Integration of neuroscience and endocrinology in hybrid PBL curriculum.

At the University of Missouri-Columbia, the medical school employs a problem-based learning curriculum that began in 1993. Since the curriculum was changed, student performance on step 1 of the United States Medical Licensing Examination has significantly increased from slightly below the national average to almost one-half a standard deviation above the national mean. In the first and second years, classes for students are organized in classes or blocks that are 8 wk long, followed by 1 wk for evaluation. Initially, basic science endocrinology was taught in the fourth block of the first year with immunology and molecular biology. Student and faculty evaluations of the curriculum indicated that endocrinology did not integrate well with the rest of the material taught in that block. To address these issues, basic science endocrinology was moved into another block with neurosciences. We integrate endocrinology with neurosciences by using the hypothalamus and its role in neuroendocrinology as a springboard for endocrinology. This is accomplished by using clinical cases with clear neuroscience and endocrinology aspects such as Cushing's disease and multiple endocrine neoplastic syndrome type 1.

Mechanisms of guanylin action via cyclic GMP in the kidney.

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.

Guanylin peptides: renal actions mediated by cyclic GMP.

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."

Guanylin peptides: cyclic GMP signaling mechanisms.

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.

Guanylin peptides: cyclic GMP signaling mechanisms

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.

Lymphoguanylin: cloning and characterization of a unique member of the guanylin peptide family.

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.

Reversal of the low-affinity neurotrophin receptor stromal-epithelial expression pattern between benign and malignant human prostate.

Reduced expression of the low-affinity p75 neurotrophin receptor (p75(NTR)) occurs in prostate epithelial cells during malignant transformation. Recent studies indicating that the p75(NTR) can transduce signals that induce apoptosis suggest that diminished p75(NTR) in transformed prostate cells may contribute to immortalization. Mutations in the transmembrane domain of the p75(NTR) gene have been associated with decreased p75(NTR) protein expression and may block the ability of the p75(NTR) to induce apoptosis. Therefore, we used Western blot to analyze prostate cancer (PC) cell lines for p75(NTR) protein expression and gene single strand conformation polymorphism (SSCP) analysis and direct DNA sequencing to analyze mutations in the transmembrane domain of the p75(NTR). p75(NTR) Protein was present in all cell lines, and mutations in the p75(NTR) gene were not detected in cDNA derived from any cell line. To define the expression pattern of p75(NTR) in PCs in vivo, we used immunohistochemical techniques to examine tissue specimens from 20 benign, 19 malignant primary, and 14 metastatic prostate specimens. In benign prostate tissues, expression of p75(NTR) was universally detected in basal cells but not in secretory epithelial or stromal cells. In both primary and metastatic PC tissues, p75(NTR) immunoreactivity could not be detected in malignant prostate epithelial cells. However, in contrast to the benign prostate, p75(NTR) protein was expressed in stromal cells surrounding malignant epithelial cells. Stromal p75(NTR) expression was present in 84% (16 of 19) primary and in 86% (12 of 14) metastatic specimens. These data show that in the benign prostate p75(NTR) protein is expressed by basal cells and not stromal cells whereas in malignant prostate p75(NTR) protein is expressed by stromal cells but not prostatic carcinoma cells. Reversal of the p75(NTR) stromal-epithelial pattern of expression between benign and malignant prostate suggests that p75(NTR) may contribute to the development and maintenance of prostate cancer.

Expression and localization of aminopeptidase A, aminopeptidase N, and dipeptidyl peptidase IV in benign and malignant human prostate tissue.

BACKGROUND: Cell-surface peptidases are ectoenzymes which regulate the access of bioactive peptides to their receptors on cell membranes. Abnormalities in their expression and function result in altered peptide activity which contribute to neoplastic transformation and/or progression. METHODS: Expression of aminopeptidase A (APA), aminopeptidase N (APN, CD13), and dipeptidyl peptidase IV (DPP IV, CD26) was immunohistochemically examined in 20 benign and 33 malignant prostate tissues (19 primaries and 14 metastases). RESULTS: Benign prostatic stroma exhibited no APA, APN, or DPP IV immunoreactivity. Stromal cells surrounding prostatic carcinoma cells demonstrated increased APA expression in 24/33 (73%) of tumors. Benign prostatic epithelial cells strongly expressed APN and DPP IV but not APA. In contrast, APN was expressed in > 80% of tumor cells in 5/33 (15%) of specimens, heterogeneously expressed (20-80% of cells positive) in 4/33 (12%) of specimens, and minimally expressed or absent in 24/33 (73%) of tumor specimens, with a similar pattern of expression in primary and metastatic tumors. DPP IV was expressed by > 80% of tumor cells in 18/19 (95%) of primary prostate cancer specimens, but in only 7/14 (50%) of metastases. CONCLUSIONS: These data show that cell-surface peptidases are differentially expressed by normal prostatic stromal and epithelial cells, with increased expression of APA in the stroma surrounding prostate cancer cells, absent APN expression in most tumor cells, and a decreased frequency of DPP IV expression in metastatic tumors. Further studies will elucidate the biological effects of the presence or loss of cell-surface peptidases in the benign and malignant prostate.

Structure and activity of uroguanylin and guanylin from the intestine and urine of rats.

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.

Signaling pathways for guanylin and uroguanylin in the digestive, renal, central nervous, reproductive, and lymphoid systems.

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.

Guanylyl cyclase receptors and guanylin-like peptides in reptilian intestine.

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.

The guanylin and uroguanylin peptide hormones and their receptors.

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.

Opossum colonic mucosa contains uroguanylin and guanylin peptides.

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.

Uroguanylin: cloning of preprouroguanylin cDNA, mRNA expression in the intestine and heart and isolation of uroguanylin and prouroguanylin from plasma.

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.

Distribution of Escherichia coli heat-stable enterotoxin/guanylin/uroguanylin receptors in the avian intestinal tract.

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.

Induction of differentiation and growth arrest associated with nascent (nonoligosomal) DNA fragmentation and reduced c-myc expression in MCF-7 human breast tumor cells after continuous exposure to a sublethal concentration of doxorubicin.

The effects on DNA integrity of continuous (72-h) exposure of human MCF-7 breast adenocarcinoma cells to 50 nM doxorubicin (a concentration which can be maintained in the plasma by continuous infusion) were characterized by bisbenzimide spectrofluorophotometry, cell flow cytometry, agarose gel electrophoresis, and neutral elution. Spectrofluorophotometry and cell flow cytometry indicated the presence of DNA fragmentation, which was maximal at 24 h. Resolution of these fragments on agarose gels failed to demonstrate "laddered" oligosomal profiles. Neutral elution analysis at 24 h indicated that doxorubicin induced fragmentation of nascent, but not mature, double-stranded DNA. Drug-treated cells exhibited endoreduplication and significant shifts in cell cycle distribution, (i.e., increased G0/G1 and G2/M fractions and a markedly reduced S-phase fraction). These alterations occurred without inhibiting the incorporation of [3H]dThd into cellular DNA; in fact, both the rate and magnitude of [3H]dThd incorporation increased progressively. Doxorubicin also produced a sustained decline in c-myc mRNA levels that paralleled both growth arrest and induction of DNA fragmentation. Ultrastructural examination revealed morphological alterations consistent with the induction of differentiation (e.g., increased lipid content and mitochondrial density, appearance of tight junctions, and secretory ducts) and further suggested the possibility of autocatalysis (e.g., lipofuschin-containing vacuoles). A gradual decline in cell number was observed, with loss of approximately 35% of the cell population after 72 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Uroguanylin: structure and activity of a second endogenous peptide that stimulates intestinal guanylate cyclase.

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.

Guanylin stimulation of Cl- secretion in human intestinal T84 cells via cyclic guanosine monophosphate.

Intestinal salt and fluid secretion is stimulated by Escherichia coli heat-stable enterotoxins (ST) through activation of a membrane guanylate cyclase found in the intestine. Guanylin is an endogenous intestinal peptide that has structural similarity to the bacterial peptides. Synthetic preparations of guanylin or E. coli ST 5-17 stimulated Cl- secretion in T84 cells cultured on semipermeable membranes as measured by increases in short circuit current (Isc). The guanylin/ST receptors appeared to be on the apical surface of T84 cells, since addition of guanylin to the apical, but not basolateral, reservoir stimulated Isc. Bumetanide added to the basolateral side effectively inhibited the Isc responses of T84 cells to either guanylin or ST 5-17. Guanylin appeared to be about one-tenth as potent as ST in stimulating transepithelial Cl- secretion. Guanylin and E. coli ST 5-17 both caused massive (> 1,000-fold) increases in cGMP levels in T84 cells, but guanylin was less potent than ST. Both peptides fully inhibited the binding of 125I-ST to receptor sites on intact T84 cells. The radioligand binding data obtained with guanylin or ST 5-17 best fit a model predicting two receptors with different affinity for these ligands. The Ki values for guanylin were 19 +/- 5 nM and 1.3 +/- 0.5 microM, whereas the Ki values for ST 5-17 were 78 +/- 38 pM and 4.9 +/- 1.4 nM. We conclude that guanylin stimulated Cl- secretion via the second messenger, cGMP, in T84 human colon cells. At least two guanylin receptors with different affinities for these ligands may exist in the cultured T84 cells. It may be postulated that guanylin is an endogenous hormone that controls intestinal Cl- secretion by a paracrine mechanism via cGMP and that E. coli ST stimulates Cl- secretion by virtue of an opportunistic mechanism through activation of guanylin receptors.

Stimulation of intestinal Cl- transport by heat-stable enterotoxin: activation of cAMP-dependent protein kinase by cGMP.

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)