Showing papers by "Franz Hofmann published in 1985"

On the mechanism of β-adrenergic regulation of the Ca channel in the guinea-pig heart

Abstract: Dose-response relations for the increase in the amplitude of Ca current (I Ca) on external application of isoprenaline (ISP) and internally applied cyclic AMP (cAMP) or catalytic subunit of cAMP-dependent protein kinase (C subunit) were established in single ventricular cells of the guinea pig. An intracellular dialysis technique was used. The threshold concentration was for ISP 10−9 M, for cAMP 3 μM (pipette concentration to which 10−5 M 3-isobutyl-1-methylxanthine was added) and for C subunit around 0.4 μM (pipette concentration). The concentrations for the half-maximal effect were 3.7×10−8 M (ISP), 5.0 μM (cAMP) and 0.95 μM (C subunit) and for the maximum effect 10−6 M (ISP), 15–20 μM (cAMP) and 3–4 μM (C subunit). For all three agents the maximum increase in the Ca current density was similar (a factor of 3–4), suggesting that they converge on the same site of the Ca channel. Accordingly, the effects of cAMP and C subunit onI Ca were non-additive to those of ISP. From these data the relationship both between concentrations of ISP and cAMP and between those of cAMP and active C subunit in terms of their effects onI Ca could be estimated and were compared with those obtained in broken cell preparations. A competitive inhibitor of phosphorylation, 5′-adenylyl-imidodiphosphate (5 mM), greatly reduced the effects of ISP and C subunit onI Ca. Cell dialysis with 3 mM adenosine-5′-(γ-thio)-triphosphate, which produces a dephosphorylationresistant phosphorylation, markedly potentiated the effects of ISP and cAMP onI Ca. The results support the hypothesis that phosphorylation of a protein within, or close to, the Ca channel by cAMP-dependent protein kinase is the mechanism of β-adrenergic stimulation.

cGMP-dependent protein kinase: autophosphorylation changes the characteristics of binding site 1

Abstract: cGMP-dependent protein kinase binds 4 mol cGMP/mol enzyme to two different sites. Binding to site 1 (apparent Kd 17 nM) shows positive cooperativity and is inhibited by Mg . ATP, whereas binding to site 2 (apparent Kd 100-150 nM) is non-cooperative and not affected by Mg . ATP. Autophosphorylation of the enzyme abolishes the cooperative binding to site 1 and the inhibitory effect of Mg . ATP. The association (K1) and dissociation (K-1) rate constant for site 2 and K1 for site 1 are not affected significantly by Mg . ATP or autophosphorylation. The dissociation rate from site 1 measured in the presence of 1 mM unlabelled cGMP is decreased threefold and over tenfold by Mg . ATP and autophosphorylation, respectively. In contrast, the dissociation rate from site 1 measured after a 500-fold dilution of the enzyme-ligand complex is 100-fold faster than that determined in the presence of 1 mM cGMP and is only slightly influenced by Mg . ATP or autophosphorylation. Only Kd values calculated with the latter K-1 values are similar to the Kd values obtained by equilibrium binding. These results suggest that autophosphorylation of cGMP-dependent protein kinase affects mainly the binding characteristics of site 1.

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

Abstract: 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 5mM 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 μM. Diltiazem up to concentrations of 10 μM did not affect the amount of nimodipine bound at equilibrium at 20±C. However, but in agreement with this result, diltiazem decreased threefold at 20±C the dissociation and association rates for the high-affinity nimodipine receptor. These rates were only marginally affected at 4±C and 37±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 μM. 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. Binding of (−)-desmethoxyverapamil was not affected by these adenosine uptake inhibitors. These results show that bovine cardiac sarcolemma contains different high-affinity receptors for dihydropyridines and phenylalkylamines which interact by allosteric mechanisms with each other and with diltiazem. The low-affinity site for nimodipine may be identical with part of the adenosine transporter.

Relaxation of skinned coronary arteries depends on the relative concentrations of Ca2+, calmodulin and active cAMP-dependent protein kinase

Abstract: Maximally contracted detergent skinned coronary smooth muscle fibres are relaxed by lowering the concentration of free Ca2+. The extent and rate of relaxation depends on the concentration of free Ca2+ and calmodulin (CaM) suggesting that it is the Ca2+. CaM complex which is responsible for maintaining tension. At a fixed concentration of Ca2+ and CaM further relaxation can be achieved by addition of the catalytic subunit of the cAMP-dependent protein kinase (cAMP-kinase). The extent as well as the relaxation rate depend on the concentration of cAMP-kinase (0.01–0.5 μM) and both are antagonized by high concentrations of Ca2+ and CaM. The Ca2+-requirement for obtaining half maximal concentration is shifted from 1.1 μM to 6.3 μM Ca2+ in the presence of 0.5 μM cAMP-kinase. These data indicate that the response of the contractile apparatus to a change in the free [Ca2+] can be modulated by cAMP-kinase at the level of the contractile proteins. It is further suggested that the tone of coronary smooth muscle is determined by the relative and not by the absolute concentrations of Ca2+, CaM and cAMP-kinase.

Diastereomers of adenosine 3',5'-monothionophosphate (cAMP[S]) antagonize the activation of cGMP-dependent protein kinase.

Abstract: cGMP-dependent protein kinase contains four cGMP-binding sites which are homologeous to the four cAMP-binding sites of cAMP-dependent protein kinase. The interaction of the diastereomers of adenosine 3′,5′-thionophosphate, (PS)-cAMP[S] and (PR)-cAMP[S], with cGMP-dependent protein kinase has been studied. 1. Autophosphorylation of cGMP-dependent protein kinase is stimulated by cAMP and (PS)-cAMP[S] with apparent KA values of 7 μM and 94 μM, respectively. 2. cAMP-stimulated autophosphorylation is inhibited competitively by (PR)-cAMP[S] with a Ki value of 15 μM. 3. The phosphorylation of the peptide substrate (Leu-Arg-Arg-Ala-Ser-Leu-Gly) is stimulated by cGMP (approx. KA 1 μM) and cAMP (approx. KA 98 μM) but neither by the (PR) nor (PS) stereomer of cAMP[S]. 4. (PR)-cAMP[S] and (PS)-cAMP[S] inhibit competitively cAMP- or cGMP-stimulated phosphorylation of the peptide substrate with Ki values of 52 μM and 73 μM, respectively. 5. (PS)-cAMP[S] stimulates the phosphorylation of the peptide substrate by an autophosphorylated enzyme. 6. Binding of [3H]cGMP to cGMP-dependent protein kinase is inhibited by (PS)-cAMP[S] and (PR)-cAMP[S] with IC50 values of 200 μM and 15 μM, respectively. 7. These results show that both diasteromers of cAMP[S] bind to cGMP-dependent protein kinase. (PR)-cAMP[S] has properties of a pure antagonist whereas (PS)-cAMP[S] has properties of a partial agonist. The results provide further evidence that autophosphorylation of the enzyme affects the interaction between the cGMP-binding sites and the catalytic center of the enzyme by facilitating the activation of the phosphotransferase reaction.

The molecular basis of second messenger systems for regulation of smooth muscle contractility.

Abstract: Smooth muscle contractility is regulated by the intracellular signals calcium, cAMP and cGMP. The possible mechanisms for generating these signals, the intracellular receptors for these signals, and their potential intracellular targets are discussed briefly.

The calmodulin-dependent phosphorylation of cardiac myosin.

Abstract: Cardiac myosin light chains are phosphorylated in vivo and in vitro. The enzyme, myosin light-chain kinase, has been purified and found to be very specific for cardiac myosin light chains. Experiments with skinned cardiac fibers suggest that phosphorylation of myosin light chain-2 decreases ATP consumption, presumably by lowering the cross-bridge cycle. These results are discussed in this chapter.