Nurettin Abacioglu

Circadian variation of nitric oxide synthase activity in mouse tissue

Endogenous nitric oxide (NO) is an important mediator in the processes that control biological clocks and circadian rhythms. The present study was designed to elucidate if NO synthase (NOS) activity in the brain, kidney, testis, aorta, and lungs and plasma NOx levels in mice are controlled by an endogenous circadian pacemaker. Male BALB/c mice were exposed to two different lighting regimens of either light–dark 14:10 (LD) or continuous lighting (LL). At nine different equidistant time points (commencing at 09:00h) blood samples and tissues were taken from mice. The plasma and tissue homogenates were used to measure the levels of NO2+ NO3− (NOx) and total protein. The NOx concentrations were determined by a commercial nitric oxide synthase assay kit, and protein content was assessed in each homogenate tissue sample by the Lowry method. Nitric oxide synthase activity was calculated as pmol/mg protein/h. The resulting patterns were analyzed by the single cosinor method for pre-adjusted periods and by curve-fitting programs to elucidate compound rhythmicity. The NOS activity in kidneys of mice exposed to LD exhibited a circadian rhythm, but no rhythmicity was detected in mice exposed to LL. Aortic NOS activity displayed 24h rhythmicity only in LL. Brain, testis, and lung NOS activity and plasma NOx levels displayed 24h rhythms both in LD and LL. Acrophase values of NOS activity in brain, kidney, testis, and lungs were at midnight corresponding to their behavioral activities. Compound rhythms were also detected in many of the examined patterns. The findings suggest that NOS activity in mouse brain, aorta, lung, and testis are regulated by an endogenous clock, while in kidney the rhythm in NOS activity is synchronized by the exogenous signals.

Participation of the components of L-arginine/nitric oxide/ cGMP cascade by chemically-induced abdominal constriction in the mouse

The purpose of this study was to investigate the role of the L-arginine/nitric oxide (NO)/cGMP pathway in p-benzoquinone-induced writhing model in mouse. L-arginine, a NO precursor, displayed antinociceptive effects at the doses of 0.125-1.0 mg/kg. When the doses of L-arginine were increased gradually to 10-100 mg/kg, a dose-dependent triphasic pattern of nociception-antinociception-nociception was obtained. The NO synthase (NOS) inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) (18.7515 mg/kg), possessed antinociceptive activity. Methylene blue (MB), a guanylyl cyclase and/or NOS inhibitor, (5-160 mg/kg) also produced a dose-dependent triphasic response. When L-arginine (50 mg/ kg) was combined with L-NAME (75 mg/kg). L-arginine-induced antinociception did not change significantly. Cotreatment of L-arginine with 5 mg/kg MB significantly decreased MB-induced antinociception and reversed the nociception induced by 40 mg/kg MB to antinociception. It is concluded that the components of L-arginine/nitric oxide/cGMP cascade may participate in nociceptive processes both peripherally and centrally by a direct effect on nociceptors or by the involvement of other related pathways of nociceptive processes induced by NO.

Nitric-oxide production by human umbilical vessels in severe pre-eclampsia.

Objective: Pre-eclampsia is characterized by an increased vascular tone which might be related to an abnormal endothelial cell function. As representatives of the fetal circulation, we compared the nitric oxide (NO)-releasing capacity of human umbilical vessels from normal and pre-eclamptic pregnancies. Methods: Normal and pre-eclamptic umbilical vessels were mounted in parallel in an organ chamber with three perfusion lines superfusing the same detector tissue (rubbed rat aortic ring). In this cascade system the capacity of the umbilical vessels to release NO was measured under basal conditions and after stimulation with histamine, bradykinin or calcium ionophore A 23187. Results: Relaxations dependent on basal NO release were found to be significantly higher in pre-eclamptic vessels (especially in veins) than in normal vessels. Conversely, stimulaled NO release in response to histamine or bradykinin was significantly decreased in pre-eclamptic umbilical arteries, but not in veins, compared with normal vessels. However, there was no significant difference in the release of NO in response to A 23187 between normal and pre-eclamptic vessels. Conclusions: The NO-releasing and NO-producing capacity in the vessels from fetal circulation is not diminished in pre-eclampsia. However, in pre-eclamptic umbilical arteries the NO release in response to certain stimuli (histamine or bradykinin) is diminished, probably as a result of alterations in the receptor function.

Effects of cyclooxygenase inhibitors on nitric oxide production and survival in a mice model of sepsis.

The effects of selective ((5,5-dimethyl-3-(3-florophenyl)-4-(4-methylsulphonyl-2(5H)-furanon); DFU) and (N-(2-cyclohexyloxy-4-nitrophenyl)-methansulphonamide; NS 398)) or non-selective (diclophenac and proquazon) inducible cyclooxygenase (COX-2) inhibitors on the survival, nitrite (stable product of nitric oxide (NO) as an index for inducible NO synthase (iNOS) activity) and 6-keto-prostaglandin F(1alpha) (6-keto-PGF(1alpha), stable product of prostacyclin as an index for COX-2 activity) production in serum, lungs, brain and/or kidney were investigated in endotoxin-induced sepsis model in mice. Endotoxin (10 mg x kg(-1), i.p.)-induced mortality was prevented by DFU, NS 398 and proquazon (0.1, 10 and 1 mg x kg(-1), respectively) and enhanced 2.6-fold with 0.1mg x kg(-1) diclophenac. Endotoxin-induced increase in the serum levels of nitrite was only inhibited by 10 mg x kg(-1) diclophenac. Endotoxin caused a significant decrease only in the brain levels of nitrite without affecting 6-keto-PGF(1alpha) levels in all tissues. The decreased levels of nitrite induced by endotoxin is further reduced by 0.1mg x kg(-1) DFU and 1 and 10mg x kg(-1) diclophenac while 10 mg x kg(-1) DFU and 1mg x kg(-1) proquazon increased it. On the other hand, 1mg x kg(-1) diclophenac and proquazon, and 10 mg x kg(-1) NS 398 increased the endotoxin-induced lung levels of 6-keto-PGF(1alpha). The results suggest that the COX inhibitors may have different effects on the survival and NO production depending on tissue and dose.

Estimation of anti-inflammatory, antinociceptive and antioxidant activities on Arctium minus (Hill) Bernh. ssp. minus.

ETHNOPHARMACOLOGICAL RELEVANCE Arctium minus (Hill) Bernh. ssp. minus (Asteraceae) leaves are used to alleviate rheumatic pain, against fever and sunstroke with externally application in Turkish folk medicine. AIM OF THE STUDY To evaluate the anti-inflammatory, antinociceptive and antioxidant activities of aqueous and ethanol extracts prepared from the leaves of Arctium minus ssp. minus. MATERIALS AND METHODS The ethanolic and aqueous extracts from the leaves of Arctium minus ssp. minus were evaluated in mice for anti-inflammatory activity using carrageenan-induced hind paw edema model and for antinociceptive activity using p-benzoquinone-induced abdominal contractions test. Moreover, the antioxidant power of the extracts has been determined by using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and flow injection analysis-luminol chemiluminescence (FIA-CL). In addition, the total phenolic content in both extracts was determined with spectrophotometric method. RESULTS Our results showed that only the ethanol extract exhibited a dose-dependent anti-inflammatory activity ranging between 11.1 and 23.6% at 200mg/kg dose as well as displayed a significant antinociceptive activity without inducing any gastric damage. Although, both extracts were shown to possess significant DPPH radical-scavenging activity, that of aqueous extract was found to have more pronounced activity. In FIA-CL system, the ethanol extract was shown to possess a significant scavenger activity against H(2)O(2) while the aqueous extract was much more potent antioxidant activity against HOCl-luminol CL than ethanol extract. CONCLUSION According to our results, it was concluded that Arctium minus ssp. minus contains potent natural antioxidants. In this study, in vivo experimental results have also supported the folk medicinal utilization of Arctium minus ssp. minus.

Time-Dependent Variations in Serum Nitrite, 6-Keto-Prostaglandin F1α and Thromboxane B2 Levels Induced by Lipopolysaccharide in Mice

Clinical features of certain immuno-inflammatory disorders exhibit time-dependent fluctuations, which could be related to circadian rhythmicity of proinflammatory mediator production. Many biologically active substances including nitric oxide (NO) and eicosanoids are released into the circulation in sepsis. Increased NO and eicosanoid levels have been reported to be responsible from death in septic shock. The aim of this study was to investigate the variations in the NO and eicosanoid production and mortality induced by bacterial endotoxin, lipopolysaccharide (LPS) injected either in the morning or in the evening. Experiments were performed on mice synchronised to 12 h light and 12 h dark (lights on at 09:00 h). Animals were injected intraperitoneally with LPS (10 mg/kg) at 09:00 (morning) and 21:00 h (evening) alone or in combination with aminoguanidine (NO synthase (NOS) inhibitor) (100 mg/kg) or indomethacin (cyclooxygenase (COX) inhibitor) (100 mg/kg). The serum was separated from blood samples obtained at nine different time points. Nitrite (stable product of NO), 6-keto-prostaglandin F1α (6-keto-PGF1α, stable product of prostacyclin) and thromboxane B2 (TxB2, stable product of thromboxane) concentrations in serum samples were measured. Serum nitrite levels showed a 24 h circadian rhythmicity depending on LPS injection time. Morning injection caused a peak after 15 h, while evening injection had two peaks after 9 and 18 h. The peak values obtained from morning and evening injections were significantly decreased by aminoguanidine and indomethacin. When LPS injected to mice in the morning and in the evening, it gradually increased the mortality rate within 24 h which could be abolished by aminoguanidine, but not indomethacin. Indomethacin-induced inhibition on LPS-induced nitrite levels was higher in the morning than in the evening. 6-keto-PGF1α and TxB2 levels were decreased by indomethacin when injected with LPS at both injection times, but not aminoguanidine. These results showed that there is an interaction between NO and eicosanoids, and LPS may produce different effects on NOS activity, but not eicosanoid production and mortality, depending on injection time in the experimental septic shock model in mice. Chronopharmacological manipulations of NOS and COX pathways and interactions between them could lead to novel therapeutic approaches for the treatment of septic shock.

In Vitro Evidence of Tissue Susceptibility Rhythms. I. Temporal Variation in Effect of Potassium Chloride and Phenylephrine on Rat Aorta

In this study, time-dependent variations in the in vitro sensitivity of rat thoracic aorta rings to potassium chloride (KCl) and phenylephrine (Phe) were investigated. Animals were synchronized with a 12h light and 12h darkness (lights on 08:00-20:00) schedule, and thoracic aortas were obtained at six different times of the day (1, 5, 9, 13, 17, and 21 hours after lights on). In order to avoid endothelial influence, all experiments were performed in endothelium-denuded preparations. Responses to KCI showed time-dependent variations in all the concentrations used. Phe-induced contractions also exhibited time-dependent differences. The rhythmic pattern of Phe responses did not change with the presence of the alpha1-adrenergic antagonist prazosin. In addition, both the EC50 values of KCl and Phe, and also the K(B) values of prazosin, displayed rhythmicity. In conclusion, time of obtaining tissues is an important factor for experimental standardization in, at least, vascular smooth muscle preparations.

Circadian-rhythm-dependent effects of l-nG-nitroarginine methyl ester (L-name) on morphine-induced analgesia

Circadian changes in the interactions between L-NG-nitroarginine methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor, and morphine-induced antinociception were investigated by the mouse hot-plate test. Both the basal pain sensitivity and morphine-induced analgesia undergo significant 24 h variations. L-NAME (40 mg/kg, i.p.) alone did not show any antinociceptive activity, but potentiated morphine-induced analgesia when combined with morphine at all injection times. In terms of percentage absolute potentiation (%AP), L-NAME dramatically augmented the analgesic effect of morphine in the late dark period at 19 hours after lights on (HALO). It is concluded that nitric oxide (NO) is involved in the modulation of the analgesic effect of morphine; thus, the L-NAME and morphine combination might be beneficial in alleviating pain.

In Vitro Susceptibility Rhythms. II. Biological-Time-Dependent Differences in Effect of βr and β2-Adrenergic Agonists of Rat Aorta and Influence of Endothelium

Time-dependent variations in the vasodilator effects of β-adrenergic agonists terbutaline (Ter) and dobutamine (Dob) were studied in isolated rings of rat thoracic aorta in both endothelium-intact and endothelium-de-nuded preparations. Rats were housed in light from 08: 00 to 20: 00 and in darkness from 20: 00 to 08: 00 and sacrificed at six different times of the day. In endothelium-intact and endothelium-denuded aortic rings precontracted submaximally with phenylephrine (Phe), addition of Ter and Dob produced concentration-dependent relaxations. Removal of endothelium reduced the relaxant responses and area under curve (AUC) values and augmented the EC50 values to Ter and Dob at most, but not all, time points. Two-way analysis of variance (ANOVA) revealed that AUCs, maximum responses, and EC50values significantly depended on both treatment (endothelium intact/endothe-lium denuded) and time of sacrifice. Results of the present study clearly show that in vitro sensitivity of rat thoracic aorta to P-adrene...

Antinociceptive effect of some Amaryllidaceae plants in mice

The antinociceptive effects of ethanolic extracts of Pancratium maritimum L., Narcissus tazetta subspecies tazetta and Leucojum aestivum L. bulbs have been investigated in mice using the p‐benzoquinone‐induced abdominal constriction and hot‐plate tests.