Site-specific effects of dietary salt intake on guanylin and uroguanylin mRNA expression in rat intestine.

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.

Renal effects of serine-7 analog of lymphoguanylin in ex vivo rat kidney.

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.

Guanylyl cyclase-C receptor mRNA distribution along the rat nephron.

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.

Increased urinary excretion of uroguanylin in patients with congestive heart failure.

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

Renal effects of uroguanylin and guanylin in vivo.

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.

Renal effects of uroguanylin and guanylin in vivo

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 g-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 receptors are coupled to multiple mitogen-activated protein kinase cascades by distinct signal transduction pathways: role in activation of reduced nicotinamide adenine dinucleotide oxidase.

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 is a selective marker for metastatic colorectal tumors in human extraintestinal tissues.

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.

Hypotensive mechanisms of amifostine.

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.

Escherichia coli heat-stable enterotoxin receptors. A novel marker for colorectal tumors.

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 E. coli ST mediated by guanylyl cyclase C in T84 human colon carcinoma cells.

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.

Rat guanylyl cyclase C expressed in COS-7 cells exhibits multiple affinities for Escherichia coli heat-stable enterotoxin.

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.

Escherichia coli heat-stable toxin receptors in human colonic tumors.

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 regulate guanylyl cyclase C in rat intestine.

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.

Sequential methylation of 2-mercaptoethanol to the dimethyl sulfonium ion, 2-(dimethylthio)ethanol, in vivo and in vitro.

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.