RESUMO
Optical magnetometers based on electromagnetically induced transparency (EIT) in atomic vapor can in principle achieve outstanding sensitivity and accuracy in a small volume. Up until now, however, the predicted fundamental quantum-noise-limited sensitivity has not been achieved in practice due to various technical limitations associated with the measurement apparatus. Here we analyze these restrictions and propose viable mitigation strategies for performance optimization. As an example, we show that an EIT magnetometer can theoretically achieve sensitivity better than 100 fT at 1 s measurement time under realistic conditions.
RESUMO
BACKGROUND: Irritable bowel syndrome is characterised by chronic abdominal pain and frequent comorbid anxiety. The substance P / neurokinin-1 receptor system is implicated in the regulation of both pain and anxiety, suggesting a potential therapeutic target in IBS. AIM: To determine whether inhibition of the neurokinin-1 receptor (NK1R) will change pain ratings and brain responses to experimental visceral pain and anxiety symptoms in women with IBS or not. METHODS: Rome II positive IBS women were recruited for a double-blind, placebo-controlled, cross-over study of NK1R antagonist AV608. Treatment periods were 3 weeks with a 2-week washout period. Functional MRI during a visceral distension paradigm was performed before first treatment and after treatment blocks. SPM8 was used to compare brain activity during painful and nonpainful visceral stimuli in regions associated with emotional arousal and interoception. Negative affect, anxiety symptoms and pain ratings were assessed. RESULTS: Eleven subjects completed the study and eight subjects provided fMRI data. AV608, compared with placebo, was associated with reduced anxiety, negative affect, and pain ratings. During AV608 treatment, the amygdala, hippocampus and anterior cingulate gyrus showed decreased activity during visceral distension. AV608 was also associated with decreases in activity in brain regions associated with interoception (posterior insula, anterior mid-cingulate gyrus). CONCLUSIONS: Chronic treatment with AV608 in IBS is associated with improved mood and pain ratings and activity of emotional arousal related brain regions. This suggests that further exploration of NK1R antagonists is warranted in visceral pain disorders, particularly in patients with comorbid anxiety symptoms.
Assuntos
Ansiedade/psicologia , Nível de Alerta/fisiologia , Emoções/fisiologia , Síndrome do Intestino Irritável/fisiopatologia , Antagonistas dos Receptores de Neurocinina-1 , Dor/tratamento farmacológico , Dor Visceral/fisiopatologia , Dor Abdominal/etiologia , Adolescente , Adulto , Idoso , Estudos Cross-Over , Dilatação Patológica , Método Duplo-Cego , Feminino , Humanos , Pessoa de Meia-Idade , Medição da Dor , Limiar da Dor/efeitos dos fármacos , Projetos Piloto , Piperidinas , Receptores da Neurocinina-1/uso terapêutico , Estresse Psicológico , Adulto JovemRESUMO
Apoptosis is an important physiological process that promotes tissue homeostasis by eliminating unnecessary or malfunctioning cells. Abnormality in this process contributes to tumorigenesis, as well as the resistance to cancer treatment by radiation and chemotherapy. Restoration of normal apoptosis would not only promote cancer cell death and halt tumor progression, but also increase the response to many current cancer therapies. Although apoptosis induction is an important principle of currently used radiation and chemotherapy treatment, uncovering the mechanisms that govern this process, and which are lost during transformation, represents an important direction for realizing improved therapies for the future. This article first briefly reviews aspects of current discovery strategies for new anticancer therapeutics based on intervening in cell death pathways, and then discusses in more detail several cancer-relevant death pathways, which are disabled during transformation and which can be targeted therapeutically. These include anoikis/cell adhesion; energy metabolism and the unfolded protein response. Finally, we introduce a new concept, which utilizes cancer-specific apoptosis induced by oncolytic viruses. The discussion of these topics involves novel targets, compounds and virotherapy.
Assuntos
Antineoplásicos/uso terapêutico , Apoptose , Transformação Celular Neoplásica , Neoplasias/metabolismo , Neoplasias/terapia , Animais , Anoikis/efeitos dos fármacos , Antineoplásicos/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Adesão Celular , Hipóxia Celular , Metabolismo Energético/efeitos dos fármacos , Glucose/metabolismo , Humanos , Mitocôndrias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Terapia Viral Oncolítica , Dobramento de ProteínaRESUMO
Tiagabine is a new antiepileptic drug which acts by a novel mechanism, inhibiting the reuptake of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) into neurons and glia. A double-blind, placebo-controlled, crossover trial was undertaken, based upon a response-dependent design. Ninety-four patients with complex partial seizures with or without secondary generalised tonic-clonic seizures were recruited into an open screening phase and tiagabine was added to their existing drug therapy in doses titrated to reduce seizure frequency by > or = 25% or to the limit of tolerance. Forty-six responders were subsequently randomised to a double-blind crossover trial in which tiagabine was compared with placebo. Forty-two patients completed the trial. A significant reduction in the frequency of complex partial and secondary generalised tonic clonic seizures was seen. Twenty-six percent had a reduction of > or = 50% in the frequency of their complex partial seizures, and of the 27 patients who also had secondary generalised tonic clonic seizures, 63% experienced a reduction of > or = 50%. No interactions with baseline antiepileptic drugs were detected and no serious adverse reactions occurred. The commonest adverse events were tiredness, dizziness and headache. We conclude that tiagabine has promising antiepileptic effects. Further trials are underway.
Assuntos
Anticonvulsivantes/uso terapêutico , Epilepsias Parciais/tratamento farmacológico , Ácidos Nipecóticos/uso terapêutico , Adolescente , Adulto , Idoso , Método Duplo-Cego , Humanos , Pessoa de Meia-Idade , Placebos , Tiagabina , Resultado do TratamentoRESUMO
The mechanism of low-affinity NGF receptor (LNGFR) truncation was investigated in cultured Schwann cells. Affinity labeling of Schwann cells with 125I-NGF or metabolic labeling with 35S-cysteine showed that truncated NGF receptor (NGF-Rt) was derived from the cell surface form of the receptor. Addition of full-length, exogenous NGF receptor (M(r) = 80 kDa) to Schwann cell membranes resulted in cleavage of the exogenous substrate to NGF-Rt. Investigations into the mechanism of truncation revealed that metalloprotease inhibitors such as phenanthroline, bathophenanthroline, and 8-hydroxyquinoline (8-OHQ) blocked LNGFR truncation in a concentration-dependent fashion. Inhibitors of other protease classes had no effect on truncation. In addition, truncation did not occur at 4 degrees C. It was found that truncation could also occur in Schwann cell membrane preparations, indicating that the putative protease was membrane bound and closely associated with the LNGFR. Metal reconstitution experiments revealed a strong preference toward zinc for the truncating activity, with iron and manganese having slight reconstitution activity in phenanthroline-quenched membranes. To determine if apparent truncation could be inhibited in vivo, the metalloprotease inhibitor 8-OHQ was administered to neonatal rats. 8-OHQ resulted in decreased urine and blood NGF-Rt levels and increased the sciatic nerve LNGFR content; this effect was dose dependent. In adult rats with sciatic nerve crush lesions, 8-OHQ (30-300 mg/kg, t.i.d.) significantly enhanced the rate of sensory neuron regeneration as assessed by the nerve pinch assay. This was accompanied by increased levels of LNGFR in distal nerve segments. These results suggest that Schwann cells possess a metalloprotease-like activity that serves to cleave LNGFR from the surface of these cells. We propose that the putative metalloprotease represents a novel mechanism by which the Schwann cell regulates this particular cell surface protein. Furthermore, increasing the amount of Schwann cell surface LNGFR appears to be of functional significance in that sensory nerve regeneration can be enhanced by inhibition of truncation.
Assuntos
Metaloendopeptidases/fisiologia , Receptores de Fator de Crescimento Neural/fisiologia , Marcadores de Afinidade , Animais , Metaloendopeptidases/antagonistas & inibidores , Compressão Nervosa , Regeneração Nervosa/efeitos dos fármacos , Neurônios Aferentes/efeitos dos fármacos , Neurônios Aferentes/fisiologia , Oxiquinolina/farmacologia , Fenantrolinas/farmacologia , Receptores de Fator de Crescimento Neural/química , Células de Schwann/metabolismo , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/metabolismoRESUMO
The balance between catecholamine (CA) release and reuptake is closely regulated and determines the effective level of transmitter at the synaptic cleft. Drugs that block CA uptake have potential utility as antidepressant medications. One such drug is racemic (+/-)-(1' R*,3R*)-3-phenyl-1-[1',2',3',4'-tetrahydro-5',6'- methylenedioxy-1'-naphthalenyl-methyl]-pyrrolidine methanesulfonate (A-7500), a novel polycyclic compound developed at Abbott Laboratories. This compound is known to bind to CA transporters in the central nervous system, however, its effects on an intact neurosecretory system have not been studied. In this regard, norepinephrine (NE) release from bovine adrenal chromaffin cells (BACC) is a classic model system for CA release and is an excellent system in which to examine the effects of drugs which modulate neurotransmitter release. We compared the effects of A-75200 and its two constituent enantiomers, A-74111 and A-74112, to the effects of three well-characterized uptake inhibitors, desipramine (DMI), nomifensine and cocaine. We found that the Abbott compounds inhibit [3H]norepinephrine ([3H]NE) uptake with an EC50 comparable to cocaine. In addition, unlike nomifensine and cocaine, these compounds inhibited nicotine- and K(+)-stimulated NE release, whereas histamine-stimulated release was preserved. Thus, the Abbott compounds block the effects on secretion of two agonists (nicotine and K+) which depend on a depolarization-dependent influx of extracellular calcium. We conclude that in addition to blocking NE uptake by inhibiting the NE transporter, the Abbott compounds may modulate peripheral NE release by inhibiting calcium flux through voltage-gated channels. This study demonstrates the utility of bovine adrenal chromaffin cells for preclinical trials of drugs that affect catecholaminergic neurotransmission.
Assuntos
Medula Suprarrenal/efeitos dos fármacos , Grânulos Cromafim/efeitos dos fármacos , Inibidores da Captação de Neurotransmissores/farmacologia , Norepinefrina/metabolismo , Pirrolidinas/farmacologia , Tetra-Hidronaftalenos/farmacologia , Medula Suprarrenal/metabolismo , Animais , Cálcio/metabolismo , Bovinos , Células Cultivadas , Grânulos Cromafim/metabolismo , Relação Dose-Resposta a Droga , Nicotina/farmacologia , Potássio/farmacologiaAssuntos
Anticonvulsivantes/síntese química , Animais , Anticonvulsivantes/farmacocinética , Anticonvulsivantes/farmacologia , Anticonvulsivantes/uso terapêutico , Encéfalo/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Epilepsia/sangue , Epilepsia/tratamento farmacológico , Humanos , Relação Estrutura-Atividade , Transmissão Sináptica/efeitos dos fármacosRESUMO
Hormonal feedback regulation of hypothalamic peptides putatively involved in growth hormone (GH) regulation has been studied by measurement of steady-state mRNA levels in male hypophysectomized rats with or without thyroid hormone, corticosterone, testosterone or GH replacement. Hypothalamic GH-releasing factor (GRF) mRNA levels increased progressively following hypophysectomy to 420% of sham levels after 15 days while hypothalamic insulin-like growth factor I (IGF-I) and insulin-like growth factor II (IGF-II) mRNA levels decreased to less than 40% of sham levels. Whole hypothalamic somatostatin mRNA levels were not significantly different from sham. One week of continuous GH infusion restored hypothalamic IGF-I mRNA to levels (95%) indistinguishable from those in sham-operated controls but had no effect on either IGF-II or GRF mRNA. Thyroid hormone, corticosterone and testosterone treatment without GH had no effect on the hypophysectomy-induced reduction of either IGF-I or IGF-II mRNA levels but reversed the elevation of GRF mRNA. We conclude that hypothalamic IGF-I may be involved in GH feedback regulation and thus may function as a hypothalamic modulator of GH. In contrast, IGF-II may be regulated by one of the pituitary trophic hormones but not by GH or the target hormones tested. Finally, hypothalamic GRF mRNA regulation appears to be complex and may include target hormone feedback.
Assuntos
Hormônio Liberador de Hormônio do Crescimento/genética , Hipofisectomia , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like I/genética , RNA Mensageiro/metabolismo , Somatostatina/genética , Animais , Corticosterona/farmacologia , Retroalimentação , Hormônio do Crescimento/farmacologia , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Masculino , Ratos , Ratos Endogâmicos , Testosterona/farmacologia , Tiroxina/farmacologiaAssuntos
Anticonvulsivantes/uso terapêutico , Drogas em Investigação/uso terapêutico , Epilepsia/tratamento farmacológico , Ácidos Nipecóticos/uso terapêutico , Animais , Anticonvulsivantes/efeitos adversos , Anticonvulsivantes/farmacocinética , Encéfalo/efeitos dos fármacos , Drogas em Investigação/efeitos adversos , Drogas em Investigação/farmacocinética , Eletroencefalografia/efeitos dos fármacos , Epilepsia/sangue , Potenciais Evocados/efeitos dos fármacos , Humanos , Camundongos , Ácidos Nipecóticos/efeitos adversos , Ácidos Nipecóticos/farmacocinética , Ratos , TiagabinaRESUMO
A68930 (5,6-dihydroxy-3-phenyl-1-aminomethyl-isochroman) is a potent (EC50 = 2.5 nmol/L) partial agonist at the D-1 dopamine receptor. In contrast, A68930 is a much weaker agonist (EC50 = 3,920 nmol/L) at the D-2 dopamine receptor. The orientation of the 3-phenyl substituent in the molecule is critical for the affinity and selectivity of the molecule towards the D-1 receptor in vitro and in vivo. The role of the D-1 receptor in the functioning of the basal ganglia is discussed.
Assuntos
Benzopiranos/farmacologia , Cromanos/farmacologia , Receptores Dopaminérgicos/fisiologia , Animais , Corpo Estriado/fisiologia , Denervação , Dopamina/fisiologia , Hidroxidopaminas , Oxidopamina , Doença de Parkinson/tratamento farmacológico , Ratos , Receptores de Dopamina D1 , EstereoisomerismoRESUMO
We have used the polymerase chain reaction technique (PCR) to clone the cDNA of the D2 dopamine receptor from rat striatal mRNA. Two major PCR products were produced; one product was identical to a previously published rat cDNA, while the other, more abundant product differed only by an 87-nucleotide insert located in the region of the putative third cytoplasmic loop of the D2 receptor. A PCR approach for determining message abundance was used to determine the relative message abundance of the two forms of the D2 receptor in a variety of tissues. Possible implications of the two forms of the D2 receptor for dopamine-mediated signal transduction are discussed.
Assuntos
Encéfalo/metabolismo , Corpo Estriado/metabolismo , Receptores Dopaminérgicos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Fígado/metabolismo , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Especificidade de Órgãos , Hipófise/metabolismo , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/genética , Ratos , Receptores Dopaminérgicos/fisiologia , Receptores de Dopamina D2 , Mapeamento por Restrição , Transdução de SinaisRESUMO
A ligand affinity matrix has been developed and utilized to purify the dopamine D2 receptor approx. 2100 fold from bovine striatal membranes. 3-[2-Aminoethyl]-8-[3-(4-fluorobenzoyl)propyl]-4-oxo-1-phenyl-1,3,8- triazaspiro[4.5]decan-4-one (AES) was synthesized and used to prepare the affinity matrix by coupling to epoxy-activated Sepharose 6B (AES-Sepharose). AES (Ki approximately 1.7 nM) is similar in potency to the parent compound, spiperone (Ki approximately 0.8 nM), in competing for [3H]spiperone-binding activity. AES has no significant potency in competing for the dopamine D1 receptor as assessed by competition for [3H]SCH23390 binding (Ki greater than 1 microM). Covalent photoaffinity labeling of the dopamine D2 receptor in bovine striatal membranes with N-(p-azido-m-[125I]iodophenethyl)spiperone [( 125I]N3-NAPS) was prevented by AES at nanomolar concentrations. The dopamine D2 receptor was solubilized from bovine striatal membranes using 0.25% cholate in the presence of high ionic strength, followed by precipitation and subsequent treatment with 0.5% digitonin. Nearly 100% of the [3H]spiperone-binding activity in the cholate-digitonin solubilized preparation was absorbed at a receptor-to-resin ratio of 2:1 (v/v). Dopamine D2 receptor was eluted from the affinity resin using a competing dopaminergic antagonist molecule, haloperidol. Recovery of dopamine D2 receptor activity from the affinity matrix was approx. 9% of the activity adsorbed to the resin. The [3H]spiperone-binding activity in AES-Sepharose affinity purified preparations is saturable and of high affinity (0.2 nM). Affinity-purified preparations maintain the ligand-binding characteristics of a dopamine D2 receptor as assessed by agonist and antagonist competition for [3H]spiperone binding.
Assuntos
Cromatografia de Afinidade , Corpo Estriado/análise , Receptores Dopaminérgicos/isolamento & purificação , Marcadores de Afinidade , Animais , Benzazepinas/metabolismo , Ligação Competitiva , Bovinos , Membrana Celular/análise , Ácido Cólico , Ácidos Cólicos , Digitonina , Estrutura Molecular , Fotoquímica , Receptores Dopaminérgicos/metabolismo , Receptores de Dopamina D2 , Sefarose , Espiperona/análogos & derivados , Espiperona/síntese química , Espiperona/metabolismoRESUMO
The glycoprotein nature of the binding subunit of the dopamine D2 receptor in rat striatum has been examined by photoaffinity labeling receptor preparations with N-(p-azido-m-[125I]iodophenethyl)spiperone followed by treatment of crude membrane receptor or receptor fractions isolated from sodium dodecyl sulfate (SDS) polyacrylamide gels with endo- and exoglycosidases. The major photoaffinity labeled protein migrates as a heterogeneous species on 10% SDS polyacrylamide gels and ranges from 130,000 to 75,000 relative molecular mass (Mr). This heterogeneity can be explained by glycosylation of the receptor by complex-type N-linked oligosaccharides. Three fractions of labeled receptor were isolated from SDS polyacrylamide gels over a range of 130,000 to 75,000 Mr; after digestion with peptide-N4-[N-acetyl-beta-glucosaminyl] asparagine amidase, all fractions yielded a single peptide approximately 40,000 Mr. Treatment of photoaffinity labeled membranes with alpha-mannosidase was without effect. The dopamine D2 receptor appears to contain substantial amounts of sialic acid as treatment of photoaffinity labeled membranes with neuraminidase increased the receptor mobility on SDS polyacrylamide gels to a species of 50,000-54,000 Mr. Treatment of the receptor with neuraminidase followed by endo-alpha-N-acetylgalactosaminidase did not change the electrophoretic migration pattern from that seen after neuraminidase treatment alone, suggesting that the binding peptide contains no serine- or threonine-linked oligosaccharides. A smaller binding peptide of approximately 31,000 Mr is also apparent in crude photoaffinity labeled membranes. This material also contains N-linked oligosaccharide. Complete removal of N-linked oligosaccharide from the dopamine D2 receptor did not change the rank order potency of agonist and antagonist compounds to compete for [3H]spiperone binding to crude membrane fractions. The dopamine D2 receptor represents a highly glycosylated neural receptor.
Assuntos
Corpo Estriado/metabolismo , Glicoproteínas de Membrana/metabolismo , Oligossacarídeos , Receptores Dopaminérgicos/metabolismo , Marcadores de Afinidade/metabolismo , Animais , Autorradiografia , Azidas/metabolismo , Membrana Celular/metabolismo , Glicosilação , Radioisótopos do Iodo , Cinética , Masculino , Glicoproteínas de Membrana/isolamento & purificação , Peso Molecular , Oligossacarídeos/isolamento & purificação , Ratos , Receptores Dopaminérgicos/isolamento & purificação , Receptores de Dopamina D2 , Espiperona/análogos & derivados , Espiperona/metabolismoRESUMO
The effects of thyrotropin releasing hormone (TRH) on spinal cord growth were evaluated using the in oculo transplant model. The growth of fetal spinal cord allografts, placed into the anterior eye chamber of Sprague-Dawley rats, was markedly augmented by acute exposure of the graft and host animal to TRH at the time of transplantation. No significant growth augmentation was seen after equimolar administration of a mixture of the amino acids that comprise the TRH molecule. It is concluded that acutely administered TRH, at the time of grafting, elicits a significant stimulation of the growth of spinal cord tissue. Our data strengthen the rationale for continued clinical trials of this peptide in spinal cord injury.
Assuntos
Fenômenos Fisiológicos Oculares , Medula Espinal/transplante , Hormônio Liberador de Tireotropina/farmacologia , Aminoácidos/farmacologia , Animais , Feminino , Feto/fisiologia , Ratos , Ratos Endogâmicos , Medula Espinal/embriologia , Medula Espinal/crescimento & desenvolvimento , Fatores de TempoRESUMO
Many parvocellular neurons in the paraventricular nucleus of the hypothalamus express high levels of corticotropin releasing factor (CRF) or vasopressin following adrenalectomy. To determine whether glucocorticoids feed back directly on these neurons, a mouse monoclonal antibody directed against the rat liver glucocorticoid receptor was used in combination with polyclonal antisera directed against either vasopressin or CRF to permit simultaneous visualization of either peptide with glucocorticoid receptor-like immunoreactivity (IR). Rats were adrenalectomized (ADX) for 2 weeks to optimize numbers of vasopressin - and CRF-IR neurons. Six hours prior to sacrifice, a separate group of adrenalectomized rats was treated with corticosterone (40 mg/kg). This short-term replacement resulted in nuclear localization of glucocorticoid receptor-like-IR but did not attenuate the increased numbers of CRF- and vasopressin-IR neurons observed after adrenalectomy. It was therefore possible to visualize vasopressin- or CFR-IR and nuclear glucocorticoid receptor-like-IR simultaneously. Cell counts of double-labeled neurons in the paraventricular nucleus of the hypothalamus (PVH) demonstrated that glucocorticoid receptor-like-IR is colocalized in virtually all the CRF and vasopressin immunoreactive parvocellular neurons studied, which respond to adrenalectomy by increased peptide expression. These data suggest that a major feedback effect of glucocorticoids on the hypothalamic-pituitary-adrenal axis is exerted directly within nuclei of CRF and vasopressin neurons.
Assuntos
Hormônio Liberador da Corticotropina/análise , Núcleo Hipotalâmico Paraventricular/análise , Receptores de Glucocorticoides/análise , Vasopressinas/análise , Adrenalectomia , Animais , Anticorpos Monoclonais , Contagem de Células , Imuno-Histoquímica , Masculino , Ratos , Ratos EndogâmicosRESUMO
Decreasing transsynaptic activity through surgical adrenal denervation or by medullary explantation, increases Leu-enkephalin immunoreactivity (Leu-Enk) and preproenkephalin mRNA (prepro-EK). Membrane depolarization prevents this rise. To determine whether depolarizing effects are mediated by intracellular movement of calcium ions, explanted medullae were depolarized in the presence of EGTA or the calcium ion 'channel' blockers D600 or verapamil. Inhibition of Ca2+ influx prevented the effects of KCl-induced depolarization on the rise in Leu-Enk and on prepro-EK. Increasing intracellular Ca2+ with the ionophore A23187, in the absence of depolarizing agents, reproduced the effects of depolarization. By contrast, medullae grown in the presence of A23187, but in Ca2+-free medium, showed similar increases in prepro-EK mRNA and Leu-Enk, indicating an absolute requirement for Ca2+. In addition, KCl-inhibitory effects could be partially blocked by the calmodulin and protein kinase-C antagonist, trifluoperazine. However, KCl effects were not antagonized by the preferential calmodulin inhibitors W7, W13 or calmidizolium even at doses 10-fold higher than required to prevent calmodulin-dependent effects. Thus, these data suggest that inhibitory effects of transsynaptic activity and membrane depolarization on adrenal enkephalin occurs through Ca2+ and perhaps through a protein kinase-C dependent pathway, mechanisms known to augment catecholamine biosynthesis. It appears then that the same or similar molecular mechanisms can result in differential regulation of these co-localized transmitter systems.
Assuntos
Medula Suprarrenal/fisiologia , Encefalina Leucina/genética , Encefalinas/genética , Precursores de Proteínas/genética , Animais , Calcimicina/farmacologia , Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Masculino , Técnicas de Cultura de Órgãos , Proteína Quinase C/antagonistas & inibidores , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos , Transcrição Gênica/efeitos dos fármacos , Trifluoperazina/farmacologiaRESUMO
The biosynthesis and posttranslational processing of proenkephalin and the level of preproenkephalin mRNA were investigated in the mossy fiber system of the granule cells of the hippocampus in the presence or absence of a unilateral lesion of the hilus, a procedure that produces an episode of recurrent bilateral hippocampal seizures lasting several hours. Both immunocytochemistry and radioimmunoassay (RIA) have demonstrated that the hilus lesion leads to large bilateral increases in enkephalin immunoreactivity in the mossy fiber system. In the present study, RIA data indicate that following an initial decline in immunoreactivity, enkephalin content within the mossy fibers first begins to increase between 18 and 24 hr after lesioning. Using the technique of in vivo radiolabeling and high-performance liquid chromatographic purification of identified radiolabeled peptides, we observed a 14-fold increase in incorporation of radiolabeled methionine into Met5-enkephalin at 24-30 hr postlesion, as compared with control animals, when Met5-enkephalin was purified from the mossy fiber terminal fields. To examine the posttranslational proteolytic processing of proenkephalin, the biosynthesis of 5 additional Met5-enkephalin-containing peptides was also examined. We determined that the posttranslational processing of proenkephalin did not yield exclusively penta-, hepta-, and octapeptides but larger opioid peptides as well in both control and lesioned animals, and that the ratio of the enkephalin peptides was not altered following the lesion. Measurement of preproenkephalin messenger RNA levels in the granule cells by Northern analysis revealed a marked increase following the lesion. Compared with the control animals, preproenkephalin mRNA was 8.5-fold higher in the contralateral dentate gyrus at 12 hr postlesion and 14- to 15-fold higher by 24 hr.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Encefalinas/biossíntese , Regulação da Expressão Gênica , Hipocampo/fisiologia , Fibras Nervosas/fisiologia , Animais , Encefalinas/genética , Masculino , Precursores de Proteínas/genética , RNA Mensageiro/análise , Ratos , Ratos EndogâmicosAssuntos
Encéfalo/fisiologia , Neuropeptídeos/biossíntese , Animais , Comunicação Celular , Encefalinas/biossíntese , Regulação da Expressão Gênica , Neurônios/fisiologia , Neuropeptídeos/genética , Hipófise/fisiologia , Pró-Opiomelanocortina/biossíntese , Ratos , Receptores de Neurotransmissores/fisiologia , Transmissão SinápticaRESUMO
The biosynthesis and posttranslational proteolytic processing of proenkephalin was studied in three projection systems in the rat central nervous system--the caudate-putamen to the globus pallidus, the paraventricular nucleus of the hypothalamus to the median eminence, and the mossy fiber system of the granule cells of the hippocampus. By using the techniques of in vivo radiolabeling and sequential high-performance liquid chromatographic purification coupled with chemical modification, the biosynthesis of six radiolabeled [Met]enkephalin-containing peptides--[Met5]enkephalin, [Met5,Arg6,Gly7,Leu8]enkephalin, [Met5,Arg6,Phe7]enkephalin, metorphamide, peptide E, and BAM 18P--was followed. In each projection system, radiolabeled enkephalins were purified to constant radiochemical specific activity. However, the posttranslational processing of proenkephalin was found to differ between these three systems, as judged by the relative ratio of these peptides. These findings imply that specific, different physiologies and behaviors may be elicited by the enkephalins based upon the specific [Met]enkephalin-containing peptides that are cleaved from proenkephalin and released in synaptic terminal fields.
Assuntos
Encéfalo/metabolismo , Encefalinas/metabolismo , Precursores de Proteínas/metabolismo , Animais , Corpo Estriado/metabolismo , Encefalinas/biossíntese , Hipocampo/metabolismo , Hipotálamo/metabolismo , Masculino , Ratos , Ratos EndogâmicosRESUMO
The present study examines the relative levels of vasopressin (AVP) mRNA within the paraventricular (PVN), supraoptic (SON), and suprachiasmatic (SCN) nuclei of the rat hypothalamus, and details the rates at which these levels change over the course of a 6 d salt-loading regimen. The quantitation of vasopressin mRNA was achieved by using three different procedures: (1) cell-free translation in rabbit reticulocyte lysate or (2) Northern analysis of poly(A)RNAs isolated from micro-punch dissected SON, PVN, and SCN, and (3) in situ hybridization histochemistry. The former involved the quantitative immunoprecipitation of the neurophysin precursors containing arginine8-vasopressin (AVP) or oxytocin, and the latter two techniques employed a radiolabeled synthetic oligodeoxynucleotide complementary to the 3' region of the AVP mRNA. Both the cell-free studies and the Northern gel analyses detected a sevenfold increase of AVP mRNA in the SON, a fivefold increase in the PVN, and no significant change in the SCN following 6 d of salt-loading. After the initiation of salt-drinking, these increases were shown to occur between 24 and 48 hr in the SON and between 48 and 72 hr in the PVN. The in situ hybridization studies revealed the anatomically correct hybridization of either 32P- or 3H-labeled AVP oligonucleotide to magnocellular perikarya within both the SON and PVN. Autoradiographic grains could be shown to be confined to the cytoplasm of these cells, and could be co-localized with immunoreactivity directed against the carboxy terminus of the AVP percursor. Comparison of x-ray level autoradiograms of control and 6 day salt-loaded SON revealed up to a sevenfold increase in specific signal in the salt-loaded sections. It is concluded that the response of AVP mRNA to osmotic stimuli in the three hypothalamic nuclei is heterogeneous, and that this heterogeneity can be explained by separating AVP neurons into two systems: one responsible for eliciting the antidiuretic actions of AVP via plasma AVP levels, and the other involved in CNS activities not directly involved with antidiuresis.