Diminution by benzodiazepines of the chronotropic responses to noradrenaline in rat isolated atria

European Journal of Pharmacology, 164 (1989) 467-478

467

Elsevier EJP 50787

Diminution by benzodiazepines of the chronotropic responses to noradrenaline in rat isolated atria Belrn Elgoyhen * and Edda Adler-Graschinsky Instituto de lnvestigaciones Farmocolbgicas, CON1CET, Junln 956, Buenos Aires 11 l 3, Argentina

Received 10 January 1989, accepted 21 February 1989

The effects of various benzodiazepines on chronotropic responses were assayed in spontaneously beating rat isolated atria. The increases in atrial rate obtained from concentration-response curves to noradrenafine were reduced dose dependently by both the peripheral agonist, Ro 5-4864 5 and 10/~M, and the mixed agonist, diazepam 5, 10 and 50 /~M, but not by the central benzodiazepine agonist, clonazepam 10 and 30 /~M. The inhibitory effects of the benzodiazepines on the atrial responses to noradrenaline were not counteracted by either the peripheral benzodiazepine antagonist, PK 11195 10 ttM, or the central benzodiazepine antagonist, Ro 15-1788 10 and 100/~M. Both 10 ~M Ro 5-4864 and 10 ~M diazepam also reduced the increases in atrial rate produced by either the phosphodiesterase inhibitor, 3-isobutyl-l-methylxanthine, or the adenylate cyclase activator, forskolin. On the contrary, diazepam and Ro 5-4864 did not modify the chronotropic responses of the atria either to direct exposure to CaC12 or to the calcium agonist, BAY K 8644. The increases in the intracellular levels of cAMP induced by noradrenaline were not modified by Ro 5-4864 and were even increased by 50% in the presence of diazepam. It is concluded that benzodiazepines probably reduce the chronotropic responses to noradrenaline in rat isolated atria through the interaction with the cAMP-linked chain of events that follows the activation of fl-adrenoceptors. Benzodiazepines; Sympathetic neurotransmission; Atrial responses; Norepinephrine; (Peripheral effects)

1. Introduction Drugs of the benzodiazepine group have long been used for their anxiolytic, anticonvulsant and hypnotic-sedative properties. In addition, several peripheral a u t o n o m i c functions such as gastric secretion, blood pressure and heart rate are affected by benzodiazepines (for review see Haefely et al., 1983) As concerns cardiovascular responses, evidence obtained in conscious dogs has shown that the oral administration of diazepam and b r o m a z e p a m causes a direct decrease in the pacemaker rate (Gerold et al., 1976) in addition to

* To whom all correspondence should be addressed: Junin 956, 5 o Piso, 1113 Buenos Aires, Argentina.

a central reduction in the vagal tone to the heart that leads to tachycardia. A vagolytic action of centrally acting benzodiazepines, resulting in an increase in heart rate, has also been observed in anaesthetized rats (DiMicco, 1987). O n the other hand, a slight but significant decrease in heart rate was f o u n d after the intravenous administration of diazepam and chlordiazepoxide in the open-chest dog preparation (Daniell, 1975) and a decrease in the contractility of the guinea-pig isolated ventricular papillary muscle was reported for the peripheral agonist, R o 5-4864 (Mestre et al., 1984). Moreover, although numerous studies have dealt with the direct effects of the benzodiazepines on the cardiovascular system of several m a m malian species, including m a n (for references see Gerold et al., 1976), there is a lack of information

0014-2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

468

about the actions of benzodiazepines on the responses of the heart to the physiological sympathetic neurotransmitter. Consequently, the aim of the present work was to study whether benzodiazepines modify the atrial chronotropic responses elicited by noradrenaline (NA). The experimental model selected was the rat isolated atrium and the drugs used were Ro 5-4864 (4'-chlorodiazepam) and clonazepam, which have a selective affinity for peripheral and for central receptors, respectively, and the mixed agonist, diazepam, with affinity for both sites (Mt~hier and Richards, 1983). The results obtained with the rat atria show that both Ro 5-4864 and diazepam, but not clonazepam, decreased in a concentration-dependent manner the chronotropic responses to N A and suggest that this effect is related to interference with the cAMP-linked chain of events that follows the activation of the atrial fl-adrenoceptors.

the response to the previous administration of the agonist had levelled off (i.e., at intervals of 8 min with forskolin and 4 min with other drugs). The concentration-response curves to N A and to IBMX were made twice for each preparation, 60 min apart, and the test drugs were added 30 min before and during the second curves. Only one concentration-response curve was performed with each preparation with the remaining drugs and the experimental groups were run in parallel with their respective controls. The concentration-response curves to CaC12 were done with the Krebs solution described above replaced by the following medium (mM): NaC1 118.0; KC1 4.7; CaC12 0.8; MgC12 1.2; N a H C O 3 25.0; glucose 11.1; E D T A 0.004 and ascorbic acid 0.11. The solution was buffered with Tris-HC1 (0.5 M, p H 7.8) so that the final p H of the medium bubbled with a 95% 02-5% CO 2 mixture was 7.4.

2.2. Cyclic A M P measurement 2. Materials and methods

2.1. Physiological procedures Wistar rats of weight 200-300 g and of either sex were anaesthetized with ethyl ether. The hearts were removed and both atria were dissected in a modified Krebs solution of the following composition (mmol/1): NaC1 118.0; KC1 4.7; CaC12 2.6; MgC12 1.2; N a H z P O 4 1.0; N a H C O 3 25.0; glucose 11.1; E D T A 0.004 and ascorbic acid 0.11. The temperature was kept at 3 7 ° C and the solution was bubbled with a 95% 02-5% CO 2 mixture, so that its final p H was 7.4. As previously described (Adler-Graschinsky et al., 1972; Butta and AdlerGraschinsky, 1987) the atria were set up in a 5-ml isolated organ bath and were then attached to a force-displacement transducer connected to a Grass polygraph to record the spontaneous contractions of the preparation. A period of equilibration of about 90 min was allowed to elapse until the basal resting rate varied by no more than 10 b e a t s / m i n during a 10 rain interval. Cumulative concentration-response curves to NA, 3-isobutyl-l-methylxanthine (IBMX), forskolin, CaC12 or BAY K 8644 were performed by increasing the concentration stepwise as soon as

To study the effects of the benzodiazepines on intracellular c A M P levels the atria were dissected as previously described and were allowed to stabilize for 60 min in Krebs solution. The benzodiazepines were added to the media 30 rain before and during a 30 s stimulation with 0.5 # M NA. Each atrium was then immediately homogenized in 2 ml of 5% trichloroacetic acid and centrifuged for 10 min at 3000 rpm. An aliquot of 1.5 ml of the supernatant was extracted four times with 4 ml of water-saturated diethyl ether and the residual ether was removed by heating the samples at 40 ° C for 30 min. The cyclic A M P content in the tissue was measured by the saturation assay method described by Brown et al. (1971). The results were expressed as pmol of cAMP per mg of protein determined according to Lowry et al. (1951).

2.3. Statistics The significance of differences between values was determined by means of Student's t-test, except for the values for cAMP tissue content which were analyzed with the two-way analysis of variance (ANOVA) followed by Duncan's test. A P

469

value smaller than 0.05 was regarded as significant. 2.4. Drugs (-)-NE bitartrate monohydrate, atropine sulphate, quinidine sulphate and forskolin were obtained from Sigma Chemical Co. (St. Louis, MO, USA). The following drugs were kindly donated: 3-isobutyl-l-methylxanthine by Searle & Co. (Chicago, ILL, USA), diazepam and clonazepam by Productos Roche S.A., (Buenos Aires, Argentina), Ro 5-4864 (7-chloro-5-[4-chlorophenyl]-l,3-dihydro-l-methyl-2H-1,4-benzodiazepin-2-one) and Ro 15-1788 (ethyl-8-fluoro5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate) by F. Hoffman-La Roche Inc. (Basel, Switzerland); PK 11195 (1-[2-chlorophenyl]-N-methyl-N-[1-methylpropyl] -3-isoquinolinecarboxamide) by Pharmuka Lab. (Gennevilliers, France) and BAY K 8644 (methyl-l,4-dihydro-2,6-dimethyl-3-nitro-4-[2-trifluoromethylphenyl]-pyridine-5-carboxylate) by Bayer A.G. (Leverkusen, FRG). Stock solutions were prepared daily as follows: diazepam (6 mg/ml), Ro 5-4864 (6 mg/ml), forskolin (0.74 m g / m l ) and BAY K 8644 (1 m g / m l ) in absolute ethanol; clonazepam (0.6 m g / m l ) and Ro 15-1788 (1.5 m g / m l ) in a mixture of ethanolwater (1.22 : 1). Further dilutions were performed in double-distilled water. The final concentration of ethanol in the organ both was 50 mM when a 10 /~M concentration of the benzodiazepines was used. The control experiments were therefore performed in the presence of 50 mM ethanol. The solutions of BAY K 8644 were protected from light throughout the experiments, IBMX was dissolved in Krebs solution and the remaining drugs were dissolved in double-distilled water.

3. Results

3.1. Effects of Ro 5-4864, diazepam and clonazepam on the chronotropic responses to NA of rat isolated atria Figure 1 shows the effect of the peripheral benzodiazepine agonist, Ro 5-4864, on the chrono-

tropic responses of rat atria to exogenous NA. Under control conditions (fig. 1A) there was no difference between the first and the second curves made 60 min apart. Ro 5-4864 reduced the chronotropic responses to NA in a concentration-dependent manner. Whereas 1 ~tM Ro 5-4864 (fig. 1B) was without effect on the atrial responses to NA, 5 btM Ro 5-4864 (fig. 1C) shifted the second curve to NA to the right and this displacement was even more pronounced in the presence of the 10/~M concentration of the drug (fig. 1D). At the concentration of NA that caused 60% of the maximal response, the displacement in log units between the second and first curves in the presence of Ro 5-4864 5 # M (0.42 + 0.12, n = 7) differed

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Fig. 1. Effects of Ro 5-4864 on the concentration-response curves to NA. Two consecutive concentration-response curves to NA were made 60 min apart. The second curves (O) were preceded by 30-min washes with Krebs solution followed by 30-min incubation with: 50 mM ethanol in A (controls, n = 6); 1 /~M Ro 5-4864 in B (n = 4); 5 /~M Ro 5-4864 in C (n = 7) and 10 ktM Ro 5-4864 in D (n = 6). Ordinates: increases in atrial rate as percentage of the maximum of the first curve. Abscissas: N A concentration (molar). The maximal increases in atrial rate obtained in the two consecutive curves were as follows (beats/min): in A, 153 + 10 and 161 + 10; in B, 157 + 21 and 166+19; in C, 1 5 0 + 7 and 134+6; in D, 162+9 and 128+11 (P < 0.05). Means and S.E.M. are shown.

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Fig. 2. Effects of d i a z e p a m on the c o n c e n t r a t i o n - r e s p o n s e curves to N A . T w o c o n s e c u t i v e c o n c e n t r a t i o n - r e s p o n s e curves to N A were m a d e 60 m i n apart. The second curves (O) were preceded by 30-min w a s h e s with Krebs solution f o l l o w e d by 30-min incubation with: 1 /~M d i a z e p a m in A (n = 6); 5 /tM d i a z e p a m in B (n = 4); 10 /~M d i a z e p a m in C (n = 6) and 50 /~M d i a z e p a m in D (n = 6). Ordinates: increases in atrial rate as percentage of the m a x i m u m of the first curve. Abscissas: NA concentration (molar). The maximal increases in atrial rate obtained in the two consecutive curves were as follows ( b e a t s / min): in A, 159+9 and 151+14; in B, 1625:15 and 1435:16; in C, 166+12 and 1055:17 (P <0.025), in D, 1585:10 and 89 5:16 (P < 0.01). M e a n values and S.E.M. are shown.

azepam. Diazepam 1 # M was without effect on the atrial responses and at the concentration of N A that caused 60% of the maximal responses, the differences in log units between the second and first curves were 0 . 4 3 _ 0.02 with 5 /~M diazepam (fig. 2B), 0 . 7 8 _ 0.15 with 10 /tM diazepam (fig. 2C) and 1.64 _+ 0.42 with 50 /IM diazepam (fig. 2D). On the other hand the atrial chronotropic responses to N A were not modified by either 10 or 3 0 / t M clonazepam (figs. 3A,B). A higher concentration of this drug, namely 50/zM, could not be tested because it caused arrest of the atrial beats. The inhibitory effects of the benzodiazepines were time-dependent and reversible, i.e. a 5-min incubation with 10 /~M diazepam was without effect on the concentration-response curves to N A and a 60-min wash period with K.rebs solution completely abolished the reductions caused by 10 /~M 5-4864 (data not shown). In addition, the depressant actions of the benzodiazepines were unrelated to either a quinidine-like action or a muscarine-like effect. This is because neither did 10/~M quinidine modify the responses to N A nor did 20 /xM atropine counteract the effect of 10

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Fig. 3. Effects of c l o n a z e p a m on the c o n c e n t r a t i o n - r e s p o n s e curves to N A . T w o c o n s e c u t i v e c o n c e n t r a t i o n - r e s p o n s e curves to N A were m a d e 60 m i n apart. The second curves (O) were preceded b y 3 0 - m i n w a s h e s with Krebs solution f o l l o w e d by 30-min i n c u b a t i o n with: 10 #M c l o n a z e p a m in A (n = 5) and 30 /~M c l o n a z e p a m in B (n = 4). Ordinate: increases in atrial rate as p e r c e n t a g e of the m a x i m u m of the first curve. Abscissas: N A c o n c e n t r a t i o n (molar). T h e m a x i m a l increases in atrial rate o b t a i n e d in the two c o n s e c u t i v e curves were as follows ( b e a t s / r n i n ) : in A, 167+13 and 167+15; in B, 1554-16 and 159 + 16. M e a n values and S.E.M. are shown.

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~o-a 10-7 166 1o-S ,66 1oS (-)- NA concentration (M) Fig. 4. Effects of PK 11195 and Ro 15-1788 on the inhibitory action of Ro 5-4864 and diazepam in the atrial responses to NA. Two consecutive concentration-response curves to N A were made 60 min apart. The second curves (o) were preceded by 30-min washes with Krebs solution followed by 30-mm incubation with: 1 0 / t M Ro 5-4864 plus 1 0 / x M PK 11195 in A (n = 5); 10 /~M diazepam plus 1 0 / z M P K 11195 in B (n = 5); 10 /xM PK 11195 in C (n = 4); 10 laM diazepam plus 10/~M Ro 15-1788 in D (n = 5); 1 0 / t M diazepam plus 100 ttM Ro 15-1788 in E (n = 4); 10 laM Ro 15-1788 in F (n = 7) and 100 /aM Ro 15-1788 in G (n = 5). Maximal chronotropic responses were as follows ( b e a t s / m m ) : in A, 152+11 and 113_+18; in B, 168_+12 and 109_+13 (P < 0.025); in C, 1 6 5 + 1 9 and 153_+6; in D, 169_+7 and 1 3 0 + 1 2 (P < 0.025); in E, 163_+18 and 9 9 + 1 5 (P < 0.05); in F, 147_+13 and 167___12; in G, 167 + 11 and 151 _+12. Mean values and S.E.M. are shown.

/~M Ro 5-4864 (Elgoyhen and Adler-Graschinksy, unpublished observations). Two benzodiazepine antagonists, one peripheral (PK 11195, Le Fur et al., 1983) and the other central (Ro 15-1788, Hunkeler et al., 1981), were assayed on the inhibitory actions of the benzodiazepines on the concentration-response curves to

NA. The inhibitory effect of 10 # M Ro 5-4864 on the atrial responses to N A was not counteracted by an equimolar concentration of PK 11195, either added starting 30 min before (data not shown) o r added simultaneously with the agonist (fig. 4A). Higher concentrations of PK 11195 could not be used because the atrial rate became irregular or

472

was even arrested. On the other hand, neither 10 /~M PK 11195 nor 10 and 100 /~M Ro 15-1788 prevented the decreases in atrial rate induced by 10/tM diazepam (figs. 4B, 4D and 4E). The 10 # M concentrations of the benzodiazepine antagonist did not themselves modify the atrial responses to NA (figs. 4C,F), with the exception of a slight (10%) though significant (P < 0.05) increase in the maximal responses to NA in the case of Ro 151788. A 10 times higher concentration Of Ro 151788 (100 /tM) inhibited the atrial responses to NA and displaced the second concentration-response curves to N A to the right by about 0.5 log unit (P < 0.05) (fig. 4G).

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One of the intracellular events that follows catecholamine stimulation of cardiac fl-adrenoceptots is an increase in cAMP levels (Taniguchi et al. 1977). We therefore studied the effects of the benzodiazepines on the chronotropic responses of rat atria to IBMX, a methylxanthine that increases c A M P levels through inhibition of phosphodiesterases (for review see Wells and Miller, 1983). Two consecutive concentration-response curves to IBMX were obtained 60 min apart. The maximal response to IBMX (171 _+ 7 beats/min, n = 21) did not differ from the maximal chronotropic response to NA (160 _+ 5 beats/min, n = 33). The first and the second curves were practically superimposable under control conditions (fig. 5A). As reported for the responses of the atria to NA, both 10 /~M Ro-4864 (fig. 5B) and 10 /~M diazepam (fig. 5C) but not 10 /~M clonazepam (fig. 5D) diminished the responses of the atria to IBMX. The pattern and the size of the reductions caused by the benzodiazepines in the responses to IBMX were similar to those for the responses to NA, i.e. the reductions were more pronounced for the higher than for the lower concentrations of agonist. The E C 7 0 of IBMX ( - l o g M) in the presence of either Ro 5-4864 (4.58_ 0.08) or diazepam (4.50 + 0.14) differed significantly (P < 0.05) from their respective controls (5.11 + 0.01, n = 6, for Ro 5-4864 and 5 . 1 0 _ 0.06, n = 6, for

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Fig. 5. Effects of Ro 5-4864, diazepam and clonazepam on the chronotropic responses to IBMX in rat isolated atria. Two consecutive concentration-response curves to IBMX were made before ( O ) and after (O) 30-min incubation with: 50 mM ethanol in A (controls, n = 5); 10 /aM Ro 5-4864 in B (n = 6); 10 # M diazepam in C (n = 6) and 10 /tM clonazepam in D (n = 4). Mean values are shown. The vertical lines indicate the S.E.M. Maximal increases in atrial rate in the two consecutive curves were as follows (beats/min): in A, 180 + 14 and 205 + 23; in B, 168-t-7 and 125_+7 (P<0.005); in C, 194_+12 and 140_+6 (P < 0.005); in D, 132_+17 and 151 _+22.

diazepam), whereas the EC20 was not modified by either drug. The chronotropic effect of IBMX on rat atria does not involve the release of N A from nerve terminals, as reported for the chronotropic responses of the dog heart to aminophylline (Westfall and Fleming, 1968). This is because concentrations of IBMX ranging from 0.18 to 54/~M did not increase the outflow of tritium in atria labelled with [3H]NA (data not shown).

3.3. Effects of Ro 5-4864 and diazepam on the chronotropic responses of the atria to forskolin Figure 6 shows the effects of Ro 5-4864 and of diazepam on the responses of the atria to forskolin, a drug that increases cAMP levels through the stimulation of membrane adenylate cyclase

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activity (for review see S e a m o n a n d Daly, 1983). O n l y one c o n c e n t r a t i o n - r e s p o n s e curve was perf o r m e d in each p r e p a r a t i o n in this case b e c a u s e the atrial rate d i d n o t return to its b a s a l values after the highest forskolin c o n c e n t r a t i o n . A 30-rain i n c u b a t i o n with either 1 0 / ~ M R o 5-4864 (fig. 6A) or 10 /~M d i a z e p a m (fig. 6B) significantly dim i n i s h e d the c h r o n o t r o p i c responses of the a t r i a to forskolin. A s o b s e r v e d before with N A (figs. 1 a n d 2) a n d with I B M X (fig. 5) the r e d u c t i o n s c a u s e d b y the b e n z o d i a z e p i n e s were m o r e p r o n o u n c e d for the higher t h a n for the lower forskolin c o n c e n t r a t i o n s a n d were significant for the EC70 (P < 0.05) b u t n o t for the EC20. I n a d d i t i o n , the p e r i p h e r a l agonist was m o r e p o t e n t t h a n the m i x e d agonist to decrease the responses at low forskolin c o n c e n t r a t i o n s . F o r example, the increase in atrial rate elicited b y 0 . 0 6 / ~ M forskolin, 60 + 6 b e a t s / m i n ( n = 8) was n o t m o d i f i e d b y d i a z e p a m b u t was r e d u c e d to 35 + 5 b e a t s / m i n (n = 10, P < 0.025) b y R o 5-4864.

effects of R o 5-4864 a n d d i a z e p a m on c o n c e n t r a t i o n - r e s p o n s e curves of the s p o n t a n e o u s l y b e a t i n g rat isolated atria, either to CaC12 or to a d r u g that p r o m o t e s calcium entry, B A Y K 8644 ( S c h r a m m et al., 1983). The c o n c e n t r a t i o n - r e s p o n s e curves, p r e c e d e d b y a 30-min i n c u b a t i o n with either 10 # M R o 5-4864 or 1 0 / ~ M d i a z e p a m , were o b t a i n e d o n l y once for each p r e p a r a t i o n . Second curves were n o t p e r f o r m e d b e c a u s e the s p o n t a n e o u s atrial rate either b e c a m e irregular after the highest conc e n t r a t i o n o f CaC12 or d i d not r e t u r n to the b a s a l values after the highest c o n c e n t r a t i o n of B A Y K 8644. U n d e r these e x p e r i m e n t a l c o n d i t i o n s b o t h CaC12 (fig. 7) a n d B A Y K 8644 (fig. 8) p r o d u c e d a c o n c e n t r a t i o n - d e p e n d e n t increase in atrial rate with a m a x i m a l c h r o n o t r o p i c r e s p o n s e that r e a c h e d 65% of the m a x i m u m reached with N A . T h e e x p o s u r e to 10/~M R o 5-4864 as well as to 10 /~M d i a z e p a m 30 m i n before a n d d u r i n g the c o n c e n t r a t i o n - r e s p o n s e curves d i d n o t m o d i f y the responses of the atria to either CaC12 (fig. 7) or B A Y K 8644 (fig. 8).

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Fig. 8. Effects of Ro 5-4864 and diazepam on the chronotropic responses of the rat isolated atria to BAY K 8644. Only one concentration-response curve was obtained with each preparation 30 min after the incubation with 10/~M Ro 5-4864 in A (o) and with 10 #M diazepam in B (O). The control curves (©) were preceded by 30-min incubation with 50 mM ethanol. Mean values and S.E.M. of six experiments per group are shown.

p e r i m e n t a l c o n d i t i o n s b y drugs that increase c A M P levels b u t n o t b y drugs with a n effect mediated b y the direct e n t r y of calcium, we studied whether benzodiazepines would m o d i f y either the basal or the stimulated levels of cAMP. The prod u c t i o n of c A M P was stimulated b y 0.5/~M N A , a c o n c e n t r a t i o n that causes a p p r o x i m a t e l y 70% of the m a x i m a l c h r o n o t r o p i c effect o n the entire c o n c e n t r a t i o n - r e s p o n s e curve to N A . This conc e n t r a t i o n was selected o n the basis of the depressant effect exerted b y b o t h R o 5-4864 a n d diazepam (figs. 1 a n d 2). As shown in table 1 the basal values of c A M P were n o t modified b y either

TABLE 1 Effects of Ro 5-4864 and of diazepam on the tissue levels of cAMP. The means:l:S.E.M, of cAMP levels (pmol per mg of protein) are given for four experiments per group. Drugs were added to the media 30 min before and during a 30-s incubation with either saline in the basal group or 0.5 #M NA in the stimulated groups. Drugs

Basal group

Stimulated group

Ethanol 50 mM (controls) Ro 5-4864 10 # M Diazepam 10/~M

11.86 + 1.54 13.82 + 0.36 12.43+0.92

18.74 + 1.04 a 25.33 + 2.63 a 28.94+1.40 a,b

a p < 0.05 compared with the respective basal group; b p < 0.05 compared with the control stimulated group.

R o 5-4864 or diazepam. Moreover, the 1.6 fold increase i n c A M P levels i n d u c e d b y N A was n o t d i m i n i s h e d b y these b e n z o d i a z e p i n e s a n d dia z e p a m even caused a significant increase that reached u p to 50% of the control values.

4. Discussion T h e present experiments showed that microm o l a r c o n c e n t r a t i o n s of Ro 5-4864 a n d of dia z e p a m decreased c o n c e n t r a t i o n d e p e n d e n t l y the c h r o n o t r o p i c responses elicited b y N A in atria isolated from adult W i s t a r rats. This observation suggests that, as reported for the depressant action of benzodiazepines o n guinea-pig papillary muscle (Mestre et al., 1984), some of the b e n z o d i a z c p i n e effects could be linked to a direct i n t e r a c t i o n with peripheral tissues. The fact that the i n h i b i t i o n caused by R o 5-4864 a n d b y diazepam o n the responses of the atria to N A was more pron o u n c e d for the higher t h a n for the lower conc e n t r a t i o n s of N A is consistent with the observation that b e n z o d i a z e p i n e s are useful tools to c o u n teract responses to stressful c o n d i t i o n s (Haefely et al., 1983), where elevated p l a s m a catecholamine c o n c e n t r a t i o n s coexist with a n increased sympathetic tone (for review see Axelrod a n d Reisine, 1984). I n addition, because p l a s m a c o n c e n t r a t i o n s

475

within the micromolar range are reached with diazepam after acute as well as after chronic administration of therapeutic doses to human volunteers (Berlin et al., 1972; Hillestad et al., 1974a,b) a direct inhibitory action on the cardiac responses to NA, specially under stressful situations, could contribute to the cardiovascular effects reported for the benzodiazepines in patients (for review see De Feudis, 1982 and Haefely et al., 1983). It is of interest to note that preliminary experiments have shown that 10 /~M Ro 5-4864 but not 10 ~M clonazepam also reduces the atrial inotropic responses to NA (data not shown). The reductions in the atrial chronotropic responses to NA were obtained both with a peripheral, Ro 5-4864, and with a mixed agonist, diazepam, but not with a central agonist, clonazepare. Although it is tempting to speculate that benzodiazepines may act at peripheral receptors to exert their action, the fact that the depressant effects of Ro 5-4864 and of diazepam on the concentration-response curves to NA were not counteracted by the peripheral benzodiazepine antagonist, PK 11195, does not support the likelyhood of an interaction with specific benzodiazepine receptors on rat atrial tissue. A peripheral effect of Ro 5-4864 not counteracted by PK 11195 has also been reported for the increase in coronary flow produced by Ro-4864 in the rat heart (Grupp et al., 1987). A putative interaction of benzodiazepines with peripheral receptors in the rat atria is also precluded by the observation that, under our experimental conditions, the inhibitory effects of Ro 5-4864 and of diazepam were not found at concentrations below the micromolar range despite the fact that the affinity of diazepam for peripheral benzodiazepine binding sites in the rat heart is in the nanomolar range (Taniguchi et al., 1982). A similar discrepancy between binding affinity and pharmacological activity has been reported for several benzodiazepine effects such as the enhancement of melanogenesis in mouse melanoma cells (Matthew et al., 1981) and the decrease in the electrically stimulated contractions in the guinea-pig ileum (Hullihan et al., 1983). The observation that the central benzodiazepine antagonist, Ro 15-1788, did not prevent the action of diazepam also precludes the par-

ticipation of benzodiazepine receptors of the central type under the present conditions. In addition, the fact that Ro 15-1788 itself inhibited the atrial responses to N A at a concentration of 100 /~M, is consistent with the observation that this antagonist has intrinsic benzodiazepine activity (for review see File and Pellow, 1986). As reported for mouse spinal cord neurones (McLean and Macdonald, 1988), it is possible that a reduction by benzodiazepines of sodium conductance in the rat heart could account for the inhibition of the agonist-mediated atrial responses that we now observed. However, under our experimental conditions, the atrial responses to NA were not reduced by 10 /~M quinidine although the fast sodium current is actually blocked by this drug in isolated rat cardiac cells (Lee et al., 1981). Attention has recently been focused on the possibility that many of the actions of the benzodiazepines may involve the participation of adenosine, possibly through the interaction with the uptake of this nucleoside (for review see Phillis and O'Regan, 1988). It should be noted in this regard that the potentiation by diazepam of the negative inotropic effect of adenosine in guinea-pig atria results from inhibition of adenosine uptake (Clanachan and Marshall 1980; Kenakin, 1982; Moritoki et al., 1985). Nevertheless, since the negative inotropic effect of adenosine in rat atria is not enhanced and the adenosine uptake is not decreased by diazepam (Moritoki et al., 1985) it is unlikely that the depressant effects of Ro 5-4864 and of diazepam observed with rat atria could involve the participation of adenosine. Because acetylcholine antagonizes the responses to isoproterenol in the rabbit heart and those to both isoproterenol and forskolin in the frog isolated cardiac myocytes (Inui et al., 1982; Hartzell and Fischmeister, 1987), another possibility is that the inhibitory effects of Ro 5-4864 and of diazepam that we now saw resulted from the activation of rat atrial muscarinic receptors. Nevertheless, the participation of muscarinic receptors is precluded by the observation that 20 t~M atropine did not modify the depressant effects of 10 t~M Ro 5-4864 on the responses to NA. The observation that the inhibitory effects of diazepam were observed when the drug was added

476 30 but not 5 rain before the concentration-response curves to NA suggests that the benzodiazepines must gain access to an intracellular site to exert their action. Among the possible intracellular mechanisms on which benzodiazepines could interact are those related either to the increase in cAMP levels (Taniguchi et al., 1977) or to the increase in Ca 2÷ influx (Reuter, 1967) that follows fl-adrenergic stimulation. Under our experimental conditions both Ro 5-4864 and diazepam inhibited the chronotropic responses to IBMX and to forskolin whereas they did not modify the responses to either the direct exposure to CaC12 or the direct entry of calcium promoted by the calcium agonist BAY K 8644. The lack of effect of diazepam on the chronotropic responses elicited by the entry of calcium is unrelated to the fact that the maximum reached under the latter conditions was 65% of that produced by NA. This is because the EC65 of NA was indeed reduced by diazepam. Moreover, the present results differ from those reported for other peripheral tissues i.e. that benzodiazepines interfere with responses mediated by the entry of calcium, such as the contraction of the guinea-pig ileum and papillary muscle and the reduction in the release of fl-endorphin from an anterior pituitary-derived tumor cell fine (Ishii et al., 1982; Mestre et al., 1985; Bisserbe et al., 1986). Hence, benzodiazepines appear to modify in rat atrium the responses mediated by an increase in cAMP levels rather than the responses elicited by direct entry of calcium ions. This view is further supported by the observation that neither Ro 5-4864 nor diazepam modified a neuroeffector response mediated by the activation of al-adrenoceptors as in the case of contractions elicited by N A in the nictitating membrane isolated from cats (Elgoyhen and Adler-Graschinsky, unpublished results). According to several reports (for review see Fain and Garcia-Shinz, 1980) the activation of postsynaptic al-adrenoceptors does not enhance the production of cAMP but increases the intracellular calcium concentration. Nevertheless, the fact that the NA-induced cAMP accumulation was not diminished but was even increased by diazepam suggests that there is no direct correlation between benzodiazepine effects and atrial tissue levels of cAMP. A putative

inhibition of rat atrial phosphodiesterases caused by diazepam, as described for several regions of the cat brain (Dalton et al., 1974), can be excluded because the basal cAMP levels were not modified by this drug. The possibility exists that benzodiazepines interfere with a step beyond the increase in cAMP tissue levels that follows fl-adrenergic stimulation. In this regard, by analogy with the inhibition of the calcium-calmodulin-dependent protein kinase system described for micromolar concentrations of different benzodiazepines in the rat brain (DeLorenzo et al., 1981), both Ro 5-4864 and diazepam could act as inhibitors of the cAMP-dependent protein kinases and therefore of the protein phosphorylation system in the rat atrial tissue. Another possibility is that the inhibitory effects of the benzodiazepines on the atrial responses could involve a small but significant and localized change in cAMP levels which escapes detection when total tissue cAMP is determined. The compartmentalized location of cAMP reported for rabbit and guinea-pig hearts supports the latter view (Hayes et al., 1980). Discrepancies between cAMP tissue levels and functional responses have also been reported for the negative chronotropic effect of carbachol in guinea-pig papillary muscle (Gramann and Scholz, 1984; Korth et al., 1987). It is concluded that Ro 5-4864 and diazepam probably reduce in a concentration-dependent manner the chronotropic responses to NA in the rat isolated atria through the interaction with the cAMP-linked chain of events that follows fladrenergic stimulation. The fact that the reductions in atrial responses were more pronounced for the higher than for the lower N A concentrations suggests that benzodiazepines could have a cardiovascular effect specially under those stressful conditions where catecholamine plasma levels are increased. Although the underlying mechanisms of this depressant effect require further study, they could involve either a small localized change in cAMP levels or a modification in the events that follow the increase in cAMP induced by catecholamines, namely at the level of the cAMP-dependent protein kinases. Experiments to elucidate these possibilities are included in our current investigations.

477

Acknowledgements The skillful technical assistance of Ms. Delia Marina Galli and Ms. Maria Rodriguez and the expert secretarial assistance of Ms. Isabel Mercado are gratefully acknowledged. This work was supported by Grant 3911701 form the Consejo Nacional de Investigaciones Cientlficas y T~cnicas, Argentina.

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