Hartmut Glossmann

Alpha adrenoceptors in rat brain direct identification with prazosin

SummaryTritiated prazosin was used to characterize high affinity binding sites with characteristics similar to α1 adrenoceptors in rat brain membranes. These sites were compared with α2 adrenoceptors labeled with tritiated clonidine. The prazosin sites had an association constant of 2 nM−1 and bound the ligand optimal around pH 7.0. The density of the sites was 300 fmoles per mg of protein; the half time of dissociation of prazosin was 7 min at 30° C. The order or potencies of agonists, determined from binding-inhibition experiments with labeled prazosin, was: naphazoline > clonidine > adrenaline > noradrenaline > phenylephrine > α-methylnoradrenaline > dophamine. The order of potencies of antagonists was: prazosin > phenoxybenzamine > phentolamine > clozapine > yohimbine. Sodium ions and divalent cations as well as guanyl nucleotides have little or no effect on the binding of the labeled antagonist. This is in contrast to the binding of the labeled agonist clonidine (Glossmann and Presek, 1979a, 1979b). Labeled prazosin may be a useful tool to characterize α1 adrenoceptors.

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 (K d = 1.5±0.03 nM, B max = 2.1 ± 0.25 pmol/protein, at 37°C) copurified (6‐fold) in this fraction with [3H]ouabain binding (6.6‐fold) and 125I‐α‐bungarotoxin binding (5‐fold). d‐cis‐Diltiazem (but not 1‐cis‐diltiazem) stimulated (±) [3H]nimodipine binding (ED 50 1 μM) by increasing the B max. 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.

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

SummaryHigh-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×g, 4h), passed freely through 0.2 μm nitrocellulose filters and were stable at 4° C with half-lives of >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°C. Specific binding of the 1,4-dihydropyridine calcium channel label was fully reversible (k−1=1.5 min−1, at 37°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 μmol/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-cymotrypsin 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 selectivelyeluted 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.

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°C the K D is 39 nM and B max is 11 pmol/mg protein. The binding is fully reversible (K −1 = 0.02 min−1). The binding sites discriminate between the diastereoisomers 1‐ and d‐cis‐diltiazem, recognize verapamil, gallopamil and tiapamil, and are sensitive to La3+‐inhibition. At 30°C the K D is 37 nM and the B max 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°C an enhancement of d‐cis‐diltiazem binding by the channel blockers is observed. This is attributed to a B max increase. EC 50‐values for enhancement and the maximal enhancement differ for the individual 1,4‐dihydropyridines. At 2°C 1,4‐dihydropyridines inhibit d‐cis‐[3H]diltiazem binding. This is attributed to a B max 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.

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

SummaryThe 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.

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

SummaryThe 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 μM 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.

Purified calcium channels have three allosterically coupled drug receptors.

(−)‐[3H]Desmethoxyverapamil and (+)‐[3P]PN 200‐110 were employed to characterize phenylalkylamineselective and 1,4‐dihydropyridine‐selective receptors on purified Ca2+ channels from guinea‐pig skeletal muscle t‐tubules. In contrast to the membrane‐bound Ca2+ channel, d‐cis‐diltiazem (EC50 = 4.5 ± 1.7 μM) markedly stimulated the binding of (+)‐[3H]PN 200‐110 to the purified ionic pore. In the presence of 100 μM d‐cis‐diltiazem (which binds to the benzothiazepine‐selective receptors) the B max for (+)‐[3H]PN 200‐110 increased from 497 ± 81 to 1557 ± 43 pmol per mg protein, whereas the K d decreased from 8.8 ± 1.7 to 4.7 ± 1.8 nM at 25°C. P‐cis‐Diltiazem was inactive. (−)‐Desmethoxyverapamil, which is a negative heterotropic allosteric inhibitor of (+)‐[3H]IN 200‐110 binding to membrane‐bound channels, stimulated 1,4‐dihydropyridine binding to the isolated channel. (−)‐[3H]Desmethoxyverapamil binding was stimulated by antagonistic 1,4‐dihydropyridines [(+)‐PN 200‐110 ⪢(−)(CR)‐202‐791 ⪢(+)(4R)‐Bay K 8644] whereas the agonistic enantiomers (+)(S)‐202‐791 and (−)(4S)‐Bay K 8644 were inhibitory and (−)‐PN 200‐110 was inactive. The results indicate that three distinct drug‐receptor sites exist on the purified Ca2+ channel, two of which are shown by direct labelling to be reciprocally allosterically coupled.

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 K d of 350 pM to guinea‐pig skeletal muscle membranes in the absence of ultraviolet light. The reversible [3H]azidopine binding is inhibited stereoselectively 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 M r∼145 000. The covalent labelling of the M r ∼145 ooo 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.

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

Abstract: 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 μmg‐1 of membrane protein and a KD of 0.9 nM. Bay K 8644 inhibited [3H]nitrendipine binding with a K1 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.

(—)-[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 K D of 2.2 ± 0.1 nM and a B maxof 18 ± 4 membrane protein at 25°C. Binding was stereoselectively inhibited by phenylalkylamine Ca2+ antagonists and in a mixed, non‐competitive fashion by the benzoihiazepine 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 <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.