| 1. |
- Arner, Anders, et al.
(författare)
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Intracellular calcium in hypertrophic smooth muscle from rat urinary bladder
- 2007
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Ingår i: Scandinavian Journal of Urology and Nephrology. - : Informa UK Limited. - 0036-5599 .- 1651-2065. ; 41:4, s. 270-277
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Tidskriftsartikel (refereegranskat)abstract
- Objective. To explore whether infravesical outlet obstruction is associated with alterations in calcium activation of detrusor smooth muscle. Material and methods. Outlet obstruction was created by partial ligature of the urethra in female rats. Western blotting was performed using an antibody against the cytoplasmatic region of the alpha(1c) subunit of the L- type Ca2(+) channel. Intracellular calcium was measured using Fura-2 in detrusors that had been obstructed for 10 days and activated by high K+ concentrations at different extracellular Ca2(+) concentrations. The rate of force development after rapid opening of L- type Ca2(+) channels was measured in contractions initiated by flash photolysis of nifedipine in Ca2(+) containing depolarizing solution. Results. Bladder weight increased from 6293 to 254943 mg after 10 days of obstruction. Expression of the alpha(1c) subunit increased after 3 days and continued to increase until it was about fourfold greater after 10 days; however, it had not increased further at 6 weeks. This change was reversible after removal of obstruction. Activation with K+ produced a stable force at different extracellular Ca2(+) concentrations, with no difference in response between controls and rats that had been obstructed for 10 days. Intracellular Ca2(+) concentrations were lower in the obstructed group, showing that the calcium sensitivity of the contraction force had increased. The delay between the opening of L- type channels and the onset of contraction was longer in obstructed detrusors. Conclusions. Growth of detrusor muscle following obstruction is accompanied by attenuated calcium transients following activation, despite upregulation of L- type Ca2(+) channels. The Ca2(+) sensitivity of contraction was increased in obstructed detrusors. We suggest that the decreased surface: volume ratio in hypertrophic smooth muscle cells is partly involved in the lowered Ca2(+) transients. The increases in L- type calcium channels and in calcium sensitivity may be compensatory mechanisms.
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| 2. |
- Balogh, Johanna, et al.
(författare)
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Desmin filaments influence myofilament spacing and lateral compliance of slow skeletal muscle fibres.
- 2005
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Ingår i: Biophysical Journal. - : Elsevier BV. - 1542-0086 .- 0006-3495. ; 88:2, s. 1156-1165
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Tidskriftsartikel (refereegranskat)abstract
- Intermediate filaments composed of desmin interlink Z-disks and sarcolemma in skeletal muscle. Depletion of desmin results in lower active stress of smooth, cardiac, and skeletal muscles. Structural functions of intermediate filaments in fast (psoas) and slow (soleus) skeletal muscle were examined using x-ray diffraction on permeabilized muscle from desmin-deficient mice (Des–/–) and controls (Des+/+). To examine lateral compliance of sarcomeres and cells, filament distances and fiber width were measured during osmotic compression with dextran. Equatorial spacing (x-ray diffraction) of contractile filaments was wider in soleus Des–/– muscle compared to Des+/+, showing that desmin is important for maintaining lattice structure. Osmotic lattice compression was similar in Des–/– and Des+/+. In width measurements of single fibers and bundles, Des–/– soleus were more compressed by dextran compared to Des+/+, showing that intermediate filaments contribute to whole-cell compliance. For psoas fibers, both filament distance and cell compliance were similar in Des–/– and Des+/+. We conclude that desmin is important for stabilizing sarcomeres and maintaining cell compliance in slow skeletal muscle. Wider filament spacing in Des–/– soleus cannot, however, explain the lower active stress, but might influence resistance to stretch, possibly minimizing stretch-induced cell injury.
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| 3. |
- Balogh, Johanna, et al.
(författare)
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Phospholipase C and cAMP-dependent positive inotropic effects of ATP in mouse cardiomyocytes via P2Y(11)-like receptors.
- 2005
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Ingår i: Journal of Molecular and Cellular Cardiology. - : Elsevier BV. - 1095-8584 .- 0022-2828. ; 39:2, s. 223-230
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Tidskriftsartikel (refereegranskat)abstract
- ATP is released as a cotransmitter together with catecholamines from sympathetic nerves. In the heart ATP has been shown to cause a pronounced positive inotropic effect and may also act in synergy with β-adrenergic agonists to augment cardiomyocyte contractility. The aim of the present study was to investigate the inotropic effects mediated by purinergic P2 receptors using isolated mouse cardiomyocytes. Stable adenine nucleotide analogs were used and the agonist rank order for adenine nucleotide stimulation of the mouse cardiomyocytes was AR-C67085 > ATPγS > 2-MeSATP >>> 2-MeSADP = 0, that fits the agonist profile of the P2Y11 receptor. ATPγS induced a positive inotropic response in single mouse cardiomyocytes. The response was similar to that for the β1 receptor agonist isoproterenol. The most potent response was obtained using AR-C67085, a P2Y11 receptor agonist. This agonist also potentiated contractions in isolated trabecular preparations. The adenylyl cyclase blocker (SQ22563) and phospholipase C (PLC) blocker (U73122) demonstrated that both pathways were required for the inotropic response of AR-C67085. A cAMP enzyme immunoassay confirmed that AR-C67085 increased cAMP in the cardiomyocytes. These findings are in agreement with the P2Y11 receptor, coupled both to activation of IP3 and cAMP, being a major receptor for ATP induced inotropy. Analyzing cardiomyocytes from desmin deficient mice, Des–/–, with a congenital cardiomyopathy, we found a lower sensitivity to AR-C67085, suggesting a down-regulation of P2Y11 receptor function in heart failure. The prominent action of the P2Y11 receptor in controling cardiomyocyte contractility and possible alterations in its function during cardiomyopathy may suggest this receptor as a potential therapeutic target. It is possible that agonists for the P2Y11 receptor could be used to improve cardiac output in patients with circulatory shock and that P2Y11 receptor antagonist could be beneficial in patients with congestive heart failure (CHF).
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| 4. |
- Ekberg, Olle, et al.
(författare)
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An In Vitro Model for Studying Neuromuscular Transmission in the Mouse Pharynx.
- 2009
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Ingår i: Dysphagia. - : Springer Science and Business Media LLC. - 1432-0460 .- 0179-051X. ; 24, s. 32-39
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Tidskriftsartikel (refereegranskat)abstract
- The muscles of the pharynx are controlled by networks of neurons under the control of specific regions in the brain stem, which have been fairly well studied. However, the transmission between these neurons and the pharyngeal muscles, at the motor end plates, is less well understood. Therefore, an in vitro model for the study of neuromuscular transmission in the pharyngeal muscle of the mouse was developed. Ring preparations from the inferior constrictor and the cricopharyngeus muscles were isolated and mounted for isometric force recording at physiologic temperature. Preparations from the diaphragm and the soleus muscles were examined in parallel. The muscles were stimulated at supramaximal voltage with short tetani at 100 Hz. Following direct stimulation of the muscle fibers, using a longer pulse duration, the rate of force development of the pharyngeal muscles was similar to that of the diaphragm and faster than that of the soleus muscle. By varying the duration of the stimulation pulses, conditions where the nerve-mediated activation contributed to a major extent of the contractile responses were identified. Gallamine completely inhibited the nerve-mediated responses. In separate experiments the dose dependence of gallamine inhibition was examined, showing similar sensitivity in the inferior pharyngeal constrictor compared to the diaphragm and soleus muscles. We conclude that reproducible contractile responses with an identifiable nerve-induced component can be obtained from the mouse inferior pharyngeal constrictor. The pharyngeal muscles have contractile characteristics similar to those of the faster diaphragm. The sensitivity to the neuromuscular blocking agent gallamine of the inferior pharyngeal constrictor was in the same concentration range as that of the diaphragm and soleus muscles.
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| 5. |
- Ekman, Mari, et al.
(författare)
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Decreased phosphatase activity, increased Ca2+ sensitivity, and myosin light chain phosphorylation in urinary bladder smooth muscle of newborn mice
- 2005
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Ingår i: Journal of General Physiology. - : Rockefeller University Press. - 0022-1295 .- 1540-7748. ; 125:2, s. 187-196
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Tidskriftsartikel (refereegranskat)abstract
- Developmental changes in the regulation of smooth muscle contraction were examined in urinary bladder smooth muscle from mice. Maximal active stress was lower in newborn tissue compared with adult, and it was correlated with a lower content of actin and myosin. Sensitivity to extracellular Ca2+ during high-K+ contraction, was higher in newborn compared with 3-wk-old and adult bladder strips. Concentrations at half maximal tension (EC50) were 0.57 +/- 0.01, 1.14 +/- 0.12, and 1.31 +/- 0.08 mM. Force of the newborn tissue was inhibited by similar to45% by the nonmuscle myosin inhibitor Blebbistatin, whereas adult tissue was not affected. The calcium sensitivity in newborn tissue was not affected by Blebbistatin, suggesting that noninuscle myosin is not a primary cause for increased calcium sensitivity. The relation between intracellular [Ca2+] and force was shifted toward lower [Ca2+] in the newborn bladders. This increased Ca2+ sensitivity was also found in permeabilized muscles (EC50: 6.10 +/- 0.07, 5.77 +/- 0.08, and 5.55 +/- 0.02 pCa units, in newborn, 3-wk-old, and adult tissues). It was associated with an increased myosin light chain phosphorylation and a decreased rate of dephosphorylation. No difference was observed in the myosin light chain phosphorylation rate, whereas the rate of myosin light chain phosphatase-induced relaxation was about twofold slower in the newborn tissue. The decreased rate was associated with a lower expression of the phosphatase regulatory Subunit MYPT-1 in newborn tissue. The results show that myosin light chain phosphatase activity can be developmentally regulated in mammalian urinary bladders. The resultant alterations in Ca2+ sensitivity may be of importance for the nervous and myogenic control of the newborn bladders.
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| 6. |
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| 7. |
- Ekman, Mari, et al.
(författare)
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Receptor-induced phasic activity of newborn mouse bladders is inhibited by protein kinase C and involves T-type Ca channels.
- 2009
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Ingår i: BJU International. - 1464-4096 .- 1464-410X. ; 104:5, s. 690-697
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE To investigate the mechanisms involved in the phasic contractile activity after muscarinic receptor activation of newborn urinary bladders and to compare neonatal and adult bladders. MATERIALS AND METHODS Detrusor muscle strips were isolated from newborn mice (aged 0-2 days) and compared with preparations from adult mice (aged 10-12 weeks). The effects of an activator (phorbol 12,13-dibutyrate, PDBu) and an inhibitor (GF109203X) of protein kinase C (PKC) on contractions were investigated. T-type Ca(2+) channels were blocked with NiCl(2). RESULTS The newborn urinary bladders responded with prominent phasic contractile activity in response to carbachol (1 microm). GF109203X (3 microm) reduced carbachol-induced force by approximately 60% in the newborn, compared with 30% in the adult. PDBu (1 microm) enhanced the muscarinic receptor-mediated contraction in adult bladder muscle, whereas it completely abolished the responses in the newborn. There was no inhibition after activation with depolarization (high-K(+)) or purinergic agonists (ATP, alpha,beta-methylene ATP). NiCl(2) (>30 microm) inhibited the peak responses to carbachol in the newborn and at 300 microm it completely abolished the phasic contractile response. The responses of the adult bladder muscle were only marginally affected by NiCl(2). CONCLUSIONS Muscarinic receptor stimulation recruits the PKC signalling pathway in both the adult and neonatal urinary bladder. Potent PKC activation is inhibitory on carbachol-induced activation in the newborn and stimulatory in the adult bladder. Furthermore, muscarinic receptor stimulation activates T-type Ca(2+) channels in the newborn, but not the adult bladder.
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| 8. |
- Ekman, Mari, et al.
(författare)
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Signal transduction pathways of muscarinic receptor mediated activation in the newborn and adult mouse urinary bladder.
- 2009
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Ingår i: BJU International. - 1464-4096. ; 103:1, s. 90-97
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE To study the role of M(2) and M(3) muscarinic receptor subtypes, sources of activator Ca(2+), and mechanisms involved in increased force oscillations in muscarinic contractions in the bladders of newborn and adult mice, as in the adult bladder muscarinic M(3) receptors are considered to mediate the main part of bladder contraction, and this has not been established in the newborn bladder. MATERIALS AND METHODS Bladder preparations from newborn (0-2 days) and adult (10-12 weeks) mice were mounted for in vitro force registration and activated with carbachol and high-K(+) solution in the presence of M(3) (4-DAMP 30 nm) or M(2) (methoctramine, 100 nm) receptor antagonists. Thapsigargin (1 microm) or ryanodine (10 microm) were used to inhibit sarcoplasmic reticulum Ca(2+) release. L-NAME (300 microm) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 microm) were used to inhibit nitric oxide synthase and guanylyl cyclase, respectively. Gap-junction function was inhibited with by 18-beta-glycyrrhetinic acid (18-beta-GA; 0.1-100 microm). Big-conductance (BK) and small-conductance (SK) K(+) channels were inhibited by apamine and charybdotoxin (0.3 microm), respectively. RESULTS Concentration-response relations for carbachol in the presence of 4-DAMP and methoctramine showed that M(3) receptors are the main activating pathway also in the newborn bladder. Neither thapsigargin nor ryanodine influenced the muscarinic responses of the newborn and adult bladders. Carbachol-induced contractions were not influenced by L-NAME or ODQ. The 18-beta-GA inhibited carbachol-induced contractions in both newborn and adult tissue in a similar manner. Apamine and charybdotoxin slightly increased the amplitude of the contractile responses. CONCLUSION These results suggest that in the newborn mouse bladder, as in adult bladders, the M(3) muscarinic receptor subtype is mainly responsible for carbachol-induced contractile responses. The main mechanism for muscarinic receptor-induced activation is influx of Ca(2+) from the extracellular medium, and there seems to be no major contribution of Ca(2+) release from intracellular stores. The phasic contractile activity induced by carbachol in the newborn bladder is not influenced by gap junction inhibition and does not involve SK and BK channels.
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| 9. |
- Scott, Rolf Sjuve, et al.
(författare)
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The role of desmin in active force transmission and maintenance of structure during growth of urinary bladder.
- 2008
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Ingår i: American Journal of Physiology: Cell Physiology. - : American Physiological Society. - 1522-1563 .- 0363-6143. ; 295:2, s. 324-331
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Tidskriftsartikel (refereegranskat)abstract
- The role of the intermediate filament protein desmin in hypertrophy of smooth muscle was examined in desmin deficient mice (Des -/-). A partial obstruction of the urethra was created and after 9-19 days bladder weight increased about 3-fold in both Des -/- and wild type (Des +/+) animals. Bladder growth was associated with synthesis of actin and myosin. In the hypertrophic Des +/+ bladder the relative content of desmin increased. In Des -/- mice desmin was absent. No alterations in the amount of vimentin were observed. Although Des -/- obstructed bladders were capable of growth they had structural changes with partial disruption of the wall. Des-/- bladders had slightly lower passive stress and significantly lower active stress compared to Des+/+. Des-/- preparations had lower shortening velocity. During hypertrophy these structural and mechanical alterations in the Des-/- urinary bladder became more pronounced. In conclusion, desmin in the bladder smooth muscle is not needed for growth but has a role in active force transmission and maintenance of wall structure. Key words: smooth muscle, intermediate filaments, desmin, transgenic.
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| 10. |
- Shakirova, Yulia, et al.
(författare)
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Increased Rho activation and PKC-mediated smooth muscle contractility in the absence of caveolin-1.
- 2006
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Ingår i: American Journal of Physiology: Cell Physiology. - : American Physiological Society. - 1522-1563 .- 0363-6143. ; 291:6, s. 1326-1335
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Tidskriftsartikel (refereegranskat)abstract
- Abstract in UndeterminedCaveolae are omega-shaped membrane invaginations that are abundant in smooth muscle cells. Since many receptors and signaling proteins co-localize with caveolae, these have been proposed to integrate important signaling pathways. The aim of this study was to test whether RhoA/Rho-kinase and protein kinase C (PKC)-mediated Ca2+ sensitization depends on caveolae using caveolin (Cav)-1-deficient (KO) and wild-type (WT) mice. In WT smooth muscle, caveolae were detected and Cav-1, -2 and -3 proteins were expressed. Relative mRNA expression levels were ~15:1:1 for Cav-1, -2, and -3, respectively. Caveolae were absent in KO and reduced levels of Cav-2 and Cav-3 proteins were seen. In intact ileum longitudinal muscle, no differences in the responses to 5-HT or the muscarinic agonist carbachol were found, whereas contraction elicited by endothelin-1 was reduced. Rho activation by GTP{gamma}S was increased in KO compared with WT as shown using a pull-down assay. Following {alpha}-toxin permeabilization, no difference in Ca2+ sensitivity or in Ca2+ sensitization was detected. In KO femoral arteries, phorbol 12,13-dibutyrate (PDBu)-induced and PKC-mediated contraction was increased. This was associated with increased {alpha}1-adrenergic contraction. Following inhibition of PKC, {alpha}1-adrenergic contraction was normalized. PDBu-induced Ca2+ sensitization was not increased in permeabilized femoral arteries. In conclusion, Rho activation, but not Ca2+ sensitization, depends on caveolae in the ileum. Moreover, PKC driven arterial contraction is increased in the absence of caveolin-1. This depends on an intact plasma membrane and is not associated with altered Ca2+ sensitivity.Ca2+ sensitization; Rho-associated kinase; myosin phosphatase target protein; lipid rafts; CPI-17; G protein-coupled receptor
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