Effect of platelet-derived growth factor on the development and persistence of asbestos-induced fibroproliferative lung disease.

Platelet-derived growth factor (PDGF) isoforms and PDGF receptor-alpha are upregulated in fibroproliferative lesions in response to asbestos exposure. To examine the functional role of PDGF in asbestos-induced lung disease, we have evaluated the impact of PDGF-B overexpression in the lung on the development of pulmonary fibrosis induced by asbestos inhalation. Transgenic mice expressing PDGF-B from the surfactant protein C promoter and wild-type C57BL/6 mice were exposed to aerosolized chrysotile asbestos fibers via three different exposure regimens: 3 consecutive days to 9 mg/m(3), once a week for 5 weeks to 12 mg/m(3), or once a week for 8 weeks to 11 mg/m(3). The 3-day exposure did not produce fibroproliferative lesions in SPC-PDGFB or wild-type mice, indicating that PDGF expression did not increase susceptibility to a subthreshold dose of asbestos. Transgenic and wild-type mice subjected to the 5-week exposure protocol exhibited similar fibrogenic lesions histologically 48 hours and 8 weeks postexposure, but lungs from transgenic mice had elevated lung hydroxyproline content 8 weeks postexposure relative to wild-type mice. In addition, SPC-PDGFB transgenic mice developed pronounced thickening of arterioles following the 5-week exposure regimen. Mice exposed to asbestos for 8 weeks and examined 10 months later showed pronounced, diffuse fibrotic lesions of terminal bronchioles and alveolar ducts, but no histological differences between transgenic and nontransgenic mice were observed. These results indicated that PDGF-B overexpression can stimulate increased collagen deposition and vascular smooth muscle hyperplasia following asbestos inhalation and that a limited exposure (8 times) to chrysotile aerosol can produce long-lasting fibrotic lesions. The 8-week exposure regimen provides an animal model that encompasses an important aspect of human asbestosis-i.e., persistence of fibrosis for long periods after cessation of asbestos exposure.

Neuronal modulation of lung injury induced by polymeric hexamethylene diisocyanate in mice.

1,6-Hexamethylene diisocyanate biuret trimer (HDI-BT) is a nonvolatile isocyanate that is a component of polyurethane spray paints. HDI-BT is a potent irritant that when inhaled stimulates sensory nerves of the respiratory tract. The role of sensory nerves in modulating lung injury following inhalation of HDI-BT was assessed in genetically manipulated mice with altered innervation of the lung. Knockout mice with a mutation in the low-affinity nerve growth factor receptor (NGFR), which have decreased innervation by nociceptive nerve fibers, and transgenic mice expressing nerve growth factor (NGF) from the lung-specific Clara cell secretory protein (CCSP) promoter, which have increased innervation of the airways, were exposed to HDI-BT aerosol and evaluated at various times after exposure. NGFR knockout mice exhibited significantly more, and CCSP-NGF transgenic mice exhibited significantly less injury and inflammation compared with wild-type mice, indicative of a protective effect of nociceptive nerves on the lung following HDI-BT inhalation. Transgenic mice overexpressing the tachykinin 1 receptor (Tacr1) in lung epithelial cells also showed less severe injury and inflammation compared with wild-type mice after HDI-BT exposure, establishing a role for released tachykinins acting through Tacr1 in mediating at least part of the protective effect. Treatment of lung fragments from Tacr1 transgenic mice with the Tacr1 ligand substance P resulted in increased cAMP accumulation, suggesting this compound as a possible signaling mediator of protective effects on the lung following nociceptive nerve stimulation. The results indicate that sensory nerves acting through Tacr1 can exert protective or anti-inflammatory effects in the lung following isocyanate exposure.

The absence of a Ca(2+) signal during mouse egg activation can affect parthenogenetic preimplantation development, gene expression patterns, and blastocyst quality.

A series of Ca(2+) oscillations during mammalian fertilization is necessary and sufficient to stimulate meiotic resumption and pronuclear formation. It is not known how effectively development continues in the absence of the initial Ca(2+) signal. We have triggered parthenogenetic egg activation with cycloheximide that causes no Ca(2+) increase, with ethanol that causes a single large Ca(2+) increase, or with Sr(2+) that causes Ca(2+) oscillations. Eggs were co-treated with cytochalasin D to make them diploid and they formed pronuclei and two-cell embryos at high rates with each activation treatment. However, far fewer of the embryos that were activated by cycloheximide reached the blastocyst stagecompared tothose activated by Sr(2+) orethanol. Any cycloheximide-activated embryos that reached the blastocyst stage had a smaller inner cell mass number and a greater rate of apoptosis than Sr(2+)-activated embryos. The poor development of cycloheximide-activated embryos was due to the lack of Ca(2+) increase because they developed to blastocyst stages at high rates when co-treated with Sr(2+) or ethanol. Embryos activated by either Sr(2+) or cycloheximide showed similar signs of initial embryonic genome activation (EGA) when measured using a reporter gene. However, microarray analysis of gene expression at the eight-cell stage showed that activation by Sr(2+) leads to a distinct pattern of gene expression from that seen with embryos activated by cycloheximide. These data suggest that activation of mouse eggs in the absence of a Ca(2+) signal does not affect initial parthenogenetic events, but can influence later gene expression and development.

On-filter determination of collected wood dust by diffuse reflectance infrared Fourier-transform spectroscopy (DRIFTS).

A new analytical technique based on DRIFTS spectroscopy has been developed for the specific and sensitive determination of size-fractionated wood dust from 37 mm glass fiber filter samples collected with the Respicon sampler. A translational diffuse reflectance apparatus was modified to accept filter samples by incorporating a special filter holder in the sample stage and a clockwork motor to drive the translational stage during infrared scanning, thus providing an average analysis across the filter face. Filter samples were pre-treated with ethyl acetate to uniformly redeposit dust onto the filter and extract potential chemical interferences. Two absorbance maxima (1251 and 1291 cm(-1)), corresponding to the cellulose content of the wood, were suitable for quantitation of wood dust. Analysis of seven species of wood at 1291 cm(-1) showed an equivalent quantitative response for all species except maple. The response at 1251 cm(-1) was more variable across species but more sensitive for the softwoods. There was a statistically significant effect of particle size on the analytical response, so that analytical standards should be matched to the samples in terms of particle size distribution. Analytical limit of detection was approximately 0.08 mg of wood dust per sample with overall precision of about 6%. Comparison of DRIFTS and gravimetric analyses of 51 pure wood dust samples ranging from about 0.2 to 2 mg yielded a slope of 1.08 and r2 equal to 0.9. Other particulate contaminants common in the industrial wood processing industry showed little or no interference with the determination of wood dust by this method.

Ca2+ oscillations at fertilization in mammals.

The mouse egg provided the first direct measurement of Ca(2+) oscillations in any cell type. These sperm-induced Ca(2+) oscillations occur at a relatively low frequency, and can be detected up to 18-20 h after sperm-egg fusion. The Ca(2+) oscillations consist of two series of transients; the first lasts about 4 h, from metaphase II until interphase of the first cell cycle, and the second lasts the duration of the first mitotic division. This cell-cycle-regulated aspect to the pattern of Ca(2+) signalling at fertilization is reflected in the role of the Ca(2+) transients in stimulating exit from metaphase arrest. Recent developments have started to shed light on the mechanism initiating Ca(2+) oscillations at fertilization, on how the frequency of the oscillations is set, and on what determines their temporal pattern.

Norepinephrine-induced Ca(2+) waves depend on InsP(3) and ryanodine receptor activation in vascular myocytes.

In rat portal vein myocytes, Ca(2+) signals can be generated by inositol 1,4,5-trisphosphate (InsP(3))- and ryanodine-sensitive Ca(2+) release channels, which are located on the same intracellular store. Using a laser scanning confocal microscope associated with the patch-clamp technique, we showed that propagated Ca(2+) waves evoked by norepinephrine (in the continuous presence of oxodipine) were completely blocked after internal application of an anti-InsP(3) receptor antibody. These propagated Ca(2+) waves were also reduced by approximately 50% and transformed in homogenous Ca(2+) responses after application of an anti-ryanodine receptor antibody or ryanodine. All-or-none Ca(2+) waves obtained with increasing concentrations of norepinephrine were transformed in a dose-response relationship with a Hill coefficient close to unity after ryanodine receptor inhibition. Similar effects of the ryanodine receptor inhibition were observed on the norepinephrine- and ACh-induced Ca(2+) responses in non-voltage-clamped portal vein and duodenal myocytes and on the norepinephrine-induced contraction. Taken together, these results show that ryanodine-sensitive Ca(2+) release channels are responsible for the fast propagation of Ca(2+) responses evoked by various neurotransmitters producing InsP(3) in vascular and visceral myocytes.

Ca(2+) signals mediated by Ins(1,4,5)P(3)-gated channels in rat ureteric myocytes.

Localized Ca(2+)-release signals (puffs) and propagated Ca(2+) waves were characterized in rat ureteric myocytes by confocal microscopy. Ca(2+) puffs were evoked by photorelease of low concentrations of Ins(1,4,5)P(3) from a caged precursor and by low concentrations of acetylcholine; they were also observed spontaneously in Ca(2+)-overloaded myocytes. Ca(2+) puffs showed some variability in amplitude, time course and spatial spread, suggesting that Ins(1,4,5)P(3)-gated channels exist in clusters containing variable numbers of channels and that within these clusters a variable number of channels can be recruited. Immunodetection of Ins(1,4,5)P(3) receptors revealed the existence of several spots of fluorescence in the confocal cell sections, supporting the existence of clusters of Ins(1,4,5)P(3) receptors. Strong Ins(1,4,5)P(3) photorelease and high concentrations of acetylcholine induced Ca(2+) waves that originated from an initiation site and propagated in the whole cell by spatial recruitment of neighbouring Ca(2+)-release sites. Both Ca(2+) puffs and Ca(2+) waves were blocked selectively by intracellular applications of heparin and an anti-Ins(1,4,5)P(3)-receptor antibody, but were unaffected by ryanodine and intracellular application of an anti-ryanodine receptor antibody. mRNAs encoding for the three subtypes of Ins(1,4,5)P(3) receptor and subtype 3 of ryanodine receptor were detected in these myocytes, and the maximal binding capacity of [(3)H]Ins(1,4,5)P(3) was 10- to 12-fold higher than that of [(3)H]ryanodine. These results suggest that Ins(1,4,5)P(3)-gated channels mediate a continuum of Ca(2+) signalling in smooth-muscle cells expressing a high level of Ins(1,4,5)P(3) receptors and no subtypes 1 and 2 of ryanodine receptors.

Effects of hindlimb suspension on cytosolic Ca2+ and [3H]ryanodine binding in cardiac myocytes.

Effects of a 14-day hindlimb suspension were examined on [3H]ryanodine binding to rat ventricular microsomes and on cytosolic Ca2+ concentration ([Ca2+]i) and voltage-dependent Ca2+ channels in isolated ventricular myocytes. In suspended rats, the amplitude of the twitch [Ca2+]i transient was increased without significant modifications of the basal [Ca2+]i and sarcoplasmic reticulum content. Because cell capacitance, L-type Ca2+-current density, and Ca2+-channel gating were not significantly modified after suspension, the increase in [Ca2+]i was expected to reside in a change in ryanodine receptors. Scatchard analysis of [3H]ryanodine binding revealed that suspension enhanced binding by increasing the affinity of the receptors for [3H]ryanodine without affecting the maximal binding capacity. Both Ca2+-release channel activity and [3H]ryanodine binding are modulated by Ca2+. However, the Ca2+ sensitivity of [3H]ryanodine binding remained unchanged after suspension. Taken together, these results suggest that the increase in twitch [Ca2+]i transients after suspension may result from a change in the intrinsic properties of the ryanodine receptors but not from a change in the expression level of these receptors.

Effect of a 14-day hindlimb suspension on cytosolic Ca2+ concentration in rat portal vein myocytes.

Effects of a 14-day hindlimb suspension were examined on increases in cytosolic Ca2+ concentration ([Ca2+]i) evoked by vasoactive compounds and on Ca2+ channels in rat portal vein myocytes. The maximal increases in [Ca2+]i elicited by caffeine, norepinephrine, and angiotensin II were reduced by 30-50% in suspended rats, and complete recovery was obtained 4 days after suspension removal. In contrast, voltage-gated Ca2+ channels were unaffected by hindlimb suspension. Using both confocal microscopy and the patch-clamp technique, we measured local increases in [Ca2+]i which corresponded to activation of a small number of ryanodine-sensitive Ca(2+)-release channels (Ca2+ sparks) and D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]-gated Ca2+ channels. After hindlimb suspension, these local Ca2+ events, as well as the Ca2+ sensitivity of ryanodine-sensitive Ca2+ release channels, remained unchanged. In contrast, the propagated Ca2+ responses (Ca2+ waves) were significantly reduced in parallel with a noticeable inhibition of [3H]ryanodine binding to vascular membranes. Taken together, these results suggest that inhibition of the vasoconstrictor-induced increases in [Ca2+]i during long-term suspension may be related to a reduction of the number of functional ryanodine-sensitive and Ins(1,4,5)P3-gated channels in the sarcoplasmic reticulum of rat portal vein myocytes.

An HDI polyisocyanate aerosol exposure system for large-scale animal experiments.

An exposure system that allows large-scale exposure of animals to 1,6-hexamethylene diisocyanate (HDI)-based polyisocyanates at a stable concentration and aerosol size distribution was developed. The HDI polyisocyanate aerosol is generated by nebulizing a solution of a commercial polyisocyanate product dissolved in acetone. The aerosol is delivered with a constant airflow into a horizontal flow chamber. Complete mixing of aerosol in the chamber is ensured by a circulating fan. This method has been used to generate atmospheres containing HDI polyisocyanates at a concentration of 10.46+/-0.23 mg/m(3) over a 5-hour period. The overall mass median aerodynamic equivalent diameter was found to be 1.42 microm with a geometric standard deviation of 1.26. The HDI monomer concentration was 0.15+/-0.04 mg/m(3). The average chamber acetone concentration was determined to be 2481+/-222 ppm (mean+/-standard deviation). Different HDI polyisocyanate concentrations in the chamber can be achieved by altering the concentration of the commercial polyisocyanate product in acetone and the chamber flow rate. The described exposure system will be useful for performing toxicological studies involving HDI polyisocyanates.

Pulmonary toxicity of polymeric hexamethylene diisocyanate aerosols in mice.

The acute pulmonary response of male C57BL/6 mice exposed to respirable polymeric hexamethylene diisocyanate biuret trimer aerosol (HDI-BT), a component of polyurethane spray paints, was examined. Mice were exposed to concentrations of 1 and 10 mg/m(3) HDI-BT for 5 h and were evaluated 6, 18, 42, 90, 186, and 378 h after the end of exposure. Mice exposed to 1 or 10 mg/m(3) HDI-BT exhibited dose-dependent lung function impairment, edema, neutrophilic inflammation, cellular proliferation, and histologic lesions in terminal bronchioles and alveolar ducts. Impairment of pulmonary function, indicated by decreased frequency and increased enhanced pause (Penh), was maximal immediately after exposure and progressively recovered at later time points. Lung weight and lavage fluid protein content peaked at 6 and 18 h after exposure, respectively. Total cells and macrophages recovered in lavage fluid peaked 90 h after exposure. Neutrophils recovered in lavage fluid peaked between 18 and 42 h after exposure. Proliferative lesions, as identified histologically and by bromodeoxyuridine incorporation, were maximal 90 h after exposure. In contrast, no inflammatory cell influx, protein leakage, or lung pathology were observed in mice exposed to 360 ppb HDI monomer vapor. This model will be useful for investigating molecular mechanisms by which HDI-BT causes lung injury, which is known to occur in humans exposed occupationally to this pulmonary toxicant.