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Peripheral benzodiazepine binding sites in kidney: Modifications by diabetes insipidus
Life Sciences, Vol. 35, pp. 2095-2103 Printed in the U.S.A.
PERIPHERAL BENZODIAZEPINE
Pergamon Press
BINDING SITES IN KIDNEY: MODIFICATIONS BY DIABETES
INSIPIDUS. Maria D e l 3 Z o m p o F.TalIman .
1
, Juan M.Saavedra
2
, Julian
Chevillard
2, Robert
M . P o s t 1, J o h n
I Biological Psychiatry Branch, NIMH, Bethesda, MD, USA 2 Lab.of Clinical Science, NIH, Bethesda, MD, USA. 3 Neuroscience Branch, NIMH, Bethesda, MD, USA. (Received in final form August 31, 1984) SUMMARY
Using [3HJ diazepam as ligand, it is possible to distinguish neuronal binding sites from those present on gl~al elements and in peripberal tissues (non-neuronal). The function of the "non-neuronal"~indingl sites is still obscure. Preliminary data showed a distribution of !.Hi diazepam binding sites in kidney that could suggest a localization along the renal tubules. This is the site at which a renal peptide, arginine-vasopressin (AVP) is supposed to act. In an attempt to examine the function of these "non-neuronal" sites, we studied the L ~ H 3 diazepam binding in kidney of Brattleboro rats which lack AVP and present the symptoms of diabetes insipidus. The homozygous Brattleboro rats showed an increase in the apparent number of benzodiazepine binding sites (Bmax) compared to Long-Evans control rats. Replacement of AVP in these animals results in a reversal of the electrolyte alterat~onsl_ -~ of diabetes insipidus and in an increase of the affinity of the L H~ diazepam binding. These findings may indicate a possible relationship between benzodiazepine binding sites and vasopressin action in kidney and may support receptor function of these "non-neuronal" binding sites.
INTRODUCTION The benzodiazepine receptors in brain have been studied by many authors in the last 5 years, since their discovery in 1977. Specific11..: --i and saturable benzodiazepine binding sites with high affinity for L H~ diazepam have been identified in brain (1,2,3). These sites seem to represent the receptors upon which the benzodiazepines exert their central pharmacological effects. In contrast, the peripheral or "non-neuronal" binding sites have been less well investigated even though initial studies found them in some peripheral tissues such as kidney, liver and lung (4). Recently, these peripheral sites were found on cultured cells of various types (5,6,7), particularly i n the cultures containing glia alone, in spinal cord (8), heart (9,10), mast cells (ii) and platelets (12). Reprint Requests: Dr. M. Del Zompo, Clinical Pharmacology, Cagliari, Via Porcell 4, 09100 Cagliari, ITALY. 0024-3205/84
$3.00 + .00
University of
2096
However,
Benzodiazepine Binding Sites in Kidney
Vol. 35, No. 21, 1984
the peripheral-type benzodiazepine binding sites, although having
a high affinity f°r L'~H~r'1~i~zepam, differ from those labelled by ~ -'L~HI diazepam in brain. In fact, the [ H3 diazepam binding to neuronal sites is displaced quite potently by clonazepam but n°t3bYmz-~Ro5-4864, a centrally inactive benzodiazepine. In contrast, binding o f [ H ~ diazepam to kidney and glial elements is displaced by Ro5-4864 but not by clonazepam (13). Further differences between the neuronal- and non-neuronal type binding site such as the solubilization conditions (14), the ~ -aminobutyric acid (GABA) effects (15,17) and the anion modulation (7) have been demonstrated. From studies on kidney homogenates and membrane fractions, the existence of an adenylate cyclase sensitive to arginine-vasopressin (AVP) was located along the collecting tubule (17) and more recently in the medullary portion of the rat thick ascending limb (18). In an attempt to examine the function of peripheral benzodiazepine binding sites, we evaluated the interactions between these sites and the renal peptide arginine-vasopressin on a particular kind of rat, Brattleboro, which are characterized by their inability to synthesize vasopressin (19,20,21).
MATERIALS AND METHODS In these experiments, we used male homozygous (Di/Di) or heterozygous (Di/+) Brattleboro rats for the hypothalamic diabetes insipidus and normal male Long-Evans (L.E.), the strain from which the Brattleboro were derived, as experimental animals. The groups of rats treated received iU/lOOg/day AVP for 7 days. Membrane Preparation All rats were killed by decapitation and their kidneys rapidly frozen after removal. The tissue was homogenized using a polytron in 50 volumes (w/v) of ice-cold 0.05 M Tris HCI buffer (pH 7.0). Membrane fractions were prepared by centrifuging homogenates at 15,000 x g for I0 min. Membrane pellets were washed, centrifuged and resuspended 3 times with 50 volumes (w/v) of ice-cold Tris HC1 (0.05 M). The pellets were finally resuspended in i0 volumes of buffer for the assay. Aliquots of homogenate (i00 9]1) were used for the samples. Proteins were determined according to Lowry et al (1951).
Receptor Binding Assay The d i a z e p a m b i n d i n g a s s a y on k i d n e y membranes was m e a s u r e d u s i n g _ ~ n i n c u b a t i o n volume o f 500 ~ 1 c o n s i s t i n g o f t h e a l i q u o t s o f h o m o g e n a t e a n d L ~ diazepam (specific activity 87.6 Ci/mmol, New E n g l a n d N u c l e a r , Boston, USA) a t a f i n a l c o n c e n t r a t i o n o f inN. Each a s s a y c o n t a i n e d 0 . 6 2 + 0 . 0 6 mg o f membrane protein. The assays were performed in 0.05 N Tris HCI (pH 7.0). Incubation was carried out at 4°C for 30 min and then 7 ml of ice-cold Tris HCI buffer was added to each tube and the samples were immediately filtered through Whatman GF/B filters under low vacuum. The filtration was followed by two 7 ml rinses with ice-cold buffer. The filters were suspended in scintillation fluid and radioactivity was measured with a Beckman liquid scintillation spectrometer. The assay was conducted in triplicate and individual replicates agreed to + 5%. Specific binding is defined as the difference
Vol. 35, No. 21, 1984
Benzodiazepine
Binding Sites in Kidney
2097
between total~=_i~i~ding and binding in the presence of I}/M unlabelled Ro5-4864. Non specific L HJ diazepam binding was about 8 - I0~ of total binding. Statistical Analysis Binding data were analyzed by conventional Scatchard analysis. All averages are expressed as the mean + the standard error of the mean. Regression lines were determined by the least squares method. Tests of statistical significance were calculated using t-test and analysis of variance (ANOVA).
RESULTS In order to demonstrate the function of the peripheral benzodiazepine binding sites, we evaluated the ~ 3 ~ diasepam binding in k~neYr~ ! of Brattleboro rats, suffering from diabetes insipidus. Tab.l shows the L, Ha diazepam binding in the kidney membranes of Long-Evans and Brattleboro rats, using 1 ~ M of [3H~ diazepam. The specific bound is increased and is statistically significant in the Di/Di compared to the control~; the treatment with argininevasopressin further increases the specific of rats, a l s o i n t h e i~.E. c o n t r o i r a t s .
diazepam
bound
in aI1 groups
TABLE 1 t H ~ Diazepam boro rats.
~uM
binding in the kidney membranes of control and Brattle-
GROUP (n = 8)
SPECIFIC BOUND (pmol/mg protein)
Long Evans
6.9 +
.7
Di/+
9.7 + 1.0
Di/Di
10.5 +
.9*
L.E. + AVP
14.0 +
.8**
Di/+ + AVP
14.6 +
.9**
Di/Di+ AVP
9.2 +
AVP = a r g i n i n e - v a s o p r e s s i n
iU/10Og /day
for
.7
7 days
Di/+ = Brattleboro rats heterozygous for diabetes insipidus Di/Di = Brattleboro rats homozygous for diabetes insipidus Long Evans = control rats *p = ~ . 0 1
**p = ~ .O01.
The e x p e r i m e n t
are expressed as mean + S.E.
was repeated
twice
and the
values
2098
Benzodiazepine
Binding Sites in Kidney
Vol. 35, No. 21, 1984
°.7 I 0.6
0.5
UJ UJ n," IA.
CI Z
0.4
0.3
D 0 IX}
0.2
0.1
0.0
[
I
I
I
i
1.0
2.0
3.0
4.0
\,
5.0
| \
6.0
j
7.0
BOUND (pmol/mg protein) FIG.I. Scatchard plot of ~3H~ diazepam specific binding to kidney membrane in different groups of rats; Long-Evans • , Brattleboro heterozygous for diabetes insipidus Di/+ [] , Brattleboro homozygous for diabetes insipidus Di/Di • . See 3 results for binding characteristic. Bound = pmol specific~lly~:~ bound C H~diazepam per mg.protein. Free = concentration of unbound L H~ diazepam in the incubation medium (nM). The data presented are representative of 4 experiments V3~ Fig.l shows Scatchard plot of specific L H ~ diazepam binding to kidney membranes at 4°C in the L.E., Di/Di and Di/+ rats. The regression line (r = 0.97) indicates a K D of 9.3 + 1.6 nM for the L.E., 8.2 ~ 1.9 nM for the Di/+ and of 9.9 + 1.5 nM for the Di/Di, while Bmax is 4.8 + .6 pmol/mg prot.,
Vol. 35, No. 21, 1984
Benzodiazepine
Binding Sites in Kidney
2099
5.0 + .4 pmol/mg prot. and 6.3 + .4 pmol/mg prot., respectively. The difference in Bmax between L.E. and Di/Di rats is statistically significant (p 0.001). The Scatchard plot of L°H~ diazepam binding in kidney membranes of Di/Di and Di/Di treated with arginine-vasopressin is shown in Fig.2.
0.7
-
0.6
\
\
\ \
0.5 LU I/J
U_
-
\
\
\
\
0.4
a z
m
0
0.3
nn
0.2
\\
-
0.1
0.0
1.0
1
I
2.0
3.0
i ~i 4.0
5.0
BOUND (pmol/mg protein) FIG.2. Scatchard plot of [3H]diazepam binding in kidney membranes of Di/Di • and Di/ Di treated with AVP at a dose of iU/lOOg/day for 7 days ( © ). The data presented are representative of 4 experiments.
This graph shows a K D of 10.7 ~ .8 nM in the Di/Di and of 7.6 ! i.i nM in the Di/Di + AVP, while Bmax is 4.9 Z .8 pmol/mg prot. for the former and 4~6-% .6 pmol/mg prot. for the latter. Fig.3 shows the Seatchard plot of C H~ diazepam binding in L.E. and in L.E. treated with AVP. The regression line
2100
Benzodiazepine
Binding
Sites
in K i d n e y
Vol.
35, No.
21, 1984
0.7
0.6
0.5 LU LU
~: U.
0.4
a z
:D
O
0.3
tn
0.2
0.1
0.0
1.0
2.0
3.0
4.0 5.0
BOUND (pmol/mg protein) FIG.3. S c a t c h a r d plot of [3H~ d i a z e p a m b i n d i n g
(r = 0.99) of 6.3 both a
groups.
decrease
with AVP. The
indicates,
+ 1.0 nM
the
treatment levels
in
There
in this
the
L.E.
series
KD
The decrease
to control
Tab.2.
for
In both in
in kidney m e m b r a n e s
. The data p r e s e n t e d are r e p r e s e n t a t i v e
treated with AVP ~
for the L.E.
and for
of e x p e r i m e n t s
between
of water
no
a K D of 9.0 ~
the groups
in L o n g - E v a n s
.9 nM
we found no change of animals
treated
of
and B r a t t l e b o r o
diazepam
in the Bmax and and
non-treated
significant. rats
intake and urine output of Di/Di
inhibition
and L.E.
while Bmax is 4.3 + .2 p m o l / m g prot.
in the K D was not s t a t i s t i c a l l y
with AVP
is
case,
+ AVP,
of L.E. •
of 4 experiments.
caused
a return
animals,
as shown k i d n e y by
binding
on
Vol.
35, No. 21, 1984
Benzodiazepine
arginine-vasopressin
in vitro experiments
Binding
Sites in Kidney
210]
( data not shown).
TABLE II Modification
of Water Intake and Urine Output of Brattleboro
Rats.
WATER INTAKE
URINE OUTPUT
(ml/lOOg/24h)
(ml/lOOg/24h)
L.E.
9.5 + 2
4 + 1
Di/+
8 + 3
3 + 1
Di/Di
73 + 6*
69 + 5*
Di/+ + AVP
i0 + 3
4 + 1
Di/Di + AVP
12 + 3
4 + 2
ANIMALS
*p <
.001
DISCUSSION This study demonstrates
a statistically
zepam specific bound in kidney,
significant
increase of
not only in those rats homozygous
diafor diabetes
insipidus (Di/Di) when compared to controls, but also in the same group of animals treated with arginine-vasopressin, in the same way compared to control group.
The
higher
binding
number which a
Scatchard in
analysis Di/Di
of benzodiazepine reverses
decrease
the
the K D findings
binding
and vasopressin
sites.
four
different
is
due
to
experiments an
sites;
in contrast,
diabetes
insipidus,
affinity) Also
increase
that the
the apparent
the treatment causes
and a return
the control
shows in
with AVP,
in Di/Di
animals,
to normality
group
showed
of Bmax
a decrease
in
these data were of statistical~3_%significance. These a possible interaction between L H~ diazepam binding action
of AVP on these peripheral (data not shown). Although
of
(increased
binding
," neither of could suggest
of
animals
symptoms
in the K D
of benzodiazepine
sites
the
in kidney,
receptors
it is now evident
even though there is no direct effect
as demonstrated
with in vitro experiments
that the "non-neuronal"
or peripheral
benzodia-
zepine binding sites still exist in kidney (4,10), their physiological role is still unknown. Some authors (22) reported that the number of renal benzodiazepine binding sites is higher in the deoxycorticosterone (DOCA) /salt hypertensive rats in comparison to controls. Others found a decrease on
the
number
of
the
same
binding
sites
in a different
experimental
model,
spontaneously hypertensive rats, when compared to WKY controls (23). Our data may help to find an explanation of different behaviour of the "non-neuronal" diazepam binding sites which is qualitatively lytes
(24)
in various experimental models. In fact, DOCA salt, identical to aldosterone in its effects on electro-
and AVP both play
a role
in the regulation
of water
permeability
2102
Benzodiazepine Binding Sites in Kidney
Vol. 35, No. 21, 1984
and sodium balance in kidney (17,24) and cause a modification in the peripheral benzodiazepine binding sites in the same tissue. Moreover, we should here underline the increase in the number of peripheral benzodiazepine binding sites in kidney in those rats suffering from diabetes insipidus and thus lacking in AVP. The decrease in the K D observed in all our experiments in animals of all groups treated rq~with AVP requires further investigation as AVP in vitro does not inhibit [~H~ diazepam binding on kidney. All these data may suggest the hypothesis of a coupling between these binding sites and some ion channels. Thus, this study, in agreement with other evidence suggesting the involvement of the "non-neuronal" binding sites in the modulation of phospholipid methylation (25) and prolactin release (26) may support a function of these sites. As diabetes insipidus increases the number, and AVP the affinity of 3HI diazepam binding sites in kidney, this observation may give rise to additional studies on animal to investigate whether these peripheral type sites have specific functions, by means of treating the Di/Di rats chronically with Ro5-4864 and on man in order to study their importance on diabetes insipidus.
REFERENCES i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
H.MOHLER and T.OKADA, Science. 1 9 8 : 8 4 9 (1977). R.SQUIRES and C.BRAESTRUP, Nature. 2 6 6 : 7 3 2 (1977). J.F.TALLMAN, S.M.PAUL, P.SKOLNICK and D.GALLAGER, Science. 2 0 7 : 2 7 4 (1980) C.BRAESTRUP, R.ALBECHSTEN and R.F.SQUIRES, Nature. 2 6 9 : 7 0 2 (1977). D.GALLAGER, P.MALLORGA, W.OERTEL, R.HENNEBERRY and J.F.TALLMAN. J.NeuroSci: i: 218 (1981). H.SHOEMAKER, M.BLISS and H.I.YAMAMURA, Eur.J.Pharmacol. 7 1 : 4 7 3 (1981). H.SHOEMAKER, M.BLISS and S.H.YAMAMURA, Brain Neurotransmitters and Hormones p.l15, Raven Pressp New York, 1982. M.DEL ZOMPO, R.M.POST and J.F.TALLMAN, Neuropharmacology, 2 2 : 1 1 5 (1983). W.R.ROESKE and H.I.YAMAMURA, Am.Fed.Clin.Res. In Press (1982). T.TANIGUCHI, J.K.T.WANG and S.SPECTOR, Biochem.Pharmacol. 3 1 : 5 8 9 (1982). T.TANIGUCHI, J.K.T.WANG and S.SPECTOR, Life Sci. 2 7 : 1 7 1 (1980). J.K.T.WANG, T.TANIGUCHI and S.SPECTOR, Life Sci. 2 7 : 1 8 8 1 (1980). C.BRAESTRUP and R.F.SQUIRES, Proc.Nat.Aead.Sci. U.S.A. 74 3805 (1977). J.W.THOMAS and J.F.TALLMAN, J . B i o l . C h e m . 2 5 6 : 9 8 3 8 (1981) J.PATEL and P.MARANGOS, NeuroSci.Lett. 3 0 : 1 5 7 (1982). M.HERKENHAM and C.B.PERT, J.NeuroSci. 2 (8): 1129 (1982). R.W.BERLINER and C.M.BENNET, Am.J.Med. 4 2 : 7 7 7 (1967). M.IMBERT-TEOUL, D.CHABARDES, M.MONTEGUT and F.MOREL, Endocrinology. 10__2, 1254 (1978). H.VALTIN and H.A.SCHROEDER, Am.J.Physiol. 2 0 6 : 4 2 5 (1964). H.W.SOKOL, Neuroendocrinology 6 : 9 0 (1970). F.VANDESANDE and K.DIERICKX, Cell Tiss.Res. 1 6 5 : 3 0 7 (1976). J.W.REGAN, H.I.YAMAMURA, S.YAMADA and W.R.ROESKE, Life Sci. 28: 991 (1981). T.TANIGUCHI, J.K.T.WANG and S.SPECTOR, Eur.J.Pharmacol. 70: 587 (1981). R.C.HAYNES Jr. and F.MURAD, The Pharmacological Basis of Therapeutics, p. 1466, Macmillan Pub. Inc., New York, (1980).
Vol. 35, No. 21, 1984
Benzodiazepine Binding Sites in Kidney
25. W.J.STRITT~4ATTER, F.HIRATA, J.AXELROD, P.MALLORGA, J.F.TALLMAN and R.C. HENNEBERRY, Nature (London). 2 8 2 : 8 5 7 (1979). 26. L.GRANDISON, Soe.NeuroSei Abstr. 161:: 16, 503 (1981). 27. O.H.LOWRY, N.J.ROSENBROUGH, A.L.FARR and R.J.RANDALL, J.Biol.Chem. 193: 265 (1951).
2103
PERIPHERAL BENZODIAZEPINE
Pergamon Press
BINDING SITES IN KIDNEY: MODIFICATIONS BY DIABETES
INSIPIDUS. Maria D e l 3 Z o m p o F.TalIman .
1
, Juan M.Saavedra
2
, Julian
Chevillard
2, Robert
M . P o s t 1, J o h n
I Biological Psychiatry Branch, NIMH, Bethesda, MD, USA 2 Lab.of Clinical Science, NIH, Bethesda, MD, USA. 3 Neuroscience Branch, NIMH, Bethesda, MD, USA. (Received in final form August 31, 1984) SUMMARY
Using [3HJ diazepam as ligand, it is possible to distinguish neuronal binding sites from those present on gl~al elements and in peripberal tissues (non-neuronal). The function of the "non-neuronal"~indingl sites is still obscure. Preliminary data showed a distribution of !.Hi diazepam binding sites in kidney that could suggest a localization along the renal tubules. This is the site at which a renal peptide, arginine-vasopressin (AVP) is supposed to act. In an attempt to examine the function of these "non-neuronal" sites, we studied the L ~ H 3 diazepam binding in kidney of Brattleboro rats which lack AVP and present the symptoms of diabetes insipidus. The homozygous Brattleboro rats showed an increase in the apparent number of benzodiazepine binding sites (Bmax) compared to Long-Evans control rats. Replacement of AVP in these animals results in a reversal of the electrolyte alterat~onsl_ -~ of diabetes insipidus and in an increase of the affinity of the L H~ diazepam binding. These findings may indicate a possible relationship between benzodiazepine binding sites and vasopressin action in kidney and may support receptor function of these "non-neuronal" binding sites.
INTRODUCTION The benzodiazepine receptors in brain have been studied by many authors in the last 5 years, since their discovery in 1977. Specific11..: --i and saturable benzodiazepine binding sites with high affinity for L H~ diazepam have been identified in brain (1,2,3). These sites seem to represent the receptors upon which the benzodiazepines exert their central pharmacological effects. In contrast, the peripheral or "non-neuronal" binding sites have been less well investigated even though initial studies found them in some peripheral tissues such as kidney, liver and lung (4). Recently, these peripheral sites were found on cultured cells of various types (5,6,7), particularly i n the cultures containing glia alone, in spinal cord (8), heart (9,10), mast cells (ii) and platelets (12). Reprint Requests: Dr. M. Del Zompo, Clinical Pharmacology, Cagliari, Via Porcell 4, 09100 Cagliari, ITALY. 0024-3205/84
$3.00 + .00
University of
2096
However,
Benzodiazepine Binding Sites in Kidney
Vol. 35, No. 21, 1984
the peripheral-type benzodiazepine binding sites, although having
a high affinity f°r L'~H~r'1~i~zepam, differ from those labelled by ~ -'L~HI diazepam in brain. In fact, the [ H3 diazepam binding to neuronal sites is displaced quite potently by clonazepam but n°t3bYmz-~Ro5-4864, a centrally inactive benzodiazepine. In contrast, binding o f [ H ~ diazepam to kidney and glial elements is displaced by Ro5-4864 but not by clonazepam (13). Further differences between the neuronal- and non-neuronal type binding site such as the solubilization conditions (14), the ~ -aminobutyric acid (GABA) effects (15,17) and the anion modulation (7) have been demonstrated. From studies on kidney homogenates and membrane fractions, the existence of an adenylate cyclase sensitive to arginine-vasopressin (AVP) was located along the collecting tubule (17) and more recently in the medullary portion of the rat thick ascending limb (18). In an attempt to examine the function of peripheral benzodiazepine binding sites, we evaluated the interactions between these sites and the renal peptide arginine-vasopressin on a particular kind of rat, Brattleboro, which are characterized by their inability to synthesize vasopressin (19,20,21).
MATERIALS AND METHODS In these experiments, we used male homozygous (Di/Di) or heterozygous (Di/+) Brattleboro rats for the hypothalamic diabetes insipidus and normal male Long-Evans (L.E.), the strain from which the Brattleboro were derived, as experimental animals. The groups of rats treated received iU/lOOg/day AVP for 7 days. Membrane Preparation All rats were killed by decapitation and their kidneys rapidly frozen after removal. The tissue was homogenized using a polytron in 50 volumes (w/v) of ice-cold 0.05 M Tris HCI buffer (pH 7.0). Membrane fractions were prepared by centrifuging homogenates at 15,000 x g for I0 min. Membrane pellets were washed, centrifuged and resuspended 3 times with 50 volumes (w/v) of ice-cold Tris HC1 (0.05 M). The pellets were finally resuspended in i0 volumes of buffer for the assay. Aliquots of homogenate (i00 9]1) were used for the samples. Proteins were determined according to Lowry et al (1951).
Receptor Binding Assay The d i a z e p a m b i n d i n g a s s a y on k i d n e y membranes was m e a s u r e d u s i n g _ ~ n i n c u b a t i o n volume o f 500 ~ 1 c o n s i s t i n g o f t h e a l i q u o t s o f h o m o g e n a t e a n d L ~ diazepam (specific activity 87.6 Ci/mmol, New E n g l a n d N u c l e a r , Boston, USA) a t a f i n a l c o n c e n t r a t i o n o f inN. Each a s s a y c o n t a i n e d 0 . 6 2 + 0 . 0 6 mg o f membrane protein. The assays were performed in 0.05 N Tris HCI (pH 7.0). Incubation was carried out at 4°C for 30 min and then 7 ml of ice-cold Tris HCI buffer was added to each tube and the samples were immediately filtered through Whatman GF/B filters under low vacuum. The filtration was followed by two 7 ml rinses with ice-cold buffer. The filters were suspended in scintillation fluid and radioactivity was measured with a Beckman liquid scintillation spectrometer. The assay was conducted in triplicate and individual replicates agreed to + 5%. Specific binding is defined as the difference
Vol. 35, No. 21, 1984
Benzodiazepine
Binding Sites in Kidney
2097
between total~=_i~i~ding and binding in the presence of I}/M unlabelled Ro5-4864. Non specific L HJ diazepam binding was about 8 - I0~ of total binding. Statistical Analysis Binding data were analyzed by conventional Scatchard analysis. All averages are expressed as the mean + the standard error of the mean. Regression lines were determined by the least squares method. Tests of statistical significance were calculated using t-test and analysis of variance (ANOVA).
RESULTS In order to demonstrate the function of the peripheral benzodiazepine binding sites, we evaluated the ~ 3 ~ diasepam binding in k~neYr~ ! of Brattleboro rats, suffering from diabetes insipidus. Tab.l shows the L, Ha diazepam binding in the kidney membranes of Long-Evans and Brattleboro rats, using 1 ~ M of [3H~ diazepam. The specific bound is increased and is statistically significant in the Di/Di compared to the control~; the treatment with argininevasopressin further increases the specific of rats, a l s o i n t h e i~.E. c o n t r o i r a t s .
diazepam
bound
in aI1 groups
TABLE 1 t H ~ Diazepam boro rats.
~uM
binding in the kidney membranes of control and Brattle-
GROUP (n = 8)
SPECIFIC BOUND (pmol/mg protein)
Long Evans
6.9 +
.7
Di/+
9.7 + 1.0
Di/Di
10.5 +
.9*
L.E. + AVP
14.0 +
.8**
Di/+ + AVP
14.6 +
.9**
Di/Di+ AVP
9.2 +
AVP = a r g i n i n e - v a s o p r e s s i n
iU/10Og /day
for
.7
7 days
Di/+ = Brattleboro rats heterozygous for diabetes insipidus Di/Di = Brattleboro rats homozygous for diabetes insipidus Long Evans = control rats *p = ~ . 0 1
**p = ~ .O01.
The e x p e r i m e n t
are expressed as mean + S.E.
was repeated
twice
and the
values
2098
Benzodiazepine
Binding Sites in Kidney
Vol. 35, No. 21, 1984
°.7 I 0.6
0.5
UJ UJ n," IA.
CI Z
0.4
0.3
D 0 IX}
0.2
0.1
0.0
[
I
I
I
i
1.0
2.0
3.0
4.0
\,
5.0
| \
6.0
j
7.0
BOUND (pmol/mg protein) FIG.I. Scatchard plot of ~3H~ diazepam specific binding to kidney membrane in different groups of rats; Long-Evans • , Brattleboro heterozygous for diabetes insipidus Di/+ [] , Brattleboro homozygous for diabetes insipidus Di/Di • . See 3 results for binding characteristic. Bound = pmol specific~lly~:~ bound C H~diazepam per mg.protein. Free = concentration of unbound L H~ diazepam in the incubation medium (nM). The data presented are representative of 4 experiments V3~ Fig.l shows Scatchard plot of specific L H ~ diazepam binding to kidney membranes at 4°C in the L.E., Di/Di and Di/+ rats. The regression line (r = 0.97) indicates a K D of 9.3 + 1.6 nM for the L.E., 8.2 ~ 1.9 nM for the Di/+ and of 9.9 + 1.5 nM for the Di/Di, while Bmax is 4.8 + .6 pmol/mg prot.,
Vol. 35, No. 21, 1984
Benzodiazepine
Binding Sites in Kidney
2099
5.0 + .4 pmol/mg prot. and 6.3 + .4 pmol/mg prot., respectively. The difference in Bmax between L.E. and Di/Di rats is statistically significant (p 0.001). The Scatchard plot of L°H~ diazepam binding in kidney membranes of Di/Di and Di/Di treated with arginine-vasopressin is shown in Fig.2.
0.7
-
0.6
\
\
\ \
0.5 LU I/J
U_
-
\
\
\
\
0.4
a z
m
0
0.3
nn
0.2
\\
-
0.1
0.0
1.0
1
I
2.0
3.0
i ~i 4.0
5.0
BOUND (pmol/mg protein) FIG.2. Scatchard plot of [3H]diazepam binding in kidney membranes of Di/Di • and Di/ Di treated with AVP at a dose of iU/lOOg/day for 7 days ( © ). The data presented are representative of 4 experiments.
This graph shows a K D of 10.7 ~ .8 nM in the Di/Di and of 7.6 ! i.i nM in the Di/Di + AVP, while Bmax is 4.9 Z .8 pmol/mg prot. for the former and 4~6-% .6 pmol/mg prot. for the latter. Fig.3 shows the Seatchard plot of C H~ diazepam binding in L.E. and in L.E. treated with AVP. The regression line
2100
Benzodiazepine
Binding
Sites
in K i d n e y
Vol.
35, No.
21, 1984
0.7
0.6
0.5 LU LU
~: U.
0.4
a z
:D
O
0.3
tn
0.2
0.1
0.0
1.0
2.0
3.0
4.0 5.0
BOUND (pmol/mg protein) FIG.3. S c a t c h a r d plot of [3H~ d i a z e p a m b i n d i n g
(r = 0.99) of 6.3 both a
groups.
decrease
with AVP. The
indicates,
+ 1.0 nM
the
treatment levels
in
There
in this
the
L.E.
series
KD
The decrease
to control
Tab.2.
for
In both in
in kidney m e m b r a n e s
. The data p r e s e n t e d are r e p r e s e n t a t i v e
treated with AVP ~
for the L.E.
and for
of e x p e r i m e n t s
between
of water
no
a K D of 9.0 ~
the groups
in L o n g - E v a n s
.9 nM
we found no change of animals
treated
of
and B r a t t l e b o r o
diazepam
in the Bmax and and
non-treated
significant. rats
intake and urine output of Di/Di
inhibition
and L.E.
while Bmax is 4.3 + .2 p m o l / m g prot.
in the K D was not s t a t i s t i c a l l y
with AVP
is
case,
+ AVP,
of L.E. •
of 4 experiments.
caused
a return
animals,
as shown k i d n e y by
binding
on
Vol.
35, No. 21, 1984
Benzodiazepine
arginine-vasopressin
in vitro experiments
Binding
Sites in Kidney
210]
( data not shown).
TABLE II Modification
of Water Intake and Urine Output of Brattleboro
Rats.
WATER INTAKE
URINE OUTPUT
(ml/lOOg/24h)
(ml/lOOg/24h)
L.E.
9.5 + 2
4 + 1
Di/+
8 + 3
3 + 1
Di/Di
73 + 6*
69 + 5*
Di/+ + AVP
i0 + 3
4 + 1
Di/Di + AVP
12 + 3
4 + 2
ANIMALS
*p <
.001
DISCUSSION This study demonstrates
a statistically
zepam specific bound in kidney,
significant
increase of
not only in those rats homozygous
diafor diabetes
insipidus (Di/Di) when compared to controls, but also in the same group of animals treated with arginine-vasopressin, in the same way compared to control group.
The
higher
binding
number which a
Scatchard in
analysis Di/Di
of benzodiazepine reverses
decrease
the
the K D findings
binding
and vasopressin
sites.
four
different
is
due
to
experiments an
sites;
in contrast,
diabetes
insipidus,
affinity) Also
increase
that the
the apparent
the treatment causes
and a return
the control
shows in
with AVP,
in Di/Di
animals,
to normality
group
showed
of Bmax
a decrease
in
these data were of statistical~3_%significance. These a possible interaction between L H~ diazepam binding action
of AVP on these peripheral (data not shown). Although
of
(increased
binding
," neither of could suggest
of
animals
symptoms
in the K D
of benzodiazepine
sites
the
in kidney,
receptors
it is now evident
even though there is no direct effect
as demonstrated
with in vitro experiments
that the "non-neuronal"
or peripheral
benzodia-
zepine binding sites still exist in kidney (4,10), their physiological role is still unknown. Some authors (22) reported that the number of renal benzodiazepine binding sites is higher in the deoxycorticosterone (DOCA) /salt hypertensive rats in comparison to controls. Others found a decrease on
the
number
of
the
same
binding
sites
in a different
experimental
model,
spontaneously hypertensive rats, when compared to WKY controls (23). Our data may help to find an explanation of different behaviour of the "non-neuronal" diazepam binding sites which is qualitatively lytes
(24)
in various experimental models. In fact, DOCA salt, identical to aldosterone in its effects on electro-
and AVP both play
a role
in the regulation
of water
permeability
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Benzodiazepine Binding Sites in Kidney
Vol. 35, No. 21, 1984
and sodium balance in kidney (17,24) and cause a modification in the peripheral benzodiazepine binding sites in the same tissue. Moreover, we should here underline the increase in the number of peripheral benzodiazepine binding sites in kidney in those rats suffering from diabetes insipidus and thus lacking in AVP. The decrease in the K D observed in all our experiments in animals of all groups treated rq~with AVP requires further investigation as AVP in vitro does not inhibit [~H~ diazepam binding on kidney. All these data may suggest the hypothesis of a coupling between these binding sites and some ion channels. Thus, this study, in agreement with other evidence suggesting the involvement of the "non-neuronal" binding sites in the modulation of phospholipid methylation (25) and prolactin release (26) may support a function of these sites. As diabetes insipidus increases the number, and AVP the affinity of 3HI diazepam binding sites in kidney, this observation may give rise to additional studies on animal to investigate whether these peripheral type sites have specific functions, by means of treating the Di/Di rats chronically with Ro5-4864 and on man in order to study their importance on diabetes insipidus.
REFERENCES i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
H.MOHLER and T.OKADA, Science. 1 9 8 : 8 4 9 (1977). R.SQUIRES and C.BRAESTRUP, Nature. 2 6 6 : 7 3 2 (1977). J.F.TALLMAN, S.M.PAUL, P.SKOLNICK and D.GALLAGER, Science. 2 0 7 : 2 7 4 (1980) C.BRAESTRUP, R.ALBECHSTEN and R.F.SQUIRES, Nature. 2 6 9 : 7 0 2 (1977). D.GALLAGER, P.MALLORGA, W.OERTEL, R.HENNEBERRY and J.F.TALLMAN. J.NeuroSci: i: 218 (1981). H.SHOEMAKER, M.BLISS and H.I.YAMAMURA, Eur.J.Pharmacol. 7 1 : 4 7 3 (1981). H.SHOEMAKER, M.BLISS and S.H.YAMAMURA, Brain Neurotransmitters and Hormones p.l15, Raven Pressp New York, 1982. M.DEL ZOMPO, R.M.POST and J.F.TALLMAN, Neuropharmacology, 2 2 : 1 1 5 (1983). W.R.ROESKE and H.I.YAMAMURA, Am.Fed.Clin.Res. In Press (1982). T.TANIGUCHI, J.K.T.WANG and S.SPECTOR, Biochem.Pharmacol. 3 1 : 5 8 9 (1982). T.TANIGUCHI, J.K.T.WANG and S.SPECTOR, Life Sci. 2 7 : 1 7 1 (1980). J.K.T.WANG, T.TANIGUCHI and S.SPECTOR, Life Sci. 2 7 : 1 8 8 1 (1980). C.BRAESTRUP and R.F.SQUIRES, Proc.Nat.Aead.Sci. U.S.A. 74 3805 (1977). J.W.THOMAS and J.F.TALLMAN, J . B i o l . C h e m . 2 5 6 : 9 8 3 8 (1981) J.PATEL and P.MARANGOS, NeuroSci.Lett. 3 0 : 1 5 7 (1982). M.HERKENHAM and C.B.PERT, J.NeuroSci. 2 (8): 1129 (1982). R.W.BERLINER and C.M.BENNET, Am.J.Med. 4 2 : 7 7 7 (1967). M.IMBERT-TEOUL, D.CHABARDES, M.MONTEGUT and F.MOREL, Endocrinology. 10__2, 1254 (1978). H.VALTIN and H.A.SCHROEDER, Am.J.Physiol. 2 0 6 : 4 2 5 (1964). H.W.SOKOL, Neuroendocrinology 6 : 9 0 (1970). F.VANDESANDE and K.DIERICKX, Cell Tiss.Res. 1 6 5 : 3 0 7 (1976). J.W.REGAN, H.I.YAMAMURA, S.YAMADA and W.R.ROESKE, Life Sci. 28: 991 (1981). T.TANIGUCHI, J.K.T.WANG and S.SPECTOR, Eur.J.Pharmacol. 70: 587 (1981). R.C.HAYNES Jr. and F.MURAD, The Pharmacological Basis of Therapeutics, p. 1466, Macmillan Pub. Inc., New York, (1980).
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Benzodiazepine Binding Sites in Kidney
25. W.J.STRITT~4ATTER, F.HIRATA, J.AXELROD, P.MALLORGA, J.F.TALLMAN and R.C. HENNEBERRY, Nature (London). 2 8 2 : 8 5 7 (1979). 26. L.GRANDISON, Soe.NeuroSei Abstr. 161:: 16, 503 (1981). 27. O.H.LOWRY, N.J.ROSENBROUGH, A.L.FARR and R.J.RANDALL, J.Biol.Chem. 193: 265 (1951).
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