Regulation of histamine release in rat hypothalamus and hippocampus by presynaptic galanin receptors

Peptides, Vol. 12, pp. 1113-1117. ©Pergamon Press plc, 1991. Printed in the U.S.A.

0196-9781191 $3.00 + .00

Regulation of Histamine Release in Rat Hypothalamus and Hippocampus by Presynaptic Galanin Receptors J. M. A R R A N G , C. G U L A T - M A R N A Y ,

N. D E F O N T A I N E A N D J. C. S C H W A R T Z

Unitg de Neurobiologie et Pharmacologie (U. 109) de I'INSERM, Centre Paul Broca 2ter rue d'Algsia, 75014 Paris, France R e c e i v e d 27 February 1991 ARRANG, J. M., C. GULAT-MARNAY, N. DEFONTAINE AND J. C. SCHWARTZ, Regulation of histamine release in rat hypothalamus and hippocampus by presynaptic galanin receptors. PEPTIDES 12(5) 1113-1117, 1991.--The effect of galanin, a peptide present in a subpopulation of histaminergic neurons emanating from the rat posterior hypothalamus, was investigated on K+-evoked [3H]histamine release in slices and synaptosomes from rat cerebral cortex, striatum, hippocampus and hypothalamus, Porcine galanin (0.3 ~M) significantly inhibited histamine release induced by 25 mM K ÷ in slices from hypothalamus and hippocampus, but not from cerebral cortex and striatum, i.e., only in regions in which a colocalization of histamine and galanin has been described. The inhibitory effect of galanin was concentration dependent, with an ECso value of 5.8--- 1.9 nM. The maximal inhibition was of 30--40% in hypothalamic and hippocampal slices depolarized with 25 mM K + . The galanin-induced inhibition observed in hypothalamic slices was not prevented in the presence of 0.6 p,M tetrodotoxin and also occurred in hippocampal and hypothalamic synaptosomes, strongly suggesting the activation by galanin of presynaptic receptors located upon histaminergic nerve endings. The maximal inhibitory effect of galanin in slices or synaptosomes was lower than that previously reported for histamine acting at H3-autoreceptors, possibly suggesting that not all histaminergic axon terminals, even in the hypothalamus and hippocampus, are endowed with galanin receptors. It increased progressively in hypothalamic and hippocampal synaptosomes as the strength of the depolarizing stimulus was reduced. It is concluded that galanin modulates histamine release via presynaptic receptors, presumably autoreceptors located upon nerve terminals of a subpopulation of cerebral histaminergic neurons. Galanin

Histamine release

Hippocampus

Hypothalamus

METHOD

GALANIN, a 29 amino acid peptide, is widely distributed in the central nervous system (26). Among other biological effects, galanin has a potent inhibitory effect on the release of acetylcholine in the ventral hippocampus of the rat (8). Potent inhibitory effects of galanin on serotonergic neurons in the rat brain (34) and on dopamine release from the rat median eminence (24) have also been reported. In all the previous studies, galanin presumably acts at a presynaptic level. Radioimmunological and immunohistochemical studies have shown that galanin-like immunoreactivity coexists with acetylcholine in the septum-basal forebrain (19), with serotonin in the dorsal raphe nucleus, with dopamine in the hypothalamic arcuate nucleus and with noradrenaline in the locus coeruleus (20). Galanin has also been shown to be colocalized with histamine in some tuberomammillary neurons of the posterior hypothalamus (16,33), suggesting its involvement in histaminergic neurotransmission. Presynaptic histamine H3-autoreceptors (1-4, 12, 35), M 1muscarinic heteroreceptors (9), ot2-adrenergic heteroreceptors (10,12) and K-opioid heteroreceptors (11) modulate histamine release from rat brain slices in which the endogenous pool of the amine was labeled using [3H]histidine. The same model is used here to examine whether galanin receptors regulate histamine release in various regions of the rat brain.

[3H]Histamine Release From Slices The procedure was essentially that described by Arrang et al. (1,2). Slices from rat cerebral cortex (total), striatum (total), hippocampus (total), or hypothalamus (total or dissected rostrocaudally about 2/3, 1/3 in anterior and posterior part) were preincubated for 45 min with L-[3H]histidine (1 txM). After several washing steps, aliquots of the slice suspension (1-2 mg of protein) were incubated for 2 min with 25 mM potassium (final concentration). In each experiment, burimamide, a histamine H 2and H3-receptor antagonist (1), was added 10 min before the depolarizing stimulus. When required, galanin was added together with burimamide. Incubations were stopped by rapid centrifugation, and [3H]histamine levels in tissue and medium were quantiffed after isolation by ion-exchange chromatography (27). In each set of experiments, the recovery of [3H]histamine (88 --- 3%) and the contamination by L-[3H]histidine (<0.005%) in the isolation procedure were determined, and results were corrected accordingly.

[~H]Histamine Release From Synaptosomes The procedure was that described by Arrang et al. (2). A crude synaptosomal fraction from rat cerebral cortex, striatum,

1113

1114

ARRANG ET A L

hippocampus or hypothalamus, prepared according to the method of Whittaker (36), was preincubated for 45 min with L-[3H]his tidine (2 p,M). After six successive washings and centrifugations, synaptosomes (0.3-1 mg of protein) were incubated in each experiment for 8 min with burimamide (100 p,M), bestatin (100 p,M), an aminopeptidase inhibitor, and, when required, galanin before depolarization for 2 min with potassium in various concentrations. Incubations were ended and 3H-labeled amine levels were determined as in slice experiments. Protein contents were determined according to the method of Lowry et al. (18) using bovine serum albumin as the standard.

Calculation of the Data Radioactivity (in dpm) was estimated by liquid scintillation spectrometry at 45% counting efficiency. At least 2000 disintegrations were counted for each pellet or supernatant sample over a background of 16 cpm. Values for [3H]histamine release elicited by tissue (slices or synaptosomes) depolarization were given as percentages of the total 3H-amine present in tissue plus medium and were calculated in each experiment by subtracting the mean spontaneous efflux (2 mM K ÷). For the concentration-response curve of galanin, results were expressed as percent of this difference and the ECso for galanin was obtained by fitting the data with an iterative computer leastsquares method derived from Parker and Wand (25). Statistical evaluation of the results was by Student's t-test. Any significant difference obtained for [3H]histamine release from values calculated in percentages before or after subtraction of the mean spontaneous effiux was checked by statistical analysis (t-test) of the same parameter expressed in dpm/mg protein of [3H]histamine measured in the medium. Chemicals and Drugs L-[2,5-3H]Histidine (46 Ci/mmol) was obtained from the Radiochemical Centre (Amersham, UK). It was purified before use by ion-exchange chromatography, so that its [3H]histamine content was <0.01%. [3H]Histamine of high specific radioactivity was prepared by enzymatic decarboxylation of L-[2,5-3H]histidine for 1 h at 37°C, using L-histidine decarboxylase from Clostridium welchii (Sigma), and purification onto Amberlite CG50 microcolumns (28). Tetrodoxin was from Sigma. Burimamide and bestatin were kindly donated by Smith Kline and French Laboratories (UK) and Laboratoires Roger Bellon (France), respectively. Galanin(1-29) (porcine, batch No. A22632) was purchased from Bachem (Bubendorf, Switzerland). RESULTS

The spontaneous efflux of [3H]histamine from slices of various rat brain regions into the medium in the presence of 2 mM K ÷ and 100 R,M burimamide represented 6.7 -+0.5%, 7.3 -+0.2%, 3.0 -+ 0.2% and 6.3 -+0.2% of the total 3H-labeled amine (present in tissue plus medium) in cerebral cortex, striatum, hippocampus and hypothalamus, respectively. In the presence of 25 mM K + and 100 p.M burimamide, a two-fold increase in the level of [3H]histamine in the medium was observed in each region (14.5-+0.7%, 14.7-+0.6%, 10.5-+0.2% and 14.1 -+0.4% of the total, respectively), with the release expressed over spontaneous efflux representing --7% of the total (Table 1). The K÷-evoked release was significantly inhibited (by 30--40%) in the presence of 0.3 R,M porcine galanin in slices from hippocampus and hypothalamus [8.1-+0.4% ( p < 0 . 0 0 1 ) a n d 11.2__.0.4% (p<0.01) of the total, respectively, i.e., - 5 % of the total over spontaneous efflux], but remained unaffected in slices from cerebral cor-

\.

i

*

o

;

, LOG

0*

I

I

, -6 -5 GALANIN CONCENTRATION (M)

FIG. 1. Inhibition by galanin of [3H]histamine release from slices of rat hypothalamus. Slices were allowed to synthesize [3H]histamine during a 45-min preincubation with L-[3H]histidine. After several washing steps, slices were incubated for 10 min in the presence of burimamide, a histamine H3-receptor antagonist, and when required, porcine galanin. They were subsequently depolarized for 2 min in the presence of 25 mM K ÷ (final concentration). The spontaneous effiux (2 mM K ÷) of [3H]histamine represented 6.6__-0.5% of the total (mean--+S.E.M. of 5 experiments) and was not significantly modified by galanin. In the absence of galanin, the [3H]histamine release induced by 25 mM K ÷ was calcu* lated in each experiment as the difference between mean [3H]histamine levels in 25 and 2 mM K ÷ media, respectively, and represented 10.4--_2.9% of the total 3H-labeled amine (present in tissue plus medium). Results are expressed as percentages of this value. Each point represents the results from 2-3 separate experiments with quadruplicate determinations. *p<0.05 as compared with 25 mM K ÷ alone.

tex or striatum (13.7---0.5% and 14.8-+0.6% of the total, respectively, i.e., - 7 % of the total over spontaneous efflux) (Table 1). The inhibition occurred with the same amplitude in the anterior and posterior parts of the hypothalamus (Table 1). The inhibitory effect of galanin on the K ÷-evoked [3H]histamine release from hypothalamic slices was concentration dependent and saturable. The maximal inhibition was 29 - 1% and the ECso was 5 . 8 -+ 1.9 nM (Fig. 1). The inhibition induced by 0.3 R,M galanin in hippocampal and hypothalamic slices remained unaltered in the presence of 0.3 p,M scopolamine, 3 p,M yohimbine and 30 IxM naloxone (not shown). In the presence of 0.6-1 t~M tetrodotoxin, while [3H]histamine release from hypothalamic slices was slightly reduced, the inhibitory effect of galanin (0.3 p,M) was not significantly modified (Table 2). As already observed (2,9), the spontaneous effiux of [3H]histamine from synaptosomes in 2 mM K + was significantly higher than that observed with slices in all the regions tested since it represented 1091 -+9 dpm/mg protein, i.e., 16.4-+0.1% of total in cerebral cortex, 1231-+36 dpm/mg protein, i.e., 15.0-+0.4% of total in striatum, 318-+9 dpm/mg protein, i.e., 12.8-+0.3% of total in hippocampus and 1879-+23 dpm/mg protein, i.e., 14.4-+ 0.2% of total in hypothalamus. In hippocampal and hypothalamic synaptosomes, [3H]histamine release was already significantly enhanced in the presence of 10 mM K + [380-+3 dpm/mg protein, i.e., 15.2-+0.1% of total ( + 1 9 % , p<0.01) and 2034-+39 dpm/mg protein, i.e., 15.7 -+0.3% ( + 8%, p<0.05), respectively] and rapidly increased with the potassium concentration present in the medium (Table 3). In both regions, the K+-evoked release observed in the

GALANIN EFFECT ON HISTAMINE RELEASE

1115

TABLE 1

TABLE 3

EFFECT OF GALANIN ON [3H]HISTAMINE RELEASE FROM SLICES OF VARIOUS RAT BRAIN REGIONS DEPOLARIZED WITH 25 raM K ÷

GALANIN-INDUCED INHIBITION OF [3H]HISTAMINE RELEASE FROM SYNAPTOSOMES OF RAT HYPOTHALAMUS AND HIPPOCAMPUS INDUCED BY POTASSIUM IN INCREASING CONCENTRATIONS

Percent [3H]Histamine Release Percent [3H]Histamine Release Region

Galanin = 0

Galanin = 0.3 IzM Conditions

Cerebral cortex Striatum Hippocampus

7.8 +- 0.7 7.4 __ 0.6 7.5 _+ 0.2

7.0 - 0.5 N s 7.5 - 0.5 N's" 5.1 ~- OAt ( - 32%)

Hypothalamus total

7.7 - 0.4

4.8 - 0.4¢ (-38%) 3.7 _ 0.5* (-43%) 4.0 +-- 0.6* (-38%)

anterior

6.5 ___ 0.6

posterior

6.5 - 0.6

Slices from various brain regions were preincubated for 10 min with 100 ~,M burimamide and, when required, 0.3 IJ,M galanin before being depolarized for 2 min with 25 mM K ÷ (final concentration). Release induced by 25 mM K + is expressed as the percent of total [3H]histamine over mean spontaneous efflux which represented 6.2 +_ 0.9%. Numbers in parentheses are percentage changes as compared with 25 mM K ÷ alone. Mean --- S.E.M. from two experiments with 3-8 determinations. *p<0.05; ?p<0.01; N.S. =nonsignificant as compared with respective controls ([Galanin] =0).

presence o f 100 ixM b u r i m a m i d e and 100 p,M bestatin, an aminopeptidase inhibitor, was reduced in the presence o f galanin. T h e release f r o m h i p p o c a m p a l and h y p o t h a l a m i c s y n a p t o s o m e s w a s m o r e m a r k e d l y inhibited by galanin (1 IxM) w h e n w e a k depolarizing stimuli were u s e d and represented 354-+ 3 d p m / m g protein, i.e., 1 4 . 2 + - 0 . 1 % o f total in the presence o f l0 m M K + ( p < 0 . 0 0 1 as c o m p a r e d with l 0 m M K + without galanin) and 1921 -+ 13 d p m / m g protein, i.e., 1 4 . 8 - + 0 . 1 % o f total ( p < 0 . 0 5 ) in h i p p o c a m p u s and h y p o t h a l a m u s , respectively. W h e n these values were e x p r e s s e d over s p o n t a n e o u s efflux, a 42% inhibition in h i p p o c a m p u s and a 69% inhibition in h y p o t h a l a m u s were f o u n d (Table 3). T h e inhibitory effect o f 1 ~ M galanin w a s less

(a) Hypothalamus 10 mM K ÷ 15 mM K ÷ 25 mM K ÷ (b) Hippocampus 10 mM K + 15 mM K ÷ 25mMK ÷

Galanin=0

Galanin= 1 I~M

Difference

1.3 _ 0.3 8.2 ___ 0.2 10.7 ___ 0.3

0.4 - 0.1" 6.3 ___ OAt 9.8 --- 0.3

-69% -23% -8%

2.4 ___ 0.1 13.1 + 1.0 16.5 ± 0.4

1.4 _ OAt 10.4 - 0.6 14.1 _ 0.3t

-42% -20% -14%

Values for [3H]histamine release are given as percent of total over mean spontaneous efflux (2 mM K÷), which represented 14.2 _+ 0.5% of total [3H]histamine. Synaptosomes were incubated for 2 min in the presence of KC1 in various concentrations after preincubation for 8 rain with I00 p,M burimarnide, 100 ~M bestatin and, when required, I IxM galanin. Values are mean _+ S.E.M. of 4--6 determinations from two experiments. *p<0.05; 1"p<0.01 as compared with respective controls. m a r k e d in both regions in the presence o f 15 m M K ÷ ( - 2 0 % ) and b e c a m e negligible ( - 1 0 % ) in the presence o f 25 m M K ÷ (Table 3). A s previously o b s e r v e d with slices (Table 1), no significant inhibition o f [3H]histamine release by galanin could be f o u n d in s y n a p t o s o m e s f r o m cerebral cortex or striatum, e v e n in the presence o f 10 m M K ÷ (Table 4) [ 1 3 6 4 - + 9 and 1320-+20 d p r n / m g protein in cerebral cortex, 1469-+9 and 1431 - 11 d p m / m g protein in striatum in the a b s e n c e and presence o f galanin (1 IxM), respectively]. DISCUSSION T h e m a i n finding o f the present study is that galanin inhibits h i s t a m i n e release f r o m extrinsic histaminergic nerve e n d i n g s (29) TABLE 4 INHIBITION BY GALANIN OF [3H]HISTAMINE RELEASE FROM SYNAPTOSOMES OF VARIOUS RAT BRAIN REGIONS DEPOLARIZED WITH 10 mM K +

TABLE 2 INFLUENCE OF TETRODOTOXIN ON GALANIN-INDUCED MODULATION OF [3H]HISTAMINE RELEASE FROM SLICES OF RAT HYPOTHALAMUS DEPOLARIZED WITH 25 mM K + Percent [3H]Histamine Release Galanin = 0 Control

6.3 • 0.4

Tetrodotoxin (0.6 IJ.M)

5.1 _+ 0.5

Percent [3H]Histamine Release Region

Galanin = 0

Galanin = 1 IxM

Cerebral cortex

4.1 -+ 0.1

Striatum

2.8 - 0.1

Hippocampus

2.4 -- 0.1

Hypothalamus

1.3 +- 0.3

3.5 -+ 0.3 ( - 15%) 2.4 _ 0.2 ( - 14%) 1.4 ___ OAt (-42%) 0.4 _ 0.1" ( - 69%)

Galanin = 0.3 IzM 4.6 (3.2 (-

_ 0.9 27%) --- 0.7* 37%)

Values are mean -+ S.E.M. of 4-12 determinations in one experiment in which 100 p,M burimamide and, when required, 0.6 ~M tetrodotoxin and 0.3 IJ,M galanin were added l0 min before depolarization with 25 mM K + . Similar data were obtained when 1 I.LM tetrodotoxin was added in the medium. Values in parentheses indicate percentage changes as compared with 25 mM K + alone. *p<0.05 as compared with 25 mM K + in the absence of galanin.

The spontaneous efflux of [3H]histamine (2 mM K ÷) represented 14.6 _ 0.7% of total [3H]histamine and was not significantly modified in the presence of 1 I~M galanin. Synaptosomes were preincubated for 8 min with 100 p.M burimamide, 100 I~M bestatin, and, when required, 1 izM galanin before depolarization for 2 min with 10 mM K + (final concentration). Mean ___ S.E.M. of six determinations in one typical experiment. *p<0.05; ?p<0.001 as compared with respective controls.

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ARRANG ET A L

in the hippocampus and hypothalamus but not in the cerebral cortex or striatum. This inhibition was not prevented in slices in which the traffic of action potentials was blocked by tetrodotoxin and was also evidenced in synaptosomes, two observations which strongly suggest that the inhibitory effect of galanin resulted from the activation of presynaptic receptors located upon histaminergic neurons. Interestingly, in contrast to cerebral cortex and striatum, hippocampus and hypothalamus are two regions of the rat brain known to contain high concentrations of galanin-like immunoreactivity (7, 20-22, 31) and galanin receptor sites (6, 23, 30, 32). Moreover, recent immunohistochemical studies have demonstrated that galanin and histamine coexist in some, but not all, magnocellular neurons of the rat hypothalamic tuberomammillary nucleus (16,33), the region of origin of histaminergic neurons (29). Retrograde tracing experiments have shown that this subgroup of hypothalamic neurons has efferent projections to the ventral hippocampus and probably also to other rat brain regions (16). The present findings add support to the idea of functional heterogeneity of the histaminergic neurons, already suggested by the observations that only subpopulations of the tuberomammillary neurons are immunoreactive to [Met enkephalyl]-Arg6-Phe7, substance P, monoamine oxidase or galanin (15, 16, 33). Moreover, histaminergic neurons are heterogeneous with respect to their afferent and efferent projections (14). The maximal inhibition of histamine release ( - 3 0 % , Fig. 1 and Table 1) elicited by galanin was significantly lower than that elicited by histamine acting at Ha-autoreceptors (1,12), by carbachol acting at M~-muscarinic heteroreceptors (9) and by noradrenaline acting at a2-adrenergic heteroreceptors (10,12), Although this might indicate that galanin acts as a partial agonist on this system [as dynorphin A(1-13) acting at K-opioid heteroreceptors (11)], an alternative hypothesis may be that not all histaminergic axon terminals, even in the hypothalamus and hippocampus, are endowed with galanin receptors. Also, it cannot be excluded that the peptide might have restricted access to deep layers of the slice preparation, possibly in relationship with its inactivation by peptidases. Although presynaptic interaction between coexisting messengers has been demonstrated both in the periphery and in the central nervous system (5,13), the interactions between colocalized transmitters and their respective receptors remain poorly understood. The receptors for galanin presented in this study fulfill the criteria usually required for autoreceptors. The involvement of presynaptic galanin autoreceptors has also been suggested in central cholinergic (8), serotonergic (34) and dopaminergic neurotransmission (24). Further studies are needed to know whether

galanin autoreceptors not only inhibit the release of classical aminergic cotransmitters (including histamine), but also modulate the release of galanin itself from the same nerve endings. Moreover, endogenous activation of histamine H 3 and galanin autoreceptors present on some histaminergic neurons might influence each other. The latter influence was eliminated in the present study since burimamide was added throughout all the experiments in order to ensure blockade of H3-autoreceptors, readily activated by endogenous histamine (1,2), and to make easier the characterization of galanin autoreceptors. The inhibitory effect of galanin at autoreceptors is probably even higher than presently described since it may be limited by several factors in our system. First, the limited penetration and the enzymatic degradation of this 29 amino acid peptide in tissue slices probably underestimates its affinity, as suggested by the difference observed between its ECso value on histamine release ( - 5 nM, Fig. 1) and it K D value ( - 0 . 5 nM) determined in binding (30) or autoradiographic studies (17,31). Note, however, that heterogeneity among the still poorly characterized galanin receptors cannot be ruled out at the present stage. Second, we used porcine galanin and rat brain preparations, a circumstance that may lead to lower affinity binding of the peptidergic ligand to galanin autoreceptors. Third, as previously reported for histamine acting at H3-autoreceptors (2), as well as for a variety of neurotransmitters, an inverse relationship was observed between the maximal inhibitory effect of galanin and the strength of the depolarizing stimulus (Table 3). Such an effect was interpreted as reflecting a modulation of Ca 2 ÷ influx by presynaptic receptors together with a saturation kinetic of the effect of intraneuronal Ca ÷ ÷ on stimulus-release coupling (2). Another factor is likely to be that a stronger depolarization induces release of a larger amount of endogenous galanin in hippocampus and hypothalamus, leading to a higher degree of occupation of inhibitory galanin receptors which become less available to stimulation by exogenous galanin. Related to this observation, the partial depolarization intrinsic to a crude synaptosomal preparation as compared with slices (4) probably accounts for the absence of any significant inhibitory effect of galanin in hippocampal and hypothalamic synaptosomes in the presence of 25 mM K + (Table 3). The inhibition, as high as 70% found in hypothalamic synaptosomes in the presence of 10 mM K ÷ (Tables 3 and 4), might suggest a very efficient brake triggered by galanin in in vivo physiological conditions. ACKNOWLEDGEMENT We thank Mrs Annie Galtier for typing the manuscript.

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