The furoxan nitric oxide donor,PRG150, evokes dose‐dependent analgesia in a rat model of painful diabetic neuropathy

Painful diabetic neuropathy (PDN) is a type of peripheral neuropathic pain that is often intractable. Elevated nitric oxide (NO) from neuronal and non‐neuronal sources in the somatosensory system is implicated in the pathobiology of peripheral neuropathic pain. However, in diabetes, nitrergic nerve degeneration to deplete NO bioactivity appears causal in the pathogenesis of irreversible autonomic neuropathy, another long term complication of diabetes. Hence, this study hypothesized that progressive NO depletion may underpin the pathobiology of PDN and that NO donors may alleviate PDN. Diabetes was induced in rats with intravenous streptozotocin (STZ) at 70 mg/kg and confirmed if blood glucose levels (BGLs) on day 10 post‐STZ were ≥15 mmol/L. Analgesic efficacy of subcutaneous (s.c.) bolus doses of the furoxan NO donor, PRG150 was assessed in the STZ‐diabetic rat model of PDN at 10‐, 14‐ and 24‐weeks post‐STZ relative to the sydnominine NO donor, SIN‐1 and its prodrug, molsidomine. PRG150 produced dose‐dependent analgesia in STZ‐diabetic rats whereas SIN‐1 and molsidomine evoked neuro‐excitatory side‐effects, but not analgesia. The 1000‐fold larger doses of PRG150 needed to produce analgesia at 14‐ and 24‐weeks (800 pmol/kg) c.f. 10‐weeks (8 fmol/kg) post‐STZ in rats, suggest that progressive NO depletion is also causal in PDN. Importantly, doses of PRG150 up to 10 000 fold higher than the analgesic dose did not produce hypotension in rats. The 50‐fold greater release of NO by SIN‐1 c.f. PRG150 in vitro, may underpin the neuro‐excitatory rather than analgesic effects of SIN‐1/molsidomine. PRG150 is worthy of further investigation as a potential novel analgesic for PDN.     

Nitric oxide modulates μ‐opioid receptor function in vitro

Painful diabetic neuropathy (PDN) is a type of peripheral neuropathic pain that develops as a consequence of prolonged hyperglycaemia‐induced injury to the long nerves. Apart from pain, PDN is also characterized by morphine hyposensitivity. Intriguingly, in streptozotocin (STZ)‐induced diabetic rats exhibiting marked morphine hyposensitivity, dietary administration of the nitric oxide (NO) precursor, L‐arginine at 1 g/d, progressively rescued morphine efficacy and potency over an 8‐week treatment period. In earlier work, single bolus doses of the furoxan nitric oxide (NO) donor, PRG150 (3‐methylfuroxan‐4‐carbaldehyde), evoked dose‐dependent pain relief in STZ‐diabetic rats but the efficacious doses were 3‐4 orders of magnitude higher in advanced diabetes than that required in early STZ diabetes. Together, these findings suggested a role for NO in the modulation of μ‐opioid (MOP) receptor signalling. Therefore, the present study was designed to assess a role for NO released from PRG150, in modulating MOP receptor function in vitro. Here, we show an absolute requirement for the MOP receptor, but not the δ‐opioid (DOP) or the κ‐opioid (KOP) receptor, to transduce the cellular effects of PRG150 on forskolin‐stimulated cAMP responses in vitro. PRG150 did not interact with the classical naloxone‐sensitive binding site of the MOP receptor, and its effects on cAMP responses in HEK‐MOP cells were also naloxone‐insensitive. Nevertheless, the inhibitory effects of PRG150 on forskolin‐stimulated cAMP responses in HEK‐MOP cells were dependent upon pertussis toxin (PTX)‐sensitive G_i/o proteins as well as membrane lipid rafts and src kinase. Together, our findings implicate a role for NO in modulating MOP receptor function in vivo.     

Pharmacological characterization of standard analgesics on mechanical allodynia in streptozotocin-induced diabetic rats

The present study was designed to investigate the anti-allodynic effects of current analgesic agents, such as pregabalin, amitriptyline, mexiletine, morphine, and diclofenac, in a rat model of streptozotocin (STZ)-induced diabetic neuropathy. Diabetic rats developed a sustained decrease in withdrawal threshold response to the von Frey test within 8 weeks after a single injection of STZ (45 mg/kg, i.v.). The anti-allodynic effects of analgesic agents were examined after a single oral or subcutaneous administration at 3 and 7 weeks after beginning of STZ-treatment. Pregabalin (3-30 mg/kg, p.o.), an antiepileptic agent, dose-dependently blocked the mechanical allodynia in rats treated both at 3 and 7 weeks. Mexiletine (10-100 mg/kg, p.o.), a sodium channel blocker, dose-dependently ameliorated mechanical allodynia in rats treated at 3 weeks; however, the efficacy was diminished at 7 weeks. Morphine (1-10 mg/kg, s.c.) was effective in rats treated at 3 weeks; however, it was ineffective at 7 weeks. Conversely, an antidepressant amitriptyline (0.3-3 mg/kg, p.o.) improved mechanical allodynia in rats treated at 7 weeks, whereas it was ineffective at 3 weeks. Diclofenac, a non-steroidal anti-inflammatory drug, was ineffective at both time points. These results demonstrate that, except for diclofenac, the standard analgesic agents tested can effectively alleviate the mechanical allodynia seen in STZ-induced diabetic neuropathy. Their efficacies varied depending on the duration of the diabetic condition, suggesting that temporal changes in pharmacodynamic factors could affect the responsiveness of this model to analgesic agents.     

Potential excitatory role of nitric oxide on 2‐deoxy‐d‐glucose‐induced gastric motility in rats

Previous studies have shown that 2‐deoxy‐d ‐glucose (2‐DG) increases gastric motility via the vagus nerve, but the underlying mechanism remains elusive. Since nitric oxide (NO) is involved in gastric motility, a possible interplay between 2‐DG and NO can be suggested. In the present study, Wistar rats (250‐350 g) of both sexes were intravenously injected with 2‐DG (200 mg/kg), and the effects of the intravenous injection of the nitric oxide synthase (NOS) inhibitors; nitro‐l ‐arginine methyl ester (l ‐NAME, 10 mg/kg) and N^ω‐nitro‐l ‐arginine (l ‐NNA, 10 mg/kg) were investigated. Animals were anaesthetized and cannulated for intravenous drug injections while the left vagal nerve was electrically stimulated (0.1‐10 Hz, 0.5 ms duration, 12 V, for 60 seconds), and intragastric pressure and gastric motility changes were monitored using a latex gastric balloon. 2‐DG increased the mean intragastric pressure (baseline, 5.0±0.4 cmH₂O; after 2‐DG, 14.4±1.5 cmH₂O; P=.0156) and significantly increased the gastric motility index, while NOS inhibitors significantly attenuated both parameters. However, pretreatment with NOS inhibitors significantly augmented the gastric responses to peripheral electrical vagal stimulation. These results suggest that NO plays an excitatory role in gastric responsiveness to 2‐DG and that this function may be effected in the central nervous system.     

Protective Effects of d‐Limonene on Lipid Peroxidation and Antioxidant Enzymes in Streptozotocin‐Induced Diabetic Rats

The aim of this study was to evaluate the protective effects of d ‐limonene on the levels of lipid peroxidation by‐products and antioxidant defence systems in the plasma and tissues of normal and streptozotocin (STZ)‐induced diabetes rats. The experimental diabetes was induced in rats by a single dose of STZ (40 mg/kg i.p.) injection, and treatment with d ‐limonene was continued for 45 days. After the treatment period, oxidative stress parameters such as lipid peroxidation by‐products; enzymatic antioxidants such as superoxide dismutase, catalase, glutathione peroxidase and glutathione‐S‐transferase; non‐enzymic antioxidants including reduced glutathione, Vitamins C and E were measured in the plasma and tissues of experimental rats. An increase in the levels of lipid peroxidation by‐products and significant decrease in antioxidant enzymes were observed in untreated diabetic rats. Administration of d ‐limonene to diabetic rats for 45 days caused a significant reduction in the levels of lipid peroxidation by‐products and an increase in the activities of antioxidant enzymes, when compared with the untreated diabetic group. There was no significant difference in normal treated groups, when compared with normal rats. Biochemical observations were substantiated with the help of histopathological examinations through its antioxidant properties and thereby conferred protection against STZ‐induced diabetic rats. The result of this study indicates that d ‐limonene has antioxidant potential in addition to its antidiabetic effect in experimental diabetes.     

Antihyperglycaemic action of diosmin,a citrus flavonoid,is induced through endogenous β‐endorphin in type I‐like diabetic rats

Diosmin is one of the flavonoids contained in citrus and has been demonstrated to improve glucose metabolism in diabetic disorders. However, the mechanism(s) of diosmin in glucose regulation remain obscure. Therefore, we investigated the potential mechanism(s) for the antihyperglycaemic action of diosmin in streptozotocin‐induced diabetic rats (STZ‐diabetic rats). Diosmin lowered hyperglycaemia in a dose‐dependent manner in STZ‐diabetic rats. This action was inhibited by naloxone at a dose sufficient to block opioid receptors. Additionally, we determined the changes in plasma β‐endorphin‐like immunoreactivity (BER) using enzyme‐linked immunosorbent assay (ELISA). Diosmin also increased BER dose‐dependently in the same manner. Repeated treatment of STZ‐diabetic rats with diosmin for 1 week resulted in an increase in the expression of the glucose transporter subtype 4 (GLUT 4) in the soleus muscle and a reduction in the expression of phosphoenolpyruvate carboxykinase (PEPCK) in the liver. These effects were also inhibited by naloxone at a dose sufficient to block opioid receptors. Bilateral adrenalectomy in STZ‐diabetic rats eliminated the actions of diosmin, including both the reduction in hyperglycemia and the elevation of plasma BER. In conclusion, our results suggest that diosmin may act on the adrenal glands to enhance the secretion of β‐endorphin, which can stimulate the opioid receptors to attenuate hepatic gluconeogenesis and increase glucose uptake in soleus muscle, resulting in reduced hyperglycemia in STZ‐diabetic rats.     

Asymmetric dimethylarginine does not inhibit arginase activity and is pro‐proliferative in pulmonary endothelial cells

Asymmetric dimethylarginine (ADMA) is an endogenously produced nitric oxide synthase (NOS) inhibitor. l ‐Arginine can be metabolised by NOS and arginase, and arginase is the first step in polyamine production necessary for cellular proliferation. We tested the hypothesis that ADMA would inhibit NOS but not arginase activity and that this pattern of inhibition would result in greater l ‐arginine bioavailability to arginase, thereby increasing viable cell number. Bovine arginase was used in in vitro activity assays with various concentrations of substrate (l ‐arginine, ADMA, N^G‐monomethyl‐l ‐arginine (L‐NMMA) and N^G‐nitro‐l ‐arginine methyl ester (l ‐NAME)). Only l ‐arginine resulted in measurable urea production (K_m = 6.9 ± 0.8 mmol/L; V_max = 6.6 ± 0.3 μmol/mg protein per min). We then incubated bovine arginase with increasing concentrations of ADMA, l ‐NMMA and l ‐NAME in the presence of 1 mmol/L l ‐arginine and found no effect of any of the tested compounds on arginase activity. Using bovine pulmonary arterial endothelial cells (bPAEC) we determined the effects of ADMA on nitric oxide (NO) and urea production and found significantly lower NO production and greater urea production (P < 0.003) with ADMA, without changes in arginase protein levels. In addition, ADMA treatment resulted in an approximately 30% greater number of viable cells after 48 h than in control bPAEC. These results demonstrate that ADMA is neither a substrate nor an inhibitor of arginase activity and that in bPAEC ADMA inhibits NO production and enhances urea production, leading to more viable cells. These results may have pathophysiological implications in disorders associated with higher ADMA levels, such as pulmonary hypertension.     

Effect of orchiectomy on renal function in control and diabetic rats with chronic inhibition of nitric oxide

• 1 Male gender is associated with higher blood pressure (BP) and more rapid loss of renal function in a spectrum of clinical and experimental renal diseases, including diabetic nephropathy. Consequently, modulation of testosterone levels could exert beneficial effects in the diabetic kidney.
• 2 The aim of the present study was to determine whether testosterone deficiency (orchiectomy) could influence BP and renal function in streptozotocin‐diabetic rats, with or without accelerated endothelial dysfunction achieved by chronic inhibition of nitric oxide (NO) synthesis using N^G‐nitro‐l‐ arginine methyl ester (l ‐NAME; 40–100 mg / L in the drinking water for 2 weeks), as well as in age‐matched non‐diabetic rats subjected to the same interventions.
• 3 Orchiectomy did not affect l ‐NAME‐induced increases in BP in non‐diabetic or diabetic rats. In non‐diabetic rats, orchiectomy prevented l ‐NAME‐induced increases in proteinuria. These effects on proteinuria were not observed in diabetic rats. In non‐diabetic rats, orchiectomy had no effect on renal haemodynamics in animals receiving vehicle and did not affect l ‐NAME‐induced changes in renal haemodynamics, characterized by reductions in renal plasma flow (RPF) and higher filtration fractions (FF). In intact diabetic rats, l ‐NAME treatment resulted in lower RPF. This difference was not observed in diabetic rats subjected to orchiectomy, although l ‐NAME‐treated diabetic orchiectomized rats had lower RPF and higher FF compared with vehicle‐treated intact diabetic rats.
• 4 In conclusion, we report modest beneficial effects of orchiectomy on proteinuria in normal, but not in diabetic, rats with inhibition of NO production. This suggests that testosterone reduction does not attenuate the deleterious impact of the diabetic metabolic milieu in the kidney.

     

Modification of morphine analgesia by venlafaxine in diabetic neuropathic pain model

BackgroundThe purpose of this study was to investigate the influence of single or chronic (21 days) administration of the serotonin and noradrenaline reuptake inhibitor, venlafaxine, on the antinociceptive action of the opioid receptor agonist, morphine, in streptozotocin (STZ)-induced hyperalgesia.MethodsThe studies were performed on male Wistar rats. Changes in nociceptive thresholds were determined using mechanical stimuli. Diabetes was induced by a single administration of STZ (40 mg/kg, im).ResultsVenlafaxine was shown to modulate analgesic activity of morphine in STZ-induced hyperalgesia. However, whereas acute co-administration of venlafaxine increased the analgesic activity of morphine, chronic treatment with venlafaxine attenuated opioid efficacy.ConclusionDepending on the mode of administration (single or long-term), venlafaxine modulates analgesic activity of morphine. Further investigations are necessary to clarify the mechanisms of these interactions, which may be clinically relevant.     

Serum 25‐Hydroxyvitamin D and Parathyroid Hormone Levels in Non‐Lactating Women with Post‐Partum Thyroiditis: The Effect of l‐Thyroxine Treatment

Vitamin D deficiency seems to be implicated in the onset and progression of some autoimmune disorders. No previous study has investigated vitamin D homeostasis in post‐partum thyroiditis. We compared 25‐hydroxyvitamin D and parathyroid hormone (PTH) levels between four groups of non‐lactating women who gave birth within 12 months before the beginning of the study: hypothyroid women with post‐partum thyroiditis (group A; n = 14), euthyroid females with post‐partum thyroiditis (group B; n = 14), women with non‐autoimmune hypothyroidism (group C; n = 16) and healthy euthyroid females without thyroid autoimmunity (group D; n = 15). In the second part of the study, groups A and C were treated for 6 months with l ‐thyroxine. Serum levels of 25‐hydroxyvitamin D were lower, while PTH higher in patients with post‐partum thyroiditis than in patients without thyroid autoimmunity. They were also lower (25‐hydroxyvitamin D) or higher (PTH) in group A than in group B, as well as in group C in comparison with group D. l ‐thyroxine treatment increased 25‐hydroxyvitamin D and reduced PTH levels only in hypothyroid women with post‐partum thyroiditis. Baseline levels of 25‐hydroxyvitamin D correlated with thyroid antibody titres, thyroid function and circulating PTH levels, while the effect of l ‐thyroxine on serum levels of this vitamin correlated with the changes in thyroid antibody titres and PTH levels. The results of our study suggest the association of vitamin D status with post‐partum thyroiditis and l ‐thyroxine treatment of this disorder.     

Argirein alleviates corpus cavernosum dysfunction by suppressing pro‐inflammatory factors p66Shc and ER stress chaperone Bip in diabetic rats

Objectives The aim was to investigate whether argirein, which releases rhein and l ‐arginine after medication, could improve erectile dysfunction (ED) in diabetic rats through normalising the abnormalities of nitric oxide synthase (NOS), p66Shc and immunoglobulin heavy‐chain binding protein (Bip), in the corpus cavernosum (CC). Methods SD rats were randomly divided into six groups. Except for the control group, rats were injected with streptozotocin (STZ) (60 mg/kg, i.p.) once. During weeks 5–8 following STZ injection, except for STZ‐injected untreated rats, others were treated with aminoguanidine (AMG; 100 mg/kg/day, i.g.), or argirein at three doses (50, 100 and 200 mg/kg/day, i.g.). The vascular activity and biomarkers of the cavernosum were examined. Key findings Constrictive and dilative activity was abnormal in the CC, associated with decreased nitric oxide (NO) in serum in the diabetic (DM) group. Increased expression of p66Shc, Bip and inducible nitric oxide synthase (iNOS) and decreased endothelial nitric oxide synthase (eNOS) in the CC were significant in DM rats. Argirein and AMG improved these abnormities significantly. Conclusions We concluded that vascular activity of the cavernosal tissue was impaired due to upregulated p66Shc and Bip in the diabetic CC. Argirein alleviates the vascular dysfunction of the CC by suppressing these upregulated pro‐inflammatory proteins caused by diabetic lesions.     

Minocycline Attenuates Depressive‐Like Behaviour Induced by Rat Model of Testicular Torsion: Involvement of Nitric Oxide Pathway

Testicular torsion/detorsion (T/D) can induce depression in pre‐ and post‐pubertal patients. This study was conducted to investigate the psychological impact of testicular torsion and mechanism underlying its depressive‐like behaviour, as well as antidepressant‐like activity of minocycline and possible involvement of nitric oxide (NO)/cyclic GMP pathway in this paradigm in male rats undergoing testicular T/D. Unilateral T/D was performed in 36 male adult Wistar rats, and different doses of minocycline were injected alone or combined with N^ω‐nitro‐l ‐arginine methyl ester (l ‐NAME), non‐specific NO synthase (NOS) inhibitor; aminoguanidine (AG), specific inducible NOS inhibitor; l ‐arginine, an NO precursor; and selective PDE5I, sildenafil. After assessment of locomotor activity in open‐field test, immobility times were recorded in the forced swimming test (FST). Moreover, 30 days after testicular T/D, testicular venous testosterone and serum nitrite concentrations were measured. A correlation was observed between either a decrease in plasma testosterone or an increase in serum nitrite concentrations with prolongation in immobility time in the testicular T/D‐operated rats FST. Minocycline (160 mg/kg) exerted the highest significant antidepressant‐like effect in the operated rats in the FST (p < 0.001). Furthermore, combination of subeffective doses of minocycline (80 mg/kg) and either l ‐NAME (10 mg/kg) or AG (50 mg/kg) demonstrated a significant robust antidepressant‐like activity in T/D group (p < 0.01). Consequently, NO/cGMP pathway was involved in testicular T/D‐induced depressive‐like behaviour and antidepressant‐like activity of minocycline in the animal model. Moreover, a contribution was observed between either decreased testosterone or elevated serum nitrite levels and depressive‐like behaviour following testicular T/D.     

Role of nitric oxide in the antifibrotic and anticholestatic actions of interferon‐α2b

Interferons possess important antifibrotic properties. In addition, there is evidence that they induce the production of nitric oxide (NO) and that it downregulates the synthesis of extracellular matrix by certain cells. The aim of the present work was to evaluate if L ‐arginine, the NO synthase substrate, is able to increase the antifibrotic properties of interferon‐α_2b and if L ‐NAME, an NO synthesis inhibitor, can prevent them. Fibrosis was induced by bile duct ligation (BDL) for 5 weeks in rats and interferon‐α_2b (IFN; 100,000 IU rat, s.c., daily) and/or L ‐arginine (500 mg/kg, p.o., twice daily) or L ‐NAME (100 mg/kg, p.o., twice daily) were administered. Collagen content was determined by measuring hydroxyproline in liver samples. Malondialdehyde (MDA) was used to estimate lipid peroxidation levels. Glycogen was measured colorimetrically. Serum enzyme activities and bilirubins were determined by standard procedures. Fibrosis was increased 6‐fold by BDL. L ‐arginine or IFN partially prevented the increment in collagen. Furthermore, administration of both drugs simultaneously showed an additive effect (P < 0.05), while L ‐NAME abolished the protective effect of IFN. The same effect was observed on the other markers of liver function or damage studied herein. The additive effects of L ‐arginine and IFN could be due to a synergism of both compounds by increasing NO concentration, which can act as an antifibrotic agent but also as a cytoprotective compound. These results also suggest that the protective effects of IFN are mediated by NO, since L ‐NAME prevented them. Drug Dev. Res. 48:45–52, 1999. © 1999 Wiley‐Liss, Inc.     

Oxcarbazepine antinociception in animals with inflammatory pain or painful diabetic neuropathy

1. Diabetic neuropathy is one of the most frequent complications of diabetes mellitus. However, the mechanisms underlying these disorders are not yet well defined and it has been reported that currently available analgesics have hardly any ameliorating effect on painful diabetic neuropathy. 2. The purpose of the present study was to evaluate the antinociceptive effect of oxcarbazepine (OCBZ), a keto derivative of carbamazepine (CBZ), in animal models generally used in pain research and in rats and mice with streptozotocin (STZ)-induced diabetes. In addition, we compared the effect of OCBZ with those of CBZ, mexiletine and morphine. 3. Diabetes was induced by injection of STZ at a dose of 300 mg/kg (i.p.) in mice and 50 mg/kg (i.v.) in rats. Experiments were conducted 2 weeks after STZ injection and those animals with a serum glucose level above 400 mg/dL were used for data analysis. Antinociceptive effects of the drugs were evaluated by the paw withdrawal test (normal, STZ-induced diabetic and carrageenin-injected rats), tail-flick test (normal and STZ-induced diabetic mice) and nociceptive behaviour (formalin-injected mice). 4. In the present study, diabetic mice showed thermal hyperalgesia and diabetic rats exhibited mechanical hyperalgesia. From these results, the STZ-induced diabetic animals used in the present study were found to be suitable for research on painful diabetic neuropathy. In STZ-induced diabetic animals, the antinociceptive effects of OCBZ, CBZ and mexiletine were facilitated, whereas the effect of morphine was attenuated, compared with effects in normal animals. 5. Oxcarbazepine inhibited the formalin-induced biphasic pain responses and increased the nociceptive threshold in the case of carrageenin-induced hyperalgesia. In view of these results, inhibition of substance P-mediated pain transmission may be involved in the antinociceptive action of OCBZ. 6. These results indicate that OCBZ has an analgesic action and is a possible therapeutic agent for the treatment of neuropathic pain, such as occurs in painful diabetic neuropathy.     

Ramipril and resveratrol co‐treatment attenuates RhoA/ROCK pathway‐regulated early‐stage diabetic nephropathy‐associated glomerulosclerosis in streptozotocin‐induced diabetic rats

Clinical studies have shown that hyperglycemia can induce early‐stage diabetic nephropathy (DN). Furthermore, oxidative stress, tubular epithelial‐mesenchymal transition and extracellular matrix accumulation promote the progression of DN to chronic kidney disease and tubulointerstitial fibrosis. It is necessary to initiate treatment at the early stages of DN or even during the early stages of diabetes. In this work, rats with streptozotocin (STZ)‐induced diabetes mellitus (DM) presented early DN symptoms within 45 days, and collagen accumulation in the glomerulus of the rats was primarily mediated through the RhoA/ROCK pathway instead of the TGF‐β signaling pathway. Resveratrol (15 mg/kg/day) and ramipril (10 mg/kg/day) co‐treatment of STZ‐induced DN rats showed that glomerulosclerosis in early‐stage DN was reversible (P < .05 compared with that in STZ‐induced DM rats). The results of this study support early intervention in diabetes or DN as a more efficient therapeutic strategy.     

New 1,4‐Dihydropyridines Down‐regulate Nitric Oxide in Animals with Streptozotocin‐induced Diabetes Mellitus and Protect Deoxyribonucleic Acid against Peroxynitrite Action

Diabetes mellitus (DM) and its complications cause numerous health and social problems throughout the world. Pathogenic actions of nitric oxide (NO) are responsible to a large extent for development of complications of DM. Search for compounds regulating NO production in patients with DM is thus important for the development of pharmacological drugs. Dihydropyridines (1,4‐DHPs) are prospective compounds from this point of view. The goals of this study were to study the in vivo effects of new DHPs on NO and reactive nitrogen and oxygen species production in a streptozotocin (STZ)‐induced model of DM in rats and to study their ability to protect DNA against nocive action of peroxynitrite. STZ‐induced diabetes caused an increase in NO production in the liver, kidneys, blood and muscles, but a decrease in NO in adipose tissue of STZ‐treated animals. Cerebrocrast treatment was followed by normalization of NO production in the liver, kidneys and blood. Two other DHPs, etaftorone and fenoftorone, were effective in decreasing NO production in kidneys, blood and muscles of diabetic animals. Furthermore, inhibitors of nitric oxide synthase (NOS) and an inhibitor of xanthine oxidoreductase (XOR) decreased NO production in kidneys of diabetic animals. Treatment with etaftorone decreased expression of inducible NOS and XOR in kidneys, whereas it increased the expression of endothelial NOS. In vitro, the studied DHPs did not significantly inhibit the activities of NOS and XOR but affected the reactivity of peroxynitrite with DNA. These new DHPs thus appear of strong interest for treatment of DM complications.     

Detection and identification of 2‐nitro‐morphine and 2‐nitro‐morphine‐6‐glucuronide in nitrite adulterated urine specimens containing morphine and its glucuronides

In vitro urine adulteration is a well‐documented practice adopted by individuals aiming to evade detection of drug use, when required to undergo mandatory sports and workplace drug testing. Potassium nitrite is an effective urine adulterant due to its oxidizing potential, and has been shown to mask the presence of many drugs of abuse. However, limited research has been conducted to understand its mechanism of action, and to explore the possibility of the drugs undergoing direct oxidation to form stable reaction products. In this study, opiates including morphine, codeine, morphine‐3‐glucuronide and morphine‐6‐glucuronide were exposed to potassium nitrite in water and urine to mimic the process of nitrite adulteration. It was found that two stable reaction products were detected by liquid chromatography‐mass spectrometry (LC‐MS) when morphine and morphine‐6‐glucuronide were exposed to nitrite. Isolation and elucidation using spectrometric and spectroscopic techniques revealed that they were 2‐nitro‐morphine and 2‐nitro‐morphine‐6‐glucuronide, respectively. These reaction products were also formed when an authentic morphine‐positive urine specimen was fortified with nitrite. 2‐Nitro‐morphine was found to be stable enough to undergo the enzymatic hydrolysis procedure and also detectable by gas chromatography‐mass spectrometry (GC‐MS) after forming a trimethylsilyl derivative. On the contrary, morphine‐3‐glucuronide did not appear to be chemically manipulated when exposed to potassium nitrite in urine. These reaction products are not endogenously produced, are relatively stable and can be monitored with both LC‐MS and GC‐MS confirmatory techniques. As a result, these findings have revealed the possibility for the use of 2‐nitro‐morphine and 2‐nitro‐morphine‐6‐glucuronide as markers for the indirect monitoring of morphine and morphine‐6‐glucuronide in urine specimens adulterated with nitrite. Copyright © 2013 John Wiley & Sons, Ltd.     

Antinociceptive and antidiarrheal effects of pioglitazone in a rat model of diarrhoea‐predominant irritable bowel syndrome: Role of nitric oxide

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