Life Sciences, Vol. 65, NO. 10, pp. 1059-1066, 1999 Copyright 1999 ElsevierScienceInc. 0 Printedin the USA.

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ELSEVIER

PI1SOO24-3205(99)00336-7

ANTINOCICEPTION PRODUCED BY SYSTEMIC, SPINAL AND SUPRASPINAL ADMINISTRATION OF AMILORIDE IN MICE

Juliano Ferreira, Adair R. S. Santos and *Job B. Calixto

Department OfPhannacology,

CCB, Universidade Federal de Santa Catarina, Kua Ferreim Lima, 82,

88015420, Florianopolis-SC, Brazil.

(Receivedin final form May 5, 1999) Summary

This study investigates the antinociceptive and antihyperalgesic action caused by ip., i.t. or i.c.v. injections of amiloride when assessed against formalin, capsaicin-induced licking, acetic acid-induced writhing and glutamatt&nduced hy-peralgesia in mice. The systemic, spinal and supraspinal administration of amiloride causes dose-related antinociception when assessed against acetic acid-induced writhing, formalin and capsaicin-induced licking. In addition, amiloride administered by the same routes produced graded inhibition of glutamate-induced hyperalgesia in mice. Together, these results suggest, that amiloride or its derivatives may constitute a strategy for the development of new antinociceptive drugs.
Key Words: formalin, capsaicin, acetic acid, glutamate, amiloride, antinociception, mice

Amiloride, a K+-sparing diuretic, has been reported to elicit a wide variety of actions, affecting several ionic channels and a multitude of receptors and enzymes (l-3). The amiloride sensitive Nacharmel/degenerin family constitutes a group of proteins that is thought to be involved in a wide variety of fbnctions, such as sodium and pH homeostasis, transduction of mechanical stimuli, and even nociception (4). Recently, four new mammalian I-I+-gatedcation channel subunits have been discovered. These channels are expressed in the central and peripheral nervous system. It is now believed that in sensory neurones, these channels are possibly involved in perception of pain following tissue acidosis (S-7). In the present study we sought to determine whether systemic, spinal and supraspinal injections of amiloride had antinociceptive or antihyperalgesic effects when assessed in chemical and thermal models of nociception in mice.

*To whom all correspondence should be addressed. Fax: (5548) 2224164, Tel: (5548) 3319491, 33 19764; E-mail: calixto@fkmaco.ufsc.br

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Methods

Animals. Male Swiss mice (25 - 30 g), housed at 22 * 2C under a 12 h light/l2 h dark cycle and with access to water and food adlibitum, were used. The animals were acclimatised to the laboratory for at
least 1 h before testing and were performed in accordance with current guidelines for the care of laboratory animals and the ethical guidelines for investigations of experimental pain in conscious animals (8).

Ahinistration of amiloriak For intracerebroventricular (i.c.v.) or intrathecal (i.t.) injections, the animals were anaesthetised with ether, and a volume of 5 pl of only sterile PBS (phosphate buffer saline), or containing amiloride was injected directly into the lateral ventricle (i.c.v.), or between LS and L6 (it.) using a microsyringe connected to polyethylene tubing, as described previously (9,10,11,12). Amiloride was injected ip. (11.3 - 225.5 prnovkg, 30 min prior), i.t. (11.3 - 1127.4 nmoYsite, 10 min prior) and i.c.v. (3.7 - 1127.4 nmol/site, 10 min prior). Control animals received the same volume of PBS injection.

Acetic acid-induced abdominal constrictiott. The abdominal

constrictions, which consisted of a contraction of the abdominal muscle together with a stretching of hind limbs, were induced by intraperitoneal injection of acetic acid (0.6%, i.p. pH 2.8), and were carried out according to the procedure described previously (13) A&r acetic acid injection, mice were placed in separate boxes, and the number of abdominal constrictions was counted cumulatively over a period of 20 min. The antinociceptive activity was expressed as the reduction of the numbers of abdominal constrictions in mice pre-treated with amiloride when compared with the control animals (PBS pretreated mice).

Formalin-induced licking. The procedure used for formalin-induced

licking was essentially similar to that described previously (12,14). 2.5% formalin (0.92% of formaldehyde) was prepared in PBS solution and 20 ul was injected intraplantarly in the right hindpaw using a microsyringe with a 26gauge needle. The amount of time spent licking the injected paw was timed with a chronometer and was considered as indicative of pain

Gpaicin-induced licking. The method used for capsaicin-induced licking was similar to that described previously (12,15). The animals were placed individually for adaptation, 20 min before testing, in transparent glass cylinders (20 cm in diameter), which subsequently served as observation chambers. Following the adaptation period, 20 pl of capsaicin (1.6 @paw) was injected intraplantarly in the right hindpaw using a microsyringe with a 26-gauge needle. The amount of time that animals spent licking the injected paw was timed with a chronometer and was considered as indicative of pain.

Glutamate-mduced thermal hyperalgesia. In the glutamate-induced hyperalgesia, the hot-plate (Ugo Basile, model-DS 37; 50 f 1 C) apparatus was used to measure the response latencies according to the method described previously (11,16). Animals were placed into a glass cylinder and the time between placement and shaking or licking their paws, or jumping (whichever came first), was recorded as the index of response latency. Control mice received it. injection of saline or glutamate, and typica latencies for this groups were 15.2 + 0.4 and 7.5 f 0.5 s, respectively, An automatic 30 s cut-off was used to prevent tissue damage, but the latencies of amiloride treated and control groups usually never exceeded 20 s. Following glutamate (100 nmol/site, i.t.) injection, the hot-plate latencies

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were recorded at 5, 15, 30, and 60 min in absence and in presence of several drug treatments. The maximal percentage of effect (MPE) of glutamate-induced hyperalgesia was calculated as follows: %MPE = (postdrug-predrug) / (predrug-30) x 100.

Rota-rod test. In order to evaluate the possible muscle-relaxant or sedative effects of amiloride, the mice were tested on the rota-rod apparatus as described previously (11). Animals received amiloride or saline, 30 min before being tested. Results are expressed as the time (seconds) for which animals remained on the rota-rod. The cut-off time used was 60 s.

Neonatal capsaicinpre-treatment.To explore the role of capsaicin-sensitive fibres in the

antinociceptive effect of amiloride, neonatal mice were anaesthetised with ether and received capsaicin (50 mg/kg, subcutaneously) or vehicle (10% ethanol, 10% Tween-80 and 80% physiological saline) on day 2 of life, as described previously (17). The antinociceptive effect caused by amiloride, against the algesic response induced by formalin, was analysed at 6 to 7 weeks after the administration of neonatal capsaicin or vehicle (used as control).

Statistical analysis. The results are presented as the mean f SEM, and the statistical significance of

differences between groups was calculated by means of one-way analysis of variance (ANOVA) followed by Dunnetts multiple comparison test. P-values less than 0.05 (P < 0.05) were considered as indicative of significance. The IDTO values (i.e., the dose of amiloride reducing the pain response by 50% in relation to control group values) were determined by linear regression from individual experiments using GraphPad Software, and are reported as geometric means accompanied by their respective 95% confidence limits. In the glutamat~induced hyperalgesia the II& values were calculated on the peak (5 min) of glutamate response.

Drugs. The following drugs were used: cap&in, glutamic acid, amiloride and PBS (Sigma Chemical CO., St. Louis, U.S.A.), formalin and acetic acid (Merck AG, Darmstadt, Germany). Drugs used were prepared in PBS and maintained in a freezer at -18 C. The cap&in was prepared in absolute ethanol. The final concentration of ethanol did not exceed 5% and did not cause any detectable effect.

Amiloride, given i.p, it. or icv., produced graded inhibition of acetic acid-induced abdominal constrictions (Pig. 1A) and also of capsaicin-induced licking (Pig. 1B). The mean II& values for this effect are shown in Table 1. In the it. (WO.05) and i.c.v. (p<O.Ol) routes, amiloride was about 2- to 7-fold more potent in inhibiting cap&in-induced licking than acetic acid-induced writhing, respectively. Given i.p., it. or i.c.v., amiloride dose-dependently inhibited the early (Pig. 2A) and the late (Pig. 2 B) phase of formalin-induced licking. The mean IDm values are shown in Table 1. This antinociceptive effect of amiloride developed rapidly (15 min) and lasted for up to 2 h, when administered by i.p. route (results not shown).

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Fig. 1 Effect of i.p. (W), i.t. (0) or i.c.v. (0) injection of the amiloride against the acetic acid-induced writhing (panel A) and capsaicin-induced licking (panel 8) in mice. Each point represents the mean of 6 to 10 animals and the vertical lines indicate the SEM. C, Control values (animals injected with the vehicle) and the asterisks denote the significance levels, when compared with control groups (ANOVA) *P ~0 .05, **P c 0.01. In some cases the error bars are hidden within the symbols.
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Fig. 2 Effect of i.p. 0, i.t. (0) or i.c.v. (0) injection of the amilonde against the formalin-induced licking (first phase, panel A and second phase, panel B) in mice. Each point represents the mean of 6 to IO animals and the vertical lines indicate the SEM. C, Control values (animals injected with the vehicle) and the asterisks denote the significance levels, when compared with control groups *P (ANOVA) l c 0.05, l ~0 .Ol. In some cases the error bars are hidden P within the symbols.

Neonatal pre-treatment of mice with capsaicin prevented the antinociception caused by amiloride (225.4 pmolkg, i.p.) in the early phase of formalin response (Fig. 3A). In the second phase, the

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capsaicin pre-treatment did not signifkxntly

alter either formalin response or amiloride

antinociception (Fig. 3B).

vahbh (Lp.) Amlbrldo(226.4pmoUkg, 1.~)

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Fig. 3 Effect of neonatal treatment with vehicle (closed columns) and capsaicin (hatched columns) in the antinociceptive response caused by amiloride against the formalin-induced licking (first phase, panel A and second phase, panel B) in mice. Each column represents the mean of 6 to 10 animals and the vertical lines indicate the SEM. The closed column represent the control values and asterisks denote the significance levels, when compared with control values (WO.01). ## Denote the significant levels of mice treated with amiloride when compared with vehicle (p< 0.01).

The results in Figure 4 show that amiloride, when adminiskred by i.p., it. or i.c.v. routes, dosedependently inhibited glutamate-induced hyperalgesia. The mean IDSO values are shown in Table 1. However, amiloride, in the same range of doses, had no effect on the hot plate assay (resuits not shown).
261A
Og !I -26-SO-76t, 0616 -A6 1 +Amibdda (113 pmd) +Amihide (375 pnoi) 1 1 I 50 46 80 -21-SO-7s06 I -O-Glutamate (100 rmdj -A- +Amilodda (37.6 nmol) t +Amilwi& (I 13 rrnol) A +Amiloridr 076 rmol) I 1 1 16 30 4s 60 TIME (IT&l) tl-SO-76I 0616
4

26-B

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Gltimate (100 rind) +Amlorib (37.8 nmol) +Amiluidn (113 fund) +Amilork* (376 mwl) I I 1 30 45 60 (rdll)

TIE (min)

TIME

Fig. 4 Effect of i.p. (A, 30 min prior), i.t. (B, co-administered) or i.c.v (C, 10 min prior) injection of the amiloride on the hyperalgesia caused by i.t. injection of glutamate (100 nmoi/site) in mice. Each point on the curve represents the mean of 8 to 12 animals and vertical lines show SEM. Asterisks denote the significance levels in comparison with control (glutamate) values (one-way ANOVA followed by Dun&ts test). +P ~0.05, +*P c 0.01. In some cases the error bars are hidden within the symbols.

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When analysed in the rota-rod test, amiloride (751.6 umolikg), given i.p. 30 min before, did not significantly affect the motor performance of animals. The control response in the rota-rod test was 58.2 + 1.1 s versus 57 6 + 1.9 s in the presence of the amiloride (n = 6, in each group).

TABLE I Comparison of the mean ID= values of the antinociceptive effect of the amiloride, given by i.p., i.t. and i.c.v. routes, in several modeis of nociception in mice -

i.p. (umol/kg) Acetic acid

i.t. (nmol/site) i.c.v. (nmovsite)

Capsaicin

Glutamate

Formalin

L
Each group represents the mean f SEM of 6-10 animals. ID% with their respective 95% confiience limits. bMaximal inhibitions.

Discussion

Amiloride, due to its ability to inhibit Na transport in a wide variety of cellular systems, has proven a useful drug for the study of molecular mechanisms involved in Na translocation across cell membranes (18). Gur results have provided, for the first time, unequivocal evidence indicating that amiloride, at doses that cause no side-effects (motor dysfbnction), presents systemic, spinal and supraspinal antinociceptive and antihyperalgesic actions in relation to chemical models of nociception, namely, acetic acid-induced writhing, capsaicin and formalin-induced licking and glutamate-mediated hyperalgesia. However, amiloride had no direct effect on the thermal model of pain, the hot plate test. The difference in the antinociception caused by amiloride between chemical and thermal models of nociception could be associated with a sustained input in the former model of nociception ( 19). An interesting result of the present study was the great effectiveness of amiloride, when given systemically, spinally or supraspinaliy, in blocking capsaicin-induced nociception. Capsaicin, a pungent ingredient isolated from hot peppers, administered to mammals, binds to specific membrane receptors and excites unmyelinated and poorly-myelinated A6 primary sensory neurones, causing release of neuropeptides, mainly tachykinins and calcitonin gene related peptide, and consequently

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inducing nociception (20,21). It has been suggested that acid stimuli may be endogenous activators of capsaicin-gated current, causing pain during ischemia and inflammation (22). The mechanism by which amiloride produces antinociception still remains not completely understood. It has been reported that amiloride has broad pharmacological actions, such as blockade of II+-gated cation and voltage gated Ca and Na channels, and it can also antagonise adenosine Al receptor (1,2,6). All these effects could, at least in part, contribute to the reported antinociceptive action of amiloride.

Our results show that the antinociceptive actions of amiloride involve, partly, an action on C fibres. There is now evidence indicating that both formalin and cap&in-mediated nociception are related with substance P and calcitonin gene related peptide, besides glutamate release into the dorsal spinal cord (12,23). The neonatal treatment of animals with capsaicin, in a dose known to produce a degeneration of afTerent unmyelinated fibres and decrease the SP content in the lumbar spinal cord (17), markedly attenuates amiloride-induced antinociception when assessed against the first phase of the formalin test. Also relevant are the results showing that amiloride, when given systemically, or by i.t. or i.c.v. routes, antagonises, in a graded and complete manner, glutamate-induced hyperalgesia assessed in the hot-plate test. These findings give further support for an action of amiloride on Cd and Na+ channels since glutamate action, among other effects, involves an influx of both Caf+ and Na+ ions across the cell membrane (24). The mechanism by which amiloride produces its antinociceptive action seems to be unrelated to non-specific toxic effects such as muscle relaxation or sedation of animals as revealed in the rota-rod assay, and appears to be associated, at least in part, with interference in the homeostasis and function of the somatosensory system. Thus, amiloride or its derivatives might be of potential interest in the development of new antihyperalgesic drugs.

Acknowledgements This study was supported by grants from CNPq and FINEP (Brazil). J. Ferreira and A.R.S Santos are MSc and PhD students in Pharmacology, respectively, and they thank CAPES for fellowship support.

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