Proteins reach their extracellular destination by one of two distinct secretory pathways: the constitutive secretory pathway, common to all mammalian cells, is the default pathway in which proteins are rapidly released from the cell by exocytosis; the regulated secretory pathway, present in certain cell types such as neuronal, endocrine, and exocrine cells, is characterized by storage of selected proteins in secretory granules which are released in response to appropriate external stimuli (1, 2). The coexistence of constitutive and regulated secretory pathways within the same cell implies that segregation of proteins must occur, a process which is believed to take place in the trans-Golgi network (3-6). Previous studies have identified selective aggregation and interaction of proteins with the membrane of the trans-Golgi network as important elements in the sorting of proteins away from the constitutive pathway and into the regulated secretory pathway (7-10). To date no sorting receptor has been conclusively identified; however, the ability of different cell types to target heterologous proteins to the regulated secretory pathway supports the existence of a common sorting mechanism (2). Sorting signals recognized by the putative receptor(s) are not encoded in the primary sequence of secretory proteins but are comprised of a motif(s) generated by higher order structure of the molecule (2, 11). Such a sorting signal has recently been identified for chromogranin B and shown to consist of a 20-amino acid loop stabilized by an intramolecular disulfide bond (12, 13).The alveolar type II epithelial cell is a specialized exocrine cell, which synthesizes and secretes pulmonary surfactant, a complex mixture of phospholipids and proteins required for maintenance of alveolar patency. Both the lipid and protein components of surfactant are stored in secretory granules (lamellar bodies), which are released by exocytosis in response to secretagogue stimulation (14, 15). The regulated secretory pathway of the type II cell is atypical in that the lamellar body compartment communicates extensively with the endocytic pathway; up to 85% of surfactant lipids are recycled to the lamellar body for resecretion (16). A further unique characteristic of lamellar bodies is the lysosomal nature of this compartment, including hydrolytic enzymes (17) and at least one lysosomal membrane glycoprotein (18). Sorting determinants mediating the selective transport of secretory proteins to the lamellar body have not been studied previously, and it is consequently unclear if these determinants act in a cell-specific manner. The purpose of the present study was to identify peptide domains required for targeting SP-B 1 to the lamellar body and to determine if these targeting epitopes are recognized by the sorting machinery of both endocrine and neuronal cells.Human SP-B is synthesized by the alveolar type II epithelial cell as a preproprotein of 381 amino acids. Within the proprotein the 79-residue mature peptide is flanked by propeptides of 177 and 10...

Pulmonary surfactant is a complex mixture of lipids and proteins that is synthesized exclusively by the alveolar type II epithelial cell. Surfactant is stored in large inclusions (lamellar bodies) that are secreted into the alveolar airspace by exocytosis. Newly secreted lamellar bodies unravel into a tubular network (tubular myelin) that subsequently adsorbs and spreads as a phospholipid-rich film that reduces surface tension at the air-liquid interface. Rapid adsorption and spreading of the phospholipid film are critical and require the presence of specific proteins, in particular surfactant proteins B and C (1-3).Surfactant protein B (SP-B) 1 is a hydrophobic peptide of 79 amino acids that avidly associates with surfactant phospholipids in the alveolar airspace (reviewed in Ref. 4). Homozygous mutations leading to the complete absence of SP-B in newborn human infants result in the rapid onset of respiratory distress syndrome which is refractory to mechanical ventilation and surfactant replacement (5, 6). The latter observation suggests that simple addition of mature peptide to the SP-B-deficient airway is not sufficient to restore lung function. Therefore, despite an obvious requirement for SP-B in normal lung function, the precise role(s) of SP-B in the structure, function, and metabolism of surfactant remains unclear.Human SP-B is synthesized by the type II epithelial cell as a preproprotein of 381 amino acids. The mature peptide is generated by sequential cleavage of the signal peptide (23 amino acids), the N-terminal propeptide (177 residues), and the Cterminal propeptide (102 residues) (7, 8). Propeptide cleavage occurs within the late endosome/multivesicular body which subsequently directs newly synthesized SP-B to the lamellar body (9); mature SP-B is also recycled from the alveolar airspace, via the endocytic pathway to the multivesicular body (10). Previous in vitro evidence supports the concept that trafficking of the mature peptide through the biosynthetic pathway requires the N-terminal but not the C-terminal domain of the propeptide (11,12). In the absence of both propeptide domains the mature peptide is degraded within the endoplasmic reticulum (11); however, an SP-B construct encoding both the Nterminal propeptide and the mature peptide (hSP-B ⌬c ) produces a truncated proprotein that is sorted to the multivesicular body where the propeptide is appropriately cleaved to liberate mature SP-B (12). These results clearly demonstrate that the N-terminal propeptide is required for the intracellular trafficking of SP-B; the function of the C-terminal propeptide, however, remains unknown.Lung morphogenesis and surfactant phospholipid synthesis in SP-B(Ϫ/Ϫ) mice proceed normally prior to birth (13). However, lamellar body formation is disrupted, resulting in abnormal inclusions consisting of multivesicular bodies and disorganized lamellae. Neither mature lamellar bodies nor tubular myelin were detected in the SP-B(Ϫ/Ϫ) mice. In addition to effects on lamellar body biogenesis, SP-B deficiency resulted in

This study reports the ability of rat alveolar type II cells to internalize mature bovine surfactant protein B (SP-B) in vitro. Isolated type II cells were incubated with labeled SP-B, and binding and internalization were studied biochemically and morphologically. Biochemical analyses demonstrated a time-dependent association of 125I-labeled SP-B with type II cells; binding steadily increased through 4 h and then remained constant through 20 h of incubation. The association of [3H]SP-B with type II cells was characterized via light and electron microscopic autoradiography. Significant quantities of [3H]SP-B were found at the plasma membrane, in the endocytic pathway, and in lamellar bodies. The pathway of SP-B internalization was not altered by the presence of whole rat surfactant; however, the quantity of SP-B internalized into lamellar bodies was increased. 3[H]SP-B was not associated with coated pits and colocalized with horseradish peroxidase (HRP), consistent with receptor-independent internalization. Cell-associated SP-B was not degraded and was detected in lamellar bodies undergoing exocytosis. These results suggest that SP-B may follow a recycling pathway similar to that previously reported for surfactant phospholipids.

ABSTRACT. We studied the acinar distribution for uptake of the bile acid analogue ['251]-cholylglycyltyrosine in livers from adult and 14-day-old suckling rats. Portal and peripheral (systemic) serum bile acid concentrations were also measured by combined gas chromatography-mass spectrometry as an independent index of hepatic bile acid clearance from portal blood. Utilizing light microscopic autoradiography, a steep, decreasing portal to centrilobular gradient for cl~olylglycyltyrosine uptake was noted in adult rat liver. In contrast, there was no lobular gradient for cholylglycyltyrosine uptake visible in the 14-day-rat liver; all hepatocytes within the acinus contained a similar number of silver grains. Portal vein total bile acid concentrations were significantly higher in serum of adult compared to 14-day-old rats. In contrast, bile acid concentrations were 10-fold higher in the peripheral serum of developing versus adult rats. The peripheral to portal serum bile acid concentration ratio was 0.23 in the adult and 6.48 in the 14-day-old rat. We conclude that the entire hepatic lobule participates in the uptake of bile acids in the 14-day-old rat even under the basal conditions of this study. The normal "reserve" function of centrilobular hepatocytes is not sufficient to compensate for the decreased transport capacity of the developing liver with the result that increased concentrations of bile acids enter and accumulate in the systemic circulation. (Pediatr Res 21: 417-421, 1987)

The identification of numerous cyclin-dependent kinases (cdk) and G1 cyclins suggests that cell cycle progression through G1/S may be controlled in a tissue-specific manner by various cdk/cyclin complexes. In situ hybridization was used to characterize expression of the cyclin-dependent kinase cdk4 in prenatal and postnatal rat lung and other tissues and to determine whether cdk4 expression is limited to proliferating cells, identified by BrdU incorporation and cdk1 mRNA expression. cdk4 co-localized with cdk1 in proliferating cells of both prenatal and postnatal lung and other tissues, consistent with an SPF function that is not tissue-specific. The distribution of cdk1 and cdk4 expression was identical in fetal rat tissues and was detected in lung parenchyma and throughout the airway. Pulmonary cell proliferation declined with increasing postnatal age and could be found only in focal areas of day 21 terminal and respiratory bronchiolar epithelium. Proliferation was undetectable in adult lung. Postnatal cdk4 expression was not restricted to cells expressing cdk1: cdk4 was evenly distributed in bronchiolar epithelium and was present throughout the airway and alveolar septae of day 21 lung. Expression of cdk4 was also maintained in adult bronchiolar epithelium. These studies demonstrate that although the expression of cdk1 is tightly correlated with proliferative capacity, the expression of cdk4 is not limited to proliferating cells, suggesting that cdk4 may have additional cell-specific functions unrelated to cell cycle progression.