bluuebunny/crossref_metadata_2025 huggingface dataset:
ETA and ETB receptors on single smooth muscle cells cooperate in mediating guinea pig tracheal contraction
Authors: Inui T. , James A. F. , Fujitani Y. , Takimoto M. , Okada T. , Yamamura T. , Urade Y.
Published: December 2017

We investigated the distribution of endothelin A (ETA) and ETB receptors in single smooth muscle cells and their contribution to ET-induced contractions of guinea pig trachea. ETA and ETB receptors were detected in smooth muscle membranes (maximum binding capacities of 810 and 360 fmol/mg protein and dissociation constants of 38 and 5.1 pM for 125I-labeled ET-1 and 125I-ET-3, respectively) and visualized autoradiographically in primary cultured cells. ET-1 and ET-3 evoked concentration-dependent increases in intracellular Ca2+ concentration and smooth muscle tension. The half-maximally effective concentrations of ET-1 and ET-3 at inducing contractions were 1.9 and 2.7 nM, respectively. The Ca2+ responses showed tachyphylaxis to both ETs after stimulation with ET-1, but only to ET-3 after stimulation with ET-3. Consecutive applications of ET-3 and ET-1 (10 nM each) classified the cells into ETA dominant (approximately 30%) responding to only ET-1, ETB dominant (approximately 20%) responding to only ET-3, and ETA- and ETB-possessing (approximately 50%) cells responding to both. The ETA antagonist, 10 microM BQ-123, attenuated ET-1-induced contractions but did not affect the ET-3-induced contractions. The results indicate that both receptors coexist in a major population of smooth muscle cells and cooperate in mediating ET-1-induced contractions.
Polarized distribution of Na(+)-H+ antiport activity in rat alveolar epithelial cells
Authors: Lubman R. L. , Crandall E. D.
Published: December 2017

In this study, we investigated the polarized distribution of Na(+)-H+ antiport activity in alveolar epithelial cell monolayers. Rat alveolar type II pneumocytes were grown on detachable tissue culture-treated Nuclepore filters. The membrane filters, with their adherent intact alveolar epithelial cell monolayers, were mounted in a cuvette designed to contain two fluid compartments separated by the monolayer. Cells were loaded with the pH-sensitive dye 2',7'-biscarboxyethyl-5,6-carboxylfluorescein and intracellular pH (pHi) measured spectrofluorometrically. Monolayers were studied at ambient temperature on days 3–4 in culture, coincident with the development of high tissue resistance (RT > or = 2000 omega.cm2). Cells were incubated in HCO(3-)-free Na+ buffer [(in mM) 140 NaCl, 6 HEPES, pH 7.4] and acidified by NH3 prepulse. Rates of realkalinization (JH+) were calculated as the product of the initial rate of recovery (dpHi/dt) and the intracellular buffer capacity (beta i). Under control conditions, recovery occurred with an initial JH+ of 28.4 mM/min. When 100 microM dimethylamiloride (DMA), an amiloride analogue with enhanced specificity for inhibiting the Na(+)-H+ antiporter, was present in the basolateral fluid, recovery was inhibited by > 90%. Conversely, when the monolayers were acidified in Na+ buffer containing DMA (100 microM) in the apical fluid, acidification and recovery were identical to control. Recovery from acidification was inhibited by basolateral DMA with a one-half maximal inhibitory concentration (IC50) of 100 nm and by basolateral amiloride with an IC50 of 10 microns. Recovery was completely inhibited by omission of Na+ from the basolateral fluid, but omission of Na+ from apical fluid had no effect. We conclude that Na(+)-H+ antiport activity is located exclusively on the basolateral surface of these alveolar epithelial cell monolayers, where it most likely represents the high-amiloride affinity isoform of the Na(+)-H+ antiporter, NHE-1. The Na(+)-H+ antiporter, asymmetrically distributed to the basolateral surface of the polarized alveolar epithelium, contributes to intracellular homeostasis in alveolar pneumocytes and may also play a role in signal transduction in these cells.
Serotonin increases DNA synthesis in rat proximal and distal pulmonary vascular smooth muscle cells in culture
Authors: Pitt B. R. , Weng W. , Steve A. R. , Blakely R. D. , Reynolds I. , Davies P.
Published: December 2017

Cultured smooth muscle cells obtained from rat lung periphery (RPC) and proximal pulmonary artery (RSMC) expressed mRNA for serotonin (5-HT) type 2 receptor (5-HT2) and 5-HT transporter (by Northern blot analysis). Functional expression of these genes was evident since both cell types 1) bound 125I-labeled lysergic acid diethylamide (LSD; 5-HT2 receptor antagonist) that was equally effectively displaced by either ketanserin or mianserin; and 2) transported 5-[3H]HT in an imipramine-sensitive manner. Serotonin (10(-9)-10(-5) M) stimulated DNA synthesis (as measured by [3H]thymidine uptake) in RPC and RSMC. The 5-HT-induced increase in DNA synthesis was significantly inhibited in both cell types by the 5-HT2 receptor antagonist, ketanserin (10(-7)-10(-6) M), and by fluoxetine (10(-6) M), a putative 5-HT transport inhibitor. Acute exposure to 5-HT (1–100 microM) caused an abrupt rise in intracellular calcium ([Ca2+]i) in single pulmonary vascular smooth muscle cells as microspectrofluorometrically determined using the calcium-sensitive dye, fura 2. The 5-HT-induced change in [Ca2+]i was completely abolished in the presence of 10(-6) M ketanserin as well as imipramine or fluoxetine (10(-6) M). The calcium transients due to 5-HT persisted in a Na(+)-free condition (in which the transporter activity was completely abolished) and imipramine and fluoxetine (and ketanserin) were effective inhibitors of 5-HT under these conditions. Therefore, the 5-HT2 receptor, but not the transporter, is responsible for initiating the acute effects (e.g., calcium transients) of 5-HT in cultured rat pulmonary vascular smooth muscle cells and fluoxetine (1 microM) may have 5-HT2-receptor antagonist properties.(ABSTRACT TRUNCATED AT 250 WORDS)
Hypoxic vasoconstriction in rat pulmonary and mesenteric arteries
Authors: Leach R. M. , Robertson T. P. , Twort C. H. , Ward J. P.
Published: December 2017

Hypoxic vasoconstriction was investigated in isolated pulmonary and mesenteric arteries of the rat. Experiments were performed on large (approximately 2 mm pulmonary, approximately 0.8 mm mesenteric) and small (100-350 microns) arteries. Hypoxia [oxygen partial pressure (PO2) approximately 33 mmHg] elicited a biphasic response in arteries precontracted with prostaglandin F2 alpha (10 microM). A transient contraction reaching a peak within 2-3 min was observed in both large and small pulmonary and mesenteric arteries (phase 1). In pulmonary arteries, this was followed by a slowly developing contraction over 45 min (phase 2). In mesenteric arteries, there was no phase 2 but instead a profound relaxation. Mechanical disruption of the endothelium had no significant effect on phase 1 in preconstricted large pulmonary arteries but reduced phase 1 in small arteries by 40%. Phase 2 was abolished in both large and small arteries. Inhibition of endothelium-derived relaxing factor synthesis or cyclooxygenase pathways had no effect on either phase. Verapamil substantially reduced phase 1 but abolished phase 2. In conclusion, we have found a clear biphasic response to hypoxia in pulmonary arteries of the rat, but, in contrast to some previous reports, phase 1 was only partially dependent on the endothelium, whereas phase 2 was entirely dependent on the endothelium. Small and large arteries had qualitatively similar responses. These results are consistent with the involvement of at least two mechanisms for hypoxic vasoconstriction, one of which may involve release of an as yet unidentified endothelium-derived constrictor factor.
Stimulation of lung surfactant secretion by endothelin-1 from rat alveolar type II cells
Authors: Sen N. , Grunstein M. M. , Chander A.
Published: December 2017

Endothelin-1 (ET-1), a potent vasoactive peptide released by endothelial cells, alters cytosolic Ca2+ and phosphoinositide metabolism in cells of myogenic and nonmyogenic origin. We evaluated the effect of ET-1 on surfactant phosphatidylcholine (PC) secretion from alveolar type II cells labeled with [methyl-3H]choline. ET-1 stimulated the secretion of PC in a time- and dose-dependent manner. Binding of 125I-labeled ET-1 to type II cell membranes was saturable at approximately 1 nM and suggested the presence of a single type of receptor. The secretagogue effect of ET-1 was independently inhibited by nifedipine and nitrendipine, both L-type calcium channel blockers, and removal of extracellular calcium. ET-1 also increased cellular diacylglycerol content by approximately 50% within 30 s, which could not be attenuated by pretreatment with nifedipine, suggesting an early activation of protein kinase C that was independent of Ca2+ influx. Further, ET-1-stimulated PC secretion was also blocked by inhibitors of protein kinase C. Collectively, these results indicate that the binding of ET-1 to type II cells is coupled to the activation of protein kinase C, which increases calcium influx through L-type calcium channels, and results in increased secretion of lung surfactant. Since ET-1 is released from pulmonary micro- and macrovasculature endothelial cells in close proximity to type II cells, our findings support the novel concept that endothelial cells interact with alveolar type II cells in a paracrine fashion to regulate surfactant secretion.
Characterization of mRNA transcripts and organization of human SP-A1 and SP-A2 genes
Authors: McCormick S. M. , Boggaram V. , Mendelson C. R.
Published: December 2017

In the present study, we have characterized the mRNA transcripts and intron-exon organization of the human surfactant protein (SP)A1 and SP-A2 genes. By primer extension analysis of mRNA isolated from human fetal lung explants using an oligonucleotide primer to exon II (as delineated in the SP-A1 gene), a minimum of nine primer extended transcripts was observed. Rapid amplification of cDNA ends was used to amplify the primer extended transcripts for sequence analysis. Sequence analysis of 47 full-length primer extended cDNAs and comparison with the sequences of the genes encoding SP-A1 and SP-A2 revealed four different classes of transcripts of the SP-A2 gene and five different classes of transcripts of the gene encoding SP-A1. A major difference between SP-A2 and SP-A1 mRNA transcripts is that SP-A2 transcripts are always comprised of sequences contained within six exons; the extra exon in SP-A2 (exon II of VI) encodes additional 5'-untranslated sequence and is located between exons I and II of SP-A1. By contrast, the majority of transcripts of the SP-A1 gene are comprised of sequences contained within five exons. In the cases of both SP-A1 and SP-A2 genes, a small proportion of the mRNA transcripts contain sequences present in alternate exons. In addition, the majority of the SP-A1 mRNA transcripts are initiated 5 bp downstream of the transcription initiation site of SP-A2. In our companion paper [McCormick and Mendelson. Am. J. Physiol. 266 (Lung Cell. Mol. Physiol. 10): L367-L374, 1994], we report that the SP-A1 and SP-A2 genes are differentially regulated during development and by adenosine 3',5'-cyclic monophosphate and glucocorticoids in human fetal lung in culture.
Cytokine-cytokine synergy and protein kinase C in the regulation of lung fibroblast leukemia inhibitory factor
Authors: Elias J. A. , Zheng T. , Whiting N. L. , Marcovici A. , Trow T. K.
Published: December 2017

Studies were undertaken to characterize the cytokines and cytokine-cytokine interactions that stimulate human lung fibroblast leukemia inhibitory factor (LIF) production and the mechanisms of these regulatory effects were investigated. Unstimulated fibroblasts did not produce significant amounts of LIF, whereas recombinant interleukin-1 alpha (rIL-1 alpha), transforming growth factor-beta (TGF-beta), and recombinant tumor necrosis factor (rTNF) were dose-dependent stimulators of LIF production. TGF-beta and rIL-1 alpha also interacted in a synergistic fashion to further increase LIF elaboration. Under all conditions alterations in LIF production were associated with comparable alterations in LIF mRNA accumulation. The kinetics of mRNA induction, however, differed with rIL-1-induced LIF mRNA being readily detected after 2 h, TGF-beta 1 induction peaking after 16-24 h, and the induction caused by rIL-1 alpha plus TGF-beta 1 being most prominent after 2-4 h and decreasing with additional incubation. Protein synthesis was not required for LIF induction. In addition, even though A23187 was an effective stimulator of LIF production, the calmodulin antagonists W-7 and trifluoperazone dichoride (TFP) did not significantly alter the LIF-stimulatory effects of IL-1 and TGF-beta. PKC did appear to play an important role in this induction, however, since LIF was induced by PMA and cytokine induction of LIF production was markedly diminished by chronic phorbol ester preincubation, staurosporine, and H-7, but not by HA1004. These studies demonstrate that 1) rIL-1, TGF-beta, TNF, agents that increase intracellular calcium and agents that activate PKC, stimulate lung fibroblast LIF production; 2) rIL-1 and TGF-beta interact in a synergistic fashion to further increase fibroblast LIF production; and 3) rIL-1 and TGF-beta stimulate lung fibroblast LIF production via a pretranslational activation pathway that is largely PKC-dependent and protein synthesis-, cyclic nucleotide-, and calmodulin-independent. Cytokine-stimulated LIF production may play an important role in homeostasis and repair in the human lung.
Contraction and depolarization induced by fetal bovine serum in airway smooth muscle
Authors: Abdullah N. A. , Hirata M. , Matsumoto K. , Aizawa H. , Inoue R. , Hamano S. , Ikeda S. , Xie Z. , Hara N. , Ito Y.
Published: December 2017

We investigated the effects of fetal bovine serum (FBS) on the resting membrane potential and muscle tone of canine airway smooth muscles using tension recording and microelectrode methods. At concentrations < 0.1%, FBS induced a sustained increase in muscle tone. At concentrations < 1%, FBS depolarized the resting membrane potential of dog trachea in a dose-dependent manner. When FBS was dialyzed, it failed to induce a sustained increase in muscle tone or to depolarize the membrane, indicating that FBS contained factor(s) that induced contraction of dog airway smooth muscles. When FBS was dialyzed against distilled water, the outer solution which was freeze dried and then reconstituted (< 1% in original vol) induced a sustained increase in muscle tone, indicating that FBS contained dialyzable factor(s) that increased muscle tone and depolarized the resting membrane potential of dog airway smooth muscles. Methysergide (10(-6) M) or cyproheptadine (10(-6) M), nonspecific antagonists to serotonin receptors, 5-hydroxytryptamines (5-HT1 and 5-HT2), markedly reduced the FBS-induced increase in the muscle tone to approximately 20% of the original value but did not abolish the response. On the other hand, methysergide (10(-6) M) completely suppressed the increase in the muscle tone evoked by peak fractions obtained by application of the dialysate of FBS or 5-HT to a C18 reverse-phase column, indicating the peak fractions contained only 5-HT. These observations indicate that FBS contained 5-HT and an unknown factor(s) responsible for increase in the muscle tone of the airway smooth muscle induced by FBS.
Identification of PDE isozymes in human pulmonary artery and effect of selective PDE inhibitors
Authors: Rabe K. F. , Tenor H. , Dent G. , Schudt C. , Nakashima M. , Magnussen H.
Published: December 2017

The effects of the nonselective phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) and the selective PDE inhibitors motapizone (type III), rolipram (type IV), zardaverine (type III/IV), and zaprinast (type V and I) on prostaglandin F2 alpha (PFG2 alpha)-induced tone in human pulmonary arteries was investigated. Relaxation was achieved by IBMX [concentration eliciting 50% of maximum response (EC50): 11.3 microM, n = 10], motapizone (EC50:3.0 microM, n = 7), zardaverine (EC50: 3.2 microM, n = 9), and zaprinast (EC50: 31.8 microM, n = 6), whereas rolipram was almost ineffective. The combination of motapizone and zaprinast (10 microM) was the most effective relaxant with supra-additive relaxation and a motapizone EC50 of 575 nM. Biochemical studies revealed the presence of the PDE isozymes I, III, IV and V in the cytosolic and particulate phases of arterial homogenates; PDE II was not detectable. Partial inhibition of adenosine 3',5'-cyclic monophosphate (cAMP)-hydrolyzing PDE activity was achieved with rolipram (26 +/- 2.2%) or motapizone (60 +/- 5.4%), whereas there was almost complete inhibition of total PDE activity with zardaverine (81 +/- 2.0%) or the combination of motapizone and rolipram (82 +/- 2.3%). Inhibition of guanosine 3',5'-cyclic monophosphate (cGMP)-hydrolyzing PDE activity was achieved with zaprinast (62 +/- 2.6%) and motapizone (13 +/- 2.3%), indicating the cGMP-hydrolyzing activity of PDE III. We conclude that four out of the five recognized PDE isozyme families are present in human pulmonary artery. PGF2 alpha-induced tone in this tissue is effectively relaxed through PDE inhibitors with selectivity for type III, III/IV, and type V PDE.
Ozone-induced release of cytokines and fibronectin by alveolar macrophages and airway epithelial cells
Authors: Devlin R. B. , McKinnon K. P. , Noah T. , Becker S. , Koren H. S.
Published: December 2017

Acute exposure of animals and humans to ozone results in decrements in lung function, development of airway hyperreactivity, inflammation, edema, damage to pulmonary cells, and production of several compounds with tissue damaging, fibrinogenic or fibrotic potential. The contribution of airway epithelial cells and alveolar macrophages to these processes is unclear. In this study we have directly exposed human alveolar macrophages and human airway epithelial cells to ozone in vitro and measured the cytotoxic effects of ozone, as well as the production of the inflammatory cytokines interleukin-6 (IL-6) and interleukin-8 (IL-8), and fibronectin, all of which are substantially elevated in the bronchoalveolar lavage fluid of humans exposed to ozone. Cells were grown on rigid, collagen-impregnated filter supports, and the interaction of cells with ozone facilitated by exposing them to the gas with medium below the support but no medium on top of the cells. The results show that, although macrophages are much more sensitive to ozone than epithelial cells, they do not produce increased amounts of IL-6, IL-8, or fibronectin following ozone exposure. In contrast, epithelial cells produce substantially more of all three proteins following ozone exposure, and both IL-6 and fibronectin are secreted vectorially. An immortalized human airway epithelial cell line (BEAS 2B) was used in these experiments since human airway epithelial cells are infrequently available for in vitro studies. Data from this study extend previous findings which suggest that the BEAS cell line is a useful model to study the interaction between airway epithelial cells and environmental toxicants.
Nitric oxide synthase type I and type III gene expression are developmentally regulated in rat lung
Authors: North A. J. , Star R. A. , Brannon T. S. , Ujiie K. , Wells L. B. , Lowenstein C. J. , Snyder S. H. , Shaul P. W.
Published: December 2017

The successful transition from fetal to neonatal life involves a marked decline in pulmonary vascular resistance which is modulated in part by endothelium-derived nitric oxide. To define the molecular processes which prepare the pulmonary circulation for nitric oxide mediation of vasodilatation at the time of birth, we determined the ontogeny of endothelial nitric oxide synthase (NOS-III) gene expression in lungs from fetal and newborn rats. Maturational changes in lung neuronal NOS (NOS-I) expression were also investigated; the latter isoform has been localized to rat bronchiolar epithelium. NOS proteins were examined by immunoblot analysis, and mRNA abundance was assessed in reverse transcription-polymerase chain reaction assays. Both NOS-III and NOS-I protein were detectable in 16-day fetal lung, they increased 3.8- and 3.1-fold, respectively, to maximal levels at 20 days of gestation (term = 22 day), and they fell postnatally (1-5 days). In parallel with the findings for NOS-III protein, NOS-III mRNA increased from 16 to 20 days gestation and fell after birth. In contrast, NOS-I mRNA abundance declined during late fetal life and rose postnatally. These findings were confirmed by Northern analyses. Thus NOS-III and NOS-I gene expression are developmentally regulated in rat lung, with maximal NOS-III and NOS-I protein present near term. The regulation of pulmonary NOS-III may primarily involve alterations in transcription or mRNA stability, whereas NOS-I expression in the maturing lung may also be mediated by additional posttranscriptional processes.
Characterization of muscarinic receptor subtypes in pig airways: radioligand binding and northern blotting studies
Authors: Haddad E. B. , Mak J. C. , Hislop A. , Haworth S. G. , Barnes P. J.
Published: December 2017

This study was undertaken to characterize the muscarinic receptor subtypes present in adult pig peripheral lung and airway smooth muscle. The binding of the nonselective muscarinic antagonist [N-methyl-3H]scopolamine ([3H]NMS) to pig airways showed a single class of binding sites with a maximum density of 172 and 450 fmol/mg protein in peripheral lung and airway smooth muscle, respectively. Unlike [3H]NMS, the M1-selective antagonist, [3H]telenzepine, recognized two populations of binding sites in peripheral lung. Approximately 14% of total [3H]telenzepine binding sites displayed high affinity [dissociation constant (Kd) = 0.95 nM], whereas the remaining sites showed low affinity (Kd = 14.2 nM). The high- and the low-affinity [3H]telenzepine binding sites displayed the pharmacological profile of M1 and M2 receptors, respectively. Heterogeneity of pig airways muscarinic receptor was also revealed by competitive binding experiments against [3H]NMS with the M2-selective antagonist methoctramine. This compound recognized 70 and 90% of total receptors with high affinity in airway smooth muscle (Ki = 4.44 nM) and peripheral lung (Ki = 9.82 nM), respectively. This result suggests that the dominant muscarinic receptor in pig airways is of the M2 subtype. Northern blot analysis demonstrated the presence of m1 and m2 mRNAs transcripts in peripheral lung and m2 and m3 mRNAs in airway smooth muscle with no evidence for m4 mRNA.
Induction of pulmonary Mn superoxide dismutase mRNA by interleukin-1
Authors: White J. E. , Tsan M. F.
Published: December 2017

We have previously demonstrated that intratracheal (IT) but not intraperitoneal (IP) administration of 5 micrograms tumor necrosis factor (TNF) or interleukin-1 (IL-1) selectively enhances pulmonary Mn superoxide dismutase (Mn SOD) mRNA, leading to increased Mn SOD protein and enzyme activity, and protects rats against O2 toxicity. In this study, we demonstrated that enhancement of pulmonary Mn SOD mRNA by TNF or IL-1 was highly dependent on the route of administration. IT insufflation of 5 micrograms TNF or IL-1 selectively enhanced levels of pulmonary but not splenic or renal Mn SOD mRNA. In contrast, IP or intravenous (i.v.) administration of TNF (5 micrograms) or IL-1 (5, 20, or 50 micrograms) had little or no effect on levels of Mn SOD mRNA in the lung, spleen, or kidney. Both TNF and IL-1, whether given by IT, IP, or i.v. administration, had no effect on levels of Cu, Zn SOD mRNA. IP administration of 2 mg/kg actinomycin D 2 h before IT insufflation of IL-1 paradoxically increased the level of pulmonary Mn SOD mRNA without affecting the level of Cu,Zn SOD or beta-actin mRNA in IL-1-treated rats. Nuclear runoff transcription assay revealed that IT insufflation of IL-1 enhanced the rate on MN SOD but not Cu,Zn SOD mRNA synthesis. We conclude that IL-1-induced increase in pulmonary Mn SOD mRNA is at least in part regulated at the transcriptional level.
pHi regulation in alveolar macrophages: relative roles of Na(+)-H+ antiport and H(+)-ATPase
Authors: Bidani A. , Brown S. E. , Heming T. A.
Published: December 2017

In rabbit alveolar macrophages, recovery of intracellular pH (pHi) from acid loads to pHi values > or = 6.8 at an extracellular pH (pHo) of 7.4 (nominal absence of CO2-HCO3-) is insensitive to amiloride, an inhibitor of Na(+)-H+ exchange, and abolished by bafilomycin A1, an inhibitor of vacuolar-type H(+)-ATPase [A. Bidani, S.E.S. Brown, T.A. Heming, R. Gurich, and T.D. Dubose, Jr. Am. J. Physiol. 257 (Cell Physiol. 26): C65-C76, 1989; A. Bidani and S. E. S. Brown. Am. J. Physiol. 259 (Cell Physiol. 28): C586-C598, 1990]. To further evaluate the roles of Na(+)-H+ exchange and H(+)-ATPase activity in pHi regulation in rabbit alveolar macrophages, we have investigated the effects of amiloride and bafilomycin over a greater range of pHi (6.3-7.0) and pHo (5.0-7.4). The results indicate that rabbit alveolar macrophages possess H(+)-ATPase and a Na(+)-H+ antiporter, both of which are activated by decrements in pHi. However, in all cases, H(+)-ATPase activity exclusively determined basal pHi and was the principal mechanism (> 50%) for pHi recovery from intracellular acid loads. The pHi set point for activation of Na(+)-H+ exchange was approximately 6.8 at pHo of 7.4 and approximately 6.5 at pHo of 6.8. Na(+)-H+ exchange did not contribute significantly to pHi recovery at acid-loaded pHi above these set points. At pHo of 7.4 and pHi > or = 6.8, pHi recovery displayed an activation energy of approximately 11,000 kcal/mol and temperature coefficient of approximately 2.1, which are consistent with an energy-dependent process (i.e., H+ pump).(ABSTRACT TRUNCATED AT 250 WORDS)
Confocal imaging of time-dependent internalization and localization of NBD-PC in intact rat lungs
Authors: Chinoy M. R. , Fisher A. B. , Shuman H.
Published: December 2017

Morphological localization of a fluorescent analogue of phosphatidylcholine (PC), C12-NBD-PC ([1-palmitoyl-2-[12-(7-nitro-2-1,3-benzoxadiazol-4-yl) amino] dodecanoyl]PC) was evaluated in adult rat lung. Fluorescence was observed at various times after intratracheal liposome instillation. Fluorometric analysis of C12-NBD-PC in lung homogenate showed an initial rapid uptake followed by a more gradual uptake phase. Microscopic observation of fixed lung slices showed C12-NBD-PC uptake in alveolar type II cells (identified by surfactant protein A and Maclura pomifera agglutinin colocalization), nonciliated distal airway epithelial cells, but not in subepithelial tissue. Macrophages showed an initial uptake that did not increase with time. Fluorescence measured in type II cells was diffuse at 5 min, became punctate, and intensified gradually by 2 h. Isolation of type II cells and lamellar bodies from instilled lung confirmed localization of fluorophore in them. This study shows time-dependent uptake of C12-NBD-PC by type II and bronchiolar cells and preferential internalization to lung lamellar bodies as previously described for surfactant PC.
Amiloride-sensitive Na+ channels in fetal type II pneumocytes are regulated by G proteins
Authors: MacGregor G. G. , Olver R. E. , Kemp P. J.
Published: December 2017

We have used the patch-clamp technique to record single channels in excised membrane patches from type II pneumocytes freshly isolated from fetal guinea pig lung by elastase digestion and differential filtration. The 10/56 patches exhibited spontaneous channel activity with a mean open-state probability (NPo) of 0.5 +/- 0.1. In symmetrical Na(+)-rich solutions, the channels had a unitary conductance of 11.1 +/- 0.5 pS and showed current reversal at approximately 0 mV. Superfusing the inner membrane leaflet of the patch with a K(+)-rich solution resulted in single-channel current activity with a conductance of 5.6 +/- 0.2 pS being resolved. Current reversed at +22.1 +/- 1.9 mV, which is compatible with a PNa+/PK+ of 1.8 +/- 0.1. The addition of 0.1 mM guanosine 5'-O-(3-thiotriphosphate) to the cytoplasmic face of the patch elicited channel activity in 12/31 previously quiescent patches, whereas, in spontaneously active patches, channel NPo was increased. Amiloride, in the concentration range 0.4-4 microM, reduced the frequency of observed spontaneous (or activatable) channel activity, reduced NPo, and induced flickery channel behavior. No activity was seen in the presence of 10 microM amiloride in the pipette. This is the first direct observation of a G protein regulated Na(+)-conductive pathway in alveolar epithelium, and it may represent one route by which the alveolar epithelium of the fetus can regulate the Na(+)-driven fluid reabsorption necessary for the adaptation of the newborn lung to air breathing at birth.
3'-untranslated region of SP-B mRNA mediates inhibitory effects of TPA and TNF-alpha on SP-B expression
Authors: Pryhuber G. S. , Church S. L. , Kroft T. , Panchal A. , Whitsett J. A.
Published: December 2017

Surfactant protein-B (SP-B) is a small hydrophobic polypeptide that enhances spreading and stability of surfactant phospholipids in the alveolus of the lung. Decreased expression of SP-B is associated with respiratory failure in premature infants and in adult patients with acute respiratory distress syndrome (ARDS). Tumor necrosis factor-alpha (TNF-alpha) and 12-O-tetradecanoylphorbol-13 acetate (TPA) cause ARDS-like lung injury in vivo. Inhibitory effects of TPA and TNF-alpha on SP-B mRNA expression in vitro were mediated by decreased SP-B mRNA stability rather than by decreased rate of SP-B gene transcription. In the present study, a human pulmonary adenocarcinoma cell line, NCI H441-4, was stably transfected with expression vectors consisting of the thymidine kinase (TK) promotor and human growth hormone (hGH) gene, in which the hGH 3'-untranslated region (3'-UTR) was replaced by the 2.0-kb human SP-B cDNA [pTKGH(SP-B2.0)] or the 837-bp human SP-B 3'-UTR [pTKGH(SP-B.837)]. The mRNAs and cellular growth hormone protein generated from the chimeric TKGH(SP-B2.0) and TKGH(SP-B.837) genes were each inhibited by approximately 50% by TPA and TNF-alpha. Dexamethasone decreased the inhibitory effects of TPA and TNF-alpha. The inhibition of steady-state hGH-SP-B mRNA by TPA and TNF-alpha was mediated by a cis-active element located in the 3-UTR region of SP-B mRNA.
Dexamethasone inhibits IL-1 and TNF activity in human lung fibroblasts without affecting IL-1 or TNF receptors
Authors: Monick M. M. , Aksamit T. R. , Geist L. J. , Hunninghake G. W.
Published: December 2017

Interleukin (IL-1) and tumor necrosis factor (TNF) activate human lung fibroblasts through interactions with specific receptors. One effect of this interaction of IL-1 and TNF with fibroblasts is an increased production of the cytokines, IL-6 and IL-8. Dexamethasone blocks the induction of IL-6 and IL-8 by IL-1 or TNF. In these studies, we determined whether dexamethasone interferes with the upregulation of IL-6 and IL-8 by downregulating expression of the IL-1 or TNF receptor genes. Confluent lung fibroblasts were treated with medium alone (control) or medium with dexamethasone (10(-6) M). Dexamethasone did not decrease the binding of IL-1 and TNF to their receptors, nor did it decrease amounts of IL-1 or TNF receptor RNA. Both IL-1 and TNF increased release of IL-6 and IL-8 from the cells in a dose-dependent manner and dexamethasone inhibited this effect. Dexamethasone also inhibited the induction of IL-6 and IL-8 RNA by IL-1 and TNF. The studies show that dexamethasone does not block the effects of IL-1 or TNF on fibroblasts by decreasing expression of IL-1 or TNF receptors.
Morphometric analysis of pulmonary intracapillary leukocyte pools in ex vivo-perfused rabbit lungs
Authors: Ermert L. , Duncker H. R. , Rosseau S. , Schutte H. , Seeger W.
Published: December 2017

Characterization and quantification of lung intracapillary leukocytes is of interest for a better understanding of immunological and inflammatory features in this organ. We developed a technique of computer-assisted measurement of digitalized electron-microscopic images and electronic data processing for morphometry of intracapillary leukocyte pools in rabbit lungs (L. Ermert, W. Seeger, and H.-R. Duncker, Cell Tissue Res. 271: 469-476, 1993). Measurements were undertaken in buffer-perfused isolated lungs (avoiding any reentry of washed-out cells); perfusion fixation was performed 7.5, 35, and 185 min after onset of artificial circulation (n = 5 each). Data were compared with that of nonperfused lungs fixed by tracheal instillation (baseline). Total lung capillary neutrophil counts were 1.41 x 10(9), 1.35 x 10(9), 1.37 x 10(9), and 0.69 x 10(9) (baseline, 7.5, 35, and 185 min perfusion, respectively). Corresponding data for intracapillary lymphocytes were 1.07 x 10(9), 0.84 x 10(9), 0.81 x 10(9), and 0.57 x 10(9); and for microvascular monocytes, data were 0.21 x 10(9), 0.19 x 10(9), 0.18 x 10(9), and 0.08 x 10(9). Ratios of cell volume and surface variables of the different intracapillary leukocyte types did not change during ex vivo lung perfusion. We conclude that the rabbit pulmonary capillary bed harbors large pools of different leukocytes, which surpass pool sizes of corresponding circulating cells and display very slow washout kinetics under conditions of lung-buffer perfusion. A major impact of these intracapillary leukocyte pools on immunological and inflammatory events in isolated-perfused and transplanted lungs must be assumed.
Bioelectric properties of proximal bronchiolar epithelium
Authors: Ballard S. T. , Taylor A. E.
Published: December 2017

Distal airway epithelium is widely believed to secrete ions and liquid into the airspace, although this process has never been demonstrated in intact small airways. To determine the characteristics of active ion transport in distal airway epithelium, the effects of selective inhibitors of active Na+ absorption and Cl- secretion on the bioelectric properties of intact proximal bronchiolar epithelium were evaluated. Large bronchioles (450-1,200 microns outside diameter, 1.5-5.0 mm length) were excised from 4- to 8-wk-old pigs, cannulated with glass microcannulas, and perfused. Transepithelial potential difference (PD), short-circuit current (Isc), and resistance (Rm) were measured by cable analysis. In 14 tissues, resting PD, Isc, and Rm were -3.4 +/- 0.4 mV (lumen negative), 19.6 +/- 4.7 microA/cm2, and 255 +/- 50 omega.m2, respectively. The conductive Na+ channel blocker amiloride (10 microM) significantly (P < 0.05) reduced PD and Isc by 37 and 41% and significantly increased Rm by 23% in seven tissues. Subsequent bumetanide (10 microM), a blocker of active Cl- secretion through inhibition of Na(+)-K(+)-2Cl- cotransport, significantly reduced the amiloride-insensitive PD and Isc by 41 and 50%, whereas Rm significantly increased 15%. Because amiloride is known to induce Cl- secretion, the order of drug addition was reversed to determine the fractional contribution of active Cl- secretion to the resting PD, Isc, and Rm. In seven different bronchioles, bumetanide did not significantly affect PD, Isc, or Rm.(ABSTRACT TRUNCATED AT 250 WORDS)
Butyrate modulates surfactant protein mRNA in fetal rat lung by altering mRNA transcription and stability
Authors: Peterec S. M. , Nichols K. V. , Dynia D. W. , Wilson C. M. , Gross I.
Published: December 2017

We examined the effects of Na butyrate, a known regulator of gene expression, on surfactant protein mRNA concentration, transcription, and degradation. Exposure of explants of 18-day fetal rat lung to Na butyrate resulted in a decrease in surfactant protein A (SP-A) mRNA concentration to 7% of control after 6 h and to 18% of control after 24 h. The reduction in SP-A mRNA concentration was associated with decreased mRNA transcription and stability at both these times. The effects on SP-B mRNA were similar to those on SP-A, but quantitatively less. In contrast, butyrate had a biphasic effect on SP-C mRNA concentration. There was an initial decrease to 30% of control at 6 h, followed by an increase to control levels by 24 h. Transcription of SP-C was increased at both these times, whereas degradation was enhanced at 6 h, but not at 24 h. The level of surfactant protein mRNA after butyrate treatment therefore depends on the balance between induced changes in transcription and degradation. Butyrate had no effect on gamma-actin mRNA concentration in this system. Circulating levels of butyric acid analogues are elevated in the mothers and fetuses in diabetic pregnancies. Some of these fetuses have delayed lung maturation and decreased amniotic fluid SP-A levels. We speculate that butyric acid analogues partially mediate the changes in pulmonary maturation induced by maternal diabetes.
Phorbol-stimulated influx of extracellular calcium in rat pulmonary artery endothelial cells
Authors: Murphy H. S. , Maroughi M. , Till G. O. , Ward P. A.
Published: December 2017

Stimulation of rat pulmonary artery endothelial cells (RPAEC) with phorbol 12-myristate 13-acetate (PMA) resulted in an increase in intracellular calcium ([Ca2+]i). Unlike the response to bradykinin, C5a and tumor necrosis factor-alpha (TNF-alpha) previously reported (15), the PMA-induced increase in [Ca2+]i was predominantly dependent on extracellular calcium. The PMA response paralleled the BAY K 8644-induced, extracellular calcium-dependent increase in [Ca2+]i. Pretreatment of endothelial cells with the protein kinase C inhibitor staurosporine resulted in a concentration-dependent inhibition of the increase in [Ca2+]i in response to PMA. The ability of PMA analogues to induce significant increase in [Ca2+]i paralleled their ability to induce O2- generation in neutrophils. The PMA-induced influx of extracellular Ca2+ was inhibited by the L-channel selective antagonists diltiazem, nifedipine, nicardipine, and verapamil in a dose-dependent manner. Depolarizing conditions induced by high [K+]o enhanced the calcium response to PMA. The data presented are consistent with the hypothesis that PMA-induced increases in [Ca2+]i in endothelial cells are the result of Ca2+ influx through voltage-dependent L-type Ca2+ channels.
Chloride secretion and function of serous and mucous cells of human airway glands
Authors: Finkbeiner W. E. , Shen B. Q. , Widdicombe J. H.
Published: December 2017

Cells from the acini of human tracheal glands were grown in culture to produce confluent cell sheets of mucous or mixed seromucous phenotype. Levels of mediator-induced Cl secretion in mucous cells were 2-18% those of seromucous cells. Levels of the cystic fibrosis transmembrane conductance regulator (an apical membrane Cl channel) were also much less in mucous than in seromucous cells. These results suggest that serous cells are more important than mucous cells in providing the fluid component of gland secretions.
Concurrent generation of nitric oxide and superoxide damages surfactant protein A
Authors: Haddad I. Y. , Crow J. P. , Hu P. , Ye Y. , Beckman J. , Matalon S.
Published: December 2017

The conditions under which nitric oxide (.NO) may modulate or promote lung injury have not been identified. We hypothesized that .NO-induced injury results from peroxynitrite, formed by the reaction of .NO with superoxide. The simultaneous generation of .NO and superoxide by 3-morpholinosydnonimine (SIN-1, 0.1-2 mM) resulted in oxidation of dihydrorhodamine, a marker of peroxynitrite production, and a dose-dependent decrease in the ability of SP-A to enhance lipid aggregation. Western blot analysis of SIN-1 exposed SP-A samples, overlaid with a polyclonal antibody against nitrotyrosine, were consistent with nitration of SP-A tyrosine residues. Superoxide dismutase (100 U/ml), L-cysteine (5 mM), xanthine oxidase (10 mU/ml) and xanthine (500 microM), or urate (100 microM) prevented the SIN-1-induced dihydrorhodamine oxidation and injury to SP-A. .NO alone, generated by S-nitroso-N-acetylpenicillamine plus 100 microM L-cysteine, or superoxide and hydrogen peroxide, generated by pterin and xanthine oxidase in the absence of iron, did not damage SP-A or oxidize dihydrorhodamine. We concluded that peroxynitrite, but not .NO or superoxide and hydrogen peroxide, in concentrations likely to be encountered in vivo, caused nitrotyrosine formation and decreased the ability of SP-A to aggregate lipids.
Leumedin NPC 15669 inhibits antigen-induced recruitment of inflammatory cells into the canine airways
Authors: Kaneko T. , Jorens P. G. , Richman-Eisenstat J. B. , Dazin P. F. , Nadel J. A.
Published: December 2017

We studied the effects of NPC 15669, a member of a new class of anti-inflammatory drugs called leumedins, on chemotaxis of both human eosinophils and neutrophils and on Mac-1 receptor upregulation on stimulated eosinophils in vitro. Then, we examined the effect of NPC 15669 on antigen-induced eosinophil and neutrophil recruitment and subsequent airway hypersecretion (indicated by an increase in lysozyme concentration) in the allergic dog trachea in vivo. NPC 15669 inhibited eosinophil chemotaxis in vitro at a drug concentration of 10(-5) M (mean inhibition, 48.2%) without affecting Mac-1 receptor upregulation on stimulated eosinophils. NPC 15669 also inhibited neutrophil chemotaxis: at 10(-5) M, NPC 15669 inhibited neutrophil chemotaxis by a mean of 29.7%. In allergic dogs in vivo, NPC 15669 (10(-5) M) prevented antigen-induced recruitment of eosinophils and neutrophils and prevented the increase in elastase and lysozyme concentrations. We conclude that NPC 15669 is an effective inhibitor of antigen-induced leukocyte recruitment and elastase release and subsequent hypersecretion in airways.
Characterization of vasoactive intestinal peptide receptors on rat alveolar macrophages
Authors: Sakakibara H. , Shima K. , Said S. I.
Published: December 2017

In view of the evidence that vasoactive intestinal peptide (VIP) may modulate acute inflammatory injury in the lung, we investigated the presence and characteristics of VIP receptors on alveolar macrophages (AMs). We examined the binding of monoiodinated [Tyr(125I)10]-labeled VIP (125I-VIP) to rat AMs (> 96% pure), obtained from Sprague-Dawley rats by bronchoalveolar lavage (BAL). At 23 degrees C, the interaction of 125I-VIP with AMs was rapid, reversible, saturable, and linearly proportional to the number of cells. At equilibrium, the binding was competitively inhibited by 10(-11)-10(-6) M of native peptide [half-maximal inhibition (IC50) = 0.53 +/- 0.34 nM, n = 8], with evidence for two classes of binding sites: one with a high affinity (Kd = 0.20 +/- 0.09 nM) and a low capacity (1,190 +/- 640 sites/cell) and another with a low affinity (Kd = 43.2 +/- 13.8 nM) and a high capacity (51,700 +/- 14,000 sites/cell). VIP-related peptides inhibited the binding with the order of potency: VIP > peptide histidine isoleucine > helodermin >> secretin; glucagon was ineffective. In the presence of 3-isobutyl-1-methylxanthine, VIP dose dependently stimulated adenosine 3',5'-cyclic monophosphate accumulation in intact AMs, with maximal stimulation (6.3 times basal level) at 1 nM, and half-maximal accumulation at 0.23 +/- 0.11 nM VIP (Kd for high-affinity sites). For determination of the mass of the VIP receptor, 125I-VIP was covalently bound to AMs with the cross-linking agent dithiobis succinimidyl propionate. Autoradiographic studies after sodium dodecyl sulfate/polyacrylamide gel electrophoresis of solubilized affinity-labeled cells revealed a single major band of M(r) 76,400. We conclude that VIP binds to specific receptors on rat AMs that are coupled to adenylate cyclase, through which VIP may modulate inflammatory responses within the lung.
Tumor necrosis factor-alpha decreases pulmonary artery endothelial nitrovasodilator via protein kinase C
Authors: Johnson A. , Phelps D. T. , Ferro T. J.
Published: December 2017

We postulated that tumor necrosis factor-alpha (TNF) decreases endothelium-derived nitrovasodilator(s) via a protein kinase C (PKC)-dependent pathway. Calf pulmonary artery endothelial monolayers (PAEM) were treated with TNF (10, 100, and 1,000 U/ml) for 15 min or 18 h during an 18-h incubation. At the end of the incubation, the cell lysate and supernatant were harvested. Compared with controls, an 18-h incubation with TNF (100 and 1,000 U/ml) resulted in a decrease in NO2- [the oxidation product of nitric oxide (NO)] in PAEM lysate and supernatant. TNF (100 U/ml) treatment for 15 min did not suppress NO2- levels. The decrease in NO2- and the increase in lipid peroxides in response to TNF were prevented by pretreatment (15 min prior to and throughout the incubation) with either calphostin C (1 microM; a specific PKC inhibitor) or the antioxidants N-acetylcysteine (1 mM), 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron) (10 mM), and superoxide dismutase (10 U/ml). Treatment with phorbol 12-myristate 13-acetate (PMA, 1 microM for 15 min), an activator of PKC, decreased NO2- similarly to TNF. Pretreatment with calphostin C or N-acetylcysteine prior to TNF (10 U/ml) revealed an increase in NO2- levels above control treatment. Treatment with the NO synthase antagonists NG-monomethyl-L-arginine (1 mM) and N-nitroso-L-arginine (1 mM) induced an L-arginine (1 mM)-dependent decrease in NO2- in control but not in TNF-treated PAEM. The induction of NO2- by calcium ionophore (A23187; 500 nM) was not affected by treatment with TNF.(ABSTRACT TRUNCATED AT 250 WORDS)
Structure and function of human histamine N-methyltransferase: critical enzyme in histamine metabolism in airway
Authors: Yamauchi K. , Sekizawa K. , Suzuki H. , Nakazawa H. , Ohkawara Y. , Katayose D. , Ohtsu H. , Tamura G. , Shibahara S. , Takemura M. , et al.
Published: December 2017

In mammals, histamine is inactivated principally by two enzymes: histamine N-methyltransferase (HMT; EC 2.1.1.8) and diamine oxidase (DAO; EC 1.4.3.6.). The cDNA clone of human HMT (hHMT) has been isolated from a cDNA library of human kidney and its nucleotide, and deduced amino acid sequences have been determined. One clone, phHMT-1, containing an insert of 1.4 kb, was confirmed to encode HMT by transient expression of HMT activity in COS cells. hHMT consists of 292 amino acid residues [relative molecular weight (M(r)) = 33,279] and shares 82% identity with that of rat HMT. Northern blot analysis with hHMT cDNA probe revealed that 1.6-kb HMT mRNA transcript was expressed in the lung, nasal polyps, and kidney. HMT activity was measured in human trachea and bronchi. In addition, the contractile response of isolated human bronchi to histamine was potentiated in the presence of an HMT inhibitor, SKF 91488, but a DAO inhibitor, aminoguanidine, was without effect. These results suggest that HMT plays an important role in degrading histamine and in regulating the airway response to histamine. Therefore, the level of HMT gene expression in human airway may be one of the critical factors determining the airway responsiveness to histamine. In situ chromosomal hybridization demonstrated that human HMT gene was localized in chromosome 1 p32.
Isolation, culture, and characterization of rat lung microvascular endothelial cells
Authors: Magee J. C. , Stone A. E. , Oldham K. T. , Guice K. S.
Published: December 2017

Highly pure primary cultures of rat lung microvascular endothelial cells were obtained from peripheral lung tissue using a combination of selective culture strategies. The cells had a characteristic morphology consistent with an endothelial origin and were positive for a number of endothelial cell markers, including uptake of fluorescent acetylated lactate dehydrogenase, binding of the lectin Bandeiraea simplicifolia I, and positive immunofluorescence staining with two endothelial cell monoclonal antibodies. The cells behaved as microvascular endothelial cells using an in vitro angiogenesis assay. This isolation method provides a simple method for culturing the pulmonary microvasculature of the rat and these studies support the idea that endothelial cells from different vessels exhibit phenotypic heterogeneity. This method should prove useful for studying specialized endothelial cell function and differentiation in vitro.