Autophosphorylation of the pea mitochondrial heat-shock protein homolog

Highly purified mitochondria isolated from 14-day-old pea (Pisum sativum L., cv Little Marvel) seedlings contain a homolog of the 70,000 molecular weight heat-shock protein. The amount of this heat-shock cognate (Hsc70) was not reduced by limited proteolysis of intact mitochondria or by preparation of mitoplasts, indicating that the protein is located within the matrix compartment. Pea mitochondrial Hsc70 binds to immobilized ATP and reacts on western blots with anti-tomato Hsc70 antiserum. When a mitochondrial matrix fraction was incubated with [γ-³²P]ATP, there was phosphorylation of Hsc70. The extent of phosphorylation was increased by including calcium chloride in the reactions. Phospho amino acid analysis of purified mitochondrial Hsc70, phosphorylated in the calcium-stimulated reaction, revealed only phosphothreonine. Pea mitochondrial Hsc70, purified by a combination of ATP-agarose affinity chromatography and gel permeation chromatography, was labeled when incubated with ATP plus calcium, suggesting autophosphorylation rather than phosphorylation by an associated kinase. In analogy to mammalian cells and yeast, it is likely that mitochondrial Hsc70 acts as a molecular chaperone, and it is possible that phosphorylation plays a role in chaperone function.     

Immunocytochemical studies on the distribution pattern of daunomycin in rat gastrointestinal tract

The cancer drug daunomycin is used in treatment of leukemia but possesses severe side effects that involve the gastrointestinal tract. We therefore used a newly developed immunocytochemical procedure to determine the distribution of DM in the gastrointestinal tracts of rats after i.v. injection. Two hours after injection, DM was diffusely distributed in nuclei and most parts of the cytoplasm of intestinal epithelial cells. The cytoplasmic immunoreactivity for DM was most pronounced in small granules of the apical cytoplasm. Sixteen hours after injection, DM immunostaining was by and large absent in the villous epithelium but persisted in the intestinal crypts. In addition, staining was also detected in endothelial cells, scattered cells of the lamina propria and in smooth muscle cells. After 5 days, only little staining for DM remained. Similar findings were made in the colon. In the gastric mucosa, DM accumulation persisted at 16 h in some glandular cells but was lost from the surface epithelium. No staining was detected in saline-injected control rats. The distribution of DM accumulation correlated partially with the distribution of apoptotic cells as detected by the TUNEL procedure. Our results pinpoint that DM may exert prolonged effects on glandular and regenerative cells of the gastrointestinal tract—an observation that may explain the gastrointestinal toxicity of the drug. It seems possible that DM accumulation in surface epithelial cells is rapidly cleared through drug transporters.     

Cellular distribution of prostanoid EP receptors mRNA in the rat gastrointestinal tract

The inhibition of PGE(2) synthesis resulting from sustained NSAIDs therapy has been linked to gastrointestinal irritations and ulceration. The multiple physiological effects of PGE(2) in the gut are mediated through the activation of four receptors termed EP(1-4). The aim of the study was to determine the precise distribution of the four prostaglandin E(2) receptors in the rat stomach, small intestine, and colon. We used non-radioactive in situ hybridization techniques on paraffin-embedded tissue. Mucous cells of the stomach and goblet cells of the small intestine and colon were found to express mRNA for all four EP subtypes. A positive hybridization signal for EP(1), EP(3), and EP(4) was detected in the parietal cells of the stomach whereas the chief cells expressed low levels of EP(1) and EP(3). The EP(1) and EP(3) receptor mRNA could also be detected in the muscularis mucosa, longitudinal muscle and enteric ganglias of the stomach and small intestine. However, close examination of the enteric ganglias indicated that most of the positive labeling was localized to the glial cells, although some neurons did express EP(3). In conclusion, we have detailed the distribution of prostanoid EP receptors in the gut at the cellular level, giving new insights to the role of prostaglandins in gastrointestinal functions.     

Cell-specific expression of mitochondrial carbonic anhydrase in the human and rat gastrointestinal tract.

Mitochondrial carbonic anhydrase V (CA V) in liver provides HCO3- to pyruvate carboxylase for the first step in gluconeogenesis and HCO3- to carbamyl phosphate synthetase I for the first step in ureagenesis. Because carbamyl phosphate synthetase I and ornithine transcarbamylase are also expressed in enterocytes, we tested the hypothesis that CA V is expressed in the gastrointestinal tract in addition to liver. Polyclonal rabbit antisera were raised against a polypeptide of 17 C-terminal amino acids of human CA V and against purified recombinant mouse isozyme and were used in Western blotting and immunoperoxidase staining of human and rat tissues. Immunohistochemistry showed that CA V is expressed cell-specifically in the alimentary canal mucosa from stomach to rectum. Immunoreactions for CA V were detected in the parietal cells and gastrin-producing G-cells of the stomach and in intestinal enterocytes. Western blotting of human and rat gastrointestinal tissues with isozyme-specific antibodies showed positive signals for CA V with the expected molecular mass. The findings in human tissues paralleled those in rat. The cell-specific pattern of CA V expression suggests a role for CA V in alimentary canal physiology. We propose that mitochondrial CA V participates in the detoxification of ammonia produced in the gastrointestinal tract by providing bicarbonate to carbamyl phosphate synthetase I. (J Histochem Cytochem 47:517-524, 1999)     

Characterization and distribution of bombesin-like peptides in the rat brain and gastrointestinal tract

Extracts of rat brain and gastrointestinal tract, analyzed by reverse-phase high-performance liquid chromatography and radioimmunoassay, contained two bombesin-like immunoreactivity peaks with similar retention times as porcine gastrin-releasing peptide (GRP) and its COOH-terminal decapeptide, neuromedin C or GRP(18-27). However, the GRP-like peptide peak did not elute with exactly the same retention time as porcine GRP. The highest concentration of bombesin-like immunoreactivity was found in extracts of antrum, whereas the lowest was found in whole brain. Neuromedin C was present at lower concentrations than the GRP in antrum, duodenum, and ileum, while similar amounts of each were found in brain.     

L-Carnitine dissimilation in the gastrointestinal tract of the rat

Results of previous studies in this laboratory and others have suggested that L-carnitine is degraded in the gastrointestinal tract of the rat, perhaps by the action of indigenous flora. L-[methyl-14C]Carnitine was administered to rats either orally or intravenously in doses of 86 nmol or 124 mumol, and expired air, 48-h urine and fecal collections, and selected tissues at 48 h after isotope administration were examined for radiolabeled carnitine and metabolites. Urine and feces of rats receiving oral L-[methyl-14C]carnitine consistently contained two radiolabeled metabolites which were identified as trimethylamine N-oxide (primarily in urine) and gamma-butyrobetaine (primarily in feces). In these rats, these metabolites accounted for up to 23% and 31% of the administered dose, respectively. By contrast, for rats receiving intravenous L-[methyl-14C]carnitine or germ-free rats receiving the isotope orally or intravenously, virtually all of the radioactivity recovered was in the form of carnitine. Analyses for 14CO2 and [14C]trimethylamine in expired air revealed little or no (less than 0.1% of dose) conversion to these compounds, regardless of size of dose or route of administration. Results of this study demonstrate conclusively that L-carnitine is degraded in the gastrointestinal tract of the rat and that indigenous flora are responsible for these transformations.     

Kallikrein-gene expression in the rat gastrointestinal tract.

The serine proteinase glandular kallikrein has been demonstrated in the gastrointestinal tract, although there is some doubt as to whether it is synthesized there or derives from exocrine-gland secretions. Using a rat pancreatic kallikrein cRNA probe we have demonstrated kallikrein-like gene expression in the corpus, duodenum, jejunum, ileum, caecum and colon, and compared the pattern of expression with that of the gastrointestinal peptides somatostatin, gastrin and glucagon. In addition, using a panel of oligonucleotide probes specific for various members of the rat kallikrein-gene family, we have shown that the kallikrein-like gene expressed appears to be expressed as true kallikrein.     

Spirochetes autochthonous to the rat gastrointestinal tract

Spirochetes are structurally unique microorganisms found in the gastrointestinal tracts of most mammals. In an attempt to determine the ecological status of these bacteria, enumeration and distribution of morphologically distinct spirochetes were studied in the tracts of conventional laboratory rats. Five different types were seen to colonize infant rats between 19 and 26 days of age and subsequently to form stable communities in all 15 adults examined. Two types were found predominantly in lumen contents of the large bowel. The other three were consistently seen in the mucous blanket, attached to enterocyte surfaces or deep in the glands of the cecum and proximal colon. One type inhabiting the mucosal environment was also seen to pass into and through epithelial cells with no detectable host response. We conclude that spirochetes fulfill all the criteria for autochthonicity to the rat gastrointestinal tract.     

Synaptophysin-containing microvesicles transport heat-shock protein hsp60 in insulin-secreting beta cells

58–62 kDa heat-shock proteins (hsp60) are molecular chaperonins involved in the process of protein folding, transmembrane translocation and assembly of oligomeric protein complexes. In eukaryotic cells hsp60 proteins have been found in mitochondria and chloroplasts. However, we have recently documented that, in addition to mitochondria, a hsp60-like protein is present in secretory granules of insulin-secreting beta cells. The pathway by which hsp60 is targeted to secretory granules was unknown. Here we report the existence of microvesicles involved in the transport of hsp60 protein. Immunoelectron microscopy of serial thin-sections of beta cells directly visualized stages associated with hsp60 delivery: attachment of microvesicles to a secretory granule, fusion with the secretory granule membrane and release of hsp60 molecules. Further biochemical and immunological analysis of microvesicles revealed the presence in their membrane of synaptophysin, a major component of synaptic-like microvesicles (SLMV) of neuroendocrine cells. Double immunogold labelling with antibodies to synaptophysin and hsp60 demonstrated co-localization of both proteins in the same microvesicles. Moreover, fusion of synaptophysin-positive microvesicles leaves synaptophysin incorporated, at least transiently, to secretory granule membranes. These findings suggest that, in beta cells, synaptic-like vesicles are involved in the transport and delivery of hsp60 and represent a novel pathway for protein transport and secretion.     

The effects of alpha-lactabumin and whey protein concentrate on dry matter recovery, TCA soluble protein levels, and peptide distribution in the rat gastrointestinal tract

The effects of two dietary proteins on dry matter recovery, trichloroacetic acid (TCA) soluble protein concentration, and peptide distribution in gastrointestinal contents were investigated in rats trained to consume, in a single 2-hour daily meal, diets containing alpha-lactalbumin (alpha-LA) or whey protein concentrate (WPC) for two weeks. Compared with the WPC diet, the alpha-LA diet emptied faster from the stomach. Dry matter recovery was higher in the stomach contents of rats fed the WPC diet than in those given the alpha-LA diet, but dry matter content in the small intestine was comparable. TCA soluble protein levels in the stomach and the small intestinal contents were also significantly (P < 0.001) higher in rats fed the WPC diet. The concentration of peptides having molecular weights (MW) ranging from 12,500-30,000 daltons (Da) was higher in the stomach contents of rats fed the WPC diet. Conversely, the level of peptides ranging from 5000-12,500 Da was higher in the stomach contents of rats fed the alpha-LA diet. For both diets, the small intestinal contents were characterized by high levels of amino acids and small peptides. These results suggest that the hydrolysis and absorption of alpha-LA is faster than that of WPC.     

Transmural gradient of leukocyte-endothelial interaction in the rat gastrointestinal tract

Gastrointestinal injury usually starts in the superficial mucosa. We investigated whether leukocyte-endothelial interactions were greater in the gastrointestinal mucosa than the submucosa and muscularis in control tissue and after upregulation of adhesion molecules with endotoxin and after chemical insult with nonsteroidal anti-inflammatory drugs. Inactin-anesthetized rats were given either endotoxin, flurbiprofen, or nitric oxide (NO)-flurbiprofen, after which ICAM-1 and P-selectin expression was measured with the dual-label antibody technique. Leukocyte-endothelial interactions in the different gastric layers were assessed after endotoxin using intravital microscopy. Endotoxin caused a two- to threefold increase in ICAM-1 expression in the stomach and duodenum. There was, however, a gradient in expression across the gut wall with the level of expression in the superficial mucosa (per g) being only 10-25% of that in the deeper layers in both control and endotoxin-treated animals. Constituitive expression of P-selectin in control animals was barely detectable. Endotoxin caused a modest increase in mucosal P-selectin but a very significant increase in the deeper layers. Flurbiprofen caused a slight upregulation of ICAM-1 in the gastric mucosa and duodenum, whereas NO-flurbiprofen had no affect on expression. Intravital microscopy revealed no adhesion and virtually no leukocyte rolling in the vessels of the gastric mucosa despite endotoxin treatment. There was, however, some adhesion and significant leukocyte rolling in the submucosa and muscularis. Thus the superficial gastric and duodenal mucosal microcirculations have a much lower density of ICAM-1 and P-selectin and less leukocyte-endothelial interactions than occurs in the deeper layers of the gut wall even during stimulated upregulation with endotoxin.     

Distribution of bombesin binding sites in the rat gastrointestinal tract

In an attempt to identify potential target sites for the satiety action of bombesin (BN), the distribution and pharmacological specificity of bombesin binding sites were examined in the rat gastrointestinal tract by in vitro autoradiography utilizing (125I-Tyr4) bombesin. Specific BN binding was localized to the circular muscle level of the gastric fundus and antrum, submucosal layer of the small intestine and longitudinal and circular muscle and submucosal layers of the colon. Pharmacological studies indicated that gastrin releasing peptide (GRP), Ac-GRP20-27 and BN-like compounds, litorin and ranatensin, inhibited the binding of (125I-Tyr4)BN with high affinity while compounds which lacked COOH-terminal homology with BN demonstrated a low affinity for BN binding sites. The wide distribution of BN binding sites in the gastrointestinal tract provides a number of potential sites for the mediation of the satiety action of BN.     

Immunohistochemical demonstration of GABAB receptors in the rat gastrointestinal tract

Immunohistochemical localization of GABA_B-receptors was demonstrated in the rat gastrointestinal tract using a monoclonal antibody (GB-1) raised against the purified GABA_B-receptor. Immunoreactive staining for GABA_B-receptors was found in some populations of endocrine, muscular and neuronal components in the stomach and gut wall. Positive mucosal epithelial, probably endocrine, cells were distributed throughout the stomach and intestine. Double immunostaining indicated that such positive cells for GABA_B-receptors often co-possessed serotonin in the small intestine but not in the gastric body. In the muscular layer of the digestive canal, positive staining was seen as dotty granules punctuated on the surface of muscle fibers. In the enteric nervous system, positive neuronal somata were found in both submucosal and myenteric ganglia throught the entire canal extending from the stomach to the rectum. This is the first report to visualize the cellular localization of GABA_B-receptors in the gastrointestinal system of the rat, and should provide a fundamental basis for future studies on gastrointestinal functions regulated by GABA_B-receptors. Special issue dedicated to Dr. Kinya Kuriyama.     

Distribution of prostaglandin E receptors in the rat gastrointestinal tract

: In order to study the role of prostaglandin in the regulation of the gastrointestinal functions, gene expression of prostaglandin receptors along the rat gastrointestinal tracts were investigated.

: Rats were used for the study. The combination of counterflow elutriation separation of mucosal cells and Northern blot analysis was used to detect the gene expression of prostaglandin receptors in gastrointestinal tracts.

: In small intestine and colon, prostaglandin E2 EP1 and EP3 receptor mRNAs were mainly localized in the deeper intestinal wall containing muscle layers. EP4 receptor gene expression, on the other hand, was detected in the intestinal mucosal layer.

In the stomach, EP1 mRNA was detected in gastric muscle layers, whereas EP3 and EN receptor gene expression was mainly present in the gastric mucosal layer containing epithelial cells. In gastric epithelial cells, parietal cells were found to have both EP3 and EP4 receptors. At lower concentrations, prostaglandin E2 inhibited gastric acid secretion by parietal cells probably through EP4 receptors. At higher concentrations, however, it stimulated it. On the other hand, mucous cells possessed only EP4 receptor mRNA.

: Thus, it is suggested that prostaglandin E2 modulates gastrointestinal functions through at least three different prostaglandin receptors (EP1, EP3, and EP4), each of which has a distinct distribution in the gastrointestinal tract.     

Influence of folate-binding protein from bovine milk on the absorption of folate in gastrointestinal tract of rat

The absorption of [¹⁴C]pteroylglutamic acid (PteGlu) bound to the folate-binding protein of bovine milk was investigated in rat gastrointestinal tract in vivo and in situ. When bound [¹⁴C]PteGlu was given to rat intragastrically via oral intubation, a considerable amount of PteGlu was released from folate-binding protein under the acidic conditions in stomach, and it recombined with folate-binding protein in jejunum in vivo. Compared with free PteGlu, bound PteGlu was more gradually absorved in small intestine, but finally the total amount of bound PteGlu absorved was almost the same as that of free PteGlu. In all experiments in situ, bound PteGlu was only slightly absorbed in jejunum, where free PteGlu was rapidly absorved under the same conditions. On the other hand, the absorption rates of the two forms of PteGlu were almost similar to each other in ileum. These results suggest that PteGlu bound to folate-binding protein is absorbed by a manner different from that of free PteGlu in rat gastrointestinal tract.