Biomedical subjects

D E Rannels

Publications and source records attributed to D E Rannels.

At least 73 records · Page 4Linked to original sources

PubMed1987

Multiple pathways for uptake of paraquat, methylglyoxal bis(guanylhydrazone), and polyamines.

The uptake of polyamines, methylglyoxal bis(guanylhydrazone) (MGBG), and paraquat [N,N-dimethyl-4,4'-bipyridylium] into control Chinese hamster ovary (CHO) cells and a mutant CHO cell line selected for resistance to the toxicity of MGBG was examined. In contrast to control CHO cells, the mutant cells had no detectable uptake of MGBG or any of the polyamines. There was no difference between the two cell lines in the uptake of alpha-aminoisobutyric acid (AIB), which indicates that there was no general change in membrane transport processes. The mutant cells were also found to be resistant to the toxicity of paraquat and to have a reduced capability to take up the herbicide. This finding confirms that the uptake of paraquat is necessary for the toxicity of this compound and that the paraquat is taken up by a transport system that also transports MGBG. Competition experiments showed that an excess of unlabeled paraquat inhibited uptake of MGBG and, to a lesser extent, uptake of putrescine and spermidine, but no inhibitory action on spermine uptake could be detected. Studies with type II cells isolated from rat lung also demonstrated uptake of paraquat and spermidine, but paraquat was only a weak inhibitor of spermidine uptake in this system. These results suggest that there may be multiple systems for the uptake of MGBG and polyamines and that paraquat is taken up by at least one but not by all of these systems.

Aminoisobutyric Acids↗

PubMed1987

Culture of type II pneumocytes on a type II cell-derived fibronectin-rich matrix.

Recent evidence suggests that during primary culture, type II pneumocytes synthesize and deposit components of an extracellular matrix. The present study investigated the response of freshly isolated type II cells to a preformed, fibronectin-rich matrix synthesized by type II cells over a 6-day interval of primary culture on a plastic surface. Type II cells on 6-day matrix (M6) degraded the preformed matrix and deposited newly synthesized fibronectin more rapidly than cells on plastic, suggesting that M6 itself stimulated type II cell-mediated matrix turnover. In type II cells on plastic, incorporation of radiolabeled thymidine into DNA increased 620 and 1,880% after 2 and 3 days in culture, respectively, as the cells assumed a more flattened phenotype. Although cells on M6 did not divide, both basal rates of thymidine labeling and sensitivity to serum modulators of DNA synthesis were enhanced by the M6 surface, as compared with plastic. Culture of type II cells on surfaces of purified fibronectin enhanced the rate of DNA synthesis in a manner similar to that observed on M6; this effect was blocked by antifibronectin. The data suggest that more rapid fibronectin synthesis and deposition are important components of the response of type II cells to primary culture. Extracellular matrix produced by type II cells appears to be similar to the basement membrane onto which these cells proliferate in vivo after lung injury. A fibronectin-rich surface in itself may thus induce additional extracellular matrix synthesis and further direct cellular differentiation and proliferation.

Animals↗

PubMed1987

Role of laminin in maintenance of type II pneumocyte morphology and function.

Loss of differentiated function by type II pneumocytes plated on plastic surfaces was demonstrated by decreased lamellar body content, increased cellular protein, and rapid cellular flattening, changes that were retarded modestly by plating cells on laminin-coated surfaces. Laminin surfaces also inhibited [3H]thymidine (THM) incorporation into cellular DNA by 40% compared with plastic at 40 h, but did not alter an additional mitogenic effect of rat serum over fetal calf serum. In contrast, cells plated on the laminin-rich basement membrane-like gel formed from an extract of EHS mouse sarcoma, matrix gel (MG), maintained a high content of intracellular lipids in lamellar inclusions and retained a rounded morphology for at least 3 days. MG markedly inhibited THM incorporation and morphological changes when cells were cultured on this surface or when MG was formed over cells initially plated on plastic for various intervals. The importance of the laminin component of MG was demonstrated when these surfaces were pretreated with a highly specific antilaminin serum. Type II cells commenced flattening on the treated MG surface, and THM incorporation increased with the same time course as did control cells on plastic. The data suggest that short-term culture and study of differentiated type II pneumocytes may require a laminin-rich substratum. THM incorporation into type II cell DNA provides an important early and sensitive index of cell-basement membrane interaction and subsequent maintenance of function.

Animals↗

PubMed1987

Uptake of exogenous spermidine by rat lungs perfused in situ.

Uptake of the polyamine spermidine (SPD) from the pulmonary circulation was characterized by using ventilated rat lungs perfused in situ with Krebs-Henseleit-bicarbonate buffer containing 4.5% bovine serum albumin, 5.6 mM glucose, and 20 amino acids at plasma levels. [14C]SPD was accumulated by the lungs in a time- and concentration-dependent manner. The pathway of SPD uptake exhibited saturation kinetics with an apparent Km in the range of 1 microM and a Vmax of 450-540 pmol/g lung min. SPD uptake was inhibited by the naturally occurring polyamines putrescine and spermine (SPM) and by the inhibitor of polyamine synthesis, methyglyoxal bis(guanylhydrazone) (MGBG). Inhibition of SPD uptake by SPM followed competitive kinetics; although MGBG was also a competitive inhibitor of SPD uptake, MGBG was less effective than SPM. These observations indicate that SPD is taken up from the pulmonary circulation by a carrier-mediated pathway that is inhibited by other natural polyamines and by MGBG and exhibits substrate affinity in the range of plasma SPD concentrations.

Animals↗

PubMed1987

Static mechanical properties of lungs from adrenalectomized pneumonectomized rats.

The pressure-volume characteristics of saline-filled lungs of adult rats were studied after left pneumonectomy (PNX), with or without prior bilateral adrenalectomy (ADX). Male Sprague-Dawley rats (200-240 g) remained unoperated (UNOP) or were divided into three surgical groups: left PNX, ADX alone, or PNX preceded 5 days earlier by ADX (ADX/PNX). Two weeks post-PNX, maximum lung volumes [MLV; measured at transpulmonary pressure (Ptp) of 10 cmH2O] of right lungs from PNX and ADX/PNX rats remained 20 and 37% below UNOP values, respectively, although right lung mass was equal to or exceeded that of both lungs in UNOP controls. Four weeks postoperative, MLVs approached but remained below normal values. In the ADX/PNX group, MLV was 35% below that predicted for lung mass. The effects of ADX on both lung mass and MLV were blocked by daily injections of ADX/PNX animals with hydrocortisone acetate (5 mg/kg). Neither PNX nor ADX/PNX altered lung volume at zero Ptp or changed the concentration of lung collagen or elastin. When corrected for differences in MLV, relative tissue compliance was unaffected by PNX but was reduced by ADX/PNX. These results indicate that MLV is not restored in parallel with lung mass during the rapid phase of compensatory tissue growth, which follows partial pneumonectomy, and that this discrepancy is accentuated by adrenalectomy.

Adrenalectomy↗

PubMed1987

Control of compensatory lung growth by adrenal hormones.

The effects of adrenalectomy and/or in vivo treatment with hydrocortisone acetate (HCA;5 mg X kg-1 X day-1) on lung growth were investigated in control and pneumonectomized rats of 250 g body wt. Left pneumonectomy (day 0) initiated rapid hyperplastic growth of the right lung, which was unaffected by HCA. Similarly, HCA had no effect on lung growth in unoperated control animals. Two weeks after pneumonectomy, right lung dry mass, protein, RNA, and DNA were equal to that in both lungs of unoperated rats. Adrenalectomy 5 days before (day -5) left pneumonectomy increased the rate and extent of right lung growth, but did not change its hyperplastic character. Continuous HCA treatment (days -5 to 14) prevented the adrenalectomy-mediated increase in postpneumonectomy lung growth. "Early" HCA dosing (days -5 to 6) of adrenalectomized-pneumonectomized animals suppressed lung growth to the pneumonectomy level, but from days 7 to 14 growth accelerated to the adrenalectomized-pneumonectomized rate. Conversely, "late" HCA, initiated when adrenalectomized-pneumonectomized animals had restored normal total lung mass (days 6 to 14), quickly reduced right lung growth to rates typical of unoperated controls. The latter effects were not observed unless continuous steroid treatment was provided throughout this interval. The data support a role for glucocorticosteroids in modulation of the accelerated compensatory lung growth initiated by partial resection of the tissue.

Adrenal Cortex Hormones↗

PubMed1987

Effect of altered inflation on pulmonary uptake of methylglyoxal bis(guanylhydrazone).

The effects of increased pulmonary ventilation on uptake of an exogenous, nonmetabolized polyamine analog, methylglyoxal bis(guanylhydrazone) [MGBG] were investigated in rat lungs perfused in situ with buffer containing 4.5% bovine serum albumin, 5.6 mM glucose and plasma levels of amino acids. The perfusate was equilibrated and the lungs were ventilated with warmed, humidified O2/N2/CO2 (20:75:5). A 28% increase in lung inflation rapidly accelerated MGBG uptake at low (1.5 microM) but not at high (50 microM) substrate concentration, a change which appeared to reflect a decrease in the apparent Km of the uptake pathway. This effect was not associated with acute alterations in pulmonary vascular permeability or resistance, nor with instability of the preparations. Taken with observations made previously, these results suggest that deformation of the tissue by increased inflation may account for increased MGBG uptake by lungs from rats subjected to partial pneumonectomy.

Animals↗

PubMed1987

Interactions of beta adrenergic antagonists with isolated rat alveolar type II pneumocytes. I. Analysis, characterization and regulation of specific beta adrenergic receptors.

Binding of beta adrenergic receptors (BAR) in membranes of freshly isolated type II pulmonary epithelial cells to the radioligand [125I]iodocyanopindolol was saturable, steresospecific and of high affinity. Optimal conditions for the assay of BAR on type II pneumocyte membranes are presented. Type II pneumocyte BAR are primarily of the beta-2 subtype, as indicated by inhibition of subtype-specific antagonists, ICI 118,551 and betaxolol. Maximum binding and Kd were measured (Kd, 4-20 pM; maximum binding, 30-50 fmol/mg of protein) and used to identify factors which alter BAR. The number of BAR on type II pneumocytes doubled after 42-hr culture in the presence of dexamethasone. Ligand-receptor interactions were of similar affinity to those in membrane particulates from whole lung, but maximum binding was reduced 3- to 4-fold. Less than 5% of total pulmonary BAR can be accounted for by those expressed on freshly isolated type II pneumocyte membranes. Other cell types thus probably account for the majority of specific beta adrenergic binding sites in the lung as a whole.

Adrenergic beta-Antagonists↗

PubMed1987

Interactions of beta adrenergic antagonists with isolated rat alveolar type II pneumocytes. II. Receptor-independent accumulation of beta adrenergic antagonists and other cationic amphiphilic drugs in lamellar bodies.

Accumulation of basic drugs by pulmonary tissue is well known. The cationic amphiphilic nature of many of these compounds suggests that they may be sequestered within an acidic and/or phospholipid-rich compartment. We described previously receptor-independent, concentrative, temperature- and pH-dependent sequestration of the beta adrenergic antagonists [125I]iodocyanopindolol and [125I]iodopindolol by intact rat type II pneumocytes in primary culture. The present study reveals that type II pneumocytes sequester [125I]iodocyanopindolol to an extent greater than other cell types (type II cells greater than polymorphonuclear leukocytes greater than S49) within 80-min incubations. Localization of fluorescence into large granular structures was observed after incubation of type II cells with 9-aminoacridine-propranolol (9-AAP). The distribution of fluorescence coincides with surfactant-containing lamellar bodies (LB) visualized with tannic acid/osmium staining. The large granule localization of 9-AAP fluorescence decreases with time in primary culture in parallel with changes in cell morphology and decreased numbers of LB. Comparison of patterns of fluorescence after incubation with 9-AAP, acridine orange and 9-aminoacridine (all 1 microM) indicates that localization is not a property of the acridine moiety, but requires the propranolol side-chain. Association of 9-AAP fluorescence with LB is inhibited completely by chlorpromazine (10 microM); the same concentration of propranolol or chlorquine produces less extensive, but detectable, inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic beta-Antagonists↗

PubMed1986

Effects of halothane on transport of 5-hydroxytryptamine by platelet membranes.

Na+-dependent uptake of 5-HT (5-hydroxytryptamine) into plasma membrane vesicles derived from bovine blood platelets and ATP-dependent 5-HT uptake into storage vesicles in platelet lysates were measured. Na+-dependent uptake was temperature-dependent, inhibited by imipramine and exhibited Michaelis-Menten kinetics (apparent Km, 0.12 +/- 0.02 microM; Vmax. 559 +/- 54 pmol/min per mg of protein. Halothane had no effect on Na+-dependent transport of 5-HT in plasma-membrane vesicles. ATP-dependent 5-HT transport into storage granules also exhibited Michaelis-Menten kinetics (apparent Km 0.34 +/- 0.03 microM; Vmax. 34.3 +/- 1.7 pmol/min per mg of protein) and was inhibited by noradrenaline (norepinephrine), but not by imipramine. Exposure of the granules to halothane resulted in a progressive decrease in Vmax. The results demonstrate a possible site for disruption of platelet function by anaesthetics.

Adenosine Triphosphate↗

PubMed1986

Receptor-independent sequestration of beta-adrenergic ligands by alveolar type II cells.

Numerous studies indicate that synthesis and secretion of surfactant by type II pneumocytes are modulated by the interaction of beta-adrenergic agonists with specific cell surface receptors. Two 125I-labeled beta-adrenergic ligands, l-iodopindolol (IPIN) and l-iodocyanopindolol (ICYP), were thus employed to investigate the properties of type II cell beta-receptors. Saturable, high-affinity, stereospecific binding to crude membrane fractions from whole rat lungs was exhibited by both ligands (IPIN, KD 283 pM, Bmax 508 fmol/mg protein; ICYP, KD 18 pM, Bmax 404). Type II cell membranes obtained by N2 cavitation also revealed stereospecific, saturable, high-affinity binding. In intact cells (37 degrees C) however, rapid, highly concentrative (cell/media greater than 1000), nonspecific ligand uptake compromised estimates of specific binding (specific/total binding less than 0.1). Total ligand uptake was inhibited at 4 degrees C, by decreasing pH within the physiological range (7-8) and by the lysosomotropic compound chloroquine (50-200 microM), without a detectable change in specific binding. Other basic drugs were also inhibitory at similar concentrations; acidic drugs had no effect. Even at 4 degrees C, specific binding remained low, as IPIN and ICYP were displaced less than 30% by l-alprenolol (1 microM). Physicochemical properties of IPIN and ICYP considered with the above studies suggest that passive ligand entry into intact pneumocytes and subsequent trapping of the protonated species in a cellular compartment of low pH may account for high nonspecific ligand uptake.

Animals↗

PubMed1986

Increased pulmonary uptake of exogenous polyamines after unilateral pneumonectomy.

Alterations in pulmonary uptake of the naturally occurring polyamine spermidine and of an exogenous polyamine substrate analogue, methylglyoxal bis(guanylhydrazone) (MGBG), were investigated during the early phase of compensatory lung growth after partial pneumonectomy (PNX) in rats. In lungs perfused in situ 3 days after left PNX, when a small (14%) but significant (P less than 0.01) increase in right lung mass could be detected, uptake of [14C]spermidine and of [14C]MGBG from the pulmonary circulation was increased. MGBG uptake exhibited saturation kinetics (1-50 microM MGBG), both in lungs of control animals apparent Km, 11.3 microM; Vmax, 479 pmol X g-1 X min-1) and on the 1st and 3rd post-PNX days. In both PNX groups, the apparent Km of the uptake pathway was decreased somewhat (8.5 microM), while Vmax increased progressively to 584 and 678 pmol X g-1 X min-1 at days 1 and 3, respectively. The effects of PNX on MGBG uptake were detected as early as 3 h after lung resection and were no longer evident when compensatory lung growth was completed 14 days after surgery. In rats adrenalectomized 5 days before left PNX, an accelerated onset and increased rate of lung restoration were associated with a doubling of the effect of PNX alone on MGBG uptake.

Adrenalectomy↗

PubMed1986

Keratin species in type II pneumocytes in culture and during lung injury.

A detailed understanding of alveolar epithelial cell transitions during remodeling after lung injury requires the identification of specific markers. We have developed a panel of monoclonal antibodies against species of the intermediate filament protein, keratin. These individual species are recognized markers of the state of differentiation of various epithelial cells. These and complementary protein analytic methods have been applied to studies of isolated, enriched Type II pneumocyte preparations as well as to normal and injured lung tissues. Monoclonal antibody 24A3, initially raised against Morris hepatoma 7777 keratins, decorated a filament network in isolated cultured rat Type II pneumocytes by indirect immunofluorescence; it reacts by 2-dimensional polyacrylamide gel immunoblot procedures with an acidic, 46,000-dalton keratin. Monoclonal antikeratin antibodies AE1 and AE3, raised against human epidermal keratins, reacted poorly with isolated Type II cells; however, AE3 reacted by immunoblot technique with the 55,000-dalton keratin subclass. The bronchial epithelium reacted intensely with 24A3 as well as with a mix of AE1 plus AE3 in ethanol-fixed, paraffin-embedded sections of normal and injured rat lung. Alveolar regions of normal lung reacted poorly with all 3 antibodies, however, as visualized by light microscopy. At the same time, very large, presumptive epithelial cells in the alveolar regions stained intensely with 24A3 3 days after intratracheal instillation of bleomycin, whereas thin cells lining the alveoli in injured regions were intensely reactive 14 days after bleomycin treatment. These elongated cells may represent Type II pneumocytes in the process of converting to Type I cells.

Animals↗

PubMed1986

Protein synthesis in perfused rat lungs: determinations based on incorporation of radioactive proline.

Functional compartmentation and metabolism of radioactive proline was evaluated to define conditions under which synthesis of lung proteins could be measured accurately based on proline incorporation. Rat lungs were perfused with Krebs-Henseleit bicarbonate buffer equilibrated with O2/N2/CO2 (20:75:5) and containing 4.5% (w/v) bovine serum albumin, 5.6 mM glucose and amino acids at plasma levels. Intracellular proline increased linearly as perfusate proline concentration was increased from 108 microM, the plasma level, to 540 or 1080 microM. At each concentration, the pool of proline which provided precursors to protein synthesis rapidly reached a steady-state specific radioactivity, but when extracellular proline was 108 microM, this pool was diluted significantly by proline from endogenous sources. At 540 or 1080 microM extracellular proline, the specific radioactivities of perfusate and intracellular proline approached equality and rates of protein synthesis calculated based on the specific radioactivity of extracellular proline compared favorably with those calculated from the specific radioactivity of phenylalanyl-tRNA. Similar results were obtained in lungs of two groups of rats in which intracellular proline concentration differed 3-fold. Thus, the contribution of endogenous proline to the pathway of protein synthesis was minimized when extracellular proline was present at high concentration. Under this condition, calculations of protein synthesis based on proline incorporation were most accurate.

Animals↗

PubMed1985

Effects of prior adrenalectomy on postpneumonectomy lung growth in the rat.

The effects of adrenalectomy, with and without subsequent glucocorticoid replacement therapy, on postpneumonectomy compensatory lung growth in the rat were investigated. Male Sprague-Dawley rats (200-230 g) were subjected to no operation (UNOP), left pneumonectomy (PNX), or PNX preceded by bilateral adrenalectomy 5 days earlier (ADX/PNX). At 14 days post-PNX, when compensatory lung growth is normally complete in 200-g rats, right lung (RL) dry weights of PNX (263 +/- 6 mg, n = 26) and ADX/PNX (334 +/- 13 mg, n = 25) rats were increased 58 and 101%, respectively, relative to UNOP controls (166 +/- 5 mg, n = 10). Increases in total DNA, RNA, and protein in the right lungs of PNX and ADX/PNX rats occurred in proportion to RL dry mass. The increase in all parameters examined in PNX and ADX/PNX rats at 7 days post-PNX was half that at 14 days, indicating linear lung growth in both treatment groups. The stimulatory effect of ADX on lung growth was blocked by hydrocortisone acetate (HCA), administered intraperitoneally in daily doses of 5 mg/kg, beginning on the day of PNX. The RL dry weights of HCA-treated ADX/PNX rats (241 +/- 7 mg, n = 10) did not differ significantly from the corresponding value in PNX rats (270 +/- 14 mg, n = 7). The lower RL weights in the HCA-treated rats resulted from an inhibition of cell division, as evidenced by the total RL DNA content, which was similar to that in PNX animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenalectomy↗

PubMed1985

Lung growth in response to unilateral pneumonectomy in rapidly growing rats.

The rapidity with which lung growth is initiated and completed after pneumonectomy was examined in young rats (4 wk of age; 82 g). After left pneumonectomy, the remaining lobes of the right lung grew to equal the weight of both lungs of control animals by day 7 and within 14 days increased from 366 to 968 mg. The tissue concentrations of RNA, DNA phosphate, collagen, and noncollagen proteins did not increase during the growth response. In contrast, total amounts of these constituents increased significantly in the remaining lung of pneumonectomized animals during the 1st postoperative wk and approached levels found in both lungs of sham-operated and unoperated controls by the end of the 2nd wk after pneumonectomy. Although cell size increased in control lungs during the experimental period, there was little evidence of additional cellular hypertrophy associated with compensatory lung growth. The character of the response to pneumonectomy in these rats was similar to that observed previously in older animals (320 g). Thus in spite of the higher basal rate of lung growth in the younger rats, the pattern and rapidity of compensation after pneumonectomy was similar in both age groups.

Animals↗

PubMed1985

Carrier-mediated uptake of methylglyoxal bis(guanylhydrazone) by rat lungs perfused in situ.

Rat lungs perfused in situ were employed to begin investigations of the pathways by which the tissue takes up circulating polyamines (PA). Uptake kinetics were studied using [14C]methylglyoxal bis(guanylhydrazone) (MGBG), a nonmetabolized substrate analogue thought to enter cells via the PA carrier. Lungs concentrated MGBG from the perfusate at a linear rate for at least 60 min. Uptake was saturable with respect to perfusate MGBG concentration; it exhibited an apparent Km of 12.5 microM and Vmax of 0.6 nmol X g lung-1 X min-1. MGBG (1 microM) uptake was inhibited rapidly and to a similar extent (30-40%) by the naturally occurring PAs spermidine, spermine, or putrescine (50 microM); no additional inhibition of uptake was exerted when all three compounds were present simultaneously (total concentration, 150 microM). No inhibition by 5-hydroxytryptamine was evident. Spermidine produced a half-maximal inhibitory effect at a perfusate concentration of 1.9 microM (vs. 1 microM MGBG). The spermidine-insensitive component of MGBG uptake operated at a Vmax similar to that of the control (total), 1.2 nmol X g-1 X min-1, but the apparent Km was increased 3.5-fold to 44 microM. These observations indicate that MGBG is taken up from the pulmonary circulation by a high-affinity, carrier-mediated, concentrative uptake process that is inhibited, at least in part, by naturally occurring polyamines.

Animals↗

PubMed1985

Interaction of paraquat and amine uptake by rat lungs perfused in situ.

The kinetics of [14C]paraquat (N,N-di[14C]methyl-4,4'-bipyridylium) uptake from the pulmonary circulation were investigated in rat lungs perfused in situ. During the 1st h of exposure to the herbicide paraquat entered the lungs primarily by diffusion; no evidence was obtained from concentrative uptake, saturation kinetics (1-3,500 microM paraquat), or inhibition by the amines methyl-glyoxal bis(guanylhydrazone) or spermidine, both of which were expected to compete for paraquat transport sites. In contrast, after 60 min of exposure, uptake rates increased two- to three-fold, and paraquat was accumulated to an apparent intracellular concentration greater than that in the perfusate. The latter phase of paraquat uptake was saturable and was inhibited by methylglyoxal bis(guanylhydrazone); it did not appear to reflect a progressive paraquat-induced alteration in cellular permeability but rather predominance of a rapid, carrier-mediated uptake pathway.

Animals↗