- Email: [email protected]
Selectivity of the protective effects of dihydropyridine calcium channel antagonists against the ethanol withdrawal syndrome
Brain Research 930 (2002) 111–122 www.elsevier.com / locate / bres
Research report
Selectivity of the protective effects of dihydropyridine calcium channel antagonists against the ethanol withdrawal syndrome 1
W.P. Watson , H.J. Little* Drug Dependence Unit, Psychology Department, Durham University, Science Laboratories, South Road, Durham DH1 3 LE, UK Accepted 12 November 2001
Abstract Four dihydropyridine calcium channel antagonists were compared for their ability to protect against the hyperexcitability produced in mice by withdrawal from chronic ethanol treatment and to protect against seizures due to bicuculline or pentylenetetrazol. Comparison was also made of their effects on locomotor activity, body temperature and motor co-ordination, and with the corresponding effects of the benzodiazepine, diazepam. Nitrendipine, nimodipine, nicardipine (at 50 and 10 mg / kg) and isradipine (at 10 and 4 mg / kg) decreased the withdrawal hyperexcitability, but showed no anticonvulsant action against either bicuculline or pentylenetetrazol. Diazepam (1.5 and 4 mg / kg) both protected against the withdrawal signs and decreased seizure incidence after bicuculline and pentylenetetrazol, although the latter effects were of shorter duration than those on the withdrawal signs. The four dihydropyridines decreased spontaneous locomotor activity, an effect which lasted up to 6 h. Only isradipine and diazepam had any ataxic actions at the doses tested. All the dihydropyridines had hypothermic actions, considerably shorter in duration than effects on withdrawal hyperexcitability, with little evidence of dose dependence, except for nicardipine, which had a larger, dose-related, hypothermic action. Of the four compounds, isradipine was more potent in terms of dose, but not any more selective for effectiveness against the withdrawal signs, than the other three dihydropyridines, and nicardipine was slightly less effective in protecting against the withdrawal signs. The results indicate that the anticonvulsant effects of the dihydropyridines were selective for ethanol withdrawal hyperexcitability, whereas diazepam showed no such selectivity. 2002 Published by Elsevier Science B.V. Keywords: Calcium channel; Ethanol; Dihydropyridine; Anticonvulsant
1. Introduction Voltage-dependent neuronal calcium channels are divided into several subtypes, according to their activation and inactivation characteristics in cultured cells. The original classification showed the L-subtype of channel to be selectively blocked by dihydropyridine calcium antagonists, which are used therapeutically as hypotensive and antiarrythmic agents [22]. The application of this classification to vertebrate neurones is still in progress and, although many authors have shown the dihydropyridines to be selective for L-type channels, as defined by the electrophysiological characteristics of the channels, some reports have found them to affect other channel subtypes [1]. Abbreviations: PTZ, pentylenetetrazol; GABA, aminobutyric acid *Corresponding author. Fax: 144-191-374-7774. E-mail address: [email protected] (H.J. Little). 1 Current address: Neuropharmacology Department, H. Lundbeck A / S, Ottiliavej 9, 2500 Valby, Copenhagen, Denmark.
There are high affinity binding sites in the central nervous system for dihydropyridines, and these are thought to represent the voltage-sensitive calcium channels on neurones [17]. Dihydropyridine calcium channel antagonists have few overt effects on behaviour, but have been reported to possess some anticonvulsant actions. In acute studies, nitrendipine and nimodipine potentiated the ataxic and general anaesthetic effects of ethanol [4, 5]. The calcium channel activator, Bay K 8644, decreased the general anaesthetic actions of ethanol and other sedative / hypnotics [7]. Considerable evidence has also been published indicating that dihydropyridine-sensitive calcium channels play a role in physical dependence on ethanol. Chronic administration of ethanol increased the density of the high affinity binding sites in the CNS; the effects of dihydropyridines on transmitter release and phosphatidylinositol turnover were also increased [6]. Nitrendipine prevented the hyperexcitability produced
0006-8993 / 02 / $ – see front matter 2002 Published by Elsevier Science B.V. PII: S0006-8993( 02 )02236-9
112
W.P. Watson, H. J. Little / Brain Research 930 (2002) 111 – 122
by withdrawal from long-term ethanol administration, both in vivo [15] and in isolated neuronal preparations [28]. The stereospecificity of these effects was studied with the isomers of isradipine, and found to follow the same pattern as that for calcium channel blockade [15, 28, 11]. When given chronically, concurrently with ethanol, nifedipine and nitrendipine prevented the development of ethanol tolerance [5] and nitrendipine prevented the withdrawal syndrome, effects that were not due to residual dihydropyridine in the brain at the times of testing [31]. The acute effect of nitrendipine and isradipine on ethanol withdrawal hyperexcitability is not shared by all drugs that block L-type calcium channels. The benzothiazepine, diltiazem, increased the withdrawal hyperexcitability [23]. Diltiazem binds at a different site on the L-type calcium channel complex and increases, rather than decreasing, binding of dihydropyridines [32]. This compound also increased the severity of seizures caused by bicuculline and pentylenetetrazol [24]. A different pattern was seen with the dihydropyridine, felodipine, which did not protect against the alcohol withdrawal syndrome but did possess anticonvulsant activity against bicuculline and pentylenetetrazol seizures [26]. In order to determine the extent to which the ability of dihydropyridine calcium channel antagonists to protect against ethanol withdrawal hyperexcitability is shared by other members of this drug group, the current project compared the acute effects of nitrendipine, nimodipine, isradipine and nicardipine. The ability to protect against ethanol withdrawal signs seizures was compared with effects on seizures produced by the convulsant drugs, bicuculline and pentylenetetrazol, and effects on spontaneous locomotor activity, ability to cause ataxia and body temperature. The aims were, firstly, to determine the selectivity of the ability of the compounds to prevent ethanol withdrawal signs, compared with anticonvulsant properties against the actions of the convulsant drugs, and, secondly, to see if there was any selectivity in their anticonvulsant effects compared with their sedative, ataxic and hypothermic actions. Comparison was also made with the benzodiazepine, diazepam, as this type of compound is used clinically to treat alcohol withdrawal hyperexcitability. The doses were chosen on the basis of previous experience with the drugs in the prevention of ethanol withdrawal signs in rodents.
2. Methods Male mice of the TO strain were used throughout, obtained from Bantin and Kingman, UK. All were kept in a 12 h light–dark cycle with access ad libitum to tap water and laboratory rodent chow (CRM). The weight range was 25–40 g, with not more than a 5-g range in any single experiment. In all studies involving behavioural ratings,
the observer who carried out the rating allocation was blind to the prior drug treatment.
2.1. Drugs used Nitrendipine, nimodipine, (Bayer), nicardipine (Yamanouchi) and isradipine (Sandoz) were suspended in 0.5% Tween 80 and sonicated. The dihydropyridines were kept in covered vials and protected from light. Pentylenetetrazol (PTZ) (Sigma) and diazepam (Roche) were dissolved in isotonic saline. Bicuculline (Sigma) was made up as a 10 mg / ml stock solution in 1 M HCl, and was then further dissolved in saline brought to pH 3 with HCl. All drugs were stirred before injection, and sonicated where necessary. The following doses of drugs were tested: nitrendipine, nimodipine and nicardipine 10 and 50 mg / kg; isradipine 4 and 10 mg / kg; diazepam 1.5 and 4 mg / kg. All compounds were administered intraperitoneally (i.p.) in a dose volume of 10 ml / kg. The pretreatment times are given in each methods section.
2.2. Ethanol withdrawal syndrome Ethanol was administered in a liquid diet schedule (Dyets, Pennsylvania, USA; [14]). All mice received control diet for an initial 2-day period. Ethanol-treated mice then received a diet containing 7% (v / v) ethanol for either 10 days or 3 weeks, in order to provide two levels of severity of the withdrawal syndrome. Control groups were pair-fed a control diet, balanced isocalorifically to match the ethanol-containing diet [14]. There were no differences in the weights of the ethanol-treated and control mice at the end of the treatment periods. The amount of ethanol consumed by the groups of mice was between 21 and 27 g / kg / 24 h. The dihydropyridines and diazepam were administered immediately on withdrawal of the six groups of mice from the chronic alcohol treatment. Two withdrawal studies were carried out, one after 3 weeks chronic ethanol treatment, that gave pronounced withdrawal signs, and one after 10 days chronic ethanol administration, that gave a more mild withdrawal. The severity of the withdrawal syndromes was measured by ratings of behaviour produced by handling. This method, originated by Goldstein and Pal [9], measures tremor, myoclonic jerks and clonic convulsive behaviour and was modified for the present use [15,10,25]. The mice were gently picked up by the tail for 30 s and the behaviour rated according to the scale given below. Untreated mice normally score 0 or 1 on this rating scale. The results were expressed as median values6interquartile ranges. Each treatment group contained 10–12 mice. Behavioural ratings 1. Mild tremor on lifting and turning
W.P. Watson, H. J. Little / Brain Research 930 (2002) 111 – 122
2. Continuous severe tremor on lifting and turning 3. Clonic forelimb extensor spasm on lifting 4. Clonic forelimb extensor spasm on lifting, which continued after placing mouse on cage top. 5. Spontaneous evidence of myoclonic activity followed by (4).
2.3. Anticonvulsant actions The anticonvulsant actions of the dihydropyridines were studied against bicuculline and pentylenetetrazol, measured by convulsion incidence and latency after intraperitoneal injection of the convulsants. Because the aim was to compare the effects of the dihydropyridines with their actions on ethanol withdrawal hyperexcitability, the convulsants were given 30 min after dihydropyridine administration, at which other effects of the dihydropyridines (see temperature measurement results) were known to peak and at 5 h after dihydropyridine administration, which corresponded to the time interval between administration of drugs at the beginning of the withdrawal period and the peak of the withdrawal hyperexcitability. Each group of eight mice was pretreated with either the vehicle (Tween 80) or a dihydropyridine or diazepam. The animals were then injected, after the appropriate pretreatment time, with either bicuculline, 5 mg / kg or PTZ 70 mg / kg. The number of mice exhibiting clonic seizures (resulting in a loss of posture) and the time to the beginning of a clonic convulsion (latency) were recorded for the next 60 min. The effects of the dihydropyridines were also examined on the dose response curves to bicuculline; a range of doses (4, 4.5 or 5 mg / kg) of bicuculline were given and the convulsion incidence measured as above. In all cases, the animals were humanely killed as soon as the first sign of a seizure was seen or at 60 min after administration of the convulsant.
2.4. Ataxic actions The ataxic actions of the compounds were measured by the rotorod method. Mice were placed on a rod rotating at 4.5 rpm, and the time they remained on the rod was measured automatically. A cut-off time of 180 s was used in all experiments. Measurements were made at 10-min intervals after the acute administration of the drug under test, until the ability to remain in the rod had returned to control values. Eight mice were used in each treatment group. Before the acute drug injections, the mice were tested on the rotorod to ensure that they stayed on for 180 s; only a very small number did not do so and were excluded from the studies.
2.5. Effects on spontaneous locomotor activity The effects of the compounds on spontaneous locomotor activity were determined automatically by breaking of
113
infra-red beams. Measurements were made of total activity (total beam breaks) and ambulatory activity (breaking of consecutive beams), measured for 60 min. Six mice were used for each treatment group, with the exception of the vehicle treated groups in which the n values were increased in some instances in order to maintain comparability over days. Separate groups of mice were used for each set of measurements. In order to study the time course of the drug actions, the treatments were given between 8.00 and 9.00 am. Pretreatment times used for the dihydropyridines and for diazepam were 0, 1, 3, 5 and 7 h from drug administration, and comparisons were made with control results from mice injected with the vehicle and tested concurrently.
2.6. Effects on body temperature Body temperatures were measured by rectal probe. The gentle handling of mice necessary for weighing and coding causes the body temperature to rise [21]. An experimental schedule was therefore adopted in which the temperatures of mice were measured immediately they were removed from the cages. They were then weighed and coded, and the temperatures measured again 20 min after the initial measurement. Drug injections were then given, and the temperatures measured every 20 min for 2 h. If the temperatures were still significantly altered at 2 h, they were measured again at 3 h to see if they had returned to control values. Care was taken that the room temperature was constant for all the experiments.
2.7. Statistical analysis The ratings of withdrawal hyperexcitability were compared by nonparametric two-way analysis of variance, using a format designed for repeat measurements on the same samples and multiple comparisons with a control [16]. The effects of the drugs on withdrawal hyperexcitability were also expressed as area under the curve, to evaluate the overall effectiveness of the compounds, and these results were compared by one-way analysis of variance followed by posthoc Dunnett’s test. The incidence of convulsions were compared by Fisher’s exact probability test and latencies to convulsions by one-way analysis of variance followed by posthoc Dunnett’s test. The results from the dose response studies with bicuculline were analysed by probit analysis ( MLP program, Numerical Algorithum Group; [34]). The rotorod results at individual time points were compared by the Mann–Whitney U test for comparisons with control values at each time interval. One-way analysis of variance was used for comparisons at each time interval of the locomotor activity counts and the body temperatures, followed by posthoc Dunnett’s test.
114
W.P. Watson, H. J. Little / Brain Research 930 (2002) 111 – 122
3. Results
3.1. Ethanol withdrawal syndrome The effects of the drugs on hyperexcitability in TO mice caused by withdrawal from chronic ethanol treatment are illustrated in Fig. 1, which shows the severe withdrawal signs seen after cessation of 3 weeks ethanol intake and the milder syndrome seen after only 10 days ethanol consumption. An example of the pattern of the withdrawal signs and the protection by one of the dihydropyridines (nitrendipine) is illustrated in Fig. 1a, while Fig. 1b shows the effects of all the drugs expressed as area under the curve, for both withdrawal experiments. It can be seen in Fig. 1b that the effects of the four dihydropyridines, at the doses tested in decreasing the withdrawal signs were very similar in magnitude. In the first experiment (severe withdrawal) nitrendipine, nimodipine and nicardipine, at 50 mg / kg, and isradipine, at 10 mg / kg all gave significant protection against the withdrawal hyperexcitability (P,0.05 for results up to 10 h after injection). Diazepam also gave significant protection when administered at 4 mg / kg (P,0.05). None of the lower doses of the dihydropyridines gave significant protection against the more substantial withdrawal hyperexcitability in the first experiment but the effects of these doses could be seen in the second withdrawal study (mild withdrawal). As had been found previously, the milder withdrawal signs seen after the shorter period of chronic alcohol consumption appeared earlier and were of shorter duration than the more severe syndrome. Statistical comparisons were therefore made only up to 5 h from withdrawal, up to which time there was a significant increase in hyperexcitability ratings in the mice receiving vehicle treatment, compared with values for control animals that had not been given alcohol. At 10 mg / kg, nitrendipine and nimodipine significantly decreased the withdrawal severity (P,0.05) for comparisons up to 5 h. Isradipine, at 4 mg / kg, also significantly decreased the ratings. Nicardipine, at 10 mg / kg, however, did not have any significant effect on the withdrawal while diazepam, at 1.5 mg / kg gave significant protection (P,0.05).
3.2. Anticonvulsant actions 3.2.1. Effects on the convulsant actions of bicuculline The effects of the drugs on convulsions due to bicuculline, 5 mg / kg, are shown in Table 1; the compounds were administered either 30 min or 5 h before the bicuculline. At the 30-min interval, there were no significant changes in convulsion incidence with any of the compounds, except diazepam, which provided good protection at both 1.5 and 4 mg / kg (P,0.01 for both doses). As the higher doses of the dihydropyridines (50 mg / kg of nitrendipine, nimodipine and nicardipine, 10 mg / kg of isradipine) did
not have any significant effects against bicuculline seizures, the lower doses of these drugs were not tested. At the 5-h interval, none of the dihydropyridines significantly altered the incidence of, or latencies to, clonic convulsions (P.0.05). Following administration of nitrendipine at 50 mg / kg, however, the incidence of convulsions was 3 / 8, which just failed to reach statistical significance when compared with the control value (P50.06). At this pretreatment time, diazepam, at 4 mg / kg, again protected against bicuculline convulsions (P,0.01). In order to examine their actions in more detail, the effects of the compounds on the dose response curves to bicuculline were measured 5 h after administration of the compounds. The ED50 values derived from the probit analysis are given in Table 2. Statistical analysis of these data, however, did not prove possible because the lines differed significantly from homogeneity (P,0.05).
3.2.2. Effects on the convulsant actions of PTZ Table 3 shows the effects of the compounds on convulsions due to PTZ. At the 30-min pretreatment interval, none of the dihydropyridines had any significant effects on convulsion incidence or latency. Diazepam significantly decreased convulsion incidence when given at either 1.5 or 4 mg / kg (P,0.01 for each dose). The only significant change when the drugs were administered 5 h before the PTZ, was a decrease in convulsion incidence after nitrendipine, 50 mg / kg (P,0.05). As the higher doses of the dihydropyridines did not have any significant effects against PTZ seizures, the lower doses of these drugs were not tested. 3.3. Ataxic actions At the lower doses tested (i.e. nitrendipine, nimodipine and nicardipine at 10 mg / kg, and isradipine at 4 mg / kg), none of the dihydropyridine compounds had any significant effects on rotorod performance up to 2 h after administration (data not shown). The effects of the higher doses are illustrated in Fig. 1c. At 10 mg / kg, isradipine caused significant ataxia at 20 min after injection (P,0.05, compared with control values) but not at later times, while nitrendipine, nicardipine and nimodipine had no significant effects at 50 mg / kg. Diazepam at both 1.5 (data not shown) and 4 mg / kg (Fig. 1c) significantly lowered the times the mice were able to stay on the rotating rod (P,0.05, compared with control values at 20 min for 1.5 mg / kg and 20, 40 and 60 min for the 4 mg / kg dose).
3.4. Effects on spontaneous locomotor activity The effects of the compounds on spontaneous locomotor activity are illustrated in Fig. 2 (total activity) and Fig. 3 (ambulatory activity). The times indicated on the graphs are the times at which each set of measurements was completed, i.e. the end of each 60-min measuring period.
W.P. Watson, H. J. Little / Brain Research 930 (2002) 111 – 122
115
Fig. 1. Effects of the drugs against responses to handling during ethanol withdrawal. (A) Effects of nitrendipine (j) and of Tween vehicle (n) given after withdrawal from chronic ethanol treatment. Values are median scores. (B) Effects of the drugs against ethanol withdrawal hyperexcitability expressed as mean areas under the curve for the ratings of the responses to handling. The shaded columns show the results for the more severe withdrawal syndrome and the effects of the higher doses of the compounds: nitrendipine (NITR), nimodipine (NIM) and nicardipine (NIC) at 50 mg / kg, isradipine (ISR) at 10 mg / kg and diazepam (DZ) at 4 mg / kg. The open column shows the results for the milder withdrawal syndrome and the effects of the lower doses of the compounds: nitrendipine, nimodipine and nicardipine at 10 mg / kg, isradipine at 4 mg / kg and diazepam at 1.5 mg / kg. *, Significant (P,0.05) decreases in withdrawal hyperexcitability compared with ratings after administration of vehicle. The incidence of hyperexcitability was significant for both withdrawals compared with values for mice not treated with ethanol (P,0.01). (C) Effects of the drugs on the ability to stay on a rotating rod. Values are means6standard error of the mean (S.E.M.) Nitrendipine (j), nimodipine (m), and nicardipine (h) given at 50 mg / kg, isradipine (d) at 4 mg / kg, diazepam (3) at 4 mg / kg and Tween vehicle (n).
W.P. Watson, H. J. Little / Brain Research 930 (2002) 111 – 122
116
Table 1 Seizures produced by bicuculline (5 mg / kg) with 30 min and 5 h pretreatment Treatment
Incidence
Latency
S.E.M
30 min pretreatment Tween Nit 50 Nim 50 Nic 50 ISR 10 DZ 1.5 DZ 4
8/8 7/8 7/8 6/8 7/8 1 / 8 (P,0.01) 2 / 8 (P,0.01)
252 212 227 182 238 30 93, 120
60 86 46 30 37
5 h pretreatment Tween Nit 50 Nim 50 Nic 50 ISR10 DZ 4
7/8 3 / 8 (P50.06) 5/8 4/8 5/8 2 / 8 (P,0.01)
119 123 149 149 128 128, 183
– 14 11 22 18 28
Tween, Tween vehicle; Nit, nitrendipine; Nim, nimodipine; ISR, isradipine; DZ, diazepam; numbers after the drug names indicate doses in mg / kg. Latency (min), time to start of first convulsion. Table 2 ED50 doses of bicuculline required for 50% convulsions Drug
ED50
95% Limits
Tween Nit 50 Nim 50 Nic 50 ISR 10 DZ 4
3.87 6.8 4.15 4.82 4.77 6.91
(2.48–5.36) (4.61–8.99) (2.88–5.42) (3.56–6.07) (4.46–5.59) (3.55–10.25)
Significant decreases in activity were seen with nitrendipine, for the 10 mg / kg dose only between 0 and 1 h after injection (P,0.05) and for the 50 mg / kg dose, from 0 to 6 h after injection (P,0.01 for 1–4 h; P,0.05 for 6 h) (Figs. 2a and 3a). For nimodipine, significant decreases in activity were seen for both the 10 and 50 mg / kg doses until 4 h after administration (P,0.01) (Figs. 2b and 3b). Table 3 Seizures produced by PTZ 70 (mg / kg) with 30 min and 5 h pretreatment Drug
Incidence
Latency
S.E.M
30-min Pretreatment Tween Nit 50 Nim 50 Nic 50 ISR 10 DZ 1.5 DZ 4
8/8 5/8 8/8 6/8 6/8 1 / 8 (P,0.01) 1 / 8 (P,0.01)
139 219 286 162 166 30 44
34 68 55 22 10 – –
5-h Pretreatment Tween Nit 50 Nim 50 Nic 50 ISP 10 DZ 4
8/8 4 / 9 (P,0.05) 8/8 6/8 8/8 8/8
137 168 119 128 186 219
14 36 20 18 53 54
Nicardipine, at 10 mg / kg significantly decreased the activity up to 2 h from administration (P,0.01 at 1 h; P,0.05 at 2 h), and at 50 mg / kg up to 6 h after the injections (Figs. 2d and 3d). At both 4 and 10 mg / kg, isradipine significantly decreased the activity counts up to 6 h after administration (P,0.01 at 1–4 h; P,0.05 at 6 h) (Figs. 2c and 3c). Diazepam, at 1.5 or 4 mg / kg did not alter locomotor activity (data not shown). In terms of magnitude of effect, the largest absolute decreases in activity were seen with nitrendipine and nicardipine, both at 50 mg / kg (Figs. 2a and d, 3a and d). The time of maximal effect of the higher dose of each dihydropyridine appeared to be between 2 and 4 h after administration, for both total activity measures and the measurements of ambulatory activity. The lower doses had effects that were significant for shorter times than the higher doses, as would have been expected, but this was most noticeable for nitrendipine, as the 10 mg / kg dose of this compound had significant effects only up to 1 h after injection. The activity of the control animals did not vary significantly between the different measurement times (P. 0.05).
3.5. Effects on body temperature The effects of the compounds on body temperature are illustrated in Fig. 4. Nitrendipine (Fig. 4a), significantly lowered the temperatures for up to 80 min at 10 mg / kg (P,0.05) and up to 120 min at 50 mg / kg (P,0.01 up to 80 min; P,0.05 at 120 min). Nimodipine (Fig. 4b) lowered temperatures were significant only at 20 and 40 min from injection (P,0.05, both doses, both time intervals) and the 50 mg / kg dose of nimodipine had only a little greater effect than the 10 mg / kg dose. The effects of isradipine (Fig. 4c) were similar to those of nimodipine, with both the 4 and 10 mg / kg doses having significant effects up to 80 min (both doses: P,0.01 up to 60 min; P,0.05 for 80-min interval). The maximal reductions for all three of these dihydropyridines were seen at 40 min from injection. The effect of nicardipine (Fig. 4d) differed from that of the other three dihydropyridines, in that the effect of the higher dose (50 mg / kg) was much larger and there was also a clear dose relationship. At 10 mg / kg nicardipine lowered body temperature significantly at 20 and 40 min (P,0.01) and at 50 mg / kg the effect was significant at all times up to 240 min (P,0.05) but the temperatures had returned to control values at 360 min. Diazepam did not significantly alter body temperature (data not shown).
4. Discussion The dihydropyridines, nitrendipine, nimodipine and nicardipine all provided protection against the hyperexcitability caused by withdrawal from chronic ethanol treat-
W.P. Watson, H. J. Little / Brain Research 930 (2002) 111 – 122
117
Fig. 2. Effects of the drugs on measures of total locomotor activity. Values are means6S.E.M. Controls (vehicle injection) illustrated by n throughout. (A) Effects of nitrendipine at 10 (j) and 50 (j) mg / kg; (B) effects of nimodipine at 10 (m) and 50 (m) mg / kg; (C) effects of isradipine at 4 (d) and 10 (d) mg / kg, and (D) effects of nicardipine 10 (d) and 50 (d) mg / kg.
ment. This effect was prolonged, lasting for 10 h for the effects of the higher doses (50 mg / kg of nitrendipine, nimodipine and nicardipine and 10 mg / kg of isradipine) on the more severe withdrawal syndrome and included prevention of the peak withdrawal hyperexcitability which occurred between 8 and 10 h after cessation of alcohol consumption. Isradipine was more effective in terms of dose than the other three calcium channel antagonists, providing good protection against the withdrawal syndrome when given at 10 mg / kg, rather than 50 mg / kg
that was required for the other three dihydropyridines. Nitrendipine, nimodipine and nicardipine had similar effects at the higher dose (50 mg / kg of each compound) against the more severe syndrome, and nitrendipine and nimodipine at 10 mg / kg decreased the milder withdrawal signs. Nicardipine at 10 mg / kg, however, did not have any significant effect on the milder syndrome. The protective effects against withdrawal hyperexcitability of the four dihydropyridines were selective, as they did not protect against the chemically-induced convulsions,
118
W.P. Watson, H. J. Little / Brain Research 930 (2002) 111 – 122
Fig. 3. Effects of the drugs on ambulatory locomotor activity. Values are means6S.E.M. Controls (vehicle injection) illustrated by n throughout. (A) Effects of nitrendipine at 10 (j) and 50 (j) mg / kg; (B) effects of nimodipine at 10 (m) and 50 (m) mg / kg; (C) effects of isradipine at 4 (m) and 10 (m) mg / kg and (D) effects of nicardipine 10 (d) and 50 (d) mg / kg.
in contrast to diazepam. The doses of chemical convulsants were chosen to be just submaximal for the production of clonic convulsions. It is debatable whether or not the severity of those effects can be directly compared with the withdrawal syndrome, since during the latter, full convulsions are not seen in all animals even during the more severe withdrawal. However, the selectivity of the dihydropyridines for protection against the withdrawal hyperexcitability was apparent, and this lasted for 8–10 h. The selectivity of the dihydropyridines for ethanol withdrawal hyperexcitability is compatible with the lack of
effect of nitrendipine on neuronal hyperexcitability in vitro produced by bicuculline, compared with the effectiveness of the same nitrendipine concentration to prevented neuronal excitability due to withdrawal from chronic alcohol consumption [28]. We have recorded synaptic potentials in vitro following chronic ethanol treatment in vivo and showed that the changes underlying ethanol withdrawal hyperexcitability consist of increases in excitatory amino acid transmission [30,18] and increased conductance of voltage sensitive calcium channels [29]. We found no decreases in GABA mediated inhibitory potentials [30,18].
W.P. Watson, H. J. Little / Brain Research 930 (2002) 111 – 122
119
Fig. 4. Effects of the drugs on body temperature. Values are means6S.E.M. Controls (vehicle injection) illustrated by n throughout. (A) Effects of nitrendipine at 10 (j) and 50 (j) mg / kg; (B) effects of nimodipine at 10 (m) and 50 (m) mg / kg; (C) effects of isradipine at 4 (d) and 10 (d) mg / kg and (D) effects of nicardipine 10 (h) and 50 (h) mg / kg.
120
W.P. Watson, H. J. Little / Brain Research 930 (2002) 111 – 122
The present results therefore support our earlier conclusion, that the ethanol withdrawal syndrome in mice does not involve decreases in GABA transmission. The fact that diazepam gave protection against the alcohol withdrawal signs was not evidence that these signs were caused by decreases in GABA transmission, even though the actions of diazepam are produced by potentiation of the effects of endogenous GABA. Benzodiazepines are anticonvulsant against seizures due to a variety of causes and the increases in GABA transmission that these drugs produce can balance out changes in other neurotransmitter systems. Effects of dihydropyridine calcium channel antagonists have been reported previously on PTZ seizures, but the effects are not very consistent. O’Neill and Bolger [19] found increased latency to seizure but not seizure incidence when nimodipine 10 and 20 mg / kg (30 min pretreatment) was tested against PTZ 85 mg / kg. Palmer et al. [20], however, reported protective effects of nitrendipine, nicardipine and nimodipine against 85 mg / kg PTZ, with ED50 values for the dihydropyridines of 15–30 mg / kg (30 min pretreatment times). Zapater et al. [33] found effects of nimodipine 30, 100 and 300 mg / kg on seizure latency but not seizure incidence. Chakrabarti et al. [3] reported effects of nimodipine at 5 mg / kg against PTZ doses from 40 to 75 mg / kg (15 min pretreatment time) but a synergistic interaction with the alcohol (dose 2 g / kg) used to dissolve the drug were not excluded. The reason for the differences within these reports and between these and the present study is not clear; it is possible there are species or strain differences. The effect of the dihydropyridines on locomotor activity was seen primarily at the earlier time periods, with the effect of the higher dose of each compound wearing off by the 5–6 h time interval and no longer significant by the 7–8 h time interval. This is in some contrast with the effects of the dihydropyridines on the withdrawal syndrome when protective effects were seen up to 10 h after administration. The results suggest that the mechanism by which dihydropyridine calcium channel antagonists prevent the withdrawal signs is different from that by which they decrease locomotor activity. Between the four dihydropyridines, there was little difference between the magnitude of the effects of the higher doses on locomotor activity, although nimodipine at 50 mg / kg had slightly less effect. The lower doses, however, showed more variation, with nitrendipine and nicardipine at 10 mg / kg causing smaller magnitude of activity decreases than nimodipine at 10 mg / kg or isradipine at 50 mg / kg. This might suggest that the dose response relationships for effects on locomotor activity differ between the compounds, but as only two doses were tested this is, as yet, speculation. The dihydropyridines, with the exception of isradipine, did not have effects on motor co-ordination while in contrast, diazepam had considerable ataxic effects. This suggests that the effects of the dihydropyridines on the
tremor and myoclonic jerks involved in the ethanol withdrawal syndrome are unlikely to be related to any effects on motor systems. It also shows that the effects of the dihydropyridines on exploratory locomotor activity, described above, were not due to changes in motor coordination. The hypothermic actions of all the dihydropyridine calcium channel antagonists followed completely different time courses to the protection against ethanol withdrawal hyperexcitability. The latter lasted 10 h while the hypothermic effects were maximal at 40 min and, with the exception of nicardipine, were no longer significant by 2 h after the drug administration. Nicardipine at 50 mg / kg had a considerably larger effect than the other dihydropyridines showed a clearer dose relationship. The hypothermic effects are likely to have been largely due to vasodilation, although there may have been some central component. These results therefore suggest that the effects of the compounds in ethanol withdrawal are not due to their effects on body temperature and are unlikely to be due to their vascular actions. The effects of drugs on temperature and on locomotor activity are linked in mice, as one form of thermoregulation is brought about by the animals huddling together. However, the time course of the locomotor changes far outlasted the hypothermic effects of the compounds. After injection of the drugs, the animals were kept in group cages until they were transferred to novel cages for the locomotor activity measurements. The doses of the dihydropyridines required for central actions, such as prevention of alcohol withdrawal signs, are higher than those that produce cardiovascular doses. However, the central concentrations of the drugs after administration of the doses used in the present study correspond closely to the concentrations that are required to affect neuronal activity in vitro, in electrophysiological recordings from brain slices [5,31]. This difference may be due to the differences between tissues in the time spent by cells in a depolarised state. The binding affinities of the dihydropyridines reported for rodent brain tissue are as follows: the Kd for [ 3 H]nitrendipine was 160 pM for cerebrocortical tissue [8], that of [ 3 H]-nimodipine 1.11 nM for brain membranes [2] and that of [ 3 H]-isradipine 35 pM for cerebrocortical tissue [13]. The latter authors made a direct comparison between [ 3 H]-isradipine and [ 3 H]-nitrendipine and found the Kd for the latter compound to be 372 pM. A similar comparison was made by the authors of [27] who found the Kd of rat brain membranes for [ 3 H]-isradipine was 15.6 pM and that of [ 3 H]-nitrendipine was 124 pM and that the two compounds bound to the same number of receptors. The affinities of isradipine and nitrendipine therefore parallels the difference in potency between these two compounds seen in vivo in the present studies. The reported affinity of nimodipine for the binding sites, however, is higher than that of nitrendipine, but these two compounds had very similar potencies in vivo. However, although the authors of
W.P. Watson, H. J. Little / Brain Research 930 (2002) 111 – 122
[2] reported a Kd of 1.11 nM for nimodipine, there were differences between brain regions, with some areas showing lower Kd values. A literature search failed to reveal a Kd value for [ 3 H]-nicardipine binding to brain tissue, but Kanami et al. [12] found the Ki values for competition for binding of brain tissue were very similar for nicardipine, nimodipine and nitrendipine against [ 3 H]-nitrendipine binding. Dihydropyridine binding, however, is affected by both depolarisation and by the surrounding concentrations of calcium ions. Homogenate binding is of necessity carried out on fully depolarised neurones, so direct comparisons between in vitro binding affinity and in vivo potency are not fully valid. In addition, many of the dihydropyridines are stereospecific. The drugs used in the present studies were the racemates, because it was not possible to obtain sufficient of the isomers to carry out extensive in vivo studies. We have previously shown that the effect of isradipine on alcohol withdrawal signs is selective for the (1)-isomer [15]. Of the binding affinities quoted above, nitrendipine was reported to show an approximately 20fold difference between Kd values for the two isomers, but there was only a two-fold difference between binding of the isomers of nimodipine [2]. Lee et al. [13] used (1)isradipine for their binding studies and reported a 240-fold lower affinity for the (2)-isomer of isradipine. Nicardipine showed a tenfold difference in the potency of its isomers in displacing (1)-[ 3 H]-isradipine [13]. Overall, in this study, the four dihydropyridines showed fairly similar profiles of action, but nicardipine had slightly more effect on locomotor activity, considerably more effect on body temperature and slightly less effect on the alcohol withdrawal syndrome than the other three compounds. Selectivity was clearly evident for all four compounds in protection against ethanol withdrawal seizures, compared with effectiveness against seizures produced by bicuculline or PTZ.
Acknowledgements We thank the Medical Research Council UK for financial assistance with this work.
References [1] N. Akaike, P.G. Kostyuk, Y.V. Osipchuk, Dihydropyridine sensitive low-threshold calcium channels in isolated rat hypothalamic neurones, J. Physiol. 412 (1989) 181–195. [2] P. Bellman, A. Schade, R. Towart, Dihydropyridine receptor in rat brain labelled with [ 3 H-]-nimodipine, Proc. Natl. Acad. Sci. 80 (1983) 2356–2360. [3] A. Chakrabarti, H.K Saini, S.K. Garg, Dose-finding study with nimodipine: a selective central nervous system calcium channel blocker on aminophylline induced seizure models in rats, Brain Res. Bull. 45 (1998) 495–499.
121
[4] S.J. Dolin, H.J. Little, Augmentation by calcium channel antagonists of general anaesthetic potency in mice, Br. J. Pharmacol. 88 (1986) 909–914. [5] S.J. Dolin, H.J. Little, Are changes in neuronal calcium channels involved in ethanol tolerance?, J. Pharmacol. Exp. Ther. 250 (1989) 985–991. [6] S.J. Dolin, H.J. Little, M.J. Hudspith, C. Pagonis, J.M. Littleton, Increased dihydropyridine-sensitive calcium channels in rat brain may underlie ethanol physical dependence, Neuropharmacology 26 (1987) 275–279. [7] S.J. Dolin, M.J. Halsey, H.J. Little, Effects of the calcium channel activator, Bay K 8644, on general anaesthetic potency in mice, Br. J. Pharmacol. 94 (1988) 413–422. [8] F.J. Erlert, E. Itoga, The interaction of [ 3 H]-nitrendipine with receptors for calcium antagonists in the cerebral cortex and heart of rats, Biochem. Biophys. Res. Commun. 104 (1982) 937–943. [9] D.B. Goldstein, N. Pal, Alcohol dependence produced in mice by inhalation of ethanol: Grading the withdrawal reaction, Science 172 (1971) 288–290. [10] A. Green, E.M. Davies, M.A. Whittington, H.J. Little, A.J. Cross, Action of chlormethiazole in a model of ethanol withdrawal, Psychopharmacology 102 (1990) 239–242. [11] R.P. Hof, U.T. Ruegg, A. Hof, A. Vogel, Stereoselectivity at the calcium channel: opposite actions of the enantiomers of a 1,4dihydropyridine, J. Cardiovasc. Pharmacol. 7 (1985) 689–693. [12] J. Kinami, Y-L. Qu, H. Tsuchihashi, T. Nagatomo, T. Maniwa, A. Miyagishi, Assessment of Ca 21 antagonist effects of SM-6586 and its isomers, novel 1,4-dihydropyridine derivatives, by radioligand binding assay, Jpn. J. Pharmacol. 58 (1992) 75–78. [13] H.R. Lee, W.R. Roeske, H.I. Yamamura, High affinity binding [1H]-PN 200-110 binding to dihydropyridine receptors associated with calcium channels in rat brain, Life Sci. 35 (1984) 721–732. [14] C.S. Lieber, L.M. Decarli, Liquid diet technique of ethanol administration: 1989 update, Alc. Alcohol 24 (1989) 197–211. [15] J.M. Littleton, H.J. Little, M.A. Whittington, Effects of dihydropyridine calcium channel antagonists in ethanol withdrawal; doses required, stereospecificity and actions of Bay K 8644, Psychopharmacology 100 (1990) 387–392. [16] R. Meddis, Statistics Using Ranks: A Unified Approach, Basil Blackwell, Oxford, New York, 1984. [17] R.J. Miller, Voltage-sensitive calcium channels, J. Biol. Chem. 267 (1992) 1403–1406. [18] A. Molleman, H.J. Little, Increases in non-NMDA glutaminergic transmission, but no change in GABAB transmission, during withdrawal from chronic ethanol treatment, J. Pharmacol. Exp. Ther. 274 (1995) 1035–1041. [19] S.K. O’Neill, G.T. Bolger, The effects of dihydropyridine calcium channel modulators on pentylenetetrazol convulsions, Brain Res. Bull. 25 (1990) 211–214. [20] G.C. Palmer, M.L. Stagnitto, R.K. Ray, M.A. Knowles, R. Harvey, G.E. Garske, Anticonvulsant properties of calcium channel blockers in mice: N-methyl-D,L-aspartate-and Bay K 8644-induced convulsions are potently blocked by the dihydropyridines, Epilepsia 34 (1993) 372–380. [21] S.C. Taylor, H.J. Little, D.J. Nutt, N.A. Sellars, Benzodiazepine agonist and a contragonist have hypothermic effects in mice, Neuropharmacology 24 (1985) 69–73. [22] R.W. Tsien, D. Lipscombe, D.V. Madison, K.R. Bley, A.P. Fox, Multiple types of neuronal calcium channels and their selective modulation, Trends Neurosci. 11 (1988) 431–438. [23] W.P. Watson, H.J. Little, Interactions between diltiazem and ethanol: differences from those seen with dihydropyridine calcium channel antagonists, Psychopharmacology 114 (1994a) 329–336. [24] W.P. Watson, H.J. Little, Effects of diltiazem in convulsive states differ from those previously reported for dihydropyridine calcium channel antagonists, Psychopharmacology 114 (1994b) 321–328. [25] W.P. Watson, H.J. Little, Identification of distinct components, with
122
[26]
[27]
[28]
[29]
[30]
W.P. Watson, H. J. Little / Brain Research 930 (2002) 111 – 122 different timecourses, of convulsion threshold changes during ethanol withdrawal, J. Pharmacol. Exp. Ther. 272 (1995) 876–884. W.P. Watson, A. Misra, H.J. Little, A.R. Green, The differential effects of felodipine and nitrendipine on cerebral dihydropyridine binding ex vivo and the ethanol withdrawal syndrome, Br. J. Pharmacol. 112 (1994) 1017–1024. G.A. Weiland, R.E. Oswald, The mechanism of binding of dihydropyridine calcium channel blockers to rat brain membranes, J. Biol. Chem. 260 (1985) 8456–8464. M.A. Whittington, H.J. Little, A calcium channel antagonist stereoselectively decreases ethanol withdrawal hyperexcitability, but not that due to bicuculline, in hippocampal slices, Br. J. Pharmacol. 103 (1991) 1313–1320. M.A. Whittington, H.J. Little, Changes in voltage-operated calcium channels modify ethanol withdrawal hyperexcitability in mouse hippocampal slices, Exp. Physiol. 78 (1993) 347–370. M.A. Whittington, J.D.C. Lambert, H.J. Little, Increased NMDAreceptor and calcium channel activity during ethanol withdrawal hyperexcitability, Alcohol Alcoholism 30 (1995) 105–114.
[31] M.A. Whittington, S.J. Dolin, T.L. Patch, R.J. Siarey, A.R. Butterworth, H. Little, Chronic dihydropyridine treatment can reverse the behavioural consequences of and prevent the adaptations to, chronic ethanol treatment, Br. J. Pharmacol. 103 (1991a) 1669–1676. [32] H.I. Yamamura, H. Shoemaker, R.G. Boles, W.R. Roeske, Diltiazem enhancement of [ 3 H]-nitrendipine binding to calcium channel associated drug receptor sites in rat brain synaptosomes, Biochem. Biophys. Res. Commun. 108 (1982) 640–646. [33] P. Zapater, J. Javaloy, J.F. Romain, M.T. Vigal, J.F. Horga, Anticonvulsant effects of nimodipine and two novel dihydropyridines (PCA50922 and PCA 50941) against seizures elicited by pentylenetetrazol and electroconvulsive shock in mice, Brain Res. 796 (1998) 311–314. [34] J.T. Lichfield, F. Wilcoxon, A simplified method of evaluating close–effect experiments, J. Pharmacol. Exp. Ther. 96 (1948) 99– 113.
Research report
Selectivity of the protective effects of dihydropyridine calcium channel antagonists against the ethanol withdrawal syndrome 1
W.P. Watson , H.J. Little* Drug Dependence Unit, Psychology Department, Durham University, Science Laboratories, South Road, Durham DH1 3 LE, UK Accepted 12 November 2001
Abstract Four dihydropyridine calcium channel antagonists were compared for their ability to protect against the hyperexcitability produced in mice by withdrawal from chronic ethanol treatment and to protect against seizures due to bicuculline or pentylenetetrazol. Comparison was also made of their effects on locomotor activity, body temperature and motor co-ordination, and with the corresponding effects of the benzodiazepine, diazepam. Nitrendipine, nimodipine, nicardipine (at 50 and 10 mg / kg) and isradipine (at 10 and 4 mg / kg) decreased the withdrawal hyperexcitability, but showed no anticonvulsant action against either bicuculline or pentylenetetrazol. Diazepam (1.5 and 4 mg / kg) both protected against the withdrawal signs and decreased seizure incidence after bicuculline and pentylenetetrazol, although the latter effects were of shorter duration than those on the withdrawal signs. The four dihydropyridines decreased spontaneous locomotor activity, an effect which lasted up to 6 h. Only isradipine and diazepam had any ataxic actions at the doses tested. All the dihydropyridines had hypothermic actions, considerably shorter in duration than effects on withdrawal hyperexcitability, with little evidence of dose dependence, except for nicardipine, which had a larger, dose-related, hypothermic action. Of the four compounds, isradipine was more potent in terms of dose, but not any more selective for effectiveness against the withdrawal signs, than the other three dihydropyridines, and nicardipine was slightly less effective in protecting against the withdrawal signs. The results indicate that the anticonvulsant effects of the dihydropyridines were selective for ethanol withdrawal hyperexcitability, whereas diazepam showed no such selectivity. 2002 Published by Elsevier Science B.V. Keywords: Calcium channel; Ethanol; Dihydropyridine; Anticonvulsant
1. Introduction Voltage-dependent neuronal calcium channels are divided into several subtypes, according to their activation and inactivation characteristics in cultured cells. The original classification showed the L-subtype of channel to be selectively blocked by dihydropyridine calcium antagonists, which are used therapeutically as hypotensive and antiarrythmic agents [22]. The application of this classification to vertebrate neurones is still in progress and, although many authors have shown the dihydropyridines to be selective for L-type channels, as defined by the electrophysiological characteristics of the channels, some reports have found them to affect other channel subtypes [1]. Abbreviations: PTZ, pentylenetetrazol; GABA, aminobutyric acid *Corresponding author. Fax: 144-191-374-7774. E-mail address: [email protected] (H.J. Little). 1 Current address: Neuropharmacology Department, H. Lundbeck A / S, Ottiliavej 9, 2500 Valby, Copenhagen, Denmark.
There are high affinity binding sites in the central nervous system for dihydropyridines, and these are thought to represent the voltage-sensitive calcium channels on neurones [17]. Dihydropyridine calcium channel antagonists have few overt effects on behaviour, but have been reported to possess some anticonvulsant actions. In acute studies, nitrendipine and nimodipine potentiated the ataxic and general anaesthetic effects of ethanol [4, 5]. The calcium channel activator, Bay K 8644, decreased the general anaesthetic actions of ethanol and other sedative / hypnotics [7]. Considerable evidence has also been published indicating that dihydropyridine-sensitive calcium channels play a role in physical dependence on ethanol. Chronic administration of ethanol increased the density of the high affinity binding sites in the CNS; the effects of dihydropyridines on transmitter release and phosphatidylinositol turnover were also increased [6]. Nitrendipine prevented the hyperexcitability produced
0006-8993 / 02 / $ – see front matter 2002 Published by Elsevier Science B.V. PII: S0006-8993( 02 )02236-9
112
W.P. Watson, H. J. Little / Brain Research 930 (2002) 111 – 122
by withdrawal from long-term ethanol administration, both in vivo [15] and in isolated neuronal preparations [28]. The stereospecificity of these effects was studied with the isomers of isradipine, and found to follow the same pattern as that for calcium channel blockade [15, 28, 11]. When given chronically, concurrently with ethanol, nifedipine and nitrendipine prevented the development of ethanol tolerance [5] and nitrendipine prevented the withdrawal syndrome, effects that were not due to residual dihydropyridine in the brain at the times of testing [31]. The acute effect of nitrendipine and isradipine on ethanol withdrawal hyperexcitability is not shared by all drugs that block L-type calcium channels. The benzothiazepine, diltiazem, increased the withdrawal hyperexcitability [23]. Diltiazem binds at a different site on the L-type calcium channel complex and increases, rather than decreasing, binding of dihydropyridines [32]. This compound also increased the severity of seizures caused by bicuculline and pentylenetetrazol [24]. A different pattern was seen with the dihydropyridine, felodipine, which did not protect against the alcohol withdrawal syndrome but did possess anticonvulsant activity against bicuculline and pentylenetetrazol seizures [26]. In order to determine the extent to which the ability of dihydropyridine calcium channel antagonists to protect against ethanol withdrawal hyperexcitability is shared by other members of this drug group, the current project compared the acute effects of nitrendipine, nimodipine, isradipine and nicardipine. The ability to protect against ethanol withdrawal signs seizures was compared with effects on seizures produced by the convulsant drugs, bicuculline and pentylenetetrazol, and effects on spontaneous locomotor activity, ability to cause ataxia and body temperature. The aims were, firstly, to determine the selectivity of the ability of the compounds to prevent ethanol withdrawal signs, compared with anticonvulsant properties against the actions of the convulsant drugs, and, secondly, to see if there was any selectivity in their anticonvulsant effects compared with their sedative, ataxic and hypothermic actions. Comparison was also made with the benzodiazepine, diazepam, as this type of compound is used clinically to treat alcohol withdrawal hyperexcitability. The doses were chosen on the basis of previous experience with the drugs in the prevention of ethanol withdrawal signs in rodents.
2. Methods Male mice of the TO strain were used throughout, obtained from Bantin and Kingman, UK. All were kept in a 12 h light–dark cycle with access ad libitum to tap water and laboratory rodent chow (CRM). The weight range was 25–40 g, with not more than a 5-g range in any single experiment. In all studies involving behavioural ratings,
the observer who carried out the rating allocation was blind to the prior drug treatment.
2.1. Drugs used Nitrendipine, nimodipine, (Bayer), nicardipine (Yamanouchi) and isradipine (Sandoz) were suspended in 0.5% Tween 80 and sonicated. The dihydropyridines were kept in covered vials and protected from light. Pentylenetetrazol (PTZ) (Sigma) and diazepam (Roche) were dissolved in isotonic saline. Bicuculline (Sigma) was made up as a 10 mg / ml stock solution in 1 M HCl, and was then further dissolved in saline brought to pH 3 with HCl. All drugs were stirred before injection, and sonicated where necessary. The following doses of drugs were tested: nitrendipine, nimodipine and nicardipine 10 and 50 mg / kg; isradipine 4 and 10 mg / kg; diazepam 1.5 and 4 mg / kg. All compounds were administered intraperitoneally (i.p.) in a dose volume of 10 ml / kg. The pretreatment times are given in each methods section.
2.2. Ethanol withdrawal syndrome Ethanol was administered in a liquid diet schedule (Dyets, Pennsylvania, USA; [14]). All mice received control diet for an initial 2-day period. Ethanol-treated mice then received a diet containing 7% (v / v) ethanol for either 10 days or 3 weeks, in order to provide two levels of severity of the withdrawal syndrome. Control groups were pair-fed a control diet, balanced isocalorifically to match the ethanol-containing diet [14]. There were no differences in the weights of the ethanol-treated and control mice at the end of the treatment periods. The amount of ethanol consumed by the groups of mice was between 21 and 27 g / kg / 24 h. The dihydropyridines and diazepam were administered immediately on withdrawal of the six groups of mice from the chronic alcohol treatment. Two withdrawal studies were carried out, one after 3 weeks chronic ethanol treatment, that gave pronounced withdrawal signs, and one after 10 days chronic ethanol administration, that gave a more mild withdrawal. The severity of the withdrawal syndromes was measured by ratings of behaviour produced by handling. This method, originated by Goldstein and Pal [9], measures tremor, myoclonic jerks and clonic convulsive behaviour and was modified for the present use [15,10,25]. The mice were gently picked up by the tail for 30 s and the behaviour rated according to the scale given below. Untreated mice normally score 0 or 1 on this rating scale. The results were expressed as median values6interquartile ranges. Each treatment group contained 10–12 mice. Behavioural ratings 1. Mild tremor on lifting and turning
W.P. Watson, H. J. Little / Brain Research 930 (2002) 111 – 122
2. Continuous severe tremor on lifting and turning 3. Clonic forelimb extensor spasm on lifting 4. Clonic forelimb extensor spasm on lifting, which continued after placing mouse on cage top. 5. Spontaneous evidence of myoclonic activity followed by (4).
2.3. Anticonvulsant actions The anticonvulsant actions of the dihydropyridines were studied against bicuculline and pentylenetetrazol, measured by convulsion incidence and latency after intraperitoneal injection of the convulsants. Because the aim was to compare the effects of the dihydropyridines with their actions on ethanol withdrawal hyperexcitability, the convulsants were given 30 min after dihydropyridine administration, at which other effects of the dihydropyridines (see temperature measurement results) were known to peak and at 5 h after dihydropyridine administration, which corresponded to the time interval between administration of drugs at the beginning of the withdrawal period and the peak of the withdrawal hyperexcitability. Each group of eight mice was pretreated with either the vehicle (Tween 80) or a dihydropyridine or diazepam. The animals were then injected, after the appropriate pretreatment time, with either bicuculline, 5 mg / kg or PTZ 70 mg / kg. The number of mice exhibiting clonic seizures (resulting in a loss of posture) and the time to the beginning of a clonic convulsion (latency) were recorded for the next 60 min. The effects of the dihydropyridines were also examined on the dose response curves to bicuculline; a range of doses (4, 4.5 or 5 mg / kg) of bicuculline were given and the convulsion incidence measured as above. In all cases, the animals were humanely killed as soon as the first sign of a seizure was seen or at 60 min after administration of the convulsant.
2.4. Ataxic actions The ataxic actions of the compounds were measured by the rotorod method. Mice were placed on a rod rotating at 4.5 rpm, and the time they remained on the rod was measured automatically. A cut-off time of 180 s was used in all experiments. Measurements were made at 10-min intervals after the acute administration of the drug under test, until the ability to remain in the rod had returned to control values. Eight mice were used in each treatment group. Before the acute drug injections, the mice were tested on the rotorod to ensure that they stayed on for 180 s; only a very small number did not do so and were excluded from the studies.
2.5. Effects on spontaneous locomotor activity The effects of the compounds on spontaneous locomotor activity were determined automatically by breaking of
113
infra-red beams. Measurements were made of total activity (total beam breaks) and ambulatory activity (breaking of consecutive beams), measured for 60 min. Six mice were used for each treatment group, with the exception of the vehicle treated groups in which the n values were increased in some instances in order to maintain comparability over days. Separate groups of mice were used for each set of measurements. In order to study the time course of the drug actions, the treatments were given between 8.00 and 9.00 am. Pretreatment times used for the dihydropyridines and for diazepam were 0, 1, 3, 5 and 7 h from drug administration, and comparisons were made with control results from mice injected with the vehicle and tested concurrently.
2.6. Effects on body temperature Body temperatures were measured by rectal probe. The gentle handling of mice necessary for weighing and coding causes the body temperature to rise [21]. An experimental schedule was therefore adopted in which the temperatures of mice were measured immediately they were removed from the cages. They were then weighed and coded, and the temperatures measured again 20 min after the initial measurement. Drug injections were then given, and the temperatures measured every 20 min for 2 h. If the temperatures were still significantly altered at 2 h, they were measured again at 3 h to see if they had returned to control values. Care was taken that the room temperature was constant for all the experiments.
2.7. Statistical analysis The ratings of withdrawal hyperexcitability were compared by nonparametric two-way analysis of variance, using a format designed for repeat measurements on the same samples and multiple comparisons with a control [16]. The effects of the drugs on withdrawal hyperexcitability were also expressed as area under the curve, to evaluate the overall effectiveness of the compounds, and these results were compared by one-way analysis of variance followed by posthoc Dunnett’s test. The incidence of convulsions were compared by Fisher’s exact probability test and latencies to convulsions by one-way analysis of variance followed by posthoc Dunnett’s test. The results from the dose response studies with bicuculline were analysed by probit analysis ( MLP program, Numerical Algorithum Group; [34]). The rotorod results at individual time points were compared by the Mann–Whitney U test for comparisons with control values at each time interval. One-way analysis of variance was used for comparisons at each time interval of the locomotor activity counts and the body temperatures, followed by posthoc Dunnett’s test.
114
W.P. Watson, H. J. Little / Brain Research 930 (2002) 111 – 122
3. Results
3.1. Ethanol withdrawal syndrome The effects of the drugs on hyperexcitability in TO mice caused by withdrawal from chronic ethanol treatment are illustrated in Fig. 1, which shows the severe withdrawal signs seen after cessation of 3 weeks ethanol intake and the milder syndrome seen after only 10 days ethanol consumption. An example of the pattern of the withdrawal signs and the protection by one of the dihydropyridines (nitrendipine) is illustrated in Fig. 1a, while Fig. 1b shows the effects of all the drugs expressed as area under the curve, for both withdrawal experiments. It can be seen in Fig. 1b that the effects of the four dihydropyridines, at the doses tested in decreasing the withdrawal signs were very similar in magnitude. In the first experiment (severe withdrawal) nitrendipine, nimodipine and nicardipine, at 50 mg / kg, and isradipine, at 10 mg / kg all gave significant protection against the withdrawal hyperexcitability (P,0.05 for results up to 10 h after injection). Diazepam also gave significant protection when administered at 4 mg / kg (P,0.05). None of the lower doses of the dihydropyridines gave significant protection against the more substantial withdrawal hyperexcitability in the first experiment but the effects of these doses could be seen in the second withdrawal study (mild withdrawal). As had been found previously, the milder withdrawal signs seen after the shorter period of chronic alcohol consumption appeared earlier and were of shorter duration than the more severe syndrome. Statistical comparisons were therefore made only up to 5 h from withdrawal, up to which time there was a significant increase in hyperexcitability ratings in the mice receiving vehicle treatment, compared with values for control animals that had not been given alcohol. At 10 mg / kg, nitrendipine and nimodipine significantly decreased the withdrawal severity (P,0.05) for comparisons up to 5 h. Isradipine, at 4 mg / kg, also significantly decreased the ratings. Nicardipine, at 10 mg / kg, however, did not have any significant effect on the withdrawal while diazepam, at 1.5 mg / kg gave significant protection (P,0.05).
3.2. Anticonvulsant actions 3.2.1. Effects on the convulsant actions of bicuculline The effects of the drugs on convulsions due to bicuculline, 5 mg / kg, are shown in Table 1; the compounds were administered either 30 min or 5 h before the bicuculline. At the 30-min interval, there were no significant changes in convulsion incidence with any of the compounds, except diazepam, which provided good protection at both 1.5 and 4 mg / kg (P,0.01 for both doses). As the higher doses of the dihydropyridines (50 mg / kg of nitrendipine, nimodipine and nicardipine, 10 mg / kg of isradipine) did
not have any significant effects against bicuculline seizures, the lower doses of these drugs were not tested. At the 5-h interval, none of the dihydropyridines significantly altered the incidence of, or latencies to, clonic convulsions (P.0.05). Following administration of nitrendipine at 50 mg / kg, however, the incidence of convulsions was 3 / 8, which just failed to reach statistical significance when compared with the control value (P50.06). At this pretreatment time, diazepam, at 4 mg / kg, again protected against bicuculline convulsions (P,0.01). In order to examine their actions in more detail, the effects of the compounds on the dose response curves to bicuculline were measured 5 h after administration of the compounds. The ED50 values derived from the probit analysis are given in Table 2. Statistical analysis of these data, however, did not prove possible because the lines differed significantly from homogeneity (P,0.05).
3.2.2. Effects on the convulsant actions of PTZ Table 3 shows the effects of the compounds on convulsions due to PTZ. At the 30-min pretreatment interval, none of the dihydropyridines had any significant effects on convulsion incidence or latency. Diazepam significantly decreased convulsion incidence when given at either 1.5 or 4 mg / kg (P,0.01 for each dose). The only significant change when the drugs were administered 5 h before the PTZ, was a decrease in convulsion incidence after nitrendipine, 50 mg / kg (P,0.05). As the higher doses of the dihydropyridines did not have any significant effects against PTZ seizures, the lower doses of these drugs were not tested. 3.3. Ataxic actions At the lower doses tested (i.e. nitrendipine, nimodipine and nicardipine at 10 mg / kg, and isradipine at 4 mg / kg), none of the dihydropyridine compounds had any significant effects on rotorod performance up to 2 h after administration (data not shown). The effects of the higher doses are illustrated in Fig. 1c. At 10 mg / kg, isradipine caused significant ataxia at 20 min after injection (P,0.05, compared with control values) but not at later times, while nitrendipine, nicardipine and nimodipine had no significant effects at 50 mg / kg. Diazepam at both 1.5 (data not shown) and 4 mg / kg (Fig. 1c) significantly lowered the times the mice were able to stay on the rotating rod (P,0.05, compared with control values at 20 min for 1.5 mg / kg and 20, 40 and 60 min for the 4 mg / kg dose).
3.4. Effects on spontaneous locomotor activity The effects of the compounds on spontaneous locomotor activity are illustrated in Fig. 2 (total activity) and Fig. 3 (ambulatory activity). The times indicated on the graphs are the times at which each set of measurements was completed, i.e. the end of each 60-min measuring period.
W.P. Watson, H. J. Little / Brain Research 930 (2002) 111 – 122
115
Fig. 1. Effects of the drugs against responses to handling during ethanol withdrawal. (A) Effects of nitrendipine (j) and of Tween vehicle (n) given after withdrawal from chronic ethanol treatment. Values are median scores. (B) Effects of the drugs against ethanol withdrawal hyperexcitability expressed as mean areas under the curve for the ratings of the responses to handling. The shaded columns show the results for the more severe withdrawal syndrome and the effects of the higher doses of the compounds: nitrendipine (NITR), nimodipine (NIM) and nicardipine (NIC) at 50 mg / kg, isradipine (ISR) at 10 mg / kg and diazepam (DZ) at 4 mg / kg. The open column shows the results for the milder withdrawal syndrome and the effects of the lower doses of the compounds: nitrendipine, nimodipine and nicardipine at 10 mg / kg, isradipine at 4 mg / kg and diazepam at 1.5 mg / kg. *, Significant (P,0.05) decreases in withdrawal hyperexcitability compared with ratings after administration of vehicle. The incidence of hyperexcitability was significant for both withdrawals compared with values for mice not treated with ethanol (P,0.01). (C) Effects of the drugs on the ability to stay on a rotating rod. Values are means6standard error of the mean (S.E.M.) Nitrendipine (j), nimodipine (m), and nicardipine (h) given at 50 mg / kg, isradipine (d) at 4 mg / kg, diazepam (3) at 4 mg / kg and Tween vehicle (n).
W.P. Watson, H. J. Little / Brain Research 930 (2002) 111 – 122
116
Table 1 Seizures produced by bicuculline (5 mg / kg) with 30 min and 5 h pretreatment Treatment
Incidence
Latency
S.E.M
30 min pretreatment Tween Nit 50 Nim 50 Nic 50 ISR 10 DZ 1.5 DZ 4
8/8 7/8 7/8 6/8 7/8 1 / 8 (P,0.01) 2 / 8 (P,0.01)
252 212 227 182 238 30 93, 120
60 86 46 30 37
5 h pretreatment Tween Nit 50 Nim 50 Nic 50 ISR10 DZ 4
7/8 3 / 8 (P50.06) 5/8 4/8 5/8 2 / 8 (P,0.01)
119 123 149 149 128 128, 183
– 14 11 22 18 28
Tween, Tween vehicle; Nit, nitrendipine; Nim, nimodipine; ISR, isradipine; DZ, diazepam; numbers after the drug names indicate doses in mg / kg. Latency (min), time to start of first convulsion. Table 2 ED50 doses of bicuculline required for 50% convulsions Drug
ED50
95% Limits
Tween Nit 50 Nim 50 Nic 50 ISR 10 DZ 4
3.87 6.8 4.15 4.82 4.77 6.91
(2.48–5.36) (4.61–8.99) (2.88–5.42) (3.56–6.07) (4.46–5.59) (3.55–10.25)
Significant decreases in activity were seen with nitrendipine, for the 10 mg / kg dose only between 0 and 1 h after injection (P,0.05) and for the 50 mg / kg dose, from 0 to 6 h after injection (P,0.01 for 1–4 h; P,0.05 for 6 h) (Figs. 2a and 3a). For nimodipine, significant decreases in activity were seen for both the 10 and 50 mg / kg doses until 4 h after administration (P,0.01) (Figs. 2b and 3b). Table 3 Seizures produced by PTZ 70 (mg / kg) with 30 min and 5 h pretreatment Drug
Incidence
Latency
S.E.M
30-min Pretreatment Tween Nit 50 Nim 50 Nic 50 ISR 10 DZ 1.5 DZ 4
8/8 5/8 8/8 6/8 6/8 1 / 8 (P,0.01) 1 / 8 (P,0.01)
139 219 286 162 166 30 44
34 68 55 22 10 – –
5-h Pretreatment Tween Nit 50 Nim 50 Nic 50 ISP 10 DZ 4
8/8 4 / 9 (P,0.05) 8/8 6/8 8/8 8/8
137 168 119 128 186 219
14 36 20 18 53 54
Nicardipine, at 10 mg / kg significantly decreased the activity up to 2 h from administration (P,0.01 at 1 h; P,0.05 at 2 h), and at 50 mg / kg up to 6 h after the injections (Figs. 2d and 3d). At both 4 and 10 mg / kg, isradipine significantly decreased the activity counts up to 6 h after administration (P,0.01 at 1–4 h; P,0.05 at 6 h) (Figs. 2c and 3c). Diazepam, at 1.5 or 4 mg / kg did not alter locomotor activity (data not shown). In terms of magnitude of effect, the largest absolute decreases in activity were seen with nitrendipine and nicardipine, both at 50 mg / kg (Figs. 2a and d, 3a and d). The time of maximal effect of the higher dose of each dihydropyridine appeared to be between 2 and 4 h after administration, for both total activity measures and the measurements of ambulatory activity. The lower doses had effects that were significant for shorter times than the higher doses, as would have been expected, but this was most noticeable for nitrendipine, as the 10 mg / kg dose of this compound had significant effects only up to 1 h after injection. The activity of the control animals did not vary significantly between the different measurement times (P. 0.05).
3.5. Effects on body temperature The effects of the compounds on body temperature are illustrated in Fig. 4. Nitrendipine (Fig. 4a), significantly lowered the temperatures for up to 80 min at 10 mg / kg (P,0.05) and up to 120 min at 50 mg / kg (P,0.01 up to 80 min; P,0.05 at 120 min). Nimodipine (Fig. 4b) lowered temperatures were significant only at 20 and 40 min from injection (P,0.05, both doses, both time intervals) and the 50 mg / kg dose of nimodipine had only a little greater effect than the 10 mg / kg dose. The effects of isradipine (Fig. 4c) were similar to those of nimodipine, with both the 4 and 10 mg / kg doses having significant effects up to 80 min (both doses: P,0.01 up to 60 min; P,0.05 for 80-min interval). The maximal reductions for all three of these dihydropyridines were seen at 40 min from injection. The effect of nicardipine (Fig. 4d) differed from that of the other three dihydropyridines, in that the effect of the higher dose (50 mg / kg) was much larger and there was also a clear dose relationship. At 10 mg / kg nicardipine lowered body temperature significantly at 20 and 40 min (P,0.01) and at 50 mg / kg the effect was significant at all times up to 240 min (P,0.05) but the temperatures had returned to control values at 360 min. Diazepam did not significantly alter body temperature (data not shown).
4. Discussion The dihydropyridines, nitrendipine, nimodipine and nicardipine all provided protection against the hyperexcitability caused by withdrawal from chronic ethanol treat-
W.P. Watson, H. J. Little / Brain Research 930 (2002) 111 – 122
117
Fig. 2. Effects of the drugs on measures of total locomotor activity. Values are means6S.E.M. Controls (vehicle injection) illustrated by n throughout. (A) Effects of nitrendipine at 10 (j) and 50 (j) mg / kg; (B) effects of nimodipine at 10 (m) and 50 (m) mg / kg; (C) effects of isradipine at 4 (d) and 10 (d) mg / kg, and (D) effects of nicardipine 10 (d) and 50 (d) mg / kg.
ment. This effect was prolonged, lasting for 10 h for the effects of the higher doses (50 mg / kg of nitrendipine, nimodipine and nicardipine and 10 mg / kg of isradipine) on the more severe withdrawal syndrome and included prevention of the peak withdrawal hyperexcitability which occurred between 8 and 10 h after cessation of alcohol consumption. Isradipine was more effective in terms of dose than the other three calcium channel antagonists, providing good protection against the withdrawal syndrome when given at 10 mg / kg, rather than 50 mg / kg
that was required for the other three dihydropyridines. Nitrendipine, nimodipine and nicardipine had similar effects at the higher dose (50 mg / kg of each compound) against the more severe syndrome, and nitrendipine and nimodipine at 10 mg / kg decreased the milder withdrawal signs. Nicardipine at 10 mg / kg, however, did not have any significant effect on the milder syndrome. The protective effects against withdrawal hyperexcitability of the four dihydropyridines were selective, as they did not protect against the chemically-induced convulsions,
118
W.P. Watson, H. J. Little / Brain Research 930 (2002) 111 – 122
Fig. 3. Effects of the drugs on ambulatory locomotor activity. Values are means6S.E.M. Controls (vehicle injection) illustrated by n throughout. (A) Effects of nitrendipine at 10 (j) and 50 (j) mg / kg; (B) effects of nimodipine at 10 (m) and 50 (m) mg / kg; (C) effects of isradipine at 4 (m) and 10 (m) mg / kg and (D) effects of nicardipine 10 (d) and 50 (d) mg / kg.
in contrast to diazepam. The doses of chemical convulsants were chosen to be just submaximal for the production of clonic convulsions. It is debatable whether or not the severity of those effects can be directly compared with the withdrawal syndrome, since during the latter, full convulsions are not seen in all animals even during the more severe withdrawal. However, the selectivity of the dihydropyridines for protection against the withdrawal hyperexcitability was apparent, and this lasted for 8–10 h. The selectivity of the dihydropyridines for ethanol withdrawal hyperexcitability is compatible with the lack of
effect of nitrendipine on neuronal hyperexcitability in vitro produced by bicuculline, compared with the effectiveness of the same nitrendipine concentration to prevented neuronal excitability due to withdrawal from chronic alcohol consumption [28]. We have recorded synaptic potentials in vitro following chronic ethanol treatment in vivo and showed that the changes underlying ethanol withdrawal hyperexcitability consist of increases in excitatory amino acid transmission [30,18] and increased conductance of voltage sensitive calcium channels [29]. We found no decreases in GABA mediated inhibitory potentials [30,18].
W.P. Watson, H. J. Little / Brain Research 930 (2002) 111 – 122
119
Fig. 4. Effects of the drugs on body temperature. Values are means6S.E.M. Controls (vehicle injection) illustrated by n throughout. (A) Effects of nitrendipine at 10 (j) and 50 (j) mg / kg; (B) effects of nimodipine at 10 (m) and 50 (m) mg / kg; (C) effects of isradipine at 4 (d) and 10 (d) mg / kg and (D) effects of nicardipine 10 (h) and 50 (h) mg / kg.
120
W.P. Watson, H. J. Little / Brain Research 930 (2002) 111 – 122
The present results therefore support our earlier conclusion, that the ethanol withdrawal syndrome in mice does not involve decreases in GABA transmission. The fact that diazepam gave protection against the alcohol withdrawal signs was not evidence that these signs were caused by decreases in GABA transmission, even though the actions of diazepam are produced by potentiation of the effects of endogenous GABA. Benzodiazepines are anticonvulsant against seizures due to a variety of causes and the increases in GABA transmission that these drugs produce can balance out changes in other neurotransmitter systems. Effects of dihydropyridine calcium channel antagonists have been reported previously on PTZ seizures, but the effects are not very consistent. O’Neill and Bolger [19] found increased latency to seizure but not seizure incidence when nimodipine 10 and 20 mg / kg (30 min pretreatment) was tested against PTZ 85 mg / kg. Palmer et al. [20], however, reported protective effects of nitrendipine, nicardipine and nimodipine against 85 mg / kg PTZ, with ED50 values for the dihydropyridines of 15–30 mg / kg (30 min pretreatment times). Zapater et al. [33] found effects of nimodipine 30, 100 and 300 mg / kg on seizure latency but not seizure incidence. Chakrabarti et al. [3] reported effects of nimodipine at 5 mg / kg against PTZ doses from 40 to 75 mg / kg (15 min pretreatment time) but a synergistic interaction with the alcohol (dose 2 g / kg) used to dissolve the drug were not excluded. The reason for the differences within these reports and between these and the present study is not clear; it is possible there are species or strain differences. The effect of the dihydropyridines on locomotor activity was seen primarily at the earlier time periods, with the effect of the higher dose of each compound wearing off by the 5–6 h time interval and no longer significant by the 7–8 h time interval. This is in some contrast with the effects of the dihydropyridines on the withdrawal syndrome when protective effects were seen up to 10 h after administration. The results suggest that the mechanism by which dihydropyridine calcium channel antagonists prevent the withdrawal signs is different from that by which they decrease locomotor activity. Between the four dihydropyridines, there was little difference between the magnitude of the effects of the higher doses on locomotor activity, although nimodipine at 50 mg / kg had slightly less effect. The lower doses, however, showed more variation, with nitrendipine and nicardipine at 10 mg / kg causing smaller magnitude of activity decreases than nimodipine at 10 mg / kg or isradipine at 50 mg / kg. This might suggest that the dose response relationships for effects on locomotor activity differ between the compounds, but as only two doses were tested this is, as yet, speculation. The dihydropyridines, with the exception of isradipine, did not have effects on motor co-ordination while in contrast, diazepam had considerable ataxic effects. This suggests that the effects of the dihydropyridines on the
tremor and myoclonic jerks involved in the ethanol withdrawal syndrome are unlikely to be related to any effects on motor systems. It also shows that the effects of the dihydropyridines on exploratory locomotor activity, described above, were not due to changes in motor coordination. The hypothermic actions of all the dihydropyridine calcium channel antagonists followed completely different time courses to the protection against ethanol withdrawal hyperexcitability. The latter lasted 10 h while the hypothermic effects were maximal at 40 min and, with the exception of nicardipine, were no longer significant by 2 h after the drug administration. Nicardipine at 50 mg / kg had a considerably larger effect than the other dihydropyridines showed a clearer dose relationship. The hypothermic effects are likely to have been largely due to vasodilation, although there may have been some central component. These results therefore suggest that the effects of the compounds in ethanol withdrawal are not due to their effects on body temperature and are unlikely to be due to their vascular actions. The effects of drugs on temperature and on locomotor activity are linked in mice, as one form of thermoregulation is brought about by the animals huddling together. However, the time course of the locomotor changes far outlasted the hypothermic effects of the compounds. After injection of the drugs, the animals were kept in group cages until they were transferred to novel cages for the locomotor activity measurements. The doses of the dihydropyridines required for central actions, such as prevention of alcohol withdrawal signs, are higher than those that produce cardiovascular doses. However, the central concentrations of the drugs after administration of the doses used in the present study correspond closely to the concentrations that are required to affect neuronal activity in vitro, in electrophysiological recordings from brain slices [5,31]. This difference may be due to the differences between tissues in the time spent by cells in a depolarised state. The binding affinities of the dihydropyridines reported for rodent brain tissue are as follows: the Kd for [ 3 H]nitrendipine was 160 pM for cerebrocortical tissue [8], that of [ 3 H]-nimodipine 1.11 nM for brain membranes [2] and that of [ 3 H]-isradipine 35 pM for cerebrocortical tissue [13]. The latter authors made a direct comparison between [ 3 H]-isradipine and [ 3 H]-nitrendipine and found the Kd for the latter compound to be 372 pM. A similar comparison was made by the authors of [27] who found the Kd of rat brain membranes for [ 3 H]-isradipine was 15.6 pM and that of [ 3 H]-nitrendipine was 124 pM and that the two compounds bound to the same number of receptors. The affinities of isradipine and nitrendipine therefore parallels the difference in potency between these two compounds seen in vivo in the present studies. The reported affinity of nimodipine for the binding sites, however, is higher than that of nitrendipine, but these two compounds had very similar potencies in vivo. However, although the authors of
W.P. Watson, H. J. Little / Brain Research 930 (2002) 111 – 122
[2] reported a Kd of 1.11 nM for nimodipine, there were differences between brain regions, with some areas showing lower Kd values. A literature search failed to reveal a Kd value for [ 3 H]-nicardipine binding to brain tissue, but Kanami et al. [12] found the Ki values for competition for binding of brain tissue were very similar for nicardipine, nimodipine and nitrendipine against [ 3 H]-nitrendipine binding. Dihydropyridine binding, however, is affected by both depolarisation and by the surrounding concentrations of calcium ions. Homogenate binding is of necessity carried out on fully depolarised neurones, so direct comparisons between in vitro binding affinity and in vivo potency are not fully valid. In addition, many of the dihydropyridines are stereospecific. The drugs used in the present studies were the racemates, because it was not possible to obtain sufficient of the isomers to carry out extensive in vivo studies. We have previously shown that the effect of isradipine on alcohol withdrawal signs is selective for the (1)-isomer [15]. Of the binding affinities quoted above, nitrendipine was reported to show an approximately 20fold difference between Kd values for the two isomers, but there was only a two-fold difference between binding of the isomers of nimodipine [2]. Lee et al. [13] used (1)isradipine for their binding studies and reported a 240-fold lower affinity for the (2)-isomer of isradipine. Nicardipine showed a tenfold difference in the potency of its isomers in displacing (1)-[ 3 H]-isradipine [13]. Overall, in this study, the four dihydropyridines showed fairly similar profiles of action, but nicardipine had slightly more effect on locomotor activity, considerably more effect on body temperature and slightly less effect on the alcohol withdrawal syndrome than the other three compounds. Selectivity was clearly evident for all four compounds in protection against ethanol withdrawal seizures, compared with effectiveness against seizures produced by bicuculline or PTZ.
Acknowledgements We thank the Medical Research Council UK for financial assistance with this work.
References [1] N. Akaike, P.G. Kostyuk, Y.V. Osipchuk, Dihydropyridine sensitive low-threshold calcium channels in isolated rat hypothalamic neurones, J. Physiol. 412 (1989) 181–195. [2] P. Bellman, A. Schade, R. Towart, Dihydropyridine receptor in rat brain labelled with [ 3 H-]-nimodipine, Proc. Natl. Acad. Sci. 80 (1983) 2356–2360. [3] A. Chakrabarti, H.K Saini, S.K. Garg, Dose-finding study with nimodipine: a selective central nervous system calcium channel blocker on aminophylline induced seizure models in rats, Brain Res. Bull. 45 (1998) 495–499.
121
[4] S.J. Dolin, H.J. Little, Augmentation by calcium channel antagonists of general anaesthetic potency in mice, Br. J. Pharmacol. 88 (1986) 909–914. [5] S.J. Dolin, H.J. Little, Are changes in neuronal calcium channels involved in ethanol tolerance?, J. Pharmacol. Exp. Ther. 250 (1989) 985–991. [6] S.J. Dolin, H.J. Little, M.J. Hudspith, C. Pagonis, J.M. Littleton, Increased dihydropyridine-sensitive calcium channels in rat brain may underlie ethanol physical dependence, Neuropharmacology 26 (1987) 275–279. [7] S.J. Dolin, M.J. Halsey, H.J. Little, Effects of the calcium channel activator, Bay K 8644, on general anaesthetic potency in mice, Br. J. Pharmacol. 94 (1988) 413–422. [8] F.J. Erlert, E. Itoga, The interaction of [ 3 H]-nitrendipine with receptors for calcium antagonists in the cerebral cortex and heart of rats, Biochem. Biophys. Res. Commun. 104 (1982) 937–943. [9] D.B. Goldstein, N. Pal, Alcohol dependence produced in mice by inhalation of ethanol: Grading the withdrawal reaction, Science 172 (1971) 288–290. [10] A. Green, E.M. Davies, M.A. Whittington, H.J. Little, A.J. Cross, Action of chlormethiazole in a model of ethanol withdrawal, Psychopharmacology 102 (1990) 239–242. [11] R.P. Hof, U.T. Ruegg, A. Hof, A. Vogel, Stereoselectivity at the calcium channel: opposite actions of the enantiomers of a 1,4dihydropyridine, J. Cardiovasc. Pharmacol. 7 (1985) 689–693. [12] J. Kinami, Y-L. Qu, H. Tsuchihashi, T. Nagatomo, T. Maniwa, A. Miyagishi, Assessment of Ca 21 antagonist effects of SM-6586 and its isomers, novel 1,4-dihydropyridine derivatives, by radioligand binding assay, Jpn. J. Pharmacol. 58 (1992) 75–78. [13] H.R. Lee, W.R. Roeske, H.I. Yamamura, High affinity binding [1H]-PN 200-110 binding to dihydropyridine receptors associated with calcium channels in rat brain, Life Sci. 35 (1984) 721–732. [14] C.S. Lieber, L.M. Decarli, Liquid diet technique of ethanol administration: 1989 update, Alc. Alcohol 24 (1989) 197–211. [15] J.M. Littleton, H.J. Little, M.A. Whittington, Effects of dihydropyridine calcium channel antagonists in ethanol withdrawal; doses required, stereospecificity and actions of Bay K 8644, Psychopharmacology 100 (1990) 387–392. [16] R. Meddis, Statistics Using Ranks: A Unified Approach, Basil Blackwell, Oxford, New York, 1984. [17] R.J. Miller, Voltage-sensitive calcium channels, J. Biol. Chem. 267 (1992) 1403–1406. [18] A. Molleman, H.J. Little, Increases in non-NMDA glutaminergic transmission, but no change in GABAB transmission, during withdrawal from chronic ethanol treatment, J. Pharmacol. Exp. Ther. 274 (1995) 1035–1041. [19] S.K. O’Neill, G.T. Bolger, The effects of dihydropyridine calcium channel modulators on pentylenetetrazol convulsions, Brain Res. Bull. 25 (1990) 211–214. [20] G.C. Palmer, M.L. Stagnitto, R.K. Ray, M.A. Knowles, R. Harvey, G.E. Garske, Anticonvulsant properties of calcium channel blockers in mice: N-methyl-D,L-aspartate-and Bay K 8644-induced convulsions are potently blocked by the dihydropyridines, Epilepsia 34 (1993) 372–380. [21] S.C. Taylor, H.J. Little, D.J. Nutt, N.A. Sellars, Benzodiazepine agonist and a contragonist have hypothermic effects in mice, Neuropharmacology 24 (1985) 69–73. [22] R.W. Tsien, D. Lipscombe, D.V. Madison, K.R. Bley, A.P. Fox, Multiple types of neuronal calcium channels and their selective modulation, Trends Neurosci. 11 (1988) 431–438. [23] W.P. Watson, H.J. Little, Interactions between diltiazem and ethanol: differences from those seen with dihydropyridine calcium channel antagonists, Psychopharmacology 114 (1994a) 329–336. [24] W.P. Watson, H.J. Little, Effects of diltiazem in convulsive states differ from those previously reported for dihydropyridine calcium channel antagonists, Psychopharmacology 114 (1994b) 321–328. [25] W.P. Watson, H.J. Little, Identification of distinct components, with
122
[26]
[27]
[28]
[29]
[30]
W.P. Watson, H. J. Little / Brain Research 930 (2002) 111 – 122 different timecourses, of convulsion threshold changes during ethanol withdrawal, J. Pharmacol. Exp. Ther. 272 (1995) 876–884. W.P. Watson, A. Misra, H.J. Little, A.R. Green, The differential effects of felodipine and nitrendipine on cerebral dihydropyridine binding ex vivo and the ethanol withdrawal syndrome, Br. J. Pharmacol. 112 (1994) 1017–1024. G.A. Weiland, R.E. Oswald, The mechanism of binding of dihydropyridine calcium channel blockers to rat brain membranes, J. Biol. Chem. 260 (1985) 8456–8464. M.A. Whittington, H.J. Little, A calcium channel antagonist stereoselectively decreases ethanol withdrawal hyperexcitability, but not that due to bicuculline, in hippocampal slices, Br. J. Pharmacol. 103 (1991) 1313–1320. M.A. Whittington, H.J. Little, Changes in voltage-operated calcium channels modify ethanol withdrawal hyperexcitability in mouse hippocampal slices, Exp. Physiol. 78 (1993) 347–370. M.A. Whittington, J.D.C. Lambert, H.J. Little, Increased NMDAreceptor and calcium channel activity during ethanol withdrawal hyperexcitability, Alcohol Alcoholism 30 (1995) 105–114.
[31] M.A. Whittington, S.J. Dolin, T.L. Patch, R.J. Siarey, A.R. Butterworth, H. Little, Chronic dihydropyridine treatment can reverse the behavioural consequences of and prevent the adaptations to, chronic ethanol treatment, Br. J. Pharmacol. 103 (1991a) 1669–1676. [32] H.I. Yamamura, H. Shoemaker, R.G. Boles, W.R. Roeske, Diltiazem enhancement of [ 3 H]-nitrendipine binding to calcium channel associated drug receptor sites in rat brain synaptosomes, Biochem. Biophys. Res. Commun. 108 (1982) 640–646. [33] P. Zapater, J. Javaloy, J.F. Romain, M.T. Vigal, J.F. Horga, Anticonvulsant effects of nimodipine and two novel dihydropyridines (PCA50922 and PCA 50941) against seizures elicited by pentylenetetrazol and electroconvulsive shock in mice, Brain Res. 796 (1998) 311–314. [34] J.T. Lichfield, F. Wilcoxon, A simplified method of evaluating close–effect experiments, J. Pharmacol. Exp. Ther. 96 (1948) 99– 113.