Marie-Aude Devynck

Fluorescence measurements of free Ca2+ concentration in human erythrocytes using the Ca2+-indicator fura-2

We report here the use of the fluorescent Ca2+-chelator fura-2 to directly measure free Ca2+ concentration within intact human erythrocytes and the influence of viscosity on the fluorescence of this probe. The bright fluorescence of fura-2 has permitted the use of low concentrations of indicator and cells, thus minimizing the screening effect and the intrinsic fluorescence of haemoglobin. Erythrocytes (10(8) cells/ml) were loaded with 0.5 microM fura-2AM then diluted at 10(7) cells per ml for measurements. The extracellular signal was suppressed by addition of manganese ions just before recording spectra. Under these conditions, a blood sample of 100 microliter was sufficient for analysis. To study the influence of viscosity on fura-2 fluorescence, gelatin and polyvinylpyrrolidone at various concentrations were added to a physiological buffer to perform fura-2-Ca fluorescence standard curves. Fluorescence intensities and the apparent affinity constant for Ca2+ were modified by viscosity. When intra-erythrocytic viscosity was simulated with 21 g/l polyvinylpyrrolidone to obtain a mean viscosity of 14 mPa.s similar to that observed in human erythrocytes, the mean value of free Ca2+ concentration measured in erythrocytes from healthy subjects was 78 +/- 16 nM (mean +/- S.D., n = 29).

Evidence for structural changes in erythrocyte membranes of spontaneously hypertensive rats. A fluorescence polarization study

Summary Diphenylhexatriene (DPH) was used as a fluorescent probe to detect possible structural differences between erythrocyte membranes of spontaneously hypertensive (SHR) and normotensive (WKY) rats which differ by several functional and chemical aspects. Fluorescence polarization was found constantly higher in SHR than in WKY erythrocyte membranes. In adult rats, the activation energy of the fluorescent probe motion appeared lower in SHR than in WKY erythrocyte ghosts. These results represent an additional evidence for an erythrocyte membrane abnormality in genetic hypertension of the rat, especially so as the difference is observed in young animals before the rise in blood pressure.

Variations in the number of uterine angiotensin receptors following changes in plasma angiotensin levels

3H-labelled angiotensin II binding to receptor sites was studied in plasma membranes isolated from myometrial homogenates of uterine horns. Removal of the kidneys, which results in the disappearance of plasma angiotensin II, was followed 19 h after nephrectomy by an increase in the number of uterine receptor sites without significant variation in the apparent dissociation constant. Acute pressor i.v. injection of angiotensin II into nephrectomized rats immediately before removing uteri, did not affect the number of uterine angiotensin receptors, whereas long-lasting angiotensin infusion did reduce the number of receptors. These changes cannot be accounted for by variations in the occupancy of receptor sites. These results demonstrate that the number of angiotensin receptors, at least in uterine contractile cells, is affected by chronic variations of endogenous angiotensin levels. The relation between the specific supersensitivity to angiotensin II observed in uteri from nephrectomized rats and the variations at the receptor level is discussed.

Post-nephrectomy changes in adrenal angiotensin II receptors in the rat; influence of exogenous angiotensin and a competitive inhibitor

3H-angiotensin binding sites have been studied in a particulate fraction prepared from rat adrenal glands. This binding is rapid and reversible, of high affinity (KD29 degrees C = 3-5 X 10(-9) M) and with demonstrable specificity for the angiotensin II octapeptide. The number of binding sites varies with endogenous angiotensin levels: nephrectomy is followed by an increase in number of binding sites. This increase can be prevented by chronic angiotensin II administration and, to a lesser extent, by administration of Sar1,Ala8-angiotensin II, a competitive antagonist of the hormone. No variation in the equilibrium dissociation constant accompanied these changes in binding capacity. The post-nephrectomy increase in capacity is time-dependent, with a lag period of 24-40 h. The observed changes in receptor concentration do not appear explicable on the basis of receptor occupancy. Accordingly, angiotensin II receptors in the rat adrenal appear to be dependent on circulating angiotensin levels as previously reported for rat uterus.

Stimulation by nifedipine of calcium transport by cardiac sarcolemnal vesicles from spontaneously hypertensive rats

The effects of the calcium antagonist nifedipine on the binding and ATP-dependent accumulation of calcium by cardiac plasma membranes from spontaneously hypertensive rats (SHR) and from their normotensive controls (WKY) were studied at free calcium concentrations of 2 X 10(-8) M and 4 X 10(-7) M corresponding to very high affinity and high affinity binding sites respectively. Nifedipine did not significantly modify calcium binding to either class of sites in SHR or WKY membranes. In contrast, in a free calcium concentration of 2 X 10(-8) M, nifedipine enhanced ATP-dependent Ca2+ transport. The concentration of nifedipine required for significant stimulation was smaller in SHR than in WKY membranes (10(-7) M and 10(-5) M respectively). Another calcium antagonist, D600, did not modify ATP-dependent calcium accumulation by SHR or WKY vesicles. These results raise the question of the mechanism of action of nifedipine on the calcium pump and confirm the presence of abnormalities in cardiac plasma membranes from young SHR, rendering them more sensitive than WKY membranes to the calcium antagonist nifedipine.

Angiotensin receptors in rat uterine membranes

Angiotensin II is a polypeptide hormone possessing a potent contractile effect on various smooth muscle cells [1]. Hormone-receptor interaction is the first event in the series of molecular processes leading to a hormonal effect on a target cell. Extensive investigations performed during the last decade have demonstrated that receptors for polypeptide hormones are located on the plasma membrane (for review, see [2]). Previous studies from our laboratory have characterized angiotensin II receptor sites of rabbit aortae plasma membranes [3]. In the present investigation, we report the principal properties of angiotensin II receptors of the plasma membrane of rat uterine myometrial cells which appear to be essentially similar to those of rabbit aorta smooth muscle cells.

LCEC monitoring of 5-hydroxyindolic compounds in the cerebrospinal fluid of the rat related to sleep and feeding

A new technique which allows for both the chronic withdrawal of CSF and continuous recording of EEG sleep patterns and food intake in the freely moving rat is described. Liquid chromatography with electrochemical detection (LCEC) was used for the direct assay of tryptophan metabolites in the CSF. Both 5-hydroxyindolacetic acid (5-HIAA) and 5-hydroxytryptophan (5-HTP) were easily detectable. However, serotonin (5-HT) levels were relatively low and 5-hydroxytryptophol (5-HTPhol) and N-methylserotonin (N-Me-5HT) were undetectable in several cases. The continuous monitoring of 5-HIAA and 5-HTP indicated stable values throughout the 3-hr experiments during which no food or small meals were consumed. In the rat which consumed a large meal, both 5-HIAA and 5-HTP significantly increased following that meal. This increase in metabolites may be the result of an increased availability of tryptophan to the brain as a result of the meal. Although this study is preliminary, the described technique can provide further information about the possible relationship between behavioral (sleep and/or feeding) changes and the concomitant neurochemical fluctuations.

In vivo dopamine release from the anterior hypothalamus of the rat.

The release of dopamine from the anterior hypothalamic/preoptic region of the anesthetized rat was investigated in vivo using a superfusion technique with a push-pull cannula. Dopamine was measured electrochemically after separation by liquid chromatography. The spontaneous release of dopamine was very low but detectable in some experiments. An inhibitor of monoamine oxidase (pargyline) and the immediate precursor of dopamine (L-DOPA) were added to synthetic cerebrospinal fluid superfusing the area. When these substances were present dopamine release was increased considerably and appeared to be stable for a long period of time. Mechanisms contributing to the formation of newly synthetized dopamine are discussed in relation to the releasing effect of d-amphetamine and the inhibiting effect of calcium-free medium. The functional significance of dopamine release was shown by the increased release of dopamine following an increase in blood pressure obtained by an intraarterial injection of blood. Finally, ventral noradrenergic bundle lesion on the same side of the superfusion site considerably enhanced dopamine release which may indicate an inhibitory control of dopamine release by noradrenergic neurons. Furthermore, this experimental procedure provides valuable means for analyzing the effects of pharmacological as well as other manipulations on the dopamine released from a superfused brain area in vivo.

Circulating Digitalis-Like Compounds in Essential Hypertension

Inhibitors of the Na+ pump have been proposed as participating in sodium excretion, extracellular fluid regulation, and in the rise of blood pressure. The presence of digitalis-like compounds in human plasma has been investigated by comparing the effects of plasma extracts to those of ouabain in 4 tests. - competition with ouabain for binding to the Na+ pump, - inhibition of Na+ and K+ dependent hydrolysis - inhibition of serotonin uptake by human platelets - central hypertensive effect Plasma fractions exhibited digitalis-like properties in the 4 tests. The effects of plasma extracts of 42 normotensive subjects (21 with family history of hypertension) and 38 patients with essential hypertension (15 with antihypertensive treatment) and 9 patients with chronic renal failure were compared. Plasma from Forty per cent of untreated hypertensive patients and normotensives with hypertensive heredity had a high inhibition level. Inhibition was enhanced in beta-blocker treated patients and decreased in those on diuretics. No digitalis-like activity was observed in uremic plasma. These observations strongly suggest the presence of digitalis-like compound(s) in human plasma and point to its possible association with hypertension.

Plasma Renin Activity and Adrenal Angiotensin II Receptors in Fetal, Newborn, Adult and Pregnant Rabbits

Plasma renin activity (PRA) and adrenal angiotensin II receptors have been studied simultaneously in the rabbit at various stages of development. In the fetus both parameters show very low values but increase rapidly during the last 4 days of gestation. PRA reaches a maximal level in the early post-natal period but the concentration of adrenal angiotensin II receptors continues to increase further up to the adult state. In adult females, pregnancy results in an initial rise in PRA and adrenocortical angiotensin II receptors. However, PRA remains at a high level throughout the gestation period whereas the number of adrenocortical angiotensin II receptors decreases progressively as pregnancy progresses. The role of circulating angiotensin in the regulation of the concentration and affinity of its receptor sites is discussed.