The Role of Nitric Oxide in Diabetes – induced  changes of Morphine tolerance in Rats.  Khojasteh Joharchia, Masoumeh Jorjanib*a, b Neuroscience Research Center & Department of Pharmacology,Faculty of Medicine,Shaheed Beheshti University of Medical Sciences,Tehran, Iran   b*Corresponding Author: Masoumeh Jorjani, Ph.DNeuroscience Research Center & Department of PharmacologyFaculty of Medicine,Shaheed Beheshti University of Medical SciencesEvin, Tehran, Islamic Republic of IranP.O.Box: 19835-181, Tel: 22414131-3(229), Fax: 22403154E-mail: mjorjani@sbmu.   Original articleAbstractSeveral neuroendocrine complications including diabetes change the morphine antinociception and the development of tolerance to the drug. Morphine antinociception was reduced significantly in morphine tolerant diabetic rats compared to the non-diabetic animals. The exact mechanism of this effect is not known. This study was performed to determine the role of nitric oxide (NO) on morphine tolerance in diabetic state. Nociceptive responses in alloxan-induced diabetic morphine tolerated rats were measured by the hot-plate test.

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The urinary nitric oxide level was measured spectrophotometrically with Griess reagent. For the conversion of nitrate to nitrite, vanadium chloride was used. The results showed that experimental diabetes increased morphine analgesia. Conversely, degree of tolerance to morphine was diminished in diabetic state. The urinary nitrite content in diabetic morphine tolerated rats was higher than non-diabetic groups.

L-arginine significantly increased the NO production in diabetic morphine tolerated animals, whereas aminoguanidine decreased it. Appropriately, L-arginine increased the latency time of reaction to noxious stimuli in diabetic compared to non-diabetic rats. L-arginine-treated animals also showed more tolerance to morphine analgesia. As expected, aminoguanidine deducted the level of morphine tolerance in diabetic animals.

It is suggested that NO has a modulatory role in the effects of diabetes on morphine analgesia and tolerance. Keywords: Morphine analgesia, Tolerance, Experimental diabetes, Nitric Oxide. 1.0.  IntroductionOpiates, chiefly morphine, are commonly known as the most useful drugs for relief of pain. However, the uses of opioids are restricted due to development of tolerance and the physical or mental dependency to these drugs.

It has been reported that several neuroendocrine complications including diabetes, change the morphine antinociception and the development of tolerance or dependency to this drug (Leedom and Meeran, 1989). The increase in blood glucose concentration or hyperglycemia in diabetes profoundly alters hypothalamic-pituitary function, including the activity of endogenous opiate system. Changes in the concentrations of either brain or blood glucose levels appear to modulate opioid antinociception and basal nociceptive processes (Levine et al.

, 1982). In the other hand, both clinical and animal studies show that morphine has a limited analgesia in the treatment of painful diabetic neuropathy (Galer, 1995; Ohsawa et al., 2000; Chen and Pan, 2003).

There are also several reports indicating that experimental diabetes mellitus attenuates the antinociceptive effect of morphine in animals (Simon & Dewey, 1981; Raz et al., 1988; Kamei et al., 1992; Gullapalli et al., 2002). In the previous study, we found that morphine antinociception and dependency significantly decreased in diabetic morphine tolerated animals (Joharchi & Jorjani, in press). The exact mechanisms of these diabetes-induced changes are not well known.The role of nitric oxide (NO) as a major neurotransmitter in morphine antinociception and tolerance has been established (Kolesnikov et al., 1993; Przewlocki et al.

, 1993; Brignola et al., 1994; Machelska et al., 1997).

Machelska et al reported that L-arginine, a nitric oxide precursor, reduces the antinociceptive effect of morphine (Brignola et al., 1994), whereas the constitutive nitric oxide synthase (cNOS) inhibitors potentiate the morphine analgesia in the tail-flick test (Przewlocki et al., 1993; Machelska et al., 1997). He suggested that inhibition of the spinal NO synthase potentiates the mu-, delta- and to a lesser extent, kappa opioid receptors-mediated spinal antinociception in both acute and prolonged pain. Furthermore, cNOS inhibitors were demonstrated to attenuate the tolerance developed to the analgesic effect of morphine (Majeed et al.

, 1994; Bhargava & Zhao 1996). By considerable evidences that NO modulates synaptic transmission in both the central and peripheral nervous system (Meller & Gebhart, 1993), it has been suggested that NO is involved in nociceptive processes either in the periphery or within the spinal cord (Haley & Dickenson, 1992). Enhancing of morphine antinociception after inhibition of NO synthesis in rats (Przewlocki et al., 1993) and blocking of morphine tolerance in mice indicate a selective action of NO in the mechanisms of mu receptor-mediated tolerance and dependence (Kolesnikov et al., 1993; Majeed et al., 1994). Accordingly, administration of aminoguanidine, a selective induced nitric oxide synthase (iNOS) inhibitor (Salerno et al., 2002), markedly improved the antinociceptive effect of morphine (Abdel-Zaher et al.

, 2006) and attenuated the increase in urinary nitrite concentration in diabetic mice (Grover et al., 2000).In the present study we tried to investigate the role of nitric oxide in the effects of diabetes on morphine tolerance.

 2.0. Materials and Methods2.1.

AnimalsThe male adult Sprague-Dawley rats weighing 180-250 g were used. Animals were housed in a room with ambient temperature of 22 ± 2°C, a 12-h light/dark cycle and free access to water and food. All experiments were performed in accordance with the recommendations and policies of the International Association for the Study of Pain (Zimmermann, 1983) and the Institutional Animal Welfare Law. All study protocols were approved by the internal deputy for animal research and the respective local government committee which is advised by an independent ethics committee in our Neuroscience Research Center.The experiments were designed in four main groups: diabetic, non-diabetic and their related control groups. Each subgroup consisted of 6 rats. Each animal was used only once, the total number of animals was 96. 2.

2. DrugsMorphine sulfate, alloxan and naloxone (Sigma, U.S.A.) were dissolved in distilled water, L-arginine (a NO precursor, Alexis Corporation, Switzerland) in saline and aminoguanidine (a selective iNOS inhibitor, Alexis Corporation, Switzerland) in PBS (Phosphate Buffer Saline).

Griess Reagent: 1 part of 0.1% naphthylethylene-diamine dihydrochloride in distilled water plus 1 part 1% sulfanilamide (or sulfanilic acid) in 5% concentrated H3PO4, (Alexis Corporation, Switzerland). Vanadium Chloride, (Sigma, U.S.A.) 400 mg, was prepared in 1 M HCl (50ml). 2.

3. Induction of experimental diabetesDiabetes was induced by a single injection of alloxan (100 mg/kg, s.c.

). Forty eight hours after administration, the urinary sample was collected and its glucose level was measured colorimetrically using tape-tests. The animals with urine glucose levels less than 300 mg/dl were not considered valuable for this study, those with serum glucose levels above 300 mg/dl were used as the diabetic rats.

There were not any significant differences between mean body weights of diabetic (217.3 g) versus non-diabetic (219 g) groups. 2.4. Induction of toleranceRepeated doses of morphine sulfate (7 mg/kg, i.p., once per day for 5 consecutive days) were administered to induce tolerance in animals. 2.

5. Nociceptive testingHot-plate test was used to measure the nociceptive threshold. Animals were placed on a hot plate (55 ± 0.5°C) according to the procedure described by Eddy and Leimback in 1953. The reaction time measured was either hind paw licking or jumping off the plate. The cut-off point imposed was 60 s to avoid tissue damage (Porreca et al., 1984), and we did not observe any paw edema or redness with repeated testing. Baseline latency was determined first, and hence morphine analgesia was evaluated 30 min after the injection of drug.

 2.6. Application of NO precursor and iNOS inhibitorL-arginine (100 mg/kg, i.p.) was used as a NO precursor (Brignola et al., 1994) and aminoguanidine (60 mg/kg, i.p.

) as a selective iNOS inhibitor (Griffiths et al., 1993; Salerno et al., 2002), 30 min before morphine injection, in both diabetic and non-diabetic morphine treated groups.2.7. Estimation of urinary nitriteOn the last day of experiment, each rat was placed individually in a metabolic cage and its urine was collected for 24 hours, considering that the animals were allowed to drink water ad libitum before the study, but were denied any water during the 24-h study period.

Urinary nitrate was converted to nitrite by vanadium chloride (Miranda et al., 2001). Nitrite was estimated using Griess reagent (Guevara et al., 1998) which was added to suitably diluted urine and optical density (O.D.) was measured at 545 nm (Medispec ESR 200, Elisa Plate Reader, Awareness Technology Inc. U.S.

A). 2.8. Data analysisData are expressed as the means ± S.

E.M. The One-way analysis of variance (ANOVA) followed by Tukey-Kramer and Two-way ANOVA followed by Bonferroni post test were used to calculate the statistical significance for multiple comparisons. P-value < 0.05 was considered to be significant.

 3.0. Results3.

1. Effect of experimental diabetes on morphine antinociception and tolerance The results of the hot-plate test are shown in figure 1 which indicates three facts:1)    At the baseline, even before any morphine injection, the hot plate latency time is a little higher in diabetic group but there are no significant differences between diabetic (11.17±0.87) and non-diabetic animals (8.00±0.

52) in this state. The same result (11.67±0.71 vs. 6.

83±0.60) is obtained between respective control groups.2)    Single dose administration of morphine sulfate (7 mg/kg, i.p.) induced significant analgesia in both diabetic (44.

67±3.83) and non-diabetic (22.50±0.99) rats in comparison to their baselines (P < 0.001). The interesting point is that analgesia, or increase in hot plate latency time, is significantly higher in diabetic rats compared to non-diabetic group (P < 0.001).

3)    After the induction of tolerance by chronic administration of morphine sulfate (7 mg/kg, i.p. for 5 consecutive days), the antinociceptive effect of morphine in diabetic group (14.50±1.18) is highly reduced, and becomes almost equal to non-diabetic (14.17±0.

83), and consequently, tolerance to morphine analgesia in diabetic group (P < 0.001) is more than non diabetic animals (P < 0.01) in acute pain.

 3.2. Effect of experimental diabetes on  urinary nitrite contentFigure 2 shows that the optical density (OD) at 545 nm in diabetic tolerated rats is significantly increased (349.00±1.93) comparing to its control group (339.00±2.31, P < 0.05).

It is also increased in morphine tolerated non-diabetic animals (330.67±4.3 vs. 317.17±2.73, P < 0.01).

However, this enhancement is much higher in diabetic rats comparing to non-diabetic ones, both in morphine tolerated (349.00±1.93 vs. 330.67±4.3, P < 0.001) and control groups (339.

00±2.31 vs. 317.17±2.73, P < 0.001).Administration of L-arginine (100 mg/kg, i.p.

for 5 days) as a NO precursor increased the OD in both diabetic and non-diabetic tolerated rats compared to their control groups (P < 0.001, Fig. 3A). This enhancement is more dominant in diabetic group compared with non-diabetics (P < 0.

001, Fig. 3A). Conversely, aminoguanidine (60 mg/kg, i.

p./5 days) as a selective iNOS inhibitor, decreased the OD in both groups compared with their controls (P < 0.001) and the reduction is also evident in comparison between diabetic and non-diabetic groups (P < 0.001, Fig. 3B). 3.3.

Effects of L-arginine and Aminoguanidine on morphine antinociception and tolerance in diabetic stateThe analytical results are confirmed with behavioral studies (Fig. 4 A&B). The hot plate latency time, after a single dose of morphine & L-arginine administration, significantly decreased in both diabetic and non-diabetic rats comparing to the morphine treated ones (P < 0.001 in both). This deduction was more significant in non-diabetic rats (P < 0.01). After the induction of tolerance and chronic administration of morphine & L-arginine, the potentiation of tolerance is seen in both diabetic (P < 0.

001) and non-diabetic (P < 0.001) rats compared to their morphine treated ones. In this state there is no significant respect between diabetic and no-diabetic animals, while there is a significant (P < 0.001) difference between single and chronic morphine injections in diabetic ones(Fig. 4A).

On the other hand, the hot plate latency time after administration of morphine & aminoguanidine significantly increased in both single dose and tolerant diabetic animals (P < 0.001). There is no difference in non-diabetic groups in single dose administration, but there is a significant increase in morphine analgesia in tolerant rats (P < 0.001, Fig.

4B), and there is also a considerable enhancement in latency time in diabetic groups compared with non-diabetic groups (P < 0.001) both in single dose and after the induction of tolerance (Fig. 4B). Morphine analgesia after single dose administration compared to chronic administration is not significant in diabetic groups as well as in non-diabetic animals (Fig.4B). 4.

0. DiscussionThe results of the present study demonstrate that the morphine analgesia, after a single dose administration, significantly enhanced in diabetic state comparing to non-diabetic state in experimental animals (P < 0.001, Fig.

1). This result is consistent with some earlier reports (Levine et al., 1982; Chu et al., 1986), but is conversed with some others (Simon & Dewey, 1981; Raz et al.

, 1988; Kamei et al., 1992; Gullapalli et al., 2002). After the induction of tolerance, the decrease in latency time was more predominant in diabetic group rather than in non-diabetic animals. It is proposed that in early diabetes, despite the antagonistic effect of glucose on morphine analgesia, the pain threshold is adequately maintained and even increased, partly due to a compensatory increase secretion of endogenous opioid peptides such as ß-endorphin (Leedom & Meeran, 1989; Raz et al., 1988). It has been reported that an endogenous d-opioid receptor-mediated antinociceptive system is enhanced in diabetic mice (Kamei et al.

, 1992). Others reported that the reduction of morphine antinociception in diabetic animals may not be due to changes in receptor affinity or to an alteration of opioid receptor activity (Khawaja & Green, 1992) but, may be caused by the alterations of second/ third messengers and/ or ion channels (Ohsawa et al., 2000). Experimental diabetes-induced changes in opioid receptor gene expression may also be involved (Cheng et al., 2001). Furthermore it is important to consider that diabetes mellitus constitutes a chronic stress state.

The chronic nature of ß-endorphin secretion may induce changes in opiate receptor activity within the brain, in a manner that tolerance develops to exogenous opiates. Thus, diabetic tolerated animals would be expected to be more tolerant to the analgesic effects of exogenous opiates (Leedom & Meeran, 1989).The data obtained in this study suggest that diabetes results progressive changes in the neuroendocrine system during the course of the disease. Many of the complications produced by diabetes develop over an extended period rather than as an immediate response to hyperglycemia. So the differences obtained in other studies outcomes might be attributed mainly to the time course for the development of diabetic complications, or it might be due to the differences in applied doses of morphine, types of administration (i.

c.v. or systemic), nociceptive testing or strain/gender of animals.Morphine is metabolized mostly to morphine -3-beta glucuronide in rats. This metabolite is not as potent as morphine in animal models and has no antinociceptive efficacy (Okura et al., 2007). Even though some investigators have shown that morphine-3-beta glucuronide may contribute to excitatory effects of morphine (Smith et al., 2000).

Alloxan-induced diabetes also had no effect on hepatic microsomal protein and cytochrome P-450 contents (Pachecka et al., 1993). Indeed N-demethylation, another metabolic pathway of morphine, which is prominent in rodents, is not affected by alloxan. However potential alloxan-induced differences in morphine adsorption, distribution and excretion or changes in other pharmacokinetic parameters like total clearance and volume of distribution (Courteix et al., 1998) can not be ruled out and might explain the differences observed in morphine antinociception between diabetic and non-diabetic rats.

It is shown that the experimental diabetes significantly increased urinary nitrite concentration (Grover et al., 2000). The same result was observed in this study (Fig.

2). Enhancement of urinary nitrite in morphine tolerated rats confirmed the earlier reports about the role of NO in development of tolerance to morphine in mice (Bhargava & Zhao, 1996). The fact that more increase in NO production in diabetic morphine tolerated animals was observed in this study, represent the additive effect of diabetes and morphine tolerance on NO formation, which results a markedly decrease in morphine analgesia in diabetic tolerant animals compared to non-diabetics. Note that the more enhancement of NO production after administration of L-arginine in diabetic tolerated rats compared to non-diabetics (Fig.

3A) supports this hypothesis. Aminoguanidine treatment reduces the NO production in diabetic tolerated rats (Fig. 3B). It suggests that aminoguanidine sensitive iNOS may be responsible for changes in NO formation and morphine antinociception in these diabetic morphine tolerated animals.

Some investigators have also reported that morphine tolerance is associated with increased NOS activity in laboratory animals (Przewlocki et al., 1993; Machelska et al., 1997; Majeed et al., 1994; Bhargava & Zhao 1996). Our result in non-diabetic morphine tolerated rats compared to their control group confirms this finding (Fig. 2). The significant difference between diabetic and non-diabetic with their control groups, receiving vehicle during 5 days, prove that tolerance to hot plate latency (Fig. 1) is not related to test replication or that increased nitrite production (Fig.

2) is not due to injection procedure.L-arginine potentiation of NO production is observed in both diabetic (P < 0.001) and non-diabetic (P < 0.

001) animals (Fig. 3A). Aminoguanidine markedly diminished the urinary nitrite content in non-diabetic tolerated rats (P < 0.001) as well as diabetic ones (P < 0.001). Therefore, despite of Grover’s report, we propose that iNOS may also be involved in the analgesic effect of morphine in non-diabetic animals. This proposition is supported with the very recently report which states that blockade of NO production, by aminoguanidine, via inhibition of iNOS, can attenuate the development of morphine tolerance and dependence (Abdel-Zaher et al., 2006).

This statement is similar to our results (Fig.4).  In this manner the results of behavioral studies (Fig. 4) show that L-arginine decreases the morphine analgesia and augments the tolerance to this drug both in diabetic and non-diabetic groups (P ; 0.

001, Fig. 4A). Conversely aminoguanidine potentates the antinociception of morphine and blocks the development of tolerance in both groups which is more dominant in diabetic group compared to non-diabetics (P;0.001, Fig 4B).In conclusion, it is suggested that the increased urinary nitrite content in morphine tolerated rats may be due to the increased NO production. There is the same possibility in diabetic state. Markedly decrease in morphine antinociception in morphine tolerated diabetic rats could be explained by increase of NO production compared to non-diabetic ones. Our results support the hypothesis that endogenous NO plays a role in the modulation of peripheral pain mechanism(s) and may involved in modulation of morphine effects in diabetic state, and that the L-arginine-NO pathway modulates morphine-sensitive processes.

Consequently, we come to the question; can we use inhibitors of NO formation as an adjuvant to morphine for relief of neuropathic pain? Further studies will clarify the possible mechanism(s) of the modulatory role played by NO on morphine-induced effects in diabetes.;AcknowledgementsThe authors thank Mr. Safar-Ali Ghaffari for his excellent technical assistance, and Ms. M. Zahmatkesh for her good advises in NO analysis. This research was granted by Neuroscience Research Center of Shaheed Beheshti University of Medical Sciences. Some of this work was presented at the 2nd   FAONS symposium, Tehran, Iran, May, 2004; and 11th world congress on the study of pain, Sydney, Australia, Aug., 2005.

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