Proc. Nati. Acad. Sci. USA Vol. 75, No. 12, pp. 6290-6294,, December 1978 Neurobiology Histamine H1 receptors identified in mammalian brain membranes with [3H]mepyramine (trieyclic antidepressants/antihistamine/neurotransmitter/amitriptyline) VINH TAN TRAN, RAYMOND S. L. CHANG, AND SOLOMON H. SNYDER* Departments of Pharmacology and Experimental Therapeutics, and Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 Communicated by Julius Axelrod, August 30,1978 ABSTRACT The antihistamine [3H mepyramine binds to Male Sprague-Dawley rats (150-200 g) were killed by cer- HI histamine receptors in mammalian brain membranes. vical dislocation, their brains were rapidly removed and ho- Potencies of H1 antihistamines at the binding sites correlate mogenized with a Polytron for 30 min (setting 5) in 30 vol of with their pharmacological antihistamine effects in the guinea pig ileum. Specific [3Himepyramine binding is saturable with ice-cold Na/K phosphate buffer (50 mM, pH 7.5), and the a dissociation constant of about 4 nM in both equilibrium and suspension was centrifuged (50,000 X g for 10 min). The pellet kinetic experiments and a density of 10pmolper gram ofwhole was resuspended in the same volume of fresh buffer and cen- brain. Some tricyclic antidepressants are potent inhibitors of trifuged, and the final pellet was resuspended in the original secific [3Hmepamine binding. Regional variations of volume of ice-cold buffer by Polytron homogenization. Calf [3Hjmepyramine ing do not correlate with variations in brains were obtained from a local abattoir within 2 hr after the endogeneous histamine and histidine decarboxylase activity. death of the animals and transferred to the laboratory in ice- Histamine is a neurotransmitter candidate in mammalian brain cold saline. The brain was quickly dissected into individual (1-3). Effects of histamine on cyclic AMP and cyclic GMP regions on ice and stored frozen in plastic vials at -70'C until formation may reflect H2 and HI receptor influences, respec- use. Male Hartley guinea pigs (300-400 g) were used. tively (4-8). Preliminary reports described the labeling of his- To measure specific binding of [3H]mepyramine, [f3H]- tamine HI receptors in rat brain (9) and guinea pig ileum (10) mepyramine and unlabeled substances were added to 0.45 ml with [3H]mepyramine. We now present detailed properties of of tissue homogenate with a final incubation volume of 0.5 ml. labeled with Incuation was carried out at 250C for 20-30 min and termi- histamine HI receptors in mammalian brain nated by addition of 4 ml of ice-cold buffer followed by rapid [3H]mepyramine. filtration onto glass fiber filters (GF/B) positioned over a vac- MATERIALS AND METHODS uum and subsequently with three 4-ml washes of cold buffer. Radioactivity trapped on the filters was counted in 10 ml of [3H]Mepyramine (30 Ci/mmol) was prepared by tritium re- Aquasol (New England Nuclear) at an efficiency of 38% after duction of dibrominated mepyramine at New England Nu- a 12-hr stoarge at 4VC. Specific binding was defined as radio- clear. The tritiated material migrated as a single band in thin activity bound after subtraction of nonspecific binding deter- layer chromatography on silica gel and cellulose in 1- mined in the presence of 2MiM triprolidine. Maximal reduction butanol/glacial acetic acid/water, 2:4:1 (vol/vol). a-Chymo- of binding by triprolidine to 40% of total binding levels is trypsin (EC 3.4.21.1; 60 units/mg), phospholipase C (EC 3.1.4.3; constant over the range 0.1-10 MM. Thin layer chromatography from Clostridlum perfringens, 70 units/mg proteirn), and of membranes and medium showed no metabolism of [3H]- trypsin (EC 3.4.21.4; 195 units/mg) were obtained from mepyramine. Worthington. Phospholipase A (EC 3.1.1.4; from bee venom, 1230 units/mg protein), was purchased from Sigma. Com- RESULTS pounds were obtained from the following sources: pyrathiazine Specific [3H]mepyramine binding, total minus nonspecific (Upjohn); promethazine (Wyeth Laboratories, Philadelphia, binding, is linear with tissue concentrations in the range 2-20 PA); trimeprazine, diphenylpyraline, chlorpromazine, meti- mg of wet weight and displays a sharp pH optimum at 7.5. amide, cimetidine, trifluoperazine (Smith, Kline and French Specific [3H]mepyramine binding becomes saturated with Laboratories); methdilazine (Mead Johnson, Evansville, IN); increasing concentrations (Fig. 1). With 0.3 nM receptor con- methapyraline, nortriptyline (Eli Lilly); mepyramine (Robinson centration, which is 10% of the dissociation constant (Kd) (11), Laboratory, San Francisco, CA); chlorcyclizine, triprolidine Scatchard analysis reveals a single population of binding sites (Burroughs Wellcome, Research Triangle Park, NC); antistine, with a Kd of 4.5 nM and a maximal number of binding sites tripelennamine, dimethindene (CIBA, Summit, NJ); meclizine, Bmax of 10.7 pmol per gram of tissue (Fig. 1 middle), re- doxepin (Pfizer); carbinoxamine (McNeil Laboratory, Fort sembling densities of muscarinic, cholinergic, opiate, and a- Washington, PA); phenindamine (Hoffman-LaRoche); or- and fl-adrenergic receptors (12). Hill plots are linear with a Hill phenadrine (Riker Laboratories, Northridge, CA); brom- coefficient of 1.01 (Fig. 1 bottom). pheniramine (Robins Research Laboratory, Richmond, VA); Kinetics of Specific [3HlMepyramine Binding. Specific d- and l-chlorpheniramine (Schering, Bloomfield, NJ); flu- [3H]mepyramine binding associates at 250C with a bimolecular phenazine, triflupromazine (Squibb); methysergide (Sandoz, association rate constant (k1) of 0.073 nM-1 min1 (Fig. 2 Hanover, NJ); 1,4-methylhistamine, N-acetylhistamine (Cal- upper). The dissociation of bound [3H]mepyramine at 250C biochem). Dimaprit and 4-methylhismin were generous gifts on a semilog scale is linear with a half-life of about 2.8 min (Fig. from Marshall Plaut, Johns Hopkins University. 2 lower) and a dissociation rate constant (k-1) of 0.26 min-1. The Kd estimated from the ratio (k-1/ki) is 3.6 nM, closely The publication costs of this article were defrayed in part by page similar to the equilibrium Kd. charge payment. This article must therefore be hereby marked "ad- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. * To whom correspondence should be addressed. 6290 Downloaded by guest on October 2, 2021 Neurobiology: Tran et al. Proc. Natl. Acad. Scz. USA 75(1978) 6291 (A .4-0) -. 0) o 0 D - E a c 30 *11 0. *0c .0 >0 av Ec .0 I v 0 E 0 -. co a a 10 30 IL Time, min V.6 C 70 0 .0 C 7 D 50 ,> ,5 2 E -E 301 a a, -a aU0 ID C 201 0 c- C1 . 10, - \.~~~ 4-E 0 4 6 8 0 Time, min I I FIG. 2. Kinetics of [3H]mepyramine specific binding. (Upper) I Association. Specific [3Hlmepyramine binding to rat brain mem- branes (15 mg of wet weight) at 250C was measured at various time 0 4 8 intervals after the addition of [3H]mepyramine (2 nM). Specific [3H]Mepyramine bound, binding was defined as the difference between the absence and pmol/g of tissue presence of triprolidine (2 ,M). Points shown are those from a single experiment performed in triplicate which was replicated twice. The bimolecular rate constant is calculated according to the equation k 1 = [2.203/t(a - b)]/log [b(a - x)Ia(b - x)], where a is the initial con- centration of 3H-mepyramine (2.0 nM) and b is the initial receptor concentration (0.3 nM). *, Specific binding; 0, nonspecific binding. (Lower) Dissociation. Specifically bound [3H]mepyramine was as- sayed at 250C at various intervals after incubation with [3H]mepy- ramine to equilibrium under standard assay conditions. At time zero, x triprolidine was added to the incubation mixture at a final concen- E tration of 2 gM, and the reaction was terminated by filtration at various intervals. Points shown are those from a single experiment, performed in triplicate, which was replicated twice. C values of 2-3 nM, about 5-8 times more potent than bromodi- phenhydramine and diphenhydramine. Among the ethyl- 0 enediamines, mepyramine, methapyrilene, and triprolidine In[3Hlmepyramine conc. all have Ki values of about 5 nM, whereas antazoline is only 1% FIG. 1. Specific [3H]mepyramine binding to rat brain mem- as potent. d-Chlorpheniramine is about 100 times more potent branes. (Top) Membrane preparations (10-15 mg) were incubated that its I isomer indicating stereospecificity. The H2 antihisti- with various concentrations of [3H]mepyramine at 250C for 20 min mines, burimimide and metiamide, are extremely weak com- in 0.5 ml of 50 mM Na/K phosphate buffer (pH 7.5) in the presence petitors for [3H]mepyramine binding. or absence of 2MuM triprolidine. The experiment was replicated three Histamine itself, with a Ki of 40 ,gM, is substantially weaker times. 0, Total; *, specific; X, nonspecific. (Middle) Scatchard than the H1 antihistamines. However, its effects are selective, analysis of data shown in Top. Kd = 4.5 nM and Bmax = 10.7 pmol/g of tissue. (Bottom) Hill plot of the same data. Slope = 1.01. because 1,4-methylhistamine, virtually inactive at H1 receptors in smooth muscle, is much weaker in competing for [3H]- H1 antihistamines are potent competitors for the specific mepyramine binding. Moreover, dimaprit, 4-methylhistamine, binding of [3H]mepyramine Table 1, Fig. 3). The phenothiazine and 2-aminoethylpyridine, selective H2 receptor agonists, are antihistamines, pyrathiazine and trimeprazine, are most potent substantially weaker than histamine itself. with K, values of about 1 nM, 10-100 times more potent in re- The most potent inhibitors of binding are certain tricyclic ducing [3HJmepyramine binding than are antipsychotic phe- antidepressants. Doxepin (Ki = 0.7 nM) is more potent than any nothiazines. Among the ethanolamine antihistamines, carbi- of the H1 antihistamines evaluated. Amitriptyline (Ki = 4 nM) noxamine and diphenylpyraline are the most potent with K, is comparable in potency to the most active HI antihistamines, Downloaded by guest on October 2, 2021 6292 Neurobiology: Tran et al.