RESUMO
Vasopressin is one of several small neuropeptides that are reported to be autocrine growth factors for small cell carcinoma of the lung (SCCL). It has been assumed that this peptide exercises its mitogenic influences through the vasopressin V1a receptor, and we have previously demonstrated that this receptor is expressed by classical and variant SCCL. Activation of the vasopressin V1a receptor produces changes in phospholipases C, D, and A2, in protein kinase C, and in Ca2+ mobilization. This study demonstrates that SCCL cells express not only vasopressin V1a receptors but also mRNAs and proteins representing normal V1b receptors and V2 receptors. They were also shown to express mRNA for a human form of the putative receptor rabbit vasopressin-activated calcium-mobilizing receptor (VACM-1). Additionally, SCCL tumor cells were found to express mRNA and protein representing a possible nonfunctional, shortened, "diabetic" form of the vasopressin V2 receptor that is the product of incomplete posttranscriptional splicing. At least four of these five vasopressin receptors were produced by cell lines exemplifying classical and variant forms of SCCL. No differences in the sequences for the V1 receptors between classical and variant SCCL were found. However, although the nature and expression of both vasopressin V1 receptors and human VACM are apparently unaffected by dedifferentiation in SCCL, only the abnormal (and probably nonfunctional) form of the V2 receptor could be demonstrated in variant cell line NCI H82. Functional engagement of vasopressin V2 receptors is reported to produce rises in cAMP and activation of protein kinase A, whereas stimulation of V1b receptors is believed to produce similar changes to those produced by V1a receptors, i.e., activation of phospholipases and of protein kinase C. Stimulation of VACM receptors raises intracellular free Ca2+ through currently unknown but phosphoinositide-independent mechanisms. The presence of all known vasopressin receptors that are, together, potentially capable of inducing several different transduction cascades in small cell tumor cells suggests that this peptide serves a multifaceted role in tumor physiology.
Assuntos
Carcinoma de Células Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Receptores de Vasopressinas/metabolismo , Vasopressinas/fisiologia , Animais , Sequência de Bases , Northern Blotting , Western Blotting , Primers do DNA/química , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Coelhos , Receptores de Vasopressinas/genética , Células Tumorais CultivadasRESUMO
Increasing evidence that ion channels play a key role in the modulation of cellular mitogenesis led us to investigate the membranes of T47D human breast cancer cells to identify the ion currents present. We report here the results of voltage-clamp studies in the whole-cell configuration on isolated, non-synchronized single cells obtained from a ductal breast carcinoma. In these studies we identified an outward rectifying potassium current and a chloride current. The potassium current activated at potentials more positive than -40 mV, reached an average value of 1.4 nA, and did not inactivate with time. This current was sensitive to block by extracellular tetraethylammonium chloride (TEA, IC50 = 1 micro M), was insensitive to charybdotoxin (CTX, IC50 = 7.8 micro M), and was not diminished by repetitive pulses separated by 1 s. Rapid voltage-dependent inactivation of the current was demonstrated by tail current analysis. The current appeared calcium-insensitive. Application of hyperpolarizing pulses did not elicit an inward potassium rectifier current. Treatment with tetrodotoxin did not reveal the presence of an inward sodium current. The potassium current was increased by the presence of aspartate in place of chloride and in the presence of the chloride channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). We conclude that currents present in T47D breast cancer cells include a chloride current and a voltage-gated potassium outward rectifier. We suggest that the potassium current, either alone or in conjunction with potassium currents reported in different human breast cancer cell lines by others, may play a role in the modulation of the cell cycle.
Assuntos
Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Canais de Cloreto/metabolismo , Canais de Potássio/metabolismo , Transdução de Sinais , Canais de Cálcio/metabolismo , Charibdotoxina/farmacologia , Cloretos/metabolismo , Feminino , Humanos , Técnicas de Patch-Clamp , Potássio/metabolismo , Sódio/metabolismo , Canais de Sódio/metabolismo , Tetraetilamônio , Compostos de Tetraetilamônio/farmacologia , Tetrodotoxina/farmacologia , Células Tumorais CultivadasRESUMO
Four classical and three variant small-cell carcinoma of the lung (SCCL) cell lines were examined for vasopressin and vasopressin V1a-receptor immunoreactivity. One of these classical cell lines, NCI-H345, and one variant cell line, NCI-H82, were further investigated for binding of V1 and V2 vasopressin-receptor antagonists, vasopressin-induced calcium mobilization, and vasopressin-induced thymidine uptake. All classical and variant SCCL cell lines examined contained vasopressin and vasopressin-receptors as determined by immunocytochemistry. Both NCI-H82 and NCI-H345 demonstrated similar binding patterns with the V1 and V2 vasopressin-receptor antagonists, indicating the presence of both receptor subtypes. For the classical cell line (NCI-H345), vasopressin (1 microM) induced an increase in cytosolic free calcium, while the peptide was ineffective at increasing cytosolic calcium in the variant cell line (NCI-H82). However, vasopressin (0.1 or 1 microM) was unable to stimulate thymidine uptake in the classical (NCI-H345) or variant (NCI-H82) cell lines for the conditions used. These results indicate that both classical and variant SCCL produce vasopressin, and vasopressin V1a and V2 receptors. In the variant cell line, there appears to be a disruption in the activation cascade for V1a receptors as indicated by the lack of vasopressin-induced calcium mobilization.
Assuntos
Cálcio/metabolismo , Carcinoma de Células Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Receptores de Vasopressinas/biossíntese , Timidina/farmacocinética , Vasopressinas/biossíntese , Calcimicina/análogos & derivados , Calcimicina/farmacologia , Citosol/metabolismo , Humanos , Células Tumorais CultivadasRESUMO
We have previously provided evidence that an autocrine loop involving vasopressin is present in perhaps all breast cancers. This study now shows MCF-7 breast cancer cells express mRNAs for all currently recognized vasopressin receptor subtypes (V1a, V1b, and V2). Cloning and DNA sequencing over the entire open reading frame of each mRNA revealed that normal sequences representing each receptor were present. However, in addition, an abnormal mRNA for the V2 receptor, expected to give rise to a truncated 'diabetic' protein, was also expressed. Western analysis revealed that all three normal mRNAs gave rise to proteins of sizes compatible with them being functional receptors. The abnormal V2 receptor mRNA also gave rise to proteins.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Receptores de Vasopressinas/genética , Sequência de Bases , Clonagem Molecular , Primers do DNA/genética , DNA de Neoplasias/genética , Feminino , Humanos , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Receptores de Vasopressinas/biossíntese , Receptores de Vasopressinas/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
Studies using fetal sheep, goats, and guinea pigs indicate that vasopressin may play a role in preparing the fetal lung for the transition from a uterine to an air-breathing environment by slowing lung liquid secretion. The mechanism of vasopressin action is believed to occur through V2 receptors with subsequent activation of amiloride-sensitive sodium channels. However, the presence of the V2 receptor in human lung has not yet been documented. In the present study, expression of the vasopressin V2 receptor in fetal and adult human lung was examined using reverse transcription-polymerase chain reaction (RT-PCR), Northern blot analysis, and DNA sequencing. Using RT-PCR and primer pairs specific for the human V2 receptor, PCR products of the predicted sizes of 512 and 862 bp were obtained from adult human lung. DNA sequencing of the cloned PCR products revealed exact identity with the published sequence for the V2 receptor. Northern blot analysis revealed the expression of a approximately 1.9 kb mRNA in adult human lung as well as in kidney, but not in fetal human lung at 22-24 weeks of gestation. However, using the more sensitive RT-PCR assay the 862-bp product was successfully amplified from human fetal lung, although the data indicate the mRNA for this receptor is expressed in lower levels than in adult human lung or kidney. Using RT-PCR and primers specific for the rat V2 receptor, a PCR product of the predicted size of 461 bp was amplified from adult rat lung and kidney, despite an earlier report that this receptor mRNA is absent from the lung of this species. The role for the V2 receptor in adult human lung is unknown at this time, but, as in the human kidney and lungs of fetal sheep, goats, and guinea pigs, this receptor may play a role in fluid balance.
Assuntos
Pulmão/metabolismo , RNA Mensageiro/biossíntese , Receptores de Vasopressinas/biossíntese , Adulto , Animais , Sequência de Bases , Feto/metabolismo , Expressão Gênica , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Ratos , Análise de Sequência de DNARESUMO
Vasopressin and other neuropeptides are believed to serve as autocrine growth factors for small-cell carcinoma of the lung (SCCL), and these mitogenic influences are reported to involve increases in intracellular Ca2+. Of the classical and variant forms of SCCL, the latter is not only more drug-resistant but also refractory to vasopressin, and other peptides, with respect to changes in intracellular Ca2+. It is currently unclear if this refractiveness of variant SCCL is due to the absence of involved peptide receptors, to the production of abnormal receptors, or to abnormalities in components of induced transduction cascades. In this study, the presence of structurally-normal and functional vasopressin V1a receptors, was examined in a classical SCCL cell line (NCI H345) that is Ca(2+)-responsive to vasopressin, and a variant SCCL cell line (NCI H82) that is unresponsive in this regard to the peptide. Both cell lines were shown to express an mRNA of 1.9 Kb for the vasopressin V1a receptor. RT-PCR, cloning, and DNA sequencing revealed the structure of the mRNA was identical for both cell lines, and, in turn, identical to the mRNA expressed for this receptor by human liver cells. In both cell lines and liver, this mRNA was shown by Western analysis and RIA to generate major protein products of approximately 70,000 and 43,000 daltons. Vasopressin action on NCI H82 cells resulted in a substantial rise in the levels of total inositol phosphates. However, it was reaffirmed that these changes in inositol phosphates were not accompanied by a rise in Ca2+ levels. All of these data indicate that variant SCCL, as well as classical SCCL, expresses structurally-normal and functional vasopressin V1a receptors, but their activation in variant SCCL raises IP3 levels without a corresponding rise in intracellular Ca2+. This difference between the two SCCL sub-types therefore involves either steps in the inositol triphosphate cascade beyond the activation of phospholipase C, or alternatively, components of other transduction events that might be involved with changes in intracellular Ca2+.
Assuntos
Carcinoma de Células Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Receptores de Vasopressinas/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Cálcio/metabolismo , Carcinoma de Células Pequenas/genética , Clonagem Molecular , Primers do DNA/genética , Variação Genética , Humanos , Fosfatos de Inositol/metabolismo , Neoplasias Pulmonares/genética , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Neoplásico/genética , Receptores de Vasopressinas/genética , Células Tumorais CultivadasRESUMO
Expression of the vasopressin gene appears to be a property common to all small-cell lung tumours. For some cultures of small-cell lung carcinoma (SCCL), Northern and Western Blot analyses have revealed that expression of this gene and its protein products are regulated by cAMP and glucocorticoids. In this study, these evaluations have been extended by examining the production of vasopressin-associated human neurophysin (VP-HNP) by computer-enhanced quantitative immunocytochemistry in a classical cell-line (H69) of SCCL, and defining the amount of protein in cells by area of positive staining above an arbitrarily set threshold. Intracellular cAMP was raised by incubating cells with either 8,Br-cAMP (0.5 mM) and IBMX (0.5 mM), or with forskolin (25 microM) and IBMX (0.5 mM). Both of these treatments caused a significant increase in the amount of positive VP-HNP immunoreactivity in the cells, an increase that was further enhanced by simultaneous administration of dexamethasone (0.1 microM). Addition of dexamethasone alone, however, caused a significant decrease in VP-HNP levels. Results confirm earlier findings from Western Blot analysis revealing the influence these agents have on production of vasopressin gene-related proteins by H69 cells, and indicate that computer-enhanced quantitative immunocytochemistry can be effectively used to provide a suitable index of this production.
Assuntos
Carcinoma de Células Pequenas/patologia , AMP Cíclico/fisiologia , Densitometria/métodos , Regulação Neoplásica da Expressão Gênica , Processamento de Imagem Assistida por Computador , Técnicas Imunoenzimáticas , Neoplasias Pulmonares/patologia , Proteínas de Neoplasias/biossíntese , Neurofisinas/biossíntese , Vasopressinas/farmacologia , 1-Metil-3-Isobutilxantina/farmacologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Colforsina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dexametasona/farmacologia , Ativação Enzimática/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas de Neoplasias/genética , Neurofisinas/genética , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
The effects of preincubation of human T-lymphoma cells with increasing concentrations of calcium L-threonate on the uptake of L-[1-14C]ascorbic acid were examined. Calcium L-threonate (0-1,000 mg%) stimulated ascorbic acid (1.25 mg%) uptake in a dose-dependent manner. These results indicate that calcium threonate and possibly other ascorbic acid metabolites have biological activity and potential pharmacological applications.
Assuntos
Ácido Ascórbico/farmacocinética , Butiratos/farmacologia , Linfoma de Células T/metabolismo , Análise de Variância , Relação Dose-Resposta a Droga , Humanos , Células Tumorais Cultivadas/metabolismoRESUMO
Hyperglycemia and/or hypoinsulinemia have been found to inhibit L-ascorbic acid cellular transport. The resultant decrease in intracellular ascorbic acid may de-inhibit aryl sulfatase B and increase degradation of sulfated glycosaminoglycans (sGAG). This could lead to a degeneration of the extracellular matrix and result in increased intimal permeability, the initiating event in atherosclerosis. The present studies show that the glucose transport inhibitor cytochalasin B blocked the uptake of 3H-2-deoxy-D-glucose (2.5 mg%) by mouse 3T3 fibroblasts. Cytochalasin B also blocked the uptake of 14C-L-ascorbic acid (1.25 mg%). The results of these studies further support the hypothesis that glucose and ascorbate share a common transport system. This may have important implications concerning the vascular pathology associated with diabetes mellitus.
Assuntos
Ácido Ascórbico/metabolismo , Citocalasina B/farmacologia , Desoxiaçúcares/metabolismo , Desoxiglucose/metabolismo , Diabetes Mellitus/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Relação Dose-Resposta a Droga , FibroblastosRESUMO
The Osteogenic Disorder Shionogi (ODS) rat, Clea Inc., Tokyo, Japan lacks the ability to synthesize L-ascorbic acid (AA). As with man, monkey and the guinea pig, this rat lacks L-gulonolactone oxidase necessary for the synthesis of AA from glucose. This study shows this animal to be an alternative to the guinea pig in AA studies. The anti-scorbutic potency of Ester C (EC), a calcium ascorbate and calcium threonate mixture, was compared with an AA dose of equal ascorbate activity equivalents (AAE) for anti-scorbutic activity in the ODS rat. The minimal anti-scorbutic dose of EC was determined to be 0.44 mg/kg/day (AAE), while an AA dose of 0.51 mg/kg/day (AAE) was not anti-scorbutic in a 24 day study. At 24 days EC rats gained 125% of initial body weight (BW) and the AA rats only 45% BW. Scorbutic signs at 24 days were scored on a 0 (min) to 3 (max) scale. The EC/AA ratio scores were: hemorrhage 0/1.4, behavior change 0/2.0, piloerection 0/2.2, mobility 0.4/2.2, dysbasia 0.6/2.8 and ataxia 0.4/1.0. Pearson's correlation coefficient for BW versus AAE was r = .34 for the AA group and r = .90 for the EC group. The morbidity index for EC was 0/5 and for the AA group 2/5. The AAE dose of AA which was 16% higher/day than the EC AAE dose was not anti-scorbutic, while the EC dose was anti-scorbutic. EC rats had 3.5X greater weight gain, a sensitive indicator of scurvy, than the AA rats. EC rats had 3-4 times less, if any, scorbutic signs than AA rats. The results clearly show that, based on ascorbate activity equivalents, EC has more available ascorbate activity/potency than AA. The mechanism of this increased potency is believed to be due to the facilitated transport of AAE into the cell by the threonate (a normal in vivo metabolite of AA) present in the EC product. In addition, previous studies have shown EC (AAE) to be higher in plasma and excreted less rapidly than the AAE derived from AA administered orally.
Assuntos
Ácido Ascórbico/farmacologia , Escorbuto/tratamento farmacológico , Treonina/farmacologia , Animais , Ácido Ascórbico/biossíntese , Peso Corporal/efeitos dos fármacos , Ácido Desidroascórbico/farmacologia , Combinação de Medicamentos , Distribuição Aleatória , Ratos , Ratos Mutantes , Escorbuto/metabolismoRESUMO
BACKGROUND: The excitatory amino acid domoic acid, a glutamate and kainic acid analog, is the causative agent of amnesic shellfish poisoning in humans. No studies to our knowledge have investigated the potential contribution to short-term neurotoxicity of the brain microglia, a cell type that constitutes circa 10% of the total glial population in the brain. We tested the hypothesis that a short-term in vitro exposure to domoic acid, might lead to the activation of rat neonatal microglia and the concomitant release of the putative neurotoxic mediators tumor necrosis factor-alpha (TNF-alpha), matrix metalloproteinases-2 and-9 (MMP-2 and -9) and superoxide anion (O2-). RESULTS: In vitro, domoic acid [10 microM-1 mM] was significantly neurotoxic to primary cerebellar granule neurons. Although neonatal rat microglia expressed ionotropic glutamate GluR4 receptors, exposure during 6 hours to domoic acid [10 microM-1 mM] had no significant effect on viability. By four hours, LPS (10 ng/mL) stimulated an increase in TNF-alpha mRNA and a 2,233 % increase in TNF-alpha protein In contrast, domoic acid (1 mM) induced a slight rise in TNF-alpha expression and a 53 % increase (p < 0.01) of immunoreactive TNF-alpha protein. Furthermore, though less potent than LPS, a 4-hour treatment with domoic acid (1 mM) yielded a 757% (p < 0.01) increase in MMP-9 release, but had no effect on MMP-2. Finally, while PMA (phorbol 12-myristate 13-acetate) stimulated O2- generation was elevated in 6 hour LPS-primed microglia, a similar pretreatment with domoic acid (1 mM) did not prime O2- release. CONCLUSIONS: To our knowledge this is the first experimental evidence that domoic acid, at in vitro concentrations that are toxic to neuronal cells, can trigger a release of statistically significant amounts of TNF-alpha and MMP-9 by brain microglia. These observations are of considerable pathophysiological significance because domoic acid activates rat microglia several days after in vivo administration.
Assuntos
Ácido Caínico/análogos & derivados , Ácido Caínico/toxicidade , Metaloproteinase 9 da Matriz/metabolismo , Microglia/efeitos dos fármacos , Superóxidos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Antígeno CD11b/análise , Sobrevivência Celular/efeitos dos fármacos , Cerebelo/efeitos dos fármacos , Interações Medicamentosas , Imunofluorescência , Humanos , Lipopolissacarídeos/farmacologia , Toxinas Marinhas/toxicidade , Espectrometria de Massas , Microglia/metabolismo , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/análise , Receptores de Glutamato/análiseRESUMO
Olecranization of the patella is a technique employed by some to maintain the normal anatomic relationship of the femorotibial articulation following posterior cruciate ligament (PCL) repairs and reconstructions. It involves temporarily placing a large diameter pin longitudinally through the patella and into the tibia. The patella thus becomes a bony shelf and, in theory, holds the tibia forward, protecting the knee from a posterior drawer force. This technique appears desirable in that it allows postoperative knee motion while theoretically affording protection to the PCL. Unfortunately, it is based on an erroneous concept that the spatial relationship of the patella and tibia remains constant throughout knee flexion. Negative experience at our institution with this technique prompted our investigation. Using lateral radiographs of 20 normal knees taken at four different positions of flexion, we studied the relative motion that occurs between the patella and tibia in the sagittal plane during knee flexion from 0 degrees to 90 degrees, and defined two separate arcs of patellar motion. The patellar tendon arc is a 30 degree arc through which the patella traverses relative to the tibia during knee flexion. The patellar arc is a 22 degree arc on which the patella flexes relative to itself during knee flexion. We also studied the effect that olecranization of the patella has on the PCL in six cadaver knees. Using a combination of direct tension measurement, radiographic measurement, and fluoroscopy, we found that olecranization of the patella not only fails to protect the PCL, but actually induces a detrimental posterior drawer force during knee flexion.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Patela/cirurgia , Ligamento Cruzado Posterior/cirurgia , Adulto , Fenômenos Biomecânicos , Pinos Ortopédicos , Humanos , Instabilidade Articular/fisiopatologia , Traumatismos do Joelho/reabilitação , Traumatismos do Joelho/cirurgia , Articulação do Joelho/diagnóstico por imagem , Articulação do Joelho/fisiologia , Ligamento Cruzado Posterior/lesões , Radiografia , Amplitude de Movimento Articular , Estudos RetrospectivosAssuntos
Carcinoma de Células Pequenas/genética , Neoplasias Pulmonares/genética , Pulmão/metabolismo , Receptores de Vasopressinas/genética , Transcrição Gênica , Carcinoma de Células Pequenas/metabolismo , Variação Genética , Humanos , Fígado/metabolismo , Neoplasias Pulmonares/metabolismo , RNA Mensageiro/genética , Receptores de Vasopressinas/biossíntese , Receptores de Vasopressinas/classificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasAssuntos
Cateteres de Demora/enfermagem , Infusões Parenterais/enfermagem , Enfermagem Pediátrica , Criança , Pré-Escolar , Humanos , Lactente , Recém-Nascido , Torniquetes , VeiasRESUMO
1. Previously, we reported that calcium L-threonate caused a dose-related increase in uptake of ascorbic acid (AA) by human T-lymphoma cells. Preincubation of mouse fibroblasts with calcium L-threonate also resulted in a dose-related augmentation in uptake of AA as compared to non-treated controls. 2. Potassium L-lyxonate increased AA uptake by lymphoma cells, but did not significantly affect uptake by fibroblasts. Tartaric acid decreased uptake of AA by both cell lines. 3. Ouabain and dinitrophenol had no effect on AA uptake nor on the ability of threonate to augment AA uptake by fibroblasts. However, in T-lymphoma cells ouabain and dinitrophenol reduced AA uptake and prevented augmentation of AA uptake by calcium L-threonate.
Assuntos
Células 3T3/efeitos dos fármacos , Células 3T3/metabolismo , Ácido Ascórbico/farmacocinética , Butiratos/farmacologia , Linfoma de Células T/metabolismo , Açúcares Ácidos/farmacologia , Animais , Humanos , Camundongos , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
The hairpin ribozyme is a short endonucleolytic RNA motif isolated from a family of related plant virus satellite RNAs. It consists of two independently folding domains, each comprising two Watson-Crick helices flanking a conserved internal loop. The domains need to physically interact (dock) for catalysis of site-specific cleavage and ligation reactions. Using tapping-mode atomic force microscopy in aqueous buffer solution, we were able to produce high quality images of individual hairpin ribozyme molecules with extended terminal helices. Three RNA constructs with either the essential cleavage site guanosine or a detrimental adenosine substitution and with or without a 6-nt insertion to confer flexibility to the interdomain hinge show structural differences that correlate with their ability to form the active docked conformation. The observed contour lengths and shapes are consistent with previous bulk-solution measurements of the transient electric dichroism decays for the same RNA constructs. The active docked construct appears as an asymmetrically docked conformation that might be an indication of a more complicated docking event than a simple collapse around the interdomain hinge.
Assuntos
RNA Catalítico/química , Sequência de Bases , Processamento de Imagem Assistida por Computador , Microscopia de Força Atômica , Dados de Sequência Molecular , Conformação de Ácido Nucleico , SoluçõesRESUMO
Research suggests that oxytocin acts as a growth modulating agent for breast cancer cells. However, the signaling mechanisms responsible for these modulatory effects have not been fully elucidated. In the physiological setting oxytocin is known to stimulate contraction of myometrial cells in the uterus and myoepithelial cells in the breast by increasing intracellular free calcium ([Ca2+]i). The expression of oxytocin receptor mRNA in T-47D breast cancer cells, and four additional breast cancer cell lines (BT-549, MCF-7, MDA-MB- 231, ZR-75-1), was confirmed by RT-PCR analysis. Oxytocin-induced changes in [Ca2+]i in indo-1 AM loaded T-47D breast cancer cells were monitored using flow cytometric analysis. In this cell line, oxytocin (0, 1, 10, 100, and 1,000 nM) did not induce a dose-dependent increase in the mean 405 nm/485 nm emission ratio. These results indicate that oxytocin signaling in T-47D breast cancer cells does not appear to involve an increase in [Ca2+]i.
Assuntos
Neoplasias da Mama/metabolismo , Cálcio/metabolismo , Ocitocina/farmacologia , Sequência de Bases , Neoplasias da Mama/patologia , Primers do DNA , Relação Dose-Resposta a Droga , Citometria de Fluxo , Humanos , RNA Mensageiro/genética , Receptores de Ocitocina/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
Data are presented that provide convincing evidence for the expression of structurally normal and functional NMDA receptors by acetylcholine-producing human LA-N-2 neuroblastoma cells in culture. Reverse transcription and polymerase chain reaction (RT-PCR), followed by cloning and DNA sequencing, revealed the presence in these cells of mRNA representing the key subunit, NMDAR1, of the receptor. This mRNA was further demonstrated by Northern analysis to be the same size as that described for human neurons. The neutral red cytotoxicity assay was utilized to examine the influence on these neuroblastoma cells of a 48-h incubation with either L-glutamic acid or the specific NMDA agonist N-phthalamoyl-L-glutamic acid (NPG). Cell cytotoxicity was shown by this assay to be increased through incubation with glutamate at 1 and 10 mM by 27 and 37%, and through incubation with NPG at 0.1 and 1 mM by 28 and 46%. A possible mechanism of these toxic effects was further evaluated using the whole-cell configuration of the patch-clamp technique and the specific NMDA agonists (+/-)1-aminocyclobutane-cis-1,3-dicarboxylic acid (ACDA) and NPG. Using this procedure, a voltage-dependent tetrodotoxin-sensitive inward sodium current was found to be increased (x 1.5) by L-glutamic acid and by both NMDA agonists in the presence of glycine. Another voltage-gated inward current, probably carried by calcium ions, was increased three- to fourfold. Hence, these glutamate activities observed in human LA-N-2 neuroblastoma cells appear to occur through the activation of functional NMDA receptors in much the same way as reported for neurons, and both glutamate and NMDA agonists can be toxic to these neuroblastoma cells. Our findings, therefore, suggest this cell line will provide a model suitable for investigating the mechanisms of NMDA-related long-term potentiation (LTP) in neurons and of the NMDA-related neurotoxic effects of glutamate in disease states that involve a reduction in cholinergic function.