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Effects of calcium antagonists on biogenic amines in discrete brain areas
European Journal of Pharmacology, 181 (1990) 187-197
187
Elsevier EJP 51328
Effects of calcium antagonists on biogenic amines in discrete brain areas Renato Gaggi and Anna Maria Gianni Institute of Pharmacology, Unioersityof Bologna, via Irnerio 48, 40126 Bologna, ltaly
Received 12 October 1989, revised MS received 19 February 1990, accepted 13 March 1990
Discrete brain sections were obtained from rats given i.p. verapamil, nifedipine, diltiazem or flunarizine (0, 10, 20 or 40 mg/kg). The biogenic amines and metabolites in the hypothalamus, brainstem, hippocampus, striatum, thalamus-midbrain and cortex were determined by high-performance liquid chromatography with electrochemical detection. The treatments induced several changes in the levels of neurotransmitters and metabolites, displaying regional specificity and differences according to the various compounds. It was speculated that some effects could have been due to indirect actions and/or to interactions of the compounds with receptors other than the voltage-sensitive calcium channels. However blockade of these channels could account for the following effects. (a) The nifedipine-induced increases in the 5-hydroxy-3-indoleacetic acid levels and, in general, the signs of activation of the serotonergic systems. (b) The fall in the 3,4-dihydroxyphenylacetic acid levels and, in general, the signs of attenuation of dopaminergic neurotransmission induced by nifedipine, verapamil and diltiazem. (c) The fall in the norepinephrine levels induced by all the compounds studied. C a 2+
channel antagonists; Brain sections (rat); Biogenic amines; (Biogenic amine metabolites)
1. Introduction The calcium antagonists have been recently reclassified on the basis of their effects in a number of tests. There are now two main categories, each including three subtypes (Paoletti and Govoni, 1987; Vanhoutte and Paoletti, 1987). The first category, regarding drugs selective for slow Ca 2+ channels, includes the verapamil, nifedipine and diltiazem-like subtypes. The second category includes the flunarizine-like subtype and the other two subtypes which include non-selective calcium entry blockers. Flunarizine is considered poorly selective for the voltage-sensitive calcium channel (VSCC) since it also inhibits Ca 2 ÷ influx through receptor-operated channels (Holmes et al., 1984).
Correspondence to: R. Gaggi, Institute of Pharmacology, Via Irnerio 48, 1-40126-Bologna, Italy.
Verapamil, nifedipine, diltiazem and flunarizine recognize specific binding sites in the macromolecular complex of the VSCC in various tissues and modulate [3H]nitrendipine binding to this complex (Godfraind et al., 1986). Verapamil, nifedipine and flunarizine inhibit, whereas diltiazero facilitates (Boles et al., 1984), [3H]nitrendipine binding to its receptor. The highest density of [3H]nitrendipine binding sites in the body occurs in the brain (Gould et al., 1982). It is now generally accepted that these sites, which are not exclusively associated with blood vessels (Murphy et al., 1982), represent functional VSCC (Miller, 1987). It has been hypothesized that the receptors for [3H]nitrendipine in the central nervous system are L channels. This type of channel, being preferentially localized in the cell soma, seems to be involved in the regulation of neuronal functions other than the generation of action potentials or the release of neurotransmitters (Miller, 1987).
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
mg/kg
2 2 2 2 4 8
2.26 4- 0.143 2.10±0.083 1.81 4- 0.091 a 1.824-0.068 a 1.94 4- 0.100 2.46 4- 0.102
NE
2 2 2 2 4 8
10 20 40 20 20
2 2 2 4 8
Hippocampus 0 2
0 10 20 40 20 20
0.35 + 0.026 0.32 ± 0.027 0.28 4- 0.018 0.28 4- 0.017 0.28 4- 0.020 0.34 4- 0.022
0.74 + 0.032 0.64 4- 0.030 a 0.62 4- 0.025 a 0.63 4- 0.028 a 0.68 4- 0.026 0.73 4- 0.022
0.055 + 0.007 0.062 4- 0.007 0.082 4- 0.004 a 0.0794-0.003 ~ 0.045 4- 0.003 0.053 ± 0.008
0.36 + 0.011 0.37+0.009 0.41 4- 0.023 0.45+0.017 ~ 0.33 4- 0.019 0.36 ± 0.016
DA
Tissue levels: ~ g / g wet weight
Brainstem (pons + medulla)
0 10 20 40 20 20
Hypothalamus
Hours
before killing
Doses
0.080 ___0.009 0.0674-0.010 0.064 + 0.005 0.073+0.006 0.067 4- 0.005 0.074 + 0.006
DOPAC
HVA
0.41 + 0.031 0.44 __.0.056 0.40 ± 0.049 0.41 +0.038 0.40 4- 0.024 0.47 4- 0.014
0.76 ± 0.056 0.66 ± 0.030 0.70 4- 0.031 0.744-0.026 0.71 ± 0.048 0.80 4- 0.034
1.07 4- 0.075 1.01 ± 0.049 0.93 4- 0.030 1.054-0.051 1.04 4- 0.055 1.14 4- 0.055
5-HT
0.29 4- 0.010 0.33 + 0.031 0.39 ± 0.022 a 0.454-0.040 b 0.39 + 0.020 a 0.33 4- 0.011
0.40 ± 0.031 0.40 4- 0.041 0.47 4- 0.032 0.53+_0.030 a 0.46 ± 0.053 0.39 4- 0.014
0.53 + 0.021 0.604-0.039 0.64 ± 0.029 ~ 0.724-0.025 b 0.60 4- 0.030 0.58 4- 0.010
5-HIAA
22 18 16 16 20 21
DA x 100
DOPAC
DA x 100
HVA
5-HT 100
70 76 98 110 97 70
52 61 67 72 65 49
50 59 69 68 58 51
x
5-HIAA
Content of biogenic amines and metabolites in brain sections obtained from rats treated with nifedipine. The values are means for six rats ± S.E. a p < 0.05, t, p < 0.01 significantly different from the respective control (two-tailed Dunnett's t-test).
TABLE 1
O0
+ midbrain 2 2 2 2 4 8
2 2 2 2 4 8
Cortex 0 10 20 4O 20 20
Thalamus 0 10 20 40 2O 2O
2 2 2 2 4 8
Striatum 0 10 2O 4O 20 2O b b b a
0.62 + 0.037 0.52 4- 0.026 0.45 5=0.029 a 0.52 4- 0.049 0.47 5=0.028 a 0.58 + 0.050
0.31 5:0.022 0.27 4- 0.014 0.244-0.016 a 0.25 4- 0.017 0.25 4- 0.014 0.34 4- 0.017
0.31 4- 0.019 0.23 5=0.014 0.22 4- 0.022 0.204-0.012 0.244-0.013 0.28 + 0.011
0.22 4- 0.026 0.20 4- 0.015 0.184-0.015 0.28 4- 0.030 0.18_+0.013 0.22 + 0.019
0.43 4- 0.026 0.38 5:0.027 0.41 4- 0.049 0.38 5=0.043 0.33 5=0.027 0.42 4- 0.027
9.66 4- 0.37 8.47 4- 0.45 9.31 4- 0.47 9.54 4- 0.21 8.514-0.39 10.04_+0.18
0.14 4- 0.009 0.134-0.011 0.104-0.002 " 0.11 4-0.009 0.134-0.008 0.18 4- 0.012 a
0.12 4- 0.014 0.12 4- 0.012 0.10 4- 0.008 0.13_+0.017 0.13_+0.018 0.19 4- 0.013 b
1.30 4- 0.070 1.06 4- 0.055 a 1.01 4- 0.050 a 1.00 4- 0.021 b 1.06 4- 0.089 a 1.36 4- 0.062
0.062 4- 0.012 0.057 4- 0.021 0.070 + 0.024 0.068 + 0.018 0.080 + 0.011 0.084 4- 0.016
0.048 4- 0.005 0.051 4- 0.005 0.046 4- 0.005 0.050 4- 0.006 0.048 4- 0.004 0.053 4- 0.006
0.57 4- 0.013
0.48 4- 0.033 b
0.70 + 0.042 0.55 4- 0.056 a 0.49 4- 0.026 b 0.45 4- 0.042 b
0.72 4- 0.035 0.65 + 0.037 0.64 + 0.032 0.74 + 0.063 0.63 4- 0.047 0.82 + 0.074
0.36 4- 0.033 0.39 4- 0.039 0.33 4- 0.023 0.35 4- 0.026 0.32 4- 0.016 0.43 4- 0.042
0.56 4- 0.033 0.50 4- 0.024 0.50 4- 0.012 0.58 4- 0.028 0.52 + 0.035 0.58 4- 0.026
0.52 4- 0.026 0.53 + 0.045 0.59 4- 0.032 0.82 4- 0.089 b 0.58 4- 0.036 0.60 4- 0.039
0.20 + 0.014 0.20 4- 0.015 0.21 + 0.014 0.23 4- 0.015 0.19 4- 0.010 0.24 + 0.022
0.45 4- 0.042 0.43 4- 0.042 0.51 4- 0.038 0.60 4- 0.033 a 0.48 4- 0.048 0.51 4-0.018
63 67 55 39 74 76
27 31 25 35 38 44
13 13 11 10 13 19
7.2 6.5 5.3 4.8 5.7 5.7
72 82 92 111 92 72
80 86 102 103 92 88
before killing
mg/kg
2 2 2 2 4 8
2.29+0.123 2.30_+0.081 2.19+0.128 1.82_+0.177 2.28_+0.155 2.31_+0.102
NE
2 2 2 2 4 8
2 2 2 2 4 8
2 2 2 2 8 8
0 10 20 40 20 20
2 2 2 2 4 8
Thalamus + midbrain
0 10 20 40 20 20
Striatum
0 10 20 40 20 20
Hippocampus
0 10 20 40 20 20
0.64_+0.021 0.63-+0.010 0.58_+0.014 0.55_+0.030 0.61_+0.037 0.64_+0.040
0.33_+0.016 0.24 + 0.011 0.23 +0.021 0.19_+0.007 0.25_+0.010 0.30_+0.006 b b b b
0.32_+0.013 0.31 _+0.021 0.27 _+0.010 0.25 -I-0.012 a 0.26 _+0.018 0.27 _+0.015
0.73 + 0.031 0.72 _+0.019 0.65 + 0.011 0.54 5:0.022 b 0.63 _+0.040 0.77 _+0.030
+0.007 _+0.004 -+0.020 _+0.043 b _+0.010 _+0.026
0.24 0.20 0.23 0.21 0.20 0.23
9.40 9.08 8.83 8.33 8.51 8.67
4-0.015 -+0.006 _+0.019 _+0.008 _+0.008 _+0.014
_+0.46 _+0.21 -+0.36 _+0.56 _+0.33 _+0.16
0.063 -+0.003 0.052 _+0.004 0.058 -+0.002 0.080_+0.008 0.052 _+0.004 0.059 _+0.004
0.30 0.33 0.35 0.46 0,32 0.33
DA
Tissue levels: /xg/g wet weight
Brainstem (pons + medulla)
0 10 20 40 20 20
Hypothalamus
Hours
Doses
0.13 0.10 0.12 0.12 0.11 0.13
1.51 1.36 1.31 1.28 1.11 1.02
+0.007 -I-0.002 b _+0.006 _+0.004 _+0.007 " _+0.008
_+0.125 _+0.035 -+0.059 _+0.084 +0.066 b -t-0.033 b
0.042-+0.007 0.046_+0.008 0.038-+0.009 0.086-+0.021 0.067_+0.018 0.057_+0.015
DOPAC
_+0.051 _+0.037 +0.044 _+0.056 _+0.038 +0.027
0,058_+0.015 0.047-+0.012 0.063_+0.010 0.070_+0.022 0.050_+0.019 0.064_+0.014
0.69 0.64 0.62 0.58 0.64 0.58
HVA
0.75+0.053 0.68__.0.037 0.61 _+0.034 0.66_+0.026 0.71_+0.037 0.71_+0.027
0.57_+0.025 0.59+0.021 0.56-+0.020 0.53_+0.019 0.60_+0.022 0.55-+0.030
0.37_+0.014 0.35_+0.024 0.33_+0.011 0.35_+0.010 0.34_+0.016 0.34 + 0.019
0.71 _+0.020 0.69_+0.025 0.68 _+0.024 0.70 _+0.022 0.71 _+0.027 0.71_+0.026
1.01_+0.028 1.02_+0.030 1.07_+0.053 1.14_+0.040 1.06_+0.025 1.00_+0.038
5-HT
0.53_+0.026 0.46-+0.029 0.56_+0.051 0.56_+0.025 0.68_+0,024 0.63_+0.031
0.46_+0.013 0.43+0.026 0.47-t-0.022 0.48_+0.021 0.48_+0.029 0.40-+0.014
0.31_+0.025 0.23_+0.016 0.30_+0.027 0.29_+0.012 0.34_+0.021 0.26 _+0.007
0.40 _+0.021 0.35+0.016 0.40 _+0.019 0.39 _+0.004 0.43 _+0.025 0.41_+0.013
0.58_+0.011 0.53_+0.027 0.58_+0.024 0.60_+0.018 0.62_+0.025 0.63_+0.021
5-HIAA
"
55 51 51 55 53 54
16 15 15 15 13 12
14 14 11 19 21 17
DA × 100
DOPAC DA × 100
HVA
70 67 84 85 96 88
84 64 91 83 98 75
5-HT × 100
5-HIAA
Content of biogenic amines and metabolites in brain sections obtained from rats treated with verapamil. The values are means for six rats_+ S.E. a P < 0.05, b p < 0.01 significantly different from the respective control (two-tailed Dunnett's t-test).
TABLE 2 o
191 On the other hand the calcium antagonists exert several actions in the central nervous system, e.g., flunarizine exhibits anticonvulsant properties in experimental models of epilepsy (Desmedt et al., 1975), nifedipine blocks sleep induction by flurazepam in the rat (Mendelson et al., 1984), verapamil, diltiazem and flunarizine increase morphine-induced analgesia (Del Pozo et al., 1987), nifedipine, flunarizine and verapamil antagonize morphine-tolerance in mice (Contreras et al., 1988), and verapamil and nimodipine suppress the morphine-withdrawal syndrome in rats (Bongianni et al., 1986). We now investigate the effects of a phenylalkylamine (verapamil), a dihydropyridine (nifedipine), a benzothiazepine (diltiazem) and a diphenylpiperazine (flunarizine). These represent, respectively, calcium antagonists of the first four subtypes on the biogenic amine content of discrete rat brain sections.
2. Material and methods
was analyzed on the same day, repeating complete replications sequentially.
2.2. Drugs and treatments Nifedipine and diltiazem hydrochloride were kindly supplied by Schiapparelli Farmaceutici (Turin, Italy). Verapamil hydrochloride was from Sigma Chemical Co (S. Louis, MO, U.S.A.) and flunarizine hydrochloride was kindly supplied by Italfarmaco S.p.A. (Milan, Italy). Verapamil and diltiazem were dissolved with saline while nifedipine and flunarizine were suspended in 1% Tween 80. The dose per kg body weight was contained in 3.0 ml. All the drugs were administered intraperitoneally. Two groups of six rats were treated with 20 mg/kg of nifedipine 8 or 4 h before killing. Another four groups of six rats were treated, 2 h before killing with nifedipine 0 (vehicle), 10, 20 or 40 mg/kg. The procedure was repeated with the hydrochloride salt of verapamil, diltiazem or flunarizine.
2.3. Analytical procedures 2.1. Animals and general procedure A total of 144 male Sprague-Dawley rats (Nossan, Milano, Italy) weighing 180-200 g were used. They were maintained on standard laboratory food and controlled conditions of light (7:00-19:00 h), temperature (22 __+2°C) and humidity (60%). On the day of the experiment all the rats were deprived of food at 9:00 h while water was allowed ad libitum. To avoid circadian variations in the brain content of biogenic amines, all rats were decapitated in the afternoon (16:00-18:00 h). The brain was rapidly removed and ice-cooled. The cerebellum and the olfactory tubercles were discarded whereas the hypothalamus, the hippocampus, the striatum, the brainstem (pons plus medulla oblongata) and the cortex were dissected, immediately frozen with pulverised dry ice and kept at - 2 5 ° C until the time of analysis. The remaining cerebral tissue, consisting mostly of the thalamus and midbrain, was also collected, frozen and analyzed. All the samples from the same experiment were analyzed in the 10-15 days after collection. The same brain section of each animal
The determination of norepinephrine (NE), dopamine (DA), 5-hydroxytryptamine (5-HT) and the metabolites, dihydroxphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) was performed simultaneously by high-performance liquid chromatography (HPLC) with electrochemical detection (Wagner et al., 1982; Seegal et al., 1986; Gaggi et al., 1989). The HPLC system consisted of a P 330 pump (Violet, Rome, Italy) linked to a Rheodyne 7125 injector. The column (ODS1-C18; 10 /zm; 25 cm x 4 mm i.d.) and the pre-column (ODS1-C18; 10/~m; 5 cm x 4 mm i.d.) were also from Violet (Rome, Italy). The electrochemical detector (Eldec 103, Chromatofield, Chateauneuf-Les Martigues, France) was connected with a TRIO integrator (Trivector Systems Int. Ltd., Sandy, England). The potential of the glassy-carbon electrode versus silver-silver chloride reference electrode was maintained at + 0.8 V. The mobile phase consisted of a 840 : 160 mixture of 0.1 M NaH2PO 4 and methanol. The mixture was made 2.6 x 10 -3 M in octyl sulfate,
mg/kg
0 10 20 40 20 20
Cortex
0 10 20 40 20 20
Striatum
0 10 20 40 20 20
2 2 2 2 8 8
2 2 2 2 4 8
2 2 2 2 4 8
Hypothalamus
Hours
before killing
Doses
0.34+-0.030 0.225:0,011 t, 0.21+-0.017 b 0.20+- 0.014 t' 0.21 + 0.018 b 0.22 +-0.022 b
0.32+-0.015 0.275:0.016 0.34+-0.017 0.35+-0.019 0.295:0.012 0.365:0.014
2.30 5:0,087 2.18 + 0.038 2.35 +-0.088 1.83 +0.120 b 2.22 5:0.126 1.94 + 0.099
NE
0.45+-0.052 0.64+-0.048 b 0.525:0.049 0.52+-0.028 0.46+-0.036 0.57+0.017
9.04+-0.28 8.71 5:0.29 9.435:0.53 9.35+-0.43 8.40+-0.18 10.05+-0.52
0.36 + 0.024 0.29 +-0.018 0.30 +- 0.021 0.31+-0.030 0.36 +-0.039 0.37 +- 0.023
DA
Tissue levels: /xg/g wet weight
0.13 0.14 0.14 0.11 0.10 0.11
1.46 1.32 1.29 1.37 1.17 1.50
_ 0.011 +-0.013 +-0.009 +-0.008 +-0.008 +-0.008
+-0.110 +-0.042 +-0.086 +-0.081 +-0.059 a +-0.036
0.046 +-0.003 0.036 +-0.003 0.037 + 0.007 0.025+-0.006 0.033 +-0.007 0.030 +-0.005
DOPAC
_+0.029 5:0.036 +-0.064 +-0.068 +0.034 _+0.042 a
0.053 + 0.003 0.0745:0.007 a 0.0595:0.005 0.0625:0.005 0.0505:0.007 0.0695:0.007
0.69 0.67 0.72 0.79 0.65 0.87
HVA
0.43 + 0.034 0.39+0.022 0.33+0.019 b 0.36+0.016 0.33+0.019 b 0.35+0.019
0.60+--0.019 0.48 + 0.022 b 0.52+-0.021 0.55+-0.031 0.535:0.036 0.55+-0.016
0.95+-0.029 0.98+-0.027 0.95+-0.036 0.89+-0.008 0.93 + 0.041 0.94+-0.030
5-HT
0.23 + 0.014 0.245:0.006 0.195:0.008 a 0.19+0.011 a 0.185:0.011 a 0.21+0.014
0.48+-0.041 0.50 +- 0.032 0.42 +-0.021 0.46+-0.057 0.42+-0.015 0.49+-0.018
0.57+-0.018 0.60+-0.018 0.52+-0.029 0.54+-0.018 0.54 +- 0.031 0.59+-0.032
5-HIAA
29 22 26 20 21 19
16 15 14 15 14 15
x
DA 100
DOPAC
12 12 11 12 11 12
7.7 7.7 7.6 8.4 7.8 8.7
DA x 100
HVA
5-HT 100
54 61 56 52 55 58
79 103 81 84 79 90
x
5-HIAA
Content of biogenic amines and metabolites in brain sections obtained from rats treated with diltiazem. The values are means for six rats+- S.E. a P < 0.05, ~' P < 0.01 significantly different from the respective control (two-tailed Dunnett's t-test).
TABLE 3
193 10 -4 M in E D T A and 2.5 × 10 -4 M in triethylamine and the p H was adjusted to 3.4 with 3 M H3PO 4. The flow rate was 1.5 m l / m i n . At the time of the analysis a frozen aliquot of a standard mixture was thawed and diluted 10 times with the medium used for the preparation of the tissue samples. This medium consisted of a freshly prepared solution 0.2 M in HC104, 2.7 × 10 -4 M E D T A and 1.6 × 10 -4 M in ascorbic acid. The frozen brain sections were weighed and immediately disrupted by ultrasound in 0.5-1.0 ml of the medium. After centrifugation for 10 rain at 11 000 × g, 20 #1 of the supernatant were injected into the H P L C apparatus. D H B A was used as an internal standard.
2.4. Statistical analysis Analysis of variance (ANOVA) was applied to the data, followed by the two-tailed Dunnett's t-test to compare individual groups to the control. Linear correlation ratios between an effect and the log of the drug doses administered 2 h before killing were computed as needed.
3. Results
3.1. Effects of nifedipine The treatments were well tolerated by all the rats and did not induce any gross behavioural change. All data are shown in table 1. Nifedipine induced a 16-35% reduction of the N E levels in all the brain sections with the exception of those from the hippocampus. The m a x i m u m effect was produced in the striatum. In all cases except for the hypothalamus (r16 -- 0.506, P < 0.05) the coefficient of linear correlation between the tissue levels of N E and the log of nifedipine doses did not differ significantly from zero. The increases in DA levels induced by nifedipine in the hypothalamus (up to 25%; r16 = 0.611, P < 0.01) and in the brainstem (up to 49%; ra6 = 0.476, P < 0.05), unlike those induced in the thalamus-midbrain, were linearly dose-related. The D O P A C levels were decreased, mostly in the striatum, but this effect was not linearly related to
the log of the drug doses. The striatal content of H V A was reduced (up to 35%) similarly by the treatment. The levels of 5-HT were unchanged by the treatment in all the brain sections. Finally, the treatment with nifedipine dose dependently increased the concentrations of 5 - H I A A in the hypothalamus (up to 36%; r16 = 0.556, P < 0.05), brainstem (up to 35%; r16 = 0.567, P < 0.05), hippocampus (up to 58%; r16=0.556, P < 0 . 0 5 ) , striatum (up to 33%; r16 = 0.625, P < 0.01) and thalamus-midbrain (up to 58%; r16---0.635, P < 0.01).
3.2. Effects of verapamil Verapamil induced no changes in the cortex (data not shown). This calcium antagonist had little effect on any of the brain sections (table 2) despite the fact that the doses administered were sufficient to produce clear signs of toxicity in some rats. In particular, the rats treated with 10 or 20 m g / k g of verapamil exhibited no behavioural changes or signs of slight sedation, whereas those treated with the highest dose appeared behaviourally depressed. Two of these animals were replaced because they appeared to be comatose at the time of killing and died 1 h later. Verapamil dose dependently reduced the N E levels in the striatum (up to 43%; r16 = 0.496, P < 0.05), brainstem (up to 26%; r16 = 0.859, P < 0.01) and hippocampus (up to 22%; r16 = 0.554, P < 0.05). The significant enhancement of the hypothalamic D A content was also linearly dose-related ( r 1 6 = 0.572, P < 0.05). The concentrations of D O P A C were decreased in some brain sections while the HVA levels remained unchanged. The 5-HT levels were not changed by verapamil in any of the brain sections whereas the 5-HIAA content was occasionally reduced or enhanced.
3.3. Effects of diltiazem The treatments were well tolerated by all the rats and did not induce any gross behavioural change. No changes were induced by diltiazem in the hippocampus, brainstem and thalamus-midbrain (data not shown). With regard to the other
before kilting
mg/kg
2 2 2 2 4 8
2 2 2 2 4 8
2 2 2 2 4 8
2 2 2 2 4 8
0 10 20 40 20 20
2 2 2 2 4 8
Thalamus + midbrain
0 10 20 40 20 20
Cortex
0 10 20 40 20 20
Striatum
0 10 20 40 20 20
Hippocampus
0 10 20 40 20 20
Hypothalamus
Hours
Doses
0.64+0.019 0.754-0.042 0.674-0.066 0.74_+0.023 0.70 i 0.051 0.654-0.025
0.24+0.011 0.25 4- 0.008 0.26 4- 0.019 0.26 + 0.010 0.254-0.010 0.30+0.015 b
0.304-0.015 0.304-0.019 0.284-0.020 0.28+0.007 0.284-0.010 0.324-0.008
0.34+0.018 0.35 4- 0.008 0.32 4- 0.031 0.40 4- 0.027 0.37 4- 0.038 0.394-0.019
2.42 + 0.070 2.13 ± 0.094 2.23 ± 0.151 2.23 ±0.150 2.25 + 0.077 2.38 + 0.072
NE
0.244-0.011 0.63 + 0.070 b 0.374-0.035 0.44+0.102 0.25±0.016 0.265:0.028
0.434-0.060 0.31 + 0.076 0.45 4- 0.025 0.31 _ 0.025 0.61 4-0.026 0.534-0.065
9.334-0.34 10.00+0.42 9.114-0.31 9.774-0.40 9.754-0.12 9.444-0.27
0.33 + 0.012 0.35 _+0.031 0.39 4- 0.0.29 0.494-0.037 h 0.33 4- 0.031 0.37 4- 0.030
DA
Tissue levels: p g / g wet weight
+0.061 4-0.086 +0.134 4-0.205 +0.145 +0.166 a a t, a b
0.12 0.22 0.17 0.19 0.12 0.14
4-0.009 +0.022 b 4-0.015 _+0.025 ~ _+0.005 _+0.012
0.0794-0.013 0.079 + 0.012 0.117 4- 0.010 0.097 4- 0.015 0.138+0.009 ~' 0.1324-0.022 a
1.42 2.02 2.02 2.38 2.00 2.10
0.044 4- 0.004 0.052 4- 0.004 0.048 + 0.005 0.0664-0.008 a 0.046 _ 0.004 0.060 + 0.006
DOPAC
0.052 4- 0.010 0.1174-0.023 a 0.073 4- 0.010 0.1124-0.018 a 0.055 + 0.006 0.085 ± 0.014
a b b b
4- 0.025 4-0.115 b 4-0.095 a 4-0.246 b 4- 0.074 b + 0.151 b
0.075 ± 0.007 0.1144-0.007 0.125 4- 0.006 0.141 4-0.019 0.148 4- 0.007 0.156_+0.019
0.70 1.41 1.33 1.75 1.44 1.63
HVA
0.75 ± 0.036 0.81 +0.036 0.78 + 0.049 0.85 + 0.059 0.71 + 0.030 0.75 4- 0.047
0.304-0.013 0.264-0.014 0.314-0.011 0.31 + 0.015 0.364-0.048 0.35+0.031
0.564-0.023 0.49 4- 0.025 0.54 4- 0.014 0.54 4- 0.024 0.524-0.020 0.524-0.017
0.41 _+0.012 0.374-0.036 0.38 4- 0.012 0.45 4- 0.018 0.394-0.034 0.41 4- 0.009
1.02 4- 0.031 1.004-0.063 1.08+0.028 1.134-0.048 1.034-0.025 1.06 4- 0.029
5-HT
a t, b b
0.55 4- 0.047 0.46+0.024 0.47 + 0.028 0.51 4- 0.044 0.45 4- 0.009 0.49 4- 0.022
0.23_+0.017 0.184-0.010 a 0.194-0.008 0.21 ± 0.019 0.184-0.004 a 0.184-0.007 a
0.514-0.032 0.38 + 0.015 b 0.45 4- 0.024 0.48 _+0.020 0.394-0.011 b 0.404-0.020 b
0.32 4- 0.024 0.244-0.016 a 0.26 4- 0.016 0.29 4- 0.021 0.22-t-0.015 b 0.23 4- 0.018 b
0.58 4- 0.024 0.504-0.036 0.484-0.007 0.464-0.028 0.414-0.019 0.45 4- 0.013
5-H IA A
51 34 46 43 49 52
18 26 26 31 23 25
15 20 22 24 21 22
13 15 12 14 14 16
DA × 100
DOPAC
21.9 18.5 19.9 25.6 22.0 32.4
17.4 36.8 27.8 45.5 24.3 29.4
7.5 14.1 14.6 17.9 14.8 17.3
DA × 100
HVA
76 67 60 67 49 50
91 79 83 90 76 77
78 65 68 64 56 56
57 50 45 41 40 43
5-HT × 100
5-HIAA
Content of biogenic amines and metabolites in brain sections obtained from rats treated with flunarizine. The values are means for six rats 4- S.E. a p < 0.05, b p < 0.01 significantly different from the respective control (two-tailed Dunnett's t-test).
TABLE 4
195 brain sections (table 3), the drug dose dependently reduced the N E levels only in the hypothalamus (up to 20%; q6 = 0.490, P < 0.05), and decreased the 5-HIAA levels only in the c o r t e x (r16 = 0.642, P < 0.01). Other diltiazem-induced changes in the NE, DA, DOPAC, HVA and 5HT levels were observed in the striatum a n d / o r the cortex.
3.4. Effects of flunarizine The animals treated with this compound showed slight sedation. No changes were observed in the brainstem of the treated rats (data not shown). The N E levels (table 4) were occasionally increased only in the cortex. The DA levels were dose dependently increased only in the hypothalamus (up to 48%; r16 = 0.602, P < 0.01). The DOPAC concentrations were enhanced in several brain sections but did not show a linear correlation with the flunarizine doses. The HVA levels in the striatum, the cortex and the thalamus-midbrain were changed in the same way. Flunarizine had no effect on the 5-HT content while it reduced the 5-HIAA levels in several brain sections, but without a linear correlation with the drug doses.
4. Discussion
The data obtained evidenced regional specificity and great differences in the nature and size of the drug-induced changes in brain biogenic amines and their metabolites. The regional specificity of the drug effect could be due to the fact that subtype of the calcium antagonist binds to a specific recognition site in the macromolecular complex of the VSCC (Godfraind et al., 1986) and these channels could differ according to the various brain regions. However, the most pronounced effects of the drugs studied were not observed in the hippocampus or the cortex, i.e. in the brain regions with the highest density of VSCC (Gould et al., 1985). In all cases the effects induced in the hypothalamus and the brainstem, which almost completely lack [3H]nitrendipine binding sites, would be expected to be produced by indirect actions.
It must be emphasized that all the compounds studied, at the doses administered should have produced marked effects on cardiac function and blood pressure. The potency of the peripheral effects and the relatively poor penetration of verapamil into the brain (Mcllhenny, 1971) could explain the low ratio between the toxic and the centrally active doses of this compound. Such peripheral effects could also mediate strong actions on the brain. Diltiazem, because of its lower protein binding (Piepho et al., 1982), nifedipine (Sorkin et al., 1985) and, especially, flunarizine (Holmes et al., 1984), because of their higher lipid solubility, could penetrate the bloodbrain barrier to a greater extent than verapamil. These drugs also have a weaker effect on the blood pressure a n d / o r on the heart. On the other hand, flunarizine is not selective for the brain VSCC and verapamil has been found to interact with receptors other than VSCC, in particular 5-HT receptors (De Feudis, 1987). In this regard, diltiazem, nifedipine and flunarizine have been found to be relatively inactive or much less effective than verapamil (Auguet et al., 1986). Nifedipine could potentiate the effects of adenosine or interact with an adenosine receptor (Swanson and Green, 1986). Moreover, there is evidence to support an interaction between D-2 receptors and VSCC-functions. Diltiazem and, especially verapamil but not nifedipine, have been said to compete with D-2 dopaminergic agents for a common binding site on striatal membranes (De Vries and Beart, 1985). Therefore, in the present study, the absence of a linear correlation between the dose administered and some of the effects measured could be explained at least partly by the progressive involvement of various receptor types, with different sensitivities, in the mechanism of action of the compound. However, the fall in N E levels could be mostly due to the blockade of VSCC, since all the calcium antagonists of the first category, unlike flunarizine, induced similar effects. The effects of the drugs on brain DA content consisted of a moderate and inconsistent increase in various brain regions, with the exception of the striatum. Nifedipine, verapamil and diltiazem lowered the DOPAC levels and this effect could have been
196 due mostly to the blockade of VSCC, since these compounds, which are selective for the slow channels, induced similar effects, whereas flunarizine raised the D O P A C and H V A levels. These data regarding D A and its metabolites agree with those of other authors (Stefanini, 1988). Considering the trends of the D O P A C / D A and H V A / D A ratios, it appears that verapamil and, to a lesser extent nifedipine a n d diltiazem, inhibited whereas flunarizine activated dopaminergic neurotransmission. Flunarizine, however, behaved as a neuroleptic drug and it is k n o w n that neuroleptic drugs leave the D A levels u n c h a n g e d and enhance the brain content of D O P A C and H V A (Bartholini and Lloyd, 1980). All the calcium antagonists changed the brain content of 5 - H I A A according to the drug administered and the brain region studied. Flunarizine in particular, lowered while nifedipine enhanced the metabolite levels. Since the 5 - H T levels remained substantially unchanged, the 5 - H I A A / 5 - H T ratio decreased, suggesting that flunarizine impaired serotonergic neurotransmission. The ratio increased with nifedipine, suggesting that this calcium antagonist activated the serotonergic system in all the brain regions with the possible exception of the cortex. A clear increase of the 5 - H I A A / 5 - H T ratio was also occasionally induced b y verapamil. These observations agree with the finding that nifedipine, unlike verapamil, was c o m p a r a b l e with imipramine for its ability to reverse the shuttlebox escape deficit by inescapable shock ( G e o f f r o y et al., 1988). Concerning the mechanism of action, the nifedipine-induced increase in the 5 - H I A A levels and the signs of general activation of the serotonergic systems would be mostly p r o d u c e d by V S C C blockade. In fact, we observed in a previous study (Gaggi et al., 1989) that the same effects could be p r o d u c e d by reduction of neuronal calcium influx induced by salmon calcitonin leading to a marked and longlasting hypocalcaemia. Moreover, the nifedipineinduced enhancement of 5 - H I A A levels was linearly dose-related in all the brain sections. In conclusion, the administration of calcium antagonists to rats induced marked changes in the brain of levels of biogenic amines and metabolites. These changes, displaying regional specificity and
differences according to the various c o m p o u n d s , could not have been p r o d u c e d only b y the mechanism of V S C C blockade. Interactions of the comp o u n d s with other receptors should be at least partly involved and some effects could be induced by indirect actions. Nevertheless, since several calcium antagonists have been p r o p o s e d for treatm e n t of a variety of C N S disorders ( R a e b u r n and Gonzales, 1988), the present data could be useful to facilitate the choice of the most suitable comp o u n d for each therapeutic category.
References Auguet, M., S. Delaflotte, F. Clostre and F.V. De Feudis, 1986, Verapamil as an apparent competitive antagonist of the serotonin receptor of rabbit isolated aorta, Gen. Pharmacol. 17, 133. Bartholini, G. and K.G. Lloyd, 1980, Biochemical effects of neuroleptic drugs, in: Psychotropic agents, Part I: Antipsychotics and Antidepressants, Vol. 55/1, eds. H. Hoffmeister and G. Stille (Springer-Verlag, Berlin, Heidelberg, New York) p. 193. Boles, R.G., H.I. Yamamura, H. Schoemaker and W.R. Roeske, 1984, Temperature-dependent modulation of [3H]nitrendipine binding by the calcium channel antagonists verapamil and diltiazem in rat brain synaptosomes, J. Pharmacol. Exp. Ther. 229, 33. Bongianni, F., V. Carla, F. Moroni and D.E. Pellegrini Giampietro, 1986, Calcium channel inhibitors suppress the morphine-withdrawal syndrome in rats, Br. J. Pharmacol. 88, 561. Contreras, E., L. Tamayo and M. Amigo, 1988, Calcium channel antagonists increase morphine-induced analgesia and antagonize morphine tolerance, European J. Pharmacol. 148, 463. De Feudis, F.V., 1987, Interaction of Ca 2+ antagonists at 5-HT2 and H 2 receptor and GABA uptake sites, Trends Pharmacol. Sci. 8, 200. Del Pozo, E., G. Caro and J.M. Baeyens, 1987, Analgesic effects of several calcium channel blockers in mice, European J. Pharmacol. 137, 155. Desmedt, L.K.C., G.J.E. Niemegeers and P.A.J. Janssen, 1975, Anticonvulsive properties of cinnarizine and flunarizine in rats and mice, Arzneim. Forsch. 25, 217. De Vries, D.J. and P.M. Beart, 1985. Competitive inhibition of [ 3H]spiperone binding to D-2 dopamine receptors in striatal homogenates by calcium channel antagonists and polyvalent cations, European J. Pharmacol. 106, 133. Gaggi, R., R. De Iasio and A.M. Gianni, 1989, Relationships between the effects of peripherally administered salmon calcitonin on calcaemia and brain biogenic amines, Jap. J. Pharmacol. 51, 309.
197 Geoffroy, M., E. Mogilnicka, M. Nielsen and O.J. Rafaelsen, 1988, Effect of nifedipine on the shuttlebox escape deficit induced by inescapable shock in the rat, European J. Pbarmacol. 154, 277. Godfraind, T., R. Miller and M. Wibo, 1986, Calcium antagonism and calcium entry blockade, Pharmacol. Rev. 38, 321. Gould, R.J., K.M.M. Murphy and S.H. Snyder, 1982, [3H]nitrendipine labeled calcium channels discriminate inorganic calcium agonists and antagonists, Proc. Natl. Aead. Sci. U.S.A. 79, 3656. Gould, R.J., K.M.M. Murphy and S.H. Snyder, 1985, Autoradiographic localization of calcium channel antagonist receptors in rat brain with [3H]nitrendipine, Brain Res. 330, 217. Holmes, B., R.N. Brogden, R.C. Heel, T.M. Speight and G.S. Avery, 1984, Flunarizine. A review of its pharmacodynamic and pharmacokinetic properties and therapeutic use, Drugs 27, 6. Mcllhenny, H.M., 1971, Metabolism of [14C]verapamil, J. Med. Chem. 14, 1178. Mendelson, W . B . P . Skolnick, J.V. Martin, M.D. Luu, R. Wagner and S.M. Paul, 1984, Diazepam stimulated increases in the synaptosomal uptake of 4SCa2+: reversal by dihydropyridine calcium channel antagonists, European J. Pharmacol. 104, 181. Miller, R.J., 1987, Multiple calcium channels and neuronal functions, Science 235, 46. Murphy, K.M.M., R.J. Gould and S.H. Snyder, 1982, Autoradiographic visualization of [3H]nitrendipine binding sites in rat brain: localization to synaptic zones, European J. Pharmacol. 81,517. Paoletti, R. and S. Govoni, 1987, Classification of calcium
antagonists: proposal of the WHO committee, Pharmacol. Res. Comm. 19, 195. Piepho, R.W., D.C. Bloedow, J.P. Lacz, D.J. Runser, D.C. Dimmit and R.K. Browne, 1982, Pharmacokinetics of diltiazem in selected animal species and human beings, Am. J. Cardiol. 49, 529. Raeburn, D. and R.A. Gonzales, 1988, CNS disorders and calcium antagonists, Trends Pharmacol. Sci. 9, 117. Sorkin, E.M., S.P. Clissold and R.N. Brogden, 1985, Nifedipine. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic efficacy, in ischaemic heart disease, hypertension and related cardiovascular disorders, Drugs 30, 182. Seegal, R.F., K.O. Brosch and B. Bush, 1986, High-performance liquid chromatography of biogenic amines and metabolites in brain, cerebrospinal fluid, urine and plasma, J. Chromatogr. 377, 131. Stefanini, E., 1988, Effect of calcium antagonists on brain dopaminergic activity, Neurosci. Lett. Suppl. 33, Third Meeting of the Italian Society of Neuroscience, Abstr. p. S186. Swanson, T.H. and C.L. Green, 1986, Nifedipine: more than a calcium channel blocker, Gen. Pharmacol. 17, 255. Vanhoutte, P.M. and R. Paoletti, 1987, The WHO classification of calcium antagonists, Trends Pharmacol. Sci. 8, 4. Wagner, J., P. Vitali, M.G. Palfreyman, M. Zraika and S. Huot, 1982, Simultaneous determination of 3,4-dihydroxyphenylalanine, 5-hydroxytryptophan, dopamine, 4-hydroxy-3-metoxyphenylalanine, norepinephrine, 3,4-dihydroxyphenylacetic acid, homovaniilic acid, serotonin and 5-hydroxyindoleacetic acid in rat cerebrospinal fluid and brain by high-performance liquid chromatography with electrochemical detection, J. Neurochem. 38, 1241.
187
Elsevier EJP 51328
Effects of calcium antagonists on biogenic amines in discrete brain areas Renato Gaggi and Anna Maria Gianni Institute of Pharmacology, Unioersityof Bologna, via Irnerio 48, 40126 Bologna, ltaly
Received 12 October 1989, revised MS received 19 February 1990, accepted 13 March 1990
Discrete brain sections were obtained from rats given i.p. verapamil, nifedipine, diltiazem or flunarizine (0, 10, 20 or 40 mg/kg). The biogenic amines and metabolites in the hypothalamus, brainstem, hippocampus, striatum, thalamus-midbrain and cortex were determined by high-performance liquid chromatography with electrochemical detection. The treatments induced several changes in the levels of neurotransmitters and metabolites, displaying regional specificity and differences according to the various compounds. It was speculated that some effects could have been due to indirect actions and/or to interactions of the compounds with receptors other than the voltage-sensitive calcium channels. However blockade of these channels could account for the following effects. (a) The nifedipine-induced increases in the 5-hydroxy-3-indoleacetic acid levels and, in general, the signs of activation of the serotonergic systems. (b) The fall in the 3,4-dihydroxyphenylacetic acid levels and, in general, the signs of attenuation of dopaminergic neurotransmission induced by nifedipine, verapamil and diltiazem. (c) The fall in the norepinephrine levels induced by all the compounds studied. C a 2+
channel antagonists; Brain sections (rat); Biogenic amines; (Biogenic amine metabolites)
1. Introduction The calcium antagonists have been recently reclassified on the basis of their effects in a number of tests. There are now two main categories, each including three subtypes (Paoletti and Govoni, 1987; Vanhoutte and Paoletti, 1987). The first category, regarding drugs selective for slow Ca 2+ channels, includes the verapamil, nifedipine and diltiazem-like subtypes. The second category includes the flunarizine-like subtype and the other two subtypes which include non-selective calcium entry blockers. Flunarizine is considered poorly selective for the voltage-sensitive calcium channel (VSCC) since it also inhibits Ca 2 ÷ influx through receptor-operated channels (Holmes et al., 1984).
Correspondence to: R. Gaggi, Institute of Pharmacology, Via Irnerio 48, 1-40126-Bologna, Italy.
Verapamil, nifedipine, diltiazem and flunarizine recognize specific binding sites in the macromolecular complex of the VSCC in various tissues and modulate [3H]nitrendipine binding to this complex (Godfraind et al., 1986). Verapamil, nifedipine and flunarizine inhibit, whereas diltiazero facilitates (Boles et al., 1984), [3H]nitrendipine binding to its receptor. The highest density of [3H]nitrendipine binding sites in the body occurs in the brain (Gould et al., 1982). It is now generally accepted that these sites, which are not exclusively associated with blood vessels (Murphy et al., 1982), represent functional VSCC (Miller, 1987). It has been hypothesized that the receptors for [3H]nitrendipine in the central nervous system are L channels. This type of channel, being preferentially localized in the cell soma, seems to be involved in the regulation of neuronal functions other than the generation of action potentials or the release of neurotransmitters (Miller, 1987).
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
mg/kg
2 2 2 2 4 8
2.26 4- 0.143 2.10±0.083 1.81 4- 0.091 a 1.824-0.068 a 1.94 4- 0.100 2.46 4- 0.102
NE
2 2 2 2 4 8
10 20 40 20 20
2 2 2 4 8
Hippocampus 0 2
0 10 20 40 20 20
0.35 + 0.026 0.32 ± 0.027 0.28 4- 0.018 0.28 4- 0.017 0.28 4- 0.020 0.34 4- 0.022
0.74 + 0.032 0.64 4- 0.030 a 0.62 4- 0.025 a 0.63 4- 0.028 a 0.68 4- 0.026 0.73 4- 0.022
0.055 + 0.007 0.062 4- 0.007 0.082 4- 0.004 a 0.0794-0.003 ~ 0.045 4- 0.003 0.053 ± 0.008
0.36 + 0.011 0.37+0.009 0.41 4- 0.023 0.45+0.017 ~ 0.33 4- 0.019 0.36 ± 0.016
DA
Tissue levels: ~ g / g wet weight
Brainstem (pons + medulla)
0 10 20 40 20 20
Hypothalamus
Hours
before killing
Doses
0.080 ___0.009 0.0674-0.010 0.064 + 0.005 0.073+0.006 0.067 4- 0.005 0.074 + 0.006
DOPAC
HVA
0.41 + 0.031 0.44 __.0.056 0.40 ± 0.049 0.41 +0.038 0.40 4- 0.024 0.47 4- 0.014
0.76 ± 0.056 0.66 ± 0.030 0.70 4- 0.031 0.744-0.026 0.71 ± 0.048 0.80 4- 0.034
1.07 4- 0.075 1.01 ± 0.049 0.93 4- 0.030 1.054-0.051 1.04 4- 0.055 1.14 4- 0.055
5-HT
0.29 4- 0.010 0.33 + 0.031 0.39 ± 0.022 a 0.454-0.040 b 0.39 + 0.020 a 0.33 4- 0.011
0.40 ± 0.031 0.40 4- 0.041 0.47 4- 0.032 0.53+_0.030 a 0.46 ± 0.053 0.39 4- 0.014
0.53 + 0.021 0.604-0.039 0.64 ± 0.029 ~ 0.724-0.025 b 0.60 4- 0.030 0.58 4- 0.010
5-HIAA
22 18 16 16 20 21
DA x 100
DOPAC
DA x 100
HVA
5-HT 100
70 76 98 110 97 70
52 61 67 72 65 49
50 59 69 68 58 51
x
5-HIAA
Content of biogenic amines and metabolites in brain sections obtained from rats treated with nifedipine. The values are means for six rats ± S.E. a p < 0.05, t, p < 0.01 significantly different from the respective control (two-tailed Dunnett's t-test).
TABLE 1
O0
+ midbrain 2 2 2 2 4 8
2 2 2 2 4 8
Cortex 0 10 20 4O 20 20
Thalamus 0 10 20 40 2O 2O
2 2 2 2 4 8
Striatum 0 10 2O 4O 20 2O b b b a
0.62 + 0.037 0.52 4- 0.026 0.45 5=0.029 a 0.52 4- 0.049 0.47 5=0.028 a 0.58 + 0.050
0.31 5:0.022 0.27 4- 0.014 0.244-0.016 a 0.25 4- 0.017 0.25 4- 0.014 0.34 4- 0.017
0.31 4- 0.019 0.23 5=0.014 0.22 4- 0.022 0.204-0.012 0.244-0.013 0.28 + 0.011
0.22 4- 0.026 0.20 4- 0.015 0.184-0.015 0.28 4- 0.030 0.18_+0.013 0.22 + 0.019
0.43 4- 0.026 0.38 5:0.027 0.41 4- 0.049 0.38 5=0.043 0.33 5=0.027 0.42 4- 0.027
9.66 4- 0.37 8.47 4- 0.45 9.31 4- 0.47 9.54 4- 0.21 8.514-0.39 10.04_+0.18
0.14 4- 0.009 0.134-0.011 0.104-0.002 " 0.11 4-0.009 0.134-0.008 0.18 4- 0.012 a
0.12 4- 0.014 0.12 4- 0.012 0.10 4- 0.008 0.13_+0.017 0.13_+0.018 0.19 4- 0.013 b
1.30 4- 0.070 1.06 4- 0.055 a 1.01 4- 0.050 a 1.00 4- 0.021 b 1.06 4- 0.089 a 1.36 4- 0.062
0.062 4- 0.012 0.057 4- 0.021 0.070 + 0.024 0.068 + 0.018 0.080 + 0.011 0.084 4- 0.016
0.048 4- 0.005 0.051 4- 0.005 0.046 4- 0.005 0.050 4- 0.006 0.048 4- 0.004 0.053 4- 0.006
0.57 4- 0.013
0.48 4- 0.033 b
0.70 + 0.042 0.55 4- 0.056 a 0.49 4- 0.026 b 0.45 4- 0.042 b
0.72 4- 0.035 0.65 + 0.037 0.64 + 0.032 0.74 + 0.063 0.63 4- 0.047 0.82 + 0.074
0.36 4- 0.033 0.39 4- 0.039 0.33 4- 0.023 0.35 4- 0.026 0.32 4- 0.016 0.43 4- 0.042
0.56 4- 0.033 0.50 4- 0.024 0.50 4- 0.012 0.58 4- 0.028 0.52 + 0.035 0.58 4- 0.026
0.52 4- 0.026 0.53 + 0.045 0.59 4- 0.032 0.82 4- 0.089 b 0.58 4- 0.036 0.60 4- 0.039
0.20 + 0.014 0.20 4- 0.015 0.21 + 0.014 0.23 4- 0.015 0.19 4- 0.010 0.24 + 0.022
0.45 4- 0.042 0.43 4- 0.042 0.51 4- 0.038 0.60 4- 0.033 a 0.48 4- 0.048 0.51 4-0.018
63 67 55 39 74 76
27 31 25 35 38 44
13 13 11 10 13 19
7.2 6.5 5.3 4.8 5.7 5.7
72 82 92 111 92 72
80 86 102 103 92 88
before killing
mg/kg
2 2 2 2 4 8
2.29+0.123 2.30_+0.081 2.19+0.128 1.82_+0.177 2.28_+0.155 2.31_+0.102
NE
2 2 2 2 4 8
2 2 2 2 4 8
2 2 2 2 8 8
0 10 20 40 20 20
2 2 2 2 4 8
Thalamus + midbrain
0 10 20 40 20 20
Striatum
0 10 20 40 20 20
Hippocampus
0 10 20 40 20 20
0.64_+0.021 0.63-+0.010 0.58_+0.014 0.55_+0.030 0.61_+0.037 0.64_+0.040
0.33_+0.016 0.24 + 0.011 0.23 +0.021 0.19_+0.007 0.25_+0.010 0.30_+0.006 b b b b
0.32_+0.013 0.31 _+0.021 0.27 _+0.010 0.25 -I-0.012 a 0.26 _+0.018 0.27 _+0.015
0.73 + 0.031 0.72 _+0.019 0.65 + 0.011 0.54 5:0.022 b 0.63 _+0.040 0.77 _+0.030
+0.007 _+0.004 -+0.020 _+0.043 b _+0.010 _+0.026
0.24 0.20 0.23 0.21 0.20 0.23
9.40 9.08 8.83 8.33 8.51 8.67
4-0.015 -+0.006 _+0.019 _+0.008 _+0.008 _+0.014
_+0.46 _+0.21 -+0.36 _+0.56 _+0.33 _+0.16
0.063 -+0.003 0.052 _+0.004 0.058 -+0.002 0.080_+0.008 0.052 _+0.004 0.059 _+0.004
0.30 0.33 0.35 0.46 0,32 0.33
DA
Tissue levels: /xg/g wet weight
Brainstem (pons + medulla)
0 10 20 40 20 20
Hypothalamus
Hours
Doses
0.13 0.10 0.12 0.12 0.11 0.13
1.51 1.36 1.31 1.28 1.11 1.02
+0.007 -I-0.002 b _+0.006 _+0.004 _+0.007 " _+0.008
_+0.125 _+0.035 -+0.059 _+0.084 +0.066 b -t-0.033 b
0.042-+0.007 0.046_+0.008 0.038-+0.009 0.086-+0.021 0.067_+0.018 0.057_+0.015
DOPAC
_+0.051 _+0.037 +0.044 _+0.056 _+0.038 +0.027
0,058_+0.015 0.047-+0.012 0.063_+0.010 0.070_+0.022 0.050_+0.019 0.064_+0.014
0.69 0.64 0.62 0.58 0.64 0.58
HVA
0.75+0.053 0.68__.0.037 0.61 _+0.034 0.66_+0.026 0.71_+0.037 0.71_+0.027
0.57_+0.025 0.59+0.021 0.56-+0.020 0.53_+0.019 0.60_+0.022 0.55-+0.030
0.37_+0.014 0.35_+0.024 0.33_+0.011 0.35_+0.010 0.34_+0.016 0.34 + 0.019
0.71 _+0.020 0.69_+0.025 0.68 _+0.024 0.70 _+0.022 0.71 _+0.027 0.71_+0.026
1.01_+0.028 1.02_+0.030 1.07_+0.053 1.14_+0.040 1.06_+0.025 1.00_+0.038
5-HT
0.53_+0.026 0.46-+0.029 0.56_+0.051 0.56_+0.025 0.68_+0,024 0.63_+0.031
0.46_+0.013 0.43+0.026 0.47-t-0.022 0.48_+0.021 0.48_+0.029 0.40-+0.014
0.31_+0.025 0.23_+0.016 0.30_+0.027 0.29_+0.012 0.34_+0.021 0.26 _+0.007
0.40 _+0.021 0.35+0.016 0.40 _+0.019 0.39 _+0.004 0.43 _+0.025 0.41_+0.013
0.58_+0.011 0.53_+0.027 0.58_+0.024 0.60_+0.018 0.62_+0.025 0.63_+0.021
5-HIAA
"
55 51 51 55 53 54
16 15 15 15 13 12
14 14 11 19 21 17
DA × 100
DOPAC DA × 100
HVA
70 67 84 85 96 88
84 64 91 83 98 75
5-HT × 100
5-HIAA
Content of biogenic amines and metabolites in brain sections obtained from rats treated with verapamil. The values are means for six rats_+ S.E. a P < 0.05, b p < 0.01 significantly different from the respective control (two-tailed Dunnett's t-test).
TABLE 2 o
191 On the other hand the calcium antagonists exert several actions in the central nervous system, e.g., flunarizine exhibits anticonvulsant properties in experimental models of epilepsy (Desmedt et al., 1975), nifedipine blocks sleep induction by flurazepam in the rat (Mendelson et al., 1984), verapamil, diltiazem and flunarizine increase morphine-induced analgesia (Del Pozo et al., 1987), nifedipine, flunarizine and verapamil antagonize morphine-tolerance in mice (Contreras et al., 1988), and verapamil and nimodipine suppress the morphine-withdrawal syndrome in rats (Bongianni et al., 1986). We now investigate the effects of a phenylalkylamine (verapamil), a dihydropyridine (nifedipine), a benzothiazepine (diltiazem) and a diphenylpiperazine (flunarizine). These represent, respectively, calcium antagonists of the first four subtypes on the biogenic amine content of discrete rat brain sections.
2. Material and methods
was analyzed on the same day, repeating complete replications sequentially.
2.2. Drugs and treatments Nifedipine and diltiazem hydrochloride were kindly supplied by Schiapparelli Farmaceutici (Turin, Italy). Verapamil hydrochloride was from Sigma Chemical Co (S. Louis, MO, U.S.A.) and flunarizine hydrochloride was kindly supplied by Italfarmaco S.p.A. (Milan, Italy). Verapamil and diltiazem were dissolved with saline while nifedipine and flunarizine were suspended in 1% Tween 80. The dose per kg body weight was contained in 3.0 ml. All the drugs were administered intraperitoneally. Two groups of six rats were treated with 20 mg/kg of nifedipine 8 or 4 h before killing. Another four groups of six rats were treated, 2 h before killing with nifedipine 0 (vehicle), 10, 20 or 40 mg/kg. The procedure was repeated with the hydrochloride salt of verapamil, diltiazem or flunarizine.
2.3. Analytical procedures 2.1. Animals and general procedure A total of 144 male Sprague-Dawley rats (Nossan, Milano, Italy) weighing 180-200 g were used. They were maintained on standard laboratory food and controlled conditions of light (7:00-19:00 h), temperature (22 __+2°C) and humidity (60%). On the day of the experiment all the rats were deprived of food at 9:00 h while water was allowed ad libitum. To avoid circadian variations in the brain content of biogenic amines, all rats were decapitated in the afternoon (16:00-18:00 h). The brain was rapidly removed and ice-cooled. The cerebellum and the olfactory tubercles were discarded whereas the hypothalamus, the hippocampus, the striatum, the brainstem (pons plus medulla oblongata) and the cortex were dissected, immediately frozen with pulverised dry ice and kept at - 2 5 ° C until the time of analysis. The remaining cerebral tissue, consisting mostly of the thalamus and midbrain, was also collected, frozen and analyzed. All the samples from the same experiment were analyzed in the 10-15 days after collection. The same brain section of each animal
The determination of norepinephrine (NE), dopamine (DA), 5-hydroxytryptamine (5-HT) and the metabolites, dihydroxphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) was performed simultaneously by high-performance liquid chromatography (HPLC) with electrochemical detection (Wagner et al., 1982; Seegal et al., 1986; Gaggi et al., 1989). The HPLC system consisted of a P 330 pump (Violet, Rome, Italy) linked to a Rheodyne 7125 injector. The column (ODS1-C18; 10 /zm; 25 cm x 4 mm i.d.) and the pre-column (ODS1-C18; 10/~m; 5 cm x 4 mm i.d.) were also from Violet (Rome, Italy). The electrochemical detector (Eldec 103, Chromatofield, Chateauneuf-Les Martigues, France) was connected with a TRIO integrator (Trivector Systems Int. Ltd., Sandy, England). The potential of the glassy-carbon electrode versus silver-silver chloride reference electrode was maintained at + 0.8 V. The mobile phase consisted of a 840 : 160 mixture of 0.1 M NaH2PO 4 and methanol. The mixture was made 2.6 x 10 -3 M in octyl sulfate,
mg/kg
0 10 20 40 20 20
Cortex
0 10 20 40 20 20
Striatum
0 10 20 40 20 20
2 2 2 2 8 8
2 2 2 2 4 8
2 2 2 2 4 8
Hypothalamus
Hours
before killing
Doses
0.34+-0.030 0.225:0,011 t, 0.21+-0.017 b 0.20+- 0.014 t' 0.21 + 0.018 b 0.22 +-0.022 b
0.32+-0.015 0.275:0.016 0.34+-0.017 0.35+-0.019 0.295:0.012 0.365:0.014
2.30 5:0,087 2.18 + 0.038 2.35 +-0.088 1.83 +0.120 b 2.22 5:0.126 1.94 + 0.099
NE
0.45+-0.052 0.64+-0.048 b 0.525:0.049 0.52+-0.028 0.46+-0.036 0.57+0.017
9.04+-0.28 8.71 5:0.29 9.435:0.53 9.35+-0.43 8.40+-0.18 10.05+-0.52
0.36 + 0.024 0.29 +-0.018 0.30 +- 0.021 0.31+-0.030 0.36 +-0.039 0.37 +- 0.023
DA
Tissue levels: /xg/g wet weight
0.13 0.14 0.14 0.11 0.10 0.11
1.46 1.32 1.29 1.37 1.17 1.50
_ 0.011 +-0.013 +-0.009 +-0.008 +-0.008 +-0.008
+-0.110 +-0.042 +-0.086 +-0.081 +-0.059 a +-0.036
0.046 +-0.003 0.036 +-0.003 0.037 + 0.007 0.025+-0.006 0.033 +-0.007 0.030 +-0.005
DOPAC
_+0.029 5:0.036 +-0.064 +-0.068 +0.034 _+0.042 a
0.053 + 0.003 0.0745:0.007 a 0.0595:0.005 0.0625:0.005 0.0505:0.007 0.0695:0.007
0.69 0.67 0.72 0.79 0.65 0.87
HVA
0.43 + 0.034 0.39+0.022 0.33+0.019 b 0.36+0.016 0.33+0.019 b 0.35+0.019
0.60+--0.019 0.48 + 0.022 b 0.52+-0.021 0.55+-0.031 0.535:0.036 0.55+-0.016
0.95+-0.029 0.98+-0.027 0.95+-0.036 0.89+-0.008 0.93 + 0.041 0.94+-0.030
5-HT
0.23 + 0.014 0.245:0.006 0.195:0.008 a 0.19+0.011 a 0.185:0.011 a 0.21+0.014
0.48+-0.041 0.50 +- 0.032 0.42 +-0.021 0.46+-0.057 0.42+-0.015 0.49+-0.018
0.57+-0.018 0.60+-0.018 0.52+-0.029 0.54+-0.018 0.54 +- 0.031 0.59+-0.032
5-HIAA
29 22 26 20 21 19
16 15 14 15 14 15
x
DA 100
DOPAC
12 12 11 12 11 12
7.7 7.7 7.6 8.4 7.8 8.7
DA x 100
HVA
5-HT 100
54 61 56 52 55 58
79 103 81 84 79 90
x
5-HIAA
Content of biogenic amines and metabolites in brain sections obtained from rats treated with diltiazem. The values are means for six rats+- S.E. a P < 0.05, ~' P < 0.01 significantly different from the respective control (two-tailed Dunnett's t-test).
TABLE 3
193 10 -4 M in E D T A and 2.5 × 10 -4 M in triethylamine and the p H was adjusted to 3.4 with 3 M H3PO 4. The flow rate was 1.5 m l / m i n . At the time of the analysis a frozen aliquot of a standard mixture was thawed and diluted 10 times with the medium used for the preparation of the tissue samples. This medium consisted of a freshly prepared solution 0.2 M in HC104, 2.7 × 10 -4 M E D T A and 1.6 × 10 -4 M in ascorbic acid. The frozen brain sections were weighed and immediately disrupted by ultrasound in 0.5-1.0 ml of the medium. After centrifugation for 10 rain at 11 000 × g, 20 #1 of the supernatant were injected into the H P L C apparatus. D H B A was used as an internal standard.
2.4. Statistical analysis Analysis of variance (ANOVA) was applied to the data, followed by the two-tailed Dunnett's t-test to compare individual groups to the control. Linear correlation ratios between an effect and the log of the drug doses administered 2 h before killing were computed as needed.
3. Results
3.1. Effects of nifedipine The treatments were well tolerated by all the rats and did not induce any gross behavioural change. All data are shown in table 1. Nifedipine induced a 16-35% reduction of the N E levels in all the brain sections with the exception of those from the hippocampus. The m a x i m u m effect was produced in the striatum. In all cases except for the hypothalamus (r16 -- 0.506, P < 0.05) the coefficient of linear correlation between the tissue levels of N E and the log of nifedipine doses did not differ significantly from zero. The increases in DA levels induced by nifedipine in the hypothalamus (up to 25%; r16 = 0.611, P < 0.01) and in the brainstem (up to 49%; ra6 = 0.476, P < 0.05), unlike those induced in the thalamus-midbrain, were linearly dose-related. The D O P A C levels were decreased, mostly in the striatum, but this effect was not linearly related to
the log of the drug doses. The striatal content of H V A was reduced (up to 35%) similarly by the treatment. The levels of 5-HT were unchanged by the treatment in all the brain sections. Finally, the treatment with nifedipine dose dependently increased the concentrations of 5 - H I A A in the hypothalamus (up to 36%; r16 = 0.556, P < 0.05), brainstem (up to 35%; r16 = 0.567, P < 0.05), hippocampus (up to 58%; r16=0.556, P < 0 . 0 5 ) , striatum (up to 33%; r16 = 0.625, P < 0.01) and thalamus-midbrain (up to 58%; r16---0.635, P < 0.01).
3.2. Effects of verapamil Verapamil induced no changes in the cortex (data not shown). This calcium antagonist had little effect on any of the brain sections (table 2) despite the fact that the doses administered were sufficient to produce clear signs of toxicity in some rats. In particular, the rats treated with 10 or 20 m g / k g of verapamil exhibited no behavioural changes or signs of slight sedation, whereas those treated with the highest dose appeared behaviourally depressed. Two of these animals were replaced because they appeared to be comatose at the time of killing and died 1 h later. Verapamil dose dependently reduced the N E levels in the striatum (up to 43%; r16 = 0.496, P < 0.05), brainstem (up to 26%; r16 = 0.859, P < 0.01) and hippocampus (up to 22%; r16 = 0.554, P < 0.05). The significant enhancement of the hypothalamic D A content was also linearly dose-related ( r 1 6 = 0.572, P < 0.05). The concentrations of D O P A C were decreased in some brain sections while the HVA levels remained unchanged. The 5-HT levels were not changed by verapamil in any of the brain sections whereas the 5-HIAA content was occasionally reduced or enhanced.
3.3. Effects of diltiazem The treatments were well tolerated by all the rats and did not induce any gross behavioural change. No changes were induced by diltiazem in the hippocampus, brainstem and thalamus-midbrain (data not shown). With regard to the other
before kilting
mg/kg
2 2 2 2 4 8
2 2 2 2 4 8
2 2 2 2 4 8
2 2 2 2 4 8
0 10 20 40 20 20
2 2 2 2 4 8
Thalamus + midbrain
0 10 20 40 20 20
Cortex
0 10 20 40 20 20
Striatum
0 10 20 40 20 20
Hippocampus
0 10 20 40 20 20
Hypothalamus
Hours
Doses
0.64+0.019 0.754-0.042 0.674-0.066 0.74_+0.023 0.70 i 0.051 0.654-0.025
0.24+0.011 0.25 4- 0.008 0.26 4- 0.019 0.26 + 0.010 0.254-0.010 0.30+0.015 b
0.304-0.015 0.304-0.019 0.284-0.020 0.28+0.007 0.284-0.010 0.324-0.008
0.34+0.018 0.35 4- 0.008 0.32 4- 0.031 0.40 4- 0.027 0.37 4- 0.038 0.394-0.019
2.42 + 0.070 2.13 ± 0.094 2.23 ± 0.151 2.23 ±0.150 2.25 + 0.077 2.38 + 0.072
NE
0.244-0.011 0.63 + 0.070 b 0.374-0.035 0.44+0.102 0.25±0.016 0.265:0.028
0.434-0.060 0.31 + 0.076 0.45 4- 0.025 0.31 _ 0.025 0.61 4-0.026 0.534-0.065
9.334-0.34 10.00+0.42 9.114-0.31 9.774-0.40 9.754-0.12 9.444-0.27
0.33 + 0.012 0.35 _+0.031 0.39 4- 0.0.29 0.494-0.037 h 0.33 4- 0.031 0.37 4- 0.030
DA
Tissue levels: p g / g wet weight
+0.061 4-0.086 +0.134 4-0.205 +0.145 +0.166 a a t, a b
0.12 0.22 0.17 0.19 0.12 0.14
4-0.009 +0.022 b 4-0.015 _+0.025 ~ _+0.005 _+0.012
0.0794-0.013 0.079 + 0.012 0.117 4- 0.010 0.097 4- 0.015 0.138+0.009 ~' 0.1324-0.022 a
1.42 2.02 2.02 2.38 2.00 2.10
0.044 4- 0.004 0.052 4- 0.004 0.048 + 0.005 0.0664-0.008 a 0.046 _ 0.004 0.060 + 0.006
DOPAC
0.052 4- 0.010 0.1174-0.023 a 0.073 4- 0.010 0.1124-0.018 a 0.055 + 0.006 0.085 ± 0.014
a b b b
4- 0.025 4-0.115 b 4-0.095 a 4-0.246 b 4- 0.074 b + 0.151 b
0.075 ± 0.007 0.1144-0.007 0.125 4- 0.006 0.141 4-0.019 0.148 4- 0.007 0.156_+0.019
0.70 1.41 1.33 1.75 1.44 1.63
HVA
0.75 ± 0.036 0.81 +0.036 0.78 + 0.049 0.85 + 0.059 0.71 + 0.030 0.75 4- 0.047
0.304-0.013 0.264-0.014 0.314-0.011 0.31 + 0.015 0.364-0.048 0.35+0.031
0.564-0.023 0.49 4- 0.025 0.54 4- 0.014 0.54 4- 0.024 0.524-0.020 0.524-0.017
0.41 _+0.012 0.374-0.036 0.38 4- 0.012 0.45 4- 0.018 0.394-0.034 0.41 4- 0.009
1.02 4- 0.031 1.004-0.063 1.08+0.028 1.134-0.048 1.034-0.025 1.06 4- 0.029
5-HT
a t, b b
0.55 4- 0.047 0.46+0.024 0.47 + 0.028 0.51 4- 0.044 0.45 4- 0.009 0.49 4- 0.022
0.23_+0.017 0.184-0.010 a 0.194-0.008 0.21 ± 0.019 0.184-0.004 a 0.184-0.007 a
0.514-0.032 0.38 + 0.015 b 0.45 4- 0.024 0.48 _+0.020 0.394-0.011 b 0.404-0.020 b
0.32 4- 0.024 0.244-0.016 a 0.26 4- 0.016 0.29 4- 0.021 0.22-t-0.015 b 0.23 4- 0.018 b
0.58 4- 0.024 0.504-0.036 0.484-0.007 0.464-0.028 0.414-0.019 0.45 4- 0.013
5-H IA A
51 34 46 43 49 52
18 26 26 31 23 25
15 20 22 24 21 22
13 15 12 14 14 16
DA × 100
DOPAC
21.9 18.5 19.9 25.6 22.0 32.4
17.4 36.8 27.8 45.5 24.3 29.4
7.5 14.1 14.6 17.9 14.8 17.3
DA × 100
HVA
76 67 60 67 49 50
91 79 83 90 76 77
78 65 68 64 56 56
57 50 45 41 40 43
5-HT × 100
5-HIAA
Content of biogenic amines and metabolites in brain sections obtained from rats treated with flunarizine. The values are means for six rats 4- S.E. a p < 0.05, b p < 0.01 significantly different from the respective control (two-tailed Dunnett's t-test).
TABLE 4
195 brain sections (table 3), the drug dose dependently reduced the N E levels only in the hypothalamus (up to 20%; q6 = 0.490, P < 0.05), and decreased the 5-HIAA levels only in the c o r t e x (r16 = 0.642, P < 0.01). Other diltiazem-induced changes in the NE, DA, DOPAC, HVA and 5HT levels were observed in the striatum a n d / o r the cortex.
3.4. Effects of flunarizine The animals treated with this compound showed slight sedation. No changes were observed in the brainstem of the treated rats (data not shown). The N E levels (table 4) were occasionally increased only in the cortex. The DA levels were dose dependently increased only in the hypothalamus (up to 48%; r16 = 0.602, P < 0.01). The DOPAC concentrations were enhanced in several brain sections but did not show a linear correlation with the flunarizine doses. The HVA levels in the striatum, the cortex and the thalamus-midbrain were changed in the same way. Flunarizine had no effect on the 5-HT content while it reduced the 5-HIAA levels in several brain sections, but without a linear correlation with the drug doses.
4. Discussion
The data obtained evidenced regional specificity and great differences in the nature and size of the drug-induced changes in brain biogenic amines and their metabolites. The regional specificity of the drug effect could be due to the fact that subtype of the calcium antagonist binds to a specific recognition site in the macromolecular complex of the VSCC (Godfraind et al., 1986) and these channels could differ according to the various brain regions. However, the most pronounced effects of the drugs studied were not observed in the hippocampus or the cortex, i.e. in the brain regions with the highest density of VSCC (Gould et al., 1985). In all cases the effects induced in the hypothalamus and the brainstem, which almost completely lack [3H]nitrendipine binding sites, would be expected to be produced by indirect actions.
It must be emphasized that all the compounds studied, at the doses administered should have produced marked effects on cardiac function and blood pressure. The potency of the peripheral effects and the relatively poor penetration of verapamil into the brain (Mcllhenny, 1971) could explain the low ratio between the toxic and the centrally active doses of this compound. Such peripheral effects could also mediate strong actions on the brain. Diltiazem, because of its lower protein binding (Piepho et al., 1982), nifedipine (Sorkin et al., 1985) and, especially, flunarizine (Holmes et al., 1984), because of their higher lipid solubility, could penetrate the bloodbrain barrier to a greater extent than verapamil. These drugs also have a weaker effect on the blood pressure a n d / o r on the heart. On the other hand, flunarizine is not selective for the brain VSCC and verapamil has been found to interact with receptors other than VSCC, in particular 5-HT receptors (De Feudis, 1987). In this regard, diltiazem, nifedipine and flunarizine have been found to be relatively inactive or much less effective than verapamil (Auguet et al., 1986). Nifedipine could potentiate the effects of adenosine or interact with an adenosine receptor (Swanson and Green, 1986). Moreover, there is evidence to support an interaction between D-2 receptors and VSCC-functions. Diltiazem and, especially verapamil but not nifedipine, have been said to compete with D-2 dopaminergic agents for a common binding site on striatal membranes (De Vries and Beart, 1985). Therefore, in the present study, the absence of a linear correlation between the dose administered and some of the effects measured could be explained at least partly by the progressive involvement of various receptor types, with different sensitivities, in the mechanism of action of the compound. However, the fall in N E levels could be mostly due to the blockade of VSCC, since all the calcium antagonists of the first category, unlike flunarizine, induced similar effects. The effects of the drugs on brain DA content consisted of a moderate and inconsistent increase in various brain regions, with the exception of the striatum. Nifedipine, verapamil and diltiazem lowered the DOPAC levels and this effect could have been
196 due mostly to the blockade of VSCC, since these compounds, which are selective for the slow channels, induced similar effects, whereas flunarizine raised the D O P A C and H V A levels. These data regarding D A and its metabolites agree with those of other authors (Stefanini, 1988). Considering the trends of the D O P A C / D A and H V A / D A ratios, it appears that verapamil and, to a lesser extent nifedipine a n d diltiazem, inhibited whereas flunarizine activated dopaminergic neurotransmission. Flunarizine, however, behaved as a neuroleptic drug and it is k n o w n that neuroleptic drugs leave the D A levels u n c h a n g e d and enhance the brain content of D O P A C and H V A (Bartholini and Lloyd, 1980). All the calcium antagonists changed the brain content of 5 - H I A A according to the drug administered and the brain region studied. Flunarizine in particular, lowered while nifedipine enhanced the metabolite levels. Since the 5 - H T levels remained substantially unchanged, the 5 - H I A A / 5 - H T ratio decreased, suggesting that flunarizine impaired serotonergic neurotransmission. The ratio increased with nifedipine, suggesting that this calcium antagonist activated the serotonergic system in all the brain regions with the possible exception of the cortex. A clear increase of the 5 - H I A A / 5 - H T ratio was also occasionally induced b y verapamil. These observations agree with the finding that nifedipine, unlike verapamil, was c o m p a r a b l e with imipramine for its ability to reverse the shuttlebox escape deficit by inescapable shock ( G e o f f r o y et al., 1988). Concerning the mechanism of action, the nifedipine-induced increase in the 5 - H I A A levels and the signs of general activation of the serotonergic systems would be mostly p r o d u c e d by V S C C blockade. In fact, we observed in a previous study (Gaggi et al., 1989) that the same effects could be p r o d u c e d by reduction of neuronal calcium influx induced by salmon calcitonin leading to a marked and longlasting hypocalcaemia. Moreover, the nifedipineinduced enhancement of 5 - H I A A levels was linearly dose-related in all the brain sections. In conclusion, the administration of calcium antagonists to rats induced marked changes in the brain of levels of biogenic amines and metabolites. These changes, displaying regional specificity and
differences according to the various c o m p o u n d s , could not have been p r o d u c e d only b y the mechanism of V S C C blockade. Interactions of the comp o u n d s with other receptors should be at least partly involved and some effects could be induced by indirect actions. Nevertheless, since several calcium antagonists have been p r o p o s e d for treatm e n t of a variety of C N S disorders ( R a e b u r n and Gonzales, 1988), the present data could be useful to facilitate the choice of the most suitable comp o u n d for each therapeutic category.
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