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Anesth Analg 2004;98:185-192
© 2004 International Anesthesia Research Society

PAIN MEDICINE

Reduction in [D-Ala2, NMePhe4, Gly-ol5]Enkephalin-Induced Peripheral Antinociception in Diabetic Rats: The Role of the L-Arginine/Nitric Oxide/Cyclic Guanosine Monophosphate Pathway

Arda Tasatargil, MD, and Gulay Sadan, MD

Department of Pharmacology, Faculty of Medicine, Akdeniz University, Antalya, Turkey

Address correspondence and reprint requests to Arda Tasatargil, MD, Department of Pharmacology, Faculty of Medicine, Akdeniz University, 07070 Antalya, Turkey. Address e-mail to arda{at}med.akdeniz.edu.tr or ardatas@akdeniz.edu.tr.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
To test our hypothesis that the abnormally small efficacy of µ-opioid agonists in diabetic rats may be due to functional changes in the L-arginine/nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway, we evaluated the effects of N-iminoethyl-L-ornithine, methylene blue, and 3-morpholino-sydnonimine on [D-Ala2, NMePhe4, Gly-ol5]enkephalin (DAMGO)-induced antinociception in both streptozotocin (STZ)-diabetic and nondiabetic rats. Animals were rendered diabetic by an injection of STZ (60 mg/kg intraperitoneally). Antinociception was evaluated by the formalin test. The µ-opioid receptor agonist DAMGO (1 µg per paw) suppressed the agitation response in the second phase. The antinociceptive effect of DAMGO in STZ-diabetic rats was significantly less than in nondiabetic rats. N-Iminoethyl-L-ornithine (100 µg per paw), an NO synthase inhibitor, or methylene blue (500 µg per paw), a guanylyl cyclase inhibitor, significantly decreased DAMGO-induced antinociception in both diabetic and nondiabetic rats. Furthermore, 3-morpholino-sydnonimine (200 µg per paw), an NO donor, enhanced the antinociceptive effect of DAMGO in nondiabetic rats but did not change in diabetic rats. These results suggest that the peripheral antinociceptive effect of DAMGO may result from activation of the L-arginine/NO/cGMP pathway and dysfunction of this pathway; also, events that are followed by cGMP activation may have contributed to the demonstrated poor antinociceptive response of diabetic rats to µ-opioid agonists.

IMPLICATIONS: This is the first study on the role of the nitric oxide (NO)/cyclic guanosine monophosphate pathway on [D-Ala2, NMePhe4, Gly-ol5]enkephalin (DAMGO)-induced peripheral antinociception and the effect of diabetes on this pathway. The study suggests a possible role of DAMGO as a peripherally-acting analgesic drug.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Opioids were initially believed to induce antinociceptive effects exclusively through their actions in the central nervous system (1,2). The idea that opioids act to produce antinociception primarily through central mechanisms has been challenged with studies in which the local administration of opioids into inflamed tissue results in antinociception at doses that are systemically inactive (3). It has been demonstrated that local opioid receptors are present on the peripheral terminals of primary afferents, on the basis of the direct blockade of inflammatory pain (4–7). Clinically, opioids are largely used in inhibiting pain associated with cancer, arthritis, and surgery. Especially under inflammatory conditions, peripherally administered opioids can produce potent analgesic effects by interacting with local opioid receptors (8). Inflammation occurs before drug treatment in these clinical conditions (e.g., arthritis, cancer, and postoperative pain). Therefore, to obtain similarities to these clinical inflammatory diseases, we used an inflammation model, the formalin test. Moreover, antinociception is often not observed when opiates are administered locally into uninflamed tissues (8), suggesting that the inflammatory component is necessary for the full expression of peripheral antinociception. The formalin test measures the response of a long-lasting nociceptive stimulus because noxious stimulus is tonic rather than acute and, thus, may bear a closer resemblance to clinical pain (9).

Peripheral opioid receptors are coupled with the L-arginine/nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway (10,11). NO is an endogenous activator of guanylate cyclase, which results in the accumulation of cGMP (12). The peripheral antinociceptive effect of morphine is potentiated by a specific cGMP phosphodiesterase inhibitor, MY5445 (13). Furthermore, the administration of dibutyryl-cGMP into the paw results in antinociception (14). Little is known about events after the accumulation of cGMP at peripheral sites. Aley and Levine (15,16) have suggested that the development of µ-opioid acute tolerance for peripheral antinociception involves NO. These reports clearly indicate the potential involvement of NO systems in peripheral opioid actions. However, interactions between the NO/cGMP pathway and one of the peripherally-acting µ-opioid receptor agonists, [D-Ala2, NMePhe4, Gly-ol5]enkephalin (DAMGO), have not been investigated.

However, it has been reported that mice with streptozotocin (STZ)-induced diabetes are selectively hyporesponsive to µ-opioid receptor-mediated antinociception (17). Similarly, Simon and Dewey (18) also demonstrated that the antinociceptive potency of morphine is reduced in spontaneously diabetic (C57BL/Ksj-db/db) mice. The exact mechanism responsible for this poor response is still unclear. Moreover, the effect of intraplantar (ipl) DAMGO administration in diabetic rats also has not been reported.

We have shown (19) that the induction of diabetes impairs nitrergic transmission in various tissues in rats because of impairment in synthesis, release of NO, or both. It is also possible that the antinociceptive effect of DAMGO mediated by the L-arginine/NO/cGMP pathway is impaired in diabetes. Thus, the aim of this study was to investigate the role of the L-arginine/NO/cGMP pathway on the antinociceptive effect of DAMGO and to clarify the hypothesis that dysfunction of the L-arginine/NO/cGMP pathway may be responsible for the reduction in the peripheral antinociceptive effect of DAMGO.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Male Wistar rats weighing approximately 250–300 g at the beginning of the experiments were used. They had free access to food and water in an animal room that was maintained at 22°C ± 0.5°C with a 12-h light-dark cycle. Animals were rendered diabetic by an intraperitoneal injection of STZ 60 mg/kg prepared in 0.1 M citrate buffer at pH 4.5. The STZ-treated rats received 2% sucrose in their drinking water for the first 48 h after treatment to reduce the severity of the initial hypoglycemic phase after STZ injection. Weight-matched control rats were injected with citrate buffer only and fed rat chow and plain water throughout the study. One week after STZ injection, the blood glucose levels were measured in a drop of blood obtained from a tail vein by puncturing the vein with a sterile needle using test strips (Glucostix; Bayer Diagnostics, Istanbul, Turkey) and glucometer (Glucometer Gx; Bayer Diagnostics). This was to ensure that diabetes had been induced. The experiments were conducted 6 wk after the injection of STZ or its vehicle. Rats with serum glucose levels more than 300 mg/dL were considered diabetic and therefore suitable for the study. All procedures followed the Guidelines on Ethical Standards for Investigation of Experimental Pain in Animals (IASP, 1983). Additionally, this study was approved by the local Animal Care Committee.

Antinociception was evaluated by the formalin test (20). Because it was suggested that small formalin concentrations elicit submaximal nociceptive responses (21), we used the small concentration (1%) of formalin. To perform the formalin test, the rats were briefly anesthetized with isoflurane (4%). Each rat was placed in an open Plexiglas observation chamber 10 min before the injection of formalin to allow acclimation to its surroundings, and then 50 µL of dilute formalin (1% in saline) was injected into the right hind paw by using a 30-gauge needle. Each animal was then returned to the chamber for observation. The nociceptive response induced by formalin was quantified as the number of flinches of the injected paw during 1-min periods every 5 min until 60 min after formalin injection. Flinching behavior induced by formalin shows a biphasic response. The early phase (Phase 1; 0–5 min) is followed by late phases (Phase 2a, 5–40 min; and Phase 2b, 40–60 min). After the 1-h observation period, animals were immediately killed by cervical dislocation.

This protocol was performed in both STZ-diabetic and nondiabetic rats. In the ipl administration study, drugs were injected into the right hind paw in a volume of 50 µL. DAMGO (a µ-opioid receptor agonist; 1 µg ipl) was administered 5 min before the injection of formalin. In the ipl administration of N-iminoethyl-L-ornithine (L-NIO), methylene blue (MB), or 3-morpholino-sydnonimine (SIN-1) (an NO synthase inhibitor, a soluble guanylyl cyclase inhibitor, and an NO donor, respectively), each of these drugs was injected first (100, 500, and 200 µg, respectively), followed 30 min later by the DAMGO injection and, 5 min later, by the formalin injection. Similarly, vehicle controls were conducted with the same protocol. The selection of the drug doses used in our study was based on our dose-response studies. Moreover, the doses we used in our study were consistent with the doses in other studies (7,8,10,15, 16,21–23).

To exclude the central effects of opioids, many strategies can be used (24). In this study, we used the strategy of evaluating the efficacy of ipsilateral versus contralateral hind paw administration because the route and site of administration would be the same. Equivalent doses are ineffective when administered into the contralateral hind paw. This result demonstrated that the antinociceptive effect of DAMGO at the dose of 1 µg was mediated by peripheral opioid receptors.

Although we chose a peripheral effective dose of DAMGO (1 µg per paw) in this study, we also evaluated some side effects—such as sedation, respiratory depression, and hypotension—that are related to the centrally mediated side effects of opioids. Sedation was evaluated by examining the motor function tested by examining the placing/stepping reflex, normal ambulation, and righting. The effects of DAMGO on respiratory rate were determined by visually observing the spontaneous respiratory movements. We also evaluated the effect of DAMGO on mean arterial blood pressure by using a Harvard rat tail blood pressure monitor.

The following drugs were obtained from Sigma (St. Louis, MO): DAMGO, L-NIO, MB, and SIN-1. Solutions of drugs were made fresh on the day of their use. Drugs were dissolved in the following vehicles: sterile saline (formalin and DAMGO) and distilled water (all other drugs). STZ was dissolved immediately before its injection in 0.1 M citrate buffer (pH 4.5). The drug was kept on ice at all times before its use.

The data are expressed as means ± SEM. Statistical comparisons were made by using the Kruskal-Wallis test followed by the Mann-Whitney U-test. P values less than 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
STZ-diabetic rats had an increased blood glucose level and a decreased body weight compared with age-matched control rats (P < 0.05). Table 1 shows serum glucose levels and body weight in both STZ-diabetic and nondiabetic rats. Diabetic animals exhibited many other symptoms often associated with diabetes (e.g., polyuria, polydipsia, and diarrhea).


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  		Table 1. Mean Blood Glucose and Body Weight of Rats
 
We did not observe any side effects in our experiments. The locally effective dose of DAMGO also did not cause any changes such as sedation or reduction of locomotion after ipl injections. The mean respiratory rate in control rats was not significantly different from that of rats administered DAMGO (67 ± 1.4 breaths/min and 64 ± 1.7 breaths/min, respectively). Moreover, no significant difference was observed between these groups (88.5 ± 2.2 mm Hg and 85.8 ± 3.6 mm Hg in control and DAMGO-administered groups, respectively). Although there was no significant effect on behavioral response, the resting blood pressure and respiratory rate were not significantly altered after the DAMGO administration into the paw, which again suggested no general sedative effect in these rats.

Ipl formalin (1%; 50 µL) administration into the right hind paw produced a nociceptive effect in both STZ-diabetic and nondiabetic rats. In the control studies (saline), no significant difference was observed between the groups (data not shown). No side effects were observed in either group. Figure 1 shows that ipl injections of DAMGO produced a significant (P < 0.05) antinociceptive effect on Phase 2a and 2b at the dose of 1 µg in nondiabetic rats. However, DAMGO injected 5 min before formalin did not alter the early behavioral response (Phase 1) to formalin (Fig. 1). As shown in Figure 1, DAMGO at the dose of 1 µg when injected into the left hind paw contralaterally did not produce antinociception in the right paw in nondiabetic rats.



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  		Figure 1. The antinociceptive effect of 1 µg of intraplantar [D-Ala2, NMePhe4, Gly-ol5]enkephalin (DAMGO) on the 1% formalin-induced nociception in nondiabetic rats. Each bar represents the mean number of flinches of six animals ±SEM. *Significant difference (P < 0.05) between DAMGO and saline.

 
Similarly, DAMGO (1 µg ipl) also produced a significant (P < 0.05) reduction in the number of flinches in the second phase, Phase 2a and 2b, when compared with saline in STZ-diabetic rats (Fig. 2). However, the antinociceptive potency of DAMGO in diabetic rats was significantly (P < 0.05) less than that in nondiabetic rats (Fig. 3).



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  		Figure 2. The antinociceptive effect of 1 µg of intraplantar [D-Ala2, NMePhe4, Gly-ol5]enkephalin (DAMGO) on the 1% formalin-induced nociception in streptozotocin-diabetic rats. Each bar represents the mean number of flinches of six animals ±SEM. *Significant difference (P < 0.05) between DAMGO and saline.

 


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  		Figure 3. The effect of locally administered [D-Ala2, NMePhe4, Gly-ol5]enkephalin (DAMGO) on the 1% formalin-induced nociceptive responses in nondiabetic and streptozotocin-diabetic rats. Each bar represents the mean number of flinches of six animals ± SEM. *Significant difference (P < 0.05) of the STZ-diabetic group compared with the nondiabetic group.

 
Local pretreatment with the NO synthesis inhibitor L-NIO (100 µg ipl) significantly (P < 0.05) reduced the peripheral antinociception produced by DAMGO (1 µg ipl) in both STZ-diabetic and nondiabetic rats (Figs. 4a and 5a). The other drug tested, MB (500 µg ipl), also significantly (P < 0.05) inhibited the peripheral antinociceptive effect of DAMGO in all groups (Fig. 4b and 5b). However, saline (vehicle of L-NIO and MB) showed no significant effect on DAMGO-induced peripheral antinociception in either STZ-diabetic or nondiabetic rats (data not shown).



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  		Figure 4. Contribution of the nitric oxide/cyclic guanosine monophosphate pathway to the peripheral antinociceptive effect of [D-Ala2, NMePhe4, Gly-ol5]enkephalin (DAMGO) in nondiabetic rats. (a) and (b) show the blockade by N-iminoethyl-L-ornithine (L-NIO; 100 µg per paw) and methylene blue (MB; 500 µg per paw), respectively, of the antinociceptive effect of locally administered DAMGO on the formalin-induced nociceptive responses (number of flinches). (c) shows potentiation by 3-morpholino-sydnonimine (SIN-1; 200 µg per paw) of the antinociceptive effect of locally administered DAMGO on the formalin-induced nociceptive responses (number of flinches). Each bar represents the mean number of flinches of six animals ± SEM. *Significant difference (P < 0.05) of the DAMGO group compared with the saline group. #Significant difference (P < 0.05) between DAMGO and DAMGO + L-NIO, DAMGO + MB, or DAMGO + SIN-1.

 


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  		Figure 5. Contribution of the nitric oxide/cyclic guanosine monophosphate pathway to the peripheral antinociceptive effect of [D-Ala2, NMePhe4, Gly-ol5]enkephalin (DAMGO) in streptozotocin-diabetic rats. (a) and (b) show the blockade by N-iminoethyl-L-ornithine (L-NIO; 100 µg per paw) and methylene blue (MB; 500 µg per paw), respectively, of the antinociceptive effect of locally administered DAMGO on the formalin-induced nociceptive responses (number of flinches). (c) shows potentiation by 3-morpholino-sydnonimine (SIN-1; 200 µg per paw) of the antinociceptive effect of locally administered DAMGO on the formalin-induced nociceptive responses (number of flinches). Each bar represents the mean number of flinches of six animals ± SEM. *Significant difference (P < 0.05) of the DAMGO group compared with the saline group. #Significant difference (P < 0.05) between DAMGO and DAMGO + L-NIO, DAMGO + MB, or DAMGO+SIN-1.

 
Figure 4c shows that SIN-1 (200 µg ipl) injected into the right hind paw significantly potentiated (P < 0.05) the antinociception induced by DAMGO (1 µg ipl) in nondiabetic rats. However, SIN-1 (200 µg ipl) had no significant effect on DAMGO-induced antinociception in STZ-diabetic rats (Fig. 5c). Saline (vehicle of SIN-1) showed no significant effect on DAMGO-induced peripheral antinociception in either STZ-diabetic or nondiabetic rats (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
There are sound reasons to believe that the animals treated with STZ in this study were fully diabetic. Data shown in Table 1, demonstrating the failure of STZ-treated rats to gain weight, together with the highly significant increase in blood glucose levels and the increase in urine output in these animals, are strongly indicative of the successful induction of diabetes.

In our study, ipl formalin injection induced biphasic flinching behavior comparable to other reports. After local administration, DAMGO (1 µg) inhibited the late phase (Phase 2a and 2b) of formalin-induced flinching and produced a significant antinociceptive effect. DAMGO was ineffective in the early phase of the formalin test. Indeed, this point is so interesting that it must be explained. Our results are consistent with a number of previous studies that have reported that the peripheral effective doses of opioids are insensitive in the first phase (23,25,26). In these studies, the local administration of opioids (e.g., DAMGO, loperamide, and methylmorphine) inhibited only the second phase of formalin-induced responses. The data possibly indicate that the peripheral transduction of pain in the formalin test is different in the two phases, because if similar mechanisms were involved it would be expected that DAMGO would affect them in a similar fashion. These results support the notion that the first phase is produced by direct activation of large nociceptive neurons by formalin and the second phase reflects pain generated in acutely injured tissue, possibly because of activation of small afferents (27,28). Therefore, the first and second phases may depend on distinct afferent mechanisms, and DAMGO may have different effects on these phases. However, the insensitivity is not due to the first phase involving higher pain scores, because the pain scores are lower in the first phase than in the second. Moreover, the insensitivity to DAMGO in the first phase is unrelated to drug kinetics, because DAMGO produced similar effects only in the second phase, regardless of whether DAMGO was injected 20 min before the first phase or just 5 min before the first phase. In addition, the insensitivity may be explained by the effect of DAMGO on Ca2+ channels. Taddese et al. (29) have shown that the µ-opioid agonist DAMGO inhibits calcium channels in almost all small nociceptors, which mediate persistent pain, but has minimal effects on large nociceptors, which mediate phasic pain. Similarly, these authors also reported that in a typical small nociceptor (25 µm in diameter), DAMGO (1 µM) potently inhibited a sustained Ca2+ current but had no effect on a typical large nociceptor (45 µm in diameter) (29). Opioids inhibits "second pain," the dull ache that persists after a noxious stimulus, but they have a relatively minimal effect on "first pain," the initial sharp sensation (30). The cellular mechanism for this specificity is unknown, but nociceptors may play a role because two different types of nociceptors mediate first and second pain: action potentials for first pain are transmitted by rapid-conducting, myelinated axons, and those for second pain are transmitted by slow-conducting, unmyelinated axons (31). Therefore, a mechanism for selective opioid inhibition of second pain may be suggested: activation of µ-opioid receptors by DAMGO inhibits Ca2+ currents predominantly on unmyelinated, slow-conducting nociceptors, therebyselectively suppressing Ca2+-evoked neurotransmitter release from those presynaptic terminals subserving second pain. In addition to these explanations, the lack of effects of DAMGO on the first phase or after injection into a contralateral paw supports the notion that DAMGO is a peripherally selective compound that does not act as a local anesthetic and does not produce centrally mediated analgesia after systemic absorption. The lack of effect of DAMGO on the first-phase responses in our study is consistent with the data on other opiate agonists that do not cross the blood-brain barrier. Thus, it seems likely that only second-phase responses can be suppressed by 1 µg of DAMGO.

Several authors have reported that inhibition of activation of adenylate cyclase is associated with the peripheral antinociceptive effect of opioids (4,32). However, the other reports indicate that the l-arginine/NO/cGMP pathway plays an important role in this effect (10,21,22). Granados-Soto et al. (21) demonstrated that local administration of NG-monomethyl-L-arginine, an NO synthase blocker, or MB, a soluble guanylyl cyclase inhibitor, significantly attenuated the antinociceptive effect of morphine. These results indicate that the local administration of morphine induces antinociception as a result of activation of the L-arginine/NO/cGMP pathway. In agreement with previous studies, in this study, the antinociceptive effect of locally administered DAMGO in nondiabetic rats was antagonized by L-NIO, an NO synthase inhibitor. MB also exerted similar effects and antagonized the analgesic effect of the local µ-receptor agonist DAMGO in the rat formalin test. Moreover, the antinociceptive effect of DAMGO was potentiated by SIN-1, a stimulator of NO generation, in the rat formalin test. Saline had no effect on the DAMGO-induced peripheral antinociception. These results indicate that the antinociception produced by DAMGO is sensitive to L-NIO, MB, and SIN-1, implying that L-arginine/NO/cGMP pathways are involved in this effect. This is the first study to report that the L-arginine/NO/cGMP pathway is involved in the peripheral antinociceptive effect of DAMGO. Our results agree with data reported in several other studies, in which an antinociceptive effect, via activation of the L-arginine/NO/cGMP pathway, was also demonstrated.

It has been reported that the antinociceptive potency of morphine is decreased in STZ-induced diabetes, an animal model of type 1 diabetes (18). The mechanism of a poor antinociceptive effect in diabetic animals has been questioned. However, the antinociceptive effect of DAMGO in diabetic rats has not been reported. This is the first study to indicate the effects of diabetes on DAMGO-induced peripheral antinociception and the role of the NO/cGMP pathway in these effects. In our study, it has been shown that the antinociceptive effect of DAMGO is reduced in diabetic rats compared with nondiabetic rats. The antinociceptive effect of DAMGO in diabetic rats was reversed by L-NIO or MB but was not potentiated by SIN-1. These results suggest that the peripheral antinociceptive effect of DAMGO may result from activation of the L-arginine/NO/cGMP pathway and dysfunction of this pathway; also, events that follow cGMP activation may change the antinociceptive effect of DAMGO in diabetic rats. However, further investigation is required to fully explain these mechanisms. NO can activate different types of K+ channels in different types of tissues by an increase in cGMP (33–37). Moreover, Soares et al. (38) described the role of activation of K+ channels after cGMP production in NO donor sodium nitroprusside-induced antinociception. The activation of K+ channels after cGMP production may alter threshold neuronal sensitivity to pain and produce nociceptor desensitization. Thus, the decreased peripheral antinociceptive effect of DAMGO may be related to dysfunction of adenosine triphosphate (ATP)-sensitive K+ channels. However, we still do not know whether the activation of ATP-sensitive potassium channels at the nociceptor level occurs directly via cGMP; further studies are needed to clarify this point.

There are also several alternative mechanisms that may be responsible for this effect. For example, the hyperalgesia observed in diabetic rats may result from a low threshold in these animals (39,40). The membrane excitability is regulated by different ion channels and pumps. Moreover, opioids also produce antinociceptive effects by regulating these channels (41–46). The dysfunction of these channels in diabetic rats may be responsible for the lowering of the threshold. Among them, tetrodotoxin-resistant (TTX-R) Na+ channels have been suggested to be closely related to nociceptive function and inflammatory hyperalgesia (47–49). Hirade et al. (50) have shown that prolonged hyperglycemia induced by STZ has effects on TTX-R Na+ currents in dorsal root ganglion (DRG) neurons. Therefore, in STZ- treated animals, increased activity of TTX-R Na+ channels would be responsible for increased nociceptive excitability. However, nociceptive sensory neurons also express multiple types of voltage-dependent calcium channels (51). The chronic complications associated with diabetes result, in part, from the pronounced changes in cellular calcium homeostasis (52). Likewise, Hall et al. (53) have reported that voltage-dependent calcium currents were enhanced in capsaicin-sensitive DRG neurons from diabetic rats. Moreover, the prolongation of Ca2+ transients may be a cause of the increased pain sensitivity that often accompanies diabetes mellitus (54). Recently, the role of L-type Ca2+ channels in attenuated morphine antinociception in STZ-diabetic rats has been suggested (55). Taken together, diabetes-induced alterations in neuronal Ca2+ homeostasis of primary afferent neurons might play a role in the pathologic changes of peripheral nociception induced by µ-opioids during diabetes mellitus. A possible distinct mechanism that is involved in peripheral hyperalgesia may be associated with K+ channels. There is evidence that modulation of some K+ channels at the peripheral level represents an important step in the peripheral mechanism of nociception (38,56). The activation of K+ channels may alter threshold neuronal sensitivity to pain and produce nociceptor desensitization. The dysfunction of the K+ channels at the nociceptor level may be responsible for the decreased peripheral antinociceptive effect of DAMGO. Moreover, the presence of increased activity of Ca2+ channels in diabetic rats may render the K+ outflow unable to counteract the decreased nociceptor depolarization. Moreover, it has been suggested that the increased phosphorylation of µ-opioid receptors by the activation of protein kinase C may, in part, be involved in the attenuation of the antinociception induced by DAMGO in diabetic mice (57). Protein kinase C modulates several cellular functions via phosphorylation of proteins, including some receptors and ion channels.

Many investigators have reported that hyperglycemia can increase diacylglycerol levels and activate protein kinase C in several tissues (58,59). The possible effect of protein kinase C on peripheral antinociception may result from its effects on some channels or receptors that are involved in peripheral antinociception. DRG neurons expressing TTX-R Na+ channels have been demonstrated to be responsible for nociceptive excitability (47,48). The phosphorylation of these channels by protein kinase C modulates channel functions in various tissues (60–62). Hirade et al. (50) also have suggested that the altered phosphorylation of TTX-R Na+ channels by protein kinases may contribute to hyperalgesia in diabetes. Moreover, the sensitization of Na+ channels, probably TTX-R Na+ channels, by the long-term activation of protein kinase C may play an important role in the enhancement of the duration of fenvalerate-induced nociceptive behavior in diabetic mice (63). Therefore, it is possible that the decreased peripheral analgesia after the administration of DAMGO in diabetic rats may result from a secondary effect in response to sensitization of TTX-R Na+ channels by the long-term activation of protein kinase C. In addition, protein kinase C modulates several cellular functions via phosphorylation of some receptors. It has been reported that phosphorylation of µ-opioid receptors by protein kinase C leads to desensitization of µ-opioid receptor-mediated antinociception (64). These results suggest that µ-opioid receptors can be phosphorylated by the activation of protein kinase C and that this process leads to receptor desensitization of µ-opioid receptor-mediated responses in diabetic rats. However, the exact mechanism is still unknown.

In summary, DAMGO has potential therapeutic usefulness as a peripherally selective local opiate antihyperalgesic drug that lacks many of the side effects associated with systemic opiate administration. Because DAMGO does not produce systemic effects at the doses we used in this study, it may be a potentially superior drug of choice for the treatment of inflammatory pain, where the administration of drug directly at the site of injury is possible. According to our results, local administration of DAMGO produces antinociception as a result of the activation of the L-arginine/NO/cGMP pathway. However, the antinociceptive effect of DAMGO was found to be less in diabetic than in nondiabetic rats. For these reasons, it is reasonable to suggest that dysfunction of the L-arginine/NO/GMP pathway, especially events that are followed by cGMP activation, may contribute to the demonstrated poor antinociceptive response of diabetic rats to µ-opioid agonists.


   Acknowledgments
 
Supported by the Akdeniz University Research Foundation (99.02.0103.01).


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Accepted for publication August 13, 2003.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press