Neutrophil surface presentation of the anti-neutrophil cytoplasmic antibody-antigen proteinase 3 depends on N-terminal processing.

The neutrophil serine protease proteinase 3 (PR3) is a main autoantigen in anti-neutrophil cytoplasmic antibody-associated vasculitis. PR3 surface presentation on neutrophilic granulocytes, the main effector cells, is pathogenically important. PR3 is presented by the NB1 (CD177) glycoprotein, but how the presentation develops during neutrophil differentiation is not known. An N-terminally unprocessed PR3 (proPR3) is produced early during neutrophil development and promotes myeloid cell differentiation. We therefore investigated if PR3 presentation depended on NB1 during neutrophil differentiation and if PR3 and proPR3 could both be presented by NB1. In contrast to mature neutrophils, differentiating neutrophils showed an early NB1-independent PR3 surface display that was recognized by only two of four monoclonal anti-PR3 antibodies and occurred in parallel with proPR3, but not PR3 secretion, suggesting that the NB1-independent surface PR3 was proPR3. PR3 gene expression preceeded NB1. When the NB1 receptor was detected on the surface, a mode of PR3 surface display similar to mature neutrophils developed together with the degranulation system. Ectopic expression studies showed that NB1 was a sufficient receptor for PR3 but not proPR3. ProPR3 display on the plasma membrane may influence the bone marrow microenvironment. NB1-mediated PR3 presentation depended on PR3 N-terminal processing implicating the PR3-N-terminus as NB1-binding site.

Regulation of calcium sparks and spontaneous transient outward currents by RyR3 in arterial vascular smooth muscle cells.

Intracellular Ca(2+) levels control both contraction and relaxation in vascular smooth muscle cells (VSMCs). Ca(2+)-dependent relaxation is mediated by discretely localized Ca(2+) release events through ryanodine receptor (RyR) channels in the sarcoplasmic reticulum (SR). These local increases in Ca(2+) concentration, termed sparks, stimulate nearby Ca(2+)-activated K(+) (BK) channels causing BK currents (spontaneous transient outward currents or STOCs). STOCs are hyperpolarizing currents that oppose vasoconstriction. Several RyR isoforms are coexpressed in VSMCs; however, their role in Ca(2+) spark generation is unknown. To provide molecular information on RyR cluster function and assembly, we examined Ca(2+) sparks and STOCs in RyR3-deficient freshly isolated myocytes of resistance-sized cerebral arteries from knockout mice and compared them to Ca(2+) sparks in cells from wild-type mice. We used RT-PCR to identify RyR1, RyR2, and RyR3 mRNA in cerebral arteries. Ca(2+) sparks in RyR3-deficient cells were similar in peak amplitude (measured as F/F(0)), width at half-maximal amplitude, and duration compared with wild-type cell Ca(2+) sparks. However, the frequency of STOCs (between -60 mV and -20 mV) was significantly higher in RyR3-deficient cells than in wild-type cells. Ca(2+) sparks and STOCs in both RyR3-deficient and wild-type cells were inhibited by ryanodine (10 micromol/L), external Ca(2+) removal, and depletion of SR Ca(2+) stores by caffeine (1 mmol/L). Isolated, pressurized cerebral arteries of RyR3-deficient mice developed reduced myogenic tone. Our results suggest that RyR3 is part of the SR Ca(2+) spark release unit and plays a specific molecular role in the regulation of STOCs frequency in mouse cerebral artery VSMCs after decreased arterial tone.

Simulation of voltage-sensitive optical signals in three-dimensional slabs of cardiac tissue: application to transillumination and coaxial imaging methods.

Voltage-sensitive dyes are an important tool in visualizing electrical activity in cardiac tissue. Until today, they have mainly been applied in cardiac electrophysiology to subsurface imaging. In the present study, we assess different imaging methods used in optical tomography with respect to their effectiveness in visualizing 3D cardiac activity. To achieve this goal, we simulate optical signals produced by excitation fronts initiated at different depths inside the myocardial wall and compare their properties for various imaging modes. Specifically, we consider scanning and broad-field illumination, including trans- and epi-illumination. We focus on the lateral optical resolution and signal intensity, as a function of the source depth. Optical diffusion theory is applied to derive a computationally efficient approximation of the point-spread function and to predict voltage-sensitive signals. Computations were performed both for fluorescent and absorptive voltage-sensitive dyes. Among all the above-mentioned methods, fluorescent coaxial scanning yields the best resolution (<2.5 mm) and gives the most information about the intramural cardiac activity.

Endothelin-1 in subarachnoid hemorrhage: An acute-phase reactant produced by cerebrospinal fluid leukocytes.

BACKGROUND AND PURPOSE: The most potent vasoconstrictor known, endothelin-1, is currently considered to mediate cerebral vasospasm in subarachnoid hemorrhage (SAH), which can cause delayed cerebral ischemia. In our study, we performed clinical and in vitro experiments to investigate the origin and the mechanisms of the secretion of endothelin-1 in SAH. METHODS: Endothelin-1 and markers of inflammatory host response (interleukin [IL]-1ss, IL-6, and tumor necrosis factor-alpha) were comparatively quantified in the cerebrospinal fluid (CSF) of SAH patients and control subjects, and concentrations were related to clinical characteristics. Furthermore, mononuclear leukocytes isolated from the CSF of SAH patients and control subjects were analyzed regarding their mRNA expression of endothelin-1 and inflammatory cytokines. Finally, complementary in vitro experiments were performed to investigate whether coincubation of blood and CSF can trigger leukocytic mRNA expression and release of these factors. RESULTS: Activated mononuclear leukocytes in the CSF of SAH patients synthesize and release endothelin-1 in parallel with known acute-phase reactants (IL-1ss, IL-6, and tumor necrosis factor-alpha). Complementary in vitro experiments not only further confirmed this leukocytic origin of endothelin-1 but also showed that aging and subsequent hemolysis of blood is sufficient to induce such endothelin-1 production. CONCLUSIONS: The demonstration that endothelin-1 is produced by activated CSF mononuclear leukocytes suggests that subarachnoid inflammation may represent a therapeutic target to prevent vasospasm and delayed cerebral ischemia after SAH.

From triple cysteine mutants to the cysteine-less glucose transporter GLUT1: a functional analysis.

Two triple cysteine mutants containing Cys-less N- or C-terminal halves and the Cys-less GLUT1 were generated by site-directed mutagenesis. Following expression in Xenopus oocytes, the intrinsic transport activities of the multiple cysteine mutants were slightly decreased when either the cysteine residues of the C-terminal half or all six residues were changed; substitution of serine for cysteine residues located at the N-terminal half was without consequence for the catalytic activity. The exofacial ligand ethylidene glucose inhibited 2-deoxy-D-glucose uptake of wild-type and Cys-less GLUT1-expressing Xenopus oocytes with comparable half-saturation constants (11.5 and 13.2 mM). However, each of the multiple cysteine mutants exhibited an increase in affinity for the endofacial inhibitor cytochalasin B, with the greatest effect being observed for the Cys-less construct (decrease in Ki by the factor 5-6).

The differential role of Cys-421 and Cys-429 of the Glut1 glucose transporter in transport inhibition by p-chloromercuribenzenesulfonic acid (pCMBS) or cytochalasin B (CB).

Cys-421 and Cys-429 of Glut1 were replaced by site-directed mutagenesis in order to investigate their involvement in basal glucose transport and transport inhibition. Neither of the two cysteine residues was essential for basal 2-deoxy-D-glucose uptake in Xenopus oocytes expressing the respective mutant M421 and M429. If applied from the external side, the poorly permeable sulfhydryl-reactive agent pCMBS inhibited 2-deoxy-D-glucose uptake of Glut1- and M421-expressing Xenopus oocytes but failed to affect uptake of the Cys-429 mutant. This is in agreement with the proposed two-dimensional model of Glut1 confirming that Cys-429 is the only residue exposed to the surface of the plasma membrane. The replacement of Cys-421 at the exofacial end of helix eleven caused a partial protection of 3-O-methylglucose transport inhibition by CB; this residue may thus be involved in stabilizing an adjacent local tertiary structure necessary for the full activity of this inhibitor.

GTP analogs suppress uptake but not transport of D-glucose analogs in Glut1 glucose transporter-expressing Xenopus oocytes.

A Xenopus oocyte expression-co-injection system was used to study the influence of guanine nucleotides on D-glucose uptake. GTP analogs like GTP gamma S and GppNHp had no effect on 3-O-methylglucose transport determined by zero-trans uptake or equilibrium exchange, but suppressed 2-deoxyglucose uptake into Glut1 glucose transporter-expressing oocytes by up to 86%. Both GTP analogs showed concentration dependence of their effectiveness, with GTP gamma S being more potent than GppNHp. No statistically significant differences were observed between groups of oocytes co-injected with water or GDP beta S (250 and 500 microM intracellular concentration). Glut1 transporter expression in plasma membrane was not different between water or GTP gamma S-co-injected oocytes. Thus, inhibition of hexokinase catalytic activity is the most likely causative factor for down-regulation of 2-deoxyglucose uptake.

Stretch-activated nonselective cation channels in urinary bladder myocytes: importance for pacemaker potentials and myogenic response.

Filling of the bladder with urine stretches the myocytes in the wall. Stretch activates nonselective cation channels (SACs) thereby constituting a pacemaking mechanism. Once action potentials are triggered, Ca2+ influx through nifedipine-sensitive Ca2+ channels provides activator Ca2+ for the stretch-induced increase in wall tension (myogenic response). An additional component of myogenic response is independent of nifedipine and membrane potential; Ca2+ influx through SACs is large enough to induce Ca2+ release from intracellular stores.

ET-1-induced pulmonary vasoconstriction shifts from ET(A)- to ET(B)-receptor-mediated reaction after preconstriction.

Endothelin-1 (ET-1) has been reported to induce pulmonary vasoconstriction via either ET(A) or ET(B) receptors, and vasorelaxation after ET-1 injection has been observed. Our study investigated the effects of ET-1 in isolated rabbit lungs, which were studied at basal tone (part I) and after preconstriction (U-46619; part II). Pulmonary arterial pressure (PAP) and lung weight gain were monitored continuously. In part I, ET-1 (10(-8) M; n = 6; control) was injected after pretreatment with the ET(A)-receptor antagonist BQ-123 (10(-6) M; n = 6) or the ET(B)-receptor antagonist BQ-788 (10(-6) M; n = 6). The same protocol was carried out in part II after elevation of pulmonary vascular tone. ET-1 induced an immediate PAP increase (DeltaPAP 4.3 +/- 0.4 mmHg at 10 min) that was attenuated by pretreatment with BQ-123 (P < 0.05 at 10 min and P < 0.01 thereafter) and that was more pronounced after BQ-788 (P < 0.01 at 10 min and P < 0.001 thereafter). In part II, ET-1 induced an immediate rise in PAP with a maximum after 5 min (DeltaPAP 6.3 +/- 1.4 mmHg), leveling off at DeltaPAP 3.2 +/- 0.2 mmHg after 15 min. Pretreatment with BQ-123 failed to attenuate the increase. BQ-788 significantly reduced the peak pressure at 5 min (0.75 +/- 0.4 mmHg; P < 0.001) as well as the plateau pressure thereafter (P < 0.01). We conclude that ET-1 administration causes pulmonary vasoconstriction independent of basal vascular tone, and, at normal vascular tone, the vasoconstriction seems to be mediated via ET(A) receptors. BQ-788 treatment resulted in even more pronounced vasoconstriction. After pulmonary preconstriction, ET(A) antagonism exerted no effects on PAP, whereas ET(B) antagonism blocked the PAP increase. Therefore, ET-1-induced pulmonary vasoconstriction is shifted from an ET(A)-related to an ET(B)-mediated mechanism after pulmonary vascular preconstriction.

Altered gene expression in cerebral capillaries of stroke-prone spontaneously hypertensive rats.

Stroke-prone spontaneously hypertensive rats (SHRSP) are a well-characterized, genetic model for stroke. We showed earlier that the structure and function of the tight junctions in SHRSP blood-brain barrier endothelial cells is disturbed prior to stroke. To investigate the molecular events leading to endothelial dysfunction in SHRSP cerebral capillaries, we carried out suppression subtractive hybridization (SSH) in combination with a cDNA filter screening step. We identified two cDNA fragments that were upregulated in SHRSP, compared to stroke-resistant spontaneously hypertensive rats (SHR), and found open reading frames of 133 and 138 amino acids, respectively. These peptides did not match any known proteins in public databases. A third upregulated SHRSP cDNA fragment was identified as the rat sulfonylurea receptor 2B (SUR2B). We also isolated and cloned the cDNA of the rat homologue for the mouse G-protein signaling 5 (RGS5) regulator. This regulator was downregulated in SHRSP. We used in situ hybridization to show that rat RGS5 is expressed in the brain capillary endothelium and in the choroid plexus. Our findings may lead to the identification of new stroke-related genes.

Intramural wave propagation in cardiac tissue: asymptotic solutions and cusp waves.

The cardiac muscle is well known to conduct electric impulses anisotropically, showing a larger conduction velocity along than across fibers. Fiber orientation is not uniform within the cardiac wall, but rotates by as much as 180 degrees throughout the wall thickness. Numerical simulations and experiments have indicated that this rotational anisotropy considerably affects the spread of excitation in cardiac tissue: the wave front shows a complex intramural shape with trailing cusps. The cusps can travel across layers and reach the epicardial and endocardial surfaces where they cause apparent accelerations of propagation. In the present study we provide an analytical description of the asymptotic wave front, as well as of cusp waves. We investigate the motion of cusp waves, based on the assumption that they occur at the intersection of asymptotic solutions, and we show that our theoretical analysis is in close agreement with numerical simulations. The asymptotic solutions are found to be determined purely by the fiber organization within the cardiac wall, independent of the excitable properties of cardiac tissue.

Shaping of a scroll wave filament by cardiac fibers.

Scroll waves of electrical excitation in heart tissue are implicated in the development of lethal cardiac arrhythmias. Here we study the relation between the geometry of myocardial fibers and the equilibrium shape of a scroll wave filament. Our theory accommodates a wide class of myocardial models with spatially varying diffusivity tensor, adjusted to fit fiber geometry. We analytically predict the exact equilibrium shapes of the filaments. The major conclusion is that the filament shape is a compromise between a straight line and full alignment with the fibers. The degree of alignment increases with the anisotropy ratio. The results, being purely geometrical, are independent of details of ionic membrane mechanisms. Our theoretical predictions have been verified to excellent accuracy by numerically simulating the stable equilibration of a scroll filament in a model of the FitzHugh-Nagumo type.

Adipose tissue and circulating endothelial cell specific molecule-1 in human obesity.

Adipocytes produce the endothelial-cell specific molecule-1 (ESM-1), which inhibits leukocyte adhesion and migration through the endothelium. This study investigates ESM-1 expression and regulation in human adipose tissue. Subcutaneous abdominal adipose tissue was obtained from seventy postmenopausal women. Fourteen women subsequently underwent non-pharmacological weight reduction. In vitro experiments were performed on adipocytes isolated from human mammary adipose tissue. We determined gene expression by TaqMan RT-PCR and measured ESM-1 levels in serum and cell culture medium by ELISA. Mature adipocytes produced ESM-1. ESM-1 gene expression was higher in adipocytes than in preadipocytes. Cortisol inhibited ESM-1 gene expression in preadipocytes. Insulin and cortisol inhibited adipocyte ESM-1 production in adipocytes. This inhibitory effect of insulin was attenuated by insulin resistance, as ESM-1 gene expression in subcutaneous adipose tissue was increased in obese, hyperinsulinemic women. In contrast, ESM-1 serum levels were reduced in obese women and inversely correlated to C-reactive protein levels. Five percent weight loss did not markedly change gene expression. Circulating ESM-1 levels increased significantly, albeit modestly. ESM-1 is actively produced by adipocytes. However, since ESM-1 adipocyte gene expression and circulating plasma levels are not correlated, other sources of ESM-1 may be more important. Circulating ESM-1 levels are reduced in the overweight and obese, consistent with the notion that ESM-1 may play some role in obesity-associated vascular disease.

Properties of stretch-activated channels in myocytes from the guinea-pig urinary bladder.

1. Stretch-activated channels (SACs) were analysed on patches attached to myocytes isolated from the guinea-pig urinary bladder. At 22 degrees C application of -2 to -4 kPa to the patch electrode induced SACs at a density of one to two per patch (3-5 M omega electrodes). 2. With electrodes containing 145 mM K+, 20 mM TEA and 2 mM Mg2+, the single channel current followed a linear I-V curve with a slope conductance of 39 +/- 5 pS (mean +/- S.D.) and a reversal potential of 2 +/- 6 mV. Substitution of chloride by aspartate ions left both parameters unchanged suggesting that the anions do not contribute to the currents. 3. Hyperpolarization from -30 to -80 mV did not open channels by itself but increased channel activity (NPo; where N is the number of channels in the patch and Po is the probability of the channel being open) twofold. The hyperpolarization-induced increase in NPo can be attributed to a reduction of long closures. At positive patch potentials numerous blank records strongly diminished NPo. 4. Inward currents through SACs can be carried by a variety of cations. In the presence of 2 mM Mg2+, the respective channel conductance was 40 +/- 4 pS for 140 mM K+ > 34 +/- 2 pS for 140 mM Na+ > or = 33 +/- 6 pS for 140 mM Cs+ > 19 +/- 2 pS for 110 mM Ba2+ > 17 +/- 2 pS for 110 mM Ca2+. 5. Reduction of CaCl2 from 110 to 10 mM did not change the conductance but shifted the reversal potential from +7 to -7 mV; the reversal potentials suggest that SACs are slightly more permeable for Ca2+ than for K+. 6. In the absence of divalent cations, the conductance of K+ was 82 +/- 4 pS for inward but 45 pS for outward currents. Addition of either 2 mM Ca2+ or 2 mM Mg2+ reduced the conductance for inward currents to 40 pS. 7. The change from 140 to 14 mM KCl plus 136 mM Tris-Cl reduced the conductance from 82 to 56 pS whereas the reversal potential shifted only from -4 to -9 mV. When 20 mM K+ and 300 mM sucrose were applied, the conductance fell to 39 pS and the reversal potential shifted by -30 mV. The results suggest that Tris+ can permeate through SACs when extracellular divalent cations are absent.(ABSTRACT TRUNCATED AT 400 WORDS)

The role of cysteine residues in glucose-transporter-GLUT1-mediated transport and transport inhibition.

The role of cysteine residues in transport function of the glucose transporter GLUT1 was investigated by a mutagenesis-expression strategy. Each of the six cysteine residues was individually replaced by site-directed mutagenesis. Expression of the heterologous wild-type or mutant glucose transporters and transport measurements at two hexose concentrations (50 microM and 5 mM) were undertaken in Xenopus oocytes. The catalytic activity of GLUT1 was retained, despite substitution of each single cysteine residue, which indicated that no individual residue is essential for hexose transport. This finding questions the involvement of oligomerization or intramolecular stabilization by a single disulphide bond as a prerequisite for transporter activation under basal conditions. Application of the impermeant mercurial thiol-group-reactive reagent p-chloromercuribenzenesulphonate (pCMBS) to the external or internal surface of plasma membrane demonstrated that cysteine-429, within the sixth external loop, and cysteine-207, at the beginning of the large intracellular loop which connects transmembrane segments 6 and 7, are the residues which are involved in transport inhibition by impermeant thiol-group-reactive reagents from either side of the cell. These data support the predicted membrane topology of the transport protein by transport measurements. If residues other than the cysteines at positions 429 or 207 are exposed to either side of the plasma membrane by conformational changes, they do not contribute to the transport inhibition by pCMBS. Application of pCMBS to one side of the plasma membrane also inhibited transport from the opposite direction, most likely due to the hindrance of sugar-induced interconversion of transporter conformation.

cAMP accelerates the decay of stretch-activated inward currents in guinea-pig urinary bladder myocytes.

1. Myocytes from the urinary bladder were stretched longitudinally by 5-20%. At -50 mV, stretch induced whole-cell inward currents (Iin) between -100 and -600 pA. Iin decayed slowly with time to 93 +/- 20% (mean +/- S.E.M., n = 6) of the initial value in 1 min. The mechanisms of this 'adaptation' and its modulation by dibutyryl cAMP (dBcAMP) were analysed with whole-cell and single channel currents. 2. When the cells were internally perfused with 100 microM 8-bromo-cAMP (8BrcAMP), stretch induced an Iin of the usual amplitude that decayed completely within 40 +/- 13 s. When 200 microM dBcAMP was bath applied 10 s after the start of the stretch, Iin decayed to zero within 85 +/- 18 s. 3. dBcAMP increased the K+ current through Ca(2+)-activated BK channels (IK(Ca)) at 0 mV with a time course that correlated well with the decay of Iin, and block of IK(Ca) by TEA suppressed the dBcAMP-induced decay of Iin. In the presence of intracellular BAPTA, dBcAMP increased the stretch-induced Iin. The results suggest that adaptation is caused by superimposition of IK(Ca) which is increased through elevation of near-membrane [Ca2+] and by cAMP-dependent phosphorylation. 4. Single channel analysis was carried out with 140 mM KCl electrode solution and at -50 mV. Stretch-activated channels (SACs) were recorded during pulses of negative pressures between -2 and -5 kPa. Activity (NPo) of SACs was constant for at least 4 min, e.g. evidence for adaptation was missing. dBcAMP (200 microM) increased NPo of SACs by 142 +/- 35% (n = 16). 5. dBcAMP increased NPo via frequency of openings and channel open time. In five of sixteen patches, dBcAMP induced openings without suction. Similar effects were induced by the catalytic subunit of cAMP-dependent protein kinase (PKAc), applied to inside-out patches. 6. NPo, normalized by its maximum, increased with more negative pressure along an S-shaped curve. dBcAMP increased the sensitivity of SACs to stretch by shifting the point of half-maximal activity from -3.2 to -2.6 kPa. 7. The augmentation of NPo by dBcAMP is attributed to the phosphorylation of SACs promoting their opening. Adaptation of Iin is discussed as a 'secondary' effect of stretch-activated channels: Ca2+ influx through SACs increases the Ca2+ concentration that activates BK channels whose Ca2+ sensitivity is increased by cAMP.

Stretch effects on whole-cell currents of guinea-pig urinary bladder myocytes.

1. By means of two patch-pipettes, isolated urinary bladder myocytes were longitudinally stretched up to 20% beyond slack length (delta L = 20%). 2. Experiments were conducted using both voltage and current clamp configurations. In current clamped cells at 23 degrees C, delta L depolarized the membrane from -50 to ca -15 mV, the amplitude of depolarization increasing with the extent of delta L. At 36 degrees C, delta L induced action potentials or increased the frequency of spontaneous action potentials. 3. In voltage clamped cells at a holding potential of -50 mV, stretch induced an inward current (Iin) and increased the input conductance. Both effects increased with delta L. They were blocked by 40 microM gadolinium, suggesting stretch activation of non-selective cation channels (SACs) as the underlying mechanism. 4. Stretch-induced difference currents rectified outwardly and reversed at a reversal potential (Erev) of -28 +/- 10 mV. Twenty millimolar [TEA]o suppressed the rectification and shifted Erev to 0 +/- 1 mV. The result suggests that stretch can activate not only SACs but also TEA-sensitive K+ channels. 5. Stretch changed the net current due to clamp steps to 0 mV as though it increased the potassium current (IK) and reduced the calcium current (ICa). While 20 mM intracellular BAPTA did not modify the stretch-induced whole-cell inward current (Iin) at -50 mV, it suppressed the stretch effects on IK and ICa as if these effects were mediated by an increase in the subsarcolemmal Ca2+ concentration. 6. The results support the hypothesis that longitudinal stretch can activate SACs and Ca2+ influx through them. In non-clamped cells, stretch can also modulate Ca2+ influx through L-type Ca2+ channels via changes in membrane potential.

Predicting filament drift in twisted anisotropy.

Excitable media with twisted anisotropy have recently been attracting significant interest because of their applicability to wave propagation in heart tissue. Here we consider the dynamics of an intramural scroll wave whose filament lies initially within an arbitrary layer of mutually parallel cardiac fibers, and drifts parallel to itself from layer to layer. Earlier simulations have demonstrated that such a filament stabilizes in a layer whose fiber direction is the same as its own. In the present paper we analytically derive the trajectory of the filament, and obtain good agreement with earlier numerical data. For sufficiently sparse scrolls, our analysis predicts an equilibrium alignment perpendicular rather than parallel to the fibers.

Topological constraint on scroll wave pinning.

Scroll waves in an excitable medium rotate about tubelike filaments, whose ends, when they exist, can lie on the external boundary of the medium or be pinned to an inclusion. We derive a topological rule that governs such pinning. It implies that some configurations cannot occur although they might otherwise have been expected. Heart tissue provides an application of these concepts. Computational illustrations based on a FitzHugh-Nagumo model are given.