Deletion of SM-B, the high ATPase isoform of myosin, upregulates the PKC-mediated signal transduction pathway in murine urinary bladder smooth muscle

Abstract

Hypolite JA, Chang S, LaBelle E, Babu GJ, Periasamy M, Wein AJ, Chacko S. Deletion of SM-B, the high ATPase isoform of myosin, upregulates the PKC-mediated signal transduction pathway in murine urinary bladder smooth muscle. Am J Physiol Renal Physiol 296: F658 -F665, 2009. First published December 3, 2008 doi:10.1152/ajprenal.90221.2008.-Detrusor smooth muscle (DSM) hypertrophy induced by partial bladder outlet obstruction (PBOO) is associated with changes in the NH 2-terminal myosin heavy chain isoform from predominantly SM-B to SM-A, alteration in the Ca 2ϩ sensitization pathway, and the contractile characteristics from phasic to tonic in rabbits. We utilized the SM-B knockout (KO) mouse to determine whether a shift from SM-B to SM-A without PBOO is associated with changes in the signal transduction pathway mediated via PKC and CPI-17, which keeps the myosin phosphorylation (MLC 20) level high by inhibiting the myosin phosphatase. DSM strips from SM-B KO mice generated more force in response to electrical field stimulation, KCl, carbachol, and phorbol 12,13-dibutyrate than that of age-matched wild-type mice. There was no difference in the ED 50 for carbachol but the maximum response was greater for the SM-B KO mice. DSM from SM-B KO mice revealed increased mass and hypertrophy. The KO mice also showed an overexpression of PKC-␣, increased levels of phospho-CPI-17, and an elevated level of IP 3 and DAG upon stimulation with carbachol. Two-dimensional gel electrophoresis revealed an increased level of MLC 20 phosphorylation in response to carbachol. Together, these changes may be responsible for the higher level of force generation and maintenance by the DSM from the SM-B KO bladders. In conclusion, our data show that ablation of SM-B is associated with alteration of PKC-mediated signal transduction and CPI-17-mediated Ca 2ϩ sensitization pathway that regulate smooth muscle contraction. Interestingly, similar changes are also present in PBOO-induced DSM compensatory response in the rabbit model in which SM-B is downregulated. smooth muscle contraction; alternative splicing; CPI-17; light chain phosphorylation MYOSIN II IS THE MAJOR COMPONENT of the thick filament in smooth muscles from all sources. Smooth muscle myosin II is regulated by alternative splicing at both 3Ј and 5Ј end of the myosin heavy chain (MHC) pre-mRNA producing COOH-terminal (SM1 and SM2) and NH 2 -terminal(SM-A and SM-B) MHC isoforms, respectively. Alternative splicing at the 5Ј end inserts a 21-nt insertion that encodes a seven amino acid sequence in the NH 2 -terminal head region of the myosin near the ATP-binding site (3,26). Studies have shown that smooth muscle myosin containing the SM-B isoform (with the 7-amino acid insert) have a higher actin-activated ATPase activity and a higher shortening velocity compared with those smooth muscle which predominantly consist of SM-A, lacking the seven amino acid insert (14,26,29). Studies have also shown that smooth muscles containing primarily this high-velocity isoform (SM-B), such as the urin...

SMB myosin heavy chain knockout enhances tonic contraction and reduces the rate of force generation in ileum and stomach antrum

“…5). PKC␣ downregulation in SMB (Ϫ/Ϫ) antrum is inconsistent with the study from Hypolite et al (27) reporting PKC␣ protein expression is upregulated in detrusor smooth muscle. The difference may be tissue specific regulation of PKC␣ in SMB (Ϫ/Ϫ) mice.…”

“…Results from Patzak et al (44) using the same animal model report that there is no correlation between SMB MHC protein expression and the rate of force generation in perfused afferent and efferent renal arterioles. Hypolite et al (27) reported that compared with the wild type the loss of SMB MHC protein in detrusor smooth muscle increases contraction force due to the upregulation of PKC-mediated signaling pathway. In spite of the studies on smooth muscle tissue function using this SMB MHC knockout mouse model, it remains unclear if and how SMA/B MHC protein expression relates to phasic and tonic muscle mechanics.…”

Spontaneous and evoked contractions are regulated by PKC-mediated signaling in detrusor smooth muscle: involvement of BK channels

“…Furthermore, PBOO in rats (13), rabbits, and patients with BPH are all associated with downregulation of BK channels (5) and increased DO, characterized by an increase in spontaneous and NVC. These studies lend further credence to the idea that BK channels play a vital role in regulating DSM excitability and thus may influence the development of spontaneous and NVC contractions under pathological conditions.PKC is an 80-kDa protein that is also involved in the regulation of smooth muscle tone and force maintenance (11,19,26). Recent evidence suggests that PKC can regulate the sensitivity of BK channels for calcium and other substrates in smooth muscle and nonmuscle cells (3,28).…”

“…PKC is an 80-kDa protein that is also involved in the regulation of smooth muscle tone and force maintenance (11,19,26). Recent evidence suggests that PKC can regulate the sensitivity of BK channels for calcium and other substrates in smooth muscle and nonmuscle cells (3,28).…”

GATA-6 and NF-κB Activate CPI-17 Gene Transcription and Regulate Ca2+ Sensitization of Smooth Muscle Contraction

“…Longitudinal muscle strips (2 by 5 mm) devoid of mucosa and serosa were obtained from wild-type (WT) and NF-B c-Rel KO mice and suspended in 7 ml of Tyrode's buffer (124.9 mM NaCl, 2.5 mM KCl, 23.8 mM NaHCO 3 , 0.5 mM MgCl 2 , 0.4 mM NaH 2 PO 4 , 1.8 mM CaCl 2 , 5.5 mM dextrose) in an organ bath (Radnoti) to equilibrate for 1 h in 95% O 2 and 5% CO 2 . The force generated in response to KCl (125 mM), electrical field stimulation (80 V, 32 Hz for 1 ms), phorbol dibutyrate (PDBu; 1 M), and carbachol (10 M) was measured using the AD Instruments PowerLab computerized system (AD Instruments) as described previously (33,34). The force measurements were normalized to the wet weight of the muscle strips.…”