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Abstract: Both systemically and intrathecally administered cholinergic agonists ... maintained on isoflurane anaesthesia for administration of oxotremorine ...

C Pharmacology & Toxicology 2002, 90, 187–192. Printed in Denmark . All rights reserved

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Intravenously Administered Oxotremorine and Atropine, in Doses Known to Affect Pain Threshold, Affect the Intraspinal Release of Acetylcholine in Rats Klas S. P. Abelson and A. Urban Höglund Department of Physiology, Division of Comparative Medicine, Biomedical Center, Uppsala University, Uppsala, Sweden (Received August 14, 2001; Accepted November 20, 2001) Abstract: Both systemically and intrathecally administered cholinergic agonists produce antinociception while cholinergic antagonists decrease pain threshold. The mechanism and the site of action of these substances are not known. In the present study it was hypothesized that systemically administered muscarinic agonists and antagonists modify nociceptive threshold by affecting intraspinal release of acetylcholine (ACh). Catheters were inserted into the femoral vein in rats maintained on isoflurane anaesthesia for administration of oxotremorine (10–300 mg/kg) and atropine (0.1, 10, 5000 mg/ kg). Spinal microdialysis probes were placed intraspinally at approximately the C2–C5 spinal level for sampling of acetylcholine and dialysis delivery of atropine (0.1, 1, 10 nM). Additionally, the tail-flick behaviour was tested on conscious rats injected intraperitoneally with saline, atropine (10, 100 and 5000 mg/kg), or subcutaneously with oxotremorine (30, 100, 300 mg/kg). Subcutaneous administration of oxotremorine (30, 100, 300 mg/kg) significantly increased the tail-flick latency. These doses of oxotremorine dose-dependently increased the intraspinal release of acetylcholine. Intravenously administered atropine, in a dose that produced hyperalgesia (5000 mg/kg) in the tail-flick test, significantly decreased the intraspinal release of acetylcholine. Our results suggest an association between pain threshold and acetylcholine release in spinal cord. It is also suggested that an approximately 30% increase in basal ACh release produces antinociception and that a 30% decrease in basal release produces hyperalgesia.

Systemic administration of muscarinic agonists produces an elevation of pain threshold in a variety of species including man (Flodmark & Wramner 1945; Harris et al. 1969; Pert 1975; Gower 1987; Machelska et al. 1999). It is not known whether these agonists act on supraspinal or spinal sites to produce antinociception. It is, however, likely that systemically administered muscarinic agonists produce antinociception through an activation of spinal muscarinic mechanisms since intrathecally administered muscarinic agonists increase pain threshold in several species including humans (Yaksh et al. 1985; Gillberg et al. 1989; Iwamoto & Marion 1993b; Naguib & Yaksh 1994, 1997; Abram & O’Connor 1995; Bouaziz et al. 1995; Hood et al. 1995; Eisenach 1999). Targets for muscarinic agonists would be the muscarinic acetylcholinergic receptors (mAChRs) localized in superficial laminae of the dorsal horn of spinal cord (Gillberg et al. 1988; Höglund & Baghdoyan 1997) where nociceptive Ad and C fibers terminate. The spinal cholinergic system appears to be differently organized than in brain. Administration of muscarinic agonists into different brain areas causes a decreased release of acetylcholine (ACh) (Billard et al. 1995; Baghdoyan et al. 1998) while intraspinal administration of oxotremorine or carbachol induces an increase in intraspinal ACh release Author for correspondence: A. Urban Höglund, Department of Physiology, Biomedical Center, Box 572, S-751 23 Uppsala, Sweden (fax π46 18 50 17 40, e-mail Urban.Hoglund/bmc.uu.se).

(Höglund et al. 2000). It is not known as of yet how an activation of mAChRs interferes with nociceptive signal transmission. It is likely though, that an increase in the basal release of ACh decreases the firing rate of nociceptive fibers, either through a direct stimulation of muscarinic receptors or through an indirect action on some of the other transmitter systems implicated to have an influence on the regulation on nociceptive transmission (Iwamoto & Marion 1993a; Xu & Tseng 1994; Xu et al. 1996, 1997; Baba et al. 1998; Eisenach 1999). Although it is assumable that systemic administration of muscarinic agonists increases intraspinal ACh release and consequently produces antinociception, this has never been shown. Likewise, it is not known to what extent the release of ACh needs to be increased to produce antinociception. With the working hypothesis that an increase of the spinal release of ACh increases the pain threshold, a decreased release of spinal ACh would produce a nociceptive response. Atropine produces hyperalgesia, suggestedly through a supraspinal action, when administered in high doses systemically (Ghelardini et al. 1990). Since we found that intraspinally administered scopolamine decreased the spinal release of ACh (Höglund et al. 2000), which is opposite to the effect of scopolamine in brain, it is plausible that also atropine reduces the intraspinal release of ACh, when administered systemically in a dose that produces hyperalgesia. The present study was designed to test the hypothesis

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that systemically administered oxotremorine, in doses that are known to produce antinociception, increases basal spinal ACh release, and that systemic administration of hyperalgesic doses of atropine decreases the intraspinal release of ACh. Part of this study was undertaken to further evaluate the suggestion by Gheraldini et al. (1990) that lower doses of systemically administered atropine produce antinociception.

Materials and Methods All experiments were conducted after approval of the Animal Ethics Committee in Uppsala, Sweden. Male outbred Sprague-Dawley rats (B&K Universal, Sollentuna, Sweden) (nΩ39) weighing 367–488 g were used for dialysis experiments. They had free access to food (R36, Ewos, Vadstena, Sweden) and tap water at all times. The animals were acclimatized after delivery at a room temperature of 20 ∫2æ and were kept on a 12 hr light/dark cycle for one week. Drugs and chemicals used. The drugs oxotremorine sesquifumarate, atropine, neostigmine bromide, acetylcholine chloride and choline chloride were purchased from Sigma-Aldrich Sweden AB, Stockholm, Sweden. Methohexital sodium (BrietalA) was purchased from Eli Lilly, Indianapolis, Indiana, USA. Isoflurane was purchased from Abbot Scandinavia, Kista, Sweden. The salts NaCl, CaCl2, Na2HPO4 and KCl were purchased from KEBO Lab, Spånga, Sweden. Spinal microdialysis probes were purchased from Marsil Enterprises, San Diego, California, USA. Experimental procedure. Intraspinal dialysis. For each experiment, anaesthesia was induced with methohexital sodium (40 mg/kg intraperitoneally). The rat was intubated, connected to a HarvardA (Harvard Apparatus Inc., South Natic, Massachusets, USA) ventilator, and placed on a heated pad to maintain core body temperature at 37.5æ. The methohexital sodium anaesthesia was subsequently replaced by isoflurane in 100% oxygen. During surgery, isoflurane was kept at about 2.5%, and during microdialysis sampling isoflurane was maintained at 1.3%. The end-tidal pCO2 was kept at 4 kPa. A catheter was inserted into the femoral vein, enabling intravenous injection of oxotremorine and atropine. For insertion of the microdialysis probe, a midline incision was made at the back of the skull. The neck muscles were dissected to expose the cisterna magna. The dura and pia mater were cut and a spinal microdialysis probe (Marsala et al. 1995) was inserted intraspinally so that the tip was approximately at the C5 level. The probe was perfused (3 ml/min.) with Ringer’s solution (147 mM NaCl, 2.4 mM dihydrous CaCl2, 4.0 mM KCl), containing 10 mM of the acetylcholine esterase inhibitor neostigmine to prevent the degradation of ACh (Billard et al. 1995; Roth et al. 1996; Höglund et al. 2000). After insertion of the microdialysis probe, the rats rested for 40 min. before spinal microdialysis sampling. ACh (pmol/10 min. dialysis sample) was quantified by high performance liquid chromatography (HPLC) with electrochemical detection (Antech, Leyden, The Netherlands). The mobile phase was 50 mM Na2HPO4 (pH 9.0), which enabled detection of ACh at 4.25 min. and choline after 5.5 min. The dialysis probe recovery of ACh was determined in vitro both before and after each experiment to ensure that all microdialysis measures accurately reflected the spinal ACh release and were not confounded by intra-experimental probe damage. A standard calibration curve ranging from 1 to 20 pmol ACh was established before each experiment from two samples of each concentration. Twenty ml of the 30 ml collected were analyzed for ACh directly after each sampling period was completed. Oxotremorine in doses of 10, 30, 100 and 300 mg/kg or atropine (0.1, 10 and 5000 mg/kg) were administered intravenously over 7 min. during the fifth intraspinal baseline sampling period. After

injection, the intravenous catheter was rinsed with approximately 1 ml of 0.9% NaCl to ensure that the entire quantity of the substances was administered. Five intraspinal sampling periods were used to calculate baseline release of ACh from which the percent change of release of ACh was calculated. The effect of intravenously administered oxotremorine was found to be of short duration, thus it was considered feasible to repeat the administration of the same dose three times in the same animal. After collection of five samples (50 min.) representing the effect of oxotremorine, another five 10 min. dialysis samples were collected to determine the new basal release of ACh before another dose of oxotremorine was administered. The baseline samples collected between oxotremorine administrations did not differ significantly. The action of atropine was found to cause a more long-lasting effect on the intraspinal release of ACh, thus the effect of this substance was studied for 150 min. after collection of five 10 min. baseline samples. The doses of oxotremorine and atropine were chosen based on the results presented by Gower (1987) and Gheraldini et al. (1990). Gower (1987) showed that 30, 100 and 300 mg/kg subcutaneously administered oxotremorine significantly increased tail-flick latency in rats. Gheraldini et al. (1990) showed that 10 mg/kg atropine increased licking latency and tail-flick latency, and that 5000 mg/kg administered subcutaneously decreased licking latency in rats. According to our working hypothesis, the doses 30, 100 and 300 mg/ kg of oxotremorine and 10 mg/kg of atropine would increase while the 5000 mg/kg dose would decrease the intraspinal release of ACh. To study the effect of intraspinally administered atropine on ACh release, atropine was added to the Ringer’s solution containing 10 mM neostigmine to make concentrations of 0.1, 1 and 10 nM. In these experiments, baseline release of ACh was recorded during five 10 min. sampling periods. Thereafter atropine was administered by dialysis in increasingly higher concentrations during five 10 min. sampling periods. No animal was treated with more than three concentrations of atropine. The concentrations were chosen based on the reports by Doods et al. (1987) and Bolden et al. (1992) who showed that the Ki of atropine is about 1 nM. Tail-flick tests. Male Sprague-Dawley rats were used in this experiment. These animals were kept under similar conditions as the rats used for dialysis experiments. One week before the tail-flick tests started the rats were handled daily to accommodate them to the experimental procedure. Tail-flick latency was tested using an IITC model 33 analgesia meter (IITC Inc., Woodland Hills, CA, USA), with a sensitivity setting of 10, beam at 8, and a cut-off time set at 10 sec. Baseline tail-flick measurements were obtained after administration of saline 4 ml/kg intraperitoneally in the atropine test or 1 ml/kg subcutaneously in the oxotremorine test. The effect on tailflick latency of different doses of atropine 10, 100 or 5000 mg/kg (1, 10 and 500 mg in a volume of 10 ml/kg intraperitoneally) was studied at 15, 30 and 45 min. after injection. The effect of oxotremorine 30, 100 or 300 mg/kg administered subcutaneously was studied 20 min. after injection. Statistical analysis. Intraspinal dialysis data. To calculate the statistical difference between groups of animals treated with different concentrations of substances the data was analyzed using repeated measures analysis with the Tukey post-hoc test (SPSS version 10.0). After that a significant difference within treatment groups was established with the repeated measures analysis, analysis of variance with the Dunnett’s post hoc test was performed to determine which of the time points were different from baseline release of ACh. P values ⬍0.05 were considered significant. Tail-flick tests. The atropine data was analyzed using analysis of variance (ANOVA, SPSS version 10.0). The Dunnett’s post-hoc test was used to determine the statistical difference between salinetreated animals and the change in tail-flick latencies at 15, 30 and

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45 min. after injection of atropine. The oxotremorine data was analyzed using the non-parametric Wilcoxon Signed Ranks Test because some animals treated with 100 and 300 mg/kg reached the cutoff time of 10 sec. in tail-flick latency.

Results Probe recovery of ACh averaged 32% and for each experiment, pre- and post-experimental in vitro probe recoveries were compared by t-test. For all data described there were no statistically significant changes in dialysis probe recovery during any single experiment. Five samples were taken before each individual experiment to establish the basal release from which the percent change was calculated. The basal release averaged 2.48∫0.32 (mean∫S.E.M, nΩ52) pmol/20 ml dialysate. The basal release for each experiment is presented in the figure legends. Intravenous administration of oxotremorine 30 mg/kg produced a rapid 20% significant increase of intraspinal release of ACh during the first 10 min. after administration (F(4)Ω23.84). The effects of 100 and 300 mg/kg oxotremorine at 10 min. after injection were 27% (F(4)Ω10.21) and 73% (F(4)Ω103.85) respectively and lasted longer (fig. 1). The repeated measures test with the Tukey post-hoc test showed that the effect of 300 mg/kg was significantly different from the effect of 10 and 30 mg/kg (F(3)Ω7.35). Non-linear regression analysis showed that these data fit a normally shaped dose-response curve with a calculated maximum at 177% and 134% respectively with ED50Ω500 and 630 mg/kg respectively (fig. 2). All doses of oxotremorine produced a brief, about 1æ decrease in core body temperature, which was counteracted by the heating pad which

Fig. 1. Effects of 10 (ò, number of experiments (n)Ω3), 30 (h, nΩ 9), 100 (H, nΩ10) and 300 (g, nΩ6) mg/kg oxotremorine injected intravenously on the intraspinal release of ACh. The effect of oxotremorine is expressed as percent change from baseline. Basal ACh release was 1.40∫0.02, 2.17∫0.66, 2.49∫0.35 and 0.76∫0.06 (mean pmol/20 ml∫S.E.M.), respectively. *represents P⬍0.01 versus baseline as determined with Dunnett’s post-hoc test (F(5,48) Ω12.48, F(5.30) Ω28.07 and F(5,54) Ω5.97 for 30, 100 and 300 mg/kg, respectively). Ω Y represents P⬍0.05 versus 10 mg/kg at each time point as determined with repeated measurements test followed by the Tukey’s post-hoc test (F(3,24) ⱖ14.74).

Fig. 2. Non-linear curve-fit of the effect of 10, 30, 100 and 300 mg/kg subcutaneously administered oxotremorine on the intraspinal release of acetylcholine at 10 (H) and 20 (h) min. after injection of oxotremorine. The goodness of fit (r2) was 0.98 for both curves.

the rat rested on. Normal body temperature was attained again after about 5 min. after injection. Salivation was observed after administration of 100 and 300 mg/kg intravenously. The subcutaneous administration of 100 and 300 mg/kg oxotremorine to conscious animals increased the tail-flick latency significantly. A few of the animals treated with 100 mg/kg and almost all of the animals treated with 300 mg/kg reached the cut-off time of 10 sec. (fig. 3). Animals treated with the highest dose of oxotremorine showed clear signs of muscarinic receptor activation such as extensive salivation, lacrimation and defaecation. In addition, these animals also showed a behaviour with short time lasting sudden jerks. At 100 mg/kg the only sign of activation of muscarinic receptors was an extensive salivation. Intravenous atropine 5000 mg/kg produced a long-lasting significant (F(2,9)Ω8.7) decrease in intraspinal ACh release (fig. 4). About 50 min. after injection the decrease was stabilized at about 30%. Ten and 0.1 mg/kg did not change the

Fig. 3. Effects of subcutaneously administered saline, 30, 100 or 300 mg/kg oxotremorine on the tail-flick latency. *represents P⬍0.01 versus saline as determined with the Wilcoxon Signed Ranks Test (ZΩª3.9, 100 mg/kg; ZΩª3.3, 300 mg/kg).

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parison to control animals while 10 mg/kg had no effect 15 min. after administration. The effect of 5000 mg/kg atropine 15 min. after administration was significant (F(3,100)Ω2.7, PΩ0.021). Due to the subsequent decrease in tail-flick latencies in the saline treated group at 30 and 45 min. after injection, no significant effects of the different doses of atropine were observed at these times. Discussion

Fig. 4. Effects of 0.1 (H, nΩ4), 10 (g, nΩ6) and 5000 (h, nΩ4) mg/ kg atropine injected intravenously on the intraspinal release of ACh. The effect of atropine is expressed as percent change from baseline. Basal ACh release was 2.16∫0.86, 4.26∫2.08, 4.88∫1.88 (mean pmol/20 ml ∫S.E.M.), respectively. *represents P⬍0.025 versus baseline as determined with Dunnett’s post-hoc test (F(15,43) Ω6.96). Y represents P⬍0.05 versus 0.1 mg/kg at each time point as determined with repeated measurements test followed by the Tukey’s post-hoc test (F(2,11) ⱖ6.75).

intraspinal release of ACh significantly from baseline. Note that the effect of 10 mg/kg is inexplicably less pronounced than after administration of 0.1 mg/kg. Intraspinal administration of atropine (0.1, 1 and 10 nM) produced significant decreases of intraspinal release of ACh (F(3)Ω33.2). The maximal decrease was about 30% after microdialysis of both 1 and 10 nM atropine (fig. 5). Neither the intravenous nor the intraspinal route of administration of atropine caused any changes in core body temperature. The doses 100 and 5000 mg/kg of intraperitoneally administered atropine decreased the tail-flick latency in com-

Fig. 5. Effects of intraspinally delivered 0.1 (H, nΩ5), 1 (g, nΩ5), 10 (h, nΩ5) nM atropine and controls (dialysis with Ringer’s solution only ò, nΩ5). The effect of atropine is expressed as percent change from baseline. Basal ACh release was 3.74∫1.25, 3.74∫1.25, 3.74∫1.25, and 4.14∫1.05 (mean pmol/20 ml ∫S.E.M.), respectively. *represents P⬍0.05 and **represents P⬍0.01 versus baseline as determined with Dunnett’s post-hoc test (F(5,24) Ω3.76, F(5.24) Ω14.67 and F(5,24) Ω9.25 for 0.1, 1 and 10 nM atropine, respectively).

The present data supports our working hypothesis that there is a relationship between the intraspinal release of ACh and the pain threshold. The subcutaneous administration of oxotremorine 100 and 300 mg/kg produced significant increases in tail-flick latency in accordance with previous studies (Gower 1987; Machelska et al. 1999). These doses also caused a dose-dependent increase in intraspinal release of ACh. Thus, it may be suggested that subcutaneously administered oxotremorine produces antinociception through an increase in the spinal release of ACh. Unfortunately, it is not possible to correlate the increase in tail-flick latency with the increase of spinal ACh release, since the tail-flick latency is not a fully graded response because the need for a cut-off time. The present data allow us, however, to suggest that a 27% increase in intraspinal ACh release is enough to obtain potent analgesia, since this increase was seen after subcutaneous administration of 100 mg/kg oxotremorine. The non-linear curve-fit analysis showed that the maximum increase of ACh release would be 177% 10 min. and 134% 20 min. after the subcutaneous administration of oxotremorine if a normally shaped dose-response curve is assumed. This is close to the maximal effect of 160% observed after intraspinally administered oxotremorine (Höglund et al. 2000) and thus supports the hypothesis that the subcutaneous administered oxotremorine produces its antinociceptive action through a spinal action. Since only a 27% increase in the spinal release of ACh is needed to produce antinociception, a potent means to obtain analgesia may be harboured within the spinal cholinergic nervous system. Other substances such as clonidine, charbacol, epibatidine, and morphine have also been found to enhance the release of spinal ACh (Detweiler et al. 1993; Xu et al. 1997; Höglund et al. 2000). Since all these substances are antinociceptive, the common denominator may be the up-regulation of spinal ACh release. Under the assumption that these substances act specifically on adrenergic, muscarinic, nicotinic and opiate receptors the regulation of spinal ACh release appears to be rigorously controlled. The resultant decrease in intraspinal ACh release after subcutaneous administration of 5000 mg/kg atropine further supports our working hypothesis. This dose of atropine decreased the tail-flick latency and reduced the intraspinal release of ACh with about 30% from baseline. The 30% reduction of ACh release appears to be the maximal reduction of ACh release possible to obtain with the use of atropine (and scopolamine (Höglund et al. 2000)) since the intraspinal

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microdialysis of 1 (ΩKi) and 10 nM both caused an approximately 30% reduction. This observation is in line with the above-mentioned suggestion that not only muscarinic receptors are involved in the tonic regulation of spinal ACh release. Gheraldini et al. (1990) showed that 10 mg/kg of atropine increased both licking latency in the hot-plate test and the tail-flick latency, and that 5000 mg/kg decreased both latencies. Based on these findings they concluded that atropine in low doses amplifies cholinergic transmission by a selective blockade of presynaptic autoreceptors. Their findings are not in accordance with our suggestion that spinal ACh release is not autoreceptor-regulated through muscarinic receptors (Höglund et al. 2000). The present behavioural study focused on the effect of 10 mg/ kg atropine. Although data was collected from 25 rats, we were unable to observe increased tail-flick latencies. The reason for the discrepancy between our findings and the study by Gheraldini et al. (1990) is unknown, but may reside in the use of different strains of rats. Our findings are, however, in agreement with the results presented by Zhuo & Gebhart (1991) who injected both atropine and scopolamine intrathecally in wide dose ranges and observed decreases in nociceptive threshold. The present data confirms our previous suggestion that muscarinic receptors are not participating in the presynaptic autoreceptor regulation of spinal ACh release. Previously, two studies have been performed regarding the effects of intravenously administered analgesics on the intraspinal release of ACh. Xu et al. (1997) and Bouaziz et al. (1996) found that intravenous morphine caused a dosedependent increase in intraspinal ACh release. These studies suggest that morphine activates the release of intraspinal ACh through an action on bulbospinal pathways. In relation to this suggestion, it is possible that systemically administered atropine and oxotremorine are acting on supraspinal pathways to produce the changes in intraspinal ACh release presented here. In such a case a supraspinal action of oxotremorine would result in a reduced release of ACh (Billard et al. 1995), which in turn could change the activity in descending pathways regulating the spinal release of ACh. Further studies including transection of the spinal cord might provide more insight into this possibility. In summary, it is suggested that pain threshold is regulated by changes in tonically regulated release of ACh in spinal cord. A 20–73% up-regulated release of ACh, as seen after oxotremorine administration of doses 30–300 mg/kg intravenously, is associated with an increased pain threshold. A 30% down-regulated ACh release, as observed after 5000 mg/kg atropine intravenously is associated with a lowered pain threshold. The present data confirms earlier findings that intraspinal release of ACh is not regulated by presynaptic muscarinic autoreceptors. Acknowledgements The work was supported in part by The Swedish Medical Research Council (grant no. K98-04R-12790) and Council

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for Medical Tobacco Research, Swedish Match (grant no. 200006).

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