RESUMO
Assembly strategies for functional nanowire devices that merge bottom-up and top-down technologies have been debated for over a decade. Although several breakthroughs have been reported, nanowire device fabrication techniques remain generally incompatible with large-scale and high-yield top-down microelectronics manufacturing. Strategies enabling the controlled transfer of nanowires from the growth substrate to pre-defined locations on a target surface would help to address this challenge. Based on the promising concept of mechanical nanowire transfer, we developed the technique of surface-controlled contact printing, which is based purely on dry friction between a nanowire and a target surface. Surface features, so-called catchers, alter the local frictional force or deposition probability and allow the positioning of single nanowires. Surface-controlled contact printing extends the current scope of nanowire alignment strategies with the intention to facilitate efficient nanowire device fabrication. This is demonstrated by the simultaneous assembly of 36 nanowire resistors within a chip area of greater than 2 cm(2) aided only by mask-assisted photolithography.
RESUMO
Hypertension is a common disorder of multifactorial origin that constitutes a major risk factor for cardiovascular events such as stroke and myocardial infarction. Previous studies demonstrated an enhanced signal transduction via pertussis toxin-sensitive G proteins in lymphoblasts and fibroblasts from selected patients with essential hypertension. We have detected a novel polymorphism (C825T) in exon 10 of the gene encoding the beta3 subunit of heterotrimeric G proteins (GNB3). The T allele is associated with the occurrence of a splice variant, GNB3-s (encoding G beta3-s), in which the nucleotides 498-620 of exon 9 are deleted. This in-frame deletion causes the loss of 41 amino acids and one WD repeat domain of the G beta subunit. By western-blot analysis, G beta3-s appears to be predominantly expressed in cells from individuals carrying the T allele. Significant enhancement of stimulated GTPgammaS binding to Sf9 insect cells expressing G beta3-s together with G alpha(i)2 and G gamma5 indicates that this splice variant is biologically active. Genotype analysis of 427 normotensive and 426 hypertensive subjects suggests a significant association of the T allele with essential hypertension.
Assuntos
Proteínas de Ligação ao GTP/genética , Variação Genética , Hipertensão/genética , Alelos , Processamento Alternativo , Sequência de Aminoácidos , Animais , Linhagem Celular , Proteínas de Ligação ao GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Hipertensão/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Dados de Sequência Molecular , Peptídeos/metabolismo , Peptídeos/farmacologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Spodoptera/citologiaRESUMO
CONTEXT: Risk alleles of the fat mass- and obesity-associated gene (FTO) are related not only to increased body mass index (BMI) values but also to mortality. It was speculated that cellular effects of the FTO gene affect most organs, especially their ability to maintain or regenerate proper function when afflicted by various diseases. FTO is highly expressed in the hypothalamus and also in the pituitary gland. The decrease in growth hormone (GH) secretion is known to cause a decrease in lean body mass in older subjects. OBJECTIVE: We hypothesized an association of rs9926289 with insulin-like growth factor (IGF)-I. DESIGN AND SETTING: Cross-sectional data from the Study of Health in Pomerania, a population-based study in the northeastern part of Germany, were used. PARTICIPANTS: For the final analyses, 3882 subjects aged 20-79 years were available. MAIN OUTCOME MEASURES: Continuous IGF-I, low IGF-I according to clinically meaningful age- and gender-specific reference values, and BMI were used as outcome measures. RESULTS: Over all age groups, a statistically significant relationship between FTO and IGF-I was found. In subjects younger than 55 years of age, homozygous carriers of the FTO risk allele exhibited lower serum IGF-I levels adjusted for 5-year age groups, gender and IGF-I binding protein 3 levels (linear regression, coefficient±s.e. for FTO AA genotype:-8.6±2.8; P=0.002). Further adjustments for obesity and diabetes did not suspend this association (coefficient:-7.8; P=0.005). As expected, the FTO AA genotype effect on BMI was reduced from 0.76 to 0.62 kg m(-2) by including IGF-I. No relationship between FTO and IGF-I levels was found in subjects aged 55 years or older (-2.7±2.4; P=0.260 for FTO AA genotype adjusted for age, gender and IGF-I binding protein 3 levels). CONCLUSION: We propose that the GH-IGF-I axis is a mediator for the relationship between FTO and BMI.
Assuntos
Índice de Massa Corporal , Hormônio do Crescimento Humano/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Obesidade/genética , Proteínas/genética , Adulto , Idoso , Dioxigenase FTO Dependente de alfa-Cetoglutarato , Estudos Transversais , Feminino , Variação Genética , Genótipo , Alemanha , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/sangue , Masculino , Pessoa de Meia-Idade , Obesidade/metabolismo , Adulto JovemRESUMO
Only about 70% of migraine and cluster headache (CH) patients report significant treatment responses to triptans, which are agonists at 5-HT(1B/D) receptors belonging to the family of G protein-coupled receptors. We analyzed whether a common polymorphism in the gene for the G protein beta3 subunit (GNB3 C825T) modulates responder rates to triptans among a cohort of 231 unrelated Caucasian CH patients. A total of 180 CH patients used triptans, of whom 71.1% reported treatment success. The adjusted odds ratio for treatment response to triptans for heterozygous carriers of the GNB3 825T allele was 2.96 (95% confidence interval 1.34-6.56; P=0.0074) vs carriers of the 825CC genotype. The GNB3 genotype status did not affect responses to other acute and preventive therapeutic regimes including oxygen, verapamil, and corticosteroids, i.e., drugs not directly affecting G proteins. We conclude that pain relief by triptans is significantly modulated by a common genetic GNB3 variant.
Assuntos
Cefaleia Histamínica/tratamento farmacológico , Agonistas de Dopamina/uso terapêutico , Proteínas Heterotriméricas de Ligação ao GTP/genética , Polimorfismo Genético , Agonistas do Receptor 5-HT1 de Serotonina , Triptaminas/uso terapêutico , Adulto , Cefaleia Histamínica/genética , Cefaleia Histamínica/metabolismo , Agonistas de Dopamina/farmacologia , Feminino , Frequência do Gene , Genótipo , Alemanha , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Razão de Chances , Seleção de Pacientes , Estudos Prospectivos , Receptor 5-HT1B de Serotonina/metabolismo , Receptor 5-HT1D de Serotonina/metabolismo , Inquéritos e Questionários , Resultado do Tratamento , Triptaminas/farmacologia , População Branca/genéticaRESUMO
An enhancement of sodium-proton exchange activity is a frequently observed ion transport abnormality in essential hypertension. The cellular basis for this has not yet been elucidated. Due to the lack of a specific cell culture system it has been impossible to distinguish between intrinsic cellular abnormalities and influences exerted by the hypertensive neurohumoral milieu. Using Epstein-Barr virus we have immortalized lymphocytes from controls and from patients with essential hypertension that exhibited enhanced sodium-proton exchanger activity. Sodium-proton exchanger activity was determined in cells loaded with the fluorescent cytosolic pH indicator 2'7'-biscarboxyethyl-5,6-carboxyfluorescein acetoxymethylester (BCECF) after pretreatment with 250 nM of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate for 10 min. Cell lines from hypertensive patients displayed higher Vmax values of sodium-proton exchange than those from normotensive controls (129.6 +/- 30.0 vs. 77.1 +/- 13.2 mmol H+/min.; P < 0.001). Hill coefficients for H+ were distinctly lower in hypertension compared to normotension (1.12 +/- 0.12 vs. 1.50 +/- 0.14; P < 0.0001). The enhanced antiporter activity in cell lines from hypertensive patients was not accompanied by a corresponding increase in steady-state NHE-1 mRNA transcript levels, which argues against overexpression of antiporter protein in hypertension. The cells from hypertensive patients with high sodium-proton exchange activity proliferated distinctly faster than those from normotensive controls. These human cell lines represent a novel model to study the mutual interaction between sodium-proton exchange and cell proliferation, and may provide insights into the alterations in ion transport observed in a group of patients with essential hypertension.
Assuntos
Proteínas de Transporte/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Hipertensão/metabolismo , Linfócitos/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Adulto , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/genética , Divisão Celular , Transformação Celular Viral , Células Cultivadas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Cinética , Linfócitos/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/biossíntese , Trocadores de Sódio-Hidrogênio/efeitos dos fármacos , Trocadores de Sódio-Hidrogênio/genética , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The effect of acid-base disturbances on sodium/proton (Na+/H+) exchange has been examined in animal models; however, few data are available from human studies. To test the effect of metabolic acidosis on Na+/H+ exchange in man, as well as to examine the relationship between Na+/H+ exchange and cytosolic calcium ([Ca2+]i), we measured both variables in patients with decreased renal function with mild metabolic acidosis (pH 7.34 +/- 0.06), in normal control subjects (pH 7.41 +/- 0.02), and in subjects before (pH 7.40 +/- 0.01), and after (pH 7.26 +/- 0.04) ammonium chloride (NH4Cl) 15 g for 5 d. Lymphocytes and platelets were loaded with the cytosolic pH (pHi) indicator 2'-7'-bis(carboxyethyl)-5,6-carboxyfluorescein and acidified to pH approximately 6.6 with propionic acid. To quantitate Na+/H+ exchange, dpHi/dt was determined at 1 min. [Ca2+]i was measured with fura-2. Na+/H+ exchange was significantly increased only in lymphocytes of patients with renal insufficiency. Neither intracellular pH (pHi) nor [Ca2+]i was different from controls. NH4Cl resulted in a significant increase in Na+/H+ exchange in lymphocytes, but not in platelets of normal subjects. Values of pHi and [Ca2+]i in either cell type remained unaffected. Since metabolic acidosis influenced Na+/H+ only in lymphocytes, but not in platelets, it is possible that protein synthesis may be involved in increasing Na+/H+ exchange.
Assuntos
Acidose Tubular Renal/sangue , Acidose/sangue , Plaquetas/metabolismo , Proteínas de Transporte/metabolismo , Linfócitos/metabolismo , Sódio/sangue , Doença Aguda , Dióxido de Carbono/sangue , Doença Crônica , Eletrólitos/sangue , Fluoresceínas , Corantes Fluorescentes , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Cinética , Masculino , Trocadores de Sódio-HidrogênioRESUMO
Epstein-Barr virus-immortalized B lymphoblasts obtained from hypertensive patients with enhanced Na+/H+ exchanger activity (HT cells) proliferate distinctly faster upon serum stimulation than those from normotensive controls with low exchanger activity (NT cells) (Rosskopf, D., E. Frömter, and W. Siffert. 1993. J. Clin. Invest. 92:2553-2559). Stimulation with platelet-activating factor (PAF) as well caused an enhanced proliferation of HT cells. In analyzing possible differences in signal transduction between the immortalized NT and HT lymphoblasts, we observed that cell stimulation with PAF and somatostatin caused a twofold higher increase in [Ca2+]i in HT than in NT cell lines. This difference was completely abrogated by pertussis toxin (PTX) treatment. Furthermore, PAF-stimulated formation of inositol 1,4,5-trisphosphate (IP3) was twofold enhanced in HT cell lines. On the other hand, PAF receptor density and affinity, total cellular phospholipase C activity, expression of PTX-sensitive G proteins, and control binding of the stable GTP analogue, guanosine 5'-[gamma-thio]triphosphate (GTP gamma S), to membrane G proteins were not different in NT and HT cell lines. However, PAF- and mastoparan-stimulated binding of GTP gamma S to G proteins, which was fully PTX-sensitive, was 2.5-fold higher in HT than NT cell lines. These data suggest an enhanced receptor-mediated activation of PTX-sensitive G proteins despite unchanged receptor and G protein expression. Thus, this study not only suggests that enhanced signal transduction and cell proliferation are abnormalities in a certain group of patients with essential hypertension but also explains these findings as a result of an enhanced G protein activation in this common disorder.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Hipertensão/metabolismo , Linfócitos/efeitos dos fármacos , Receptores de Superfície Celular , Receptores Acoplados a Proteínas G , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Fenômenos Fisiológicos Sanguíneos , Cálcio/metabolismo , Bovinos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Transformada , Ativação Enzimática , Proteínas de Ligação ao GTP/antagonistas & inibidores , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Herpesvirus Humano 4 , Humanos , Hipertensão/patologia , Linfócitos/metabolismo , Toxina Pertussis , Fosfatidilinositol Diacilglicerol-Liase , Diester Fosfórico Hidrolases/metabolismo , Fator de Ativação de Plaquetas/farmacologia , Glicoproteínas da Membrana de Plaquetas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Somatostatina/farmacologia , Fatores de Virulência de Bordetella/farmacologiaAssuntos
Predisposição Genética para Doença , Polimorfismo Genético/genética , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Estresse Fisiológico/genética , Estresse Psicológico/genética , Bases de Dados Bibliográficas/estatística & dados numéricos , Meio Ambiente , Feminino , Frequência do Gene , Genótipo , Humanos , Masculino , Metanálise como Assunto , Fatores SexuaisRESUMO
OBJECTIVE: The aim was to investigate the effect of ethylisopropylamiloride (EIPA), an inhibitor of Na+/H+ exchange, and of extracellular pH on the proliferation of immortalised lymphoblasts derived from patients with essential hypertension and normotensive controls. METHODS: Na+/H+ exchange activity was determined in cells loaded with the fluorescent pH indicator BCECF. Cell proliferation was determined by FACS analysis, by cell counting, and from the incorporation of [3H]-thymidine. RESULTS: EIPA inhibited the Na+/H+ exchanger with an average KI value of 14 nM in all cell lines. Cell growth and DNA synthesis were only inhibited at EIPA concentrations > 10 microM suggesting a non-specific effect independent of Na+/H+ exchanger blockade. When extracellular pH was varied from 7.1 to 7.7 by changing the HCO3- concentration at constant PCO2, cell proliferation was optimal at pH 7.4, but reduced at acidic and alkaline pH in cells from normotensive and hypertensive subjects. The increased proliferation of lymphoblasts from hypertensive subjects persisted over the whole pH range. Comparable results were obtained when pH was altered by varying the PCO2 at constant HCO3-. Preincubation of cells with pertussis toxin inhibited serum stimulated DNA synthesis by 14.5% and 23.5% (P = 0.02) in cell lines from normotensive and hypertensive subjects. CONCLUSIONS: The enhanced Na+/H+ exchanger activity in lymphoblasts from patients with essential hypertension is obviously not the major determinant of the enhanced proliferation of these cells. The increased sensitivity of the growth of "hypersensitive" cell lines to pertussis toxin suggests a cellular alteration which resides upstream of Na+/H+ exchange activity and proliferation control.
Assuntos
Amilorida/análogos & derivados , Antiarrítmicos/farmacologia , Hipertensão/metabolismo , Linfócitos/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Amilorida/farmacologia , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Humanos , Concentração de Íons de Hidrogênio , Linfócitos/efeitos dos fármacos , Trocadores de Sódio-Hidrogênio/efeitos dos fármacosRESUMO
Recent studies have revealed that an enhancement of sodium-proton exchange is a frequently observed ion transport abnormality in essential hypertension. An altered antiport activity not only is measurable in blood cells of hypertensive subjects ex vivo but also is detectable in skeletal muscle in vivo. Several lines of argument suggest that the altered antiport activity is not an epiphenomenon of hypertension: 1) the increased activity is found only in a subgroup of patients with high blood pressure, 2) it is not tightly correlated to the severity or duration of hypertension, and 3) high sodium-proton exchange activity persists over time and is not affected by antihypertensive treatment. Available evidence suggests that enhanced sodium-proton exchange is associated with or a cause for the structural alterations found in resistance vessels of hypertensive individuals (media hypertrophy) and left ventricular hypertrophy. This review summarizes some of the physiological properties and roles of the sodium-proton exchanger and discusses its kinetic properties in essential hypertension. Furthermore, the reasons for the enhanced antiport activity and its potential implications regarding the pathogenesis of hypertension are discussed.
Assuntos
Antiporters , Proteínas de Transporte/metabolismo , Hipertensão/etiologia , Animais , Proteínas de Transporte/fisiologia , Humanos , Concentração de Íons de Hidrogênio , Cinética , Membranas/metabolismo , Modelos Cardiovasculares , Trocadores de Sódio-HidrogênioRESUMO
Increased immunoglobulin levels and leukocyte counts have frequently been reported in essential hypertension. The underlying mechanisms, however, have remained obscure. Enhanced Na(+)-H+ exchanger activity is another frequently observed abnormality in essential hypertension that persists in immortalized B lymphoblasts and coincides with enhanced proliferation. We investigated the capacity of B lymphoblasts from essential hypertensive patients to synthesize and secrete immunoglobulins. Six B cell lines from essential hypertensive patients with enhanced Na(+)-H+ exchanger phenotype and six cell lines from normotensive subjects were studied. Lymphocyte markers were visualized by immunostaining. Immunoglobulin secretion was analyzed by enzyme-linked immunosorbent assay. These cell lines did not differ with respect to B cell markers. In response to 100 nmol/L platelet-activating factor, cells from hypertensive patients proliferated distinctly more quickly and their cell number increased by 3.9 +/- 0.4-fold (mean +/- SD) within 4 days, whereas the number of cells from normotensive subjects increased by only 2.6 +/- 0.1-fold. Furthermore, platelet-activating factor induced average increases in IgM and IgG formation of 13.3- and 5.4-fold, respectively, in lymphoblasts from hypertensive patients, which was significantly higher than increases in cells from normotensive subjects (1.4- and 1.2-fold, respectively). Thus, lymphoblasts from hypertensive patients proliferate more quickly and secrete more immunoglobulins in response to a physiological stimulus in vitro. This may contribute to the raised immunoglobulin levels and leukocyte counts reported in vivo.
Assuntos
Linfócitos B/metabolismo , Hipertensão/imunologia , Imunoglobulinas/biossíntese , Adulto , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Humanos , Masculino , Pessoa de Meia-Idade , Fator de Ativação de Plaquetas/farmacologiaRESUMO
Recent studies have shown that a polymorphism (C825T) in the gene encoding the G protein beta 3 subunit (GNB3) is associated with hypertension and obesity. We characterized the entire GNB3 gene, which spans 7.5 kb and is composed of 11 exons and 10 introns. Its promoter lacks a TATA box but harbors GC-rich regions. The functional activity of the GNB3 promoter was verified with reporter gene assays that also demonstrated its inducibility by phorbol esters. A novel polymorphism in the promoter region A(-350)G occurred with frequencies (G allele) of 76%, 97%, and 61% in Africans, Chinese, and Germans, respectively. Reporter gene constructs with either the A or the G allele did not differ with regard to inducement of the reporter protein. A silent nucleotide exchange in the coding region (A657T) occurred with T allele frequencies ranging from 0.5% to 2.4%. Another polymorphism (G814A) results in the replacement of glycine by serine at position 272. In Germans, the A allele occurred at a frequency of 10%. Finally, a C1429T polymorphism in the 3' untranslated region of GNB3 was identified that occurred at T allele frequencies of 38%, 17%, and 30% in Africans, Chinese, and Germans, respectively. Haplotype prediction indicated in Germans an almost complete association of GNB3 825T with 1429T, and vice versa. An analysis of these polymorphic loci in nonhuman primates revealed that the ancestral GNB3 gene harbored the (-350)G, 825C, and 1429C alleles. This is the first complete characterization of the human GNB3 gene and its promoter region, which will enable refined epidemiological and biochemical investigations of GNB3 in hypertension and obesity.
Assuntos
Proteínas de Ligação ao GTP/genética , Polimorfismo Genético , Regiões Promotoras Genéticas , Adolescente , Adulto , Sequência de Bases , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência MolecularRESUMO
Using the reverse polymerase chain reaction (RT-PCR), we have examined the expression of Na+/H+ exchanger mRNA in human buffy coat preparations, lymphocytes and neutrophils. Total RNA from all cell types was reverse transcribed specifically and then amplified by PCR. The identity of the PCR products was confirmed by restriction enzyme analysis and hybridization with a specific oligonucleotide probe. The detection of low abundance Na+/H+ antiporter specific transcripts by RT-PCR in different human blood cells ex vivo should facilitate future studies on regulatory and pathophysiological aspects of Na+/H+ exchanger mRNA expression in human cells and tissue samples.
Assuntos
Proteínas de Transporte/genética , Linfócitos/fisiologia , Neutrófilos/fisiologia , RNA Mensageiro/sangue , Sequência de Bases , Southern Blotting , Proteínas de Transporte/sangue , Clonagem Molecular , Humanos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Mapeamento por Restrição , Trocadores de Sódio-HidrogênioRESUMO
The 825T allele of a common C825T polymorphism in the gene encoding the beta3 subunit of heterotrimeric G proteins is associated with enhanced activation of pertussis toxin (PTX)-sensitive G proteins. We investigated responses of human platelets upon stimulation with epinephrine, which activates PTX-sensitive G proteins, and with agonists which activate additionally, or exclusively PTX-insensitive pathways. Slopes and maximum of the secondary aggregation were significantly enhanced in platelets from 825T allele carriers after epinephrine, and after combined epinephrine/ADP. This effect was more pronounced after inhibition of the cyclooxygenase-2 pathway by acetylsalicylic acid. This phenomenon appeared independent of platelet secretion, or inhibition of the adenylyl cyclase.
Assuntos
Toxina Adenilato Ciclase , Epinefrina/farmacologia , Proteínas Heterotriméricas de Ligação ao GTP/genética , Toxina Pertussis , Agregação Plaquetária/fisiologia , Polimorfismo Genético , Fatores de Virulência de Bordetella/farmacologia , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Difosfato de Adenosina/farmacologia , Adolescente , Adulto , Alelos , Colforsina/farmacologia , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase/farmacologia , Sinergismo Farmacológico , Humanos , Técnicas In Vitro , Isoenzimas/sangue , Masculino , Proteínas de Membrana , Selectina-P/sangue , Agregação Plaquetária/genética , Prostaglandina-Endoperóxido Sintases/sangue , Trombina/farmacologia , Vasopressinas/farmacologiaRESUMO
Accumulating evidence suggests an increased activity of the Na(+)-H+ exchanger in essential hypertension. The present investigation aimed at developing a test for routine measurements. Platelet-rich plasma was added directly to a cuvette placed into an aggregometer containing 140 mmol/l sodium propionate medium (pH 6.7, 37 degrees C). The accumulation of intracellular sodium due to activation of Na(+)-H+ exchange results in an osmotic cell swelling, which is detectable as a decrease in optical density (OD). This reaction reflects activation of the Na(+)-H+ exchanger since we observed (1) a dose-dependent inhibition by amiloride (inhibition constant, Ki = 10 mumol/l) and ethylisopropylamiloride (Ki = 0.07 mumol/l) and (2) a dependence on extracellular sodium of the OD changes. Electron microscopy of sodium propionate-treated platelets revealed a general swelling and a distinct decrease in electron density of the cytosol without other significant alterations. Quantification of Na(+)-H+ exchange activities was accomplished by calculating rate constants of the recorded changes in OD. Application of this assay to 20 essential hypertensives and 32 normotensives demonstrated an increased activity of the Na(+)-H+ exchanger in essential hypertensives (rate constants 29.8 x 10(-3) per s versus 21.7 x 10(-3) per s).
Assuntos
Plaquetas/metabolismo , Proteínas de Transporte/sangue , Hipertensão/metabolismo , Adulto , Amilorida/análogos & derivados , Amilorida/farmacologia , Feminino , Humanos , Masculino , Microscopia Eletrônica , Óptica e Fotônica , Osmose , Sódio/sangue , Trocadores de Sódio-HidrogênioRESUMO
OBJECTIVE: Primary hypertension has been reported to be associated with an enhancement of Na(+)-H+ exchange. However, details of the kinetic properties of the Na(+)-H+ exchanger in hypertensives and its dependence upon age and gender in normotensives are unknown. PARTICIPANTS: We determined the activity of the platelet Na(+)-H+ exchanger in 20 normotensives and 26 untreated primary hypertensives. INTERVENTIONS: In eight hypertensive individuals antihypertensive treatment was interrupted for 1 week. Treatment for 6 weeks with a daily single dose of 10 mg enalapril decreased mean arterial pressure to 105.7 +/- 11.6 mmHg. METHODS: Platelets were loaded with the intracellular pH (pHi) indicator 2'-7'-bis-carboxyethyl-5(6)-carboxyfluorescein (BCECF) and acidified by propionic acid. Initial velocities of pH recovery were determined and used for calculation of maximum velocity (Vmax), baseline pHi and the pHi value for half maximal activation (pH0.5) of the Na(+)-H+ exchanger in each individual. RESULTS: In normotensives, Vmax averaged 0.05 +/- 0.01 dpHi/min independently of age, gender and actual diastolic blood pressure. In hypertensives, two different subgroups were defined bearing either low or high Na(+)-H+ exchange activity. Values of pHi and pH0.5 were identical in all subgroups irrespective of Vmax. The twofold enhancement of Na(+)-H+ exchange in the second group was preserved in thrombin-stimulated platelets. Vmax values remained unaffected by enalapril treatment. CONCLUSIONS: Enhanced Na(+)-H+ exchange activity in hypertensives is primarily characterized by an increase in Vmax. This enhancement is refractory to antihypertensive treatment and therefore appears to be a relatively fixed parameter.
Assuntos
Plaquetas/metabolismo , Proteínas de Transporte/metabolismo , Enalapril/uso terapêutico , Hipertensão/tratamento farmacológico , Fatores Etários , Plaquetas/efeitos dos fármacos , Proteínas de Transporte/sangue , Proteínas de Transporte/efeitos dos fármacos , Feminino , Fluoresceínas , Humanos , Concentração de Íons de Hidrogênio , Hipertensão/metabolismo , Masculino , Pessoa de Meia-Idade , Fatores Sexuais , Trocadores de Sódio-HidrogênioRESUMO
A C825T polymorphism was recently described in GNB3, the gene encoding the Gbeta3 subunit of heterotrimeric G proteins. The 825T allele is associated with the expression of a shorter splice variant (Gbeta3-s) and enhanced signal transduction via pertussis toxin (PTX)-sensitive G proteins. Given the pivotal role of G protein betagamma dimers in chemotaxis, we related the genotype at the GNB3 locus as a marker for Gbeta3-s expression to chemotaxis of human neutrophils in response to stimulation with interleukin-8 (IL-8). IL-8, which activates a CXC receptor coupled to PTX-sensitive G proteins, induced at 10 nM an enhanced maximum chemotaxis of neutrophils from individuals with TC/TT genotype compared to CC genotype. Furthermore, migration of neutrophils from 825T allele carriers was 2.5-fold higher at 0.1 nM and 1 nM IL-8. At these concentrations of IL-8, no significant chemotaxis was observed in neutrophils from homozygous C825 allele carriers, indicating a genotype-dependent, different potency of IL-8 to chemoattract neutrophils. In contrast, IL-8-induced Ca2+ signals and O2- generation were independent of genotype. The role of Gbeta3-s in enhanced chemotaxis could be confirmed by determination of chemotaxis of COS-7 cells following transfection with either Gbeta3-s or "wild-type" Gbeta3. Upon stimulation of the transfected cells with the chemoattractant lysophosphatidic acid (LPA), we observed an enhanced chemotactic response of Gbeta3-s-transfected compared to Gbeta3-transfected COS-7 cells, confirming that Gbeta3-s actually causes enhanced chemotaxis.
Assuntos
Fatores Quimiotáticos/farmacologia , Quimiotaxia de Leucócito/genética , Proteínas de Ligação ao GTP/genética , Interleucina-8/farmacologia , Neutrófilos/fisiologia , Alelos , Processamento Alternativo , Animais , Ânions , Células COS , Cálcio/metabolismo , Humanos , Técnicas In Vitro , Lisofosfolipídeos/farmacologia , Oxigênio/metabolismo , TransfecçãoRESUMO
The 825T-allele of the C825T polymorphism in GNB3, the gene for the G protein beta3 subunit, has been reported to be associated with essential hypertension and obesity. Expression of Gbeta3s, the gene product of GNB3 associated with the GNB3 825T-allele, causes increased signal transduction which may contribute to pathogenetic mechanisms ultimately resulting in hypertension and obesity. Given the known involvement of heterotrimeric G proteins in insulin secretion and insulin action on the cellular level, we analysed insulin sensitivity in each 15 young lean normotensive males with TC- and CC-genotypes, respectively. Blood glucose and serum insulin samples were taken during a standard oral glucose tolerance test. Insulin-stimulated glucose disposal was analysed by euglycemic-hyperinsulinemic clamp. Both groups did not differ with regard to the time-courses for glucose or insulin concentrations in the oral glucose tolerance test. Furthermore, insulin-stimulated glucose disposal was virtually independent of genotype. The TC-genotype is not associated with a primary defect in insulin secretion or sensitivity suggesting that obesity and hypertension in carriers of 825T do not likely result from primary alterations in glucose and insulin homeostasis. However, GNB3 825T-associated obesity may predispose to insulin resistance, an issue which remains to be investigated. Furthermore, fasting cholesterol was significantly higher in TC compared to CC genotype (4.71 versus 3.96 mmol/l; p = 0.007) suggesting that enhanced G protein signalling might be associated with alterations of cholesterol metabolism.
Assuntos
Colesterol/metabolismo , Glucose/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/genética , Polimorfismo Genético , Adulto , Alelos , Pressão Sanguínea , Composição Corporal , Peso Corporal , Genótipo , Técnica Clamp de Glucose , Teste de Tolerância a Glucose , Humanos , Hipertensão/genética , Resistência à Insulina/genética , Masculino , Obesidade/genética , Transdução de SinaisRESUMO
BACKGROUND: Type 2 diabetes mellitus is a common late-onset disease with a strong genetic component. It is characterized by insulin resistance which results from alterations in insulin signal transduction. The G protein beta 3 subunit 825T allele was recently found to be associated with hypertension and obesity which makes it a sensible candidate gene for type 2 diabetes. METHODS: In a case-control study on 320 male patients and 962 male healthy controls we investigated the association of two candidate genes with diabetes, i.e. (i) the GNB3 825T allele, associated with a G protein beta 3 subunit splice variant and enhanced intracellular signal transduction, and (ii) the insulin receptor substrate-1 (IRS-1) 972Arg variant, which encodes a protein variant associated with cellular insulin resistance. RESULTS: The GNB3 825T allele and the IRS-1 972Arg variant were significantly associated with diabetes (odds ratios for either variant 1.4 1.8). Odds ratios were 3 4 in males carrying both alleles. CONCLUSIONS: The results document an association of a hypertension susceptibility gene with type 2 diabetes which may partially explain the frequent coexistence of both disorders.
Assuntos
Alelos , Diabetes Mellitus Tipo 2/genética , Proteínas de Ligação ao GTP/genética , Fosfoproteínas/genética , Adulto , Fatores Etários , Idoso , Índice de Massa Corporal , Estudos de Casos e Controles , Predisposição Genética para Doença , Humanos , Proteínas Substratos do Receptor de Insulina , Resistência à Insulina , Masculino , Pessoa de Meia-Idade , Transdução de SinaisRESUMO
Pharmacogenomics investigates inherited differences in drug responses including beneficial and adverse reactions. While a considerable amount of evidence for genetic influences on drug responses has been accumulated within the last decade, predominantly in small studies, its value in routine therapy is still a matter of debate. The aim of this review is to discuss well established examples where pharmacogenomic techniques can improve routine treatment. Examples include genotyping of CYP2D6 in the context of antidepressant therapy, analysis of TPMT variants for the prediction of mercaptopurine-induced bone marrow depression, VKORC1 and CYP2C9 analyses for a better control of anticoagulant administration and the SLCO1B1 variant in the context of statin-induced myopathies.