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The secretory units and duct system of the echidna sublingual glands exhibit subtle architectural modifications to accommodate the viscous secretion produced by these glands. The glands are compound tubular glands, the secretory units of which are elongate with open lumina and consist only of mucous cells. Closely packed spindle-shaped myoepithelial cells invest the secretory units, but are absent around the ducts. The branched secretory tubules open into an abbreviated duct system characterized by wide lumina. Striated ducts normally associated with the second portion of the intralobular duct system are absent. The duct system shows the most obvious modification of general salivary gland architecture presumably to accommodate the viscous secretion propelled from the secretory units by surrounding myoepithelial cells., (© 2011 Blackwell Verlag GmbH.)

The secretory units of the platypus and echidna mandibular glands consist of a single serous cell type. Secretory granules within the cells of the platypus mandibular gland stained intensely with the periodic acid-Schiff staining procedure but failed to stain with Alcian Blue, suggesting the granules contained neutral glycoproteins. Secretory granules within the mandibular glands of the echidna failed to stain with the methods used indicating little if any glycoprotein was associated with the secretory granules. Ultrastructurally, secretory granules of the platypus mandibular gland were electron dense with a central core of less electron-dense material and were membrane bound. In contrast, those of the echidna presented a lamellated appearance and also were limited by a membrane. These secretory granules appeared to form as a result of concentric layering of lamellae within cisternae of the Golgi membranes. The intralobular ductal system of the platypus was more extensively developed than that of the echidna. The striated ducts of both species were characterized by elaborate infoldings of the basolateral plasmalemma and an abundance of associated mitochondria., (© 2011 Blackwell Verlag GmbH.)

Koala mandibular (submandibular) glands are compound tubuloacinar glands, the secretory units of which consist only of serous cells. Intercellular canaliculi occur between the serous cells, which are continuous with a minute lumen that courses through the centre of each secretory unit. Intercalated ducts are abundant and join striated ducts, the latter being characterized by elaborate basolateral infoldings of the plasmalemma. Secretory granules within the serous cells fail to stain with either the PAS or Alcian Blue (pH 2.5) staining procedures. Ultrastructurally, the secretory granules are membrane bound, and consist of a homogeneous electron lucent material with a fine filamentous texture. The granules tend to coalesce into irregular shaped complexes of secretory material. Discharge of secretory material into the canalicular lumen is a common observation., (© 2010 Blackwell Verlag GmbH.)

The echidna and platypus have a crural/femoral gland that is linked by a large duct to a canalized, keratinous spur located on the medial side of the ankle. The echidna crural gland, like the femoral gland of the platypus, exhibits cyclic activity, being prominent in both monotremes when they are sexually active. In the present study, we compared the structure and histochemistry of these glands. During the active phase, the secretory epithelium forming the respective glands of both species increased in height and became packed with secretory granules that differed markedly in structure. Secretory granules of the echidna crural gland were electron dense and characterized by cores or areas of increased electron density. Those of the platypus were initially electron dense, but then became less dense and coalesced into irregular complexes of secretory material. Large cytoplasmic blebs extended from epithelial cell apices and appeared to be shed into the lumen, resulting in an apocrine mode of secretion. Exocytosis was also observed. A similar form of release of secretory product was not observed in the echidna. Secretory granules of both species were periodic acid-Schiff positive and stained for protein, suggesting that much of the secretory product was glycoprotein. Myoepithelial cells enveloped the secretory tubules of the platypus femoral gland, whereas they were not observed surrounding tubules comprising the echidna crural gland. During the quiescent phase, the epithelial cells of both species lost their secretory granules and decreased in height. As a result, the secretory tubules became smaller, intralobular connective tissue increased and the glands decreased in overall size.

Peroxiredoxin 2 (PRDX2) is a highly efficient redox protein that neutralizes hydrogen peroxide, resulting in protection of cells from oxidative damage and in regulation of peroxide-mediated signal transduction events. The oxidized form of PRDX2 is reverted back to the reduced form by the thioredoxin system. In the present study, we investigated the presence of PRDX2 in mouse and boar spermatozoa and in mouse spermatids using proteomic techniques and immunocytochemistry. Sperm and spermatid extracts displayed a 20-kDa PRDX2 band on Western blotting. PRDX2 occurred as a Triton-soluble form in spermatids and as a Triton-insoluble form in mature spermatozoa. Boar seminiferous tubule extracts were immunoprecipitated with PRDX2 antibody and separated by SDS-PAGE. Peptide mass fingerprinting by matrix-assisted laser desorption ionization-time of flight (TOF) and microsequencing by nanospray quadrupole-quadrupole TOF tandem mass spectrometry revealed the presence of PRDX2 ions in the immunoprecipitated band, along with sperm mitochondria-associated cysteine-rich protein, cellular nucleic acid-binding protein, and glutathione peroxidase 4. In mouse spermatocytes and spermatids, diffuse labeling of PRDX2 was observed in the cytoplasm and residual bodies. After spermiation, PRDX2 localization became confined to the mitochondrial sheath of the sperm tail midpiece. Boar spermatozoa displayed similar PRDX2 localization as in mouse spermatozoa. Boar spermatozoa with disrupted acrosomes expressed PRDX2 in the postacrosomal sheath region. Peroxidase enzyme activity of boar sperm extracts was evaluated by estimating the rate of NADPH oxidation in the presence or absence of a glutathione depletor (diethyl maleate) or a glutathione reductase inhibitor (carmustine). Diethyl maleate partially inhibited peroxidase activity, whereas carmustine showed an insignificant effect. These observations suggest that glutathione and glutathione reductase activity contribute only partially to the total peroxidase activity of the sperm extract. While the specific role of PRDX2 in the total peroxidase activity of sperm extract is still an open question, the present study for the first time (to our knowledge) shows the presence of PRDX2 in mammalian spermatozoa. Peroxidase activity in sperm extracts is not due to the glutathione system and therefore possibly involves PRDX2 and other peroxiredoxins.

The stable metabolite of nitric oxide in plasma is NOx, the sum of nitrite plus nitrate. Measures of plasma NOx may provide information about the nitric oxide tonus of the entire endothelium including capillary microvessels. Although data are available for mammalian species, plasma NOx measurements in early vertebrate species are scarce. The purpose of this study was to test the hypothesis that plasma NOx would be similar to the NO in the water environment for fish in early classes (Agnatha and Chondrichthye) and would exceed water NOx levels in the known nitrite-sensitive fish (Osteichthye). Plasma samples were obtained from 18 species of adult fish (n=167) and from their housing or natural water environment. NOx was measured by using chemiluminescence. Plasma NO was detected in all species and ranged from 0.5 nmol/ml (skate) to 453.9 nmol/ml (shortnose gar). Average plasma NOx was significantly higher in sea lamprey than in Atlantic hagfish whereas that of little skate was 3-fold lower than in spiny dogfish shark. Plasma NO differed significantly among early bony fish (paddlefish, pallid sturgeon, gar) yet was similar among modern bony fish, with the exception of rainbow trout. Plasma NOx reflected water NO in only 2 species (hagfish and shark), and levels did not coincide with nitrite sensitivity. This study provides an expanded comparative view of plasma NO, levels across 3 groups of early fish. The data obtained suggest a nitric oxide system in early and modern fish.

Prostaglandins can protect the in vivo gastric mucosa against necrosis produced by a variety noxious agents. Cimetidine has also been shown to have protective properties in humans and in some models of experimental injury. Whether prostaglandins or cimetidine may protect gastric mucosal cells directly in the absence of systemic factors remains controversial. In the present study, the potential protective actions of prostaglandin and cimetidine against indomethacin injury were assessed in isolated rat gastric glands. Gastric glands were pre-incubated in oxygenated medium with either placebo, 16,16 dimethyl prostaglandin E(2) (dm PGE(2)) or cimetidine and incubated at 37 degrees C in medium containing 0.5 mg/ml of indomethacin for 2, 4 and 6 hrs. Cell injury and protection was assessed by the Fast Green exclusion test (viability test), leakage of lactate dehydrogenase (LDH) into the medium, and by scanning and transmission electron microscopy. In addition, the generation of PGE(2) by the gland cells was determined using RIA assay. Indomethacin by itself significantly reduced the viability of gastric glands, increased LDH release into the medium and produced prominent ultrastructural damage. In contrast to cimetidine, co-incubation of gastric glands with dm PGE(2) added to indomethacin, significantly reduced indomethacin-induced injury, increased the number of viable cells, reduced LDH leakage and diminished the extent of ultrastructural damage. The dose of indomethacin (5 microg/ml) which significantly inhibited the generation of PGE(2) (up to 90% inhibition) had no effect on cell viability nor LDH release. We conclude that 1) exogenous PGE2 exerts a potent protective activity in vitro which is independent on neural, vascular and hormonal factors; 2) inhibition of endogenous PGs may not the primary mechanism in the deleterious action of indomethacin against damage to gastric glandular cells and 3) indomethacin can exert a direct cytotoxic effect on the mucosal cells in gastric glands.

The present study has demonstrated the immunohistochemical localization of gastrin-releasing peptide (GRP), neuronal nitric oxide synthase (nNOS) and neurone-specific enolase (NSE) in the uterus of the North American opossum. Although the presence of GRP, nNOS and NSE has been reported recently in the uterus of eutherian species this is the first description of these peptides in a metatherian species. Metatherian mammals are of interest because in these species it is the prolonged lactation phase of development that is the period of primary reproductive investment rather than intrauterine development as is true of eutherian mammals. The opossum, like other marsupial species, has a very abbreviated gestation period which in Didelphis lasts only 12.5 days. GRP was localized in the cytoplasm of cells forming the surface lining epithelium and the glandular epithelium of the opossum endometrium late in pregnancy, at 11.5 days of gestation. Likewise, immunoreactivities of nNOS and NSE were found primarily within the epithelial cells of the endometrium at 11.5 days of gestation. As these peptides and enzymes appear primarily at the time of establishment of the yolk sac placenta (between day 10 and day 12.5 gestation), the present results strongly suggest that these factors may play a fundamental role in the placentation of the opossum.

Mucins of the gastroduodenal junction are secreted by the mucous surface and mucus-producing glandular cells in the stomach, and by goblet cells and Brunner's glands in the duodenum. Developmental studies have demonstrated that Brunner's glands can arise from undifferentiated gastric epithelium and/or intestinal epithelium in the proximal duodenum. The aim of this study was to investigate the carbohydrate composition of mucins from this region and compare it with that of mucins from Brunner's glands to evaluate the probable evolution of mucins from these glands. Toward that end, paraffin sections from 13 mammalian species were stained by classic carbohydrate histochemistry and treated with 13 lectins. In general, the mucous surface cells of the stomach, pyloric glands, duodenal goblet cells, and Brunner's glands secretory epithelium had different lectin-binding patterns. However, the lectin-binding profile of the secretory epithelium of Brunner's glands resembled that of pyloric glands more closely than that of duodenal goblet cells and mucous surface cells of the stomach. Mucins from Brunner's glands and pyloric glands showed a greater terminal carbohydrate residue diversity than those of gastric mucous surface cells or duodenal goblet cells. The lectin-binding profile argues for the evolution of similar mucins from the epithelia of Brunner's glands and pyloric glands. The greater diversity of carbohydrate residues in mucins secreted by Brunner's glands suggests that their mucus is more adaptable. This may explain why Brunner's glands metaplasia rather than goblet cell metaplasia is seen in the mucosa adjacent to chronic intestinal ulcers., (Copyright 2004 Wiley-Liss, Inc.)

Redox control of cell physiology is one of the most important regulatory mechanisms in all living organisms. The thioredoxin system, composed of thioredoxin and thioredoxin reductase, has emerged as a key player in cellular redox-mediated reactions. For many years, only one thioredoxin system had been described in higher organisms, ubiquitously expressed in the cytoplasm of eukaryotic cells. However, during the last decade, we and others have identified and characterized novel thioredoxin systems with unique properties, such as organelle-specific localization in mitochondria or endoplasmic reticulum, tissue-specific distribution mostly in the testis, and features novel for thioredoxins, such as microtubule-binding properties. In this review, we will focus on the mammalian testis-specific thioredoxin system that comprises three thioredoxins exclusively expressed in spermatids (named Sptrx-1, Sptrx-2, and Sptrx-3) and an additional thioredoxin highly expressed in testis, but also present in lung and other ciliated tissues (Txl-2). The implications of these findings in the context of male fertility and testicular cancer, as well as evolutionary aspects, will be discussed.

Experiments were designed to detect and determine differences between nitrite/nitrate concentration ([NOx]) in plasma across 15 species selected from seven classes of vertebrates. Blood collected in syringes was placed immediately into ethylenediaminetetraacetic acid (EDTA)-containing tubes and was centrifuged. Plasma [NOx] was determined by measurement of chemiluminescence. Across classes of vertebrates, baseline plasma [NOx] ranged from 0.6 to 171.3 nmol/ml. Mean +/- SD plasma [NOx] was highest in a fresh-water, jawless fish (lamprey, 95.5 +/- 9.1 nmol/ml) and lowest in a saltwater cartilaginous fish (skates, 1.1 +/- 0.4 nmol/ml). Both amphibians tested had a wide range in plasma [NOx], which was explained partly by temporal changes during the year. Within the mammalian class, plasma [NOx] ranged from 3.8 to 43.2 nmol/ml. Results of this study indicate that NO is detectable in plasma of all classes of vertebrates and that baseline concentration varies among species.

The guanylin family of cGMP-regulating peptides has three subclasses of peptides containing either three intramolecular disulfides found in bacterial heat-stable enterotoxins (ST), or two disulfides observed in guanylin and uroguanylin, or a single disulfide exemplified by lymphoguanylin. These small, heat-stable peptides bind to and activate cell-surface receptors that have intrinsic guanylate cyclase (GC) activity. Two receptor GC signaling molecules have been identified that are highly expressed in the intestine (GC-C) and/or the kidney (OK-GC) and are selectively activated by the guanylin peptides. Stimulation of cGMP production in renal target cells by guanylin peptides in vivo or ex vivo elicits a long-lived diuresis, natriuresis, and kaliuresis. Activation of GC-C receptors in target cells of intestinal mucosa markedly stimulates the transepithelial secretion of Cl(-) and HCO(-)/(3), causing enhanced secretion of fluid and electrolytes into the intestinal lumen. Bacterial ST peptides act as mimics of guanylin and uroguanylin in the intestine, which provide a cellular mechanism underlying the diarrhea caused by ST-secreting strains of Escherichia coli. Uroguanylin and guanylin may participate in a novel endocrine axis linking the digestive system and kidney as a physiological mechanism that influences Na(+) homeostasis. Guanylin, uroguanylin, and/or lymphoguanylin may also serve within intrarenal signaling pathways controlling cGMP production in renal target cells. Thus we propose that guanylin regulatory peptides participate in a complex multifactorial biological process that evolved to regulate the urinary excretion of NaCl when dietary salt levels exceed the body's physiological requirements. This highly integrated and redundant mechanism allows the organism to maintain sodium balance by eliminating excess NaCl in the urine. Uroguanylin, in particular, may be a prototypical "intestinal natriuretic hormone."

Endometrial periglandular fibrosis (EPF) has been proposed as a possible aetiology for equine embryonic and fetal loss. However, the pathophysiology of EPF is not well understood. Angiotensin-converting enzyme (ACE) is found in macrophages, endothelium (during angiogenesis) and myofibroblasts at sites of fibrosis in the heart, kidneys, liver and skin in several species. An increase in local tissue ACE-binding activity appears to be a critical event in the initiation and progression of fibrosis in these tissues. The aim of this study was to investigate the correlation between ACE activity in the equine endometrium and the degree of EPF, as determined by histological evaluation and morphometry based on a collagen-specific stain. ACE-binding activity values were significantly higher in the endometrial samples with moderate EPF (modified Kenney EPF category IIB) compared with endometria in all other categories. Ultrastructurally, the fibroblasts surrounding the glandular basal laminae in modified Kenney EPF category IIB and III endometria were undergoing myofibroblastic transformation-like changes. These observations indicate a possible link between ACE activity and the onset of EPF in mares.

Brunner's glands are unique to mammalian species and in eutherians are confined primarily to the submucosa of the proximal duodenum. In the majority of species examined, they begin at the gastrointestinal junction and extend for variable distances distally in the wall of the proximal small intestine. Ducts of individual glands empty either directly into the intestinal lumen or unite with overlying intestinal glands (crypts of Lieberkühn) dependent on the species. Secretory units of Brunner's glands consist of epithelial tubules that show frequent distal branchings. The secretory units, with the exception of those found in rabbits and horses, consist primarily of a mucin producing cell type. However, other cell types normally associated with the overlying intestinal epithelium may be encountered scattered within the secretory units reflecting the developmental origin of these glands. Secretion from Brunner's glands contributes to a layer of mucus that forms a slippery, viscoelastic gel that lubricates the mucosal lining of the proximal intestinal tract. The unique capacity of this mucus layer to protect delicate underlying epithelial surfaces is due primarily to the gel-forming properties of its glycoprotein molecules. Mucin glycoproteins produced by Brunner's glands consist primarily but not exclusively of O-linked oligosaccharides attached to the central protein core of the glycoprotein molecule. Human Brunner's glands produce class III mucin glycoproteins and are thought to be the product of mucin gene MUC6 which is assigned to chromosome 11 (11p15-11p15.5 chromosome region). In addition to mucin glycoproteins and a limited amount of bicarbonate, numerous additional factors (epidermal growth factor, trefoil peptides, bactericidal factors, proteinase inhibitors, and surface-active lipids) have been identified within the secretory product of Brunner's glands. These factors, incorporated into the mucus layer, guard against the degradation of this protective barrier and underlying mucosa by gastric acid, pancreatic enzymes, and other surface active agents associated with this region. Yet other factors produced by Brunner's glands function to provide active and passive immunological defense mechanisms, promote cellular proliferation and differentiation, as well as contribute factors that elevate the pH of luminal contents of this region by promoting secretion of the intestinal mucosa, pancreatic secretion and gall bladder contraction. Additional insights concerning the role of Brunner's glands in the mammalian gastrointestinal tract as well as their possible evolution in this class of vertebrates have been gained from a basic understanding of their pathobiology.

Guanylin, uroguanylin, and lymphoguanylin are small peptides that activate cell-surface guanylate cyclase receptors and influence cellular function via intracellular cGMP. Guanylins activate two receptors, GC-C and OK-GC, which are expressed in intestine and/or kidney. Elevation of cGMP in the intestine elicits an increase in electrolyte and water secretion. Activation of renal receptors by uroguanylin stimulates urine flow and excretion of sodium, chloride, and potassium. Intracellular cGMP pathways for guanylins include activation of PKG-II and/or indirect stimulation of PKA-II. The result is activation of CFTR and/or C1C-2 channel proteins to enhance the electrogenic secretion of chloride and bicarbonate. Similar cellular mechanisms may be involved in the renal responses to guanylin peptides. Uroguanylin serves as an intestinal natriuretic hormone in postprandial states, thus linking the digestive and renal organ systems in a novel endocrine axis. Therefore, uroguanylin participates in the complex physiological processes underlying the saliuresis that is elicited by a salty meal.

Guanylate cyclases (GC) serve in two different signaling pathways involving cytosolic and membrane enzymes. Membrane GCs are receptors for guanylin and atriopeptin peptides, two families of cGMP-regulating peptides. Three subclasses of guanylin peptides contain one intramolecular disulfide (lymphoguanylin), two disulfides (guanylin and uroguanylin) and three disulfides (E. coli stable toxin, ST). The peptides activate membrane receptor-GCs and regulate intestinal Cl- and HCO3- secretion via cGMP in target enterocytes. Uroguanylin and ST also elicit diuretic and natriuretic responses in the kidney. GC-C is an intestinal receptor-GC for guanylin and uroguanylin, but GC-C may not be involved in renal cGMP pathways. A novel receptor-GC expressed in the opossum kidney (OK-GC) has been identified by molecular cloning. OK-GC cDNAs encode receptor-GCs in renal tubules that are activated by guanylins. Lymphoguanylin is highly expressed in the kidney and heart where it may influence cGMP pathways. Guanylin and uroguanylin are highly expressed in intestinal mucosa to regulate intestinal salt and water transport via paracrine actions on GC-C. Uroguanylin and guanylin are also secreted from intestinal mucosa into plasma where uroguanylin serves as an intestinal natriuretic hormone to influence body Na+ homeostasis by endocrine mechanisms. Thus, guanylin peptides control salt and water transport in the kidney and intestine mediated by cGMP via membrane receptors with intrinsic guanylate cyclase activity.

Uroguanylin, guanylin, and lymphoguanylin are small peptides that activate renal and intestinal receptor guanylate cyclases (GC). They are structurally similar to bacterial heat-stable enterotoxins (ST) that cause secretory diarrhea. Uroguanylin, guanylin, and ST elicit natriuresis, kaliuresis, and diuresis by direct actions on kidney GC receptors. A 3,762-bp cDNA characterizing a uroguanylin/guanylin/ST receptor was isolated from opossum kidney (OK) cell RNA/cDNA. This kidney cDNA (OK-GC) encodes a mature protein containing 1,049 residues sharing 72.4-75.8% identity with rat, human, and porcine forms of intestinal GC-C receptors. COS or HEK-293 cells expressing OK-GC receptor protein were activated by uroguanylin, guanylin, or ST13 peptides. The 3.8-kb OK-GC mRNA transcript is most abundant in the kidney cortex and intestinal mucosa, with lower mRNA levels observed in urinary bladder, adrenal gland, and myocardium and with no detectable transcripts in skin or stomach mucosa. We propose that OK-GC receptor GC participates in a renal mechanism of action for uroguanylin and/or guanylin in the physiological regulation of urinary sodium, potassium, and water excretion. This renal tubular receptor GC may be a target for circulating uroguanylin in an endocrine link between the intestine and kidney and/or participate in an intrarenal paracrine mechanism for regulation of kidney function via the intracellular second messenger, cGMP.

Guanylin and uroguanylin are small peptides containing two disulfide bonds that activate membrane guanylate cyclase-receptors in the intestine, kidney and other epithelia. Hybridization assays with a uroguanylin complementary DNA (cDNA) detected uroguanylin-like messenger RNAs (mRNAs) in the opossum spleen and testis, but these transcripts are larger than uroguanylin mRNAs. RT of RNA from spleen to produce cDNAs for amplification in the PCR followed by cloning and sequencing revealed a novel lymphoid-derived cDNA containing an open reading frame encoding a 109-amino acid polypeptide. This protein shares 84% and 40% of its residues with preprouroguanylin and preproguanylin, respectively. A 15-amino acid, uroguanylin-like peptide occurs at the COOH-terminus of the precursor polypeptide. However, this peptide is unique in having only three cysteine residues. We named the gene and its peptide product lymphoguanylin because the source of the first cDNA isolated was spleen and its mRNA is expressed in all of the lymphoid tissues tested. A 15-amino acid form of lymphoguanylin containing a single disulfide bond was synthesized that activates the guanylate cyclase receptors of human T84 intestinal and opossum kidney (OK) cells, although with less potency than uroguanylin and guanylin. Northern and/or RT-PCR assays detected lymphoguanylin mRNA transcripts in many tissues and organs of opossums, including those within the lymphoid/immune, cardiovascular/renal, reproductive, and central nervous organ systems. Lymphoguanylin joins guanylin and uroguanylin in a growing family of peptide agonists that activate transmembrane guanylate cyclase receptors, thus influencing target cell function via the intracellular second messenger, cGMP.

Objectives: To develop an objective, quantifiable assay for endometrial periglandular fibrosis (EPF) and correlate assay results with histologic and ultrastructural changes in equine endometrial biopsy specimens., Sample Population: Endometrial biopsy specimens from 70 mares from 3 to 27 years old in estrus., Procedure: In a double-blinded study design, endometrial biopsy specimens were graded histologically (modified Kenney classification) for EPF and inflammation. Endometrial periglandular collagen volume fraction (%EPCVF) was determined by light microscopic image analysis of picrosirius red-stained sections. Specimens from selected mares were examined by transmission electron microscopy., Results: %EPCVF values varied significantly among the 4 modified Kenney EPF categories (I, IIA, IIB, and III) and increased with increasing age of mares. Morphologically, EPF consisted of concentric layers of transformed fibroblasts with myofibroblastic features and deposition of fibrillar collagen around unaltered glandular basal laminae., Conclusions and Clinical Relevance: %EPCVF correlates well with morphologic changes in endometrial biopsy specimens. Determination of %EPCVF could be useful in evaluation and clinical management of subfertile mares and in investigations of the pathogenesis of EPF.

Guanylin and uroguanylin are peptides that stimulate membrane guanylate cyclases (GC) and regulate intestinal and renal function via cGMP. Complementary DNAs were isolated encoding opossum preproguanylin and a 279-amino acid portion of a receptor-guanylate cyclase expressed in opossum kidney (OK) cells (GC-OK). The tissue expression of messenger RNA transcripts for these signaling molecules were then compared. Northern and/or reverse transcription-PCR assays revealed that guanylin, uroguanylin, and GC-OK messenger RNAs are expressed in tissues within the digestive, renal, central nervous, reproductive, and lymphoid organ systems. Receptor autoradiography localized the receptors for uroguanylin and guanylin to renal proximal tubules and seminiferous tubules of testis. Synthetic guanylin and uroguanylin peptides activated the receptor-GCs in opossum kidney cortex and in cultured OK cells eliciting increased intracellular cGMP. Expression of agonist and receptor-GC signaling molecules provides a pathway for paracrine and/or autocrine regulation of cellular functions via cGMP in the digestive, renal, central nervous, reproductive, and lymphoid/immune organ systems. Uroguanylin also links the intestine and kidney in a potential endocrine axis that activates tubular receptor-GCs and influences renal function.

Uroguanylin and guanylin are related peptides that activate common guanylate cyclase signaling molecules in the intestine and kidney. Uroguanylin was isolated from urine and duodenum but was not detected in extracts from the colon of rats. Guanylin was identified in extracts from small and large intestine but was not detected in urine. Uroguanylin and guanylin have distinct biochemical and chromatographic properties that facilitated the separation, purification, and identification of these peptides. Northern assays revealed that mRNA transcripts for uroguanylin were more abundant in small intestine compared with large intestine, whereas guanylin mRNA levels were greater in large intestine relative to small intestine. Synthetic rat uroguanylin and guanylin had similar potencies in the activation of receptors in T84 intestinal cells. Production of uroguanylin and guanylin in the mucosa of duodenum is consistent with the postulate that both peptides influence the activity of an intracellular guanosine 3',5'-cyclic monophosphate signaling pathway that regulates the transepithelial secretion of chloride and bicarbonate in the intestinal epithelium.

Receptors for guanylin and uroguanylin were identified on the mucosal surface of enterocytes lining the intestine of the bobtail skink (Tiliqua rugosa), king's skink (Egernia kingii), and knight anole (Anolis equestris) by receptor autoradiography using 125I-ST (Escherichia coli heat-stable enterotoxin) as the radioligand. Specific, high-affinity binding of 125I-ST to receptors was found on the microvillus border of enterocytes and little or no specific binding of 125I-ST was observed in other strata comprising the gut wall. The American alligator (Alligator mississippensis) also exhibited receptor binding, but unlike the other three species had relatively high levels of apparent nonspecific binding. A comparison of intestinal cGMP accumulation responses between the American alligator and the knight anole demonstrated a greater magnitude of cGMP responses to ST and guanylin in vitro in the knight anole relative to the tissue cGMP accumulation responses of alligators. Treatment with ST resulted in markedly greater tissue cGMP accumulation responses in both species compared to treatment with guanylin. To complete a paracrine signaling pathway in reptilian intestine, guanylin-like peptides that stimulated cGMP accumulation in human T84 intestinal cells were isolated from the intestinal mucosa of alligators. We conclude that functional receptor-guanylyl cyclases and one or more endogenous guanylin/uroguanylin-like peptides occur in the intestinal tract of reptiles as well as in the intestines of mammals and birds. Thus, higher vertebrates have a conserved signaling pathway that regulates intestinal function through the first-messenger peptides, guanylin and/or uroguanylin, and the intracellular second messenger, cGMP.

Guanylin and uroguanylin are peptides that activate receptor guanylate cyclases (GCs) and elicit increased intestinal secretion. Bacteria that cause traveler's diarrhea produce heat-stable toxins (STs) that mimic this action. Investigation of the distribution and identity of receptor GCs in the gastrointestinal tract of rats revealed that receptors were localized to epithelial cells in stomach and intestine. Clusters of cells in gastric mucosa and enterocytes lining the intestine exhibited specific binding of 125I-labeled ST. Ligated loops of stomach and intestine treated with intraluminal ST had significant increases in guanosine 3',5'-cyclic monophosphate (cGMP), with duodenum exhibiting the greatest response. Expression of guanylate cyclase C (GCC) mRNA and a truncated, GCC-like mRNA was found in both stomach and intestine. Both mRNAs were isolated as cDNAs encoding the GC catalytic domain. The 0.9-kilobase (kb) cDNA is 99.8% identical to GCC, whereas the truncated, 0.75-kb GCC-like cDNA has a 159-nucleotide deletion and is 96.6% identical to GCC at the protein level. Uroguanylin and guanylin mRNAs were detected in stomach and intestine. Uroguanylin mRNA was most abundant in small intestine, whereas guanylin mRNA was highest in large intestine. Thus the stomach and intestine are targets for regulation of transport by guanylin and uroguanylin via cGMP.

Background: Uroguanylin and guanylin are intestinal peptides that activate a receptor-guanylate cyclase, which is also a receptor for Escherichia coli heat-stable enterotoxin (STa). These peptides may have a role in the body's regulation of fluid and electrolytes., Methods: STa, bioactive guanylin, and bioactive uroguanylin were evaluated for effects in: 1) the suckling mouse intestinal fluid secretion assay; 2) an in vitro suckling mouse intestinal loop assay; 3) an intestinal receptor autoradiography assay; 4) a control or agonist-stimulated assay for cGMP response in T84 cells; and 5) an in vivo renal function assay in mice., Results: In vivo, orally administered uroguanylin and STa but not guanylin, stimulated intestinal fluid secretion. All three peptides activated intestinal guanylate cyclase and had common intestinal receptors. In vitro, after pretreatment with chymotrypsin, only uroguanylin and STa retained agoinst activity. Chymostatin preserved guanylin activity. STa and uroguanylin induced diuresis, natriuresis, and kaliuresis. Guanylin was less potent than uroguanylin and STa., Conclusions: The results suggest that the endogenous intestinal peptides, uroguanylin and guanylin, regulate water and electrolyte homeostasis both through local effects on intestinal epithelia and endocrine effects on the kidney.

Guanylin and uroguanylin are newly discovered, related peptides that activate common guanylyl cyclase signaling molecules and via 3', 5'-guanosine cyclic monophosphate regulate the activity of a variety of tissues and organs. Additionally, the message for both peptides is expressed in a variety of tissues and organs, including the intestinal tract and kidney, and thus may serve as part of a functional endocrine axis linking these two major organ systems in fluid/volume homeostasis. This manuscript reviews the discovery and nature of the guanylin and uroguanylin peptides, their actions on the intestinal mucosa and kidney, the distribution and molecular biology of the guanylyl cyclase C receptor, and explores the future directions of this rapidly developing, expanding field of inquiry.

Uroguanylin and guanylin are structurally related peptides that activate an intestinal form of membrane guanylate cyclase (GC-C). Guanylin was isolated from the intestine, but uroguanylin was isolated from urine, thus a tissue source for uroguanylin was sought. In these experiments, uroguanylin and guanylin were separated and purified independently from colonic mucosa and urine of opossums. Colonic, urinary, and synthetic forms of uroguanylin had an isoelectric point of approximately 3.0, eluted from C18 reverse-phase high-performance liquid chromatography (RP-HPLC) columns at 8-9% acetonitrile, elicited greater guanosine 3', 5'-cyclic monophosphate (cGMP) responses in T84 cells at pH 5.5 than pH 8, and were not cleaved and inactivated by pretreatment with chymotrypsin. In contrast, colonic, urinary, and synthetic guanylin had an isoelectric point of approximately 6.0, eluted at 15-16% acetonitrile on C18 RP-HPLC columns, stimulated greater cGMP responses in T84 cells at pH 8 than pH 5.5, and were inactivated by chymotrypsin, which hydrolyzed the Phe-Ala or Try-Ala bonds within guanylin. Uroguanylin joins guanylin as an intestinal peptide that may participate in an intrinsic pathway for cGMP-mediated regulation of intestinal salt and water transport. Moreover, uroguanylin and guanylin in urine may be derived from the intestinal mucosa, thus implicating these peptides in an endocrine mechanism linking the intestine with the kidney.

The composition of milk samples collected from captive opossums (Didelphis virginiana) was determined at various intervals during lactation. The milk solids increased from 9% at week one to a maximum of 34% at 11 weeks post-partum. There were changes in the relative proportions of protein, lipid and carbohydrate at different stages of lactation. Lipid represented the greatest fraction of the solids except for a period at mid-lactation when there was a peak in protein concentration. The concentrations of sodium, potassium and magnesium were relatively constant, 41 +/- 4, 35 +/- 11 and 9.2 +/- 1.6 mmol respectively, although calcium increased from 13 +/- 5 mmol at week one to a peak of 112 +/- 35 mmol at 9 weeks.

Uroguanylin is a small peptide isolated from opossum urine that activates membrane guanylate cyclases. We report the isolation by molecular cloning of cDNAs encoding the 109 amino acid preprouroguanylin containing the active uroguanylin peptide at its C-terminus. Preprouroguanylin mRNAs of 1.2 kb were detected throughout the small and large intestine and in the atria and ventricles of heart, but not in kidney, stomach or liver. Transfection of COS-1 cells with the uroguanylin cDNA resulted in prouroguanylin secretion. Both uroguanylin and prouroguanylin were isolated from opossum plasma. Thus, uroguanylin is made by the intestine and heart and circulates as a bioactive form of uroguanylin and the inactive prouroguanylin.

Uroguanylin and guanylin are peptides isolated from urine and intestinal mucosa, which regulate cyclic GMP production in enterocytes by activating an apical membrane, receptor-guanylate cyclase. This study extended our previous findings, which showed that colonic mucosa of opossums contained uroguanylin and guanylin peptides, by purifying prouroguanylin and proguanylin from this tissue. Prouroguanylin and proguanylin coeluted from Sephadex G-75 gelfiltration columns with a similar molecular size between 6 and 12 kDa. Mass spectrometry indicated that proguanylin (approximately 8.7 kDa) had a 10% lower molecular mass than prouroguanylin (approximately 9.7 kDa). Isoelectric focusing separated prouroguanylin (pI approximately 4.5) from proguanylin (pI approximately 7.5). N-terminal sequence analysis of reverse phrase-HPLC purified prohormones revealed 13 amino acids in opossum proguanylin that shared 77-85% identity with human and rat proguanylin, but only 23% identity with opossum prouroguanylin. The N-terminal 19 residues obtained for opossum prouroguanylin shared 32-42% identity with rat and human proguanylin. Prouroguanylin and proguanylin were both inactive and required pretreatment with proteases to elicit cyclic GMP responses in T84 cells. V8 protease treatment of proguanylin liberated a bioactive, 16-amino acid form of guanylin. Chymotrypsin treatment activated prouroguanylin, but inactivated the bioactive peptide domain within proguanylin. In summary, colonic mucosa contains the bioactive peptide and inactive prohormone forms of uroguanylin and guanylin. Thus, after proteolytic processing of prouroguanylin and proguanylin, bioactive uroguanylin and guanylin could both function to regulate guanylate cyclase activity by autocrine and/or paracrine actions on enterocytes. Also, these peptide hormones are implicated in an intestinal-renal axis for the endocrine regulation of salt and water homeostasis.

Brunner's glands are located in the submucosa of the proximal duodenum and are unique to mammalian species. The North American opossum (Didelphis virginiana) is generally regarded as a prototype marsupial that closely resembles fossil didelphids which can be placed at the beginning of mammalian evolution. The current investigation provided an opportunity for the analysis of secretory products from these glands in a species thought to be more closely related to earlier evolutionary forms. Extracts of Brunner's glands were subjected to SDS-PAGE and Western blotting. The results indicate the presence of two high molecular weight PAS-positive glycoprotein bands. In addition to these two PAS-positive bands, several other glycoprotein bands were detected in the high molecular weight range that bind several lectins which typically recognize O-linked carbohydrates indicative of mucus type glycoproteins. The same lectins bind to glandular structures in tissue sections. Comparison of lectin binding sites with the pyloric glands of the stomach and duodenal goblet cells indicates that Brunner's glands carbohydrate residues resemble those of the pyloric glands more closely than those of the duodenal goblet cells. The low cell turnover rate in Brunner's glands is in contrast to the rapid turnover rate of goblet cell precursors in the duodenal crypts. The mucus composition and the cell turnover rate correlate well with embryological data and suggest that Brunner's glands of Didelphis evolved from an epithelium more closely associated with the stomach than that of the duodenum as the topography of the gland would suggest.

Pathogenic strains of enteric bacteria secrete small heat-stable toxins (STs) that activate membrane guanylyl cyclase receptors found in the intestine. The intestinal peptide agonists, guanylin and uroguanylin, are structurally related to STs. Receptors for 125I-ST were found throughout the entire length of the intestinal tract of all the birds examined. These receptors were restricted to intestinal epithelial cells covering villi and forming intestinal glands and were not observed in other strata of the gut wall. The most intense labeling of receptors by 125I-ST occurred in the region of the microvillus border of individual enterocytes. There appeared to be a decrease in receptor density distally along the length of the small intestine, although labeling of receptors by 125I-ST was observed throughout the small intestine and colon. Cellular cGMP accumulation responses to Escherichia coli ST and rat guanylin in the domestic turkey and duck were greater in the proximal small intestine compared to the distal small intestine or colon. Brush border membranes (BBM) isolated from the mucosa of proximal small intestine of turkeys exhibited agonist-stimulated guanylyl cyclase activity. The rank order potency for enzyme activation was E. coli ST > uroguanylin > guanylin. Competitive radioligand binding assays using 125I-ST and turkey intestine BBM revealed a similar rank order affinity for the receptors that was exemplified by the Kd values of ST 2.5 nM, uroguanylin 80 nM and guanylin 2.6 microM. It may be concluded that functional receptors for the endogenous peptides, guanylin and uroguanylin, occur in the apical membranes of enterocytes throughout the avian intestine. The receptor-guanylyl cyclase(s) of proximal small intestine were preferentially activated by uroguanylin relative to guanylin, but both endogenous peptides were less potent than their molecular mimic, E. coli ST.

General brain growth and differentiation of the neocortex have been studied in the marsupial, Didelphis virginiana from the 10.5 day embryo through adulthood. Didelphis is born after a short gestation period of about 12.5 days, at a time when the telencephalic wall consists only of two layers and is considered to be at an embryonic stage of development. The cortical plate does not appear until late in the first postnatal week, thus neocortical development is totally a postnatal phenomenon in Didelphis as has been shown in other marsupial species examined to date. The general pattern of development and the establishment of the six-layered adult neocortex in Didelphis is similar to that described in eutherian mammals. Signs of cortical lamination can be seen as early as postnatal day 35 and the cytoarchitecture of a typical mammalian neocortex is well defined by postnatal day 60 in Didelphis prior to the onset of weaning.

The human intestinal tract, as well as that of several eutherian and metatherian mammals, was examined for the distribution of heat-stable enterotoxin (ST)/guanylin receptors. These receptors were confined to the intestinal epithelium lining the lumen and forming the intestinal glands throughout the length of both the small intestine and colon of all species examined. In man and most other mammalian species, there appeared to be a decrease in receptor density distally along the longitudinal axis of the small intestine. ST/guanylin receptors were not observed in other strata forming the gut wall. Along the vertical axis of the human small intestine (villus/crypt unit), as well as that of most other mammals, receptor density was greatest in enterocytes located near the base of villi and in those forming the proximal portion of the intestinal glands. ST/guanylin receptors were for the most part confined to the region of the plasmalemma forming the microvillus border. In the colon of man and the other species examined, receptor density was greatest in enterocytes forming the proximal region of the intestinal glands. Receptors were present in the intestinal epithelium lining the lumen of the colon, but generally were fewer in number. The distribution of cellular cGMP accumulation responses to E. coli ST and guanylin in the opossum (Didelphis virginiana) and raccoon (Procyon lotor) revealed that proximal small intestine had greater magnitudes of cGMP responses than did the distal small intestine. Proximal colon had greater cGMP responses than distal colon, which had no significant cGMP responses to either ST or guanylin.

The intestinal hormone guanylin and bacterial heat-stable enterotoxins (STs) are members of a peptide family that activates intestinal membrane guanylate cyclase. Two different peptides that activate the human intestinal T84 cell guanylate cyclase have been purified from urine and intestinal mucosa of opossums (Didelphis virginiana). The highly acidic peptide, QEDCELCINVACTGC, was named uroguanylin because it was isolated from urine and shares 53% identity with guanylin. A second peptide, SHTCEICAFAACAGC, was purified from urine and intestinal mucosa. This alanine-rich peptide was 47% identical to uroguanylin and 73% identical to human guanylin, suggesting that it may be an opossum homologue of guanylin. Synthetic uroguanylin-(2-15) (i.e., EDCELCINVACTGC) was 10-fold more potent than synthetic rat guanylin, but both peptides were less potent than Escherichia coli ST in the T84 cell cGMP bioassay. Uroguanylin-(2-15) and guanylin inhibited 125I-ST binding to T84 cell receptors in competitive radioligand binding assays. Transepithelial Cl- secretion was stimulated by 1 microM uroguanylin, indicated by an increase in the short circuit current of T84 cells. Thus, uroguanylin is another paracrine hormone in the emerging peptide family that activates intestinal membrane guanylate cyclase. The second peptide may be the opossum form of guanylin, or perhaps, it is still another member of this peptide family. The presence of uroguanylin and guanylin in urine and receptors in proximal tubules suggests that these peptides may also originate from renal tissue and may regulate kidney function.

Marsupials are unique models for developmental biology-oriented research because of the immature state of their development at birth. The North American opossum (Didelphis virginiana) has several advantages over other marsupials, including large litter size, short prenatal period (12.5 d), an extended postnatal period while accessible in the pouch, and its ability to reproduce reliably in captivity. Studies of ocular development in this species have not been reported previously. The aim of the present investigation was therefore to document the major landmarks in prenatal and postnatal development of the cornea, lens, iris, ciliary body and retina. Fifteen embryos (10.5, 10.7 and 11 d postconception and 6 h after birth [12 d]) were studied by paraffin histology. Eyes of pouch young at 8 d, 2, 6, 9 and 13 wk were studied by transmission electron microscopy and light microscopy. The study revealed a similar pattern of ocular development in Didelphis to other metatherian and eutherian mammals. Differentiation of the eye is particularly rapid in the 2 d before birth. For example, although the lens vesicle separates from the surface ectoderm on d 10, by birth (2.5 d later) a primitive cornea and fused eyelids have formed, presumably to protect the eye during migration to the pouch. At birth the retinal pigment epithelium (RPE) contains melanin; however, the inner layer of the optic cup does not differentiate into an inner and outer neuroblastic layer until 8 d after birth. Around 6 wk after birth most components of the adult eye are identifiable, albeit in an immature form. These include the corneal layers, the iris (including the sphincter pupillae), ciliary processes, RPE tapetum, and a fully laminated retina with immature photoreceptors. A knowledge of the timing of major events in eye development in Didelphis and their comparison with equivalent events in human eye development should allow the appropriate choice of stages for any future experimental studies utilising this marsupial species.

Light-microscopic and ultrastructural analysis of the ocular tissues of the North American opossum (Didelphis virginiana) revealed that the arterial and venous segments of retinal vessels, including capillaries of the smallest calibre, occur in pairs. They do not form anastomotic networks, the common pattern in mammals with vascularised retinae, but instead the two segments of the pair join to form hairpin end loops. The paired vessels, with the arteriolar limb usually on the vitread aspect, penetrate the retina and branch to form three distinct layers of capillaries. The most superficial lies in the nerve fiber layer, the middle is situated in the inner nuclear layer and the deepest extends to the external limiting membrane, which is considerably deeper than in normal mammalian holangiotic retinae. The paired capillaries display classical morphological features of central nervous system capillaries, i.e., they are lined by continuous endothelial cells united by tight junctions. The lining endothelium is supported by a distinct basal lamina that splits to envelop pericytes. The latter, although abundant, are invariably interposed between the two vessels that form each vascular unit. Phylogenetic and functional aspects of this unique form of retinal vascularisation are discussed.

Heat-stable enterotoxins activate guanylate cyclase, whereas heat-labile enterotoxins stimulate adenylate cyclase. Both classes of toxins cause secretory diarrhea at least in part by stimulating Cl- secretion in the intestine. The mechanism for regulation of Cl- secretion by guanosine 3',5'-cyclic monophosphate (cGMP) was investigated using cultured T84 intestinal cells as a model for intestinal crypt cells. Escherichia coli heat-stable enterotoxin (ST) markedly stimulated cGMP production in T84 cells. Cl- secretion across T84 cell monolayers cultured on permeable filters was stimulated by E. coli ST, cholera toxin, or 8-BrcAMP, but 8-BrcGMP was ineffective. cGMP analogues that are known to be potent and specific activators of cGMP-dependent protein kinase (cG-kinase) also had little effect on 36Cl- uptake by T84 cells cultured in plastic dishes. E. coli ST, forskolin, cholera toxin, or membrane-permeant cAMP analogues markedly increased 36Cl- uptake into T84 cells. The general protein kinase inhibitor, staurosporine, inhibited the stimulation of Cl- permeability elicited by E. coli ST, vasoactive intestinal peptide (VIP), or 8-BrcAMP. DEAE-Sephacel chromatography revealed a predominant type II isoform of cAMP-dependent protein kinase (cA-kinase) in T84 cells, whereas little or no cytosolic cG-kinase activity was found. Treatment of T84 cells with E. coli ST or VIP resulted in an increase in the cA-kinase activity ratio (-cAMP/+cAMP) if the cytosolic enzyme was assayed at reduced temperature (on ice).(ABSTRACT TRUNCATED AT 250 WORDS)

Relaxin-immunoreactivity was demonstrated in the cytoplasm in the luteal cells from pregnant and lactating opossums. Immunoreactivity for relaxin was not demonstrated elsewhere in the ovary, in the reproductive tract or in the placenta. The corpus luteum is thought to be the primary source of relaxin in Didelphis and in this regard is similar to several eutherian mammals including man.

The Harderian gland of the North American opossum (Didelphis virginiana) is large and well developed, despite the absence of a nictitating membrane in the adult of this species. The elongate glands are surrounded by a delicate connective tissue capsule from which thin septae extend, subdividing the gland into numerous lobules. The secretory units of the opossum Harderian gland are drained by a well defined but not extensive intralobular and interlobular duct system. Most of the secretory end pieces consist of tubulo-alveolar units with widely dilated lumina filled with secretory product. Numerous intact lipid vesicles suspended within an amorphous material constitute the luminal contents. Cells lining the tubulo-alveolar secretory end-pieces are usually columnar in shape, and characterized by numerous lipid-containing secretory vesicles and aggregations of poly-tubular complexes 40-60 nm in diameter. In addition, these cells contain numerous large irregularly shaped mitochondria, whose matrix is of considerable electron density. Intralobular and interlobular ducts are lined by electron-lucent epithelial cells that lack both the lipid-containing vesicles and the large mitochondria, although typical smaller mitochondria are found scattered within the cytoplasm. Both secretory end-pieces and ductal elements are invested by an abundance of myoepithelial cells. A second, smaller serous type of secretory unit may occur near the centre of some Harderian gland lobules. In these units secretory tubules and acini are compactly arranged surrounding a narrow lumen. Serous cells are pyramidal in shape and the cytoplasm is characterized by numerous electron-dense secretory granules and scattered profiles of rough endoplasmic reticulum.(ABSTRACT TRUNCATED AT 250 WORDS)

We studied the effect of either placebo, 16,16-dimethyl-prostaglandin E2 (16,16-dimethyl-PGE2), or cimetidine on spontaneous degeneration of isolated rat gastric glands maintained in vitro in a basic oxygenated medium for 24 h. We assessed the viability of gland cells with fast green exclusion, measured release of lactate dehydrogenase (LDH) into the medium, and assessed the cell ultrastructure using a scanning electron microscope. Gastric glands incubated in medium for 6, 12, and 24 h underwent spontaneous degeneration reflected by a decrease in cell viability, increase in LDH release into the medium, and ultrastructural cell damage. 16,16-Dimethyl-PGE2 either at 0.1, 1, or 10 micrograms/ml significantly reduced the decrease in cell viability, increasing cell survival; reduced LDH release into the medium; and ultrastructural damage. Incubation with cimetidine at 1 or 10 micrograms/ml did not affect cell viability at 6, 12, or 24 h, whereas 100 micrograms/ml reduced cell viability (vs. placebo) at 12 and 24 h. LDH release and ultrastructural damage were not affected (not reduced) by cimetidine. Our study indicates that 16,16-dimethyl-PGE2, but not cimetidine, directly protects isolated gastric gland cells against degeneration in vitro, under conditions independent of systemic, neural, and hormonal factors.

The nasal cavities of opossums prior to and shortly after birth were examined by scanning electron microscopy. Numerous morphologically mature olfactory receptor neurons are observed in the dorso-rostral-most extent of the olfactory epithelium positioned adjacent to the opening of the nares in all prenatal stages and newborn animals examined. The remainder of the olfactory epithelium, occupying a more dorso-caudal position within the nasal cavity, is undifferentiated, and lacks morphologically mature receptor neurons. A short transition zone of stratified squamous epithelium lies between the epithelium lining the nares and olfactory epithelium. It forms an abrupt junction with the latter. The remainder of the nasal cavity in this group of animals is lined by a non-ciliated pseudostratified type (undifferentiated respiratory) of epithelium. By the end of the second postnatal week the morphologically mature olfactory epithelium is no longer observed in the vestibular area of the nasal cavity, which is lined by stratified squamous epithelium at this time. Mature receptor neurons are now observed within the olfactory epithelium lining the roof of the nasal cavity and covering the turbinates. The greater part of the nasal cavity is lined by a ciliated respiratory epithelium. It is proposed that the precocious differentiation of mature olfactory receptor neurons within the rostral-most extent of the olfactory epithelium just prior to birth is important in guiding the newborn young to the pouch.

Corpora lutea from opossums late in pregnancy were examined by immunohistochemistry for the presence of oxytocin. Oxytocin-immunoreactivity was observed in all corpora lutea examined but not elsewhere in ovarian tissue. The immunoreactive staining observed was confined primarily to the perinuclear cytoplasm of reactive luteal cells. Not all luteal cells showed oxytocin-immunoreactivity. The immunohistochemical localization of oxytocin in the pregnant opossum corpus luteum demonstrates for the first time this peptide in a metatherian ovary. Its presence in this primitive species suggests that oxytocin has a fundamental role in the physiology of the mammalian ovary.

Assessment of gastric ulcer healing is usually based on a visual examination (by endoscopy in patients, or the evaluation of ulcer size in experimental studies), and not on histologic and ultrastructural assessment of subepithelial mucosal healing. This approach has led to the assumption that the mucosa of grossly "healed" gastric and/or duodenal ulcers returns to normal, either spontaneously or following treatment. However, the re-epithelialized mucosa of grossly "healed" experimental gastric ulcer has recently been found to have prominent histologic and ultrastructural abnormalities, including reduced height, marked dilation of gastric glands, poor differentiation and/or degenerative changes in glandular cells, increased connective tissue, and disorganized microvascular network. It has been postulated that these residual abnormalities might interfere with mucosal defense and may be the basis of ulcer recurrence. In the present article, the ulcer healing process and the role of luminal factors, transitional zone at the ulcer margin, and granulation tissue are discussed. The healing of an ulcer is accomplished by filling of the mucosal defect with epithelial cells and connective tissue to reconstruct mucosal architecture. Under influence of growth factors [predominantly epidermal growth factor (EGF) and transforming growth factor (TGF alpha)], the epithelial cells at the ulcer margin dedifferentiate and proliferate, supplying cells for re-epithelialization of the mucosal scar surface and reconstruction of glandular structures. Granulation tissue at the ulcer base supplies connective tissue cells to restore the lamina propria and endothelial cells and microvessels for mucosal microvasculature reconstruction. The final outcome of healing reflects a dynamic interaction between an "epithelial" component from the ulcer margin and a connective tissue component including microvessels originating from granulation tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

It has long been assumed that the mucosa in areas of grossly 'healed' gastric or duodenal ulcers returns to normal, either spontaneously or after treatment. This assumption is based almost entirely upon visual, superficial examination by endoscopy. Few, if any, histological and ultrastructural studies examined the deeper mucosa in the areas of grossly healed ulcers. In several experimental studies, we analysed the development, evolution, and healing of acetic acid-induced gastric ulcers in rats and assessed the histological and ultrastructural features (structure and cellular composition) of the gastric mucosa in areas of grossly healed ulcers. The gastric mucosa of grossly 'healed' ulcers showed re-epithelialization of the mucosal surface at every study interval (2 weeks, 2, 3, and 4 months), but the subepithelial mucosa displayed prominent abnormalities. Two patterns of scarring were distinguished: (a) the mucosa in the area of healed ulcer was thinner (25-45% thinner than normal mucosa) with increased connective tissue and poor differentiation and/or degenerative changes in the glandular cells; and (b) the mucosa displayed a marked dilation of gastric glands with poor differentiation of the glandular cells and a reduction in the supportive microvascular network. It is theorized that these abnormalities could interfere with oxygenation, nutrient supply, and mucosal resistance and defence; therefore, they could be a basis for ulcer recurrence. These observations indicate that the quality of mucosal structural restoration rather than the speed of ulcer healing is the most important factor in determining risk of ulcer recurrence. The clinical relevance of these findings is supported by a preliminary study in which marked histological abnormalities were found in the subepithelial mucosa in patients with 'healed' duodenal ulcers.

Pancreatic endocrine cells were examined in a primitive egg-laying mammal, the echidna, using immunohistochemistry. Immunoreactive endocrine cells were observed using antisera to insulin, glucagon, somatostatin, avian pancreatic polypeptide and bovine pancreatic polypeptide. In addition, motilin-immunoreactive cells were identified in both the endocrine and exocrine pancreas of pouch-young and adult echidnas using three types of motilin antisera. Since the motilin-immunoreactive cells did not cross-react with any other pancreatic hormones tested, they are identified as an independent endocrine cell type.

In the North American opossum, heat-stable specific binding sites for E. coli enterotoxin are observed (i) in epithelial cells lining the small intestine, colon, gall bladder, cystic duct, common bile duct and trachea, and (ii) in epithelial cells forming the duodenal (Brunner's) glands, liver, kidneys (metanephros, mesonephros) and testis, as demonstrated by autoradiography. Enterotoxin-specific binding sites in the intestinal tract are only found in intestinal epithelial cells with the highest concentration in the microvillus border. Enterotoxin-specific binding sites also occur in epithelial cells comprising the secretory tubules and ducts of the duodenal glands. In the kidneys (metanephros and mesonephros), enterotoxin-specific binding sites are confirmed primarily to the proximal tubules, whereas in the testis they are localized in seminiferous tubules. In the liver, enterotoxin-specific binding sites are confined primarily to hepatocytes. E. coli enterotoxin caused a 7-fold increase of cGMP in the liver and a 30-fold increase in the duodenal glands. The liver responded in about half of the animals studied, whereas the duodenal glands gave a consistent response in each case. Likewise, the duodenal glands consistently showed strong labelling for 125I-enterotoxin, whereas receptor labelling of hepatocytes was inconsistent in nearly half the incubations and corresponds to the observed cGMP measurements.