Deletion of Orai2 augments endogenous CRAC currents and degranulation in mast cells leading to enhanced anaphylaxis

All three members of the Orai family of cation channels–Orai1, Orai2 and Orai3–are integral membrane proteins that can form store-operated Ca²⁺ channels resembling endogenous calcium release-activated channels (CRAC) in many aspects. Loss of function studies in human and murine models revealed many functions of Orai1 proteins not only for Ca²⁺ homeostasis, but also for cellular and systemic functions in many cell types. By contrast, the knowledge regarding the contribution of Orai2 and Orai3 proteins in these processes is sparse. In this study, we report the generation of mouse models with targeted inactivation of the Orai2 gene to study Orai2 function in peritoneal mast cells (PMC), a classical cell model for CRAC channels and Ca²⁺-dependent exocytosis of inflammatory mediators. We show that the Ca²⁺ rise triggered by agonists acting on high-affinity Fc receptors for IgE or on MAS-related G protein-coupled receptors is significantly increased in Orai2-deficient mast cells. Ca²⁺ entry triggered by depletion of intracellular stores (SOCE) is also increased in Orai2^−/− PMCs at high (2 mM) extracellular Ca²⁺ concentration, whereas SOCE is largely reduced upon re-addtion of lower (0.1 mM) Ca²⁺ concentration. Likewise, the density of CRAC currents, Ca²⁺-dependent mast cell degranulation, and mast cell-mediated anaphylaxis are intensified in Orai2-deficient mice. These results show that the presence of Orai2 proteins limits receptor-evoked Ca²⁺ transients, store-operated Ca²⁺ entry (SOCE) as well as degranulation of murine peritoneal mast cells but also raise the idea that Orai2 proteins contribute to Ca²⁺ entry in connective tissue type mast cells in discrete operation modes depending on the availability of calcium ions in the extracellular space.     

Mitochondrial free [Ca2+] levels and the permeability transition

Mitochondrial Ca²⁺ activates many processes, from mitochondrial metabolism to opening of the permeability transition pore (PTP) and apoptosis. However, there is considerable controversy regarding the free mitochondrial [Ca²⁺] ([Ca²⁺]_M) levels that can be attained during cell activation or even in mitochondrial preparations. Studies using fluorescent dyes (rhod-2 or similar), have reported that phosphate precipitation precludes [Ca²⁺]_M from increasing above 2–3 μM. Instead, using low-Ca²⁺-affinity aequorin probes, we have measured [Ca²⁺]_M values more than two orders of magnitude higher. We confirm here these values by making a direct in situ calibration of mitochondrial aequorin, and we show that a prolonged increase in [Ca²⁺]_M to levels of 0.5–1 mM was actually observed at any phosphate concentration (0–10 mM) during continuous perfusion of 3.5–100 μM Ca²⁺-buffers. In spite of this high and maintained (>10 min) [Ca²⁺]_M, mitochondria retained functionality and the [Ca²⁺]_M drop induced by a protonophore was fully reversible. In addition, this high [Ca²⁺]_M did not induce PTP opening unless additional activators (phenyl arsine oxide, PAO) were present. PAO induced a rapid, concentration-dependent and irreversible drop in [Ca²⁺]_M. In conclusion [Ca²⁺]_M levels of 0.5–1 mM can be reached and maintained for prolonged periods (>10 min) in phosphate-containing medium, and massive opening of PTP requires additional pore activators.     

Nickel induces intracellular calcium mobilization and pathophysiological responses in human cultured airway epithelial cells

Environmental exposure to nickel is associated to respiratory disorders and potential toxicity in the lung but molecular mechanisms remain incompletely explored. The extracellular Ca²⁺-sensing receptor (CaSR) is widely distributed and may be activated by divalent cations. In this study, we investigated the presence of CaSR in human cultured airway epithelial cells and its activation by nickel. Nickel transiently increased intracellular calcium (?log EC₅₀ = 4.67 ± 0.06) in A549 and human bronchial epithelial cells as measured by epifluorescence microscopy. Nickel (20 μM)-induced calcium responses were reduced after thapsigargin or ryanodine exposure but not by Ca²⁺-free medium. Inhibition of phospholipase-C or inositol trisphosphate release reduced intracellular calcium responses to nickel indicating activation of G_q-signaling. CaSR mRNA and protein expression in epithelial cells was demonstrated by RT-PCR, western blot and immunofluorescence. Transfection of specific siRNA inhibited CaSR expression and suppressed nickel-induced intracellular calcium responses in A549 cells thus confirming nickel-CaSR activation. NPS2390, a CaSR antagonist, abolished the calcium response to nickel. Nickel-induced contraction, proliferation, α₁(I)collagen production and inflammatory cytokines mRNA expression by epithelial cells as measured by traction microscopy, BrdU assay and RT-PCR, respectively. These responses were blocked by NPS2390. In conclusion, micromolar nickel concentrations, relevant to nickel found in the lung tissue of humans exposed to high environmental nickel, trigger intracellular Ca²⁺ mobilization in human airway epithelial cells through the activation of CaSR which translates into pathophysiological outputs potentially related to pulmonary disease.     

High extracellular Ca2+ enhances the adipocyte accumulation of bone marrow stromal cells through a decrease in cAMP

Bone marrow stromal cells (BMSCs) are common progenitors of both adipocytes and osteoblasts. We recently suggested that increased [Ca²⁺]_o caused by bone resorption might accelerate adipocyte accumulation in response to treatment with both insulin and dexamethasone. In this study, we investigated the mechanism by which high [Ca²⁺]_o enhances adipocyte accumulation.We used primary mouse BMSCs and evaluated the levels of adipocyte accumulation by measuring Oil Red O staining. CaSR agonists (both Ca²⁺ and Sr²⁺) enhanced the accumulation of adipocytes among BMSCs in response to treatment with both insulin and dexamethasone. We showed that high [Ca²⁺]_o decreases the concentration of cAMP using ELISA. Real-time RT-PCR revealed that increasing the intracellular concentration of cAMP (both chemical inducer (1 μM forskolin and 200 nM IBMX) and a cAMP analog (10 μM pCPT-cAMP)) suppressed the expression of PPARγ and C/EBPα. In addition, forskolin, IBMX, and pCPT-cAMP inhibited the enhancement in adipocyte accumulation under high [Ca²⁺]_o in BMSCs. However, this inhibited effect was not observed in BMSCs that were cultured in a basal concentration of [Ca²⁺]_o. We next observed that the accumulation of adipocytes in the of bone marrow of middle-aged mice (25–40 weeks old) is higher than that of young mice (6 weeks old) based on micro CT. ELISA results revealed that the concentration of cAMP in the bone marrow mononuclear cells of middle-aged mice is lower than that of young mice. These data suggest that increased [Ca²⁺]_o caused by bone resorption might accelerate adipocyte accumulation through CaSR following a decrease in cAMP.     

Investigating the function of Gdt1p in yeast Golgi glycosylation

The Golgi ion homeostasis is tightly regulated to ensure essential cellular processes such as glycosylation, yet our understanding of this regulation remains incomplete. Gdt1p is a member of the conserved Uncharacterized Protein Family (UPF0016). Our previous work suggested that Gdt1p may function in the Golgi by regulating Golgi Ca^2 +/Mn^2 + homeostasis. NMR structural analysis of the polymannan chains isolated from yeasts showed that the gdt1Δ mutant cultured in presence of high Ca^2 + concentration, as well as the pmr1Δ and gdt1Δ/pmr1Δ strains presented strong late Golgi glycosylation defects with a lack of α-1,2 mannoses substitution and α-1,3 mannoses termination. The addition of Mn^2 + confirmed the rescue of these defects. Interestingly, our structural data confirmed that the glycosylation defect in pmr1Δ could also completely be suppressed by the addition of Ca^2 +. The use of Pmr1p mutants either defective for Ca^2 + or Mn^2 + transport or both revealed that the suppression of the observed glycosylation defect in pmr1Δ strains by the intraluminal Golgi Ca^2 + requires the activity of Gdt1p. These data support the hypothesis that Gdt1p, in order to sustain the Golgi glycosylation process, imports Mn^2 + inside the Golgi lumen when Pmr1p exclusively transports Ca^2 +. Our results also reinforce the functional link between Gdt1p and Pmr1p as we highlighted that Gdt1p was a Mn^2 + sensitive protein whose abundance was directly dependent on the nature of the ion transported by Pmr1p. Finally, this study demonstrated that the aspartic residues of the two conserved motifs E-x-G-D-[KR], likely constituting the cation binding sites of Gdt1p, play a crucial role in Golgi glycosylation and hence in Mn^2 +/Ca^2 + transport.     

The alkaloid matrine of the root of Sophora flavescens prevents arrhythmogenic effect of ouabain

Matrine, a alkaloid of the root of Sophora flavescens, has multiple protective effects on the cardiovascular system including cardiac arrhythmias. However, the molecular and ionic mechanisms of matrine have not been well investigated. Our study aimed at to shed a light on the issue to investigate the antiarrhythmic effects of matrine by using ouabain to construct an arrhythmic model of cardiomyocytes. In this experiment, matrine significantly and dose-dependently increased the doses of ouabain required to induce cardiac arrhythmias and decreased the duration of arrhythmias in guinea pigs. In cardiomyocytes of guinea pigs, ouabain 10 μM prolonged action potential duration by 80% (p < 0.05) and increased L-type Ca²⁺ currents and Ca²⁺ transients induced by KCl (p < 0.05). Matrine 100 μM shortened the prolongation of APD and prevented the increase of L-type Ca²⁺ currents and Ca²⁺ transients induced by ouabain. Taken together, these findings provide the first evidence that matrine possessed arrhythmogenic effect of ouabain by inhibiting of L-type Ca²⁺ currents and Ca²⁺ overload in guinea pigs.     

Stress-induced dissociations between intracellular calcium signaling and insulin secretion in pancreatic islets

In healthy pancreatic islets, glucose-stimulated changes in intracellular calcium ([Ca²⁺]_i) provide a reasonable reflection of the patterns and relative amounts of insulin secretion. We report that [Ca²⁺]_i in islets under stress, however, dissociates with insulin release in different ways for different stressors. Islets were exposed for 48 h to a variety of stressors: cytokines (low-grade inflammation), 28 mM glucose (28G, glucotoxicity), free fatty acids (FFAs, lipotoxicity), thapsigargin (ER stress), or rotenone (mitochondrial stress). We then measured [Ca²⁺]_i and insulin release in parallel studies. Islets exposed to all stressors except rotenone displayed significantly elevated [Ca²⁺]_i in low glucose, however, increased insulin secretion was only observed for 28G due to increased nifedipine-sensitive calcium-channel flux. Following 3–11 mM glucose stimulation, all stressors substantially reduced the peak glucose-stimulated [Ca²⁺]_i response (first phase). Thapsigargin and cytokines also substantially impacted aspects of calcium influx and ER calcium handling. Stressors did not significantly impact insulin secretion in 11 mM glucose for any stressor, although FFAs showed a borderline reduction, which contributed to a significant decrease in the stimulation index (11:3 mM glucose) observed for FFAs and also for 28G. We also clamped [Ca²⁺]_i using 30 mM KCl + 250 μM diazoxide to test the amplifying pathway. Only rotenone-treated islets showed a robust increase in 3–11 mM glucose-stimulated insulin secretion under clamped conditions, suggesting that low-level mitochondrial stress might activate the metabolic amplifying pathway. We conclude that different stressors dissociate [Ca²⁺]_i from insulin secretion differently: ER stressors (thapsigargin, cytokines) primarily affect [Ca²⁺]_i but not conventional insulin secretion and ‘metabolic’ stressors (FFAs, 28G, rotenone) impacted insulin secretion.     

Rho-kinase inhibition reverses impaired Ca2+ handling and associated left ventricular dysfunction in pressure overload-induced cardiac hypertrophy

Recent studies have implicated a relationship between RhoA/ROCK activity and defective Ca²⁺ homeostasis in hypertrophic hearts. This study investigated molecular mechanism underlying ROCK inhibition-mediated cardioprotection against pressure overload-induced cardiac hypertrophy, with a focus on Ca²⁺ homeostasis.Cardiac hypertrophy model was established by performing transverse aortic constriction (TAC) in 8-week-old male rats. Groups were assigned as SHAM, TAC and TAC + Fas (rats undergoing TAC and treated with fasudil). Rats in the TAC + Fas group were administered fasudil (5 mg/kg/day), and rats in the SHAM and TAC groups were treated with vehicle for 10 weeks. Electrophysiological recordings were obtained from isolated left ventricular myocytes and expression levels of proteins were determined using western blotting. Rats in the TAC group showed remarkable cardiac hypertrophy, and fasudil treatment significantly reversed this alteration. TAC + Fas myocytes showed significant improvement in reduced contractility and Ca²⁺ transients. Moreover, these myocytes showed restoration of slow relaxation rate and Ca²⁺ reuptake. Although L-type Ca²⁺ currents did not change in TAC group, there was a significant reduction in the triggered Ca²⁺ transients which was reversed either by long-term fasudil treatment or incubation of TAC myocytes with fasudil. The hearts of rats in the TAC group showed a significant decrease in ROCK1, ROCK2, RyR2 protein levels and p-PLB^S16/T17/SERCA2 ratio and increase in RhoA expression and MLC phosphorylation. However, fasudil treatment largely reversed TAC-induced alterations in protein expression.Thus, our findings indicate that upregulation of the RhoA/ROCK pathway is significantly associated with cardiac hypertrophy-related Ca²⁺ dysregulation and suggest that ROCK inhibition prevents hypertrophic heart failure.     

Calcium-mediated pore expansion and cell death following nanoelectroporation

Opening of long-lived pores in the cell membrane is the principal primary effect of intense, nanosecond pulsed electric field (nsPEF). Here we demonstrate that the evolution of pores, cell survival, the time and the mode of cell death (necrotic or apoptotic) are determined by the level of external Ca^2 + after nsPEF. We also introduce a novel, minimally disruptive technique for nsEP exposure of adherent cells on indium tin oxide (ITO)-coated glass coverslips, which does not require cell detachment and enables fast exchanges of bath media. Increasing the Ca^2 + level from the nominal 2–5 μM to 2 mM for the first 60–90 min after permeabilization by 300-nsPEF increased the early (necrotic) death in U937, CHO, and BPAE cells. With nominal Ca^2 +, the inhibition of osmotic swelling rescued cells from the early necrosis and increased caspase 3/7 activation later on. However, the inhibition of swelling had a modest or no protective effect with 2 mM Ca^2 + in the medium. With the nominal Ca^2 +, most cells displayed gradual increase in YO-PRO-1 and propidium (Pr) uptake. With 2 mM Ca^2 +, the initially lower Pr uptake was eventually replaced by a massive and abrupt Pr entry (necrotic death). It was accompanied by a transient acceleration of the growth of membrane blebs due to the increase of the intracellular osmotic pressure. We conclude that the high-Ca^2 +-dependent necrotic death in nsPEF-treated cells is effected by a delayed, sudden, and osmotically-independent pore expansion (or de novo formation of larger pores), but not by the membrane rupture.     

Increase of intracellular Ca2+ by adenine and uracil nucleotides in human midbrain-derived neuronal progenitor cells

Nucleotides play an important role in brain development and may exert their action via ligand-gated cationic channels or G protein-coupled receptors. Patch-clamp measurements indicated that in contrast to AMPA, ATP did not induce membrane currents in human midbrain derived neuronal progenitor cells (hmNPCs). Various nucleotide agonists concentration-dependently increased [Ca²⁺]_i as measured by the Fura-2 method, with the rank order of potency ATP > ADP > UTP > UDP. A Ca²⁺-free external medium moderately decreased, whereas a depletion of the intracellular Ca²⁺ storage sites by cyclopiazonic acid markedly depressed the [Ca²⁺]_i transients induced by either ATP or UTP. Further, the P2Y₁ receptor antagonistic PPADS and MRS 2179, as well as the nucleotide catalyzing enzyme apyrase, allmost abolished the effects of these two nucleotides. However, the P2Y_1,2,12 antagonistic suramin only slightly blocked the action of ATP, but strongly inhibited that of UTP. In agreement with this finding, UTP evoked the release of ATP from hmNPCs in a suramin-, but not PPADS-sensitive manner. Immunocytochemistry indicated the co-localization of P2Y_1,2,4-immunoreactivities (IR) with nestin-IR at these cells. In conclusion, UTP may induce the release of ATP from hmNPCs via P2Y₂ receptor-activation and thereby causes [Ca²⁺]_i transients by stimulating a P2Y₁-like receptor.     

Magnolol inhibits colonic motility through down-regulation of voltage-sensitive L-type Ca2+ channels of colonic smooth muscle cells in rats

This study aimed to investigate the effect of magnolol (5,5′-diallyl-2,2′-biphenyldiol) on contraction in distal colonic segments of rats and the underlying mechanisms. Colonic segments were mounted in organ baths for isometric force measurement. Whole-cell voltage-sensitive L-type Ca²⁺ currents were recorded on isolated single colonic smooth muscle cells using patch-clamp technique. The spontaneous contractions and acetylcholine (ACh)- and Bay K 8644-induced contractions were inhibited by magnolol (3–100 μM). In the presence of Bay K8644 (100 nM), magnolol (10–100 μM) inhibited the contraction induced by 10 μM ACh. By contrast, tetrodotoxin (100 nM) and N_ώ-nitro-l-arginine methyl ester (l-NAME 100 μM) did not change the inhibitory effect of magnolol (10 μM). In addition, magnolol (3–100 μM) inhibited the L-type Ca²⁺ currents. The present results suggest that magnolol inhibits colonic smooth muscle contraction through downregulating L-type Ca²⁺ channel activity.     

Involvement of plasma membrane Ca2+ channels,IP3 receptors,and ryanodine receptors in the generation of spontaneous rhythmic contractions of the cricket lateral oviduct

In the present study, the isolated cricket (Gryllus bimaculatus) lateral oviduct exhibited spontaneous rhythmic contractions (SRCs) with a frequency of 0.29 ± 0.009 Hz (n = 43) and an amplitude of 14.6 ± 1.25 mg (n = 29). SRCs completely disappeared following removal of extracellular Ca²⁺ using a solution containing 5 mM EGTA. Application of the non-specific Ca²⁺ channel blockers Co²⁺, Ni²⁺, and Cd²⁺ also decreased both the frequency and amplitude of SRCs in dose-dependent manners, suggesting that Ca²⁺ entry through plasma membrane Ca²⁺ channels is essential for the generation of SRCs. Application of ryanodine (30 μM), which depletes intracellular Ca²⁺ by locking ryanodine receptor (RyR)-Ca²⁺ channels in an open state, gradually reduced the frequency and amplitude of SRCs. A RyR antagonist, tetracaine, reduced both the frequency and amplitude of SRCs, whereas a RyR activator, caffeine, increased the frequency of SRCs with a subsequent increase in basal tonus, indicating that RyRs are essential for generating SRCs. To further investigate the involvement of phospholipase C (PLC) and inositol 1,4,5-trisphosphate receptors (IP₃Rs) in SRCs, we examined the effect of a PLC inhibitor, U73122, and an IP₃R antagonist, 2-aminoethoxydiphenyl borate (2-APB), on SRCs. Separately, U73122 (10 μM) and 2-APB (30–50 μM) both significantly reduced the amplitude of SRCs with little effect on their frequency, further indicating that the PLC/IP₃R signaling pathway is fundamental to the modulation of the amplitude of SRCs. A hypotonic-induced increase in the frequency and amplitude of SRCs and a hypertonic-induced decrease in the frequency and amplitude of SRCs indicated that mechanical stretch of the lateral oviduct is involved in the generation of SRCs. The sarcoplasmic reticulum Ca²⁺-pump ATPase inhibitors thapsigargin and cyclopiazonic acid impaired or suppressed the relaxation phase of SRCs. Taken together, the present results indicate that Ca²⁺ influx through plasma membrane Ca²⁺ channels and Ca²⁺ release from RyRs play an essential role in pacing SRCs and that Ca²⁺ release from IP₃Rs may play a role in modulating the amplitude of SRCs, probably via activation of PLC.     

Opposite regulation of KCNQ5 and TRPC6 channels contributes to vasopressin-stimulated calcium spiking responses in A7r5 vascular smooth muscle cells

Physiologically relevant concentrations of [Arg⁸]-vasopressin (AVP) induce repetitive action potential firing and Ca²⁺ spiking responses in the A7r5 rat aortic smooth muscle cell line. These responses may be triggered by suppression of KCNQ potassium currents and/or activation of non-selective cation currents. Here we examine the relative contributions of KCNQ5 channels and TRPC6 non-selective cation channels to AVP-stimulated Ca²⁺ spiking using patch clamp electrophysiology and fura-2 fluorescence measurements in A7r5 cells. KCNQ5 or TRPC6 channel expression levels were suppressed by short hairpin RNA constructs. KCNQ5 knockdown resulted in more positive resting membrane potentials and induced spontaneous action potential firing and Ca²⁺ spiking. However physiological concentrations of AVP induced additional depolarization and increased Ca²⁺ spike frequency in KCNQ5 knockdown cells. AVP activated a non-selective cation current that was reduced by TRPC shRNA treatment or removal of external Na⁺. Neither resting membrane potential nor the AVP-induced depolarization was altered by knockdown of TRPC6 channel expression. However, both TRPC6 shRNA and removal of external Na⁺ delayed the onset of Ca²⁺ spiking induced by 25 pM AVP. These results suggest that suppression of KCNQ5 currents alone is sufficient to excite A7r5 cells, but AVP-induced activation of TRPC6 contributes to the stimulation of Ca²⁺ spiking.     

Endothelium-independent vasorelaxant effect of sodium ferulate on rat thoracic aorta

AimsThis study was designed to investigate the effects of sodium ferulate (SF) on rat isolated thoracic aortas and the possible mechanisms.Main methodsIsometric tension was recorded in response to drugs in organ bath. Cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) was measured using Fluo-3 in cultured rat aortic smooth muscle cells (RASMC).Key findingsSF (0.1–30 mM) relaxed the isolated aortic rings precontracted with phenylephrine (PE) and high-K⁺ in a concentration-dependent manner with respective pD₂ of 2.7 ± 0.02 and 2.6 ± 0.06. Mechanical removal of endothelium did not significantly modify the SF-induced relaxation. In Ca²⁺-free solution, SF noticeably inhibited extracellular Ca²⁺-induced contraction in high-K⁺ and PE pre-challenged rings, and suppressed the transient contraction induced by PE and caffeine. The vasorelaxant effect of SF was unaffected by various K⁺ channel blockers such as tetraethylammonium, glibenclamide, 4-aminopyridine, and barium chloride. In addition, SF concentration-dependently reduced the contraction induced by phorbol-12-myristate-13-acetate, an activator of protein kinase C (PKC), in the absence of extracellular Ca²⁺, with the pD₂ of 2.9 ± 0.03. In RASMC, SF had no effect on PE- or KCl-induced [Ca²⁺]_i increase either in the presence or in the absence of external Ca²⁺.SignificanceThese results indicate that SF acts directly as a non-selective relaxant to vascular smooth muscle. The direct inhibition of the common pathway after [Ca²⁺]_i increase may account for the SF-induced relaxation in Ca²⁺-dependent contraction, while the blockage of the PKC-mediated contractile mechanism is likely responsible for the SF-induced relaxation in Ca²⁺-independent contraction.     

Micromechanical measurements on P-protein aggregates (forisomes) from Vicia faba plants

Forisomes are chemomechanically active P-protein aggregates found in the phloem of legumes. They can convert chemical energy into mechanical work when induced by divalent metal ions or changes in pH, which control the folding state of individual forisome proteins. We investigated the changing geometric parameters of individual forisomes and the strength and dynamics of the forces generated during this process. Three different divalent ions were tested (Ca²⁺, Sr²⁺ and Ba²⁺) and were shown to induce similar changes to the normalized length and diameter. In the concentration range from 0.1 to 4 M, K⁺ and Cl^? ions had no influence on the contraction behaviour of the forisomes induced by 10 mM Ca²⁺. In the absence of dissolved oxygen, these changes were independent of the radius of the metal ion, water uptake and the strength of binding between the selected metal ions and those protein molecules responsible for forisome conformational transformation. In the absence of any load, bound Ca²⁺, Sr²⁺ and Ba²⁺ ions showed apparent and averaged dissociation constants of 14, 62 and 1070 µM, respectively. Various forisomes generated bending on a quartz glass fibre with a diameter of 9 µm. The fibre bending was measured microscopically also by correlation between the digital patterns of a predefined window of observation before and after bending. Similar bending forces of approximately 90 nN were measured for a single forisome sequentially exposed to 10 mM Ca²⁺, Sr²⁺ and Ba²⁺. In the absence of dissolved oxygen, the same conditions resulted in averaged bending forces of (93 ± 40) nN, (58 ± 20) nN, and (91 ± 20) nN after contacting different forisomes with 10 mM Ca²⁺, 10 mM Sr²⁺, and 10 mM Ba²⁺ respectively, demonstrating that the force generated was independent on ion concentrations above a certain threshold value.     

KMUP-1 ameliorates monocrotaline-induced pulmonary arterial hypertension through the modulation of Ca2+ sensitization and K+-channel

AimsThis study investigates the actions of KMUP-1 on RhoA/Rho-kinase (ROCK)-dependent Ca²⁺ sensitization and the K⁺-channel in chronic pulmonary arterial hypertension (PAH) rats.Main methodsSprague–Dawley rats were divided into control, monocrotaline (MCT), and MCT + KMUP-1 groups. PAH was induced by a single intraperitoneal injection (i.p.) of MCT (60 mg/kg). KMUP-1 (5 mg/kg, i.p.) was administered once daily for 21 days to prevent MCT-induced PAH. All rats were sacrificed on day 22.Key findingsMCT-induced increased right ventricular systolic pressure (RVSP) and right ventricular hypertrophy were prevented by KMUP-1. In myograph experiments, KCl (80 mM), phenylephrine (10 µM) and K⁺ channel inhibitors (TEA, 10 mM; paxilline, 10 µM; 4-AP, 5 mM) induced weak PA contractions in MCT-treated rats compared to controls, but the PA reactivity was restored in MCT + KMUP-1-treated rats. By contrast, in β-escin- or α-toxin-permeabilized PAs, CaCl₂-induced (1.25 mM, pCa 5.1) contractions were stronger in MCT-treated rats, and this action was suppressed in MCT + KMUP-1-treated rats. PA relaxation in response to the ROCK inhibitor Y27632 (0.1 μM) was much higher in MCT-treated rats than in control rats. In Western blot analysis, the expression of Ca²⁺-activated K⁺ (BK_Ca) and voltage-gated K⁺ channels (Kv2.1 and Kv1.5), and ROCK II proteins was elevated in MCT-treated rats and suppressed in MCT + KMUP-1-treated rats. We suggest that MCT-treated rats upregulate K⁺-channel proteins to adapt to chronic PAH.SignificanceKMUP-1 protects against PAH and restores PA vessel tone in MCT-treated rats, attributed to alteration of Ca²⁺ sensitivity and K⁺-channel function.     

Semi-automated program for analysis of local Ca2+ spark release with application for classification of heart cell type

Local Ca²⁺ spark releases are essential to the Ca²⁺ cycling process. Thus, they play an important role in ventricular and atrial cell contraction, as well as in sinoatrial cell automaticity. Characterizing their properties in healthy cells from different regions in the heart can reveal the basic biophysical differences among these regions. We designed a semi-automatic Matlab Graphical User Interface (called Sparkalyzer) to characterize parameters of Ca²⁺ spark release from any major cardiac tissue, as recorded in line-scan mode with a confocal laser-scanning microscope. We validated the algorithm on experimental images from rabbit sinoatrial, atrial, and ventricular cells loaded with Fluo-4 AM. The program characterizes general image parameters of Ca²⁺ transients and sparks: spark duration, which indicates for how long the spark provides Ca²⁺ to the closed intracellular mechanisms (typical value: 25 ± 1, 23 ± 1, 26 ± 1 ms for sinoatrial, atrial, and ventricular cells, respectively); spark amplitude, which indicates the amount of Ca²⁺ released by a single spark (1.6 ± 0.1, 1.6 ± 0.2, 1.4 ± 0.1 F/F₀ for sinoatrial, atrial, and ventricular cells, respectively); spark length, which is the length of the Ca²⁺ wavelets fired out of a row of ryanodine receptors (5 ± 0.1, 5 ± 0.2, 3.4 ± 0.3 μm for sinoatrial, atrial, or ventricular cells, respectively) and number of sparks (0.14 ± 0.02, 0.025 ± 0.01, 0.02 ± 0.01 for 1 μm in 1 s for sinoatrial, atrial, and ventricular cells, respectively). This method is reliable for Ca²⁺ spark analysis of sinoatrial, atrial, or ventricular cells. Moreover, by examining the average value of Ca²⁺ spark characteristics and their scattering around the mean, atrial, ventricular and sinoatrial cells can be differentiated.     

Multiple effects of anthracene-9-carboxylic acid on the TMEM16B/anoctamin2 calcium-activated chloride channel

Ca^2 +-activated Cl⁻ currents (CaCCs) play important roles in many physiological processes. Recent studies have shown that TMEM16A/anoctamin1 and TMEM16B/anoctamin2 constitute CaCCs in several cell types. Here we have investigated for the first time the extracellular effects of the Cl⁻ channel blocker anthracene-9-carboxylic acid (A9C) and of its non-charged analogue anthracene-9-methanol (A9M) on TMEM16B expressed in HEK 293T cells, using the whole-cell patch-clamp technique. A9C caused a voltage-dependent block of outward currents and inhibited a larger fraction of the current as depolarization increased, whereas the non-charged A9M produced a small, not voltage dependent block of outward currents. A similar voltage-dependent block by A9C was measured both when TMEM16B was activated by 1.5 and 13 μM Ca^2 +. However, in the presence of 1.5 μM Ca^2 + (but not in 13 μM Ca^2 +), A9C also induced a strong potentiation of tail currents measured at − 100 mV after depolarizing voltages, as well as a prolongation of the deactivation kinetics. On the contrary, A9M did not produce potentiation of tail currents, showing that the negative charge is required for potentiation. Our results provide the first evidence that A9C has multiple effects on TMEM16B and that the negative charge of A9C is necessary both for voltage-dependent block and for potentiation. Future studies are required to identify the molecular mechanisms underlying these complex effects of A9C on TMEM16B. Understanding these mechanisms will contribute to the elucidation of the structure and functional properties of TMEM16B channels.     

F281, synthetic agonist of the sigma-2 receptor,induces Ca2+ efflux from the endoplasmic reticulum and mitochondria in SK-N-SH cells

We demonstrate that F281, a synthetic agonist of the sigma-2 receptor (s2R), induces a non transient increase in intracellular [Ca²⁺] ([Ca²⁺]_i) and cell death in SK-N-SH cells. Sigma receptors are classified into two subtypes, with different molecular weight and tissue distribution. While the sigma-1 receptor has been cloned, the s2r is less characterized and its physiological ligand and role need further investigation. In tumour cell lines, synthetic agonists of the s2R trigger apoptosis and modulate [Ca²⁺]_i. In particular, CB-64D induces a Ca²⁺ response while PB28 supresses Ca²⁺ signalling. We have recently synthesized F281, by replacing the 5-methoxytetraline moiety of PB28 with a carbazole nucleus. Although this bioisosteric substitution should not affect the ligand affinity at the receptor, F281 (after 24 h incubation) was more cytotoxic than PB28 (EC₅₀ values 65.4 nM and 8.13 μM, respectively) in SK-N-SH cells. We used the fluorescent probes fura-2, rhod-2 and JC-1. F281 mobilizes Ca²⁺ from mitochondria and from the endoplasmic reticulum, by opening its inositol 1,4,5-trisphosphate receptor; Ca²⁺-entry through the channels activated by store depletion was also observed. After the increase in [Ca²⁺]_i and within 10 min, we observed a sudden drop in metabolic activity and intracellular [ATP] leading to cell death.