Immunohistochemical localization of glycosaminoglycans and proteoglycans in predentin and dentin of rat incisors.

We examined immunocytochemically the type and distribution of glycosaminoglycans and proteoglycans (PG) in predentin and dentin demineralized with EDTA after aldehyde fixation of rat incisors using (a) four monoclonal antibodies (1-B-5,9-A-2,3-B-3, and 5-D-4) which recognize epitopes in unsulfated chondroitin (C0-S), chondroitin 4-sulfate (C4-S), chondroitin 6-sulfate (C6-S), and keratan sulfate (KS) associated with the PG, and (b) monoclonal (5-D-5) and polyclonal antibodies specific for the core protein of large and small dermatan sulfate (DS) PG. Light microscope immunoperoxidase staining after pre-treatment of tissue sections with chondroitinase ABC localized the majority of stainable PG (C4-S, KS, DSPG, C0-S, and C6-S) in predentin and, to a lesser extent (C4-S and small DSPG), in the dentin matrix. The former site demonstrated relatively homogeneous PG distribution, whereas the latter site revealed that strong staining of C4-S and small DSPG was confined mostly to dentinal tubules surrounding odontoblastic processes, with only weak staining in the rest of the dentin matrix. These results indicate that there is not only a definite difference between PG of predentin and dentin but also a selective decrease in the concentration or alteration of these macromolecules during dentinogenesis and mineralization.     

Schistosoma mansoni: eggshell formation is regulated by pH and calcium

The protein precursors of the schistosome eggshell are synthesized and packaged into secretory vesicles in the vitelline cells. These vesicles appear to contain an emulsion of eggshell precursor material. Evidence is presented to show that these secretory vesicles are acidic as in other systems and that this acidity stabilizes the emulsion and prevents the eggshell cross-linking reactions from occurring. Alkalinizing treatments trigger eggshell formation within the secretory vesicles as shown by (1) the induction of autofluorescence and (2) by electron microscopy which shows that the eggshell precursors have aggregated within the secretory vesicles into spherical particles bearing microspines. These aggregates formed in the secretory vesicles were isolated and shown to have the same protease resistance and amino acid composition as authentic eggshell. The calcium ionophore A23187 induces scattered autofluorescence in intact female worms which electron micrographs show to be due to exocytosis of eggshell material. Based on these observations we propose a model for the formation of schistosome eggshell and suggest that it may apply to all trematodes in which the eggshell precursors are present as stable emulsions in the secretory vesicles of the vitelline cells.     

Nature and distribution of chondroitin sulphate and dermatan sulphate proteoglycans in rabbit alveolar bone

Summary The type and distribution of mineral binding and collagenous matrix-associated chondroitin sulphate and dermatan sulphate proteoglycans in rabbit alveolar bone were studied biochemically and immunocytochemically, using three monoclonal antibodies (mAb 2B6, 3B3, and 1B5). The antibodies specifically recognize oligosaccharide stubs that remain attached to the core protein after enzymatic digestion of proteoglycans and identify epitopes in chondroitin 4-sulphate and dermatan sulphate; chondroitin 6-sulphate and unsulphated chondroitin; and unsulphated chondroitin, respectively. In addition, mAb 2B6 detects chondroitin 4-sulphate with chondroitinase ACII pre-treatment, and dermatan sulphate with chondroitinase B pre-treatment. Bone proteins were extracted from fresh specimens with a three-step extraction procedure: 4m guanidine HCl (G-1 extract), 0.4m EDTA (E-extract), followed by guanidine HCl (G-2 extract), to characterize mineral binding and collagenous matrix associated proteoglycans in E- and G2-extracts, respectively. Biochemical results using Western blot analysis of SDS-polyacrylamide gel electrophoresis of E- and G2-extracts demonstrated that mineral binding proteoglycans contain chondroitin 4-sulphate, chondroitin 6-sulphate, and dermatan sulphate, whereas collagenous matrix associated proteoglycans showed a predominance of dermatan sulphate with a trace of chondroitin 4-sulphate and no detectable chondroitin 6-sulphate or unsulphated chondroitin. Immunocytochemistry showed that staining associated with the mineral phase was limited to the walls of osteocytic lacunae and bone canaliculi, whereas staining associated with the matrix phase was seen on and between collagen fibrils in the remainder of the bone matrix. These results indicate that mineral binding proteoglycans having chondroitin 4-sulphate, dermatan sulphate, and chondroitin 6-sulphate were localized preferentially in the walls of the lacunocanalicular system, whereas collagenous associated dermatan sulphate proteoglycans were distributed over the remainder of the bone matrix.     

Ultrastructure and innervation of water buffalo (Bubalus bubalis) seminal vesicle.

The lining epithelium of secretory end pieces and central glandular duct in the seminal vesicle of the water buffalo (Bubalus bubalis) consists of columnar principal and small polymorphous basal cells. A system of intercellular and even intracellular canaliculi enlarges the secretory surface. The most prominent organelle of the columnar principal cells is the granular endoplasmic reticulum, forming large aggregates of parallel lamellae. Using antibodies against the neural cell adhesion molecule L1 and the neural marker protein gene product 9.5 (PGP 9.5), the innervation pattern of the seminal vesicle becomes evident. The muscular layer surrounding the propria contains a dense network of unmyelinated fibers. Thicker bundles traverse the muscular layer to reach the propria. Around glandular secretory tubules and below the epithelial lining of the glandular duct a tightly woven subepithelial plexus is observed which sends short penetrating branches into the basal zone of the epithelium. These intraepithelial nerves are devoid of Schwann cells and basal lamina (naked axons) and are situated within the intercellular spaces between principal and basal cells. Acetylcholinesterase histochemistry with short (1-2 h) incubation times, dopamine-beta-hydroxylase immunohistochemistry and ultrastructural study of transmitter-containing vesicles was performed. The results suggest that muscular contraction in the seminal vesicle is predominantly under the influence of the sympathetic nervous system, whereas secretory epithelial function is regulated by both sympathetic and parasympathetic fibers.     

Partial biomimetic reconstitution of avian eggshell formation

The avian eggshell is a biocomposite ceramic consisting of minute amounts of organic matrix and a crystalline calcium carbonate (calcite) filler. It is formed by a well regulated spatio-temporal assembling process, where extracellular matrix proteins, especially the sulfated glycosaminoglycan anionic sites of specific proteoglycans, have been involved in nucleation and growth of the inorganic crystalline phase. Together with such extracellular matrix molecules, the activity of carbonic anhydrase, is crucial for the normal eggshell formation. Here, we studied the effect of dermatan sulfate and carbonic anhydrase on the in vitro calcification of non-mineralized eggshell membrane-mammillae substrate at different pH and incubation times. Crystal morphology was analyzed by scanning electron microscopy. Crystal nucleation and growth was delayed at lower pH. Dermatan sulfate modified crystal morphology producing aggregates of large calcite crystals exhibiting a columnar morphology, contributing to the eggshell texture development. Carbonic anhydrase increased the velocity of crystal growth and eventually contributed to the fusion of the crystal aggregates to each other. Although, the effect of other macromolecules could not be ruled out, the combinatory effect of proteoglycans and carbonic anhydrase seems to be important for the control of eggshell formation.     

骨质中硫酸软骨素类蛋白多糖的类型和特征

采用骨质蛋白质的三步 (盐酸胍 - EDTA-盐酸胍 )提取法 ,较完全地提取兔长骨和人牙槽骨骨质中各类蛋白多糖 ( PGs) ,并采用凝胶过滤和离子交换柱层析等方法进行纯化 ,再用单克隆抗体 ( MAb2 B6、MAb3B3和 MAb1 B5)检测、分析其中 PGs的类型和性质 .结果表明 ,兔长骨中 PGs的主要类型为 DS类 ( 4 5k D)、C6S类 ( 2 0 0 k D)、C4S类 ( 4 5k D)和 COS类 ( 2 0 0 k D) PG;人牙槽骨中则主要含 DS类 PG( 4 5k D) ,和少量 COS类 PG( 4 5k D和 1 1 0 k D) ,未发现 C4S类 PG.根据此结果可以推测 ,兔长骨以混合方式 (软骨成骨和类骨质成骨 )骨化 ,而人牙槽骨则以类骨质成骨为主 .两者骨质结构和损伤后修复方式可能也有一定的差异 .     

The fine structure of the secretory cells of the uterus (shell gland) of the chicken

The secretory processes in the shell gland of laying chickens were the subject of this study. Three cell types contribute secretory material to the forming egg: ciliated and non-ciliated columnar cells of the uterine surface epithelium, and cells of tubular glands in the mucosa. The ciliated cells as well as the non-ciliated cells have microvilli, which undergo changes in form and extent during the secretory cycle. At the final stages of shell formation they resemble stereocilia. It is postulated that the microvilli of both cells are active in the production of the cuticle of the shell. The ciliated cell which has both cilia and microvilli manufactures secretory granules which arise from the Golgi complex in varying amounts throughout the egg laying cycle. Granule production reaches its greatest intensity during the early stages of shell deposition. The ciliated cell probably supplies proteinaceous material to the matrix of the forming egg shell. The non-ciliated cell has only microvilli. Secretory granules, containing an acid mucopolysaccharide, arise from the Golgi complex. Some granules are extruded into the uterine lumen where they supply the egg shell with organic matrix. Others migrate towards the supranuclear zone. Here a number of them disintegrate. This is accompanied by the formation of a large membraneless space, which is termed “vacuoloid.” Subsequently the vacuoloid regresses and during regression an extensive rough endoplasmic reticulum with numerous polyribosomes of spiral configuration appears. It is suggested that material in the vacuoloid originating from the disintegrating granules is resynthesized and utilized for the formation of secretory product. The uterine tubular gland cells have irregular, frondlike microvilli. During egg shell deposition, these microvilli form large blebs and are probably related to the elaboration of a watery, calcium-containing fluid.     

Microstructure of matrix and mineral components of eggshells from White Leghorn chickens (Gallus gallus)

The avian eggshell is a composite structure of organic matrix and mineral (calcium carbonate) that is rapidly and sequentially fabricated in the oviduct in <24 hr. The eggshell is an excellent vehicle for the study of biomineralization processes and the role of the organic matrix in the mineral-matrix composite. The organic matrix components of eggshells from White Leghorn chickens (Gallus gallus) were examined by transmission electron microscopy (TEM) and optical microscopy. The mineral phase was analyzed by TEM, scanning electron microscopy (SEM), X-ray compositional microanalysis, and electron diffraction. Ultrastructural examination of the matrices within the calcified eggshell reveals a complex architecture that differs within each of the major zones of the eggshell: the shell membranes, the mammillary zone, the palisade region, and the cuticle. The mammillary layer consists of the calcium reserve assembly (CRA) and crown region, each with a unique substructure. TEM images show that the matrix of the CRA consists of a dense, flocculent material partially embedded within the outer shell membrane (a mostly noncalcified region of the shell). The mantle of the collagen fibers of the shell membranes is rich in polyanions (cuprolinic blue-positive), as is the CRA matrix. The CRA is capped by a centrally located calcium reserve body sac (CRB sac) that contains numerous 300–400 nm, electron-dense, spherical vesicles. Directly above the CRB sac is a zone of matrix consisting of stacks of interconnected vesicles (similar in morphology to CRA vesicles) that are interspersed with a granular material. The palisade region, the largest of the mineralized zones, contains hollow vesicles ∼450 nm (s.d. = 75 nm) in diameter, with a crescent-shaped, electron-dense fringe. An interconnecting matrix material is also found between the vesicles in the palisades region. The cuticle is composed of two layers, a mineralized inner layer and an outer layer consisting of only organic matrix. The bulk of the mineral within the eggshell is calcite, with small amounts of needlelike hydroxyapatite in the inner cuticle and occasionally, vaterite micro crystals found at the base of the palisade (cone) region. The well-crystallized calcite crystals within the palisade are columnar, typically ∼20 μm wide by 100–200 μm long; aside from numerous entrapped vesicles and occasional dislocations, they are relatively defect-free. The bulk of the matrix found in the palisade and crown regions are thought to be residual components of the rapid mineralization process. The unique matrix structure within the CRB corresponds to the region of preferentially solubilized calcite used by the developing embryo and the hydroxyapatite found in the inner cuticle may play a role in the cessation of mineral growth. © 1996 Wiley-Liss, Inc.     

Localization and synthesis of type III collagen and fibronectin in human reparative dentine. Immunoperoxidase and immunogold staining

The injury of dental pulp tissue, following caries, is accompanied by the deposit of a typical hard scar tissue known as reparative dentine which should be regarded as the mineralization of a new organic matrix. Highly purified antibodies were used in combination with immunoperoxidase or immunogold technique at the ultrastructural level to reveal the distribution and synthesis of types I and III collagen and fibronectin elaborated by typical matrix-forming cells in the new tissue. Specific immunoperoxidase labelling, on demineralized teeth, clearly demonstrated that type I collagen represents the main type of collagen (88%). It is associated with bundles of fine striated fibrils of type III collagen and in close vicinity with fibronectin and constituted, at least, the new organic matrix of reparative dentine. Immunogold staining gave precise localization mainly over Golgi apparatus for the 3 components, thus suggesting that the cells concerned should not be considered as new odontoblasts but rather as pulpal cells in the process of differentiation participating in the formation of new dentine. Moreover, these events are very similar to those observed during wound healing in other tissues.     

Ultrastructure of the reproductive system of the black swamp snake (Seminatrix pygaea). II. Annual oviducal cycle

This article is the first ultrastructural study on the annual oviducal cycle in a snake. The ultrastructure of the oviduct was studied in 21 females of the viviparous natricine snake Seminatrix pygaea. Specimens were collected and sacrificed in March, May, June, July, and October from one locale in South Carolina during 1998-1999. The sample included individuals: 1) in an inactive reproductive condition, 2) mated but prior to ovulation, and 3) from early and late periods of gravidity. The oviduct possesses four distinct regions from cranial to caudal: the anterior infundibulum, the posterior infundibulum containing sperm storage tubules (SSTs), the uterus, and the vagina. The epithelium is simple throughout the oviduct and invaginations of the lining form tubular glands in all regions except the anterior infundibulum and the posterior vagina. The tubular glands are not alveolar, as reported in some other snakes, and simply represent a continuation of the oviducal lining with no additional specializations. The anterior infundibulum and vagina show the least amount of variation in relation to season or reproductive condition. In these regions, the epithelium is irregular, varying from squamous to columnar, and cells with elongate cilia alternate with secretory cells. The secretory product of the infundibulum consists largely of lipids, whereas a glycoprotein predominates in the vagina; however, both products are found in these regions and elsewhere in the oviduct. In the SST area and the anterior vagina, tubular glands are compound as well as simple. The epithelium of the SST is most active after mating, and glycoprotein vacuoles and lipid droplets are equally abundant. When present, sperm form tangled masses in the oviducal lumen and glands of the SST area. The glands of the uterus are always simple. During sperm migration, a carrier matrix composed of sloughed epithelial cells, a glycoprotein colloid, lipids, and membranous structures surround sperm in the posterior uterus. During gravidity, tubular glands, cilia, and secretory products diminish with increasing development of the fetus, and numerous capillaries abut the basal lamina of the attenuated epithelial lining of the uterus.     

Localization and synthesis of type III collagen and fibronectin in human reparative dentine

Summary The injury of dental pulp tissue, following caries, is accompanied by the deposit of a typical hard scar tissue known as reparative dentine which should be regarded as the mineralization of a new organic matrix. Highly purified antibodies were used in combination with immunoperoxidase or immunogold technique at the ultrastructural level to reveal the distribution and synthesis of types I and III collagen and fibronectin elaborated by typical matrix-forming cells in the new tissue.Specific immunoperoxidase labelling, on demineralized teeth, clearly demonstrated that type I collagen represents the main type of collagen (88%). It is associated with bundles of fine striated fibrils of type III collagen and in close vicinity with fibronectin and constituted, at least, the new organic matrix of reparative dentine.Immunogold staining gave precise localization mainly over Golgi apparatus for the 3 components, thus suggesting that the cells concerned should not be considered as new odontoblasts but rather as pulpal cells in the process of differentiation participating in the formation of new dentine. Moreover, these events are very similar to those observed during wound healing in other tissues.     

Chromogranin A in the pancreatic islet: cellular and subcellular distribution

Chromogranin A (CGA) is the major soluble protein within secretory vesicles of chromaffin cells. A polyclonal antiserum was raised against bovine CGA and characterized in two-dimensional immunoblots. Cellular and subcellular distribution of CGA in bovine pancreatic islet was investigated by immunocytochemistry. At the light microscopic level, CGA-like immunoreactivity was found in the same cells that react with antibodies against insulin, glucagon, and somatostatin. A minority of cells containing pancreatic polypeptide also showed faint immunostaining. At the ultrastructural level (protein A-gold technique), CGA-like immunoreactivity was confined exclusively to the secretory vesicles. Whereas the hormones were localized mainly in the central part of the secretory vesicles, CGA was present predominantly in the periphery. These findings indicate that a CGA-like protein is a regular constituent of the matrix of secretory vesicles in pancreatic endocrine cells.     

Spectrin-like proteins in green algae (Desmidiaceae)

Immunochemical detection of actin as well as spectrin-like proteins have been carried out in the green algae Micrasterias denticulata, Closterium lunula, and Euastrum oblongum. In these algae, actin is detected on Western blots at 43 kDa with antibodies to actin from higher plant and animal origin. By use of antibodies to human and chicken erythrocyte spectrin a cross-reactivity with desmid proteins is found at about the molecular mass of 220 kDa, where also human erythrocyte spectrin is detected. Additional bands are present at 120 kDa and 70 kDa, which are probably breakdown products. An antibody against chicken alpha-actinin, a small protein of the spectrin superfamily, recognizes bands at 90 kDa, where it is expected, and 70 kDa, probably the same breakdown product as mentioned for spectrin. Isoelectric focusing provides staining at pI 4.6 with antibodies against spectrin. Immunogold labelling of spectrin and alpha-actinin antigens on high-pressure frozen, freeze-substituted Micrasterias denticulata cells with the same antibodies exhibits staining, especially at membranes of different populations of secretory vesicles, at dictyosomes, and the plasma membrane. However, no clear correlation to the growth pattern of the cell could be observed. Taken together, our results demonstrate the presence of spectrin-like proteins in desmid cells which are probably functional in exocytosis.     

Partial biochemical and immunochemical characterization of avian eggshell extracellular matrices.

There is evidence to suggest that extracellular matrix molecules, such as proteoglycans, are involved in the regulation of mineral deposition in calcifying tissues. One mineralizing system which is characterized by extremely rapid mineralization is the hen eggshell. This eggshell consists of a pair of nonmineralized eggshell membranes subjacent to the calcified eggshell proper; the eggshell proper is organized into palisades (columns) of mineralized matrix separated by pores. Between the membranes and the shell proper are compacted foci of tissue called mammillary knobs, which are thought to be sites where mineralization is initiated. Previous work from this laboratory has shown the presence of types I, V, and X collagen in the shell membranes. To address the question of the possible role of proteoglycans and glycosaminoglycans in mineralization of the eggshell, two approaches were used. First, immunohistochemistry was performed with monoclonal antibodies to various proteoglycan and glycosaminoglycan epitopes. This analysis indicates that different glycosaminoglycans are localized to discrete regions within the eggshell. Dermatan sulfate is present within the matrix of the shell proper and, to a lesser extent, the mammillary knobs and the outer portion of the shell membranes. In contrast, keratan sulfate is found in the shell membranes and prominently in the mammillary knobs. Interestingly, different keratan sulfate antibodies immunostain distinct regions of the eggshell, which suggests that various types of keratan sulfate are distributed differently. The second approach utilized was to extract the eggshell membranes and recover anionic molecules by anion-exchange chromatography. This resulted in the extraction of material which was recognized by antibodies to keratan sulfate, but not to chondroitin sulfate. This material was very large, as evidenced by its elution in the void volume of a Sepharose CL-2B column. The large size may be due to the extensive cross-links known to occur in the eggshell. If eggshell membranes are extracted at elevated temperature, the material recovered is of much smaller size. These results indicate that molecules recognized by antibodies to glycosaminoglycans are present in the eggshell, and their localized distribution relative to the calcified matrix suggests that they may be involved in the regulation of mineral deposition.     

Ultrastructural localization of the major proteoglycan and type II procollagen in organelles and extracellular matrix of cultured chondroblasts

The mechanisms of synthesis and intracellular routing of the various cartilage matrix macromolecules are still unclear. We have studied this problem in cultured chondroblasts at the ultrastructural level using monospecific antibodies against the core protein of the keratan sulfate/chondroitin sulfate-rich cartilage proteoglycan (KS:CS-PG) or Type II procollagen, and cuprolinic blue, a cationic dye that binds to the glycosaminoglycan chains of proteoglycans. Intracellularly, the proteoglycan antibodies localized KS:CS-PG and its precursors primarily in the Golgi complex and secretory vesicles. In contrast, the bulk of Type II procollagen was found within the rough endoplasmic reticulum (ER). While devoid of collagen, the extracellular matrix was rich in KS:CS-PG molecules some of which studded the chondroblast plasmalemma. Cuprolinic blue staining indicated that the proteoglycans present in the Golgi complex fell into a predominant class of large proteoglycans, probably representing KS:CS-PG, and a minor class of smaller proteoglycans. Groups of these divergent proteoglycans often occupied distinct Golgi subcompartments; moreover, single large proteoglycans appeared to align along the luminal surface of Golgi cisternae and secretory vesicles. These results suggest that in cultured chondroblasts KS:CS-PG and Type II procollagen are differentially distributed both in organelles and in the extracellular matrix, and that different proteoglycan types may occupy distinct subcompartments in trans Golgi.     

Ultrastructural localization of the major proteoglycan and type II procollagen in organelles and extracellular matrix of cultured chondroblasts

Summary The mechanisms of synthesis and intracellular routing of the various cartilage matrix macromolecules are still unclear. We have studied this problem in cultured chondroblasts at the ultrastructural level using (i) monospecific antibodies against the core protein of the keratan sulfate/chondroitin sulfate-rich cartilage proteoglycan (KS:CS-PG) or Type II procollagen, and (ii) cuprolinic blue, a cationic dye that binds to the glycosaminoglycan chains of proteoglycans. Intracellularly, the proteoglycan antibodies localized KS:CS-PG and its precursors primarily in the Golgi complex and secretory vesicles. In contrast, the bulk of Type II procollagen was found within the rough endoplasmic reticulum (ER). While devoid of collagen, the extracellular matrix was rich in KS:CS-PG molecules some of which studded the chondroblast plasmalemma. Cuprolinic blue staining indicated that the proteoglycans present in the Golgi complex fell into a predominant class of large proteoglycans, probably representing KS:CS-PG, and a minor class of smaller proteoglycans. Groups of these divergent proteoglycans often occupied distinct Golgi subcompartments; moreover, single large proteoglycans appeared to align along the luminal surface of Golgi cisternae and secretory vesicles. These results suggest that in cultured chondroblasts KS:CS-PG and Type II procollagen are differentially distributed both in organelles and in the extracellular matrix, and that different proteoglycan types may occupy distinct subcompartments in trans Golgi.     

The gallbladder of the Capivara Hydrochoerus hydrochoeris (Mammalia: Rodentia)

The authors studied morphological and histochemically the mucopolysaccharides and proteins in the gallbladder tubular glands and epithelial cells of the capivara Hydrochoerus hydrochoeris. Based on the results the authors concluded: 1. the gallbladder single columnar epithelium consists of secretory, migrating, and goblet cells; 2. in the lamina propria are single coiled tubular glands; 3. goblet and tubular gland cells show neutral and sulphated mucopolysaccharides and sialic acid; 4. columnar cells show neutral mucopolysaccharides and protein radicals; 5. migrating cells show only protein radicals.     

Co-solubilization of asymmetric acetylcholinesterase and dermatan sulfate proteoglycan from the extracellular matrix of rat skeletal muscles

We have previously communicated that heparin released asymmetric acetylcholinesterase (AChE) from cholinergic synapses. Here we report studies showing that heparin, besides releasing asymmetric AChE from the skeletal muscle extracellular matrix (ECM), specifically solubilizes a dermatan sulfate proteoglycan (DSPG) which accounts for more than 95% of the 35S-released material. The co-solubilization of AChE and the proteoglycan opens up the possibility that both macromolecules could be involved in the formation of the soluble AChE complex observed after incubation of muscle homogenate with heparin. Our results suggest a possible association between asymmetric AChE and DSPG at the muscle ECM, moreover this work is the first report of the existence of DSPG at the skeletal muscle cell surface.     

Fine structure of the ventral and dorsal lobes of the prostate in the young adult Syrian hamster, Mesocricetus auratus

The normal ventral and dorsal prostatic lobes of the young adult Syrian hamster were examined at the light and electron microscopic levels. Each lobe is composed of branched tubular secretory units separated from each other by loose interacinar connective tissue and draining into the urethra. The lumen of each acinus is lined by a simple epithelium composed of columnar secretory cells with occasional small basal cells. The epithelial layer, with the thin underlying lamina propria, forms a mucosa that is often highly folded. The whole acinus is bounded by a thick muscular stroma. In each of the ventral lobes, there are three main ducts, each one formed of tubular branched tributary secretory units. The walls of the secretory acini are moderately folded. Microvilli dominate the lumenal surface of the secretory epithelial cells. The Golgi complex is very extensive and shows dilated cisternae and secretory vesicles and vacuoles of various sizes. Membrane-bounded secretory granules populate the Golgi and apical areas and are released into the acinar lumen by exocytosis. The rough endoplasmic reticulum is dispersed throughout the cytoplasm, except in the region of the Golgi apparatus. In each of the dorsal lobes, there are several main tubular ducts that open into the urethra. Both proximal (ductal) and distal portions of the glandular tree are secretory in nature. Microvilli and cytoplasmic bulges and blebs dominate the lumenal surface of the secretory cells. The cells are also characterized by highly dilated cisternae of rough endoplasmic reticulum. The secretory cells show heterogeneity in the degree of dilation and distribution of rough endoplasmic reticulum, and this heterogeneity may reflect location in the glandular tree.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subcellular Distribution of 65,000 Calmodulin-Binding Protein (p65) and Synaptophysin (p38) in Adrenal Medulla

Both neuronal and endocrine cells contain secretory vesicles that store and release neurotransmitters and peptides. Neuronal cells release their secretory material from both small synaptic vesicles and large dense-core vesicles (LDCVs), whereas endocrine cells release secretory products from LDCVs. Neuronal small synaptic vesicles are known to express three integral membrane proteins: 65,000 calmodulin-binding protein (65-CMBP) (p65), synaptophysin (p38), and SV2. A controversial question surrounding these three proteins is whether they are present in LDCV membranes of endocrine and neuronal cells. Sucrose density centrifugation of adrenal medulla was performed to study and compare the subcellular distribution of two of these small synaptic vesicle proteins (65-CMBP and synaptophysin). Subsequent immunoblotting and 125I-Protein A binding experiments performed on the fractions obtained from sucrose gradients showed that 65-CMBP was present in fractions corresponding to granule membranes and intact chromaffin granules. Similar immunoblotting and 125I-Protein A binding experiments with synaptophysin antibodies showed that this protein was also present in intact granules and granule membrane fractions. However, an additional membrane component, equilibrating near the upper portion of the sucrose gradient, also showed strong immunoreactivity with anti-synaptophysin and high 125I-Protein A binding activity. In addition, immunoblotting experiments on purified plasma and granule membranes demonstrated that 65-CMBP was a component of both membranes, whereas synaptophysin was only present in granule membranes. Thus, there appears to be a different subcellular localization between 65-CMBP and synaptophysin in the chromaffin cell.