RESUMEN
Essential hypertension is often associated with high levels of plasma cholesterol or triglycerides. The relationships between plasma lipids and platelet lipids, membrane fluidity and functions in untreated hypertensive patients were investigated by measuring the fluorescence anisotropies of two fluorescent dyes (DPH and its cationic derivative, TMA-DPH, with different subcellular localization), cytosolic Ca2+ and pH, cyclic AMP content and aggregation to ADP and collagen. Hypercholesterolemia was found to be accompanied by a rise in platelet cholesterol content without changes in TMA-DPH or DPH anisotropies whereas hypertriglyceridemia was associated with a decreased cholesterol to phospholipid molar ratio, a decreased DPH anisotropy and a tendency of the cytosol to alkalinize. These results point out the differences between the effects of an acute cholesterol load and those of chronic hypercholesterolemia on platelet membrane microviscosity and aggregation. They demonstrate a strong association between plasma triglyceride levels and platelet membrane structure.
Asunto(s)
Plaquetas/fisiología , Hipertensión/sangre , Lípidos/sangre , Fluidez de la Membrana/fisiología , Adulto , Anciano , Plaquetas/metabolismo , Calcio/sangre , AMP Cíclico/sangre , Citosol/metabolismo , Femenino , Humanos , Concentración de Iones de Hidrógeno , Hipercolesterolemia/sangre , Hipertrigliceridemia/sangre , Masculino , Persona de Mediana Edad , Agregación Plaquetaria/fisiologíaRESUMEN
OBJECTIVE: To verify that platelet cytosolic pH is altered in essential hypertension and to investigate the mechanisms involved. METHODS: Cytosolic pH was determined in unstimulated platelets by the fluorescent indicator 2,7-bis-carboxyethyl-5(6)-carboxyfluorescein (BCECF). Membrane microviscosity was evaluated by the fluorescence anisotropies of diphenylhexatriene (DPH) and its cationic derivative trimethylamino-diphenylhexatriene (TMA-DPH). RESULTS: The cytosolic alkalinization previously observed in platelets from untreated hypertensive patients was confirmed. The buffering capacity appeared unaltered and the cytosolic pH was not modified by 50 mumol/l N-5-ethylisopropylamiloride, a specific inhibitor of the Na(+)-H+ exchange. Exposure to external Na(+)-free media produced an intracellular acidification that was similar in hypertensive and normotensive donors and maintained the cytosolic pH difference between the two groups. In the two blood pressure groups platelet cytosolic pH varied inversely with the steady-state anisotropy of TMA-DPH but not with that of DPH. Experimentally induced acidification of the cytosol by Na+ removal with or without nigericin treatment was accompanied by rises in TMA-DPH anisotropy. CONCLUSIONS: This study of platelet intracellular pH in essential hypertension confirms cytosolic alkalinization and demonstrates its association with changes in the dynamic properties of the platelet plasma membrane.
Asunto(s)
Plaquetas/metabolismo , Hipertensión/sangre , Fluidez de la Membrana/fisiología , Membrana Celular/fisiología , Citosol/metabolismo , Difenilhexatrieno/análogos & derivados , Femenino , Fluoresceínas , Polarización de Fluorescencia , Humanos , Concentración de Iones de Hidrógeno , Hipertensión/metabolismo , Masculino , Persona de Mediana EdadRESUMEN
Newborn spontaneously hypertensive rats (SHR) develop cardiac hypertrophy before a rise in blood pressure. Cytosolic pH (pHi) has been discovered to modulate cell growth and proliferation; therefore, we have investigated pHi in myocytes and fibroblasts from 3- to 4-day-old SHR and normotensive Wistar (W) and Wistar-Kyoto controls (WKY). The ratio of heart to body weight was higher in SHR than in W and WKY (7.56 +/- 0.10 v 6.21 +/- 0.10 and 5.98 +/- 0.14 mg/g in 10, 5, and 7 groups of 20 to 40 animals; P less than .001 for both). Cytosolic pH, determined with the fluorescent probe BCECF, was measured from the sixth to the eighth day in culture on confluent cells. The mean pHi was higher in myocytes from SHR than in those from W or WKY rats (7.19 +/- 0.03, N = 30, v 7.09 +/- 0.03 and 7.11 +/- 0.02, N = 25 and 30; P = .008 and .024, respectively). In contrast, pHi was similar in fibroblasts from the three strains (7.21 +/- 0.03, 7.18 +/- 0.03, and 7.19 +/- 0.02, N = 15, 15, and 14, in SHR, W, and WKY rats, respectively). External acidification induced similar decreases in pHi from SHR and WKY myocytes, maintaining higher pHi values in SHR myocytes along the entire external pH (pHo) range studied. The inhibition of Na+/H+ exchange by the amiloride derivative, ethylisopropylamiloride, decreased the steady-state pHi of myocytes independently of the initial pHi values. This study demonstrated a cytosolic alkalinization in contractile cardiac cells from SHR before a significant rise in blood pressure and in the absence of hemodynamic influences and specific plasma factors.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Fibroblastos/fisiología , Corazón/fisiología , Hipertensión/fisiopatología , Miocardio/citología , Animales , Cardiomegalia/fisiopatología , Células Cultivadas , Citoplasma/fisiología , Concentración de Iones de Hidrógeno , Hipertensión/patología , Contracción Miocárdica , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKYRESUMEN
Since Ca2+ ions seem to directly participate in the control of erythrocyte membrane structure and deformability and because cell Ca2+ metabolism has been repeatedly proposed to be modified in hypertension, the intracellular calcium ion concentration ([Ca2+]i) was investigated in red blood cells from hypertensive and normotensive subjects. [Ca2+]i was measured by using the fluorescent Ca2+ chelator fura-2. Red blood cell [Ca2+]i was increased in hypertensive compared with normotensive subjects in the whole population and further increased when hypertensive were compared with age-matched normotensive subjects. An inverse relation between age and [Ca2+]i was observed when calculated with blood pressure adjusted. In hypertensive patients, high [Ca2+]i values were associated with a reduced erythrocyte deformability. The initial rate of 45Ca2+ uptake did not differ between the two blood pressure groups. Similarly, when the extracellular Ca2+ concentration was elevated from 1 to 2 mmol/l, [Ca2+]i increased by 16 +/- 4% (p less than 0.03) in red blood cells from both groups, thus maintaining a significant difference between hypertensive and normotensive subjects. Under these conditions, the addition of 10(-7) mol/l nicardipine, a dihydropyridine Ca2+ antagonist, decreased [Ca2+]i by 15 +/- 4% (p less than 0.05) and 7 +/- 5% in erythrocytes from hypertensive and normotensive subjects, respectively, thereby reducing the difference in [Ca2+]i observed between these two groups. This nicardipine effect was positively correlated to the initial [Ca2+]i. In the presence of 5 mumol/l W7, a calmodulin antagonist, [Ca2+]i increased significantly only in erythrocytes from hypertensive patients (26 +/- 6%, p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Calcio/sangre , Eritrocitos/metabolismo , Hipertensión/sangre , Calmodulina/antagonistas & inhibidores , Calmodulina/sangre , Femenino , Humanos , Membranas Intracelulares/metabolismo , Masculino , Persona de Mediana Edad , Concentración Osmolar , Valores de Referencia , Reología , Sulfonamidas/farmacologíaRESUMEN
The influence of transmembrane Na+ and Ca2+ gradients on cytosolic pH (pHi) and free Ca2+ concentration ([Ca2+]i) have been examined in unstimulated human platelets with the aid of BCECF and Fura-2 fluorescent dyes. The removal of external Na+ (Na+o) acidified the cytosol in a pHo-dependent manner which was insensitive to EIPA and DIDS, the inhibitors of the Na+/H+ exchanger and bicarbonate transporters. Na+o removal also increased [Ca2+]i by 17 +/- 5%, but the amplitude of the concomitant acidification was independent on Ca2+ influx or cytosolic Ca2+ concentration. In contrast, in the presence of 145mM Na+o, a rise in external Ca2+ concentration from 1 to 2mM increased [Ca2+]i by 38 +/- 11% and acidified the cytosol by 0.16 +/- 0.04 pH units. These results indicated that, in resting human platelets, the transmembrane Na+ gradient is a major determinant of pHi. Two Na(+)-dependent processes have been found: one is triggered by an external acidification and the other activated by a rise in Ca2+ influx or cytosolic concentration.
Asunto(s)
Plaquetas/fisiología , Calcio/fisiología , Sodio/fisiología , Citosol/metabolismo , Homeostasis/fisiología , Humanos , Concentración de Iones de HidrógenoRESUMEN
Both marked hypercholesterolemia and severe hypertension have been reported to be associated with an enhanced sensitivity of blood platelets to activating agents. To investigate a possible mutual synergistic effect of moderate hypercholesterolemia and mild hypertension on platelet reactivity, we studied in 29 patients the response to aggregating agents, ADP and collagen, and the intracellular cyclic AMP content and cytosolic Ca2+ concentration that participate, respectively, as inhibitory and stimulatory mediators in platelet responses. When compared to age- and blood pressure-matched patients with normal or slightly elevated plasma cholesterol, the patients with total platelet cholesterol higher than 6.4 mM were characterized by a decreased response to collagen and ADP (14.5 +/- 3.0 vs. 23.8 +/- 2.0 a.u. and 17.7 +/- 4.5 vs. 26.9 +/- 2.7 a.u., respectively), a tendency to a reduced cAMP content both in the basal state and after phosphodiesterase inhibition by Ro-15 2041 (2.83 +/- 0.18 vs. 3.26 +/- 0.22 mumol/10(8) cells and 4.57 +/- 0.29 vs. 5.38 +/- 0.36 mumol/10(8) cells, respectively), and no change in cytosolic Ca2+ concentration (190 +/- 11 vs. 203 +/- 13 nM). After a chronic treatment with nitrendipine (20 mg/day for 6 months), blood pressure, platelet [Ca2+]i and cAMP content decreased in the patients with normal or moderately elevated hypercholesterolemia (p less than 0.001, less than 0.001, and less than 0.05, respectively), but these effects were attenuated or absent in the patients with higher hypercholesterolemia. Plasma lipids and the platelet-aggregating response to ADP and collagen were unchanged by this long-term nitrendipine treatment in both groups.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Plaquetas/efectos de los fármacos , Presión Sanguínea/efectos de los fármacos , Hipercolesterolemia/fisiopatología , Hipertensión/fisiopatología , Nitrendipino/farmacología , Plaquetas/química , Calcio/sangre , AMP Cíclico/sangre , Femenino , Humanos , Concentración de Iones de Hidrógeno , Hipercolesterolemia/sangre , Lípidos/sangre , Masculino , Persona de Mediana Edad , Nitrendipino/administración & dosificación , Agregación Plaquetaria/efectos de los fármacosRESUMEN
Variations of Ca2+ influx (evaluated by the initial rate of 45Ca2+ uptake) and cytosolic free Ca2+ concentration ([Ca2+]i, measured with fura-2) were investigated in human erythrocytes. When external Ca2+ concentration ([Ca2+]o) rose from 1 to 2 mM, the initial rate of Ca2+ influx nearly doubled whereas [Ca2+]i increased only by 15%. Nicardipine dose-dependently decreased both initial rate of Ca2+ influx and [Ca2+]i (up to 53 and 18%. respectively at 10(-6) M). The less marked changes in [Ca2+]i than in Ca2+ influx indicate a partial adjustment of the Ca2+ extruding-pump activity to of Ca2+ influx. In vivo administration of nicardipine reduced [Ca2+]i only when its initial value exceeded 80 nM and prevented the rise in [Ca2+]i induced by the increase in [Ca2+]o. Our results indicate that nicardipine may reduce Ca2+ influx in human erythrocytes and participate in the control of [Ca2+]i when elevated.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/sangre , Eritrocitos/metabolismo , Nicardipino/farmacología , Canales de Calcio/efectos de los fármacos , Citosol/efectos de los fármacos , Citosol/metabolismo , Relación Dosis-Respuesta a Droga , Eritrocitos/efectos de los fármacos , Humanos , Nicardipino/administración & dosificación , Vanadatos/farmacologíaRESUMEN
The kinetic properties of the Na+/H+ exchanger in human platelets were investigated by direct measurements of pHi as detected with the fluorescent dye, BCECF. In acid-loaded cells, the antiporter displayed a hyperbolic dependence regarding external Na+ with an apparent Km of 38 +/- 4 mM (pHo 7.2 at 25 degrees C) whereas its pHi-dependent activation between 7.3 to 6.4 did not obey a Michaelian model. External acidification from 7.7 to 6.5 decreased significantly the initial rate of Na(+)-dependent H+ efflux. The amiloride derivative, ethylisopropylamiloride blocked this exchanger and exerted a non-competitive inhibition with respect to Na+o (Ki = 17 nM). The cation selectivity of the external site of the antiporter was Na+ greater than Li+ greater than K+ and choline. These results indicate that the BCECF technique allows to evaluate the main features of the Na+/H+ exchanger in human platelets, which possesses kinetic properties similar to those reported in other cell types.
Asunto(s)
Plaquetas/metabolismo , Proteínas Portadoras/metabolismo , Concentración de Iones de Hidrógeno , Sodio/metabolismo , Amilorida/análogos & derivados , Amilorida/farmacología , Fluoresceínas/química , Humanos , Técnicas In Vitro , Cinética , Nigericina/química , Intercambiadores de Sodio-HidrógenoRESUMEN
Inhibition of the Na(+)-K+ pump by digitalis compounds has been reported to increase intracellular Na+ and Ca2+ concentrations and to stimulate Na(+)-H+ exchange. The activity of endogenous digitalis-like compounds, proposed to promote natriuresis and to raise blood pressure, has been found to be increased in volume expansion and hypertension. The enhanced cytosolic [Ca2+] present in platelets from hypertensive patients may thus originate from inhibition of the Na(+)-K+ pump by endogenous inhibitors, enhanced mobilization of internal Ca2+ stores due to phospholipase C activation and/or structural membrane defects. In unstimulated platelets from essential hypertensives, the increase in [Ca2+]i depends on external Ca2+, thereby underlining the importance of Ca2+ influx. The observation that [Ca2+]i was also enhanced in erythrocytes (p = 0.03) demonstrates that intracellular stores are not required for this rise. Plasma digitalis-like activity was positively correlated with platelet [Ca2+]i (inhibition of renal Na+,K(+)-ATPase, competition with ouabain binding, p less than 0.01). Platelet [Ca2+]i also rose during chronic digoxin administration (p less than 0.02) but not after acute in vitro ouabain treatment. The alkalinisation of platelet cytosol (p = 0.005) also agrees with the stimulation of the Na(+)-H(+)-exchange. In conclusion, these results are compatible with a participation of endogenous Na(+)-K+ pump inhibitors in the control of cytoplasmic [Ca2+] and cell excitability.
Asunto(s)
Plaquetas/metabolismo , Proteínas Sanguíneas/metabolismo , Calcio/sangre , Digoxina , Hipertensión/sangre , Saponinas , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Adulto , Cardenólidos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Potasio/sangre , Potasio/metabolismo , Serotonina/sangre , Sodio/sangre , Sodio/metabolismoRESUMEN
Essential hypertension is associated with various cell abnormalities, including alterations in the metabolism of intracellular messengers, such as cytosolic free Ca2+ and cyclic AMP or IP3. Intracellular pH is implicated in the regulation of number vital functions, including cell metabolism, division and response to various stimuli. We have measured cytosolic H+ concentration ([H+]i) in human platelets and investigated a possible relationship with free Ca2+ concentrations ([Ca2+]i) in hypertensive patients. [H+]i was determined with the pH sensitive fluorescent probe BCECF in platelets from 15 normotensive subjects and 15 patients with mild to moderate essential hypertension, free from medication, il any, for at least two weeks. Donor characteristics are indicated in the table. (table; see text) [H+] cytosolic from hypertensive patients was significantly lowered by 21 p. 100 compared to normotensive values (table). Cytosolic free Ca2+ concentrations, measured with the Ca2+ fluorescent probe Fura2, were significantly increased by 19 p. 100 in platelets from hypertensive patients when compared to those of normotensive donors. Taken all together, [H+]i and [Ca2+]i varied inversely (r = -0.421, p = 0.02) in these thirty donors and tends to be correlated in the essential hypertensive patients (r = 0.490, p = 0.06). This correlation remained significant at constant age systolic and diastolic blood pressures. The simultaneous rise in [H+]i and [Ca2+]i in platelets from essential hypertensive patients are compatible with their observed enhanced sensitivity to several aggregating agents. This alkalinisation, if present in the vascular smooth muscle cells, may also reflect facilitated cell proliferation and increased sensitivity to stimulating agents, two parameters implicated in the rise of arterial blood pressure.
Asunto(s)
Plaquetas/metabolismo , Calcio/metabolismo , Concentración de Iones de Hidrógeno , Hipertensión/sangre , Adulto , Citosol/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Alterations in the metabolism of intracellular messengers, such as calcium and cyclic adenosine 5'-phosphate (cAMP), have been reported in essential hypertension. Since intracellular pH (pHi) participates in the control of fundamental cell functions, we looked for changes in platelet cytosolic H+ concentration [( H+]i) in hypertension and investigated whether or not its impaired metabolism is linked to the calcium handling abnormalities. The fluorescent pH indicator BCECF has been used to evaluate intracellular H+ concentration in platelets, unstimulated ex vivo, from normotensive (n = 20) and hypertensive patients (n = 20). Cytosolic [H+] was 20% lower in hypertensive than in normotensive subjects (49.5 +/- 3.4 and 61.8 +/- 2.2 nmol/l cells, respectively, P less than 0.005; mean pHi values were 7.21 and 7.33, respectively). Platelet cytosolic H+ and free Ca2+ concentrations ([Ca2+]i) were determined in parallel in 15 normotensive and 15 hypertensive patients. [Ca2+]i was found to be 19% higher (P less than 0.01), and [H+]i 22% lower (P less than 0.02), in the hypertensive patients compared with the normotensive subjects. Platelet pHi and [Ca2+]i were increased simultaneously in some hypertensive patients. These results are compatible with the hypothesis of an in vivo activation of platelets in hypertension. If a similar alkalinization exists in smooth muscle cells, it may participate in cell proliferation and in an enhanced sensitivity to agonists, two parameters thought to be involved in blood pressure elevation.