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G Zernig

Publications and source records attributed to G Zernig.

51 records · Page 3Linked to original sources
PubMed1990

Mitochondrial Ca2+ antagonist binding sites are associated with an inner mitochondrial membrane anion channel.

The inner mitochondrial membrane contains specific Ca2+ antagonist binding sites unrelated to the L-type Ca2+ channel. The mitochondrial 1,4-dihydropyridine (DHP) and phenylalkylamine sites are reciprocally allosterically coupled, require anions (e.g., Cl-, No3-) for optimal binding, and are inhibited by purine and pyrimidine nucleotides in a noncompetitive manner. In mitochondrial swelling experiments, a concentration-dependent inhibition of an inner mitochondrial membrane anion channel (IMAC) by Ca2+ antagonists from different chemical classes can be demonstrated. Under the conditions of the swelling experiments, affinity of different Ca2+ antagonists and amiodarone, a known IMAC inhibitor, for the mitochondrial (+/-)-[3H]nitrendipine binding site (Kd, 7.2 +/- 2.0 microM; Bmax, 1.03 +/- 0.37 nmol/mg of protein) strongly correlated with their inhibitory potency for the IMAC. Linear regression of pIC50 values for IMAC-induced swelling versus pIC50 values for (+/-)-[3H]nitrendipine binding inhibition yielded a correlation coefficient of 0.91 for all tested DHPs (n = 12, p less than 0.001). Amiodarone inhibited (+/-)-[3H]nitrendipine binding and IMAC-induced swelling with pIC50 values of 6.11 and 5.93, respectively. The correlation coefficient between binding and inhibition of IMAC-induced swelling for amiodarone and all tested Ca2+ antagonists (including non-DHP compounds) was 0.76 (n = 20, p less than 0.001), with the slopes approaching unity. These results suggest the association of the mitochondrial Ca2+ antagonist binding sites with an IMAC.

Amiodarone↗
PubMed1989

Hydrophobic calcium channel ligands: methodical problems and their solution.

Niguldipine is a 1,4-dihydropyridine derivative that combines L-type Ca2+ channel-blocking effects and alpha 1-adrenolytic activity within a single molecule, exemplifying a novel approach in the treatment of hypertension. As niguldipine is a very hydrophobic compound, it (1) readily adsorbs to surfaces of the plastic-ware often used in radioligand binding assays and (2) partitions into the hydrophobic membrane compartments. Both phenomena decrease the actual free drug concentration in radioligand-binding assays and lead to gross underestimation of the affinity of niguldipine (and other hydrophobic ligands) for the 1,4-dihydropyridine binding domain of the L-type Ca2+ channel or for alpha 1A adrenoceptors, respectively. Partitioning of the hydrophobic molecules into the membrane phase leads to a dependence of the Ki value on "total receptor" concentration despite mathematic corrections of the experimentally determined IC50 values. The Ki dependence was mimicked by adding denatured membranes (devoid of high-affinity receptor-binding activity) to native membrane preparations. Loss to pipet tips and tubes was avoided by a special dilution protocol. Partitioning into the hydrophobic membrane compartments needed more elaborate correction procedures.

Adsorption↗
PubMed1989

Stereoselective binding of niguldipine enantiomers to alpha 1A-adrenoceptors labeled with [3H]5-methyl-urapidil.

[3H]5-Methyl-urapidil, a potent antihypertensive derivative of urapidil, binds to alpha 1A-adrenoceptors in rat brain cortex membranes with a dissociation constant (KD) of 0.89 nM and a Bmax of 116 fmol/mg protein. The ligand does not bind to purified liver cell membranes (alpha 1B-adrenoceptors). [3H]5-Methyl-urapidil also labels 5-HT1A receptors in brain membranes (KD: 0.84 nM and Bmax: 235 fmol/mg protein). (+/-)-Niguldipine, a novel 1,4-dihydropyridine with Ca2+-antagonistic as well as alpha 1A-adrenoceptor blocking properties, is a competitive inhibitor of [3H]5-methyl-urapidil binding to alpha 1A-adrenoceptors. In contrast to those for prazosin, the Ki values for niguldipine were highly dependent on the membrane protein concentration, indicating partitioning of niguldipine into hydrophobic compartments unavailable for alpha-adrenoceptor interaction. The extrapolated, 'true' Ki values were as follows: (+/-)-niguldipine: 0.298 nM, (-)-niguldipine: 3.12 nM, (+)-niguldipine: 0.145 nM.

Animals↗
PubMed1989

Stereoselective hemodynamic effects of (R)-and (S)-propranolol in man.

In a randomized, double-blind, placebo-controlled, cross-over study 24 healthy volunteers were examined before and 2 h after oral administration of 80 mg (R,S)-, 40 mg (R)- and 40 mg (S)-propranolol.HCl; 8 of them received placebo in an additional run. During exercise on a bicycle ergometer and a rest period the rate pressure product was decreased by 80 mg (R,S)-propranolol.HCl (-32.8% p less than 0.0001) and 40 mg (S)-propranolol.HCl (-32.3%; p less than 0.0001), whereas 40 mg (R)-propranolol.HCl as well as placebo showed no effect. Corresponding binding inhibition experiments using (-)-(125I)iodocyanopindolol in a sarcolemma-enriched cardiac membrane preparation yielded a eudismic ratio of 179 for (S)- over (R)-propranolol. 2 h after oral application, stereospecific HPLC analysis revealed different individual concentrations in plasma of (R)- 22.3 +/- 21.7 ng/ml) and (S)-propranolol (30.4 +/- 26.9 ng/ml) when 80 mg of (R,S)-propranolol.HCl was administered. The plasma levels were similar when 40 mg of the pure enantiomer of (R)- (22.7 +/- 20.3 ng/ml) or (S)-propranolol.HCl (28.7 +/- 22.5 ng/ml) was applied. (R)- and (S)-propranolol are two substances with different pharmacodynamic and pharmacokinetic properties. As there are methods available to produce the optically pure enantiomers, they should be used rather than the racemic mixture.

Adult↗
PubMed1988

The mitochondrial high-capacity low-affinity (+/-)-[3H]nitrendipine binding site is regulated by nucleotides.

The high-capacity, low-affinity (+/-)-[3H]nitrendipine binding site in the inner mitochondrial membrane from guinea-pig heart is regulated by purine and pyrimidine nucleotides. The rank order in (+/-)-[3H]nitrendipine binding inhibition assays (with decreasing potency) was: ATP (IC50 11.8 microM) = adenosine 5'-O-(2-thiotriphosphate (ATP gamma S) greater than 5'-adenylylimidodiphosphate (AppNHp) greater than ADP much greater than GTP = ITP = CTP greater than UTP greater than guanosine 5'-tetraphosphate (GT4P) greater than guanosine 5'-O-(2-thiotriphosphate) (GTP gamma S) greater than 5'-guanylylimidodiphosphate (GppNHp) greater than IDP greater than CDP greater than GDP. There was no effect of AMP, adenosine 3':5'-cyclic monophosphate (cAMP), adenosine, UDP, NAD, and NADP. The ATP effect was fully reversible upon wash-out. Adenine nucleotides and analogs had a (+/-)-[3H]nitrendipine binding inhibition profile in mitochondrial membranes from guinea-pig liver or kidney similar to that obtained in heart mitochondrial membranes. In heart mitochondria, 0.3 mM ATP decreased the Bmax from 1.69 +/- 0.04 nmol/mg protein to 0.73 +/- 0.24 nmol/mg protein whilst it decreased the KD only moderately, from 521 +/- 50 to 352 +/- 43 nM, in equilibrium saturation studies. In kinetic studies, ATP slowed down the dissociation rate of the (+/-)-[3H]nitrendipine binding site complex from 0.016 +/- 0.004 to 0.0042 +/- 0.0002 min-1 but it also decreased the association rate constant from 1.52 +/- 0.15 to 0.41 +/- 0.28 10(4).M-1.min-1, yielding a kinetically determined KD (1024 nM) identical to the control KD (1053 nM).(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1988

A novel 1,4-dihydropyridine-binding site on mitochondrial membranes from guinea-pig heart, liver and kidney.

The 1,4-dihydropyridine (+/-)-[3H]nitrendipine reversibly binds to mitochondrial preparations from guinea-pig heart with a dissociation constant (Kd) of 593 +/- 77 nM and a maximum density of binding sites (Bmax.) of 1.75 +/- 0.27 nmol/mg of protein. This low-affinity high-capacity 1,4-dihydropyridine-binding site does not discriminate between the enantiomers of nitrendipine and is also found in mitochondrial membranes from guinea-pig liver (Kd 586 +/- 91 nM; Bmax. 0.36 +/- 0.04 nmol/mg of protein) and kidney (Kd 657 +/- 149 nM; Bmax. 0.56 +/- 0.12 nmol/mg of protein). Phenylalkylamines (e.g. verapamil) inhibit ( +/- )-[3H]nitrendipine binding with micromolar inhibition constants, but the benzothiazepine D-cis-diltiazem, a potent Ca2+-channel blocker, is without effect. The binding is heat-stable, shows a V-shaped pH-dependence with a minimum around pH 7.0, and is strongly dependent on ionic strength in the incubation medium. The cations La3+ greater than Cd2+ much greater than Co2+ greater than Ca2+ much greater than Ba2+ greater than Mg2+ greater than Li+ greater than Na+ and the anions NO3- greater than C1- greater than or equal to F- stimulate the binding, whereas PO4(3-) greater than SO4(2-) slightly inhibit it. The low-affinity ( +/- )-[3H]nitrendipine-binding site located on the mitochondrial inner membrane is biochemically and pharmacologically different from the 1,4-dihydropyridine-receptor domain of the L-type Ca2+ channel. Furthermore, it is not identical with any of the low-affinity 1,4-dihydropyridine-binding sites described so far.

Animals↗
PubMed1986

Stereospecific regulation of [3H]inositol monophosphate accumulation by calcium channel drugs from all three main chemical classes.

Depolarization of [3H]inositol-prelabelled rat cortical slices through the elevation of extracellular K+ levels leads to increased accumulation of [3H]inositol phosphates. In the presence of 18 mM K+, Ca2+ channel activators selectively stimulated the formation of [3H]inositol monophosphate ([3H]IP1) whereas Ca2+ channel blockers were inhibitory. Blockade of the Na+ channel by 1 microM tetrodotoxin had no effect but chelation of extracellular Ca2+ abolished the response. The enantiomers of the benzoxadiazol 1,4-dihydropyridine 202-791 showed opposite stereospecific regulation of [3H]IP1 formation: (+)-(S)-202-791 stimulated (252%; ED50: 88 nM), whereas (-)-(R)-202-791 inhibited (65% inhibition, ED50: 602 nM). The (-) enantiomer of Bay K 8644 was a potent [3H]IP1 stimulator (258%; ED50: 82 nM). While (+)-Bay K 8644 was inactive in the presence of 18 mM K+, it completely inhibited the (-)-Bay K 8644-induced stimulation with a Ki of 103 nM. Representatives of the other two main classes of Ca2+ channel blockers (phenylalkylamines and benzothiazepines) inhibited K+ depolarization-induced and (-)-Bay K 8644 enhanced [3H]IP1 formation in a dose-dependent, stereospecific manner. The results show that Ca2+ channel blockers are efficient modulators of depolarization-induced and Ca2+ channel activator-induced [3H]inositol monophosphate formation in brain, and demonstrate the functional coupling of three distinct drug receptor sites on neuronal Ca2+ channels.

Animals↗
PubMed1985

Human red-blood-cell Ca2+-antagonist binding sites. Evidence for an unusual receptor coupled to the nucleoside transporter.

The human red blood cell ghost Ca2+-antagonist binding sites were characterized with (+/-)-[3H]nimodipine. The labelled 1,4-dihydropyridine bound in a non-cooperative, reversible manner with a Kd of 52 nM at 25 degrees C to 9.65 pmol sites/mg ghost protein. The stereochemistry of the binding domain was evaluated with the optically pure enantiomers of chiral 1,4-dihydropyridines. In contrast to the 1,4-dihydropyridine-selective receptors on Ca2+ channels in electrically excitable tissues, the (+) enantiomer of nimodipine and the (-) enantiomer of the benzoxadiazol 1,4-dihydropyridine (PN 200-110) were bound with higher affinity than the respective optical antipodes. The human red blood cell ghost [3H]nimodipine-labelled sites also interacted with the inorganic Ca2+-antagonist La3+ (increase in the number of binding sites), and were allosterically regulated by the optical enantiomers of the phenylalkylamine-type Ca2+-antagonists (e.g. verapamil, desmethoxyverapamil, methoxyverapamil). The benzothiazepines d- or l-cis-diltiazem were without effect. Nucleosides (adenosine approximately equal to inosine greater than cytidine) were inhibitory at the nimodipine-labelled site, as were the nucleoside uptake inhibitors dipyridamole, hexobendine, dilazep, nitrobenzylthioinosine and nitrobenzylthioguanosine. The binding sites have essential sulfhydryl groups, show trypsin sensitivity, but are relatively heat stable. When nitrobenzylthioinosine was employed as a covalent probe to inactivate the red blood cell ghost nucleoside carrier, [3H]nimodipine binding was irreversibly lost. (+)-Nimodipine greater than (-)-nimodipine inhibited [14C]adenosine transport into human red blood cells. A good correlation between IC50 values for inhibition of [3H]nimodipine binding and IC50 values for inhibition of [14C]adenosine uptake was found for 18 compounds. Sheep red blood cells (which lack the nucleoside transporter) had no detectable [3H]nimodipine binding sites. It is concluded that the Ca2+-antagonist receptor sites of the human erythrocyte are coupled to the nucleoside transporter.

Adenosine↗
PubMed1985

Simultaneous measurements of action potentials and contractions in single cultured adult and embryonic heart muscle cells.

Using both a microphotometrical technique and conventional microelectrodes, excitation and contractions have been measured simultaneously in spontaneously active and electrically driven isolated cultured heart muscle cells of embryonic chicks as well as adult rats and guinea-pigs. This experimental arrangement makes it possible to measure continuously and with high accuracy, excitation and E.C. coupling at the single cell level. The contraction time course of spontaneously active embryonic cells and electrically driven adult cells resembles contractions of macroscopic heart muscle preparations against various preloads; mostly a nearly isometric contraction form is found. The effects of various inotropic factors on excitation and contraction (beat frequency, Ca, epinephrine) were studied and it could be shown that the embryonic cells show different features of E.C. coupling. The activity of adult cells resembles very strongly that characteristically found in macroscopic preparations. This proved the feasibility of the method in studies of cardiac E.C. coupling at the single cell level.

Animals↗
PubMed1985

Single voltage-dependent and outward rectifying K+-channels in isolated rat heart cells.

Studies on single K+-channel currents recorded from isolated rat heart muscle cells, in which early repolarization is known to be exceptionally fast, are reported here. A K+-channel which is blocked by TEA (tetraethylammonium) from the inside only has been found. The total open time of the channel, measured in steady-state after activation, indicated outward rectifying properties. The single channel conductance increases with depolarization from 25 pS at -70 mV to 75 pS at + 70 mV. Selectivity of the channel has also been measured and it was found that only Rb+ and K+ can permeate the channel, whereas the permeability (P) for Li+, Na+, Cl-, Mg2+, and Ca2+ is less than 0.05 times PK+. Ba2+ and CS+ block the channel activity. These results clearly demonstrate the existence of K+-selective outward rectifying conductance pathways in rat ventricular myocytes.

Animals↗
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↗
PubMed1985

Morphological integrity of single adult cardiac myocytes isolated by collagenase treatment: immunolocalization of tubulin, microtubule-associated proteins 1 and 2, plectin, vimentin, and vinculin.

Single cardiac myocytes were isolated from hearts of 9 to 12-week-old rats by means of collagenase (100 U/ml). After assessment of their functional integrity they were processed for immunofluorescence microscopy of the cytoskeletal proteins tubulin, microtubule-associated proteins 1 and 2 (MAP-1 and MAP-2), plectin, vimentin, and vinculin. Antibodies to tubulin decorated a delicate filamentous network that apparently was unrelated to any sarcomeric organization. The distribution of MAP-1 and MAP-2 was strikingly different from that of tubulin, as both antigens were confined to Z-line structures. These structures were also prominently stained by affinity-purified antibodies to plectin and a monoclonal antibody to vimentin. Co-distribution of plectin and vimentin was also observed at the former intercalated disk region of the heart cell. Anti-vinculin antibodies decorated an intricate meshwork consisting of delicate filaments with predominantly irregular orientation and occasional assembly into whorls. These immunolocalization data indicate that the cell shape and cytoskeletal architecture characteristic of cardiac myocytes in tissues is maintained in single isolated cells. Furthermore, intermediate filaments rather than microtubules seem to be instrumental in the preservation of cell morphology.

Animals↗
PubMed1984

A study of the mode and site of action of capsaicin in guinea-pig heart and rat uterus.

Capsaicin (CAP) has been shown to exert a selective neurotoxic effect on peptidergic primary afferent neurons. The effect of CAP on the isolated guinea-pig left auricle and papillary muscle preparations and on the isolated rat uterus was used to elucidate its mode and site of action with regard to cellular Ca2+ utilization. In the electrically driven left auricle CAP first increased and then decreased the size of the contractions while in the electrically driven papillary muscle CAP caused only a decrease in the contractions. Electrophysiological measurements showed that the initial increase in contraction size coincided with a decrease in the upstroke velocity of the action potential. This membrane-stabilizing effect of CAP seemed also responsible for the decrease in contractile activity. The positive inotropic effect of CAP on the left auricle was concentration-dependent (0.03-6.5 microM). The positive inotropic effect of 0.33 microM CAP was reproducible at intervals of 15 min, whereas tachyphylaxis developed at shorter intervals or higher concentrations of CAP. The percent increase in the size of contractions by 0.33 microM CAP was smaller when [Ca2+]e was doubled but larger when 2.2 microM verapamil or 0.1 mM La3+ was present. The increase in contractions by 0.4-400 microM isoproterenol was greatly reduced by 0.33 microM CAP in a noncompetitive manner. The positive inotropic effect of 2.9 microM glucagon was also inhibited by 0.33 microM CAP. In the isolated anoestrous rat uterus 0.03-3.3 microM CAP caused a transient inhibition of the spontaneous contractions similarly to the effect of 2.2 microM verapamil.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗