Distribution and characterization of diazepam binding inhibitor (DBI) in peripheral tissues of rat

We studied the expression and distribution of the polypeptide diazepam binding inhibitor (DBI) in rat peripheral organs by immunocytochemistry, radioimmunoassay, Northern blot analysis and binding assay. Variable amounts of the DBI peptide and DBI mRNA were found in all the tissues examined (liver, duodenum, testis, kidney, adrenal gland, heart, ovary, lung, skeletal muscle and spleen), with the highest level of expression in liver (220 pmol of DBI/mg protein) and the lowest in spleen (11 pmol of DBI/mg protein). A good correlation between DBI-like immunoreactivity (DBI-LI) and mRNA content was found in all tissues except the heart. The immunohistochemical analysis revealed discrete localization of DBI-LI in cell types with specialized functions: for example, the highest DBI-LI content was found in steroid-producing cells (glomerulosa and fasciculata cells of adrenal cortex, Leydig cells of testis); lower DBI-LI immunostaining was found in epithelial cells specialized for water and electrolyte transport (intestinal mucosa, distal convoluted tubules of kidney). Hepatic cells contained moderate immunoreactivity however the total content of DBI in liver is relatively high and is due to the diffuse presence of DBI in every hepatocyte. Cells with high expression of DBI have been shown to contain a high density of mitochondrial benzodiazepine (BZ) binding sites. This observation led us to perform a competitive binding assay between DBI and [3H]PK11195 (a ligand for the mitochondrial BZ binding sites) on mitochondrial membranes of adrenal cortical cells. In this experiment, DBI yielded an apparent competitive inhibition of the binding of PK11195 to the BZ binding sites. Our data support a possible role for DBI as endogenous regulator of intracellular metabolic functions, such as steroidogenesis, via the mitochondrial BZ receptors.     

Tissue distribution of immunoreactive mouse extracellular superoxide dismutase

Protein content and mRNA expression ofextracellular superoxide dismutase (EC-SOD) were investigated in 16 mouse tissues. We developed a double-antibody sandwich ELISA using theaffinity-purified IgG against native mouse EC-SOD. EC-SOD could bedetected in all of the tissues examined (lung, kidney, testis, brownfat, liver, adrenal gland, pancreas, colon, white fat, thymus, stomach,spleen, heart, skeletal muscle, ileum, and brain, in decreasing order of content measured as µg/g wet tissue). Lung showed a markedly higher value of EC-SOD than other tissues. Interestingly, white fat hada high content of EC-SOD in terms of micrograms per milligram protein,which corresponded to that of lung. Kidney showed the strongestexpression of EC-SOD mRNA. Relatively strong expression of the mRNA wasobserved in lung, white fat, adrenal gland, brown fat, and testis.Heart and brain showed only weak signals, and no such expression couldbe detected in either digestive organs or skeletal muscle.Immunohistochemically, EC-SOD was localized mainly to connectivetissues and vascular walls in the tissues examined. Deep staining inthe cytosol was observed in the cortical tubular cells of kidney. Theseresults suggest that EC-SOD is distributed systemically inmice and that the physiological importance of this enzyme may be acompensatory adaptation to oxidative stress, particularly in lung andkidney.

     

Cell type-specific expression of beta-carotene 9',10'-monooxygenase in human tissues.

The symmetrically cleaving beta-carotene 15,15'-monooxygenase (BCO1) catalyzes the first step in the conversion of provitamin A carotenoids to vitamin A in the mucosa of the small intestine. This enzyme is also expressed in epithelia in a variety of extraintestinal tissues. The newly discovered beta-carotene 9',10'-monooxygenase (BCO2) catalyzes asymmetric cleavage of carotenoids. To gain some insight into the physiological role of BCO2, we determined the expression pattern of BCO2 mRNA and protein in human tissues. By immunohistochemical analysis it was revealed that BCO2 was detected in cell types that are known to express BCO1, such as epithelial cells in the mucosa of small intestine and stomach, parenchymal cells in liver, Leydig and Sertoli cells in testis, kidney tubules, adrenal gland, exocrine pancreas, and retinal pigment epithelium and ciliary body pigment epithelia in the eye. BCO2 was uniquely detected in cardiac and skeletal muscle cells, prostate and endometrial connective tissue, and endocrine pancreas. The finding that the BCO2 enzyme was expressed in some tissues and cell types that are not sensitive to vitamin A deficiency and where no BCO1 has been detected suggests that BCO2 may also be involved in biological processes other than vitamin A synthesis.     

Oxidative Myocytes of Heart and Skeletal Muscle Express Abundant Sarcomeric Mitochondrial Creatine Kinase

Sarcomeric mitochondrial creatine kinase catalyzes the reversible transfer of a high energy phosphate between ATP and creatine. To study cellular distribution of the kinase, we performed immunocytochemical studies using a peptide antiserum specific for the kinase protein. Our results demonstrated that the sarcomeric mitochondrial creatine kinase gene is abundantly expressed in heart and skeletal muscle, with no protein detected in other tissues examined, including brain, lung, liver, spleen, kidney, bladder, testis, stomach, intestine, and colon. RNA blot study showed that there is no detectable expression of the kinase mRNA in the thymus gland. In heart and skeletal muscle, the kinase protein is expressed in atrial and ventricular cardiomyocytes and a subpopulation of skeletal myofibres. In skeletal muscle, fast myosin heavy chain co-localization studies demonstrated that the sarcomeric mitochondrial creatine kinase is highly expressed in type 1, slow-oxidative and type 2A, fast-oxidative-glycolytic myofibres. We conclude that the kinase gene is abundantly expressed in oxidative myocytes of heart and skeletal muscle and may contribute to oxidative capacity of these cells.     

Specific protein binding to a conserved region of the ornithine decarboxylase mRNA 5'-untranslated region.

An RNA gel retardation assay was used to identify one or more cellular protein(s) (ornithine decarboxylase mRNA 5'-UTR binding protein (ODCBP)) that bind specifically to a conserved region of the 5'-untranslated region (5'-UTR) of rat ornithine decarboxylase (ODC) mRNA. Ultraviolet light cross-linking demonstrated that this protein has an apparent Mr = 58,000 in mammalian cells. Treatment with the oxidizing agent diamide prevented binding of the ODCBP to ODC mRNA; addition of beta-mercaptoethanol reversed this inhibition and permitted mRNA.ODCBP complex formation. Cytoplasmic extracts from a variety of animal cells and tissues demonstrated similar binding activities; however, there was marked tissue-specific expression of the protein in the rat, with brain, heart, lung, and testis containing large amounts, and kidney, spleen, and skeletal muscle expressing negligible amounts. Binding was completely prevented by several mutations within a highly conserved heptanucleotide region (CCAU/ACUC) that was within 61 bases of the initiation codon in ODC mRNAs from mammals, Xenopus, and Caenorhabditis elegans; mutations 5' and 3' of the conserved heptanucleotide domain had no effect on binding activity. Binding was not affected by manipulation of cellular polyamine levels or by treatment of cells with agents that stimulate ODC biosynthesis. Thus, we have identified a widely distributed cellular protein that binds to a conserved domain within the 5'-UTR of ODC mRNA from many animal species; functional consequences of this binding remain to be determined.     

Tissue-specific expression of a novel GTP-binding protein (smg p25A) mRNA and its increase by nerve growth factor and cyclic AMP in rat pheochromocytoma PC-12 cells

We have purified a novel GTP-binding protein, designated as the smg-25A protein (smg p25A), from bovine brain membranes and determined its primary structure. In the present studies, the smg-25A mRNA levels in various tissues have been studied. The 1.6-kilobase smg-25A mRNA is detected in rat brain by Northern blot analysis. This mRNA is not detected in other rat tissues including thymus, lung, heart, liver, small intestine, kidney, and skeletal muscle. The 1.6-kilobase smg-25A mRNA is also detected in bovine adrenal medulla but not in the cortex. Moreover, this mRNA is detected in rat pheochromocytoma PC-12 cells and its level increases after differentiation of the cells into sympathetic neuron-like cells in response to nerve growth factor or dibutyryl cyclic AMP. This mRNA level does not increase in response to 12-O-tetradecanoylphorbol-13-acetate incapable of inducing differentiation. These results suggest that the smg-25A gene is specifically expressed in nerve tissues and that smg p25A plays a role in some neuronal functions.     

Localization of mitochondrial 60-kD heat shock chaperonin protein (Hsp60) in pituitary growth hormone secretory granules and pancreatic zymogen granules.

We used quantitative immunogold electron microscopy and biochemical analysis to evaluate the subcellular distribution of Hsp60 in rat tissues. Western blot analysis, employing both monoclonal and polyclonal antibodies raised against mammalian Hsp60, shows that only a single 60-kD protein is reactive with the antibodies in brain, heart, kidney, liver, pancreas, pituitary, spleen, skeletal muscle, and adrenal gland. Immunogold labeling of tissues embedded in the acrylic resin LR Gold shows strong labeling of mitochondria in all tissues. However, in the anterior pitutary and in pancreatic acinar cells, Hsp60 also localizes in secretory granules. The labeled granules in the pituitary and pancreas were determined to be growth hormone granules and zymogen granules, respectively, using antibodies to growth hormone and carboxypeptidase A. Immunogold labeling of Hsp60 in all compartments was prevented by preadsorption of the antibodies with recombinant Hsp60. Biochemically purified zymogen granules free of mitochondrial contamination are shown by Western blot analysis to contain Hsp60, confirming the morphological localization results in pancreatic acinar cells. In kidney distal tubule cells, low Hsp60 reactivity is associated with infoldings of the basal plasma membrane. In comparison, the plasma membrane in kidney proximal tubule cells and in other tissues examined showed only background labeling. These findings raise interesting questions concerning translocation mechanisms and the cellular roles of Hsp60.     

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.     

Quantitation of apolipoprotein E mRNA in the liver and peripheral tissues of nonhuman primates

A sensitive DNA-excess solution hybridization assay was used to quantitate apo-E mRNA in the liver and peripheral tissues of two nonhuman primates, Macaca fascicularis and Cercopithecus aethiops. When expressed on the basis of total RNA, apo-E mRNA values for M. fascicularis adrenal, brain, testis, and spleen ranged from 17-52% of the liver value. Apo-E mRNA values for mesenteric lymph node, kidney, thymus, and skeletal muscle were 1-5% of the liver value. When expressed on a cellular basis, apo-E mRNA was most abundant in the liver at approximately 1200 molecules/cell. Peripheral tissues showed a continuous range of apo-E mRNA from 1.5 molecules/cell in the thymus up to 350 molecules/cell in the brain. Similar results were obtained with peripheral tissues from C. aethiops in which case apo-E mRNA also was found in skin, lung, skeletal muscle, small intestine, and vascular tissues such as heart, aorta, and brachial artery. Calculation of the total apo-E mRNA/organ showed that most of the apo-E mRNA was present in the liver. However, summation of apo-E mRNA in peripheral tissues indicated that 20-40% of total body apo-E mRNA was extrahepatic. This results indicates that apo-E made in peripheral tissues may play a quantitatively important role in cholesterol metabolism since peripheral tissues have the potential to contribute a significant fraction of plasma apo-E.     

Comparative localization of aldehyde oxidase and xanthine oxidoreductase activity in rat tissues

The distribution of aldehyde oxidase activity was evaluated in unfixed cryostat sections from tissues of male Wistar rats using a tissue protectant, polyvinyl alcohol, with Tetranitro BT as a final electron acceptor. The distribution of aldehyde oxidase activity was compared with that of xanthine oxidoreductase. The enzyme histochemical method demonstrated aldehyde oxidase activity in the epithelium of the tongue, renal tubules and bronchioles, as well as in the cytoplasm of liver cells. Such activity was not detected in oesophagus, stomach, spleen, adrenal glands, small or large intestine or skeletal and heart muscle fibres. In contrast, xanthine oxidoreductase activity was demonstrated in the tongue, renal tubules, bronchioles, oesophageal, gastric, small and large intestinal epithelial cells, adrenal glands, spleen and liver cytoplasm but not in skeletal and heart muscle fibres. The significance of the ubiquitous distribution of aldehyde oxidase activity, especially in surface epithelial cells from various tissues, except for the gastrointestinal tract, is unclear. However, aldehyde oxidase may possess some physiological activity other than in the metabolism of N-heterocyclics or of certain drugs. © 1998 Chapman & Hall     

Quantitation of muscle glycogen phosphorylase mRNA and enzyme amounts in adult rat tissues

Mammalian glycogen phosphorylases comprise a family of isozymes that are expressed selectively in a variety of cell types. As an initial step towards understanding the molecular processes that regulate the differential expression of the phosphorylase family, we have begun a quantitative examination of isozyme expression in vivo. In this paper, we report quantitative estimates of the amounts of the muscle (M) isozyme and its mRNA in adult rat tissues. Quantitative estimates of the amount of M-phosphorylase were obtained by an analysis involving electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose filters and sequential treatment with M-isozyme specific antibody and radioactively- labeled protein A. M-phosphorylase mRNA amounts were determined by an analysis involving transfer of RNA from agarose gels to nitrocellulose filters and subsequent hybridization with radioactively labelled rat M-phosphorylase cDNA. These studies indicate that M-phosphorylase is present in all tissues tested with the possible exception of liver. These are skeletal muscle, heart, brain, stomach, lung, kidney, spleen and testis. Quantitation of M-phosphorylase amounts indicate that there is a wide spectrum of variation (over 1000-fold range) in the relative amounts of the M-isozymes in these tissues. Relative mRNA levels parallel isozyme levels indicating that the major control of expression of this isozyme is governed by mRNA accumulation.     

Expression of plasma prekallikrein mRNA in human nonhepatic tissues and cell lineages suggests special local functions of the enzyme.

At present it is generally accepted that plasma prekallikrein (PPK) is synthesized in the liver and secreted into the bloodstream. Surprisingly, it has recently been shown that PPK mRNA is present also in RNA from the kidney, adrenal gland and placenta. In spite of its novelty and possible important physiological implications this finding has been neglected. Here we report that PPK mRNA is expressed also in the human brain, heart, lung, trachea, endothelial cells and leukocytes as well as in a variety of fibroblast and epithelial cell lines. Expression of PPK mRNA in fibroblasts, endothelial cells and leukocytes suggests that PPK mRNA detected in RNA preparations from whole tissue may originate solely from these ubiquitously occurring cells. However, PPK mRNA expression in various epithelial cell lines demonstrates that tissue-specific cells also transcribe the PPK gene. The presence of PPK mRNA in nonhepatic tissues and cells indicates that they have the capacity to synthesize the protein. The physiological role of PPK synthesized in extrahepatic tissue is unknown. It may participate in local actions within tissues as well as contributing to the PPK pool in blood plasma. Cultured cells will provide a valuable model for exploring the physiological significance of extrahepatic PPK expression.     

Heat shock protein induction in the freshwater prawn Macrobrachium malcolmsonii: acclimation-influenced variations in the induction temperatures for Hsp70

The intracellular build-up of thermally damaged proteins following exposure to heat stress results in the synthesis of heat shock proteins (Hsps). In the present study, the upper thermal tolerance and expression of heat shock protein 70 (Hsp70) were examined in juveniles of the freshwater prawn Macrobrachium malcolmsonii that had been acclimated at two different temperatures, i.e. 20 degrees C (group A) and 30 degrees C (group B), in the laboratory for 30 days. Upper thermal tolerance was determined by a standard method. For heat-shock experiments, prawns in groups A and B were exposed to various elevated temperatures for 3 h each, followed by 1 h recovery at the acclimation temperature. Endogenous levels of Hsp70 were determined in the gill, heart, hepatopancreas and skeletal muscle tissues by Western blotting analysis of one dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The critical thermal maximum (CT max) for prawns in groups A and B was 37.7+/-0.27 degrees C and 41.41+/-0.16 degrees C, respectively. In general, Western blotting analysis for Hsp70 revealed one band at the 70 kDa region, containing both constitutive (Hsc70) and inducible (Hsp70) isoforms, in the gill and heart tissues; these were not detected in the hepatopancreas and skeletal muscle tissues. The onset temperature for Hsp70 induction in both gill and heart tissues was 30 degrees C for prawns in group A and 34 degrees C for those in group B. The optimum induction temperatures (at which Hsp70 induction was maximum) were found to be 34 degrees C and 32 degrees C, respectively, in the gill and heart tissues of group A prawns, and 38 degrees C and 36 degrees C, respectively, for group B prawns. These results suggest that the temperature at which acclimation occurs influences both upper thermal tolerance and Hsp70 induction in M. malcolmsonii.     

Expression of PDE11A in normal and malignant human tissues.

Cyclic nucleotide phosphodiesterase 11A (PDE11A) is the newest member in the PDE family. Although the tissue distribution of PDE11A mRNA has been shown, its protein expression pattern has not been well studied. The goal of this report is to investigate the distribution of PDE11A proteins in a wide range of normal and malignant human tissues. We utilized a polyclonal antibody that recognized all four PDE11A isoforms. Its specificity was demonstrated by Western blot analysis on a recombinant human PDE11A protein and native PDE11A proteins in various human tissues. Immunohistochemistry showed that PDE11A is widely expressed. Various degrees of immunoreactivity were observed in the epithelial cells, endothelial cells, and smooth muscle cells of all tissues examined. The highest expression was in the epithelial, endothelial, and smooth muscle cells of the prostate, Leydig, and spermatogenic cells of the testis, the tubule epithelial cells in the kidney, the epithelial and endothelial cells in the adrenal, the epithelial cells and macrophages in the colon, and the epidermis in the skin. Furthermore, PDE11A expression was also detected in several human carcinomas. Our results suggest that PDE11A might be involved in multiple physiological processes in various organs via its ability to modulate intracellular cAMP and cGMP levels.     

Tissue-Specific Induction of Hsp90 mRNA and Plasma Cortisol Response in Chinook Salmon following Heat Shock, Seawater Challenge, and Handling Challenge

In studying the whole-body response of chinook salmon (Oncorhynchus tshawytscha) to various stressors, we found that 5-hour exposure to elevated temperature (mean 21.6°C; + 10.6°C over ambient) induced a marked increase in Hsp90 messenger RNA accumulation in heart, brain, gill, muscle, liver, kidney, and tail fin tissues. The most vital tissues (heart, brain, gill, and muscle) showed the greatest Hsp90-mRNA response, with heart tissue increasing approximately 35-fold. Heat shock induced no increase in plasma cortisol. In contrast, a standard handling challenge induced high plasma cortisol levels, but no elevation in Hsp90 mRNA in any tissue, clearly separating the physiological and cellular stress responses. We saw no increase either in tissue Hsp90 mRNA levels or in plasma cortisol concentrations after exposing the fish to seawater overnight. Received October 1, 1999; accepted January 21, 2000     

Expression studies of neogenin and its ligand hemojuvelin in mouse tissues.

Juvenile hemochromatosis is a severe hereditary iron overload disease caused by mutations in the HJV (hemojuvelin) and HAMP (hepcidin) genes. Hepcidin is an important iron regulatory hormone, and hemojuvelin may regulate hepcidin synthesis via the multifunctional membrane receptor neogenin. We explored the expression of murine hemojuvelin and neogenin mRNAs and protein. Real-time RT-PCR analysis of 18 tissues from male and female mice was performed to examine the mRNA expression profiles. To further study protein expression and localization we used immunohistochemistry on several tissues from three mouse strains. Mouse Neo1 mRNA was detectable in the 18 tissues tested, the highest signals being evident in the ovary, uterus, and testis. Neogenin protein was observed in the brain, skeletal muscle, heart, liver, stomach, duodenum, ileum, colon, renal cortex, lung, testis, ovary, oviduct, and uterus. The spleen, thymus, and pancreas were negative for neogenin. The highest signals for Hjv mRNA were detectable in the skeletal muscle, heart, esophagus, and liver. The results indicate that Neo1 mRNA is widely expressed in both male and female mouse tissues with the highest signals detected in the reproductive system. Moreover, Hjv and Neo1 mRNAs are simultaneously expressed in skeletal muscle, heart, esophagus, and liver.     

Apolipoprotein gene expression in the rabbit: abundance, size, and distribution of apolipoprotein mRNA species in different tissues

We have used human apolipoprotein cDNAs as hybridization probes to study the relative abundance and distribution of apolipoprotein mRNAs in rabbit tissues by RNA blotting analysis. The tissues surveyed included liver, intestine, lung, pancreas, spleen, stomach, skeletal muscle, testis, heart, kidney, adrenal, aorta, and brain. We found that liver is the sole or major site of synthesis of apoA-II, apoA-IV, apoB, apoC-I, apoC-II, apoC-III, and apoE, and the intestine is a major site of synthesis of apoA-I, apoA-IV, and apoB. Minor sites of apolipoprotein mRNA synthesis were as follows: apoA-I, liver and skeletal muscle; apoA-IV, spleen and lung; apoB, kidney; apoC-II and apoC-III, intestine. ApoE mRNA was detected in all tissues surveyed with the exception of skeletal muscle. Sites with moderate apoE mRNA (10% of the liver value) were lung, brain, spleen, stomach, and testis. All rabbit mRNAs had forms with sizes comparable to their human counterparts. In addition, hybridization of hepatic and intestinal RNA with human apoA-IV and apoB probes produced a second hybridization band of approximately 2.4 and 8 kb, respectively. Similarly, hybridization of rabbit intestinal RNA with human apoC-II produced a hybridization band of 1.8 kb. The 8 kb apoB mRNA form may correspond to the apoB-48 mRNA, whereas the apoA-IV- and apoC-II-related mRNA species have not been described previously. This study provides a comprehensive survey of the sites of apolipoprotein gene expression and shows numerous differences in both the abundance and the tissue distribution of several apolipoprotein mRNAs between rabbit and human tissues. These findings and the observation of potentially new apolipoprotein mRNA species are important for our understanding of the cis and trans acting factors that confer tissue specificity as well as factors that regulate the expression of apolipoprotein genes in different mammalian species.