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Biomedical subjects

D Palm

Publications and source records attributed to D Palm.

At least 55 records · Page 3Linked to original sources
PubMed1991

Chemical and functional analysis of components of adenylyl cyclase from human platelets treated with phorbolesters.

Human platelets, prelabeled with [32P]phosphate were treated with tetradecanoylphorbol acetate (TPA) for 5 min at 37 degrees C. Phosphorylation of the components of adenylyl cyclase was determined in membranes using specific antibodies against G-proteins and the catalytic moiety. Less than 0.01 mol of [32P]phosphate/mol could be detected in immunoprecipitates using antibodies against sequences within the alpha-subunit of the GTP binding protein Gi. TPA, however, caused the incorporation of 0.67-1.1 mol of [32P]phosphate per mol of catalyst while 0.13-0.2 mol were found in the absence of TPA. Lack of modification of the alpha-subunit of Gi was also indicated by the results of reconstitution experiments with purified Gi alpha from bovine brain: adenylyl cyclase in membranes from untreated platelets was significantly more inhibited by added G1 alpha, than that from TPA treated cells. While beta, gamma-subunits were like-wise inhibitory no difference dependent on platelet-pretreatment could be observed.

Adenylyl Cyclase Inhibitors↗
PubMed1991

Multisite contacts involved in coupling of the beta-adrenergic receptor with the stimulatory guanine-nucleotide-binding regulatory protein. Structural and functional studies by beta-receptor-site-specific synthetic peptides.

Synthetic peptides, 12-22 amino acid residues long, comprising the presumed coupling sites of the beta-adrenergic receptor with the stimulatory guanine-nucleotide-binding regulatory protein (Gs), were examined for their ability to modulate Gs activation in turkey erythrocyte membranes. Three peptides corresponding to the second cytoplasmic loop, the N-terminal region of the third cytoplasmic loop, and the N-terminal region of the putative fourth cytoplasmic loop, compete synergistically with the hormone-stimulated receptor for Gs activation with median effector concentrations of 15-35 microM, or 3-4 microM for combinations of two peptides. One peptide, corresponding to the C-terminal region of the third cytoplasmic loop, carries the unique ability to activate the Gs-adenylate-cyclase complex independent of the signalling state of the receptor. These observations are consistent with a dynamic model of receptor-mediated G-protein activation in membranes, where domains composed of the second, third and fourth intracellular loop of the receptor bind to and are interactive with the G-protein heterotrimer, resulting in ligand-induced conformational changes of the receptor. In response to hormone binding, the extent or the number of sites involved in interaction with Gs may be readjusted using a fourth site. Modulation of coupling sites may elicit congruent conformational changes within the Gs heterotrimer, with qualitatively different effects on GTP/GDP exchange in the alpha subunit of Gs and downstream effector regulation. This model corroborates and expands a similar model suggested for activated rhodopsin-transducin interaction [König, B., Arendt, A., McDowell, J. H., Kahlert, M., Hargrave, P. A. & Hofmann, K. P. (1989) Proc. Natl Acad. Sci. USA 86, 6878-6882].

Aluminum↗
PubMed1991

Monoclonal antibodies against various forms of the adenylyl cyclase catalytic subunit and associated proteins.

Monoclonal antibodies against partially purified adenylyl cyclase from bovine brain cortex were raised in mice. Three types of antibody were obtained. Type 1 was specific for the calmodulin-sensitive enzyme. Type II also recognized this enzyme, but recognized the calmodulin-insensitive enzymes from a variety of species and tissues as well. Type I antibodies precipitated their antigens in both the native and denatured forms, while type II strongly favored the denatured forms. Type III antibodies precipitated adenylyl cyclase activity, but as shown by Western blot analysis, were directed against 38-kDa and 45-kDa glycoproteins. The 38-kDa protein was identified as synaptophysin.

Adenylyl Cyclases↗
PubMed1991

AF-DX 116, a cardioselective muscarinic antagonist in humans: pharmacodynamic and pharmacokinetic properties.

Effects of AF-DX 116, a cardioselective antagonist, on M cholinergic receptors (M-ChR) were studied in healthy volunteers. Occupancy of M-ChR subtypes by drug present in plasma samples (radioreceptor assay) was compared with these effects. After an intravenous dose of AF-DX 116 saturating greater than 90% of cardiac M2-ChR, an increase in heart rate by 25 beats/min was observed. This cardiac receptor occupancy and effect wore off with a parallel time course within 10 hours. No inhibition of salivary flow was observed, coinciding with a lack of M3-ChR blockade in the radioreceptor assay. Beta-adrenergic receptor blockade by propranolol did not affect either of the effects. No indication for active metabolites or stereoselective drug metabolism was found comparing HPLC and receptor assay for drug concentrations in plasma. We conclude that AF-DX 116 may be a useful drug for the treatment of bradycardia. Its lack of troublesome side effects is the result of its selectivity for cardiac M-ChR.

Adult↗
PubMed1991

Bioequivalence of two atenolol formulations in healthy volunteers. Evaluation and prediction of effect kinetics at beta-adrenoceptors in vivo by means of a radioreceptor assay.

Pharmacokinetic and pharmacodynamic effects of single oral doses of 200 mg of two atenolol formulations (Tenormin, atenolol (T) and Duraatenolol, atenolol (D)) were assessed in eight male healthy volunteers for 48 h (double-blind crossover design). Both formulations were bioequivalent in terms of pharmacokinetic parameters (AUC, tmax, Cmax, t1/2) as detected by gas liquid chromatography. By means of a radioreceptor assay (RRA) any significant contribution of beta 2-adrenoceptors and active metabolites to effects observed in vivo was ruled out. Based on parameters derived from RRA a prediction of the time course of any beta 1-adrenoceptor-mediated effect in vivo was made. During the first 12 h after intake of atenolol (T) and atenolol (D) effect kinetics simulated from RRA data corresponded very well to the inhibition of exercise-induced tachycardia observed in vivo. Thus, RRA is an in vitro method enabling assessment and prediction of relative bioequivalence of 2 atenolol formulations in vivo. Moreover, it enables establishment of a causal relationship between pharmacodynamic and pharmacokinetic parameters.

Adolescent↗
PubMed1990

Escherichia coli maltodextrin phosphorylase: contribution of active site residues glutamate-637 and tyrosine-538 to the phosphorolytic cleavage of alpha-glucans.

The role of Escherichia coli maltodextrin phosphorylase (EC 2.4.1.1) active site residues Glu637 and Tyr538 which line the sugar-phosphate contact region of the enzyme was investigated by site-directed mutagenesis. Substitution of Glu637 by an Asp or Gln residue reduced kcat to approximately 0.2% of wild-type activity, while the Km values were affected to a minor extent. This indicated participation of Glu637 in transition-state binding rather than in ground-state binding. 31P NMR analysis of the ionization state of enzyme-bound pyridoxal phosphate suggested that Glu637 is also involved in modulation of the protonation state of the coenzyme phosphate observed during catalysis. Despite loss of proposed hydrogen-bonded substrate contacts, the Tyr538Phe mutant enzyme retained more than 10% activity; the apparent affinity of all substrates was slightly decreased. Mutations at either site affected the error rate of the enzyme (ratio of hydrolysis/phosphorolysis). Besides a role in substrate binding, the hydrogen-bond network of Tyr538 supports the exclusion of water from the active site.

Amino Acid Sequence↗
PubMed1990

Identification of a Gs-protein coupling domain to the beta-adrenoceptor using site-specific synthetic peptides. Carboxyl terminus of Gs alpha is involved in coupling to beta-adrenoceptors.

Competition between Gs-protein and the synthetic peptide, GSA 379-394, derived from the carboxyl-terminal region of the alpha s-subunit, led to complete inhibition of receptor-mediated adenylate cyclase activation in turkey erythrocyte membranes. Related peptides corresponding to the homologous carboxyl-terminal region of alpha t-, alpha il- or alpha o-subunits did not interfere with beta-receptor-Gs coupling. The direct coupling between Gs and adenylate cyclase was not influenced by any of these peptides. These results emphasize the important role of the carboxyl-terminus of G-protein alpha-subunits for the specific recognition of their corresponding receptors and for signal transduction.

Adenylyl Cyclases↗
PubMed1990

Sensorineural hearing loss owing to deficient G proteins in patients with pseudohypoparathyroidism: results of a multicentre study.

Pseudohypoparathyroidism (PHP) is a rare disorder that might be caused by an hereditary defect in the G protein system. These membrane-bound proteins are responsible for the transduction of biological signals through the outer cell membrane. The investigation of 22 patients with PHP showed a symmetric sensorineural hearing loss in 63.6% of the subjects. In erythrocyte membrane preparations from blood samples of 15 of these patients, we measured the concentration of the stimulatory Gs protein and the inhibitory Gi protein by means of the Western blot analysis using selective antibodies against alpha-subunits of G proteins. In nine of the 15 cases (60%), we found a distinct decrease in the amount of the Gs protein with a partial preponderance of the Gi protein. These patients had a considerable symmetric sensorineural hearing loss. Pathophysiological mechanisms and the possible role of G proteins in the inner ear are discussed.

Adolescent↗
PubMed1990

[Decreased vagal tone in hyperkinetic heart syndrome?].

Increasing amounts of atropine (0.03 mg + 0.07 mg + 0.01 mg + 0.25 mg + 0.5 mg + 1.0 mg + 1.5 mg; sigma = 3.5 mg, interval between injections: 30 min) were injected intravenously in a cumulative manner in five patients with hyperkinetic heart syndrome and in seven healthy volunteers. The heart rate increased from 93 to 127 beats per min in patients, and from 63 to 104 beats per min. in controls. The diastolic blood pressure slightly increased in both groups, whereas the systolic blood pressure did not change significantly. The accommodation diminished from 6 to 2 dioptries in patients, and from 8 to 5 dioptries in controls. Salivary flow fell from 1.6 to 0.1 g/min in patients with hyperkinetic heart syndrome, and from 1.3 to 0.1 g/min in control persons, respectively. The maximum occupancy of muscarinic receptors (m2-glandular), as measured in plasma specimens by means of a radioreceptor assay, reached more than 80% in each group. The dose-response-curves of atropine were highly comparable in patients and controls. It is concluded that the effects of atropine are essentially the same in patients with hyperkinetic heart syndrome and in healthy control persons. There is no evidence for a diminished vagal tone in such patients.

Adult↗
PubMed1989

Mapping of beta-adrenoceptor coupling domains to Gs-protein by site-specific synthetic peptides.

Peptides corresponding to the known sequence of turkey erythrocyte beta 1-adrenergic receptor were synthesized and the effects on receptor-mediated cyclase activation were measured. Peptides corresponding to the first and second intracellular loops (T61-71 and T138-159) inhibited at micromolar concentrations the hormone-dependent cyclase activation in turkey erythrocyte membranes. In contrast, the peptide corresponding to the C-terminal part of the third intracellular loop (T284-295) increased the cyclase activity in a hormone-independent manner. Peptides T338-353 and T2-10 and a number of synthetic peptides unrelated to the beta-adrenoceptor had no effect.

Adenylyl Cyclases↗
PubMed1989

AF-DX 116 discriminates heart from gland M2-cholinoceptors in man.

The M2-cholinoceptor subtype selective antagonist AF-DX 116 was compared with atropine with respect to effects on heart rate and salivary flow in healthy volunteers. These effects were related with in vitro occupancy of M-cholinoceptor subtypes in radioreceptor assays of plasma samples. Radioreceptor assays comprised M1-cholinoceptors in bovine cerebral cortex and M2-cholinoceptors in pig heart and rat salivary gland membranes. 3H-pirenzepine served as a label in the cerebral cortex 3H-N-methyl-scopolamine in the heart and gland preparations. Oral administration of 240 mg AF-DX 116 led to a time dependent increase in heart rate with a maximum effect comparable to atropine 40 micrograms/kg i.v. The effects of both drugs on heart rate were matched by a greater than 80% occupancy of heart M2-cholinoceptors in the radioreceptor assay of plasma samples. In contrast to the complete inhibition of salivary flow after atropine, AF-DX 116 induced an increase of salivation. The effects on salivary flow coincided with a greater than 80% occupancy of glandular M2-cholinoceptors after atropine but no detectable occupancy after AF-DX 116. Occupancy of the M1-subtype amounted to 61.7% after AF-DX 116 and a blockade of inhibitory, presynaptic M1-autoreceptors at missing postsynaptic blockade of glandular M2-cholinoceptors might explain the hypersalivation induced by AF-DX 116.

Adult↗
PubMed1989

Radioreceptor assay of metoprolol in human plasma: comparison with an enantiospecific high-performance liquid chromatographic (HPLC) procedure.

Plasma concentrations of metoprolol after acute and repetitive administration of R/S-metoprolol to healthy volunteers were measured by a beta-adrenoceptor subtype-specific radioreceptor assay (RRA) and by an enantiospecific high-performance liquid chromatographic (HPLC) method. In the RRA, R/S-metoprolol showed a 20-fold beta 1-subtype selectivity: the S-(-)-enantiomer was 35-fold more potent than the R-(+)-enantiomer. A comparison between S-(-)-metoprolol concentrations detected in the plasma samples by HPLC and those detected by RRA yielded a 1/1 relationship, indicating that active metabolites are not present to a significant extent. These results were independent of the widely scattering metabolic clearance of metoprolol (with the potential of differences in the rate and extent of formation of active metabolites) in the volunteers. In general, HPLC methods can be validated by comparison with RRA in order to clarify whether active metabolites are present and--on the basis of the Ki value from RRA--whether the detection limit of the physicochemical procedure is sufficient to cover the therapeutically relevant range.

Animals↗
PubMed1989

Selective antagonists reveal different functions of M cholinoceptor subtypes in humans.

Effects of atropine and of the subtype selective mAChR antagonists pirenzepine (PZ) and AF-DX 116 were studied in humans. Dose- or time-response curves were established for heart rate and salivary flow. Plasma samples were drawn in parallel with the effect measurements and analysed for drug concentrations. Subtype-selective radioreceptor assays of the samples served to estimate the respective receptor occupancy in vivo. It is shown that low doses of PZ (M1-selective blockade) cause cholinomimetic effects indicated by bradycardia and increase in salivary flow. After high doses of PZ or atropine, tachycardia and inhibition of salivary flow are observed in parallel with occupancy of both the M2 and M3 subtypes. AF-DX 116 induces a tachycardia together with an increased salivary flow in agreement with its selectivity profile (M2 greater than M1 greater than M3). The diagnostic and therapeutic applications of M1- or M2-selective blockade by low dose PZ or AF-DX 116 respectively are discussed.

Humans↗
PubMed1988

Penbutolol: pharmacokinetics, effect on exercise tachycardia, and in vitro inhibition of radioligand binding.

The pharmacokinetics of penbutolol 40 mg, its reduction in exercise-induced tachycardia, and the in vitro inhibition of radioligand binding to beta-adrenoceptors by plasma have been investigated in 7 healthy volunteers. The peak penbutolol concentration of 285 ng/ml was observed 1.2 h after administration, and the maximum of 4'-OH-penbutolol of 4.76 ng/ml was found after 1.64 h. Penbutolol was detected for up to 48 h, and 4'-OH-penbutolol dropped below the limit of detection after about 10 h. The terminal plasma concentration of penbutolol declined with an average half-life of 19 h. The maximum reduction in exercise-induced tachycardia was 33 beats/min 2.6 h after taking penbutolol. There was still a significant reduction of about 7 beats/min after 48 h. This effect could be adequately explained by the concentration-time course of penbutolol in combination with Clark's model of the concentration-effect relationship. Antagonist activity in plasma caused 91% inhibition of radioligand binding in vitro to beta 2-adrenoceptors on rat reticulocyte membranes 1.6 h after intake of penbutolol. By 48 h after intake, radioligand binding was still significantly inhibited (23%). The in vitro inhibition of radioligand binding by plasma showed a linear correlation with the reduction in exercise-induced tachycardia for all phases of the workload. The time course of the reduction in heart rate was completely explained by the in vitro inhibition of radioligand binding. However, it was not possible to explain the in vitro inhibition of radioligand binding by the concentration-time course of penbutolol using a simple competition model, although both variables were based on the same sampling site. When the in vitro inhibition of radioligand binding was plotted against the penbutolol concentration at the same sampling times (with both variables transformed to multiples of the apparent inhibition constant) the discrepancy became even more apparent as time-related counterclockwise hysteresis. None of the known metabolites of penbutolol can explain the discrepancy between the penbutolol concentration and the inhibition of radioligand binding in vitro. It appears that an other active metabolite is formed, which contributes to the effect in vitro and in vivo and so can explain the observed discrepancy.

Adrenergic beta-Antagonists↗