Cellular Localization of Transforming Growth Factor-α mRNA in Rat Forebrain

Abstract: The cellular localization of transforming growth factor-α (TGFa) mRNA in juvenile and adult rat forebrain was examined using in situ hybridization with a ³⁵S-labeled cRNA probe. TGFα cRNA-labeled neuronal perikarya were distributed across many forebrain regions including the olfactory bulb, caudate-putamen, nucleus accumbens, olfactory tubercle, ventral pallidum, amygdala, hippocam-pal stratum granulosum and CA3 stratum pyramidale, and piriform, entorhinal, and retrosplenial cortices. TGFα cRNA-hybridizing cells were also localized to several thalamic nuclei and to the suprachiasmatic, dorsomedial, and ventromedial nuclei of the hypothalamus. In addition, labeled cells were present in regions of white matter including the corpus callosum, anterior commissure, internal and external capsules, optic tract, and lateral olfactory tract. Thus, both neurons and glia appear to synthesize TGFα in normal brain. Hybridization densities were greater in neuronal fields at 2 weeks of age compared with the adult, suggesting a role for TGFα in the development of several forebrain systems. Our results demonstrating the prominent and widespread expression of TGFα mRNA in forebrain, combined with the extremely low abundance of epidermal growth factor mRNA in brain, support the argument that TGFα is the principal endogenous ligand for the epidermal growth factor receptor in normal brain.     

Cross-reactivity of an antiserum to the α-subunit of the Na+, K+-ATPase of toad (Bufo marinus) kidney with basal and apical membranes of transporting epithelia of the rat

Summary An antibody to the 96 kD -subunit of the Na⁺, K⁺ -ATPase from Bufo marinus has been used in immunostaining rat kidney and salivary glands. Intense staining was observed on basolateral membranes of distal tubules of the kidney and striated ducts of the three major salivary glands. Less intense staining was seen on the basolateral membranes of parotid acinar cells, but no staining was seen on the acinar cells of submandibular or sublingual glands. These sites of staining have been shown, by other methods, to posses substantial Na⁺, K⁺ -ATPase, indicating that the antibody recognizes antigenic determinants of the sodium pump highly conserved in the course of evolution. In addition, staining with this antibody was observed at the apical region of cells of the proximal straight tubule and of the papillary collecting duct in the kidney. Absorption studies suggest that the apical antigenic determinants are the same or closely related to each other but are distinct from basolateral antigenic determinants.     

Immunohistochemical localization of the metalloproteinase meprin in salivary glands of male and female mice.

Meprin is a membrane-bound metalloproteinase which is expressed at high levels in the brush border membrane of proximal tubules of kidneys of some mouse strains (referred to as high meprin-activity mice). The mature active proteinase is not present in kidneys of many inbred strains of mice; however, these low meprin-activity mice possess a kidney protein that crossreacts with polyclonal antibodies prepared against meprin. In the present studies, immunohistochemical methods were used to determine the presence of meprin in liver, pancreas, spleen, testis, thymus, kidney, salivary glands, stomach, duodenum, and skin. Meprin crossreactivity was observed only in kidney and salivary glands. In salivary glands, the enzyme was found on the luminal surface of intercalated and striated ducts of submandibular and parotid glands and on interlobular ducts of the latter. In both kidney and salivary glands, the intensity of immunochemical staining was greater in males compared with females. For both sexes, immunoreactivity was markedly greater in the high meprin-activity mice compared to the low meprin-activity mice. These studies indicate that meprin has a limited tissue distribution, and that genetic and hormonal factors that regulate the proteinase are similar in kidney and salivary glands. The localization of the proteinase implies that the enzyme functions in modifying proteins and peptides that are secreted or re-absorbed in the ducts of these tissues.     

Carbonic anhydrase activity in kidney and lower intestine of the European starling

A histochemical investigation of kidney and lower intestine of the European starling (Sturnus vulgaris) shows no carbonic anhydrase activity in proximal convoluted tubules, although activity is seen in similarly prepared sections of rat proximal tubules. Early distal tubule cells in the starling are stained throughout the cytoplasm and at the apical and highly infolded basolateral membranes. Late distal tubules lose apical activity and have reduced basolateral infolding, resulting in less intense staining. Darkly stained intercalated cells appear in the connecting tubules and cortical collecting ducts. Both of these segments also show intense basolateral staining. Medullary cones of the starling are highly organized, with central zones containing unstained thin descending limbs of loops of Henle, surrounded by both medullary collecting ducts with only scattered cells staining for enzyme, and by thick ascending limb segments. The latter contain many uniformly stained cells intermingled with occasional unstained cells. Scattered cells of the starling colonic villi demonstrate intense apical brush border membrane staining as well as cytoplasmic staining. Cells lining the cloaca stain less intensely. A biochemical assay for carbonic anhydrase was used to quantify enzyme activity in these tissues. Starling kidney contained 1.96 ± 0.33 (mean ± SEM) enzyme units/mg protein, less than half the activity seen in rat kidney. Stripped colonic epithelium contained 0.66 ± 0.15 enzyme units/mg protein. These quantitative results correlate well with the interpretations derived from the histochemical observations. The lack of proximal tubule carbonic anhydrase activity suggests that the avian kidney relies more on distal nephron segments to achieve net acidification of the urine.     

Monoclonal antibodies against rat kidney antigens.

The proximal tubule of the nephron is subdivided into three structurally and functionally distinct segments, which can be differentiated with the help of special methods. With the aim of producing selective markers for these three portions of the proximal tubule, we raised monoclonal antibodies against the brush border membranes of the rat kidney. Immunohistochemistry was carried out with eleven different monoclonal antibodies to sections of rat kidney and other tissues at the light- and electron-microscopical level. These monoclonal antibodies mainly detect antigens located on the brush border of the proximal tubule, and they allow a distinction between its three segments. However, some antibodies also recognize other portions of the nephron, or even the glomerulus or stromal elements. Sites recognized by the antibodies are not limited to the kidney, but staining is observed on the intestinal brush border, the intralobular ducts of the pancreas, the bile canaliculi of the liver and on the macrophage clusters of the spleen. These antibodies are interesting reagents which can be applied to study biochemical differences between brush border membranes. In addition, they recognize antigenically related sites in other organs with reabsorptive or secretory tasks.     

Expression of cytochrome P-450 4 enzymes in the kidney and liver: Regulation by PPAR and species-difference between rat and human

Members of the cytochrome P-450 4 (CYP4) family catalyze the ω-hydroxylation of fatty acids, and some of them have the PPAR response element in the promoter area of the genes. The localization of CYP4A and PPAR isoforms and the effect of PPAR agonists on CYP4A protein level and activity were determined in rat kidney and liver. Immunoblot analysis showed that CYP4A was expressed in the liver and proximal tubule, with lower expression in the preglomerular microvessel, glomerulus and thick ascending limb (TAL), but the expression was not detected in the collecting duct. PPARα was expressed in the liver, proximal tubule and TAL. PPARγ was expressed in the collecting duct, with lower expression in the TAL, but no expression in the proximal tubule and liver. The PPARα agonist clofibrate induced CYP4A protein levels and activity in the renal cortex and liver. The PPARγ agonist pioglitazone did not modulate them in these tissues. The localization of CYP4A and CYP4F were further determined in human kidney and liver by immunohistochemical technique. Immunostainings for CYP4A and CYP4F were observed in the hepatocytes of the liver lobule and the proximal tubules, with lower stainings in the TALs and collecting ducts, but no staining in the glomeruli or renal vasculatures. These results indicate that the inducibility of CYP4A by PPAR agonists in the rat tissues correlates with the expression of the respective PPAR isoforms, and that the localization of CYP4 in the kidney has a species-difference between rat and human.     

Comparative distribution of carbonic anhydrase isozymes III and II in rodent tissues

Carbonic anhydrase (CA) III was demonstrated immunocytochemically in epithelium in some regions of salivary gland ducts, colon, bronchi, and male genital tract and in adipocytes, in addition to skeletal muscle and liver where the isozyme was previously localized. Basal cells beneath the submandibular gland's excretory ducts in guinea pig stained for CA III. Carbonic anhydrase III occurred alone in some and with CA II in other sites but was often absent from CA-II-containing types of cells. This was exemplified by CA III's abundance in CA-II-positive proximal colon and its sparsity in the CA-II-rich distal colon of the mouse. Striated ducts in guinea pig, but not mouse salivary glands, stained darker for CA and appeared accordingly to function more actively in ion transport compared with excretory ducts. Carbonic anhydrase content varied among genera in liver and pancreas and between mouse species and strains in salivary glands and kidney. Newly observed murine sites of CA II activity included Auerbach's plexus and a population of leukocytes infiltrating the lamina propria in small intestine, and several types of cells in the male genital tract. In immunoblot tests, antisera to CA III showed no cross reactivity with antisera to CA II, but those to CA II disclosed weak cross reactivity with CA III.     

The relation between structure and function of bile ducts in man, some laboratory animals and the Adelie penguin.

The biliary trees of man, dog, cat, rabbit, rat, guinea pig and penguin were examined in histological sections and by latex casts. The trees of man, dog, and cat were similar with only minor differences. Tubulo-alveolar glands were present in all three species around large intrahepatic ducts and in large portal tracts there were zones of ductules (areas with many small bile ducts), surrounded by small vessels with no apparent relation to hepatocytes. Both these features were present in the guinea pig and tubulo-alveolar glands were present in the penguin liver. The biliary epithelium of the rat was comparatively simple but that of the rabbit appeared to be highly specialized. An estimation of the complexity of the biliary tree was obtained in the mammals by comparing the circumference of small portal venous branches with the circumference of the accompanying bile ducts, and obtaining a ratio. Man, dog, and cat had fewer and smaller bile ducts than the other species. The literature on the rate of formation and composition of bile in the species studied here was reviewed and it appears that the physiology of bile secretion can be related to the morphology of the biliary tree.     

Keratin 19-like immunoreactivity in receptor cells of mammalian taste buds

Three monoclonal antibodies, 4.62, LPZK and 170.2.14, were usedto evaluate keratin 19-like immunoreactivity in gustatory epithelia.Keratin 19-like immunoreactivity was restricted to the intragemmalcells for all types of mammalian taste buds examined. Thesetaste buds included fungiform, foliate and vallate taste budsin rat, gerbil and rabbit, and nasopalatine, epiglottal andpalatine taste buds in rat. There was no keratin 19-like immunoreactivityin basal cells or in perigemmal cells lateral to the immunoreactivetaste receptor cells. Denervation of the rat vallate papillaeliminated all taste buds, as well as all immunoreactive tastecells. That the immunoreactive material in the taste cells waskeratin 19 was supported by the comparable staining of rat tastebuds with each of three monoclonal antibodies specific for keratin19. Furthermore, as predicted, these antibodies selectivelystained luminal cells of ral bile ducts, bladder, salivary ducts,trachea, ureter and uterus. It was concluded that monoclonalantibodies against keratin 19 can usefully distinguish intragemmaltaste receptor cells from keratinocytes, and from the perigemmaland basal cells of gustatory epithelia. Anti-keratin 19 antibodiesmay serve to identify differentiated taste cells in gustatoryepithelia undergoing taste bud development, renewal, degenerationor regeneration.     

Intralobular distribution of albumin synthetic activity in the hepatocytes of the normal and regenerating mouse liver

Immunolocalization of albumin was investigated in normal and regenerating adult mouse liver after perfusion fixation with saponin. Light and electron microscopy have demonstrated that in normal liver the intralobular activity of albumin synthesis was distributed along the gradient decreasing from the portal tract to central vein. The uniform intensive staining of all hepatocytes was observed in regenerating liver. In addition, albumin synthesis was expressed in the epithelial cells of portal bile ducts.     

Localization of cAMP-dependent signal transducers in early rat liver carcinogenesis

 Cyclic AMP (cAMP) is an important regulator of liver growth and differentiation. The main intracellular cAMP receptor, cAMP-dependent protein kinase (PKA), consists of two regulatory (R) and two catalytic (C) subunits. There are two classes, RI and RII, of the regulatory subunit, giving rise to type I (RI₂C₂) and type II (RII₂C₂) PKA. The RI/RII ratio generally decreases during organ development, and increases during carcinogenesis. Alterations in this ratio have been implicated as an important factor in experimental and clinical carcinogenesis. We have studied the expression of RIα, RIIα, Cα, and an important substrate of PKA, the cAMP-response element binding protein, during rat liver carcinogenesis. Two-color immunofluorescence and confocal laser scan microscopy were used to characterize localization of the cAMP-dependent signal transducers in hepatocytes, bile ducts, oval cells, and preneoplastic lesions. We found that bile ducts and oval cells (putative liver stem cells) contained a higher RI/RII ratio than hepatocytes and preneoplastic lesions. Thus, an altered RI/RII ratio was not detected during early rat liver carcinogenesis, but may contribute to differentiation of putative liver stem cells to hepatocytes. Accepted: 19 August 1997     

Immunohistochemical localisation of pterin-4α-carbinolamine dehydratase in rat peripheral organs

 The bifunctional protein PCD/DCoH is both a pterin-4α-carbinolamine dehydratase (PCD) involved in the recycling of tetrahydrobiopterin (BH₄) and a dimerisation cofactor (DCoH) of the hepatic nuclear factor 1α (HNF-1α). An antiserum raised against rat PCD/DCoH was used to localise the protein in peripheral organs. In liver, all the hepatocytes but not the other cell types are immunoreactive. In kidney, the protein is prevalent in the proximal and distal convoluted tubules. In adrenals, all the cells of the medulla are labelled. Positive nerve cells occur in myenteric ganglia of the whole gastrointestinal tract and in the intestinal submucous ganglia. Many positive endocrine cells are present in the epithelium. The immunoreactivity is either cytoplasmic (hepatocytes, convoluted tubules of the kidney and part of the gastrointestinal endocrine cells) or prominently nuclear (kidney collecting tubules, adrenals, intestinal neural plexuses and part of the gastrointestinal endocrine cells). Our results show that PCD/DCoH is present in cells expressing enzymes that use BH₄ as a cofactor and/or HNF-1α. In addition, PCD/DCoH is present in other cells, for example, neurons in the submucosal plexus. This fact and the prominent nuclear immunoreactivity found in all the positive cells derived from the neural crests argue in favour of a new, still unknown function for the protein. Accepted: 18 January 1999     

The passage of radioactive lanthanum from the biliary to the vascular system

Summary Radioactive lanthanum nitrate, an electron opaque tracer, was injected into the common bile duct of rats. Two minutes following the end of injection, samples of blood for radioactivity counts, and of liver and kidney for electron microscopic studies were taken. High levels of radioactivity found in the blood, and demonstration of lanthanum in the kidney by electron microscopy, indicated that this tracer entered the blood stream in vivo. No lanthanum was seen in the cytoplasm of liver cells, and there was no evidence of rupture in bile ducts, junctional ducts, or liver cells. Tight junctions connecting parenchymal cells and cholangiolar cells appeared well preserved. Lanthanum was seen in bile canaliculi, interspaces between liver cells, portions of the extracellular space of the liver, lumina of cholangioles and lumina of portal venules and sinusoids. It is postulated that lanthanum passed from the biliary tract, the site of injection, through the tight junction between liver cells and cholangiolar cells. It is suggested that such passage may represent a physiologic pathway, but the possibility of a chemical action of lanthanum on the tight junction can not be ruled out.This study was supported by the A. D. Williams Fund of Virginia Commonwealth University and the U. S. Veterans Administration. Radiation Counting Equipment was loaned by Dr. F. O'Foghludha, Department of Radiation Physics, Virginia Commonwealth University.     

Kallikrein-like activity in salivary glands using a new tripeptide substrate, including preliminary secretory studies and observations on mast cells

Summary The tripeptide substrated-val-leu-arg-4-methoxy-2-naphthylamine gave a precise localization of reaction product in cryostat sections of aldehyde-fixed salivary glands from a number of species, with Fast Blue B as the capture reagent. In submandibular glands, there was strong staining of the granules in granular tubules of rats and hamsters and somewhat less in mice. Submandibular striated ducts showed variable periluminal staining in a finer granular form; it was abundant in guinea-pigs, strong in cats but somewhat less pronounced in dogs. Parotid glands contained less reactivity with none detectable in hamsters and guinea-pigs. In the rabbit, neither gland showed any reaction. Mast cells were densely stained in glands from cats and dogs; they were less reactive in rats and unstained in the other species.The closely related 7-amino-4-trifluoromethylcoumarin derivative of the tripeptide has been found highly satisfactory for assessing activity in submandibular saliva from cats. Preliminary functional studies indicate that an extensive rapid secretion of enzyme occurs into saliva on sympathetic stimulation, with a corresponding depletion of reactive material from the striated ducts in tissue sections. Far less mobilization of enzyme occurs into saliva on parasympathetic stimulation with no obvious change in the histochemical reaction of striated ducts. The possible significance of these findings in cats is discussed.Extensive qualitative and quantitative studies are required to evaluate enzyme and substrate specificities in each species. Nevertheless, derivatives ofd-val-leu-arg offer great promise for the functional testing of kallikrein-like reactivity both by histochemical means on cells and biochemically in their secretions.     

Glycoconjugate with terminal α galactose

Summary Glycoconjugates associated with the basal cell layer of various types of epithelia in the mouse and rat were examined histochemically with a battery of lectinhorseradish peroxidase (HRP) conjugates of differing sugar binding specificities. Basal cells in paraffin sections of composite tissue blocks stained with an isolectin from Griffonia simplicifolia (GSA I-B₄) specific for terminal -galactose residues but failed to react with the other lectins. Basal cells in epithelium lining striated and excretory ducts of salivary and lacrimal glands, tongue, esophagus, trachea, renal calyx, ureter, urinary bladder, urethra, epididymis and vas deferens stained selectively and intensely for content of a glycoconjugate with terminal -galactose. This galactoconjugate appeared associated with the plasmalemma of basal cells. Basal cells with a galactocalyx formed an intermittent to continuous layer generally increasing in prevalence distally in glandular duct systems. A minor population of pyramido-columnar cells with cytosolic GSA I-B₄ reactivity occurred in striated ducts and appeared less numerous in intralobular excretory ducts and more prevalent in extraglandular ducts. In trachea and renal pelvis, the GSA I-B₄ positive cell profiles ranged from low cuboidal to tall pyramidal in contour, but the latter appeared not to reach the lumen. In contrast, no GSA I-B₄ positive basal cells were seen in any segment of the pancreatic or bile ducts or in the epithelium of the gastrointestinal tract. These findings suggest that the basal cells found in similar sites in different epithelia and possessing in common a unique -galactoconjugate may function in a manner common to all and not simply in providing progenitor cells for epithelial renewal. The location and distribution of GSA I-B₄ reactive basal cells in diverse epithelia suggests that through their -galactocalyx they serve in maintaining the established composition of luminal fluid perhaps by impeding the transepithelial movement of fluid and ions.In honour of Prof. P. van DuijnThis research was supported by National Institutes of Health Grants HL-29775 and AM-10956     

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.     

Distribution of S-100 protein and its subunits in bovine exocrine glands

 The distribution of S-100 protein and its α- and β-subunits in bovine exocrine glands was studied by indirect immunohistochemistry. The entire spectrum of salivary glands, glands of the respiratory tract, intestinal glands, male and female genital glands, and skin glands was examined. S-100 and its β-subunit were identified in most serous secretory cells of mixed salivary glands, although secretory acini in some serous glands remained unreactive for these antigens. Mucous cells were constantly negative; mucoid cells were positive in the lacrimal and Harderian gland. The α-subunit of S-100 protein was identified in serous cells but the staining reaction was faint. Subunits of S-100 showed a characteristic distribution along the excretory duct systems of compound glands: S-100 and the β-subunit were present in intercalated duct epithelium, while striated duct epithelium stained for S100-α. Therefore, it is suggested that S100-α is related to resorption and secretion in striated ducts, while S100-β may govern acinar exocytosis and probably regulates proliferation and differentiation of glandular cells. Differing staining intensities for S-100 and its subunits in secretory cells of exocrine glands most probably indicate functional differences with regard to secretory activity and the cell cycle. Accepted: 11 February 1997