Bioisosteres at C9 of 2-Deoxy-2,3-didehydro-N-acetyl Neuraminic Acid Identify Selective Inhibitors of NEU3.

Human neuraminidases play critical roles in many physiological and pathological processes. Humans have four isoenzymes of NEU, making selective inhibitors important tools to investigate the function of individual isoenzymes. A typical scaffold for NEU inhibitors is 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA) where C9 modifications can be critical for potency and selectivity against human NEU. To design improved DANA analogues, we generated a library of compounds with either a short alkyl chain or a biphenyl substituent linked to the C9 position through one of six amide bioisosteres. Bioisostere linkers included triazole, urea, thiourea, carbamate, thiocarbamate, and sulfonamide groups. Within this library, we identified a C9 biphenyl carbamate derivative (963) that showed high selectivity and potency for NEU3 (Ki = 0.12 ± 0.01 µM). In contrast, NEU1 and NEU4 isoenzymes preferred amide and triazole linkers, respectively. Finally, analogues with urea, sulfonamide, and amide linkers showed enhanced inhibitory activity for a bacterial NEU, NanI from Clostridium perfringens.

Proteomic insights into Helcococcus kunzii in a diabetic foot ulcer-like environment.

PURPOSE: Helcococcus kunzii is a skin commensal, Gram-positive bacterium, mostly isolated from infected chronic wounds. This opportunistic pathogen is usually co-isolated with Staphylococcus aureus. The present dataset explores the production and secretion of H. kunzii bacterial virulence interacting proteins in a growth medium mimicking chronic wounds in exponential and stationary growth phases. EXPERIMENTAL DESIGN: The H. kunzii cellular proteome and exoproteome were assessed by analyzing three biological replicates per condition tested. Samples were analyzed using a Q-Exactive HF mass spectrometer. Comparative and functional analyses were performed to profile the identified protein set. RESULTS: The H. kunzii's cellular proteome encompassed 969 proteins, among which 64 and 53 were specifically identified in the exponential and stationary phase of growth, respectively. Its exoproteome comprised 58 proteins, among which 16 and 14 were characteristic of each growth stage. Metabolic differences between the two phases of growth are discussed. Besides, the production of previously shortlisted and novel putative H. kunzii targets involved in modulating the virulence of S. aureus is investigated. CONCLUSION AND CLINICAL RELEVANCE: This work, pioneering the study of H. kunzii physiology in a chronic wound-like environment, should assist future research on this opportunistic pathogen and the search for innovative approaches for wound management.

Bacterial Interactions in the Context of Chronic Wound Biofilm: A Review.

Chronic wounds, defined by their resistance to care after four weeks, are a major concern, affecting millions of patients every year. They can be divided into three types of lesions: diabetic foot ulcers (DFU), pressure ulcers (PU), and venous/arterial ulcers. Once established, the classical treatment for chronic wounds includes tissue debridement at regular intervals to decrease biofilm mass constituted by microorganisms physiologically colonizing the wound. This particular niche hosts a dynamic bacterial population constituting the bed of interaction between the various microorganisms. The temporal reshuffle of biofilm relies on an organized architecture. Microbial community turnover is mainly associated with debridement (allowing transitioning from one major representant to another), but also with microbial competition and/or collaboration within wounds. This complex network of species and interactions has the potential, through diversity in antagonist and/or synergistic crosstalk, to accelerate, delay, or worsen wound healing. Understanding these interactions between microorganisms encountered in this clinical situation is essential to improve the management of chronic wounds.

Comparative genomics analysis of two Helcococcus kunzii strains co-isolated with Staphylococcus aureus from diabetic foot ulcers.

Helcococcus kunzii is a commensal Gram-positive bacterial species recovered from the human skin microbiota and considered as an opportunistic pathogen. Although little is known about its clinical significance, its increased abundance has been reported in infected wounds, particularly in foot ulcers in persons with diabetes. This species is usually detected in mixed cultures from human specimens and frequently isolated with Staphylococcus aureus. Modulation of staphylococci virulence by H. kunzii has been shown in an infection model of Caenorhabditis elegans. The aim of this study was to compare the genomes of two H. kunzii strains isolated from foot ulcers -isolate H13 and H10 showing high or low impact on S. aureus virulence, respectively- and the H. kunzii ATCC51366 strain. Whole genome analyses revealed some differences between the two strains: length (2.06 Mb (H13) and 2.05 Mb (H10) bp), GC content (29.3% (H13) and 29.5% (H10)) and gene content (1,884 (H13) and 1,786 (H10) predicted genes). The core-proteome phylogenies within the genus characterised H. kunzii H13 and H10 as genetically similar to their ancestor. The main differences between the strains were mainly in sugar-associated transporters and various hypothetical proteins. Five targets were identified as potentially involved in S. aureus virulence modulation in both genomes: the two-component iron export system and three autoinducer-like proteins. Moreover, H13 strain harbours a prophage inserted in 1,261,110-1,295,549 (attL-attR), which is absent in H10 strain. The prophage PhiCD38_2 was previously reported for its ability to modulate secretion profile, reinforcing the autoinducer-like hypothesis. In the future, transcriptomics or metaproteomics approaches could be performed to better characterize the H13 strain and possibly identify the underlying mechanism for S. aureus virulence modulation.

Effects of Chlorides and Sulphates on Heavy Metal Leaching from Mortar with Raw and Electrodialytically Treated MSWI Fly Ash.

Municipal solid waste incineration (MSWI) fly ash could be used as supplementary cementitious material in cement-based materials. However, heavy metal leaching, such as Cd, Cr, Cu, Pb and Zn, both from the MSWI fly ash and cement-based materials containing MSWI fly ash, remains a persistent obstacle. Here, an up-scaled electrodialytic treatment was used as a pre-treatment to remove heavy metals from MSWI fly ash before using the fly ash in mortar. Mortar samples with 10 wt% replacement of cement with either raw or elecrtodialytically treated MSWI fly ash were subjected to monolithic (in-use scenario) and crushed mortar (end-of-life scenario) leaching tests. The environmental conditions (e.g., exposure to chlorides or sulfates) at the surface of cement-based materials can affect leaching. Acidified H2O, NaCl or Na2SO4 solutions were, therefore, used for the leaching tests. Up to 80% heavy metal removal by the up-scaled electrodialytic pre-treatment was feasible. Regulatory limits for disposing of the MSWI fly ash in non-hazardous waste landfills were exceeded, even if the electrodialytic treatment removed heavy metals. However, leaching from monolithic mortar samples complied with the regulatory limits, while Cr leaching exceeded the regulatory limits for all crushed mortar samples when using NaCl or Na2SO4. Both NaCl and Na2SO4 generally increased the heavy metal leaching yield from fly ash and mortar compared to leaching with acidified H2O. The results of the study suggest that environmental conditions should be taken into account when assessing leaching from cement-based materials with MSWI fly ash.

Strengthening the Reporting of Observational Studies in Epidemiology Using Mendelian Randomization: The STROBE-MR Statement.

Importance: Mendelian randomization (MR) studies use genetic variation associated with modifiable exposures to assess their possible causal relationship with outcomes and aim to reduce potential bias from confounding and reverse causation. Objective: To develop the STROBE-MR Statement as a stand-alone extension to the STROBE (Strengthening the Reporting of Observational Studies in Epidemiology) guideline for the reporting of MR studies. Design, Setting, and Participants: The development of the STROBE-MR Statement followed the Enhancing the Quality and Transparency of Health Research (EQUATOR) framework guidance and used the STROBE Statement as a starting point to draft a checklist tailored to MR studies. The project was initiated in 2018 by reviewing the literature on the reporting of instrumental variable and MR studies. A group of 17 experts, including MR methodologists, MR study design users, developers of previous reporting guidelines, and journal editors, participated in a workshop in May 2019 to define the scope of the Statement and draft the checklist. The draft checklist was published as a preprint in July 2019 and discussed on the preprint platform, in social media, and at the 4th Mendelian Randomization Conference. The checklist was then revised based on comments, further refined through 2020, and finalized in July 2021. Findings: The STROBE-MR checklist is organized into 6 sections (Title and Abstract, Introduction, Methods, Results, Discussion, and Other Information) and includes 20 main items and 30 subitems. It covers both 1-sample and 2-sample MR studies that assess 1 or multiple exposures and outcomes, and addresses MR studies that follow a genome-wide association study and are reported in the same article. The checklist asks authors to justify why MR is a helpful method to address the study question and state prespecified causal hypotheses. The measurement, quality, and selection of genetic variants must be described and attempts to assess validity of MR-specific assumptions should be well reported. An item on data sharing includes reporting when the data and statistical code required to replicate the analyses can be accessed. Conclusions and Relevance: STROBE-MR provides guidelines for reporting MR studies. Improved reporting of these studies could facilitate their evaluation by editors, peer reviewers, researchers, clinicians, and other readers, and enhance the interpretation of their results.

Metabolic adaption to extracellular pyruvate triggers biofilm formation in Clostridioides difficile.

Clostridioides difficile infections are associated with gut microbiome dysbiosis and are the leading cause of hospital-acquired diarrhoea. The infectious process is strongly influenced by the microbiota and successful infection relies on the absence of specific microbiota-produced metabolites. Deoxycholate and short-chain fatty acids are microbiota-produced metabolites that limit the growth of C. difficile and protect the host against this infection. In a previous study, we showed that deoxycholate causes C. difficile to form strongly adherent biofilms after 48 h. Here, our objectives were to identify and characterize key molecules and events required for biofilm formation in the presence of deoxycholate. We applied time-course transcriptomics and genetics to identify sigma factors, metabolic processes and type IV pili that drive biofilm formation. These analyses revealed that extracellular pyruvate induces biofilm formation in the presence of deoxycholate. In the absence of deoxycholate, pyruvate supplementation was sufficient to induce biofilm formation in a process that was dependent on pyruvate uptake by the membrane protein CstA. In the context of the human gut, microbiota-generated pyruvate is a metabolite that limits pathogen colonization. Taken together our results suggest that pyruvate-induced biofilm formation might act as a key process driving C. difficile persistence in the gut.

Perhalogenated Tetraazaperopyrenes and Their Corresponding Mono- and Dianions.

Chlorination and bromination of 2,9-perfluoropropyl-substituted tetraazaperopyrenes (TAPPs) under forcing conditions resulted in fully core-halogenated TAPP derivatives, devoid of hydrogen atoms at the polycyclic aromatic core. The octahalogenation stabilized the reduced mono- and dianionic compounds sufficiently to allow for their characterization. The additional ortho-chlorination led to an improvement of the electron mobility compared to the bay-substituted tetrachloro-TAPP when employed as an n-channel semiconductor in thin-film transistors.

Twisting the TAPPs: Bay-Substituted Non-planar Tetraazapero-pyrenes and their Reduced Anions.

A new synthesis of tetraazaperopyrenes (TAPPs) starting from a halogenated perylene derivative 3,4,9,10- tetrabromo-1,6,7,12-tetrachloroperylene (1) gave access to bay-substituted TAPPs for the first time. Selective lithiation of the bromine-positions and subsequent addition of tosyl azide led to the formation of the tetraazidotetrachloroperylene (2), which was subsequently reduced by addition of sodium borohydride to the corresponding tetraaminotetrachloroperylene (3). Oxidation to its semiquinoidal form 4 and subsequent cyclization with acid chlorides gave rise to a series of bay-chlorinated TAPPs. Whereas the aromatic core of the previously studied ortho-substituted TAPPs was found to be planar, the steric pressure of the two chlorine substituents on each side leads to the twist of the peropyrene core of approximately 30 degrees, a structural feature also observed in other bay-substituted perylene derivatives. An experimental and computational analysis reveals that introducing chloride substituents at these positions leads to slightly increased electron affinities (EA) enabling the selective generation and characterization of the reduced mono-anionic radicals and closed shell di-anionic species. These anions were isolated and characterized by UV/Vis spectroscopy and EPR or NMR, respectively. Processing of the bay-chlorinated TAPPs in n-channel organic TFTs revealed electron mobilities of 0.001 to 0.003 cm2 V-1 s-1 . These reduced electron mobilities compared to the ortho-halogenated TAPPs are thought to be rooted in the less densely packed solid-state structures.

Understanding UV-Vis Spectra of Halogenated Tetraazaperopyrenes (TAPPs): A Computational Study.

The UV-vis absorption and emission spectra of halogenated tetraazaperopyrenes (TAPPs) have been investigated employing second-order approximate coupled cluster (CC2) and (time-dependent) density functional theory (DFT). We have found that the qualitative estimates of (vertical) absorption and excitation energies are possible within a single particle picture based on frontier orbitals, but the single particle picture is not sufficient to achieve quantitative accuracy. Going from the single-particle picture to the many-particle picture improves the agreement with experimental results, but still no satisfying correlation of theory and experiment is obtained. The comparison of CC2- and DFT-based methods reveals that deviations from the experimental results cannot be explained by deficiencies of the electronic-structure methods but rather stem from neglecting vibrational effects. An agreement of theoretical results and experimental spectra is found for adiabatic excitation energies, which are given as energy differences of vibronic states, which are directly accessible using both theoretical and experimental methods. The most pronounced vibronic influence is found for the Stokes shifts, which are significantly overestimated by computing the vertical electronic transitions only. Based on the vibronic contributions, the small Stokes shift of the TAPP compounds can be explained by the temperature dependence of the vibrationally resolved UV-vis spectra.

A comparison of recommendations and received treatment for mood and anxiety disorders in a representative national sample.

BACKGROUND: The exact nature of treatment and management recommendations made, and received, for mood and anxiety disorders in a community population is unclear. In addition, there is limited evidence on the impact of recommendations on actual receipt of treatment or implementation of management strategies. We aim to describe the frequency with which specific recommendations were made and implemented; and thus assess the size of any gap between the recommendation and implementation of treatments and management strategies. METHODS: We used the Survey 'Living with a Chronic Condition in Canada - Mood and Anxiety Disorders (SLCDC-MA), a unique crossectional survey of a large (N = 3358) and representative sample of Canadians with a diagnosed mood or anxiety disorder, which was conducted by Statistics Canada. The survey collected information on recommendations for medication, counselling, exercise, reduction of alcohol consumption, smoking cessation and reduction of street drug use. We also estimate the frequency that recommendations are made and followed, as well the impact of the prior on the latter. We consulted people with lived experience of the disorders to help interpret our results. RESULTS: The results generally showed that most people would receive recommendations, almost all for antidepressant medications (94.6%), with lower proportions for the other treatment and management strategies (e.g. 62.1 and 66% for counselling and exercise). Most recommendations were implemented and had an impact on behaviour. The exception to this was smoking reduction/cessation, which was often not recommended or followed through. Other than with medication, at least 20% of the population, for each recommendation, would not have their recommendation implemented. A substantive group also exists who access treatments, and employ various management strategies, without a recommendation. CONCLUSIONS: The results indicate that there is a gap between recommendations made and the implementation of treatments. However, its size varies substantially across treatments.

Contrasting Frustrated Lewis Pair Reactivity with Selenium- and Boron-Based Lewis Acids.

The activation of π-bonds in diynyl esters has been investigated by using soft and hard Lewis acids. In the case of the soft selenium Lewis acid PhSeCl, sequential activation of the alkyne bonds leads initially to an isocoumarin (1 equiv PhSeCl) and then to a tetracyclic conjugated structure with the isocoumarin subunit fused to a benzoselenopyran (3 equiv PhSeCl). Conversely, the reaction with the hard Lewis acidic borane B(C6 F5 )3 initiates a cascade reaction to yield a complex π-conjugated system containing phthalide and indene subunits.

Chiral carbene-borane adducts: precursors for borenium catalysts for asymmetric FLP hydrogenations.

The carbene derived from (1R,3S)-camphoric acid was used to prepare the borane adduct with Piers' borane 7. Subsequent hydride abstraction gave the borenium cation 8. Adducts with 9-BBN and the corresponding (1R,3S)-camphoric acid-derived carbene bearing increasingly sterically demanding N-substituents (R = Me 9, Et 10, i-Pr 11) and the corresponding borenium cations 12-14 were also prepared. These cations were not active as catalysts in hydrogenation, although 9-11 were shown to undergo carbene ring expansion reactions at 50 °C to give species 15-17. The IBOX-carbene precursors 18 and 19 derived from amino alcohols (S)-valinol and (S)-tert-leucinol (R = i-Pr, t-Bu) were used to prepare borane adducts 20-23. Reaction of the carbenes 1,3-dimethylimidazol-2-ylidene (IMe), 1,3-di-iso-propylimidazol-2-ylidene (IPr) 1-benzyl-3-methylimidazol-2-ylidene (IBnMe), 1-methyl-3-phenylimidazol-2-ylidene (IPhMe) and 1-tert-butyl-3-methylimidazol-2-ylidene (ItBuMe) with diisopinocampheylborane (Ipc2BH) gave chiral adducts: (IMe)(Ipc2BH) 24, (IPr)(Ipc2BH) 25, (IBnMe)(Ipc2BH) 26, (IPhMe)(Ipc2BH) 27, and (ItBuMe)(Ipc2BH) 28. Triazolylidene-type adducts including the (10)-phenyl-9-borabicyclo [3.3.2]decane adduct of 1,3,4-triphenyl-1H-1,2,3-triazolium, rac-29 and the 9-BBN derivative of (S)-2-amino-2'-methoxy-1,1'-binaphthalene-1,2,3-triazolium 34a/b were also prepared. In catalytic studies of these systems, while several species were competent catalysts for imine reduction, in general, low enantioselectivities, ranging from 1-20% ee, were obtained. The implications for chiral borenium cation catalyst design are considered.

Primary determinants of ischaemic stroke/brain abscess risks are independent of severity of pulmonary arteriovenous malformations in hereditary haemorrhagic telangiectasia.

BACKGROUND: Brain abscesses and ischaemic strokes complicate pulmonary arteriovenous malformations (PAVMs). At risk individuals are poorly recognised. Stroke/abscess risk factors have not been defined. METHODS: A cohort study of 323 consecutive individuals with PAVMs (n = 219) and/or the commonly associated condition hereditary haemorrhagic telangiectasia (HHT, n = 305) was performed. Most of the 201 individuals with PAVMs and HHT had no respiratory symptoms, and were unaware they had HHT. Anderson-Gill models assessed constant and time dependent potential predictive variables for stroke/abscess, and rate reduction by PAVM embolisation. RESULTS: 57 individuals with PAVMs and HHT experienced brain abscess or ischaemic stroke, usually prior to the diagnosis of underlying PAVMs/HHT. The primary determinants of stroke and abscess risks were unrelated to severity of PAVMs. Males had higher brain abscess rates (hazard ratio 3.61 (95% CI 1.58, 8.25), p = 0.0024); interventional histories and bacteriological isolates suggested dental sources. Once adjusted for gender, there was a marginal association between brain abscess and low oxygen saturation. For ischaemic stroke, there was no association with any marker of PAVM severity, or with conventional neurovascular risk factors. Surprisingly, low mean pulmonary artery pressure was strongly associated with ischaemic stroke (hazard ratio 0.89 (95% CI 0.83, 0.95) per mm Hg increase; p = 6.2x10(-5)). PAVM embolisation significantly reduced ischaemic stroke rate (p = 0.028); no strokes/abscesses occurred following obliteration of all angiographically visible PAVMs. The mean PAVM diagnosis-treatment interval was longer, however, when neurological risks were unrecognised. CONCLUSIONS: Ischaemic strokes and brain abscesses occur commonly in undiagnosed HHT patients with PAVMs. Risk reduction could be improved.

The independence of lung liquid absorption in postnatal sheep on pulmonary blood flow, blood gases or perfusion pressure.

This study was performed to determine whether the absorption of liquid from the lungs of postnatal sheep is dependent on pulmonary perfusion pressure, blood gases or blood flow. Relationships between perfusion pressure, rate of lung liquid absorption and perfusate PO2, PCO2 and pH were examined by linear regression analysis from in situ perfused lungs from sheep aged 6 weeks to 6 months. The airspaces of the lungs were filled with liquid containing an impermeant tracer, to allow measurement of the rate of liquid absorption. There was no significant relationship between the rate of lung liquid absorption and pulmonary blood flow (n = 36, r = -0.01, P > 0.1), pulmonary perfusion pressure (n = 36, r = 0.28, P > 0.05) or perfusate PO2, PCO2 or pH. No significant relationships were found between pulmonary blood flow and perfusate PO2, PCO2 or pH. There was no evidence to suggest that the absorption of liquid from the lungs of postnatal sheep is dependent on pulmonary blood flow, blood gases or perfusion pressure, within the limits studied, indicating that lung liquid absorption is dependent on the pulmonary epithelium and not on the pulmonary vasculature. The findings that lung liquid absorption continues in hypoxic environments and despite severe reductions in blood flow may be relevant to the field of transplant surgery.

Lack of a role for cyclic nucleotide gated cation channels in lung liquid absorption in fetal sheep.

1. Late gestation fetal sheep were chronically catheterised in utero to allow measurement of the rate of production of lung liquid (Jv) from 132-143 days gestation (term, 147 days), and to test the hypothesis that cyclic nucleotide gated cation channels mediate a component of fetal lung liquid absorption. 2. In eight experiments, 0.5 microg min-1 adrenaline caused a significant (P < 0.005) reduction in Jv from +18. 12 +/- 3.52 to -10.27 +/- 5.26 ml h-1. Dichlorobenzamil (a blocker of cyclic nucleotide gated cation channels) at 1.5 x 10-5 M did not significantly inhibit the adrenaline-induced lung liquid absorption (Jv dichlorobenzamil, -5.77 +/- 2.78 ml h-1; P > 0.1) when the data were grouped, but did exert a significant gestational effect (r = 0. 90, P < 0.01). Subsequent addition of 10-4 M amiloride (a blocker of epithelial sodium channels) abolished the adrenaline-induced absorption of lung liquid (mean Jv amiloride, +6.45 +/- 1.59 ml h-1; P < 0.01 relative to Jv adrenaline and P < 0.005 relative to Jv dichlorobenzamil). 3. In seven experiments, 0.5 microg min-1 adrenaline caused a significant (P < 0.0005) reduction in Jv from +18.95 +/- 2. 98 to -10.08 +/- 3.75 ml h-1. Amiloride (10-4 M) inhibited the adrenaline response (Jv amiloride, +5.46 +/- 1.09 ml h-1; P < 0.005). However, subsequent addition of 1.5 x 10-5 M dichlorobenzamil had no additive effect to that of amiloride (Jv dichlorobenzamil, +4.58 +/- 0.93 ml h-1; P > 0.1). 4. In six experiments, the cGMP analogue 8-Br-cGMP at 10-4 M caused a significant (P < 0.05) reduction in Jv from +15.20 +/- 2.81 to +11.63 +/- 1.71 ml h-1. Amiloride (10-4 M) did not block the effect of 8-Br-cGMP (Jv amiloride, +14.00 +/- 2.49 ml h-1; not significantly different from 8-Br-cGMP). Subsequent addition of 1.5 x 10-5 M dichlorobenzamil also did not block the effect of 8-Br-cGMP (Jv dichlorobenzamil, +11.37 +/- 1.22 ml h-1; not significantly different from either Jv amiloride or Jv 8-Br-cGMP). 5. We conclude that, in fetal sheep, neither adrenaline nor cGMP stimulate lung liquid absorption by actions on cyclic nucleotide gated cation channels, and that the effect of cGMP on fetal lung liquid secretion is minor and does not involve epithelial sodium channels. The effect of dichlorobenzamil, when given before amiloride, was probably due to an action on amiloride sensitive epithelial sodium channels.

A novel role for cyclic nucleotide-gated cation channels in lung liquid homeostasis in sheep.

1. Sheep lungs were artificially perfused in situ with warmed whole oxygenated sheep blood. The airspaces of the lungs were filled with liquid containing an impermeant tracer, to allow measurement of the rate of net transepithelial liquid movement under various conditions. 2. Dichlorobenzamil (1.5 x 10-5 M), a blocker of cyclic nucleotide-gated cation channels, inhibited the resting absorption of lung liquid in sheep aged 6 months (n = 5) (from -36.47 +/- 4.62 to -4.36 +/- 5.27 ml h-1, means +/- s.e.m.; P < 0.005, paired t test). Amiloride (10-4 M), a blocker of epithelial sodium channels, had no additive effect to that of dichlorobenzamil. 3. In the lungs of sheep aged 6 months (n = 4), amiloride (10-4 M) partially inhibited the resting absorption of liquid (from -35.21 +/- 8.57 to -11.05 +/- 4.91 ml h-1; P < 0.05, one-tailed paired t test), and dichlorobenzamil (1.5 x 10-5 M) exerted an additive effect to that of amiloride resulting in secretion at +6.29 +/- 3.05 ml h-1 (P < 0. 01, paired t test). 4. In the lungs of sheep aged 6 weeks (n = 3), amiloride (10-4 M) also inhibited the resting absorption of liquid (from -26.36 +/- 14.05 to -5.17 +/- 8.27 ml h-1; P < 0.05, one-tailed paired t test); however, dichlorobenzamil (1.5 x 10-5 M) did not exert an additive effect to that of amiloride. 5. In the lungs of sheep aged 6 months (n = 4), amiloride (10-4 M) partially inhibited the resting absorption of liquid (from -35.70 +/- 8.58 to -6.79 +/- 4.28 ml h-1; P < 0.05, paired t test), and pimozide (1.5 x 10-4 M), another blocker of cyclic nucleotide-gated cation channels, also exerted an additive effect to that of amiloride, resulting in secretion of lung liquid at +15.36 +/- 9.14 ml h-1 (P < 0.05, paired t test). 6. We conclude that cyclic nucleotide-gated cation channels mediate a component of lung liquid absorption in sheep aged 6 months (but not in sheep aged 6 weeks), and that a mechanism for lung liquid secretion (present in fetuses) is retained at 6 months of age.

The regulation of lung liquid absorption by endogenous cAMP in postnatal sheep lungs perfused in situ.

1. The lungs of two groups of lambs aged 0-2 weeks and 6-12 weeks were artificially perfused in situ with warmed and oxygenated sheep blood. The airspaces of the lungs were filled with liquid containing an impermeant tracer to allow estimation of net liquid movement across the pulmonary epithelium at rest and after administration of certain drugs. 2. Dibutyryl cAMP (dB-cAMP, 10-4 M) stimulated the rate of lung liquid (LL) absorption in the lungs of four neonatal sheep aged 9-12 days, from -1.43 +/- 0.2 to -2.75 +/- 0.3 ml h-1 (kg body wt)-1 (P < 0.05, comparison of regression lines by Student's t test), but had no effect in four juvenile sheep aged 6-12 weeks (P > 0.10). 3. Theophylline, a non-selective phosphodiesterase (PDE) inhibitor (5 x 10-4 M), increased LL absorption from a resting rate of -1.55 +/- 0.3 to -3.62 +/- 0.5 ml h-1 kg-1 in the lungs of four sheep aged 1-12 days and from -1.47 +/- 0.3 to -3.73 +/- 0.4 ml h-1 kg-1 in four sheep aged 6-12 weeks (P < 0.05, Student's paired t test). 4. The beta-adrenergic antagonist sotalol (10-4 M) reduced LL absorption rate from -1.47 +/- 0.1 to -1.22 +/- 0.1 ml h-1 kg-1 (P < 0.01) in the lungs of four sheep aged 4-13 days, while theophylline given after sotalol had no effect. In four sheep aged 6-12 weeks, sotalol had no effect on LL absorption rate, whereas theophylline given after sotalol increased LL absorption rate from -1.06 +/- 0.1 to -1.92 +/- 0.2 ml h-1 kg-1 (P < 0.05). 5. The A1/A2 purinergic receptor blocker 7-(beta-chloroethyl) theophylline (CET; given at 5 x 10-6 M and 10-4 M) had no effect on LL absorption rate in the lungs of four sheep aged 6-12 weeks, confirming that theophylline produced its effect of increasing LL absorption by inhibiting PDE hydrolytic activity. 6. The selective PDE IV (cAMP-specific) PDE inhibitor rolipram was given in the perfused lungs of seven sheep aged 6-12 weeks at doses between 10-8 and 10-4 M, increasing LL absorption rate at concentrations of 10-6 M and above; the half-maximal effective concentration was estimated to be 5.9 x 10-7 M. 7. Rolipram (10-5 M) increased LL absorption rate from -1.99 +/- 0.2 to -3.18 +/- 0.5 ml h-1 kg-1 in the perfused lungs of four sheep aged 6-11 days, and from -1.21 +/- 0.4 to -3.45 +/- 0.3 ml h-1 kg-1 in the perfused lungs of four sheep aged 6-12 weeks (P < 0.05). Sotalol (10-4 M) reduced LL absorption rate from -3.39 +/- 0.8 to -2. 18 +/- 0.4 ml h-1 kg-1 (P < 0.05) in four sheep aged 10-14 days, while rolipram given after sotalol had no effect. In four sheep aged 6-12 weeks, sotalol had no effect on resting LL absorption rate, whereas rolipram given after sotalol increased absorption rate from -1.27 +/- 0.1 to -2.02 +/- 0.6 ml h-1 kg-1 (P < 0.05). 8. We conclude that cAMP mediates a component of LL absorption postnatally, and that while beta-adrenergic stimulation was the sole source of endogenous cAMP in neonates, this was not the case in juveniles, in whom cAMP originated, at least in part, from other sources.