Evidence for the presence of immunoreactive histidyl-proline diketopiperazine [Cyclo (His-Pro)] in the adult human brain

Peptides. Vol. 4, pp. 87%881, 1983. ©AnkhoInternationalInc. Printed in the U.S.A.

Evidence for the Presence of Immunoreactive Histidyl-Proline Diketopiperazine [Cyclo (His-Pro)] in the Adult Human Brain C. R I C H A R D P A R K E R , JR., M A T S A T O M O M O R I , * J O Y C E P E G U E S , * C H A N D A N P R A S A D * A N D J O H N F. W I L B E R *

Cecil H. and Ida Green Center for Reproductive Biology Sciences and the Departments o f Obstetrics and Gynecolog~v and Physiology, University of Texas Southwestern Medical School 5323 Haro, Hines Boulevard, Dallas, TX 75235 *The Department o f Medicine, L S U School of Medicine, New Orleans, LA R e c e i v e d 7 July 1983 PARKER, C. R., JR., M. MORI, J. PEGUES, C. PRASAD AND J. F. WILBER. Evidence fi~r the presence of immunoreactive histidyl-proline diketopiperazine [Cyclo (His-Pro)] in the adult human brain. PEPTIDES 4(6) 87%881, 1983.--The current study was undertaken to evaluate the presence of cyclo (His-Pro) in adult human brain tissues obtained at autopsy. We found evidence for immunoreactive cyclo (His-Pro), which diluted in parallel to the radioimmunoassay standard curve and which had mobility on HPLC that was similar to synthetic cyclo (His-Pro), in several regions of the adult human brain. Whereas the levels of cyclo (His-Pro) in the pituitary stalk-median eminence were high (2.2 ng/mg protein), the concentrations in the whole hypothalamus were much lower (0.105 ng/mg protein). Among the extrahypothalamic brain regions examined, the levels of cyclo (His-Pro) were highest in the cerebellar hemisphere (0.168 ng/mg protein) and olfactory bulbs (0.180 ng/mg protein) and were lowest in the hippocampus (0.080 ng/mg protein) and occipital cortex (0.079 ng/mg protein). Thus, immunoreactive cyclo (His-Pro) has widespread distribution in the adult human brain and the potential exists for this cyclic diepeptide to play a role in human brain function. Cyclo (His-Pro) Human brain

Histidyl-proline diketopiperazine Thyrotropin-releasing hormone Central nervous system Neuropeptide Neurotransmitter

TRH

result of trauma or cardiovascular accident. Brain tissues (1-6 regions per brain) were extracted by homogenization at 4°C in 10 vols of acidified ethanol (1 M acetic acid:ethanol:water, 75:15:10). The extracts were centrifuged at 2,000×g for 10 min at 4°C to sediment debris, and the supernatant fluids were removed and evaporated to dryness under a stream of air. The dried residue was suspended in phosphate buffered saline (0.01 M phosphate in 0.14 M NaC1, pH 7.0) containing 0.1% procine gelatin. The extracts were assayed for cyclo (His-Pro) according to the methods of Mori et al. [8]. Additional aliquots of tissue extracts were assayed for protein by the method of Lowry et al. [6]. To characterize the immunoreactive cyclo (His-Pro) in human brain tissues, we tested for parallelism between serial dilutions of extracts and the assay standard curve. In addition, several brain tissue extracts were pooled, mixed with trace amounts of [3H[cyclo (His-Pro), and subjected to high performance liquid chromatography (HPLC). HPLC was performed using a Waters HPLC and /xBondapak/Cl8 columns: isocratic elution (1 ml/min) was achieved using 22%

CYCLO (His-Pro), or histidyl-proline diketopiperazine, is a cyclic dipeptide that may derive from the metabolism of thyrotropin releasing hormone (TRH). Cyclo (His-Pro) has been found in numerous regions of the brains of experimental animals [9,10], and also has been found to exhibit several biologic properties [1-4, 7, 15-17], some of which are opposite to those of TRH [2, 16, 17]. Recently, several groups of investigators have reported that TRH has widespread distribution within the human central nervous system [5, 12, 13]. Since cyclo (His-Pro) may be a naturally occurring metabolite of TRH, it seemed likely that this cyclic dipeptide also is present in the human brain. The current investigation was undertaken to evaluate this possibility. METHOD Brains of adult humans (n=27; 18--82 years of age) were dissected at the time of autopsy (6--28 hr after death; mean postmortem interval= 14.3 hr). Tissues were obtained only in those instances in which death occurred suddenly as the

1Supported, in part, by NIH Grants AG02295, AG00306, and NS19885.

879

880

PARKER E7 AL. TABLE 1

600

3OO

REGIONAL

400

200

Brain Region

\

g_

LOCALIZATION OF CYCLO (HIS-PRO) P O S T M O R T E M A D U L ] H U M A N BRAIN

IN

THE

cyclo (His-Pro) Concentration* 32 u

ii

<[

g :~

ng/mg protein

4 11 9 6 3 6 4 9 6 4 4 ,+ 2 I I I

2.18 + 1.32 0.105 + 0.014 0.168 ± 0.022 0.180 + 0.038 0.123 + 0.030 0.109 ~ 0.041 0.101 + 0.048 (I.083 + 0.017 [).082 ± 0.030 0.080 + [).016 0.079 + 0.034 0.225 0.475 0.026 0.18() 0.070

ng/mg wet wt

o

o 200

o

N

Xo~i X

'

,.×

....

....

×

400

Y: (D

X "''X

u r~

o

0

4

0 HPLC

6 ELUTION

8

~0

VOL (ML)

FIG. I. Representative HPLC mobility of immunoreactive cyclo (His-Pro) and PH]cyclo (His-Pro). Extracts of several human brain tissues were pooled, mixed with trace amounts of pH]cyclo (HisPro) and evaporated to dryness. The dried residue was suspended in HPLC buffer and chromatographed as detailed in the text. After aliquots of each column fraction were taken for counting radioactivity, fractions 2-4, 5 and 6, and 7 and 8 were pooled, evaporated and then analyzed for content of immunoreactive cyclo (His-Pro). Similar results were obtained when a second pool of brain extracts were processed for HPLC as above. The elution profile of synthetic pHlcyclo (His-Pro), when chromatographed in the absence of tissue extract, was identical to that shown in the presence of brain tissue extracts.

E t O H in 0.01 M a m m o n i u m acetate, p H 4.0 as solvent. Column fractions (1.0 ml) were assayed for radioactivity and certain fractions were pooled and e v a p o r a t e d to dryness. The dried residues were resuspended in assay buffer and analyzed for content of cyclo (His-Pro) immunoreactivity.

Stalk-medianeminence Hypothalamus Cerebellum Olfactory Bulb Temporal Cortex Amygdahl Corpus Striatum Medulla Pons Hippocampus Occipital Cortex Optic Chiasm Mammillary Bodies Thalamus Substantia Nigra Pineal

39.29 + 10.26 _~ 16.01 ~ 13.25 + 11.24 + 9.82 : 6.74 ~ 8.10 + 8.32 + 9.95 + 7.8[) + 14.86 45.8[) 2.22 3 I. 14 4.50

11.[5 2.[)2 2.50 1.97 2.28 3.36 2.59 1.29 2.54 1.57 3.60

*Data are presented as the mean + SE except fiw tissues with less than 3 observalions.

p o c a m p u s and occipital cortex. We found that the concentrations of cyclo (His-Pro) in the hypothalamus as well as in e x t r a h y p o t h a l a m i c brain tissues (olfactory bulbs, cerebellum) were not correlated to the postmortem interval, suggesting that this peptide is stable in the postmortem human brain.

RESULTS

We found i m m u n o r e a c t i v e cyclo (His-Pro) to be present in n u m e r o u s regions of the adult human brain. In all tissues tested, the i m m u n o r e a c t i v e cyclo (His-Pro) in tissue extracts was parallel to the assay standard curve (data not shown). The mobilities during H P L C of [aH[cyclo (His-Pro) and the i m m u n o r e a c t i v e c y c l o (His-Pro) in a pool prepared from extracts of several brain regions were found to be similar (Fig. 1). The H P L C mobility o f p H ] c y c l o (His-Pro), when processed in the absence of tissue, was identical to that shown in Fig. 1. T h e s e results are suggestive that i m m u n o r e a c t i v e cyclo (His-Pro) in human brain tissues is similar to or identical with synthetic cyclo (His-Pro). The regional localization of i m m u n o r e a c t i v e cyclo (HisPro) in the brains of several adult humans is summarized in Table 1. The highest c o n c e n t r a t i o n of cyclo (His-Pro) (2.18_+ 1.32 ng/mg protein; mean_+SE) was found in the pituitary stalk-median e m i n e n c e region. Yet, the concentration o f cyclo (His-Pro) in the whole human hypothalamus, inclusive of the pituitary stalk, was much lower (0.105_+0.014 ng/mg protein). Indeed, the levels of cyclo (His-Pro) in the hypothalamus w e r e a m o n g the lowest of all brain tissues tested. Substantial quantities of this peptide appear to be present in the mammillary bodies and the optic chiasm. A m o n g the e x t r a h y p o t h a l a m i c tissues tested, the concentrations of cyclo (His-Pro) were highest in the olfactory bulbs and cerebellum (anterior cerebellar hemisphere). The lowest levels of cyclo (His-Pro) were contained in the pons, medulla, hip-

DISCUSSION

In the current study, we detected immunoreactive cyclo (His-Pro) in several regions of the adult human brain. Based on its parallelism to the radioimmunoassay standard curve and on its co-migration with synthetic cyclo (His-Pro) during H P L C , we suggest that the i m m u n o r e a c t i v e cyclo (His-Pro) in extracts of adult human brain tissues is similar to the authentic cyclic dipeptide. H o w e v e r , a more complete analysis of the biophysical properties of the immunoreactive substance in brain tissues of the human brain will be required before it can be fully characterized. The i m m u n o r e a c t i v e cyclo (His-Pro) in brain tissue extracts of the rat brain has previously been shown to be similar, if not identical, to the synthetic peptide [8]. In agreement with reports concerning the rat [9] and m o n k e y [10] central nervous system, we found that cyclo (His-Pro) is widely distributed throughout the adult human brain. With the exception of the stalk-median e m i n e n c e region, wherein quite high levels (2.2 ng/mg protein) of cyclo (His-Pro) were found, the mean concentrations of cyclo (His-Pro) were much lower in other brain regions, ranging from 0.026 to 0.475 ng/mg protein. The adult human cerebellum, which contains among the lowest levels of T R H in the human brain [5, 12, 13], was found to have levels of cyclo (His-Pro) that were among the highest of the extrahypothalamic brain regions. In contrast, the levels of cyclo (His-Pro) in the hypothalamus, a brain tissue known to contain high

C Y C L O ( H I S - P R O ) IN H U M A N B R A I N

881

concentrations o f T R H [5, 12, 13], were l o w e r than those in many non-hypothalarnic brain regions. Such a dissociation b e t w e e n cyclo (His-Pro) and T R H also has been noted to obtain in the brains of rats and m o n k e y s [9,10], raising the possibility that cyclo (His-Pro) may not arise as a product of T R H metabolism. The widespread distribution o f cyclo (His-Pro) in the adult human brain, taken together with the d e m o n s t r a t i o n s of various biological properties of cyclo (His-Pro) in the brains of experimental animals [1-4, 7, 15-17], are suggestive that this substance may play a role in the function o f the human brain. Despite the fact that s o m e actions of cyclo (His-Pro) are similar to those of T R H [15], it is unlikely that cyclo (His-Pro) acts via an interaction with the putative T R H r e c e p t o r in humans [14] and experimental animals [11, 17, 18], T h e r e f o r e , independent binding sites for this peptide

probably exist in the pituitary and the central nervous system. It is clear that m a n y questions remain concerning the role of cyclo (His-Pro) in brain function. Further study should resolve many of these issues, including the regulation of production o f cyclo (His-Pro), the sites and m e c h a n i s m s o f its action, and the factors contributing to the divergence in the relative distribution of cyclo (His-Pro) and its putative precursor, T R H , in the central nervous system.

ACKNOWLEDGEMENTS The authors thank Sylvia Williams and Kristine Drake for excellent editorial assistance and Antonieta Capdevila for technical help in this study.

REFERENCES

I. Battaini, F. and A. Peterkofsky. Histidyl-proline diketopiperazine, and endogenous brain peptide that inhibits (Na+-K)-ATPase. Biochem Biophys Res Commtttl 94: 240--247, 1980. 2. Bauer, K., J. K. Graf, A. Faivre-Bauman, S. Beier, A. TixierVidal and H. Kleinhauf. Inhibition of prolactin secretion by histidyl-proline diketopiperazine. Nature 274: 174-175, 1978. 3. Bhargava, H. N. Inhibition of abstinence syndrome in opiate dependent mice by cyclo (His-Pro). Ltilb Sci 28: 1261-1267, 1981. 4. Jacobs, J. J., C. Prasad and J. F. Wilber. Cyclo (His-Pro~: Mapping hypothalamic sites for its hypothermic action. Brain Res 250: 205-209, 1982. 5. Kubek, M. J., M. A. Lorincz and J. F. Wilber. The indentification of thyrotropin releasing hormone (TRH) in hypothalamic and extrahypothalamic loci of the human nervous system. Brain Res 126: 196-200, 1977. 6. Lowry. O. H., N. J. Rosenbrough, A. L. Farr and R. J. Randall. Protein measurement with the folin phenol reagent. J Biol ('hem 193: 265-275, 1951. 7. Morley, J. E., A. S. Levine and C. Prasad. Histidyl-proline diketopiperazine decreases food intake in rats. Brain Res 210: 475-478, 1981. 8. Mori. M., C. Prasad and J. F. Wilber. Specific radioimmunoassay of cyclo (His-Pro), a biologically active metabolite of thyrotropin-releasing hormone. Endocrinology 108: 1995-1997, 1981. 9. Mori, M., C. Prasad and J. F. Wilber. Regional dissociation of histidyl-proline diketopiperazine [Cyclo (His-Pro)] and thyrotropin-releasing hormone (TRH) in the rat brain. Brain Res 231: 451-453, 1982.

10. Mori, M., A. Jayaraman, C. Prasad, J. Pegues and J. F. Wilber. Cyclo (His-Pro): A thyrotropin-releasing hormone-derived neuropeptide distributed ubiquitously throughout the primate brain. Clin Res 29: 872A, 1981. 11. Ogowa, N., Y. Yamawaki, H. Kurada, 1. Nukira, Z. Ota, M. Fujino and N. Yanaihara. Characteristics of thyrotropin releasing hormone (TRH) receptors in rat brain. Peptides 3: 669--677, 1982. 12. Okon, E. and Y. Koch. Localization of gonadotropin releasing and thyrotropin-releasing hormones in human brain by radioimmunoassay. Nature 263: 345-347, 1976. 13. Parker, C. R., Jr. and J. C. Porter. Regional localization and subcellular compartmentalization of thyrotropin releasing hormone (TRH) in adult human brain. J Nearoehem, in press. 14. Parker, C. R., Jr. and A. Capdevila. Evidence for the presence of TRH binding sites in the human brain. Peptides. submitted. 15. Prasad, C., T. Matsui and A. Peterkofsky. Antagonism of ethanol narcosis by histidyl-proline diketopiperazine, Nature 268: 142-144, 1977. 16. Prasad, C., T. Matsui, J. Williams and A. Peterkofsky. Thermoregulation in rats: opposing effects of thyrotropin-releasing hormone and its metabolite histidyl-proline diketopiperazine. Bi~chem Biophys Res Commun 85: 1582-1587, 1978. 17. Prasad, C., J. F. Wilber, V. Akerstrom and A. Banergi. Cyclo (His-Pro): a selective inhibitor of rat prolactin secretion in vitro. L~fe Sei 27: 1979-1983, 1980. 18. Taylor, R. L. and D. R. Burr. Properties of [:~HI (3-Me-His e) TRH binding to apparent TRH receptors in the sheep central nervous system, Brain Res 218: 207-217, 1981,