Acta Physiol Scand 1991, 141, 573-574
ADONIS
000167729100082K
The adenosine analogue R-PIA interacts with substance P binding in rat brain and spinal cord C.-0. S T I L L E R , J. FASTBOM", B. B. FREDHOLM and E. B R O D I N Department of Pharmacology, Karolinska Institute and li Department of Clinical Pharmacology, Karolinska Hospital, Stockholm, Sweden
Adenosine analogues have antinociceptive effects in rats and mice when injected systemically, intracerebroventricularly or intrathecally, and adenosine has been suggested to play a role in endogenous antinociception (Sawynok & Sweeney 1989). Adenosine has been reported to be released by potassium, capsaicin and morphine from primary afferents (Sweeney et al. 1989) and to inhibit the transmission of nociceptive information within the spinal cord (Sawynok & Sweeney 1989). However, while adenosine has been long known to inhibit the release of various transmitters in a number of brain regions (Fredholm & Hedqvist 1980), inhibition of release of nociceptive transmitter candidates, for example substance P (SP), from terminals of primary afferents, has not received experimental support (Vasko & Ono 1990). Further autoradiographic studies indicate that adenosine A, and A, receptors are not located on the terminals of capsaicin-sensitive, S P containing primary afferents, but predominantly on intrinsic neurons in the substantia gelatinosa (Choca et al. 1988). It was recently reported that the behaviour indicative of pain, induced by intrathecally administered SP, can be inhibited by coadministration of adenosine analogues (DeLander & Wahl 1988), thus suggesting that adenosine inhibits nociceptive transmission by an action at sites postsynaptic to primary afferent terminals. One possibility that does not seem to have been tested so far, is that adenosine interacts with the SP-receptor. SP (or neurokinin 1, NK,) receptors are highly concentrated on superficial layers of the dorsal spinal cord and in this region also adenosine (A, and A,) receptors have been demonstrated (Buck et al. 1986, Choca et al. 1987, 1988). In the present study we have used autoradiographic techniques to investigate the effect of the adenosine analogue N6-(R-phenylisopropy1)-adenosine (R-PIA) Received 9 January 1991, accepted 11 January 1991. Key words :adenosine, brain, rat, receptor binding, spinal cord, substance P. Correspondence : Ernst Brodin, Department of Pharmacology, Karolinska Institutet, Box 60400, S104 01 Stockholm, Sweden.
on SP receptor binding in the dorsal horn of the spinal cord of the rat. Since adenosine and SP receptors have a somewhat similar distribution also in the brain, the effect of R-PIA on SP receptor binding was studied also in the striatum and dentate gyrus of the hippocampus. Sagittal sections of rat brain (1.9 mm lateral to the midline) and transverse sections of the lumbar (LCLS) spinal cord (20 pm) were thaw-mounted on gelatin-coated slides and stored at -80 "C. All incubations were performed at room temperature, in 50 mM Tris-HCI pH 7.4 containing 0.1 yo bovine
0
1 0 7
4
1 0 7 4
1 0 7 4
-Log R-PIA concentration (M)
Fig. 1. The effect of various concentrations of N6-(Rphenylisopropy1)-adenosine (R-PIA) on the binding of '251-Bolton-Hunter labelled substance P to rat striatum (a), dentate gyrus (b) and lamina I-IV of the lumber spinal cord (c), expressed in per cent of total specific binding. The specific binding of substance P was 1.4f0.16 fmol mg-' in the striatum, 1.9k0.28 fmol mg-l in the dentate gyrus and 0.8f0.16 fmol mg-' in the dorsal horn. Each column represents the mean of data from 6 rats and the vertical bars the SEM. * indicates significant difference at P < 0.05, ** at P < 0.01 and *** at P < 0.001 (Friedmans test).
573
574
C.-0. Stiller et al.
serum albumin (BSA). After 25 min preincubation the sections were incubated for 120 min with lZ5Ilabelled Bolton-Hunter-SP (200,000 cpm ml..', specific activity 2000 Ci mmol-'), 3 mM MnCI, and 0.01 04 Bacitracin, without or with different concentrations of R-PIA. Non-specific binding was achieved in the presence of 1 PM SP. Following the incubation, the sections were washed, dried and exposed to Hyperfilm3H (Amersham) for 6 days. Autoradiograms were analysed by quantitative densitometry, using a computerized image analyser. For calibration "'1 standards (Amersham) were used. Statistical significance was assessed by Friedman's non-parametric test. Data are expressed as a percentage of the total specific binding. The distribution of [12'I]BH-SP binding in rat brain and spinal cord was in accordance with previous reports on the distribution of NK, receptors (Buck et al. 1986). Incubation with R-PIA decreased the binding of [1251]BH-SPin the striatum and the dentate gyrus, whereas the binding in the dorsal spinal cord was increased at low concentrations and decreased at high concentrations of R-PJA. Results are shown in Figure 1. In the striatum incubation with lo-'", lo-' M R-PIA reduced the binding to 7 6 k 14% and (mean fSEM, P < 0.05), 58 f 11 yo ( P < 0.001) and 23*8% ( P < 0.001) of total specific binding respectively. In the dentate gyrus R-PJA reduced the specific binding significantly ( P < 0.01) only at the highest concentration tested (28 k 7 yo of total binding). In the dorsal horn of the lumbar spinal cord (lamina I-IV), R-PIA increased the specific binding of ['251]BH-SP to 148 32 Yo (not significant) and 144f 16% ( P < 0.01) at lo-'" and lo-' M respectively, and decreased binding to 42 f 9 y o ( P < 0,001) of total specific binding at lo-' M. The present results demonstrate that an adenosine receptor agonist influences SP receptor binding in nitro in certain regions of the rat brain and spinal cord. Interaction with the NK, receptor could be a mechanism by which endogenous adenosine may interfere with the effect of SP released from nerve terminals in both brain and spinal cord. Such interaction may also be involved in morphine analgesia, which appears to be mediated by adenosine to a considerable extent (Sweeney et al. 1987). Further experiments are required to determine whether the
altered binding of SP in the presence of R-PIA is due to a direct interaction of R-PIA with NK, receptors or if the effect is due to an interaction between adenosine receptors and NK, receptors. This study was supported by the Swedish medical research council (project no 14X-6386 and 14X-2553), the 1987 year fund against stroke, the Ostermans foundation, AB Astra, Magnus Bergvall's foundation, Ake Wiberg's foundation and the Karolinska Institutet.
REFERENCES BUCK,S.H., HELKE,C.J., BURCHER, E., SHULTS,C.W. & O'DONOHUE, T.L. 1986. Pharmacological characterization and autoradiographic distribution of binding sites for iodinated tachykinins in the rat central nervous system. Peptides 7, 1109-1 120. R.D. & PROUDFIT, H.K. 1988. CHOCA,J.I., GREEN, Adenosine A, and A, receptors of the substantia gelatinosa are located predominantly on intrinsic neurons : an autoradiographic study. 3 Pharmacol Exp Ther 247, 757-764. CHOCA,J.I., PROUDFIT, H.K. & GREEN,R.D. 1987. Identification of A, and A, receptors in the rat spinal cord. 3 Pharmacol Exp Ther 242(3), 905-910. DELANDER, G.E. & WAHL, J.J. 1988. Behaviour induced by putative nociceptive neurotransmitters is inhibited by adenosine or adenosine analogues coadministered intrathecally. 3Pharmacol Exp Ther 246(2), 565-570. FREDHOLM, B.B. & HEDQVIST, P. 1980. Modulation of neurotransmission by purine nucleotides and nucleosides. Biochem Pharmacol 29, 1635-1643. SAWYNOK, J. & SWEENEY, M.I. 1989. The role of purines in nociception. Neuroscience 32(3), 557-569. SWEENEY, M.I., WHITE,T.D. & SAWYNOK, J. 1987. Involvement of adenosine in the spinal antinociceptive effects of morphine and noradrenaline. J Pharmacol Exp Ther 243(2), 657-665. SWEENEY, M.J., WHITE,T.D. & SAWYNOK, J. 1988. Morphine, capsaicin and K' release purines from capsaicin-sensitive primary afferent nerve terminals in the spinal cord. 3 Pharmacol Exp Ther 248(1), 447454. VASKO,M.R. & ONO, H . 1990. Adenosine analogues do not inhibit the potassium-stimulated release of substance P from rat spinal cord slices. NaunynSchmiedeberg's Arch Pharmacol 324, 441446.
ADONIS
000167729100082K
The adenosine analogue R-PIA interacts with substance P binding in rat brain and spinal cord C.-0. S T I L L E R , J. FASTBOM", B. B. FREDHOLM and E. B R O D I N Department of Pharmacology, Karolinska Institute and li Department of Clinical Pharmacology, Karolinska Hospital, Stockholm, Sweden
Adenosine analogues have antinociceptive effects in rats and mice when injected systemically, intracerebroventricularly or intrathecally, and adenosine has been suggested to play a role in endogenous antinociception (Sawynok & Sweeney 1989). Adenosine has been reported to be released by potassium, capsaicin and morphine from primary afferents (Sweeney et al. 1989) and to inhibit the transmission of nociceptive information within the spinal cord (Sawynok & Sweeney 1989). However, while adenosine has been long known to inhibit the release of various transmitters in a number of brain regions (Fredholm & Hedqvist 1980), inhibition of release of nociceptive transmitter candidates, for example substance P (SP), from terminals of primary afferents, has not received experimental support (Vasko & Ono 1990). Further autoradiographic studies indicate that adenosine A, and A, receptors are not located on the terminals of capsaicin-sensitive, S P containing primary afferents, but predominantly on intrinsic neurons in the substantia gelatinosa (Choca et al. 1988). It was recently reported that the behaviour indicative of pain, induced by intrathecally administered SP, can be inhibited by coadministration of adenosine analogues (DeLander & Wahl 1988), thus suggesting that adenosine inhibits nociceptive transmission by an action at sites postsynaptic to primary afferent terminals. One possibility that does not seem to have been tested so far, is that adenosine interacts with the SP-receptor. SP (or neurokinin 1, NK,) receptors are highly concentrated on superficial layers of the dorsal spinal cord and in this region also adenosine (A, and A,) receptors have been demonstrated (Buck et al. 1986, Choca et al. 1987, 1988). In the present study we have used autoradiographic techniques to investigate the effect of the adenosine analogue N6-(R-phenylisopropy1)-adenosine (R-PIA) Received 9 January 1991, accepted 11 January 1991. Key words :adenosine, brain, rat, receptor binding, spinal cord, substance P. Correspondence : Ernst Brodin, Department of Pharmacology, Karolinska Institutet, Box 60400, S104 01 Stockholm, Sweden.
on SP receptor binding in the dorsal horn of the spinal cord of the rat. Since adenosine and SP receptors have a somewhat similar distribution also in the brain, the effect of R-PIA on SP receptor binding was studied also in the striatum and dentate gyrus of the hippocampus. Sagittal sections of rat brain (1.9 mm lateral to the midline) and transverse sections of the lumbar (LCLS) spinal cord (20 pm) were thaw-mounted on gelatin-coated slides and stored at -80 "C. All incubations were performed at room temperature, in 50 mM Tris-HCI pH 7.4 containing 0.1 yo bovine
0
1 0 7
4
1 0 7 4
1 0 7 4
-Log R-PIA concentration (M)
Fig. 1. The effect of various concentrations of N6-(Rphenylisopropy1)-adenosine (R-PIA) on the binding of '251-Bolton-Hunter labelled substance P to rat striatum (a), dentate gyrus (b) and lamina I-IV of the lumber spinal cord (c), expressed in per cent of total specific binding. The specific binding of substance P was 1.4f0.16 fmol mg-' in the striatum, 1.9k0.28 fmol mg-l in the dentate gyrus and 0.8f0.16 fmol mg-' in the dorsal horn. Each column represents the mean of data from 6 rats and the vertical bars the SEM. * indicates significant difference at P < 0.05, ** at P < 0.01 and *** at P < 0.001 (Friedmans test).
573
574
C.-0. Stiller et al.
serum albumin (BSA). After 25 min preincubation the sections were incubated for 120 min with lZ5Ilabelled Bolton-Hunter-SP (200,000 cpm ml..', specific activity 2000 Ci mmol-'), 3 mM MnCI, and 0.01 04 Bacitracin, without or with different concentrations of R-PIA. Non-specific binding was achieved in the presence of 1 PM SP. Following the incubation, the sections were washed, dried and exposed to Hyperfilm3H (Amersham) for 6 days. Autoradiograms were analysed by quantitative densitometry, using a computerized image analyser. For calibration "'1 standards (Amersham) were used. Statistical significance was assessed by Friedman's non-parametric test. Data are expressed as a percentage of the total specific binding. The distribution of [12'I]BH-SP binding in rat brain and spinal cord was in accordance with previous reports on the distribution of NK, receptors (Buck et al. 1986). Incubation with R-PIA decreased the binding of [1251]BH-SPin the striatum and the dentate gyrus, whereas the binding in the dorsal spinal cord was increased at low concentrations and decreased at high concentrations of R-PJA. Results are shown in Figure 1. In the striatum incubation with lo-'", lo-' M R-PIA reduced the binding to 7 6 k 14% and (mean fSEM, P < 0.05), 58 f 11 yo ( P < 0.001) and 23*8% ( P < 0.001) of total specific binding respectively. In the dentate gyrus R-PJA reduced the specific binding significantly ( P < 0.01) only at the highest concentration tested (28 k 7 yo of total binding). In the dorsal horn of the lumbar spinal cord (lamina I-IV), R-PIA increased the specific binding of ['251]BH-SP to 148 32 Yo (not significant) and 144f 16% ( P < 0.01) at lo-'" and lo-' M respectively, and decreased binding to 42 f 9 y o ( P < 0,001) of total specific binding at lo-' M. The present results demonstrate that an adenosine receptor agonist influences SP receptor binding in nitro in certain regions of the rat brain and spinal cord. Interaction with the NK, receptor could be a mechanism by which endogenous adenosine may interfere with the effect of SP released from nerve terminals in both brain and spinal cord. Such interaction may also be involved in morphine analgesia, which appears to be mediated by adenosine to a considerable extent (Sweeney et al. 1987). Further experiments are required to determine whether the
altered binding of SP in the presence of R-PIA is due to a direct interaction of R-PIA with NK, receptors or if the effect is due to an interaction between adenosine receptors and NK, receptors. This study was supported by the Swedish medical research council (project no 14X-6386 and 14X-2553), the 1987 year fund against stroke, the Ostermans foundation, AB Astra, Magnus Bergvall's foundation, Ake Wiberg's foundation and the Karolinska Institutet.
REFERENCES BUCK,S.H., HELKE,C.J., BURCHER, E., SHULTS,C.W. & O'DONOHUE, T.L. 1986. Pharmacological characterization and autoradiographic distribution of binding sites for iodinated tachykinins in the rat central nervous system. Peptides 7, 1109-1 120. R.D. & PROUDFIT, H.K. 1988. CHOCA,J.I., GREEN, Adenosine A, and A, receptors of the substantia gelatinosa are located predominantly on intrinsic neurons : an autoradiographic study. 3 Pharmacol Exp Ther 247, 757-764. CHOCA,J.I., PROUDFIT, H.K. & GREEN,R.D. 1987. Identification of A, and A, receptors in the rat spinal cord. 3 Pharmacol Exp Ther 242(3), 905-910. DELANDER, G.E. & WAHL, J.J. 1988. Behaviour induced by putative nociceptive neurotransmitters is inhibited by adenosine or adenosine analogues coadministered intrathecally. 3Pharmacol Exp Ther 246(2), 565-570. FREDHOLM, B.B. & HEDQVIST, P. 1980. Modulation of neurotransmission by purine nucleotides and nucleosides. Biochem Pharmacol 29, 1635-1643. SAWYNOK, J. & SWEENEY, M.I. 1989. The role of purines in nociception. Neuroscience 32(3), 557-569. SWEENEY, M.I., WHITE,T.D. & SAWYNOK, J. 1987. Involvement of adenosine in the spinal antinociceptive effects of morphine and noradrenaline. J Pharmacol Exp Ther 243(2), 657-665. SWEENEY, M.J., WHITE,T.D. & SAWYNOK, J. 1988. Morphine, capsaicin and K' release purines from capsaicin-sensitive primary afferent nerve terminals in the spinal cord. 3 Pharmacol Exp Ther 248(1), 447454. VASKO,M.R. & ONO, H . 1990. Adenosine analogues do not inhibit the potassium-stimulated release of substance P from rat spinal cord slices. NaunynSchmiedeberg's Arch Pharmacol 324, 441446.
