The permeability of junctional complexes to ultrastructural tracers of different molecular weight and the freeze-fracture appearance of junctional structure were investigated in the resting and stimulated rat parotid gland . Tracers were administered retrogradely via the main excretory duct, and allowed to flow by gravity (16 mmHg) into the gland for 15-60 min . Secretion was induced in some animals by intraperitoneal injection of isoproterenol . In resting glands, the tracers microperoxidase, cytochrome c, myoglobin, tyrosinase (subunits), and hemoglobin were restricted to the luminal space of the acini and ducts . i n glands stimulated 1-4 h before tracer administration, reaction product for microperoxidase, cytochrome c, myoglobin, and tyrosinase was found in the intercellular and interstitial spaces, whereas hemoglobin was usually retained in the lumina. In contrast, horseradish peroxidase and lactoperoxidase appeared to penetrate the tight junctions and reaction product was localized in the extracellular spaces in both resting and stimulated glands . Diffuse cytoplasmic staining for horseradish peroxidase and lactoperoxidase was frequently observed in acinar and duct cells . The distribution of horseradish peroxidase was similar in both Sprague-Dawley and Wistar-Furth rats, and at concentrations of 0.1-10 mg/ml in the tracer solution . Freeze-fracture replicas of stimulated acinar cells revealed an increased irregularity of the tight junction meshwork, but no obvious gaps or discontinuities were observed. These findings indicate that (a) tight junctions in the resting rat parotid gland are impermeable to tracers of molecular weight ?1,900 ; (b) stimulation with isoproterenol results in a transient increase in junctional permeability allowing passage of tracers of molecular weight x34,500; (c) junctional permeability cannot be directly correlated with junctional structure; and (d) the behavior of horseradish peroxidase and lactoperoxidase in the rat parotid gland is inconsistent with their molecular weights . Cell membrane damage due to the enzymatic activity or binding of these two tracers may account for the observed distribution .

The ability of the intralobular duct cells of the rat parotid gland to take up protein from the lumen was examined by retrograde infusion of exogenous proteins and by immunogold localization of endogenous secretory proteins. Small amounts of native horseradish peroxidase (HRP) were taken up by intercalated and striated duct cells, and were present in small vesicles, multivesicular bodies, and lysosomes. In contrast, HRP modified by periodate oxidation was avidly internalized by the duct cells and was present in large apical vacuoles that acquired lysosomal hydrolase activity. Native and cationized ferritin were taken up in a similar manner when infused at a high concentration (up to 10 mg/mL). At lower concentrations (0.3-1.0 mg/mL), endocytosis of cationized ferritin occurred mainly in small apical tubules and vesicles in striated duct cells. Little native ferritin was taken up at these concentrations. After stimulation of acinar cell secretion by isoproterenol, similar vacuoles were occasionally observed in both intercalated and striated duct cells. Labeling of thin sections with antibodies to amylase and to a 26,000-dalton secretory protein (protein B1), followed by protein A-gold, revealed the presence of these proteins in the vacuoles, indicating endocytosis of acinar secretory proteins by the duct cells. Although uptake of acinar proteins by duct cells occurs at a low rate in normal animals, previous work suggests that extensive endocytosis may occur in certain pathological conditions. This may be a mechanism for removing abnormal or modified proteins from saliva before it reaches the oral cavity.

The biogenesis of basement-membrane components was investigated in the endodermal cells of the rat parietal yolk sac in 12.5-day pregnant rats; 3H-proline was injected into conceptuses. After various time intervals, the parietal yolk sac, including endodermal cells and the associated Reichert's membrane, was removed and processed for electron-microscopic radioautography. Silver grains were counted over endodermal cell organelles and Reichert's membrane. At 2 and 5 min after 3H-proline injection, endodermal cells showed heavy labeling in rough endoplasmic reticulum (rER). Silver grain density over the rER decreased from 2 to 20 min and then remained at a plateau. Grain density was moderate over the Golgi apparatus initially but rose to a peak at 2 hr and decreased by 4 hr and later. Grain density was negligible over secretory granules at 2 and 5 min and increased moderately with time to reach a maximum at 8 hr. Thus, radioautographic peaks occurred sequentially in rER, Golgi apparatus, and secretory granules. By 4 hr and later, silver grains accumulated over Reichert's membrane. These results indicated that endodermal cells incorporated labeled proline into substances which were processed from the rER through the Golgi apparatus, transported from there to the cell surface by secretory granules, and released for export to Reichert's membrane. To clarify the nature of the exported substances, the amount of label present in proline and hydroxyproline residues after 3H-proline injection was measured in Reichert's membrane with or without the associated endodermal cells. Within the cells, 61.8% of the labeled proteins were classified as "sedentary" and 38.2% as "exportable." Of the label exported to Reichert's membrane, 66.3% consisted of type IV collagen and the rest of other basement-membrane components. The results obtained with this model suggest that basement-membrane proteins, including type IV collagen, are elaborated by the associated cells through the classical pathway: rER-Golgi apparatus-secretory granules.

The permeability of tight junctions in the rat parotid gland to the ultrastructural tracer myoglobin (m.w. 17,800) was investigated after in vivo stimulation by autonomic drugs. At various times after administration of beta-adrenergic (isoproterenol), alpha-adrenergic (methoxamine), or cholinergic (methacholine) agonists, the parotid duct was cannulated, and a solution of myoglobin was allowed to flow by gravity (16 mm Hg) into the gland for one hr. In resting glands, cytochemical reaction product for myoglobin was localized in the luminal space of acini and ducts. The tracer was also localized to the luminal space after stimulation with methacholine and methoxamine. In contrast, one to four hr after isoproterenol stimulation, reaction product was present in the intercellular and interstitial spaces of the gland. At later times after stimulation (from six to 24 hr), the tracer was again restricted to the luminal space of the acini and ducts. These results indicate that isoproterenol stimulation causes a transient increase in tight junctional permeability, whereas stimulation with methoxamine or methacholine does not change junctional permeability to myoglobin.