Ruthenium Red Research Articles

Physiological Characterization of a Plant Mitochondrial Calcium Uniporter in Vitro and in Vivo.

Over the recent years, several proteins that make up the mitochondrial calcium uniporter complex (MCUC) mediating Ca2+uptake into the mitochondrial matrix have been identified in mammals, including the channel-forming protein MCU. Although six MCU gene homologs are conserved in the model plant Arabidopsis (Arabidopsis thaliana) in which mitochondria can accumulate Ca2+, a functional characterization of plant MCU homologs has been lacking. Using electrophysiology, we show that one isoform, AtMCU1, gives rise to a Ca2+-permeable channel activity that can be observed even in the absence of accessory proteins implicated in the formation of the active mammalian channel. Furthermore, we provide direct evidence that AtMCU1 activity is sensitive to the mitochondrial calcium uniporter inhibitors Ruthenium Red and Gd3+, as well as to the Arabidopsis protein MICU, a regulatory MCUC component. AtMCU1 is prevalently expressed in roots, localizes to mitochondria, and its absence causes mild changes in Ca2+ dynamics as assessed by in vivo measurements in Arabidopsis root tips. Plants either lacking or overexpressing AtMCU1 display root mitochondria with altered ultrastructure and show shorter primary roots under restrictive growth conditions. In summary, our work adds evolutionary depth to the investigation of mitochondrial Ca2+ transport, indicates that AtMCU1, together with MICU as a regulator, represents a functional configuration of the plant mitochondrial Ca2+ uptake complex with differences to the mammalian MCUC, and identifies a new player of the intracellular Ca2+ regulation network in plants.

Functional involvement of protein kinase C, Rho-kinase and TRPC3 decreases while PLC increases with advancement of pregnancy in mediating oxytocin-induced myometrial contractions in water buffaloes (Bubalus bubalis)

Present study unravels the involvement of different calcium signaling pathways in oxytocin-induced contractions in myometrium of non-pregnant and pregnant buffaloes during early and mid-pregnancy stages. Uteri of pregnant animals were more sensitive than of non-pregnant buffaloes. Phasic contractions and frequency of contraction significantly increased with advancement of pregnancy, while tonic contractions non-significantly and amplitude significantly decreased from six months pregnancy onward. Oxytocin produced concentration-dependent-contraction on isolated myometrial strips of pregnant and non-pregnant buffaloes and the dose response curves (DRCs) of oxytocin were significantly (P < 0.05) shifted to right in the presence of nifedipine (1 μM), in Ca2+-free Ringer Locke solution (RLS), ruthenium red (30 μM), ruthenium red + nifedipine, cyclopiazonic acid (CPA; Ca2+ free RLS as well as RLS), CPA (10 μM)+nifedipine, U-73122 (1 μM) + nifedipine and SKF96365 (25 μM) on uteri of non-pregnant and pregnant (early and mid) animals. The DRCs were also significantly shifted towards right in the presence of Y-27632 (10 μM), GF109203X (5 μM) and Pyr3 (10 μM) on uteri of non-pregnant and early pregnancy stage buffaloes while only in the presence of U-73122 (1 μM) on uteri of mid-pregnancy stage buffaloes. Our finding suggest that and L-type Ca2+ channels, IP3-RyR-gated, and store-operated calcium channels including transient receptor potential channel (TRPC) pathways play significant role in mediating oxytocin-induced contractions in myometrium of pregnant and non-pregnant buffaloes. SERCA plays major role only during early-pregnancy while functional role of protein kinase C (PKC), Rho-kinase and TRPC3 pathways decreased and role of G-protein coupled receptor-phospholipase C (GPCR-PLC) pathway increased with advancement of pregnancy.

The role of peltate scales in desiccation tolerance of Pleopeltis polypodioides.

The extreme drought tolerance of the resurrection fern is in part the result of the dorsal scales that assist in water distribution and controlled desiccation. We studied the effect of peltate scales on water uptake and loss of the desiccation-tolerant epiphytic fern Pleopeltis polypodioides using optical and FTIR microscopy and staining with calcofluor, solophenyl flavine7GFE, and Ruthenium Red. We provide information on structure, property, and function of the scales by measuring water uptake and dehydration, contact angles, and metabolic activity. Peltate scales mainly contain cellulose, xylogalactans, and pectin. Water is absorbed from the center of scales, and the overlapping arrangement of scales facilitates surface spreading of water. Intact fronds hydrated fully within 5h of imbibition of the apical pinna, without scales water uptake stopped after 1h. Hydration rates via rhizomes followed a longer time course but also improved in the presence of scales. Fronds with and without scales lost half of their water content in 15 or 4h, respectively. The overall metabolism of rapidly dehydrated fronds was significantly reduced compared with slowly dehydrated fronds. Thus, water management and metabolism of Pleopeltis are dependent on surface properties determined by peltate scales.

Mitochondrial permeability transition pore induction is linked to formation of the complex of ATPase C-subunit, polyhydroxybutyrate and inorganic polyphosphate.

Mitochondrial permeability transition pore (mPTP) opening allows free movement of ions and small molecules leading to mitochondrial membrane depolarization and ATP depletion that triggers cell death. A multi-protein complex of the mitochondrial ATP synthase has an essential role in mPTP. However, the molecular identity of the central 'pore' part of mPTP complex is not known. A highly purified fraction of mammalian mitochondria containing C-subunit of ATPase (C-subunit), calcium, inorganic polyphosphate (polyP) and polyhydroxybutyrate (PHB) forms ion channels with properties that resemble the native mPTP. We demonstrate here that amount of this channel-forming complex dramatically increases in intact mitochondria during mPTP activation. This increase is inhibited by both Cyclosporine A, an inhibitor of mPTP and Ruthenium Red, an inhibitor of the Mitochondrial Calcium Uniporter. Similar increases in the amount of complex formation occurs in areas of mouse brain damaged by ischemia-reperfusion injury. These findings suggest that calcium-induced mPTP is associated with de novo assembly of a channel comprising C-subunit, polyP and PHB.

Multiple target of hAmylin on rat primary hippocampal neurons

Alzheimer's disease (AD) and type II diabetes mellitus (DM2) are the most common aging-related diseases and are characterized by β-amyloid and amylin accumulation, respectively. Multiple studies have indicated a strong correlation between these two diseases. Amylin oligomerization in the brain appears to be a novel risk factor for developing AD. Although amylin aggregation has been demonstrated to induce cytotoxicity in neurons through altering Ca2+ homeostasis, the underlying mechanisms have not been fully explored. In this study, we investigated the effects of amylin on rat hippocampal neurons using calcium imaging and whole-cell patch clamp recordings. We demonstrated that the amylin receptor antagonist AC187 abolished the Ca2+ response induced by low concentrations of human amylin (hAmylin). However, the Ca2+ response induced by higher concentrations of hAmylin was independent of the amylin receptor. This effect was dependent on extracellular Ca2+. Additionally, blockade of L-type Ca2+ channels partially reduced hAmylin-induced Ca2+ response. In whole-cell recordings, hAmylin depolarized the membrane potential. Moreover, application of the transient receptor potential (TRP) channel antagonist ruthenium red (RR) attenuated the hAmylin-induced increase in Ca2+. Single-cell RT-PCR demonstrated that transient receptor potential vanilloid 4 (TRPV4) mRNA was expressed in most of the hAmylin-responsive neurons. In addition, selective knockdown of TRPV4 channels inhibited the hAmylin-evoked Ca2+ response. These results indicated that different concentrations of hAmylin act through different pathways. The amylin receptor mediates the excitatory effects of low concentrations of hAmylin. In contrast, for high concentrations of hAmylin, hAmylin aggregates precipitated on the neuronal membrane, activated TRPV4 channels and subsequently triggered membrane voltage-gated calcium channel opening followed by membrane depolarization. Therefore, our data suggest that TRPV4 is a key molecular mediator for the cytotoxic effects of hAmylin on hippocampal neurons.

Calcium-Sensing Receptor and Transient Receptor Ankyrin-1 Mediate Emesis Induction by Deoxynivalenol (Vomitoxin).

The common foodborne mycotoxin deoxynivalenol (DON, vomitoxin) can negatively impact animal and human health by causing food refusal and vomiting. Gut enteroendocrine cells (EECs) secrete hormones that mediate DON's anorectic and emetic effects. In prior work utilizing a cloned EEC model, our laboratory discovered that DON-induced activation of calcium-sensing receptor (CaSR), a G-coupled protein receptor (GPCR), and transient receptor ankyrin-1 (TRPA1), a transient receptor potential (TRP) channel, drives Ca2+-mediated hormone secretion. Consistent with these in vitro findings, CaSR and TRPA1 mediate DON-induced satiety hormone release and food refusal in the mouse, an animal model incapable of vomiting. However, the roles of this GPCR and TRP in DON's emetic effects remain to be determined. To address this, we tested the hypothesis that DON triggers emesis in mink by activating CaSR and TRPA1. Oral gavage with selective agonists for CaSR (R-568) or TRPA1 (allyl isothiocyanate; AITC) rapidly elicited emesis in the mink in dose-dependent fashion. Oral pretreatment of the animals with the CaSR antagonist NPS-2143 or the TRP antagonist ruthenium red (RR), respectively, inhibited these responses. Importantly, DON-induced emesis in mink was similarly inhibited by oral pretreatment with NPS-2143 or RR. In addition, these antagonists suppressed concurrent DON-induced elevations in plasma peptide YY3-36 and 5-hydroxytryptamine-hormones previously demonstrated to mediate the toxin's emetic effects in mink. Furthermore, antagonist co-treatment additively suppressed DON-induced emesis and peptide YY 3-36 release. To summarize, the observations here strongly suggest that activation of CaSR and TRPA1 might have critical roles in DON-induced emesis.

Silibinin triggers yeast apoptosis related to mitochondrial Ca2+ influx in Candida albicans

Candida albicans is a common yeast that resides in the human body, but can occasionally cause systemic fungal infection, namely candidiasis. As this infection rate is gradually increasing, it is becoming a major problem to public health. Accordingly, we for the first time investigated the antifungal activity and mode of action of silibinin, a natural product extracted from Silybum marianum (milk thistle), against C. albicans. On treatment with 100μM silibinin, generation of reactive oxygen species (ROS) from mitochondria, which can cause yeast apoptosis via oxidative stress, was increased by 24.17% compared to that in untreated cells. Subsequently, we found disturbances in ion homeostasis such as release of intracellular K+ and accumulation of cytoplasmic and mitochondrial Ca2+. Among these phenomena, mitochondrial Ca2+ overload particularly plays a crucial role in the process of apoptosis, promoting the activation of pro-apoptotic factors. Therefore, we investigated the significance of mitochondrial Ca2+ in apoptosis by employing 20mM ruthenium red (RR). Additional apoptosis hallmarks such as mitochondrial membrane depolarization, cytochrome c release, caspase activation, phosphatidylserine (PS) exposure, and DNA damage were observed in response to silibinin treatment, whereas RR pre-treatment seemed to block these responses. In summary, our results suggest that silibinin induces yeast apoptosis mediated by mitochondrial Ca2+ signaling in C. albicans.

Cellular response to high pulse repetition rate nanosecond pulses varies with fluorescent marker identity

Nanosecond electric pulses (nsEP's) are a well-studied phenomena in biophysics that cause substantial alterations to cellular membrane dynamics, internal biochemistry, and cytoskeletal structure, and induce apoptotic and necrotic cell death. While several studies have attempted to measure the effects of multiple nanosecond pulses, the effect of pulse repetition rate (PRR) has received little attention, especially at frequencies greater than 100 Hz. In this study, uptake of Propidium Iodide, FM 1–43, and YO-PRO-1 fluorescent dyes in CHO-K1 cells was monitored across a wide range of PRRs (5 Hz–500 KHz) using a laser-scanning confocal microscope in order to better understand how high frequency repetition rates impact induced biophysical changes. We show that frequency trends depend on the identity of the dye under study, which could implicate transmembrane protein channels in the uptake response due to their chemical selectivity. Finally, YO-PRO-1 fluorescence was monitored in the presence of Gadolinium (Gd3+), Ruthenium Red, and in calcium-free solution to elucidate a mechanism for its unique frequency trend.

Sarcolemmal ATP-sensitive potassium channel protects cardiac myocytes against lipopolysaccharide-induced apoptosis.

The sarcolemmal ATP-sensitive K+ (sarcKATP) channel plays a cardioprotective role during stress. However, the role of the sarcKATP channel in the apoptosis of cardiomyocytes and association with mitochondrial calcium remains unclear. For this purpose, we developed a model of LPS-induced sepsis in neonatal rat cardiomyocytes (NRCs). The TUNEL assay was performed in order to detect the apoptosis of cardiac myocytes and the MTT assay was performed to determine cellular viability. Exposure to LPS significantly decreased the viability of the NRCs as well as the expression of Bcl-2, whereas it enhanced the activity and expression of the apoptosis-related proteins caspase-3 and Bax, respectively. The sarcKATP channel blocker, HMR-1098, increased the apoptosis of NRCs, whereas the specific sarcKATP channel opener, P-1075, reduced the apoptosis of NRCs. The mitochondrial calcium uniporter inhibitor ruthenium red (RR) partially inhibited the pro-apoptotic effect of HMR-1098. In order to confirm the role of the sarcKATP channel, we constructed a recombinant adenovirus vector carrying the sarcKATP channel mutant subunit Kir6.2AAA to inhibit the channel activity. Kir6.2AAA adenovirus infection in NRCs significantly aggravated the apoptosis of myocytes induced by LPS. Elucidating the regulatory mechanisms of the sarcKATP channel in apoptosis may facilitate the development of novel therapeutic targets and strategies for the management of sepsis and cardiac dysfunction.

Extensive Natural Variation in Arabidopsis Seed Mucilage Structure.

Hydrated Arabidopsis thaliana seeds are coated by a gelatinous layer called mucilage, which is mainly composed of cell wall polysaccharides. Since mucilage is rich in pectin, its architecture can be visualized with the ruthenium red (RR) dye. We screened the seeds of around 280 Arabidopsis natural accessions for variation in mucilage structure, and identified a large number of novel variants that differed from the Col-0 wild-type. Most of the accessions released smaller RR-stained capsules compared to the Col-0 reference. By biochemically characterizing the phenotypes of 25 of these accessions in greater detail, we discovered that distinct changes in polysaccharide structure resulted in gelatinous coatings with a deceptively similar appearance. Monosaccharide composition analysis of total mucilage extracts revealed a remarkable variation (from 50 to 200% of Col-0 levels) in the content of galactose and mannose, which are important subunits of heteromannan. In addition, most of the natural variants had altered Pontamine Fast Scarlet 4B staining of cellulose and significantly reduced birefringence of crystalline structures. This indicates that the production or organization of cellulose may be affected by the presence of different amounts of hemicellulose. Although, the accessions described in this study were primarily collected from Western Europe, they form five different phenotypic classes based on the combined results of our experiments. This suggests that polymorphisms at multiple loci are likely responsible for the observed mucilage structure. The transcription of MUCILAGE-RELATED10 (MUCI10), which encodes a key enzyme for galactoglucomannan synthesis, was severely reduced in multiple variants that phenocopied the muci10-1 insertion mutant. Although, we could not pinpoint any causal polymorphisms in this gene, constitutive expression of fluorescently-tagged MUCI10 proteins complemented the mucilage defects of a muci10-like accession. This leads us to hypothesize that some accessions might disrupt a transcriptional regulator of MUCI10. Therefore, this collection of publicly-available variants should provide insight into plant cell wall organization and facilitate the discovery of genes that regulate polysaccharide biosynthesis.

Potential roles for calcium-sensing receptor (CaSR) and transient receptor potential ankyrin-1 (TRPA1) in murine anorectic response to deoxynivalenol (vomitoxin).

Food contamination by the trichothecene mycotoxin deoxynivalenol (DON, vomitoxin) has the potential to adversely affect animal and human health by suppressing food intake and impairing growth. In mice, the DON-induced anorectic response results from aberrant satiety hormone secretion by enteroendocrine cells (EECs) of the gastrointestinal tract. Recent in vitro studies in the murine STC-1 EEC model have linked DON-induced satiety hormone secretion to activation of calcium-sensing receptor (CaSR), a G-coupled protein receptor, and transient receptor potential ankyrin-1 (TRPA1), a TRP channel. However, it is unknown whether similar mechanisms mediate DON's anorectic effects in vivo. Here, we tested the hypothesis that DON-induced food refusal and satiety hormone release in the mouse are linked to activation of CaSR and TRPA1. Oral treatment with selective agonists for CaSR (R-568) or TRPA1 (allyl isothiocyanate (AITC)) suppressed food intake in mice, and the agonist's effects were suppressed by pretreatment with corresponding antagonists NPS-2143 or ruthenium red (RR), respectively. Importantly, NPS-2143 or RR inhibited both DON-induced food refusal and plasma elevations of the satiety hormones cholecystokinin (CCK) and peptide YY3-36 (PYY3-36); cotreatment with both antagonists additively suppressed both anorectic and hormone responses to DON. Taken together, these in vivo data along with prior in vitro findings support the contention that activation of CaSR and TRPA1 contributes to DON-induced food refusal by mediating satiety hormone exocytosis from EEC.

Multiple Sources of Ca2+ Contribute to Methylmercury-Induced Increased Frequency of Spontaneous Inhibitory Synaptic Responses in Cerebellar Slices of Rat.

We previously showed that elevated intracellular Ca(2+) ([Ca(2+)]i) in the molecular layer and granule cells in cerebellar slices is responsible for the initial increases in frequency of spontaneous or miniature inhibitory postsynaptic currents (sIPSCs or mIPSCs) of Purkinje cells following methylmercury (MeHg) treatment. To identify the contribution of different Ca(2+) sources to MeHg-induced stimulation of spontaneous GABA release, we examined sIPSC or mIPSC frequency of Purkinje cells in acutely prepared cerebellar slices using whole-cell patch-clamp recording techniques under conditions of lowered [Ca(2+)]o, pretreatment with caffeine, cyclopiazonic acid (CPA), thapsigargin or ruthenium red (RR) to deplete ryanodine-sensitive and insensitive intracellular Ca(2+) stores or mitochondria, or a combination of lowering [Ca(2+)]o and increased BAPTA buffering. Lowering [Ca(2+)]o significantly reduced sIPSC or mIPSC frequency and amplitudes, but failed to completely prevent MeHg-induced increase in these events frequency. Caffeine, CPA, or thapisgargin also minimized MeHg-induced increase in sIPSC frequency, yet none of them completely blocked MeHg-induced increase in sIPSC frequency. Similarly, the mitochondrial Ca(2+) transport inhibitor RR, or a combination of lowering [Ca(2+)]o and BAPTA buffering reduced but did not prevent MeHg-induced changes in mIPSC frequency. Consistently, confocal Ca(2+) imaging under low [Ca(2+)]o conditions or in the presence of caffeine or CPA exhibited a marked reduction of MeHg-induced increases in [Ca(2+)]i in both molecular and granule layers. Thus, these results verify that a combination of extracellular Ca(2+) influx and Ca(2+) release from different intracellular Ca(2+) pools all contribute to MeHg-induced increase in [Ca(2+)]i and spontaneous GABA release, although extracellular Ca(2+) appears to be the primary contributor.

Effect of TRPV4-p38 MAPK Pathway on Neuropathic Pain in Rats with Chronic Compression of the Dorsal Root Ganglion.

The aim of this study was to investigate the relationships among TRPV4, p38, and neuropathic pain in a rat model of chronic compression of the dorsal root ganglion. Mechanical allodynia appeared after CCD surgery, enhanced via the intrathecal injection of 4α-phorbol 12,13-didecanoate (4α-PDD, an agonist of TRPV4) and anisomycin (an agonist of p38), but was suppressed by Ruthenium Red (RR, an inhibitor of TRPV4) and SB203580 (an inhibitor of p38). The protein expressions of p38 and P-p38 were upregulated by 4α-PDD and anisomycin injection but reduced by RR and SB203580. Moreover, TRPV4 was upregulated by 4α-PDD and SB203580 and downregulated by RR and anisomycin. In DRG tissues, the numbers of TRPV4- or p38-positive small neurons were significantly changed in CCD rats, increased by the agonists, and decreased by the inhibitors. The amplitudes of ectopic discharges were increased by 4α-PDD and anisomycin but decreased by RR and SB203580. Collectively, these results support the link between TRPV4 and p38 and their intermediary role for neuropathic pain in rats with chronic compression of the dorsal root ganglion.

Arabidopsis CML38, a Calcium Sensor That Localizes to Ribonucleoprotein Complexes under Hypoxia Stress.

During waterlogging and the associated oxygen deprivation stress, plants respond by the induction of adaptive programs, including the redirected expression of gene networks toward the synthesis of core hypoxia-response proteins. Among these core response proteins in Arabidopsis (Arabidopsis thaliana) is the calcium sensor CML38, a protein related to regulator of gene silencing calmodulin-like proteins (rgsCaMs). CML38 transcripts are up-regulated more than 300-fold in roots within 6 h of hypoxia treatment. Transfer DNA insertional mutants of CML38 show an enhanced sensitivity to hypoxia stress, with lowered survival and more severe inhibition of root and shoot growth. By using yellow fluorescent protein (YFP) translational fusions, CML38 protein was found to be localized to cytosolic granule structures similar in morphology to hypoxia-induced stress granules. Immunoprecipitation of CML38 from the roots of hypoxia-challenged transgenic plants harboring CML38pro::CML38:YFP followed by liquid chromatography-tandem mass spectrometry analysis revealed the presence of protein targets associated with messenger RNA ribonucleoprotein (mRNP) complexes including stress granules, which are known to accumulate as messenger RNA storage and triage centers during hypoxia. This finding is further supported by the colocalization of CML38 with the mRNP stress granule marker RNA Binding Protein 47 (RBP47) upon cotransfection of Nicotiana benthamiana leaves. Ruthenium Red treatment results in the loss of CML38 signal in cytosolic granules, suggesting that calcium is necessary for stress granule association. These results confirm that CML38 is a core hypoxia response calcium sensor protein and suggest that it serves as a potential calcium signaling target within stress granules and other mRNPs that accumulate during flooding stress responses.

The speed of swelling kinetics modulates cell volume regulation and calcium signaling in astrocytes: A different point of view on the role of aquaporins.

Regulatory volume decrease (RVD) is a process by which cells restore their original volume in response to swelling. In this study, we have focused on the role played by two different Aquaporins (AQPs), Aquaporin-4 (AQP4), and Aquaporin-1 (AQP1), in triggering RVD and in mediating calcium signaling in astrocytes under hypotonic stimulus. Using biophysical techniques to measure water flux through the plasma membrane of wild-type (WT) and AQP4 knockout (KO) astrocytes and of an astrocyte cell line (DI TNC1) transfected with AQP4 or AQP1, we here show that AQP-mediated fast swelling kinetics play a key role in triggering and accelerating RVD. Using calcium imaging, we show that AQP-mediated fast swelling kinetics also significantly increases the amplitude of calcium transients inhibited by Gadolinium and Ruthenium Red, two inhibitors of the transient receptor potential vanilloid 4 (TRPV4) channels, and prevented by removing extracellular calcium. Finally, inhibition of TRPV4 or removal of extracellular calcium does not affect RVD. All together our study provides evidence that (1) AQP influenced swelling kinetics is the main trigger for RVD and in mediating calcium signaling after hypotonic stimulus together with TRPV4, and (2) calcium influx from the extracellular space and/or TRPV4 are not essential for RVD to occur in astrocytes.

C-type natriuretic peptide prevents activation of perivascular mast cells and inflammation in the postischemic microvasculature

Background Atrial- and C-type- natriuretic peptides (ANP, CNP) are both vasodilatatory hormones which are mainly secreted from cardiac atria and vascular endothelium, respectively. Both peptides activate cyclic GMP (cGMP) formation by binding to their specific receptors: the particulate guanylyl cyclases A (GC-A) and B (GC-B). Cyclic GMP via cGMP-dependent protein kinase I (cGKI) was shown to inhibit mast cell (MC) degranulation [1]. During postischemic reperfusion, the activation of MC is detrimental [2]. Due to pleiotropic effects, cytokines released from MC cause tissue damage and vascular leakage. It is unclear whether and how ANP and/or CNP regulate the activity of MC and, thereby, influence ischemia-reperfusion-induced disruption of the microvascular barrier. Methods and results Intravital microscopy was used to visualize the microcirculation within the cremaster muscle of anesthetized mice, and Fluorescein isothiocyanate (FITC)-Dextran was injected intravenously to quantify the microvascular leakage [3,4]. Cremaster blood flow was temporally blocked with an arterial clamp to provoke 30 min of tissue ischemia. During the following 30 min of postischemic reperfusion, the vascular leakage was measured by the ratio of the fluorescent (FITC-Dextran) intensity (RFI) between the adjacent interstitial space and the intravascular lumen. Subsequently resident perivascular degranulated MCs were stained by superfusing Ruthenium Red solution and thereafter counted (number per field). In wildtype mice, ischemia-reperfusion (compared to sham operated cremaster muscles) provoked marked MC degranulation and robust vascular leakage of FITCDextran (as indicated by higher RFI). Local CNP superfusion during reperfusion significantly reduced the amount of degranulated MCs and concomitantly vascular leakage. These effects were mimicked by the membrane-permeable cGKI activator 8-Br-cGMP. The protective effects of 8-Br-cGMP were endothelium-independent, since they were preserved in mice with conditional, endothelial deletion of cGKI. Quantitative real-time RT-PCR revealed local CNP-mRNA expression in the m. cremaster, which was not altered by acute ischemia. In contrast to CNP, ANP superfusion did not significantly alter the amount of degranulated MCs, but still effectively reduced FITC-Dextran leakage resulted from ischemia-reperfusion. Accordingly, in vitro, in cultured human MCs (HMC-1.1, originally developed by Dr. Butterfield and a gift from Dr. Drube) and primary cultured murine bone marrow MC, CNP, but not ANP, induced a marked increase in intracellular cGMP levels and enhanced the phosphorylation of the cytoskeleton-associated vasoactive stimulated phosphoprotein (VASP) at Ser237. Conclusions Our study shows divergent effects of NPs in the postischemic microcirculation. CNP inhibited the ischemic activation of resident, perivascular MCs and consequently the associated microvascular leakage, possibly via MC specific cGMP/cGKI signaling. At difference, ANP had no effect on MCs but still attenuated the postischemic vascular barrier disruption via direct effects on endothelial cells. Our future studies aim to dissect the

Toxicity of polyhydroxylated fullerene to mitochondria

Mitochondrial dysfunction is considered as a crucial mechanism of nanomaterial toxicity. Herein, we investigated the effects of polyhydroxylated fullerene (C60(OH)44, fullerenol), a model carbon-based nanomaterial with high water solubility, on isolated mitochondria. Our study demonstrated that fullerenol enhanced the permeabilization of mitochondrial inner membrane to H+ and K+ and induced mitochondrial permeability transition (MPT). The fullerenol-induced swelling was dose-dependent and could be effectively inhibited by MPT inhibitors such as cyclosporin A (CsA), adenosine diphosphate (ADP), ruthenium red (RR) and ethylenediaminetetraacetic acid (EDTA). After treating the mitochondria with fullerenol, the mitochondrial membrane potential (MMP) was found collapsed in a concentration-independent manner. The fluorescence anisotropy of hematoporphyrin (HP) changed significantly with the addition of fullerenol, while that of 1,6-diphenyl-hexatriene (DPH) changed slightly. Moreover, a decrease of respiration state 3 and increase of respiration state 4 were observed when mitochondria were energized with complex II substrate succinate. The results of transmission electron microscopy (TEM) provided direct evidence that fullerenol damaged the mitochondrial ultrastructure. The investigations can provide comprehensive information to elucidate the possible toxic mechanism of fullerenols at subcellular level.

Role of mitochondrial calcium uniporter in lung ischemia-reperfusion injury in rats

Objective To evaluate the role of mitochondrial calcium uniporter in lung ischemia-reperfusion(I/R)injury in rats. Methods Thirty healthy male Wistar rats, weighing 250-300 g, were randomly divided into 3 groups(n=10 each)using a random number table: sham operation group(group S), group I/R and ruthenium red(mitochondrial calcium uniporter inhibitor)group(group RR). Lung I/R was produced by clamping the left pulmonary hilum for 45 min followed by 2 h reperfusion in I/R and RR groups.In addition, ruthenium red 2.5 mg/kg was injected via the tail vein at 1 h before occlusion of the left pulmonary hilum in RR group.Arterial blood samples were obtained at 2 h of reperfusion for blood gas analysis, and oxygenation index(PaO2/FiO2)was calculated.The rats were sacrificed, and then the lungs were removed for determination of wet/dry lung weight ratio(W/D ratio). Results Compared with group S, oxygenation index was significantly decreased, and W/D ratio was increased in I/R and RR groups.Oxygenation index was significantly higher, and W/D ratio was lower in group RR than in group I/R. Conclusion The mechanism underlying lung I/R injury is related to the opening of mitochondrial calcium uniporter in rats. Key words: Mitochondrial membrane transport proteins; Reperfusion injury; Lung

The influence of ruthenium on vascular tone.

Over the past few years, ruthenium has been under attention for development of organometallic drugs with various therapeutic applications. Because of its favourable characteristics, ruthenium is perfectly suitable for drug design. Ruthenium-containing complexes exert a wide range of biological effects. However, so far, the influence of ruthenium itself on vascular tone has never been studied. The effect of ruthenium was analysed through organ bath studies measuring isometric tension on mice thoracic aorta. After obtaining a stable contraction plateau, cumulative concentration-response curves of the ruthenium-compounds (RuCl3 , Ruthenium Red, [RuCl2 (CO)3 ]2 and RuCl2 (DMSO)4 ) (30-600 μmol/l) were performed. The effect of RuCl3 after contraction with different contractile agents was evaluated. Furthermore, the influence of ruthenium-containing molecules on endogenous (acetylcholine) and exogenous (sodium nitroprusside) NO-mediated relaxations was determined. All studied ruthenium compounds elicit, to some extent, a decrease of the contraction level. Looking further into the underlying mechanism, we found that RuCl3 relaxes aortic rings only when contracted with norepinephrine. This RuCl3 -induced relaxation can be prevented by the antioxidants ascorbic acid and N-acetyl L-cysteine. In addition, ruthenium compounds may diminish acetylcholine- or sodium nitroprusside-induced relaxations. Ruthenium-containing molecules can influence vascular tone induced by norepinephrine due to oxidative inactivation. Moreover, they can undermine NO-mediated responses. This should be considered when developing ruthenium-containing drugs.

C‐Type Natriuretic Peptide as Mediator of an Endothelial Cell to Mast Cell Communication

Mast cells (MC) play a critical role in leukocyte recruitment to sites of inflammation after ischemia/reperfusion (I/R). They reside in the perivascular tissue and release proteases and chemokines having detrimental effects on their immediate surroundings, such as endothelial cells. The triggers and counterregulators of MC activation are still enigmatic. We investigated whether endothelial C‐type natriuretic peptide (CNP) via its cGMP forming guanylyl cyclase B (GC‐B) receptor modulates the activity of MC and inflammation after I/R.Intravital microscopy was used to visualize the microcirculation within the cremaster muscle of anesthetized mice. Blood flow was temporally blocked for 30 min of ischemia. FITC‐Dextran was used to quantify macromolecule leakage during reperfusion. Ruthenium Red was used to stain perivascular degranulated MC. I/R provoked marked degranulation of perivascular resident MC (~7‐fold vs controls) and prominent leakage of FITC‐Dextran from postcapillary venules to the adjacent interstitial space. Local superfusion of CNP during reperfusion, significantly reduced the number of degranulated MC (to ~1.8‐fold vs controls) and concomitantly prevented vascular leakage. These effects were mimicked by the membrane‐permeable cGMP analog, 8‐bromo cGMP. In cultured murine bone marrow and human MC CNP activated GC‐B/cGMP signaling and cGMP protein kinase I‐dependent phosphorylation of the cytoskeleton‐associated vasoactive stimulated phosphoprotein (VASP) at Ser237.Our study indicates that the endothelial peptide CNP inhibits I/R‐induced degranulation of perivascular MC and thereby attenuates microvascular inflammation.Supported by SFB 688 and CHFC Würzburg