RESUMO
Cytoskeletal F-actin associated with synaptic vesicles and granules plays an important role during Ca(2+)-mediated exocytosis. In the present work, we have used amperometry and confocal fluorescence to study the role of internal Ca(2+) in the rearrangement of F-actin (visualised with phalloidin-Alexa 546) during exocytosis in rat mast cells. The F-actin-depolymerising drug, latrunculin A, and the ryanodine receptor agonists ryanodine and caffeine that, per se did not induce exocytosis, enhanced the exocytotic responses elicited by compound 48/80 (C48/80). They also induced cortical actin depolymerisation in the presence or absence of external Ca(2+). Degranulation induced by C48/80 was accompanied by the formation of a cytoplasmic F-actin network. Depletion of internal Ca(2+) with cyclopiazonic acid inhibited latrunculin potentiation of C48/80-stimulated exocytosis and completely blocked the formation of the cytoplasmic F-actin network. This indicates that the mobilisation of Ca(2+) from ryanodine-sensitive intracellular stores plays an important role in the depolymerisation of the cortical F-actin barrier and possibly in the formation of the internal F-actin network during exocytotic activation of peritoneal mast cells.
Assuntos
Citoesqueleto de Actina/fisiologia , Cálcio/metabolismo , Exocitose/fisiologia , Mastócitos/metabolismo , Polímeros/metabolismo , Rianodina/farmacologia , Citoesqueleto de Actina/metabolismo , Actinas/efeitos dos fármacos , Actinas/metabolismo , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Cafeína/farmacologia , Degranulação Celular , Citoplasma/metabolismo , Exocitose/efeitos dos fármacos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Masculino , Mastócitos/efeitos dos fármacos , Mastócitos/fisiologia , Peritônio/citologia , Ratos , Ratos Sprague-Dawley , Tiazolidinas/farmacologia , p-Metoxi-N-metilfenetilamina/farmacologiaRESUMO
Grain protein content is one of the major determinants of the baking and nutritional quality of wheat. It has previously been reported that the ditelosomic line of wheat (Triticum aestivum L.) CSDT7BL, where the short arm of chromosome 7B is missing, shows a lower grain protein concentration than the normal line, but a similar grain yield. In the present paper the growth and nitrogen (N) metabolism of wheat plants cv. Chinese Spring (CS) and its ditelosomic line CSDT7BL were compared. When plants were grown to maturity in pots with different N supplements, the wild-type line showed a higher grain protein concentration and a lower straw N concentration than the ditelosomic line at every N level analysed, suggesting a deficiency in the N remobilization capacity. When 15-d-old plants were grown in a growth cabinet in pots with sand, and supplied with nutrient solutions of different nitrate concentrations, the ditelosomic line showed no differences in N uptake per unit of root dry weight, nitrate reductase activity, nitrate, total N concentration or free amino acid concentration. However, the ditelosomic line showed a decreased capacity to export amino acids in the phloem under high N, independently of the N source. This deficiency was also observed under dark-induced senescence. The diminished export of amino acids to the phloem was principally caused by a decrease in the export of Glu, Asp, and Gln. It is suggested that the decrease in grain protein concentration in the ditelosomic line is a consequence of defective export in the phloem of these amino acids.
Assuntos
Aminoácidos/metabolismo , Nitrogênio/metabolismo , Proteínas de Plantas/metabolismo , Triticum/metabolismo , Transporte Biológico Ativo , Senescência Celular , Cromossomos , Ambiente Controlado , Nitrato Redutase , Nitrato Redutases/metabolismo , Nitratos/farmacologia , Caules de Planta/enzimologia , Caules de Planta/genética , Caules de Planta/crescimento & desenvolvimento , Caules de Planta/metabolismo , Compostos de Amônio Quaternário/farmacologia , Triticum/enzimologia , Triticum/genética , Triticum/crescimento & desenvolvimento , Ureia/farmacologiaRESUMO
Mast cells exocytotically release histamine/serotonin in response to different secretagogues. We have used substance P and compound 48/80 to study the Ca++ dependency of serotonin exocytosis from peritoneal mast cells using carbon fiber amperometric techniques. The exocytotic release pattern consists of a burst of events superimposed on a slow, transient, amperometric current baseline increase. Cellular re lease parameters (number, frequency and total charge of amperometric events) and individual event characteristics (charge integral, half width and peak amplitude) were similar for the two secretagogues used. Zero Ca++ conditions greatly reduced, without completely abolishing,cellular release parameters. Cyclopiazonic acid, an inhibitor of the endoplasmatic Ca++ ATPase, reduced the cellular exocytotic capacity and diminished the amplitude of individual exocytotic events more effectively than the 0 Ca++ condition. The cyclopiazonic acid effects occurred in the presence of external Ca++, indicating that this condition is not sufficient for maintaining full exocytotic capacity. The results confirm the importance of intracellular Ca++ for exocytotic activation. For the first time evidence is presented that the integrity of intracellular Ca++ pools determines the amplitude and frequency of individual exocytotic events. Saponin, a non-specific detergent, also induced quantal release similar to that obtained with substance P and compound 48/80. This release was not dependent on extracellular Ca++, but cyclopiazonic acid significantly reduced individual exocytotic release.
Assuntos
Cálcio/fisiologia , Exocitose/fisiologia , Mastócitos/fisiologia , Animais , Células Cultivadas , Transporte de Íons/fisiologia , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Fluorescence signals from the calcium sensitive dyes Fluo-3 or Rhod-2 were obtained simultaneously with isometric tension in single fibres isolated from the anterior tibialis muscle of Leptodactylus insularis (20-22 degrees C). Fluo-3 fluorescence signals were transformed into [Ca2+]i transients as previously described. Most of the decay phase of single twitch transient is well fitted by a single exponential (tau of about 10 ms), followed by a slower declining component lasting tens of milliseconds. During short periods, 10 to 20 s, of low frequency stimulation, between 0.2 and 5 Hz, the basal [Ca2+]i increased slowly from 0.1 to about 0.4 microM, with only minor changes in the exponentially decaying phase. In fibres poisoned with thapsigargin or cyclopiazonic acid (1-2 microM) the tau of decay of fluorescence or Ca2+ transients of single twitches was very similar to that observed in non-poisoned fibres. Nevertheless, in poisoned fibres challenged with repetitive stimulation. the tau of Ca2+ transients decay increased from about 10 ms to >40 ms, while the basal [Ca2+]i increased from 0.1 to 2 microM. Short rest periods (about 5 min) could reverse these effects, indicating that they were not a direct consequence of SR Ca 2+ -ATPase inhibition. The correlation coefficient between tau of decay and basal [Ca2+]i was >0.8 (P<0.0001). Qualitatively similar results were obtained measuring Rhod-2 fluorescence signals. A lumped, two-compartment model could account for these results. Loading the fibres with EGTA-AM, diminished the effects of prolonged stimulation observed in poisoned fibres. Moreover, we show that the Na+ - Ca2+ exchange mechanism does not participate appreciably in fast Ca2+ removal.
Assuntos
Cálcio/metabolismo , Contração Isométrica/fisiologia , Músculo Esquelético/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Compostos de Anilina , Animais , Anuros , Sítios de Ligação/fisiologia , Soluções Tampão , ATPases Transportadoras de Cálcio/antagonistas & inibidores , ATPases Transportadoras de Cálcio/metabolismo , Estimulação Elétrica , Eletrofisiologia , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes , Cinética , Magnésio/metabolismo , Microscopia de Fluorescência , Modelos Biológicos , Fibras Musculares Esqueléticas/enzimologia , Músculo Esquelético/citologia , Parvalbuminas/fisiologia , Tempo de Reação/fisiologia , Sódio/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Tapsigargina/farmacologia , XantenosRESUMO
Cooling increases the twitch force of frog skeletal muscle (Rana temporaria; Rana pipiens), but decreases the twitch force of tropical toad muscle (Leptodactylus insularis). Action potentials and intramembranous charge movement in frog and toad fibers were slowed identically by cooling. Cooling increased the integral of twitch Ca2+ detected by aequorin in frog fibers (1.4-fold), while also decreasing the peak and slowing the rate of decay. Conversely, cooling decreased the integral (0.6-fold) and the peak of twitch Ca2+ in toad fibers, without affecting the rate of decay. The difference in entire Ca2+ transients may account for cold-induced twitch potentiation in frogs and twitch paralysis in toads. In sustained contractions of toad fibers, cooling markedly decreased maximum force caused by: (i) tetanic stimulation, (ii) two-microelectrode voltage clamp steps, (iii) high [K+], or (iv) caffeine. Maximum force in sustained contractions was decreased moderately by cooling frog fibers. Rapid rewarming and simultaneous removal of high [K+] or caffeine during a sustained contraction, caused toad muscle force to rise towards the value corresponding to the warm temperature. This did not occur after removing high [K+] or caffeine from toad fibers kept in the cold. Transmission electron micrographs showed no relevant structural differences. Parvalbumins are thought to promote relaxation of frog muscle in the cold. The unique parvalbumin isoforms in toad muscle apparently lack this property.
Assuntos
Anuros/fisiologia , Contração Muscular/fisiologia , Músculo Esquelético/fisiologia , Ranidae/fisiologia , Animais , Cafeína/farmacologia , Cálcio/fisiologia , Temperatura Baixa , Estimulação Elétrica/métodos , Eletrofisiologia , Técnicas In Vitro , Membranas/fisiologia , Microscopia Eletrônica , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/ultraestrutura , Potássio/farmacologia , Rana pipiens/fisiologia , Rana temporaria/fisiologia , DescansoRESUMO
Cut twitch muscle fibers, mounted in a triple Vaseline-gap chamber, were used to study the effects of temperature on intramembranous charge movement and, in particular, on the repriming of charge 1 (the intramembranous charge that normally moves in the potential range between -100 and +40 mV). Changing the holding potential from -90 to 0 mV modified the voltage distribution of charge movement but not the maximum movable charge. Temperature changes between 16 and 5 degrees C did not modify the fiber linear capacitance, the maximum nonlinear intramembranous charge, or the voltage distribution of charge 1 and charge 2 (the intramembranous charge moving in the membrane potential range between approximately -4 and -160 mV). We used a pulse protocol designed to study the repriming time course of charge 1, with little contamination from charge 2. The time course of charge movement repriming at 15 degrees C is described by a double exponential with time constants of 4.2 and 25 s. Repriming kinetics were found to be highly temperature dependent, with two rate-limiting steps having Q10 (increase in rate of a process by raising temperature 10 degrees C) values of 1.7 and 7.1 above and below 11.5 degrees C, respectively. This is characteristic of processes with a high energy of activation and could be associated with a conformational change of the voltage sensor or with the interaction between the voltage sensor and the calcium release channel.
Assuntos
Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/fisiologia , Animais , Anuros , Técnicas In Vitro , Cinética , Potenciais da Membrana , Movimento , Técnicas de Patch-Clamp , Temperatura , Termodinâmica , Fatores de TempoRESUMO
Cut twitch muscle fibers mounted in a triple Vaseline-gap chamber were used to study the effects of ryanodine on intramembranous charge movement, and in particular on the repriming of charge 1. Charge 1 repriming was measured either under steady-state conditions or by using a pulse protocol designed to study the time course of repriming. This protocol consisted of repolarizing the fibers to -100 mV from a holding potential of 0 mV, and then measuring the reprimed charge moving in the potential range between -40 and +20 mV. Ryanodine at a high concentration (100 microM) did not affect the maximum amount of movable charge 1 and charge 2, or their voltage dependence. This indicates that the alkaloid does not interact with the voltage sensor molecules. However, ryanodine did reduce the amount of reprimed charge 1 by approximately 60% suggesting the possibility of a retrograde interaction between ryanodine receptors and voltage sensors.
Assuntos
Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Rianodina/farmacologia , Animais , Anuros , Fenômenos Biofísicos , Biofísica , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , Relação Dose-Resposta a Droga , Técnicas In Vitro , Cinética , Potenciais da Membrana , Proteínas Musculares/efeitos dos fármacos , Proteínas Musculares/metabolismo , Rianodina/administração & dosagem , Canal de Liberação de Cálcio do Receptor de RianodinaRESUMO
STUDY OBJECTIVE: To assess patients' comprehension of their emergency department discharge instructions and to determine if inner-city patients' literacy levels are adequate to comprehend written discharge instructions. DESIGN: Prospective, observational study. SETTING: The ED of an inner-city university hospital. PARTICIPANTS: Two hundred seventeen patients consecutively discharged from the ED during 12 separate time slots. INTERVENTIONS: Patients were interviewed after discharge from the ED and asked to state their diagnosis, medication instructions, and follow-up instructions. Comparisons between patient recall and instructions as written in the chart were assessed by independent raters and scored from poor to excellent. Patients were administered a standardized test of reading ability. RESULTS: Overall comprehension rates were judged to be good, although 23% of patients exhibited no understanding of at least one component of their discharge instructions. Mean reading ability of the patients was at the sixth-grade level. The ED's printed discharge instructions were written at an 11th-grade reading level. Patients with low literacy scores were more likely to have poor comprehension of instructions. CONCLUSION: Overall comprehension rates in this population were good despite the fact that ED instruction sheets were written at an inappropriately high reading level. Verbal instructions given by the discharging physician likely have a significant effect on patients' comprehension of instructions.
Assuntos
Cognição , Serviço Hospitalar de Emergência , Alta do Paciente , Educação de Pacientes como Assunto , População Urbana , Adulto , Escolaridade , Feminino , Hospitais com mais de 500 Leitos , Hospitais Universitários , Humanos , Masculino , Pessoa de Meia-Idade , Philadelphia , Estudos ProspectivosRESUMO
We have studied the effects of the sulfhydryl reagents on contractile responses, using either electrically stimulated single muscle fibers or short muscle fibers that were voltage-clamped with a two-microelectrode voltage-clamp technique that allows the fiber tension in response to membrane depolarization to be recorded. The sulfhydryl inhibitors para-chloromercuribenzoic acid (PCMB) and parahydroximercuriphenyl sulfonic acid (PHMPS), at concentrations from 0.5 to 2 mM, cause loss of the contractile ability; however, before this effect is completed, they change the fiber contractile behavior in a complex way. After relatively short exposure to the compounds, < 20 min, before the fibers lose their contractile capacity, secondary tension responses may appear after electrically elicited twitches or tetani. After losing their ability to contract in response to electrical stimulation, the fibers maintain their capacity to develop caffeine contractures, even after prolonged periods (120 min) of exposure to PHMPS. In fibers under voltage-clamp conditions, contractility is also lost; however, before this happens, long-lasting (i.e., minutes) episodes of spontaneous contractile activity may occur with the membrane polarized at -100 mV. After more prolonged exposure (> 30 min), the responses to membrane depolarization are reduced and eventually disappear. The agent DTT at a concentration of 2 mM appears to protect the fibers from the effects of PCMB and PHMPS. Furthermore, after loss of the contractile responses by the action of PCMB or PHMPS, addition of 2 mM DTT causes recovery of tension development capacity.
Assuntos
Contração Muscular/efeitos dos fármacos , Músculos/efeitos dos fármacos , Fármacos Neuromusculares Despolarizantes/farmacologia , Reagentes de Sulfidrila/farmacologia , Animais , Anuros , Cafeína/farmacologia , Cloromercurobenzoatos/farmacologia , Ditiotreitol/farmacologia , Estimulação Elétrica , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Microeletrodos , Músculos/citologia , Compostos de Fenilmercúrio/farmacologia , Rana pipiens , Ácido p-CloromercurobenzoicoRESUMO
The effect of sulhydryl reagents on nonlinear membrane currents of frog skeletal muscle fibers has been studied using the triple Vaseline gap voltage-clamp technique. These compounds, which are known to interfere with depolarization contraction coupling, also appear to diminish intramembranous charge movement recorded with fibers polarized to -100 mV (charge 1). This effect, however, is accompanied by changes in the fiber membrane conductance and in most cases by the appearance of an inwardly directed current in the potential range between -60 and +20 mV. This current is reduced by both cadmium and nifedipine and does not occur in Ca-free solution, suggesting that it is carried by calcium ions flowing through regular calcium channels that are more easily activated in the presence of SH reagent. These changes in the membrane electrical active and passive properties decrease the quality and reliability of the P/n pulse subtracting procedure normally used for charge movement measurements. These effects can be substantially reduced by cadmium ions (0.1 mM), which has no effect on charge movement. When SH reagents are applied in the presence of cadmium, no effects are observed, indicating that this cation may protect the membrane from the reagent effects. The effects of -SH reagents can be observed by applying them in the absence of cadmium, followed by addition of the cation. Under these conditions the conductance changes are reversed and the effects of the SH reagents on charge movement can be measured with a higher degree of confidence. Maximum charge is reduced by 32% in the presence of 1.5 mM PCMB and by 31% in the presence of 2 mM PHMPS. These effects do not occur in the presence of DTT and in some cases they may be reversed by this agent. Charge 2, recorded in depolarized muscle fibers, is also reduced by these agents.
Assuntos
Canais Iônicos/efeitos dos fármacos , Músculos/fisiologia , Reagentes de Sulfidrila/farmacologia , Animais , Anuros , Cádmio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Ditiotreitol/farmacologia , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Músculos/citologia , Músculos/efeitos dos fármacos , Fibras Nervosas/efeitos dos fármacos , Nifedipino/farmacologia , Compostos Organomercúricos/farmacologia , Rana pipiensRESUMO
PURPOSE: A randomized trial was conducted to evaluate the impact of a telephone counseling intervention to improve patient adherence to colposcopic examination for suspected cervical intraepithelial neoplasia (CIN). METHODS: Subjects were lower-income, minority women who missed a scheduled initial appointment for colposcopy at an urban medical clinic. Patients were randomly assigned to either a control condition (n = 42) or a telephone counseling condition (n = 48). The 15-minute, structured telephone counseling intervention protocol addressed educational, psychosocial, and practical barriers to colposcopy adherence. RESULTS: The most common patient-reported barriers to colposcopy adherence included a lack of understanding of the purpose of colposcopy (50%), worry about or fear of cancer (25%), and forgetting (23%). Telephone counseling was found to be highly effective in addressing these barriers and improving adherence to diagnostic follow-up and treatment. Of patients in the control condition, 43% complied with a rescheduled colposcopy appointment, compared with 67% in the telephone counseling condition. Logistic regression analysis indicated that the effect of telephone counseling was independent of sociodemographic confounder variables (odds ratio = 2.6; P less than .003). Additionally, 74% of patients who received the initial telephone counseling adhered to recommended treatment, compared with 53% of patients in the control condition. CONCLUSION: Brief, structured telephone contact may be a cost-effective mechanism for improving adherence to diagnostic follow-up and treatment for a variety of cancer screening tests.
Assuntos
Colposcopia , Aconselhamento , Cooperação do Paciente , Neoplasias do Colo do Útero/diagnóstico , Adolescente , Adulto , Colposcopia/psicologia , Aconselhamento/métodos , Escolaridade , Feminino , Humanos , Renda , Modelos Logísticos , Grupos Minoritários/psicologia , Aceitação pelo Paciente de Cuidados de Saúde , Educação de Pacientes como Assunto , Telefone , População Urbana , Neoplasias do Colo do Útero/etnologiaRESUMO
This study identified factors associated with cervical cancer screening patterns among lower income primary care patients. One hundred forty-one women completed a self-administered questionnaire before their medical visit. The results indicated that 71 percent had a Papanicolaou (Pap) test in the past year, 14 percent had one between 1 and 3 years ago, and 15 percent had not had a Pap test for 3 or more years. Advanced age was associated with a reduced likelihood of adequate screening; 21 percent of women aged 50-64 years and 39 percent of those aged 65 years and older had not had a Pap test in the past 3 years. Factors positively associated with screening included perceived susceptibility to cervical cancer and the belief in the efficacy of Pap tests and benefits of screening. Fear of finding cancer was a significant barrier to cervical screening in this population. Of those women who had not been screened adequately, 58 percent were interested in obtaining Pap tests in the primary care setting. The implications for promoting cervical cancer screening in primary care practices are discussed.
Assuntos
Atitude Frente a Saúde , Programas de Rastreamento/normas , Atenção Primária à Saúde/normas , Neoplasias do Colo do Útero/prevenção & controle , Adulto , Idoso , Escolaridade , Emprego , Feminino , Humanos , Renda , Programas de Rastreamento/métodos , Pessoa de Meia-Idade , Aceitação pelo Paciente de Cuidados de Saúde , Cooperação do Paciente , Philadelphia , Atenção Primária à Saúde/métodos , Fatores Socioeconômicos , Inquéritos e Questionários , Neoplasias do Colo do Útero/psicologiaAssuntos
Galopamil/farmacologia , Canais Iônicos/efeitos dos fármacos , Lantânio/farmacologia , Junção Neuromuscular/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Animais , Canais de Cálcio/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Rana pipiens , Retículo Sarcoplasmático/efeitos dos fármacosRESUMO
We have developed a quick-freezing method, using a copper block cooled with liquid helium or nitrogen, which permits us to freeze muscles without any cryoprotectant at predetermined, precisely measured points in the recorded tension time-course of a single twitch or tetanus. Our aim is to arrest structural intermediates of the cross-bridge cycle for observation in the electron microscope. Chemically stimulated, demembranated muscles as well as electrically stimulated, live muscles can be frozen on the same apparatus. Good freezing of relaxed and contracting muscles has been obtained to a depth of 10-20 microns, with excellent structural preservation after freeze-substitution.
Assuntos
Contração Muscular , Músculos/ultraestrutura , Animais , Congelamento , Secções Congeladas , Microscopia Eletrônica , Músculos/fisiologia , Rana pipiensAssuntos
Animais , Galopamil/farmacologia , Canais Iônicos/efeitos dos fármacos , Lantânio/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Canais de Cálcio/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Junção Neuromuscular/efeitos dos fármacos , Rana pipiens , Retículo Sarcoplasmático/efeitos dos fármacosRESUMO
Ca2+ selective microelectrodes have been used to determine the free myoplasmic [Ca2+] in human skeletal muscle obtained from patients who had developed early signs associated with malignant hyperthermia (MH) during anesthesia. Intercostal muscle biopsies were performed under local anesthesia in four MH patients 15 days to 4 months after developing the MH crisis and in three control subjects. We used only microelectrodes that showed a Nernstian response between pCa3 and pCa7 (30.5 mV per decade at 37 degrees C). Membrane resting potential (V(m)) and calcium potential (V(Ca)) were obtained from superficial fibers. The free cytosolic [Ca2+] was 0.39 +/- 0.1 microM (mean +/- SEM, n = 18) in muscle fibers obtained from malignant hyperthermic patients, whereas in control subjects it was 0.11 +/- 0.02 microM (n = 10). These results suggest that this syndrome might be related to an abnormally high myoplasmic free resting calcium concentration, probably due to a defective function of the plasma membrane or the sarcoplasmic reticulum.