- Email: [email protected]
Regional dissociation of histidyl-proline diketopiperazine (cyclo-(His-Pro)) and thyrotropin-releasing hormone (TRH) in the rat brain
Brain Research, 231 (1982) 451-453 Elsevier Biomedical Press
451
Regional dissociation of histidyl-proline diketopiperazine (cyclo-(His-Pro)) and thyrotropin-releasing hormone (TRH) in the rat brain
MASATOMO MOR|, CHANDAN PRASAD and JOHN F. WILBER Section of Endocrinology, Department of Medicine and Department of Biochemistry, Louisiana State University Medical Center, 1542 Tulane Avenue, New Orleans, LA 70112 (U.S.A.)
(Accepted October 8th, 1981) Key words: cyclo-(His Pro) - - thyrotropin-releasing hormone - - regional distribution
The concentrations of cyclo-(His-Pro) and its precursor, thyrotropin-releasinghormone (TRH) were measured in seven different areas of rat brain using specific radioimmunoassays. Although the concentration of both of these peptides was highest in the hypothalamus, their distribution patterns in all other loci of the brain were dissimilar. These results suggest that factors in addition to TRH concentrations are important in determining the unique concentration pattern of cyclo-(His-Pro) in the brain. Cyclo-(His-Pr o) is a cyclic dipeptide which can be for med in vitl 0 7 or in vivo 8 by the limited proteolysis of T R H by the enzyme pyroglutamate aminopeptidase. This peptide exhibits a number of neuropharmacologic properties not dependent upon integrity of the endocrine system 10, including antagonism of ethanol narcosis 8, stimulation of brain c G M P levels t2, inhibition of striatal Na+-K + ATPase 1, production of hypothermia in rats 9, and reduction of food-intake 5. We therefore have developed a specific radioimmunoassay for cyclo-(His-Pro) to investigate whether or not it may be present in the rat central nervous system and have documented that this peptide is present in the whole rat brain in concentrations of 35-61 pmol/brain 4. That cyclo-(His-Pro) is present in the rat brain has been reported also recently by Yanagisawa et al. ~3. Because cyclo-(His-Pro) is derived from TRH, which is distributed unevenly in the rat brain 11, the studies herein were carried out to investigate the relationship between the concentrations of these two peptides in 7 different regions of the rat brain. The data show that the concentrations of T R H and cyclo(His-Pro) in different regions of rat brain are dissociated. Male Sprague-Dawley 1ats (200-250 g) supplied by Charles River Breeding Labs were used. They were given free access to food and water and maintained on a 12 h light-dark cycle at an ambient temperature of 23 °C. Animals were killed by decapitation. Brains were removed and dissected into 7 regiens 2, and then homogenized in ice-cold 0.4 M perchloric acid (hypothalamus and striatum in 1 ml, midbrain and hippocampus in 2 ml, pons medulla and cerebellum in 3 ml, cortex in 5 ml). The homogenates were centrifuged at 10,000 g for 20 min. The supernatants were 0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press
452 "FABLE 1 Regional distribution ~f cyclo-( His-Pro) and TRH in rat brain
Rat brains (n 21) were dissected into the following regions by the technique of Glowinski et aP ~. The tissues were extracted and then assayed for cyclo(His-Pro) and TRH by radioimmunoassay as described in the text. The data are presented as mean -~ S.E.M. Region
Hypothalamus Cortex Hippocampus Striatum Midbrain Cerebellum Ponsmedulla
,[molpeptide/mg protein Cyelo-(His -Pro)
TRH
832.6 :%:58.6 601.6 ~ 33.2 571.7 ! 51.4 500.5 ~ 44.8 478.3:4:28.4 414.9 i 26.0 397.9 ]: 27.6
2208.5 :i: 78.1 ! 50.5 i 126.0 i 351.2 31.5 ! 269.1 !
Molar rati~ Cyclo-f His Pro)/TRH
155.3 9.2 10.4 7.6 36.7 4.0 15.1
0.40 :j: 0.03 8.91 ! 0.90 21.70 436 4.28 0.45 1.58 i 0.14 18.99 _[: 4.14 159 ' (!16
neutralized with saturated KHCO3 and then recentrifuged. The clear neutralized supernatants were iyophilized and then resuspended in phosphate-buffered saline containing 0.25 ~,, bovine serum albumin. Cyclo-(His-Pro) and T R H radioimmunoassays were performed as described by us previously 4. The recovery of both exogenous cyclo-(His-Pro) and T R H in the above extraction procedure was 90°~i. Protein measurements were performed according to Lowry et al. a. The data were analyzed using multivariate analysis of variance 6. The concentrations of T R H and cyclo-(His-Pro)in the 7 regions of the rat brain are summarized in Table I. ttighest concentrations for both peptides were identified in hypothalamus. However, the hypothalamic concentrations of cyclo-(His-Pro) were only slightly but significantly (P < 0.01) higher than those of the next highest region (cortex). By contrast, T R H concentrations were strikingly higher in the hypothalamus than in all other regions (P < 0.01). The distribution pattern of the two peptides, moreover, were quite dissimilar. The lowest concentration of cyclo-(His-Pro) was found in pons medulla (397.9 27.6 fmol/mg protein, 48 0/o of hypothalamic concentration), which was similar to cerebellum, midbrain, and striatal cyclo-(His-Pro) concentrations (P > 0.2), but statistically lower than hypothalamic, cortical, and hippocampal concentrations of this peptide (P < 0.01). By contrast, the lowest concentrations of Tt/.H, found in cortex, hippocampus, and cerebellum, were statistically all lower than those of striatum, midbrain, and pons medulla (P < 0.01). This regional dissociation of T R H and cyelo-(His-Pro) is dramatized further by scrutiny of the cyclo-(His-Pro)/TRH molar ratios. The ratios varied widely, from 0.4 for hypothalamus to 21.7 for hippocampus. Molar ratios were different for regions statistically, excepting midbrain, pons medulla, and hippocampus, cerebellum (P > 0.2). The order of the distribution of T R H (hypothalamus > midbrain > ports medulla > striatum > cortex > hippocampus > cerebellum) in brain was opposite to the order of distribution of cyclo( H i s - P r o ) / T R H ratios.
453 The biochemical basis for the dissociation o f the c o n c e n t r a t i o n o f these two peptides is presently u n k n o w n . However, if the c o n c e n t r a t i o n o f precursor ( T R H ) were the only rate-limiting determinant, the ratios should have been constant. In contrast, c y c l o - ( H i s - P r o ) / T R H ratios decreased with increasing T R H concentrations. Possible a d d i t i o n a l factors c o n t r i b u t i n g to this dissociation could include: (1) regional differences in the activities o f the enzymes imidopeptidase, which cleaves H i s P r o N H ~ to His a n d ProNH~, a n d pyroglutamate aminopeptidase, (2) regional variations in the catabolic rate o f c y c l o - ( H i s - P r o ) release. Studies are presently in progress to elucidate which o f these factor(s) m a y be involved in d e t e r m i n i n g the steady-state concentrations o f c y c l o - ( H i s - P r o ) in the rat brain. This research was s u p p o r t e d by the Office o f N a v a l Research ( C o n t r a c t N0001480-C-0416) a n d N I H ( G r a n t AM25482). We t h a n k Dr. R o b e r t Elston for the statistical analyses o f the d a t a a n d Mrs. Betty Douglas for superb secretarial help.
1 Battaini, F. and Peterkofsky, A., Histidyl-proline diketopiperazine, and endogenous brain peptide that inhibits (Na+-K+)-ATPase, Biochem. biophys. Res. Commun., 94 (1980) 240. 2 GIowinski, J. and Iversen, L. L., Regional studies of catecholamines in the rat brain, J. Neurochem., 13 (1966) 655. 3 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the folin reagent, J. biol. Chem., 193 (1951) 265. 4 Mori, M., Prasad, C. and Wilber, J. F., Specific radioimmunoassay of cyclo-(His-Pro), A biologically active metabolite of thyrotropin-releasing hormone, Endocrinology, 108 (1981) 1995. 5 Morley, J. E., Levine, A. S. and Prasad, C., Histidyl-proline diketopiperazine decreases food intake in rats, Brain Research, 210 (1981) 475. 6 Morrison, D. E., Multivariate Statistical Methods, McGraw-Hill, New York, Chapter 4. 7 Prasad, C. and Peterkofsky, A., Demonstration of two separate enzymatic activities for the degradation of thyrotropin-releasing hormone in hamster hypothalamic extracts, J. biol. Chem., 251 (1976) 3229. 8 Prasad, C., Matsui, T. and Peterkofsky, A., antagonism of ethanol narcosis by histidyl-proline diketopiperazine, Nature (Lond.), 268 (1977) 142. 9 Prasad, C., Matsui, T., Williams, J. and Peterkofsky, A., Thermoregulation in rats: opposing effects of thyrotropin-releasing hormone and its metabolite histidyl-proline diketopiperazine, Biochem. biophys. Res. Commun., 85 (1978) 1582. 10 Prasad, C., Wilber, J., Akerstrom, V. and Banerji, A., Cyclo-(His-Pro) : A selective inhibitor of rat prolactin secretion in vitro, Life Sci., 27 (1980) 1979. 11 Winkour, A. and Utiger, R. D., Thyrotropin-releasing hormone: regional distribution in rat brain, Science, 185 (1974) 265. 12 Yanagisawa, T., Prasad, C., Williams, J. and Peterkofsky, A., Antagonism of ethanol-induced decrease in rat brain cGMP concentrations by histidyl-proline diketopiperazine, a thyrotropinreleasing hormone metabolite, Biochem. biophys. Res. Commun., 86 (1979) 1146. 13 Yanagisawa, T., Prasad, C. and Peterkofsky, A., The subcellular and organ distribution and natural form of histidyl-proline diketopiperazine in rat brain determined by a specific radioimmunoassay, J. bioL Chem., 255 (1980) 1290.
451
Regional dissociation of histidyl-proline diketopiperazine (cyclo-(His-Pro)) and thyrotropin-releasing hormone (TRH) in the rat brain
MASATOMO MOR|, CHANDAN PRASAD and JOHN F. WILBER Section of Endocrinology, Department of Medicine and Department of Biochemistry, Louisiana State University Medical Center, 1542 Tulane Avenue, New Orleans, LA 70112 (U.S.A.)
(Accepted October 8th, 1981) Key words: cyclo-(His Pro) - - thyrotropin-releasing hormone - - regional distribution
The concentrations of cyclo-(His-Pro) and its precursor, thyrotropin-releasinghormone (TRH) were measured in seven different areas of rat brain using specific radioimmunoassays. Although the concentration of both of these peptides was highest in the hypothalamus, their distribution patterns in all other loci of the brain were dissimilar. These results suggest that factors in addition to TRH concentrations are important in determining the unique concentration pattern of cyclo-(His-Pro) in the brain. Cyclo-(His-Pr o) is a cyclic dipeptide which can be for med in vitl 0 7 or in vivo 8 by the limited proteolysis of T R H by the enzyme pyroglutamate aminopeptidase. This peptide exhibits a number of neuropharmacologic properties not dependent upon integrity of the endocrine system 10, including antagonism of ethanol narcosis 8, stimulation of brain c G M P levels t2, inhibition of striatal Na+-K + ATPase 1, production of hypothermia in rats 9, and reduction of food-intake 5. We therefore have developed a specific radioimmunoassay for cyclo-(His-Pro) to investigate whether or not it may be present in the rat central nervous system and have documented that this peptide is present in the whole rat brain in concentrations of 35-61 pmol/brain 4. That cyclo-(His-Pro) is present in the rat brain has been reported also recently by Yanagisawa et al. ~3. Because cyclo-(His-Pro) is derived from TRH, which is distributed unevenly in the rat brain 11, the studies herein were carried out to investigate the relationship between the concentrations of these two peptides in 7 different regions of the rat brain. The data show that the concentrations of T R H and cyclo(His-Pro) in different regions of rat brain are dissociated. Male Sprague-Dawley 1ats (200-250 g) supplied by Charles River Breeding Labs were used. They were given free access to food and water and maintained on a 12 h light-dark cycle at an ambient temperature of 23 °C. Animals were killed by decapitation. Brains were removed and dissected into 7 regiens 2, and then homogenized in ice-cold 0.4 M perchloric acid (hypothalamus and striatum in 1 ml, midbrain and hippocampus in 2 ml, pons medulla and cerebellum in 3 ml, cortex in 5 ml). The homogenates were centrifuged at 10,000 g for 20 min. The supernatants were 0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press
452 "FABLE 1 Regional distribution ~f cyclo-( His-Pro) and TRH in rat brain
Rat brains (n 21) were dissected into the following regions by the technique of Glowinski et aP ~. The tissues were extracted and then assayed for cyclo(His-Pro) and TRH by radioimmunoassay as described in the text. The data are presented as mean -~ S.E.M. Region
Hypothalamus Cortex Hippocampus Striatum Midbrain Cerebellum Ponsmedulla
,[molpeptide/mg protein Cyelo-(His -Pro)
TRH
832.6 :%:58.6 601.6 ~ 33.2 571.7 ! 51.4 500.5 ~ 44.8 478.3:4:28.4 414.9 i 26.0 397.9 ]: 27.6
2208.5 :i: 78.1 ! 50.5 i 126.0 i 351.2 31.5 ! 269.1 !
Molar rati~ Cyclo-f His Pro)/TRH
155.3 9.2 10.4 7.6 36.7 4.0 15.1
0.40 :j: 0.03 8.91 ! 0.90 21.70 436 4.28 0.45 1.58 i 0.14 18.99 _[: 4.14 159 ' (!16
neutralized with saturated KHCO3 and then recentrifuged. The clear neutralized supernatants were iyophilized and then resuspended in phosphate-buffered saline containing 0.25 ~,, bovine serum albumin. Cyclo-(His-Pro) and T R H radioimmunoassays were performed as described by us previously 4. The recovery of both exogenous cyclo-(His-Pro) and T R H in the above extraction procedure was 90°~i. Protein measurements were performed according to Lowry et al. a. The data were analyzed using multivariate analysis of variance 6. The concentrations of T R H and cyclo-(His-Pro)in the 7 regions of the rat brain are summarized in Table I. ttighest concentrations for both peptides were identified in hypothalamus. However, the hypothalamic concentrations of cyclo-(His-Pro) were only slightly but significantly (P < 0.01) higher than those of the next highest region (cortex). By contrast, T R H concentrations were strikingly higher in the hypothalamus than in all other regions (P < 0.01). The distribution pattern of the two peptides, moreover, were quite dissimilar. The lowest concentration of cyclo-(His-Pro) was found in pons medulla (397.9 27.6 fmol/mg protein, 48 0/o of hypothalamic concentration), which was similar to cerebellum, midbrain, and striatal cyclo-(His-Pro) concentrations (P > 0.2), but statistically lower than hypothalamic, cortical, and hippocampal concentrations of this peptide (P < 0.01). By contrast, the lowest concentrations of Tt/.H, found in cortex, hippocampus, and cerebellum, were statistically all lower than those of striatum, midbrain, and pons medulla (P < 0.01). This regional dissociation of T R H and cyelo-(His-Pro) is dramatized further by scrutiny of the cyclo-(His-Pro)/TRH molar ratios. The ratios varied widely, from 0.4 for hypothalamus to 21.7 for hippocampus. Molar ratios were different for regions statistically, excepting midbrain, pons medulla, and hippocampus, cerebellum (P > 0.2). The order of the distribution of T R H (hypothalamus > midbrain > ports medulla > striatum > cortex > hippocampus > cerebellum) in brain was opposite to the order of distribution of cyclo( H i s - P r o ) / T R H ratios.
453 The biochemical basis for the dissociation o f the c o n c e n t r a t i o n o f these two peptides is presently u n k n o w n . However, if the c o n c e n t r a t i o n o f precursor ( T R H ) were the only rate-limiting determinant, the ratios should have been constant. In contrast, c y c l o - ( H i s - P r o ) / T R H ratios decreased with increasing T R H concentrations. Possible a d d i t i o n a l factors c o n t r i b u t i n g to this dissociation could include: (1) regional differences in the activities o f the enzymes imidopeptidase, which cleaves H i s P r o N H ~ to His a n d ProNH~, a n d pyroglutamate aminopeptidase, (2) regional variations in the catabolic rate o f c y c l o - ( H i s - P r o ) release. Studies are presently in progress to elucidate which o f these factor(s) m a y be involved in d e t e r m i n i n g the steady-state concentrations o f c y c l o - ( H i s - P r o ) in the rat brain. This research was s u p p o r t e d by the Office o f N a v a l Research ( C o n t r a c t N0001480-C-0416) a n d N I H ( G r a n t AM25482). We t h a n k Dr. R o b e r t Elston for the statistical analyses o f the d a t a a n d Mrs. Betty Douglas for superb secretarial help.
1 Battaini, F. and Peterkofsky, A., Histidyl-proline diketopiperazine, and endogenous brain peptide that inhibits (Na+-K+)-ATPase, Biochem. biophys. Res. Commun., 94 (1980) 240. 2 GIowinski, J. and Iversen, L. L., Regional studies of catecholamines in the rat brain, J. Neurochem., 13 (1966) 655. 3 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the folin reagent, J. biol. Chem., 193 (1951) 265. 4 Mori, M., Prasad, C. and Wilber, J. F., Specific radioimmunoassay of cyclo-(His-Pro), A biologically active metabolite of thyrotropin-releasing hormone, Endocrinology, 108 (1981) 1995. 5 Morley, J. E., Levine, A. S. and Prasad, C., Histidyl-proline diketopiperazine decreases food intake in rats, Brain Research, 210 (1981) 475. 6 Morrison, D. E., Multivariate Statistical Methods, McGraw-Hill, New York, Chapter 4. 7 Prasad, C. and Peterkofsky, A., Demonstration of two separate enzymatic activities for the degradation of thyrotropin-releasing hormone in hamster hypothalamic extracts, J. biol. Chem., 251 (1976) 3229. 8 Prasad, C., Matsui, T. and Peterkofsky, A., antagonism of ethanol narcosis by histidyl-proline diketopiperazine, Nature (Lond.), 268 (1977) 142. 9 Prasad, C., Matsui, T., Williams, J. and Peterkofsky, A., Thermoregulation in rats: opposing effects of thyrotropin-releasing hormone and its metabolite histidyl-proline diketopiperazine, Biochem. biophys. Res. Commun., 85 (1978) 1582. 10 Prasad, C., Wilber, J., Akerstrom, V. and Banerji, A., Cyclo-(His-Pro) : A selective inhibitor of rat prolactin secretion in vitro, Life Sci., 27 (1980) 1979. 11 Winkour, A. and Utiger, R. D., Thyrotropin-releasing hormone: regional distribution in rat brain, Science, 185 (1974) 265. 12 Yanagisawa, T., Prasad, C., Williams, J. and Peterkofsky, A., Antagonism of ethanol-induced decrease in rat brain cGMP concentrations by histidyl-proline diketopiperazine, a thyrotropinreleasing hormone metabolite, Biochem. biophys. Res. Commun., 86 (1979) 1146. 13 Yanagisawa, T., Prasad, C. and Peterkofsky, A., The subcellular and organ distribution and natural form of histidyl-proline diketopiperazine in rat brain determined by a specific radioimmunoassay, J. bioL Chem., 255 (1980) 1290.