Husamettin Gul

Topical cannabinoid enhances topical morphine antinociception

Opioids and cannabinoids produce antinociception through both spinal and supraspinal action. Both opioids and cannabinoids also have important peripheral action. Many previous studies indicate that systemically administered cannabinoids enhance antinociceptive properties of opioids. Experiments were conducted to test the hypothesis that topical cannabinoids would enhance the topical antinociceptive effects of morphine. Antinociception was measured in the radiant tail‐flick test after immersion of the tail of mice into a solution of dimethyl sulfoxide (DMSO) containing WIN 55, 212‐2, a cannabinoid agonist and morphine, an opioid agonist. Morphine and WIN 55, 212‐2 produce time dependent topical analgesic effects limited to the portion of the tail exposed to drugs. WIN 55, 212‐2 had a potency lower than that of morphine. The topical antinociceptive effects of WIN 55, 212‐2 were blocked by systemic pretreatment of cannabinoid CB1 receptor selective antagonist, AM 251. This suggests that topical antinociceptive effects of WIN 55, 212‐2 involve CB1 receptors. Combination of topical WIN 55, 212‐2 with topical morphine yielded significantly greater analgesic effects than that of topical morphine alone. The ability of the CB1 receptor antagonist AM 251 to antagonize the enhancement of antinociception of morphine by WIN 55, 212‐2 indicates that WIN 55, 212‐2 acts through a CB1 receptor to enhance the potency of topical morphine. Additionally, spinally administered ineffective doses of WIN 55, 212‐2 potentiated the antinociceptive effects of topical morphine. These results demonstrate an antinociceptive interaction between topical opioids with topical, and spinal cannabinoids. These observations are significant in using of topical combination of cannabinoid and morphine in the management of pain.

Topical cannabinoid antinociception: synergy with spinal sites

Analgesic effects of cannabimimetic compounds have been known to be related to their central effects. Cannabinoid receptors also exist in the periphery but their role in pain perception has been remained to be clarified. Therefore, we assessed topical antinociceptive effects of WIN 55, 212‐2, a mixed CB1 and CB2 receptors agonist, in mice using tail‐flick test. Immersion of the tail of mouse into the WIN 55, 212‐2 solution produced dose‐dependent antinociception. This antinociceptive activity was limited to the portion of the tail exposed to WIN 55, 212‐2. The antinociceptive response was dependent on duration of exposure to WIN 55, 212‐2 solution. The topical antinociceptive effects of WIN 55, 212‐2 were dose dependently blocked by topical pretreatment of CB1 receptor‐selective antagonist, AM 251. Thus, topical antinociceptive action of WIN 55, 212‐2 involve CB1 receptors. Intrathecal (i.th.) administration of WIN 55, 212‐2 produced a dose‐dependent antinociceptive effect. Interestingly, ineffective i.th. doses of WIN 55, 212‐2 produced a marked antinociception when combined with topical application of WIN 55, 212‐2 and topical antinociceptive effect was potentiated. The dose–response curve of i.th. WIN 55, 212‐2 was shifted to the left 15‐fold by topical WIN 55, 212‐2. This finding suggests that there is an antinociceptive synergy between peripheral and spinal sites of cannabinoid action and it also implicates that local activaton of cannabinoid system may regulate pain initiation in cutaneous tissue. Our findings support that cannabinoid system participates in buffering the emerging pain signals at the peripheral sites in addition to their spinal and supraspinal sites of action. In addition, an antinociceptive synergy between topical and spinal cannabinoid actions exists. These results also indicate that topically administered cannabinoid agonists may reduce pain without the dysphoric side effects and abuse potential of centrally acting cannabimimetic drugs.

The interaction between IL-1β and morphine: possible mechanism of the deficiency of morphine-induced analgesia in diabetic mice

&NA; It is known that diabetic mice are less sensitive to the analgesic effect of morphine. Some factor(s) derived from mononuclear cells, e.g. interleukin‐1&bgr; (IL‐1&bgr;), may be responsible for the diminished analgesic effect of morphine in diabetic mice. Therefore, we examined direct effects of IL‐1&bgr;, intracerebroventricularly (i.c.v.), on morphine‐induced analgesia, subcutaneously (s.c.), in diabetic and control mice by using the tail‐flick test. Morphine at doses of 1, 2 and 5 mg/kg (s.c.) produced dose‐dependent analgesia in diabetic and control mice but diabetic mice were less sensitive to the analgesic effect of morphine when compared to the controls. IL‐1&bgr; at a dose of 0.1 ng/mouse produced analgesia in control mice but not in diabetics, whereas IL‐1&bgr; at a dose of 10 ng/mouse produced a hyperalgesic effect both in diabetic and control mice. IL‐1&bgr; at a dose of 1 ng/mouse has neither an analgesic nor a hyperalgesic effect in control and diabetic mice. Administration of a neutral (neither analgesic nor hyperalgesic) dose of IL‐1&bgr;, 1 ng/mouse (i.c.v.), just prior to administration of morphine (s.c.) abolished the analgesic effect of morphine at doses of 1, 2 and 5 mg/kg in control mice and the analgesic effect of morphine became similar to that in diabetics. The diminished analgesic effect of morphine in diabetes was attenuated further with IL‐1&bgr; at a dose of 1 ng/mouse (i.c.v.). These results suggest that the decreased analgesic effect of morphine in diabetes may be related to IL‐1&bgr;.

Testosterone relaxes human internal mammary artery in vitro.

Preliminary clinical studies of testosterone therapy in male patients with coronary artery disease raised promising results. However, there is no study on in vitro effects of testosterone in human isolated arteries. We investigated the effect of testosterone on contractile tone of human isolated internal mammary artery. The responses in human internal mammary artery (IMA) were recorded isometrically by a force-displacement transducer in isolated organ baths. Testosterone (10 nM to 100 μM) was added cumulatively to organ baths either at rest or after precontraction with KCl (68 mM) and PGF2α (10 μM). Testosterone-induced relaxations were tested in the presence of cyclooxygenase inhibitor indomethacin (10 μM), nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME, 1 μM), nonselective large-conductance Ca2+-activated and voltage-sensitive K+ channel inhibitor tetraethylammonium (TEA, 1 mM), ATP-sensitive K+ channel inhibitor glibenclamide (GLI, 100 μM), and voltage-sensitive K+ channel inhibitor 4-aminopyridine (4-AP, 1 mM). Testosterone produced relaxation in human IMA (Emax 33% and 41% of KCl- and PGF2α-induced contraction, respectively). Vehicle had no significant relaxant effect. Except for TEA, the relaxation at low concentrations is not affected by either K+ channel inhibitors (GLI and 4-AP) or L-NAME and indomethacin. We report for the first time that supraphysiological concentrations of testosterone induce relaxation in IMA. This response may occur in part via large-conductance Ca2+-activated K+ channel-opening action.

Beneficial effects of N-acetylcysteine on sodium taurocholate-induced pancreatitis in rats

BackgroundAcute pancreatitis (AP) is a complex disease associated with significant complications and a high rate of mortality. Although several mechanisms are put forward, oxidative stress seems the most important early event in the pathophysiology of AP. Therefore, we evaluated the beneficial effects of N-acetylcysteine (NAC), a strong antioxidant, in experimental AP.MethodsForty-nine Sprague-Dawley rats were used. Acute pancreatitis (AP) was induced by the intraductal infusion of sodium taurocholate. Rats were divided into seven groups (each containing seven rats): control, sham-operated (saline-treated, 3.5 and 12 h), non-treated AP (3.5 and 12 h) and NAC-treated AP (3.5 and 12 h). Treated rats received intraperitoneal (i.p.) NAC 1000 mg/kg 24 h before and just before the induction of pancreatitis.ResultsRats with AP had extensive parenchymal and fat necrosis and NAC treatment at 12 h reduced tissue necrosis significantly (P ≪ 0.05). NAC treatment at 12 h reduced leukocytic infiltration significantly (P ≪ 0.05). Edema and hemorrhage were significantly increased in the AP groups when compared to controls (P ≪ 0.001). NAC treatment reduced edema and hemorrhage at both 3.5 and 12 h slightly but not significantly. The total pathological mean score was significantly increased in the AP groups (P ≪ 0.05) and it was reduced by NAC treatment (P ≪ 0.05). NAC treatment decreased plasma amylase and lipase levels significantly (P ≪ 0.05). While glutathione peroxidase (GPx) activity of pancreatic tissue was similar in the NAC-treated and AP groups, hepatic tissue GPx activity was lower in the AP groups, and NAC treatment restored it (P ≪ 0.05). NAC had no effect on pancreatic superoxide dismutase level. In the NAC-treated rats, the serum NO2/NO3 (nitrite/nitrate) level was significantly increased in the 3.5-h group when compared to the respective AP group (P ≪ 0.05). NAC treatment also significantly reduced the serum concentration of the lipid peroxidation product, malondialdehyde, at 12 h (P ≪ 0.05).ConclusionsNAC treatment had beneficial effects in sodium taurocholate-induced AP in rats. It reduced pancreatic tissue necrosis and lipid peroxidation. In our study, the mechanism underlying the beneficial effects of NAC seemed to be its antioxidant activity, either by increasing hepatic GPx activity, or by a direct scavenging effect on free radicals, thus enhancing the production of and/or inhibiting the degradation of nitric oxide.

Pharmacologic characterization of contractile serotonergic receptors in human isolated mesenteric artery.

The 5-hydroxytryptamine (5-HT) receptors mediating contraction in human isolated mesenteric arteries were characterized. Endothelium-denuded human isolated mesenteric arteries were used. 5-HT induced concentration-dependent contractions in mesenteric arteries (Emax, 127.37 ± 7.61% of 80 m M KCl maximal contraction; pD2, 6.73 ± 0.09 [−logEC50]). Sumatriptan, a selective 5-HT1B/1D receptor agonist, induced concentration-dependent contractions in some of the arteries (Emax, 61.82 ± 10.04%; pD2, 6.56 ± 0.21, n = 9) but not in the others (Emax < 5%, n = 13), suggesting that functional 5-HT1B/1D receptors exist in some but not in all mesenteric arteries. GR127935 (a selective 5-HT1B/1D receptor antagonist, 3 n M) inhibited sumatriptan-induced contractions in arteries in which sumatriptan responses were strong in an insurmountable manner. GR127935 (10 n M) also inhibited 5-HT responses and shifted the concentration–response curve of 5-HT to the right significantly (p < 0.05; pD2s were 6.54 ± 0.18 and 5.93 ± 0.11 in the presence of vehicle and GR127935, respectively). Ketanserin (0.01–1 &mgr;M) competitively antagonized 5-HT responses in human mesenteric arteries: pA2 value was 8.40 ± 0.25 (slope of Schild regression, 1.43 ± 0.18; r2, 0.98). Tropisetron (5-HT3 receptor antagonist) and prazosin (&agr;1-adrenoceptor antagonist) did not affect the contractions induced by 5-HT. These results suggest that 5-HT2A and 5-HT1B/1D receptors, but not 5-HT3 and &agr;1-adrenoceptors, are involved in the 5-HT–induced contractions in human isolated mesenteric arteries. Sumatriptan-induced and 5-HT1B/1D receptor-mediated responses vary greatly among patients.

The local antinociceptive actions of nonsteroidal antiinflammatory drugs in the mouse radiant heat tail-flick test

BACKGROUND:While many preclinical models detect the analgesic activity of nonsteroidal antiinflammatory drugs (NSAIDs), the radiant heat tail-flick response has repeatedly been insensitive to this class of drugs. As the tail-flick test involves nociceptive processing at spinal circuits with supraspinal modulation, it seems reasonable to assume that the NSAIDs should not modify strong nociceptive stimuli, since the primary site of action of NSAIDs is likely to be in the periphery. METHODS:We injected 3–300 &mgr;g of diclofenac, dipyrone, ketorolac, lysine acetyl salicylate, and sodium salicylate intradermally into mice tails and evaluated the tail-flick response to radiant heat. These results were compared with intraperitoneally injected controls. We also evaluated the ability of naloxone to reverse the observed effects. RESULTS:Intradermal injection of each NSAID produced a dose-dependent increase in tail-flick latency. Intraperitoneal NSAIDs injection produced no antinociceptive effects. Naloxone pretreatment had no effect on the antinociceptive effects of intradermal diclofenac, ketorolac, lysine acetyl salicylate, and sodium salicylate. Naloxone completely blocked the antinociceptive effects of intradermal dipyrone. CONCLUSIONS:Local, but not systemic, administration of NSAIDs produced antinociception in the tail-flick thermal assay. The endogenous opioid system contributes to the peripheral antinociceptive effects of dipyrone, but not to that of diclofenac, ketorolac, lysine asetyl salicylate, or sodium salicylate, suggesting differences in the mechanisms of action among the NSAIDs.

The relation between oxidative stress, inflammation, and neopterin in the paraquat-induced lung toxicity

Paraquat (PQ) is a well-known quaternary nitrogen herbicide. The major target organ in PQ poisoning is the lung. Reactive oxygen species (ROS) and inflammation play a crucial role in the development of PQ-induced pulmonary injury. Neopterin is synthesized in macrophage by interferon γ and other cytokines. We aimed to evaluate the utility of neopterin as a diagnostic marker in PQ-induced lung toxicity. Sprague Dawley rats were randomly divided into two groups (sham and PQ), administered intraperitoneally 1 mL saline and PQ (15 mg/kg/mL) respectively. Blood samples and lungs were collected for analyses. Lung injury and fibrosis were seen in the PQ group. Serum total antioxidant capacity, lactate dehydrogenase (LDH), and lung transforming growth factor-1β (TGF-1β) levels were significantly higher than the sham group (in all, p < 0.001). In addition, in the PQ group, serum neopterin and lung malondialdehyde (MDA) levels were also significantly higher than the sham group (in all, p = 0.001). Serum neopterin levels were correlated with LDH activities, lung MDA, lung TGF-1β levels, and the degree of lung injury. These findings demonstrated that oxidative stress, reduction of antioxidant capacity, and inflammation play a crucial role in the PQ-induced lung injury. Elevated serum neopterin levels may be a prognostic parameter to determine extends of PQ-induced lung toxicity. Further studies may be performed to clarify the role of neopterin by different doses of PQ.

Simultaneous determination of cyclosporine A, tacrolimus, sirolimus, and everolimus in whole-blood samples by LC-MS/MS.

Objectives. Cyclosporine A (CyA), tacrolimus (TRL), sirolimus (SIR), and everolimus (RAD) are immunosuppressive drugs frequently used in organ transplantation. Our aim was to confirm a robust sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determination of CyA, TRL, SIR, and RAD in whole-blood samples. Materials and Methods. We used an integrated online solid-phase extraction-LC-MS/MS system and atmospheric pressure ionization tandem mass spectrometry (API-MS/MS) in the multiple reaction monitoring (MRM) detection mode. CyA, TRL, SIR, and RAD were simultaneously analyzed in whole blood treated with precipitation reagent taken from transplant patients. Results. System performance parameters were suitable for using this method as a high-throughput technique in clinical practice. The high concentration of one analyte in the sample did not affect the concentration of other analytes. Total analytical time was 2.5 min, and retention times of all analytes were shorter than 2 minutes. Conclusion. This LC-MS/MS method can be preferable for therapeutic drug monitoring of these immunosuppressive drugs (CyA, TRL, SRL, and RAD) in whole blood. Sample preparation was too short and simple in this method, and it permits robust, rapid, sensitive, selective, and simultaneous determination of these drugs.

Increased vasoconstrictor reactivity and decreased endothelial function in high grade varicocele; functional and morphological study

The pathophysiology of human varicocele is not fully understood. We investigated vasoconstrictor reactivity, endothelial function and morphological changes in different grades of varicocele to clarify the pathophysiology. Contractile responses to phenylephrine, norepinephrine, serotonin and histamine were determined in isolated human varicose spermatic veins using the organ bath technique. Endothelial function was tested with acetylcholine-induced relaxation after phenylephrine-induced precontraction in the absence and presence of nitric oxide synthase inhibitor, L-NAME, and cyclooxygenase inhibitor, indomethacin. The cyclic guanosine monophosphate (cGMP) level was measured in the spermatic vein and peripheral plasma. Morphological changes were evaluated with light microscopy. Phenylephrine, norepinephrine, serotonin and histamine induced concentration-dependent contractions. The maximum contractions for all of these agents except norepinephrine were significantly higher in grade III than grade I and II (P<0.05). The sensitivity to phenylephrine was significantly higher in grades II and III than in grade I (P<0.05). In the presence of L-NAME and indomethacin, the difference from respective control phenylephrine-induced contractions was higher in grade I and II than grade III. Acetylcholine did not induce stable relaxation but the level of cGMP, which is responsible for the vasorelaxant effect of NO, in veins was lower in grades II and III than grade I (P<0.05). Vessel wall thickness increased in grade II and dilatation developed in grade III when compared to grade I (P<0.05). Our findings suggest that endothelium produces less vasorelaxant which results in the more enhanced effects of vasoconstrictor substances in grade III, indicating that endothelial dysfunction develops at high grades of varicocele.