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Studies on the analgesic effects of lanthanides
Journal of the Less-Common
STUDIES ZHANG
ON THE ANALGESIC
ZUXUAN,
Department CHEN
ZHANG
401 - 409
EFFECTS
YUANZHEN
and WANG
401
OF LANTHANIDES* XINGUANG
of Biology, Nanjing University (China)
RONGSAN
Coordination (Received
Metals, 112 (1985)
and REN ZHENZHANG
Chemistry Research Institute, Nanjing University (China)
January
10,1985)
Summary The analgesic effects of ten lanthanides have been examined. The intraventricularly administered lanthanides produced a long-lasting analgesia. These effects were antagonized by divalent cations. Yttrium had no analgesic effect and was unaffected by the divalent cations, indicating that the analgesia is possibly a mutual characteristic of the lanthanide series of elements, but not of the rare earths. Naloxone fully or partly reverses the analgesic effects of the lanthanides, morphine or electroacupuncture, and at the same time increases the Ca*+ levels in the brain. Naloxone, however, has no effect on the EGTA-induced analgesia and the Ca *+ levels in the brain. All these findings indicate that the lanthanides are likely to share a mutual antinociceptive site and analgesic mechanism with those of morphine and electroacupuncture.
1. Introduction Our previous studies showed that intracisternally administered lanthanum chloride produces analgesic effects as measured by the mouse tail-flick method, and the characteristics of the analgesic effects are similar to those of morphine and electroacupuncture [ 11. It was found that the actions of Pr3+ in vitro resemble those of La 3+ but the former ion exerts a dual action on the transmitter release of the neuromuscular synapse [2]. There are 15 lanthanides whose chemical and physical properties are very similar [ 31. It follows that other lanthanides, in addition to La and Pr, will probably produce analgesia by the same mechanism. In order to confirm this, we have continued to study the correlation of divalent cations with the lanthanide series of elements emphasis being placed on a comparison of the characteristics of *Paper presented at the International land, March 4 - 8, 1985.
Rare Earth Conference,
0022-5088/85/$3.30
@ Elsevier Sequoia/Printed
ETH Zurich,
Switzer-
in The Netherlands
402
the analgesic effects of Tb3+ and Pr3+ as well as those of EGTA, morphine and electroacupuncture.
2. Experimental
details
Mice weighing 25 - 30 g were used. They were divided at random into a variety of experimental groups. The animals were treated on the basis of the electroacupuncture analgesia model proposed by our laboratory [4]. The isotope 45Ca was prepared in 0.9% NaCl. The agent was administered intraventricularly (20 PCi kg-‘, 0.5 mCi ml-’ of specific radioactivity) or intraperitoneally (80 PCi kg-‘) 10 - 30 min before electroacupuncture. After the final pain threshold was measured, each of the mice was decapitated immediately and the brain was dissected into discrete regions and weighed. Each brain region was put into a vial, then 0.3 ml of H202 and 0.4 ml of formic acid were added to the vial. After incubation at 80 “C for 1 h, and cooling, 5 ml of scintillation fluid (PPO, POPOP) and 4 ml of ethylene glycol ether were added. The radioactivity in the transparent homogenetic solution was determined with an FJ-2101 model, double-channel, liquid scintillation spectrometer. The front segment brain Ca2+ contents were measured with a specific ion electrode [ 51. The chlorides used in the present work were analytical reagents, prepared in saline before use. The lanthanide solution, 1 - 8 pmol kg-’ in a volume of 5 ~1 per mouse, was injected intraventricularly (i.vt.). Naloxone, 0.2 mg kg-’ in a volume of 0.5 ml per mouse, was injected intraperitoneally (i.p.). The corresponding control group of animals received saline of equivalent volume by the same method. The degree of ‘analgesia of either electroacupuncture or the lanthanides was expressed as a percentage of maximum possible effect according to the equation : DA = lOO(TL - BL)/(lO
- BL)
where DA is the degree of analgesia; line latency.
TL, the test latency;
and BL, the base-
3. Results and discussion 3.1. Survey on the analgesic effects of lanthanides In the present experiment, the analgesic effects of La3+, Ce3+, Pr3+, Nd3+, Sm 3+, Eu3+, Gd3+, Tb3+, Dy3+ and Ho3+ were compared. The results are shown in Table 1. These data show that 10 lanthanides of the same dosage (1 - 8 pmol kg-‘) had analgesic effects which were enhanced with increased dosage. Of the 10 lanthanides, Sm3+, Dy3+, Ho3+ and La3+ gave higher analgesic effects and Ce3+ the least.
403 TABLE
1
Comparison
Ion
of the analgesic
effect
of lanthanides
Degree of analgesia (%) 1 pmol kg-’ 1.8 f 1.0 (10)
2 pmol kg-’
4 pmol kg-
1.2 28.8 27.5 20.7 25.9 28.5 19.0 31.2 44.1 22.4 16.0
1.7 84.2 21.8 36.4 30.7 42.0 25.7 70.7 66.2 41.8 15.6
+ + + f f + f f f + t
1.6 (10) 4.4 (8) 5.7 (7) 8.6a (10) 8.6 (10) 12.0 (8) 5.9 (8) 11.8 (10) 13.0 (6) 10.6a (9) 3.0 (10)
-I + f It + f + It f f +
3.1 10.5 11.5 5.8 13.4 13.3 3.4 14.7 16.3 17.2 5.4
1 (10) (8) (8) (10) (9) (8) (10) (9) (6) (8) (9)
8 pmol kg-. 4.9 90.8 52.6 66.1 67.0 96.6 64.5 70.5 72.7 95.3 91.9
f. + + f + + + f f + +
1.8 8.9 12.7 6.4 12.2 1.4 14.7 12.2 12.9 4.7 8.1
1 (10) (9) (9) (1%) (10) (8) (10) (9) (7) (10) (9)
Saline Las+ Ce3+ Bra+ Nd3+ Sm3+ Eu 3+ Gd 3+ Tb3+ Dy3+ Ho3+
18.3 21.6 16.5 17.0 7.9 10.3 15.8 20.8 2.6 20.1
The saline parentheses different,p
and ions were injected intraventricularly 15 min before testing. Numbers in indicate the numbers of animals used. The values, except a are significantly < 0.05. Each value represents the fS.E.M.
f 5.5 (10) Y7.7 (8) f 4.5 (10) f 4.5 (8) f 2.7a (8) zk 3.0 (10) f 3.4 (9) + 7.8 (7) f 2.8 .(lO) + 9.0a (10)
of Ca2+ on analgesic effects of lanthanides The animals were divided into two groups: (i) lanthanide plus saline, (ii) lanthanide plus Ca 2+. The results are shown in Table 2. The above tests show that intraventricular injection of saline had no effect on pain threshold, and after administration of lanthanides the analgesic effects were markedly enhanced, particularly with Sm3+, Dy3+, Ho3+ and La3+. After addition of Ca2+, their analgesic effects were antagonized, and the animals displayed obvious nociception. The pain threshold of the Ho3+ plus Ca2+ group was still higher than that of the control group, but compared with that of the Ho3+ -treated group, it was markedly reduced. The results showed that the antagonism of Ca2+ on the analgesic effects of these 10 lanthanides was remarkable. 3.2. Antagonism
3.3. Antagonism of divalent cations on analgesic effects of lanthanides Yttrium and ten lanthanides were tested in our study. These elements were injected intraventricularly in a concentration of 4 pmol kg-‘. Five minutes later divalent cations such as Ca2+, Mg2+ or Mn2+, 0.0125 mol/mouse, were injected intraventricularly. The results are shown in Table 3. The rare earths apart from yttrium had marked analgesic effects. These effects were antagonized by Ca2+, Mg2+ or Mn’+. Y3+ itself had no analgesic effect and was not affected by the divalent cations. Although the physical and chemical characteristics of Y 3+ are very similar to those of the lanthanides, their pharmacological effects are very different, indicating that the analgesic effect is possibly a mutual characteristic of lanthanides.
404 TABLE 2 Antagonism of CaZ+ on the analgesic effect of lanthanides Ion
8 pmol kg-’
Added Ca2+ 8 Mmol kg-’
Pain threshold (s)
Saline La3+ Ce3+ Bra+ Nd 3+ Sm 3+ Eu3+ Gd3+ Tb3+ Dy 3+ Ho3+
DA (%)
Pain threshold (s)
Before
After
treat-
treat-
treot-
treat-
ment
ment
ment
ment
3.4 + 0.1 3.3 f 0.1 3.5 f 0.1 3.4 + 0.2 3.3+ 0.1 3.3 + 0.1 3.22 0.1 3.3 f 0.8 3.5?: 0.1 3.2 f 0.1 3.4 + 0.1
3.5 9.3 6.9 7.7 7.9 9.9 8.1 8.1 9.2 9.4 9.5
+ 0.1 + 0.7 f 0.8 f 0.5 + 0.8 + 0.1 + 0.9 f 0.8 f0.4 + 0.6 f 0.5
Be fore
0.5 + 2.0 (10) 90.8 + 8.9 (9) 52.6f 12.7 (9) 66.1 It 6.4 (10) 67.0+ 12.2 (10) 98.6 rl 1.4 (8) 64.5x 14.7 (10) 70.5f 12.2 (9) 72.7 + 12.9 (7) 95.3 + 4.7 (10) 91.9 + 8.1 (9)
3.1 f 3.4 + 3.0 + 3.3 + 3.3 f 3.4 f 3.4+ 3.1 + 3.2f 3.4 f 3.1 f
After
0.1 0.2 0.1 0.2 0.2 0.2 0.1 0.1 0.1 0.2 0.1
2.9 f 3.4 f 3.0f 3.2 + 3.1 + 3.3 + 3.3 f 3.7 + 3.7 f 3.8 + 4.0 +
0.3 0.1 0.1 0.2 0.2 0.1 0.2 0.3 0.1 0.5 0.7
DA W)
-3.8 0.2 -0.6 0.4 -1.8f -1.7 -1.2 8.1 6.7 6.5 13.6
+ 3.2 (10) + 2.4 (8) + 1.5 (8) It 2.2 (10) 2.1 (8) + 2.4 (8) +4.3 (10) + 2.3 (10) f 3.4 (8) f 10.5 (10) + 10.Oa (10)
Saline (10 /.d/mouse) or saline (5 pi/mouse) plus Ca 2+ (5 pi/mouse) injected intraventricularly 15 min before testing for the control. Numbers in parentheses indicate the number of animals used. Each value represents + S.E.M. aSignificantly different from saline, p < 0.05.
TABLE 3 Antagonism of divalent ions on the analgesic effect of lanthanides Ion
Degree of analgesia (9~) Saline
Saline La3+ Ce3+ Prs+ Nd3+ Sm3+ Eu3+ Gd3+ l-bs+ Dy3+ Ho3+ Yj+
1.2 24.4 18.4 28.7 24.6 64.6 17.4 33.3 34.1 16.1 26.4 -3.9
+ + f f + + + + f + + +
Ca2+ 1.6 (9) 3.6 (9) 3.1 (10) 7.0 (20) 3.2 (10) 3.4 (10) 1.4 (10) 5.0 (10) 11.2 (10) 3.1 (10) 4.2 (9) 1.6 ( 10)a
-3.3 -6.0 -4.5 2.8 -3.8 -0.5 -6.9 -5.3 -0.4 -7.6 -8.7 -11.6
Mn2+
Mg2+ + f f + + f f + + + + *
2.7 1.6 0.9 2.6 1.6 2.2 2.4 2.0 2.7 2.6 3.2 1.3
(10)” (10) (10) (10) (10) (9) (10) (10) (10) (10) (10) (lO)a
-4.8 0.3 0.5 3.0 -3.6 13.7 -2.8 2.5 4.4 -8.1 0.8 -6.3
f f + + f + * + f f f +
1.6 1.1 2.3 1.8 3.2 7.0 1.4 1.4 1.9 2.3 3.2 1.7
(lO)a (10) (10) (10) (10) (9) (10) (10) (8) (10) (10) (lO)a
7.8 -11.1 -8.3 -3.0 -11.2 -8.0 -13.2 -8.1 -8.7 -18.1 -12.0 -14.0
+ 1.7 + 2.3 + 1.4 f 3.1 + 1.2 & 1.2 f 2.0 + 1.0 + 2.2 zt 1.9 + 3.2 + 1.6
(8)a (9) (10) (10) (10) (10) (10) (9) (8) (10) (10) (lo)=
The saline or rare earth (4 pmol kg-‘) injected i.vt. The divalent ions (0.0125 pmol/mouse) were injected i.vt. 5 min after administration of the rare earth. The values, except a are significantly different, p < 0.05 or p < 0.01. Each value represents the f S.E.M. Number of mice used shown in parentheses.
405
3.4. Effect of Pr3’ on electroacupuncture of various brain regions
analgesia and change
in 45Ca levels
The test was divided into 6 groups. Each group consisted of 9 - 10 mice. Pr3” was injected intraventricularly 10 min before electroacupuncture, and Ca2+ (3.75 mmol kg-‘) containing 80 FCi kg-’ of 45Ca or naloxone (0.2 mg kg-r) containing 80 j&i kg-’ of 45Ca was injected i.p. 30 min before electroacupuncture. The corresponding control groups of animals received saline of equivalent volume containing the same dose of 45Ca by the same method. The results are shown in Table 4. TABLE
4
Effect of Pr3+ on electroacupuncture brain regions
analgesia
Group
DA
Control
Route
(10)
i. ut.
i.p.
Striatum
Hippocampus
HYPOthalnmus
3.5 f 0.5
4.2 f 0.4
8.2 f 0.4
2.6 * 0.4
3.6 + 0.7
5.7 + 1.0a
32.4
f 7.0b
2.4 I! 0.2
2.6 f 0.3a
3.5 f 0.5b
85.3
+ 3.4b
2.1 + 0.4a
2.6 + 0.4a
4.9 f 0.6b
4.6 + 5.4
4.3 f 0.3
4.6 + 0.6
6.5 f 4.4
5.8 * 0.8”
5.1 f 0.6a
saline saline
46.0
Pr3+ (10)
Prc13
saline
Pr3+ + EA (9)
PrCls
saline
Pr3+ + EA + CaZ+ (10)
PrCls
CaClz
Prc13
naloxone
+ naloxone
x 10e3)
+ 8.1b
saline
Pr3+ + EA
in the 45Ca levels of various
45Ca (cpmlbrain
(%I
saline
EA (10)
and changes
1.0 + 0.5
11.3
f 1.0a
8.5 + 0.9
(9)
Ca*+ (3.75 Note. Pr 3+ (4 pmol kg-‘) was injected i.vt. 10 min before electroacupuncture. mmol kg-‘) containing 80 pCi kg-’ of 45Ca o r naloxone (0.2 mg kg-‘) containing 80 pCi Each value represents kg-’ of 45Ca was injected i.p. 30 min before electroacupuncture. f S.E.M. Numbers in parentheses indicate the numbers of animals used. aSignificantly different from control, p < 0.05. bSignificantly different from control,p < 0.01. EA = electroacupuncture; DA = degree of analgesia.
The above six series of tests show that the saline containing 80 @i kg-’ of 45Ca had no effect on the baseline pain threshold of animals. After intraventricular pminjection of Pr3+, electroacupuncture analgesic effects were greatly enhanced and at the same time, 45Ca levels in the discrete brain regions were greatly reduced. Both Ca2+ and naloxone reversed the analgesic effects of the Pr3+ plus electroacupuncture group, and the 45Ca levels in the discrete brain regions increased, whereas those in the striatum and hippocampus of the CaCl,-treated group, as well as in the hypothalamus of the naloxone-treated group showed no obvious changes. To summarize, alterations in the 45Ca levels in the hypothalamus closely parallel the analgesic effects of the Pr3+ and electroacupuncture.
406
3.5. Comparison of Tb 3’, Pr3’, morphine, electroacupuncture and EGTAinduced analgesia as well as brain Ca” levels The analgesic responses to Tb3+, Pr3+, morphine, electroacupuncture and EGTA with and without naloxone pretreatment were compared. Naloxone, 4 mg kg-‘, in a volume of 0.5 ml/mouse, was injected i.p. 15 min before a treatment with Tb’+, Pr3+, m orphine, EGTA or electroacupuncture. After measurement of the final pain threshold, each of the animals was decapitated immediately and the front segment brain including the hypothalamus was taken out. The brain Ca2+ levels were measured with a specific ion electrode. The results, given in Table 5, show that intraperitoneal pretreatment of naloxone could fully or partly reverse the analgesic effects of Tb3+, Pr3+ morphine or electroacupuncture (the animals displayed obvious nociceptioh) and concurrently increase the brain Ca2+ levels. On the other hand, naloxone had no effect on EGTA-induced analgesia and the brain Ca2+ levels. These findings indicate that Tb3+, Pr3+, morphine and electroacupuncture can produce analgesia by analogous mechanisms. TABLE 5 Comparison of Tba+, Pr3+ EGTA, morphine, and electroacupuncture as well as brain calcium levels Treatment
Naloxone
Saline DA (%I
Morphine (20 pg/mouse) Tb3+ (4 pmol kg-l) Br3+ (4 pmol kg-‘) EGTA (4 pmol kg-l) Electroacupuncture
67.5 80.6 42.9 39.1 70.0
f * + + f
induced analgesia
6.5 8.3 5.8 6.3 7.6
Ca2+ (mg 100 g)
DA (%I
2.78 2.94 3.04 2.35 2.30
1.3 2.9 5.1 38.6 17.8
k f r f f
0.15 0.11 0.09 0.22 0.61
Ca2+ (w 100 g) + + + f +
1.4b 4.2b 6.0a 5.9 5.5=
5.36 4.97 5.37 2.51 5.23
+ f. + f +
0.14b 0.20n 0.22b 0.25 0.2gb
Morphine, Tb*, Pr3+, EGTA were injected i.vt. 10 min after administration of the saline or naloxone (4 mg kg-l) i.p. aSignificantly different from saline, p < 0.05. bSignificantly different from saline, p < 0.01.
3.6. Time course of Ca2’ antagonism of electroacupuncture analgesia and decay of 45Ca levels in the discrete bmin regions In the test under discussion the duration of Ca2+ antagonism of electroacupuncture and the time course of the removal of 45Ca from the discrete brain regions were observed. The test consisted of 6 phases. Each phase involved 8 - 10 mice. Calcium chloride (8 pmol kg-‘) containing 20 /.L!i kg-’ of 45Ca was injected i.vt. 0.5, 2, 4, 6 and 24 h before electroacupuncture. The latency was measured once every 10 min on three occasions, and the average was taken as the baseline threshold. Thirty minutes after electroacupuncture, the latency was again determined. The degree of analgesia was assessed according to the above procedure. After measurement of the final
407
pain threshold, each of the animals was decapitated immediately and the discrete brain regions were dissected. The radioactivity in the solutions was measured. No noticeable changes in the degree of analgesia were found among the corresponding 6 phase control groups. Their values were in the rangeof73*8-81*10. As can be seen in Fig. 1, an injection of Ca2+ 0.5 h before testing greatly decreased the electroacupuncture effects, indicating that the electroacupuncture analgesia was fully antagonized by Ca2+, and the 45Ca levels in the discrete brain regions were increased. The degree of electroacupuncture analgesia was enhanced gradually with time and 45Ca was concurrently removed from the regions. The decreased analgesic effect virtually returned to levels of electroacupuncture control 6 h after intraventricular injection. It was ineffective when Ca2+ was administered intraventricularly 24 h before electroacupuncture testing, and 45Ca levels of the brain regions were hardly detectable. These findings indicate that electroacupuncture analgesia may be associated with a calcium-depleted state within nerve cells or inhibition of Ca2+ movement across cell membranes. Kx-500 7 0 x4oo
-
c B 300 \ v
s
s 3
-
200
-
100
-
O112
2
4
6
time (h
)
24
Fig. 1. Time course of Ca2+ antagonism of electroacupuncture and decay of 45Ca levels in the brain. Ca 2+ (8 pmol kg-‘) containing 20 /.Zi kg-’ of 45Ca (n = 8 - 10 per group) was injected i.vt. 0.5, 2, 4, 6 and 24 h before electroacupuncture. Vertical lines indicate ? S.E.M. Each point, except *, is significantly different, p < 0.05 or p < 0.01. DA = degree of analgesia. 0, hypothalamus; A, hippocampus; 0, striatum.
3.7. Antagonism of calcium on analgesic effects of Tb3’ and Pr3+ The antagonism of Ca2+ (1 - 4 pmol kg-‘) to the analgesic effects of Tb3+ and Pr3+ was observed. Ca2+ and Tb3” or Pr3+ (8 pmol kg-‘) were injected i.vt. simultaneously. The latency before and after treatment was measured once every 10 min on three separate occasions, and the average was taken as the baseline threshold. The degree of analgesia was assessed according to the above procedure. The results are shown in Fig. 2. As can be seen in Fig. 2, the lanthanides Tb3+ and Pr3+themselves had significant analgesic effects which could be antagonized by Ca2+. The degree of antagonism was gradually enhanced with dosage.
408 100
0
N.S.
DA P/J
0
0
I
2 Co’&
4 (pmollkg)
Fig. 2. Antagonism of Ca2+ on analgesic effects of Tb3+ and Pr3+. DA = degree of analgesia. Ca2+ (1 - 4 pmol kg-‘) and Tb3+ or Pr3+ (8 pmol kg-‘) were injected simultaneously. *p < 0.05. $3 < 0.01. n=7 -8.
4. General discussion The biological effects on lanthanides, besides La3+ and Pr3+, have so far not been studied in depth. Ten of the lanthanides presented in Table 1 have marked analgesic effects which might be antagonized by i.vt. or i.p. injection of Ca*+. It was found that intracisternal injection of Ce3+ (0.005 I.tmol kg-‘) did not produce an analgesic effect, whereas a dose of 0.002 pmol kg-’ of La3+ induced an obvious analgesic effect, [5]. The ionic radii of La3’ and Ce3+ are very similar [ 31, but their analgesic effects are different. It was observed that intraventricular injection of the lanthanides shown in Table 3, together with La3+ and Pr3+, also had marked analgesic effects which could be antagonized by divalent cations of Ca*+, Mg*+ or Mn*+. By contrast, Y3+ had no analgesic effect,, and the addition of divalent cations did not lead to any improvement. This indicates that for antinociception the lanthanides indeed possess special properties. On the other hand, the lanthanides Tb3+ and Pr3+, EGTA, morphine and electroacupuncture were shown to enhance the pain threshold without interference of motor coordination. However, naloxone could fully or partly reverse the analgesia of Tb3+, Pr3+, morphine or electroacupuncture and concurrently increased the brain Ca 2+ levels in animals. Naloxone had no effect on EGTA-induced analgesia and the brain Ca*+ levels, however. These findings indicate that there are different calcium pools which will affect analgesic responsiveness in CNS. It has been reported in recent years that additional similarities between La3+ and morphine include: (i) the antinociceptive effects of La3’, (ii) animals tolerant to the effects of morphine are also tolerant to the effects withdrawal in of La3+, (iii) La3+ suppresses both abrupt and precipitated morphine-depend&t mice, and (iv) La3+ and morphine share the same neuroanatomical site of antinociceptive activity, the periaqueductal grey [ 1,6, 71. In the present study, it has been observed that the intraventricular injection of Pr3+ enhances electroacupuncture analgesia, and the effects are antagonized by naloxone and Ca*+. Moreover, the analgesic effects of Pr3+ are very similar to those of La3+ and morphine. In our study, it was found that for
409
analgesic effects both electroacupuncture and morphine probably share the same neuroanatomical site, and thus the analgesic effects of La3+ and the other lanthanides are likely to share the mutual neuroanatomical site and the mechanism of antinociceptive activity with those of morphine and electroacupunctl Ire.
References 1 Zhang Zuxuan, Zhang Yuanzhen, Yu Qixjang, Li Chunbao and Chen Rongsan, Kexue Tongbao, 25 (1980) 872. 2 E. Alneas and R. Rahamimoff, Nature (London), 247 (1974) 478. 3 T. Moeller, The Chemistry of the Lanthanides, Reinhold, New York, 1963. 4 Zhang Zuxuan, Tong Jingbo, Ge Ming and Chen Rongsan, Acta Phys. Sin., 35 (1933) 172. 5 Zhang Zuxuan, Zhang Yuanzhen and Chen Rongsan, Kexue Tongbao, 27 (1979) 471. 6 E. T. Iwamoto, R. A. Harris, H. H. Loh and E. L. Way, J. Pharmacol. Exp. Ther., 206 (1978) 46. 7 R. Rahamimoff and E. Alneas, Proc. N&l. Acad. Sci. U.S.A., 70 (1973) 3613.
STUDIES ZHANG
ON THE ANALGESIC
ZUXUAN,
Department CHEN
ZHANG
401 - 409
EFFECTS
YUANZHEN
and WANG
401
OF LANTHANIDES* XINGUANG
of Biology, Nanjing University (China)
RONGSAN
Coordination (Received
Metals, 112 (1985)
and REN ZHENZHANG
Chemistry Research Institute, Nanjing University (China)
January
10,1985)
Summary The analgesic effects of ten lanthanides have been examined. The intraventricularly administered lanthanides produced a long-lasting analgesia. These effects were antagonized by divalent cations. Yttrium had no analgesic effect and was unaffected by the divalent cations, indicating that the analgesia is possibly a mutual characteristic of the lanthanide series of elements, but not of the rare earths. Naloxone fully or partly reverses the analgesic effects of the lanthanides, morphine or electroacupuncture, and at the same time increases the Ca*+ levels in the brain. Naloxone, however, has no effect on the EGTA-induced analgesia and the Ca *+ levels in the brain. All these findings indicate that the lanthanides are likely to share a mutual antinociceptive site and analgesic mechanism with those of morphine and electroacupuncture.
1. Introduction Our previous studies showed that intracisternally administered lanthanum chloride produces analgesic effects as measured by the mouse tail-flick method, and the characteristics of the analgesic effects are similar to those of morphine and electroacupuncture [ 11. It was found that the actions of Pr3+ in vitro resemble those of La 3+ but the former ion exerts a dual action on the transmitter release of the neuromuscular synapse [2]. There are 15 lanthanides whose chemical and physical properties are very similar [ 31. It follows that other lanthanides, in addition to La and Pr, will probably produce analgesia by the same mechanism. In order to confirm this, we have continued to study the correlation of divalent cations with the lanthanide series of elements emphasis being placed on a comparison of the characteristics of *Paper presented at the International land, March 4 - 8, 1985.
Rare Earth Conference,
0022-5088/85/$3.30
@ Elsevier Sequoia/Printed
ETH Zurich,
Switzer-
in The Netherlands
402
the analgesic effects of Tb3+ and Pr3+ as well as those of EGTA, morphine and electroacupuncture.
2. Experimental
details
Mice weighing 25 - 30 g were used. They were divided at random into a variety of experimental groups. The animals were treated on the basis of the electroacupuncture analgesia model proposed by our laboratory [4]. The isotope 45Ca was prepared in 0.9% NaCl. The agent was administered intraventricularly (20 PCi kg-‘, 0.5 mCi ml-’ of specific radioactivity) or intraperitoneally (80 PCi kg-‘) 10 - 30 min before electroacupuncture. After the final pain threshold was measured, each of the mice was decapitated immediately and the brain was dissected into discrete regions and weighed. Each brain region was put into a vial, then 0.3 ml of H202 and 0.4 ml of formic acid were added to the vial. After incubation at 80 “C for 1 h, and cooling, 5 ml of scintillation fluid (PPO, POPOP) and 4 ml of ethylene glycol ether were added. The radioactivity in the transparent homogenetic solution was determined with an FJ-2101 model, double-channel, liquid scintillation spectrometer. The front segment brain Ca2+ contents were measured with a specific ion electrode [ 51. The chlorides used in the present work were analytical reagents, prepared in saline before use. The lanthanide solution, 1 - 8 pmol kg-’ in a volume of 5 ~1 per mouse, was injected intraventricularly (i.vt.). Naloxone, 0.2 mg kg-’ in a volume of 0.5 ml per mouse, was injected intraperitoneally (i.p.). The corresponding control group of animals received saline of equivalent volume by the same method. The degree of ‘analgesia of either electroacupuncture or the lanthanides was expressed as a percentage of maximum possible effect according to the equation : DA = lOO(TL - BL)/(lO
- BL)
where DA is the degree of analgesia; line latency.
TL, the test latency;
and BL, the base-
3. Results and discussion 3.1. Survey on the analgesic effects of lanthanides In the present experiment, the analgesic effects of La3+, Ce3+, Pr3+, Nd3+, Sm 3+, Eu3+, Gd3+, Tb3+, Dy3+ and Ho3+ were compared. The results are shown in Table 1. These data show that 10 lanthanides of the same dosage (1 - 8 pmol kg-‘) had analgesic effects which were enhanced with increased dosage. Of the 10 lanthanides, Sm3+, Dy3+, Ho3+ and La3+ gave higher analgesic effects and Ce3+ the least.
403 TABLE
1
Comparison
Ion
of the analgesic
effect
of lanthanides
Degree of analgesia (%) 1 pmol kg-’ 1.8 f 1.0 (10)
2 pmol kg-’
4 pmol kg-
1.2 28.8 27.5 20.7 25.9 28.5 19.0 31.2 44.1 22.4 16.0
1.7 84.2 21.8 36.4 30.7 42.0 25.7 70.7 66.2 41.8 15.6
+ + + f f + f f f + t
1.6 (10) 4.4 (8) 5.7 (7) 8.6a (10) 8.6 (10) 12.0 (8) 5.9 (8) 11.8 (10) 13.0 (6) 10.6a (9) 3.0 (10)
-I + f It + f + It f f +
3.1 10.5 11.5 5.8 13.4 13.3 3.4 14.7 16.3 17.2 5.4
1 (10) (8) (8) (10) (9) (8) (10) (9) (6) (8) (9)
8 pmol kg-. 4.9 90.8 52.6 66.1 67.0 96.6 64.5 70.5 72.7 95.3 91.9
f. + + f + + + f f + +
1.8 8.9 12.7 6.4 12.2 1.4 14.7 12.2 12.9 4.7 8.1
1 (10) (9) (9) (1%) (10) (8) (10) (9) (7) (10) (9)
Saline Las+ Ce3+ Bra+ Nd3+ Sm3+ Eu 3+ Gd 3+ Tb3+ Dy3+ Ho3+
18.3 21.6 16.5 17.0 7.9 10.3 15.8 20.8 2.6 20.1
The saline parentheses different,p
and ions were injected intraventricularly 15 min before testing. Numbers in indicate the numbers of animals used. The values, except a are significantly < 0.05. Each value represents the fS.E.M.
f 5.5 (10) Y7.7 (8) f 4.5 (10) f 4.5 (8) f 2.7a (8) zk 3.0 (10) f 3.4 (9) + 7.8 (7) f 2.8 .(lO) + 9.0a (10)
of Ca2+ on analgesic effects of lanthanides The animals were divided into two groups: (i) lanthanide plus saline, (ii) lanthanide plus Ca 2+. The results are shown in Table 2. The above tests show that intraventricular injection of saline had no effect on pain threshold, and after administration of lanthanides the analgesic effects were markedly enhanced, particularly with Sm3+, Dy3+, Ho3+ and La3+. After addition of Ca2+, their analgesic effects were antagonized, and the animals displayed obvious nociception. The pain threshold of the Ho3+ plus Ca2+ group was still higher than that of the control group, but compared with that of the Ho3+ -treated group, it was markedly reduced. The results showed that the antagonism of Ca2+ on the analgesic effects of these 10 lanthanides was remarkable. 3.2. Antagonism
3.3. Antagonism of divalent cations on analgesic effects of lanthanides Yttrium and ten lanthanides were tested in our study. These elements were injected intraventricularly in a concentration of 4 pmol kg-‘. Five minutes later divalent cations such as Ca2+, Mg2+ or Mn2+, 0.0125 mol/mouse, were injected intraventricularly. The results are shown in Table 3. The rare earths apart from yttrium had marked analgesic effects. These effects were antagonized by Ca2+, Mg2+ or Mn’+. Y3+ itself had no analgesic effect and was not affected by the divalent cations. Although the physical and chemical characteristics of Y 3+ are very similar to those of the lanthanides, their pharmacological effects are very different, indicating that the analgesic effect is possibly a mutual characteristic of lanthanides.
404 TABLE 2 Antagonism of CaZ+ on the analgesic effect of lanthanides Ion
8 pmol kg-’
Added Ca2+ 8 Mmol kg-’
Pain threshold (s)
Saline La3+ Ce3+ Bra+ Nd 3+ Sm 3+ Eu3+ Gd3+ Tb3+ Dy 3+ Ho3+
DA (%)
Pain threshold (s)
Before
After
treat-
treat-
treot-
treat-
ment
ment
ment
ment
3.4 + 0.1 3.3 f 0.1 3.5 f 0.1 3.4 + 0.2 3.3+ 0.1 3.3 + 0.1 3.22 0.1 3.3 f 0.8 3.5?: 0.1 3.2 f 0.1 3.4 + 0.1
3.5 9.3 6.9 7.7 7.9 9.9 8.1 8.1 9.2 9.4 9.5
+ 0.1 + 0.7 f 0.8 f 0.5 + 0.8 + 0.1 + 0.9 f 0.8 f0.4 + 0.6 f 0.5
Be fore
0.5 + 2.0 (10) 90.8 + 8.9 (9) 52.6f 12.7 (9) 66.1 It 6.4 (10) 67.0+ 12.2 (10) 98.6 rl 1.4 (8) 64.5x 14.7 (10) 70.5f 12.2 (9) 72.7 + 12.9 (7) 95.3 + 4.7 (10) 91.9 + 8.1 (9)
3.1 f 3.4 + 3.0 + 3.3 + 3.3 f 3.4 f 3.4+ 3.1 + 3.2f 3.4 f 3.1 f
After
0.1 0.2 0.1 0.2 0.2 0.2 0.1 0.1 0.1 0.2 0.1
2.9 f 3.4 f 3.0f 3.2 + 3.1 + 3.3 + 3.3 f 3.7 + 3.7 f 3.8 + 4.0 +
0.3 0.1 0.1 0.2 0.2 0.1 0.2 0.3 0.1 0.5 0.7
DA W)
-3.8 0.2 -0.6 0.4 -1.8f -1.7 -1.2 8.1 6.7 6.5 13.6
+ 3.2 (10) + 2.4 (8) + 1.5 (8) It 2.2 (10) 2.1 (8) + 2.4 (8) +4.3 (10) + 2.3 (10) f 3.4 (8) f 10.5 (10) + 10.Oa (10)
Saline (10 /.d/mouse) or saline (5 pi/mouse) plus Ca 2+ (5 pi/mouse) injected intraventricularly 15 min before testing for the control. Numbers in parentheses indicate the number of animals used. Each value represents + S.E.M. aSignificantly different from saline, p < 0.05.
TABLE 3 Antagonism of divalent ions on the analgesic effect of lanthanides Ion
Degree of analgesia (9~) Saline
Saline La3+ Ce3+ Prs+ Nd3+ Sm3+ Eu3+ Gd3+ l-bs+ Dy3+ Ho3+ Yj+
1.2 24.4 18.4 28.7 24.6 64.6 17.4 33.3 34.1 16.1 26.4 -3.9
+ + f f + + + + f + + +
Ca2+ 1.6 (9) 3.6 (9) 3.1 (10) 7.0 (20) 3.2 (10) 3.4 (10) 1.4 (10) 5.0 (10) 11.2 (10) 3.1 (10) 4.2 (9) 1.6 ( 10)a
-3.3 -6.0 -4.5 2.8 -3.8 -0.5 -6.9 -5.3 -0.4 -7.6 -8.7 -11.6
Mn2+
Mg2+ + f f + + f f + + + + *
2.7 1.6 0.9 2.6 1.6 2.2 2.4 2.0 2.7 2.6 3.2 1.3
(10)” (10) (10) (10) (10) (9) (10) (10) (10) (10) (10) (lO)a
-4.8 0.3 0.5 3.0 -3.6 13.7 -2.8 2.5 4.4 -8.1 0.8 -6.3
f f + + f + * + f f f +
1.6 1.1 2.3 1.8 3.2 7.0 1.4 1.4 1.9 2.3 3.2 1.7
(lO)a (10) (10) (10) (10) (9) (10) (10) (8) (10) (10) (lO)a
7.8 -11.1 -8.3 -3.0 -11.2 -8.0 -13.2 -8.1 -8.7 -18.1 -12.0 -14.0
+ 1.7 + 2.3 + 1.4 f 3.1 + 1.2 & 1.2 f 2.0 + 1.0 + 2.2 zt 1.9 + 3.2 + 1.6
(8)a (9) (10) (10) (10) (10) (10) (9) (8) (10) (10) (lo)=
The saline or rare earth (4 pmol kg-‘) injected i.vt. The divalent ions (0.0125 pmol/mouse) were injected i.vt. 5 min after administration of the rare earth. The values, except a are significantly different, p < 0.05 or p < 0.01. Each value represents the f S.E.M. Number of mice used shown in parentheses.
405
3.4. Effect of Pr3’ on electroacupuncture of various brain regions
analgesia and change
in 45Ca levels
The test was divided into 6 groups. Each group consisted of 9 - 10 mice. Pr3” was injected intraventricularly 10 min before electroacupuncture, and Ca2+ (3.75 mmol kg-‘) containing 80 FCi kg-’ of 45Ca or naloxone (0.2 mg kg-r) containing 80 j&i kg-’ of 45Ca was injected i.p. 30 min before electroacupuncture. The corresponding control groups of animals received saline of equivalent volume containing the same dose of 45Ca by the same method. The results are shown in Table 4. TABLE
4
Effect of Pr3+ on electroacupuncture brain regions
analgesia
Group
DA
Control
Route
(10)
i. ut.
i.p.
Striatum
Hippocampus
HYPOthalnmus
3.5 f 0.5
4.2 f 0.4
8.2 f 0.4
2.6 * 0.4
3.6 + 0.7
5.7 + 1.0a
32.4
f 7.0b
2.4 I! 0.2
2.6 f 0.3a
3.5 f 0.5b
85.3
+ 3.4b
2.1 + 0.4a
2.6 + 0.4a
4.9 f 0.6b
4.6 + 5.4
4.3 f 0.3
4.6 + 0.6
6.5 f 4.4
5.8 * 0.8”
5.1 f 0.6a
saline saline
46.0
Pr3+ (10)
Prc13
saline
Pr3+ + EA (9)
PrCls
saline
Pr3+ + EA + CaZ+ (10)
PrCls
CaClz
Prc13
naloxone
+ naloxone
x 10e3)
+ 8.1b
saline
Pr3+ + EA
in the 45Ca levels of various
45Ca (cpmlbrain
(%I
saline
EA (10)
and changes
1.0 + 0.5
11.3
f 1.0a
8.5 + 0.9
(9)
Ca*+ (3.75 Note. Pr 3+ (4 pmol kg-‘) was injected i.vt. 10 min before electroacupuncture. mmol kg-‘) containing 80 pCi kg-’ of 45Ca o r naloxone (0.2 mg kg-‘) containing 80 pCi Each value represents kg-’ of 45Ca was injected i.p. 30 min before electroacupuncture. f S.E.M. Numbers in parentheses indicate the numbers of animals used. aSignificantly different from control, p < 0.05. bSignificantly different from control,p < 0.01. EA = electroacupuncture; DA = degree of analgesia.
The above six series of tests show that the saline containing 80 @i kg-’ of 45Ca had no effect on the baseline pain threshold of animals. After intraventricular pminjection of Pr3+, electroacupuncture analgesic effects were greatly enhanced and at the same time, 45Ca levels in the discrete brain regions were greatly reduced. Both Ca2+ and naloxone reversed the analgesic effects of the Pr3+ plus electroacupuncture group, and the 45Ca levels in the discrete brain regions increased, whereas those in the striatum and hippocampus of the CaCl,-treated group, as well as in the hypothalamus of the naloxone-treated group showed no obvious changes. To summarize, alterations in the 45Ca levels in the hypothalamus closely parallel the analgesic effects of the Pr3+ and electroacupuncture.
406
3.5. Comparison of Tb 3’, Pr3’, morphine, electroacupuncture and EGTAinduced analgesia as well as brain Ca” levels The analgesic responses to Tb3+, Pr3+, morphine, electroacupuncture and EGTA with and without naloxone pretreatment were compared. Naloxone, 4 mg kg-‘, in a volume of 0.5 ml/mouse, was injected i.p. 15 min before a treatment with Tb’+, Pr3+, m orphine, EGTA or electroacupuncture. After measurement of the final pain threshold, each of the animals was decapitated immediately and the front segment brain including the hypothalamus was taken out. The brain Ca2+ levels were measured with a specific ion electrode. The results, given in Table 5, show that intraperitoneal pretreatment of naloxone could fully or partly reverse the analgesic effects of Tb3+, Pr3+ morphine or electroacupuncture (the animals displayed obvious nociceptioh) and concurrently increase the brain Ca2+ levels. On the other hand, naloxone had no effect on EGTA-induced analgesia and the brain Ca2+ levels. These findings indicate that Tb3+, Pr3+, morphine and electroacupuncture can produce analgesia by analogous mechanisms. TABLE 5 Comparison of Tba+, Pr3+ EGTA, morphine, and electroacupuncture as well as brain calcium levels Treatment
Naloxone
Saline DA (%I
Morphine (20 pg/mouse) Tb3+ (4 pmol kg-l) Br3+ (4 pmol kg-‘) EGTA (4 pmol kg-l) Electroacupuncture
67.5 80.6 42.9 39.1 70.0
f * + + f
induced analgesia
6.5 8.3 5.8 6.3 7.6
Ca2+ (mg 100 g)
DA (%I
2.78 2.94 3.04 2.35 2.30
1.3 2.9 5.1 38.6 17.8
k f r f f
0.15 0.11 0.09 0.22 0.61
Ca2+ (w 100 g) + + + f +
1.4b 4.2b 6.0a 5.9 5.5=
5.36 4.97 5.37 2.51 5.23
+ f. + f +
0.14b 0.20n 0.22b 0.25 0.2gb
Morphine, Tb*, Pr3+, EGTA were injected i.vt. 10 min after administration of the saline or naloxone (4 mg kg-l) i.p. aSignificantly different from saline, p < 0.05. bSignificantly different from saline, p < 0.01.
3.6. Time course of Ca2’ antagonism of electroacupuncture analgesia and decay of 45Ca levels in the discrete bmin regions In the test under discussion the duration of Ca2+ antagonism of electroacupuncture and the time course of the removal of 45Ca from the discrete brain regions were observed. The test consisted of 6 phases. Each phase involved 8 - 10 mice. Calcium chloride (8 pmol kg-‘) containing 20 /.L!i kg-’ of 45Ca was injected i.vt. 0.5, 2, 4, 6 and 24 h before electroacupuncture. The latency was measured once every 10 min on three occasions, and the average was taken as the baseline threshold. Thirty minutes after electroacupuncture, the latency was again determined. The degree of analgesia was assessed according to the above procedure. After measurement of the final
407
pain threshold, each of the animals was decapitated immediately and the discrete brain regions were dissected. The radioactivity in the solutions was measured. No noticeable changes in the degree of analgesia were found among the corresponding 6 phase control groups. Their values were in the rangeof73*8-81*10. As can be seen in Fig. 1, an injection of Ca2+ 0.5 h before testing greatly decreased the electroacupuncture effects, indicating that the electroacupuncture analgesia was fully antagonized by Ca2+, and the 45Ca levels in the discrete brain regions were increased. The degree of electroacupuncture analgesia was enhanced gradually with time and 45Ca was concurrently removed from the regions. The decreased analgesic effect virtually returned to levels of electroacupuncture control 6 h after intraventricular injection. It was ineffective when Ca2+ was administered intraventricularly 24 h before electroacupuncture testing, and 45Ca levels of the brain regions were hardly detectable. These findings indicate that electroacupuncture analgesia may be associated with a calcium-depleted state within nerve cells or inhibition of Ca2+ movement across cell membranes. Kx-500 7 0 x4oo
-
c B 300 \ v
s
s 3
-
200
-
100
-
O112
2
4
6
time (h
)
24
Fig. 1. Time course of Ca2+ antagonism of electroacupuncture and decay of 45Ca levels in the brain. Ca 2+ (8 pmol kg-‘) containing 20 /.Zi kg-’ of 45Ca (n = 8 - 10 per group) was injected i.vt. 0.5, 2, 4, 6 and 24 h before electroacupuncture. Vertical lines indicate ? S.E.M. Each point, except *, is significantly different, p < 0.05 or p < 0.01. DA = degree of analgesia. 0, hypothalamus; A, hippocampus; 0, striatum.
3.7. Antagonism of calcium on analgesic effects of Tb3’ and Pr3+ The antagonism of Ca2+ (1 - 4 pmol kg-‘) to the analgesic effects of Tb3+ and Pr3+ was observed. Ca2+ and Tb3” or Pr3+ (8 pmol kg-‘) were injected i.vt. simultaneously. The latency before and after treatment was measured once every 10 min on three separate occasions, and the average was taken as the baseline threshold. The degree of analgesia was assessed according to the above procedure. The results are shown in Fig. 2. As can be seen in Fig. 2, the lanthanides Tb3+ and Pr3+themselves had significant analgesic effects which could be antagonized by Ca2+. The degree of antagonism was gradually enhanced with dosage.
408 100
0
N.S.
DA P/J
0
0
I
2 Co’&
4 (pmollkg)
Fig. 2. Antagonism of Ca2+ on analgesic effects of Tb3+ and Pr3+. DA = degree of analgesia. Ca2+ (1 - 4 pmol kg-‘) and Tb3+ or Pr3+ (8 pmol kg-‘) were injected simultaneously. *p < 0.05. $3 < 0.01. n=7 -8.
4. General discussion The biological effects on lanthanides, besides La3+ and Pr3+, have so far not been studied in depth. Ten of the lanthanides presented in Table 1 have marked analgesic effects which might be antagonized by i.vt. or i.p. injection of Ca*+. It was found that intracisternal injection of Ce3+ (0.005 I.tmol kg-‘) did not produce an analgesic effect, whereas a dose of 0.002 pmol kg-’ of La3+ induced an obvious analgesic effect, [5]. The ionic radii of La3’ and Ce3+ are very similar [ 31, but their analgesic effects are different. It was observed that intraventricular injection of the lanthanides shown in Table 3, together with La3+ and Pr3+, also had marked analgesic effects which could be antagonized by divalent cations of Ca*+, Mg*+ or Mn*+. By contrast, Y3+ had no analgesic effect,, and the addition of divalent cations did not lead to any improvement. This indicates that for antinociception the lanthanides indeed possess special properties. On the other hand, the lanthanides Tb3+ and Pr3+, EGTA, morphine and electroacupuncture were shown to enhance the pain threshold without interference of motor coordination. However, naloxone could fully or partly reverse the analgesia of Tb3+, Pr3+, morphine or electroacupuncture and concurrently increased the brain Ca 2+ levels in animals. Naloxone had no effect on EGTA-induced analgesia and the brain Ca*+ levels, however. These findings indicate that there are different calcium pools which will affect analgesic responsiveness in CNS. It has been reported in recent years that additional similarities between La3+ and morphine include: (i) the antinociceptive effects of La3’, (ii) animals tolerant to the effects of morphine are also tolerant to the effects withdrawal in of La3+, (iii) La3+ suppresses both abrupt and precipitated morphine-depend&t mice, and (iv) La3+ and morphine share the same neuroanatomical site of antinociceptive activity, the periaqueductal grey [ 1,6, 71. In the present study, it has been observed that the intraventricular injection of Pr3+ enhances electroacupuncture analgesia, and the effects are antagonized by naloxone and Ca*+. Moreover, the analgesic effects of Pr3+ are very similar to those of La3+ and morphine. In our study, it was found that for
409
analgesic effects both electroacupuncture and morphine probably share the same neuroanatomical site, and thus the analgesic effects of La3+ and the other lanthanides are likely to share the mutual neuroanatomical site and the mechanism of antinociceptive activity with those of morphine and electroacupunctl Ire.
References 1 Zhang Zuxuan, Zhang Yuanzhen, Yu Qixjang, Li Chunbao and Chen Rongsan, Kexue Tongbao, 25 (1980) 872. 2 E. Alneas and R. Rahamimoff, Nature (London), 247 (1974) 478. 3 T. Moeller, The Chemistry of the Lanthanides, Reinhold, New York, 1963. 4 Zhang Zuxuan, Tong Jingbo, Ge Ming and Chen Rongsan, Acta Phys. Sin., 35 (1933) 172. 5 Zhang Zuxuan, Zhang Yuanzhen and Chen Rongsan, Kexue Tongbao, 27 (1979) 471. 6 E. T. Iwamoto, R. A. Harris, H. H. Loh and E. L. Way, J. Pharmacol. Exp. Ther., 206 (1978) 46. 7 R. Rahamimoff and E. Alneas, Proc. N&l. Acad. Sci. U.S.A., 70 (1973) 3613.