| 1. |
- Barg, Sebastian, et al.
(författare)
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Different interactions of cardiac and skeletal muscle ryanodine receptors with FK-506 binding protein isoforms
- 1997
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Ingår i: American Journal of Physiology: Cell Physiology. - 1522-1563. ; 272:5 Pt 1, s. C1726-C1733
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Tidskriftsartikel (refereegranskat)abstract
- In the present study, we compare functional consequences of dissociation and reconstitution of binding proteins FKBP12 and FKBP12.6 with ryanodine receptors from cardiac (RyR2) and skeletal muscle (RyR1). The skeletal muscle RyR1 channel became activated on removal of endogenously bound FKBP12, consistent with previous reports. Both FKBP12 and FKBP12.6 rebind to FKBP-depleted RyR1 and restore its quiescent channel behavior by altering ligand sensitivity, as studied by single-channel recordings in planar lipid bilayers, and macroscopic behavior of the channels (ryanodine binding and net energized Ca2- uptake). By contrast, removal of FKBP12.6 from the cardiac RyR2 did not modulate the function of the channel using the same types of assays as for RyR1. FKBP12 or FKBP12.6 had no effect on channel activity of FKBP12.6-depleted cardiac RyR2, although FKBP12.6 rebinds. Our studies reveal important differences between the two ryanodine receptor isoforms with respect to their functional interaction with FKBP12 and FKBP12.6.
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| 2. |
- Dreja, Karl, et al.
(författare)
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Differential modulation of caffeine- and IP3-induced calcium release in cultured arterial tissue
- 1999
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Ingår i: American Journal of Physiology: Cell Physiology. - 1522-1563. ; 276:5, s. 1115-1120
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Tidskriftsartikel (refereegranskat)abstract
- To investigate the Ca2+-dependent plasticity of sarcoplasmic reticulum (SR) function in vascular smooth muscle, transient responses to agents releasing intracellular Ca2+ by either ryanodine (caffeine) or D-myo-inositol 1,4,5-trisphosphate [IP3; produced in response to norepinephrine (NE), 5-hydroxytryptamine (5-HT), arginine vasopressin (AVP)] receptors in rat tail arterial rings were evaluated after 4 days of organ culture. Force transients induced by all agents were increased compared with those induced in fresh rings. Stimulation by 10% FCS during culture further potentiated the force and Ca2+ responses to caffeine (20 mM) but not to NE (10 microM), 5-HT (10 microM), or AVP (0.1 microM). The effect was persistent, and SR capacity was not altered after reversible depletion of stores with cyclopiazonic acid. The effects of serum could be mimicked by culture in depolarizing medium (30 mM K+) and blocked by the addition of verapamil (1 microM) or EGTA (1 mM) to the medium, lowering intracellular Ca2+ concentration ([Ca2+]i) during culture. These results show that modulation of SR function can occur in vitro by a mechanism dependent on long-term levels of basal [Ca2+]i and involving ryanodine- but not IP3 receptor-mediated Ca2+ release.
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| 3. |
- Gomez, Maria, et al.
(författare)
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Long-term regulation of contractility and calcium current in smooth muscle
- 1997
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Ingår i: American Journal of Physiology: Cell Physiology. - 1522-1563. ; 273:5, s. 1714-1720
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Tidskriftsartikel (refereegranskat)abstract
- Longitudinal smooth muscle strips from guinea pig ileum were cultured in vitro for 5 days, and the relationship between extracellular Ca2+ and force in high-K+ medium was evaluated. In strips cultured with 10% fetal calf serum (FCS), this relationship was shifted to the right (50% effective concentration changed by 2-3 mM) compared with strips cultured without FCS. The shift was prevented by inclusion of verapamil (1 microM) during culture and mimicked by ionomycin in the absence of FCS. The intracellular Ca2+ concentration ([Ca2+]i) during stimulation with high-K+ solution or carbachol was reduced after culture with FCS, whereas the [Ca2+]i-force relationship was unaffected. Cells were isolated from cultured strips, and whole cell voltage-clamp experiments were performed. Maximum inward Ca2+ current (10 mM Ba2+), normalized to cell capacitance, was almost three times smaller in cells isolated from strips cultured with FCS. Culture with 1 microM verapamil prevented this reduction. These results suggest that increased [Ca2+]i during culture downregulates Ca2+ current density, with associated effects on contractility.
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| 4. |
- Labotka, R. J., et al.
(författare)
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Ammonia permeability of erythrocyte membrane studied by 14N and 15N saturation transfer NMR spectroscopy
- 1995
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Ingår i: American Journal of Physiology - Cell Physiology. - 0363-6143 .- 1522-1563. ; 268:3, s. C686-699
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Tidskriftsartikel (refereegranskat)abstract
- The permeability of biological membranes to the rapidly penetrating compound ammonia is extremely difficult to study due to the lack of readily available radionuclides. 14N and 15N saturation transfer nuclear magnetic resonance (NMR) experiments were used to measure the erythrocyte membrane permeability of ammonia under equilibrium exchange conditions. When 14N spectra from erythrocytes suspended in NH4Cl solution were obtained in the presence of the extracellular shift reagent dysprosium tripolyphosphate, intracellular and extracellular ammonia signals were readily resolved. Comparison with 15N spectra from erythrocyte suspensions containing 15N4Cl revealed that the intracellular [14N]ammonia signals were 100% NMR visible. 14N and 15N saturation transfer NMR experiments showed similar influx rates and permeabilities, indicating no loss of saturation transfer due to quadrupolar relaxation of 14N nuclei upon membrane passage. Ammonia influx was directly proportional to concentration (0.39 +/- 0.012 fmol.cell-1.s-1.mM-1 at pH 7.0) and not saturable, which is consistent with passive diffusion. Apparent ammonia permeability increased with pH over the range of pH 6-8 as the fraction of free NH3 increased. However, diffusion through unstirred layers became increasingly rate limiting. The permeability of the unstirred layers (1.1 +/- 0.45 x 10(-3) cm/s) was considerably lower than that of NH3 (0.21 +/- 0.014 cm/s). The Arrhenius activation energy for NH3 permeability was 49.5 +/- 11.8 kJ/mol. No evidence for NH+4 influx over the time domain of these experiments was found.
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| 5. |
- Lindqvist, Anders, et al.
(författare)
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Long-term effects of Ca(2+) on structure and contractility of vascular smooth muscle
- 1999
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Ingår i: American Journal of Physiology: Cell Physiology. - 1522-1563. ; 277:1, s. 64-73
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Tidskriftsartikel (refereegranskat)abstract
- Culture of dispersed smooth muscle cells is known to cause rapid modulation from the contractile to the synthetic cellular phenotype. However, organ culture of smooth muscle tissue, with maintained extracellular matrix and cell-cell contacts, may facilitate maintenance of the contractile phenotype. To test the influence of culture conditions, structural, functional, and biochemical properties of rat tail arterial rings were investigated after culture. Rings were cultured for 4 days in the absence and presence of 10% FCS and then mounted for physiological experiments. Intracellular Ca(2+) concentration ([Ca(2+)](i)) after stimulation with norepinephrine was similar in rings cultured with and without FCS, whereas force development after FCS was decreased by >50%. The difference persisted after permeabilization with beta-escin. These effects were associated with the presence of vasoconstrictors in FCS and were dissociated from its growth-stimulatory action. FCS treatment increased lactate production but did not affect ATP, ADP, or AMP contents. The contents of actin and myosin were decreased by culture but similar for all culture conditions. There was no effect of FCS on calponin contents or myosin SM1/SM2 isoform composition, nor was there any appearance of nonmuscle myosin. FCS-stimulated rings showed evidence of cell degeneration not found after culture without FCS or with FCS + verapamil (1 microM) to lower [Ca(2+)](i). The decreased force-generating ability after culture with FCS is thus associated with increased [Ca(2+)](i) during culture and not primarily caused by growth-associated modulation of cells from the contractile to the synthetic phenotype.
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| 6. |
- Salehi, S Albert, et al.
(författare)
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Islet constitutive nitric oxide synthase: biochemical determination and regulatory function
- 1996
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Ingår i: American Journal of Physiology: Cell Physiology. - 1522-1563. ; 270:6 Pt 1, s. 1634-1641
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Tidskriftsartikel (refereegranskat)abstract
- Recent immunohistochemical findings suggested that a constitutive nitric oxide synthase (cNOS) resides in endocrine pancreas. Here we provide direct biochemical evidence for the presence of cNOS activity in isolated islets. The regulating influence of this nitric oxide synthase (NOS) activity for islet hormone release was also investigated. We observed that cNOS activity could be quantitated in islet homogenates by monitoring the formation of L-citrulline from L-arginine using an Amprep CBA cation-exhange minicolumn before derivatization with o-phthaldialdehyde and subsequent high-performance liquid chromatography analysis. The islet NOS was dependent on both Ca2+ and calmodulin and suppressed by the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME). This effect was enantiomerically specific. Islet insulin release induced by a mixture of L-arginine and glucose was enhanced by L-NAME, whereas L-arginine-induced glucagon release was inhibited. The effect of L-NAME on insulin release was dose dependently potentiated by increasing glucose concentrations, suggesting that glucose is an important regulator of islet NO production. Complementary in vivo studies showed similar results, i.e., the insulin secretory response to a mixture of glucose and L-arginine was extremely enhanced by pretreatment with L-NAME, whereas L-arginine-stimulated glucagon response was suppressed. Finally, in isolated islets, the intracellular nitric oxide (NO) donor hydroxylamine suppressed insulin release and increased glucagon release. In summary, the islets of Langerhans contain a constitutive, Ca2+/calmodulin-dependent isoform of NOS. Islet NO suppressed insulin but enhanced glucagon secretion. The data also suggest a negative feedback by NO on glucose-induced insulin release. The islet NO system is a novel and important regulatory factor in insulin and glucagon secretion.
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| 7. |
- Swärd, Karl, et al.
(författare)
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Inhibition of polyamine synthesis influences contractility of intestinal smooth muscle in culture
- 1997
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Ingår i: American Journal of Physiology: Cell Physiology. - 1522-1563. ; 273:1, s. 77-84
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Tidskriftsartikel (refereegranskat)abstract
- Smooth muscle strips from guinea pig ileum were cultured for 5 days and then tested for contractile properties to investigate whether endogenous polyamines influence excitation-contraction coupling. Inhibition of spermidine and spermine synthesis by culture in the presence of the adenosylmethionine decarboxylase (EC4.1.1.50) inhibitor CGP-48664 (1-10 microM) decreased spermidine and spermine levels by 50% and increased putrescine by 20-fold. After culture with 10 microM, but not 1 microM, CGP-48664, the relationship between extracellular Ca2+ concentration and force in high K(+)-depolarized strips was shifted to the right, and phasic contractile activity as well as sensitivity to muscarinic stimulation was enhanced. When spermidine and spermine (each 50 microM) were available for cellular uptake during culture in the presence of 10 microM CGP-48664, spermidine and spermine concentrations were increased, and the effect on Ca2+ sensitivity was reversed. In strips cultured with 0 or 1 microM CGP-48664 in the presence of 50 microM spermidine and 50 microM spermine, no effect on Ca2+ sensitivity was observed. Force development relative to intracellular Ca2+ concentration was decreased in CGP-48664 (10 microM)-treated strips. The results suggest that endogenous polyamines influence excitation-contraction coupling in smooth muscle, although overall tissue concentrations may not reflect the polyamine pools responsible for this effect.
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| 8. |
- Swärd, Karl, et al.
(författare)
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Polyamines inhibit myosin phosphatase and increase LC20 phosphorylation and force in smooth muscle
- 1995
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Ingår i: American Journal of Physiology: Cell Physiology. - 1522-1563. ; 269:3, s. 563-571
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Tidskriftsartikel (refereegranskat)abstract
- The increase in Ca(2+)-activated force caused by polyamines in beta-escin-permeabilized guinda pig ileum is shown to be associated with increased myosin 20-kDa light chain (LC20) phosphorylation and shortening velocity. Myosin LC20 dephosphorylation with arrested kinase activity was slower in the presence of 1 mM spermine. Smooth muscle phosphatases (SMP-I, -II, -III, and -IV) isolated from turkey gizzard are all active against phosphorylated LC20, but only SMP-III and -IV dephosphorylate heavy meromyosin (HMM). Spermine inhibited SMP-III activity toward LC20 but stimulated HMM dephosphorylation, whereas SMP-IV was inhibited with both substrates. In contrast, SMP-I and -II were stimulated by spermine. The relative effects of different polyamines correlated with an increasing number of positive charges. Spermine did not affect binding of SMP-IV to myosin and did not dissociate any of the subunits of the enzyme. Incubation of permeabilized strips with SMP-IV resulted in attenuated responses to Ca2+, an effect that was opposed by spermine and abolished by microcystin-LR. We conclude that spermine selectively inhibits myosin phosphatase activity and suggest that polyamines function as endogenous myosin phosphatase inhibitors.
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