RESUMEN
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.
Asunto(s)
GMP Cíclico/orina , Riñón/efectos de los fármacos , Péptidos/farmacología , Serina/análogos & derivados , Cloruro de Sodio/orina , Animales , Línea Celular , Femenino , Glucosa/farmacología , Humanos , Riñón/fisiología , Masculino , Péptidos Natriuréticos , Zarigüeyas , Péptidos/química , Ratas , Ratas Endogámicas WKY , Trometamina/farmacologíaRESUMEN
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.
Asunto(s)
GMP Cíclico/fisiología , Hormonas Gastrointestinales , Guanilato Ciclasa/fisiología , Péptidos/fisiología , Transducción de Señal , Animales , Guanilato Ciclasa/metabolismo , Mucosa Intestinal/metabolismo , Riñón/metabolismo , Ratones , Péptidos Natriuréticos , Zarigüeyas , ARN Mensajero/metabolismo , Ratas , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores de Enterotoxina , Receptores Acoplados a la Guanilato-Ciclasa , Receptores de Péptidos/metabolismoRESUMEN
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.
Asunto(s)
Activadores de Enzimas/farmacología , Hormonas Gastrointestinales , Riñón/efectos de los fármacos , Péptidos/farmacología , Animales , Animales Recién Nacidos , Células Cultivadas , GMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Guanilato Ciclasa/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/efectos de los fármacos , Riñón/fisiología , Masculino , Ratones , Ratones Endogámicos ICR , Ratones Noqueados , Natriuresis/efectos de los fármacos , Péptidos Natriuréticos , Péptidos/fisiología , ARN Mensajero/metabolismo , Receptores de Enterotoxina , Receptores Acoplados a la Guanilato-Ciclasa , Receptores de Péptidos/metabolismo , OrinaRESUMEN
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.
Asunto(s)
Animales , Ratas , Ratones , GMP Cíclico , Péptidos , Transducción de Señal , Guanilato Ciclasa , Intestinos/metabolismo , Riñón/metabolismo , Zarigüeyas , Péptidos/fisiologíaRESUMEN
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.
Asunto(s)
Animales , Masculino , Ratones , Activadores de Enzimas/farmacología , Riñón/efectos de los fármacos , Péptidos/farmacología , GMP Cíclico , Guanilato Ciclasa , Intestinos , Natriuresis/efectos de los fármacos , Péptidos/fisiología , ARN MensajeroRESUMEN
Guanylin and uroguanylin are peptides that bind to and activate guanylate cyclase C and control salt and water transport in many epithelia in vertebrates, mimicking the action of several heat-stable bacteria enterotoxins. In the kidney, both of them have well-documented natriuretic and kaliuretic effects. Since atrial natriuretic peptide (ANP) also has a natriuretic effect mediated by cGMP, experiments were designed in the isolated perfused rat kidney to identify possible synergisms between ANP, guanylin and uroguanylin. Inulin was added to the perfusate and glomerular filtration rate (GFR) was determined at 10-min intervals. Sodium was also determined. Electrolyte dynamics were measured by the clearance formula. Guanylin (0.5 microg/ml, N = 12) or uroguanylin (0.5 microg/ml, N = 9) was added to the system after 30 min of perfusion with ANP (0.1 ng/ml). The data were compared at 30-min intervals to a control (N = 12) perfused with modified Krebs-Hanseleit solution and to experiments using guanylin and uroguanylin at the same dose (0.5 microg/ml). After previous introduction of ANP in the system, guanylin promoted a reduction in fractional sodium transport (%TNa+, P<0.05) (from 78.46 +/- 0.86 to 64.62 +/- 1.92, 120 min). In contrast, ANP blocked uroguanylin-induced increase in urine flow (from 0.21 +/- 0.01 to 0.15 +/- 0.007 ml g-1 min-1, 120 min, P<0.05) and the reduction in fractional sodium transport (from 72.04 +/- 0. 86 to 85.19 +/- 1.48, %TNa+, at 120 min of perfusion, P<0.05). Thus, the synergism between ANP + guanylin and the antagonism between ANP + uroguanylin indicate the existence of different subtypes of receptors mediating the renal actions of guanylins.
Asunto(s)
Factor Natriurético Atrial/metabolismo , Hormonas Gastrointestinales , Riñón/metabolismo , Péptidos/metabolismo , Animales , Sinergismo Farmacológico , Péptidos Natriuréticos , Ratas , Ratas WistarRESUMEN
Guanylin and uroguanylin are peptides that bind to and activate guanylate cyclase C and control salt and water transport in many epithelia in vertebrates, mimicking the action of several heat-stable bacteria enterotoxins. In the kidney, both of them have well-documented natriuretic and kaliuretic effects. Since atrial natriuretic peptide (ANP) also has a natriuretic effect mediated by cGMP, experiments were designed in the isolated perfused rat kidney to identify possible synergisms between ANP, guanylin and uroguanylin. Inulin was added to the perfusate and glomerular filtration rate (GFR) was determined at 10-min intervals. Sodium was also determined. Electrolyte dynamics were measured by the clearance formula. Guanylin (0.5 µg/ml, N = 12) or uroguanylin (0.5 µg/ml, N = 9) was added to the system after 30 min of perfusion with ANP (0.1 ng/ml). The data were compared at 30-min intervals to a control (N = 12) perfused with modified Krebs-Hanseleit solution and to experiments using guanylin and uroguanylin at the same dose (0.5 µg/ml). After previous introduction of ANP in the system, guanylin promoted a reduction in fractional sodium transport (TNa+, P<0.05) (from 78.46 + or - 0.86 to 64.62 = or - 1.92, 120 min). In contrast, ANP blocked uroguanylin-induced increase in urine flow (from 0.21 = or - 0.01 to 0.15 + or - 0.007 ml g-1 min-1, 120 min, P<0.05) and the reduction in fractional sodium transport (from 72.04 + or - 0.86 to 85.19 + or - 1.48, TNa+, at 120 min of perfusion, P<0.05). Thus, the synergism between ANP + guanylin and the antagonism between ANP + uroguanylin indicate the existence of different subtypes of receptors mediating the renal actions of guanylins
Asunto(s)
Ratas , Animales , Factor Natriurético Atrial/metabolismo , Riñón/metabolismo , Péptidos/metabolismo , Sinergismo Farmacológico , Ratas WistarRESUMEN
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.