RESUMO
We report on laser cooling of a large fraction of positronium (Ps) in free flight by strongly saturating the 1^{3}S-2^{3}P transition with a broadband, long-pulsed 243 nm alexandrite laser. The ground state Ps cloud is produced in a magnetic and electric field-free environment. We observe two different laser-induced effects. The first effect is an increase in the number of atoms in the ground state after the time Ps has spent in the long-lived 2^{3}P states. The second effect is one-dimensional Doppler cooling of Ps, reducing the cloud's temperature from 380(20) to 170(20) K. We demonstrate a 58(9)% increase in the fraction of Ps atoms with v_{1D}<3.7×10^{4} ms^{-1}.
RESUMO
The phylogeography of typical alpine plant species is well understood in Europe. However, the genetic patterns of boreo-montane species are mostly unstudied. Therefore, we analysed the AFLPs of 198 individuals of Polygonatum verticillatum over a major part of its European distribution. We obtained a total of 402 reproducible fragments, of which 96.8% were polymorphic. The average Phi(ST) over all samples was high (73.0%). The highest number of private fragments was observed in the Cantabrian Mountains; the highest genetic diversities of the populations were detected in populations from the Alps. BAPS, Principal Coordinates and Cluster analyses revealed a deep split between the Cantabrian population and all other samples. The latter further distinguished two major groups in western and eastern Europe. These results suggest a complex biogeographical history of P. verticillatum. The Cantabrian population was most probably isolated for the longest time. Furthermore, putative glacial survival centres might have existed in the western group around the glaciated Alps and in the eastern group in the foothills of the Carpathian and Balkan mountain systems. The origin of the Scandinavian populations is still unresolved, but an origin from the southeastern Alps or the western Balkans appears the most likely scenario.
Assuntos
Aclimatação , Clima , Ecossistema , Variação Genética , Camada de Gelo , Filogenia , Polygonatum/genética , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , DNA de Plantas , Europa (Continente) , Genética Populacional , Genoma de Planta , Geografia , Fenótipo , Polimorfismo de Fragmento de Restrição , ÁrvoresRESUMO
Mice with deletions of nicotinic ACh receptor (nAChR) subunit genes are valuable models for studying nAChR functions. We could previously show in the mouse superior cervical ganglion (SCG) that the absence of distinct subunits affects the functional properties of receptors. Here, we have addressed the question of whether deletions of the subunits alpha5, alpha7, or beta2 are compensated at the mRNA level, monitored by reverse transcription and quantitative real-time polymerase chain reaction. Relative to our reference gene, alpha3, which is expressed in all SCG nAChRs, mRNA levels of beta4 showed little change from birth until adult ages in intact ganglia of wild-type mice. In contrast, alpha4 declined sharply after birth and was barely detectable in adult animals. alpha5, alpha7, and beta2 subunit message levels also declined, though more slowly and less completely than alpha4. The subunits alpha6 and beta3 were detected by conventional polymerase chain reaction at very low levels, if at all, whereas alpha2 was never seen in any of our samples. The developmental profile of nAChR mRNA levels in the three knockout strains did not differ markedly from that of wild-type mice. Likewise, message levels of nAChR subunits were similar in cultures prepared from either wild-type or knockout animals. Our observations indicate a developmental regulation of nAChR subunit mRNAs in the SCG of mice after birth that was not affected by the three knockouts under investigation.
Assuntos
Neurônios/metabolismo , RNA Mensageiro/genética , Receptores Nicotínicos/genética , Gânglio Cervical Superior/crescimento & desenvolvimento , Gânglio Cervical Superior/metabolismo , Acetilcolina/metabolismo , Animais , Diferenciação Celular/genética , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/citologia , Subunidades Proteicas/genética , RNA Mensageiro/metabolismo , Especificidade da Espécie , Gânglio Cervical Superior/citologia , Transmissão Sináptica/genética , Receptor Nicotínico de Acetilcolina alfa7RESUMO
BACKGROUND AND PURPOSE: Mice with targeted deletions of neuronal nicotinic acetylcholine receptor (nAChR) subunit genes are valuable models to study nAChR function such as catecholamine outflow by presynaptic receptor activation. Contrary to the rat, our present knowledge on presynaptic nAChRs in mice primarily relies on observations made with synaptosomes. We have now used brain slices to investigate nicotine-induced catecholamine outflow in wild type (WT) and nAChR (beta2 and alpha5) knockout mice for a comparison with rat brain slice preparations. EXPERIMENTAL APPROACH: Brain slices from rat and mouse hippocampus, parieto-occipital neocortex, and corpus striatum were loaded with either [3H]-noradrenaline or [3H]-dopamine. We provoked catecholamine outflow by electrical field stimulation and nicotinic agonists. KEY RESULTS: When set in relation to electrical field stimulation, nicotine-evoked catecholamine release was sizeable in the striatum but low in the neocortex of both rats and mice. [3H]-noradrenaline outflow was, on the other hand, substantial in the rat but low in the mouse hippocampus. About 10% (or less) of nicotine-induced catecholamine release persisted in the presence of tetrodotoxin in all our preparations. CONCLUSIONS AND IMPLICATIONS: Targeted deletion of the beta2 subunit gene essentially abolished the effect of nicotine, indicating that this subunit is an essential constituent of nAChRs that indirectly (via action potentials) induce catecholamine release from hippocampal and striatal slices in mice. The impact of nAChRs in catecholaminergic projection areas differs between species and has thus to be considered when extrapolating results from animal models to human conditions.
Assuntos
Encéfalo/efeitos dos fármacos , Catecolaminas/metabolismo , Agonistas Nicotínicos/farmacologia , Receptores Nicotínicos/fisiologia , Alcaloides/farmacologia , Animais , Azocinas/farmacologia , Encéfalo/metabolismo , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Iodeto de Dimetilfenilpiperazina/farmacologia , Dopamina/metabolismo , Relação Dose-Resposta a Droga , Estimulação Elétrica , Feminino , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neocórtex/efeitos dos fármacos , Neocórtex/metabolismo , Nicotina/farmacologia , Norepinefrina/metabolismo , Subunidades Proteicas/agonistas , Subunidades Proteicas/genética , Subunidades Proteicas/fisiologia , Quinolizinas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Nicotínicos/genética , Tetrodotoxina/farmacologiaRESUMO
Primary cultures of postganglionic sympathetic neurons were established more than 30 years ago. More recently, these cultures have been used to characterize various neurotransmitter receptors that govern sympathetic transmitter release. These receptors may be categorized into at least three groups: (1) receptors which evoke transmitter release: (2) receptors which facilitate; (3) receptors which inhibit, depolarization-evoked release. Group (1) comprises nicotinic and muscarinic acetylcholine receptors, P2X purinoceptors and pyrimidinoceptors. Group (2) currently harbours beta-adrenoceptors, P2 purinoceptors, receptors for PACAP and VIP, as well as prostanoid EP1 receptors. In group (3), muscarinic cholinoceptors, alpha 2- and beta-adrenoceptors, P2 purinoceptors, and receptors for the neuropeptides NPY, somatostatin (SRIF1) and LHRH, as well as opioid (delta and kappa) receptors can be found. Receptors which regulate transmitter release from neurons in cell culture may be located either at the somatodendritic region or at the sites of exocytosis, i.e. the presynaptic specializations of axons. Most of the receptors that evoke release are located at the soma. There ionotropic receptors cause depolarizations to generate action potentials which then trigger Ca(2+)-dependent exocytosis at axon terminals. The signalling mechanisms of metabotropic receptors which evoke release still remain to be identified. Receptors which facilitate depolarization-evoked release appear to be located preferentially at presynaptic sites and presumably act via an increase in cyclic AMP. Receptors which inhibit stimulation evoked release are also presynaptic origin and most commonly rely on a G protein-mediated blockade of voltage-gated Ca2+ channels. Results obtained with primary cell cultures of postganglionic sympathetic neurons have now supplemented previous data about neurotransmitter receptors involved in the regulation of ganglionic as well as sympatho-effector transmission. In the future, this technique may prove useful to identify yet unrecognized receptors which control the output of the sympathetic nervous system and to elucidate underlying signalling mechanisms.
Assuntos
Neurônios/fisiologia , Neurotransmissores/metabolismo , Receptores de Neurotransmissores/fisiologia , Sistema Nervoso Simpático/fisiologia , Animais , Receptores Adrenérgicos beta/fisiologia , Receptores Muscarínicos/fisiologia , Receptores de Neuropeptídeos/fisiologia , Receptores Nicotínicos/fisiologia , Receptores Opioides/fisiologia , Receptores de Prostaglandina/fisiologia , Receptores Purinérgicos/fisiologia , Transdução de SinaisRESUMO
Immunization with dendritic cells and unfractionated MHC class I-binding peptides derived from autologous tumor cells has been shown to induce effective antitumor immunity. However, the importance of the relative abundance of tumor peptides on the surface of tumor cells is not known. We have addressed this question using peptides isolated from three tumor cell lines transfected with a minigene encoding amino acids 33-41 of the lymphocytic choriomeningitis virus glycoprotein (LCMV(33-41)). The three cell lines expressed different levels of MHC class I molecules and had different abilities to stimulate proliferation of LCMV(33-41)-specific T cells in vitro. We found that antitumor immune responses were best elicited by immunizing mice with dendritic cells and synthetic LCMV(33-41) peptide. Peptide preparations from a given tumor cell line conferred protection against challenge with the same tumor cell line. However, protective immunity to a different tumor could be induced only if the cell line used for peptide preparation presented a high relative proportion of LCMV(33-41) in association with MHC class I. Our results suggest that multiple peptide epitopes are required for the induction of an effective antitumor immune response using MHC class I-binding peptides from tumor cells. Also, the ability to induce an antitumor immune response appears to correlate with the proportion, rather than the absolute amount, of tumor-specific peptide in the mixture used for immunization.
Assuntos
Antígenos de Neoplasias/imunologia , Antígenos Virais , Células Dendríticas/imunologia , Glicoproteínas/imunologia , Proteínas de Neoplasias/imunologia , Fragmentos de Peptídeos/imunologia , Proteínas Virais , Animais , Antígenos de Neoplasias/metabolismo , Carcinoma Pulmonar de Lewis/imunologia , Carcinoma Pulmonar de Lewis/prevenção & controle , Carcinoma Pulmonar de Lewis/terapia , Células Cultivadas , Feminino , Glicoproteínas/antagonistas & inibidores , Glicoproteínas/genética , Antígenos H-2/biossíntese , Antígenos H-2/imunologia , Antígeno de Histocompatibilidade H-2D , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Imunoterapia Adotiva , Ativação Linfocitária/imunologia , Masculino , Melanoma Experimental/imunologia , Melanoma Experimental/prevenção & controle , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas de Neoplasias/isolamento & purificação , Proteínas de Neoplasias/uso terapêutico , Oligopeptídeos/imunologia , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/genética , Linfócitos T/imunologia , Transfecção , Células Tumorais CultivadasRESUMO
GABA(A) receptors are the major inhibitory transmitter receptors in the CNS. Recombinant GABA(A) receptors composed of alpha(1)beta(3)gamma(2) subunits have been demonstrated to assemble as pentamers consisting of two alpha(1), two beta(3), and one gamma(2) subunit. Using truncated and chimeric alpha(1) subunits, we identified the alpha(1)(80-100) sequence as a major binding site for gamma(2) subunits. In addition, we demonstrated its direct interaction with gamma(2)(91-104), a sequence that previously has been identified to form the contact to alpha(1) subunits. The observation that the amino acid residues alpha(1)P96 and alpha(1)H101, which can be photolabeled by [(3)H]flunitrazepam, are located within or adjacent to the alpha(1)(80-100) sequence, indicates that the benzodiazepine binding site of GABA(A) receptors is located close to this intersubunit contact. The observation that alpha(1)(80-100) interacts with gamma(2) but not with beta(3) subunits indicates the existence of an additional beta(3) binding site on alpha(1) subunits. The preferred alternate use of the gamma(2) and beta(3) binding sites in two different alpha(1) subunits of the same receptor ensures the incorporation of only a single gamma(2) subunit and thus, determines subunit stoichiometry of alpha(1)beta(3)gamma(2) receptors. Distinct binding sites and their alternate use can therefore explain how subunits of hetero-oligomeric transmembrane proteins assemble into a defined protein complex.
Assuntos
Subunidades Proteicas , Receptores de GABA-A/biossíntese , Sequência de Aminoácidos/fisiologia , Animais , Western Blotting , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Imunofluorescência , Expressão Gênica , Humanos , Rim/citologia , Rim/metabolismo , Peso Molecular , Técnicas de Patch-Clamp , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/metabolismo , Testes de Precipitina , Ligação Proteica/fisiologia , Ratos , Receptores de GABA-A/química , Receptores de GABA-A/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Relação Estrutura-Atividade , TransfecçãoRESUMO
In the human T-cell rearranging gamma (TRG gamma) locus, five joining (J) segments have been identified: J1, J2 and three additional segments JP, JP1 and JP2. We report the sequence of the germline JP1 segment and compare it with the other human and mouse J gamma segments. We also demonstrate that rearrangements to the three additional J gamma segments can be identified by hybridization of the KpnI digests to the J gamma 1 probe pH60. Since rearrangements to J1 or J2 can be assigned, using the same pH60 probe, to one of the nine variable (V) gamma genes known to rearrange [(1987) EMBO J. 6, 1945-1950], our results show that a unique probe can detect all the TRG gamma rearrangements and be particularly useful for assessing the preferential usage of V gamma and J gamma segments in the TRG gamma-expressing cells.
Assuntos
Receptores de Antígenos de Linfócitos T/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Genes , Humanos , Camundongos , Dados de Sequência Molecular , Receptores de Antígenos de Linfócitos T gama-delta , Sequências Reguladoras de Ácido NucleicoRESUMO
We report the first specific human immunoglobulin subclass probe which was obtained by subcloning the gamma 3 hinge region. This specific gamma 3 probe allowed us to identify with certainty the C gamma 3 gene on Southern genomic blots, to describe the first C gamma 3 restriction fragment length polymorphism (EZZ gamma 3 RF) and to show that an IgG3 selective deficiency, previously described serologically, was not due to a deletion of the C gamma 3 gene. Such a probe should be particularly useful for screening libraries from individuals with IgG3 immunodeficiencies or presenting unusual C gamma 3 genes and, consequently, for studying the C gamma gene evolution.
Assuntos
Regiões Constantes de Imunoglobulina/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias gama de Imunoglobulina/genética , Imunoglobulinas/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Éxons , Éxons Codificadores da Região de Dobradiça , Humanos , Alótipos de Imunoglobulina , Isotipos de Imunoglobulinas , Cadeias gama de Imunoglobulina/deficiência , Linhagem , Polimorfismo de Fragmento de RestriçãoRESUMO
The effects of somatostatin and analogues were investigated in cultures of chick sympathetic neurons. Electrically evoked tritium overflow from cultures labelled with [3H]noradrenaline was reduced by somatostatin-14 in a concentration-dependent manner, with half maximal effects at 0.3 nM and a maximum of 45% inhibition. Somatostatin-28 was equipotent to somatostatin-14 (half maximal concentration at 0.5 nM), and seglitide was less potent, the effects being half maximal at 4.2 nM. The inhibitory action of somatostatin-14 on stimulation-evoked overflow desensitized within minutes at 100 nM, but not at 10 nM, and was abolished by a pretreatment of neurons with pertussis toxin. All somatostatin analogues reduced voltage-activated Ca2+ currents recorded in the whole-cell configuration of the patch-clamp technique, with somatostatin-14 being equipotent to somatostatin-28, but more potent than seglitide. However, the inhibition of Ca2+ currents occurred at concentrations more than ten-fold higher than those required for the reduction of stimulation evoked 3H overflow. The action of somatostatin upon Ca2+ currents was also abolished by pertussis toxin and desensitized within minutes. In preceding experiments, alpha 2-adrenoceptor activation had been found to reduce transmitter release and Ca2+ currents of chick sympathetic neurons through a pertussis toxin-sensitive mechanism. In the present study, the alpha 2-adrenergic agonist UK 14,304 completely occluded the inhibition of Ca2+ currents and of electrically evoked overflow by somatostatin-14. Neither UK 14,304 nor somatostatin affected the resting membrane potential or voltage-dependent K+ currents. These results demonstrate that chick sympathetic neurons possess SRIF1 type somatostatin receptors which control transmitter release. This effect is mediated by pertussis toxin-sensitive GTP binding proteins and apparently involves an inhibition of voltage-activated Ca2+ channels, but not a modulation of K+ channels. Since alpha 2-adrenergic agonists share all of these actions and occlude the effects of somatostatin, alpha 2-adrenoceptors and SRIF1 receptors seem to regulate sympathetic transmitter release via common signalling mechanisms.
Assuntos
Gânglios Simpáticos/fisiologia , Neurônios/fisiologia , Norepinefrina/metabolismo , Toxina Pertussis , Receptores Adrenérgicos alfa 2/fisiologia , Receptores de Somatostatina/fisiologia , Somatostatina/farmacologia , Fatores de Virulência de Bordetella/farmacologia , Agonistas alfa-Adrenérgicos/farmacologia , Animais , Tartarato de Brimonidina , Células Cultivadas , Embrião de Galinha , Relação Dose-Resposta a Droga , Estimulação Elétrica , Antagonistas de Hormônios/farmacologia , Cinética , Potenciais da Membrana/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Peptídeos Cíclicos/farmacologia , Quinoxalinas/farmacologia , Receptores Adrenérgicos alfa 2/efeitos dos fármacos , Receptores de Somatostatina/efeitos dos fármacos , Somatostatina-28RESUMO
[3H]Noradrenaline release was studied in cultured sympathetic neurons derived from superior cervical ganglia of neonatal rats. Acetylcholine elicited a concentration- and time-dependent increase in 3H outflow which was half-maximal at about 300 microM and within 5 s. The overflow induced by 10 s exposure to 300 micro A acetylcholine was reduced by the nicotinic antagonist hexamethonium, but increased by the muscarinic antagonist atropine. Cd2+ (300 microM) prevented the overflow evoked by electrical field stimulation, but reduced acetylcholine-induced overflow by less than 50%. Removal of extracellular Ca2+ abolished stimulation-evoked tritium overflow irrespective of the stimulus. The selective alpha2-adrenoceptor agonist UK 14,304 inhibited acetylcholine-evoked overflow to a significantly smaller extent (approximately 25% maximal inhibition) than electrically induced overflow ( > or = 45% maximal inhibition). These inhibitory effects were antagonized by the alpha2-adrenoceptor antagonist yohimbine. Noradrenaline (0.1 microM) reduced acetylcholine-evoked overflow to the same extent as did UK 14,304 (0.1 microM). UK 14,304 had no effect when 3H overflow was evoked by acetylcholine in the presence of 300 microM Cd2+. Currents through nicotinic acetylcholine receptors and voltage-activated Ca2+ currents were studied with the whole-cell variant of teh patch-clamp technique. UK 14,304 reduced nicotinic acetylcholine receptor currents and voltage-activated Ca2+ currents with similar potency and efficacy. Yohimbine, however, antagonized only the inhibition of voltage-activated Ca2+ currents, but not the effects of UK 14,304 on nicotinic receptor currents. Furthermore, yohimbine per se reduced currents through nicotinic receptors. Noradrenaline (10 microM) inhibited voltage-dependent Ca2+ currents just as did UK 14,304 (10 microM), but failed to reduce currents through nicotinic acetylcholine receptor channels. Cd2+ (300 microM) abolished voltage-activated Ca2+ currents and reduced nicotinic acetylcholine receptor currents by 65%. These results indicate that acetylcholine evokes noradrenaline release from rat sympathetic neurons by activation of nicotinic receptors and restricts this release via muscarinic receptors. The acetylcholine-induced transmitter release is based on two mechanisms, one involving and the other one bypassing voltage-dependent Ca2+ channels. alpha2-Adrenoceptor activation reduces voltage-activated Ca2+ currents and effects exclusively the component of acetylcholine-induced release which involves voltage-dependent Ca2+ channels. These results support the hypothesis that voltage-activated Ca2+ channels are the sole site of autoinhibitory alpha2-adrenergic effects on transmitter release from rat sympathetic neurons. The inhibitory effects of alpha2-adrenoceptor agonists and antagonists on currents through nicotinic acetylcholine receptors are not mediated by an alpha2-adrenoceptor.
Assuntos
Acetilcolina/farmacologia , Gânglios Simpáticos/metabolismo , Inibição Neural/fisiologia , Neurônios/metabolismo , Norepinefrina/metabolismo , Receptores Adrenérgicos alfa/fisiologia , Animais , Cádmio/farmacologia , Canais de Cálcio/fisiologia , Células Cultivadas , Estimulação Elétrica , Eletrofisiologia , Gânglios Simpáticos/citologia , Ativação do Canal Iônico , Ligantes , Ratos , Ratos Sprague-Dawley , Receptores Nicotínicos/fisiologiaRESUMO
The cytotoxic effects of DL-, D- and L-alpha-aminoadipic acid, a six-carbon homologue of glutamate, were investigated in cell cultures of dissociated postnatal mouse cerebellum. Treatment with alpha-aminoadipic acid resulted in rapid nuclear and cytoplasmic swelling and, after longer periods of exposure, karyopyknosis of astrocytes, identified by indirect immunofluorescence labelling with anti-human glial fibrillary acidic protein antiserum. The number of astrocytes with pyknotic nuclei depended on the concentration of alpha-aminoadipic acid as well as on the duration of drug action. The presence of 0.21 mM DL-alpha-aminoadipic acid or 0.10 mM L-alpha-aminoadipic acid for 40 h caused karyopyknosis in 50% of the astrocytes. In contrast, D-alpha-aminoadipic acid, had little gliotoxic activity. None of the cytotoxic effects of DL-alpha-aminoadipic acid or L-alpha-aminoadipic acid observed for astrocytes were seen for the neurons present in the cultures when the drug was added after 4 days in vitro. Neurotoxic effects were evident, however, when alpha-aminoadipic acid was included in the culture medium at plating. These results indicate that alpha-adminoadpic acid can be used to substantially reduce the number of astroglia in cerebellar cultures and that dissociated cell cultures will provide a useful model with which to study the mechanisms of alpha-aminoadipic acid induced glial toxicity.
Assuntos
Ácido 2-Aminoadípico/toxicidade , Aminoácidos Dicarboxílicos/toxicidade , Astrócitos/efeitos dos fármacos , Animais , Núcleo Celular/efeitos dos fármacos , Células Cultivadas , Camundongos , Conformação MolecularRESUMO
The accumulation and retention mechanisms of 99mTc-d, 1-hexamethylpropyleneamine oxime (99mTc-d, 1-HMPAO) were investigated in cultures of the dissociated rat cerebellum. Our experiments indicate a linear dependency of the uptake on incubation time and on the concentration of the radioligand. Upon chloroform extraction and distribution between the lipophilic and the hydrophilic phases, we located 69.1% of the retained radioactivity in the hydrophilic phase, 24.1% in a bound state and 6.8% in the lipophilic phase. The water-soluble, unbound radioactive contents of the cultures were identified as 99mTcO4- by HPLC analysis. Treatment of cultures with diethyl maleate (DEM) inhibited the accumulation of radioactivity along with a reduction of the GSH contents of the cultures. However, even in the absence of GSH, significant amounts of radioactivity were accumulated. DEM reduced the radioactive contents of cultures predominantly by diminishing the aqueous phase of the chloroform-extracted material. By contrast, the metabolic state, manipulated by treating the cultures with oligomycin B or 2,4-dinitrophenol, had no significant effect on the accumulation of radioactivity. Our experiments suggest two major mechanisms for the retention of radioactivity following the exposure of neuronal tissue to 99mTc-d, 1-HMPAO: Conversion of the lipophilic complex to the hydrophilic product, 99mTcO4-, and binding to non-diffusible cell components.
Assuntos
Cerebelo/metabolismo , Compostos de Organotecnécio/farmacocinética , Oximas/farmacocinética , 2,4-Dinitrofenol , Animais , Células Cultivadas , Cerebelo/diagnóstico por imagem , Cromatografia Líquida de Alta Pressão , Dinitrofenóis/farmacologia , Meia-Vida , Maleatos/farmacologia , Oligomicinas/farmacologia , Cintilografia , Ratos , Tecnécio/farmacocinética , Tecnécio Tc 99m Exametazima , Fatores de TempoRESUMO
1. The role of bradykinin receptors in the regulation of sympathetic transmitter release was investigated in primary cultures of neurones dissociated from superior cervical ganglia of neonatal rats. These cultures were loaded with [3H]-noradrenaline and the outflow of radioactivity was determined under continuous superfusion. 2. Bradykinin (100 nmol l[-1] applied for 10 min) caused a transient increase in tritium outflow that reached a peak within four minutes after the beginning of the application and then declined towards the baseline, despite the continuing presence of the peptide. ATP (100 micromol l[-1]) and nicotine (10 micromol l[-1]) caused elevations in 3H outflow with similar kinetics, whereas outflow remained elevated during a 10 min period of electrical field stimulation (0.5 ms, 50 mA, 50 V cm[-1], 1.0 Hz). 3. When bradykinin was applied for periods of 2 min, the evoked 3H overflow was half-maximal at 12 nmol l(-1) and reached a maximum of 2.3% of cellular radioactivity. The preferential B1 receptor agonist des-Arg9-bradykinin failed to alter 3H outflow. The B2 receptor antagonists, [D-Phe7]-bradykinin (1 micromol l[-1]) and Hoe 140 (10 nmol l[-1]), per se did not alter 3H outflow, but shifted the concentration-response curve for bradykinin-evoked 3H overflow to the right by a factor of 7.9 and 4.3, respectively. 4. Bradykinin-induced overflow was abolished in the absence of extracellular Ca2+ and in the presence of either 1 micromol l(-1) tetrodotoxin or 300 micromol l(-1) Cd2+, as was electrically-induced overflow. Activation of alpha2-adrenoceptors by 1 micromol l(-1) UK 14,304 reduced both bradykinin- and electrically-triggered overflow. The Ca2+-ATPase inhibitor thapsigargin (0.3 micromol l[-1]) failed to alter either type of stimulated overflow. Caffeine (10 mmol l[-1]) enhanced bradykinin-induced overflow, but reduced overflow triggered by electrical field stimulation. 5. Inclusion of Ba2+ (0.1 to 1 mmol l[-1]) in the superfusion medium enhanced electrically induced overflow by approximately 100% and potentiated bradykinin-triggered overflow by almost 400%. Application of 1 mmol l(-1) Ba2+ for periods of 2 min triggered 3H overflow, and this overflow was abolished by 1 micromol l(-1) tetrodotoxin and enhanced by 10 mmol l(-1) caffeine. In contrast, inclusion of tetraethylammonium (0.1 to 1 mmol l[-1]) in the superfusion buffer caused similar increases of bradykinin- and electrically evoked 3H overflow (by about 100%), and tetraethylammonium, when applied for 2 min, failed to alter 3H outflow. 6. Treatment of cultures with 100 ng ml(-1) pertussis toxin caused a significant increase in bradykinin-, but not in electrically-, evoked tritium overflow. Treatment with 100 ng ml(-1) cholera toxin reduced both types of stimulated 3H overflow. 7. These data reveal bradykinin as a potent stimulant of action potential-mediated and Ca2+-dependent transmitter release from rat sympathetic neurones in primary cell culture. This neurosecretory effect of bradykinin involves activation of B2-receptors, presumably linked to pertussis- and cholera toxin-insensitive G proteins, most likely members of the Gq family. Results obtained with inhibitors of muscarinic K+ (KM) channels, like caffeine and Ba2+, indicate that the secretagogue action of bradykinin probably involves inhibition of these K+ channels.
Assuntos
Neurotransmissores/metabolismo , Norepinefrina/metabolismo , Receptores da Bradicinina/fisiologia , Gânglio Cervical Superior/metabolismo , Agonistas alfa-Adrenérgicos/farmacologia , Animais , Bradicinina/farmacologia , Tartarato de Brimonidina , Cádmio/farmacologia , Cálcio/farmacologia , Células Cultivadas , Técnicas In Vitro , Bloqueadores dos Canais de Potássio , Quinoxalinas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor B2 da Bradicinina , Receptores Adrenérgicos alfa 2/efeitos dos fármacos , Receptores da Bradicinina/efeitos dos fármacos , Receptores da Bradicinina/metabolismo , Tetrodotoxina/farmacologia , Tapsigargina/farmacologiaRESUMO
In rat cultured sympathetic neurones, UDP, UTP and ATP at micromolar concentrations triggered Ca(2+)-dependent and tetrodotoxin-sensitive [3H]-noradrenaline release. The overflow evoked by UTP or ATP was similar at 100 mumol l-1, the concentration used in all subsequent experiments. Pre-exposure of the neurones to 100 mumol l-1 UTP significantly reduced ensuing secretory effects of UTP but not of ATP. Conversely, pre-exposure to ATP diminished the overflow due to ATP but not that due to UTP. In the presence of 10 mumol l-1 pyridoxal-5'-phosphate or 30 mumol l-1 suramin, the secretory response to ATP was reduced, but the effect of UTP was unaltered. Zn2+ (10 mumol l-1) reduced the overflow triggered by UTP, but increased the overflow due to ATP. These results indicate the presence of separate receptors for pyrimidine nucleotides and for purine nucleotides which both trigger transmitter release.
Assuntos
Trifosfato de Adenosina/farmacologia , Neurônios/metabolismo , Neurotransmissores/metabolismo , Receptores de Neurotransmissores/metabolismo , Sistema Nervoso Simpático/metabolismo , Animais , Células Cultivadas , Técnicas In Vitro , Neurônios/efeitos dos fármacos , Norepinefrina/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Neurotransmissores/efeitos dos fármacos , Gânglio Cervical Superior/citologia , Gânglio Cervical Superior/efeitos dos fármacos , Gânglio Cervical Superior/metabolismo , Sistema Nervoso Simpático/citologia , Sistema Nervoso Simpático/efeitos dos fármacos , Difosfato de Uridina/farmacologia , Uridina Trifosfato/farmacologiaRESUMO
1. The release of previously incorporated [3H]-noradrenaline was investigated in cultures of dissociated chick or rat sympathetic neurones and in cerebrocortical slices from neonatal or adult rats. Noradrenaline, in the presence of 10 mumol l-1 of the uptake inhibitor, cocaine, or the selective alpha 2-adrenoceptor agonist, 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK 4,304), was applied for different periods of time in order to detect a possible time-dependence of the alpha 2-adrenoceptor-mediated inhibition of electrically evoked tritium outflow. 2. In chick sympathetic neurones, stimulation-evoked overflow was reduced to 30%, 42%, or 56% of control when noradrenaline (1 mumol l-1) was present for 2, 8, or 16 min, respectively. Likewise, UK 14,304 (1 mumol l-1) present for these periods of time reduced 3H overflow to 35%, 51%, and 53% of control, respectively. Addition of 1 nmol l-1 to 10 mumol l-1 UK 14,304 for either 2 or 16 min did not produce significantly different IC50 values, but the inhibitory effects were smaller with 16 min as compared to 2 min exposure at concentrations > or = 10 nmol l-1. 3. In rat sympathetic neurones, noradrenaline (100 nmol l-1) reduced stimulation-evoked overflow to 33%, 56%, or 57% of control, when present for 2, 8, or 16 min, respectively. Addition of UK 14,304 (1 mumol l-1) for these periods of time caused inhibition to 11%, 41%, and 46% of control. Applying UK14,304 for either 2 or 16 min did not result in significantly different IC5o values, but the inhibition induced by 16 min as compared to 2 min exposure was smaller at concentrations > 10 nmol 1-1.4. In cerebrocortical slices from either neonatal or adult rats, exposure to 0.1 to 1.0 micromol 1-1 UK14,304 for 16 min never caused a smaller inhibition than a corresponding 3 min exposure, although various experimental conditions were investigated.5 The results demonstrate that alpha 2-adrenoceptors which regulate noradrenaline release from sympathetic neurones undergo agonist-induced desensitization within minutes. Such rapid desensitization of alpha 2-autoreceptors was not detected in brain slice preparations.
Assuntos
Autorreceptores/efeitos dos fármacos , Norepinefrina/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Agonistas alfa-Adrenérgicos/farmacologia , Animais , Tartarato de Brimonidina , Células Cultivadas , Embrião de Galinha , Estimulação Elétrica , Técnicas In Vitro , Quinoxalinas/farmacologia , Ratos , Ratos Sprague-Dawley , Sistema Nervoso Simpático/citologia , Sistema Nervoso Simpático/efeitos dos fármacos , Sistema Nervoso Simpático/metabolismo , Fatores de TempoRESUMO
B cell receptor (BcR) signaling requires a tight regulation of several protein tyrosine kinases and phosphatases, and associated co-receptors. Mounting evidence indicates that abnormal BcR signaling, such as occurs in SHP-1 and Lyn-deficient mice, results in production of pathogenic autoantibodies and lupus-like glomerulonephritis, suggesting that altered signaling thresholds could underlie the development of systemic autoimmunity. To test this hypothesis, we investigated expression of BcR-associated signaling molecules in lymphocytes from patients with systemic lupus erythematosus (SLE) during inactive phases of the disease. We found that the transmembrane regulatory protein tyrosine phosphatase CD45 is expressed at abnormal levels. Strikingly, this reduction persisted during four months of follow-up. By contrast, despite its potent role as a regulator of thymus-independent immune responses and of B cell life span, the CD22 co-receptor is expressed at normal levels in B lymphocytes isolated ex vivo from SLE patients. We also noted unusual levels of the cytosolic protein tyrosine kinase Lyn and the protein tyrosine phosphatase SHP-1 in the lymphocytes of the patients. Since in normal B cells Lyn and SHP-1 act in concert within a common negative pathway in which CD45 counteracts SHP-I regulatory role, we propose that this feedback regulatory pathway is crippled to different degrees in human SLE B cells. Break of the balance between positive and negative signaling molecules likely modifies the BcR signaling thresholds. Such alterations, together with other factors, may contribute to the disruption of self-tolerance in this disease.
Assuntos
Moléculas de Adesão Celular , Lectinas , Lúpus Eritematoso Sistêmico/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos B/metabolismo , Linfócitos B/enzimologia , Linfócitos B/imunologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Antígenos Comuns de Leucócito/metabolismo , Lúpus Eritematoso Sistêmico/enzimologia , Ativação Linfocitária , Camundongos , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/metabolismo , Tolerância a Antígenos Próprios , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico , Transdução de Sinais , Quinases da Família src/metabolismoRESUMO
EverCare represents a creative approach to providing medical services to long-stay nursing home patients. It offers a capitated package of Medicare-covered services with more intensive primary care provided by nurse practitioners. The program's underlying premise is that better primary care will result in reduced hospital use. This work examines the implementation of the program in six locations. It identifies some of the issues that must be addressed if the program is to succeed both operationally and financially.
Assuntos
Assistência de Longa Duração , Programas de Assistência Gerenciada , Casas de Saúde , Idoso , Capitação , Serviços Contratados/economia , Medicina de Família e Comunidade , Administração Financeira , Geriatria , Sistemas Pré-Pagos de Saúde , Hospitalização , Humanos , Assistência de Longa Duração/classificação , Assistência de Longa Duração/economia , Assistência de Longa Duração/organização & administração , Programas de Assistência Gerenciada/economia , Programas de Assistência Gerenciada/organização & administração , Medicare/economia , Medicare/organização & administração , Profissionais de Enfermagem , Casas de Saúde/classificação , Casas de Saúde/economia , Casas de Saúde/organização & administração , Equipe de Assistência ao Paciente , Atenção Primária à Saúde/economia , Atenção Primária à Saúde/organização & administração , Desenvolvimento de Programas , Avaliação de Programas e Projetos de Saúde , Estados UnidosRESUMO
gamma-Aminobutyric acid (GABA) and glycine receptor channels have been investigated using the patch-clamp technique. Six out of 12 membrane patches exposed to GABA as well as to glycine responded to both substances, 3 to only GABA, two to only glycine and one was insensitive to both transmitters which were applied in micromolar concentrations. Channel currents reversed at the potential predicted by the constant field equation (using Cl- activities), indicating that the charge was carried by Cl-. Although multiple-conductance states were induced by GABA as well as by glycine, the phenomenon was much more pronounced after glycine applications. GABA channel permeabilities ranged from 3.7 X 10(-14) to 6.0 X 10(-14) cm3 s-1. With Cl- distributed symmetrically at 145 mM, these values would correspond to conductances of 19 and 31 pS, respectively. Glycine channel permeabilities ranged from 3.3 X 10(-14) to 14.7 X 10(-14) cm3 s-1 (corresponding conductances 17 and 76 pS).
Assuntos
Cerebelo/fisiologia , Glicina/fisiologia , Canais Iônicos/fisiologia , Ácido gama-Aminobutírico/fisiologia , Animais , Células Cultivadas , Cerebelo/citologia , Glicina/metabolismo , Canais Iônicos/classificação , Canais Iônicos/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Ácido gama-Aminobutírico/metabolismoRESUMO
The estimation of the survival rate of tetanus-intoxicated mice is suggested to be a useful animal model for assaying the anti-tetanus potency of drugs. This model was tested with the four central depressants phenobarbitone, chlorpromazine, diazepam and halothane. In agreement with their clinical value, diazepam appeared to be the most, phenobarbitone the least effective agent.