Abstract
Regulation of myosin light chain phosphatase (MLCP) via protein kinase C (PKC) and the 17 kDa PKC-potentiated inhibitor of myosin light chain phosphatase (CPI-17) has been reported as a Ca2+ sensitization signaling pathway in smooth muscle (SM), and thus may be involved in tonic vs. phasic contractions. This study examined the protein expression and spatial-temporal distribution of PKCα and CPI-17 in intact SM tissues. KCl or carbachol (CCh) stimulation of tonic stomach fundus SM generates a sustained contraction while the phasic stomach antrum generates a transient contraction. In addition, the tonic fundus generates greater relative force than phasic antrum with 1 µM phorbol 12, 13-dibutyrate (PDBu) stimulation which is reported to activate the PKCα – CPI-17 pathway. Western blot analyses demonstrated that this contractile difference was not caused by a difference in the protein expression of PKCα or CPI-17 between these two tissues. Immunohistochemical results show that the distribution of PKCα in the longitudinal and circular layers of the fundus and antrum do not differ, being predominantly localized near the SM cell plasma membrane. Stimulation of either tissue with 1 µM PDBu or 1 µM CCh does not alter this peripheral PKCα distribution. There are no differences between these two tissues for the CPI-17 distribution, but unlike the PKCα distribution, CPI-17 appears to be diffusely distributed throughout the cytoplasm under relaxed tissue conditions but shifts to a primarily peripheral distribution at the plasma membrane with stimulation of the tissues with 1 µM PDBu or 1 µM CCh. Results from double labeling show that neither PKCα nor CPI-17 co-localize at the adherens junction (vinculin/talin) at the membrane but they do co-localize with each other and with caveoli (caveolin) at the membrane. This lack of difference suggests that the PKCα - CPI-17 pathway is not responsible for the tonic vs. phasic contractions observed in stomach fundus and antrum.
Highlights
The mechanism(s) determining tonic vs. phasic smooth muscle (SM) contractions remains unresolved
The difference between the sustained and transient responses of these two tissues to KPSS and CCh, and the presence or absence of a contractile response to PDBu stimulation could be due to the expression and/or spatial-temporal distribution of the downstream second messengers (PKCa and CPI-17) that are purported to be responsible for myosin light chain phosphatase (MLCP) inhibition with
One of the major pathways proposed to be involved in Ca2+ sensitization is via G-protein coupled receptors (GPCR)/ phospholipase C (PLC)/diacylglycerol (DAG)/protein kinase C (PKC)/CPI-17 to inhibit MLCP
Summary
The mechanism(s) determining tonic (sustained) vs. phasic (transient) smooth muscle (SM) contractions remains unresolved. There are multiple proposed hypotheses to explain tonic force maintenance in SM including: slowly- or non-cycling smooth muscle myosin cross-bridges called latch bridges [1,2,3,4]; recruitment of non-muscle myosin II [5,6,7]; formation of caldesmon or calponin dependent actin-to-myosin cross-links [8,9]; formation of cytoskeletal force-bearing structures [10,11,12]; and regulation of myosin LC20 phosphorylation via second messenger pathways affecting myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activity [13,14,15,16,17,18] This last category is of interest because of the reported differential expression, localization and regulation of these second messenger proteins in various SM tissues. Stomach fundus SM generates a tonic (sustained) contraction while stomach antrum SM generates a phasic (transient) contraction in response to a variety of stimuli
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