0022-3565/02/3023-1062–1069
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
U.S. Government work not protected by U.S. copyright
JPET 302:1062–1069, 2002
Vol. 302, No. 3
36129/1001749
Printed in U.S.A.
Differential Transport of a Secretin Analog across the BloodBrain and Blood-Cerebrospinal Fluid Barriers of the Mouse
WILLIAM A. BANKS, MARTIN GOULET, JAMES R. RUSCHE, MICHAEL L. NIEHOFF, and RICHARD BOISMENU
Geriatric Research, Education, and Clinical Center, Veterans Affairs Medical Center, and Department of Internal Medicine, Division of
Geriatrics, St. Louis University School of Medicine, St. Louis, Missouri (W.A.B., M.L.N.); and Repligen Corporation, Needham, Massachusetts
(M.G., J.R.R., R.B.)
Received March 15, 2002; accepted May 8, 2002
Secretin was the first hormone to be discovered, its action
being elucidated by Bayliss and Starling (1902). It is released
into the blood from the proximal small intestine and stimulates the pancreas to secrete bicarbonate and water. Secretin
also inhibits gastric acid secretion and may influence the
release of other hormones (Strand, 1999). It is a member of a
large and important family of hormones that includes vasoactive intestinal peptide, glucagon, growth hormone-releasing hormone, and pituitary adenylate cyclase-activating
polypeptide (PACAP). To date, only a single receptor unique
to secretin has been cloned (Ishihara et al., 1991).
Secretin was first found in the brain (Mutt et al., 1979;
O’Donohue et al., 1981) and shown to have effects on the
central nervous system (Ishibashi et al., 1979; Becker et al.,
1982; Charlton et al., 1983) over 20 years ago. Brain recep-
This study was supported by in part by VA Merit Review, R01 NS41863,
R01 AA12743, and by Repligen Corporation.
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
DOI: 10.1124/jpet.102.036129.
mance liquid chromatography were used to confirm the passage of intact I-SA across the BBB. I-SA entered every brain
region, with the highest uptake into the hypothalamus and
cerebrospinal fluid (CSF). Unlabeled SA (10 ␮g/mouse) did not
inhibit uptake by brain but did inhibit clearance from blood and
uptake by the CSF, colon, kidney, and liver. The decreased
clearance of I-SA from blood increased the percentage of the
i.v. injected dose taken up per brain (%Inj/g) from about 0.118
to 0.295%Inj/g. In conclusion, SA crosses the vascular barrier
by a nonsaturable process and the choroid plexus by a saturable process in amounts that for other members of its family
produce central nervous system (CNS) effects. This passage
provides a pathway through which peripherally administered
SA could affect the CNS.
tors specific for secretin are coupled to adenylate cyclase and
occur in many brain regions (Fremeau et al., 1983, 1986;
Yung et al., 2001). The cAMP levels in the hippocampus and
hypothalamus especially are stimulated by secretin (Karelson et al., 1995). Secretin (Strand, 1999) is much more potent
in stimulating pancreatic secretion when given directly into
the brain than when given intravenously (Cotner et al.,
1996). Additionally, secretin may affect cerebellar neural
transmission and stabilize Purkinje cells (Yung et al., 2001).
Recently, secretin has been suggested to have beneficial
effects in autism. Autism is characterized by a markedly
abnormal or impaired development in social interactions and
communication that begins in childhood. Autistic children
also tend to have gastrointestinal complaints. Autism has
strong but complex patterns of inheritance, with the distal q
region of chromosome 7 likely involved (Wassink and Piven,
2000). A spectrum of disease is thought to occur, with autism
being the most severe form and Asperger’s syndrome a much
milder form. In an open trial, three children were noted to
have a significant improvement in gastrointestinal symp-
ABBREVIATIONS: PACAP, pituitary adenylate cyclase-activating polypeptide; BBB, blood-brain barrier; CNS, central nervous system; SA,
secretin analog; HPLC, high-performance liquid chromatography; I-SA, secretin analog radioactively labeled with 131I; T-Alb, albumin radioactively
labeled with 99mTc; Ki, unidirectional influx rate; Vi, initial volume of distribution within brain; %Inj/g, percentage of the intravenously injected dose
present in 1 g of brain; PE, polyethylene; CSF, cerebrospinal fluid.
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ABSTRACT
Secretin is a gastrointestinal peptide belonging to the vasoactive intestinal peptide (VIP)/glucagon/pituitary adenylate cyclase-activating polypeptide (PACAP) family recently suggested to have therapeutic effects in autism. A direct effect on
brain would require secretin to cross the blood-brain barrier
(BBB), an ability other members of the VIP/PACAP family have.
Herein, we examined whether a secretin analog (SA) radioactively labeled with 131I (I-SA) could cross the BBB of 4-week-old
mice. We found I-SA was rapidly cleared from serum with
fragments not precipitating with acid appearing in brain and
serum. Levels of radioactivity were corrected to reflect only
intact I-SA as estimated by acid precipitation. After i.v. injection, I-SA was taken up by brain at a modest rate of 0.9 to 1.5
␮l/g-mm. Capillary depletion, brain perfusion, and high-perfor-
Secretin and the BBB
Materials and Methods
Synthesis and Radioactive Labeling
The analog [Tyr10]secretin-27 (SA) was synthesized by solid-phase
methods and purified to homogeneity by HPLC (tyrosine substituted
for leucine). SA was radioactively labeled with the method previously
used to characterize its receptor binding activity (Dong et al., 1999).
In brief, iodo-bead (Pierce Chemical, Rockford, IL) was incubated
with Na 131I for 10 s, and the mixture was purified on a C18 column
with reverse-phase HPLC. Specific activity of the radioactively labeled SA (I-SA) was 165 Ci/mmol, assuming a 20% counting efficiency. Amounts of radioactivity injected below are not corrected for
counting efficiency of the gamma counter. Albumin was labeled with
99m
Tc (T-Alb) by mixing with stannous tartrate, adjusting to pH 2.0
to 3.0 with 0.2 M HCl, and incubating for 20 min.
Pharmacokinetic Analysis after Intravenous Injection
Intravenous Injection Method. Male ICR mice (21–28 day old)
from our breeding colony were used for all studies. Mice were anesthetized with 0.15 ml of 40% ethyl carbamate, and the right carotid
and left jugular were exposed. Lactated Ringer’s solution (0.2 ml/
mouse) containing 106 each of I-SA (43 ng/mouse) and T-Alb was
injected into the jugular vein. Whole blood was collected 2 to 30 min
later from a cut in the carotid artery, centrifuged, and the serum
counted in a gamma counter. The brain was immediately removed
after collection of blood, the pineal and pituitary discarded, and the
remaining whole brain counted in a gamma counter.
Stability of I-SA in Blood and Brain Assessed by Acid Precipitation. Other mice were anesthetized and I-SA (3 ⫻ 106 cpm/
mouse) injected into the left jugular vein in a volume of 0.2 ml of
lactated Ringer’s solution. Serum from the carotid artery and the
whole brain were obtained as described above. Serum (50 ␮l) was
mixed thoroughly with 0.25 ml of lactated Ringer’s solution containing 1% bovine serum albumin and 0.25 ml of 30% trichloroacetic acid,
centrifuged at 5000g at 4°C for 10 min, and the resulting pellet and
supernatant counted. Each whole brain was mechanically homogenized in 2 ml of water containing 0.25 mM each of EDTA, L-thyroxine, N-ethylmaleimide, and 1,10-phenanthroline. The homogenate
was centrifuged at 5000g for 10 min, and the supernatant was
collected. Brain supernatant (0.25 ml) was vigorously mixed with
0.25 ml of 30% trichloroacetic acid, centrifuged at 5000g for 10 min,
and the resulting supernatant and pellet collected. To determine
degradation of I-SA that occurred ex vivo (processing controls), 100
␮l of I-SA in lactated Ringer’s and BSA solution was placed on the
surface of a nonradioactive mouse brain or in a tube used to obtain
carotid blood and the samples processed as described above. The
percentage of radioactivity precipitated by acid was calculated as the
percentage of total counts per minute (supernatant ⫹ pellet counts
per minute) found in the pellet. Results for the brain and blood
samples obtained after i.v. injection of I-SA were expressed as a
percentage of the results for the processing controls. Brain and
serum levels of radioactivity used in pharmacokinetic analyses were
corrected to reflect only the portion that could be precipitated by
acid.
Multiple Time Regression Analysis. Mice were anesthetized
and the right carotid artery and left jugular vein isolated and exposed. Lactated Ringer’s (0.2 ml) containing 106 cpm of I-SA was
injected into the jugular vein. To test for saturation, some mice had
10 ␮g of unlabeled SA included in the injection. Brain and serum
samples were obtained 1 to 10 min later, as described above, and
counted in a gamma counter, and the results were corrected to reflect
only that portion precipitated by acid. The unidirectional influx rate
(Ki, expressed in units of microliters per gram-minute) and the
apparent volume of distribution at time 0 (Vi, expressed in units of
microliters per gram) were determined from the following equation.
Am/Cpt ⫽ Ki
冋冕
册
t
0Cp(␶)d␶ /Cpt ⫹ Vi
(1)
where Am is counts per minute per gram of brain, Cpt is counts per
minute per milliliter of arterial serum at time t, and exposure time
(Expt, expressed in minutes) is measured by the term [兰0t Cp(␶)d␶]/
Cpt. Only the linear portion of the relation between Am/Cpt and
Expt was used to calculate Ki and Vi.
Clearance from Blood. The percentage of the intravenously
injected dose of I-SA in a milliliter of arterial serum (%Inj/ml) was
calculated from the following equation:
%Inj/ml ⫽ 100共Cpt)/Inj
(2)
where Inj is the total counts per minute injected i.v. The log of
%Inj/ml was plotted against time and the half-time disappearance
calculated from the slope, and the volume of distribution was calculated from the intercept of the resulting line.
Percentage of Injected Dose Taken Up by Brain. The percentage of the intravenously injected dose of I-SA taken up by each
gram of brain (%Inj/g) was then calculated from the following equation:
%Inj/g ⫽ 共Am/Cpt ⫺ Vi兲/103 共%Inj/ml)
(3)
where Vi corrects the brain/serum ratio for the amount of I-SA in the
vascular space.
Uptake by Various Tissues. Mice were anesthetized as described above, the left jugular vein and right carotid artery exposed,
and 0.2 ml of lactated Ringer’s solution containing 106 cpm of I-SA
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toms and social interaction skills within 5 weeks of a single
secretin infusion of 0.4 ␮g/kg (Horvath et al., 1998). Controlled trials have found no improvement 3 weeks (DunnGeier et al., 2000) and 4 weeks (Sandler et al., 1999; Chez et
al., 2000) after a single infusion of secretin. A double-blind,
placebo-controlled trial in which multiple doses of secretin
were given has detected an improvement in the social interactions of 3- to 4-year-old autistic children (M. Goulet, J. R.
Rusche, R. Boismenu, unpublished observations).
The putative effects of secretin on autism could be mediated through actions on peripheral tissues or by affecting
brain function. Peripherally circulating hormones can affect
brain function indirectly through several mechanisms, such
as affecting afferent nerve transmission or releasing other
centrally active substances. Little or no secretin is made in
the brain (Kopin et al., 1990; Yung et al., 2001). This suggests
that brain secretin is of peripheral origin, but this would
require secretin to cross the blood-brain barrier (BBB). Other
members of the secretin family have effects on the CNS
(Kastin et al., 2001) and can cross the BBB. PACAP, for
example, crosses the BBB by way of a saturable transport
system termed peptide transport system-6 (Banks et al.,
1993). About 0.11% of an intravenous dose of PACAP crosses
the BBB by way of peptide transport system-6 to enter each
gram of brain. This is an amount sufficient to reverse hippocampal neuronal loss caused by four-vessel occlusion when
PACAP is given systemically within 24 h after the ischemia
(Uchida et al., 1996). Furthermore, peripherally administered secretin can induce Fos expression in distinct brain
regions in the rat (Goulet et al., 2001).
The ability of related peptides to cross the BBB raises the
possibility that secretin analog (SA) might also be able to
cross. This would provide a mechanism by which the peptide
could exert effects on the brain. Herein, we examined the
ability of an SA to cross the BBB of mice.
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Banks et al.
and of T-Alb injected into the jugular vein. Five minutes later,
arterial blood was collected from the carotid artery and the mouse
immediately decapitated. The kidney, spleen, testes, right and left
ventricle of the heart, stomach, duodenum, jejunum, ileum, colon,
adrenal gland, pancreas, and whole brain (free of pituitary and
pineal), and pieces of lung, liver, and thigh muscle were collected and
weighed. The gastrointestinal tract organs were opened lengthwise,
their luminal contents washed out, and the tissues padded dry before
weighing. The level of radioactivity in the serum and tissue samples
was determined in a gamma counter. The results are expressed as
(counts per minute per gram of tissue)/(counts per minute per microliter of serum) ⫽ microliters per gram. Acid precipitation was
performed on the adrenal, kidney, and colon and on processing
controls for those tissues.
Capillary Depletion
Ratio ⫽ 共cpm Fr)/(w兲共cpm/␮l serum)
(4)
where cpm Fr is the counts per minute in either the parenchyma or
supernatant fraction, w is the weight of the cortex, and cpm/␮l serum
is the level counts per minute in a microliter of serum.
Brain Perfusion
Mice were anesthetized, the thorax opened, and the heart exposed.
Both jugulars were severed and the descending thoracic aorta was
clamped. A 26-gauge butterfly needle was inserted into the left
ventricle of the heart and Zlokovic’s buffer (7.19 g/l NaCl, 0.3 g/l KCl,
0.28 g/l CaCl2, 2.1 g/l NaHCO3, 0.16 g/l KH2PO4, 0.17 g/l anhydrous
MgCl2, 0.99 g/l D-glucose, and 10 g/l bovine serum albumin added the
day of perfusion) containing 105 cpm/ml was infused at a rate of 2
ml/min for 1 to 5 min (Zlokovic et al., 1988). This rate of perfusion
quickly fills the brain’s vascular space without disrupting the BBB
(Shayo et al., 1996). An injection check of 10 ␮l of the buffer solution
was taken before and after perfusion, so exact concentration of radioactivity could be calculated. After perfusion, the needle was removed, and the mouse was decapitated. Acid precipitation was performed on the brains and the counts per minute per whole brain
corrected accordingly. Brain/perfusion ratios were calculated by dividing the counts per minute per brain by the weight in grams of the
brain and by the counts per minute in a microliter of perfusion fluid
Stability of I-SA
HPLC Analysis. Mice were anesthetized and I-SA (3 ⫻ 106 cpm/
mouse) injected into the left jugular vein in a volume of 0.2 ml of
lactated Ringer’s solution. Serum from the carotid artery and the
whole brain were obtained 2, 5, or 8 min later. Each whole brain was
mechanically homogenized in 2 ml of water containing 0.25 mM each
of EDTA, L-thyroxine, N-ethylmaleimide, and 1,10-phenanthroline,
and the homogenate centrifuged at 5000g for 10 min at 4°C. The
samples were stored at ⫺70°C until assay. Samples were separated
by reversed-phase HPLC on a C18 column. Mobile phase (acetonitrile
with 0.1% trifluoroacetic acid) was against water with 0.1% trifluoroacetic acid and increased from 0 to 60% in 60 min. Fractions were
collected every minute for 60 min and counted on the gamma
counter. Processing controls were also analyzed and used to correct
results as described above for acid precipitation.
Octanol/Buffer Partition Coefficient. The lipid solubility of
I-SA was measured by determining its octanol/buffer partition coefficient. I-SA (105 cpm) was added to 0.5 ml of 0.25 M chloride-free
phosphate-buffered solution and 0.5 ml of octanol. This was vigorously mixed for 1 min and the two phases separated by centrifugation at 4500g for 10 min. Aliquots of 100 ␮l were taken in triplicate
from each phase and counted. The mean partition coefficient was
expressed as the log of the ratio of counts per minute (octanol phase)
to counts per minute (phosphate-buffered saline phase).
Entry into Cerebrospinal Fluid. Mice were anesthetized, prepared as described above, and given an injection into the jugular vein
of 106 cpm of I-SA with or without 10 ␮g/mouse of unlabeled SA. Five
minutes later, the scalp was removed from the posterior aspect of the
head, exposing the muscles overlying the posterior fossa. A 30-gauge
needle connected to a length of PE-10 tubing was inserted into the
posterior fossa with the head in a dependent position. CSF was
collected into the PE tubing by capillary action. After collecting about
10 ␮l of CSF, the tubing was removed, arterial blood collected from
the previously exposed carotid artery, the mouse decapitated, and
the whole brain removed. The exact amount in microliters of CSF
collected was determined by measuring the length in centimeters of
PE tubing filled with CSF and multiplying by 0.668. Only CSF that
was absolutely clear was analyzed. The CSF, brain, and serum were
counted in a gamma counter. Acid precipitation was performed on
CSF, brain, and serum and each sample’s level of radioactivity (I)
were corrected. The results were expressed as brain/serum (microliters per gram), CSF/serum (microliters per milliliter), and brain/CSF
(milliliters per gram) ratios.
Uptake into Brain Regions. Mice were anesthetized, prepared
as described above, and given an i.v. injection of 106 cpm of I-SA. Five
minutes later, arterial serum was obtained from the carotid artery,
and the brain was removed and dissected into 11 regions after the
manner of Glowinski and Iversen (1966). Each region was weighed,
and its level of radioactivity was determined in a gamma counter.
The results were expressed as (counts per minute per brain region)/
(regional wt in grams)(counts per minute per microliter of serum) ⫽
microliters per gram of brain region.
Statistics
Means are reported with their standard errors and the number (n)
per group. Two group comparisons were performed using Student’s
two-tailed, unpaired t test with comparisons considered significant
at p ⬍ 0.05 level. When more than two means were compared,
analysis of variance was performed followed by Newman-Keuls post
test. Regression lines were calculated by the least-squares method
with the Prism 3.0 program (GraphPad Software, San Diego, CA).
The slope (Ki) with its error term, the intercept (Vi) with its error
term, the regression coefficient (r), the n value, and the p value are
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In other mice (n ⫽ 4), the relative distribution of I-SA between the
cerebral cortex and capillaries was assessed by the method of
Triguero et al. (1990) as modified for mice by Gutierrez et al. (1993).
After anesthesia, mice received an injection into the jugular vein of
0.2 ml of lactated Ringer’s solution containing 1% bovine serum
albumin containing 1 ⫻ 106 cpm of I-SA and 1 ⫻ 106 cpm of T-Alb.
Five minutes later, the abdomen was opened, and blood was collected
from the abdominal aorta. The thorax was opened, the thoracic
descending aorta clamped, the left and right jugular veins severed,
and the brain flushed of its intravascular contents by injecting 20 ml
of lactated Ringer’s solution over 1 min into the left ventricle of the
heart. The mouse was decapitated and the brain harvested. The
cerebral cortex was isolated and weighed and placed in ice-cold
physiological buffer (10 mM HEPES, 141 mM NaCl, 4 mM KCl, 2.8
mM CaCl2, 1 mM MgSO4, 1 mM NaH2PO4, and 10 mM D-glucose
adjusted to pH 7.4). The cortex was then homogenized using a glass
tissue grinder (10 strokes) in 0.8 ml of physiological buffer. Dextran
solution, 1.6 ml of a 26% solution in physiological buffer, was added
to the homogenate, mixed vigorously, and homogenized (three
strokes). The homogenate was centrifuged at 5400g for 15 min at 4°C
in a swing bucket rotor. The pellet, which contains the brain vasculature, and the supernatant, which contains the brain parenchyma,
were carefully separated and the radioactivity of each component
determined using a gamma counter. The parenchyma/serum and
capillary/serum ratios (microliters per gram) were calculated by the
following equation:
to yield units of microliters per gram. Influx rate (Ki) was determined
from the slope of the relation between brain/perfusion ratios and
time in minutes.
Secretin and the BBB
1065
TABLE 1
HPLC characterization for radioactivity recovered from brain and
serum after intravenous injection of I-SA
The percentage of radioactivity eluting in the position of intact I-SA is given. Means
with S.D. (n) are shown.
Time
Serum
Brain
86.5 ⫾ 0.3 (2)
50.4 ⫾ 16.6 (2)
68.6 (1)
70.1 ⫾ 4.3 (2)
107 ⫾ 49.0 (2)
80.8 ⫾ 15.9 (2)
min
2
5
8
reported. Regression lines were compared for statistically significant
differences with the Prism 3.0 program, which first compares slopes,
and if there is no statistically significant differences (p ⬍ 0.05), then
compares intercepts.
Results
Stability of I-SA in brain and serum after i.v. injection was
first determined. The portion of radioactivity in serum and
brain precipitated by acid decreased with time (Fig. 1). These
values were not different in mice that received unlabeled SA
(data not shown). These results were used to correct the
brain and serum values of the other experiments. HPLCs of
radioactivity recovered from processing controls and from
brain and serum 5 min after i.v. injection of I-SA are shown
in Fig. 2. Table 1 shows the summary of HPLC results for
Fig. 2. Representative HPLCs for serum and brain. Results for radioactivity
extracted 5 min after i.v. injection of
I-SA and for processing controls is similar.
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Fig. 1. Relation between time and the log of the percentage of radioactivity extracted from brain or serum that could be precipitated with acid.
The amount of radioactivity that could be precipitated decreased with
time in both serum and brain.
brain and serum over time. In general, acid precipitation
overestimated degradation of I-SA, especially in brain.
The top panels of Fig. 3 show the results of multiple-time
regression analysis for I-SA. The top left panel shows an
experiment measuring the unidirectional influx rate of I-SA
at 0.949 ⫾ 0.0388 ␮l/g-mm. The top right panel shows that
unlabeled SA (10 ␮g/mouse) did not inhibit the influx of I-SA;
the influx rate for I-SA was 1.53 ⫾ 0.221 ␮l/g-mm and for
I-SA ⫹ SA was 1.50 ⫾ 0.046 ␮l/g-mm. Therefore, the dominant mechanism of passage across the vascular BBB is likely
the nonsaturable process of transmembrane diffusion. The
log of the octanol/buffer partition coefficient was ⫺2.27. Capillary depletion showed that over 90% of the I-SA taken up by
brain had entered the parenchymal space (Table 2).
Unlabeled SA did saturate the clearance of I-SA from
blood, decreasing the volume of whole body distribution from
8.4 to 1.97 ml (Fig. 3, bottom left). The half-time disappearance of I-SA was 3.58 min. The increase in serum levels
resulting from the injection of SA means that more of the
injected dose is presented to the brain. As a result, brain
uptake increased (Fig. 3, bottom right). The results for %Inj/g
were fitted to a one-site binding model (hyperbola). This
model gave a maximum uptake of 0.118%Inj/g in the absence
of SA and a maximum uptake of 0.295%Inj/g in its presence;
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Banks et al.
TABLE 2
Capillary depletion
Vascular space was washed out and fractions corrected for any contamination by
residual serum as measured with T-Alb. Means are given with their standard errors.
n ⫽ 4/group.
Fraction
␮l/g
Parenchyma
Capillaries
10.7 ⫾ 1.2
0.91 ⫾ 0.14
half of this maximum value was reached 1.8 to 1.9 min after
i.v. injection.
The unidirectional influx rate measured during brain perfusion was 2.73 ⫾ 0.69 ␮l/g-mm (Fig. 4, top). Acid precipitation showed that about 70% of the radioactivity from blood
was precipitated by acid with no change in the amount of
material degraded over time. HPLC, however, found less
degradation on average than with processing controls. A
representative HPLC for radioactivity extracted from brain
after 5 min of perfusion is shown in the bottom panel of Fig.
4.
Figure 5, top, shows that I-SA entered the CSF. All results
were corrected for acid precipitation, including those for I-SA
in CSF and for T-Alb in brain, serum, and CSF. All CSF
samples were clear without evidence of blood. Acid precipi-
tation of I-SA gave results similar to those described above.
For CSF, about 90% of the 131I radioactivity precipitated
with acid, indicating that it was largely I-SA. Similarly,
about 90% of the Tc radioactivity found in brain and serum
was precipitated by acid, but only 49% of the Tc radioactivity
in CSF precipitated. The CSF/serum ratio was much higher
for I-SA than for T-Alb, demonstrating an uptake not accounted for by leakage, extracellular pathways, or traumatic
tap. For comparison, the brain/serum ratios from the same
mice are shown. The CSF/brain ratio after subtracting the
T-Alb values from both compartments was 16.0, indicating
that I-SA enters the CSF much more rapidly than the parenchyma.
The bottom panel of Fig. 5 shows the effects of including 10
␮g/mouse of unlabeled SA in the injection. Unlabeled SA had
no effect on the T-Alb brain/serum or CSF/serum ratios (data
not shown), and these values have been subtracted from the
I-SA ratios shown in the bottom panel of Fig. 5. A t test
showed that the CSF/serum ratio for I-SA was significantly
decreased by unlabeled SA (p ⬍ 0.01). There was no effect on
the brain/serum ratio. Inclusion of unlabeled SA decreased
the CSF/brain ratio to 0.2.
Figure 6 shows uptake into various brain regions. The
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Fig. 3. Top left panel shows initial experiment measuring blood-to-brain unidirectional influx rate (Ki) of I-SA. Ki ⫽ 0.949 ⫾ 0.0388 ␮l/g-mm.
Remaining panels shows results for experiment with or without 10 ␮g of unlabeled SA/mouse included in the i.v. injection of I-SA. Top right panel
shows lack of inhibition for brain uptake, with a Ki of about 1.5 ␮l/g-mm for both groups. Bottom left panel shows that this dose altered clearance of
I-SA from blood. Bottom right panel shows results expressed as the percentage of the i.v. injected dose taken up per gram of brain. For I-SA, the
maximum value was calculated to be 0.118%Inj/g; with inclusion of 10 ␮g of SA, the maximum value was calculated to be 0.295%Inj/g.
Secretin and the BBB
1067
Fig. 4. Brain perfusion studies for I-SA. Top, Ki as measured by brain
perfusion was 2.73 ⫾ 0.69 ␮l/g-mm. Bottom, HPLC of radioactivity extracted from brain at end of 5-mm perfusion. Proportion of radioactivity
eluting as I-SA was higher than found for processing controls (compare
with Fig. 2, top right). Overestimation of degradation products by processing controls can occur when degradation products are cleared in vivo
from brain.
highest uptake was into the hypothalamus and the slowest
was into the frontal cortex.
Figure 7, top, shows the distribution of I-SA among 15
tissues (n ⫽ 5). These results have been corrected for vascular space by subtraction of T-Alb. The bottom panel shows
those tissues for which inclusion of unlabeled SA (n ⫽ 3)
decreased the tissue/serum ratio for I-SA; they have also
been corrected for vascular space. A trend (p ⬍ 0.10) toward
a decrease was noted for heart, jejunum, pancreas, testis, and
thigh muscle. Inclusion of unlabeled SA did not affect the
T-Alb tissue/serum ratio for any tissue except kidney (p ⬍
0.05), which decreased from 160 ⫾ 15 (n ⫽ 5) to 97 ⫾ 11 (n ⫽
3). There were no trends toward a decrease for any of the
other T-Alb tissue/serum ratios. The percentage of extracted
radioactivity that was precipitated by acid was, after correction with processing controls, 97 ⫾ 2% for the adrenals, 32 ⫾
3% for the colon, and 23 ⫾ 2% for kidney (n ⫽ 2/tissue).
Discussion
These results show that I-SA completely crosses the BBB
as an intact molecule at a modest rate. The amount of I-SA
taken up by the brain after i.v. injection is in the range seen
for other peptides and regulatory proteins that affect brain
Fig. 5. Top, uptake of I-SA into brain and CSF. T-Alb is included for
comparison, which for CSF measures entry by extracellular pathways or
from BBB disruption and for brain measures vascular space. The uptake
rate into the CSF compartment was about 16 times faster than uptake by
brain. Bottom, effect of including unlabeled SA in the i.v. injection of I-SA
on CSF/serum and brain/serum ratios. The unlabeled SA inhibited uptake of I-SA into CSF but not into brain.
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 18, 2017
function by crossing the BBB. I-SA entered all regions of the
CNS with uptake by the CSF, hypothalamus, hippocampus,
and olfactory bulb being particularly high. Uptake into the
CSF but not by brain was inhibited by 10 ␮g/mouse of unlabeled SA, strongly suggesting that this peptide crosses the
choroid plexus by a saturable transport system and crosses
the vascular BBB by transmembrane diffusion. Clearance
from blood and uptake by some peripheral tissues was inhibited by the 10-␮g dose of SA, resulting in increases in serum
levels, which, in turn, increased the percentage of the injected dose entering the brain. These findings support the
hypothesis that peripherally administered SA can affect CNS
function. These findings and their implications are discussed
in detail below.
I-SA injected intravenously crossed the BBB with a unidirectional influx rate (Ki) of about 1 to 1.5 ␮l/g-mm. In all of
these studies, we used mice that were 3 to 4 weeks of age.
This precedes puberty in mice and so parallels the use of
secretin in childhood autism. By 21 days of age, the BBB in
mice and rats has largely matured and has a permeability to
serum albumin similar to that of adult rodents (Davson and
Segal, 1996). Brain and serum levels of radioactivity had to
be corrected for accumulation of degradation products in
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Banks et al.
Fig. 6. Uptake of I-SA into various brain regions. Dotted line indicates
value for whole brain. Means are given with their S.E. (n ⫽ 7/group). a,
p ⬍ 0.05 versus hypothalamus; b, p ⬍ 0.05 versus hypothalamus, hippocampus, or olfactory bulbs; and c, p ⬍ 0.05 versus whole brain.
Fig. 7. Uptake of I-SA by various tissues after i.v. injection. Top, uptake
for 15 tissues; H indicates only I-SA was included in injection. Bottom,
three tissues for which tissue/serum ratio was lower when unlabeled SA
was included in the injection (C-tissue). 多, p ⬍ 0.05; 多多, p ⬍ 0.005.
studies, and the large difference between CSF/serum ratios
for I-SA and T-Alb confirms that the results are not due to
traumatic taps. The CSF/serum ratio for I-SA was about 16
times greater than the brain/serum ratio, showing that uptake into CSF is much greater than uptake into brain. Unlabeled SA inhibited uptake into CSF but had no effect on
brain uptake. Therefore, I-SA is transported into the CSF by
a saturable transport system.
When 106 cpm of I-SA was injected intravenously, about
0.118% of the injected dose entered the brain. Because 106
cpm of I-SA was estimated to contain about 43 ng of SA,
about 0.051 ng was taken up per gram of brain. Inhibition of
clearance resulted in higher serum levels of I-SA; that is, a
greater percentage of the injected dose remained in blood
longer. This increased presentation to brain and, because
blood-to-brain passage was not saturable, resulted in more
I-SA entering brain. When 10 ␮g of SA was included in the
I-SA intravenous injection, about 0.295% of the injected dose
entered the brain, or about 30 ng of SA. This exceeds the KD
of 0.2 nM (0.6 ng/ml for SA with a molecular weight of 3089)
for the brain secretin receptor (Fremeau et al., 1983). These
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these compartments. Acid precipitation, which can be a reliable estimate of the amount of radioactivity representing
intact peptide, was used to make these corrections. In this
case, however, acid precipitation underestimated the amount
of intact peptide, especially at later time points for brain.
This may have led to an underestimation of Ki by as much as
2-fold.
To negate the effects of degradation in the circulation, we
measured Ki by brain perfusion and found that uptake was
indeed higher, with a value of 2.73 ␮l/g-mm. Brain perfusion
sometimes produces higher Ki values when a transporter is
partially saturated or when uptake by brain is retarded by
binding to serum proteins. The Ki from the brain perfusion
experiment was also corrected based on acid precipitation,
which indicated that about 70% of the radioactivity in brain
was intact I-SA. HPLC showed little or no degradation in
brain after correction by processing control, and so Ki may
have been underestimated by about 30%. The Ki for I-SA,
therefore, likely is between 2 and 3 ␮l/g-mm.
Capillary depletion and CSF sampling showed that I-SA
completely crossed the BBB to enter the CNS. The CSF/
serum ratio was about 16 times higher than the brain/serum
ratio. Because the time between injection and sampling of
brain and CSF was short, the results suggest that I-SA
crossed at the choroid plexus more rapidly than at the brain
vasculature.
I-SA uptake by brain was not inhibited by a dose of 10
␮g/mouse, or about 500 ␮g/kg. It is possible that a higher
dose may have unmasked saturable transport, but it exceeds
the dose used in autism of about 0.4 ␮g/kg. The 10-␮g/mouse
dose was also sufficient to saturate clearance from blood and
uptake by the colon, kidney, and liver. Therefore, it is probable that the major mechanism by which I-SA crosses the
vascular BBB is by transmembrane diffusion. The log of the
octanol/buffer partition coefficient for I-SA was (⫺)2.27,
which is in the lower range for peptides (Banks and Kastin,
1985).
I-SA uptake by CSF exceeded that of T-Alb, demonstrating
that entry into the CSF was not caused by residual leakage of
serum proteins. Only absolutely clear CSF was used in these
Secretin and the BBB
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Address correspondence to: Dr. W. A. Banks, St. Louis University School of
Medicine, 915 N. Grand Blvd., St. Louis, MO 63106. E-mail: [email protected]
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levels of uptake are similar to those for other peptides and
regulatory proteins that affect brain function after crossing
the BBB. For example, the effect of intravenously administered interleukin-1␣ on cognition is partially mediated by
cytokine, which has crossed the BBB to act within the posterior division of the septum. The uptake also exceeds that
for morphine, which is less than 0.02%Inj/g.
Uptake of I-SA was not uniform throughout the CNS but
varied about 3- to 4-fold. The areas with the fastest uptake
were the hypothalamus and hippocampus and the areas with
the slowest were the frontal and parietal cortexes. Abnormalities in both these regions, especially the hippocampus, have
been reported in autism. The hippocampus and other limbic
structures are proportionately smaller in autism (Aylward et
al., 1999). Hippocampal neurons tend to be smaller, more
tightly packed, and to have less branching of the dendrites
(Raymond et al., 1996). The pattern of uptake differs from
that of secretin receptor distribution, suggesting that initial
passage across the BBB is independent of receptor location
(Fremeau et al., 1983). The ability of secretin to stimulate
cAMP is, however, highest for the hypothalamus and hippocampus and lowest for the frontal cortex (Karelson et al.,
1995).
In conclusion, these studies show that an analog of secretin
crosses the BBB to enter the CSF and the parenchymal space
of the brain. Entry is at a modest rate and the amount of SA
crossing the BBB is sufficient to affect brain function. Entry
is most likely to be mediated by nonsaturable transmembrane diffusion at the vascular BBB but by a saturable transporter at the choroid plexus. I-SA entered every region of the
brain but uptake was highest for hypothalamus and hippocampus. We conclude that the therapeutic effect of SA seen
in autism could be due to its ability to cross the BBB and so
act at sites within the CNS.
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