Rapid determination of the elevated Na(+)-H+ exchange in platelets of patients with essential hypertension using an optical swelling assay.

Accumulating evidence suggests an increased activity of the Na(+)-H+ exchanger in essential hypertension. The present investigation aimed at developing a test for routine measurements. Platelet-rich plasma was added directly to a cuvette placed into an aggregometer containing 140 mmol/l sodium propionate medium (pH 6.7, 37 degrees C). The accumulation of intracellular sodium due to activation of Na(+)-H+ exchange results in an osmotic cell swelling, which is detectable as a decrease in optical density (OD). This reaction reflects activation of the Na(+)-H+ exchanger since we observed (1) a dose-dependent inhibition by amiloride (inhibition constant, Ki = 10 mumol/l) and ethylisopropylamiloride (Ki = 0.07 mumol/l) and (2) a dependence on extracellular sodium of the OD changes. Electron microscopy of sodium propionate-treated platelets revealed a general swelling and a distinct decrease in electron density of the cytosol without other significant alterations. Quantification of Na(+)-H+ exchange activities was accomplished by calculating rate constants of the recorded changes in OD. Application of this assay to 20 essential hypertensives and 32 normotensives demonstrated an increased activity of the Na(+)-H+ exchanger in essential hypertensives (rate constants 29.8 x 10(-3) per s versus 21.7 x 10(-3) per s).

Platelet Na(+)-H+ exchanger activity in normotensive and hypertensive subjects: effect of enalapril therapy upon antiport activity.

OBJECTIVE: Primary hypertension has been reported to be associated with an enhancement of Na(+)-H+ exchange. However, details of the kinetic properties of the Na(+)-H+ exchanger in hypertensives and its dependence upon age and gender in normotensives are unknown. PARTICIPANTS: We determined the activity of the platelet Na(+)-H+ exchanger in 20 normotensives and 26 untreated primary hypertensives. INTERVENTIONS: In eight hypertensive individuals antihypertensive treatment was interrupted for 1 week. Treatment for 6 weeks with a daily single dose of 10 mg enalapril decreased mean arterial pressure to 105.7 +/- 11.6 mmHg. METHODS: Platelets were loaded with the intracellular pH (pHi) indicator 2'-7'-bis-carboxyethyl-5(6)-carboxyfluorescein (BCECF) and acidified by propionic acid. Initial velocities of pH recovery were determined and used for calculation of maximum velocity (Vmax), baseline pHi and the pHi value for half maximal activation (pH0.5) of the Na(+)-H+ exchanger in each individual. RESULTS: In normotensives, Vmax averaged 0.05 +/- 0.01 dpHi/min independently of age, gender and actual diastolic blood pressure. In hypertensives, two different subgroups were defined bearing either low or high Na(+)-H+ exchange activity. Values of pHi and pH0.5 were identical in all subgroups irrespective of Vmax. The twofold enhancement of Na(+)-H+ exchange in the second group was preserved in thrombin-stimulated platelets. Vmax values remained unaffected by enalapril treatment. CONCLUSIONS: Enhanced Na(+)-H+ exchange activity in hypertensives is primarily characterized by an increase in Vmax. This enhancement is refractory to antihypertensive treatment and therefore appears to be a relatively fixed parameter.

[A membrane-thin-layer oxygenator for experimental organ perfusions]. / Ein Membran-Dünnschichtoxygenator zur experimentellen Organperfusion.

The authors describe device and functioning of a new efficacious membrane thin-layer oxygenator, specially developed for experiments on the isolated hearts of small animals. Compared with customary oxygenators with direct oxygenation, advantages arise from the separation of gas and perfusion solution, and its small volume for filling (7,5 ml). The diffusion capacity of the oxygenator (exchange surface 980 cm2) was tried by means of a hemoglobin-free modified Krebs-Henseleit solution with various flow values at 37 degrees C, depending on the gage of the layer. At the optimal gage of 0,15 mm and a flow value of 15 ml/minute a perfusion solution can be oxygenated from 0 to 570 torr, and an airequilibrated solution up to 646 torr. Measurements of oxygen consumption in the isolated perfused rat heart proved that the diffusion capacity of the membrane thin-layer oxygenator will suffice the oxygenation of the coronary minute volume of the arrested heart according to Langendorff as well as of the working heart according to Morgan. Primarily this oxygenator is useful for experimental organ perfusion in metabolic and functional tests, and organ preservation. Its functioning principles, however, also permit its use as thins-layer dialysator.

Determination of infarct size in rats with 86rubidium.

A quick method is presented which allows the determination of infarct sizes within hearts of experimental animals like rats. The method is based on the drop of 86Rb uptake in infarcted hearts, and has been compared with infarct sizes determined morphometrically. The standard deviation of this method is low (6.5%) because an internal standard in used (noninfarcted myocard - usually right ventricle tissue - has been used).

[Coronary flow measurements on the isolated rat heart following experimental coronary artery stenosis]. / Koronarflussmessungen am isolierten Rattenherzen nach experimenteller Koronararterienstenose

A stenosing proliferation of the vascular wall was produced by exposing the Ramus descendens of the rat's left coronary artery to continued mechanical stimulation. Two weeks after the operation the hearts were perfused according to a modified Langendorff method, and the flow rate was measured under aerobic conditions and following short-time anoxia. Under either experimental conditions, the flow rate proved to be significantly decreased.

Chemical and morphological studies of rat myocardium after application of a necrogenic dose of isoproterenol.

In male Wistar rats myocardial necroses were produced by application of 80 mg of isoproterenol per kg of body weight. 2 and 48 hours after isoproterenol administration the Na+ and K+ contents were determined. At these times Na+ presented increases of 128 and 83% respectively, and K+ of 13 and 8% respectively as compared to the controls. These results were related to the morphological changes appearing at the same time, and were discussed with references to the literature. Taking edema and vasodilatation into account, the degree of cell damage can be estimated on the basis of the chemical data available.