Biomedical subjects

H Glossmann

Publications and source records attributed to H Glossmann.

At least 73 records · Page 4Linked to original sources

PubMed1993

Evidence for an external location of the dihydropyridine agonist receptor site on smooth muscle and skeletal muscle calcium channels.

1. The location of the binding domain for agonist dihydropyridines (DHP) has been studied by comparing the action of (+)-202,791 and (-)-Bay K 8644 on Ba2+ currents (IBa) in whole cell patch clamp experiments. Drug effects were examined upon internal and external (extracellular) application in A7r5 smooth muscle cells and BC3H1 cells, a cell line expressing Ca channels of the skeletal muscle type. 2. Efficiency of internal drug application in the whole cell studies was demonstrated by inhibition of potassium currents and barium currents (IBa) upon internal perfusion with tetraethylammonium (TEA+) (10 mM) and the permanently charged phenylalkylamine, D 890 (100 microM) respectively. The uncharged DHP, (-)-STBODIPY-DHP (2 microM) was used to estimate the time course of internal perfusion by monitoring its fluorescence. 3. Intracellular application of (+)-202,791 and (-)-Bay K 8644 (5 microM) in patch clamp experiments was ineffective in stimulating Ca2+ channel currents in both cell lines. In contrast a 50 fold lower agonist concentration (0.1 microM (-)-Bay K 8644) applied to the external face of the membrane induced typical changes in tail currents and a current increase under conditions when up to 10 microM of the agonist was present in the intracellular perfusion solution. 4. In cell-attached patches in A7r5 cells, (-)-Bay K 8644 increased and (+)-PN 200,110 inhibited single channel activity when applied via the bath solution. This suggests partitioning and lateral diffusion of the DHPs in the lipid of the plasma membrane. 5. We conclude that the binding site for agonist DHPs on Ca2+ channels in A7r5 and BC3H1 cells is located close to the external surface of the membrane. The DHP binding domain can be reached by agonists and antagonists from the extracellular but not from the intracellular face of the membrane.

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

PubMed1993

Identification of a 27-kDa high affinity phenylalkylamine-binding polypeptide as the sigma 1 binding site by photoaffinity labeling and ligand-directed antibodies.

The verapamil-like arylazide (-)-[3H]azidopamil specifically photoaffinity labeled two low molecular mass polypeptides, with apparent molecular masses of 22 and 27 kDa, in the endoplasmic reticulum of guinea pig liver, kidney, adrenal gland, and lung. It was recently shown that the 22-kDa polypeptide binds the anti-ischemic phenylalkylamine (-)-[3H]emopamil and other anti-ischemic drugs with high affinity. We now provide evidence that the photolabeling of the 27-kDa polypeptide is blocked by nanomolar concentrations of sigma ligands [order of potency, haloperidol > pentazocine > 1,3-ditolylguanidine > dextromethorphan > (+)-SKF10,047]. The apparent affinities of these and other drugs closely corresponded to those for 1,3-[3H]ditolylguanidine-labeled sigma binding sites. Based on its high affinity for the (+)-enantiomer [but not the (-)-enantiomer] of SKF10,047 (Ki = 51 nM), pentazocine (Ki = 3 nM), and dextromethorphan (Ki = 30 nM), the (-)-[3H]azidopamil-labeled site on the 27-kDa polypeptide was classified as being of the sigma 1 subtype. Using antiphenylalkylamine antibodies, we developed a novel immunological detection method that allows the rapid and sensitive staining of the photolabeled 27-kDa polypeptide after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We conclude that the phenylalkylamines emopamil and azidopamil represent a novel class of sigma ligands, highly suitable for the further structural characterization of polypeptides carrying sigma 1 binding sites.

Affinity Labels↗

PubMed1993

Heterogeneity of L-type calcium channel alpha 1 subunits: stereoselective discrimination of different populations by the novel 1,4-dihydropyridine B 874-67.

The basic (pKa = 8.49) 1,4-dihydropyridine B 874-67 [[3-(C1R,2S)- 2-methylamino-1-phenylpropyl]-5-methyl-1,4-dihydro-2,6-dimethyl-(4R)-4-( 3-nitrophenyl)pyridine-3,5-dicarboxylate hydrochloride] has unique properties; it can discriminate two populations of alpha 1 subunits in 1,4-dihydropyridine-sensitive calcium channels labeled with the neutral 1,4-dihydropyridine (+)-[3H]PN 200-110. The two populations, which occur in proportions of approximately 2:1 in rabbit skeletal muscle membranes and highly purified calcium channel preparations, differ approximately 20-fold in their affinity. The corresponding diastereomer, B 874-66, and other 1,4-dihydropyridines (neutral, basic, or permanently charged) do not share this property. The two populations were observed at 2 degrees, 22 degrees, and 37 degrees in similar proportions. Heterogeneity was also observed for guinea pig heart membrane calcium channels labeled with (+)-[3H]PN 200-110. Heterotropic allosteric regulators, Ca2+, and Mg2+, but not Ba2+ and Ni2+, abolished the discriminatory activity of B 874-67 at 2 degrees and 22 degrees, regardless of whether binding of the neutral 1,4-dihydropyridine was stimulated or inhibited. It is proposed that the two alpha 1 subunit populations differ with respect to their 1,4-dihydropyridine binding domain. The structural basis for the two populations is unclear but may relate to the functional heterogeneity of membrane-bound and highly purified calcium channel preparations previously observed by others.

Allosteric Regulation↗

PubMed1993

Biochemical characterization of a 22-kDa high affinity antiischemic drug-binding polypeptide in the endoplasmic reticulum of guinea pig liver: potential common target for antiischemic drug action.

The phenylalkylamine emopamil prevents brain damage due to experimental cerebral ischemia. Stereoselective, high affinity, binding sites for (-)-[3H]emopamil in guinea pig brain cortex and liver membranes have been proposed to mediate its antiischemic effect. Using [N-methyl-3H]LU49888 as a photoaffinity probe we now provide evidence that the cation-sensitive emopamil binding site is localized on a 22-kDa polypeptide in guinea pig liver, kidney, lung, and adrenal gland. This 22-kDa polypeptide binds other antiischemic drugs with high affinity and is a nonglycosylated integral membrane protein of the endoplasmic reticulum. It can be solubilized with digitonin without changes in its drug-binding properties. The solubilized binding activity has a sedimentation coefficient of 12.0 +/- 0.4 S and an apparent Stokes radius of 6.0 +/- 0.1 nm. From these data it is concluded that the 22-kDa polypeptide is associated in a larger oligomeric complex with a molecular mass of at least 84 kDa. [N-methyl-3H]LU49888 also specifically labels a second 27-kDa polypeptide in the endoplasmic reticulum, which can be distinguished from the 22-kDa polypeptide by its pharmacological and hydrodynamic properties. The photolabeled 22-kDa polypeptide was partially purified under denaturating conditions. This will allow the further structural analysis of this putative target for antiischemic drugs.

Affinity Labels↗

PubMed1992

A unique fluorescent phenylalkylamine probe for L-type Ca2+ channels. Coupling of phenylalkylamine receptors to Ca2+ and dihydropyridine binding sites.

The first fluorescently labeled phenylalkylamine, DMBODIPY-PAA (5-(3-[3-(4,4-difluoro-5,7-dimethyl-3a, 4a-diaza-4-bora-indacen-3-yl)propionamido] phenethyl-N-methylamino)-2-isopropyl-2-(3,4,5-trimethoxyphenyl)-valer onitrile) has been introduced for L-type Ca2+ channel research. DMBODIPY-PAA binds reversibly to L-type Ca2+ channels purified from rabbit skeletal muscle microsomes by wheat germ agglutinin-Sepharose chromatography. In this preparation DMBODIPY-PAA labels 412 pmol of phenylalkylamine receptors/mg of protein with a Kd of 6.82 nM and a favorable signal-to-noise ratio. Therefore DMBODIPY-PAA has a higher affinity for purified Ca2+ channels than the commonly employed radioligands and consequently has assisted in channel purification after prelabeling by simply monitoring receptor-bound fluorescence. (+)-PN200-110 (which is stimulatory for (-)-[3H]desmethoxyverapamil binding to purified Ca2+ channels) inhibits DMBODIPY-PAA labeling. Since these drug interactions are reciprocal, the phenylalkylamine and dihydropyridine binding sites of the alpha 1-subunit are tightly coupled. Kinetic and equilibrium binding studies with (-)-[3H]desmethoxyverapamil and DMBODIPY-PAA show that phenylalkylamine binding to L-type Ca2+ channels is dependent on Ca2+. Chelation of divalent metal ions converts phenylalkylamine receptors into a very low affinity state. This conversion is temperature- and time-dependent and completely reversible (K0.5 for free Ca2+ = 58 nM). This study demonstrates the utility of fluorescent ligands for binding studies with L-type Ca2+ channels and provides evidence for coupling between Ca2+ binding sites and phenylalkylamine receptors.

Animals↗

PubMed1992

The dihydropyridine niguldipine inhibits T-type Ca2+ currents in atrial myocytes.

The whole-cell tight seal recording technique was used to investigate the effects of niguldipine, a novel dihydropyridine, on Ca2+ currents in guinea pig atrial cells. Ca2+ currents were separated into T-type and L-type components by an appropriate voltage protocol. Extracellular application of 1 microM (+/-)-niguldipine (NIG) resulted in a pronounced blockade of both T-type (to 20 +/- 10% of control, n = 5) and L-type Ca2+ currents (to 28 +/- 12% of control, n = 5). Current to voltage relationships clearly showed that both Ca2+ currents were blocked over the whole voltage range examined (-60 to +40 mV). The inhibitory effect of niguldipine on T-type Ca2+ currents was found to be voltage-dependent, i.e. prolonged hyperpolarization to -90 mV led to a partial and transient removal of NIG block. The IC50 for T-type Ca2+ current inhibition by (+/-)-NIG was determined as 0.18 microM. NIG action is stereospecific. (+)-niguldipine was found to be more potent than (-)-niguldipine in blocking both Ca2+ currents. This study demonstrates the Ca2+ antagonistic action of the dihydropyridine NIG, which may not discriminate between T- and L-type Ca2+ channels.

Animals↗

PubMed1992

[35S]sadopine, a novel high affinity, high specific activity, L-type Ca2+ channel probe: characterization of two equipotent diastereomers with opposite allosteric properties.

A novel 35S-labeled dihydropyridine (DHP), 1,4-dihydro-2,6-dimethyl-4-(2-trifluoromethylphenyl)-pyridine-3,5-dic arboxyl-3- [2-(N-tert-butoxycarbonyl-L-[35S]methionyl)-aminoethyl]-ester-5-ethyl ester, ([35S]sadopine) (800-1400 Ci/mmol), the respective (+)- and (-)-diastereomers, and unlabeled (+/- )-, (-)-, and (+)-sadopine were synthesized. [35S]Sadopine is an excellent high affinity, high specific activity radioligand to label selectively the DHP receptor of L-type Ca2+ channels in tissue sections as well as in membrane fragments. Both diastereomers bind to the DHP receptors in a saturable and reversible manner, with equal, subnanomolar, dissociation constants. Despite their similar affinities, (+)- and (-)-sadopine differ with respect to their kinetic properties [the association and dissociation rate constants are 10-fold higher for (+)-[35S]sadopine at 22 degrees] and their allosteric modulation of the phenylaklylamine or benzothiazepine binding domain. (+)-Sadopine is a negative but (-)-sadopine a positive allosteric modulator of (-)-[N-methyl-3H]LU49888 or (+)-cis-[3H] diltiazem binding at 30 degrees. Both diastereomers act as L-type Ca2+ channel blockers in cardiac and smooth muscle cells. Computer-based analysis of the electrostatic potentials of the two diastereomers and calculation of the interaction energies with a hypothetical DHP receptor model predicted not only the similar affinities of (+)- and (-)-sadopine but also their Ca2+ channel-blocking effects. The temperature-dependent allosteric differences between the diastereomers suggest that two distinct conformational states of the DHP receptor are stabilized in vitro, both corresponding to a nonconducting state of the channel. Our data indicate that access to the DHP receptor site, but not binding affinity, is a function of the opposite stereochemistry of the sadopine diastereomers. Therefore, labeled and unlabeled (+)- and (-)-sadopine will be useful probes to further characterize the molecular basis of DHP-Ca2+ channel interaction and the pharmacological and physiological significance of the different allosteric conformations of the channel induced by Ca2+ channel-active drugs.

Animals↗

PubMed1991

Identification of 1,4-dihydropyridine binding regions within the alpha 1 subunit of skeletal muscle Ca2+ channels by photoaffinity labeling with diazipine.

To identify regions that are involved in the formation of the dihydropyridine receptor site of skeletal muscle L-type Ca2+ channels, the alpha 1 subunit of the channel complex was specifically labeled with the 1,4-dihydropyridine-receptor-selective photoaffinity probe [3H]diazipine. Photoaffinity-labeled regions were identified by probing labeled proteolytic fragments with several anti-peptide antibodies recognizing different segments of the alpha 1 sequence. Forty to 50% of the alpha 1-associated [3H]diazipine label was contained in the tryptic fragment between Arg-988 and Ala-1023 derived from the loop between segments S5 and S6 in domain III. This region corresponds to a portion of the channel that is believed to contribute to formation of the transmembrane pore. Twenty to 30% of the labeling occurred in a V8 protease fragment between Glu-1349 and Trp-1391. This fragment contains transmembrane segment S6 of domain IV and has previously been shown to form part of the drug receptor for phenylalkylamine Ca2+ antagonists. Our data suggest that the dihydropyridine receptor is formed by close apposition of two discontinuous regions of the alpha 1 subunit sequence in domains III and IV. In light of previous work localizing this receptor site to the extracellular surface of the lipid bilayer, it is proposed that amino acid residues at the extracellular surface in the loop connecting segments IIIS5 and IIIS6 and at the extracellular end of segment IVS6 contribute to formation of the dihydropyridine receptor site.

Affinity Labels↗

PubMed1991

Novel sites for phenylalkylamines: characterisation of a sodium-sensitive drug receptor with (-)-[3H]emopamil.

(-)-Emopamil ((S)-emopamil, (2S)-2-isopropyl-5-(methylphenethylamino)- 2-phenylvaleronitrile hydrochloride) is a Ca(2+)-antagonistic phenylalkylamine which also blocks serotonin (5-HT2) receptors and has antiischemic properties. The (-)-[3H]emopamil tissue distribution profile of specific binding is in striking contrast to that observed for (+)-[3H]PN 200-110 or (-)-[3H]desmethoxyverapamil: (-)-[3H]emopamil labels membrane fractions from guinea-pig liver much greater than adrenal gland greater than kidney approximately lung approximately ductus deferens approximately brain approximately skeletal muscle. Binding to liver membrane was saturable (KD = 12.8 nM, Bmax = 35 pmol/mg of protein), stereoselective, reversible (K-1 = 0.22 min-1 at 25 degrees C) and inhibited by tetraethylammonium (IC50: 1.8 mM) greater than Li+ (IC50: 12.5 mM) approximately Na+ (IC50: 13.6 mM) and [NH4+] (IC50: 79.3 mM) but not by Rb+, Cs+ or K+. The high-affinity liver membrane binding sites have a pharmacological profile that is distinct from the phenylalkylamine receptor domain of the voltage-dependent L-type Ca2+ channel. Similar sites exist in brain and other tissues, albeit with a lower density. Amiodarone, butoprozine and amiloride derivatives bind with high affinity whereas 1,4-dihydropyridines do not interact at all. It is suggested that the novel phenylalkylamine site is linked to a sodium-dependent carrier or transport system.

Animals↗

PubMed1991

Positive heterotropic allosteric regulators of dihydropyridine binding increase the Ca2+ affinity of the L-type Ca2+ channel. Stereoselective reversal by the novel Ca2+ antagonist BM 20.1140.

The alpha 1-subunit of the voltage-dependent L-type Ca2+ channel has distinct, allosterically coupled binding domains for drugs from different chemical classes (dihydropyridines, benzothiazepines, phenylalkylamines, diphenylbutylpiperidines). (-)-BM 20.1140 (ethyl-2,2-di-phenyl-4-(1-pyrrolidino)-5-(2-picolyl)- oxyvalerate) is a novel Ca2+ channel blocker which potently stimulates dihydropyridine binding (K0.5 = 2.98 nM) to brain membranes. This property is shared by (+)-cis-diltiazem, (+)-tetrandrine, fostedil and trans-diclofurime, but (-)-BM 20.1140 does not bind in a competitive manner to the sites labeled by (+)-cis-[3H]diltiazem. (+)-cis-Diltiazem and (-)-BM 20.1140 have differential effects on the rate constants of dihydropyridine binding. (+)-BM 20.1140 reverses the stimulation of the positive allosteric regulators (pA2 value for reversal of (-)-BM 20.1140 stimulation = 7.4, slope 0.72). The underlying molecular mechanism of the potentiation of dihydropyridine binding has been clarified. The K0.5 for free Ca2+ to stabilize a high affinity binding domain for dihydropyridines on purified L-type channels from rabbit skeletal muscle is 300 nM. (+)-Tetrandine (10 microM) increases the affinity 8-fold (K0.5 for free Ca2+ = 30.1 nM) and (+)-BM 20.114 (10 microM) inhibits the affinity increase (K0.5 for free Ca2+ = 251 nM). Similar results were obtained with membrane-bound Ca(2+)-channels from brain tissue which have higher affinity for free Ca2+ (K0.5 for free Ca2+ = 132 nM) and for dihydropyridines compared with skeletal muscle. It is postulated that the dihydropyridine and Ca(2+)-binding sites are interdependent on the alpha 1-subunit, that the different positive heterotropic allosteric regulators (by their differential effects on Ca2+ rate constants) optimize coordination for Ca2+ in the channel pore and, in turn, increase affinity for the dihydropyridines.

Alkaloids↗

PubMed1991

A rapid procedure for the purification of cardiac 1,4-dihydropyridine receptors from porcine heart.

Highly purified porcine cardiac sarcolemma was used as a source for purification of mammalian cardiac 1,4-dihydropyridine receptors associated with the voltage-dependent Ca2+ channel. The cardiac digitonin-solubilized receptor prelabeled with (+)-[3H]PN 200-110 was enriched at least 236-fold using an improved, rapid three-step purification protocol which could be completed within 12 h. The purity of the preparation was at least 22%, the yield of the receptors 24%. Photoaffinity labeling experiments with (-)-[3H]azidopine allowed the identification of the cardiac alpha 1 subunit. In contrast to the purified rabbit or guinea-pig skeletal muscle Ca2+ channel complex, none of the purified polypeptides underwent rapid and substantial phosphorylation by the catalytic subunit of the cyclic AMP-dependent protein kinase in vitro.

Affinity Labels↗

PubMed1991

Effect of alpha-adrenoceptor agonists on cell membrane potential in renal epitheloid Madin Darby canine kidney cells.

In Madin Darby canine kidney (MDCK) cells epinephrine--via alpha-adrenoceptors--leads to sustained hyperpolarization of the cell membrane. The present study defined the subtype of alpha-adrenoceptor involved. Both the alpha 1-adrenoceptor agonist, phenylephrine, and alpha 2-adrenoceptor agonists (BHT 920, guanfacine, moxonidine, oximetazoline) were able to hyperpolarize the cell membrane. The effect of epinephrine was only transient in the presence of the alpha 2-adrenoceptor antagonist, yohimbine, whereas the effect persisted in the presence of the alpha 1-adrenoceptor antagonist, prazosin. The epinephrine-induced hyperpolarization was almost abolished in the presence of both prazosin and yohimbine. In conclusion, both, alpha 1- and alpha 2-receptors are involved in the hyperpolarizing action of epinephrine.

Adrenergic alpha-Agonists↗

PubMed1991

Calcium channels from Cyprinus carpio skeletal muscle.

The complete amino acid sequence of the L-type calcium channel alpha 1 subunit from the carp (Cyprinus carpio) white skeletal muscle was deduced by cDNA cloning and sequence analysis. The open reading frame encodes 1852 amino acids (Mr 210,060). A 155-amino acid COOH-terminal sequence (after the fourth internal repeat) is evolutionarily preserved (90% homology) and may represent an important functional domain of L-type calcium channels. The photolabeled, membrane-bound, and purified carp alpha 1 subunits have masses of 211 and 190 kDa. The purified channel could not be phosphorylated by cAMP-dependent protein kinase. Two glycoproteins (alpha 2 subunits) are associated with the alpha 1 subunit and change their apparent masses from 235 and 220 kDa to 159 kDa upon reduction of disulfide bonds. Nucleic acid hybridization with alpha 2 cDNA revealed an 8.0-kilobase transcript in carp skeletal muscle. Evidence for a copurification of subunits similar in size to mammalian beta or gamma subunits was not obtained.

Affinity Labels↗

PubMed1991

Naftopidil, a new alpha-adrenoceptor blocking agent with calcium antagonistic properties: characterization of Ca2+ antagonistic effects.

The newly developed antihypertensive agent naftopidil blocks alpha 1-adrenoceptors and inhibits Ca2+ entry via potential-dependent channels in vascular muscle. The aim of our study was to detect possible Ca2+ channel blocking activity in various isolated preparations of the guinea pig heart. Prazosin and verapamil were used for reference. In papillary muscles, 10 microM of all drugs reduced the force of contraction Fc. The action potential duration and the refractory period were hardly affected by naftopidil, decreased by verapamil, and slightly increased by prazosin. In constant-flow Langendorff hearts, the drugs reduced the perfusion pressure, decreased the Fc, and slowed the spontaneous heart rate (order of potency: verapamil much greater than naftopidil greater than prazosin). In voltage-clamped ventricular cardiomyocytes, the calcium current ICa was completely inhibited by verapamil (pD2 value of 6.9) and to 53.5% by naftopidil (pD2 value of 6.4). Prazosin (10 microM) decreased ICa by little more than 10%. There were no differences in the steady-state inhibition of ICa by the two enantiomers of naftopidil. The block of ICa was clearly use dependent. Radioligand binding studies with (+)-[3H]PN 200-110. (-)-[3H]desmethoxy-verapamil, and (+)-cis-[3H]diltiazem in guinea pig skeletal muscle T-tubulus membranes demonstrated that racemic naftopidil exhibited some affinity for the three distinct drug receptor domains of the L-type Ca2+ channel. In conclusion, the present data are consistent with the hypothesis that naftopidil is a weak ligand for L-type calcium channels. It partially blocks ICa and shows no stereoselectivity.

Action Potentials↗

PubMed1991

Very high affinity interaction of DPI 201-106 and BDF 8784 enantiomers with the phenylalkylamine-sensitive Ca2(+)-channel in Drosophila head membranes.

1. Piperazinylindoles (DPI 201-106, BDF 8784), drugs known to act on voltage-dependent Na(+)-channels, bind with very high affinity to a Ca2(+)-channel-associated phenylalkylamine receptor in Drosophila melanogaster head membranes. These compounds and (+)-tetrandrine, a naturally occurring Ca2(+)-antagonist, were the most selective inhibitors for phenylalkylamine-labelled Drosophila Ca2(+)-channels compared to mammalian L-type Ca2(+)-channels. 2. Replacement of the cyano group by a methyl group in (+)-DPI 201-106 ((+)-BDF 8784) increases the IC50 value for inhibition of phenylalkylamine labelling of Drosophila Ca2(+)-channels from 0.29 to 2.1 nM but decreases the IC50 value for inhibition of phenylalkylamine labelling of mammalian skeletal muscle Ca2(+)-channels from 3480 to 49 nM. 3. DPI 201-106 enantiomers completely block (at 0.1 microM) phenylalkylamine photolabelling of a 136 K polypeptide in Drosophila head membranes whereas 10 microM aconitine or lidocaine are without effect. 4. Assessment of the Ca2(+)-antagonist effects of the substituted DPI 201-106 enantiomers in K(+)-depolarized taenia strips from guinea-pig caecum yielded pA2 values of 6.33 +/- 0.07 for (-)-BDF 8784 and 6.99 +/- 0.17 for (+)-BDF 8784, respectively. 5. Piperazinylindoles, previously believed to act nonspecifically on voltage-dependent mammalian L-type Ca2(+)-channels, therefore have stereoselectivity for a novel binding site and chemical selectivity unrelated to local anaesthetic activity. 6. It is proposed that a very high affinity piperazinylindole-selective site is coupled to the phenylalkylamine receptor of Drosophila Ca2(+)-channels. These sites are still present on mammalian L-type Ca2(+)-channels but have lower affinity and/or are less tightly coupled to phenylalkylamine receptors on the alpha 1-subunit.

Affinity Labels↗

PubMed1990

Uneventful self poisoning with a very high dose of captopril.

A 43-year-old patient with mild heart failure attempted suicide by ingesting between 5000 and 7500 mg of captopril. Blood pressure oscillated around 100-120/50-75 mmHg and pulse rate showed no tendency to accelerate (75-100/min). The psychiatric examination showed no drug induced psychopathological symptoms. The calculated half-life of captopril was 4.4 h. Seven hours after ingestion of approximately 50 times the maximal therapeutic daily dose of captopril the serum concentration reached 20 micrograms/ml. The calculated amount of absorbed captopril was approximately 5400 mg. Atrial natriuretic factor (ANF) plasma levels were slightly elevated and showed no tendency to increase or to fall.

Adult↗

PubMed1990

Heparin binds with high affinity to voltage-dependent L-type Ca2+ channels. Evidence for an agonistic action.

Heparin and related polyanions are a new class of compounds interacting with 1,4-dihydropyridine-sensitive L-type Ca2+ channels in a tissue-specific manner. Labeling of membrane-bound Ca2+ channels in rabbit skeletal muscle transverse tubules at the phenylalkylamine, benzothiazepine, and 1,4-dihydropyridine-selective domains was inhibited reversibly by a noncompetitive mechanism as shown by equilibrium saturation analysis and kinetic studies. (+)-cis-diltiazem but not (-)-cis-diltiazem reduced the inhibitory potency of heparin for 1,4-dihydropyridines. Antagonistic but not agonistic 1,4-dihydropyridines reversed heparin inhibition at the benzothiazepine site. Heparin forms a tight complex with the purified Ca2+ channel which is highly sensitive with respect to heparin inhibition (IC50 value: 0.05 microgram/ml) of 1,4-dihydropyridine binding. Reconstituted channel complexes have completely lost 1,4-dihydropyridine binding-inhibition by heparin and are not retained by lectin or heparin affinity columns. In whole cell patch clamp experiments with guinea-pig cardiac myocytes heparin increased the current through L-type Ca2+ channels when applied extracellulary. Synthetic peptides (representing putative heparin binding domains) which were derived from the rabbit skeletal muscle alpha 1-subunit reversed the inhibitory effects of heparin on 1,4-dihydropyridine receptors. Reversal for a peptide representing an extracellular domain occurred by an apparently competitive mechanism. It is suggested that heparin and related polyanions may interact with an evolutionary conserved cluster of basic amino acids in the large putative extracellular domain connecting the fifth and sixth putative transmembrane segment in the first motif of the ionic pore-forming alpha 1-subunit from skeletal muscle.

Allosteric Regulation↗

PubMed1990

Identification of the benzothiazepine-binding polypeptide of skeletal muscle calcium channels with (+)-cis-azidodiltiazem and anti-ligand antibodies.

The purified dihydropyridine-sensitive calcium channel from skeletal muscle transverse tubules consists of several subunits, termed alpha 1, alpha 2, beta, gamma and delta. From its associated drug receptors, those for 1,4-dihydropyridines and phenylalkylamines have been shown previously by photoaffinity labeling to reside on the alpha 1 subunit. In the present study the arylazide photo-affinity ligand, (+)-cis-azidodiltiazem ((+)-cis-(2S,3S)-5-[2-(4- azidobenzoyl)aminoethyl]-2,3,4,5-tetrahydro-3-hydroxy-2-(4-methoxyphenyl )-4- oxo-1,5-benzothiazepine), and the respective tritiated derivative, (+)-cis-[3H]azidodiltiazem (45 Ci/mmol), were developed to identify directly the benzothiazepine binding subunit. (+)-cis-Azidodiltiazem binds competitively to the benzothiazepine receptor in rabbit skeletal muscle transverse tubule membranes. Upon ultraviolet irradiation of the (+)-cis-[3H]azidodiltiazem-purified calcium channel complex, the ligand photoincorporates exclusively into the alpha 1 subunit. Photoincorporation is protected by 100 microM (-)-desmethoxyverapamil and 100 microM (+)-cis-diltiazem. A polyclonal antiserum directed against (+)-cis-azidodiltiazem was employed to detect (+)-cis-azidodiltiazem immunoreactivity photoincorporated into the purified calcium channel complex, confirming the exclusive labeling of the alpha 1 subunit. Our data provide direct evidence that, together with the drug receptors for 1,4-dihydropyridines and phenylalkylamines, the benzothiazepine binding domain of skeletal muscle calcium channels is located on the alpha 1 subunit. We conclude that our anti-ligand antibodies could be used successfully to affinity purify the photolabeled proteolytic fragments of the alpha 1 subunit which are expected to form part of the benzothiazepine binding domain.

Affinity Labels↗