Are the glandular trichomes in Jacquinia armillaris (Theophrastoideae-Primulaceae) salt glands?

Salt stress is harmful to plants, especially for those that live under conditions of intense salt aport. For this reason, several species present alternatives to prevent or diminish the damages that high salt concentrations may cause to the cells. Salt glands are one of these alternatives once they are specialized structures that secrete salt. Here, we aimed to investigate if the glandular trichomes in the leaves of Jacquinia armillaris are salt glands. Anatomical and ultrastructural observations showed that the glandular trichomes in J. armillaris resemble the salt glands from other recretohalophytes Primulaceae, such as, their occurrence in sunken regions in the leaf epidermis, the presence of a large basal cell that acts as a collecting cell, the detachment of the cuticle from the outer periclinal walls forming a cuticular chamber, the thickness of the cuticle in the stalk portion of the trichome, and the presence of sodium and chloride ions in the secretion and in the xylem. Altogether, the gathered results support the hypothesis that the glandular trichomes in J. armillaris are adapted to salt secretion, thus characterizing as salt glands.

The Nasal Gland in Turkeys (Meleagris gallopavo): Anatomy, Histology, and Ultrastructure.

This study provides a detailed description of the major morphoanatomic and ultrastructural features of the nasal gland in turkeys. In this avian species, nasal or salt glands are bilateral, pale pink, elongated to spindle-shaped, serous, tubuloalveolar structures, with a mean length ranging from 0.64 ± 0.15 cm in poults of 4 days of age to 2.15 ± 0.17 cm at 22 weeks. Instead of having a supraorbital location as commonly seen in waterfowl and other avian species, these glands run underneath the lacrimal, frontal, and nasal bones in turkeys. The reference point for sample collection for histologic examination is just before the rostral edge of the eyelid. Each gland adheres to the surrounding bone through a thick capsule of dense connective tissue merging with the skull periosteum. Histologically, the salt gland consists of secretory tubuloalveolar structures, lined by cuboidal epithelial cells with a central canaliculus and ducts. There are small and large ducts lined by a bilayered epithelium consisting of large apical columnar secretory cells occasionally admixed with rare cuboidal cells. These cells are periodic acid Schiff negative and slightly Alcian blue positive. Both alveolar and secretory ductal cells contain slightly electrondense granular vesicles, highly folded lateral surfaces, and large numbers of mitochondria, characteristic of ion-transporting epithelia. This study provides valuable information for the accurate identification and localization of the nasal gland during necropsy, as well as its correct histologic interpretation, ultimately improving our understanding of the role of this gland in the pathophysiology of specific diseases in turkeys.

La glándula nasal en pavos (Meleagris gallopavo): Anatomía, histología y ultraestructura Este estudio proporciona una descripción detallada de las principales características morfo-anatómicas y ultraestructurales de la glándula nasal en pavos. En esta especie aviar, las glándulas nasales o glándulas salinas son estructuras bilaterales, tubuloalveolares de color rosa pálido, alargadas y serosas, con una longitud media que varía de 0.64 ± 0.15 centímetros en los pavipollos de 4 días de edad hasta 2.15 ± 0.17 centímetros en aves a las 22 semanas. En lugar de tener una ubicación supraorbital como se ve comúnmente en las aves acuáticas y otras especies de aves, estas glándulas corren por debajo de los huesos lagrimales, frontales y nasales en los pavos. El punto de referencia para la recolección de muestras para el examen histológico es justo antes del borde rostral del párpado. Cada glándula se adhiere al hueso circundante a través de una gruesa cápsula de tejido conectivo denso que se fusiona con el periostio del cráneo. Histológicamente, la glándula salina consiste en estructuras tubulo-alveolares secretoras, revestidas por células epiteliales cuboidales con un canalículo central y conductos. Hay conductos pequeños y grandes revestidos por un epitelio de dos capas que consiste en grandes células secretoras columnares apicales ocasionalmente mezcladas con escasas células cuboidales. Estas células son ácido periódico de Schiff negativas y ligeramente positivas para el azul de alcián. Las células ductales alveolares y secretoras contienen vesículas granulares ligeramente electrondensas, superficies laterales altamente plegadas y grandes cantidades de mitocondrias, características de los epitelios transportadores de iones. Este estudio proporciona información valiosa para la identificación y localización exacta de la glándula nasal durante la necropsia, así como su correcta interpretación histológica, mejorando en última instancia nuestra comprensión del papel de esta glándula en la fisiopatología de enfermedades específicas en pavos.

The rectal glands of Heterorhabditis bacteriophora (Rhabditida: Heterorhabditidae) hermaphrodites and their role in symbiont transmission.

Differential interference contrast, transmission electron and epifluorescence microscopy techniques were employed to examine the ultrastructure of the rectal glands in Heterorhabditis bacteriophora hermaphrodites, with special attention to the location of Photorhabdus bacteria symbionts within these structures. Three rectal glands were clearly visualized in all examined specimens, with two glands positioned sub-ventrally and another gland located dorsally. The dorsal rectal gland in all examined specimens is larger than the subventral ones. Our observations indicate that Photorhabdus bacteria do not colonize the rectal glands of H. bacteriophora hermaphrodites, but rather are present in the most posterior-intestinal cells.

Functional and morphological plasticity of crocodile (Crocodylus porosus) salt glands.

The estuarine crocodile, Crocodylus porosus, inhabits both freshwater and hypersaline waterways and maintains ionic homeostasis by excreting excess sodium and chloride ions via lingual salt glands. In the present study, we sought to investigate the phenotypic plasticity, both morphological and functional, in the lingual salt glands of the estuarine crocodile associated with chronic exposure to freshwater (FW) and saltwater (SW) environments. Examination of haematological parameters indicated that there were no long-term disruptions to ionic homeostasis with prolonged exposure to SW. Maximal secretory rates from the salt glands of SW-acclimated animals (100.8+/-14.7 micromol 100 g(-0.7) body mass h(-1)) were almost three times greater than those of FW-acclimated animals (31.6+/-6.2 micromol 100 g(-0.7) body mass h(-1)). There were no differences in the mass-specific metabolic rate of salt gland tissue slices from FW- and SW-acclimated animals (558.9+/-49.6 and 527.3+/-142.8 microl O(2) g(-1) h(-1), respectively). Stimulation of the tissue slices from SW-acclimated animals by methacholine resulted in a 33% increase in oxygen consumption rate. There was no significant increase in the metabolic rate of tissues from FW-acclimated animals in response to methacholine. Morphologically, the secretory cells from the salt glands of SW-acclimated animals were larger than those of FW-acclimated animals. In addition, there were significantly more mitochondria per unit volume in secretory tissue from SW-acclimated animals. The results from this study demonstrate that the salt glands of C. porosus are phenotypically plastic, both morphologically and functionally and acclimate to changes in environmental salinity.

Rectal gland of Bactrocera papayae: ultrastructure, anatomy, and sequestration of autofluorescent compounds upon methyl eugenol consumption by the male fruit fly.

Sexually mature males of Bactrocera papayae are strongly attracted to and consume methyl eugenol (ME). Upon consumption, ME is biotransformed to two phenylpropanoids, 2-allyl-4,5-dimethoxyphenol (DMP) and (E)-coniferyl alcohol (CF), that are transported in the hemolymph, sequestered and stored in the rectal glands, and subsequently released as sex and aggregation pheromones during courtship. To date, very little work on the ultrastructure and anatomy of the rectal gland has been done, and the accumulation of phenylpropanoids in the rectal glands of males has not been observed visually. Our objectives are to describe the anatomy and fine structures of the rectal glands of males and females and to observe the accumulation of autofluorescent compounds in the rectal glands of males. The rectal glands of males and females have four rectal papillae with each papilla attached to a rectal pad. The rectal pads protrude from the rectal gland as the only surfaces of the gland that are not surrounded by muscles. The rectal papillae of ME-fed males had oil droplets and autofluorescent compounds that were absent from those of ME-deprived males. The autofluorescent compounds accumulated in the rectal sac, which is an evagination that is not found in rectal glands of females. The accumulation of these compounds increased with time and reached maximum at a day post-ME feeding and decreased thereafter. This trend is similar to the accumulation pattern of phenylpropanoids, CF and DMP in the rectal gland.

The microanatomy of the rectal salt gland of the Port Jackson Shark, Heterodontus portusjacksoni (Meyer) (Heterodontidae): suggestions for a counter-current exchange system.

A comprehensive anatomical study was undertaken to examine the rectal salt gland in the Port Jackson shark, Heterodontus portusjacksoni, a shark known to invade estuarine environments. The microstructure and vascular organisation of the rectal salt gland was investigated using histological observation and scanning electron microscopy of vascular corrosion casts. Cellular specialisation was observed in the lining of the central lumen of this gland. This may indicate that there is some modification of the principal product of the gland prior to its secretion. The rectal salt gland has a complex structure related to its function. Contrary to previous reports, the flow in secretory tubules is in the opposite direction to that of the capillaries and thus constitutes a counter-current arrangement. The similarity in the organisation of the counter-current and lobulate arrangement of salt-secreting glands through phylogenetically diverse organisms, such as sharks and birds, suggests that this arrangement is important in achieving efficient salt secretion.

Preparation of Na+,K+-ATPase with near maximal specific activity and phosphorylation capacity: evidence that the reaction mechanism involves all of the sites.

The phosphorylation capacity of Na+,K+-ATPase preparations in common use is much less than expected on the basis of the molecular weight of the enzyme deduced from cDNA sequences. This has led to the popularity of half-of-the-sites or flip-flop models for the enzyme reaction mechanism. We have prepared Na+,K+-ATPase from nasal salt glands of salt-adapted ducks which has a phosphorylation capacity and specific activity near the theoretical maxima. Preparations with specific activities of >60 micromol (mg of protein)-1 min-1 at 37 degrees C had phosphorylation capacities of >60 nmol/mg of protein, and the rate of turnover of the enzyme was 9690 min-1, within the range reported for the enzyme from other sources. The fraction of the maximal specific activity of the enzyme compared well with the fraction of the protein on SDS-PAGE which was alpha and beta chains, especially at the highest specific activity which indicates that all of the alphabeta protomers are active. The gels of the most reactive preparations contained only alpha and beta chains, but less active preparations contained a number of extraneous proteins. The major contaminant was actin. The preparation did not contain any protein which migrated in the molecular weight range of the gamma subunit. The subunit composition of the enzyme was alpha1 and beta1 only. This is the first report of a pure, homogeneous, fully active preparation of the protein. Reaction models which incorporate a half-of-the-sites or flip-flop mechanism do not apply to this enzyme.

Ultrastructural and cytochemical characterization of cultured dogfish shark rectal gland cells.

The dogfish shark rectal gland (SRG) is histologically complex, containing connective, nerve, and smooth muscle tissue and at least three types of epithelial cells: secretory tubule, central duct, and endothelial. This cellular heterogeneity precludes studies of the intact SRG from distinguishing direct tubular effects of mediators that modulate chloride secretion from their indirect effects on nonepithelial cells such as neurons. Primary SRG cultures express high levels of secretagogue-stimulated chloride secretion, suggesting that SRG cells retain a significant level of cytodifferentiation in vitro. However, because nontubular cells could contaminate these cultures, the question of whether secretagogues activate chloride secretion through direct or indirect effects on tubular epithelial cells remains unresolved. To address this issue, detailed ultrastructural and cytochemical analyses of SRG cultures were carried out to assess the level of cellular heterogeneity and the degree of cytodifferentiation expressed by SRG cells in vitro. The results demonstrate that, after 15 days, primary SRG monolayer cultures are composed exclusively of tubular epithelial cells with no detectable contamination by central duct cells, fibroblasts, smooth muscle cells, endothelial cells, or neurons. Tubular epithelial cells express most of the structural features of native SRG cells, including numerous mitochondria, massive basolateral surface amplification, complex tight junctions, and an extensive tubulovesicular system in the apical cytoplasm. Cultured SRG cells also display a striking level of polarization of cytoplasmic organelles and plasma membrane secretagogue receptors. These results account for the exceptionally high rates of electrogenic chloride secretion by SRG tubular epithelial cells in vitro and confirm that the effects of secretagogues on transport activity reflect their direct interaction with tubular epithelial cells.

Properties of single- and double-barreled Cl channels of shark rectal gland in planar bilayers.

Chloride channels from the apical plasma membrane fraction of rectal gland of Squalus acanthias were characterized by incorporation into planar bilayers in the presence of cAMP-PK/ATP. In a total of 80 bilayer preparations, 21 Cl-selective channels were observed as single channels and 13 as pairs. This was a significantly greater number of double Cl channels than expected from a binomial distribution. The double Cl channels were divided into two groups based on kinetic and voltage-dependent behavior. One group had properties identical to the single channels (gb1) while the other was consistent with a double-barreled channel (gb2) with coordinated activity between proto-channels. The single-channel slope conductances of gb1 and gb2 from -60 to +20 mV with a 250/70 mM KCl gradient were 41 and 75 pS, respectively. With symmetrical 250 mM KCl, the I-V relation of gb1 showed outward rectification with 47.8 +/- 6.6 pS at cis negative potentials and 68.9 +/- 6.1 pS at cis positive potentials. gb1 was open from 70 to 95% at all electrochemical potentials from -80 to +40 mV. gb2 was steeply voltage dependent between -80 and -20 mV. Both gb1 and gb2 were insensitive to Ca (from 100 nm to 1 microM), blocked by 0.1 mM DIDS and highly selective for chloride. These data suggest that double-barreled Cl channels are related to the family of small, outwardly rectifying Cl channels of epithelial membranes.

The duct system of the avian salt gland as a transporting epithelium: structure and morphometry in the duck Anas platyrhynchos.

The duct system of the nasal salt gland of the duck comprises central central canals, secondary ducts and main ducts. The secondary and main ducts consist of a layer of columnar cells overlying a layer of small cuboidal cells. The columnar cells have complex intercellular spaces showing evidence of Na+K+-ATPase at the apical regions. Approximately 70% of surface area of the duct system is external to the gland. During adaptation to salt water the duct system increases in size as does the gland. Although the components of the gland of adapted ducks, including the duct system within the gland, increase in size compared with normal ducks, the percentage volume densities of the components remain similar in both categories of ducks, i.e. the duct system increases in size in proportion to the glandular tissue. The volume of the duct system external to the gland is six to seven times larger than the volume within the gland. Thus, if ductal modification of secreted fluid occurs, it will be most likely to take place in the ducts external to the gland. Total surface areas of the duct system were measured from serial sections of glands and ducts from one normal and one adapted duck. These were used to calculate possible flux rates of water and sodium across the duct epithelium, assuming the occurrence of either water reabsorption of sodium secretion. Although these flux rates are high it is shown that they are similar to calculated flux rates across the luminal surface of the secretory tubules.

Resolution of apical from basolateral membrane of shark rectal gland.

Membrane fractions were isolated from the rectal gland of Squalus acanthias using differential centrifugation and a sucrose gradient run in the presence of 1 M KBr. Using the basolateral membrane marker Na+-K+-ATPase, we obtained a sixfold purification with the most highly purified fraction from the gradient (sp act = 336 +/- 37 mumol X mg protein-1 X h-1). Electrogenic Br- transport was used as a marker activity of the apical membrane, which enabled the identification and purification of a membrane fraction that is highly resolved from the basolateral membrane. The most active fraction was purified approximately 50-fold compared with the crude homogenate. In this fraction, the specific activity of electrogenic anion transport was 296 +/- 87 nmol X mg protein-1 X min-1, whereas the ATPase was only 17.6 +/- 5.7 mumol X mg protein-1 X h-1, representing about a 4-5% contamination of the apical fraction with the basolateral membrane.

Correlation of Na+,K+-ATPase content and plasma membrane surface area in adapted and de-adapted salt glands of ducklings.

During salt-water adaptation, an increase occurs in Na+,K+-ATPase content and surface area of the basolateral plasma membrane of the principal cell of the duck salt gland. To determine the degree to which these changes are correlated, accepted morphometric methods were used to determine numerical cell densities and plasma membrane surface densities of peripheral and principal cells. After adaptation, the plasma membrane surface area per principal cell was five times greater than in controls. Following de-adaptation, the plasma membrane content in principal cells returned to 1.9 times control levels. Two other cell constituents, mitochondria and lipid droplets, displayed similar quantitative changes. Na+,K+-ATPase content increased about fourfold with adaptation and decreased to near control levels with de-adaptation. Thus, changes in Na+,K+-ATPase content and basolateral plasma membrane surface area in adapting and de-adapting secretory epithelia of the salt gland occur nearly in parallel. These quantitative data enable Na+,K+-ATPase synthesis and degradation to be investigated in relation to membrane biogenesis.

Plasma membrane biogenesis in the avian salt gland: a biochemical and quantitative electron microscopic autoradiographic study.

The avian salt gland provides an ideal system for the study of plasma membrane (PM) biogenesis. Feeding ducklings 1% sodium chloride (salt stress) induces the secretory cells of the gland to synthesize large amounts of PM, which forms an extensive basolateral PM domain after 7-9 days of treatment. In the present study, the initial biosynthetic events following salt stress were investigated. In vivo studies using 3H-uridine indicated that increased rates of RNA synthesis could be detected by 2 hr after the beginning of salt stress and continued through at least 12 hr. Under in vitro conditions, increased rates of protein and glycoprotein synthesis (as monitored by 3H-leucine and 3H-fucose incorporation, respectively) were also detected after 2 hr and continued through 7-9 days. Increased levels of Na,K-ATPase, a specific secretory cell PM marker, were detected after 8 hr of treatment as monitored by specific activity and 3H-ouabain binding. Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis coupled with fluorography indicated that both 3H-leucine and 3H-fucose were incorporated into partially purified preparations of Na,K-ATPase isolated after 12 hr. Light microscopic autoradiographic analysis of pulse-chase experiments indicated that in secretory cells of 12-hr salt-stressed glands, 3H-leucine- and 3H-fucose-labelled products reached the cell periphery by 1-2 hr after the initial pulse. The incorporation of both tritiated precursors was predominantly associated with the secretory cells. Quantitative electron microscopic autoradiography indicated that 3H-leucine is initially taken up by elements of the rough endoplasmic reticulum (RER) and cytoplasm (5 min postpulse), subsequently transported to and concentrated within components of the Golgi apparatus (10 min of chase), and ultimately incorporated into all domains of the plasma membrane of secretory cells by 1-2 hr of chase. The data is consistent with a flow of newly synthesized membrane components from RER to Golgi to plasma membrane and is analogous to the pattern previously found for the synthesis and processing of PM proteins in a wide variety of cell types.

Electron microscopic morphometric studies on the resting and secreting nasal (salt) glands of the domestic duck. I. Standardisation of the fixation procedure.

In order to perform morphometric analysis of the nasal (salt) glands of domestic duck it was necessary to standardise the regime of fixation and processing for this tissue. This paper reports the results of these experiments in which the nasal glands of nine groups of ducks were perfused by fixatives containing various concentrations of glutaraldehyde; each group included three secreting ducks and three non-secreting control ducks. Fixative osmolality ranged from 300-825 m-osmol. The resulting effects on the electron microscopical appearances of mitochondria and intercellular spaces were assessed. The fixative finally selected as most satisfactory for both control and secreting tissue contained 3.5% glutaraldehyde and 3% dextran in sodium cacodylate buffer, giving an osmolality range of 580-650 m-osmol.

Primary culture of duck salt gland. I. Morphology of confluent cell layers.

Dissociated avian salt gland secretory cells were maintained in primary culture after plating on hydrated collagen gels. When seeded at 3 X 10(6) cells/cm2, confluent cell sheets formed within 2-3 days, whereas cultures seeded at lower densities formed a complex reticulum of cell aggregates, which remained nonconfluent even after 7 days. Scanning electron microscopy showed that the free surface of 3-day confluent cultures consisted of intermixed convex and flattened cell membranes with prominent junctional boundaries and abundant microvilli. Transmission electron microscopy indicated that these cultures were multilayers of 1-4 cells in thickness. The plasma membranes of the superficial cells were polarized into apical and basolateral regions displaying, respectively, microvilli and interdigitating lateral membrane folds. These membrane domains were separated by shallow occluding junctions, which consisted of both single strands and simple net-like arrays in freeze-fracture images. Underlying epithelial cells retained lateral membrane folds and formed desmosomal contacts with superficial and neighboring cells. These cultures, unlike the intact tissue, allow direct access to the apical and basolateral cell surfaces for electrophysiological analysis of transmural active ion transport.

Activation and inhibition of Na/K-ATPase by filipin-cholesterol complexation. A correlative biochemical and ultrastructural study on the microsomal and purified enzyme of the avian salt gland.

The Na/K-ATPase-rich microsomal fraction and purified Na/K-ATPase membranes of the salt-stressed avian salt gland were studied at defined filipin/cholesterol molar ratios (F/C) using enzyme assay and electron microscopy including negative staining, thin sectioning and freeze fracturing. Comparative examinations of detergent-treated microsomal fractions and the use of electron microscopic tracers revealed that F/C up to 2 activated latent Na/K-ATPase in sealed right-side-out vesicles by increasing membrane permeability without disrupting the vesicular membrane. Therefore, filipin offers an alternative to the detergents for the activation of latent vectorial membrane enzymes and a possible tool to examine their subcellular localization and sidedness in the membrane. The same F/C had no stimulatory effect on the microsomal anion-ATPase suggesting that the 2 ATPases are not located in the same membrane. Increasing F/C applied to the unfixed Na/K-ATPase membranes caused an increase in the number of structural F-C-complexes and a progressive lateral displacement of the enzyme particles which finally led to a separation of the areal distribution of these structures at F/C = 10. Such displacements did not occur in unfixed microsomes and were prevented by glutaraldehyde fixation of the purified membranes. F/C exceeding 2 progressively and temperature-dependently inhibited the Na/K-ATPase in its membrane-bound states, whereas the solubilized enzyme was rather insensitive. The structural and biochemical data suggest that inhibition results from the perturbation of the lipidic microenvironment of the enzyme caused by filipin-cholesterol complexation.

The distribution of intracellular ions in the avian salt gland.

To investigate the mechanism of salt secretion in the avian salt gland, we used quantitative electron probe microanalysis to measure the intracellular elemental concentrations in dry cryosections of unspecialized and partially specialized secretory epithelial cells from fresh water- and salt water-adapted ducklings, respectively. In conjunction with this, human and duckling erythrocytes were also analyzed, since these provided the experimental basis for using in situ erythrocytes as standards for determining the local water content of epithelia from the analysis of dried cryosections. The microprobe results from both types of erythrocytes compared favorably with chemical determinations of elemental concentrations. The nucleated avian erythrocytes, whose wet-weight elemental concentrations were determined by a compartmental analysis that required neither a peripheral standard nor a measure of the local mass, revealed a marked accumulation of P and K in the nucleus (388 and 190 mmol/kg wet wt, respectively) relative to the cytoplasm (67 and 85 mmol/kg wet wt). In both developmental states of the epithelial cells, the nucleus and apical cytoplasm had essentially similar and unremarkable concentrations of Na (76 and 83 mmol/kg dry wt, respectively, in the adapted cells vs. 72 and 81 mmol/kg dry wt in the control cells) and K (602 and 423 mmol/kg dry wt vs. 451 and 442 mmol/kg dry wt). Chloride, however, which was in general rather high, was significantly depressed in the apical cytoplasm of adapted cells only (164 and 124 mmol/kg dry wt in the nucleus and cytoplasm, respectively, of adapted cells (P less than 0.05) vs. 138 and 157 mmol/kg dry wt for control cells (P less than 0.05). Cation concentrations (Na + K) were elevated approximately 15% in the basal regions of adapted cells as compared with apical cytoplasm. When tissue water variations are accounted for, the results suggest that: (a) an active, energy-requiring process is responsible for chloride accumulation in this cell; (b) the apical membrane is a regulatory site for secretion; and (c) there are regional distinctions in the distribution of ions and water, particularly in the salt water-adapted cell. These conclusions are consistent with active chloride transport as the basis for salt secretion in this tissue.

Ultrastructure of the purified and reconstituted Na/K-ATPase of the avian salt gland.

Na/K-ATPase of salt-stressed salt glands of the domestic duck (Anas platyrhynchos) was purified in membrane-bound form by incubation of the microsomal fraction with sodium dodecylsulphate and ATP followed by discontinuous sucrose gradient centrifugation. Gel electrophoresis of the purified plasma membrane preparation substantially showed the two polypeptide subunits of the Na/K-ATPase both of which stained with the periodic acid-Schiff reagent. About 99% of the total ATPase activity was ouabain-inhibitable amounting to 1300 mumol Pi/(mg protein X h) of specific activity. The anion-stimulated, ouabain-insensitive ATPase increased parallel to the Na/K-ATPase up to the microsomal fraction until it totally vanished during SDS incubation. Electron microscopy of thin sections revealed that the purified fraction consisted of flat and cup-shaped triple-layered membrane fragments. Particles arranged into clusters and strands were visible as 3 to 5 nm surface particles in negatively stained suspensions and as 8 to 10 nm intramembraneous particles in freeze fracture replicas. The differential distribution of the intramembraneous particles on the fracture faces reflected the structural membrane asymmetry. Solubilization of Na/K-ATPase led to the disappearance of intramembraneous particles. Incorporation of the solubilized enzyme into phosphatidylcholine vesicles again showed 8 to 10 nm particles apparently orientated at random in the artificial membrane. Control liposomes prepared in the absence of solubilized enzyme were devoid of intramembraneous particles. These results clearly demonstrate that the avian salt gland Na/K-ATPase exists as 8 to 10 nm particles in both the purified plasma membrane and the artificial phospholipid membrane.

Structure of tight junctions during Cl secretion in the perfused rectal gland of the dogfish shark.

In epithelia that secrete sodium chloride, high-conductance tight junctions between cells have been proposed as the primary pathway for transepithelial sodium flux. We examined the properties of tight junctions in the perfused rectal gland of the dogfish shark during basal secretion and following adenosine 3',5'-cyclic monophosphate stimulation of sodium chloride secretion. Freeze-fracture electron microscopy revealed extensive interdigitation of adjacent cells with an associated amplification in the length of tight junctions per area of luminal surface, averaging 102 +/- 4.7 m/cm2 in outer regions of 80 +/- 6.7 in inner regions of the gland. Marked heterogeneity of junctional structure was present with junctional elements varying from single strands to three duplex elements and junctional depth varying from 15 to 60 nm. In glands perfused with lanthanum chloride, ionic lanthanum filled the intercellular space up to but not through the tight junctions. Characteristics of tight junctions were not different during basal and maximally stimulated sodium chloride secretion. These studies define tight junctions in the rectal gland as an anatomical barrier capable of restricting the passage of relatively small molecules such as urea while providing a greatly amplified junctional area for the passive diffusion of sodium and water.