Relationship of intracellular free Ca2+ concentration and calcium-activated chloride channels of pulmonary artery smooth muscle cells in rats under hypoxic conditions

Summary To investigate the relationship between intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and calcium-activated chloride (Cl_CB) channels of pulmonary artery smooth muscle cells (PASMCs) in rats under acute and chronic hypoxic conditions, acute hypoxia-induced contraction was observed in rat pulmonary artery by using routine blood vascular perfusionin vitro. The fluorescence Ca²⁺ indicator Fura-2/AM was used to observe [Ca²⁺]_i of rat PASMCs under normal and chronic hypoxic condition. The effect of Cl_CB channels on PASMCs proliferation was assessed by MTT assay. The Cl_CB channel blockers niflumic acid (NFA) and indaryloxyacetic acid (IAA-94) exerted inhibitory effects on acute hypoxia-evoked contractions in the pulmonary artery. Under chronic hypoxic condition, [Ca²⁺]_i was increased. Under normoxic condition, [Ca²⁺]_i was (123.63±18.98) nmol/L, and in hypoxic condition, [Ca²⁺]_i was (281.75±16.48) nmol/L (P<0.01). Under normoxic condition, [Ca²⁺]_i showed no significant change and no effect on Cl_CB channels was observed (P>0.05 Chronic hypoxia increased [Ca²⁺]_i which opened Cl_CB channels. The NFA and IAA-94 blocked the channels and decreased [Ca²⁺]_i from (281.75±16.48) nmol/L to (117.66±15.36) nmol/L (P<0.01). MTT assay showed that under chronic hypoxic condition NFA and IAA-94 decreased the value of absorbency (A value) from 0.459±0.058 to 0.224±0.025 (P<0.01). Hypoxia increased [Ca²⁺]_i which opened Cl_CB channels and had a positive-feedback in [Ca²⁺]_i. This may play an important role in hypoxic pulmonary hypertension. Under chronic hypoxic condition, Cl_CB channel may play a part in the regulation of proliferation of PASMCs. YANG Zhao, female, born in 1967, Doctor in Charge     

Effect of SJAMP on ATP Release of Platelet

Ｓｅｃｒｅｔｉｏｎｏｆｐｌａｔｅｌｅｔｓｉｓａｎａｇｏｎｉｓｔ－ｉｎ－ｄｕｃｅｄｒｅｓｐｏｎｓｅ，ｗｈｉｃｈｉｓｏｆｉｍｐｏｒｔａｎｃｅｆｏｒｔｈｅｅｎｈａｎｃｅｍｅｎｔｏｆｐｌａｔｅｌｅｔａｃｔｉｖａｔｉｏｎａｎｄｆｏｒｔｈｅｖａｒｉｏｕｓｅｆｅｃ...     

Hypoxia increases cytosolic free calcium in porcine pulmonary arterial endothelial cells

Summary Alteration of cytosolic free calcium ([Ca²⁺ ₁]) in hypoxia was studied with fluorescent probe, Fura-2 in cultured porcine pulmonary arterial endothelial cells. It was found that hypoxia caused by bubbling the cell suspension with 100% N₂ increased [Ca²⁺ ₂] in endothelial cells by 90±20% (n=8,P<0.05), but didn’t affect that in a Ca²⁺-free buffer, La³⁺ (2×10⁻⁵ mol/L) inhibited the hypoxia-induced increment in [Ca²⁺]₁, whereas verapamil (10⁻⁸ mol/L) didn’t. These findings suggest that hypoxia could induce Ca²⁺ influx in pulmonary arterial endochelial cells, which might play an important role in vascular response to hypoxia.     

Mechanism of anti-β-adrenoceptor antibody mediated myocardial damage in dilated cardiomyopathy

Summary Antibodies against β₁-adrenoceptor can be detected in serum of patients with dilated cardiomyopathy (DCM), which have β-agonist-like activity, and induce a positive chronotropic effect on cardiac myocytes by its persistence at full strength. Effects of the antibodies against β-adrenoceptor from sera of patients with DCM on myocardial cytotoxicity and cytoplasmic free Ca²⁺-concentration ([Ca²⁺]_i) were observed in the cultured single layer SD rat ventricular cells by using the cytotoxicity assay and fluorescent Ca²⁺- indicator fura-2/AM. The positive sera of the anti-β-adrenoceptor antibodies from patients with DCM markedly enhanced myocardial [Ca²⁺]_i. Betaloc, a β_i-receptor blocker, might inhibit the increase of the antibody-mediated myocardial [Ca²⁺]_i, and the sera from healthy donors had no effect on myocardial [Ca²⁺]_i. Our results suggest that the anti-β-adrenoceptor antibody might increase myocardial [Ca²⁺]_i and result in myocardial damage. The antibodies might activate receptor-gating [Ca²⁺]-channel, thereby causing myocardial [Ca²⁺]_i rise and calcium overload. Early use of betaloc is recommended in the treatment of dilated cardiomyopathy. This project was supported by the National Natural Science Fundation of China (No. 39370317).     

2-甲基-3-羟基蒽醌诱导人乳腺癌MCF-7细胞凋亡的机制

为探讨2-甲基-3-羟基蒽醌抗肿瘤作用及其机制,本研究采用锥虫蓝法检测细胞活力,流式细胞仪检测细胞周期变化、细胞凋亡率、线粒体膜电位及细胞内游离钙的变化,Western blot方法检测凋亡相关蛋白caspase-4、caspase-7、caspase-9、Bcl-2、Bax、JNK、细胞色素C的表达。结果发现：2-甲基-3-羟基蒽醌时间依赖性地抑制乳腺癌细胞的生长,升高细胞内游离钙含量,降低线粒体膜电位并诱导其凋亡;药物上调Bax并下调Bcl-2蛋白的表达;诱导caspase-4、caspase-7、caspase-9、calpain的活化及细胞色素C的释放。结果提示2-甲基-3-羟基蒽醌可能通过Ca²⁺/calpain/caspase-4途径诱导人乳腺癌MCF-7细胞凋亡。     

Effect of Interleukin-1β on the Elevation of Cytoplasmic Free Calcium of the Cultured Hippocampal Neurons Induced by L-Glutamate

Ｃｏｍｐｌｉｃａｔｅｄｍｏｄｕｌａｔｉｎｇｍｅｃｈａｎｉｓｍｓａｒｅｉｎｖｏｌｖｅｄｉｎｔｈｅｎｅｔｗｏｒｋｂｅｔｗｅｅｎｎｅｒ－ｖｏｕｓａｎｄｉｍｍｕｎｅｓｙｓｔｅｍ．Ｉｎｔｅｒｌｅｕｋｉｎ－１（ＩＬ－１）,ａｓａｎｉｍｐｏｒｔａｎｔｃｙｔｏｋｉｎ...     

Effects of L-THP on Ca2+ overload of cultured rat cardiomyocytes during hypoxia and reoxygenation

Summary The effects of L-tetrahydropalmatine (L-THP) on the cultured rat cardiomyocytes during hypoxia and reoxygenation and the mechanism of L-THP treating reperfusion-arrythmias were studied. The concentration of intracellular free calcium ([Ca²⁺]_i) of single cultured ventricular myocyte was determined by using EPC-9 light-electricity measurement system. It was found that L-THP (100 μmol/L) could reduce the [Ca²⁺]_i augmentation in single cultured ventricular myocyte during hypoxia and reoxygenation. Verapamil (10 μmol/L) had the similar effect. It was concluded that LTHP could inhibit the Ca²⁺ overload of cultured rat cardiomyocytes during hypoxia and reoxygenation. This study was supported by a grant from the National Research Foundation of the Ministry of Public Health (No. 96-Q-025).     

Effects of Total Flavonoids ofHippophae Rhamnoides L. on intracellular free calcium in cultured vascular smooth muscle cells of spontaneously hypertensive rats and Wistar-Kyoto rats

Objective: To explore the effects of total flavonoids ofHippophae rhamnoides L. (TFH), quercetin (Que) and isorhamnetin (Isor) on the intracellular free calcium ([Ca²⁺ ]_i) in vascular smooth muscle cells (VSMC) of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY).Methods: Fluo 3-acetoxymethylester(Fluo-3/AM) was used to observe the effects of TFH (100mg/L) and its essential monomers, namely Que (l0^-4 mol/L) and Isor (l0^-4 mol/L) on changes of [Ca²⁺]_i in cultured SHR and WKY VSMC (abbr. to Ca-SHR & Ca-WKY) following exposure to high K⁺, norepinephrine (NE) and angiotensin II (Ang II), and to compare with the effects of verapamil (Ver).Results: (1) TFH, Que and Isor had inhibitory effects on resting Ca-SHR (P<0.05), but had no significant effects on Ca-WKY (P>0.05). (2) High K⁺ could increase Ca-SHR more significantly than Ca-WKY (P<0.05); TFH, Que and Isor could inhibit the elevation of [Ca²⁺]_i induced by high K⁺-depolarization, with the effects similar to that of Ver, and the effect on Ca-SHR was more significant than that on Ca-WKY (P<0.05). (3) NE and Ang II could increase Ca-SHR more significantly than Ca-WKY (P<0.05), TFH, Que and Isor had remarkably inhibitory effect on the elevation of Ca-SHR and Ca-WKY induced by NE or Ang II. (4) In the absence of extracellular Ca²⁺, TFH, Que and Isor also had certain inhibitory effect on Ca-SHR and Ca-WKY induced by NE, and the effect on the former was more significant than that on the latter(P<0. 05).Conclusion: TFH, Que and lsor might decrease the levels of [Ca²⁺]_i in VSMCs by blocking both voltage-dependent calcium channels (VDC) and receptor-operated calcium channels (ROC) in physiological or pathological state, which may be one of the important mechanisms of their hypotensive and protective effects on target organs in patients with hypertension. Supported by One-hundred-people Plan of Hygiene System in Shanghai (No. 990122)     

针刺对局灶性脑缺血大鼠海马神经元细胞[Ca2+]i的影响

[目的]研究脑缺血不同时点大鼠海马神经元内游离Ca²⁺浓度[Ca²⁺]_i及醒脑开窍针刺对其影响,探讨醒脑开窍针刺法早期干预对脑缺血的治疗作用。[方法]建立大脑中动脉所致局灶性脑缺血(MCAO)模型,采用醒脑开窍针刺法治疗,采用激光扫描共聚焦显微技术动态观察脑组织海马CA1区锥体细胞[Ca²⁺]_i在不同时间点的变化。[结果]与正常组大鼠相比,模型组大鼠脑组织海马CA1区锥体细胞内游离[Ca²⁺]_i(以细胞内Ca²⁺相对荧光强度表示)在局灶性脑缺血1 h时升高,持续升高至缺血24 h时(P<0.05)。假手术组与正常组相比[,Ca²⁺]_i变化差异无统计学意义(P>0.05)。相应时间点非穴位针刺组与模型组[Ca²⁺]_i比较,无统计学意义(P>0.05)。在相应时间点,醒脑开窍针刺组[Ca²⁺]_i低于模型组(P<0.01)。[结论]急性局灶性脑缺血后大鼠海马神经细胞[Ca²⁺]_i随缺血时间的延长而持续升高,提示细胞内钙超载;醒脑开窍针刺组能有效调节缺血区的[Ca²⁺]_i,提示在缺血后针刺治疗效果越早越好,为临床及早应用针刺治疗缺血性脑血管病提供了理论依据。     

辣椒素对背根神经节神经元钙离子浓度和线粒体膜电位的影响

目的探讨辣椒素对体外原代培养的胎鼠背根神经节（dorsal root ganglion,DRG）神经元钙离子浓度（［Ca²⁺］_i）和线粒体膜电位（mitochondiral membrane potential,MMP）的影响作用。方法分散培养的胎鼠DRG神经元用不同浓度的辣椒素（0.001μmol/L, 0.01μmol/L, 0.1μmol/L, 1μmol/L, 10μmol/L）孵育1min,用共聚焦激光扫描显微镜检测神经元［［Ca²⁺］_i的改变。对于能引起［Ca²⁺］_i升高的浓度组,在辣椒素孵育1min时,用流式细胞术检测神经元MMP的变化;并且在移去辣椒素后10min,重新检测神经元［Ca²⁺］_i的变化。结果0.001μmol/L、0.01μmol/L辣椒素孵育1min,神经元胞内［［Ca²⁺］_i没有变化;0.1μmol/L、1μmol/L和10μmol/L辣椒素孵育1min,神经元胞内［Ca²⁺］_i升高,移去辣椒素后10min,0.1μmol/L、1μmol/L辣椒素孵育的DRG神经元胞内［Ca²⁺］_i恢复到基础水平,10μmol/L辣椒素孵育的标本升高的［Ca²⁺］_i没有明显改变。辣椒素孵育时用无钙溶液,则神经元胞内［Ca²⁺］_i不升高。10?μmol/L辣椒素孵育1min的DRG神经元MMP降低,0.1μmol/L和1μmol/L辣椒素孵育的标本MMP无明显变化。结论一定浓度的辣椒素可使原代培养的DRG神经元胞内［Ca²⁺］_i升高,MMP降低。低浓度辣椒素引起的［Ca²⁺］_i的升高在10min内可以恢复到基础水平,而高浓度辣椒素引起的［Ca²⁺］_i的升高在10min内则不能恢复。辣椒素所致的胞内［Ca²⁺］_i升高可能是由于胞外钙离子内流引起的。     

Effects of cyclosporine A on angiotensin II-induced cardiomyocyte hypertrophy in neonatal rats

Summary The effects of cyclosporine A (CsA) on Angiontensin II (Ang II)-induced protein contents, c-fos protein levels and cytosolic Ca²⁺ level ([Ca²⁺]i) in cultured cardiomyocytes of neonatal rats were observed. Total protein contents were determined by Bradford method. The expression of c-fos protein was detected by Western blot. [Ca²⁺]i labeled with fluorescent probe Fluo-3/AM was measured under a laser scanning confocal microscope. The results revealed that as compared with control, the total protein contents were increased in cardiomyocytes treated with Ang II (10⁻⁷ mol/ L), which could be inhibited by CsA in a dose-dependent manner. It was found that Ang II could increase the c-fos protein expression, which could be inhibited by CsA in a dose-dependent manner. Ang II induced the [Ca²⁺]i elevation in cardiomyocytes. CsA did not influence the resting intracellular Ca²⁺, but inhibited significantly the Ang II-induced [Ca²⁺]i elevation. It was concluded that CsA can suppress the Ang II-induced c-fos protein expression and [Ca²⁺]i elevation in single cardiomyocyte, which might pay a role in the prevention of Ang II-induced cardiomyocyte hypertrophy by CsA. Han Zhaomin, female, born in 1974, Pharmacist     

Rapid Inhibition of the Glutamate-induced Increase of Intracellular Free Calcium by Magnesium in Rat Hippocampal Neurons

Inrecentyears ,largenumbersofrelativestud iesonthemagnesium (Mg2 )andneurologicaldis easesverifiedinmanyneurologicaldiseases ,suchascerebrovasculardiseases,chronicalcoholpoisoning ,Wilsondiseaseetc .,wererelatedwiththeMg2 defi ciency .Magnesiumreagentsc…     

大承气颗粒药物血清对小鼠肠上皮内淋巴细胞增殖的作用*

[目的]探讨大承气颗粒药物血清对小鼠肠上皮内淋巴细胞(IELs)的增殖作用及其作用途径.[方法]分离、鉴定、培养Balb/c小鼠IELs;制备不同浓度的大承气颗粒单次灌胃大鼠的药物血清和临床等效剂量的大承气颗粒多次灌胃大鼠的药物血清,并与大黄素、大黄酚等比较,观测其对IELs增殖及IELs[Ca2+]i的作用.[结果]IELs是CD3阳性细胞为(82.76±2.61),CD8阳性细胞为(72.48±3.57),CD4阳性细胞为(9.91±2.52)的淋巴细胞.单次灌胃的药物血清和多次灌胃的药物血清均能显著刺激IELs增殖.单次灌胃的药物血清对IELs的作用与大承气颗粒的浓度、采血时间、药物血清的作用时间等有关.稀释的药物血清比其原液及正常血清原液作用强.等摩尔浓度的大黄酚、大黄素、厚朴酚的作用以大黄酚较强.大鼠正常血清、药物血清和植物血凝素均能增加IELs[Ca2+]i.[结论]大承气颗粒药物血清能显著刺激IELs增殖,其作用途径之一是增加IELs[Ca2+]i,对维护肠免疫屏障有重要意义.     

K<Superscript>+</Superscript> channels and their effects on membrane potential in rat bronchial smooth muscle cells

Summary In order to investigate the K⁺ channels and their effects on resting membrane potential (Em) and excitability in rat bronchial smooth muscle cells (BSMCs), the components of outward K⁺ channel currents and the effects of K⁺ channels on Em and tension in rat bronchial smooth muscle were observed by using standard whole-cell recording of patch clamp and isometric tension recording techniques. The results showed that under resting conditions, total outward K⁺ channel currents in freshly isolated BSMCs were unaffected by ATP-sensitive K⁺ channel blocker. There were two types of K⁺ currents: voltage-dependent delayed rectifier K⁺ channel (K_v) and large conductance calcium-activated K⁺ channel (BK_Ca) currents. 1 mmol/L 4-aminopyridine (4-AP, an, inhibitor of K_V) caused a significant depolarization (from −8.7±5.9 mV to −25.4±3.1 mV,n=18,P<0.001). In contrast, 1 mmol/L tetraethylammonium (TEA, an inhibitor of BK_Ca) had no significant effect on Em (from −37.6±4.8 mV to −36.8±4.1 mV,n=12,P>0.05). 4-AP caused a concentration-dependent contraction in resting bronchial strips. TEA had no effect on resting tension, but application of 5 mmol/L TEA resulted in a left shift with bigger pD₂ (the negative logarithm of the drug concentration causing 50% of maximal effect) (from 6.27±0.38 to 6.89±0.54,n=10,P<0.05) in the concentration-effect curve of endothine-1, and a right shift with smaller pD₂ (from 8.10±0.23 to 7.69±0.08,n=10,P<0.05) in the concentration-effect curve of isoprenaline. It was suggested that in rat BSMCs there may be two types of K⁺ channels, K_v and BK_Ca, which serve distinct roles. K_v participates in the control of resting Em and tension. BK_Ca is involved in the regulation of relaxation or contraction associated with excitation. LIU Xiansheng, male, born in 1969, M. D., Ph. D. This project was supported by a grant from the National Natural Sciences Foundation of China (No. 30270583).     

Effects of Benzo(a)pyrene on the Contractile Function of the Thoracic Aorta of Sprague-dawley Rats

ObjectiveTo evaluate the possible vascular effects of an environment carcinogen benzo(a)pyrene (BaP).MethodsThe cytotoxicit of BaP and rat liver S9 (0.25 mg/mL)-activated BaP were examined by MTT assay. Thoracic aortic rings were dissected from Sprague-Dawley rats. Contraction of aortic rings was induced by 60 mmol/L KCl or 10^?6 mol/L phenylephrine (PE) in an ex-vivo perfusion system after BaP (100 μmol/L) incubation for 6 h. [Ca²⁺]_i was measured using Fluo-4/AM. For in-vivo treatment, rats were injected with BaP for 4 weeks (10 mg/kg, weekly, i.p.).ResultsBaP (1–500 μm) did not significantly affect cell viability; S9-activated BaP stimulated cell proliferation. BaP did not affect the contractile function of endothelium-intact or -denuded aortic rings. BaP did not affect ATP-induced ([Ca²⁺]_i) increases in human umbilical vein endothelial cells. In BaP-treated rats, heart rate and the number of circulating inflammatory cells were not affected. Body weight decreased while blood pressure increased significantly. The maximum aortic contractile responses to PE and KCl and the maximum aortic relaxation response to acetylcholine were significantly decreased by 25.0%, 34.2%, and 10.4%, respectively.ConclusionThese results suggest, in accordance with its DNA-damaging properties, that metabolic activation is a prerequisite for BaP-induced cardiovascular toxicity.     

Investigation on signal transduction pathways after H1 receptor activated by histamine in C6 glioma cells: Involvement of phosphatidylinositol and arachidonic acid metabolisms

BackgroundInformation related to histamine-induced cellular responses in C6 glioma cells through second messenger pathways has not been fully studied, especially the involvement of arachidonic acid (AA) metabolism. In addition, specific labeled ligand binding to histamine receptor sites still needs to be clarified.MethodsLabeled mepyramine ligand was used to study its binding sites; [³H] inositol was used to detect inositol 4-phosphate (IP₁) formation, and fura-2/AM was used to detect intracellular free calcium ion ([Ca²⁺]i) level activated by the phosphatidylinositol-phospholipase C (PI-PLC) pathway. Also, labeled AA was used to detect the metabolism of AA and its metabolites release via the activation of phospholipase A2 in the presence of histamine.ResultsC6 glioma cells incubated with histamine in the presence of 10 mM LiCl for 60 minutes induced an increase of IP₁ and glycerophosphoric-inositol (GPI) accumulation. In addition, histamine caused an increase of extracellular AA with its metabolite release, eliciting a transient and sustained increase of free [Ca²⁺]i. The sustained increase of [Ca²⁺]i was almost or completely blocked by La³⁺ and excess ethylene diamine tetraacetic acid. The calcium ion influx associated with the sustained phase required the presence of histamine on the receptor sites, and could be blocked by a H₁ antagonist, chlorpheniramine.ConclusionC6 glioma cells possess histamine H₁ receptors that have affinity towards [³H]mepyramine binding, and are coupled to PI-PLC to generate inositol phosphates and to increase [Ca²⁺]i, and they are coupled to phospholipase A2 (PLA2) to generate GPI and AA with its metabolite release. The transient increase in [Ca²⁺]i can be attributed to Ca²⁺ release from intracellular stores, whereas the sustained increase in [Ca²⁺]i is due to influx of extracellular calcium ions. The sustained increase in [Ca²⁺]i plays a role in the activation of histamine receptor-coupled PLA2.     

Cyclic AMP dynamics in the pancreatic β-cell

Abstract

Insulin secretion from pancreatic β-cells is tightly regulated by glucose and other nutrients, hormones, and neural factors. The exocytosis of insulin granules is triggered by an elevation of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) and is further amplified by cyclic AMP (cAMP). Cyclic AMP is formed primarily in response to glucoincretin hormones and other G_s-coupled receptor agonists, but generation of the nucleotide is critical also for an optimal insulin secretory response to glucose. Nutrient and receptor stimuli trigger oscillations of the cAMP concentration in β-cells. The oscillations arise from variations in adenylyl cyclase-mediated cAMP production and phosphodiesterase-mediated degradation, processes controlled by factors like cell metabolism and [Ca²⁺]_i. Protein kinase A and the guanine nucleotide exchange factor Epac2 mediate the actions of cAMP in β-cells and operate at multiple levels to promote exocytosis and pulsatile insulin secretion. The cAMP signaling system contains important targets for pharmacological improvement of insulin secretion in type 2 diabetes.     

低氧通过Ca~(2+)-NFAT信号通路促进肺动脉平滑肌细胞增生

目的通过观察低氧对肺动脉平滑肌细胞增生的影响及作用机制,探讨低氧在肺动脉高压发病过程中的作用,为肺动脉高压的进一步干预治疗提供可靠的理论依据。方法培养人肺动脉平滑肌细胞(human pulmonary artery smooth muscle cell,HPASMCs)并分为以下几组:正常对照组、低氧组(24h、48h、72h)、低氧并处理组(低氧48h并在细胞外液中加入EDTA2mmol/L或VIVIT4μmol/L)。分别应用细胞计数法检测细胞的增生情况、荧光钙成像法测定细胞内游离钙离子浓度([Ca2+]i)、激光共聚焦显微镜观察活化T细胞核因子c3(nuclear factor of activated Tcells,NFATc3)在细胞核内外分布的变化。结果低氧呈时间依赖性促进HPASMCs增生;低氧处理的细胞静息[Ca2+]i和库容性钙内流(capacitative calcium entry,CCE)显著升高,NFATc3的核转位增多。EDTA(Ca2+螯合剂)或VIVIT(NFAT特异性抑制剂)能够明显抑制低氧诱导的细胞增生现象。结论低氧能促进HPASMCs的增生,其机制可能是通过增加静息[Ca2+]i和库容性钙内流,使HPASMCs内的Ca2+异常升高并导致NFATc3转位至核内,进而促进与增生有关的基因的表达。     

Cyclic AMP dynamics in the pancreatic β-cell

Insulin secretion from pancreatic β-cells is tightly regulated by glucose and other nutrients, hormones, and neural factors. The exocytosis of insulin granules is triggered by an elevation of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) and is further amplified by cyclic AMP (cAMP). Cyclic AMP is formed primarily in response to glucoincretin hormones and other G_s-coupled receptor agonists, but generation of the nucleotide is critical also for an optimal insulin secretory response to glucose. Nutrient and receptor stimuli trigger oscillations of the cAMP concentration in β-cells. The oscillations arise from variations in adenylyl cyclase-mediated cAMP production and phosphodiesterase-mediated degradation, processes controlled by factors like cell metabolism and [Ca²⁺]_i. Protein kinase A and the guanine nucleotide exchange factor Epac2 mediate the actions of cAMP in β-cells and operate at multiple levels to promote exocytosis and pulsatile insulin secretion. The cAMP signaling system contains important targets for pharmacological improvement of insulin secretion in type 2 diabetes.