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A Goll

Publications and source records attributed to A Goll.

30 records · Page 2Linked to original sources
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

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

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

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↗
PubMedDate not supplied

[Prevention of heterotopic ossification after THA with indomethacin: analysis of risk factors].

AIM: Several risk factors are associated with heterotopic bone formation following total hip replacement. All these risk factors were defined in cohorts without any treatment against postoperative ectopic bone. The aim of this prospective study was to reveal risk factors for the development of postoperative ossifications in patients who underwent a 7-day course of indomethacin therapy. METHODS: 211 consecutive patients with 217 cementless total hip arthroplasties were included. Patients were given 100 mg indomethacin daily in a 7-day course. RESULTS: Grade 2 or 3 ossifications were observed in 13 (5%) of the male and 3 (2%) of the female Individuals postoperatively (p = 0.0043; odds ratio = 0.45). Older individuals (p = 0.0021; odds ratio = 1.03) as well as patients with primary osteoarthritis (p = 0.0307; odds ratio = 0.28) also showed a higher risk for developing ectopic bone formations. CONCLUSION: With a 7-day course of indomethacin after total hip arthroplasty, male and elderly individuals, as well as patients with primary osteoarthritis, are considered to be at high risk to develop heterotopic ossifications postoperatively. Indomethacin reduces the incidence of postoperative ectopic ossifications, but not the patient's risk.

Anti-Inflammatory Agents, Non-Steroidal↗