. Moreover, d-glucose-evoked increases in cytosolic ATP and d-glucose-stimulated insulin secretion were diminished in MICU1- or MCU-silenced cells. Our data highlight the crucial role of MICU1 and MCU in mitochondrial Ca(2+) uptake in pancreatic ß-cells and their involvement in the positive feedback required for sustained insulin secretion.

The vascular barrier-protecting hawthorn extract WS® 1442 raises endothelial calcium levels by inhibition of SERCA and activation of the IP3 pathway.

WS® 1442 has been proven as an effective and safe therapeutical to treat mild forms of congestive heart failure. Beyond this action, we have recently shown that WS® 1442 protects against thrombin-induced vascular barrier dysfunction and the subsequent edema formation by affecting endothelial calcium signaling. The aim of the study was to analyze the influence of WS® 1442 on intracellular calcium concentrations [Ca(2+)](i) in the human endothelium and to investigate the underlying mechanisms. Using ratiometric calcium measurements and a FRET sensor, we found that WS® 1442 concentration-dependently increased basal [Ca(2+)](i) by depletion of the endoplasmic reticulum (ER) and inhibited a subsequent histamine-triggered rise of [Ca(2+)](i). Interestingly, the augmented [Ca(2+)](i) did neither trigger an activation of the contractile machinery nor led to a barrier breakdown (macromolecular permeability). It also did not impair endothelial cell viability. As assessed by patch clamp recordings, WS® 1442 did only slightly affect endothelial Na(+)/K(+)-ATPase, but increased [Ca(2+)](i) by inhibiting the sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (SERCA) and by activating the inositol 1,4,5-trisphosphate (IP(3)) pathway. Most importantly, WS® 1442 did not induce store-operated calcium entry (SOCE), but even irreversibly prevented histamine-induced SOCE. Taken together, WS® 1442 prevented the deleterious hyperpermeability-associated rise of [Ca(2+)](i) by a preceding, non-toxic release of Ca(2+) from the ER. WS® 1442 interfered with SERCA and the IP(3) pathway without inducing SOCE. The elucidation of this intriguing mechanism helps to understand the complex pharmacology of the cardiovascular drug WS® 1442.