κ-阿片受体激动剂U50488H对内皮素-1所致大鼠室性心律失常的影响

目的:研究κ-阿片受体选择性激动剂U50488H是否可通过调节内皮素-1(ET-1)表达的水平,进而影响c-Src蛋白酪氨酸激酶(PTK)的表达以实现抗心律失常的作用。方法:将42只大鼠随机分为7组(每组6只):正常对照组、U50488H组、U50488H+nor-BNI组、nor-BNI组、ET-1组、ET-1+U50488H组及ET-1+U50488H+nor-BNI组。左心室及股动脉插管观测大鼠心率(HR)、动脉压(ABP)、左心室内压(LVP)及心脏收缩和舒张功能(±LVdp/dtmax)等血流动力学指标,并计算大鼠室性心律失常的发生情况和大鼠的死亡率。实时荧光定量PCR检测ET-1及ET-1受体(ETRA)mRNA表达;Western blot测定ETRA及下游分子c-Src PTK的表达水平。结果:U50488H可显著抑制ET-1所致大鼠ABP、LVP以及心脏收缩、舒张功能的升高,并可显著降低ET-1所致大鼠室性心律失常的发生率和死亡率(P<0.01),以及抑制心肌ET-1 mRNA的水平(P<0.01)。给予ET-1后,磷酸化(P)-c-Src PTK的表达水平升高,U50488H可显著降低c-Src PTK的水平以及ET-1引起的P-c-Src PTK表达的水平(P<0.05),此作用可被κ-阿片受体阻断剂nor-BNI所阻断。结论:κ-阿片受体选择性激动剂U50488H可抑制ET-1所致大鼠室性心律失常发生。该作用可能与抑制ET-1及其下游分子c-Src PTK的表达有关。     

连接蛋白43表达的变化与心律失常的关系

近年研究表明,多种途径都可以改变连接蛋白43（connexin43,Cx43）的表达和分布,进而影响到心律失常的发生与进展。本文主要综述在房颤和骨骼肌成肌细胞心脏移植中,Cx43表达的变化,以及体外定向重离子流放射、缺血预处理、缝隙连接阻滞剂和阿片类物质对Cx43的影响,说明Cx43表达的变化与心律失常的关系。     

缝隙连接、连接蛋白43及其与心律失常的关系

缝隙连接(GJ)通道是介导心肌细胞间电化学信息交流,保证心脏整体活动的协调性和同步性的特殊通道。在致心律失常发生上,GJ通道介导的细胞间电耦联障碍甚至比膜离子通道功能紊乱起了更重要的作用。连接蛋白43(Cx43)是心室GJ通道的主要构成成份,其表达和分布的异常将导致心室肌细胞的整体性异常,从而传导速度和传导各向异性发生改变,产生折返和传导阻滞。以GJ通道为作用靶点的新一代抗心律失常药的出现将为心律失常的治疗带来新的希望。     

к阿片受体介导的抗心律失常作用

目的:研究激动к阿片受体对大鼠心肌缺血性心律失常的影响. 方法:在结扎大鼠冠状动脉的同时,静脉注射选择性к阿片受体激动剂U50488H,观察其对大鼠心率(HR)、左心室内压(LVSP)和室性心律失常发生的影响;分离大鼠心室肌细胞,观察U50488H对细胞内钙和L-型钙电流的影响. 结果:心肌缺血后,大鼠HR无明显变化,LVSP 显著降低,心电图显示了缺血性改变和心律失常发生;心肌缺血并给予U50488H后,HR显著减慢,LVSP进一步降低,大鼠心肌缺血性心律失常的发生率以及心律失常评分明显降低.U50488H可明显降低心肌细胞的钙瞬变,并能显著降低L-型钙电流.U50488H的作用均可被选择性к阿片受体阻断剂nor-BNI阻断.结论:激动к阿片受体可减少大鼠心肌缺血性心律失常的发生,其机制可能与其抑制L-型钙通道和细胞内钙有关.     

κ-阿片受体和缝隙连接蛋白Cx43与心律失常的关系

心肌缺血时,交感神经兴奋可激活β受体介导cAMP依赖的信号途径导致心肌细胞内的钙升高,以及Cx43的结构和功能改变;而Cx43蛋白的改变已被证明是导致心律失常的重要原因。研究发现,κ-阿片受体激活后,对缺血心肌具有明确的保护作用和抗缺血性心律失常的作用,但κ-阿片受体是否可能通过调节Cx43蛋白进而产生抗心律失常的作用几无报道。本文就κ-阿片受体以及Cx43蛋白与心律失常的关系做一综述。     

缝隙连接蛋白43与心肌缺血再灌注心律失常的研究进展

近年来,心肌缺血再灌注损伤(myocardial ischemic-reperfusion injury,MIRI)的治疗和预防是心血管领域新兴的研究课题之一。缝隙连接(gap junction,GJ)是心脏电生理的基本结构。缝隙连接蛋白(connexin,Cx)是缝隙连接的基本单位。缝隙连接蛋白43(connexin 43,Cx43)是心脏Cx家族中最丰富的成员,Cx43的正常表达对于心脏发育、电耦联的心肌细胞活性和心肌功能的协调至关重要。Cx43与MIRI之间的关系已成为当前研究的重点。该文就Cx43与心肌缺血再灌注心律失常的关系作一综述。     

U50,488H对高脂大鼠血管内皮功能的影响

目的 探讨κ-阿片受体（κ-OR）选择性激动剂U50,488H对高脂大鼠血管内皮功能的影响及其机制。方法 成年SD大鼠分别饲以正常和高脂饲料14周,腹腔隔日注射U50,488H和κ-OR阻断剂nor-BNI,麻醉后下腔静脉取血,检测总胆固醇（TC）及低密度脂蛋白（LDL）的水平。透射电子显微镜观察动脉内皮细胞和平滑肌细胞的超微结构改变。观察胸主动脉对内皮依赖性血管舒张剂乙酰胆碱（ACh）及内皮非依赖性血管舒张剂（SNAP）的舒张反应。结果 高脂饲料喂养可引起大鼠血清TC和LDL显著升高;主动脉血管内皮依赖性舒张功能显著降低。透射电镜下可见动脉内皮细胞和平滑肌细胞的超微结构出现损伤。U50,488H可显著减轻高脂血症引起的血管内皮依赖性舒张功能障碍和动脉内皮细胞与平滑肌细胞超微结构损伤,κ-OR阻断剂nor-BNI可以阻断U50,488H的上述作用。结论 高脂血症大鼠存在内皮功能障碍,U50,488H可通过激活κ-OR改善高脂血症导致的内皮结构改变和功能障碍。     

胺碘酮与抗心律失常肽合用对陈旧性心肌梗死兔心缝隙连接蛋白43和室性心律失常诱发率的影响

目的 研究胺碘酮与抗心律失常肽合用抗心律失常的效果.方法 日本长耳兔200只,随机分为假手术组20只(A组)和结扎冠状动脉的心肌梗死(心梗)模型组180只.术后8 周末,再将存活的124只心梗模型兔随机分为对照组31只(B组)、胺碘酮组31只(C组)、抗心律失常肽(AAP10)组31只(D组)、胺碘酮+AAP10组31只(E组).第9周开始给A、B及D组兔口饲生理盐水2 ml、1次/d,给C、E组兔口饲含胺碘酮的生理盐水2 ml、1次/d,胺碘酮用量100 mg·kg-1·d-1.12周末,A、B、C、D、E组各存活20、24、26、25、27只.查超声心动图,麻醉后取左室心肌块动脉插管灌流,A、B及C组灌流台氏液,D、E组灌流加入500 nmol/L AAP10的台氏液,程序刺激同时记录容积心电图及QT、QRS、ERP、Tp-e及室性心律失常诱发率,计算Tp-e/QT.灌流完成后取心肌组织,利用Western blot和免疫荧光技术检测缝隙连接蛋白(Cx)43.组间计数资料统计学分析采用bonferroni方法校正的卡方检验或精确检验,以P＜0.05为差异有统计学意义.组间计量资料统计分析采用完全随机设计资料的方差分析;另B、C、D、E组间计量资料采用析因设计的方差分析,以评价两种药物之间的交互作用,以P＜0.05为差异有统计学意义.结果 A、B、C、D、E组心律失常诱发率分别为0、62.5%、26.9%、40.0%、22.2%,E组较B组诱发率减小.B组较A组Tp-e/QT增大,E组较B组Tp-e/QT减小.B组较A组Cx43表达明显减少.C、E组较B组Cx43表达增加,差异有统计学意义.结论 陈旧性心梗兔离体心肌块模型可用于室性心律失常的研究.胺碘酮慢性应用能上调心梗兔Cx43,与AAP10合用上调心梗兔Cx43作用加强,并进一步减小Tp-e/QT,抗心律失常作用进一步加强.     

Cx43mRNA、miR-210、VEGF在心律失常患者中的表达及其与心律失常的相关性

目的:探讨间隙连接蛋白43(Cx43)mRNA、微小RNA-210(miR-210)、血管内皮生长因子(VEGF)在心律失常患者中的表达及其与心律的相关性。方法:选择我院99例心律失常患者为心律失常组,同期健康体检者46例为健康对照组。心律失常组治疗1个月后复查,并随访2年,根据是否复发心律失常分为复发组(43例)和未复发组(53例);检测各组Cx43mRNA、miR-210、VEGF水平及心电图相关指标,分析血Cx43mRNA、miR-210、VEGF水平与心电图相关指标间的相关性及对心律失常的预测价值。结果:与健康对照组比较,心律失常组入院时、复诊时Cx43mRNA均显著降低,miR-210、VEGF、QTc、Tp-Tec水平均显著增加,P均=0.001;与入院时比较,心律失常组复诊时Cx43mRNA水平显著升高,miR-210、VEGF、QTc、Tp-Tec水平显著降低,P均=0.001;Pearson相关分析显示,复诊时Cx43mRNA水平与QTc、Tp-Tec呈显著负相关(r=-0.562、-0.363,P均=0.001),miR-210、VEGF水平与QTc、Tp-Tec呈显著正相关(r=0.328~0.498,P均=0.001);Cox分析显示,复诊时Cx43mRNA是影响心律失常预后的独立保护因素(HR=0.417,P=0.002),miR-210、VEGF是其独立危险因素(HR=3.675,3.697,P均=0.001);血Cx43mRNA、miR-210、VEGF联合预测心律失常患者预后的曲线下面积(AUC=0.928)显著大于单一指标预测,P均=0.001。结论:Cx43mRNA、miR-210、VEGF在心律失常患者中表达异常且与心律失常关系密切,可为临床评估病情及预后提供参考。     

H(+)-induced vasodilation of rat aorta is mediated by alterations in intracellular calcium sequestration

Acidosis induces vasodilation both in vivo and in vitro. Although it is commonly surmised that acidosis alters contractility by affecting contractile proteins and calcium entry, the exact role of these mechanisms in acidosis-induced vasodilation has not been determined. In the present study, we demonstrated that a novel mechanism, involving increased calcium sequestration into intracellular sites sensitive to norepinephrine, mediates the vasodilation associated with relatively modest decreases in pH. The effects of changing pH from 7.4 to 7.0 on tension development, 45Ca fluxes, and the norepinephrine-releasable intracellular calcium stores were studied in isolated rat aorta. Acute acidification produced marked endothelium-independent dilations of aortic rings that had been precontracted with norepinephrine. In contrast, this maneuver had only modest effects on contractions elicited by 80 mM KCl or phorbol ester. Acidification in this range did not alter basal or norepinephrine-stimulated undirectional 45Ca influx, nor did it reduce the norepinephrine-induced net gain in 45Ca content. Furthermore, neither norepinephrine-stimulated 45Ca efflux nor the peak contractile response to norepinephrine in calcium-free buffer was affected, although in this setting, the duration of the phasic contractile response was shortened. When calcium was restored to tissues exposed to norepinephrine in calcium-free buffer, acidification slowed the rate of tension development without altering 45Ca uptake, thus changing the relation between tension development and calcium entry. These effects of acidification were shown to be associated with an increase in the amount of calcium sequestered into the norepinephrine-sensitive intracellular calcium store. These findings clearly indicate that acidification, within a range that has no effect on other aspects of smooth muscle activation, elicits vasodilation by stimulating intracellular calcium sequestration. This action may represent a predominant mechanism whereby acidosis alters vascular smooth muscle contractility.     

Autocrine regulation of myocyte Cx43 expression by VEGF

Cardiac myocytes can rapidly adjust their expression of gap junction channel proteins in response to changes in load. Previously, we showed that after only 1 hour of linear pulsatile stretch (110% of resting cell length; 3 Hz), expression of connexin43 (Cx43) by cultured neonatal rat ventricular myocytes is increased by approximately 2-fold and impulse propagation is significantly more rapid. In the present study, we tested the hypothesis that vascular endothelial growth factor (VEGF), acting downstream of transforming growth factor-beta (TGF-beta), mediates stretch-induced upregulation of Cx43 expression by cardiac myocytes. Incubation of nonstretched cells with exogenous VEGF (100 ng/mL) or TGF-beta (10 ng/mL) for 1 hour increased Cx43 expression by approximately 1.8-fold, comparable to that observed in cells subjected to pulsatile stretch for 1 hour. Stretch-induced upregulation of Cx43 expression was blocked by either anti-VEGF antibody or anti-TGF-beta antibody. Stretch-induced enhancement of conduction was also blocked by anti-VEGF antibody. ELISA assay showed that VEGF was secreted into the culture medium during stretch. Furthermore, stretch-conditioned medium stimulated Cx43 expression in nonstretched cells. This effect was also blocked by anti-VEGF antibody. Upregulation of Cx43 expression stimulated by exogenous TGF-beta was blocked by anti-VEGF antibody, but VEGF-stimulation of Cx43 expression was not blocked by anti-TGF-beta antibody. Thus, stretch-induced upregulation of Cx43 expression is mediated, at least in part, by VEGF, which acts downstream of TGF-beta. Because the cultures contained only approximately 5% nonmyocytic cells, these results indicate that myocyte-derived VEGF, secreted in response to stretch, acts in an autocrine fashion to enhance intercellular coupling.     

U50,488H对大鼠肺动脉的舒张作用及机制

目的 :观察κ阿片受体选择性激动剂U5 0 ,4 88H对大鼠肺动脉的舒张作用并探讨其机制。方法 :分离正常大鼠肺动脉 ,采用离体血管灌流实验 ,观察U5 0 ,4 88H对大鼠肺动脉血管环舒缩功能的影响。结果 :①U5 0 ,4 88H在30～ 14 0 μmol/L范围内对大鼠肺动脉呈现明显的剂量依赖性舒张效应 ,该作用可被κ阿片受体选择性阻滞剂nor BNI所阻断 ;②经去内皮处理后 ,U5 0 ,4 88H对肺动脉的舒张效应显著减弱 ;③预先用NO合成酶抑制剂左旋硝基精氨酸甲酯 （L NAME）处理可显著降低U5 0 ,4 88H对肺动脉的舒血管效应 ;而 β受体阻断剂心得安、M受体阻断剂 （阿托品 ）、KATP通道阻断剂 （优降糖 ）、前列腺素合成抑制剂 （消炎痛 ）等对U5 0 ,4 88H舒张肺动脉的作用无明显影响。结论 :本研究首次证明κ阿片受体激动剂U5 0 ,4 88H可内皮依赖性地舒张大鼠肺动脉 ,其舒张肺动脉的效应可能与NO途径有关。     

Cardioprotective and antiarrhythmic effect of U50,488H in ischemia/reperfusion rat heart

The objective of this study was to investigate the protective effect of U50,488H, a selective κ-opioid receptor agonist, in the ischemia/reperfusion (I/R) rat and to delineate the underlying mechanism. Rat heart I/R injury was induced by occluding the left anterior descending coronary artery for 45 min and restoring perfusion for 120 min. U50,488H or vehicle was intravenously injected before ischemia. Electrocardiogram, heart rate (HR), arterial blood pressure (ABP), left ventricular pressure (LVP), systolic function (+dp/dt _max), and diastolic function (−dp/dt _max) were monitored in the course of the experiment. Myocardial infarction size was evaluated. Plasma concentrations of cardiac troponin T (cTnT), creatine kinase (CK), and lactate dehydrogenase (LDH) were measured. Single rat ventricular myocyte was obtained by enzymatic dissociation method. The potassium currents (I _K) of isolated ventricular myocytes were recorded with the whole-cell configuration of the patch-clamp technique. Compared with the sham control group, no significant change was found in HR, while ABP, LVP and ±dp/dt _max were significantly reduced in the I/R group. Administration of U50,488H significantly lowered HR in both control and I/R groups. Compared with the vehicle-treated I/R group, administration of U50,488H had no significant effect on I/R-induced reduction in ABP, LVP, and ±dp/dt _max. However, this treatment significantly reduced the myocardial infarction size, and markedly decreased the contents of plasma cTnT, CK and LDH. During ischemia and reperfusion, the incidence of ventricular arrhythmia in U50,488H-treated rats was significantly reduced. These effects were independent of the bradycardia induced by U50,488H, as the reducing infarct size and antiarrhythmic effect of U50,488H were still observed in animals in which heart rate was kept constant by electrical pacing. U50,488H and BRL-52537 still produced an antiarrhythmic effect when the rat heart was subjected to a shorter ischemic period of 10 min occlusion of coronary artery, which produced no infarction. I _K of the myocytes were inhibited by U50,488H in a dose-dependent manner in normal and hypoxic rat ventricular myocytes. However, the effects of U50,488H on I _K did not show any significant difference in normal and hypoxic myocytes. The above-described effects of U50,488H were totally blocked by nor-Binaltorphimine, a selective κ-opioid receptor antagonist. The results suggest that κ-opioid agonist U50,488H exerts its direct cardioprotective and antiarrhythmic effects against I/R via κ-opioid receptor, which participates in the regulation of potassium channels in normal and hypoxic ventricular myocytes. The first two authors contributed equally to this project.     

Regulation of connexin43 protein complexes by intracellular acidification

Ischemia-induced acidification of astrocytes or cardiac myocytes reduces intercellular communication by closing gap junction channels and subsequently internalizing gap junction proteins. To determine whether such coupling changes might be attributable to altered interactions between connexin43 (Cx43) and other proteins, we applied the nigericin/high K+ method to vary intracellular pH (pHi) in cultured cortical astrocytes. Intracellular acidification was accompanied by internalization of Cx43 with retention of Cx43 scaffolding protein Zonula Occludens-1 (ZO-1) at cell surfaces, suggesting that ZO-1 and Cx43 dissociate at low pHi. Coimmunoprecipitation studies revealed decreased binding of ZO-1 and increased binding of c-Src to Cx43 at low pHi. Resonant mirror spectroscopy was used to quantify binding of the SH3 domain of c-Src and the PDZ domains of ZO-1 to the carboxyl terminal domain of Cx43 (Cx43CT). Data indicate that the c-Src/Cx43CT interaction is highly pH dependent whereas the ZO-1/Cx43CT interaction is not. Moreover, binding of c-Src to Cx43CT prevented and reversed ZO-1/Cx43CT binding. We hypothesize that increased affinity of c-Src for Cx43 at low pHi aids in separation of Cx43 from ZO-1 and that this may facilitate internalization of Cx43. These data suggest that protracted acidification may remodel protein-protein interactions involving Cx43 and thus provide an important protective mechanism to limit lesion spread after ischemic injury.     

During U937 monocytic differentiation repression of the CD43 gene promoter is mediated by the single-stranded DNA binding protein Purα

Human CD43 is an abundant, heavily glycosylated molecule expressed exclusively on the surface of leucocytes. When leucocytes are at rest, CD43 acts to prevent intercellular interaction but during leucocyte differentiation such cell-cell interaction is facilitated by CD43. This change in the function of CD43 is mediated in part by a reduction in its level of expression. Previous studies have implicated proteolytic cleavage events at the cell surface in causing such reduction. Here, we report that, in an in vitro model of leucocyte differentiation, CD43 mRNA levels were also subject to reduction. Specifically, we demonstrated that within 48 h of the cell line U937 being induced to differentiate along the monocytic pathway, CD43 mRNA levels were reduced by 69%. This decline coincided with a decrease in the activity of the CD43 gene promoter mediated by the single-stranded DNA binding protein Pur alpha. Previously, we have demonstrated that Pur alpha mediates induction of the CD11c beta 2 integrin promoter during U937 differentiation. Consequently, Pur alpha represents a potential means by which the induction of pro-adhesive molecules and the repression of anti-adhesive molecules is co-ordinated during leucocyte differentiation.     

The pressor effect of sodium-volume expansion is calcium mediated.

To investigate the calcium dependence of salt-induced hypertension we concurrently measured blood pressure and serum ionized calcium in conscious normotensive female dogs undergoing five infusions: 1) sodium chloride (0.9%) 2) calcium chloride (10 mg/kg), 3) combined sodium chloride and calcium chloride, 4) nicardipine (1 micrograms/kg/min), and 5) combined sodium chloride and calcium chloride in the presence of nicardipine. While saline and calcium chloride infusions individually did not affect blood pressure, saline combined with calcium chloride significantly and consistently raised mean arterial pressure (MAP) (delta MAP = 7 +/- 2 mm Hg, P less than .001 v baseline). Serum ionized calcium (Caio) levels increased within the normal range with the infusion of calcium alone (1.32 +/- 0.03 to 1.48 +/- 0.01 mmol/L, P less than .005). Extracellular Caio rose less with the combined NaCl-CaCl2 infusion (delta Caio 0.10 +/- 0.01 v 0.16 +/- 0.02 mmol/L, P less than .02). The difference in calcium elevations could not be attributed to volume expansion alone, since saline infusion itself did not affect serum ionized calcium (1.32 +/- 0.3 to 1.31 +/- 0.01 mmol/L, P = NS). Furthermore, nicardipine prevented the pressor effect of the combined saline-calcium infusion. (delta MAP = -2 +/- 3 v 7 +/- 2 mm Hg, P less than .001), and restored the rise in extracellular Caio to that seen with the nonpressor calcium infusion (delta Caio 0.15 +/- 0.01 mmol/L v 0.16 +/- 0.02 mmol/L, P = NS). Altogether, these data demonstrate that the rise in blood pressure and ionized calcium following an acute infusion of sodium and calcium chloride are interdependent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Angiotensin II-induced extracellular signal-regulated kinase 1/2 activation is mediated by protein kinase Cdelta and intracellular calcium in adult rat cardiac fibroblasts

Angiotensin II (Ang II)-induced proliferation of cardiac fibroblasts is a major contributing factor to the pathogenesis of cardiac fibrosis. Ang II activates extracellular signal-regulated kinase (ERK) 1/2 to induce cardiac fibroblast proliferation, but the signaling pathways leading to ERK 1/2 activation have not been elucidated in these cells. The goal of the current study was to identify the intracellular mediators of Ang II-induced ERK 1/2 activation in adult rat cardiac fibroblasts. We determined that 100 nmol/L of Ang II-induced ERK 1/2 phosphorylation is inhibited by simultaneous chelation of cytosolic calcium and downregulation of protein kinase C (PKC) by phorbol ester or by the specific PKCdelta inhibitor rottlerin, as well as PKCdelta small interfering RNA, but not by inhibition of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate, phorbol ester, rottlerin, or PKCdelta small interfering RNA alone. We also found that Ang II does not transactivate the epidermal growth factor receptor in adult cardiac fibroblasts, because pretreatment with 1 mumol/L of AG 1478 did not significantly inhibit [(3)H]-thymidine incorporation or ERK 1/2 activation. In addition, immunoprecipitation of the epidermal growth factor receptor demonstrated no significant Ang II-induced phosphorylation of tyrosine residues. Inhibition of phosphatidylinositide 3-kinase, PKCzeta, and src tyrosine kinase had no effect on Ang II-induced ERK 1/2 activation. Collectively, these data demonstrate that Ang II does not transactivate the epidermal growth factor receptor in adult rat cardiac fibroblasts to activate ERK 1/2, a common pathway described in vascular smooth muscle and other cell types, but rather occurs via activation of distinct parallel signaling pathways mechanistically controlled by intracellular Ca(2+) and PKCdelta.     

Calcium regulation of chloroplast protein translocation is mediated by calmodulin binding to Tic32

The import of nuclear-encoded proteins into chloroplasts is tightly controlled on both sides of the envelope membranes. Regulatory circuits include redox-control as well as calcium-regulation, with calmodulin being the likely mediator of the latter. Using affinity-chromatography on calmodulin-agarose, we could identify the inner envelope translocon component Tic32 as the predominant calmodulin-binding protein of this membrane. Calmodulin-binding assays corroborate the interaction for heterologously expressed as well as native Tic32. The interaction is calcium-dependent and is mediated by a calmodulin-binding domain between Leu-296 and Leu-314 close to the C-proximal end of the pea Tic32. We furthermore could establish Tic32 as a bona fide NADPH-dependent dehydrogenase. NADPH but not NADH or NADP(+) affects the interaction of Tic110 with Tic32 as well as Tic62. At the same time, dehydrogenase activity of Tic32 is affected by calmodulin. In particular, binding of NADPH and calmodulin to Tic32 appear to be mutually exclusive. These results suggest that redox modulation and calcium regulation of chloroplast protein import convene at the Tic translocon and that both could be mediated by Tic32.