505: Effect of Partial Outlet Obstruction on the Expression of Smooth Muscle Myosin Isoforms in Rat Ureter

The alternatively spliced SM1 and SM2 smooth muscle myosin heavy chains differ at their respective carboxyl termini by 43 versus 9 unique amino acids. To determine whether these tailpieces affect filament assembly, SM1 and SM2 myosins, the rod region of these myosin isoforms, and a rod with no tailpiece (tailless), were expressed in Sf 9 cells. Paracrystals formed from SM1 and SM2 rod fragments showed different modes of molecular packing, indicating that the tailpieces can influence filament structure. The SM2 rod was less able to assemble into stable filaments than either SM1 or the tailless rods. Expressed full-length SM1 and SM2 myosins showed solubility differences comparable to the rods, establishing the validity of the latter as a model for filament assembly. Formation of homodimers of SM1 and SM2 rods was favored over the heterodimer in cells coinfected with both viruses, compared with mixtures of the two heavy chains renatured in vitro. These results demonstrate for the first time that the smooth muscle myosin tailpieces differentially affect filament assembly, and suggest that homogeneous thick filaments containing SM1 or SM2 myosin could serve distinct functions within smooth muscle cells.

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Increased corpus cavernosum smooth muscle tone associated with partial bladder outlet obstruction is mediated via Rho-kinase

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00717.2003 ◽

2005 ◽

Vol 289 (4) ◽

pp. R1124-R1130 ◽

Cited By ~ 33

Author(s):

Shaohua Chang ◽

Joseph A. Hypolite ◽

Stephen A. Zderic ◽

Alan J. Wein ◽

Samuel Chacko ◽

...

Keyword(s):

Smooth Muscle ◽

Bladder Outlet Obstruction ◽

Rabbit Model ◽

Corpus Cavernosum ◽

Rho Kinase ◽

Outlet Obstruction ◽

Smooth Muscle Myosin ◽

Partial Bladder Outlet Obstruction ◽

Kinase Expression ◽

Muscle Myosin

Numerous studies have now demonstrated that lower urinary tract symptoms (LUTS) are associated with erectile dysfunction (ED) in men independent of age or general health. Because one-third of men over the age of 50 will develop LUTS and a recent study showed ED in 62% of patients presenting for LUTS, the importance of determining the mechanistic link between these two pathologies is clear. Using a rabbit model of partial bladder outlet obstruction (PBOO), a primary cause of LUTS, we have identified an increased basal corpus cavernosum smooth muscle (CCSM) tone associated with an elevated level of smooth muscle myosin (SMM) phosphorylation in PBOO compared with sham-operated control rabbits (sham). Results from in vitro kinase and phosphatase assays using purified smooth muscle myosin showed increased kinase and decreased phosphatase activities in cellular extracts from corpora cavernosa isolated from PBOO compared with sham rabbits. Increased Rho-kinase expression in the CCSM of PBOO rabbits was suggested by the observations that Rho-kinase inhibitors attenuated the increased kinase activity and were less effective in relaxing CCSM strips from PBOO vs. sham rabbits. This hypothesis was then confirmed by RT-PCR and Western blotting, which demonstrated increased expression of both isoforms of Rho-kinase (ROKα and ROKβ). Increased SMM basal phosphorylation (necessary for SM contraction) in the CCSM of PBOO rabbits, mediated via an increase in Rho-kinase expression/activity, would be expected to make the CCSM more difficult to relax (necessary for erection), which suggests that the RhoA/Rho-kinase pathway as being involved in the mechanism for LUTS-associated ED.

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Human Tonic and Phasic Smooth Muscle Myosin Isoforms Are Unresponsive to the Loop 1 Insert

ISRN Structural Biology ◽

10.1155/2013/634341 ◽

2013 ◽

Vol 2013 ◽

pp. 1-6 ◽

Cited By ~ 1

Author(s):

Katalin Ajtai ◽

Azad Mayanglambam ◽

Yihua Wang ◽

Thomas P. Burghardt

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Myosin ◽

Myosin Isoforms ◽

Muscle Myosin ◽

Phasic Smooth Muscle

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Smooth muscle myosin heavy chains combine to form three native myosin isoforms

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1993.264.5.h1653 ◽

1993 ◽

Vol 264 (5) ◽

pp. H1653-H1662 ◽

Cited By ~ 1

Author(s):

A. E. Tsao ◽

T. J. Eddinger

Keyword(s):

Smooth Muscle ◽

Sodium Dodecyl ◽

Coiled Coil ◽

Smooth Muscle Myosin ◽

Polyacrylamide Gel ◽

Myosin Isoforms ◽

Myosin Heavy Chains ◽

Polyacrylamide Gels ◽

Heavy Chains ◽

Muscle Myosin

Two smooth muscle myosin heavy chains (MHC; SM1 and SM2) of approximately 204 and 200 kDa exist in smooth muscle cells and can be visualized on reducing sodium dodecyl sulfate (SDS)-polyacrylamide gels. Chymotryptic digestion of the native myosin molecule results in two fragments: heavy meromyosin (HMM) and light meromyosin (LMM). LMM is the alpha-helical coiled-coil carboxy terminal half of the molecule containing the difference peptide between SM1 and SM2. Electrophoresis of the LMM fragments on a reducing SDS-polyacrylamide gel resolves two subunits from the two MHC [LM1 from SM1 (approximately 100 kDa) and LM2 from SM2 (approximately 95 kDa), where LM1 and LM2 are LMM from SM1 and SM2, respectively]. CuCl2 oxidation of the LMM fragment forms intramolecular disulfide bonds between adjacent cysteines on the two LMM fragments. When the native LMM is oxidized with CuCl2 and run on a nonreducing SDS-polyacrylamide gel, three bands are observed, which migrate at approximately 195, 190, and 185 kDa (bands 1, 2, and 3). Excision of these bands and electrophoresis on a reducing SDS-polyacrylamide gel show their subunit composition. Band 1 is composed solely of LM1. Band 2 is composed of an equal ratio of LM1 and LM2, and band 3 is composed solely of LM2. Using a variety of biochemical procedures, along with nonreducing SDS-polyacrylamide gels, we interpret these results to indicate that there are three smooth muscle myosin isoforms that result from the various combinations of the two smooth muscle MHC (SM1 homodimer, SM1-SM2 heterodimer, and SM2 homodimer).

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