PubMed Health⌕ Search

Biomedical subjects

A V Somlyo

Publications and source records attributed to A V Somlyo.

At least 19 recordsLinked to original sources
PubMed1992

Kinetics of Ca2+ release and contraction induced by photolysis of caged D-myo-inositol 1,4,5-trisphosphate in smooth muscle. The effects of heparin, procaine, and adenine nucleotides.

The kinetics of Ca2+ release and contraction induced by photolytic release of inositol 1,4,5-trisphosphate (InsP3) were determined in permeabilized smooth muscle. The rate of Ca2+ release was half-maximal at 1 microM InsP3. The concentration-dependent delay of Ca2+ release at saturating InsP3 concentration was approximately 10 ms and within the uncertainty of the measurements. The relationship between the delay and InsP3 concentration showed no evidence of a high level (n = 4 or higher) of cooperativity but could not distinguish between no cooperativity (n = 1) or a low level (n = 2) of cooperativity. Submaximal [InsP3] caused only partial Ca2+ release from the InsP3-sensitive stores. InsP3-induced Ca2+ release was markedly potentiated by ATP or by adenosine 5'-(beta,gamma-methylene-triphosphate), but neither the rate nor the amplitude of release was significantly affected by procaine (2-5 mM). Heparin increased the delay between photolysis and Ca2+ release, indicating that the off rate of inert ligand(s) bound to InsP3 receptors may contribute to the physiological delay in Ca2+ release. There was a much longer (370 ms +/- 45 S.E.) delay between the rise of Ca2+ and force development, presumably reflecting events preceding and associated with myosin light chain phosphorylation.

Adenine Nucleotides↗
PubMed1992

Arachidonic acid inhibits myosin light chain phosphatase and sensitizes smooth muscle to calcium.

Arachidonic acid (AA) increased, at constant Ca2+, the levels of force and 20-kDa myosin light chain (MLC20) phosphorylation in permeabilized smooth muscle, and slowed relaxation and MLC20 dephosphorylation. The Ca(2+)-sensitizing effect of AA was not inhibited by inhibitors of AA metabolism (indomethacin, nordihydroguaiaretic acid, or propyl gallate), of protein kinase C (pseudopeptide) or by guanosine-5'-O-(beta-thiodiphosphate) and was abolished by oxidation of AA in air. A non-metabolizable AA analog, 5,8,11,14-eicosatetraynoic acid) also had Ca(2+)-sensitizing effects. Extensive treatment with saponin abolished the Ca(2+)-sensitizing effects of phorbol 12,13-dibutyrate and guanosine-5'-O-(gamma-thiotriphosphate), but not that of AA. A purified, oligomeric MLC20 phosphatase isolated from gizzard smooth muscle was dissociated into subunits by AA, and its activity was inhibited toward heavy meromyosin but not phosphorylase. We conclude that AA may act as a messenger-promoting protein phosphorylation through direct inhibition of the form of protein phosphatase(s) that dephosphorylate MLC20 in vivo.

Animals↗
PubMed1992

Myosin light chain phosphatase activities and the effects of phosphatase inhibitors in tonic and phasic smooth muscle.

Phosphatase inhibitors microcystin-LR, tautomycin, and okadaic acid caused contraction and increased 20-kDa myosin light chain (MLC20) phosphorylation in Ca(2+)-free solutions in both phasic and tonic smooth muscle permeabilized with beta-escin, and inhibited the heavy meromyosin (HMM) phosphatase activity of smooth muscle homogenates with the same potency sequence: microcystin-LR greater than tautomycin greater than okadaic acid. The sensitivity to all three inhibitors was significantly higher, the half-times of relaxation and dephosphorylation were 4-6 times longer, and the HMM phosphatase and MLC20 kinase activity/smooth muscle cell wet weight was 2.0- and 1.9-fold lower in the tonic, femoral artery, than in the phasic, ileum or portal vein, smooth muscle. Preincubation with 0.2 microM inhibitor-2 decreased the HMM phosphatase activity by 35% in the ileum and by 60% in the femoral artery. The results suggest that the HMM phosphatases of smooth muscle have properties common to type 1 protein phosphatases, but are inhibited only partially by high concentrations of inhibitor-2, and that the lower HMM phosphatase activity of tonic smooth muscle may contribute to its greater sensitivity to phosphatase inhibitors and its slower rate of relaxation.

Animals↗
PubMed1992

Beta-adrenergic effects on cellular Na, Mg, Ca, K and Cl in vascular smooth muscle: electron probe analysis of rabbit pulmonary artery.

The effects of beta-adrenergic stimulation on the cellular content and subcellular distribution of Na, Mg, Ca, K and Cl were determined by electron probe X-ray microanalysis of muscles stimulated with 5-hydroxytryptamine. Isoproterenol caused a significant decrease in cytoplasmic and mitochondrial Na and Cl, and an increase in cytoplasmic Mg. Isoproterenol also significantly decreased total cytoplasmic Ca measured with small diameter probes, without affecting cellular Ca measured with large probes that included the sarcoplasmic reticulum (SR). The decrease in cytoplasmic Na and the effects on cytoplasmic and cellular Ca are consistent with, respectively, beta-adrenergic stimulation of the Na-pump and of Ca-uptake into the SR, but the beta-adrenergic increase in cytoplasmic Mg also raises the possibility of stimulated Na/Mg exchange.

Animals↗
PubMed1992

The giant cardiac membrane patch method: stimulation of outward Na(+)-Ca2+ exchange current by MgATP.

1. A giant patch method was used to study the stimulatory effect of cytoplasmic MgATP on outward Na(+)-Ca2+ exchange current in inside-out cardiac membrane patches (1-10 G omega seals with 14-24 microns pipette tip diameters) excised from guinea-pig, rabbit and mouse myocytes. 2. To establish the validity of the method with respect to structure, bleb formation was examined with electron microscopy and with confocal fluorescence light microscopy. The blebs, which form as the sarcolemma detaches, excluded intracellular organelles and transverse tubules. The blebbed cells contained normal sarcomeres, sarcoplasmic reticulum, triads and diads. 3. To further establish the validity of the method for ion transport studies, measurements of Na(+)-K+ pump currents and charge movements are described briefly which demonstrate (i) free access to the cytoplasmic membrane side, (ii) MgATP dependence comparable to reconstituted pump (Kd, 94 microns), (iii) fast, rigorous concentration control and (iv) Na(+)-K+ pump densities in the range of whole-cell densities. 4. Stimulation of outward Na(+)-Ca2+ exchange current by MgATP attenuated exchange current decay during step increments of cytoplasmic sodium, shifted the secondary activation of outward exchange current by cytoplasmic calcium to lower free calcium concentrations and, particularly in mouse cardiac sarcolemma, induced cytoplasmic calcium-independent current. 5. Upon removal of MgATP the stimulatory effect usually decayed with a t50 (half-time) of about 3 min. However, the reversal took place much more rapidly (t50, 5-20 s) in patches from individual guinea-pig and rabbit myocyte batches. When decay was rapid, secondary activation by cytoplasmic calcium was shifted to higher free cytoplasmic calcium concentrations (Kd, 10-65 microns-free calcium). 6. With repeated applications of MgATP the rate and magnitude of the stimulatory effect progressively decreased. 7. The Kd for MgATP of the initial rate of stimulation of outward exchange current was 3 mM or greater. When decay was rapid, the steady-state dependence of exchange current on MgATP also had a Kd of 3 mM or greater. 8. Stimulation of Na(+)-Ca2+ exchange current by MgATP occurred in the absence of cytoplasmic calcium with 9 mM-EGTA. 9. The stimulatory effect of 2 mM-MgATP was not inhibited by up to 200 microM of the protein kinase inhibitor 1-(5-isoquinoline sulphonyl)-2-methylpiperazine (H7), or by peptide inhibitors of cyclic AMP-dependent protein kinase, protein kinase C and calcium-calmodulin-dependent protein kinase II.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1991

Electron probe microanalysis of calcium release and magnesium uptake by endoplasmic reticulum in bee photoreceptors.

Honey bee photoreceptors contain large sacs of endoplasmic reticulum (ER) that can be located unequivocally in freeze-dried cryosections. The elemental composition of the ER was determined by electron probe x-ray microanalysis and was visualized in high-resolution x-ray maps. In the ER of dark-adapted photoreceptors, the Ca concentration was 47.5 +/- 1.1 mmol/kg (dry weight) (mean +/- SEM). During a 3-sec nonsaturating light stimulus, approximately 50% of the Ca content was released from the ER. Light stimulation also caused a highly significant increase in the Mg content of the ER; the ratio of Mg uptake to Ca released was approximately 0.7. Our results show unambiguously that the ER is the source of Ca2+ release during cell stimulation and suggest that Mg2+ can nearly balance the charge movement of Ca2+.

Animals↗
PubMed1991

Ca2+ channel blockers distinguish between G protein-coupled pharmacomechanical Ca2+ release and Ca2+ sensitization.

The effects of Ca2+ channel blockers on two modes of G protein-mediated pharmacomechanical coupling, Ca2+ release and modulation of Ca2+ sensitivity of the contractile apparatus, were investigated. Smooth muscles were permeabilized with Staphylococcal alpha-toxin or with beta-escin to avoid effects due to block of sarcolemmal Ca2+ channels, while retaining receptor/G protein coupling. In permeabilized portal vein smooth muscle, verapamil and nifedipine inhibited Ca2+ release induced by an alpha 1-adrenergic agonist (phenylephrine) and by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), but not that induced by inositol 1,4,5-trisphosphate (InsP3). These Ca2+ channel blockers also did not block the phenylephrine- or GTP gamma S-induced force development at constant cytoplasmic Ca2+ ("Ca2+ sensitization"). An alpha 1-blocker (prazosin) inhibited both the Ca2(+)-releasing and Ca2(+)-sensitizing effects of phenylephrine, but not those of GTP gamma S, nor did it block InsP3-induced Ca2+ release. We conclude that Ca2+ channel blockers selectively uncouple the Ca2(+)-releasing, but not the Ca2(+)-sensitizing, component of pharmacomechanical coupling. These findings raise the possibility that pharmacomechanical Ca2+ release may be modulated by dihydropyridine binding proteins at the level of G proteins/phospholipase C and also indicate a divergence of the Ca2(+)-releasing and Ca2(+)-sensitizing effects at some step prior to phospholipase C.

Animals↗
PubMed1990

Flash photolysis studies of excitation-contraction coupling, regulation, and contraction in smooth muscle.

1. Flash photolysis of caged compounds of phenylephrine, inositol 1, 4, 5 trisphosphate (InsP3), GTP gamma S, ATP, and CTP has been successfully used to study excitation-contraction coupling, contractile regulation, and contraction in smooth muscle. Major processes explored with this method were (a) the delay between agonist-receptor interaction and contraction and between the rise in InsP3, Ca2+ release and contraction; (b) the effect of myosin light chain phosphorylation on the rate of force development and the respective contributions of phosphorylation and crossbridge kinetics to differences between phasic and tonic smooth muscles; (c) the kinetics of the crossbridge cycle. We have also reviewed recent results obtained by other methods and bearing on the mechanisms of pharmacomechanical Ca2+ release and modulation of the Ca2+ sensitivity of the regulatory/contractile apparatus. 2. The long delay (1.5 at 22 degrees C) following activation of alpha 1-adrenergic receptors through photolysis of caged phenylephrine and the high Q10 of this process are consistent with the hypothesis that activation of phospholipase C is the major mechanism of alpha-adrenergic pharmacomechanical Ca2+ release. 3. The delay between photolysis of caged InsP3 and Ca2+ release is short: 30 ms or less, while the latency of contraction is significant (0.3-0.5 s at 22 degrees C) and similar to the lag between the rise in [Ca2+]i and force development in intact smooth muscles. The latency of contraction following photolysis of caged ATP in permeabilized muscles in rigor, in the presence of Ca2+ and calmodulin, is similar, about 0.2-0.5 s at 22 degrees C. 4. In muscles in which the myosin light chains are maintained in a phosphorylated state during rigor, photolysis of caged ATP initiates contractions with a short delay (10 ms or less). This result and those summarized above (2 and 3) suggest that the major portion of the delay between agonist-receptor interaction and contraction is due to activation of phospholipase C and InsP3 production, and about 0.2-0.5 s of the delay (22 degrees C) can be ascribed to prephosphorylation reactions between Ca2+, calmodulin, and myosin light chain kinase, and/or to mechanical processes, or to the chemical kinetics of two-step reactions. 5. Force development from rigor, initiated by photolysis of caged ATP in the presence of Ca2(+)-calmodulin, is rate-limited by myosin light chain phosphorylation; it is significantly accelerated if the myosin light chains are already phosphorylated prior to photolysis.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Cytosolic heparin inhibits muscarinic and alpha-adrenergic Ca2+ release in smooth muscle. Physiological role of inositol 1,4,5-trisphosphate in pharmacomechanical coupling.

In order to test the physiological significance of inositol 1,4,5-trisphosphate (InsP3) in pharmacomechanical coupling, we have utilized two near-physiological systems, in which relatively high molecular weight solutes can be applied intracellularly and receptor coupling is retained: beta-escin permeabilization and reversible permeabilization. We showed that in smooth muscle permeabilized with beta-escin, one of the saponin esters, alpha 1-adrenergic (phenylephrine) and muscarinic (carbachol) agonists, as well as caffeine and InsP3, cause contractions mediated by Ca2+ release. These contractions were calmodulin-dependent and blocked by depletion of Ca2+ stored in the sarcoplasmic reticulum. Intracellular heparin (Mr = about 5000), a blocker of InsP3 binding to its receptor and a specific inhibitor of InsP3-induced Ca2+ release in smooth muscles, inhibited the responses to the agonists and to InsP3, but not those to caffeine, nor did it block the enhanced contractile response to cytoplasmic Ca2+ induced by agonists and by GTP gamma S. Neomycin blocked Ca2+ release induced by carbachol, but not by caffeine. In reversibly permeabilized ileum smooth muscle cells, loaded with Fura-2 acid and heparin, the intracellular heparin inhibited Ca2+ release and contractions induced by carbachol in Ca2+-free, high K+ solution. Heparin did not inhibit the high K+ contractions (with 1.2 mM Ca2+) and had no significant inhibitory effects on carbachol-induced responses in the presence of extracellular Ca2+. These results, obtained under near-physiological conditions, support the conclusion that InsP3 is the major physiological messenger of the Ca2+ release component of pharmacomechanical coupling, but not of the components mediated by Ca2+ influx or by potentiation of the contractile response to Ca2+.

Animals↗
PubMed1989

Protein phosphatase composition in the smooth muscle of guinea-pig ileum studied with okadaic acid and inhibitor 2.

Using okadaic acid, a potent inhibitor of type 2A and type 1 protein phosphatases, and inhibitor 2, an intrinsic inhibitory factor of type 1 phosphatase, we characterized the phosphorylated myosin light-chain (PMLC) phosphatase activity in the smooth-muscle extracts of guinea-pig ileum. In the intact fibres the control activity was 254 +/- 13 nmol of Pi/min per g wet wt. (n = 15) against 32P-labelled PMLC (4 microM) from chicken gizzard. The following phosphatase fractions were identified: an inhibitor-2-sensitive (type 1) fraction (fractional activity = 35%), a Mg2+-dependent and okadaic acid-insensitive (type 2C) fraction (17%), and two type 2A-like fractions that had different susceptibility to okadaic acid. The type 2A-like fraction with lower affinity to okadaic acid accounted for 30% of the control activity. After the cell membrane was permeabilized by Triton X-100, more than 60% of this fraction remained and accounted for about 90% of the total activity, whereas the other fractions were nearly abolished. The type 2A-like fraction may be bound to some intracellular structure such as contractile proteins.

Animals↗
PubMed1989

Receptor-coupled, permeabilized smooth muscle. Role of the phosphatidylinositol cascade, G-proteins, and modulation of the contractile response to Ca2+.

alpha-Adrenergic (phenylephrine) and muscarinic (carbachol) agonists and inositol 1,4,5-trisphosphate caused calcium release and contractions in smooth muscle strips permeabilized with Staphylococcus aureus alpha-toxin. The responses to phenylephrine and carbachol required or were potentiated by added GTP and could be inhibited by GDP beta S. GTP and phenylephrine also increased the contractile response of permeabilized portal vein smooth muscle to cytoplasmic Ca2+. We conclude that while the G-protein-coupled phosphatidylinositol cascade, through inositol 1,4,5-trisphosphate-induced calcium release, is a major mechanism of pharmacomechanical coupling, a second G-protein-mediated pathway that modulates the calcium sensitivity of the regulatory contractile proteins also exists.

Animals↗
PubMed1989

Dynamics of the endoplasmic reticulum in living non-muscle and muscle cells.

The dynamic changes of the endoplasmic reticulum (ER) in interphase and mitotic cells was detected by the vital fluorescent dye 3,3'-dihexyloxacarbocyanine iodide. Two types of arrays characterize the continuous ER system in the non-muscle PtK2 cell: 1) a lacy network of irregular polygons and 2) long strands of ER that are found aligned along stress fibers. In cross-striated myotubes there was a periodic localization of fluorescence over each I-band corresponding to the positions of the terminal cisternae of the sarcoplasmic reticulum (SR). In contrast to the arrangement in muscle cells, the alignment of the long strands of ER alon stress fibers showed no strict periodicity that could be correlated with the sarcomeric units of the stress fibers. The ER and SR arrays seen in living cells were also detected in fixed cells stained with antibodies directed against proteins of the endoplasmic reticulum and sarcoplasmic reticulum, respectively. Observations of vitally stained PtK2 cells at 1 to 2 minute intervals using low light level video cameras and image processing techniques enabled us to see the polygonal ER units form and undergo changes in their shapes. During cell division, the ER, rhodamine 123-stained mitochondria, and phagocytosed fluorescent beads were excluded from the mitotic spindle while soluble proteins were not. No obvious concentration or alignment of membranes could be found associated with the contractile proteins in the cleavage furrow. After completion of cell division there was a redeployment of the ER network in each daughter cell.

Animals↗
PubMed1989

Kinetics of contraction initiated by flash photolysis of caged adenosine triphosphate in tonic and phasic smooth muscles.

Laser flash photolysis of caged adenosine triphosphate (ATP), in the presence of Ca2+, was used to examine the time course of isometric force development from rigor states in glycerinated tonic (rabbit trachealis) and phasic (guinea-pig ileum and portal vein) smooth muscles. Photolytic liberation of ATP from caged ATP initiated force development, at 20 degrees C, with half-time (t1/2) of 5.4 s in trachealis and 1.2-2.2 s in the phasic muscles. Prior to photolysis, some muscles were phosphorylated with ATP plus okadaic acid (an inhibitor of myosin light-chain phosphatase) or thiophosphorylated with ATP gamma S to fully activate the regulatory system, before turning on the contractile apparatus. In these prephosphorylated muscles, force development, after caged ATP photolysis, was more rapid than in the unphosphorylated muscles, but the t1/2 values for trachealis (0.8-1.1 s) were still longer than for ileum and portal-vein muscles (0.20-0.25 s). The results suggest that both the contractile machinery and the regulatory system are slower in the tonic than in the phasic smooth muscles. The time course of force development for each muscle type was sigmoidal, with an initial delay (td) of approximately 10% of the t1/2 value. Some possible chemical and mechanical origins of the delay are discussed.

Adenosine Triphosphate↗