bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of selenium deficiency on glutathione-induced protection from hyperbaric hyperoxia in rat
Authors: Jenkinson S. G. , Jordan J. M. , Duncan C. A.
Published: December 2017

Exposure of rats to 100% O2 at high pressure (greater than 2.0 ATA) results in generalized convulsions and death within several hours. The tripeptide, glutathione, has been shown to protect rats exposed to hyperbaric hyperoxia with delayed onset of seizures and prolonged survival. To investigate the hypothesis that glutathione exerts its protective effects via the glutathione redox cycle, we injected selenium-deficient rats and their selenium-supplemented controls with either glutathione (1 mmol/kg) or an equivolume of saline before exposure to 100% O2 at 4 ATA. Selenium-deficient rats exhibit marked reduction in liver glutathione peroxidase activity (GSH-Px). Glutathione administration significantly delayed both the onset of seizures and time to death in the control animals. In selenium-deficient rats, however, glutathione administration was not protective, having no significant effects on time to seizure or time to death. We also measured changes in glutathione concentrations in lung, liver, and brain of these same groups of animals exposed either to hyperbaric hyperoxia or to room air. In control rats, lung and brain glutathione concentrations did not change with the hyperbaric exposure regardless of glutathione pretreatment status, but hepatic glutathione concentration declined significantly during the exposure when glutathione was not supplied. If these animals were pretreated with glutathione, the decline in hepatic glutathione concentrations did not occur. In selenium-deficient rats, the hyperbaric exposure did not result in changes in lung, brain, or liver glutathione concentrations either in the glutathione-pretreated or in the saline-pretreated animals. Exogenous GSH administration does not protect selenium-deficient rats from hyperbaric hyperoxia.(ABSTRACT TRUNCATED AT 250 WORDS)
Oxygen tolerance in neonatal rats: role of subcellular superoxide generation
Authors: Ischiropoulos H. , Nadziejko C. E. , Kumae T. , Kikkawa Y.
Published: December 2017

We compared the superoxide anion generating capacity of subcellular fractions from the lungs of neonatal and adult rats. Microsomal and mitochondrial fractions from adult rats produced approximately three times more superoxide (nanomoles per minute per milligram protein) than fractions from neonatal rats in the presence of 100% O2. Subcellular superoxide anion generating capacity was also examined in adult and neonatal rats exposed to greater than 95% fractional concentration of O2 in inspired gas. The O2- produced by mitochondrial and microsomal fractions of adult and neonatal rats increased above control levels for the first 24 h and declined below control values after 48 h of exposure in adults, whereas the elevated O2- production was sustained in microsomal fractions of neonates through 60 h. During the course of hyperoxic exposure, the largest difference in the superoxide generating capacity between adult and neonate was observed after 8-24 h of hyperoxia. The microsomal and mitochondrial fractions from adult rats produced three to seven times more O2- compared with neonatal rats. Cu,Zn superoxide dismutase (SOD) increased during the course of hyperoxia only in neonates at 8, 24, and 48 h of exposure. No change was observed in the activity of Mn SOD. The ratio of SOD activity (units per lung) to subcellular superoxide generating capacity (nanomoles per minute per lung) was calculated for the normal adults and neonates. The ratio for adult rats averaged 23 and 17 for mitochondrial and microsomal fractions, respectively, and 51 for neonatal rats for both subcellular fractions under normoxic conditions. These results suggest that O2- tolerance of neonates may be explained by the favorable balance between antioxidant defenses and subcellular superoxide generating capacity. The role of increased activity of Cu,Zn SOD as an accompanying or a causative phenomenon in O2 tolerance of neonates could not be determined from these experiments.
On the nature of enduring modifications induced in cells and organisms
Authors: Rubin H.
Published: December 2017

The clarity of Mendelian genetics and the elegance of the molecular mechanisms of replication and readout of DNA have tended to obscure a solid body of evidence demonstrating that nongenetic, enduring modifications can be induced in the behavior of cells, modifications that continue to be expressed for many divisions after withdrawal of the inducing stimulus. The most prosaic case is the differentiated state of metazoan cells, which persists throughout the lifetime of the organism. Much less widely known but well-characterized examples are also cited for bacteria, protozoa, and cultured cells of higher plants and animals. The spontaneous neoplastic transformation of cultured mouse NIH 3T3 cells is introduced as an enduring adaptive response to moderate growth constraints. Evidence in support of the thesis that physiological adaptation is the driving force for chemically induced carcinogenesis in the intact animal is also presented. The cases described here involve integrated responses of many if not all of the regulatory components of the cell, rather than singular molecular mechanisms. The continuous generation of phenotypic heterogeneity, a process observed readily in cell culture, provides the basis for a model that accounts for enduring modifications. This model, designated progressive state selection, makes no attempt at a detailed biochemical explanation of heterogeneity, but uses it as a fundamental postulate to represent the adaptive behavior of the cells.
Interaction of native and chemically modified albumin with pulmonary microvascular endothelium
Authors: Schneeberger E. E. , Lynch R. D. , Neary B. A.
Published: December 2017

The ability of native and chemically modified bovine serum albumin (BSA) to maintain normal pulmonary microvascular permeability was tested in "bloodless," fluorocarbon emulsion exchange transfused rats. Wet-to-dry weight ratios (W/D) of whole lung and morphometric estimates of the amount of ferritin transported to basement membrane were used to assess changes in water flux and macromolecular transport, respectively. Native and modified BSA in capillary walls were localized by immunogold techniques. Arginine residues of BSA were blocked with cyclohexanedione (CHD-BSA), and lysine residues were modified either by succinylation (Succ-BSA) or reductive methylation. Succinylation and CHD modification of BSA caused alterations in antigenicity and trypsin sensitivity; succinylation reduced the isoelectric point (pI). Whereas administration of either CHD-BSA or Succ-BSA increased the W/D, transport of ferritin to basement membrane was greater in the presence of Succ-BSA than CHD-BSA. By contrast, infusion of reductively methylated BSA in which modified lysines altered neither antigenicity nor pI, resulted in a W/D and amount of ferritin in basement membranes comparable to that of BSA. Binding of CHD-BSA and Succ-BSA to endothelial glycocalyx appeared to be reduced relative to native BSA and reductively methlyated BSA. The lowered pI of Succ-BSA may have contributed to its reduced binding; reductively methylated BSA with an unaltered pI was present in the glycocalyx. These data are consistent with a role for positively charged arginine residues in the interaction of albumin with the glycocalyx. The W/D of animals perfused with BSA was higher than those reported for rats perfused with complete rat serum proteins. This is consistent with the notion that serum factors, in addition to albumin, are required to maintain normal microvascular permeability.
Neurokinin receptors mediating substance P-induced contraction in adult rabbit airways
Authors: Cook J. A. , Brunner S. L. , Tanaka D. T.
Published: December 2017

Airway contractile responses to substance P (SP) were examined in isolated adult rabbit bronchial (BSM) and tracheal smooth muscle (TSM) segments. The tissues were placed in organ baths containing modified Krebs-Ringer solution, and isometric contractions to SP were monitored in the presence of phosphoramidon, an inhibitor of neutral endopeptidase (NEP). Under these conditions, BSM segments were significantly more reactive and more sensitive to SP than TSM segments. Removal of SPs cholinergic component with atropine (ATP) eliminated these regional differences in reactivity without affecting sensitivity. In considering the basis for these observations, it has been suggested that SP activates up to three different neurokinin (NK) subset receptors. Accordingly, we examined the regional airway contractile responses to Senktide, a selective NK-3 receptor agonist, and Septide, a selective NK-1 receptor agonist. In the presence of ATR, Senktide was inactive in both BSM and TSM, whereas Septide produced significantly greater contractions in BSM than in TSM. Subsequent desensitization of NK-1 receptors with Septide virtually eliminated the regional differences in airway sensitivity to SP. These findings indicate that 1) endogenous NEP activity can mask significant regional airway differences in SP-mediated contraction; and 2) these latter differences are the result of cholinergic, NK-1, and NK-2 receptor influences.
Coupling mechanisms in airway smooth muscle
Authors: Coburn R. F. , Baron C. B.
Published: December 2017

This review documents available information about coupling mechanisms involved in airway smooth muscle force development and maintenance and relaxation of force. Basic concepts, obtained from experiments performed on many different mammalian cell types, are in place regarding coupling between surface membrane receptors and cell function; these concepts are considered as a framework for understanding coupling between receptors and contractile proteins in smooth muscles and in airway smooth muscles. We have divided various components of coupling mechanisms into those dependent on changes in the surface membrane potential (electromechanical coupling) and those independent of the surface membrane potential (pharmacomechanical coupling). We have, to some degree, emphasized modulation of coupling mechanisms by intrasurface membrane microprocessing or by second messengers. A challenge for the future is to obtain a better understanding of how coupling mechanisms are altered or modulated during different phases of contractions evoked by a single agonist and under conditions of multiple agonist exposure to airway smooth muscle cells.
Isolation and culture of alveolar type II cells
Authors: Dobbs L. G.
Published: December 2017

The alveolar type II cell performs many important functions within the lung, including regulation of surfactant metabolism, ion transport, and alveolar repair. Because type II cells comprise only 15% of all lung cells, it is difficult to attribute specific functions to type II cells from studies of whole lungs or mixed cell cultures. At the present time, there is no passaged line that exhibits the full range of known type II cell functions. For these reasons, investigators have used isolated type II cells to study alveolar cell biology, biochemistry, and molecular biology. This review addresses many of the issues involved in isolating and culturing type II cells, including the choice of a method of isolating cells, the importance of using specific markers of the differentiated type II cell phenotype, and the problems of maintaining these differentiated phenotypic characteristics in tissue culture.
Alpha-skeletal actin messenger RNA increases in acute right ventricular hypertrophy
Authors: Bakerman P. R. , Stenmark K. R. , Fisher J. H.
Published: December 2017

Newborn calves exposed to hypobaric hypoxia develop severe pulmonary hypertension. Right ventricular hypertrophy and failure occur as a consequence of the increased pressure load. Alterations in right ventricular myocyte performance or differentiation could be reflected by the changes in the expression of contractile protein genes. We studied expression of contractile actin isotypes by measuring mRNA levels in total cellular RNA purified from right (RV) and left ventricles (LV) of calves with severe pulmonary hypertension after a 2-wk exposure to hypobaric hypoxia and age-matched controls. alpha-Skeletal actin mRNA was increased greater than 10-fold in the RV of hypertensive animals, whereas alpha-cardiac actin mRNA did not appear to change. alpha-Skeletal actin mRNA and alpha-cardiac actin mRNA did not increase in the LV of any of the hypoxic animals. After a 2-wk hypoxic exposure, calves were removed from the chamber. Two days later, RV alpha-skeletal actin mRNA decreased dramatically but was apparently elevated above that of an age-matched control. Thirty days after hypoxia, there appeared to be a persistent increase in RV alpha-skeletal actin mRNA. Although the physiological significance of these changes are unknown, an alteration in the RV myocyte phenotype has occurred.
Regulation of lung surfactant secretion
Authors: Chander A. , Fisher A. B.
Published: December 2017

Secretion of lung surfactant is the direct step in release of the lipoprotein-like product, synthesized in lung epithelial type II cells, onto the alveolar surface. Release of surfactant phosphatidylcholine (PC) proceeds via formation of surface pores during exocytosis of lamellar bodies. Surfactant secretion is regulated locally in the lung by changes in ventilation rate, possibly mediated by distension and altered intracellular pH. Secretion is also stimulated by various agents, including agonists for beta-adrenergic, purinoceptors, and vasopressin receptors and is associated with increased cytosolic Ca2+, cellular adenosine 3',5'-cyclic monophosphate, and activation of protein kinases. Limited studies suggest that secretion of surfactant protein A may be regulated by both cAMP-dependent and protein kinase C-dependent pathways. The integration of these various mechanisms for the in vivo regulation of surfactant secretion remains largely unexplored. Future research into the mechanisms involved in lamellar body fusion with the plasma membrane, role of protein phosphorylation, transient changes in cAMP and Ca2+, and coordination between the secretion of phospholipid and protein components of surfactant should enhance our understanding of secretion of surfactant “lipoprotein.”
Cellular basis of transduction in carotid chemoreceptors
Authors: Biscoe T. J. , Duchen M. R.
Published: December 2017

Understanding transduction mechanisms is central to much of sensory physiology. The carotid chemoreceptors monitor the PO2 of arterial blood en route to the brain and are powerfully excited when the arterial PO2 falls to less than 60 mmHg. The type I cell is generally believed to be the transducer. These cells release catecholamines in response to agents that excite the receptor (hypoxia, cyanide, K(+)-induced depolarization, etc.). Adherent to the cells are the saucer-shaped nerve endings of the axons of the sinus nerve. We and others have used patch-clamp techniques to study the electrophysiological properties of the type I cells. We have also investigated type I cell chemistry with microfluorometric techniques, to measure intracellular Ca2+ concentration ([Ca2+]i), mitochondrial NADH, and mitochondrial membrane potential (delta psi m). During hypoxia there are graded increases in NADH, [Ca2+]i (which presumably will promote transmitter release), and graded depolarization of delta psi m. These results suggest that the Ca2+ is largely derived from an intracellular store, probably from mitochondria, and that release is entrained to delta psi m. Comparative studies with other cells indicate an increased sensitivity of the mitochondria of type I cells to changes in PO2. The data suggest that the electrophysiological responses of type I cells to hypoxia are not central to the response, although the excitability of the cells may provide a mechanism for the modulation of the response by varying voltage-gated Ca2+ influx.
Vasopressin signal transduction in rat type II pneumocytes
Authors: Brown L. A. , Chen M.
Published: December 2017

Previous studies demonstrate that in cultured type II pneumocytes, [Arg8]-vasopressin (AVP) stimulates surfactant secretion independent of adenosine 3',5'-cyclic monophosphate (cAMP). In the current study AVP stimulated a 50% loss of radioactive phosphatidylinositol 4,5-bisphosphate (PIP2) within 15 s. Consistent with AVP-induced PIP2 hydrolysis was an increased appearance of the two breakdown products 1,2-diacylglycerol (1,2-DAG) and inositol 1,4,5-trisphosphate (IP3). Also, AVP stimulated the appearance of radiolabel in phosphatidic acid (PA) suggesting that the conversion of 1,2-DAG to PA could be used for PIP2 resynthesis. The effects of AVP on PIP2 and IP3 were mimicked by the bioactive AVP fragment and inhibited by the specific AVP1 antagonist. The EC50 for AVP on IP3 production was 6 nM. AVP stimulated protein kinase C (PK-C) activity twofold over the basal activity of 0.74 +/- 0.07 nmol P.min-1.mg protein-1 but did not activate cAMP-dependent protein kinase activity. The AVP1 antagonist inhibited AVP activation of PK-C. Therefore, activation of the AVP1 receptor resulted in PIP2 hydrolysis for signal transduction, PK-C activation, and surfactant secretion.
Relaxation of canine airway smooth muscle by the heparin preservative benzyl alcohol
Authors: Tamaoki J. , Kanemura T. , Horii S. , Chiyotani A. , Sakai N. , Kobayashi K. , Takizawa T.
Published: December 2017

To characterize the actions of heparin and its common preservative benzyl alcohol (BA) on airway smooth muscle functions, we studied the effects of purified heparin, commercial heparin salt solution (HSS) containing heparin and BA, and BA on the contractile responses of canine bronchial segments to various agonists under isometric conditions in vitro. Addition of HSS or the equivalent volume of BA reversibly depressed acetylcholine-induced contraction in a dose-dependent fashion, IC50 values being 5.7 +/- 1.1 (SE) mM of BA, whereas purified heparin had no effect. This depression was not affected by pretreatment of tissues with propranolol, indomethacin or ouabain, or removal of epithelium, and BA at sufficient concentrations to cause muscle relaxation did not alter intracellular adenosine 3',5'-cyclic monophosphate contents. BA also attenuated the contractile responses to electrical field stimulation, histamine, and serotonin, but it was without effect on those to KCl. In addition, the acetylcholine-induced enhancement of hydrolysis of phosphatidylinositol 4,5-biphosphate in the lipid fraction and the resultant production of phosphatidic acid were inhibited in the presence of BA. These results suggest that the heparin preservative BA but not heparin relaxes airway smooth muscle, probably through the decrease in intracellular Ca2+ release by inhibiting agonist-mediated phosphatidylinositol turnover.
Clearance and recycling of pulmonary surfactant
Authors: Wright J. R.
Published: December 2017

In a steady state the rate of secretion of pulmonary surfactant lipids and proteins into the alveolar airspace must be balanced by the rate of removal. Several potential pathways for clearance have been identified including uptake by alveolar type II cells, which also synthesize and secrete surfactant components, uptake by other epithelial cells, and internalization by alveolar macrophages. A small amount of surfactant moves up the airways and through the epithelium-endothelium barrier into the blood. Some of the surfactant lipids and proteins that are cleared from the alveolar airspace appear to be “recycled” in that they appear in the lamellar body, a surfactant secretory granule found in the type II cell. Some surfactant lipids are degraded, probably intracellularly, and the degradation products are reutilized to synthesize new lipids. Several factors have been shown to affect internalization by the type II cell and/or alveolar clearance including the surfactant proteins, lipids, and known stimuli of surfactant secretion. Surfactant proteins may be involved in regulating pool size by modulating both secretion rates and uptake rates, possibly by a receptor-mediated process, although such receptors have not yet been identified or isolated. Clearance of surfactant lipids from the alveolar airspace is more rapid than clearance from the whole lung, and these two processes may be regulated by different factors. Elucidation of the factors that fine tune the balance between synthesis, secretion, and clearance of the lipid and protein components of surfactant awaits further investigation
Protection of alveolar macrophages from hyperoxia by gamma-glutamyl transpeptidase
Authors: Forman H. J. , Skelton D. C.
Published: December 2017

Exposure to hyperoxia causes loss of alveolar macrophage cell function. Toxicity was measured as suppression of the respiratory burst stimulated by phorbol myristate acetate subsequent to exposure (43.5% depression by 2-h exposure to 5 atm absolute O2 vs. controls). The presence of extracellular glutathione significantly protected these cells (7% loss). gamma-Glutamyl transpeptidase, a membrane enzyme with its active site directed outward, was necessary for use of extracellular glutathione. This was demonstrated using the gamma-glutamyl transpeptidase inhibitor, serine-borate complex, which significantly blocked both protection of cells by extracellular glutathione and extracellular glutathione-dependent synthesis of glutathione. The principal use of glutathione in antioxidant defense is as a substrate for glutathione peroxidase. The apparent Km for glutathione of glutathione peroxidase of rat alveolar macrophages was determined to be 2 mM; however, rat alveolar macrophages have approximately 1.3 mM intracellular glutathione, which is insufficient for maximal enzymatic activity. During hyperoxic exposure, this deficit would probably be more significant. Thus the ability of extracellular glutathione along with gamma-glutamyl transpeptidase activity to provide amino acids for de novo glutathione synthesis appears to be a potentially important component of antioxidant defense.
Neural control of goblet cell secretion in guinea pig airways
Authors: Tokuyama K. , Kuo H. P. , Rohde J. A. , Barnes P. J. , Rogers D. F.
Published: December 2017

We studied the neural control of goblet cell secretion in the lower airways of anesthetized guinea pigs using a semiquantitative morphometric technique. The magnitude of discharge of intracellular mucus was determined in histological sections of the trachea and main bronchi stained for mucus glycoproteins. Bilateral electrical stimulation of the cervical vagus nerves induced goblet cell secretion. The magnitude of the effect was dependent on the frequency, voltage, and pulse width of the stimulus, and the duration of stimulation. At 10 Hz, 5 V, and 5 ms for 3 min, there was a 62% decrease in the amount of intracellular mucus below that with sham stimulation. The secretion was blocked either by atropine or by pretreatment with capsaicin but was not significantly inhibited by idazoxan, an alpha-adrenoceptor antagonist. The magnitude of goblet cell discharge in animals pretreated with propranolol was intermediate between that in controls and that with nerve stimulation, although not significant to either. These results demonstrate that goblet cell secretion is under neural control in guinea pig airways and suggest that cholinergic, nonadrenergic-noncholinergic, and possibly adrenergic neural pathways, may contribute to the secretion.
Myosin isoforms in neonatal rat extensor digitorum longus, diaphragm, and soleus muscles
Authors: LaFramboise W. A. , Daood M. J. , Guthrie R. D. , Butler-Browne G. S. , Whalen R. G. , Ontell M.
Published: December 2017

The postnatal elimination of embryonic and neonatal myosin isoforms in the rat extensor digitorum longus, diaphragm, and soleus muscles was compared using electrophoresis and immunohistochemical techniques. Electrophoresis of native myosin showed that neonatal bands were present in all three muscles on day 4 but were absent from the day 21 extensor digitorum longus muscle that exhibited its adult electrophoretic pattern. Mature electrophoretic banding patterns were present on days 60 and 125 in the diaphragm and soleus muscles, respectively. Immunohistochemical analysis indicated that embryonic myosin heavy chain was present in all day 4 samples but absent by day 21. Quantitative evaluation determined that the rate of elimination of neonatal myosin heavy chain (MHCneo) was faster in the extensor digitorum longus muscle than in the diaphragm, with the soleus muscle having the slowest rate of elimination of this isoform. Embryonic myosin light chain was detected by two-dimensional electrophoresis through day 8 in each of the muscles. These data indicate that postnatal elimination of MHCneo is tissue specific and time dependent but not governed by either activity level or rostral-caudal position.
Maturation of acetylcholinesterase expression in tracheal smooth muscle contraction
Authors: Mitchell R. W. , Murphy T. M. , Kelly E. , Leff A. R.
Published: December 2017

We examined postganglionic development of acetylcholinesterase (AChase) activity and tracheal smooth muscle (TSM) contraction elicited by cholinomimetic activation and electrical field depolarization in vitro. Epithelium-intact tracheal strips excised from 21 2-wk-old swine (2ws) and 19 10-wk-old swine (10ws) were tethered isometrically at optimal resting length, and responses were expressed as percent of the maximum to 63 mM potassium-chloride (%KCl). Cumulative concentration-response curves to KCl were equivalent for TSM from 2 and 10ws. However, maximal contraction to ACh in 2ws (168 +/- 8.4 %KCl) was greater than for 10ws (142 +/- 2.3 %KCl; P less than 0.02). Stimulus-response curves (field electrodes; AC source) demonstrated greater sensitivity for TSM in 10ws (stimulus causing 50% of the maximal response = 3.32 +/- 0.13 V in 2ws vs. 2.25 +/- 0.12 V in 10ws; P less than 0.001), indicating that the greater cholinomimetic responsiveness of 2ws did not result from augmented presynaptic nerve conduction. The AChase inhibitor, physostigmine, caused 1) greater sensitivity of responses elicited by electrical field stimulation in 2ws (P less than 0.05) but not in 10ws (P = NS), 2) augmentation of maximal responses to exogenous ACh in 10ws (27% increase; P less than 0.01) but not 2ws (2% increase; P = NS), and 3) a greater increase in sensitivity to cholinomimetic activation in 2ws compared with 10ws (P less than 0.02). These data demonstrate increased cholinergic contraction of TSM in 2 vs. 10ws that results at least in part from reduced AChase activity in the trachea of immature animals.
Cellular LTB4 production differs in allergic sheep with and without late airway responses
Authors: Chapman G. A. , Signoretti F. , Lauredo I. T. , Torrealba P. , Sielczak M. W. , Ahmed A. , Abraham W. M.
Published: December 2017

We tested the hypothesis that allergic sheep that develop both early and late airway responses to inhaled Ascaris suum antigen (late responders) have an increased capacity to generate leukotrienes (LTs) compared with allergic sheep that show only early responses to inhaled antigen (acute responders). To test this hypothesis, we measured LTB4 production, in vitro, by granulocytes isolated from peripheral blood and by macrophages isolated from bronchoalveolar lavage (BAL) from both groups of sheep greater than or equal to 2 wk after the animal's last antigen challenge; LTB4 production by granulocytes isolated from BAL from both groups of sheep 6 and 48 h after local airway challenge with A. suum antigen was also measured. LTB4 production was induced by incubating cells (i.e., either granulocytes or macrophages) with calcium ionophore (A23187, 2 microM) and arachidonic acid (30 microM). LTB4 production was quantitated by high-performance liquid chromatography and verified by radioimmunoassay (RIA). On stimulation peripheral blood granulocytes from late responders (n = 7) produced (means +/- SD/10(6) cells) 13.3 +/- 5.2 ng LTB4 compared with 5.3 +/- 1.5 ng LTB4 (P less than 0.05) for acute responders (n = 7). This increased LTB4 production did not result from variations in granulocyte differential or cyclooxygenase activity (as indicated by RIA measurements of prostaglandin E2 production).(ABSTRACT TRUNCATED AT 250 WORDS)
Vasoactive intestinal peptide prevents lung injury due to xanthine/xanthine oxidase
Authors: Berisha H. , Foda H. , Sakakibara H. , Trotz M. , Pakbaz H. , Said S. I.
Published: December 2017

Reactive oxygen species mediate injury and inflammation in many tissues. The addition of xanthine and xanthine oxidase to perfused rat lungs led to increases in peak airway pressure and perfusion pressure, pulmonary edema, and increased protein content in bronchoalveolar lavage fluid. Treatment with 1-10 micrograms.kg-1.min-1 of vasoactive intestinal peptide (VIP), a widely distributed neuropeptide, markedly reduced or totally prevented all signs of injury. Simultaneously, VIP also diminished or abolished the associated generation of arachidonate products. Similar protection was provided by catalase (100 micrograms/ml) but not by the VIP-related peptides secretin or glucagon. The pulmonary vasodilator papaverine (0.15 mg/ml) was also ineffective. Injured lungs that were not treated with VIP released large amounts of this peptide in the perfusate. The results indicate that VIP has potent protective activity against injury triggered by xanthine/xanthine oxidase and may be a physiological modulator of inflammatory tissue damage associated with toxic oxygen metabolites.
Bleomycin and cyclophosphamide increase pulmonary type IV procollagen mRNA in mice
Authors: Hoyt D. G. , Lazo J. S.
Published: December 2017

Constant 7-day subcutaneous infusion of bleomycin (100 mg/kg) induces pulmonary fibrosis in C57Bl/6N mice, whereas BALB/cN mice are relatively resistant. In contrast, cyclophosphamide (200 mg/kg, ip) induces fibrosis in BALB/cN mice, whereas C57Bl/6N mice are resistant. The effect of these drugs on the pulmonary levels of mRNA encoding the major basement membrane components, laminin and type IV collagen, relative to poly (A+)RNA was determined in both C57Bl/6N and BALB/cN mice. In the sensitive C57Bl/6N mice, bleomycin increased alpha 1IV and alpha 2IV procollagen mRNA/poly (A+)RNA twofold in the absence of increases in laminin A, B1, and B2 mRNA/poly (A+)RNA. In the relatively resistant BALB/cN mice, bleomycin did not alter alpha 1IV procollagen mRNA/poly (A+)RNA and only transiently increased laminin A, B1, B2, and alpha 2IV procollagen mRNA/poly (A+)RNA. Similarly, cyclophosphamide increased alpha 1IV and alpha 2IV procollagen mRNA/poly (A+)RNA twofold in the sensitive BALB/cN mice and not in C57Bl/6N mice. Laminin mRNAs/poly (A+)RNA were not increased by cyclophosphamide in either strain. Thus, in these models, pulmonary fibrosis is preceded by a coordinate increase in steady-state levels of mRNA encoding basement membrane procollagen but is not associated with an increase in laminin gene expression
Increased myosin phosphorylation in sensitized canine tracheal smooth muscle
Authors: Kong S. K. , Halayko A. J. , Stephens N. L.
Published: December 2017

We have reported that the maximal velocity of shortening and myofibrillar adenosine triphosphatase (ATPase) activity of antigen-sensitized airway smooth muscle are higher than that of nonsensitized airway smooth muscle (Kong, S. K., R. P. C. Shiu, and N. L. Stephens. J. Appl. Physiol. 60: 92–94, 1986). To extend these studies, we attempted to determine whether the increased myofibrillar ATPase activity from sensitized airway smooth muscle was associated with either a change in distribution of two myosin heavy chain isozymes or an increase in myosin light chain phosphorylation. Myosin heavy chain isozymes from both control and sensitized airway smooth muscle were separated by 4% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gels were analyzed by densitometry, which indicated that isozyme band pattern of sensitized airway smooth muscle was not different from that of the control. The maximal levels of phosphorylated myosin light chain from whole cell homogenates of sensitized and control tracheal smooth muscles were 0.65 +/- 0.029 (n = 6) and 0.40 +/- 0.025 mol Pi/mol light chain (n = 6), respectively. The degree of phosphorylation of myosin light chain of sensitized airway smooth muscle was significantly higher than that of the control (P less than 0.05). This study also indicated that increased myofibrillar ATPase activity in sensitized tracheal smooth muscle was correlated with phosphorylation of myosin light chain.
Calcium and alpha-adrenergic regulation of Na-Cl(K) cotransport in rabbit tracheal epithelial cells
Authors: Liedtke C. M.
Published: December 2017

The marked sensitivity of Cl and fluid secretion in mammalian airways to basolateral application of loop diuretics has led to postulates that electrically neutral Na-Cl entry plays a critical role during secretion. Electrically neutral Na-Cl(K) cotransport was investigated by determining the initial rate of 22Na and 36Cl efflux in epithelial cells isolated from rabbit trachea and preequilibrated with radioactive tracer at 25 degrees C. Tracer transport was initiated by 10-fold dilution of an aliquot of cells in radioisotope-free medium. The initial rate of radiolabeled ion transport was calculated from the linear portion of efflux curves. Base-line Na and Cl transport rates were not affected by furosemide or bumetanide. l-Epinephrine stimulated Na and Cl transport rates 1.8-fold each in Ca2(+)-replete and 2.6- and 2.3-fold, respectively, in Ca2(+)-deficient transport medium. Loop diuretics and yohimbine, an alpha 2-adrenergic antagonist, blocked the effects of l-epinephrine, and, clonidine, an alpha 2-adrenergic agonist, stimulated yohimbine- and furosemide-sensitive Cl transport. The beta-adrenergic agonist l-isoproterenol alone did not affect tracer transport and, in combination with clonidine, did not affect the response to clonidine. Elevation of intracellular Ca2+ with ionomycin stimulated tracer transport, and buffering of intracellular Ca2+ with 1,2-bis(aminophenoxy)ethane- N,N,N',N'-tetraacetic acid blocked the stimulatory effects of alpha-adrenergic agents. These results indicate an alpha 2-adrenergic stimulation of loop diuretic-sensitive Na and Cl transport that requires elevated intracellular Ca2+ as the second messenger. The transport mechanism is probably a Na-Cl or Na-K-2Cl cotransport located in the basolateral membrane.
Pathobiology of pulmonary fibrosis
Authors: Crouch E.
Published: December 2017

Pulmonary fibrosis is characterized by an increase in lung matrix and alterations in the numbers and spatial relationships of lung parenchymal cells. The increase in matrix results from a proliferation and “activation” of fibroblasts (FB) with increased production and deposition of matrix macromolecules at sites of lung injury. Connective tissue cell activation is associated with increased gene expression of collagens, fibronectin, proteoglycans and other matrix components; cytoskeletal alterations; and probably also with changes in the expression of matrix receptors and matrix-degrading enzymes and inhibitors. The fibroproliferative reaction involves the participation of a variety of cytokines and inflammatory mediators by resident and inflammatory cells at sites of lung injury. Thickening of the alveolar wall can result secondary to matrix deposition within the interstitium and as a result of “mural incorporation” of organized airspace exudate. However, marked structural remodeling of the gas-exchange tissues, with the development of honeycomb lung, involves airspace fibrosis and alveolar collapse. The latter processes lead to areas of airspace obliteration secondary to airspace filling, and to fibrous adhesion of collapsed septa. The extent of airspace obliteration is determined largely by the severity or extent of epithelial injury. Although lung fibrosis is usually irreversible, the activated state is reversible after clearance of exudate and reepithelialization. A continuing and seemingly autonomous fibroproliferative reaction can result in the face of ongoing injury and delayed repair.
Uptake of palmitic acid by rabbit alveolar type II cells
Authors: Maniscalco W. M. , Stremmel W. , Heeney-Campbell M.
Published: December 2017

Alveolar type II cells require a source of palmitic acid for synthesis of dipalmitoyl phosphatidylcholine (DPPC), a major constituent of pulmonary surfactant. Previous studies indicated that maximal rates of DPPC synthesis are achieved only if exogenous palmitate is available to the type II cell. Little is known of the mechanisms by which fatty acids enter type II cells. To determine if uptake is mediated by a membrane carrier system, as described in other cell types, we examined the kinetics of palmitate uptake. Using freshly isolated rabbit type II cells, we demonstrated that radiolabeled palmitate uptake was maximal and linear for 45 s; after 1 min the apparent rate of uptake declined. The initial uptake phase was taken as a measure of cellular fatty acid influx because intracellular radiolabeled palmitate remained 80% nonesterified at this time but was 55% esterified by 2 min. Cellular influx of palmitate showed saturation kinetics with increasing concentration of nonalbumin bound palmitate. Michaelis constant was 52.6 nM, and maximum velocity was 152 pmol.10(6) cells-1.min-1. The hypothesis that saturable cellular influx of palmitate is likely linked to the previously identified membrane fatty acid binding protein (MFABP) was supported by Western-blot analysis of rat lung tissue with an antibody to MFABP that demonstrated the presence of this carrier protein in lung tissue. These data suggest that palmitate uptake by type II cells is saturable and may be mediated by a membrane-associated carrier as described in other cell types.
Aldosterone regulation of basolateral potassium channels in alveolar epithelium
Authors: Illek B. , Fischer H. , Clauss W.
Published: December 2017

To reveal the regulatory mechanism of the mineralocorticoid aldosterone on basolateral K+ channels, the aldosterone-sensitive lung epithelium of Xenopus laevis was investigated in Ussing chambers under voltage-clamp conditions. Transepithelial measurements were supplemented by current fluctuation analysis of short-circuit current noise in nonstimulated and aldosterone-stimulated lung tissues. The addition of 10(-6) M aldosterone stimulated short-circuit current from 11.3 +/- 2.0 to 27.8 +/- 4.8 microA/cm2 (n = 11) within 4–5 h. In the presence of an alveolar-to-pleural K+ gradient, transepithelial K+ currents were induced by permeabilizing the apical membrane with the pore-forming antibiotic amphotericin B. When the local anesthetic lidocaine (25-1,000 microM) was added to the pleural solution, macroscopic K+ current was dose dependently depressed. Lidocaine induced a Lorentzian component in the power density spectra, and the corner frequency increased linearly with blocker concentration. Aldosterone treatment did not affect mean single K+ channel current, which was 1.5 +/- 0.12 pA corresponding to a 15-pS channel conductance, whereas the number of basolateral K+ channels doubled. We conclude that the basolateral K+ channels in alveolar epithelia are a target site of aldosterone action.
Lung fibroblasts from animals breathing 100% oxygen produce growth factors for alveolar type II cells
Authors: Everett M. M. , King R. J. , Jones M. B. , Martin H. M.
Published: December 2017

Type II cells were isolated from rats with a purity of 80–95% with less than 4% macrophages. These cells, after plating for approximately 16 h, were cultured with 50% RPMI 1640 and 50% (vol/vol) conditioned medium obtained from confluent hamster lung fibroblasts, together with 0.1% fetal calf serum (FCS). Conditioned media were obtained from either fibroblasts derived from normal hamsters breathing room air [normoxic-conditioned medium (NCM)] or from hamsters exposed for 4 days to 100% O2 [hyperoxic-conditioned medium (HCM)]. Controls consisted of 100% minimal essential medium (MEM) containing 0.1% FCS. Over a 96-h culture period, NCM stabilized cell populations but was unable to induce proliferation. In contrast, at low cell densities, HCM could cause a two- to threefold increase in type II cell number within 24–48 h after introduction. This effect could not be demonstrated at high cell densities. When tested with FCS concentrations ranging from 0 to 10%, maximum effects were obtained using 0.1–0.2% FCS. We conclude that lung fibroblasts from oxidant-injured hamsters produce growth factors that can stimulate at least one mitotic division in cultured type II cells, which are plated at low density. These factors are absent, or present in much lower concentration, in lung fibroblasts from normal animals.
Pre- and postjunctional muscarinic receptors in canine bronchi
Authors: Janssen L. J. , Daniel E. E.
Published: December 2017

Cholinergic receptors on canine bronchial smooth muscle (mediating excitation) or on the cholinergic nerve endings (modulating acetylcholine release) have not yet been characterized. In this study, the effects of agents selective for the various muscarinic receptors on mechanical and electrical responses in isolated segments of canine bronchial smooth muscle (3rd-6th order) were investigated. Carbachol (Cch), bethanechol (Bch), oxotremorine (Oxo), and McNeil A343 (McN) produced membrane depolarization and contractions with a ranked potency order of Oxo approximately Cch greater than Bch greater than McN. Oxo-induced contractions were antagonized by pirenzepine (Pir), AF-DX 116 (AF-DX), or hexahydrosiladifenidol (Hexa); Schild analysis yielded pA2 values of 6.61, 6.81, and 7.40, respectively. Contractions induced by field stimulation (FS) were antagonized by Pir, AF-DX, or Hexa with a potency order of Hexa greater than AF-DX approximately Pir. FS responses were potentiated by lower (i.e., M1-selective) concentrations of Pir, and antagonized by McN. The non-M1 agonists and antagonists had no such effects. These findings indicate that the postjunctional receptors are of the M3 subtype, and that the inhibitory receptors on the cholinergic nerve endings are of the M1 subtype.
Mechanisms of lung polyamine accumulation in chronic hypoxic pulmonary hypertension
Authors: Shiao R. T. , Kostenbauder H. B. , Olson J. W. , Gillespie M. N.
Published: December 2017

Chronic hypoxia causes polyamine-dependent hypertensive pulmonary vascular remodeling (J. E. Atkinson. J. W. Olson, R. J. Altierre, and M. N. Gillespie, J. Appl. Physiol. 62: 1562–1568, 1987), but mechanisms by which lung polyamine contents are elevated have not been established. This study measured polyamine contents, biosynthetic activities, and transport in lungs of rats exposed to hypobaric hypoxia (simulated altitude: 4,570 m) for 4–14 days. Hypoxia increased lung contents of spermidine and spermine within 40 h and of putrescine within 4 days. These changes preceded hypoxia-induced increases in pulmonary arterial pressure and development of right ventricular hypertrophy. Additional experiments determined whether increased lung polyamine contents could be ascribed to elevated activity of ornithine decarboxylase (ODC), the rate-limiting enzyme in conversion of ornithine to putrescine. Lung ODC activity did not differ from controls at 40 h posthypoxia and was reduced below control levels from 4–14 days of exposure. Putrescine transport kinetics were assessed in isolated, salt solution-perfused lungs. Apparent Km for putrescine uptake was increased from 10.4 microM in control lungs to 16.9 microM in lungs from rats maintained for 7 days in an hypoxic environment. Maximal velocity (Vmax) of lung putrescine transport was increased from 1.67 nmol.g-1.min-1 in controls to 2.65 in hypoxic lungs. Putrescine efflux also was altered by hypoxic exposure; T1/2 for loss of diamine from a slowly effluxing pool was increased from 60.6 min in controls to 91.5 min in hypoxic lungs.(ABSTRACT TRUNCATED AT 250 WORDS)
A novel epithelial cell from neonatal rat lung: isolation and differentiated phenotype
Authors: Roberts P. E. , Phillips D. M. , Mather J. P.
Published: December 2017

A novel epithelial cell from normal neonatal rat lung has been isolated, established, and maintained for multiple passages in the absence of serum, without undergoing crisis or senescence. By careful manipulation of the nutrition/hormonal microenvironment, we have been able to select, from a heterogeneous population, a single epithelial cell type that can maintain highly differentiated features in vitro. This cell type has characteristics of bronchiolar epithelial cells. A clonal line, RL-65, has been selected and observed for greater than 2 yr in continuous culture. It has been characterized by ultrastructural, morphological, and biochemical criteria. The basal medium for this cell line is Ham's F12/Dulbecco's modified Eagle's (DME) medium plus insulin (1 micrograms/ml), human transferrin (10 micrograms/ml), ethanolamine (10(-4) M), phosphoethanolamine (10(-4) M), selenium (2.5 x 10(-8) M), hydrocortisone (2.5 x 10(-7) M), and forskolin (5 microM). The addition of 150 micrograms/ml of bovine pituitary extract to the defined basal medium stimulates a greater than 10-fold increase in cell number and a 50- to 100-fold increase in thymidine incorporation. The addition of retinoic acid results in further enhancement of cell growth and complete inhibition of keratinization. We have demonstrated a strategy that may be applicable to isolating other cell types from the lung and maintaining their differentiated characteristics for long-term culture in vitro. Such a culture system promises to be a useful model in which to study cellular events associated with differentiation and proliferation in the lung and to better understand the molecular mechanisms involved in these events.
Lung injury in Fischer but not Sprague-Dawley rats after short-term hyperoxia
Authors: He L. S. , Chang S. W. , Ortiz de Montellano P. , Burke T. J. , Voelkel N. F.
Published: December 2017

The Fischer rat is known for its susceptibility to develop liver necrosis when challenged with paraquat (Smith et al., J. Pharmacol. Exp. Ther. 235: 172-177, 1985). We postulated that other organs, specifically the lung, may also be more susceptible to injury and examined whether lungs from Fischer (F) rats were injured more easily when challenged with active oxygen species than Sprague-Dawley (SD) rat lungs. We aimed to investigate whether increased susceptibility to oxidant injury was related to differences in lung antioxidant defenses. Perfused lungs from both rat strains were challenged by addition of H2O2 to the perfusate or by short-term hyperoxic ventilation. To assess nonoxidant modes of lung injury, we examined lung responses after exposure to protamine sulfate or neutrophil elastase. Intravascular H2O2 or 3 h in vitro hyperoxia caused lung edema in F but not SD rats, and elastase injured F rat lungs more than the lungs from SD rats. Protamine, however, injured the lungs from both strains to a similar degree. Catalase, but not superoxide dismutase or allopurinol, protected F rat lungs against edema, resulting from 3 h in vitro hyperoxia. The lung homogenate levels for reduced glutathione or conjugated dienes and the activities of lung tissue catalase, glutathione peroxidase, and cytochrome P-450 were not different between the two strains. Lung tissue ATP levels, however, were lower in F than in SD rats. Although the F rat strain appears to have an altered oxidant-antioxidant defense balance, the exact cause of the greater susceptibility to oxidant stress of the F rat strain remains elusive.