RESUMEN
Na,K-ATPase is the main system effectively excluding the superfluous sodium out of the cells on the expense of energy derived from hydrolysis of ATP. In brain 3 different isoforms of the catalytic α-subunit are known. The present study was focused to energy utilization and ability to bind sodium by the Na,K-ATPase as well as expression of all 3 isoforms of the catalytic α-subunit concerning its sex specificity in two selected regions of the brain, in cortex and in cerebellum of rats. Western blot analysis showed higher expression of all 3 catalytic α-subunits of Na,K-ATPase in cerebellum when compared to cortex which was not followed by higher activity. On contrary the total activity of the enzyme was lower in cerebellum comparing with cortex in females with no significant localization dependent differences of activities in males. Concerning sex dependence only the expression of α3 isoform was higher in cortex of male rats with no differences in the levels of α1 and α2 isoforms. However, the total activity of Na,K-ATPase in cortex was similar in male and female groups. On the other hand in cerebellum of females the total activity of Na,K-ATPase was significantly lower as compared with males. The obtained data indicate localization and sex dependent variations in maintenance of sodium homeostasis in the brain.
Asunto(s)
Encéfalo/enzimología , Cerebelo/enzimología , Corteza Cerebral/enzimología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Sodio/metabolismo , Animales , Femenino , Homeostasis , Masculino , Ratas , Ratas Wistar , Factores SexualesRESUMEN
Na,K-ATPase represents the key enzyme maintaining the ionic gradient across plasma membrane. It was documented that in directly irradiated organs the activity of this enzyme is decreased. The aim of present study was to clarify the remote effect of irradiation in mediastinal area on the activity of the Na,K-ATPase in selected brain regions in rats. Ionizing radiation in single dose 25 Gy induced alterations in oxidative status of blood plasma. Irradiation also decreased the activity of the Na,K-ATPase in cerebral cortex. Measurements of kinetic properties of the enzyme dependently on the concentration of energy substrate ATP or cofactor Na+ indicated that the lowered enzyme activity is probably a consequence of decreased number of active molecules of the enzyme, as suggested by lowered Vmax values (by 13 - 14%). Immunoblot analysis revealed that this effect is connected namely to decreased presence of α2 and α3 subunits (by 25% and 30% respectively). Considering the current concepts about involvement of the malfunction of α2 α3 subunits in development of primary brain dysfunctions, it may be hypothesized that the lowered functionality of those subunits of Na,K-ATPase may represent a predisposition to neurodegenerative disorders after irradiation. The observed effect seems to be localization dependent as the enzyme in cerebellum resisted to irradiation.
Asunto(s)
Corteza Cerebral/efectos de la radiación , Subunidades de Proteína/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Cerebelo/metabolismo , Cerebelo/efectos de la radiación , Corteza Cerebral/metabolismo , Masculino , Estrés Oxidativo/efectos de la radiación , Ratas Wistar , Rayos XRESUMEN
In rodents, increased angiotensin 2 (Ang2) during pregnancy increases blood pressure and decreases salt sensitivity in the offspring. To explore the underlying mechanisms, this study evaluated the effects of prenatal Ang2 exposure on the activity of renal Na,K-ATPase, which is one of the main systems that maintains sodium ion homeostasis in an organism. Moreover, this study also investigated the impact of a higher-salt diet on the enzyme activity in the offspring in a sex-dependent manner. Pregnant Wistar rats were implanted with osmotic minipumps that continuously released Ang2 (2 µg/kg/h) for 2 weeks. Male and female offspring of treated and control females were allocated to groups fed with normal or high-salt diets. In the offspring prenatally treated with Ang2, a significant Vmax increase (23 - 36%) was observed in females fed with both a normal and high-salt diet. In addition, a significant increase in Km (20 - 26%) was also observed in the female groups, compared to respective male groups, independently of their diet. Evaluation of KNa showed significantly lower values (13 - 17%) in female offspring fed with a high-salt diet, independent of the prenatal treatment. In conclusion, these data suggest that increased prenatal Ang2 has a predominant impact on the properties of renal Na,K-ATPase in both sexes. Moreover, the enzyme is resistant to higher salt intake in offspring prenatally exposed to Ang2.
Asunto(s)
Angiotensina II/farmacología , Riñón/efectos de los fármacos , Efectos Tardíos de la Exposición Prenatal , Cloruro de Sodio Dietético , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Femenino , Riñón/metabolismo , Masculino , Intercambio Materno-Fetal , Embarazo , Ratas Wistar , Caracteres SexualesRESUMEN
The present study was oriented to gender specificity of Na,K-ATPase in cerebellum, the crucial enzyme maintaining the intracellular homeostasis of Na ions in healthy and diabetic Wistar rats. The effects of diabetes on properties of the Na,K-ATPase in cerebellum derived from normal and streptozotocin (STZ)-diabetic rats of both genders were investigated. The samples were excised at different time intervals of diabetes induced by STZ (65 mg kg-1) for 8 days and 16 weeks. In acute 8-day-lasting model of diabetes, Western blot analysis showed significant depression of α1 isoform of Na,K-ATPase in males only. On the other hand, concerning the activity, the enzyme seems to be resistant to the acute model of diabetes in both genders. Prolongation of diabetes to 16 weeks was followed by increasing the number of active molecules of Na,K-ATPase exclusively in females as indicated by enzyme kinetic studies. Gender specificity was observed also in nondiabetic animals revealing higher Na,K-ATPase activity in control males probably caused by higher number of active enzyme molecules as indicated by increased value of V max when comparing to control female group. This difference seems to be age dependent: at the age of 16 weeks, the V max value in females was higher by more than 90%, whereas at the age of 24 weeks, this difference amounted to only 28%. These data indicate that the properties of Na,K-ATPase in cerebellum, playing crucial role in maintaining the Na+ and K+ gradients, depend on gender, age, and duration of diabetic impact.
Asunto(s)
Envejecimiento/metabolismo , Cerebelo/enzimología , Diabetes Mellitus Experimental/enzimología , Proteínas del Tejido Nervioso/metabolismo , Caracteres Sexuales , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Enfermedad Aguda , Envejecimiento/patología , Animales , Cerebelo/patología , Diabetes Mellitus Experimental/patología , Femenino , Masculino , Ratas , Ratas WistarRESUMEN
Deformability of red blood cells (RBC) is the ability of RBC to change their shape in order to pass through narrow capillaries in circulation. Deterioration in deformability of RBC contributes to alterations in microcirculatory blood flow and delivery of oxygen to tissues. Several factors are responsible for maintenance of RBC deformability. One of them is the Na,K-ATPase known as crucial enzyme in maintenance of intracellular ionic homeostasis affecting thus regulation of cellular volume and consequently RBC deformability. Decreased deformability of RBC has been found to be the marker of adverse outcomes in cardiovascular diseases (CVD) and the presence of cardiovascular risk factors influences rheological properties of the blood. This review summarizes knowledge concerning the RBC deformability in connection with selected risk factors of CVD, including hypertension, hyperlipidemia, and diabetes mellitus, based exclusively on papers from human studies. We attempted to provide an update on important issues regarding the role of Na,K-ATPase in RBC deformability. In patients suffering from hypertension as well as diabetes mellitus the Na,K-ATPase appears to be responsible for the changes leading to alterations in RBC deformability. The triggering factor for changes of RBC deformability during hypercholesterolemia seems to be the increased content of cholesterol in erythrocyte membranes.
Asunto(s)
Diabetes Mellitus/sangre , Eritrocitos/citología , Hipercolesterolemia/sangre , Hipertensión/sangre , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Diabetes Mellitus/enzimología , Eritrocitos/enzimología , Humanos , Hipercolesterolemia/enzimología , Hipertensión/enzimología , Factores de RiesgoRESUMEN
Measurements of red blood cell (RBC) deformability together with estimation of NO-synthase activity and Na,K-ATPase activity were used for characterization of RBC functionality in rats subjected to single dose of Escherichia coli lipopolysaccharides (LPS) at a dose of 1âmg/kg. We hypothesized that LPS might initiate a malfunction of RBC. We also investigated the potential effect of carotenoids (10âmg/kg/day) produced in red yeast biomass of Rhodotorula glutinis on RBC in LPS-challenged rats. LPS significantly reduced the deformability of RBC (by 14%) together with decrease of NO-synthase activity by 20%. Daily supplementation of carotenoids for 10 days attenuated the LPS-induced injury, as observed by 22% increase of RBC deformability and 23% increase of NO-synthase activity. The activity of Na,K-ATPase was also improved probably due to increased number of active enzyme molecules as indicated by 66% enhancement of Vmax value, hence maintaining the activity of erythrocyte Na,K-ATPase to the level even higher as compared with healthy control animals. It may be concluded that administration of yeast biomass with high content of carotenoids resulted in advanced function of erythrocytes as concerns their ability to squeeze through narrow capillaries of the circulation, better intrinsic production of NO and improvement of intracellular homeostasis of sodium.
Asunto(s)
Carotenoides/metabolismo , Eritrocitos/efectos de los fármacos , Lipopolisacáridos/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Biomasa , Modelos Animales de Enfermedad , Deformación Eritrocítica/efectos de los fármacos , Lipopolisacáridos/farmacología , Masculino , Óxido Nítrico , Ratas , LevadurasRESUMEN
Time sequence study was performed to characterize the effects of diabetes mellitus type 1 on properties of the Na, K-ATPase in cerebral cortex derived from normal and streptozotocin (STZ)-diabetic rats of both genders. The samples were excised at varying time intervals of diabetes induced by STZ (65 mg kg(-1)) for 8 days, and 8 and 16 weeks. Expression of α1-3 isoforms of Na, K-ATPase was not altered in statistically significant level during all stages of diabetes neither in female nor in male rats as revealed from Western blot analysis. Studies of kinetic properties of the enzyme resulted in variations in active number of Na, K-ATPase molecules as well as its qualitative properties. Sixteen-week-old control male rats showed better affinity to substrate as indicated by 13 % decrease of K m value. The effect persisted also in males subjected to 8 days lasting diabetes; however, in males subjected to 8 weeks lasting diabetes, the effect was lost. In 25-week-old rats, the Na, K-ATPase revealed again altered properties in males and females but the mechanism of the variation was different. In females, the number of active molecules of Na, K-ATPase was higher by 32 % in controls and by 17 % in rats with chronic diabetes when comparing to respective male groups as suggested by increased value of V max. So the properties of Na, K-ATPase in cerebral cortex, playing crucial role in maintaining intracellular homeostasis of Na(+) ions, depend on gender, age, and duration of diabetic insult.
Asunto(s)
Corteza Cerebral/enzimología , Corteza Cerebral/metabolismo , Diabetes Mellitus Experimental/enzimología , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/enzimología , Diabetes Mellitus Tipo 1/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Tipo 1/inducido químicamente , Femenino , Masculino , Ratas , Estreptozocina/farmacologíaRESUMEN
Previous studies showed that adverse effect of ionizing radiation on the cardiovascular system is beside other factors mostly mediated by reactive oxygen and nitrogen species, which deplete antioxidant stores. One of the structures highly sensitive to radicals is the Na,K-ATPase the main system responsible for extrusion of superfluous Na(+) out of the cell which utilizes the energy derived from ATP. The aim of present study was the investigation of functional properties of cardiac Na,K-ATPase in 20-week-old male rats 6 weeks after γ-irradiation by a dose 25 Gy (IR). Irradiation induced decrease of systolic blood pressure from 133 in controls to 85 mmHg in IR group together with hypertrophy of right ventricle (RV) and hypotrophy of left ventricle (LV). When activating the cardiac Na,K-ATPase with substrate, its activity was lower in IR in the whole concentration range of ATP. Evaluation of kinetic parameters revealed a decrease of the maximum velocity (V max) by 40 % with no changes in the value of Michaelis-Menten constant (K m). During activation with Na(+), we observed a decrease of the enzyme activity in hearts from IR at all tested Na(+) concentrations. The value of V max decreased by 38 %, and the concentration of Na(+) that gives half maximal reaction velocity (K Na) increased by 62 %. This impairment in the affinity of the Na(+)-binding site together with decreased number of active Na,K-ATPase molecules, as indicated by lowered V max values, are probably responsible for the deteriorated efflux of the excessive Na(+) from the intracellular space in hearts of irradiated rats.
Asunto(s)
Rayos gamma/efectos adversos , Corazón/efectos de la radiación , Sarcolema/efectos de la radiación , ATPasa Intercambiadora de Sodio-Potasio/efectos de la radiación , Animales , Presión Sanguínea/efectos de la radiación , Hipertensión/fisiopatología , Hipertrofia Ventricular Izquierda , Hipertrofia Ventricular Derecha , Masculino , Miocardio/enzimología , Neoplasias/radioterapia , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/efectos adversos , Sarcolema/enzimologíaRESUMEN
This study deals with changes, induced by hypertension and its treatment, in the function and properties of mitochondria in the heart and kidneys. Male, 16-week-old hypertensive rats were allocated to 3 groups: (i) animals treated daily for 4 weeks with captopril (CAP, 80 mg·(kg body mass)(-1), n = 45), (ii) animals treated with CAP + nifedipine (NIF, 10 mg·kg(-1), n = 45), or (iii) untreated hypertensive controls (n = 96). Wistar rats (n = 96) were used as normotensive controls. Systolic blood pressure (SBP), heart rate (HR), and heart mass / body mass (HW/BW) ratio were measured at the beginning and end of the experiments; measurements for mitochondrial Mg(2+)-ATPase activity, O(2)-consumption (QO(2)), respiratory control index (RCI), ADP/O, oxidative phosphorylation rate (OPR), conjugated diene content (CD), and membrane fluidity (MF) were also taken at different time intervals. In the heart, elevated SBP, HR, and HW/BW accompanied increased QO(2), OPR, and Mg(2+)-ATPase activity, indicating an adaptive response to hypertension-induced increase in the energy demands of the myocardium. Treatments with CAP or with CAP + NIF were very similar in their prevention of increase in SBP, HR, HW/BW, and the rise in OPR (all p < 0.05-0.01). In the kidneys, hypertension induced a drop in OPR; however, antihypertensive therapy aggravated the resulting energy deficiency, whereby treatment with CAP + NIF was more detrimental than treatment with CAP alone. Heart and kidney mitochondria exhibited negligible changes in CD and moderately increased MF, which was more potentiated by treatment with CAP alone than with CAP + NIF.
Asunto(s)
Adaptación Fisiológica/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Antihipertensivos/uso terapéutico , Hipertensión/tratamiento farmacológico , Riñón/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Membranas Mitocondriales/efectos de los fármacos , Miocardio/metabolismo , Animales , Antihipertensivos/administración & dosificación , Antihipertensivos/farmacología , Presión Sanguínea/efectos de los fármacos , Captopril/administración & dosificación , Captopril/farmacología , Captopril/uso terapéutico , Quimioterapia Combinada , Frecuencia Cardíaca/efectos de los fármacos , Hipertensión/metabolismo , Hipertensión/fisiopatología , Riñón/metabolismo , Masculino , Fluidez de la Membrana/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/metabolismo , Membranas Mitocondriales/metabolismo , Nifedipino/administración & dosificación , Nifedipino/farmacología , Nifedipino/uso terapéutico , Ratas , Ratas Endogámicas SHR , Ratas WistarRESUMEN
For better understanding of pathophysiological processes leading to increased retention of sodium as a consequence of hyperlipidemia, the properties of renal Na,K-ATPase, a key enzyme involved in maintaining sodium homeostasis in the organism, were studied. Enzyme kinetics of renal Na,K-ATPase were used for characterization of ATP- and Na(+)-binding sites after administration of fish oil (FO) (30 mg·day(-1)) or atorvastatin (0.5 mg·100 g(-1)·day(-1)) to healthy Wistar rats and rats with hereditary hypertriglyceridemia of both genders. Untreated healthy Wistar and also hypertriglyceridemic female rats revealed higher Na,K-ATPase activity as compared to respective untreated male groups. Hypertriglyceridemia itself was accompanied with higher Na,K-ATPase activity in both genders. Fish oil improved the enzyme affinity to ATP and Na(+), as indicated by lowered values of K(m) and K(Na) in Wistar female rats. In Wistar male rats FO deteriorated the enzyme in the vicinity of the Na(+)-binding site as revealed from the increased K(Na) value. In hypertriglyceridemic rats FO induced a significant effect only in females in the vicinity of the sodium binding sites resulting in improved affinity as documented by the lower value of K(Na). Atorvastatin aggravated the properties of Na,K-ATPase in both genders of Wistar rats. In hypertriglyceridemic rats protection of Na,K-ATPase was observed, but this effect was bound to females only. Both treatments protected renal Na,K-ATPase in a gender specific mode, resulting probably in improved extrusion of excessive intracellular sodium out of the cell affecting thus the retention of sodium in hHTG females only.
Asunto(s)
Aceites de Pescado/farmacología , Ácidos Heptanoicos/farmacología , Hipertrigliceridemia/metabolismo , Riñón/enzimología , Pirroles/farmacología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Anticolesterolemiantes , Atorvastatina , Femenino , Aceites de Pescado/uso terapéutico , Hipertrigliceridemia/tratamiento farmacológico , Riñón/metabolismo , Masculino , Ratas , Ratas Endogámicas , Ratas Wistar , Factores SexualesRESUMEN
The effect of quercetin, a plant-derived bioflavonoid with documented positive effect on the cardiovascular system, was examined after 4-week supplementation in the dose of 20 mg kg(-1) x day(-1) to young male normotensive control (C) and to spontaneously hypertensive rats (SHR) over the period of their 5(th)-8(th) week of age. The study was focused on the influence of quercetin on properties of the renal Na,K-ATPase, a key system in maintaining the homeostasis of sodium in the organism. Spontaneous hypertension by itself enhanced the activity of Na,K-ATPase probably as a consequence of a higher number of active enzyme molecules, as suggested by the 15% increase of V(max), along with improved affinity to ATP, as indicated by the 30% decrease in the value of Michaelis-Menten constant K(m) in untreated SHR vs. untreated normotensive rats. Quercetin induced a decrease of Na,K-ATPase activity in the presence of all ATP and Na(+) concentrations investigated. Evaluation of kinetic parameters resulted in a constant V(max) value. The ATP-binding properties of the enzyme were not influenced by quercetin, as suggested by statistically insignificant changes in the value of K(m) both in controls and in SHR. On the other hand, the affinity to sodium decreased, as suggested by an increase in the K(Na) value by 22% and 31% in normotensive and hypertensive groups, respectively. This impairment in the affinity of the Na(+)-binding site of Na,K-ATPase molecules was probably responsible for the deteriorated enzyme function in the kidneys of quercetin treated animals.
Asunto(s)
Antioxidantes/farmacología , Hipertensión/enzimología , Riñón/efectos de los fármacos , Riñón/enzimología , Quercetina/farmacología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Presión Sanguínea , Peso Corporal/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Flavonoides/farmacología , Hipertensión/fisiopatología , Riñón/metabolismo , Cinética , Masculino , Ratas , Ratas Endogámicas SHR , Ratas Wistar , Sodio/metabolismoRESUMEN
The present study was focused on regulatory role of nitric oxide on functional properties of the cardiac Na, K-ATPase in three various animal models of hypertension: spontaneously hypertensive male rats (SHR) with increased activity of nitric oxide synthase (NOS) by 60 % (Sh1), SHR with decreased activity of NOS by 40 % (Sh2) and rats with hypertension induced by L-NAME (40 mg/kg/day) with depressed activity of NOS by 72 % (LN). Studying the utilization of energy substrate we observed higher Na, K-ATPase activity in the whole concentration range of ATP in Sh1 and decreased activity in Sh2 and LN. Evaluation of kinetic parameters revealed an increase of Vmax value by 37 % in Sh1 and decrease by 30 % in Sh2 and 17 % in LN. The KM value remained unchanged in Sh2 and LN, but was lower by 38 % in Sh1 indicating increased affinity of the ATP binding site, as compared to controls. During the activation with Na+ we observed increased Vmax by 64 % and increased KNa by 106 % in Sh1. In Sh2 we found decreased Vmax by 40 % and increased KNa by 38 %. In LN, the enzyme showed unchanged Vmax with increased KNa by 50 %. The above data indicate a positive role of increased activity of NOS in improved utilization of ATP as well as enhanced binding of Na+ by the cardiac Na, K-ATPase.
Asunto(s)
Hipertensión/enzimología , Miocardio/enzimología , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Sitios de Unión , Presión Sanguínea , Modelos Animales de Enfermedad , Metabolismo Energético , Inhibidores Enzimáticos , Hipertensión/inducido químicamente , Hipertensión/genética , Hipertensión/fisiopatología , Cinética , Masculino , NG-Nitroarginina Metil Éster , Óxido Nítrico Sintasa/antagonistas & inhibidores , Ratas , Ratas Endogámicas SHR , Cloruro de Sodio/metabolismoRESUMEN
The effect of the pyridoindole antioxidant stobadine on diabetes-induced changes of Na,K-ATPase, especially those concerning the utilisation of its substrate ATP, was investigated. Sixteen weeks of streptozotocin-induced diabetes (single i.v. dose of streptozotocin; 55 mg/kg) was followed by decrease in the enzyme activity. This effect was emphasised in the presence of higher concentrations of substrate and in the presence of 8 mmol x l(-1) ATP it represented 20%. It might be a consequence of altered functional properties of Na,K-ATPase as suggested by 20% decrease in the V(max) value along with decrease in the K(m) value by 20%. Administration of 0.05% (w/w) stobadine in the diet to diabetic rats improved the function of renal Na,K-ATPase with respect to utilisation of ATP as suggested by significant increase in the enzyme activity in the whole concentration range of ATP investigated as a consequence of V(max) elevation to the level comparable to absolute controls. In conclusion, stobadine may play a positive role in restoring the functional properties of renal Na,K-ATPase, especially concerning the utilisation of energy derived from hydrolysis of ATP, improving thus the maintenance of ionic homeostasis during diabetes.
Asunto(s)
Adenosina Trifosfato/metabolismo , Carbolinas/administración & dosificación , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/metabolismo , Riñón/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Estreptozocina , Animales , Antioxidantes/administración & dosificación , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Indoles/metabolismo , Riñón/efectos de los fármacos , Masculino , Ratas , Ratas WistarRESUMEN
In the present study we examined the effect of dietary supplementation with the pyridoindole antioxidant stobadine on functional properties of the cardiac Na(+),K(+)-ATPase in diabetic rats. Diabetes lasting sixteen weeks which was induced by a single i.v. dose of streptozotocin (55 mg x kg(-1)) was followed by decrease in the enzyme activity. Evaluation of kinetic parameters revealed a statistically significant decrease in the maximum velocity (Vmax) (32% for ATP-activation, 33% for Na(+)-activation), indicating a diabetes-induced diminution of the number of active enzyme molecules in cardiac sarcolemma. The ATP-binding properties of the enzyme were not affected by diabetes as suggested by statistically insignificant changes in the value of Michaelis-Menten constant, K(M (ATP)). On the other hand, the affinity to sodium decreased as suggested by 54% increase in the K(M (Na+)) value. This impairment in the affinity of the Na(+)-binding site together with decreased number of active Na(+),K(+)-ATPase molecules are probably responsible for the deteriorated enzyme function in hearts of diabetic animals. Administration of stobadine to diabetic rats dramatically improved the function of cardiac Na(+),K(+)-ATPase with regard to Na(+)-handling, as documented by statistically significant elevation of Vmax by 66 and 47% decrease in K(M (Na+)). Our data suggest that stobadine may prevent the diabetes-induced deterioration of cardiac Na(+),K(+)-ATPase, thus enabling to preserve its normal function in regulation of intracellular homeostasis of Na(+) and K(+) ions.
Asunto(s)
Antioxidantes/farmacología , Carbolinas/farmacología , Diabetes Mellitus Experimental/tratamiento farmacológico , Indoles/química , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Estreptozocina/farmacología , Animales , Sitios de Unión , Membrana Celular/metabolismo , Masculino , Miocardio/patología , Ratas , Ratas Wistar , Cloruro de Sodio/farmacologíaRESUMEN
(2-benzyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole-8-yl)-acetic acid (compound 1), a novel aldose reductase inhibitor, was assayed for efficacy and selectivity to inhibit rat lens aldose reductase under in vitro conditions by using enzyme preparations obtained from diabetic animals. The inhibitory efficiency was characterized by IC(50) in micromolar region. Enzyme kinetics analysis revealed uncompetitive type of inhibition, both in relation to the D,L-glyceraldehyde substrate and to the NADPH cofactor. In testing for selectivity, comparisons to rat kidney aldehyde reductase, an enzyme with the highest homology to aldose reductase, was used. The inhibition selectivity of the compound tested was characterized by selectivity factor around 20 and was even slightly improved under conditions of prolonged experimental diabetes. These findings were identical with those in the control rats. To conclude, the inhibitory mode, efficacy and selectivity of compound 1, a novel aldose reductase inhibitor, was preserved even under the conditions of prolonged STZ-induced experimental diabetes of rats.
Asunto(s)
Acetatos/farmacología , Aldehído Reductasa/antagonistas & inhibidores , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/enzimología , Indoles/farmacología , Riñón/enzimología , Cristalino/enzimología , Animales , Femenino , Masculino , Ratas , Ratas Wistar , EstreptozocinaRESUMEN
Overload of reactive oxygen species during diabetes is known to impair cellular homeostasis and to promote deterioration of membrane function in the organism. The aim of the present study was to examine the effect of dietary supplementation with the pyridoindole atioxidant stobadine on functional properties of the renal Na, K-ATPase in diabetic rats. After 16 weeks of streptozotocin-induced diabetes (single intravenous dose of streptozotocin; 55 mg/kg), a significant inhibition (by 35%-42%) of the enzyme was observed throughout the range of NaCl 2-100 mmol/l, probably as an event of altered functional properties of Na,K-ATPase, suggested by the 42% decrease of the V(max) value. Administration of 0.05% (w/w) stobadine in the diet dramatically improved the function of renal Na,K-ATPase in diabetic rats with regard to sodium handling, as suggested by significant stimulation (by 104%-77% in accordance with increasing concentration of NaCl) of the enzyme over the whole NaCl concentration range investigated. This stimulatory effect was accompanied by an increase of V(max) value to the level of nondiabetic rats on standard diet. In conclusion, stobadine was found to antagonise the negative effects of diabetes on the renal Na,K-ATPase, preserving its normal function in regulation of intracellular homeostasis of Na(+) and K(+) ions.
Asunto(s)
Antioxidantes/farmacología , Carbolinas/farmacología , Diabetes Mellitus Experimental/metabolismo , Riñón/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Sodio/metabolismo , Animales , Antioxidantes/administración & dosificación , Carbolinas/administración & dosificación , Diabetes Mellitus Experimental/enzimología , Dieta , Riñón/enzimología , Cinética , Masculino , Ratas , Ratas WistarRESUMEN
The aim of this study was to assess the molecular basis of renal Na,K-ATPase disturbances in response to NO-deficient hypertension induced in rats by NO-synthase inhibition with 40 mg/kg/day N(G)-nitro-L-arginine methyl ester (L-NAME) for four weeks. After 4-week administration of L-NAME, the systolic blood pressure (SBP) increased by 30 %. Three weeks after terminating the treatment, SBP recovered to control value. When activating the Na,K-ATPase with its substrate ATP, a 36 % increase in K(m) and 29 % decrease in V(max) values were observed in NO-deficient rats. During activation with Na+, the V(max) was decreased by 20 % and the K(Na) was increased by 111 %, indicating a profound decrease in the affinity of the Na+-binding site in NO-deficient rats. After spontaneous recovery from hypertension, the V(max) remained at the level as in hypertension for both types of enzyme activation. However, in the presence of lower concentrations of substrate which are of physiological relevance an improvement of the enzyme activity was observed as documented by return of K(m) for ATP to control value. The K(Na) value for Na+ was decreased by 27 % as compared to hypertension, but still exceeded the corresponding value in the control group by 55 % thus resulting in a partial restoration of Na+ affinity of Na,K-ATPase which was depressed as a consequence of NO-dependent hypertension.
Asunto(s)
Adenosina Trifosfato/metabolismo , Hipertensión Renal/enzimología , Óxido Nítrico/deficiencia , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Sodio/metabolismo , Animales , Presión Sanguínea/efectos de los fármacos , Membrana Celular/enzimología , Inhibidores Enzimáticos/farmacología , Hipertensión Renal/inducido químicamente , Riñón/enzimología , Masculino , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Ratas , Ratas Endogámicas WKYRESUMEN
It was previously shown that 4 hours lasting inhibition of nitric oxide synthesis by administration of an L-arginine analogue, the A(G)-nitro-L-arginine methyl ester (L-NAME) changed the affinity of the Na-binding site of Na,K-ATPase thus resulting in elevation of enzyme activity especially at higher concentrations of sodium. Using the same experimental model, we focused our attention in the present study to the question of binding of ATP to the enzyme molecule in the left ventricle (LV), ventricular septum (S) and the right ventricle (RV) of the dog heart. Activation of the enzyme by increasing concentrations of ATP revealed a significant increase of the Vmax only in septum (by 38 %). The K(M) increased significantly in septum (by 40 %) and in left ventricle (by 56 %) indicating an altered sensitivity of the ATP-binding site of Na,K-ATPase in the hearts of NO-deficient animals. The alterations of Na,K-ATPase in its ability to bind and hydrolyze ATP are localized to the tissue surrounding the cavity of the left ventricle.
Asunto(s)
Miocardio/enzimología , Óxido Nítrico Sintasa/deficiencia , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Perros , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Ventrículos Cardíacos/enzimología , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/efectos de los fármacosRESUMEN
The Na(+),K(+)-ATPase is postulated to be involved in systemic vascular hypertension through its effects on smooth muscle reactivity and cardiac contractility. Investigating the kinetic properties of the above enzyme we tried to assess the molecular basis of alterations in transmembrane Na(+)-efflux from cardiac cells in spontaneously hypertensive rats (SHR). In the investigated group of SHR the systolic blood pressure and the heart weight were increased by 48% and by 60%, respectively. Upon activating the cardiac Na(+),K(+)-ATPase with substrate, its activity was lower in SHR in the whole concentration range of ATP. Evaluation of kinetic parameters revealed a decrease of the maximum velocity (Vmax) by 28% which was accompanied with lowered affinity of the ATP-binding site as indicated by the increased value of Michaelis-Menten constant (Km) by 354% in SHR. During activation with Na(+), we observed an inhibition of the enzyme in hearts from SHR at all tested Na(+) concentrations. The value of Vmax decreased by 37%, and the concentration of Na(+) that gives half maximal reaction velocity (KNa) increased by 98%. This impairment in the affinity of the Na(+)-binding site together with decreased affinity to ATP in the molecule of the Na(+),K(+)-ATPase are probably responsible for the deteriorated efflux of the excessive Na(+) from the intracellular space in hearts of SHR.
Asunto(s)
Adenosina Trifosfato/metabolismo , Hipertensión/fisiopatología , Miocardio/enzimología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Sodio/metabolismo , Adenosina Trifosfato/química , Animales , Presión Sanguínea , Peso Corporal , Activación Enzimática , Cinética , Masculino , Tamaño de los Órganos , Unión Proteica , Ratas , Ratas Endogámicas WKY , Valores de Referencia , Sarcolema/enzimología , Sodio/química , ATPasa Intercambiadora de Sodio-Potasio/químicaRESUMEN
The role that Na,K-ATPase plays in Na+ and K+ antiport through the sarcolemma, in cation-homeostasis in cardiomyocytes as well as in excitation-contraction coupling and cell signalling in the myocardium is now widely recognized. It was its key importance for the cell membrane function that kept this enzyme intensively studied during the last three decades and finally brought to its discoverer the deserved Nobel Prize. Almost weekly are appearing new data concerning structure, function, regulation and role of the Na,K-ATPase in different physiological and pathological conditions. The special importance of the enzyme for heart function as well as the great amount of data that is concerned specifically with the heart Na,K-ATPase and accumulated since yet, started to call for setting them in order. The present paper updates basically important data on the cardiac Na,K-ATPase in relation to its specific properties, molecular mechanisms of function, mode of action, humoral and pharmacological modulation, adaptability, physiological role and clinical aspects.