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Effects of morphine and (D-Ala, Met)-enkephalinamide on 45Ca-uptake to rat brain striatal slices
Neuropeptides5: 57-60, 1984
EFFECTS OF MORPHINE AND (D-ALA, MET)-ENKEPHALINAMIDE UPTAKE TO RAT BRAIN STRIATAL SLICES
ON 45Ca-
K. Saito, N. Fujita*, M. Nakahiro* and R. Inoki. Department of Pharmacology, Osaka University, Faculty of Dentistry, Yamadaoka l-8, Suita, Osaka 565 and * Department of PharmacolgY 1, Osaka University, Faculty of Medicine, Nakanoshima 4-3-57, Kitaku, Osaka 530, Japan (reprint requests to RI) ABSTRACT Effects of morphine and (D-Ala, Met)-enkephalinamide on 45Ca-uptake to rat striatal slices were examined. While morphine didn't give any effect on 45Ca-uptake to striatal slices in normal Krebs-Ringer solution, it inhibited K+ (4OmM) -stimulated 45Ca-uptake to slices. In high K+ medium, 45Cauptake was enhanced to 170 8 and this was reduced to 147 and 137 % by 10e5 and 10W4M morphine, respectively. Furthermore, the effect of morphine was antagonized by 10T5M naloxone. (D-Ala, Met)-enkephalinamide (10-4M) also inhibited K+-stimulated 45Ca-uptake to the slices to the same extent as was observed by morphine. K+-stimulated 45Ca-uptake to striatal slices obtained from chronically morphine administered rat (6 mg/kg, twice/day, 7 days) was not inhibited by morphine. INTRODUCTION Ca++ plays an important function in neurotransmission wherein the release of transmitters is observed following the increase of intracellular Ca++. It has been suggested that morphine exhibits its analgesic activity by inhibiting the increase of intracellular Ca++. A supportive evidence for this is the antagonistic effect of Ca++ on morphine analgesia (1). Furthermore, it has been shown that morphine inhibits 45Ca-uptake to synaptosomal fraction of rat brain (2,3). In the present study, the effects of morphine and (D-Ala, Met)-enkephalinamide on 45Ca-uptake to rat striatal slices were examined. MATERIALS AND METHODS Rats were killed by decapitation and their brains were quickly removed. Parasagittal slices were made at 57
the thickness of 350 ,,m and preincubated in modified oxygenated Krebs-Ringer solution (composition: NaCl; 124 mM, KCl; 5 mM, KHzP04; 1.24 mM, MgS04; 1.3 mM, CaClz; 2.6 mM, NaHC03; 26 mM, Glucose; 10 mM) for about 1 hr at 35OC. Striata were dissected and uptake of 45Ca was measured at 35 'C by adding 45Ca (1.3 FCi). RESULTS In normal Krebs-Ringer solution,45Ca was gradually taken up by slices at 35 'C. When 40 mM out of 124 mM NaCl in Krebs-Ringer solution was replaced by 40 mM KCl, 45Ca-uptake to striatal slices increased rapidly within 20 seconds and gradual increment was observed thereafter. In the following experiments,45Ca-uptake was measured for 20 seconds. Table 1. Effects of morphine on45Ca-uptake striatal slices Addition or replacementa None Morphine (10e4M) KC1 (40 mM)a KC1 (40 mM)a+Morphine (10w5M) KC1 (40 mM)a+Morphine (10T4M) KC1 (40 mM)a+Morphine (10s4M) +Naloxone (10m5M)
to rat brain
45Ca-uptake 100 92.7 169.0 147.0 137.2
+ 7.1 r15.9 r17.5 k20.4
(n=3) (n=ll) (n=S)* (n=ll)**
164.3 k21.5 (n=4)***
45Ca-uptake was expressed as % of that observed in a:KCl (40 mM) was replaced for normal Krebs-Ringer solution. 40 mM out of 124 mM NaCl in Krebs-Ringer solution. *, **: Significantly different from that observed in the presence of 40 mM KC1 (*P
58
Table 2. Effect of (D-Ala, Met)-enkephalinamide uptake to rat brain striatal slices
on 45Ca-
Addition or replacement
45Ca-uptake
None KC1 (40 mM) KC1 (40 mM)+(D-Ala, Met)enkephalinamide (10m4M)
100 178.2 215.2 (n=4) 138.8 +12.8 (n=4)**
**:Significantly different from that observed in the presence of 40 mM KC1 (P
None KC1 (40 mM) KC1 (40 mM)+Morphine (10m4M) Chronic None morphine KC1 (40 mM) KCl+Morphine (10m4M)
45Ca-uptake 100 133.1 104.6 100 153.0 147.8
211.1 (n=3) + 6.6 (n=3)* 234.0 (n=4) k24.5 (n=4)
*:Significantly different from that observed in the presence of 40 mM KC1 (PcO.05). (see details in legend for Table 1). Effect of morphine on 45Ca-uptake to striatal slices prepared from rats chronically treated with morphine (6 Although K+-stimumg/kg, twice a day, 7 days) was examined. lation on 45Ca-uptake was not so high in this series of experiments, morphine (10m4M) inhibited 45Ca-uptake to slices prepared from saline-treated group. On the contrary, morphine was without effect of Kc-stimulated 45Ca-uptake in slices prepared from chronically morphine administered group. DISCUSSION It has been shown that morphine inhibits synaptosomal 45Ca-uptake which is reversed by naloxone (2,3). In brain slice preparation, however, morphine (10e3M) inhibited K+-stimulated 45Ca-uptake only in the presence of low concentration of Ca (0.1 mM) (4). In the present study, 10e4 and 10s5M morphine inhibited K+-stimulated 45Ca-uptake significantly in the presence of 2.6 mM CaC12. The difference might be attributable to the concentration of K+-stimulating 45Ca influx. In former experiment, 100 mM KC1 was employed while 40 mM KC1 was replaced for NaCl in the present study. The reversal of the effect of morphine by naloxone indicated its specific action. The inhibition of K+-stimulated 59 NEUR.-
D
45Ca-uptake by (D-Ala, Met)-enkephalinamide further supports the specific action of opiate agonist. Thus, it is shown that brain slices are also feasible for studying the mechanism of action of morphine and opioid peptides. In the present study, chronic morphine treatment diminished the effect of morphine on 45Ca-uptake as shown in synaptosomal preparation (3). Repeated treatments of animals with morphine developed desensitization to the drugs and revealed the increase in number of opioid receptors (5) instead of expecting decrease of its number. Thus, the observed desensitization to morphine might be phenomena involving Since sequences following the activation of receptors. morphine was without effect in 45Ca-uptake after chronic treatment of rats by itself, changes of Ca movement might also be involved in desensitization phenomena. REFERENCES 1.
Kakunaga, T., Kaneto, H., and Hano, K. (1966) Pharmacologic studies on analgesics -VII. Significance of the calcium ion in morphine analgesia. J. Pharmacol. Exp. Ther. 153: 134-141.
2.
Guerrero-Munoz, F., Cerreta, K.V., Guerrero, M.L. and Way, E.1,. (1979) Effect of morphine on synaptosomal Ca++ uptake. J. Pharmacol. Exp. Ther. 209 : 132-136.
3.
Konno, F. and Takayanagi, I. (1982) Effect of morphine, codeine and codeine-epoxide on calcium uptake into the synaptosomes isolated from naive and tolerant rats. Japan. J. Pharmacol. 32: 1143-1150.
4.
Kakunaga, T. (1966) Pharmacological studies on analgesics (IX) Significance of calcium in the mechanisms of morphine analgesia. Folia Pharmacol. Jap. 62: 40-50.
5.
Pert, C.B. and Snyder, S.H. (1976) Opiate receptors binding: enhancement by opiate administration in vivo. Biochem. Pharmacol. 25: 847-853.
60
EFFECTS OF MORPHINE AND (D-ALA, MET)-ENKEPHALINAMIDE UPTAKE TO RAT BRAIN STRIATAL SLICES
ON 45Ca-
K. Saito, N. Fujita*, M. Nakahiro* and R. Inoki. Department of Pharmacology, Osaka University, Faculty of Dentistry, Yamadaoka l-8, Suita, Osaka 565 and * Department of PharmacolgY 1, Osaka University, Faculty of Medicine, Nakanoshima 4-3-57, Kitaku, Osaka 530, Japan (reprint requests to RI) ABSTRACT Effects of morphine and (D-Ala, Met)-enkephalinamide on 45Ca-uptake to rat striatal slices were examined. While morphine didn't give any effect on 45Ca-uptake to striatal slices in normal Krebs-Ringer solution, it inhibited K+ (4OmM) -stimulated 45Ca-uptake to slices. In high K+ medium, 45Cauptake was enhanced to 170 8 and this was reduced to 147 and 137 % by 10e5 and 10W4M morphine, respectively. Furthermore, the effect of morphine was antagonized by 10T5M naloxone. (D-Ala, Met)-enkephalinamide (10-4M) also inhibited K+-stimulated 45Ca-uptake to the slices to the same extent as was observed by morphine. K+-stimulated 45Ca-uptake to striatal slices obtained from chronically morphine administered rat (6 mg/kg, twice/day, 7 days) was not inhibited by morphine. INTRODUCTION Ca++ plays an important function in neurotransmission wherein the release of transmitters is observed following the increase of intracellular Ca++. It has been suggested that morphine exhibits its analgesic activity by inhibiting the increase of intracellular Ca++. A supportive evidence for this is the antagonistic effect of Ca++ on morphine analgesia (1). Furthermore, it has been shown that morphine inhibits 45Ca-uptake to synaptosomal fraction of rat brain (2,3). In the present study, the effects of morphine and (D-Ala, Met)-enkephalinamide on 45Ca-uptake to rat striatal slices were examined. MATERIALS AND METHODS Rats were killed by decapitation and their brains were quickly removed. Parasagittal slices were made at 57
the thickness of 350 ,,m and preincubated in modified oxygenated Krebs-Ringer solution (composition: NaCl; 124 mM, KCl; 5 mM, KHzP04; 1.24 mM, MgS04; 1.3 mM, CaClz; 2.6 mM, NaHC03; 26 mM, Glucose; 10 mM) for about 1 hr at 35OC. Striata were dissected and uptake of 45Ca was measured at 35 'C by adding 45Ca (1.3 FCi). RESULTS In normal Krebs-Ringer solution,45Ca was gradually taken up by slices at 35 'C. When 40 mM out of 124 mM NaCl in Krebs-Ringer solution was replaced by 40 mM KCl, 45Ca-uptake to striatal slices increased rapidly within 20 seconds and gradual increment was observed thereafter. In the following experiments,45Ca-uptake was measured for 20 seconds. Table 1. Effects of morphine on45Ca-uptake striatal slices Addition or replacementa None Morphine (10e4M) KC1 (40 mM)a KC1 (40 mM)a+Morphine (10w5M) KC1 (40 mM)a+Morphine (10T4M) KC1 (40 mM)a+Morphine (10s4M) +Naloxone (10m5M)
to rat brain
45Ca-uptake 100 92.7 169.0 147.0 137.2
+ 7.1 r15.9 r17.5 k20.4
(n=3) (n=ll) (n=S)* (n=ll)**
164.3 k21.5 (n=4)***
45Ca-uptake was expressed as % of that observed in a:KCl (40 mM) was replaced for normal Krebs-Ringer solution. 40 mM out of 124 mM NaCl in Krebs-Ringer solution. *, **: Significantly different from that observed in the presence of 40 mM KC1 (*P
58
Table 2. Effect of (D-Ala, Met)-enkephalinamide uptake to rat brain striatal slices
on 45Ca-
Addition or replacement
45Ca-uptake
None KC1 (40 mM) KC1 (40 mM)+(D-Ala, Met)enkephalinamide (10m4M)
100 178.2 215.2 (n=4) 138.8 +12.8 (n=4)**
**:Significantly different from that observed in the presence of 40 mM KC1 (P
None KC1 (40 mM) KC1 (40 mM)+Morphine (10m4M) Chronic None morphine KC1 (40 mM) KCl+Morphine (10m4M)
45Ca-uptake 100 133.1 104.6 100 153.0 147.8
211.1 (n=3) + 6.6 (n=3)* 234.0 (n=4) k24.5 (n=4)
*:Significantly different from that observed in the presence of 40 mM KC1 (PcO.05). (see details in legend for Table 1). Effect of morphine on 45Ca-uptake to striatal slices prepared from rats chronically treated with morphine (6 Although K+-stimumg/kg, twice a day, 7 days) was examined. lation on 45Ca-uptake was not so high in this series of experiments, morphine (10m4M) inhibited 45Ca-uptake to slices prepared from saline-treated group. On the contrary, morphine was without effect of Kc-stimulated 45Ca-uptake in slices prepared from chronically morphine administered group. DISCUSSION It has been shown that morphine inhibits synaptosomal 45Ca-uptake which is reversed by naloxone (2,3). In brain slice preparation, however, morphine (10e3M) inhibited K+-stimulated 45Ca-uptake only in the presence of low concentration of Ca (0.1 mM) (4). In the present study, 10e4 and 10s5M morphine inhibited K+-stimulated 45Ca-uptake significantly in the presence of 2.6 mM CaC12. The difference might be attributable to the concentration of K+-stimulating 45Ca influx. In former experiment, 100 mM KC1 was employed while 40 mM KC1 was replaced for NaCl in the present study. The reversal of the effect of morphine by naloxone indicated its specific action. The inhibition of K+-stimulated 59 NEUR.-
D
45Ca-uptake by (D-Ala, Met)-enkephalinamide further supports the specific action of opiate agonist. Thus, it is shown that brain slices are also feasible for studying the mechanism of action of morphine and opioid peptides. In the present study, chronic morphine treatment diminished the effect of morphine on 45Ca-uptake as shown in synaptosomal preparation (3). Repeated treatments of animals with morphine developed desensitization to the drugs and revealed the increase in number of opioid receptors (5) instead of expecting decrease of its number. Thus, the observed desensitization to morphine might be phenomena involving Since sequences following the activation of receptors. morphine was without effect in 45Ca-uptake after chronic treatment of rats by itself, changes of Ca movement might also be involved in desensitization phenomena. REFERENCES 1.
Kakunaga, T., Kaneto, H., and Hano, K. (1966) Pharmacologic studies on analgesics -VII. Significance of the calcium ion in morphine analgesia. J. Pharmacol. Exp. Ther. 153: 134-141.
2.
Guerrero-Munoz, F., Cerreta, K.V., Guerrero, M.L. and Way, E.1,. (1979) Effect of morphine on synaptosomal Ca++ uptake. J. Pharmacol. Exp. Ther. 209 : 132-136.
3.
Konno, F. and Takayanagi, I. (1982) Effect of morphine, codeine and codeine-epoxide on calcium uptake into the synaptosomes isolated from naive and tolerant rats. Japan. J. Pharmacol. 32: 1143-1150.
4.
Kakunaga, T. (1966) Pharmacological studies on analgesics (IX) Significance of calcium in the mechanisms of morphine analgesia. Folia Pharmacol. Jap. 62: 40-50.
5.
Pert, C.B. and Snyder, S.H. (1976) Opiate receptors binding: enhancement by opiate administration in vivo. Biochem. Pharmacol. 25: 847-853.
60