Psychopharmacology (1993) 110:497-499

Psychopharmacology © S p r i n g e r - V e r l a g 1993

Modulation of nicotine-induced analgesia by calcium agonist and antagonist in adult rats* Christina W.Y. Chin, Robert C. Block, Wen Hsien Wu, and Vlasta K. Zbuzek Department of Anesthesiology, UMD-NJ Medical School, 185 South Orange Avenue, Newark, NJ 07103-2714, USA Received July 28, 1992 / Final version September 3, 1992

Abstract. T h e i n v o l v e m e n t of calcium in nicotine-induced analgesia in male rats was e x p l o r e d using the tail-flick test. A single dose of nicotine (1 m g / k g SC) p r o d u c e d a maxim a l effect on tail-flick latency (15 s) within 8 - 1 0 r a i n , which lasted for 4 min. P r e t r e a t m e n t with the calcium chelator, E D T A (250 g M / k g SC four injections at 15 rain intervals), before the single dose of nicotine accelerated the onset a n d p r o l o n g e d the d u r a t i o n of the nicotine-induced analgesia. T h e m a x i m a l effect on tail-flick latency occurred within 2 m i n a n d lasted for 10-20 rain. Conversely, p r e t r e a t m e n t with calcium chloride (1.5 m M / k g IP) att e n u a t e d n i c o t i n e - i n d u c e d analgesia. It is suggested that nicotine m a y exert its antinociceptive effects via m o d u l a tion of calcium fluxes across the n e u r a l m e m b r a n e . Key words: N i c o t i n e analgesia - C a l c i u m - E D T A - Rats Tail-flick

D u r i n g the last 2 decades, evidence has s u p p o r t e d the view t h a t a single dose of nicotine has an antinociceptive effect ( M a t t i l a et al. 1968; Aceto et al. 1983; Christensen a n d Smith 1990) a n d t h a t r e p e a t e d injections of nicotine induce tolerance ( P h a n et al. 1973). T h e m e c h a n i s m s involved in nicotine-induced analgesia are n o t well u n d e r s t o o d . It is generally recognized t h a t nicotine affects different n e u r o t r a n s m i t t e r s , which may, at least in part, c o n t r i b u t e to nicotine-induced analgesia. F o r example, centrally a d m i n i s t e r e d nicotine interacts with the e n d o g e n o u s o p i o i d s ( M o l i n e r o a n d Rio 1987; Naftchi et al. 1988; D a v e n p o r t et al. 1990) while opiatei n d u c e d analgesia seems to be well c o r r e l a t e d with m e t a l ions, especially calcium ( C h a p m a n a n d W a y 1980). Since calcium blockers seem to p o t e n t i a t e the analgesic effects of several drugs such as m o r p h i n e and fentanyl ( M i r a n d a a n d Paeile 1990), we e x p l o r e d w h e t h e r calcium ions m a y also be involved in n i c o t i n e - i n d u c e d analgesia.

* Presented in part, as a poster at the 75th Annual Meeting of FASEB, Atlanta, Georgia, April 21-25, 1991. Correspondence to. V.K. Zbuzek

Materials and methods Animals' The research protocol was approved by the Institutional Animal Care and Use Committee. Adult male Sprague-Dawley rats, 5 months old and weighing 250-265 g, were used in all experiments. They were housed three per cage with free access to food and water at constant temperature of 22 ± 0.5 °C and 12 h light/dark cycle.

Pain measurement In this study, the tail-flick (TF) latency was recorded as the measurement of pain response, A tail-flick analgesia meter (model 33, Innovators in Instrumentation Inc., Landing, N J, USA) was used. This device consists of a photoelectric lamp as the heat source, a photoelectric sensor to detect the tail-flick and an automatic timing device. The beam intensity dial was set to obtain basal latencies of 6-8 s. The rats were kept in a transparent restrainer with their tails free to move outside. The tip of the tail was exposed to a spot of radiant heat from the photoelectric lamp above at a fixed distance. When the rat felt pain, it flicked its tail away from the heat. The time elapsed from the exposure to the flick of the tail (TF latency) was recorded automatically in 0.1 s. The cut-off time was set to 15 s to prevent thermal injury to the tail.

Test compounds and dosage 1. 0.9% normal saline, was injected SC in the volume of 1 ml/kg body weight, as a control to nicotine. 2. Nicotine free base (Sigma) was tested at doses of 0.5, 1.0 and 1.5 mg/kg SC. The dose of 1.0 mg was selected and injected in a volume of 1 ml/kg. 3. Ethylenediamine-tetraacetic acid (EDTA) (Sigma) was injected SC at a dose of 250 gM in a volume of 2 ml/kg body weight, 4 times at 15 rain intervals, (modified from Kakunga et al. 1966). 4. CaClz was tested at the doses of 2.5, 1.5 and 1.25 mM/kg IP in single and divided doses at different time intervals. A dose of 1.5 mM in a volume of 2 ml/kg, injected 1 h before nicotine, was selected in this study.

Procedure All rats were acclimated in restrainers 2 h a day for 3 days, and the experiments were carried out at the same period of time during the

498 day. Before each experiment, the rats were allowed to rest in the restrainers for 40 min.

Table 1. Dose-related analgesic effect of nicotine and its side effects Dose mg/kg SC

ED a

Side effects

Experimental protocols

Tremor

Convulsion

60% 16% 0

60% 0 0

Experiment 1. After habituation in the restrainer, three consecutive baseline TF latencies were measured in 20 rats. After injection of saline, TF latencies were measured every 2 min for 20 min, followed by every 5 rain up to 1 h. One week later, nicotine was injected and the same paradigm was used to measured TF latency. Twelve rats which responded to nicotine-induced analgesia (responders) were assigned randomly to two groups (with equal numbers) for experiments 2 and 3, which were carried out 1 week later.

1.5 mg 1.0 mg 0.5 mg

Experiment 2. In this experiment, rats (n = 6) were pretreated with EDTA (250 gM/kg) 4 times at 15 rain intervals, then injected with saline and TF was measured as in experiment 1. One week later, the same experiment was repeated and nicotine was injected instead of saline. The same paradigm was followed as in experiment 1.

induced analgesia. CaCI2 was injected at 60, 30, 15 and 0 min prior to the injection of nicotine (1 mg/kg SC)

Experiment 3. In this experiment, rats (n = 6) were pretreated with CaC12 (1.5 m M / k g IP) 1 h before the nicotine was injected. The same paradigm was carried out as experiment 1.

Calculation and statistical analysis The values of the TF latency are presented as medians; the area under the curve (AUC) was calculated for each rat. Statistical analyses of mean AUC areas were assessed by one-way analysis of variance. Fisher's LSD (least significant difference) test was used to define the difference between groups.

Results

100% 60% 0

" Effective dose

Table 2. Dose-related inhibitory effect of calcium on nicotine-

Dosage mM/kg IP Effect

60 min

30 rain

15 rain

0 rain

2.5 mM

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--90% 10% 0 0

--62% 13% 0 0

0 80% 38% 13% 0 0

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1.5 mM 1.25 mM "Effective dose bLethal dose

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In the present study, injection of 1 m g / k g nicotine revealed 60% responders. All animals used in experiments 2 and 3 were responders. Occasionally, this dose p r o d u c e d tremors within the first minute after the injection, which disappeared during the next 1 rain. A similar post-nicotine reaction has been described (Tripathi et al. 1982). Higher dosage with nicotine (1.5 mg/kg) p r o d u c e d longer lasting tremors which m a d e the measurement of T F impossible and a lower dosage (0.5 mg/kg) was not effective (Table 1). Calcium chloride at a dose of 2.5 m M / k g IP, as originally used in mice by K a k u n g a et al. (1966) injected either in single or divided doses, caused 80% mortality in rats, in our experiments. While the dose of 1.25 m M had no effect, the dose of 1.5 m M / k g IP, injected 60 rain before nicotine, had an optimal antagonizing effect on nicotine-induced analgesia, without mortality (Table 2).

Nicotine alone Nicotine alone p r o d u c e d a maximal T F latency increase (15 s) within 8-10 min, which lasted for 4 min (Fig. 1). The m e a n A U C was 42.19 and 137.36 in saline and nicotine treatment, respectively (P < 0.0001).

EDTA pretreatment As with saline, E D T A alone (250 g M / k g 4 times) did not change T F latency. However, rats pretreated with E D T A

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Fig. 1. Median TF latency measured in naive rats following a single dose of saline (- - -) and nicotine ( ), injected at time 0, indicated by an arrow

responded to nicotine by a maximal T F latency increase within 2 rain, which lasted for 14 min (Fig. 2). The mean A U C was 55.25 and 206.42 in E D T A alone and nicotine with E D T A pretreatment (P < 0.0001), respectively. In this group, the analgesic response increased 1.5 times c o m p a r e d to nicotine alone (P < 0.01).

Calcium pretreatment A single dose of CaC12 (1.5 m M / k g ) did not change the T F latency measured for 2 h (data not shown). However, the analgesic effect of nicotine was completely abolished by the pretreatment with CaC12 1 h before (Fig. 3). The mean

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Fig. 2. Median TF latency in rats pretreated four times with EDTA as indicated by four perpendicular arrows. At time 0, the rats were injected with saline (- - -) or nicotine ( )

nicotine, as shown not only for morphine ( K a k u n a g a et al. 1966) and endogenous opiates ( C h a p m a n and W a y 1980) but also for prolactin ( R a m a s w a m y et al. 1986). The present findings m a y indicate that nicotine, as with morphine, produces an analgesic effect by altering the influx of calcium across the membrane. In a recent review, evidence was b r o u g h t to support the existence of a relationship between calcium modulators and opioid drugs (Miranda and Paeile 1990). Apparently, the analgesic effect of non-opioid drugs, such as prolactin (Ramaswamy et al. 1986) and nicotine, (present study) m a y also, at least in part, be modulated by calcium. If our hypothesis is correct, then calcium channel blockers, drugs used in the treatment of cardiac patients, should potentiate the antinociceptive effect of nicotine. Appropriate experiments are in progress to answer this question. Acknowledgements. The authors wish to thank Dr. Stanley Von

Hagen for statistical analysis, Dr. John Katz for invaluable comments and Ms. Doric Johnson for her excellent aid in the preparation of this manuscript.

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Fig. 3. Median TF latency in rats pretreated with calcium 1 h before nicotine ( ) and nicotine alone (- - -) injected at time 0 A U C was 162.58 and 66.58 in nicotine alone and nicotine with CaC12 pretreatment (P < 0.0001), respectively.

Discussion The present study was designed to test, in rats, the hypothesis that calcium is involved in the antinociceptive effect of nicotine. Indeed, the calcium chelator E D T A potentiated whereas calcium ions attenuated the antinociceptive effect of nicotine. Although this pilot study is the first report on this subject, some similar findings are described in the literature. F o r example, in mice, morphine-induced analgesia was potentiated by E D T A ( K a k u n a g a et al. 1966) and endorphin/enkephalin analgesia by E G T A ( C h a p m a n and W a y 1982). O n the contrary, pretreatment with calcium chloride attenuated the analgesic effect of

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