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Effects of phenobarbitone on hepatic microsomal enzyme activity and liver blood flow in spontaneously hypertensive rats
Pergamon Press
Life Sciences, Vol . 24, pp . 535-540 Printed in the U.S .A .
EFFECTS OF PHENOBARBITONE ON HEPATIC MICROSOMAL ENZYME ACTIVITY AND LIVER BLOOD FLOW IN SPONTANEOUSLY HYPERTENSIVE RATS M .S . Yates, C .R . Hiley and D .J . Back
Department of Pharmacology and Therapeutics, University of Liverpool, Liverpool L69 3BX . England (Received in final form December 28, 1979)
Summary Hepatic microsomal enzyme activity, liver blood flow and phenobarbitone sleeping time were determined in spontaneously hypertensioe rats (SHR) and normotensive Wistar rats (NR) after pretreatment with saline or phenobarbitone . In NR and SHR the increases in total liver blood flow produced by phenobarbitone were sufficient to maintain liver perfusion despite the increase in liver weight and in both strains of rat the increase was entirely due to increased portal venous return . Saline pretreated SHR had shorter phenobarbitone sleeping times than control NR and their livers had greater total cytochrome c reductase activities and total microsomal protein than those of NR but cytochrome P-450 contents were not significantly different . Phenobarbitone significantly shortened sleeping times in both strains but NR still slept longer than SHR . Total microsomal protein,cytochrome P-450 content and cytochrome c reductase activity were increased by phenobarbitone in both SHR and NR but the increases in cytochrome P-450 and cytochrome c reductase were greater in the hypertensioe rats . The Japanese strain of spontaneously hypertensioe rat (SHR) originated by Okamoto and Aoki (1) has become a widely used model of human essential hypertension . Hiley and Yates (2) have investi gated the pattern of cardiac output distribution in anaesthetised SHR and reported that, although the liver receives the same total proportion of cardiac output in SHR as in normotensive Wistar controls (NR), a greater percentage passes through the hepatic artery in SHR and less through the portal vein . It is known that administration of phenobarbitone, a potent inducer of hepatic microsomal drug metabolising enzymes,increasesliver blood flow in NR and that this is a result of increased portal venous return (3,4). Since the different pattern of hepatic blood supply in SHR might affect the response to phenobarbitone, we have investigated the effects of phenobarbitone on liver blood flow in SHR and compared the response with that of NR . We have also studied hepatic microsomal drug metabolising activity in untreated and phenobarbitone treated SHR . Compatisons of SHR and NR drug metabolising activity have been made by previous workers but the results have been conflicting . Vainionpaa et al ., (5) found no significant differences in cytochrome P-450 content, in vitro microsomal enzyme activity and hexobarbitone sleeping time between SHR and NR whilst Willis and Queener (6) reported 0300-9653/79/0205-0535$02 .00/0 Copyright (c) 1979 Pergamon Press
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statistically shorter phenobarbitone sleeping time in SHR than in normotensive Wistar-Kyoto and Wistar controls . This communication reports the effects of phenobarbitone on cytochrome P-450 content, cytochrome c reductase activity, hepatic microsomal protein content and phenobarbitone sleeping time in SHR and NR . Materials and Methods Groups of age-matched (10-14 week old), male normotensive Wistar rats from our own colony and spontaneously hypertensive rats (OLAC 1976 Ltd , Bicester, Oxfordshire) were given twice daily intraperitoneal injections of phenobarbitone (80 mg kg -lday-1 ) or 0 .9% NaCl (w/v) for 5 days . The phenobarbitone was dissolved in 0 .9% NaCl and all injections were given as 100 X1/1008 body weight (bw) . On the seventh day after commencement of drug administration,~ rats were allocated randomly to blood flow studies, biochemical investigation or determination of phenobarbitone sleeping time . Measurement of liver blood flow : The method used was essentially that of McDevitt and Nies 7 . Rats from treatment groups were anaesthetised with ketamine (120mg kg -1 ) . Blood préssure was recorded from one femoral artery using a Bell & Howell pressure transducer (type 4-422-0001) connected to a Grass model 79 polygraph . The other femoral artery was cannulated in order to allow withdrawal of blood at a constant rate (0 .6m1 min-1 ) by a Perfusor IV pump (Braun, Melsungen, Germany) . A third cannula was passed down the right carotid artery and into the left ventricle ; pressure monitoring was used to ensure the tip was placed in the ventricle . 60,000-80,000 carbonised plastic microspheres (15+5~m) labelled with 85Sr (3M Co ., St . Paul, Minnesota), suspended by ultrasonication in 0 .6m1 0 .9% NaCl containing 0 .02% Tween 80 were injected through the left ventricular cannula over 20 sec . Simultaneously, and for 70 sec after the injection, blood was withdrawn from the femoral artery . The animal was killed with an overdose of sodium phenobarbitone and the placement of the ventricular cannula confirmRadioactivity in the .liver, gastrointesed by visual inspection . tinal tract, spleen and blood sample were measured in a gamma counter (Intertechnique CG 4000) . Cardiac output was determined as (total counts injected x blood sample withdrawal rate/counts in blood sample) and organ blood flow as (counts in organ x blood sample withdrawal rate/counts in blood sample) . The counts trapped in the liver give hepatic arterial flow whilst portal blood flow is obtained by adding the flows through the spleen and gastrointestinal tract (3) . Biochemical studies : Rats were killed by cervical dislocation, their livers removed and a 30% (w/v) homogenate in ice-cold 1 .15% The (w/v) KC1 obtained by use of a Potter-Elvehjem homogeniser . homogenate was centrifuged at 100008 for 20 min at 4oC . The super natant was decanted and further centrifuged at 1050008 for 60 min at 4°C . The resulting microsomal pellet was resuspended in 10 ml Protein and cytochrome P-450 con0 .2M phosphate buffer, pH7 .4 . tents were determined respectively by the methods of Lowry, et al . Cytochrome c reductase activity was (8), and Omura and Sato (9) . measured by the method of Williams and Kamin (10) . Pentobarbitone slee~inq time : Pre-treated rats were injected intraperitoneally with sodium phenobarbitone (40 mg kg -1 ) . Sleeping time was defined as the time between loss and regain of
Vol . 24, No . 6, 1979
Phenobarbitone in SHR
537
the righting reflex . Statistical comparisons were made by analysis of variance (11 ). Results Total liver blood flow per unit weight of liver in saline treated SHR was not significantly different from that found in However, it can be seen that hepatic control NR (Table 1) . arterial flow was significantly greater and portal venous flow significantly lower in the control SHR than in the NR which were pre-treated with saline alone . Furthermore, pre-treatment of the TABLE I Liver Blood Flow in NR and SHR
Body weight (g) Cardiac output (ml/min/100g bw) Mean arterial pressure (mm Hg) Liver weight (g)
Treated with Saline or Phenobarbitone
Saline
Phenobarb .
Saline
(n -- 6)
(n = 6)
(n = 6)
Phenobarb . (n = 6)
305+16
300±8
306+10
301+10
21 .4+0 .6
22 .0+0 .5
21 .5+0 .5
21 .5+0 .4
116+4
115+4
158+4++
156+5
f f
11 .7+0 .5
15 .1+0 .7"
11 .3+0 .4
16 .4+0 .4
Total Liver (ml/mm/100g bw)
4 .01+0 .15
5 .21+0 .1 "
3 .61+0 .14
5 .00+0 .23
Hepatic artery (ml/min/g liver)
0 .13+0 .02
0 .10+0 .01
0 .32+0 .04+
0 .23+0 .04 "
Portal vein (ml/min/g liver)
0 .93+0 .06
0 .96+0 .05
0 .58+0 .04 ++
0 .68+0 .04 "
Blood flow
(ml/min/g liver)
1 .06+0 .05
1 .06+0 .05
0 .90+0 .06
0 .90+0 .07
All values are given as mean _+ S .E .M . n represent the number of animals in the sample . Statistically significant differences between Columns 1 and 2, "p < 0 .01 ; between Columns 1 and 3, +p < 0 .05, ++p < 0 .01 ; between Columns 3 and 4 f ~p< 0 .01 ; between Columns 2 and 4, " p< 0 .01 . two strains of rat with phenobarbitone did not change this pattern of liver blood supply although it did result in greater total liver blood flows relative to body weight in both NR and SHR . In NR the total liver blood flow was 30% greater than the control whereas the
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Phenobarbitone in SHR
Vol . 24, No . 6, 1979
phenobarbitone - pre-treated SHR had a total liver blood flow 39% greater than the corresponding saline-treated animals . Since after phenobarbitone the liver weights of the NR were 30% greater and those of the SHR 45% greater than the respective controls, the rate of perfusion per unit weight of liver was unchanged by the drug treatment . TABLE 2 Hepatic Microsomal Enzymes and Sleeping Time in NR and SHR Treated with Saline or Phenobarbitone
NR
Body weight (g) Microsomal protein (mg/g liver)
SHR
Saline
Phenobarb .
Saline
(n = 6)
(n = 4)
(n = 4)
300+10
303+13
309+17
(n = 4) 305+11
16 .4_+0 .8
20 .4_+1 .7 "
Cytochrome P-450 (pmol/liver)
174+15
454+7 ""
Cytochrome c reductase (mol/ min/liver)
8 .1+0 .4
16 .7_+1 .8 "
10 .0_+0 .5++
30 .9_+2 .7'
72 .3+3 .2
1 DNS ""
48 .4+1 .3 ++
3 DNS' 1 .0 (1)
(mg/liver)
Sleeping Time (min)
163+14
276+30'
8 .8+2 .3 (3)
25 .0_+0 .4++
Phenobarb .
249+13 + 193+12
24 .0_+1 .7 372+18' 794+40'
All values are given as mean _+ S .E .M . n represents the number of animals in the sample . DNS = Did not sleep . Statistical comparisons between Columns 1 and 2 ; " p < 0 .05, " ~< 0 .01 1 and 3 ; +p < 0 .05, +ip< 0 .01 'Column 4 significantly different from Column 2 (p < 0 . 05) and Column 3 (p< 0 .01) . Furthermore, in both strains of animal the overall increase in liver. blood flow, necessary to compensate for the greater liver weight after phenobarbitone was entirely the result of increased portal venous return . Thus, in NR 2 .3+0 .4% and 2 .2+0 .2% of the cardiac output passed through the hepatic artery in the saline and phenobarbitone - treated groups respectively whereas in SHR the corresponding figures were 5 .7+0 .6% and 5 .9+1 .0% . In contrast, portal venous return was significantly greaterin those NR which had received phenobarbitone being 21 .9+0 .9% of the cardiac output compared to the control value of 17 .3+3 .4% (p < 0 .05) . Similarly, in control SHR the portal vein carriéd 10 .3+0 .6% of the cardiac output compared to 18 .0_+1 .1% in those SHR which were given phenobarbitone (p< 0 .01) . The livers of SHR that had received saline
Vol . 24, No . 6, 1979
Phenobarbitone in SHR
539
injections had greater cytochrome c reductase activities than the NR as well as more microsomal protein but total cytochrome P-450 content was not significantly different between the strains of rat (Table 2) . Phenobarbitone sleeping time was significantly shorter for saline - treated SHR than the equivalent NR . Phenobarbitone greatly decreased sleeping time in both strains of animal . Cytochrome P-450 content and cytochrome c reductase activity were increased in both NR and SHR as was total liver microsomal protein . However, microsomal protein content per unit weight of liver was increased only in NR . The responses of cytochrome P-450 and cytochrome c reductase to phenobarbitone were greater in SHR (p < 0 .05 and p < 0 .01 respectively) . Liver cytochrome P-450 content was 4 times greater in phenobarbitone - pretreated SHR compared to control SHR whereas the difference was 2 .6 fold in NR . In the case of cytochrome c reductase, activity was 209% greater in SHR after phenobarbitone compared to an increase of 106% in the normotensive animals . The increases in total liver microsomal protein were not significantly different between the strains and were less dramatic ; in SHR the increase was 49% compared to 69% in NR . Discussion Both SHR and NR responded to phenobarbitone with an increase in the proportion of cardiac output passing through those tissues which drain into the hepatic portal vein . This increase in portal venous return was sufficient in both strains of animals to compensate for the greater liver weight in the phenobarbitone - treated rats . Thus the blood flow per unit weight of liver was maintained in SHR and NR . Since there was no change in the proportion of cardiac output passing through the hepatic artery, the effects of phenobarbitone were entirely due to changes in the splanchnic vascular bed . Although strain difference did not affect the pattern of hepatic blood flow response, there were greater increases in cytochrome P-450 content and cytochrome c reductase activity in the Japanese strain . That there should be a difference between SHR and the Wistar stock from which they are derived is not surprising since there are known to be other differences between them apart from the hypertension ; for example, SHR gain weight more slowly after gaining adulthood than NR (12, 13) . Another difference between the strains lies in the shorter phenobarbitone sleeping times observed in the SHR . In previous studies sleeping times in untreated SHR have been reported to be both the same as and shorter than in NR (5,6) . The results obtained in this study are in agreement with those of Willis and Queener (6) who also found that phenobarbitone sleeping time was significantly shorter in SHR . Vainionpaa et al ., (5) determined hexobarbitone sleeping time to be not signif candy different between the strains . The observations of shorter sleeping times may have a pharmacokinetic basis since the same blood concentrations of phenobarbitone have been found on .waking in SHR as in NR (6) and this eliminates the possibility of different sensitivities to the drug in the central nervous systems of the two strains .
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Ossenberg et al ., (14) have studied the pharmacokinetics of Their data suggest that pentobarbitone in the normotensive rat . blood clearance is 9 .64 ml/min/kg and that almost all the meta bolism occurs in the liver . If this value of clearance is valid for the strains of rat used in this study then our blood flow data Thus would suggest that hepatic extraction is approximately 25%. the extraction and clearance of pentobarbitone by the liver would be largely independent of liver blood flow (15) and any differences in pharmacokinetics would have to be due to different metabolic activity in the two strains of rat . The data presented in this paper do not reveal any significant difference in cytochrome P-450 activity between control SHR and NR . However, the significant differences in cytochrome c reductase and microsomal protein content show that hepatic biochemistry does differ between the two strains . Therefore, it is possible that the explanation for shorter sleeping times in the SHR does lie in altered metabolic activity affecting the pharmacokinetics of pentobarbitone . Nevertheless, both cytochrome c reductase activity and cytochrome P-450 content are significantly greater in the phenobarbitone - pre-treated SHR than those NR receiving the drug and this is consistent with the observed shorter sleeping times in the induced SHR . Acknowledgements This work was supported by grants from ICI Pharmaceuticals Divr sion and the Mersey Regional Health Authority . We are indebted to Mr . P.J . Roberts for technical assistance . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 .
K . OKAMOTO and K . AOKI, Jap . Circulation J. _27282-293 (1963) . C .R . HILEY and M .S . YATES, Clin . Sci . Mol . Med . 5 5 317-320 (1978 A .S . NIES, G .R . WILKINSON, B .D . RUSH, J .T . STRÔTHER and D .G . McDevitt, Biochem . Pharmacol . 25 1991-1993 (1976) . M.S . YATES, C .R . HILEY, P .J . RÔBERTS, D .J . BACK and F .E . CRAWFORD, Biochem . Pharmacol . In press . (1978) . V . VAINIONPAA, E .R . HEIKKINEN and H . VAAPATALO, Pharmacol . Res . Commun . 6 343-346 (1974) . L.R . WILLIS and S .F . QUEENER, Can . J . Physiol . Pharmacol . 5 5 1205--1207 (1977) . D .G . McDEVITT and A .S . NIES, Cardiovasc . Res . _10 494-498 (1976) . O .H . LOWRY, N .J . ROSEBROUGH, A .L . FARR and R .J . RANDALL, J . Biol . Chem . 193 265-275 (1951) . SATO, J . Biol . Chem . 239 2370-2378 (1964) . T . OMURA and C .H . WILLIAMS and H . KAMIN, J . Biol . Chem . 237 587-595 (1962). V .H . DENENBERG, Statistics and Experimental Design for Be havioral and Biological Researchers , pp 125-221, Hemisphere Publishing Corp ., Washington, 1976 . M .A . PFEFFER and E .D . FROHLICH, Circulation Res . 32 and 33 1-28 - 1-38 (1973) . K . NISHIYAMA, A. NISHIYAMA and E .D . FROHLICH, Am . J . Physiol . 230 691-698 (1976) . -W FF . OSSENBERG, M. PEIGNOUX, D . BOURDIAU AND J .-P . BENHAMOU, J . Pharmacol . Exptl . Therap . 19 4 111-116 (1975) . G .R . WILKINSON and D .G . SHAND,Clin . Pharmacol . Therap . 18 377 -390 (1975) .
R.
Life Sciences, Vol . 24, pp . 535-540 Printed in the U.S .A .
EFFECTS OF PHENOBARBITONE ON HEPATIC MICROSOMAL ENZYME ACTIVITY AND LIVER BLOOD FLOW IN SPONTANEOUSLY HYPERTENSIVE RATS M .S . Yates, C .R . Hiley and D .J . Back
Department of Pharmacology and Therapeutics, University of Liverpool, Liverpool L69 3BX . England (Received in final form December 28, 1979)
Summary Hepatic microsomal enzyme activity, liver blood flow and phenobarbitone sleeping time were determined in spontaneously hypertensioe rats (SHR) and normotensive Wistar rats (NR) after pretreatment with saline or phenobarbitone . In NR and SHR the increases in total liver blood flow produced by phenobarbitone were sufficient to maintain liver perfusion despite the increase in liver weight and in both strains of rat the increase was entirely due to increased portal venous return . Saline pretreated SHR had shorter phenobarbitone sleeping times than control NR and their livers had greater total cytochrome c reductase activities and total microsomal protein than those of NR but cytochrome P-450 contents were not significantly different . Phenobarbitone significantly shortened sleeping times in both strains but NR still slept longer than SHR . Total microsomal protein,cytochrome P-450 content and cytochrome c reductase activity were increased by phenobarbitone in both SHR and NR but the increases in cytochrome P-450 and cytochrome c reductase were greater in the hypertensioe rats . The Japanese strain of spontaneously hypertensioe rat (SHR) originated by Okamoto and Aoki (1) has become a widely used model of human essential hypertension . Hiley and Yates (2) have investi gated the pattern of cardiac output distribution in anaesthetised SHR and reported that, although the liver receives the same total proportion of cardiac output in SHR as in normotensive Wistar controls (NR), a greater percentage passes through the hepatic artery in SHR and less through the portal vein . It is known that administration of phenobarbitone, a potent inducer of hepatic microsomal drug metabolising enzymes,increasesliver blood flow in NR and that this is a result of increased portal venous return (3,4). Since the different pattern of hepatic blood supply in SHR might affect the response to phenobarbitone, we have investigated the effects of phenobarbitone on liver blood flow in SHR and compared the response with that of NR . We have also studied hepatic microsomal drug metabolising activity in untreated and phenobarbitone treated SHR . Compatisons of SHR and NR drug metabolising activity have been made by previous workers but the results have been conflicting . Vainionpaa et al ., (5) found no significant differences in cytochrome P-450 content, in vitro microsomal enzyme activity and hexobarbitone sleeping time between SHR and NR whilst Willis and Queener (6) reported 0300-9653/79/0205-0535$02 .00/0 Copyright (c) 1979 Pergamon Press
53 6
Phenobarbitone in SHR
Vol . 24, No . 6, 1979
statistically shorter phenobarbitone sleeping time in SHR than in normotensive Wistar-Kyoto and Wistar controls . This communication reports the effects of phenobarbitone on cytochrome P-450 content, cytochrome c reductase activity, hepatic microsomal protein content and phenobarbitone sleeping time in SHR and NR . Materials and Methods Groups of age-matched (10-14 week old), male normotensive Wistar rats from our own colony and spontaneously hypertensive rats (OLAC 1976 Ltd , Bicester, Oxfordshire) were given twice daily intraperitoneal injections of phenobarbitone (80 mg kg -lday-1 ) or 0 .9% NaCl (w/v) for 5 days . The phenobarbitone was dissolved in 0 .9% NaCl and all injections were given as 100 X1/1008 body weight (bw) . On the seventh day after commencement of drug administration,~ rats were allocated randomly to blood flow studies, biochemical investigation or determination of phenobarbitone sleeping time . Measurement of liver blood flow : The method used was essentially that of McDevitt and Nies 7 . Rats from treatment groups were anaesthetised with ketamine (120mg kg -1 ) . Blood préssure was recorded from one femoral artery using a Bell & Howell pressure transducer (type 4-422-0001) connected to a Grass model 79 polygraph . The other femoral artery was cannulated in order to allow withdrawal of blood at a constant rate (0 .6m1 min-1 ) by a Perfusor IV pump (Braun, Melsungen, Germany) . A third cannula was passed down the right carotid artery and into the left ventricle ; pressure monitoring was used to ensure the tip was placed in the ventricle . 60,000-80,000 carbonised plastic microspheres (15+5~m) labelled with 85Sr (3M Co ., St . Paul, Minnesota), suspended by ultrasonication in 0 .6m1 0 .9% NaCl containing 0 .02% Tween 80 were injected through the left ventricular cannula over 20 sec . Simultaneously, and for 70 sec after the injection, blood was withdrawn from the femoral artery . The animal was killed with an overdose of sodium phenobarbitone and the placement of the ventricular cannula confirmRadioactivity in the .liver, gastrointesed by visual inspection . tinal tract, spleen and blood sample were measured in a gamma counter (Intertechnique CG 4000) . Cardiac output was determined as (total counts injected x blood sample withdrawal rate/counts in blood sample) and organ blood flow as (counts in organ x blood sample withdrawal rate/counts in blood sample) . The counts trapped in the liver give hepatic arterial flow whilst portal blood flow is obtained by adding the flows through the spleen and gastrointestinal tract (3) . Biochemical studies : Rats were killed by cervical dislocation, their livers removed and a 30% (w/v) homogenate in ice-cold 1 .15% The (w/v) KC1 obtained by use of a Potter-Elvehjem homogeniser . homogenate was centrifuged at 100008 for 20 min at 4oC . The super natant was decanted and further centrifuged at 1050008 for 60 min at 4°C . The resulting microsomal pellet was resuspended in 10 ml Protein and cytochrome P-450 con0 .2M phosphate buffer, pH7 .4 . tents were determined respectively by the methods of Lowry, et al . Cytochrome c reductase activity was (8), and Omura and Sato (9) . measured by the method of Williams and Kamin (10) . Pentobarbitone slee~inq time : Pre-treated rats were injected intraperitoneally with sodium phenobarbitone (40 mg kg -1 ) . Sleeping time was defined as the time between loss and regain of
Vol . 24, No . 6, 1979
Phenobarbitone in SHR
537
the righting reflex . Statistical comparisons were made by analysis of variance (11 ). Results Total liver blood flow per unit weight of liver in saline treated SHR was not significantly different from that found in However, it can be seen that hepatic control NR (Table 1) . arterial flow was significantly greater and portal venous flow significantly lower in the control SHR than in the NR which were pre-treated with saline alone . Furthermore, pre-treatment of the TABLE I Liver Blood Flow in NR and SHR
Body weight (g) Cardiac output (ml/min/100g bw) Mean arterial pressure (mm Hg) Liver weight (g)
Treated with Saline or Phenobarbitone
Saline
Phenobarb .
Saline
(n -- 6)
(n = 6)
(n = 6)
Phenobarb . (n = 6)
305+16
300±8
306+10
301+10
21 .4+0 .6
22 .0+0 .5
21 .5+0 .5
21 .5+0 .4
116+4
115+4
158+4++
156+5
f f
11 .7+0 .5
15 .1+0 .7"
11 .3+0 .4
16 .4+0 .4
Total Liver (ml/mm/100g bw)
4 .01+0 .15
5 .21+0 .1 "
3 .61+0 .14
5 .00+0 .23
Hepatic artery (ml/min/g liver)
0 .13+0 .02
0 .10+0 .01
0 .32+0 .04+
0 .23+0 .04 "
Portal vein (ml/min/g liver)
0 .93+0 .06
0 .96+0 .05
0 .58+0 .04 ++
0 .68+0 .04 "
Blood flow
(ml/min/g liver)
1 .06+0 .05
1 .06+0 .05
0 .90+0 .06
0 .90+0 .07
All values are given as mean _+ S .E .M . n represent the number of animals in the sample . Statistically significant differences between Columns 1 and 2, "p < 0 .01 ; between Columns 1 and 3, +p < 0 .05, ++p < 0 .01 ; between Columns 3 and 4 f ~p< 0 .01 ; between Columns 2 and 4, " p< 0 .01 . two strains of rat with phenobarbitone did not change this pattern of liver blood supply although it did result in greater total liver blood flows relative to body weight in both NR and SHR . In NR the total liver blood flow was 30% greater than the control whereas the
538
Phenobarbitone in SHR
Vol . 24, No . 6, 1979
phenobarbitone - pre-treated SHR had a total liver blood flow 39% greater than the corresponding saline-treated animals . Since after phenobarbitone the liver weights of the NR were 30% greater and those of the SHR 45% greater than the respective controls, the rate of perfusion per unit weight of liver was unchanged by the drug treatment . TABLE 2 Hepatic Microsomal Enzymes and Sleeping Time in NR and SHR Treated with Saline or Phenobarbitone
NR
Body weight (g) Microsomal protein (mg/g liver)
SHR
Saline
Phenobarb .
Saline
(n = 6)
(n = 4)
(n = 4)
300+10
303+13
309+17
(n = 4) 305+11
16 .4_+0 .8
20 .4_+1 .7 "
Cytochrome P-450 (pmol/liver)
174+15
454+7 ""
Cytochrome c reductase (mol/ min/liver)
8 .1+0 .4
16 .7_+1 .8 "
10 .0_+0 .5++
30 .9_+2 .7'
72 .3+3 .2
1 DNS ""
48 .4+1 .3 ++
3 DNS' 1 .0 (1)
(mg/liver)
Sleeping Time (min)
163+14
276+30'
8 .8+2 .3 (3)
25 .0_+0 .4++
Phenobarb .
249+13 + 193+12
24 .0_+1 .7 372+18' 794+40'
All values are given as mean _+ S .E .M . n represents the number of animals in the sample . DNS = Did not sleep . Statistical comparisons between Columns 1 and 2 ; " p < 0 .05, " ~< 0 .01 1 and 3 ; +p < 0 .05, +ip< 0 .01 'Column 4 significantly different from Column 2 (p < 0 . 05) and Column 3 (p< 0 .01) . Furthermore, in both strains of animal the overall increase in liver. blood flow, necessary to compensate for the greater liver weight after phenobarbitone was entirely the result of increased portal venous return . Thus, in NR 2 .3+0 .4% and 2 .2+0 .2% of the cardiac output passed through the hepatic artery in the saline and phenobarbitone - treated groups respectively whereas in SHR the corresponding figures were 5 .7+0 .6% and 5 .9+1 .0% . In contrast, portal venous return was significantly greaterin those NR which had received phenobarbitone being 21 .9+0 .9% of the cardiac output compared to the control value of 17 .3+3 .4% (p < 0 .05) . Similarly, in control SHR the portal vein carriéd 10 .3+0 .6% of the cardiac output compared to 18 .0_+1 .1% in those SHR which were given phenobarbitone (p< 0 .01) . The livers of SHR that had received saline
Vol . 24, No . 6, 1979
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injections had greater cytochrome c reductase activities than the NR as well as more microsomal protein but total cytochrome P-450 content was not significantly different between the strains of rat (Table 2) . Phenobarbitone sleeping time was significantly shorter for saline - treated SHR than the equivalent NR . Phenobarbitone greatly decreased sleeping time in both strains of animal . Cytochrome P-450 content and cytochrome c reductase activity were increased in both NR and SHR as was total liver microsomal protein . However, microsomal protein content per unit weight of liver was increased only in NR . The responses of cytochrome P-450 and cytochrome c reductase to phenobarbitone were greater in SHR (p < 0 .05 and p < 0 .01 respectively) . Liver cytochrome P-450 content was 4 times greater in phenobarbitone - pretreated SHR compared to control SHR whereas the difference was 2 .6 fold in NR . In the case of cytochrome c reductase, activity was 209% greater in SHR after phenobarbitone compared to an increase of 106% in the normotensive animals . The increases in total liver microsomal protein were not significantly different between the strains and were less dramatic ; in SHR the increase was 49% compared to 69% in NR . Discussion Both SHR and NR responded to phenobarbitone with an increase in the proportion of cardiac output passing through those tissues which drain into the hepatic portal vein . This increase in portal venous return was sufficient in both strains of animals to compensate for the greater liver weight in the phenobarbitone - treated rats . Thus the blood flow per unit weight of liver was maintained in SHR and NR . Since there was no change in the proportion of cardiac output passing through the hepatic artery, the effects of phenobarbitone were entirely due to changes in the splanchnic vascular bed . Although strain difference did not affect the pattern of hepatic blood flow response, there were greater increases in cytochrome P-450 content and cytochrome c reductase activity in the Japanese strain . That there should be a difference between SHR and the Wistar stock from which they are derived is not surprising since there are known to be other differences between them apart from the hypertension ; for example, SHR gain weight more slowly after gaining adulthood than NR (12, 13) . Another difference between the strains lies in the shorter phenobarbitone sleeping times observed in the SHR . In previous studies sleeping times in untreated SHR have been reported to be both the same as and shorter than in NR (5,6) . The results obtained in this study are in agreement with those of Willis and Queener (6) who also found that phenobarbitone sleeping time was significantly shorter in SHR . Vainionpaa et al ., (5) determined hexobarbitone sleeping time to be not signif candy different between the strains . The observations of shorter sleeping times may have a pharmacokinetic basis since the same blood concentrations of phenobarbitone have been found on .waking in SHR as in NR (6) and this eliminates the possibility of different sensitivities to the drug in the central nervous systems of the two strains .
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Ossenberg et al ., (14) have studied the pharmacokinetics of Their data suggest that pentobarbitone in the normotensive rat . blood clearance is 9 .64 ml/min/kg and that almost all the meta bolism occurs in the liver . If this value of clearance is valid for the strains of rat used in this study then our blood flow data Thus would suggest that hepatic extraction is approximately 25%. the extraction and clearance of pentobarbitone by the liver would be largely independent of liver blood flow (15) and any differences in pharmacokinetics would have to be due to different metabolic activity in the two strains of rat . The data presented in this paper do not reveal any significant difference in cytochrome P-450 activity between control SHR and NR . However, the significant differences in cytochrome c reductase and microsomal protein content show that hepatic biochemistry does differ between the two strains . Therefore, it is possible that the explanation for shorter sleeping times in the SHR does lie in altered metabolic activity affecting the pharmacokinetics of pentobarbitone . Nevertheless, both cytochrome c reductase activity and cytochrome P-450 content are significantly greater in the phenobarbitone - pre-treated SHR than those NR receiving the drug and this is consistent with the observed shorter sleeping times in the induced SHR . Acknowledgements This work was supported by grants from ICI Pharmaceuticals Divr sion and the Mersey Regional Health Authority . We are indebted to Mr . P.J . Roberts for technical assistance . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 .
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