A comparative assessment of the acute inhalation toxicity of vanadium compounds.

Vanadium compounds have become important in industrial processes, resulting in workplace exposure potential and are present in ambient air as a result of fossil fuel combustion. A series of acute nose-only inhalation toxicity studies was conducted in both rats and mice in order to obtain comparative data on the acute toxicity potential of compounds used commercially. V2O3, V2O4, and V2O5, which have different oxidation states (+3, +4, +5, respectively), were delivered as micronized powders; the highly water-soluble and hygroscopic VOSO4 (+4) could not be micronized and was instead delivered as a liquid aerosol from an aqueous solution. V2O5 was the most acutely toxic micronized powder in both species. Despite its lower overall percentage vanadium content, a liquid aerosol of VOSO4 was more toxic than the V2O5 particles in mice, but not in rats. These data suggest that an interaction of characteristics, i.e., bioavailability, solubility and oxidation state, as well as species sensitivity, likely affect the toxicity potential of vanadium compounds. Based on clinical observations and gross necropsy findings, the lung appeared to be the target organ for all compounds. The level of hazard posed will depend on the specific chemical form of the vanadium. Future work to define the inhalation toxicity potential of vanadium compounds of various oxidation states after repeated exposures will be important in understanding how the physico-chemical and biological characteristics of specific vanadium compounds interact to affect toxicity potential and the potential risks posed to human health.

The National Environmental Respiratory Center (NERC) experiment in multi-pollutant air quality health research: II. Comparison of responses to diesel and gasoline engine exhausts, hardwood smoke and simulated downwind coal emissions.

The NERC Program conducted identically designed exposure-response studies of the respiratory and cardiovascular responses of rodents exposed by inhalation for up to 6 months to diesel and gasoline exhausts (DE, GE), wood smoke (WS) and simulated downwind coal emissions (CE). Concentrations of the four combustion-derived mixtures ranged from near upper bound plausible to common occupational and environmental hotspot levels. An "exposure effect" statistic was created to compare the strengths of exposure-response relationships and adjustments were made to minimize false positives among the large number of comparisons. All four exposures caused statistically significant effects. No exposure caused overt illness, neutrophilic lung inflammation, increased circulating micronuclei or histopathology of major organs visible by light microscopy. DE and GE caused the greatest lung cytotoxicity. WS elicited the most responses in lung lavage fluid. All exposures reduced oxidant production by unstimulated alveolar macrophages, but only GE suppressed stimulated macrophages. Only DE retarded clearance of bacteria from the lung. DE before antigen challenge suppressed responses of allergic mice. CE tended to amplify allergic responses regardless of exposure order. GE and DE induced oxidant stress and pro-atherosclerotic responses in aorta; WS and CE had no such effects. No overall ranking of toxicity was plausible. The ranking of exposures by number of significant responses varied among the response models, with each of the four causing the most responses for at least one model. Each exposure could also be deemed most or least toxic depending on the exposure metric used for comparison. The database is available for additional analyses.

Health effects of subchronic inhalation exposure to gasoline engine exhaust.

Gasoline engine emissions are a ubiquitous source of exposure to complex mixtures of particulate matter (PM) and non-PM pollutants; yet their health hazards have received little study in comparison with those of diesel emissions. As a component of the National Environmental Respiratory Center (NERC) multipollutant research program, F344 and SHR rats and A/J, C57BL/6, and BALBc mice were exposed 6 h/day, 7 days/week for 1 week to 6 months to exhaust from 1996 General Motors 4.3-L engines burning national average fuel on a simulated urban operating cycle. Exposure groups included whole exhaust diluted 1:10, 1:15, or 1:90, filtered exhaust at the 1:10 dilution, or clean air controls. Evaluations included organ weight, histopathology, hematology, serum chemistry, bronchoalveolar lavage, cardiac electrophysiology, micronuclei in circulating cells, DNA methylation and oxidative injury, clearance of Pseudomonas aeruginosa from the lung, and development of respiratory allergic responses to ovalbumin. Among the 120 outcome variables, only 20 demonstrated significant exposure effects. Several statistically significant effects appeared isolated and were not supported by related variables. The most coherent and consistent effects were those related to increased red blood cells, interpreted as likely to have resulted from exposure to 13-107 ppm carbon monoxide. Other effects supported by multiple variables included mild lung irritation and depression of oxidant production by alveolar macrophages. The lowest exposure level caused no significant effects. Because only 6 of the 20 significant effects appeared to be substantially reversed by PM filtration, the majority of effects were apparently caused by non-PM components of exhaust.

Blunted ventilatory response to hypoxia/hypercapnia in mice with cigarette smoke-induced emphysema.

It has been reported that the degree of emphysema induced by chronic cigarette smoke (CS) is greater in female C3H/HeN mice as compared to other mouse strains. We hypothesized that these mice would develop the similar major characteristics seen in hypercapnic patients with chronic obstructive pulmonary disease (COPD), including emphysema, pulmonary inflammation, hypercapnia/hypoxemia, rapid breathing, and attenuated ventilatory response (AVR). Mice were exposed either to CS or filtered air (FA) for 16 weeks. After exposure, arterial blood gases and minute ventilation were measured before and during chemical challenges in anesthetized and spontaneously breathing mice. We found that as compared to FA, CS exposure caused emphysema and pulmonary inflammation associated with: (1) hypercapnia and hypoxemia, (2) rapid breathing, and (3) AVR to 25 breaths of pure N(2), 5% CO(2) alone, and 5% CO(2) coupled with 10% O(2). The similarity of these pathophysiological characteristics between our mouse model and COPD patients suggests that this model could be effectively applied to study COPD pathophysiology, especially central mechanisms of the AVR genesis.

Health effects of subchronic exposure to environmental levels of hardwood smoke.

Hardwood smoke is a contributor to both ambient and indoor air pollution. As part of a general health assessment of multiple anthropogenic source emissions conducted by the National Environmental Respiratory Center, a series of health assays was conducted on rodents exposed to environmentally relevant levels of hardwood smoke. This article summarizes the study design and exposures, and reports findings on general indicators of toxicity, bacterial clearance, cardiac function, and carcinogenic potential. Hardwood smoke was generated from an uncertified wood stove, burning wood of mixed oak species. Animals were exposed to clean air (control) or dilutions of whole emissions based on particulate (30, 100, 300, and 1000 micromg/m3). F344 rats, SHR rats, strain A/J mice, and C57BL/6 mice were exposed by whole-body inhalation 6 h/day, 7 days/wk, for either 1 wk or 6 mo. Effects of exposure on general indicators of toxicity, bacterial clearance, cardiac function, and carcinogenic potential were mild. Exposure-related effects included increases in platelets and decreases in blood urea nitrogen and serum alanine aminotransferase. Several other responses met screening criteria for significant exposure effects but were not consistent between genders or exposure times and were not corroborated by related parameters. Pulmonary histopathology revealed very little accumulation of hardwood smoke particulate matter. Parallel studies demonstrated mild exposure effects on bronchoalveolar lavage parameters and in a mouse model of asthma. In summary, the results reported here show few and only modest health hazards from short-term to subchronic exposures to realistic concentrations of hardwood smoke.

Health effects of subchronic exposure to environmental levels of diesel exhaust.

Diesel exhaust is a public health concern and contributor to both ambient and occupational air pollution. As part of a general health assessment of multiple anthropogenic source emissions conducted by the National Environmental Respiratory Center (NERC), a series of health assays was conducted on rats and mice exposed to environmentally relevant levels of diesel exhaust. This article summarizes the study design and exposures, and reports findings on several general indicators of toxicity and carcinogenic potential. Diesel exhaust was generated from a commonly used 2000 model 5.9-L, 6-cylinder turbo diesel engine operated on a variable-load heavy-duty test cycle burning national average certification fuel. Animals were exposed to clean air (control) or four dilutions of whole emissions based on particulate matter concentration (30, 100, 300, and 1000 microg/m(3)). Male and female F344 rats and A/J mice were exposed by whole-body inhalation 6 h/day, 7 days/wk, for either 1 wk or 6 mo. Exposures were characterized in detail. Effects of exposure on clinical observations, body and organ weights, serum chemistry, hematology, histopathology, bronchoalveolar lavage, and serum clotting factors were mild. Significant exposure-related effects occurring in both male and female rats included decreases in serum cholesterol and clotting Factor VII and slight increases in serum gamma-glutamyl transferase. Several other responses met screening criteria for significant exposure effects but were not consistent between genders or exposure times and were not corroborated by related parameters. Carcinogenic potential as determined by micronucleated reticulocyte counts and proliferation of adenomas in A/J mice were unaffected by 6 mo of exposure. Parallel studies demonstrated effects on cardiac function and resistance to viral infection; however, the results reported here show few and only modest health hazards from subchronic or shorter exposures to realistic concentrations of contemporary diesel emissions.

Effects of concurrent ozone exposure on the pathogenesis of cigarette smoke-induced emphysema in B6C3F1 mice.

Episodic elevation of air pollutants may exacerbate respiratory distress associated with chronic obstructive pulmonary disease (COPD), yet few experiments have been performed to determine how continuously polluted atmospheres may contribute to the etiology of COPD, in general and pulmonary emphysema in particular. This study describes the effects of concurrent exposure to ozone (O(3)) in the pathogenesis of cigarette smoke (CS)-induced emphysema in the mouse. Female B6C3F1 mice were whole-body exposed either to filtered air (FA) or to mainstream CS at a concentration of 250 mg total particulate material/m(3) for 6 h/day, 5 days/wk for 15 or 32 wk. Concurrently, mice were exposed either to FA or to O(3) at 0.3 ppm for 8 h/night, 5 nights/wk for the same time periods. At necropsy, mouse lungs were lavaged, and bronchoalveolar lavage fluid (BALF) was analyzed for inflammatory cell numbers, total protein, lactate dehydrogenase (LDH) and alkaline phosphatase (AP) activities, superoxide production by isolated alveolar macrophages, glutathione content, inflammatory cytokines, and proteolytic activity. Other lungs were inflated at constant pressure for 6 h with formalin for fixation, routine histopathology, and stereology. After 32 wk of exposure, CS with or without concurrent O(3) exposure produced stereologic evidence of emphysema as previously described. Concurrent O(3) exposure did not worsen any of these parameters, nor did O(3) by itself cause stereologic changes that were consistent with emphysema. The O(3) exposure caused only slight elevations of BALF macrophages, while CS exposure caused marked increases in the numbers of both BALF macrophages and neutrophils. Neutrophils in the BALF in response to CS exposure were also more numerous at 32 wk than at 15 wk. Exposure to CS caused an increase in BALF total protein, LDH, AP, and interleukin (IL)-1beta. After 32 wk, CS exposure was associated with decreased superoxide production from isolated alveolar macrophages. The CS exposure elevated BALF total glutathione primarily at 15 wk. Overall, O(3) had little effect on endpoints that were significantly affected by CS exposure. We conclude that concurrent O(3) exposure has no effect on the induction of emphysema by CS in this animal model.

Interactions between benzo[a]pyrene and UVA light affecting ATP levels, cytoskeletal organization, and resistance to trypsinization.

Polycyclic aromatic hydrocarbons affect cells in many ways, including covalent modifications of DNA, participation in redox cycling, and alterations in cellular signaling pathways. Similarly, exposure to ultraviolet (UV) light may modify DNA, generate reactive oxygen species, and alter signaling. Because environmental conditions may interact to affect cellular functions, we investigated the combined effects of benzo[a]pyrene (BaP) and UV light in a cell line in which BaP-induced alterations in Ca(2+) homeostasis have previously been shown. Exposure of MCF-10A cells to BaP (18 h) followed by a brief (5 min) exposure to UVA resulted in resistance to trypsinization of cells grown on type I collagen (Vitrogen). This effect was not seen following treatment with BaP or UVA alone nor with benzo(e)pyrene (BeP)+UVA. BaP+UVA light also caused actin filaments to reorganize from typical stress fibers to substrate-associated aggregates of actin and caused depletion of cellular adenosine triphosphate (ATP). The effects of BaP+UVA on adhesion and actin aggregate formation were partially prevented by treatment with reduced glutathione. Depletion of cellular ATP affected resistance to trypsinization and actin organization in a similar manner. Thus, these studies suggest a redox-sensitive interaction between BaP+UVA light to deplete cellular ATP levels, resulting in resistance to trypsinization and actin filament reorganization in MCF-10A cells.

The role of a mitochondrial pathway in the induction of apoptosis by chemicals extracted from diesel exhaust particles.

We are interested in the cytotoxic and proinflammatory effects of particulate pollutants in the respiratory tract. We demonstrate that methanol extracts made from diesel exhaust particles (DEP) induce apoptosis and reactive oxygen species (ROS) in pulmonary alveolar macrophages and RAW 264.7 cells. The toxicity of these organic extracts mimics the cytotoxicity of the intact particles and could be suppressed by the synthetic sulfhydryl compounds, N-acetylcysteine and bucillamine. Because DEP-induced apoptosis follows cytochrome c release, we studied the effect of DEP chemicals on mitochondrially regulated death mechanisms. Crude DEP extracts induced ROS production and perturbed mitochondrial function before and at the onset of apoptosis. This mitochondrial perturbation follows an orderly sequence of events, which commence with a change in mitochondrial membrane potential, followed by cytochrome c release, development of membrane asymmetry (annexin V staining), and propidium iodide uptake. Structural damage to the mitochondrial inner membrane, evidenced by a decrease in cardiolipin mass, leads to O-*2 generation and uncoupling of oxidative phosphorylation (decreased intracellular ATP levels). N-acetylcysteine reversed these mitochondrial effects and ROS production. Overexpression of the mitochondrial apoptosis regulator, Bcl-2, delayed but did not suppress apoptosis. Taken together, these results suggest that DEP chemicals induce apoptosis in macrophages via a toxic effect on mitochondria.

Effect of inhaled residual oil fly ash on the electrocardiogram of dogs.

Epidemiology studies have found associations between increases in air pollutants and increases in morbidity and mortality from cardiovascular disease. The 1995 finding by Godleski et al. at Harvard that inhalation exposures of dogs to high concentrations of residual oil fly ash (ROFA) caused changes in the ST segment and T waves in the electrocardiogram (ECG) suggested a potential mechanism, and also suggested that inhaled metals might contribute to the effect. We conducted the present study to establish a baseline correspondence to the Godleski et al. findings in preparation for studies of the cardiac effects of specific particle-borne metals. The ROFA used in this study consisted of 45% carbon and 15.5% transition metals by mass. In vitro assays using cultured A549 cells and rat alveolar macrophages demonstrated that the ROFA was biologically active but was not highly cytotoxic. Four 10.5-yr-old beagles were exposed by oral inhalation to 3 mg/m3 of aerosolized ROFA for 3 h/day on 3 consecutive days. During the exposures, ECGs were continuously recorded from leads I, II, III, and V4. ECG data were also collected during three control exposures to clean air, during one of which changes were induced using drugs as a positive control. The ROFA exposures caused no consistent changes in the amplitude of the ST segment, the form or amplitude of the T wave, or arrhythmias. The data suggested a slight slowing of heart rate during exposure. Whether the difference between the present and previous findings resulted from differences in the composition of the two batches of ROFA or differences in methodology could not be determined by the study. This study did not address the cardiac effects of ROFA in subjects having preexisting cardiac susceptibility factors, nor was it a rigorous evaluation of effects on the frequency distribution of heart rate. Our results indicate that healthy dogs can inhale high concentrations of ROFA without changes in cardiac electrophysiology, which are detectable by clinical evaluations.

Multiple modes of responses to air pollution particulate materials in A549 alveolar type II cells.

Exposure to components of air pollution may cause adverse effects on lung cellular and organ functions through several mechanisms. Cell death, altered gene expression including production of cytokines, and modifications of normal cellular processes are possible outcomes that may be independent or coupled. To assess the effects of materials representative of a variety of particulate components of air pollution on lung epithelium, a human cell line of type II origin (A549 cells) was exposed to these materials in vitro. Materials tested included carbon black (CB), diesel soot from two sources (DS), residual oil fly ash (ROFA), Ottowa Ambient Air particulate (OAA), silicon dioxide (SiO2), and nickel subsulfide (Ni3S2). Endpoints included loss of adherence measured by crystal violet staining (CV), lactate dehydrogenase release (LDH), release of interleukin-8 (IL-8) measured by ELISA, and alkaline phosphatase activity in the cells (APc) and released into the supernatant (APS). Nuclear morphology was also examined. SiO2 and Ni3S2 both caused dose-dependent acute toxicity as assessed by LDH and CV, and caused alterations in nuclear morphology consistent with apoptosis. However, much more IL-8 was released into the tissue culture supernatant by SiO2 at the same levels of cytotoxicity than by Ni3S2. Neither of these acutely toxic materials increased APc or APS, but the less cytotoxic materials caused very significant release of AP in the order OAA > DS > ROFA >> SiO2 = Ni3S2. OAA and, to a lesser extent, DS caused increases in mitotic fraction and increased CV staining, consistent with stimulation of proliferation. These results suggest multiple modes of responses to toxic materials and imply that a toxicological screening process should address these and possibly other endpoints.

Responses of salivary acinar cells to intracellular alkalinization.

Responses of rat submandibular acini to intracellular alkalinization were investigated. Intracellular alkalinization was induced by addition of NH4Cl or methyl amines, or by prepulse with Na butyrate. Only partial recovery occurred following Na butyrate prepulse or methylated amine addition, but full recovery was observed following addition of NH4Cl. The latter recovery was DIDS and dimethylamiloride-insensitive but was inhibited by bumetanide or high [K+] and stimulated in Na(+)-free buffer and by ouabain. Acetylcholine stimulated recovery from NH4Cl- or Na butyrate pre-pulse-induced alkalinization and reduced the extent of alkalinization induced by methylated amines. Acetylcholine-stimulated recovery from NH4Cl-induced alkalinization was mimicked by substance P or ionomycin and was partially Ca(2+)-dependent. This stimulated recovery was bumetanide-insensitive but was partially sensitive to charybdotoxin. Taken together, these data indicate that in unstimulated cells, recovery from alkalinization induced by NH4Cl occurs by bumetanide-sensitive transport of the NH4+ ion, that DIDS-inhibitable anion transport contributes little to this recovery, and that acetylcholine and other Ca(2+)-elevating agents accelerate recovery from NH4Cl-induced alkaline challenge by a mechanism insensitive to bumetanide, DIDS, ouabain, and dimethylamiloride but sensitive to extracellular Ca2+ and to charybdotoxin. Partial recovery from alkaline challenge can also occur in the absence of NH4+ ions, and acetylcholine also stimulates this mode of recovery. Together, these data suggest that these cells have little intrinsic ability to recover from intracellular alkalinization and that the NH4+ ion may be a surrogate for K+ in at least two ion transport pathways.

Muscarinic signaling pathway in submandibular cells of adult and early postnatal rats.

Elements of the muscarinic signal transduction pathway were compared in submandibular acinar cells of 1-day-old, 1-week-old, and adult rats after exposure to concentrations of acetylcholine ranging from 0.05 to 10 microM. Formation of inositol trisphosphate (IP3) and intracellular Ca2+ were comparable in cells from the three age groups after exposure to agonist concentrations < 1 microM. At higher agonist concentrations, IP3 generation and peak initial changes in [Ca2+]i were significantly greater in cells of newborn animals. In cells of 1-week-old animals, increased peak [Ca2+]i responses were seen even at low agonist concentrations, although IP3 production was not increased when compared with fully mature cells. Increased initial [Ca2+]i peaks, but comparable subsequent plateau [Ca2+]i values, were seen in the immature cells in both Ca(2+)-containing and Ca(2+)-free solutions. Permeabilized cells of early postnatal animals took up less 45Ca2+ into nonmitochondrial Ca2+ pools in the presence of 1.5 mM ATP and also released less tracer in response to intermediate IP3 concentrations than adult cells. Developing salivary cells thus show differences in important functional linkages of the muscarinic signal transduction pathway, including those between receptor activation and phosphoinositide turnover and between IP3 and release of internally stored Ca2+. Differences in the Ca2+ stores or in their sensitivity to IP3 may account for the latter observation. Immature salivary cells seem to have adequate mechanisms for Ca2+ entry.

Intracellular [Ca2+] and K+ and Cl- efflux responses in submandibular cells of neonatal and adult rats.

The effects of graded doses (5 x 10(-8) to 10(-5)) acetylcholine on intracellular Ca2+ and on 86Rb and 36Cl efflux were compared in submandibular cell clusters of 1 and 7 day-old and adult rats. Initial Ca2+ peaks were similar at agonists concentrations lower than 10(-7) M but the release of Rb+ and Cl- were smaller in cells of young animals. At higher agonist concentrations, Ca2+ peaks were higher in immature cells; however, initial Cl- (but not Rb+) efflux was similar to that of mature cells. Plateau Ca2+ levels were independent of age and agonist concentrations but the content of Cl- and Rb+ varied greatly and differences between age groups were less evident. These data confirm a dissociation between intracellular Ca2+ levels and Ca(2+)-mediated ion transport in immature salivary cells.

Effects of NH4Cl and dimethylamine on Cl- fluxes in resting and stimulated rat submandibular acinar cells.

Transmembrane movements of K+ and Cl- in salivary acinar cells are important in the formation of saliva, and may be affected by changes in intracellular pH (pHi). Exposure to NH4Cl increases pHi transiently, but NH4+ may have effects independent of pHi. To investigate how Cl- transport may be altered under these conditions, rat submandibular acini were exposed to NH4Cl, and transmembrane Cl- transport was studied with 36Cl-. NH4Cl increased intracellular Cl- in these cells. The initial phase of this increase was partially HCO(3-)-dependent and was inhibited by 1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), while the sustained phase was inhibited by 0.1 mM bumetanide. NH4Cl also inhibited acetylcholine-induced Cl- efflux from tracer preloaded cells. Changes in pH did not always correlate in time or extent with those of Cl- transport. We conclude that 1) exposure to NH4Cl increases Cl-uptake primarily by a bumetanide-sensitive transport system that did not reach steady state during the experiment, 2) exposure to NH4Cl also stimulates Cl- uptake by a DIDS-sensitive mechanism, and 3) only the latter is pHi sensitive.

Intracellular pH changes induced by exposure to weak acids and bases in submandibular cells of early postnatal rats.

The intracellular pH of submandibular cells of adult, 1-day-old and 1-week-old rats was measured with the pH sensitive fluorimetric indicator SNARF-1, following shifts induced by exposure to a weak acid (sodium butyrate) or a weak base (NH4Cl). The effects of several ion transport inhibitors and agonists on both the pH change and the rate of recovery were examined. The results indicate that, in most respects, the processes involved in pH regulation were similar in the neonatal and mature cells. However, cholinergic and peptidergic stimulation accelerated recovery from sodium butyrate-induced acidification more, and from NH4Cl-induced alkalinization less, in cell preparations from 1-day-old animals.

The effect of NH4Cl on Rb+ fluxes in resting and stimulated rat submandibular acinar cells.

Dispersed salivary acini isolated from the rat submandibular gland were incubated in a HEPES-buffered Krebs-Ringer solution or in the same buffer containing 20 mM NH4Cl and the accumulation and efflux of K+ were measured with the radiotracer 86Rb+, in the presence and absence of acetylcholine and of transport inhibitors. Exposure to NH4Cl caused a significant (greater than 50%) reduction in tracer accumulation. This effect was blocked by 0.1 mM bumetanide, but not by 1 mM ouabain. The effect of NH4Cl was, on the other hand, nearly additive with that of 1 microM acetylcholine. In cells preincubated with tracer, acute addition of NH4Cl caused a significant net efflux of isotope, so that the tracer content fell to 45% of the control value within 10 min. Bumetanide added to preloaded cells in the same fashion had no effect on tracer content and did not modify the efflux of 86Rb+ induced by 1 microM acetylcholine. However, this inhibitor essentially abolished the NH4Cl-induced tracer efflux. Exposure to NH4Cl during tracer loading did not appear to affect subsequent agonist-stimulated tracer efflux. These results suggest that: (1) the inhibition of K+ entry by NH4Cl is due to an effective competition by the NH4+ ion with Rb+ (and K+) for uptake via a bumetanide-sensitive Na+/K+/2Cl- contransporter.(ABSTRACT TRUNCATED AT 250 WORDS)

Mapping gold-labeled receptors on cell surfaces by backscattered electron imaging and digital image analysis: studies of the IgE receptor on mast cells.

The response of cells to signaling molecules such as hormones, growth factors, and immune mediators that bind to cell-surface receptors depends in part on the density and distribution of the relevant receptors. We have developed methods to map the distribution of IgE receptors on RBL-2H3 mast cells at high resolution in the scanning electron microscope (SEM). The key elements of our procedure are a new fixative that preserves receptor binding activity; a family of colloidal gold-conjugated probes that bind directly or indirectly to the IgE-receptor complex; an SEM with detectors for both secondary and backscattered electrons (to observe surface topography and gold particles, respectively); and an image processor that can average, digitize, and store these images. Topographical maps are generated by processing and superimposing the digitized images. The methods we describe can be applied to study the density and distribution of any membrane receptor that can be labeled with colloidal gold particles.

The blocked pinocytic activity of mitotic cells is restored in mitotic-interphase hybrids.

During mitosis there is an abrupt inhibition of a wide range of membrane functions, including fluid-phase and adsorptive pinocytosis. We have used cell hybrids formed between mitotic and interphase cells to approach the mechanism of this inhibition. We report that fluid pinocytosis is reactivated in the mitotic partner of hybrids formed between mitotic and interphase Chinese hamster ovary (CHO) cells. It thus appears that the interphase cell provides some necessary element(s) for membrane activity during mitosis. This dominance of interphase membrane properties stands in contrast with earlier evidence that mitotic nuclear properties dominate in similar mitotic-interphase hybrids.

DNP-phycobiliproteins, fluorescent antigens to study dynamic properties of antigen-IgE-receptor complexes on RBL-2H3 rat mast cells.

In RBL-2H3 rat mucosal mast cells, the crosslinking of cell-surface IgE-receptor complexes by multivalent antigens initiates a sequence of responses leading to degranulation. We have developed a family of dinitrophenol (DNP)-conjugated fluorescent antigens to study dynamic membrane events associated with these responses. Lysyl groups on the phycobiliproteins, B-phycoerythrin and C-phycocyanin, were labelled with DNP, yielding fluorescent conjugates that cause the release of [3H]serotonin from anti-DNP-IgE-primed RBL-2H3 cells. The binding of these antigens to IgE-receptor complexes was observed by fluorescence microscopy and quantified by flow cytometry. Incubation with 1 microgram/ml DNP42-B-phycoerythrin stimulates maximum degranulation from IgE-saturated cells. Under these conditions, approximately 26 X 10(3) molecules of DNP42-B-phycoerythrin are bound per cell at equilibrium. The rate and extent of antigen binding and of antigen-stimulated mediator release decrease in parallel as the concentration and DNP:protein ratio of the fluorescent conjugates is reduced. Secretion stops immediately when the nonfluorescent monovalent antigen, DNP-lysine, is added to degranulating cell suspensions. DNP-lysine also displaces surface-bound antigen when added during the first minutes after multivalent antigen. However, the ability of DNP-lysine to displace surface-bound DNP42-B-phycoerythrin from IgE-receptor complexes decreases progressively with time. Treatment with dihydrocytochalasin B and several analogs that prevent antigen-stimulated F-actin assembly enhances secretion and delays the transition of antigen to its DNP-lysine-resistant form. Cytochalasin treatment also permits the long-range movement of antigen into surface caps.(ABSTRACT TRUNCATED AT 250 WORDS)