Chlorpropamide upregulates antidiuretic hormone receptors and unmasks constitutive receptor signaling.

The mechanism by which chlorpropamide (CP) treatment promotes antidiuresis is unknown. CP competitively inhibited antidiuretic hormone (ADH) binding and adenylyl cyclase (AC) stimulation (inhibition constants K(i) and K'(i) of 2.8 mM and 250 microM, respectively) in the LLC-PK(1) cell line. CP (333 microM) increased the apparent K(a) of ADH for AC activation (0.31 vs. 0.08 nM) without affecting a maximal response, suggesting competitive antagonism. Because CP lowers "basal" AC activity and the AC activation-ADH receptor occupancy relationship (A-O plots), it is an ADH inverse agonist. Twenty-four-hour CP exposure (100 microM) upregulated the ADH receptors without affecting affinity. This lowered K(a) and increased basal AC activity and maximal response (1. 86 vs. 1.35 and 14.9 vs. 10.6 fmol cAMP. min(-1). 10(3) cells(-1), n = 6, P<0.05). NaCl, which potentiates ADH stimulation, also increased basal AC activity. This, together with the CP-ADH inverse agonism and increased basal AC activity at higher receptor density, unmasks constitutive receptor signaling. The CP-ADH inverse agonism explains receptor upregulation and predicts the need for residual ADH with functional isoreceptors for CP-mediated antidiuresis. This could be why CP ameliorates partial central diabetes insipidus but not nephrogenic diabetes insipidus.

Localization of a cell adhesion site on collagen XIV (undulin).

Cell adhesion to collagen XIV is implied to be mediated by proteoglycans as cellular receptors (T. Ehnis et al., 1996, Exp. Cell Res. 229, 388-397). In order to define the cell binding region(s), fusion proteins expressed in Escherichia coli and covering the large noncollagenous domain NC3 of collagen XIV were used as substrates for the adhesion of skin fibroblasts. A prominent cell binding site could be localized in the N-terminal fibronectin type III repeat of collagen XIV and its immediate C-terminal extension. Since this region also mediates the binding of the small chondroitin/dermatan sulfate proteoglycan decorin (T. Ehnis et al., 1997, J. Biol. Chem. 272, 20414-20419), our finding could provide the molecular basis for the observation that decorin serves as inhibitor and potential modulator of cellular interactions with collagen XIV.

Identification of the autoantigen of celiac disease.

Tissue transglutaminase is demonstrated to be the unknown endomysial autoantigen by means of immunoprecipitations from a fibrosarcoma cell culture. A novel hypothesis for the pathogenesis of celiac disease is formulated: The mainly intracellular tissue transglutaminase is released from cells during wound healing where it aids in stabilizing the wound area by cross-linking a small set of extracellular matrix components.

Localization of a binding site for the proteoglycan decorin on collagen XIV (undulin).

Through its ability to bind extracellular matrix constituents and growth factors the small leucine-rich chondroitin/dermatan sulfate proteoglycan decorin which is present in many types of connective tissues may play an important biological role in remodeling and maintenance of extracellular matrices during inflammation, fibrosis, and cancer growth. In this study we investigated the known binding of decorin to human collagen XIV. This binding was unaffected when the small collagenous moiety of collagen XIV was removed with collagenase. Therefore, fragments covering the large noncollagenous domain NC3 of collagen XIV were expressed in Escherichia coli, each fused to a 26-kDa fragment of glutathione S-transferase. Using radioiodinated decorin as ligand for the immobilized fusion proteins, a binding site that interacted with the decorin core protein could be assigned to the NH2-terminal fibronectin type III repeat of collagen XIV. In addition, an auxiliary binding site located COOH-terminal to this fibronectin type III repeat interacted with the glycosaminoglycan component of decorin.

Identification of tissue transglutaminase as the autoantigen of celiac disease.

Celiac disease is characterized by small intestinal damage with loss of absorptive villi and hyperplasia of the crypts, typically leading to malabsorption. In addition to nutrient deficiencies, prolonged celiac disease is associated with an increased risk for malignancy, especially intestinal T-cell lymphoma. Celiac disease is precipitated by ingestion of the protein gliadin, a component of wheat gluten, and usually resolves on its withdrawal. Gliadin initiates mucosal damage which involves an immunological process in individuals with a genetic predisposition. However, the mechanism responsible for the small intestinal damage characteristic of celiac disease is still under debate. Small intestinal biopsy with the demonstration of a flat mucosa which is reversed on a gluten-free diet is considered the main approach for diagnosis of classical celiac disease. In addition, IgA antibodies against gliadin and endomysium, a structure of the smooth muscle connective tissue, are valuable tools for the detection of patients with celiac disease and for therapy control. Incidence rates of childhood celiac disease range from 1:300 in Western Ireland to 1:4700 in other European countries, and subclinical cases detected by serological screening revealed prevalences of 3.3 and 4 per 1000 in Italy and the USA, respectively. IgA antibodies to endomysium are particularly specific indicators of celiac disease, suggesting that this structure contains one or more target autoantigens that play a role in the pathogenesis of the disease. However, the identification of the endomysial autoantigen(s) has remained elusive. We identified tissue transglutaminase as the unknown endomysial autoantigen. Interestingly, gliadin is a preferred substrate for this enzyme, giving rise to novel antigenic epitopes.

Complete primary structure of human collagen type XIV (undulin).

A partial cDNA sequence coding for the human extracellular matrix protein undulin has been completed. The completed sequence provides conclusive evidence for the suggested identity of undulin and collagen type XIV. Two differently sized polyproteins of 1780 and 1796 amino acids, with an overall amino acid sequence identity of 75% compared to chicken CXIV, emerge from variant 3' sequence ends encoding the C-terminal non-collagenous (NC) NC1 domain of human collagen type XIV.

A chondroitin/dermatan sulfate form of CD44 is a receptor for collagen XIV (undulin).

Collagen XIV, a fibril-associated collagen with interrupted triple helices, is expressed in differentiated soft connective tissues and in cartilage. However, a cellular receptor for this protein has not been identified. Here we show that human placental collagen XIV, isolated by a mild and simple two-step method, serves as adhesive protein for a variety of mesenchymal and some epithelial cells. Cell adhesion could be inhibited by preincubation of the collagen XIV substrate with heparin or with the chondroitin/dermatan sulfate proteoglycan decorin and by pretreatment of cells with chondroitinase ABC or heparinase III, suggesting a cell membrane proteoglycan as receptor. Affinity chromatography of 125I-labeled fibroblast cell surface proteins on collagen XIV-Sepharose yielded a chondroitin/dermatan sulfate proteoglycan with a molecular mass of 97-105 kDa after chondroitinase ABC digestion and of 60-70 kDa after further treatment with N-glycosidase F. The eluates contained also some high-molecular-weight material that was susceptible to digestion with heparinase but no detectable integrins. Immunoprecipitation with a specific monoclonal antibody identified the prominent chondroitin/dermatan sulfate proteoglycan as a member of the CD44 family. The interaction between collagen XIV and cells appears to be finely tuned, since matrix-associated glycosaminoglycans, and particularly proteoglycans like decorin, could compete with cells for the binding site(s) on collagen XIV under physiological conditions.

Expression of vasopressin receptors (V2-subtype) on LLC-PK1 cells during cell culture.

Vasopressin receptor expression on LLC-PK1-cells (a porcine renal tubular cell line) during cell culture is still not fully understood. We studied receptor expression using a novel vasopressin analogue with high specific radioactivity ([125I][8-p-hydroxy-phenylpropionyl]-lys8-vasopressin, 74EBq/mol (2000 Ci/mmol)). LLC-PK1 cells were grown in monolayers for 1 to 6 days. Scatchard analysis performed with membranes of LLC-PK1 cells revealed a single binding site with a binding constant (Kd) of 0.46 +/- 0.04 nmol/l. During cell culture, the binding constant (Kd) was not altered, but receptor density increased significantly (21,115 +/- 645 receptors per cell, day 2; 42,315 +/- 1512 receptors per cell, day 6). A receptor occupancy of about 30% was found to be associated with a cAMP stimulation of 50%. The receptor reserve might be even higher because, by using a highly specific oxytocin antagonist, we found that 20% of the occupied [125I][8-p-hydroxy-phenylpropionyl]-lys8-vasopressin-binding sites are oxytocin receptors. For lys8-vasopressin receptor studies, great care has to be taken to examine cells in identical culture phases.