Important Clearance Mechanism Research Articles

Enhanced disulphide bond stability contributes to the once-weekly profile of insulin icodec

Insulin icodec is a once-weekly insulin analogue that has a long half-life of approximately 7 days, making it suitable for once weekly dosing. The Insulin icodec molecule was developed based on the hypothesis that lowering insulin receptor affinity and introducing a strong albumin-binding moiety would result in a long insulin half-life, provided that non-receptor-mediated clearance is diminished. Here, we report an insulin clearance mechanism, resulting in the splitting of insulin molecules into its A-chain and B-chain by a thiol–disulphide exchange reaction. Even though the substitutions in insulin icodec significantly stabilise insulin against such degradation, some free B-chain is observed in plasma samples from minipigs and people with type 2 diabetes. In summary, we identify thiol–disulphide exchange reactions to be an important insulin clearance mechanism and find that stabilising insulin icodec towards this reaction significantly contributes to its long pharmacokinetic/pharmacodynamic profile.

Effect of expiratory muscle training on peak cough flow in children and adolescents with cystic fibrosis: A randomized controlled trial.

Cough is an important defense and airway clearance mechanism for removing thick and viscous secretions in cystic fibrosis (CF). The primary aim of this study was to investigate the effect of expiratory muscle training (EMT) on peak cough flow (PCF) and secondly on respiratory muscle functions, functional exercise capacity, and quality of life (QoL) in CF. Thirty patients were randomized as training and sham groups. Both groups were trained with the EMT protocol, which involved twice per day for at least 5 days per week for 6 weeks. The training intensity in the training group was 30% of the maximal expiratory pressure (MEP). In the sham group, it remained at the lowest pressure (5 cmH2 O). The primary outcome was PCF. The secondary outcomes were MEP, maximal inspiratory pressure (MIP), spirometric measures, six-minute walking distance (6MWD), and QoL (Cystic Fibrosis Questionnaire-Revised). Twenty-eight patients completed the study. Changes in PCF (p = .041) and MEP (p = .003) were higher in the training group than the sham group. Also, treatment burden (p = .008), digestive symptoms (p = .019), and vitality (p = .042) in QoL were more improved in the training group. MIP (p = .028) and 6MWD (p = .035) changed significantly only in the training group. Spirometric measurements did not change (p > .05). The results of the study show that EMT could improve PCF, MEP, treatment burden, digestive symptoms, and vitality domains of QoL in patients with CF. Moreover, MIP and functional exercise capacity improved in the training group with EMT.

Pharmacogenomics of poor drug metabolism in Greyhounds: Cytochrome P450 (CYP) 2B11 genetic variation, breed distribution, and functional characterization

Greyhounds recover more slowly from certain injectable anesthetics than other dog breeds. Previous studies implicate cytochrome P450 (CYP) 2B11 as an important clearance mechanism for these drugs and suggest Greyhounds are deficient in CYP2B11. However, no CYP2B11 gene mutations have been identified that explain this deficiency in Greyhounds. The objectives of this study were to provide additional evidence for CYP2B11 deficiency in Greyhounds, determine the mechanisms underlying this deficiency, and identify CYP2B11 mutations that contribute to this phenotype in Greyhounds. Greyhound livers metabolized CYP2B11 substrates slower, possessed lower CYP2B11 protein abundance, but had similar or higher mRNA expression than other breeds. Gene resequencing identified three CYP2B11 haplotypes, H1 (reference), H2, and H3 that were differentiated by mutations in the gene 3′-untranslated region (3′-UTR). Compared with 63 other dog breeds, Greyhounds had the highest CYP2B11-H3 allele frequency, while CYP2B11-H2 was widely distributed across most breeds. Using 3′-UTR luciferase reporter constructs, CYP2B11-H3 showed markedly lower gene expression (over 70%) compared to CYP2B11-H1 while CYP2B11-H2 expression was intermediate. Truncated mRNA transcripts were observed in CYP2B11-H2 and CYP2B11-H3 but not CYP2B11-H1 transfected cells. Our results implicate CYP2B11 3′-UTR mutations as a cause of decreased CYP2B11 enzyme expression in Greyhounds through reduced translational efficiency.

Identification of glucuronidation and biliary excretion as the main mechanisms for gossypol clearance: in vivo and in vitro evidence

1. The natural polyphenol gossypol possesses many therapeutic benefits. Here we aim to determine the elimination pathways of gossypol in vivo and in vitro.2. Metabolite elucidation of gossypol was performed using UPLC-QTOF/MS coupled with Metabolynx analysis. Clearance of gossypol was evaluated in bile duct cannulated rats and in the single-pass perfused rat intestine model. In vitro glucuronidation of gossypol was characterized using liver and intestine microsomes as well as recombinant UDP-glucuronosyltransferase (UGT) enzymes.3. Analysis of rat plasma, urine, and feces revealed glucuronidation as the only metabolic pathway for gossypol. In bile duct cannulated rats, considerable amounts of glucuronides (G1, G2 and G3; 58.8–83.2% of dose) and parent compound (5.0–20%) were excreted into bile after IV administration. In the perfused rat intestine model, gossypol was well absorbed with a (the dimensionless effective permeability) value of 4.4. Significant amounts of glucuronides (G1, G2 and G3) were excreted into the gut lumen (2.5%) and into the bile (4.8%). Biliary excretion of unchanged gossypol (6.0%) was comparable to that of glucuronides. Further, gossypol was subjected to rapid glucuronidation by liver and intestine microsomes. Reaction phenotyping showed that multiple UGT1A enzymes (including UGT1A1, 1A3, 1A7 and 1A8) are mainly responsible for gossypol metabolism.4. In conclusion, glucuronidation was the only metabolic pathway for gossypol in rats. Excretion of unchanged gossypol into bile was also an important clearance mechanism.

Amyloid β peptide cleavage by kallikrein 7 attenuates fibril growth and rescues neurons from Aβ-mediated toxicity in vitro

The gradual accumulation and assembly of β-amyloid (Aβ) peptide into neuritic plaques is a major pathological hallmark of Alzheimer disease (AD). Proteolytic degradation of Aβ is an important clearance mechanism under normal circumstances, and it has been found to be compromised in those with AD. Here, the extended substrate specificity and Aβ-degrading capacity of kallikrein 7 (KLK7), a serine protease with a unique chymotrypsin-like specificity, was characterized. Preferred peptide substrates of KLK7 identified using a bacterial display substrate library were found to exhibit a consensus motif of RXΦ(Y/F)↓(Y/F)↓(S/A/G/T) or RXΦ(Y/F)↓(S/T/A) (Φ=hydrophobic), which is remarkably similar to the hydrophobic core motif of Aβ (K16L17V18F19F20 A21) that is largely responsible for aggregation propensity. KLK7 was found to cleave after both Phe residues within the core of Aβ42 in vitro, thereby inhibiting Aβ fibril formation and promoting the degradation of preformed fibrils. Finally, the treatment of Aβ oligomer preparations with KLK7, but not inactive pro-KLK7, significantly reduced Aβ42-mediated toxicity to rat hippocampal neurons to the same extent as the known Aβ-degrading protease insulin-degrading enzyme (IDE). Taken together, these results indicate that KLK7 possesses an Aβ-degrading capacity that can ameliorate the toxic effects of the aggregated peptide in vitro.

Quantitative Prediction of Glucuronidation in Humans Using the In Vitro- In Vivo Extrapolation Approach

Glucuronidation has been recognized as an important clearance mechanism in humans. Therefore, knowledge about the contribution of glucuronidation to clearance of drug candidates is of great value in early drug development. In this article, we discuss the recent progress made to predict in vivo glucuronidation parameters (e.g., hepatic clearance, and intestinal availability) using in vitro data, which are readily obtained using microsomes and hepatocytes, so called "in vitro- in vivo extrapolation" (IVIVE). Of note the intrinsic clearances obtained from microsomal incubations in the presence of bovine serum albumin (BSA) provide accurate predictions of the in vivo clearances in addition to those from hepatocytes. Further, we describe the lack of correlation between cellular and microsomal production of glucuronide and provide possible reasons. Due to the high prediction accuracy, those who study in vitro glucuronidation are encouraged to map their data to in vivo using IVIVE strategy for more informative data interpretation.

Sa1454 Correlation of the Threshold Volume to Trigger Reflexive Pharyngeal Swallow With the Hypopharyngeal Safe Volume in Awake State Can Predict Aspiration During Sleep

BACKGROUND: Cough and swallowing are important protective mechanisms that maintain the integrity of the aero-digestive tract across the age spectrum. Premature neonates commonly experience swallowing and breathing problems. Consequently, anterograde or retrograde aspiration is a frequent concern in neonates with chronic lung disease such as bronchopulmonary dysplasia (BPD). AIMS: Our aims were to define the cough-initiating and subsequent post-tussive mechanisms that restore aero-digestive normalcy (defined as return of respiratory rhythm to baseline along with esophageal quiescence) in human premature infants suffering from BPD. METHODS: Ten premature neonates (born at 25.5 ± 0.6 wks gestation, studied at 42.2 ± 1.2 wks post-menstrual age), diagnosed with BPD underwent concurrent pharyngo-esophageal manometry, respiratory inductance plethysmography (using Respitrace) and nasal air flow (using thermistor) methods to determine the relationships between esophageal motility and respiratory phases using a specially designed manometric catheter with upper and lower esophageal sphincter (UES and LES) sleeves and 5 recording pressure ports from pharynx, proximal-, middle-, and distalesophagus, and stomach. Cough was recognized as forceful exhalation preceded by deep inhalation on the respiratory waveforms along with audible cough marked during the study. The dynamic characteristics were analyzed preceding and following cough events defined by manometry and Respitrace. RESULTS: 1) Cough initiating mechanisms during 59 cough episodes are shown (Table). 2) Post-tussive mechanisms that restored aero-digestive normalcy were primary peristalsis in 81.4% (48/59), secondary peristalsis in 15.3 % (9/59), and not recognizable in 3.4 % (2/59). CONCLUSIONS: 1) In premature neonates with BPD, non-propagating swallows and UES contractile reflexes are the dominant reasons for the origin of the cough reflex, indicating the majority of cough events have upper aero-digestive origins. 2) Primary peristalsis is the most important clearance mechanism for post-tussive restoration of respiratory normalcy and pharyngo-esophageal quiescence. 3) These phenomena underscore the importance of postnatal development of esophagus-airway reciprocal communications mediated by Glossopharyngeal and Vagal nerves in human premature neonates with BPD. *Supported by PPG-PO1 DK 068051 Mechanisms triggering cough in neonates.

Sa1453 Mechanisms of Origin and Resolution of Cough Reflex in Human Neonates

BACKGROUND: Cough and swallowing are important protective mechanisms that maintain the integrity of the aero-digestive tract across the age spectrum. Premature neonates commonly experience swallowing and breathing problems. Consequently, anterograde or retrograde aspiration is a frequent concern in neonates with chronic lung disease such as bronchopulmonary dysplasia (BPD). AIMS: Our aims were to define the cough-initiating and subsequent post-tussive mechanisms that restore aero-digestive normalcy (defined as return of respiratory rhythm to baseline along with esophageal quiescence) in human premature infants suffering from BPD. METHODS: Ten premature neonates (born at 25.5 ± 0.6 wks gestation, studied at 42.2 ± 1.2 wks post-menstrual age), diagnosed with BPD underwent concurrent pharyngo-esophageal manometry, respiratory inductance plethysmography (using Respitrace) and nasal air flow (using thermistor) methods to determine the relationships between esophageal motility and respiratory phases using a specially designed manometric catheter with upper and lower esophageal sphincter (UES and LES) sleeves and 5 recording pressure ports from pharynx, proximal-, middle-, and distalesophagus, and stomach. Cough was recognized as forceful exhalation preceded by deep inhalation on the respiratory waveforms along with audible cough marked during the study. The dynamic characteristics were analyzed preceding and following cough events defined by manometry and Respitrace. RESULTS: 1) Cough initiating mechanisms during 59 cough episodes are shown (Table). 2) Post-tussive mechanisms that restored aero-digestive normalcy were primary peristalsis in 81.4% (48/59), secondary peristalsis in 15.3 % (9/59), and not recognizable in 3.4 % (2/59). CONCLUSIONS: 1) In premature neonates with BPD, non-propagating swallows and UES contractile reflexes are the dominant reasons for the origin of the cough reflex, indicating the majority of cough events have upper aero-digestive origins. 2) Primary peristalsis is the most important clearance mechanism for post-tussive restoration of respiratory normalcy and pharyngo-esophageal quiescence. 3) These phenomena underscore the importance of postnatal development of esophagus-airway reciprocal communications mediated by Glossopharyngeal and Vagal nerves in human premature neonates with BPD. *Supported by PPG-PO1 DK 068051 Mechanisms triggering cough in neonates.

Autophagy in Dementias

Dementias are a varied group of disorders typically associated with memory loss, impaired judgment and/or language and by symptoms affecting other cognitive and social abilities to a degree that interferes with daily functioning. Alzheimer's disease (AD) is the most common cause of a progressive dementia, followed by dementia with Lewy bodies (DLB), frontotemporal dementia (FTD), (VaD) and HIV-associated neurocognitive disorders (HAND). The pathogenesis of this group of disorders has been linked to the abnormal accumulation of proteins in the brains of affected individuals, which in turn has been related to deficits in protein clearance. Autophagy is a key cellular protein clearance pathway with proteolytic cleavage and degradation via the ubiquitin-proteasome pathway representing another important clearance mechanism. Alterations in the levels of autophagy and the proteins associated with the autophagocytic pathway have been reported in various types of dementias. This review will examine recent literature across these disorders and highlight a common theme of altered autophagy across the spectrum of the dementias.

Endocytosis of Advanced Glycation End-Products in Bovine Choriocapillaris Endothelial Cells

Age-related changes in Bruch's membrane (BM), situated between the retina and the choroid, include the accumulation of advanced glycation end-products (AGEs) and other modified macromolecules. An important clearance mechanism for such molecules is endocytosis via macrophages and specialized endothelial cells. This study examined the endocytic clearance of AGEs in choriocapillaris endothelial (CCE) cells, which are strategically located beneath the BM. Bovine CCE cultures were incubated with radiolabeled or fluorescently labeled AGE-modified bovine serum albumin (AGE-BSA). Cells and tissues were examined for the expression of two AGE-binding scavenger receptors, stabilin-1 and -2, by immunohistochemistry and Western blotting, and with antibody inhibition studies. CCE cells effectively endocytosed AGE-BSA via a scavenger receptor-mediated pathway. A polyclonal antibody against stabilin-2 significantly inhibited endocytosis (40%). Tissue sections stained positive for stabilin-2 and -1 in the choriocapillaris layer, which was confirmed by immunoblots of cell extracts. Colocalization of stabilin-1 and -2 and internalized AGE-BSA was seen in early endosomes. CCEs actively endocytose AGE-BSA and express the scavenger receptors, stabilin-1 and -2, of which at least stabilin-2 is involved in AGE-BSA uptake. Impairment of this stabilin-mediated clearance function may contribute to depositions of waste macromolecules in BM and subsequent retinopathy.

Substance P and acetylcholine are co-localized in the pathway mediating mucociliary activity in Rana pipiens

Mucociliary activity is an important clearance mechanism in the respiratory system of air breathing vertebrates. Substance P (SP) and acetylcholine play a key role in the stimulation of the mucociliary transport in the frog palate. In this study, retrograde neuronal tracing was combined with immunocytochemistry for SP and choline acetyl transferase (ChAT) in the trigeminal ganglion and for neurokinin-1 receptor (NK1R) in the palate of Rana pipiens. The cells of origin of the palatine nerve were identified in the trigeminal ganglion using the retrograde tracer Fluorogold (FG). Optimal labeling of FG cells in the trigeminal ganglion was obtained at 96 h of exposure. Immunoflorescent shows that SP and acetylcholine are co-localized in 92% of the cells labeled with FG in the trigeminal ganglion. NK1 receptors were found in the membrane of epithelial and goblet cells of the palate. Ultrastructural study of the palate showed axonal-like endings with vesicles in connection with epithelial and goblet cells. These results further support the concerted action of both neurotransmitters in the regulation of mucociliary activity in the frog palate.

Effects of chemotherapy on endogenous erythropoietin levels and the pharmacokinetics and erythropoietic response of darbepoetin alfa: A randomised clinical trial of synchronous versus asynchronous dosing of darbepoetin alfa

The introduction of longer-acting erythropoietic agents into the practice of oncology has demanded an understanding of the interaction of chemotherapy with the pharmacokinetics and haematological effects of these erythropoietins. We report results of a randomised trial comparing the haematological effects of darbepoetin alfa, 6.75 μg/kg, administered once every 3 weeks to anaemic cancer chemotherapy patients on either an asynchronous (day 15) or synchronous (day 1) schedule relative to their every-3-week chemotherapy. A total of 81 patients were randomised and received the study drug (43 asynchronous; 38 synchronous). No difference was observed between groups in the primary endpoint of mean haemoglobin change after 6 weeks of therapy ( P = 0.45) and change scores were similar to those observed with standard weekly darbepoetin alfa therapy. In a subset of patients evaluated with intensive pharmacokinetic sampling, an increase in endogenous erythropoietin concentration (up to 4-fold) lasting approximately 1 week following chemotherapy administration was observed in both groups. Synchronous administration of darbepoetin alfa was associated with a 1.3-fold increase in the area under the darbepoetin alfa concentration–time curve compared with asynchronous administration. Our data suggest that darbepoetin alfa is effective administered every 3 weeks regardless of timing of administration with respect to chemotherapy and that receptor-mediated uptake by the erythron may be an important clearance mechanism for erythropoietic proteins.

Pharmacokinetic and pharmacodynamic considerations when treating patients with sepsis and septic shock.

Sepsis and septic shock are accompanied by profound changes in the organism that may alter both the pharmacokinetics and the pharmacodynamics of drugs. This review elaborates on the mechanisms by which sepsis-induced pathophysiological changes may influence pharmacological processes. Drug absorption following intramuscular, subcutaneous, transdermal and oral administration may be reduced due to a decreased perfusion of muscles, skin and splanchnic organs. Compromised tissue perfusion may also affect drug distribution, resulting in a decrease of distribution volume. On the other hand, the increase in capillary permeability and interstitial oedema during sepsis and septic shock may enhance drug distribution. Changes in plasma protein binding, body water, tissue mass and pH may also affect drug distribution. For basic drugs that are bound to the acute phase reactant alpha(1)-acid glycoprotein, the increase in plasma concentration of this protein will result in a decreased distribution volume. The opposite may be observed for drugs that are extensively bound to albumin, as the latter protein decreases during septic conditions. For many drugs, the liver is the main organ for metabolism. The determinants of hepatic clearance of drugs are liver blood flow, drug binding in plasma and the activity of the metabolic enzymes; each of these may be influenced by sepsis and septic shock. For high extraction drugs, clearance is mainly flow-dependent, and sepsis-induced liver hypoperfusion may result in a decreased clearance. For low extraction drugs, clearance is determined by the degree of plasma binding and the activity of the metabolic enzymes. Oxidative metabolism via the cytochrome P450 enzyme system is an important clearance mechanism for many drugs, and has been shown to be markedly affected in septic conditions, resulting in decreased drug clearance. The kidneys are an important excretion pathway for many drugs. Renal failure, which often accompanies sepsis and septic shock, will result in accumulation of both parent drug and its metabolites. Changes in drug effect during septic conditions may theoretically result from changes in pharmacodynamics due to changes in the affinity of the receptor for the drug or alterations in the intrinsic activity at the receptor. The lack of valid pharmacological studies in patients with sepsis and septic shock makes drug administration in these patients a difficult challenge. The patient's underlying pathophysiological condition may guide individual dosage selection, which may be guided by measuring plasma concentration or drug effect.

INVOLVEMENT OF α-2-MACROGLOBULIN RECEPTOR IN CLEARANCE OF INTERLEUKIN 8–α-2-MACROGLOBULIN COMPLEXES BY HUMAN ALVEOLAR MACROPHAGES

The purpose of this study was to determine if interleukin 8 (IL-8) in complex with α2-macroglobulin (α-2-M) can be taken up by human alveolar macrophages. First, we demonstrated that human alveolar macrophages have receptors for α-2-M but not IL-8. The binding of125I-labeled α-2-M to the cells was specific and saturable, whereas125I-labeled recombinant human IL-8 (rhIL-8) did not bind to macrophages. However,125I-rhIL-8–α-2-M complexes bound to macrophages, and unlabeled α-2-M competed for the binding. We then cultured the cells in the presence of125I-rhIL-8–α-2-M complexes,125I-rhIL-8 alone or buffer for 24h. Macrophages were lysed, and the released radioactivity measured. IL-8 concentrations in supernatants and cells were also measured using an IL-8 ELISA. When the macrophages were incubated with125I-rhIL-8–α-2-M complexes there was a significant amount of IL-8 associated with the cells. However, this was not the case when the cells were incubated with125I- rhIL-8 alone suggesting that only these complexes were taken-up by human alveolar macrophages. Furthermore, the clearance of complexes was specifically inhibited by a monoclonal antibody against the 515-kDa subunit of the α-2-M receptor (α-2-MR) but not by an isotopic mouse IgG1. The study shows an important clearance mechanism for IL-8 in the lung.

Reduced binding and phagocytosis of Pneumocystis carinii by alveolar macrophages from persons infected with HIV-1 correlates with mannose receptor downregulation.

The macrophage mannose receptor, a pattern recognition molecule and component of innate immunity, mediates binding and phagocytosis of Pneumocystis carinii and likely represents an important clearance mechanism in the lungs of immunocompetent hosts. The purpose of this study was to examine the ability of alveolar macrophages from HIV-infected individuals to bind and phagocytose P. carinii, and to investigate the role of the macrophage mannose receptor in mediating this interaction. Compared with healthy individuals, alveolar macrophage phagocytosis of P. carinii from HIV+ persons was reduced up to 74% (P = 0.02), primarily reflecting a reduction in the number of organisms associated with each macrophage (P = 0.019). Furthermore, macrophages from HIV+ individuals demonstrated up to an 80% (P < 0.05) reduction in mannose receptor surface expression and endocytosis. Mannose receptor affinity was unaltered, and mRNA levels were modestly reduced (P < 0.05). Cells from HIV+ individuals with CD4(+) counts < 200 cells/mm3 (representing individuals at high clinical risk for P. carinii pneumonia) demonstrated the lowest levels of P. carinii phagocytosis and mannose receptor endocytosis. In vitro HIV infection of alveolar macrophages from healthy individuals reduced mannose receptor endocytosis to 53.2% (P < 0.05) and P. carinii binding and phagocytosis to 67.4% (P < 0.05) of control. Our studies suggest that HIV infection may alter innate immunity in the lungs, and that impaired alveolar macrophage mannose receptor-mediated binding and phagocytosis of P. carinii may contribute to the susceptibility of HIV-infected individuals to this opportunistic pulmonary pathogen.

Effect of Adrenergic Stimulation on Clearance from Small Ciliated Airways in Healthy Subjects

Mucociliary transport is an important clearance mechanism of larger airways, but in the smallest ciliated airways (bronchioles) it may be less effective. The present study aimed at investigating whether clearance from the bronchioles in subjects tvith healthy airways was stimulated by an adrenergic agonist (terbutaline sulphate). Tracheobronchial clearance was studied twice in 10 healthy subjects after inhalation of 6-μm (aerodynamic diameter) monodisperse Teflon particles labeled with 111In. At one exposure, oral treatment with terbutaline sulphate, known to stimulate clearance in large airways, began immediately after inhalation of the particles. The other exposure was a control measurement. The particles were inhaled at an extremely slow flow, 0.05 L/s, which gave deposition mainly in the small ciliated airways (bronchioles). Lung retention was measured at 0, 24, 48, and 72 h. Clearance was significant every 24 hfor both exposures (p <. 05, two-tailed paired t-test), with similar fractions of retained particles at all time points. During treatment with terbutaline sulphate, the subjects' pulse rates tended to be higher, but clearance rates did not increase. We found, as expected, no significant correlation between lung retention and lung function in either exposure. This study shows that an adrenergic agonist does not significantly influence overall clearance from the bronchiolar region in healthy subjects. This suggests that mucociliary transport does not significantly contribute to clearance from the smallest ciliated airways. Other mechanisms may be more important for the transportation of mucus from these airways.

Human surfactant protein A enhances attachment of Pneumocystis carinii to rat alveolar macrophages.

Pneumocystis carinii (PC) pneumonia remains one of the most important opportunistic pulmonary infections. The alveolar macrophage (AM) is likely the primary cell for recognition and removal of PC. The histopathology of PC pneumonia is characterized by a surfactant-like alveolar exudate. We hypothesize that surfactant protein A(SP-A), the major apoprotein of surfactant, mediates attachment of PC to rat AMs by acting as a ligand between the organism and the AM. In this study, attachment of PC was determined using (51)Cr-labeled PC incubated at 4 degrees C with normal rat AM monolayers in the presence or absence of human SP-A. SP-A significantly enhanced attachment of PC from 14.2 +/- 1.2% to 42.0 +/- 3.8% (P<0.05). This enhanced attachment was visualized and quantified morphologically with confocal microscopy. PC attachment by SP-A was calcium- and mannose-dependent as SP-A-mediated attachment was significantly reduced in the presence of EGTA and mannose to 13.1 +/- 1.6% and 19.3 +/- 2.6%, respectively (P<0.05). Addition of type V collagen and antibodies to SP-A also significantly reduced SP-A-mediated attachment to 4.9 +/- 1.2% and 10.1 +/- 1.2%, respectively (P<0.05). We conclude that SP-A can function as a ligand between PC and the AM and may represent an important detection and clearance mechanism of PC from the alveolar spaces.

A simple assay for ecto-5′-nucleotidase using intact pulmonary artery endothelial cells: Effect of endotoxin-induced cell injury

Adenosine may be protective in acute vascular injury by inhibiting platelet aggregation and neutrophil oxidant release. In contrast, adenine nucleotides, which may be released with acute vascular injury, stimulate platelet aggregation and neutrophil oxidant release. Ectonucleotidases, membrane enzymes that catabolize extracellular nucleotides, are the primary mechanism for degrading circulating nucleotides to adenosine. Ecto-5′-nucleotidase converts extracellular AMP to adenosine. We hypothesized that endothelial cell injury alters ecto-5′-nucleotidase activity. Using a novel assay first reported by Jamal et al. ( Biochem J 250: 369–373, 1988) with rat adipocytes, we studied the properties of ecto-5′-nucleotidase in intact monolayers of cultured bovine pulmonary artery endothelial cells (BPAEC) and examined the effect of endotoxin on enzyme activity. The assay uses a fluorescent analog of AMP, 1, N 6-etheno-AMP (E-AMP), as the substrate for ecto-5′-nucleotidase, and measures ethenoadenosine (E-Ado) formation. Etheno-AMP in Hepes buffer, pH7.4, at 22°, was added to confluent monolayers of BPAEC; samples of supernatant were collected after various intervals, and E-AMP and E-Ado were quantitated by HPLC. Using these methods we found a K m of 15 ± 6 μM, a pH optimum of 7.48, minimal effect of MgCl 2 or CaCl 2 at physiologic pH, and inhibition by α,β-methylene ADP, a known 5′-nucleotidase inhibitor. We established that the monolayer assay was indeed measuring cell surface associated 5′-nucleotidase. To determine the effect of endotoxin, we incubated confluent monolayers with endotoxin in Minimal Essential Medium plus 10% fetal bovine serum for 24 hr, washed them, and assessed the conversion of E-AMP to E-Ado by the endotoxin-injured cells. Endotoxin stimulated endothelial ecto-5′-nucleotidase activity. This increase in 5′-nucleotidase activity in response to endotoxin injury may represent an important clearance mechanism for circulating adenine nucleotides and may be protective in acute vascular injury by increasing adenosine production.

Intracellular Particle Dissolution in Alveolar Macrophages

Aerosol particles deposited in the lungs that are not readily soluble in the epithelial lining fluid will be phagocytized by alveolar macrophages (AM). Inside the phagolysosomal vacuole, the constituents of the plasma allow dissolution of a variety of compounds at a higher rate than dissolution in extracellular lung fluids. Chelator concentration and a pH value of about 5 were found to control intracellular particle dissolution (IPD). Hence, IPD is the initial step of translocation of dissolved material to blood, which is an important lung clearance mechanism for particles retained long term. IPD rates of uniform test particles determined in human, baboon, and canine AM cultures were similar to initial translocation rates determined in lung clearance studies of the same species after inhalation of the same test particles. IPD rate in cultured AM proved to be a sensitive functional parameter of AM, which was used to identify changes in the clearance mechanism of translocation during different exposure conditions.

Intracellular particle dissolution in alveolar macrophages.

Aerosol particles deposited in the lungs that are not readily soluble in the epithelial lining fluid will be phagocytized by alveolar macrophages (AM). Inside the phagolysosomal vacuole, the constituents of the plasma allow dissolution of a variety of compounds at a higher rate than dissolution in extracellular lung fluids. Chelator concentration and a pH value of about 5 were found to control intracellular particle dissolution (IPD). Hence, IPD is the initial step of translocation of dissolved material to blood, which is an important lung clearance mechanism for particles retained long term. IPD rates of uniform test particles determined in human, baboon, and canine AM cultures were similar to initial translocation rates determined in lung clearance studies of the same species after inhalation of the same test particles. IPD rate in cultured AM proved to be a sensitive functional parameter of AM, which was used to identify changes in the clearance mechanism of translocation during different exposure conditions.