Freeze-fracture and lead ion tracer evidence for a paracellular fluid secretory pathway in rat parotid glands.код для вставкиСкачать
THE ANATOMICAL RECORD 208169-80 (1984) Freeze-Fracture and Lead Ion Tracer Evidence for a Paracellular Fluid Secretory Pathway in Rat Parotid Glands J.A.V. SIMSON AND H.L. BANK Departments ofAnatomy (J.A.VS.) and Pathology (H.L.B.), Medical University of South Carolina, Charleston, S C 29425 ABSTRACT The morphology and permeability of tight junctions of the three major epithelial constituents of rat parotid gland-acinus, intercalated duct, and striated duct-have been examined ultrastructurally. Acinar and intercalated duct junctions (including those surrounding intercellular canaliculi) averaged two to three sealing strands, whereas striated duct junctions had five to eight sealing strands. When the permeability of the junctional complex was probed by means of a recently devised lead ion tracer technique, acinar junctions were found to be very permeable, intercalated duct junctions were somewhat permeable, and striated duct junctions were essentially impermeable to the tracer. Thus, by both morphological and tracer-permeability criteria, acinar tight junctions appear to be “leaky.” These data provide strong evidence that, in rat parotid glands, a potential paracellular secretory pathway exists in the acinar region for the transepithelial passage of fluid. Saliva consists of a “carrier fluid” (composed ofwater and small inorganic ions) which contains a variety of dissolved macromolecules. The carrier fluid is essential for the solubilization of secretory macromolecules and for delivery of active secretory components to their site of action. The intracellular synthesis and transfer of the macromolecular components of exocrine secretions have been studied in detail (Castle et al., 1972; Palade, 1975);however, the routes and mechanisms of transfer of water and ions (the “micromolecular components” of secretion) are less well understood. Certain diseases such as cystic fibrosis and glaucoma apparently involve abnormalities of water and ion transfer into or out of secretory fluid. In order to understand the morphological basis of secretory alterations in these diseases, it is essential that the routes and mechanisms of fluid and ion transfer be elucidated. Rodent salivary glands are a n excellent model for dissecting the mechanisms for secretion of fluid and macromolecules, since P-adrenergic stimulation of these glands elicits a predominantly macromolecular secretory response, whereas cholinergic and a-adrenergic stimulation elicit a copious secretion which is low in macromolecules (see Peterson, 1973; Young et al., 1979; Abe and Dawes, 1980; Simson, 1982). 0 1984 ALAN R. LISS, INC. The morphology of the three main epithelial segments of rodent parotid gland has been previously described (Scott and Pease, 1959; Rutberg, 1961; Simson, 1969a) and is diagramed in Figure l.The acinus is composed of pyramidal cells containing abundant rough endoplasmic reticulum and stored secretory granules. These granules are rapidly discharged into the lumen in response to secretory stimuli such as isoproterenol (Simson, 1969b). The intercalated duct is a short segment of low cuboidal cells which often contain a few apical granules that are not discharged in response to isoproterenol. This segment is surrounded by myoepithelial cells which are presumed to contract during a secretory response (see Young and van Lennep, 1978; Emmelin, 1981). The morphology of the striated duct is typical of the morphology of cells specialized for ion transport (see Berridge and Oschman, 1972).Specifically, the cells of this segment possess extensively invaginated and interdigitated basolateral plasma membranes lined by numerous mitochondria. This segment has been demonstrated to reabsorb sodium from the luminal fluid (see Schneyer et al., 1972).Salivary gland acinar cells do not conform to the morphology expected of iontransporting epithelia. However, the primary Received November 1, 1982; accepted September 2, 1983. 70 J.A.V. SIMSON AND H.L. BANK Acinus Intercalated Duct Striated Duct Fig. 1. Diagram of the three major epithelial cell types in the salivary gland secretory unit. The acinus forms the initial segment of the secretory lumen, which has extensions between acinar cells known as intercellular canaliculi (*I. Acinar cells possess abundant secretory granules, while intercalated duct cells have few granules. The nuclei of striated duct cells are displaced api- cally by the elaborate basal membrane infoldings, which are lined by mitochondria. Occluding junctions (tight junctions) are indicated by a thickening of the membrane line between cells; adhering junctions (zonulae adherentes and desmosomes) are indicated by the “whiskered’ regions. secretory product of these glands, including the carrier fluid, is apparently elaborated in the vicinity of the acinus-intercalated duct junction (Martinez et al., 1966; Young, 1973; Mangos et al., 1981). Recent evidence has suggested that, in certain epithelia, a paracellular (extracellular) pathway probably serves as a route for ion and water flow (Fromter and Diamond, 1972; Spring and Hope, 1978; Jansen et al., 1980; Ernst et al., 1980). In the present study, we have used both freeze-fracture and a lead-ion tracer tracer techniques (Simson and Dom, 1983) to investigate junctional morphology and permeability in the three main epithelial segments of the rat parotid gland. Based upon the number of tight junctional sealing strands observed in freeze-fracture preparations, we conclude that a potential paracellular secretory pathway exists between acinar cells and may exist between intercalated duct cells, but does not exist between cells of the striated duct. In addition, lead tracer deposition patterns indicate that junctions between acinar cells are highly permeable, junctions between intercalated duct cells are less permeable, and junctions between striated duct cells are essentially impermeable to this inorganic cationic tracer. MATERIALS AND METHODS Routine ultrastructural morphology was obtained from parotid glands of perfusionfixed, anesthetized female rats (SpragueDawley) weighing about 200 gm. The primary fixative was 1%glutaraldehyde, 3% paraformaldehyde in 0.1 M sodium phosphate, pH 7.0. After initial perfusion of the fixative (8 minutes) via the aorta, glands were removed and immersed in fresh fixative for 23 hours, rinsed briefly (2 minutes) in phosphate buffer, then transferred to 1% OsO4 in 0.1 M collidine buffer for 1 hour. Tissues were rinsed briefly in distilled water, dehydrated in graded alcohols and propylene oxide, infiltrated and embedded in epoxy resin. Thin sections were stained with uranyl acetate and lead citrate (UA-LC). The lead ion tracer technique has been described in detail elsewhere (Simson and Dom, 1983). This method utilizes a soluble, inorganic cation as a n in vivo tracer. Since this tracer is much smaller than the enzymes (e.g., horseradish peroxidase or microperoxidase) usually used in in vivo tracer studies, it rapidly distributes into compartments in functional continuity with the vasculature and the extracellular space. The technique PARACELLULAR SECRETORY PATHWAY IN PAROTID involves whole animal perfusion with a blunted, 18-gauge needle via the ascending aorta. The tracer sequence is designed to 1) flush the cells from the vasculature and replace reactive anions (e.g., Cl-), with a 0.15 M sodium acetate preperfusion fluid 2) briefly (1 minute) perfuse with a lead ioncontaining tracer fluid (0.02 M Pb acetate in 0.13 M sodium acetate); 3) perfuse the vasculature to remove the free tracer with a 0.15 M sodium acetate rinse fluid; and 4)fix the tissue and simultaneously capture the lead ion tracer, with 2% glutaraldehyde buffered with 0.1 M sodium phosphate. All perfusion solutions were between pH 7.0 and 7.4 and were brought to room temperature before use. Perfusion solutions were maintained a t a constant height above the animal such that the hydrostatic pressure at delivery was between 85-110 mm Hg (see Simson and Dom, 1983).After 5 minutes tissues were removed and fixed for a n additional 55 minutes in the same fixative as that used for perfusion. This was followed by postfixation in phosphatebuffered osmium tetroxide. Dehydration, infiltration, and embedding were performed as described above. Either en bloc or thin section staining with uranyl acetate was performed on some of the material. To quantify permeability of junctions to the lead tracer, all micrographs of material perfused with tracer for 1 minute were scored for luminal deposits in acini, intercalated ducts, and striated ducts. In acini, most lumina were intercellular canaliculi and hence, bounded by only two junctions. In this segment, the numbers of tracer-containing and empty lumina were counted and the percentage of lumina which contained tracer deposits was calculated. In intercalated ducts and striated ducts, the number of tracer deposits clearly on the luminal side of a junction (anywhere within the lumen) were counted and expressed as a percentage of the total number of junctions surrounding each lumen. Freezefracture studies were carried out on tissues that had been taken from a second series of animals fixed by perfusion with 1% paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.0, at room temperature. Tissues were fixed for 1hour, rinsed for ?hhour in 0.1 M phosphate buffer and gradually infiltrated in ascending concentrations of glycerol. Tissues were equilibrated overnight in 30% glycerol a t 4"C,then cut or teased into very small pieces, loaded in gold hinges and quench-frozen in resolidifying 71 propane cooled by liquid nitrogen. The individual sample hinges were inserted into a multiple sample holder under the surface of liquid nitrogen and the holder transferred to the precooled support stage of the freeze-etching unit. Freeze-etching was performed in a modified Denton DFE-2 freeze-etch unit mounted on a Varian ion pumping station. The samples were fractured a t -125°C 5°C and immediately replicated (Bank and Robertson, 1976). After replication, the samples were warmed to -3O"C, removed from the vacuum system, and the adhering biological material digested in sodium hypochlorite. Following extensive washing, the samples were recovered on 75-mesh Formvar-coated grids (Bank et al., 1978; Briggman et al., 1981). Sections and replicas were viewed on either a n Hitachi HS-9 or a JEOL 100 S electron microscope operated a t 60 or 80 kV, photographed at magnifications ranging from 7,000 to 40,000, and photographically enlarged between two- and sixfold. Well-focused,freeze-fracture micrographs of junctions were used for measuring junctional depth and for counting sealing strands (Table 1).A line was drawn perpendicular to the luminal surface at the midpoint of the exposed junction. In very extensive junctions (more than 3 pm in length parallel to the luminal surface), two such lines were drawn, each a third of the way from each end of the junction. The distance between the luminal strand (usually a t the luminal surface) and the basal edge of the basalmost sealing strand was measured with a n ocular micrometer graduated with a 0.1-mm scale. These measurements were converted to pm (the junctional depth) using a conversion factor appropriate for the magnification of the micrograph. The number of sealing strands intersecting the perpendicular line (including the most luminal and basal strands) was also counted. RESULTS Normal Morphology Junctional complexes between acinar cells (Fig. 2 ) separated the lateral intercellular space from the acinar lumen and from intercellular canaliculi. These canaliculi (see Fig. 1)are fingerlike extensions of the acinar lumen between acinar cells. Between acinar cells, the region of close apposition of cell membranes (tight junction or zonula occludens) was short and was usually accom- 72 J.A.V. SIMSON AND H.L. BANK TABLE 1. Characterization of salivary gland junctions Number of sealing strandsa Epithelial segment Depth (pmIa X+O X f O Acinus 0.25 5 0.15 Intercalated duct (N = 28) 0.21 0.05 Striated duct (N = 14) 0.43 k 0.11 (N = 13) 2.5 f 0.8 (N = 28) 2.5 0.6 (N = 14) 6.0 5 1.6 (N = 13) + Percent of junctions with precipitatesb 63% = 178) 9% (N = 64) 5% (N = 40) (N aN is the number of identifiable junctions analyzed. The means of the measurements on striated duct junctions were significantly different (c< 0.01%confidence interval) from the means of acinar and intercalated duct junctions in terms of both depth of junctzon and number of sealing strands. There were no significant differences between acinar and intercalated duct cell junctions for either arameter. 'The method used for evaluating permeable junctions is described in Materials and Methods. N is either the total number of lumens (intercellular canaliculi) of acini or the total number of apical junctions in the ductal segments. panied by a single underlying desmosome. Between intercalated duct cells, the region of close apposition was also short (Fig. 3) and numerous desmosomal attachments were seen basal to the tight junctions. Between the cells of the striated duct, a long region of close apposition characterized apical tight junctions (Fig. 4).Basal to the tight junction was a well-developed zonula adherens (ZA), with numerous underlying desmosomal attachments; a mat of cytoplasmic filaments radiated from the ZA and desmosomes. There was also considerable variability in the depth of the junctions in this segment (Table 1). Because intercalated ducts were the shortest glandular segment, only five examples of intercalated ducts with a total of 14 demonstrable junctions were unequivocally identified in freeze-fracture replicas. These junctions were characterized by two parallel sealing strands (Table 1) with intermediate strands anastomosing at perpendicular angles rather than oblique angles as occurred in acinar cell junctions (Fig. 11).DiscontinuLead Ion Tracer Technique ities were not observed in the ridges of sealThe lead ion readily penetrated the junc- ing strands of these junctions, and there was tions between acinar cells, gained access to less variability in the junctional depth than most of acinar lumina, and was visible in was seen in acinar junctions (Table 1). sections as luminal precipitates (Figs. 5, 6; Junctions between striated duct cells were Table 1). Tracer deposits were sometimes much more extensive than in the other two present on the luminal side of junctional epithelial segments examined. These junccomplexes between intercalated duct cells tions consisted of five to eight sealing strands (Fig. 7). However, these junctions appeared arranged in a bifurcating and anastomosing to be much less permeable to the tracer than meshwork oriented roughly parallel to the acinar junctions (Table 1).The lead tracer lumen (Fig. 12). The depth of the junction rarely penetrated junctions between striated was about twice that of acinar and intercaduct cells (Fig. 8, Table 11, although it was lated duct junctions, and the sealing strands present between the extensive basal and lat- were more tightly packed (Table 1).The depth eral membrane infoldings (Fig. 9). and number of sealing strands of striated duct junctions were statistically significantly Freeze Fracture different (P < .01) from those parameters in In junctions between acinar cells, few seal- junctions of either acini or intercalated ducts. ing strands were present (Fig. 10, Table 1). DISCUSSION These sealing strands consisted of ridges (P face) and grooves (E face) arranged in a loose In this study, the permeability of junctions meshwork with, usually, two strands paral- to an ionic tracer has been correlated with lel t o the lumen and incomplete, auxillary the freeze-fracture morphology of tight juncstrands anastomosing at oblique angles. tions in three separate epithelial segments of Ridges of sealing strands of acinar cell junc- rat parotid gland. Most junctions between tions were often discontinuous (Fig. lob). acinar cells permitted ready passage of the PARACELLULAR SECRETORY PATHWAY IN PAROTID Fig. 2. Junctional region between two acinar cells. The tight junction (arrow) is located subjacent to the lumen (Lu) of an intercellular canaliculus. Two short desmosomal junctions lie between the tight junction and the intercellular space (ICS).Uranyl acetate, lead citrate (UA-LC)-stainedthin section, ~ 5 6 , 0 0 0 . Fig. 3. Junctional region between two intercalated duct cells. The tight junction is indicated by the arrow. Because of extensive desmosonal attachments between 73 these cells, the lumen (Lu) and intercellular space (ICS) are more widely separated than in acini. UA-LC stain, ~56,000. Fig. 4. Junctional region between two striated duct cells illustrating the long tight junction (arrow) characteristic of this segment. The zonula adherens (ZA) and its associated mat of filaments are cut tangentially; basal to it lies a well-defined desmosome (D). UA-LC stained, ~56,000. 74 J.A.V. SIMSON AND H.L. BANK Fig. 5. Lead tracer method, illustrating tracer (arrow) in an acinar lumen Gu). The tracer has apparently penetrated a tight junction between adjacent acinar cells. UA-stained thin section, ~40,000. Fig. 6. Lead tracer method illustrating tracer in the lumen (Lu) of a n intercellular canaliculus between two acinar cells. Lead deposits are also present at the sites of the tight junctions (arrows). En bloc UA-stained, x21,OOO. Fig. 7. Lead tracer method illustrating a rare tracer deposit (arrow) in the lumen (Lu)of an intercalated duct. Most of the lead deposits are trapped within the intercellular space (arrowhead) basal to the junctional complex. Unstained, X30,OOO. PARACELLULAR SECRETORY PATHWAY IN PAROTID 75 Fig. 8. Lead tracer method illustrating tracer deposits between striated duct cells (arrows) but not in the lumen (Lu).Unstained, ~ 2 0 , 0 0 0 . Fig. 9. The lead tracer method illustrating that tracer readily enters the labyrinthine plasmalemmal folds of striated ducts from the basal (B) extracellular space. Unstained, ~ 2 0 , 0 0 0 . lead tracer, and these junctions had few (two to three) loosely-arranged, frequently discontinuous sealing strands, characteristic of “leaky” tight junctions. In contrast, junctions between intercalated duct cells stopped passage of most lead tracer ions and had two to three well-ordered junctional sealing strands with continuous apical and basal strands. Most junctions between striated duct cells were composed of several (five to eight) closely packed sealing strands. These junctions almost totally impeded passage of the lead tracer during the brief perfusion. Although acinar and intercalated duct junctions possessed approximately equal numbers of sealing strands, the observed differences 76 J.A.V. SIMSON AND H.L. BANK Fig. 10. Freeze-fracture images of acinar tight junctions adjacent to the lumen (Lu).Circled arrows indicate direction of shadow. a) The sealing strands form ridges on the P face (upper arrow) and grooves on the E face (lower arrow). A large secretory granule (SG)identifies this as an acinus. b) This micrograph illustrates the variable and often discontinuous (arrows) nature of the particle-studded ridges of acinar tight junction sealing strands. An open sealing strand is indicated by the upper arrow. ~60,000. Fig. 11. Freeze-fracture images of intercalated duct tight junctions parallel to the lumen (TAU). Circled arrows indicate direction of shadow. The fracture face often passes from one membrane face to the other (*) in the junctional region of this segment. Note the continuity of the apical and basal sealing strands, as well as the regularity of cross strands between them. a) E face (* on P face). b) P face (* on E face). x60.000. PARACELLULAR SECRETORY PATHWAY IN PAROTID 77 Fig. 12. Freeze-fracture images of striated duct junctions illustrating the width and complexity of the tight junctions (arrows) between these cells. Circled arrows indicate direction of shadow. a) Tight junction illustrating E face grooves. b) A complex junction illustrating a probable intersection between three cells. c) A junction in which the fracture plane exposes both the E face (apical, near arrow) and P face (more basal, near asterisk). ~60,000. in the paracellular permeability of lead ion tracer through the two types of junctions may result from differences in the thickness and continuity of apical and basal sealing strands and the presence of intermediate cross strands in the intercalated duct junctions. Our data may underestimate the junctional permeability in the various segments for two reasons: 1)in thin sections, the probability of encountering sparse precipitates is less than one, and 2) we cannot be certain that all tracer ions penetrating a junction have been precipitated. It is possible that lead ions which penetrated junctions in duct regions were less readily precipitated than those in the acinar intercellular canaliculi, because of the larger fluid volume which may have diluted the tracer in the ductal lumen. The differential permeability to the tracer in the various gland segments cannot be accounted for on the basis of differences in flow rates, since the present studies were per- 78 J.A.V. SIMSON AND H.L. BANK formed on nonsecreting glands, and there is no basal (unstimulated) secretion in these glands. Studies are currently under way to determine the influence of secretogogues on junctional morphology and tracer permeability of the three main epithelial segments. Our results basically conform to the model initially proposed by Claude and Goodenough (1973) and supported by others (Humbert et al., 1976) that a direct correlation exists between the “tightness” (i.e., impermeability to water and ions) of tight junctions and the number of sealing strands visualized by freeze fracture. However, it has become clear that other factors besides number of sealing strands-for example, sealing strand continuity and geometry-are also important in determining permeability characteristics (Claude, 1978;Briggman et al., 1981; Cereijido et al., 1981). Claude (1978) has suggested that the proportion of “open strands” in a junction more strongly influences junctional permeability than does the total number of sealing strands. Our results are also in general agreement with previous freeze-fractureand tracer studies of salivary glands. De Camilli et al. (1976) illustrated, but did not describe or discuss, the freeze-fracture morphology of junctions of the three epithlial segments of rat parotid gland. Their images conform to those obtained in the present study. Shimono et al. (1980) examined junctional morphology as well as lanthanum and ruthenium red impregnation in rat sublingual gland. Their findings were similar t o those in the present study, in that junctions of acini had less depth and possessed fewer sealing strands than those of striated ducts. However, their data on intercalated duct junctions were intermediate between those of acini and striated ducts, whereas, in the present study, intercalated duct junctions more closely resembled acinar junctions. This discrepancy could reflect a real difference in the intercalated ducts in the two glands (sublingual vs. parotid), or could result from sampling difficulties in obtaining identifiable fractures through intercalated duct junctions. Garrett et al. (1982) found a very slight but detectable passage of vascularly perfused horseradish peroxidase (MW - 40,000) tracer into saliva in actively secreting dog salivary glands, and provided cytochemical evidence that this may occur via “leaky” tight junctions between acinar cells. Oliver and Hand (1978) observed acinar junctional permeabil- ity to luminally administered horseradish peroxidase in isoproterenol-stimulated rat parotid glands. These results bear upon the question of the route and mechanism of passage of ions and water into the salivary secretory fluid. Two classical models have been proposed to account for net transfer of fluid across an epithelium (Ogilvie et al., 1963; Diamond and Bossert, 1967). Both models involve transcellular flow (across two cell membranes and the intervening cytoplasm) of ions and water. The model of Ogilvie et al. (1963) involves differential permeabilities of basal and luminal membranes. This model is interesting in that it requires little energy, but it has been difficult to test in biological systems. The Diamond and Bossert (1967) “standinggradient” model requires substantial energy to drive the postulated ion pumps necessary for ion movement across a membrane against a concentration gradient. Recent evidence from microprobe analysis (Gupta and Hall, 1979) not only lends credence to the standing-gradient model in certain epithelia, but also suggests, at least in insect salivary glands, a paracellular contribution to the generation of isotonic secretions. In fact, it is clear that, in some epithelia, a major route for the flux of ions and small molecules is via “leaky” tight junctions (Fromter and Diamond, 1972; Wright and Pietras, 1978; Cereijido et al., 1981). Additional evidence for a route for passive transfer of small molecules in salivary glands comes from earlier studies by Martin and Burgen (19621, who found that low molecular weight sugars can enter the saliva passively and their concentration in the saliva is inversely correlated with molecular size. Furthermore, permeability to these sugars was increased by sympathetic stimulation. The present study was designed to determine if paracellular transport could occur in the parotid gland by employing analysis of both junctional morphology by freeze-fracture and junctional permeability to an ionic tracer. Although results from either method alone must be interpreted cautiously, we believe that, taken together, the data from the two methods used in this study strongly support an interpretation of differential permeability in the three major epithelial segments of the rat parotid gland. Based on the freezefracture and lead tracer evidence obtained, we predict that a paracellular secretory pathway exists between parotid acinar cells. Such PARACELLULAR SECRETORY PATHWAY IN PAROTID a pathway would provide a low-resistance route for the passage of water, inorganic ions, and small organic molecules into the saliva. Control of the fluid flux through this potential paracellular pathway (and hence control of the rate of secretion) could be achieved in a t least three ways. 1) Junctional sealing strand organization could be altered by altering intracellular calcium concentration (Cereijido et al., 1981; Pinto da Silva and Kachar, 1982). 2) Blood flow or blood pressure could be altered by modifying local intravascular pressure (Shannon et al., 1974). 3) Osmotic and hydrostatic pressure gradients across the junction may be varied physiologically. Such a model for a paracellular secretory mechanism has been proposed recently (Simson, 1982). From our results it is clear that a potential paracellular secretory pathway exists between parotid acinar cells. Further physiological studies will be required to determine the contribution of paracellular fluid flux to the total secretion and to investigate the postulated control mechanisms. ACKNOWLEDGMENTS We would like to express our sincere appreciation to Ms. Jan Condon for excellent technical assistance, to Ms. Marion Hinson for skilled secretarial assistance, and to Dr. Patrick Moore for careful reading and helpful discussion of the manuscript. This work was supported in part by NIH grant RR05767 to the College of Dental Medicine (J.A.V.S.) and NIH grant AM 18115 (H.L.B.). LITERATURE CITED Abe, K., and C. Dawes (1980) The secretion of protein and of some electrolytes in response to a-and Padrenergic agonists by rat parotid and submandibular salivary glands enlarged by chronic treatment with isoproterenol. J. Dent. Res., 59t1081-1089. Bank, H.L., and J.D. Robertson (1976) A simple electrode for metallic replication. J. Microscopy, 106:343-350. Bank, H., C. Wise, K. Hargrove, and S.S. Spicer (1978) Intramembranous particles in erythrocyte, reticulocyte and erythroblastic leukemic cells of the rat: A model system for erythrocyte maturation. Exp. Hematol., 6:528-538. Berridge, M.J., and J.L. Oschman (1972) Transporting Epithelia. Academic Press, New York. Briggman, J.V., H.L. Bank, J.B. Bigelow, J.S. Graves, and S.S. Spicer (1981) Structure of the tight junctions of the human eccrine sweat gland. Am. J. Anat., 162r357-368. Castle, J.D., J.D. Jamieson, and G.E. Palade (1972) Radioautographic analysis of the secretory process in the parotid acinar cell of the rabbit. J. Cell Biol., 53: 290-311. Cereijido, M., I. Meza, and A. Martinez-Palomo (1981) 79 Occluding junctions in cultured epithelial monolayers. Am. J. Physiol., 2403296-102. Claude, P. (1978) Morphological factors influencing transepithelial permeability: A model for the resistance of the zonula occludens. J. Membr. Biol., 39t219232. Claude, P., and D.A. Goodenough (1973) Fracture faces of zonulae occludentes from “tight” and “leaky” epithelia. J. Cell Biol., 58:390-400. De Camilli, P., D. Peluchetti, and J. Meldolsi (1976) Dynamic changes of the luminal plasmalemma in stimulated parotid acinar cells. A freeze-fracture study. J. Cell Biol., 70:59-74. Diamond, J.M., and W.H. Bossert (1967) Standing-gradient osmotic flow. J. Gen. Physiol., 50t2061-2083. Emmelin, N. (1981) Nervous control of mammalian salivary glands. Philos. Trans. R. Soc. Lond. [Bioll, 296: 27-35. Ernst, S.A., W.C. Dodson, and K.J. Karnaky (1980) Structural diversity of occluding junctions in the lowresistance chloride-secreting opercular epithelium of seawater-adapted killfish (Funculus heteroclitus). J. Cell Biol., 87r488-497. Fromter, E., and J. Diamond (1972) Route of passive ion permeation in epithelia. Nature New Biol., 2359-13. Garrett, J.R., A.H. Klinger, and P.A. Parsons (1982) Permeability of canine submandibular glands to bloodborne horseradish peroxidase during parasympathetic secretion. Quart. J. Exp. Physiol., 67:31-39. Gupta, B.L., and T.A. Hall (1979) Quantitative electron probe x-ray microanalysis of electrolyte elements within epithelial tissue compartments. Fed. Proc., 38: 144-153. Humbert, F., A. Grandchamp, C. Precami, A. Perrelet, and L. Orci (1976)Morphological changes in tight junctions of necturns maculonus proximal tubules undergoing saline diureses. J. Cell Biol., 69:90-96. Jansen, J.W.C.M., V.V.A.M. s h r e w s , H.G.P. Swartz, A.M.M. Fleuren-Jakobs, J.J.H.H.M. DePont, and S.L. Bonting (1980) Role of calcium in exocrine pancreatic secretion. VI. Characteristics of the paracellular pathway for divalent cations. Biochim. Biophys. Acta, 599:316-323. Mangos, J.A., R.L. Boyd, G.M. Loughlin, A. Cockrell, and R. Fucci (1981) Secretion of monovalent ions and water in ferret salivary glands: a micropuncture study. J. Dent. Res., 60:733-737. Martin, K., and A.S.V. Burgen (1962) Changes in the permeability of the salivary gland caused by sympathetic stimulation and in catecholamines. J. Gen. Physiol., 46:225-243. Martinez, J.R., H. Holzgreve, and A. Freck (1966) Micropuncture study of the submaxillary gland of adult rats. Pflugers Arch., 290:124-133. Ogilvie, J.T., J.R. McIntosh, and P.F. Curran (1963) Volume flow in a series-membrane system. Biochim. Biophys. Acta, 66:441-444. Oliver, C., and A.R. Hand (1978) Uptake and fate of luminally administered horseradish in resting and isoproterenol-stimulated rat parotid acinar cells. J. Cell Biol., 76:207-220. Palade, G. (1975) Intracellular aspects of the process of protein synthesis. Science, 289:347-358. Petersen, O.H. (1973) Electrophysiological study of ionic transports in pancreatic and salivary acinar cells. In: Transport Mechanisms in Epithelia. Alfred Bergon Symposium V. H.H. Ussing and N.A. Thorn, eds. Academic Press, New York, pp. 478-489. Pinto da Silva, P., and B. Kachar (1982) On tight-junction structure. Cell, 28:441-450. Rutberg U. (1961) Ultrastructure and secretory mecha- 80 J.A.V. SIMSON AND H.L. BANK nism of the parotid gland. Acta. Odontol. Scand., lS(Supp1 3O):l-69. Schneyer, L.H., J.A. Young, and C.A. Schneyer (1972) Salivary secretion of electrolytes. Physiol. Rev., 52:720-770. Scott. B.L., and D.C. Pease (1959) Electron microscopy of the’ salivary and lacrimal glands of the rat. Am. J. Anat., 104:115-161. Shannon, I.L., R.P. Suddich, and F.J. Dowd, Jr. (1974) Saliva: Composition and Secretion. Karger, Basel, pp. 67-77. Shimono, M., T. Yamamura, and G. Fumagalli (1980) Intercellular junctions in salivary glands: Freeze-fracture and tracer studies of normal rat sublingual gland. J. Ultrastruct. Res., 72286-299. Simson, J.A.V. (1969a) A study of the effects of isoproterenol on the cytology of the parotid and submandibular glands of the rat. Thesis, State University of New York, Upstate Medical Center, Syracuse, New York. Simson, J.A.V. (1969b) Discharge and restitution of secretory material in the rat parotid gland in response to isoproterenol. Z. Zellforsch., 101:175-191. Simson, J.A.V. (1982) Morphological evidence for a paracellular fluid pathway in parotid acini. In: Fluid and Electrolyte Abnormalities in Exocrine Glands in Cystic Fibrosis. P.M. Quinton, J.R. Martinez and U.Hopper, eds. San Francisco Press, Inc: San Francisco, pp. 79-101. Simson, J.A.V., and R.M. Dom (1983) The use of lead as an ionic tracer for investigating routes of passive fluid transfer “in vivo.” J. Histochem. Cytochem., 31t675-683. Spring, K.R., and A. Hope (1978) Size and shape of the lateral intercellular spaces in a lining epithelium. Science, 20054-58. Wright, E.M., and R.J. Pietras (1978) Routes of nonelectrolyte permeation across epithelial membranes. J. Membr. Biol., 17:298-312. Young, J.A. (1973) Electrolyte transport by salivary epithelia. Proc. Aust. Physiol. Pharmacol. Soc., 4:lOl-121. Young, J.A., D.I. Cook, G. Jones, J. McGerr, and C. Thompson (1979) The effect of phenylephrine on excretion of fluid and electrolytes by the parotid and mandibular glands of the rat. Aust. J. Exp. Biol. Med. Sci., 57.555-562. Young, J.A., and E.W. Van Lennep (1978) The Morphology of Salivary Glands. Academic Press, London, pp 93-99.