Comparative aspects of brain TRH inactivation in different vertebrate species

GENERAL

AND

COMPARATIVE

Comparative E . C . GRIFFITHS,*~’

ENDOCRINOLOGY

Aspects

47, 508-515 (1982)

of Brain TRH Inactivation Vertebrate Species

T. J. VISSER,? W. KLOOTWIJK,? A. I. SMITHS

in Different

J. R. MCDERMOTT,~ AND

*Department of Physiology, Stopford Building, University of Manchester, Manchester Ml3 9PT. England; TDepartment of Internal Medicine III and Clinical Endocrinology, Erasmus University, Faculty of Medicine, Rotterdam, The Netherlands; and SMRC Neuroendocrinology Unit. Newcastle General Hospital Newcastle-upon-Tyne NE4 6BE, England

Accepted October 14, 1981 The ability of peptidase enzymes in two subcellular fractions prepared from the whole brain of several vertebrates to degrade thyrotrophin-releasing hormone (TRH) in vitro has been investigated by using radioimmunoassays for TRH and its deamidated metabolite, TRH-OH. Peptide products formed by the fractions were identified by high-pressure liquid chromatography (HPLC). The soluble fraction from rat, mouse, dove, lizard, frog, and goldfish rapidly inactivated TRH to only one product, TRH-OH, whereas the particulate fraction produced the histidylproline diketopiperazine, cyclo(His-Pro), and a small amount of TRIFOH, in all the vertebrates. In the case of the goldfish, the reverse was found, with TRH-OH as the major particulate product. This would indicate the presence of a proline endopeptidase deamidating TRH in the soluble/supernatant fraction and both the proline endopeptidase and a pyroglutamyYaminopeptidase in the particulate fraction. The demonstration of these peptidases forming the two metabolites from TRH in the brain of lower vertebrates provides further evidence for the role of TRH as a central neurotransmitter and indicates that cyclo(His-Pro) and TRH-OH could have properties in lower vertebrates similar to those that have already been demonstrated in the hypothalamus-pituitary endocrine system and brain of mammals.

In mammals, thyrotrophin-releasing hormone (TRH; pGlu-His-Pro-NH2) is believed to have both an endocrine function in stimulating thyrotrophin and prolactin release from the anterior pituitary and a neurotransmitter or neuromodulator function in the central nervous system (Morley, 1979). This tripeptide is widely distributed in the brain of various vertebrate species (Jackson, 1978), but there are doubts about its exact role in the control of pituitary function in lower vertebrates (Goos, 1978; Crim et al., 1978; Ball, 1981). It has been suggested that TRH was originally active as a neurotransmitter in the brain and developed its more specialized properties in the pituitary-thyroid axis during evolution (Metcalf and Dettmar, 1981). One of the ’ To whom correspondence

should be addressed.

criteria used to identify TRH as a central neurotransmitter is the presence in the brain of enzymes inactivating the tripeptide: a pyroglutamyl aminopeptidase, a histidyl-prolineamide imidopeptidase, and a proline endopeptidase inactivating TRH have been characterized in mammalian brain (Prasad and Peterkofsky, 1976; Matsui et al., 1979; Rupnow et al., 1979). However, the mechanism of TRH inactivation in the central nervous system of other vertebrates is unknown. Radioimmunoassays for TRH and one metabolite, TRH-OH or deamido-TRH (Visser et al., 1975, 1977), have been used in conjunction with highpressure liquid chromatography (HPLC) to investigate TRH inactivation by two subcellular fractions prepared from the brain of rat (Sprague-Dawley), mouse (albino), dove (Streptopelia risoria), lizard (Gekko 508

0016~6480/82/080508-08$01.00/0 Copyright 0 1982 by Academic Press, Inc. All riShts of reproduction in any form reserved.

509

BRAIN TRH INACTIVATION

gecko and Agama caudospinosa), frog (Rana pipiens), and goldfish (Carassius auratus). MATERIALS

AND METHODS

TRH and related pcptides were obtained from Uniscience, Cambridge, England, and Ria UK, Sunderland, England. Male or female animals of each species were killed by decapitation at the same time of day (0930- 1030), and the whole brain rapidly dissected out and homogenized in 0.25 M sucrose at 2” using a hand-held homogenizer (Gallenkamp TKW-300-030T). Two subcellular fractions were prepared from the homogenate, as previously described (Grifftths et al., 198Qa): a supematant (soluble/cytoplasmic) fraction and a particulate fraction containing microsomes and mitochondria. After overnight dialysis against double-distilled water at 2” and protein estimation by a microkjeldahl procedure, aliquots of the fractions were incubated in a total volume of 1 ml with either 100 ng TRH (for radioimmunoassay) or 100 pg TRH (for HPLC) at 37” and pH 7.38 for 60 min. For radioimmunoassay, the protein concentration was 100 pg N/ml, and for HPLC, 200 and 400 pg N/ml for supernatant and particulate fractions, respectively. Enzyme activity was terminated by placing the samples incubated in boiling water for 10 min. Control incubations were prepared with each experiment by preboiling the fractions for 10 min and then incubating with TRH. Samples were freeze dried, and residual TRH and TRH-OH formed were measured by radioimmunoassay (Visser er al., 1975, 1977). For HPLC, the freezedried samples were extracted with 2 ml 80% methanol containing 0.1% trifluoracetic acid (TFA). The methanolic extracts were dried down using a vortex evaporator and the residues were redissolved in 200 ~1 0.08% TFA. A 40-~1 sample was applied to a PBondapak C-18 reverse-phase column and eluted with a linear gradient of 5 to 100% B for 10 min at a flow rate of 1 ml/ml, as previously described (McDermott et al., 1981). Solvent A was 0.08% TFA and Solvent B was 0.08% TFA in 7% acetonitrile; the eluant was monitored at 206 nm. Peptides were identified by comparing their retention times of those of standards.

RESULTS Brain TRH inactivation: Product identification by HPLC. Typical results for

HPLC identification of the products formed from TRH by goldfish, frog, dove, gecko, agama, and mouse are shown in Fig. I. In the control sample, TRH was unaffected by the incubation procedure, but after incubation with the supernatant and particulate

fractions, products formed were clearly separated from TRH (retention time, 12.4 min). In the supernatant fraction, only one product was formed from TRH in all the species studied (Table 1) and this was identified as TRH-OH from the retention time (14.0 min). In the particulate fraction, however, the histidyl-proline diketopiperazine, cyclo(His-Pro), with a retention time of 10.9 min was formed together with varying amounts of TRH-OH, suggesting that the two enzymes forming these products from TRH, the pyroglutamyl aminopeptidase and proline endopeptidase, were present in this fraction. Brain TRH inactivation: quantification by radioimmunoassay. The rates of TRH

inactivation by the two subcellular brain fractions from the different vertebrates are shown in Table 2. In the supernatant fraction, there was rapid TRH inactivation, with residual TRH varying from 11 .OO ng/ 100 pg N in the frog to 80.5 ng/lOO pg N, in the goldfish. Except in gecko and goldfish, the sum of residual TRH and TRH-OH formed was approximately 100 &ml, and this compared well with control sample values of 102.75 + 3.86 rig/ml TRH (12 = 8 2 SEM). The particulate fraction also showed rapid TRH inactivation (control TRH 100.25 t 2.78; II = 8 ? SEM), though TRH-OH formation was only small, except in goldfish. Of the vertebrate brains studied, the highest rate of TRH inactivation by this fraction was in the dove (see Table 2). DISCUSSION

The combined radioimmunoassay and HPLC data demonstrate the presence of the proline endopeptidase deamidating TRH in the supernatant fraction from all the species. With the exception of frog and gecko, the TRH-OH formed by this fraction appeared to be stable over the incubation period used, as previously found with rat brain (Griffiths et al., 1980a). In the case

GRIFFITHS

510

ET AL.

GECKO

.I 3

I 2

\

t s

4 t

t

t

C

P

S

FIG. 1. High-pressure liquid chromatography separation of products formed from TRH by subcellular fractions of brain from different vertebrate species. 1, TRH; 2, TRH-OH; 3, cyclo(His-Pro); C, control; P, particulate fraction; S, supematant fraction.

BRAIN

TRH

511

INACTIVATION

FROG

J 31

f-

?-

C

P

&IL

A 2

1

?--

2

S

GOLDFISH

I

I t C

?--

?--

P

S

FIG.

l--Continued

Lb2

512

GRIFFITHS

ET AL.

AGAMA

t-

C

t-

d

I;

3 1

2

t-

S

P

FIG.

I-Continued

_,I 1

513

BRAIN TRH INACTIVATION TABLE

1

PRODUCTS FORMED FROM TRH BY BRAIN FRACTIONS FROM DIFFERENT VERTEBRATE SPECIES, AS IDENTIFIED BY HIGH-PRESSURE LIQUID CHROMATOGRAPHY

Supematant fraction

Species Rat Mouse Dove Agama

1 1 1 1 1 1 1

Gecko

Frog Goldfish Note.

Particulate fraction

1, TRH-OH;

2; [II 2; [ll 2; [II 2; [II 2; [ll 2; [ll 1; PI

2, cycle (His-Pro).

of frog and gecko, TRH-OH may be further degraded to its constituent amino acids, since no peptide products other than TRH-OH were found by HPLC. In the particulate fraction, TRH was rapidly inac-

tivated, principally to cyclo(His-Pro) with a small amount of TRH-OH also formed, suggesting the presence of the pyroglutamyl peptidase (and some proline endopeptidase) in this fraction. Some reduction of TRH to its constituent amino acids by pyroglutamyl- and imidopeptidases may also occur (Griffiths et al., 1980b). Considering the use of enzymic inactivation as one of several criteria for identifying TRH as a central neurotransmitter, the information provided here would certainly complement this theory, especially when the presence of TRH in different brain areas of several vertebrates (Jackson, 1978) and of TRH receptors in the brain of one avian species, Gallus gallus (Thompson et al., 1981) are taken into account. In mammals, TRH may be a prohormone or precursor for other biologically active peptides (Griffiths and Webster, 1981).

TABLE TRH INACTIVATION FROM

Species Rat S P Mouse S

P Dove S

P Agama S P Gecko S P Frog S P Goldfish S

P

2

AND TRH-OH FORMATION BY SUBCELLULAR BRAIN OF DIFFERENT VERTEBRATE SPECIES

FRACTIONS

Residual TRH (ng/60 min/lOO pg N)

TRH-OH formed (ngb0 min/lOO pg N)

41.00 ? 2.64 36.75

+ 3.06

54.72 k 0.84 4.50 r 0.50

33.75 36.25

k 1.84 -+ 1.25

63.75 k 1.18 4.75 t 0.47

30.00 f 2.51 9.25 f 1.88

77.75 c 4.02 6.75 k 0.47

5.66 + 3.17

89.50 + 2.21 8.00 f 0.40

41.75 + 2.17 19.33 2 1.45 43.00

k 1.41

11.00 + 0.70

36.66 k 0.88 8.50 + 0.70

35.75

+ 2.01

44.50 * 0.86 8.00 f 2.00

80.50 24.25

2 3.40 + 1.31

27.50 -c 2.95 22.25 k 0.69

Note. S, Supematant fraction; P, particulate fraction; n = 4-6 f SEM.

514

GRIFFITHS

Cyclo(His -Pro) formed enzymically from the tripeptide by a process called biotransformation has both endocrine and central actions related to those of TRH (Peterkofsky and Battaini, 1980), whereas TRH-OH can stimulate “wet-dog” shakes after intraventricular administration in rats (Boschi et al., 1980). Because the ability to produce both these brain TRH metabolites has now been shown in other vertebrates, the possibility exists that TRH-OH, and especially cyclo(His-Pro), may have similar effects in the various species studied. For example, the role of thyroid hormones and prolactin in amphibian metamorphosis is well documented (see King and Millar, 1981): TRH will stimulate the release of these hormones in mammals and might do the same in other vertebrates. Cyclo(HisPro), one TRH metabolite, can inhibit prolactin release in mammals (Peterkofsky and Battaini, 1980), and because this cyclical dipeptide can be formed by frog brain, it might have some role in controlling the hormonal events of metamorphosis. This suggestion is given further support by the recent demonstration by Wilber et al. (1981) of increased cyclo(His -Pro) formation during ontogeny by the brain of Rana catesbeiana and the increased brain TRH content in Xenopus faevis during metamorphosis (King and Millar, 1981). However, the exact functional significance of TRH-OH and cyclo(His-Pro) in lower vertebrates remains to be determined. These peptides can clearly be formed from TRH by enzymes in the brain of the vertebrates studied, and a further indication of their role could be obtained by measuring both in different brain regions. Specific radioimmunoassays are available (Visser et al., 1975; Yanagisawa et ul., 1980), which could be used to measure changes in the metabolites under different physiological conditions. This approach to the metabolites’ properties may be assisted by the HPLC system used here: it has the ability to readily and rapidly separate all

ET AL.

three related peptides and thus overcome the small (~0.7%) cross-reactivity between TRH and TRH-OH in the TRH-OH radioimmunoassay (Visser et al., 1973, as well as any interactions in the cyclo(HisPro) assay. It should also be mentioned that the incubation temperature of 37” may not necessarily be the physiological temperature of the poikilotherms used. Incubation of frog brain fractions with TRH at 20”, however, gives results very similar to those at 37” (Griffiths and Visser, unpublished observations). Obviously, a considerable amount of work remains to be done in the future to investigate the functions of TRH-OH and cyclo(His-Pro) in lower vertebrates and to determine how these metabolites might interact with TRH in its endocrine and neurotransmitter properties. ACKNOWLEDGMENTS We wish to thank Miss C. Baris, Miss S. Kershaw, and Mr. S. Leeson for technical assistance, Dr. Balmant for his help in obtaining the animals used, and Miss J. A. King for helpful comments in the preparation of this manuscript.

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515

of pyroglutamyl peptidase and amidase activities in hamster hypothalamic extracts. J. Biol. Chem. 251, 3229-3234. Rupnow, J. H., Taylor, W. L., and Dixon, J. E. (1979). Purification and characterization of a thyrotropin-releasing hormone deamidase from rat brain. Biochemistry 18, 1206-1212. Thompson, D. F., Taylor, R. L., and Burt, D. R. (1981). TRH receptor binding in avian pituitary and brain. Gen. Comp. Endocrinol. 44, 77-81. Visser, T. J., Klootwijk. W., Dotter, R., and Hennemann, G. (1975). A radioimmunoassay for the measurement of pyroglutamyl-histidyl-proline, a proposed thyrotropin-releasing hormone metabolite. J. C/in. Endocrinol. Metab. 40, 742-745. Visser, T. J., Klottwijk, W., Dotter, R., and Hennemann, G. (1977). A new radioimmunoassay of thyrotropin-releasing hormone. FEBS Left. 83, 37-40. Wilber, J. F., Prasad, C., and Amborski, R. (1981). Histidylproline diketopiperazine Cyclo(His-Pro), a regulator of amphibian metamorphosis. Proc. Endocrine Sot., 63rd Ann. Meeting, Cincinnati, p. 323, abstr. 962. Yanagisawa, T., Prasad, C., and Peterkofsky, A. (1980). The subcellular and organ distribution and natural form of histidyl-proline diketopiperazine in rat brain determined by a specific radioimmunoassay. J. Biol. Chem. 255, 10290-10298.