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Biomedical subjects

F Hofmann

Publications and source records attributed to F Hofmann.

At least 325 records · Page 18Linked to original sources
PubMed1986

Modulation of Ca current during the phosphorylation cycle in the guinea pig heart.

The calcium current (ICa) in the heart is increased by phosphorylation of a protein which is part of, or close to, the Ca channel. The phosphorylation is catalysed by cAMP-dependent protein kinase (cAMP-PK). The question whether dephosphorylated channels are available to open on depolarization was examined in ventricular myocytes of guinea pig by recording whole cell ICa during dialysis with either regulatory (R) subunit of cAMP-PK or protein kinase inhibitor (PKI) or adenosine-5'-(gamma-thio)-triphosphate (ATP gamma S). The following results were obtained: 1) R subunit reduced and PKI reversed the isoprenaline (ISP)-induced enhancement of ICa, suggesting their ability to inhibit cAMP-PK. 2) R subunit and PKI, however, reduced basal (i.e. non beta-adrenergically stimulated) ICa only by about 20%. 3) Dialysis with ATP gamma S resulted in a slow increase in basal ICa, presumably due to dephosphorylation-resistant thiophosphorylation. 4) When, however, the cell was dialyzed with PKI the effect of ATP gamma S was almost completely suppressed, suggesting no detectable phosphorylation related to the channel activity in this condition. These results support the view that even in the dephosphorylated state Ca channels are available to open on depolarization and that phosphorylation by cAMP-PK increases the opening probability.

Adenosine Triphosphate↗
PubMed1986

Cyclic GMP-dependent protein kinase relaxes skinned fibers from guinea pig taenia coli but not from chicken gizzard.

The effect of cGMP and cGMP-dependent protein kinase (cG-PK) on contraction and relaxation was studied in skinned smooth muscle fibers from guinea pig taenia coli and chicken gizzard. At a fixed [Ca2+] relaxation was significantly enhanced by activated cG-PK in fibers from guinea pig taenia coli, but not in those from chicken gizzard. The Ca2+-requirement for half maximal tension maintenance was shifted to the right. Relaxation was associated with a decline in phosphorylated myosin light chain-2 from 34% to 25%. Similarly to relaxation activated cG-PK inhibited tension development only in fibers from taenia coli. These results suggest that mammalian and chicken smooth muscle fibers respond differently to cG-PK.

Animals↗
PubMed1986

Barium can replace calcium in calmodulin-dependent contractions of skinned renal arteries of the rabbit.

Renal arteries of the rabbit were chemically skinned using Triton X-100. In EGTA-buffered solutions containing calmodulin and ATP, small strips of the skinned preparations were found to develop contractile force which was dependent on the concentrations of either free calcium or of free barium. However, a 220 times greater concentration of barium than of calcium was necessary for comparable effects. Quantitatively, the response to barium was dependent on the concentration of calmodulin added to the test solutions. The contractile effect of barium was partly antagonized by the calmodulin antagonist, trifluoperazine.

Animals↗
PubMed1986

Mode of action of nitrates at the cellular level.

Nitrates probably induce vasorelaxation via a rise of cytosolic cGMP, and subsequent phosphorylation of target proteins by cGMP-dependent protein kinase. A dual type of action by this mechanism seems likely: cGMP-dependent protein kinase relaxes chemically skinned vascular smooth muscle which has no functioning cell membrane. Thus, the contractile apparatus with its regulatory and contractile proteins may be one of the targets for their action. Calcium visualization techniques using aequorin or quin-2, and ion flux studies showing suppression of Ca2+-dependent 86Rb efflux by nitrates and 8-Br-cGMP suggest that the cytosolic calcium level is another target for their action. Whether this lowering of intracellular calcium occurs via cGMP-dependent activation of the sarcolemmal Ca2+ extrusion ATPase, requires confirmation.

Animals↗
PubMed1986

[Antiviral action of lysolecithin analogs against human pathogenic viruses].

The virucidal effect, the inhibition of virus adsorption and penetration and the influence on later phases of replication of human pathogenic viruses were studied. The compounds showed a significant virucidal effect to enveloped viruses (measles virus, herpes virus type 1 and 2), whereas the compounds were ineffective against nonenveloped viruses (Coxsackie virus A9 and B1, attenuated poliovirus type 1, adenovirus type 1). Interactions with the viral envelope are supposed.

Antiviral Agents↗
PubMed1985

The influence of P-light chain phosphorylation by myosin light chain kinase on the calcium sensitivity of chemically skinned heart fibres.

Phosphorylation of the P-light chain of myosin might be involved in the regulation of cardiac contractility. Thus an enhanced phosphorylation level of the P-light chain catalyzed by Ca2+-calmodulin-dependent myosin light chain kinase (MLCK) increased significantly the Ca2+ sensitivity of chemically skinned ventricular fibre bundles of the pig. This effect was reversible. Whereas force development at submaximal Ca2+ concentration (pCa 5.5) increased by approximately 50% in the presence of MLCK, maximum tension achieved at maximum Ca2+-concentration (pCa 4.3) was not affected.

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↗
PubMed1985

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

cGMP-dependent protein kinase contains four cGMP-binding sites which are homologous 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. Autophosphorylation of cGMP-dependent protein kinase is stimulated by cAMP and (PS)-cAMP[S] with apparent KA values of 7 microM and 94 microM, respectively. cAMP-stimulated autophosphorylation is inhibited competitively by (PR)-cAMP[S] with a Ki value of 15 microM. The phosphorylation of the peptide substrate (Leu-Arg-Arg-Ala-Ser-Leu-Gly) is stimulated by cGMP (approx. KA 1 microM) and cAMP (approx. KA 98 microM) but neither by the (PR) nor (PS) stereoisomer of cAMP[S]. (PR)-cAMP[S] and (PS)-cAMP[S] inhibit competitively cAMP-or cGMP-stimulated phosphorylation of the peptide substrate with Ki values of 52 microM and 73 microM, respectively. (PS)-cAMP[S] stimulates the phosphorylation of the peptide substrate by an autophosphorylated enzyme. Binding of [3H]cGMP to cGMP-dependent protein kinase is inhibited by (PS)-cAMP[S] and (PR)-cAMP[S] with IC50 values of 200 microM and 15 microM, respectively. These results show that both diastereomers 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.

Binding, Competitive↗
PubMed1985

cGMP-dependent protein kinase. Autophosphorylation changes the characteristics of binding site 1.

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.

Adenosine Triphosphate↗
PubMed1985

The calmodulin-dependent phosphorylation of cardiac myosin.

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.

Animals↗
PubMed1985

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

Dose-response relations for the increase in the amplitude of Ca current (ICa) 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 microM (pipette concentration to which 10(-5) M 3-isobutyl-1-methylxanthine was added) and for C subunit around 0.4 microM (pipette concentration). The concentrations for the half-maximal effect were 3.7 X 10(-8) M (ISP), 5.0 microM (cAMP) and 0.95 microM (C subunit) and for the maximum effect 10(-6) M (ISP), 15-20 microM (cAMP) and 3-4 microM (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 on ICa 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 on ICa 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 on ICa. Cell dialysis with 3 mM adenosine-5'-(gamma-thio)-triphosphate, which produces a dephosphorylation-resistant phosphorylation, markedly potentiated the effects of ISP and cAMP on ICa.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

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

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+ X 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 microM) and both are antagonized by high concentrations of Ca2+ and CaM. The Ca2+-requirement for obtaining half maximal concentration is shifted from 1.1 microM to 6.3 microM Ca2+ in the presence of 0.5 microM 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.

Animals↗