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Biomedical subjects

M Wehling

Publications and source records attributed to M Wehling.

At least 109 records · Page 6Linked to original sources
PubMed1996

Nongenomic effects of aldosterone on intracellular pH in vascular smooth muscle cells.

The aim of the present study was to investigate rapid effects of aldosterone and other steroids on intracellular pH of vascular smooth muscle cells and to compare these effects with those of peptide hormones. After addition of 100 nmol/L aldosterone, initial acidification is followed by significant alkalinisation occurring within two minutes, while 1 mumol/L hydrocortison does not affect intracellular pH. The initial response to 100 nmol/L angiotensin II is similar; however, subsequent alkalinization is not seen for this agonist. PDGF induces an initial acidification followed by a minor recovery so that cells remain acidified for eight minutes. Both pH recovery after angiotensin II and alkalinization after aldosterone were blocked in sodium-free medium. These results demonstrate rapid effects of aldosterone on intracellular pH in vascular smooth muscle cells, which include final alkalinization not seen after angiotensin II or PDGF.

Aldosterone↗
PubMed1996

Biotin-labelled and photoactivatable aldosterone and progesterone derivatives as ligands for affinity chromatography, fluorescence immunoassays and photoaffinity labelling.

New derivatives of progesterone and aldosterone were synthesized and functionally tested with commercially available antibodies. The covalent labelling of antibodies specific for aldosterone and progesterone was detected by SDS/PAGE analysis and subsequent autoradiography after using 3-(O-carboxymethyl)-oximino-(3-[125I]iodo-4-azidosalicylamidobu tylamine) derivatives of aldosterone and progesterone, respectively, as photoactivatable radioligands. Labelling was not observed in the presence of an excess of the unlabelled steroid. Aldosterone was labelled with biotin and used as a tracer in a time-resolved fluorescence immunoassay. The nonradioactive tracer is highly selective for its antibody-binding site, with almost no detectable cross-reactivity for other steroids. Biotin-labelled progesterone was immobilized by avidin-agarose and used for affinity chromatography. This yielded a more than 20-fold enrichment of an anti-progesterone polyclonal antibody. These results demonstrate that derivatives of steroids are particularly useful for the development of nonradioactive assays for the determination of natural steroids and may be also useful for the detection of specific binding sites in biological material such as plasma membranes.

Affinity Labels↗
PubMed1996

Spontaneous changes of heart rate, blood pressure, and ischemia-type ST-segment depressions in patients with hypertension without significant coronary artery disease: beneficial effects of beta-blockade.

In hypertensives, a reduced coronary flow reserve is observed and may contribute to angina pectoris and silent (painless) myocardial ischemia, which frequently occur in these patients even in the absence of coronary artery disease (CAD). To assess the frequency of ischemia-type ST-segment depressions in these patients and the influence of heart rate (HR) and blood pressure (BP) as major determinants of myocardial oxygen demand and to test the effects of beta-blocker therapy (10-20 mg betaxolol/day for 4 weeks) on these variables, simultaneous 24-h Holter and 24-h ambulatory BP monitoring was performed in 19 patients with hypertension (age, 43-71 years; nine women, 10 men) without CAD (stenosis < 50% in angiography). Before treatment, 25 periods of significant ST-segment depressions with a total duration of 470 min were observed in nine patients. ST-segment depressions were significantly correlated with preceding increases in HR and the rate-pressure product. The majority (79%) of episodes with ST-segment depression were clinically painless. In this open study, beta-blockade significantly decreased the number of episodes with ST-segment depressions to six in four of 15 patients and the total duration to 38 min (p < 0.05). The data demonstrate that HR seems to be associated with the development of ischemic ST-segment deviations in patients with hypertension without CAD. Antihypertensive therapy in these patients should target not only sufficient BP control, but also reduction of ischemic events.

Adrenergic beta-Antagonists↗
PubMed1996

Nongenomic effects of aldosterone on phosphocreatine levels in human calf muscle during recovery from exercise.

Nongenomic in vitro effects of aldosterone on the sodium-proton antiport and intracellular second messengers have been described in human mononuclear leukocytes, vascular smooth muscle cells, and endothelial cells. To test the potential physiological relevance of these effects, an in vivo 31P magnetic resonance spectroscopy study on the human calf at rest and during exercise was performed in 10 healthy volunteers receiving either 1 mg aldosterone or placebo iv in a double blind, randomized, cross-over trial. Spectra were analyzed for phosphocreatine, ATP, phosphomonoesters, inorganic intracellular phosphate, and intracellular pH. Resting values remained unchanged by aldosterone. After isometric contraction of the calf (50% body weight for 3 min), phosphocreatine recovered to significantly higher levels after application of aldosterone compared with placebo. Other parameters were not significantly changed by aldosterone. Effects appeared immediately after isometric contraction and, thus, occurred within 8 min of aldosterone administration. They are, therefore, likely to represent the first contemporary evidence of nongenomic in vivo effects of aldosterone in man. These findings also point to an involvement of aldosteron in the acute stress adaptation of cellular oxidative metabolism in human muscle physiology.

Adult↗
PubMed1995

Rapid aldosterone signaling in vascular smooth muscle cells: involvement of phospholipase C, diacylglycerol and protein kinase C alpha.

Rapid in vitro effects of aldosterone (ALDO) on intracellular sodium, potassium and calcium, cell volume and the sodium-proton-antiport have been described in human mononuclear leukocytes and rat vascular smooth muscle cells (VSMC). These nongenomic effects are signaled through membrane receptors with a high affinity for aldosterone, but not for hydrocortisone. Effects of ALDO on the production of diacylglycerol (DAG) and protein kinase C alpha (PKC) were measured in VSCM by enzymatic assay and immunoblotting. DAG production was stimulated twofold by ALDO (> or = 1 nM) within 30 sec while hydrocortisone was inactive at concentrations of up to 1 microM. The inhibitors of phospholipase C, neomycin and U-73122 completely blocked this effect. PKC translocation from cytosol to membranes by ALDO occurred within 5 min, the extent of this effect was comparable to that of angiotensin II. These data demonstrate rapid intracellular signaling for ALDO in VSMC through phospholipase C, DAG and PKC in addition to calcium and inositol-1,4,5-trisphosphate as determined earlier.

Aldosterone↗
PubMed1995

Characterization and solubilization of novel aldosterone-binding proteins in porcine liver microsomes.

Using the radioligand [1,2,6,7-3H]aldosterone ([3H]aldosterone), specific binding sites for aldosterone were identified and characterized in microsomal preparations from porcine liver. The maximum binding capacity is approximately 700 fmol x mg-1 microsomal protein. The reversible binding of [3H]aldosterone was saturable and Scatchard analysis revealed two apparent dissociation constants (Kd), Kd1 < or = 11 nM and Kd2 = 118 nM. Binding was optimal at pH 7.2, thermolabile, and was reduced by more than 70% when membrane vesicles were pretreated with trypsin. Binding was selective for aldosterone with cortisol being a weak agonist at 1000-fold higher concentrations only. Among those detergents tested to optimize conditions for solubilization, n-octylglucoside (75 mM) was most favorable and solubilized 25% of the radioligand-binding protein complex in the undissociated form. These binding sites have unique pharmacological properties, which are similar to those found for aldosterone membrane binding in human lymphocytes and pig kidney, and for rapid aldosterone effects on sodium-proton exchange.

Aldosterone↗
PubMed1995

Looking beyond the dogma of genomic steroid action: insights and facts of the 1990s.

The genomic theory of steroid action has been the unquestioned dogma for the explanation of steroid effects over the past four decades. Despite early observations on rapid steroid effects being clearly incompatible with this theory, only recently has nongenomic steroid action been more widely recognized and led to a critical reappraisal of unsolved questions about this dogma. Evidence for nongenomic steroid effects is now coming from all fields of steroid research, and mechanisms of agonist action are being studied with regard to the membrane receptors and second messengers involved. A prominent example of a receptor/effector cascade for nongenomic steroid effects has been described for rapid aldosterone effects in various cell types, including lymphocytes and vascular smooth muscle cells. Rapid in vitro effects of aldosterone on the sodium proton antiport have been found in human lymphocytes, cultured vascular smooth muscle, and endothelial cells involving non-classical membrane receptors with a high affinity for aldosterone, but not for cortisol, and phosphoinositide turnover. Another important second messenger, [Ca2+]i, is consistently increased by aldosterone within 1-2 min. In vascular smooth muscle cells, calcium is released from perinuclear stores while in endothelial cells a predominant increase of subplasmalemmal calcium is seen. Effects are half-maximal at physiological concentrations of free aldosterone (0.1 nM), while cortisol is inactive up to 0.1 microM; the classical mineralocorticoid antagonist canrenone is ineffective in blocking the action of aldosterone. The data show that intracellular signaling for nongenomic aldosterone effects also involves calcium, but pathways of cell activation may vary between different cell types. Further evidence for nongenomic steroid effects is encountered presently for various groups of steroids such as neurosteroids, mineralocorticoids, vitamin D3, and sex hormones. Future research will have to target the cloning of the first membrane receptor for steroids and evaluate the clinical relevance of these rapid steroid effects.

Animals↗
PubMed1995

Nongenomic aldosterone effects: the cell membrane as a specific target of mineralocorticoid action.

Studies in extrarenal, nonepithelial cells such as human lymphocytes and smooth muscle cells indicate that aldosterone produces not only delayed genomic effects, but also rapid, non-genomic effects on transmembrane electrolyte movements. These non-genomic events involve the immediate activation of the sodium/proton-exchanger of the cell membrane at very low, physiological concentrations of aldosterone in both lymphocytes and cultured rat vascular smooth muscle cells. This new pathway for mineralocorticoid action is further characterized by a 10,000-fold selectivity for aldosterone over cortisol and the ineffectiveness of spironolactones, classical mineralocorticoid antagonists, as antagonists of the response. Aldosterone-specific binding sites have been demonstrated in the plasma membrane of human lymphocytes, with features identical to those seen for the rapid aldosterone effects in the same cells. As second messenger the inositol-1,4,5-trisphosphate pathway has been identified both in human lymphocytes and vascular smooth muscle cells, which respond over the same rapid time course. In addition, the aldosterone effect on inositol-1,4,5-trisphosphate production in vascular smooth muscle cells is sensitive to pertussis toxin, but not to cholera toxin, pointing to a possible involvement of G-proteins in the cellular signalling. This article reviews the data supporting a new, two-step model for successive non-genomic and genomic mineralocorticoid effects.

Aldosterone↗
PubMed1995

Rapid effects of aldosterone on sodium transport in vascular smooth muscle cells.

Increasing evidence has accumulated for rapid nongenomic steroid actions in various cell systems and, more recently, for rapid aldosterone effects on the Na(+)-H+ antiport in human mononuclear leukocytes. The aim of the present study was to demonstrate a rapid, nongenomic aldosterone action in rat vascular smooth muscle cells as a key effector cell in cardiovascular regulation. Basal 22Na+ influx in quiescent vascular smooth muscle cells was 22.1 +/- 1.9 nmol/mg protein per minute (mean +/- SEM, n = 9). Aldosterone (1 nmol/L) stimulated influx to 28.6 +/- 1.5 nmol/mg protein per minute after 4 minutes (n = 9, P < .05), with a half-maximal effect between 0.1 and 0.5 nmol/L; the effects were inhibited by ethylisopropylamiloride, the specific inhibitor of the Na(+)-H+ exchanger, demonstrating the involvement of this transport system in rapid effects of aldosterone. Hydrocortisone (1 mumol/L) was ineffective, and fludrocortisone and deoxycorticosterone increased influx with half-maximal effects at approximately 0.5 nmol/L. Canrenone, a classic antagonist of aldosterone action, did not inhibit stimulation by aldosterone at a 1000-fold excess concentration. Aldosterone significantly stimulated intracellular inositol 1,4,5-trisphosphate levels (P < .05) after 30 seconds; the inhibitors of phospholipase C, neomycin and U-73122, inhibited aldosterone-stimulated Na+ influx and increase of intracellular inositol 1,4,5-trisphosphate. The rapid stimulation of sodium transport in vascular smooth muscle cells and the pharmacological characteristics of this effect are clearly incompatible with the classic, genomic pathway of steroid action and represent further evidence for nongenomic effects of aldosterone.

Aldosterone↗
PubMed1995

Nongenomic effects of aldosterone on intracellular Ca2+ in vascular smooth muscle cells.

Genomic mechanisms of steroid action have been increasingly elucidated over the past four decades. In contrast, rapid steroid actions have been widely recognized only recently, and detailed analysis of the mechanisms involved are still lacking. The present article describes rapid effects of mineralocorticoid hormones on free intracellular calcium in vascular smooth muscle cells as determined by fura 2 spectrofluorometry in single cultured cells from rat aorta. These effects are almost immediate and reach a plateau after only 3 to 5 minutes and are characterized by high specificity for mineralocorticoids versus glucocorticoids. The potent mineralocorticoids aldosterone and fludrocortisone are agonists with estimated apparent EC50 values of approximately 0.1 to 0.5 nmol/L; deoxycorticosterone acetate is an agonist with an EC50 of approximately 5 nmol/L; and progesterone, cortisol, corticosterone, and estradiol have much lower potency (EC50 values of approximately 0.5 to 5 mumol/L). The effect of aldosterone is blocked by neomycin and short-term treatment with phorbol esters but augmented by staurosporine, indicating an involvement of phospholipase C and protein kinase C. The Ca2+ effect appears to involve the release of intracellular Ca2+, as shown by the inhibitory effect of thapsigargin; intriguingly, a relatively small maximum effect (approximately 40 nmol/L increase) is consistently seen. This mechanism operates at physiological subnanomolar aldosterone concentrations and appears to be a likely candidate for rapid fine tuning of cardiovascular responsivity. It may also contribute to known clinical features of mineralocorticoid action that are difficult to explain by the traditional genomic mechanism alone.

Aldosterone↗
PubMed1995

Left ventricular hypercontractility in hypertensive patients with anginal pain and normal coronary angiograms.

This study was designed to assess left ventricular contractility in hypertensive patients with normal coronary angiography and anginal pain. An abnormally high percentage of hypertensive patients (approximately 30%) undergoing cardiac catheterization because of anginal pain and/or exercise-induced ST-segment depressions has angiographically normal coronary arteries. Possible reasons for these signs of ischemia include a microvasculopathy, metabolic abnormalities and an increased oxygen consumption as a result of left ventricular hypercontractility which was studied here. Left ventricular volumes and ejection fraction were determined in 50 patients with arterial hypertension (23 men, 27 women, age 60 +/- 8 years, RR 154 +/- 24/91 +/- 12 mm Hg) by cardiac catheterization and computerized analysis of laevocardiographies. The control group were 50 normotensives (30 men, 20 women, age 57 +/- 12 years, RR 128 +/- 12/76 +/- 8 mm Hg) without coronary artery disease. The angiographical data were correlated with age, sex. ECG, echocardiography, laboratory findings, medication and duration of hypertension. The left ventricular ejection fraction was significantly increased in the group of hypertensives (75.8 +/- 6.3 vs. 67.7 +/- 5.0%, p < 0.001). This difference was mainly due to a significantly reduced endsystolic left ventricular volume (37.1 +/- 15.3 vs. 47.7 +/- 10.8 ml, p < 0.001); enddiastolic left ventricular volume was not significantly different (140.5 +/- 26.8 vs. 149.0 +/- 27.5 ml, p > 0.1). A hyposystolic form of hypertensive heart disease was not observed in this group of patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1995

Aldosterone specific membrane receptors, rapid activation of the sodium-hydrogen exchanger, and cardiovascular implications.

Functional studies in extrarenal non-epithelial cells such as smooth muscle cells and easily accessible human lymphocytes have demonstrated that aldosterone not only produces classical genomic effects, but also rapid non-genomic effects on transmembrane electrolyte movements. These involve the activation of the Na+/H+ exchanger of the cell membrane at very low, physiological concentrations of aldosterone with an acute onset within one to two minutes. They cannot therefore be explained by genomic mechanisms which appear to succeed the membrane related rapid responses as late effects. The mechanisms underlying these fast aldosterone electrolyte effects have been studied extensively in human lymphocytes and rat vascular smooth muscle cells representing attractively valuable tools in the delineation of the receptor-effector mechanisms involved. This includes the demonstration of membrane binding sites in lymphocytes which are highly specific for aldosterone and transmit the rapid non-genomic responses to aldosterone, as suggested by the intriguing similarities of binding kinetics and pharmacology in the receptor and effector assays. The unique characteristics of this new pathway for steroid action includes its rapid time course, 10,000-fold selectivity for aldosterone over cortisol, and the ineffectiveness of spironolactones, the classical mineralocorticoid antagonists. The two step model for the membrane action of mineralocorticoids developed here shares many features with that developed for other steroid hormones, for example on their neural activity. Since not only lymphocytes but also vascular smooth muscle cells are equipped with the rapid aldosterone effector mechanism, this new model may become the basis for the developing concept of steroids as regulators of cardiovascular functions.

Animals↗
PubMed1994

Rapid effects of aldosterone on free intracellular calcium in vascular smooth muscle and endothelial cells: subcellular localization of calcium elevations by single cell imaging.

Rapid in vitro effects of aldosterone on the intracellular concentrations of sodium, potassium and calcium, cell volume and the sodium-proton-antiport have been described in human mononuclear leukocytes and vascular smooth muscle cells (VSMC). These nongenomic effects are signalled through membrane receptors with a high affinity for aldosterone, but not for cortisol, and through the phosphoinositide pathway. In the present study, we demonstrate that free intracellular calcium is increased rapidly by aldosterone in VSMC and endothelial cells (EC) as determined by single cell imaging of Fura2-fluorescence. In VSMC, calcium elevation is localized to the perinuclear region whereas in EC, a predominant increase of subplasmalemmal calcium is seen. In VSMC, effects are half maximal at 0.1 nM aldosterone; cortisol is inactive up to 0.1 microM. These data show that intracellular signalling for aldosterone also involves calcium, but the subcellular localization of this signal varies between cell types.

Aldosterone↗
PubMed1994

Distinction of weakly homologous cDNA amplificates by single-strand conformation polymorphism analysis: application to guanylyl cyclase isozymes.

By use of the polymerase chain reaction (PCR), uniform amplification products of 225 to 240 bp length were obtained from five cDNA clones representing different types of guanylyl cyclases. These short DNA double strands were differentiated by single-strand conformation polymorphism (SSCP), using polyacrylamide gel electrophoresis with the Pharmacia Phast-System. Following heat denaturation, the samples were separated on native polyacrylamide gels at different running temperatures. Nucleic acids on the gel were detected by an automated silver stain procedure. Using 7.5% homogeneous or 4-15% gradient polyacrylamide gels at a temperature of 12 degrees C, single-strand conformations of amplificates, representing three different particulate guanylyl cyclases and the two subunits of soluble guanylyl cyclase, were differentiated. The characteristic banding patterns resulting from dissimilar migration of the single-strand conformations were assigned to different guanylyl cyclase types. For the enzyme family of guanylyl cyclases, the feasibility of a combined PCR and electrophoresis approach for analyzing the expression of related genes was demonstrated. This application of the PCR-SSCP technique provided a rapid and sensitive tool for the characterization of PCR products obtained with a common primer pair and suggested its use for investigating the tissue distribution of gene expression within a class of homologous proteins.

Animals↗
PubMed1994

Volumes and Na+/H+ antiporter activity of lymphocytes in patients with congestive heart failure.

Previous studies in patients with congestive heart failure (CHF) treated with diuretics and/or digoxin have shown abnormalities of cellular volume and electrolytes in biopsies of skeletal muscle. These abnormalities seem to play an important role with regard to the dysregulation of peripheral vascular resistance and characteristic clinical features of CHF, for example, muscular weakness. This study assessed the effect of angiotension-converting enzyme (ACE) inhibitor therapy on cell volume and cell volume regulation in patients with CHF. Cell diameters of human mononuclear leukocytes (HML) were determined electronically by a Coulter Counter. Cell diameters for 19 patients with decreased left ventricular ejection fraction (determined via levocardiography) on therapy with ACE inhibitors (group 1) were compared to those of HML from patients on diuretics alone (group 2, n = 16). The activity of the Na+/H+ antiporter was determined by cell swelling in isotonic propionate. The control group consisted of 20 normal, age- and sex-matched volunteers. HML diameters were significantly increased from 7.16 +/- 0.07 in normals to 7.24 +/- 0.08 microns (group 1; P < 0.01) and 7.23 +/- 0.11 microns (group 2; P < 0.05), indicating an abnormal regulation of cell volume. There were no statistically significant correlations between the individual ejection fraction or digoxin therapy and average cell diameters. In both patient groups ethylisopropylamiloride-sensitive swelling rates were normal compared to the control group indicating a normal activity of the Na+/H+ antiporter. In conclusion, increased cell sizes reflect a structural change in HML rather than a rapidly reversible functional abnormality which was not affected different by ACE inhibition and diuretic therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗