Involvement of the Peripheral µ-Opioid Receptor in Tramadol-Induced Constipation in Rodents.

Tramadol is a weak opioid that produces analgesic effect via both the µ-opioid receptor (MOR) and non-opioid targets. Constipation is the most common opioid-related side effect in patients with cancer and non-cancer pain. However, the contribution of MOR to tramadol-induced constipation is unclear. Therefore, we used naldemedine, a peripherally acting MOR antagonist, and MOR-knockout mice to investigate the involvement of peripheral MOR in tramadol-induced constipation using a small intestinal transit model. A single dose of tramadol (3-100 mg/kg, per os (p.o.)) inhibited small intestinal transit dose-dependently in rats. Naldemedine (0.01-10 mg/kg, p.o.) blocked the inhibition of small intestinal transit induced by tramadol (30 mg/kg, p.o.) in rats. The transition rate increased dose-dependently over the range of naldemedine 0.01-0.3 mg/kg, and complete recovery was observed at 0.3-10 m/kg. Additionally, tramadol (30 and 100 mg/kg, subcutaneously (s.c.)) inhibited small intestinal transit in wild-type mice but not in MOR-knockout mice. These results suggest that peripheral MOR participates in tramadol-induced constipation.

The antagonistic activity profile of naloxone in µ-opioid receptor agonist-induced psychological dependence.

Naloxone is a µ-opioid receptor antagonist that has been used to prevent overdose-related respiratory depression and deaths by the illicit use of opioids. Naloxone can also deter the abuse potential of opioids, but little has been reported regarding its antagonistic activity profile against opioid-induced psychological dependence. This study aimed to confirm the antagonistic activity profile of naloxone against several µ-opioid receptor agonists and investigate whether naloxone could affect the psychological dependence induced by widely used µ-opioid receptor agonist, oxycodone. In the Guanosine-5'-o-(3-thio) triphosphate (GTPγS) binding assay, naloxone (30-30,000 nM) inhibited the GTPγS binding induced by oxycodone, hydrocodone, morphine, and fentanyl. It elicited parallel rightward shifts in the concentration-response curves, indicating that naloxone possessed a competitive antagonistic activity profile against these µ-opioid receptor agonists. In the conditioned place preference test, oxycodone (0.01-1 mg/kg, i.v.) produced dose-dependent increases in place preference. The increased place preference induced by oxycodone (1 mg/kg) was significantly attenuated by co-administration of naloxone at a dose of 0.5 mg/kg but not 0.01 mg/kg. Naloxone (0.5 mg/kg, i.v.) also blocked oxycodone (1 mg/kg)-induced dopamine release in nucleus accumbens; however, at a lower dose (0.01 mg/kg), it did not affect the intrinsic dopamine release by oxycodone. These results indicate that the psychological dependence of oxycodone could be antagonized by naloxone, depending on the dose. This characterization might lead to a better understanding of the competitive antagonistic activity profile of naloxone for µ-opioid receptor in the brain.

Sphingomyelin Regulates the Activity of Secretory Phospholipase A2 in the Plasma Membrane.

We examined the effect of the cellular sphingolipid level on the release of arachidonic acid (AA) and the activity of secretory phospholipase A2 (sPLA2 ) using two Chinese hamster ovary (CHO)-K1 cell mutants, LY-B and LY-A cells, deficient in sphingolipid synthesis. In LY-B cells, deficiency of sphingolipids enhanced the release of AA induced by bee venom sPLA2-III or human sPLA2-V. These alterations were reversed by replenishment of exogenous sphingomyelin (SM). In LY-A cells, deficiency of SM increased the release of AA induced by sPLA2. In CHO-K1 cells, decrease and increase of SM level in the plasma membrane by pharmacological methods increased and inhibited the release of AA, respectively. SM inhibited the activity of sPLA2 in vitro. Niemann-Pick disease type C (NPC) is a lysosomal storage disorder caused by mutation of either the NPC1 or NPC2 gene, and is characterized by accumulation of cholesterol and sphingolipids including SM in late endosomes/lysosomes. Increased levels of AA and sPLA2 activity are involved in various neurodegenerative diseases. In CHO cells lacking NPC1 (A101 cells), SM level was lower in the plasma membrane, while it was higher in late endosomes/lysosomes. The release of AA induced by sPLA2 was increased in A101 cells than that in parental cells (JP17 cells), which was attenuated by adding exogenous SM. In addition, sPLA2 -III-induced cytotoxicity in A101 cells was much higher than that in JP17 cells. These results suggest that SM in the plasma membrane plays important roles in regulating sPLA2 activity and the enzyme-induced cytotoxicity in A101 cells.

Arachidonic acid metabolism via cytosolic phospholipase A2 α induces cytotoxicity in niemann-pick disease type C cells.

Niemann-Pick disease type C (NPC) is a neurodegenerative lipid storage disorder caused by mutations in NPC1 or NPC2 genes. Loss of function of either protein results in the endosomal accumulation of cholesterol and other lipids. Here, we report that NPC1-deficient Chinese hamster ovary cells exhibit increased release of arachidonic acid (AA) and synthesis of prostaglandin E(2) compared with wild-type cells. The enhanced release of AA was inhibited by both treatment with the selective inhibitor of cytosolic phospholipase A(2) α (cPLA(2) α) and cultivation in lipoprotein-deficient medium. There was no difference in the expression of both cyclooxygenase-1 and -2 between NPC cells and wild-type cells. U18666A, a cholesterol transport-inhibiting agent commonly used to mimic NPC, also increased the release of AA in L929 mouse fibrosarcoma cells. Furthermore, U18666A-induced formation of reactive oxygen species (ROS) resulted in the induction of cell death and cell cycle delay/arrest in L929 cells. Interestingly, these responses induced by U18666A were much weaker in cPLA(2) α knockdown L929 cells. These results suggest that cPLA(2) α-AA pathway plays important roles in the cytotoxicity and the ROS formation in NPC cells.

Role of cytosolic phospholipase A(2)α in cell rounding and cytotoxicity induced by ceramide-1-phosphate via ceramide kinase.

Ceramide-1-phosphate (C1P), produced by ceramide kinase (CERK), is implicated in the regulation of many biological functions including cell growth and inflammation. C1P is a direct activator of group IVA cytosolic phospholipsase A(2) (PLA2G4A or cPLA(2)α). Although activation of the CERK-C1P pathway causes mitogenic and cytoprotective responses in many cells, the pathway shows cytotoxicity in several cells and the precise mechanism has not been elucidated. In the present study, we examined the effect of human CERK (hCERK) expression on cytotoxicity in two cell lines. Expression of hCERK in CHO cells caused cell rounding and lactate dehydrogenase (LDH) leakage, and co-addition of ceramide enhanced these responses. Expression of hCERK enhanced C1P formation and release of arachidonic acid in Ca(2+) ionophore-stimulated cells. Treatment with 20µM C2-C1P for 24 h caused cell rounding, and the response was significantly decreased by an inhibitor of cPLA(2)α. In L929 cells, expression of hCERK with and without ceramide caused cell rounding and LDH leakage, respectively, and the responses were significantly less in a stable clone of L929 cells lacking cPLA(2)α. These findings suggest the involvement of cPLA(2)α in CERK-C1P pathway-induced cytotoxicity.

Effects of ceramide, ceramidase inhibition and expression of ceramide kinase on cytosolic phospholipase A2alpha; additional role of ceramide-1-phosphate in phosphorylation and Ca2+ signaling.

Ceramide and the metabolites including ceramide-1-phosphate (C1P) and sphingosine are reported to regulate the release of arachidonic acid (AA) and/or phospholipase A(2) (PLA(2)) activity in many cell types including lymphocytes. Recent studies established that C1P, a product of ceramide kinase, interacts directly with Ca(2+) binding regions in the C2 domain of alpha type cytosolic PLA(2) (cPLA(2)alpha), leading to translocation of the enzyme from the cytosol to the perinuclear region in cells. However, a precise mechanism for C1P-induced activation of cPLA(2)alpha has not been well elucidated; such as the phosphorylation signal caused by the extracellular signal-regulated kinases (ERK1/2) pathway, a downstream of the protein kinase C activation with 4beta-phorbol myristate acetate (PMA), is required or not. In the present study, we showed that the increase in intracellular ceramide levels (exogenously added cell permeable ceramides and an inhibition of ceramidase by (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol and the increase in C1P formation by transfection with the vector for human ceramide kinase significantly enhanced the Ca(2+) ionophore (A23187) -induced release of AA via cPLA(2)alpha's activation in CHO cells. Ceramides did not show additional effects on the release from the cells treated with the inhibitor of ceramidase. Ceramides and C2-C1P neither had effect on the intracellular mobilization of Ca(2+) nor the phosphorylation of cPLA(2)alpha in cells. A23187/PMA-induced release of AA was enhanced by ceramides and C2-C1P and by expression of ceramide kinase. Our findings suggest that C1P is a stimulatory factor on cPLA(2)alpha that is independent of the Ca(2+) signal and the PKC-ERK-mediated phosphorylation signal.