Role of PKC and RhoA/ROCK pathways in the spontaneous phasic activity in the rectal smooth muscle

Singh, Jagmohan; Rattan, Satish

doi:10.1152/ajpgi.00473.2012

Cited by 16 publications

(21 citation statements)

References 43 publications

(67 reference statements)

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“…Tonic or phasic characteristics of SM have been associated with the variation in the contractile proteins contents (6,9,19,24,26,29,40). Previously we have reported higher expression of RhoA/Rho kinase (ROCK) in tonic IAS vs. phasic RSM SM and smooth muscle cells (SMC) (25,26,32,38).Preliminary proteomics experiments (data not presented here) revealed transgelin (SM22) to be consistently downregulated in the IAS vs. RSM SMC. In these exploratory studies, we used two-dimensional differential in gel electrophoresis (2D-DIGE), an advance discovery proteomics technique (1, 10, 11), coupled with ProteomeX Electrospray Ion-Trap (ThermoElectron) MS based on Peptide Mass Fingerprinting techniques (16).…”

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confidence: 73%

“…Adventitious structures connected to the IAS and the RSM were removed carefully by sharp dissection. After this, the mucosa was removed using sharp dissection, and circular SM strips (ϳ1 ϫ 7 mm) of the IAS and RSM were prepared as explained previously (28,38). The experimental protocols were approved by the institutional animal care and use committee of Thomas Jefferson University in accordance with the recommendations of the American Association for the Accreditation of Laboratory Animal Care.…”

Section: Methodsmentioning

confidence: 99%

“…Each SM strip was initially stretched to a tension of 0.7 g. The muscle strips were then given at least 1 h to equilibrate during which they were washed with KPS every 20 min. For IAS only the SM strips that developed spontaneous tone and responded by relaxation to electrical field stimulation were used for the study (31,38). The RSM was characterized by the presence of a low-grade tone with the superimposed phasic contractions.…”

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

“…IAS and RSM representing tonic and phasic SM, respectively (38), are located adjacently, and yet have different physiological properties. Therefore, these SM offer a unique opportunity to examine the differences between tonic and phasic SM (29).…”

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Role of SM22 in the differential regulation of phasic vs. tonic smooth muscle

Rattan

Ali

2015

American Journal of Physiology-Gastrointestinal and Liver Physi

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Preliminary proteomics studies between tonic vs. phasic smooth muscles identified three distinct protein spots identified to be those of transgelin (SM22). The latter was found to be distinctly downregulated in the internal anal sphincter (IAS) vs. rectal smooth muscle (RSM) SMC. The major focus of the present studies was to examine the differential molecular control mechanisms by SM22 in the functionality of truly tonic smooth muscle of the IAS vs. the adjoining phasic smooth muscle of the RSM. We monitored SMC lengths before and after incubation with pFLAG-SM22 (for SM22 overexpression), and SM22 small-interfering RNA. pFLAG-SM22 caused concentration-dependent and significantly greater relaxation in the IAS vs. the RSM SMCs. Conversely, temporary silencing of SM22 caused contraction in both types of the SMCs. Further studies revealed a significant reverse relationship between the levels of SM22 phosphorylation and the amount of SM22-actin binding in the IAS and RSM SMC. Data showed higher phospho-SM22 levels and decreased SM22-actin binding in the IAS, and reverse to be the case in the RSM SMCs. Experiments determining the mechanism for SM22 phosphorylation in these smooth muscles revealed that Y-27632 (Rho kinase inhibitor) but not Gö-6850 (protein kinase C inhibitor) caused concentration-dependent decreased phosphorylation of SM22. We speculate that SM22 plays an important role in the regulation of basal tone via Rho kinase-induced phosphorylation of SM22.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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Role of SM22 in the differential regulation of phasic vs. tonic smooth muscle

Rattan

Ali

2015

American Journal of Physiology-Gastrointestinal and Liver Physi

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PKCα-Mediated Signals Regulate the Motile Responses of Cochlear Outer Hair Cells

Park

Kalinec

2015

Biophysical Journal

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There is strong evidence that changes in the actin/spectrin-based cortical cytoskeleton of outer hair cells (OHCs) regulate their motile responses as well as cochlear amplification, the process that optimizes the sensitivity and frequency selectivity of the mammalian inner ear. Since a RhoA/protein kinase C (PKC)-mediated pathway is known to inhibit the actin-spectrin interaction in other cell models, we decided to investigate whether this signaling cascade could also participate in the regulation of OHC motility. We used high-speed video microscopy and confocal microscopy to explore the effects of pharmacological activation of PKCα, PKCβI, PKCβII, PKCδ, PKCε, and PKCζ with lysophosphatidic acid (LPA) and their inhibition with bisindolylmaleimide I, as well as inhibition of RhoA and Rho-associated protein kinase (ROCK) with C3 and Y-27632, respectively. Motile responses were induced in isolated guinea pig OHCs by stimulation with an 8 V/cm external alternating electrical field as 50 Hz bursts of square wave pulses (100 ms on/off). We found that LPA increased expression of PKCα and PKCζ only, with PKCα, but not PKCζ, phosphorylating the cytoskeletal protein adducin of both Ser-726 and Thr-445. Interestingly, however, inhibition of PKCα reduced adducin phosphorylation only at Ser-726. We also determined that LPA activation of a PKCα-mediated signaling pathway simultaneously enhanced OHC electromotile amplitude and cell shortening, and facilitated RhoA/ROCK/LIMK1-mediated cofilin phosphorylation. Altogether, our results suggest that PKCα-mediated signals, probably via adducin-mediated inhibition of actin-spectrin binding and cofilin-mediated depolymerization of actin filaments, play an essential role in the homeostatic regulation of OHC motility and cochlear amplification.

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