Tonic and Phasic Smooth Muscle Contraction Is Not Regulated by the PKCα - CPI-17 Pathway in Swine Stomach Antrum and Fundus

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.

Smooth Muscle‐Protein Translocation and Tissue Function

“…Based on this large body of data showing spatial‐temporal redistribution of PKC from the cytosol to the plasma membrane occurs with activation in isolated SMCs, investigators infer that this also occurs and is a (the) mechanism whereby PKC‐CPI‐17 regulates MLCP activity in intact SM tissues (i.e., PKC is a cytosolic protein that, upon tissue activation, translocates to the plasmalemma where it is activated and then translocates back to the cytosol where it activates CPI‐17 which decreases MLCP activity and thus affects MLC 20 phosphorylation levels). In contrast to this, we have reported that PKCα has a preferential peripheral localization at the plasma membrane in unstimulated SMCs in tissues and this does not change with agonist activation (Zhang et al, ) and (Fig. ).…”

Protein Kinase C as Regulator of Vascular Smooth Muscle Function and Potential Target in Vascular Disorders

“…However, PKC may be localized in the cell periphery in both resting and stimulated tissues. Immunohistochemical studies have shown that the distribution of PKCα in the longitudinal and circular layers of the swine stomach tonic fundus and phasic antrum under resting conditions does not differ, being predominantly localized near the smooth muscle plasma membrane, and stimulation of either tissue with PDBu or carbachol does not alter this peripheral PKCα distribution (Zhang et al, 2013). Also, PKC is found in other cell compartments like the mitochondria and soluble fraction of cells subjected to oxidative stress, a known activator of PKC (Konishi et al, 2001; Steinberg, 2015).…”

Diversity and plasticity in signaling pathways that regulate smooth muscle responsiveness: Paradigms and paradoxes for the myosin phosphatase, the master regulator of smooth muscle contraction

“…The functional pleiotropy of CPI-17 may explain why CPI-17 signaling is silenced in the larger arteries, smooth muscle from genetically hypertensive rats and from other organs and models (90, 109,110,111,112). The multi-functionality may be a common feature of the endogenous regulator proteins for kinases/phosphatases, such as DARPP32, phosphatase inhibitor-1, inhibitor-2, a Raf kinase inhibitor protein (RKIP), and cyclin-dependent kinase inhibitors (p21CIP/WAF, p27KIP1, and p57) (113,114,115,116,117,118,119,120,121,122,123).…”