RESUMO
OBJECTIVE: The objective of this study was to analyze the uptake of rubidium in malignant tumors. PARTICIPANTS AND METHODS: Sixteen malignant lesions were included. Two radiologists compared each lesion to four references (subcutaneous fat, lung, mediastinal blood pool, and liver) at rest and stress and scored as 1-4. Maximum standardized uptake value (SUV) in each lesion and four references, as well as ratios of lesion SUV to SUV of each of the references, were calculated at rest and stress. We assessed an agreement for scores of reader 1 versus reader 2 (inter-reader) at rest and stress, scores at rest versus stress (intrapatient) for reader 1 and reader 2, and lesion SUV and respective ratios at rest and stress using paired t-test and Bland-Altman analyses. RESULTS: Fifteen (94%) out of 16 lesions had a score of 3 or 4 at rest or stress or both by at least one reviewer. We did not find evidence of inter-reader bias at rest or stress or intrapatient (rest vs. stress) bias for either reader. SUV ranged from 1.0 to 8.1 at rest and from 0.7 to 6.7 at stress. There was an excellent agreement between ratios of lesion SUV to lung SUV at rest versus stress. On the extreme, there was a poor agreement between ratios of lesion SUV to liver SUV at rest versus stress. Otherwise, the agreement was good for the majority of the results, and moderate for a few others. CONCLUSION: Malignant tumors can be readily depicted and quantified on rubidium PET/CT. Further research is needed.
Assuntos
Neoplasias/diagnóstico por imagem , Neoplasias/metabolismo , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Radioisótopos de Rubídio/metabolismo , Transporte Biológico , Humanos , Processamento de Imagem Assistida por ComputadorRESUMO
BACKGROUND: It has not been possible to study the pumping and signaling functions of Na/K-ATPase independently in live cells. RESULTS: Both cell-free and cell-based assays indicate that the A420P mutation abolishes the Src regulatory function of Na/K-ATPase. CONCLUSION: A420P mutant has normal pumping but not signaling function. SIGNIFICANCE: Identification of Src regulation-null mutants is crucial for addressing physiological role of Na/K-ATPase. The α1 Na/K-ATPase possesses both pumping and signaling functions. However, it has not been possible to study these functions independently in live cells. We have identified a 20-amino acid peptide (Ser-415 to Gln-434) (NaKtide) from the nucleotide binding domain of α1 Na/K-ATPase that binds and inhibits Src in vitro. The N terminus of NaKtide adapts a helical structure. In vitro kinase assays showed that replacement of residues that contain a bulky side chain in the helical structure of NaKtide by alanine abolished the inhibitory effect of the peptide on Src. Similarly, disruption of helical structure by proline replacement, either single or in combination, reduced the inhibitory potency of NaKtide on Src. To identify mutant α1 that retains normal pumping function but is defective in Src regulation, we transfected Na/K-ATPase α1 knockdown PY-17 cells with expression vectors of wild type or mutant α1 carrying Ala to Pro mutations in the region of NaKtide helical structure and generated several stable cell lines. We found that expression of either A416P or A420P or A425P mutant fully restored the α1 content and consequently the pumping capacity of cells. However, in contrast to A416P, either A420P or A425P mutant was incapable of interacting and regulating cellular Src. Consequently, expression of these two mutants caused significant inhibition of ouabain-activated signal transduction and cell growth. Thus we have identified α1 mutant that has normal pumping function but is defective in signal transduction.
Assuntos
Transdução de Sinais , ATPase Trocadora de Sódio-Potássio/genética , Quinases da Família src/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Caveolina 1/metabolismo , Linhagem Celular , Proliferação de Células , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ouabaína/farmacologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína , Ratos , Radioisótopos de Rubídio/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/metabolismo , Quinases da Família src/químicaRESUMO
Nicotine and opioids share several behavioral and rewarding properties. Although both opioids and nicotine have their own specific mechanism of action, there is empirical and experimental evidence of interactions between these drugs. We studied receptor-level interactions of nicotine and morphine at α4ß2, α7 and α3(â) nicotinic acetylcholine receptors. [(3)H]epibatidine displacement was used to determine if morphine binds competitively to nicotinic acetylcholine receptors. Functional interactions of morphine and nicotine were studied with calcium fluorometry and (86)Rb(+) efflux assays. Morphine displaced [(3)H]epibatidine from nicotinic agonist binding sites in all cell lines studied. The Ki values for morphine were 13.2µM in SH-EP1-hα4ß2 cells, 0.16µM and 126µM in SH-SY5Y cells and 43.7µM in SH-EP1-hα7 cells. In SH-EP1-hα4ß2 cells expressing α4ß2 nicotinic acetylcholine receptors, morphine acted as a partial agonist of (86)Rb(+) efflux comparable to cytisine (with EC50 values of 53.3µM for morphine and 5.38µM for cytisine). The effect of morphine was attenuated concentration-dependently by the nicotinic antagonist mecamylamine. In the SH-SY5Y cell line expressing several subtypes of nicotinic acetylcholine receptors morphine had an inhibitory effect on nicotine induced (86)Rb(+) ion efflux mediated by α3(â) nicotinic acetylcholine receptors. These results suggest that morphine acts as a partial agonist at α4ß2 nicotinic acetylcholine receptors and as a weak antagonist at α3(â) nicotinic acetylcholine receptors.
Assuntos
Morfina/farmacologia , Nicotina/farmacologia , Receptores Nicotínicos/metabolismo , Ligação Competitiva , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Cálcio/metabolismo , Linhagem Celular Tumoral , Interações Medicamentosas , Humanos , Morfina/metabolismo , Nicotina/metabolismo , Piridinas/metabolismo , Radioisótopos de Rubídio/metabolismo , Receptor Nicotínico de Acetilcolina alfa7RESUMO
Astrocytes are electrically nonexcitable cells that display increases in cytosolic calcium ion (Ca²+) in response to various neurotransmitters and neuromodulators. However, the physiological role of astrocytic Ca²+ signaling remains controversial. We show here that astrocytic Ca²+ signaling ex vivo and in vivo stimulated the Na+,K+-ATPase (Na+- and K+-dependent adenosine triphosphatase), leading to a transient decrease in the extracellular potassium ion (K+) concentration. This in turn led to neuronal hyperpolarization and suppressed baseline excitatory synaptic activity, detected as a reduced frequency of excitatory postsynaptic currents. Synaptic failures decreased in parallel, leading to an increase in synaptic fidelity. The net result was that astrocytes, through active uptake of K+, improved the signal-to-noise ratio of synaptic transmission. Active control of the extracellular K+ concentration thus provides astrocytes with a simple yet powerful mechanism to rapidly modulate network activity.
Assuntos
Astrócitos/fisiologia , Cálcio/metabolismo , Potássio/metabolismo , Transmissão Sináptica/fisiologia , Trifosfato de Adenosina/farmacologia , Animais , Animais Recém-Nascidos , Astrócitos/citologia , Astrócitos/metabolismo , Transporte Biológico/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Células Cultivadas , Potenciais Pós-Sinápticos Excitadores/fisiologia , Espaço Extracelular/metabolismo , Hipocampo/citologia , Hipocampo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Rede Nervosa/metabolismo , Rede Nervosa/fisiologia , Neurônios/citologia , Neurônios/metabolismo , Neurônios/fisiologia , Ouabaína/farmacologia , Ratos , Ratos Wistar , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/fisiologia , Radioisótopos de Rubídio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
Na(+),K(+)-ATPase is a heterodimer consisting of catalytic α1-α4 and regulatory ß1-ß3 subunits. Recently, we reported that transfection with ouabain-resistant α1R-Na(+),K(+)-ATPase rescues renal epithelial C7-MDCK cells exclusively expressing the ouabain-sensitive α1S-isoform from the cytotoxic action of ouabain. To explore the role of α2 subunit in ion transport and cytotoxic action of ouabain, we compared the effect of ouabain on K(+) ((86)Rb) influx and the survival of ouabain-treated C7-MDCK cells stably transfected with α1R- and α2R-Na(+),K(+)-ATPase. α2R mRNA in transfected cells was â¼8-fold more abundant than α1R mRNA, whereas immunoreactive α2R protein content was 5-fold lower than endogenous α1S protein. A concentration of 10 µmol/L ouabain led to complete inhibition of (86)Rb influx both in mock- and α2R-transfected cells, whereas maximal inhibition of (86)Rb influx in α1R-transfectd cells was observed at 1000 µmol/L ouabain. In contrast to the massive death of mock- and α2R-transfected cells exposed to 3 µmol/L ouabain , α1R-cells survived after 24 h incubation with 1000 µmol/L ouabain. Thus, our results show that unlike α1R, the presence of α2R-Na(+),K(+)-ATPase subunit mRNA and immunoreactive protein does not contribute to Na(+)/K(+) pump activity, and does not rescue C7-MDCK cells from the cytotoxic action of ouabain. Our results also suggest that the lack of impact of transfected α2-Na(+),K(+)-ATPase on Na(+)/K(+) pump activity and cell survival can be attributed to the low efficiency of its translation and (or) delivery to the plasma membrane of renal epithelial cells.
Assuntos
Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Ouabaína/efeitos adversos , Biossíntese de Proteínas/genética , Biossíntese de Proteínas/fisiologia , Isoformas de RNA/genética , ATPase Trocadora de Sódio-Potássio/biossíntese , ATPase Trocadora de Sódio-Potássio/fisiologia , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Morte Celular/fisiologia , Células Cultivadas , Cães , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/genética , Transporte de Íons/fisiologia , Isoenzimas/genética , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/fisiologia , Potássio/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Radioisótopos de Rubídio/metabolismo , ATPase Trocadora de Sódio-Potássio/genética , Transfecção/métodosRESUMO
The previously undescribed heterozygous missense mutation E758K was discovered in the human AE1/SLC4A1/band 3 gene in two unrelated patients with well-compensated hereditary spherostomatocytic anemia (HSt). Oocyte surface expression of AE1 E758K, in contrast to that of wild-type AE1, required coexpressed glycophorin A (GPA). The mutant polypeptide exhibited, in parallel, strong GPA dependence of DIDS-sensitive (36)Cl(-) influx, trans-anion-dependent (36)Cl(-) efflux, and Cl(-)/HCO(3)(-) exchange activities at near wild-type levels. AE1 E758K expression was also associated with GPA-dependent increases of DIDS-sensitive pH-independent SO(4)(2-) uptake and oxalate uptake with altered pH dependence. In marked contrast, the bumetanide- and ouabain-insensitive (86)Rb(+) influx associated with AE1 E758K expression was largely GPA-independent in Xenopus oocytes and completely GPA-independent in Ambystoma oocytes. AE1 E758K-associated currents in Xenopus oocytes also exhibited little or no GPA dependence. (86)Rb(+) influx was higher but inward cation current was lower in oocytes expressing AE1 E758K than previously reported in oocytes expressing the AE1 HSt mutants S731P and H734R. The pharmacological inhibition profile of AE1 E758K-associated (36)Cl(-) influx differed from that of AE1 E758K-associated (86)Rb(+) influx, as well as from that of wild-type AE1-mediated Cl(-) transport. Thus AE1 E758K-expressing oocytes displayed GPA-dependent surface polypeptide expression and anion transport, accompanied by substantially GPA-independent, pharmacologically distinct Rb(+) flux and by small, GPA-independent currents. The data strongly suggest that most of the increased cation transport associated with the novel HSt mutant AE1 E758K reflects activation of endogenous oocyte cation permeability pathways, rather than cation translocation through the mutant polypeptide.
Assuntos
Anfíbios/metabolismo , Anemia Hemolítica Congênita/metabolismo , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Bicarbonatos/metabolismo , Cloretos/metabolismo , Glicoforinas/metabolismo , Mutação de Sentido Incorreto , Oócitos/metabolismo , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Ambystoma mexicanum/metabolismo , Sequência de Aminoácidos , Anfíbios/genética , Anemia Hemolítica Congênita/sangue , Anemia Hemolítica Congênita/genética , Animais , Proteína 1 de Troca de Ânion do Eritrócito/antagonistas & inibidores , Proteína 1 de Troca de Ânion do Eritrócito/genética , Bumetanida/farmacologia , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Clonagem Molecular , Análise Mutacional de DNA , Feminino , Glicoforinas/genética , Heterozigoto , Humanos , Concentração de Íons de Hidrogênio , Cinética , Masculino , Potenciais da Membrana , Pessoa de Meia-Idade , Dados de Sequência Molecular , Ouabaína/farmacologia , Ácido Oxálico/metabolismo , Radioisótopos de Rubídio/metabolismo , Índice de Gravidade de Doença , Inibidores de Simportadores de Cloreto de Sódio e Potássio/farmacologia , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/metabolismo , Sulfatos/metabolismo , Xenopus laevis/metabolismoRESUMO
Oligomerization, function, and regulation of unmodified mouse Kcc1 K-Cl cotransporter were studied by chemical crosslinking. Treatment of Xenopus oocytes and 293T cells expressing K-Cl cotransporter Kcc1 with several types of chemical cross-linkers shifted Kcc1 polypeptide to higher molecular weight forms. More extensive studies were performed with the amine-reactive disuccinyl suberate (DSS) and with the sulfhydryl-reactive bis-maleimidohexane (BMH). Kcc1 cross-linking was time-dependent in intact oocytes, and was independent of protein concentration in detergent lysates from oocytes or 293T cells. Kcc1 cross-linking by the cleavable cross-linker DTME was reversible. The N-terminal and C-terminal cytoplasmic tails of Kcc1 were not essential for Kcc1 crosslinking. PFO-PAGE and gel filtration revealed oligomeric states of uncrosslinked KCC1 corresponding in mobility to that of cross-linked protein. DSS and BMH each inhibited KCC1-mediated (86)Rb(+) uptake stimulated by hypotonicity or by N-ethylmaleimide (NEM) without reduction in nominal surface abundance of KCC1. These data add to evidence supporting the oligomeric state of KCC polypeptides.
Assuntos
Reagentes de Ligações Cruzadas/farmacologia , Simportadores/química , Animais , Linhagem Celular , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Transporte de Íons/efeitos dos fármacos , Rim/citologia , Rim/embriologia , Camundongos , Microscopia de Fluorescência , Peso Molecular , Oócitos , Estrutura Terciária de Proteína , RNA Complementar/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Radioisótopos de Rubídio/metabolismo , Simportadores/efeitos dos fármacos , Xenopus laevis , Cotransportadores de K e Cl-RESUMO
We examined for immediate and persistent changes in nAChRs in cerebral cortex, thalamus and striatum of male rats caused by prenatal exposure to nicotine from gestational day 3 to postnatal day 10 (PN10), and how such exposure affected the responses of adolescents to subsequent nicotine challenge. Receptor numbers were assessed by [(3)H]epibatidine binding and receptor function was measured by acetylcholine-stimulated (86)Rb efflux (cerebral cortex and thalamus) and nicotine-stimulated dopamine release (striatum). Immediate effects of prenatal nicotine, assessed in PN10 animals, were not detected for any parameter. A subsequent 14 day nicotine exposure in adolescence revealed persistent changes caused by prenatal nicotine exposure. Nicotine exposure in adolescents caused up-regulation of binding in all three regions; however, this up-regulation was lost in thalamus from animals prenatally exposed to nicotine. Nicotine exposure in adolescents caused decreased nicotine-stimulated dopamine release in striatum; this effect was lost in animals prenatally exposed to nicotine. Comparison of parameters in PN10 and PN42 rats revealed developmental changes in the CNS cholinergic system. In thalamus, binding increased with age, as did the proportion of (86)Rb efflux with high sensitivity to acetylcholine. In cortex, binding also increased with age, but there was no change in total (86)Rb efflux, and the proportion of high to low sensitivity efflux declined with age. Nicotine-stimulated striatal dopamine release (both total and alpha-conotoxin MII-resistant release) increased with age in naïve animals, but not in those prenatally exposed to nicotine. These findings demonstrate that prenatal exposure to nicotine causes alterations in nAChRs and in their regulation by nicotine that persist into adolescence. These changes may play a role in the increased risk for nicotine addiction observed in adolescent offspring of smoking mothers.
Assuntos
Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Nicotina/administração & dosagem , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Receptores Nicotínicos/metabolismo , Acetilcolina/farmacologia , Envelhecimento , Animais , Animais Recém-Nascidos , Berberina/análogos & derivados , Berberina/farmacologia , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/embriologia , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/metabolismo , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/embriologia , Corpo Estriado/crescimento & desenvolvimento , Corpo Estriado/metabolismo , Dopamina/metabolismo , Feminino , Masculino , Nicotina/farmacologia , Gravidez , Ratos , Ratos Sprague-Dawley , Radioisótopos de Rubídio/metabolismo , Sinaptossomos/metabolismo , Tálamo/efeitos dos fármacos , Tálamo/embriologia , Tálamo/crescimento & desenvolvimento , Tálamo/metabolismo , Trítio/farmacologia , Regulação para CimaRESUMO
A panel of 18 protein tyrosine kinase antagonists were tested for their inhibitory effect on human P2X(7) receptor-mediated (86)Rb(+) (K(+)) efflux. The most potent compound (compound P), a phthalazinamine derivative and an inhibitor of vascular endothelial growth factor receptor kinase, blocked ATP-induced (86)Rb(+)-efflux in human B-lymphocytes and erythrocytes by 76% and 66%, respectively. This inhibition was dose-dependent in both cell types with an IC(50) of approximately 5muM. Kinetic analysis showed compound P was a non-competitive inhibitor of P2X(7). This compound also inhibited ATP-induced ethidium(+) influx into B-lymphocytes and P2X(7)-transfected-HEK-293 cells, as well as ATP-induced (86)Rb(+)-efflux from canine erythrocytes. Externally, but not internally, applied compound P impaired ATP-induced inward currents in P2X(7)-transfected-HEK-293 cells. This study demonstrates that a novel protein tyrosine kinase antagonist directly impairs native and recombinant human P2X(7) receptors. The data suggests that antagonists which target ATP-binding sites of kinases may potentially block the P2X(7) receptor.
Assuntos
Compostos de Anilina/química , Ftalazinas/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Antagonistas do Receptor Purinérgico P2 , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Compostos de Anilina/farmacologia , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Linhagem Celular , Cães , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Etídio/metabolismo , Humanos , Concentração Inibidora 50 , Ftalazinas/farmacologia , Potássio/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores Purinérgicos P2X7 , Radioisótopos de Rubídio/metabolismoRESUMO
When cardiomyocytes were subjected to hypoxia, tumor necrosis factor-alpha (TNF-alpha; 3-50 ng/ml) or adenosine (1-100 microM), decreased hypoxic damage as was detected by lactate dehydrogenase (LDH) release, MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) absorbance, ROS (reactive oxygen species) measurement or desmin immunostaining. This cardioprotection was not prevented in TNF-alpha-treated cultures by 5-hydroxydecanoic acid (5-HD). Our aim was to elucidate whether adenosine and TNF-alpha mediate a similar protective mechanism against hypoxia in primary heart cultures and in H9c2 cardiomyocytes. Adenosine and TNF-alpha are known for their negative inotropic effects on the heart. We have suggested that deoxyglucose uptake reflects heart contractility in cell cultures; therefore, we assayed its accumulation under various conditions. Treatment for 20 min with adenosine, R-PIA [(-)-N(6)-phenylisopropyladenosine] (10 microM), or TNF-alpha reduced (3)H-deoxyglucose uptake in primary heart cultures and also in H9c2 cardiomyocytes by 30-50%. Isoproterenol accelerated (3)H-deoxyglucose uptake by 50%. Adenosine, R-PIA, or TNF-alpha attenuated the stimulatory effect of isoproterenol on (3)H-deoxyglucose uptake to control levels. Hypoxia reduced (3)H-deoxyglucose uptake by 50%, as in the treatment of the hypoxic cultures with TNF-alpha or adenosine. Glibenclamide (2 microM), 5-HD (300 microM), or diazoxide (50 microM) increased (3)H-deoxyglucose uptake by 50-80%. Adenosine (100 microM) and TNF-alpha (50 ng/ml) stimulated (86)Rb efflux. Glibenclamide attenuated this effect. We demonstrate that TNF-alpha, like adenosine, accelerated Ca(2+) uptake into the sarcoplasmic reticulum (SR) by 50-100% and therefore prevented cardiomyocyte Ca(2+) overload. Our findings further suggest that TNF-alpha, as well as adenosine, may mediate an adaptive effect in the heart by preventing Ca(2+) overload via activation of SR Ca-ATPase (SERCA(2)a).
Assuntos
Adenosina/farmacologia , Antiarrítmicos/farmacologia , Coração/efeitos dos fármacos , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Adenosina/análogos & derivados , Animais , Animais Recém-Nascidos , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular , Desoxiglucose/metabolismo , Diazóxido/farmacologia , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Interações Medicamentosas , Glibureto/farmacologia , Coração/fisiologia , Isoproterenol/farmacologia , Contração Miocárdica/fisiologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/ultraestrutura , Fármacos Neuroprotetores/farmacologia , Ratos , Radioisótopos de Rubídio/metabolismo , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismoRESUMO
Maternal smoking is a risk factor associated with nicotine abuse, so the effect of perinatal nicotine exposure was studied on the responsiveness to nicotine across adolescence in the rat. Pregnant Sprague-Dawley rats were implanted with s.c. Alzet osmotic minipumps delivering nicotine (L-nicotine hydrogen tartrate, 2 mg/kg/day free base) or vehicle (0.9% saline) on gestational day 7. There was no effect of nicotine on dam weight gain, food consumption, or water consumption or on the number of live pups or weights at the time of birth. Pups were cross-fostered to obtain the following prenatal/postnatal exposure groups: control/control, nicotine/nicotine, nicotine/control, and control/nicotine. On postnatal days 28, 35, 49, and 63, nicotine-stimulated (86)Rb(+) efflux was measured in synaptosomes prepared from the frontal cortex, hippocampus, striatum (STR), and thalamus (THL), using a previously developed method. Significant effects of treatment and concentration were detected in all four brain regions, and significant effects of age were observed in the STR and THL. Significant interactions of age and treatment were observed in each of the four brain regions. Nicotine-stimulated (86)Rb(+) efflux peaked during adolescence in control rats. However, perinatal exposure to nicotine eliminated this peak during adolescence. These results are consistent with recent behavioral and receptor binding results from other laboratories and are the first direct evidence at the cellular level that the nicotinic acetylcholine receptor response varies during adolescence and is affected by perinatal nicotine exposure.
Assuntos
Feto/efeitos dos fármacos , Nicotina/farmacologia , Receptores Nicotínicos/efeitos dos fármacos , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Feminino , Masculino , Ratos , Ratos Sprague-Dawley , Receptores Nicotínicos/fisiologia , Radioisótopos de Rubídio/metabolismo , Sinaptossomos/metabolismoRESUMO
Lipophilic monocations can pass through phospholipid bilayers and accumulate in negatively-charged compartments such as the mitochondrial matrix, driven by the membrane potential. This property is used to visualize mitochondria, to deliver therapeutic molecules to mitochondria and to measure the membrane potential. In theory, lipophilic dications have a number of advantages over monocations for these tasks, as the double charge should lead to a far greater and more selective uptake by mitochondria, increasing their therapeutic potential. However, the double charge might also limit the movement of lipophilic dications through phospholipid bilayers and little is known about their interaction with mitochondria. To see whether lipophilic dications could be taken up by mitochondria and cells, we made a series of bistriphenylphosphonium cations comprising two triphenylphosphonium moieties linked by a 2-, 4-, 5-, 6- or 10-carbon methylene bridge. The 5-, 6- and 10-carbon dications were taken up by energized mitochondria, whereas the 2- and 4-carbon dications were not. The accumulation of the dication was greater than that of the monocation methyltriphenylphosphonium. However, the uptake of dications was only described by the Nernst equation at low levels of accumulation, and beyond a threshold membrane potential of 90-100 mV there was negligible increase in dication uptake. Interestingly, the 5- and 6-carbon dications were not accumulated by cells, due to lack of permeation through the plasma membrane. These findings indicate that conjugating compounds to dications offers only a minor increase over monocations in delivery to mitochondria. Instead, this suggests that it may be possible to form dications within mitochondria that then remain within the cell.
Assuntos
Membranas Intracelulares/metabolismo , Lipídeos/química , Mitocôndrias/metabolismo , Compostos Organofosforados/metabolismo , Compostos de Terfenil/metabolismo , Trifosfato de Adenosina/metabolismo , Algoritmos , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Cátions Bivalentes/química , Cátions Bivalentes/metabolismo , Humanos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/fisiologia , Ionóforos/farmacologia , Células Jurkat , Bicamadas Lipídicas/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/fisiologia , Nigericina/farmacologia , Oniocompostos/química , Oniocompostos/metabolismo , Compostos Organofosforados/química , Cloreto de Potássio/farmacologia , Ratos , Rotenona/farmacologia , Radioisótopos de Rubídio/metabolismo , Compostos de Terfenil/química , Trítio/metabolismo , Compostos de Tritil/química , Compostos de Tritil/metabolismo , Desacopladores/farmacologiaRESUMO
D-fructose (10 mM) augments, in rat pancreatic islets, insulin release evoked by 10 mM D-glucose. Even in the absence of D-glucose, D-fructose (100 mM) displays a positive insulinotropic action. It was now examined whether the insulinotropic action of D-fructose could be attributed to an increase in the ATP content of islet cells. After 30-60 min incubation in the presence of D-glucose and/or D-fructose, the ATP and ADP content was measured by bioluminescence in either rat isolated pancreatic islets (total ATP and ADP) or the supernatant of dispersed rat pancreatic islet cells exposed for 30 s to digitonine (cytosolic ATP and ADP). D-fructose (10 and 100 mM) was found to cause a concentration-related decrease in the total ATP and ADP content and ATP/ADP ratio below the basal values found in islets deprived of exogenous nutrient. Moreover, in the presence of 10 mM D-glucose, which augmented both the total ATP content and ATP/ADP ratio above basal value, D-fructose (10 mM) also lowered these two parameters. The cytosolic ATP/ADP ratio, however, was increased in the presence of D-glucose and/or D-fructose. Under the present experimental conditions, a sigmoidal relationship was found between such a cytosolic ATP/ADP ratio and either (86)Rb net uptake by dispersed islet cells or insulin release from isolated islets. These data provide, to our knowledge, the first example of a dramatic dissociation between changes in total ATP content or ATP/ADP ratio and insulin release in pancreatic islets exposed to a nutrient secretagogue. Nevertheless, the cationic and insulinotropic actions of d-glucose and/or d-fructose were tightly related to the cytosolic ATP/ADP ratio.
Assuntos
Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Citosol/metabolismo , Frutose/farmacologia , Ilhotas Pancreáticas/metabolismo , Animais , Citosol/efeitos dos fármacos , Feminino , Glucose/farmacologia , Técnicas In Vitro , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/ultraestrutura , Ratos , Ratos Wistar , Radioisótopos de Rubídio/metabolismoRESUMO
Cardiotonic glycosides, like ouabain, inhibit Na(+)-K(+)-ATPase. Recent evidence suggests that low molar concentrations of ouabain alter cell growth. Studies were conducted to examine the effect of ouabain on Akt phosphorylation and rate of cell proliferation in opossum kidney (OK) proximal tubule cells. Cells exposed to 10 nM ouabain displayed increased Akt Ser(473) phosphorylation, as evidenced by an increase in phospho-Akt Ser(473) band density. Ouabain-stimulated Akt Ser(473) phosphorylation was inhibited by pretreatment with phosphatidylinositol 3-kinase (PI3K) inhibitors (LY294002 and wortmannin), a PLC inhibitor (edelfosine), and an Akt inhibitor. Moreover, ouabain-mediated Akt Ser(473) phosphorylation was suppressed by reduction of extracellular calcium (EGTA) or when intracellular calcium was buffered by BAPTA-AM. An inhibitor of calcium store release (TMB-8) and an inhibitor of calcium entry via store-operated calcium channels (SKF96365) also suppressed ouabain-mediated Akt Ser(473) phosphorylation. In fura-2 AM-loaded cells, 10 nM ouabain increased capacitative calcium entry (CCE). Ouabain at 10 nM did not significantly alter baseline cytoplasmic calcium concentration in control cells. However, treatment with 10 nM ouabain caused a significantly higher ATP-mediated calcium store release. After 24 h, 10 nM ouabain increased the rate of cell proliferation. The Akt inhibitor, BAPTA-AM, SKF96365, and cyclopiazonic acid suppressed the increase in the rate of cell proliferation caused by 10 nM ouabain. Ouabain at 10 nM caused a detectable increase in (86)Rb uptake but did not significantly alter Na(+)-K(+)-ATPase (ouabain-sensitive pNPPase) activity in crude membranes or cell sodium content. Taken together, the results point to a role for CCE and Akt phosphorylation, in response to low concentrations of ouabain, that increase the rate of cell proliferation without inhibiting Na(+)-K(+)-ATPase-mediated ion transport.
Assuntos
Cálcio/metabolismo , Proliferação de Células , Inibidores Enzimáticos/metabolismo , Túbulos Renais Proximais/citologia , Ouabaína/metabolismo , Animais , Linhagem Celular , Didelphis , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Radioisótopos de Rubídio/metabolismo , Serina/metabolismo , Fosfolipases Tipo C/metabolismoRESUMO
Pyridine N-n-alkylation of S(-)-nicotine (NIC) affords N-n-alkylnicotinium analogs, previously shown to competitively inhibit [(3)H]NIC binding and interact with alpha4beta2* nicotinic receptors (nAChRs). The present study determined the ability of the analogs to inhibit NIC-evoked (86)Rb(+) efflux from rat thalamic synaptosomes to assess functional interaction with alpha4beta2* nAChRs. In a concentration-dependent manner, NIC evoked (86)Rb(+) efflux (EC(50) = 170 nmol/L). Analog-induced inhibition of NIC-evoked (86)Rb(+) efflux varied over a approximately 450-fold range. Analogs with long n-alkyl chain lengths (C(9)-C(12)) inhibited efflux in the low nmol/L range (IC(50) = 9-20 nmol/L), similar to dihydro-beta-erythroidine (IC(50) = 19 nmol/L). Compounds with shorter n-alkyl chain lengths (C(1)-C(8)) produced inhibition in the low micromol/L range (IC(50) = 3-12 micromol/L). C(10) and C(12) analogs completely inhibited NIC-evoked efflux, whereas C(1-9) analogs produced maximal inhibition of only 10% to 60%. While the C(10) analog N-n-decylnicotinium iodide (NDNI) did not produce significant inhibition of NIC-evoked dopamine release in previously reported studies, NDNI possesses high affinity for [(3)H]NIC binding sites (K(i) = 90 nmol/L) and is a potent and efficacious inhibitor of NIC-evoked (86)Rb(+) efflux as demonstrated in the current studies. Thus, NDNI is a competitive, selective antagonist at alpha4beta2* nAChRs.
Assuntos
Nicotina/antagonistas & inibidores , Antagonistas Nicotínicos/farmacologia , Receptores Nicotínicos/metabolismo , Radioisótopos de Rubídio/metabolismo , Tálamo/metabolismo , Animais , Relação Dose-Resposta a Droga , Masculino , Nicotina/metabolismo , Antagonistas Nicotínicos/química , Ratos , Ratos Sprague-Dawley , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo , Tálamo/efeitos dos fármacosRESUMO
RATIONALE: Individuals vary in their susceptibility to nicotine addiction. However, there is little evidence that behavioral sensitivity to nicotine is dependent upon the functional state of nicotinic cholinergic receptors (nAChRs). OBJECTIVE: To determine the relationship between in vivo pharmacological desensitization (in other words, acute tolerance) and brain regional nAChR function. METHODS: Male Sprague-Dawley rats, trained to discriminate nicotine (0.4 mg/kg free base) from saline in a two-lever drug discrimination task, were tested for the development of acute tolerance. Rats were injected with 0.4 mg/kg nicotine, tested for nicotine discrimination for 2 min, then injected with the same dose of nicotine 90 min, 180 min, and 270 min after the first injection and tested for nicotine discrimination after each injection. These subjects were separated into two groups, desensitizers (DZ) and nondesensitizers (NDZ), based upon performance in the repetitive dosing drug discrimination paradigm. The sensitivity of nAChRs in specific brain regions of these two groups was assessed by the use of an 86Rb+ efflux assay using synaptosomes prepared from the frontal cortex, hippocampus, striatum, and "thalamus," which included the midbrain and hypothalamus as well as the thalamus. RESULTS: The nicotine-induced increase in 86Rb+ efflux was significantly greater in NDZ as compared to DZ in the "thalamus." There was no statistically significant difference in the effects of nicotine in the frontal cortex, hippocampus, and striatum of these two groups. A significant correlation was observed between thalamic 86Rb+ efflux and the rate of behavioral desensitization of individual rats. CONCLUSION: These findings are consistent with the concept that the production of acute tolerance by nicotine in vivo correlates directly with its ability to induce nAChR desensitization at the cellular level.
Assuntos
Encéfalo/efeitos dos fármacos , Nicotina/farmacologia , Receptores Nicotínicos/efeitos dos fármacos , Animais , Mapeamento Encefálico , Condicionamento Operante/efeitos dos fármacos , Aprendizagem por Discriminação/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Tolerância a Medicamentos , Masculino , Neurônios/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Radioisótopos de Rubídio/metabolismoRESUMO
Serum- and glucocorticoid-induced kinase 1 (SGK1) is thought to be an important regulator of Na(+) reabsorption in the kidney. It has been proposed that SGK1 mediates the effects of aldosterone on transepithelial Na(+) transport. Previous studies have shown that SGK1 increases Na(+) transport and epithelial Na(+) channel (ENaC) activity in the apical membrane of renal epithelial cells. SGK1 has also been implicated in the modulation of Na(+)-K(+)-ATPase activity, the transporter responsible for basolateral Na(+) efflux, although this observation has not been confirmed in renal epithelial cells. We examined Na(+)-K(+)-ATPase function in an A6 renal epithelial cell line that expresses SGK1 under the control of a tetracycline-inducible promoter. The results showed that expression of a constitutively active mutant of SGK1 (SGK1(T)(S425D)) increased the transport activity of Na(+)-K(+)-ATPase 2.5-fold. The increase in activity was a direct consequence of activation of the pump itself. The onset of Na(+)-K(+)-ATPase activation was observed between 6 and 24 h after induction of SGK1 expression, a delay that is significantly longer than that required for activation of ENaC in the same cell line (1 h). SGK1 and aldosterone stimulated the Na(+) pump synergistically, indicating that the pathways mediated by these molecules operate independently. This observation was confirmed by demonstrating that aldosterone, but not SGK1(T)(S425D), induced an approximately 2.5-fold increase in total protein and plasma membrane Na(+)-K(+)-ATPase alpha(1)-subunit abundance. We conclude that aldosterone increases the abundance of Na(+)-K(+)-ATPase, whereas SGK1 may activate existing pumps in the membrane in response to chronic or slowly acting stimuli.
Assuntos
Células Epiteliais/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Túbulos Renais Distais/citologia , Proteínas Serina-Treonina Quinases/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Aldosterona/metabolismo , Animais , Calnexina/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Ativação Enzimática , Inibidores Enzimáticos/metabolismo , Células Epiteliais/citologia , Proteínas Imediatamente Precoces/genética , Ouabaína/metabolismo , Proteínas Serina-Treonina Quinases/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Radioisótopos de Rubídio/metabolismo , ATPase Trocadora de Sódio-Potássio/genética , Xenopus laevisRESUMO
Multiple ion channels have now been shown to be regulated by phosphatidylinositol 4,5-bisphosphate (PIP2) at the cytoplasmic face of the membrane. However, direct evidence for a specific interaction between phosphoinositides and ion channels is critically lacking. We reconstituted pure KirBac1.1 and KcsA protein into liposomes of defined composition (3:1 phosphatidylethanolamine:phosphatidylglycerol) and examined channel activity using a 86Rb+ uptake assay. We demonstrate direct modulation by PIP2 of KirBac1.1 but not KcsA activity. In marked contrast to activation of eukaryotic Kir channels by PIP2, KirBac1.1 is inhibited by PIP2 incorporated in the membrane (K(1/2) = 0.3 mol %). The dependence of inhibition on the number of phosphate groups and requirement for a lipid tail matches that for activation of eukaryotic Kir channels, suggesting a fundamentally similar interaction mechanism. The data exclude the possibility of indirect modulation via cytoskeletal or other intermediary elements and establish a direct interaction of the channel with PIP2 in the membrane.
Assuntos
Proteínas de Escherichia coli/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Canais de Potássio/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias , Membrana Celular/metabolismo , Citoplasma/metabolismo , Citoesqueleto/metabolismo , Relação Dose-Resposta a Droga , Histidina/química , Íons , Cinética , Lipossomos/metabolismo , Micelas , Dados de Sequência Molecular , Fosfatos/química , Fosfatos/metabolismo , Fosfatos de Fosfatidilinositol/química , Canais de Potássio Corretores do Fluxo de Internalização/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Estrutura Terciária de Proteína , Radioisótopos de Rubídio/metabolismoRESUMO
Although the amiloride-sensitive epithelial sodium channel (ENaC) plays an important role in the modulation of alveolar liquid clearance, the precise mechanism of its regulation in alveolar epithelial cells is still under investigation. Protein kinase C (PKC) has been shown to alter ENaC expression and activity in renal epithelial cells, but much less is known about its role in alveolar epithelial cells. The objective of this study was to determine whether PKC activation modulates ENaC expression and transepithelial Na+ transport in cultured rat alveolar epithelial cells. Alveolar type II cells were isolated and cultured for 3 to 4 d before they were stimulated with phorbol 12-myristate 13-acetate (PMA 100 nmol/L) for 4 to 24 h. PMA treatment significantly decreased alpha, beta, and gammaENaC expression in a time-dependent manner, whereas an inactive form of phorbol ester had no apparent effect. This inhibitory action was seen with only 5-min exposure to PMA, which suggested that PKC activation was very important for the reduction of alphaENaC expression. The PKC inhibitors bisindolylmaleimide at 2 micromol/L and Gö6976 at 2 micromol/L diminished the PMA-induced suppression of alphaENaC expression, while rottlerin at 1 micromol/L had no effect. PMA elicited a decrease in total and amiloride-sensitive current across alveolar epithelial cell monolayers. This decline in amiloride-sensitive current was not blocked by PKC inhibitors except for a partial inhibition with bisindolylmaleimide. PMA induced a decrease in rubidium uptake, indicating potential Na+-K+-ATPase inhibition. However, since ouabain-sensitive current in apically permeabilized epithelial cells was similar in PMA-treated and control cells, the inhibition was most probably related to reduced Na+ entry at the apical surface of the cells. We conclude that PKC activation modulates ENaC expression and probably ENaC activity in alveolar epithelial cells. Ca2+-dependent PKC is potentially involved in this response.
Assuntos
Células Epiteliais/efeitos dos fármacos , Proteína Quinase C/metabolismo , Canais de Sódio/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Amilorida/farmacologia , Animais , Transporte Biológico , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Células Epiteliais/fisiologia , Canais Epiteliais de Sódio , Regulação da Expressão Gênica , Masculino , Proteína Quinase C/antagonistas & inibidores , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/fisiologia , Ratos , Ratos Sprague-Dawley , Radioisótopos de Rubídio/metabolismo , Sódio/metabolismo , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio/genética , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
The present study was undertaken to define the nature of key transport processes for sodium, glucose, proline, and sulfate in primary culture of canine anterior cruciate ligament (ACL) and medial collateral ligament (MCL) cells. Uptake studies using radiolabeled isotopes were performed and Na,K-ATPase activity was determined in cell lysates. At 25 degrees C both ACL and MCL cells showed a significant uptake of 86Rb. Ouabain inhibited Rb uptake by 55% in ACL cells and by 60% in MCL cells. The transport activity of Na,K-ATPase in intact cells was calculated to be 57 and 71 nmol.(mg protein)-1.(15 min)-1, respectively. The enzymatic activity of Na,K-ATPase in cell lysates was observed to be 104 for ACL cells and 121 nmol.(mg protein)-1.(15 min)-1 for MCL cells. Cytochalasin B, a known inhibitor of sodium-independent D-glucose transport, completely inhibited D-glucose uptake in ACL and MCL cells. Removal of Na+ or addition of 10-5 mol/L phlorizin, a potent inhibitor of the sodium-D-glucose cotransporter, did not alter D-glucose uptake, suggesting that glucose entered the cells using a sodium-independent pathway. Both ACL and MCL cells exhibited high sulfate uptake that was not altered by replacement of Na+ by N-methyl-D-glucamine, whereas DIDS, an inhibitor of sulfate/anion exchange abolished sulfate uptake in both cell types. Thus, neither cell type seems to possess a sodium-sulfate cotransport system. Rather, sulfate uptake appeared to be mediated by sulfate/anion exchange. Proline was rapidly taken up by ACL and MCL cells and its uptake was reduced by 85% when Na+ was replaced by N-methyl-D-glucamine, indicating that proline entered the cells via sodium-dependent cotransport systems. The data demonstrate that both ACL and MCL cells possess a highly active sodium pump, a secondary active sodium-proline cotransport system, and sodium-independent transport systems for D-glucose and sulfate.