Calprotectin inhibits matrix metalloproteinases by sequestration of zinc.

BACKGROUND/AIMS: Calprotectin, a 36 kDa protein present in neutrophil cytoplasm, has antimicrobial and apoptosis inducing activities, which are reversed by the addition of zinc. Matrix metalloproteinases (MMPs), a family of zinc dependent enzymes, are important in many normal biological processes including embryonic development, angiogenesis, and wound healing, but also pathological processes such as inflammation, cancer, and tissue destruction. The aim of this study was to investigate whether calprotectin can inhibit MMP activity, and whether such inhibition could be overcome by the addition of zinc. METHODS: MMP activity was measured by the degradation of substrates precoated on to microwells, and visualised by Coomassie blue staining of residual substrate. Seven metalloproteinases (MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, and MMP-13) were tested against two substrates: gelatin and alpha-casein. RESULTS: All MMPs except MMP-1 were active against gelatin, whereas MMP-7 was the only enzyme active against alpha-casein. The addition of calprotectin inhibited the activity of all the MMPs, but different concentrations of the protein, from 0.3 microM to > 11microM, were necessary to produce a 50% inhibition of the MMPs. Inhibition by calprotectin was largely overcome by the addition of zinc. CONCLUSIONS: The findings suggest that calprotectin inhibits MMPs by sequestration of zinc. The data also suggest that MMPs have different affinities for zinc and that calprotectin has a lower zinc affinity than the MMPs.     

Tyrosine kinase and phosphatase regulation of slow delayed-rectifier K+ current in guinea-pig ventricular myocytes

The objective of this study was to investigate the involvement of tyrosine phosphorylation in the regulation of the cardiac slowly activating delayed-rectifier K⁺ current ( I _Ks) that is important for action potential repolarization. Constitutive I _Ks recorded from guinea-pig ventricular myocytes was suppressed by broad-spectrum tyrosine kinase (TK) inhibitors tyrphostin A23 (IC₅₀, 4.1 ± 0.6 μ m ), tyrphostin A25 (IC₅₀, 12.1 ± 2.1 μ m ) and genistein (IC₅₀, 64 ± 4 μ m ), but was relatively insensitive to the inactive analogues tyrphostin A1, tyrphostin A63, daidzein and genistin. I _Ks was unaffected by AG1478 (10 μ m ), an inhibitor of epidermal growth factor receptor TK, and was strongly suppressed by the Src TK inhibitor PP2 (10 μ m ) but not by the inactive analogue PP3 (10 μ m ). The results of experiments with forskolin, H89 and bisindolylmaleimide I indicate that the suppression of I _Ks by TK inhibitors was not mediated via inhibition of ( I _Ks-stimulatory) protein kinases A and C. To evaluate whether the suppression was related to lowered tyrosine phosphorylation, myocytes were pretreated with TK inhibitors and then exposed to the phosphotyrosyl phosphatase inhibitor orthovanadate (1 m m ). Orthovanadate almost completely reversed the suppression of I _Ks induced by broad-spectrum TK inhibitors at concentrations around their IC₅₀ values. We conclude that basal I _Ks is strongly dependent on tyrosine phosphorylation of Ks channel (or channel-regulatory) protein.     

Adenosine inhibition via A1 receptor of N-type Ca2+ current and peptide release from isolated neurohypophysial terminals of the rat

Effects of adenosine on voltage-gated Ca²⁺ channel currents and on arginine vasopressin (AVP) and oxytocin (OT) release from isolated neurohypophysial (NH) terminals of the rat were investigated using perforated-patch clamp recordings and hormone-specific radioimmunoassays. Adenosine, but not adenosine 5'-triphosphate (ATP), dose-dependently and reversibly inhibited the transient component of the whole-terminal Ba²⁺ currents, with an IC₅₀ of 0.875 μ m. Adenosine strongly inhibited, in a dose-dependent manner (IC₅₀= 2.67 μ m ), depolarization-triggered AVP and OT release from isolated NH terminals. Adenosine and the N-type Ca²⁺ channel blocker ω-conotoxin GVIA, but not other Ca²⁺ channel-type antagonists, inhibited the same transient component of the Ba²⁺ current. Other components such as the L-, Q- and R-type channels, however, were insensitive to adenosine. Similarly, only adenosine and ω-conotoxin GVIA were able to inhibit the same component of AVP release. A₁ receptor agonists, but not other purinoceptor-type agonists, inhibited the same transient component of the Ba²⁺ current as adenosine. Furthermore, the A₁ receptor antagonist 8-cyclopentyltheophylline (CPT), but not the A₂ receptor antagonist 3, 7-dimethyl-1-propargylxanthine (DMPGX), reversed inhibition of this current component by adenosine. The inhibition of AVP and OT release also appeared to be via the A₁ receptor, since it was reversed by CPT. We therefore conclude that adenosine, acting via A₁ receptors, specifically blocks the terminal N-type Ca²⁺ channel thus leading to inhibition of the release of both AVP and OT.     

3, 3', 4, 4'-Tetrachlorobiphenyl Inhibits Proliferation of Immature Thymocytes in Fetal Thymus Organ Culture

The environmental pollutant 3, 3', 4, 4'-tetrachlorobiphenyl (TCB) leads to thymic atrophy and immuno-suppression, the former possibly causing the latter. TCB binds lo the cytosolic aryl-hydrocarbon receptor (AhR) and transforms it into a DNA-binding state. The development of fetal thymocyles is severely affected by TCB and other AhR-binding xenobiotics, leading to a skewed pattern of thymocyte maturation stages. Murine thymocyte proliferation after exposure to TCB was studied in fetal thymus organ culture (FTOC). C57BL/6 fetus thymic lobes from day 15 of gestation were explanted and grown for 2, 4, 6. and 8 days in organ culture in the presence or absence of 3.3 μM TCB. Subsets of thymocytes were defined by CD4 and CD8 surface markers, and their cell cycle was analysed by DNA staining with 7-amino-actinomycin D (7-AAD). Exposure of fetal thymi in vitro to 3.3 μM TCB significantly reduced the total number of thymocytes. and fewer thymocytes were in S/G₂M phase. The inhibition of cell proliferation induced by TCB treatment affected mainly the CD4⁻ CD8⁻ (double-negative, DN) and CD4⁻ CD8⁺ (single-positive, SP) subsets, and these inhibition appeared mainly in more immature thymocytes, i. e. DNCD3⁻ and CD8⁺CD3⁻ subpopulations, whereas no effect of TCB on CD4⁺CD8⁺ (double-positive, DP) cell proliferative activity was observed. Analysis of the relation of cell proliferation and development of subsets in differentiating fetal ihymocytes suggests that TCB enhanced thymocyte differentiation into mature CD8⁺ cells.     

Effect of protease-activated receptor 2 activation on single TRPV1 channel activities in rat vagal pulmonary sensory neurons

Protease-activated receptor 2 (PAR₂) is involved in airway inflammation and airway hyperresponsiveness; both are the prominent features of asthma. Transient receptor potential vanilloid receptor 1 (TRPV1) is expressed in pulmonary sensory nerves, functions as a thermal and chemical transducer and contributes to neurogenic inflammation. Using cell-attached single-channel recordings we investigated the effect of PAR₂ activation on single TRPV1 channel activities in isolated pulmonary sensory neurons. Our immunohistochemical study demonstrated the expression of PAR₂ in rat vagal pulmonary sensory neurons. Our patch-clamp study further showed that intracellular application of capsaicin (0.75 μ m ) induced single-channel current that exhibited outward rectification in these neurons. The probability of the channel being open ( P _o) was significantly increased after the cells were pretreated with PAR₂-activating peptide (100 μ m , 2 min). Pretreatment with trypsin (0.1 μ m , 2 min) also increased the single-channel P _o, and the effect was completely inhibited by soybean trypsin inhibitor (0.5 μ m , 3 min). In addition, the effect of PAR₂ activation was abolished by either U73122 (1 μ m , 4 min), a phospholipase C inhibitor, or chelerythrine (10 μ m , 4 min), a protein kinase C inhibitor. In conclusion, our data demonstrated that activation of PAR₂ upregulated single-channel activities of TRPV1 and that the effect was mediated through the protein kinase C-dependent transduction pathway.     

The role of histidine residues in modulation of the rat P2X2 purinoceptor by zinc and pH

P2X₂ receptor currents are potentiated by acidic pH and zinc. To identify residues necessary for proton and zinc modulation, alanines were singly substituted for each of the nine histidines in the extracellular domain of the rat P2X₂ receptor. Wild-type and mutant receptors were expressed in Xenopus oocytes and analysed with two-electrode voltage clamp. All mutations caused less than a 2-fold change in the EC₅₀ of the ATP concentration-response relation. Decreasing the extracellular pH from 7.5 to 6.5 potentiated the responses to 10 μ m ATP of wild-type P2X₂ and eight mutant receptors more than 4-fold, but the response of the mutant receptor H319A was potentiated only 1.4-fold. The H319A mutation greatly attenuated the maximal potentiation that could be produced by a drop in pH, shifted the p K _a (-log of dissociation constant) of the potentiation to a more basic pH as compared with P2X₂ and revealed a substantial pH-dependent decrease in the maximum response with a p K _a near 6.0. Substituting a lysine for H319 reduced the EC₅₀ for ATP 40-fold. Zinc (20 μ m ) potentiated the responses to 10 μ m ATP of wild-type P2X₂ and seven histidine mutants by ∼8-fold but had virtually no effect on the responses of two mutants, H120A and H213A. Neither H120A nor H213A removed the voltage-independent inhibition caused by high concentrations of zinc. The observation that different mutations selectively eliminated pH or zinc potentiation implies that there are two independent sites of action, even though the mechanisms of pH and zinc potentiation appear similar.     

Mechanism of extracellular release of human neutrophil calprotectin complex.

Calprotectin is an abundant cytosolic protein complex of human neutrophils with in vitro extracellular antimicrobial activity. Studies suggest that calprotectin may be actively secreted from intact HL-60 cells and that it can be translocated to polymorphonuclear neutrophil (PMN) cell membranes. To examine whether calprotectin is secreted extracellularly, we incubated soluble and particulate stimuli, including live and heat-inactivated Candida albicans, with whole blood and measured calprotectin levels in the plasma. We compared the release of calprotectin to that of lactoferrin, a protein known to be secreted by PMNs. Extracellular lactoferrin was detected after incubation with any of the particulate stimuli. In contrast, a significant increase in extracellular calprotectin was found only after incubation with live C. albicans. Specifically, the increase in extracellular calprotectin correlated directly with a proportional decrease in PMN viability. Our results indicate that human PMN calprotectin is not secreted extracellularly except as a result of cell disruption or death.     

Neurite outgrowth-regulating properties of GABA and the effect of serum on mouse spinal cord neurons in culture

Time-lapse photography was used to examine the effects of γ-aminobutyric acid (GABA) on the outgrowth and motility of neurites in cultures from mouse spinal cord. GABA at concentrations of 100, 10 and 1 μ m caused significant inhibition of neurite outgrowth and the motility of growth cones was significantly reduced by treatment with 100 and 10 μ m GABA. This effect was mimicked by the GABA_B receptor agonist baclofen, whereas the GABA_A receptor agonist muscimol had no effect. The effect of GABA on outgrowth and motility seems to be dependent on the type of serum employed. The results reported here were obtained only when heat-inactivated serum was used and not when non heat-inactivated serum was added to the culture medium. They suggest that GABA has a role in the regulation of process outgrowth within the embryonic mouse spinal cord.     

Theophylline and cAMP inhibit lysophosphatidic acid-induced hyperresponsiveness of bovine tracheal smooth muscle cells

We have established an in vitro model of airway hyperresponsiveness, using a bovine tracheal smooth muscle cell (BTSMC)-embedded collagen gel lattice. When the gel was pretreated with lysophosphatidic acid (LPA), which activates the small G protein RhoA, ATP- and high K⁺ solution-induced gel contraction was significantly augmented. This was not due to the modulation of Ca²⁺ mobilizing properties, since ATP- and high K⁺-induced Ca²⁺ transients were not significantly different between control and LPA-treated BTSMC. Y-27632, an inhibitor of Rho-kinase, suppressed the LPA-induced augmentation of gel contraction, whereas it did not inhibit the contraction of control gels. Theophylline (> 1 μ m ) reversed the LPA-induced augmentation of gel contraction, whereas it inhibited control gel contraction only with a very high concentration (100 μ m ). We confirmed that theophylline increased the intracellular concentration of cAMP ([cAMP]_i) in BTSMC. Elevation of [cAMP]_i with dibutyryl cAMP or forskolin also reversed the LPA-induced augmentation of gel contraction. Furthermore, theophylline, as well as dibutyryl cAMP and forskolin, suppressed the LPA-induced membrane translocation of RhoA, indicating that they prevented airway hyperresponsiveness by inhibiting RhoA. We conclude from these results that theophylline inhibits LPA-induced, RhoA/Rho-kinase-mediated hyperresponsiveness of tracheal smooth muscle cells due to the accumulation of cAMP.     

Cyclic AMP-dependent Cl− secretion induced by thromboxane A2 in isolated human colon

Increased release of thromboxane A₂ (TXA₂) has been shown to be involved in inflammatory bowel diseases. In the present study, we have investigated the effect of a stable TXA₂ analogue (STA₂) on the electrical parameters in isolated human colonic mucosa. In the human mucosa set between Ussing chambers, STA₂ stimulated Cl⁻ secretion in a concentration-dependent manner with an EC₅₀ of 0.06 μ m . The STA₂-induced Cl⁻ secretion was significantly inhibited by ONO-3708 (10 μ m ), a specific TXA₂ receptor antagonist. The effect of STA₂ (0.3 μ m ) was independent of the colonic segment from which the tissue was obtained, from caecum to rectum. Chromanol 293B, an inhibitor of the cAMP-dependent KvLQT1 channel, attenuated the STA₂-induced Cl⁻ secretion in the human colonic mucosa (IC₅₀ value 1.18 μ m ). We found that KvLQT1 mRNA and protein were expressed in all the tested segments of the human colon. The STA₂-induced Cl⁻ secretion was significantly inhibited by 8-bromo-2'-monobutyryladenosine-3',5'-cyclic monophosphorothioate (50 μ m ), a membrane-permeant cAMP antagonist. STA₂ (0.3 μ m ) significantly increased the intracellular cAMP levels and the short-circuit current via TXA₂ receptor in a human colonic cell line. These results suggest that the TXA₂-induced Cl⁻ secretion in the colon is mediated via the cAMP pathway in addition to the Ca²⁺–calmodulin pathway which was previously reported.     

Hypersensitivity of pulmonary chemosensitive neurons induced by activation of protease-activated receptor-2 in rats

This study was carried out to determine the effect of protease-activated receptor-2 (PAR₂) activation on the pulmonary chemoreflex responses and on the sensitivity of isolated rat vagal pulmonary chemosensitive neurons. In anaesthetized, spontaneously breathing rats, intratracheal instillation of trypsin (0.8 mg ml⁻¹, 0.1 ml), an endogenous agonist of PAR₂, significantly amplified the capsaicin-induced pulmonary chemoreflex responses. The enhanced responses were completely abolished by perineural capsaicin treatment of both cervical vagi, suggesting the involvement of pulmonary C-fibre afferents. In patch-clamp recording experiments, pretreatment with trypsin (0.1 μ m , 2 min) potentiated the capsaicin-induced whole-cell inward current in isolated pulmonary sensory neurons. The potentiating effect of trypsin was mimicked by PAR₂-activating peptide (PAR₂-AP) in a concentration-dependent manner. PAR₂-AP pretreatment (100 μ m , 2 min) also markedly enhanced the acid-evoked inward currents in these sensory neurons. Furthermore, the sensitizing effect of PAR₂ was completely abolished by pretreatment with either U73122 (1 μ m , 4 min), a phospholipase C inhibitor, or chelerythrine (10 μ m , 4 min), a protein kinase C (PKC) inhibitor. In summary, our results have demonstrated that activation of PAR₂ upregulates the pulmonary chemoreflex sensitivity in vivo and the excitability of isolated pulmonary chemosensitive neurons in vitro , and this effect of PAR₂ activation was mediated through the PKC-dependent transduction pathway. These results further suggest that the hypersensitivity of these neurons may play a part in the development of airway hyper-responsiveness resulting from PAR₂ activation.     

Leukocyte Migration Agarose Test for the Assessment of Human Neutrophil Chemotaxis

To apply the leukocyte migration agrose test (LMAT) to the in vitro assessment of human neutrophil chemotaxis, effects of different culture conditions on neutrophil migration under agarose were, studied. Presence of either serum or human serum albumin (HSA) in the culture medium was necessary for detectable neutrophil migration. HSA was preferred since heat-stabile chemotactic agents were found to be generated from fresh serum in the presence of agarose additional CO₂ in the assay milieu could be replaced by decreasing the NaHCO₃ concentration of the culture medium. Both the directed and the spontaneous migration rates of neutrophil leukocytes increased when the concentration of agarose was decreased. Area and distance of migration and cumulative cell count of migrated neutrophil leukocytes were suitable for quantitative the neutrophil migration rate.     

Molecular determinants of glycine receptor αβ subunit sensitivities to Zn2+-mediated inhibition

Glycine receptors exhibit a biphasic sensitivity profile in response to Zn²⁺-mediated modulation, with low Zn²⁺ concentrations potentiating (< 10 μ m ), and higher Zn²⁺ concentrations inhibiting submaximal responses to glycine. Here, a substantial 30-fold increase in sensitivity to Zn²⁺-mediated inhibition was apparent for the homomeric glycine receptor (GlyR) α1 subunit compared to either GlyR α2 or α3 subtypes. Swapping the divergent histidine (H107) residue in GlyR α1, which together with the conserved H109 forms part of an intersubunit Zn²⁺-binding site, for the equivalent asparagine residue present in GlyR α2 and α3, reversed this phenotype. Co-expression of heteromeric GlyR α1 or α2 with the ancillary β subunit yielded receptors that maintained their distinctive sensitivities to Zn²⁺ inhibition. However, GlyR α2β heteromers were consistently 2-fold more sensitive to inhibition compared to the GlyR α2 homomer. Comparative studies to elucidate the specific residue in the β subunit responsible for this differential sensitivity revealed instead threonine 133 in the α1 subunit as a new vital component for Zn²⁺-mediated inhibition. Further studies on heteromeric receptors demonstrated that a mutated β subunit could indeed affect Zn²⁺-mediated inhibition but only from one side of the intersubunit Zn²⁺-binding site, equivalent to the GlyR α1 H107 face. This strongly suggests that the α subunit is responsible for Zn²⁺-mediated inhibition and that this is effectively transduced, asymmetrically, from the side of the Zn²⁺-binding site where H109 and T133 are located.     

Role of cyclic nucleotide phosphodiesterase isoforms in cAMP compartmentation following β2-adrenergic stimulation of ICa,L in frog ventricular myocytes

The role of cyclic nucleotide phosphodiesterase (PDE) isoforms in the β₂-adrenergic stimulation of the L-type Ca²⁺ current ( I _Ca,L) was investigated in frog ventricular myocytes using double patch-clamp and double-barrelled microperfusion techniques. Isoprenaline (ISO, 1 nM to 10 μM) was applied on one half of the cell, either alone or in the presence of PDE inhibitors, and the local and distant responses of I _Ca,L were used to determine the gradient of local vs. distant cAMP concentration (α). IBMX (100 μM), a non-selective PDE inhibitor, reduced α from 40 to 4.4 indicating a 9-fold reduction in intracellular cAMP compartmentation when all PDE activity was blocked. While PDE1 and PDE2 inhibition had no effect, PDE3 inhibition by milrinone (3 μM) or PDE4 inhibition by Ro 20-1724 (3 μM) reduced α by 6- and 4-fold, respectively. A simultaneous application of milrinone and Ro 20-1724 produced a similar effect to IBMX, showing that PDE3 and PDE4 were the major PDEs accounting for cAMP compartmentation. Okadaic acid (3 μM), a non-selective phosphatase inhibitor, or H89 (1 μM), an inhibitor of cAMP-dependent protein kinase (PKA), had no effect on the distant response of I _Ca,L to ISO indicating that PDE activation by PKA played a minor role in cAMP compartmentation. Our results demonstrate that PDE activity determines the degree of cAMP compartmentation in frog ventricular cells upon β₂-adrenergic stimulation. PDE3 and PDE4 subtypes play a major role in this process, and contribute equally to ensure a functional coupling of β₂-adrenergic receptors with nearby Ca²⁺ channels via local elevations of cAMP.     

Influence of Tumour Condition on the Macrophage Activity in Candida albicans Infection

To better understand the interactions between opportunistic fungi and their hosts, we investigated hydrogen peroxide (H₂O₂)_, nitric oxide and TNF-α production by peritoneal macrophages from Ehrlich tumour-bearing mice (TBM) during microbial infections. For this purpose, TBM at days 7, 14 and 21 of tumour progression were inoculated intraperitoneally with C. albicans and evaluated after 24 and 72 h. We observed that TBM showed significant increases in H₂O_2, TNF-α levels and fungal clearance at day 7 after C. albicans infection. However, as the tumour advanced, there was a progressive decline in the release of H₂O₂ and TNF-α that was paired with the dissemination of C. albicans . These results demonstrate that protective macrophage activities against Candida albicans are limited to the initial stages of tumour growth; continued solid tumour growth weakened the macrophage response and as a consequence, weakened the host's susceptibility to opportunistic infections.     

Immunosuppressive Properties of α1-Microglobulin

α₁-Microglobulin (α₁m), a serum glycoprotein (26,000 d). was found to impede the proliferative response of human lymphocytes to purified protein derivative (PPD) and tetanus toxoid. The data suggest that, α₁m operates through an unstable suppressor mechanism, which no longer can function after 24 h of preculturing. This effect of α₁m on antigen stimulation did not seem to be due to binding of α₁m to PPD or cells, to altered kinetics of the PPD response, or to non-specific cytotoxicity. In contrast, PPD-induced leucocyte migration inhibition was not reversed by α₁m. α₁m did not cause significant inhibition in experiments in which lymphocytes were stimulated by the mitogens phytohaemagglutinin or concanavalin A. Finally, α₁m had its own leucocyte migration inhibitory effect.     

Cholinergic control of excitability of spinal motoneurones in the salamander

The cholinergic modulation of the electrical properties of spinal motoneurones was investigated in vitro , with the use of the whole-cell patch-clamp recording technique in lumbar spinal cord slices from juvenile urodeles ( Pleurodeles waltlii ). Bath application of acetylcholine (20 μ m ) with eserine (20 μ m ) induced an increase in the resting membrane potential, a decrease of the input resistance, a decrease of the action potential amplitude, and a reduction of the medium afterhyperpolarization (mAHP) that followed each action potential. Moreover, the firing rate of motoneurones during a depolarizing current pulse and the slope of their stimulus current–spike frequency relation were increased. All of these effects were mimicked by extracellular application of muscarine (20 μ m ), and blocked by application of the muscarinic receptor antagonist atropine (0.1–1 μ m ). They were not observed during bath application of nicotine (10 μ m ). These results suggest that the cholinergic modulation of spinal motoneurone excitability was mediated by activation of muscarinic receptors. Our results further show that the muscarinic action primarily resulted from a reduction of the Ca²⁺-activated K⁺ current responsible for the mAHP, an inhibition of the hyperpolarization-activated cation current, I _h, and an enhancement of the inward rectifying K⁺ current, I _Kir. We conclude that cholinergic modulation can contribute significantly to the production of motor behaviour by altering several ionic conductances responsible for the repetitive discharge of motoneurones.     

纳米氧化锌对白色念珠菌的抗菌作用

背景：纳米氧化锌作为无机抗菌剂,与有机杀菌剂的抗菌机制明显不同。 目的：探讨纳米氧化锌对白色念珠菌等真菌的抗菌作用。 方法：分别配制成100,75,50,25,10 g/L的纳米氧化锌与普通氧化锌悬浊液,以生理盐水作为阴性对照,采用KB纸片扩散法将30 μL不同浓度悬浊液分别作用于白色念珠菌,将培养平板上置于37 ℃恒温箱培养24 h观察并测量抑菌圈大小。 结果与讨论：随着纳米氧化锌浓度的提高,对白色念珠菌的抑制作用逐渐增强,抑菌圈直径逐渐增大。普通氧化锌的无明显抑菌作用。证实纳米氧化锌能够抑制白色念珠菌等真菌的生长。     

Effects of TGF-β on Eosinophil Chemotaxis

Transforming growth factor-β (TGF-β), which can decrease the effects of interleukin (IL)-3, IL-5 and granulocyte–macrophage colony-stimulating factor (GM-CSF) on eosinophil viability, has been shown to be chemotactic for neutrophils. However, there is little information on its effects on eosinophil chemotaxis. Because TGF-β has recently been found in increased concentrations in asthmatic sputum, we investigated whether TGF-β could influence eosinophil migration and eosinophil viability. Purified eosinophils from normal donors were incubated with increasing concentrations of TGF-β. Chemotaxis was measured with a modified Boyden chamber technique. In addition, eosinophils were incubated for 96 h with either IL-3, IL-5 or GM-CSF (1 ng/ml) together with increasing concentrations of TGF-β. Eosinophil viability was then determined with propidium jodide and flowcytometry. Eosinophil chemotaxis was significantly increased in the presence of TGF-β in concentrations between 10⁻⁹ and 10⁻⁴ μg/ml. The optimal concentration of TGF-β in this assay was between 10⁻⁹ and 10⁻⁸ μg/ml. The chemotactic effect of TGF-β diminished when higher as well as lower concentrations (between 10⁻¹² and 10⁻³ μg/ml) were employed. In contrast, inhibition of eosinophil survival induced by IL-3, IL-5 and GM-CSF reached its maximum at concentrations of TGF-β between 10⁻⁴ and 10⁻³ μg/ml. From these data we conclude that TGF-β in low concentrations can induce eosinophil chemotaxis whereas higher concentrations reduce eosinophil survival mediated by IL-3, IL-5 and GM-CSF.     

Voltage independence of vasomotion in isolated irideal arterioles of the rat

The cellular mechanisms underlying vasomotion of irideal arterioles from juvenile rats have been studied using electrophysiological methods, ratiometric calcium measurements and video microscopy. Vasomotion was not affected by removal of the endothelium. Spontaneous contractions were preceded by spontaneous depolarizations. Both were abolished by the intracellular calcium chelator, BAPTA AM (20 μ m ), but not by ryanodine (10 μ m ), suggesting a dependence on the cyclical release of calcium from intracellular stores, other than those operated by ryanodine receptors. Oscillations were little changed when the membrane potential of short segments of arteriole was either depolarized or hyperpolarized. When the segments were voltage clamped, oscillating inward currents were recorded, indicating that the changes in membrane potential were voltage independent. Vasomotion was preceded by intracellular calcium oscillations and both were abolished by inhibitors of phospholipase C (U73122, 10 μ m ), phospholipase A₂ (AACOCF₃, 30 μ m ) and protein kinase C (chelerythrine chloride, 5 μ m , and myristoylated protein kinase C peptide, 10 μ m ). Inhibition of vasomotion by the dual lipoxygenase and cyclo-oxygenase inhibitor, NDGA (10 μ m ), the lipoxygenase inhibitor, ETI (1 μ m ) but not by the cyclo-oxygenase inhibitors, aspirin (10 μ m ) and indomethacin (10 μ m ), or the cytochrome P450 inhibitor 17-ODYA (10 μ m ), suggested an involvement of the lipoxygenase pathway. The observations suggest that vasomotion of iris arterioles is voltage independent and results from the cyclical release of calcium from IP₃-sensitive stores which are activated by cross talk between the phospholipase C and phospholipase A₂ pathways in vascular smooth muscle.