Cerebral microvascular dilation during hypotension and decreased oxygen tension: a role for nNOS

Endothelial (eNOS) and neuronal nitric oxide synthase (nNOS) are implicated as important contributors to cerebral vascular regulation through nitric oxide (NO). However, direct in vivo measurements of NO in the brain have not been used to dissect their relative roles, particularly as related to oxygenation of brain tissue. We found that, in vivo, rat cerebral arterioles had increased NO concentration ([NO]) and diameter at reduced periarteriolar oxygen tension (Po(2)) when either bath oxygen tension or arterial pressure was decreased. Using these protocols with highly selective blockade of nNOS, we tested the hypothesis that brain tissue nNOS could donate NO to the arterioles at rest and during periods of reduced perivascular oxygen tension, such as during hypotension or reduced local availability of oxygen. The decline in periarteriolar Po(2) by bath manipulation increased [NO] and vessel diameter comparable with responses at similarly decreased Po(2) during hypotension. To determine whether the nNOS provided much of the vascular wall NO, nNOS was locally suppressed with the highly selective inhibitor N-(4S)-(4-amino-5-[aminoethyl]aminopentyl)-N'-nitroguanidine. After blockade, resting [NO], Po(2), and diameters decreased, and the increase in [NO] during reduced Po(2) or hypotension was completely absent. However, flow-mediated dilation during occlusion of a collateral arteriole did remain intact after nNOS blockade and the vessel wall [NO] increased to approximately 80% of normal. Therefore, nNOS predominantly increased NO during decreased periarteriolar oxygen tension, such as that during hypotension, but eNOS was the dominant source of NO for flow shear mechanisms.     

Shear stress and 17beta-estradiol modulate cerebral microvascular endothelial Na-K-Cl cotransporter and Na/H exchanger protein levels

Ion transporters of blood-brain barrier (BBB) endothelial cells play an important role in regulating the movement of ions between the blood and brain. During ischemic stroke, reduction in cerebral blood flow is accompanied by transport of Na and Cl from the blood into the brain, with consequent brain edema formation. We have shown previously that a BBB Na-K-Cl cotransporter (NKCC) participates in ischemia-induced brain Na and water uptake and that a BBB Na/H exchanger (NHE) may also participate. While the abrupt reduction of blood flow is a prominent component of ischemia, the effects of flow on BBB NKCC and NHE are not known. In the present study, we examined the effects of changes in shear stress on NKCC and NHE protein levels in cerebral microvascular endothelial cells (CMECs). We have shown previously that estradiol attenuates both ischemia-induced cerebral edema and CMEC NKCC activity. Thus, in the present study, we also examined the effects of estradiol on NKCC and NHE protein levels in CMECs. Exposing CMECs to steady shear stress (19 dyn/cm(2)) increased the abundance of both NKCC and NHE. Estradiol abolished the shear stress-induced increase in NHE but not NKCC. Abrupt reduction of shear stress did not alter NKCC or NHE abundance in the absence of estradiol, but it decreased NKCC abundance in estradiol-treated cells. Our results indicate that changes in shear stress modulate BBB NKCC and NHE protein levels. They also support the hypothesis that estradiol attenuates edema formation in ischemic stroke in part by reducing the abundance of BBB NKCC protein.     

Callipeltin A, a cyclic depsipeptide inhibitor of the cardiac sodium-calcium exchanger and positive inotropic agent

Callipeltin A, a cyclic depsipeptide from the New Caledonian Lithistida sponge Callipelta sp., is a macrocyclic lactone containing four amino acids in the L configuration, Ala, Leu, Thr (2 residues); one (Arg) in the D configuration; two N-methyl amino acids, N-MeAla and N-MeGln; a methoxy tyrosine, a 3, 4-dimethyl-l-glutamine; and a 4-amino-7-guanidino-2,3 dihydroxypentanoic acid (AGDHE), formally derived from L-Arg. In cardiac sarcolemmal vesicles Callipeltin A induces a powerful (IC(50) = 0.85 microM) and selective inhibition of the Na(+)/Ca(2+) exchanger. In electrically driven guinea-pig atria, at concentrations ranging between 0.7 and 2.5 microM, Callipeltin A induces a positive inotropic effect, which at the highest concentrations is accompanied by a rise in resting tension. It is suggested that the positive inotropic effect is linked to the inhibition of the Na(+)/Ca(2+) exchanger and that Callipeltin A may be an useful tool to study the role of the cardiac Na(+)/Ca(2+) exchanger in physiological and pathological conditions.     

Mad2 and BubR1 function in a single checkpoint pathway that responds to a loss of tension

The spindle checkpoint monitors microtubule attachment and tension at kinetochores to ensure proper chromosome segregation. Previously, PtK1 cells in hypothermic conditions (23 degrees C) were shown to have a pronounced mitotic delay, despite having normal numbers of kinetochore microtubules. At 23 degrees C, we found that PtK1 cells remained in metaphase for an average of 101 min, compared with 21 min for cells at 37 degrees C. The metaphase delay at 23 degrees C was abrogated by injection of Mad2 inhibitors, showing that Mad2 and the spindle checkpoint were responsible for the prolonged metaphase. Live cell imaging showed that kinetochore Mad2 became undetectable soon after chromosome congression. Measurements of the stretch between sister kinetochores at metaphase found a 24% decrease in tension at 23 degrees C, and metaphase kinetochores at 23 degrees C exhibited higher levels of 3F3/2, Bub1, and BubR1 compared with 37 degrees C. Microinjection of anti-BubR1 antibody abolished the metaphase delay at 23 degrees C, indicating that the higher kinetochore levels of BubR1 may contribute to the delay. Disrupting both Mad2 and BubR1 function induced anaphase with the same timing as single inhibitions, suggesting that these checkpoint genes function in the same pathway. We conclude that reduced tension at kinetochores with a full complement of kinetochore microtubules induces a checkpoint dependent metaphase delay associated with elevated amounts of kinetochore 3F3/2, Bub1, and BubR1 labeling.     

NIDD, a novel DHHC-containing protein, targets neuronal nitric-oxide synthase (nNOS) to the synaptic membrane through a PDZ-dependent interaction and regulates nNOS activity

Targeting of neuronal nitric-oxide synthase (nNOS) to appropriate sites in a cell is mediated by interactions with its PDZ domain and plays an important role in specifying the sites of reaction of nitric oxide (NO) in the central nervous system. Here we report the identification and characterization of a novel nNOS-interacting DHHC domain-containing protein with dendritic mRNA (NIDD) (GenBank accession number AB098078), which increases nNOS enzyme activity by targeting the nNOS to the synaptic plasma membrane in a PDZ domain-dependent manner. The deduced NIDD protein consisted of 392 amino acid residues and possessed five transmembrane segments, a zinc finger DHHC domain, and a PDZ-binding motif (-EDIV) at its C-terminal tail. In vitro pull-down assays suggested that the C-terminal tail region of NIDD specifically interacted with the PDZ domain of nNOS. The PDZ dependence was confirmed by an experiment using a deletion mutant, and the interaction was further confirmed by co-sedimentation assays using COS-7 cells transfected with NIDD and nNOS. Both NIDD and nNOS were enriched in synaptosome and synaptic plasma membrane fractions and were present in the lipid raft and postsynaptic density fractions in the rat brain. Co-localization of these proteins was also observed by double staining of the proteins in cultured cortical neurons. Thus, NIDD and nNOS were co-localized in the brain, although the colocalizing regions were restricted, as indicated by the distribution of their mRNA expression. Most important, co-transfection of NIDD and nNOS increased NO-producing nNOS activity. These results suggested that NIDD plays an important role in the regulation of the NO signaling pathway at postsynaptic sites through targeting of nNOS to the postsynaptic membrane.     

Angiotensin II induces complex fractionated electrogram in a cultured atrial myocyte monolayer mediated by calcium and sodium-calcium exchanger

Angiotensin II (AngII) has been implicated in the mechanism of atrial fibrillation (AF). There may be calcium-dependent pro-fibrillatory effect of AngII on atrial myocytes. We used cultured confluent HL-1 atrial myocyte monolayer with spontaneously propagated depolarization to study direct pro-fibrillatory effect of AngII and its molecular mechanism. AngII stimulation induced fibrillatory-like complex electrogram and calcium wave propagation. AngII shortened action potential duration and augmented calcium transient, thus increasing electrochemical gradient of forward-mode sodium-calcium exchanger (NCX) current and induced frequent irregular afterdepolarizations. AngII increased expression of sodium-calcium exchanger (NCX), further increasing calcium-membrane voltage coupling gain. The fibrillatory effect of AngII was attenuated by NCX blocker SEA0400 and NCX siRNA knockdown. AngII increased expression of L-type calcium channel and augmented calcium transient through PKC and CREB. The fibrillatory effect of AngII was also attenuated by PKC inhibitor chelerythrine and dominant negative form of CREB. In conclusions, AngII itself may electrically contribute to the mechanism of AF through increasing NCX expression and augmenting calcium transient, which is PKC and CREB dependent. Specific genetic knockdown of NCX attenuated calcium mediated afterdepolarization and complex electrogram.     

Purification and amino-terminal sequence of the bovine cardiac sodium-calcium exchanger: evidence for the presence of a signal sequence

The Na(+)-Ca2+ exchange carrier was purified from bovine cardiac tissue by a new procedure which relies principally upon anion-exchange chromatography. The purified protein exhibited two major bands on sodium dodecyl sulfate gels, at 120 and 160 kDa. The relative intensities of the two bands could be altered by variations in the procedures used for preparing the samples for electrophoresis, suggesting that they represent two different conformational states of the same protein. The NH2-terminal amino acid sequences of the 120- and 160-kDa bands were identical and agreed closely with a region of the deduced amino acid sequence of the recently cloned canine cardiac exchanger. The NH2-terminal sequence was preceded in the deduced sequence by a 32-residue segment that exhibited the characteristics of a signal sequence; the initial amino acid in the NH2-terminal sequence followed immediately after the predicted cleavage site for the signal sequence. The Na(+)-Ca2+ exchanger appears to be unique among membrane transport carriers in encoding a cleaved signal sequence. The characteristics of the sequences flanking the first putative transmembrane segment of the mature exchanger suggest that the signal sequence is necessary to ensure the correct topological orientation of the exchanger in the membrane.     

Use of a modified oxygen microelectrode and laser-Doppler flowmetry to monitor changes in oxygen tension and microcirculation in a flap

Flap failure is a clinical problem in free tissue transfer, and there is no reliable device for monitoring the tissue. Differentiating between an arterial occlusion and venous congestion is also a problem. A study was undertaken to monitor viability in a pedicled groin flap and to compare two different monitoring methods. The oxygen tension in the flap, measured with a modified Clark-type microelectrode (tip diameter = 3 to 8 microm; 90 percent response within 2.6 +/- 0.5 seconds), was compared with changes in blood flow in the flap, measured with a laser-Doppler probe. In 11 Sprague-Dawley rats, the changes in oxygen tension and blood flow in the pedicled groin flap were studied after clamping and subsequent reperfusion of the artery or vein. After occlusion of the artery to the flap, oxygen tension decreased to a stable value (i.e., the recording level remained unchanged for 30 seconds), from 19.7 +/- 1.8 to 0.3 +/- 0.1 mmHg, after 193 +/-25 seconds; blood flow decreased to a stable value, from 117 +/- 21 to 54 +/- 18 perfusion units, after 26 +/- 6 seconds. Clamping of the vein resulted in a decrease in oxygen tension, from 17.1 +/- 1.8 to 1.4 +/- 0.7 mmHg, after 416 +/- 67 seconds, and blood flow decreased to a stable value, from 90 +/- 14 to 35 +/- 6 perfusion units, after 107 +/- 27 seconds. The results of this study show that there is a difference in oxygen tension and blood flow responses between arterial and venous occlusion and that it may be possible with both methods to distinguish arterial from venous occlusion. However, although oxygen tension measurements are slightly slower in response than laser-Doppler measurements, the values are more reliable as a diagnostic tool for interpretation of a vessel occlusion.     

Attitude of Baikal and Palearctic amphipods to oxygen as a factor of environment and mechanism of adaptation to its lowered level

The paper presents the materials of a comparative study of characteristics of anaerobic mechanisms use under the conditions of lowered oxygen content by Baikal amphipods Eulimnogammarus vittatus (Dyb.), E. cyaneus (Dyb.), as well as by Palearctic Gammarus lacustris Sars. It is shown that the system of anaerobic metabolism works most effectively for Palearctic G. lacustris. This species is characterized by the least degree of use of low-efficient way of energy production—anaerobic lipolysis, and the highest degree of use of the most efficient way of energy production under the conditions of low oxygen content-anaerobic succinate production. For the Baikal species that are less resistant to the lowered level of oxygen we show more expressed induction of anaerobic lipolysis and lower degree of engagement of anaerobic succinate production.     

Decreased oxygen tension lowers reactive oxygen species and apoptosis and inhibits osteoblast matrix mineralization through changes in early osteoblast differentiation

Accumulating data show that oxygen tension can have an important effect on cell function and fate. We used the human pre-osteoblastic cell line SV-HFO, which forms a mineralizing extracellular matrix, to study the effect of low oxygen tension (2%) on osteoblast differentiation and mineralization. Mineralization was significantly reduced by 60-70% under 2% oxygen, which was paralleled by lower intracellular levels of reactive oxygen species (ROS) and apoptosis. Following this reduction in ROS the cells switched to a lower level of protection by down-regulating their antioxidant enzyme expression. The downside of this is that it left the cells more vulnerable to a subsequent oxidative challenge. Total collagen content was reduced in the 2% oxygen cultures and expression of matrix genes and matrix-metabolizing enzymes was significantly affected. Alkaline phosphatase activity and RNA expression as well as RUNX2 expression were significantly reduced under 2% oxygen. Time phase studies showed that high oxygen in the first phase of osteoblast differentiation and prior to mineralization is crucial for optimal differentiation and mineralization. Switching to 2% or 20% oxygen only during mineralization phase did not change the eventual level of mineralization. In conclusion, this study shows the significance of oxygen tension for proper osteoblast differentiation, extra cellular matrix (ECM) formation, and eventual mineralization. We demonstrated that the major impact of oxygen tension is in the early phase of osteoblast differentiation. Low oxygen in this phase leaves the cells in a premature differentiation state that cannot provide the correct signals for matrix maturation and mineralization.     

ArfGAP1 responds to membrane curvature through the folding of a lipid packing sensor motif

ArfGAP1 promotes GTP hydrolysis in Arf1, a small G protein that interacts with lipid membranes and drives the assembly of the COPI coat in a GTP-dependent manner. The activity of ArfGAP1 increases with membrane curvature, suggesting a negative feedback loop in which COPI-induced membrane deformation determines the timing and location of GTP hydrolysis within a coated bud. Here we show that a central sequence of about 40 amino acids in ArfGAP1 acts as a lipid-packing sensor. This ALPS motif (ArfGAP1 Lipid Packing Sensor) is also found in the yeast homologue Gcs1p and is necessary for coupling ArfGAP1 activity with membrane curvature. The ALPS motif binds avidly to small liposomes and shows the same hypersensitivity on liposome radius as full-length ArfGAP1. Site-directed mutagenesis, limited proteolysis and circular dichroism experiments suggest that the ALPS motif, which is unstructured in solution, inserts bulky hydrophobic residues between loosely packed lipids and forms an amphipathic helix on highly curved membranes. This helix differs from classical amphipathic helices by the abundance of serine and threonine residues on its polar face.     

FHL5, a novel actin-binding protein, is highly expressed in eel gill pillar cells and responds to wall tension

Supporting evidence for the contractile nature of fish branchial pillar cells was provided by demonstrating the presence of actin fibers and a novel four-and-a-half LIM (FHL) protein in which expression is specific for contractile tissues and sensitive to the tension applied to the pillar cell. When eel gill sections were stained with rhodamine-phalloidin, a selective fluorescent probe for fibrous actin, a strong bundle-like staining was observed around collagen columns in pillar cells, suggesting the presence of abundant actin fibers. A cDNA clone encoding a novel member of the actin-binding FHL family, FHL5, was isolated from a subtracted cDNA library of eel gill. Northern analysis revealed that FHL5 mRNA is highly expressed only in gills, heart, and skeletal muscle. In gills, FHL5 was found to be confined to pillar cells by immunohistochemistry. Confocal fluorescence microscopy showed that FHL5 is present in both cytosol and nucleus; within the cytosol, a large portion of FHL5 is colocalized with the phalloidin-positive actin bundles. Furthermore, transfection of myogenic C2C12 cells with FHL5 cDNA demonstrated, in addition to its interaction with actin stress fibers, a nuclear shuttling activity of FHL5. The mRNA and protein levels were found to be elevated on 1) transfer of eels from seawater to freshwater, 2) volume expansion by infusion of isotonic dextran-saline, and 3) constriction of gill vasculature by bolus injection of endothelin-1. These results suggest contractile nature of pillar cells and a role of FHL5 in maintaining the integrity and regulating the dynamics of pillar cells.     

Identification of the sodium-calcium exchanger as the major ricin-binding glycoprotein of bovine rod outer segments and its localization to the plasma membrane

After neuraminidase treatment the Na+/Ca2+ exchanger of bovine rod outer segments was found to specifically bind Ricinus communis agglutinin. SDS gel electrophoresis and Western blotting of ricin-binding proteins purified from rod outer segment membranes by lectin affinity chromatography revealed the existence of two major polypeptides of Mr 215K and 103K, the former of which was found to specifically react with PMe 1B3, a monoclonal antibody specific for the 230-kDa non-neuraminidase-treated Na+/Ca2+ exchanger. Reconstitution of the ricin affinity-purified exchanger into calcium-containing liposomes revealed that neuraminidase treatment had no significant effect on the kinetics of Na+/Ca2+ exchange activation by sodium. We further investigated the density of the Na+/Ca2+ exchanger in disk and plasma membrane preparations using Western blotting, radioimmunoassays, immunoelectron microscopy, and reconstitution procedures. The results indicate that the Na+/Ca2+ exchanger is localized in the rod photoreceptor plasma membrane and is absent or present in extremely low concentrations in disk membranes, as we have previously shown to be the case for the cGMP-gated cation channel. Previous reports describing the existence of Na+/Ca2+ exchange activity in rod outer segment disk membrane preparations may be due to the fusion of plasma membrane components and/or the presence of contaminating plasma membrane vesicles.     

Enhancing oxygen tension and cellular function in alginate cell encapsulation devices through the use of perfluorocarbons

Encapsulation devices are often hindered by the inability to achieve sufficient oxygen levels for sustaining long-term cell survival both in vivo and in vitro. We have investigated the use of synthetic oxygen carriers in alginate gels to improve metabolic activity and viability of HepG2 cells over time. Perfluorocarbons (PFCs), specifically perfluorotributylamine (PFTBA) and perfluorooctylbromide (PFOB), were emulsified with alginate and used to encapsulate HepG2 cells in a spherical geometry. Cellular state was assessed using the MTT assay and Live/Dead stain as well as through analysis of both lactate and lactate dehydrogenase (LDH) levels which are indirect indicators of oxygen availability. Addition of 1% surfactant resulted in stable emulsions with evenly dispersed PFC droplets of the order of 1-2 microm in diameter, with no influence on cell viability. Both PFCs evaluated were effective in increasing cellular metabolic activity over alginate-only gels. The presence of 10% PFOB significantly increased cellular growth rate by 10% and reduced both intracellular LDH and extracellular lactate levels by 20-40%, improving glucose utilization efficiency. The characteristic drop in cellular metabolic activity upon encapsulation was eliminated with addition of 10% PFC and viability was better maintained throughout the bead, with a significant decrease in necrotic core size. Results were consistent under a physiologically relevant 5% oxygen environment. The incorporation of PFC synthetic oxygen carriers into encapsulation matrices has been successfully applied to improve cell function and viability with implication for a variety of tissue engineering applications.