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V Flockerzi

Publications and source records attributed to V Flockerzi.

84 records · Page 5Linked to original sources
PubMed1986

Cardiac sarcoplasmic reticulum contains a low-affinity site for phenylalkylamines.

The distribution of the bovine cardiac binding sites for the organic calcium-channel blockers was studied. Crude microsomal membranes were separated into three fractions, which contained mainly membranes derived from sarcolemma, 'junctional' sarcoplasmic reticulum containing transversal tubuli, and free sarcoplasmic reticulum. The high-affinity binding site for the dihydropyridines, determined in the presence of nitrobenzylthioinosine, was enriched 12-fold and 17-fold in sarcolemma and junctional sarcoplasmic reticulum. The binding sites for the phenylalkylamines, determined with [3H]verapamil or [3H](-)desmethoxyverapamil, were enriched 1.5-3.4-fold in sarcolemma and junctional sarcoplasmic reticulum but 6-10-fold in free sarcoplasmic reticulum. The phenylalkylamine-binding site, present in free sarcoplasmic reticulum, was partially destroyed by chymotrypsin or phospholipase A2 and C treatment. Specific binding was proportional to the concentration of the added membrane protein. The binding of (-)desmethoxyverapamil was half-maximally inhibited by 6.5 mM calcium chloride and was optimal in the presence of 5 mM EGTA. In three out of five preparations (-)desmethoxyverapamil bound to a single site with an apparent Kd value of 191 +/- 42.8 nM and a density of 34.5 +/- 7.7 pmol/mg protein. In two out of five preparations an additional high-affinity site (Kd approximately 0.67 nM) was detected. The low-affinity site bound other phenylalkylamines, but stereospecific binding of phenylalkylamines was not observed. Binding of phenylalkylamines to the low-affinity site was inhibited by some but not all calmodulin 'antagonists'. Furthermore dihydropyridines did not affect the binding of (--)desmethoxyverapamil suggesting that the low-affinity site differs considerably from the high-affinity sarcolemmal site. These results suggest that free sarcoplasmic reticulum contains a binding site for phenylalkylamines at a relative high density, which is not related to the high-affinity site present in the voltage-dependent calcium channel.

Amines↗
PubMed1986

Solubilization of the bovine cardiac sarcolemmal binding sites for calcium channel blockers.

Nonionic and ionic detergents were used to solubilize the bovine cardiac sarcolemmal binding sites for nimodipine and (-)desmethoxyverapamil in the absence of added ligand. Only Chaps, digitonin and sucrose monolauryl ester were able to solubilize the binding sites in a form that bound radioligands. About 45% of each of the membrane-bound high-affinity site was solubilized by 0.4% Chaps (w/v) in the presence of 48% (w/v) glycerol. The solubilized binding sites were destroyed by trypsin or by a 10-min incubation at 50 degrees C. Calcium stimulated nimodipine binding slightly at 0.3 mM and inhibited (-)desmethoxyverapamil binding completely with an IC50 of 1.2 mM. Nimodipine binding was reduced by 20% in the presence of EGTA. The solubilized receptors sedimented in sucrose density gradients with an apparent s20,w of 21 S. An identical sedimentation value was obtained for the cardiac sarcolemmal and skeletal transverse tubulus receptor which were prelabeled with nitrendipine and solubilized by digitonin. Solubilization reduced the affinity of nimodipine for its high-affinity site slightly from 0.35 nM to 1.2 nM and that for its low-affinity site from 33 nM to 130 nM. Solubilization did not affect significantly the specific density of these sites. Binding of nimodipine to the low-affinity site was completely abolished by 0.1 microM nitrobenzylthioinosine. After solubilization only the high-affinity site for (-)desmethoxyverapamil could be measured with tenfold reduced affinity (Kd = 15.3 nM) but unchanged specific density. Binding to the solubilized high-affinity site for nimodipine and (-)desmethoxyverapamil was stereospecific and showed a similar rank order as the particulate binding sites. Binding of nimodipine was inhibited allosterically by phenylalkylamines. Similarly, (+)PN200-110 inhibited allosterically (-)desmethoxyverapamil binding. d-cis-Diltiazem stimulated nimodipine binding at 20 degrees C 1.2-fold, reduced the dissociation rate from 0.018 min-1 to 0.0083 min-1 and had no effect on the association rate (0.173 min-1. nM-1). The Kd calculated from the rate constants was 0.1 nM and in close agreement with the value of 0.49 nM measured under equilibrium conditions in the presence of nitrobenzylthioinosine. In contrast, desmethoxyverapamil increased the dissociation rate of nimodipine to 0.03 min-1. The association and dissociation rate constants for (-)desmethoxyverapamil were 0.024 min-1. nM-1 and 0.025 min-1, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Characterization of the binding sites for nimodipine and (-)-desmethoxyverapamil in bovine cardiac sarcolemma.

The bovine cardiac sarcolemmal binding sites for the dihydropyridine nimodipine and the phenylalkylamine (-)-desmethoxyverapamil were studied. The density of the nimodipine and (-)-desmethoxyverapamil binding sites increased 8.3-fold and 3.4-fold with the sarcolemma. The binding sites for both compounds were destroyed by trypsin. Nimodipine bound in the presence of 1 mM free calcium to a high-affinity and a low-affinity site with apparent Kd values of 0.35 +/- 0.09 nM (n = 9) and 33 +/- 6.0 nM (n = 9) and with apparent densities of 0.3 +/- 0.05 pmol/mg (n = 9) and 8.2 +/- 1.0 pmol/mg (n = 9). The binding to the high-affinity site was abolished by 1 mM EGTA. The binding sites were specific for dihydropyridines. The (-)-isomers of several phenylalkylamines inhibited nimodipine binding by an apparent allosteric mechanism. (-)-Desmethoxyverapamil bound in the presence of 5 mM EGTA to a high-affinity and a low-affinity site with apparent Kd values of 1.4 +/- 0.3 nM (n = 6) and 171 +/- 26 nM (n = 6) and with apparent densities of 0.16 +/- 0.02 pmol/mg (n = 6) and 13.6 +/- 2.7 pmol/mg (n = 6). The binding to both sites was inhibited by calcium with a half-maximal concentration of 4.3 mM. The binding sites were specific for the other phenylalkylamines and had a higher affinity for the (-)-isomers than for the (+)-isomers. Nimodipine inhibited the binding of (-)-desmethoxyverapamil by an apparent allosteric mechanism. d-cis-Diltiazem inhibited non-competitively the binding of (-)-[3H]desmethoxyverapamil with a Ki of 3.7 microM. Diltiazem up to concentrations of 10 microM did not affect the amount of nimodipine bound at equilibrium at 20 degrees C. However, but in agreement with this result, diltiazem decreased threefold at 20 degrees C the dissociation and association rates for the high-affinity nimodipine receptor. These rates were only marginally affected at 4 degrees C and 37 degrees C. d-cis-Diltiazem reversed in a competitive manner the inhibition of nimodipine binding elicited by the addition of (-)-desmethoxyverapamil with a Ka value of 1.6 microM. The amount of nimodipine bound was inhibited by 50% by the adenosine uptake inhibitors nitrobenzylthioinosine and hexobendine with apparent median inhibitory concentrations of 1 nM and 3 nM, respectively. Nitrobenzylthioinosine completely abolished binding of nimodipine to the low-affinity site, but did not affect binding to the high-affinity site.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1983

Phosphorylation of purified bovine cardiac sarcolemma and potassium-stimulated calcium uptake.

Sarcolemmal vesicles were prepared from bovine cardiac muscle by differential and discontinuous sucrose density gradient centrifugation. Na+/K+-ATPase was purified 33-fold to a specific activity of 53 +/- 0.5 (12) mumol Pi X mg-1 X h-1, binding sites for strophantin 20-fold to a density of 56.3 +/- 5.3 (14) pmol/mg and that for the calcium antagonist nitrendipine 5.5-fold to a density of 0.72 +/- 0.07 (6) pmol/mg. The specific activity of the Na+/Ca2+ exchanger was 61.1 +/- 3.7 (6) nmol/mg. The vesicles had an intravesicular volume of 20 +/- 4 (4) microliter/mg and 56.9 +/- 6 (4)% of the vesicles were right-side-out oriented. Several peptides of the purified membranes were phosphorylated in the presence of Mg . ATP and EGTA. Most of the radioactive phosphate was incorporated into a peptide with an apparent molecular mass of 22 kDa. Denaturation of the membranes at 100 degrees C changed the mobility of this peptide to 15 kDa and 11 kDa. This peptide could not be distinguished from a sarcoplasmic reticulum peptide of similar molecular mass. The phosphorylation of the sarcolemmal peptide was stimulated by Ca2+/calmodulin, cAMP and the catalytic subunit of cAMP-dependent protein kinase. A comparison of the phosphorylation of sarcolemmal membranes with that of sarcoplasmic reticulum showed that Ca2+/calmodulin stimulated in each membrane, the phosphorylation of the 22-kDa peptide and a 44-kDa peptide, and in the sarcoplasmic reticulum the phosphorylation of an additional peptide of 55-kDa. Ca2+/calmodulin-dependent phosphorylation of a 55-kDa peptide could not be demonstrated in sarcolemma, regardless if sarcolemmal membranes were incubated together with sarcoplasmic reticulum or if the phosphorylation was carried out in the presence of purified cardiac myosin light chain kinase or phosphorylase kinase. 'Depolarization' induced Ca2+ uptake which was measured according to Bartschat, D.K., Cyr, D.L. and Lindenmayer, G.E. [(1980) J. Biol. Chem. 255, 10044-10047] was 5 nmol/mg protein. This uptake was not enhanced after preincubation of the vesicles with Mg . ATP or Mg . ATP and cAMP-dependent protein kinase. The value of 5 nmol/mg protein is in agreement with the theoretical amount of Ca2+ which can be accumulated by the bovine cardiac sarcolemma in the absence of a driving force other than the Ca2+ gradient. The potassium-stimulated Ca2+ uptake was not blocked by the organic Ca2+ channel blockers. Prolonged incubation of Mg . ATP with sarcolemmal vesicles in the presence of various ATPase inhibitors led to the hydrolysis of ATP. The liberated phosphate precipitated with Ca2+ in the presence of LaCl3. These precipitates amounted to an apparent Ca2+ uptake ranging from 50 to over 1000 nmol/mg. The results suggest that potassium-stimulated Ca2+ uptake of bovine cardiac sarcolemmal vesicles is not enhanced in the presence of ATP or by phosphorylation of a 22-kDa peptide.

Adenosine Triphosphate↗
PubMed1983

Injection of catalytic subunit of cAMP-dependent protein kinase into isolated cardiac myocytes.

Cyclic adenosine 3',5'-monophosphate (cAMP) or the free catalytic subunit (C) of the cAMP-dependent protein kinase were pressure injected into single guinea pig ventricular cells. The following results were obtained: Injection of cAMP prolonged the action potential and shifted the action potential plateau to a more positive level. Under voltage clamp, cAMP injection increased the amplitude of the slow inward calcium current (Isi). Injection of C permanently prolonged the action potential and enhanced the amplitude of Isi by a factor of 2-4, depending on the amount of injected C. In the current-voltage relations the potential of maximum Isi and the apparent current reversal did not change. After maximum prolongation of the action potential due to repeated injections of C, even high concentrations of adrenaline did not further change the configuration of the action potential. In many experiments transient depolarizations appeared after the injection. Correspondingly, under voltage clamp transient inward currents occurred. C injection increased both the time-dependent and time-independent potassium outward current. In response to injection of the catalytic subunit, the isotonic contraction was larger in amplitude and relaxation was faster. It is concluded that the cAMP-dependent protein kinase increases the slow inward calcium current in the heart, presumably by phosphorylation of some membrane proteins.

Action Potentials↗
PubMed1982

cGMP-dependent protein kinase decreases calcium sensitivity of skinned cardiac fibers.

Chemically skinned (Lubrol WX) cardiac muscle fibers produce half-maximum isometric tension at pCa 6.18 (pH 6.7) in presence of MgATP (10 mM). After addition of cGMP (5 microM) and cGMP-dependent protein kinase (0.1 microM), the pCa required for half-maximum activation is 5.96, while maximum tension is not affected. Similar shifts in the tension/pCa-relationship have been observed after incubation of skinned cardiac muscle fibers with cAMP of catalytic subunit of the cAMP-dependent protein kinase. The shift in the Ca2+-sensitivity is associated with an increased incorporation of radioactivity into a Mr 28000 band (presumably troponin-I) and a Mr 145000 band.

1-Methyl-3-isobutylxanthine↗
PubMed1982

Injection of subunits of cyclic AMP-dependent protein kinase into cardiac myocytes modulates Ca2+ current.

beta-Adrenergic stimulation of the heart is thought to increase cardiac muscle contractility by activation of cyclic AMP-dependent protein kinase and concomitant increase in the phosphorylation of certain proteins (for refs see refs 1-6). Electrophysiological studies have shown that the stimulation of cardiac beta-adrenoreceptors, the external application of cyclic AMP or its analogues to Purkinje fibres, or the injection of cyclic AMP into single myocytes can increase the slow inward current (Isi) during the plateau phase of the action potential (AP). In heart muscle this current is mainly carried by Ca2+ (refs 10, 11) and it has been suggested that cyclic AMP-dependent phosphorylation of some component of the calcium channel increases the amount of Ca2+ which enters the cell during depolarization. We have investigated this hypothesis by examining the electrical responses of isolated guinea pig ventricular myocytes to pressure injections of subunits of the cyclic AMP-dependent protein kinase. We report here that injection of the catalytic subunit (C) resulted in a lengthening of the action potential duration (APD) and an increase in the height of the plateau as well as the amplitude of Isi. By contrast, the injection of regulatory subunit (R) shortened the APD of fast and slow response APs, an effect which was reversed by adrenaline.

Action Potentials↗
PubMedDate not supplied

Purified dihydropyridine-binding site from skeletal muscle t-tubules is a functional calcium channel.

Many excitable cells contain at least two different voltage-dependent Ca channels (L- and T-type). The cardiac, slow, L-type Ca channel is further modulated by cyclic AMP-dependent phosphorylation, which increases the probability of it being open, and is readily blocked by Ca channel blockers including dihydropyridines and phenylalkylamines. The tritiated congeners of these blockers bind in vitro to sites which have the same pharmacological characteristics as those observed in vivo, that is, stereospecific and allosteric interaction between distinct sites. The dihydropyridine-binding site purified from skeletal muscle t-tubules contains three peptides of relative molecular mass (Mr) 142,000 (142K), 56K and 31K. The cAMP kinase incorporates one mol phosphate per mol of the 142K peptide and binding of (+)PN-200/110, a potent Ca antagonist, is allosterically affected by D-cis-diltiazem and verapamil. The purified dihydropyridine-receptor complex has also been incorporated into phospholipid bilayer membranes. Here, we show for the first time that the complex can be reconstituted to form a functional 20-pS Ca channel that retains the principal regulatory, biochemical and pharmacological properties of membrane-bound L-type Ca channels.

Allosteric Regulation↗
PubMedDate not supplied

Primary structure of the receptor for calcium channel blockers from skeletal muscle.

The complete amino-acid sequence of the receptor for dihydropyridine calcium channel blockers from rabbit skeletal muscle is predicted by cloning and sequence analysis of DNA complementary to its messenger RNA. Structural and sequence similarities to the voltage-dependent sodium channel suggest that in the transverse tubule membrane of skeletal muscle the dihydropyridine receptor may act both as voltage sensor in excitation-contraction coupling and as a calcium channel.

Amino Acid Sequence↗
PubMedDate not supplied

Mutations of calcium channel beta subunit genes in mice.

Ca2+ influx through high voltage activated Ca2+ channels initiates a number of physiological processes including e.g. excitation-contraction coupling in cardiac myocytes and excitation-transcription coupling in neurones. The Ca2+ channels involved are complexes of a pore-forming alpha1 subunit, a transmembrane delta subunit disulfide-linked to an extracellular alpha2 subunit, a intracellular beta subunit and, at least in some tissues, a gamma subunit. Experimental analysis of beta subunit function comprises functional coexpression of its cDNA together with the cDNAs of the other subunits. This experimental approach can be supplemented by investigating functional alterations that result from the genetic elimination of Ca2+ channel beta genes in mice. Here we summarize the phenotype of mice deficient in the beta1 subunit, the beta3 subunit or the beta4 subunit, respectively.

Animals↗