Permeation of Growth Hormone across the Blood-Brain Barrier
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《内分泌学杂志》
Pennington Biomedical Research Center (W.P., Y.Y., C.M.C., A.J.K.), Louisiana State University System, Baton Rouge, Louisiana 70808
Department of Pharmaceutical Biosciences (F.N.), Uppsala University, Uppsala S-75124, Sweden
Institute Cochin (P.O.C.), Department of Cellular Biology, Paris 75654, France
Abstract
Exogenous GH can affect central nervous system function when given peripherally to animals and as a supplemental therapy to humans. This study tested whether GH crosses the blood-brain barrier (BBB) by a specific transport system and found that both mice and rats have small but significant uptake of GH into the brain without a species difference. Determined by multiple-time regression analysis, the blood-to-brain influx transfer constants of 125I-labeled rat GH in mice (0.23 ± 0.07 μl/g·min) and rats (0.32 ± 0.04 μl/g·min) were comparable to those of some cytokines of similar size, with a half-time disappearance of 125I-GH of 3.8–7.6 min in blood. Intact 125I-GH was present in both serum and brain homogenate 20 min after iv injection. At this time, about 26.8% of GH in brain entered the parenchyma, whereas 10% was entrapped in endothelial cells. Neither excess GH nor insulin showed acute modulation of the influx, indicating lack of a saturable transport system for GH at the BBB. Binding and cellular uptake studies in cultured cerebral microvessel endothelial cells (RBE4) further ruled out the presence of high-capacity adsorptive endocytosis. The brain influx of GH by simple diffusion adds definitive value to the long-disputed question of whether and how GH crosses the BBB. The central nervous system effects of peripheral GH can be attributed to permeation of the BBB despite the absence of a specific transport system.
Introduction
THE PRINCIPAL FORM of GH is a large polypeptide of 191 amino acids (22 kDa). By existing dogma, it would be considered too large a protein to cross the blood-brain barrier (BBB), the communicating interface between the cerebral microcirculation (the periphery) and central nervous system (CNS) parenchyma. Among many restrictive factors, the presence of tight junctions between the capillary endothelial cells composing the BBB and the underlying continuous basement membrane substantially reduces paracellular permeation of large proteins. However, specific transport systems at the BBB have been identified for many peptides and proteins. This growing list includes some ILs (1, 2, 3, 4), tumor necrosis factor (5, 6), and other cytokines that are even larger than GH (7).
The speculation that GH crosses the BBB comes from several lines of evidence. First, there are studies showing that exogenous GH used as a supplementary therapy can improve human cognitive function, mood, memory, and sleep (8, 9). Second, not only can GH affect cerebrospinal fluid (CSF) levels of various neuropeptides, amino acids, and monoamine metabolites (10, 11), but it also can be recovered from the CSF after peripheral administration (12). Third, GH receptors are present in the CNS (13). Recently, the distribution of mRNA for the GH receptor was mapped in rat brainstem and spinal cord (14). GH also can cause age-dependent up-regulation of its own receptor and change N-methyl-D-aspartate receptor subunit gene transcripts in the hippocampus (15). Thus, it is possible that GH penetrates the BBB to exert its effects directly upon the brain. If so, a saturable transport system for GH would be more likely than the process of simple diffusion to be subject to regulation in pathophysiological states, thereby providing a target for therapeutic intervention.
The many CNS effects of GH provide a good reason to determine its mechanism of passage across the BBB. In addition to its effects on cognitive function, mood, memory, and sleep in humans (8, 9), GH also can exert neuroprotective actions in animal models of spinal cord injury (16, 17), and hypoxic ischemia (18). After spinal cord injury in the rat by thoracic dorsal horn incision, there is an age-dependent increase in trauma-induced permeability to GH (19). If the increased entry of GH to the injured spinal cord is beneficial for neuroregeneration, delivery of therapeutic doses of GH by way of its transport system could further facilitate locomotor recovery.
In addition, GH alters appetite and feeding behavior (20). It is not known whether these effects occur outside or within the BBB, and if inside, whether by direct action or indirect alteration of other neurotransmitters in response to GH. Thus, a better understanding of the interactions of GH with the BBB could assist in the design of strategies to control feeding.
Despite considerable indirect evidence suggesting that GH may cross the BBB, the possible penetration of GH into the CNS has not been established by pharmacokinetic quantification. In this study, we determined the pharmacokinetics of BBB permeation of radioactively labeled GH by use of multiple-time regression analysis (21) designed to assess the influx transfer of slowly penetrating substances across the BBB. We found that GH had significant entry from blood to brain while remaining intact, and that significantly more of the injected GH entered brain parenchyma than was associated with the cerebral vasculature. However, the entry of GH was not mediated by a saturable transport system. The effects of protamine and poly-L-lysine to increase the internalization of GH by RBE4 cells suggest that heparin sulfate proteoglycan is involved in the interactions between GH and the BBB. Thus, simple diffusion of GH across the BBB provides an explanation of the dose-dependent effect of peripheral GH on CNS function.
Materials and Methods
Adult male CD1 mice of 5–6 wk of age were used following the protocol approved by the Institutional Animal Care and Use Committee. The mice were studied after anesthesia induced by ip injection of ketamine. Recombinant human and rat nonglycosylated GH were obtained from Diagnostic Systems Laboratories (Webster, TX). Recombinant human insulin, BSA, and chloramine-T radiolabeling reagents were purchased from Sigma (St. Louis, MO). GH was radioactively labeled with 125I by the chloramine-T method with the reaction stopped at 1 min by addition of sodium metabisulfate and purified on Sephadex G-10. The specific activity of 125I-GH was about 200 Ci/g. Albumin was radioactively labeled with 131I by the chloramine-T method with a specific activity of about 60 Ci/g. Most studies involved rat125I-GH in mice unless otherwise specified.
Multiple-time regression analyses
To determine whether rat GH enters the mouse brain and whether it does so by saturable transport, two groups of mice were studied: one with radioactively labeled compounds only and a similar group with inclusion of 1 μg/mouse of unlabeled GH. The injection solution of lactated Ringer’s with 1% BSA (LR/BSA) was prepared fresh each time. There were seven mice in each group. A mixture of 30,000 cpm/μl each of 125I-GH and 131I-albumin in 100 μl of injectate was delivered to the left jugular vein at time 0. At various times between 1 and 30 min, blood was collected from a cut in the right common carotid artery and the mouse was decapitated immediately afterwards. The radioactivity in the whole brain and 50 μl of serum was measured, and the brain/serum ratio of 125I-GH and 131I-albumin in each gram of brain was calculated separately. Based on the exponential decay pattern of serum radioactivity, the exposure time was calculated for each time point. The exposure time is the integral of serum radioactivity over time divided by the serum radioactivity at a given time (21). The linear regression correlation between the brain/serum ratio and exposure time was determined by use of GraphPad Statistical Software (GraphPad, San Diego, CA). The unidirectional influx transfer constant (Ki) was determined from the slope of the linear regression line, and the initial volume of distribution (Vi) was determined from the intercept. Differences of the regression lines between the two groups were compared by the least square method by use of the GraphPad program.
To determine the dose of excess unlabeled GH required for potential saturation, the experiment was repeated with higher doses of GH. To test the species specificity of BBB permeation, human and rat 125I-GH were used separately in the studies on mice, and rat 125I-GH was used in both young male CD1 mice and Wistar rats.
In addition to multiple-time regression analyses, single-time uptake of 125I-GH was determined at 10 min after iv injection in mice and the potential modulatory effect of excess GH and insulin was determined by one-way ANOVA (n = 6/group).
Degradation assays by HPLC, acid precipitation, and gel autoradiography
Each mouse received an iv injection of 3–4 μCi of 125I-GH in 100 μl of injection at time 0. At 20 min, arterial blood and brain were obtained and processed on ice. The brain was homogenized in 1 ml of LR/BSA in the presence of Complete Protease Inhibitor Cocktail (Sigma). To assess the extent of ex vivo degradation, a processing control was generated by addition of 125I-GH into the blood-collection tube and brain homogenate. About 30,000 cpm of brain supernatant or serum was injected onto the reversed phase HPLC or precipitated by 15% of trichloroacetic acid. The mobile phase of HPLC was acetonitrile with 0.1% trifluoroacetic acid that was increased from 10–100% over 40 min.
For gel autoradiography, protein from the serum and brain homogenate was fractionated with 8% SDS-PAGE. There were five groups: serum and brain samples of the processing control and mice receiving 125I-GH iv, and the stock solution. The proteins were transferred to a nitrocellulose membrane, and exposed to x-ray film at –80 C.
In situ brain perfusion
The composition of the perfusion buffer and the conditions for perfusion have been described previously (22). In brief, a group of eight to 10 mice that received 125I-GH and 131I-albumin was studied in parallel with a group that received additional unlabeled GH at 20 μg/ml. After clamping of the abdominal aorta and severing of the jugular veins of the anesthetized mice, the oxygenated perfusate was delivered at 2 ml/min for various time intervals between 1 and 10 min. The perfusion buffer contained about 2000 cpm/μl of 125I-GH and 131I-albumin. Each mouse received a prewash of 2 min to clear the cerebral vasculature and 1 min of postwash to remove radioisotopes that had not entered the brain. At the end of the procedure at each time point, the mouse was decapitated, and the brain/perfusate ratio of radioactivity per gram of brain was measured. Statistical analysis was performed as above.
Capillary depletion
Four groups of mice (n = 4/group) were studied. The groups were mice receiving 125I-GH and 131I-albumin with or without cardiac perfusion to wash out residual radioactivity in the cerebral vasculature, and mice receiving additional excess GH at 10 μg/mouse with or without cardiac perfusion with 20 ml of LR. At the end of the study (10 min after iv injection), blood and brain were collected. The cerebral cortex was homogenized in capillary buffer (22) and mixed thoroughly with dextran to yield a final concentration of dextran of about 18.4%. The mixture was centrifuged at 9000 x g for 30 min at 4 C with a swing bucket rotor to achieve effective separation of brain parenchyma from the capillaries. After measurement in a -counter, the ratios of tissue/serum radioactivity for 125I-GH and 131I-albumin were calculated and expressed per gram of cerebral cortex. Group means are presented with their SEs, statistically significant differences being determined by ANOVA followed by Tukey’s post hoc test.
Cellular uptake assays
The control group with 125I-GH only was studied simultaneously (triplicated wells/group) with the following potential modulators: excess unlabeled GH (1 mg/ml), 500 mM monodansylcadeverine (an inhibitor of adsorptive endocytosis), 300 mM protamine, 300 mM poly-L-lysine (both being positively charged proteins that also inhibit adsorptive endocytosis), and hypertonic sucrose (1.2 M). Equal numbers of immortalized rat brain microvessel endothelial cells RBE4 (kind gift from NeuroTech, Paris, France) were seeded to six-well plates precoated with rat tail collagen. Radiotracer uptake assays were performed when the cells grew confluent. The cells were preequilibrated for 15 min in 1 ml of transport buffer that contained equal amounts of MEM and F10, supplemented with 20 mM HEPES and 0.05% BSA. 125I-GH (700,000 cpm/ml) was added in 1 ml of transport buffer prewarmed to 37 C, and the plates were gently agitated for 20 min. At this time, internalization of 125I-GH was terminated by transferring the plates to ice, followed by rapid removal of the transport buffer and three washes with ice-cold PBS. Afterwards, specific cell surface binding was determined by incubation of the cells with ice-cold stop-strip buffer [0.2 N acetic acid in PBS (pH 2.5)] for 10 min and collection of this and subsequent washes with stop-strip buffer. The cells were then lysed and collected. The maximal potential internalization represents the amount of radioactivity from the acid-resistant binding combined with the amount internalized at the same time point. The percent of surface binding and maximal potential internalization were determined by normalization of the values of individual wells to the total amount of 125I-GH added at time 0.
Separate groups were studied at 0 C to inhibit endocytosis and compared with groups studied at 37 C. For the 0 C groups, the plates were kept on ice the entire time. Group means were expressed with their SEs, and statistically significant changes were determined by an overall ANOVA for each fraction, followed by Tukey’s post hoc test.
In contrast to continuous uptake of 125I-GH for 20 min, the radiotracer pulse-chasing study was performed to determine the internalization at 20 min after binding equilibrium. The control group with 125I-GH only was studied simultaneously with the following five groups: 1 mg/ml of unlabeled GH, 300 mM of protamine, both protamine and unlabeled GH, 50 μg/ml of heparin, and 300 mM of poly-L-lysine. After equilibration of cells in ice-cold transport buffer for 15 min, the cells were incubated in ice-cold transport buffer containing 125I-GH with or without the above potential modulators (700,000 cpm/ml) at 4 C for 1 h. Afterwards the radioactively labeled tracer was removed by two washes with ice-cold PBS, and the plates were rapidly warmed up to 37 C in the presence of prewarmed transport buffer. At 20 min, surface binding and maximal potential internalization were determined as above.
Results
Influx transfer of 125I-GH from blood to brain
The blood-to-brain influx of 125I-GH was linear during the study period (1–30 min after iv injection in mice). As shown in Fig. 1A, the influx transfer constant was 0.23 ± 0.07 μl/g·min and the initial volume of distribution was 15.5 μl/g. Of the total amount injected iv, the percent of brain uptake was about 0.1%/gram of brain at 20 min. Given the specific activity of 200 Ci/g of 125I-GH and the injection amount of 1.36 μCi, about 6.8 pg of GH was present in a gram of brain tissue. The results indicated that a significant amount of 125I-GH reached the brain, and this was supported by results from degradation studies showing that most of the measured radioactivity represented intact GH (see below). Addition of 1 μg/mouse of unlabeled GH (about 150-fold excess) to the 125I-GH injection solution did not significantly affect the rate of entry. The lack of inhibitory effect of unlabeled GH was reproduced in subsequent experiments with various doses of GH and shorter time intervals. The serum half-life of 125I-GH ranged from 3.8–7.6 min in different experiments.
To further determine the possible presence of a saturable transport system as well as modulation by insulin, which affects GH, three groups of mice were studied (n = 6/group). At 10 min after iv injection of 125I-GH, the volume of distribution of 125I-GH in the brain was 14.80 ± 0.80 μl/g. It was not significantly changed by the addition of 200 μg/mouse (about 30,000-fold excess) of unlabeled GH (Vi = 15.50 ± 0.95 μl/g) or 10 μg/mouse of unlabeled insulin (Vi = 14.20 ± 0.22 μl/g). This is shown in Fig. 1B. Similarly, the influx transfer constant of 125I-insulin, which was 0.97 ± 0.14 μl/g·min, was not significantly changed by the presence of 10 μg/mouse of unlabeled GH (Ki = 0.71 ± 0.18 μl/g·min).
A further study was performed with radioactively labeled rat GH injected iv into rats. The influx transfer constant to the whole brain was 0.32 ± 0.04 μl/g·min, and the initial volume of distribution was 19.27 ± 0.74 μl/g. Addition of 80 μg/rat of excess unlabeled GH did not cause a significant change in the influx rate [F (1, 19) = 0.6, P > 0.05] (Fig. 1C). There was no significant regional difference in the influx transfer constant of 125I-GH among the dissected brain regions (frontal, parietal, occipital cortices, striatum, thalamus, hypothalamus, brainstem, and cerebellum). In each region, there was no significant effect of excess unlabeled GH that would have indicated saturable transport.
Degradation assays to determine that the radioactivity measured represented intact 125I-GH
After 20 min of iv circulation, HPLC showed that intact 125I-GH accounted for 91% of the total radioactivity in serum, similar to that in the serum of the processing control in which 125I-GH was mixed with blood in the test tube only. In serum samples, up to 90% in mouse 20 min after iv injection of 125I-GH and 95% of radioactivity in the processing control were acid precipitable. In the supernatant of brain homogenate, up to 81% in mouse 20 min after iv injection of 125I-GH and 94% in the processing control were also acid precipitable. Similarly, the radioactivity in the brain after 10 min of in situ brain perfusion mainly represented intact 125I-GH. The acid precipitable radioactivity was 90% in brain after 10 min of in situ perfusion of 125I-GH, and 89.4% in brain after perfusion of 125I-GH along with excess unlabeled GH. The results indicate that intact 125I-GH was present in the brain after iv delivery. This is further supported by the presence of a radioactive band at molecular mass of 22 kDa in gel autoradiography in all samples, correlating with intact 125I-GH.
In situ brain perfusion studies to determine the transfer of 125I-GH across the BBB
To eliminate the possibility that serum GH binding proteins interfered with detection of a saturable transport system, and to confirm that the radioactivity measured in the whole brain of mice after iv injection of 125I-GH had reached the brain compartment rather than remaining in the vasculature, in situ brain perfusion was performed with serum-free buffer. The control group received 125I-GH and 131I-albumin whereas the experimental group received 20 μg/ml of unlabeled GH in addition. The influx transfer constant of 125I-GH was 0.84 ± 0.08 μl/g·min without excess GH and 0.82 ± 0.10 μl/g·min in the presence of excess GH; there was no significant difference between the two groups (Fig. 2). Therefore, the lack of saturation was not explained by the presence of serum binding proteins. This further supports the conclusion that the substantial entry of rat GH across the mouse BBB did not occur by a saturable transport system.
Differentiation of the amount of 125I-GH taken up by the brain vs. that trapped in the cerebral vasculature
As shown in Fig. 3, the nonperfused brain had an apparently high volume of distribution of 125I-GH when the brain was obtained 10 min after iv injection (n = 4 /group). This reflects high uptake in the brain parenchymal fraction (12.60 ± 0.61 μl/g) and low uptake in the capillary fraction (3.63 ± 0.76 μl/g). However, the vascular marker 131I-albumin had a parenchymal uptake of 6.77 ± 0.50 μl/g and capillary uptake of 0.75 ± 0.08 μl/g. These values roughly represent the radioactivity that is either in the capillary lumen or loosely bound to the capillary walls because albumin is considered to have minimal transcellular permeation of an intact BBB. Thus, the actual uptake of 125I-GH into the brain parenchyma, as shown after perfusion to wash out the vascular space, was only 4.35 ± 0.32 μl/g, or 26.8% of the total radioactivity measured in the whole brain. The actual amount of 125I-GH trapped in the cerebral vasculature was 1.63 ± 0.39 μl/g at 10 min, or 10% of the total radioactivity. This is significantly less than that reaching the brain parenchyma [F (1, 8) = 117.5, P < 0.001].
Uptake of 125I-GH by RBE4 cells and its modulation by protamine and poly-L-lysine
The concentration of 125I-GH in the transport buffer (700,000 cpm/ml, with specific activity of 200 Ci/g) was about 1.6 ng/ml. Therefore, 1 mg/ml of excess unlabeled GH represented over 60,000 times excess. At this dose, there was no significant inhibition of either surface binding or internalization of 125I-GH. Thus, there was no saturable transport system for GH in the cultured RBE4 cells. To further rule out adsorptive endocytosis, which can occur with glycoproteins and has a lower affinity and higher capacity than receptor-mediated endocytosis, specific inhibitors for adsorptive endocytosis (monodansylcadeverine, poly-L-lysine, and protamine)were also included in the study. Monodansylcadeverine had no significant effect on surface binding or internalization of 125I-GH after 20 min of coincubation. Unexpectedly, the percent of 125I-GH at the cell surface at 20 min was significantly increased in the presence of protamine (P < 0.05), whereas that internalized was also significantly increased by both protamine and poly-L-lysine (P < 0.01). Hypertonic buffer significantly decreased (P < 0.05) surface binding without affecting maximal potential internalization. Both binding and internalization were significantly decreased in low temperature at this time (P < 0.01). The lack of inhibitory effects of protamine, poly-L-lysine, and monodansylcadeverine ruled out the possibility of adsorptive endocytosis (Fig. 4A).
The significantly less binding of 125I-GH at 4 C in contrast to that at 37 C suggests that binding equilibrium had not been achieved during the incubation period. Thus, a further study was performed by pulse-chasing, in which the cells were incubated with 125I-GH at 4 C for 1 h and then internalization of the bound 125I-GH at 20 min was determined. Similar to what was seen in Fig. 4A, the surface binding and internalization were both significantly increased by protamine (P < 0.01). This increase was not affected by addition of excess unlabeled GH. In contrast, poly-L-lysine and heparin showed no significant effects (Fig. 4B).
Discussion
It is well known that GH in the peripheral circulation can exert multiple CNS effects. The recovery of GH in the CSF (12) suggested that GH crosses the BBB. Here we provide the first direct evidence that recombinant, nonglycosylated human and rat GH could be stable enough in the blood and cerebral microcirculation to permeate the BBB of mouse and rat. With a sensitive radiotracer uptake method, we showed that the polypeptide GH had an influx transfer constant of 0.23 ± 0.07 μl/g·min in mice and 0.32 ± 0.04 μl/g·min in rats, a value comparable to that of some neurotropins and cytokines, polypeptides known to cross the BBB (7, 22, 23, 24, 25). HPLC, acid precipitation, and gel autoradiography confirmed that the radioactivity measured in the brain was mainly intact 125I-GH rather than products of degradation.
GH had a moderately high initial volume of distribution in the brain. The majority of the peripherally injected 125I-GH was retained in the cerebral vasculature 10 min after iv injection, although a significant amount (26.8%) entered brain parenchyma, whereas 10% was retained in the microvessel endothelial cells composing the BBB. Consistent with the capillary depletion studies, in situ brain perfusion showed that the perfusate-to-brain transfer of 125I-GH was about 35 times lower than that of nerve growth factor (22) and about 250 times lower than that of brain-derived neurotrophic factor (26). Similar to what we have seen for other proteins, the primary structure or the presence of receptors may not be the determining factor for BBB permeability. Regardless, a significant amount of GH can cross the BBB to exert CNS effects directly.
The permeation of GH, however, is a nonsaturable process, as shown both in vivo and in vitro. In studies with cultured cerebral microvessel endothelial cells (major components of the BBB), surface binding of 125I-GH did not seem to be mediated by specific GH receptors because excess unlabeled GH failed to inhibit the binding or internalization of 125I-GH. Although it is possible that glycosylated GH might be internalized by adsorptive endocytosis, modulators of adsorptive endocytosis (dansylcadeverine, protamine, and poly-L-lysine) failed to decrease the uptake of nonglycosylated GH in this study, thus ruling out the presence of adsorptive endocytosis (27) for this commonly used form of the hormone. The enhancing effect of protamine might be explained by its binding to GH, thereby increasing its positive charge so as to facilitate adsorptive endocytosis, as it does for albumin (28). However, adsorptive endocytosis is usually a specific process, but the uptake of 125I-GH was not saturable by coadministration of excess unlabeled GH. Nonetheless, naturally occurring GH may be glycosylated, which increases its chance of adsorptive endocytosis. Endogenous GH can exist in different forms due to posttranslational modifications, including deamidation, acylation, glycosylation, and oligomerization, thereby resulting in distinct kinetic patterns of clearance, receptor binding, and complexing with binding proteins (29). Although there do not seem to be major differences in biological activity among these forms (30), it is possible that different forms of GH penetrate the BBB in different ways.
The plasma concentration of GH has a distinctive circadian rhythm and ranges from 0–250 ng/ml. Therefore, although the trace amount of I-GH would not increase the blood level of GH, the excess exogenous GH injected to test for saturation might affect the concentration of GH and the subsequent mediators of its function. This could be an issue for long-term effects in survival studies. Nonetheless, it should not affect the conclusions from the acute in vivo studies.
Based on the lack of inhibition by a large excess of GH in the in vivo and in vitro experiments, we conclude that it is unlikely the GH receptor or other carrier systems are involved in the blood-to-brain transfer of GH. This means that the permeation of GH probably occurs by the process of simple diffusion dependent on the physiochemical properties of GH. In pathological conditions in which the BBB is partially disrupted, such as CNS trauma, hypoxia/ischemia, tumor, and certain developmental or degenerative stages, more GH could reach its brain targets than by a process limited by saturation. The permeation of peripheral GH across the BBB does not override the importance of neuronal sources of GH, such as in the hypothalamic region of rats (31) and retina of embryonic chicks (32), which play important roles in neuromodulation.
It has been suggested that peripheral GH can be detected in the brain and CSF; however, there has not been direct demonstration whether and how the BBB is involved. Because the choroid plexus has a high level of expression of GH receptors, receptor-mediated transport is possible there. Despite the theoretical possibility that the blood-CSF barrier may possess a specific transport system, our pharmacokinetic study on mice and rats ruled out receptor-mediated transport for GH at the BBB. Results from cultured cerebral microvessel endothelial cells also ruled out high-capacity adsorptive endocytosis. Despite the lack of a specific transport system, GH was relatively stable in blood within the first 10 min after iv delivery and underwent limited simple diffusion across the BBB. The majority of GH entering the cerebral circulation was retained in the cerebral vasculature, but significant penetration to brain parenchyma did occur. These studies add definitive value to the long dispute whether and how GH crosses the BBB.
Footnotes
This work was supported by the National Institutes of Health [NS45751 and NS46528 (to W.P.) and DK54880 and AA12865 (to A.J.K.)].
Abbreviations: BBB, Blood-brain barrier; CNS, central nervous system; CSF, cerebrospinal fluid; Ki, unidirectional influx rate; LR, lactated Ringer’s; Vi, initial volume of distribution.
References
Banks WA, Kastin AJ, Durham DA 1989 Bidirectional transport of interleukin-1 across the blood-brain barrier. Brain Res Bull 23:433–437
Banks WA, Ortiz L, Plotkin SR, Kastin AJ 1991 Human interleukin (IL)1, murine IL-1 and murine IL-1 are transported from blood to brain in the mouse by a shared saturable mechanism. J Pharmacol Exp Ther 259:988–996
Banks WA, Kastin AJ, Gutierrez EG 1994 Penetration of interleukin-6 across the murine blood-brain barrier. Neurosci Lett 179:53–56
Banks WA, Kastin AJ, Ehrensing CA 1994 Blood-borne interleukin-1 is transported across the endothelial blood-spinal cord barrier in mice. J Physiol 479:257–264
Gutierrez EG, Banks WA, Kastin AJ 1993 Murine tumor necrosis factor is transported from blood to brain in the mouse. J Neuroimmunol 47:169–176
Pan W, Banks WA, Kastin AJ 1997 Permeability of the blood-brain and blood-spinal cord barriers to interferons. J Neuroimmunol 76:105–111
Pan W, Kastin AJ 1999 Penetration of neurotrophins and cytokines across the blood-brain/blood-spinal cord barrier. Adv Drug Deliv Rev 36:291–298
Nyberg F 2000 Growth hormone in the brain: characteristics of specific brain targets for the hormone and their functional significance. Front Neuroendocrinol 21:330–348
Burman P, Broman JE, Hetta J, Wiklund I, Erfurth EM, Hagg E, Karlsson FA 1995 Quality-of-life in adults with growth-hormone (GH) deficiency—response to treatment with recombinant human GH in a placebo-controlled 21-month trial. J Clin Endocrinol Metab 80:3585–3590
Johansson JO, Larsson G, Elmgren A, Hynsj L, Lindahl A, Lundberg PA, Isaksson O, Lindstedt S, Bengtsson B 1995 Treatment of growth hormone-deficient adults with recombinant human growth hormone increases the concentration of growth hormone in the cerebrospinal fluid and affects neurotransmitters. Neuroendocrinology 61:57–66
Nyberg F, Burman P 1996 Growth hormone and its receptors in the central nervous system—location and functional significance. Horm Res 45:18–22
Coculescu M 1999 Blood-brain barrier for human growth hormone and insulin-like growth factor-I. J Pediatr Endocrinol Metab 12:113–124
Lai ZN, Emtner M, Roos P, Nyberg F 1991 Characterization of putative growth-hormone receptors in human choroid-plexus. Brain Res 546:222–226
Kastrup Y, Le Greves M, Nyberg F, Blomqvist A 2005 Distribution of growth hormone receptor mRNA in the brain stem and spinal cord of the rat. Neuroscience 130:419–425
Le Greves M, Steensland P, Le Greves P, Nyberg F 2002 Growth hormone induces age-dependent alteration in the expression of hippocampal growth hormone receptor and N-methyl-D-aspartate receptor subunits gene transcripts in male rats. Proc Natl Acad Sci USA 99:7119–7123
Hanci M, Kuday C, Oguzoglu SA 1994 The effects of synthetic growth hormone on spinal cord injury. J Neurosurg Sci 38:43–49
Winkler T, Sharma HS, Stalberg E, Badgaiyan RD, Westman J, Nyberg F 2000 Growth hormone attenuates alterations in spinal cord evoked potentials and cell injury following trauma to the rat spinal cord. An experimental study using topical application of rat growth hormone. Amino Acids 19:363–371
Gustafson K, Hagberg H, Bengtsson BA, Brantsing C, Isgaard J 1999 Possible protective role of growth hormone in hypoxia-ischemia in neonatal rats. Pediatr Res 45:318–323
Mustafa A, Sharma HS, Olsson Y, Gordh T, Thoren P, Sjoquist PO, Roos P, Adem A, Nyberg F 1995 Vascular-permeability to growth-hormone in the rat central-nervous-system after focal spinal-cord injury—influence of a new antioxidant H-290/51 and age. Neurosci Res 23:185–194
Wang X, Day JR, Zhou Y, Beard JL, Vasilatos-Younken R 2000 Evidence of a role for neuropeptide Y and monoamines in mediating the appetite-suppressive effect of GH. J Endocrinol 166:621–630
Kastin AJ, Akerstrom V, Pan W 2001 Validity of multiple-time regression analysis in measurement of tritiated and iodinated leptin crossing the blood-brain barrier: meaningful controls. Peptides 22:2127–2136
Pan W, Banks WA, Kastin AJ 1998 Permeability of the blood-brain barrier to neurotrophins. Brain Res 788:87–94
Poduslo JF, Curran GL 1996 Permeability at the blood-brain and blood-nerve barriers of the neurotrophic factors: NGF, CNTF, NT-3, BDNF. Mol Brain Res 36:280–286
Pan W, Kastin AJ, Brennan JM 2000 Saturable entry of leukemia inhibitory factor from blood to the central nervous system. J Neuroimmunol 106:172–180
Pan W, Kastin AJ 2003 Transport of cytokines and neurotrophins across the BBB and their regulation after spinal cord injury. In: Shanker H, Westman J, eds. Blood-spinal cord and brain barriers in health and disease. San Diego: Academic Press; 395–407
Pan W, Banks WA, Fasold MB, Bluth J, Kastin AJ 1998 Transport of brain-derived neurotrophic factor across the blood-brain barrier. Neuropharmacology 37:1553–1561
Deguchi Y, Okutsu H, Okura T, Yamada S, Kimura R, Yuge T, Furukawa A, Morimoto K, Tachikawa M, Ohtsuki S, Hosoya K, Terasaki T 2002 Internalization of basic fibroblast growth factor at the mouse blood-brain barrier involves perlecan, a heparan sulfate proteoglycan. J Neurochem 83:381–389
Pardridge WM, Buciak JL, Kang Y-S, Boado RJ 1993 Protamine-mediated transport of albumin into brain and other organs of the rat. J Clin Invest 92:2224–2229
Baumann G 1991 Growth hormone heterogeneity: genes, isohormones, variants, and binding proteins. Endocr Rev 12:424–449
Berghman LR, Buyse J, Huybrechts LM, Darras VM, Vandesande F, Kuhn ER, Decuypere E, Scanes CG 1994 Disappearance rate of glycosylated and non-glycosylated chicken growth hormone: influence on biological activity. Comp Biochem Physiol Pharmacol Toxicol Endocrinol 108:161–169
Lechan RM, Nestler JL, Molitch ME 1981 Immunohistochemical identification of a novel substance with human growth hormone-like immunoreactivity in rat brain. Endocrinology 109:1950–1962
Harvey S, Kakebeeke M, Sanders EJ 2004 Growth hormone localization in the neural retina and retinal pigmented epithelium of embryonic chicks. J Mol Neurosci 22:139–145(Weihong Pan, Yongmei Yu, )
Department of Pharmaceutical Biosciences (F.N.), Uppsala University, Uppsala S-75124, Sweden
Institute Cochin (P.O.C.), Department of Cellular Biology, Paris 75654, France
Abstract
Exogenous GH can affect central nervous system function when given peripherally to animals and as a supplemental therapy to humans. This study tested whether GH crosses the blood-brain barrier (BBB) by a specific transport system and found that both mice and rats have small but significant uptake of GH into the brain without a species difference. Determined by multiple-time regression analysis, the blood-to-brain influx transfer constants of 125I-labeled rat GH in mice (0.23 ± 0.07 μl/g·min) and rats (0.32 ± 0.04 μl/g·min) were comparable to those of some cytokines of similar size, with a half-time disappearance of 125I-GH of 3.8–7.6 min in blood. Intact 125I-GH was present in both serum and brain homogenate 20 min after iv injection. At this time, about 26.8% of GH in brain entered the parenchyma, whereas 10% was entrapped in endothelial cells. Neither excess GH nor insulin showed acute modulation of the influx, indicating lack of a saturable transport system for GH at the BBB. Binding and cellular uptake studies in cultured cerebral microvessel endothelial cells (RBE4) further ruled out the presence of high-capacity adsorptive endocytosis. The brain influx of GH by simple diffusion adds definitive value to the long-disputed question of whether and how GH crosses the BBB. The central nervous system effects of peripheral GH can be attributed to permeation of the BBB despite the absence of a specific transport system.
Introduction
THE PRINCIPAL FORM of GH is a large polypeptide of 191 amino acids (22 kDa). By existing dogma, it would be considered too large a protein to cross the blood-brain barrier (BBB), the communicating interface between the cerebral microcirculation (the periphery) and central nervous system (CNS) parenchyma. Among many restrictive factors, the presence of tight junctions between the capillary endothelial cells composing the BBB and the underlying continuous basement membrane substantially reduces paracellular permeation of large proteins. However, specific transport systems at the BBB have been identified for many peptides and proteins. This growing list includes some ILs (1, 2, 3, 4), tumor necrosis factor (5, 6), and other cytokines that are even larger than GH (7).
The speculation that GH crosses the BBB comes from several lines of evidence. First, there are studies showing that exogenous GH used as a supplementary therapy can improve human cognitive function, mood, memory, and sleep (8, 9). Second, not only can GH affect cerebrospinal fluid (CSF) levels of various neuropeptides, amino acids, and monoamine metabolites (10, 11), but it also can be recovered from the CSF after peripheral administration (12). Third, GH receptors are present in the CNS (13). Recently, the distribution of mRNA for the GH receptor was mapped in rat brainstem and spinal cord (14). GH also can cause age-dependent up-regulation of its own receptor and change N-methyl-D-aspartate receptor subunit gene transcripts in the hippocampus (15). Thus, it is possible that GH penetrates the BBB to exert its effects directly upon the brain. If so, a saturable transport system for GH would be more likely than the process of simple diffusion to be subject to regulation in pathophysiological states, thereby providing a target for therapeutic intervention.
The many CNS effects of GH provide a good reason to determine its mechanism of passage across the BBB. In addition to its effects on cognitive function, mood, memory, and sleep in humans (8, 9), GH also can exert neuroprotective actions in animal models of spinal cord injury (16, 17), and hypoxic ischemia (18). After spinal cord injury in the rat by thoracic dorsal horn incision, there is an age-dependent increase in trauma-induced permeability to GH (19). If the increased entry of GH to the injured spinal cord is beneficial for neuroregeneration, delivery of therapeutic doses of GH by way of its transport system could further facilitate locomotor recovery.
In addition, GH alters appetite and feeding behavior (20). It is not known whether these effects occur outside or within the BBB, and if inside, whether by direct action or indirect alteration of other neurotransmitters in response to GH. Thus, a better understanding of the interactions of GH with the BBB could assist in the design of strategies to control feeding.
Despite considerable indirect evidence suggesting that GH may cross the BBB, the possible penetration of GH into the CNS has not been established by pharmacokinetic quantification. In this study, we determined the pharmacokinetics of BBB permeation of radioactively labeled GH by use of multiple-time regression analysis (21) designed to assess the influx transfer of slowly penetrating substances across the BBB. We found that GH had significant entry from blood to brain while remaining intact, and that significantly more of the injected GH entered brain parenchyma than was associated with the cerebral vasculature. However, the entry of GH was not mediated by a saturable transport system. The effects of protamine and poly-L-lysine to increase the internalization of GH by RBE4 cells suggest that heparin sulfate proteoglycan is involved in the interactions between GH and the BBB. Thus, simple diffusion of GH across the BBB provides an explanation of the dose-dependent effect of peripheral GH on CNS function.
Materials and Methods
Adult male CD1 mice of 5–6 wk of age were used following the protocol approved by the Institutional Animal Care and Use Committee. The mice were studied after anesthesia induced by ip injection of ketamine. Recombinant human and rat nonglycosylated GH were obtained from Diagnostic Systems Laboratories (Webster, TX). Recombinant human insulin, BSA, and chloramine-T radiolabeling reagents were purchased from Sigma (St. Louis, MO). GH was radioactively labeled with 125I by the chloramine-T method with the reaction stopped at 1 min by addition of sodium metabisulfate and purified on Sephadex G-10. The specific activity of 125I-GH was about 200 Ci/g. Albumin was radioactively labeled with 131I by the chloramine-T method with a specific activity of about 60 Ci/g. Most studies involved rat125I-GH in mice unless otherwise specified.
Multiple-time regression analyses
To determine whether rat GH enters the mouse brain and whether it does so by saturable transport, two groups of mice were studied: one with radioactively labeled compounds only and a similar group with inclusion of 1 μg/mouse of unlabeled GH. The injection solution of lactated Ringer’s with 1% BSA (LR/BSA) was prepared fresh each time. There were seven mice in each group. A mixture of 30,000 cpm/μl each of 125I-GH and 131I-albumin in 100 μl of injectate was delivered to the left jugular vein at time 0. At various times between 1 and 30 min, blood was collected from a cut in the right common carotid artery and the mouse was decapitated immediately afterwards. The radioactivity in the whole brain and 50 μl of serum was measured, and the brain/serum ratio of 125I-GH and 131I-albumin in each gram of brain was calculated separately. Based on the exponential decay pattern of serum radioactivity, the exposure time was calculated for each time point. The exposure time is the integral of serum radioactivity over time divided by the serum radioactivity at a given time (21). The linear regression correlation between the brain/serum ratio and exposure time was determined by use of GraphPad Statistical Software (GraphPad, San Diego, CA). The unidirectional influx transfer constant (Ki) was determined from the slope of the linear regression line, and the initial volume of distribution (Vi) was determined from the intercept. Differences of the regression lines between the two groups were compared by the least square method by use of the GraphPad program.
To determine the dose of excess unlabeled GH required for potential saturation, the experiment was repeated with higher doses of GH. To test the species specificity of BBB permeation, human and rat 125I-GH were used separately in the studies on mice, and rat 125I-GH was used in both young male CD1 mice and Wistar rats.
In addition to multiple-time regression analyses, single-time uptake of 125I-GH was determined at 10 min after iv injection in mice and the potential modulatory effect of excess GH and insulin was determined by one-way ANOVA (n = 6/group).
Degradation assays by HPLC, acid precipitation, and gel autoradiography
Each mouse received an iv injection of 3–4 μCi of 125I-GH in 100 μl of injection at time 0. At 20 min, arterial blood and brain were obtained and processed on ice. The brain was homogenized in 1 ml of LR/BSA in the presence of Complete Protease Inhibitor Cocktail (Sigma). To assess the extent of ex vivo degradation, a processing control was generated by addition of 125I-GH into the blood-collection tube and brain homogenate. About 30,000 cpm of brain supernatant or serum was injected onto the reversed phase HPLC or precipitated by 15% of trichloroacetic acid. The mobile phase of HPLC was acetonitrile with 0.1% trifluoroacetic acid that was increased from 10–100% over 40 min.
For gel autoradiography, protein from the serum and brain homogenate was fractionated with 8% SDS-PAGE. There were five groups: serum and brain samples of the processing control and mice receiving 125I-GH iv, and the stock solution. The proteins were transferred to a nitrocellulose membrane, and exposed to x-ray film at –80 C.
In situ brain perfusion
The composition of the perfusion buffer and the conditions for perfusion have been described previously (22). In brief, a group of eight to 10 mice that received 125I-GH and 131I-albumin was studied in parallel with a group that received additional unlabeled GH at 20 μg/ml. After clamping of the abdominal aorta and severing of the jugular veins of the anesthetized mice, the oxygenated perfusate was delivered at 2 ml/min for various time intervals between 1 and 10 min. The perfusion buffer contained about 2000 cpm/μl of 125I-GH and 131I-albumin. Each mouse received a prewash of 2 min to clear the cerebral vasculature and 1 min of postwash to remove radioisotopes that had not entered the brain. At the end of the procedure at each time point, the mouse was decapitated, and the brain/perfusate ratio of radioactivity per gram of brain was measured. Statistical analysis was performed as above.
Capillary depletion
Four groups of mice (n = 4/group) were studied. The groups were mice receiving 125I-GH and 131I-albumin with or without cardiac perfusion to wash out residual radioactivity in the cerebral vasculature, and mice receiving additional excess GH at 10 μg/mouse with or without cardiac perfusion with 20 ml of LR. At the end of the study (10 min after iv injection), blood and brain were collected. The cerebral cortex was homogenized in capillary buffer (22) and mixed thoroughly with dextran to yield a final concentration of dextran of about 18.4%. The mixture was centrifuged at 9000 x g for 30 min at 4 C with a swing bucket rotor to achieve effective separation of brain parenchyma from the capillaries. After measurement in a -counter, the ratios of tissue/serum radioactivity for 125I-GH and 131I-albumin were calculated and expressed per gram of cerebral cortex. Group means are presented with their SEs, statistically significant differences being determined by ANOVA followed by Tukey’s post hoc test.
Cellular uptake assays
The control group with 125I-GH only was studied simultaneously (triplicated wells/group) with the following potential modulators: excess unlabeled GH (1 mg/ml), 500 mM monodansylcadeverine (an inhibitor of adsorptive endocytosis), 300 mM protamine, 300 mM poly-L-lysine (both being positively charged proteins that also inhibit adsorptive endocytosis), and hypertonic sucrose (1.2 M). Equal numbers of immortalized rat brain microvessel endothelial cells RBE4 (kind gift from NeuroTech, Paris, France) were seeded to six-well plates precoated with rat tail collagen. Radiotracer uptake assays were performed when the cells grew confluent. The cells were preequilibrated for 15 min in 1 ml of transport buffer that contained equal amounts of MEM and F10, supplemented with 20 mM HEPES and 0.05% BSA. 125I-GH (700,000 cpm/ml) was added in 1 ml of transport buffer prewarmed to 37 C, and the plates were gently agitated for 20 min. At this time, internalization of 125I-GH was terminated by transferring the plates to ice, followed by rapid removal of the transport buffer and three washes with ice-cold PBS. Afterwards, specific cell surface binding was determined by incubation of the cells with ice-cold stop-strip buffer [0.2 N acetic acid in PBS (pH 2.5)] for 10 min and collection of this and subsequent washes with stop-strip buffer. The cells were then lysed and collected. The maximal potential internalization represents the amount of radioactivity from the acid-resistant binding combined with the amount internalized at the same time point. The percent of surface binding and maximal potential internalization were determined by normalization of the values of individual wells to the total amount of 125I-GH added at time 0.
Separate groups were studied at 0 C to inhibit endocytosis and compared with groups studied at 37 C. For the 0 C groups, the plates were kept on ice the entire time. Group means were expressed with their SEs, and statistically significant changes were determined by an overall ANOVA for each fraction, followed by Tukey’s post hoc test.
In contrast to continuous uptake of 125I-GH for 20 min, the radiotracer pulse-chasing study was performed to determine the internalization at 20 min after binding equilibrium. The control group with 125I-GH only was studied simultaneously with the following five groups: 1 mg/ml of unlabeled GH, 300 mM of protamine, both protamine and unlabeled GH, 50 μg/ml of heparin, and 300 mM of poly-L-lysine. After equilibration of cells in ice-cold transport buffer for 15 min, the cells were incubated in ice-cold transport buffer containing 125I-GH with or without the above potential modulators (700,000 cpm/ml) at 4 C for 1 h. Afterwards the radioactively labeled tracer was removed by two washes with ice-cold PBS, and the plates were rapidly warmed up to 37 C in the presence of prewarmed transport buffer. At 20 min, surface binding and maximal potential internalization were determined as above.
Results
Influx transfer of 125I-GH from blood to brain
The blood-to-brain influx of 125I-GH was linear during the study period (1–30 min after iv injection in mice). As shown in Fig. 1A, the influx transfer constant was 0.23 ± 0.07 μl/g·min and the initial volume of distribution was 15.5 μl/g. Of the total amount injected iv, the percent of brain uptake was about 0.1%/gram of brain at 20 min. Given the specific activity of 200 Ci/g of 125I-GH and the injection amount of 1.36 μCi, about 6.8 pg of GH was present in a gram of brain tissue. The results indicated that a significant amount of 125I-GH reached the brain, and this was supported by results from degradation studies showing that most of the measured radioactivity represented intact GH (see below). Addition of 1 μg/mouse of unlabeled GH (about 150-fold excess) to the 125I-GH injection solution did not significantly affect the rate of entry. The lack of inhibitory effect of unlabeled GH was reproduced in subsequent experiments with various doses of GH and shorter time intervals. The serum half-life of 125I-GH ranged from 3.8–7.6 min in different experiments.
To further determine the possible presence of a saturable transport system as well as modulation by insulin, which affects GH, three groups of mice were studied (n = 6/group). At 10 min after iv injection of 125I-GH, the volume of distribution of 125I-GH in the brain was 14.80 ± 0.80 μl/g. It was not significantly changed by the addition of 200 μg/mouse (about 30,000-fold excess) of unlabeled GH (Vi = 15.50 ± 0.95 μl/g) or 10 μg/mouse of unlabeled insulin (Vi = 14.20 ± 0.22 μl/g). This is shown in Fig. 1B. Similarly, the influx transfer constant of 125I-insulin, which was 0.97 ± 0.14 μl/g·min, was not significantly changed by the presence of 10 μg/mouse of unlabeled GH (Ki = 0.71 ± 0.18 μl/g·min).
A further study was performed with radioactively labeled rat GH injected iv into rats. The influx transfer constant to the whole brain was 0.32 ± 0.04 μl/g·min, and the initial volume of distribution was 19.27 ± 0.74 μl/g. Addition of 80 μg/rat of excess unlabeled GH did not cause a significant change in the influx rate [F (1, 19) = 0.6, P > 0.05] (Fig. 1C). There was no significant regional difference in the influx transfer constant of 125I-GH among the dissected brain regions (frontal, parietal, occipital cortices, striatum, thalamus, hypothalamus, brainstem, and cerebellum). In each region, there was no significant effect of excess unlabeled GH that would have indicated saturable transport.
Degradation assays to determine that the radioactivity measured represented intact 125I-GH
After 20 min of iv circulation, HPLC showed that intact 125I-GH accounted for 91% of the total radioactivity in serum, similar to that in the serum of the processing control in which 125I-GH was mixed with blood in the test tube only. In serum samples, up to 90% in mouse 20 min after iv injection of 125I-GH and 95% of radioactivity in the processing control were acid precipitable. In the supernatant of brain homogenate, up to 81% in mouse 20 min after iv injection of 125I-GH and 94% in the processing control were also acid precipitable. Similarly, the radioactivity in the brain after 10 min of in situ brain perfusion mainly represented intact 125I-GH. The acid precipitable radioactivity was 90% in brain after 10 min of in situ perfusion of 125I-GH, and 89.4% in brain after perfusion of 125I-GH along with excess unlabeled GH. The results indicate that intact 125I-GH was present in the brain after iv delivery. This is further supported by the presence of a radioactive band at molecular mass of 22 kDa in gel autoradiography in all samples, correlating with intact 125I-GH.
In situ brain perfusion studies to determine the transfer of 125I-GH across the BBB
To eliminate the possibility that serum GH binding proteins interfered with detection of a saturable transport system, and to confirm that the radioactivity measured in the whole brain of mice after iv injection of 125I-GH had reached the brain compartment rather than remaining in the vasculature, in situ brain perfusion was performed with serum-free buffer. The control group received 125I-GH and 131I-albumin whereas the experimental group received 20 μg/ml of unlabeled GH in addition. The influx transfer constant of 125I-GH was 0.84 ± 0.08 μl/g·min without excess GH and 0.82 ± 0.10 μl/g·min in the presence of excess GH; there was no significant difference between the two groups (Fig. 2). Therefore, the lack of saturation was not explained by the presence of serum binding proteins. This further supports the conclusion that the substantial entry of rat GH across the mouse BBB did not occur by a saturable transport system.
Differentiation of the amount of 125I-GH taken up by the brain vs. that trapped in the cerebral vasculature
As shown in Fig. 3, the nonperfused brain had an apparently high volume of distribution of 125I-GH when the brain was obtained 10 min after iv injection (n = 4 /group). This reflects high uptake in the brain parenchymal fraction (12.60 ± 0.61 μl/g) and low uptake in the capillary fraction (3.63 ± 0.76 μl/g). However, the vascular marker 131I-albumin had a parenchymal uptake of 6.77 ± 0.50 μl/g and capillary uptake of 0.75 ± 0.08 μl/g. These values roughly represent the radioactivity that is either in the capillary lumen or loosely bound to the capillary walls because albumin is considered to have minimal transcellular permeation of an intact BBB. Thus, the actual uptake of 125I-GH into the brain parenchyma, as shown after perfusion to wash out the vascular space, was only 4.35 ± 0.32 μl/g, or 26.8% of the total radioactivity measured in the whole brain. The actual amount of 125I-GH trapped in the cerebral vasculature was 1.63 ± 0.39 μl/g at 10 min, or 10% of the total radioactivity. This is significantly less than that reaching the brain parenchyma [F (1, 8) = 117.5, P < 0.001].
Uptake of 125I-GH by RBE4 cells and its modulation by protamine and poly-L-lysine
The concentration of 125I-GH in the transport buffer (700,000 cpm/ml, with specific activity of 200 Ci/g) was about 1.6 ng/ml. Therefore, 1 mg/ml of excess unlabeled GH represented over 60,000 times excess. At this dose, there was no significant inhibition of either surface binding or internalization of 125I-GH. Thus, there was no saturable transport system for GH in the cultured RBE4 cells. To further rule out adsorptive endocytosis, which can occur with glycoproteins and has a lower affinity and higher capacity than receptor-mediated endocytosis, specific inhibitors for adsorptive endocytosis (monodansylcadeverine, poly-L-lysine, and protamine)were also included in the study. Monodansylcadeverine had no significant effect on surface binding or internalization of 125I-GH after 20 min of coincubation. Unexpectedly, the percent of 125I-GH at the cell surface at 20 min was significantly increased in the presence of protamine (P < 0.05), whereas that internalized was also significantly increased by both protamine and poly-L-lysine (P < 0.01). Hypertonic buffer significantly decreased (P < 0.05) surface binding without affecting maximal potential internalization. Both binding and internalization were significantly decreased in low temperature at this time (P < 0.01). The lack of inhibitory effects of protamine, poly-L-lysine, and monodansylcadeverine ruled out the possibility of adsorptive endocytosis (Fig. 4A).
The significantly less binding of 125I-GH at 4 C in contrast to that at 37 C suggests that binding equilibrium had not been achieved during the incubation period. Thus, a further study was performed by pulse-chasing, in which the cells were incubated with 125I-GH at 4 C for 1 h and then internalization of the bound 125I-GH at 20 min was determined. Similar to what was seen in Fig. 4A, the surface binding and internalization were both significantly increased by protamine (P < 0.01). This increase was not affected by addition of excess unlabeled GH. In contrast, poly-L-lysine and heparin showed no significant effects (Fig. 4B).
Discussion
It is well known that GH in the peripheral circulation can exert multiple CNS effects. The recovery of GH in the CSF (12) suggested that GH crosses the BBB. Here we provide the first direct evidence that recombinant, nonglycosylated human and rat GH could be stable enough in the blood and cerebral microcirculation to permeate the BBB of mouse and rat. With a sensitive radiotracer uptake method, we showed that the polypeptide GH had an influx transfer constant of 0.23 ± 0.07 μl/g·min in mice and 0.32 ± 0.04 μl/g·min in rats, a value comparable to that of some neurotropins and cytokines, polypeptides known to cross the BBB (7, 22, 23, 24, 25). HPLC, acid precipitation, and gel autoradiography confirmed that the radioactivity measured in the brain was mainly intact 125I-GH rather than products of degradation.
GH had a moderately high initial volume of distribution in the brain. The majority of the peripherally injected 125I-GH was retained in the cerebral vasculature 10 min after iv injection, although a significant amount (26.8%) entered brain parenchyma, whereas 10% was retained in the microvessel endothelial cells composing the BBB. Consistent with the capillary depletion studies, in situ brain perfusion showed that the perfusate-to-brain transfer of 125I-GH was about 35 times lower than that of nerve growth factor (22) and about 250 times lower than that of brain-derived neurotrophic factor (26). Similar to what we have seen for other proteins, the primary structure or the presence of receptors may not be the determining factor for BBB permeability. Regardless, a significant amount of GH can cross the BBB to exert CNS effects directly.
The permeation of GH, however, is a nonsaturable process, as shown both in vivo and in vitro. In studies with cultured cerebral microvessel endothelial cells (major components of the BBB), surface binding of 125I-GH did not seem to be mediated by specific GH receptors because excess unlabeled GH failed to inhibit the binding or internalization of 125I-GH. Although it is possible that glycosylated GH might be internalized by adsorptive endocytosis, modulators of adsorptive endocytosis (dansylcadeverine, protamine, and poly-L-lysine) failed to decrease the uptake of nonglycosylated GH in this study, thus ruling out the presence of adsorptive endocytosis (27) for this commonly used form of the hormone. The enhancing effect of protamine might be explained by its binding to GH, thereby increasing its positive charge so as to facilitate adsorptive endocytosis, as it does for albumin (28). However, adsorptive endocytosis is usually a specific process, but the uptake of 125I-GH was not saturable by coadministration of excess unlabeled GH. Nonetheless, naturally occurring GH may be glycosylated, which increases its chance of adsorptive endocytosis. Endogenous GH can exist in different forms due to posttranslational modifications, including deamidation, acylation, glycosylation, and oligomerization, thereby resulting in distinct kinetic patterns of clearance, receptor binding, and complexing with binding proteins (29). Although there do not seem to be major differences in biological activity among these forms (30), it is possible that different forms of GH penetrate the BBB in different ways.
The plasma concentration of GH has a distinctive circadian rhythm and ranges from 0–250 ng/ml. Therefore, although the trace amount of I-GH would not increase the blood level of GH, the excess exogenous GH injected to test for saturation might affect the concentration of GH and the subsequent mediators of its function. This could be an issue for long-term effects in survival studies. Nonetheless, it should not affect the conclusions from the acute in vivo studies.
Based on the lack of inhibition by a large excess of GH in the in vivo and in vitro experiments, we conclude that it is unlikely the GH receptor or other carrier systems are involved in the blood-to-brain transfer of GH. This means that the permeation of GH probably occurs by the process of simple diffusion dependent on the physiochemical properties of GH. In pathological conditions in which the BBB is partially disrupted, such as CNS trauma, hypoxia/ischemia, tumor, and certain developmental or degenerative stages, more GH could reach its brain targets than by a process limited by saturation. The permeation of peripheral GH across the BBB does not override the importance of neuronal sources of GH, such as in the hypothalamic region of rats (31) and retina of embryonic chicks (32), which play important roles in neuromodulation.
It has been suggested that peripheral GH can be detected in the brain and CSF; however, there has not been direct demonstration whether and how the BBB is involved. Because the choroid plexus has a high level of expression of GH receptors, receptor-mediated transport is possible there. Despite the theoretical possibility that the blood-CSF barrier may possess a specific transport system, our pharmacokinetic study on mice and rats ruled out receptor-mediated transport for GH at the BBB. Results from cultured cerebral microvessel endothelial cells also ruled out high-capacity adsorptive endocytosis. Despite the lack of a specific transport system, GH was relatively stable in blood within the first 10 min after iv delivery and underwent limited simple diffusion across the BBB. The majority of GH entering the cerebral circulation was retained in the cerebral vasculature, but significant penetration to brain parenchyma did occur. These studies add definitive value to the long dispute whether and how GH crosses the BBB.
Footnotes
This work was supported by the National Institutes of Health [NS45751 and NS46528 (to W.P.) and DK54880 and AA12865 (to A.J.K.)].
Abbreviations: BBB, Blood-brain barrier; CNS, central nervous system; CSF, cerebrospinal fluid; Ki, unidirectional influx rate; LR, lactated Ringer’s; Vi, initial volume of distribution.
References
Banks WA, Kastin AJ, Durham DA 1989 Bidirectional transport of interleukin-1 across the blood-brain barrier. Brain Res Bull 23:433–437
Banks WA, Ortiz L, Plotkin SR, Kastin AJ 1991 Human interleukin (IL)1, murine IL-1 and murine IL-1 are transported from blood to brain in the mouse by a shared saturable mechanism. J Pharmacol Exp Ther 259:988–996
Banks WA, Kastin AJ, Gutierrez EG 1994 Penetration of interleukin-6 across the murine blood-brain barrier. Neurosci Lett 179:53–56
Banks WA, Kastin AJ, Ehrensing CA 1994 Blood-borne interleukin-1 is transported across the endothelial blood-spinal cord barrier in mice. J Physiol 479:257–264
Gutierrez EG, Banks WA, Kastin AJ 1993 Murine tumor necrosis factor is transported from blood to brain in the mouse. J Neuroimmunol 47:169–176
Pan W, Banks WA, Kastin AJ 1997 Permeability of the blood-brain and blood-spinal cord barriers to interferons. J Neuroimmunol 76:105–111
Pan W, Kastin AJ 1999 Penetration of neurotrophins and cytokines across the blood-brain/blood-spinal cord barrier. Adv Drug Deliv Rev 36:291–298
Nyberg F 2000 Growth hormone in the brain: characteristics of specific brain targets for the hormone and their functional significance. Front Neuroendocrinol 21:330–348
Burman P, Broman JE, Hetta J, Wiklund I, Erfurth EM, Hagg E, Karlsson FA 1995 Quality-of-life in adults with growth-hormone (GH) deficiency—response to treatment with recombinant human GH in a placebo-controlled 21-month trial. J Clin Endocrinol Metab 80:3585–3590
Johansson JO, Larsson G, Elmgren A, Hynsj L, Lindahl A, Lundberg PA, Isaksson O, Lindstedt S, Bengtsson B 1995 Treatment of growth hormone-deficient adults with recombinant human growth hormone increases the concentration of growth hormone in the cerebrospinal fluid and affects neurotransmitters. Neuroendocrinology 61:57–66
Nyberg F, Burman P 1996 Growth hormone and its receptors in the central nervous system—location and functional significance. Horm Res 45:18–22
Coculescu M 1999 Blood-brain barrier for human growth hormone and insulin-like growth factor-I. J Pediatr Endocrinol Metab 12:113–124
Lai ZN, Emtner M, Roos P, Nyberg F 1991 Characterization of putative growth-hormone receptors in human choroid-plexus. Brain Res 546:222–226
Kastrup Y, Le Greves M, Nyberg F, Blomqvist A 2005 Distribution of growth hormone receptor mRNA in the brain stem and spinal cord of the rat. Neuroscience 130:419–425
Le Greves M, Steensland P, Le Greves P, Nyberg F 2002 Growth hormone induces age-dependent alteration in the expression of hippocampal growth hormone receptor and N-methyl-D-aspartate receptor subunits gene transcripts in male rats. Proc Natl Acad Sci USA 99:7119–7123
Hanci M, Kuday C, Oguzoglu SA 1994 The effects of synthetic growth hormone on spinal cord injury. J Neurosurg Sci 38:43–49
Winkler T, Sharma HS, Stalberg E, Badgaiyan RD, Westman J, Nyberg F 2000 Growth hormone attenuates alterations in spinal cord evoked potentials and cell injury following trauma to the rat spinal cord. An experimental study using topical application of rat growth hormone. Amino Acids 19:363–371
Gustafson K, Hagberg H, Bengtsson BA, Brantsing C, Isgaard J 1999 Possible protective role of growth hormone in hypoxia-ischemia in neonatal rats. Pediatr Res 45:318–323
Mustafa A, Sharma HS, Olsson Y, Gordh T, Thoren P, Sjoquist PO, Roos P, Adem A, Nyberg F 1995 Vascular-permeability to growth-hormone in the rat central-nervous-system after focal spinal-cord injury—influence of a new antioxidant H-290/51 and age. Neurosci Res 23:185–194
Wang X, Day JR, Zhou Y, Beard JL, Vasilatos-Younken R 2000 Evidence of a role for neuropeptide Y and monoamines in mediating the appetite-suppressive effect of GH. J Endocrinol 166:621–630
Kastin AJ, Akerstrom V, Pan W 2001 Validity of multiple-time regression analysis in measurement of tritiated and iodinated leptin crossing the blood-brain barrier: meaningful controls. Peptides 22:2127–2136
Pan W, Banks WA, Kastin AJ 1998 Permeability of the blood-brain barrier to neurotrophins. Brain Res 788:87–94
Poduslo JF, Curran GL 1996 Permeability at the blood-brain and blood-nerve barriers of the neurotrophic factors: NGF, CNTF, NT-3, BDNF. Mol Brain Res 36:280–286
Pan W, Kastin AJ, Brennan JM 2000 Saturable entry of leukemia inhibitory factor from blood to the central nervous system. J Neuroimmunol 106:172–180
Pan W, Kastin AJ 2003 Transport of cytokines and neurotrophins across the BBB and their regulation after spinal cord injury. In: Shanker H, Westman J, eds. Blood-spinal cord and brain barriers in health and disease. San Diego: Academic Press; 395–407
Pan W, Banks WA, Fasold MB, Bluth J, Kastin AJ 1998 Transport of brain-derived neurotrophic factor across the blood-brain barrier. Neuropharmacology 37:1553–1561
Deguchi Y, Okutsu H, Okura T, Yamada S, Kimura R, Yuge T, Furukawa A, Morimoto K, Tachikawa M, Ohtsuki S, Hosoya K, Terasaki T 2002 Internalization of basic fibroblast growth factor at the mouse blood-brain barrier involves perlecan, a heparan sulfate proteoglycan. J Neurochem 83:381–389
Pardridge WM, Buciak JL, Kang Y-S, Boado RJ 1993 Protamine-mediated transport of albumin into brain and other organs of the rat. J Clin Invest 92:2224–2229
Baumann G 1991 Growth hormone heterogeneity: genes, isohormones, variants, and binding proteins. Endocr Rev 12:424–449
Berghman LR, Buyse J, Huybrechts LM, Darras VM, Vandesande F, Kuhn ER, Decuypere E, Scanes CG 1994 Disappearance rate of glycosylated and non-glycosylated chicken growth hormone: influence on biological activity. Comp Biochem Physiol Pharmacol Toxicol Endocrinol 108:161–169
Lechan RM, Nestler JL, Molitch ME 1981 Immunohistochemical identification of a novel substance with human growth hormone-like immunoreactivity in rat brain. Endocrinology 109:1950–1962
Harvey S, Kakebeeke M, Sanders EJ 2004 Growth hormone localization in the neural retina and retinal pigmented epithelium of embryonic chicks. J Mol Neurosci 22:139–145(Weihong Pan, Yongmei Yu, )