| 1. |
- Arheden, Håkan, et al.
(författare)
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Dissociation between force and [Ca2+]i during extra systoles in guinea-pig ventricular muscle microinjected with fura-2
- 1999
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Ingår i: Acta Physiologica Scandinavica. - : Wiley. - 0001-6772. ; 165:1, s. 1-8
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Tidskriftsartikel (refereegranskat)abstract
- Thin trabeculae were dissected from the right ventricle of guinea-pig heart and stimulated to contract isometrically at 0.5 Hz (26 degrees C). Rapid and transient changes of force were obtained by inducing three extra systoles (ES1-3) at 450-ms intervals. The two regular contractions (P1-2) following (ES1-3) were potentiated. Fura-2 salt was microinjected into the preparation to monitor intracellular calcium ([Ca2+]i). Three distinct phases of [Ca2+]i were seen: (1) a rapid rising phase to about 200 nmol L(-1), (2) a slower rising phase to a peak at 400 nmol L(-1), and (3) a slow decline to about 50 nmol L(-1). During ES1, there was a discrepancy between force, which decreased, and peak [Ca2+]i, which increased to 600 nmol L(-1). It is likely that the increased [Ca2+]i during the extra systoles reflects increased sarcolemmal calcium inflow, causing post-extra-systolic potentiation. Ryanodine (1-2 microM) was added to inhibit the intracellular calcium release and thus reduce the intracellular [Ca2+]i gradients following excitation. Ryanodine inhibited phase 1 of [Ca2+]i and abolished post-extra-systolic potentiation. There was a close relationship between dF/dt and [Ca2+]i with ryanodine during control and ES1-3. It is likely that fura-2 reports a spatially averaged [Ca2+]i and that phase 1 of the signal therefore apparently underestimates activator calcium in the close vicinity of the contractile elements.
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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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Effects of polyamines on voltage-activated calcium channels in guinea-pig intestinal smooth muscle
- 1995
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Ingår i: Pflügers Archiv. - 0031-6768. ; 430:4, s. 501-507
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Tidskriftsartikel (refereegranskat)abstract
- Effects of polyamines on the spontaneous mechanical and electrical activity of guinea-pig intestinal smooth muscle were studied. Spermine and spermidine inhibited action potential generation and contractions, while putrescine had no effect. Single smooth muscle cells were isolated from the longitudinal muscle layer of the guinea-pig ileum. Whole-cell voltage-clamp experiments were carried out to investigate the effects of polyamines on current through voltage-activated Ca2+ channels. Spermine and spermidine (0.1-1 mM) reduced the inward current in a concentration-dependent manner. Spermine blocked current activated by the dihydropyridine agonist BAY K 8644 (1 microM), whereas no additional inhibition by spermine was seen after blockage of dihydropyridine-sensitive channels by nifedipine (0.1 microM). Inhibition by spermine or spermidine did not shift the peak of the current voltage relation of the inward current. Steady-state activation and inactivation relationships were not affected and thus the amplitude, but not the voltage dependence, of the window current responsible for Ca2+ inflow during sustained depolarization was affected. Putrescine (1 mM) had no significant effect on the inward current. These results suggest that spermine and spermidine inhibit contraction in spontaneously active intestinal smooth muscle by inhibiting Ca2+ current responsible for generation of action potentials.
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| 4. |
- Gomez, Maria, et al.
(författare)
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Endogenous polyamines modulate Ca2+ channel activity in guinea-pig intestinal smooth muscle
- 1999
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Ingår i: Pflügers Archiv. - : Springer Science and Business Media LLC. - 0031-6768. ; 438:4, s. 445-451
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Tidskriftsartikel (refereegranskat)abstract
- The polyamines spermine and spermidine inhibit L-type Ca2+ channels in whole-cell recordings from guinea-pig ileum cells (Gomez and Hellstrand, Pflugers Arch, 430:501-507, 1995 [4]). To study whether they modulate channel activity under physiological conditions, we further investigated their actions on Ca2+ channels and the effects of altered cellular polyamine contents. In inside-out patches, spermine (0.1-1 mM) inhibited channel activity without affecting the amplitude of unitary currents. In cell-attached recordings, addition of spermine to the bath did not influence channel activity in the patch, indicating that its extracellular action is direct and not mediated via passage of the polyamine through the cell membrane. Cellular contents of spermidine and spermine were decreased by about 50% by organ culture of ileum strips for 5 days with the adenosylmethionine decarboxylase inhibitor CGP 48664 (10 microM). This caused enhanced channel activity in cell-attached recordings, suggesting a reduced level of channel block by endogenous polyamines compared with control cells. Whole-cell recordings in the perforated patch mode showed increased current in polyamine-depleted cells, while this was not seen when cells were dialysed with the pipette solution. We conclude that polyamines block Ca2+ channels from the inside as well as the outside of the cell membrane, and that endogenous polyamines in smooth muscle modulate Ca2+ channel activity.
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| 5. |
- Hellstrand, Per
(författare)
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Long-term effects of intracellular calcium and growth factors on excitation and contraction in smooth muscle
- 1998
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Ingår i: Acta Physiologica Scandinavica. - 0001-6772. ; 164:4, s. 637-644
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Tidskriftsartikel (refereegranskat)abstract
- Modulation of vascular smooth muscle cells from a contractile to a synthetic phenotype is thought to be important in the development of the atherosclerotic lesion. Such modulation depends on growth factors and is influenced by cell-cell and cell-matrix interactions. Whereas smooth muscle cells in the vessel wall are contractile, dispersed cells in culture rapidly modulate to synthetic phenotype, which complicates long-term in vitro studies. In contrast, vascular segments or smooth muscle strips in organ culture can maintain contractility for at least a week, sufficient for studies involving altered metabolism or protein expression. Examples are effects of endogenous polyamines on membrane ion channels and excitation-contraction coupling. While smooth muscle tissue is well preserved in serum-free culture, growth stimulation with fetal calf serum (FCS) causes multiple effects, including decreased contractility, ultrastructural changes, decreased expression of L-type Ca2+ channels, and increased SR release of Ca2+ via ryanodine receptors. These are all consequences of increased basal [Ca2+]i caused by FCS, as they are reversed by culture with verapamil in a concentration (1 microM) that does not inhibit stimulation of DNA and protein synthesis by FCS. The effects of FCS on contractility and Ca2+ channel expression are mimicked in serum-free culture with increased [Ca2+]i. Contractile protein patterns, including myosin isoform composition, are unaffected by FCS, suggesting that reversal to synthetic phenotype is limited and not the immediate cause of decreased contractility.
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| 6. |
- Lindqvist, Anders, et al.
(författare)
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Inhibition of calcium entry preserves contractility of arterial smooth muscle in culture
- 1997
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Ingår i: Journal of Vascular Research. - 1423-0135. ; 34:2, s. 103-108
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Tidskriftsartikel (refereegranskat)abstract
- The addition of the growth stimulator fetal calf serum (FCS, 10%) to rings of rat tail artery causes an increase in [Ca2+]i, accompanied by contraction. This response was inhibited by the calcium entry blocker verapamil (1 microM). To investigate the effect of Ca2+ entry blockade on growth and contractility, rings of rat tail artery were cultured for 4 days in medium with or without FCS and then mounted for tension registration and stimulated with noradrenaline (NA) or high-K+ solution. In cultured rings growth was quantitated by [3H]-thymidine incorporation and increase in protein contents. FCS in the medium stimulated DNA synthesis by about 2-fold and increased protein contents by about 70%. The growth-stimulated cultured rings developed less force than freshly prepared rings (2.2 +/- 0.3 vs. 8.3 +/- 1.0 mN/mm). The addition of 1 microM verapamil to the medium during culture increased maximal NA-evoked force to 5.0 +/- 0.4 mN/mm but had no effect on the increases in DNA synthesis and protein contents. Force developed by growth-arrested rings, cultured in the absence of FCS, was not different from that of freshly prepared rings (7.2 +/- 0.6 mM/mm). Verapamil did not affect maximal force in these rings. Similar responses were seen when contraction was elicited by high-K+ solution. We conclude that verapamil, present during culture, preserves contractility of arterial smooth muscle, and that this effect is not parallel to inhibition of growth.
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| 7. |
- 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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| 8. |
- Swärd, Karl, et al.
(författare)
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Contractile effects of polycations in permeabilized smooth muscle
- 1998
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Ingår i: Journal of Muscle Research and Cell Motility. - 0142-4319. ; 19:5, s. 463-472
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Tidskriftsartikel (refereegranskat)abstract
- The polycations spermine, neomycin and polylysine potentiated Ca(2+)-activated force in beta-escin permeabilized guinea-pig ileum strips. The effect was inhibited by the calmodulin antagonists trifluoperazine, mastoparan and W13. Potentiation was slow or absent in chi-toxin permeabilized strips, indicating dependence on penetration of the polycations into cells. The effects of spermine and neomycin were maintained after extensive permeabilization by beta-escin, which eliminated the contractile effect of GTPgammaS. Replacement of ATP by CTP, which is not a substrate for myosin light chain kinase, inhibited contractile potentiation. Potentiation of Ca(2+)-activated contractions was associated with increased phosphorylation of the myosin regulatory light chains (LC20). A contractile effect of polylysine and neomycin was also seen in Ca(2+)-free medium and after partial LC20 thiophosphorylation, indicating that phosphorylation-independent processes may contribute to the response. Although spermine does not cause contraction in Ca(2+)-free medium at physiological [MgATP], it did so when [MgATP] was lowered to 40 micron. Similar to high-[Mg2+], the rate of contraction on addition of ATP to strips incubated with microcystin-LR in inhibit phosphatase activity was increased by the polycations, but only at [Ca2+] < 0.3 micron. The results suggest that polycations increase Ca(2+)-activated force by inhibiting myosin phosphatase activity, thereby increasing myosin LC20 phosphorylation. However, additional activation mechanisms, evident at low [Ca2+] and at low [ATP] and possibly involving direct activation of myosin, contribute to their effect.
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| 9. |
- 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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| 10. |
- 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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