Antinociceptive interactions between intrathecally administered α noradrenergic agonists and′5-N-ethylcar☐amide adenosine

Brain Research, 519 (1990) 287-293 Elsevier

287

BRES 15567

Antinociceptive interactions between intrathecally administered a noradrenergic agonists and 5'-N-ethylcarboxamide adenosine Sofia Aran* and Herbert K. Proudfit Department of Pharmacology, University of lllinois at Chicago, Chicago, IL 60680 (U.S.A.) (Accepted 5 December 1989) Key words: Adenosine; N-Ethyicarboxamide adenosine; Clonidine, antinociception; Spinal cord; Intrathecal; a2; Phenylephrine Recently, it has been shown that intrathecal injection of norepinephrine and the mixed A 1 / A 2 adenosine agonist 5'-N-ethylcarboxamide adenosine (NECA) interact in a supra-additive manner to produce antinociception. The present studies were designed to determine whether a 1 or a 2 noradrenergic receptors are involved in producing the antinociception induced by NECA and norepinephrine. The results indicated that intrathecal injection of NECA (0.97-4.9 nmol), the ct2 noradrenergic agonist clonidine (3.8-375 nmol), or the a 1 agonist phenylephrine (4.9-73.4 nmol) produced dose-dependent antinociception in rats. Furthermore, intrathecal injection of subeffective doses of NECA and clonidine interacted supra-additively to produce potent antinociception. In contrast, no supra-additive interaction was observed between NECA and phenylephrine. The supra-additive interaction of NECA and clonidine did not appear to result from alterations in cardiovascular tone because changes in blood pressure and nociceptive thresholds were not correlated in time. These results suggest that the noradrenergic component of the supra-additive interaction between adenosine A 2 receptor agonists and noradrenergic agonists is mediated by a 2 noradrenergic receptors.

INTRODUCTION Adenosine and adenosine analogs appear to modulate nociceptive information in the spinal cord since intrathecal administration of adenosine agonists produces antinociception that can be blocked by adenosine receptor antagonists 1-3A3'14'2°'3°'33. Furthermore, adenosine receptors have been demonstrated in the spinal cord dorsal horn 16"19 and are concentrated in the superficial laminae 7'8 where many of these receptors are located on spinothalamic tract neurons 9. Recent evidence suggests that adenosine interacts in a supra-additive manner with norepinephrine in the spinal cord to produce potent antinociception, For example, intrathecal administration of norepinephrine and Nethylcarboxamide adenosine ( N E C A ) , a mixed A1/A 2 adenosine agonist, produces antinociception by a supraadditive mechanism in the spinal cord 1'3. However, R-phenylisopropyl adenosine ( R - P I A ) , a selective adenosine A 1 receptor agonist, does not interact in a supraadditive manner with norepinephrine, which indicates that this supra-additive interaction is mediated either by adenosine A 2 receptors alone or by a combined action at both A 1 and A 2 receptors 1"3. There is a large amount of convincing evidence that noradrenergic a 2 receptors mediate the antinociceptive actions of intrathecally-

administered norepinephrine in the spinal cord (for review see Proudfit 31 or Yaksh41). However, the noradrenergic receptors that modulate the supra-additive antinociceptive interactions between norepinephrine and adenosine have not been clearly defined. There is some evidence that a 2 noradrenergic receptors may mediate this interaction 2'14, but the nature of the interactions was not clearly defined in these reports because the methods of analysis were not appropriate. The purpose of the present study was to identify the noradrenergic receptor subtype involved in producing the supra-additive interaction between N E C A and norepinephrine. In addition, isobolographic analysis was used to more accurately determine the nature of the interaction between noradrenergic and purinergic agonists. The results indicated that the noradrenergic component involved in the supra-additive interaction between N E C A and noradrenergic agonists is mediated by the a2 receptor subtype. A preliminary report of these results has been previously presented 2. MATERIALS AND METHODS Surgical procedures Male Sprague-Dawley rats (400-450 g) were anesthetized with ketamine (100 mg/kg, i.p.) and implanted with chronic intrathecai catheters using a modification of the technique described by Yaksh

* Present address: Department of Anesthesia and Critical Care, University of Chicago, 5841 S Maryland, Box 428, Chicago, IL 60637, U.S.A. Correspondence: H.K. Proudfit, Department of Pharmacology, University of Illinois at Chicago, P.O. Box 6998, Chicago, IL 60680, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

288 and Rudy 4°. The cannula tip lay on the dorsal surface of the spinal cord at the level of the lumbar segments. Seven days after surgery, one group of these animals was anesthetized again and implanted with chronic indwelling cannulae in the carotid artery for measurement of blood pressure. All animals were housed individually. After cannulation of the carotid artery, an additional two days were allowed for recovery before experimental procedures were performed.

saline; clonidine + saline; or clonidine + NECA. Tail flick and hot plate latencies were determined 15, 30, 60, and 90 min after coadministration of drugs. The effects of NECA and phenylephrine on nociception were similarly determined in a separate group of animals. Animals in this group were randomly assigned to one of three treatment groups: NECA + saline; phenylephrine + saline; or NECA + phenylephrine. Tail flick and hot plate latencies were determined at 5, 10, 15, and 30 min after coadministration of drugs.

Analgesiometric testing procedures

Statistical methods

The effects of intrathecal drug injections on nociception were determined using both the tail flick and hot plate tests. For the tail flick test, a high intensity light beam was focused on the dorsal surface of the rat's blackened tail. The time interval between the application of the light and the tail flick response was measured electronically and the stimulus was terminated at 14 s in the absence of a response. The average of three such measurements was defined as the tail flick latency (TFL). The hot plate response was determined by placing the rat on a 55 °C copper plate and the time taken to respond by either licking the hind paw or jumping was defined as the hot plate latency (HPL). Animals were removed from the hot plate at 40 s in the absence of a response.

Comparisons between treatment group means were made using two-way analysis of variance for repeated measures. Comparisons between individual means were made using the Newman-Keuls test for multiple post-hoc comparisons 25. Statistical comparisons of the slopes of dose-response curves to determine parallelism were made using linear regression and Student's t-test 35. The statistical method employed to determine the mode of drug interaction was a modification of isobolographic analysis j7'18'27'38. Regression analysis was used to estimate the doses of NECA, clonidine or phenylephrine, given alone, that produce the same effect as both drugs given together. For example, if the combination of drugs produced an increase in mean response latency that was 60% of maximum (the cutoff value), then the ED60 (effective dose) for each drug given alone would be determined from the individual doseresponse curves for these two drugs. The resulting ED values are plotted with their corresponding 95% confidence limits. The line connecting the two ED values is termed the 'dose-additive' line. The doses for the two drugs given in combination are plotted as a single point. If the point for the combined drugs falls below the dose-additive line the interaction is considered to be supra-additive, and if it lies above this line, an infra-additive effect is indicated 3s. Points for drug combinations which fall within the 95% confidence limits around the dose-additive line reflect an additive interaction.

Blood pressure measurements Blood pressure changes produced by intrathecal administration of drugs were determined using animals implanted with both an intrathecal catheter and a carotid cannula. The purpose of these experiments was to determine whether drug-induced changes in nociception are associated with changes in blood pressure. Blood pressure was recorded in unrestrained rats by connecting the indwelling cannula to a pressure transducer using a 60 cm length of PE-20 tubing. Baseline measurements were continuously monitored until blood pressure values were stable. Intrathecal injections were then made without restraining the animals. Tail flick responses were measured in the same manner as the other experimental groups. The hot plate test was not performed using these animals.

RESULTS

lntrathecal drug injection All drugs were injected into the spinal cord subarachnoid space in a volume of 15 ~1 followed by 10/~1 of saline at a rate of 15/d/min using an electric gear-driven syringe pump. Each drug was dissolved in physiological saline and filtered using a 0.2/~m filter (Millipore) immediately before injection. The pH of drug solutions ranged from 5.0 to 7.0, and was not adjusted to pH 7.3 because, in our experience, injections of solutions in this pH range have no discernible effects on either nociception or the antinociceptive actions of various drugs. The following drugs were used: 5'N-ethylcarboxamide adenosine (NECA), clonidine hydrochloride and phenylephrine hydrochloride. These agents were obtained from Sigma Chemical Co., St. Louis, MO.

Experimental protocol (1) Drug dose-response relationships. The purpose of this experiment was to determine the doses of NECA, clonidine and phenylephrine that would produce minimal changes in nociceptive thresholds. Tail flick and hot plate latencies were measured before and again at intervals of 15, 30, 60 and 90 min after intrathecal injection of various doses of NECA (0.97, 2.3 or 4.9 nmol) and clonidine (3.8, 11.3, 150.0 or 375.0 nmol). Response latencies were determined at 5, 10, 15 and 30 min after intrathecal injection of phenylephrine (4.9, 49.0 or 73.6 nmol). Each animal received a single drug dose. (2) Interactions between drugs. The purpose of this experiment was to determine whether NECA would interact supra-additively with the a 2 noradrenergic receptor agonist clonidine or the a I noradrenergic receptor agonist phenylephrine. Subeffective doses of NECA (2.3 nmol), clonidine (11.3 nmol) and phenylephrine (73.6 nmol) were used in these experiments. NECA and clonidine were coadministered because peak antinociceptive effects of each drug were observed within 30 min after the injection. Animals were randomly assigned to one of three treatment groups: NECA +

Time course of drug effects The antinociceptive effects of intrathecally administered NECA, clonidine and phenylephrine were assessed u s i n g t h e tail flick a n d h o t p l a t e tests. T h e effects o f various doses of these three drugs on response latencies as a f u n c t i o n o f t i m e is i l l u s t r a t e d in Figs. respectively.

The

latency

to

peak

i n j e c t i o n was 30 m i n f o r N E C A ,

effect

1 a n d 2, after

drug

and 15-30 min for

clonidine and phenylephrine. The duration of action was d o s e - d e p e n d e n t a n d r a n g e d f r o m 30 to 90 m i n f o r N E C A a n d c l o n i d i n e a n d f r o m 15 t o 30 m i n f o r p h e n y l e p h r i n e .

Dose-response relationships Dose-response

curves for the antinociceptive actions

of c l o n i d i n e , N E C A a n d p h e n y l e p h r i n e a r e i l l u s t r a t e d in Fig. 3. B o t h c l o n i d i n e a n d N E C A

p r o d u c e d statistically

s i g n i f i c a n t d o s e - d e p e n d e n t a n t i n o c i c e p t i o n as d e t e r m i n e d b y b o t h a n a l g e s i o m e t r i c t e s t s ( P <- 0.01, A N O V A ) .

In

contrast, intrathecal injection of phenylephrine produced statistically s i g n i f i c a n t a l t e r a t i o n s in n o c i c e p t i o n t h a t were dependent on the test used. Thus, phenylephrine produced a dose-dependent enhancement of nociception u s i n g t h e tail flick t e s t ( P -< 0.01, A N O V A ) ,

but had no

statistically s i g n i f i c a n t d o s e - d e p e n d e n t e f f e c t o n h o t p l a t e l a t e n c i e s ( P > 0.05, A N O V A ) .

However,

the highest

289

A

B

~

14

"~'12

C

4.9

375.0

>- 10 z w

8

5~ O -...1 LL

~2.3

~

150.0

0.97 •

4

11.3 • 3.8 •

I

_--a 2

T 0

?

?

I

I

I

I

I

I

0

30

60

0

30

60

TIME

I

i

i

90

I

,

i

0

I

i

,

15

I

30

(rain)

Fig. 1. Time course of alterations in tail flick latencies produced by intrathecal injection of: (A) NECA, (B) clonidine and (C) phenylephrine. Ordinate: tail flick latency (s). Abscissa: time (min). Drug injections were made at zero time (arrow) following baseline determinations of tail flick latency. The doses of each drug (nmol) are indicated to the right of each line. Values indicate means _+ S.E.M. for 6 to 9 rats. In this and subsequent figures, the error bars for some data points are obscured by the plotting symbol. Also, in some cases the symbols for baseline values are superimposed. latencies. However, significant increases in both tail flick and hot plate latencies were observed following the injection of both N E C A and clonidine (P -< 0.05, ANOVA). The peak effect occurred within 30 min after the injection and began to recover toward preinjection levels by 60 to 90 min. Isobolographic analysis indicated that low doses of N E C A (2.3 nmol) and clonidine (11.3 nmol) increase tail flick and hot plate latencies in a supra-additive manner when administered together (see inset, Fig. 4). In contrast to the supra-additive effects of N E C A and clonidine on nociceptive response latencies, phenylephrine failed to produce significant increases in response latencies when coadministered intrathecally with N E C A (Fig. 5). Thus, the mean tail flick and hot plate latencies

dose of phenylephrine (73.6 nmol) produced an increase in mean hot plate latency that was significantly different from the baseline control values (P -< 0.05, ANOVA). Statistical comparisons of the slopes of the doseresponse curves indicated that the lines for clonidine and N E C A were not parallel (P -< 0.01, Student's t-test) using either the tail flick or the hot plate test.

Interactions between drugs The capacity of subeffective doses of N E C A and clonidine to alter nociception was determined at 15, 30, 60, and 90 min following intrathecal coadministration of N E C A and clonidine (Fig. 4). Neither N E C A (2.3 nmol) nor clonidine (11.3 nmol) injected alone produced significant increases in either tail flick or hot plate

A

40

B

C

O

v09 >- 30

4.9

0 z U.l

~

375.0

2.4

20

150.0 11.3 I

W

o.g7 ~ I..O "1-

10 • ill

•

? 0

t 0

~

~./T

3.a

4.9

? I

I

t

30

60

0

I 73.6 49.0

? i

I

30

~

I

60

190

i

i

I

0

,

,

l

15

,

,

I

30

TIME (rnin) Fig. 2. Time course of alterations in hot plate ]atencies produced by intrathecal injection of: ( A ) N E C A , (B) clonidine and (C) phenylephrine.

See legend for Fig. 1.

290 at both 15 and 30 min after the coadministration of NECA and phenylephrine were not significantly greater than the mean response latencies after either NECA alone or phenylephrine alone (P -> 0.05, ANOVA). Isobolographic analysis could not be used to analyze the interactions between NECA and phenylephrine since the two agonists had opposite actions using the tail flick test and phenylephrine did not produce a significant doseresponse relationship using the hot plate test.

Blood pressure effects Because all of the drugs used in these experiments can produce alterations in cardiovascular function, control experiments were designed to determine whether the changes in nociceptive response latencies are related to concomitant changes in blood pressure. To this end, blood pressure and tail flick latencies were simultaneously monitored before and after intrathecal injections of NECA alone and of clonidine and NECA together. Intrathecal injection of NECA (2.3 nmol) produced a 14 o) ~t) "'12 >-

~

o 14 (/] v12 >(J Z iii

10

NECA (nmol)

8

_o

4

__1 LL .._1 2

T i

I

i

[

0

30

60

90

TIME (min)

•

0 lo Z LU I.-- 8

5

transient and statistically significant decrease in mean arterial blood pressure from a mean control value of 144 + 4 mm Hg (n --- 7) to 102 + 5 mm Hg (P -< 0.05, ANOVA) at 15 rain after drug injection. The mean arterial pressure returned to baseline values by 30 min after drug injection (P -> 0.05, ANOVA), the time at which maximum elevations of tail flick latencies occurred. In a second group of animals (n = 7) injections of clonidine (11.3 nmol) and NECA (2.3 nmol) admin-

o

6

>-

~ 4 u_ ._.1 2

Z UJ

40

30

.... , 1

........ 3

, 10

........ 40

t 100

I

~

2

NE~CA(nmol) 6 7 8

20

.....

60o

Log Dose (nmol) I---

O

40

0 Z W

I

i

i

i

30

60

90

TIME (min) 30

I.U

O T

0

0

, , , i

.

1

.

.

.

.

4

.

.

.

,

10

.

.

.

.

.

.

.

.

40

i

100

.

.

.

.

.

600

Log Dose (nmol) Fig. 3. Dose-response relationships for the alterations in nociception produced by intrathecal injection of NECA, clonidine and phenylephrine. Each point represents the mean + S.E.M. of the maximum increase in tail flick and hot plate latencies for 6 to 9 animals measured approximately 30 min after intrathecal drug injection. Ordinates: tail flick latency (s) (upper panel) or hot plate latency (s) (lower panel). Abscissae: drug doses (nmol) plotted on a log scale. Symbols: circles, NECA (0.97, 2.3 or 4.9 nmol); squares, clonidine (3.8, 11.3, 150.0 or 375 nmol); triangles, phenylephrine (4.9, 49.0 or 73.6 nmol).

Fig. 4. Effect of intrathecal injection of submaximal doses of clonidine and NECA on tail flick and hot plate latencies. Ordinates: tail flick latency (s) (upper panel) and hot plate latency (s) (lower panel). Abscissae: time (min). Each point represents the mean + S.E.M. for 7 to 9 rats. A single injection containing either a combination of both drugs or a single drug was made following baseline determinations (arrow). Symbols: triangles, (clonidine 11.3 nmol + saline); squares, (NECA, 2.3 nmol + saline); circles, (clonidine, 11.3 nmol + NECA, 2.3 nmol).Inset: isobolograms for the antinociceptive interaction between clonidine and N E C A using the tail flick (upper panel) and the hot plate (lower panel) tests. Ordinates: doses of clonidine (nmol). Abscissae: doses of NECA (nmol). Dose-combinations of clonidine and NECA representing EDs( J doses for tail flick and hot plate tests are represented by the solid dose-additive lines. The 95% confidence limits are represented by the dashed line. The dose-combination of clonidine (11.3 nmol) and NECA (2.3 nmol) used in this experiment is represented by a filled circle. This dose-combination produced increases in response latencies that were 80% of the maximum effect for the tail flick and hot plate tests. For the tail flick and hot plate tests, the experimental point fell below the lower confidence limit for the dose-additive line which indicates a supra-additive interaction.

291 istered together also produced a statistically significant decrease in mean arterial blood pressure from a control value of 132 + 6.0 to 104 + 4 mm Hg at 15 min after drug injection (P -< 0.05, ANOVA). The mean arterial blood pressure returned to baseline levels 20 min after drug injection. However, the peak increase in tail flick responses was not observed until after the blood pressure had nearly returned to baseline values.

after the antinociception had returned to baseline levels. Dose-dependent motor effects were also observed following the administration of phenylephrine, and consisted of hyperreflexia, serpentine movement of the tail and rigidity. Tail flick latencies could not be reliably determined with doses of phenylephrine higher than 74 nmol.

Effects of drugs on spontaneous motor activity

Previous reports have demonstrated that intrathecal administration of norepinephrine and the adenosine agonist NECA interact supra-additively to produce antinociception 1"3'~4. The purpose of the present studies was to use a dose-additive model and isobolographic analysis to determine the nature of the interaction between norepinephrine and NECA, and to determine whether the noradrenergic component of this interaction is mediated by a 1 or a 2 adrenergic receptor subtypes. The first major finding of these experiments was the observation that subeffective doses of NECA and clonidine, an a 2 adrenergic agonist, interacted in a supraadditive manner to produce antinociception. This observation suggests that ct2 noradrenergic receptors are involved in mediating the supra-additive interaction between norepinephrine and NECA that was previously observed 1'3. This conclusion is supported by evidence which indicates that clonidine acts at a 2 receptors in the spinal cord to produce antinociception 21,32,4~. The observation that a supra-additive interaction was not produced by the administration of phenylephrine, the selective a~ noradrenergic agonist, and NECA also supports the conclusion that a 2 noradrenergic receptors are involved in mediating the supra-additive interaction between norepinephrine and NECA ~'3. Although the mechanism of the supra-additive interaction between NECA and clonidine is not clear, there are several possible mechanisms which could explain this interaction. In general, a supra-additive interaction may result from the administration of two drugs that produce a similar effect by different mechanisms of action. Conversely, an additive interaction may be produced by two drugs that act by a common mechanism of action, or within a common pathway 29'38. This latter case would be equivalent to administering two doses of the same drug. Noradrenergic agonists appear to produce antinociception by at least two different mechanisms that are mediated by a 2 receptors in the spinal cord dorsal horn: (1) direct postsynaptic inhibition of dorsal horn interneurons and nociceptive projection neurons12,15'44; and (2) a direct action on primary afferent terminals to diminish the release of nociceptive transmitters 23'26'28. Antinociceptive actions of a 2 agonists at these sites is supported by autoradiographic evidence which demonstrates the

Motor impairment in the form of hind limb adduction was consistently observed in animals receiving the highest dose of NECA (4.9 nmol). Motor effects were variable with lower doses of NECA (0.97-2.4 nmol). However, hind limb adduction could be dissociated from antinociception since the onset of antinociception occurred before the onset of motor effects and could be demonstrated using drug doses that had no effect on motor function. Furthermore, hind limb adduction persisted "-"14 ~12

>(.3 10 Z I..U 8

~

-J

4

..-1 2 m

i

i

i

10

20

30

TIME (rain)

o 40

z

30

LU 2o

W ~

0 -1-

lO

o

i

I

i

10

20

30

TIME (rain) Fig. 5. Effect of intrathecal injection of submaximai doses of phenylephrine and NECA on tail flick and hot plate latencies. Ordinates: tail flick latency (s) (upper panel) and hot plate latency (s) (lower panel). Abscissae: time (rain). Each point represents the mean + S.E.M. for 6 to 8 rats. A single injection containing either a combination of both drugs or a single drug was made following baseline determinations (arrow). Symbols: triangles, (phenyiephrine, 73.6 nmol + saline); circles, (NECA, 2.3 nmol + phenylephrine, 73.6 nmol); squares, (NECA, 2.3 nmol + saline).

DISCUSSION

292 existence of a a receptors on dorsal horn neurons ~1,34,36,43. Reductions in the binding of a 2 agonists to dorsal horn membranes following dorsal root rhizotomies indicates that a 2 adrenoceptors are also located on primary afferent terminals in the spinal cord dorsal horn 22. Although the mechanisms by which adenosine neurons inhibit pain transmission in the spinal cord are not known, there is evidence that suggests some possible mechanisms. For example, adenosine and NECA can hyperpolarize unidentified dorsal horn neurons in the spinal cord under in vitro conditions 24. Such inhibition appears to be mediated by adenosine receptors since this effect is antagonized by theophyUine. An inhibitory action of adenosine agonists on nociceptive dorsal horn neurons is supported by anatomical evidence which indicates that both adenosine A 1 and A 2 receptors are located on spinothalamic tract neurons 9. These observations indicate that adenosine agonists and noradrenergic agonists, which act at a 2 adrenoceptors, both produce antinociception by inhibiting nociceptive spinal cord dorsal horn neurons. However, unlike norepinephrine, adenosine and N E C A do not inhibit substance P release in the spinal cord 37. This conclusion is supported by autoradiographic evidence which indicates that adenosine receptors are not located on primary afferent terminals in the superficial dorsal horn 8. Thus, there appear to be both similar and dissimilar mechanisms by which noradrenergic and purinergic agonists act to produce antinociception. These observations support the conclusion that the supra-additive interaction between NECA and clonidine is a consequence of these agonists acting by different neuronal mechanisms to produce antinociception. An additional observation from the present studies was that intrathecal injection of the aj noradrenergic agonist phenylephrine produced a dose-dependent decrease in tail flick latencies. However, phenylephrine had no dose-dependent effect on hot plate latencies. In addition to the elevated tail flick latencies, motor effects such as rigidity, serpentine movements of the tail and hyperreflexia were also produced by intrathecal administration of phenylephrine. This observation is consistent with the report that injection of al adrenergic receptor agonists into the ventral horn facilitates ventral root reflexes 4. The inconsistent effects of phenylephrine on tail flick and hot plate latencies may be due to the effects of phenylephrine on spinal cord motor reflexes. Since the tail flick response is primarily a spinally-mediated flexor reflex 39, an increase in the excitability of motoneurons would be expected to enhance this reflex and produce shorter tail REFERENCES 1 Aran, S., Porter, N.M. and Proudfit, H.K., Potentiation of the

flick latencies. In contrast, the hot plate response is mediated by supraspinal neurons 39 and presumably should not be affected to the same extent by alterations in spinal cord motor refexes. These results are in conflict with those of others who have reported antinociceptive effects of a 1 agonists injected intrathecally 32. Although the reason for these conflicting results is not clear, one possible explanation may be the difference in doses of phenylephrine used. In the present studies, doses of phenylephrine less than 74 nmol produced decreases in tail flick latencies, while doses greater than 74 nmol produced severe motor disturbances that precluded nociceptive testing. However, antinociceptive actions of phenylephrine have been observed following intrathecal injection of doses of 100 nmol or greater 32. These observations suggest that low doses of phenylephrine enhance tail flick responses, while high doses produce prolonged tail flick latencies that may be the result of motor impairment. The results of the experiments designed to determine whether o~1 receptors mediate the supra-additive effects of combined administration of norepinephrine and N E C A indicated that phenylephrine had no significant effect on antinociception when coadministered with NECA. Thus, a I receptors do not appear to interact with the adenosine agonist N E C A in a supra-additive fashion to produce antinociception. The interpretation of these results may be confounded by alterations in cardiovascular reflexes produced by the drug combinations used in these experiments. For example, adenosine analogs produce hypotension 5 that may be due to arterial vasodilation 6. In addition, intrathecal injection of clonidine decreases arterial blood pressure 1°' 42 and induces antinociception when given in similar doses 42. However, the antinociceptive interaction between clonidine and N E C A does not appear to result from cardiovascular changes, since the time course of changes in blood pressure values and nociception were not correlated. In summary, the present studies have demonstrated the following: (1) adenosine and norepinephrine agonists interact in a supra-additive mode to produce potent antinociception; (2) The noradrenergic component of the supra-additive interaction between adenosine and norepinephrine agonists is mediated by ct2 receptors; and (3) The supra-additive interaction can be dissociated from changes in blood pressure. Acknowledgement. This work was supported by USPHS Grant

DA03980. antinociceptive effect of norepinephrine by the adenosine analog 5'-N-ethylcarboxamide adenosine, Soc. Neurosci. Abstr., 11 (1985) 130.

293 2 Aran S. and Proudfit,' H.K., Antinociception induced by intrathecal injection of sub-effective doses of clonidine and 5'-N-ethylcarboxamide adenosine, Soc. Neurosci. Abstr., 12 (1986) 1017. 3 Aran, S. and Proudfit, H.K., Antinociception produced by interactions between intrathecally-administered adenosine agonists and norepinephrine, Brain Research, in press. 4 Bell, J.A. and Matsumiya, T., Inhibitory effects of dorsal horn and excitant effects of ventral horn intraspinal microinjections of norepinephrine and serotonin in the cat, Life Sci., 29 (1981) 1507-1514. 5 Barraco, R.A., Aggarwal, A.K, Phillis, J.W., Moran, M.A. and Wu, P.H., Dissociation of the locomotor and hypotensive effects of adenosine analogues in the rat, Neurosci. Lett., 48 (1984) 139-144. 6 Berne, R.M., Winn, H.R., Knabb, R.M., Ely, S.W. and Rubio, R., In R.M. Berne, T.W. Rail and R. Rubio (Eds.), Regulatory Function of Adenosine, Nijhoff, Boston, 1983, pp. 293-317. 7 Choca, J.I., Proudfit, H.K. and Green, R.D., Identification of A 1 and A z adenosine receptors in the rat spinal cord, J. Pharmacol. Exp. Ther., 242 (1987) 905-910. 8 Choca, J.I., Green, R.D, and Proudfit, H.K., Adenosine A 1 and A 2 receptors of the substantia gelatinosa are located predominantly on intrinsic neurons: an autoradiography study, J. Pharmacol. Exp. Ther., 247 (1988) 757-764. 9 Choca, J.I., Green, R.D. and Proudfit, H.K., Autoradiographic evidence that spinothalamic tract neurons possess adenosine receptors, Soc. Neurosci. Abstr., 14 (1988) 776, 10 Connor, H.E., Drew, G.M., Finch, L. and Hicks, P.E., Pharmacological characteristics of spinal a-adrenoreceptors in rats, J. Auton. Pharmacol., 1 (1981) 149-156. 11 Dashwood, M.R., Gilbey, M.P. and Spyer, K.M., The localization of adrenoceptors and opiate receptors in regions of the cat central nervous system involved in cardiovascular control, Neuroscience, 15 (1985) 537-551. 12 Davies, J. and Quinlan, J.E., Selective inhibition of responses of feline dorsal horn neurones to noxious cutaneous stimuli by Tizanidine (DS103-282) and noradrenaline: involvement of a2-adrenoreceptors, Neuroscience, 16 (1985) 673-683. 13 DeLander, G.E. and Hopkins, C.J., Involvement of A z adenosine receptors in spinal mechanisms of antinociception, Eur. J. Pharmacol., 139 (1987) 215-223. 14 DeLander, G.E. and Hopkins, C.J., Interdependence of spinal adenosinergic, serotonergic and noradrenergic systems mediating antinociception, Neuropharmacology, 26 (1987) 1791-1794. 15 Fleetwood-Walker, S.M., Mitchell, R., Hope, P.J., Molony, V. and Iggo, A., An a z receptor mediates the selective inhibition by noradrenaline of nociceptive responses of identified dorsal horn neurons, Brain Research, 334 (1985) 243-254. 16 Geiger, J.D., Labella, F.S. and Nagy, J.I., Characterization and localization of adenosine receptors in rat spinal cord, J. Neurosci., 4 (1984) 2303-2310. 17 Gessner, P.K and Cabana, B.E., A study of the interaction of the hypnotic effects and of the toxic effects of chloral hydrate and ethanol, J. Pharmacol. Exp. Ther., 174 (1970) 247-259. 18 Gessner, P.K., The isobolographic method applied to drug interactions. In P.L. Morelli, S. Garattini, S.N. Cohen (Eds.), Drug Interactions, Raven Press, New York, p. 349, 1974. 19 Goodman, R.R. and Snyder, S.H., Autoradiographic localization of adenosine receptors in rat brain using [3H]cyclohexyl adenosine, J. Neurosci., 2 (1982) 1230-1241. 20 Holmgren, M., Hedner, J., Mellstrand, T., Norberg, G. and Hedner, T., Characterization of the antinociceptive effects of some adenosine analogs in the rat, Naunyn-Schmied. Arch. Pharmacol., 334 (1986) 290-293. 21 Howe, J.R., Wang, J.-Y. and Yaksh, T.L., Selective antagonism of the antinociceptive effect of intrathecally applied a-adrenergic agonists by intrathecal prazosin and intrathecal yohimbine, J. Pharmacol. Exp. Ther., 224 (1983) 552-558. 22 Howe, J.R., Yaksh, T.L. and Go, V.L.W., The effect of unilateral dorsal root ganglionectomies or ventral rhizotomies on a2-adrenoceptor binding to, and the substance p, enkephalin,

and neurotensin content of, the cat lumbar spinal cord, Neuroscience, 21 (1987) 385-394. 23 Jeftinija, S., Semba, K. and Randic, M., Norepinephrine reduces excitability of single cutaneous primary afferent C and A fibers the cat spinal cord, Adv. Pain Res. Ther., 5 (1983) 271-276. 24 Kangrga, I., Randic, M. and Jeftinija, S., Adenosine and (--) Baclofen have a neuromodulatory role in the rat spinal dorsal horn, Soc. Neurosci. Abstr., 13 (1987) 1134. 25 Keppel, G., Design and Analysis: a Researcher's Handbook, Prentice-Hall, Englewood Cliffs, N J, 1973. 26 Kuraishi, Y., Hirota, N., Sato, Y., Kaneko, S., Satoh, M. and Takagi, H., Noradrenergic inhibition of the release of substance P from primary afferents in the rabbit spinal dorsal horn, Brain Research, 359 (1985) 177-182. 27 Loewe, S., The problem of synergism and antagonism of combined drugs, Arz.-Forsch., 3 (1953) 285-290. 28 Pang, I.H. and Vasko, M.R., Morphine and norepinephrine but not 5-hydroxytryptamine and y-amino butyric acid inhibit the potassium-stimulated release of substance P from rat spinal cord slices, Brain Research, 6 (1986) 268-279. 29 Poch, G. and Holzmann, S., Quantitative estimation of overadditive and underadditive drug effects by means of theoretical, additive dose-response curves, J. Pharmacol. Methods, 4 (1980) 179-188. 30 Post, C., Antinociceptive effects in mice after intrathecal injection of 5'-N-ethylcarboxamide adenosine, Neurosci. Lett., 51 (1984) 325-330. 31 Proudfit, H.K., Pharmacologic evidence for the modulation of nociception by noradrenergic neurons, Prog. Brain Res., 77 (1988) 357-370. 32 Reddy, S.V., Maderdrut, J.L. and Yaksh, T.L., Spinal cord pharmacology of adrenergic agonist-mediated antinociception, J. Pharmacol. Exp. Ther., 213 (1980) 525-533. 33 Sawynok, J., Sweeney, M.I. and White, T.D., Classification of adenosine receptors mediating antinociception in the rat spinal cord, Br. J. Pharmacol., 88 (1986) 923-930. 34 Seybold, V.S. and Elde, R.P., Receptor autoradiography in thoracic spinal cord: correlation of neurotransmitter binding sites with sympathoadrenal neurons, J. Neurosci., 4 (1984) 2533-2542. 35 Tallarida, R.J. and Murray, R.B., Manual of Pharmacologic Calculations, Springer, New York, 1987. 36 Unnerstall, J.R., Kopajtic, T.A. and Kuhar, M.J., Distribution of a 2 agonist binding sites in the rat and human central nervous system: analysis of some functional, anatomic correlates of the pharmacologic effects of clonidine and related adrenergic agents, Brain Res. Rev., 7 (1984) 69-101. 37 Vasko, M. R., Cartwight, S. and Ono, H., Adenosine agonists do not inhibit the potassium-stimulated release of substance P from rat spinal cord slices, Soc. Neurosci. Abstr., 12 (1986) 799. 38 Wessinger, W.D., Approaches to the study of drug interactions in behavioral pharmacology, Neurosci. Biobehav. Rev., 10 (1986) 103-113. 39 Willis, W.D., Sensory Physiology, Springer, New York, 1982, 9-11 pp. 40 Yaksh,T.L. and Rudy, T., Chronic catheterization of the spinal subarachnoid space, Physiol. Behav., 17 (1976) 1031-1036. 41 Yaksh T.L., Pharmacology of spinal adrenergic systems which modulate spinal nociceptive processing, Pharmacol. Biochem. Behav., 22 (1985) 845-858. 42 Yasuoka, S. and Yaksh, T.L., Effects on nociceptive threshold and blood pressure of intrathecally administered morphine and a-adrenergic agonists, Neuropharmacology, 22 (1983) 309-315. 43 Young, W.S., III and Kuhar, M.J., Noradrenergic a I and a e receptors: light microscopic and autoradiographic localization, Proc. Natl. Acad. Sci. U.S.A., 77 (1980) 1696-1700. 44 Zhao, Z.-Q. and Duggan, A.W., Idazoxan blocks the action of noradrenaline but not spinal inhibition from electrical stimulation of the locus coeruleus and nucleus Kolliker-Fuse of the cat, Neuroscience, 25 (1988) 997-1005.