RESUMO
A gastrointestinal-renal natriuretic signaling axis has been proposed to regulate sodium excretion in response to acute sodium ingestion. Such an axis is thought to be regulated by a gastrointestinal sodium sensor coupled to the activation/release of a natriuretic signal and could have important clinical and scientific implications. Here we systematically tested for this putative axis and the potential involvement of the gastrointestinal-derived natriuretic prohormones prouroguanylin and proguanylin in 15 healthy volunteers. There was no difference in sodium excretion following equivalent oral or intravenous sodium loads during either high- or low-sodium diets. Furthermore, serum concentrations of prouroguanylin and proguanylin did not increase, did not differ following oral or intravenous sodium, and did not correlate with sodium excretion. Thus, our results do not support an acute gastrointestinal-renal natriuretic axis or a central role for prouroguanylin or proguanylin in humans. If such an axis does exist, it is not characterized by a significant difference in the pattern of sodium excretion following either an oral or intravenous sodium load.
Assuntos
Trato Gastrointestinal/metabolismo , Rim/metabolismo , Natriurese , Transdução de Sinais , Cloreto de Sódio/sangue , Administração Oral , Adulto , Aldosterona/sangue , Pressão Sanguínea , Creatinina/sangue , Dieta Hipossódica , Feminino , Florida , Hormônios Gastrointestinais/sangue , Humanos , Infusões Intravenosas , Masculino , Precursores de Proteínas/sangue , Cloreto de Sódio/administração & dosagem , Cloreto de Sódio na Dieta/sangue , Comprimidos , Fatores de Tempo , Adulto JovemRESUMO
Analogues of the E. coli heat-stable enterotoxin (STh) are currently under study as both imaging and therapeutic agents for colorectal cancer. Studies have shown that the guanylate cyclase C (GC-C) receptor is commonly expressed in colorectal cancers. It has also been shown that STh peptides inhibit the growth of tumor cells expressing GC-C. The ability to determine GC-C status of tumor tissue using in vivo molecular imaging techniques would provide a useful tool for the optimization of GC-C-targeted therapeutics. In this work, we have compared receptor binding affinities, internalization/efflux rates, and in vivo biodistribution patterns of an STh analogue linked to N-terminal DOTA, TETA, and NOTA chelating moieties and radiolabeled with Cu-64. The peptide F(19)-STh(2-19) was N-terminally labeled with three different chelating groups via NHS ester activation and characterized by RP-HPLC, ESI-MS, and GC-C receptor binding assays. The purified conjugates were radiolabeled with Cu-64 and used for in vitro internalization/efflux, in vivo biodistribution, and in vivo PET imaging studies. In vivo experiments were carried out using SCID mice bearing T84 human colorectal cancer tumor xenografts. Incorporation of DOTA-, TETA-, and NOTA-chelators at the N-terminus of the peptide F(19)-STh(2-19) resulted in IC(50)s between 1.2 and 3.2 nM. In vivo, tumor localization was similar for all three compounds, with 1.2-1.3%ID/g at 1 h pi and 0.58-0.83%ID/g at 4 h pi. The principal difference between the three compounds related to uptake in nontarget tissues, principally kidney and liver. At 1 h pi, (64)Cu-NOTA-F(19)-STh(2-19) demonstrated significantly (p < 0.05) lower uptake in liver than (64)Cu-DOTA-F(19)-STh(2-19) (0.36 +/- 0.13 vs 1.21 +/- 0.65%ID/g) and significantly (p < 0.05) lower uptake in kidney than (64)Cu-TETA-F(19)-STh(2-19) (3.67 +/- 1.60 vs 11.36 +/- 2.85%ID/g). Use of the NOTA chelator for coordination of Cu-64 in the context of E. coli heat-stable enterotoxin analogues results in higher tumor/nontarget tissue ratios at 1 h pi than either DOTA or TETA macrocycles. Heat-stable enterotoxin-based radiopharmaceuticals such as these provide a means of noninvasively determining GC-C receptor status in colorectal cancers by PET.
Assuntos
Toxinas Bacterianas/química , Neoplasias Colorretais/diagnóstico , Radioisótopos de Cobre/química , Enterotoxinas/química , Tomografia por Emissão de Pósitrons/métodos , Animais , Proteínas de Escherichia coli , Feminino , Humanos , Camundongos , Camundongos Endogâmicos ICR , Camundongos SCID , Transplante de Neoplasias , Coloração e RotulagemRESUMO
BACKGROUND: Uroguanylin is an endogenous peptide agonist that binds to the guanylate cyclase C receptor (GC-C). GC-C is overexpressed in human colorectal cancer (CRC), and exposure of GC-C-expressing cells to GC-C agonists results in cell cycle arrest and/or apoptosis, highlighting the therapeutic potential of such compounds. This study describes the first use of radiolabeled uroguanylin analogs for in vivo detection of CRC. MATERIALS AND METHODS: The peptides uroguanylin and E(3)-uroguanylin were N-terminally labeled with the DOTA chelating group via NHS ester activation and characterized by RP-HPLC, ESI-MS, and GC-C receptor binding assays. The purified conjugates were radiolabeled with In-111 and used for in vivo biodistribution and SPECT imaging studies. In vivo experiments were carried out using SCID mice bearing T84 human colorectal cancer tumor xenografts. RESULTS: Alteration of the position 3 aspartate residue to glutamate resulted in increased affinity for GC-C, with IC(50) values of 5.0+/-0.3 and 9.6+/-2.9 nM for E(3)-uroguanylin and DOTA-E(3)-uroguanylin, respectively. In vivo, (111)In-DOTA-E(3)-uroguanylin demonstrated tumor uptake of 1.17+/-0.23 and 0.61+/-0.07% ID/g at 1 and 4 h post injection, respectively. The specificity of tumor localization was demonstrated by coinjection of 3 mg/kg unlabeled E(3)-uroguanylin, which reduced tumor uptake by 69%. Uptake in kidney, however, was dramatically higher for the uroguanylin peptides than for previously characterized radiolabeled E. coli heat-stable enterotoxin (STh) analogs targeting GC-C, and was also inhibited by coinjection of unlabeled peptide in a fashion not previously observed. CONCLUSION: Use of uroguanylin-targeting vectors for in vivo imaging of colorectal cancers expressing GC-C resulted in tumor uptake that paralleled that of higher affinity heat-stable enterotoxin peptides, but also resulted in increased kidney uptake in vivo.
Assuntos
Neoplasias Colorretais/diagnóstico por imagem , Radioisótopos de Índio , Peptídeos Natriuréticos , Compostos Radiofarmacêuticos , Sequência de Aminoácidos , Animais , Neoplasias Colorretais/metabolismo , Feminino , Compostos Heterocíclicos com 1 Anel/química , Compostos Heterocíclicos com 1 Anel/farmacocinética , Humanos , Radioisótopos de Índio/química , Radioisótopos de Índio/farmacocinética , Camundongos , Camundongos Endogâmicos ICR , Camundongos SCID , Dados de Sequência Molecular , Peptídeos Natriuréticos/química , Peptídeos Natriuréticos/farmacocinética , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/farmacocinética , Distribuição Tecidual , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Transplante HeterólogoRESUMO
BACKGROUND: Radiolabeled analogs of the E. coli heat-stable enterotoxin (ST(h)) are currently under study as imaging and therapeutic agents for colorectal cancer. The aim of these studies is to compare in vitro and in vivo characteristics of two novel ST(h) analogs with appended DOTA chelating moieties. MATERIALS AND METHODS: ST(h) analogs were synthesized with pendant N-terminal DOTA moieties and radiolabeled with indium-111. In vitro cell binding was studied using cultured T-84 human colorectal cancer cells, and in vivo biodistribution studies were carried out using T-84 human colorectal tumor xenografts in SCID mice. RESULTS: Competitive radioligand binding assays employing T-84 human colon cancer cells demonstrated similar IC50 values for the F19-ST(h)(2-19) and F9-ST(h)(6-19) analogs. Addition of DOTA to the N-terminus of these peptides elicited distinctly different effects on binding affinities in vitro, effects that were largely unchanged by metallation with nonradioactive (nat)In. In vivo pharmacokinetic studies in SCID mice bearing T-84 human colon cancer-derived tumor xenografts demonstrated tumor uptake of 0.74 +/- 0.1% ID/g at 4 h post-injection (p.i.) for the 111In-DOTA-F19-ST(h)(2-19) analog, and significantly reduced tumor localization (0.27 + 0.08 % ID/g) for the 111In-DOTA-F9-ST(h)(6-19) analog. CONCLUSION: These results demonstrate that placement of a DOTA moiety immediately adjacent to Cys 6 in ST(h) significantly inhibits receptor binding in vitro and in vivo, highlighting the need for intervening spacer residues between the pharmacophore and the DOTA chelating moiety in effective ST(h)-based radiopharmaceutical constructs.
Assuntos
Toxinas Bacterianas/farmacocinética , Neoplasias do Colo/tratamento farmacológico , Enterotoxinas/farmacocinética , Proteínas de Escherichia coli/farmacocinética , Temperatura Alta , Animais , Toxinas Bacterianas/uso terapêutico , Ligação Competitiva , Neoplasias do Colo/diagnóstico por imagem , Neoplasias do Colo/metabolismo , Enterotoxinas/química , Enterotoxinas/uso terapêutico , Proteínas de Escherichia coli/uso terapêutico , Feminino , Compostos Heterocíclicos com 1 Anel , Humanos , Radioisótopos de Índio/farmacocinética , Radioisótopos de Índio/uso terapêutico , Ligantes , Camundongos , Camundongos Endogâmicos ICR , Camundongos SCID , Ligação Proteica , Desnaturação Proteica , Cintilografia , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Compostos Radiofarmacêuticos/uso terapêutico , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
Escherichia coli heat-stable enterotoxin (STa), guanylin and uroguanylin are novel natriuretic and kaliuretic peptides that bind to and activate membrane guanylate cyclase (GC) receptors such as GC-C and OK-GC that are expressed in the kidney and intestine. Atrial natriuretic peptide (ANP) and its renal form (urodilatin, UROD) elicit natriuretic effects by activation of a different membrane guanylate cyclase, GC-A. Experiments were done in perfused rat kidneys to search for possible synergistic interactions between ANP, UROD, guanylin and uroguanylin on renal function. Pretreatment with ANP (0.03 nM) enhanced guanylin (0.19 microM) natriuretic activity (%ENa(+); from 18.5+/-4.25 to 31.5+/-1.69, P<0.05, 120 min) and its kaliuretic activity (%EK(+); from 24.5+/-4.43 to 50.6+/-3.84, P<0.05, 120 min). Furthermore, ANP increased the natriuretic (29.05+/-3.00 to 37.8+/-2.95, P<0.05, 120 min) and kaliuretic (from 33.2+/-3.52 to 42.83+/-2.45, P<0.05, 120 min) responses of perfused kidneys treated with low-dose (0.06 microM) uroguanylin. In contrast, ANP clearly inhibited the uroguanylin-induced (0.31 microM) increase in %ENa(+) (from 35.9+/-2.37 to 14.8+/-1.93, P<0.05, 120 min), and in %EK(+) (from 51.0+/-4.43 to 38.8+/-3.61, P<0.05, 120 min). UROD (0.03 nM) also enhanced the guanylin-induced natriuresis (to %ENa(+)=31.0+/-1.93, P<0.05, 120 min) and kaliuresis (to %EK(+)=54.2+/-3.61, P<0.05, 120 min), and inhibited the %ENa(+) of uroguanylin (0.31 microM) to 17.9+/-1.67 as well as its %EK(+) to 24.3+/-3.13 (both at 120 min, P<0.05). The synergism between ANP and UROD with either guanylin or uroguanylin at sub-threshold doses and the unexpected antagonism between ANP and UROD with uroguanylin at a pharmacological dose point to possible interactions between natriuretic peptide receptor (NPR) and uroguanylin/guanylin receptor signaling pathways. The interactions herein described may play a contributory role in the regulation of kidney function in many pathophysiological states, such as in the saliuresis following ingestion of salty meals.
Assuntos
Fator Natriurético Atrial/metabolismo , Hormônios Gastrointestinais/metabolismo , Rim/metabolismo , Peptídeos Natriuréticos/metabolismo , Animais , GMP Cíclico/metabolismo , Taxa de Filtração Glomerular , Guanilato Ciclase/metabolismo , Masculino , Gambás , Fragmentos de Peptídeos/metabolismo , Peptídeos/química , Perfusão , Ratos , Ratos Wistar , Fatores de TempoRESUMO
The human E. coli heat-stable enterotoxin (ST(h), amino acid sequence N1SSNYCCELCCNPACTGCY19) binds specifically to the guanylate cyclase C (GC-C) receptor, which is present in high density on the apical surface of normal intestinal epithelial cells as well as on the surface of human colon cancer cells. Analogs of ST(h) are currently being used as vectors targeting human colon cancers. Previous studies in our laboratory have focused on development of 111Indium-labeled ST(h) analogs for in vivo imaging applications. Here, we extend the scope of this work to include targeting of the therapeutic radionuclides 90Y and 177Lu. The peptide DOTA-F19-ST(h)(1-19) was synthesized using conventional Fmoc-based solid-phase techniques and refolded in dilute aqueous solution. The peptide was purified by RP-HPLC and characterized by MALDI-TOF MS and in vitro receptor binding assay. The DOTA-conjugate was metallated with nonradioactive Lu(III)Cl3 and Y(III)Cl3, and IC50 values of 2.6+/-0.1 and 4.2+/-0.9 nM were determined for the Lu- and Y-labeled peptides, respectively. 177Lu(III)Cl3 and 90Y(III)Cl3 labeling yielded tracer preparations that were inseparable by C18 RP-HPLC, indicating that putative differences between Lu-, Y- and In coordination spheres are not observed in the context of labeled ST(h) peptides. In vivo biodistribution studies of the 177Lu-labeled peptide in severe combined immunodeficient (SCID) mice bearing T-84 human cancer tumor xenografts showed rapid clearance from the bloodstream, with >90 %ID in the urine at 1 h pi. Localization of the tracer within tumor xenografts was 1.86+/-0.91 %ID/g at 1 h pi, a value higher than for all other tissues with the exception of kidney (2.74+/-0.24 %ID/g). At 24 h pi, >98 %ID was excreted into the urine, and 0.35+/-0.23 %ID/g remained in tumor, again higher than in all other tissues except kidney (0.91+/-0.46 %ID/g). Biodistribution results at 24 h pi for the 90Y-labeled peptide mirrored those for the 177Lu analog, in agreement with the identical behavior of the labeled analogs by C18 RP-HPLC. These results demonstrate the ability of 177Lu- and 90Y-labeled ST(h) molecules to specifically target GC-C receptors expressed on T-84 human colon cancer cells.
Assuntos
Toxinas Bacterianas/farmacocinética , Neoplasias do Colo/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Escherichia coli , Lutécio/farmacocinética , Peptídeos Natriuréticos/metabolismo , Radioisótopos de Ítrio/farmacocinética , Animais , Toxinas Bacterianas/química , Toxinas Bacterianas/uso terapêutico , Linhagem Celular Tumoral , Neoplasias do Colo/diagnóstico por imagem , Neoplasias do Colo/radioterapia , Avaliação Pré-Clínica de Medicamentos , Estabilidade de Medicamentos , Feminino , Temperatura Alta , Humanos , Marcação por Isótopo/métodos , Lutécio/química , Lutécio/uso terapêutico , Taxa de Depuração Metabólica , Camundongos , Camundongos Endogâmicos ICR , Radioisótopos/química , Radioisótopos/farmacocinética , Radioisótopos/uso terapêutico , Cintilografia , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/farmacocinética , Compostos Radiofarmacêuticos/uso terapêutico , Receptores de Superfície Celular/metabolismo , Distribuição Tecidual , Radioisótopos de Ítrio/química , Radioisótopos de Ítrio/uso terapêuticoRESUMO
Research into the interaction between the E. coli heat-stable enterotoxin (STh) and the guanylin receptor guanylate cyclase C (GC-C) has generated >100 synthetic analogs of the peptide, several of which have been investigated as imaging or therapeutic agents for colorectal cancers. The evidence presented here suggests that in addition to STh binding to GC-C expressing cell lines derived from human colon, STh also specifically binds to an as yet unidentified receptor expressed in high densities on the surface of cell lines derived from human breast cancers. In vitro whole-cell crosslinking studies using 125I-labeled F19-STh(1-19) demonstrate that the putative STh binding protein migrates as an approximately 120-125 kDa species by SDS-PAGE, significantly smaller than the glycosylated GC-C molecule found in the T84 human colon cancer cell line. RT-PCR using total RNA isolated from breast and colon cancer cell lines indicates that GC-C transcripts are undetectable in human breast cancer cell lines and abundant in human colon cancer cell lines. In vitro competitive binding studies using STh analogs and the estrogen receptor positive (ER+) T-47D cell line demonstrated IC50 values between 2.6 and 8.5 nM. Similar studies on the estrogen receptor negative (ER-) cell line MDA-MB-231 showed IC50's between 5.6 and 9.9 nM. Saturation binding analysis revealed receptor expression to fall between 40,000 and 120,000 sites per cell in these cell lines, receptor abundances equal to or greater than the abundance of GC-C in colorectal cancer cell lines. STh binding to these cells, although of similar affinity to STh binding to GC-C, is distinguishable from it on the basis of its ligand specificity. The characteristics of STh analogs as radiopharmaceutical agents were tested in an in vivo model utilizing T-47D human breast cancer cell xenografts in SCID mice. Clearance of STh analogs was rapid, primarily via renal excretion into the urine, with >85% ID excreted into the urine at 1 h p.i. Tumor uptake at 1 h p.i. in T-47D tumor cell xenografts was 0.67+/-0.23% ID/g, and was significantly decreased (p<0.05) upon co-administration of 4 mg/kg unlabeled STh. These results suggest that STh may find application for the imaging and treatment of breast cancer.
Assuntos
Neoplasias da Mama/terapia , Ensaios de Seleção de Medicamentos Antitumorais , Enterotoxinas/uso terapêutico , Escherichia coli/metabolismo , Radioisótopos de Índio/farmacologia , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Reagentes de Ligações Cruzadas/farmacologia , Feminino , Humanos , Técnicas In Vitro , Concentração Inibidora 50 , Camundongos , Camundongos SCID , Transplante de Neoplasias , Ligação ProteicaRESUMO
In February 2003, a pool of 6 Culex pipiens mosquitoes collected in Lehigh County, PA, tested positive for West Nile Virus (WNV). West Nile viral RNA was detected by reverse transcriptase-polymerase chain reaction. This is the first time in Pennsylvania and the third time in North America that WNV has been found in overwintering adult mosquitoes.
Assuntos
Culex/virologia , Vírus do Nilo Ocidental/fisiologia , Animais , Pennsylvania , Reação em Cadeia da Polimerase , Estações do AnoRESUMO
Three human Escherichia coli heat-stable peptide (STh) analogues, each containing a DOTA chelating group, were synthesized by SPPS and oxidative refolding and compared in in vitro and in vivo systems. One analogue, DOTA-F19-STh(1-19), contains an N-terminal DOTA group attached via an amide bond linkage to an STh moiety which is essentially wild-type except for a Tyr to Phe alteration at position 19 of the molecule. A second analogue, DOTA-R1,4,F19-STh(1-19), differs from the first in that asparagine residues in positions 1 and 4 have been altered to arginine residues in order to examine the effect of positively charged groups in the linker domain. A third analogue, DOTA-11AUN-F19-STh(1-19), differs from the first in that it incorporates an 11-aminoundecanoic acid spacer group between the DOTA group and the first asparagine residue. In vitro competitive binding assays utilizing T-84 human colon cancer cells demonstrated that significant alterations to the N-terminal region of the STh molecule were well tolerated and did not significantly affect binding affinity of STh for the guanylyl cyclase C (GC-C) receptor. Internalization and efflux studies of the indium-labeled species demonstrated that inclusion of positive charge in the linker moiety inhibits internalization of the compound within tumor cells. The characteristics of the three analogues were compared in an in vivo model utilizing T-84 human colon cancer cell xenografts in SCID mice. Clearance of all analogues was rapid, primarily via renal excretion into the urine, with >89% ID excreted into the urine at 1 h pi for all analogues. The 111In-DOTA-R1,4,F19-STh(1-19) and 111In-DOTA-11AUN-F19-STh(1-19) analogues both had longer residence times in the blood than did the 111In-DOTA-F19-STh(1-19) analogue, probably accounting for increased %ID/g values for tumors and nontarget tissues at 1 h pi. At 4 h pi, significant differences between analogues were only seen with respect to metabolic routes of excretion, indicating that increased blood residence time did not result in increased tumor residualization. Reduction of hepatic uptake of these compounds, however, could have significance in the development of agents for the imaging of hepatic metastases. The ability to manipulate in vivo pharmacodynamics and tumor uptake of radiolabeled STh peptides through modification of linker moieties is under continuing investigation in order to produce optimal imaging and therapeutic radiopharmaceuticals.
Assuntos
Quelantes/química , Proteínas de Escherichia coli/química , Temperatura Alta , Animais , Linhagem Celular Tumoral , Quelantes/metabolismo , Quelantes/farmacocinética , Cromatografia Líquida de Alta Pressão , Proteínas de Escherichia coli/administração & dosagem , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/farmacocinética , Feminino , Humanos , Radioisótopos de Índio , Concentração Inibidora 50 , Camundongos , Estrutura Molecular , Peso Molecular , Transplante de Neoplasias , Ligação Proteica , Desnaturação Proteica , Renaturação Proteica , Distribuição TecidualRESUMO
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.
Assuntos
Hormônios Gastrointestinais/farmacologia , Guanilato Ciclase/genética , Natriurese/efeitos dos fármacos , Natriurese/fisiologia , Peptídeos/farmacologia , Receptores de Peptídeos/genética , Animais , Animais Lactentes , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/farmacologia , Northern Blotting , Enterotoxinas/metabolismo , Enterotoxinas/farmacologia , Proteínas de Escherichia coli , Hormônios Gastrointestinais/metabolismo , Guanilato Ciclase/metabolismo , Injeções Intravenosas , Camundongos , Camundongos Endogâmicos , Camundongos Mutantes , Peptídeos Natriuréticos , Peptídeos/metabolismo , RNA Mensageiro/análise , Receptores de Enterotoxina , Receptores Acoplados a Guanilato Ciclase , Receptores de Peptídeos/metabolismoRESUMO
Uroguanylin is a peptide hormone that regulates sodium excretion by the kidney when excess NaCl is consumed. A new study demonstrates that mice deficient in uroguanylin have blunted urinary sodium excretion responses to oral sodium loads in addition to elevated blood pressure (see related article beginning on page 1244). A physiological role for uroguanylin is discussed, linking the intestine and kidney in an endocrine axis for the maintenance of sodium balance.
Assuntos
Peptídeos/fisiologia , Sódio/metabolismo , Sequência de Aminoácidos , Animais , Modelos Animais de Doenças , Hormônios Gastrointestinais/química , Hormônios Gastrointestinais/fisiologia , Humanos , Camundongos , Dados de Sequência Molecular , Peptídeos Natriuréticos , Peptídeos/química , Equilíbrio HidroeletrolíticoRESUMO
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.
Assuntos
Diuréticos/farmacologia , Hormônios Gastrointestinais/farmacologia , Rim/efeitos dos fármacos , Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Animais Lactentes , Diuréticos/síntese química , Diuréticos/farmacocinética , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Hormônios Gastrointestinais/síntese química , Hormônios Gastrointestinais/farmacocinética , Técnicas In Vitro , Rim/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Dados de Sequência Molecular , Peptídeos Natriuréticos , Oligopeptídeos/farmacologia , Peptídeos/síntese química , Peptídeos/farmacocinética , Perfusão , Ratos , Ratos Endogâmicos WKY , Fatores de TempoRESUMO
Guanylyl cyclase C (GC-C) is a membrane-associated form of guanylyl cyclase and serves as the receptor for the heat-stable enterotoxin (ST) peptide and endogenous ligands guanylin, uroguanylin, and lymphoguanylin. The major site of expression of GC-C is the intestinal epithelial cell, although GC-C is also expressed in extraintestinal tissue such as the kidney, airway epithelium, perinatal liver, stomach, brain, and adrenal glands. Binding of ligands to GC-C leads to accumulation of intracellular cGMP, the activation of protein kinases G and A, and phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel that regulates salt and water secretion. We examined the expression of GC-C and its ligands in various tissues of the reproductive tract of the rat. Using reverse transcriptase and the polymerase chain reaction, we demonstrated the presence of GC-C, uroguanylin, and guanylin mRNA in both male and female reproductive organs. Western blot analysis using a monoclonal antibody to GC-C revealed the presence of differentially glycosylated forms of GC-C in the caput and cauda epididymis. Exogenous addition of uroguanylin to minced epididymal tissue resulted in cGMP accumulation, suggesting an autocrine or endocrine activation of GC-C in this tissue. Immunohistochemical analyses demonstrated expression of GC-C in the tubular epithelial cells of both the caput epididymis and cauda epididymis. Our results suggest that the GC-C signaling pathway could converge on CFTR in the epididymis and perhaps control fluid and ion balance for optimal sperm maturation and storage in this tissue.
Assuntos
Epididimo/enzimologia , Expressão Gênica , Guanilato Ciclase/genética , Guanilato Ciclase/fisiologia , Receptores de Peptídeos/genética , Receptores de Peptídeos/fisiologia , Transdução de Sinais , Animais , Anticorpos Monoclonais , GMP Cíclico/biossíntese , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Feminino , Hormônios Gastrointestinais/genética , Imuno-Histoquímica , Ligantes , Masculino , Peptídeos Natriuréticos , Especificidade de Órgãos , Peptídeos/genética , Fosforilação , RNA Mensageiro/análise , Ratos , Receptores de Enterotoxina , Receptores Acoplados a Guanilato Ciclase , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
New human Escherichia coli heat-stable peptide (ST(h)) analogues containing a DOTA chelating group were synthesized by sequential and selective formation of disulfides bonds in the peptide. This synthetic approach utilizes three orthogonal thiol-protecting groups, Trt, Acm, and t-Bu, to form three disulfide bonds by successive reactions using 2-PDS, iodine, and silyl chloride-sulfoxide systems. The DOTA-ST(h) conjugates exhibiting high guanylin/guanylate cyclase-C (GC-C) receptor binding affinities were obtained with >98% purity. In vitro competitive binding assays, employing T-84 human colon cancer cells, demonstrated the IC(50) values of <2 nM for GC-C receptor binding suggesting that the new synthetic ST(h) analogues are biologically active. In vitro stability studies of the (111)In-DOTA-Phe(19)-ST(h) conjugate incubated in human serum at 37 degrees C under 5% CO(2) atmosphere revealed that this conjugate is extremely stable with no observable decomposition at 24 h postincubation. HPLC analysis of mouse urine at 1 h pi of the (111)In-DOTA-Phe(19)-ST(h) conjugate showed only about 15% decomposition suggesting that the (111)In-DOTA-Phe(19)-ST(h) conjugate is highly stable, even under in vivo conditions. In vivo pharmacokinetic studies of the (111)In-DOTA-Phe(19)-ST(h) conjugate in T-84 human colon cancer derived xenografts in SCID mice conducted at 1 h pi showed an initial tumor uptake of 2.04 +/- 0.30% ID/g at 1 h pi with efficient clearance from the blood pool (0.23 +/- 0.14% ID/g, 1 h pi) by excretion mainly through the renal/urinary pathway (95.8 +/- 0.2% ID, 1 h pi). High tumor/blood, tumor/muscle, and tumor/liver ratios of approximately 9:1, 68:1, and 26:1, respectively, were achieved at 1 h pi The specific in vitro and in vivo uptake of the radioactivity by human colonic cancer cells highlights the potential of radiometalated-DOTA-ST(h) conjugates as diagnostic/therapeutic radiopharmaceuticals.