THE ROLE OF TRANSIENT RECEPTOR POTENTIAL VANILLOID 1 RECEPTOR IN DESIPRAMINE INDUCED ANALGESIC EFFECT IN DIABETIC MICE

THE ROLE OF TRANSIENT RECEPTOR POTENTIAL VANILLOID 1 RECEPTOR IN DESIPRAMINE INDUCED ANALGESIC EFFECT IN DIABETIC MICE

Sana Shaikh, Shrikalp Deshpande and Priyanshee Gohil*

Department of Pharmacology, K.B. Institute of Pharmaceutical Education and Research, Kadi Sarvavishvawidyalaya, Gandhinagar, Gujarat, India

ABSTRACT

Hyperalgesia is one of the debilitating complications of diabetes. The thermal allodynia and hyperalgesia in diabetic mice may be due to the hyperactivity of C-fiber in the spinal cord. Transient receptor potential vanilloid type 1 (TRPV1) present in spinal cord and activation of C-fibre may involve in hyperalgesia in diabetic mice. Desipramine is one of the tricyclic antidepressants, effective in diabetic neuropathy. The intravenous administration of desipramine depresses the C-fibre reflex that will involve in activation of convergent neurons of the spinal cord. Thus, the present study was carried out to find out the role of TRPV1 in desipramine induced analgesic effect in diabetic hyperalgesia. Mice were administered capsaicin (1 mg kg^-1), capsazepine (15 mg kg^-1), desipramine (10 mg kg^-1) from day 4 to day 11 after induction of diabetes and the nociceptive threshold was measured in terms of reaction time, tail flick latency and tail withdrawal latency. The nociceptive threshold was significantly (p < 0.05) lower in diabetic mice as compared with control group. Capsaicin produced a significant (p < 0.05) decrease in reaction time, tail flick latency and tail withdrawal latency as compare to diabetic group. Desipramine caused a significant (p < 0.05) increase in nociceptive threshold in diabetic group as well as capsaicin treated diabetic group. It was concluded that desipramine produced analgesic effect in diabetic hyperalgesia and TRPV1 might be involved in desipramine induced analgesic effect.

Hyperalgesia, TRPV1, Nociceptive threshold, Desipramine, Diabetes mellitus, Mice

INTRODUCTION: Antidepressants are widely used in chronic pain states including both inflammatory and neuropathic pain conditions.  It has been suggested that antidepressant drugs have specific analgesic properties, and various clinical ^{1, 2} and experimental ^3-5 types of evidence together demonstrate that the analgesic effect may be independent of the antidepressant effects. Interactions of antidepressants with biogenic amines, opioid systems, excitatory amino acid receptors, substance P and calcium and sodium channels have been considered to be involved pain relieving properties of antidepressants ⁶.

Desipramine (Figure 1) is one of the tricyclic antidepressants, which block the uptake of amines by nerve terminals, by competition for the binding site of the amine transporters. Oral desipramine enhances the analgesic effects of morphine for post-operative pain ⁷, reduces pain intensity in post-herpetic neuralgia ⁸, and produces pain relief in diabetic neuropathy patients ⁹.

Acute administration of desipramine is antinociceptive in animal models of inflammatory pain ¹⁰ and attenuates thermal hyperalgesia in models of neuropathic pain ^{11, 12}.

FIGURE 1: STRUCTURE OF DESIPRAMINE

TRPV1 is considered as key molecules of nociception and a correlation between TRPV1 expression and inflammatory thermal hyperalgesia has been established in rats ¹³. TRPV1 may involve in transduction of chemical and thermal hyperalgesia in a model of diabetic neuropathy ¹⁴. TRPV1 is a non-selective cation channel of TRP family located on sensory neurons. There are seven subfamily of TRP channel in which TRPV1 is associated with thermal hyperalgesia ¹⁵. Hyperalgesia is one of the common complications of diabetes mellitus. Streptozotocin (STZ) induced diabetes neuropathy also demostrate thermal hyperalgesia and mechanical allodynia ¹⁶. In experimental diabetic neuropathy, the development of hyperalgesia is due to increased expression of TRPV1 on neurons that do not normally express TRPV1 ¹⁷.

It was suggested that the thermal allodynia and hyperalgesia in diabetic mice may be due to the hyperactivity of C-fiber in the spinal cord. The TRPV1 receptor is expressed predominantly by primary sensory neurons in the spinal cord, probably in unmyelinated C-fibers and sensitization of TRPV1 receptors might be involved in the mechanism of thermal hyperalgesia and allodynia seen in diabetic mice ^{17, 18}. Desipramine, inhibit the spinal processing of C inputs by acting directly at the spinal cord level ¹⁹.

In light of above facts, the present study was designed to find out the role of TRPV1 in desipramine induced analgesic effect in STZ induced diabetic mice model.

MATERIALS AND METHODS:

Animals: Healthy albino mice of either sex, weighing 25–30 g were procured from Zydus Research Centre, India. The animals were housed and maintained at 298 K, 50 ± 15 % RH for 12 hour light-dark cycles, in polypropylene cages with free access to food and water ad libitum.

The experimental protocol (KBIPER/2011/247) was approved by the Institutional (K.B. Institute of Pharmaceutical Education and Research) Animal Ethics Committee (IAEC) under the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guideline, before carrying out the project.

Chemicals: Streptozotocin, desipramine and capsaicin were obtained from Sigma-Aldrich, USA while capsazepine obtained from Cayman chemical, China.  Dimethylsulphoxide (DMSO) was purchased from Sujan Chemials, India.

Desipramine was dissolved in saline. Capsaicin and capsazepine was dissolved in 30% DMSO to prepare stock solution and it was diluted further in saline to prepare working solutions.

Induction of type 1 diabetes: Diabetes mellitus was induced in mice using STZ. Streptozotocin was freshly prepared by dissolving 1 g STZ in 10 mL 0.1 N cold citrate buffer (1.45 g citric acid and 1.05 g sodium citrate dissolved in 100 mL distilled water) at a pH of 4.5. Single dose of STZ (25 mg kg^-1, i.p.) was administration for induction of diabetes and 10% W/V oral glucose was given to all animals after 6 h of STZ administration to prevent the hypoglycemic shock.  Control animals received an equivalent volume of saline. Confirmation of hyperglycemia was made 3 days later by measurement of the fasting blood glucose (FBG) level. Mice with FBG level >14 mmol L^-1 were considered diabetic and were included for further study ²⁰.

Estimation of blood glucose: Blood glucose levels were estimated spectrophotometrically by glucose-oxidase methodusing a commercially available enzymatic kit (Span Diagnostics Ltd., India) ²¹.

Experimental design:

The mice were divided into the following groups:

• GROUP I (Non-Diabetic control group)
• GROUP II (STZ-induced diabetic group) to serve as diabetic control animals.
• GROUP III (Capsaicin treated diabetic group: 1 mg kg^-1, i.p)
• GROUP IV (Capsazepine treated diabetic group:15 mg kg^-1, i.p)
• GROUP V (Desipramine treated diabetic group: 10 mg kg^-1, i.p)
• GROUP VI (Capsaicin (1 mg kg^-1, i.p) + Desipramine (10 mg kg^-1, i.p)
• GROUP VII (Capsaicin (1 mg kg^-1, i.p) ₊ Capsazepine (15 mg kg^-1, i.p)

Mice of group I were administered normal saline solution to serve as a non-diabetic group. Desipramine, capsaicin and capsazepine were administered to their respected groups, daily for 8 days, starting on day 3 after STZ administration. In case of group VI and group VII, desipramine and capsazepine were administered 30 min before capsaicin administration. Nociceptive threshold was noted on day 4, 8 and 11 in all groups.

Measurement of Nociceptive Threshold: For the measurement of nociceptive threshold, the hot plate, tail immersion test and the tail flick test were carried out at 0, 30, 60 and 120 minutes intervals.

1. Hot plate method: The mice were placed individually on a hot plate (Max enterprise/ HP99M, India) maintained at 328 K and the time taken by the animal for the reaction either by licking the paw or jumping or raising the limbs which ever was observe first taken as the end point. Reaction time was note down before and at 0, 30, 60 and 120 min after the drug or saline administration in each animal. Cut off time of 60 s was followed to avoid any thermal injury to the paws ²².
2. Radiant heat tail-flick method: Tail-flick latency was assessed by the analgesiometer (Instruments manufacturing corporation -model/MC-102413, India). The strength of the current passing through the naked nichrome wire was kept constant at 2 A. The distance between heat source and the tail was 1.5 cm and the application site of the heat on the tail was maintained within 2 cm, measured from the root of the tail. Cut-off reaction time was 30 s to avoid any tissue injury during the process. Tail-flick latency was measured before and 0, 30, 60 and 120 min after the drug administration.

The time taken by mice to withdraw (flick) the tail was taken as the reaction time. The animals were subjected to the same test procedure at 0, 30, 60, 120, and 180 min after the administration of drug ²².

3. Tail immersion test: Mice were held in position in a suitable restrainer with the tail extending out. 3-4 cm area of the tail was marked and immersed in the water bath thermo-statistically maintained at 328 K. The withdrawal time of the tail from hot water was noted as the reaction time or tail flick latency. The maximum cut off time for immersion was 30 s to avoid the injury of the tissues of tail ²².

Statistical analysis: All the values were expressed as Mean±SEM. The data were analyzed by One-way Analysis of Variance (ANOVA) followed by Tukey's multiple range test. The level of significance was expressed at p < 0.05.

RESULTS:

Effect of STZ on Fasting blood glucose level: FBG level was significantly (p < 0.05) increase in STZ treated mice as compare with control mice (Figure 2).

FIGURE 2: FASTING BLOOD GLUCOSE LEVEL IN CONTROL AND STZ TREATED GROUPS Each bar are expressed as mean ± SEM. (n=6); * p<0.05 as compared with control group; (One way ANOVA followed by Tukey’s Test)

Measurement of Nociceptive Threshold: The diabetic group showed a significant (p < 0.05) decrease in reaction time, tail- flick latency, tail withdrawal latency as compared to control group on day 11. Reaction time, tail- flick latency, tail withdrawal latency was significantly (p < 0.05) decrease in capsaicin treated group than diabetic mice on day 11.

Nociceptive threshold was significantly (p < 0.05) increase in desipramine as well as capsazepine treated mice than diabetic group at 11 day. In animals pretreated with desipramine and capsazepine before capsaicin administration, there was a significant (p < 0.05) increase in reaction time, tail flick latency and tail withdrawal latency as compared with capsaicin treated diabetic mice (Figure 3).

FIGURE 3: EFFECT OF CAPSAICIN, DESIPRAMINE AND CAPSAZEPINE ON REACTION TIME, TAIL FLICK LATENCY, TAIL WITHDRAWAL LATENCY AT 60 MIN ON DAY 11. Each bar are expressed as mean ± SEM. (n=6); * p<0.05 as compared with control group; # p<0.05 as compared with diabetic group; @ p<0.05 as compared with Capsaicin group; (One way ANOVA followed by Tukey’s Test)

DISCUSSION: The present work aimed to study the involvement of TRPV1 in analgesic effect of desipramine in diabetic hyperalgesic mice.

The transient receptor potential vanilloid 1 (TRPV1) receptor, previously known as the vanilloid receptor 1 (VR1) or capsaicin receptor, is a ligand-gated, non-selective cation channel expressed predominately by primary nociceptive sensory neurons ¹⁴. It activated by the pungent component of hot chilli peppers, capsaicin, as well as heat, protons and some endogenous substances known to be associated with tissue inflammation. TRPV1 has, therefore, been suggested to be a molecular integrator of chemical and physical stimuli that elicit pain ²³. It has been shown that TRPV1 play a significant role in nociception and in the pathogenesis of experimental diabetic hyperalgesia and the development of its complications ¹⁶.

Streptozotocin (STZ) is a glucosamine nitrosourea compound used for the induction of the diabetes mellitus in animals and also used to study the associated complications. The induction of diabetes by STZ is characterized by initial phase of hyperglycemia, then hypoglycemia and then it will cause hyperglycemic effect in animal. This effect will cause diabetic mellitus after 3 days of STZ administration ²⁴.

In present study, the STZ treated mice showed significantly (p<0.05) increase in FBG level and became diabetic. The diabetogenic action of STZ was also accompanied by the development of persistent hyperalgesia by enhancing expression and function of TRPV1 receptor in DRG neurons ²⁵.

The present study demonstrated that diabetic mice induced by a single injection of STZ developed significant hyperalgesia. The nociceptive threshold was significantly lower in diabetic mice as compared with the control group. Hyperalgesia was evident on Day 4 and the maximum decrease in pain threshold was observed on Day 11 after STZ injection.

The modulation of nociception by antidepressants is mainly centrally mediated and involved central serotonergic, noradrenergic, and opioidergic systems in the modulation of pain threshold caused by antidepressants.

Most pharmacological data so far revealed that facilitation of central serotonergic and noradrenergic transmission is potentially anti-nociceptive, whereas inhibition of serotonergic and noradrenergic activity increases the sensitivity to noxious stimuli ^{26, 27}.

Tricyclic antidepressants exhibit anti-nociceptive properties in neuropathic, nociceptive and inflammatory models of pain ²⁸ but this effect through the TRPV1 receptor is not clear. It has been demonstrated that desipramine is effective in diabetic neuropathy and can serve as a relatively selective adrenergic probe of analgesic mechanisms ⁹.

Desipramine, which is a powerful reuptake inhibitor of norepinephrine, has been found to have marked antinociceptive properties in some investigations ^{3, 29}.

Oral desipramine reduces pain intensity in post-herpetic neuralgia ⁸ and produces pain relief in diabetic neuropathy patients ⁹. The intravenous administration of desipramine depresses the C-fibre reflex that will involve in activation of convergent neurons of the spinal cord ^{28, 30, 31}. The thermal allodynia and hyperalgesia in diabetic mice may be due to the hyperactivity of C-fiber in the spinal cord ^{32, 33}.

TRPV1 present in spinal cord and activation of C-fibre activity may involve in hyperalgesia in diabetic mice. The anti-nociceptive effect of desipramine was measured by hot plate method, tail flick method, tail immersion method. In present study, desipramine showed significant increase in reaction time, tail flick latency and tail withdrawal latency as compare to diabetic group. This suggests that desipramine gives analgesic effect in diabetes mellitus induced hyperalgesia.

Capsaicin, the active agent found in hot chilli peppers is powerful agonist of TRPV1. Capsaicin accomplishes its effect by evoking sharp burning pain sensation. Capsaicin induces influx of cations in nociceptors. Capsaicin activates TRPV1, that produce hyperalgesia ³⁴. Capsazepine is a powerful antagonist of TRPV1 receptor. Capsazepine, the competitive capsaicin antagonist significantly reduces inflammatory heat hyperalgesia ^{35, 36}.

In present investigation, capsaicin produced a significant decrease in reaction time, tail flick latency and tail withdrawal latency as compare to diabetic group. Capsazepine antagonized the hyperalgesic effect of capsaicin. Likewise, desipramine caused a significant increase in reaction time, tail flick latency and tail withdrawal latency of capsaicin treated group compare to diabetic group.

This suggests the involvement of TRPV1 in desipramine induced analgesic effect in diabetic hyperalgesia.

CONCLUSION: In conclusion, desipramine produced analgesic effect in diabetic hyperalgesia and TRPV1 might be involved in desipramine induced analgesic effect.

REFERENCES:

1. Magni G, Conlon P, Arsie D: Tricyclic antidepressants in the treatment of cancer pain: a review. Pharmacopsychiatry 1987; 20(4):160-164.
2. Magni G: The use of antidepressants in the treatment of chronic pain. A review of the current evidence. Drugs 1991; 42(5):730-748.
3. Spiegel K, Kalb R, Pasternak GW: Analgesic activity of tricyclic antidepressants. Ann Neurol 1983; 13(4):462-465.
4. Tura B, Tura SM: The analgesic effect of tricyclic antidepressants. Brain Res 1990; 518(1-2):19-22.
5. Casas J, Gibert-Rahola J, Chover AJ, Mico JA: Test-dependent relationship of the antidepressant and analgesic effects of amitriptyline. Methods Find Exp Clin Pharmacol 1995; 17(9):583-588.
6. Eschalier A, Mestre C, Dubray C, Ardid D: Why are antidepressants effective as pain relief? CNS Drugs 1994; 2(4):261-267.
7. Max MB, Zeigler D, Shoaf SE, Craig E, Benjamin J, Li SH, Buzzanell C, Perez M, Ghosh BC: Effects of a single oral dose of desipramine on postoperative morphine analgesia. J Pain Symptom Manage 1992; 7(8):454-462.
8. Rowbotham MC, Reisner LA, Davies PS, Fields HL: Treatment response in antidepressant-naive postherpetic neuralgia patients: double-blind, randomized trial. J Pain 2005; 6(11):741-746.
9. Max MB, Kishore-Kumar R, Schafer SC, Meister B, Gracely RH, Smoller B, Dubner R: Efficacy of desipramine in painful diabetic neuropathy: a placebo-controlled trial. Pain 1991; 45(1):3-9.
10. Jones CK, Eastwood BJ, Need AB, Shannon HE: Analgesic effects of serotonergic, noradrenergic or dual reuptake inhibitors in the carrageenan test in rats: evidence for synergism between serotonergic and noradrenergic reuptake inhibition. Neuropharmacology 2006; 51(7-8):1172-1180.
11. Sawynok J, Esser MJ, Reid AR: Peripheral antinociceptive actions of desipramine and fluoxetine in an inflammatory and neuropathic pain test in the rat. Pain 1999; 82(2):149-158.
12. Jett M-F, McGuirk J, Waligora D, Hunter JC: The effects of mexiletine, desipramine and fluoxetine in rat models involving central sensitization. Pain 1997; 69(1–2):161-169.
13. Luo H, Cheng J, Han JS, Wan Y: Change of vanilloid receptor 1 expression in dorsal root ganglion and spinal dorsal horn during inflammatory nociception induced by complete Freund's adjuvant in rats. Neuroreport 2004; 15(4):655-658.
14. Palazzo E, Luongo L, de Novellis V, Rossi F, Marabese I, Maione S: Transient receptor potential vanilloid type 1 and pain development. Curr Opin Pharmacol 2012; 12(1):9-17.
15. Pedersen SF, Owsianik G, Nilius B: TRP channels: an overview. Cell Calcium 2005; 38(3-4):233-252.
16. Kamei J, Zushida K, Morita K, Sasaki M, Tanaka S: Role of vanilloid VR1 receptor in thermal allodynia and hyperalgesia in diabetic mice. Eur J Pharmacol 2001; 422(1-3):83-86.
17. Hong S, Wiley JW: Early painful diabetic neuropathy is associated with differential changes in the expression and function of vanilloid receptor 1. J Biol Chem 2005; 280(1):618-627.
18. Pabbidi RM, Cao DS, Parihar A, Pauza ME, Premkumar LS: Direct role of streptozotocin in inducing thermal hyperalgesia by enhanced expression of transient receptor potential vanilloid 1 in sensory neurons. Mol Pharmacol 2008; 73(3):995–1004.
19. Mestre C, Hernández A, Eschalier A, Pelissier T: Effects of clomipramine and desipramine on a C-fiber reflex in rats. Eur J Pharmacol 1997; 335(1):1-8.
20. Brosky G, Logothetopoulos J: Streptozotocin diabetes in mouse and g. pig. Diabetes 1969; 18:606-611.
21. Trinder P:  Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor.  Ann Clin Biochem 1969; 6:24-25.
22. Vogel H. (ed): Radient heat method, hot plate method and tail immersion method. In: Drug discovery and evaluation: Pharmacological assays. 2^nd ed,  Springer-Verlag, Berlin 1997: 694-697.
23. Tominaga M, Caterina MJ, Malmberg AB, Rosen TA, Gilbert H, Skinner K, Raumann BE, Basbaum AI, Julius D: The cloned capsaicin receptor integrates multiple pain-producing stimuli. Neuron 1998; 21(3):531-534
24. Szkuddski T: The mehanism of alloxan and STZ action in B ells of rat pancreas. Physiol Res 2001; 50:537-546.
25. Pabbidi RM, Cao DS, Parihar A, Pauza ME, Premkumar LS: Direct role of streptozotocin in inducing thermal hyperalgesia by enhanced expression of transient receptor potential vanilloid 1 in sensory neurons. Mol Pharmacol 2008; 73(3):995–1004.
26. de Felipe MC, de Ceballos ML, Fuentes JA: Hypoalgesia induced by antidepressants in mice: a case for opioids and serotonin. Eur J Pharmacol 1986; 125(2):193-199.
27. Valverde O, Mico JA, Maldonado R, Mellado M, Gibert-Rahola J. Participation of opioid and monoaminergic mechanisms on the antinociceptive effect induced by tricyclic antidepressants in two behavioural pain tests in mice. Prog Neuropsycho-pharmacol Biol Psychiatry 1994; 18(6):1073-1092.
28. Carter GT, Sullivan MD; Antidepressants in pain management. Curr Opin Investig Drugs 2002; 3(3):454-458.
29. Besson JM, Chaouch A; Peripheral and spinal mechanisms of nociception. Physiol Rev 1987; 67(1):167-186.
30. Rigal F, Eschalier A, Devoize JL, Pechadre JC: Activities of five antidepressants in a behavioral pain test in rats. Life Sci 1983; 32(26):2965-2971.
31. Eschalier A, Fialip J, Varoquaux O, Makambila MC, Marty H, Bastide P: Pharmacokinetic patterns of repeated administration of antidepressants in animals. I. Implications for antinociceptive action of clomipramine in mice. J Pharmacol Exp Ther 1988; 245(3):963-968.
32. Danysz W, Minor BG, Post C, Archer T: Chronic treatment with antidepressant drugs and the analgesia induced by 5-methoxy-N,N-dimethyltryptamine: attenuation by desipramine. Acta Pharmacol Toxicol 1986; 59(2):103-112.
33. Simon B, Xue JL, Chun K, Michael WS: Peripheral nerve injury and TRPV1-expressing primary afferent C-fibers cause opening of the blood-brain Barrier. Mol Pain 2010; 6:74.
34. Oh U, Hwang SW, Kim D: Capsaicin activates a nonselective cation channel in cultured neonatal rat dorsal root ganglion neurons. J Neurosci 1996; 16(5):1659-1667.
35. Kwak JY, Jung JY, Hwang SW, Lee WT, Oh U: A capsaicin-receptor antagonist, capsazepine, reduces inflammation-induced hyperalgesic responses in the rat: evidence for an endogenous capsaicin-like substance. Neuroscience 1998; 86(2):619-626.
36. Walker KM, Urban L, Medhurst SJ, Patel S, Panesar M, Fox AJ: The VR1 antagonist capsazepine reverses mechanical hyperalgesia in models of inflammatory and neuropathic pain. J Pharmacol Exp Ther 2003; 304(1):56-62.How to cite this article:

    Shaikh S, Deshpande S and Gohil P: The Role of Transient Receptor Potential Vanilloid 1 Receptor in Desipramine induced Analgesic Effect in Diabetic Mice. Int J Pharm Sci Res. 3(12); 4882-4887.

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