Expression of myosin isoforms in smooth muscle cells  in the corpus cavernosum penis

Corpus cavernosum smooth muscle (CCSM) in the penis is unique in that it exhibits a high resting tone and, on stimulation, the muscle cells relax, allowing cavernous spaces to fill with blood, which results in an erection (tumescence). During detumescence, the muscle cells contract and return to the state of high resting tone. This study was undertaken to determine whether CCSM with these unique properties contains myosin isoforms typical of aorta or bladder smooth muscles, muscles that exhibit tonic and phasic characteristics, respectively. RT-PCR revealed that normal CCSM contains an SM2/SM1 mRNA ratio of 1.2:1 (similar to the rabbit aorta). Approximately 31% of the myosin heavy chain transcripts possess a 21-nt insert (predominant in bladder smooth muscle but not expressed in aorta) that encodes the seven-amino acid insert near the NH2-terminal ATP binding region in the head portion of the myosin molecule found in SMB, with the remaining mRNA being noninserted (SMA). Quantitative competitive RT-PCR revealed that the CCSM possesses ∼4.5-fold less SMB than the bladder smooth muscle. Western blot analysis using an antibody specific for the seven-amino acid insert reveals that both SM1 and SM2 in the CCSM contain the seven-amino acid insert. Furthermore, SMB containing the seven-amino acid insert was localized in the CCSM by immunofluorescence microscopy using this highly specific antibody. The analysis of the expression of LC17isoforms a and b in the CCSM revealed that it is similar to that of bladder smooth muscle. Thus the CCSM possesses an overall myosin isoform composition intermediate between aorta and bladder smooth muscles, which generally express tonic- and phasiclike characteristics, respectively. Two-dimensional gel electrophoresis showed a relatively low level (∼10%) of Ca2+-dependent light-chain (LC20) phosphorylation at the basal tone, which reaches ∼23% in response to maximal stimulation. The presence of noninserted and inserted myosin isoforms with low and high levels of actin-activated ATPase activities, respectively, in the CCSM may contribute to the ability of the CCSM to remain in a state of high resting tone and to relax rapidly for normal penile function.

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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.

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LPP, a LIM protein highly expressed in smooth muscle

AJP Cell Physiology ◽

10.1152/ajpcell.00608.2002 ◽

2003 ◽

Vol 285 (3) ◽

pp. C674-C685 ◽

Cited By ~ 31

Author(s):

Isabelle Gorenne ◽

Robert K. Nakamoto ◽

Clayton P. Phelps ◽

Mary C. Beckerle ◽

Avril V. Somlyo ◽

...

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Smooth Muscles ◽

Rho Kinase ◽

Focal Adhesions ◽

Muscle Cells ◽

Nuclear Accumulation ◽

Cell Periphery ◽

Bladder Smooth Muscle ◽

Western Blots

An 80-kDa protein, prominently expressed in smooth muscle, was microsequenced and identified as LPP, the product of the lipoma-preferred partner gene (Petit MMR, Mols R, Schoenmakers EFPM, Mandahl N, and Van de Ven WJM. Genomics 36: 118–129, 1996). Using a specific anti-LPP antibody, we showed, in Western blots and with immunofluorescence microscopy, the selective expression of LPP in vascular and visceral smooth muscles (∼0.5–1 ng/μg total protein). In other mature (noncultured) tissues, including heart and skeletal muscle, the protein is present only in trace amounts and is closely correlated with the levels of the smooth muscle marker α-actin. In freshly isolated guinea pig bladder smooth muscle cells, immunofluorescence images showed LPP as linear arrays of punctate, longitudinally oriented staining superimposed with vinculin staining on the plasma membrane surface. A corresponding pattern of periodic labeling at the membrane in transverse sections of bladder smooth muscle suggested an association of LPP with peripheral dense bodies. In cultured rat aortic smooth muscle cells, LPP colocalized with vinculin at focal adhesions but not with p120 catenin or α-actinin. Overexpression of the protein increased EGF-stimulated migration of vascular smooth muscle cells in Transwell assays, suggesting the participation of LPP in cell motility. The Rho-kinase inhibitor Y-27632 dissociated focal adhesions and LPP staining at the cell periphery and enhanced the nuclear accumulation of LPP induced by leptomycin B, indicating that LPP has a potential for relocating to the nucleus through a shuttling mechanism that is sensitive to inhibition of Rho-kinase.

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1708: Transfection of Bladder Smooth Muscle Cells with Telokin Alters their Smooth Muscle Phenotype

The Journal of Urology ◽

10.1016/s0022-5347(18)38900-6 ◽

2004 ◽

Vol 171 (4S) ◽

pp. 452-452

Author(s):

Grace Hyun ◽

Shaohua Chang ◽

Stephen A. Zderic ◽

Alan J. Wein ◽

Samuel Chacko ◽

...

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Muscle Cells ◽

Bladder Smooth Muscle

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β‐Adrenoceptor signaling regulates proliferation and contraction of human bladder smooth muscle cells under pathological hydrostatic pressure

Journal of Cellular Biochemistry ◽

10.1002/jcb.29056 ◽

2019 ◽

Vol 120 (10) ◽

pp. 17872-17886 ◽

Cited By ~ 2

Author(s):

Junyu Lai ◽

Jianzhong Ai ◽

Deyi Luo ◽

Tao Jin ◽

Banghua Liao ◽

...

Keyword(s):

Smooth Muscle ◽

Hydrostatic Pressure ◽

Smooth Muscle Cells ◽

Muscle Cells ◽

Bladder Smooth Muscle ◽

Human Bladder

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Plasticity of KIR channels in human smooth muscle cells from internal thoracic artery

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00559.2002 ◽

2003 ◽

Vol 284 (6) ◽

pp. H2325-H2334 ◽

Cited By ~ 25

Author(s):

Tom Karkanis ◽

Shaohua Li ◽

J. Geoffrey Pickering ◽

Stephen M. Sims

Keyword(s):

Smooth Muscle ◽

Current Density ◽

Internal Thoracic Artery ◽

Smooth Muscles ◽

Vascular Smooth Muscles ◽

Rt Pcr ◽

Regulation Of Proliferation ◽

Negative Membrane Potential ◽

Inwardly Rectifying ◽

Contractile Cells

Inwardly rectifying K+ (KIR) currents are present in some, but not all, vascular smooth muscles. We used patch-clamp methods to examine plasticity of this current by comparing contractile and proliferative phenotypes of a clonal human vascular smooth muscle cell line. Hyperpolarization of cells under voltage clamp elicited a large inward current that was selective for K+ and blocked by Ba2+. Current density was greater in proliferative compared with contractile cells (−4.5 ± 0.9 and −1.4 ± 0.3 pA/pF, respectively; P < 0.001). RT-PCR of mRNA from proliferative cells identified transcripts for Kir2.1 and Kir2.2 but not Kir2.3 potassium channels. Western blot analysis demonstrated greater expression of Kir2.1 protein in proliferative cells, consistent with the higher current density. Proliferative cells displayed a more negative membrane potential than contractile cells (−71 ± 2 and −35 ± 4 mV, respectively; P < 0.001). Ba2+ depolarized all cells, whereas small increases in extracellular K+ concentration elicited hyperpolarization only in contractile cells. Ba2+ inhibited [3H]thymidine incorporation, indicating a possible role for KIR channels in the regulation of proliferation. The phenotype-dependent plasticity of KIR channels may have relevance to vascular remodeling.

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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.

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