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Pharmacology of cerebral histamine Cumming, Paul Kenneth 1990

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P H A R M A C O L O G Y OF C E R E B R A L H I S T A M I N E  by  PAUL KENNETH  CUMMING  B.Sc, The University of Alberta, 1984 M . S c , The University of British Columbia, 1986  A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T T H E R E Q U I R E M E N T S F O R T H E D E G R E E OF D O C T O R OF PHILOSOPHY in T H E F A C U L T Y OF G R A D U A T E STUDIES Division of Neurological Sciences  We accept this thesis as conforming to the required standard  T H E U N I V E R S I T Y OF BRITISH C O L U M B I A August 1990 ©Paul Kenneth Cumming, 1990  OF  In presenting this thesis in partial fulfilment  of the  requirements for an advanced  degree at the University of British Columbia, I. agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or  by his  or  her  representatives.  It  is  understood  that  copying or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department The University of British Columbia Vancouver, Canada  DE-6 (2/88)  ii  Abstract  Four aspects of the function of histaminergic systems were studied in the rat brain: toxicology, catabolism, release in vivo, and high affinity binding of histamine. Preparations of histamine-N-methyltransferase ( H N M T ) derived from kidney and brain were employed in the radioenzymatic quantification of histamine in biological samples. Tritiated S-adenosyl-Lmethionine ([ H ] - S A M ) served as the co-substrate. A toxicological study was conducted to determine the sensitivity of the H A innervation to prenatal treatment with methylazoxymethanol ( M A M ) , an inhibitor of mitosis. In adult rats, the M A M treatment was without effect on cerebral histamine content, although forebrain H N M T activity was 50% reduced. In another study, C-57 mice were treated with the selective dopamine neurotoxin l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP). Substantial dopamine depletions were not associated with alterations in the cerebral histamine content. In a study of the structural requirements for H N M T inhibition, 9-amino-1,2,3,4tetrahydroacridine ( T H A ) , was found to be one of the most potent inhibitors yet described. The /?-carboline alkaloids, of which harmaline is the prototype, were also found to be moderately potent H N M T inhibitors. Because of the lack of high-affinity re-uptake and the absence of alternate catabolic pathways/blockade of H N M T can potentially alter central histaminergic tone. Peripheral administration of T H A was able to produce dose-dependent increases in cerebral histamine content, as was the more potent H N M T inhibitor, metoprine. The issue of structural requirements for H N M T inhibition are discussed in the light of these results. The in vivo release of histamine was studied by the cerebral microdialysis technique. After chronic implantation of horizontal probes, TTX-insensitive and partially calciumsensitive efflux of histamine was detected in the dorsal striatum and the bed nucleus of the stria terminalis. In striatum, histamine efflux was elevated 50% after peripheral histidine loading (500 mg/kg, i.p.). After synthesis blockade with a-fluoromethylhistidine (100 mg/kg,  i.p.), extracellular histamine levels in striatum disappeared in a bi-exponential manner.  The  half-lives of this disappearance, 32 minutes and 7 hours, indicate the presence of at least two histamine pools. Striatal histamine efflux was elevated by yohimbine treatment (10 mg/kg, i.p.), suggesting the presence of a tonic a^-adrenergic inhibition of histamine release in vivo. In addition to the classical  and  receptors, histamine is able to bind to a  pharmacologically distinct site, H-j, recently characterized as an autoreceptor regulating the synthesis and release of histamine. The binding properties of the  ligand [ ^ H ] - N Q  methylhistamine ([ H ] - N - M e H A ) were studied in forebrain cryostat sections by autoradiography.  Determination of B  m a x  (25 fmole/section) and displacement studies  3 3 indicated that [ H ] - N - M e H A bound to the same site as [ H]-histamine: the high affinity histamine binding site. Binding was greatest in the basal ganglia and had a complex distribution within the cerebral cortex. Quinolinic acid lesion studies indicated that the majority of the binding in the basal ganglia was on striato-nigral projection neurons. binding was also sensitive to local excitotoxic lesions. Therefore, the majority of  Cortical binding  is located on postsynaptic structures intrinsic to these brain regions, rather than on presynaptic autoreceptors on terminals of histamine neurons.  iv  T a b l e of Contents Page  I II  III  Abstract  ii  L i s t o f Tables  vi  L i s t o f Figures  vii  Acknowledgements  ix  Introduction  1  T h e A n a l y s i s of H i s t a m i n e i n Cerebral Extracts ( A ) Introduction  9  (B) R a d i o e n z y m a t i c Assay  10  N e u r o t o x i c o l o g i c a l Studies ( A ) Introduction  21  (i) M A M  21  Methods  (ii) M P T P Methods  21  (B) Results (ii) M A M  IV  V  Results  23  (ii) M P T P Results  24  Inhibition of Histamine-N-methyltransferase ( H N M T ) ( A ) Introduction  29  (B) M a t e r i a l and Methods  30  (C) H N M T I n h i b i t i o n in vitro  34  (D) H N M T I n h i b i t i o n in vivo  47  Cerebral Microdialysis ( A ) Introduction  52  (B) Materials and Methods  53  (C) Results  55  (D) Discussion  66  V  VI  VII  Histamine Receptors (A) Introduction  70  (B) Materials and Methods  70  (C) Results  72  (D) Discussion  99  General Discussion (A) Neurotoxicology  103  (B) H N M T Inhibition  104  (C) Cerebral Microdialyisis  109  (D) Histamine Binding  112  VIII References  116  vi  List of Tables  Page  Table I  Purification of HNMT from porcine kidney  Table II  Effect of prenatal M A M exposure on histamine content and HNMT activity in the adult rat brain  Table III  17  23  Effect of MPTP treatment on histamine levels in the  mouse brain  27  Table IV  Inhibition of cortical HNMT activity  49  Table V  The effect of neurochemical lesions on the binding of [ H]-N-MeHA to rat brain 3  99  vii  List of Figures Page Figure 1  The metabolism of histamine  :  Figure 2  The linearity of HNMT, and H N M T 3 assays  12  Figure 3  Purification of HNMT by DE AE chromatography  15  Figure 4  Linearity of the HNMT assay  18  Figure 5  Alterations in dopamine and metabolites after  2  2  MPTP lesions Figure 6  2  25  Linearity of the HNMT assay in presence of 2  inhibitors  32  Figure 7  Inhibition of HNMTj by some compounds  35  Figure 8  Logit-log analysis of the inhibition of HNMTj by THA and related compounds  Figure 9  Logit-log analysis of the inhibition of HNMTj by some /S-carbolines  Figure 10  56  The effect of the omission of calcium.from the perfusion solution on the in vivo release of histamine  Figure 15  50  The effect of TTX on the in vivo efflux of histamine  Figure 14  45  HNMT inhibitors elevate cerebral histamine levels in the rat  Figure 13  43  Double reciprocal plots of the inhibition of HNMTj by harmaline  Figure 12  41  Double reciprocal plots of the inhibition of HNMTj by THA  Figure 11  37  58  The effect of a-fluoromethylhistidine (a-FMH) on extracellular histamine in the striatum  60  viii  Figure 16  Striatal extracellular histamine efflux after peripheral histidine loading  Figure 17  62  Histamine efflux in the BNST after administration of a^-adrenergic drugs  64 •3  Figure 18  Scatchard analysis of [ H ] - H A binding in rat forebrain J  sections  73 •3  Figure 19  Scatchard analysis of [ H ] - N - M e H A binding in rat forebrain J  sections Figure 20  75  H i l l plots of the binding of H3 ligands to rat forebrain sections  77 •3  Figure 21  The effect of guanylyl nucleotide on [ - ' H J - N - M e H A binding in forebrain sections  Figure 22  80  The displacement of H3 ligands by various histaminergic drugs  82 •3  Figure 23  The displacement of [ H ] - N - M e H A from rat forebrain J  sections by various psychoactive drugs Figure 24  84  Autoradiographs of [ H ] - N - M e H A binding J  in the rat brain  86  •3  Figure 25  Binding of [ H ] - N - M e H A in the basal ganglia  Figure 26  The effects of striatal lesions on the binding of [ H ] - N - M e H A  95  The effects of cortical QA-lesion on the binding of [ H]-N-MeHA  97  3  Figure 27  91  3  ix  Acknowledgements  The assistance of many friends in the production of this work was greatly appreciated. The author wishes to thank especially Chui-Se Tham for crafty surgery, Esther Leung for tireless slicing, and Bruce Hope for challenging debate. Sandra Sturgeon's compassion in the preparation of manuscripts was appreciated. I am bound also to thank Chris Shaw. Special thanks to my supervisor Steve Vincent, who provided a rash of ideas about histamine pharmacology, of which I have only been able to scratch the surface. I wish to thank my parents for giving me art supplies when I was quite small.  1 I Introduction  A considerable body of evidence suggests a neurotransmitter role for histamine in the brain. [ H]-Histamine is synthesized in cortical slices from labelled histidine and may be released by depolarization (Verdiere et al., 1975). Reductions in the synthesis of histamine in forebrain regions following lesions to the medial forebrain bundle suggested the presence of a histaminergic projection ascending in that pathway (Garbarg et al., 1976). However, until the recent localization of histamine within specific neurons, histamine had remained a neurotransmitter without a home. Even prior to the demonstration of neuronal localization, histamine had been implicated in the regulation of a wide variety of functions in the mammalian brain including arousal, water balance, body temperature and analgesia (see Hough, 1988). However, the functions of histamine in the nervous system are still poorly understood, so the present studies were carried out to characterize better some of the factors involved in the metabolism and functional aspects of histamine in the rat nervous system. The pathway for synthesis and catabolism of histamine is illustrated in Figure 1. Histamine is formed from the essential amino acid histidine by the action of a specific enzyme, histidine decarboxylase ( H D C , E C 4.1.1.22), which requires pyridoxal phosphate as the cofactor. H D C has a molecular weight of 54,000 when purified from fetal rat liver (Taguchi et al., 1984). A c D N A has recently been cloned, indicating a 73,450 molecular weight protein (Joseph et al., 1990). The discrepancy in the molecular weight may be related to post transcriptional or post translational modification of the gene product. The amino acid sequence derived from the c D N A is similar to that of other amino acid decarboxylases. The mammalian H D C has a steep p H dependence curve: at p H 7, the optimum, the affinity for histidine was about 100 uM (Hakanson, 1967). Therefore, the enzyme is probably not saturated at normal physiological concentrations of histidine, which are on the order of 100 /iM. Indeed, peripheral loading with large doses of the amino acid is able to increase  2  Figure 1. The Metabolism of Histamine  Histamine (HA) is formed from histidine by the action of histidine decarboxylase (HDC). In the periphery, HA may be deaminated by diamine oxidase (DAO), but in the central nervous system, catabolism involves the sequential action of two enzymes: histamineN-methyltransferase (HNMT) and monoamine oxidase-B (MAO-B). The aldehyde intermediate formed by MAO is unstable and the carboxylic acid forms rapidly in the presence of oxygen. Histamine may act through at least three distinct receptor types.  3  L-histidine HO-C-CHCH. I  Imidazoleacetic acid H O - C - C H .' 2  HDC  DAO  H NCH CH 2  2  N  2 V  histamine H HNMT  H  H NCH CH . 2  2  — N  2  methylhistamine  l M A O - B  HO-C-CH  •N  2  methylimidazole ^ acetic acid C  J H  H  ^  N  4  cerebral histamine content (Schwartz et al., 1972). Several studies have indicated that H D C is regulated in an inhibitory manner by c A M P (Huszti and Magyar, 1984, Huszti and Magyar, 1985), although phosphorylation of the enzyme has not been formally demonstrated. However, histamine synthesis in slices is apparently not acutely regulated by alterations in H D C (Chudomelka and M u r r i n , 1989). In the mouse kidney, H D C activity is induced by thyroxin and oestrogen and repressed by androgens. The mouse H D C gene seems to be associated with a testosterone-sensitive regulatory site (Middleton et al., 1987) responsible for the regulation of expression in kidney by steroid hormones. Castration results in two-fold increased brain levels of histamine (Orr and Quay, 1975), possibly related to elevated H D C activity. H D C and ornithine decarboxylase expression are induced by interleukin-1 and tumor necrosis factor in mouse peripheral tissues (Endo, 1989) but effects of these substances on cerebral H D C have not been studied. The cerebral metabolism of histamine differs from that of other aromatic amine neurotransmitters in that a high affinity, saturable neuronal uptake mechanism appears to be absent (Schwartz et al., 1980, Smits et al., 1988), although some uptake may occur in glia (Rafalowska et al., 1987). Histamine-N-niethyltransferase ( H N M T , E C 2.1.1.8), a specific histamine methylating enzyme, was initially detected in mammalian brain (Brown et al., 1959). Brain is rich in H N M T , which specifically catalyzes methylation of the imidazole ring of histamine, with S-adenosyl-L-methionine (SAM) serving as the methyl-donating co-substrate. H N M T has since been purified from guinea pig brain and partially characterized (Matuszewska and Borchardt, 1983). The enzyme is highly specific for histamine: only histamine and a small number of side-chain modified compounds are substrates (Dent et al., 1982).  The  compartmental distribution of H N M T remains unclear: the enzyme is present in cultured glioma cells (Garbarg et al., 1975), but a substantial proportion of striatal H N M T is sensitive to kainic acid and electrolytic lesions of the medial forebrain bundle (Sperk et al., 1981), which is indicative of a neuronal localization.  5  Diamine oxidase, the enzyme mainly responsible for the catabolism of histamine in peripheral tissues, is virtually absent from the rat brain (Shaff and Beaven, 1976). Because of the absence of catabolic pathways other than N-methylation (Schwartz et al., 1971), and the lack of a robust histamine uptake mechanism, as described above, cerebral histamine content is potentially sensitive to H N M T inhibition. The product of H N M T , tele-methylhistamine ( r - M e H A ) , is a substrate for monoamine oxidase-B ( M A O - B ) with a K  m  of 4 m M (Hough and Domino, 1978). The aldehyde formed  by deamination is unstable and spontaneously oxidizes to r-methylimidazoleacetic acid (rM e l A A ) in the presence of oxygen.  r - M e l A A is presumably removed from the brain by an  acidic metabolite transport system. As brain levels of r - M e l A A are unaffected by probenecid treatment (Khandelwal et al., 1984), the metabolite is evidently removed by a mechanism other than the probenecid-sensitive acidic metabolite transport. Inhibition of M A O - B with pargyline results in an accumulation of r - M e H A in brain which is linear over a period of several hours (Hough et al., 1984). The accumulation of this metabolite has been used as a measure of histamine turnover. Regional turnover rates so calculated are altered by treatment with a variety of substances. For example, diazepam (Oishi et al., 1986) and barbiturates (Hough, 1987) substantially reduce the apparent turnover of cerebral histamine, effects which are presumably related to the depressant properties of drugs potentiating G A B A e r g i c transmission. Morphine treatment may either stimulate or inhibit the pargyline-induced accumulation of r - M H A in different regions of mouse brain, an observation probably pertinent to the mechanism of the antinociceptive property of morphine (Licata et al., 1990). The presence of histamine-containing neurons in the caudal hypothalamus of the rat has been demonstrated by immunohistochemical studies employing antibodies raised against purified H D C (Watanabe et al., 1983, 1984) or conjugates of histamine (Panula et al., 1984). In these immunohistochemical studies, histamine-containing neurons were localized in the tuberomammillary nucleus (TM), a magnocellular nucleus in the caudal hypothalamus.  The  6  T M neurons have been shown also to contain a number of other neurochemical markers in addition to H D C , notably glutamic acid decarboxylase, which is generally a marker of G A B A e r g i c neurons (Vincent et al., 1983, Takeda et al., 1984), adenosine deaminase (Senba et al., 1985), and M A O - B (Vincent, 1989). A number of neuropeptides are found in the T M neurons, including galanin (Staines et al., 1986), T R H (Shirouzu et al., 1983), substance P (Kohler et al.,1985) and brain natriuretic peptide (Saper et al., 1989). The functional significance of the coexistence of so many substances remains to be determined. In addition to the T M nucleus, histamine neurons have been observed in the horizontal layer of the guinea pig retina (Yamamoto et al., 1987), in several sympathetic ganglia in the rat (Happola, et al., 1985) and in neuronal elements of the gut (Ekblad et al., 1985). The T M neurons provide a diffuse innervation of forebrain structures, including the cerebral cortex (Takeda et al., 1984, Watahabe et al., 1984), and striatum (Vincent et al., 1983, Steinbusch et al., 1986). A relatively dense innervation from the histaminergic T M neurons is present in the anterior hypothalamus of the rat (Staines et al., 1987) and specific structures such as the mesencephalic nucleus of the trigeminal nerve (Inagaki et al., 1987). Histamine fibres also project to the spinal cord (Wahlstedt et al., 1985), apparently without numerous axonal collateral projections to the forebrain (Takada et al., 1987). Because of the technical problems related to the small volume of the T M nucleus, few afferents to the T M neurons have been clearly identified. Retrograde tracer studies have shown an innervation of the T M from the medial preoptic nucleus (Wouterlood and Gaykema, 1988), the diagonal band of Broca (Wouterloud et al., 1988), and the prefrontal cortex (Wouterlood et al., 1987). Immunoelectron microscopic studies have demonstrated a number of synaptic inputs to T M neurons, including substance P (Tamiya et al., 1990), neuropeptide Y (Tamiya et al., 1989), and G A B A (Ericson et al., 1989). The net effect of these identified inputs presumably contributes to the regulation of histaminergic tonus. Histamine is able to interact with nervous tissue through at least three pharmacologically distinct binding sites in mammalian brain. Agonists of the H2 receptor stimulate the  7  formation of cyclic A M P (Hegstrand et al., 1976) in a manner which is synergistic with the action of noradrenaline (Sek et al., 1988). In monkey cortex, the half-maximal stimulation of adenylate cyclase occurs at a histamine concentration of 10 /iM (Newton et al., 1982).  The  histamine-sensitive adenylate cyclase is found in glia and the meninges as well as in neurons (Ebersolt et al., 1981). Activation of the H| receptor potentiates the effect of  agonists on  cyclic A M P formation in brain tissue (Palacios et al., 1978, Daly et al., 1980) and stimulates the hydrolysis of phosphatidylinositol (Fisher and Agranoff, 1987). A wide variety of antidepressant and antipsychotic drugs are able to block potently the histamine-stimulated formation of adenylate cyclase (Kanof and Greengard, 1978). Such effects are generally considered to be more closely related to side effects, rather than therapeutic actions, of these medications. Given the close proximity of the T M nucleus to the median eminence and pituitary, it is not surprising that important aspects of the regulation of neuroendocrine function in rat brain should be regulated via histamine receptors. Basal and oestrogen-stimulated L H release (Miyaki et al., 1987) are increased by activation of H| receptors. In contrast, TSH release is inhibited by H2 receptor activation (Di Renzo et al., 1987). The release of vasopressin is greatly enhanced by histamine (Dogterom et al., 1976), an effect mediated by both H j and H2 receptors (Cacabelos et al., 1987). Likewise, basal prolactin release is stimulated by either H j or H2 receptors, depending on the route of drug administration and the site of action (Knigge et al., 1988). The stress-induced release of prolactin is mediated by histamine, mostly via H2 receptors (Knigge et al., 1988). Thus, H j and H2 histamine receptors exert a variety of actions on the basal and evoked release of neurohormones. The H3 receptor, which is pharmacologically distinct from the classical H j and H2 sites, has recently been described as a presynaptic receptor site which controls the synthesis and release of histamine, i.e. an autoreceptor (Arrang et al., 1983). The potassium evoked release of [ H]-histamine is an index of autoreceptor activation. In such experiments, N a  methylhistamine was characterized as a prototypic H3 agonist (Arrang et al., 1983, van der  8 Wolf et al., 1987, Arrang et al., 1990) while thioperamide was described as an antagonist (Arrang et al., 1987). If the  binding site served principally as an autoreceptor, its  distribution in brain would be expected to correlate highly with the density of histaminergic innervation. However, a single autoradiograph of H^ binding (Arrang et al., 1987) suggests a pattern of binding inconsistent with the innervation pattern. Electrophysiological studies indicate that the post synaptic effect of histamine may be either excitatory or inhibitory (Geller, 1980), with the predominant action being inhibitory in cortical neurons (Haas and Wolf, 1977). Histamine is excitatory to supraoptic neurons via the H j receptor (Armstrong and Sladek, 1985), but excitation of hippocampal pyramidal cells is apparently produced via the H  2  receptor (Tagami et al., 1984). The latter excitation is based  upon the blockade of a calcium-dependent potassium current, which attenuates the afterhyperpolarization (Haas, 1984). The electrophysiological effects of specific H^ drugs have not yet been characterized.  9  II: The Analysis of Histamine in Cerebral Extracts  (A) Introduction  The quantitative analysis of trace levels of histamine present in cerebral extracts may be considered a classical problem in neurochemistry. Histamine does not have a distinctive ultraviolet absorption spectrum, nor is it, in contrast to the catecholamines, highly active electrochemically.  Histamine will react with formaldehyde to form a fluorescent species, but  the spectral characteristics are not readily distinguished from those of the catecholamineformaldehyde adducts, which are generally present in greater quantities. Most efforts to quantify histamine have relied upon derivatization procedures such as the o-phthaldialdehyde (OPA) method of Shore (1959). Histamine, when exposed to O P A at a basic p H , forms a fluorescent adduct. However, as O P A forms derivatives with many primary and secondary amines, it is important to avoid interference from compounds such as amino acids, catecholamines, and the polyamines, especially spermine and spermidine. Simple ion exchange purification of the extracts may remove the amino acids, but the highly basic polyamines are difficult to separate from histamine in a one-step procedure. Therefore, high performance liquid chromatography (HPLC) has been used to facilitate the analysis of histamine. In H P L C assays of histamine, the derivatization may be performed either before or after the analytical separation. In the present study, initial efforts were directed towards replication of literature methods for the analysis of histamine in biological samples by H P L C with precolumn derivatization (Tsuruta et al, 1981, Skofitsch et al., 1981). Cerebral extracts were passed through a strong cation exchanger (Cellex-P), amines were eluted with hydrochloric acid and the samples were lyophilized. After addition of O P A solution to the dried extract, portions were analyzed by reverse phase H P L C with fluorescence detection. However, because of variation in the recovery of histamine, the results were too unreliable for routine analysis. The OPA-histamine adduct being difficult to separate from other mono- and polyamine  ao  derivatives by means of H P L C , post-column derivatization methods have been developed (Yamatodani et al., 1985). Because equipment for the post-column method was not readily available, it was determined to employ a radioenzymatic assay for the analysis of histamine.  (B) Radioenzymatic Assay of Histamine and H N M T  H N M T , histamine and [ H]-labelled S A M ( [ H ] - S A M ) may be used to form radio3  3  labeled product, T - M e H A , which is readily separated from the methyl donor. As the enzyme recognizes only histamine and a few side-chain analogues (Dent et al., 1982), the specificity depends largely on the purity of the enzyme preparation. In the general method for the H N M T assay, samples (5-100 izl) were incubated in 15 ml plastic tubes (Falcon) in the presence of an H N M T preparation, [ H ] - S A M (New England Nuclear, 55-80 Ci/mmole), and 100 m M sodium phosphate buffer (pH 7.9). In some •3  experiments, the specific activity of [ H ] - S A M was reduced by the addition of various amounts of cold S A M (para-toluenesulfonate salt, Sigma). After incubation for between 15-60 minutes, reactions were stopped by the addition of 80 /xl potassium borate (1 M , p H 10) followed by the addition of 4 ml toluene/isoamyl alcohol (3:1, BDH).  The tubes were mixed  on a vortex (20 sec). After a brief centrifugation, 3 ml of the organic phase was extracted with 300 fi\ of potassium phosphate buffer (1 M , p H 7.1) by mixing on a vortex (20 sec).  The  tubes were recentrifuged and the organic phase was removed by aspiration. The aqueous phase was re-extracted with 2 ml toluene/isoamyl alcohol (vortex, 10 sec), briefly centrifuged, and the organic phase was removed by aspiration. 250 /il of the remaining aqueous phase was placed in a 20 ml plastic vial to which was added 6 ml of scintillation counting cocktail (Amersham, Aqueous Counting Scintillant). In order to permit the extraction of the tritiated product out of the strongly buffered aqueous phase and into the organic counting phase, the scintillant had been modified by the addition of 1.2% v/v bis (2-ethylhexyl)-hydrogen  11 phosphate (Aldrich), an organic anion. Radioactivity was determined by five minutes counting in a Packard Tricarb 4530 liquid scintillation counter with the windows set for tritium. In the present biochemical studies, four different H N M T preparations were utilized: a simple homogenate of rat brain ( H N M T ) , an ammonium sulfate fraction from rat brain ( H N M T j ) , a D E A E purified fraction from porcine kidney ( H N M T 2 ) , and a highly purified commercial enzyme prepared from foetal rat kidney ( H N M T 3 ) . Sensitivity and conditions for linear assay were determined for each preparation. Protein contents of the various preparations were determined by the method of Lowry et al. (1951). In some ex vivo experiments, H N M T activity was determined in crude homogenates from rat brain. Whole brain or specific regions were homogenized in four volumes of ice cold sodium phosphate buffer (100 m M , p H 7.9). 80 ul portions were incubated at 37 °C in the presence of 10 uM S A M (0.1 /tCi) and 10 uM H A in a final volume of 150 ul buffer.  The  enzyme activity was calculated in units of nmole product/mg wet weight-hour. H N M T j consisted of a crude ammonium sulfate fraction from whole rat brain (Brown et al., 1959, Taylor and Snyder, 1972). Whole rat brain was mechanically homogenized in 10 volumes of ice cold 0.25 M sucrose and centrifuged (37,000 x 60 min). From the supernatant, the 45-70% saturated ammonium sulfate fraction was taken. The final precipitate was dissolved in 2 ml phosphate buffer (10 m M , p H 7.4), dialyzed overnight at 4 °C in 4 1 of buffer and stored at - 2 0 °C. This preparation was stable for at least six months under these conditions. H N M T j was employed for H N M T inhibition assays to be described in Chapter IV, where the kinetic properties of the enzyme are also described. Under standard assay conditions, a mixture consisting of 2.5 /d H N M T j (20 ug protein) and histamine (10 uM, Sigma) and 0.1 uCi  H - S A M (10 /iM) in a final volume of 150 ul sodium phosphate (100 juM,  pH 7.9) were incubated at 37 °C. Under these conditions, the assay was linear for at least 30 minutes (Figure 2 - A ) .  12  Figure 2. The Linearity of H N M T Assays  (A) H N M T |, the rat brain fraction, produced a linear assay at times up to 30 minutes in the presence of 10 /zM histamine and 10 /xM S A M .  (B) H N M T 3 , the commerical preparation, was linear up to 250 pg H A when incubated for 60 minutes at 4 °C, as suggested by the manufacturer. Standard assay conditions were as specified in the text.  Histamine (pg)  14  H N M T 2 , which was used for the determination of histamine in tissue extracts, was prepared according to the initial steps of the method described by Harvima et al. (1985). Briefly, 100 g porcine kidney cortex were mechanically homogenized in 400 ml of ice-cold 30% sucrose. After centrifugation (37,000g x 60 min), the 45-70% ammonium sulfate fraction was taken from the crude supernatant and, after overnight dialysis, purified by D E A E sepharose ion exchange chromatography as follows: a D E A E column (20 x 1.5 cm) was equilibrated with 10 m M potassium phosphate (pH 7.4), 10% glycerol, 1 m M E D T A and 1 m M dithiothreitol (DTT, Sigma). After washing the column with 400 ml of the buffer, the kidney protein was eluted with a salt gradient (0-1 M NaCl) lasting eight hours at a flow rate of 67 ml/hour. The A gQ of each 15 ml fraction was determined (Spectronic 20, Bausch & Lomb) 2  and H N M T activity was estimated by incubation with H A (10 uM) and  H - S A M (0.1 uCi) for  30 minutes at room temperature. Results, as illustrated in Figure 3, indicate a substantial separation of H N M T from the main protein peak under these conditions. The D E A E peak fraction (65 ml) was concentrated on a 2 ml D E A E column and stored at - 2 0 °C in the presence of 20% glycerol; H N M T activity was stable for at least six months. The activities of the H N M T  2  fractions, calculated in units of rimole product/hour, determined by incubation  with 10 JUM histamine and 0.1 uCi [ H ] - S A M for 30 minutes at room temperature, indicated a 90-fold purification with an overall yield of 9% (Table 1). The H N M T  2  preparation was  devoid of detectable catechol-O-methyltransferase and indole-methyltransferase activities. The H N M T  2  assay was linear to 1000 pg H A (Figure 4 - A ) .  Addition of various  amounts of external H A standard to the extracts likewise produced a linear assay (Figure 4-B). In routine assays, the H A was quantified by means of a two point standard curve with triplicate determinations of buffer and buffer plus 100 pg H A standard. In three separate experiments, the sensitivity of the H N M T  2  assay was 25 ± 5 pg H A as defined by twice blank  and 7 ± 3 pg as defined by blank plus two standard deviations of the mean blank. H N M T j was a commercial preparation (New England Nuclear), purified approximately 200-fold from foetal rat kidney by the method of Verburg et al. (1983). Standard assay  15  Figure  3.  Purification of  HNMT2  by D E A E Chromatography  Crude ammonium sulfate fraction containing porcine kidney protein was eluted from a 25 cm D E A E column by a linear salt gradient to 1 M NaCl. H N M T activity (arbitrary units) and ^280  w  e  r  e  determined for each fraction. The fractions between the bars were pooled and  concentrated.  Table I. Purification of HNMT from Porcine Kidney  Crude supernatant Ammonium sulfate Unconcentrated D E A E Concentrated D E A E  Yield  Recovery  807 310 78 75  100% 39% 10% 9%  Activity (units/mg) 0.4 0.25 22 35  The yield at each stage is reported in activity units of nmole product formed per hour under standard conditions. Activity is calculated from the yield in units of nmole product hour ^ mg protein" . -  Figure  4.  Linearity of the Assay with  HNMT2  (A) H N M T 2 provided a linear assay for H A up to 1000 pg/sample.  (B) The addition of various amounts of external standard resulted in a linear increase in H A content of rat brain extracts, as determined with the H N M T 2 preparation. Each determination performed in triplicate.  19  Histamine Spike (ng/g)  20  conditions consisted of 25 ul sample, 2 ul enzyme, 25 ul buffer (100 m M , p H 7.9), and 1 uCi [ H J - S A M . According to the suppliers, optimum sensitivity was obtained with 1 hour incubations at 4 °C. In three separate experiments the sensitivty of H N M T j was 7 ± 2 pg/sample by the criterion of twice blank and 1.2 ± 0.3 pg/sample by the criterion of two standard deviations above the blank. The assay was linear to at least 200 pg H A (Figure 2B). The moderately greater sensitivity of H N M T j with respect to H N M T determination of the H A content of cerebral microdialysis samples.  2  indicated its use in the  21  III: Neurotoxicological Studies  (A) Introduction  Little is known about the sensitivity of the histamine neurons to neurotoxic challenge. If a selective neurotoxin were available, the biochemical and behavioral consequences of histaminergic deficiency could be better characterized. In the present study, attempts were made to interfere with histaminergic neurogenesis by exposure of foetal rats in utero to methylazoxymethanol ( M A M ) , an inhibitor of mitosis (Nagati and Matsumoto, 1969).  Another  toxicological study concerned the sensitivity of the mouse histaminergic system to the neurotoxin, 1 -methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).  i) MAM Methods  Because specific classes of neurons differentiate at different ontogenic stages, it is possible to block the development of specific lineages of neurons by administration of a mitotic inhibitor such as M A M . In the present study, precisely timed female Sprague-Dawley rats were administered M A M (25 mg/kg, i.p.) on G15 (Vincent et al., in press), while control dams received saline. Progeny were sacrificed at the age of six months. In six normal and six M A M - t r e a t e d animals the regional content of histamine was determined in striatum, cortex, and hypothalamus and the H N M T activity was determined in neocortex.  ii) MPTP Methods  M P T P produces long lasting depletions of forebrain dopamine (Burns et al., 1983). The complete mechanism of toxicity remains to be elucidated, but an essential step involves the production of the metabolite N-methyl-4-phenylpyridinium ion ( M P P ) by the action of +  22  Table II. Effect of Prenatal MAM Exposure on Histamine Content and HNMT Activity in the Rat Brain Tissue  Histamine (ng/g)  mg sample  ng total  33.9±2.5 44.1+1.8 (130%)  a  63.7+1.2 36.5+2. P (57%)  2.15±0.14 1.6±0.08 (75%)  1.68±0.06 0.92+0.07 (55%)  53.8±4.1 66.5±1.8 (124%)  a  37.6±1.6 27.3+1.4 (73%)  2.02+0.19 1.82+0.11 (90%)  0.87+0.07 0.58±0.05 (67%)  30.1±3.5 28.1+3.4 (93%)  1.19+0.16 1.27+0.09 (107%)  H N M T Activity nmoles/mg/hour  Hippocampus Control MAM  a  t  Striatum Control MAM  l  l  Hypothalamus Control MAM  696±72 561±49 (81%)  40+3.9 50.1±4.6 (124%)  The histamine content in control (n=8) and M A M treated (n=6) animals is reported in ng/g wet weight. Values are mean ± S.E.M. of n determinations. The third column inducates the ng total histamine corrected for the weights of the samples. M A M values as a percentage of control values are indicated in parenthesis. a  p<0.025;  b  p<0.001, Student's two-tailed t-test.  23  MAO-B.  The toxic metabolite subsequently accumulates within dopamine terminals via the  high affinity dopamine uptake system (Sundstrom and Jonsson, 1986) and ultimately destroys these neurons. The site of generation of the M P P  +  relevent to neurotoxicity remains  uncertain, but M A O - B dependant oxidation of M P T P has been demonstrated to occur in serotonin and histamine neurons (Vincent, 1989). Since the histamine neurons contain M A O - B and provide a widespread innervation throughout the brain, it is a possibility that they may participate in the production of M P P  +  within the basal ganglia.  In order to approach this issue, experiments were conducted to determine the sensitivity of the H A neurons to a dose of M P T P previously shown to deplete striatal dopamine (Radke et al., 1987). Young male C-57 mice weighing 25 g were given either 50 mg/kg M P T P hydrochloride (i.p., Research Biochemicals) or saline on two consecutive days and were decapitated two weeks after the last injection. Striatum, hypothalmus, neocortex and hippocampal samples were dissected, weighed and frozen at -70 °C until analysis. Striatal dopamine ( D A ) , serotonin (5-HT) and their metabolites were determined as described previously (Jakubovic et al., 1987). Briefly, tissues were extracted in 1 ml 0.2 M perchloric acid, centrifuged and analyzed by reverse-phase H P L C with electrochemical detection (Nucleosil C j g , 150 x 4.6 mm, Pharmacia). Histamine content of the other three tissues was determined using the H N M T 2 radioenzymatic assay.  (B) Results of Toxicological Studies  i) M A M Results  The regional histamine content and the cortical H N M T activity determined in the adult rat after M A M treatment at embryonic day 15 are indicated in Table II. Histamine concentration was elevated in forebrain structures, but these effects seemed to be due to reduced tissue volumes in forebrain structures of the adult M A M - t r e a t e d brains.  HNMT  24  activites were reduced in hippocampus (-45%) and striatum (-33%), but were not significantly affected in hypothalamus. In tritiated thymidine studies, neurogenesis of H D C immunoreactive neurons occurred as early as embryonic day 13, but the peak of final mitosis is on day 16, which is rather late in gestation of the rat (Reiner et al., 1988). In the present study, M A M was administered on day 15, which should have killed any neuroblasts which would give rise to the histamine lineage dividing at that time. However, MAM-treatment kills actively dividing cells, not cells at other stages of the mitotic cycle. Therefore, the preservation of histamine content in the M A M rats suggests that a population of histamine neuroblasts was not dividing or was otherwise M A M resistant on embryonic day 15. If H A neurons were reduced in number by prenatal M A M treatment, the residual neurons were presumably able to provide a proportionally greater H A innervation, perhaps in response to trophic factors in the forebrain. However, the effect of prenatal M A M on the number of H A neurons in the adult was not determined in the present study. Interestingly, H N M T activity in the M A M - t r e a t e d brain seemed to be more decreased by the MAM-treatment than were the histamine levels. As discussed above, the cellular localization of H N M T has not been well characterized. However, the M A M data suggest that a large proportion of H N M T activity in the adult forebrain appears to be derived from cells with final mitotis on or after embryonic day 15, the day of M A M administration.  ii) M P T P Results  The dose-dependent striatal dopamine depletions in mice receiving M P T P are illustrated in Figure 5 (Cumming et al., 1989). In the M P T P animals, D A declined to 28% of normal values and the concentrations of the dopamine metabolites, dihydroxyphenylacetic acid (DOPAC), homovanillic acid ( H V A ) , and 3-methoxytyramine ( 3 - M T ) , were reduced to a lesser extent.  5 - H T and its metabolite 5-hydroxyindoleacetic acid ( 5 - H I A A ) were not significantly  2'5  Figure 5. Alterations in Dopamine and Metabolites After MPTP Lesions  The catecholamine and indoleamine content in mouse striatum, determined two weeks after saline (n = 8), or M P T P poisoning (n = 6), is represented as percentage of control values. Control values (ng/g) are: Dopamine: 11860 ± 290, D O P A C : 1860 ± 140 ng/g, H V A : 1543 ± 40, 3 - M T : 877 ± 47, 5 - H T : 408 ± 13, 5 - H I A A : 426 + 42. Results are indicated ± S.E.M.  (a) p<0.05, (b) p<0.001, Student's two-tailed t-test.  Table III. Effect of MPTP Treatment on Histamine Levels in the Mouse Brain.  Saline MPTP  Hypothalmus  Hippocampus  Neocortex  155 ± 17 170 ± 19 (110%)  42.6 ± 3.8 52.7 + 7.7 (124%)  29.9 ± 2.3 28.8 ± 2.7 (96%)  Histamine (ng/g) in three brain regions two weeks following two daily injections of either saline (n = 8) or M P T P (2 x 50 mg/kg i.p., n = 6). Results are reported as ng/g wet weight S.E.M. M P T P values are also reported as a percentage of control values.  28 altered by the treatment. Histamine levels, also, were not significantly affected by M P T P treatment (Table III), although there was a trend towards elevated concentrations in the hippocampus. Concentrations of striatal dopamine, serotonin and metabolites in normal animals and the dopamine depletions produced in M P T P animals are similar to those reported in an earlier study (Radke et al., 1987) in which striatal noradrenaline was also determined and found to be 50% decreased in M P T P animals. However, in the present study, cerebral levels of histamine and the indoleamines were unaffected by M P T P treatment. The selectivity of M P T P for some of the ascending aminergic projections may be relevant to the relationship between the M P T P model and idiopathic Parkinson's disease, although it must be noted that C-57 mice are much less sensitive to M P T P than are the primates.  29  IV: Inhibtion of H N M T  (A) Introduction  Because of the lack of high affinity uptake mechanisms or alternate catabolic routes, the major route for inactivation of cerebral histamine appears to involve the enzyme H N M T . Therefore, this enzyme serves as a metabolic bottleneck through which all cerebral histamine must pass. Thus, inhibitors of H N M T , of which there are many, have the potential to alter histamine levels. Indeed, metoprine, a diaminopyridine which potently inhibits H N M T , produces a long-lasting elevation of rat brain histamine levels (Duch et al., 1978).  Lengthy  lists of H N M T inhibitors have been compiled and attempts have been made to account for inhibition in terms of common structural features (Tachibani et al., 1988, Harle and Baldo, 1988). However, the diversity of known H N M T inhibitors suggests that the binding site or sites for substrates and inhibitors may have complex properties. 9-Amino-l,2,3,4-tetrahydroacridine ( T H A ) is structurally similar to the quinacrinederived antimalarial alkaloids, which are potent inhibitors of H N M T (Harle and Baldo, 1988). T H A has been long known to be an acetylcholinesterase inhibitor (Kaul, 1962), which was the rationale for its experimental use as a therapeutic drug in Alzheimer's disease (Summers et al., 1986). Inhibition of H N M T by molecules with an aminoacridine nucleus has been previously noted, but not characterized (Duch et al., 1979). A radioenzymatic assay was employed to study the sensitivity of rat brain H N M T activity to inhibition by T H A , which was found to be one of the most potent H N M T inhibitors yet described. This inhibition was compared to that produced by various structural fragments and analogues of T H A as well as other compounds, including metoprine, a potent H N M T inhibitor (Duch et al., 1980), and physostigmine, a classical acetylcholinesterase inhibitor.  30  Based on the results of the study of the structural requirements for H N M T inhibition, a number of ^-carboline alkaloids were screened for their ability to inhibit H N M T . The pcarbolines, of which harmaline is a prototype, were found to be a new class of moderately potent H N M T inhibitors. Subsequent to the characterization in vitro of the inhibition of H N M T by T H A , the degree of H N M T inhibition produced in rat cerebral cortex was determined ex vivo at two hours after the peripheral administration of metoprine or T H A . The effects of blockade of cerebral H N M T with T H A or metoprine administration on the histamine content of cortex, striatum and hypothalamus were determined.  (B) Materials and Methods  The following compounds were employed in H N M T inhibition studies: metoprine (Burroughs-Wellcome, prepared in 5% lactic acid), physostigmine HC1, (Aldrich), 9-amino1,2,3,4-tetrahydroacridine hydrochloride ( T H A , Sigma), 9-aminoacridine hydrochloride (Fluka), 4-aminopyridine (4-AP) and 3-aminopyridine (Sigma), quinidine, 4-aminoquinaldine, 6-aminoquinoline, 8-aminoquinoline, and 8-amino-tetrahydroquinoline (Aldrich). carboline compounds,' harmalol hydrochloride dihydrate b]indole), harmol (l-methyl-9H-pyrido[3,4-b]indole),  The f$-  (4,9-dihydro-l-methyl-3H-pyrido[3,4-  harmine hydrochloride hydrate (7-  methoxy-1 -methyl-9H-pyrido[3,4-b]-indole), harmaline (4,9-dihydro-7-methoxy-1 - m e t h y l 3H-pyrido[3,4-b]indole), harmane (l-methyl-9H-pyrido[3,4-b]-indole, aribine), norharman (9H-pyrido[3,4-b]indole) and 3-amino-l-methyl-5H-pyrido[3,4-b]indole were from Aldrich. Inhibition studies were conducted using rat brain H N M T j under standard conditions as described in Chapter II. H N M T j activity was determined in the absence of inhibitors and at 3  various final inhibitor concentrations ranging between 10 concentrations for 50% inhibition  (IC^Q) were  lines of the logit-log inhibition plots.  9  and 10  M . Inhibitor  determined by interpolation of the regression  31  Kinetic parameters for the inhibition by T H A were determined by Lineweaver-Burke analysis (Lineweaver and Burke, 1934). Rat brain H N M T j was employed under standard assay conditions, with one substrate at a fixed final concentration of 6.7 uM and the other substrate at five concentrations between 1-10 uM. In the kinetic studies, T H A was included at four different concentrations between 50 and 200 n M , while harmaline was included at three concentrations between 0.5 and 1.5 uM. Kjs were calculated from the apparent K s in the m  presence of the various inhibitor concentrations according to the expression K M / K m = {1 + m  [I]/Kj]} where K  m  ^  is the apparent K  m  in the presence of the inhibitor (Gutfreund, 1965).  Replotting the data by the method of Dixon yielded almost identical results (Dixon, 1953). Each assay was conducted in triplicate. Statistics, where reported, are ± S.E.M. of (n) determinations. For in vivo experiments, groups of five male Long-Evans rats weighing between 200 and 250 grams were administered either saline, T H A (5 and 10 mg/kg), or metoprine (10 mg/kg) by the intraperitoneal route. The rats were killed two hours later by cervical dislocation and samples of neocortex, striatum and hypothalamus were dissected, weighed and stored at -70 °C until analysis, no more than two weeks later. H N M T activity in cortex samples was calculated as nmole product formed/minute-20 mg tissue and reported as the mean ± S.E.M. of five determinations. Activity in samples containing H N M T inhibitors was also calculated as a percentage of the mean activity in control cortex samples. Concentrations of histamine in cortex, striatum and hypothalamus after administration of T H A or metoprine were determined employing a radioenzymatic assay using H N M T as 2  described above (Cumming et al., 1989). It was necessary to determine the linearity of the assay in the presence of known H N M T inhibitors. Therefore, between 0 and 250 pg of H A standard was added to portions of the pooled extracts of two whole rat brains. Two hours prior to sacrifice, the rats had been treated with either saline (1 ml/kg, i.p.), T H A (10 mg/kg, i.p.) or metoprine (10 mg/kg, i.p.). The histamine assay was found to be linear both in the  32  Figure 6. Linearity of the H N M T 2 Assay in Presence of Inhibitors.  H A was determined in brain extracts to which had been added various amounts of exogenous histamine. Extracts were derived from animals which, two hours earlier, had received either saline, T H A (10 mg/kg) or metoprine (10 mg/kg). Results are ± S.E.M. of four determinations.  34  presence and absence of inhibitors (Figure 6). The slopes of the standard curves determined in the presence of inhibitors were considerably reduced with respect to the slope of the assay of histamine standards alone, but the assay still had sufficient sensitivity to permit quantification of the histamine present in the extracts. In the study of the effect of T H A and metoprine on brain H A levels, the apparent histamine content of each extract was determined. Each sample was re-analyzed after the addition of 100 pg external standard H A .  The recovery of the standard was quantitative in  the case of samples from control animals and ranged from 30-75% in the samples containing the H N M T inhibitors. Linearity of the assay under these conditions having been demonstrated, histamine concentrations were corrected for individual standard recoveries and reported as ng/g.  (C) Results of H N M T Inhibition in vitro  The inhibition of H N M T by metoprine, T H A , physostigmine and 4 - A P are illustrated in Figure 7. Metoprine was the most potent inhibitor tested, with an IC^Q of 56 n M , while T H A had an IC^Q of 74 n M . Physostigmine inhibited H N M T with an IC^Q of 8 fiM and 4 - A P with an I C  5 0  of 28  uM.  The log-logit analysis of the inhibition of H N M T by T H A and eight related compounds is illustrated in Figure 8. IC^QS were determined by interpolation of the regression lines. T H A was the most potent inhibitor in the series, with an IC^g of 130 n M , which is similar to the value determined from Figure 7. The T H A fragment 4-aminoquinaldine, (IC^Q = 790 nM) was f i v e - f o l d less potent than T H A itself and 9-aminoacridine (IC^Q = 2.8 uM) was 20 fold less potent than T H A .  Quinidine, a quinoline alkaloid structurally related to quinacrine and  quinine, was a moderately potent inhibitor, with an IC^Q of 8 uM. 4-Aminopyridine (IC^Q = 25 /zM) was nearly 100 fold more potent than was 3-aminopyridine (IC^Q of 1.9 mM).  The  Figure 7. Inhibition of H N M T by some compounds.  Inhibition of H N M T , by, from left to right, metoprine, T H A , physostigmine and 4 aminopyridine. Each point represents the mean of at least three determinations. S.E.M.s, omitted for clarity, were always less than five percentage units. Enzyme activity in the absence of inhibitors (V ) was 150 pmole/min/mg protein.  HNMT activity (% Inhibition) ro  9e  (ji O  -vj Oi  O O  Figure 8. Logit-log analysis of the inhibition of rat brain H N M T by T H A and related compounds.  Rat brain H N M T was incubated in the presence of histamine (6.7 m M ) , [ H ] - S A M (10 mM) J  and various concentrations of the following inhibitors: (a) T H A , IC^Q = 130 n M , (b) 4aminoquinaldine, IC^Q = 790 n M , (c) 9-aminoacridine, IC^Q = 2.8 /iM, (d) quinidine, IC^Q = /iM, (e) 4-aminopyridine, IC^Q = 24 / i M , (f) 6-aminoquinoline, IC^Q = 540 m M , (g) 8aminoquinoline, IC^Q = 890 m M , (h) 8-amino-tetrahydroquinoline, IC^Q = 1.3 m M , and (i) 3 aminopyridine, IC^Q = 1.9 m M . Enzyme activity in the absence of inhibitors ( V ) was 100 Q  pmole/min-mg protein. IC^Q values were estimated by interpolation of the linear regression lines.  OJ 00  39'  three aminoquinolines studied, in which the amine was in the 6 - or 8 - positions, were all poor inhibitors, with IC^gS in the m M range. H N M T inhibition plots for different concentrations of some ^-carbolines are illustrated in Figure 9, Harmine (IC^Q = 1.9 uM) and harmaline (IC^Q = 4.4 uM) differ only with respect to the partial saturation in the 3-4 position. The saturation is evidently without great influence on H N M T inhibition. Likewise, harmol (IC^Q = 2.4 uM) and harmalol (IC^Q = 1.7 uM) were nearly equipotent H N M T inhibitors. The similar results obtained for the above four molecules indicate that the presence of the 7-methoxy group does not contribute to H N M T inhibition. However, harmane (IC^Q = 7 /iM), which is unsubstituted in the 7-position, seems to be a somewhat poorer inhibitor than the others and norharman (IC^Q = 23 /iM) , which lacks the 1-methyl group, was poorer still. The least effective inhibitor in the series was 3 a m i n o - l - m e t h y l - 5 H - p y r i d o - [3,4-b]indole (IC^Q = 41 /iM), which is actually not a )9-carboline as it lacks a ring nitrogen in the 2-position.  Among the ^-carbolines, neither substituents in  the 7 position nor the partial saturation of the first ring greatly change the H N M T inhibition. However, the 1-methyl group and the orientation of the aromatic nitrogens seem to influence potency as H N M T inhibitors. The K  m  of the rat brain enzyme preparation for H A was 3.3 ± 0.3 /iM (Figures 1 0 - A ,  11-A) and the K_  m  for S A M was 4.3 ± 1.0 uM (Figures 10-B, 1 IB).  These data are the mean  of, respectively, three and four determinations, of which two are illustrated. The V  m  a  x  was  197 ± 4 pmoles/minute-mg protein. As H N M T was subject to substrate inhibition at histamine concentrations above 10 /iM, all assays were conducted in the appropriate range of concentrations. The above kinetic properties are comparable to those reported for purified guinea pig brain H N M T (Matuszewska et al., 1983). T H A inhibited H N M T in a manner which was competitive with respect to histamine (Figure 1 0 - A , K j = 35 ± 6 n M , n=3) and displayed mixed competition with respect to S A M (Figure 1 0 - B , K j = 39 ± 5 n M , n=4). The results of the kinetic study of the inhibition of H N M T by harmaline indicate that harmaline is a competitive inhibitor with respect to H A  40  (Figure 1 1 - A , 1.4 ± 0.4 /iM, n=3) and also S A M (Figure 1 1 - B , 1.4 ± 0.2 / i M , n=3).  Analysis  of all these data by the method of Dixon yielded nearly identical results. The structures of some of the H N M T inhibitors used in the present study are illustrated in Figures 7 and 9. S-Adenosyl-L-homocysteine is reported to be a potent H N M T inhibitor, but the corresponding deaminated compound S-inosyl-homocysteine is a poor inhibitor (Zappia et al., 1969). This illustrates the importance of the 6-amino group on the basic moiety for cosubstrate binding (Figure 9, inset). H N M T inhibition is insensitive to the configuration of the asymmetric carbon in S-adenosylhomocysteine, while other methyltransferases were much less inhibited by D-analogs (Borchardt and Wu, 1974). These findings are further indication of the importance of the basic portion of S A M for binding to H N M T . Examination of some of the structures in Figures 7 and 8 suggests that T H A is an H N M T inhibitor by virtue of the 4-aminoquinoline structure, which may be superimposed on the 6-membered ring of the adenine moiety of S A M (Figure 9, inset). In contrast, 6 - and 8 aminoquinolines were poor inhibitors, as was 3-aminopyridine. It is apparent that the integrity of the ring system is necessary for full H N M T inhibition by molecules in the T H A series. As 9-aminoacridine was much less potent than T H A , it appears that the degree of saturation of the acridine ring system can greatly alter the IC^Q for H N M T .  The  aminopyridine and aminoquinoline results indicate the importance of the orientation of the amino group with respect to the aromatic ring: only 4-amino compounds were good inhibitors. However, 4-aminopyridine was not a very good H N M T inhibitor. The importance of the complete T H A ring structure for full inhibition suggests that hydrophobic interactions are involved in inhibitor binding. The competitive inhibition of H N M T by harmaline (Figure 11), which also may be superimposed on the basic portion of S A M and on a conformation of histamine (Figure 9, inset), suggests that the /3-carbolines may directly interfere with the binding of both H N M T substrates.  41  Figure 9. Logit-log analysis of the inhibition of rat brain HNMT by some /3-carbolines.  Rat brain H N M T was incubated in the presence of histamine (6.7 /iM), [ H ] - S A M (10 /xM) J  and various concentrations of the following inhibitors: (a) harmalol, IC^Q = 1.7 /xM, (b) harmol, IC^Q = 2.4 /xM, (c) harmine, IC^Q = 1.9 /iM, (d) harmaline, IC^Q = 4.4 /xM, (e) harmane, IC^Q = 7.0 / i M , (f) norharmane, IC^Q = 23 /xM, and (g) 3 - a m i n o - l - m e t h y l (pyrido)indole, IC^Q = 41 /xM. Enzyme activity in the absence of inhibitors ( V ) was 100 Q  pmole/min/mg protein. IC^Q values were determined by interpolation of the linear regression lines. Inset: structures of histamine and the adenine moiety of S A M are illustrated.  43  Figure 10. Double reciprocal plots of the inhibition of H N M T by  THA  (A) Double reciprocal plots of the inhibition of H N M T by 55 n M and 110 n M T H A with [ H]-S-adenosylmethionine held at 10 uM and histamine varying between 1 and 10 uM.  Each  point represents the mean of three determinations which varied by less than five percent.  (B) Double reciprocal plots of the inhibition of H N M T by 100 n M and 200 n M T H A with histamine held at 6.7 fiM and [ H]-S-adenosylmethionine varying between 1 and 10 uM. Each point represents the mean of three determinations which varied by less than five percent.  45  Figure 11.  Double reciprocal plots of the inhibition of rat brain HNMT by harmaline.  (A) The concentration of histamine was varied between 1-10 /xM and [ H ] - S A M held at 10 3  fiM. Harmaline was included at 0, 0.75, and 1.0 ixM in this experiment. In a separate experiment, inhibition was determined in the presence of 1.5 /xM harmaline. The structure of harmaline is illustrated.  (B) The concentration of [ H ] - S A M was varied between 1-10 /xM and histamine held at 6.7 J  xzM. Harmaline was included at 0, 0.75 and 1.0 and 1.5 /xM.  47  (D) Results of H N M T inhibition in vivo  The ex vivo inhibition of cortical H N M T activity by T H A and metoprine is illustrated in Table IV.  T H A produced a dose dependent inhibition of cortical H N M T .  Metoprine was  more potent than T H A , producing nearly complete inhibition. This could be due to its slightly greater intrinsic potency or to greater penetration into brain tissue. The concentrations of T H A and metoprine present in the in vitro assay following in vivo administration could be estimated by interpolation of the percentage inhibition from the inhibition curves for rat brain H N M T (Figure 7). Given a ten-fold dilution of inhibitors in cortex under the conditions of the assay, an approximation of the inhibition in vivo could be made by interpolation one logjQ unit to the right on the inhibition curves. This provides only an estimation of the inhibition in vivo, but it is apparent that substantial inhibition of H N M T in cerebral cortex occurs at two hours after i.p. administration of moderate doses of the H N M T inhibitors. The results of the determination of histamine concentrations in brain tissues after peripheral administration of H N M T inhibitors are illustrated in Figure 12. Histamine concentrations for control rat brain areas were similar to those reported previously (Oishi et al., 1984). T H A produced a dose-dependent, approximately two-fold increase in cortical histamine. Metoprine produced a nearly three-fold increase in cortical histamine, consistent with its greater potency as an H N M T inhibitor. The drugs also elevated histamine in striatum, but the magnitude of the increases were lower than in cortex.  In hypothalamus, a trend  towards increased histamine levels did not reach significance in the T H A groups and was marginally significant in the metoprine group. The ability of a compound to inhibit H N M T in vitro does not necessarily indicate that it will discernibly influence histamine metabolism in vivo (Hough et al., 1988). The above data indicate that T H A , as well as metoprine, are able to inhibit cerebral H N M T and greatly increase cerebral histamine levels at two hours after a peripheral dose. Given that T H A is a lipophilic molecule, it seems likely that repeated adminstration of moderate doses of T H A , as  48  has been proposed in the experimental treatment of Alzheimer's disease (Summers et al., 1986), could produce a cumulative inhibition of H N M T in the central nervous system.  49  Table IV.  Inhibition of Cortical H N M T Activity Activity nmole/min-20 mg  % Inhibition in vitro  % Inhibiti :ion in vivo^ ' a  Control  14.5 ± 0.9  THA 5 mg/kg  8.9 ± 0.4  39%  75%  THA 10 mg/kg  6.4 ± 0.8  56%  85%  Metoprine 10 mg/kg  1.9 ± 0.2  87%  97%  Two hours after i.p. drug injections, H N M T activity was determined in cortical homogenates. Values are in units of nmole/min-20 mg ± S.E.M. (n=5). Inhibition in vitro is also reported as a percentage of control activity, (a) Inhibition in vivo was estimated from the inhibition curves in Figure 7 as follows: To correct for ten-fold dilution of the inhibitors during the assay, the concentrations of inhibitors in vitro were estimated by interpolation of the inhibition curves. The in vivo inhibitions were taken from interpolations of the curves at concentrations one log unit to the right. b  P<0.001, Student's two-tailed t-test.  Figure 12.  H N M T inhibitors elevate cerebral histamine levels  Animals were sacrificed at two hours after saline, T H A (5 and 10 mg/kg) or metoprine (lOmg/kg).  Histamine content in brain tissues from control animals were: neocortex (36.7  1.0 ng/g), striatum (36.5 ± 1.6 ng/g), and hypothalamus (349 % 25 ng/g).  (a) P<0.1, (b) P<0.025, (c) P<0.001 Student's two-tailed t-test.  51  Histamine Levels at Two Hours After i.p. HNMT Inhibitors  600 550 500  C =. control 1 = THA, 5 mg/kg  450  2 = THA, 10 mg/kg 3 = metoprine, 10 mg/kg  o> 400 <D  .E E  350  <o  «—> vt  T  * *  150  •3C*  •X- " T •X- *  100  *  50 0  ~i  i  i  C  1 2 3 Cortex  r  l  C  1 2 3  Striatum  C  r  1 2 3  Hypothalamus  52"  V:  Cerebral Microdialysis  (A) Introduction  Brain histamine metabolism has generally been studied in  ex vivo  experiments employing  •3  tissue extracts and slice preparations. The synthesis and release of [ H]-histamine by brain slices has been shown to be under the regulation of a variety of factors including histamine H3 autoreceptors (Arrang et al., 1987), and a^-adrenergic receptors (Gulat-Marnay, 1989).  In vivo  release of histamine has been demonstrated by means of push-pull cannulae in the hypothalamus of conscious rabbits and anesthetized cats (Philippu et al., 1982, Prast et al., 1989). The study of cerebral histamine by microdialysis has recently been reported in acute anesthetized preparations (Yamatodani et al., 1989, Russell et al., 1990). In the present study, cerebral microdialysis was employed in order to characterize some of the factors regulating the extracellular levels of histamine in the brain of freely-moving, unanesthetized rats. To this end, a sensitive radioenzymatic assay was employed in order to quantify histamine in cerebral dialysate samples. The dorsal striatum was chosen as the principle target of these studies because it receives innervation from the tuberomammilary nucleus (Steinbusch et al., 1986, Vincent et al., 1983), and because of possible interactions between histamine and psychomotor behavior (White and Rumbold, 1988). Some studies were conducted in the bed nucleus of the stria terminalis (BNST) because this region contains one of the highest histidine decarboxylase (HDC) activities in the rat nervous system ( B e n - A r i et al., 1977). (B) Materials and Methods  The protocol for cerebral microdialysis was based on methods described previously for the study of dopamine and its metabolites (Imperato and DiChiara, 1984, Westerink and  53  Tuinte, 1986) Briefly, male rats of the Wistar strain (200-300 g) were anesthetized with sodium pentobarbitol (70 mg/kg i.p.) and placed in the stereotaxic apparatus (Kopf).  After  exposure of the skull, holes were drilled in the temporal bones so as to permit the placement of a horizontal dialysis probe. Two brain areas were targeted in this study: dorsal striatum (V: -4.75, A: +0.7) and the BNST (V:-6.60, A:-0.5) according to the coordinates of Paxinos and Watson (1986). Each dialysis fibre, consisting of saponified cellulose ester (o.d. = 0.27 mm, 10 k D cutoff, Cordis Dow Medical), was covered with glue except for two 3.5 mm lengths for the dorsal striata and two 1.7 mm lengths for the BNST.  Stainless steel cannulae, glued to both  ends of the dialysis fibre, were secured with dental cement to three anchoring screws on the top of the skull. After surgery, rats were singly housed in cubical Perspex cages (40 cm) and allowed free access to food and water. Microdialysis perfusion experiments were carried out 24-72 hours after surgery. Experiments began at 11 A . M . and lasted no more that five hours, during which time the rats were unrestrained and allowed free access to food and water. The steel cannulae were connected to polyethylene tubing (i.d. = 0.28 mm, length = 80 cm) and the dialysis fibres were perfused with a solution containing 147 m M N a C l , 3 m M K C 1 , 1.3 m M C a C ^ , 1.0 m M M g C ^ and 1.0 m M sodium phosphate (pH 7.4) at a rate of either 3 or 5 /il/min. Samples were collected for 20 minute periods in Eppendorf tubes and frozen at -20 °C until the day of analysis. Some brains were taken for histological verification of the membrane placement. In the cerebral microdialysis study, the highly purified H N M T ^ was used because of its greater sensitivity. The histamine content in 25 n\ volumes was determined and calculated as pg/minute sample. Because of the nature of a radioenzymatic assay, the possibility of interference from inhibitors must always be considered. Therefore, in experiments where the composition of the perfusion solution was changed, linearity of the assay was redetermined. In experiments involving peripheral administration of drugs, 100 pg H A was added to portions  54  of perfusion samples taken at one hour after the drugs. The percentage recovery of the external standard was used in order to assess possible interference in the assay. Because within-group variation in basal histamine release was considerable (two-fold range), data were normalized to the percentage of mean basal values for each animal. Each drug treatment group contained four or five animals, and results are reported as the mean ± S.E.M. The results of some experiments were analysed by two-way analysis of variance ( A N O V A ) with time as the repeated measure. Initial cerebral microdialysis studies were carried out to determine the effect of tetrodotoxin on extracellular histamine. Animals with implants in striatum (n = 4) or BNST (n = 5) were dialyzed as above. After the collection of four basal samples, tetrodotoxin  (TTX,  Sigma, 300 nM) was introduced into the perfusion solution. Thirty minute transitional samples were discarded prior to the collection of four 20 minute samples. On the day after the T T X experiments, the same animals were used in experiments to determine the calcium dependence of extracellular histamine. After the collection of four basal samples with normal perfusion solution (Ca^ = 1.3 m M ) , animals were perfused with an otherwise identical calcium-free +  solution. A thirty minute transitional sample was discarded prior to collection of four further samples taken for analysis. To study the turnover of extracellular histamine in striatum, animals were treated with the H D C suicide substrate a-fluoromethylhistidine ( a - F M H , 100 mg/kg, i.p.), which irreversibly inhibits the enzyme. Samples were collected at 20 minute intervals for a period of six hours after synthesis blockade, and additional samples were collected 24 hours later. In order to determine the effect of amino acid precursor loading on striatal histamine release, L-histidine was administered at a dose of 500 mg/kg as the free amino acid (Sigma, i.p., 200 mg/ml in 2.5% lactic acid). Samples were collected for four hours after histidine adminstration. To study the effect of adrenergic drugs on extracellular histamine levels in the BNST, five 20 minute basal samples were collected, followed by the administration of the  agonist  55  c l o n i d i n e (0.3 mg/kg i.p., Sigma). A f t e r collection o f f i v e f u r t h e r samples, the  antagonist  y o h i m b i n e was administered (10 mg/kg i.p., Sigma) and f i v e f i n a l samples were collected and f r o z e n f o r subsequent analysis.  (C) Results  The results o f dialysis experiments i n w h i c h striatum and B N S T were perfused w i t h a solution containing T T X at a concentration o f 300 n M are illustrated i n F i g u r e 13.  TTX,  w h i c h blocks voltage-dependent sodium channels, was without effect on the extracellular histamine concentration i n either striatum or B N S T .  R e c o v e r y of the added histamine i n  T T X - c o n t a i n i n g samples was quantitative. T h e mean basal histamine content of the dialysates were 3.8 ± 0.7 (n=14) pg/minute i n the striatum and 6 ± 0.9 pg/minute (n=8) i n the B N S T . The omission o f C a ^  +  f r o m the perfusion solution (Figure 14) produced a 2 0 % decrease  i n extracellular histamine levels i n B N S T (p = 0.004) and also i n striatum (p = 0.013). O m i s s i o n of c a l c i u m d i d not alter the sensitivity o f the histamine assay. A f t e r i n j e c t i o n o f a - F M H there was a r a p i d , long-lasting disappearance of histamine o u t f l o w i n striatum (Figure 15), w h i c h resolved into a b i - e x p o n e n t i a l f u n c t i o n . T h e fast component, w h i c h had a h a l f - l i f e of 32 ± 4 minutes, was calculated f r o m the residuals of the partial decomposition after subtraction of the slow component, w h i c h had a h a l f - l i f e 7.7 ± 2.2 hours. E x t r a p o l a t i o n of the two regression lines to t  Q  indicated that the r a p i d l y t u r n i n g - o v e r  pool was approximately three times larger than the slow pool. Thus, after two hours, histamine declined to 2 5 % of basal values.  A t 24 hours after synthesis blockade, histamine  levels had returned to 6 7 % of basal values.  T h e recovery of the added histamine i n a dialysate  taken at one hour after drug administration was 104 ± 1 1 % , i n d i c a t i n g a lack of interference f r o m a - F M H i n the assay. A f t e r peripheral histidine loading, histamine o u t f l o w i n striatum increased to 180% of basal levels (Figure 16) over a period of one hour.  Thereafter, values declined nearly to basal  56  Figure 13. The effect of T T X on the in vivo efflux of histamine.  After the collection of four 20 minute basal samples, the perfusion solution was modified by the addition of 300 nM TTX. The thirty minute transitional sample was discarded and four subsequent samples were collected for analysis. Data were normalized to the percentage of the mean basal value and are here reported as the mean of four separate determinations.  58  Figure 14. The effect of the omission of calcium from the perfusion solution on the in vivo efflux of histamine.  After collection of four 20 minute basal samples, calcium was omitted from the perfusion solution. A thirty minute transitional sample was discarded and four subsequent samples were collected for analysis. Data are normalized to the mean basal value and reported as the mean of five separate determinations.  59  125 - i  100  Striatum  4  :S 75 H 0)  CO CO  CQ  Calcium free 50 -J  25  4  0 6  125 T  BNST  Sample Number  7  8  60  Figure 15.  The effect of a-fluoromethylhistidine (a-FMH) on extracellular histamine in the  striatum.  After collection of three 20 minute basal samples, animals were administered a-FMH (100 mg/kg i.p.). Values were normalized to mean basal histamine values and plotted as the mean of the natural logarithm of four separate determinations. Open squares are the residuals of the partial decomposition of the bi-exponential function with half-lives of 32 minutes and 7 hours.  The normalized histamine levels were fit to an equation of the form: % [ basal HA] = A e ^ ~  A = 109, Aj = 1.33 h r  A l t  ) + Be(  -1  B = 39, A = 0.104 hr"  1  2  _ A 2 t  ) , where:  In (% Baseline)  62  Figure 16. Striatal extracellular histamine efflux after peripheral histidine loading.  After collection of three 20 minute basal samples, animals were administered histidine (500 mg/kg, i.p.). Data were normalized to the mean basal value and each point represents the mean of four separate determinations.  CTl  64  Figure 17. Histamine efflux in the BNST after administration of o^-adrenergic drugs.  After collection of five 20 minute basal values, animals were administered clonidine (0.3 mg/kg, i.p.), followed 100 minutes later by yohimbine (10 mg/kg, i.p.). Data were normalized to the mean basal values and each point represents the mean of four separate determinations.  200 n  50 H  o  J  i  1  1  1  i  1  2  3  4 5  1  1  6 7  i  1  1  i  r—i  r—i  8 9 10 11 12 13 14 15  Sample Number  66  values at four hours after injection. Recovery of the added histamine was 92 ± 2%, indicating the possibility of minor interference with the assay in the presence of histidine. In the BNST, administration of clonidine, (Figure 17), produced a 10-15% decrease in the extracellular histamine content which just failed to reach significance (p = 0.058). Subsequent administration of yohimbine resulted in an increased histamine signal, elevated to 155% of the mean basal levels (p = 0.047) or 180% with respect to the mean clonidine levels. After administration of clonidine, all animals exhibited a marked behavioral sedation which was reversed by yohimbine. Spike recovery was 97 ± 6% in the clonidine samples and 110 ± 15% in the yohimbine samples.  (D) Discussion  The present data demonstrate the presence of histamine in dialysates from the rat striatum and BNST. In some respects, this extracellular histamine signal appears to be related to neurotransmitter pools. However, inclusion of T T X in the perfusion solution was without effect on histamine levels, suggesting that the extracellular histamine signal sampled by microdialysis may be largely independant of the local action potentials. Under similar conditions, T T X was reported to produce a rapid and complete reduction in the signal for extracellular dopamine (Westerink et al., 1987, Drew et al., 1989), acetylcholine (Damsma et al., 1987) and excitatory amino acids (Westerink et al., 1987). However, insensitivity of extracellular levels of a neurotransmitter to T T X is not without precedent; the level of extracellular GABA in striatum was unaffected by T T X (Drew et al., 1989). Tuberomammillary neurons were spontaneously active in urethane anesthetized rats, firing at a mean rate of 2-3 Hz (Reiner and McGeer, 1987). In rat hypothalamic slices, histamine neurons displayed a similar rate of activity, which was blocked by the local application of T T X (Haas and Reiner, 1988). It is therefore surprising that extracellular histamine levels are T T X insensitive in the present study. It may be that histamine release in  67  striatum and BNST is largely regulated by presynaptic mechanisms such that neurotransmitter release is functionally uncoupled from the action potential. Another possible explanation for the lack of T T X sensitivity would involve the metabolism of histamine. If uptake and metabolism were in some way linked to depolarization, then local application of T T X could disable histamine clearance as well as release, producing the appearance of unaltered histamine release. In BNST and striatum, there was a slight, but significant, decrease in histamine outflow after omission of calcium from the perfusion solution, indicating a partial calcium dependence for extracellular histamine. The potassium-evoked release of histamine from hypothalamic slices was blocked by w-conotoxin, an antagonist of the N - and L-type voltage-sensitive calcium channels, but not by dihydropyridines which are selective for the L-type channel (Takemura et al., 1989). In striatal microdialysis experiments, omission of calcium caused the disappearance of the signals for dopamine (Imperato and di Chiara, 1984, Drew et al., 1989) and acetylcholine (Damsma et al., 1987). However, extracellular G A B A in the striatum was elevated to 140% of basal levels by the omission of calcium (Drew et al., 1989) while E G T A , a calcium chelator, increased G A B A outflow from synaptosomes in a manner sensitive to calcium channel blockers (Arias et al., 1984). Therefore, outflow of some neurotransmitters in the presence of reduced extracellular calcium may involve other factors, such as intracellular calcium stores. The results of dialysis experiments after a - F M H indicate the presence of at least two pools of extracellular histamine, which is roughly in agreement with ex vivo studies conducted employing this drug. We observed that the major component of striatal histamine outflow is in a pool with a 32 minute half-life. Data reported for the disappearance of whole brain histamine in mast cell-deficient mice after administration of a - F M H (Maeyama et al., 1983) can also be analyzed to yield a bi-exponential function with half-lives of about 35 minutes and four hours.  68  The above data for the rapidly turning over pool of striatal histamine are in the range of half-lives reported for dopamine turnover in striatum, 12-36 minutes (Lane et al., 1982, Miyauchi et al, 1988). The half-life of histamine in striatum has previously been calculated on the basis of temporal changes in tissue content of histamine after synthesis blockade (8 minutes, Oishi et al., 1984) and by the accumulation of the metabolite r - M e H A  after  pargyline (11 minutes, Hough et al., 1984). However, neglecting the presence of the slow pool could lead to error in the estimation of the turnover rate for the fast pool. The concentration of histidine in rat brain is in the order of 100  (Taylor and  Snyder, 1972). Although the substrate affinity of H D C in vivo is not known with precision, the enzyme is evidently not saturated, as a peripheral load of histidine (500 mg/kg) increased brain histamine content by 80% (Schwartz et al., 1972). The magnitude and time course of this effect was similar to the present results for extracellular histamine in striatum. This provides further evidence for a functional relationship between total brain histamine content and histamine outflow as determined by cerebral microdialysis. The increased striatal histamine outflow observed after histidine loading was not seen in a previous study (Russell et al., 1990), a discrepancy which may have been due to the use of U-shaped cannula in an acute anesthetized preparation, rather than at least 24 hours after implantation of transtriatal probes. Westerink and de Vries (1988) have pointed out the difficulties in interpreting results from this type of acute experiment. In addition, Russell et al. (1990) employed a calcium-free perfusion solution which would, on the basis of the present results, be expected to alter the basal outflow of histamine. A recent report on the regulation of [ H]-histamine release from cortical slices suggested that presynaptic adrenergic receptors were involved (Gulat-Marnay et al., 1989). The basal release was reported to be 40% decreased by prior administration of clonidine at 0.3 mg/kg. Yohimbine, a relatively specific  antagonist, was without effect alone, but blocked the  clonidine-induced decrease in histamine release, suggesting that adrenergic tonus may contribute to the regulation of histamine release in the cortex. The present microdialysis data  69  are in agreement with the ex vivo results in that the  agonist tended to decrease extracellular  histamine levels, an effect which was reversed by the antagonist. The elevation of extracellular histamine above basal levels by yohimbine administration suggests that histamine release in the BNST may be subject to tonic adrenergic inhibition in vivo. It follows that some of the physiological effects of a^-adrenergic drugs may be mediated via central histaminergic neurons.  70  VI:  (A)  Histamine Receptors  Introduction  As discussed in the general introduction, histamine may act through three receptor types in the nervous system. The  distribution and functions of the classical H j and H  2  receptors  have been better characterized than has the H 3 site, originally described as a histamine autoreceptor.  In a review of the literature on the binding of histamine to cerebral membranes,  it became apparent that the observations were inconsistent with the pharmacological specificities of either the H j o r Kandel,  et  prototypic H 3  3  al. ,  H  1980) .  2  receptor  (Palacios  et  a l . ,  1978,  In the present study the binding properties of the  agonist [ H]-N -methylhistamine ( [ H ] - N - M e H A , Arrang et al., 1983), and of 3  a  3  3  [ H]-histamine ([ H ] - H A ) itself were characterized in rat forebrain sections. The two ligands were found to bind with high affinity to an identical site: the high affinity histamine binding site. Lesion studies suggest that the majority of this H^ binding is associated with postsynaptic elements rather than with presynaptic histaminergic terminals. (B)  Material and Methods  Adult male Wistar rats (250-300 g) were killed by cervical dislocation and the brain quickly removed, blocked and frozen. For binding studies, 25 Aim thick cryostat sections, each weighing 2.0 ± 0.1 mg, were prepared in the coronal plane. Sections were taken at the level of the greatest dimension of the striatum, mounted on chrom-alum coated glass slides and stored at -70 °C for no more than one week. Sections were thawed and then incubated for 45 minutes at room temperature in 150 m M sodium phosphate buffer (pH 7.5) containing 100 fiM dithiothreitol (Sigma), 2 m M M g C l and varying concentrations of the radioligands. [ H ] - H A 3  2  (32 Ci/mmole, New England Nuclear) was included at 0.4-80 n M and [ H ] - N - M e H A (80 3  71  Ci/mmole, New England Nuclear) was included at 0.15-25 n M . Nonspecific binding was determined by the addition of 5 m M of the H j agonist R-(a)-methylhistamine oxalate (aM e H A , Research Biochemicals). Sections were washed three times in 4 °C buffer (20 sec), dried under an air line, and scraped from the slides with a razor blade. The radioactivity in each section was determined by liquid scintillation counting (Packard Tri-carb 4530) after the addition of 5 ml aqueous cocktail (ACS, Amersham). The Scatchard analysis. In some experiments, B  m a x  and B  m a x  were determined by  was estimated from specific binding at  radioligand concentrations ten times the respective K^s. To examine possible regulation of binding by guanylyl nucleotides, rat forebrain sections from three rats were incubated in the presence of 100 /iM 5'-guanylyl-imidodiphosphate (GppNHp) in addition to [ 3 H ] - N - M e H A at concentrations from 1-500 n M . A t ligand concentrations between 50 and 500 n M , specific activity was reduced by the addition of j  unlabelled N - M e H A (Calbiochem).  Non-specific binding was determined by the addition of  10 /tM a - M e H A in these experiments. In displacement studies, radioligands were included at their respective K^s as determined 3 3 above: 8 n M in the case of [ H ] - H A and 2 n M in the case of [ H ] - N - M e H A . Displacement curves were produced by the addition of competitors at various final concentrations ranging -3 from 10  11 to 10"  M . The competitors used were histamine hydrochloride, mepyramine,  cimetidine, and sulpiride (all from Sigma), t - M e H A (Calbiochem), impromidine (Smith, K l i n e , and French), harmaline hydrochloride (Aldrich), trazadone (Bristol), desmethylimipramine (Merle Dow), haloperidol (McNeil Labs), thioperamine (gift of Dr. J . - C . Schwartz) and phencyclidine hydrochloride (gift of Dr. C. Blaha). In some animals neurochemical lesions were produced with either quinolinic acid (QA) or 6-hydroxydopamine ( 6 - O H D A ) in order to determine the anatomical localization of histamine binding. Four male Wistar rats (250-300 g) were placed in a stereotaxic apparatus while under pentobarbital anesthesia (60 mg/kg i.p.). Using the coordinates of Paxinos and Watson, a Hamilton syringe was placed in either the sensorimotor cerebral cortex (n = 2, A P : -  0.26, D V : - 2 . 5 - 0 . 5 , M L : -3.4) or the dorsal striatum (n = 2, A R - 0 . 2 6 , D V : - 5 . 5 , M L : +3.4) and 1 /xi of 300 m M Q A (Sigma, p H 8) was infused. One rat had both cortical and striatal lesions. QA-lesioned animals were sacrificed four days after surgery at which time neuronal destruction is complete in the zone of the lesion while axon terminals from outside the lesion remain largely intact (Schwarcz et al., 1983, Shaw et al., 1989). Four other animals received 1 ul of 18 m M 6 - O H D A (Sigma) in ascorbic acid solution directed towards the left medial forebrain bundle ( M F B , A P : +4.5, M L : +1.5, D V : +1.5) and were sacrificed seven days after the 6 - O H D A lesions. Portions of striatum from the 6 - O H D A lesioned animals were taken for determination of catecholamine content by reversed phase H P L C with electrochemical detection as described previously (Cumming et al., 1988). In the lesion studies, 25 /xm cryostat sections were prepared at the level of striatum and mesencephalon. Slides containing four sections each were incubated in [ H ] - N - M e H A at concentrations of 0.5, 1, 2, 5, and 12 n M and washed as described above. The saturation binding curves in forebrain hemisections from some 6-OHDA-lesioned animals were determined directly by liquid scintillation counting. Other slides were exposed to tritiumsensitive film (Amersham Hyperfilm) for a 6 week period after which time the films were conventionally developed and fixed. Regional optical densities were quantified by autoradiographic image analysis (Imaging Research Inc., St. Catharines, Ontario) employing callibrated standards (Amersham).  (B) Results  A representative Scatchard analysis of [ H ] - H A binding in rat forebrain (Figure 18) 3  yielded a K.^ of 8 n M and B  m a x  of 22 fmoles/section, while Scatchard analysis of [ H ] - N -  M e H A binding (Figure 19) yielded a mean of four estimations of B  of 2 n M and a B  m a x  of of 26 fmole/section. The  was 19.5 ± 2.2 fmole/2 mg section for [ H ] - H A and 24.9 ± J  73  Figure 18. Scatchard analysis of [ H ] - H A binding in rat forebrain sections.  Each point represents the mean of four determinations which differed by less than 10%. Inset: the corresponding saturation binding curves.  Ligand Bound (fmole/section)  75  Figure 19. Scatchard analysis of [ H ] - N - M e H A binding in rat forebrain sections.  Each point represents the mean of four determinations which differed by less than 10%. Inset: the corresponding saturation binding curves.  Ligand Bound (fmole/section)  Figure 20. Hill plots of the binding of I H]-N-MeHA and [ H]-HA to cryostat sections of rat forebrain.  Binding data were taken from the previous two figures.  1.5  -i  1H  7  l ° 9 i o [Ligand] 00  79 •3  3.3 fmole/2 mg section for [ H ] - N - M e H A . H i l l coefficients for the two ligands did not differ J  significantly from unity (Figure 20), indicating a lack of cooperativity in binding. •3  Addition of 100  G p p N H p greatly interfered with specific [ H ] - N - M e H A binding:  94% of the high affinity binding disappeared with the residual component having an apparent 3 n M affinity (Figure 21). A t ligand concentrations above 25 n M , a new binding site appeared with  of approximately 200 n M and B  m a x  of approximately 50 fmole/section.  Unfavorable signal-to-noise ratio at high ligand concentrations made it difficult to determine these quantities with precision. Sections incubated in parallel with 20 n M [ H ] - N - M e H A bound 30 fmole/2 mg section in the absence of G p p N H p , which is within the normal range of •p max" In the displacement studies, nonspecific binding in forebrain sections was 12% of the total at 2 n M [ H ] - N - M e H A and 25% of the total at 8 n M [ H ] - H A . I C s for the 3  3  5Q  •3  displacement of [ H ] - N - M e H A by some histamine ligands (Figure 2 2 - A ) and by various other J  psychoactive drugs (Figure 23) were estimated from the interpolation of the curves at halfmaximal specific binding after subtraction of the nonspecific binding. The displacment of •3  [ H ] - H A by some competitors is illustrated in Figure 2 2 - B . •3  Autoradiographic images of [ H ] - N - M e H A binding in rat brain indicate that the highest J  binding was in the nucleus accumbens, striatum and substantia nigra pars compacta, i.e. the basal ganglia (see Figure 24). Maximal binding seemed to respect a dorsal/ventral gradient such that levels were two-fold higher in the nucleus accumbens than on the dorsal striatum (Figure 25). Dense binding was also noted in the globus pallidus, amygdala and pyriform cortex.  In the diencephalon, moderate binding was present in the anterior hypothalamus and  in the midline thalamic nuclei (Figure 24-C). In the cerebral cortex, there was considerable heterogeneity in the maximal binding. Maximal binding in the anterior cingulate cortex was 30% higher than in the adjacent sensorimotor cortex (Table VI, see also Figure 2 4 - A ) , but binding was very low in the retrosplenial cingulate cortex (Figure 24-D). Binding in the insular cortex appeared to be  80  Figure 21. The Effect of Guanylyl Nucleotide on [ H ] - N - M e H A Binding in Forebrain Sections.  Scatchard analysis of [ H ] - N - M e H A binding in rat forebrain sections in the presence of 100 uM 5'-guanylyl-imidodiphosphate (GppNHp). Each point is the mean of 4-8 determinations. A small fraction (4%) of total binding remained in a high affinity state (3 nM). A t high ligand concentrations, a low affinity state appeared, the parameters of which coould not be determined with precision because of the poor signal-to-noise ratio.  0 H 0  1  1  1  1  1  1  2  3  4  5  Ligand Bound (fmole/section)  82  Figure 2 2 . The displacement of H-j Ligands by Various Histaminergic Drugs.  The displacment of (A) [ H]-N-MeHA, and ( B ) [ H]-HA from rat forebrain sections by J  J  various histaminergic drugs. Each point is the mean of four separate determinations.  IC^QS,  determined by interpolation at half-specific binding are: (Part A), ( 1 ) a-MeHA, I C ^ Q = 0 . 4 nM, ( 2 ) thioperamide, I C ^ Q = 3 nM, ( 3 ) burimamide, I C ^ Q = 2 0 nM, ( 4 ) histamine, I C ^ Q = 4 5 nM, (5) impromidine, I C ^ Q = 79 nM, (6) mepyramine, I C ^ Q = 79 /zM, (7) cimetidine, I C ^ Q = 1 0 0 fjM, and ( 8 ) r-methylhistamine, I C  = 1 0 0 /iM.  5 Q  •3  Drugs employed i n the displacement of [ H]-HA (Part B ) are thioperamide, I C ^ Q = 2 . 4 nM, histamine, I C ^ Q = 1 8 nM, and mepyramine, I C ^ Q = 4 1 /tM.  83  84  Figure 23. The displacement of [ H]-N-MeHA from rat forebrain sections by various psychoactive drugs.  Each point is the mean of four determinations which differed by less than 10%.  IC^QS,  determined by interpolation at half-specific binding, were : haloperidol (25 /iM), sulpiride (45 /iM), trazadone (45 /iM), phencyclidine (70 /tM), desmethylimipramine (DMI, 100 uM) and harmaline ( > 100 /iM).  o PCP  -8  -7  -6  -5  -4  -3  log Competitor Concentration CD  Figure 24. Autoradiographs of [ H ] - N - M e H A binding in the rat brain.  Sections were incubated in [ H]-N-MeHA (4 nM) washed and placed on film for six weeks.  (A) The normal striatum. The upper arrow indicates the anterior cingulate cortex and the lower arrow indicates the barrel field of somatosensory cortex. (B) Striatum at the same level as above after 6-OHDA lesion to the left medial forebrain bundle.  (C) Normal diencephalon. (D) The normal mesencephalon. The top arrow indicates the retrosplenial cortex and the bottom arrow indicates the boundary of primary and secondary visual cortex.  (E) Striatum after quinolinic acid lesion (F) Nissl staining of the above section.  (G) The mesencephalon after unilateral QA lesion to the right striatum. (H) Typical nonspecific binding in mesencephalon sections.  91  3 Figure 25. Binding of [ Ff]-N-MeHA in some regions of the basal ganglia 3  The binding of the H3 ligand [ H]-N-MeHA is heterogenous within the basal ganglia and related structures. B  m a x  is nearly two-fold higher in the nucleus accumbens than in the  dorsal striatum. The binding in the substantia nigra pars compacta is similar to that in the striatum. In this animal, there was evidence of some heterogeneity in affinity state. Each point is the mean of three determinations.  93  almost equal to that in the dorsal striatum. In the parietal cortex, binding seemed to be greater in the barrel fields than in surrounding cortex (Figure 2 4 - A ) . In some cortical areas, binding had a laminar distribution, with relatively higher binding in layers 1, 3 and 5. However, there was little specific binding in layer 3 of the primary visual cortex (Figure 24D, arrow).  There was relatively little binding in the dorsal hippocampus and moderate binding -3  in the dentate gyrus of the ventral hippocampus (Figure 24-C). Non-specific [ H ] - N - M e H A binding was very low in the mesencephalon (Figure 24-H) and all other areas examined. In the autoradiographic study, the unilateral 6 - O H D A - l e s i o n was without apparent effect on the binding in either forebrain (Figure 24-B) or mesencephalon. Scatchard analysis of autoradiographic [ H ] - N - M e H A binding in the dorsal striatum after a 6 - O H D A lesion to the left M F B does not suggest any effect of this treatment (Figure 26-B). H P L C analysis indicated a 95% depletion of striatal dopamine in the 6 - O H D A lesioned striata. In three separate determinations of total specific binding to forebrain sections, the dopamine depletions were without significant effect on binding parameters (Table V). In the autoradiographic studies, striatal QA-lesions produced great reductions in binding in striatum (Figure 24-E) and in the substantia nigra pars reticulata ipsilateral to the Q A •3  lesion (Figure 2 4 - G ) . A representative Scatchard plot of autoradiographic [ H ] - N - M e H A J  binding in dorsal striatum after a unilateral striatal QA-lesion is indicative of a major reduction in maximal binding with no apparent change in affinity (Figure 26-B). treatment produced a 70% decrease in striatal B affinity (Table V).  m a x  The  without significantly altering the apparent  Nissl-staining of the QA-lesioned forebrain section (Figure 24-F) showed  the tissue to be mostly intact, but devoid of neurons in the region of the lesion. •3  The effect of the QA-lesion on binding of [ H ] - N - M e H A in cerebral cortex is illustrated in Figure 27. The unilateral QA-lesion in sensorimotor cortex produced a 70% decrease in maximal binding without altering the affinity (Table V).  The autoradiographic  binding in cingulate cortex ipsilateral to the lesion was not altered with respect to the  contralateral side. The autoradiographic appearance of [ H ] - N - M e H A binding in QA-lesioned cerebral cortex is illustrated in Figure 2 4 - E .  Figure 26. The Effects of Striatal Lesions on the Binding of [ H ] - N - M e H A . 3  Scatchard analysis of [ H ] - N - M e H A binding in rat striatum after (A) unilateral 6 - O H D A lesions to the medial forebrain bundle and (B) unilateral QA-lesion in striatum. Each point the mean of four determinations within an individual animal.  9'S  Ligand Bound (fmole/mg)  97  Figure 27. The Effects of Cortical QA-lesion on the Binding of [ F f ] - N - M e H A .  Scatchard analysis of the autoradiographic [ H ] - N - M e H A binding i n , from left to right, Q A lesioned sensorimotor cortex, sensorimotor cortex contralateral to the lesion, cingulate cortex adjeacent and ipsilateral to the lesion, and cingulate cortex contralateral to the lesion.  9  n  fmole/mg co  Table V.  The effect of neurochemical lesions on the binding of [ H ] - N - M e H A to rat brain.  B  max  ( a )  K  d  Striatum OA-lesion contralateral ipsilateral  14.2 ± 2.g 4.4 ±1.6  1.7 ± 0.1 1.7 ± 0.6  14 ± 0.8 > 13 ± 0.3( )  3.5 ± 0.7 3.3 ± 0.9  6.7 ± 0.8, 1.6 ± 0.5 9.7 ± 1.5 9.4 ± 0.7  0.9 ± 0.9 ± 1.6 ± 1.8 ±  6-OHDA-lesions ipsilateral contralateral  (b  b  Cerebral Cortex OA-lesions contralateral sensorimotor ipsilateral sensorimotor contralateral cingulate ipsilateral cingulate  0.4 0.4 0.2 0.4  (a) B , in units of fmole/mg, was determined by regional Scatchard analysis of autoradiographs, except in (b) where the binding was determined in whole forebrain sections weighing 2 mg each. (c) is in units of nM. Note that the striatal B and are similar when determined by regional autoradiography or from the binding in whole forebrain sections. Each reported quantity is the mean of three separate determinations ± SEM. m  m  *p < 0.05, Student's two-tailed t-test  100  (D) Discussion  [ H ] - H A binds to a single high affinity site in forebrain with an affinity of 8 n M , in 3  agreement with previous reports (Palacios et al., 1978, Barbin et al., 1980). The binding of [ H ] - N - M e H A also indicates a single high-affinity site, as previously reported for another H j 3  agonist, [ H ] - a - M e H A (West et al., 1989). The H i l l coefficients indicate lack of cooperativity 3  in the binding of both ligands used in the present study. The addition of G p p N H p greatly reduced the number of high affinty binding sites for [ H ] - N - M e H A as has been reported for [ H ] - H A (Barbin et al., 1980) and [ H ] - a - M e H A , (Arrang et al., 1990). Previously, G p p N H p 3  3  3  has been shown to decrease the affinity of [ H ] - H A for membranes by accelerating  k^ Q  (Cybulsky et al., 1981). The signal-to-noise ratio in the present study was such that the low affinity  and B  m a x  values for [ H ] - N - M e H A binding could not be determined with  precision in the presence of GppNHp.  However, the present results are evidence for linkage  of the majority of the [ H ] - N - M e H A binding sites to a G-protein.  It is not known which  second messenger systems may be coupled to the H j binding site, although the effects on synthesis and release are sensitive to the extracellular C a ^ concentration (Arrang et al., 1985). +  •3  In the present study the most potent displacers of [ H ] - N - M e H A binding (Figure 21) J  were the H j agonist a - M e H A (IC^Q = 0.4 nM) and the H j antagonist thioperamide (IC^Q =3 nM). Impromidine, an H  2  and H j agonist, had an IC^g of 79 n M , while burimamide, an H  2  •3  and H j antagonist, was a relatively more potent displacer of [ H ] - N - M e H A (IC^Q = 20 nM). r-Methylhistamine, the ring-methylated histamine metabolite, had little ability to displace  3  3  [ H]-N-MeHA.  The displacement curves indicate that [ H ] - N - M e H A behaves as a typical and  potent H j ligand, the rank order of the  IC^QS  for various compounds being similar to those in  •3  previous reports employing [ H ] - a - M e H A (Arrang et al., 1983, Arrang et al., 1987, West et al., 1989). •3  The pharmacological specificity of [ H ] - H A binding has previously been found to lack correlation with classical H , and H  2  sites (Barbin et al., 1980, Kandel et al., 1980).  Inhibition  101  of [ H ] - H A binding to rat cortex correlated better with the subsequently characterized H3 properties of some drugs than with pharmacological potency at H  2  sites (Steinberg et al.,  1985). In the present study, [ H ] - H A was displaced by histamine, the selective H J  3  antagonist  thioperamide (Arrang et al., 1987) and mepyramine at IC^g values very similar to those •3  determined for the displacement of [ H ] - N - M e H A by the same competitors. In addition, the B max „ values determined for the two radioligands in forebrain sections were nearly identical, r  o v  Therefore, we conclude that the high affinity histamine binding site is identical to the H3 site. The psychomimetic substance phencyclidine is able to displace [ H ] - N - M e H A binding J  •3  with an I C ^ Q of 70 /tM, which is similar to its reported ability to displace [ H ] - a - M e H A (Arrang et al., 1988). The other psychoactive competitors in this study, including both typical •3  and atypical antidepressants and neuroleptics, were all able to displace the [ H ] - N - M e H A J  binding with  IC^QS  in the range of 25-100 i t M , with the exception of harmaline, a  hallucinogen. As it seems unlikely that such high concentrations could be attained at normal dosages, it is improbable that important effects of these drugs could be mediated by the H3 receptor. However, since these drugs were moderately potent in spite of their differing structures, it remains a possibilty that some psychoactive drugs may be found to interact significantly with H3 receptors. -3  Examination of a single autoradiogram of [ H ] - a - M e H A binding suggested that high levels were present in the striatum and substantia nigra (Arrang et al.,1987). Likewise, studies of [ H ] - H A binding in rat brain regions indicated highest binding in striatal tissue (Palacios et 3  al., 1978, Barbin et al., 1980). The present autoradiographic study confirms and extends these observations. In the present lesion studies, destruction of the striatal catecholaminergic innervation with 6 - O H D A was without effect on striatal binding, whereas binding was greatly reduced after destruction of neurons resident in the striatum with Q A .  Similarly, Barbin et  al., found [ H ] - H A binding in striatum to be 50% reduced by a local kainic acid lesion (1980). 3  Thus, much of the striatal H3 binding appears to be on intrinsic neurons, rather than on terminals of dopaminergic afferents.  102  The pattern of distribution of H j binding in cortex is complex, varying from one cortical area to another and across the cortical laminae. QA-lesions, if presumed to be without great effect on presynaptic terminals, showed the majority of the [ H ] - N - M e H A  binding to  be on intrinsic cortical neurons. A small fraction of the total H j binding sites in the forebrain could be on afferent terminals.  103  VII: General Discussion  (A) Neurotoxicology  Administration of M A M on prenatal day 15 was apparently unable to prevent the development of the forebrain histamine innervation. The increases in tissue histamine levels that were detected are more reflective of decreased mass of the target structures rather than damage to histamine neurons. Similarly, Jonsson and Hallman (1982) found a two-fold increase in the rat forebrain content of serotonin, dopamine and noradrenaline after prenatal M A M treatment. These results were interpreted as evidence that M A M treatment results in a "hyperinnervation in the atrophic regions" without reduction in the total number of monoaminergic terminals. The choice of time for the M A M treatment seemed appropriate given what is known about the ontogeny of the histamine neurons, which have the peak final mitosis on embryonic day 16 (Reiner et al., 1988). Therefore, it is not clear why M A M treatment on day 15 was unable to interfere in the subsequent development of the histamine innervation. Treatment with anti-mitotic agents prior to embryonic day 15 might interfere more effectively with the subsequent development of the H A innervation. It is interesting that M A M treatment was able to reduce H N M T levels greatly in the adult rat. The lineage of HNMT-containing cells, either neurons or glia, seems to have been largely sensitive to MAM-treatment on day 15. Alternately, expression of the catabolic enzyme in the adult may have been down-regulated in compensation for a damaged histamine innervation. Better characterization of the cellular distribution of H N M T in the adult would be pertinent to the observed MAM-senistivity. Location of the M A O - B relevant to neurotoxicity of M P T P remains unknown, but may be in astrocytes, and/or serotonin and histamine neurons (Vincent, 1989). Although these neurons are not destroyed by M P T P , they may nonetheless contribute to the formation of MPP  +  without themselves accumulating toxic concentrations of the metabolite. Pretreatment  104  with fluoxetine, a 5 - H T uptake inhibitor, is reported either to protect against (Brooks et al., 1988), or to be without effect on (Melamed et al., 1985), MPTP-induced dopamine lesions. Also, prior lesions to the dorsal raphe 5 - H T neurons with 5,7-dihydroxytryptamine did not attenuate the M P T P toxicity for dopamine neurons (Melamed et al., 1986). If both histamine and 5 - H T neurons could be selectively destroyed, it would be possible to ascertain the contribution of M A O - B in these neurons to the production of neurotoxic M P P . +  Although M A O - B in histamine neurons may contribute to formation of M P P , the +  mouse brain H A concentrations were unaffected by M P T P doses sufficient to deplete striatal dopamine greatly. The cerebral activity of H D C , the enzyme which synthesizes H A , is reported to be similar in cases of Parkinson's disease and in post mortem specimens from neurologically normal patients (Garbarg et al., 1983). Thus, the subacute M P T P model in mice resembles Parkinson's disease with respect to the apparent preservation of histamine in the central nervous system.  (B) H N M T Inhibition  H N M T is inhibited by a wide variety of compounds. Indeed, in an initial study of the enzyme, chlorpromazine and bromo-lysergic acid diethylamine were found to be H N M T inhibitors (Brown et al., 1959). Among the other compounds which inhibit H N M T are the biogenic amine, dopamine, and the amine metabolite, N,N-dimethyltryptamine (Sellinger et al., 1978). Other H N M T inhibitors include H , antagonists such as mepyramine (Taylor and Snyder, 1972, Tachibani et al., 1988), impromidine, an H  2  agonist and H j antagonist (Beaven  and Roderick, 1980), other thiourea derivatives (Beaven and Shaff, 1979), and zolantidine, a benzthiazole H  2  antagonist (Hough et al., 1988). Among the most potent H N M T inhibitors are  the dihydrofolate reductase inhibitors such as metoprine (Duch et al., 1980), antimalarial drugs such as quinacrine, which contain the aminoquinoline ring structure (Harle and Baldo, 1988), and T H A , an anticholinesterase (Cumming et al., 1990).  105  The present in vitro study shows T H A and metoprine to be nearly equipotent H N M T inhibitors; few compounds are more potent. Among these are quinacrine, which inhibits HNMT  from guinea pig skin with a K j of 20 n M (Tachibana et al., 1988) and T M Q , an  aminoquinazoline, which inhibits bovine brain H N M T with a K j of 7 n M (Duch et al., 1980). The mechanism of H N M T inhibition by T H A should presumably involve structural features common to these molecules. H N M T is inhibited by a plethora of compounds with different structural features, but many of the most effective inhibitors have in common an aromatic nitrogen and a basic nitrogen separated by one or more carbons (Harle and Baldo, 1988, Duch et al., 1980, Tachibana et al., 1988). These appear to be necessary but not sufficient requirements for H N M T inhibition, since 4 - A P was 400 fold less potent than T H A .  It may be  that, for high affinity inhibition, a side-chain on the aromatic ring or the presence of an ethylamine moiety is necessary (Tachibani et al., 1988). From the studies of H N M T inhibition, it is clear that the catalytic region of the enzyme has complex properties. A ping-pong mechanism has been proposed involving transfer of methyl from S A M to the enzyme (Thithipandha and Cohn, 1978), but because the methylated enzyme has not been isolated, direct transfer of methyl to histamine has also been suggested (Gustafsson and Forshell, 1964). Others have determined an apparent "ordered B i - B i " type reaction mechanism for rat brain H N M T (Orr and Quay, 1978) in which histamine and S A M bind sequentially to the enzyme. In this model, first methylhistamine and then S-adenosylhomocysteine dissociate from the enzyme after catalysis. The precise mechanism of action of the enzyme has bearing on the sites of action of enzyme inhibitors because inhibitors could potentially interfere with the binding of histamine, S A M , or both molecules. Many inhibitors which are competitive with respect to histamine seem also to be nearly equipotent competitive inhibitors with respect to S A M (Tachibani et al., 1988). Because two molecules cannot simultaneously occupy the identical site, one cannot conclude that S A M and histamine share a common site on the enzyme, but it seems possible that H N M T inhibitors compete with the binding of both histamine and S A M by virtue of structural features common  106  to both substrates. One conformation of histamine (Figure 9 , inset) has similarities with the adenine moiety of S A M . Harmaline also has some structural similarities with adenine (Figure 9 , inset), but has a 1-methyl substitution. If /9-carbolines inhibit H N M T by interfering with binding of the adenine moiety of S A M , then 1-amino analogs would be expected to be even more potent H N M T inhibitors. Harmaline and various other ^-carbolines are natural products with a number of physiological actions. Harmaline is a reversible M A O - A inhibitor with an of  10  n M (Nelson et al.,  1979).  benzodiazepine binding with  on the order  Some /9-carbolines, such as harmane and harmine, inhibit  IC^Q  values from  1 0 - 1 0 0  acid ethyl ester is much more potent (Fehske et al., displace G A B A binding from membranes with 1978).  ICJQ  uM, although norharman-3-carboxylic  1981).  IC^QS  In addition, harmine and harmaline  on the order of  10  uM (Roberts et al.,  A l l the /J-carbolines used in the present study inhibit dopamine uptake into rat striatal  synaptosomes with  IC^Q  values in the range of  1 0 - 1 0 0  uM (Drucker et al.,  1990).  The present  results indicate that /9-carbolines have an ability to inhibit H N M T which is intermediate in the range of potencies for these previously decribed pharmacological properties. Harmaline and related compounds may be extracted from plants such as Peganum harmala and members of genus Banisteriopsis.  Such extracts are reported to be hallucinogenic  and have been employed as such among some inhabitants of South America (Schultes,  1969).  Harmaline is found to be a competitive inhibitor of H N M T with respect to both histamine and S A M at concentrations in the uM range. Drugs are seldom entirely specific in their actions, and inhibition of H N M T is a property of many alkaloids and synthetic nitrogenous compounds. Some compounds, such as ^-carbolines, interact with such a wide variety of neurochemical systems that it is difficult to be certain which are responsible for particular physiological actions. In addition, many therapeutic agents and experimental substances such as T H A are also H N M T inhibitors. Therefore, it is important to consider effects on histamine metabolism in the central nervous system as possible contributing factors to the side effects of medication and the actions of experimental substances. For example, the antimalarial  107  prophylactic drug mefloquine, a probable H N M T inhibitor, has been reported to produce neuropsychiatric side effects, including psychosis (Sturchler et al.,  1 9 9 0 ) ,  which might possibly  be related to effects on histaminergic transmission. Metoprine, a dihydrofolate reductase inhibitor derived from diaminopyrimidine, is a very potent inhibitor of H N M T (Duch et al.,  1980).  Peripheral administration of  10  mg/kg  metoprine has been previously shown to inhibit H N M T in the rat brain by more than 8 0 % (Hough et al., 1 9 8 6 ) and produce a long-lasting twofold increase in whole rat brain histamine levels (Duch et al.,  1 9 7 8 ) .  The rates of accumulation of histamine after metoprine  adminstration, if assumed to be linear, may be calculated from data in Table V.  This yields  rates of 0 . 3 1 nmole/g-hour for cortex, 0 . 2 1 nmole/g-hour for striatum, and 0 . 7 1 nmole/g-hour for hypothalamus. These turnover rates are similar to, but somewhat lower than, those rates calculated from the accumulation of r - M e H A subsequent to pargyline adminstration (Oishi et al.,  1984,  Hough et al.,  1984).  The inhibition of M A O by pargyline, an irreversible inhibitor,  may be more rapid and complete than the competitive inhibition of H N M T by metoprine, resulting in a probable underestimation of turnover as calculated from the accumulation of histamine after partial inhibition of H N M T . Alzheimer's disease is a dementia often associated with degeneration of the cortical cholinergic innervation (Coyle et al., 1 9 8 3 ) in addition to the classical neuropathological features such as neuritic plaques and tangles. The belief that a cholinergic deficit contributes to the clinical state in Alzheimer's disease has led to attempts at neurotransmitter replacement therapy, in analogy to the use of L - D O P A for Parkinson's disease. Recent interest has focused on inhibition of acetylcholinesterase (AChE), the enzyme which inactivates acetylcholine. T H A inhibits A C h E with an  IC^Q  on the order of  100  n M (Kaul,  1962).  Improved cognitive  performance has been reported in some mildly demented Alzheimer's patients undergoing clinical trials with T H A (Summers et al.,  1986).  It is not certain that the inhibition of A C h E is the only property of T H A pertinent to its reported clinical efficacy and/or side effects. For example, T H A shares with its structural  108  fragment 4-amine-pyridine (4-AP) the ability to block certain classes of potassium channels (Rogawski, 1987), producing spike broadening, which could possibly potentiate various neurotransmitter systems. T H A and related molecules are also inhibitors of monoamine oxidase (Kaul, 1962). These latter properties of T H A , occurring at concentrations on the order of 100 fiM, seem unlikely to be significant in clinical studies employing oral T H A doses in the range of 100-200 mg per diem (Summers et al., 1986). Oral physostigmine is apparently without benefit for Alzheimer's disease (Stern et al., 1987). If T H A is a superior treatment to other A C h E inhibitors, additional physiological and biochemical properties of T H A could be responsible. The K- of T H A for H N M T is well within the therapeutic range of plasma T H A concentrations, which was on the order of 100 n M (Summers et al., 1986). The low dose of T H A used in the present pharmacological study was able to inhibit H N M T substantially in the cerebral cortex and also to produce robust increases in cerebral histamine levels. Thus, potentiation of cerebral histaminergic transmission could be an important aspect of the actions of T H A on the central nervous system. The cholinergic deficit may be sine qua non for Alzheimer's disease, but degeneration of other non-thalamic cortically-projecting neuron systems, such as the noradrenergic locus coeruleus, is well documented (German et al., 1987). In Alzheimer's cases, high densities of neurofibrillary tangles have also been found in the vicinity of the cortically-projecting histamine neurons (Saper and German, 1987). Cortical histamine levels have been reported to be either reduced (Mazurkiewicz-Kwilecki and Nsonwah, 1989) or increased (Cacabelos et al., 1989) in studies of Alzheimer's cases. The reason for this disagreement is not clear at this time. However, it is possible that a histaminergic dysfunction may contribute to aspects of the clinical condition. Potentiation of cerebral histaminergic transmission by T H A , especially in conjunction with A C h E inhibition, may improve cognitive performance.  (C) Cerebral Microdialysis  The presence of mast cells in brain has often been cited as a possible interference in the study of neuronal histamine. In a number of rodent species, mast cells are present in variable numbers in forebrain structures, especially in the dorsal thalamus, where they were in close association with blood vessels (Dropp, 1972). In the thalamus of individual adult rats, histamine content correlated highly with mast cell numbers, which ranged from 1,000-30,000 (Goldschmidt et al., 1985). However, in one mast cell-deficient strain of mice, total brain histamine content was unrelated to the mast cell phenotype (Orr and Pace, 1984). It seems that mast cell populations in brain may reflect idiosyncratic developmental processes subject to variability in different brain regions of an individual and within strains. The residual histamine in brain several hours after synthesis blockade with a - F M H is often considered to be present in mast cells (Oishi et al., 1988). However, histamine in neonatal rat brain mast cells is reported to turn over with a four day half-life (Martres et al., 1975), which is 15-fold longer than the apparent half-life for the slow pool of extracellular histamine determined in the present striatal microdialysis experiments. Histamine leakage from immature mast cells (WoldeMussie et al., 1986), or from the leakage of blood and/or basophiles into the region of the dialysis probe, could be a factors in these experiments, given the possibility of acute trauma associated with probe implantation. However, the rapid decline in striatal histamine outflow after systemic administration of a histamine synthesis inhibitor is strong evidence for neurogenic origin. The factors regulating mast cell exocytosis are complex, but increased intracellular calcium is a necessary step (Douglas and Kagayama, 1977). Thus, the single criterion of calcium sensitivity is insufficient to discriminate between the possible neuronal and mast cell contributions to extracellular histamine. In the absence for any evidence to the contrary, the most simple explaination would be a two compartment model for cerebral histamine: fast and slow turnover neuronal pools, as has been suggested for striatal dopamine (Ewing et al., 1983), or a rapid neuronal pool and a slow mast cell histamine pool.  110  However, a temporal control would be necessary to determine if the apparent slow pool was simply related to declining basal histamine release. Some of the pharmacological results here presented support the neurogenic hypothesis for extracellular histamine in the rat striatum and BNST.  However, a number of interesting  points may be raised. The first concerns the levels of histamine detected in dialysate samples. In striatum the outflow was close to 4 pg/min. Given that recovery of molecules by this type of transstriatal dialysis probe is proportional to molecular weight (Nomikos et al., 1990), the recovery of histamine in striatum may be estimated to be 25%; this extrapolates to an extracellular concentration for histamine of about 50 n M , which is comparable to that seen for dopamine in the striatum. However, the total tissue concentration of histamine in striatum is appproximately 300 fold lower than that of dopamine (Cumming et al., 1989), perhaps relating to the lack of uptake mechanisms for extracellular histamine. The dialysis membrane area was two times greater in striatum than in BNST.  Thus, the  outflow of 6 pg/min in BNST would correspond to 12 pg/min for a probe as large as that used in the striatum. Histamine outflow was greater in BNST than in striatum, as one would expect on the basis of the relatively sparse histamine innervation of the striatum (Watanabe et al., 1984). Russell et al. (1990) found histamine outflow to be greater in hypothalamus than in striatum, but absolute values cannot be compared because of the differing probe geometry and experimental design used in the two studies. Furthermore, the recovery of histamine by probes used in the present study has not been determined. The histamine outflow was insensitive to the local application of T T X and only somewhat sensitive to the reduction of calcium around the probe. These results may reflect the range of penetration of treatments, such as local application of T T X or local removal of calcium, as compared to the tissue volume from which histamine is sampled. Histamine, lacking an uptake system, may be free to diffuse in from a range greater than that perturbed by the calcium and T T X treatments. The present study suggests that the extracellular space might be tonically flooded with histamine, such that levels are insensitive to the local  application of T T X .  This hypothesis could be tested by the infusion of T T X into one medial  forebrain bundle. The histamine efflux, simultaneously determined in the ipsilateral striatum should be responsive to the complete disruption of the action potential in ascending histamine fibres. Extracellular levels of histamine in striatum are higher than what might be expected on the basis of the sparse density of innervation. Thus, the contribution of factors such as catabolism and glial uptake towards net histaminergic tonus requires further study. Given the importance of such factors as dialysis buffer composition and mode of probe implantation, the experimental conditions must be carefully chosen so as to resolve the uncertainty regarding the origin of extracellular histamine. Histamine may be behaving as a neurohumoral transmitter, acting at a considerable distance from the point of release in some brain regions. A major limitation of the present cerebral microdialysis study was the problem of possible interference with the radioenzymatic assay. As discussed above, a wide variety of substances are inhibitors of H N M T . H N M T inhibitors, which might greatly increase the extracellular histamine levels, are certain to interfere with assay. Other molecules potentially capable of altering histamine release, H j drugs for example, are also likely to interfere in the radioenzymatic assay. It is not always possible to correct determined histamine concentrations for the effect of H N M T inhibition and it is difficult to attempt purification of the samples in order to remove inhibitors. The use of better analytical techniques, such as H P L C with postcolumn derivatization (Yamatodani et al., 1985), could facilitate the more complete characterization of the outflow of histamine in cerebral microdialysis. In particular, the effects on histamine release in vivo of drugs known to alter histamine turnover should be determined.  112  (C) Histamine Binding  Pharmacological studies have previously indicated that the H  3  receptor could inhibit the  release of serotonin (Schlicker et al., 1988) and noradrenaline (Schlicker et al., 1988) in cerebral cortex and could inhibit the acetylcholine-mediated contraction of the trachea following vagal stimulation (Ichinose et al., 1989). Thus, H^ receptors in the nervous system cannot be solely autoreceptors. Indeed, Barbin et al. (1980) found a small increase in [ H ] - H A J  binding sites in striatum after a 60% destruction of the histamine innervation. This is consistent with the largely post-synaptic localization of H^ receptors in brain observed in the present study. The structural requirements for H-j activity and specificity have not been fully elucidated, but it should be noted that impromidine, which contains a guanidine moiety, is also an H  2  agonist, while burimamide, which contains a thiourea, is a weak H  2  antagonist  (Timmerman, 1990). The possibility that some compounds may act through multiple histamine receptors must be a consideration in the interpretation of pharmacological studies. Other compounds, such as the H  2  antagonist cimetidine and the H j antagonist mepyramine, are less  likely to interact with the H^ site. A n additional factor in the pharmacology of histamine is that some histamine receptor ligands could alter histaminergic transmission indirectly by inhibiting H N M T , the catabolic enzyme. [ H ] - N - M e H A binding has recently been reported to bind to a single high-affinity site in membranes from guinea pig brain (K^ = 0.4 n M ) , which, on the basis of competitive binding studies, was found to be an H-j site (Korte et al., 1990). Korte et al. (1990) reported •3  [ H ] - N - M e H A binding to be greater in the cortex than in striatum. This difference from the result reported here could reflect species differences or the use of membranes rather than brain sections. The rank order of displacers of [ H ] - N - M e H A reported by Korte et al. (1990) was also partially reversed with respect to the present results. In guinea pig membranes, thioperamide, an H^ antagonist, was a less potent displacer than was histamine, an agonist.  113  Burimamide, another H j antagonist, was a less potent displacer of [ H ] - N - M e H A than impromidine, an agonist. These differences from the present results may be related to the composition of buffers; whereas Korte et al. (1990) employed T r i s - H C l , we used 150 m M sodium phosphate, suggesting that H j antagonist binding may be favored in the presence of sodium, as has been shown for the binding of opiate antagonists (Pert and Snyder, 1974). This would have interesting implications for the mechanism by which ligand binding is able to transfer information to H j receptors. Whereas the binding of opiate receptor antagonists is driven by a change in enthalpy (Hitzmann et al., 1985), dopamine D  2  antagonist binding can  be driven by changes in either entropy or enthalpy (Testa et al., 1987). Van't H o f f analysis of the temperature dependence of H j binding would reveal the thermodynamic factors involved in ligand binding. If the entropic changes following receptor-ligand interactions are related to the organization of water molecules on the binding site, the addition of sodium, a chaotropic agent, might specifically influence the binding of H j drugs for which the dominant thermodynamic factor involved an increase in entropy.  Such a phenomenon could be useful in  the screening for potential agonists and antagonists. The present autoradiographic results indicate that the highest levels of H j binding were in the basal ganglia. The QA-lesion produced a nearly complete destruction of specific H j binding in the striatum and in the ipsilateral substantia nigra pars reticulata. Therefore, the H j binding site is present in large part on striatonigral projection neurons. These neurons are known to contain G A B A and the neuropeptides dynorphin and substance P (Semba et al., 1987, review). It follows that histamine and H j drugs could potentially regulate the release of some of these neurotransmitters and thus directly modulate outflow of the basal ganglia. A sparse histaminergic innervation of the striatum has been observed (Steinbusch et al., 1986). In an electron microscopic study, histidine decarboxylase-immunoreactive axonal varicosities were sometimes seen on spines, i.e. medium spiny striatonigral neurons (Takagi et al., 1986). One might therefore expect H j drugs to have behavioral effects related to actions on the basal ganglia. Indeed, intra-accumbens injections of N - M e H A and other H j agonists have been  114  found to produce thioperamide-sensitive hypoactivity in rats (Bristow and Bennet, 1988a, 1988b). Thioperamide enhances wakefulness in the cat, while  agonists increase slow wave  sleep (Lin et al., 1990), although effects on sleep-waking paramaters need not be mediated by the basal ganglia. The regional distribution of  binding in rat brain is different from that of the other  two histamine binding sites. In contrast to the H  3  distribution, the binding of [ H ] -  mepyramine, an H j ligand, is relatively low in the dorsal striatum and high in hypothalamic structures (Palacios et al., 1981). A selective photoaffinity ligand for the H  2  site (Ruat et al.,  1990) labels most intensely the striatum and the superficial layer but not deeper layers of the cerebral cortex. In that the basal ganglia have few histamine-containing fibres, the pattern of histaminergic innervation of the forebrain (Steinbusch et al., 1986) does not match the distribution of H^ binding. The functions of histamine in the brain must eventually be understood in terms of the polymorphic distribution of different binding sites. We have estimated using cerebral microdialysis the extracellular concentration of histamine to be 50 n M in striatum. Although the influence of G T P on the affinity of histamine binding in vivo is not known, it seems possible that the affinity of histamine for the H j site could be in the range of this extracellular histamine concentration. Thus, the H^ receptor may be an important site for the post-synaptic actions of histamine in the basal ganglia and other areas. The functions of histamine in the brain must be understood in the light of post-synaptic functions. For example, the specific G-protein(s) involved in signal transduction at the H-j receptor must be determined in order to clarify the functional roles of histamine. Systems known to be regulated by histamine, such as the neuroendocrine axis, have mostly been studied through the use of drugs more or less specific for the classical H j and H  2  receptors.  Therefore, the contribution of H^ receptors to the regulation of the neuroendocrine system is as yet unknown. 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