Is neurotransmitter histamine predominantly inactivated in astrocytes?

106

Neuroscience Letters, 80 (1987) 106-110 Elsevier Scientific Publishers Ireland Ltd.

NSL 04808

Is neurotransmitter histamine predominantly inactivated in astrocytes? Urszula Rafatowska l, Jolanta Wa~kiewicz 1 and Jan Albrecht 2 Departments of INeurochemistry and 2Neuropathology, Medical Research Centre, Polish Academy of Sciences, Warsaw (Poland)

(Received 5 March 1987; Revised version received 11 May 1987; Accepted 27 May 1987) Key words: Rat; Astrocyte; Synaptosome; Neurotransmitter; Histamine; Histidine; Histidine decarboxylase; Histamine methyltransferase

Rat synaptosomes and astroglia cell-enriched fraction were tested for the uptake of histamine (HA) and its precursor histidine, and the activities of the HA-synthesizing enzyme, histidine decarboxylase (HD) and HA-metabolizing enzyme, histamine methyltransferase (HMT). While histidine uptake was more active into synaptosomes than into astrocytes, only astrocytes were capable of a significant HA uptake. Kinetic analysis of the astrocytic HA uptake revealed a high affinity-low capacity system (Kin= 5"10-7 M, Vmax--1.6'10 ~2mol.min ~-mg ~) similar to the astroglial transport systems for other neurotransmitters. HMT was 70% more active in astrocytes than in synaptosomes, whereas HD activity was not different in these two preparations. The results indicate that astrocytes could be the major site of neurotransmitter HA inactivation.

A substantial b o d y o f evidence suggests t h a t the i n a c t i v a t i o n o f n e u r o t r a n s m i t t e r s following their release is n o t confined to the n e u r o n a l c o m p a r t m e n t b u t involves the interaction with s u r r o u n d i n g astroglia. A s t r o c y t e s have been d e m o n s t r a t e d to possess high affinity u p t a k e systems for virtually all a m i n o acid n e u r o t r a n s m i t t e r s a n d c a t e c h o l a m i n e s (reviews in refs. 11 a n d 18) a n d these systems m a y c o m p l e m e n t o r substitute for n e u r o n a l reuptake. Several lines o f evidence p o i n t to h i s t a m i n e ( H A ) as a p o t e n t i a l n e u r o t r a n s m i t t e r in the m a m m a l i a n brain. T h e a m i n e is synthesized by a specific enzyme, L-histidine d e c a r b o x y l a s e ( H D ) localized in the c y t o p l a s m o f nerve endings, a n d is p a r t l y s t o r e d in s y n a p t i c vesicles [10, 20]. D u r i n g K + - i n d u c e d d e p o l a r i z a t i o n o f b r a i n slices H A is released a n d its synthesis accelerated [22, 24]. M o r e o v e r , two specific cellular receptor sites for H A have been identified b o t h in s y n a p t o s o m a l p r e p a r a t i o n s [8, 19] a n d o r g a n o t y p i c nerve tissue culture [9]. H o w e v e r , the site o f H A i n a c t i v a t i o n and, in particular, the role o f the n e u r o n a l a n d glial c o m p a r t m e n t in this process have not been dealt with. T h e e x p e r i m e n t a l a p p r o a c h to this p r o b l e m e m p l o y e d in the present s t u d y Correspondence." J. Albrecht, Department of Neuropathology, Medical Research Centre, Polish Academy of Sciences, 00-784 Warsaw, Dworkowa 3, Poland.

0304-3940/87/$ 03.50 © 1987 Elsevier Scientific Publishers Ireland Ltd.

107 consisted in investigating separately a cell fraction enriched in astrocytes and a highly pure preparation of nerve endings (synaptosomes) from rat brain. These fractions were compared for (a) the uptake of histamine and its precursor, histidine, and (b) the activities of the HA synthesizing enzyme, HD (E.C. 4.1.1.22) and the HA-metabolizing enzyme, histamine methyltransferase (HMT, Soadenosylmethionine histamine N-methyltransferase, E.C. 2.1.1.8). Adult male rats (approx. 200 g) were used in all the experiments. Highly pure synaptosomes were prepared from the fore- and midbrains of the rats using discontinuous Ficoll-sucrose gradient centrifugation as described previously [5, 14]. The fraction enriched in astrocytes (some 75% enrichment) was isolated with the trypsinaspiration-differential centrifugation method [1]. The final pellets of synaptosomes and astroglia were suspended at 5 mg/ml in Krebs-Henseleit medium in mM: NaCI 140; KCI 5, MgSO4 1.3, Pi 1, HEPES 10 (pH 7.4). Histidine and HA uptake were measured as described before for other amino acids [23]. The measurements started with the addition of radioactive reagents: ring [14C]histamine (spec.act. 2.07 GBq/mmol) or [U-14C]histidine (spec.act. 8.880 GBq/ mmol). Incubations were performed at 37°C and at times indicated, 300/~1 were withdrawn and rapidly centrifuged (Beckman microfuge) through a layer of silicone oil (specific gravity 1.03). Radioactivity of the pellets was then counted. The values were corrected for uptake at 0°C. HD and H M T activities were determined using the radioenzymatic microassay as described by Taylor and Snyder [21]. The preparation of H M T used for HD determination through HA level was purified from guinea pig brain by the method of Brown et al. [4]. While histidine was more actively taken up into synaptosomes than into astrocytes (Fig. 1A), the opposite was observed with HA (Fig. I B). The HA uptake into synaptosomes was too low to permit a meaningful analysis of its kinetics. The astroglial HA uptake, though less active than the histidine uptake, turned out to be sufficiently active for kinetic evaluation (Fig. 2). The kinetic constants derived from this analysis

125 100' ~75'

/ I

*

25

._c

20 1s

25'

5,

Fig. 1. Uptake of histidine (A) and histamine(B) (each I/~M) by synaptosomes(circles) and astrocytes (triangles)from rat brain. Resultsare means+ S.D. for 4 experiments.

108 '5-

2-

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llsl~uM }-1 Fig. 2. Kinetics of histamine uptake into astrocytes. Uptake was measured using 0.4 mg prot./ml, incubation time 5 min and concentrations of histamine between 0.25 and 5/~M. Each point represents a mean of 4 independent experiments where individual values were within 5% of each other.

(Kin = 5" 10 -7 M, Vmax --1.6.10-12 m o l . m i n - t . m g - i ) indicate the presence o f a high affinity-low capacity transport system, closely resembling the G A B A transport system identified in the cerebral [6] and cerebellar [15] bulk isolated glia, and the 5-hydroxytryptamine and catecholamine uptake systems in primary astrocyte cultures from different rat brain regions [12]. The results tend to suggest that astroglia rather than the nerve terminals m a y be responsible for H A clearance from the synaptic cleft. The results o f enzyme activity determinations (Table I) are consistent with this suggestion. The H M T activity in astrocytes was markedly higher than in synaptosomes or in the cerebral homogenate, indicating that H A m a y be m o r e actively catabolized in the astrogliai compartment. In contrast to H M T , the H D activity was not different in astrocytes and synaptosomes; noteworthy is that both preparations were highly enriched in H D with regard to the cerebral h o m o g e n a t e which, in agreement with other reports [25], showed an extremely low H D activity. The procedure to isolate astroglia-enriched fractions has a drawback o f causing cytoplasmic m e m b r a n e d a m a g e in these cells, which has been held responsible for TABLE 1 HMT AND lid ACTIVITY IN THE CEREBRAL HOMOGENATE, SYNAPTOSOMES AND ASTROCYTES Values are means + S.D. for the number of experiments in brackets. Fraction

Homogenate Synaptosomes Astrocytes

Activity (pmol/mg prot./h) HMT

HD

161 (157, 166) (2) 144 ± 18 (4) 253 _+52 (4)

< 1.0" 11.9 +4.0 (9) 11.4± 2.0 (3)

~Threshold of detection at 1 mg prot./sample employed in the tests for either fraction.

109

their much lower metabolic and amino acid transport activity than of astrocytes cultured in vitro [7, 17]. By comparison, the method used to isolate synaptosomes yields a fraction with energetic parameters resembling the intact whole brain [14], and amino acid transport properties comparable with brain slices or nerve cells grown in vitro [23]. It is thus tempting to assume that the authentic in situ differences in the HA uptake and HMT activity between the astroglial and neuronal compartment are more in favor of astrocytes than measured in the present study. Studies with astrocytes cultured in vitro will help to verify this assumption. The active histidine uptake by synaptosomes would indicate the possibility that a portion of the HA precursor may be derived from extraneuronal sources. In this context, it may be recalled from our earlier study that astrocytes preloaded with histidine vigorously release this amino acid on stimulation with high potassium [3]. Clearly, a different experimental strategy will have to be designed to establish whether astrogila-derived histidine is shuttled to the nerve endings similarly as suggested for ctketoglutarate, the precursor of amino acid neurotransmitter pools [16]. We thank Ms. Aleksandra Lenkiewicz and Ms. Inez Kolsicka for skillful assistance. Supported by Grant 06.02. from the Polish Academy of Sciences. 1 Albrecht, J., Hilgier, W., U|as, J. and Wysmyk-Cybula, U., Some properties of a 'crude' fraction of astrocytes prepared with trypsin, Neurochem. Res., 7 (1982) 519-524. 2 Albrecht, J., Wysmyk-Cybula, U. and Rafatowska, U., The Na÷/K ÷ ATPase activity and GABA uptake in astroglial cell-enriched fraction and synaptosomes derived from rats in the early stage of experimental hepatogenic encephalopathy, Acta Neurol. Scand., 72 (1985) 317 320. 3 Albrecht, J. and Rafalowska, U., Enhanced potassium-stimulated GABA release by astrocytes derived from rats with early hepatogenic encephalopathy, J. Neurochem., in press. 4 Brown, D.D., Tomchick, R. and Axelrod, J., Distribution and properties of a histamine methylating enzyme, J. Biol. Chem., 234 (1959) 2948-2950. 5 Deutsch, C., Brown, C., Rafatowska, U. and Silver, 1.A., Synaptosomes from rat brain: morphology, compartmentation and transmembrane pH and electrical gradients, J. Neurochem., 36 (1981) 2063 2072. 6 Henn, F.A., Goldstein, M.N. and Hamberger, A., Uptake of the neurotransmitter candidate glutamate by glia, Nature (London), 249 (1974) 663~64. 7 Hertz, L., Energy metabolism of glial cells. In E. Schoffeniels, G. Franck, D. Tower and L. Hertz (Eds.), Dynamic Properties of Glia Cells, Pergamon, New York, 1978, pp. 121-132. 8 Hough, B. and Green, J.P., Histamine and its receptors in the nervous system, In A. Lajtha (Ed.), Handbook of Neurochemistry, Vol. 6, Plenum, New York, 1984. 9 Hrsli, E. and Hrsli, L., Autoradiographic localization of binding sites for 3H histamine and H~ and H2 antagonists on cultured neurons and glial cells, Neuroscience, 13 (1984) 863-870. 10 Kataoka, K. and De Robertis, E., Histamine in isolated small nerve endings and synaptic vesicles of rat brain cortex, J. Pharmacol. Exp. Ther., 156 (1967) 114-125. 11 Kimelberg, H.K., Occurrence and functional significance of serotonin and catecholamine uptake by astrocytes, Biochem. Pharmacol., 35 (1986) 2273-2281. 12 Kimelberg, H.K. and Katz, D.M., Regional differences in 5-hydroxytryptamine and catecholamine uptake in primary astrocyte cultures, J. Neurochem., 47 (1986) 1647-1652. 13 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 14 Rafatowska, U., Erecifiska, M. and Wilson, D., Energy metabolism in rat brain synaptosomes from nembutal-anesthetized and nonanesthetized animals, J. Neurochem., 34 (1980) 1380-1386.

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