Human Lung Membranes Research Articles

Nanosponges intercept coronavirus infection

Nanoparticles cloaked in human lung and immune cell membranes act as decoys to neutralize SARS-CoV-2 in cell culture, preventing host cell infection.

Quantification of membrane transporter proteins in human lung and immortalized cell lines using targeted quantitative proteomic analysis by isotope dilution nanoLC–MS/MS

Information is needed on the expression of transporters in lung to inform drug development and therapeutic decisions. Much of the information currently available is from semiquantitative gene expression or immunometric densitometry studies reported in the literature. NanoLC–MS/MS (MRM mode) isotope dilution targeted quantitative proteomics was used here to quantify twelve selected transporters in fresh human lung membrane fraction samples and in the membrane fraction of selected immortalized human lung epithelial cell line samples. Fractionation was undertaken by homogenization in crude membrane lysis buffer followed by differential centrifugation of the homogenate. In lung membranes we found OATPs to be the most highly expressed transporters of those measured, followed by PEPT2 and ABCs (P-gp & BCRP). SLC22A transporters (OCTs 2 & 3 and OCTN1) were also found to be expressed. OATP2A1, also known as the prostaglandin transporter, was the most highly expressed transporter, being low in two subjects who were at least occasional smokers. One subject, a non-smoker, had an OATP2A1 concentration that was 8.4 times higher than the next nearest concentration, which itself was higher than the concentration of any other transporter. OATP2A1 is known, from gene expression and animal functional studies, to be present in lung. These results inform the understanding of xenobiotic disposition in the lung and show the distinct profile of transporters in lung compared to other tissues.

Improvement of right heart structure and function by BAY 41-8543 in pulmonary artery banded mice

Background Receptors for the natriuretic peptides (NPs) ANP, BNP and CNP are highly expressed in lung, suggesting that this organ is an important physiological target for NP signalling. By stimulating vasoand bronchodilation and inhibiting cell proliferation and fibrotic processes, NPs may have therapeutic potential in various lung diseases. Mechanisms responsible for the relatively short half-life (few min) of NPs and the control of their local concentrations include (i) receptor-mediated internalization by the so called clearance receptor (NPR-C) and (ii) degradation by a membrane metalloprotease, called neutral endopeptidase (NEP) or neprilysin [1]. The velocity of peptide degradation/inactivation differs between ANP, BNP and CNP. Interestingly, cleavage of NPs by insulindegrading enzyme (IDE) [2] may have a particular role in NP signalling by generating peptide fragments that are hyperactive in receptor stimulation [3]. The physiological significance of NEP was supported by several studies in rodents showing that NEP inhibition leads to increased NP concentrations and activity. Moreover, we found that NEP inhibition is necessary and sufficient for detection of GC-A and GC-B by affinity labelling experiments with radioactive ANP or CNP in mouse and rat lung membrane preparations. Analogous assays, however, failed to label these receptors in human lung membranes, suggesting potent NP-degrading activity of NEP inhibitor-insensitive proteases. Methods and results ANP degradation by lung membranes in either the absence or presence of NEP inhibitors was analyzed by thin-layer-chromatography and mass spectrometry [2]. We found that NEP inhibition strongly reduces ANP degradation by rat and mouse but not human membranes. ANP-degrading activity in human lung membranes under conditions of NEP inhibition was very potent and even detectable with 1 ng of membrane protein. Like ANP, CNP was rapidly hydrolyzed. In both peptides, initial cleavage occurred at the same position within the conserved peptide ring structure being essential for biological activity. A second cleavage each was localized to the amino-terminus (behind Arg-4 or Lys-4, respectively). The cleavage sites are unrelated to those by NEP and IDE and indicate trypsin-like enzyme activity. Unlike ANP and CNP, BNP is a poor substrate and shows a completely different and complex cleavage pattern after prolonged incubation. The NEP inhibitor-insensitive protease was also detectable, albeit at much lower levels, in membranes from human aorta and mesenteric arteries, but not at all in placenta. Studies with various protease inhibitors revealed that leupeptin exposure potently inhibits NP degradation by this activity.

A novel natriuretic peptide

Background Receptors for the natriuretic peptides (NPs) ANP, BNP and CNP are highly expressed in lung, suggesting that this organ is an important physiological target for NP signalling. By stimulating vasoand bronchodilation and inhibiting cell proliferation and fibrotic processes, NPs may have therapeutic potential in various lung diseases. Mechanisms responsible for the relatively short half-life (few min) of NPs and the control of their local concentrations include (i) receptor-mediated internalization by the so called clearance receptor (NPR-C) and (ii) degradation by a membrane metalloprotease, called neutral endopeptidase (NEP) or neprilysin (1). The velocity of peptide degradation/inactivation differs between ANP, BNP and CNP. Interestingly, cleavage of NPs by insulindegrading enzyme (IDE) (2) may have a particular role in NP signalling by generating peptide fragments that are hyperactive in receptor stimulation (3). The physiological significance of NEP was supported by several studies in rodents showing that NEP inhibition leads to increased NP concentrations and activity. Moreover, we found that NEP inhibition is necessary and sufficient for detection of GC-A and GC-B by affinity labelling experiments with radioactive ANP or CNP in mouse and rat lung membrane preparations. Analogous assays, however, failed to label these receptors in human lung membranes, suggesting potent NP-degrading activity of NEP inhibitor-insensitive proteases.

Pharmacological characterisation of the β-adrenoceptor expressed by human lung mast cells

The nonselective β-adrenoceptor agonist, isoprenaline (p D 2; 8.8±0.2), and selective β 2-adrenoceptor agonists, clenbuterol (9.2±0.4) and salbutamol (7.1±0.1), inhibited the immunoglobulin E-mediated release of histamine from human lung mast cells in a concentration-dependent manner. The β 2-adrenoceptor-selective antagonist, ICI118551 (erythro-(±)-1-(7-methylindan-4-yloyl)-3-isopropylaminobutan-2-ol HCl), antagonised the isoprenaline inhibition of histamine release from human lung mast cells with high affinity (apparent p K B; 9.5±0.2), whereas high concentrations of the β 1-adrenoceptor-selective antagonist, CGP20712A (2-hydroxy-5-(2-(hydroxy-3-(4((1-methyl-4-trifluoromethyl)-1- H-imidazol-2-yl)-phenoxy)-propyl)-aminoethoxyl)-benzamide), were required to reverse the isoprenaline inhibition (apparent p K B; 6.5±0.3). Radioligand binding studies using [ 125I]-iodocyanopindolol ([ 125I]CYP) were performed on membranes derived from purified mast cells (>90% purity). Binding of [ 125I]CYP to mast cell membranes was displaced from a single binding site with a high affinity for ICI118551 (p K i; 8.9±0.1) and low affinity for CGP20712A (p K i; 6.0±0.03), indicative of a homogeneous population of β 2-adrenoceptors. In contrast, in human lung membranes, these antagonists displaced [ 125I]CYP from two sites indicative of a heterogeneous population of β 1-adrenoceptors (20%) and β 2-adrenoceptors (80%). These data indicate that the β-adrenoceptor expressed by human lung mast cells and mediating inhibition of mediator release from these cells is the β 2-adrenoceptor.

Physiological evidence for a P2Y receptor responsive to diadenosine polyphosphates in human lung via Ca 2+ release studies in bronchial epithelial cells

P2Y 2 receptors that are activated by the extracellular nucleotides ATP or UTP mediate Cl − secretion via an increase in [Ca 2+] i (intracellular calcium concentration). Therefore, in the lung of patients suffering from cystic fibrosis, inhalation of aerosolized UTP offers a way to circumvent the defect in Cl − secretion by the cystic fibrosis transmembrane conductance regulator. A possible alternative for the relatively unstable UTP in inhalation therapy is the more resistant diadenosine tetraphosphate (Ap 4A). In human and rat lung membranes, Ap 4A binds to P2 receptor sites coupled to G proteins. Here, we showed that Ap 4A caused an increase in [Ca 2+] i with an ec 50 of 17 μM in human bronchial epithelial cells (HBE1). The [Ca 2+] i rise evoked by ATP and UTP was completely, but that induced by Ap 4A only partially, caused by release of Ca 2+ from internal stores. Moreover, the potency of Ap 4A to mobilize Ca 2+ was lower than that of ATP and UTP ( ec 50 1.5 and 1.8 μM, respectively), and the maximal increase in [Ca 2+] i was considerably smaller than that after ATP or UTP. In accordance with our previous results providing evidence for a common binding site for various diadenosine polyphosphates in lung membranes, all Ap nA analogues tested (n = 3 to 6) caused a comparable [Ca 2+] i increase. Homologous or heterologous prestimulation largely diminished the increase in [Ca 2+] i found after a second pulse of either UTP or Ap 4A. Although specific binding characteristics and functional responses of Ap 4A on lung cells are in favor of a distinct receptor for Ap 4A, the cross-talk between UTP and Ap 4A in HBE1 cells and the only slight differences in Ca 2+ mobilization by ATP or UTP and Ap 4A render it impossible at this point to state unequivocally whether there exists a distinct P2Y receptor specific for diadenosine polyphosphates in lung epithelia or whether Ap 4A activates one of the nucleotide receptors already described.

In human and rat lung membranes [ 35s]GTPγS binding is a tool for pharmacological characterization of G protein-coupled devucleotide receptors

The P2Y receptor family is activated by extracellular nucleotides such as ATP and UTP. P2Y receptors regulate physiological functions in numerous cell types. In lung, the P2Y 2 receptor subtype plays a role in controlling Cl − and fluid transport Besides ATP or UTP, also diadenosine tetraphosphate (Ap 4A), a stable nucleotide, seems to be of physiological importance. In membrane preparations from human and rat lung we applied several diadenosine polyphosphates to investigate whether they act as agonists for G protein-coupled receptors. We assessed this by determining the stimulation of [ 35S]GTPγS binding. Stimulation of [ 35S]GTPγS binding to G proteins has already been successfully applied to elucidate agonist binding to various G proteincoupled receptors. Ap nA ( n = 2 to 6) enhanced [ 35S]GTPγS binding similarly in human and rat lung membranes, an indication of the existence of G protein-coupled receptor binding sites specific for diadenosine polyphosphates Moreover, in both human and rat lung membranes comparable pharmacological properties were found for a diadenosine polyphosphate ([ 3H]Ap 4A) binding site. The affinity for Ap 2A, Ap 3A, Ap 4A, Ap 5A, and Ap 6A was also comparable. 8-Diazido-Ap 4A and ATP were less potent, whereas the pyrimidine nucleotide UTP showed hardly any affinity. Thus, we present evidence that different diadenosine polyphosphates bind to a common G protein-coupled receptor binding site in membranes derived either from human or rat lung.

Production of Leukotriene C4 in Different Human Tissues Is Attributable to Distinct Membrane Bound Biosynthetic Enzymes

Microsomal glutathione S-transferase-II (GST-II) has recently been discovered and characterized as a member of the 5-lipoxygenase-activating protein (FLAP)/5(S)-hydroxy-6(R)-S-glutathionyl-7,9-trans-11, 14-cis-eicosatetraenoic acid (LTC4) synthase gene family, which also includes microsomal glutathione S-transferase-I (GST-I) as a distant member of this gene family. This new enzyme is unique as it is the only member of this family capable of efficiently conjugating reduced glutathione to both 5,6-oxido-7,9,11,14-eicosatetraenoic acid (LTA4) and 1-chloro-2,4-dinitrobenzene. Although microsomal GST-II has been demonstrated to display both general glutathione S-transferase (GST) and specific LTC4 synthase activities, its biological function remains unknown. In this study, we investigated the physiological location of microsomal GST-II as well as the relative importance of this enzyme versus LTC4 synthase for the production of LTC4 in various human tissues and cells that have been previously demonstrated to possess LTC4 synthase activity. As determined by Western blot, microsomal GST-II was predominantly expressed in human liver microsomes, human endothelial cell membranes, and sparsely detected in human lung membranes. In contrast, LTC4 synthase was prevalent in human lung membranes, human platelet homogenates, and human kidney tissue. Concomitant to the formation of LTC4, microsomal GST-II also produces a new metabolite of LTA4, a postulated LTC4 isomer. This isomer was used to distinguish between microsomal GST-II and LTC4 synthase activities involved in the biosynthesis of LTC4. Based on the relative production of LTC4 to the LTC4 isomer, microsomal GST-II was demonstrated to be the principal enzyme responsible for LTC4 production in human liver microsomes and human endothelial cells and played a minor role in the formation of LTC4 in human lung membranes. In comparison, LTC4 synthase was the main enzyme capable of catalyzing the conjugation of reduced glutathione to LTA4 in human lung membranes and human platelet homogenates. Therefore, microsomal GST-II appears to be an integral component in the detoxification of biological systems due to its marked presence in human liver, in accordance with its known GST activity. Microsomal GST-II, however, may also be pivotal for cysteinyl leukotriene formation in endothelial cells, and this could change our current understanding of the regulation of leukotriene biosynthesis in inflammatory disorders such as asthma.

Conversion of angiotensin I to angiotensin II by chymase activity in human pulmonary membranes.

An aprotinin-insensitive, angiotensin II (Ang II)-forming chymase has recently been identified in human heart tissue. We studied the hydrolysis of Ang I in human lung membranes. The hydrolysis products Ang II, Ang III, Ang-(1-9), Ang-(2-9), Ang-(1-7) and Ang-(8-10) appeared in membrane preparations from four patients. Two metabolic pathways for the formation of Ang II were identified; one depending on ACE activity (1.4 nmol Ang II/min/mg membrane protein) and the other on serine protease activity (2.1 nmol/min/mg). The serine protease activity was inhibitable to only 30 +/- 8% (mean +/- SEM) by aprotinin, suggesting chymase activity to play a role in the Ang I-conversion of human lung.

Inhibition of ferrous-induced lipid peroxidation by dipyridamole, RA-642 and mopidamol in human lung tissue

1. 1. The in vitro production of ferrous-induced lipid peroxidation was 5.71 times higher in rat lung tissue than in human lung membranes. 2. 2. The pyrimido-pyrimidine derivative RA-642 shows a more potent inhibition of ferrous-induced lipid peroxidation than dipyridamole; mopidamol had no effect. All the compounds showed higher anti-peroxidative effect in rat than in human lung tissue.

Is endothelin-1 release at reperfusion of the ischaemic human heart due to cold-induced displacement of endothelin from binding sites?

Following reperfusion of ischaemic human hearts subjected to cold (4° C) cardioplegia during coronary bypass surgery, there was an increase in cardiac outflow of endothelin-1 but not the pro-peptide big endothelin-1. Furthermore, specific endothelin-1 binding in human lung membrane preparations was displaced by incubation in buffer medium at 4° C. The present results thus indicate that cold-induced displacement of endothelin-1 binding, rather than increased synthesis, may explain the cardiac release of endothelium-1 following ischaemia during heart surgery in which cold cardioplegia has been used.

Gastrin releasing peptide-preferring bombesin binding sites in human lung

Characterization of bombesin binding sites in healthy human lung was performed through direct binding techniques. There was limited binding in the absence of trypsin and chymotrypsin inhibitors, suggesting important activities of both enzymes in human lung and/or increased sensitivity of the bombesin sites toward them. In human lung membranes, bombesin, gastrin releasing peptide (GRP) and GRP-preferring bombesin receptor antagonists displaced [ 125I-Tyr 4]bombesin binding with high affinities (36–177 nM), whereas neuromedin B possessed a lower affinity of 2878 nM. [ D-F 5Phe 6, D-Ala 11]bombesin--(6–13)-methyl ester, the most active GRP-preferring bombesin antagonist as yet reported, had the highest affinity among all antagonists tested whereas neuromedin B had the lowest affinity. These data demonstrate that the bombesin binding sites in the human lung are of the GRP-preferring type.

High affinity [ 3H]formoterol binding sites in lung: characterization and autoradiographic mapping

Agonist binding to the β 2-adrenoceptors and its mapping were studied using the newly developed radioligand [ 3H]formoterol. The results of [ 3H]formoterol saturation binding and formoterol inhibition of [ 3H]formoterol binding were consistent with binding to a single class of receptors ( K d = 1.34 ± 0.15 nM, B max = 154.9 ± 8.0 fmol/mg protein in guinea pig lung membranes, n = 8; K d = 1.05 ± 0.17 nM, B max = 67.8 ± 8.1 fmol/mg protein in human lung membranes, n = 5) and competition assays with other agonists and antagonists disclosed only a single class of site. The nonhydrolyzable GTP analogue GTPγS caused a reduction in both K d and B max, indicating that the receptors labelled by [ 3H]formoterol are coupled to a guanine nucleotide binding regulatory protein. Receptor mapping of [ 3H]formoterol binding sites shows that β 2-adrenoceptors were widely distributed in both guinea pig and human lung, with dense labelling over airway epithelium and uniformly over alveolar walls, and sparse labelling of airway and vascular smooth muscle. In addition, submucosal glands were also sparsely labelled in human bronchus. The distribution of β 2-adrenoceptors was similar to the pattern previously described with non-selective radiolabelled antagonists in the presence of selective β 1-adrenoceptor antagonists.

Expression of platelet-activating factor receptor mRNA in human and guinea pig lung.

Platelet-activating factor (PAF) has been implicated in the pathogenesis of several inflammatory pulmonary diseases, and specific binding sites have been demonstrated in human and guinea pig lung membranes by radioligand binding experiments. Both human and guinea pig PAF receptors (PAFR) have recently been cloned. We have used molecular probes to study the gene expression of PAFR in human and animal lung and in situ hybridization to determine the distribution of PAFR mRNA in peripheral lung. Northern blot analysis of total lung RNA from human lung parenchyma, using a 1.1-kb SmaI-EcoRI fragment of human PAFR cDNA or a 0.9-kb SmaI-SmaI fragment of guinea pig PAFR cDNA, demonstrated the expression of PAFR mRNA in human lung, with a single transcript of 4 kb. There was a significant increase in PAFR mRNA in the lungs of asthmatic patients and a significant decrease in PAFR mRNA in the lungs of cigarette smokers compared with normal patients. Similarly, the expression of PAFR mRNA on guinea pig and rat lung was detected as a single transcript of 3 kb, using both guinea pig and human PAFR cDNA probes. A full-length 1.8-kb human leukocyte PAFR cDNA probe was used as the DNA template for producing 35S-labeled antisense and sense cRNA probes for use in in situ hybridization studies of human peripheral lung. These studies revealed high levels of PAFR mRNA hybridization in blood vessels, moderate levels of hybridization in alveolar walls and peripheral airway smooth muscle, but no specific signal in airway epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine triphosphate-dependent transport of doxorubicin, daunomycin, and vinblastine in human tissues by a mechanism distinct from the P-glycoprotein.

Previous studies have demonstrated that a human glutathione conjugate transporter, designated as dinitrophenyl-S-glutathione ATPase (DNP-SG ATPase), catalyzed ATP hydrolysis in the presence of several amphiphilic compounds other than glutathione conjugates (Singhal, S. S., R. Sharma, S. Gupta, H. Ahmad, P. Zimniak, A. Radominska, R. Lester, and Y. C. Awasthi. 1991. FEBS [Fed. Eur. Biochem. Soc.] Lett. 281:255-257). We now demonstrate that DNP-SG ATPase purified from human lung and erythrocyte membranes catalyzed the hydrolysis of ATP in the presence of doxorubicin and its metabolites. Doxorubicin-stimulated ATP hydrolysis by DNP-SG ATPase was saturable with respect to doxorubicin (Km 1.2 and 2.8 microM for the lung and erythrocyte enzymes, respectively). Antibodies against DNP-SG ATPase immunoprecipitated the ATP hydrolyzing activity stimulated by doxorubicin, its metabolites, and glutathione conjugates. Inside our vesicles prepared from erythrocyte membranes took up doxorubicin, daunomycin, and vinblastine in an ATP-dependent manner. The uptake was linear with respect to time and vesicle protein, was dependent on ATP and magnesium, was inhibited by heavy metal salts or by heating the vesicles, and was sensitive to both osmolarity and orientation of the vesicles. The transport had an activation energy of 13 kcal/mol, was saturable with respect to both doxorubicin and ATP (Km values of 1.8 microM and 1.9 mM, respectively), and was competitively inhibited by glutathione conjugates as well as by a number of amphiphiles such as daunomycin or vinblastine. Transport was diminished upon coating the vesicles with antibodies against DNP-SG ATPase. Incorporation of increasing amounts of purified DNP-SG ATPase into the vesicles resulted in a linear increase in transport of doxorubicin. These studies demonstrated for the first time that a membrane protein that catalyzed the transport of anionic amphiphilic molecules such as glutathione conjugates could also mediate the transport of weakly cationic antitumor antibiotic, doxorubicin. Notably, the Km of transport was in the range of doxorubicin concentration achievable in human serum after intravenous dosing of doxorubicin.

High Concentration of Carboxypeptidase M in Lungs: Presence of the Enzyme in Alveolar Type I Cells

The presence of high concentrations of membrane-bound carboxypeptidase M in human, baboon, dog, and rat lung was established by employing a variety of techniques. The activity of the enzyme in the membrane-enriched fractions of human, baboon, dog, and rat lung, measured with fluorescent dansyl substrate (DNS-Ala-Arg), was 198, 261, 484, and 153 nmol/h/mg protein, respectively. This activity in the lung was much higher than that found in the heart, liver, or kidney. The enzyme, optimally active around neutral pH, was completely inhibited by 10 microM 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid and was activated by 1 mM CoCl2 to 170%. Antibody to human carboxypeptidase M immunoprecipitated the solubilized carboxypeptidase from human (98%), baboon (81%), and dog (88%) lung membrane fractions. Carboxypeptidase M is attached to lung membranes by a phosphatidylinositol glycan anchor; thus, it is released with bacterial phospholipase C. Membrane fractions from cultured human pulmonary arterial endothelial cells also contained high carboxypeptidase M activity (254 nmol/h/mg protein). A Northern blot of poly(A)+ RNA from various human tissues showed the presence of a high level of carboxypeptidase M mRNA in human lung and placenta. Finally, immunohistochemistry, employing purified antibody to the enzyme, revealed in fluorescent light microscopy that carboxypeptidase M is present in alveolar type I pneumocytes and in macrophages in apparently lower concentration. In contrast, type II alveolar epithelial cells gave negative results. Because carboxypeptidase M cleaves a variety of active peptides (e.g., bradykinin, anaphylatoxins), it may protect the alveolar surface from the effects of these peptides. In addition, carboxypeptidase M could be a marker enzyme for type I cells.

Existence of PAF receptors in human platelets and human lung tissue but not in the human myocardium

The aim of this study was to investigate whether platelet-activating factor (PAF), PAF receptors, and PAF receptor-mediated effects in the human myocardium play a role in cardiac depression during anaphylaxis or septic shock. The effects of PAF, the biologically inactive derivative lyso-PAF, and the specific PAF antagonist WEB 2086 were studied in human myocardial tissue, in human coronary arteries, in human platelets, and in human lung tissue. PAF (C 16-PAF, C 18-PAF; 0.000001 to 1 μmol/L) had no effect on isometric force of contraction of electrically driven right atrial trabeculae (patients undergoing aortocoronary bypass surgery) and left ventricular papillary muscle strips (mitral valve replacement). PAF (0.2 μmol/L) did not influence the concentration-response curve of either the β-adrenoceptor agonist isoprenaline (ISO, 0.0001 to 1 μmol/L) or the m-cholinoceptor agonist carbachol (CARB, 0.0001 to 10 μmol/L). The effectiveness (ISO +4.7 ± 0.7 mN, PAF + ISO + 4.3 ± 0.44 mN, CARB −2.7 ± 1.06 mN; PAF + CARB −2.6 ± 0.52 mN) and the potency—as indicated by the EC 50 values—of both isoprenaline and carbachol were identical with and without pretreatment with PAF (0.2 μmol/L). PAF at concentrations of 0.000001 to 10 μmol/L exerted no effect on force of contraction either precontracted (prostaglandin F 2α, 0.3 μmol/L) or unprecontracted in human coronary artery rings. Histamine (0.01 to 100 μmol/L) and noradrenalin (0.001 to 30 μmol/L) initiated concentration-dependent contraction in human coronary artery rings (EC 50: histamine, 1.86 μmol/L; noradrenalin, 0.69 μmol/L). At lower concentrations (PAF, 0.01 μmol/L) PAF produced complete aggregation of human platelets. In human platelet membranes and lung membranes, 3H-WEB 2086 exhibited saturable high-affinity binding (K D 14.4 nmol/L and 14.3 nmol/L). The maximal binding capacity was 292 fmol/mg protein and 268 fmol/mg protein, respectively. In displacement experiments PAF (0.01 to 10000 nmol/L) and WEB 2086 (0.01 to 10000 nmol/L), but not lyso-PAF, completely displaced 3H-WEB 2086 from its binding sites on human platelet and lung membranes. In contrast, neither in left ventricular membranes nor in right atrial membranes was specific binding of 3H-WEB 2086 observed. These results suggest that there are neither specific PAF receptors nor direct PAF-mediated actions in human myocardial tissue or human coronary artery rings. The effects of PAF on myocardial function may be due to the activation of mediators (e.g., histamine).

Comparative study of the binding of prolactin and growth hormone by rabbit and human lung cell membrane fractions.

The specific binding capacities for human prolactin (hPRL) and human growth hormone (hGH) were examined in human lung membrane preparations at different developmental stages. A parallel study was carried out on rabbit lung preparations to compare binding parameters. Lung tissues were obtained from 15 fetuses of 16-38 weeks after spontaneous or therapeutic abortion and from 7 adults (lobar resection surgery). A histological study was systematically performed with a radial alveolar count in the fetuses with suspected hypoplasia. Binding analysis was performed on both intact membrane preparations and MgCl2-treated membranes, using [125I]hGH and [125I]hPRL as tracers. In the rabbit lung, specific [125I]hGH binding was found. Scatchard analysis revealed a single class of binding sites (affinity constant: 2.6 +/- 0.8 x 10(9) l/nmol and number of binding sites: 9.5 +/- 4.3 fmol/mg protein for adult rabbit; 1.85 +/- 0.5 x 10(9) l/nmol and 27.6 +/- 3.0 fmol/mg protein for 25-day-old rabbit fetuses, respectively). In contrast, [125I]hPRL did not specifically bind to lung membrane preparations. In the human lung, no consistent specific binding sites for [125I]hPRL or [125I]hGH (less than 0.5%/mg protein) were detected in adults and in 11 of the 15 fetuses. In 4 fetuses, little specific binding was observed (0.59-1.9%/mg protein) for [125I]hGH and (1.5%/mg protein) for [125I]hPRL. There was no correlation with histological lung structure. Our findings confirm the presence of specific binding sites for GH in the adult rabbit lung and demonstrate such binding in the fetal rabbit lung. In contrast, our results showed no significant binding for PRL and GH in the human lung, suggesting that these hormones do not play a direct physiological role in human lung growth and maturation.

Primate vascular responses to octimibate, a non‐prostanoid agonist at the prostacyclin receptor

1. Octimibate is a potent inhibitor of human platelet aggregation, and appears to act (at least in part) through the prostacyclin receptor, as described in the preceding paper. Here, the vascular effects, both in vitro and in vivo, of octimibate have been compared to those of the stable prostacyclin (PGI2) mimetic, iloprost. Since octimibate shows extensive species variation and is potent at inhibiting platelet aggregation in primates, all of the experiments reported here have been carried out with primate tissue or in vivo in cynomolgus monkeys. 2. Activation of adenylyl cyclase in human lung membranes appears to involve stimulation of the vascular PGI2 receptor. Octimibate, as well as iloprost, stimulates adenylyl cyclase in this preparation. The EC50 values for iloprost and octimibate are 50 nM and 340 nM respectively. These values are similar to those seen with human platelet membranes. As with platelets, the maximal activation achievable with octimibate is 60% of that seen with iloprost. This result suggests that octimibate is a partial agonist for stimulation of adenylyl cyclase. 3. Iloprost (10-100 nM) relaxes human coronary and mesenteric artery precontracted with KCl, and also relaxes cynomolgus monkey aorta precontracted with phenylephrine. Octimibate appears to be a partial agonist for relaxation of human coronary artery precontracted with KCl; the intrinsic activity of octimibate (10 microM) is 0.15 compared to iloprost, and octimibate surmountably antagonizes the relaxant effects of iloprost with a Kp of 200 nM. Octimibate (up to 10 microM) evokes only weak relaxation of human mesenteric artery (precontracted with KCl) and cynomolgus monkey aorta (precontracted with phenylephrine). 4. The effects of iloprost and octimibate were compared in vivo in cynomolgus monkeys. In addition to inhibiting ex vivo platelet aggregation, both compounds cause hypotension with little effect on heart rate. The dose-response curves for inhibition of ex vivo platelet aggregation and a fall in mean arterial blood pressure were compared. The dose-separation (i.e., the relative differences in effective concentrations) for the two responses is similar with both iloprost and octimibate. 5. Since the pern; beral resistance vessels are intimately involved in regulation of systemic arterial blood pressure, the effects of both agents were tested on human peripheral resistance vessels (150-400pm diameter) in vitro. These vessels are relaxed by both iloprost and octimibate following precontraction with KCI. The IC50 value for iloprost is 44nM, and 1.7 microM octimibate evokes 50% of the maximal relaxation obtained with iloprost. Thus, the relative potencies of the two compounds in relaxing human subcutaneous resistance vessels are similar to their relative potencies in inhibiting platelet responses. This result correlates with the lack of platelet versus vascular selectivity seen with the in vivo monkey studies. 6. These results suggest that octimibate, a partial agonist at the prostacyclin receptor, is unable to discriminate between platelet and vascular prostacyclin receptors in primates.

Carbonic anhydrase IV from human lung. Purification, characterization, and comparison with membrane carbonic anhydrase from human kidney.

We have purified carbonic anhydrase (CA) IV from human lung membranes to apparent homogeneity in a form which is catalytically active and stable to storage. It has an apparent molecular mass of 35 kDa, is insensitive to endoglycosidases, and seems to contain no N-linked or O-linked oligosaccharide chains. Reduction of disulfide linkages led to altered migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and loss of catalytic activity. CA IV resembles CA II in being a "high activity" isozyme, relatively resistant to inhibition by halide ions and sensitive to inhibition by sulfonamides. Application of this purification to human kidney membranes produced homogeneous enzyme with nearly identical properties. Amino acid compositions of both lung and kidney CA IV were similar, as were tryptic peptide patterns resolved on high performance liquid chromatography (HPLC). Amino-terminal sequences of native enzyme from lung and kidney were identical, as were amino-terminal sequences of the three major tryptic peptides resolved on reverse phase HPLC. Isoelectric focusing revealed microheterogeneity in enzyme from both sources. Antibody raised to human lung CA IV reacted equally strongly with CA IV from kidney, but very weakly or not at all with other CAs. Treatment of lung membranes and kidney membranes with phosphatidylinositol-specific phospholipase C released over half of the membrane-bound CA IV, suggesting that at least half of the CA IV in both organs is anchored to membranes by phosphatidylinositol-glycan linkages.