RESUMO
Routine determination of mutations in cystic fibrosis requires accurate, rapid, reliable and low-cost methods, permitting the simultaneous detection of multiple mutations. The Elucigene CF20 kit developped by Cellmark Diagnostics, uses multiplex ARMS, which allows the screening for 20 CFTR gene mutations (deltaF508, G542X, N1303K, 1717-1G>A, G551D, W1282X, R553X, deltaI507, 1078delT, 2183AA>G, 3849+10kbC>T, R1162X, 621+1G>T, R334W, R347P, 3659delC, R117H, S1251N, E60X, A455E ) in a work day without specific instrumentation. The kit distinguishes between homozygotes and heterozygotes for deltaF508, but not for rare mutations. The kit detects from 68 to 92% of defective alleles in Caucasians. We evaluate the kit in a blind study in two independent laboratories. Thirty blood samples and thirty mouthwash samples from CF patients, carriers and unaffected individuals were analysed by the Elucigene CF20 kit. All the samples were previously analysed by denaturing gradient gel electrophoresis and sequencing. The Elucigene CF20 kit consists of three multiplexes. Each mutiplex contains ARMS specific primers for six to eight mutations and two control reactions. The absence of the upper control fragment indicates that a repeat test is required. We demonstrated a first time amplification rate of 98.3%: of the 60 samples tested, one required a reamplification. Results compared with the reference method demonstrated that in all cases where one or more of the 20 mutations detected by the kit were present in the test set, the kit accurately identified them. Reproducibility was assessed by repeating the analysis of a blood and mouthwash sample five times. Cross reactivity between R117C and R117H, R117P and R117H, R347P and R347H, deltaI507 and deltaF508, G551D and R553X were evaluated. Only a cross reactivity between R347P and R347H was observed. The kit is specially useful for first line study of patients and carrier identification.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/diagnóstico , Fibrose Cística/genética , Testes Genéticos/métodos , Mutação , Substituição de Aminoácidos , Fibrose Cística/sangue , DNA/sangue , DNA/genética , Testes Genéticos/instrumentação , Humanos , Laboratórios , Mucosa Bucal/patologia , Kit de Reagentes para Diagnóstico , Reprodutibilidade dos Testes , Deleção de Sequência , População Branca/genéticaRESUMO
Clonidine and benazoline are two structurally related imidazolines. Whereas clonidine binds both to alpha(2)-adrenoceptors (alpha(2)R) and to I(1) imidazoline receptors (I(1)R), benazoline showed a high selectivity for imidazoline receptors. Although the alpha(2)R are negatively coupled to adenylate cyclase, no effect on cAMP level by activation of I(1)R has been reported so far. We therefore aimed to compare the effects of clonidine and benazoline on forskolin-stimulated cAMP levels in cell lines expressing either I(1)R only (PC12 cells), alpha(2)R only (HT29 cells), or I(1)R and alpha(2)R together (NG10815 cells). Clonidine proved able to decrease the forskolin-stimulated cAMP level in the cells expressing alpha(2)R and this effect could be blocked by rauwolscine. In contrast, in cells lacking these adrenoceptors, clonidine had no effect. On the other hand, benazoline and other I(1) receptor-selective imidazolines decreased forskolin-stimulated cAMP level in the cells expressing I(1)R, in a rauwolscine- and pertussis toxin-insensitive manner. These effects were antagonized by clonidine. According to these results, we demonstrated that 1) alpha(2)R and I(1)R are definitely different entities because they are expressed independently in different cell lines; 2) alpha(2)R and I(1)R are both implicated in the cAMP pathway in cells (one is sensitive to pertussis toxin and the other is not); and 3) I(1)R might be coupled to more then one transduction pathway. These new data will be essential to further understand the physiological implications of the I(1)R and the functional interactions between I(1) receptors and alpha(2)-adrenoceptors.
Assuntos
AMP Cíclico/metabolismo , Imidazóis/farmacologia , Receptores de Droga/metabolismo , Toxina Adenilato Ciclase , Agonistas alfa-Adrenérgicos/farmacologia , Antagonistas Adrenérgicos alfa/farmacologia , Animais , Clonidina/farmacologia , Células HT29 , Humanos , Receptores de Imidazolinas , Ligantes , Células PC12 , Toxina Pertussis , Ratos , Receptores Adrenérgicos alfa 2/metabolismo , Fatores de Virulência de Bordetella/farmacologia , Ioimbina/farmacologiaAssuntos
Receptores Adrenérgicos alfa 2/metabolismo , Receptores de Droga/metabolismo , Animais , Ligação Competitiva , Membrana Celular/metabolismo , Clonidina/metabolismo , Glioma , Células Híbridas , Idazoxano/metabolismo , Receptores de Imidazolinas , Cinética , Neuroblastoma , Neuroglia , Receptores Adrenérgicos alfa 2/genética , Receptores de Droga/genética , Ioimbina/metabolismoRESUMO
I1 imidazoline receptors (I1R) were defined as receptors insensitive to catecholamines and highly sensitive to [3H]clonidine and analogs. By contrast, the I2R subtype is more sensitive to [3H]idazoxan. [3H]clonidine and [3H]idazoxan imidazoline specific binding sites (IBS) have been detected in crude human membranes. Pharmacologic characterization by binding assays clearly differentiates IBS from alpha 2-adrenoceptors, whereas differences between [3H]clonidine and [3H]idazoxan IBS are less clear in crude preparations. In fact, only moderate affinity for [3H]clonidine was detectable in such preparations. However, purification procedures allowed detection of high affinity [3H]clonidine IBS in the human brain, corresponding to the I1R. Difficulties in the characterization of the I1R in crude membranes are due to multiple factors including heterogeneity of IBS, their low Bmax value, the existence of allosteric modulation, and possibly the presence of natural binding inhibitors. Immunologic studies with specific anti-idiotypic antibodies revealed a 43-kD protein as the best candidate for I1R as binding activity coincides with immunodetection. No cross-reaction was found with anti-monoamine oxidase (MAO) A/B antibodies and the 43-kD protein, ruling out the possibility of this protein being an MAO-associated I2R. Neither anti-alpha 2A- nor anti-alpha 2B-specific antibodies were able to immunodetect the 43-kD protein in crude membrane preparations or in purified fractions. These results and further biochemical characterization (pHi, N-glycosylation) of the 43-kD protein definitely assessed that human brain I1R and alpha 2-adrenoceptors clearly differ physically. However, coexpression of I1R and alpha 2-adrenoceptors in synaptic plasma membranes of the bovine brainstem reinforce the possibility of a functional relationship between the two types of receptor.
Assuntos
Encéfalo/metabolismo , Clonidina/farmacocinética , Idazoxano/farmacocinética , Receptores Adrenérgicos alfa 2/metabolismo , Receptores de Droga/análise , Receptores de Droga/metabolismo , Animais , Sítios de Ligação , Bovinos , Humanos , Receptores de Imidazolinas , Cinética , Especificidade de Órgãos , CoelhosRESUMO
This study investigated the possible active principles which support the endothelial nitric oxide-dependent relaxation produced by red wine and other plant polyphenolic compounds in thoracic aorta from male Wistar rats (12-14 wk old). Relaxation experiments were recorded isometrically on vessels precontracted with norepinephrine. Ten different chromatographic fractions (3-18 mg) isolated from red wine polyphenolic compounds (RWPC) and some available defined polyphenols (10-15 mg) were tested. Fractions enriched into either anthocyanins or oligomeric condensed tannins exhibited endothelium-dependent vasorelaxant activity (maximal relaxation in the range of 59-77%) comparable to the original RWPC. However, polymeric condensed tannins elicited a weaker vasorelaxant activity than the original RWPC (maximal relaxation ranged between 20-47%, P < 0.01). Moreover, the representative of either phenolic acid derivatives (benzoic acid, vanillic acid, gallic acid), hydroxycinnamic acid (p-coumaric acid, caffeic acid) or the flavanol [(+)-epicatechin] classes failed to induce this type of response. Among the anthocyanins, delphinidin (maximal relaxation being 89%), but not malvidin or cyanidin, showed endothelium-dependent vasorelaxation. These results show that anthocyanins and oligomeric-condensed tannins exhibited a pharmacological profile comparable to the original RWPC. These compounds may be involved in the reduction of cardiovascular mortality related to the presence of wine, fruits and vegetables in the diet.
Assuntos
Flavonoides , Músculo Liso Vascular/efeitos dos fármacos , Óxido Nítrico , Fenóis/farmacologia , Polímeros/farmacologia , Vasodilatação/efeitos dos fármacos , Animais , Aorta Torácica , Dieta , Relação Dose-Resposta a Droga , Masculino , Fenóis/administração & dosagem , Fenóis/isolamento & purificação , Polímeros/administração & dosagem , Polímeros/isolamento & purificação , Polifenóis , Ratos , Ratos Wistar , Relação Estrutura-Atividade , VinhoAssuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Kit de Reagentes para Diagnóstico , Fibrose Cística/sangue , Regulador de Condutância Transmembrana em Fibrose Cística/sangue , DNA/sangue , DNA/genética , Humanos , Mutação , Reprodutibilidade dos TestesRESUMO
The relationship between Rap1 proteins and cell proliferation was assessed by investigating the effect of isoforms AA and BB of platelet-derived growth factor (PDGF) on Rap1 protein and mRNA expression throughout the smooth muscle cell cycle. Firstly, PDGF BB-induced cell cycle traverse was studied, thus demonstrating entry into S phase at 18 to 20 h. Western blotting carried out on total Rap1 proteins showed that 5 ng/ml of PDGF BB instigated a biphasic induction of total Rap1 proteins during the cell cycle. This involved a 2.1 +/- 0.4-fold increase at 6 h (early G1) and a 2.8 +/- 0.6-fold increase at 20 to 24 h (G1/S transition). Such an up-regulation was abolished by addition of 1 ng/ml of transforming growth factor-beta 1 (TGF-beta 1), which inhibited up to 80% of the PDGF BB-induced entry into S phase. Comparative RT-PCR of both rap1a and rap1b mRNAs throughout the cell cycle allowed us to differentiate between the two rap1a and rap1b species. PDGF BB induced a 1.9 +/- 0.3-fold increase at 4 h and a 2.4 +/- 0.2-fold relative increase at 16 h for rap1b mRNA, whereas a unique 1.9 +/- 0.5-fold increase in rap1a mRNA was observed at 14 h. Again, this induction of rap1a and rap1b mRNAs by PDGF BB was totally abolished by TGF-beta 1. We conclude that the differential up-regulation of Rap1a and Rap1b proteins during the smooth muscle cell cycle is directly linked to cell proliferation.
Assuntos
Ciclo Celular/fisiologia , Proteínas de Ligação ao GTP/genética , Músculo Liso Vascular/química , Proteínas Proto-Oncogênicas/genética , Animais , Anticoagulantes/farmacologia , Aorta/citologia , Becaplermina , Southern Blotting , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Cinética , Músculo Liso Vascular/citologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-sis , RNA Mensageiro/metabolismo , Fase S/efeitos dos fármacos , Fase S/genética , Suínos , Fator de Crescimento Transformador beta/farmacologia , Regulação para Cima/fisiologia , Proteínas rap de Ligação ao GTPRESUMO
Although the interrelationship between the two messengers Ca2+ and cyclic AMP in platelet function is well documented, its mechanism of action still remains to be established. We investigated here the question of the regulation of platelet Ca(2+)-ATPases by cyclic AMP through the phosphorylation of the Rap1 protein using a pathological model. We first found experimental conditions where Ca(2+)-transport by platelet membrane vesicles appeared to be dependent on the phosphorylation of the Rap1 protein. Then, we studied platelets of patients with congestive heart failure for their expression of the potential 97 kDa Ca(2+)-ATPase target of regulation through the Rap1 protein as well as the phosphorylation of the Rap1 protein using the catalytic subunit of the cyclic AMP-dependent protein kinase (C. Sub.). In the first patients studied, we found no significant modification in the expression of the 97 kDa Ca(2+)-ATPase by Western blotting using the PL/IM 430 monoclonal antibody which specifically recognized this isoform. In contrast, the Rap1 protein was differentially phosphorylated when using 15 micrograms/ml of the C. Sub. These results allowed us to use these pathological platelets to study the relationship between the expression of Rap1 protein and the regulation of Ca2+ transport by selecting a patient with severe heart failure. We could show a decrease in the expression as well as in the phosphorylation of Rap1 protein and demonstrate a lower effect of C. Sub. on Ca2+ transport. Finally, by studying a further series of patients, we could confirm that the decrease in Rap1 protein expression in heart failure, whatever its extent, was variable, and could strictly correlate the expression of Rap1 protein with the stimulatory effect of C. Sub. on Ca2+ transport. Besides the evidence for regulation of the expression of the Rap1 protein in platelets from patients with heart failure, these findings constitute a new approach in favour of the regulation of platelet Ca2+ transport through the phosphorylation of the Rap1 protein.
Assuntos
Plaquetas/metabolismo , Cálcio/sangue , Proteínas de Ligação ao GTP/metabolismo , Insuficiência Cardíaca/sangue , Trifosfato de Adenosina/sangue , Adulto , Transporte Biológico , Western Blotting , Membrana Celular/metabolismo , AMP Cíclico/sangue , Proteínas de Ligação ao GTP/biossíntese , Proteínas de Ligação ao GTP/isolamento & purificação , Expressão Gênica , Humanos , Cinética , Fosfoproteínas/sangue , Fosfoproteínas/isolamento & purificação , Fosforilação , Valores de Referência , Fatores de Transcrição/sangue , Proteínas rap de Ligação ao GTPRESUMO
1. The contribution of sarco-endoplasmic reticulum Ca(2+)-ATPases (SERCA)-regulated Ca2+ stores to the increase in intracellular free calcium ([Ca2+]i) induced by bradykinin (BK) was investigated in fura-2 loaded human tracheal smooth muscle cells (TSMC). For this purpose, we used thapsigargin, a selective inhibitor of Ca(2+)-ATPases of intracellular organelles. 2. Thapsigargin (10(-9) to 10(-6) M) induced a dose-dependent increase in [Ca2+]i in the presence of external Ca2+ with an EC50 value of 7.33 +/- 1.26 nM. In Ca(2+)-free conditions, the addition of Ca2+ (1.25 mM) caused an increase in [Ca2+]i which was directly proportional to the pre-incubation time of the cells with thapsigargin. Net increases of 60 +/- 9, 150 +/- 22 and 210 +/- 27 nM were obtained after 1, 3 and 5 min, respectively. 3. In the presence of extracellular Ca2+, BK induced a typical biphasic increase in [Ca2+]i with a fast transient phase and a sustained phase. The sustained component was reversed by addition of a bradykinin B2-receptor antagonist (Hoe 140, 10(-6) M) to the buffer as well as by deprivation of Ca2+. The transient phase induced by BK, histamine and carbachol was inhibited in a time-dependent way by preincubation of the cells with thapsigargin. 4. Comparative western blotting of human TSMC membranes using anti-SERCA2 isoform-specific antibodies clearly showed the greater expression of the 100-kDa SERCA2-b isoform compared with the SERCA2-a isoform. 5. Our data show that thapsigargin-sensitive Ca2+ stores contribute significantly to the activation of human TSMC which suggests a role for these stores in the subsequent induction of Ca2+ influx. These stores appear to be controlled by the Ca2+-ATPases (SERCA2-b isoform) which could also participate in the regulation of Ca2+ influx through the plasma membrane.
Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Cálcio/metabolismo , Músculo Liso/metabolismo , Retículo Sarcoplasmático/enzimologia , Traqueia/metabolismo , Transporte Biológico , Bradicinina/análogos & derivados , Bradicinina/farmacologia , Antagonistas dos Receptores da Bradicinina , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Carbacol/farmacologia , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Histamina/farmacologia , Humanos , Músculo Liso/citologia , Receptor B2 da Bradicinina , Terpenos/farmacologia , Tapsigargina , Traqueia/citologiaRESUMO
The effect of platelet-derived growth factor (PDGF) on Rap1 expression was investigated in rat vascular smooth muscle cells (SMC). First, evidence for Rap1 proteins was shown by their: (i) detection in membranes using a specific anti-Rap1 antibody, (ii) typical shift in electrophoretic mobility as a consequence of reduction, and (iii) cAMP-induced phosphorylation and immunoprecipitation. Then, the mitogenic activity of 10 ng/ml PDGF AA and BB for 48 h, resulting in a 2- and 5-fold increase in [3H]thymidine incorporation, was correlated with that of total Rap1 protein expression which was found to be 99% +/- 36% and 260% +/- 70%, respectively. Further time-course studies established that this up-regulation of Rap1 proteins was only observed after 48 h of PDGF BB treatment. Lastly, comparative RT-PCR of both rap1a and rap1b mRNAs showed that PDGF BB also up-regulated the rap1a mRNA species, which was 1.5-fold increased in contrast with the rap1b mRNA species. It is concluded that the PDGF BB-induced SMC proliferation is associated with an up-regulation of Rap1a protein.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Animais , Becaplermina , Células Cultivadas , DNA/biossíntese , Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Cinética , Proteínas Proto-Oncogênicas c-sis , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Proteínas Recombinantes/farmacologia , Timidina/metabolismo , Proteínas rap de Ligação ao GTPRESUMO
Two sarco/endoplasmic reticulum Ca(2+)-ATPases (SERCAs) have been previously identified in platelets: the 100-kDa SERCA2b and the 97-kDa SERCA3 isoforms. Analysis of the acylphosphate intermediate (E-P) formation and the immunoreactivity of the platelet Ca(2+)-ATPases and their proteolytic fragments upon controlled trypsinolysis revealed the presence of an additional 97-kDa Ca(2+)-ATPase that comigrates with SERCA3 on SDS-polyacrylamide gels. At a trypsin/membrane protein ratio of 0.025 at 4 degrees C, tryptic fragments of 73-, 68- and 40-kDa, previously unknown in the SERCA family, could be detected by using the PL/IM 430 anti-Ca(2+)-ATPase antibody that had been shown to recognize a 97-kDa Ca(2+)-ATPase. The 73- and 68-kDa fragments were precursors of the 40-kDa one. Ca(2+)-dependent phospholabeling of the 73-kDa fragment and immunostaining of all these proteolytic products by another antibody raised against SERCA1 established the SERCA nature of the 97-kDa parent enzyme. The SERCA3-related E-P-forming 80-kDa tryptic fragment appeared during trypsinolysis with a different time course from that of the 73-, 68-, and 40-kDa ones. At a trypsin/membrane protein ratio of 0.125 at 37 degrees C, it reached its maximum level at 5 min of digestion, while the 73-, 68-, and 40-kDa fragments were fully degraded at 2 min of trypsinization. This 80-kDa species was immunostained neither with the PL/IM 430, nor with the anti-SERCA1 antibodies. Similar results were found in some megakaryoblastoid and lymphoblastoid cell lines. All these data indicate the presence of two distinct tryptic fragmentation patterns attributed to two 97-kDa SERCA isoforms and point to the existence of a multi-SERCA system in different human non-muscle cells.
Assuntos
Plaquetas/enzimologia , ATPases Transportadoras de Cálcio/metabolismo , Retículo Endoplasmático/enzimologia , Retículo Sarcoplasmático/enzimologia , Membrana Celular/enzimologia , Células Cultivadas , Humanos , Hidrólise , Isoenzimas/metabolismo , Cinética , Mapeamento de Peptídeos , Tripsina , Células Tumorais CultivadasRESUMO
We previously demonstrated abnormal Ca2+ transport by microsomes in platelets from a grey platelet syndrome patient. Here, we investigated the platelet Ca2+ ATPases that mediate this transport, as well as its possible regulation by rap 1 protein. We showed that grey platelet syndrome platelets expressed the same two distinct Ca2+ ATPases as those recently described in normal platelets; the 100 kD SERCA2-b isoform (Sarco/Endoplasmic Reticulum Ca2+ATPase) and a new 97 kD SERCA isoform. The two Ca2+ATPases formed similar amounts of transient phosphorylated intermediates. The expression of these two Ca2+ATPases was compared by Western blotting using specific antibodies, which again emerged in similar amounts in normal and grey platelet syndrome platelets. As regards the protein phosphorylated by cAMP, it was found to be identical to rap 1 protein when it was immunoprecipitated with an antibody raised against a synthetic peptide specific for rap 1 protein. Although the expression of rap 1 protein was similar in membranes isolated from grey platelet syndrome and normal platelets, its exogenous phosphorylation by cAMP was abnormal, with a concentration (10 micrograms/ml) of the catalytic subunits of the cAMP-dependent protein kinase (C.Sub.), as it decreased to half the control level. It is concluded that the abnormal Ca2+ transport found in grey platelet syndrome platelets is not due to the abnormal expression of the Ca2+ATPases, but is associated with an abnormality of rap 1 protein phosphorylation by cAMP.
Assuntos
Transtornos Plaquetários/sangue , Plaquetas/metabolismo , AMP Cíclico/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Adulto , Plaquetas/enzimologia , Western Blotting , ATPases Transportadoras de Cálcio/sangue , Membrana Celular/metabolismo , Humanos , Fosforilação , Síndrome , Proteínas rap de Ligação ao GTPRESUMO
We recently showed that human and rat platelets express two types of SERCAs (Sarco Endoplasmic Reticulum Ca2+ATPases): a 100-kDa SERCA2b isoform and a 97-kDa SERCA isoform. Here, we explored the possibility that the rat 97-kDa isoform is identical to the SERCA3 protein. For this purpose, we first attempted to detect SERCA3 mRNA in rat platelet total RNA by reverse transcription-polymerase chain reaction using SERCA3-specific primers, and demonstrated the presence of this mRNA species by sequencing the amplification product. We then searched for a relationship between the expression of the SERCA3 mRNA and of the 97-kDa protein using either rat aortic smooth muscle cells, previously found not to express the 97-kDa SERCA isoform (negative model), or platelets of spontaneously hypertensive rats (SHR), which overexpress this isoform (overexpression model) but express the 100-kDa SERCA2b isoform normally. No expression of SERCA3 mRNA was detectable by analysis of smooth muscle cell RNA, but comparison by reverse transcription-polymerase chain reaction of the SERCA2b and SERCA3 mRNAs from the platelets of normotensive (Wistar-Kyoto, WKY) rats and SHR clearly demonstrated a 238 +/- 43% increase in the expression of the SERCA3 mRNA in SHR platelets only. Last, by comparative Western blotting of WKY rat and SHR platelet membranes using a recently developed polyclonal anti-SERCA3 antibody, we established that the 97-kDa SERCA and the SERCA3 protein are identical, as immunostaining of the 97-kDa protein revealed a 230 +/- 25% increase in the expression of this protein in SHR versus WKY rat platelets. It is concluded that the 97-kDa platelet SERCA isoform, which is up-regulated in SHR, is the SERCA3 protein. As far as we know, this constitutes the first demonstration of the actual presence of this Ca2+ATPase isoform in normal cells, in addition to the artificial transfection systems.
Assuntos
Plaquetas/enzimologia , ATPases Transportadoras de Cálcio/metabolismo , Retículo Endoplasmático/enzimologia , Retículo Sarcoplasmático/enzimologia , Animais , Western Blotting , ATPases Transportadoras de Cálcio/genética , Expressão Gênica , Hipertensão/enzimologia , Peso Molecular , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos WKYRESUMO
Evidence has accumulated that cyclic AMP (cAMP)-induced phosphorylation of a Ras-related protein (Rap1) regulates platelet Ca2+ transport. As this transport was recently found to be controlled by two isoforms of sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA), the 100 kDa SERCA2b and the newly identified 97 kDa SERCA, we attempted to establish which isoform is involved in this regulation. For this purpose, we studied the expression and regulation of both the SERCA and Rap1 isoforms in platelets, haemopoietic cells and various cancer cell lines. SERCA2b was shown to be equally expressed in all the cell lines tested, as determined by detection of its phosphoenzyme formation and by Western blotting using an isoform-specific antibody. In contrast, the expression of the 97 kDa SERCA, studied by the same methods, varied from total absence in the cancer cells to high levels in the megakaryocytic cell lines. With regard to the potential regulatory Rap1 proteins, Western blotting showed different expression of total Rap1 isoforms among the cell lineages, thus ruling out any possible relationship between Rap1 and SERCA2b. However, the expression of Rap1 proteins correlated with that of the 97 kDa SERCA isoform. More refined analysis of the rap1A and rap1B isoforms by reverse transcription PCR and by determining cAMP-induced phosphorylation of Rap1B, i.e. its functional mechanism, confirmed the correlation between Rap1B and the 97 kDa SERCA expression. This relationship was also established by the concerted up-regulation of these two proteins demonstrated in the pathological model of platelets from hypertensive rats. It is concluded that the expressions of 97 KDa SERCA and Rap1B are related, suggesting that regulation of the platelet Ca(2+)-ATPase system by cAMP-induced phosphorylation of Rap1B specifically involves the 97 kDa SERCA.
Assuntos
Plaquetas/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Animais , Western Blotting , Linhagem Celular , AMP Cíclico/metabolismo , Retículo Endoplasmático/enzimologia , Expressão Gênica , Humanos , Membranas Intracelulares/metabolismo , Isoenzimas/metabolismo , Proteínas Quinases/metabolismo , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Retículo Sarcoplasmático/enzimologia , Proteínas rap de Ligação ao GTPRESUMO
The use of platelets instead of smooth muscle cells (SMC) to study the abnormal Ca2+ handling found in hypertension was investigated using spontaneously hypertensive rats (SHR). We studied the regulation of platelet Ca(2+)-ATPases, as we have recently demonstrated that human platelets, like SMC, contain the Ca(2+)-ATPase isoform termed SERCA2-b (sarco-endoplasmic reticulum Ca(2+)-ATPase). In mixed membranes isolated from platelets of normotensive Wistar-Kyoto (WKY) rats and SHR, total Ca(2+)-ATPase activity was found to be 43% higher in SHR than in WKY rats. By the use of autophosphorylation of rat platelet Ca(2+)-ATPases with [gamma-32P]ATP, followed by SDS/PAGE and Western blotting, we found that rat platelets express two distinct Ca(2+)-ATPases: a 100 kDa isoform, recognized by a SERCA2-b-specific anti-peptide antibody, and a 97 kDa isoform, specifically recognized by a polyclonal anti-SERCA antibody. Comparative analysis of platelet membrane Ca(2+)-ATPases from WKY rats and SHR demonstrated that the expression of the SERCA2-b isoform did not change significantly (128 +/- 22%), whereas that of the 97 kDa isoform reached 300 +/- 35% in SHR when compared with WKY rats. We concluded that the upregulation of total platelet Ca(2+)-ATPases in SHR is not due to the 100 kDa SERCA2-b isoform found in SMC, but is specific to the 97 kDa Ca(2+)-ATPase isoform which is not present in SMC. Therefore platelets should be used with extreme caution as a surrogate model of vascular smooth muscle Ca2+ homeostasis.
Assuntos
Plaquetas/enzimologia , ATPases Transportadoras de Cálcio/sangue , Hipertensão/enzimologia , Animais , Western Blotting , Membrana Celular/enzimologia , Retículo Endoplasmático/enzimologia , Cinética , Peso Molecular , Músculo Liso/enzimologia , Fosforilação , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKYRESUMO
Phosphorylation, immunoblotting, limited proteolysis and drug-sensitivity analysis were used to characterize the sarcoendoplasmic-reticulum Ca2+ ATPases in a variety of human cell types. In platelets, several megakaryoblastoid and lymphoblastoid cell lines two distinct autophosphorylated forms of these ATPases with molecular mass of 100 and 97 kDa could be observed, whereas in several other cell types the 97 kDa form was absent. On immunoblots the 97 kDa species was specifically recognized by an inhibitory monoclonal antibody raised against the Ca2+ pump of platelet internal membranes, yielded on trypsinolysis a major fragment of 80 kDa, exhibited a distinct electrophoretic migration pattern as compared with the skeletal-, cardiac- and smooth-muscle Ca2+ pumps, and its autophosphorylation was strongly inhibited by the Ca(2+)-mobilizing agent 2,5-di-(t-butyl)-1,4-benzohydroquinone (tBHQ). The 100 kDa species reacted with an antibody specific for the cardiac- and smooth-muscle Ca2+ pumps, yielded on trypsinolysis fragments of 55 and 35 kDa, and its autophosphorylation was much less sensitive to tBHQ inhibition. These findings indicate the simultaneous presence of two different endoplasmic-reticulum Ca2+ pumps in a variety of human cell types, and may explain the previously observed differences in the Ca(2+)-handling characteristics of different intracellular Ca2+ pools and cell types.
Assuntos
ATPases Transportadoras de Cálcio/análise , ATPases Transportadoras de Cálcio/fisiologia , Retículo Endoplasmático/enzimologia , Hidroquinonas/farmacologia , Anticorpos Monoclonais , Plaquetas/enzimologia , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Humanos , Immunoblotting , Linfócitos/enzimologia , Megacariócitos/enzimologia , Peso Molecular , Músculo Liso/enzimologia , Miocárdio/enzimologia , Fragmentos de Peptídeos/metabolismo , Fosforilação , Tripsina/metabolismo , Células Tumorais CultivadasRESUMO
The role of the sarco-endoplasmic reticulum Ca(2+)-ATPases (SERCA) in the regulation of cell proliferation by Ca2+ was investigated by testing the effect of platelet-derived growth factor (PDGF) on cultured pig aorta smooth muscle cells. For this purpose, the PDGF-mediated rise in the Ca2+ concentration was first examined for its ability to induce the formation of prostaglandins from the specific membrane enzyme, cyclooxygenase. In parallel experiments, similar conditions (10 ng/ml PDGF for 24 h) were used to investigate the smooth muscle cell membrane SERCA2 isoforms. Total SERCA2 activity rose by 472% as reflected by their specific formation of phosphorylated intermediate (E approximately P). This rise correlated with an increase in the amount of SERCA2 proteins (100 kDa) as shown by Western blotting. With isoform-specific anti-SERCA2-a and anti-SERCA2-b antibodies, we demonstrated that the increase in total SERCA2 proteins concerned the minor isoform SERCA2-a, which rose 10-fold, whereas SERCA2-b proteins were not affected. Lastly, Northern blotting using riboprobes showed that PDGF treatment increased the SERCA2-a mRNA species by 82%, and concomitantly decreased the SERCA2-b mRNA by 28%, as a result of isoform switching. We conclude that up-regulation of the SERCA2-type Ca(2+)-ATPases occurs in PDGF-treated smooth muscle cells, which suggests that this enzymatic system plays an essential part in cell proliferation.
Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Cálcio/metabolismo , Divisão Celular/efeitos dos fármacos , Retículo Endoplasmático/enzimologia , Músculo Liso Vascular/enzimologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Retículo Sarcoplasmático/enzimologia , 6-Cetoprostaglandina F1 alfa/metabolismo , Actinas/genética , Animais , Aorta/citologia , Aorta/efeitos dos fármacos , Aorta/enzimologia , Western Blotting , ATPases Transportadoras de Cálcio/genética , Membrana Celular/metabolismo , Células Cultivadas , Dinoprostona/metabolismo , Regulação Enzimológica da Expressão Gênica , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Fosforilação , Prostaglandina-Endoperóxido Sintases/genética , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , SuínosRESUMO
We describe turbidimetric methods for simultaneously determining C-reactive protein and orosomucoid in 33 microL of undiluted serum in the Technicon RA-1000, with multipoint calibration. Precision is good and results correlate favorably with those of nephelometry with the Beckman ICS (r = 0.98 for CRP, 0.99 for orosomucoid). Analyses are easy to perform, require infrequent calibration, and are suitable for routine and emergency analysis.