RESUMO
AIMS: Patients with Type 1 and Type 2 diabetes mellitus show altered platelet function including decreased nitric oxide synthase (NOS) activity and increased peroxynitrite production. Gestational diabetes mellitus (GDM) is a clinical condition which is ideal for evaluating short-term effects of impaired glucose metabolism, ruling out the possibility that the platelet abnormalities are a consequence of diabetic complications. The aim of the present work was to study NO metabolism in platelets from pregnant women with GDM. The production of peroxides was also studied as it is strongly involved in peroxynitrite formation. METHODS: Platelet NOS activity and peroxynitrite production, levels of hydroperoxides and thiobarbituric acid reactive substances (TBARS) in platelet membranes in the basal state and after in vitro peroxidative stress with phenylhydrazine were determined in 40 pregnant women with GDM, 40 healthy pregnant women (pregnant controls) of comparable age and gestational age, and 15 healthy non-pregnant women (controls). RESULTS: NOS activity was significantly increased in both groups of pregnant women compared with non-pregnant ones, and in GDM women compared with pregnant controls. Production of peroxynitrite was higher in GDM women than in pregnant controls, who also had significantly reduced production compared with non-pregnant women. Basal levels of peroxidation of the platelet membranes evaluated either by hydroperoxide content and TBARS levels or the susceptibility to peroxidation were increased in GDM patients in comparison with both control groups. CONCLUSIONS: We have shown a modification in platelet NO and peroxynitrite production and an increase in platelet indicators of oxidative stress in GDM women compared with healthy pregnant women which might be at the basis of a cellular dysfunction.
Assuntos
Plaquetas/metabolismo , Diabetes Gestacional/metabolismo , Óxido Nítrico Sintase/metabolismo , Adulto , Plaquetas/enzimologia , Membrana Celular/metabolismo , Diabetes Gestacional/sangue , Diabetes Gestacional/enzimologia , Feminino , Humanos , Oxirredução , Ácido Peroxinitroso/biossíntese , Gravidez , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
Sialic acid (SA) content, membrane fluidity, and Na(+)/K(+)-adenosine triphosphatase (ATPase) activity were determined in erythrocyte membrane from 10 nonpregnant women (HNPW), 16 pregnant women affected by gestational diabetes mellitus (GDM), and 25 healthy pregnant women (HPW). In GDM patients the membrane erythrocyte SA content was significantly increased compared with HNPW and membrane fluidity was significantly increased in comparison with HPW. Erythrocyte membrane Na(+)/K(+)-ATPase activity was significantly reduced in GDM patients compared both to HNPW and to HPW subjects. A significant inverse correlation was found between 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) anisotropy and erythrocyte membrane SA content in HNPW and in HPW, while this significant correlation was not observed in GDM. The present results indicate that in comparison with normal pregnancy GDM is characterized by deep alterations of the erythrocyte plasma membrane physicochemical properties (increased fluidity) and functional activities (reduced Na(+)/K(+)-ATPase activity). These modifications might be at the basis of the altered blood viscosity and placental perfusion observed under such conditions. Moreover, these results show that in physiological pregnancy and in the nonpregnant state, the erythrocyte surface membrane fluidity is inversely correlated with SA content, while in GDM there is an unbalance of this relation, which might be associated with the microcirculatory abnormality present in this disease.
Assuntos
Diabetes Gestacional/sangue , Membrana Eritrocítica/química , Ácido N-Acetilneuramínico/sangue , Adulto , Membrana Eritrocítica/enzimologia , Membrana Eritrocítica/fisiologia , Feminino , Polarização de Fluorescência , Humanos , Fluidez de Membrana , Gravidez , ATPase Trocadora de Sódio-Potássio/sangueRESUMO
The aim of the present work was to analyze the effect of LDL obtained from type 1 diabetic patients in good metabolic control on human umbilical vein endothelial cells (HUVECs) after a short incubation period to detect possible atherogenic modifications of endothelial properties. Cultured HUVECs were incubated for 3 h with culture medium alone (control HUVEC), with native LDL from 12 healthy men (control LDL), or with native LDL from 12 type 1 diabetic men (type 1 LDL) (100 pg/ml). After the incubation, the following parameters were evaluated: nitric oxide synthase (NOS) activity, cytoplasmic Ca2+ levels, Na+-K+-ATPase activity, plasma membrane fluidity determined by means of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), and plasma membrane conjugated diene (CD) content. The same experiments were repeated after bradykinin stimulation or in the presence of the antioxidant butylated hydroxytoluene (BHT), and nitric oxide (NO) production in intact HUVECs was also evaluated. HUVECs incubated with control LDL in comparison with control HUVECs showed a decreased fluidity of the membrane surface evaluated by TMA-DPH and a higher CD content. These alterations were prevented by the presence of BHT. HUVECs incubated with type 1 LDL in comparison with both control HUVECs and cells incubated with control LDL showed 1) increased NOS and Na+-K+-ATPase activity, cytoplasmic Ca2+ levels, and CD content, and 2) decreased fluidity of the membrane surface evaluated by TMA-DPH. These modifications were blunted--but not abolished--by the presence of BHT. After bradykinin stimulation either in the absence or in the presence of BHT, both cytoplasmic Ca2+ levels and NO production were increased in control HUVECs and in HUVECs incubated with control LDL, while a reduced response was observed in HUVECs incubated with type 1 LDL. The alterations observed in the endothelial function after the cell-LDL interaction might play a central role in the atherogenic process in diabetes.
Assuntos
Diabetes Mellitus Tipo 1/sangue , Endotélio Vascular/fisiologia , Lipoproteínas LDL/sangue , Adulto , Cálcio/metabolismo , Membrana Celular/metabolismo , Colesterol/sangue , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Difenilexatrieno/análogos & derivados , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Corantes Fluorescentes , Humanos , Lipoproteínas LDL/farmacologia , Masculino , Fluidez de Membrana/fisiologia , Óxido Nítrico Sintase/metabolismo , Fosfolipídeos/sangue , Valores de Referência , ATPase Trocadora de Sódio-Potássio/metabolismo , Triglicerídeos/sangue , Veias UmbilicaisRESUMO
The aim of the present study was to evaluate the action of plasma from insulin-dependent diabetic (IDDM) pregnant women on nitric oxide synthase (NOS) activity in cultured human umbilical vein endothelial cells (HUVECs). We also studied the effect of the plasma on cytosolic calcium and on Na+/K+-adenosine triphosphatase (ATPase) activity. Dynamic fluorescence studies of membrane fluidity were contemporarily performed to detect a direct effect of plasma on the endothelial cell membrane. We observed a significant increase in NOS activity, intracellular calcium, and Na+/K+-ATPase activity in cultured HUVECs exposed to IDDM plasma. Our dynamic fluorescence study showed a different microenvironmental organization of the cellular membrane after incubation with plasma from IDDM pregnant women, with a marked decrease in microheterogeneity as evaluated in terms of 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) lifetime distribution width. The present investigation suggests that plasma from IDDM pregnant women can cause a generalized disturbance in the function of endothelial cells cultured from healthy subjects. Such a modification might play a central role in the pathogenesis of the vascular complications of the disease.
Assuntos
Diabetes Mellitus Tipo 1/sangue , Endotélio Vascular/metabolismo , Gravidez em Diabéticas/sangue , Veias Umbilicais/metabolismo , Adulto , Fenômenos Fisiológicos Sanguíneos , Cálcio/metabolismo , Membrana Celular/fisiologia , Células Cultivadas , Citosol/metabolismo , Difenilexatrieno/análogos & derivados , Endotélio Vascular/citologia , Feminino , Corantes Fluorescentes , Fluorometria , Humanos , Fluidez de Membrana/fisiologia , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo III , Gravidez , ATPase Trocadora de Sódio-Potássio/metabolismo , Veias Umbilicais/citologiaRESUMO
Growth retardation and low birth weight represent an important factor associated with the high risk of mortality and morbidity, particularly in twin pregnancy, since twins are frequently characterised by fetal growth retardation and sometimes by a discordant growth between the twins. The present work sets out to elucidate the role of growth discordance between twins in the behaviour of human umbilical vein endothelial cells; biochemical, ultrastructural and immunohistochemical data obtained from a discordant twin pregnancy are discussed. Endothelial cells were obtained from umbilical cord of 5 singleton pregnancies and from 5 dichorionic twin pregnancies, among which was one discordant twin pregnancy. Membrane fluidity was assayed using a fluorescent probe and each sample was immunohistochemically processed employing specific monoclonal antibodies. An increased fluidity was observed in endothelial cells from the smaller twin as compared with the larger one. As concerns electron microscopy, the features of endothelial cells from the smaller twin were similar to those of twins with similar growth, while endothelial cells from the larger one were more similar to those from singleton pregnancies. Our findings confirm the presence of the feto-fetal transfusion as a pathogenetic mechanism involved in twin growth discordancy.
Assuntos
Retardo do Crescimento Fetal , Gravidez Múltipla , Cordão Umbilical/metabolismo , Adulto , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Endotélio Vascular/ultraestrutura , Feminino , Humanos , Imuno-Histoquímica , Células de Langerhans/citologia , Fluidez de Membrana , Microscopia Eletrônica/métodos , Gravidez , Linfócitos T/citologia , Cordão Umbilical/citologia , Cordão Umbilical/ultraestruturaRESUMO
To investigate the molecular mechanisms of the inhibition of Na+,K(+)-adenosine triphosphatase (Na+,K(+)-ATPase) in diabetes mellitus, we incubated Na+,K(+)-ATPase purified from human placenta of six healthy nondiabetic women with plasma from six insulin-dependent diabetic (IDDM) men and six healthy controls and with different concentrations of lysophosphatidylcholine (LPC). We determined the enzyme activity, anthroyl ouabain-binding capacity, dissociation constant (Kd), and average lifetime values (tau) by the static and dynamic fluorescence of anthroyl ouabain. The lipid annulus of the enzyme was studied by static and dynamic fluorescence of 1-(4-trimethylamino-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH). Moreover, we studied the lipid microenvironment surrounding the Na+,K(+)-ATPase purified from the placentas of six healthy women and six insulin-dependent diabetic women, determining the percent composition of phospholipids of the lipid annulus. The addition of total and protein-free IDDM plasma to normal Na+,K(+)-ATPase significantly inhibited the enzymatic activity even at the lowest concentration studied (1: 100), whereas the ouabain-binding capacity, Kd, and tau were not affected by IDDM plasma. The fluorescence polarization and lifetime values of TMA-DPH were significantly decreased by diabetic plasma. The incubation of Na+,K(+)-ATPase with LPC caused an inhibition of the enzymatic activity without modifications of the anthroyl ouabain-binding capacity and dissociation constant. The fluorescence polarization and lifetime values of TMA-DPH were significantly decreased by 5 mumol/L LPC. The study of the phospholipids surrounding Na+,K(+)-ATPase demonstrated a significant increase in the percent LPC content in IDDM patients compared with controls together with a concomitant decrease in phosphatidylcholine. These observations indicate that the inhibition caused by diabetic plasma on Na+,K(+)-ATPase is not dependent on a modification of the ouabain-binding site and that it seems to mimic the effect of LPC addition. A link between modification of the lipid moiety of the enzyme and Na+,K(+)-ATPase inhibition might be hypothesized.
Assuntos
Diabetes Mellitus Tipo 1/sangue , Lisofosfatidilcolinas/farmacologia , Plasma/fisiologia , ATPase Trocadora de Sódio-Potássio/efeitos dos fármacos , Adulto , Estudos de Casos e Controles , Feminino , Polarização de Fluorescência , Humanos , Masculino , Fosfolipídeos/análise , Gravidez , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
BACKGROUND: The action of plasma from women affected by gestational hypertension (GH) on nitric oxide synthase (NOS) activity in cultured human umbilical vein endothelial cells (HUVECs) was evaluated in the present study, together with the effect on cytosolic calcium, on Na+, K+-ATPase activity and on membrane fluidity. METHODS: At 80% confluence, cultured HUVECs were incubated for 3 h at 37 degreesC with fresh culture medium (control samples) or with 20% (v/v) plasma (from five healthy non-pregnant women, five healthy pregnant women and five pregnant women affected by GH). RESULTS: After incubation with GH plasma, we observed a significant reduction in NOS activity, intracellular calcium concentrations and Na+, K+-ATPase activity. CONCLUSIONS: The present work gives further support to the hypothesis that a circulating factor in gestational hypertension, possibly produced by the fetoplacental unit, causes dysfunction of the vascular endothelial cells and NO reduction, resulting in a loss of vascular refractoriness to vasoactive agents.
Assuntos
Endotélio Vascular/metabolismo , Hipertensão/sangue , Plasma/fisiologia , Complicações na Gravidez/sangue , Adulto , Cálcio/metabolismo , Endotélio Vascular/efeitos dos fármacos , Feminino , Humanos , Fluidez de Membrana/efeitos dos fármacos , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo III , Gravidez , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
The causes of the reduced activity of Na+/K+-adenosine triphosphatase (ATPase) in human diabetes are still the object of controversy. The aim of this work was to investigate the mechanisms of inhibition by means of the study of the Na+/K+-ATPase purified from human placenta. We purified Na+/K+-ATPase from term placentas of six healthy women and six age-matched women with insulin-dependent diabetes mellitus (IDDM) in good metabolic control. The enzymatic activity was reduced in both the microsomal fraction and the purified Na+/K+-ATPase obtained from diabetic women, whereas no difference was found in the number of active molecules determined by anthroyl ouabain binding. The Na+/K+-ATPase purified from women with IDDM did not show any modification in the ouabain affinity or changes in the physicochemical structure of the ouabain binding site investigated by dynamic fluorescence or alterations in lateral diffusion. The activation energy of the enzyme was increased, whereas the tryptophan accessibility of the enzyme was lower in women with IDDM. The fluidity of the lipid anulus of the enzyme was higher in women with IDDM than in control women, as suggested by fluorescence polarization of 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene. The adenosine triphosphate-binding site, investigated by anisotropy decay studies of the fluorescent probe pyrene isothiocyanate, was modified in women with IDDM. It appears that the Na+/K+-ATPase of human placenta is altered in its disposition in IDDM.
Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Placenta/enzimologia , Gravidez em Diabéticas/enzimologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Acrilamida , Acrilamidas/farmacologia , Adulto , Antracenos/metabolismo , Sítios de Ligação , Difusão , Difenilexatrieno/análogos & derivados , Difenilexatrieno/metabolismo , Ativação Enzimática , Feminino , Polarização de Fluorescência , Corantes Fluorescentes , Humanos , Isotiocianatos/metabolismo , Cinética , Fluidez de Membrana , Microssomos/enzimologia , Ouabaína/análogos & derivados , Ouabaína/metabolismo , Gravidez , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/isolamento & purificação , Espectrometria de Fluorescência , Triptofano/metabolismoRESUMO
Between February 1993 and May 1994 we studied the prevalence of fungal vulvovaginitis among women attending the Obstetric and Gynecology Clinic of the University of Ancona. Out of the 222 patients, 18 (8.2%) women had symptomatic vaginitis and 24 (10.8%) were carriers. Candida albicans was the species most frequently isolated (44.2%), followed by Torulopsis glabrata (28%) and Saccharomyces cerevisiae (16.2%), from symptomatic and carrier patients. The activity of acid proteinase was determined for C. albicans isolated from both symptomatic and carrier patients. All 13 carriers showed low activity for aspartyl proteinase (score 1+), while 5 of 6 symptomatic patients showed higher activity (score 2+), with a significant difference (p = 0.026). In general, isolates of T. glabrata and S. cerevisiae were less susceptible in vitro to fluconazole than isolates of C. albicans. We did not find any differences in fluconazole MIC results among the C. albicans strains isolated from symptomatic and carrier patients. On the other hand, the fluconazole MICs of T. glabrata and S. cerevisiae isolates showed statistically significant differences between symptomatic and carrier patients (p = 0.009 and p = 0.000, respectively). The differences in proteinase secretion between the isolates from symptomatic and carrier patients suggest a correlation between proteinase production and vaginal candidiasis caused by C. albicans. Torulopsis glabrata, however, was found to be the most common causative agent of vaginitis (7 out 19 episodes), followed by C. albicans (6 out of 19 episodes). Due to the varying patterns of antifungal susceptibility, mainly to fluconazole for the yeast isolates considered in this study, an in vitro susceptibility testing program might be useful for monitoring the outcome of this infection.
Assuntos
Candida/classificação , Candidíase Vulvovaginal/microbiologia , Portador Sadio/microbiologia , Micoses/microbiologia , Saccharomyces cerevisiae , Doenças Vaginais/microbiologia , Adulto , Idoso , Ácido Aspártico Endopeptidases/metabolismo , Candida/enzimologia , Resistência Microbiana a Medicamentos , Feminino , Humanos , Itália , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Ambulatório Hospitalar , Prevalência , Estudos ProspectivosRESUMO
In order to investigate the molecular mechanisms of the inhibition of Na+/K+-ATPase in Gestational Hypertension (GH), we incubated Na+/K+-ATPase purified from human placenta of 6 healthy normotensive women with plasma from 6 GH women and 6 healthy controls. We determined the enzyme activity by the method of Esman, and the anthroyl-ouabain-binding capacity, dissociation constant (Kd) and average lifetime values (tau) by the static and dynamic fluorescence of anthroyl-ouabain. The lipid annulus of the enzyme was studied by static and dynamic fluorescence of 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5- hexatriene (TMA-DPH). The addition of total and protein-free GH plasma to normal Na+/K+-ATPase significantly inhibited the enzymatic activity even at the lowest concentration studied (1:100), as well as the ouabain-binding capacity, Kd and tau. GH plasma significantly decreased the fluorescence polarization and lifetime values of TMA-DPH. These observations indicate that the inhibition caused by GH plasma on Na+/K+-ATPase might be due to a reduction of the number of active molecules or a modification of the ouabain-binding site suggesting the existence of digitalis-like factor. A link between the modification of the lipid moiety of the enzyme and the Na+/K+-ATPase inhibition might be hypothesized.
Assuntos
Inibidores Enzimáticos/sangue , Hipertensão/sangue , Placenta/enzimologia , Complicações Cardiovasculares na Gravidez/sangue , Gravidez/sangue , ATPase Trocadora de Sódio-Potássio/metabolismo , Adulto , Proteínas Sanguíneas , Feminino , Humanos , Cinética , Ouabaína/metabolismo , Valores de Referência , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/isolamento & purificação , Espectrometria de FluorescênciaRESUMO
1. Na+,K(+)-ATPase is the membrane enzyme catalysing the active transport of Na+ and K+ across the plasma membrane of animal cells. A reduced activity of Na+,K(+)-ATPase has been described in gestational hypertension in a variety of cell types, in agreement with the hypothesis that gestational hypertension can induce membrane transport modifications similar to those reported for essential hypertension. The causes of the reduced Na+,K(+)-ATPase activity are still debated. 2. The aim of the present work was to investigate the molecular mechanism of the reduced enzymic activity in gestational hypertension using as a model Na+,K(+)-ATPase purified from human placenta. Na+,K(+)-ATPase obtained from term placentas of eight healthy pregnant women and eight age-matched women with gestational hypertension was purified as previously described. 3. We observed in gestational hypertension: (i) a significant increase in the activation energies above transition temperature; (ii) a significant decrease in the fluorescence polarization of 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (i.e. increased fluidity) and an increase in the mean lifetime (modified hydrophobicity); (iii) a lower Kq, suggesting an enzymic structural modification; and (iv) an increased mean lifetime and rotational relaxation time of pyrene isothiocyanate, indicating a modified ATP binding site.
Assuntos
Hipertensão/enzimologia , Placenta/enzimologia , Complicações Cardiovasculares na Gravidez/enzimologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Adulto , Fenômenos Biofísicos , Biofísica , Feminino , Polarização de Fluorescência , Humanos , Gravidez , Temperatura , TriptofanoRESUMO
Plasma membrane lipid dynamics and cellular morphology were evaluated in endothelial cells obtained from umbilical cords of five women affected by insulin-dependent diabetes mellitus (IDDM) and six healthy pregnant women of similar age and gestational age. Endothelial cells were prepared by an adaptation of the method of Jaffe et al. Membrane fluidity was studied by means of the steady-state fluorescence anisotropy (r) of 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), a fluorescent probe specifically anchoring at the membrane surface. Fluid phase endocytosis was evaluated by the measurement of the changes in fluorescence intensity of TMA-DPH at various times, owing to the internalization of the fluorescent marker in endocytic vesicles. The morphological and morphometric studies were performed by means of transmission electron microscopy (TEM). Endothelial cells obtained from IDDM women showed: (a) increased fluidity of the superficial region of the plasma membrane; (b) a more active fluid phase endocytosis compared with cells from healthy women; (c) increase in mitochondrial area, Weibel-Palade bodies and rough reticulum with wide cisternae. No statistically significant correlation was found between metabolic control and membrane fluidity and endocytosis. All the observed modifications suggest the presence of endothelial cell activation with membrane reshaping during IDDM. These alterations might play a central role in the pathophysiology of atherosclerosis and microangiopathy associated with diabetes mellitus.
Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Adulto , Estudos de Casos e Controles , Angiopatias Diabéticas/etiologia , Endocitose , Retículo Endoplasmático Rugoso/patologia , Feminino , Humanos , Técnicas In Vitro , Fluidez de Membrana , Lipídeos de Membrana/metabolismo , Microscopia Eletrônica , Mitocôndrias/patologia , Gravidez , Gravidez em Diabéticas/metabolismo , Gravidez em Diabéticas/patologia , Cordão Umbilical/metabolismo , Cordão Umbilical/patologiaRESUMO
A platelet (PLT) function modification has been reported in normal pregnancies compared with the nonpregnant condition and it has been hypothesized to play a central role in the pathogenesis of pregnancy-induced hypertension (PIH). The aims of the present study were (i) to evaluate the lipid composition, fluidity at different depths, transport functions, and ultrastructural features of the PLT membrane in PIH and (ii) to ascertain whether similar modifications may be determined by the state of pregnancy in comparison with the nonpregnant condition. The platelets of healthy pregnant women (HPW) showed: (i) an increase in Ca2+ ATPase activity, (ii) a decreased fluidity of the deeper site of the membrane, (iii) a reduced cholesterol concentration, with an increased ratio between unsaturated and saturated fatty acids; (iv) a decreased intramembranous particles (IMP) distribution factor (DF) of the plasma membrane E face in comparison with healthy nonpregnant women. When comparing women affected by PIH with HPW, we observed (i) reduced Na+/K+ ATPase activity and enhanced Ca2+ ATPase activity and intraplatelet calcium concentrations, (ii) an increased membrane fluidity, (iii) an increased cholesterol concentration and ratio between unsaturated and saturated fatty acids, (iv) a reduction of the IMP number and the DF. Pregnancy produces a deep modification of the platelet plasma membrane, and PIH produces a more pronounced alteration of the maternal platelets, which can be responsible for the observed modifications in placental blood flow and in the fetomaternal exchange.
Assuntos
Plaquetas/fisiologia , Hipertensão/patologia , Complicações Cardiovasculares na Gravidez/patologia , Plaquetas/ultraestrutura , Cálcio/sangue , ATPases Transportadoras de Cálcio/sangue , Citosol/metabolismo , Feminino , Técnica de Fratura por Congelamento , Humanos , Hipertensão/fisiopatologia , Fluidez de Membrana , Lipídeos de Membrana/sangue , Gravidez , ATPase Trocadora de Sódio-Potássio/sangueRESUMO
A decrease of Na+/K(+)-ATPase activity has been reported in syncytiotrophoblast plasma membrane (SPM) obtained from pregnancy induced hypertensive (PIH) women. The aim of the present work was to verify if the reported modifications in activity are due to a decreased number of enzymatic molecules or to a conformational change of the enzyme itself. Morphological studies were performed in order to better understand the relations between the enzymatic protein and the lipid bilayer. Kinetic studies were also performed. SPM obtained from PIH showed: i) an increased affinity of Na+/K(+)-ATPase for ouabain binding, ii) a significant change in the maximum velocity of the enzyme, iii) a higher distribution factor (DF) of intramembrane particles (IMPs) in the exoplasmic face of the membrane, iv) a decreased mean diameter of IMPs both in the protoplasmic and exoplasmic faces, v) a decreased number of IMPs in the exoplasmic face. In conclusion, a conformational modification seems to be at the basis of the decreased Na+/K(+)-ATPase activity during PIH as suggested by binding, ultrastructural and kinetic data herein reported.
Assuntos
Células Gigantes/patologia , Hipertensão/patologia , Complicações Cardiovasculares na Gravidez/patologia , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Trofoblastos/patologia , Adulto , Estudos de Casos e Controles , Membrana Celular/efeitos dos fármacos , Inibidores Enzimáticos/metabolismo , Feminino , Técnica de Fratura por Congelamento , Células Gigantes/metabolismo , Humanos , Hipertensão/etiologia , Hipertensão/metabolismo , Cinética , Ouabaína/metabolismo , Gravidez , Complicações Cardiovasculares na Gravidez/metabolismo , Conformação Proteica , Trofoblastos/metabolismoRESUMO
We have evaluated the effects of the different components of hormone replacement therapy (HRT) on the production of free radicals in platelet membranes from menopausal women. The study included 12 women in menopause for at least 6 months to a maximum of 4 years. First, the effect was determined of progestin only during the administration of 20 mg/day medroxyprogesterone acetate for 5 days. The peroxide production level was measured on day 0 and day 5. The second set of experiments was carried out in the first month of cyclic HRT with transdermal estradiol 50 micrograms/day from day 1 to day 25 and medroxy-progesterone acetate from day 13 to day 25. In this experiment, the peroxide level was evaluated on days 0, 12 and 25. A significant reduction of peroxide level was observed after oral medroxyprogesterone acetate administration. During HRT, we observed a similar reduction in lipid oxidation at the peak of the estrogen effect, and a further decrease with the administration of medroxyprogesterone acetate. It is concluded that reduction of lipid peroxidation during HRT is not only due to estrogens, but also depends upon the combined action of sex steroids. This observation justifies not only the combined regimen (estrogens plus progestin) in HRT, but also the positive effects of progestins alone on patients who cannot use estrogens.
Assuntos
Antioxidantes/uso terapêutico , Estradiol/uso terapêutico , Terapia de Reposição de Estrogênios/normas , Acetato de Medroxiprogesterona/uso terapêutico , Congêneres da Progesterona/uso terapêutico , Administração Cutânea , Antioxidantes/administração & dosagem , Plaquetas/metabolismo , Plaquetas/ultraestrutura , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Estradiol/administração & dosagem , Feminino , Radicais Livres , Humanos , Peroxidação de Lipídeos , Acetato de Medroxiprogesterona/administração & dosagem , Lipídeos de Membrana/metabolismo , Pessoa de Meia-Idade , Congêneres da Progesterona/administração & dosagem , Progestinas/farmacologiaRESUMO
Investigations on singleton and twin pregnancies show different functional behaviour on maternal-fetal relationship. In some ways twin pregnancies may be considered at risk and they may develop associated pathologies such as hypertension. The aim of this work was to evaluate the morpho-functional behaviours of umbilical cord veins in twin and singleton gestations to better understand the role of these extra-embryonic tissues in the regulation of pregnancies. The umbilical cords were studied from singleton pregnancies and from dichorionic twin pregnancies. Biochemical and morphological investigations were carried out. A significant decrease in the anisotropy values was observed in endothelial cells from twins compared with singletons. Our ultrastructural data show immaturity features at the vein vessel wall level in twins. Furthermore, immunohistochemical investigations showed a lower degree of expressivity concerning adhesion molecules such as ICAM-1 and ELAM. Morphogenetic extracellular glycoproteins like fibronectin and tenascin seem over-expressed in twin pregnancies. Our morpho-functional data well testify the lower maturation degree of umbilical cord veins in twins with respect to singletons.
Assuntos
Gravidez Múltipla , Gêmeos Dizigóticos , Veias Umbilicais/anatomia & histologia , Anisotropia , Endotélio/ultraestrutura , Proteínas da Matriz Extracelular/análise , Feminino , Glicoproteínas/análise , Humanos , Troca Materno-Fetal , Mitocôndrias Musculares/ultraestrutura , Gravidez , Tenascina/análise , Cordão Umbilical/ultraestrutura , Veias Umbilicais/química , Veias Umbilicais/fisiologiaRESUMO
Mother-fetus exchanges at the placental level are found to be altered in women affected by hypertensive or diabetic pregnancies following the onset of microenvironmental, circulatory, trophic or tissue disorders. Our aim was therefore to assess the alterations occurring within the umbilical cord, particularly its venous endothelial component and underlying smooth muscle layer, using transmission (TEM) and scanning electron microscopy (SEM) and immunohistochemical analyses. Immunohistochemical data appear to support the ultrastructural evidence for an activated state of these vascular structures, in both conditions (hypertension and diabetes). Furthermore, mainly during diabetic pregnancies, extracellular matrix molecules such as tenascin and fibronectin also quantitatively increase at the vein wall level. The umbilical cord seems to be a structure capable of responding actively to abnormal microenvironmental conditions which seriously threaten the health of the fetus and also the mother herself.
Assuntos
Hipertensão/patologia , Gravidez em Diabéticas/patologia , Veias Umbilicais/patologia , Moléculas de Adesão Celular/análise , Moléculas de Adesão Celular Neuronais/análise , Selectina E , Endotélio Vascular/patologia , Proteínas da Matriz Extracelular/análise , Feminino , Fibronectinas/análise , Antígenos HLA-DR/análise , Humanos , Imuno-Histoquímica , Molécula 1 de Adesão Intercelular/análise , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Músculo Liso Vascular/patologia , Gravidez , Tenascina , Veias Umbilicais/químicaRESUMO
Placentas of women suffering from pregnancy-induced hypertension (PIH) were found to contain a greater amount of Na,K-ATPase molecules, estimated from anthroyl ouabain binding, than normotensive individuals. Both the microsomal fraction of placental cells and purified Na,K-ATPase showed an increased affinity for the specific inhibitor ouabain which, in the case of the microsomes, bound with a dissociation constant of 0.9 nM as compared with 3.4 nM in the controls. Likewise, the dissociation constant of the ouabain complex with purified Na,K-ATPase was about 3.5 times lower in the hypertensive patients. The differences are apparently caused by a different microenvironment of the ouabain-binding site, as reflected in the quantum yield of bound anthroyl ouabain. If an endogenous digitalis-like factor is present in the body fluids to regulate Na,K-ATPase activity, the present results render its role quite plausible.
Assuntos
Glicosídeos Cardíacos/metabolismo , Hipertensão/enzimologia , Placenta/enzimologia , Placenta/metabolismo , Adulto , Feminino , Humanos , Hipertensão/fisiopatologia , Microssomos/enzimologia , Microssomos/metabolismo , Ouabaína/metabolismo , Placenta/ultraestrutura , Gravidez , Complicações Cardiovasculares na Gravidez/enzimologia , Complicações Cardiovasculares na Gravidez/metabolismo , Complicações Cardiovasculares na Gravidez/fisiopatologiaRESUMO
The microvillus plasma membrane of the human placental syncytiotrophoblast at term has been extensively studied, while little is known about the characteristics of its development. The aim of the present work was to compare functional and structural properties of this membrane at early and term gestational age. Ten normal term placentas (40 weeks) and ten placentas at 10 weeks of gestational age were studied. The Na+/K+-ATPase activity is significantly decreased in the syncytiotrophoblast plasma membrane obtained from term placentas as compared to the early ones, with significant variation of maximum velocity (Vmax). The microviscosity, evaluated by the P parameter of DPH and Sn parameters of 5- and 16-NS, is increased in the term placentas compared to the early placentas. This alteration is accompanied by an increased cholesterol to phospholipids ratio in term placentas, while there is a decreased unsaturated to saturated fatty acid ratio. As follows from morphological studies, an increased mean diameter in the E face was observed in the term placenta with respect to the early placenta. The distribution factor DF, which indicates the particle aggregation state, decreased in the E face in the term placenta as compared to the early one. The present biochemical morphological study shows that a deep modification of the membrane is at the basis of the syncytiotrophoblast plasma membrane development.
Assuntos
Microvilosidades/metabolismo , Trofoblastos/citologia , Trofoblastos/metabolismo , Feminino , Humanos , Fluidez de Membrana , Microvilosidades/ultraestrutura , Gravidez , Primeiro Trimestre da Gravidez , Terceiro Trimestre da Gravidez , ATPase Trocadora de Sódio-Potássio/metabolismo , Trofoblastos/ultraestruturaRESUMO
It has been recently hypothesized that in PIH a placental oxidant-antioxidant imbalance might cause the release of lipoperoxidation products into the circulation, with subsequent damage of endothelial cell membranes. In this hypothesis the endothelial cell and further increase in circulating lipoperoxide levels, which are by themselves able to induce smooth muscle constriction and increased pressor responsiveness to angiotensin II. In order to investigate this issue, we studied the basal content of lipid peroxides in terms of malondialdehyde (MDA) in the syncytiotrophoblast plasma membranes (SPM) from PIH women. Moreover, we investigated the susceptibility to peroxidation of SPM using an in vitro oxidative stress as a tool to verify the predisposition to the in vivo development of peroxidation products. The fatty acid composition of the membranes was also analyzed. Microvillus membrane lipoperoxide concentrations were significantly increased in PIH women (62.8 +/- 7.6 ng MDA/mg prot) compared with healthy pregnant subjects (37.6 +/- 4.8 ng MDA/mg prot; p < 0.01). The formation of TBARS under the action of phenylhydrazine was significantly greater in PIH women (90.3 +/- 7.4 mmol MDA/mol cholesterol) than in normal pregnant subjects (68.6 +/- 6.4 mmol MDA/mol cholesterol; p < 0.01). In PIH microvillus membrane we also observed a significant increase of the content of polyunsaturated arachidonic acid. The increased susceptibility to oxidative stress of SPMs from PIH women might be due either to reduced antioxidant systems or to an abnormality of the lipid composition of the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)