RESUMEN
Growth retardation occurs frequently in renal transplanted children (RTx) and can be improved by growth hormone (GH) treatment. This study retrospectively examines the insulin-like growth factor-1 (IGF-1) and IGF binding protein (IGFBP) profile of ten growth-retarded children previously given renal allografts, after 1 year of GH treatment period. Ten prepubertal patients (nine boys and one girl) were investigated. They had a mean chronological age (CA) of 11.4 +/- 1.1 years and a mean bone age (BA) of 7.3 +/- 0.9 years. Mean height was -3.9 +/- 0.4 SD units below the mean for CA. The mean body mass index (BMI) was 16.9 +/- 0.6 and the mean inulin clearance was 36.5 +/- 4.9 ml/min/1.73 m2. Recombinant hGH was given at 4 IU/m2/day. Plasma GH, total and free IGF-1, IGFBP-2 and -3 were measured by specific radioimmunoassay (RIA). IGFBPs were characterized by SDS PAGE techniques and ligand and immunoblot analyses. Mean velocity was markedly increased (P < 0.01) after 1 year of GH therapy, expressed as SD score for BA. The range of growth response was wide. The total and free plasma IGF-1 increased (P < 0.01) by about 100% (mean values after GH therapy: 95.9 +/- 2.1 nM and 165 +/- 29 pM, respectively). Plasma IGFBP-3 concentrations increased by about 40% (mean value: 148 +/- 18 pM, P < 0.01), with a concomitant increase in both intact IGFBP-3 and its 30-kDa proteolytic fragment. There was no change in plasma IGFBP-2 concentration. Both mean values of inulin clearance and BMI were unchanged during the treatment. In view of the IGF-1/IGFBP concentration changes, there should have been an even better growth response to GH therapy in these patients. This strongly suggests IGF-1 insensitivity, probably as a result of corticosteroid therapy.
Asunto(s)
Hormona del Crecimiento/farmacología , Sustancias de Crecimiento/metabolismo , Trasplante de Riñón/fisiología , Adolescente , Western Blotting , Estatura/efectos de los fármacos , Índice de Masa Corporal , Niño , Preescolar , Electroforesis en Gel de Poliacrilamida , Femenino , Tasa de Filtración Glomerular , Crecimiento/efectos de los fármacos , Humanos , Indicadores y Reactivos , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Masculino , Somatomedinas/metabolismoRESUMEN
Nitric oxide (NO) exerts cytoprotective effects against hepatic ischemia-reperfusion damage. This study was designed to evaluate which isoform of NO synthase (NOS) is implicated in the generation of cytoprotective NO and to investigate whether NO effects are mediated by cyclic GMP (cGMP). After partial ischemia for 45 min, liver damage was estimated by the release into plasma of cytolytic enzymes. Ischemia-reperfusion induced marked increases in plasma creatine kinase and lactate dehydrogenase after 1 h of reperfusion and of aminotransferases after 6 h of reperfusion. The pretreatment of ischemic rats with 8-bromo-cGMP (16 mg/kg i.v. 30 min before ischemia) or with L-arginine (the endogenous precursor of NO, 100 mg/kg i.v.) significantly diminished the ischemia-reperfusion-induced release of all these enzymes. This demonstrates that cGMP possesses hepatoprotective properties. By immunohistochemistry, we observed, after 6 h of reperfusion, an increase in endothelial NOS-III immunoreactivity, particularly in the small arteries and sinusoids. This NOS-III accumulation in endothelial cells could protect the liver against ischemia-reperfusion by the local generation of NO probably via cGMP.
Asunto(s)
GMP Cíclico/fisiología , Hígado/irrigación sanguínea , Óxido Nítrico Sintasa/fisiología , Óxido Nítrico/farmacología , Daño por Reperfusión/prevención & control , Animales , Arginina/farmacología , Creatina Quinasa/sangre , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacología , Endotelio Vascular/enzimología , Inmunohistoquímica , L-Lactato Deshidrogenasa/sangre , Masculino , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/análisis , Óxido Nítrico Sintasa de Tipo III , Ratas , Ratas Sprague-DawleyRESUMEN
The aim of this study was to evaluate the protective or deleterious effects of endogenous nitric oxide (NO) on liver cells during hepatic ischemia-reperfusion (IR) in the rat. Injury to hepatocytes and endothelial cells was evaluated by determining cytolysis-marker activity in plasma (alanine transaminase [ALT]; aspartate transaminase [AST]) and plasma hyaluronic acid (HA) concentration. Clamping the hepatic pedicle for 45 minutes caused a significant increase in plasma AST and ALT activity after 30 minutes of reperfusion, which reached a maximum (+270% and +740%, respectively) after 6 hours of reperfusion. Plasma HA concentration was significantly higher (+130%) only after 6 hours of reperfusion. Administration of a nonselective NO synthase (NOS) inhibitor, Nomega-nitro-L-arginine (L-NNA; 10 mg/kg iv), 30 minutes before IR, caused marked aggravation of postischemic liver injury, as shown by plasma ALT and AST activity and HA concentration. This deleterious effect was partially prevented by the simultaneous injection of L-arginine, the endogenous NO precursor (100 mg/kg iv). Interestingly, L-arginine alone limited postischemic damage (AST, -25%; ALT, -45%; HA, -21% vs. untreated IR rats at 6 hours reperfusion). Pretreatment with the Guanosine 3':5'-cyclic monophosphate-independent vasodilator, prazosin, partially reversed L-NNA effects, but it did not protect untreated IR animals. Pretreatment with aminoguanidine, a selective inhibitor of inducible NOS, did not aggravate hepatic IR injury. Thus, endogenous NO, probably produced by an early and transient activation of a constitutive NOS, protects both hepatocytes and endothelial cells against liver ischemia-reperfusion injury, and this effect is not entirely a result of vasorelaxation.
Asunto(s)
Isquemia/fisiopatología , Circulación Hepática/fisiología , Hígado/fisiopatología , Óxido Nítrico/fisiología , Daño por Reperfusión/fisiopatología , Animales , Citoprotección/fisiología , Endotelio/patología , Isquemia/patología , Hígado/patología , Masculino , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/patologíaRESUMEN
The ability of Ca2+/phospholipid-dependent protein kinase (protein kinase C, PKC) to stimulate cAMP phosphodiesterase (PDE) activity in a liver Golgi-endosomal (GE) fraction was examined in vivo and in a cell-free system. Injection into rats of 4 beta-phorbol 12-myristate 13-acetate, a known activator of PKC, caused a rapid and marked increase in PKC activity (+325% at 10 min) in the GE fraction, along with an increase in the abundance of the PKC alpha-isoform as seen on Western immunoblots. Concurrently, 4 beta-phorbol 12-myristate 13-acetate treatment caused a time-dependent increase in cAMP PDE activity in the GE fraction (96% at 30 min). Addition of the catalytic subunit of protein kinase A (PKA) to GE fractions from control and 4 beta-phorbol 12-myristate 13-acetate-treated rats led to a comparable increase (130-150%) in PDE activity, suggesting that PKA is probably not involved in the in-vivo effect of 4 beta-phorbol 12-myristate 13-acetate. In contrast, addition of purified PKC increased (twofold) PDE activity in GE fractions from control rats but affected only slightly the activity in GE fractions from 4 beta-phorbol 12-myristate 13-acetate-treated rats. About 50% of the Triton-X-100-solubilized cAMP PDE activity in the GE fraction was immunoprecipitated with an anti-PDE3 antibody. On DEAE-Sephacel chromatography, three peaks of PDE were sequentially eluted: one early peak, which was stimulated by cGMP and inhibited by erythro-9 (2-hydroxy-3-nonyl) adenine (EHNA); a selective inhibitor of type 2 PDEs; and two retarded peaks of activity, which were potently inhibited by cGMP and cilostamide, an inhibitor of type 3 PDEs. Further characterization of peak I by HPLC resolved a major peak which was activated (threefold) by 5 microM cGMP and inhibited (87%) by 25 microM EHNA, and a minor peak which was insensitive to EHNA and cilostamide. 4 beta-Phorbol 12-myristate 13-acetate treatment caused a selective increase (2.5-fold) in the activity associated with DEAE-Sephacel peak I, without changing the K(m) value. These results suggest that PKC selectively activates a PDE2, cGMP-stimulated isoform in the GE fraction.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , GMP Cíclico/farmacología , Endosomas/enzimología , Aparato de Golgi/enzimología , Hígado/enzimología , Proteína Quinasa C/metabolismo , Adenina/análogos & derivados , Adenina/farmacología , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Activación Enzimática , Inhibidores Enzimáticos , Masculino , Naftalenos/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Quinolonas/farmacología , Ratas , Ratas Sprague-Dawley , Acetato de Tetradecanoilforbol/farmacología , Factores de TiempoRESUMEN
1. The effects of 24R,25-dihydroxyvitamin D3 [24,25(OH)2D3] on alkaline phosphatase activity (ALP) were evaluated in pig kidney LLC-PK1 cells in culture. 2. The vitamin D3 metabolite increased ALP activity in these cells, whereas no effect of the hormone was observed on gamma-glutamyltranspeptidase and acid phosphatase activities. 3. ALP activity was stimulated after 3- to 12-hr incubation in the presence of 10(-9) mol/l 24,25(OH)2D3 with a maximum after 6 hr. 4. The hormonal induction of ALP activity was prevented by pretreatment of cells by actinomycin D. 5. It is proposed that 24,25(OH)2D3 could increase ALP activity by de novo protein synthesis.
Asunto(s)
24,25-Dihidroxivitamina D 3/farmacología , Fosfatasa Alcalina/metabolismo , Túbulos Renales Proximales/efectos de los fármacos , Fosfatasa Ácida/biosíntesis , Fosfatasa Ácida/metabolismo , Fosfatasa Alcalina/biosíntesis , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Activación Enzimática , Inducción Enzimática , Células Epiteliales/enzimología , Hormonas/farmacología , Túbulos Renales Proximales/enzimología , Células LLC-PK1 , Microvellosidades/efectos de los fármacos , Microvellosidades/enzimología , Ratas , Porcinos , gamma-Glutamiltransferasa/biosíntesis , gamma-Glutamiltransferasa/metabolismoRESUMEN
Ca2+-dependent protein kinase C (cPKC) activity and expression have been studied in livers from hypoinsulinemic streptozotocin (STZ)-induced diabetic and untreated control rats. In diabetic rats, cPKC activity was slightly decreased in liver total particulate and nuclear fractions but was unchanged in mitochondrial-lysosomal, microsomal and cytosolic fractions. On Western immunoblot analysis, PKC alpha was identified as two distinct proteins of 90 and 81 kDa. In diabetic rats, the abundance of the 90 kDa protein was increased in most subcellular fractions with a maximum in the cytosolic and microsomal fractions (180%) but that of the 81 kDa protein was unchanged. PKC beta2 was detected as a single 81 kDa protein in cytosolic and microsomal fractions with unchanged levels in diabetic rats. Liver PKC alpha mRNA levels as measured by reverse transcription and competitive PCR amplification were similar in diabetic and control rats. The increased expression of PKC alpha protein in diabetic rats was reversed by insulin but not by phlorizin, suggesting that it did not result from hyperglycemia. We conclude that STZ-induced diabetes induces the expression of a biologically inactive form of PKC alpha which differs from active PKC alpha by an undefined post-translational modification, possibly an increase in phosphorylation state.
Asunto(s)
Diabetes Mellitus Experimental/enzimología , Expresión Génica , Insulina/sangre , Isoenzimas/genética , Hígado/enzimología , Proteína Quinasa C/genética , Animales , Glucemia/metabolismo , Núcleo Celular/enzimología , Citosol/enzimología , Diabetes Mellitus Experimental/sangre , Insulina/farmacología , Isoenzimas/análisis , Hígado/ultraestructura , Masculino , Microsomas Hepáticos/enzimología , Mitocondrias Hepáticas/enzimología , Florizina/farmacología , Proteína Quinasa C/análisis , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The effects of 1 alpha,25-dihydroxyvitamin D3 (1,25 (OH)2D3) and human growth hormone (hGH) on the activity of alkaline phosphatase (ALP) were evaluated in pig kidney LLC-PK1 cultured cells. 1,25(OH)2D3 (10(-9)-10(-6) mol L-1) and hGH (10(-7) mol L-1) increased ALP activity in these cells while no hormonal effect was detected on two other enzyme activities: gamma-glutamyl transferase and acid phosphatase. ALP activity was maximally increased after 4.5-6 h incubation with both hormones. The hormonal induction of ALP activity was prevented by a pretreatment of cells with actinomycin D. Thus, 1,25 (OH)2D3 and may be hGH could stimulate ALP activity via a transcription of some gene.
Asunto(s)
Fosfatasa Alcalina/metabolismo , Calcitriol/farmacología , Riñón/enzimología , Fosfatasa Ácida/metabolismo , Animales , Antibacterianos/farmacología , Calcitriol/antagonistas & inhibidores , Dactinomicina/farmacología , Hormona del Crecimiento/antagonistas & inhibidores , Hormona del Crecimiento/farmacología , Humanos , Riñón/efectos de los fármacos , Células LLC-PK1 , ARN/biosíntesis , Estimulación Química , Porcinos , gamma-Glutamiltransferasa/metabolismoRESUMEN
The signal transduction pathway involved in the activation of pyruvate dehydrogenase (PDH) by insulin is still unknown. In this study, we have examined the possible involvement of protein kinase C (PKC) in the process. In addressing this question, we examined (1) the insulin-like effects of the PKC activator 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) on the PDH complex, (2) the effects of various PKC inhibitors on the PDH activation by insulin, and (3) the response of PKC-depleted cells to insulin. We used as an experimental model Zajdela hepatoma cultured (ZHC) cells, which have been demonstrated to be responsive to physiological doses of insulin. Half-maximal and maximal stimulations of the PDH complex by insulin were observed at 0.05 and 5 nmol/L, respectively. Stimulation of PDH activity by insulin (5 nmol/L) occurred within 5 minutes of incubation and was maximal (+70%) at 7.5 minutes. In the presence of PMA (162 nmol/L), enzyme activity increased within 30 seconds, was maximal (+90%) at 5 minutes, and was no longer detectable after 10 minutes. Total PDH activity was unchanged by insulin or PMA treatment. The effects of PMA and insulin on basal PDH activity were not additive. Moreover, various inhibitors of PKC--staurosporine, sphingosine, acridine orange--completely blocked the stimulation of PDH activity induced by insulin or PMA. A 17-hour treatment of ZHC cells with 500 nmol/L PMA efficiently downregulated PKC, as attested by the marked decrease in the enzyme activity and the loss of phorbol 12,13-dibutyrate binding to intact cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Insulina/farmacología , Neoplasias Hepáticas Experimentales/enzimología , Proteína Quinasa C/fisiología , Complejo Piruvato Deshidrogenasa/efectos de los fármacos , Análisis de Varianza , Animales , Regulación hacia Abajo , Activación Enzimática/efectos de los fármacos , Complejo Piruvato Deshidrogenasa/metabolismo , Ratas , Células Tumorales CultivadasRESUMEN
We studied the GH-insulin-like growth factor-I (IGF-I) axis serially over 24-36 months in six patients with medulloblastoma who underwent surgical removal of the tumor followed by craniospinal irradiation therapy for 6 weeks and then chemotherapy for 42 weeks. Eighteen and 24 months after beginning irradiation there was a decline in the peak GH secretory response to acute stimulation with arginine/insulin hypoglycemia. Six months after irradiation and during chemotherapy there was a transient decline in IGF-I, IGF binding protein-3 (IGFBP-3), and GH-BP values (respective mean values of 56.1 +/- 9.0 ng/mL, 1.1 +/- 0.2 microgram/mL, and 7.6 +/- 3.3% of radioactivity as compared to time 0 values: 13%%o/- 15 ng/mL, 2.2 +/- 0.2 micrograms/mL, and 20.0 +/- 4.0%, P < 0.001), although provoked GH secretion was normal at this time. The IGF-I, IGFBP-3, and GH-BP returned to pretreatment ranges by 12-36 months after initiation of the study. There was also a decline in body mass index and serum protein values at 6 months, suggesting suboptimal nutrition during this period. Six months after irradiation in ligand and immunoblot analysis there was a decline in IGFBP-3 and an abnormal electrophoretic mobility of IGFBP-2 that were both normalized at 36 months. In one patient we observed a high level of IGFBP-3 proteolysis at this time. This study demonstrates that before the decrease of GH secretion in patients receiving cranial irradiation there is a transient phase of GH insensitivity that may be characteristic of the acute therapeutic phase including the chemotherapy. This partial insensitivity may explain the early growth retardation observed in these patients.
Asunto(s)
Antineoplásicos/uso terapéutico , Irradiación Craneana , Hormona del Crecimiento/sangre , Somatomedinas/análisis , Médula Espinal/efectos de la radiación , Adolescente , Western Blotting , Índice de Masa Corporal , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/radioterapia , Proteínas Portadoras/sangre , Niño , Irradiación Craneana/efectos adversos , Femenino , Trastornos del Crecimiento/etiología , Humanos , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina , Factor I del Crecimiento Similar a la Insulina/análisis , Masculino , Meduloblastoma/tratamiento farmacológico , Meduloblastoma/radioterapia , RadioinmunoensayoRESUMEN
The mechanism of action of growth hormone (GH) is not known, although indirect evidence suggests that protein kinase C (PKC) might play an important role in the insulin-like actions of GH. In this investigation, we directly examined the effects of GH relative to those of insulin on PKC activity in isolated rat hepatocytes. Human GH (10(-7) mol/L) significantly increased the activity of PKC in both cytosolic and particulate fractions. The effect was maximal at 1 minute, disappeared at 5 minutes, and then increased again at 30 minutes in both fractions. At 1 minute, maximal and half-maximal stimulation of PKC activity occurred at hGH concentrations of 10(-7) and 5 x 10(-9) mol/L, respectively. Insulin (10(-7) mol/L) also induced a significant and transient increase in enzyme activity at 2 minutes in cytosolic and particulate fractions; at 30 minutes, PKC activity was decreased in the soluble fraction (-17%) and increased in the particulate fraction (+65%). Measurement of specific [3H]-phorbol dibutyrate (PDBu) binding suggested translocation of PKC from the cytosol to the membrane fraction after 30 minutes of incubation, only after insulin treatment. The early effects of GH and insulin on PKC activity were additive in both the particulate and cytosolic fractions. Although the later effects of GH and insulin on PKC were quite different, both hormones rapidly activated PKC in isolated hepatocytes, suggesting that PKC might be involved in triggering the insulin-like actions of GH.
Asunto(s)
Hormona del Crecimiento/farmacología , Hígado/enzimología , Proteína Quinasa C/metabolismo , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Insulina/farmacología , Hígado/citología , Masculino , Forbol 12,13-Dibutirato/metabolismo , Proteína Quinasa C/análisis , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , TritioRESUMEN
We report the case of a boy who developed a motor neuropathy during infectious episodes at 18 mo and 3 y of age. When he was 7 y old, he suffered persistent weakness and areflexia; his resting lactate and pyruvate values were 3.65 mM and 398 microM, respectively (controls: 1.1 +/- 0.3 mM and 90 +/- 22 microM), and an exercise test demonstrated a lactic acidosis (13.6 mM; controls: 6.4 +/- 1.3 mM) with a high pyruvate level (537 microM; controls: 176 +/- 15 microM) and a low lactate/pyruvate ratio (24.2; controls: 35 +/- 2). The results of polarographic studies on muscle mitochondria suggested a defect in pyruvate oxidation (pyruvate 17 ng atom O/min/mg protein; controls: 115 +/- 42), whereas glutamate, palmitoylcarnitine, and succinate were good respiratory substrates. The activity of total pyruvate dehydrogenase complex (PDHC) in muscle mitochondria and in fresh mononuclear cells was markedly decreased (9.7 and 0.054 nmol 14CO2/min/mg protein, respectively; controls: 123 +/- 4.5 and 0.733 +/- 0.03, respectively). Immunochemical analysis in muscle mitochondria demonstrated an absence of the alpha and beta E1 PDHC subunits. After 2 y of treatment with 500 mg/d thiamine, the patient was clinically improved. A genetic study of the main regions of mutations (exon 10 and 11) in the X chromosome encoding for the E1 alpha subunit of PDHC did not show any mutation. These data indicate that, although genetically different, this case enters in a very rare category of patients with PDHC deficiency without cerebral dysfunction and improved by thiamine + L-carnitine therapy.
Asunto(s)
Enfermedad de la Neurona Motora/enzimología , Enfermedad por Deficiencia del Complejo Piruvato Deshidrogenasa/complicaciones , Secuencia de Bases , Carnitina/administración & dosificación , Carnitina/uso terapéutico , Niño , ADN/genética , Análisis Mutacional de ADN , Quimioterapia Combinada , Humanos , Linfocitos/enzimología , Masculino , Mitocondrias Musculares/enzimología , Datos de Secuencia Molecular , Enfermedad de la Neurona Motora/tratamiento farmacológico , Enfermedad de la Neurona Motora/etiología , Complejo Piruvato Deshidrogenasa/genética , Complejo Piruvato Deshidrogenasa/inmunología , Enfermedad por Deficiencia del Complejo Piruvato Deshidrogenasa/tratamiento farmacológico , Enfermedad por Deficiencia del Complejo Piruvato Deshidrogenasa/genética , Tiamina/administración & dosificación , Tiamina/uso terapéutico , Cromosoma XRESUMEN
Human peripheral mononuclear cells (PMC) were used to examine the effects of hGH and insulin on the activity of the pyruvate dehydrogenase (PDH) complex. Incubation of PMC with 10(-7) mol/L hGH or insulin increased basal PDH activity. Hormonal effects were maximal (50-60% above control values) at 15 min. Later on, activation progressively decreased and was no longer detectable at 30 min. Total PDH activity was unaffected by hormonal treatment. PMC were subfractionated into lymphocytes and monocytes to assess the sensitivity of each cell types to the hormones. hGH significantly increased basal PDH activity in lymphocytes and monocytes (38% and 70% above control values, respectively), whereas insulin increased basal PDH activity only in monocytes (151% above control value). PMC from healthy subjects aged 1-45 yr were incubated for 15 min with 10(-7) mol/L hGH or insulin before PDH measurement. An increase of enzyme activity higher than 20% was observed in 26 patients out of 29 with hGH, and in 15 out of 18 with insulin. In conclusion, hGH is able to stimulate PDH activity of human mononuclear cells. This hormonal effect allows rapid evaluation of the cellular responsiveness of hGH in various pathophysiologic situations.
Asunto(s)
Hormona del Crecimiento/farmacología , Leucocitos Mononucleares/enzimología , Complejo Piruvato Deshidrogenasa/sangre , Adolescente , Adulto , Factores de Edad , Niño , Preescolar , Enanismo/sangre , Enanismo/enzimología , Humanos , Técnicas In Vitro , Insulina/farmacología , Cinética , Leucocitos Mononucleares/efectos de los fármacos , Linfocitos/enzimología , Persona de Mediana EdadRESUMEN
A technique using high pressure liquid chromatography gel filtration was used to evaluate GH-binding proteins (GH-BPs) in human plasma; eluate was monitored for radioactivity in a gamma-detection system connected to a computer. Plasma (200 microliters) was incubated with 125I-human (h) GH (200,000 cpm) at 4 degrees C for 20 hours. Our GH binding assay offers important gains in terms of rapidity and resolution; it has permitted a clear separation and characterization of the two GH-binding components present in human plasma.
Asunto(s)
Proteínas Portadoras/sangre , Hormona del Crecimiento/metabolismo , Cromatografía Líquida de Alta Presión , Hormona del Crecimiento/sangre , Humanos , Unión ProteicaRESUMEN
Possible regulation of GH-binding proteins (GH-BPs) in human plasma was examined. Eight children with isolated GH deficiency had a very low level of plasma GH-binding activity (10.2 +/- 1.1% of radioactivity). Under GH treatment the hormone binding to the high affinity BP (peak II-BP) increased in every patient to reach the mean value of 18.5 +/- 1.4%. In one patient, Scatchard plot analysis indicated that this increase was related to a higher binding capacity without any significant change in the binding affinity. A positive correlation existed between the GH-binding activity and insulin-like growth factor-I plasma levels. In nine boys with pubertal delay, the GH-specific binding to peak II-BP was normal (30.6 +/- 3.7% of radioactivity); it decreased significantly after testosterone treatment. In four boys with precocious puberty, the specific GH binding to peak II-BP was low (16.6 +/- 1.1%). It increased significantly to 21.6 +/- 1.1% of radioactivity after treatment with a LHRH analog. A negative correlation existed between plasma testosterone levels and GH binding to peak II-BP in boys presenting pubertal delay or precocious puberty. The high affinity GH-BP is regulated, and among the factors that play a role in this regulation, GH and testosterone have opposite effects.
Asunto(s)
Proteínas Portadoras/sangre , Hormona del Crecimiento/fisiología , Testosterona/fisiología , Adolescente , Niño , Preescolar , Hormona del Crecimiento/sangre , Hormona del Crecimiento/deficiencia , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Masculino , Pubertad Tardía/sangre , Pubertad Tardía/tratamiento farmacológico , Pubertad Precoz/sangre , Testosterona/sangre , Testosterona/uso terapéuticoRESUMEN
The plasma high affinity growth hormone binding protein corresponds to the extracellular binding domain of the liver membrane receptor. A distinct higher molecular weight protein, which binds GH with low affinity and high capacity, is present in the human plasma. In man, the biosynthesis and the exact functions of the GH binding proteins have to be clarified. The GH binding proteins are differently regulated; they are under a multihormonal control. The high affinity binding protein is low in neonates, increases after the first year of life, and reaches its highest value in young adult, without sex differences. GH is able to induce the high affinity binding protein, whereas sex steroids can decrease it. States of GH resistance, such as chronic renal failure, and certain idiopathic short statures, are associated with low levels of GH binding protein; in Laron-type dwarfism a defect of the GH receptor gene probably results in the absence of plasma GH binding activity. Recent findings on the regulation of the GH binding protein in man support a parallel regulation of liver membrane GH receptors and plasma binding protein.
Asunto(s)
Proteínas Portadoras/sangre , Hormona del Crecimiento/sangre , Homeostasis , Envejecimiento/fisiología , Animales , Hormonas Esteroides Gonadales/fisiología , Humanos , Receptores de Somatotropina/metabolismoRESUMEN
A technique using high pressure liquid chromatography gel filtration was used to evaluate GH-binding proteins (BP) in human plasma; eluate was monitored for radioactivity in a gamma-detection system connected to a computer. Plasma (200 microL) was incubated with [125I]human (h) GH (200,000 cpm) at 4 C for 20 h. The main GH-BP (peak II) was well separated from free [125I]hGH (peak III) and from a higher mol wt complex (peak I), which was minor. In our control plasma, the specific binding of [125I]hGH to peak II BP (II-BP) was 32.2 +/- 0.6% of the radioactivity. Scatchard analyses indicate an association constant of 3.6-7.4 X 10(8) M-1 and a binding capacity ranging from 24-86 ng/mL for peak II-BP in five normal adult plasma samples. Peak I material, separated from plasma of boys with pubertal delay, bound hGH with a low affinity (3 x 10(6) M-1) and a very high capacity (2 micrograms/mL). In cross-linking experiments, peak I appeared as two proteins of 165 and 174 kD; these mol wt were much higher than that of peak II-BP, previously estimated at 53,000. hGH complexed to peak II-BP remained fully immunoreactive with use of the anti-hGH antibodies of our assay. In plasma containing 10-20 micrograms/L hGH, the proportion of bound hormone (peak II) was 44.5 +/- 2.3%, whereas the amount of hGH in peak I was very low or undetectable. Specific binding of hGH to II-BP was lowest during the first year of life and highest in adulthood. No sex difference was found. I-BP is differentially regulated, since its binding activity was significantly lower in adults than in prepubertal children. Normal values for age should be taken into account to interpret GH-binding activity, particularly in children 2 yr of age or younger. Our GH binding assay offers important gains in terms of rapidity and resolution; it has permitted a clear separation and characterization of the two GH-binding components present in human plasma.
Asunto(s)
Proteínas Portadoras/sangre , Hormona del Crecimiento/sangre , Resinas Acrílicas , Adulto , Envejecimiento/sangre , Autorradiografía , Sitios de Unión , Cromatografía en Gel , Cromatografía Líquida de Alta Presión/métodos , Femenino , Humanos , RadioinmunoensayoRESUMEN
The degradation products generated from A14 and B26 125I-labelled insulins in liver endosomes in vivo and in vitro have been isolated by high-performance liquid chromatography and cleavages in the B chain have been identified by automated radiosequence analysis. In rats sacrificed various times after injection of each of the 125I-labelled insulins, two major degradation products slightly less hydrophobic than intact iodoinsulins were identified; these accounted, at 8 min. for about 45% (A14 125I-labelled insulin) and 15% (B26 125I-labelled insulin) of the total radioactivity recovered, respectively. The products generated from A14 125I-labelled insulin contained an intact A chain, whereas those generated from B26 125I-labelled insulin contained a B chain cleaved at the B16-B17 bond. With B26 125I-labelled insulin, two minor products, with cleavages at the B23-B24 and B24-B25 bonds, were also observed. In vivo chloroquine treatment did not alter the nature but caused a decrease in the amount of insulin degradation products associated with endosomes. When endosomal fractions isolated from iodoinsulin injected rats were incubated at 30 degrees C in isotonic KCl, a rapid degradation of iodoinsulin, maximal at pH 6, was observed. With A14 125I-labelled insulin, the two major degradation products identified in vivo were generated along with monoiodotyrosine, but with B26 125I-labelled insulin monoiodotyrosine was the main product formed. Addition of ATP, presumably by decreasing the endosomal pH, shifted the medium pH for maximal iodoinsulin degradation to about 7-8. These studies have allowed a direct identification of two previously suggested cleavage sites in the B chain. They have also shown that the degradation products generated in cell-free endosomes under conditions that promote endosomal acidification are similar to those identified in vivo.
Asunto(s)
Insulina/metabolismo , Hígado/metabolismo , Animales , Cloroquina/farmacología , Cromatografía Líquida de Alta Presión , Aparato de Golgi/metabolismo , Insulisina/metabolismo , Hígado/efectos de los fármacos , Masculino , Fragmentos de Péptidos/análisis , Ratas , Ratas Endogámicas , Fracciones Subcelulares/metabolismoRESUMEN
The authors report a retrospective series of 82 cases of sigmoid diverticulosis which revealed the following points: surgery is rarely performed on "cold" disease but for acute or chronic complications, diverticular disease is very often present for a very long time and the doctor or the patient has refused operation for various reasons, whenever possible, infection must be eradicated as first line treatment and continuity must be restored under protection of a right transverse colostomy which is closed 4 to 6 weeks later. The authors prefer this approach to Hartmann's operation, in which the restoration of colonic continuity appears to be more delicate and which tends to prevent the surgeon from performing a sufficiently low inferior section, the source of poor long-term results. There was no postoperative mortality in this series, which may be due to the special environment in which surgery was performed. It is preferable to operate patients with "cold" diverticulosis before the development of complications, which allows simpler one-stage surgery requiring a shorter and therefore less expansive hospital stay.