RESUMEN
PURPOSE: Recent reports have indicated the benefit of anesthesia during prostate biopsy. To assess this finding objectively we performed a prospective randomized double-blind study to compare patient pain with and without local anesthesia during transrectal ultrasound guided prostate biopsies. MATERIALS AND METHODS: Between August 2000 and March 2001, 108 men undergoing transrectal ultrasound guided biopsy of the prostate were randomized in double-blind fashion to receive intrarectal 2% lidocaine gel or intrarectal lubricant alone. No patient received pre-procedure narcotics or sedation. Pain associated with biopsy was determined using a horizontal linear visual analog pain scale. Pain scores in the 2 treatment groups were compared and possible predictors of increased pain were examined. RESULTS: The 2 groups were similar in demographic characteristics. There was no significant difference in pain score in the 2% lidocaine and lubricant alone groups (28.3 versus 28.9 mm., p = 0.88). Previous biopsy, time since previous biopsy, physician, number of biopsies and prostate volume did not correlate with pain score, while age correlated negatively with the score (r = -0.27, p = 0.005). A single complication involving a vasovagal episode resolved spontaneously. CONCLUSIONS: Intrarectal lidocaine gel provides no significant therapeutic or analgesic benefit compared with lubricant alone for transrectal ultrasound guided biopsy of the prostate. In younger patients more discomfort is associated with this procedure.
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Anestésicos Locales/administración & dosificación , Lidocaína/administración & dosificación , Próstata/patología , Administración Rectal , Anciano , Anciano de 80 o más Años , Biopsia/métodos , Método Doble Ciego , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , RectoRESUMEN
Human 5-HT7A receptors positively modulated adenylyl cyclases via Gs subtypes of G proteins in human embryonic kidney 293 cells, and bound 5-hydroxytryptamine (HT) with high and low affinity (K(I) values of 1.5 +/- 0.3 and 93 +/- 4 nM). More than 60% of 5-HT7A receptors, however, displayed the high-affinity 5-HT binding with no sensitivity to 5'-guanylylimidodiphosphate. In this study, we found that select amphipathic agents affected the high-affinity 5-HT binding to 5-HT7A. Oleic acid at low concentrations (<15 microM), but not palmitic, stearic, and arachidonic acids, increased maximal [3H]5-HT binding without affecting its K(D) value and [3H]mesulergine (antagonist) binding. Fatty acid-free bovine serum albumin (FF-BSA), a scavenger of fatty acids and lipid metabolites, substantially reduced maximal [3H]5-HT binding (no change in K(D) value and antagonist binding) but lost its action upon treatment with inactive stearic acid. FF-BSA and oleic acid produced no appreciable effects on [3H]5-HT binding to analogous 5-HT receptors 5-HT1D and 5-HT2C. Among various lysophospholipids, lysophosphatidyl choline (50 microM) decreased maximal [3H]5-HT binding, and a similar zwitterion, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS; 0.1%), increased it (no change in K(D)). Functionally, 5-HT-induced guanosine-5'-O-(3-[35S]thio)triphosphate (GTPgamma35S) binding was enhanced by oleic acid and CHAPS, but reduced by FF-BSA and lysophosphatidyl choline; the amphipathic agents and FF-BSA did not affect dopamine-induced GTPgamma35S binding at D1, a prototypic Gs-coupled receptor. At 5-HT7A, oleic acid, FF-BSA, CHAPS, and lysophosphatidyl choline also brought about corresponding changes in the half-maximal 5-HT concentration for cAMP production, without affecting the maximal and basal levels. We propose that endogenous, amphipathic lipid metabolites may modulate 5-HT7A receptors allosterically to promote high-affinity 5-HT binding and to enable receptors to couple more efficiently to Gs subtypes of G proteins.
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Ácido Oléico/metabolismo , Receptores de Serotonina/metabolismo , Regulación Alostérica , Células Cultivadas , Ergolinas/farmacología , Células HeLa , Humanos , Ácidos Oléicos/metabolismo , Receptores de Serotonina/efectos de los fármacos , Serotonina/metabolismo , Antagonistas de la Serotonina/farmacología , TritioRESUMEN
BACKGROUND AND PURPOSE: The fragility of the <9F flexible ureteroscope limits its availability to general urology practice. The purpose of this study was to determine whether the technique used to clean the flexible ureteroscope or the number of persons handling the instrument during the cleaning process influenced endoscope breakage or deterioration during regular endourologic use. PATIENTS AND METHODS: A new Olympus URF/P3 flexible 7.5F ureteroscope was used for each of two 30-day study periods during which a single surgeon used the endoscope for a variety of upper urinary tract procedures. During the first 30-day period (Group 1), the endoscope was leak-proof-pressure tested and cleaned by the endourology support team using the Steris 20 (peroxyacetic acid 35%) technique. During the second 30-day period (Group 2), the endoscope was leak-proof tested and cleaned only by the surgeon using the Cidex (glutaraldehyde 2.4%) technique. A record was kept for each ureteroscopic case to document the patient position, access technique, time the endoscope was in the urinary tract, instruments passed through the ureteroscope, and the maximum irrigant pressure used. In addition, a record was made of the number of broken fibers, the degree of flexion and deflexion of the endoscope, and the problems encountered with the endoscope during the case. RESULTS: The two study groups were similar in terms of the total number of cases performed, the mean time the endoscope was in the urinary tract per case, the access approach used, and the use of the ureteral access sheath and ancillary equipment. In Group 2, the endoscope was used for a longer total time (618 minutes v 457 minutes), and access to a lower pole calix was more than twice as common as in Group 1. This may explain why more broken fibers were noted in the instrument used in Group 2 over the study period (eight v four broken fibers) than in Group 1. The only breakage occurred as a result of the surgeon accidentally activating the laser probe inside the working channel of the endoscope in Group 2. CONCLUSION: The technique and number of personnel involved in the maintenance and cleaning of the flexible ureteroscope does not have a significant effect on the durability and function of these instruments. It is the arduous demands of the endourologic procedure that influence the durability of these fragile endoscopes.
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Desinfectantes , Endoscopios , Equipo Reutilizado , Ácido Peracético , Diseño de Equipo , HumanosRESUMEN
PURPOSE: Radical cystectomy is standard treatment for bladder cancer in healthy individuals. We determined the safety of radical cystectomy in elderly patients at high risk. MATERIALS AND METHODS: We reviewed the records of all patients who underwent radical cystectomy at our institution between January 1994 and June 2000. Of these 382 patients we identified 44 who were elderly and at high risk, as defined by age 75 years or greater and American Society of Anesthesiologists classification 3 or greater. We examined postoperative care, perioperative minor/major complications, the mortality rate and the need for rehospitalization. RESULTS: Median age of the 44 patients was 77.5 years (range 75 to 87). American Society of Anesthesiologists class was 3 in 40 patients and 4 in 4. Median hospitalization was 7 days (range 4 to 20). Postoperatively 31 of the 44 patients (70%) were transferred directly to the general urology floor, while cardiac monitoring was required postoperatively in 30%. Nine of these patients were transferred to a step-down unit and the remaining 4 required surgical intensive care unit admission. Minor and major complications developed in 10 (22.7%) and 2 (4.5%) cases, respectively. No patients died in the perioperative period and 4 patients were hospitalized within 6 months of discharge home. CONCLUSIONS: Our results support the safety of radical cystectomy in elderly patients at high risk. Acceptable perioperative morbidity and mortality may be achieved without routine intensive monitoring postoperatively.
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Cistectomía/efectos adversos , Factores de Edad , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Complicaciones Posoperatorias/epidemiología , Factores de RiesgoRESUMEN
The human D2long dopamine receptor when expressed heterologously in a human neuronal cell line, SH-SY5Y, produced more robust functional signals than when expressed in a human embryonic kidney cell line, HEK293. Quinpirole (agonist)-induced GTPgamma(35)S binding and high affinity sites were 3 - 4 fold greater in SH-SY5Y than in HEK293 cells. N-type Ca(2+) channel currents present in SH-SY5Y cells, but not HEK293 cells, were inhibited potently by quinpirole with a half-maximal inhibitory concentration of 0.15+/-0.03 nM. Inhibition of adenylyl cyclases by agonists, on the other hand, was of similar potency and efficacy in the two cell lines. GTPgamma(35)S-Bound Galpha subunits from quinpirole-activated and solubilized membranes were monitored upon immobilization with various Galpha-specific antibodies. Galpha(i) and Galpha(o) subunits were highly labelled with GTPgamma(35)S in SH-SY5Y cells, but only Galpha(i) subunits were labelled in HEK293 cells. The additional G(o) coupling in SH-SY5Y cells could arise, at least in part, from the presence of G(o) coupled-effectors, such as the N-type Ca(2+) channel, and may contribute to robust agonist-induced GTPgamma(35)S binding, which is a reliable means for measuring ligand intrinsic efficacy. It appears that expression of neuronal G protein-coupled receptors in neuronal environments could reveal additional functional characteristics that are absent in non-neuronal cell lines. This appears to be due to, at least in part, to the presence of neuron-specific effectors. These findings underscore the importance of the cellular environment in which drug actions are examined, particularly in the face of intensive efforts to develop drugs for G protein-coupled receptors of various origins.
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Clonación Molecular/métodos , Riñón/metabolismo , Neuronas/metabolismo , Receptores de Dopamina D2/biosíntesis , Canales de Calcio/metabolismo , Células Cultivadas , Antagonistas de Dopamina/farmacología , Antagonistas de los Receptores de Dopamina D2 , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Humanos , Riñón/citología , Neuroblastoma , Racloprida/farmacología , Proteínas Recombinantes/metabolismo , Espiperona/farmacología , Células Tumorales CultivadasRESUMEN
BACKGROUND: Elevated concentrations of norepinephrine (NE) have been observed in ischemic myocardium. We investigated the magnitude and mechanism of catecholamine release in the myocardial interstitial fluid (MIF) during ischemia and reperfusion in vivo through the use of microdialysis. METHODS AND RESULTS: In 9 anesthetized pigs, interstitial catecholamine concentrations were measured in the perfusion areas of the left anterior descending coronary artery (LAD) and the left circumflex coronary artery. After stabilization, the LAD was occluded for 60 minutes and reperfused for 150 minutes. During the final 30 minutes, tyramine (154 nmol. kg(-1). min(-1)) was infused into the LAD. During LAD occlusion, MIF NE concentrations in the ischemic region increased progressively from 1. 0+/-0.1 to 524+/-125 nmol/L. MIF concentrations of dopamine and epinephrine rose from 0.4+/-0.1 to 43.9+/-9.5 nmol/L and from <0.2 (detection limit) to 4.7+/-0.7 nmol/L, respectively. Local uptake-1 blockade attenuated release of all 3 catecholamines by >50%. During reperfusion, MIF catecholamine concentrations returned to baseline within 120 minutes. At that time, the tyramine-induced NE release was similar to that seen in nonischemic control animals despite massive infarction. Arterial and MIF catecholamine concentrations in the left circumflex coronary artery region remained unchanged. CONCLUSIONS: Myocardial ischemia is associated with a pronounced increase of MIF catecholamines, which is at least in part mediated by a reversed neuronal reuptake mechanism. The increase of MIF epinephrine implies a (probably neuronal) cardiac source, whereas the preserved catecholamine response to tyramine in postischemic necrotic myocardium indicates functional integrity of sympathetic nerve terminals.
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Infarto del Miocardio/metabolismo , Isquemia Miocárdica/metabolismo , Norepinefrina/metabolismo , Sistema Nervioso Simpático/metabolismo , Animales , Presión Sanguínea/fisiología , Circulación Coronaria/fisiología , Femenino , Corazón/inervación , Corazón/fisiología , Frecuencia Cardíaca/fisiología , Masculino , Microdiálisis , Infarto del Miocardio/fisiopatología , Isquemia Miocárdica/fisiopatología , Reperfusión Miocárdica , Terminaciones Nerviosas/metabolismo , Volumen Sistólico/fisiología , Porcinos , Sistema Nervioso Simpático/efectos de los fármacos , Simpatomiméticos/farmacología , Tiramina/farmacología , Fibrilación Ventricular/metabolismoRESUMEN
5-HT(1) receptor subtypes ((1B), (1D) and (1F)) have been implicated in migraine pathophysiology and their ligands have been examined for pharmacological actions in various experimental animal models. Considerable divergences exist, however, in their primary sequences between experimental animals and human, and additional models closer to human, such as non-human primates seem to be useful for migraine research. Earlier, we cloned the 5-HT(1D), and here 5-HT(1B) and 5-HT(1F) receptors from the chimpanzee, gorilla and Rhesus monkey, via polymerase chain reactions with their genomic DNAs and primers designed from the corresponding human receptors. Direct sequencing of PCR products showed that the 5-HT(1B) receptors from the chimpanzee, gorilla and monkey differ from the human receptor by 0, 1 and 7 residues, respectively while 5-HT(1F) receptors differ by 0, 3 and 10 residues, respectively. These divergent residues are mostly conservatively substituted and also largely confined to the N-terminal region and the 3rd intracellular loop, away from transmembrane segments and intracellular loops near membrane which are critical for ligand binding and G protein coupling. The chimpanzee 5-HT(1D), 5-HT(1B) and monkey 5-HT(1F) receptors, as heterologously expressed in human embryonic kidney 293 cells, showed robust agonist-induced guanosine 5'gamma (35)S triphosphate (GTPgamma(35)S) binding through activation of G proteins containing Ggamma(i) subunits. Moreover, pronounced inhibition of basal GTPgamma(35)S binding by methiothepin (an antagonist), representing constitutively active receptors, was observed with only 5-HT(1D). Overall, ligand binding and GTPgamma(35)S binding profiles for these primate receptors are comparable to those for the human receptors and validate non-human primates as useful models for human migraine research.
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Guanosina 5'-O-(3-Tiotrifosfato)/farmacocinética , Receptores de Serotonina/metabolismo , Agonistas de Receptores de Serotonina/farmacología , Animales , Clonación Molecular , Gorilla gorilla , Humanos , Cinética , Ligandos , Macaca mulatta , Pan troglodytes , Receptor de Serotonina 5-HT1B , Receptor de Serotonina 5-HT1D , Receptores de Serotonina/genética , Proteínas Recombinantes/metabolismo , Radioisótopos de Azufre , Receptor de Serotonina 5-HT1FRESUMEN
1 The D3 dopamine receptor presumably activates Gi/Go subtypes of G-proteins, like the structurally analogous D2 receptor, but its signalling targets have not been clearly established due to weak functional signals from cloned receptors as heterologously expressed in mostly non-neuronal cell lines. 2 In this study, recombinant human D3 receptors expressed in a human neuroblastoma cell line, SH-SY5Y, produced much greater signals than those expressed in a human embryonic kidney cell line, HEK293. Quinpirole, a prototypic agonist, markedly inhibited forskolin-stimulated cyclic AMP production and Ca2+-channel (N-type) currents in SH-SY5Y cells, and enhanced GTPgamma35S binding in isolated membranes, nearly ten times greater than that observed in HEK293 cell membranes. 3 GTPgamma35S-bound Galpha subunits from quinpirole-activated and solubilized membranes were monitored upon immobilization with various Galpha-specific antibodies. Galphao subunits (not Galphai) were highly labelled with GTPgamma35S in SH-SY5Y, but not in HEK293 cell membranes, despite their abundance in the both cell types, as shown with reverse transcription-polymerase chain reaction and Western blots. N-type Ca2+ channels and adenylyl cyclase V (D3-specific effector), on the other hand, exist only in SH-SY5Y cells. 4 More efficient coupling of the D3 receptor to Go subtypes in SH-SY5Y than HEK293 cells may be attributed, at least in part, to the two D3 neuronal effectors only present in SH-SY5Y cells (N-type Ca2+-channels and adenylyl cyclase V). The abundance of Go subtypes in the both cell lines seems to indicate their availability not a limiting factor.
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Proteínas de Unión al GTP/metabolismo , Receptores de Dopamina D2/metabolismo , Adenilil Ciclasas/genética , Unión Competitiva/efectos de los fármacos , Canales de Calcio/efectos de los fármacos , Canales de Calcio/metabolismo , Línea Celular , Colforsina/farmacología , AMP Cíclico/metabolismo , Agonistas de Dopamina/metabolismo , Agonistas de Dopamina/farmacología , Relación Dosis-Respuesta a Droga , Proteínas de Unión al GTP/genética , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Potenciales de la Membrana/efectos de los fármacos , Quinpirol/metabolismo , Quinpirol/farmacología , ARN/genética , ARN/metabolismo , Ensayo de Unión Radioligante , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/fisiología , Receptores de Dopamina D3 , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Radioisótopos de Azufre , Tritio , Células Tumorales CultivadasRESUMEN
The 5-HT(2C) receptor as heterologously expressed in various mammalian cells mediates inositol 1,4,5-triphosphate (IP(3)) signal by activating G(q/11) subtypes of G proteins, but minimally promotes agonist-induced GTPgamma35S binding in membranes due to slow GTP turnover rates of the G proteins. Here we discovered robust (over 200%) agonist-induced GTPgamma35S binding mediated by the human receptor expressed in human embryonic kidney (HEK) 293 cells, and investigated its pharmacology. Agonists concentration-dependently increased GTPgamma35S binding in isolated membranes, which was competitively blocked by antagonists. Intrinsic efficacies of agonists from GTPgamma35S binding were comparable to those from IP(3) measurement. Pertussis toxin treatment largely blocked serotonin-induced GTPgamma35S binding, serotonin high affinity sites by 70% without altering the total binding sites, and reduced IP(3) release by 40%. GTPgamma35S-bound Galpha subunits from serotonin-activated membranes were mainly Galpha(i), judging from immobilization studies with various Galpha-specific antibodies. Inhibition of forskolin-stimulated cAMP formation, however, was not observed. Apparently, the 5-HT(2C) receptor-mediated GTPgamma35S binding is a unique phenotype observed in HEK293 cells, reflecting its coupling to pertussis toxin-sensitive G(i) subtypes, which contribute to the IP(3) signal, along with pertussis toxin-insensitive G(q/11) subtypes.
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Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Inositol 1,4,5-Trifosfato/farmacocinética , Receptores de Serotonina/metabolismo , AMP Cíclico/metabolismo , Ergolinas/metabolismo , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Riñón/citología , Riñón/embriología , Riñón/metabolismo , Toxina del Pertussis , Receptor de Serotonina 5-HT2C , Receptores de Serotonina/efectos de los fármacos , Serotonina/metabolismo , Antagonistas de la Serotonina/metabolismo , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
The binding of polypeptide growth factors to their appropriate cell surface transmembrane receptors triggers numerous biochemical responses, including the transcriptional activation of specific genes. We have used a differential display approach to identify fibroblast growth factor-1-inducible genes in murine NIH 3T3 cells. Here, we report that the fibroblast growth factor-inducible-14 (Fn14) gene is a growth factor-regulated, immediate-early response gene expressed in a developmental stage- and adult tissue-specific manner in vivo. This gene, located on mouse chromosome 17, is predicted to encode an 129-amino acid type Ia membrane protein with no significant sequence similarity to any known protein. We have used two experimental approaches, direct fluorescence microscopy and immunoprecipitation analysis of biotinylated cell surface proteins, to demonstrate that Fn14 is located on the plasma membrane. To examine the biological consequences of constitutive Fn14 expression, we isolated NIH 3T3 cell lines expressing variable levels of epitope-tagged Fn14 and analyzed their phenotypic properties in vitro. These experiments revealed that Fn14 expression decreased cellular adhesion to the extracellular matrix proteins fibronectin and vitronectin and also reduced serum-stimulated cell growth and migration. These results indicate that Fn14 is a novel plasma membrane-spanning molecule that may play a role in cell-matrix interactions.
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Proteínas de la Membrana/genética , Receptores del Factor de Necrosis Tumoral , Células 3T3 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Adhesión Celular/genética , División Celular/genética , Movimiento Celular/genética , Mapeo Cromosómico , Clonación Molecular , Factor de Crecimiento Epidérmico/genética , Matriz Extracelular/metabolismo , Factor 1 de Crecimiento de Fibroblastos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Hemaglutininas/genética , Hibridación in Situ , Proteínas de la Membrana/química , Ratones , Microscopía Fluorescente , Mitógenos/farmacología , Datos de Secuencia Molecular , ARN Mensajero/genética , Proteínas Recombinantes de Fusión/genética , Receptor de TWEAK , TransfecciónRESUMEN
Experimental findings suggest a pronounced concentration gradient of norepinephrine (NE) between the intravascular and interstitial compartments of the heart, compatible with an active neuronal reuptake (U1) and/or an endothelial barrier. Using the microdialysis technique in eight anesthetized pigs, we investigated this NE gradient, both under baseline conditions and during increments in either systemic or myocardial interstitial fluid (MIF) NE concentration. At steady state, baseline MIF NE (0.9 +/- 0.1 nmol/l) was higher than arterial NE (0.3 +/- 0.1 nmol/l) but was not different from coronary venous NE (1.5 +/- 0.3 nmol/l). Local U1 inhibition raised MIF NE concentration to 6.5 +/- 0.9 nmol/l. During intravenous NE infusions (0.6 and 1.8 nmol. kg(-1). min(-1)), the fractional removal of NE by the myocardium was 79 +/- 4% to 69 +/- 3%, depending on the infusion rate. Despite this extensive removal, the quotient of changes in MIF and arterial concentration (DeltaMIF/DeltaA ratio) for NE were only 0.10 +/- 0.02 for the lower infusion rate and 0.11 +/- 0.01 for the higher infusion rate, whereas U1 blockade caused the DeltaMIF/DeltaA ratio to rise to 0.21 +/- 0.03 and 0.36 +/- 0.05, respectively. From the differences in DeltaMIF/DeltaA ratios with and without U1 inhibition, we calculated that 67 +/- 5% of MIF NE is removed by U1. Intracoronary infusion of tyramine (154 nmol. kg(-1). min(-1)) caused a 15-fold increase in MIF NE concentration. This pronounced increase was paralleled by a comparable increase of NE in the coronary vein. We conclude that U1 and extraneuronal uptake, and not an endothelial barrier, are the principal mechanisms underlying the concentration gradient of NE between the interstitial and intravascular compartments in the porcine heart.
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Catecolaminas/metabolismo , Miocardio/metabolismo , Porcinos/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Espacio Extracelular/metabolismo , Femenino , Hemodinámica/efectos de los fármacos , Isoproterenol/sangre , Isoproterenol/farmacocinética , Isoproterenol/farmacología , Masculino , Microdiálisis , Norepinefrina/sangre , Norepinefrina/farmacocinética , Norepinefrina/farmacología , Tiramina/sangre , Tiramina/farmacocinética , Tiramina/farmacologíaRESUMEN
The relatively new technique of microdialysis provides new possibilities for investigating in vivo the functioning of the sympathetic nervous system. The small sample volumes obtained, however, are a great challenge for analytical chemists. We report here a HPLC method for measuring in one run both natural and synthetic catecholamines [dopamine, (nor)epinephrine, alpha-methylnorepinephrine, isoproterenol and epinine] and the intraneuronal metabolite 3,4-dihydroxyphenylglycol in small microdialysis samples after derivatization with the fluorogenic agent 1,2-diphenylethylenediamine. No prior clean-up step is necessary. N-Ethylmaleimide is necessary for preventing an inhibitory action on derivatization occurring in in vivo microdialysis samples. The method can handle large numbers of samples, is sensitive (on-column detection limits 30 to 200 fg) and reproducible (RSD 1 to 7%). Recovery characteristics of the commercial microdialysis probe used (CMA/20) were extensively investigated both in vitro and in vivo at various perfusion rates; for practical purposes a rate of 2 microl/min and sampling at 10-min intervals was found to be workable and to give good and reproducible recoveries (50 to 70%).
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Catecolaminas/análisis , Metoxihidroxifenilglicol/análogos & derivados , Animales , Humanos , Metoxihidroxifenilglicol/análisis , Microdiálisis , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , PorcinosRESUMEN
PURPOSE: We report on color and power Doppler ultrasound to study cavernosal arterial anatomy, and evaluate the impact of vascular anatomy on the measurement of hemodynamic parameters. MATERIALS AND METHODS: Cavernosal arterial anatomy of 42 patients with erectile dysfunction was evaluated using color and power Doppler ultrasound. A computerized waveform analysis was used to measure peak systolic velocity, end diastolic velocity and resistive indexes at various sites, including the penile crura, and proximal mid and distal penile shaft. Hemodynamic parameters were measured in each artery in cases of bifurcated or multiple cavernosal arteries. RESULTS: A total of 80 corpora were adequately evaluated. We observed a single artery without major proximal branches in 37 corpora, a single artery with major proximal branches in 17, bifurcated arteries in 15, 2 cavernosal arteries in 4 and marked arterial tortuosity in 1. In 6 corpora the main cavernosal artery arose from the superficial dorsal artery. The peak systolic velocity was highest at the proximal and decreased progressively at the distal site. The peak systolic velocity plus or minus standard deviation at the mid shaft averaged 69.3+/-30.0% of that at the proximal penile shaft. Of the 15 corpora with bifurcated arteries 67% had a 40% or greater difference in peak systolic velocity between the branches. Complete or partial occlusion of the cavernosal artery was identified in 3 corpora, and a dramatic difference in peak systolic velocity proximal and distal to the stenotic area was demonstrated. CONCLUSIONS: Cavernosal arterial anatomy is variable and hemodynamic parameters differ at various sites of measurement. Parameters should be measured at a consistent proximal site to obtain a reliable assessment. Variations in vascular anatomy and cavernosal artery pathology should be considered when interpreting color Doppler sonography and before penile vascular surgery.
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Arterias/diagnóstico por imagen , Disfunción Eréctil/diagnóstico por imagen , Hemodinámica , Pene/irrigación sanguínea , Pene/diagnóstico por imagen , Ultrasonografía Doppler en Color , Adulto , Anciano , Humanos , Masculino , Persona de Mediana EdadRESUMEN
1. Both the 5-HT1D and 5-HT1B receptors are implicated in migraine pathophysiology. Recently isochromans have been discovered to bind primate 5-HT1D receptors with much higher affinity than 5-HT1B receptors. In the guinea-pig, a primary animal model for anti-migraine drug testing, however, isochromans bound the 5-HT1D receptor with lower affinity than the gorilla receptor. 2. This species-specific pharmacology was investigated, using site-directed mutagenesis on cloned guinea-pig receptors heterologously expressed in human embryonic kidney 293 cells. Mutations of threonine 100 and arginine 102 at the extracellular side of transmembrane II of the guinea-pig 5-HT1D receptor to the corresponding primate residues, isoleucine and histidine, respectively, enhanced its affinity for isochromans to that of the gorilla receptor, with little effects on its affinities for serotonin, sumatriptan and metergoline. Free energy change from the R102H mutation was about twice as much as that from the T100I mutation. 3. For G protein-coupling, serotonin marginally enhanced GTPgamma35S binding in membranes expressing the guinea-pig 5-HT1D receptor and its mutants, but robustly in membranes expressing the gorilla receptor. Sumatriptan enhanced GTPgamma35S binding in the latter nearly as much as serotonin, and several isochromans by 30-60% of serotonin. 4. We discovered key differences in the function and binding properties of guinea-pig and gorilla 5-HT1D receptors, and identified contributions of I100 and H102 of primate 5-HT1D receptors to isochroman binding. Among common experimental animals, only the rabbit shares I100 and H102 with primates, and could be useful for studying isochroman actions in vivo.
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Cromanos , Gorilla gorilla/fisiología , Receptores de Serotonina/efectos de los fármacos , Serotoninérgicos , Animales , Unión Competitiva/efectos de los fármacos , Clonación Molecular , Cobayas , Técnicas In Vitro , Ligandos , Mutación , Receptor de Serotonina 5-HT1D , Receptores de Serotonina/genética , Especificidad de la EspecieRESUMEN
1. Cysteine 114 (C114) of the human dopamine D3 receptor is located at the helical face of transmembrane segment III (TMIII) near aspartate 110, a counterion for the amine group of catecholamines. The contributions of C114 to receptor function were investigated here using site-directed mutagenesis of C114 to serine. 2. The C114S mutant, as expressed in Sf-9 cells, bound aminotetralin antagonists (UH-232 and AJ-76) and several agonists ((-)3-PPP, apomorphine, pramipexole and quinpirole) with markedly lower affinities as compared to the wild type D3 receptor, but bound other structurally diverse dopaminergic ligands with only minor changes in affinity. Because an N-propyl substituent is the only common structural feature among most affected ligands, we propose that the mutation alters 'a propyl cleft' on the receptor. The mutation hardly affected quinpirole-dependent [35S]-GTPgammaS binding, suggesting C114 plays a minimal role in receptor-G-protein coupling. 3. N-Ethylmaleimide(NEM), a sulfhydryl modifying agent, blocked ligand binding to the D3 receptor, but not to the C114S mutant. We infer that C114 is the primary residue on the D3 receptor vulnerable to external oxidizing agents. Dopamine D2long and D4(2) receptors contain highly homologous TMIII sequences including an equivalent cysteine residue. However, only the D2long receptor, not the D4(2) receptor, displayed NEM sensitivity similar to that of the D3 receptor. 4. We conclude that C114 is critical for high affinity interactions between the D3 receptor and ligands containing an N-propyl substituent, and unlike its counterpart in the D4(2) receptor, is highly susceptible to external oxidizing agents.
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Cisteína/fisiología , Oxidantes/farmacología , Receptores Dopaminérgicos/fisiología , Células Cultivadas , Cisteína/efectos de los fármacos , Humanos , Ligandos , Mutación Puntual , Unión Proteica , Receptores Dopaminérgicos/ultraestructuraRESUMEN
To investigate the roles of individual transmembrane segments (TM) of the human D3 dopamine receptor in its ligand-receptor interactions, we generated chimeric receptors in which its TMs were replaced, one at a time, partially or entirely, by the corresponding TM of the homologous human D1 receptor. Ligand binding properties of the chimeras, as expressed heterologously in Sf9 cells using recombinant baculoviruses, indicate that the critical binding regions for D3-selective (over D1) ligands reside at narrow regions (6 to 8 residues) near the extracellular surface for TMI, II, IV and VI, while TMV seems to be minimally involved in the ligand selectivity. For TMIII and TMVII, the critical regions seem to be deeper, involving at least the 10 residues near the extracellular surface for TMIII, and the entire TM segment for TMVII. This is based on our current observations that the chimeras with the D3 sequence in the critical regions, although the rest of the TM is of D1 origin (except TMVII), showed the binding properties indistinguishable from those of the wild-type receptor. The chimeras with the D1 sequence in the regions, on the other hand, showed ligand binding characteristics wildly variable depending on substituted TMs: Most marked decreases in ligand affinities were observed with the chimeras of TMIII and VII, and intermediate changes with those of TMIV and VI. Replacements of TMV produced no appreciable effects on the affinities of 14 test ligands (except for one). The chimeras of TMI and II with the D1 sequence in the critical regions showed no appreciable specific binding for several radioactive D3-selective ligands, possibly reflecting their critical roles in assembly and folding of the receptor. These critical regions of the D3 receptor were highly homologous to those of the D2 receptor, except for several nonconservatively substituted residues, which could be exploited to develop ligands selective for the D3 over D2 dopamine receptor or vice versa.
Asunto(s)
Membrana Celular/metabolismo , Receptores de Dopamina D2/metabolismo , Secuencia de Aminoácidos , Humanos , Ligandos , Datos de Secuencia Molecular , Mutación , Conformación Proteica , Receptores de Dopamina D1/genética , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/química , Receptores de Dopamina D2/genética , Receptores de Dopamina D3 , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The 5-HT1D receptor is a potential target of anti-migraine drugs, and here its genes were cloned from chimpanzee, gorilla and rhesus monkey, via polymerase chain reactions with their genomic DNAs and the primers designed from the 5' and 3' untranslated regions of the human receptor. Direct sequencing of the polymerase chain reaction (PCR) products revealed high degrees of identity between their deduced amino acid sequences (the chimpanzee, gorilla and rhesus monkey) and that of human, differing by two, four and 11 residues, respectively. The binding properties of the receptors, as expressed in human embryonic kidney 293 cells, were compared to those obtained with the human and guinea pig receptors, the latter differing by 33 residues from the human receptor. Standard serotonergic ligands including several indoles, ergots and methiothepin bound all the cloned primate and guinea pig receptors with comparable, low nanomolar affinities, leading to high correlation coefficients among their Ki values. R(+)-8-Hydroxydipropylaminotetralin, on the other hand, bound the human receptor with the affinity higher than those for the primates and guinea pig receptors. This indicates that certain chemical templates may differentiate the molecular divergences among the 5-HT1D receptors of various animal species, and the use of the non-human primates will be beneficial for pharmacological characterizations, more relevant to the human receptor, of future novel ligands for the 5-HT1D receptor, which are potential anti-migraine drugs.
Asunto(s)
Cobayas/metabolismo , Primates/metabolismo , Receptores de Serotonina/metabolismo , Animales , Línea Celular , Clonación Molecular , Gorilla gorilla , Humanos , Ligandos , Macaca mulatta , Pan troglodytes , Especificidad de la EspecieRESUMEN
Keratinocyte growth factor (KGF) is a secreted member of the fibroblast growth factor (FGF) family of heparin-binding proteins. Studies reported to date indicate that it functions primarily as an important paracrine mediator of epithelial cell growth and differentiation. KGF appears to act via binding to a specific FGF receptor-2 isoform generated by an alternative splicing mechanism. To determine whether KGF may play a role in vascular smooth muscle cell (SMC) biology, we investigated KGF and KGF receptor gene expression in human SMC cultured in vitro as well as in several human nonatherosclerotic artery and atheroma specimens. KGF mRNA but not KGF receptor mRNA was expressed by SMCs, as determined by Northern blot hybridization analysis or reverse transcription-polymerase chain reaction assays, respectively. Additional experiments demonstrated that 1) human SMCs produce and secrete mitogenically active KGF and that 2) the cytokine interleukin-1 increases KGF mRNA and protein levels in human SMCs. We also found that KGF transcripts but not KGF receptor transcripts were expressed in control and atherosclerotic human arteries. Taken together, these results indicate that KGF is unlikely to be involved in SMC growth regulation unless it can function intracellularly or interact with a presently unidentified KGF receptor.
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Arteriosclerosis/metabolismo , Arterias Carótidas/metabolismo , Enfermedades de las Arterias Carótidas/metabolismo , Factores de Crecimiento de Fibroblastos , Sustancias de Crecimiento/biosíntesis , Músculo Liso Vascular/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento/biosíntesis , Transcripción Genética , Animales , Células Cultivadas , Clonación Molecular , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Factor 10 de Crecimiento de Fibroblastos , Factor 7 de Crecimiento de Fibroblastos , Regulación de la Expresión Génica , Sustancias de Crecimiento/farmacología , Humanos , Ratones , Ratones Endogámicos BALB C , Reacción en Cadena de la Polimerasa , ARN Mensajero/biosíntesis , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos , Venas UmbilicalesRESUMEN
In the membranes of Spodoptera frugiperda (Sf-9) insect cells heterologously expressing the human D3 dopamine receptor, agonists selective for the receptor, but not antagonists, robustly enhanced [35S]GTPgammaS binding. Quinpirole, for instance, dose-dependently enhanced [35S]GTPgammaS binding with a half-maximal concentration of 2.3 +/- 0.2 nM. Its action was absent in the cells infected with wild type viruses, and competitively blocked by an antagonist, YM-09151-2. A number of known agonists enhanced [35S]GTPgammaS binding to variable degrees, probably reflecting their differential efficacy to activate target G-proteins via the receptor. This agonist-induced [35S]GTPgammaS binding was abolished by N-ethylmaleimide, a selective blocking agent for Gi/Go proteins, with no appreciable effect on ligand binding. We propose coupling of the cloned D3 receptor to endogenous G-proteins in Sf-9 cells, probably homologs of mammalian Gi/Go proteins. Despite the apparent coupling of the D3 receptor to G-proteins, GTPgammaS (10 microM) failed to decrease agonist binding ([3H]dopamine) to the D3 receptor, probably due to small affinity differences between low and high affinity states for agonists in the D3 receptor, as well as due to high receptor density in Sf-9 cells. We conclude that agonist-induced [35S]GTPgammaS binding for the D3 receptor is suitable for estimating ligand intrinsic efficacy and pharmacological characterizations of ligand-receptor interactions.
Asunto(s)
Agonistas de Dopamina/farmacología , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Quinpirol/farmacología , Receptores de Dopamina D2/metabolismo , Animales , Benzamidas/metabolismo , Benzamidas/farmacología , Línea Celular , Membrana Celular/metabolismo , Antagonistas de Dopamina/metabolismo , Antagonistas de Dopamina/farmacología , Etilmaleimida/farmacología , Humanos , Cinética , Receptores de Dopamina D2/biosíntesis , Receptores de Dopamina D3 , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Spodoptera , Radioisótopos de Azufre , TransfecciónRESUMEN
1. The benzodiazepine site of the alpha 6 beta 2 gamma 2 subtype of gamma-aminobutyric acidA (GABAA) receptors is distinguishable from that of the alpha 1 beta 2 gamma 2 subtype by its inability to interact with classical benzodiazepines (i.e., diazepam) and its agonistic response to Ro 15-1788, which behaves as an antagonist in the alpha 1 beta 2 gamma 2 subtype. 2. The point mutation of Arg 100 of the alpha 6 subunit to histidine (the corresponding residue in alpha 1) has been shown to enable the alpha 6 beta 2 gamma 2 subtype to interact with diazepam but failed in this study to abolish the ability of Ro 15-1788 to enhance GABA-induced Cl- currents. 3. Here we identified the segment of P161 to L187 of alpha 6 to contain the functional region responsible for the agonistic action of Ro 15-1788. Its replacement with the corresponding alpha 1 sequence abolished the ability of Ro 15-1788 to enhance GABA currents without appreciable effects on its binding affinity to the benzodiazepine site or on the functionality of the other benzodiazepine site ligands such as diazepam, U-92330 and 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate. These data support the evidence that the functionality of a given ligand could arise from a single region of the benzodiazepine site, not shared by others. 4. In addition we have learned that several residues in the N-terminal region of alpha 6, such as R100, V142 and N143, have the ability to influence GABA-dependent Cl- current induction probably by allosterically modulating low affinity GABA sites.