Vijayakumar Boggaram

9 Hormonal and developmental regulation of pulmonary surfactant synthesis in fetal lung

Pulmonary surfactant, a unique developmentally regulated, phospholipid-rich lipoprotein, is synthesized by the type II cells of the pulmonary alveolus, where it is stored in organelles termed lamellar bodies. The principal surface-active component of surfactant, dipalmitoylphosphatidylcholine, a disaturated form of phosphatidylcholine, acts in concert with the surfactant-associated proteins to reduce alveolar surface tension. Relatively large amounts of phosphatidylglycerol also are present in lung surfactants of a number of species, including man. The role of phosphatidylglycerol in surfactant function has not been elucidated; however, its presence in increased amounts in pulmonary surfactant is correlated with enhanced fetal lung maturity. Surfactant glycerophospholipid synthesis in fetal lung tissue is regulated by a number of hormones and factors, including glucocorticoids, prolactin, insulin, oestrogens, androgens, thyroid hormones, and catecholamines acting through cyclic AMP. In studies with human fetal lung in organ culture, we have observed that glucocorticoids, in combination with prolactin and/or insulin, increase the rate of lamellar body phosphatidylcholine synthesis and alter lamellar body glycerophospholipid composition to one reflective of surfactant secreted by the human fetal lung at term. Four surfactant-associated proteins, SP-A, SP-B, SP-C and SP-D, have recently been characterized. Recognition of their potential importance in the reduction of alveolar surface tension and in endocytosis and reutilization of secreted surfactant by type II cells has stimulated rapid advancement of knowledge concerning the structures of the surfactant proteins and their genes, as well as their developmental and hormonal regulation in fetal lung tissue. The genes encoding SP-A, SP-B and SP-C are expressed in a cell-specific manner and are independently regulated in fetal lung tissue during development. SP-A gene expression occurs exclusively in the type II cell and is initiated after 75% of gestation is complete. In the human fetus, expression of the SP-B and SP-C genes is detectable much earlier in development than SP-A, before the time of appearance of differentiated type II cells. It is apparent from studies using human and rabbit fetal lung in culture that cyclic AMP and glucocorticoids serve important roles in the regulation of SP-A gene expression. While the effects of cyclic AMP are exerted primarily at the level of gene transcription in human fetal lung tissue, glucocorticoids have stimulatory effects on SP-A gene transcription and inhibitory effects on SP-A mRNA stability. In addition, cyclic AMP and glucocorticoids act synergistically to increase SP-A gene transcription in human fetal lung in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of ACTH on the stability of mRNAs encoding bovine adrenocortical P-450scc, P-45011β, P-45017α, P-450C21 and adrenodoxin

ACTH treatment of bovine adrenocortical cells in primary culture causes increased accumulation of mRNAs encoding cytochromes P-450 scc , P-450 11 β , P-450 17 α , P-450 C21 and adrenodoxin as well as increased transcriptional activity of their respective genes. In this study we have shown that ACTH does not greatly affect the half-life of mRNAs encoding P-450 11 β , P-450 17 α , P-450 C21 and adrenodoxin. However, in the case of P-450 scc mRNA, ACTH causes a five-fold increase in the half-life leading to a significant stabilization of P450 scc mRNA. Thus it appears that the levels of mRNAs encoding P-450 11 β , P-450 17 α , P-450 C21 and adrenodoxin are regulated by ACTH primarily at the transcriptional level, while that for P-450 scc is regulated at both the transcriptional and post-transcriptional levels.

Regulation of pulmonary surfactant protein synthesis in fetal lung: A major role of glucocorticoids and cyclic AMP

Augmented synthesis of the lipoprotein, pulmonary surfactant, is initiated in fetal lung toward the end of-gestation. Inadequate surfactant synthesis by the lungs of premature infants can result in respiratory distress syndrome, the leading cause of neonatal morbidity and mortality in developed countries. The surfactant-associated proteins act with surfactant glycerophospholipids to reduce alveolar surface tension, and mediate the reutilization of secreted surfactant components by type II cells. Genes encoding the surfactant proteins SP-A, SP-B, and SP-C have been isolated and characterized. Recent findings suggest that surfactant protein gene expression in fetal lung is under multifactortal control and is regulated by glucocorticoids, cAMP, growth factors, and insulin.

Induction of synthesis of bovine adrenocortical cytochromes P-450scc, P-45011β, P-450c21, and adrenodoxin by prostaglandins E2 and F2α and cholera toxin

To further elucidate the mechanisms by which ACTH (adrenocorticotropin) exerts its long-term action to maintain normal levels of adrenocortical cytochromes P-450 and related enzymes, the abilities of cholera toxin and prostaglandins E2 and F2 alpha to induce the synthesis of cytochromes P-450scc, P-45011 beta, and P-450C21 and adrenodoxin have been examined. These effectors stimulate the production of cyclic AMP and thus steroidogenesis in the adrenal cortex. Using bovine adrenocortical cells in primary monolayer culture, we have shown that treatment with cholera toxin results in increased synthesis of cytochromes P-450scc and P-45011 beta and adrenodoxin, similar to the effect observed upon ACTH treatment. Prostaglandins E2 and F2 alpha are less effective at inducing the synthesis of the mitochondrial cytochromes P-450, and do not seem to induce the synthesis of adrenodoxin. Furthermore, cholera toxin was found to be less effective at inducing the synthesis of microsomal cytochrome P-450C21 than ACTH, and no more effective than the prostaglandins. Thus, while it appears that elevation of cyclic AMP levels is a necessary step leading to increased synthesis of adrenocortical forms of cytochrome P-450, the detailed mechanism of this induction will be found to be different for each of the different enzymes.

Studies on rhodanese synthesis in bovine adrenocortical cells.

The synthesis of adrenodoxin, a mitochondrial iron-sulfur protein required for adrenocortical steroidogenesis, is known to be regulated chronically by ACTH. Rhodanese, also a mitochondrial enzyme, is thought to be required for synthesis of iron-sulfur centers, such as those contained in adrenodoxin. In this study it has been found that rhodanese synthesis and activity are not regulated by ACTH, under the same conditions whereby ACTH induces adrenodoxin synthesis. In addition, unlike adrenodoxin, rhodanese is found to be synthesized in the mature form rather than as a higher molecular weight precursor protein.

Molecular Cloning of Steroid Hydroxylases

Recombinant plasmids specific for bovine adrenocortical cytochromes P-450scc and P-45011 beta have been identified and characterized. Using these cDNA inserts as probes, it is found that tissue specificity of gene expression for these two proteins is as expected. Cytochrome P-450scc mRNA is found in adrenocortical and corpus luteum RNA while cytochrome P-45011 beta mRNA is found only in adrenocortical RNA. Neither mRNA was detected in heart, liver or kidney RNA. Cytochrome P-450scc mRNA is found to be 1.9 kb in length while cytochrome P-45011 beta mRNA is found to be 4 kb in length. Treatment of bovine adrenocortical cells with ACTH or other modulators results in increased levels of cytochrome P-450scc and cytochrome P-45011 beta mRNAs. Cytochrome P-450scc is found to be encoded by a limited number or even a single gene.

β-ADRENERGIC AGONISTS AND cAMP ANALOGUES MARKEDLY INCREASE THE LEVELS OF SURFACTANT APOPROTEIN AND ITS mRNA AND INDUCE DRAMATIC MORPHOLOGIC CHANGES IN HUMAN FETAL LUNG IN VITRO

The use of β-adrenergic agonists for treatment of preterm labor is associated with a decreased incidence of RDS in premature newborns. In the present study, antibodies and a cDNA probe specific for the major surfactant apoprotein were used to evaluate the effects of Bt2 cAMP and the β-agonist, terbutallne, on the levels of surfactant apoproteln and its mRNA in human fetal lung in organ culture, using Western and Northern blotting. A marked stimulatory effect of Bt2 cAMP on the levels of surfactant apoproteln and mRNA in the fetal lung tissue was observed within 48 h of its addition to the medium. Terbutaline caused a dose-dependent increase in surfactant apoproteln and mRNA to levels comparable to those observed after treatment with Bt2cAMP. Using light and electron microscopy, we found that after 48 h incubation with Bt2cAMP, a significantly greater proportion of the ductular epithelium of the fetal lung explants was comprised of type II cells; these ducts were enlarged greatly and the interalveolar connective tissue reduced as compared to control explants. Abundant secretory material (lamellar bodies and tubular myelin) was observed within the ducts of the Bt2cAMP-treated tissue; little secretory material was observed in control tissues. Thus, catecholamines acting through β-adrenergic receptors and cAMP, may serve an important role in surfactant apoprotein gene expression and in morphologic development of the human fetal lung.