Bradykinin inhibits M current via phospholipase C and Ca2+ release from IP3-sensitive Ca2+ stores in rat sympathetic neurons

A variety of intracellular signaling pathways can modulate the properties of voltage-gated ion channels. Some of them are well characterized. However, the diffusible second messenger mediating suppression of M current via G protein-coupled receptors has not been identified. In superior cervical ganglion neurons, we find that the signaling pathways underlying M current inhibition by B2 bradykinin and M1 muscarinic receptors respond very differently to inhibitors. The bradykinin pathway was suppressed by the phospholipase C inhibitor U-73122, by blocking the IP3 receptor with pentosan polysulfate or heparin, and by buffering intracellular calcium, and it was occluded by allowing IP3 to diffuse into the cytoplasm via a patch pipette. By contrast, the muscarinic pathway was not disrupted by any of these treatments. The addition of bradykinin was accompanied by a [Ca2+]i rise with a similar onset and time to peak as the inhibition of M current. The M current inhibition and the rise of [Ca2+]i were blocked by depletion of Ca2+ internal stores by thapsigargin. We conclude that bradykinin receptors inhibit M current of sympathetic neurons by activating phospholipase C and releasing Ca2+ from IP3-sensitive Ca2+ stores, whereas muscarinic receptors do not use the phospholipase C pathway to inhibit M current channels.

NADPH-oxidase and a hydrogen peroxide-sensitive K+ channel may function as an oxygen sensor complex in airway chemoreceptors and small cell lung carcinoma cell lines

Pulmonary neuroepithelial bodies (NEB) are widely distributed throughout the airway mucosa of human and animal lungs. Based on the observation that NEB cells have a candidate oxygen sensor enzyme complex (NADPH oxidase) and an oxygen-sensitive K+ current, it has been suggested that NEB may function as airway chemoreceptors. Here we report that mRNAs for both the hydrogen peroxide sensitive voltage gated potassium channel subunit (KH2O2) KV3.3a and membrane components of NADPH oxidase (gp91phox and p22phox) are coexpressed in the NEB cells of fetal rabbit and neonatal human lungs. Using a microfluorometry and dihydrorhodamine 123 as a probe to assess H2O2 generation, NEB cells exhibited oxidase activity under basal conditions. The oxidase in NEB cells was significantly stimulated by exposure to phorbol esther (0.1 μM) and inhibited by diphenyliodonium (5 μM). Studies using whole-cell voltage clamp showed that the K+ current of cultured fetal rabbit NEB cells exhibited inactivating properties similar to KV3.3a transcripts expressed in Xenopus oocyte model. Exposure of NEB cells to hydrogen peroxide (H2O2, the dismuted by-product of the oxidase) under normoxia resulted in an increase of the outward K+ current indicating that H2O2 could be the transmitter modulating the O2-sensitive K+ channel. Expressed mRNAs or orresponding protein products for the NADPH oxidase membrane cytochrome b as well as mRNA encoding KV3.3a were identified in small cell lung carcinoma cell lines. The studies presented here provide strong evidence for an oxidase-O2 sensitive potassium channel molecular complex operating as an O2 sensor in NEB cells, which function as chemoreceptors in airways and in NEB related tumors. Such a complex may represent an evolutionary conserved biochemical link for a membrane bound O2-signaling mechanism proposed for other cells and life forms.

Voltage-controlled gating in a large conductance Ca2+-sensitive K+channel (hslo)

Large conductance calcium- and voltage-sensitive K+ (MaxiK) channels share properties of voltage- and ligand-gated ion channels. In voltage-gated channels, membrane depolarization promotes the displacement of charged residues contained in the voltage sensor (S4 region) inducing gating currents and pore opening. In MaxiK channels, both voltage and micromolar internal Ca2+ favor pore opening. We demonstrate the presence of voltage sensor rearrangements with voltage (gating currents) whose movement and associated pore opening is triggered by voltage and facilitated by micromolar internal Ca2+ concentration. In contrast to other voltage-gated channels, in MaxiK channels there is charge movement at potentials where the pore is open and the total charge per channel is 4–5 elementary charges.

Autoinhibition of serotonin cells: An intrinsic regulatory mechanism sensitive to the pattern of usage of the cells

After periods of high-frequency firing, the normal rhythmically active serotonin (5HT)-containing neurosecretory neurons of the lobster ventral nerve cord display a period of suppressed spike generation and reduced synaptic input that we refer to as “autoinhibition.” The duration of this autoinhibition is directly related to the magnitude and duration of the current injection triggering the high-frequency firing. More interesting, however, is that the autoinhibition is inversely related to the initial firing frequency of these cells within their normal range of firing (0.5–3 Hz). This allows more active 5HT neurons to resume firing after shorter durations of inhibition than cells that initially fired at slower rates. Although superfused 5HT inhibits the spontaneous firing of these cells, the persistence of autoinhibition in saline with no added calcium, in cadmium-containing saline, and in lobsters depleted of serotonin suggests that intrinsic membrane properties account for the autoinhibition. A similar autoinhibition is seen in spontaneously active octopamine neurons but is absent from spontaneously active γ-aminobutyric acid cells. Thus, this might be a characteristic feature of amine-containing neurosecretory neurons. The 5HT cells of vertebrate brain nuclei share similarities in firing frequencies, spike shapes, and inhibition by 5HT with the lobster cells that were the focus of this study. However, the mechanism suggested to underlie autoinhibition in vertebrate neurons is that 5HT released from activated or neighboring cells acts back on inhibitory autoreceptors that are found on the dendrites and cell bodies of these neurons.

Identification and properties of the crenarchaeal single-stranded DNA binding protein from Sulfolobus solfataricus

Single-stranded DNA binding proteins (SSBs) play central roles in cellular and viral processes involving the generation of single-stranded DNA. These include DNA replication, homologous recombination and DNA repair pathways. SSBs bind DNA using four ‘OB-fold’ (oligonucleotide/oligosaccharide binding fold) domains that can be organised in a variety of overall quaternary structures. Thus eubacterial SSBs are homotetrameric whilst the eucaryal RPA protein is a heterotrimer and euryarchaeal proteins vary significantly in their subunit compositions. We demonstrate that the crenarchaeal SSB protein is an abundant protein with a unique structural organisation, existing as a monomer in solution and multimerising on DNA binding. The protein binds single-stranded DNA distributively with a binding site size of ~5 nt per monomer. Sulfolobus SSB lacks the zinc finger motif found in the eucaryal and euryarchaeal proteins, possessing instead a flexible C-terminal tail, sensitive to trypsin digestion, that is not required for DNA binding. In comparison with Escherichia coli SSB, the tail may play a role in protein–protein interactions during DNA replication and repair.

Immune-type receptor genes in zebrafish share genetic and functional properties with genes encoded by the mammalian leukocyte receptor cluster

An extensive, highly diversified multigene family of novel immune-type receptor (nitr) genes has been defined in Danio rerio (zebrafish). The genes are predicted to encode type I transmembrane glycoproteins consisting of extracellular variable (V) and V-like C2 (V/C2) domains, a transmembrane region and a cytoplasmic tail. All of the genes examined encode immunoreceptor tyrosine-based inhibition motifs in the cytoplasmic tail. Radiation hybrid panel mapping and analysis of a deletion mutant line (b240) indicate that a minimum of ≈40 nitr genes are contiguous in the genome and span ≈0.6 Mb near the top of zebrafish linkage group 7. One flanking region of the nitr gene complex shares conserved synteny with a region of mouse chromosome 7, which shares conserved synteny with human 19q13.3-q13.4 that encodes the leukocyte receptor cluster. Antibody-induced crosslinking of Nitrs that have been introduced into a human natural killer cell line inhibits the phosphorylation of mitogen-activated protein kinase that is triggered by natural killer-sensitive tumor target cells. Nitrs likely represent intermediates in the evolution of the leukocyte receptor cluster.

Prion protein (PrP) synthetic peptides induce cellular PrP to acquire properties of the scrapie isoform.

Conversion of the cellular isoform of prion protein (PrPC) into the scrapie isoform (PrPSc) involves an increase in the beta-sheet content, diminished solubility, and resistance to proteolytic digestion. Transgenetic studies argue that PrPC and PrPSc form a complex during PrPSc formation; thus, synthetic PrP peptides, which mimic the conformational pluralism of PrP, were mixed with PrPC to determine whether its properties were altered. Peptides encompassing two alpha-helical domains of PrP when mixed with PrPC produced a complex that displayed many properties of PrPSc. The PrPC-peptide complex formed fibrous aggregates and up to 65% of complexed PrPC sedimented at 100,000 x g for 1 h, whereas PrPC alone did not. These complexes were resistant to proteolytic digestion and displayed a high beta-sheet content. Unexpectedly, the peptide in a beta-sheet conformation did not form the complex, whereas the random coil did. Addition of 2% Sarkosyl disrupted the complex and rendered PrPC sensitive to protease digestion. While the pathogenic A117V mutation increased the efficacy of complex formation, anti-PrP monoclonal antibody prevented interaction between PrPC and peptides. Our findings in concert with transgenetic investigations argue that PrPC interacts with PrPSc through a domain that contains the first two putative alpha-helices. Whether PrPC-peptide complexes possess prion infectivity as determined by bioassays remains to be established.

Redistribution of synaptic vesicles and their proteins in temperature-sensitive shibire(ts1) mutant Drosophila.

From an extract of Drosophila melanogaster head homogenates, a membrane fraction can be isolated that has the same sedimentation properties as vertebrate synaptic vesicles and contains Drosophila synaptotagmin. The fraction disappears from homogenates of temperature-sensitive (ts) mutant shibire(ts1) (shi(ts1)) flies paralyzed by exposure to non-permissive temperatures, and reappears on return to permissive temperatures. Since reversible, temperature-dependent depletion of synaptic vesicles is known to occur in shibire(ts1) flies, we conclude that the fraction we have identified contains synaptic vesicles. We have examined the fate of synaptic vesicle membrane proteins in shibire flies at nonpermissive temperatures and found that all of these vesicle antigens are transferred to rapidly sedimenting membranes and codistribute with a plasma membrane marker by both glycerol velocity and metrizamide density sedimentation and by confocal microscopy. Three criteria were used to establish that other neuron-specific antigens--neuronal synaptobrevin and cysteine-string proteins--are legitimate components of synaptic vesicles: cosedimentation with Drosophila synaptotagmin, immunoadsorption, and disappearance of these antigens from the vesicle fractions in paralyzed shibire flies.

Differentiation of short-wavelength-sensitive cones by NADPH diaphorase histochemistry.

NADPH diaphorase (NADPH dehydrogenase; EC 1.6.99.1) histochemistry labels neurons that synthesize the neurotransmitter nitric oxide (NO). In retina, it has been demonstrated that NO can affect the metabolism of cGMP in rod photoreceptors. To investigate potential involvement of NO in cone photoreceptor activity, we utilized NADPH diaphorase histochemistry to study the cone-dominated retina of the tree shrew (Tupaia belangeri). Unexpectedly, our results revealed different NADPH diaphorase activity in the cellular subcompartments of the spectral classes of cone photoreceptors. Although all cones showed intense labeling of inner segment ellipsoids, the short-wavelength-sensitive (SWS or "blue-sensitive") cones and the rods displayed intense staining of the myoid inner segment subcompartment as well. Furthermore, only SWS cones and rods displayed surface labeling of their nuclei. These findings indicate a manner in which SWS cones differ biochemically from other cone types and in which they are more similar to rods. Such differences may underlie some of the unusual functional properties of the SWS cone system, which have been attributed to postreceptoral processes.

Electrical and synaptic properties of embryonic luteinizing hormone-releasing hormone neurons in explant cultures.

Voltage- and ligand-activated channels in embryonic neurons containing luteinizing hormone-releasing hormone (LHRH) were studied by patch-pipette, whole-cell current and voltage clamp techniques. LHRH neurons were maintained in explant cultures derived from olfactory pit regions of embryonic mice. Cells were marked intracellularly with Lucifer yellow following recording. Sixty-two cells were unequivocally identified as LHRH neurons by Lucifer yellow and LHRH immunocytochemistry. The cultured LHRH neurons had resting potentials around -50 mV, exhibited spontaneous discharges generated by intrinsic and/or synaptic activities and contained a time-dependent inward rectifier (Iir). Voltage clamp analysis of ionic currents in the LHRH neuron soma revealed a tetrodotoxin-sensitive Na+ current (INa) and two major types of K+ currents, a transient current (IA), a delayed rectifier current (IK) and low- and high-voltage-activated Ca2+ currents. Spontaneous depolarizing synaptic potentials and depolarizations induced by direct application of gamma-aminobutyrate were both inhibited by picrotoxin or bicuculline, demonstrating the presence of functional gamma-aminobutyrate type A synapses on these neurons. Responses to glutamate were found in LHRH neurons in older cultures. Thus, embryonic LHRH neurons not yet positioned in their postnatal environment in the forebrain contained a highly differentiated repertoire of voltage- and ligand-gated channels.

Strain and damage sensing properties on multifunctional cement composites with CNF admixture

Both strain and damage sensing properties on carbon nanofiber cement composites (CNFCC) are reported in the present paper. Strain sensing tests were first made on the material’s elastic range. The applied loading levels have been previously calculated from mechanical strength tests. The effect of several variables on the strain-sensing function was studied, e.g. cement pastes curing age, current density, loading rate or maximum stress applied. All these parameters were discussed using the gage factor as reference. After this first set of elastic experiments, the same specimens were gradually loaded until material’s failure. At the same time both strain and resistivity were measured. The former was controlled using strain gages, and the latter using a multimeter on a four probe setup. The aim of these tests was to prove the sensitivity of these CNF composites to sense their own damage, i.e. check the possibility of fabricating structural damage sensors with CNFCC’s. All samples with different CNF dosages showed good strain-sensing capacities for curing periods of 28 days. Furthermore, a 2%CNF reinforced cement paste has been sensitive to its own structural damage.

Paleomagnetic and rock magnetic properties of ODP Leg 173 sites, west Iberia continental margin

We present detailed paleomagnetic and rock magnetic results of rock samples recovered during Leg 173. The Leg 173 cores display a multicomponent magnetization nature. Variations in magnetic properties correlate with changes in lithology that result from differences in the abundance and size of magnetic minerals. The combined investigation suggests that the magnetic properties of the "fresher" peridotite samples from Site 1070 are controlled mainly by titanomagnetite, with a strong Verwey transition in the vicinity of 110 K, and with field- and frequency-dependent susceptibility curves that resemble those of titanomagnetites. These results are in excellent agreement with thermomagnetic characteristics where titanomagnetites with Curie temperature ~580°C were identified from the "fresher" peridotites. In contrast to the magnetic properties observed from the "fresher" peridotites, the low-temperature curves for the "altered" peridotites did not show any Verwey transition. Thermomagnetic analysis using the high-temperature vibrating sample magnetometer also failed to show evidence for titanomagnetites. The remanent magnetization is carried by a thermally unstable mineral that breaks down at ~420°C, probably maghemite. The field- and frequency-dependent relationships are also directly opposite to those in the reversal zone, with no signs of titanomagnetite characteristics. Altogether, these rock magnetic data seem to be sensitive indicators of alteration and support the contention that maghemite is responsible for the magnetic signatures displayed in the altered peridotites of the upper section. The magnetic minerals of the basement rocks from Sites 1068, 1069, and 1070 are of variable particle size but fall within the pseudo-single-domain size range (0.2-14 µm). The average natural remanent magnetization (NRM) intensity of recovered serpenitinized peridotite is typically on the order of 20 mA/m for samples from Site 1068, but ~120 mA/m for samples from Site 1070. The much stronger magnetization intensity of Site 1070 is apparently in excellent agreement with the observed magnetic anomaly high. Nearly half of the NRM intensity remained after 400°C demagnetization, suggesting that the remanence can contribute significantly to the marine magnetic anomaly.

(Table 1) Magnetic characteristic properties of selected samples of ODP Hole 134-831B

During Ocean Drilling Program Leg 134 (Vanuatu), geological high sensitivity magnetic tools (GHMT) developed by CEA-LETI and TOTAL were used at two drill sites. GHMT combine two sensors, a proton magnetometer for total magnetic field measurements with an operational accuracy of 0.1 nanoteslas (nT), and a highly sensitive induction tool to measure the magnetic susceptibility with an operational accuracy of a few 10**-6 SI units. Hole 829A was drilled through an accretionary prism and the downhole measurements of susceptibility correlate well with other well-log physical properties. Sharp susceptibility contrasts between chalk and volcanic silt sediment provide complementary data that help define the lithostratigraphic units. At Hole 831B magnetic susceptibility and total field measurements were performed through a 700-m reef carbonate sequence of a guyot deposited on top of an andesitic volcano. The downhole magnetic susceptibility is very low and the amplitude of peak-to-peak anomalies is less than a few 10**-5 SI units. Based on the repeatability of the measurements, the accuracy of the magnetic logging measurements was demonstrated to be excellent. Total magnetic field data at Hole 831B reveal low magnetic anomalies of 0.5 to 5 nT and the measurement of a complete repeat section indicates an accuracy of 0.1 to 0.2 nT. Due to the inclination of the earth's magnetic field in this area (~-40°) and the very low magnetic susceptibility of the carbonate, the contribution of the induced magnetization to the total field measured in the hole is negligible. Unfortunately, because the core recovery was extremely poor (<5%) no detailed comparison between the core measurements and the downhole magnetic data could be made. Most samples have a diamagnetic susceptibility and very low intensity of remanent magnetization (< 10**-4 A/m), but a few samples have a stable remanent magnetization up to 0.005 A/m. These variations of the intensity of the remanent magnetization suggest a very heterogeneous distribution of the magnetization in the carbonate sequence that could explain the magnetic field anomalies measured in these weakly magnetized rocks.

Afterglows, redshifts, and properties of swift gammaray bursts

We present optical, near-IR, and radio follow-up of 16 Swift bursts, including our discovery of nine afterglows and a redshift determination for three. These observations, supplemented by data from the literature, provide an afterglow recovery rate of 52% in the optical/near-IR, much higher than in previous missions (BeppoSAX, HETE-2, INTEGRAL, and IPN). The optical/near-IR afterglows of Swift events are on average 1.8 mag fainter at t = 12 hr than those of previous missions. The X-ray afterglows are similarly fainter than those of pre-Swift bursts. In the radio the limiting factor is the VLA threshold, and the detection rate for Swift bursts is similar to that for past missions. The redshift distribution of pre-Swift bursts peaked at z similar to 1, whereas the six Swift bursts with measured redshifts are distributed evenly between 0.7 and 3.2. From these results we conclude that ( 1) the pre-Swift distributions were biased in favor of bright events and low-redshift events, ( 2) the higher sensitivity and accurate positions of Swift result in a better representation of the true burst redshift and brightness distributions ( which are higher and dimmer, respectively), and (3) similar to 10% of the bursts are optically dark, as a result of a high redshift and/or dust extinction. We remark that the apparent lack of low-redshift, low-luminosity Swift bursts and the lower event rate than prelaunch estimates ( 90 vs. 150 per year) are the result of a threshold that is similar to that of BATSE. In view of these inferences, afterglow observers may find it advisable to make significant changes in follow-up strategies of Swift events. The faintness of the afterglows means that large telescopes should be employed as soon as the burst is localized. Sensitive observations in RIz and near-IR bands will be needed to discriminate between a typical z similar to 2 burst with modest extinction and a high-redshift event. Radio observations will be profitable for a small fraction (similar to 10%) of events. Finally, we suggest that a search for bright host galaxies in untriggered BAT localizations may increase the chance of finding nearby low-luminosity GRBs.