Search PubMed⌕ Search

Biomedical subjects

M Wehling

Publications and source records attributed to M Wehling.

At least 91 records · Page 5Linked to original sources
PubMed1998

Characterization of high affinity progesterone-binding membrane proteins by anti-peptide antiserum.

A chemically synthesized 15-mer oligopeptide derived from the N terminus of high affinity progesterone-binding membrane site(s) from porcine liver was used to generate site-specific antibodies. Western blotting experiments confirmed the specificity of the anti-peptide serum obtained. In further investigations this antiserum was used for the identification of the native progesterone-binding membrane protein complex that represents an oligomer with an apparent molecular mass of about 200 kDa. In temperature-induced Triton X-114 phase separation experiments combined with Western-blotting, the progesterone-binding site was identified as an hydrophobic (integral) membrane protein. In addition, in Western blotting analyses the antiserum reacted with the progesterone-binding or related proteins in membrane fractions from a wide array of different tissues in various species.

Amino Acid Sequence↗
PubMed1998

Sparing of mdx extraocular muscles from dystrophic pathology is not attributable to normalized concentration or distribution of neuronal nitric oxide synthase.

Previous findings have led to speculations that decreased concentration of nNOS (neuronal nitric oxide synthase) may underlie some aspects of the pathophysiology of dystrophic muscle. We have tested whether the sparing of extraocular muscles (EOM) in muscular dystrophy is attributable to the presence of normal nNOS concentration and distribution in these muscles. Measurements of total nNOS concentration in control muscle showed that total nNOS comprises approximately 0.05% of total muscle protein, indicating a molar stoichiometry of approximately 60 and 20 to total dystrophin and syntrophin, respectively. Thus, most muscle nNOS is either not associated with the dystrophin complex, or binds to yet unidentified sites in the complex. nNOS concentration was at least two-fold greater in C57 EOM and tibialis anterior (TA) compared with mdx samples. No significant differences in nNOS concentration in EOM versus TA in either mdx or C57 mice were observed, nNOS was concentrated at the sarcolemma of all C57 samples, while mdx nNOS displayed a cytosolic distribution, except in fibers that reverted to express dystrophin. These data show that mdx EOM are spared by a mechanism other than normalized concentration and location of nNOS.

Aging↗
PubMed1998

Nongenomic steroid actions: completing the puzzle. Aldosterone as an example.

In the common unidimensional theory of steroid action, steroids bind to intracellular receptors and modulate nuclear transcription and thus protein synthesis. These genomic steroid effects, being characterized by their delayed onset of action and their dependence on transcription and protein synthesis, have been known for several decades. In contrast, very rapid actions of steroids, which are considered to be of nongenomic origin, have been recognized more widely and characterized in detail only during the past ten years. Specific rapid effects of steroids and related hormones like vitamin D3 and thyroid hormones on cellular function involve a conventional second messenger cascade which in most cases includes phospholipase C, phosphoinositide turnover, intracellular pH and intracellular calcium ([Ca2+]i), and protein kinase C. Furthermore, binding sites in membranes have been characterized exposing binding features compatible with an involvement in rapid-steroid signaling. Characteristics of putative membrane receptors are completely different from those of classic intracellular steroid receptors; this also includes the inability of classic steroid receptor antagonists to inhibit those rapid nongenomic steroid actions. The physiological and pathophysiological relevance of these effects is still largely unclear, but their existence has been proven recently even under in vivo conditions. New drugs modulating nongenomic steroid actions may find applications in various areas such as the cardiovascular and central nervous systems, infertility and electrolyte homeostasis. This short review focuses mainly on the nongenomic actions of aldosterone and their cardiovascular implications.

Aldosterone↗
PubMed1998

Mechanical loading regulates NOS expression and activity in developing and adult skeletal muscle.

The hypothesis that changes in muscle activation and loading regulate the expression and activity of neuronal nitric oxide (NO) synthase (nNOS) was tested using in vitro and in vivo approaches. Removal of weight bearing from rat hindlimb muscles for 10 days resulted in a significant decrease in nNOS protein and mRNA concentration in soleus muscles, which returned to control concentrations after return to weight bearing. Similarly, the concentration of nNOS in cultured myotubes increased by application of cyclic loading for 2 days. NO release from excised soleus muscles was increased significantly by a single passive stretch of 20% or by submaximal activation at 2 Hz, although the increases were not additive when both stimuli were applied simultaneously. Increased NO release resulting from passive stretch or activation was dependent on the presence of extracellular calcium. Cyclic loading of cultured myotubes also resulted in a significant increase in NO release. Together, these findings show that activity of muscle influences NO production in the short term, by regulating NOS activity, and in the long term, by regulating nNOS expression.

Aging↗
PubMed1998

Rapid cardiovascular action of aldosterone in man.

Rapid nongenomic in vitro effects of aldosterone have been demonstrated recently in cultured vascular smooth muscle and endothelial cells. But there is, as yet, little evidence for corresponding in vivo effects. The present study thus investigates the rapid nongenomic effects of aldosterone on human cardiovascular function. In a double-blind placebo-controlled randomized parallel trial on 17 patients with suspected coronary heart disease, the effect of 1 mg aldosterone iv on cardiovascular function was assessed during cardiac catheterization. Hemodynamic parameters (such as heart rate, left ventricular and atrial pressures, arterial pressures, vascular resistances, and cardiac output) were measured before and 3 and 10 min after administration of aldosterone or placebo. Significant changes were found for systemic vascular resistance, cardiac output, and cardiac index, compared with the placebo group (Wilcoxon test, P < 0.02-0.05). The effect of aldosterone dissipated within 10 min. The results are in line with the in vitro data cited above and consistent with earlier findings on acute cardiovascular effects of aldosterone, which have now been confirmed and extended by contemporary techniques. The hypotheses of rapid nongenomic in vivo effects of aldosterone are further substantiated by this study.

Aldosterone↗
PubMed1998

Cloning and tissue expression of two putative steroid membrane receptors.

We have cloned two human putative steroid binding membrane proteins, termed Hpr6.6 and Dg6. Hpr6.6 is the human homolog of a previously cloned porcine progesterone binding protein. Both proteins contain a putative transmembrane domain and a highly conserved stretch of 58 amino acids. Hpr6.6 mRNA is expressed predominantly in liver and kidney, whereas Dg6 mRNA is preferentially expressed in placenta. Hpr6.6 is located on the X chromosome and dg6 on chromosome 4. The two proteins are the first putative steroid membrane receptors cloned from man.

Amino Acid Sequence↗
PubMed1998

[The relative bioavailability and pharmacokinetics of chloral hydrate and its metabolites].

Two open, randomized cross-over trials were performed in 18 healthy volunteers each to evaluate the relative bioavailability and the pharmacokinetics of chloral hydrate (CAS 302-17-0), the active ingredient of Chloraldurat 500 (immediate release capsules, CH), Chloraldurat rot (immediate release capsules, CR) and Chloraldurat blau (enteric-coated modified release capsules, CB). In the first study the male subjects, aged 21 to 31 years, were randomly given one capsule of CH or 500 mg of chloral hydrate as drinking solution. In the second study the volunteers, aged 20 to 28 years, received either one capsule of CR or one capsule of CB or 250 mg of chloral hydrate as drinking solution. The time of administration was between 6:30 and 7:30 a.m. and the capsules had to be swallowed with 150 ml water. The reference medication consisted of 150 ml drinking solution. The wash out time in both studies was 4 weeks. Prior to the administration and (2, 4, 6, only for CH) 8, 10, 15, 20, 40, 60 min and 1.5, 2, 4, 6, 8, 12, 24, 36, 48, 72, 96, 144, 192, 240 (and 408 only for CR/CB) h afterwards blood samples of 4.5 ml were taken from the antecubital vein. Additional 4.5 ml were drawn before and 10, 20, 40 and 60 min after administration to detect unchanged chloral hydrate. In the second study times of blood sampling were modified up to 4 h after administration due to the estimated later onset of release from CB in comparison to CR. Blood samples were centrifuged within 20 min, the plasma was separated and immediately frozen at -20 degrees C. Due to the extremely short terminal half-life of chloral hydrate its active metabolite trichloroethanol is regarded as the pharmacokinetically relevant parameter for the assessment of the bioavailability of the parent substance. Compared to the reference formulation (drinking solution) the bioavailability of trichloroethanol was 94.8% (CH), 100.7% (CR) and 101.6 (CB), respectively. The maximum plasma concentrations (Cmax) of trichloroethanol were 5176 ng/ml after intake of CH (reference 6131 ng/ml), after intake of CR 3241 ng/ml and CB 3279 ng/ml (reference 2993 ng/ml). Maximum plasma concentrations (tmax) of trichloroethanol were reached after 0.67 h (reference) and after 0.98 (CH), 0.76 (CR) and 2.38 h (CB), respectively. The terminal half-life for trichloroethanol was calculated to be 9.3 to 10.2 h, for the inactive metabolite trichloracetic acid the half-life ranged from 89 to 94 h. Chloral hydrate itself could be detected only 8 to 60 min after application at very low concentrations in some of the plasma samples. It is justified to characterize its bioavailability by the active metabolite trichloroethanol due to the extremely short terminal half-life and high variability of the parent substance.

Adult↗
PubMed1998

Localization of a putative progesterone membrane binding protein in porcine hepatocytes.

A putative membrane bound steroid receptor was localized using a peptide specific antibody. Surprisingly, the distribution of immunocytochemical staining in porcine hepatocytes cells provides evidence for the localization to endomembranes (endoplasmic reticulum, Golgi apparatus). Immunofluorescence experiments with HEK cells, which were transfected with a pcDNA3.1 vector containing the coding sequence of the putative progesterone binding protein shows staining within the cells supporting these results. Additionally, 3H-progesterone binding and glucose-6-phosphatase activity as marker enzyme for endoplasmic reticulum were closely correlated in subcellular fractions of porcine liver cells.

Animals↗
PubMed1997

Specific, nongenomic actions of steroid hormones.

Traditionally, steroid hormone action has been described as the modulation of nuclear transcription, thus triggering genomic events that are responsible for physiological effects. Despite early observations of rapid steroid effects that were incompatible with this theory, nongenomic steroid action has been widely recognized only recently. Evidence for these rapid effects is available for steroids of all clones and for a multitude of species and tissues. Examples of nongenomic steroid action include rapid aldosterone effects in lymphocytes and vascular smooth muscle cells, vitamin D3 effects in epithelial cells, progesterone action in human sperm, neurosteroid effects on neuronal function, and vascular effects of estrogens. Mechanisms of action are being studied with regard to signal perception and transduction, and researchers have developed a patchy sketch of a membrane receptor-second messenger cascade similar to those involved in catecholamine and peptide hormone action. Many of these effects appear to involve phospholipase C, phosphoinositide turnover, intracellular pH and calcium, protein kinase C, and tyrosine kinases. The physiological and pathophysiological relevance of these effects is unclear, but rapid steroid effects on cardiovascular, central nervous, and reproductive functions may occur in vivo. The cloning of the cDNA for the first membrane receptor for steroids should be achieved in the near future, and the physiological and clinical relevance of these rapid steroid effects can then be established.

Animals↗
PubMed1997

Nongenomic effects of aldosterone on intracellular calcium in porcine endothelial cells.

Rapid in vitro effects of aldosterone on intracellular electrolytes, cell volume, and the sodium-proton antiport have been described in human mononuclear leukocytes and vascular smooth muscle cells. In the present study, we demonstrate rapid aldosterone effects on free intracellular calcium as determined by fura 2 fluorometry in single porcine endothelial cells. After addition of 100 nmol/l aldosterone, cells respond with a sustained rise in free intracellular calcium by approximately 50% of initial levels within 1-5 min. Elevations are predominantly seen in the subplasmalemmal space. Effective half-maximal concentration values for aldosterone are approximately 1 pmol/l and for cortisol approximately 1 nmol/l. These effects are blunted in calcium-free medium and absent after pretreatment by thapsigargine. They remain unchanged by a >1,000-fold excess of spironolactone. These findings indicate the existence of a nongenomic pathway for aldosterone action in porcine endothelial cells and may be related to known rapid cardiovascular effects of aldosterone in vivo mediated through the baroreceptor reflex.

Aldosterone↗
PubMed1997

24-hour recording of intragastric pH in clinical trials: preconditions, methodology, and standardization strategies.

At present, intragastric pH recording by stomach probe is the standard method for the assessment of the pharmacodynamic effect of newly developed antisecretory drugs, and it is being used increasingly as a diagnostic method. Intraluminal pH can be measured by a variety of different electrode systems, systematic differences among these systems require international standardization of the method. In clinical trials, some recommendations should be followed to standardize the study conditions to assure that repeated measurements are comparable. Standardization of food and liquid intake and a correct positioning of the stomach probe are of paramount importance in assuring that the data are reliable; but there are other factors that may influence the study results, for example, the daily activities of the study participants and the method of data processing. Furthermore, several technical aspects must be considered to guarantee accurate and reproducible recordings, including the type of pH-sensitive electrode and the method of calibration used. Further efforts should be made to create an international standard for the method to assure more comparable study results.

Biosensing Techniques↗
PubMed1997

Impaired rapid mineralocorticoid action on free intracellular calcium in pseudohypoaldosteronism.

Earlier observations on impaired in vitro effects of aldosterone on lymphocytic sodium and potassium pointed to the involvement of a defective nongenomic rather than genomic effector in pseudohypoaldosteronism. In this study, we investigated nongenomic aldosterone action in five patients with pseudohypoaldosteronism with regard to a rapid increase of free intracellular calcium [Ca2+]i in cultured nasal epithelial cells, assumably reflecting calcium influx through calcium channels. Patients were defined by episodes of salt loss despite high plasma aldosterone and renin levels. Four unaffected members of the families and four independent subjects served as controls. Considering an aldosterone-induced increase of [Ca2+]i by at least 10 nm as positive response, only 12% of cells from patients were responsive compared with 25% in normal subjects (P < 0.05). In terms of absolute changes, mean increase of [Ca2+]i was 1.6 +/- 1.1 nm in the patients (range-1-4) and 9.5 +/- 2.7 nm (P < 0.025) in the controls (range 1-25). Basal [Ca2+]i was not different between both groups (167 +/- 5 vs. 169 +/- 8 nm, mean +/- SE). These findings show an impaired nongenomic mineralocorticoid effector in patients with pseudohypoaldosteronism, which is in line with a defective sodium channel as shown recently by molecular cloning, and also with the fact that the classical, genomic intracellular receptor is structurally normal in these patients.

Adult↗
PubMed1997

Characterization of progesterone membrane binding sites from porcine liver probed with a novel azido-progesterone radioligand.

A new derivative of progesterone was synthesized for photoaffinity labelling of specific binding sites in porcine liver microsomes. Using progesterone-3-(O-carboxymethyl)-oximino-(3-125I-iodo-4-azidosa licylamidobutylamine) as a photoactivatable radioligand, selective covalent labelling of proteins was detected in porcine liver microsomes at apparent molecular weights of 90-100 kDa and 60-65 kDa by SDS-PAGE and subsequent radioautography. These proteins showed different ligand specifity for various steroids tested. On blue native polyacrylamide-gels three selectively labelled proteins were found corresponding to apparent molecular weights of approximately 310 kDa, approximately 215 kDa and approximately 75 kDa, respectively. Using two-dimensional electrophoresis for the analysis of these protein complexes, the 215 kDa-band could be correlated to the 90-100 kDa-band, while the 75 kDa-band may correspond to the 60-65 kDa-band at one-dimensional SDS-PAGE, respectively. One or more of these proteins may be involved in the rapid progesterone-induced increase of intracellular Ca2+ described previously in cultured hepatic cells.

Affinity Labels↗
PubMed1996

Full-length cDNA sequence of a progesterone membrane-binding protein from porcine vascular smooth muscle cells.

A full-length cDNA clone for a progesterone membrane binding protein from porcine vascular smooth muscle cells was isolated and the complete nucleotide sequence determined. The cDNA encodes a protein of 194 amino acids with a transmembrane segment. This protein is likely to represent the first steroid membrane receptor or a part of it for which sequence information is available.

Amino Acid Sequence↗
PubMed1996

Purification and partial sequencing of high-affinity progesterone-binding site(s) from porcine liver membranes.

High-affinity progesterone-binding sites have been identified, characterized in and purified from porcine liver membranes. They were functionally solubilized by the non-denaturing zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (Chaps, 20 mM. detergent/protein mass ratio 4:1) at a yield of 75-80%. Using [3H]progesterone as radioligand, binding studies showed high-affinity and low-affinity binding sites in microsomal preparations with an apparent Kd2 of 11 nM and an apparent Kd2 of 286 nM. In solubilized fractions the high-affinity binding sites were present at an apparent Kd2 of 69 nM. In both preparations, progesterone binding was time-dependent, saturable, reversible, and showed a similar hierachy of affinities for related steroids. A purification scheme was developed based on anion-exchanger procedures. The purified fraction as identified by maximum specific progester-one-binding activity contained two major polypeptides of apparent molecular masses (SDS/PAGE) of 28 kDa and 56 kDa, respectively. Sequencing of both polypeptides showed an identical amino terminus without significant identity in the amino acid sequence to any known protein primary structure.

Amino Acid Sequence↗