The sphingosine‐1‐phosphate pathway is upregulated in response to partial urethral obstruction in male rats and activates RhoA/Rho‐kinase signalling

Abstract

contractility studies, and for mRNA and protein analyses of major S1P/ROK pathway constituents via real-time polymerase chain reaction and Western blotting, respectively. In addition, early-passage SM cells were transfected with recombinant sphingosine kinase (SPHK, the enzyme that converts sphingosine to S1P). RESULTSBladders from PUO rats had greater mRNA expression of the S1P2 and S1P3 receptors, as well as SPHK1, than the sham controls (4.78, 2.04 and 2.72 times, respectively). PUO rats also had significantly greater expression of RhoA and ROK α (1.76 and 2.19 times, respectively). Western blotting and organbath contractility studies showed similar changes at the protein and in vitro functional level, with an increased contractility of bladder strips from PUO rats to exogenous S1P. Transfection of SPHK into isolated SM cells increased ROK expression. CONCLUSIONSWe show for the first time that the S1P signalling pathway is significantly upregulated in response to PUO in male rats at both the molecular and in vitro functional levels, correlating with an activation of the RhoA/ROK pathway. Further, we provide novel data that SPHK overexpression increases ROK expression in vitro , suggesting a novel hypothesis of S1P-induced bladder overactivity in the mechanism for PUOinduced bladder dysfunction and the S1P signalling pathway as a possible therapeutic target for bladder overactivity. KEYWORDSbladder, outlet obstruction, sphingosine-1-phosphate, Rho-kinase OBJECTIVESTo examine the effect of partial urethral obstruction (PUO) on the sphingosine-1-phosphate (S1P, a bioactive lipid shown to modulate smooth muscle, SM) pathway in the bladders of male rats, and to determine the effect of PUO on the RhoA/Rho-kinase (ROK) pathway, and whether there is a molecular cross-talk with the S1P pathways associated with bladder overactivity (S1P1-S1P3, where S1P1 is associated with nitric oxide-mediated SM relaxation, and S1P2 and S1P3 receptors are associated more with SM contraction via the ROK pathway). MATERIALS AND METHODSIn all, 20 male rats were divided into two groups and underwent PUO or a sham operation (control). After 2 weeks all rats were killed humanely and bladder specimens used for in vitro organ-bath physiological

Testosterone regulates smooth muscle contractile pathways in the rat prostate: emphasis on PDE5 signaling

“…RPL19 was determined to be more stable than either ␤-actin or GAPDH in a rat model of partial bladder outlet obstruction. However, we did not find any effect of aging, diabetes, or castration on these three housekeeping genes (8).…”

4F, apolipoprotein AI mimetic peptide, attenuates acute lung injury and improves survival in endotoxemic rats

“…15 In brief, total RNA was extracted from 100 Kg of lung tissues using an RNeasy total RNA extraction kit (Qiagen, Chatsworth, CA). Then, 2 Kg of total RNA was used in a complementary DNA (cDNA) reaction.…”

The effect of sphingosine-1-phosphate on colonic smooth muscle contractility: Modulation by TNBS-induced colitis

“…One or more of these receptors have been reported to mediate the contractile effect of S1P in a variety of smooth muscle preparations. It has been reported that S1P-induced contractions were mediated via S1PR2 receptors in the urinary bladder [28, 31, 37], myometrium [25], airway smooth muscle [26], esophageal [27] and gastric smooth muscle [29, 46]. S1P-induced contraction of gastric smooth muscle is also mediated in part by S1PR1 receptors [29, 46].…”

“…According to Chiba et al (2010), S1P-induced contraction of murine bronchial smooth muscle was abolished by Y-27632, an inhibitor of Rho kinase indicating a role for Rho-kinase pathway of calcium sensitization in S1P-induced contraction of the bronchial smooth muscle. The Rho kinase pathway of calcium sensitization has also been implicated in S1P-induced contraction of guinea-pig trachea [38], urinary bladder [31, 37], human airway smooth muscle [26] and porcine retinal arterioles [51]. Kim et al have reported a role for the PKC pathway of calcium sensitization in S1P-induced contraction of feline esophageal smooth muscle cells [56].…”

“…S1P also induced relaxation of vascular smooth muscles [32, 33]. S1P-induced modulation of smooth muscle contractility has been implicated in some pathological conditions including hypertension [34–36], bladder overreactivity [28, 37] and asthma [26, 29, 38–42]. Even though elevated levels of S1P were reported in patients with IBD and confirmed in experimental models of ulcerative colitis [43], the effect of S1P on colonic smooth muscle in control and colitic rats has not been investigated.…”