Keratinocytes Proteinase-activated Receptor1Contributed To Elevated Enkephalin Synthesis In Patients With Obstructive Jaundice

It is well known that cholestatic liver disease is associated with increased levels ofcirculating endogenous opioids. In cholestatic patients and rodents, previous studiesreported that the magnitude of the increase in plasma methionine-enkephalin levels rangedfrom two to six folds. Elevated plasma endogenous opioid peptides not only impair thecardiovascular, liver and renal functions, but also induce pruritus and antinociception incholestatic liver disease. In previous studies, we have also shown that the intraoperativerequirements of desflurane, isoflurane and remifentanil in patients with obstructivejaundice were decreased significantly compared with those in nonjaundiced controls.However, little is still known about the mechanism of increased endogenous opioidssynthesis in cholestatic liver diseases.Recently, Nelson et. al reported that increased synthesis of enkephalin in the skin, thebody’s largest organ, may play a vital role in cholestasis-associated antinociception.Endogenous opioid peptides like methionine-enkephalin was found in normal human skinkeratinocytes and its expression could be upregulated by chemical or physical stimuli. Nohuman studies have reported yet skin enkephalin expression pattern in cholestatic patients.Previous studies have also shown that thrombin, a pluripotent serine protease that isgenerated in acute and chronic liver injury, can specifically increase skin keratinocyteproenkephalin mRNA expression through activation of protease-activated receptors-1(PAR-1). In fact, thrombin generation was increased in patients with obstructive cholestasisand it could be decreased by biliary drainage. In bile duct-ligated (BDL) rodents, PAR-1antagonist could even protect against liver fibrosis, suggesting that PAR-1was potentiallyactivated in cholestatic liver diseases. These findings prompted us to test the hypothesisthat thrombin, which is generated in cholestatic liver disease, can activate downstreamPAR-1signaling pathways and contribute to the increased peripheral endogenous opioidsin cholestatic liver diseases.In this study, Skin enkephalin expression was measured in jaundiced andnon-jaundiced patients. Male Sprague-Dawley rats and human keratinocyte cell lineHaCaT were used in vivo and in vitro studies respectively. Nociceptive thresholds, plasmaand skin levels of methionine-enkephalin were compared in PAR-1antagonized andcontrol BDL rats. In vitro study, the effect on thrombin-induced enkephalin expression was examined and the role of extracellular regulated protein kinases1/2and p38wasinvestigated. Results are as follows:1. Electrical skin methionine-enkephalin expression in patients with obstructivejaundiceImmunohistochemistry analysis of the skin tissues revealed that in patients withobstructive jaundice, methionine-enkephalin was strongly expressed in the stratumspinosum and stratum basale of the epidermis, which are mainly constituted by thekeratinocytes.2. PAR-1antagonism reduced skin enkephalin expression, nociceptive thresholds andplasma levels of methionine-enkephalin in the BDL ratsImmunoblotting and PCR results suggested that both skin proenkephalin mRNA andprotein expression in the BDL control group significantly increased as compared withthose of the sham control group, while PAR-1antagonism (SCH79797,1μg·kg-1·day-1,4d)reduced the increased skin proenkephalin synthesis in the BDL rats.Immunohistochemistry on the sections of rat glabrous hindpaw skin revealed thatmethionine-enkephalin was strongly expressed in the keratinocytes of the epidermis in theBDL control rats, evidenced by the brown stripe spanning through the stratum spinosumand extending into the stratum basale, while PAR-1antagonism treatment (SCH79797,1μg·kg-1·day-1,4d) reduced methionine-enkephalin expression in the keratinocytes of theepidermis of the BDL rats.As previously described, BDL caused increased nociceptive threshold in response toboth thermal and mechanical stimulation. We further tested whether PAR-1antagonist canlower the nociceptive thresholds in BDL rats. Sham surgery did not significantly modifynociceptive response to thermal or mechanical stimuli through the study period in the shamcontrol rats, while mechanical and thermal nociceptive thresholds of the BDL control ratssignificantly increased from day6to day8as compared with baseline (day0). Aftersubcutaneous injection of PAR-1antagonist SCH79797(1μg·kg-1·day-1) starting on day4for four consecutive days, both thermal and mechanical nociceptive thresholds weredecreased in the BDL group rats with no significant difference between day8and baselinefor both thermal and mechanical thresholds. SCH79797at a dose of0.3μg·kg-1·day-1) did not reduce the increased nociceptive thresholds in BDL rats compared with baseline.SCH79797at1μg·kg-1·day-1did not alter nociceptive thresholds in the sham-operated rats.Plasma level of methionine-enkephalin was significantly higher in the BDL controlrats than that of the sham control rats at day8. After subcutaneous injection of PAR-1antagonist SCH79797(1μg·kg-1·day-1) for four consecutive days, the BDL rats showedreduced plasma methionine-enkephalin level compared with the BDL control group, whilethe comparison between the BDL group2(0.3μg·kg-1·day-1) and the BDL control groupshowed no statistical significance.3. Thrombin induced proenkephalin expression through PAR-1in culturedkeratinocytesNext, we investigated whether PAR-1agonist thrombin was able to increase theexpression of the three endogenous opioid precursors, namely, proenkephalin,proopiomelanocortin and prodynorphin in HaCaT cells. Thrombin at concentrations of0.1,1and5U/mL provoked a dose-dependent increase in proenkephalin mRNA expression incultured HaCaT cells following6and24hour incubation periods. An approximately4.5-fold increase in proenkephalin mRNA expression was observed when cells wereincubated with5U/mL of thrombin for24hour. Thrombin at the concentration of5U/mLfailed to stimulate proopiomelanocortin and prodynorphin mRNA expressions following a6or24hour incubation period. Immunoblotting analysis in HaCaT cells also revealed thatthrombin increased proenkephalin protein synthesis in a dose-dependent manner following6and then24hour incubation periods.As thrombin could also activate protease-activated receptors-2and3, we usedTFLLR-NH2, an selective agonist peptide of PAR-1at a concentration of100μM, whichwas able to induce an up to three-fold increase in proenkephalin protein expression incultured HaCaT cells following a6-hour incubation period, whereas PAR-1antagonistSCH79797at a concentration of5μM abolished both thrombin-(5U/mL) andTFLLR-NH2-induced expression of proenkephalin mRNA and protein at6hours followingincubation. 4. PAR-1activation induced proenkephalin expression through ERK1/2and P38MAPK pathways in HaCaT cellsThrombin (5U/mL) and TFLLR-NH2(100μM) induced phosphorylation of ERK1/2and p38MAPK in cultured HaCaT cells following a6-hour incubation period. PAR-1antagonist SCH79797(5μM) not only inhibited thrombin-and TFLLR-NH2-inducedphosphorylation of ERK1/2, but also abolished thrombin-and TFLLR-NH2-inducedphosphorylation of p38MAPK. To examine whether PAR-1induced proenkephalinexpression through activating ERK1/2and p38MAPK, cultures of HaCaT cells wereincubated with U0126or SB203580, then challenged with thrombin or TFLLR-NH2. Aftera6-hour incubation period, U0126, an ERK inhibitor at the concentration of10μM,attenuated both thrombin-(5U/mL) and TFLLR-NH2-(100μM) induced proenkephalinexpression. SB203580(20μM), a selective inhibitor of p38MAPK, also showed inhibitoryaction on thrombin-or TFLLR-NH2-induced proenkephalin expression.In conclusion, the present study demonstrates that skin enkephalin levels wereincreased in both jaundiced patients and cholestatic models. These results may have directrelevance to elevated pain thresholds during cholestasis. Our data also suggest that PAR1activation in keratinocytes may play an important role in the local synthesis of endogenousopioids during cholestasis, and support the hypothesis that PAR1antagonists may be apotential therapeutic approach to prevent increased opioidergic tone associatedcomplications in cholestatic patients.