Alterations in alveolar basement membranes during postnatal lung growth

We studied the ultrastructural characteristics of alveolar basement membranes (ABM) and capillary basement membranes (CBM) in rat lungs at birth, at 8-10 d of age, during alveolar formation, and at 6-10 wk of age, after most alveoli have formed. We also measured in vitro lung proteoglycan and heparan sulfate synthesis at each age. We noted three major age-related changes in pulmonary basement membranes. (a) Discontinuities in the ABM through which basilar cytoplasmic foot processes extend are present beneath alveolar type-2 cells but not alveolar type-1 cells. These discontinuities are most prevalent at birth but also exist in the adult. (b) Discontinuities are also present in CBM at the two earliest time points but are maximal at 8 d of age rather than at birth. Fusions between ABM and CBM are often absent at 8 d of age, but CBM and CBM/ABM fusions were complete in the adult. (c) Heparan sulfate proteoglycans identified with ruthenium red and selective enzyme degradation are distributed equally on epithelial and interstitial sides of the ABM lamina densa at birth, but decrease on the interstitial side with age. In vitro proteoglycan and heparan sulfate accumulation at birth was two times that at 8 d and five times that in the adult. Discontinuities in ABM allow epithelial-mesenchymal interactions that may influence type-2 cells cytodifferentiation. Discontinuities in CBM suggest that capillary proliferation and neovascularization are associated with alveolar formation at 8 d. When CBM becomes complete and forms junctions with ABM, lung neovascularization likely ends as does the ability to form new alveoli.     

Diaphragmatic activity and lung liquid flow in the unanesthetized fetal sheep.

For some time it has been suggested that breathing movements are made "in utero" and recently measurements of tracheal pressure and lung liquid flow in chronic fetal preparations have led to the hypothesis that rapid changes in these parameters are the result of respiratory muscle activity. To test this hypothesis diaphragmatic electrical activity was measured in seven chronic unanesthetized fetal sheep preparations and correlated with lung liquid flow and tracheal pressure. Diaphragmatic activity led to a fall of tracheal pressure and movement of a small volume of lung liquid into the lung. After the activity ceased, tracheal pressure returned to normal and flow diminished to zero or was directed out of the lung. The breathing pattern was unassociated with the net movement of lung liquid out of the lung. A histogram of the interval between breaths revealed a changing pattern of activity throughout gestation. The pattern was significantly altered after premature delivery of one animal with a respiratory problem. These observations provide evidence that respiratory muscles are active "in utero" and that the pattern of activity changes throughout gestation.     

Upregulation of erythropoietin receptor during postnatal and postpneumonectomy lung growth

Circulating erythropoietin (EPO) stimulates erythrocytosis, whereas organ-specific local EPO receptor (EPOR) expression has been linked to angiogenesis, tissue growth, and development. On the basis of the observation of concurrent enhancement of lung growth and erythrocyte production during exposure to chronic hypoxia, we hypothesized that a paracrine EPO system is involved in mediating lung growth. We analyzed EPOR protein expression in normal dog lung tissue during postnatal maturation and during compensatory lung growth after right pneumonectomy (PNX). Membrane-bound EPOR was significantly more abundant in the immature lung compared with mature lung and in the remaining lung 3 wk after PNX compared with matched sham controls. COOH-terminal cytosolic EPOR peptides, which were even more abundant than membrane-bound EPOR, were also upregulated in immature lung but differentially processed after PNX. Apoptosis was enhanced during both types of lung growth in direct relationship to cellular proliferation and EPOR expression. We conclude that both developmental and compensatory lung growth involve paracrine EPO signaling with parallel upregulation but differential processing of EPOR.     

The lid is a structural and functional determinant of lipase activity and selectivity

In several lipases access to the enzyme active site is regulated by the position of a mobile structure named the lid. The role of this region in modulating lipase function is reviewed in this paper analysing the results obtained with three different recombinant lipases modified in the lid sequence: Candida rugosa lipase isoform 1 (CRL1), Pseudomonas fragi lipase (PFL) and Bacillus subtilis lipase A (BSLA). A CRL chimera enzyme obtained by replacing its lid with that of another C. rugosa lipase isoform (CRL1LID3) was found to be affected in both activity and enantioselectivity in organic solvent. Variants of the PFL protein in which three polar lid residues were replaced with amino acids strictly conserved in homologous lipases displayed altered chain length preference profile and increased thermostability. On the other hand, insertion of lid structures from structurally homologous enzymes into BSLA, a lipase that naturally does not possess such a lid structure, caused a reduction in the enzyme activity and an altered substrate specificity. These results strongly support the concept that the lid plays an important role in modulating not only activity but also specifity, enantioselectivity and stability of lipase enzymes.     

Expression of epidermal growth factor and surfactant proteins during postnatal and compensatory lung growth

We examined whether lung growth after pneumonectomy (PNX) invokes normal signaling pathways of postnatal development. We qualitatively and quantitatively assessed the immunoexpression of epidermal growth factor (EGF), its receptor (EGFR), surfactant proteins (SP) [SP-A and -D and surfactant proproteins (proSP)-B and -C] and proliferating cell nuclear antigen (PCNA) in immature and mature dog lung. We also assayed these proteins in lungs of immature dogs 3 wk or 10 mo after they underwent right PNX compared with simultaneous matched sham controls. During maturation, alveolar cell proliferation is regionally regulated in parallel with EGF and EGFR levels and inversely correlated with SP-A and proSP-C levels. In contrast, post-PNX lung growth is not associated with EGF or EGFR upregulation but with markedly increased SP-A level and moderately increased SP-D level; proSP-B and proSP-C levels did not change. We conclude that 1) signaling of EGF axis and differential regulation of SPs persist during postnatal lung development, 2) post-PNX lung growth is not a simple recapitulation of maturational responses, and 3) SP-A and SP-D may modulate post-PNX lung growth.     

Effects of corticosteroids on postnatal lung and airway growth in the ferret

To investigate the influence of corticosteroids on postnatal lung and airway growth, young male ferrets were given cortisone acetate (20 mg/kg im daily) beginning at 8 wk of age. At 19 wk of age pulmonary function was measured. The lungs were excised for measurements of recoil pressures and wet and dry weights. The dimensions of central and peripheral airways were estimated from analysis of bronchial casts. Corticosteroid-treated animals were shorter and tended to be lighter than control animals but were heavier in relation to length. Total lung capacity was reduced in proportion to the reduction in body size. Lung recoil and wet-to-dry weight ratios were nearly identical. Maximal expiratory flows were reduced in proportion to the reduction in body size. Size-corrected airway conductance was reduced, suggesting a sensitivity of central airways to growth suppression by corticosteroids. Peripheral airways, on the other hand, were not smaller in treated animals and were larger in proportion to body size. In the ferret corticosteroid administration is associated with a suppression of lung parenchymal growth similar to that of overall body growth. The peripheral airways may be less sensitive and the central airways more sensitive to the effect of corticosteroids on growth.     

The collagen repeat sequence is a determinant of the degree of herpesvirus saimiri STP transforming activity

Herpesvirus saimiri (HVS) is divided into three subgroups, A, B, and C, based on sequence divergence at the left end of genomic DNA in which the saimiri transforming protein (STP) resides. Subgroup A and C strains transform primary common marmoset lymphocytes to interleukin-2-independent growth, whereas subgroup B strains do not. To investigate the nononcogenic phenotype of the subgroup B viruses, STP genes from seven subgroup B virus isolates were cloned and sequenced. Consistent with the lack of oncogenic activity of HVS subgroup B viruses, STP-B was deficient for transforming activity in rodent fibroblast cells. Sequence comparison reveals that STP-B lacks the signal-transducing modules found in STP proteins of the other subgroups, collagen repeats and an authentic SH2 binding motif. Substitution mutations demonstrated that the lack of collagen repeats but not an SH2 binding motif contributed to the nontransforming phenotype of STP-B. Introduction of the collagen repeat sequence induced oligomerization of STP-B, resulting in activation of NF-kappaB activity and deregulation of cell growth control. These results demonstrate that the collagen repeat sequence is a determinant of the degree of HVS STP transforming activity.     

Mycobacterial phosphodiesterase Rv0805 is a virulence determinant and its cyclic nucleotide hydrolytic activity is required for propionate detoxification

Cyclic AMP (cAMP) signaling is essential to Mycobacterium tuberculosis (Mtb) pathogenesis. However, the roles of phosphodiesterases (PDEs) Rv0805, and the recently identified Rv1339, in cAMP homeostasis and Mtb biology are unclear. We found that Rv0805 modulates Mtb growth within mice, macrophages and on host-associated carbon sources. Mycobacterium bovis BCG grown on a combination of propionate and glycerol as carbon sources showed high levels of cAMP and had a strict requirement for Rv0805 cNMP hydrolytic activity. Supplementation with vitamin B12 or spontaneous genetic mutations in the pta-ackA operon restored the growth of BCGΔRv0805 and eliminated propionate-associated cAMP increases. Surprisingly, reduction of total cAMP levels by ectopic expression of Rv1339 restored only 20% of growth, while Rv0805 complementation fully restored growth despite a smaller effect on total cAMP levels. Deletion of an Rv0805 localization domain also reduced BCG growth in the presence of propionate and glycerol. We propose that localized Rv0805 cAMP hydrolysis modulates activity of a specialized pathway associated with propionate metabolism, while Rv1339 has a broader role in cAMP homeostasis. Future studies will address the biological roles of Rv0805 and Rv1339, including their impacts on metabolism, cAMP signaling and Mtb pathogenesis.     

N-linked protein glycosylation is a major determinant for basal TRPC3 and TRPC6 channel activity

The TRPC family of receptor-activated cation channels (TRPC channels) can be subdivided into four subfamilies based on sequence homology as well as functional similarities. Members of the TRPC3/6/7 subfamily share common biophysical characteristics and are activated by diacylglycerol in a membrane-delimited manner. At present, it is only poorly understood whether members of the TRPC3/6/7 subfamily are functionally redundant or whether they serve distinct cellular roles. By electrophysiological and fluorescence imaging strategies we show that TRPC3 displays considerable constitutive activity, while TRPC6 is a tightly regulated channel. To identify potential molecular correlates accounting for the functional difference, we analyzed the glycosylation pattern of TRPC6 compared with TRPC3. Two NX(S/T) motifs in TRPC6 were mutated (Asn to Gln) by in vitro mutagenesis to delete one or both extracellular N-linked glycosylation sites. Immunoblotting analysis of HEK 293 cell lysates expressing TRPC6 wild type and mutants favors a model of TRPC6 that is dually glycosylated within the first (e1) and second extracellular loop (e2) as opposed to the monoglycosylated TRPC3 channel (Vannier, B., Zhu, X., Brown, D., and Birnbaumer, L. (1998) J. Biol. Chem. 273, 8675-8679). Elimination of the e2 glycosylation site, missing in the monoglycosylated TRPC3, was sufficient to convert the tightly receptor-regulated TRPC6 into a constitutively active channel, displaying functional characteristics of TRPC3. Reciprocally, engineering of an additional second glycosylated site in TRPC3 to mimic the glycosylation status in TRPC6 markedly reduced TRPC3 basal activity. We conclude that the glycosylation pattern plays a pivotal role for the tight regulation of TRPC6 through phospholipase C-activating receptors.