Purinergic receptors on insulin-secreting cells

Summary— The insulin secreting B cell is fitted with the two types of purinergic receptors: P₂ (for ATP and/or ADP) and P₁ (for adenosine). The activation of P₂ purinoceptors by ATP or ADP evokes a biphasic stimulation of insulin secretion from isolated perfused rat pancreas; this stimulation is dose-dependent between 10^?6 and 10^?4 M. Non hydrolysable structural analogues are also effective, and the relative potency of various agonists (2-methylthio ATP ? ATP = ADP = α, β-methylene ATP ? AMP) gave evidence for a P_2y purinoceptor subtype. Proposed mechanisms include both an increased Ca²⁺ uptake and an increased intracellular Ca²⁺ mobilization via the hydrolysis of polyphosphoinositides. ATP (or ADP) potentiates physiological insulin-secreting agents (glucose and acetylcholine) and P₂ purinoceptors could play a physiological role in the stimulation of insulin secretion. The activation of P₁ purinoceptors (adenosine receptors) decreases insulin secretion. Using structural analogues of adenosine, the receptor was characterized as an A₁ subtype; it is coupled to a pertussis toxin sensitive G protein and it inhibits adenylate cyclase. It is of physiological relevance that the B cell has the two types of purinoceptors with opposite effects. Recently, a metabolically stable structural analogue of ADP, adenosine-5′-0-(2-thiodiphosphate) or ADPßS, has been described as a potent secretory agent, effective at nanomolar concentrations on isolated perfused rat pancreas. In vivo, this substance is able to increase insulin secretion and to improve glucose tolerance after IV administration in rats and oral administration in dogs. Furthermore in streptozotocin-induced diabetes, ADPßS retains its insulin secreting effects. These results suggest that P_2y purinoceptors could be a new target for antidiabetic drugs.     

Potassium channels in vascular smooth muscle: a pathophysiological and pharmacological perspective

Potassium (K⁺) ion channel activity is an important determinant of vascular tone by regulating cell membrane potential (MP). Activation of K⁺ channels leads to membrane hyperpolarization and subsequently vasodilatation, while inhibition of the channels causes membrane depolarization and then vasoconstriction. So far five distinct types of K⁺ channels have been identified in vascular smooth muscle cells (VSMCs): Ca⁺²‐activated K⁺ channels (BK_Ca), voltage‐dependent K⁺ channels (K_V), ATP‐sensitive K⁺ channels (K_ATP), inward rectifier K⁺ channels (K_ir), and tandem two‐pore K⁺ channels (K₂P). The activity and expression of vascular K⁺ channels are changed during major vascular diseases such as hypertension, pulmonary hypertension, hypercholesterolemia, atherosclerosis, and diabetes mellitus. The defective function of K⁺ channels is commonly associated with impaired vascular responses and is likely to become as a result of changes in K⁺ channels during vascular diseases. Increased K⁺ channel function and expression may also help to compensate for increased abnormal vascular tone. There are many pharmacological and genotypic studies which were carried out on the subtypes of K⁺ channels expressed in variable amounts in different vascular beds. Modulation of K⁺ channel activity by molecular approaches and selective drug development may be a novel treatment modality for vascular dysfunction in the future. This review presents the basic properties, physiological functions, pathophysiological, and pharmacological roles of the five major classes of K⁺ channels that have been determined in VSMCs.     

氯胺酮在细胞膜内对大鼠皮层细胞延迟整流钾通道的影响

[目的]测定高浓度氯胺酮在细胞膜内对大鼠皮层细胞延迟整流钾通道电导和动力学的影响.[方法]酶解法分离新生SD大鼠大脑皮层细胞,利用自行建立的膜片箝单离子通道检测系统内面向外模式检测其钾通道的特性,挑选出单个的延迟整流钾通道,并观测高浓度氯胺酮经细胞膜内通道内口对该通道的电导及动力学的影响.[结果]钳制电压 80 mV条件下,高浓度(0.5 mg/ml)氯胺酮在细胞膜内面作用前后其延迟整流钾通道的电流,开放、关闭常数及平均开放、关闭时间分别为:(-12.12±1.49)pA、5.490 mS、7.711mS、(12.50±21.71)mS、(8.91±10.34)mS和(-5.01±0.75)PA、0.686mS、3.252mS、(1.74±2.39)mS、(3.72±3.83)mS.[结论]高浓度氯胺酮在细胞膜内面直接使SD大鼠皮层细胞延迟整流钾通道的电导降低,开放、关闲常数及平均开放、关闭时间均减小.     

衰老大鼠动脉超微结构及平滑肌钾通道反应性变化

背景：K+通道是调节血管平滑肌的收缩性与舒张性的主要离子通道,与血管张力息息相关,但对K+目的：观察衰老对大鼠动脉超微结构和平滑肌钾通道反应性的影响及可能的作用机制。通道在机体衰老过程中的作用鲜有报道。方法：健康雄性Wistar大鼠16只,19月龄设为老年组(n=8),2月龄设为青年组(n=8)。两组各随机抽取6只大鼠进行胸主动脉血管环张力测定,分别给予钙激活钾通道特异性阻断剂TEA、电压依赖性钾通道特异性阻断剂4-AP、ATP敏感性钾通道特异性阻断剂glibenclamide、内向整流钾通道的特异性阻断剂BaCl 2结果与结论：与青年组大鼠比较,老年组胸主动脉内皮细胞和平滑肌细胞等结构发生衰老性变化;KCl诱发大鼠胸主动脉达到最大收缩张力后恢复基础张力所需时间,老年组明显长于青年组;4种阻断剂均诱发血管张力增加,且TEA和4-AP诱发的胸主动脉收缩反应,老年组显著低于青年组;glibenclamide和BaCl等药物刺激,观察各阻断剂引起的动脉反应性变化。余下每组2只,取胸主动脉以透射电镜观察动脉超微结构的变化。2诱发的血管收缩两组间无显著性差异。说明衰老可引起大鼠动脉超微结构发生改变,血管舒张能力下降,其中平滑肌钾通道尤其是钙激活钾通道和电压依赖性钾通道功能下降可能是其重要机制之一。     

Low-intensity ultrasound-exposed microbubbles provoke local hyperpolarization of the cell membrane via activation of BK(Ca) channels

Ultrasound (US) contrast agents have gained wide interest in gene therapy as many researchers reported increased membrane permeability and transfection efficiency by sonoporation in the presence of US contrast agents. We recently demonstrated an increase in cell membrane permeability for Ca2+ in rat cardiomyoblast (H9c2) cells insonified in the presence of microbubbles. In the present study, we specifically investigated whether US-exposed microbubbles have an effect on the cell membrane potential and whether Ca2+-dependent potassium (BK(Ca)) channels are involved. We particularly focused on local events where the microbubble was in contact with the cell membrane. H9c2 cells were cultured on US transparent membranes. US exposure consisted of bursts with a frequency of 1 MHz with a peak-to-peak pressure of 0.1 or 0.5 MPa. Pulse repetition frequency was set to 20 Hz, with a duty cycle of 0.2%. Cells were insonified during 30 s in the presence of Sonovue(trade mark) microbubbles. The membrane potential was monitored during US exposure using the fluorescent dye di-4-aminonaphtylethenylpyridinium (di-4-ANEPPS). The experiments were repeated in the presence of iberiotoxin (100 nM), a specific inhibitor of BK(Ca) channels. Surprisingly, despite the previously reported Ca(2+) influx, we found patches of hyperpolarization of the cell membrane, as reflected by local increases in di-4-ANEPPS mean intensity of fluorescence (MIF) to 118.6 +/- 2.5% (p < 0.001, n = 267) at 0.1 MPa and 125.7 +/- 5.9% (p < 0.001, n = 161) at 0.5 MPa at t = 74 s, respectively, compared with "no US" (100.3 +/- 3.4%, n = 52). This hyperpolarization was caused by the activation of BK(Ca) channels, as iberiotoxin completely prevented hyperpolarization. (MIF(t74) = 100.6 +/- 1.4%; p < 0.001, n = 267) and 0.5 MPa (MIF(t74) = 88.8 +/- 2.0%; p< 0.001, n = 193), compared with 0.1 and 0.5 MPa microbubbles without iberiotoxin. In conclusion, US-exposed microbubbles elicit a Ca2+ influx, which leads to activation of BK(Ca) channels and a subsequent, local hyperpolarization of the cell membrane. This local hyperpolarization of the cell membrane may facilitate uptake of macromolecules through endocytosis and macropinocytosis. (E-mail: ljm.juffermans@vumc.nl).     

Interleukin-4 stimulates immunoglobulin secretion by Epstein-Barr virus (EBV)-activated tonsillar B cells,and by EBV-transformed lymphoblastoid B cell lines without increasing cell division

Summary Freshly prepared Epstein-Barr virus-transformed B lymphoblastoid cell lines derived from five different donors were tested for their responses to recombinant human interleukin-4 and to low molecular weight B cell growth factor. In the absence of either cytokine, all five lines secreted immunoglobulin of more than one isotype (IgM, IgG, and IgA, but not IgE). Stimulation with interleukin-4 resulted in a significant increase in immunoglobulin secretion, but did not enhance cell division measured by tritiated-thymidine uptake or cell counts. In contrast, low molecular weight B cell growth factor increased both immunoglobulin secretion and cell division. The increase in immunoglobulin secretion stimulated by interleukin-4 occurred for each of the different isotypes (IgM, IgG and IgA) produced by the unstimulated line. No IgE secretion was detected for any of the five lines. It was also found that low (5 units/ml), but not high (100 units/ml), concentrations of interleukin-4 increased IgM, IgG and IgA secretion by tonsillar B cells polyclonally activated with Epstein-Barr virus. Again, no IgE was detected at any time. These results suggest that interleukin-4 can function as a late-acting B cell differentiation factor as well as a growth factor for human B cells.     

K+channels and control of ventricular repolarization in the heart

Summary— K+ channels form a large family, in which voltage-operated and ligand-operated channels can be distinguished. Under physiological conditions, four K+ currents contribute to the repolarization process and their role is discussed: i) the transient outward current (i_1o) is responsible for the rapid initial repolarization process from the crest of the action potential to the plateau level; ii) the delayed K+ current (i_K) is involved in the overall repolarization process during the plateau; iii) the inward rectifier (i_K1) is responsible for the final rapid repolarization and the maintenance of the resting potential; iv) a ligand-operated channel activated by acetylcholine and adenosine participates in the repolarization process and the maintenance of the resting potential in nodal, atrial and Purkinje cells. In the context of antiarrhythmic interventions, block of outward K+ current and prolongation of refractoriness is currently considered as an alternative to block of the Na+ current and reduction of conduction velocity. Although some of these drugs show use-dependent block, the frequency-dependent changes in current and action potential duration are not ideal.     

Loss of Bim allows precursor B cell survival but not precursor B cell differentiation in the absence of interleukin 7

Interleukin (IL)-7 is a stromal cell-derived cytokine required for the survival, proliferation, and differentiation of B cell precursors. Members of the Bcl-2 family of proteins are known to have profound effects on lymphocyte survival, but not lymphocyte differentiation. To distinguish the relative dependence on IL-7 of B cell precursor survival versus B cell differentiation, the combined effects of lack of IL-7 and lack of the proapoptotic Bcl-2 relative, Bim, were studied. Bim is expressed to varying degrees in all B cell precursors and B cells. Lack of Bim compensated for lack of IL-7 in the survival of pro-, pre-, and immature B cells; however, lack of Bim did not substitute for the requirement for IL-7 in B cell precursor differentiation or B cell precursor proliferation. Precursor B cell survival is more dependent on sufficient levels of IL-7 than precursor B cell differentiation because the number of B cells and their precursors were reduced by half in mice heterozygous for IL-7 expression, but were restored to normal numbers in mice also lacking Bim. Hence, Bim and IL-7 work together to control the survival of B cell precursors and the number of B cells that exist in animals.     

Differential effects of cromakalim on pancreatic vascular resistance and insulin secretion in vitro

Summary— The effects of the potassium channel opener cromakalim on vascular resistance and insulin output were investigated in vitro within the same experimental preparation, the isolated rat pancreas perfused at a constant pressure with a physiological solution containing 8.3 mM glucose. Cromakalim induced a clear and concentration-dependent dilatory response of pancreatic vessels; the concentration-response curve obtained in the range of 10⁻⁸-10⁻⁵ M had a sigmoidal shape with a linear part between 10⁻⁷ and 10⁻⁶ M. Cromakalim did not inhibit insulin release at these concentrations. These results differ from those obtained with diazoxide, which has been previously shown both to inhibit insulin secretion and induce vasodilatation of the pancreatic vascular bed in a similar range of concentrations (10⁻⁶ – 10⁻⁵ M). The data presented provide evidence for a selective effect of cromakalim on pancreatic vascular resistance. Our present and previous results support the view that cromakalim is effective on K+ channels of vascular smooth muscle that differ from the ATP-sensitive K+ channel opened by diazoxide in insulin-secreting B-cells.     

Mitochondrial Potassium Channels as Pharmacological Target for Cardioprotective Drugs

Brief periods of ischemia are known to confer to the myocardium an increased resistance to the injury due to a later and more prolonged ischemic episode. This phenomenon, known as ischemic preconditioning (IPreC), is ensured by different biological mechanisms. Although an exhaustive comprehension of them has not been reached yet, it is widely accepted that mitochondria are pivotally involved in controlling cell life and death, and thus in IPreC. Among the several signaling pathways involved, as triggers and/or end effectors, in the mitochondrial mechanisms of cardioprotection, an important role is played by the activation of potassium channels located in the mitochondrial inner membrane (mitoK) of cardiomyocytes. Presently, different types of mitoK channels have been recognized in the heart, such as ATP‐sensitive (mitoK_ATP) and calcium‐activated (mitoBK_Ca and mitoSK_Ca) potassium channels. Consistently, drugs modulating mitoK, on one hand, have been employed as useful experimental tools for early basic studies on IPreC. On the other hand, activators of mitoK are promising and innovative therapeutic agents for limiting the myocardial injury due to ischemic episodes. In this review, we report the experimental evidence supporting the role of mitoK in signaling pathways in the mechanisms of cardioprotection and an overview on the most important molecules acting as modulators of these channels, with their profiles of selectivity. Some innovative pharmaceutical strategies for mitochondriotropic drugs have been also reported. Finally, an appendix describing the main experimental approaches usually employed to study mitoK in isolated mitochondria or in intact cells has been added.     

Potential Usefulness of Early Potassium Supplementation for Preventing Severe Hypokalemia Induced by Liposomal Amphotericin B in Hematologic Patients: A Retrospective Study

Purpose

Liposomal amphotericin B (L-AMB) is an essential antifungal agent for patients with hematologic diseases; however, the drug causes severe hypokalemia at a high frequency. Meanwhile, there is little evidence regarding the risk factors for L-AMB–induced severe hypokalemia, and the prevention protocol has not been established. The goal of this study was to identify the risk factors related to severe hypokalemia induced by L-AMB in hematologic patients.

A B cell receptor with two Igalpha cytoplasmic domains supports development of mature but anergic B cells

B cell receptor (BCR) signaling is mediated through immunoglobulin (Ig)alpha and Igbeta a membrane-bound heterodimer. Igalpha and Igbeta are redundant in their ability to support early B cell development, but their roles in mature B cells have not been defined. To examine the function of Igalpha-Igbeta in mature B cells in vivo we exchanged the cytoplasmic domain of Igalpha for the cytoplasmic domain of Igbeta by gene targeting (Igbetac-->alphac mice). Igbetac-->alphac B cells had lower levels of surface IgM and higher levels of BCR internalization than wild-type B cells. The mutant B cells were able to complete all stages of development and were long lived, but failed to differentiate into B1a cells. In addition, Igbetac-->alphac B cells showed decreased proliferative and Ca2+ responses to BCR stimulation in vitro, and were anergic to T-independent and -dependent antigens in vivo.     

Light-stimulated insulin secretion from pancreatic islet-like organoids derived from human pluripotent stem cells

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优降糖对KATP通道介导缺血预适应心肌再灌注损伤的影响

目的 :探讨优降糖在完整大鼠心脏模型中 ,对 ATP敏感钾通道 (KATP)介导心肌缺血预适应作用的影响。方法 :将 4 4只大鼠随机分为 4组 :心肌缺血预适应组 (IPC组 )、优降糖组 (GL I组 )、优降糖 IPC组 (G P组 )和对照组 (C组 )。心肌缺血预适应由 3次 10分钟缺血和 10分钟再灌注组成。所有大鼠均接受 30分钟缺血和 6 0分钟再灌注。梗死范围由饱和曲利本蓝和红四氮唑蓝染色判定 ,并以坏死区占缺血区的百分比表示。 导联记录心脏室性心律失常。结果 :IPC能显著缩小缺血再灌注后的心肌梗死范围 ,且这种作用能被 KATP通道阻滞剂优降糖完全取消。 IPC可减少缺血再灌注所致的室性心律失常的发生 ,但这种保护作用不能被优降糖所阻断。结论 :优降糖对 KATP介导 IPC的心肌保护作用有影响。     

慢性乙型肝炎患者病程中CD5^＋B细胞及CD4／CD8的变化研究

目的研究CD5^＋B细胞和CIM／CD8在慢性乙型肝炎发病中的作用，以及他们之间的相关性。方法采用免疫荧光双标记技术和流式细胞仪对48例慢性乙型肝炎患者发病前后周围血中CIM／CD8比例和CD5^＋B细胞的百分率和37例健康对照者进行比较。结果在慢性乙型肝炎患者发病高峰期周围血CIM／CD8比例显著低于病情恢复期，且和健康对照组比较差异有显著性；而CD5^＋B细胞在慢性乙型肝炎患者发病高峰期，显著高于健康对照组和慢性乙型肝炎患者恢复期，差异有显著性。结论慢性乙型肝炎的发病和T细胞免疫功能的紊乱相关，而CD5^＋B可能在乙型肝炎的慢性化机制中发挥作用。     

红细胞内钾与血浆钾比值(RPRP)的测定方法及其临床意义

目的探讨红细胞内钾与血浆钾比值(RPRP)的测定方法及其临床意义。方法收集111名无继发性高血压者(具不同程度腹部胀气、精神不振、四肢无力、原发性低血压等),取肘静脉血样,经样品特殊处理后用电极法测定血浆钾、红细胞内钾,计算出RPRP,用多元回归方法比较RPRP与症状之间的关系。结果RPRP与经常性腹部胀气、经常性四肢无力、经常性精神不振、原发性低血压等的回归系数分别为0.195,0.232,0.270,0.315,OR值分别为1.215(1.029~1.434,95%CI),1.261(1.059~1.503,95%CI),1.311(1.065~1.616,95%CI),1.371(1.175~1.567,95%CI)。结论RPRP是经常性腹部胀气、经常性四肢无力、经常性精神不振、原发性低血压的敏感指标,为其危险因素,RPRP的正常值上限在27.57~30.92之间。提示RPRP可能是人体细胞内外钾分布紊乱的诊断指标。     

Polymorphism at the 5'' end flanking region of the insulin gene is associated with reduced insulin secretion in healthy individuals

Sixty-four unrelated healthy subjects were studied for the detection of a DNA polymorphism at the 5' end of the insulin gene. No significant difference between the groups was found in blood glucose values at fasting and after an oral glucose load. A significant association was found between fasting (P less than 0.05) and after load plasma C-peptide levels (P less than 0.01) and the presence of a 1.6 Kb insertion at the 5' end of the insulin gene. A gene dose-dependent effect was noted, class 3/3 individuals having the lowest after-load C-peptide concentration and class 1/3 an intermediate level (F for the linear trend: P = 0.007). This might suggest that insulin gene polymorphism affects insulin secretion in healthy individuals. In order to confirm this, a subgroup of six class 3/3 and eight class 1/1 individuals subsequently underwent a hyperglycaemic clamp. The tissue sensitivity to insulin was similar in the two groups but glucose-stimulated insulin secretion was markedly impaired in homozygotes for the class 3 allele. In this group, insulin secretion was, on average, only one-third of that in class 1/1 individuals (P less than 0.02). Similarly impaired in class 3/3 persons was the glucose + arginine-stimulated insulin secretion (P less than 0.05). We conclude that the polymorphism at the 5' end of the insulin gene is associated with variations in insulin secretion in healthy humans.     

梭形细胞变异型非特指性弥漫性大B细胞性淋巴瘤2例临床病理观察

目的 探讨梭形细胞变异型非特指性弥漫性大B细胞性淋巴瘤(DLBCL)的临床病理特点及鉴别诊断.方法 对2例梭形细胞变异型非特指性DLBCL进行组织形态学观察及免疫组化、原位杂交、基因重排检测并文献复习.结果 2例均为女性,年龄分别为15岁和22岁.例1发生于腹腔肠系膜及大网膜,例2发生于前上纵隔.镜下肿瘤细胞肉瘤样生长,交错呈束状排列,间质有明显胶原纤维;肿瘤细胞胞质较少,核呈梭形或卵圆形,染色质粗颗粒状.免疫组化显示肿瘤细胞vimentin、CD45、CD20、CD79a、Pax5和MUM-1弥漫(+),例1 CD5(+),例2CD5(-),而其他抗体2例均(-).IgH基因重排可见较强克隆性重排条带.结论 梭形细胞变异型非特指性DLBCL非常罕见,需要与多种梭形细胞肿瘤鉴别.准确的诊断需要病理形态学观察与免疫组化和基因重排检测相结合,治疗以放、化疗为主,预后较好.     

Loss of precursor B cell expansion but not allelic exclusion in VpreB1/VpreB2 double-deficient mice

The pre-B cell receptor consists of immunoglobulin (Ig) mu heavy chains and surrogate light chain, i.e., the VpreB and lambda5 proteins. To analyze the role of the two VpreB proteins, mice lacking the VpreB1 and VpreB2 genes were generated. VpreB1(-/-) VpreB2(-/-) mice were impaired in their B cell development at the transition from pre-BI to large pre-BII cells. Pre-BII cells did not expand by proliferation, consequently 40-fold less small pre-BII and immature B cells were found in bone marrow, and the generation of immature and mature conventional B cells in spleen appeared reduced. In addition, only low numbers of B-1a cells were detected in the peritoneum. Surprisingly, Ig heavy chain allelic exclusion was still active, apparently ruling out a signaling role of a VpreB1/VpreB2-containing receptor in this process.     

B cell development in the spleen takes place in discrete steps and is determined by the quality of B cell receptor-derived signals.

Only mature B lymphocytes can enter the lymphoid follicles of spleen and lymph nodes and thus efficiently participate in the immune response. Mature, long-lived B lymphocytes derive from short-lived precursors generated in the bone marrow. We show that selection into the mature pool is an active process and takes place in the spleen. Two populations of splenic B cells were identified as precursors for mature B cells. Transitional B cells of type 1 (T1) are recent immigrants from the bone marrow. They develop into the transitional B cells of type 2 (T2), which are cycling and found exclusively in the primary follicles of the spleen. Mature B cells can be generated from T1 or T2 B cells. Mice with genetic deletions of elements participating in the B cell receptor signaling cascade display developmental arrest at the T1 or T2 stage. The analysis of these defects showed that the development of T2 and mature B cells from T1 precursors requires defined qualitative and quantitative signals derived from the B cell receptor and that the induction of longevity and maturation requires different signals.