Phorbol 12,13-Dibutyrate-Induced, Protein Kinase C-Mediated Contraction of Rabbit Bladder Smooth Muscle

Abstract

Contraction of bladder smooth muscle is predominantly initiated by M3 muscarinic receptor-mediated activation of the Gq/11-phospholipase C β-protein kinase C (PKC) and the G12/13-RhoGEF-Rho kinase (ROCK) pathways. However, these pathways and their downstream effectors are not well understood in bladder smooth muscle. We used phorbol 12,13-dibutyrate (PDBu), and 1,2-dioctanoyl-sn-glycerol (DOG), activators of PKC, in this investigation. We were interested in dissecting the role(s) of PKC and to clarify the signaling pathways in bladder smooth muscle contraction, especially the potential cross-talk with ROCK and their downstream effectors in regulating myosin light chain phosphatase activity and force. To achieve this goal, the study was performed in the presence or absence of the PKC inhibitor bisindolylmaleimide-1 (Bis) or the ROCK inhibitor H-1152. Phosphorylation levels of Thr38-CPI-17 and Thr696/Thr850 myosin phosphatase target subunit (MYPT1) were measured during PDBu or DOG stimulation using site specific antibodies. PDBu-induced contraction in bladder smooth muscle involved both activation of PKC and PKC-dependent activation of ROCK. CPI-17 as a major downstream effector, is phosphorylated by PKC and ROCK during PDBu and DOG stimulation. Our results suggest that Thr696 and Thr850-MYPT1 phosphorylation are not involved in the regulation of a PDBu-induced contraction. The results also demonstrate that bladder smooth muscle contains a constitutively active isoform of ROCK that may play an important role in the regulation of bladder smooth muscle basal tone. Together with the results from our previous study, we developed a working model to describe the complex signaling pathways that regulate contraction of bladder smooth muscle.

Sphingosine-1-phosphate induced contraction of bladder smooth muscle

“…Inhibition of ROCK by the addition of 1 μM H-1152 significantly decreased force production compared to S1P alone at all time points measured after the addition of S1P (1, 2, and 5 min). Consistent with previous results from our laboratory, inhibition of ROCK tended to decrease baseline force (Wang et al, 2009; Wang et al, 2012), although the H-1152-induced decrease in baseline force was not significant in this set of experiments. The decrease in S1P-induced isometric force production by inhibition of ROCK was significantly greater than that produced by inhibition of PKC.…”

“…The decrease in S1P-induced isometric force production by inhibition of ROCK was significantly greater than that produced by inhibition of PKC. It is important to note, that previous studies in our laboratory have shown that 3 μM Bis-1 inhibits PKC-dependent pathways and 1 μM H-1152 inhibits ROCK-dependent pathways in rabbit bladder smooth muscle (Wang et al, 2009; Wang et al, 2012). Preliminary studies from our laboratory demonstrated that these two compounds at these concentrations had no effect on MLC phosphorylation levels in a Triton X-100 skinned preparation of rabbit bladder smooth muscle, suggesting Bis and H-1152 have no non-specific effects on the MLC kinase (unpublished results).…”

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

“…However, increased expression of ROCK␤ in c-Rel KO BSM was not associated with increased CPI-17 phosphorylation in the same tissues, suggesting that PKC, rather than ROCK␤, plays a major role in CPI-17 phosphorylation in BSM. This is in agreement with an earlier report that the ROCK isoform that phosphorylates MYPT1 is not involved in in vivo phosphorylation of CPI-17 in BSM (5,39).…”

“…8G; compare the spots for phosphorylated MLC 20 and the graph). Together, these observations suggest that the PKC-mediated BSM contraction that occurs through CPI-17 expression in response to agonist stimulation is independent of MLC 20 phosphorylation, in accordance with earlier observations in rabbit vascular smooth muscle (39,40).…”

Modulation of Purinergic Neuromuscular Transmission by Phorbol Dibutyrate is Independent of Protein Kinase C in Murine Urinary Bladder

“…In addition to the PKC-dependent postjunctional effects of phorbol esters on bladder smooth muscle contraction (Wang et al, 2012), phorbol esters have been found to exert prejunctional effects. Thus, phorbol esters were found to increase [ 14 C]ACh overflow in the rat urinary bladder through a M 1 muscarinic receptor-dependent PKC pathway, with the effects of phorbol esters inhibited by both PKC inhibitors and atropine (Somogyi et al,1997).…”