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Uroguanylin (UGN) and guanylin (GN) are the endogenous intestinal ligands for guanylyl cyclase C (GC-C). We examined the circadian expression of UGN, GN, and GC-C in the jejunum, ileum, and proximal colon of young adult rats by Northern blot analyses. These assays revealed that UGN is more abundant in the proximal small intestine, whereas GN and GC-C are more abundant in the proximal colon. mRNA levels showed significant circadian variation for UGN (3- to 18-fold peak/trough difference), GN (2.1- to 2.8-fold peak/trough difference), and GC-C (3- to 5-fold peak/trough difference). The maximal abundance occurred in the dark period for all three mRNAs, although peak UGN and GN expression occurred later in the dark period in the jejunum relative to the ileum and colon. Immunoblot analyses using monospecific polyclonal antibodies against UGN and GN prohormones confirmed the regional and circadian variation detected by Northern assays. Thus the expression of these genes is regulated not only by histological position but also by circadian time.

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 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.

Many strains of enterotoxigenic Escherichia coli produce a heat-stable peptide enterotoxin (STa) that binds to the intestinal receptor guanylyl cyclase C (GC-C). STa receptors are structurally heterogeneous, but the molecular events causing this heterogeneity remain obscure. We examined the influence of cell position along the villus-crypt axis on STa receptor heterogeneity by fractionating EDTA-dissociated cells that detached in a villus-to-crypt direction. STa affinity labeling experiments revealed that the initially released villus "tip" fraction had four major STa binding proteins (STBPs), with relative molecular weight (M(r)) of 150,000, 135,000, 125,000, and 95,000, that did not react with a GC-C carboxy-terminal antibody. Yet succeeding villus cell fractions had major immunoreactive STBPs with M(r) of 275,000 and 250,000. Limited proteolysis of these larger GC-C isoforms produced 1) smaller STBPs that had M(r) similar to those in the initial villus fraction, 2) a 65,000 M(r) protein GC-C isoform that did not bind STa, and 3) elevated basal and STa-induced cyclase activity. Our data show that STBP structural heterogeneity in the intact intestine arises largely from multisite proteolytic processing of GC-C.

Guanylyl cyclase C (GC-C), an intestinal receptor guanylyl cyclase, binds diarrhea-producing bacterial ligands such as the Escherichia coli heat stable enterotoxin. We examined the regulatory influence of feeding and fasting on the expression, structure, and biochemical properties of GC-C. When solubilized at 4 degrees C under nonreducing conditions, GC-C from both fed and fasted rats migrated on 7% sodium dodecyl sulfate-polyacrylamide electrophoretic gels as two extremely large aggregates that barely penetrated the stacking and resolving gels. Chemical reduction of disulfide linkages disaggregated GC-C in fed but not fasted rat samples, causing it to migrate as smaller forms (approximately 220 and 240 kDa). Although GC-C aggregates from fasted rats resisted this disaggregating effect of chemical reduction, they rapidly acquired it within 90 min of refeeding. When solubilized at denaturing temperatures (95 degrees C) under reducing conditions, GC-C aggregates largely disassembled into four smaller proteins (relative molecular weight approximately 140,000, 131,000, 85,000, and 65,000). However, the 131-kDa glycoprotein was disproportionately increased in fasted rat membranes. This unit and the 220-kDa unit were sensitive to endoglycosidase H. Subcellular fractionation and immunohistochemical studies revealed a major redistribution of GC-C from surface to intracellular enterocyte sites during fasting.

Guanylate cyclase C (GC-C) is a transmembrane protein that serves as a receptor for the recently characterized endogenous ligand guanylin and for Escherichia coli heat-stable toxin (STa). Binding of either guanylin or STa to intestinal GC-C results in net chloride secretion. Although GC-C is expressed in the rat intestine throughout life, its expression in the rat liver has previously been shown to occur only during the perinatal period. As a step toward elucidating the role of this receptor in the liver, we tested the hypothesis that GC-C mRNA expression could be induced in the adult rat liver following 1) partial hepatectomy, a stimulus for hepatocyte proliferation; 2) intraperitoneal carbon tetrachloride injection, a model of hepatocyte regeneration in the presence of inflammatory changes; and 3) subcutaneous turpentine injection, which generates an acute phase response without hepatocyte proliferation. We demonstrated expression of GC-C mRNA in the regenerating rat liver following either partial hepatectomy or CCl4-induced hepatic necrosis. We have also shown that GC-C mRNA expression occurred in association with an acute phase reaction. Coordinate with the expression of GC-C mRNA, there was upregulation of radiolabeled STa binding to liver plasma membranes prepared from turpentine-treated rats. Maximal expression of GC-C occurred in preparations enriched for the canalicular domain. Although the function of GC-C in the liver is unknown, localization to the canalicular domain would be consistent with a role for GC-C in hepatic chloride secretion, especially in the perinatal liver and during hepatocyte regeneration.

Enterotoxigenic Escherichia coli elaborate a heat-stable enterotoxin that causes diarrhea in humans and animals. The primary event in the diarrheal cascade is the binding of this enterotoxin to specific receptors on enterocytes and activation of guanylyl cyclase. Two intestinal cell lines, Caco-2 and IEC-6, were tested for the presence of these receptors. Although both cell lines exhibited specific binding, only the Caco-2 cell line responded to heat-stable enterotoxin with increased guanylyl cyclase activity. Cloning and expression studies confirmed that the receptor present in Caco-2 cells is a homologue of guanylyl cyclase C, a known transmembrane heat-stable enterotoxin receptor. Expression of the receptor in differentiating Caco-2 cells increases with cell maturation, indicating that these cells are a suitable model for future studies. However, Northern and polymerase chain reaction analyses demonstrated that guanylyl cyclase C is not expressed in IEC-6 cells, strongly suggesting the presence of a novel heat-stable enterotoxin receptor that is not coupled to guanylyl cyclase activity.

Escherichia coli heat-stable enterotoxin (STa) mediates diarrheal disease by binding to and activating an intestinal transmembrane receptor, guanylate cyclase C (GC-C). To test the hypotheses that there was 1) increased perinatal expression of GC-C in rat intestine and 2) GC-C expression and STa binding in extraintestinal tissues of immature rat, we prepared whole cell membranes and total RNA from jejunum, ileum, colon, liver, kidney, heart, lung, brain, testis, and placenta of rats ranging in age from 12 days gestation to adult. Northern analysis demonstrated the presence of a unique 3.8-kb mRNA transcript at all ages in the jejunum, ileum, colon, and, to a lesser degree, in the testis. GC-C was also detected by Northern analysis in liver (from gestational age 18 days through 14 days postnatal) and in placenta. Steady-state mRNA encoding GC-C was not detected by Northern analysis in the other organs examined. GC-C-specific mRNA expression was greatest in the perinatal period in the jejunum, ileum, and liver. Specific binding of 125I-labeled STa was found in each of the tissue membranes in which GC-C mRNA was present; binding was not present in those tissues that had no detectable GC-C mRNA. The existence of GC-C in extraintestinal organs in the rat, and the development changes in GC-C expression support our hypothesis that GC-C, apart from its role as an STa receptor in mediating diarrheal disease, also serves as a receptor for an endogenous ligand.

Receptors for the heat-stable enterotoxin produced by Escherichia coli were found in the kidney and intestine of the North American opossum and in cultured renal cell lines. The enterotoxin markedly increased guanosine 3',5'-cyclic monophosphate (cGMP) production in slices of kidney cortex and medulla, in suspensions of intestinal mucosa, and in the opossum kidney (OK) and rat kangaroo kidney (PtK-2) cell lines. In contrast, atrial natriuretic factor elicited much smaller increases in cGMP levels of kidney, intestine, or cultured kidney cell lines. The enterotoxin receptors in OK cells had a molecular mass of approximately 120 kDa when measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of receptors crosslinked with 125I-enterotoxin. The occurrence of receptors for the E. coli peptide in OK implies that these receptors may be involved in the regulation of renal tubular function in the opossum. E. coli enterotoxin caused a much larger increase in urine cGMP excretion (10- to 50-fold over control) than did atrial natriuretic factor when these peptides were injected intravenously into opossums. However, atrial natriuretic factor elicited a marked diuresis, natriuresis, and increased urinary excretion of calcium, phosphate, potassium, and magnesium. In contrast, the enterotoxin did not acutely influence OK fluid and electrolyte excretion. Thus the substantial increase in cGMP synthesis produced by the bacterial peptide in OK cortex and medulla in vitro and the increased renal excretion of cGMP in vivo were not associated with changes in electrolyte or water excretion. Whether cGMP represents a second messenger molecule in the kidney is an interesting question that was raised but not answered in this series of experiments.

Escherichia coli heat-stable enterotoxin (STa) induces intestinal secretion by binding to enterocyte receptors and activating the guanylate cyclase-guanosine 3',5'-cyclic monophosphate (cGMP) system. The intermediate steps between binding of STa and secretion are poorly understood, due in part to the lack of a convenient system to study the effects of STa at the cellular level. To establish such a model, we investigated the binding of 125I-STa, STa activation of guanylate cyclase, and STa-induced increase in cGMP production in a well-characterized human colonic cell line, T84. Binding was specific, linear with cell number, and time, temperature and pH dependent, and reversible. ST may also be internalized by these cells. Addition of unlabeled STa competitively inhibited binding of 125I-STa. These parameters closely resemble those described in intact rat enterocytes and cell-free membrane preparations. STa stimulated guanylate cyclase and cGMP production in a dose-related manner. The similar dose-response relationships for binding, guanylate cyclase stimulation by STa, and cGMP production suggest that the guanylate cyclase-cGMP system is coupled to ST occupancy of specific receptors. These data, together with the fact that STa induces chloride secretion from T84 cells suggest that T84 cells are a suitable and convenient system to study the cellular mechanism of action of STa.

The distribution of receptors for Escherichia coli enterotoxin were examined in opossum kidney, intestine, and testis. E. coli enterotoxin stimulated guanosine 3',5'-cyclic monophosphate (cGMP) production in renal cortex, testis, and small intestinal mucosa but had only a small effect in the colon. Atrial natriuretic factor enhanced the cGMP content of renal cortex and small intestine but had no effect on testis or colon. The enterotoxin receptors were observed to be localized in proximal tubules, to epithelial cells of crypts and villi of small intestine, to crypts of colon, and in seminiferous tubules. Both convoluted and straight portions of proximal tubules exhibited specific binding sites for 125I-labeled enterotoxin. Glomeruli and distal tubules did not have receptors. Binding of 125I-enterotoxin to brush-border membranes of kidney cortex or intestinal mucosa and to testis membranes was markedly temperature dependent. The binding affinities of these receptors for E. coli enterotoxin were similar (i.e., IC50 approximately equal to 0.4-0.5 nM). Daily administration of 20 micrograms of enterotoxin intramuscularly to opossums increased urine cGMP excretion with no apparent changes in urine volume, Na+, or K+ excretion. Thus receptors for heat-stable enterotoxins are localized to proximal tubules of kidney and to enterocytes and seminiferous tubules of intestine and testis, respectively. Apical membranes may be the site of enterotoxin receptors in these epithelia.

This study was performed to determine whether receptors for Escherichia coli heat-stable enterotoxin (ST) exist on intestinal epithelial cells. Binding sites for 125I-ST were found on rat jejunal and ileal villus cells. Binding was rapid, reversible, linear with cell number, saturable, and temperature dependent. Significant degradation of 125I-ST occurred when incubated with cells at 37 degrees C but not at 25 degrees C. Binding was specific to ST since binding of 125I-ST was competitively inhibited by increasing concentrations of human or porcine ST but not by E. coli heat-labile, cholera, or staphylococcal enterotoxins. Addition of excess unlabeled ST to cells preincubated with 125I-ST resulted in dissociation of much but not all of the bound 125I-ST. Binding of 125I-ST to jejunal and ileal cells occurs with two affinities, and this is due to the phenomenon of negative cooperativity. The potency of ST for inhibiting the binding of 125I-ST was identical to the potency of ST in stimulating cGMP production. These data support the existence of receptors for ST on intestinal cells, and these receptors may be involved in the action of ST.