Europcan Journal of Pharmacolo.w - llh decu&i ,',,. ' ,uacologv Section ~_¢~ ( 1 ~,)_) 189-190
189
g, 1992 Elsevier Science Publishers B.V. All rights reserved 0922-4106/92/$05.00
EJPMOL OIt3R
Rapid communication
Angiotensin II rapidly modulates the renal peripheral benzodiazepine receptor Philip V. H o l m e s a n d R o b e r t C. D r u g a n ' Schrier Research Laboraton" Department o f Psychology, Brown University Procidence, RI 02912, U&4
Received 27 April 1992, accepted 28 April 1992
The effects of acute exposure !o angiotensin II (All) ~,~ ~he renal peripheral benzodiazcpine rcceptcr were studied in rats. As little as 37.5 #g of AII injected s.c. over an 80 min period caused immediate reductions in [3H]Ro5-4864 binding. Scatchard analysis rcveaicd that the rcCuction in [3H]Ro5-4864 binding induced by AII was due to a drop in receptor density or B,~. The influence of Aii on the peripheral benzodiazepine receptor is similar to tha! of stress. Kidncy (r::t); Benzodiazcpine receptors (peripheral):. Angiotensiq ii
The peripheral benzodiazepine receptor is an outer All or vehicle were injected s.c. at 0, 30, 60 min. Rats mitochondrial membrane receptor found in high denwere killed by decapitation in a counterbalanced order sity in adrenal gland and kidney. The peripheral benzoat 80 min and tissues were dissected, fast-frozen and diazepine receptor is also found in the brain (Anholt, stored at -80°C. Tissues were thawed, weighed, dis!986; Benavides et al., !083) The distribution of perupted in 50 volumes of 50 mM Tris-HCl buffer (pH = riphera! benzodiazepine receptors in the kidney and 7.4), and centrifuged at 20,000 × g for 20 rain. The brain is similar to that of angiotensin II (All) receptissues were resuspended in 200 volumes of buffer, and tots. Highest densities of these receptors are localized the binding of [3H]Ro5-4864 at a 1 nM concentration in the inner stripe of the outer medulla and cortex in ,:,as determined as previously described (Drugan et al., kidney and circumventricular organs in brain (BeI9%). navides et al., 1983; Basile et al., 1987; Mendelsohn et Acute exposure to AlI caused an immediate doseal., 1988). The renal peripheral benzodiazepine recepdependent decrease in renal peripheral benzoditot is under hormonal control. Adrenalectomy causes a azepine receptor binding (one-way ANOVA, F = 6.63, reduction in renal peripheral benzodiazepine receptor P < 0.001; see fig. 1). Post-hoe Newman-Keuls tests density after 2 weeks, and this decrease is reversed revealed that the 37.5 /.tg and 75 pog doses of All following one week of a!dosterone therapy suggesting a significantly reduced [-~H]Ro5-4864 binding compared tonic influence of mineralocorticoids (Basile et al., to controls. To characterize the nature of this reduc1987). We presently report that acute AII administration the procedure above was repeated using eight tion causes as immediate reduction in renal peripheral additional rats treated with either the 75 /xg dose of benzodiazepine receptor density in rats. These experiAIi or vehicle. Scatchard analysis included a range of ments are the first demonstration of a rapid hormonal [3H]RoS-4864 concentratiov~ from 0.25 to 16 nM. The regulation of the peripheral benzodiazepine receptor inset in fig. 1 is a rcpresmitative Scatcha~d plot revealin vivo and provide further evidence for the sensitivity ing that the reduction in [3H]Ro5-4864 binding in rats ot this receptor to c~docrine control, treated :dth 75 /xg of All over an 80 m i , period was Male Sprague-Dawley-derb,ed rats v.eighing 250due to a drop in receptor density o~ B, ,,:. A separate 325 g at the tinre of experimemation wc,c ~,muo,,,,!,. . . . . . . ,-~v,-.-' . ,,,~,,.'~'~'~ was ,'ond,,eted ....... r~vealing that the time divided into four groups: vehicie, AII-18.75/xg, A]!-37.5 course o{" ~hese changes is rapid, with re ~._c;;ons evi/,g, and ~,-,~/~g./'" v~ Drug administration was conducted dent as sot,a as 5 rain following a ~ingic, _~"'~#~z s.c. over an 80 mm period. One third of the total doses of injection, of .MI (data not shown). The hypothesis that ~hc decrease in renal perioheral bcnzodiazepir~e receptor density v:as related to increases in systemic arterial pres;ure was tes!ed by CorresF~ ,,lcnce re: R.C. Drugan. Schrier Research kaboratoD', Department of Psycholngy, Box 1853, Brown University, Providence, RI examining tile ,2fleets of a l l equipressor close oi n o t e 02912. USA. Tel. (401) 8{)3-2727.Fax (401}863-13t}1). pinephrine (NE,". The effects of the 75 p.g cumulative
190
ioo4 °~--t~Q_ ~ "-~.~ ~-'~-~'--~.~'~-....~ 125 _~ ~, ,~ 2 ~ ~°° o ~ § ~ O ~ e e ~O 75 := -' "-
~'oT t ~
_
~
~
.
50
.
ANG~e) !8.75
:'~.
0 ~ 2 3 4 s 6 y 8 8o.~d t°,,o~,~g p~ot~,~ ~ ~L
~
*
)
ANGc8):37.5 AN'tT~ 575 Drugtreatment(l~g)
I
. . . .
:4E 225
~o>
Fig, 1, Renal peripheral benzodi2zepine receptor density is rapidly decreased fol!~wing acute All treatment, Data are presented us mean percent of control [3H]RoS-4864 binding +S.E.M. The 37.5 and 75 .ug cumulative doses of All administered s,c. over an 80 min period significantly reduced [3H]RoS-4864 binding tP < 0.(11). A cumulative 225 ~g s.c. dose of NE exerted pressor effects equivalent to that of 75 kLg At| but failed 1o influence the renal peripheral benzodiazepine receptor. Number of rats per group is shown in parentheses. All and arterenol bitartrate salt (NE) were dissolved in distilled water. [3H]Ro5-4864 binding was determined in the first experiment using a l nM concentration of radioligand. Nonspecific binding was determined in the presence of 100 ~M Ro5-4864 and accounted for < 12% of total binding. The ellect ol the 73 /xg dose of All was replicated using eight rats and Scatchard analysis was conducted (inset). Filled symbols represent rats treated with vehicle,; open symbols, All 75 p,g. Mean Bin,~ values for vehicle-treated (7.9 pmol/mg protein_+ 0.66) and All-treated rats (6.3 pmol/mg protein _+0.i2) were significantly different (t = 2.447, P < 0.05). Mean Kt) values for vehicle (2.1 nM+0.1) and All (2.5 nM_+0.21) did not differ. dose of A l l on arterial p r e s s u r e were first c h a r a c t e r ized in six rats having carotid arterial c a t h e t e r s imp l a n t e d u n d e r controlled h a l o t h a n e a n e s t h e s i a . T h e 75 /zg dose of A l l caused stable increases in arterial pressure for 80 rain in all rats. T h e m e a n increase in
arterial pressure
from baseline was 56.7 m m
l-lg
(S.D. = 9.8). U s i n g this physiological p r e p a r a t i o n it was d e t e r m i n e d t h a t a 225 /~g c u m u l a t i v e dose of N E a d m i n i s t e r e d in the same fashion as A l l c a u s e d a stable elevation in arterial p r e s s u r e similar to t h a t c a u s e d by A l l (increase of 61.77 m m Hg, S.D. = 12.0; n = 4), N E did not intlctence renal p e r i p h e r a l b c n z o d i a z e p i n e r e c e p t o r b i n d i n g (sec fig. 1). T h e recluetion in renal p e r i p h e r a l b e n z o d i a z e p i n e r e c e p t o r density ind u c e d by A l l is a p p a r e n t l y not a direct c o n s e q u e n c e of the p r e s s e r effects associated with A l l . A n y increases
in e n d o g e n o u s A l l w h i c h m a y have b e e n c a u s e d by t h e systemic a d m i n i s t r a t i o n of N E w e r e a p p a r e n t ! v not a d e q u a t e to i n f l u e n c e the p e r i p h e r a l b e n z o d i a z e p i n e receptor. T h e rapid or phasic r e g u l a t i o n o f p e r i p h e r a l b e n z o d i a z e o i n e r e c e p t o r s by A I I a p p e a r s to b e distinct f r o m thel°ng'term°rt°nicregulati°n°ftheserecept°rsby a l d o s t e r o n e (Basile et al., 1987). F u r t i l e r m o r e , the effeet o f a c u t e A l l a d m i n i s t r a t i o n o n r e n a l p e r i p h e r a l b e n z o d i a z e p i n e r e c e p t o r s is similar to t h e effect of stress. B o t h 80 rain of i n e s c a p a b l e stress ( D r u g a n et al., 1986) a n d 80 rain of A l l t r e a t m e n t ( p r e s e n t study) c a u s e i m m e d i a t e r e d u c t i o n s in t h e density o f t h e s e r e c e p t o r s in kidney. T h e s e findings suggest t h a t A l l IIIt~ may b e a critical f a c t o r in tk~ rapid s t r e s s - i n d u c e d r e g u l a t i o n o f r e n a l p e r i p h e r a l b e n z o O a z e p i n e receptors.
Acknowledgements Submitted as partial fulfillment of requirements for Ph.D. (P.V.H.). Supported by an Alfred P. Sloan Research Fellowship BR 2852 (to R.C.D.). We thank Drs. Peter Suzdak (Novo lndustri) and Peter Sorter (Hoffmann-LaRocbe) for supplying Ro5-4864. All procedures approved by Brown Univ. I.A.C.U.C. Angiotensin It (human) and arterenol bitartrate sah (norepinephrine) were purchased from Sigma Chemical Co., St. Louis, MO, USA. [3H]Ro5-4864 (spec. act. 79.7-84.5 Ci/mM) was purchased from New Enghmd Nuclear, Boston, MA, USA.
References Anholt, R.R.H., 1986, Milochondrial benzodiazepine _,'eceplor~ as potential modulators of intermediary metabolism, Trends Pharmacol. Sci. 7, 506. Basile, A.S, N,L. Ostrowski and P. Skolnick, 1987, Aldosterone-reversible decrease in the density el r,mal periphera! benzod[o azepinc receptors in the rat after adrenalectomy, J. Pharmacol. Exp. Ther. 240, 1006. Benavides, J., D. Quarteronet, F. lmbault, C. Malgouris, A. Uzan. C. Renault, M.C. Dubroeucq, C. Gueremy and G. Le Fur, 1983, Labelling of 'peripheral-type" beuzodiazepine binding sites in the rat brain by using [3H]PK 11195, an isoquinoline earhoxamide derivative: kinetic studies and autoradiographic localization. J. Neurochenr. 41, 1744. Drt.gan, R.C., A.S. Basile, J.N. Crawley, S,M. Paul and P. Skoh;ick, 1986, Inescapable shock reduces ~t1t]Ro3-1864" bimfiny to 'periphcral-type' benzodiazepine receptors in the rat, Pharmacol. Biochem. Behav. 24, 1673. Mcndelsohn, F.A.O., R. Quirion, A.M. Allen, G, Aguilera, M.A. Millan ar,C K.J. Ca~t, 1988, Localization of angiotensin i'l recepters in rat kidney and brain, in: Angiotensin and BloNd Pressure Regulation, eds. J.W. Harding, J.W. Wright, R.C, Speth and C.D. Barne.~,(Academic Press, San Diego) p. 61.
189
g, 1992 Elsevier Science Publishers B.V. All rights reserved 0922-4106/92/$05.00
EJPMOL OIt3R
Rapid communication
Angiotensin II rapidly modulates the renal peripheral benzodiazepine receptor Philip V. H o l m e s a n d R o b e r t C. D r u g a n ' Schrier Research Laboraton" Department o f Psychology, Brown University Procidence, RI 02912, U&4
Received 27 April 1992, accepted 28 April 1992
The effects of acute exposure !o angiotensin II (All) ~,~ ~he renal peripheral benzodiazcpine rcceptcr were studied in rats. As little as 37.5 #g of AII injected s.c. over an 80 min period caused immediate reductions in [3H]Ro5-4864 binding. Scatchard analysis rcveaicd that the rcCuction in [3H]Ro5-4864 binding induced by AII was due to a drop in receptor density or B,~. The influence of Aii on the peripheral benzodiazepine receptor is similar to tha! of stress. Kidncy (r::t); Benzodiazcpine receptors (peripheral):. Angiotensiq ii
The peripheral benzodiazepine receptor is an outer All or vehicle were injected s.c. at 0, 30, 60 min. Rats mitochondrial membrane receptor found in high denwere killed by decapitation in a counterbalanced order sity in adrenal gland and kidney. The peripheral benzoat 80 min and tissues were dissected, fast-frozen and diazepine receptor is also found in the brain (Anholt, stored at -80°C. Tissues were thawed, weighed, dis!986; Benavides et al., !083) The distribution of perupted in 50 volumes of 50 mM Tris-HCl buffer (pH = riphera! benzodiazepine receptors in the kidney and 7.4), and centrifuged at 20,000 × g for 20 rain. The brain is similar to that of angiotensin II (All) receptissues were resuspended in 200 volumes of buffer, and tots. Highest densities of these receptors are localized the binding of [3H]Ro5-4864 at a 1 nM concentration in the inner stripe of the outer medulla and cortex in ,:,as determined as previously described (Drugan et al., kidney and circumventricular organs in brain (BeI9%). navides et al., 1983; Basile et al., 1987; Mendelsohn et Acute exposure to AlI caused an immediate doseal., 1988). The renal peripheral benzodiazepine recepdependent decrease in renal peripheral benzoditot is under hormonal control. Adrenalectomy causes a azepine receptor binding (one-way ANOVA, F = 6.63, reduction in renal peripheral benzodiazepine receptor P < 0.001; see fig. 1). Post-hoe Newman-Keuls tests density after 2 weeks, and this decrease is reversed revealed that the 37.5 /.tg and 75 pog doses of All following one week of a!dosterone therapy suggesting a significantly reduced [-~H]Ro5-4864 binding compared tonic influence of mineralocorticoids (Basile et al., to controls. To characterize the nature of this reduc1987). We presently report that acute AII administration the procedure above was repeated using eight tion causes as immediate reduction in renal peripheral additional rats treated with either the 75 /xg dose of benzodiazepine receptor density in rats. These experiAIi or vehicle. Scatchard analysis included a range of ments are the first demonstration of a rapid hormonal [3H]RoS-4864 concentratiov~ from 0.25 to 16 nM. The regulation of the peripheral benzodiazepine receptor inset in fig. 1 is a rcpresmitative Scatcha~d plot revealin vivo and provide further evidence for the sensitivity ing that the reduction in [3H]Ro5-4864 binding in rats ot this receptor to c~docrine control, treated :dth 75 /xg of All over an 80 m i , period was Male Sprague-Dawley-derb,ed rats v.eighing 250due to a drop in receptor density o~ B, ,,:. A separate 325 g at the tinre of experimemation wc,c ~,muo,,,,!,. . . . . . . ,-~v,-.-' . ,,,~,,.'~'~'~ was ,'ond,,eted ....... r~vealing that the time divided into four groups: vehicie, AII-18.75/xg, A]!-37.5 course o{" ~hese changes is rapid, with re ~._c;;ons evi/,g, and ~,-,~/~g./'" v~ Drug administration was conducted dent as sot,a as 5 rain following a ~ingic, _~"'~#~z s.c. over an 80 mm period. One third of the total doses of injection, of .MI (data not shown). The hypothesis that ~hc decrease in renal perioheral bcnzodiazepir~e receptor density v:as related to increases in systemic arterial pres;ure was tes!ed by CorresF~ ,,lcnce re: R.C. Drugan. Schrier Research kaboratoD', Department of Psycholngy, Box 1853, Brown University, Providence, RI examining tile ,2fleets of a l l equipressor close oi n o t e 02912. USA. Tel. (401) 8{)3-2727.Fax (401}863-13t}1). pinephrine (NE,". The effects of the 75 p.g cumulative
190
ioo4 °~--t~Q_ ~ "-~.~ ~-'~-~'--~.~'~-....~ 125 _~ ~, ,~ 2 ~ ~°° o ~ § ~ O ~ e e ~O 75 := -' "-
~'oT t ~
_
~
~
.
50
.
ANG~e) !8.75
:'~.
0 ~ 2 3 4 s 6 y 8 8o.~d t°,,o~,~g p~ot~,~ ~ ~L
~
*
)
ANGc8):37.5 AN'tT~ 575 Drugtreatment(l~g)
I
. . . .
:4E 225
~o>
Fig, 1, Renal peripheral benzodi2zepine receptor density is rapidly decreased fol!~wing acute All treatment, Data are presented us mean percent of control [3H]RoS-4864 binding +S.E.M. The 37.5 and 75 .ug cumulative doses of All administered s,c. over an 80 min period significantly reduced [3H]RoS-4864 binding tP < 0.(11). A cumulative 225 ~g s.c. dose of NE exerted pressor effects equivalent to that of 75 kLg At| but failed 1o influence the renal peripheral benzodiazepine receptor. Number of rats per group is shown in parentheses. All and arterenol bitartrate salt (NE) were dissolved in distilled water. [3H]Ro5-4864 binding was determined in the first experiment using a l nM concentration of radioligand. Nonspecific binding was determined in the presence of 100 ~M Ro5-4864 and accounted for < 12% of total binding. The ellect ol the 73 /xg dose of All was replicated using eight rats and Scatchard analysis was conducted (inset). Filled symbols represent rats treated with vehicle,; open symbols, All 75 p,g. Mean Bin,~ values for vehicle-treated (7.9 pmol/mg protein_+ 0.66) and All-treated rats (6.3 pmol/mg protein _+0.i2) were significantly different (t = 2.447, P < 0.05). Mean Kt) values for vehicle (2.1 nM+0.1) and All (2.5 nM_+0.21) did not differ. dose of A l l on arterial p r e s s u r e were first c h a r a c t e r ized in six rats having carotid arterial c a t h e t e r s imp l a n t e d u n d e r controlled h a l o t h a n e a n e s t h e s i a . T h e 75 /zg dose of A l l caused stable increases in arterial pressure for 80 rain in all rats. T h e m e a n increase in
arterial pressure
from baseline was 56.7 m m
l-lg
(S.D. = 9.8). U s i n g this physiological p r e p a r a t i o n it was d e t e r m i n e d t h a t a 225 /~g c u m u l a t i v e dose of N E a d m i n i s t e r e d in the same fashion as A l l c a u s e d a stable elevation in arterial p r e s s u r e similar to t h a t c a u s e d by A l l (increase of 61.77 m m Hg, S.D. = 12.0; n = 4), N E did not intlctence renal p e r i p h e r a l b c n z o d i a z e p i n e r e c e p t o r b i n d i n g (sec fig. 1). T h e recluetion in renal p e r i p h e r a l b e n z o d i a z e p i n e r e c e p t o r density ind u c e d by A l l is a p p a r e n t l y not a direct c o n s e q u e n c e of the p r e s s e r effects associated with A l l . A n y increases
in e n d o g e n o u s A l l w h i c h m a y have b e e n c a u s e d by t h e systemic a d m i n i s t r a t i o n of N E w e r e a p p a r e n t ! v not a d e q u a t e to i n f l u e n c e the p e r i p h e r a l b e n z o d i a z e p i n e receptor. T h e rapid or phasic r e g u l a t i o n o f p e r i p h e r a l b e n z o d i a z e o i n e r e c e p t o r s by A I I a p p e a r s to b e distinct f r o m thel°ng'term°rt°nicregulati°n°ftheserecept°rsby a l d o s t e r o n e (Basile et al., 1987). F u r t i l e r m o r e , the effeet o f a c u t e A l l a d m i n i s t r a t i o n o n r e n a l p e r i p h e r a l b e n z o d i a z e p i n e r e c e p t o r s is similar to t h e effect of stress. B o t h 80 rain of i n e s c a p a b l e stress ( D r u g a n et al., 1986) a n d 80 rain of A l l t r e a t m e n t ( p r e s e n t study) c a u s e i m m e d i a t e r e d u c t i o n s in t h e density o f t h e s e r e c e p t o r s in kidney. T h e s e findings suggest t h a t A l l IIIt~ may b e a critical f a c t o r in tk~ rapid s t r e s s - i n d u c e d r e g u l a t i o n o f r e n a l p e r i p h e r a l b e n z o O a z e p i n e receptors.
Acknowledgements Submitted as partial fulfillment of requirements for Ph.D. (P.V.H.). Supported by an Alfred P. Sloan Research Fellowship BR 2852 (to R.C.D.). We thank Drs. Peter Suzdak (Novo lndustri) and Peter Sorter (Hoffmann-LaRocbe) for supplying Ro5-4864. All procedures approved by Brown Univ. I.A.C.U.C. Angiotensin It (human) and arterenol bitartrate sah (norepinephrine) were purchased from Sigma Chemical Co., St. Louis, MO, USA. [3H]Ro5-4864 (spec. act. 79.7-84.5 Ci/mM) was purchased from New Enghmd Nuclear, Boston, MA, USA.
References Anholt, R.R.H., 1986, Milochondrial benzodiazepine _,'eceplor~ as potential modulators of intermediary metabolism, Trends Pharmacol. Sci. 7, 506. Basile, A.S, N,L. Ostrowski and P. Skolnick, 1987, Aldosterone-reversible decrease in the density el r,mal periphera! benzod[o azepinc receptors in the rat after adrenalectomy, J. Pharmacol. Exp. Ther. 240, 1006. Benavides, J., D. Quarteronet, F. lmbault, C. Malgouris, A. Uzan. C. Renault, M.C. Dubroeucq, C. Gueremy and G. Le Fur, 1983, Labelling of 'peripheral-type" beuzodiazepine binding sites in the rat brain by using [3H]PK 11195, an isoquinoline earhoxamide derivative: kinetic studies and autoradiographic localization. J. Neurochenr. 41, 1744. Drt.gan, R.C., A.S. Basile, J.N. Crawley, S,M. Paul and P. Skoh;ick, 1986, Inescapable shock reduces ~t1t]Ro3-1864" bimfiny to 'periphcral-type' benzodiazepine receptors in the rat, Pharmacol. Biochem. Behav. 24, 1673. Mcndelsohn, F.A.O., R. Quirion, A.M. Allen, G, Aguilera, M.A. Millan ar,C K.J. Ca~t, 1988, Localization of angiotensin i'l recepters in rat kidney and brain, in: Angiotensin and BloNd Pressure Regulation, eds. J.W. Harding, J.W. Wright, R.C, Speth and C.D. Barne.~,(Academic Press, San Diego) p. 61.
