Ca2+ handling of rat pancreatic beta-cells exposed to ryanodine,caffeine, and glucagon

Reported species differences in the stimulus-secretion coupling of insulin release made it important to compare the Ca²⁺ handling of rat β-cells with that previously observed in mice. Single β-cells and small aggregates were prepared from pancreatic islets of Wistar rats, attached to cover slips and then used for measuring the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) with the ratiometric fura-2 technique. Glucose (11 mM) induced slow oscillations of [Ca²⁺]_i similar to those seen in other species, including humans. Comparison of the oscillations in rat β-cells with those previously described in mouse revealed that there was a slightly lower frequency and an increased tendency to transformation into sustained [Ca²⁺]_i in response to glucagon or caffeine. Ryanodine (5–20 μM) did not affect existing oscillations but sometimes restored rhythmic activity in the presence of caffeine. Stimulation with glucose resulted not only in oscillations but also in transients of [Ca²⁺]_i sometimes appearing in synchrony in adjacent β-cells and disappearing after the addition of 200 nM thapsigargin 20 mM caffeine. The frequency of transients recorded in a medium containing glucagon and methoxyverapamil was higher than seen under similar conditions in mouse β-cells. Although exhibiting some differences compared with mouse β-cells, rat β-cells also have an intrinsic ability to oscillate and to generate the transients of [Ca²⁺]_i that are supposed to synchronize the rhythmicity of the islets in the pancreas.     

早期持续高容量血液滤过治疗重症急性胰腺炎的临床研究

 目的 研究早期持续高容量血液滤过（HVHF）对重症急性胰腺炎(SAP)患者的临床疗效。方法 以2005年1月至2011年7月南昌大学第一附属医院重症医学科收治的SAP患者60例为研究对象,随机分为血液滤过 (血滤) 组和对照组,血滤组在采用常规治疗方法的同时,使用HVHF进行早期干预,比较两组患者的腹部症状、生命体征、氧合指数、肝肾功能、急性生理与慢性健康（APACHEⅡ）评分变化。结果 与对照组相比,血滤组早期HVHF治疗后患者发热、心动过速、呼吸窘迫、腹痛、腹胀等症状明显缓解,APACHEⅡ评分［(13.3±1.0)分比(14.1±1.2)分］、血TBil［(20.4±11.3) μmol/L比 (28.1±10.9) μmol/L］、血肌酐［(178.7±71.8) μmol/L比 (215.6±51.3) μmol/L］、血尿素氮［(10.1±5.6) mmol/L比 (13.2±3.8) mmol/L］、血ALT［(51.3±13.2) U/L比(62.5±14.3) U/L］均显著下降(P值均<0.05),氧合指数(PaO₂/FiO₂)（197.3±32.4 比 178.3±31.7）有显著提高(P<0.05),且在血滤治疗过程中患者平均动脉压逐渐上升,心率逐渐下降,与对照组相比差异有统计学意义(P<0.05)。结论 早期持续性HVHF治疗SAP能早期缓解症状,改善病情,阻止全身炎症反应综合征发展为多器官功能障碍综合征;并能改善脏器功能,减少并发症,在SAP早期治疗中具有重要地位。     

胰淀素受体在胰淀素抑制胰岛β细胞功能中的作用

目的分析胰淀素受体在胰淀素抑制胰岛β细胞功能中的作用。方法胰淀素、胰淀素受体拮抗剂AC253单独及联合短时间作用于大鼠胰岛β细胞系INS-1细胞后,ELISA及RT-PCR检测葡萄糖刺激胰岛素分泌及mRNA转录情况,共聚焦显微镜下观察高糖、磺脲类药物及KCl刺激下细胞内Ca2+浓度的变化,RT-PCR检测胰淀素短时间作用对胰淀素受体成分受体活性修饰蛋白(RAMP)mRNA表达的影响。结果胰淀素短时间孵育高糖刺激下胰岛素分泌由(2.08±0.35)ng/ml降至(0.73±0.24)ng/ml(P0.01),mRNA转录由(1.00±0.12)降至(0.39±0.09)(P0.01),加用AC253后胰岛素分泌升至(1.41±0.32)ng/ml,mRNA转录升至(0.62±0.08),与单用胰淀素组比较,差异有统计学意义(P0.05)。AC253可缓解胰淀素对高糖刺激下Ca2+升高的抑制作用,Ca2+总体变化水平和峰值由(650.155±4.818)和(1.154±0.011)升至(769.795±4.956)和(1.589±0.013)(P均0.01),同样AC253也可缓解胰淀素对磺脲类药物及KCl刺激下细胞内Ca2+升高的抑制作用。胰淀素短时间孵育未改变RAMP mRNA的表达。结论胰淀素受体可能参与了胰淀素抑制胰岛β细胞的胰岛素分泌、合成及对细胞内Ca2+浓度的调节。     

Ghrelin reduces voltage-gated potassium currents in GH3 cells via cyclic GMP pathways

Ghrelin is an endogeneous growth hormone secretagogue (GHS) causing release of GH from pituitary somatotropes through the GHS receptor. Secretion of GH is linked directly to intracellular free Ca²⁺ concentration ([Ca²⁺]_i), which is determined by Ca²⁺ influx and release from intracellular Ca²⁺ storage sites. Ca²⁺ influx is via voltage-gated Ca²⁺ channels, which are activated by cell depolarization. Membrane potential is mainly determined by transmembrane K⁺ channels. The present study investigates the in vitro effect of ghrelin on membrane voltage-gated K⁺ channels in the GH₃ rat somatotrope cell line. Nystatin-perforated patch clamp recording was used to record K⁺ currents under voltage-clamp conditions. In the presence of Co²⁺ (1 mM, Ca²⁺ channel blocker) and tetrodotoxin (1 μM, Na⁺ channel blocker) in the bath solution, two types of voltage-gated K⁺ currents were characterized on the basis of their biophysical kinetics and pharmacological properties. We observed that transient K⁺ current (I _A) represented a significant proportion of total K⁺ currents in some cells, whereas delayed rectifier K⁺ current (I _K) existed in all cells. The application of ghrelin (10 nM) reversibly and significantly decreased the amplitude of both I _A and I _K currents to 48% and 64% of control, respectively. Application of apamin (1 μM, SK channel blocker) or charybdotoxin (1 μM, BK channel blocker) did not alter the K⁺ current or the response to ghrelin. The ghrelin-induced reduction in K⁺ currents was not affected by PKC and PKA inhibitors. KT5823, a specific PKG inhibitor, totally abolished the K⁺ current response to ghrelin. These results suggest that ghrelininduced reduction of voltage-gated K⁺ currents in GH₃ cells is mediated through a PKG-dependent pathway. A decrease in voltage-gated K⁺ currents may increase the frequency, duration, and amplitude of action potentials and contribute to GH secretion from somatotropes.     

GABA inhibition of immortalized gonadotropin-releasing hormone neuronal excitability involves GABA(A) receptors negatively coupled to cyclic adenosine monophosphate formation

γ-Aminobutyric acid (GABA) has been implicated in the regulation of reproduction, particularly in the developmental modulation of gonadotropin-releasing hormone (GnRH) secretion. GnRH neurons are innervated by GABA-containing processes, and the administration of GABA stimulates and inhibits GnRH secretion in vivo and in vitro. We have previously shown that GABA can exert both of these actions in sequence, by acting directly on immortalized GnRH neurons. While the stimulation is the result of a GABA_A receptor-mediated depolarization of the plasma membrane, the mechanism involved in the delayed inhibition is the subject of the present investigation. GABA (1 nM-10 μM) decreased the intracellular concentration of cyclic adenosine monophosphate (cAMP) in a dose- and time-dependent fashion. This effect was blocked by bicuculline and mimicked by muscimol but not by baclofen. To analyze the effect of GABA on cellular excitability, we used fura-2 loaded GT1-7 cells. Activation of voltage-sensitive calcium channels by high K⁺-induced depolarization (35 mM) increased [Ca²⁺]_i. GABA (10μM) and muscimol (10 μM) reduced the amplitude of K⁺-induced [Ca²⁺]_i transients. This inhibition was blocked by forskolin (20μM) or 8-Br-cAMP (1 mM). Altogether, these results show that GABA_A receptors mediate a sustained inhibitory effect of GABA on GnRH neurons, and suggest the involvement of the cAMP pathway decreasing cellular excitability.     

Inhibitory effect of insulin on vasopressin-induced intracellular calcium response is blunted in hyperinsulinemic hypertensive patients: role of membrane fatty acid composition

Impaired insulin-mediated vasodilation has been implicated in hypertension that is associated with the metabolic syndrome. The aim of this study was to determine whether an abnormality in membrane fatty acid composition was related to a weakening of insulin's inhibitory effect on agonist-stimulated intracellular free calcium elevation. Mild to moderate hypertensive patients (n = 27) and normotensive controls (n = 11) were studied. Hypertensive patients were divided into normoinsulinemic patients (n = 14) and hyperinsulinemic patients (n = 13) according to the area under the curve of plasma insulin concentrations during a 75-g oral glucose tolerance test. Nonstimulated and arginine-vasopressin (AVP) (1 μmol/l)-stimulated intraplatelet free calcium concentrations (p[Ca²⁺]_i) were measured with or without insulin (100 μU/ml) preincubation. Platelet membrane fatty acid composition, intraerythrocyte sodium content, and the ouabain-sensitive sodium efflux rate constant (K _os) of erythrocytes were also determined. Insulin preincubation reduced AVP-stimulated p[Ca²⁺]_i elevation in both normotensive controls and hypertensive patients. The inhibitory effect of insulin on AVP-stimulated elevation of p[Ca²⁺]_i (%Inhibition) was significantly (P < 0.05) blunted in hyperinsulinemic hypertensive patients (9.7% ± 2.4%) as compared to normoinsulinemic hypertensive patients (17.4% ± 2.7%) and normotensive controls (16.9% ± 1.7%). In hypertensive patients, the %Inhibition was correlated negatively with saturated fatty acids (SFA) (r = −0.51, P < 0.05) and systolic blood pressure (r = −0.44, P < 0.05), and correlated positively with membrane polyunsaturated fatty acids (PUFA) (r = 0.53, P < 0.01) and K _os (r = 0.53, P < 0.005). Multiple regression analysis showed that SFA, PUFA, and K _os were the significant variables for %Inhibition. These findings indicate that an increase in SFA and a decrease in PUFA may cause insulin insensitivity in cellular calcium and sodium handling in hypertension with hyperinsulinemia.     

血管紧张素Ⅱ对大鼠肾小球内皮细胞单核细胞趋化蛋白1表达的影响

目的 观察血管紧张素Ⅱ（AngⅡ）对大鼠肾小球内皮细胞（GEC）单核细胞趋化蛋白1（MCP-1）表达的影响,探讨其相关作用机制。方法 对大鼠GEC进行分离培养与鉴定;采用Western blot法检测大鼠GEC炎性因子MCP-1蛋白的表达;RT-PCR和Western blot法检测血管紧张素Ⅱ 1型受体（AT1R）的mRNA和蛋白水平。结果 与正常对照组比较,施加AngⅡ刺激因素后MCP-1表达量明显增高,呈剂量（10^-7 mol/L～10^-5 mol/L）依赖效应;10^-5 mol/L AngⅡ作用下,大鼠GEC的AT1R mRNA和蛋白水平明显增加,成时间依赖效应;10^-6 mol/L AT1R拮抗剂替米沙坦（TEL）可以抑制AngⅡ（10^-5 mol/L）的作用,MCP-1的表达量下降。结论 AngⅡ通过上调大鼠GEC的AT1R水平,使大鼠GEC的MCP-1表达量增加。     

口服降糖药治疗失效的2型糖尿病患者使用地特胰岛素的安全性和有效性SOLVETM国际临床观察研究中国结果报道

 目的 诺和平1天1次注射研究（Study of Once-daily LeVEmir^®,SOLVE^TM）是一项为期24周、多中心、开放的观察性研究,旨在评价口服降糖药治疗失效的2型糖尿病患者中起始加用每天1次地特胰岛素（诺和平^®）治疗后的安全性和有效性。
方法 本研究来源于SOLVE^TM国际研究的中国结果。共有3272例使用口服降糖药治疗失效的2型糖尿病患者纳入本研究。参与研究的医生处方地特胰岛素,并在基线和治疗后12周、24周分别收集患者的临床数据,以评价药物的安全性和疗效。
结果 入选的3272例患者年龄（56.2±10.8）岁,糖尿病病程（7.1±5.2）年,基线BMI（25.3±3.3）kg/m²。治疗期间未观察到重度低血糖事件和夜间重度低血糖事件。治疗24周后,糖化血红蛋白（HbA1c）从基线的（8.33±1.69）%下降到（7.16±1.18）%,空腹血糖（FPG）从（9.52±2.59）mmol/L下降到（6.84±1.42）mmol/L,全天7个时点血糖全面改善,HbA1c<7％的患者达标率为49.1％。患者平均体重下降0.15 kg。
结论 口服降糖药失效的2型糖尿病患者,起始加用1天1次地特胰岛素治疗未观察到重度低血糖事件,同时有效改善血糖控制水平,提高治疗达标率,且对体重影响为中性。     

慢性心力衰竭患者体重指数与运动耐量的相关性研究

  目的 探讨慢性收缩性心力衰竭（心衰）患者BMI与运动耐量的关系。方法 收集慢性收缩性心衰患者,计算BMI,心肺运动试验测定运动峰耗氧量（PVO₂）,公斤体重耗氧量（PKVO₂）,每搏耗氧量（VO₂/HR）和每分通气量/每分CO₂产生量（VE/VCO₂）。结果 273例慢性收缩性心衰患者中,消瘦者（BMI＜18.5 kg/m²）6例,体重正常者（BMI 18.5~<24.0 kg/m²）113例,超重者（BMI 24.0~<28.0 kg/m²）116例,肥胖者（BMI≥28 kg/m²）38例。肥胖组和超重组患者PVO₂显著高于消瘦组和正常体重组患者［（1077.2±30.9）、（1095.3±54.3）ml/min比（550.2±192.1）、（886.0±31.2）ml/min］,而PKVO₂和VE/VCO₂显著低于消瘦组和正常体重组［（14.6±2.2）、（16.5±0.5）ml·min^-1 ·kg^-1比（14.4±0.5）、（11.6±0.9）ml·min^-1·kg^-1 ,43.4±6.1、42.3±1.5比42.3±1.5、38.6±1.6,P<0.05］。在不同心功能状态下,单相关分析显示,BMI和PVO₂呈正相关（r=0.40, P<0.01）,与PKVO₂和VE/VCO₂分别呈负相关（r=-0.15、-0.25,P值均<0.01）。多元逐步回归分析显示,年龄、性别、BMI和LVEF是PKVO₂的独立影响因素,而年龄和BMI是VE/VCO₂的独立影响因素（P<0.05）。结论 慢性收缩性心衰患者BMI与运动耐量显著相关,且是运动耐量的独立危险因素。     

马利兰和氟达拉滨与马利兰和环磷酰胺预处理方案在急性髓性白血病异基因造血干细胞移植中的比较

 目的 比较马利兰（Bu）和氟达拉滨（Flu）组成的预处理方案（Bu/Flu）与Bu和环磷酰胺（Cy）组成的预处理方案（Bu/Cy）在急性髓性白血病第一次完全缓解（AML-CR1）患者异基因造血干细胞移植（allo-HSCT）中的移植相关毒性和疗效的差异。方法 32例接受allo-HSCT的AML-CR1患者按移植顺序交替分至Bu/Cy组（Bu 3.2 mg·kg^-1·d^-1,移植前第7~4天;Cy 60 mg·kg^-1·d^-1,移植前第3~2天）或Bu/Flu组（Bu 3.2 mg·kg^-1·d^-1,移植前第5～2天;Flu 30 mg·m^-2·d^-1,移植前第6～2天）。评价两组预处理相关毒性（RRT）、移植物抗宿主病（GVHD）发生率与严重程度、3年累积复发率、非复发死亡率（NRM）、3年无病生存（EFS）率和总生存（OS）率等方面的差异。结果 中位随访时间为617.5（6~1261）d。两组中性粒细胞和血小板中位重建时间无明显差异（P=0.121和P=0.171）,移植后30 d嵌合状态分析提示两组患者均达到完全植入。Bu/Cy组预处理后中性粒细胞持续<0.1×10⁹/L和血小板持续﹤20×10⁹/L中位时间明显长于Bu/Flu组［6（3~14）d比2.5（1~9）d,P=0.000;3（1~36）d比1（0~4）d,P=0.047］。Bu/Cy组与Bu/Flu组Ⅱ～Ⅳ度RRT发生率分别为68.8%和25.0%（P=0.032）;急性GVHD发生率分别为46.7%和75.0%（P=0.149）,慢性GVHD发生率分别为46.7%和80.0%（P=0.149）;NRM分别为25.0%和6.3%（P=0.333）;3年累积复发率分别为（17.9±11.7）%和（14.1±9.3）%（P=0.834）;3年EFS率分别为（65.5±12.7）%和（80.2±10.3）%（P=0.362）;3年OS率分别为（68.8±11.6）%和（87.5±8.3）%（P=0.111）。结论 Bu/Flu是一种清髓性预处理方案,与Bu/Cy方案比较具有低骨髓抑制毒性及RRT。Bu/Flu作为AML-CR1患者allo-HSCT预处理方案其疗效不低于Bu/Cy。     

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.     

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.     

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.     

Influence of Infrasound Exposure on the Whole L-type Calcium Currents in Rat Ventricular Myocytes

This study was designed to examine the effect of infrasound exposure (5 Hz at 130 dB) on whole-cell L-type Ca²⁺ currents (WLCC) in rat ventricular myocytes and the underlying mechanism(s) involved. Thirty-two adult Sprague-Dawley rats were randomly assigned to infrasound exposure and control groups. [Ca²⁺]_i, WLCC, mRNA expression of the a_1c subunit of L-type Ca²⁺ channels (LCC), and SERCA2 protein were examined on day 1, 7, and 14 after initiation of infrasound exposure. Fluo-3/AM fluorescence and the laser scanning confocal microscope techniques were used to measure [Ca²⁺]_i in freshly isolated ventricular myocytes. The Ca²⁺ fluorescence intensity (FI), denoting [Ca²⁺]_i in cardiomyocytes, was significantly elevated in a time-dependent manner in the exposure groups. There was a significant increase in WLCC in the 1-day group and a further significant increase in the 7- and 14-day groups. LCC mRNA expression measured by RT-PCR revealed a significant rise in the 1-day group and a significant additional rise in the 7- and 14-day groups compared with control group. SERCA2 expression was significantly upregulated in the 1-day group followed by an overt decrease in the 7- and 14-day groups. Prolonged exposure of infrasound altered WLCC in rat cardiomyocytes by shifting the steady-state inactivation curves to the right (more depolarized direction) without altering the slope and biophysical properties of I _Ca,L. Taken together, our data suggest that changes in [Ca²⁺]_I levels as well as expression of LCC and SERCA2 may contribute to the infrasound exposure-elicited cardiac response. Zhaohui Pei and Zhiqiang Zhuang contributed equally to this work.     

Passive transport pathways for Ca and Co in human red blood cells. Co as a tracer for Ca influx

The passive transport of calcium and cobalt and their interference were studied in human red cells using ⁴⁵Ca and ⁵⁷Co as tracers. In ATP-depleted cells, with the ATP concentration reduced to about 1 μM, the progress curve for ⁴⁵Ca uptake at 1 mM rapidly levels off with time, consistent with a residual Ca-pump activity building up at increasing [Ca^T]_c to reach at [Ca^T]_c about 5 μmol (l cells)^{− 1} a maximal pump rate that nearly countermands the passive Ca influx, resulting in a linear net uptake at a low level. In ATP-depleted cells treated with vanadate, supposed to cause Ca-pump arrest, a residual pump activity is still present at high [Ca^T]_c. Moreover, vanadate markedly increases the passive Ca²⁺ influx. The residual Ca-pump activity in ATP-depleted cells is fuelled by breakdown of the large 2,3-DPG pool, rate-limited by the sustainable ATP-turnover at about 40–50 μmol (l cells)^{− 1} h^{− 1}. The apparent Ca²⁺ affinity of the Ca-pump appears to be markedly reduced compared to fed cells. The 2,3-DPG breakdown can be prevented by inhibition of the 2,3-DPG phosphatase by tetrathionate, and under these conditions the ⁴⁵Ca uptake is markedly increased and linear with time, with the unidirectional Ca influx at 1 mM Ca²⁺ estimated at 50–60 μmol (l cells)^{− 1} h^{− 1}. The Ca influx increases with the extracellular Ca²⁺ concentration with a saturating component, with K_½(Ca) about 0.3 mM, plus a non-saturating component. From ⁴⁵Ca-loaded, ATP-depleted cells the residual Ca-pump can also be detected as a vanadate- and tetrathionate-sensitive efflux. The ⁴⁵Ca efflux is markedly accelerated by external Ca²⁺, both in control cells and in the presence of vanadate or tetrathionate, suggesting efflux by carrier-mediated Ca/Ca exchange.The ⁵⁷Co uptake is similar in fed cells and in ATP-depleted cells (exposed to iodoacetamide), consistent with the notion that Co²⁺ is not transported by the Ca-pump. The transporter is thus neither SH-group nor ATP or phosphorylation dependent. The ⁵⁷Co uptake shows several similarities with the ⁴⁵Ca uptake in ATP-depleted cells supplemented with tetrathionate. The uptake is linear with time, and increases with the cobalt concentration with a saturating component, with J_max about 16 μmol (l cells)^{− 1} h^{− 1} and K_½(Co) about 0.1 mM, plus a non-saturating component. The ⁵⁷Co and ⁴⁵Ca uptake shows mutual inhibition, and at least the stochastic Ca²⁺ influx is inhibited by Co²⁺. The ⁵⁷Co and ⁴⁵Ca uptake are both insensitive to the 1,4-dihydropyridine Ca-channel blocker nifedipine, even at 100 μM. The ⁵⁷Co uptake is increased at high negative membrane potentials, indicating that the uptake is at least partially electrogenic. The ⁵⁷Co influx amounts to about half the ⁴⁵Ca influx in ATP-depleted cells. It is speculated that the basal Ca²⁺ and Co²⁺ uptake could be mediated by a common transporter, probably with a channel-like and a carrier-mediated component, and that ⁵⁷Co could be useful as a tracer for at least the channel-like Ca²⁺ entry pathway in red cells, since it is not itself transported by the Ca-pump and, moreover, is effectively buffered in the cytosol by binding to hemoglobin, without interfering with Ca²⁺ buffering. The molecular identity of the putative common transporter(s) remains to be defined.     

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.     

Diabetes-induced activation of protein kinase C inhibits store-operated Ca<Superscript>2+</Superscript> uptake in rat retinal microvascular smooth muscle

Aims/Hypothesis To assess the effects of diabetes-induced activation of protein kinase C (PKC) on voltage-dependent and voltage-independent Ca²⁺ influx pathways in retinal microvascular smooth muscle cells.Methods Cytosolic Ca²⁺ was estimated in freshly isolated rat retinal arterioles from streptozotocin-induced diabetic and non-diabetic rats using fura-2 microfluorimetry. Voltage-dependent Ca²⁺ influx was tested by measuring rises in [Ca²⁺]_i with KCl (100 mmol/l) and store-operated Ca²⁺ influx was assessed by depleting [Ca²⁺]_i stores with Ca²⁺ free medium containing 5 µmol/l cyclopiazonic acid over 10 min and subsequently measuring the rate of rise in Ca²⁺ on adding 2 mmol/l or 10 mmol/l Ca²⁺solution.Results Ca²⁺ entry through voltage-dependent L-type Ca²⁺ channels was unaffected by diabetes. In contrast, store-operated Ca²⁺ influx was attenuated. In microvessels from non-diabetic rats 20 mmol/l D-mannitol had no effect on store-operated Ca²⁺ influx. Diabetic rats injected daily with insulin had store-operated Ca²⁺ influx rates similar to non-diabetic control rats. The reduced Ca²⁺ entry in diabetic microvessels was reversed by 2-h exposure to 100 nmol/l staurosporine, a non-specific PKC antagonist and was mimicked in microvessels from non-diabetic rats by 10-min exposure to the PKC activator phorbol myristate acetate (100 nmol/l). The specific PKC antagonist LY379196 (100 nmol/l) also reversed the poor Ca²⁺ influx although its action was less efficacious than staurosporine.Conclusion/interpretation These results show that store-operated Ca²⁺ influx is inhibited in retinal arterioles from rats having sustained increased blood glucose and that PKC seems to play a role in mediating this effect.Abbreviations DAG Diacylglycerol - PKC protein kinase C - [Ca²⁺]_i intracellular calcium concentration - STZ streptozotocin - SPP staurosporine - SR sarcoplasmic reticulum - MVSM microvascular smooth muscle - CPA cyclopiazonic acid - PMA phorbol myristate acetate - VDCC voltage-dependent Ca²⁺ channels     

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.     

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.     

Ca2+ sparks and cellular distribution of ryanodine receptors in developing cardiomyocytes from rat

Although abundant ryanodine receptors (RyRs) exist in cardiomyocytes from newborn (NB) rat and despite the maturity of their single-channel properties, the RyR contribution to excitation–contraction (E-C) coupling is minimal. Immature arrangement of RyRs in the Ca²⁺ release site of the sarcoplasmic reticulum and/or distant RyRs location from the sarcolemmal Ca²⁺ signal could explain this quiescence. Consequently, Ca²⁺ sparks and their cellular distribution were studied in NB myocytes and correlated with the formation of dyads and transverse (T) tubules. Ca²⁺ sparks were recorded in fluo-4-loaded intact ventricular myocytes acutely dissociated from adult and NB rats (0–9 days old). Sparks were defined/compared in the center and periphery of the cell. Co-immunolocalization of RyRs with dihydropyridine receptors (DHPR) was used to estimate dyad formation, while the development of T tubules was studied using di-8-ANEPPS and diIC12. Our results indicate that in NB cells, Ca²⁺ sparks exhibited lower amplitude (1.7 ± 0.5 vs. 3.6 ± 1.7 F/F₀), shorter duration (47 ± 3.2 vs. 54.1 ± 3 ms), and larger width (1.7 ± 0.8 vs. 1.2 ± 0.4 μm) than in adult. Although no significant changes were observed in the overall frequency, central sparks increased from ~ 60% at 0–1 day to 82% at 7–9 days. While immunolocalization revealed many central release sites at 7–8 days, fluorescence labeling of the plasma membrane showed less abundant internal T tubules. This could imply that although during the first week, release sites emerge forming dyads with DHPR-containing T tubules; some of these T tubules may not be connected to the surface, explaining the RyR quiescence during E-C coupling in NB.