Bladder smooth muscle organ culture preparation maintains the contractile phenotype

Smooth muscle cells, when subjected to culture, modulate from a contractile to a secretory phenotype. This has hampered the use of cell culture for molecular techniques to study the regulation of smooth muscle biology. The goal of this study was to develop a new organ culture model of bladder smooth muscle (BSM) that would maintain the contractile phenotype and aid in the study of BSM biology. Our results showed that strips of BSM subjected to up to 9 days of organ culture maintained their contractile phenotype, including the ability to achieve near-control levels of force with a temporal profile similar to that of noncultured tissues. The technical aspects of our organ culture preparation that were responsible, in part, for the maintenance of the contractile phenotype were a slight longitudinal stretch during culture and subjection of the strips to daily contraction-relaxation. The tissues contained viable cells throughout the cross section of the strips. There was an increase in extracellular collagenous matrix, resulting in a leftward shift in the passive length-tension relationship. There were no significant changes in the content of smooth muscle-specific α-actin, calponin, h-caldesmon, total myosin heavy chain, protein kinase G, Rho kinase-I, or the ratio of SM1 to SM2 myosin isoforms. Moreover the organ cultured tissues maintained functional voltage-gated calcium channels and large-conductance calcium-activated potassium channels. Therefore, we propose that this novel BSM organ culture model maintains the contractile phenotype and will be a valuable tool for the use in cellular/molecular biology studies of bladder myocytes.

Download Full-text

Stretch-induced Calcium Release in Smooth Muscle

The Journal of General Physiology ◽

10.1085/jgp.20028514 ◽

2002 ◽

Vol 119 (6) ◽

pp. 533-543 ◽

Cited By ~ 76

Author(s):

Guangju Ji ◽

Robert J. Barsotti ◽

Morris E. Feldman ◽

Michael I. Kotlikoff

Keyword(s):

Smooth Muscle ◽

Urinary Bladder ◽

Smooth Muscle Cells ◽

Calcium Release ◽

Muscle Cells ◽

Intraluminal Pressure ◽

Bladder Smooth Muscle ◽

Urinary Bladder Smooth Muscle ◽

Inward Currents ◽

Longitudinal Stretch

Smooth muscle cells undergo substantial increases in length, passively stretching during increases in intraluminal pressure in vessels and hollow organs. Active contractile responses to counteract increased transmural pressure were first described almost a century ago (Bayliss, 1902) and several mechanisms have been advanced to explain this phenomenon. We report here that elongation of smooth muscle cells results in ryanodine receptor–mediated Ca2+ release in individual myocytes. Mechanical elongation of isolated, single urinary bladder myocytes to ∼120% of slack length (ΔL = 20) evoked Ca2+ release from intracellular stores in the form of single Ca2+ sparks and propagated Ca2+ waves. Ca2+ release was not due to calcium-induced calcium release, as release was observed in Ca2+-free extracellular solution and when free Ca2+ ions in the cytosol were strongly buffered to prevent increases in [Ca2+]i. Stretch-induced calcium release (SICR) was not affected by inhibition of InsP3R-mediated Ca2+ release, but was completely blocked by ryanodine. Release occurred in the absence of previously reported stretch-activated currents; however, SICR evoked calcium-activated chloride currents in the form of transient inward currents, suggesting a regulatory mechanism for the generation of spontaneous currents in smooth muscle. SICR was also observed in individual myocytes during stretch of intact urinary bladder smooth muscle segments. Thus, longitudinal stretch of smooth muscle cells induces Ca2+ release through gating of RYR. SICR may be an important component of the physiological response to increases in luminal pressure in smooth muscle tissues.

Download Full-text

Expression and functional role of Rho-kinase in rat urinary bladder smooth muscle

British Journal of Pharmacology ◽

10.1038/sj.bjp.0705109 ◽

2003 ◽

Vol 138 (5) ◽

pp. 757-766 ◽

Cited By ~ 125

Author(s):

Alexandra Wibberley ◽

Zunxuan Chen ◽

Erding Hu ◽

J Paul Hieble ◽

Timothy D Westfall

Keyword(s):

Smooth Muscle ◽

Urinary Bladder ◽

Functional Role ◽

Rho Kinase ◽

Bladder Smooth Muscle ◽

Rat Urinary Bladder ◽

Urinary Bladder Smooth Muscle

Download Full-text

Regional variation in myosin isoforms and phosphorylation at the resting tone in urinary bladder smooth muscle

AJP Cell Physiology ◽

10.1152/ajpcell.2001.280.2.c254 ◽

2001 ◽

Vol 280 (2) ◽

pp. C254-C264 ◽

Cited By ~ 39

Author(s):

Joseph A. Hypolite ◽

Michael E. DiSanto ◽

Yongmu Zheng ◽

Shaohua Chang ◽

Alan J. Wein ◽

...

Keyword(s):

Smooth Muscle ◽

Urinary Bladder ◽

Smooth Muscles ◽

Urinary Continence ◽

Myosin Isoforms ◽

Bladder Smooth Muscle ◽

Rt Pcr ◽

Total Rna ◽

Bladder Dome ◽

Significant Difference

Urinary bladder filling and emptying requires coordinated control of bladder body and urethral smooth muscles. Bladder dome, midbladder, base, and urethra showed significant differences in the percentage of 20-kDa myosin light chain (LC20) phosphorylation (35.45 ± 4.6, 24.7 ± 2.2, 13.6± 2.1, and 12.8 ± 2.7%, respectively) in resting muscle. Agonist-mediated force was associated with a rise in LC20 phosphorylation, but the extent of phosphorylation at all levels of force was less for urethral than for bladder body smooth muscle. RT-PCR and quantitative competitive RT-PCR analyses of total RNA from bladder body and urethral smooth muscles revealed only a slight difference in myosin heavy chain mRNA copy number per total RNA, whereas mRNA copy numbers for NH2-terminal isoforms SM-B (inserted) and SM-A (noninserted) in these muscles showed a significant difference (2.28 × 108 vs. 1.68 × 108 for SM-B and 0.12 × 108 vs. 0.42 × 108 for SM-A, respectively), which was also evident at the protein level. The ratio of COOH-terminal isoforms SM2:SM1 in the urethra was moderately but significantly lower than that in other regions of the bladder body. A high degree of LC20phosphorylation and SM-B in the bladder body may help to facilitate fast cross-bridge cycling and force generation required for rapid emptying, whereas a lower level of LC20 phosphorylation and the presence of a higher amount of SM-A in urethral smooth muscle may help to maintain the high basal tone of urethra, required for urinary continence.

Download Full-text

Pelvic Arterial Occlusive Disease Affects the RhoA/Rho-Kinase Pathway in Bladder Smooth Muscle

The Journal of Urology ◽

10.1016/j.juro.2014.09.102 ◽

2015 ◽

Vol 193 (2) ◽

pp. 706-713 ◽

Cited By ~ 8

Author(s):

Hidenori Akaihata ◽

Masanori Nomiya ◽

Junya Hata ◽

Michihiro Yabe ◽

Norio Takahashi ◽

...

Keyword(s):

Smooth Muscle ◽

Rho Kinase ◽

Arterial Occlusive Disease ◽

Occlusive Disease ◽

Bladder Smooth Muscle ◽

Kinase Pathway

Download Full-text

MP42-10 AUGMENTED BLADDER SMOOTH MUSCLE RESPONSIVENESS TO HYPERGLYCEMIA THROUGH A CAVEOLAE-DEPENDENT ACTIVATION OF RHO KINASE PATHWAY

The Journal of Urology ◽

10.1016/j.juro.2017.02.1299 ◽

2017 ◽

Vol 197 (4S) ◽

Author(s):

Vivian Cristofaro ◽

Josephine A. Carew ◽

Suhas P. Dasari ◽

Raj K. Goyal ◽

Maryrose P. Sullivan

Keyword(s):

Smooth Muscle ◽

Rho Kinase ◽

Bladder Smooth Muscle ◽

Kinase Pathway

Download Full-text

Uni-axial stretch induces actin stress fiber reorganization and activates c-Jun NH2 terminal kinase via RhoA and Rho kinase in human bladder smooth muscle cells

BMC Urology ◽

10.1186/s12894-016-0127-9 ◽

2016 ◽

Vol 16 (1) ◽

Cited By ~ 5

Author(s):

Nobuhiro Kushida ◽

Osamu Yamaguchi ◽

Yohei Kawashima ◽

Hidenori Akaihata ◽

Junya Hata ◽

...

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Rho Kinase ◽

Muscle Cells ◽

Stress Fiber ◽

Actin Stress Fiber ◽

Bladder Smooth Muscle ◽

Human Bladder ◽

Axial Stretch

Download Full-text

Phosphorylation of CPI‐17 and MYPT in rabbit bladder smooth muscle: Role of PKC, Rho kinase, or both

The FASEB Journal ◽

10.1096/fasebj.21.6.a1300-d ◽

2007 ◽

Vol 21 (6) ◽

Author(s):

Tanchun Wang ◽

Derek M. Kendig ◽

Elaine M. Smolock ◽

Robert S. Moreland

Keyword(s):

Smooth Muscle ◽

Rho Kinase ◽

Bladder Smooth Muscle ◽

Rabbit Bladder

Download Full-text

Caldesmon ablation in mice causes umbilical herniation and alters contractility of fetal urinary bladder smooth muscle

The Journal of General Physiology ◽

10.1085/jgp.202012776 ◽

2021 ◽

Vol 153 (7) ◽

Author(s):

Sandra Pütz ◽

Lisa Sophie Barthel ◽

Marina Frohn ◽

Doris Metzler ◽

Mohammed Barham ◽

...

Keyword(s):

Smooth Muscle ◽

Umbilical Hernia ◽

Structural Integrity ◽

Smooth Muscle Contraction ◽

Myosin Isoforms ◽

Bladder Smooth Muscle ◽

Splice Isoforms ◽

Calmodulin Binding ◽

Urinary Bladder Smooth Muscle

The actin-, myosin-, and calmodulin-binding protein caldesmon (CaD) is expressed in two splice isoforms: h-CaD, which is an integral part of the actomyosin domain of smooth muscle cells, and l-CaD, which is widely expressed and is involved in many cellular functions. Despite extensive research for many years, CaD's in vivo function has remained elusive. To explore the role of CaD in smooth muscle contraction in vivo, we generated a mutant allele that ablates both isoforms. Heterozygous animals were viable and had a normal life span, but homozygous mutants died perinatally, likely because of a persistent umbilical hernia. The herniation was associated with hypoplastic and dysmorphic abdominal wall muscles. We assessed mechanical parameters in isometrically mounted longitudinal strips of E18.5 urinary bladders and in ring preparations from abdominal aorta using wire myography. Ca2+ sensitivity was higher and relaxation rate was slower in Cald1−/− compared with Cald1+/+ skinned bladder strips. However, we observed no change in the content and phosphorylation of regulatory proteins of the contractile apparatus and myosin isoforms known to affect these contractile parameters. Intact fibers showed no difference in actin and myosin content, regardless of genotype, although KCl-induced force tended to be lower in homozygous and higher in heterozygous mutants than in WTs. Conversely, in skinned fibers, myosin content and maximal force were significantly lower in Cald1−/− than in WTs. In KO abdominal aortas, resting and U46619 elicited force were lower than in WTs. Our results are consistent with the notion that CaD impacts smooth muscle function dually by (1) acting as a molecular brake on contraction and (2) maintaining the structural integrity of the contractile machinery. Most importantly, CaD is essential for resolution of the physiological umbilical hernia and ventral body wall closure.

Download Full-text