209
Brain Research, 554 (1991) 209-216 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 000689939116797A BRES 16797
On the blood-brain barrier to peptides: specific binding of atrial natriuretic peptide in vivo and in vitro A r m i n Ermisch 1, Hans-Joachim Rfihle 1, Roswitha Kretzschmar I and A l e x a n d e r B a e t h m a n n 2 1Department of Cell Biology and Regulation, Section of Biosciences, University of Leipzig, Leipzig (ER. G.) and 2Institute of Surgical Research, Ludwig Maximilians University, Munich (E R. G.) (Accepted 19 February 1991)
Key words: Atrial natriuretic peptide; Blood-brain barrier; Circumventricular organ; Microvessel; Receptor
Using the intracarotid bolus injection technique, a saturable binding of [125I]atrial natriuretic peptide (ANP) was found in 8 blood-brain barrier (BBB)-protected rat brain regions as well as in the pineal gland, choroid plexus, neurointermediate and anterior lobes of the pituitary, i.e. structures lacking a BBB. The presence of specific ANP binding on the BBB, here shown for the first time by an in vivo approach, was evidenced concomitantly in vitro by incubation of isolated microvessels. A single-class high affinity binding without regional differences was obtained with K d = 0.23 nM and Bmax = 120 fmoi/mg protein. From that a density of 1,400 binding sites per endothelial cell was calculated, thought to be localized predominantly in the luminal membranes. In the in vivo study, the portion of the extracted peptide that, under the conditions used, may have crossed the BBB by passive diffusion amounted to less than 0.4% of the labeled ANP administered. ANP itself did not change the tightness of the BBB to the non-diffusible reference molecule [14C]inulin. In the BBB-free areas, ANP enhanced the inulin space by nearly 50%. INTRODUCTION Recently, a family of natriuretic peptides ( A N P ) was isolated and sequenced. The peptides, primarily detected in cells of the cardiac atria, are p r o d u c e d by m a n y cell populations 2'12'26'47'69. A N P induces vascular effects associated with systemic b l o o d pressure homeostasis and the extracellular fluid composition 4A2'25'44'66. The effects are m e d i a t e d by highly selective and specific A N P receptors13,27,32,46,58,59. Specific A N P binding sites have been detected also in the brain vasculature, especially in isolated microvessels and cultured brain capillary endothelial cells 8,9,29,48,61. The endothelial cells of the microvessels constitute in vivo the b l o o d - b r a i n barrier (BBB). A n implication of a specific A N P binding on the endothelial cells in water and electrolyte balance of the brain has been discussed 8,9. Q u i t e recently, effects of A N P on the sodium transport of isolated brain microvessels were described 3°,31, and the effect of the p e p t i d e on the permeability of the BBB to a tracer p r o t e i n and ionic lanthanum was studied in vivo by electron microscopy 45. Nonetheless, the d a t a concerning the specific binding of A N P on the BBB as well as the effect of A N P on transport processes across the BBB are still scarce and partially contradictory. Thus, a convincing demonstration
of a saturable binding of the p e p t i d e on the B B B in vivo is lacking. Levin et al. 41, for e x a m p l e , failed to detect specific binding sites on the B B B of rabbits by an in vivo approach, in contrast to the results of in vitro studies on rats m e n t i o n e d above. Therefore, the present p a p e r reports on the binding p r o p e r t i e s of the brain microvasculature in various brain areas, both in vivo and in vitro. D a t a are given regarding the tightness of the b a r r i e r to A N P in comparison to the m a r k e r molecule inulin, n o r m a l l y excluded from the transport through the BBB. A differentiation was m a d e between ' B B B - p r o t e c t e d ' brain regions with a tight capillary e n d o t h e l i u m , and circumventricular organs ( C V O ) being ' B B B - f r e e ' , i.e. possessing capillaries with a leaky e n d o t h e l i u m 1°. We are aware of the fact that such basic studies might also attain significance for the explanation of clinical problems such as the dysfunction of the BBB and the pathogenesis of cerebral e d e m a as well 1.
MATERIALS AND METHODS
Retention of ANP by various brain regions Experiments were performed on 22 male Wistar rats weighing 130-220 g. They were divided into 4 groups. The experimental design and detailed data can be seen in Table I. The retention of
Correspondence: A. Ermisch, Sektion Biowissenschaften, Universit~it Leipzig, Talstrasse 33, 0-7010 Leipzig, F.R.G.
210 TABLE I
Experimental design for the in vivo studies of ANP retention by brain regions Experiment number
n
4 8 5
1
2 3
5
Tracer
[125I]ANP [14C]inulin ['25I]ANP + [14C]inulin [125I]ANP + [14C]inulin + unlabeled ANP (2.2/~g = 3.5 x 103nM)
radioactive tracers by the brain regions was measured using the intracarotid bolus injection technique as previously described 39'49. Briefly, the rats were anesthetized with 20 mg/kg of ketamine hydrochloride (Velonarcon 0.5, VEB Berlin Chemic) intraperitoneally. The right common carotid artery was cannulated and a 0.2 ml-bolus of phosphate-buffered saline (pH 7.4) containing the tracers (3-[12sI]iodotyrosyl2s) rat atrial natriuretic peptide 28 (Amersham; spec. act. 1730 or 1980 Ci/mmol) or (hydroxyl[14C]methyl)inulin (Amersham; spec. act. 9.45 mCi/mmol), or both, was rapidly injected. In Expt. 4 an excess of unlabeled ANP (rat a-ANP, Serva, Heidelberg) was injected together with the radiolabeled tracers. The animals were decapitated 15 s after injection, the brains quickly removed and the hemisphere ipsilateral to the injection side dissected into 12 regions. Additionally, the ipsilateral half of the anterior pituitary was included in the study. The tissue samples were weighed and counted for 1251by gamma spectrometry (Expt. 1). In order to determine the 14C radioactivity (Expt. 2), the samples were dissolved in Soluene 350 (Packard, Groningen) before counting by liquid scintillation spectrometry. In Expts. 3 and 4 the samples were first counted for 1251 and afterwards dissolved and counted for 14C. Aliquots of respective injection mixtures were measured in the same manner. Accumulation of radioactivity in a tissue sample (/ix) was calculated by the equation:
A,-
Rbr - - x Rin j X Wb,
100%
(1)
where Rbr represents radioactivity of the sample, Wbr its weight, and Rinj the injected amounts of radioactivity. Furthermore, the brain uptake index (BUI) 49 was calculated using the formula:
Injected tracer amount per animal
Tracer concentration
(uCi)
(ng)
(nM)
0.78 5.8 1.2 3.0 1.4 3.0
1.4 3.2 X 106 1.9 1.7 × 106 2.2 1.7 × 106
2.2 3.1 × 106 3.(1 1.6 × lff' 3.5 1.6 × 10"
where A w represents the accumulation of tritiated water in the region 2°'3s. The fraction of water extracted was assumed to be 43% 24"
Specific ANP binding sites m isolated microvessels Isolation of brain microvessels. Cerebral microvessels were prepared by mechanical homogenization and centrifugation33'36. Brains of Wistar rats were immersed in 0.25 M sucrose solution. After removal of the meninges and white matter, tissue of neocortex, hippocampus, and striatum were separately minced and homogenized through nylon meshes with 225/zm and 120/~m pore size. The homogenates were then centrifuged at 100 g for 10 min. The pellets, which were resuspended in 0.25 M sucrose, were layered twice above a discontinuous gradient of 1.8, 1.5, 1.3, 1.0 M sucrose and fractionated at 50,000 g for 30 min. The resulting pellets containing isolated cerebral microvessels were observed by light microscopy. Radioligand-binding assay. The microvessels isolated from the 3 brain regions (approx. 200/~g protein per sample) were incubated at room temperature in an isotonic Tris-HCI buffer at pH 7.436 with 10 pM [125I]ANP for 60 min in a total volume of 200/~1. The binding reaction was terminated by centrifugation at 18,000 g for 1 min. After washing, the bound radioactivity of the microvessel pellets was determined by gamma spectrometry. Competition experiments were made by addition of various concentrations of unlabeled rat ANP in a range between 10 pM and 100/~M. Studies on saturation of ANP binding were carried out with neocortex microvessels using concentrations of [125I]ANP from 10 pM to 1 nM. Specific binding of [125I]ANP was calculated by subtraction of non-specific binding determined in the presence of 1 ~M unlabeled ANP from the total binding rate.
Statistics BUI
=
AANP x 100% Ai
(2)
Thus, the BUI is equivalent to the fractional extraction of [125I]ANP relative to the reference material [14C]inulin. Inulin has traditionally been used as a substance which is nearly unable to cross the BBB. Hence, values of A i indicate the amount of free test substance remaining in the lumina of the brain vessels at the time of decapitation. They are used to calculate the extracted fraction of ANP (EANP) according to the equation:
All data are presented as means + S.D. Statistical significance was determined using two-tailed, non-parametric tests (MannWhitney U-test, matched-pairs Wilcoxon test). The parameters of specific ANP binding (Kd, Bmax) were calculated by a non-linear regression computer program.
RESULTS
Retention o f A N P and o f inulin by various brain regions Summarizing the data gained by the 4 experiments, the
AANp--Ai EANP -- - - ' ×
Aw-Ai
43%
(3)
following results were obtained for the 8 BBB-protected b r a i n r e g i o n s p r o p e r : (1) a c c u m u l a t i o n (Fig. 1) o f A N P
211 concentrated in the injected bolus in a n a n o m o l a r range, a m o u n t e d to a m e a n value of 0.419 + 0.115% dpm/g. It notably exceeded the accumulation of the space marker inulin ( m e a n value 0.144 _+ 0.0305% dpm/g). That is, A N P was taken up into the BBB-protected brain regions at a rate of more than twice that of inulin. This difference was highly significant ( P < 0.001). (2) The accumulation of A N P is saturable (Fig. 1). The addition of excess unlabeled A N P in a micromolar concentration lowered the m e a n value to 0.180 _+ 0.0256% dpm/g (P < 0.002). Table II shows in its lower part the retention values from Expts. 3 and 4, expressed as the B U I of [125I]ANP
100000 [ZZ3[12St]AN P ~ E 10.000 d)_
[ 1291]ANP+cold ANP
[]~] E14C]lnulin
iii infl iili I ~ [ 14C]lnulin +cold ANP
g
1.000
_o E
0.100
0.010
relative to [14C]inulin. The 1000-fold excess of unlabeled
AP NL CP PI
OL FC VC ST HI TH HY CO
BBB free regions
BBB protected regions
Fig. 1. Regional accumulation of [125I]ANPand of [14C]inuhnby rat brain in the absence or presence of excess unlabeled ANP. BBB-free and BBB-protected regions are depicted separately. [125I]ANP:n = 9 (Expts. 1 and 3); [125I]ANP + cold ANP: n = 5 (Expt. 4); [14C]inulin:n = 13 in BBB-free regions (Expts. 2 and 3), n = 18 in BBB-protected regions (Expts. 2,3 and 4), [14C]inulin+ cold ANP: n = 5 (Expt. 4). For experimental design cf. Table I. The hatched bars for [~4I]inulin in the BBB-protected regions involve the 5 animals of Expt. 4 injected with excess unlabeled ANP (hatched bars in BBB-free regions). This pooling of values was possible since both groups did not differ significantly in their inulin uptake. For mean values of the groups and their standard deviations see text. AP, anterior pituitary; CO, colliculi; CP, choroid plexus IV; FC, frontal cortex; HI, hippocampus; HY, hypothalamus; NL, pituitary neurointermediate lobe; OL, olfactory lobe; PI, pineal gland; ST, striatum; TH, thalamus; VC, visual cortex.
A N P diminshed the m e a n B U I by 64.2% from 391 _ 107% to 140 _+ 19.1%. The values calculated for the extracted fraction of [125I]ANP are plotted in Fig. 2. The unlabeled peptide in the injected bolus reduced E A N P from 2.79% to 0.385%, that is up to nearly one-seventh (14.7%). (3) The presence of low (Expt. 3) and high (Expt. 4) concentrations of A N P in the injected mixture did not change the accumulation of inulin. The values for A i o b t a i n e d in Expt. 2 with inulin only in the bolus (mean 0.148 +_ 0.0349% dpm/g) did not differ from A i values of Expts. 3 and 4 ( m e a n 0.141 _+ 0.0281% dpm/g). C o n c e r n i n g regional differences in the retention of ANP, we can see from Fig. 2 that two regions are distinctly above the other ones. Thus, the fraction of A N P extracted by the visual cortex is 4-fold, and that by the olfactory lobe more than 2-fold higher than the extraction by the striatum, the region with the lowest
value. The results obtained for samples of the 4 regions with leaky capillary endothelium can be summarized as follows: (1) A c c u m u l a t i o n of A N P in C V O and anterior EAN P
TABLE II Brain uptake index o f [1251]ANP relative to the extracellular space marker [14C]inulin in the absence (column A) or presence (column B) o f excess unlabeled A N P
The BUI was calculated from the accumulation values of Expts. 3 and 4 (cf. Table I). For abbreviations see Fig. 1. Region
BBB-free regions
BBB-protected regions
a One-tailed test.
B U1 (%)
Ratio B/A (%)
P
A
B
(n = 5)
(n = 5)
AP NL CP PI
202 + 57.1 208 + 38.2 127 + 21.6 217 + 56.0
149 + 30.5 139 + 15.5 104 + 23.3 160 + 29.0
73.8 66.8 81.9 73.7
n.s,
Mean
188 + 32.0
136 + 11.9
72.3
Brain Research, 554 (1991) 209-216 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 000689939116797A BRES 16797
On the blood-brain barrier to peptides: specific binding of atrial natriuretic peptide in vivo and in vitro A r m i n Ermisch 1, Hans-Joachim Rfihle 1, Roswitha Kretzschmar I and A l e x a n d e r B a e t h m a n n 2 1Department of Cell Biology and Regulation, Section of Biosciences, University of Leipzig, Leipzig (ER. G.) and 2Institute of Surgical Research, Ludwig Maximilians University, Munich (E R. G.) (Accepted 19 February 1991)
Key words: Atrial natriuretic peptide; Blood-brain barrier; Circumventricular organ; Microvessel; Receptor
Using the intracarotid bolus injection technique, a saturable binding of [125I]atrial natriuretic peptide (ANP) was found in 8 blood-brain barrier (BBB)-protected rat brain regions as well as in the pineal gland, choroid plexus, neurointermediate and anterior lobes of the pituitary, i.e. structures lacking a BBB. The presence of specific ANP binding on the BBB, here shown for the first time by an in vivo approach, was evidenced concomitantly in vitro by incubation of isolated microvessels. A single-class high affinity binding without regional differences was obtained with K d = 0.23 nM and Bmax = 120 fmoi/mg protein. From that a density of 1,400 binding sites per endothelial cell was calculated, thought to be localized predominantly in the luminal membranes. In the in vivo study, the portion of the extracted peptide that, under the conditions used, may have crossed the BBB by passive diffusion amounted to less than 0.4% of the labeled ANP administered. ANP itself did not change the tightness of the BBB to the non-diffusible reference molecule [14C]inulin. In the BBB-free areas, ANP enhanced the inulin space by nearly 50%. INTRODUCTION Recently, a family of natriuretic peptides ( A N P ) was isolated and sequenced. The peptides, primarily detected in cells of the cardiac atria, are p r o d u c e d by m a n y cell populations 2'12'26'47'69. A N P induces vascular effects associated with systemic b l o o d pressure homeostasis and the extracellular fluid composition 4A2'25'44'66. The effects are m e d i a t e d by highly selective and specific A N P receptors13,27,32,46,58,59. Specific A N P binding sites have been detected also in the brain vasculature, especially in isolated microvessels and cultured brain capillary endothelial cells 8,9,29,48,61. The endothelial cells of the microvessels constitute in vivo the b l o o d - b r a i n barrier (BBB). A n implication of a specific A N P binding on the endothelial cells in water and electrolyte balance of the brain has been discussed 8,9. Q u i t e recently, effects of A N P on the sodium transport of isolated brain microvessels were described 3°,31, and the effect of the p e p t i d e on the permeability of the BBB to a tracer p r o t e i n and ionic lanthanum was studied in vivo by electron microscopy 45. Nonetheless, the d a t a concerning the specific binding of A N P on the BBB as well as the effect of A N P on transport processes across the BBB are still scarce and partially contradictory. Thus, a convincing demonstration
of a saturable binding of the p e p t i d e on the B B B in vivo is lacking. Levin et al. 41, for e x a m p l e , failed to detect specific binding sites on the B B B of rabbits by an in vivo approach, in contrast to the results of in vitro studies on rats m e n t i o n e d above. Therefore, the present p a p e r reports on the binding p r o p e r t i e s of the brain microvasculature in various brain areas, both in vivo and in vitro. D a t a are given regarding the tightness of the b a r r i e r to A N P in comparison to the m a r k e r molecule inulin, n o r m a l l y excluded from the transport through the BBB. A differentiation was m a d e between ' B B B - p r o t e c t e d ' brain regions with a tight capillary e n d o t h e l i u m , and circumventricular organs ( C V O ) being ' B B B - f r e e ' , i.e. possessing capillaries with a leaky e n d o t h e l i u m 1°. We are aware of the fact that such basic studies might also attain significance for the explanation of clinical problems such as the dysfunction of the BBB and the pathogenesis of cerebral e d e m a as well 1.
MATERIALS AND METHODS
Retention of ANP by various brain regions Experiments were performed on 22 male Wistar rats weighing 130-220 g. They were divided into 4 groups. The experimental design and detailed data can be seen in Table I. The retention of
Correspondence: A. Ermisch, Sektion Biowissenschaften, Universit~it Leipzig, Talstrasse 33, 0-7010 Leipzig, F.R.G.
210 TABLE I
Experimental design for the in vivo studies of ANP retention by brain regions Experiment number
n
4 8 5
1
2 3
5
Tracer
[125I]ANP [14C]inulin ['25I]ANP + [14C]inulin [125I]ANP + [14C]inulin + unlabeled ANP (2.2/~g = 3.5 x 103nM)
radioactive tracers by the brain regions was measured using the intracarotid bolus injection technique as previously described 39'49. Briefly, the rats were anesthetized with 20 mg/kg of ketamine hydrochloride (Velonarcon 0.5, VEB Berlin Chemic) intraperitoneally. The right common carotid artery was cannulated and a 0.2 ml-bolus of phosphate-buffered saline (pH 7.4) containing the tracers (3-[12sI]iodotyrosyl2s) rat atrial natriuretic peptide 28 (Amersham; spec. act. 1730 or 1980 Ci/mmol) or (hydroxyl[14C]methyl)inulin (Amersham; spec. act. 9.45 mCi/mmol), or both, was rapidly injected. In Expt. 4 an excess of unlabeled ANP (rat a-ANP, Serva, Heidelberg) was injected together with the radiolabeled tracers. The animals were decapitated 15 s after injection, the brains quickly removed and the hemisphere ipsilateral to the injection side dissected into 12 regions. Additionally, the ipsilateral half of the anterior pituitary was included in the study. The tissue samples were weighed and counted for 1251by gamma spectrometry (Expt. 1). In order to determine the 14C radioactivity (Expt. 2), the samples were dissolved in Soluene 350 (Packard, Groningen) before counting by liquid scintillation spectrometry. In Expts. 3 and 4 the samples were first counted for 1251 and afterwards dissolved and counted for 14C. Aliquots of respective injection mixtures were measured in the same manner. Accumulation of radioactivity in a tissue sample (/ix) was calculated by the equation:
A,-
Rbr - - x Rin j X Wb,
100%
(1)
where Rbr represents radioactivity of the sample, Wbr its weight, and Rinj the injected amounts of radioactivity. Furthermore, the brain uptake index (BUI) 49 was calculated using the formula:
Injected tracer amount per animal
Tracer concentration
(uCi)
(ng)
(nM)
0.78 5.8 1.2 3.0 1.4 3.0
1.4 3.2 X 106 1.9 1.7 × 106 2.2 1.7 × 106
2.2 3.1 × 106 3.(1 1.6 × lff' 3.5 1.6 × 10"
where A w represents the accumulation of tritiated water in the region 2°'3s. The fraction of water extracted was assumed to be 43% 24"
Specific ANP binding sites m isolated microvessels Isolation of brain microvessels. Cerebral microvessels were prepared by mechanical homogenization and centrifugation33'36. Brains of Wistar rats were immersed in 0.25 M sucrose solution. After removal of the meninges and white matter, tissue of neocortex, hippocampus, and striatum were separately minced and homogenized through nylon meshes with 225/zm and 120/~m pore size. The homogenates were then centrifuged at 100 g for 10 min. The pellets, which were resuspended in 0.25 M sucrose, were layered twice above a discontinuous gradient of 1.8, 1.5, 1.3, 1.0 M sucrose and fractionated at 50,000 g for 30 min. The resulting pellets containing isolated cerebral microvessels were observed by light microscopy. Radioligand-binding assay. The microvessels isolated from the 3 brain regions (approx. 200/~g protein per sample) were incubated at room temperature in an isotonic Tris-HCI buffer at pH 7.436 with 10 pM [125I]ANP for 60 min in a total volume of 200/~1. The binding reaction was terminated by centrifugation at 18,000 g for 1 min. After washing, the bound radioactivity of the microvessel pellets was determined by gamma spectrometry. Competition experiments were made by addition of various concentrations of unlabeled rat ANP in a range between 10 pM and 100/~M. Studies on saturation of ANP binding were carried out with neocortex microvessels using concentrations of [125I]ANP from 10 pM to 1 nM. Specific binding of [125I]ANP was calculated by subtraction of non-specific binding determined in the presence of 1 ~M unlabeled ANP from the total binding rate.
Statistics BUI
=
AANP x 100% Ai
(2)
Thus, the BUI is equivalent to the fractional extraction of [125I]ANP relative to the reference material [14C]inulin. Inulin has traditionally been used as a substance which is nearly unable to cross the BBB. Hence, values of A i indicate the amount of free test substance remaining in the lumina of the brain vessels at the time of decapitation. They are used to calculate the extracted fraction of ANP (EANP) according to the equation:
All data are presented as means + S.D. Statistical significance was determined using two-tailed, non-parametric tests (MannWhitney U-test, matched-pairs Wilcoxon test). The parameters of specific ANP binding (Kd, Bmax) were calculated by a non-linear regression computer program.
RESULTS
Retention o f A N P and o f inulin by various brain regions Summarizing the data gained by the 4 experiments, the
AANp--Ai EANP -- - - ' ×
Aw-Ai
43%
(3)
following results were obtained for the 8 BBB-protected b r a i n r e g i o n s p r o p e r : (1) a c c u m u l a t i o n (Fig. 1) o f A N P
211 concentrated in the injected bolus in a n a n o m o l a r range, a m o u n t e d to a m e a n value of 0.419 + 0.115% dpm/g. It notably exceeded the accumulation of the space marker inulin ( m e a n value 0.144 _+ 0.0305% dpm/g). That is, A N P was taken up into the BBB-protected brain regions at a rate of more than twice that of inulin. This difference was highly significant ( P < 0.001). (2) The accumulation of A N P is saturable (Fig. 1). The addition of excess unlabeled A N P in a micromolar concentration lowered the m e a n value to 0.180 _+ 0.0256% dpm/g (P < 0.002). Table II shows in its lower part the retention values from Expts. 3 and 4, expressed as the B U I of [125I]ANP
100000 [ZZ3[12St]AN P ~ E 10.000 d)_
[ 1291]ANP+cold ANP
[]~] E14C]lnulin
iii infl iili I ~ [ 14C]lnulin +cold ANP
g
1.000
_o E
0.100
0.010
relative to [14C]inulin. The 1000-fold excess of unlabeled
AP NL CP PI
OL FC VC ST HI TH HY CO
BBB free regions
BBB protected regions
Fig. 1. Regional accumulation of [125I]ANPand of [14C]inuhnby rat brain in the absence or presence of excess unlabeled ANP. BBB-free and BBB-protected regions are depicted separately. [125I]ANP:n = 9 (Expts. 1 and 3); [125I]ANP + cold ANP: n = 5 (Expt. 4); [14C]inulin:n = 13 in BBB-free regions (Expts. 2 and 3), n = 18 in BBB-protected regions (Expts. 2,3 and 4), [14C]inulin+ cold ANP: n = 5 (Expt. 4). For experimental design cf. Table I. The hatched bars for [~4I]inulin in the BBB-protected regions involve the 5 animals of Expt. 4 injected with excess unlabeled ANP (hatched bars in BBB-free regions). This pooling of values was possible since both groups did not differ significantly in their inulin uptake. For mean values of the groups and their standard deviations see text. AP, anterior pituitary; CO, colliculi; CP, choroid plexus IV; FC, frontal cortex; HI, hippocampus; HY, hypothalamus; NL, pituitary neurointermediate lobe; OL, olfactory lobe; PI, pineal gland; ST, striatum; TH, thalamus; VC, visual cortex.
A N P diminshed the m e a n B U I by 64.2% from 391 _ 107% to 140 _+ 19.1%. The values calculated for the extracted fraction of [125I]ANP are plotted in Fig. 2. The unlabeled peptide in the injected bolus reduced E A N P from 2.79% to 0.385%, that is up to nearly one-seventh (14.7%). (3) The presence of low (Expt. 3) and high (Expt. 4) concentrations of A N P in the injected mixture did not change the accumulation of inulin. The values for A i o b t a i n e d in Expt. 2 with inulin only in the bolus (mean 0.148 +_ 0.0349% dpm/g) did not differ from A i values of Expts. 3 and 4 ( m e a n 0.141 _+ 0.0281% dpm/g). C o n c e r n i n g regional differences in the retention of ANP, we can see from Fig. 2 that two regions are distinctly above the other ones. Thus, the fraction of A N P extracted by the visual cortex is 4-fold, and that by the olfactory lobe more than 2-fold higher than the extraction by the striatum, the region with the lowest
value. The results obtained for samples of the 4 regions with leaky capillary endothelium can be summarized as follows: (1) A c c u m u l a t i o n of A N P in C V O and anterior EAN P
TABLE II Brain uptake index o f [1251]ANP relative to the extracellular space marker [14C]inulin in the absence (column A) or presence (column B) o f excess unlabeled A N P
The BUI was calculated from the accumulation values of Expts. 3 and 4 (cf. Table I). For abbreviations see Fig. 1. Region
BBB-free regions
BBB-protected regions
a One-tailed test.
B U1 (%)
Ratio B/A (%)
P
A
B
(n = 5)
(n = 5)
AP NL CP PI
202 + 57.1 208 + 38.2 127 + 21.6 217 + 56.0
149 + 30.5 139 + 15.5 104 + 23.3 160 + 29.0
73.8 66.8 81.9 73.7
n.s,
Mean
188 + 32.0
136 + 11.9
72.3
