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Spinal dynorphin involvement in the analgesia of pregnancy: effects of intrathecal dynorphin antisera
European Journal of Pharmacology, 159 (1989) 205-209
205
Elsevier EJP 20277 Short communication
Spinal dynorphin involvement in the analgesia of pregnancy: effects of intrathecal dynorphin antisera H o w a r d W. S a n d e r 1, R i c h a r d M. K r e a m 2 a n d A l a n R. G i n t z l e r * Department of Biochemistry, State University of New York, Health Science Center at Brooklyn, Brooklyn, N Y 11203, and 2 Department of Anesthesiology, Tufts University, School of Medicine, Boston, M/~ 02111, U.S.A.
Received 7 October 1988, accepted 8 November 1988
In both rats and humans there is an analgesia associated with pregnancy. This analgesia is spinally mediated and involves the K type of opiate receptor. The current study demonstrates that intrathecal administration of high affinity dynorphin antibodies produces a significant reduction in jump thresholds during pregnancy (day 20). The administration of pre-adsorbed antisera fails to produce this effect. These results support the hypothesis that a spinal dynorphin/K opiate receptor system is activated during gestation. Analgesia; Pregnancy; Spinal cord; r Opioid receptors; Dynorphin A
1. Introduction The terminal stages of pregnancy in rats and humans are associated with elevated pain thresholds that are opioid mediated. This analgesia of pregnancy involves central (spinal) but not peripheral (adrenal and pituitary) opioid systems (see Sander and Gintzler, 1987 for references). Recently, this laboratory provided evidence that is consistent with the involvement of spinal x receptors in the mediation of this analgesia (Sander et al., in press). The data supporting this inference derived from experiments demonstrating that the intrathecal (i.t.) administration of a recently developed x-selective opioid antagonist, nor-BNI, would lower pain thresholds in pregnant but not non-
Current address: Department of Medicine, Beth Israel Medical Center, New York, NY 10003, U.S.A. * To whom all correspondence should be addressed at first address.
pregnant animals. The involvement of the x type of opiate receptor in mediating the analgesia of pregnancy suggests but does not unequivocally establish that dynorphin (x-selective) is the endogenous opioid ligand that acts through that receptor. This is because other opioid ligands that are less x-selective than dynorphin, if released in sufficient quantities, might still exert their effects through the x receptor. Consequently, it is necessary to satisfy a criterion that is independent of receptor type in order to identify the endogenous opioid ligand that mediates this effect. In the present study, the involvement of dynorphin was inferred by determining the effect on jump thresholds during pregnancy (day 20) of the i.t. administration of antibodies that are specific for this opioid peptide. The rationale for this approach is predicated on the fact that most agonists lose their pharmacological activity subsequent to being bound by antibodies. Thus, highly specific antibodies can be used as highly selective antagonists of specific agonists.
0014-2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
206 2. Materials and methods
2.1. Animals Female Sprague-Dawley rats (220-250 g, Charles River), were housed individually in a room with a light period of 14 h per day (05:00-19:00 h). Food and water were provided ad libitum. Rats were time mated, and the first day on which sperm were detected in the vaginal smear was designated as day 1 of pregnancy. 2.2. lntrathecal cannulation On day 9 of pregnancy, a catheter was inserted into the lumbar intrathecal space by a modification of the procedure originally described by Yaksh and Rudy (1976). Animals were anesthetized with a combination of ketamine (96 mg/kg i/p., Parke Davis) and xylazine (4.8 mg/kg i/p., Haver Lockhart) following pretreatment with atropine (0.85 mg/kg i.p., Elkins-Sinn). A saline filled catheter (PE-10 tubing, Clay Adams) was then inserted (7.5 cm) into the spinal subarachnoid space through an incision in the atlanto-occipital membrane and secured by suturing a pre-formed loop between this membrane and the overlying muscle. The free end of the cannula was placed beneath the skin and allowed to exit in the parietal area where it was relatively inaccessible to the paws. After surgery all animals were treated intramuscularly with 30 000 units of a mixture of penicillin G benzathine and penicillin G procaine (Wyeth Laboratories) and topically with Panalog ointment. All animals appeared to be free of infection upon gross inspection.
were increased in 0.05 mA steps at 10 s intervals. To acclimate the animals to the flinch jump procedure practice trials were conducted on days 14, 16 and 18 of gestation. In addition, on day 18, the rats were placed for 5 min in the restrainer used for i.t. injections. 2.4. L t. injections On day 20 of pregnancy, baseline flinch jump thresholds were determined as described above, after which each animal was restrained in a rat restrainer (Stoelting Co.). Affinity purified dynorphin antisera in 5 #1 of saline was administered over a 50 s period to the subarachnoid space of the lumbar spinal cord via the permanent indwelling i.t. cannula. Complete delivery was insured by flushing the cannula with an additional 2 p,1 of saline. Jump thresholds were determined 10, 30 and 60 min following i.t. antisera administration. 2.5. Control In order to determine whether the effects of the antisera were specifically due to antibodies selective for dynorphin as opposed to other immunoglobulins or contaminants, pre-adsorbed antisera (antisera from which antibodies to dynorphin were removed) were utilized. All acclimation and testing procedures were identical to those used for the experimental group. Since this laboratory has previously demonstrated that the i.t. administration of 12 #1 of saline (the same volume that was used to deliver the antisera) does not affect jump thresholds, this control was not repeated in the present study (Sander and Gintzler, 1987).
2.3. Flinch jump procedure
2.6. Further procedures
Reflexive jumping in response to electric footshock was used as an index of pain threshold and was performed as described previously (Gintzler, 1980). Briefly, the jump threshold was defined in milliamperes as the lowest of two consecutive intensities that elicited simultaneous withdrawal of both front paws from the charged grids. Each trial began with the animal receiving a 300 ms footshock (0.1 mA). Subsequent shocks
In all cases, mean jump thresholds obtained at various intervals following the experimental procedure were compared with the baseline jump threshold obtained in the same animal 30 rain prior to experimental or control injections. The data for the experimental and each control group were analyzed by using a two-tailed paired Student's t-test. All time-mated rats that were included in the analysis had normal gestational
207
lengths and normal litters. P < 0.05 is considered significant. Dynorphin antisera were generated using a conjugate of synthetic dynorphin A-(1-13) and BSA. These antibodies react with dynorphin-(1-13) and dynorphin-(1-17) on an equimolar basis. The opioids [MetS]enkephalin, [MetS]enkephalin sulfoxide, [LeuS]enkephalin and B-endorphin display less than 0.01% cross reactivity in a radioimmunoassay. The dynorphin antisera were purified using an Affigel 10 column (BioRad) to which dynorphin-(1-13) was covalently attached. Following the application of dynorphin-specific antibodies, the column was washed with high salt (1 M NaCl) until the eluate did not contain any detectable protein. Dynorphin-specific antibodies were eluted in 5 ml of acetic acid (2 N) which was then dialyzed against phosphate buffer (0.1 M, pH 7.4). In order to concentrate the antisera, the dialysate was placed over a 1 ml protein A column (Pharmacia). Dynorphin antibodies were eluted in 2 ml of acetic acid (2) and dialyzed against phosphate buffer (0.1 M, pH 7.4). The resulting dialysate was tested against dynorphin-(1-17) to confirm the presence of dynorphin-specific antibodies. Alic~
I00 o
i:,~i:i:i: :
o "1"
~,:
Iz.I
.-
-r-
8o-
~
~i!!!
,~
60 -
" ~ 4o
iii!ii!i!i iiiiiCil .....
"" O.
i~ii~i~ ::::.-:::::
, • ,,, ,,
20
A
B
Fig. 1. Effect of the intrathecal (i.t.) application of (A) dynorphin antisera (n = 4) and (B) pre-adsorbed dynorphin antisera (devoid of antibodies to dynorphin) (n = 5) on the threshold for responsiveness to aversive stimuli in pregnant rats (day 20 of gestation). The data represent the m a x i m u m reduction in j u m p threshold that was observed at either 10 or 30 min after i.t. injection. Data are plotted as the m e a n s + S.E.M. percent of the baseline pre-injection thresholds that were obtained 30 min prior to injection. * P < 0.01. (ll) I.T. dynorphin antisera, (1~) i.t. pre-adsorbed dynorphin antisera.
quots of dialysate were lyophilized and taken up in 50/~1 of 0.9% NaC1 just prior to injection. The binding capacity of the purified antisera was determined to be approximately 0.5 nmol per ml (2.5 pmol per animal). In order to obtain preadsorbed antisera, an aliquot of the above lyophilized sample was taken up in water and incubated with the Affigel 10 column, to which was covalently attached dynorphin-(1-13) for 72 h at 4°C. Following this incubation, the mixture was centrifuged in a microfuge and the supernatant was collected. In order to maximize the recovery of any unbound dynorphin antibody, the Affigel 10 column was washed 3 × with equal volumes of distilled water. All the resulting supernatants were pooled, lyophilized to dryness and reconstituted in 0.9% NaC1 immediately prior to i.t. application such that the concentration of protein in the pre-adsorbed and non-adsorbed antisera was identical.
3. Results
On day 20 of gestation, following i.t. dynorphin antisera administration, there occurred a significant reduction in jump threshold relative to the mean threshold obtained 30 min prior to its injection (fig. 1A). In all animals, a reduction in jump threshold was observed at all time points tested (10, 30 and 60 min). The reductions observed at 10 and 30 min reached statistical significance (P < 0.02 and 0.01, respectively). The time, however, at which the peak inhibition occurred, varied among the animals. In contrast, the jump thresholds of pregnant rats were not reduced at any of the time points tested following the i.t. administration of pre-adsorbed antisera (devoid of antibodies to dynorphin) (fig. IB; n-4, P > 0.1).
4. Discussion
This report confirms and extends previous resuits from this laboratory in rats and those of Rust et al. (1985) and Cogan and Spinnato (1986) in women that during the third trimester of pregnancy there is an increase in maternal pain
208
threshold that is opioid-mediated. Additionally, these data support the finding that the analgesia of pregnancy is mediated via a spinal x opioid pathway(s) (Sander et al., in press). In this study, the effects of i.t. administration of affinity purified dynorphin antibodies to pregnant rats was used to assess whether dynorphin is the endogenous opioid ligand responsible for mediating the analgesia of pregnancy. The present results indicate that i.t. application of dynorphin antibodies reduce the jump thresholds of pregnancy to an extent that is comparable to that which has been observed following the i.t. administration of naltrexone. The lack of an effect following i.t. application of pre-adsorbed antisera demonstrates that the present results are due to the effects of dynorphin-specific antibodies and not to other constituents or impurities. The effect of i.t. dynorphin antisera on the jump thresholds of nonpregnant rats was not determined in the present study. Previous results, however, have indicated that i.t. naltrexone and i.t. nor-binaltorphimine at concentrations that are effective in lowering pain thresholds of pregnancy, are without any effect when administered to non-pregnant animals (Sander and Gintzler, 1987; Sander et al., in press). Therefore, it seems highly unlikely that the present results are being confounded by a hyperalgesic effect of dynorphin antisera in non-pregnant rats. Thus, the significant reduction in jump thresholds observed following the i.t. application of the dynorphin antisera most probably results from the neutralization of the physiological effects of dynorphin and indicates that dynorphin is a major opioid substrate for the spinal analgesia that occurs during pregnancy. The involvement of spinal dynorphin in mediating pregnancy-induced analgesia is consonant with the previous implication of x opiate receptors in this phenomenon, and with the known neuroanatomical distribution of these receptors in the spinal cord. x Opiate receptors are present in high density in both the rat and human lumbosacral spinal cord. These receptors seem especially concentrated in regions involved in the processing of nociceptive stimuli, such as the substantia gelatinosa (lamina II) and the grey matter surrounding the central canal (lamina X) (See Gouarderes,
1986 for references). This localization of K receptors parallels the distribution of dynorphin in lamina II as well as other regions of the the dorsal horn of the spinal cord. Reports that the i.t. administration of dynorphin can induce antinociception in rats are consistent with these anatomical observations (see Herman and Goldstein, 1984 for references). These data therefore suggest that an endogenous dynorphin/x opioid receptor system could be an important constituent of spinal analgesia. Therefore, the current demonstration that such a system mediates the spinal analgesia of pregnancy has considerable neurochemical and neuroanatomical underpinnings and emphasizes the potential importance of the spinal cord dynorphin/x system in painful situations. Consistent with this in the recent report of increased biosynthesis of spinal cord dynorphin in a rat model of peripheral inflammation and hyperalgesia (Ruda et al., 1987). In summary, the current demonstration that spinal dynorphin is involved in mediating the analgesia of pregnancy, as well as the previous implication of spinal r receptors in this phenomenon, suggests that a spinal cord dynorphin/K opiate receptor system is activated in reaction to the stress of pregnancy. This system may be physiologically relevant in a variety of other stressful situations. Thus, it may be therapeutically advantageous to selectively activate this system and thereby stimulate the mechanism(s) that underlies the naturally occurring analgesia to treat the pain of labor and delivery and possibly other types of pain as well.
References Cogan, R. and J.A. Spinnato, 1986, Pain and discomfort thresholds in late pregnancy, Pain 27, 63. Gintzler, A.R., 1980, Endorphin mediated increase in pain threshold during pregnancy, Science 219, 193. Gouarderes, C., N. Kopp, J. Cros and R. Quirion, 1986, Kappa opioid receptors in human lumbo-sacral spinal cord, Brain Res. Bull. 16, 355. Herman, B.H. and A. Goldstein, 1984, Antinociception and paralysis induced by intrathecal dynorphin A, J. Pharmacol. Exp. Ther. 232, 27. Ruda, M.A., M.J. ladarola, L.V. Cohen and W.S. Young III, 1987, In situ hydridization histochemistry and immunocy-
209 tochemistry reveal an increase in spinal dynorphin biosynthesis in a rat model of peripheral intimation and hyperalgesia, Proc. Natl. Acad. Sci. 85, 626. Rust, M., M. Keller, R. Egbert and H. Graeff, 1985, Eleventh World Congress on Gynecology and Obstetrics. Sander, H.W. and A.R. Gintzler, 1987, Spinal cord mediation of the opioid analgesia of pregnancy, Brain Res. 408, 389.
Sander, H.W., P.S. Portoghese and A.R. Gintzler, Spinal kappa opiate receptor involvement in the analgesia of pregnancy: Effects of intrathecal nor-binaltorphimine, a kappa selective antagonist, Brain Res. (in press). Yaksh, T.L. and T.A. Rudy, 1976, Chronic catherization of the spinal subarachnoid space, Physiol. Behav. 17, 1031.
205
Elsevier EJP 20277 Short communication
Spinal dynorphin involvement in the analgesia of pregnancy: effects of intrathecal dynorphin antisera H o w a r d W. S a n d e r 1, R i c h a r d M. K r e a m 2 a n d A l a n R. G i n t z l e r * Department of Biochemistry, State University of New York, Health Science Center at Brooklyn, Brooklyn, N Y 11203, and 2 Department of Anesthesiology, Tufts University, School of Medicine, Boston, M/~ 02111, U.S.A.
Received 7 October 1988, accepted 8 November 1988
In both rats and humans there is an analgesia associated with pregnancy. This analgesia is spinally mediated and involves the K type of opiate receptor. The current study demonstrates that intrathecal administration of high affinity dynorphin antibodies produces a significant reduction in jump thresholds during pregnancy (day 20). The administration of pre-adsorbed antisera fails to produce this effect. These results support the hypothesis that a spinal dynorphin/K opiate receptor system is activated during gestation. Analgesia; Pregnancy; Spinal cord; r Opioid receptors; Dynorphin A
1. Introduction The terminal stages of pregnancy in rats and humans are associated with elevated pain thresholds that are opioid mediated. This analgesia of pregnancy involves central (spinal) but not peripheral (adrenal and pituitary) opioid systems (see Sander and Gintzler, 1987 for references). Recently, this laboratory provided evidence that is consistent with the involvement of spinal x receptors in the mediation of this analgesia (Sander et al., in press). The data supporting this inference derived from experiments demonstrating that the intrathecal (i.t.) administration of a recently developed x-selective opioid antagonist, nor-BNI, would lower pain thresholds in pregnant but not non-
Current address: Department of Medicine, Beth Israel Medical Center, New York, NY 10003, U.S.A. * To whom all correspondence should be addressed at first address.
pregnant animals. The involvement of the x type of opiate receptor in mediating the analgesia of pregnancy suggests but does not unequivocally establish that dynorphin (x-selective) is the endogenous opioid ligand that acts through that receptor. This is because other opioid ligands that are less x-selective than dynorphin, if released in sufficient quantities, might still exert their effects through the x receptor. Consequently, it is necessary to satisfy a criterion that is independent of receptor type in order to identify the endogenous opioid ligand that mediates this effect. In the present study, the involvement of dynorphin was inferred by determining the effect on jump thresholds during pregnancy (day 20) of the i.t. administration of antibodies that are specific for this opioid peptide. The rationale for this approach is predicated on the fact that most agonists lose their pharmacological activity subsequent to being bound by antibodies. Thus, highly specific antibodies can be used as highly selective antagonists of specific agonists.
0014-2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
206 2. Materials and methods
2.1. Animals Female Sprague-Dawley rats (220-250 g, Charles River), were housed individually in a room with a light period of 14 h per day (05:00-19:00 h). Food and water were provided ad libitum. Rats were time mated, and the first day on which sperm were detected in the vaginal smear was designated as day 1 of pregnancy. 2.2. lntrathecal cannulation On day 9 of pregnancy, a catheter was inserted into the lumbar intrathecal space by a modification of the procedure originally described by Yaksh and Rudy (1976). Animals were anesthetized with a combination of ketamine (96 mg/kg i/p., Parke Davis) and xylazine (4.8 mg/kg i/p., Haver Lockhart) following pretreatment with atropine (0.85 mg/kg i.p., Elkins-Sinn). A saline filled catheter (PE-10 tubing, Clay Adams) was then inserted (7.5 cm) into the spinal subarachnoid space through an incision in the atlanto-occipital membrane and secured by suturing a pre-formed loop between this membrane and the overlying muscle. The free end of the cannula was placed beneath the skin and allowed to exit in the parietal area where it was relatively inaccessible to the paws. After surgery all animals were treated intramuscularly with 30 000 units of a mixture of penicillin G benzathine and penicillin G procaine (Wyeth Laboratories) and topically with Panalog ointment. All animals appeared to be free of infection upon gross inspection.
were increased in 0.05 mA steps at 10 s intervals. To acclimate the animals to the flinch jump procedure practice trials were conducted on days 14, 16 and 18 of gestation. In addition, on day 18, the rats were placed for 5 min in the restrainer used for i.t. injections. 2.4. L t. injections On day 20 of pregnancy, baseline flinch jump thresholds were determined as described above, after which each animal was restrained in a rat restrainer (Stoelting Co.). Affinity purified dynorphin antisera in 5 #1 of saline was administered over a 50 s period to the subarachnoid space of the lumbar spinal cord via the permanent indwelling i.t. cannula. Complete delivery was insured by flushing the cannula with an additional 2 p,1 of saline. Jump thresholds were determined 10, 30 and 60 min following i.t. antisera administration. 2.5. Control In order to determine whether the effects of the antisera were specifically due to antibodies selective for dynorphin as opposed to other immunoglobulins or contaminants, pre-adsorbed antisera (antisera from which antibodies to dynorphin were removed) were utilized. All acclimation and testing procedures were identical to those used for the experimental group. Since this laboratory has previously demonstrated that the i.t. administration of 12 #1 of saline (the same volume that was used to deliver the antisera) does not affect jump thresholds, this control was not repeated in the present study (Sander and Gintzler, 1987).
2.3. Flinch jump procedure
2.6. Further procedures
Reflexive jumping in response to electric footshock was used as an index of pain threshold and was performed as described previously (Gintzler, 1980). Briefly, the jump threshold was defined in milliamperes as the lowest of two consecutive intensities that elicited simultaneous withdrawal of both front paws from the charged grids. Each trial began with the animal receiving a 300 ms footshock (0.1 mA). Subsequent shocks
In all cases, mean jump thresholds obtained at various intervals following the experimental procedure were compared with the baseline jump threshold obtained in the same animal 30 rain prior to experimental or control injections. The data for the experimental and each control group were analyzed by using a two-tailed paired Student's t-test. All time-mated rats that were included in the analysis had normal gestational
207
lengths and normal litters. P < 0.05 is considered significant. Dynorphin antisera were generated using a conjugate of synthetic dynorphin A-(1-13) and BSA. These antibodies react with dynorphin-(1-13) and dynorphin-(1-17) on an equimolar basis. The opioids [MetS]enkephalin, [MetS]enkephalin sulfoxide, [LeuS]enkephalin and B-endorphin display less than 0.01% cross reactivity in a radioimmunoassay. The dynorphin antisera were purified using an Affigel 10 column (BioRad) to which dynorphin-(1-13) was covalently attached. Following the application of dynorphin-specific antibodies, the column was washed with high salt (1 M NaCl) until the eluate did not contain any detectable protein. Dynorphin-specific antibodies were eluted in 5 ml of acetic acid (2 N) which was then dialyzed against phosphate buffer (0.1 M, pH 7.4). In order to concentrate the antisera, the dialysate was placed over a 1 ml protein A column (Pharmacia). Dynorphin antibodies were eluted in 2 ml of acetic acid (2) and dialyzed against phosphate buffer (0.1 M, pH 7.4). The resulting dialysate was tested against dynorphin-(1-17) to confirm the presence of dynorphin-specific antibodies. Alic~
I00 o
i:,~i:i:i: :
o "1"
~,:
Iz.I
.-
-r-
8o-
~
~i!!!
,~
60 -
" ~ 4o
iii!ii!i!i iiiiiCil .....
"" O.
i~ii~i~ ::::.-:::::
, • ,,, ,,
20
A
B
Fig. 1. Effect of the intrathecal (i.t.) application of (A) dynorphin antisera (n = 4) and (B) pre-adsorbed dynorphin antisera (devoid of antibodies to dynorphin) (n = 5) on the threshold for responsiveness to aversive stimuli in pregnant rats (day 20 of gestation). The data represent the m a x i m u m reduction in j u m p threshold that was observed at either 10 or 30 min after i.t. injection. Data are plotted as the m e a n s + S.E.M. percent of the baseline pre-injection thresholds that were obtained 30 min prior to injection. * P < 0.01. (ll) I.T. dynorphin antisera, (1~) i.t. pre-adsorbed dynorphin antisera.
quots of dialysate were lyophilized and taken up in 50/~1 of 0.9% NaC1 just prior to injection. The binding capacity of the purified antisera was determined to be approximately 0.5 nmol per ml (2.5 pmol per animal). In order to obtain preadsorbed antisera, an aliquot of the above lyophilized sample was taken up in water and incubated with the Affigel 10 column, to which was covalently attached dynorphin-(1-13) for 72 h at 4°C. Following this incubation, the mixture was centrifuged in a microfuge and the supernatant was collected. In order to maximize the recovery of any unbound dynorphin antibody, the Affigel 10 column was washed 3 × with equal volumes of distilled water. All the resulting supernatants were pooled, lyophilized to dryness and reconstituted in 0.9% NaC1 immediately prior to i.t. application such that the concentration of protein in the pre-adsorbed and non-adsorbed antisera was identical.
3. Results
On day 20 of gestation, following i.t. dynorphin antisera administration, there occurred a significant reduction in jump threshold relative to the mean threshold obtained 30 min prior to its injection (fig. 1A). In all animals, a reduction in jump threshold was observed at all time points tested (10, 30 and 60 min). The reductions observed at 10 and 30 min reached statistical significance (P < 0.02 and 0.01, respectively). The time, however, at which the peak inhibition occurred, varied among the animals. In contrast, the jump thresholds of pregnant rats were not reduced at any of the time points tested following the i.t. administration of pre-adsorbed antisera (devoid of antibodies to dynorphin) (fig. IB; n-4, P > 0.1).
4. Discussion
This report confirms and extends previous resuits from this laboratory in rats and those of Rust et al. (1985) and Cogan and Spinnato (1986) in women that during the third trimester of pregnancy there is an increase in maternal pain
208
threshold that is opioid-mediated. Additionally, these data support the finding that the analgesia of pregnancy is mediated via a spinal x opioid pathway(s) (Sander et al., in press). In this study, the effects of i.t. administration of affinity purified dynorphin antibodies to pregnant rats was used to assess whether dynorphin is the endogenous opioid ligand responsible for mediating the analgesia of pregnancy. The present results indicate that i.t. application of dynorphin antibodies reduce the jump thresholds of pregnancy to an extent that is comparable to that which has been observed following the i.t. administration of naltrexone. The lack of an effect following i.t. application of pre-adsorbed antisera demonstrates that the present results are due to the effects of dynorphin-specific antibodies and not to other constituents or impurities. The effect of i.t. dynorphin antisera on the jump thresholds of nonpregnant rats was not determined in the present study. Previous results, however, have indicated that i.t. naltrexone and i.t. nor-binaltorphimine at concentrations that are effective in lowering pain thresholds of pregnancy, are without any effect when administered to non-pregnant animals (Sander and Gintzler, 1987; Sander et al., in press). Therefore, it seems highly unlikely that the present results are being confounded by a hyperalgesic effect of dynorphin antisera in non-pregnant rats. Thus, the significant reduction in jump thresholds observed following the i.t. application of the dynorphin antisera most probably results from the neutralization of the physiological effects of dynorphin and indicates that dynorphin is a major opioid substrate for the spinal analgesia that occurs during pregnancy. The involvement of spinal dynorphin in mediating pregnancy-induced analgesia is consonant with the previous implication of x opiate receptors in this phenomenon, and with the known neuroanatomical distribution of these receptors in the spinal cord. x Opiate receptors are present in high density in both the rat and human lumbosacral spinal cord. These receptors seem especially concentrated in regions involved in the processing of nociceptive stimuli, such as the substantia gelatinosa (lamina II) and the grey matter surrounding the central canal (lamina X) (See Gouarderes,
1986 for references). This localization of K receptors parallels the distribution of dynorphin in lamina II as well as other regions of the the dorsal horn of the spinal cord. Reports that the i.t. administration of dynorphin can induce antinociception in rats are consistent with these anatomical observations (see Herman and Goldstein, 1984 for references). These data therefore suggest that an endogenous dynorphin/x opioid receptor system could be an important constituent of spinal analgesia. Therefore, the current demonstration that such a system mediates the spinal analgesia of pregnancy has considerable neurochemical and neuroanatomical underpinnings and emphasizes the potential importance of the spinal cord dynorphin/x system in painful situations. Consistent with this in the recent report of increased biosynthesis of spinal cord dynorphin in a rat model of peripheral inflammation and hyperalgesia (Ruda et al., 1987). In summary, the current demonstration that spinal dynorphin is involved in mediating the analgesia of pregnancy, as well as the previous implication of spinal r receptors in this phenomenon, suggests that a spinal cord dynorphin/K opiate receptor system is activated in reaction to the stress of pregnancy. This system may be physiologically relevant in a variety of other stressful situations. Thus, it may be therapeutically advantageous to selectively activate this system and thereby stimulate the mechanism(s) that underlies the naturally occurring analgesia to treat the pain of labor and delivery and possibly other types of pain as well.
References Cogan, R. and J.A. Spinnato, 1986, Pain and discomfort thresholds in late pregnancy, Pain 27, 63. Gintzler, A.R., 1980, Endorphin mediated increase in pain threshold during pregnancy, Science 219, 193. Gouarderes, C., N. Kopp, J. Cros and R. Quirion, 1986, Kappa opioid receptors in human lumbo-sacral spinal cord, Brain Res. Bull. 16, 355. Herman, B.H. and A. Goldstein, 1984, Antinociception and paralysis induced by intrathecal dynorphin A, J. Pharmacol. Exp. Ther. 232, 27. Ruda, M.A., M.J. ladarola, L.V. Cohen and W.S. Young III, 1987, In situ hydridization histochemistry and immunocy-
209 tochemistry reveal an increase in spinal dynorphin biosynthesis in a rat model of peripheral intimation and hyperalgesia, Proc. Natl. Acad. Sci. 85, 626. Rust, M., M. Keller, R. Egbert and H. Graeff, 1985, Eleventh World Congress on Gynecology and Obstetrics. Sander, H.W. and A.R. Gintzler, 1987, Spinal cord mediation of the opioid analgesia of pregnancy, Brain Res. 408, 389.
Sander, H.W., P.S. Portoghese and A.R. Gintzler, Spinal kappa opiate receptor involvement in the analgesia of pregnancy: Effects of intrathecal nor-binaltorphimine, a kappa selective antagonist, Brain Res. (in press). Yaksh, T.L. and T.A. Rudy, 1976, Chronic catherization of the spinal subarachnoid space, Physiol. Behav. 17, 1031.