The H2O2-Generating Enzyme,Xanthine Oxidase,Decreases Luminal Ca2+ Content of the IP3-Sensitive Ca2+ Store in Vascular Endothelial Cells

Objective: Xanthine oxidase inhibits agonist-stimulated Ca²⁺ signaling in calf pulmonary artery endothelial cells by an H₂O₂-dependent mechanism. We investigated the effect of xanthine oxidase on luminal Ca²⁺ content of the inositol-1,4,5-trisphosphate (IP₃)-sensitive Ca²⁺ store. Methods: Luminal Ca²⁺ content was estimated from the net release of Ca²⁺ activated by 2,5-di-t-butylhydroquinone (BHQ), an inhibitor of microsomal Ca²⁺ pumps. Results: Initially, xanthine oxidase depleted the IP₃-sensitive Ca²⁺ store of releasable Ca²⁺, but with more prolonged incubation, the enzyme also depleted non-IP₃-sensitive stores. In addition, xanthine oxidase inhibited capacitative Ca²⁺ influx. Similar results were observed when thapsigargin was substituted for BHQ. Conclusions: Depletion of luminal Ca²⁺ content within the IP₃-sensitive Ca²⁺ store contributes to xanthine oxidase inhibition of Ca²⁺ signaling in vascular endothelial cells.     

L-type Ca channel facilitation mediated by H2O2-induced activation of CaMKII in rat ventricular myocytes

The Ca²⁺-dependent facilitation (CDF) of L-type Ca²⁺ channels, a major mechanism for force-frequency relationship of cardiac contraction, is mediated by Ca²⁺/CaM-dependent kinase II (CaMKII). Recently, CaMKII was shown to be activated by methionine oxidation. We investigated whether oxidation-dependent CaMKII activation is involved in the regulation of L-type Ca²⁺ currents (I_Ca,L) by H₂O₂ and whether Ca²⁺ is required in this process. Using patch clamp, I_Ca,L was measured in rat ventricular myocytes. H₂O₂ induced an increase in I_Ca,L amplitude and slowed inactivation of I_Ca,L. This oxidation-dependent facilitation (ODF) of I_Ca,L was abolished by a CaMKII blocker KN-93, but not by its inactive analog KN-92, indicating that CaMKII is involved in ODF. ODF was not affected by replacement of external Ca²⁺ with Ba²⁺ or presence of EGTA in the internal solutions. However, ODF was abolished by adding BAPTA to the internal solution or by depleting sarcoplasmic reticulum (SR) Ca²⁺ stores using caffeine and thapsigargin. Alkaline phosphatase, β-iminoadenosine 5′-triphosphate (AMP-PNP), an autophosphorylation inhibitor autocamtide-2-related inhibitory peptide (AIP), or a catalytic domain blocker (CaM-KIINtide) did not affect ODF. In conclusion, oxidation-dependent facilitation of L-type Ca²⁺ channels is mediated by oxidation-dependent CaMKII activation, in which local Ca²⁺ increases induced by SR Ca²⁺ release is required.     

Intracellular Ca- and PKC-dependent upregulation of T-type Ca channels in LPC-stimulated cardiomyocytes

Lysophosphatidylcholine (LPC) accumulation in intracellular and/or interstitial space in cardiomyocytes may underlie as a mechanism for tachycardia and various arrhythmias during cardiac ischemia, which is usually accompanied by elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i). The present study was therefore designed to investigate possible mechanisms responsible for [Ca²⁺]_i elevation by LPC focusing on T-type Ca²⁺ channel current (I_Ca.T). LPC as well as phorbol 12-myristate 13-acetate (PMA) significantly accelerated the beating rates of neonatal rat cardiomyocytes. Augmentation of I_Ca.T by LPC was dependent on the intracellular Ca²⁺ concentration: an increase of I_Ca.T was significantly larger in high [Ca²⁺]_i condition (pCa = 7) than those in low [Ca²⁺]_i condition (pCa = 11). In heterologous expression system by use of human cardiac Ca_V3.1 and Ca_V3.2 channels expressed in HEK293 cells, LPC augmented Ca_V3.2 channel current (I_Cav3.2) in a concentration-dependent manner but not Ca_V3.1 channel current (I_Cav3.1). Augmentation of I_Cav3.2 by LPC was highly [Ca²⁺]_i dependent: I_Cav3.2 was unchanged when pCa was 11 but was markedly increased when [Ca²⁺]_i was higher than 10⁻¹⁰ M (pCa ≤ 10) by LPC application (10-50 μM). A specific inhibitor of protein kinase Cα (Ro-32-0432) attenuated the increase of I_Cav3.2 by LPC. LPC stimulates I_Ca.T in a [Ca²⁺]_i-dependent manner via PKCα activation, which may play a role in triggering arrhythmias in pathophysiological conditions of the heart.     

Changes of intra-mitochondrial Ca in adult ventricular cardiomyocytes examined using a novel fluorescent Ca indicator targeted to mitochondria

In this study a Ca²⁺ sensitive protein was targeted to the mitochondria of adult rabbit ventricular cardiomyocytes using an adenovirus transfection technique. The probe (Mitycam) was a Ca²⁺-sensitive inverse pericam fused to subunit VIII of human cytochrome c oxidase. Mitycam expression pattern and Ca²⁺ sensitivity was characterized in HeLa cells and isolated adult rabbit cardiomyocytes. Cardiomyocytes expressing Mitycam were voltage-clamped and depolarized at regular intervals to elicit a Ca²⁺ transient. Cytoplasmic (Fura-2) and mitochondrial Ca²⁺ (Mitycam) fluorescence were measured simultaneously under a range of cellular Ca²⁺ loads. After 48 h post-adenoviral transfection, Mitycam expression showed a characteristic localization pattern in HeLa cells and cardiomyocytes. The Ca²⁺ sensitive component of Mitycam fluorescence was 12% of total fluorescence in HeLa cells with a K_d of  220 nM. In cardiomyocytes, basal and beat-to-beat changes in Mitycam fluorescence were detected on initiation of a train of depolarizations. Time to peak of the mitochondrial Ca²⁺ transient was slower, but the rate of decay was faster than the cytoplasmic signal. During spontaneous Ca²⁺ release the relative amplitude and the time course of the mitochondrial and cytoplasmic signals were comparable. Inhibition of mitochondrial respiration decreased the mitochondrial transient amplitude by  65% and increased the time to 50% decay, whilst cytosolic Ca²⁺ transients were unchanged. The mitochondrial Ca²⁺ uniporter (mCU) inhibitor Ru360 prevented both the basal and transient components of the rise in mitochondrial Ca²⁺. The mitochondrial-targeted Ca²⁺ probe indicates sustained and transient phases of mitochondrial Ca²⁺ signal, which are dependent on cytoplasmic Ca²⁺ levels and require a functional mCU.     

The store-operated Ca2+ entry complex comprises a small cluster of STIM1 associated with one Orai1 channel

Increases in cytosolic Ca²⁺ concentration regulate diverse cellular activities and are usually evoked by opening of Ca²⁺ channels in intracellular Ca²⁺ stores and the plasma membrane (PM). For the many signals that evoke formation of inositol 1,4,5-trisphosphate (IP₃), IP₃ receptors coordinate the contributions of these two Ca²⁺ sources by mediating Ca²⁺ release from the endoplasmic reticulum (ER). Loss of Ca²⁺ from the ER then activates store-operated Ca²⁺ entry (SOCE) by causing dimers of STIM1 to cluster and unfurl cytosolic domains that interact with the PM Ca²⁺ channel, Orai1, causing its pore to open. The relative concentrations of STIM1 and Orai1 are important, but most analyses of their interactions use overexpressed proteins that perturb the stoichiometry. We tagged endogenous STIM1 with EGFP using CRISPR/Cas9. SOCE evoked by loss of ER Ca²⁺ was unaffected by the tag. Step-photobleaching analysis of cells with empty Ca²⁺ stores revealed an average of 14.5 STIM1 molecules within each sub-PM punctum. The fluorescence intensity distributions of immunostained Orai1 puncta were minimally affected by store depletion, and similar for Orai1 colocalized with STIM1 puncta or remote from them. We conclude that each native SOCE complex is likely to include only a few STIM1 dimers associated with a single Orai1 channel. Our results, demonstrating that STIM1 does not assemble clusters of interacting Orai channels, suggest mechanisms for digital regulation of SOCE by local depletion of the ER.

In generating the cytosolic Ca²⁺ signals that regulate cellular activities, cells call upon two sources of Ca²⁺: the extracellular space, accessed through Ca²⁺ channels in the plasma membrane (PM), and Ca²⁺ sequestered within intracellular stores, primarily within the endoplasmic reticulum (ER). In animal cells, the many receptors that stimulate formation of inositol 1,4,5-trisphosphate (IP₃) provide coordinated access to both Ca²⁺ sources (1). IP₃ stimulates the opening of IP₃ receptors (IP₃R), which are large Ca²⁺-permeable channels expressed mostly within ER membranes. IP₃ thereby triggers Ca²⁺ release from the ER (2, 3). The link to extracellular Ca²⁺ is provided by store-operated Ca²⁺ entry (SOCE), which is activated by loss of Ca²⁺ from the ER. The reduction in ER free-Ca²⁺ concentration causes Ca²⁺ to dissociate from the luminal Ca²⁺-binding sites of stromal interaction molecule 1 (STIM1), a dimeric protein embedded in ER membranes. This loss of Ca²⁺ causes STIM1 to unfurl cytosolic domains that interact with the PM Ca²⁺ channel, Orai1, causing its pore to open and Ca²⁺ to flow into the cell through the SOCE pathway (Fig. 1A) (4, 5). Available evidence suggests that STIM1 must bind to the C-terminal tail of each of the six subunits of an Orai1 channel for optimal activity, with lesser occupancies reducing activity and modifying channel properties (6–10). The interactions between STIM1 and Orai1 occur at membrane contact sites (MCS), where the two membranes are organized to provide a gap of about 10–30 nm, across which the two proteins directly interact (11–13). Orai channels are unusual in having no structural semblance to other ion channels and in having their opening controlled by direct interactions between proteins in different membranes (Fig. 1A). Competing models suggest that dimeric STIM1 binds either to a pair of C-terminal tails within a single channel (6 STIM1 molecules per hexameric Orai1 channel) (Fig. 1 B, a), or that each dimer interacts with only a single C-terminal tail leaving the remaining STIM1 subunit free to cross-link with a different Orai1 channel (12 STIM1 molecules around a single Orai1 channel) (Fig. 1 B, b) (see references in ref. 14). The latter arrangement has been proposed to allow assembly of close-packed Orai1 clusters (Fig. 1 B, c) and to explain the variable stoichiometry of Orai1 to STIM1 at MCS (14).Open in a separate windowFig. 1.SOCE is unaffected by tagging of endogenous STIM1. (A) SOCE is activated when loss of Ca²⁺ from the ER, usually mediated by IP₃Rs, causes Ca²⁺ to dissociate from the EF hands of dimeric STIM1. This causes STIM1 to unfurl its cytosolic domain, unmasking the C-terminal polybasic tail (PBT) and CRAC (Ca²⁺-release-activated channel)-activation domain (CAD) Association of the PBT with PM phosphoinositides causes STIM1 to accumulate at MCS, where the CAD captures the C-terminal tail of Orai1. Binding of STIM1 to each of the six subunits of Orai1 opens the Ca²⁺ channel, allowing SOCE to occur (9). (B) Orai1 is a hexamer, comprising three pairs of dimers (33). Dimeric STIM1 may activate Orai1 by binding as three dimers (B, a), or as six dimers (B, b) with the residual STIM1 subunit free to interact with another Orai1 channel (B, c) (14). (C) Structure of the edited STIM1-EGFP. (D) TIRF images of STIM1-EGFP HeLa cells treated with STIM1 or nonsilencing (NS) shRNA before emptying of Ca²⁺ stores. (Scale bar, 10 µm.) (E) Summary results (individual values, mean ± SD, n = 3 independent experiments, each with ∼30 cells analyzed) show whole-cell fluorescence intensities from TIRF images of STIM1-EGFP HeLa cells treated with the indicated shRNA. Results from WT cells are also shown (n = 4). ****P < 0.0001, ANOVA with Bonferroni test, relative to WT cells. (F) In-gel fluorescence of lysates from WT or STIM1-EGFP HeLa cells (protein loadings in μg). The STIM1-EGFP band (arrow) and molecular mass markers (kDa) are shown. Similar results were obtained in four independent analyses. (G) WB for STIM1 and β-actin for WT and STIM1-EGFP HeLa cells. Protein loadings (μg) and molecular mass markers (kDa) are shown. Arrows show positions of native and EGFP-tagged STIM1. (H) Summary results (individual values, mean ± SD, n = 9) show expression of STIM1-EGFP relative to all STIM1 in STIM1-EGFP HeLa cells (red), and total STIM1 expression in WT and edited cells (black). (I) Effects of histamine in Ca²⁺-free HBS on the peak increase in [Ca²⁺]_c (Δ[Ca²⁺]_c) in populations of WT and STIM1-EGFP HeLa cells. Mean ± SEM from four experiments, each with six determinations. (J) Effects of CPA in Ca²⁺-free HBS on the peak increase in [Ca²⁺]_c (Δ[Ca²⁺]_c) in populations of WT and STIM1-EGFP HeLa cells. Mean ± SEM from four experiments, each with six determinations. (K) Populations of cells were treated (5 min) with CPA in Ca²⁺-free HBS to evoke graded depletion of ER Ca²⁺ stores before addition of extracellular Ca²⁺ (final free [Ca²⁺] ∼10 mM). Results (mean ± SEM, n = 6, each with six determinations) show the amplitude of the SOCE in WT and STIM1-EGFP HeLa cells. See also SI Appendix, Figs. S1 and S2.Opening of most ion channels is regulated by changes in membrane potential or by binding of soluble stimuli, where the relationship between stimulus intensity and response is readily amenable to experimental analysis. The unusual behavior of SOCE, where direct interactions between proteins embedded in different membranes control channel opening (Fig. 1A), makes it more difficult to define stimulus–response relationships and highlights the need to understand the amounts of STIM1 and Orai1 within the MCS where the interactions occur. When STIM1 or Orai1 are overexpressed their behaviors are perturbed, yet most analyses of their interactions have involved overexpression of the proteins. These difficulties motivated the present study, which was designed to determine the number of native STIM1 molecules associated with each SOCE signaling complex.     

Characterization of ionic currents and electrophysiological properties of goldfish somatotropes in primary culture

Growth hormone release in goldfish is partly dependent on voltage-sensitive Ca²⁺ channels but somatotrope electrophysiological events affecting such channel activities have not been elucidated in this system. The electrophysiological properties of goldfish somatotropes in primary culture were studied using the whole-cell and amphotericin B-perforated patch-clamp techniques. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) of identified somatotropes was measured using Fura-2/AM dye. Goldfish somatotropes had an average resting membrane potential of −78.4 ± 4.6 mV and membrane input resistance of 6.2 ± 0.2 GΩ. Voltage steps from a holding potential of −90 mV elicited a non-inactivating outward current and transient inward currents at potentials more positive than 0 and −30 mV, respectively. Isolated current recordings indicate the presence of 4-aminopyridine- and tetraethylammonium (TEA)-sensitive K⁺, tetrodotoxin (TTX)-sensitive Na⁺, and nifedipine (L-type)- and ω-conotoxin GVIA (N-type)-sensitive Ca²⁺ channels. Goldfish somatotropes rarely fire action potentials (APs) spontaneously, but single APs can be induced at the start of a depolarizing current step; this single AP was abolished by TTX and significantly reduced by nifedipine and ω-conotoxin GVIA. TEA increased AP duration and triggered repetitive AP firing resulting in an increase in [Ca²⁺]_i, whereas TTX, nifedipine and ω-conotoxin GVIA inhibited TEA-induced [Ca²⁺]_i pulses. These results indicate that in goldfish somatotropes, TEA-sensitive K⁺ channels regulate excitability while TTX-sensitive Na⁺ channels together with N- and L-type Ca channels mediates the depolarization phase of APs. Opening of voltage-sensitive Ca²⁺ channels during AP firing leads to increases in [Ca²⁺]_i.     

Hyperpolarization induced by K+ channel openers inhibits Ca2+ influx and Ca2+ release in coronary artery

The vasodilating mechanisms of the K⁺ channel openers—cromakalim, pinacidil, nicorandil, KRN2391, and Ki4032—were examined by measurement of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) using the fura-2 method in canine or porcine coronary arterial smooth muscle. The five K⁺ channel openers all produced a reduction of [Ca²⁺]_i in 5 and 30 mM KCl physiological salt solution (PSS), the effects of which were antagonized by tetrabutylammonium (TBA) or glibenclamide, but failed to affect [Ca²⁺]_i in 45 and 90 mM MCl-PSS. Cromakalim and Ki4032 only partially inhibited the 30 mM KCl-induced contractures, whereas pinacidil, nicorandil, and KRN2391 nearly abolished contractions produced by high KCl-PSS. The increased [Ca²⁺]_i and force produced by a thromboxane A₂ analogue, U46619, were inhibited by K⁺ channel openers and verapamil. In the absence of extracellular Ca²⁺, U46619 induced a transient increase in [Ca²⁺]_i with a contraction, which is effectively inhibited by cromakalim and Ki4032. Their inhibitory effects were blocked by TBA and counteracted by 20 mM KCl-induced depolarization. Cromakalim and Ki4032 did not affect caffeine-induced Ca²⁺ release. Cromakalim reduced U46619-induced IP₃ production and TBA blocked this inhibitory effect. Thus, cromakalim and Ki4032 are more specific K⁺ channel openers than pinacidil, nicorandil, and KRN2391. The vasodilation related with a reduction of [Ca²⁺]_i produced by K⁺ channel openers is due to the hyperpolarization of the plasma membrane resulting in not only the closure of voltage-dependent Ca²⁺ channels but also inhibition of the production of IP₃ and Ca²⁺ release from intracellular stores related to stimulation of the thromboxane A₂ receptor.     

Ca2+ release-activated Ca2+ channel blockade as a potential tool in antipancreatitis therapy

Alcohol-related acute pancreatitis can be mediated by a combination of alcohol and fatty acids (fatty acid ethyl esters) and is initiated by a sustained elevation of the Ca²⁺ concentration inside pancreatic acinar cells ([Ca²⁺]_i), due to excessive release of Ca²⁺ stored inside the cells followed by Ca²⁺ entry from the interstitial fluid. The sustained [Ca²⁺]_i elevation activates intracellular digestive proenzymes resulting in necrosis and inflammation. We tested the hypothesis that pharmacological blockade of store-operated or Ca²⁺ release-activated Ca²⁺ channels (CRAC) would prevent sustained elevation of [Ca²⁺]_i and therefore protease activation and necrosis. In isolated mouse pancreatic acinar cells, CRAC channels were activated by blocking Ca²⁺ ATPase pumps in the endoplasmic reticulum with thapsigargin in the absence of external Ca²⁺. Ca²⁺ entry then occurred upon admission of Ca²⁺ to the extracellular solution. The CRAC channel blocker developed by GlaxoSmithKline, GSK-7975A, inhibited store-operated Ca²⁺ entry in a concentration-dependent manner within the range of 1 to 50 μM (IC₅₀ = 3.4 μM), but had little or no effect on the physiological Ca²⁺ spiking evoked by acetylcholine or cholecystokinin. Palmitoleic acid ethyl ester (100 μM), an important mediator of alcohol-related pancreatitis, evoked a sustained elevation of [Ca²⁺]_i, which was markedly reduced by CRAC blockade. Importantly, the palmitoleic acid ethyl ester-induced trypsin and protease activity as well as necrosis were almost abolished by blocking CRAC channels. There is currently no specific treatment of pancreatitis, but our data show that pharmacological CRAC blockade is highly effective against toxic [Ca²⁺]_i elevation, necrosis, and trypsin/protease activity and therefore has potential to effectively treat pancreatitis.     

Unique characteristics of Ca2+ homeostasis of the trans-Golgi compartment

Taking advantage of a fluorescent Ca²⁺ indicator selectively targeted to the trans-Golgi lumen, we here demonstrate that its Ca²⁺ homeostatic mechanisms are distinct from those of the other Golgi subcompartments: (i) Ca²⁺ uptake depends exclusively on the activity of the secretory pathway Ca²⁺ ATPase1 (SPCA1), whereas the sarco-endoplasmic reticulum Ca²⁺ ATPase (SERCA) is excluded; (ii) IP₃ generated by receptor stimulation causes Ca²⁺ uptake rather than release; (iii) Ca²⁺ release can be triggered by activation of ryanodine receptors in cells endowed with robust expression of the latter channels (e.g., in neonatal cardiac myocyte). Finally, we show that, knocking down the SPCA1, and thus altering the trans-Golgi Ca²⁺ content, specific functions associated with this subcompartment, such as sorting of proteins to the plasma membrane through the secretory pathway, and the structure of the entire Golgi apparatus are dramatically altered.     

Complex and rate-dependent beat-to-beat variations in Ca transients of canine Purkinje cells

Purkinje fibers play an essential role in transmitting electrical impulses through the heart, but they may also serve as triggers for arrhythmias linked to defective intracellular calcium (Ca²⁺) regulation. Although prior studies have extensively characterized spontaneous Ca²⁺ release in nondriven Purkinje cells, little attention has been paid to rate-dependent changes in Ca²⁺ transients. Therefore we explored the behaviors of Ca²⁺ transients at pacing rates ranging from 0.125 to 3 Hz in single canine Purkinje cells loaded with fluo3 and imaged with a confocal microscope. The experiments uncovered the following novel aspects of Ca²⁺ regulation in Purkinje cells: 1) the cells exhibit a negative Ca²⁺-frequency relationship (at 2.5 Hz, Ca²⁺ transient amplitude was 66 ± 6% smaller than that at 0.125 Hz); 2) sarcoplasmic reticulum (SR) Ca²⁺ release occurs as a propagating wave at very low rates but is localized near the cell membrane at higher rates; 3) SR Ca²⁺ load declines modestly (10 ± 5%) with an increase in pacing rate from 0.125 Hz to 2.5 Hz; 4) Ca²⁺ transients show considerable beat-to-beat variability, with greater variability occurring at higher pacing rates. Analysis of beat-to-beat variability suggests that it can be accounted for by stochastic triggering of local Ca²⁺ release events. Consistent with this hypothesis, an increase in triggering probability caused a decrease in the relative variability. These results offer new insight into how Ca²⁺ release is normally regulated in Purkinje cells and provide clues regarding how disruptions in this regulation may lead to deleterious consequences such as arrhythmias.     

胰淀素抑制高糖刺激INS-1细胞内钙离子浓度升高的作用机制

 目的 分析阐明胰淀素短时间作用于INS-1细胞、抑制高糖刺激的细胞内钙离子浓度（［Ca²⁺］_i）升高的机制。方法 采用钙离子荧光指示剂Fluo-4/AM负载细胞后,激光共聚焦显微镜下连续动态观察INS-1细胞经胰淀素孵育前后在葡萄糖、KCl、咖啡因和卡巴胆碱存在条件下［Ca²⁺］_i荧光强度变化。结果 （1）单纯16.7 mmol/L葡萄糖刺激使细胞内钙离子荧光强度变化的曲线下面积（AUC）为990±16;0.5 μmol/L胰淀素使16.7 mmol/L葡萄糖刺激的胞内荧光强度有所下降（AUC为831±10）, 1.0、5.0、10.0 μmol/L胰淀素均可使细胞内荧光强度显著降低（AUC分别为555±9、535±6、531±5）,与单纯葡萄糖刺激组比较差异有统计学意义,且呈现剂量依赖趋势。（2）10.0 μmol/L胰淀素可使30 mmol/L KCl刺激的荧光强度明显减低,与单纯KCl刺激组相比（AUC：168±5比311±11）差异有统计学意义。（3） 10.0 μmol/L胰淀素预处理后咖啡因和卡巴胆碱刺激的胞内荧光强度与单纯咖啡因和卡巴胆碱刺激组相比差异无统计学意义。结论 高浓度胰淀素的短时间作用对INS-1细胞经咖啡因和卡巴胆碱刺激的内质网鱼尼丁受体(ryanodine receptor,RyR)、三磷酸肌醇(IP₃)钙库释放没有影响,但可使高糖及KCl刺激的胞内荧光强度降低。推测胰淀素短时间作用使 ［Ca²⁺］_i减少的现象,主要是通过影响膜上钙通道实现的,与胞内钙库的释放无直接相关性。     

Wide long lasting perinuclear Ca2+ release events generated by an interaction between ryanodine and IP3 receptors in canine Purkinje cells

The purpose of this study was to determine whether IP₃Rs contribute to the generation of wide long lasting perinuclear Ca²⁺ release events in canine Purkinje cells. Spontaneous Ca²⁺ release events (elevations of basal [Ca²⁺] equivalent to F/F₀ 3.4SD over F₀) were imaged using Fluo-4AM and 2D confocal microscope. Only cells free of Ca²⁺ waves were analyzed. Subsarcolemmal region (SSL) was defined as 5 μm from cell edges. Core was the remaining cell. The majority of events (94%, 0.0035 ± 0.0007 events (ev)/μm²/s, N = 34 cells) were detected within a single frame (typical events, TE). However, a subpopulation (6.0%, 0.00022 ± 0.00005 ev/μm²/s, N = 41 cells: wide long lasting events, WLE) lasted for several frames, showed a greater spatial extent (51.0 ± 3.9 vs. TE 9.0 ± 0.3 μm², P < 0.01) and higher amplitude (F/F₀ 1.38 ± 0.02 vs. TE 1.20 ± 0.003, P < 0.01). WLE event rate was increased by phenylephrine (10 μM, P < 0.01), inhibited by 2APB and U73122 (P < 0.05), and abolished by tetracaine (1 mM) and ryanodine (100 μM). While SSL WLEs were scattered randomly, Core WLEs (n = 69 events) were predominantly distributed longitudinally 18.2 ± 1.6 μm from the center of nuclei. Immunocytochemistry showed that IP₃R1s were located not only at SSL region but also near both ends of nucleus overlapping with RyRs. In Purkinje cells, wide long lasting Ca²⁺ release events occur in SSL and in specific perinuclear regions. They are likely due to RyRs and IP₃R1s evoked Ca²⁺ release and may play a role in Ca²⁺ dependent nuclear processes.     

SR Ca store refill—a key factor in cardiac pacemaking

This study presents a theoretical analysis of the role of store Ca²⁺ uptake on sinoatrial node (SAN) cell pacemaking. Two mechanisms have been shown to be involved in SAN pacemaking, these being: 1) the membrane oscillator model where rhythm generation is based on the interaction of voltage-dependent membrane ion channels and, 2) the store oscillator model where cyclical release of Ca²⁺ from intracellular Ca²⁺ stores depolarizes the membrane through activation of the sodium-calcium exchanger (NCX). The relative roles of these oscillators in generation and modulation of pacemaker rate have been vigorously debated and have many consequences. The main new outcomes of our study are: 1) uptake of Ca²⁺ by intracellular Ca²⁺ stores increases the maximum diastolic potential (MDP) by reducing the cytosolic Ca²⁺ concentration [Ca²⁺]_c and hence decreasing the NCX current; 2) this hyperpolarization enhances recruitment of key pacemaker currents (e.g. the hyperpolarization-activated HCN current (I_f) and T-type Ca²⁺ current (I_T-Ca)); 3) the resultant enhanced Ca²⁺ entry during the pacemaker depolarization increases [Ca²⁺]_c causing advancement of the store Ca²⁺ release cycle and increased NCX current. In overview, the novel feature of our study is an investigation of the role of store Ca²⁺ uptake on SAN pacemaking. This occurs during the early diastolic period and causes enhanced I_f, I_T-Ca and store release (and hence I_NCX) during the later diastolic period. There is thus a symbiotic interaction between the two pacemaker “clocks” over the entire diastolic period, this providing robust and highly malleable SAN pacemaking. Accounting for store Ca²⁺ uptake also provides insight into hitherto unexplained SAN behaviour, as we exemplify for the sinus bradycardia exhibited in catecholaminergic polymorphic ventricular tachycardia (CPVT).     

Theoretical investigation of action potential duration dependence on extracellular Ca in human cardiomyocytes

Reduction in [Ca²⁺]_o prolongs the AP in ventricular cardiomyocytes and the QT_c interval in patients. Although this phenomenon is relevant to arrhythmogenesis in the clinical setting, its mechanisms are counterintuitive and incompletely understood. To evaluate in silico the mechanisms of APD modulation by [Ca²⁺]_o in human cardiomyocytes. We implemented the Ten Tusscher-Noble-Noble-Panfilov model of the human ventricular myocyte and modified the formulations of the rapidly and slowly activating delayed rectifier K⁺ currents (I_Kr and I_Ks) and L-type Ca²⁺ current (I_CaL) to incorporate their known sensitivity to intra- or extracellular Ca²⁺. Simulations were run with the original and modified models at variable [Ca²⁺]_o in the clinically relevant 1 to 3 mM range. The original model responds with APD shortening to decrease in [Ca²⁺]_o, i.e. opposite to the experimental observations. Incorporation of Ca²⁺ dependency of K⁺ currents cannot reproduce the inverse relation between APD and [Ca²⁺]_o. Only when I_CaL inactivation process was modified, by enhancing its dependency on Ca²⁺, simulations predict APD prolongation at lower [Ca²⁺]_o. Although Ca²⁺-dependent I_CaL inactivation is the primary mechanism, secondary changes in electrogenic Ca²⁺ transport (by Na⁺/Ca²⁺ exchanger and plasmalemmal Ca²⁺-ATPase) contribute to the reversal of APD dependency on [Ca²⁺]_o. This theoretical investigation points to Ca²⁺-dependent inactivation of I_CaL as a mechanism primarily responsible for the dependency of APD on [Ca²⁺]_o. The modifications implemented here make the model more suitable to analyze repolarization mechanisms when Ca²⁺ levels are altered.     

Spontaneous and agonist-induced calcium oscillations in single human nonfunctioning adenoma cells

The effects of gonadotropin-releasing hormone (GnRH) and GnRH-associated peptide (GAP) on cytosolic free calcium concentration ([Ca²⁺]_i) were investigated in 20 human nonfunctioning pituitary adenomas. We divided these tumors into three classes according to their response pattern to hypothalamic peptides. In type I adenomas (8 out of 20 adenomas), GnRH and GAP mobilized intracellular calcium ions stored in a thapsigargin (TG)-sensitive store. For the same concentration of agonist, two distinct patterns of GnRH-GAP-induced Ca²⁺ mobilization were observed (1) sinusoidal oscillations, and (2) monophasic transient. The latter is followed by a protein kinase C (PKC)-dependent increase in calcium influx through L-type channels. In type II adenomas (7 out of 20 adenomas), GnRH and GAP only stimulate calcium influx through dihydropyridine-sensitive Ca²⁺ channels by a PKC-dependent mechanism. TG (1 μM) did not affect [Ca²⁺]_i in these cells, suggesting that they do not possess TG-sensitive Ca²⁺ pools. All the effects of GnRH and GAP were blocked by an inhibitor of phospholipase C (PLC), suggesting that they were owing to the activation of the phosphoinositide turnover. Type I and type II adenoma cells showed spontaneous Ca²⁺ oscillations that were blocked by dihydropyridines and inhibition of PKC activity. GnRH and GAP had no effect on the [Ca²⁺]_i of type III adenoma cells that were also characterized by a low resting [Ca²⁺]_i and by the absence of spontaneous Ca²⁺ fluctuations. K⁺-induced depolarization provoked a reduced Ca²⁺ influx, whereas TG had no effect on the [Ca²⁺]_i of type III adenoma cells. The variety of [Ca²⁺]_i response patterns makes these cells a good cell model for studying calcium homeostasis in pituitary cells.     

Intracellular Ca2+ signaling and store-operated Ca2+ entry are required in Drosophila neurons for flight

Neuronal Ca²⁺ signals can affect excitability and neural circuit formation. Ca²⁺ signals are modified by Ca²⁺ flux from intracellular stores as well as the extracellular milieu. However, the contribution of intracellular Ca²⁺ stores and their release to neuronal processes is poorly understood. Here, we show by neuron-specific siRNA depletion that activity of the recently identified store-operated channel encoded by dOrai and the endoplasmic reticulum Ca²⁺ store sensor encoded by dSTIM are necessary for normal flight and associated patterns of rhythmic firing of the flight motoneurons of Drosophila melanogaster. Also, dOrai overexpression in flightless mutants for the Drosophila inositol 1,4,5-trisphosphate receptor (InsP₃R) can partially compensate for their loss of flight. Ca²⁺ measurements show that Orai gain-of-function contributes to the quanta of Ca²⁺-release through mutant InsP₃Rs and elevates store-operated Ca²⁺ entry in Drosophila neurons. Our data show that replenishment of intracellular store Ca²⁺ in neurons is required for Drosophila flight.     

Normal passive viscoelasticity but abnormal myofibrillar force generation in human hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy, increased ventricular stiffness and impaired diastolic filling. We investigated to what extent myocardial functional defects can be explained by alterations in the passive and active properties of human cardiac myofibrils. Skinned ventricular myocytes were prepared from patients with obstructive HCM (two patients with MYBPC3 mutations, one with a MYH7 mutation, and three with no mutation in either gene) and from four donors. Passive stiffness, viscous properties, and titin isoform expression were similar in HCM myocytes and donor myocytes. Maximal Ca²⁺-activated force was much lower in HCM myocytes (14 ± 1 kN/m²) than in donor myocytes (23 ± 3 kN/m²; P < 0.01), though cross-bridge kinetics (k_tr) during maximal Ca²⁺ activation were 10% faster in HCM myocytes. Myofibrillar Ca²⁺ sensitivity in HCM myocytes (pCa₅₀ = 6.40 ± 0.05) was higher than for donor myocytes (pCa₅₀ = 6.09 ± 0.02; P < 0.001) and was associated with reduced phosphorylation of troponin-I (ser-23/24) and MyBP-C (ser-282) in HCM myocytes. These characteristics were common to all six HCM patients and may therefore represent a secondary consequence of the known and unknown underlying genetic variants. Some HCM patients did however exhibit an altered relationship between force and cross-bridge kinetics at submaximal Ca²⁺ concentrations, which may reflect the primary mutation. We conclude that the passive viscoelastic properties of the myocytes are unlikely to account for the increased stiffness of the HCM ventricle. However, the low maximum Ca²⁺-activated force and high Ca²⁺ sensitivity of the myofilaments are likely to contribute substantially to any systolic and diastolic dysfunction, respectively, in hearts of HCM patients.     

Fibronectin increases the force production of mouse papillary muscles via α5β1 integrin

The extracellular matrix (ECM) protein-integrin-cytoskeleton axis plays a central role as a mechanotransducing protein assemblage in many cell types. However, how the process of mechanotransduction and the mechanically generated signals arising from this axis affect myofilament function in cardiac muscle are not completely understood. We hypothesize that ECM proteins can regulate cardiac function through integrin binding, and thereby alter the intracellular calcium concentration ([Ca²⁺]_i) and/or modulate myofilament activation processes. Force measurements made in mouse papillary muscle demonstrated that in the presence of the soluble form of the ECM protein, fibronectin (FN), active force was increased significantly by 40% at 1 Hz, 54% at 2 Hz, 35% at 5 Hz and 16% at 9 Hz stimulation frequencies. Furthermore, increased active force in the presence of FN was associated with 12-33% increase in [Ca²⁺]_i and 20-50% increase in active force per unit Ca²⁺. A function blocking antibody for α5 integrin prevented the effects of the FN on the changes in force and [Ca²⁺]_i, whereas a function blocking α3 integrin antibody did not reverse the effects of FN. The effects of FN were reversed by an L-type Ca²⁺ channel blocker, verapamil or PKA inhibitor. Freshly isolated cardiomyocytes exhibited a 39% increase in contraction force and a 36% increase in L-type Ca²⁺ current in the presence of FN. Fibers treated with FN showed a significant increase in the phosphorylation of phospholamban; however, the phosphorylation of troponin I was unchanged. These results demonstrate that FN acts via α5β1 integrin to increase force production in myocardium and that this effect is partly mediated by increases in [Ca²⁺]_i and Ca²⁺ sensitivity, PKA activation and phosphorylation of phospholamban.     

Brain-derived neurotrophic factor stimulates growth of pituitary melanotrope cells in an autocrine way

Brain-derived neurotrophic factor (BDNF) is expressed in the mammalian pituitary gland, in both the anterior and intermediate lobes, where its functional significance is unknown. Melanotrope cells in the intermediate pituitary lobe of the amphibian Xenopus laevis also produce BDNF, which co-exists in secretory granules with α-melanophore-stimulating hormone (α-MSH), a peptide that causes pigment dispersion in dermal melanophores during adaptation of the toad to a dark background. Xenopus melanotropes are highly plastic, undergoing very strong growth to support the high biosynthesis and release of α-MSH in black-adapted animals. In this study we have tested our hypothesis that this enhanced growth of the melanotrope is maintained by autocrine release of BDNF. Furthermore, since the extracellular-regulated kinase (ERK) pathway is a major component of BDNF signaling in neuronal plasticity, we investigated its involvement in melanotrope cell growth. For these purposes melanotropes were treated for 3 days in vitro, with either an anti-BDNF serum or a recombinant tropomyosin-receptor kinase B (TrkB) receptor fragment to eliminate released BDNF, or with the ERK inhibitor U0126. We also applied a novel inhibitor of the TrkB receptor, cyclotraxin-B, to test this receptor’s involvement in melanotrope cell growth regulation. All treatments markedly reduced melanotrope cell growth. Therefore, we conclude that autocrine release of BDNF and subsequent TrkB-dependent ERK-mediated signaling is important for melanotrope cell growth during its physiologically induced activation.     

Aging differentially modifies agonist-evoked mouse detrusor contraction and calcium signals

Although aging-induced changes in urinary bladder neurotransmission have been studied in some detail, information regarding alterations in detrusor muscle is scanty and addresses only partial aspects of the myogenic response of detrusor. Rodent bladder aging shows several features similar to those reported in humans. The aim of this study was to characterize in aged mouse the alterations of detrusor muscle contraction and the putative underlying changes in Ca²⁺ signals. We studied in vitro the myogenic contraction induced by agonists in detrusor strips from adult (3 months old) or aged (23–25 months old) mice. In addition, we determined the agonist-induced [Ca²⁺]_i signals by epifluorescence microscopy in fura-2 loaded isolated detrusor cells. Aging impaired the contractile response of bladder strips to cholinergic stimulation with bethanechol and to chemical depolarization with KCl-containing solutions. On the contrary, the response to purinergic stimulation (ATP) was enhanced. Aging also diminished the transient Ca²⁺ signal evoked by bethanechol and the Ca²⁺ influx induced by KCl in bladder strips. Treatments aimed to release calcium from intracellular stores (caffeine and a low level of ionomycin in Ca²⁺-free medium) showed that aging reduces the size of agonist-releasable stores. Similar to contraction, the mobilization of Ca²⁺ by ATP was increased in aged cells. Therefore, the differential effects of aging on detrusor contraction are associated to alterations of [Ca²⁺]_i signals: the cholinergic inhibition is due to inhibition of voltage-operated Ca²⁺ influx and reduction of the size of intracellular Ca²⁺ stores, while the age-induced ATP response is accompanied by an enhanced Ca²⁺ mobilization.