Veterans Affairs Medical Center and Tulane University School of Medicine, New Orleans, Louisiana 70146
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ABSTRACT |
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The blood-brain
barrier (BBB) regulates the passage of substances between the brain and
the periphery. It has not been shown that the secretion from the brain
of a small amount of a substance can directly affect the periphery by
transport across the BBB. We found that central injection of
radioactively labeled corticotropin-releasing hormone (CRH) resulted in
the accumulation of intact CRH in the spleen. CRH also increased
splenic -endorphin, an effect not blocked by pretreatment with
dexamethasone. Inhibition of the secretion of CRH from the brain by
colchicine resulted in decreased accumulation of CRH in the spleen and
also decreased splenic
-endorphin. Similar findings occurred in the
pituitary gland. The results show that the passage of labeled CRH from
the brain can directly affect a peripheral organ, thus emphasizing the
regulatory function of the BBB.
corticotropin-releasing hormone; blood-brain barrier; neuroimmunology; peptides
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INTRODUCTION |
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BRAIN corticotropin-releasing hormone (CRH) mediates the integrated stress response and initiates adequate behavioral, endocrinologic, vegetative, autonomic, and immunological adaptation to physical and psychological challenges (7, 12). CRH plays a fundamental role in the homeostasis of the immune response. Antigenic stimuli, by means of lymphocyte production of cytokines, can induce CRH release, whereas CRH and other regulatory peptides and steroids of the hypothalamic-pituitary-adrenal axis (HPA) modulate the immune response (6, 11, 30). The effects of CRH are both central, through activation of the sympathetic nervous system and the HPA, and peripheral, because CRH acting directly on peripheral mononuclear blood cells can induce lymphocyte proliferation and the release of cytokines and proopiomelanocortin-derived (POMC) peptides (8, 28).
The anatomic and physiological characteristics of the blood-brain barrier (BBB) impose a separation between central and peripheral compartments in both the production and action of regulatory peptides. However, contrary to previous views, the BBB is not an absolute barrier, and selective passage through it has been shown to occur for small and large peptides (2, 3, 13). The physiological significance and pathological implications of such passage have not been fully studied.
In the rodent, we have shown previously that, whereas peripherally produced CRH is not able to reach the brain, a specific and active carrier-mediated transport system exists for the rapid transport of CRH from brain to blood (18, 19). This system was shown to be acutely modulated by adrenal steroids, cytokines, endogenous opiates, and opiate regulatory peptides, indicating it to be an integral part of the HPA and suggesting its relevance in the control of the stress response (18).
There is a widespread distribution of CRH within the central nervous system, including the cortex, limbic system, basal ganglia, brainstem gray matter, and cerebellum (29). The transport system from brain to blood for CRH could allow central sources of this peptide to contribute to peripheral blood levels.
Several direct peripheral effects of CRH have been described on peripheral mononuclear blood cells, testis, ovary, and placenta (8, 14, 20, 24, 31). Although it is generally assumed that these effects depend on peripheral, locally produced CRH by a paracrine or autocrine action of the peptide, the possibility remains that centrally produced CRH could cross the BBB and reach these peripheral sites in amounts sufficient to cause these effects.
To elucidate the physiological relevance of the unidirectional brain-to-blood transport system for CRH, we established three objectives for the present work: 1) to quantitatively evaluate the fraction of centrally administered CRH that reaches the blood, spleen, and pituitary; 2) to evaluate the effects of modulation of the brain-to-blood transport system for CRH on the amount reaching those target organs; and 3) to show peripheral effects of centrally administered CRH and its modulation by the BBB transport system.
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METHODS |
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Adult male ICR mice (Charles River, Wilmington, MA), weighing 19-21 g, were used in all experiments, which were approved by the Institutional Animal Care and Use Committee. The mice were housed in group cages in rooms with controlled temperature, humidity, and light. Laboratory food and tap water were continuously available.
Human/rat CRH (hCRH) was obtained from Bachem (Torrance, CA). Iodine-125 (125I, sodium salt) was obtained from Amersham (Arlington Heights, IL). All other reagents were obtained from Sigma Chemical (St. Louis, MO).
Isotopic labeling of hCRH.
Unaltered hCRH was labeled with
125I by the iodogen method
described by Salacinsky et al. (25) and modified by Linton
and Lowry (16), as previously used by us (18, 19), with the
commercially available iodo-bead reagent (Pierce, Rockford, IL). The
labeled peptide was separated from free
125I by chromatography on a C8
silica column (Variant Sample Preparation Products, Harbor City, CA),
as previously described (18, 19), diluted in Ringer's lactate, stored
at 70°C, and used within 2 wk.
Transport out of the brain and pituitary and spleen uptake. The previously described intracerebroventricular injection method was used (1). Briefly, mice were anesthetized by the intraperitoneal injection of 0.2 ml of 40% urethan. The anterior cervical region was dissected and the right carotid artery was isolated. The scalp was exposed, and a hole was made with a 26-gauge guarded needle, 1 mm lateral and 1 mm posterior to the bregma and 3.5 mm deep, to reach the lateral ventricle. At time 0, 1 µl of a solution of 125I-hCRH in Ringer's lactate [50,000 counts/min (cpm)] was injected into the lateral ventricle with a Hamilton syringe (Hamilton, Reno, NV). After 1.5, 5, 10, 20, 30, 40, 50, 60, and 90 min, the carotid artery was cut and 1 ml of arterial blood was collected. The animals were decapitated, and the brain (except for the pineal and pituitary), the pituitary, and the spleen were collected. The whole procedure was accomplished in <4 min for each animal. Radioactivity (cpm) in the brain, pituitary, and spleen and radioactivity (cpm/ml) in serum were counted for 1 min in a gamma counter. Results were expressed as the percentage of the administered dose and as counts per minute per whole organ. Ten animals were used at each time point.
The linear regression of brain radioactivity (log cpm) vs. experimental time (t) was computed: log cpm =Chromatography of pituitary and spleen extracts. To characterize the radioactivity taken up by the pituitary and spleen after intracerebroventricular administration of 125I-hCRH, an extraction procedure followed by chromatography was used.
Mice were injected intracerebroventricularly with 1 µl of 125I-hCRH at time 0 as before. After 5 or 30 min, the animals were decapitated and the pituitaries and spleens were collected. To control for in vitro degradation, a group was used in which no intracerebroventricular injection of the labeled peptide was performed, but 1 µl of the 125I-hCRH was added directly to the spleen after organ collection. Ten animals were used for each experimental and control group. For extraction, the pituitary and spleens were collected on dry ice, minced into small pieces, and boiled for 10 min; acetic acid was added to a final concentration of 0.1 M, and the mixture was homogenized and centrifuged for 20 min at 4°C. The supernatant was collected, and the pellet was again solubilized with 10 ml of a solution of 0.1 M acetic acid, followed by sonication and centrifugation. This second supernatant was added to the first, and the mixture was lyophilized. The extract was reconstituted in 250 µl of 0.1 M acetic acid and applied to the chromatography column. The chromatographic procedure was the same as that used for the separation of free 125I and 125I-hCRH after isotopic labeling (18, 19). A silica column (Variant C8) was equilibrated by a wash with 20 ml of 1% trifluoroacetic acid (TFA), then with 20 ml of 80% methanol (MeOH), and finally with 20 ml of 1% bovine serum albumin (BSA) in Ringer's lactate. The extracted sample was eluted by gravity with 5 ml of 1% TFA, followed by 10 ml of 80% MeOH and then 10 ml of 1% TFA-80% MeOH. Fractions of 0.5 ml were collected, and the radioactivity was counted for 1 min in a gamma counter.Pituitary and spleen uptake after inhibition of brain efflux. The same methodology as that in the initial experiments was used. However, at time 0, one group of 10 animals (controls) received 1 µl of a solution of 125I-hCRH as before, and the other group of 10 animals (experimental) also received 1 µl of a solution of 100 nmol colchicine in the contralateral ventricle. At 10, 20, and 30 min the brain, pituitary, and spleen were collected as before; the radioactivity was counted and expressed as a percentage of the administered dose. This dose of colchicine previously has been shown to inhibit by >50% the transport of CRH out of the brain (18). The area under the curve between 10 and 30 min was calculated by the trapezoidal rule (5).
Pituitary and spleen uptake after intravenous injection of 125I-hCRH. To evaluate the effect of intracerebroventricularly administered colchicine on the uptake of 125I-hCRH in the spleen and pituitary after intravenous injection of the labeled peptide, two groups of animals were used. One group (control) was given 1 µl of Ringer's lactate solution intracerebroventricularly 3 h before the intravenous injection, and the other group (experimental) was given 1 µl of a solution of 100 nmol colchicine 3 h before the injection.
Multiple time-regression analysis after intravenous injection of the labeled peptide was used as previously described (23). At time 0, 200 µl of 125I-hCRH (500,000 cpm) were injected into the left jugular vein. Blood was collected from the right carotid artery at 1, 5, 10, 20, 30, and 50 min. Levels of radioactivity in serum (cpm/ml) and brain and spleen (cpm/g) were counted for 1 min. The parameter exposure time (ET) was derived from the equation
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Peripheral effects of centrally administered CRH.
-endorphin levels in pituitary and spleen extracts were measured by
radioimmunoassay (INCSTAR, Stillwater, MN) to evaluate the peripheral
effects of hCRH. The assay has less than 1% cross-reactivity with
other POMC-derived peptides except for
-lipotropin (5.6%). The
sensitivity is 0.2 pmol/organ.
Statistical analysis. Results are expressed as means ± SE. A statistical software package (BMDP Statistical Software, Los Angeles, CA) was used for analysis. Differences between groups were evaluated by one-way analysis of variance followed by post hoc Newman-Keuls multiple comparison tests when appropriate. Simple linear regression was used to relate the continuous variables and to compare regression lines.
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RESULTS |
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125I-hCRH transport out of the brain.
After intracerebroventricular administration of
125I-hCRH (50,000 cpm; <1 pmol),
the percentage of the administered dose remaining in the brain
decreased exponentially in relation to time: log(%dose) = 1.773 0.010 time (n = 9, r = 0.975, P < 0.0001; Fig.
1). For the
transport out of brain for
125I-hCRH, with the assumptions of
first-order kinetics and a single-compartment model, the estimated
half-life of 125I-hCRH in brain
was 30 (range of 25-38) min, the apparent initial volume of
distribution at time 0 in brain was
0.618 (0.513-0.745) g, and the clearance rate was 0.014 (0.008-0.023) g/min.
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125I-hCRH: Blood levels.
After intracerebroventricular administration of the labeled compound,
the percentage of the administered dose in the total blood volume
(estimated as 6.8 ml, with no significant differences in the
radioactivity distributed between plasma and cellular fractions) showed
a parabolic relation to time, defined by a second-degree equation:
%dose = 27 + 0.486 time 0.005 (time)2
(n = 9, r = 0.772, P < 0.01; Fig. 2).
125I-hCRH: Pituitary levels.
Figure 3 shows the relationship between
pituitary levels of the labeled peptide vs. time after
intracerebroventricular administration of
125I-hCRH. Elimination kinetics
were expressed by the regression equation log cpm = 2.413 0.010 time (n = 6, r = 0.963, P < 0.002), resulting in a computed
half-life of the labeled peptide in the pituitary of 30 (22-49)
min.
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125I-hCRH: Spleen levels.
After intracerebroventricular injection of the labeled peptide, there
was a cyclic pattern in the levels of
125I-hCRH in the spleen (Fig.
4). If only the first seven time points are
considered, 1.5-50 min, the percentage of the administered dose in
spleen followed a parabolic relationship to time: %dose = 0.074 + 0.027 time 0.001 (time)2
(n = 7, r = 0.813, P < 0.05).
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Identity of the material taken up by pituitary and spleen. Chromatography of the control sample extracts of spleen revealed that, when the labeled material was added after organ collection but before the extraction procedure, 65% of the material eluted at the position of intact 125I-hCRH, and 26% eluted at the position of free 125I. There were no peaks eluting at the position of labeled peptide fragments.
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Pituitary and spleen uptake after inhibition of brain efflux. In relation to controls, animals treated with 100 nmol colchicine intracerebroventricularly 3 h before the administration of 125I-hCRH in the contralateral ventricle showed significantly (P < 0.05) greater retention of radioactivity in the brain at 10, 20, and 30 min. For the pituitary, the values for radioactivity after intracerebroventricular administration of 125I-hCRH between 10 and 30 min were significantly lower after intracerebroventricular colchicine (0.79 ± 0.12 vs. 0.45 ± 0.08%, P < 0.05). Similarly for the spleen, colchicine significantly lowered the uptake of peptide (0.55 ± 0.05 vs. 0.46 ± 0.05%, P < 0.05).
Organ uptake after intravenous injection of 125I-hCRH. There was no evidence for brain uptake of 125I-hCRH after intravenous injection of the labeled peptide. Multiple regression analysis did not show any significant difference in animals treated 3 h before with colchicine (100 nmol icv) compared with controls injected intracerebroventricularly with Ringer's lactate.
After intravenous injection of the labeled peptide, there was significant uptake of 125I-hCRH by the pituitary. In relation to controls, animals treated with colchicine (100 nmol icv) 3 h before showed a significantly (P < 0.05) greater uptake (higher Ki) and also a significantly (P < 0.05) greater initial volume of distribution (higher Vi) within the organ, as determined by multiple regression analysis. Splenic uptake of the labeled peptide after intravenous injection was significant vs. time but not significantly different between animals previously treated with colchicine intracerebroventricularly and controls, as shown by multiple regression analysis.-Endorphin content in the pituitary and spleen.
The content of
-endorphin in the pituitary and spleen was
significantly increased after administration of 1 µg hCRH
intracerebroventricularly. Previous administration of intraperitoneal
dexamethasone significantly reduced the content of
-endorphin in the
pituitary but not in the spleen. Previous intracerebroventricular
injection of colchicine significantly reduced
-endorphin in the
pituitary and spleen to levels not significantly different from basal
values (Fig. 6).
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DISCUSSION |
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Although CRH from the hypothalamus can reach the periphery, this probably is not a major source of peripherally circulating CRH. CRH from other central sites such as the cortex and limbic system probably contributes more to peripheral levels. The release of this CRH appears to be controlled differently from that of the hypothalamus, as further shown by differences in diurnal rhythm and response to stress (22, 27, 32).
Centrally produced CRH also could reach the periphery by the slow reabsorption of CSF. However, this is a nonspecific process with a half-life of ~45 min. The fastest way for central CRH to reach the periphery is through rapid passage across the BBB by the previously described specific, active, carrier-mediated unidirectional transport system from brain to blood (18, 19).
Our first experiment showed that, after central administration of CRH, the portion reaching the peripheral circulation is relatively large. After intracerebroventricular injection of 125I-hCRH, the amount in brain decreased exponentially, with an apparent half-life of 30 min. This agrees well with previous work (18, 19), although the time is at the end of the range. This can be readily explained by two experimental factors. First, in this study, experimental times were longer (up to 90 min), so that the slow reabsorption of CRH along with CSF could be relevant. Consideration of only the first five time points (1.5-30 min) shows an apparent half-life of 23 min, closer to the middle of the previously reported range. Second, we used a larger dose of 125I-hCRH in this report; because the transport system has limited capacity, this might have exerted a small saturating effect.
The amount of the labeled peptide in the estimated whole blood volume after central administration followed a clear parabolic relationship vs. experimental time, similar to what would result from other routes of administration. Basic kinetic parameters deduced from these blood levels suggest a first-order absorption process with an estimated half-life of 9 min, comparing well with the elimination half-life from brain. This indicates a rapid process that is more compatible with the brain-to-blood transport system described than with the reabsorption of CSF.
The computed elimination half-life in blood also is longer than
previously reported (19, 26). This may depend on the long time period
used in its computation (30-90 min) and on the different route of
administration (icv vs. iv). Under physiological conditions at steady
state, given the relationship between absorption and elimination
constants for blood, it can be estimated that the concentration of
peptide in blood will approach one-third the concentration in CSF.
Human studies have reported plasma levels of CRH in the range of
2-28 pg/ml (4, 17, 27), whereas concentrations in human CSF are at
least 3-4 times higher [9-17 pmol/l 40-80 pg/ml (10, 15)]. Thus central CRH and the
transport system previously described can account for all the reported
blood levels of the peptide.
After central administration of 125I-hCRH, the labeled peptide quickly reached the pituitary and spleen, where peak values were found at 10 and 30 min, respectively. Basic kinetic analysis indicated low-order absorption kinetics (half-life <1.5 min) for the pituitary and first-order absorption kinetics for the spleen (half-life = 9 min). It is estimated that, at steady state, the content of CRH in spleen is ~1.5 times the levels in blood, whereas maximal content in pituitary is ~23 times blood levels.
Results from the second set of experiments indicate that the material taken up by the pituitary and spleen after intracerebroventricular administration of the labeled peptide represents mostly intact peptide. Five minutes after intracerebroventricular administration of 125I-hCRH, more than one-half of the radioactivity in the pituitary and more than two-thirds of the radioactivity in the spleen eluted at the position of the intact peptide.
The much shorter absorption half-lives of CRH for both the pituitary and spleen in relation to the longer half-lives of elimination from blood reported in other studies (19, 26) also are consistent with the amount taken up corresponding to most of the intact peptide. Supporting this is the smaller percentage found in the pituitary, expected from its faster uptake than in the spleen.
Previous work (18) suggested that the brain-to-blood transport system for CRH probably corresponds to a transcytotic process, because it can be inhibited by intracerebroventricular administration of colchicine, the prototypic drug known to interfere with the microtubular system. In the third set of experiments, intracerebroventricular administration of colchicine in a dose known to inhibit the transport of CRH out of the brain significantly increased the amount of the intracerebroventricularly administered 125I-hCRH retained in the brain at 10, 20, and 30 min.
This set of experiments showed that the effect of colchicine on the uptake of CRH by the pituitary and spleen was not exerted directly on these peripheral organs. As was also found previously (18, 19), there was no evidence of brain uptake of 125I-hCRH after intravenous injection, and this was not influenced by the previous administration of intracerebroventricular colchicine. Thus the results indicate that the decreased uptake of labeled CRH by the pituitary and spleen after pretreatment with colchicine is explained by the known ability of colchicine to decrease the transport of CRH out of the brain.
The last set of experiments demonstrated that intracerebroventricular
administration of CRH at the low dose of 1 µg (0.2 nmol) significantly increased the content of -endorphin in the spleen and
pituitary. The effect was rapid, being evident 1 h after
intracerebroventricular injection of the peptide. This supports the
reports that subcutaneous CRH and intracerebroventricular
interleukin-1
significantly increase the concentration of
-endorphin in the spleen (21, 24). The effect of the
intracerebroventricular interleukin-1
was probably mediated by CRH,
because it could be blocked by previous administration of a specific
CRH antagonist.
The dose of dexamethasone we used reduced the pituitary content of
-endorphin, as might be expected from its known (9) ability to
completely suppress levels of corticosterone in the serum. Yet the
dexamethasone did not significantly reduce the splenic content of
-endorphin, suggesting different regulatory systems in these organs.
Central administration of colchicine, in doses that inhibited the
transport of CRH out of the brain, reduced the content of -endorphin
in both the spleen and the pituitary to levels indistinguishable from
basal values.
-endorphin in the spleen probably is produced there,
because hypophysectomy does not affect its content (24). Because
colchicine did not affect the uptake of intravenous CRH, the evidence
indicates that it was the decreased release of CRH from the brain that
resulted in the reduction in splenic
-endorphin.
In conclusion, kinetic analysis of a single central intracerebroventricular injection of a small amount of 125I-hCRH shows that the brain-to-blood transport system for CRH can result in meaningful amounts of this peptide in blood, pituitary, and spleen. Modulation of the release of central CRH can alter its effects on peripheral organs. This indicates that endogenous central CRH can easily and quickly cross the BBB to reach the periphery in amounts sufficient to produce physiological effects.
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ACKNOWLEDGEMENTS |
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We thank Melita B. Fasold for help in preparation of the manuscript.
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FOOTNOTES |
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J. Martin Martins is a visiting endocrinologist from Curry Cabral Hospital, Lisbon, Portugal.
This study was supported by the Veterans Administration and the Office of Naval Research.
Address for reprint requests: W. A. Banks, VA Medical Center, 1601 Perdido St., New Orleans, LA 70146.
Received 20 March 1997; accepted in final form 8 August 1997.
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