scholarly journals Calcium sensitivity of the contractile system and phosphorylation of troponin in hyperpermeable cardiac cells.

1980 ◽  
Vol 75 (3) ◽  
pp. 271-282 ◽  
Author(s):  
L Mope ◽  
G B McClellan ◽  
S Winegrad
Keyword(s):  
Cyclic Nucleotides ◽  
Polyacrylamide Gel Electrophoresis ◽  
Calcium Sensitivity ◽  
Ventricular Myocardium ◽  
Cardiac Cells ◽  
Contractile System ◽  
Maximum Activation ◽  
Nonionic Detergent ◽  
Inhibitory Subunit Of Troponin ◽  
Ca Sensitivity

Bundles of cells from rat right ventricular myocardium were made "hyperpermeable" by an overnight soak in 10 mM EGTA (McClellan and Winegrad. 1978. J. Gen. Physiol. 72:737-764). In this preparation the cytoplasmic concentration of Ca++ and ATP could be controlled while sarcolemmal receptors and enzymes were retained. The Ca sensitivity of the tissues (as indicated by the pCa for 50% maximum activation) was altered to different extents in the presence of [32Pgamma]ATP by treatment with cyclic nucleotides, catecholamines, or a low concentration of nonionic detergent. The proteins of the tissue were then isolated by SDS-polyacrylamide gel electrophoresis, and the identity of 32P-labeled proteins was determined. The Ca sensitivity is inversely related to the relative amount of 32P incorporated into the inhibitory subunit of troponin (TNI). Extrapolation of the relation to the lowest Ca sensitivity observed gives a stoichiometry of about 0.8 mol PO4 per mol TNI. These results support the hypothesis that Ca sensitivity of cardiac myofibrils is regulated by a phosphrylation of TNI that is stimulated by cyclic AMP (cAMP) and inhibited by cGMP.

scholarly journals The regulation of the calcium sensitivity of the contractile system in mammalian cardiac muscle.

1978 ◽  
Vol 72 (6) ◽  
pp. 737-764 ◽  
Author(s):  
G B McClellan ◽  
S Winegrad
Keyword(s):  
Phosphodiesterase Inhibitor ◽  
Regulatory System ◽  
Calcium Sensitivity ◽  
Cardiac Cells ◽  
General Shape ◽  
Contractile System ◽  
Ventricular Cells ◽  
Phosphate Donor ◽  
Nonionic Detergent ◽  
Ca Sensitivity

Treatment of rat ventricular cells with 10 mM EGTA makes the sarcolemma highly permeable to small ions and molecules without removing its restriction of the diffusion of larger molecules or inactivating all of its enzymatic functions. These hyperpermeable cardiac cells have been used to study the regulation of the range of concentration of Ca over which activation of the contractile proteins occurs (Ca sensitivity). The Ca sensitivity can varied from three- to sixfold without any significant alteration in the general shape of the relation between force and Ca concentrations. Although cyclic nucleotides in concentrations of 10(-9) to 10(-5) M do not influence Ca sensitivity, in the presence of a phosphodiesterase inhibitor, cGMP increases and cAMP decreases Ca sensitivity. Treatment of the hyperpermeable cells with a nonionic detergent raises Ca sensitivity as does removal of the phosphate donor by complete substitution of CTP for ATP. These data indicate that Ca sensitivity is probably modulated by a cAMP-dependent phosphorylation that decreases Ca sensitivity. The sarcolemma is required for this reaction to take place. The effect of this reaction is antagonized by a cGMP-dependent reaction occurring inside the cell. Studies involving the perfusion of the heart with and without epinephrine before the exposure to EGTA indicate that epinephrine can regulate this system of control of Ca sensitivity. The functional considerations of this regulatory system are discussed.

scholarly journals Cyclic nucleotide regulation of the contractile proteins in mammalian cardiac muscle.

1980 ◽  
Vol 75 (3) ◽  
pp. 283-295 ◽  
Author(s):  
G B McClellan ◽  
S Winegrad
Keyword(s):  
Cardiac Muscle ◽  
Inotropic Effect ◽  
Bathing Solution ◽  
Contractile System ◽  
Nonionic Detergent ◽  
Nucleotide Regulation ◽  
Inhibitory Subunit Of Troponin ◽  
Concentration Changes ◽  
Significant Interference ◽  
Ca Sensitivity

The contractile system of rat cardiac muscle that has been made hyperpermeable by soaking the tissue in EGTA (McClellan and Winegrad. 1978. J. Gen. Physiol. 72:737-764) can be probed directly with Ca buffer from the bathing solution without significant interference from either sarcoplasmic reticulum or mitochondria on the Ca concentration. Changes in Ca-activated force are due therefore to changes in the properties of the contractile system itself and not to regulation of Ca concentration. The addition of cAMP, cGMP, and GTP, guanylyl imidodiphosphate (GMP-PNP), or epinephrine to the bath does not alter maximum Ca-activated force, but when these drugs are added with 1% nonionic detergent to the bath, contractility increases by as much as 180%. An inhibitor of phosphodiesterase must be present for the inotropic effect of cAMP but not cGMP, GTP, GMP-PNP, or epinephrine. The inotropic response to cAMP is independent of the Ca sensitivity of the contractile system, but guanine nucleotides enhance contractility only when Ca sensitivity is not high. The inotropic effect of epinephrine is inhibited to a large extent by cGMP but not by GMP-PNP. These data can be explained by a model in which contractility is enhanced by a cAMP-regulated phosphorylation that can be controlled through the beta-receptor adenylate cyclase complex in the sarcolemma. The regulation involves two reactions, one a phosphorylation and a second that occurs in the presence of detergent. Phosphorylation of neither the myosin light chain nor the inhibitory subunit of troponin appears to be involved in this mechanism for regulating contractility.

scholarly journals Regulation of calcium sensitivity in perforated mammalian cardiac cells.

1983 ◽  
Vol 81 (2) ◽  
pp. 195-211 ◽  
Author(s):  
A Weisberg ◽  
G McClellan ◽  
M Tucker ◽  
L E Lin ◽  
S Winegrad
Keyword(s):  
Right Ventricular ◽  
Surface Membrane ◽  
Calcium Sensitivity ◽  
Cardiac Cells ◽  
Target Molecule ◽  
Regulatory Systems ◽  
Ventricular Cells ◽  
Ca Sensitivity

Sarcolemmal perforations can be produced in bundles of rat right ventricular cells by either perfusion of the heart or soaking of the bundles with a solution containing 10 mM EGTA. All cells are affected and lose approximately 40% of the surface membrane. In these cells it is possible to show cAMP regulation of contractility (maximum Ca-activated force) without cAMP regulation of Ca sensitivity (pCa for 50% of maximum Ca-activated force). Therefore, the target molecule for cAMP is different for the two regulatory systems. Both regulatory systems can be slowly washed out of the cell by 10 mM EGTA solution but not by relaxing or contraction solutions. A model for regulation of Ca sensitivity is proposed.

The effects of Acute and Chronic β-Adrenoceptor Blockade on the Myofibrillar Contractile System in the Dog Heart

1984 ◽  
Vol 67 (2) ◽  
pp. 259-267 ◽  
Author(s):  
M. K. Davies ◽  
P. Cummins ◽  
W. A. Littler
Keyword(s):  
Calcium Sensitivity ◽  
Maximal Activity ◽  
Biopsy Specimens ◽  
Contractile System ◽  
Chronic Therapy ◽  
Before And After ◽  
Adaptive Effect ◽  
Electrophoretic Techniques ◽  
Myocardial Contractile ◽  
And Function

1. Electrophoretic and enzyme techniques have been used to study the structure and function of the contractile protein system in the myocardium of dogs before and after β-adrenoceptor blockade. Animals were examined after acute β-adrenoceptor blockade by using intravenous atenolol (0.2 mg/kg) and following chronic therapy with oral atenolol (100 mg twice daily) for a mean period of 106 days. 2. Two-dimensional polyacrylamide-gel electrophoretic techniques were used to examine the myocardial contractile and regulatory proteins present in endomyocardial biopsy specimens obtained after acute and chronic β-adrenoceptor blockade. No differences in charge, molecular weight or the relative proportions of actin, myosin light chains, tropomyosin or troponin-C were seen after either acute or chronic β-adrenoceptor blockade. 3. The maximal activity and calcium sensitivity of the myofibrillar adenosine triphosphatase (ATPase) was also unchanged after acute and chronic atenolol therapy. 4. It is concluded that β-adrenoceptor blockade has no significant adaptive effect on the structural or functional properties of the myofibril.

scholarly journals Further studies on the interaction between human platelet membrane glycoproteins IIb and IIIa in triton X-100

Blood ◽  
1982 ◽  
Vol 60 (4) ◽  
pp. 894-904 ◽  
Author(s):  
D Pidard ◽  
JP Rosa ◽  
TJ Kunicki ◽  
AT Nurden
Keyword(s):  
Membrane Proteins ◽  
Divalent Cation ◽  
Human Platelet ◽  
Polyacrylamide Gel Electrophoresis ◽  
Indirect Evidence ◽  
Human Platelets ◽  
Platelet Membrane ◽  
Membrane Glycoproteins ◽  
Nonionic Detergent ◽  
Triton X

Abstract Analysis of human platelet membrane proteins by crossed immunoelectrophoresis (CIE) in the presence of Triton X-100 (TX-100) has previously shown that glycoproteins (GP) IIb and IIIa are located in a single immunoprecipitate, band 16.2 To investigate whether IIb and IIIa are associated in a complex, we have analyzed TX-100-solubilized 125I-labeled membrane proteins by density gradient ultracentrifugation using 10%-40% sucrose gradients containing the nonionic detergent. studies were performed using soluble proteins derived from membranes isolated in the presence or absence of EDTA. Analysis of gradient fractions by SDS-polyacrylamide gel electrophoresis showed that in the absence of divalent cation chelation, GP IIb and IIIa penetrated well into the gradient (fractions 15–17). Analysis of fractions 15–17 by CIE revealed the presence of band 16. In contrast, when the membrane proteins were incubated with EDTA prior to or after TX-100 solubilization, IIb and IIIa remained near the top of the gradient (fractions 8–11) and gave separate immunoprecipitates during CIE. Incubation of washed platelet lysates with leupeptin, an inhibitor of the Ca2+-dependent protease of human platelets, had no effect on the shape of the band 16 immunoprecipitate. Thus, for the first time, direct evidence has been obtained that GP IIb and IIIa may form a divalent cation-mediated complex. Calibration of the sedimentation profiles using proteins of known molecular weight suggests that the complex is of limited size. Indirect evidence suggests that the complex is a heterodimer.

Effects of forskolin and cyclic nucleotides on isometric force in rat aorta

1986 ◽  
Vol 250 (3) ◽  
pp. C468-C473 ◽  
Author(s):  
E. G. McMahon ◽  
R. J. Paul
Keyword(s):  
Cyclic Nucleotides ◽  
Cyclic Nucleotide ◽  
Isometric Force ◽  
Rat Aorta ◽  
Isometric Tension ◽  
Tension Development ◽  
Nucleotide Analogues ◽  
Cyclic Monophosphate ◽  
Contractile System ◽  
Rat Thoracic Aorta

The present study was undertaken to determine the extent to which cyclic nucleotide-induced relaxation in the intact rat aorta is mediated at the level of the contractile system. The relaxant effects of the cyclic nucleotide analogues [8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) and dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP)] and forskolin were examined in both the intact vessel and a Triton X-100-skinned preparation of rat thoracic aorta. Relaxation of a norepinephrine-induced contraction was essentially complete 30 min after the addition of 50 microM 8-BrcGMP [% relaxation = 87.2 +/- 4.4% (n = 4)], 100 microM DBcAMP [98.2 +/- 1.2% (n = 4)], and 1 microM forskolin [107.0 +/- 3.3% (n = 5)]. These same doses were ineffective in relaxing precontracted skinned rat aortic rings compared with the relaxation achieved in the intact vessel. The largest relaxation in the skinned aortas was achieved with the addition of 1 microM forskolin [17.4 +/- 1.5% (n = 4)]. The addition of catalytic subunit of cAMP-dependent protein kinase had no effect on isometric tension in the precontracted skinned aorta. Preincubation with the cyclic nucleotide analogues or forskolin in a low-Ca2+ solution (pCa less than 8) was also ineffective in inhibiting subsequent isometric tension development. Our results suggest that only a very small fraction of the relaxation with cyclic nucleotides and forskolin in the intact rat aorta is due to the action of these agents at the level of the contractile system.

scholarly journals Isolation of the intercellular glycoproteins of desmosomes.

1981 ◽  
Vol 90 (1) ◽  
pp. 243-248 ◽  
Author(s):  
G Gorbsky ◽  
M S Steinberg
Keyword(s):  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Insoluble Residue ◽  
Differential Centrifugation ◽  
Electron Microscopic ◽  
Nonionic Detergent ◽  
Cell Layers ◽  
Membrane Attachment

To characterize the desmosome components that mediate intercellular adhesion and cytoskeletal-plasma membrane attachment, we prepared whole desmosomes and isolated desmosomal intercellular regions (desmosomal "cores") from the living cell layers of bovine muzzle epidermis. The tissue was disrupted in a nonionic detergent at low pH, sonicated, and the insoluble residue fractionated by differential centrifugation and metrizamide gradient centrifugation. Transmission electron microscopic analyses reveal that a fraction obtained after differential centrifugation is greatly enriched in whole desmosomes that possess intracellular plaques. Metrizamide gradient centrifugation removes most of the plaque material, leaving the intercellular components and the adjoining plasma membranes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis coupled with methods that reveal carbohydrate-containing moieties on gels demonstrate that certain proteins present in whole desmosomes are glycosylated. These glycoproteins are specifically and greatly enriched in the desmosome cores of which they are the principal protein constituents, and thus may function as the intercellular adhesive of the desmosome.

Calcium sensitivity of skinned ferret EDL, soleus, and cremaster fibers

1993 ◽  
Vol 264 (5) ◽  
pp. R867-R870
Author(s):  
C. Huchet ◽  
C. Leoty
Keyword(s):  
Calcium Sensitivity ◽  
Breeding Period ◽  
Cremaster Muscle ◽  
Contractile System ◽  
Slow Twitch Muscle ◽  
Fast Twitch Muscle ◽  
Data Points ◽  
Slow Twitch ◽  
Triton X ◽  
Fast Twitch

The properties of the contractile system at different times of the year in the ferret extensor digitorum longus (EDL), soleus and cremaster muscles were examined by using chemically skinned (Triton X-100) preparations. The results show clear differences in calcium sensitivity between these skeletal muscles. The apparent calcium threshold for activation was lower in soleus than in EDL, while calcium concentrations ([Ca2+]) required to obtain the half-maximal tension, expressed as pCa50 (-log[Ca2+]), was lower in EDL than in soleus muscle. In fact, pCa50 obtained in fast and slow fibers by fitting the experimental data points by a modified Hill equation was 5.92 +/- 0.02 (n = 9) and 6.09 +/- 0.03 (n = 11) respectively. So EDL appears to be a typical fast-twitch muscle and soleus a typical slow-twitch muscle. Adult ferret cremaster muscle was composed of two types of fibers during the quiescent period similar to EDL and soleus, and only one type that was intermediate between EDL and soleus in the breeding period, as assessed by pCa50 values. These annual modifications in calcium activation of adult ferret cremaster muscle could be related to changes in the function of these muscles and may be correlated with seasonal variations of sexual activity.

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