Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by DUKE UNIVERSITY - PERKINS LIBRARY on 12/30/14 For personal use only.
Increase in Catecholamine Content sf the Rat Adrenals After Pretreatment with Diazepam A. L. GASCONA N D y. EELOIPIER' Dkpc~rtementdc pharmacologic~,FacultP de mkdecine, C'nivrrsitd de MontrBal, Montrr'ul, QrrPhec Received September 11, 8975 GASCOW, A. Ha.. and I>ELCPRIER. 9. 1975. Increase in catecholamine content of the rat adreiaals after pretreatment with diazapam. Can. J . Physiol. Pharmacaal. 53, 1 198-1 199. The effect of t'salhacute pretreatment with diazepam was studied on the catecholamine content of the heart and adrenals of the rat. The results show that at all doses studied. subacute treatment with diazepam increases the catecholamine content of the adrenal glands and there is no change in the noradrenaline content of the heart and brain.
Introdaactioa Recently, it has been shown that chronic treatment with rnicrosomal enzyme inducers such as phenobarbital, chlorophenothane (DDT) and 3-methylcholanthrene produces an increase in the catecholami~~e content of the guinea-pig seminal vesicles (Cloutier and Gascon 1971 ) . Furthermore, it has been reported that this class of agent also increases the eatecholamine content of the adrenal glands in the rat, the rabbit, the guinea-pig and the hamster (Brassard and Gascon 1975). Since diazepam is also known to induce microsomal enzymes (Jori et al. 1969: Valerino et al. B973), we have studied the effect of subacute treatment with diazepam on the catecholamine content of various tissues of the rat. Materids and R3ethods 'pra%ue-Dawley rats weighin& 190-210 g were used in our experiments. The animals were divided at random into four groups and treated as ~ ~ ~ I o w those s: of the first group were injected intraperitoneally with diuepam (150 mg/kg) once a day for 5 days; those of the second and third groups received diazepam (50 and 25 mg/kg) once a dav for 7 days; those of the ]ad group served as and received the vehicle. carboxvmethvlcelI~&, intraperitoneally during the same pe;iod (4 days). The animals were stunned and bled 24 h after the last injection. The brains, hearts and adreraals were reanoved. The organs were homogenized in 5 volumes of cold perchloric acid (0.4 N ) . Homogenates were centrifuged at 9080 g at 8 "G for IQ min and the 'Scholar of La Fondation Monast.
supernatant fractions were used for catecholamine determinations by the method of Euler and Lishajks (1959). The last step of this method is the reading of each sample in an Amit~co-Bowman spectrophotofluororneker. Finally, the concentrations sf noradrenaline were measured sirnultmnec~uslyaccording to the equation of Cohera and Goldenberg ( 1 957a, 1957h). The statistical analyses rased were the standard error of the mean and Student's t test.
Results and Discussion As seen in Tablc 1, thc 5-day pretreatment ( 150 mg/kg) and the 7-day prctr-eatnsent (25 and 50 mg/kg) with diazepam produced a significant increase ( P < 0.01 ) in the catecholamine content of the adrenal glands. In contrast to this result, diazepam did not alter the noradrenaline levels ila the brains and hearts. The lack of effect of diazepam pretreatment on the nsradrei~alinecontent of the brain has been reported by Taylor and Laverty In Our experiments9 we also shown that diazepam does not change the level of laoradrenaline in the heart but i~acreasesthe catceholamine cofntcnt of the adrenal is This last effect of other lnk~osomalenzyme inducers (Cloutier and Gascon 1971 ) . At this point, several questions can be raised. ( I ) 1s this excess in adrenal catecholaminc easily releasable? ( 2 ) the result of such a release of adrenaline during acute stress on the heart rate and rhythm? This last possibility may be of major impor-
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by DUKE UNIVERSITY - PERKINS LIBRARY on 12/30/14 For personal use only.
C0MMUNIC:ATIONS
TABLE 1 . Effect of diazepam treatment on the catecholamine content of various rat tissues Brain noradrenaline ( ~ g / gtissue)
Heart noradrenaline (Pg/g tissue)
Adrenal gland adrenaline (pg/g tissue)
Controls
0.326k0.054
0.547k8.0480
757.3k72.9
Diazepam, 158 rng/kg per day for 5 days
0.347i-0.058
0.572.5-0.0375
B032.5285.8*
Diazepam, 50 mg/kg per day for 9 days
8.320+0.062
0.542k0.036
1081.9+67.8*
Diazepam, 25 mg/kg per day for 7 days
0.339~k0.068
0.495+0.041
937.2k59.2*
tance for patients with cardiac disease. In such a patient, a massive release of adrenal catecholarnine during an acute stress may precipitate fatal cardiac arrhythmias. Other studies are presently in progress to measure the effect of acute stress on heart rate and rhythm in animals pretreated with diazepam. We would Bike to thank Mrs. Claude Denniel and Miss M. Y. Saintonge for their skillfuB technical assistance as well as Hoffrnann - La Roche Ltd. (MontreaI) for their generous supply of diazepam. This research was supported by la Fondation Rougier Armandie Earnsntagne and by La Fondation PhCnix. BROSS.LBKD, J . J., dnd GASCON,A . L. 1975. lnfluence of phenobarbit:al pretreatment on tissue catecholamine content of various anima[ species. Res. Commun. Chern. Pathol. Pharmacol. 18,619-632. CLOIJTIER, G., and GASCON, A. I,. 1971. Modification of catecholamine content of male acxessory sexual tissues of guinea-pig after pretreatment with microsomaB enzyme inducers. Biochem. Pharmacol. 20, 23192325.
COHEN,G.. and GOH.DF,NBERG, M. 1 9 5 7 ~The . simultaneoils fluorimetric determination of adrenaline and noradrenaline in plasma. I. The fluorescence characteristics of adrenaline and noradrenolutine and their simultaneous determination in mixtures. J . Neurochem. %,58-70. 1957h. 'khe sim~litaneolmsdetermination of adrenaline and noradrenaline in plasma. 11. Peripheral venous plasma concentration in normal sub-jectsand in patients with pheochromscytoma. J. Nenrochem. %,71-80. EUI.ER, U . S . ,and L'ISHAJKO, F. 1961. Improved technique for the fluorimetric determination of catecholamines. Acta Physiol. Scand. 51,348-356. Jorms, A., PRFSTINI, P. E., and PUGLIA-r-rr, C. 1969. Effect of diazepam and chloridiazepoxide on the metabolism of other drugs. 9 . Pharm. ~harrnacoi.21, 387-3961. T ~ y n o a ,K. M . , and I,AVEWTY, R . 1969. The effect of' chlordiazepoxidc, diazepam and nitrazepam on catecholamine metabolism in regions of the rat brain. Enr. J . Pharmacol. 8,296-381. V~LEWIN N.QM., ~ , VESS~L @., S . . JOHNSON,,A. 0., and AUWORI, K . C. 1973. Effects s f various centrally active drugs on the hepatic microsomal enzymes: A comparative study. Metab. Dispos. 1, 669-678.
Increase in Catecholamine Content sf the Rat Adrenals After Pretreatment with Diazepam A. L. GASCONA N D y. EELOIPIER' Dkpc~rtementdc pharmacologic~,FacultP de mkdecine, C'nivrrsitd de MontrBal, Montrr'ul, QrrPhec Received September 11, 8975 GASCOW, A. Ha.. and I>ELCPRIER. 9. 1975. Increase in catecholamine content of the rat adreiaals after pretreatment with diazapam. Can. J . Physiol. Pharmacaal. 53, 1 198-1 199. The effect of t'salhacute pretreatment with diazepam was studied on the catecholamine content of the heart and adrenals of the rat. The results show that at all doses studied. subacute treatment with diazepam increases the catecholamine content of the adrenal glands and there is no change in the noradrenaline content of the heart and brain.
Introdaactioa Recently, it has been shown that chronic treatment with rnicrosomal enzyme inducers such as phenobarbital, chlorophenothane (DDT) and 3-methylcholanthrene produces an increase in the catecholami~~e content of the guinea-pig seminal vesicles (Cloutier and Gascon 1971 ) . Furthermore, it has been reported that this class of agent also increases the eatecholamine content of the adrenal glands in the rat, the rabbit, the guinea-pig and the hamster (Brassard and Gascon 1975). Since diazepam is also known to induce microsomal enzymes (Jori et al. 1969: Valerino et al. B973), we have studied the effect of subacute treatment with diazepam on the catecholamine content of various tissues of the rat. Materids and R3ethods 'pra%ue-Dawley rats weighin& 190-210 g were used in our experiments. The animals were divided at random into four groups and treated as ~ ~ ~ I o w those s: of the first group were injected intraperitoneally with diuepam (150 mg/kg) once a day for 5 days; those of the second and third groups received diazepam (50 and 25 mg/kg) once a dav for 7 days; those of the ]ad group served as and received the vehicle. carboxvmethvlcelI~&, intraperitoneally during the same pe;iod (4 days). The animals were stunned and bled 24 h after the last injection. The brains, hearts and adreraals were reanoved. The organs were homogenized in 5 volumes of cold perchloric acid (0.4 N ) . Homogenates were centrifuged at 9080 g at 8 "G for IQ min and the 'Scholar of La Fondation Monast.
supernatant fractions were used for catecholamine determinations by the method of Euler and Lishajks (1959). The last step of this method is the reading of each sample in an Amit~co-Bowman spectrophotofluororneker. Finally, the concentrations sf noradrenaline were measured sirnultmnec~uslyaccording to the equation of Cohera and Goldenberg ( 1 957a, 1957h). The statistical analyses rased were the standard error of the mean and Student's t test.
Results and Discussion As seen in Tablc 1, thc 5-day pretreatment ( 150 mg/kg) and the 7-day prctr-eatnsent (25 and 50 mg/kg) with diazepam produced a significant increase ( P < 0.01 ) in the catecholamine content of the adrenal glands. In contrast to this result, diazepam did not alter the noradrenaline levels ila the brains and hearts. The lack of effect of diazepam pretreatment on the nsradrei~alinecontent of the brain has been reported by Taylor and Laverty In Our experiments9 we also shown that diazepam does not change the level of laoradrenaline in the heart but i~acreasesthe catceholamine cofntcnt of the adrenal is This last effect of other lnk~osomalenzyme inducers (Cloutier and Gascon 1971 ) . At this point, several questions can be raised. ( I ) 1s this excess in adrenal catecholaminc easily releasable? ( 2 ) the result of such a release of adrenaline during acute stress on the heart rate and rhythm? This last possibility may be of major impor-
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by DUKE UNIVERSITY - PERKINS LIBRARY on 12/30/14 For personal use only.
C0MMUNIC:ATIONS
TABLE 1 . Effect of diazepam treatment on the catecholamine content of various rat tissues Brain noradrenaline ( ~ g / gtissue)
Heart noradrenaline (Pg/g tissue)
Adrenal gland adrenaline (pg/g tissue)
Controls
0.326k0.054
0.547k8.0480
757.3k72.9
Diazepam, 158 rng/kg per day for 5 days
0.347i-0.058
0.572.5-0.0375
B032.5285.8*
Diazepam, 50 mg/kg per day for 9 days
8.320+0.062
0.542k0.036
1081.9+67.8*
Diazepam, 25 mg/kg per day for 7 days
0.339~k0.068
0.495+0.041
937.2k59.2*
tance for patients with cardiac disease. In such a patient, a massive release of adrenal catecholarnine during an acute stress may precipitate fatal cardiac arrhythmias. Other studies are presently in progress to measure the effect of acute stress on heart rate and rhythm in animals pretreated with diazepam. We would Bike to thank Mrs. Claude Denniel and Miss M. Y. Saintonge for their skillfuB technical assistance as well as Hoffrnann - La Roche Ltd. (MontreaI) for their generous supply of diazepam. This research was supported by la Fondation Rougier Armandie Earnsntagne and by La Fondation PhCnix. BROSS.LBKD, J . J., dnd GASCON,A . L. 1975. lnfluence of phenobarbit:al pretreatment on tissue catecholamine content of various anima[ species. Res. Commun. Chern. Pathol. Pharmacol. 18,619-632. CLOIJTIER, G., and GASCON, A. I,. 1971. Modification of catecholamine content of male acxessory sexual tissues of guinea-pig after pretreatment with microsomaB enzyme inducers. Biochem. Pharmacol. 20, 23192325.
COHEN,G.. and GOH.DF,NBERG, M. 1 9 5 7 ~The . simultaneoils fluorimetric determination of adrenaline and noradrenaline in plasma. I. The fluorescence characteristics of adrenaline and noradrenolutine and their simultaneous determination in mixtures. J . Neurochem. %,58-70. 1957h. 'khe sim~litaneolmsdetermination of adrenaline and noradrenaline in plasma. 11. Peripheral venous plasma concentration in normal sub-jectsand in patients with pheochromscytoma. J. Nenrochem. %,71-80. EUI.ER, U . S . ,and L'ISHAJKO, F. 1961. Improved technique for the fluorimetric determination of catecholamines. Acta Physiol. Scand. 51,348-356. Jorms, A., PRFSTINI, P. E., and PUGLIA-r-rr, C. 1969. Effect of diazepam and chloridiazepoxide on the metabolism of other drugs. 9 . Pharm. ~harrnacoi.21, 387-3961. T ~ y n o a ,K. M . , and I,AVEWTY, R . 1969. The effect of' chlordiazepoxidc, diazepam and nitrazepam on catecholamine metabolism in regions of the rat brain. Enr. J . Pharmacol. 8,296-381. V~LEWIN N.QM., ~ , VESS~L @., S . . JOHNSON,,A. 0., and AUWORI, K . C. 1973. Effects s f various centrally active drugs on the hepatic microsomal enzymes: A comparative study. Metab. Dispos. 1, 669-678.
