THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 257, No. 22, Iwue of November 25, pp. 13593-13601, 1982 Printed in U S.A.

Solubilization and Characterization of Histamine HI Receptors in Brain*

(Received for publication, November 25, 1981)

Lawrence Toll$ and Solomon H. Snyder8 From the Departments of Neuroscience, Pharmacology, and Experimental Therapeutics, and Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

C3H]Doxepin, a tricyclic antidepressant, binds with high affinity to guinea pig brain membranes with a drug specificity indicating an association with HI his- tamine receptors. The r3H]doxepin binding site has been solubilized, with digitonin being the only deter- gent able to maintain specific binding after solubiliza- tion. After solubilization, the kinetics and drug speci- ficity of binding are virtually identical with those ob- tained in the intact membranes, indicating a conser- vation of the transmitter binding site after removal of the receptor from its lipid environment.

The regulation of agonist affinity for the histamine HI receptor by cations is maintained after solubiliza- tion. Sodium and to a similar extent lithium, but not potassium, rubidium, or cesium, decrease the affinity of agonists for the receptor. The divalent cations man- ganese and magnesium maintain their ability to in- crease agonist affinity after solubilization. Guanine nu- cleotides, however, lose their ability to decrease ago- nist affinity for the histamine HI receptor after solubil- ization.

Histamine receptors in rat brain differ from guinea pig brain receptors in the potency of several antihis- tamines. The difference is maintained in the solubilized receptors. Sucrose gradient and gel filtration experi- ments indicated M, - 430,000 for the receptor-digitonin complex. The isoelectric point of the receptor is 4.8. None of these physical techniques distinguishes be- tween guinea pig and rat brain receptors.

Besides its role in mediating allergic responses and gastric acid secretion, histamine appears to serve as a neurotransmit- ter in the brain (1-3). Most actions of histamine are mediated by two discrete receptors. The histamine HI sites are involved in allergic and some cardiovascular actions, while histamine HP receptors mediate influences on gastric acid secretion. Pharmacological evidence indicates that both HI and Hz re- ceptor sites function in mammalian brain (4-8).

Histamine HI receptors in brain (9-11) and peripheral tis- sues (12,13) can be labeled by the binding of [3H]mepyramine. This binding is regulated in a selective fashion by certain

* This work was supported in part by United States Public Health Service Grants MH-18501 and NS-16375 and a grant from the Mc- Knight Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Park, CA 94025. $ Present address, Life Sciences Division, SRI International, Menlo

0 Recipient of United States Public Health Service Research Sci- entist Award DA-00074. To whom all correspondence should be addressed.

monovalent and divalent cations as well as guanine nucleo- tides (14). In preliminary experiments, we showed that it was feasible to solubilize HI receptors from mammalian brain, while retaining the ability to bind [3H]mepyramine selectively (15). Because of our initial observation that the antidepressant doxepin has the highest affinity for H1 receptor binding sites of any drug examined, we have developed [3H]doxepin as a ligand to provide levels of specific binding with a slower dissociation rate, thus permitting a detailed characterization of HI receptors (16). In the present paper, we describe physical properties of H1 receptors solubilized from guinea pig brain and rat brain and labeled with r3H]doxepin and compare the effects of cations and guanine nucleotides on receptor binding in the soluble and membrane-bound states.

MATERIALS AND METHODS

Membrane Preparati~n-[~H]Doxepin was prepared by New Eng- land Nuclear (Boston, MA). Frozen guinea pig brains were from Pel- Freeze. All of the detergents tried as well as ATP, GTP, bovine serum albumin, and activated charcoal were from Sigma. The sources of the drugs were the same as described by Tran et al. (11). Frozen guinea pig brains were thawed on ice and then homogenized with a Polytron (Brinkmann Instruments) in 40 volumes of Tris-HC1, pH 7.7. The homogenate was then centrifuged at 50,000 X g for 10 min. The pellet was resuspended in the same volume of ice-cold Tris-buffer and centrifuged again. The final pellet was resuspended in 80 volumes of 10 mM sodium phosphate buffer, pH 7.5, by Polytron homogenization. For experiments on rat brain membranes, male Sprague-Dawley rats were decapitated, and the whole brains were rapidly removed, ho- mogenized in 40 volumes of Tris-HC1, and treated as described for guinea pig brains.

To measure specific binding of r3H]doxepin, 100 pl of 3H-iigand and 100 p1 of the appropriate unlabeled displacer were incubated with 800 pl of the tissue homogenate to provide 10 mg of tissue/tube. The incubation was carried out at 25 “C for the appropriate length of time, usually 1 h. The reaction was terminated by placing the samples on ice followed by the addition of 4 ml of ice-cold phosphate buffer. The samples were rapidly fitered onto glass fiber filters (Whatman GF/ B) with a 3 X 4 ml wash of ice-cold buffer. Radioactivity trapped on the fiters was counted in 10 ml of Formula 947 (New England Nuclear, Boston, MA).

Specific binding was defined as radioactivity bound after subtract- ing nonspecific binding assayed in the presence of 2 PM triprolidine.

Solubilization Procedures-Preparation of membranes prior to solubilization was as described above. The final pellet containing approximately 400 mg of protein was then homogenized with a glass- Teflon homogenizer in approximately 12.5 volumes (50 m l ) of 10 mM sodium phosphate buffer, pH 7.5, containing 1% digitonin or other detergent as indicated. The homogenate was then left on ice for 30- 60 min prior to centrifugation at 100,000 X g for 60 min. The super- natant which contained approximately 1 mg of protein/& was rou- tinely diluted 2-fold with sodium phosphate buffer and used for the binding assay. In experiments to study ion or nucleotide effects on the affinity of histamine, the membranes were solubilized in 50 mM Tris-buffer, pH 7.7, containing 1% digitonin. For gel fitration, isoelec- tric focusing, and sucrose gradient centrifugation experiments, the soluble receptor preparation was concentrated approximately 40-fold

13593

13594 Histamine HI Receptors

by ultrafdtration through a Diaflo XM-100 membrane (Amicon Cor- poration) and stored up to 1 month at -70 "C.

Assay for j3H]Doxepin Binding to Solubilized Histamine HI Receptors-To measure binding to the solubilized preparations, in- cubations were the same as those described for [3H]doxepin binding to brain membranes. With the solubilized preparations, nonspecific binding was determined using 2 p~ triprolidine. High concentrations of all potent inhibitors of [3H]doxepin binding inhibited binding to the same extent as 2 p~ triprolidine. For routine experiments, after the incubation, the samples were placed on ice for 5 rnin prior to the addition of 100 pl of 10% activated charcoal and 2% bovine serum albumin in distilled water. Five min after the addition of charcoal, the samples were centrifuged for 4 min in a Brinkmann Microfuge. Eight hundred p1 of the supernatant were added to I0 ml of Formula 947 and radioactivity was assayed.

In some experiments, bound radioactivity was assayed by filtration after precipitation of the protein either with ammonium sulfate or a combination of y-globulin plus polyethylene glycol in the concentra- tions indicated in the table. After precipitation with ammonium sulfate, samples were filtered under vacuum over GF/B filters which were then washed twice with 5 ml of 50% ammonium sulfate. Five min after precipitation by polyethylene glycol, the samples were filtered and washed with 2 X 5 ml of 8.5% polyethylene glycol. Filters were assayed for radioactivity as described above.

The experimental binding data for saturation of triprolidine-sensi- tive doxepin binding were subjected to a nonlinear least squares fitting algorithm using a model for association of doxepin to two independent receptor sites (17). The model used to determine the number and affinity of the two binding sites is given by the equation

(DPH (DBL (D) -+ KH (D) -+ KI,

Dr=----"t+

where D, represents total bound doxepin, D represents free doxepin, KH and KL are the equilibrium affinity constants of doxepin to each receptor, and BH and BL are the total concentration of the two receptor sites.

Gel Filtration-Gel filtration chromatography of the soluble his- tamine receptor was accomplished in two ways. Five ml of the concentrated stock solution were applied to a column (2.5 X 60 cm) of Bio-Gel A-5m (Bio-Rad) which had been previously equilibrated and was then eluted with sodium phosphate buffer, pH 7.4, 100 mM NaCI, 0.1% digitonin. The column was eluted at approximately 20 ml/ h and 4-ml fractions were collected. Each fraction was assayed for C3H]doxepin binding to soluble receptor in duplicate as described above. The absorbance of each fraction at 280 nm was also deter- mined.

Conversely, the concentrated stock solution of guinea pig or rat brain HI receptor was diluted 10-fold into 10 mM sodium phosphate buffer, pH 7.4, with 100 m~ NaCI. This was incubated with 1 nM [3H] doxepin with or without 2 p~ triprolidine for 60 min at 25 "C. The solution was then placed on ice for 5 min, whereupon 0.35 ml of the [3H]doxepin-receptor complex was applied to a column of Bio-Gel A- 5m. The column had been previously equilibrated with and was eluted with 10 m sodium phosphate, pH 7.4, with 100 mM NaC1, 0.1% digitonin at a flow rate of 7 ml/h. The absorbance at 280 nm was monitored as 0.25-ml fractions were being collected which were as- sayed for radioactivity to locate the [3H]doxepin-receptor complex.

In one experiment, material isolated from separate sides of the peak of binding activity from the column (2.5 X 60 cm) was pooled and concentrated to 10 ml through a Diaflo XM-100 membrane. This solution was then incubated with 1 nM r3H]doxepin and gel filtration was performed as just described.

Sucrose Gradient Centrifugation-Thirty-six-ml linear gradients were prepared from 5 and 20% sucrose in H20 and DzO containing 10 mM sodium phosphate and 0.1% digitonin. The stock-concentrated HI receptor (0.25 ml) was diluted to 1 ml with phosphate buffer and layered on the top of the gradient. Aliquots (1 ml) of appropriate

were centrifuged at 100,OOO X g for 18 h in a Beckman SW 27 rotor. standard proteins were layered on identical gradients. The gradients

After centrifugation, a capillary was lowered to the bottom of the centrifuge tubes, the gradient was pumped out, and 1.5-ml fractions were collected. Each fraction was diluted to 3.5 ml with phosphate buffer and assayed for ['H]doxepin binding to soluble receptor in duplicate as described above.

Isoelectric Focusing-Gels (10 X 20 X 0.3 cm) were poured between two glass plates using 1% Isogel Agarose-EF (LKB) in distilled water, and 1% pH 4-9 ampholytes. Bio-Rad 4-6, LKB 4-6, Serva 4-6, and Servalyt 4-9 ampholytes were tested on r3H]doxepin binding. Servalyt

4-9 ampholytes were the only ones that did not inhibit ["Hldoxepin binding or increase dissociation. The agarose gel was run on a Bio- Rad horizontal electrophoresis cell cooled to 2 "C. The concentrated stock solution of HI receptor was labeled with 1 nM [3H]doxepin with or without 2 p~ triprolidine. Aliquots (0.5 m l ) of [3H]doxepin-receptor were mixed with 100 pg of hemoglobin and adsorbed onto pieces (0.5 X 1 cm) of filter paper and then placed on the gel. The gel was run approximately 2 h until the hemoglobin was sharply focused. It was then removed from the chamber and individual tracks were cut. The tracks were either stained for protein with Coomassie bdlant blue, or sliced into 4-mm fractions. T h e fractions were put into scintillation vials and 1 ml of distilled water was added to each vial. The vials were briefly shaken and the pH of each fraction was determined. Ten ml of liquid scintillation mixture were then added and the vials were left shaking overnight before counting.

Protein was assayed by the method of Lowry et al. (18) using bovine serum albumin as a standard.

RESULTS

Conditions for Detection of [3H]Doxepin Binding to Soluble HI Receptors

Membrane proteins vary in their response to surfactants, depending on their ionic or nonionic properties, with some membrane proteins retaining biological activity following treatment with a wide range of detergents while others lose activity with most detergents. Accordingly, we evaluated a wide range of detergents for use in solubilizing H, receptors (Table I). When binding is assayed by centrifugation following adsorption of unbound ligand to charcoal, substantial specific binding occurs following digitonin solubilization. However, little or no specific binding is apparent with a variety of other ionic or nonionic detergents. With 1% digitonin, specific t3H] doxepin binding to soluble preparations is 50-75% of total binding, which is somewhat less than in membranes, but still ample for routine assays. When the digitonin concentration is reduced to 0.5%, more than half the amount of protein is solubilized, but only about 10% of the specific binding is detected. One per cent digitonin solubilizes approximately half of the membrane protein, as well as removing half of the membrane-bound [3H]doxepin binding sites. Of the sites not remaining in the membranes, about 25% are detected as solubilized H, receptors. It is unclear whether the remainder

TABLE I Effect of various detergents for solubilization of histamine H I

receptors Guinea pig brain membranes were homogenized in 1% of the proper

detergent (unless stated otherwise). After centrifugation at 100,OOO x g for 1 h, the supernatants were assayed for specific binding as described under "Materials and Methods." The pellets were washed superflcidy with sodium phosphate buffer and then homogenized in 80 volumes of the same buffer. Specific binding was assayed as described for brain membranes. Values are means of triplicate deter- minations from a single experiment. The experiment was repeated, giving virtually the same results. "

I

binding

Digitonin (0.5%)

Cholate 0.5 Deoxycholate 0.2 Octylglucose- 0.7

Tween-20 0.5 Tergitol 0 Nonidet P-40 0 Triton X-100 0

pyranoside

I

pernatant I ___ specific >inding

mol 229 109 177 176 89 15 14

112 35 10 30

__

Pellet __

Protein

!J4? 800 437 692 896 447 412 671

683 462 430 440

__

___

~

Specific tctivity

nol/mg 260 249 256 196 199 36 21

164 76 23 68

Histamine HI Receptors 13595

of the sites have been denatured by the detergent treatment or whether native sites are not detected by our receptor assay.

Soluble neurotransmitter receptors have been assayed by precipitation with a combination of polyethylene glycol and a-globulin or ammonium sulfate followed by filtration over glass filtration filters (15). We evaluated these techniques as well as adsorption of unbound [3H]doxepin to charcoal fol- lowed by centrifugation to assay soluble histamine receptor binding. The highest ratio of specific to nonspecific binding occurred with the precipitation methods. However, the amount of specific binding varied considerably from experi- ment to experiment indicating a variability in the degree of precipitation of receptor-bound ligand or dissociation of the ligand-receptor complex. Assay by adsorption of unbound ligand to charcoal followed by centrifugation provides a suf- ficient ratio of specific to nonspecific binding as well as the most consistently high recovery of receptor-bound ligand. Accordingly, for routine experiments, C3H]doxepin binding to soluble receptors was assayed by charcoal adsorption and centrifugation.

Saturation and Kinetic Analysis of [3H]Doxepin Binding to Soluble Histamine H I Receptors

Saturation studies using [3H]doxepin binding to guinea pig brain membranes showed two independent specific binding sites (16). The high affinity site which had a KD of 0.26 nM and B,,, of 6.7 pmol/g of tissue, wet weight, represented binding to the histamine HI receptor. The low affinity binding could not be attributed to any known binding site.

[3H]Doxepin binding to solubilized guinea pig brain resem- bles very closely the binding to brain membranes. Scatchard analysis of binding of various ["Hldoxepin concentrations to solubilized brain membranes reveals two distinct components using a computer fit model (Fig. 1). The high and low affinity components have dissociation constants of 0.98 f 0.32 and 15.4 f 7.2 n ~ , respectively, and maximal number of binding sites of 84 f 12 and 464 f 78 fmol/mg of protein.

The rate of association of [3H]doxepin to soluble HI recep- tors was evaluated by incubating 0.5 rm [3H]doxepin with soluble preparations for varying intervals (Fig. 2). At 25 "C, binding reaches plateau values at 45-60 min. Half-maximal binding is apparent at about 8 min. The time course of association fits biomolecular kinetics. The rate constant for association ( k l ) is calculated as 0.06 min" nM". This is essentially identical with the kl for association of [3H]doxepin to intact membranes assessed in parallel experiments.

Dissociation of ['Hldoxepin from receptors was determined by incubating 0.5 nM ['Hldoxepin with the soluble membrane preparations at 25 "C for 1 h, whereupon 2 PM triprolidine was added and residual binding was monitored at varying intervals at 0 or 25 "C (Fig. 2). When plotted on a semilogarithmic scale, there appears to be a very small component with rapid dissociation. The majority of the receptors dissociate with a half-life of 45 min at 25 "C, essentially the same as in the membrane-bound state. At 0 "C, there is virtually no dissocia- tion up to 3 h. However, dissociation is complete by 24 h (data not shown). The calculated rate constant for dissociation ( L l )

at 25 "C is 0.014 min" KIM". The KD value calculated from the ratio k - l / k l for the soluble HI receptor is 0.43 r m , similar to that obtained in saturation experiments and similar to both kinetic and saturation analysis obtained in the membrane- bound state in parallel experiments.

It is unclear why dissociation experiments reveal only a small percentage of low affinity binding sites. The reason for this discrepancy may be inherent in the resolution of the nonlinear Scatchard plot. Unfortunately, even a computer- generated fit of saturation data into two straight lines for a

10 20 30 40 50

[%-I] WXEPIN (nM)

1 0 0 h

[3M]OOXEPlN BOUND I frol I rnp proleml

FIG. 1. Saturation of specific [3H]doxepin binding to soluble histamine HI receptor. A, the curue represents a computer fit of experimental data from five separate experiments, all conducted in triplicate. [3H]Doxepin at various concentrations was incubated with soluble receptor as described under "Materials and Methods" in the presence or absence of 2 p~ triprolidine. B, Scatchard analysis of the same data.

Scatchard plot does not have perfect accuracy. For the KD and B,, values determined by the computer, approximately 33% of the binding would be to the low affinity component of 0.5 nM [3H]doxepin. However, if the outer limits of the com- puter-derived standard deviations for KD and B,,, are used, only about 16% of the binding would be to the low affinity site. In fact, it is possible that the amount of low affinity binding is represented just as well by the small amount we see in the dissociation curve as the amount indicated in the Scatchard analysis.

Drug Specificity of [3H]Doxepin Binding to Soluble HI Receptors

To determine whether sites labeled in the soluble state represent pharmacologically relevant H1 receptors, we evalu- ated the potencies of a series of drugs at these sites. Because [3H]doxepin is apparently binding at more than one site, interpretation of the inhibition data is difficult. The drug specificity of the inhibition of [3H]doxepin binding appears to be similar in soluble and membrane-bound states, and similar to values for inhibition of ['Hlmepyramine binding to brain membranes (11). However, inhibition of [3H]doxepin binding does not conform to mass action kinetics for a single binding site. In all cases, the pseudo-Hill coefficients were determined to be approximately 0.7-0.8. Due to this difficulty, the values given in Table I1 are the concentrations needed to inhibit 50% of the triprolidine-sensitive C3H]doxepin binding.

Although the numerical values are not in perfect agreement with the molar potencies of some drugs in inhibiting hista- mine-induced contraction of the guinea pig ileum (19,20), the ICao values listed in Table I1 show a good correlation and indicate that the majority of ["Hldoxepin binding is to hista- mine HI receptors. Thus, in all circumstances, binding is stereospecific with respect to the isomers of the HI antihista- mine chlorpheniramine. H1 antihistamines which are potent

13596 Histamine HI Receptors

A 50r

C

2 e, 40-

F

7 SPECIFIC BINDING

15 30 45 60

TIME.rnln

I ‘ I A IO 20 30 40 50 60 70 80 90

TIME, rnin FIG. 2. Kinetics of [’Hldoxepin binding to solubilized hista-

mine HI receptor. A, association. Binding was measured as described under “Materials and Methods,” except charcoal was added and the samples were centrifuged immediately following incubation for the desired time. Specific binding was defined as the difference between the binding in the presence (blank) and absence (total) of 2 p~ triprolidine. The bimolecular rate constant is calculated according to the equation -kl = [2.203/(a - b)]/log[b(a - x)/a(b - x ) ] where a is the initial concentration of [3H]doxepin (0.5 nm) and b is the initial receptor concentration. B, dissociation. Specifically bound [3H]doxe- pin was assayed after various intervals a t 25 and 0 ”C after incubation with [3H]doxepin to equilibrium (60 min) under standard assay con- ditions. At time zero, 2 PM triprolidine was added to the incubation. The reaction was terminated by the addition of each sample to a centrifuge tube containing charcoal and by their immediate centrifu- gation. Points were means of triplicates of a single experiment repli- cated twice.

in pharmacological studies such as mepyramine, triprolidine, pyrathiazine, and methapyrilene display high affinities for both soluble and membrane-bound states with IC50 values of 0.5-4 nM. The most potent agent examined is doxepin itself with ICm values in the soluble and membrane-bound state of about 1.0 m. Among histamine-related agonists, the H1-spe- cific agonists aminoethylpyridine and methylaminoethylpyri- dine display substantial affinity, although about 4-fold less than the affinity of histamine itself. By contrast, the Hz- specific agonist dimaprit is only 1% as potent as histamine (data not shown). A variety of substances unrelated to hista- mine receptors display no affinity for these sites.

Comparison of Cation and Guanine Nucleotide Effects on Soluble and Membrane- bound H I Receptors

In the membrane-bound state, sodium fairly selectively decreases the affinities of agonists but not antagonists for

opiate (21), a-adrenergic (22), and histamine HI (14) receptors. It has been postulated that this effect may reflect an associa- tion of sodium permeability and neurotransmitter recognition sites in synaptic membranes. Accordingly, it would be of considerable interest to determine whether such an effect is retained in the soluble HI receptor. At 100 l l l ~ Concentration, sodium decreases the affinity of histamine for soluble Hi receptor binding sites about 5-fold, quite similar to the influ- ence of sodium in parallel experiments with membrane-bound HI receptors (Table 111). This effect is obtained to a similar extent with lithium, but is not manifested with the other monovalent cations examined, potassium, rubidium, and ce- sium. In the membrane-bound as well as in the soluble state, lithium has a similar effect as sodium, while potassium, rubid- ium, and cesium are inactive. Sodium decreases the affinity of the HI agonists aminoethylpyridine and methylaminoethyl- pyridine to a similar extent as histamine itself.

To determine the concentration dependence of the sodium effect, we evaluated the extent of reduction of [3H]doxepin binding to soluble receptors in the presence of 10 /.LM histamine and varying sodium chloride concentrations (Fig. 3). The extent of inhibition of [3H]doxepin binding by histamine varies with sodium concentration. Half of the maximal reduction in the potency of histamine is apparent with 25 mM sodium, similar to that observed previously with membrane-bound histamine (14), opiate (21), and a-adrenergic (22) receptors.

Receptor binding for numerous neurotransmitters is regu- lated by divalent cations (23-26) and guanine nucleotides (27- 33). In some instances, there appear to be interactions between the regulation by divalent cations and guanine nucleotides (32). Guanine nucleotide effects are thought to involve a GTP binding protein which is separate and distinct from the neu- rotransmitter receptor itself. Accordingly, in a solubilized

TABLE I1 Drug influences on f Hlmepyramine and Hldoxepin binding to

membrane-bound or soluble HI receptors Specific binding was determined as described under “Materials and

Methods.” The inhibition of specific [3H]doxepin binding to guinea pig brain membranes and soluble HI receptors was determined with 0.5 nM [3H]doxepin and 5-7 concentrations of competing drugs as- sayed in triplicate. ICso values were determined by log probit analysis. NE = less than 15% displacement observed at 10 mM. Values are the mean from at least two separate experiments conducted in triplicate.

IC, Drug Membrane-bound Soluble

[3H]doxepin [3H]doxepin nM

Antihistamines Mepyrmine Triprolidine Pyrathiazine Methapyrilene d-Chlorpheniramine I-Chlorpheniramine

Tricyclic antidepressants Doxepin Amitriptyline

H2 antagonists Metiamide Cimetidine

5-Hydroxytryptamine Propranolol Clonidine Histidine Carbamylcholine y-Aminobutyric acid Lysergic acid diethylamide Cyclohexyladenosine

Others

0.9 0.9 2.5 6.6 3.6

330

0.7 5.7

465,000 NE

NE

225,000 NE

435,000 NE

4,500 NE

0.8 0.7 2.5 5.2 2.8

315

0.7 2.7

345,000 462,000

NE 195,000 270,000

NE 450,000

NE 14,700

NE

Histamine HI Receptors

TABLE I11 Effect of monovalent ions on agonist inhibition of fH]doxepin binding

13597

Specific [3H]doxepin binding and ICso values were determined as described in Table 11. The concentration of the indicated ion was 100 mM in each case. Values are mean f S.D. from at least three separate experiments conducted in triplicate.

ICs0

Control NaCl KC1 LiCl RbCl CSCl

Soluble Histamine 12.3 f 0.6 75 f 6 Aminoethylpyridine 63 f 10 260 f 24 Methylaminoethylpyridine 54 f 12 162 f 48

Histamine 10 f 3 80 f 19 Membrane bound

100.

90 -

80 -

70 -

60.

50 ~

40”3 IO 20 50 100

[NaCI]

FIG. 3. Effect of various concentrations of NaCl on inhibition by histamine of specific C3Qdoxepin binding. Specific [3H]dox- epin binding was determined as described under “Materials and Methods.” Specific [3H]doxepin binding at various concentrations of NaCl in the presence of 10 PM histamine was expressed as the percentage of specific binding in the absence of histamine. Values are mean 2 S.D. from two separate experiments, each conducted in triplicate.

receptor preparation, one would not expect to detect an influ- ence of guanine nucleotides on receptor binding. In instances where the divalent cation effect is closely linked to the GTP binding protein, one would also not anticipate observing such influences in the soluble state. With solubilized @-receptors, neither GTP nor divalent cation influences are detectable (34).

In brain membranes, we earlier found that guanine nucleo- tides selectively decrease the affinity of histamine for [3H] mepyramine binding sites, while the affinities of HI antago- nists were not markedly affected (14). This effect was selective, being manifested by GTP, its nonmetabolized analog 5“guan- yly1-/3,yimidodiphosphate and GDP, but not by GMP, ATP, ADP, or AMP. Divalent cations, on the other hand, enhanced the affinity of H1 agonists for receptor sites.

In the present study, we confirm the enhancement of his- tamine’s affinity for HI receptors in the presence of divalent cations with the effects being most marked using manganese and magnesium (Table IV). In contrast to findings with solu- bilized @-receptors (34), the enhanced affinity of histamine is apparent in the solubilized as well as in the membrane-bound HI receptor. By contrast, while GTP decreases histamine’s affinity in the membrane-bound state, an effect not apparent with ATP, neither GTP nor ATP alters histamine’s potency at HI receptors in the soluble state.

P M

18 f 4 84 f 3 15 k 3 13.5 f 2

28 f 4 165 f 19 16 f 3

TABLE IV Influence of GTP, ATP, MnCZ2, MgCL, and CaC& on inhibition by

histamine of [3H]doxepin binding Specific [3H]doxepin binding and IC, values were determined as

described in Table 11. Values were means f S.D. from at least three separate experiments conducted in triplicate

Histamine IC,

Soluble Membrane hound

P M

Control 12.3 f 0.6 10.3 & 3.0 MnCI2 (1 mM) 6.0 f 0.4 3.1 t 0.3 MgC12 (1 mM) 9.3 f 1.5 4.2 f 0.6 CaClz (1 mM) 12.0 f 0.7 5.4 k 0.4 GTP (1 mM) 13.3 f 2.0 23.0 k 3.0 ATP (1 mM) 13.5 f 3.0 11.0 f 3.0

TABLE V Drug effects on fH]doxepin binding to soluble and membrane-

bound histamine H, receptors in rat and guinea pig brain Specific [3H]doxepin binding and IC50 values were determined as

described in Table 11. Values are mean k S.D. from two separate experiments conducted in triplicate.

IC%

Rat brain Guinea pig brain nM

Membranes Triprolidine 13.5 f 1.2 0.9 f 0.2 d-Chlorpheniramine 31 f 1.5 4.6 2 0.9 Pyrathiazine 30 f 2.4 3.0 1 0.6 Doxepin 1.0 f 0.3 0.7 f 0.2

Triprolidine 52 f 18 2.7 t 1.5 d-Chlorpheniramine 67 f 10 6.3 f 1.8 Pyrathiazine 30 f 10 3.0 -+ 0.2 Doxepin 1.5 f 0.3 0.6 k 0.2

Soluble

Species Comparisons of the HI Receptor in Soluble and Membrane- bound States

Several antihistamines differ markedly in affinities for HI receptor binding sites in guinea pig and rat brain membranes, suggesting receptor heterogeneity (35). Interestingly, not all antihistamines show this heterogeneity, nor does any other class of drug.

Such apparent receptor heterogeneity could relate to the receptor’s lipid environment or to the primary structure of the receptor protein itself. To distinguish between these possibil- ities, we compared the ability of several drugs to displace [3H] doxepin from the HI receptor in the membrane and in soluble form in guinea pig and rat brain (Table V).

Several antihistamines, but not the tricyclic antidepressant doxepin, are substantially more potent in displacing [3H]dox- epin from the guinea pig HI receptor than from the rat HI

13598 Histamine HI Receptors

. .

E 0

5 10 5 20 25 30 35 40 L

FRACTION NUMBER

9

8

7

6 %

5

4

53

FIG. 4. Isoelectric focusing of solubilized [3H]doxepin-receptor complex. The H, receptor from guinea pig brain was bound with ['H]doxepin in the presence (A) and absence (A) of 2 ~ L M triprolidine. Receptor from rat brain was incubated in the absence of triprolidine (0). Radioactivity was extracted from 4-mm slices of the gel.

receptor. This difference is maintained upon solubilization, indicating that it is not simply a membrane phenomenon,

Molecular Parameters of the Solubilized HI Receptors In light of the difference in binding properties of the hista-

mine Hi receptor from the guinea pig and rat brains, some physical properties of the H1 receptor binding sites were compared in the two species.

Isoelectric Focusing-The isoelectric point of the HI recep- tor was determined by isoelectric focusing of the digitonin- solubilized guinea pig and rat brain membranes through an agarose gel containing 0.1% digitonin. The receptor was lo- cated by binding ['Hldoxepin in the presence or absence of triprolidine (for total and blank) prior to focusing and then slicing and counting the individual tracks of the focused gel. Focusing is complete in 2 h at 2 "C, in which time there is virtually no dissociation of [3H]doxepin from the receptor.

The radioactivity in each slice of the gel is shown in Fig. 4. It is interesting that there are consistently two peaks of radioactivity for both rat and guinea pig in samples corre- sponding to total ['Hldoxepin binding. When the receptor is incubated in the presence of ["Hldoxepin and triprolidine, only the peak at pH 6.5 remains. The peak representing specific binding and, thus, the H1 receptor therefore disap- pears in the presence of triprolidine and has an isoelectric point of 4.8. Furthermore, there is absolutely no difference in the isoelectric point of the HI receptor solubilized from guinea pig or rat brain.

Gel Filtration-To determine an approximation of the size of the histamine HI receptor, the solubilized receptor was run on a gel filtration column to determine the Stokes radius and sedimented through a sucrose gradient to find its sedimenta- tion coefficient.

To determine the Stokes radius of the H1 receptor-digitonin complex, the solubilized receptor was treated in two ways. In one experiment, the solubilized receptor was layered on a column (2.5 X 60 cm) of Bio-Gel A-5m, followed by elution in the presence of 0.1% digitonin. The collected fractions were assayed for ['Hldoxepin binding (Fig. 5A). In another experi-

ment, ['Hldoxepin was bound fwst to the solubilized receptor followed by addition of the receptor-['Hldoxepin complex to a column (1 X 28 cm) of Bio-Gel A-5m (Fig. 5B). The time needed for completion of this column elution is less than 3 h, in which time there is virtually no dissociation of [3H]doxepin from the receptor.

The peak of activity representing the receptor-digitonin complex elutes at the same rate relative to calibrating proteins in both column assay conditions (Fig. 5). In both cases, doxe- pin binding activity moves slightly faster than the main pro- tein peak. The Stokes radius is estimated at 6.5 nm.

In columns equilibrated and eluted with 0.05% or less digi- tonin, no binding activity elutes from the column. When ["HI doxepin is bound to the receptor prior to gel filtration, most of the protein and all of the receptor-["Hldoxepin complex appears in the void volume, indicating aggregation of the receptor.

Because the peaks representing H, receptor eluted from the column are so broad, it is possible that receptors from the sides of the peak represent distinct proteins. To examine this more closely, we pooled fractions with ['Hldoxepin binding activity from the sides of the peak eluted from the column (2.5 X 60 cm). These were then concentrated, bound with ["HI doxepin, and run on a second column (1 x 28 cm). The peak of radioactivity eluted has fewer counts but the same shape as material not run over the initial gel filtration column. This indicates that the broadness of the peak is probably a function of the detergent-protein complex rather than due to distinct proteins.

Sucrose Density Gradient-Molecular weight of HI recep- tors was also estimated using a gradient of 5-20% sucrose (Fig. 6). Because [3H]doxepin would dissociate from the receptor during the 18-h centrifugation, unlabeled receptor was layered onto the gradients and each fraction was assayed separately for [3H]doxepin binding. The peak of ["Hldoxepin binding is well separated from the majority of the protein with a sedi- mentation coefficient of 14.4 S .

The molecular weight of a detergent-solubilized membrane protein in a crude solution can be determined from the Stokes

Histamine H I Receptors 13599

A I

0 (D

2 0.5

AI _IProtc’”

?

i‘

t I 200

n

150

100

5 0

20 30 40 50 60 70 80 90 1 0 0

i”

FRACTION NUMBER

4 + I 1

Specific 4 I I

Binding \ / I !

FRACTION NUMBER FIG. 5. Gel filtration of solubilized histamine HI receptor. A,

gel filtration of solubilized receptor from guinea pig brain followed by assay for [3H]doxepin binding in each fraction. Fractions were assayed for specific binding (A) and absorbance at 280 nm (0). B, soluble HI receptor was labeled in the membrane stated with [3H]doxepin.

radius and the sedimentation coefficients determined in Hz0 and D20 (36-38). To do this, the partial specific volume of the protein-detergent complex is estimated from its sedimentation properties, the molecular weight of the complex is determined, and the amount of detergent bound is calculated from the known values for partial specific volume of detergent and protein. The molecular weight of the protein component of

1.0

B

D.5

0

Receptar from guinea pig was incubated in the presence (0 blank) or absence (A total) of triprolidine, and rat brain receptor in the absence of triprolidine (A). Binding and filtration conditions were as described under “Materials and Methods.” Calibrating proteins are 1, thyro- globulin; 2, ferritin, 3, catalase; 4, hemoglobin.

the protein-detergent complex can then be calculated. For this procedure to give meaningful results, the detergent used for solubilization must have a partial specific volume signifi- cantly different than that of the protein in question. Unfor- tunately for the histamine HI receptor, digitonin is the only detergent of use, and it has a partial specific volume of 0.738 ml/g (39), similar to the partial specific volume of protein.

Histamine H I Receptors

L

a

I 1 12 1314

- u X n

IO 20

FRACTION NUMBER

5

ii P

25

FIG. 6. Sucrose density gradient centrifugation of solubi- lized histamine HI receptor. Centrifugation was performed as described under “Materials and Methods.” Fractions were assayed for specific [3H]doxepin binding (A) and protein (0). Protein markers for the determination of s20.~ were as follows: 1, thyroglobulin, 19.3 S; 2, catalase, 11.3 S; 3, aldolase, 7.35 S; and 4, hemoglobin, 4.2 S. Points for [3H]doxepin binding were mean & S.D. for duplicate determina- tions from a single experiment replicated four times.

TABLE VI Molecular parameters of soluble histamine HI receptor-digitonin

complex Data were obtained as described in the text. Calculations of partial

specific volume, molecular weight, and frictional ratio were as de- scribed bv Neer (37).

Parameter

Stokes radius ( A (nm)) 6.5 k 0.3 Partial specific volume (J (ml/g)) 0.73 k 0.05 Sedimentation coefficient (SZO,~,) 14.4 f 1.2 M , 430,000 Frictional ratio 2.3 Isoelectric point 4.6 zk 0.2

Because of this, the partial specific volume of the protein- detergent complex does not change with increasing detergent bound. One would then expect no difference in the observed sedimentation coefficient in the H20 and DzO, which in fact, has been found (data not shown). Consequently, the amount of detergent bound and the molecular weight of the protein component of the protein detergent complex cannot be deter- mined. Nevertheless, the molecular parameters of the protein digitonin complex can be calculated (Table VI).

DISCUSSION

The present study shows the feasibility of measuring brain histamine HI receptor binding in the soluble state. Receptor binding can be detected in solubilized preparations, but only under very specific conditions. Of numerous detergents eval- uated, only digitonin permits the retention of ligand binding to soluble receptors. Although it was possible to detect specific binding in solubilized preparations with various assays, highly reproducible levels with good recovery of receptor binding could be obtained only by using charcoal and centrifugation.

Digitonin solubilized half of the membrane protein and removed half of the (3H]doxepin binding sites from the mem- brane. About 25% of those sites could be recovered as soluble HI receptor sites. It is possible that digitonin is solubilizing a subpopulation of Hl receptors. Conversely, the similarity be- tween binding characteristics in soluble and membrane-bound forms may suggest that our “solubilized” receptor may still be attached to small membrane fragments which do not sediment a t 100,000 X g for 1 h. This possibility was discounted by sucrose gradient sedimentation experiments and gel filtration of the soluble receptor. Results of these studies indicate a truly soluble binding site.

It is unclear why digitonin is uniquely capable of solubilizing HI receptors with retention of binding activity. Other recep- tors that maintain binding activity after solubilization by digitonin but not other detergents include &adrenergic recep- tors in erythrocytes (34,40), muscarinic cholinergic receptors (41, 42), and dopamine (43, 44) receptors. Digitonin is a mild detergent which acts by binding to cholesterol. This may conserve the proper receptor conformation or possibly leave in place phospholipids which may be necessary for receptor function.

One of the more interesting aspects of the solubilized HI receptor is the extreme similarity of drug influences, satura- tion, and kinetic properties in soluble and particulate condi- tions. Other receptors whose properties seem relatively un- changed in the solubilized compared to the membrane-bound state include ,&adrenergic (34), 7-aminobutyric acid, and ben- zodiazepine (15), and dopamine (43) receptors. Similarly, the influence of monovalent and divalent cations is the same in the soluble and membrane-bound states. If alterations in sodium conductance are related to the synaptic actions of histamine HI receptors, it would be possible that such an ion conductance channel occurs on the same or very closely associated molecule as the histamine recognition site. In this case, the sodium recognition site might be expected to remain associated with the HI receptor even after solubilization and extensive purification.

There is evidence that, at a-adrenergic receptor binding sites, guanine nucleotides and divalent cations interact in regulating binding so that their combined effects are not simply the algebraic sum of their individual influences (32). By contrast, the influences of divalent cations and guanine nucleotides on histamine H1 receptor binding in intact brain membranes seem to be the sum of opposing effects with no unique type of synergistic interactions (14). Our findings that manganese and magnesium enhance agonist affinity a t soluble HI receptors, despite the loss of guanine nucleotide effects, indicate that divalent cations and guanine nucleotides influ- ence histamine HI receptors at distinct sites.

Phylogenetic studies with a number of receptors have in- dicated a distinct conservation of receptor properties among varying species. For this reason, the finding of different bind- ing properties of the histamine HI receptor in guinea pig and rat brain (35) as well as in different tissues (13) raises the possibility of multiple histamine HI receptors. Conversely, differences between guinea pig and rat brain binding affinities could simply be due to membrane effects and not to the receptor itself. Our finding that the solubilized HI receptor from rat and guinea pig both retain their binding properties indicates real differences in the receptor proteins.

In attempting to distinguish between the two HI receptors on a molecular level, we characterized the receptors in terms of isoelectric point, sedimentation properties, and Stokes re- sults. In general, isoelectric focusing is extremely sensitive in distinguishing among proteins. However, slight spreading of membrane proteins in the presence of detergent is not unusual.

Histamine H I Receptors 13601

The isoelectric point determined for the HI receptor from both guinea pig and rat brain was pH 4.6-4.8, slightly more acidic than the majority of the membrane proteins.

Utilizing sedimentation and gel filtration experiments, we were able to determine the sue, shape, and molecular weight of the receptor-digitonin complex. We are not, however, able to make a determination about the amount of digitonin bound to the receptor and, thus, the molecular weight of the receptor itself. Smith and Pickels (45) showed that digitonin itself sediments as micelles with a particle weight of at least 75,000 daltons containing at least 60 molecules of digitonin. Hubbard (39) found 180-200 molecules of digitonin bound to rhodopsin upon solubilization. It is quite likely that as many as 200 molecules of digitonin representing about 240,000 daltons comprise the detergent portion of the 440,000-dalton deter- gent-receptor complex. With a large portion of the receptor- digitonin complex being due to detergent, it is not surprising that we did not find differences in sedimentation or Stokes radius between guinea pig and rat HI receptor. A more exten- sive characterization of the possible multiple receptors will entail purification of the receptor.

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