脾虚大鼠壁细胞钙调素活性及黄芪作用的研究

目的：观察大黄、利血平两种脾虚模型大鼠胃壁细胞内钙调素活性变化以及黄芪注射液对其的作用。方法：胃壁细胞分离采用Lewin胃袋法Percoll密度梯度离心，细胞内钙调素活性测定用PDE酶法，结果：大黄和利血平脾虚模型大鼠胃壁细胞钙调素活性（ng/mg)明显下降，黄芪注射液治疗后钙调素活性明显上升，结论：脾虚大鼠壁细胞钙调素活性明显下降，黄芪注射液对壁细胞内钙调素活性这一病理变化有明显调整作用。     

Engulfment and intracellular killing of F9 teratocarcinoma cells by non-activated murine macrophages

Activated macrophages kill several types of tumor cells in vitro, whereas non-activated macrophages lack this capacity. We, however, observed that non-activated macrophages efficiently kill F9 teratocarcinoma as well as other teratocarcinoma cell lines. Dexamethasone, a glucocorticoid known to prevent macrophage activation, did not perturb the killing of F9 teratocarcinoma cells. Neither tumor necrosis factor alpha, nor the reactive oxygen intermediates, i.e. hydrogen peroxide, superoxide anion, and hydroxyl radical, nor serine proteases participated in this killing, shown by employing various agents which interfere with their production, secretion, or function. Using acridine orange/ethidium bromide vitality staining, the F9 teratocarcinoma cells were shown to be phagocytized alive by macrophages and subsequently killed intracellularly. Intact lysosomal function is required for the killing of F9 cells, as the lysosomotropic drugs chloroquine and ammonium chloride markedly inhibited this killing without perturbing their engulfment. The signal transduction pathway induced in the macrophages upon interaction with F9 teratocarcinoma cells seems to differ from that induced by macrophage activation. Neither the protein kinase C inhibitors polymyxin B and H-7 [1-(5-isoquinolinylsulfonyl)-2-methyl piperazine] nor the protein kinase C activator phorbol 12-myristate-13-acetate affected the killing of F9 cells. However, chlorpromazine (a powerful inhibitor of calmodulin), dibutyryl cAMP (a cAMP analog), and prostaglandin E2 inhibited the macrophage-mediated killing of F9 cells. In vivo studies indicate that an increased number of macrophages at the F9 tumor inoculation site (the peritoneal cavity) as a result of elicitation by thioglycollate prevents F9 tumor development. Our findings indicate that non-activated macrophages kill teratocarcinoma cells using a mechanism which differs from that employed by activated macrophages in the killing of other tumor cells.     

Phosphorylation and function of cardiac myosin binding protein-C in health and disease

During the past 5 years there has been an increasing body of literature describing the roles cardiac myosin binding protein C (cMyBP-C) phosphorylation play in regulating cardiac function and heart failure. cMyBP-C is a sarcomeric thick filament protein that interacts with titin, myosin and actin to regulate sarcomeric assembly, structure and function. Elucidating the function of cMyBP-C is clinically important because mutations in this protein have been linked to cardiomyopathy in more than sixty million people worldwide. One function of cMyBP-C is to regulate cross-bridge formation through dynamic phosphorylation by protein kinase A, protein kinase C and Ca²⁺-calmodulin-activated kinase II, suggesting that cMyBP-C phosphorylation serves as a highly coordinated point of contractile regulation. Moreover, dephosphorylation of cMyBP-C, which accelerates its degradation, has been shown to associate with the development of heart failure in mouse models and in humans. Strikingly, cMyBP-C phosphorylation presents a potential target for therapeutic development as protection against ischemic-reperfusion injury, which has been demonstrated in mouse hearts. Also, emerging evidence suggests that cMyBP-C has the potential to be used as a biomarker for diagnosing myocardial infarction. Although many aspects of cMyBP-C phosphorylation and function remain poorly understood, cMyBP-C and its phosphorylation states have significant promise as a target for therapy and for providing a better understanding of the mechanics of heart function during health and disease. In this review we discuss the most recent findings with respect to cMyBP-C phosphorylation and function and determine potential future directions to better understand the functional role of cMyBP-C and phosphorylation in sarcomeric structure, myocardial contractility and cardioprotection.     

Role of calcium/calmodulin signaling pathway in Vibrio vulnificus cytolysin-induced hyperpermeability

Endothelial hyperpermeability, a hallmark of septicemia, is induced by stress fiber formation, which is primarily regulated by the calcium/calmodulin signaling pathway in endothelial cells. We previously reported that trifluoperazine, a calcium/calmodulin antagonist, blocks Vibrio vulnificus cytolysin (VVC) -induced lethality at in vivo animal model. The object of this study was therefore to examine whether VVC induces stress fiber formation through calcium/calmodulin signaling in endothelial cells. Here, we monitored calcium-influx after treatment of VVC using confocal microscopy in CPAE cells, pulmonary endothelial cell line. Interestingly, we found that VVC-induced dose-dependently increases of [Ca²⁺]_i in CPAE cells. Moreover, VVC-induced stress fiber formation as well as phosphorylation of myosin light chain (MLC) in a dose- and time-dependent manner, which was completely blocked by trifluoperazine. These results suggest that the calcium/calmodulin signaling pathway plays a pivotal role in VVC-induced hyperpermeability.     

Characterization of the microtubule-binding activity of kinesin-like calmodulin binding protein from Dunaliella salina

Although the C-terminal motor and the N-terminal myosin-like domains of KCBP in Dunaliella salina (DsKCBP) are implicated in interaction with the microtubules, its microtubule binding property has not been addressed. It has been shown that several calmodulin isoforms suppress the microtubule binding activity of KCBP, but whether the calmodulin-like protein (CLP) has this ability remains unknown. The results of our previous study showed that there are two microtubule binding sites in DsKCBP, motor domain at the C-terminus and MyTH4-FREM at the N-terminus. In the present study, MyTH4, without the companion of FERM, was identified as the minimal domain responsible for interaction with the microtubules in the N-terminal of DsKCBP. CLP interacted with the calmodulin-binding domain of DsKCBP in the presence of Ca²⁺, and inhibited the microtubule-binding activity of motor domain but not MyTH4 domain. Furthermore, MyTH4 domain in the N-terminus of DsKCBP was responsible for binding to the microtubules, and had 10-fold weaker affinity to the microtubules than the motor domain.     

Precision Medicine in Catecholaminergic Polymorphic Ventricular Tachycardia: JACC Focus Seminar 5/5

In this final of a 5-part Focus Seminar series on precision medicine, we focus on catecholaminergic polymorphic ventricular tachycardia (CPVT). This focus on CPVT allows us to take a “deep dive” and explore the full extent of the precision medicine opportunities for a single cardiovascular condition at a level that was not possible in the preceding articles. As a new paradigm presented in this article, it has become clear that CPVT can occur as either a typical or atypical form. Although there is a degree of overlap between the typical and atypical forms, it is notable that they arise due to different underlying genetic changes, likely exhibiting differing mechanisms of action, and presenting with different phenotypic features. The recognition of these differing forms of CPVT and their different etiologies and mechanisms is an important step toward implementing rapidly emerging precision medicine approaches that will tailor novel therapies to specific gene defects.     

钙通道和Na+/H+交换在表皮生长因子促肝癌细胞生长中的作用

目的许多生长因子如表皮生长因子(EGF),与肿瘤的发生密切相关.EGF与表皮生长因子受体(EGFR)结合,通过一系列的信息传导,导致肝癌细胞的增生.但受体后的信息传导机制尚不清楚.本实验探讨酪氨酸激酶、蛋白激酶C、Na+/H+交换、钙调蛋白和电压依赖性钙通道在EGF促肝癌细胞生长中的作用.方法本研究于无血清RPMI1640中培养肝癌细胞SMMC7721,采用3H-Thymidine(3H-TdR)掺入的方法,检测肝癌细胞DNA合成速率,研究酪氨酸激酶、蛋白激酶C、Na+/H+交换、钙调蛋白和电压依赖性钙通道在EGF促肝癌细脆生长中的作用.结果EGF 10-9M对肝癌细脆的生长有极显著促进作用,与对照组比较差异有显著意义(P＜0.05),酪氮酸激酶阻滞剂Genistein对EGF的促肝癌细胞生长作用具有极显著抑制作用(P＜0.001).钙调蛋白阻滞剂W-7、蛋白激酶C阻滞剂H-7和Na+/H+交换阻滞剂amiloride对EGF的促肝癌细胞生长作用具有显著抑制作用(P＜0.001,P＜0.01,P＜0.05),而对基础状态细胞的3H-TdR掺入值无显著影响(P＞0.05).电压依赖性钙通道阻滞剂Varapamil对BGF的促肝癌细胞生长作用无显著抑制作用(P＞0.05),对基础状态细胞的3H-TdR掺入值亦无显著影响(P＞0.05).结论结果显示,酪氨酸激酶、蛋白激酶C、Na+/H+交换及依赖钙-钙调蛋白途径在BGF的作用中起关键作用.电压依赖性钙通道与EGF的作用无关.     

Elementary mechanisms of calmodulin regulation of NaV1.5 producing divergent arrhythmogenic phenotypes

In cardiomyocytes, Na_V1.5 channels mediate initiation and fast propagation of action potentials. The Ca²⁺-binding protein calmodulin (CaM) serves as a de facto subunit of Na_V1.5. Genetic studies and atomic structures suggest that this interaction is pathophysiologically critical, as human mutations within the Na_V1.5 carboxy-terminus that disrupt CaM binding are linked to distinct forms of life-threatening arrhythmias, including long QT syndrome 3, a “gain-of-function” defect, and Brugada syndrome, a “loss-of-function” phenotype. Yet, how a common disruption in CaM binding engenders divergent effects on Na_V1.5 gating is not fully understood, though vital for elucidating arrhythmogenic mechanisms and for developing new therapies. Here, using extensive single-channel analysis, we find that the disruption of Ca²⁺-free CaM preassociation with Na_V1.5 exerts two disparate effects: 1) a decrease in the peak open probability and 2) an increase in persistent Na_V openings. Mechanistically, these effects arise from a CaM-dependent switch in the Na_V inactivation mechanism. Specifically, CaM-bound channels preferentially inactivate from the open state, while those devoid of CaM exhibit enhanced closed-state inactivation. Further enriching this scheme, for certain mutant Na_V1.5, local Ca²⁺ fluctuations elicit a rapid recruitment of CaM that reverses the increase in persistent Na current, a factor that may promote beat-to-beat variability in late Na current. In all, these findings identify the elementary mechanism of CaM regulation of Na_V1.5 and, in so doing, unravel a noncanonical role for CaM in tuning ion channel gating. Furthermore, our results furnish an in-depth molecular framework for understanding complex arrhythmogenic phenotypes of Na_V1.5 channelopathies.

Voltage-gated sodium channels (Na_V) are responsible for the initiation and spatial propagation of action potentials (AP) in excitable cells (1, 2). Na_V channels undergo rapid activation that underlie the AP upstroke while ensuing inactivation permits AP repolarization. The Na_V1.5 channel constitutes the predominant isoform in cardiomyocytes, whose pore-forming α-subunit is encoded by the SCN5A gene. Na_V1.5 dysfunction underlies diverse forms of cardiac disease including cardiomyopathies, arrhythmias, and sudden death (3–6). Human mutations in Na_V1.5 are associated with two forms of inherited arrhythmias–congenital long QT syndrome 3 (LQTS3) and Brugada syndrome (BrS) (7). LQTS3 stems from delayed or incomplete inactivation of Na_V1.5 that causes persistent Na influx that prolongs AP repolarization—a “gain-of-function” phenotype (7–9). BrS predisposes patients to sudden death and is associated with a reduction in the peak Na current that may slow cardiac conduction or cause region-specific repolarization differences—a “loss-of-function” phenotype (10, 11). Genetic studies have identified an expanding array of mutations in multiple Na_V1.5 domains, including the channel carboxy-terminus (CT) that is a hotspot for mutations linked to both LQTS3 and BrS (12, 13). This domain interacts with the Ca²⁺-binding protein calmodulin (CaM), suggesting that altered CaM regulation of Na_V1.5 may be a common pathophysiological mechanism (12, 14–16). More broadly, human mutations in the homologous regions of neuronal Na_V1.1 (17, 18), Na_V1.2 (19, 20), and Na_V1.6 (21) as well as skeletal muscle Na_V1.4 (22) are linked to varied clinical phenotypes including epilepsy, autism spectrum disorder, neurodevelopmental delay, and myotonia (23). Taken together, a common Na_V mechanistic deficit—defective CaM regulation—may underlie these diverse diseases.CaM regulation of Na_V channels is complex, isoform specific, and mediated by multiple interfaces within the channel (14–16). The Na_V CT consists of a dual vestigial EF hand segment and a canonical CaM-binding “IQ” (isoleucine–glutamine) domain (24, 25) (Fig. 1A). The IQ domain of nearly all Na_V channels binds to both Ca²⁺-free CaM (apoCaM) and Ca²⁺/CaM, similar to Ca_V channels (26–31). As CaM is typically a Ca²⁺-dependent regulator, much attention has been focused on elucidating Ca²⁺-dependent changes in Na_V gating. For skeletal muscle Na_V1.4, transient elevation in cytosolic Ca²⁺ causes a dynamic reduction in the peak current, a process reminiscent of Ca²⁺/CaM-dependent inactivation of Ca_V channels (32). Cardiac Na_V1.5 by comparison exhibits no dynamic effect of Ca²⁺ on the peak current (32–34). Instead, sustained Ca²⁺ elevation has been shown to elicit a depolarizing shift in Na_V1.5 steady-state inactivation (SSI or h_∞), although the magnitude and the presence of a shift have been debated (32, 35). Additional CaM-binding sites have been identified in the channel amino terminus domain (36) and the III-IV linker near the isoleucine, phenylalanine, and methionine (IFM) motif that is well recognized for its role in fast inactivation (35, 37). However, recent cryogenic electron microscopy structures, biochemical, and functional analyses suggest that both the III-IV linker and the Domain IV voltage-sensing domain might instead interact with the channel CT in a state-dependent manner (38–43).Open in a separate windowFig. 1.Absence of dynamic Ca²⁺/CaM effects on WT Na_V1.5 SSI. (A, Left) Structure of Na_V1.5 transmembrane domain (6UZ3) (70) juxtaposed with that of Na_V1.5 CT–apoCaM complex (4OVN) (28). (Right) Arrhythmia-linked CT mutations highlighted in Na_V1.5 CT–apoCaM structure (LQTS3, blue; BrS, magenta; mixed syndrome, purple). (B) Dynamic Ca²⁺-dependent changes in Na_V1.5 SSI probed using Ca²⁺ photouncaging. Na currents specifying h_∞ at ∼100 nM (Left) and ∼4 μM Ca²⁺ step (Right). (C) Population data for Na_V1.5 SSI under low (black, Left) versus high (red, Right) intracellular Ca²⁺ reveal no differences (P = 0.55, paired t test). Dots and bars are mean ± SEM (n = 8 cells). (D) FRET two-hybrid analysis of Cerulean-tagged apoCaM interaction with various Venus-tagged Na_V1.5 CT (WT, black; IQ/AA, red; S[1904]L, blue). Each dot is FRET efficiency measured from a single cell. Solid line fits show 1:1 binding isotherm.Beyond Ca²⁺-dependent effects, the loss of apoCaM binding to the Na_V1.5 IQ domain increases persistent current (34, 44), suggesting that apoCaM itself may be pathophysiologically relevant. Indeed, Na_V1.5 mutations in the apoCaM-binding interface are associated with LQTS3 and atrial fibrillation (7), as well as a loss-of-function BrS phenotype and a mixed-syndrome phenotype whereby some patients present with BrS while others with LQTS3 (Fig. 1A) (13, 45). How alterations in CaM binding paradoxically elicits both gain-of-function and loss-of-function effects is not fully understood, though important to delineate pathophysiological mechanisms and for personalized therapies.Here, using single- and multichannel recordings, we show that apoCaM binding elicits two distinct effects on Na_V1.5 gating: 1) an increase in the peak channel open probability (P_O/peak) and 2) a reduction in the normalized persistent channel open probability (R_persist), consistent with previous studies (34, 44). The two effects may explain how mixed-syndrome mutations in the Na_V1.5 CT produce either BrS or LQTS3 phenotypes. On one hand, the loss of apoCaM association may diminish P_O/peak and induce BrS by shunting cardiac AP. On the other hand, increased R_persist may prevent normal AP repolarization, resulting in LQTS3. Analysis of elementary mechanisms suggests that these changes relate to a switch in the state dependence of channel inactivation. Furthermore, dynamic changes in Ca²⁺ can inhibit persistent current for certain mutant Na_V1.5 owing to enhanced Ca²⁺/CaM binding that occurs over the timescale of a cardiac AP. This effect may result in beat-to-beat variability in persistent Na current for some mutations. In all, these findings explain how a common deficit in CaM binding can contribute to distinct arrhythmogenic mechanisms.     

Serotonin inhibits Na+/H+ exchange activity via 5-HT4 receptors and activation of PKC alpha in human intestinal epithelial cells

BACKGROUND & AIMS: Increased serotonin levels have been implicated in the pathophysiology of diarrhea associated with celiac and inflammatory diseases. However, the effects of serotonin on Na+ /H+ exchange (NHE) activity in the human intestine have not been investigated fully. The present studies examined the acute effects of 5-hydroxytryptamine (5-HT) on NHE activity using Caco-2 cells as an in vitro model. METHODS: Caco-2 cells were treated with 5-HT (.1 micromol/L, 1 h) and NHE activity was measured as ethyl-isopropyl-amiloride (EIPA)-sensitive 22Na uptake. The effect of 5-HT receptor-specific agonists and antagonists was examined. The role of signaling intermediates in 5-HT-mediated effects on NHE activity was elucidated using pharmacologic inhibitors and immunoblotting. RESULTS: NHE activity was inhibited significantly (approximately 50%-75%, P < .05) by .1 micromol/L 5-HT via inhibition of maximal velocity (Vmax) without any changes in apparent affinity (Km) for the substrate Na+ . NHE inhibition involved a decrease of both NHE2 and NHE3 activities. Studies using specific inhibitors and agonists showed that the effects of 5-HT were mediated by 5-HT4 receptors. 5-HT-mediated inhibition of NHE activity was dependent on phosphorylation of phospholipase C gamma 1 (PLC gamma 1) via activation of src-kinases. Signaling pathways downstream of PLC gamma 1 involved increase of intracellular Ca 2+ levels and subsequent activation of protein kinase C alpha (PKC alpha). The effects of 5-HT on NHE activity were not cell-line specific because T84 cells also showed NHE inhibition. CONCLUSIONS: A better understanding of the regulation of Na+ absorption by 5-HT offers the potential for providing insights into molecular and cellular mechanisms involved in various diarrheal and inflammatory disorders.