| 51. |
- Gezelius, Henrik, 1977-, et al.
(författare)
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Conditional genetic labeling of the Renshaw cell population for functional studies of motor control
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The Renshaw cells were among the first interneurons to be characterized in the mammalian spinal cord. Although the basic function of recurrent inhibition to motor neurons, as well as the Renshaw cell connectivity to other neurons have been thoroughly studied, the exact functional role of the Renshaw cells in motor control is still unknown. To further characterize the role of Renshaw cells in spinal cord circuitry, we searched for candidate genes useful in the Cre-loxP system. It has been reported that the mRNA expression of nicotinic cholinergic receptor alpha 2 (Chrna2) is found in a restricted number of cells at the ventral rim in adult rat and mouse spinal cord. In our own search for genes with distinct ventral expression, we noted a similar restricted Chrna2 mRNA expression pattern in the mouse spinal cord at postnatal day (P) 11 and during development at embryonic day 14.5. Based on the fact that the gene product is a cholinergic receptor and the pattern of expression, the neurons are predicted to be Renshaw cells. The possibility that these cells were motor neurons was excluded, since Chrna2 and Vesicular acetylcholine were not co-expressed at P11. To further study this cell population, we have generated a transgenic mouse expressing Cre recombinase (Cre) under the control of the Chrna2 promoter region. To visualize the Cre-expressing cells, the Chrna2-Cre transgenic mouse were bred with a reporter mouse expressing β-galactosidase (β-gal) in the nucleus after loxP excision. As expected, spinal cord β-gal immunoreactivity was observed in a limited number of ventrally located cells in the Cre-bearing offspring. Co-labeling of β-gal with calbindin-28K, a known marker for Renshaw cells, indicated that a majority of the calbindin positive cells were also β-gal positive at the ventral rim where calbindin is specific. In addition, β-gal positive cells without observable calbindin were also detected. It is conceivable that Chrna2 is expressed in additional cells apart from Renshaw cells or that a previously unidentified Renshaw cell subpopulation does not express calbindin. Nonetheless, a mouse with Cre-activity restricted to Chrna2-expressing cells opens the possibility to functionally study a limited population of spinal cord interneurons through genetic techniques, with the ambition to explore the specific role of Renshaw cells in spinal cord circuitry and motor control.
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| 52. |
- Gomez-Pinilla, Pedro J, et al.
(författare)
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Melatonin restores impaired contractility in aged guinea pig urinary bladder
- 2008
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Ingår i: Journal of Pineal Research. - : Blackwell Publishing Ltd. - 1600-079X .- 0742-3098. ; 44:4, s. 416-425
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Tidskriftsartikel (refereegranskat)abstract
- Urinary bladder disturbances are frequent in the elderly population but the responsible mechanisms are poorly understood. This study evaluates the effects of aging on detrusor myogenic contractile responses and the impact of melatonin treatment. The contractility of bladder strips from adult, aged and melatonin-treated guinea pigs was evaluated by isometric tension recordings. Cytoplasmatic calcium concentration ([Ca2+](i)) was estimated by epifluorescence microscopy of fura-2-loaded isolated detrusor smooth muscle cells, and the levels of protein expression and phosphorylation were quantitated by Western blotting. Aging impairs the contractile response of detrusor strips to cholinergic and purinergic agonists and to membrane depolarization. The impaired contractility correlates with increased [Ca2+](i) in response to the stimuli, suggesting a reduced Ca(2+)sensitivity. Indeed, the agonist-induced contractions in adult strips were sensitive to blockade with Y27362, an inhibitor of Rho kinase (ROCK) and GF109203X, an inhibitor of protein kinase C (PKC), but these inhibitors had negligible effects in aged strips. The reduced Ca2+ sensitivity in aged tissues correlated with lower levels of RhoA, ROCK, PKC and the two effectors CPI-17 and MYPT1, and with the absence of CPI-17 and MYPT1 phosphorylation in response to agonists. Interestingly, melatonin treatment restored impaired contractility via normalization of Ca2+ handling and Ca2+ sensitizations pathways. Moreover, the indoleamine restored age-induced changes in oxidative stress and mitochondrial polarity. These results suggest that melatonin might be a novel therapeutic tool to palliate aging-related urinary bladder contractile impairment.
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| 53. |
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| 54. |
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| 55. |
- 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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| 56. |
- Löfgren, Mia, et al.
(författare)
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Substrate and product dependence of force and shortening in fast and slow smooth muscle
- 2001
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Ingår i: Journal of General Physiology. - : Rockefeller University Press. - 0022-1295 .- 1540-7748. ; 117:5, s. 407-418
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Tidskriftsartikel (refereegranskat)abstract
- To explore the molecular mechanisms responsible for the variation in smooth muscle contractile kinetics, the influence of MgATP, MgADP, and inorganic phosphate (P(i)) on force and shortening velocity in thiophosphorylated "fast" (taenia coli: maximal shortening velocity Vmax = 0.11 ML/s) and "slow" (aorta: Vmax = 0.015 ML/s) smooth muscle from the guinea pig were compared. P(i) inhibited active force with minor effects on the V(max). In the taenia coli, 20 mM P(i) inhibited force by 25%. In the aorta, the effect was markedly less (< 10%), suggesting differences between fast and slow smooth muscles in the binding of P(i) or in the relative population of P(i) binding states during cycling. Lowering of MgATP reduced force and V(max). The aorta was less sensitive to reduction in MgATP (Km for Vmax: 80 microM) than the taenia coli (Km for Vmax: 350 microM). Thus, velocity is controlled by steps preceding the ATP binding and cross-bridge dissociation, and a weaker binding of ATP is not responsible for the lower V(max) in the slow muscle. MgADP inhibited force and V(max). Saturating concentrations of ADP did not completely inhibit maximal shortening velocity. The effect of ADP on Vmax was observed at lower concentrations in the aorta compared with the taenia coli, suggesting that the ADP binding to phosphorylated and cycling cross-bridges is stronger in slow compared with fast smooth muscle.
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| 57. |
- Malmqvist, Ulf, et al.
(författare)
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Contractile and cytoskeletal proteins in smooth muscle during hypertrophy and its reversal
- 1991
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Ingår i: American Journal of Physiology: Cell Physiology. - 1522-1563. ; 260:5, s. 1085-1093
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Tidskriftsartikel (refereegranskat)abstract
- Hypertrophy of rat urinary bladder smooth muscle was induced by partial urethral obstruction. Bladder weight increased from 70 to 240 mg after 10 days and to 700 mg after 7 wk. Removal of the obstruction after 10 days caused a regression of bladder weight to 130 mg. The relative volume of smooth muscle in the bladder wall increased during hypertrophy. The concentration of myosin in the smooth muscle cells decreased in 10-day hypertrophied bladders, whereas the concentration of actin was unchanged. The actin-myosin ratio was 2.3 in controls, 3.3 in 10-day obstructed bladders, and 2.9 in 7-wk obstructed bladders. After removal of obstruction, the ratio was normalized. Two isoforms of myosin heavy chains were identified (SM1 and SM2). The relative amount of SM2 decreased during hypertrophy. The relative proportion of actin isoforms (alpha, beta, and gamma) was altered toward more gamma and less alpha. These changes were reversible upon removal of the obstruction. Desmin was the dominating intermediate filament protein. The concentration of desmin and filamin increased in the hypertrophic bladders. The increased desmin-actin and filamin-actin ratios in obstructed bladders were normalized after removal of the obstruction. The results suggest that the turnover of contractile and cytoskeletal proteins is fast and can be regulated in response to changes in the functional demands in smooth muscle.
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| 58. |
- Malmqvist, Ulf, et al.
(författare)
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Contractile properties during development of hypertrophy of the smooth muscle in the rat portal vein
- 1988
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Ingår i: Acta Physiologica Scandinavica. - 0001-6772. ; 133:1, s. 49-61
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Tidskriftsartikel (refereegranskat)abstract
- Structural and mechanical alterations during hypertrophy of the rat portal vein were investigated. Growth of the vessel was induced by a partial ligature of the vessel causing an increased transmural pressure. Vessel segments from animals kept with ligature for 1, 3, 5 and 7 days, were compared with vessels from sham-operated animals. Maximal active force and vessel cross-sectional area increased with time in the ligated group. On day 7, force and cross-sectional area at the optimal length, were markedly increased in the ligated group (21.1 +/- 1.0 mN, 0.55 +/- 0.04 mm2, n = 9) compared with the control vessels (11.7 +/- 1.0 mN, 0.30 +/- 0.02 mm2, n = 7). Light and electron microscopy of preparations fixed at optimal length showed that the amount of smooth muscle and the cross-sectional area of cell profiles were almost doubled in the ligated group on day 7, consistent with hypertrophy of the smooth muscle. The force per smooth muscle cell area was similar in the two groups (ligated: 132 +/- 15; control: 145 +/- 16 mN mm-2, n = 4-5). The maximal shortening velocity was significantly lower in the hypertrophied group (ligated: 0.28 +/- 0.02; control: 0.41 +/- 0.01 optimal length s-1, n = 6). In chemically skinned preparations, activated by maximal thiophosphorylation of the myosin light chains, force was higher in the ligated group compared to the controls but no difference in maximal shortening velocity was observed. In conclusion, the increased transmural pressure is associated with a rapid increase in the amount of smooth muscle in the portal vein. The mechanical data show that after 7 days the force generating ability of the contractile system has increased in proportion to the smooth muscle cell mass. The unaltered maximal shortening velocity in the skinned hypertrophied preparations suggests that the kinetic properties of the maximally activated contractile system are unaltered. The decreased maximal shortening velocity in the intact hypertrophied preparations may reflect alterations in the excitation-contraction coupling.
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| 59. |
- Malmqvist, Ulf, et al.
(författare)
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Cytoskeletal and contractile proteins in detrusor smooth muscle from bladders with outlet obstruction--a comparative study in rat and man
- 1991
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Ingår i: Scandinavian Journal of Urology and Nephrology. - : Informa UK Limited. - 0036-5599 .- 1651-2065. ; 25:4, s. 261-267
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Tidskriftsartikel (refereegranskat)abstract
- Detrusor biopsies were obtained from patients with urinary outlet obstruction due to prostatic enlargement and from age-matched control patients. The relative amounts of actin and myosin and their isoforms, as well as desmin and filamin were determined and compared with corresponding results from bladders from control rats and rats with 10 days of experimental outlet obstruction of the urinary bladder. In the human control detrusor the actin/myosin ratio was similar to that in the control rat. The isoform distribution of the myosin heavy chains differed between man and rat. In the biopsies from the patients with outlet obstruction and in the obstructed rat bladders the actin/myosin ratio was increased. A change in the myosin heavy chain distribution in the obstructed bladders was observed for both species. The filamin/actin ratio increased significantly in the obstructed rat bladders and tended to increase in the obstructed human bladders. Desmin was the dominating intermediate filament protein. The desmin/actin ratio increased in obstructed bladders in man and in rat.
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| 60. |
- Malmqvist, Ulf, et al.
(författare)
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Mechanics and Ca(2+)-sensitivity of human detrusor muscle bundles studied in vitro
- 1991
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Ingår i: Acta Physiologica Scandinavica. - 0001-6772. ; 143:4, s. 373-380
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Tidskriftsartikel (refereegranskat)abstract
- Mechanical properties of isolated smooth muscle strips from human urinary bladder were investigated in vitro. Bladder tissue was obtained from tumour-free wall regions of bladders from male patients undergoing cystectomy for bladder carcinoma. In intact muscle strips, activated with high-K+ solution, half-maximal force occurred at about 0.9 mM extracellular [Ca2+]. The length-active force relation was determined and the muscle strips were fixed for light and electron microscopy at optimal length for active force (1o). The maximal active force per unit smooth muscle cross-sectional area was 208 +/- 49 mN/mm2, n = 6. Chemically skinned preparations were obtained by treatment with triton X-100. These preparations had a steep [Ca2+]-force relation in the micromolar range which was influenced by calmodulin. The skinned preparations could be maximally activated by irreversible thiophosphorylation of the regulatory light chains. The force-velocity relation was determined in the maximally activated skinned muscle at 22 degrees C at 0.51o. When the muscle was shortened by 10%, force was reduced by 35% whereas the maximal shortening velocity was little affected.
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