ADENOSINE TRIPHOSPHATASE LOCALIZATION IN AMPHIBIAN EPIDERMIS

The localization of ATPase1 activity has been studied by light and electron microscopy in the epidermis of Rana pipiens, Rana catesbiana, and Bufo marinus. The reaction was carried out on skin (glutaraldehyde-fixed or fresh) sectioned with or without freezing. Best results were obtained with nonfrozen sections of fixed tissue. The incubation mixture was either a Wachstein-Meisel medium, or a modification which approximates assay systems used in biochemical studies of transport ATPases. The reaction product was found localized in contact with the outer leaflet of all cell membranes facing the labyrinth of intercellular spaces of the epidermis. It was absent from: (a) membrane areas involved in cell junctions (desmosomes, zonulae and maculae occludentes); (b) cell membranes facing the external medium (i.e., those on the distal aspect of the ultimate cell layer in s. corneum); (c) cell membranes facing the dermis (those on the proximal aspect of cells in s. germinativum). In the presence of (Na+ + K+) the localization did not change, but the reaction was not appreciably activated. A similar though less intense reaction was obtained with ITP, but not with ADP, AMP, and GP as substrates. The results are discussed in relation to available data on transport ATPases in general, and on the morphology and physiology of amphibian skin in particular. Assuming that the ATPase studied is related to transport ATPase, the findings suggest a series of modifications to the frog skin model proposed by Koefoed-Johnsen and Ussing. The salient feature of this modified model is the localization of the Na+ pump along all cell membranes facing the intercellular spaces of the epidermis

FUNCTIONS OF COATED VESICLES DURING PROTEIN ABSORPTION IN THE RAT VAS DEFERENS

The role of coated vesicles during the absorption of horseradish peroxidase was investigated in the epithelium of the rat vas deferens by electron microscopy and cytochemistry. Peroxidase was introduced into the vas lumen in vivo. Tissue was excised at selected intervals, fixed in formaldehyde-glutaraldehyde, sectioned without freezing, incubated in Karnovsky's medium, postfixed in OsO4, and processed for electron microscopy. Some controls and peroxidase-perfused specimens were incubated with TPP,1 GP, and CMP. Attention was focused on the Golgi complex, apical multivesicular bodies, and two populations of coated vesicles; large (> 1000 A) ones concentrated in the apical cytoplasm and small (<750 A) ones found primarily in the Golgi region. 10 min after peroxidase injection, the tracer is found adhering to the surface plasmalemma, concentrated in bristle-coated invaginations, and within large coated vesicles. After 20–45 min, it is present in large smooth vesicles, apical multivesicular bodies, and dense bodies. Peroxidase is not seen in small coated vesicles at any interval. Counts of small coated vesicles reveal that during peroxidase absorption they first increase in number in the Golgi region and later, in the apical cytoplasm. In both control and peroxidase-perfused specimens incubated with TPP, reaction product is seen in several Golgi cisternae and in small coated vesicles in the Golgi region. With GP, reaction product is seen in one to two Golgi cisternae, multivesicular bodies, dense bodies, and small coated vesicles present in the Golgi region or near multivesicular bodies. The results demonstrate that (a) this epithelium functions in the absorption of protein from the duct lumen, (b) large coated vesicles serve as heterophagosomes to transport absorbed protein to lysosomes, and (c) some small coated vesicles serve as primary lysosomes to transport hydrolytic enzymes from the Golgi complex to multivesicular bodies

SEGREGATION AND PACKAGING OF GRANULE ENZYMES IN EOSINOPHILIC LEUKOCYTES

During their differentiation in the bone marrow, eosinophilic leukocytes synthesize a number of enzymes and package them into secretory granules. The pathway by which three enzymes (peroxidase, acid phosphatase, and arylsulfatase) are segregated and packaged into specific granules of eosinophils was investigated by cytochemistry and electron microscopy. During the myelocyte stage, peroxidase is present within (a) all rough ER cisternae, including transitional elements and the perinuclear cisterna; (b) clusters of smooth vesicles at the periphery of the Golgi complex; (c) all Golgi cisternae; and (d) all immature and mature specific granules. At later stages, after granule formation has ceased, peroxidase is not seen in ER or Golgi elements and is demonstrable only in granules. The distribution of acid phosphatase and arylsulfatase was similar, except that the reaction was more variable and fully condensed (mature) granules were not reactive. These results are in accord with the general pathway for intracellular transport of secretory proteins demonstrated in the pancreas exocrine cell by Palade and coworkers. The findings also demonstrate (a) that in the eosinophil the stacked Golgi cisternae participate in the segregation of secretory proteins and (b) that the entire rough ER and all the Golgi cisternae are involved in the simultaneous segregation and packaging of several proteins

LYSOSOME FUNCTION IN THE REGULATION OF THE SECRETORY PROCESS IN CELLS OF THE ANTERIOR PITUITARY GLAND

The nature and content of lytic bodies and the localization of acid phosphatase (AcPase) activity were investigated in mammotrophic hormone-producing cells (MT) from rat anterior pituitary glands. MT were examined from lactating rats in which secretion of MTH1 was high and from postlactating rats in which MTH secretion was suppressed by removing the suckling young. MT from lactating animals contained abundant stacks of rough-surfaced ER, a large Golgi complex with many forming secretory granules, and a few lytic bodies, primarily multivesicular bodies and dense bodies. MT from postlactating animals, sacrificed at selected intervals up to 96 hr after separation from their suckling young, showed (a) progressive involution of the protein synthetic apparatus with sequestration of ER and ribosomes in autophagic vacuoles, and (b) incorporation of secretory granules into multivesicular and dense bodies. The content of mature granules typically was incorporated into dense bodies whereas that of immature granules found its way preferentially into multivesicular bodies. The secretory granules and cytoplasmic constituents segregated within lytic bodies were progressively degraded over a period of 24 to 72 hr to yield a common residual body, the vacuolated dense body. In MT from lactating animals, AcPase reaction product was found in lytic bodies, and in several other sites not usually considered to be lysosomal in nature, i.e., inner Golgi cisterna and associated vesicles, and around most of the immature, and some of the mature secretory granules. In MT from postlactating animals, AcPase was concentrated in lytic bodies; reaction product and incorporated secretory granules were frequently recognizable within the same multivesicular or dense body which could therefore be identified as "autolysosomes" connected with the digestion of endogenous materials. Several possible explanations for the occurrence of AcPase in nonlysosomal sites are discussed. From the findings it is concluded that, in secretory cells, lysosomes function in the regulation of the secretory process by providing a mechanism which takes care of overproduction of secretory products

ORIGIN OF GRANULES IN POLYMORPHONUCLEAR LEUKOCYTES : Two Types Derived from Opposite Faces of the Golgi Complex in Developing Granulocytes

The origin, nature, and distribution of polymorphonuclear leukocyte (PMN) granules were investigated by examining developing granulocytes from normal rabbit bone marrow which had been fixed in glutaraldehyde and postfixed in OsO4. Two distinct types of granules, azurophil and specific, were distinguished on the basis of their differences in size, density, and time and mode of origin. Both types are produced by the Golgi complex, but they are formed at different stages of maturation and originate from different faces of the Golgi complex. Azurophil granules are larger (∼800 mµ) and more dense. They are formed only during the progranulocyte stage and arise from the proximal or concave face of the Golgi complex by budding and subsequent aggregation of vacuoles with a dense core. Smaller (∼500 mµ), less dense specific granules are formed during the myelocyte stage; they arise from the distal or convex face of the Golgi complex by pinching-off and confluence of vesicles which have a finely granular content. Only azurophil granules are found in progranulocytes, but in mature PMN relatively few (10 to 20%) azurophils are seen and most (80 to 90%) of the granules present are of the specific type. The results indicate that inversion of the azurophil/specific granule ratio occurs during the myelocyte stage and is due to: (a) reduction of azurophil granules by multiple mitoses; (b) lack of new azurophil granule formation after the progranulocyte stage; and (c) continuing specific granule production. The findings demonstrate the existence of two distinct granule types in normal rabbit PMN and their separate origins from the Golgi complex. The implications of the observations are discussed in relationship to previous morphological and cytochemical studies on PMN granules and to such questions as the source of primary lysosomes and the concept of polarity within the Golgi complex

DIFFERENCES IN ENZYME CONTENT OF AZUROPHIL AND SPECIFIC GRANULES OF POLYMORPHONUCLEAR LEUKOCYTES : II. Cytochemistry and Electron Microscopy of Bone Marrow Cells

In the previous paper we presented findings which indicated that enzyme heterogeneity exists among PMN leukocyte granules. From histochemical staining of bone marrow smears, we obtained evidence that azurophil and specific granules differ in their enzyme content. Moreover, a given enzyme appeared to be restricted to one of the two types. Clear results were obtained with alkaline phosphatase, but those with a number of other enzymes were suggestive rather than conclusive. Since the approach used previously was indirect, it was of interest to localize the enzymes directly in the granules. Toward this end, we carried out cytochemical procedures for five enzymes on normal rabbit bone marrow cells which had been fixed and incubated in suspension. The localization of reaction product in the granules was determined by electron microscopy. In accordance with the results obtained on smears, azurophil granules were found to contain peroxidase and three lysosomal enzymes: acid phosphatase, arylsulfatase, and 5'-nucleotidase; specific granules were found to contain alkaline phosphate. Specific granules also contained small amounts of phosphatasic activity at acid pH. Another finding was that enzyme activity could not be demonstrated in mature granules with metal salt methods (all except peroxidase); reaction product was seen only in immature granules. The findings confirm and extend those obtained previously, indicating that azurophil granules correspond to lysosomes whereas specific granules represent a different secretory product

THE PERMEABILITY OF GLOMERULAR CAPILLARIES TO GRADED DEXTRANS : Identification of the Basement Membrane as the Primary Filtration Barrier

Graded dextrans have been used as tracers to identify the primary permeability barrier(s) to macromolecules among the structural elements (endothelium, mesangium, basement membrane, epithelium) of the glomerular capillary wall. Three narrow-range fractions of specified molecular weights and Einstein-Stokes radii (ESR) were prepared by gel filtration: (a) 32,000 mol wt, ESR = 38 Å; (b) 62,000 mol wt, ESR = 55 Å; and (c) 125,000 mol wt, ESR = 78 Å. These fractions are known to be extensively filtered, filtered in only small amounts, and largely retained, respectively, by the glomerular capillaries. Tracer solutions were infused i.v. into Wistar-Furth rats, and the left kidney was fixed after 5 min to 4 h. The preparations behaved as predicted: initially, all three fractions appeared in the urinary spaces, with 32,000 > 62,000 » 125,000. The smallest fraction was totally cleared from the blood and urinary spaces by 2.5 h, whereas the intermediate and largest fractions were retained in the circulation at high concentrations up to 4 h. With all fractions, when particles occurred in high concentration in the capillary lumina, they were present in similarly high concentrations in the endothelial fenestrae and inner (subendothelial) portions of the basement membrane, but there was a sharp drop in their concentration at this level—i.e., between the inner, looser portions of the basement membrane and its outer, more compact portions. With the two largest fractions, accumulation of particles occurred against the basement membrane in the mesangial regions with time. No accumulation was seen with any of the fractions in the epithelial slits or against the slit membranes. Dextran was also seen in phagosomes in mesangial cells, and in absorption droplets in the glomerular and proximal tubule epithelium. It is concluded that the basement membrane is the main glomerular permeability barrier to dextrans, and (since their behavior is known to be similar) to proteins of comparable dimensions (40,000–200,000 mol wt). The findings are discussed in relation to previous work using electron-opaque tracers to localize the glomerular permeability barrier and in relation to models proposed for the functions of the various glomerular structural elements

FUNCTIONAL EVIDENCE FOR THE EXISTENCE OF A THIRD CELL TYPE IN THE RENAL GLOMERULUS : Phagocytosis of Filtration Residues by a Distinctive "Third" Cell

Two types of cells can be recognized on the luminal side of the glomerular basement membrane: the superficial endothelial cells which directly line the lumen and are comparable to endothelia lining the capillaries of other tissues, and the deep cells, ordinarily not in contact with the lumen, which are distinguished by their long cytoplasmic arms extending for some distance in several directions along the capillary wall, numerous spinous processes, and occasional intraluminal pseudopodia. Experiments carried out with electron-opaque tracers indicated that a functional distinction, based on extent of phagocytosis, can be made between the superficial and deep cells, thus supporting the existence of a distinctive "third" cell (in addition to endothelium and epithelium) in the renal glomerulus. Ferritin, colloidal gold, or thorotrast was administered intravenously to normal and, in the case of ferritin, to nephrotic rats. Kidney tissue was fixed at selected intervals from 1 hour to 10 days after the injection and studied by electron microscopy. Within 1 to 4 hours after tracer administration, the particles which did not traverse the glomerular capillary wall gradually accumulated in the less compact, inner strata of the basement membrane and the large spongy areas of axial regions. After 1 day the concentration of circulating tracer declined and the peripheral areas of the capillaries became relatively free of particles while large accumulations developed in the axial regions. During this period increasing quantities of ferritin were taken up by the deep cells and were found within large and small sized invaginations of their cell membrane or concentrated within cytoplasmic vesicles, vacuoles, multivesicular and dense bodies. At the same time the deep cells showed increased numbers of intraluminal pseudopodia. Within 2 to 4 days the deposits in the spongy areas were cleared and concomitantly increased quantities of tracer appeared in the deep cells within dense cytoplasmic bodies, some of which were more compact than before. When ferritin was given to nephrotic animals the sequence of events was generally the same except that the ferritin deposits at any given period were more massive, their incorporation into the deep cells occurred primarily by means of large pockets 1 to 2 µ in diameter and their clearance from the spongy areas was slower. In normal as well as in nephrotic animals, the phagocytic activity of the superficial endothelium was negligible when compared to that of the deep cells

GLOMERULAR PERMEABILITY : II. FERRITIN TRANSFER ACROSS THE GLOMERULAR CAPILLARY WALL IN NEPHROTIC RATS

Ferritin was used as a tracer to investigate glomerular permeability in the nephrotic rat. The results were compared with those previously obtained in normal animals. A nephrotic syndrome was induced by 9 daily injections of the aminonucleoside of puromycin. Ferritin was administered intravenously on the 10th day, and kidney tissue was fixed at intervals of 5 minutes to 44 hours after injection of the tracer and examined by electron microscopy. The observations confirmed that at this stage of the experimental nephrotic syndrome the changes affect predominantly the visceral epithelium (loss of foot processes, reduction and modification of urinary slits, and intracellular accumulation of vacuoles and protein absorption droplets). Less extensive changes were found in other layers (reduction of endothelial fenestrae, an increase in the population of "deep" cells, and a thinning and "loosening" of the basement membrane.) At short intervals (5 to 15 minutes) after ferritin administration, the tracer was found at high concentration in the lumen and endothelial fenestrae, and at decreasing concentrations embedded throughout the basement membrane and incorporated into the epithelium (within cytoplasmic vesicles and within invaginations of the plasmalemma facing the basement membrane). After longer intervals (1 to 3 hours) the distribution of the tracer within the capillary wall was similar except that its concentration in the epithelium was higher, and, in addition to plasma membrane invaginations and small vesicles, ferritin also marked larger vacuoles, dense bodies, and intermediate forms. Large accumulations of tracer typically occurred in the spongy areas of the basement membrane, especially in the axial regions. Ferritin also appeared in the endothelium within membrane-limited vacuoles and dense bodies, particularly in the deep cells. After 6 to 44 hours the tracer still occurred in the lumen and throughout the basement membrane. The ferritin deposits in the spongy areas as well as the ferritin-containing vacuoles of the deep endothelium were larger and more numerous. In the epithelium ferritin was found not only within various membrane-limited bodies, but also "free" within the cytoplasmic matrix. These observations indicate that in the nephrotic glomerulus, as in the normal, the basement membrane functions as the main filtration barrier; however, in nephrosis, the basement membrane is defective and allows leakage of increased quantitites of ferritin and presumably plasma proteins. The basement membrane defect appears to be fine and widespread, occurring at or near the molecular level of organization of the filter. The accumulation of unfiltered ferritin in axial regions together with the demonstration of its subsequent phagocytosis by the "deep" endothelial cells suggest that the latter may function in the removal of filtration residues. Finally, the findings indicate that in the nephrotic, as in the normal animal, the epithelium acts as a monitor that recovers, at least in part, the protein which leaks through the filter, and that in nephrosis, the recovering activities of the epithelium are greatly enhanced because of the increased permeability of the basement membrane

HORMONE SECRETION BY CELLS DISSOCIATED FROM RAT ANTERIOR PITUITARIES

A new procedure has been developed for dissociating anterior pituitary tissue and producing a viable suspension of single cells. The procedure involves incubation of small tissue blocks in 1 mg/ml trypsin (15 min), followed by incubation in 8 µg/ml neuraminidase and 1 mM EDTA (15 min), followed by mechanical dispersion. Cell yields are ∼55%, based on recovered DNA. By electron microscopy five types of secretory cells (somatotrophs, mammotrophs, thyrotrophs, gonadotrophs, and corticotrophs) plus endothelial and follicular cells can be identified and are morphologically well preserved up to 20 h after dissociation. Throughout this period, the cells incorporate linearly [3H]leucine into protein for up to 4 h at a rate 90% greater than hemipituitaries, and they synthesize, transport intracellularly, and release the two major pituitary secretory products, growth hormone and prolactin. Immediately after dissociation the cells' ability to respond to secretogogues (high K+ and dibutyryl cyclic AMP) is impaired, but after a 6–12-h culture period, the cells apparently recover and discharge 24% and 52%, respectively, of their content of prelabeled growth hormone over a 3-h period in response to these two secretogogues. This represents a stimulation of 109% and 470% over that released by cells incubated in control medium. The results demonstrate that function and morphologic integrity are preserved in this cell system. Therefore it is suitable for the study of various aspects of pituitary secretion and its control