Apical expression of functional asialoglycoprotein receptor in the human intestinal cell line HT-29.

BACKGROUND & AIMS: The asialoglycoprotein receptor localizes to the basolateral membrane of hepatocytes and to the apical membrane of enterocytes. The aim of this study was to examine HT-29 cells as a polarized cell model for studying apically localized endogenous asialoglycoprotein receptor. METHODS: Subunits H1 and H2 (human) were detected by Western blot and immunoprecipitated using subunit-specific antisera against hepatic receptor peptides. Receptor function was assessed by uptake of iodinated asialo-orosomucoid, immunoglobulin (Ig) A1, and haptocorrin. Immunocytochemistry was analyzed by standard light and confocal microscopy. RESULTS: Receptor content of the minor subunit, H2, was predominant. HT-29 cells mediated specific uptake and degradation of 125I-asialo-orosomucoid. A high-affinity (0.6 x 10(-9) mmol/L) and a low-affinity binding site were present. The specific ligand binding capacity of the apical surface was approximately twice that of the basolateral surface. Immunocytochemistry revealed a predominant apical membrane location of the minor receptor subunit, with some intracellular receptor. The apical H2 subunit was preferentially labeled with amino acid precursors compared with basolaterally located subunit. Human IgA1 bound specifically to HT-29 cells with a molar ratio of 0.26 compared with asialo-orosomucoid; porcine haptocorrin bound with a molar ratio of 1.35. CONCLUSIONS: HT-29 cells produce a functional apically located asialoglycoprotein receptor and provide a model for receptor trafficking in the enterocyte.

Expression of an endogenous asialoglycoprotein receptor in a human intestinal epithelial cell line, Caco-2.

We have previously shown that rat asialoglycoprotein receptor expressed in the intestine and liver differ in mRNA size, cell surface distribution, and ratio of compositional protein subunits. In this study, we examined a well characterized intestinal epithelial cell line, Caco-2, as a potential model for studying endogenous receptor in a polarized cell line. Both subunits H1 and H2 of human asialoglycoprotein receptor were detected in Caco-2 cells by Western blots using subunit-specific antisera raised against the hepatic receptor. Antigenic receptor level in fully differentiated Caco-2 cells was approx. 1/3 to 1/2 the level of hepatic HepG2 cells H1 was the dominant subunit in both cell lines. The apparent size of H1 and H2 in Caco-2 cells was not the same as that in HepG2 cells, due to differences in N-linked glycosylation. Consistent with this finding, Northern blot analysis showed that receptor mRNA in the two cell types was of identical size. In pulse-chase experiments H1 was first detected as a 'high-mannose' precursor (40 kDa) in Caco-2 cells that was converted to mature H1 (43 kDa) with a half-life of approx. 60 min. Antigenic levels of H1 and H2 in undifferentiated Caco-2 cells were low, but increased rapidly during cell differentiation, reaching a peak level at 7 days after confluence. Immunocytochemical staining and domain-selective cell surface biotinylation assays showed that the ASGP-R was predominantly localized in the basolateral domain. The receptor in Caco-2 cells was capable of mediating specific uptake and degradation of [125I]asialoorosomucoid. The ligand uptake capacity of the basolateral surface of was approx. 10-fold higher than the apical. These characteristics (H1 subunit and basolateral predominance) of the receptor in Caco-2 cells, resembles the hepatic receptor. We conclude that Caco-2 cells endogenously express in ectopic hepatic-type functional asialoglycoprotein receptor.

Asialoglycoprotein receptor mRNAs are expressed in most extrahepatic rat tissues during development.

The expression of an asialoglycoprotein receptor, comprised of a major (R-1) and minor (R-2/3) component, was characterized in various rat tissues during postnatal development. Two R-1 mRNA subspecies were identified in the liver by Northern blots. The 1.4-kb form, which was the predominant subspecies in the liver, was detected in small amounts only in the stomach and kidney. The 1.7-kb mRNA, however, was present in all extrahepatic tissues examined. R-1 mRNA content in the liver increased by only 38% during the suckling period, but the extrahepatic R-1 mRNA had a dramatic biphasic developmental pattern with the highest levels at birth and after weaning. The R-2/3 mRNA was present as a single species on Northern blot in the liver and in a restricted number of extrahepatic tissues (salivary glands, ileum, and kidney) but was detected in most tissues by polymerase chain reaction. The R-2/3 mRNA content in the liver increased fourfold after birth but was consistently low in the other tissues. [57Co]cobalamin-haptocorrin binding activity was present in all extrahepatic tissues examined, although only at a level 2-5% of hepatic binding. Binding affinities of the adult kidney brush-border membranes (1.9 x 10(9) M-1) and the adult liver plasma membranes (2.4 x 10(9) M-1) were comparable. On Western blot, < 4% of adult liver R-1 content and a relatively larger amount of R-2/3 were detected in all four extrahepatic tissues examined. Thus the low binding activity in extrahepatic tissues correlates with the presence of both receptor subunits, which are closely related to or identical with the hepatic receptor subunits.

Theophylline and prostaglandin E2 on duodenal bicarbonate secretion: role for 5'-cyclic adenosine monophosphate.

Cyclic adenosine monophosphate (cAMP) has been implicated as an intracellular "second" messenger in duodenal mucosal bicarbonate secretion in animals. The purpose of this study was to determine whether cAMP may mediate duodenal mucosal bicarbonate secretion in humans. In healthy volunteers, a 4-cm segment of proximal duodenum was isolated from gastric and pancreaticobiliary secretions. Either the phosphodiesterase inhibitor theophylline, prostaglandin (PG) E2, or a combination thereof was administered topically to the isolated duodenal mucosa. Theophylline (10(-2) mol/L) and PGE2 (10(-5)-10(-4) mol/L) each significantly increased bicarbonate secretion and transmucosal potential difference. Moreover, when theophylline and PGE2 were administered in combination, the duodenal bicarbonate output was additive compared to either agent alone. When theophylline was infused with increasing doses of PGE2, the dose-response curve was shifted to the left. Furthermore, increases in bicarbonate secretion and transmucosal potential difference were correlated significantly. These results suggest that cAMP may act as an intracellular mediator of human duodenal mucosal bicarbonate secretion.

The minor components of the rat asialoglycoprotein receptor are apically located in neonatal enterocytes.

Because specific uptake of the asialoglycoprotein haptocorrin has been reported in suckling distal intestine, evidence of the asialoglycoprotein receptor in rat ileum was sought. On Western blot, two different polyclonal antisera against purified rat holoreceptor recognized only proteins of the size of the minor receptor components (51 and 55 kilodaltons) in both suckling and adult rat intestine. Messenger RNA encoding the minor component (RHL-2/3) was detected in total RNA extracted from rat ileum. Calcium-specific binding of porcine or rat haptocorrin-[57Co]cobalamin complexes was detected in the brush border membranes of distal suckling rat and porcine small intestine. This binding was almost completely blocked by another asialoglycoprotein, asialofetuin. The pH optimum for binding was 6-9, with a Ka of 0.25 nmol/L and a capacity of 4.6 fmol/mg protein. These properties, with the exception of the low capacity, are all consistent with those of the intact receptor on hepatocytes. The intestinal receptor was localized not to the basolateral membrane, as in the liver, but to the apical brush border, as suggested by the binding data. Furthermore, immunoperoxidase stains of paraffin-embedded tissue detected strong binding to the brush border and apical phagolysosome of mid and distal suckling rat intestine. These data show that, contrary to expectations, the minor components of the asialoglycoprotein receptor are functional and expressed in the apical membrane of the suckling intestine. The abundance of the protein by Western blot suggests possible roles for it in the neonatal gut other than haptocorrin binding.

[Diagnosis of contaminated small bowel syndrome following subtotal gastrectomy: a comparative study of mannitol-BHT and jejunal bacteriology].

Recent reports from abroad showed that lactulose breath hydrogen test (BHT) is a noninvasive and an accurate method for diagnosing contaminated small bowel syndrome (CSBS). In this study, BHT was carried out with mannitol, an inexpensive and rich-in-source sugar, instead of lactulose. In comparison with the "gold standard" of jejunal bacteriology, it was disclosed that a double peak pattern of breath hydrogen response after oral administration of mannitol was characteristic of CSBS with a sensitivity of 71.4% and a specificity of 66.7%, which are about same as those of the lactulose-BHT used abroad.