钙调蛋白抑制剂对小鼠精子获能影响的研究

目的:探讨钙调蛋白是否参与小鼠精子获能过程。方法:用50、100、200μmol/L浓度的钙调蛋白抑制剂W7和10、20、30μmol/L浓度的钙调蛋白抑制剂卡米达佐(CZ)分别与小鼠精子孵育2h,通过金霉素染色法计算出B型精子百分率。并用100μmol/L钙调蛋白抑制剂W7和10μmol/LCZ分别与小鼠精子孵育2h后,再加入5μmol/L孕酮诱发顶体反应,计算精子顶体反应百分率。结果:不同浓度的W7和CZ作用小鼠精子,B型精子百分率呈浓度依赖性方式下降,与对照组比较,均有显著性差异(P<0.05)。顶体反应率与对照组相比均有极显著差异(P<0.01)。结论:钙调蛋白参与小鼠精子获能,是精子获能过程中的关键蛋白。     

The dystrophin–glycoprotein complex,cellular signaling,and the regulation of cell survival in the muscular dystrophies

Mutations of different components of the dystrophin–glycoprotein complex (DGC) cause muscular dystrophies that vary in terms of severity, age of onset, and selective involvement of muscle groups. Although the primary pathogenetic processes in the muscular dystrophies have clearly been identified as apoptotic and necrotic muscle cell death, the pathogenetic mechanisms that lead to cell death remain to be determined. Studies of components of the DGC in muscle and in nonmuscle tissues have revealed that the DGC is undoubtedly a multifunctional complex and a highly dynamic structure, in contrast to the unidimensional concept of the DGC as a mechanical component in the cell. Analysis of the DGC reveals compelling analogies to two other membrane‐associated protein complexes, namely integrins and caveolins. Each of these complexes mediates signal transduction cascades in the cell, and disruption of each complex causes muscular dystrophies. The signal transduction cascades associated with the DGC, like those associated with integrins and caveolins, play important roles in cell survival signaling, cellular defense mechanisms, and regulation of the balance between cell survival and cell death. This review focuses on the functional components of the DGC, highlighting the evidence of their participation in cellular signaling processes important for cell survival. Elucidating the link between these functional components and the pathogenetic processes leading to cell death is the foremost challenge to understanding the mechanisms of disease expression in the muscular dystrophies due to defects in the DGC. © 2001 John Wiley & Sons, Inc. Muscle Nerve 24: 1575–1594, 2001     

Solution structure of the regulatory domain of human cardiac troponin C in complex with the switch region of cardiac troponin I and W7: The basis of W7 as an inhibitor of cardiac muscle contraction

The solution structure of Ca²⁺-bound regulatory domain of cardiac troponin C (cNTnC) in complex with the switch region of troponin I (cTnI_147-163) and the calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfinamide (W7), has been determined by NMR spectroscopy. The structure reveals that the W7 naphthalene ring interacts with the terminal methyl groups of M47, M60, and M81 as well as aliphatic and aromatic side chains of several other residues in the hydrophobic pocket of cNTnC. The H3 ring proton of W7 also contacts the methyl groups of I148 and M153 of cTnI_147-163. The N-(6-aminohexyl) tail interacts primarily with the methyl groups of V64 and M81, which are located on the C- and D-helices of cNTnC. Compared to the structure of the cNTnC•Ca²⁺•W7 complex (Hoffman, R. M. B. and Sykes, B. D. (2009) Biochemistry 48, 5541-5552), the tail of W7 reorients slightly toward the surface of cNTnC while the ring remains in the hydrophobic pocket. The positively charged -NH₃⁺ group from the tail of W7 repels the positively charged R147 of cTnI_147-163. As a result, the N-terminus of the peptide moves away from cNTnC and the helical content of cTnI_147-163 is diminished, when compared to the structure of cNTnC•Ca²⁺•cTnI_147-163 (Li, M. X., Spyracopoulos, L., and Sykes B. D. (1999) Biochemistry 38, 8289-8298). Thus the ternary structure cNTnC•Ca²⁺•W7•cTnI_147-163 reported in this study offers an explanation for the ∼ 13-fold affinity reduction of cTnI_147-163 for cNTnC•Ca²⁺ in the presence of W7 and provides a structural basis for the inhibitory effect of W7 in cardiac muscle contraction. This generates molecular insight into structural features that are useful for the design of cTnC-specific Ca²⁺-desensitizing drugs.     

Effect of Ace-Inhibitors,Calmodulin Antagonists,Acetylcholine Receptor Blocking,and Alpha Receptor Blocking Agents on Motility of Human Sperm

The purpose of this study was to investigate the influence of several pharmacological compounds on the motility and velocity of washed human spermatozoa. Results were evaluated by multiple exposure photography and computer-aided picture analysis. The motility-inhibiting effect of the antifertility drug gossypol was confirmed. Gossypol proved to be a potent inhibitor of the angiotensin converting enzyme (ACE) detectable in high concentrations in seminal plasma. However, human sperm motility was not inhibited during incubation with two other specific ACE-inhibitors (captopril, enalapril). On the contrary, high concentrations of captopril even showed a slight motility-stimulating effect. These results indicate no direct involvement of ACE in the regulation of sperm motility but suggest a direct interaction of gossypol with the plasma membrane of spermatozoa. To clarify whether or not gossypol blocks membranous ion transport, the effect of well-defined ion transport blocking agents on sperm motility was investigated. It was determined that the acetylcholine receptor blocker alpha-bungarotoxin and trifluoperazin, a specific calmodulin antagonist, inhibit sperm motility completely. Since stimulation of sperm motility by captopril may be due to an alpha-mimetic action of this compound, the influence of two alpha receptor blockers (bromocriptine, lisuride) on sperm motility was studied. Although lisuride inhibited sperm motility completely, bromocriptine revealed no influence. A temporary and reversible intervention with membrane transport processes could be a suitable way to regulate human sperm motility and male fertility.     

Nuclear magnetic resonance imaging of tumour growth and neovasculature performance in vivo reveals Grb7 as a novel antiangiogenic target

Development of neovasculature is a necessary requirement for tumour growth and it provides additional opportunities for therapeutic intervention. However, current antiangiogenic therapies have limited efficacy, mostly because of the development of resistance. Hence, characterization of new antiangiogenic molecular targets is of considerable clinical interest. We report that a calmodulin‐binding domain (CaM‐BD) deletion mutant of the growth factor receptor bound protein 7 (Grb7) (denoted Grb7Δ) impairs tumour growth and associated angiogenesis in vivo. We implanted glioma C6 cells in rat brains transfected with an enhanced yellow fluorescent protein (EYFP) chimera of Grb7?, its EYFP‐Grb7 wild type counterpart, and EYFP alone. We systematically followed intracerebral growth of the tumours, their cellularity and the functional performance of tumour‐associated microvasculature using magnetic resonance imaging, including anatomical T1W and T2W images and functional diffusion and perfusion parameters. Tumours grown from implanted C6 cells expressing EYFP‐Grb7Δ developed slower, became smaller and presented lower apparent cellularity than those derived from cells expressing EYFP‐Grb7 and EYFP. Vascular perfusion measurements within tumours derived from EYFP‐Grb7?‐expressing cells showed significantly lower cerebral blood flow (CBF), cerebral blood volume (CBV) and mean transit time (MTT) values. These findings were independently validated by histological and immunohistochemical techniques. Taken together, these findings confirm that the CaM‐BD of Grb7 plays an important role in tumour growth and associated angiogenesis in vivo, thus identifying this region of the protein as a novel target for antiangiogenic treatment. Copyright © 2013 John Wiley & Sons, Ltd.     

Migration of primary cultured rabbit gastric epithelial cells requires intact protein kinase C and Ca2+/calmodulin activity

Superficial gastric mucosal injury is rapidly repaired by epithelial cell migration. This study aims to characterize the intracellular signal transduction pathways underlying the repair process. Primary monolayer cultures of rabbit gastric epithelial cells were wounded. The measured spontaneous cell migration speed at the edge of the wound was 457 ± 89 m/24 hr. Epidermal growth factor stimulated and genistein (receptor tyrosine protein kinase inhibitor) inhibited cell migration significantly. Down-regulation of protein Kinase C (PKC) with long-term phorbol 12-myristate 13-acsetate or inhibition with calphostin-C significantly inhibited cell migration. Blocking of Ca²⁺ channels with verapamil and endogenous Ca²⁺ release with TMB-8 or inhibition of the Ca²⁺/calmodulin complex with calmidazolium likewise significantly inhibited migration speed and also abolished the rise of [Ca²⁺]_i, which was measured in migrating cells. Modulation of the cAMP-PKA pathway or prostaglandin synthesis had no influence on cell migration. Gastric epithelial cell migration implies activation of receptor tyrosine kinase. It is associated with increased [Ca²⁺]_i and requires an intact Ca²⁺/calmodulin complex. Intact PKC activity also is needed.     

慢性缺氧对大鼠血浆内皮素及心肌ETRa、CaM和CaMKⅡ的影响

目的：研究慢性缺氧对大鼠血浆内皮素和心肌酶的影响。方法：以常压缺氧方式建立缺氧动物模型，SD幼鼠被随机分为3组：正常组、缺氧1周组和缺氧3周组，通过酶联免疫吸附试验检测不同组动物血浆内皮素变化，通过RT—PCR方法检测心肌组织中ETRa、CaM、CaMKⅡ的mRNA表达。结果：血浆内皮素水平随缺氧时间延长而升高，正常组、缺氧1周组和缺氧3周组比较均有差异（P〈0．05）。同时，心肌细胞ETRa、CaM、CaMKⅡ mRNA的转录水平也随缺氧时问延长而增加，缺氧1周组和正常组比较有差异（P〈0．05）；但缺氧3周组和缺氧1周组比较有所不同，CaM和CaMKⅡδ转录水平表现有差异（P〈0．05），而ETRa和CaMKⅡγ转录水平无差异（P〉0．05）。结论：慢性缺氧使动物血浆中内皮素水平升高，心肌细胞ETRa、CaM和CaMKⅡ mRNA转录增加，可能会对心脏功能产生影响。     

L-selectin shedding is activated specifically within transmigrating pseudopods of monocytes to regulate cell polarity in vitro

L-selectin is a cell adhesion molecule that tethers free-flowing leukocytes from the blood to luminal vessel walls, facilitating the initial stages of their emigration from the circulation toward an extravascular inflammatory insult. Following shear-resistant adhesion to the vessel wall, L-selectin has frequently been reported to be rapidly cleaved from the plasma membrane (known as ectodomain shedding), with little knowledge of the timing or functional consequence of this event. Using advanced imaging techniques, we observe L-selectin shedding occurring exclusively as primary human monocytes actively engage in transendothelial migration (TEM). Moreover, the shedding was localized to transmigrating pseudopods within the subendothelial space. By capturing monocytes in midtransmigration, we could monitor the subcellular distribution of L-selectin and better understand how ectodomain shedding might contribute to TEM. Mechanistically, L-selectin loses association with calmodulin (CaM; a negative regulator of shedding) specifically within transmigrating pseudopods. In contrast, L-selectin/CaM interaction remained intact in nontransmigrated regions of monocytes. We show phosphorylation of L-selectin at Ser 364 is critical for CaM dissociation, which is also restricted to the transmigrating pseudopod. Pharmacological or genetic inhibition of L-selectin shedding significantly increased pseudopodial extensions in transmigrating monocytes, which potentiated invasive behavior during TEM and prevented the establishment of front/back polarity for directional migration persistence once TEM was complete. We conclude that L-selectin shedding directly regulates polarity in transmigrated monocytes, which affirms an active role for this molecule in driving later stages of the multistep adhesion cascade.The passage of leukocytes from the circulation toward the surrounding extravascular space is a critical event in the inflammatory response (1–3). The multistep adhesion cascade defines the increasingly adhesive steps leukocytes make with the endothelium to exit the circulation and enter the surrounding microenvironment (4). Each step of the cascade [tethering, rolling, slow rolling, firm adhesion, and transendothelial migration (TEM)] critically depends on cell adhesion molecules expressed on both leukocytes and endothelial cells. Such cell adhesion molecules have been identified and characterized to act in a sequential and interdependent manner. The molecular mechanism driving leukocyte integrin function during the multistep adhesion cascade is relatively well defined (4–6). In contrast, mechanisms underpinning the regulation of nonintegrin receptors are only beginning to emerge. One such example, L-selectin, is known to promote the initial tethering and subsequent rolling of leukocytes along activated endothelial cells (7). Knocking out L-selectin in mice has provided compelling evidence for this cell adhesion molecule in directing neutrophils toward sites of inflammation (8). Intriguingly, knocking out L-selectin has a dramatic impact on neutrophil chemotaxis in vivo, but the molecular basis for this observation remains elusive (9). Following firm adhesion, L-selectin is broadly considered to be proteolytically cleaved (or shed) at a defined extracellular membrane/proximal domain by membrane-associated matrix metalloproteinases (10). The best-characterized “sheddase” is a disintegrin and metalloproteinase (ADAM17; or TNF-α concerting enzyme) (11). Shedding of L-selectin hinges on its interaction with calmodulin (CaM); binding of CaM to L-selectin protects from shedding, and leukocyte activation leads to CaM dissociation from the L-selectin tail to drive shedding (12). What promotes CaM dissociation is not known, but mutating the L-selectin tail can have a profound impact on the shedding response (13–15). L-selectin shedding is a rapid event, and the biological significance of its outcome may be numerous (10). In respect to leukocyte recruitment, L-selectin shedding would rapidly halt any further contribution of this molecule toward cell adhesion, signaling, or migration. The timing of L-selectin shedding is therefore critical in relation to input signals derived from other surface receptors contributing to the same cellular event (7). Our understanding of coordinated receptor signaling during TEM is extremely poor, mainly because insight into the molecular regulation of each individual cell adhesion molecule is still lacking.The contribution of L-selectin to the multistep adhesion cascade has been defined through two major experimental approaches: function-blocking studies (using soluble ligand or monoclonal antibody) and gene KO/knock-in mice. Such studies have built the foundation of our knowledge of the adhesion cascade. However, these approaches limit our understanding of where the targeted molecule’s first nonredundant step is required. Direct imaging of cell adhesion molecules in space and time, during recruitment under flow conditions, can provide additional clues beyond the first nonredundant point of execution. Here, we have used a series of advanced imaging techniques to pinpoint where and when L-selectin is cleaved during the multistep adhesion cascade. Using primary human monocytes, we reveal the shedding event was occurring specifically during TEM and not before. Through stable expression of WT and mutant forms of L-selectin tagged to GFP or red fluorescent protein (RFP), we could define the shedding event on a mechanistic level in THP-1 cells. Fluorescence lifetime imaging microscopy (FLIM) enabled quantitative measurement of the fluorescence resonance energy transfer (FRET) efficiency between L-selectin–GFP and CaM-RFP, allowing us to monitor the subcellular distribution of their interaction during TEM. We show, for the first time to our knowledge, that L-selectin shedding can regulate the invasive behavior of monocytes crossing activated endothelial monolayers under flow. Furthermore, we show that polarity in transmigrated monocytes is disrupted if shedding of L-selectin is blocked. Taken together, these results reveal previously unidentified roles for L-selectin that extend beyond tethering and rolling.     

Membrane translocation of TRPC6 channels and endothelial migration are regulated by calmodulin and PI3 kinase activation

Lipid oxidation products, including lysophosphatidylcholine (lysoPC), activate canonical transient receptor potential 6 (TRPC6) channels leading to inhibition of endothelial cell (EC) migration in vitro and delayed EC healing of arterial injuries in vivo. The precise mechanism through which lysoPC activates TRPC6 channels is not known, but calmodulin (CaM) contributes to the regulation of TRPC channels. Using site-directed mutagenesis, cDNAs were generated in which Tyr⁹⁹ or Tyr¹³⁸ of CaM was replaced with Phe, generating mutant CaM, Phe⁹⁹-CaM, or Phe¹³⁸-CaM, respectively. In ECs transiently transfected with pcDNA3.1-myc-His-Phe⁹⁹-CaM, but not in ECs transfected with pcDNA3.1-myc-His-Phe¹³⁸-CaM, the lysoPC-induced TRPC6-CaM dissociation and TRPC6 externalization was disrupted. Also, the lysoPC-induced increase in intracellular calcium concentration was inhibited in ECs transiently transfected with pcDNA3.1-myc-His-Phe⁹⁹-CaM. Blocking phosphorylation of CaM at Tyr⁹⁹ also reduced CaM association with the p85 subunit and subsequent activation of phosphatidylinositol 3-kinase (PI3K). This prevented the increase in phosphatidylinositol (3,4,5)-trisphosphate (PIP3) and the translocation of TRPC6 to the cell membrane and reduced the inhibition of EC migration by lysoPC. These findings suggest that lysoPC induces CaM phosphorylation at Tyr⁹⁹ by a Src family kinase and that phosphorylated CaM activates PI3K to produce PIP3, which promotes TRPC6 translocation to the cell membrane.Endothelial cell (EC) migration is required for healing after arterial injuries, such as those that occur with angioplasties. Oxidized low-density lipoprotein and lysophosphatidylcholine (lysoPC), the major lysophospholipid of oxidized low-density lipoprotein, are abundant in plasma and atherosclerotic lesions and inhibit EC migration (1). A brief influx of calcium is required to initiate EC migration (2), but lysoPC causes a prolonged influx of Ca²⁺ that disrupts the cytoskeletal dynamics required for normal EC migration (3, 4). Specifically, lysoPC activates canonical transient receptor potential 6 (TRPC6) channels, as shown by patch clamp recording, with Ca²⁺ influx (3). The increased [Ca²⁺]_i initiates events that result in TRPC5 channel activation (3). The later activation of TRPC5 compared with TRPC6 and the failure of TRPC6 and TRPC5 to coimmunoprecipitate indicates that they do not form a heteromeric complex. The importance of this pathway is found in TRPC6-deficient EC, where lysoPC has little effect on EC migration (3). Furthermore, a high cholesterol diet markedly inhibits endothelial healing in wild-type (WT) mice, but has no effect in TRPC6-deficient (TRPC6^−/−) mice (5). The mechanism of TRPC6 activation by lysoPC is not fully elucidated, limiting the ability to block this important pathway.Calmodulin (CaM), a small, highly conserved, intracellular calcium-binding protein (6), binds to TRPC channels and regulates their activation. TRPC proteins, including TRPC6, possess a C-terminal CaM-binding domain that overlaps with a binding site for the inositol trisphosphate receptor, and CaM and the inositol triphosphate receptor compete for binding at this site (7). Removal of CaM from the common binding site results in activation of TRP3 channels (8). TRPC proteins contain additional binding sites for CaM and other Ca²⁺-binding proteins, indicating a complex regulatory mechanism in response to changes in [Ca²⁺]_i that includes positive and negative regulation of channels (9). In addition to CaM regulating TRPC proteins by direct binding, CaM-dependent kinases activate TRPC channels (10). CaM activity and peptide binding affinity is altered by its phosphorylation state and bound Ca²⁺, and Ca²⁺ can regulate the phosphorylation of CaM (11). LysoPC activates tyrosine kinases, including Src family tyrosine kinases (12), and Src family kinases can phosphorylate CaM (13). The role of CaM and CaM phosphorylation in TRPC6 channel activation or in EC migration is incompletely understood.TRPC6 channel activation generally requires externalization; however, the mechanism of TRPC6 channel translocation to the plasma membrane is not clear. In HEK cells overexpressing TRPC6, stimulation of Gq protein-coupled receptors causes TRPC6 externalization and localization to caveolae or lipid rafts by an exocytotic mechanism (14). In smooth muscle cells, phosphatidylinositol 3-kinase (PI3K) is involved in carbachol-induced TRPC6 externalization (15). The mechanism by which lysoPC induces TRPC6 externalization in EC is unknown.PI3K produces phosphatidylinositol (3,4,5)-trisphosphate (PIP3) from phosphatidylinositol (4,5)-bisphosphate (PIP2) in the inner leaflet of the plasma membrane and participates in numerous intracellular signaling processes, including TRPC6 activation (16). PI3K is composed of a p85 regulatory subunit (p85α, p85β, or p55γ) and a p110 catalytic subunit (p110α, p110β, p110γ, or p110δ), and activity can be influenced by CaM association (17).The purpose of the present study is to explore the underlying mechanism of lysoPC-induced TRPC6 activation. We identify a mechanism in which phosphorylation of CaM at Tyr⁹⁹ plays a key role in lysoPC-induced TRPC6 externalization and inhibition of EC migration.