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Biomedical subjects

H Glossmann

Publications and source records attributed to H Glossmann.

At least 145 records · Page 8Linked to original sources
PubMed1983

Radiation inactivation of alpha 1-adrenoceptors.

Radiation inactivation of alpha 1-adrenoceptors in rat cerebral cortex membranes has been performed with 10 MeV electrons from a linear accelerator at temperatures less than or equal to -100 degrees C. Alpha 1-adrenoceptor inactivation was monitored with [ 3H ]-prazosin and [( 125I ]-2-(beta-4-hydroxylphenyl)ethylaminomethyl)tetralone [( 125I ]-HEAT). Saturation analysis of irradiated membranes with both ligands indicated that a decrease in alpha-adrenoceptor density occurred with increasing radiation dose. The dissociation constants of [ 3H ]-prazosin and [ 125I ]-HEAT were not markedly changed by the irradiation. Application of the target volume theory gave molecular weights of 91,500 +/- 1,700 (S.D.) (D37: 19,6 +/- 0.36 Mrad) with [ 125I ]-HEAT as ligand, and 77,000 +/- 18,000 (S.D.) (D37: 23.3 +/- 4.6 Mrad) with [ 3H ]-prazosin, respectively, when an empirical temperature correction factor of 2.8 was used. [ 3H ]-flunitrazepam-labelled benzodiazepine receptor target size was used as an internal control. The molecular weight of the alpha 1-adrenoceptors, corrected for this internal control, was 85,000 +/- 1.600 [( 125I ]-HEAT) and 71,500 +/- 17,000 [( 3H ]-prazosin).

Animals↗
PubMed1983

Identification of voltage operated calcium channels by binding studies: differentiation of subclasses of calcium antagonist drugs with 3H-nimodipine radioligand binding.

3H-Nimodipine (3H-NIM) is a high affinity radioligand suitable to study Ca2+ -channels in a variety of tissues. The binding is saturable, reversible, and stereospecific in purified bovine heart and partially purified guinea-pig brain membranes. In the latter a Bmax of 600fmol/mg protein, dissociation constants (KD) of 0.4-0.8nM and a Hill slope of 1.0 are found. At 37 degrees C the optimal pH in 50mM TRIS-HCl buffer is 7.1-7.4. The calcium channel is a metalloprotein, and the divalent cation which is essential for the binding of 3H-NIM can be removed by EDTA (EC50 20 microM); the nimodipine binding site of the channel may then be reconstituted by divalent cations with Mn2+ greater than Ca2+ greater than Mg2+ greater than Sr2+. Ca2+ -antagonist drugs can be divided into three main classes based on their interaction with the 3H-NIM binding site: Class I has one site law of mass action-displacement isotherms with 3H-NIM, Class II exhibits complex biphasic inhibition profiles and Class III drugs increase the affinity of 1,4 dihydropyridines for the Ca2+ -channel. Diltiazem is a Class III Ca2+ -antagonist. Our in vitro studies lead us to conclude that the Ca2+ -channel contains multiple regulatory sites at which drugs can act.

Animals↗
PubMed1982

Identification of putative calcium channels in skeletal muscle microsomes.

Saturable binding sites for the labelled calcium antagonist (+/-)[3H]nimodipine were found in guinea-pig hind limb skeletal muscle homogenates. Binding sites were enriched in a microsomal pellet by differential centrifugation of the homogenate. [3H]Nimodipine binding (Kd = 1.5 +/- 0.03 nM, Bmax = 2.1 +/- 0.25 pmol/protein, at 37 degrees C) copurified (6-fold) in this fraction with [3H]ouabain binding (6.6-fold) and 125I-alpha-bungarotoxin binding (5-fold). d-cis-Diltiazem (but not 1-cis-diltiazem) stimulated (+/-) [3H]nimodipine binding (ED50 1 microM) by increasing the Bmax. Binding sites discriminated between the optical enantiomers of 1.4-dihydropyridine calcium antagonists and the optically pure enantiomers of D-600. The data confirm, with biochemical techniques, the presence of 1,4-dihydropyridine and (+/-) D-600 inhibitable calcium channels in skeletal muscle, previously found with electrophysiological techniques.

Animals↗
PubMed1982

The increase in hormone-stimulated adenylate cyclase activity following Rous sarcoma virus transformation.

Rous sarcoma virus (RSV)-infected chicken embryo cells were used to study the effect of viral transformation on the hormone-stimulated synthesis of cyclic AMP. Transformation by RSV greatly increased the cells' ability to synthesize and accumulate cyclic AMP in response to the beta-adrenergic agonist isoproterenol as compared to untransformed cells. This enhancement was observed in both intact cells and in membranes prepared from these cells. The inclusion of guanosine 5'-0-(3-thiotriphosphate), a nonhydrolyzable analogue of GTP, in assays of adenylate cyclase activity did not abolish the quantitative differences between the transformed and normal cell membranes. Infection of cells by Rous-associated virus, which lacks the oncogene src, did not induce this hyperresponsiveness thus indicating the probable involvement of the src gene product in this phenomenon. The duration of the isoproterenol-induced cyclic AMP elevation was longer in the transformed than in the untransformed cells; transformed cells, unlike untransformed cells, required at least 120 min before full desensitization became established. Membranes prepared from transformed cells specifically bound more than 5 times the quantity of the beta-adrenergic radiolabeled antagonist (-)3H-dihydroalprenolol and 125I-iodocyanopindolol compared to the untransformed cell membranes. Thus, it appears that major differences between the transformed and normal phenotypes reside in the concentration of membrane beta-adrenergic receptors and the inability of RSV-transformed cells to self-limit their response to specific external stimuli.

Adenylyl Cyclases↗
PubMed1982

[125I]-HEAT:fifty percent of the ligand can bind to the alpha1-adrenoceptors with extremely high affinity.

[125]-HEAT, 125iodo-2-[Beta-(4-hydroxyphenyl)-ethyl-aminomethyl]tetralone, is a novel alpha1-adrenoceptor ligand which labels alpha1-adrenoceptors in peripheral tissues as well as in the central nervous system. Using the technique of ligand saturation by receptors, we find that only 50% of the 125I-labeled HEAT molecules bind with high affinity to receptors from a variety of tissues. This was observed with partially purified rat brain membranes and highly purified rat liver plasma membranes in the absence or presence of sodium ion (as NaCl, 150 mM) which stimulated 125I-HEAT binding, by increasing the affinity. If the bindability of [125I]-HEAT is taken into account, KD values as low as 7-8 pM (at 30 degrees C) are found in equilibrium binding experiments and optimally stimulating concentrations of sodium ion. The limited high affinity binding of [125I]-HEAT could not be explained by radiochemical impurities. Instead, we suggest that only one enantiomer of the racemic ligand is preferentially bound to the receptors with a KD in the picomolar range. Since the enantiomers are in dynamic equilibrium in solution (via keto-enol tautomerism) [125-I]-HEAT is a unique radioligand which makes it unlikely that the respective isomers can be separated by successive depletion with receptors.

Animals↗
PubMed1982

Evidence of multiple receptor sites within the putative calcium channel.

[3H]-Nimodipine a potent calcium channel blocker, binds to an apparently homogenous population of receptors in guinea-pig brain membranes (KD=0.62 nM, Hill coefficient or approximately 1.0). Diltiazem (10(-5) M) lowers the KD for [3H]-nimodipine by a factor of 3 without changing the maximum number of binding sites. Diltiazem decreased the dissociation rate constant of the nimodipine-receptor complex from 0.18 min-1 to 0.049 min-1 and altered the pharmacological profile as revealed by displacement studies with (-) and (+) verapamil, (-) and (+) prenylamine and 1,4 dihydropyridines. In conclusion [3H]-nimodipine binding can be utilized as a tool to evaluate complex molecular interactions between calcium channel blockers.

Animals↗
PubMed1982

[125I] N6-p-Hydroxyphenylisopropyladenosine, a new ligand for Ri adenosine receptors.

N6-p-Hydroxyphenylisopropyladenosine (HPIA) has been labelled with carrier-free Na[125I] to very high specific activity (2,175 Ci/mmol) and used as an agonist ligand to characterize Ri adenosine receptors in rat cerebral cortex membranes. The binding is saturable, reversible, stereospecific and dependent on protein concentration. The specific binding at 37 degrees C was of high affinity with an equilibrium dissociation constant KD of 0.48 nmol/l and was saturable with 0.23 pmol of [125I]HPIA per mg of protein. The rate constant of association, k1, was 3.25 x 10(8) l mol-1 min-1 and that of dissociation, k2 0.0110 min-1 yielding at t1/2 of 63 min. In competition experiments the (-)isomer of N6-phenylisopropyladenosine (PIA) was 16-fold more potent than the (+)isomer in competing for binding sites. Specific binding was most effectively displaced by N6-cyclohexyladenosine (CHA, ki=0.26 nmol/l), (-)PIA (ki= 0.33 nmol/l) and HPIA (ki=0.52 nmol/l), whereas 5'-N-ethylcarboxamidoadenosine (NECA, ki=1.42 nmol/l) was less effective. The methylxanthines 3-isobutyl-1-methylxanthine (IBMX), theophylline and caffeine which have been classified as adenosine antagonists had ki values between 5-43 mumol/l. Binding of [125I]HPIA was regulated by guanine nucleotides and divalent cations. The results indicate that [125I]HPIA labels Ri adenosine receptors in rat brain membranes.

Adenosine↗
PubMed1982

Ionic modulation of alpha-adrenoceptors.

Effects of cations and nucleotides on the in vivo binding properties of alpha-adrenoceptors in rat cerebral cortex membranes are described. Na+ ion converts a large fraction alpha-adrenoceptors in the absence or presence of Mg2+ into a low affinity state. The effects of 10 mM Na+ can be observed in 250 mM Tris-HCL buffer. It is suggested that alpha 2-adrenoceptor agonists alter the affinity of Na+ for a receptor-associated membrane component. alpha 2-Adrenoceptors are linked to guanylnucleotide binding proteins, which may be involved in signal transfer. These sites are altered in their nucleotide binding properties by pretreatment of the membranes with a medium that allows for ADP ribosylation or/and phosphorylation. alpha 1-Adrenoceptors were probed with a new, high affinity ligand, 125J-HEAT. 125J-HEAT binds (30 degrees C) with Kd values between 6 and 8 pM to alpha 1-adrenoceptors if Na+ slows down the dissociation of 125J-HEAT in comparison with MG2+. Na+ as well as Mg2+ reverse the inhibitory action of phosphatidic acid on the alpha 1-adrenoceptors labeled by 125J-HEAT. Data on the target size, obtained by radiation inactivation, of the alpha 1-adrenoceptors are presented.

Adrenergic alpha-Antagonists↗
PubMed1982

[3H]-Nimodipine and [3H]-nitrendipine as tools to directly identify the sites of action of 1,4-dihydropyridine calcium antagonists in guinea-pig tissues. Tissue-specific effects of anions and ionic strength.

We have performed a comparative binding study with tissues from the guinea-pig with the tritiated calcium antagonists [3H]-nimodipine (isopropyl-[2-methoxy-ethyl]-1,4-dihydro-2,6-dimethyl-4- [3-nitrophenyl]-3,5-pyridinedicarboxylate, Bay e 9736) and [3H]-nitrendipine (1,4-dihydro-2,6-dimethyl-4-[3-nitrophenyl]-3,5-pyridine carboxylic acid, 3-ethyl-5-methyl ester, Bay 3 5009). These compounds are potent nifedipine analogues. Binding of both tritiated calcium antagonists to heart, kidney, lung and brain membranes was evaluated under four different buffer conditions, namely TrisCl and TrisNO3, present at low and high ionic strength (50 and 500 mmol/l). Effects of anions, independent of ionic strength, were observed in brain membranes. In the lung membranes no [3H]-nitrendipine binding in excess above 10 mumol/l unlabelled calcium antagonist was observed at low ionic strength in either TrisCl or TrisNO3. The pharmacological profile of [3H]-nimodipine binding in brain membranes was that expected of a potent 1,4-dihydropyridine calcium antagonist. The apparent dissociation constant (KD) of [3H]-nimodipine for binding sites in brain membranes, determined in TrisNO3 buffer (50 mmol/l, pH = 7.4), was 0.3-0.4 nmol/l at 37 degrees C. The maximum number of binding sites (Bmax) was 300-350 fmol/mg of protein and is in the same range as is commonly observed for neurotransmitters, hormones or channel toxins.

Animals↗
PubMed1981

Mammalian beta-adrenoceptors: concomitant biospecific elution with protein kinase activity from Sepharose-alprenolol.

The mammalian beta-adrenoceptors were solubilized from crude guinea pig lung membranes with the plant glycoside digitonin. The solubilized receptors were absorbed by affinity gels with (+/-)alprenolol as ligand and could be eluted biospecifically in good yield with either agonists or antagonists. A more than 100-fold purification can be achieved in a one-step procedure. Antagonist-eluted beta-adrenoceptors have affinities for antagonists similar to those of particulate or solubilized receptors. Their binding constants for agonists, however, are comparable to those of the particulate receptor. A cAMP-independent protein kinase activity was eluting concomitantly with the beta-adrenoceptors. The protein kinase phosphorylated endogenous substrates with 50 000 and greater than 150 000 subunit molecular weight.

Adenylyl Cyclases↗
PubMed1981

Cation sensitivity of [125I]heat binding to alpha 1-adrenoceptors in rat cerebral cortex membranes.

The alpha 1-adrenoceptor antagonist 2-[beta-(4-hydroxyphenyl)-ethylaminomethyl] tetralone (HEAT) was radiolabeled with 125I and purified to the maximal specific radioactivity of 2200 Ci/mmol. [125 I]HEAT selectively labels alpha 1-adrenoceptors (KD approximately 0.1 nM at 30 degrees C) in rat cerebral cortex membranes. Divalent cations stimulated [125 I]HEAT binding to the alpha 1-adrenoceptors. The rank order of potencies was Ni2+ greater than Mn2+ greater than Mg2+ greater than Ca2+ approximately Sr2+. The effect of Mg2+ was mainly on the Bmax of the alpha I-adrenoceptors. Monovalent cations also stimulated [125I]HEAT binding. Maximal stimulation (up to 3-fold) was seen with Na+ (optimal concentration at approximately 150 mM). Li+ and NH4+ were less effective whereas K+ was ineffective. The stimulation of [125I]HEAT binding to alpha I-adrenoceptors by Na+ was temperature-dependent.

Adrenergic alpha-Antagonists↗
PubMed1981

Quercetin inhibits tyrosine phosphorylation by the cyclic nucleotide-independent, transforming protein kinase, pp60src.

The bioflavonoid quercetin is a potent inhibitor of a cyclic nucleotide-independent, tumor virus-coded protein kinase which phosphorylates tyrosine residues and acts as a cellular transforming protein. Half-maximal inhibition of the protein kinase occurred at 3-4 microM quercetin whereas rutin was much less effective. The finding, that quercetin inhibits a cyclic nucleotide-independent protein kinase activity, may provide clues to the diverse pharmacological effects of the bioflavonoids.

Cyclic AMP↗
PubMed1980

Calcium- and potassium-channel blockers interact with alpha-adrenoceptors.

The so-called calcium antagonist D-600 and agents that enhance the influx of calcium ions into nerve terminals during action potentials (4-aminopyridine, tetraethylammonium, guanidine) interact with alpha-adrenoceptors. The interaction was revealed by binding experiments in vitro with the alpha 2- and alpha 1-specific ligands [3H]clonidine and [3H]prazosin, resp. D-600 binds in a competitive manner to alpha 2- and alpha 1-adrenoceptors in rat-brain membranes. [3H]Prazosin was used to identify alpha 1-adrenoceptors in rat-heart-membrane fragments. D-600 inhibited the binding of the tritiated antagonist, in a manner similar to that seen in rat-brain membranes. 4-Aminopyridine, guanidine and tetraethylammonium blocked no-competitvely the binding of [3H]clonidine in brain membranes. There ws little or no effect of these agents on the binding of [3H]prazosin to alpha 1-adrenoceptors in rat heart or brain. The results indicate that D-600 binds to a region common to alpha 1- and alpha 2-adrenoceptors, whereas the potassium-channel blockers reveal a structural feature of the alpha 2-adrenoceptors in brain membranes which is also present in voltage-dependent potassium channels but is not shared by alpha 1-adrenoceptors.

Aminopyridines↗