Nonsaturable entry of neuropeptide Y into brain

Abba J. Kastin and Victoria Akerstrom

Veterans Affairs Medical Center and Tulane University School of Medicine, New Orleans, Louisiana 70112-1262


    ABSTRACT
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Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

Neuropeptide Y (NPY) is found and is active both in the periphery and brain, but its crossing of the blood-brain barrier (BBB) in either direction has not been measured. We used multiple time-regression analysis to determine that radioactively labeled NPY injected intravenously entered the brain much faster than albumin, with an influx constant of 2.0 × 10-4 ml · g · -1 · min-1. However, this rate of entry was not significantly changed by injection of 10 µg/mouse of excess NPY, by leptin, or by food deprivation. HPLC showed that most of the NPY entering the brain was intact, and capillary depletion with and without washout showed that the NPY did not remain bound to endothelial cells or associated with vascular elements. Perfusion in a blood-free solution eliminated binding to serum proteins as an explanation for the lack of saturation. Efflux of labeled NPY from the brain occurred at the same rate as albumin, reflecting the normal rate of reabsorption of cerebrospinal fluid. Thus NPY can readily enter the brain from blood by diffusion across the BBB.

neuropeptide Y; blood-brain barrier; leptin; peptides


    INTRODUCTION
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Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

ALTHOUGH MANY PEPTIDES affecting ingestion of food cause decreased intake, neuropeptide Y (NPY) dramatically increases eating (1, 13, 16). Hypoglycemia results in increased NPY levels in plasma (17), but whether NPY in the blood reaches the brain has not been determined.

Entry into the brain of many blood-borne peptides has been studied by sensitive methods permitting determination of the self-inhibition required to show saturability of transport. Blood-to-brain penetration of the blood-brain barrier (BBB) by some substances involved in ingestive behavior, such as insulin (3, 11, 15), leptin (9), and pancreatic polypeptide (7), occurs by saturable transport systems, whereas entry of other ingestive substances, such as amylin (10) and cyclo(His-Pro) (8), is not saturable. Yet the brain takes up a larger percentage of a peripherally injected dose of the nonsaturably transported amylin than of the saturably transported insulin (4).

In this study, we quantified the rate of entry of 125I-NPY into the brain and determined whether it showed saturation with unlabeled NPY and was affected by fasting. HPLC was used to determine the integrity of the peptide reaching the brain, capillary depletion with washout was used to determine any confounding association with the vasculature, and 99mTc-albumin was used to determine vascular changes that might alter penetration of the BBB. Perfusion in blood- free solution and efflux from the brain were also tested.


    METHODS
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Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

Multiple time-regression analysis of entry into the brain. Adult male albino ICR mice (Charles River, Wilmington, MA; 10 mice/group), each weighing ~22 g, were anesthetized with urethan (4 g/kg ip). NPY was radiolabeled with 125I by chloramine T and purified on a column of Sephadex G-10. Acid precipitation showed incorporation of 125I into NPY of 95-98% for the iodinations used in these studies. HPLC of the 125I-NPY immediately before use showed ~90% purity each time.

The 125I-NPY was injected in a dose of 0.36 µCi/mouse (3.94 pmol/mouse) through the isolated left jugular vein along with 3 µCi/mouse of 99mTc-albumin in 200 µl of lactated Ringer solution containing 1% albumin (LR-BSA). At 2, 3, 4, 5, 6, 8, 10, 20, and 30 min after intravenous injection, blood was collected from a cut in the right carotid artery, and the mouse was immediately decapitated. Serum and brain samples were obtained and counted in a dual-channel gamma counter. The ratio of the radioactivity of brain tissue to that of serum was calculated, and multiple time-regression analysis was applied to study the relationship between the ratios and exposure time. In the study involving fasting, food was removed for 48 h while water remained freely available.

Exposure time is the theoretical steady-state value of circulation time after the decay of the NPY in blood is corrected. Because NPY has slow penetration into the brain, unaffected by cerebral blood flow, a linear relationship was present between the tissue-to-serum radioactivity ratio and exposure time. The slope of this regression line represents the influx rate (Ki) of NPY. To determine whether the entry of 125I-NPY is saturable, self-inhibition was tested by addition of unlabeled NPY (10 µg/mouse) in the injected solution (6). Leptin was tested at a dose of 10 µg/mouse.

HPLC of NPY in blood and brain after 30-min circulation in the blood. Brain and blood samples were obtained 30 min after intravenous injection of 125I-NPY. The pooled brain samples were homogenized in phosphate-buffered saline with a glass homogenizer. After centrifugation, the supernatant was lyophilized and rehydrated 10 min before elution on a reversed-phase C18 column. The gradient consisted of 25% acetonitrile in water with 0.1% trifluoroacetic acid increasing to 65% over 40 min. Flow rate on the HPLC was 1 ml/min, and 1-ml fractions were collected. The values in blood and brain of mice injected 30 min earlier with 125I-NPY and 99mTc-albumin were corrected for processing as determined by addition of 125I-NPY to blood and brain samples of uninjected mice.

Capillary depletion with and without perfusion to determine the compartmental distribution of NPY. The capillary depletion method was used to separate cerebral capillaries from the parenchyma. In addition, the NPY remaining in the vascular space was identified by application of an intracardial perfusion method. Each mouse received an intravenous injection of 125I-NPY and 99mTc-albumin in 200 µl of LR-BSA at time 0. At 10 min, blood was collected from the abdominal aorta. Then the four mice in the washout group were perfused intracardially with 20 ml of LR solution within 30 s while the descending aorta was blocked and bilateral jugular veins were severed. The four mice in the nonwashout group received no perfusion. The mice were decapitated, and brain samples were collected.

The cerebral cortex was homogenized in a glass homogenizer in physiological buffer and mixed thoroughly with 26% dextran to yield a final concentration of dextran slightly over 15.5%, which is necessary for proper separation of the vasculature from the brain parenchyma. An aliquot of the homogenate was centrifuged at 5,400 g for 15 min at 4°C. The pellet, containing the capillaries, and the supernatant, representing the brain parenchymal-interstitial fluid space, were carefully separated; the percentage of contamination of the supernatant by the vasculature was assessed by measurement of the specific activity of the vascular enzyme marker gamma -glutamyl transpeptidase (kit from Sigma, St. Louis, MO); and protein levels were assayed (Bio-Rad, Richmond, CA).

The ratios of radioactivity of 125I-NPY in the supernatant (parenchyma) or pellet (capillary) to that in serum, corrected by subtraction of 99mTc-to-albumin ratios of radioactivity representing vascular space, were used to determine the compartmental distributions: the reversible capillary binding, the NPY retained in endothelial cells, and the NPY crossing the BBB to arrive at the parenchyma of the brain tissue.

Perfusion in a blood-free solution. 125I-NPY and 99mTc-albumin were added to buffer by the method of Zlokovic et al. (18) modified for use in mice (14). Another group of mice received unlabeled NPY, which was added to the perfusate at a concentration of 5.0 µg/ml. This was perfused through the left ventricle of the heart at a rate of 2 ml/min up to 5 min in anesthetized mice in which the thoracic aorta had been clamped and both jugular veins had been severed immediately before the perfusion. A 20-ml wash followed. The rate of entry was calculated from the slope of the regression line plotted for the brain-to-perfusate ratio of radioactivity vs. time (14).

Efflux of NPY from the brain. About 25,000 cpm of both 125I-NPY and 99mTc-albumin were simultaneously injected into the brain of mice anesthetized with urethan at a site 1 mm lateral and 1 mm posterior to the bregma through a 26-gauge needle, with all but the last 3.5 mm covered with polyethylene tubing (2). Mice were studied (n = 5/group) at 0, 2, 5, 10, and 20 min after injection. The 0-min value was determined in mice overdosed with anesthesia before injection, as previously explained (5). The half-time of disappearance was determined from the regression line obtained from the plot of the logarithm of brain radioactivity against time.


    RESULTS
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Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

Entry into brain. As shown in Fig. 1, the unidirectional rate of entry (Ki) of 125I-NPY was 1.94 × 10-4 ml · g-1 · min-1. Inclusion of 10 µg per mouse of unlabeled NPY in the intravenous injection did not significantly reduce the Ki for 125I-NPY (1.88 × 10-4 ml · g-1 · min-1). Leptin, 10 µg/mouse, also did not affect the entry rate of 125I-NPY. There was no substantial entry of 99mTc-albumin into the brain, and its penetration was not affected by the addition of 10 µg NPY in the intravenous injection (Fig. 1).


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Fig. 1.   Blood-to-brain influx of 125I-neuropeptide Y (I-NPY) and 99mTc-albumin (Alb) with addition of 10 µg/mouse of unlabeled NPY plotted against exposure time (t). Entry of 125I-NPY was faster than that of 99mTc-albumin but was not significantly inhibited by excess NPY, indicating a lack of saturation. , I-NPY; , I-NPY + NPY; down-triangle, Alb; black-down-triangle , Alb + NPY.

In the 10 fasted mice weighing 20.6 ± 0.5 g, the Ki was 1.69 × 10-4 ml · g-1 · min-1 compared with a Ki in the 10 fed mice weighing 27.2 ± 0.8 g of 1.92 × 10-4 ml · g-1 · min-1, a nonsignificant difference (Fig. 2). Again, entry of 125I-NPY was much faster than that of the 99mTc-albumin.


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Fig. 2.   Blood-to-brain influx of 125I-NPY and 99mTc-albumin (Alb) plotted against exposure time (t) in mice fasted for 48 h and in fed controls. Entry of 125I-NPY was faster than that of 99mTc-albumin, but fasting did not affect entry into brain. , I-NPY; , I-NPY + NPY; down-triangle, Alb; black-down-triangle , Alb + NPY.

With inclusion of the preliminary studies involving smaller doses of unlabeled NPY and a shorter time of food deprivation, a total of four experiments was performed in which the Ki of 125I-NPY was determined. The resulting mean Ki was 1.98 ± 0.11 × 10-4 ml · g · -1 · min-1.

HPLC for 125I-NPY. The percentage of recovered radioactive material eluting at the same position as the NPY standard was determined in serum and brain of the processed control samples and in the serum and brain samples obtained 30 min after intravenous injection that were processed identically. This comparison revealed essentially no decrease in the percentage of total radioactivity that represented intact 125I-NPY in the sample of serum obtained at 30 min and only a 10% decrease in the corresponding brain sample.

Capillary depletion with washout. At 10 min after intravenous injection of 125I-NPY, the parenchyma-to-serum ratio was much higher than the capillary-to-serum ratio, regardless of whether cardiac perfusion was used to remove any vascular binding. Figure 3 shows the tissue-to-serum ratios after subtraction of the values for the coadministered 99mTc-albumin that represent the volume of the vascular space. The value for the total, nonperfused cortex is composed of the three components shown in Fig. 3. Uptake by brain parenchyma was 13 times greater than entrapment by the capillaries and 5 times greater than any NPY loosely associated with the endothelial cells of the cerebral vasculature or with circulating cellular elements as determined by washout of the vascular space.


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Fig. 3.   Tissue-to-serum ratios (means ± SE) for mice injected with 125I-NPY corrected for simultaneously injected 99mTc-albumin. Values for cortex are composed of 3 components: 1) vascular: loose and reversible association of 125I-NPY with endothelial cells of cerebral vasculature and any associated circulating cellular elements; 2) capillaries: binding or entrapment of 125I-NPY by capillary endothelial cells; and 3) parenchyma: 125I-NPY in brain parenchyma.

Perfusion in blood-free buffer. To eliminate the possibility that the lack of self-inhibition in the in vivo study was caused by interactions of 125I-NPY with blood components, in situ perfusion of 125I-NPY in physiological buffer was used. When the ratio of radioactivity of brain over perfusate was plotted against perfusion time, the slopes of the regression lines did not show a significant difference between the groups with and without the added unlabeled NPY. The results, corrected for albumin, are shown in Fig. 4.


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Fig. 4.   Blood-to-brain influx of 125I-NPY (I-NPY, ) by in situ perfusion plotted against time (t) in blood-free buffer with addition of 5 µg/ml of unlabeled NPY (I-NPY + NPY, ). This was corrected for simultaneously injected 99mTc-albumin that reflects cerebral vascular space and any leakage across a potentially disrupted blood-brain barrier. No saturation was observed.

Efflux from brain. The half-time of disappearance of 125I-NPY from brain was 23.3 min. The half-time of disappearance of 99mTc-albumin from brain was 23.1 min. As seen in Fig. 5, these were not significantly different.


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Fig. 5.   Efflux of 125I-NPY (I-NPY; ) and 99mTc-albumin (Alb; ) from brain after central injection. Data are means ± SE. Their rates of disappearance were the same. cpm, Counts/min.


    DISCUSSION
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Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

The results show that NPY entered the brain of mice significantly faster than did the albumin control. The Ki of 2.0 × 10-4 ml · g · -1 · min-1 is in the general range seen with several nonsaturable and even some saturable systems for peptides and polypeptides.

Cyclo(His-Pro), a peptide involved in food ingestion, also enters the brain by a nonsaturable mechanism. Even though a slightly slower Ki has been found for cyclo(His-Pro) than for NPY, the penetration of the BBB by cyclo(His-Pro) is sufficient to reverse ethanol-induced narcosis (8). Thus a substance need not be transported across the BBB by a saturable system to exert a biological effect.

The nonsaturable entry of NPY, however, was slower than that of the saturable transport of some ingestive substances of larger molecular size than NPY, such as leptin (9). A substance with high blood concentrations might be expected to have a high-capacity transport system requiring larger amounts than we tested to show saturation. A comparison with leptin, however, makes this an unlikely explanation for our inability to find a saturable transport system into the brain for NPY: the dose of NPY we used to assess self-inhibition in the intravenous bolus injection was 10 times larger than that used for leptin (9), and circulating leptin levels are higher than those of NPY (12). Leptin did not affect the entry of NPY. In situ brain perfusion of NPY in physiological buffer also did not show saturation, removing the possibility that interactions of NPY with blood components explained the results.

Radioactively labeled NPY injected peripherally might not be found in brain if there were an efflux system rapidly removing it from the brain. Such a facilitated brain-to-blood transport could make the rate of entry appear artifactually slow. This did not happen with NPY, which was removed from the brain at the same rate as albumin, reflecting the normal rate of reabsorption of cerebrospinal fluid.

Additional methods were used to establish that NPY reached the parenchyma of the brain. Capillary depletion studies, which effectively separate the vascular and parenchymal compartments without cross contamination, showed that the material reached the brain without being bound to the endothelial cells of the capillaries comprising the BBB or to the loosely adherent vascular components. HPLC showed that most of the 125I-NPY reaching the brain remained intact.

Fasting, expected to upregulate the production of NPY in the periphery, did not change the entry of NPY into brain. Although it is possible that increased transport into the brain after fasting might have been obscured by increased saturation of a transport system resulting from elevated blood concentrations, we could not demonstrate a saturable transport system. More likely, it is the simple diffusion of NPY across the BBB that explains its relative ease of entry into the brain from the blood.


    ACKNOWLEDGEMENTS

We thank Dr. Weihong Pan and Melita B. Fasold for editorial assistance.


    FOOTNOTES

This research was supported by the US Dept. of Veterans Affairs and the National Institutes of Health.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: A. J. Kastin, VA Medical Center, 1601 Perdido St., New Orleans, LA 70112-1262.

Received 6 July 1998; accepted in final form 9 November 1998.


    REFERENCES
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Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

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Am J Physiol Endocrinol Metab 276(3):E479-E482