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Department of Pharmacology and Toxicology, Medical College of Virginia/Virginia Commonwealth University. Richmond, Virginia Accepted for publication August 19, 1994

ABSTRACT Dose-effect

Administration to Mice: Possible Mechanisms for Interaction with Morphine’
SANDRA P. WELCH, CHARITY THOMAS and GRAY S. PATRICK THC and tracerebroventricularly (icv.)] and compared with those previously generated after administration intrathecally The ED,, values after administration of 56,667 was significantly more potent after icv. administration than administration, and was nearly 10 times more potent than CP 55,940 (icv.j. CP 55,940 and for 7 days totally abolished the antinociception produced by the cannabiABBREVIATIONS: sulfoxide. 310 manner nearly 1 O-fold after icv. administration. The or Cl-CAMP (10 the cannabinoids. Recent work has shown that the cannabinoids produce potent antinociceptive effects in mice and rats through both spinal and supraspinal mechanisms (Welch and Stevens, 1992; Welch, 1993; Smith and Martin, 1992; Lichtman and Martin, the salt; DMSO,

Modulation of Cannabinoid-Induced Antinociception After

56,667, which did not produce greater than additive effects in combination with shifted phine when the drugs were administered the morphine (icv.) dose-effect curve in a nabinoids (icv.) were not blocked by ICI 174,864 (20 mouse), significantly attenuated the antinociception produced by the and CP 55,940. However, when administered icv., forskolin and Cl-CAMP produced antinociception, but did not block or produce greater than additive effects with the possible effect; THC, tetrahydrocannabinol; monosodium salt; 4-AP, 4-aminopyridine; TEA, tetraethylammonium; or naloxone (20 kg/mouse or 10 Received for publication September This CL, confidence limit; AD,,, dose producing 50% antagonism; Cl-c-AMP, antinociception in the rat has been proposed to (icv. and i.t.). Pretreatment of the mice with forskolin the mechanism by which cannabinoids produce supported by the Nationai Pertussis toxin pretreatment Although the spinal mechanism of cannabi(79

were generated for the canrrabinoids . VS icv. did not differ significantly. intrathecally; icv, intracerebroventricularly; and receptor i.t.), which produced no 1993. of Drug Abuse Grants

and Martin, 55,940, of the intravenously; tration of the cannabinoids. Conversely. the antinociceptive effects of the cannabinoids. The AZ. ues antinociception produced by the cannabinoids failed to alter the antinociception produced by the which did not alter the antinociceptive effects of Thapsigargin (icv.) blocked the antinociception icv. The CP 55,940 were 215

nmollmouse, respectively. effect curves of ATP- and apamin-sensitive potassium channels, these intracellular systems may be common points of nificantly reversed the calcium-induced blockade of sium channels, produced a parallel rightward shift in the apamin (5 nabinoids. Because acute administration of tinociception has not been tion of a specific cannabinoid receptor has been the to] intense investigation leading to the cloning of a have been shown to interact with In receptor addition. an endoge ligand for the cannabinoid receptor has been identified ane et al., tion-What are the transduction mechanisms with the activation of the cannabinoid receptor’? modulation of adenylate sible transduction mechanisms have been proposed calcium. prostaglandin opioids. The role of adenylate cyclase, calcium and potassium channel modulation, and to a lesser decrease calcium entry to and content of neurons. levels and produce hyperpolarization of neurons dibutyryl cyclic

and CP 55,940. Apamin, blocker for calcium-induced nor-binaitorphimine; ICI 174,864. administration of calcium and calcium subcutaneously: These reports led to the next logical icv.) failed to block icv. 176 (122-253) and and determined. The monophosphate percentage of the of protein-coupled 55,940 (1

1995 role of

of the cannabinoids is the focus of this paper. These mechanisms will be discussed individually, although the interplay of these various systems in the transduction the effects of the cannabinoids is clearly a possibility. C AMP. The cloning of the cannabinoid receptor was performed in modulation after receptor activation Numerous studies have shown the involvement of the cannabinoids in the modulation of amide, the endogenous cannabinoid ligand, has also been shown to inhibit levels in cells of various types in culture as well as in homogenates of brain regions (Kelly and Butcher. 1979; Zimmerman et al.. et

al., 1986). The potency of numerous cannabinoids to inhibit
and Ng, 1984; Howlett and Fleming, 1984; Howlett, 1984; Howlett, The interaction of the cannabinoids with a membrane protein bosylated protein was identified as the formation in the neuroblastoma cells was found to correlate to the antinociceptive effects of the drugs in The effects of the cannabinoids on spinal accumulation have not been examined. This study utilizes modulators of adenylate cyclase or levels in to evaluate the effects of such treatments on the antinociceptive effects of the cannabinoids in the brain and in the spinal cord. transmission and antinociception. Previous work has indicated that An alteration of intracellular cal-

cium by the cannabinoids could be the trigger for a events leading to decreased decreases the release of acetylcholine presynaptically by a decrease in the influx of calcium into presynaptic nerve terminals studies of the involvement of calcium in the antinociceptive effects of the cannabinoids have not been reported previously. In addition, potassium channel opening and the resultant hyperpolarization of 19821 found that cannabinoids decrease calcium uptake to several brain regions, an effect which did not correlate to the psychoactivity of the drugs. Direct measurement of the effects of cannabinoids on free intracellular calcium has shown that depolarization-induced rises in intracellular calcium are attenuated by vitro studies indicate a role for calcium in the effects of the cannabinoids, in membrane could account for the antinociceptive effects of cannabinoids. The modulation of potassium channels by the cannabinoids may differ in the brain and in the spinal cord. The involvement of potassium channels in nabinoid-induced effects has been reported in although the nature of the interaction of the drugs with potassium channels has not been determined. et al., 1973; Chesher and Jackson, 1985; Sanders et al., 1979; Martin, fails to block the effects of various parenterally administered cannabinoids also failed to block the antinociception induced by a variety of al., is unclear. Many investigators have shown that

icv. or in concentrations of 1 Welch, using et The role of opioids in cannabinoid antinocicepas possible mechanisms in the antinoeiceptive 1981; Gilbert, 1981; Welch and Stevens, It is intriguing that, despite the data with an evaluation of and ADP-ribosylation was shown. The production in icv. or spinally administered or higher et al., protein

Naloxone Stokes 1988 brain

311 suggesting that cannabinoids and opiates have distinct of action, the effects of morphine have been found to be enhanced by crude cannabis extract and Bhattacharya, et al., 1984). Intrathecal administration of several cannabinoids leads to synergism with istered morphine in the production of antinociception in mice binoids intraventricular injections. Intrathecal injections were performed according to the protocol of Hylden and Wilcox antinociception, but not catalepsy, or hypoactivity has been also reported Although we have evaluated the effects of opioid antagonists on the antinociceptive effects of (Welch and Stevens, 1992; Welch, with icv. administered opioids and opioid antagonists has not been reported previously. Thus, the research presented in this manuscript was designed to evaluate the involvement of the cannabinoids routes both of administration) on the modulation of in the production ofantinociception. In addition, we wanted to compare and contrast the interaction of the carmabinoids with morphine in the brain and in the spinal cord. Finally, from our results we hoped to generate a hypothesis as to the possible Mice were lightly anesthetized with ether and an incision was made in the scalp such that the bregma was exposed. Injections were performed using a 26-gauge needle with a sleeve of PE produced significant antinociceptive effects

alone in the tail flick test and were not used as the cannabinoid vehicle when performing i.t. injections. Preliminary time course studies of the various drugs (at the highest dose which exhibited no toxicity) or their respective controls were initially performed in combination with the ED,, dose of each cannabinoid. Once the time of the peak effect of those drugs which altered cannabinoid were administered. lntraventricular injections were performed according to the method of Pedigo et al. tubing to control the depth of the injection, An injection volume of 5 DMSO. Nor-BNI, ICI 174.864, naloxone hydrochloride, morphine sulfate, apamin, pertussis toxin, calcium chloride, magnesium chloride. and verapamil were dissolved in distilled water. The cannabinoids or DMSO vehicle min before determination of the response latency of the mice in the tail flick test. This time point represents the peak DMSO vehicle produced scratching behavior in mice which lasted 2 min after injection. Other vehicles have been tested previously in our laboratory. needle. and L6 area of the spinal cord with a Injection volumes of 5 at a site 2-mm rostra1 and 2-mm to the bregma at a depth of 2 mm was administered to the mice. The cannabinoids, charybdotoxin. TEA. of the drugs as determined in previous studies in our laboratory (Welch and Stevens, 1992 spinal and supraspinal sites.

Intrathecal administered cannabinoids, and the interaction of

by K 8644, ryanodine and nimodipine were prepared in was determined. all further testing was performed at that and Stevens, 1992; Smith and Martin, 1992; Smith et and of the A”-THC-induced Recently, the blockade of cannabinoid antinocicep-

kappa opioid antagonist,
with morphine Unanesthetized mice were injected between the been reported et the interaction of icv. administered Cannabinoid-Induced 1: interaction of the opiates and the and potassium channels in and by orally administered Cl-CAMP, forskolin. 1993 were administered The blockade by and emulphor!

thapsigargin, 1975 and

Welch et al. time point. With few exceptions, the time of the peak effects of the

drugs was 5 to 10 before the administration of the cannabinoids. The exceptions include glyburide pretreatment; thapsigargin, which produced peak blockade after a 1-hr pretreatment and pertussis toxin, which was administered i. t. (0.5 potassium channel openers and morphine (Welch and days before testing in combination with the cannabinoids. Thapsigargin, Bay K 8644 and calcium produced antinociception when administered i,t. Forskolin, 1993) using a 15 and Cl-CAMP and Bay K 8644 produced antinociception when administered To test for greater than additive effects of the intrinsically antinociceptive drugs in combination with the cannabinoids, doses of the drugs were chosen which produced no significant antinociception. These doses were then tested in combination with a wide range of doses of the cannabinoids. For the studies of the enhancement of the effects of morphine nabinoids before morphine. The mice were tested 10 min after the administration of morphine using the tail flick test. To study the effect of on calcium-induced blockade of the cannabinoids, the was administered icv. at 5 min before the calcium which was administered at 10 min before the cannabinoid The mice were tested 15 min later using the tail flick test. The tail flick test. The tail flick procedure used was that of and a cut-off time of 10 were empioyed. Antinociception was quantified as the %MPE as developed by Harris and Pierson using the following formula: Percent MPE was calculated for each mouse using at least 12 mice

per dose. Using the modification of the were determined using the method of Litchfield and as well as the

Using at least three doses of blocker, for each mouse, the mean effect and standard error of the mean was calculated for each dose. Dose-response curves were generated using at least three doses of test drug. method omitting doses producing 100% or 0% MPE) and 95% confidence limits et al., 1977). The AD,, for blockade the cannabinoid-induced antinociception was determined by calculation of the percent antagonism of ciception (using a dose of the cannabinoid that resulted in at least 80% MPE) by the blockers according to the following formula: % antagonism = 100 values were determined by log-probit analysis (a modification of the method omitting doses producing 100% or 0% 1949). were At least 12 mice per dose were used for all determinations. Statistical analysis. Significant differences between treatment and control groups were determined using analysis of variance and the Dunnett’s 19551. The dose-response curves were evaluated for the parallelism of shifts using the method of Tallarida and Murray Drugs. All of the cannabinoids were obtained from the National Institute on Drug Abuse with the exception of CP 55.940, CP 56,667, levonantradol and dextronantradol which were obtained from Dr.

Lawrence Melvin, Pfizer Central Research. Nor-BNI, Bay K 8644, MA). chemicals, Inc. ICI 174,864 was purchased from Cambridge Research and Smith and forskolin were purchased from Research test the cannabinoids were administered 10 values were determined by log-probit analysis control\ %MPE = 100 x using the method of Litchfield and program as well as the 1 Control reaction times of (test (Valley Stream, NY). Calcium of antagonist + cannabinoid) of vehicle + cannabinoid) which blocks both or control) program

and 95% to 4 by et monosodium was provided by Miles, Inc. Pharmaceutical Division (West Haven, CN). Thapsigargin was purchased from LC GVIA was purchased from Peninsula (Belmont, CA). Ryanodine and pertussis toxin were purchased from Calbiochem (San Diego, CA).

values for a series of cannabinoids are listed in table 1. The ED,, values of the cannabinoids after administration were taken from Welch and Stevens and were compared with the ED,, values generated for the icv. administration. The dose-effect curves used to generate the of the cannabinoids were full agonists in the tail flick test precluded but the solubility of the the use of higher doses. The did not differ significantly after administration after icv. administration. Conversely, the us. and CP icv. were nearly identical. The ED,, values for or icv.. although a trend toward higher potency of the drug was

did not differ significantly after administration or icv., although a trend toward higher potency of the drug was observed after i.t. administration. CP 56,667 was significantly more potent after icv. administration than administration, and was nearly 10 times more potent than CP 55,940 and CP 56,667 are stereoisomers and CP 55,940 has been described to be the active isomer in many tests (Johnson and Melvin, 1986; Little icv. administration. However, 25 &mouse produced 44% MPE in the tail flick test. Dextronantradol was inactive at 25 and icv. administration. Interaction with opioids. Table 2 lists the ED,, values for morphine (taken from Welch and Stevens, 1992) and the ED,, values for morphine (icv.) and levonantradol produced greater than additive effects in combination with TABLE 1 ED, values and 95% confidence limits for various cannabinoids in the tail flick test in mice The cannabinoids were administered erther using the tail flick test. The ED,, values and 95% 2.89 (1.6-5.1) CP 56,667 4.17 Levonantradol 0.04 Dextronantradol Inactive at 25 11 shown in figure 1. 55,940 2.28

IN). Drug which produced only an average of 68% MPE at under “Methods.” magnesium chloride, verapamil. glyburide. al., to complete a dose-effect curve of the drug and apamin were purchased from Sigma values for after both We tried to administer higher doses of was MA). As-THC, values after administration of obtained from Boehringer Mannheim after pretreatment with cannabinoids We were unable to obtain enough 44.97 (22.96-88.09) 16.4 (1 l-24.8) 72.07 (36.06-l 44) 125.8 14.67 pretreatment with cannabinoids values for the cannabinoids

CP both or and icv. at the exception of 0.02 0.18 were determined 25 min before = 44% 4-U


Fig. 1. Dose-effect icv. administration. Cannabinoids were injected icv. and tested for the production of tinociception using the tail flick test and the ED,, values were determined as described under “Methods.” The following cannabinoids were tested: A = levonantradol; B At least 12 mice were used per dose. morphine when the drugs were administered nantradol) failed to produce greater than additive effects with morphine CP 55,940, CP 56,667 and dextronantradol and CP 56,667 produced greater than additive effects with morphine when the drugs were administered icv. The values for morphine were shifted from 2.4 produced greater than additive effects with morphine when the drugs were administered either i.t. or icv. The shifts in the morphine dose-effect curve in combination with the mouse by CP nabinoids were parallel. Various opioid antagonists were administered before icv. administered cannabinoids and did not alter noid-induced antinociceptive effects. These drugs are listed in Table 3 and include the delta opioid antagonist. ICI 174,864 (20 the kappa opioid antagonist, nor-BNI (70 (Takemori et al., 1988) and naloxone (20 Interaction with C for 7 days, which ribosylates

doses used blocks mu, delta and kappa receptors

et al., 1987

0.001 0.01 0.1 10 100 1000

AMP. Pertussis toxin pretreatment Conversely, CP for the cannabinoids and CP 56,667, respectively. CP 56,667: C = CP 55,940; D = or 10 et al., and/or inactive isomer of to 0.28 and 0.65 proteins (for a which at

TABLE 2 ED, values for morphine various cannabinoids in the tail in combination with and icv. administration Mice were injected with cannabinoids either

binoid pretreatment or vehicle pretreatment. Cannabinoids were administered 10 min before morphine and the mice were tested using the tail flick test at 10 min after morphine administration. The were determined as described under “Methods.” Pretreatment ED,, of morphine review see Brown and Birnbaumer, 19901, totally abolished the antinociception produced by the cannabinoids and significantly attenuated or blocked the antinociception produced by the cannabinoids administered i.t. was least affected by the pertussis toxin pretreatment. Pretreatment with pertussis toxin did not alter the baseline latencies for the mice in the tail flick test and did not produce any overt toxicity in the mice. However, pretreatment with pertussis toxin icv. (0.5 for 7 days) led to loss in body weight, and lethality in greater than 30% of the animals. Pretreatment of the mice with 5 and 25 &mouse forskolin which produced no antinociception, significantly attenuated the antinociception produced by the A similar effect was observed using CP 55,940. Forskolin (25 &mouse pretreatment significantly reduced the antinociceptive effects of CP 55,940 forskolin MPE. However, when administered produced antinociception. The ED,, of forskolin which produced no antinociceptive effects, did not block or produce greater than additive effects with the antinociception produced by the cannabinoids administered icv. Administration of partially, but significantly, blocked the antinociceptive effects produced by the administration of ED,, doses the three cannabinoids tested (fig. failed to alter the antinociception produced by the cannabinoids administered i.t. How-

ever, produced antinociceptive effects when administered icv. [ED,, values = 0.5 was obtained using higher doses of Cl-CAMP. Both Cl-CAMP and dibutyryl and 2.3) dibutyryl respectively) did not block or produce greater than additive effects with the antinociception produced by the cannabinoids administered icv. Interaction with calcium. Various calcium modulators were tested in combination with the cannabinoids (table The 1.6) None 1 (0.6-l 2.7 (1.7-4.3)

DMSO 0.61 (0.26-I
Levonantradol 0.06 (0.015.24) 0.66 N.T. 11 Dextronantradoi 0.51 morphine. The dose-effect curve for morphine was determined after

55.940 0.3
However, no greater blockade of the

(3.13 (6.25
(25 administration of calcium

administered (10 Forskolin (0.1 Cannabinoid-Induced Antinociception respectively]. Inactive doses of (0.02 and 0.05

0.15 0.05

0.08 0.26
(fig. from 83 values and 95% test in mice after before morphine or icv. before MPE to 16 (fig. 2.4 (1.7-3.3)

and 0.3 was 0.5



itself did not alter the effects of the cannabinoids, it did significantly block calcium-induced blockade of the cannabinoids.

3 gargin (0.1 &mouse), which increases intracellular calcium

314 Welch et al.
TABLE 3 cannabinoids after before cannabinoids and the time pools of the endoplasmic reticulum by blocking activity (Bian et al., 1991; Koshiyama and Tashjian, 1991); Bay K 8644 (0.3 &mouse), which predominantly increases calcium entry through dihydropyridine-sensitive calcium channels; verapamil (30 &mouse), which block voltage-gated which alters calcium flux from the intracellular caffeine-sensitive (but not sensitive) calcium pool (Thayer et al., 1988; caltoxin (1 &mouse) which blocks both cium channels and ryanodine

et al., 1989) failed to
produce antinociception or to alter the antinociception administration of the cannabinoids. The doses listed represent the highest non-antinociceptive dose for each of the drugs. At higher doses, calcium, thapsigargin and BAY K 8644, which increase intracellular calcium, produced antinociception after blocked the antinociceptive effects of the cannabinoids (fig. 4, panel A). The AD,, values for calcium-induced block of

mouse, respectively. Higher doses of calcium (greater than 450 because we could not use higher doses of calcium. This difficulty is reflected in the large confidence limits for the AD,, of calcium us. which did not alter the antinociceptive effects of and CP 55,940 were 215 produced hyperactivity in the mice. It was difficult to generate an icv. Opioid antagonists Naloxone No block No block ICI 174,864 No block No block nor-BNI Blocked No block Calcium modulators Calcium No block Blocked Verapamil No block No block No block No block Nimodipine No block No block Thapsigargin No block Blocked BAY-K No block No block Ryanodine No block No block Modulators of Blocked No block Forskolin Blocked No block Magnesium Chloride No block No block Modulators of potassium channels No block No block Apamin Blocked No block l administration. Calcium No block No block TEA No block No block Glyburide No block No block blockade of entry of calcium into the

by the et al. were administered of blockade of the antinociceptive effects of toxin Blocked Blocked as described under “Methods” 1986; Antinociception was determined using the tail flick Drugs and icv. administration 176 (122-253) and 123

et al., 1987; Reynolds et al.,
et al., 1987; Zemig, and nimodipine before (1 &mouse icv. calcium channels (Godfor calcium us. No block No block and significantly or

Fig. 2. The effect of pertussis toxin pretreatment (i.t.) on the antinoci (panel A) c icv. (panel 7 vehicle was administered days before the administration of th cannabinoids or icv.) in distilled water-pretreated mice (the vehicle for the At least 12 mice per treatment group were used. l vehicle. Th mice were tested for antinociceptive effects after either administration of the cannabinoids or DMSO. The clear bar denote 6% (icv.), respectively. Bars denoted represent the antinociceptive effects of those cannabinoids in mic pretreated for 7 days i.t. with distilled water vehicle. The bars denote PANEL B 100 80 group. respective distilled reversed the calcium-induced blockade of blocked the antinociception produced by gargin us. panel B!. Pretreatment of the mice icv. with thapsigargin for 1 55,940, data not shown) administered icv. Other calcium modulators were tested including: or tion after either icv. administration. All of these dru: were tested at the highest nontoxic doses in combination

the cannabinoids administered either i.t. or icv. and failed alter cannabinoid-induced antinociception (table 3). PANEL A represents the antinociception produced by the administration 40 vehicle in PT-treated mice. Not shown is the effect of DMSO indicate the effects of the cannabinoids in PT-pretreated generated in those treatment groups were 5 effects of the cannabinoids administered either 0 and nimodipine omega conotoxin None of these drugs produced = 0.003 toxin or CP 55,940) or (0.5 &mouse) or distilled and magnesiu: and “CP verapamil 3% P 55940

for 0.01 (fig. and 8 (fig. or Th


Fig. 3. The effect of forskolin pretreatment (i.t.) (panel A) or Cl-c-AMP [panel B) on the antinociceptive effects of the cannabinoids administered or distilled water vehicle was administered or DMSO vehicle. The mice were tested for antinociceptive effects after represents the antinociception produced by the administration of distilled water in distilled water-pretreated mice (the vehicle for the forskolin). The 55,940” represent the antinociceptive effects of those Cannabinoids in mice pretreated which block voltage-gated potassium channels (North, 1992; mouse), which blocks ATP-gated potassium channels mouse), which blocks the large with distilled water vehicle. The bars denoted with potassium channels. Various potassium channel blockers were tested: TEA sett et al., 1988; Gaines et al., 1988; at these doses, which are the highest nontoxic doses of the drugs, failed to alter cannabinoid-induced mice per treatment group were used. were used for all treatment groups. Panel and However. apamin. blocker of Panel A, Forskolin potassium channels (Smith et al., 1986;

et al., 1988). These drugs administered either effects of distilled water administration before DMSO. At least and Lazdunski, generated in that treatment group was of the cannabinoids or i.t.)-pretreated mice. “VEH” denotes the before the administration of indicate the effects of the cannabinoids in before 1 5 25 @mouse, and charybdotoxin fast) conductance 1992); glyburide P Not shown is the effect of

The dark bar denoted
0.01. Bars denoted et al., 1989; and I 4%. At

conductance and

or Fig. 4. The effect of calcium pretreatment (icv.) (panel A) or w-conotoxin CP 55,940 or DMSO vehicle. The mice were tested for antinociceptive effects after icv. administration of the cannabinoids or DMSO. The bar denoted represents the antinociception produced by the administration of distilled water icv. before DMSO icv. The AD,, values for calcium blockade of the cannabinoids were determined as described under “Methods.” At least 12 mice per treatment group were used. Mice were pretreated with dual injections icv. of vehicle (v), calcium (Ca, 300 binoids were shifted from or distilled water vehicle was administered icv. before the icv. administration of et al., 1982; Castle et as described under “Methods.” The mice were tested 15 min after the injection of At least 12 mice per treatment group were used. w-Conotoxin significantly reversed the calcium-induced blockade of calcium-gated potassium channels produced a parallel rightward shift in the dose-effect curves of was shifted from 6 failed respectively. However, apamin (5 to block icv. administered cannabinoids.

and CP 55,940 (fig. 6, panels A-C). The ED,, values for the to 33-fold by administration of apamin P 120, 300

PANEL A (OC) icv. before calcium pretreatment (panel B) on the -Calcium Calcium Calcium PANEL 100 80 0.01 from 60 40 20 effects of the cannabinoids administered icv. Panel A. Calcium antinociception. and CP 55,940 were shifted from 21 . 1989; Strong, &mouse to 123 to 82 Cannabinoid-Induced P

The ED,, of and/or 0.01 from Panel and 20 or The ED,, values for P 0.05 from P and 0.6 0.05;

to greater than lo-fold by the pretreatment with cannabinoids

at doses which did not have any antinociceptive effects. W’hen we compared the enhancement of morphine-induced administration ciception by the cannabinoids with those data previously generated after i.t. administration, we found that some cannabinoids enhanced morphine in the 1981; Gilbert, 1981; Lichtman and Martin, 1991, a and b; Welch and Stevens, 1992). Intrathecally nabinoids seem to act at predominantly spinal sites in the production of antinociception (Smith and Martin, 1992). It is not clear whether the icv. administration of the cannabinoids activates both spinal and supraspinal sites in the production of antinociception, Due to the lypophilicity of the drugs, passage to the spinal cord is very likely. In summary, the cannabinoids seem to produce potent antinociceptive effects at both spinal and supraspinal sites, However, the mechanism by which the cannabinoids produce antinociceptive effects at these two sites seemed to differ and is the focus of the remainder of this manuscript. Interaction with opiates. We have previously that the ED,, values for morphine were shifted from

316 Welch et al.
The mice were tested 15 min later in the tail flick test. The clear bar denoted represents the antinociceptive effects of DMSO icv. administered 1 hr before binoids produced antinociception after both i.t. and icv. administration and are approximately at either site with the exception of CP 56,667. These results are consistent with previous studies which indicate that the cannabinoids are active as antinociceptive drugs when injected

Fig. 5. Blockade of gargin (icv.). Thapsigargin, at the doses listed, or vehicle was injected at 1 hr before The remaining bars represent thapsigargin administration before

At least 12 mice were used per dose. l P represents

VEH 0.001


(icv.)-induced antinociception by of the cannabinoids. at 1 hr before 0.05 from icv. The bar labeled P 0.01 from been shown to be dense in the striatum and the which are associated with dense binding of the opiatt et al., 1988; Gamse et al., 1979). Thus, neuroanatom

cal location of the receptors does not seem to account for
differences observed between the effects of CP 55,940 were determined 15 min later using the tail flick test. The ED values and the parallelism of the shifts in the dose-effect curves we determined as described under “Methods” using 12 mice per dose. brain whereas others enhanced the effects of morphine in

bility that subtypes of cannabinoid receptors may exist brain and spinal cord and differ in their interactions opiate-sensitive pathways either via their neuroanatomic: proximity to opiate receptors or differ in the convergence second messenger systems with those of the opiates. Hot; ever, in the case of one cannabinoid, CP 55,940, binding gelatinosa of the spinal cord are administered spinal cord. Our data are suggestive of the intriguing Fig. 6. Blockade of the antinociceptive effects of min before the cannabinoids. The dose-effect curves of the PANEL A i.t.) PANEL C (panel B and CP 55,940 (panel C) by apamin when all 10 Apamin (5 1000 A8 1 10 doses doses doses

was administered at et A9 + apamin ED50 = 82 (panel

in the spinal cord.

opioid or the direct interaction with opiate receptors. Thus, the enhancement of opiate antinociception by binoids icv. may be due to interactions with common intracellular second messenger systems. Some possible points of interaction include the modulation of calcium, sensitive, high-voltage-activated calcium channel, an effect that is blocked by the administration of pertussis toxin and independent of the formation of C AMP. Because the L-type calcium channel blocker, nitrendipine, fails to alter such an effect, the cannabinoids were hypothesized to interact with an N-type calcium channel (Mackie and Hille, 1992). A similar study produced similar results. The cannabinoids were found to inhibit I,, current in newoblastoma cells, an effect which was not. dose related, but was pertussis toxin- and administration of calcium to mice administration of the cannabinoids is not sensitive to calcium modulation and thus may not directly involve calcium modulation. Conversely, the icv. administered cannabinoids were sensitive to blockade by calcium icv. and the AD,, values for calcium icv. us. the cannabinoids of the cannabinoids at supraspinal sites are not mediated ciceptive effects of icv. administered cannabinoids. These data suggest that the icv. administration of the cannabinoids does not result in the release of an antinociceptive and potassium flux. Modulation of calcium and enhance or fail blastoma cells indicate cannabinoids inhibit an 1986) or by modulators of intracellular pools of calcium such as et al., 1988; Welch et al., 1992; Smith and

Dewey, 19923, as well as Bay K 8644 and thapsigargin (Damaj et before the cannabinoids did not produce greater than additive effects with the cannabinoids. These data indicate that the antinociception produced by the did not differ significantly from that previously shown for calcium blockade of calcium entry to a variety of tissues. Electrophysiological studies in All such previous studies were performed in Based on the results of such studies we hypothesized that the antinocicepproduced by the cannabinoids might be sensitive to calcium, or modulators of calcium. administration of calcium chloride or a series of modulators of calcium flux across voltage-gated calcium channels ciceptive effects result from the 1993). Administration of non-antinociceptive doses of these drugs effects of the cannabinoids administered i.t. were not nor-BNI administered icv. failed to block the have previously reported that the kappa antagonist, and ICI 174,864 data support the hypothesis that the antinociceptive 1990; by the produced by the cannabinoids administered interaction with nor-BNI-sensitive receptors.

Thayer et al., 1988; et al., 1991; Koshiyama and Tashjian, 1991 (Godfraind et but not other opioid antagonists, blocks alter antinociception in the brain

et (Caulfield and Brown antinociception (Welch, 1993).

1988; Harris and Stokes,
1987; Reynolds et also failed to block the et al., nimodipine, Bay K 8644 or 1986; Cannabinoids either the antinocicep1989 et al., 1987; or morphine icv. (Harris et al., 1975; Chapman and Way, 1980;

Guerrero-Munoz and and stimulates calcium influx into cells (for a review see Irvine, also blocked the antinociception produced by which increases intracellular calcium (Koshiyama and Tashjian, which increases calcium entry to cells via the voltage-gated calcium channel, as well as via nonspecific mechanisms (Bouchelouche et nabinoids is due to calcium entry through voltage-gated calcium channels because induced blockade (fig. alone did not alter cannabinoid-induced antinociception. Thus, it appears that the entry of calcium to neurons must be stimulated by the administration of calcium to observe effects of omega calcium channels, and found no reversal of the effects of icv. calcium (data not shown). Thus, the calcium-induced blockade of a similar logic we attempted to reverse the calcium-induced blockade of cannabinoids with both pamil and nimodipine, which block calcium noid-induced antinociception seems to be due entry through the tinociception by thapsigargin, which raises intracellular calcium levels, indicates that the cannabinoids may decrease and Stokes (1982) and Martin et al. (1988). In addition, the stimulation of calcium entry to neurons after thapsigargin treatment would further increase calcium concentrations intsacellularly, an effect insensitive to blockade by classic calcium channel blockers (for review, see Irvine? 1992; Jackson et al., 1988; Takemura et al., 1991) and consistent with our data. Our data therefore suggest that, formation follows a structure-activity relationship and

stereoselectivity similar to that observed for behavioral measures (Howlett, 1985; Dill and Howlett, 1988; Howlett et al., 1986; Howlett and Fleming, 1984; Howlett, 1987; Howlett et al., 1990; Devane et 1989 calcium channel. These results are consistent with reported effects observed in vitro (Mackie and Hille, 1992). The blockade of cannabinoid-induced entry to neurons which is consistent with the in vitro work nabinoids produce antinociception at supraspinal, but not spinal, sites by decreasing intracellular calcium levels via blockade of calcium entry through calcium channels. Consistent with the report of Mackie and Hills antinociception resulting from supraspinal administration of the cannabinoids seems to involve interaction with a due to the blockade of antinociception by pertussis toxin pretreatment which inactivates lation (for a review, see Brown and proteins, nabinoids with but indicating a lack of involvement of induced antinociception or other behavioral effects of the cannabinoids (Hillard and Bloom, 1983; Little and Martin, 1991; Dolby and Kleinsmith, 1974; Dolby and Kleinsmith, 1977; Askew and Ho, 1974). In studies indicate a somewhat different profile of cannabinoid action. Studies in 1986; Bidaut-Russell et al., 1991; Howlett et al., Our data agree with studies indicating an interaction of the did not alter cannabinoid antinociception. It is likely that the calcium-induced blockade of the

or doses of BAY K 8644 protein and be independent of changes in cells show that cannabinoid-induced inhibition of in the brain in the modulation of Cannabinoid-induced Antinociception and is mediated via coupling to the although and CP 55,940. and 1982; proteins via. reversed the et al., in

protein because pertussis toxin attenuates binding of CP

55,940 These studies, along with supporting work evaluating the stereoselectivity and the Hill coefficient for binding of the synthetic ylation of calcium channels nabinoid receptor linked through a our studies indicate that modulation of tion by alteration of calcium entry to neurons protein to the modulation of C AMP. is not directly involved in the production of antinociception, we can not rule out the possibility that and Birnbaumer, 1990; Schultz et cannabinoids could decrease calcium entry to neurons. In the spinal cord, the cannabinoids seem to produce protein tinociception in conjunction with the modulation of because the antinociceptive effects ofi.t. cannabinoids are blocked by pertussis toxin, forskolin and Cl-CAMP, The lack of blockade of antinociception by may be due to the lack of penetration of the Gimenez-Gallego et Morphine has been shown to modulate ratios, High ATP levels close these channels leading to a less negative membrane potential and depolarization. Opening-of these channels leads to hyperpolarization. Pharmacologically, these channels are blocked by the oral hypoglycemic agents, such as glyburide, which stimulate the entry of

to the site of action. The effects of on antinociception are the opposite in the brain us. in the spinal cord, which is similar to the opposite effects of calcium observed in brain administration may reflect differences in the function of in the brain us. in the spinal cord in the- modulation of antinociception.

Modulation of potassium.
basic classes of potassium channels exist in the central nervous system (for a review, see Aronsen, 1992). This classification is clearly a generalization and the drugs discussed often interact with

more than one

the channels. In general these potassium
channels are either voltage-activated, calcium-gated, gated or ligand-activated. The nels are either opened or closed in response to membrane electrical activity and are blocked pharmacologically by TEA

proteins or other second messenger systems,

Calcium-gated potassium channels have been studied using the bee venom apamin, which blocks the small (low) conductance (SK or
et al., 1982; Castle et al., 1989; Strong, 1990). An endogenous ligand for the apamin receptor has been found in brain tive calcium-gated potassium channels in peripheral sensory neurons channels are gated by changes in intracellular et al., 1988: Howlett et al.. spinal cord The calcium-gated potassium channels are activated

by alteration in intracellular calcium and are thought to be coupled to

et al., 1987). Charybdotoxin, a scorpion venom, blocks predominantly the large 1988). Charybdotoxin does not interact with apamin-sensitive sites in rat brain as well as several toxins (North, 1992; Aronsen, 1992 indirectly alters icv. cannabinoid-induced in cannabinoid-induced antinociception following icv. 19883 by insertion into the mouth of the channel preparations et al,, 1988). Thus, differences in the 19901. By decreasing et

cannabinoid. CP 55,940 et fast) conductance

1988; Bidaut-Russell and Howlett, I 1992). The ATP-gated support the hypothesis of a

et al., channel potassium et al., channel in of et Anatomically, these channels exist in areas where high levels of opiate binding occur and Lazdunski, 1990). ATP-gated potassium channels have been found in the brain, with particularly high levels in the substantia nigra, hippocampus, basal ganglia and thalamus. and to a lesser degree in the spinal cord, although the function of such channels remains unknown et al., 1988). Opiates also interact with l&and-activated potassium channels, a diverse collection of channels which are modulated by a variety of neurotransmitters and drug classes. These channels are most effects produced by the cannabinoids, although the is blocked by potassium channel blockers and conversely, that potassium channel openers administered produce antinociception that is blocked by opiate antagonists (Welch and The administration of apamin. a blocker of calcium-gated potassium channels, produced a complete blockade of morphine-induced antinociception. Evaluation of several potassium channel blockers blocked the antinociceptive effects of the cannabinoids producing parallel rightward shifts in the dose-effect curves of the cannabinoids. the parallel shifts could be coincidental and the

could be nonspecific, this is unlikely because parallel shifts were observed with three cannabinoids. it is likely that cannabinoids interact spinally with a gated, apamin-sensitive potassium channel in the production of antinociception. Cannabinoids administered icv. appear to produce antinociception which does not involve on cannabinoid-induced antinociception can not be attributed to lack of binding of the apamin. Summary. Cannabinoids produce antinociception after administration to both spinal and supraspinal sites; however, modulation of antinociception by calcium, and potassium seems to differ in the brain in the spinal cord. Additionally, the cannabinoid-induced modulation of opiate antinociception seems to differ in the brain and the spinal

cord. Based on our data, we hypothesize that in the brain, cannabinoids produce antinociception decreasing calcium levels in neurons through receptor linkage to proteins involved in the modulation of the function of the calcium channel. Cyclic
seem to not be involved in the production of antinociceptior induced by icv. administered cannabinoids. In the ing in a decreased calcium-gated potassium channels. The involvement of tion with G proteins has the potential to alter both and potassium channel function. Because acutely administered have beer shown to interact with of apamin in sitive potassium channels. Since apamin binding has been shown in the brain

the lack of effects of apamin or to play an indirect role. In addition, potassium cord, our data indicate that the cannabinoids produce ciceptive 1978; Childers and Snyder, We have shown previously that opiate-induced in combination with the cannabinoids into the ceils of both the pancreas and the brain appears to not play a direct role in the 1988; Gaines that only apamin coupled to a G protein (Brown and Birnbaumer. via the interaction with G,, proteins 1988; protein-coupled receptors production and interaction et t. seems to either not be decrease calcium entry et al., and

et and cal

and content of neurons (Harris et al., 1976; Cardenas and R

OSS , 1976; Welch and Olson, levels (Collier and Roy, 1974; 1978) and produce hyperpolarization of neurons these three intracellular systems may be common points of interaction with the cannabinoids. For example, it has been hypothesized that the end result of bination may be the phosphorylation of similar proteins which are proposed to be synapsins I and II which are volved in the release of (Childers et al., 1992). Finally, the data suggest that cannabinoids interact spinally with nor-BNI-sensitive receptors, This could indicate some interaction of the cannabinoids directly with opiate-sensitive pathways. Kappa receptor-selective opioids have not been shown to be displaced by the cannabinoids. Thus, although we can hypothesize as be done to delineate conclusively the mechanisms underlying the greater than additive effects observed using cannabinoids and opiates in combination. Acknowledgments The author wishes to thank Dr. Bill R. Martin, Department of the points of interaction ofcannabinoids and opiates based on the in mechanisms of action of the drugs indicated by this work and that and Toxicology, Medical College of Virginia for his assistance and collaboration on this project. References levels in rat brain areas. calcium-activated potassium channels: Effects of channel gating, voltage, and ionic strength. J. Gen. Physiol. 91: 317333, 1988.

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