Differential gene expression of 36-kDa microfibril-associated glycoprotein (MAGP-36/MFAP4) in rat organs

By using quantitative Western blot analysis and the real time polymerase chain reaction technique, we investigated the differential gene expression of microfibril-associated glycoprotein (MAGP-36) in rat organs. The gene was expressed highly in sites rich in elastic fibers, such as aorta, skin, and esophagus. However, MAGP-36 was also expressed highly in some other sites containing no elastic fibers. In lung and trachea, the expression levels of MAGP-36 mRNA were about seven times higher than those in other elastic tissues, although the protein abundances were almost at the same levels as other elastic tissues. MAGP-36 seemed to be secreted outside these organs. In brain, kidney, and spleen, although the expression levels of MAGP-36 mRNA were low, substantial amounts of MAGP-36 protein were detected. An immunohistochemical study revealed that MAGP-36 was present at the brush border of the S3 segment of proximal tubules in kidney. Since MAGP-36 is known to bind to mannan, MAGP-36 might be involved in mannose transport in the S3 segment. Thus, MAGP-36 might be multifunctional and present in a wide variety of sites in various organs.     

Expression of intestinal alkaline phosphatase in human organs

Human intestinal alkaline phosphatase was immunohistochemically identified and localized in the pancreas, liver and kidney by use of a monoclonal antibody specific for intestinal alkaline phosphatase isozyme and by amplified biotin-streptavidin staining. In all the examined organs, the intestinal isozyme was found to be localized in the epithelial cells of ducts: bile ducts in the liver, distal convoluted tubules and collecting tubules in the kidney and ducts in the secretory epithelium in the pancreas. In the liver the antibody also stained some sinus-lining cells. In all the examined organs the endothelial cells of the capillaries and some vessels were stained. By use of immunoelectron microscopy, intestinal alkaline phosphatase was, as expected, found to be localized to the microvillar region of the small intestine. The isozyme was abundantly expressed in the apical area of the microvilli and in membrane remnants in the fuzzy coat. Capillaries and vessels in the submucosa were also stained, as well as small vesicles in the endothelial cells. The present investigation demonstrates the expression and localization of the intestinal alkaline phosphatase in several organs, though previously believed to be expressed only in the intestine.     

Expression of intestinal alkaline phosphatase in human organs.

Human intestinal alkaline phosphatase was immunohistochemically identified and localized in the pancreas, liver and kidney by use of a monoclonal antibody specific for intestinal alkaline phosphatase isozyme and by amplified biotin-streptavidin staining. In all the examined organs, the intestinal isozyme was found to be localized in the epithelial cells of ducts: bile ducts in the liver, distal convoluted tubules and collecting tubules in the kidney and ducts in the secretory epithelium in the pancreas. In the liver the antibody also stained some sinus-lining cells. In all the examined organs the endothelial cells of the capillaries and some vessels were stained. By use of immunoelectron microscopy, intestinal alkaline phosphatase was, as expected, found to be localized to the microvillar region of the small intestine. The isozyme was abundantly expressed in the apical area of the microvilli and in membrane remnants in the fuzzy coat. Capillaries and vessels in the submucosa were also stained, as well as small vesicles in the endothelial cells. The present investigation demonstrates the expression and localization of the intestinal alkaline phosphatase in several organs, though previously believed to be expressed only in the intestine.     

Notes on Technic: Differential staining of Fungus and host cells Using a modifciation of Pianeze IIIB

Intercellular secretory capillaries in parotid glands, eccrine sweat glands and intracellular secretory capillaries in parietal cells of gastric glands were demonstrated histo-chemically by the use of the Wachstein-Meisel adenosinetriphosphatase (ATPase) technique in the rabbit, rat and guinea pig. However, with the Wachstein-Meisel 5-nucleotidase technique, secretory capillaries were not stained. For parotid glands, optimal incubation in ATPase substrate mixture was: in rabbit, 15 min; in rat, 2.5 hr; and in guinea pig, 2 hr. For eccrine sweat glands, optimal incubation was 15 min in rabbit, 30 min in rat and 15 min in guinea pig. For parietal cells of gastric glands, optimal incubation was 3 hr for all three species. Secretory capillaries were best demonstrated in the parotid by using rabbit tissue; in eccrine sweat glands, with rat tissue, and in parietal cells, guinea pig tissue. Since ATPase activity in cell membranes of secretory cells may play a part in the mechanism of transport of secretory products from their place of formation in the acini to the excretory ducts, the Wachstein-Meisel ATPase technique can therefore be used successfully for staining secretory capillaries in many of the exocrine glands of laboratory mammals.     

Staining Secretory Capillaries of Exocrine Glands with Techniques for Specific Phosphatases

Intercellular secretory capillaries in parotid glands, eccrine sweat glands and intracellular secretory capillaries in parietal cells of gastric glands were demonstrated histo-chemically by the use of the Wachstein-Meisel adenosinetriphosphatase (ATPase) technique in the rabbit, rat and guinea pig. However, with the Wachstein-Meisel 5-nucleotidase technique, secretory capillaries were not stained. For parotid glands, optimal incubation in ATPase substrate mixture was: in rabbit, 15 min; in rat, 2.5 hr; and in guinea pig, 2 hr. For eccrine sweat glands, optimal incubation was 15 min in rabbit, 30 min in rat and 15 min in guinea pig. For parietal cells of gastric glands, optimal incubation was 3 hr for all three species. Secretory capillaries were best demonstrated in the parotid by using rabbit tissue; in eccrine sweat glands, with rat tissue, and in parietal cells, guinea pig tissue. Since ATPase activity in cell membranes of secretory cells may play a part in the mechanism of transport of secretory products from their place of formation in the acini to the excretory ducts, the Wachstein-Meisel ATPase technique can therefore be used successfully for staining secretory capillaries in many of the exocrine glands of laboratory mammals.     

Vasopressin and oxytocin as neuromediators

Vasopressin and oxytocin are peptide hormones which act on a variety of target organs, including kidney, smooth muscle, liver, and anterior pituitary. During the last decade, it has become apparent that these two neuropeptides may in addition act as neurotransmitters. We review a number of arguments which support this conjecture: 1) Vasopressin and oxytocin are not only synthesized in hypothalamoneurohypophysial neurones, but also in other--hypothalamic and extrahypothalamic--cell bodies whose axon projects to the limbic system, the brainstem and the spinal cord. 2) Vasopressin and oxytocin can be shed from central axons by the same secretory mechanism as are classical neurotransmitters. 3) Specific binding sites having a high affinity for vasopressin and/or oxytocin are present in the central nervous system. These binding sites represent functional receptors, because agonist binding leads to an increase in membrane phosphatidylinositol turnover. 4) Receptors, or at least part of them, are localized on neurones, since application of exogenous vasopressin and oxytocin alters the rate of firing of single neurones present in regions where binding sites have been detected autoradiographically. 5) Central vasopressin and oxytocin may play a role in brain functions, since in situ injection of antagonists interferes with physiological regulations.     

Identification and characterization of telocytes in the uterus of the oviduct in the Chinese soft‐shelled turtle,Pelodiscus sinensis: TEM evidence

Telocytes (Tcs) are cells with telopodes (Tps), which are very long cellular extensions with alternating thin segments (podomers) and dilated bead‐like thick regions known as podoms. Tcs are a distinct category of interstitial cells and have been identified in many mammalian organs including heart, lung and kidney. The present study investigates the existence, ultrastructure, distribution and contacts of Tcs with surrounding cells in the uterus (shell gland) of the oviduct of the Chinese soft‐shelled turtle, Pelodiscus sinensis. Samples from the uterine segment of the oviduct were examined by transmission electron microscopy. Tcs were mainly located in the lamina propria beneath the simple columnar epithelium of the uterus and were situated close to nerve endings, capillaries, collagen fibres and secretory glands. The complete morphology of Tcs and Tps was clearly observed and our data confirmed the existence of Tcs in the uterus of the Chinese soft‐shelled turtle Pelodiscus sinensis. Our results suggest these cells contribute to the function of the secretory glands and contraction of the uterus.     

Cytochemical and electron-microscopic study of the paraoesophageal bodies and related nerves in Schizophyllum sabulosum (L.), diplopoda julidae

The ultrastructural study of the paraoesophageal bodies of Schizophyllum sabulosum reveals the occurrence of two axonal types (ax 1 and ax 2) near secretory cells. Two possibilities exist for the functional role of the nerves related to these paraoesophageal bodies. The results of treatment with proteases (pronase, pepsin, trypsin) and the identification of glycogen in both the paraoesophageal bodies and the nerves that link them to the brain and Gabe organs, suggest transport of at least part of the secretions from the paraoesophageal bodies to the Gabe organs.     

Choline transporter as a novel target for molecular imaging of cancer

Abnormalities of choline processing in cancer cells have been used as a basis for imaging of cancer with positron emission tomography and magnetic resonance spectroscopy. In this study, the transport mechanism for choline was investigated in cultured PC-3 prostate cancer cells. Furthermore, tritiated hemicholinium 3 (HC-3), a well-known inhibitor of choline transport, was studied as a prototypic molecular imaging probe in PC-3 cells and 9L glioma-bearing rats. [(3)H]Choline uptake by PC-3 cells was found to have both facilitative and nonfacilitative components. Facilitative transport was characterized by partial sodium dependence and intermediate affinity (K(M) = 9.7 +/- 0.8 microM). HC-3 inhibited choline with a K(I) of 10.5+/- 2.2 microM. Ouabain (1 mM) caused a 94% reduction in choline uptake. At physiologic choline concentration, phosphocholine was the rapid and predominant metabolic fate. The binding of [(3)H]HC-3 to PC-3 cells was rapid and specific (competitively blocked with unlabeled HC-3). Biodistribution of [(3)H]HC-3 in 9L glioma-bearing rats showed the ranking of uptake to be kidney > lung > tumor > liver > skeletal muscle congruent with blood > brain. In comparison with [(14)C]choline, [(3)H]HC-3 showed over twofold higher tumor uptake and favorable uptake ratios of tumor to blood, tumor to muscle, tumor to lung, and tumor to liver. The data demonstrate the quantitative importance of an intermediate-affinity, partially sodium-dependent choline transport system on choline processing in PC-3 cancer cells. The biodistribution properties of [(3)H]HC-3 in tumor-bearing rats encourage the development of molecular imaging probes based on choline transporter binding ligands.     

Atorvastatin activates heme oxygenase-1 at the stress response elements

Statins are known to inhibit growth of a number of cancer cells, but their mechanism of action is not well established. In this study, human prostate adenocarcinoma PC-3 and breast adenocarcinoma MCF-7 cell lines were used as models to investigate the mechanism of action of atorvastatin, one of the statins. Atorvastatin was found to induce apoptosis in PC-3 cells at a concentration of 1 μM, and in MCF-7 cells at 50 μM. Initial survey of possible pathway using various pathway-specific luciferase reporter assays showed that atorvastatin-activated antioxidant response element (ARE), suggesting oxidative stress pathway may play a role in atorvastatin-induced apoptosis in both cell lines. Among the antioxidant response genes, heme oxygenase-1 (HO-1) was significantly up-regulated by atorvastatin. Pre-incubation of the cells with geranylgeranyl pyrophosphate blocked atorvastatin-induced apoptosis, but not up-regulation of HO-1, suggesting that atorvastatin-induced apoptosis is dependent on GTPase activity and up-regulation of HO-1 gene is not. Six ARE-like elements (designated StRE1 [stress response element] through StRE6) are present in the HO-1 promoter. Atorvastatin was able to activate all of the elements. Because these StRE sites are present in clusters in HO-1 promoter, up-regulation of HO-1 by atorvastatin may involve multiple StRE sites. The role of HO-1 in atorvastatin-induced apoptosis in PC-3 and MCF-7 remains to be studied.     

Ultrastructural study of tracer permeability through the cat and ferret enamel organ

The access of exogenous materials to the developing enamel surface has been intensively studied in rodents, but not in other mammalian species. This ultrastructural study investigates the permeability of injected horseradish peroxidase (HRP) and lanthanum tracers in cat and ferret tooth buds. In cat enamel organs fixed by immersion, lanthanum did not escape the capillaries overlying secretory stage tooth buds, but it did permeate up to the distal junctions of ruffle-ended (RA) and the proximal junctions of smooth-ended (SA) ameloblasts. Perfusion fixation with lanthanum compromised junctional integrity of cat ameloblasts at all stages of development. Similarly, HRP rarely escaped the capillaries associated with cat secretory stage enamel organs. However, unlike lanthanum, HRP was mostly confined to the vasculature of maturation stage enamel organs in immersion fixed cats at all time intervals examined. In ferrets, HRP penetrated up to, but not beyond, the distal junctional complexes of secretory ameloblasts. In maturation stage enamel organs, HRP coated the papillary and RA cells, but did not penetrate the RA distal cell junctions. HRP did permeate the extracellular spaces of SA to reach the underlying enamel surface. Ameloblasts in transitional phases of SA and RA endocytosed HRP at the distal cell surface. This data leads to several conclusions. First, HRP localization in the ferret paralleled that observed in rodents. Second, the results of cat enamel organs substantiate previous studies showing perfusion fixation can increase vascular and intercellular permeability to lanthanum. However, in cats fixed by immersion, both lanthanum and HRP were restricted to capillaries associated with the secretory stage enamel organ, and only lanthanum escaped maturation stage capillaries. It is suggested that variations in the fenestrations and distribution of capillaries associated with the cat enamel organ may differentially retain some materials and permit other materials to escape with relative ease.     

VEGF-C and VEGF-D expression in neuroendocrine cells and their receptor, VEGFR-3, in fenestrated blood vessels in human tissues.

Recently, vascular endothelial growth factor receptor 3 (VEGFR-3) has been shown to provide a specific marker for lymphatic endothelia in certain human tissues. In this study, we have investigated the expression of VEGFR-3 and its ligands VEGF-C and VEGF-D in fetal and adult tissues. VEGFR-3 was consistently detected in the endothelium of lymphatic vessels such as the thoracic duct, but fenestrated capillaries of several organs including the bone marrow, splenic and hepatic sinusoids, kidney glomeruli and endocrine glands also expressed this receptor. VEGF-C and VEGF-D, which bind both VEGFR-2 and VEGFR-3 were expressed in vascular smooth muscle cells. In addition, intense cytoplasmic staining for VEGF-C was observed in neuroendocrine cells such as the alpha cells of the islets of Langerhans, prolactin secreting cells of the anterior pituitary, adrenal medullary cells, and dispersed neuroendocrine cells of the gastrointestinal tract. VEGF-D was observed in the innermost zone of the adrenal cortex and in certain dispersed neuroendocrine cells. These results suggest that VEGF-C and VEGF-D have a paracrine function and perhaps a role in peptide release from secretory granules of certain neuroendocrine cells to surrounding capillaries.     

Cholesterol regulates prostasome release from secretory lysosomes in PC-3 human prostate cancer cells

Prostasomes are vesicles secreted by epithelial cells of the prostate gland. However, little is known about the mechanism and the regulation of prostasome secretion. Since endocytic organelles may be involved in prostasome release, PC-3-derived prostasomes were investigated by Western blot analysis for the presence of marker proteins normally associated with these organelles. Prostasomes secreted by PC-3 cells contain clathrin, Tsg101, Hrs, Rab11, Rab5, LAMP-1, LAMP-2, LAMP-3/CD63, and annexin II. Moreover, electron microscopy of PC-3 cells revealed the presence of characteristic multivesicular body-like secretory lysosomes containing vesicles with the same size-distribution as released prostasomes. Ultrastructural immunogold labelling showed that LAMP-1, LAMP-2 and LAMP-3/CD63 were associated with these vesicles. In addition, we have investigated whether cholesterol plays a role in prostasome release by the human prostate cancer cell line PC-3. Interestingly, prostasome release was significantly increased when the cholesterol levels of PC-3 cells were reduced by the cholesterol-sequestering agent methyl-beta-cyclodextrin (MBCD), or by treatment with lovastatin and mevalonate. In conclusion, these studies indicate that cholesterol plays an important role in the release of prostasomes by the human prostate cancer PC-3 cells, and suggest that prostasomes may be released after fusion of secretory lysosomes with the plasma membrane.     

Aquaporins: water channel proteins of the cell membrane

Aquaporins (AQP) are integral membrane proteins that serve as channels in the transfer of water, and in some cases, small solutes across the membrane. They are conserved in bacteria, plants, and animals. Structural analyses of the molecules have revealed the presence of a pore in the center of each aquaporin molecule. In mammalian cells, more than 10 isoforms (AQP0-AQP10) have been identified so far. They are differentially expressed in many types of cells and tissues in the body. AQP0 is abundant in the lens. AQP1 is found in the blood vessels, kidney proximal tubules, eye, and ear. AQP2 is expressed in the kidney collecting ducts, where it shuttles between the intracellular storage sites and the plasma membrane under the control of antidiuretic hormone (ADH). Mutations of AQP2 result in diabetes insipidus. AQP3 is present in the kidney collecting ducts, epidermis, urinary, respiratory, and digestive tracts. AQP3 in organs other than the kidney may be involved in the supply of water to them. AQP4 is present in the brain astrocytes, eye, ear, skeletal muscle, stomach parietal cells, and kidney collecting ducts. AQP5 is in the secretory cells such as salivary, lacrimal, and sweat glands. AQP5 is also expressed in the ear and eye. AQP6 is localized intracellular vesicles in the kidney collecting duct cells. AQP7 is expressed in the adipocytes, testis, and kidney. AQP8 is expressed in the kidney, testis, and liver. AQP9 is present in the liver and leukocytes. AQP10 is expressed in the intestine. The diverse and characteristic distribution of aquaporins in the body suggests their important and specific roles in each organ.     

Palmitoylation sites and processing of synaptotagmin I,the putative calcium sensor for neurosecretion

Synaptotagmin I, the calcium sensor for neurotransmission, is palmitoylated. We have identified the palmitoylation sites as five cysteine residues located between the transmembrane and cytoplasmic regions. In contrast to wild-type synaptotagmin, the non-acylated mutant is not converted to the endoglycosidase-H-resistant form after expression in CV-1 cells. This indicates a block in transport through the Golgi complex. However, when expressed in PC-12 and RBL cells non-acylated synaptotagmin is targeted to the plasma membrane and to secretory granules. No significant cleavage of [(3)H]palmitate from synaptotagmin was observed in pulse-chase experiments. This indicates that the majority of fatty acids are structural rather than dynamic components.     

Licht- und elektronenmikroskopische Untersuchung von neurosekretorischen Zellen im Gehirn der Eintagsfliege Ephemera danica Müll. (Ephemeroptera: Ephemeridae)

In the brains of the males the amount of stained secretory material was nearly constant during the last four instars. In the females a decrease in this neurosecretory product in the last nymphal and subimaginal stage could be observed, followed by an increase in the imagines. In the final nymphal stage four types of neurosecretory cells (nsc) were found in the medial protocerebral cell group, showing differences in shape, size, and the contents of the cytoplasm, especially of the secretory granules. In addition to the medial nsc, some cells in the frontal part of the brain and in the deutocerebrum are described. They contain electron-opaque granules and probably have a neurosecretory function. The secretory product of the medial nsc is transported along the axons of the nervus corporis cardiaci 1 (ncc1), an unpaired nerve tract on the ventral side of the brain. Leaving the brain the ncc1 immediately enters the corpus cardiacum. Connections between secretory granules and neurotubuli point to an important role for the neurotubuli in the transport of secretory material.     

A regulatory role of polycystin-1 on cystic fibrosis transmembrane conductance regulator plasma membrane expression.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by genetic mutations in either PKD1 or PKD2, the genes that encode polycystin-1 (PC-1) and polycystin-2 (PC-2), respectively. ADPKD is characterized by the formation of multiple, progressive, fluid-filled renal cysts. To elucidate the mechanism of fluid secretion by ADPKD cysts, we examined the effect of PC-1 on the plasma membrane expression of cystic fibrosis transmembrane conductance regulator (CFTR), a key Cl(-) secretory protein. Five stably transfected MDCK lines were used in this study: two transfected with empty vector (control cells) and three expressing human PC-1 (PC-1 cells). The cAMP-induced endogenous short circuit currents (I(sc)) were smaller in PC-1 cells than in control cells. Compared to control cells, PC-1 cells transiently expressing pEGFP-CFTR showed significant reduction of whole cell cAMP-activated Cl(-) currents. Cell surface biotinylation experiments also indicated a reduction in surface expression of CFTR in PC-1 cells compared to control. Furthermore, studies using CHO cells transiently expressing PC-1 and CFTR suggest the importance of the PC-1 COOH-terminus in the observed reduction of CFTR plasma membrane expression. No differences in either endogeneous K(+) currents or P2Y receptor responses were observed between PC-1 and control cells, indicating the specificity of PC-1's action. These results indicate that PC-1 selectively maintains low cell surface expression of CFTR. Moreover, these findings suggest that the malfunction of PC-1 enhances plasma membrane expression of CFTR, thus causing abnormal Cl(-)secretion into the cyst lumen.     

Immunohistochemical localization of annexin V (CaBP33) in rat organs.

We investigated the cellular distribution of annexin V (CaBP33) in rat tissues by immunohistochemistry. Several cell types were shown to express the protein. Glial cells in the cerebellum and in the optic nerve, the corneal epithelium, the posterior epithelium in the iris, chondrocytes, skeletal muscle cells and cardiomyocytes, the capillary endothelial cells in many organs, the muscularis mucosae and the muscular layer in the intestinal tract, hepatocytes, Müller cells in the retina, the lens fibers, Sertoli and Leydig cells in the testis, and smooth muscle cells in the epididymis and bronchi displayed intense immunostaining. In the adrenal gland, only the cortex showed immunoreaction product. In the kidney, no apparent staining of renal cells was observed, whereas endothelial cells of peritubular capillaries were stained. In the heart, annexin V was found associated exclusively with the sarcolemma and intercalated discs, as opposed to the diffuse distribution of the protein in skeletal muscle cells. In the spleen, only reticular elements in the white pulp and endothelial cells in the red pulp appeared to be immunostained. The present data complement the biochemical work thus far done on annexin V and suggest that the protein is neither restricted to secretory cells nor exclusively related to exocytotic events in secretory cells.     

Localization of NO-Ergic Structures in Oral Lobes, Labia, and Esophagus of the Bivalve Mollusc Crenomytilus grayanus

Localization of NO-ergic elements in oral lobes, labia, and esophagus in the bivalve mollusc, mussel Crenomytilus grayanus was studied using histochemical technique [1] for detection of NADPH-diaphorase (EC 1.6.99.1). The NO-producing elements were revealed in all studied parts of the digestive system. NADPH-diaphorase was found in nerve and secretory cells as well as in nerve plexuses. Numerous NO-ergic nerve cells were observed in the basal part of epithelium of labia and of the initial part of esophagus as well as in the subepithelial area of these organs. In the middle and posterior parts of esophagus, only subepithelially located NO-ergic nerve cell are present. Basiepithelial NO-producing secretory cells are found in all the parts, but most of these cells are observed in labia and the initial part of esophagus. Subepithelial secretory cells labeled with diformazan granules are spread from the folded surface of oral lobes to the initial part of esophagus; no such cells were found on the smooth surface of the lobes. The deposit labeled basi- and subepithelial nerve plexuses in all studied organs except for oral lobes. These plexuses are the most developed in labia and the initial part of esophagus of the studied mollusc.