Proteomic Analysis of Bladder Smooth Muscle Cell Response to Cyclic Hydrostatic Pressure

Abstract

We and other laboratories previously demonstrated that multiple signaling pathways are at work in SMC responses to applied mechanical stimuli, indicating the need for a high-throughput approach to study the complex mechanotransduction mechanism. In the present study, the feasibility of proteomic techniques to examine the mechanotransduction of bladder smooth muscle cells (SMC) was tested for the first time. Specifically, two-dimensional (2D) gel electrophoresis and image analyses were employed to separate and identify protein profiles in control and in cells exposed to cyclic pressure (0.1 Hz, 40 cmH 2 O) for 1 h. The results provide evidence that 20 (out of 155) spots exhibited changes (up-or down-regulation) in intensity between control and cyclic pressure groups. The mass spectrometry analysis provided sequences for 118 tryptic peptides from these 20 spots, and peptide mapping provided a positive match of peptide sequences to 24 distinct proteins. Moreover, the results of Western analysis independently confirmed up-regulation of nuclear matrix protein, lamin A/C in SMC exposed to cyclic pressure. Since the protein samples were predominantly of the cytosolic fraction, we concluded that the observed up-regulation was due to the translocation of either membrane-bound or nuclear localized proteins into the cytosol. Based on these observations, the present study demonstrated that the proteomic approach can be a powerful analytical tool for mechanobiology research that allows analysis of a large number of variables, thereby supporting development of new hypotheses to guide future investigation.

Cyclic Stretch Promotes Proliferation and Contraction of Human Bladder Smooth Muscle Cells by Cajal-Mediated c-kit Expression in Interstitial Cells

“…Some researchers have reported that mechanical stimulation affects smooth muscle cells [19–24,28–30]. Nagatomi et al [30] reported that when rat BSMCs are exposed to cyclic pressure (0.1 Hz, 40 cmH 2 O) for 1 h, multiple signaling pathways respond to mechanical stimuli.…”

“…Some researchers have reported that mechanical stimulation affects smooth muscle cells [19–24,28–30]. Nagatomi et al [30] reported that when rat BSMCs are exposed to cyclic pressure (0.1 Hz, 40 cmH 2 O) for 1 h, multiple signaling pathways respond to mechanical stimuli. Lee et al [31] also reported that the expression of prolyl 4-hydroxylase is significantly increased when BSMCs are exposed to pressures of 40 cmH 2 O or less for up to 72 h. In other words, cyclic pressure represents mechanical pressure, and mechanical stretch stimulates the proliferation of BSMCs.…”

Elucidating the Process of Activation of Methyl-Coenzyme M Reductase

“…For protein identification, in-gel tryptic digestion was performed according to the literature (25). Digested peptides were analyzed using an Acquity ultraperformance liquid chromatography (UPLC) system coupled to a quadrupole-time of flight mass spectrometer (Q-Tof premier; Waters) using electrospray ionization (ESI) in the positive-ion mode operated by Masslynx software (V4.1).…”

Effects of Different Sustained Hydrostatic Pressures on Connexin 43 in Human Bladder Smooth Muscle Cells

“…There have a few reports on effects of mechanical stimulation on smooth muscle cells. Nagatomi et al [14] reported rat BSMCs exposed to cyclic pressure (0.1 Hz, 40 cm H 2 O) for 1 h, multiple signaling pathways are responses to mechanical stimuli. Lee et al [15] reported that hBSMCs were exposed to pressures of 40 cm H 2 O or less for up to 72 h, and the expression of prolyl 4-hydroxylase was significantly increased.…”