RESUMO
ON and OFF bipolar cells were identified in the light-adapted carp retina by means of intracellular recording and Lucifer yellow dye injection. The receptive field centers, determined by measuring the response amplitudes obtained by centered spots of different diameters, were 0.3-1.0 mm for ON bipolar cells and 0.3-0.4 mm for OFF bipolar cells. These central receptive field values were much larger than the dendritic field diameters measured by histological methods. Simultaneous intracellular recordings were made from pairs of neighboring bipolar cells. Current of either polarity injected into one member of a bipolar cell pair elicited a sign-conserving, sustained potential change in the other bipolar cell. The coupling efficiency was nearly identical for both depolarizing and hyperpolarizing currents. The maximum separation of coupled bipolar cells was approximately 130 microns. This electrical coupling was reciprocal and summative, and it was observed in cell types of similar function and morphology. Dye coupling was observed in 4 out of 34 stained cells. These results strongly suggest that there is a spatial summation of signals at the level of bipolar cells, which makes their central receptive fields much larger than their dendritic fields.
Assuntos
Retina/fisiologia , Animais , Carpas , Comunicação Celular , Eletrofisiologia , Junções Intercelulares/fisiologia , Estimulação Luminosa , Células Fotorreceptoras/fisiologia , Retina/citologiaRESUMO
Simultaneous intracellular recordings were made from a bipolar cell and a horizontal cell in the carp retina. The properties of the bipolar cell were studied while injecting current into the horizontal cell. Hyperpolarization of horizontal cells, irrespective of their type, elicited a hyperpolarizing response in on-center bipolar cells and a depolarizing response in off-center bipolar cells. Analyses of the ionic mechanisms of bipolar cell responses revealed that depolarization of horizontal cells simulated and hyperpolarization opposed the effect of central illumination. The effect of polarization was exerted in such a manner that each type of horizontal cells modified the transmission from those photoreceptors from which they receive main inputs. In on-center bipolar cells, for example, the L-type horizontal cells receiving inputs mainly from red cones modified the cone-bipolar transmission accompanied by a conductance change of K+ and/or Cl- channels, and the intermediate horizontal cells receiving inputs from rods modified the rod-bipolar transmission accompanied by a conductance change of Na+ channels. In off-center bipolar cells, the effect of polarization of any type of horizontal cells was mediated mainly by conductance changes of Na+ channels. Feedback mechanisms from horizontal cells to photoreceptors could explain these results reasonably well.
Assuntos
Potenciais de Ação , Carpas/fisiologia , Cyprinidae/fisiologia , Canais Iônicos/metabolismo , Retina/fisiologia , Animais , Carpas/metabolismo , Cloretos/metabolismo , Estimulação Elétrica , Técnicas In Vitro , Potássio/metabolismo , Retina/metabolismo , Sódio/metabolismoRESUMO
To elucidate the synaptic transmission between bipolar cells and amacrine cells, the effect of polarization of a bipolar cell on an amacrine cell was examined by simultaneous intracellular recordings from both cells in the isolated carp retina. When either an ON or OFF bipolar cell was depolarized by an extrinsic current step, an ON-OFF amacrine cell was transiently depolarized at the onset of the current but no sustained polarization during the current was detected. The current hyperpolarizing the OFF bipolar cell also produced the transient depolarization of the amacrine cell at the termination of the current. These responses had a latency of approximately 10 ms. The amplitude of the current-evoked responses changed gradually with current intensity within the range used in these experiments. They were affected by polarization of the amacrine cell membrane; the amplitude of the current-evoked responses as well as the light-evoked responses was increased when the amacrine cell membrane was hyperpolarized, while the amplitude was decreased when the cell was depolarized. These results confirm directly that ON-OFF amacrine cells receive excitatory inputs from both ON and OFF bipolar cells: the ON transient is due to inputs from ON bipolar cells, and the OFF transient to inputs from OFF bipolar cells. The steady polarization of bipolar cells is converted into transient signals during the synaptic process.
Assuntos
Retina/citologia , Sinapses/fisiologia , Animais , Eletrofisiologia , Potenciais Evocados , Peixes , Transmissão SinápticaRESUMO
Cholesteryl ester transfer protein (CETP) transfers cholesteryl ester (CE) from HDL to apolipoprotein (apo) B-containing lipoproteins and plays a crucial role in reverse cholesterol transport, which is a major protective system against atherosclerosis. Genetic CETP deficiency is the most common cause of a marked hyperalphalipoproteinemia (HALP) in the Japanese, and various mutations have been identified in the coding region as well as in the exon/intron boundaries in the CETP gene. In the present study, we identified a novel mutation in the promoter region of the CETP gene. This mutation was a G-to-A substitution at the -69 nucleotide of the promoter region (-69 G-->A), corresponding to the second nucleotide of the PEA3/ETS binding site (CGGAA) located upstream of the putative TATA box. Four (2.0%) of 196 unrelated subjects with a marked HALP (HDL cholesterol >/=2.59 mmol/L=100 mg/dL) were revealed to be heterozygous for the -69 G-->A mutation, and the allelic frequency of the mutant was 0.0102 in the subjects with a marked HALP. The subjects with the -69 G-->A mutation had low plasma CETP levels. Reporter gene assay showed that this mutation markedly reduced the transcriptional activities in HepG2 cells (8% of wild type). These results suggested that this mutation would be dominant negative. In conclusion, a novel -69 G-->A mutation in the CETP gene causes the decreased transcriptional activity leading to HALP.
Assuntos
Proteínas de Transporte/genética , Glicoproteínas , Hiperlipoproteinemias/genética , Mutação Puntual , Regiões Promotoras Genéticas , Proteínas de Transporte/sangue , Linhagem Celular , Proteínas de Transferência de Ésteres de Colesterol , Feminino , Frequência do Gene , Humanos , Hiperlipoproteinemias/sangue , Japão , Lipídeos/sangue , Masculino , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Two types of on-center bipolar cells, rod- and cone-dominant bipolars have been identified in the dark-adapted retina of the carp by means of intracellular recordings and Lucifer-yellow dye injection. They differ physiologically and morphologically in the following respects: 1) responses of rod-dominant cells to bright lights are characterized by a transient depolarization followed by a smaller sustained depolarization, while those of cone-dominant cells are approximately rectangular; 2) the cone-dominant cells are about 1.5 log units less sensitive to light than the rod-dominant cells; 3) the latency of the response is shorter in the cone-dominant cells than in the rod-dominant cells; 4) the mean diameters of the cone-dominant cell receptive field (0.7 mm) and dendritic field (90 micrometers) are larger than those of the rod-dominant cell receptive field (0.5 mm) and dendritic field (56 micrometers); 5) the mean diameter of the cone-dominant cell soma (8 micrometers) is smaller than that of the rod-dominant cell soma (13 micrometers); and 6) the terminations of the cone-dominant cell axons form a ramification (67 micrometers mean diameter) in contrast to a big terminal swelling of the rod-dominant cell axons (37 micrometers mean diameters). At least two ionic mechanisms are responsible for generating the depolarizing response of on-center bipolar cells, one having a reversal at a positive potential and the other at a negative potential. Responses with a negative reversal potential only are obtained from some of cone-dominant cells and responses with a positive reversal potential only are obtained from some other cone-dominant cells and the rod-dominant cells. There are a large number of bipolar cells that respond by both ionic mechanisms, although the ratio between them varied considerably in different cells.
Assuntos
Carpas/anatomia & histologia , Cyprinidae/anatomia & histologia , Retina/citologia , Visão Ocular/fisiologia , Animais , Eletrofisiologia , Neurônios/citologia , Neurônios/fisiologia , Células Fotorreceptoras/fisiologia , Retina/anatomia & histologia , Retina/fisiologia , Campos VisuaisRESUMO
Electrical membrane properties of carp off-center bipolar cells were studied by injecting a ramp of current (changing at a rate of about 0.3 nA/s) through one barrel of a double-barreled electrode and recording the voltage drop through the other barrel. The light-elicited response reversed the polarity at a positive membrane potential. The current-voltage curve showed inward and outward rectifications which modified the amplitude of the light-elicited response. The degree of membrane rectifications varied considerably among the cells sampled. Off-center bipolar cells were stained by iontophoretic injections of Lucifer-yellow dye after studying their electrical properties. There was a significant correlation between the difference in cell morphology and the degree of membrane rectification. The cells, which were characterized by small cell bodies lying close to the proximal part of the inner nuclear layer and by thin primary dendrites, tended to show rectifying membrane properties, compared to those characterized by large cell bodies lying close to the distal part of the inner nuclear layer and by thick primary dendrites. A site showing rectifying membrane properties and the origins of a large variability in membrane rectifications are discussed in detail.
Assuntos
Carpas/fisiologia , Cyprinidae/fisiologia , Retina/citologia , Animais , Membrana Celular/fisiologia , Condutividade Elétrica , Corantes Fluorescentes , Potenciais da Membrana , Neurônios/citologia , Neurônios/fisiologia , Retina/fisiologiaRESUMO
On- and off-center bipolar cells were identified in the carp retina by means of intracellular recording, intracellular injection of HRP, and Golgi silver-chromate impregnation. Light and electron microscopy revealed that these functionally different bipolar cells make synaptic contacts with both rods and cones, and that both on- and off-center cells can be further divided into two subtypes (I and II) according to the relationship between the position of their dendritic processes and the synaptic ribbons in the photoreceptor terminal. The type I on-center bipolar cell is characterized by a large cell body, a thick primary dendrite, and a big swelling of the axon terminal in the innermost part of the inner plexiform layer (IPL). Dendritic processes of this cell type make predominantly ribbon contacts with rods and nonribbon contacts with cones. The type II on-center cell, having a large dendritic tree in the outer plexiform layer and a large ramification of the axon terminal extending over the inner part of the IPL makes mostly nonribbon contacts with rods and cones. Many of these type II cell processes, however, terminate very close to cone synaptic ribbons. The type I off-center cell shows two varieties in the axon terminal structure; a large terminal swelling or a large flat ramification of the terminal in the outermost part of the IPL. These cells make predominantly ribbon contacts with rods and cones. Usually, but not always, the process of a type I off-center cell runs parallel to the synaptic ridge apex of cones. The type II off-center cell, showing a large ramification of the axon terminal extending over the outer half of the IPL, makes mainly nonribbon contacts with rods and cones. The results from the HRP-EM study generally agree with those from the Golgi-EM study. A few discrepancies between the results obtained with these two techniques are noted and their implication is discussed.
Assuntos
Peixes/anatomia & histologia , Complexo de Golgi/ultraestrutura , Células Fotorreceptoras/ultraestrutura , Retina/ultraestrutura , Animais , Histocitoquímica , Peroxidase do Rábano Silvestre/metabolismo , Microscopia EletrônicaRESUMO
The responses of a ganglion and an amacrine cell were recorded simultaneously in the carp retina. Sinusoidal current injected into amacrine cells modulated ganglion cell discharges either in phase (excitation) or in opposite phase (inhibition). ON-center ganglion cells received excitatory inputs and OFF-center ganglion cells received inhibitory inputs from ON-center amacrine cells. They received inputs of opposite polarity from OFF-center amacrine cells. Namely, inputs from ON-center and OFF-center amacrine cells augment the responses of ON-center and OFF-center ganglion cells in a push-pull manner.
Assuntos
Carpas/fisiologia , Retina/fisiologia , Células Ganglionares da Retina/fisiologia , Animais , Polaridade Celular , Eletrofisiologia , Técnicas In Vitro , Inibição Neural/fisiologia , Estimulação Luminosa , Retina/citologiaRESUMO
We describe two physiologically and morphologically distinct types of on-center bipolar cell in the carp retina. These cells can be distinguished from one another in the following respects: (1) "type I" cells respond to moderate light intensities with a transient depolarization followed by a plateau, while the response of "type II" cells is approximately rectangular in shape; (2) latency of type II cell responses is shorter than that of type I cell responses; (3) mean diameter of type II cell dendritic fields (114 micron) is larger than that of type I cell dendritic fields (65 micron) in the outer plexiform layer; (4) mean diameter of type II cell bodies (7 micron) is smaller than that of type I cell bodies (9 micron); (5) axons of type II cells show extensive arborizations (51 micron), while those of type I cells show a single swelling with a few short collaterals (31 micron) in the proximal half of inner plexiform layer. HRP-injected on- and off-center bipolars were examined by electron microscopy. The dendrites of these bipolar cells invaginate into rod spherules and often occupy the central position of the triad. The dendrites of such bipolar cells also invaginate into cone pedicles, but in quite distinct patterns. Type I on-center bipolars do not send processes to the synaptic ribbons. In contrast, off-center bipolars and type II on-center bipolars send processes to the synaptic ribbons. The dendrites of off-center bipolars occupy the central position of the triad. Although type II on-center bipolar dendrites approach the ribbon very closely, there is usually a process from an off-center bipolar interposed between them.
Assuntos
Carpas/anatomia & histologia , Cyprinidae/anatomia & histologia , Células Fotorreceptoras/ultraestrutura , Retina/ultraestrutura , Sinapses/ultraestrutura , Animais , Dendritos/ultraestrutura , Peroxidase do Rábano Silvestre , Microscopia Eletrônica , Neurônios/ultraestrutura , Retina/citologiaRESUMO
Cadmium mainly accumulates in the kidney and causes renal injury. To clarify the mechanism of Cd nephrotoxicity, we investigated the effects of this element on intercellular communication through gap junction channels in primary cultures of rat renal proximal tubular cells. Sixty minutes after exposure to 100 microM Cd, dye coupling experiments showed that gap junctional intercellular communication (GJIC) was significantly inhibited. This inhibition occurred before the appearance of cytotoxicity. Intracellular calcium concentrations [Ca2+]i, which modulate the function of gap junctions, gradually increased after exposure to Cd and reached a maximum after 60 minutes. These results suggest that the inhibition of GJIC as a result of Cd exposure is related to an increase in [Ca2+]i, and that GJIC inhibition may be an indicator of nephrotoxicity.
Assuntos
Cloreto de Cádmio/toxicidade , Junções Comunicantes/efeitos dos fármacos , Túbulos Renais Proximais/efeitos dos fármacos , Acetilglucosaminidase/metabolismo , Animais , Comunicação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Imunofluorescência , Junções Comunicantes/ultraestrutura , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , L-Lactato Desidrogenase/metabolismo , Masculino , Nitratos/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
Intracellular recordings were made simultaneously from pairs of neighboring bipolar cells by advancing two independent microelectrodes into retinas of carp (Cyprinus carpio). Bipolar cells were identified by their response properties and in several samples were verified by intracellular injection of Lucifer yellow. Current of either polarity injected into one member of the bipolar cell pair elicited a signconserving, sustained potential change in the other bipolar cell without any significant delay. This electrical coupling was reciprocal, and it was observed between cell types similar in function and in morphology. Our results strongly suggest that there is a spatial summation of signals at the level of bipolar cells, which makes central receptive field areas much larger than their dendritic fields.
RESUMO
The physiological and morphological properties of color-opponent bipolar cells in the carp retina were studied. Fifty nine OFF-center bipolar cells and 63 ON-center bipolar cells out of about 500 total bipolar cells recorded showed color-opponent responses. The OFF-center color-opponent bipolar cells were classified into three subgroups according to their spectral and spatial responses. Fifty OFF-center color-opponent cells responded with depolarization to a blue light spot and with hyperpolarization to a red spot in the receptive-field center. The polarity of the surround response was opposite to that of center response at each wavelength. Therefore these cells were classified as OFF double-opponent cells (OFF-DO). Eight cells responded with hyperpolarization to a blue and green spot and with depolarization to a red spot. The surround responses of those cells were depolarizing at any wavelength (R+G- cell). One responded with hyperpolarization to a blue and red spot and with depolarization to a green spot. The surround response showed a different spectral characteristic from that of the center response. It responded with depolarization to a blue and green annulus and with hyperpolarization to a red annulus (R-G+B- cell). The ON-center color-opponent bipolar cells were similarly classified into three subgroups. Sixty of ON-center color-opponent cells were the double color-opponent type (ON-DO cell), showing the responses of opposite polarity to the OFF-DO cells. Two cells were classified as R- G+ cell, and one cell as R+G-B+ cell. Both OFF- and ON-DO cells were identified by their morphology as Cajal's giant bipolar cells, and R+G-, R-G+, R-G+B-, and R+G-B+ cells as Cajal's small bipolar cells. The analysis of the latency and the ionic mechanisms of their responses suggest that DO cells under light-adapted conditions receive direct inputs from long-wavelength (red) cones, RG cells from middle-wavelength (green) cones, and RGB cells from short-wavelength (blue) cones. Possible mechanisms of the opponent inputs to these bipolar cells are discussed.
Assuntos
Carpas/fisiologia , Percepção de Cores/fisiologia , Interneurônios/fisiologia , Retina/fisiologia , Animais , Membrana Celular/fisiologia , Peroxidase do Rábano Silvestre , Interneurônios/citologia , Potenciais da Membrana , Retina/citologiaRESUMO
Newly prepared phosphatidylethanolamine (PE) conjugates of glycosaminoglycans (GAGs) enabled us to immobilize GAGs to solid phase through hydrophobic interaction. Using these compounds, called GAG-PEs, we studied the effects of GAGs immobilized to culture plates coated with various extracellular matrix (ECM) proteins in terms of cell-substrate adhesion, DNA synthesis, tyrosine aminotransferase (TAT) induction, and intercellular communication in primary rat hepatocytes. Treatment with chondroitin sulfate (CS)-PE at 10 micrograms/ml made laminin, fibronectin, vitronectin, and collagens type I-V less adhesive as substrates for cell attachment and inhibited cell spreading on these substrates. The effect on cell attachment was lost after long incubations over 2 h. Dermatan sulfate (DS)-PE was also inhibitory, but less effective. The conjugates of heparan sulfate (HS), heparin, and hyaluronan were much less effective. DNA synthesis initiated by EGF in the culture on laminin substrates was inhibited most strongly by CS-PE, as well as by DS-PE and hyaluronan-PE, but not by either HS-PE or heparin-PE. With type I collagen substrates, GAG-PEs had similar effects but to lesser extent. TAT induction in the culture on laminin substrates but not on type I collagen substrates was significantly enhanced by CS-PE. In terms of DNA synthesis and TAT induction, the culture on laminin substrates treated with CS-PE was comparable to that at higher cell density on the non-treated laminin substrates. Intercellular communication assessed by dye coupling was maintained longer on the substrates treated with CS-PE. Taken together, our results demonstrate that CS immobilized especially onto laminin substrates inhibits the growth of hepatocytes and enhances their differentiated functions by modulating cell-ECM protein interactions.
Assuntos
Comunicação Celular/efeitos dos fármacos , Sulfatos de Condroitina/farmacologia , Replicação do DNA/efeitos dos fármacos , Fígado/metabolismo , Tirosina Transaminase/biossíntese , Animais , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Sulfatos de Condroitina/fisiologia , Proteínas da Matriz Extracelular/farmacologia , Proteínas da Matriz Extracelular/fisiologia , Masculino , Ratos , Ratos WistarRESUMO
Methylmercury (MeHg) causes renal injury in addition to central and peripheral neuropathy. To clarify the mechanism of nephrotoxicity by MeHg, we investigated the effect of this compound on intercellular communication through gap junction channels in primary cultures of rat renal proximal tubular cells. Twenty minutes after exposure to 30 microM MeHg, gap junctional intercellular communication (GJIC), which was assessed by dye coupling, was markedly inhibited before appearance of cytotoxicity. When the medium containing MeHg was exchanged with MeHg-free medium, dye coupling recovered abruptly. However, the dye-coupling was abolished again 30 min after replacement with control medium, and the cells were damaged. Intracellular calcium concentration, [Ca2+]i, which modulates the function of gap junctions, significantly increased following exposure of the cells to 30 microM MeHg and returned to control level following replacement with MeHg-free medium. These results suggest that the inhibiting effect of MeHg on GJIC is related to the change in [Ca2+]i, and may be involved in the pathogenesis of renal dysfunction.
Assuntos
Cálcio/metabolismo , Junções Comunicantes/efeitos dos fármacos , Túbulos Renais Proximais/efeitos dos fármacos , Compostos de Metilmercúrio/toxicidade , Óxido Nítrico/biossíntese , Animais , Comunicação Celular , Células Cultivadas , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Serum paraoxonase (PON) is associated with plasma high density lipoproteins, and prevents the oxidative modification of low density lipoproteins. We have developed a sensitive sandwich enzyme-linked immunosorbent assay (ELISA), using two monoclonal antibodies against PON, to measure serum PON concentration. The concentration of PON in healthy Japanese subjects was 59.3 +/- 1.3 microgram/mL (mean +/- SEM; n = 87). Serum PON concentrations in relation to the PON 192 genetic polymorphism were: 69.5 +/- 2.9 microgram/mL in the QQ genotype; 63.0 +/- 1.9 microgram/mL in the QR genotype; and 52.8 +/- 1.7 microgram/mL in the RR genotype. Concentrations were significantly lower in the RR than in the QQ genotype (P < 0.01). Serum paraoxonase specific activity was higher in RR than in QQ subjects (18.6 +/- 0.40 vs. 2. 56 +/- 0.05 nmol/min/microgram, P < 0.01), but arylesterase specific activity was unrelated to genotype. PON concentration was positively associated (P < 0.001) with both serum arylesterase activity and, after adjusting for the effect of the position 192 polymorphism, with serum paraoxonase activity. Subjects with angiographically verified coronary heart disease had significantly lower PON concentrations than the healthy controls (52.0 +/- 2.3 microgram/mL; n = 35, P < 0.01). This association was independent of the position 192 genotype. Our new ELISA should be of value for epidemiologic and clinical studies of serum PON concentration. immunosorbent assay for human serum paraoxonase concentration.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Esterases/sangue , Idoso , Animais , Anticorpos Monoclonais , Arildialquilfosfatase , HDL-Colesterol/sangue , Doença das Coronárias/enzimologia , Esterases/genética , Feminino , Genótipo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Polimorfismo Genético , Valores de Referência , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Plasma phospholipid transfer protein (PLTP) plays a central role in the remodeling of HDLs. Reliable and accurate methods for assaying PLTP concentration are required. METHODS: A sandwich ELISA for PLTP has been developed, using two monoclonal antibodies against recombinant human PLTP (rhPLTP) expressed in Chinese hamster ovary cells. The ELISA allows for the quantification of PLTP in the range 0.625-15.0 ng/assay (1.2-30.0 mg/L). Intra- and interassay CVs were <3.0% and <4.2% respectively. The assay was used to quantify plasma PLTP concentrations in 132 Japanese subjects (75 males and 57 females). RESULTS: PLTP concentrations were 12.0 +/- 3. 0 mg/L (mean +/- SD; range, 4.9-20.5 mg/L). No sex difference was observed. Plasma PLTP concentration was positively correlated with HDL-cholesterol (r = 0.72; P: <0.001), apolipoprotein (apo) A-I (r = 0.62; P: <0.001) and HDL(2)-cholesterol (r = 0.72; P: <0.001), and was negatively correlated with triacylglycerol (r = -0.45; P: <0. 001). There was no correlation with plasma apo A-II. These results agree with other evidence that plasma PLTP is associated with large apo A-I-containing lipoproteins. There was no correlation (r = -0. 01) between plasma PLTP and plasma phosphatidylcholine transfer activity (range, 3.5-10.5 micromol. mL(-1). h(-1)), suggesting that PLTP may exist in active and inactive forms. CONCLUSION: This new ELISA will be of value for further studies of PLTP in health and disease.
Assuntos
Proteínas de Transporte/sangue , Proteínas de Membrana/sangue , Proteínas de Transferência de Fosfolipídeos , Fosfolipídeos/metabolismo , Adulto , Animais , Anticorpos Monoclonais , Células CHO , Calibragem , Proteínas de Transporte/imunologia , Proteínas de Transporte/metabolismo , Cricetinae , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Humanos , Masculino , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Proteínas Recombinantes/imunologiaRESUMO
Plasma phospholipid transfer protein (PLTP) plays an important role in the maintenance of plasma high-density lipoprotein (HDL) content and remodeling of HDL in the circulation. In the present study we have used different fractionation methods to investigate the distribution of PLTP in human plasma. A novel enzyme-linked immunosorbent assay developed during the study allowed for simultaneous assessment of both PLTP mass and activity in the fractions obtained. Size-exclusion chromatography and plasma fractionation by nondenaturing polyacrylamide gel electrophoresis (PAGE) yielded similar results demonstrating that PLTP associates in native plasma with two distinct particle populations, while ultracentrifugation with high salt leads to detachment of PLTP from lipoprotein particles and loss of a majority of its phospholipid transfer activity. Interestingly, analysis of the size-exclusion chromatography fractions demonstrated that PLTP exists in the circulation as an active population that elutes in the position of HDL corresponding to an average molecular mass of 160+/-40 kDa and an inactive form with an average mass of 520+/-120 kDa. The inactive fraction containing approximately 70% of the total PLTP protein eluted between HDL and low density lipoprotein (LDL). Thus, the two PLTP pools are associated with different types of lipoprotein particles, suggesting that the PLTP activity in circulation is modulated by the plasma lipoprotein profile and lipid composition.