Effect of phorbol 12,13-dibutyrate on smooth muscle tone in rat stomach fundus

Matsumoto, Takayuki; Nishiyama, Misaki; Kobayashi, Tsuneo; Kasuya, Yutaka; Kamata, Katsuo

doi:10.1540/jsmr.41.107

Cited by 6 publications

(3 citation statements)

References 33 publications

(39 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Likewise, the effects of the PKC activator PDBu resemble those of intact human IAS here (Fig. 3, A-C), showing a biphasic effect (contraction in the lower concentrations and relaxation in the higher), and other tonic gastrointestinal smooth muscle preparations (8,19,20) depending on the level of the basal tone. However, these data are different from the earlier studies in human IAS reconstructs showing a frank increase in the tone (38).…”

Section: Ias Reconstructsupporting

confidence: 58%

Bioengineered human IAS reconstructs with functional and molecular properties similar to intact IAS

Singh

Rattan

2012

American Journal of Physiology-Gastrointestinal and Liver Physi

View full text Add to dashboard Cite

Because of its critical importance in rectoanal incontinence, we determined the feasibility to reconstruct internal anal sphincter (IAS) from human IAS smooth muscle cells (SMCs) with functional and molecular attributes similar to the intact sphincter. The reconstructs were developed using SMCs from the circular smooth muscle layer of the human IAS, grown in smooth muscle differentiation media under sterile conditions in Sylgard-coated tissue culture plates with central Sylgard posts. The basal tone in the reconstructs and its changes were recorded following 0 Ca2+, KCl, bethanechol, isoproterenol, protein kinase C (PKC) activator phorbol 12,13-dibutyrate, and Rho kinase (ROCK) and PKC inhibitors Y-27632 and Gö-6850, respectively. Western blot (WB), immunofluorescence (IF), and immunocytochemical (IC) analyses were also performed. The reconstructs developed spontaneous tone (0.68 ± 0.26 mN). Bethanechol (a muscarinic agonist) and K+depolarization produced contraction, whereas isoproterenol (β-adrenoceptor agonist) and Y-27632 produced a concentration-dependent decrease in the tone. Maximal decrease in basal tone with Y-27632 and Gö-6850 (each 10−5M) was 80.45 ± 3.29 and 17.76 ± 3.50%, respectively. WB data with the IAS constructs′ SMCs revealed higher levels of RhoA/ROCK, protein kinase C-potentiated inhibitor or inhibitory phosphoprotein for myosin phosphatase (CPI-17), phospho-CPI-17, MYPT1, and 20-kDa myosin light chain vs. rectal smooth muscle. WB, IF, and IC studies of original SMCs and redispersed from the reconstructs for the relative distribution of different signal transduction proteins confirmed the feasibility of reconstruction of IAS with functional properties similar to intact IAS and demonstrated the development of myogenic tone with critical dependence on RhoA/ROCK. We conclude that it is feasible to bioengineer IAS constructs using human IAS SMCs that behave like intact IAS.

show abstract

Section: Ias Reconstructsupporting

confidence: 58%

Bioengineered human IAS reconstructs with functional and molecular properties similar to intact IAS

Singh

Rattan

2012

American Journal of Physiology-Gastrointestinal and Liver Physi

View full text Add to dashboard Cite

show abstract

“…To probe the enhanced force seen in the SM from SM-B null bladders, we used the synthetic PKC activator PDBu to stimulate the smooth muscle in our physiological studies. PDBu is a well-known activator of PKC signaling and contraction in a variety of smooth muscles (1,12,33,53). Our results indicate a remarkable difference with bladders from SM-B null, recording substantially higher force levels than WT bladders when corrected for cross-sectional area.…”

Section: Discussionmentioning

confidence: 99%

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

Hypolite¹,

Chang²,

LaBelle³

et al. 2009

American Journal of Physiology-Renal Physiology

View full text Add to dashboard Cite

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...

show abstract

“…(6;7) Protein kinase C (PKC) is another important regulatory protein in smooth muscle contractility. (8-10) PDBu induces rabbit DSM contraction by activating PKC. (11;12) The activated PKC can phosphorylate a 17kDa protein called PKC potentiated inhibitor protein-17 kDa (CPI-17).…”

Section: Introductionmentioning

confidence: 99%

Alteration of the PKC-mediated signaling pathway for smooth muscle contraction in obstruction-induced hypertrophy of the urinary bladder

Chang

Hypolite

Mohanan

et al. 2009

Laboratory Investigation

View full text Add to dashboard Cite

Normal urinary bladder function requires contraction and relaxation of the detrusor smooth muscle (DSM). The DSM undergoes compensatory hypertrophy in response to partial bladder outlet obstruction (PBOO) in both men and animal models. Following bladder hypertrophy, the bladder either retains its normal function (compensated) or becomes dysfunctional (decompensated) with increased voiding frequency and decreased void volume. We analyzed the contractile characteristics of DSM in a rabbit model of PBOO. The protein kinase C (PKC) agonist Phorbol 12, 13-dibutyrate (PDBu) elicited similar levels of contraction of DSM strips from normal or compensated bladders. However, PDBu-induced contraction decreased significantly in DSM strips from decompensated bladders. The expression and activity of PKC α were also lowest in decompensated bladders. The PKC specific inhibitor bisindolylmaleimide-1 (Bis) blocked PDBu-induced contraction and PKC activity in all three groups. Moreover, the phosphorylation of the phosphoprotein inhibitor CPI-17 was diminished in DSM from the decompensated bladder, which would result in less inhibitory potency of CPI-17 on myosin light chain phosphatase activity and contribute to less contractility. Immunostaining revealed the co-localization of PKC and phosphorylated CPI-17 in the DSM and confirmed the decreases of these signaling proteins in the decompensated bladder. Our results show a differential PKC-mediated DSM contraction with corresponding alterations of PKC expression, activity and the phosphorylation of CPI-17. Our finding suggests a significant correlation between bladder function and PKC pathway. An impaired PKC pathway appears to be correlated with bladder severe dysfunction observed in decompensated bladders.

show abstract

Effect of phorbol 12,13-dibutyrate on smooth muscle tone in rat stomach fundus

Cited by 6 publications

References 33 publications

Bioengineered human IAS reconstructs with functional and molecular properties similar to intact IAS

Bioengineered human IAS reconstructs with functional and molecular properties similar to intact IAS

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

Alteration of the PKC-mediated signaling pathway for smooth muscle contraction in obstruction-induced hypertrophy of the urinary bladder

Contact Info

Product

Resources

About