PubMed Health⌕ Search

Biomedical subjects

D R Ferry

Publications and source records attributed to D R Ferry.

At least 73 records · Page 4Linked to original sources
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↗
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

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↗
PubMedDate not supplied

Determination of quercetin in human plasma by HPLC with spectrophotometric or electrochemical detection.

A reversed-phase high-performance liquid chromatographic method for the determination of quercetin in human plasma following intravenous infusion is described. Quercetin in plasma was extracted with methanol-dimethyl sulphoxide (4:1 v/v) and separated on a C18 Hypersil-BDS column with 44% (v/v) methanol in 0.1 M ammonium acetate (pH 5.15) containing 0.27 mM EDTA as the mobile phase. The drug was detected specifically and sensitively at its absorption maximum of 375 nm, or electrochemically, with a detection limit of 80 ng/mL and 2 ng/mL, respectively.

Chromatography, High Pressure Liquid↗