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G Pfitzer

Publications and source records attributed to G Pfitzer.

54 records · Page 3Linked to original sources
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↗
PubMed1985

Modulation of calcium sensitivity in guinea pig taenia coli: skinned fiber studies.

In summary then, skinned fiber experiments have provided evidence showing that the relationship between force development and free calcium ion concentration may be variable. The cyclic-nucleotides c-GMP (cf 38) and c-AMP in particular might be important in modulating calcium sensitivity of skinned fibers possibly via an alteration of myosin phosphorylation. Additionally, the coupling between myosin phosphorylation and the ATPase activity of actomyosin or tension generation may itself be modulated in actomyosin contractile systems. The recognition of the physiological relevance of these modulating mechanisms however must await experiments in which the relationship between force development, free calcium ion concentration and myosin phosphorylation is studied in intact fibers. With the advent of more sophisticated calcium measuring techniques, such information is now available.

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↗
PubMed1985

The positive inotropic dihydropyridine Bay K 8644 does not affect calcium sensitivity or calcium release of skinned cardiac fibres.

BAY K 8644 is a positive inotropic dihydropyridine which concentration-dependently increased contractile force in guinea-pig atria at 10 to 1000 nmol/l. In chemically skinned cardiac fibres from guinea-pig hearts the substance did not change the tension induced by calcium. Also BAY K 8644 did not release calcium from intracellular myocardial stores like caffeine since it failed to evoke a contractile response in saponin-treated, calcium-loaded cardiac muscle. Thus, the drug exerts its effects neither by sensitizing the contractile apparatus to calcium nor by a caffeine-like action.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1984

The variable relation between myosin light-chain phosphorylation and actin-activated ATPase activity in chicken gizzard smooth muscle. Modulation by tropomyosin.

Using pure myosin light-chain kinase and myosin from chicken gizzard and rabbit skeletal actin, we have established the relationship between myosin phosphorylation and actin-activated ATPase activity by varying the concentration of Ca2+ or calmodulin (CaM). By increasing Ca2+ or CaM the extent of myosin phosphorylation increased from 10% to about 100%, the requirements for half-maximal activation being 0.17 microM Ca2+ and 1 nM CaM. ATPase was activated only when a phosphorylation 'threshold' of about 60% was surpassed. By increasing phosphorylation from 60% to 100%, ATPase activity was further stimulated, the relationship between myosin phosphorylation and ATPase activity being curvilinear. Addition of 1 microM tropomyosin lowered the Ca2+ requirement for half-maximal ATPase activity in the prescence of 0.05 microM CaM from 0.4 microM to 0.2 microM but did not affect the maximal ATPAse activity. In addition, the CaM requirement of ATPase activity was also lowered by gizzard tropomyosin at 9 microM Ca2+. The Ca2+/CaM requirement for myosin phosphorylation remained unaffected by tropomyosin. Thus, tropomyosin altered the relationship between myosin phosphorylation and ATPase activity: the phosphorylation 'threshold' was lowered from 60% to about 10%, hence causing a leftward shift of the relationship which becomes similar to that in the case of native actomyosin.

Actins↗
PubMed1984

Forskolin inhibits tension development in detergent-treated cardiac muscle fibers.

In detergent-treated cardiac muscle fibers, forskolin, a potent activator of adenylate cyclases, inhibits tension development elicited with submaximal [Ca2+] and increases incorporation of 32P into troponin-I. A similar reduced tension development has been observed after treatment with cAMP or the catalytic subunit of the cAMP-dependent protein kinase. It is concluded that these fibers still contain much of the enzyme cascade involved in evoking a contractile response to beta-adrenergic stimulation.

1-Methyl-3-isobutylxanthine↗
PubMed1984

Tension transients in skinned muscle fibres of insect flight muscle and mammalian cardiac muscle: effect of substrate concentration and treatment with myosin light chain kinase.

Glycerinated single fibres from the dorsal longitudinal muscle of Lethocerus maximus were isometrically contracted in MgATP-salines (10 microM Ca2+; 1.5 mM Mg2+; pH 6.7; 22 degrees C and 20 mM PEP; 100 U/ml pyruvate kinase). The ratio of ATPase activity to tension decreased by a factor of 2 after reducing the ATP-concentration from 15 to 0.5 mM. At all ATP-concentrations (0.5-15 mM), the fibres showed tension adjustments in response to small step changes in length characteristic to an actively contracting muscle: i) an elastic phase which did not depend on ATP-concentration ii) a quick phase of stress relaxation with at least two exponential components; iii) a phase of delayed tension generation. An increase in size of the length step and/or a decrease of ATP-concentration slowed the quick phase and the delayed phase. Similar results have been obtained with skinned cardiac muscle (pig right ventricle). To see, how the isolated contractile system is affected by an increase in the light chain phosphorylation, tension transients were studied in skinned right ventricular muscle fibres before and after incubation with ATP gamma S (2 mM), pure myosin light chain kinase (9 micrograms/ml), Calmodulin (1 microM) and Ca2+ (0.8 microM). While isometric tension development elicited by 20 microM Ca2+ in the ATP salt solution was barely affected in presence of the enzyme, the ATPase activity was decreased by about 25% of the control. There was also a marked decrease (about 50%) in the contraction velocity as determined by the recovery of tension following a quick release. Quick stretches cause an immediate increase in tension followed by a rapid fall and a subsequent rise in tension. The velocity of this tension rise decreased by approximately 30% after incubation with myosin light chain kinase.

Adenosine Triphosphate↗
PubMed1984

cGMP and cAMP inhibit tension development in skinned coronary arteries.

The effects of physiological concentrations of cGMP and cAMP on tension development in skinned coronary arteries (Triton X-100) were studied. cGMP inhibited tension elicited at intermediate Ca2+ concentrations at pH 7.0 but not at more acidic or alkaline pH values. cAMP, on the other hand, decreased submaximal tension development independent of pH (from pH 6.5 to pH 7.2). Neither nucleotide affected tension development at maximally activating Ca2+ concentrations.

Animals↗
PubMed1984

Effect of calmodulin, Ca2+, and cAMP protein kinase on skinned tracheal smooth muscle.

The characteristics of contraction and relaxation of membrane skinned smooth muscle from guinea pig trachealis muscle are described. Micromolar Ca2+ elicited reproducible contractions in Mg-ATP salt solution at 20 degrees C. The speed of contraction was much faster at 30 and 37 degrees C, enabling cumulative concentration-response curves to be obtained. At these temperatures, a progressive increase in basal tension occurred in the absence of Ca2+. This tension was active and developed more rapidly at pH 6.7 than at pH 7.0. Calmodulin (0.1-10 microM) greatly increased the speed of contraction and lowered the threshold Ca2+ concentration ([Ca2+]) required to initiate contraction from 0.13 to 0.02 microM Ca2+. Trifluoperazine antagonized responses to Ca2+. Thiophosphorylation with adenosine 5'-O-(3-thiotriphosphate) produced maximum tension development, which was Ca2+-independent. This effect was reversible. The results are compatible with myosin-linked regulation of contraction in which a Ca2+ X calmodulin complex activates myosin light chain kinase to phosphorylate myosin. The catalytic subunit of cAMP-dependent protein kinase strongly inhibited tension development and slowly relaxed fibers contracted with threshold [Ca2+] consistent with an action via phosphorylation of myosin light chain kinase. This effect was extremely slow compared with the rate of relaxation by Ca2+ withdrawal or with relaxation of intact smooth muscle by beta-adrenergic agonists.

Animals↗
PubMed1984

Effect on contractility of skinned fibres from mammalian heart and smooth muscle by a new benzimidazole derivative, 4,5-dihydro-6-[2-(4-methoxyphenyl)-1H-benzimidazol-5-yl]-5-methy l-3(2H )- pyridazinone.

The positive inotropic drug 4,5-dihydro-6-[2-(4-methoxyphenyl)-1H-benzimidazol-5-yl]methyl-3(2 H)- pyridazinone (UD-CG 115 BS) enhances calcium induced contraction of skinned fibres from cardiac muscle probably by increasing the calcium sensitivity of troponin. Submaximal calcium induced contraction of skinned fibres from a smooth muscle (guinea-pig taenia coli) are slightly inhibited.

Animals↗
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

Length dependence of calcium activated isometric force and immediate stiffness in living and glycerol extracted vascular smooth muscle.

Vascular smooth muscle series elasticity was examined in living and glycerinated preparations as a function of the tissue length or calcium elicited force. Isometrically contracted smooth muscle strips were submitted to small quick stretches and releases (rise time 1.5 ms). The resulting immediate tension changes were proportion to the length changes for length steps ranging from -0.5% L0 to +2% L0. Plotting the immediate tension changes versus the length steps resulted in force-extension diagrams of the series elasticity (T1-curves). The linear parts of the T1-curves extrapolated to a common abscissa intercept of about -1% to -2% L0 irrespective of the tissue length or the degree of calcium activated force. The slopes of the T1-curves taken as the stiffness of the series elasticity increased in proportion to the isometric tension and depended on the tissue length or the degree of calcium activation in a similar way as tension. It is concluded that tension changes due to changes in the calcium concentration or the tissue length are caused by a change in the number of attached crossbridges. Results obtained in "skinned" fibres were similar to the one obtained in living fibres indicating that electromechanical coupling was not a major factor in determining the decrease in isometric tension and stiffness at short lengths.

Adenosine Triphosphate↗
PubMed1982

Calcium antagonists affect calmodulin-dependent contractility of a skinned smooth muscle.

The calcium antagonist prenylamine which had previously been found to inhibit fully the calmodulin myosin light chain kinase systems in concentrations of 200 mumol/l has now been found to inhibit the calmodulin-dependent contraction of skinned smooth muscle (guinea pig taenia coli). The inhibition by prenylamine is itself calcium-dependent. At maximum activation with Ca2+ and calmodulin, little inhibition is observed. Verapamil as well as prenylamine in small concentrations (less than 10 mumol/l) have been found to increase the rate of calcium-induced isometric tension development.

Animals↗
PubMed1981

Cyclic AMP inhibits contractility of detergent treated glycerol extracted cardiac muscle.

Glycerinated myocardial fibres treated with a detergent (Lubrol WX) and suspended in ATP salt solution produce half maximum isometric tension at pCa 6.2 (at pH 6.7). After addition of cyclic AMP (1-100 microM), the pCa required for half maximum activation is 5.9. c-AMP in concentrations of 1-100 microM induces a dose dependent inhibition (up to 40% at pCa6), and this effect can be amplified by the phosphodiesterase inhibitor IBMX (3-isobutyl-1-methylxanthine) 10(-4) M. The effect is similar in presence and absence of sodium fluoride 10 mM. Since in detergent treated skinned fibres the cell membrane and the sarcoplasmic reticulum are extracted and since the Ca2+ ion concentration was kept constant and buffered, we propose that c-AMP does not act via the cell membrane or the sarcoplasmic reticulum, but via phosphorylation of troponin I. The latter is the only component which becomes phosphorylated in skinned fibres during c-AMP induced relaxation, an effect which is also responsible for the inhibition of actomyosin ATPase at constant Ca2+ ion concentration (cf. Ray and England 1976).

Animals↗
PubMedDate not supplied

Skinned coronary smooth muscle: calmodulin, calcium antagonists, and cAMP influence contractility.

The effects of Ca2+, calmodulin, cAMP, the catalytic subunit of cAMP-dependent protein kinase (CSU) and some Ca2+ antagonists were studied in chemically (Triton X-100) skinned coronary smooth muscle. Calmodulin increased the Ca2+ responsiveness of the muscle fiber as indicated by the reduction in the threshold as well as the half-maximal activating Ca2+ concentration. Trifluoroperazine, a calmodulin antagonist, inhibited Ca2+-calmodulin-induced contraction. Both cAMP and CSU were effective inhibitors of contraction induced at an intermediate Ca2+ concentration. Fendiline, a Ca2+-antagonist, at 2 x 10(-4) M produced a significant inhibitory effect, which was reduced by increasing the Ca2+ concentration. From other Ca2+ antagonists tested, W-7, but not D600 and verapamil, produced some inhibitory effect. The data indicate that the response of skinned coronary smooth muscle to Ca2+, calmodulin and cAMP are similar to those obtained with other skinned smooth muscles. Furthermore, skinned fiber preparation can serve as a useful tool to investigate possible direct effects of drugs on the activating and regulatory systems in smooth muscle.

Animals↗
PubMedDate not supplied

[Urocortin decreases phosphorylation of MYPT1 and increases the myosin phosphatase activity via elevation of the intracellular level of cAMP].

Urocortin, a peptide hormone related to the corticotropin releasing factor, is suggested to be involved in blood pressure regulation by dilating the peripheral blood vessels. In rat tail arteries, urocortin-induced vasodilation is due to a decrease in myofilament Ca2+ sensitivity the mechanism of which is still unclear. In this study, the hypothesis was tested that the decrease in Ca2+ sensitivity in mouse tail arteries results from the activation of myosin light chain phosphatase. The relaxation of KCl-precontracted (42 mM) intact mouse tail arteries by urocortin (1 nM and 10 nM) was significantly inhibited by 1 microM antisauvagine30, a CRF-2 receptor antagonist (p < 0.05, n = 3). The addition of 1 microM KT 5720, an inhibitor of PKA, to intact rat tail arteries did not affect the KCl-induced force but significantly attenuated the urocortin-induced relaxation (n = 5). In alpha-toxin permeabilized mouse tail arteries, urocortin relaxed submaximally activated preparations at constant pCa 6.1 by 37.6 +/- 8.2% (n = 5) as compared to control vessels (n = 5, p < 0.001). The relaxation in permeabilized vessels was inhibited by pre-treatment with 30 microM Rp-8-CPT-cAMPS, an inactive analogue of cAMP. In permeabilized mouse tail arteries, treatment with 100 nM urocortin was associated with dephosphorylation of MLC20(Ser19) and MYPT1(Thr696/Thr850). The effect of urocortin on MYPTI dephosphorylation was completely abolished by 30 M Rp-8-CPT-cAMPS and mimicked by the cAMP analogue Sp-5,6-DCI-cBiMPS. Based on these findings, we propose that the urocortin-induced relaxation is due to a decrease in calcium sensitivity mediated by a cAMP-dependent increase in the activity of MLCP.

Animals↗