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H Glossmann

Publications and source records attributed to H Glossmann.

At least 127 records · Page 7Linked to original sources
PubMed1985

Relationship between the stereoselective negative inotropic effects of verapamil enantiomers and their binding to putative calcium channels in human heart.

Ventricular preparations from patients with mitral disease and hypertrophic obstructive cardiomyopathy (HOCM) were set up to contract isometrically. Ventricular membrane particles were also prepared and putative calcium channels were labelled with [3H]-nimodipine. Positive staircase was induced by varying the rate of stimulation of isolated strips from 6 min-1 to 120 min-1 in the presence of 6-60 microM (-)-adrenaline or (-)-noradrenaline. (-)-Verapamil 3-5 microM or (+)-verapamil 20-30 microM reversed the force-frequency relationship (i.e. caused negative staircase) in preparations from patients with mitral disease or HOCM. In subendocardial strips of ventricular septum from 5 patients with HOCM paced at 60 min-1, both (-)-verapamil and (+)-verapamil caused cardiodepression. Half-maximal cardiodepression was observed with 0.4 microM (-)-verapamil and with 3 microM (+)-verapamil. [3H]-nimodipine bound to ventricular membrane particles in a saturable, reversible fashion to a high affinity site with an equilibrium dissociation constant of 0.23 nM. The density of these sites was 95 fmol mg-1 of membrane protein. Binding of the tritiated 1,4-dihydropyridine was stereoselectively inhibited by 1,4-dihydropyridine enantiomers and nifedipine. (-)-Verapamil and (+)-verapamil inhibited high affinity [3H]-nimodipine binding in a negative heterotropic allosteric manner with (-)-verapamil being 5 times more potent than (+)-verapamil on an IC50 basis. At a given [3H]-nimodipine concentration, (+)-verapamil inhibited a greater fraction of specific [3H]-nimodipine binding. The allosteric mode of (+)-verapamil inhibition of [3H]-nimodipine binding was confirmed by kinetic studies. (-)-Verapamil shifted (+)-verapamil-binding inhibition curves to the right in an apparently competitive fashion. The inversion of staircase caused by both verapamil enantiomers suggests that they cause a use-dependent channel blockade. The similar potency ratios for binding and for cardiodepression are indicative of a common locus of action for both verapamil enantiomers within the calcium channel.

Aged↗
PubMed1985

Calcium channels: basic properties as revealed by radioligand binding studies.

The structural features and molecular properties of calcium channels have been explored with 1,4-dihydropyridines, d-cis diltiazem and verapamil in radiolabeled form. The concept of multiple (at least three) drug receptor sites of the channel (sites 1, 2, and 3) has been proposed. Site 1 is labeled by 1,4 dihydropyridines, site 2 by (+/-) verapamil (in skeletal muscle) and by (-) desmethoxyverapamil (in brain, skeletal muscle, and heart), site 3 by d-cis diltiazem (in skeletal muscle). The three distinct drug receptor sites communicate with each other via reciprocal allosterism and are linked to divalent cation binding sites. The concept of the reciprocal allosteric communication between the receptor sites has been proven by individual labeling of each of the drug receptor sites in the skeletal muscle t-tubule membrane where the density of calcium channels is extremely high. In brain and heart, labeling of the receptor sites 1 and 2 has also been successful. Three novel ligands for the calcium channel are presented: 125I-iodipine, a 1,4 dihydropyridine ligand, which has an exceptionally high specific activity (2,200 Ci/mmol), 3H-azidopine, a high-affinity, photoaffinity ligand and finally (-) 3H-desmethoxyverapamil, a high-affinity radioligand for receptor site 2. These ligands will aid in the isolation and final characterisation of the calcium channel. 125I-iodipine, 3H-azidopine and (-) 3H-desmethoxyverapamil bind to detergent-solubilized channels from skeletal muscle t-tubules. Whereas the 1,4-dihydropyridines label only a 12.9 S macromolecule, (-) 3H-desmethoxyverapamil additionally labels a approximately 5.0 S peak, where almost no high-affinity 1,4 dihydropyridine binding is found. Taken together with the results from target-size analysis experiments and photoaffinity labeling, an oligomeric structure of the channel is most likely. One polypeptide chain (Mr = 145,000) in reduced form has been unambiguously identified and is currently further characterized. It remains to be seen if the all of the earlier postulated four elements of the channel (alpha, beta, delta, gamma) are on different polypeptide chains or if the 145,000 peptide corresponds to the (beta, delta) or the (delta, gamma) element.

Affinity Labels↗
PubMed1985

Calcium channels and calcium channel drugs: recent biochemical and biophysical findings.

The biochemical and biophysical features of the voltage-dependent calcium channels, as discovered in vitro by means of radiolabelled drugs, are presented. The concept of distinct but reciprocally allosterically coupled drug receptor domains linked to calcium binding sites is explained. The evidence for the existence of isochannels (and isoreceptors) is reviewed and the voltage-dependence of 1,4-dihydropyridine binding and action is discussed. The structure of the channel is investigated by radiation-inactivation and by photoaffinity labelling. Low affinity binding sites for calcium channel drugs are shown to reside on the nucleoside carrier.

Adenosine↗
PubMed1984

(-)-[3H]Desmethoxyverapamil, a novel Ca2+ channel probe. Binding characteristics and target size analysis of its receptor in skeletal muscle.

(-)-[3H]Desmethoxyverapamil (2,7-dimethyl-3-(3,4-dimethoxyphenyl)-3-cyan- 7-aza-9-(3-methoxyphenyl)-nonanhydrochloride) was used to label putative Ca2+ channels in guinea pig skeletal muscle. The binding sites for (-)-[3H]desmethoxyverapamil co-purified with t-tubule membrane markers in an established subcellular fractionation procedure. (-)-[3H]Desmethoxyverapamil bound to partially purified t-tubule membranes with a KD of 2.2 +/- 0.1 nM and a Bmax of 18 +/- 4 pmol/mg membrane protein at 25 degrees C. Binding was stereoselectively inhibited by phenylalkylamine Ca2+ antagonists and in a mixed, non-competitive fashion by the benzothiazepine Ca2+ antagonist d-cis-diltiazem and the 1,4-dihydropyridine Ca2+ antagonist (+)-PN 200-110. Target size analysis of the (-)-[3H]desmethoxyverapamil drug receptor site revealed a molecular mass of 107 +/- 2 kDa. In contrast, the target size of the allosterically coupled benzothiazepine drug receptor site, labelled by d-cis-[3H]diltiazem, was 130.5 +/- 4 kDa (p less than 0.01) and of the 1,4-dihydropyridine binding site 179 kDa, when labelled with [3H]nimodipine. It is concluded that (-)-[3H]desmethoxyverapamil is an extremely useful radioligand for the phenylalkylamine-selective receptor site of the t-tubule localized Ca2+ channel which is allosterically linked to two other distinct drug receptor sites.

Animals↗
PubMed1984

Target size analysis and molecular properties of Ca2+ channels labelled with [3H]verapamil.

[3H]Verapamil was employed to label the drug receptor sites within the Ca2+-channel in skeletal muscle microsomes which are coupled in a negative heterotropic allosteric manner to the previously characterized 1,4-dihydropyridine receptors. At 2 degrees C the Kd of a high-affinity receptor site is 45 nM and the maximum density of binding sites is 37 pmol/mg of protein. Established subcellular fractionation procedures were used to isolate transverse tubule membranes from rabbit and guinea-pig skeletal muscle. [3H]Verapamil, d-cis-[3H]diltiazem as well as 1,4-[3H]dihydropyridine receptors copurify with t-tubule membranes. The ratio of high-affinity verapamil: 1,4-dihydropyridine d-cis-diltiazem Ca2+ channel receptor sites is 4:2:1. The verapamil drug receptors are heat-labile and have essential sulfhydryl groups since they are inactivated by p-chloromercuriphenylsulfonic acid and N-ethylmaleimide. The receptors recognize the main classes of Ca2+ antagonists and agonists in a stereoselective manner. Divalent cations (Mn2+ greater than Ca2+ greater than Mg2+) are inhibitory. Target size analysis with high-energy electrons was performed and the Mr of the verapamil drug receptor site is 110000.

Alamethicin↗
PubMed1984

Photoaffinity labelling of Ca2+ channels with [3H]azidopine.

A 1,4-dihydroypyridine arylazide photoaffinity ligand, [3H]azidopine (50.6 Ci/mmol), has been synthesized. [3H]Azidopine binds reversibly with a Kd of 350 pM to guinea-pig skeletal muscle membranes in the absence of ultraviolet light. The reversible [3H]azidopine binding is inhibited steroselectively by 1,4-dihydropyridines, phenylalkylamine Ca2+ channel blockers and La3+. Covalent incorporation into membrane proteins after photolysis was investigated by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis. [3H]Azidopine is photoincorporated specifically into a protein of Mr approximately 145 000. The covalent labelling of the Mr approximately 145 000 band is inhibited stereoselectively by drugs and cations which block the reversible [3H]azidopine binding. It is suggested that [3H]azidopine is photoincorporated into a subunit of the putative Ca2+ channel.

Affinity Labels↗
PubMed1984

(-)-3H-desmethoxyverapamil labelling of putative calcium channels in brain: autoradiographic distribution and allosteric coupling to 1,4-dihydropyridine and diltiazem binding sites.

The optically pure phenylalkylamine Ca2+-antagonist (-)-3H-desmethoxyverapamil has been used to directly label putative Ca2+ channels in thaw-mounted guinea-pig brain sections and in hippocampus homogenates. The autoradiographic distribution of (-)-3H-desmethoxyverapamil binding shows the highest density of binding sites in the molecular layer of the hippocampus, medium levels in the cerebral cortex and low levels in the cerebellum. This anatomical distribution is the same as that found for 1,4-dihydropyridine binding sites, labelled by 3H-nimodipine. (-)-3H-desmethoxyverapamil binds to hippocampus membranes with a KD value of 1.6 +/- 0.2 nmol/l and a Bmax of 870 +/- 175 fmol per mg of protein. Binding is stereospecifically inhibited by the phenylalkylamines desmethoxyverapamil, D-600 and verapamil. 1,4-Dihydropyridines regulate (-)-3H-desmethoxyverapamil binding in a negative heterotropic allosteric manner, depending on the availability of free divalent cations. The potency series of phenylalkylamine Ca2+ antagonists in inhibiting high affinity (-)-3H-desmethoxyverapamil binding to hippocampus membranes and the allosteric regulation by chemically different classes of Ca2+ antagonists suggest that the (-)-3H-desmethoxyverapamil binding sites in hippocampus are associated with putative Ca2+ channels.

Animals↗
PubMed1984

125I-iodipine, a new high affinity ligand for the putative calcium channel.

The compound 1,4-dihydro-2,6-dimethyl-4-(2-trifluoromethylphenyl)-3, 5-pyridinecarboxylic acid 2-(amino-ethyl)ethylester was reacted with N-succinimidyl 3-(4-hydroxy, 5-125I-iodophenyl)propionate (2,200 Ci/mmol. The purified, 125I-labelled product of this reaction is termed 125I-iodipine and has a specific activity of approximately 2,200 Ci/mmol on the date of preparation. 125I-Iodipine binds reversibly in a saturable manner and with high affinity to skeletal muscle and brain membranes with KD values at 25 degrees C of 400 and 60 pmol/l, respectively. The binding of 125I-iodipine is stereoselectively inhibited by chiral 1,4-dihydropyridine calcium channel blockers. 125I-Iodipine labelled sites also recognize the recently described 1,4-dihydropyridine calcium channel activators and discriminate the chemically unrelated compounds (-)- and (+)verapamil, (-)- and (+)methoxyverapamil and d- and l-cis diltiazem. It is concluded that 125I-iodipine labels the putative calcium channel.

Animals↗
PubMed1984

Novel 1,4-dihydropyridine (Bay K 8644) facilitates calcium-dependent [3H]noradrenaline release from PC 12 cells.

The effects of the novel 1,4-dihydropyridine Bay K 8644 [methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine- 5-carboxylate] on the release of [3H]noradrenaline in cultured PC 12 cells were investigated. K+ in a concentration-dependent manner evoked 3H-transmitter release with an EC50 of 50-56 mM. Bay K 8644 at 30 nM potentiated the K+-evoked [3H]noradrenaline release; however, in the absence of calcium neither K+ evoked nor Bay K 8644 enhanced [3H]noradrenaline release. At a K+ concentration of 25 mM, Bay K 8644 stimulated [3H]noradrenaline release fivefold, with an EC50 of 10 nM, and 100 nM of the calcium channel blocker nitrendipine shifted the concentration response curve of Bay K 8644 to the right in an apparently competitive fashion. Nitrendipine blocked the Bay K 8644-potentiated release with an EC50 of 700 nM in the presence of 500 nM Bay K 8644. [3H]Nitrendipine bound to a saturable population of binding sites on PC 12 cell membranes with a Bmax of 180 fmol X mg-1 of membrane protein and a KD of 0.9 nM. Bay K 8644 inhibited [3H]nitrendipine binding with a Ki of 16 nM. It is concluded that Bay K 8644 binds to, and stabilizes, the open state of calcium channels and thus acts as a "calcium agonist" to mediate calcium-dependent cellular events such as catecholamine release from PC 12 cells.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1984

Molecular pharmacology of the calcium channel: evidence for subtypes, multiple drug-receptor sites, channel subunits, and the development of a radioiodinated 1,4-dihydropyridine calcium channel label, [125I]iodipine.

Radiolabeled Ca2+ antagonists (1,4-dihydropyridines, verapamil, and D-cis-diltiazem) were used to study voltage-operated Ca2+ channels in different excitable tissues. The concept of three subtypes of Ca2+ channels, represented by brain, heart, and skeletal-muscle isoreceptors for 1,4-dihydropyridines, is developed. The three subtypes are characterized by a variety of criteria. Despite the biochemical differences between the subtypes, they have the same Mr in situ by target-size analysis (Mr approximately equal to 180,000, when evaluated by [3H]nimodipine). The concept of the metalloprotein nature of the channel and the interaction of channel drugs with the Me2+ binding sites of the ionic pore is demonstrated. Distinct but interacting drug-receptor sites of the Ca2+ channel are found by direct labeling as well as indirectly by drug competition studies. We distinguish between the 1,4-dihydropyridine site, the verapamil site, and the D-cis-diltiazem site. Each receptor site can exist in high and low-affinity state; the distribution of receptor sites in these states is regulated by temperature, ions, and drugs. The concept of intrinsic activity of drugs to stabilize the high-affinity state is exemplified for the 1,4-dihydropyridines. A change in the channel architecture is induced by binding of D-cis-diltiazem to its drug receptor site. This is proven by target-size analysis of the channel in situ. Partially purified t-tubule membranes from skeletal muscle are an extremely rich source of Ca2+ channel drug-receptor sites. The stoichiometry was determined in this preparation and found to be four verapamil:two 1,4-dihydropyridine:one D-cis-diltiazem site. A novel Ca2+ channel probe, [125I]iodipine (2,200 Ci/mmol), was synthetized, and the properties of this ligand are presented.

Animals↗
PubMed1983

Temperature-dependent regulation of d-cis-[3H]diltiazem binding to Ca2+ channels by 1,4-dihydropyridine channel agonists and antagonists.

The binding of the Ca2+-channel blocker d-cis-[3H]diltiazem to guinea pig skeletal muscle microsomes is temperature-dependent. At 2 degrees C the KD is 39 nM and Bmax is 11 pmol/mg protein. The binding is fully reversible (K -1 = 0.02 min -1). The binding sites discriminate between the diastereoisomers l- and d-cis-diltiazem, reconize verapamil, gallopamil and tiapamil, and are sensitive to La3+-inhibition. At 30 degrees C the KD is 37 nM and the Bmax is 2.9 pmol/mg protein. D-cis-diltiazem-labelling is regulated by the 1,4-dihydropyridine Ca2+-channel blockers and a novel Ca2+-channel activator in a temperature-dependent manner. At 30 degrees C an enhancement of d-cis-diltiazem binding by the channel blockers is observed. This is attributed to a Bmax increase. EC50-values for enhancement and the maximal enhancement differ for the individual 1,4-dihydropyridines. At 2 degrees C 1,4-dihydropyridines inhibit d-cis-[3H]diltiazem binding. This is attributed to a Bmax decrease. We have directly labelled one of the drug receptor sites within the Ca2+-channel which can allosterically interact with the 1,4-dihydropyridine binding sites.

Animals↗
PubMed1983

Target size analysis of skeletal muscle Ca2+ channels. Positive allosteric heterotropic regulation by d-cis-diltiazem is associated with apparent channel oligomer dissociation.

[3H]PN 200-110, a potent chiral benzoxadiazol 1,4-dihydropyridine Ca2+ antagonist was used to label guinea pig skeletal muscle Ca2+ channels. [3H]PN 200-110 binds with a Kd of approximately 1 nM to a homogeneous population of non-interacting binding sites; d-cis-diltiazem, but not l-cis-diltiazem increases the Bmax of [3H]PN 200-110 by 25% and slows the dissociation rate 3-fold at 37 degrees C. Target size analysis of the [3H]PN 200-110-labelled Ca2+ channels with 10 MeV electrons gave an Mr of 136 000 which was reduced to 75 000 by d-cis-diltiazem treatment of membranes. It is concluded that positive heterotropic allosteric regulation by d-cis-diltiazem is accompanied by channel oligomer dissociation.

Allosteric Regulation↗
PubMed1983

Calcium channels: evidence for oligomeric nature by target size analysis.

Radiation inactivation was employed to measure the molecular size of calcium channels in guinea-pig skeletal muscle membranes, labelled by the potent 1,4-dihydropyridine calcium antagonist [3H]nimodipine. The molecular size was decreased when the membranes were preincubated and assayed with d-cis-diltiazem, a calcium channel blocker, which is structurally unrelated to the 1,4-dihydropyridines. d-cis-Diltiazem, which is a positive heterotropic regulator of 1,4-dihydropyridine calcium channel binding in vitro, reduced the molecular size from 178 000 to 111 500. 1-cis-Diltiazem, the diastereoisomer, which is devoid of calcium antagonistic action, did not decrease the molecular size of the 1,4-dihydropyridine binding site. Neither diastereoisomer affected the molecular size of the membrane-bound acetyl-cholinesterase, indicating that a stereospecific interaction with the calcium channel structure is the basis for these observations. It is concluded that this decrease in size is indicative of the oligomeric nature of the calcium channel and that calcium channel blockers, acting via different, but interacting drug receptor sites, induce different conformations of the channel structure, resulting in altered conductivity for ions.

Animals↗
PubMed1983

Differential labelling of putative skeletal muscle calcium channels by [3H]-nifedipine, [3H]-nitrendipine, [3H]-nimodipine and [3H]-PN 200 110.

Four 1,4-dihydropyridine calcium channel blockers [3H]-nifedipine, [3H]-nitrendipine, [3H]-nimodipine and [3H]-PN 200 110 (Isopropyl 4-(2,1,3,benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5-methoxy- carbonylpyridine-3-carboxylate) were employed to label putative calcium channels in guinea-pig hind limb skeletal muscle membranes. The four radioligands differed with respect to the number of sites (Bmax) which were labelled. The following rank order of Bmax values was found: PN 200 110 greater than nimodipine = nitrendipine greater than nifedipine. d-cis-Diltiazem caused an increase in the density of high-affinity binding sites for all four calcium channel blockers. The relative stimulation was smallest for PN 200 110. It is suggested that 1,4-dihydropyridines exhibit different efficacies for induction of a channel state with high affinity for these drugs.

Animals↗
PubMed1983

Solubilization and partial purification of putative calcium channels labelled with [3H]-nimodipine.

High-affinity binding sites for the potent 1,4-dihydropyridine calcium channel blocker [3H]-nimodipine were solubilized from guinea-pig skeletal muscle microsomes with digitonin and CHAPS [3-(3-cholamidopropyl)-dimethyl-ammonio-l-propanesulfonate]. Detergent-solubilized binding sites could not be sedimented by centrifugation (50,000 X g, 4 h), passed freely through 0.2 micron nitrocellulose filters and were stable at 4 degrees C with half-lives of greater than 60 h. The solubilized 1,4-dihydropyridine binding sites were precipitable with polyethyleneglycol 6000 on Whatman GF/C filters. Saturation analysis of solubilized microsomes with [3H]-nimodipine revealed a single class of binding sites (Bmax = 0.5 to 1.7 pmol per mg of protein) with a KD of 2.2-3.6 nmol/l at 37 degrees C. Specific binding of the 1,4-dihydropyridine calcium channel label was fully reversible (k-1 = 1.5 min-1, at 37 degrees C). The solubilized drug receptors discriminated between the optical enantiomers of chiral 1,4-dihydropyridine calcium channel blockers, (-)- and (+)D-600 as well as between l-cis and d-cis-diltiazem. d-cis-Diltiazem stimulated the binding of [3H]-nimodipine (ED50:3.6 mumol/l), by increasing the Bmax and slowed the dissociation rate of the labelled 1,4-dihydropyridine calcium channel blocker. The solubilized binding sites were sensitive to pronase, alpha-chymotrypsin and phospholipases A and C indicating their protein nature as well as their lipid requirement. Chelation of endogeneous divalent cations by EDTA, EGTA or CDTA inhibits high-affinity [3H]-nimodipine binding, demonstrating that divalent cations are required for high affinity [3H]-nimodipine binding. Detergent-solubilized binding sites are adsorbed by several sepharose-immobilized lectins, including concanavalin A, wheat germ agglutinin and lentil-lectin but not by helix pomatia lectin. Preparative chromatography on concanavalin A sepharose was performed and the adsorbed [3H]-nimodipine binding sites were selectively eluted by alpha-methylmannoside; NaCl (1 mol/l) being completely ineffective as elutant. The purification factors by this method were 17-40-fold. The binding sites could be also purified (up to 10-fold) by sucrose density centrifugation. The S20, w value of the drug receptors is 12.9 s. It is concluded that the 1,4-dihydropyridine binding sites of the putative calcium channel are intimately associated with carbohydrate containing structures. Since the detergent-solubilized material shows allosteric regulation of 1,4-dihydropyridine binding, interaction with chemically different classes of calcium channel blockers, metalloprotein nature and a S20, w value which is indicative of structure large enough to span the membrane, we conclude that we have solubilized and partially purified the putative calcium channel.

Animals↗
PubMed1983

Putative calcium channel molecular weight determination by target size analysis.

The molecular weight of the putative calcium channel in guinea-pig brain membranes, labelled with [3H]-nimodipine, has been determined in situ by target size analysis. Irradiation of guinea-pig brain membranes at 153-173 K with 10 MeV electrons reduced the density of [3H]-nimodipine binding sites without affecting the equilibrium dissociation constant. The decay of the calcium channel blocker binding site density as a function of radiation dose fits to a monoexponential function. Application of the target size theory gives a molecular weight of 185,000. [3H]-Flunitrazepam labelled benzodiazepine receptor target size was measured as an internal control. The molecular weight of the benzodiazepine receptors was 76,000, which is in good agreement with published results.

Animals↗