At a late stage of fetal development, the mammalian alveolar epithelium undergoes an abrupt differentiation as a part of the preparation of the lung for the postnatal demands of gas exchange. Some of the most striking changes occur in the type II pneumocytes as they lose their glycogen and start to produce the lamellated inclusion granules that contain pulmonary surfactant. Premature birth before adequate type II cell maturation results in the neonatal respiratory distress syndrome, which is frequently fatal. We have used serial ultrathin sectioning, electron microscopy, and three-dimensional reconstructions to study the ultrastructural features of maturation of rat type II cells from a single rat each at age gestational day 20 through adult stages. We found evidence over this time span for compartmentation of several secretory granule precursors within type II cells. Changes in the polarization of lamellar bodies were observed over the time period studied. We also found marked gestational changes in the number and morphology of type II cell cytoplasmic processes that perforate the basement membrane. Type II cell mitochondria changed in shape during postnatal development from single, spherical to complex, branched structures. Volume composition obtained from serial sections of a small number of type II cells agreed closely with published morphometric data, indicating that throughout the animal's lifespan, type II cells are a homogenous population.

ABSTRACT. Preparation by the developing alveolar epithelium for the transition to air breathing and surfactant secretion at birth are critical components of neonatal survival. We combined morphometric analysis and biochemical assays of lung phospholipids to measure the amount and redistribution of lung surfactant during the perinatal period of rats. Within 10 min of the start of air breathing, there was a small increase in type I1 cell lamellar body content by morphometric and biochemical estimates. By 24 h, the whole lung and alveolar extracellular pool surfactant lipid had substantially increased. Subfractionation of the alveolar surfactant pool obtained at four times, from birth to 24 h of life, demonstrated a 20-fold increase in the ratio of phospholipid in a tubular myelin-rich fraction compared to a unilamellar vesicle-rich fraction. We conclude that packaging of surfactant may be very active immediately postbirth. Our results also indicate a major shift in the physical forms of extracellular surfactant during the first hours of air breathing. (Pediatr Res 21: 5-9, 1987) Abbreviations quantifying changes in the intracellular and extracellular pools of surfactant-associated phospholipid in the late fetal and newborn period, using morphometric and biochemical assays. METHODS Animals.Timed pregnant (previously virgin) Sprague-Dawley females (Zivic-Miller, Allison Park, PA) were received 5 to 7 days prior to parturition. During their late stages of pregnancy, they were allowed food and water ad libitum and were kept in 12-h light-dark cycles. The onset of labor was defined as behavior including nest building and agitation along with vaginal bleeding. Birth began shortly after nesting was complete and was allowed to progress uninterrupted until three to six fetuses were delivered. The air-breathing newborns were kept with the unanesthetized mother. The mothers were then anesthetized with 30-50 mg/kg pentobarbital intraperitoneally; the remaining fetuses were delivered individually through a hysterotomy. These fetuses (birth group) were more lethargic, but made limb motions. They were still connected to the placenta and made no respiratory efforts. The mothers continued spontaneous breathing during the entire procedure. DSPC, disaturated phosphatidylcholineNo more than 10 min passed before the newborns (+lo min PL, phospholipid group) were also anesthetized with intraperitoneal pentobarbital, LDH, lactate dehydrogenase the thoracic contents were rapidly removed and lung wet weight was obtained. Two or three unlavaged lungs were pooled for the LB DSPC' lamellar body disaturated phosphatidylcholine ~h o s~h o l i p i d determinations in all fetal animals. Pooled s a m~l e s -A -from these animals were regarded as a single determination. Since the +10 min group were expected to have developed in the lower poles of the uterus, we obtained lungs from fetuses in Immediately after birth, the newborn must transition to air the two lowest and the two highest uterine positions of four breathing and the effects this has on...

V5Z lL7.Specific binding of a2-adrenergic ligands f3H] clonidine and [~HIRX 781097 was observed w i t h p l a m manbrane fragments isolated from interscapular brawn adipose tissue of 7-day-old rats.Scatchard and W o l f analyses of the data and results on specific d i s p l a c m t of DI HI norepinephrine by l m wncentrations of epinephrine and yohimbine were wnsistent with the existence ill brown adipocytes of a haegeneous class of the binding sites. Campetition experiments on b t h a-agonists and a-antagonists binding were consistent with pharmacological definition of the a2-adrenoreceptor subtype. Chemical denervation by 6-hydroxydopamine resulted in a more pronounced distinction of the hu affinity states of the receptors. A tvc-slope Scatchard plot showed mean apparent I(D of 29 and 255 M for the c i a and a 2~, respectively. The maxirmm nunber of sites was scmwhat higher (0.58 pl/nq protein) than in control animals. Pre-treatment of infant rats with yohimbine (7 days of 10 mJ/kg b.w. daily) has resulted in increased specific binding of [ 3~ yohimblne prenolol seen to indicate "hybrid" characteristics. Campetition binding studies m y be interpreted in terms that toth the aand @-adrenoreceptors in brown adipocytes of infant rats are "hybrid" species which m y in fact reside within integrated protein molecules.(Supported by Canadian Medical Research Council Grant MA-7217).-. INDUCTION O F T H E M A J O R S U R F A C T A N T APOPRO-MENT. Jeanne M. Sn der and Carole R. The major apoprotein associated with lung surfactant is a 35 kDa, sialoglycoprotein with an isoelectric point (pl) of 4.5.We used antibodies directed against the major rabbit lung surfactant apoprotein to characterize the induction of this protein during rabbit fetal lung development. Proteins from homogenates of fetal lung tissue were separated by two-dimensional (2D) gel electrophoresis, transferred to nitrocellulose paper and analyzed using an immunoblot technique. A n immunoreactive apoprotein of 26 kDa, pl 4.5, was first detectable on day 24 of gestation. Based on its migration in 2D gels, the protein did not appear to contain sialic acid. The 35 kDa form of the protein was first detectable in lung homogenates from day 30 fetal rabbits. Lamellar bodies (LB), the storage form of surfactant in the type I I cell, are first observed on day 26 of gestation. L B were purified from lung tissues of fetuses of 28 and 30 days gestation and their apoproteins were analyzed using immunoblot techniques. The apoprotein associated with the purified LB was the 35 kDa form of the protein. Therefore, a post-translational modification of the 26 kDa protein, possibly the addition of sialic acid residues, may be required for the association of the surfactant apoprotein with LB in the fetal lung type II cell. Newborn offspring of streptozotocin-diabetic rats have surprisingly superior tolerance to hyperoxic exposure compared to control offspring (Fed. Res. g:298A,1984). MECHANISMS FOR SUPERIOR HYPEROXIC TOLERANCE IN NEW-By day 13 of exposure to 95-100% 02, 79% of s...