RESUMO
At the neuromuscular junction (NMJ), changes to the size of the postsynaptic potential induce homeostatic compensation. At the Drosophila NMJ, increased glutamate release causes a compensatory decrease in quantal content, but it is unknown if this mechanism operates at the cholinergic mammalian NMJ. We addressed this question by recording endplate potentials (EPP) and muscle contraction in 3-month and 24-month ChAT-ChR2-EYFP mice that overexpress vesicular acetylcholine transporter and release more acetylcholine per vesicle. At 3 months, the quantal content of EPPs from ChAT-ChR2-EYFP mice were not different from WT controls, however tetanic depression was greater, and quantal size during high-frequency stimulation and the size of the readily releasable pool (RRP) were decreased. At 24 months of age, quantal content was reduced in ChAT-ChR2-EYFP mice, which normalized synaptic depression despite smaller RRP. The effect of pancuronium on indirect evoked muscle twitch was not different between groups. These results indicate that an increase in the amount of acetylcholine per vesicle induces two distinct age-dependent homeostatic mechanisms compensating excessive acetylcholine release.
Assuntos
Acetilcolina/metabolismo , Envelhecimento/metabolismo , Envelhecimento/fisiologia , Homeostase/fisiologia , Junção Neuromuscular/metabolismo , Transmissão Sináptica/fisiologia , Animais , Potenciais Pós-Sinápticos Excitadores/fisiologia , Expressão Gênica , Camundongos , Contração Muscular/fisiologia , Potenciais Sinápticos/fisiologia , Proteínas Vesiculares de Transporte de Acetilcolina/genética , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismoRESUMO
Antimicrobial peptides (AMPs) represent a large and ubiquitous group of peptides. The current crisis in antibiotic therapy has led to an intensified search for new antimicrobial agents. In this regard, scorpion venom constitutes a rich source of biologically active peptides including AMPs. In the present study, the purification of a novel peptide with antimicrobial activity against the Gram-negative bacteria Klebsiella pneumoniae is described. This antimicrobial peptide, named Cm38, was purified from Centruroides margaritatus scorpion venom using a two-step chromatographic strategy using C8 and C18 columns. This toxin inhibits the proliferation of the Gram-negative bacteria Klebsiella pneumoniae with a Minimal Inhibitory Concentration (MIC) of 64 µM. An analysis of the N-terminal sequence of Cm38 revealed a close structural relationship to Cn11, a Na+-channel modulator toxin previously isolated from Centruroides noxius scorpion venom. Therefore, to test Cm38 for effects on ion channels, we measured its effects on action potential firing in cultured dorsal root ganglion neurons. Cm38 depolarized and increased action potential firing in a subset of neurons tested. The present work reports a new peptide related to Cn11 with antimicrobial properties that is also active in neurons.
Assuntos
Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Gânglios Espinais/efeitos dos fármacos , Klebsiella pneumoniae/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Venenos de Escorpião/química , Sequência de Aminoácidos , Animais , Anti-Infecciosos/química , Anti-Infecciosos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Eletrofisiologia , Gânglios Espinais/citologia , Hemólise/efeitos dos fármacos , Humanos , Masculino , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Neurônios/citologia , Ratos , Ratos Wistar , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Neurotoxicity is a major symptom of envenomation caused by Brazilian coral snake Micrurus frontalis. Due to the small amount of material that can be collected, no neurotoxin has been fully sequenced from this venom. In this work we report six new three-finger like toxins isolated from the venom of the coral snake M. frontalis which we named Frontoxin (FTx) I-VI. Toxins were purified using multiple steps of RP-HPLC. Molecular masses were determined by MALDI-TOF and ESI ion-trap mass spectrometry. The complete amino acid sequence of FTx II, III, IV and V were determined by sequencing of overlapping proteolytic fragments by Edman degradation and by de novo sequencing. The amino acid sequences of FTx I, II, III and VI predict 4 conserved disulphide bonds and structural similarity to previously reported short-chain alpha-neurotoxins. FTx IV and V each contained 10 conserved cysteines and share high similarity with long-chain alpha-neurotoxins. At the frog neuromuscular junction FTx II, III and IV reduced miniature endplate potential amplitudes in a time-and concentration-dependent manner suggesting Frontoxins block nicotinic acetylcholine receptors.
Assuntos
Venenos Elapídicos/química , Elapidae , Potenciais Pós-Sinápticos em Miniatura/efeitos dos fármacos , Placa Motora/efeitos dos fármacos , Neurotoxinas/toxicidade , Proteínas de Répteis/toxicidade , Alquilação , Sequência de Aminoácidos , Animais , Fracionamento Químico , Cisteína/análise , Venenos Elapídicos/toxicidade , Técnicas In Vitro , Dados de Sequência Molecular , Peso Molecular , Placa Motora/fisiologia , Neurotoxinas/química , Neurotoxinas/isolamento & purificação , Neurotoxinas/metabolismo , Concentração Osmolar , Oxirredução , Músculos Peitorais/efeitos dos fármacos , Músculos Peitorais/fisiologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Isoformas de Proteínas/química , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/toxicidade , Rana catesbeiana , Proteínas de Répteis/química , Proteínas de Répteis/isolamento & purificação , Proteínas de Répteis/metabolismo , Alinhamento de SequênciaRESUMO
OBJECTIVE: The aim of the study was to evaluate clinical and laboratorial features of 1234 patients with different etiologies of hyperprolactinemia, as well as the response of 388 patients with prolactinomas to dopamine agonists. DESIGN, SETTING, AND PATIENTS: A total of 1234 hyperprolactinemic patients from 10 Brazilian endocrine centers were enrolled in this retrospective study. MAIN OUTCOME MEASURE: PRL measurement, thyroid function tests, and screening for macroprolactin were conducted. RESULTS: Patients were subdivided as follows: 56.2% had prolactinomas, 14.5% drug-induced hyperprolactinemia, 9.3% macroprolactinemia, 6.6% non-functioning pituitary adenomas, 6.3% primary hypothyroidism, 3.6% idiopathic hyperprolactinemia, and 3.2% acromegaly. Clinical manifestations were similar irrespective of the etiology of the hyperprolactinemia. The highest PRL levels were observed in patients with prolactinomas but there was a great overlap in PRL values between all groups. However, PRL>500 ng/ml allowed a clear distinction between prolactinomas and the other etiologies. Cabergoline (CAB) was more effective than bromocriptine (BCR) in normalizing PRL levels (81.9% vs 67.1%, p<0.0001) and in inducing significant tumor shrinkage and complete disappearance of tumor mass. Drug resistance was observed in 10% of patients treated with CAB and in 18.4% of those that used BCR (p=0.0006). Side-effects and intolerance were also more common in BCR treated patients. CONCLUSION: Prolactinomas, drug induced hyperprolactinemia, and macroprolactinemia were the 3 most common causes of hyperprolactinemia. Although PRL levels could not reliably define the etiology of hyperprolactinemia, PRL values >500 ng/ml were exclusively seen in patients with prolactinomas. CAB was significantly more effective than BCR in terms of prolactin normalization, tumor shrinkage, and tolerability.
Assuntos
Agonistas de Dopamina/uso terapêutico , Hiperprolactinemia/diagnóstico , Hiperprolactinemia/tratamento farmacológico , Adolescente , Adulto , Idoso , Brasil , Bromocriptina/uso terapêutico , Feminino , Humanos , Hiperprolactinemia/sangue , Hiperprolactinemia/etiologia , Masculino , Pessoa de Meia-Idade , Prolactina/sangue , Estudos Retrospectivos , Resultado do TratamentoRESUMO
BACKGROUND: In patients with acromegaly, sleep apnea-related hypoxemia results in considerable morbidity and mortality. AIMS: To evaluate the relative weight of pathogenic factors in predicting such hypoxemia. METHODS: In this cross-sectional study, 34 acromegaly patients were submitted to clinical evaluation, nocturnal oximetry, and nasolaryngeal airway tomography. GH, IGF-I, and its upper limit normal value were measured. Nocturnal hypoxemia was defined as >5 episodes of desaturation/h of sleep. Craniofacial abnormalities were expressed using a linear parameter index (LPI). Nocturnal hypoxemia was predicted using logistic regression, including the variables markers of craniofacial abnormality, hormonal alteration, and obesity. Coefficients were standardized in order to determine their effect magnitudes relative to the outcome. The best model included the variables gender, age, LPI, body mass index (BMI), and IGFI upper limit normal value. MAIN RESULTS: In the absence of the age and gender variables, the odds ratio for the LPI (1.60) was slightly higher than those found for BMI (1.49) and upper limit normal value (1.40). When the data were adjusted for age, the hormone upper limit normal value presented little alteration (1.49), although the decrease in the LPI was considerable (1.21), as was the increase in the BMI (2.18). The relative weight of the LPI was age-dependent. The gender variable did not alter the relevance of the others. CONCLUSIONS: The effects that craniofacial aspect, obesity, and hormonal alterations have on nocturnal hypoxemia are of similar magnitude.
Assuntos
Acromegalia/complicações , Anormalidades Craniofaciais/complicações , Doenças do Sistema Endócrino/complicações , Hipóxia/etiologia , Obesidade/complicações , Adulto , Idoso , Índice de Massa Corporal , Ritmo Circadiano/fisiologia , Anormalidades Craniofaciais/sangue , Anormalidades Craniofaciais/diagnóstico por imagem , Anormalidades Craniofaciais/epidemiologia , Estudos Transversais , Doenças do Sistema Endócrino/sangue , Doenças do Sistema Endócrino/epidemiologia , Feminino , Hormônio do Crescimento Humano/sangue , Humanos , Hipóxia/sangue , Hipóxia/diagnóstico por imagem , Hipóxia/epidemiologia , Fator de Crescimento Insulin-Like I/análise , Masculino , Pessoa de Meia-Idade , Obesidade/sangue , Obesidade/epidemiologia , Oxigênio/análise , Oxigênio/sangue , Radiografia , Caracteres Sexuais , Síndromes da Apneia do Sono/complicações , Síndromes da Apneia do Sono/diagnóstico por imagem , Síndromes da Apneia do Sono/epidemiologia , Síndromes da Apneia do Sono/etiologia , Adulto JovemRESUMO
OBJECTIVE: To evaluate the role of non-invasive dynamic tests in the diagnosis and differential diagnosis of Cushing's syndrome (CS). METHODS: We studied laboratory features of 74 patients with endogenous CS, subdivided as follows: 46 (62.1%) with Cushing's disease (CD), 21 (28.3%) with an adrenal tumor, and 7 (9.5%) with ectopic ACTH syndrome (EAS). RESULTS: In 100% of cases of CS we found serum cortisol levels greater than 1.8 microg/dl after low-dose dexamethasone suppression tests (LDDST), as well as elevation of midnight serum or salivary cortisol. However, urinary free cortisol was normal in 11.5% of patients. ACTH levels were suppressed in patients with adrenal tumors, normal or high in CD and invariably increased in EAS. After the 8-mg overnight dexamethasone suppression test (HDDST), serum cortisol suppression >50% was observed in 79.5% of cases of CD and in 28.6% of subjects with EAS, whereas cortisol suppression >80% was only found in CD. After stimulation with CRH or desmopressin an ACTH rise > or =35% occurred in 86.5% of individuals with CD and 14.3% of those with EAS, whereas an ACTH rise > or =50 achieved 100% specificity. Moreover, the combination of serum cortisol suppression >50% after HDDST and an ACTH increase > or =35% after the administration of CRH or desmopressin only occurred in CD. CONCLUSION: Our findings demonstrate that LDDST had 100% sensitivity for the diagnosis of CS and that HDDST and stimulation tests with CRH or desmopressin may be very useful for confirmation of CS etiology when analyzed together or when more stringent cut-offs are used.
Assuntos
Síndrome de ACTH Ectópico/diagnóstico , Síndrome de Cushing/diagnóstico , Neoplasias das Glândulas Suprarrenais/diagnóstico , Hormônio Liberador da Corticotropina , Desamino Arginina Vasopressina , Dexametasona , Diagnóstico Diferencial , Humanos , Hidrocortisona/sangue , Imageamento por Ressonância Magnética , Hipersecreção Hipofisária de ACTH/diagnóstico , Hipófise/patologia , Valor Preditivo dos Testes , Estudos Retrospectivos , Saliva/química , Sensibilidade e EspecificidadeRESUMO
AIM: The aim of this study was to evaluate the clinical and laboratory features of 64 patients with macroprolactinemia and to compare them to those of individuals with monomeric hyperprolactinemia. METHODS: The study included 64 patients (54 women and 10 men) with macroprolactinemia and 96 patients (70 women and 26 men) with monomeric hyperprolactinemia (32 with prolactinomas). RESULTS: Symptoms related to prolactin (PRL) excess were found in about 44% of individuals from the macroprolactinemia group and in 88.5% of patients with monomeric hyperprolactinemia (P<0.0001). However, the frequency of menstrual disturbances (oligomenorrhea or amenorrhea), galactorrhea and erectile dysfunction did not differ in both groups. In contrast, the association of galactorrhea and menstrual disturbances was significantly more prevalent in women with monomeric hyperprolactinemia. Although mean PRL levels were higher in patients with monomeric hyperprolactinemia (565.9+/-2726.4 vs 113.3+/-94.5 ng/mL, P<0.001), there was a great overlap between both groups. Among macroprolactinemic patients, pituitary magnetic resonance imaging revealed an image suggestive of a microadenoma in 7 (10.9%) and a macroadenoma in 1 (1.6%). Normalization of PRL levels during therapy with dopamine agonists was significantly more frequent in patients with monomeric hyperprolactinemia than in subjects with macroprolactinemia (78.6% vs 32%, P=0.0006). CONCLUSION: Our data show that symptoms related to PRL excess are frequently found in subjects with macroprolactinemia. Moreover, no clinical or laboratory features could reliably differentiate macroprolactinemic patients from those with monomeric hyperprolactinemia. Therefore, the screening for macroprolactin should not be restricted to asymptomatic patients.
Assuntos
Hiperprolactinemia/diagnóstico , Neoplasias Hipofisárias/diagnóstico , Prolactina/sangue , Prolactinoma/diagnóstico , Adulto , Biomarcadores/sangue , Antagonistas de Dopamina/uso terapêutico , Disfunção Erétil/etiologia , Feminino , Galactorreia/etiologia , Humanos , Hiperprolactinemia/sangue , Hiperprolactinemia/complicações , Masculino , Programas de Rastreamento , Distúrbios Menstruais/etiologia , Pessoa de Meia-Idade , Neoplasias Hipofisárias/sangue , Neoplasias Hipofisárias/complicações , Neoplasias Hipofisárias/tratamento farmacológico , Valor Preditivo dos Testes , Prolactinoma/sangue , Prolactinoma/complicações , Prolactinoma/tratamento farmacológico , Reprodutibilidade dos Testes , Resultado do TratamentoRESUMO
AIM: The aim of this study was to evaluate the positive predictive value of two growth hormone stimulation tests (insulin-induced hypoglycemia and clonidine) for stature below percentile 10 in patients treated for acute lymphoblastic leukemia in childhood. METHODS: The study population was a cohort of 30 patients (aged 14.1+/-2.9 years; 20 male) treated for acute lymphoblastic leukemia during childhood and then examined after insulin-induced hypoglycemia (30 patients) and clonidine (16 patients) tests. The follow-up time was 7.7+/-2.8 years since treatment and 2.3+/-1.3 years after administration of the tests. RESULTS: In the last evaluation, 12 patients (40%) were below and 18 (60%) were above percentile 10. The insulin-induced hypoglycemia test response was: 9 patients (30%) had growth hormone peak <5 ng/mL and 19 (63.3%) <7 ng/mL. The clonidine test response was: 7 patients had growth hormone peak <5 ng/mL and 8 (50%) <7 ng/mL. For stature below of the percentile 10, the positive predictive values of insulin-induced hypoglycemia test (33%) and clonidine (28%) were low when growth hormone peak <5 ng/mL was considered; however, when growth hormone peak <7 ng/mL was considered, the positive predictive values were 83% and 50% for the insulin-induced hypoglycemia and clonidine tests, respectively. CONCLUSIONS: In patients treated for acute lymphoblastic leukemia in childhood, the positive predictive values for statural deficit of both tests were low, except for the insulin-induced hypoglycemia test when a growth hormone peak <7 ng/mL was considered.
Assuntos
Estatura/efeitos dos fármacos , Estatura/efeitos da radiação , Hormônio do Crescimento Humano/deficiência , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Adolescente , Agonistas alfa-Adrenérgicos , Criança , Pré-Escolar , Clonidina , Estudos de Coortes , Quimioterapia Combinada , Feminino , Hormônio do Crescimento Humano/sangue , Humanos , Hipoglicemiantes , Insulina , Masculino , Valor Preditivo dos TestesRESUMO
Ischemic pain occurs when there is insufficient blood flow for the metabolic needs of an organ. The pain of a heart attack is the prototypical example. Multiple compounds released from ischemic muscle likely contribute to this pain by acting on sensory neurons that innervate muscle. One such compound is lactic acid. Here, we show that ASIC3 (acid-sensing ion channel #3) has the appropriate expression pattern and physical properties to be the detector of this lactic acid. In rats, it is expressed only in sensory neurons and then only on a minority (approximately 40%) of these. Nevertheless, it is expressed at extremely high levels on virtually all dorsal root ganglion sensory neurons that innervate the heart. It is extraordinarily sensitive to protons (Hill slope 4, half-activating pH 6.7), allowing it to readily respond to the small changes in extracellular pH (from 7.4 to 7.0) that occur during muscle ischemia. Moreover, both extracellular lactate and extracellular ATP increase the sensitivity of ASIC3 to protons. This final property makes ASIC3 a "coincidence detector" of three molecules that appear during ischemia, thereby allowing it to better detect acidosis caused by ischemia than other forms of systemic acidosis such as hypercapnia.
Assuntos
Isquemia/fisiopatologia , Ácido Láctico/metabolismo , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neurônios Aferentes/fisiologia , Dor/fisiopatologia , Canais de Sódio/fisiologia , Canais Iônicos Sensíveis a Ácido , Animais , Concentração de Íons de Hidrogênio , RatosRESUMO
Ischemic pain occurs when there is insufficient blood flow for the metabolic needs of an organ. The pain of a heart attack is the prototypical example. Multiple compounds released from ischemic muscle likely contribute to this pain by acting on sensory neurons that innervate muscle. One such compound is lactic acid. Here, we show that ASIC3 (acid-sensing ion channel #3) has the appropriate expression pattern and physical properties to be the detector of this lactic acid. In rats, it is expressed only in sensory neurons and then only on a minority (40 percent) of these. Nevertheless, it is expressed at extremely high levels on virtually all dorsal root ganglion sensory neurons that innervate the heart. It is extraordinarily sensitive to protons (Hill slope 4, half-activating pH 6.7), allowing it to readily respond to the small changes in extracellular pH (from 7.4 to 7.0) that occur during muscle ischemia. Moreover, both extracellular lactate and extracellular ATP increase the sensitivity of ASIC3 to protons. This final property makes ASIC3 a "coincidence detector" of three molecules that appear during ischemia, thereby allowing it to better detect acidosis caused by ischemia than other forms of systemic acidosis such as hypercapnia.
Assuntos
Animais , Ratos , Ácido Láctico/metabolismo , Canais de Sódio/fisiologia , Dor/fisiopatologia , Isquemia/fisiopatologia , Neurônios Aferentes/fisiologia , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Concentração de Íons de HidrogênioRESUMO
Hyponatremia is a frequent occurrence after pituitary surgery, having been described in 9% to 35% of the patients. It is produced by the syndrome of inappropriate secretion of antidiuretic hormone (SIADH) or, more frequently, by the cerebral salt-wasting syndrome (CSWS). The clinical presentation of both syndromes is identical and the differential diagnosis can be difficult. The determination of the volemic state is essential for the diagnosis, since the patients with the SIADH are euvolemic or hypervolemic, while those with CSWS are hypovolemic. Several methods can be used to detect the volemic state in these patients and, among them, the furosemide test can best discriminate between SIADH and CSWS. A furosemide infusion (20 mg) normalizes sodium serum levels in SIADH patients, but not in CSWS patients who remain hyponatremic. The differentiation between the 2 syndromes is clinically relevant since their treatment is antithetical. SIADH patients need liquid restriction of liquids and/or furosemide to reduce the volume of extracellular water, while CSWS patients need volume replacement with sodium supplementation (or fludrocortisone can be a good alternative). The diagnosis and treatment of these syndromes are discussed on the basis of the literature reports.
Assuntos
Hiponatremia/diagnóstico , Hiponatremia/etiologia , Hiponatremia/terapia , Doenças da Hipófise/cirurgia , Diagnóstico Diferencial , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
We investigated how elevated quantal release produced by motor nerve stimulation affects the size of the quanta. The motor nerve was stimulated at 10 Hz in preparations in which excitation-contraction coupling was disrupted. Two hundred stimuli reduced the size of the time integrals of the miniature endplate currents ([integral]MEPCs), measured at the same junction immediately after stimulation, by 16 %. Three thousand stimuli reduced size by 23 %. When the solution contained 10 microM neostigmine (NEO) 3000 stimuli reduced [integral]MEPCs by 60 %, because with acetylcholinesterase (AChE) inhibited, [integral]MEPC size is more sensitive to changes in acetylcholine (ACh) content. Similar decreases in miniature endplate potential size ([integral]MEPP) followed repetitive stimulation of contracting preparations. The depolarization produced by iontophoretic pulses of ACh was scarcely changed by 3000 nerve stimuli at 10 Hz, suggesting that the decreases in miniature sizes are largely due to less ACh released per quantum. Following 3000 stimuli at 10 Hz the sizes of the [integral]MEPCs increased back to pre-stimulus values with a half-time of 8-10 min. Recovery was blocked by (-)-vesamicol (VES), by hemicholinium-3 (HC3) and by nicotinic cholinergic agonists - all of which inhibit ACh loading into synaptic vesicles. The number of quanta in the total store was estimated by releasing them with carbonyl cyanide m-chlorophenylhydrazone (CCCP). CCCP releases fewer quanta after stimulation than from unstimulated controls. After resting for hours following stimulation, the releasable number increased, even when ACh loading inhibitors were present. We conclude that the inhibitors do not block a significant fraction of the ACh loading into reformed reserve vesicles and propose that ACh can be loaded in a series of steps.
Assuntos
Acetilcolina/metabolismo , Neurônios Motores/metabolismo , Junção Neuromuscular/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Colinérgicos/farmacologia , Inibidores da Colinesterase/farmacologia , Iodeto de Dimetilfenilpiperazina/farmacologia , Estimulação Elétrica , Hemicolínio 3/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Neurônios Motores/efeitos dos fármacos , Neostigmina/farmacologia , Fármacos Neuromusculares Despolarizantes/farmacologia , Junção Neuromuscular/efeitos dos fármacos , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Técnicas de Patch-Clamp , Piperidinas/farmacologia , Rana pipiens , Vesículas Sinápticas/efeitos dos fármacos , Desacopladores/farmacologiaRESUMO
Acute or chronic zinc administration may cause hyperglycemia in experimental animals. These findings are attributed to permissive actions of glucocorticoids and glucagon upon hepatic gluconeogenesis and glycogenolysis. The effect of Zn(+)+ on plasma glucose, C-peptide, glucagon, and cortisol was investigated in healthy and insulin-dependent diabetes mellitus (IDDM) patients. Ten normal individuals (5 of each sex, aged 24.10 +/- 1.96) and 10 IDDM (5 of each sex, aged 25.20 +/- 8.10) were tested at 7:00 AM after 12-h fast. Twenty-five mg of Zn(+)+ were administered intravenously during 1 min, and blood samples were collected from the contralateral arm at 0, 3, 30, 60, 90 and 120 min after Zn(+)+ injection. The plasma levels of glucose, C-peptide, and glucagon remained constant throughout the experimental period in both groups studied. Plasma cortisol levels decreased significantly, which is consistent with our previous findings. These results suggest that, in contrast to experimental animals, acute Zn(+)+ administration, despite decreasing cortisol levels, does not change carbohydrate metabolism in human beings.
Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Glucose/metabolismo , Zinco/farmacologia , Adulto , Glicemia/análise , Peptídeo C/análise , Feminino , Glucagon/sangue , Humanos , Hidrocortisona/sangue , Masculino , Zinco/sangueRESUMO
Nicotinic cholinergic agonists are known to decrease synchronous evoked quantal output at the frog neuromuscular junction [Van der Kloot 1993, J Physiol (Lond) 468:567-589]. Here we also show that carbachol decreases the frequency of miniature endplate potentials (FMEPP) in solutions containing elevated levels of K+ and Ca2+. Carbachol did not decrease FMEPP in hypertonic solutions or in solutions containing the Ca2+ ionophore ionomycin and Ca2+. We conclude that the nicotinic agonists decrease Ca2+ influx through voltage-gated Ca2+ channels. Carbachol did not alter two-pulse facilitation. A blocker of N-type Ca2+ channels, omega-conotoxin GVIA, antagonized the nicotinic agonist-induced decrease in evoked quantal output. The effect of carbachol was not altered by omega-conotoxin MVIIC, a blocker of P-type and certain other Ca2+channels. The Ca2+ channel targeted by the nicotinic agonists appears to be of the N-type.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Canais de Cálcio/fisiologia , Colinérgicos/farmacologia , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/fisiologia , ômega-Conotoxinas , Animais , Eletrofisiologia , Soluções Hipertônicas/farmacologia , Ionomicina/farmacologia , Ionóforos , Placa Motora/efeitos dos fármacos , Placa Motora/fisiologia , Peptídeos/farmacologia , Potássio/farmacologia , Rana pipiens , Soluções , ômega-Conotoxina GVIARESUMO
1. We spatially localized the origins of quantal currents by recording simultaneously with two intracellular electrodes and employing the prediction of the one-dimensional cable equations that the time integrals of the resulting voltage changes fall off exponentially with distance. 2. Miniature endplate potentials (MEPPs) were more frequent near the centre of the endplate. In contrast to some work using other methods, we did not find MEPPs originating at the margins of the endplate to be strikingly smaller. 3. Spontaneous MEPPs and uniquantal endplate potentials (EPPs) were released over the same length of endplate and with the same relative probabilities at different regions. 4. Nicotinic agonists decreased evoked quantal output, but did not change the length over which uniquantal EPPs were generated. We conclude they do not block nerve conduction in the terminals. 5. Data sets were obtained with an extracellular electrode and two intracellular electrodes. The extracellular electrode was invariably near the centre of the region in which congruous MEPPs appeared to be generated. However, the range in the calculated positions of the synchronous MEPPs was as long as 0.8 mm. Therefore, it may be possible that extracellular electrodes have a longer recording range than commonly assumed.
Assuntos
Potenciais Evocados/efeitos dos fármacos , Músculo Esquelético/fisiologia , Junção Neuromuscular/metabolismo , Animais , Carbacol/farmacologia , Eletrofisiologia , Potenciais Evocados/fisiologia , Gluconatos/farmacologia , Agonistas Nicotínicos/farmacologia , Rana pipiensRESUMO
1. This investigation was undertaken to explore an unexpected effect of vesamicol, an agent that inhibits active acetylcholine (ACh) uptake into isolated synaptic vesicles. Previous studies at the neuromuscular junction showed that vesamicol makes miniature end-plate currents (MEPCs) smaller only after tens of thousands of quanta have been released. Inhibiting acetylcholinesterase (AChE) makes the MEPCs larger than normal. Our unexpected finding was that with the AChE inhibitor present, adding 2 microM (-)-vesamicol decreases the size of the MEPCs by approximately 30%. The decrease was apparent within 15-30 min, during which only a few thousand quanta had been released. 2. Experimental tests showed that the (-)-vesamicol treatment is unlikely to be acting postsynaptically. For example, it did not slow the rise of MEPCs, which would occur if the endplate receptors were blocked. 3. When AChE was inhibited, three treatments expected to block active choline (Ch) uptake into the presynaptic terminals decreased MEPC size: 1) elevating extracellular K+ to diminish the Na+ electrochemical gradient required for Ch uptake; 2) replacing extracellular Na+ with methylamine+; and 3) adding hemicholinium-3 (HC-3), an inhibitor of the Ch transporter. These treatments did not act by reactivating AChE, blocking the endplate ACh receptor, or by enhancing the desensitization of the ACh receptor. 4. Previous evidence suggests that synaptic vesicles are formed and partially filled with ACh in the cytoplasm and then receive additional ACh when they attach to the active zones, a process that is called second-stage loading. We conclude that the MEPCs are becoming smaller when second-stage loading is blocked by (-)-vesamicol or when the supply of ACh in the cytoplasm of the motor nerve terminal is depleted. 5. To follow the time course of second-stage loading, we used the false transmitter precursor monoethylcholine (MECh). It enters the terminal and is transformed into acetylmonoethylcholine (AMECh). When 200 microM MECh was placed in the extracellular solution and the AChE was inhibited, MEPC size was significantly smaller after 10 min. MEPC size increased once again over a period of time when MECh was removed from the extracellular solution and replaced with Ch. 6. We conclude that at the neuromuscular junction second-stage loading is responsible for loading a significant fraction of the ACh into the quanta.
Assuntos
Acetilcolina/metabolismo , Inibidores da Colinesterase/farmacologia , Placa Motora/efeitos dos fármacos , Fármacos Neuromusculares Despolarizantes/farmacologia , Piperidinas/farmacologia , Análise de Variância , Animais , Colina/metabolismo , Placa Motora/metabolismo , Neostigmina/farmacologia , Rana pipiensRESUMO
Monoethylcholine (MECH) enters motor nerve terminals where it is made into acetylmonoethylcholine (AMECH). AMECH opens endplate channels for about half of the average duration observed where they are opened by acetylcholine (ACH). Therefore when AMECH is present in a quantum the endplate currents decay more rapidly. MECH has been used to measure quantal turnover in motor nerve terminals. We find that the incorporation of AMECH into quanta is blocked by vesamicol, an inhibitor of ACH transport into synaptic vesicles. AMECH is incorporated more rapidly when acetylcholinesterase is inhibited, when the choline uptake inhibitor, hemicholinium-3, is present or when extracellular Na+ (required for active CH uptake) is replaced with methylamine. This suggests that in the absence of these inhibitors CH obtained from released ACH is recycled. Therefore, experiments on the rate of incorporation of MECH are misleading unless CH recycling is prevented. Previous work also suggested that MECH is incorporated at a faster rate into those quanta which are released by stimulation than into those released spontaneously. We conclude that quanta released spontaneously and following nerve stimulation probably come from the same pool. The distribution of t1/2's during the incorporation of MECH can be accounted for in the framework of recent studies of the recycling of synaptic vesicles. We conclude that false transmitter is a valuable tool for studying the loading of quanta, but that there are several complications to be considered when trying to use it to measure the turnover of the population of quanta.
Assuntos
Colina/análogos & derivados , Junção Neuromuscular/metabolismo , Neurotransmissores/metabolismo , Pró-Fármacos/metabolismo , Animais , Colina/metabolismo , Inibidores da Colinesterase/farmacologia , Edrofônio/farmacologia , Estimulação Elétrica , Hemicolínio 3/farmacologia , Metilaminas/farmacologia , Camundongos , Camundongos Endogâmicos , Neostigmina/farmacologia , Fármacos Neuromusculares Despolarizantes/farmacologia , Inibidores da Captação de Neurotransmissores/farmacologia , Piperidinas/farmacologia , Rana pipiensRESUMO
The current model does not account adequately for the characteristics of miniature endplate currents (MEPCs). We do not understand their relatively slow rise, the shape of their rise, their variable and sometimes prolonged decay, and the correlation between amplitude and decay time. If we assume that ACh is released from the vesicle through a pore and that the vesicle enlarges as it takes on additional transmitter, the predictions are more like MEPCs. However, previous measurements showed that after quantal size was increased the vesicles in the terminal were not enlarged. This need not be a problem, because some of the ACh is added to vesicles positioned at the active zones, a process known as second-stage loading. By using the false transmitter precursor monoethylcholine we provide additional evidence for second-stage loading. The distribution of quantal sizes at the junction usually does not follow a normal probability distribution; it is skewed to the right. The skew can be accounted for by a model incorporating second-stage loading in which the vesicles are released randomly, without regard to their ACh content. If the vesicles increase in size when they contain more transmitter, only vesicles at the active zone need swell.
Assuntos
Placa Motora/fisiologia , Músculo Esquelético/inervação , Acetilcolina/fisiologia , Animais , Gluconatos , Soluções Hipertônicas , Potenciais da Membrana , Modelos Biológicos , Modelos Teóricos , Músculo Esquelético/fisiologia , Técnicas de Patch-Clamp , Probabilidade , Teoria Quântica , Rana pipiens , Fatores de TempoRESUMO
Fluctuation analysis was used to estimate the mean single-channel conductance and the mean channel duration of opening. Miniature endplate currents (MEPCs) were measured with the voltage-clamp technique. The timing of endplate channel opening during the generation of the MEPC was estimated by a deconvolution method. Often all of the channels opened during the rise of the MEPC, but in about half of the examples some 10% of the channels opened after the peak. We studied the effects of acetylcholinesterase (AChE) inhibition with neostigmine, diisopropyl fluorophosphate (DFP) and fasciculin-2. With AChE largely inhibited, the number of channels opening increased as much as fourfold, largely by channels opening in the "tail" that follows the peak of the MEPC. The results were compared to models of MEPC generation. Models did not account well for the pattern of channel opening, particularly after AChE inhibition. In the presence of fasciculin-2, the addition of 2 microM (-)-vesamicol reduced the number of channels opening and shortened the period over which channels were open. One interpretation is that quantal ACh release is not almost instantaneous, but that some of the ACh is released over a period of a millisecond or more and that some of the release is blocked by (-)-vesamicol.
Assuntos
Inibidores da Colinesterase/farmacologia , Venenos Elapídicos/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Placa Motora/efeitos dos fármacos , Piperidinas/farmacologia , Acetilcolina/antagonistas & inibidores , Animais , Feminino , Técnicas In Vitro , Isoflurofato/farmacologia , Masculino , Matemática , Potenciais da Membrana , Camundongos , Modelos Neurológicos , Placa Motora/metabolismo , Rana pipiensRESUMO
The influence of 6-hydroxydopamine (6-OHDA) pretreatment on xylazine (XLZ)-induced antinociception was studied in mice using the writhing test (60 mg/kg acetic acid, ip, as the algogenic compound administered 10 min after 0.5 and 0.75 mg/kg XLZ, sc). 6-OHDA (100 mg kg-1 injection-1 administered ip on days 1, 3, 5, 7, 9 and 11 after birth) did not modify XLZ-induced antinociception, suggesting that this effect is mediated by postsynaptic alpha-2 adrenoceptors.