RESUMO
This guideline was developed to identify evidence-based best practices in haemophilia care delivery, and discuss the range of care providers and services that are most important to optimize outcomes for persons with haemophilia (PWH) across the United States. The guideline was developed following specific methods described in detail in this supplement and based on the GRADE (Grading of Recommendations, Assessment, Development and Evaluation approach). Direct evidence from published literature and the haemophilia community, as well as indirect evidence from other chronic diseases, were reviewed, synthesized and applied to create evidence-based recommendations. The Guideline panel suggests that the integrated care model be used over non-integrated care models for PWH (conditional recommendation, moderate certainty in the evidence). For PWH with inhibitors and those at high risk for inhibitor development, the same recommendation was graded as strong, with moderate certainty in the evidence. The panel suggests that a haematologist, a specialized haemophilia nurse, a physical therapist, a social worker and round-the-clock access to a specialized coagulation laboratory be part of the integrated care team, over an integrated care team that does not include all of these components (conditional recommendation, very low certainty in the evidence). Based on available evidence, the integrated model of care in its current structure, is suggested for optimal care of PWH. There is a need for further appropriately designed studies that address unanswered questions about specific outcomes and the optimal structure of the integrated care delivery model in haemophilia.
Assuntos
Gerenciamento Clínico , Hemofilia A/terapia , Autoanticorpos/sangue , Atenção à Saúde/métodos , Atenção à Saúde/organização & administração , Atenção à Saúde/normas , Medicina Baseada em Evidências , Hemofilia A/patologia , Humanos , Pesquisa , Fatores de RiscoRESUMO
The addition of the calcium ionophore A23187 to rabbit neutrophils increases the amount of actin associated with the cytoskeleton regardless of the presence or absence of calcium in the incubation medium. In the presence of extracellular calcium, the effect of A23187 is biphasic with respect to concentration. The action of the ionophore is rapid, transient, and is inhibited by pertussis toxin, hyperosmolarity, and quinacrine. On the other hand, the addition of pertussis toxin or hyperosmolarity has small if any, effect on the rise in intracellular calcium produced by A23187. While quinacrine does not affect the fMet-Leu-Phe-induced increase in cytoskeletal actin and the polyphosphoinositide turnover, its addition inhibits completely the stimulated increase in Ca-influx produced by the same stimulus. The results presented here suggest that a rise in the intracellular concentration of free calcium is neither necessary nor sufficient for the stimulated increase in cytoskeletal-associated actin. A possible relationship between the lipid remodeling stimulated by chemoattractants and the increased cytoskeletal actin is discussed.
Assuntos
Actinas/metabolismo , Cálcio/metabolismo , Citoesqueleto/metabolismo , Neutrófilos/metabolismo , Aminoquinolinas , Animais , Ácido Araquidônico , Ácidos Araquidônicos/farmacologia , Calcimicina/farmacologia , Proteínas do Citoesqueleto/isolamento & purificação , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/efeitos dos fármacos , Corantes Fluorescentes , Cinética , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Coelhos , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Stimulation of rabbit neutrophils by the chemotactic factors fMet-Leu-Phe and leukotriene B4, by platelet activating factor, or by arachidonic acid produces a rapid and dose-dependent increase in the amounts of actin and of a 65,000-mol-wt protein associated with the cytoskeleton. Phorbol 12-myristate, 13-acetate, the calcium ionophore A23187 in the presence or absence of EGTA, and the fluorescent calcium chelator quin-2 also cause an increase in cytoskeletal actin. The stimulated increases in the cytoskeletal actin are not dependent on a rise in the intracellular concentration of free calcium and are not mediated by an increase in the intracellular pH or activation of protein kinase C. The increases in the cytoskeletal actin produced by fMet-Leu-Phe and leukotriene B4, but not by phorbol 12-myristate, 13-acetate, are inhibited by high osmolarity. The effect of hyperosmolarity requires a decrease in cell volume, is not mediated by an increase in basal intracellular concentration of free calcium, and is not prevented by pretreating the cells with amiloride. Preincubation of the cells with hyperosmotic solution also inhibits degranulation produced by all the stimuli tested. The inhibitory action of high osmolarity on the fMet-Leu-Phe and leukotriene B4 induced stimulation of cytoskeletal actin is discussed in terms of the possibility that the addition of high osmolarity, either directly or through activation of protein kinase C, causes receptor uncoupling.
Assuntos
Actinas/metabolismo , Citoesqueleto/ultraestrutura , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/ultraestrutura , Aminoquinolinas/farmacologia , Animais , Ácido Araquidônico , Ácidos Araquidônicos/farmacologia , Calcimicina/farmacologia , Citoesqueleto/efeitos dos fármacos , Glucose/farmacologia , Humanos , Leucotrieno B4/farmacologia , Peso Molecular , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Coelhos , Acetato de Tetradecanoilforbol/farmacologia , Ureia/farmacologia , Equilíbrio HidroeletrolíticoRESUMO
Cytochemical techniques have been employed to study the localization of adenylate cyclase and (Ca2+ + Mg2+)-stimulated ATPase activities in platelets after fixation. Biochemical analysis of adenylate cyclase demonstrated a 70% reduction in activity in homogenates from fixed cells, but the residual activity could be stimulated 10--20 times by prostaglandin E1 (1 micrometer) under the same incubation conditions as employed in the cytochemical studies (e.g. media containing 2 mM lead nitrate and 10 mM NaF). Adenylate cyclase activity employing 5'-adenylyl-imiodiphosphate (AMP-P(NH)P) as substrate was found to be associated with the dense tubular system (smooth endoplasmic reticulum) in intact fixed platelets, and was apparent only when the cells were incubated with prostaglandin E1. Less activity was found along the membranes of the surface connected open canalicular system and occasionally at the outer cell surface. Enzymatic activity was blocked by the adenylate cyclase inhibitor 9-(tetrahydro-2-furyl) adenine and was not due to AMP-P(NH)P phosphohydrolase activity. The low adenylate cyclase activity in the surface membranes may be due to enzyme inactivation as a result of fixation, since a surface membrane fraction obtained by the glycerol lysis technique from unfixed cells had an adenylate cyclase specific activity equivalent to that in the microsomal membrane fraction. (Ca2+ + Mg2+)-stimulated ATPase activity was found associated with the membranes of the surface connected open canalicular system in unfixed cells. After brief fixation (5--15 min) with glutaradehyde, strong (Ca2+ + Mg2+)ATPase activity became apparent in the dense tubular system. Longer periods of fixation inactivated enzymatic activity. Addition of Ca2+ (1.0 mM) to incubation medium with low Mg2+ (0.2 mM), or increasing Mg2+ to 4.0 mM, in both cases strongly stimulated enzyme activity. The ATPase activity in the platelet membranes was not inhibited by ouabain. It is suggested that the Ca2+-stimulated ATPase and adenylate cyclase activities in the dense tubules may possibly be involved in regulation of intracellular Ca2+ transport.
Assuntos
Adenilil Ciclases/sangue , Plaquetas/enzimologia , ATPases Transportadoras de Cálcio/sangue , Transporte Biológico Ativo , Plaquetas/ultraestrutura , Cálcio/sangue , Ativação Enzimática/efeitos dos fármacos , Humanos , Membranas Intracelulares/enzimologia , Magnésio/farmacologia , Ouabaína/farmacologiaRESUMO
Platelets were briefly fixed in paraformaldehyde/glutaraldehyde and then incubated with 5'-adenylyl imidodiphosphate under conditions suitable for the cytochemical detection of adenylate cyclase activity. The adenylate cyclase activity of these platelets retains the ability to respond to prostaglandins E1, D2, I2 (prostacyclin), forskolin and fluoride. Sites of stimulated adenylate cyclase activity were localized cytochemically by the reaction of lead with the reaction product imidodiphosphate to form deposits of lead imidodiphosphate that are visible in the electron microscope. Reaction product deposition was seen only in the dense tubule system of human platelets when the incubation medium contained forskolin, prostacyclin, or prostaglandin D2 at concentrations known to stimulate the enzyme in intact platelets. Epinephrine, an antagonist of adenylate cyclase inhibited the cytochemical reaction stimulated by prostacyclin. The fact that the cytochemical reaction was induced by agonists that stimulate the enzyme through two different types of prostaglandin receptors and by forskolin, which acts distal to the receptors, confirms that the method specifically detects adenylate cyclase. The presence of adenylate cyclase in the dense tubules may be significant for the regulation of intracellular Ca2+ and arachidonic acid metabolism by this membrane system.
Assuntos
Adenilil Ciclases/sangue , Plaquetas/efeitos dos fármacos , Diterpenos/farmacologia , Prostaglandinas/farmacologia , Plaquetas/metabolismo , Plaquetas/ultraestrutura , Cálcio/sangue , Colforsina , Epoprostenol/farmacologia , Histocitoquímica , Humanos , Técnicas In Vitro , Membranas Intracelulares/metabolismo , Microscopia Eletrônica , Prostaglandina D2 , Prostaglandinas D/farmacologiaRESUMO
Activators of protein kinase C, such as tumor-promoting phorbol esters (e.g., phorbol myristate acetate), mezerein, (-)-indolactam V and 1-oleoyl 2-acetoyl glycerol, potentiate arachidonic acid release caused by elevation of intracellular Ca2+ with ionophores. This action of protein kinase C-activators required protein phosphorylation, and was attributed to enhanced hydrolysis of phospholipids by phospholipase A2 (Halenda, et al. (1989) Biochemistry 28, 7356-7363). Recently Fuse et al. ((1989) J. Biol. Chem 264, 3890-3895) reported that the apparent enhanced release of arachidonate was actually due to inhibition of the processes of re-uptake and re-esterification of released arachidonic acid. They attributed this to loss of arachidonyl-CoA synthetase and arachidonyl-CoA lysophosphatide acyltransferase activities, which were measured in membranes obtained from phorbol myristate acetate-treated platelets. In this paper, we show that phorbol myristate acetate, at concentrations that strongly potentiate arachidonic acid release, does not inhibit either arachidonic acid uptake into platelets or its incorporation into specific phospholipids. Furthermore, the fatty acid 8,11,14-eicosatrienoic acid, a competitive substrate for arachidonyl-CoA synthetase, totally blocks arachidonic acid uptake into platelets, but, unlike phorbol myristate acetate, does not potentiate arachidonic acid release by Ca2+ ionophores. We conclude that the action of phorbol myristate acetate is to promote the process of arachidonic acid release by phospholipase A2.
Assuntos
Ácidos Araquidônicos/sangue , Plaquetas/efeitos dos fármacos , Fosfolipídeos/sangue , Acetato de Tetradecanoilforbol/farmacologia , Ácido 8,11,14-Eicosatrienoico/farmacologia , Ácido Araquidônico , Plaquetas/metabolismo , Calcimicina/farmacologia , Humanos , Técnicas In VitroRESUMO
The microviscosity of artificial lipid membranes and natural membranes was measured by the fluorescence polarization technique employing perylene as the probe. Lipid dispersions composed of brain gangliosides exhibited greater microviscosity than phosphatidylserine (268 cP vs 173 cP, at 25 degrees C). Incorporation of cholesterol (30-50%) increased the microviscosity of lipid phases by 200-500 cP. Cholesterol's effect on membrane fluidity was completely reversed by digitonin but not by amphotericin B. Incorporation of membrane proteins into lipid vesicles gave varying results. Cytochrome b5 did not alter membrane fluidity. However, myelin proteolipid produced an apparent increase in microviscosity, but this effect might be due to partitioning of perylene between lipid and protein binding sites since tha latter have a higher fluorescence anisotropy than the lipid. The local anesthetics tetracain and butacaine increased the fluidity of lipid dispersions, natural membranes and intact ascites tumor cell membranes. The effect of anesthetics appears to be due to an increased disordering of lipid structure. The fluidity of natural membranes at 25 degrees C varied as follows: polymorphonuclear leukocytes, 335 cP; bovine brain myelin, 270 cP; human erythrocyte, 180 cP; rat liver microsomes, 95 cP; rat liver mitochondria, 90 cP. In most cases the microviscosity of natural membranes reflects their cholesterol: phospholipid ratio. The natural variations in fluidity of cellular membranes probably reflect important functional requirements. Similarly, the effects of some drugs which alter membrane permeability may be the result of their effects on membrane fluidity.
Assuntos
Membrana Celular/ultraestrutura , Colesterol , Gangliosídeos , Membranas Artificiais , Membranas/ultraestrutura , Fosfatidiletanolaminas , Fosfatidilserinas , Proteínas , Tetracaína , Animais , Membrana Celular/efeitos dos fármacos , Colesterol/farmacologia , Leucócitos/efeitos dos fármacos , Membranas/efeitos dos fármacos , Microssomos Hepáticos/ultraestrutura , Mitocôndrias Hepáticas/ultraestrutura , Modelos Biológicos , Proteínas/farmacologia , Coelhos , Ratos , Espectrometria de Fluorescência , Temperatura , Tetracaína/farmacologia , Termodinâmica , ViscosidadeRESUMO
The regulation of the phosphorylation of the acetylcholine receptor in electroplax membranes from Torpedo californica and of purified acetylcholine receptor was investigated. The phosphorylation of the membrane-bound acetylcholine receptor was not stimulated by Ca2+/calmodulin, nor was it inhibited by EGTA, but it was stimulated by the catalytic subunit of cAMP-dependent protein kinase, and was blocked by the protein inhibitor of cAMP-dependent protein kinase. Purified acetylcholine receptor was not phosphorylated by Ca2+/calmodulin-dependent protein kinase activity in electroplax membranes, nor by partially purified Ca2+/calmodulin-dependent protein kinases from soluble or particulate fractions from the electroplax. Of the four acetylcholine receptor subunits, termed alpha, beta, gamma and delta, only the gamma- and delta-subunits were phosphorylated by the cAMP-dependent protein kinase (+ cAMP), or by its purified catalytic subunits.
Assuntos
Cálcio/metabolismo , Calmodulina/metabolismo , AMP Cíclico/metabolismo , Órgão Elétrico/metabolismo , Receptores Colinérgicos/metabolismo , Animais , Centrifugação com Gradiente de Concentração , Eletroforese em Gel de Poliacrilamida , Peso Molecular , Fosforilação , Proteínas Quinases/metabolismo , TorpedoRESUMO
Ca2+, Mg2+-ionophores X537A and A23,187 (10(-7)-10(-6) M) induced the release of adenine nucleotides adenosine diphosphate (ADP, adenosine triphosphate (ATP), serotonin, beta-glucuronidase, Ca2+, and Mg2+ from washed human platelets. Enzymes present in the cytoplasm or mitochondria, and Zn2+ were not released. The rate of ATP and Ca2+ release measured by firefly lantern extract and murexide dye, respectively, was equivalent to that produced by the physiological stimulant thrombin. Ionophore-induced release of ADP, and serotonin was substantially (approximately 60%) but not completely inhibited by EGTA, EDTA, and high extracellular Mg2+, without significant reduction of Ca2+ release. The ionophore-induced release reaction is therefore partly dependent upon uptake of extracellular Ca2+ (demonstrated using 45Ca), but also occurs to a significant extent due to release into the cytoplasm of intracellular Ca2+. The ionophore-induced release reaction and aggregation of platelets could be blocked by prostaglandin E1 (PGE1) or dibutyryl cyclic AMP. The effects of PGE1, and N6, O2-dibutyryl adenosine 3':5'-cyclic monophosphoric acid (dibutyryl cAMP) were synergistically potentiated by the phosphodiesterase inhibitor theophylline. It is proposed that Ca2+ is the physiological trigger for platelet secretion and aggregation and that its intracellular effects are strongly modulated by adenosine 3':5'-cyclic monophosphoric acid (cyclic AMP).
Assuntos
Antibacterianos/farmacologia , Plaquetas/efeitos dos fármacos , Bucladesina/farmacologia , Calcimicina/farmacologia , Lasalocida/farmacologia , Prostaglandinas E/farmacologia , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Plaquetas/enzimologia , Plaquetas/metabolismo , Calcimicina/antagonistas & inibidores , Cálcio/metabolismo , Ácido Edético/farmacologia , Ácido Egtázico/farmacologia , Glucuronidase/metabolismo , Humanos , Lasalocida/antagonistas & inibidores , Magnésio/metabolismo , Magnésio/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Serotonina/metabolismo , Teofilina/farmacologia , Trombina/farmacologia , Zinco/metabolismoRESUMO
To study the role of the inositol 1,3,4,5-trisphosphate-binding protein GAP1(IP4BP) in store-operated Ca2+ entry, we established a human erythroleukemia (HEL) cell line in which the expression of GAP1(IP4BP) was substantially reduced by transfection with a vector containing antisense DNA under control of a Rous Sarcoma virus promoter and the Escherichia coli LacI repressor (AS-HEL cells). Control cells were transfected with vector lacking antisense DNA (V-HEL cells). GAP1(IP4BP) protein, which is a member of the GTPase-activating protein (GAP1) family, was reduced by 85% in AS-HEL cells and was further reduced by 96% by treatment with isopropylthio-beta-D- galactoside to relieve LacI repression. The loss of GAP1(IP4BP) was associated with both a membrane hyperpolarization and a substantially increased Ca2+ entry induced by thrombin or thapsigargin. The activation of intermediate conductance Ca2+-activated K+ channels in AS-HEL cells (not seen in V-HEL cells) was responsible for the membrane hyperpolarization and the enhanced Ca2+ entry, and both were blocked by charybdotoxin. Stimulated V-HEL cells did not hyperpolarize and basal Ca2+ influx was unaffected by charybdotoxin. In V-HEL cells hyperpolarized by removal of extracellular K+, the thapsigargin-stimulated Ca2+ influx was increased. Expression of mRNA for the human Ca2+-activated intermediate conductance channel KCa4 was equivalent in both AS-HEL and V-HEL cells, suggesting that the specific appearance of calcium-activated potassium current (IK(Ca)) in AS-HEL cells was possibly due to modulation of preexisting channels. Our results demonstrate that GAP1(IP4BP), likely working through a signaling pathway dependent on a small GTP-binding protein, can regulate the function of K(Ca) channels that produce a hyperpolarizing current that substantially enhances the magnitude and time course of Ca2+ entry subsequent to the release of internal Ca2+ stores.
Assuntos
Cálcio/metabolismo , DNA Antissenso/genética , Canais de Potássio/metabolismo , Receptores Citoplasmáticos e Nucleares/biossíntese , Receptores Citoplasmáticos e Nucleares/genética , Western Blotting , Linhagem Celular , DNA Antissenso/biossíntese , Estimulação Elétrica , Eletrofisiologia , Fura-2 , Humanos , Leucemia Eritroblástica Aguda/metabolismo , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
The aim of this study is to determine if COPD patients undergoing lung resection with perioperative ß-blocker use are more likely to suffer postoperative COPD exacerbations than those that did not receive perioperative ß-blockers. Methods. A historical cohort study of COPD patients, undergoing lung resection surgery at Memorial Sloan-Kettering Cancer Center between 2002 and 2006. Primary outcomes were the rate of postoperative COPD exacerbations, defined as any initiation or increase of glucocorticoids for documented bronchospasm. Results. 520 patients with COPD were identified who underwent lung resection. Of these, 205 (39%) received perioperative ß-blockers and 315 (61%) did not. COPD was mild among 361 patients (69% of all patients), moderate in 117 patients (23%), and severe in 42 patients (8%). COPD exacerbations occurred among 11 (5.4%) patients who received perioperative ß-blockers and among 20 (6.3%) patients who did not. Secondary outcomes, which included respiratory failure, 30-day mortality, and the presence or absence of any cardiovascular complication, ICU transfer, cardiovascular complication, or readmission within 30 days, did not differ in prevalence between the two groups. Conclusions. This study implies that perioperative ß-blockers use among COPD patients undergoing lung resection surgery does not impact the rate of exacerbations.
RESUMO
A case of false hyperthyrotropinemia was investigated. The serum of the patient contained heterophilic antibodies against rabbit immunoglobulins secondary to immunization with rabbit serum. Some vaccines against viral or bacterial diseases contain animal serum capable of inducing heterophilic antibodies in man. This technical problem can be prevented by the routine addition of control rabbit serum or immunoglobulins to the TSH RIA.
Assuntos
Anticorpos Heterófilos , Imunoglobulina G/imunologia , Tireotropina/sangue , Animais , Reações Falso-Positivas , Feminino , Humanos , Imunoglobulinas , Pessoa de Meia-Idade , Coelhos/imunologia , RadioimunoensaioRESUMO
This report concerns the evaluation of various estrogens, estrone (El), estradiol (E2), and estrone sulfate (E1S), as well as E1S-sulfatase and aromatase activities in pre- and postmenopausal women with breast cancer. The levels (in picomoles per g; mean +/- SEM) of the various estrogens in the breast tissue from premenopausal patients (n = 11) are: El, 1.4 +/- 0.5; E2, 1.2 +/- 0.6; and E1S, 1.2 +/- 0.3. In postmenopausal patients (n = 23), the values are, respectively, 1.0 +/- 0.4, 1.4 +/- 0.7, and 3.3 +/- 1.9. These concentrations of estrogens in the tumors of postmenopausal patients are significantly higher than those found in plasma. The activity of E1S-sulfatase in both pre- and postmenopausal patients was 50-200 times higher than that of aromatase. E1S-sulfatase and aromatase activities are significantly higher in post-menopausal than in cycling patients. It is concluded that despite the low levels of circulating estrogens in postmenopausal patients, the tissue concentrations of these steroids are several-fold higher than those in plasma, suggesting tumor accumulation of these estrogens. The physiopathology and clinical significance of these high levels of the various estrogens (E1, E2, and E1S) as well as sulfatase and aromatase activities in postmenopausal patients with breast cancer is yet to be explored.
Assuntos
Aromatase/metabolismo , Neoplasias da Mama/química , Neoplasias da Mama/enzimologia , Mama/química , Estrogênios/análise , Sulfatases/metabolismo , Adulto , Idoso , Mama/enzimologia , Neoplasias da Mama/sangue , Estradiol/análise , Estradiol/sangue , Estrogênios/sangue , Estrona/análogos & derivados , Estrona/análise , Estrona/sangue , Feminino , Humanos , Ciclo Menstrual , Pessoa de Meia-Idade , Pós-Menopausa , Pré-Menopausa , Valores de Referência , Sensibilidade e EspecificidadeRESUMO
Short term treatment with GnRH agonists has been reported to increase plasma gonadotropin alpha-subunit (Gn alpha) levels while decreasing plasma immunoreactive LH (IR-LH) levels. In this study we examined the effect of D-Trp6-LHRH (LHRH-A) in microcapsules (60 micrograms/kg, im, every 28 days for 1 yr) in 13 girls suffering from precocious puberty. Plasma IR-Gn alpha was measured by RIA; plasma IR-LH and IR-FSH were measured by both polyclonal RIAs and monoclonal immunoradiometric assays (IRMA). Before treatment, basal IR-LH and IR-FSH levels and peak responses to LHRH measured by both RIA and IRMA were similar, and the Gn alpha response paralleled that of LH. After the first injection of LHRH-A, RIA LH levels were significantly higher than pretreatment levels until day 21, while IRMA LH levels transiently increased, but returned to pretreatment levels by day 7 and became lower thereafter (P less than 0.005). Plasma IR-Gn alpha levels increased from days 3-21 (P less than 0.05). After 1.5 months of treatment, basal RIA LH levels remained detectable and not different from pretreatment levels; IRMA LH levels were very low. The mean RIA and IRMA LH responses to LHRH were decreased at 1.5 and 12 months (P less than 0.01). Basal plasma RIA and IRMA FSH levels were similar during treatment (P greater than 0.05) and significantly lower than pretreatment values (P less than 0.01). The mean RIA and IRMA FSH responses to LHRH decreased significantly at 1.5 months (P less than 0.001). After 12 months, both RIA and IRMA FSH responses were increased, but IRMA values were significantly lower than RIA values. A sustained increase in basal Gn alpha values occurred, but there was a tendency for the peak levels after LHRH treatment to decrease, becoming significantly lower than pretreatment peak levels after 1 yr. The chromatographic analysis on Sephadex G-100 of a pool of plasma samples collected during a LHRH test in three children treated for 6 months indicated that IR-Gn alpha coeluted with [125I]Gn alpha. The large discrepancy between RIA and IRMA LH values suggests the secretion of unusual LH molecules which are recognized by RIA but not by IRMA. The sustained release of large amounts of IR-Gn alpha indicates dissociated effects of LHRH-A on alpha- and beta-subunit secretion by the gonadotrophs. The sustained response of Gn alpha to LHRH demonstrates that gonadotroph cell LHRH receptors are still responsive to LHRH during treatment with a LHRH agonist.
Assuntos
Hormônio Liberador de Gonadotropina/análogos & derivados , Gonadotropinas/metabolismo , Hipófise/efeitos dos fármacos , Puberdade Precoce/tratamento farmacológico , Cápsulas , Criança , Pré-Escolar , Cromatografia , Estudos de Avaliação como Assunto , Feminino , Hormônio Liberador de Gonadotropina/uso terapêutico , Gonadotropinas/classificação , Humanos , Puberdade Precoce/sangue , Fatores de Tempo , Pamoato de TriptorrelinaRESUMO
TRH is present in human normal pituitaries and in pituitary adenomas. In this study we demonstrated that the same tissues can release TRH in vitro. Fragments from seven normal pituitaries (10-15 mg/syringe) and dispersed cells from eight prolactinomas, four GH-secreting and two nonsecreting adenomas (1-3 x 10(6) cells/syringe) were perifused using a Krebs-Ringer culture medium. After 1 h of equilibration the perifusion medium was collected every 2 min (1 mL/fraction) for 3 h. TRH, PRL, and GH were measured by RIA under basal conditions and in the presence of 10(-10) to 10(-6) mol/L dopamine (DA), alone or concomitant with haloperidol, or in the presence of 10(-10) or 10(-6) mol/L somatostatin. Both normal pituitary fragments and pituitary adenomatous cells (from all types of adenomas studied) spontaneously released TRH in vitro. TRH was detected in the perifusion medium either immediately after the end of the equilibration period or 30-60 min later. The molecular identity of TRH was assessed by high pressure liquid chromatography. There was no difference in the profile and the rate of TRH secretion between normal and tumoral tissues, and no correlation was found between the level of TRH release and that of PRL or GH secretion. DA stimulated TRH release from normal pituitaries and from PRL- and GH-secreting adenomas at doses as low as 10(-10) mol/L. A concomitant decrease in PRL and GH release was observed from adenomatous cells and in one case of normal tissue. Haloperidol (10(-7) mol/L) antagonized the effect of 10(-8) mol/L DA on both TRH and PRL secretion in normal pituitary and in prolactinomas. DA had no effect on TRH release from two nonsecreting tumors. The amounts of TRH released during 1 h of perifusion were 60-1640 pg/2 mg wet wt tissue in normal pituitaries and 54-2174 pg/10(6) cells in adenomas; these values were very high compared to those precedently reported within the tissues. These results indicate that pituitary cells can release TRH in vitro and suggest that TRH might be synthesized in situ. We suggest that TRH could act on pituitary hormone secretion and/or cell proliferation via a paracrine and/or an autocrine mechanism.
Assuntos
Adenoma/metabolismo , Dopamina/farmacologia , Haloperidol/farmacologia , Hipófise/metabolismo , Neoplasias Hipofisárias/metabolismo , Somatostatina/farmacologia , Hormônio Liberador de Tireotropina/metabolismo , Adenoma/patologia , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Técnicas In Vitro , Cinética , Masculino , Pessoa de Meia-Idade , Perfusão , Hipófise/efeitos dos fármacos , Hipófise/patologia , Neoplasias Hipofisárias/patologia , Valores de Referência , Hormônio Liberador de Tireotropina/isolamento & purificaçãoRESUMO
In a TSH screening program for congenital hypothyroidism we detected seven newborn infants with normal plasma T4 and T3 levels but high immunoassayable TSH. Similar findings were obtained in their mothers. Serial plasma dilution curves, with and without the addition of normal rabbit serum to the samples, showed that the result of TSH assay performed with antihuman TSH rabbit antiserum was falsely elevated in mothers and infants by an interfering factor. Follow-up of the infants demonstrated that the falsely elevated plasma TSH levels returned to normal within the first 6 months of life. On the contrary, plasma TSH levels remained high in the mothers. These results suggested a placental transfer of maternal antibodies. Indeed, the analysis of the mothers anamnesis revealed that all had previously received injections of a microbial vaccine cultured on a rabbit lung-containing medium. We conclude that placental transfer of a maternal antirabbit factor may cause an artefactual hyperthyrotropinemia in the newborn and the incorrect diagnosis of neonatal hypothyroidism. This can be avoided by the addition of normal rabbit serum or immunoglobulin to the TSH RIA tubes.
Assuntos
Hipotireoidismo/diagnóstico , Recém-Nascido , Troca Materno-Fetal , Tireotropina/sangue , Animais , Reações Falso-Positivas , Feminino , Humanos , Doenças do Recém-Nascido/diagnóstico , Cinética , Gravidez , Coelhos , RadioimunoensaioRESUMO
Intact platelets were stimulated with thrombin and the amount of GTP-binding protein (G-protein) oligomers was assessed by measuring ADP ribosylation of 40-41 kDa protein by pertussis toxin in isolated membranes. The toxin substrate fell by 57-62% in 10-60 s, but then returned towards normal over 5 min. Recovery was greatly enhanced by removal of thrombin from receptors with hirudin. Phorbol myristate acetate increased ADP-ribosylatable protein, but only back to initial levels prior to PMA. In contrast prostaglandin D2 plus theophylline (which increase cyclic AMP) did not increase ADP ribosylation, but could completely block the fall of the toxin substrate caused by thrombin. These results indicate that activation of thrombin receptors promotes the dissociation of G-protein oligomers to release free alpha-subunits, and this effect can be modulated by protein kinase C and cyclic AMP-dependent protein kinase. The possible relationships of these findings to the regulation of stimulus-response coupling in platelets is discussed.
Assuntos
Plaquetas/efeitos dos fármacos , Glicoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Forbóis/farmacologia , Prostaglandinas D/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Trombina/farmacologia , Adenosina Difosfato Ribose/metabolismo , Hirudinas/farmacologia , Humanos , Peso Molecular , Toxina Pertussis , Glicoproteínas da Membrana de Plaquetas , Prostaglandina D2 , Proteína Quinase C/farmacologia , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Trombina , Trombina/metabolismo , Fatores de Virulência de Bordetella/farmacologiaRESUMO
The Ca2+-mobilizing action of thrombin was demonstrated in a cell-free platelet membrane system consisting of open sheets of plasma membrane plus sealed membrane vesicles that accumulate Ca2+ and release Ca2+ in response to IP3. Thrombin plus GTP, acting on plasma membrane (not vesicles), produced a soluble factor (destroyed by alkaline phosphatase) that released Ca2+ from the vesicles. This effect of thrombin/GTP was blocked by a monoclonal antibody that binds to vesicles and prevents Ca2+ release by IP3. Pertussis toxin plus NAD ADP-ribosylated plasma membrane polypeptides of 39 and 41 kDa and blocked Ca2+ release by thrombin/GTP, but not by IP3.
Assuntos
Plaquetas/metabolismo , Cálcio/metabolismo , Adenosina Difosfato Ribose/metabolismo , Anticorpos Monoclonais , Plaquetas/efeitos dos fármacos , Membrana Celular/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/farmacologia , Humanos , Técnicas In Vitro , Inositol 1,4,5-Trifosfato , Fosfatos de Inositol/farmacologia , Toxina Pertussis , Trombina/farmacologia , Fatores de Virulência de Bordetella/farmacologiaRESUMO
The effects of pretreatment of rabbit neutrophils with phorbol 12-myristate 13-acetate on the ability of pertussis toxin to catalyze ADP-ribosylation and of fMet-Leu-Phe to activate a high-affinity GTPase in these cell homogenates were examined. The addition of phorbol 12-myristate 13-acetate, but not 4 alpha-phorbol 12,13-didecanoate, to intact cells was found to stimulate by more than 100% the pertussis toxin-dependent ribosylation of a 41 kDa protein (either the alpha-subunit of the 'inhibitory' guanine nucleotide-binding protein N or a closely analogous protein) and to inhibit by more than 60% the activation by fMet-Leu-Phe of the GTPase of the neutrophil homogenates. The addition of fMet-Leu-Phe to intact cells increases the ADP-ribosylation catalyzed by pertussis toxin of the 41 kDa protein. On the other hand, the exposure of neutrophil homogenates to fMet-Leu-Phe results in a decreased level of ADP-ribosylation. This decreased ribosylation reflects a dissociation of the GTP-binding protein oligomer that is not followed by association, possibly because of the release of the alpha-subunit into the suspending media. The implications of these results for the understanding of the mechanism of inhibition of cell responsiveness by phorbol esters and the heterologous desensitization phenomenon are discussed. Prominent among these are the possibilities that (i) the rate of dissociation of the Ni oligomer is affected by the degree of its phosphorylation by protein kinase C, and/or (ii) the dissociated phosphorylated alpha-subunit (the 41 kDa protein) is functionally less active than its dephosphorylated couterpart.
Assuntos
Adenosina Difosfato Ribose/metabolismo , GTP Fosfo-Hidrolases/análise , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Açúcares de Nucleosídeo Difosfato/metabolismo , Toxina Pertussis , Forbóis/farmacologia , Monoéster Fosfórico Hidrolases/análise , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Virulência de Bordetella/farmacologia , Animais , GTP Fosfo-Hidrolases/antagonistas & inibidores , Proteínas de Ligação ao GTP/metabolismo , Técnicas In Vitro , Neutrófilos/metabolismo , Fosforilação , Proteína Quinase C/análise , CoelhosRESUMO
The ultrastructural cytochemical localization of a potassium-dependent oubain-sensitive nitrophenyl phosphatase (transport ATPase) activity in human blood platelets is described. This potassium-dependent nitrophenyl phosphatase activity was not affected by 5 mM levamisole, indicating that the reaction product identified was not due to nonspecific alkaline phosphatase activity. The K+-dependent nitrophenyl phosphatase was strictly localized to the platelet plasma membrane, while the open canalicular system and dense tubular system were devoid of reaction product. In contrast, (Ca2+,Mg2+)-activated ATPase activity was predominantly localized in the open canalicular system and dense tubular system with very little cytochemical activity expressed at the plasma membrane. These data demonstrate a relative segregation of these enzymes into unique membrane compartments of the human platelet. Such data may be useful with regard to identification of purified membrane fractions from platelets and may be significant with regard to the understanding of the function(s) of the different membrane compartments of the human platelet.