YFa and analogs:Investigation of opioid receptors in smooth muscle contraction

AIM:To study the pharmacological profile and inhibition of smooth muscle contraction by YFa and its analogs in conjunction with their receptor selectivity. METHODS:The effects of YFa and its analogs (D-Ala2) YFa, Y (D-Ala2) GFMKKKFMRF amide and Des-Phe-YGGFMKKKFMR amide in guinea pig ileum (GPI) and mouse vas deferens (MVD) motility were studied using an isolated tissue organ bath system, and morphine and DynA (1-13) served as controls. Acetylcholine was used for muscle stimulation. The observations were validated by specific antagonist pretreatment experiments using naloxonazine, naltrindole and norbinaltor-phimine norBNI. RESULTS:YFa did not demonstrate significant inhibition of GPI muscle contraction as compared with mor-phine (15% vs 62%, P = 0.0002), but moderate inhibition of MVD muscle contraction, indicating the role of κ opioid receptors in the contraction. A moderate inhibition of GPI muscles by (Des-Phe) YFa revealed the role of anti-opiate receptors in the smooth muscle contraction. (D-Ala-2) YFa showed significant inhibition of smooth muscle contraction, indicating the involvement of mainly δ receptors in MVD contraction. These results were supported by specific antagonist pretreatment assays. CONCLUSION:YFa revealed its side-effect-free analgesic properties with regard to arrest of gastroin-testinal transit. The study provides evidences for the involvement of κ and anti-opioid receptors in smooth muscle contraction.

Effect of the ginsenoside Rb1 on the spontaneous contraction of intestinal smooth muscle in mice

AIM: To investigate the effect and the possible mechanism of ginsenoside Rb1 on small intestinal smooth muscle motility in mice. METHODS: Intestinal smooth muscle strips were isolated from male ICR mice (5 wk old), and the effect of ginsenoside Rb1 on spontaneous contraction was recorded with an electrophysiolograph. The effect of ginsenoside Rb1 on ion channel currents, including the voltage-gated K + channel current (IK V ), calcium-activated potassium channel currents (IK Ca ), spontaneous transient outward currents and ATP-sensitive potassium channel current (IK ATP ), was recorded on freshly isolated single cells using the whole-cell patch clamp technique. RESULTS: Ginsenoside Rb1 dose-dependently inhibited the spontaneous contraction of intestinal smooth muscle by 21.15% ± 3.31%, 42.03% ± 8.23% and 67.23% ± 5.63% at concentrations of 25 μmol/L, 50 μmol/L and 100 μmol/L, respectively (n=5,P<0.05). The inhibitory effect of ginsenoside Rb1 on spontaneous contraction was significantly but incompletely blocked by 10 mmol/L tetraethylammonium or 0.5 mmol/L 4-aminopyridine, respectively (n=5, P<0.05). However, the inhibitory effect of ginsenoside Rb1 on spontaneous contraction was not affected by 10 μmol/L glibenclamide or 0.4 μmol/L tetrodotoxin. At the cell level, ginsenoside Rb1 increased outward potassium currents, and IK V was enhanced from 1137.71 ± 171.62 pA to 1449.73 ± 162.39 pA by 50 μmol/L Rb1 at +60 mV (n=6, P<0.05). Ginsenoside Rb1 increased IK Ca and enhanced the amplitudes of spontaneous transient outward currents from 582.77 ± 179.09 mV to 788.12 ± 278.34 mV (n=5, P<0.05). However, ginsenoside Rb1 (50 μmol/L) had no significant effect on IK ATP (n=3, P<0.05). CONCLUSION: These results suggest that ginsenoside Rb1 has an inhibitory effect on the spontaneous contraction of mouse intestinal smooth muscle mediated by the activation of IK V and IK Ca , but the K ATP channel was not involved in this effect.

The role of TRPP2 in agonist-induced gallbladder smooth muscle contraction

TRPP2 channel protein belongs to the superfamily of transient receptor potential(TRP) channels and is widely expressed in various tissues, including smooth muscle in digestive gut. Accumulating evidence has demonstrated that TRPP2 can mediate Ca^(2+) release from Ca^(2+) stores. However, the functional role of TRPP2 in gallbladder smooth muscle contraction still remains unclear. In this study, we used Ca^(2+) imaging and tension measurements to test agonist-induced intracellular Ca^(2+) concentration increase and smooth muscle contraction of guinea pig gallbladder, respectively. When TRPP2 protein was knocked down in gallbladder muscle strips from guinea pig, carbachol(CCh)-evoked Ca^(2+) release and extracellular Ca^(2+) influx were reduced significantly, and gallbladder contractions induced by endothelin 1 and cholecystokinin were suppressed markedly as well. CCh-induced gallbladder contraction was markedly suppressed by pretreatment with U73122, which inhibits phospholipase C to terminate inositol 1,4,5-trisphosphate receptor(IP3) production, and 2-aminoethoxydiphenyl borate(2APB), which inhibits IP3 recepor(IP3R) to abolish IP3R-mediated Ca^(2+) release. To confirm the role of Ca^(2+) release in CCh-induced gallbladder contraction, we used thapsigargin(TG)-to deplete Ca^(2+) stores via inhibiting sarco/endoplasmic reticulum Ca^(2+)-ATPase and eliminate the role of store-operated Ca^(2+) entry on the CCh-induced gallbladder contraction. Preincubation with 2 μmol L^(-1) TG significantly decreased the CCh-induced gallbladder contraction. In addition, pretreatments with U73122, 2APB or TG abolished the difference of the CCh-induced gallbladder contraction between TRPP2 knockdown and control groups. We conclude that TRPP2 mediates Ca^(2+) release from intracellular Ca^(2+) stores, and has an essential role in agonist-induced gallbladder muscle contraction.

Circular smooth muscle contributes to esophageal shortening during peristalsis

AIM:To study the angle between the circular smooth muscle(CSM) and longitudinal smooth muscle(LSM) fibers in the distal esophagus.METHODS:In order to identify possible mechanisms for greater shortening in the distal compared to proximal esophagus during peristalsis,the angles between the LSM and CSM layers were measured in 9 cadavers.The outer longitudinal layer of the muscularis propria was exposed after stripping the outer serosa.The inner circular layer of the muscularis propria was then revealed after dissection of the esophageal mucosa and the underlying muscularis mucosa.Photographs of each specimen were taken with half of the open esophagus folded back showing both the outer longitudinal and inner circular muscle layers.Angles were measured every one cm for 10 cm proximal to the squamocolumnar junction(SCJ) by two independent investigators.Two human esophagi were obtained from organ transplant donors and the angles between the circular and longitudinal smooth muscle layers were measured using micro-computed tomography(micro CT) and Image J software.RESULTS:All data are presented as mean ± SE.The CSM to LSM angle at the SCJ and 1 cm proximal to SCJ on the autopsy specimens was 69.3 ± 4.62 degrees vs 74.9 ± 3.09 degrees,P = 0.32.The CSM to LSM angle at SCJ were statistically significantly lower than at 2,3,4 and 5 cm proximal to the SCJ,69.3 ± 4.62 degrees vs 82.58 ± 1.34 degrees,84.04 ± 1.64 degrees,84.87 ± 1.04 degrees and 83.72 ± 1.42 degrees,P = 0.013,P = 0.008,P = 0.004,P = 0.009 respectively.The CSM to LSM angle at SCJ was also statistically significantly lower than the angles at 6,7 and 8 cm proximal to the SCJ,69.3 ± 4.62 degrees vs 80.18 ± 2.09 degrees,81.81 ± 1.75 degrees and 80.96 ± 2.04 degrees,P = 0.05,P = 0.02,P = 0.03 respectively.The CSM to LSM angle at 1 cm proximal to SCJ was statistically significantly lower than at 3,4 and 5 cm proximal to the SCJ,74.94 ± 3.09 degrees vs 84.04 ± 1.64 degrees,84.87 ± 1.04 degrees and 83.72 ± 1.42 degrees,P = 0.019,P = 0.008,P = 0.02 respectively.At 10 cm above SCJ the angle was 80.06 ± 2.13 degrees which is close to being perpendicular but less than 90 degrees.The CSM to LSM angles measured on virtual dissection of the esophagus and the stomach on micro CT at the SCJ and 1 cm proximal to the SCJ were 48.39 ± 0.72 degrees and 50.81 ± 1.59 degrees.Rather than the angle of the CSM and LSM being perpendicular in the esophagus we found an acute angulation between these two muscle groups throughout the lower 10 cm of the esophagus.CONCLUSION:The oblique angulation of the CSM may contribute to the significantly greater shortening of distal esophagus when compared to the mid and proximal esophagus during peristalsis.

Inhibition of LIM kinase reduces contraction and proliferation in bladder smooth muscle

Overactive bladder(OAB)is the most bothersome symptom in lower urinary tract symptoms(LUTS).Current pharmacologic treatment aims to inhibit detrusor contraction;however,shows unsatisfied efficacy and high discontinuation rate.LIM kinases(LIMKs)promote smooth muscle contraction in the prostate;however,their function in the bladder smooth muscle remains unclear.Here,we studied effects of the LIMK inhibitors on bladder smooth muscle contraction and proliferation both in vitro and in vivo experiments.Bladder expressions of LIMKs are elevated in OAB rat detrusor tissues.Two LIMK inhibitors,SR7826 and LIMKi3,inhibit contraction of human detrusor strip,and cause actin filament breakdown,as well as cell proliferation reduction in cultured human bladder smooth muscle cells(HBSMCs),paralleled by reduced cofilin phosphorylation.Silencing of LIMK1 and LIMK2 in HBSMCs resulted in breakdown of actin filaments and decreased cell proliferation.Treatment with SR7826 or LIMKi3 decreased micturition frequency and bladder detrusor hypertrophy in rats with bladder outlet obstruction.Our study suggests that LIMKs may promote contraction and proliferation in the bladder smooth muscle,which could be inhibited by small molecule LIMK inhibitors.LIMK inhibitors could be a potential therapeutic strategy for OAB-related LUTS.

Serum response factor:Look into the gut

Serum response factor(SRF) is a transcription factor that regulates many genes involved in cellular activities such as proliferation,migration,differentiation,angiogenesis,and apoptosis.Although it has only been known for about two decades,SRF has been studied extensively.To date,over a thousand SRF studies have been published,but it still remains a hot topic.Due to its critical role in mesoderm-derived tissues,most of the SRF studies focused on muscle structure/function,cardiovascular development/maintenance,and smooth muscle generation/repair.Recently,SRF has received more attention in the digestive field and several important discoveries have been made.This review will summarize what we have learned about SRF in the gastrointestinal tract and provide insights into possible future directions in this area.