Leonard R. Forte

Guanylin regulatory peptides: structures, biological activities mediated by cyclic GMP and pathobiology.

The guanylin family of bioactive peptides consists of three endogenous peptides, including guanylin, uroguanylin and lymphoguanylin, and one exogenous peptide toxin produced by enteric bacteria. These small cysteine-rich peptides activate cell-surface receptors, which have intrinsic guanylate cyclase activity, thus modulating cellular function via the intracellular second messenger, cyclic GMP. Membrane guanylate cyclase-C is an intestinal receptor for guanylin and uroguanylin that is responsible for stimulation of Cl- and HCO3- secretion into the intestinal lumen. Guanylin and uroguanylin are produced within the intestinal mucosa to serve in a paracrine mechanism for regulation of intestinal fluid and electrolyte secretion. Enteric bacteria secrete peptide toxin mimics of uroguanylin and guanylin that activate the intestinal receptors in an uncontrolled fashion to produce secretory diarrhea. Opossum kidney guanylate cyclase is a key receptor in the kidney that may be responsible for the diuretic and natriuretic actions of uroguanylin in vivo. Uroguanylin serves in an endocrine axis linking the intestine and kidney where its natriuretic and diuretic actions contribute to the maintenance of Na+ balance following oral ingestion of NaCl. Lymphoguanylin is highly expressed in the kidney and myocardium where this unique peptide may act locally to regulate cyclic GMP levels in target cells. Lymphoguanylin is also produced in cells of the lymphoid-immune system where other physiological functions may be influenced by intracellular cyclic GMP. Observations of nature are providing insights into cellular mechanisms involving guanylin peptides in intestinal diseases such as colon cancer and diarrhea and in chronic renal diseases or cardiac disorders such as congestive heart failure where guanylin and/or uroguanylin levels in the circulation and/or urine are pathologically elevated. Guanylin peptides are clearly involved in the regulation of salt and water homeostasis, but new findings indicate that these novel peptides have diverse physiological roles in addition to those previously documented for control of intestinal and renal function.

Natriuretic and kaliuretic activities of guanylin and uroguanylin in the isolated perfused rat kidney

Guanylin and uroguanylin are novel peptides that activate membrane guanylate cyclases found in the kidney and intestine. We compared the effects of these peptides in the isolated perfused rat kidney. Both peptides are natriuretic and kaliuretic in this preparation. Uroguanylin (0.19-1.9 μM) increased glomerular filtration rate from 0.77 ± 0.07 to 1.34 ± 0.3 ml ⋅ g-1 ⋅ min-1at the highest concentration. A maximal increase in Na+ excretion was achieved at 0.66 μM uroguanylin, with a reduction in fractional Na+ reabsorption from 78.7 ± 1.7 to 58.8 ± 4.4%. The highest dose of uroguanylin increased kaliuresis by 50%. Osmolar clearance doubled at the highest concentration of uroguanylin tested ( P< 0.05). Guanylin also elicited a natriuresis and kaliuresis but appeared to be less potent than uroguanylin. The highest concentration of guanylin (1.3 μM) decreased fractional Na+ reabsorption from 73.9 ± 2.4 to 64.5 ± 4.0%, but lower doses were ineffective. Guanylin stimulated urine K+ excretion at the lowest concentration tested (0.33 μM) without any effect on Na+ excretion. These peptides may influence salt and water homeostasis by biological effects in the kidney that are mediated by the intracellular second messenger, cGMP.Guanylin and uroguanylin are novel peptides that activate membrane guanylate cyclases found in the kidney and intestine. We compared the effects of these peptides in the isolated perfused rat kidney. Both peptides are natriuretic and kaliuretic in this preparation. Uroguanylin (0.19-1.9 microM) increased glomerular filtration rate from 0.77 +/- 0.07 to 1.34 +/- 0.3 ml . g-1 . min-1 at the highest concentration. A maximal increase in Na+ excretion was achieved at 0. 66 microM uroguanylin, with a reduction in fractional Na+ reabsorption from 78.7 +/- 1.7 to 58.8 +/- 4.4%. The highest dose of uroguanylin increased kaliuresis by 50%. Osmolar clearance doubled at the highest concentration of uroguanylin tested (P < 0.05). Guanylin also elicited a natriuresis and kaliuresis but appeared to be less potent than uroguanylin. The highest concentration of guanylin (1.3 microM) decreased fractional Na+ reabsorption from 73. 9 +/- 2.4 to 64.5 +/- 4.0%, but lower doses were ineffective. Guanylin stimulated urine K+ excretion at the lowest concentration tested (0.33 microM) without any effect on Na+ excretion. These peptides may influence salt and water homeostasis by biological effects in the kidney that are mediated by the intracellular second messenger, cGMP.

Regulation of intestinal Cl- and HCO3- secretion by uroguanylin

Uroguanylin is an intestinal peptide hormone that may regulate epithelial ion transport by activating a receptor guanylyl cyclase on the luminal surface of the intestine. In this study, we examined the action of uroguanylin on anion transport in different segments of freshly excised mouse intestine, using voltage-clamped Ussing chambers. Uroguanylin induced larger increases in short-circuit current ( I sc) in proximal duodenum and cecum compared with jejunum, ileum, and distal colon. The acidification of the lumen of the proximal duodenum (pH 5.0-5.5) enhanced the stimulatory action of uroguanylin. In physiological Ringer solution, a significant fraction of the I sc stimulated by uroguanylin was insensitive to bumetanide and dependent on[Formula: see text] in the bathing medium. Experiments using pH-stat titration revealed that uroguanylin stimulates serosal-to-luminal [Formula: see text]secretion ([Formula: see text]) together with a larger increase in I sc. Both[Formula: see text]and I sc were significantly augmented when luminal pH was reduced to pH 5.15. Uroguanylin also stimulated the[Formula: see text]and I sc across the cecum, but luminal acidity caused a generalized decrease in the bioelectric responsiveness to agonist stimulation. In cystic fibrosis transmembrane conductance regulator (CFTR) knockout mice, the duodenal I sc response to uroguanylin was markedly reduced, but not eliminated, despite having a similar density of functional receptors. It was concluded that uroguanylin is most effective in acidic regions of the small intestine, where it stimulates both [Formula: see text] and Cl- secretion primarily via a CFTR-dependent mechanism.

The hemolytic activity of some trialkyltin and triphenyltin compounds

Abstract Triphenyltin chloride caused the rapid hemolysis of dog, hog, rabbit, or rat blood; however, human blood was resistant to hemolysis by triphenyltin chloride. Trialkytin compounds with alkyl groups having 3–6 carbon atoms were hemolytic to both human and rat blood. Tri-n-butyltin derivatives in which the fourth group or atom bonded to tin was acetate, methoxide, hydroxide, oxygen, bromide or chloride were all hemolytic; however, bis(triphenyltin) sulfide was not hemolytic. Butyltins having 1, 2 or 4 carbon-tin bonds, trimethyltin chloride and triethyltin bromide all exhibited low hemolytic activity. It was demonstrated that the rate of hemolysis of human blood by tri-n-butyltin chloride was reduced when the solutes of the medium were predominantly nonelectrolytes and that potassium leaked from the cells faster than hemoglobin during tri-n-butyltin induced hemolysis. A nitrogen atmosphere or the addition of low concentrations of dl -α-tocopherol also reduced the rate of hemolysis of human blood in the presence of tri-n-butyltin chloride. Human plasma, but not human serum albumin, was partially effective in protecting washed, human red cells from tri-n-butyltin induced hemolysis. Sodium sulfide and several sulfhydryl compounds, in concentrations about equal to that of the organotin, protected human and rat blood from the hemolytic activity of tri-n-butyltin chloride, tricyclohexyltin hydroxide, or triphenyltin chloride. It was concluded that the most hemolytic organotins were the trialkyltins with alkyl groups having 3–6 carbon atoms and that some sulfhydryl compounds can react chemically with the organotins to reduce hemolytic activity.

Characterization of the adenyl cyclase of rat kidney plasma membranes

Abstract 1. 1. This paper examines the characteristics of a renal plasma membrane adenyl cyclase with respect to NaF and hormonal activation, enzyme kinetic parameters, effects of solubilization procedures on adenyl cyclase activity, and gel-filtration chromatography of a “soluble” membrane adenyl cyclase. 2. 2. Renal membrane adenyl cyclase was stimulated by NaF, vasopressin and parathyroid hormone in a concentration-dependent manner. 3. 3. A K m for ATP of 0.1 mM was obtained for both the renal cortex and medulla membrane adenyl cyclase whereas the divalent cation dependency was found to be: cortex, Mg 2+ > Mn 2+ > Co 2+ ; medulla, Mg 2+ = Mn 2+ > Co 2+ , for sustaining adenyl cyclase activity in the presence of NaF. 4. 4. The particulate plasma membrane adenyl cyclase was solubilized by use of the detergent, Lubrol WX or by intense shear forces with a French pressure cell. Addition of NaF to the membrane suspension prior to solubilization was necessary for protection of adenyl cyclase activity. Gel-filtration chromatography with 6 % agarose gave two adenyl cyclase peaks of different specific activity and molecular size.

Structure and activity of OK-GC: a kidney receptor guanylate cyclase activated by guanylin peptides

Uroguanylin, guanylin, and lymphoguanylin are small peptides that activate renal and intestinal receptor guanylate cyclases (GC). They are structurally similar to bacterial heat-stable enterotoxins (ST) that cause secretory diarrhea. Uroguanylin, guanylin, and ST elicit natriuresis, kaliuresis, and diuresis by direct actions on kidney GC receptors. A 3,762-bp cDNA characterizing a uroguanylin/guanylin/ST receptor was isolated from opossum kidney (OK) cell RNA/cDNA. This kidney cDNA (OK-GC) encodes a mature protein containing 1,049 residues sharing 72.4-75.8% identity with rat, human, and porcine forms of intestinal GC-C receptors. COS or HEK-293 cells expressing OK-GC receptor protein were activated by uroguanylin, guanylin, or ST13 peptides. The 3.8-kb OK-GC mRNA transcript is most abundant in the kidney cortex and intestinal mucosa, with lower mRNA levels observed in urinary bladder, adrenal gland, and myocardium and with no detectable transcripts in skin or stomach mucosa. We propose that OK-GC receptor GC participates in a renal mechanism of action for uroguanylin and/or guanylin in the physiological regulation of urinary sodium, potassium, and water excretion. This renal tubular receptor GC may be a target for circulating uroguanylin in an endocrine link between the intestine and kidney and/or participate in an intrarenal paracrine mechanism for regulation of kidney function via the intracellular second messenger, cGMP.Uroguanylin, guanylin, and lymphoguanylin are small peptides that activate renal and intestinal receptor guanylate cyclases (GC). They are structurally similar to bacterial heat-stable enterotoxins (ST) that cause secretory diarrhea. Uroguanylin, guanylin, and ST elicit natriuresis, kaliuresis, and diuresis by direct actions on kidney GC receptors. A 3,762-bp cDNA characterizing a uroguanylin/guanylin/ST receptor was isolated from opossum kidney (OK) cell RNA/cDNA. This kidney cDNA (OK-GC) encodes a mature protein containing 1,049 residues sharing 72.4-75.8% identity with rat, human, and porcine forms of intestinal GC-C receptors. COS or HEK-293 cells expressing OK-GC receptor protein were activated by uroguanylin, guanylin, or ST13 peptides. The 3.8-kb OK-GC mRNA transcript is most abundant in the kidney cortex and intestinal mucosa, with lower mRNA levels observed in urinary bladder, adrenal gland, and myocardium and with no detectable transcripts in skin or stomach mucosa. We propose that OK-GC receptor GC participates in a renal mechanism of action for uroguanylin and/or guanylin in the physiological regulation of urinary sodium, potassium, and water excretion. This renal tubular receptor GC may be a target for circulating uroguanylin in an endocrine link between the intestine and kidney and/or participate in an intrarenal paracrine mechanism for regulation of kidney function via the intracellular second messenger, cGMP.

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.

Alpha-2 adrenergic activation inhibits forskolin-stimulated adenylate cyclase activity and lipolysis in human adipocytes

Forskolin at 10 muM caused a 100-fold increase in the intracellular concentration of cyclic AMP and a 6-fold increase in glycerol release in the human adipocyte. These responses are comparable to those prompted by 10 muM isoproterenol. The effects of forskolin on cyclic AMP and lipolysis were dose-dependent. Alpha-2 adrenergic activation, achieved with 10 muM epinephrine and 30 muM propranolol, significantly inhibited forskolin-stimulated cyclic AMP accumulation and glycerol release, shifting the dose-response curves to the right. Forskolin at 10 muM caused a 4.5-fold increase in the adenylate cyclase activity of human adipocyte membranes. When either isoproterenol or epinephrine (0.1 mM) was combined with forskolin, the magnitude of response was substantially greater than the sum of responses achieved by each agent incubated alone.

Opossum kidney contains a functional receptor for the Escherichia coli heat-stable enterotoxin

The Escherichia coli heat-stable enterotoxin (ST1 or STa) binds to specific receptors on mammalian intestinal brush border membranes, and stimulates guanylate cyclase in those membranes. We have found a similar signal transduction system in brush border membranes prepared from kidney cortex of the American opossum (Didelphis virginiana, and in a cell line (OK cell) derived from that tissue. Activation of guanylate cyclase by ST1 is therefore not limited to intestinal cells. Furthermore, since it is unlikely that ST1 which is produced in the intestinal lumen, would have access to kidney receptors, this suggests the existence of an endogenous peptide resembling ST1, at least in marsupials.

Effect of mineralocorticoid agonists and antagonists on binding of 3H-aldosterone to adrenalectomized rat kidney plasma membranes

Abstract Binding of 3H-aldosterone to plasma membranes of adrenalectomized rat kidney has been demonstrated in vivo and in vitro . Steroids with mineralocorticoid activity, deoxycorticosterone or 9-α-fluorohydrocortisone (agonists), or aldosterone antagonists (spironolactone, progesterone) markedly reduced the binding of 3H-aldosterone to renal plasma membranes. Steroids without agonist or antagonist properties (testosterone, 17-β-estradiol) were not effective. The 3H-aldosterone-membrane complex was resistent to solubilization and subsequent chromatography employing Sephadex G-25. These experiments indicate that binding of 3H-aldosterone to adrenalectomized rat kidney plasma membranes may be involved in the cellular mode of action of aldosterone.