Vasopressin analgesia: Specificity of action and non-opioid effects

Peptides. Vol. 5. pp. 747-756, 1984. ' Ankho International Inc. Printed in the U.S.A.

0196-9781/84 $3.00 + .00

Vasopressin Analgesia: Specificity of Action and Non-Opioid Effects J E F F R E Y H. K O R D O W E R ' A N D R I C H A R D J. B O D N A R "

Department of Psychology, Queens College, CUN Y. Flushing, N Y 11367 Received 2 S e p t e m b e r 1983 KORDOWER. J. H. AND R. J. BODNAR. Vasopressin analgesia: Specificity of action and n,m-opioid effects. PEPTIDES ~4) 747-756, 1984.--Recent neuroanatomical and behavioral evidence has indicated that vasopressin (VP) increases pain thresholds. In the present study intracerebroventricular (ICV) administration of both arginine VP (AVP: 75--500 ng) and I-deamino-8-D-arginine vasopressin (DDAVP: 150-500 ng) elevated tail flick latencies. Oxytocin (OXY, 1CV). also elevated tail-flick latencies {150-1000 ng); however this increase was accompanied by "'barrel-roll" seizure activity. VP analgesia was eliminated by pretreatment with l-deamino-penicillamine-2(O-methyl)tyrosine-AVP (dPTyr(me}AVP: 500 ng, ICVL a VP antagonist, but not naloxone (l or l0 ~g, ICV), suggesting that VP modulates nonciceptive thresholds through its own binding sites. Conversely, pretreatment with naloxone (I p.g, ICV) but not dFTyr(me)AVP ( l #.g, ICV) attenuated the analgesic efficacy of systemic morphine (10 mg/kg), further dissociating VP and central opiate analgesic processes. Finally, systemic pretreatment with dexamethasone potentiated VP analgesia. These data support the notion that VP is a specific non-opioid pain inhibitor. Arginine vasopressin Analgesia Rats

Oxytocin

dPTytlme)AVP

I-deamino-8-D-arginine vasopressin

IN addition to their well known neural projections to the posterior lobe of the pituitary gland, neurons containing vasopressin (VP) and oxytocin (OXY) also project to extrahypothalamic structures including some that modulate noxious input. These include the sensory nuclei of the trigeminal and vagus nerves and the substantia gelatinosa of the dorsal horn of the spinal cord [10, 41. 45]. In this regard, central [6.31] and systemic [6.7] injections of lysine VP (LVP) increase tail-flick latencies in rats. While systemic administration of AVP increases writhing thresholds and hot-plate latencies in mice 15]. similar injections of l-
Pain

descending extrahypothalamic magnoceilular projections contain OXY, as compared to VP [41], this peptide might also modulate pain thresholds. While systemic administration of OXY has been reported to be without effect upon pain thresholds [5], its central effects have yet to be explored. Thus the first experiment compared dose and duration properties of AVP, DDAVP, and OXY respectively upon tailflick latencies. While VP analgesia appears not to be mediated by the endogenous opioids [5, 6, 7, 43], it is still not known whether it is acting through its own putative binding sites. Specific pharmacological antagonists to VP have recently been developed including 1-deamino-penicillamine-2(O-methyi)tyrosine-AVP (dVryr(me)AVP), which antagonizes the pressor, but not the antidiuretic activity of VP [3]. Additionally. dPTyr(me)AVP possesses behavioral effects opposite to those of VP in active avoidance paradigms [30] and it reverses VP's facilitory effects upon extinction on pole-jump avoidance [33,34], and appetitive learning and memory tasks [20]. Thus, the second experiment investigated whether central pretreatment with the AVP antagonist dPTyr(me)AVP, but not naloxone would block AVP and DDAVP analgesia respectively. The third experiment addressed the complementary relationship, that is. whether central injections of naloxone, but not dPTyr(me)AVP, would attenuate systemic morphine analgesia. Since VP releases adrenocorticotrophic hormone (ACTH) (see review: [39]) and potentiates corticotropin releasing factor's (CRF) release of ACTH [22], and since

'Present address: Department of Anatomy. Box 603. University of Rochester School of Medicine. 601 Elmwood Ave., Rochester, NY 14(-.,42. :Requests for reprints should be addressed to R. J. Bodnar.

747

748

KORDOWER AND BODNAR TABLE 1

OkVP) C ) ~ ) Vehicle ~,, "__ 75 ng

Ar g~nJne V e s o p r e s l l l n

ALTERATIONS IN TAIL-FLICK LATENCIES FOLLOWING INTRACEREBROVENTRICULAR (ICV) ADMINISTRATION OF ARGININE VASOPRESSIN (AVP)

Post-Injection (rain) Dose (ng)

Pre

5

~

15

30

45

60

0

Mean SEM

2.68 0.19

2.41 0.16

2.53 0.50

2.30 0.18

2.78 0.36

2.43 0.25

75

Mean SEM

2.48 O. 17

3.27t 0.35

3.33 + 0.37

2.64 0.19

3.20 0.41

2.79 0.17

150

Mean SEM

2.66 0.18

3.85* 0.31

3.35 e 0.36

3.0It 0.46

2.75 0.43

2.35 0.21

500

Mean SEM

2.32 0.15

4.47* 0.63

4.35* 0.31

2.66 0.25

2.99 0.70

2.40 0.49

Hlsong N~zB 50Ong

I 4

¢II

~2 tL

m

j-.

Note I: In this and all subsequent experiments, the pre-injection values failed to differ from each other across doses or across times. To conserve space, these values are pooled (Pre). Also, vehicle injections failed to alter post-injection tail-flick latencies. Note 2: Significant differences from corresponding vehicle values-Dunnett comparison* (.o<0.05) and *(.o<0.01). Note 3: Significant effects were observed across the time course, F(7,35)=8.50, p<0.001, and for the interaction between doses and time, F(21,105)= 1.89, p<0.019.

PreInjection

Post-Injection

(m,n)

FIG. 1. Mean alterations in tail-flick latencies (sec) following intracerebroventricular (ICV) administration of arginine vasopressin (AVP: 75, 150, 500 ng) or vehicle. Ix: Significantly different p<0.05) from pre-injection values.)

stained with cresyl violet for cell body visualization. Coronal sections through the lateral ventricle were analyzed for cannula placement. Only animals with cannula tips located in the lateral ventricle were included for data analysis.

Protocol ACTH increases pain thresholds [2, 49, 50], it is conceivable that VP analgesia is mediated through ACTH release. Thus the fourth experiment examined whether pretreatment with the synthetic glucocorticoid dexamethasone would alter VP analgesia. EXPERIMENT 1 METHOD

Surgery Twenty-four female albino Sprague-Dawley rats (250-350 g) were administered chlorpromazine HCI (3 mg/mi normal saline/kg body weight, IP) 20 rain before anesthetization with Ketamine HCI (95 mg/ml sterile water/kg body weight, IM). One stainless steel 22 gauge guide cannula (Plastic Products), was stereotaxically (Kopf) aimed so that its tip was positioned 0.3 mm above the left lateral ventricle. With the incisor bar set at +5 ram, coordinates were 0.5 mm anterior to the bregma suture, 1.3 mm lateral to the sagittal suture, and 3.6 mm from the top o f the skull. The cannula was secured to three stainless steel anchor screws with dental acrylic. All animals were allowed 10 days to recover from surgery before behavioral testing began. Rats were maintained on a 12 hr light: 12 hr dark lighting schedule, and were housed individually with food and water available ad lib.

Histology Following exprimental testing in this and all subsequent experiments, all animals were anesthetized with sodium pentobarbital (100 rag/2 ml normal saline/kg body weight, IP} and perfused through transcardiac puncture with 0.9% saline followed by I ( ~ buffered formalin. Each brain was removed, blocked, sliced into 40/zm sections, mounted and

All rats were tested for their responsiveness to radiant heat in a modification of the procedure of D ' A m o u r and Smith [19]. The stimulus source (IITC Company) was mounted 8 cm above the dorsum of and 6 cm proximal to the tip of the tail of a lightly restrained animal. The intensity of the thermal stimulus was set to produce stable basal tail-flick latencies between 2.0 and 3.0 sec. To avoid tissue damage, a trial was automatically terminated if a response did not occur within 6 sec. Three groups of rats, matched for baseline tail-flick latencies, received ICV injections of AVP (Peninsula Labs: 0, 75, 150. 500 ng), DDAVP (Peninsula Labs: 0, 150, 500 ng) and OXY (Peninsula Labs: 0, 150, 500, 1000 ng) respectively. Half of the rats in each group received the injection doses in an ascending order while the remaining rats received the injection doses in a descending order. In this, and all subsequent analyses, the order of injections failed to show any main or interaction effects. A minimum of 72 hr elapsed between each injection. All peptides were dissolved in a 5 ~1 vehicle solution which contained 0.5% chlorbutanol and 0.05 M acetic acid in saline. This solvent ensures the stability of AVP, DDAVP. and OXY over extended periods (J. Haldar and W. H. Sawyer, personal communication). Infusions were made at a rate of I t~i every 20 see through a stainless steel 28 gauge internal cannula. Eight blocks of tail-flick latencies were determined 10.5, and 0 rain before, and 5, 15. 30.45. and 60 min following each ICV injection. Each block consisted of two latency determinations separated by a 20 sec intertrial interval. RESULTS

As summarized in Table I and Fig. I. AVP significantly elevated tail-flick latencies above pre-injection levels at 15 rain following the 75 ng dose. at 5 rain following the 150 ng

VASOPRESSIN AND PAIN PERCEPTION

749

TABLE 2

Oeelmino-O"Arginine VelOprel i i n(DDAYP) Vehicle

ALTERATIONS IN TAIL-FLICK LATENCIES FOLLOWING ICV ADMINISTRATION OF I-DEAMINO-g-D-ARGININE VASOPRESSIN (DDAVP)

i

Ng~=8 1SOng = 600no

Post-Injection (min) Dose

(ng)

Pre

5

15

30

45

60

0

Mean SEM

3.07 0.32

3.03 0.22

2.81 0.19

3.19 0.41

3.03 0.42

2.97 0.23

150

Mean SEM

2.75 0.27

3.84~ 0.60

3.02 0.39

3.05 0.38

2.87 0.46

2.% 0.31

500

Mean SEM

2.63 0.22

3.65~ 0.40

3.91" 0.50

3.58 ~, 0.45

3.61~ 0.55

3.22 0.18

Note 1: Significant difference from corresponding vehicle valueDunnett comparison .~(p<0.05) and *(p<0.01). Note 2: Significant differences were observed across the time course, F(7,42)=2.36, p<0.04.

TABLE 3

t

I-1

P r ,s ,'5 el) ,5' 6o Inie¢tion Post-lnle(tion (rain) FIG. 2. Mean alterations in tail-flick latencies (see) following ICV desamino-D-arginine vasopressin (DDAVP: 150, 500 rig) or vehicle. Ix: Significantly different (p<0.05) from pre-injection tail-flick values.)

Oxytocln

u

ALTERATIONS IN TAIL-FLICK LATENCIES FOLLOWING ICV ADMINISTRATION OF OXYTOC1N

soo-g

N,,8 lO00ng

Post-Injection (min) Dose Ing)

Pre

5

15

30

0'--0 V150119 ehicle

45

60

0

Mean SEM

2.10 0.12

2.10 0.17

2.27 0.22

2.16 0.08

2.09 0.16

2.44 0.21

150

Mean SEM

2.23 0.10

2.91" 0.30

2.04 0.16

1.84 0.14

2.07 0.06

1.80 0.10

500

Mean SEM

2.16 0.14

2.750.29

2.51 0.22

2.43 0.42

2.08 0.12

1.87 0.06

1000

Mean SEM

2.24 0.14

3.%* 0.47

3.04* 0.43

2.70* 0.33

2.23 0.12

2.50 0.40

i-i Pr

e-

Injection

Post*

Injection

train)

Note 1: Significant difference from corresponding vehicle values--;Ip<0.051 and *(p<0.01). Note 2: Significant differences were observed among doses, F(3.18)=5.24. p<0.009, across the time course, F{7,42)=5.95, p < 0 . 0 0 1 , and for the interaction b e t w e e n d o s e s and times, F(21.126)= 2.04. p <0.009.

FIG. 3. Mean alterations in tail*flick latencies (see) following ICY oxytocin (OXY: 150, 500, 1000 rig) or vehicle. Barrel-roll seizure activity accompanied alterations in latencies following the 500 and 1000 ng dose. Ix: Significantly different (/,<0.05) from pre-injection values.)

dose, and at 5 and 15 min following the 500 ng dose. AVP also significantly elevated tail-flick iatencies above corresponding vehicle values at 15 min following the 75 ng dose, at 5, 15, and 30 min following the 150 ng dose, and at 5 and 15 following the 500 ng dose. As summarized in Table 2 and Fig. 2. D D A V P significantly elevated tail-flick latencies above pre-injection levels at 3 rain following the 150 ng dose. and at 5. 15.30. and 45 rain following the 500 ng dose. DDAVP also significantly elevated tail-flick latencies above corresponding vehicle values at 5 min following the 150 ng dose, and at 15 min following the 500 ng dose. As summarized in Table 3 and Fig. 3. OXY significantly elevated tail-flick latencies above pre-injection levels and above corresponding vehicle

values at 5 min following both the 150 and 500 ng doses, and at 5, 15, and 30 min following the 1000 ng dose. EXPERIMENT 2 METHOD

Baseline tail-frick latencies of four groups of carmulated rats were determined as described in Experiment 1. The first group of 8 rats received four series of ICV injections: (a) saline-vehicle; (b) saline-AVP (500 ng); (c) naloxone (Endo Labs: I /~g)-AVP (500 ng); and (d) dPTyr(me)AVP (Dr. M. Manning, Medical College of Ohio: 500 ng)-AVP

750

KORDOWER AND BODNAR TABLE 4 R E V E R S A L O F A V P (500 rig) A N A L G E S I A BY I - D E A M I N O - P E N I C I L L A M I N E - 2 ( O - M E T H Y L )

TYROSINE-AVP(dPTyr~me)AVP:500 rig). BUT NOT BY NALOXONE (NAL: I. l0 #.g) Post-Injection {min) Condition

Pre

5

15

30

45

60

Mean SEM

2.42 0.10

2.36 0.15

2.32 0.15

2.24 0.13

2.48 0.14

0.13

SAL-AVP

Mean SEM

2.54 0.14

3.84* 0.36

3.86* 0.19

3.59* 0.46

2.86 0.11

2.23 0.13

NAL I-AVP

Mean SEM

2.51 0. I I

3.59* 0.57

3.77* 0.42

3.23* 0.26

2.93 0.23

3.12 0.38

NAL 10-AVP

Mean SEM

2.48 0.08

3.41" 0.45

3.89* 0.36

3.95* 0.45

2.64 0.15

2.63 0.22

dPTyr(me)AVPAVP

Mean SEM

2.30 0.07

2.57 0.22

2.28 0.13

2.48 0.19

2.31 0.15

2.43 0.27

SAL-VEH

2.33

Note 1: Significant differences from corresponding saline-vehicle values tSALVEH)-Dunnett comparison fop<0.05) and *(p<0.01}. Note 2: Significant differences were observed across experimental treatments. F13,35)=6.64, p<0.0004, across the time course, F17,28)= 13.18. p<0.0001, and for the interaction between treatments and times, F128,245)=2.94, p<0.0001.

(500 ng). There was a 10 min interinjection interval within each pair of injections. The treatment order, infusion rate, and time interval between the four series of drug administrations were as described previously. The vehicle solution for naloxone and dPTyr(me)AVP was normal saline while the vehicle for AVP was 5% chlorobutanol and 0.05 M acetic acid dissolved in saline. A second group of eight rats received naloxone (10 ~g) followed 10 rain later by AVP (500 ng). A third group of 13 rats received an identical injection sequence as the first group except that DDAVP (500 rig) was administered in lieu of AVP. A fourth group of 8 rats received dPTyr(me)AVP (0,500 ng) followed 10 min later by an ICV injection of the 5% chlorbutanoi and 0.05 M acetic acid vehicle. Eight blocks of tall-flick latencies were determined for all animals in all groups at 10, .5, and 0 rain before the first ICV injection, and 5, 15, 30.45, and 60 min following the second ICV injection. RESULTS

dPTyr~me)A VP. Naloxone. and A VP Analgesia As summarized in Table 4 and Fig. 4, the saline-AVP condition significantly elevated latencies at 5, 15, and 30 rain following injection relative to both pre-injection levels and corresponding saline-vehicle values. Tail-flick latencies following the dPTyr(me)AVP-AVP condition failed to differ from those elicited by rats following the saline-vehicle condition and were significantly lower than the saline-AVP condition at 5, 15, and 30 rain after injection. In contrast, latencies following the naloxone (I /xg)-AVP and naloxone ( l0/~g)-AVP conditions failed to differ from those elicited by rats following saline-AVP treatment, and were significantly higher than the saline-vehicle condition at 5, 15, and 30 rain ~ t e r injection. Latencies were also significantly elevated above pre-injection levels 60 rain following the halo×one I 1 /~g)-AVP condition.

Antogon,,l

•

,g

"~ AVP ( $ 0 0 n 9 )

~

S o l , h e - Veh,t I¢

~

N O lot o n e( IOug~AV P

t---o

~ f f . g . . . . . Avp

c l l ~ n r -

AvP

(SOOng)

:2

p,. P

•

Injection

i 15 Post-injection

3~)

i 45

f

6G

Cm,-)

FIG. 4. Elimination of AVP analgesia by ICV pretreatment 10 rain earlier with dPTyr(me)AVP (500 ng). In contrast similar pretreatment with naloxone (I, 10 p,g) failed to alter AVP analgesia, ix: Significantly different Ip<0.05) from pre-injection values.I

dPTyr(me)A VP. Naloxone and DDA VP Analgesia As summarized in Table 5 and Fig. 5 the saline-DDAVP condition significantly elevated latencies at 5 and 15 rain following injection relative to pre-injection and corresponding saline-vehicle values. Tail-flick latencies following the dPTyr~me)AVP-DDAVP condition failed to differ from those following the saline-vehicle condition and were significantly lower than the saline-DDAVP condition at 5 and 15 rain after injection. In contrast, latencies following the halo×one (I /~g)-DDAVP condition failed to differ from those elicited by the saline-DDAVP condition and were significantly higher

VASOPRESSIN AND PAIN PERCEPTION

751 TABLE 5

REVERSAL OF DDAVP (500 ng) ANALGESIA BY dPTyr(me)AVP (500 ng), BUT NOT BY NAL (I /,tg)

Post-Injection (min) Condition

Ire

5

15

30

45

60

SAL-VEH

Mean SEM

2.37 0.05

2.72 0.28

2.46 0.24

2.24 0.34

2.59 0.47

2.60 0.23

SAL-DDAVP

Mean SEM

2.45 0.12

3.55* 0.37

3.22t 0.27

2.92 0.27

2.90 0.18

2.73 0.24

NAL-DDAVP

Mean SEM

2.70 0.15

3.62* 0.03

3.33¢ 0.33

2.85 0.18

2.79 0.17

3.07 0.17

dPTyr(me)AVPDDAVP

Mean SEM

2.62 0.17

2.93 0.36

2.90 0.21

3.08 0.24

2.82 0.23

3.07 0.28

Note l: Significant difference from corresponding saline-vehicle values (SAL-VEH)Dunnetl comparison t'(p<0.05)and *[p<0.01). Note 2: Significant differences were observed across the lime course. F(8,88)=7.64, p<0.0001, and for the interaction between times and experimental treatments, F(24,264)--- 1.67, p <0.036.

Antagonists

r I.

PreInjection

vs

DDAVP(500)

~,--~ Saline--Vehicle ~.-~Sal t ne~ODAVP ~b--~,N,qoxohi(lag)-. DDAVP ~ ~lmtlgo nilt--ODAVP N. 1(~3°(~ng)

Post-Injection

train)

~ 6f

( ~ . ~ $aiine--Sat~ne

0

I

PreInjection

N,=a

Post-

Injection

s,,,.e

o~i.~

FIG. 5. Elimination of DDAVP analgesia I~y ICV pretreatment 10 rain earlier with dPTyrlme)AVP (500 ng). In contrast, similar pretreatment with naloxone ( I/,Lg) failed to alter DDAVP analgesia. (x: Significantly different (p<0.05) from pre-injection values.)

FIG. 6. Failure of dPTyr(me)AVP (500 ng) to alter basal tail-flick latencies.

than the saline-vehicle condition at 5 and 15 min after injection.

EXPERIMENT 3 METHOD

dPT.vr(me)A VP and Tail-Flick Latencies While significant differences were observed between dPTyr(me)AVP-vehicle and saline-vehicle treatments, F(1,5)=6.65, p < 0 . 0 5 , they failed to differ across blocks, F(7.35)=0.96, or for the interaction between blocks and treatment. F(7,39)=0.61. Dunnett comparisons failed to reveal post-injection latency differences for either group (Fig. 6).

Baseline tail-flick latencies of three groups of eight rats each were determined following implantation of a lateral ventricle cannula. A single 1CV injection of either dPTyr(me)AVP (1 ~.g), naloxone (1 ~g), or saline was administered to each group respectively, i0 min prior to a subcutaneous injection of morphine (Pennick Labs: 10 mg morphine/ml buffered solution/kg body weight). Tail-flick latency determinations were made 10, 5. and 0 min before the

752

K O R D O W E R A N D BODNAR TABLE 6 REVERSAL OF MORPHINE (MOR: I0 mg/kg) ANALGESIA BY NAL (1 v.g). BUT NOT BY dPTyr(me)AVP ( I V.g) Post-Injection (min) Condition

Pre

5

15

30

45

60

120

VEH-MOR

Mean SEM

2.32 0.15

3.02 0.40

4.44 0.47

5.66 0.18

5.82 0.15

6.00 0.00

5.55 0.30

NAL-MOR

Mean SEM

2.25 0.16

2.46 0.38

2.71" 0.36

4.63* 0.34

5.17" 0.34

5.46 0.29

4.73* 0.49

dPTyr(me)AVPMOR

Mean SEM

2.24 0.13

2.79 0.31

4.85 0.46

5.74 0.13

6.00 0.00

5.98 0.02

5.69 0.22

Note I: Significant difference from corresponding value in VEH-MOR condition: Dunnett comparison tip<0.05)and *(p<0.01). Note 2: Significant differences were observed across experimental treatments. F(2,21)= 6.59. p<0.05, across the time course, F(8,168)= 129.57, p<0.0001, and for the interaction between treatments and times, F(16,168) =2.44, p <0.0025.

first injection, and 5, 15, 30, 45, 60, and 120 min following the second injection.

e_

RESULTS As summarized in Table 6 and Fig. 7, tail-flick latencies of the saline-morphine and dPTyr~mejAVP-morphine groups were significantly and similarly elevated in all post-injection test intervals. In contrast, while the naloxone-morphine group displayed analgesia at 15, 30, 45, 60, and 120 min after injection, these effects were significantly less than the saline-morphine group at 15, 30, 45, and 60 min following injection.

Baseline tail-flick latencies of four groups of eight rats each were determined following implantation of a lateral ventricle cannula. The first and second groups received dexamethasone injections at 24 hr (Sigma Co.: 0.4 mg/kg body weight, IP) and at I hr (0.2 mg/kg body weight, IP) prior to an ICV injection of either AVP (500 ng) or vehicle respectively. The third and fourth groups received vehicle (20% ethanol solution) injections at 24 hr and I hr prior to an ICV injection of AVP or vehicle respectively. Tail-flick determinations were made 10, 5, and 0 min prior to the ICV injection and 5, 15, 30. 45, 60 rain and 24 hr thereafter.

¥ehiC ie'~

Moc'phlne

•

AVl I J Antloon, st I

10 mg/k s

oug)

t~

n:8

EXPERIMENT 4 METHOD

0

J

Pr

e-

l niection

,k

3"

Post-

Iniection

2s

.b.-" ,[o

(rn,n~

FIG. 7. Attenuation of systemic morphine ( 10 mg/kg) analgesia by ICV pretreatment 10 min earlier with naloxone (I ~,g) but not dPTytlme)AVP ( I V.g). (x: Significantly different (p<0.05) from the vehicle-morphine condition. )

latencies at 5, 15 and 30 min after injection relative to the pre-injection, baseline, and corresponding dexamethasonevehicle levels. Tukey comparisons revealed that dexamethasone significantly potentiated the magnitude of AVP analgesia at 5, 15. and 30 rain after injection.

RESULTS As summarized in Table 7 and Fig. 8, significant decreases in latencies were observed relative to baseline levels at 15 and 30 rain following the dexamethasone-vehicle condition and at 30 and 45 min following the vehicle-vehicle condition. However. latencies of these groups failed to differ when comparing pre-injection and post-injection values. The vehicle-AVP groups displayed significantly elevated latencies at 5 and 15 min after injection relative to pre-injection and baseline levels and at 3. 15, 30, and 45 min after injection relative to corresponding vehicle-vehicle latencies. The dexamethasone-AVP group displayed significantly elevated

DISCUSSION Both central and systemic administration of VP and certain analogs elevate nociceptive thresholds on the tail-flick [6, 7, 31], hot-plate [5]. and writhing [5] assays, but not on the flinch-jump test [31]. In addition, decreased availability of VP, either by central administration of a VP antiserum to normal rats [9] or use of the Brattleboro rat [8], decreases basal pain thresholds. Since VP also projects to sites involved in pain perception, including the sensory nuclei of the trigeminal and vagus nerves, and lamina I! of the spinal cord dorsal horn [ 10, 41. 45], these findings taken together indi-

VASOPRESSIN AND PAIN PERCEPTION

753 TABLE 7

P O T E N T I A T I O N O F AVP (500 ng) A N A L G E S I A BY D E X A M E T H A S O N E (DEX) P R E T R E A T M E N T

Post-Injection (min) Condition

Pre

5

15

30

45

60

24 hr

VEH-VEH

Mean SEM

2. I 1 0.14

2.18 0.18

1.98 0.13

1.86 0.17

1.81 0.17

1.94 0.23

2.66 0.34

VEH-AVP

Mean SEM

1.97 0.15

3.18" 0.56

3.27* 0.49

2.45+ 0.44

2.40+ 0.38

2.09 0.32

2.10 0.15

DEX-VEH

Mean SEM

1.91 0.17

1.90 0.17

1.80 0.11

1.66 0.19

2.06 0.26

2.17 0.25

2.21 0.12

DEX-AV P

Mean SEM

2.06 0.16

4.56~ 0.52

4.200.51

3.47~ 0.61

2.14 0.25

2.11 0.31

2.43 0.23

Note I: Significant difference from corresponding VEH-VEH values: Dunnett comparison -ip<0.051 and *lp<0.01) and from corresponding VEH-AVP values: Dunnett comparison ~(p
care that one action of extrahypothalamic VP is to inhibit pain. The present study demonstrates that VP's modulation of pain thresholds appears to be specific to its actions upon putative binding sites, and appears not to be acting through endogenous opioid or ACTH neurons. The first experiment indicated that equimolar administration of ng doses of AVP and DDAVP elevate tail-flick latencies. but it should be noted that the route of administration plays a crucial role as to whether, and to what extent, a particular form of VP or its analogues produce analgesia. While the present study showed that central administration of AVP (75-500 ng) increased tail-flick iatencies for 15-30 min in rats, Berkowitz and Sherman [5] found that intravenous AVP (120/~g) administration failed to alter tail-flick latencies while subcutaneous AVP (400/zg) produced analgesia on this measure. Intravenous AVP injections at doses of 30. but not 5 gg increased hot plate latencies for up to 60 min and writhing thresholds for up to 15 min in mice [5]. While the present study showed that central administration of DDAVP (150500 ngl increased tail-flick latencies for up to 45 rain in rats, Berson and co-workers [7] found that subcutaneous DDAVP (128 gg) injections produced marginal, but significant analgesia on this measure. Moreover, while central LVP (500 ng) injections increased tail-flick latencies, but not flinchjump thresholds for up to 15 rain in rats [31], subcutaneous LVP injections increased latencies only at 16-128/zg [7] but not lower [ 3 1 ] doses. Finally, while central desglycinamide-8-LVP (500 rig) increased tail-flick iatencies, but not fiinch jump thresholds for up to 60 rain [31]. Berson and co-workers [7] found that this analogue was without effect at a 128 gg dose. From these data. it appears that while the threshold dose of various VP peptide forms and analogues necessary to elicit a significant effect is lower following central administration, the magnitude of analgesia appears larger following the larger systemic dose. Thus parametric variables are important in analyzing VP analgesia. ,just as they are for analyzing other forms of non-

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FIG. 8. Potentiation of AVP analgesia by systemic pretreatment at 24 hr (0.4 mg/kg) and 1 hr (0.2 mg/kg) with dexamethasone. (x: Significantly different (.0<0.05) from ore-injection levels: t: significantly higher (p<0.05) than the saline-AVP condition.)

opioid peptide analgesia, including neurotensin [13, 14, 37, 40] and substance P [23, 36, 42, 44]. OXY is synthesized in the same brain nuclei as VP, projects to similar hypothalamo-hypophyseal (see reviews: [57.58]) and extrahypothalamic brain [10,11] regions as VP and differs structurally from VP only in two of the nine amino acids in the peptide chain. Though OXY elevates tail-flick latencies, this effect appears not to be a direct consequence of activation of a pain-inhibitory system, but rather an epiphenomenon of concomittant seizure activity. In the present study, seizure activity was observed in five rats following the 1000 ng OXY dose and in one rat following the 500 ng OXY dose. These seizures resembled closely those re-

754

K O R D O W E R A N D BODNAR

ported previously for ICV OXY [32] administration which are characterized by forelimb and trunk rigidity followed by tilting ipsilateraily to the injection side. This is followed by rapid "barrel roll" seizures in which the rat maintains the rigid body posture and quickly rotates in the direction ipsilateral to the injection site. These seizures last for a few minutes after which the rat's trunk becomes slightly flaccid. While the animals in the present study appeared to have recovered prior to the onset o f behavioral testing, the increase in tail-flick latencies following ICV OXY administration might presently best be interpreted in terms of the analgesic properties of seizures [25,35] and rotation [24]. Interactions between peripheral VP and the endogenous opioids have been demonstrated with beta-endorphin and morphine inhibiting plasma VP release following electrical stimulation to the medial basal hypothalamus [28], following exposure to foot shock [291, or following isoprenaline administration [26]. Moreover, some endogenous opioids and VP co-exist within the same neurons [38,511. Despite such interactions in the periphery, the second and third experiments provide further evidence that VP analgesia acts independently of the endogenous opioids. Central administration of either I or 10 #,g doses of naloxone failed to alter the analgesic effects of AVP or DDAVP even though ICV injection of a 10 ~tg dose of naloxone is capable of eliminating analgesia induced by a systemic 75 mg/kg morphine dose [53]. In contrast, dPTyr(me)AVP failed to alter systemic morphine analgesia. These data support and extend previous findings showing that systemic administration of either naloxone [6,7] or naltrexone [5] failed to alter VP analgesia, that morphine tolerant rats displayed normal VP analgesia [7], that Brattleboro rats exhibit normal morphine analgesia [8], and that systemic VP administration had no effect upon morphine analgesia [43]. The occurrance of VP analgesia is dependent upon interaction with its putative binding sites. The VP antagonist dPTyr(me)AVP blocks the pressor but not the antidiuretic response of AVP [3] and exerts effects opposite to those of VP on the pole jump task [30]. It also reverses VP's effects upon appetitive [20] and aversive learning and memory paradigms [33,34], and blocks the isoprenaline-induced release of VP [27]. The present data demonstrate that central pretreatment with an equimolar dose of dPTyr(me)AVP completely blocked the analgesia following both ICV AVP .and DDAVP. This elimination of AVP and DDAVP analgesia was not due to a compensatory decrease in basal latencies induced by dPTyr(me)AVP since the dose sufficient to block VP analgesia failed to alter tail-flick latencies.

Berkowitz and Sherman [4] have reported that systemic administration of a similarly structured VP antagonist reversed systemic VP analgesia in mice. Recently. specific binding sites for (3H)-AVP have been characterized and localized in the nucleus tractus solitarius, the lateral septum. the magnocellular hypothalamic nuclei, and in the anterior and neural lobes of the hypophysis [4. 17, 48,521. This effect was specific since the ability of non-labeled AVP to displace ('~H)-AVP from binding was superior than desglycinimideA V P or OXY [17]. The search for the precise site of action for the analgesic response following AVP administration has been complicated further by the recent observations of VPimmunoreactive perikarya localized in the bed nucleus of the stria terminalis, the dorsomedial hypothalamic region, the medial amygdaloid nucleus, and the locus coeruleus [12. 16. 47] in addition to the well documented paraventricular, supraoptic, and suprachiasmatic hypothalamic nuclei. Since dPTyr(me)AVP also blocks the pressor [3] as well as the analgesic effects of VP, this experiment does not eliminate the possibility that VP analgesia is the consequence of hypertension (see reivew: [I ]), a condition known to increase pain thresholds [18.54.55,561. However, VP analogues that are known to possess minimal pressor activity [ 15] elevates pain thresholds following central administration [311. Moreover, while VP, vasotocin, and phenylephrine each elicit similar pressor effects, only VP produced an antinociceptive response [5,7]. In conclusion, the present series of experiments strongly support the notion that VP mediates central nociceptive processes through its own binding sites. While AVP. DDAVP, and OXY elevated pain thresholds, the latter effect appear to be secondary to its seizure producing activity. Moreover, while central administration of dPTyr~me)AVP. but not naloxone, eliminated central VP analgesia, central naloxone, but not dPTyr(me)AVP attenuated morphine analgesia. Finally, the unexpected finding that dexamethasone potentiated AVP analgesia will need further investigation into the precise mechanism producing this effect, as well as finding the pathway(s) responsible for VP analgesia itself. ACKNOWLEDGEMENTS This research was supported in part by NIH GRSG 5S05-RR07064 and PSC/CUNY Grant 6-63210 to RJB. We thank Dr. M. Manning for his generous gift of the dPTyr(me)AVP. Endo Laboratories for the naloxone hydrochloride, and Pennick inc. for the morphine sulfate. We also thank Drs. M. Manning. W. H. Sawyer. and G. Nilaver for helpful comments and criticisms.

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