川芎嗪对猪冠状动脉平滑肌细胞大电导钙激活钾通道的作用

本工作旨在研究川芎嗪对猪冠状动脉平滑肌细胞钾通道的作用,为阐明其扩张冠状动脉血管的机制提供实验依据。采用膜片钳细胞贴附式和内面向外式记录方式观察川芎嗪对猪冠状动脉平滑肌细胞大电导钙激活钾通道(large-conductance Ca2+- activated potassium channels,BKCa channels)的作用,分别用蛋白激酶A(protein kinase A,PKA)抑制剂H-89和蛋白激酶G (protein kinase G,PKG)抑制剂KT-5823处理细胞,再观察川芎嗪对BKCa通道作用的变化。结果表明在研究的0．73-8．07 mmol/L浓度范围,川芎嗪可以剂量依赖性地激活BKCa通道,使通道的开放概率从(0．01±0．003)增加到(0．03±0．01)-(．21± 0．18)(P<0．01,n=10),使通道平均关闭时间从(732．33±90．67)ms降低到(359．67±41．30)-(2．96±0．52)ms(P<0．01, n=10)。川芎嗪的这种激活作用在浴液游离钙离子浓度接近0 mmol/L时也存在。PKA的特异性抑制剂H-89(3 μmol/L)和 PKG的特异性抑制剂KT-5823(1 μmol/L)对川芎嗪激活BKCa通道的作用无影响。以上结果提示:川芎嗪能直接激活冠状动脉平滑肌BKCa通道,这种作用可能是川芎嗪扩张冠状动脉血管的一种重要机制。     

尼古丁调节大鼠冠状动脉平滑肌细胞大电导钙激活钾通道

目的:研究尼古丁对Wistar大鼠冠状动脉平滑肌大电导钙激活钾通道(BKca)活性的抑制作用及其细胞信号转导机制。方法:8周雄性Wistar大鼠随机分为两组:生理盐水组和尼古丁组;分别予以生理盐水和尼古丁2mg/(kg.d)注射21 d,蛋白酶法分离冠状动脉血管平滑肌细胞,将两组平滑肌细胞分别以对氯苯硫基环腺苷酸(CPT-cAMP,100μmol/L)和佛司可林(forskolin,10μmol/L)干预,单通道膜片钳记录干预前后平滑肌细胞单通道电流的平均开放时间(To)、平均关闭时间(Tc)、平均开放概率(Po)。结果:CPT-cAMP和Forskolin均能显著延长生理盐水组大鼠BKca的平均开放时间,缩短平均关闭时间,增加通道开放概率(P均<0.01)。对尼古丁组BKca的To、Tc、Po均无明显影响。结论:尼古丁促使冠状动脉血管收缩的生理机制是通过抑制cAMP/PKA途径诱导的大电导钙激活钾通道活性增加实现的。     

大电导钙激活钾通道（BKCa）及其开放剂研究进展

大电导钙激活钾通道（BKCa）广泛分布在哺乳动物各种组织（不含心肌细胞）中,并参与细胞内信号转导、细胞的兴奋及代谢调节等生理过程。BKCa功能异常牵涉到特发性癫痫、高血压等疾病的发生。BKCa通道是治疗高血压、尿失禁、哮喘、冠心病及缺血性脑中风等疾病的潜在药物靶点。探索高活性、高选择性、细胞通透性优良、类药性好的BKCa通道开放剂,不仅有助于阐明BKCa通道在生理病理条件下的作用机制,而且为治疗心脑血管疾病的药物研发奠定基础。对各类BKCa通道开放剂做一概述。     

血管平滑肌细胞中大电导钙激活钾通道的调节作用

大电导钙激活钾通道(BKCa)广泛分布于血管平滑肌细胞(VSMCs).由通道组成亚基α和调节亚基β构成.BKCa在细胞膜电位以及血管张力方面有重要的调节作用,并且与高血压等心血管类疾病的发生也有密切的关系.本文就BKCa通道的分子结构、生理功能及其与心血管疾病的关系研究进展作一综述.     

下丘脑神经元钙激活钾通道的整流现象

目的研究SD乳鼠下丘脑神经元中钙激活钾通道的整流现象.方法采用膜片钳内面向外式记录方式.结果记录到一种大电导钙激活钾通道(KCa),在对称140mmol/L[K+]时内向电导为(171±12)pS,不随[Ca2+]变化而改变,而外向电导可受[Ca2+]调控,当[Ca2+]为500μmol/L时,外向电导为(76±14)pS.[Ca2+]越大,整流现象越明显,Mg2+对这种KCa的整流作用不明显.结论下丘脑神经元中KCa具有Ca2+依赖性整流现象,它可能与神经元的兴奋性和稳定性有关.     

NO调控下丘脑钙激活钾通道的机制研究

目的 :研究NO对下丘脑神经元钙激活钾通道 (KCa)的作用及其机制。方法 :采用膜片钳技术内面向外式及细胞贴附式。结果 :NO可直接或通过升高cGMP来提高KCa通道的开放概率 (Po) ,这种增强作用是因为通道开放时间延长及开放频率增加。结论 :下丘脑神经元中NO可通过不同机制激活KCa。     

三磷酸肌醇对猪冠状动脉平滑肌细胞大电导钙激活钾通道的作用

应用膜片钳单通道电流记录技术,研究三磷酸肌醇(trisphosphateinositol,IP3)对猪冠状动脉平滑肌细胞大电导钙激活钾通道(large-conductanceCa2+-activatedpotassiumchannels,BKchannels)的作用。结果显示:在内面向外式(inside-out)膜片下,IP3(10~50μmol/L)可以浓度依赖性地增加通道的开放概率,而对电流幅值无明显影响,开放概率的增加是通过明显缩短平均关闭时间实现的(n=11,P<0.01);洗去药物后通道活性可以恢复到对照水平;IP3对通道的激活作用不随时间而衰减;IP3的降解产物对通道没有明显的激活作用。结果表明:在inside-out膜片下,IP3能够激活猪冠状动脉平滑肌细胞BK通道。     

大电导钙激活钾通道对大鼠骨髓间充质干细胞增殖的影响

为了观察开放和拮抗大电导钙激活钾通道（bigconductanceCa2＋-activated砧channel．BKca）对大鼠骨髓间充质干细胞（bonemarrowmesenchymalstemcells,BMSCs）增殖的影响并探讨其机制,该研究分离培养了大鼠BMSCs,采用BKca通道特异性开放剂msl619）和拮抗剂（IBTX）干预,MTT、平板克隆测定细胞增殖活力及细胞克隆形成能力;流式细胞术分析细胞凋亡及细胞周期分布;Westernblot、定量PCR检测周期蛋白cyclinD1基因和蛋白表达水平;整细胞膜片钳技术分析细胞膜电生理特性。结果显示,NS1619干预组与对照组相比,BMSCs~胞膜科通道外向电流振幅增大,细胞增殖能力和克隆形成能力增强,凋亡减少。此外,开放BKca通道明显促进细胞从G1期向S期过渡,cyclinD1蛋白7LmRNA表达上调,而拮抗BKca通道则相反。推测,BKca通道通过调节细胞周期进程最终影响细胞增殖,该作用可能与其具有调控细胞膜心电流的电生理特性有关。     

钙激活钾通道在黎芦碱致神经细胞损伤中的作用

目的:观察新生SD大鼠原代培养皮层神经元的钙激活钾通道(Kca)在黎芦碱致神经元损伤模型上的激活、抑制效应.方法:采用细胞贴附和内面向外两种膜片钳单通道记录方法记录新生SD大鼠原代培养皮层神经元的Kca电生理活动.结果:黎芦碱在胞外可激活Kca.在有钙浴液内,细胞贴附式,钳制膜电位 30 mV,加入不同浓度黎芦碱(μmol/L:15、25、50、75),通道开放概率由0.005分别增加为0.014±0.003、0.085±0.010、0.132±0.016、0.059±0.006(P＜0.01),在50μmol/L以内表现出浓度依赖性.无钙浴液内,细胞贴附式膜片上,钳制膜电位 50 mV,随药物浓度(μmol/L)增加为15、40、60、100时,通道开放概率由0.005分别增加为0.014±0.010、0.113±0.006、0.141±0.004、0 295±0.009(P＜0.05).6例内面向外式膜片上,钳制膜电位 40 mV,分别加入黎芦碱25 μmol/L、50μmol/L 3 min后,通道开放概率由0.011±0.008分别增加为0.010±0.010、0.012±0.007(P＞0.05).黎芦碱在胞内Kca开放概率,平均开放/关闭时间,电流幅值均无明显变化.结论:黎芦碱通过影响胞内游离钙水平间接调节Kca,在缺血缺氧早期,胞内游离钙增高激活Kca开放.     

线粒体ATP敏感钾通道与钙激活钾通道激活对正常与缺血脑线粒体通透性转变的影响

目的：明确线粒体ATP敏感钾通道与钙激活钾通道对正常和缺血脑线粒体渗透性转变的作用。方法：实验采用分光光度法,在分离的线粒体上分别观察两种线粒体钾通道激动剂对正常与缺血脑线粒体肿胀的影响。结果：在正常脑线粒体,diazoxide与NSl619能有效抑制由钙诱导的线粒体氏20下降,但其效应可被atractyloside所阻断。与正常相比,缺血损伤后的脑线粒体在钙离子诱导下线粒体A520下降较快,diazoxide与NS1619仍可抑制由钙诱导的线粒体A520下降,其作用同样为atractykxside所阻断。结论：线粒体ATP敏感钾通道与钙激活钾通道激活在离体条件均具有保护脑线粒体的作用,其作用可能是通过影响线粒体通透性转变而实现。     

重叠PCR法构建人心房肌SK2(KCNN2)基因表达质粒及鉴定和序列分析

目的:小电导钙激活钾通道亚型2(SK2)在心房肌功能活动中起重要作用,但是由于其表达密度低,直接进行RT-PCR一步法无法得到该基因(KCNN2)的编码区全长序列,本研究旨在采用Overlapping PCR(重叠PCR)法进行基因全长序列的扩增和表达质粒的构建,探讨其在长片段基因扩增的应用。方法:收集人心房肌标本,采用提取总RNA之后逆转录为cDNA,分三段设计KCNN2基因(AY258141)引物进行分段扩增,同时进行分段测序,然后采用Overlapping PCR得到KCNN2基因编码区全长序列,通过限制性酶切位点定向克隆到表达载体pIRES-hrGFP上。采用酶切法和测序法进行鉴定。结果:三段KCNN2基因扩增产物大小与预测值一致,最后得到的表达质粒测序结果与基因库数据基本一致。结论:成功构建人心房肌SK通道基因表达质粒pIRES-hrGFP-SK2,Overlapping PCR能够很好的用于长片段基因扩增。     

H_2O_2对人心房肌细胞I_(Kur)和I_(Ca,L)的浓度依赖性双向影响

本实验旨在观察活性氧（reactive oxygen species,ROS）对人心房肌细胞电生理活动特性的影响。取有心房颤动（atrial fibrillation,AF）和非AF心脏手术患者（各12例）右心耳组织,用酶消化法得到单个心房肌细胞。两组细胞（每组n=75）分别随机分为三个亚组：对照组（n=12）、H2O2组（0．1、0．2、0．5、0．75、1、2、5、10μmol／LH2O2,每个浓度n=7）和维生素C（ROS清除30）组（1gmol／L维生素C,n=7）。实验采用全细胞膜片钳方法记录电生理活动。与非AF对照组相比,AF对照组超快速延迟整流钾电流（ultrarapid delayed rectifier K^＋current,KKw）和L-型钙电流（L—type calcium current,ICaL）电流密度（pA／pF）均明显降低（6．27&#177;0．67VS3．77&#177;0．56,P〈0．05;6．31&#177;0．60 vs 3．34&#177;0．32,P〈0．05）,动作电位时程（action potential duration,APD）（ms）也明显缩短（405&#177;13 vs 354&#177;12,P〈0．05）。在非AF和AF组中,H2O2对心房肌细胞,IKw和,ICa,L的电流密度均有浓度依赖性双向影响——高抑低促。非AF组中,H2O2浓度为0．2gmol／L时有最大增强作用,而0．75Bmol／L为分界浓度,人于0．75Bmol／L时,随H2O2浓度增加IKw和,ICa,L的电流密度逐渐降低;在另一方面,0．2、1、2、5和10μmol／LH2O2孵育的心房肌细胞APD90与同组对照组相比均明显缩短（P〈0．05）,而与AF对照组相比无明显差异。在AF组中,H2O2的最大效应浓度为0．5Bmol／L,而1gmol／L为分界浓度。维生素C可以逆转H2O2的上述作用,但单独给予维生素C并不改变通道特性。H2O2诱导正常人心房肌细胞发生电生理活动特性改变与AF时心肌电重构（atrial electrical remodeling,AER）相似,显示ROS可能诱发AF;同时,H2O2又能加重AF时AER,对AF有维持作用。以上结果提示ROS清除剂可能对预防和治疗AF有重要意义。     

The effect of ranolazine on maintaining sinus rhythm in patients with resistant atrial fibrillation

Background

Atrial fibrillation (AF) may arise out of anomalous impulse activity at atrial venous junctions. Triggered activity may be a source of abnormal impulse activity. Ranolazine is an anti-anginal agent, which inhibits normal and abnormal late Na+ channel current in the ventricle and peak Na+ channel current in the atrium. This produces an energy sparing effect and stabilizes cardiac membranes. Ranolazine is a potent inhibitor of triggered activity. The purpose of this report is to describe our initial experience with ranolazine used in patients with resistant AF.

Methods

Seven patients (4 males, 3 females, 67 ± 9 years) who developed recurrent AF within hours to a few days of restoring sinus rhythm despite AF ablation and /or failing one or more anti-arrhythmic agents were started on ranolazine (500-1000 mg/twice/day) after stopping all other anti-arrhythmic therapy. All but one patient had some form of associated structural heart disease.

Results

Two patients received no apparent benefit from ranolazine developing recurrent AF within 2 days. All other patients derived significant benefit. Four patients have experienced no recurrent AF. The other patient relapsed at 3 months and again at 6 months. The mean time in sinus rhythm to date, or to the first relapse, for the five responders was 27 ± 11 weeks. No clinically evident pro-arrhythmic episodes occurred.

Conclusion

Ranolazine was helpful in maintaining sinus rhythm in the majority of patients in which more established measures had failed. A controlled prospective trial is warranted to further investigate the efficacy of ranolazine in AF.     

Functional interactions between the SK2 channel and the nicotinic acetylcholine receptor in enteric neurons of the guinea pig ileum

The neurotransmitter acetylcholine (ACh) plays a critical role in gastrointestinal function. The role of the small conductance Ca²⁺-activated K⁺ (SK) channel in ACh release was examined using myenteric plexus preparations of guinea pig ileum. Apamin, an inhibitor of the SK channel, significantly enhanced nicotine-induced ACh release, but neither electrical field stimulation- nor 5-hydroxytryptamine-induced ACh release, suggesting that SK channels might be selectively involved in the regulation of nicotine-induced ACh release. Therefore, we investigated the distribution of SK2 and SK3 subunits and the interaction between SK2 channels and nicotinic ACh receptors (nAChRs) in the guinea pig ileum. The immunoreactivity of SK2 subunits was located in enteric neuronal cells. Furthermore, SK2-immunoreactive cells stained with an antibody for choline acetyltransferase, a marker for cholinergic neurons, and with an antibody for the α3/5 subunits of nAChR. In contrast, immunoreactivity of SK3 subunits was not found in enteric neurons. A co-immunoprecipitation assay with Triton X-100-soluble membrane fractions prepared from the ileum revealed an association of the SK2 subunit with the α3/5 subunits of nAChR. These results suggest that SK2 channels negatively regulate the excitation of enteric neurons via functional interactions with nAChRs.     

Effects of hyperthermia on intracellular calcium concentration and responses of cancerous mammary cells in culture.

Effects of hyperthermia on the intracellular calcium concentration (Cai) of an established mouse breast cancer cell line, MMT060562, were studied using fura-2 fluorescence microscopy and the whole-cell clamp technique. A sudden change of temperature from 37 to 45 degrees C induced a transient increase in the fluorescence ratio permeability of the cell membrane and inward current. Deletion of extracellular calcium abolished the fluorescence ratio response to the rise in temperature. Cai of some cells increased after hyperthermia treatment at 44-48 degrees C for 20 min, but the average increase of Cai was negligible. After hyperthermia treatment, spontaneous oscillation of Cai, chemical responses to ATP and bradykinin and the mechanically-induced spreading response diminished. However, the mechanically induced increase of Cai within the stimulated cell remained even after hyperthermia treatment. Suppression of the ATP-induced Cai response recovered to about half the original level within 12 h. Blockage of protein synthesis with cycloheximide (100 microM) had no effect on the recovery. The D-myo-inositol 1,4,5-triphosphate (IP3)-dependent increase of Cai remained intact even after hyperthermia treatment. It is concluded that hyperthermia treatment increases both the permeability of the cell membrane and Cai, but decreases the sensitivity of cells to ATP and bradykinin, presumably due to modification of the signal transduction mechanism.     

Incidence and predictors of left atrial thrombus in patients with atrial fibrillation prior to ablation in the real world of China

BackgroundThe present study was to evaluate the value of CHADS2 and CHA2DS2VASC scores on predicting left atrial (LA) or left atrial appendage (LAA) thrombus in atrial fibrillation (AF) patients prior to ablation in the real world of China.Methods and resultsA total of 397 patients with non-valvular AF were analyzed to determine the relationship between CHADS2 and CHA2DS2VASC scores and LA/LAA thrombus identified on transesophageal echocardiography prior to radiofrequency ablation(RFA). LA/LAA thrombus was present in 38 patients (9.6%). There was a strong association between higher CHADS2 score or CHA2DS2VASC score and LA/LAA thrombus. No thrombus was identified in patients with CHA2DS2VASC score of 0 regardless of anticoagulation status. However, LA/LAA thrombus was detected in 2.9% patients with CHADS2 score of 0 without adequate anticoagulation, while no thrombus was present in the patients with CHADS2 score of 0 with adequate anticoagulation. Univariate analysis showed that heart failure (LVEF＜50%), LA≥40 mm, diabetes mellitus, previous stroke or TIA, CAD, hypertension, inadequate anticoagulation therapy, CHADS2 score of ≥2 and CHA2DS2VASC score of ≥2 were significantly associated with LA/LAA thrombus. Multivariable Cox regression analysis demonstrated that CHA2DS2VASC score of ≥2 (p = 0.02) and previous stroke or TIA (p = 0.04) were independently associated with LA/LAA thrombus regardless of anticoagulation status. ROC curve analysis showed that higher CHADS2 score and CHA2DS2VASC score could be similarly used to predict the presence of LA thrombus.ConclusionsBoth higher CHA2DS2VASC and CHADS2 scores were associated with LA/LAA thrombus in non-valvular AF patients prior to ablation. Although CHA2DS2VASC score and CHADS2 score had similar value to predict LA/LAA thrombus, CHA2DS2VASc score was better to identify low-risk patients for LA/LAA thrombus than CHADS2 score without anticoagulation. There will be a possibility of performing AF ablation or cardioversion in patients with a CHA2DS2VASC of 0 without TEE or anticoagulation therapy. The safety need to be verified by more multicentre randomized controlled clinical trails.     

白介素-2对心肌细胞[Ca~(2 )]_i的作用及其信号转导途径

为研究白介素 2 (interleukin 2 ,IL 2 )对心肌细胞内钙浓度 ([Ca2 ]i)的影响及其信号转导途径 ,实验采用酶解法分离成年大鼠心室肌细胞 ,以Fura 2 /AM为钙探针 ,用细胞内双波长钙荧光系统检测细胞 [Ca2 ]i 的变化。结果发现 :(1)IL 2 (0 5～ 2 0 0U/ml)浓度依赖性地降低单个心室肌细胞内钙瞬态 ,IL 2 (2 0 0U/ml)对咖啡因诱导的肌浆网内储钙的释放无影响 ;(2 )纳洛酮 (naloxone ,Nal) (10 -8mol/L)和nor binaltorphimine (nor BNI,10 -8mol/L)可阻断IL 2对心肌细胞钙瞬态的作用 ,而纳曲吲哚 (naltrindole ,NTI) (10 -6mol/L)不能阻断此作用 ;(3)κ阿片受体激动剂U5 0 488H (10 -6mol/L)降低心肌细胞钙瞬态 ,nor BNI (10 -8mol/L)可阻断此作用 ;(4 ) 5mg/L百日咳毒素 (PTX)预处理可取消IL 2降低心肌细胞钙瞬态的作用 ,而酪氨酸激酶抑制剂genistein (10 -4 mol/L)不能取消IL 2的作用 ;(5 )U7312 2预处理可阻断IL 2的作用。研究结果表明 ,IL 2降低心肌细胞钙瞬态的作用 ,是通过心肌细胞上κ阿片受体介导的 ,其下游途径包括PTX敏感的G蛋白和磷脂酶C。     

胍丁胺对大鼠心室肌细胞内游离钙浓度的影响

本研究旨在观察胍丁胺 (agmatine ,Agm)对分离大鼠心室肌细胞内游离钙浓度 ( [Ca2 +]i)的影响。用酶解方法分离大鼠心室肌细胞 ,用Fluo 3 AM负载 ,然后用激光共聚焦法测定单个心室肌细胞 [Ca2 +]i 的荧光强度 (fluorescenceintensity ,FI) ,结果以FI或相对荧光强度 (F/F0 % )表示。实验结果表明 ,在正常台氏液 (含钙 1 0mmol/L)和无钙台氏液中 ,单个大鼠心室肌细胞的荧光密度分别为 12 8 8± 13 8和 119 6± 13 6,两者无差异。Agm 0 1、1和 10mmol/L浓度依赖性地显著降低细胞的钙浓度 ;在正常台氏液中加入EGTA 3mmol/L ,Agm同样降低细胞的钙浓度。KCl 60mmol/L ,PE 3 0 μmol/L ,和Bay K 864 410 μmol/L均升高心室肌细胞的[Ca2 +]i。Agm同样降低高浓度KCl、Bay K 864 4和PE诱发的心室肌细胞 [Ca2 +]i 升高。当细胞外液钙浓度由 1mmol/L增加到 10mmol/L时 ,诱发心室肌细胞钙超载 ,同时部分心室肌细胞产生可传播的钙波 (Ca2 +wave) ,Agm 1mmol/L降低钙波的传播速度和持续时间 ,最终阻断钙波。以上结果提示 ,Agm对心室肌细胞的胞浆[Ca2 +]i具有抑制作用 ,此作用通过阻断电压依赖性钙通道而实现 ;并可能与抑制大鼠心室肌细胞内钙释放有关     

西洛他唑对人心房肌细胞瞬间外向钾电流的影响

目的:观察西洛他唑对人心房肌细胞瞬间外向钾电流(Ito1)的影响,探讨该药抗心律失常作用的机制.方法:二步酶解法分离人单个右心房肌细胞,应用全细胞膜片钳技术记录人心房肌细胞Ito1.结果:在保持电位-50 mV和去极化脉冲为 50 mV条件下,30 μmol/L西洛他唑显著降低Ito1,使Ito1幅值由加药前(8.16±0.70)pA/pF降至(4.84±0.60)pA/pF(P<0.01).西洛他唑在1～50 μmol/L范围内呈浓度依赖性的抑制Ito1,1 μmol/L时即产生作用,50 μmol/L时达最大效应(降低51.09%±3.00%),IC50为(13.18±2.60)μmol/L.此外,该药对Ito1的电压依赖性激活和失活曲线以及恢复曲线均无显著影响.结论:本实验结果表明西洛他唑浓度依赖性地阻滞人心房肌细胞的Ito1.     

Effects of cholinergic agonists on diacylglycerol and intracellular calcium levels in pancreatic β-cells

We have studied the effects of cholinegic agonists on the rates of insulin release and the concentrations of diacylglycerol (DAG) and intracellular free Ca²⁺ ([Ca²⁺]_i) in the β-cell line MIN6. Insulin secretion was stimulated by glucose, by glibenclamide and by bombesin. In the presence of glucose, both acetylcholine (ACh) and carbachol (CCh) produced a sustained increase in the rate of insulin release which was blocked by EGTA or verapamil. The DAG content of MIN6 β-cells was not affected by glucose. Both CCh and ACh evoked an increase in DAG which was maximal after 5 min and returned to basal after 30 min; EGTA abolished the cholinergic-induced increased in DAG. ACh caused a transient rise in [Ca²⁺]_i which was abolished by omission of Ca²⁺ or by addition of devapamil. Thus, cholinergic stimulation of β-cell insulin release is associated with changes in both [Ca²⁺]_i and DAG. The latter change persists longer than the former and activation of protein kinase C and sensitization of the secretory process to Ca²⁺ may underlie the prolonged effects of cholinergic agonists on insulin release. However, a secretory response to CCh was still evident after both [Ca²⁺]_i and DAG had returned to control values suggesting that additional mechanisms may be involved.