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Guanylin (GN) and uroguanylin (UGN) are low-molecular-weight peptide hormones produced mainly in the intestinal mucosa in response to oral salt load. GN and UGN (guanylin peptides) induce secretion of electrolytes and water in both intestine and kidney. Thought to act as "intestinal natriuretic factors", GN and UGN modulate renal salt secretion by both endocrine mechanisms (linking the digestive system and kidney) and paracrine/autocrine (intrarenal) mechanisms. The cellular function of GN and UGN in intestine and proximal tubule is mediated by guanylyl cyclase C (GC-C)-, cGMP-, and G protein-dependent pathways, whereas, in principal cells of the cortical collecting duct (CCD), these peptide hormones act via GC-C-independent signaling through phospholipase A2 (PLA2). The Cl(-)/HCO(-)3 exchanger pendrin (SLC26A4), encoded by the PDS gene, is expressed in non-α intercalated cells of the CCD. Pendrin is essential for CCD bicarbonate secretion and is also involved in NaCl balance and blood pressure regulation. Our recent studies have provided evidence that pendrin-mediated anion exchange in the CCD is regulated at the transcriptional level by UGN. UGN exerts an inhibitory effect on the pendrin gene promoter likely via heat shock factor 1 (HSF1) action at a defined heat shock element (HSE) site. Recent studies have unraveled novel roles for guanylin peptides in several organ systems including involvement in appetite regulation, olfactory function, cell proliferation and differentiation, inflammation, and reproductive function. Both the guanylin system and pendrin have also been implicated in airway function. Future molecular research into the receptors and signal transduction pathways involved in the action of guanylin peptides and the pendrin anion exchanger in the kidney and other organs, and into the links between them, may facilitate discovery of new therapies for hypertension, heart failure, hepatic failure and other fluid retention syndromes, as well as for diverse diseases such as obesity, asthma, and cancer., (© 2014 S. Karger AG, Basel.)

The pendrin/SLC26A4 Cl(-)/HCO(3)(-) exchanger, encoded by the PDS gene, is expressed in cortical collecting duct (CCD) non-A intercalated cells. Pendrin is essential for CCD bicarbonate secretion and is also involved in NaCl balance and blood pressure regulation. The intestinal peptide uroguanylin (UGN) is produced in response to oral salt load and can function as an "intestinal natriuretic hormone." We aimed to investigate whether UGN modulates pendrin activity and to explore the molecular mechanisms responsible for this modulation. Injection of UGN into mice resulted in decreased pendrin mRNA and protein expression in the kidney. UGN decreased endogenous pendrin mRNA levels in HEK293 cells. A 4.2-kb human PDS (hPDS) promoter sequence and consecutive 5' deletion products were cloned into luciferase reporter vectors and transiently transfected into HEK293 cells. Exposure of transfected cells to UGN decreased hPDS promoter activity. This UGN-induced effect on the hPDS promoter occurred within a 52-bp region encompassing a single heat shock element (HSE). The effect of UGN on the promoter was abolished when the HSE located between nt -1119 and -1115 was absent or was mutated. Furthermore, treatment of HEK293 cells with heat shock factor 1 (HSF1) small interfering RNA (siRNA) reversed the UGN-induced decrease in endogenous PDS mRNA level. In conclusion, pendrin-mediated Cl(-)/HCO(3)(-) exchange in the renal tubule may be regulated transcriptionally by the peptide hormone UGN. UGN exerts its inhibitory activity on the hPDS promoter likely via HSF1 action at a defined HSE site. These data define a novel signaling pathway involved in the enterorenal axis controlling electrolyte and water homeostasis.

Pendrin (SLC26A4), a Cl(-)/anion exchanger encoded by the gene PDS, is highly expressed in the kidney, thyroid and inner ear epithelia and is essential for bicarbonate secretion/chloride reabsorption, iodide accumulation and endolymph ion balance, respectively. The molecular mechanisms controlling pendrin activity in renal, thyroid and inner ear epithelia have been the subject of recent studies. The effects of ambient pH, the hormone aldosterone and the peptide uroguanylin (UGN; the "intestinal natriuretic hormone"), known modulators of electrolyte balance, on transcription of the pendrin gene, have been investigated. Luciferase reporter plasmids containing different length fragments of the human PDS (hPDS) promoter were transfected into renal HEK293, thyroid LA2, and inner ear VOT36 epithelial cells. Acidic pH decreased and alkaline pH increased hPDS promoter activity in transfected HEK293 and VOT36, but not in LA2 cells. Aldosterone reduced hPDS promoter activity in HEK293 but had no effect in LA2 and VOT36 cells. These pH and aldosterone-induced effects on the hPDS promoter occurred within 96-bp and 89-bp regions, respectively, which likely contain distinct response elements to these modulators. Injection of UGN into mice resulted in decreased pendrin mRNA and protein expression in the kidney. Exposure of transfected HEK293 to UGN decreased hPDS promoter activity. The findings provided evidence for the presence of a UGN response element within the 96-bp region overlapping with the pH response element on the hPDS promoter. Pendrin is also expressed in airway epithelium. The cytokins interleukin 4 (IL-4) and interleukin-13 (IL-13), known regulators of airway surface function, have been shown to increase hPDS promoter activity by a STAT6-dependent mechanism. In conclusion, systemic pH, the hormone aldosterone, and the peptide UGN influence renal tubular pendrin gene expression and, perhaps, pendrin-mediated Cl(-)/HCO(3)(-) exchange at the transcriptional level. Pendrin-driven anion transport in the endolymph and at the airway surface may be regulated transcriptionally by systemic pH and IL-3/IL-4, respectively. The distinct response elements and the corresponding transcription factors mediating the effect of these modulators on the PDS promoter remain to be identified and characterized., (Copyright © 2011 S. Karger AG, Basel.)

Background: Guanylin (GN) and uroguanylin (UGN) are intestinally derived peptide hormones that are similar in structure and activity to the diarrhea-causing Escherichia coli heat-stable enterotoxins (STa). These secretagogues have been shown to affect fluid, Na+, K+, and Cl- transport in both the intestine and kidney, presumably by intracellular cyclic guanosine monophosphate (cGMP)-dependent signal transduction. However, the in vivo consequences of GN, UGN, and STa on renal function and their mechanism of action have yet to be rigorously tested., Methods: We hypothesized that intravenous administration of GN, UGN, or STa would cause an increase in natriuresis in wild-type mice via cGMP and guanylyl cyclase-C (GC-C, Gucy2c), the only known receptor for these peptide-hormones, and that the peptide-induced natriuresis would be blunted in genetically altered mice devoid of GC-C receptors (GC-C(-/-) null)., Results: In wild-type mice using a modified renal clearance model, GN, UGN, and STa elicited significant natriuresis, kaliuresis, and diuresis as well as increased urinary cGMP levels in a time- and dose-dependent fashion. Absolute and fractional urinary sodium excretion levels were greatest approximately 40 minutes following a bolus infusion with pharmacologic doses of these peptides. Unexpectedly, GC-C(-/-) null mice also responded to the GN peptides similarly to that observed in wild-type mice. Glomerular filtration rate (GFR), blood pressure, and plasma cGMP in the mice (wild-type or GC-C(-/-) null) did not significantly vary between the vehicle- and peptide-treatment groups. The effects of UGN may also influence long-term renal function due to down-regulation of the Na+/K+ ATPase gamma-subunit and the Cl- channel ClC-K2 by 60% and 75%, respectively, as assessed by differential display polymerase chain reaction (PCR) (DD-PCR) and Northern blot analysis of kidney mRNA from mice treated with UGN., Conclusion: GN, UGN, and STa act on the mouse kidney, in part, through a cGMP-dependent, GC-C-independent mechanism, causing significant natriuresis by renal tubular processes. UGN may have further long-term effects on the kidney by altering the expression of such transport-associated proteins as Na+/K+ ATPase and ClC-K2.

Guanylin and uroguanylin are two novel peptides that activate membrane-bound guanylate cyclases found in the kidney and intestine, influencing fluid and electrolyte homeostasis by cyclic GMP. Their natriuretic and kaliuretic activities are well documented. Since guanylin is inactivated by chymotrypsin in vitro, experiments were designed to evaluate the role of chymotrypsin-like proteases in renal metabolism of guanylin. Using the isolated perfused rat kidney, guanylin and a recombinant derivative containing a lysine residue in the N-terminus of the native peptide was tested. There were three experimental groups. In the first group, lys-guanylin (0.1-2.5 microg/ml) was placed into perfusate reservoir. In the second group, chymostatin (6 microg/ml), a chymotrypsin inhibitor, was placed into solution. In the third group, after 30 min. of perfusion with chymostatin (6 microg/ml), guanylin (0.3 microg/ml) was placed into solution. A maximal decrease in fractional Na+ reabsorption (%TNa+) was achieved at 1.0 microg/ml of lys-guanylin (from 73.25+/-2.29 to 54.97+/-0.10, P<0.05). Lys-guanylin (1.0 microg/ml) also decreased fractional K+ reabsorption (%TK+) from 59.26+/-3.93 to 30.75+/-0.78 (P<0.05). Chymostatin had no detectable effects in electrolyte reabsorption in this assay. When introduced after chymostatin, guanylin lowered %TNa+ (from 81.2+/-1.86 to 72.6+/-2.45, P<0.05) and %TK+ (from 69.4+/-4.12 to 65.8+/-2.81, P<0.05). At this subthreshold concentration, guanylin alone lacks effects in %TNa+ or %TK+. Furthermore, the ability of both peptides to promote increases in intestinal fluid secretion was evaluated in the in vivo suckling mouse model. When administered per os, guanylin failed to stimulate intestinal secretion. When chymostatin was present in the test solution, guanylin induced intestinal secretion in this assay. In marked contrast, lys-guanylin alone induced diarrhoea in the suckling mouse. The present paper concludes that guanylin undergoes metabolism in target tissues such as the intestine and kidney and its lysine-containing analogue retains full biological activity.

Guanylin and uroguanylin are newly discovered intestinal peptides that have been shown to affect NaCl transport in both the intestine and kidney. The present study tests the hypothesis that guanylin and uroguanylin mRNA expression in each major region of the intestine is regulated by NaCl intake. Semiquantitative multiplex RT-PCR analysis was used to determine the molecular expression of guanylin and uroguanylin in the duodenum, jejunum, ileum, and colon in rats maintained on low (LS), normal (NS), or high (HS) NaCl intake for 4 days. LS intake reduced the expression of uroguanylin, and to a lesser degree, guanylin mRNA in all intestinal segments compared to NS intake. The duodenum was the site of the greatest decrease for both. In contrast, HS intake significantly increased the expression of guanylin mRNA only in the duodenum and jejunum and had minimal effect on uroguanylin mRNA. The minimum time required for altered gene expression was determined by delivering an oral NaCl challenge directly to the gastrointestinal tract by oro-gastric administration to LS or NS animals. In LS rats, NaCl oro-gastric administration significantly increased mRNA expression of both peptides in all intestinal segments. Furthermore, the increases in guanylin and uroguanylin mRNA were detected within 4 h and plateaued by 8 h. Conversely, acute oro-gastric administration of the same NaCl solution to NS rats caused elevations of guanylin mRNA only in the duodenum and jejunum, and of uroguanylin mRNA only in the ileum and colon. In conclusion, the data demonstrate that variations in NaCl intake lead to intestinal segment-specific changes in guanylin and uroguanylin mRNA expression.

Guanylin and uroguanylin compose a family of natriuretic, diuretic, and kaliuretic peptides that bind to and activate apical membrane receptor guanylyl cyclase signaling molecules in renal and intestinal epithelia. Recently, a complementary DNA encoding an additional member of the guanylin family of cGMP-regulating peptides was isolated from lymphoid tissues of the opossum and was termed lymphoguanylin (LGN). A peptide analog of opossum LGN was synthesized containing a single disulfide bond with the internal cysteine-7 replaced by a serine residue (LGN(Cys7-->Ser7)). The biological activity of LGN(Ser) was tested by using a cGMP bioassay with cultured T84 (human intestinal) cells and opossum kidney (OK) cells. LGN(Ser) has potencies and efficacies for activation of cGMP production in the intestinal and kidney cell lines that are 100- and 1,000-fold higher than LGN, respectively. In the isolated perfused rat kidney, LGN(Ser) stimulated a maximal increase in fractional Na+ excretion from 24.8 +/- 3.0 to 36.3 +/- 3.3% 60 min after administration and enhanced urine flow from 0.15 +/- 0.01 to 0.24 +/- 0.01 ml. g(-1). min(-1). LGN(Ser) (0.69 microM) also increased fractional K+ excretion from 27.3 +/- 2.3 to 38.0 +/- 3.0% and fractional Cl- excretion from 26.1 +/- 0.8 to 43.5 +/- 1.9. A ninefold increase in the urinary excretion of cGMP from 1.00 +/- 0.04 to 9.28 +/- 1.14 pmol/ml was elicited by LGN(Ser), whereas cAMP levels were not changed on peptide administration. These findings demonstrate that LGN(Ser), which contains a single disulfide bond like native LGN, activates guanylyl cyclase-C (GC-C) receptors in T84 and OK cells and may be very helpful in studying the physiological importance of activation of GC-C in vivo. LGN(Ser) also exhibits full activity in the isolated perfused kidney equivalent to that observed previously with opossum uroguanylin, suggesting a physiological role for LGN in renal function. Thus the single amino acid substitution enhances the activity and potency of LGN.

Guanylin (GN) and uroguanylin (UGN) are two recently identified peptides that have been shown to affect water and electrolyte transport in both the intestine and the kidney. Mechanistically, the effects of both peptides are thought to be mediated by intracellular cGMP which results from ligand binding to a plasma membrane guanylyl cyclase-C (GC-C) receptor. To date, the specific intrarenal site(s) of GN and UGN action have not been established. To begin to address this issue, the present studies utilized semi-quantitative RT-PCR to assess the distribution of GC-C mRNA in specific microdissected segments of the rat nephron. GC-C mRNA expression was highest in the cortical collecting tubule, followed by the proximal convoluted tubule, medullary thick ascending limb and collecting tubule, and thin limbs of Henle's loop. Expression levels were significantly lower in all other segments tested, including the glomerulus. The renal tubular expression pattern for cGMP-dependent protein kinase II (cGK-II) mRNA, which is activated in response to GN/UGN-dependent cGMP accumulation, was similar to that for GC-C. Notably, both GN and UGN mRNAs were also expressed along the nephron. The highest levels of expression for both peptides were detected in the medullary collecting tubule. Lower, but comparable levels of GN and UGN expression also occurred in the cortical collecting tubule, cortical and medullary thick ascending limb, and thin limbs of Henles loop. In the proximal convoluted tubule, GN mRNA expression was also quite high, while UGN mRNA was almost undetectable. The presence of renal GC-C and cGK-II in the kidney are consistent with a proposed endocrine function for GN and UGN. In addition however, the present data suggest that intrarenally synthesized GN and UGN may also contribute to the regulation of renal tubular transport.

Uroguanylin is a small-molecular-weight peptide that activates membrane-bound receptor-guanylate cyclases in the intestine, kidney, and other epithelia. Uroguanylin has been shown to participate in the regulation of salt and water homeostasis in mammals via cGMP-mediated processes, bearing a distinct similarity to the action of the atriopeptins, which play a defined role in natriuresis and act as prognostic indicators of severe congestive heart failure (CHF). The objectives of this study were to measure the urinary levels of uroguanylin and the circulating plasma levels of atrial natriuretic peptide (ANP) in healthy individuals (n = 53) and patients with CHF (n = 16). Urinary excretion of uroguanylin was assessed by a cGMP accumulation bioassay employing human T84 intestinal cells. In individuals without CHF, the concentration of uroguanylin bioactivity was 1.31 +/- 0.27 nmol cGMP/ml urine and 1.73 +/- 0.25 micromol cGMP/24-h urine collection. The urinary bioactivity of uroguanylin in males (1.74 +/- 0.55 nmol cGMP/ml urine; n = 27) tended to be higher than the excretion levels in females (0.94 +/- 0.16 nmol cGMP/ml urine; n = 26) over a 24-h period but did not achieve statistical significance. Both male and female groups showed 24-h temporal diurnal variations with the highest uroguanylin levels observed between the hours of 8:00 AM and 2:00 PM. The circulating level of ANP was 12.1 +/- 1.6 pg/ml plasma and did not significantly vary with respect to male/female population or diurnal variation. In patients with CHF, the concentration of plasma ANP and urinary uroguanylin bioactivity increased substantially (7.5-fold and 70-fold, respectively, both P

Uroguanylin and guanylin are newly discovered endogenous heat-stable peptides that bind to and activate a membrane bound guanylyl cyclase signaling receptor (termed guanylyl cyclase C; GC-C). These peptides are not only found in blood but are secreted into the lumen of the intestine and effect a net secretion of electrolytes (Na+, K+, Cl-, HCO3-) and fluid into the intestine via a cyclic guanosine-3', 5'-monophosphate (cGMP) mechanism. GC-C is also the receptor for Escherichia coli heat-stable enterotoxin (STa) and activation by STa results in a diarrheal illness. Employing mouse renal in vivo models, we have demonstrated that uroguanylin, guanylin, and STa elicit natriuretic, kaliuretic, and diuretic effects. These biological responses are time- and dose-dependent. Maximum natriuretic and kaliuretic effects are observed within 30-40 min following infusion with pharmacological doses of the peptides in a sealed-urethra mouse model. Our mouse renal clearance model confirms these results and shows significant natriuresis following a constant infusion of uroguanylin for 30 min, while the glomerular filtration rate, plasma creatinine, urine osmolality, heart rate, and blood pressure remain constant. These data suggest the peptides act through tubular transport mechanisms. Consistent with a tubular mechanism, messenger RNA-differential display PCR of kidney RNA extracted from vehicle- and uroguanylin-treated mice show the message for the Na+/K+ ATPase gamma-subunit is down-regulated. Interestingly, GC-C knockout mice (Gucy2c -/-) also exhibit significant uroguanylin-induced natriuresis and kaliuresis in vivo, suggesting the presence of an alternate receptor signaling mechanism in the kidney. Thus, uroguanylin and guanylin seem to serve as intestinal and renal natriuretic peptide-hormones influencing salt and water transport in the kidney through GC-C dependent and independent pathways. Furthermore, our recent clinical probe study has revealed a 70-fold increase in levels of urinary uroguanylin in patients with congestive heart failure. In conclusion, our studies support the concept that uroguanylin and guanylin are endogenous effector peptides involved in regulating body salt and water homeostasis.

Formyl peptide receptor activation of three mitogen-activated protein kinase (MAPK) cascades, extracellular signal-regulated kinases (ERKs), N-terminal kinases (JNKs), and p38 MAPK was examined in differentiated HL-60 granulocytes. FMLP stimulated a concentration- and time-dependent increase in ERK, JNK, and p38 MAPK activities, all of which were dependent on a pertussis toxin-sensitive G protein. Pharmacologic inhibitors were used to examine the roles of tyrosine kinases, phosphatidylinositol 3-kinase, protein kinase C, and phospholipase C. FMLP-stimulated ERK activity was dependent on tyrosine kinases, phosphatidylinositol 3-kinase, protein kinase C, and phospholipase C; p38 MAPK activation was dependent on phosphatidylinositol 3-kinase and phospholipase C; while JNK activation was independent of all of these signaling components. The mitogen-activated protein kinase/ERK kinase inhibitor PD098059 reduced ERK activation by 90%, while an inhibitor of p38 MAPK, SB203580, inhibited p38 MAPK activation by 80%. Both PD098059 and SB203580 inhibited FMLP-stimulated superoxide release, as did inhibitors directed against protein kinase C, tyrosine kinases, and phosphatidylinositol 3-kinase. We conclude that formyl peptide receptors are coupled to three MAPK cascades by Gi proteins. ERKs, p38 MAPK, and JNKs are each activated by distinct proximal signal transduction pathways. Activation of p38 MAPK is necessary for FMLP stimulation of respiratory burst activity; however, a second signal that may involve ERK is also required for this activity.

Guanylyl cyclase C (GCC) has been detected only in intestinal mucosa and colon carcinoma cells of placental mammals. However, this receptor has been identified in several tissues in marsupials, and its expression has been suggested in tissues other than intestine in placental mammals. Selective expression of GCC by colorectal tumor cells in extraintestinal tissues would permit this receptor to be employed as a selective marker for metastatic disease. Thus, expression of GCC was examined in human tissues and tumors, correlating receptor function with detection by PCR. GCC was detected by ligand binding and catalytic activation in normal intestine and primary and metastatic colorectal tumors, but not in extraintestinal tissues or tumors. Similarly, PCR yielded GCC-specific amplification products with specimens from normal intestine and primary and metastatic colorectal tumors, but not from extraintestinal tissues or tumors. Northern blot analysis employing GCC-specific probes revealed an approximately 4-kb transcript, corresponding to recombinant GCC, in normal intestine and primary and metastatic colorectal tumors, but not in extraintestinal tissues. Thus, GCC is selectively expressed in intestine and colorectal tumors in humans and appears to be a relatively specific marker for metastatic cancer cells in normal tissues. Indeed, PCR of GCC detected tumor cells in blood from some patients with Dukes B colorectal cancer and all patients examined with Dukes C and D colorectal cancer, but not in that from normal subjects or patients with Dukes A colon carcinoma or other nonmalignant intestinal pathologies.

Amifostine, a chemo- and radioprotective agent developed as adjunctive therapy for malignancies, induces hypotension after approximately 20% of patient administrations. This study examines the molecular mechanisms underlying hypotension induced by amifostine. Amifostine and its metabolite, WR-1065, induced dose-dependent hypotension in anesthetized rats that was not blocked by N(G)-methyl L arginine (L-NAME), an NO synthase inhibitor. WR-1065 but not amifostine induced concentration-dependent relaxation of isolated rat aortic rings in an endothelium-independent fashion. Relaxation was not associated with increases in cGMP or cAMP and could not be blocked by L-NAME or indomethacin. Similarly, neither amifostine or WR-1065 activated adenylyl, particulate guanylyl, or soluble guanylyl cyclases. WR-1065 relaxed rat aortic rings precontracted with norepinepherine, suggesting alpha-adrenergic blocking activity. However, neither amifostine nor WR-1065 altered the ability of prazosin or phentolamine to bind to alpha-adrenergic receptors. Further, WR-1065 had no effect on receptor-mediated increases in intracellular calcium in BAL 17 murine B lymphocytes in vitro. Thus, hypotension after administration of amifostine is mediated by WR-1065 and appears to result from direct relaxation of vascular smooth muscle. Smooth muscle relaxation induced by WR-1065 is not related to production of nitric oxide, prostaglandins, or cyclic nucleotides; alpha-adrenergic receptor antagonism; or interference with receptor-dependent increases in intracellular calcium. Administration of ephedrine, an efficacious adrenergic agonist, attenuated hypotension induced by amifostine in anesthetized rats and may be useful in alleviating hypotension associated with amifostine administration in patients.

Purpose: Receptors for Escherichia coli heat-stable toxin (ST) are selectively expressed in membranes of intestinal mucosa cells and colon carcinoma cells in vitro, suggesting their use as a marker for colorectal tumors in vivo. The present studies examined the expression and function of ST receptors in normal human tissues and primary and metastatic colorectal tumors obtained from patients at surgery., Methods: Surgical specimens were obtained as follows: from normal colon; from primary adenocarcinomas from all anatomic divisions of the colon and rectum; from gallbladder, kidney, liver, lung, lymph node, ovary, peritoneum, stomach; and from colon carcinomas metastatic to liver, lung, lymph node, ovary, and peritoneum. Membranes prepared from these specimens were assessed for the presence and functional characteristics of ST receptors., Results: ST bound specifically to membranes from each division of normal colon and rectum and all primary and metastatic colorectal tumors examined. The affinity and density of ST receptors were similar in tumors of different grades and from various metastatic sites. ST-receptor interaction was coupled to activation of guanylyl cyclase in all normal samples of colon and rectum and all primary and metastatic colorectal tumors examined. In contrast, neither ST binding nor ST activation of guanylyl cyclase was detected in any extraintestinal tissues examined., Conclusions: Functional ST receptors are expressed in normal colonic tissue and primary and metastatic colorectal tumors but not by extraintestinal tissues in humans. Expression of ST receptors does not vary as a function of the metastatic site or grade of these tumors. Receptors expressed by colorectal tumors retain their characteristic function, with binding of ST coupled to activation of guanylyl cyclase. These studies support the suggestion that ST receptors represent a specific marker for human colorectal tumors that may have use as a target for directing diagnostics and therapeutics to these tumors in vivo.

Internalization of Escherichia coli heat-stable enterotoxin (ST) mediated by guanylyl cyclase C was examined in T84 human colon carcinoma cells. Surface-associated, receptor-bound ST was quantitatively separated from intracellular ligand employing acidic guanidine-HCl. ST was internalized in a time-, temperature-, and ligand concentration-dependent fashion only by cells specifically expressing guanylyl cyclase C. Only receptors which bound reversibly to ST appeared to mediate endocytosis. The rate of internalization of ST empirically determined in these studies was 0.23 min-1. The density of surface receptors for ST was similar at 4 degrees C and 37 degrees C, suggesting that these receptors recycle back to the cell surface following internalization of ligand. Similarly, internalized ST was rapidly cleared from the intracellular compartment following endocytosis. These studies demonstrate that ST undergoes ligand-dependent receptor-mediated endocytosis in human colon carcinoma cells.

Intestinal cells exhibit binding sites with different affinities for Escherichia coli heat-stable enterotoxin (ST) and guanylin, suggesting the existence of different receptors for these peptides. Guanylyl cyclase C from intestinal cells has been identified as one receptor for these peptides. Equilibrium and kinetic binding characteristics of rat guanylyl cyclase C expressed in COS-7 cells were examined, employing ST, to determine if this receptor exhibited multiple affinities. Scatchard analysis of equilibrium binding yielded curvilinear isotherms consistent with the presence of high (pM) and low (nM) affinity sites. Kinetic analysis of binding demonstrated that these sites exhibited similar dissociation but different association kinetics. In addition, two distinct affinity states of low affinity sites were identified with dissociation constants of 0.15 and 5.85 nM. Association of ST and low affinity sites was biphasic, while dissociation from these sites was unimodal. Close agreement of equilibrium and kinetic dissociation constants suggested that low affinity sites were in the lowest affinity state at equilibrium. Comparison of the ligand dependence of guanylyl cyclase activity (EC50 = 110 nM) with receptor occupancy revealed that binding of ST to the lowest affinity state of low affinity sites (EC50 = 80 nM) is directly coupled to catalytic activation. These studies suggest that binding sites with different affinities for ST exhibited by intestinal cells reflect the expression of a single gene product, guanylyl cyclase C, rather than different receptors for the ligand. The shift in affinity state of low affinity sites and its correlation with catalytic activation suggest a central role for this phenomenon in mechanisms mediating receptor-effector coupling of membrane guanylyl cyclases.

Background/aims: Escherichia coli heat-stable enterotoxins (ST) are small peptides of 18 or 19 amino acids that bind to specific cell surface receptors located on the intestinal brush border and activate guanylate cyclase, resulting in an increase in the intracellular cyclic guanosine 3',5'-monophosphate content of the cell. The present study examined whether receptors for ST are expressed by primary and metastatic human colonic tumors in vivo., Methods: Plasma membranes prepared from surgical tissue samples from normal colon, liver and lung, primary colonic adenocarcinomas, and colon carcinomas metastatic to lung and liver were analyzed for the structural and functional characteristics of constituent ST receptors., Results: All primary and metastatic colonic tumors examined bound ST, showing receptors of high (pmol/L) and low (nmol/L) affinity with densities that were similar to those in normal colon. Also, affinity cross-linking of labeled ST to membranes showed similar binding proteins in primary and metastatic tumors and normal colon. ST binding and affinity-labeled proteins were not detected in normal extraintestinal tissues. Guanylate cyclase was activated by ST in membranes from all colonic tumors studied, with efficacies and potencies that were similar to those in normal colon. ST did not activate this enzyme in normal extraintestinal tissues., Conclusions: Receptors for ST are expressed by primary and metastatic human colonic tumors in vivo, with structural and functional characteristics that are similar to those in normal human colon.

Opposing adenine nucleotide-dependent pathways regulating guanylyl cyclase C (GC-C) in rat intestinal membranes have been identified and characterized. ATP analogues substituted in the 2-position were potent inhibitors of basal and Escherichia coli heat-stable enterotoxin (ST)-stimulated GC-C, independent of the metal cation cofactor present. Inhibition of GC-C was associated with large changes in Vmax but only small changes in the S0.5, suggesting a noncompetitive mechanism. Also, inhibition of GC-C was associated with a concentration-dependent shift from positive to negative cooperativity when manganese served as the cation cofactor. These data support the existence of a noncompetitive allo steric regulatory mechanism mediating adenine nucleotide-dependent inhibition of GC-C. Adenine nucleotides not substituted in the 2-position potentiated the activation of GC-C by ST in intestinal membranes. The potentiating and inhibitory pathways regulating GC-C enzyme activity were separate and distinct. A specific inhibitor (2-chloroadenosine 5'-triphosphate (2ClATP)), was without effect on the potency of a selective activator (adenosine 5'-O-thiomonophosphate (AMPS)) of GC-C. Similarly, AMPS was without effect on the potency of 2ClATP to inhibit GC-C. These data suggest that adenine nucleotide-dependent activation and inhibition are mediated by independent sites which may modulate the second messenger response of GC-C to ST.

Thioether methyltransferase (S-adenosyl-L-methionine: thioether S-methyltransferase; EC 2.1.1.96) catalyzes the methylation of X in compounds of the type R-X-R'(X = S, Se, Te), yielding a methyl onium ion. Previous results using mice have demonstrated a role for thioether methyltransferase in the conversion and clearance of thioethers by methylation to more water-soluble methyl sulfonium ions suitable for excretion in the urine. A potential major physiological source of thiethers is reactions catalyzed by microsomal thiol methyltransferase (S-adenosyl-L-methionine: thiol S-methyltransferase; EC 2.1.1.9), which has been shown to methylate a diverse range of aliphatic sulfhydryl compounds. This study provides evidence for the sequential methylation of the aliphatic thiol, 2-mercaptoethanol, first to the methyl thioether, 2-(methylthio)ethanol, by thiol methyltransferase followed by methylation of this methyl thioeter to the dimethyl sulfonium ion, 2-(-dimethylthio)ethanol, by thioether methyltransferase. This sequence of reactions was demonstrated in vivo by injecting mice i.p. with radioactive 2-mercaptoethanol and analyzing the labeled methylated products, 2-(methylthio)ethanol and 2(dimethylthio)ethanol, in the urine by HPLC. In addition, the system converting 2-mercaptoethanol to 2-(dimethylthio)ethanol was reconstituted in vitro using solubilized mouse liver microsomes as a source of thiol methyltransferase and purified thioether methyltransferase from mouse lung. The results of these in vivo and in vitro studies established the sequential methylation of 2-mercaptoethanol by these two enzymes.