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M W Roe

Publications and source records attributed to M W Roe.

27 records · Page 2Linked to original sources
PubMed1993

Voltage-dependent intracellular calcium release from mouse islets stimulated by glucose.

Glucose-activated beta-cell insulin secretion depends upon elevation of intracellular calcium concentration, [Ca2+]i, which is thought to arise from Ca2+ influx through voltage-dependent calcium channels. Using fura-2-loaded mouse islets, we demonstrate, in fact, that the major component of the glucose-activated [Ca2+]i rise represents voltage-dependent intracellular Ca2+ release. Furthermore, the Ca2+ release pool possesses a novel pharmacology in that it is caffeine-sensitive but ryanodine-insensitive. In the absence of external Ca2+, glucose still caused intracellular Ca2+ release, an effect blockable by tetrodotoxin. However, depolarization of the islet with KCl in low Ca(2+)-containing solutions induced intracellular Ca2+ release, which was resistant to tetrodotoxin. We conclude that glucose release of intracellular Ca2+ is dependent upon depolarization alone, possibly through increasing inositol 1,4,5-trisphosphate production.

Animals↗
PubMed1991

Epidermal growth factor and other mitogens induce binding of a protein complex to the c-fos serum response element in human astrocytoma and other cells.

Rapid stimulation of c-fos transcription by many agonists requires the serum response element (SRE), which binds at least two distinct nuclear proteins, p67SRF and p62TCF. Using nuclear protein extracts from 1321-N1 human astrocytoma cells, we investigated ligand-induced changes in binding of these proteins to SRE probes. In these cells c-fos mRNA expression can be induced by epidermal growth factor (EGF) through protein kinase C-independent pathways and by phorbol esters through protein kinase C. We detected two DNA-protein complexes that formed specifically with the SRE (bands 1 and 2). Band 2 formation was increased 4-6 min after stimulation with EGF as well as serum and phorbol esters; this peaked at 10-30 min and returned to basal levels by 60 min. Induction of band 2 formation preceded the onset and peak accumulation of c-fos mRNA (15 and 30 min after EGF stimulation, respectively) and its return to basal levels (by 1-2 h). Band 2 formation was also increased A431 cells stimulated with EGF and in HeLa and Swiss-3T3 cells stimulated with serum. We found that band 1 contained p67SRF bound to the SRE; band 2 contained p67SRF and a second protein. Gel shift analyses using [35S]methionine-labeled p67SRF and nonradioactive DNA probes suggested that hormone treatment most likely modified the second protein component of band 2. Transient transfection of 1321-N1 cells with plasmids containing point mutations that prevented band 2 formation in vitro also abolished induction of c-fos transcription in vivo as assayed by RNase protection analysis. Thus, hormone-stimulated formation of the protein-DNA complex represented by band 2 may be involved in the activation of c-fos transcription.

Astrocytoma↗
PubMed1989

Platelet-derived growth factor and angiotensin II cause increases in cytosolic free calcium by different mechanisms in vascular smooth muscle cells.

Platelet-derived growth factor (PDGF) and angiotensin II (AII) are thought to mediate their biological effects in vascular smooth muscle cells (VSMCs) by causing alterations in cytosolic free calcium ([ Ca2+]i). In this study we examine the pathways by which PDGF and AII alter [Ca2+]i in VSMCs. Addition of PDGF resulted in a rapid, transient, concentration-dependent increase in [Ca2+]i; this rise in [Ca2+]i was blocked completely by preincubation of cells with ethylene glycol-bis (beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) or CoCl2, by the voltage-sensitive Ca2+-channel antagonists verapamil or nifedipine, by 12-O-tetradecanoylphorbol-13-acetate (TPA), or by pertussis toxin. AII also caused an increase in [Ca2+]i; however, AII-stimulated alterations in [Ca2+]i displayed different kinetics compared with those caused by PDGF. Pretreatment of cells with 8-(diethylamine)-octyl-3,4,5-trimethyoxybenzoate hydrochloride (TMB-8), almost totally inhibited AII-induced increases in [Ca2+]i. EGTA or CoCl2 only slightly diminished AII-stimulated increases in [Ca2+]i. Nifedipine, verapamil, TPA, and pertussis toxin pretreatment were without effect on AII-induced increases in [Ca2+]i. PDGF and AII both stimulated increases in total inositol phosphate accumulation, although the one-half maximal concentration (ED50) for alterations in [Ca2+]i and phosphoinisitide hydrolysis differed by a factor of 10 for PDGF (3 X 10(-10) M for Ca2+ vs. 2.5 X 10(-9) M for phosphoinositide hydrolysis), but they were essentially identical for AII (7.5 X 10(-9) M for Ca2+ vs. 5.0 X 10(-9) M for phosphoinositide hydrolysis). PDGF stimulated mitogenesis (as measured by [3H]-thymidine incorporation into DNA) in VSMCs with an ED50 similar to that for PDGF-induced alterations in phosphoinositide hydrolysis. PDGF-stimulated mitogenesis was blocked by pretreatment of cells with voltage-sensitive Ca2+ channel blockers, TPA, or pertussis toxin. These results suggest that PDGF and AII cause alterations in [Ca2+]i in VSMCs by at least quantitatively distinct mechanisms. PDGF binding activates a pertussis-toxin-sensitive Ca2+ influx into cells via voltage-sensitive Ca2+ channels (blocked by EGTA, verapamil, and nifedipine), as well as stimulating phosphoinositide hydrolysis leading to release of Ca2+ from intracellular stores. AII-induced alterations in [Ca2+]i are mainly the result of phosphoinositide hydrolysis and consequent entry of Ca2+ into the cytoplasm from intracellular stores. Our data also suggest that changes in [Ca2+]i caused by PDGF are required for PDGF-stimulated mitogenesis.

Angiotensin II↗
PubMed1987

Platelet-derived growth factor-induced alterations in vinculin distribution in porcine vascular smooth muscle cells.

Exposure of porcine vascular smooth muscle cells to platelet-derived growth factor (PDGF; 18-180 ng/ml) but not epidermal growth factor (EGF; 30 ng/ml), somatomedin C (SmC; 30 ng/ml), or insulin (10 microM), results in a rapid, reversible, time- and concentration-dependent disappearance of vinculin staining in adhesion plaques; actin-containing stress fibers also become disrupted following exposure of cells to PDGF. Disappearance of vinculin staining from adhesion plaques is also caused by 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 200-400 nM), though the time course of the disappearance of vinculin staining under these conditions takes longer than in cells exposed to PDGF. The PDGF-induced removal of vinculin from adhesion plaques was inhibited in a concentration-dependent fashion by 8-(N,N-diethylamino) octyl-3,4,5-trimethoxybenzoate (TMB-8; 0.25-4 microM) and leupepetin (2-300 microM), and by n-alpha-tosyl-L-lysine chloromethylketone (TLCK; 100 microM) and trifluoperazine (TFP; 2.5 microM). Addition of PDGF to vascular smooth muscle cells caused a rapid, transient increase in cytosolic free calcium, from a basal resting level of 146 +/- 6.9 nM (SEM, n = 62) to 414 +/- 34 nM (SEM, n = 22) as determined using the calcium-sensitive indicator Fura-2 and Digitized Video Microscopy. This increase in cellular calcium preceded the disappearance of vinculin from adhesion plaques and was partially blocked by pretreatment of cells with TMB-8 but not leupeptin. This rise in cytosolic free calcium was found to occur in approximately 80% of the sample population and displayed both spatial and temporal subcellular heterogeneity. Exposure of cells to TPA (100 nM) did not result in a change in cytosolic free calcium. Both PDGF (20 ng/ml) and TPA (100 nM) caused cytosolic alkalinization which occurred after PDGF-induced disruption of vinculin from adhesion plaques, as determined using the pH-sensitive indicator BCECF and Digitized Video Microscopy. PDGF stimulated DNA synthesis and vinculin disruption in a similar dose-dependent fashion. Both could be inhibited by leupeptin or TMB-8. These results suggest that 1) exposure of vascular smooth muscle cells to PDGF is associated with the disruption of vinculin from adhesion plaques, 2) PDGF-induced vinculin disruption is regulated by an increase in cytosolic calcium (but not cytosolic alkalinization), and involves proteolysis; 3) activation of protein kinase C also causes vinculin removal from adhesion plaques but by a calcium-independent mechanism, and 4) the cellular response to PDGF-stimulated increases in cytosolic free calcium is heterogeneous.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1983

Deposition pattern of inorganic particles at the alveolar level in the lungs of rats and mice.

The early pathogenesis of particle-induced lung disease is likely to be determined in large part by the initial pattern of dust deposition at the alveolar level. We have studied the deposition pattern of 5 aerosolized dusts (chrysotile and crocidolite asbestos, fiber glass, alpha-quartz, and ash from Mt. St. Helens) in the lungs of rats. Mice were exposed to chrysotile asbestos. Quantitative electron microscopy was carried out on tissues fixed by vascular perfusion. Immediately after a brief exposure, significantly (p less than or equal to 0.01) greater numbers of particulates had deposited on alveolar duct bifurcations when compared with the number of particles on duct surfaces adjacent to the bifurcations. Few particles were counted at midpoints between bifurcations, and particles rarely were observed within alveoli. Our data show that regardless of mineral nature, shape, or concentration, inhaled particulates small enough to pass through the conducting airways are deposited primarily at alveolar duct bifurcations. We proposed that the alveolar deposition patterns observed are the result of air-flow characteristics that cause enhanced deposition of particles at alveolar duct bifurcations intersecting the flow. This is similar to deposition patterns that occur at bifurcations of conducting airways.

Aerosols↗
PubMed1982

Deposition and translocation of inhaled silica in rats. Quantification of particle distribution, macrophage participation, and function.

Chronic exposure to silica dust causes fibrotic lung disease. Using brief exposures, we have attempted to define the initial patterns of dust deposition, anatomical compartments through which silica is translocated, and the participation of pulmonary macrophages in clearing the inhaled dust. To accomplish this, particle distribution and translocation at the alveolar level were studied in rats exposed to aerosolized alpha-quartz. Animals were exposed to 109 mg. per cu. m. of crystalline silica in inhalation chambers for 3 hours and sacrificed at varying times after exposure. The lungs were fixed by vascular perfusion through the right ventricle and tissue blocks were prepared for transmission and scanning electron microscopy. Lungs of additional animals were lavaged to recover populations of pulmonary macrophages for in vitro studies. Scanning electron microscopy in concert with back-scattered electron imaging showed that 24 hours postexposure there was a significant decrease in the number of silica particles per unit area of alveolar duct surface when compared with lung tissue from animals sacrificed immediately after exposure. Transmission electron microscopy revealed that silica crystals had been translocated to alveolar type I cells, interstitium, and macrophages. The percentage of silica-containing macrophages on alveolar surfaces increased from 36 +/- 2 per cent (mean +/- S.E.) immediately after exposure to 66 +/- 2 per cent during the 24 hours following exposure. This high percentage of macrophage participation was maintained through a 24-day postexposure period and then returned to 25 +/- 2 per cent 42 days after exposure. The percentage of silica-containing macrophages recovered by lavage were remarkably similar to those studied in situ: 24 +/- 4 per cent immediately postexposure, 62 +/- 3 per cent from 12 hours through 24 days, and 28 +/- 4 per cent 42 days postexposure. Although the percentage of macrophages with silica remained steady, the amount of silica per cell decreased during this 12-hour to 24-day period. Metabolic and viability studies of lavaged macrophages in vitro showed no differences between sham and silica-exposed animals. We propose that the events reported here represent normal, steady state clearance of a subpathogenic dose of potentially toxic particulates.

Animals↗
PubMed1980

Use of backscattered electron imaging to quantify the distribution of inhaled crystalline silica.

Inhalation of crystalline silica causes fibrotic pulmonary disease. The lung pathology of silicosis is well characterized and predictable, but the initial patterns of particle deposition and translocation are unknown. Scanning electron microscopy and backscattered electron imaging were utilized to quantify silica particle distribution in the distal air spaces of rats following a three-hour exposure to silica dust at a concentration of 100mg/m3. Lungs were perfused through the vasculature with 2% Karnovsky's fixative at a pressure of 15cm of water for 30 minutes. Blocks of tissue were dissected from five predetermined regions of the left lung and critical point dried. Mounted blocks were further dissected to reveal terminal bronchioles and their attached alveolar ducts. The tissue was sputter-coated with gold. Silica particles were visualized on the alveolar duct surfaces, then counted using negative backscattered electron imaging. The precise area of alveolar duct surfaces was calculated by using a standard magnification of 10,000X and a grid of 64cm2 over the viewing cathode ray tube. Thus, quantitation of silica particles could be expressed as number of particles per square micron. Our data show that there were fewer particles on the alveolar duct surfaces and in alveolar spaces of animals 8 hours after exposure when compared with animals 3 hours post-exposure.

Animals↗
PubMedDate not supplied

Assessment of Fura-2 for measurements of cytosolic free calcium.

Fura-2 has become the most popular fluorescent probe with which to monitor dynamic changes in cytosolic free calcium in intact living cells. In this paper, we describe many of the currently recognized limitations to the use of Fura-2 in living cells and certain approaches which can circumvent some of these problems. Many of these problems are cell type specific, and include: (a) incomplete hydrolysis of Fura-2 acetoxymethyl ester bonds by cytosolic esterases, and the potential presence of either esterase resistant methyl ester complexes on the Fura-2/AM molecule or other as yet unidentified contaminants in commercial preparations of Fura-2/AM; (b) sequestration of Fura-2 in non-cytoplasmic compartments (i.e. cytoplasmic organelles); (c) dye loss (either active or passive) from labeled cells; (d) quenching of Fura-2 fluorescence by heavy metals; (e) photobleaching and photochemical formation of fluorescent non-Ca2+ sensitive Fura-2 species; (f) shifts in the absorption and emission spectra, as well as the Kd for Ca2+ of Fura-2 as a function of either polarity, viscosity, ionic strength or temperature of the probe environment; and (g) accurate calibration of the Fura-2 signal inside cells. Solutions to these problems include: (a) labeling of cells with Fura-2 pentapotassium salt (by scrape loading, microinjection or ATP permeabilization) to circumvent the problems of ester hydrolysis; (b) labeling of cells at low temperatures or after a 4 degrees C pre-chill to prevent intracellular organelle sequestration; (c) performance of experiments at lower than physiological temperatures (i.e. 15-33 degrees C) and use of ratio quantitation to remedy inaccuracies caused by dye leakage; (d) addition of N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) to chelate heavy metals; (e) use of low levels of excitation energy and high sensitivity detectors to minimize photobleaching or formation of fluorescent non-Ca2+ sensitive forms of Fura-2; and (f) the use of 340 nm and 365 nm (instead of 340 nm and 380 nm) for ratio imaging, which diminishes the potential contributions of artifacts of polarity, viscosity and ionic strength on calculated calcium concentrations, provides a measure of dye leakage from the cells, rate of Fura-2 photobleaching, and can be used to perform in situ calibration of Fura-2 fluorescence in intact cells; however, use of this wavelength pair diminishes the dynamic range of the ratio and thus makes it more sensitive to noise involved in photon detection. Failure to consider these potential problems may result in erroneous estimates of cytosolic free calcium.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗