Reciprocal modulation of calcium dynamics at rod and cone photoreceptor synapses by nitric oxide

The abundance of nitric oxide (NO) synthesizing enzymes identified in the vertebrate retina highlight the importance of NO as a signaling molecule in this tissue. Here we describe opposing actions of NO on the rod and cone photoreceptor synapse. Depolarization-induced increases of calcium concentration in rods and cones were enhanced and inhibited, respectively, by the NO donor S-nitrosocysteine. NO suppressed calcium current in cones by decreasing the maximum conductance, whereas NO facilitated rod Ca channel activation. NO also activated a nonselective voltage-independent conductance in both rods and cones. Suppression of NO production in the intact retina with N(G)-nitro-l-arginine favored cone over rod driven postsynaptic signals, as would be expected if NO enhanced rod and suppressed cone synaptic activity. These findings may imply involvement of NO in regulating the strength of rod and cone pathways in the retina during different states of adaptation.     

The G12 family of heterotrimeric G proteins and Rho GTPase mediate Sonic hedgehog signalling

Sonic hedgehog (Shh) is a secreted morphogen crucial for cell fate decision, cellular proliferation, and patterning during vertebrate development. The intracellular Shh signalling is transduced by Smoothened (Smo), a seven-transmembrane spanning protein that belongs to the G-protein coupled receptor family. Among four families of Galpha subunits, Galphai has been thought to be responsible for transducing Shh signalling, while several lines of evidence indicated that other signalling pathways may be involved. We found that the G12 family of heterotrimeric G proteins and the small GTPase RhoA are involved in Shh/Smo-mediated cellular responses, including stimulation of target gene promoter and inhibition of neurite outgrowth of neuroblastoma cells. We also found that the G12/RhoA pathway is responsible for Smo-induced nuclear import of GLI3 which is thought to transduce Shh signals to nucleus. Furthermore, misexpression of a G12-specific GTPase-activating protein in rat neural tubes leads to pertubation of motor neurone and interneurone development, mimicking the effects of decreased Shh signalling. These results show that Shh signalling is mediated in part by activating G12 family coupled signalling pathways. The participation of RhoA, a pivotal molecular switch in many signal transduction pathways, may help explain how Shh can trigger a variety of cellular responses.     

Rho GTPase在信号转导和细胞骨架中的作用

Rho GTPases参与多种重要的细胞生命活动,如肌动蛋白细胞骨架的重构、细胞黏附、细胞运动、囊泡运输、转录激活、基因表达和细胞周期的调控等。当Rho GTPase蛋白水平改变,活性状态改变,效应蛋白丰度改变后,出现异常的Rho 信号,从而影响细胞骨架重组使细胞迁移。调节这些生物信号的转导通路非常复杂, 因此,Rho GTPases已成为近年来的研究热点。     

FMRP interferes with the Rac1 pathway and controls actin cytoskeleton dynamics in murine fibroblasts

Fragile X syndrome, the most common form of inherited mental retardation, is caused by absence of FMRP, an RNA-binding protein implicated in regulation of mRNA translation and/or transport. We have previously shown that dFMR1, the Drosophila ortholog of FMRP, is genetically linked to the dRac1 GTPase, a key player in actin cytoskeleton remodeling. Here, we demonstrate that FMRP and the Rac1 pathway are connected in a model of murine fibroblasts. We show that Rac1 activation induces relocalization of four FMRP partners to actin ring areas. Moreover, Rac1-induced actin remodeling is altered in fibroblasts lacking FMRP or carrying a point-mutation in the KH1 or in the KH2 RNA-binding domain. In absence of wild-type FMRP, we found that phospho-ADF/Cofilin (P-Cofilin) level, a major mediator of Rac1 signaling, is lowered, whereas the level of protein phosphatase 2A catalytic subunit (PP2Ac), a P-Cofilin phosphatase, is increased. We show that FMRP binds with high affinity to the 5'-UTR of pp2acbeta mRNA and is thus a likely negative regulator of its translation. The molecular mechanism unraveled here points to a role for FMRP in modulation of actin dynamics, which is a key process in morphogenesis of dendritic spines, synaptic structures abnormally developed in Fragile X syndrome patient's brain.     

Impaired cytoskeletal organization and membrane integrity in lens fibers of a Rho GTPase functional knockout transgenic mouse

To investigate the effects of Rho GTPase inactivation on lens fiber cell cytoskeletal and morphological integrity, a transgenic mouse model expressing C3-exoenzyme (a bacterial toxin) in a lens-specific manner was utilized. Cryosections of whole eyes from C3 transgenic mice and littermate controls were stained for F-actin with rhodamine-phalloidin or immunostained for beta-catenin, aquaporin-0 or connexin-50, and confocal images were recorded. Lens fiber cell morphology was examined at both light and electron microscopic levels. To investigate the influence of Rho GTPase inactivation on the profiles of gene expression, cDNA libraries generated from transgenic and littermate control mouse lenses were screened by cDNA microarray analysis. In contrast to the wild-type lens, fiber cells of the transgenic lens were grossly swollen and disorganized, with abnormal membrane architecture. Staining of F-actin, beta-catenin, aquaporin-0 and connexin-50 was reduced dramatically in the C3 transgenic lens as compared to controls. Western blot analysis and cDNA microarray analysis did not reveal any noticeable decreases in actin, beta-catenin and aquaporin-0 protein levels or expression in C3 transgenic lenses, indicating that altered cytoskeletal organization in response to Rho GTPase inactivation might underlie the noted changes in staining for these proteins. Additionally, cDNA microarray analysis of C3 lens revealed altered expression (at least two-fold, compared to littermate controls) of 44 genes. These include genes encoding extracellular matrix and basement membrane proteins, cell survival and apoptotic pathways, and ion and protein transport. These data indicate that disruption of Rho GTPase function in the developing mouse lens results in abnormal cytoskeletal organization, fiber cell interactions, impaired lens fiber cell morphology and altered gene expression of cellular proteins involved in diverse functions. This work reveals that the morphological and cytoskeletal abnormalities triggered upon Rho GTPase inactivation in lens could be one of the important insults associated with cataract formation in C3 transgenic mouse lens.     

Rapid and persistent modulation of actin dynamics regulates postsynaptic reorganization underlying bidirectional plasticity

The synapse is a highly organized cellular specialization whose structure and composition are reorganized, both positively and negatively, depending on the strength of input signals. The mechanisms orchestrating these changes are not well understood. A plausible locus for the reorganization of synapse components and structure is actin, because it serves as both cytoskeleton and scaffold for synapses and exists in a dynamic equilibrium between F-actin and G-actin that is modulated bidirectionally by cellular signaling. Using a new FRET-based imaging technique to monitor F-actin/G-actin equilibrium, we show here that tetanic stimulation causes a rapid, persistent shift of actin equilibrium toward F-actin in the dendritic spines of rat hippocampal neurons. This enlarges the spines and increases postsynaptic binding capacity. In contrast, prolonged low-frequency stimulation shifts the equilibrium toward G-actin, resulting in a loss of postsynaptic actin and of structure. This bidirectional regulation of actin is actively involved in protein assembly and disassembly and provides a substrate for bidirectional synaptic plasticity.     

Deficiency of the hematopoietic cell-specific Rho family GTPase Rac2 is characterized by abnormalities in neutrophil function and host defense

In mammals, the Rho family GTPase Rac2 is restricted in expression to hematopoietic cells, where it is coexpressed with Rac1. Rac2-deficient mice were created to define the physiological requirement for two near-identical Rac proteins in hematopoietic cells. rac2-/- neutrophils displayed significant defects in chemotaxis, in shear-dependent L-selectin-mediated capture on the endothelial substrate Glycam-1, and in both F-actin generation and p38 and, unexpectedly, p42/p44 MAP kinase activation induced by chemoattractants. Superoxide production by rac2-/- bone marrow neutrophils was significantly reduced compared to wild type, but it was normal in activated peritoneal exudate neutrophils. These defects were reflected in vivo by baseline neutrophilia, reduced inflammatory peritoneal exudate formation, and increased mortality when challenged with Aspergillus fumigatus. Rac2 is an essential regulator of multiple specialized neutrophil functions.     

A GAL4‐inducible transgenic tool kit for the in vivo modulation of Rho GTPase activity in zebrafish

Background: Rho GTPases are small monomeric G‐proteins that play key roles in many cellular processes. Due to Rho GTPases' widespread expression and broad functions, analyses of their function during late development require tissue‐specific modulation of activity. The GAL4/UAS system provides an excellent tool for investigating the function of Rho GTPases in vivo. With this in mind, we created a transgenic tool kit enabling spatial and temporal modulation of Rho GTPase activity in zebrafish. Results: Transgenic constructs were assembled driving dominant‐negative, constitutively active, and wild‐type versions of Cdc42, RhoA, and Rac1 under 10XUAS control. The self‐cleaving viral peptide F2A was utilized to allow bicistronic expression of a fluorescent reporter and Rho GTPase. Global heat shock of hsp70l:gal4⁺ transgenic embryos confirmed GAL4‐specific construct expression. Western blot analysis indicated myc‐tagged Rho GTPases were expressed only in the presence of GAL4. Construct expression was confined to proper cells when combined with pou4f3:gal4 or ptf1a:gal4. Finally, transgene expression resulted in reproducible defects in lens formation, indicating that the transgenes are functional in vivo. Conclusions: We generated and validated 10 transgenic lines, creating a versatile tool kit for the temporal‐spatial modulation of Cdc42, RhoA, and Rac1 activity in vivo. These lines will enable systematic analysis of Rho GTPase function in any tissue of interest. Developmental Dynamics 245:844–853, 2016. © 2016 Wiley Periodicals, Inc.     

Myenteric denervation in gastric carcinogenesis: differential modulation of nitric oxide and annexin-A1

This study evaluated the properties of endogenous nitric oxide synthases (NOS) and annexin-A1 (ANXA1) and determined how they can be exploited in the N-methyl-N-nitro-N-nitrosoguanidine (MNNG)-induced gastric carcinogenesis and myenteric denervation model. Male Wistar rats were treated with MNNG and/or aminoguanidine (AG) for 20 weeks. In another set of experiments, rats with nondenervated and denervated stomachs were treated with MNNG or water for 28 weeks. Fragments of the pyloric region were processed for histopathology, NOS activity, and immunohistochemistry to explore the activity and expression of constitutive (cNOS) and inducible (iNOS) NO synthase and their relationship with annexin-A1 (ANXA1) expression. NO inhibition by AG increased the percentage of animals with adenocarcinomas (~29%) compared with the untreated MNNG group (~4%). Myenteric denervation did not alter NOS activity. cNOS activity was significantly greater in nondernervated and denervated stomachs with or without lesions (P<0.001) than iNOS activity (P<0.01), as confirmed by immunohistochemistry. Further, cNOS activity in normal stomachs and outside the lesion area was considerably higher than inside it (P<0.01). By densitometric analysis of nondenervated and denervated stomachs, ANXA1 expression was modulated in epithelial and inflammatory cells (mast cells and neutrophils), wherein significant alterations were induced by lesion development and myenteric denervation. In conclusion, NO protects against the development of gastric adenocarcinomas. The pattern of ANXA1 expression was not associated with NOS activity or expression, suggesting that NO and ANXA1 act in gastric tumors in disparate pathways.     

Role of the monomeric GTPase Rho in hematopoietic progenitor cell migration and transplantation

To investigate the role of the monomeric guanosine triphosphatase (GTPase) Rho on migration of hematopoietic progenitor cells (HPC), we employed different clostridial toxins which inhibit the Rho family of GTPases. Pretreatment with C2I-C3, a cell-accessible C3 transferase fusion protein that targets Rho, increased chemokinetic migration of the factor-dependent multipotent cell line Factor Dependent Cell Paterson with mixed lineage differentiation potential (FDCP-mix) and of primary lineage marker-depleted HPC in vitro. In contrast, treatment with lethal toxin (LT) from Clostridium sordellii, which predominantly inactivates Rac, and with toxin B from C. difficile, which inactivates Rho, Rac and Cdc42, decreased in vitro migration. When HPC pretreated with LT or toxin B were transplanted into mice, homing to the bone marrow was impaired, whereas C2I-C3 treatment did not alter HPC homing. However, in a competitive hematopoietic repopulation experiment in C57BL/6 mice, pretreatment of bone marrow cells with any of the inhibitors, including the Rho inhibitor C2I-C3, resulted in suppressed donor-type hematopoiesis. Our data indicate that whereas Rac supports HPC cell cycling, migration, short-term homing and hematopoietic regeneration, Rho coordinates down-regulation of HPC migration and is required for hematopoietic regeneration.     

Involvement of rostral ventromedullar neuronal structures in nitric oxide modulation of central chemosensitive drive

Surface perfusion of the rostral ventromedullar cerebral subdivisions with artificial cerebrospinal fluid containing exogenous NO donor sodium nitroprusside (0.1 mM) increased the discharge rate of the phrenic nerve and potentiated the response of the respiratory center to hypercapnia in narcotized mature rats. The latter reaction was prevented by blockage of NO-synthase in rostral ventromedullar neural structures with N-nitro-L-arginine methyl ester (L-NAME, 0.3 mM). It was hypothesized that rostral ventromedullar neural structures are involved in modulatory action of NO on central chemosensitive drive.     

一氧化氮在新生大鼠败血症中的含量变化及其抑制剂的治疗作用

本文观察了一氧化氮在１０ｄ龄新生大鼠败血症中的含量变化，并探讨了其抑制剂美蓝或／和地塞米松的治疗作用。结果表明：新生大鼠血浆一氧化氮水平于细菌注射后６ｈ开始明显升高（Ｐ＜０．０５）。用美蓝或／和地塞米松治疗可显著改善代谢紊乱指标低葡萄糖血症和高乳酸血症，降低模拟病情严重程度的丧失疼痛反对发生率以及延长存活时间（Ｐ＜０．０５）；地塞米松治疗还可明显降低２４ｈ病死率（Ｐ＜０．０５）．一氧化氮及其抑制剂的临床应用前景值得重视。     

Antidiabetogenic effect of pentoxifylline is associated with systemic and target tissue modulation of cytokines and nitric oxide production

We have shown recently that xanthine derivative pentoxifylline (PTX) downregulates an inflammatory autoimmune process triggered in genetically susceptible Dark Agouti rats by multiple low doses of streptozotocin (MLD-SZ, 20 mg/kg/day ip for 5 days). We studied the cellular and molecular consequences of PTX treatment during MLD-SZ-induced diabetes with special emphasis on local vs. systemic production of inflammatory mediators. Administration of PTX (200 mg/kg/day for 10 days) during induction of the disease reduced clinical signs of diabetes and protected rats from development of destructive intrainsulitis. Pentoxifylline did not affect diabetogenic effect of single high dose of SZ (100 mg/kg SZ). Ex vivo analysis of the islets of Langerhans performed in early disease development revealed that PTX downregulates production of proinflammatory cytokines IFN-gamma and TNF, as well as inducible nitric oxide synthase (iNOS) expression and NO production. In addition, PTX treatment suppressed splenocyte autoreactivity, as well as the frequency of cells expressing IL-2R and MHC class II antigens. There was no evidence of any changes in proportion of ICAM-1 and LFA-1 expressing splenocytes in comparison to control MLD-SZ-treated animals. In contrast to suppressed intraislet production, high peripheral expression of both iNOS mRNA and NO was found in MLD-SZ rats treated with PTX. Taken together, the data indicate that the effect on both systemic and intra-islet production of NO, suppression of autoreactive cell activation and of local type 1 cytokine release may contribute to the therapeutic benefit achieved by PTX in the rat.     

Expression and modulation of Rho kinase in human pregnant myometrium

There is little information outlining the role of Rho kinase, RhoA, and calcium sensitization in regulation of human uterine contractility during pregnancy. The aims of this study were to investigate the expression of RhoA, and the Rho kinases ROCK I and ROCK II in human pregnant myometrium, to evaluate the effects of Rho kinase inhibition on pregnant human myometrial contractility in vitro, and to compare these effects with those of the calcium channel blocker nifedipine. RT-PCR using primers for RhoA, ROCK I and ROCK II was performed on mRNA isolated from human pregnant myometrium. Isometric recording was performed in isolated myometrial strips obtained at Caesarean section. The effects of the Rho kinase inhibitor Y-27632 (1 nmol/l to 10 mmol/l), and nifedipine (1 nmol/l to 10 mmol/l), on oxytocin (0.5 nmol/l) induced contractions were measured and compared. Expression of RhoA, ROCK I and ROCK II mRNA was identified in human pregnant myometrium (n = 3). Y-27632 exerted a potent relaxant effect on myometrial contractility with a pD(2) value (+/- SEM) of 7.63 +/- 0.38 (n = 6). The maximum net relaxant effect (+/- SEM) was 72.3 +/- 6.1% (n = 6). Corresponding values for nifedipine were 7.24 +/- 0.48 (n = 6; P = 0.469) and 93.40 +/- 3.1% (n = 6; P = 0.028). Rho A/Rho kinase-mediated calcium sensitization may play role in the physiology of human parturition, and pharmacological inhibition of this pathway may therefore provide a novel approach to tocolysis for pre-term labour.     

Substrate-dependent cellular behavior of Swiss 3T3 fibroblasts and activation of Rho family during adhesion and spreading processes

Recent biochemical studies revealed that intracellular Rho guanosine triphosphatases (Rho, Rac1, and Cdc42) are key regulatory molecules that link surface receptors to cytoskeletal organization and regulation of cell shape/morphology/motility. In this study, Swiss 3T3 fibroblasts were cultured on three representative substrates [tissue culture polystyrene dishes, nontreated polystyrene, and poly(ethylene terephthalate)] for 24 h after plating. Time-dependent changes in cell shape, morphology, cytoskeletal dynamics, and motility as well as Rho family activities were determined on each substrate. The cells on tissue culture polystyrene and on poly(ethylene terephthalate), which induced rapid and relatively rapid cell spreading, respectively, expressed Rac1 and Cdc42 activities continuously during the observation period. In contrast, such activities were suppressed in cells on polystyrene, which induced slow spreading but the highest cell motility compared with the other two substrates. Although a clear-cut relationship between cellular behavior and Rho family activation was not obtained, substrate-dependent coordinated control of cellular activities by Rho family is discussed.     

小肠内NOS分布及NO作用研究进展

一氧化氮作为一种细胞间和细胞内的信息物质 ,在小肠的生理病理过程中有重要作用。本文着重介绍小肠内的一氧化氮合酶分布NO与小肠运动、消化吸收、粘膜保护的关系等方面的研究进展。     

Efficacy and economy of inhaled nitric oxide in neonates accepted for extra-corporeal membrane oxygenation

OBJECTIVE: To study the clinical efficacy as well as the cost-effectiveness of inhaled nitric oxide (NO) compared with extra-corporeal membrane oxygenation (ECMO) in neonates with pulmonary hypertension and hypoxic respiratory failure accepted for ECMO treatment. DESIGN: Retrospective study of the first 10 consecutive neonatal patients treated with inhaled NO during 1992-94. Inhaled NO was administered after failure of conventional treatment as a last resort before initiating ECMO. For cost-effectiveness calculations the above-described patient population was compared with ECMO patients having a very favourable ECMO course. Setting: Twelve-bed neonatal and paediatric intensive care unit with ECMO capabilities at a tertiary university referral hospital. RESULTS: Inhaled NO was found to significantly decrease the oxygenation index (OI pre-NO: median 80.5; OI post-NO: median 22.5; P=0.003) and five of the patients could successfully be handled without the use of ECMO. ECMO was found to be more than four times as expensive as inhaled NO treatment on a per hour basis ($244 vs. $53 per hour) and the net savings from being able to avoid ECMO in five patients was calculated to be $156 200. CONCLUSION: Initiation of inhaled NO caused a significant improvement in oxygenation index and reduced the need for ECMO by 50% in critically ill neonatal patients with hypoxic respiratory failure. Inhaled NO was also found to compare very favourably with ECMO regarding cost-effectiveness ($53 vs. $244 per treatment hour).