Phosphoinositide hydrolysis mediated by histamine H1-receptors in rat brain cortex

European Journal of Pharmacology, 123 (1986) 187-196

187

Elsevier

PHOSPHOINOSITIDE HYDROLYSIS MEDIATED BY HISTAMINE Hi-RECEPTORS I N RAT BRAIN C O R T E X ENRIQUE CLARO *, LOURDES ARBONI~S, AGUSTINA GARCiA and FERNANDO PICATOSTE Instituto de Biologia Fundamental "Vicent Villar Palasi', and Departamento de Bioquimica, Facultad de Medicina, Universidad A utbnoma de Barcelona, Bellaterra, Barcelona, Spain

Received 31 July 1985, revised MS received 29 November 1985, accepted 14 January 1986

E. CLARO, L. ARBONlkS, A. GARCiA and F. PICATOSTE, Phosphoinositide hydrolysis mediated by histamine Hi-receptors in rat brain cortex, European J. Pharmacol. 123 (1986) 187-196. Histamine stimulated the accumulation of [3H]inositol 1-phosphate in the presence of lithium in [3H]inositol-prelabelled slices from rat brain cortex in a concentration-dependent manner, with an ECs0 value of 94.7 #M. High concentrations of antagonists of histamine H 2 receptors, muscarinic receptors, aa-adrenoceptors and serotonin receptors did not inhibit the effect. The histamine Hi-receptor antagonists mepyramine, triprolidine, promethazine, d-chlorpheniramine and the tricyclic antidepressant doxepin inhibited the response with K i values corresponding to an interaction with histamine Hi-receptors. The ECso for the response was about three times lower than the K i value (approximately 300 #M) for the inhibition by histamine of [3H]mepyramine binding to membranes from rat brain cortex. Partial inactivation of Ha-receptors with the alkylating antagonist phenoxybenzamine resulted in similar reductions in [3H]mepyramine binding sites and in the maximal histamine-induced [3H]inositol 1-phosphate accumulation, without affecting the K D for the radioligand or the ECs0 for the response. The apparent dissociation constant for histamine calculated from these experiments ( K A = 92.2 #M) was not different from the ECs0 value. The present results indicate that histamine-stimulated phosphoinositide hydrolysis in rat brain cortex is mediated by Ha-receptors and that no receptor reserve is present. Phosphoinositide hydrolysis

Histamine Hi-receptors

1. Introduction Histamine (HA) Hi-receptors are present in mammalian brain where they have been localized and characterized mainly by means of binding techniques using the radiolabelled antagonist [3H]mepyramine (for reviews see Schwartz et al., 1982; Green, 1983). Moreover, it has been shown that brain Ha-receptors mediate some HA-induced biochemical responses such as the stimulation of glycogen hydrolysis in mouse brain cortex (Quach et al., 1980) and the potentiation of cyclic A M P accumulation elicited by H A H2-receptor agonists (Palacios et al., 1978) and adenosine ( D a u m et al., 1982) in guinea-pig brain. These responses have * To whom all correspondence should be addressed. 0014-2999/86/$03.50 © 1986 Elsevier SCience Publishers B.V.

Rat brain cortex

[3H]Mepyramine

been suggested to be secondary to changes in intracellular free calcium concentration. Activation of receptors which alter intracellular free calcium concentration also stimulates phosphoinositide hydrolysis, generating diacylglycerol and inositol phosphates thought to act as second messengers. This effect has been proposed as a general transducing mechanism for this type of receptors (for a review see Berridge, 1984). Preliminary evidence for the ability of H A to stimulate phosphoinositide hydrolysis in brain tissue came from studies showing that intracerebral administration of the amine increased the in vivo incorporation of 32pi into rat brain phospholipids (Friedel and Schanberg, 1975; Subramanian et al., 1980). More recently, using a more direct method to measure phosphoinositide breakdown devel-

188 oped by Berridge et al. (1982) i.e. measurement of [3H]inositol 1-phosphate ([3H]IP) accumulation in the presence of lithium in [3H]inositol prelabelled tissue samples, Daum et al. (1984) demonstrated that HA stimulates phosphoinositide hydrolysis in guinea-pig brain slices through Hi-receptors. HA has also been shown to elicit phosphoinositide breakdown in rat brain cortical slices (Berridge et al., 1982; Brown et al., 1984), although no detailed pharmacological characterization of the receptor involved was performed in this case. In the present study we have characterized the HA-induced phosphoinositide hydrolysis in rat brain cortical slices demonstrating that the effect is mediated by Hi-receptors. We also showed the absence of a receptor reserve for this effect. Part of this study was presented in abstract from (Claro et al., 1985).

2. Materials and methods

2.1. Measurement of [3H]inositol 1-phosphate accumulation The HA-induced [3H]IP accumulation in rat brain cortical slices was measured by the method described by Berridge et al. (1982) with some modifications. Male Sprague-Dawley rats (200-300 g) were killed by decapitation and the brain quickly removed. Cerebral cortices, dissected free of white matter, were sliced with a Mcllwain tissue chopper. The slices (350 × 350 /~m) were dispersed and washed in Krebs-Henseleit (KH) buffer (in mM: 116 NaC1, 4.7 KC1, 1.2 MgSO 4, 1.2 KH2PO 4, 2.5 CaCI 2, 25 N a H C O 3, 11 glucose) pH 7.4, equilibrated with O2/CO 2 (95:5) and incubated at 37°C for 30 min. The slices were washed twice and incubated further in 5 volumes of the same buffer containing 0.32 /~M [3H]inositol (16.5 Ci/mmol) at 37°C for 120 min. At the end of this incubation, prelabelled slices were washed extensively with fresh K H buffer and allowed to settle. Portions (50/zl) of packed slices were incubated at 37°C for 60 min in a modified K H buffer containing 10 mM LiC1 and the corrected NaC1 concentration, in the presence or absence of HA and the indicated drugs, in a final volume of 250 /~1.

When present, antagonists were preincubated with slices for 15 min. Incubations were stopped by adding 0.94 ml ice-cold chloroform/methanol (1 : 2 v/v). After 15 min on ice, 0.31 ml of chloroform and 0.31 ml of water were added and the phases separated by centrifugation at 2000 × g for 5 min. Aliquots (0.75 ml) of the aqueous phases were diluted with 3 ml of water and passed through columns containing 0.3 ml of Dowex l-X8 (100-200 mesh) formate form. After washing the columns with 4 ml of water and 4 ml of 5 mM disodium tetraborate/60 mM sodium formate [3H]IP was eluted with 4 ml of 0.1 M formic acid/0.2 M ammonium formate. This fraction was shown to contain most of the [3H]IP since, in routine controls of chromatographic recovery, more than 95% of a load of a [14C]IP standard (7 nCi) co-eluted in the same fraction. When more polar [3H]inositol phosphates were to be eluted, 4 ml of 0.1 M formic acid/0.4 M ammonium formate followed by the same volume of 0.1 M formic acid/1 M ammonium formate were used as eluents. The radioactivity present in eluates was counted in the gel phase after addition of 10 ml of a toluene/triton X100 based scintillation cocktail. Tritium incorporated into lipids was determined by counting aliquots (100 /~1) of the organic phases. The measurement of the radioac= tivity of the column eluates and organic phases was done in a Packard Scintillation Counter with 40 and 50% efficiency respectively. Radioactivity in the [3H]IP fraction was routinely expressed as percent of total label incorporated into lipids in order to correct for variations in [3H]inositol incorporation and sample size.

2.2. Binding of [~H]mepyramine to particulate fractions from rat cerebral cortex Binding of [3H]mepyramine (0.5-14.0 nM) to membranes from rat brain cortex was assayed in 50 mM N a / K phosphate buffer pH 7.5 at 25°C for 40 min or in lithium-containing K H buffer at 37°C for 15 min, by the filtration method previously described (Codolh and Garcia, 1985). Nonspecific binding was determined in the presence of 6 × 1 0 - 7 M promethazine.

189

2.3. Irreversible inhibition of [3H]mepyramine binding and HA-stimulated [3H]IP accumulation in rat brain cortex by phenoxybenzamine Aliquots of packed slices were incubated in 5 volumes of KH buffer with phenoxybenzamine (10-8-10 -5 M), at 37°C for 45 min. The medium was aspirated off at the end of the incubation and the slices were washed extensively with ice-cold phosphate buffer (50 mM, pH 7.5) then homogenized in 10 volumes of the same buffer. Particulate fractions were obtained and [3H]mepyramine binding was assayed in phosphate buffer. When the effect of phenoxybenzamine pretreatment on the HA-induced [3H]IP accumulation in cortical slices was to be studied, the slices were prelabelled with 0.32/~M [3H]inositol at 37°C for 120 min, washed and incubated again in 5 volumes of KH buffer with or without 300 nM phenoxybenzamine at 37°C for 45 min. After extensive washing with fresh KH buffer, control and pretreated slices were used to measure the effect of HA on [3H]IP accumulation as described in section 2.1.

2.4. Chemicals Myo-[2 -3 H]inositol (16.5 Ci/mmol) and [3n]mepyramine (24.1 Ci/mmol) were obtained from New England Nuclear, myo-[U24 C]inositol 1phosphate (55 mCi/mmol) from Amersham, Dowex 1-X8 (100-200 mesh, formate form) from Biorad, histamine dihydrochloride, mepyramine and promethazine from Sigma, methysergide from Sandoz and atropine from Merck. Generous gifts of dimaprit, cimetidine, metiamide and phenoxybenzamine (Smith, Kline & French), doxepin (Pfizer), d-chlorpheniramine (Essex S.A.) and 1chlorpheniramine (Schering) are acknowledged.

3. Results

3.1. Characteristics of the histamine-induced [ 3H]IP accumulation in rat brain cortical sfices Under our experimental conditions, the incorporation of [3H]inositol into lipids of cortical slices

[3H]IP ACCUMULATION (PERCENT OF CONTROL)

/t/t

25(3

2O<3

+cm .,'~f +,#+

+~ -LOG [HA]

Fig. 1. Concentration-effect relationship for HA-stimulated [ 3H]IP accumulation. Rat brain cortical slices were labelled for 120 min with 0.32 ~M [3H]inositol and washed. Aliquots of prelabeiled slices were incubated for 60 min in the presence of 10 mM LiCl and in the absence (control, B) or presence of increasing HA concentrations. Incubations were terminated by adding chloroform/methanol and [3H]IP was separated from the aqueous phases by chromatography on Dowex 1 columns. For each sample [3H]IP accumulation was expressed as percent of [3H]inositol incorporated into lipids. The results, presented as percent of controls, are means__. S.E.M. of quadruplicate determinations in 7 separate experiments performed with the same HA concentrations. Average control value was 5.85 +0.51.

was linear for at least 120 min of incubation. At this time (used in routine experiments) the radioactivity incorporated ranged from 15 000 to 25 000 cpm per 50 #l of packed slices. In the presence of 10 mM LiC1, HA stimulated the accumulation of [3H]IP but not that of inositol bisphosphate and inositol trisphosphate and the response was linear for at least 60 min. The HA-induced [3H]IP accumulation was concentration-dependent (fig. 1), reaching maximal stimulation (124.1 _+ 14.2% over controls, means _+ S.E.M., n = 9) at HA concentrations from 1 to 3 mM, with an average ECs0 value of 94.7 _+ 11.8

190 TABLE 1

gM. Linear Hill transformation of the data allowed to estimate Hill coefficients which averaged a value (1.04 + 0.05) not significantly different from unity, indicating that the response was mediated by a single receptor population. The response caused by 1 mM HA was blocked by micromolar concentrations of the Hi-receptor antagonists mepyramine, triprolidine, promethazinc and d-chlorpheniramine and the tricyclic antidepressant doxepin (table 1). The same concentration of the inactive isomer 1-chlorpheniramine did not inhibit the response, indicating a stereospecific interaction. The participation of HA H2-receptors in the effect was excluded, since dimaprit (1 mM did not induce the response and metiamide (100 #M) and cimetidine (100 gM) were unable to block it. Finally, micromolar concentrations of serotonin receptor, al-adrenoceptor and muscarinic receptor antagonists also failed to block the HA-induced response. As shown in fig. 2 (left panel), mepyramine caused parallel shifts to the right of the concentration-effect curve for HA, suggesting competitive

Effect of various drugs on HA-stimulated [ 3H]IP accumulation in rat brain cortical slices. Prelabelled slices were incubated in the presence of the indicated compounds and the [3H]IP accumulation was determined. Results are means+ S.E.M. of quadruplicate determinations from a single experiment. Drug

[3H]IP as % of total label incorporated into lipids

None Histamine 1 mM Dimaprit 1 mM HA 1 mM + mepyramine 1 ~tM HA 1 mM + triprolidine 1/~M HA 1 mM + promethazine 1 #M HA 1 mM + d-chlorpheniramine 1 ~tM HA 1 mM + 1-chlorpheniramine 1 jaM HA 1 mM +doxepin 1 ~aM HA 1 mM +cimetidine 100 ~M HA 1 mM + metiamide 100 #M HA 1 mM + methysergide 1/~M HA 1 mM +prazosin 1/~M HA 1 mM+atropine 1 ~M

6.87 + 0.14 30.51 5-1.21 9.45 5- 0.37 6.67 5- 0.37 6.80 5- 0.37 7.545:0.14 9.64 5- 0.03 34.47 5- 0.29 7.39 5- 0.62 32.92 5- 0.37 25.50 5- 0.20 31.52 5- 0.46 32.63 5- 0.68 31.89 5-1.62

PERCENT OF MAXIMAL EFFECT

PERCENT OF MAXIMAL EFFECT

t

100

75

/

75

t/'

50

2~

t/ /1 ,

-LOG tHAI

50

25

--/J -LOG [MEPYRAMINE]

Fig. 2. Antagonism by mepyramine of HA-stimulated [3H]IP accumulation. Left panel: concentration-effect curves for the HA-induced response in the absence (e) or presence of 25 nM (O) and 50 nM (A) mepyramine. The results were normalized to percent of the stimulation produced by 3 mM HA. Right panel: inhibition by increasing mepyramine concentrations of the response induced by 1 mM HA. Results are expressed as percent of the stimulation produced by 1 mM HA. In both experimental procedures mepyramine was added to the tissue samples 15 min before the addition of HA. All points are means5-S.E.M, of 3 separate experiments, each performed in quadruplicate.

191 TABLE 2 Inhibition of HA-stimulated [3H]IP accumulation by several antihistamines. Results are means+S.E.M, of values obtained in N independent experiments like the one represented in fig. 2 (right panel). IC50 and n H values were estimated from linear Hill transformations of the data. K i values were calculated from the corresponding IC50 values using the equation K i = IC5o/(1 + HA/ECso), where HA = 10- 3 M and ECs0 = 94.7 × 10- 6 M.

Mepyramine Triprolidine Promethazine d-Chlorpheniramine 1-Chlorpheniramine Doxepin

IC50 (nM)

nH

K i (nM)

N

43.6 + 21.1 32.8 + 1.2 79.5 + 21.5 176.0 + 57.5 50 100 10.6

1.00 + 0.18 1.17 + 0.20 1.08 + 0.12 0.88 + 0.15 0.94 0.96

3.8 + 1.8 2.8 + 0.1 6.9 + 1.9 15.2 + 5.5 4 334 0.9

3 3 3 3 1 1

antagonism. The dissociation c o n s t a n t ( K i ) for m e p y r a m i n e , calculated from the relationship dose ratio - 1 = [ m e p y r a m i n e ] / K i, was 8.2 + 1.2 n M (n = 3), which is of the same order of m a g n i t u d e as that reported for [3H]mepyramine b i n d i n g to rat b r a i n Hi-receptors ( T r a n et al. 1978; Hill a n d Young, 1980). K i values for other antihistaminic c o m p o u n d s were o b t a i n e d by measuring the inh i b i t i o n of the [3H]IP a c c u m u l a t i o n i n d u c e d by 1 m M H A in rat cortical slices b y increasing concentrations of antagonist in experiments like those presented in fig. 2 (right panel) for mepyramine. Values for the IC50 a n d Hill coefficients (n H) were o b t a i n e d from linear Hill transformations of the data a n d the K i values were calculated from the c o r r e s p o n d i n g IC50 utilizing the Cheng-Prusoff e q u a t i o n (Cheng a n d Prusoff, 1973) (table 2). The K i values o b t a i n e d for the classical antihistamines m e p y r a m i n e , triprolidine, p r o m e t h a z i n e a n d dc h l o r p h e n i r a m i n e a n d for the tricyclic antidepressant doxepin were in the n a n o m o l a r range a n d similar to those reported for the i n h i b i t i o n of specific [3H]mepyramine b i n d i n g to rat b r a i n m e m b r a n e s ( T r a n et al., 1978; Hill a n d Young, 1980). The Hill coefficients were not significantly different from unity. The stereospecificity of the i n t e r a c t i o n was d e m o n s t r a t e d by the fact that 1-chlorpheniramine was a b o u t three h u n d r e d times less p o t e n t than d-chlorpheniramine.

3.2. Histamine inhibition of [~H]mepyramine binding to rat brain cortical membranes T h e i n h i b i t i o n b y H A of [3H]mepyraminespecific b i n d i n g to rat b r a i n cortical m e m b r a n e s

was assessed in order to correlate the effect of H A on [3H]IP a c c u m u l a t i o n in rat b r a i n cortex with the agonist occupancy of Ht-receptors. As described in Methods, the b i n d i n g experiments were carried out both in phosphate buffer at 25°C, as routinely performed, a n d in K H buffer at 37°C in order to reproduce the m e d i u m a n d temperature c o n d i t i o n s used in experiments where the response was measured. Scatchard analysis on the saturation curves (not shown) gave the same value for the dissociation c o n s t a n t (KD) for [3H]mepyram i n e u n d e r both c o n d i t i o n s (5.7 a n d 5.9 n M , respectively). I n h i b i t i o n b y H A of [3H]mepyram i n e b i n d i n g was also similar in both experimental situations (table 3), presenting Hill coefficients TABLE 3 HA inhibition of specific [3H]mepyraminebinding to rat brain cortical membranes. Aliquots (0.7 mg of protein) of the membrane fraction were incubated with 1.9 nM [3H]mepyramine and 13 different HA concentrations. Incubations were carried out in phosphate buffer at 25°C for 40 min or in lithium-containing KH buffer at 37°C for 15 min. Non-specific binding was defined in the presence of 6)<10-7 M promethazine. Linear Hill transformation of the data was used to estimate Hill coefficients and IC50 values. Calculation of K i w a s done using the equation K i = IC50/(1 + [3H]mepyramine/KD) (Cheng and Prusoff, 1973) where K D is the equilibrium dissociation constant for [3H]mepyramineobtained in saturation experiments. Results are means+S.E.M, of 3 independent experiments for each incubation condition. Incubation buffer

Inhibition of [3H]mepyramine binding by HA nH

K i

Na/K phosphate KH

0.72 _+0.03 0.82 + 0.08

265 _ 70 310 + 56

(p.M)

192

of less than unity and apparent K~ values about three times greater than the EC50 value for the stimulation of [ 3H]IP accumulation. 3.3. Irreversible inactivation of histamine Hl-receptors with phenoxybenzamine The difference between the ECs0 for the HA stimulation of [3H]IP accumulation and the apparent K i for inhibition of [3H]mepyramine binding prompted us to study the effect of the irreversible antagonist phenoxybenzamine on specific [3H]mepyramine binding and HA-induced [3H]IP accumulation. Incubation of rat brain cortical slices with phenoxybenzamine, as described in Methods, was shown to reduce [3H]mepyramine (1.9 nM)-specific binding in a concentration-dependent manner (fig. 3, left panel). Saturation experiments performed with control and phenoxybenzamine (300 nM)-pretreated slices revealed PERCENT OF UNINHIBITED [aH]MEPYRAMINE SPECIFIC BINDING

that the alkylating agent reduced the total number of specific binding sites without affecting their affinity for [3H]mepyramine (fig. 3, right panel). The same concentration of phenoxybenzamine reduced the maximal response to HA to a similar extent (fig. 4) without altering the ECs0 value, indicating the absence of 'spare' receptors. The linear plot of l/[HA] vs. 1/[HA'], where [HA] and [HA'] are the HA concentrations giving an equal response before and after partial inactivation of receptors (fig. 4, insert), allows the calculation of the equilibrium dissociation constant (KA) from: K A = (slope- 1)/ordinate intercept (Furchgott, 1978). The average K Awas 92.2 + 15.7 ~M (n = 3), a value closely similar to the EC50. 4. Discussion

The results obtained in this study provide strong evidence that HA induces phosphoinositide BOUND/FREE (hnol/mg prote4n.nM)

100

75

5,0

25

\, \. ~e\ .

\ 20

-LOG [ 1:~,4ENOXYBENZ.AMINE]

40

60

80

I~-IIMEPYRAMINE il(XA~O (fn~/mg Wot~m)

Fig. 3. Inhibition of [3H]mepyramine binding to rat brain cortical membranes by pretreatment with phenoxybenzamine. Left panel: aliquots (250 FI) of packed slices were incubated in KH buffer (1.25 ml final volume) containing increasing concentrations of 'phenoxybenzamine, at 37°C for 45 min. The slices were then washed extensively with ice-cold 50 mM phosphate buffer, pH 7.5, and homogenized. Particulate fractions were obtained and binding assays were performed in phosphate buffer using 1.9 nM [ 3H]mepyramine. Results, expressed as percent of the specific binding found in phenoxybenzamine-untreated tissue, are means _+S.E. of triplicate determinations in a single experiment. Right panel: Scatchard plots of saturation isotherms of specific [3H]mepyramine binding to membranes from control (e) and phenoxybenzamine (300 nM)-pretreated slices (C)). Results are means of triplicate determinations in a single experiment.

193 PERCENT OF MAXIMAL EFFECT 100

I /.

/I.

t[HA](.~0-4)

~.o

/o/"

l"

~,'[MA,](.~o-O)

/

i

-LOG [HA]

Fig. 4. Effect of phenoxybenzamine on HA-stimulated [3H]IP accumulation in rat brain cortical slices. Prelabelled slices were incubated in KH buffer in the absence (e) or presence (O) of 300 nM phenoxybenzamine at 37°C for 45 rain. After extensive washings with KH buffer, control and phenoxybenzaminetreated slices were incubated without (B) or with the indicated concentrations of HA and [3H]IP accumulation was determined. Results, expressed as percent of the stimulation produced by 3 mM HA in control slices, are means + S.E.M. of 3 different experiments each performed in quadruplicate. Inset represents the plot of the reciprocals of the isoactive HA concentrations in control (HA) and phenoxybenzamine (HA')pretreated slices, from which the equilibrium dissociation constant (KA) for HA was estimated according to Furchgott (1978).

breakdown in rat brain cortex through activation of Hi-receptors. Using essentially the same experimental procedure as described by Berridge et al. (1982), we have shown that, in the presence of lithium, HA elicits the accumulation of [3H]IP in [3H]inositol-prelabelled rat brain cortical slices. This effect demonstrates the ability of HA to induce phosphoinositide hydrolysis in a more direct way than did the measurement of the HAstimulated 32p1 incorporation into rat brain phospholipids used in previous studies (Friedel and Schanberg, 1975; Subramanian et al., 1980), since this latter effect reflects phosphoinositide resynthesis, a less direct consequence of the initial

agonist-induced phosphoinositide breakdown. Under our experimental conditions, the maximal response elicited by 1-3 mM HA represents on the average a 124% increase over basal levels, a value similar to or slightly higher than that reported by Berridge et al. (1982) and Brown et al. (1984) who measured the same HA-induced effect in rat brain cortex with a method similar to that used by us. Compared to the effect of other biogenic amines on phosphoinositide hydrolysis in rat brain cortex, the magnitude of the response elicited by HA is in the same order as the response elicited by serotonin (Brown et al., 1984; Kendall and Nahorski, 1985) and much lower than the one found upon stimulation of muscarinic receptors (Berridge et al., 1982; Brown et al., 1984; Gonzales and Crews, 1984; Jacobson et al., 1985) and al-adrenoceptors (Brown et al., 1984; Schoepp et al., 1984; Minneman and Johnson, 1984; Kendall et al., 1985). Direct mediation of Hi-receptors in HA-induced phosphoinositide hydrolysis is indicated by the failure of specific antagonists for other receptors (atropine, prazosin and methysergide) to block the response at micromolar concentrations. Also, the lack of effect of dimaprit as agonist, and of metiamide and cimetidine as inhibitors, excludes the participation of HA H2-receptors. Furthermore, mediation by Hi-receptors, first indicated by the blockade of the response by micromolar concentrations of H~-receptor antagonists, was confirmed by further pharmacological characterization. Mepyramine caused parallel shifts to the right of the concentration-response curve for HA, indicating competitive antagonism. Also the K i value obtained was similar to that reported for the binding of [3H]mepyramine to H~-receptors in rat brain membranes (Tran et al., 1978; Hill and Young, 1980). Assuming competitive antagonism, we estimated inhibition constants from the inhibition of the response to 1 mM HA by increasing concentrations of mepyramine, triprolidine, promethazine and d-chlorpheniramine. The K i values calculated were similar to those reported for the inhibition of [3H]mepyramine binding to rat brain HFreceptors (Tran et al., 1978; Hill and Young, 1980). Furthermore, the 300-fold higher potency of d-chlorpheniramine versus the inactive isomer 1-chlorpheniramine indicated a stereospecific in-

194 teraction. The tricyclic antidepressant doxepin, which radioligand binding studies have shown to have a higher affinity for Hi-receptors than do other H~-antagonists (Tran et al., 1978) was included in this study since its inhibitory potency had not been determined by measuring a functional response in brain tissue. As shown, the K i for doxepin inhibition of HA-stimulated phosphoinositide breakdown in rat brain cortex is lower than the K i values for the other H~-antagonists, in agreement with binding studies in rat brain (Tran et al., 1978; Aceves et al., 1985) and with the inhibition of Hi-receptor-mediated contraction of guinea-pig ileum (Aceves et al., 1985). Hill coefficients for the inhibition curves of the H~antagonists and doxepin were not different from unity, indicating interactions with a single receptor' population. Phosphoinositide hydrolysis coupled to activation of Hi-receptors has been also demonstrated in guinea-pig brain (Daum et al., 1983; 1984). However, there are differences between guinea-pig and rat brain regarding both the potency of HA in eliciting the response and the relationship of this potency with the apparent HA affinity for the H~-receptors determined in binding studies. The ECs0 values for the HA-induced [3H]IP accumulation in rat brain cortex is about seven times higher than the values reported for guinea-pig (Daum et al., 1984), indicating a different sensitivity to HA of the same tissue from the two species. Differences in the characteristics of the H~-receptors in guinea-pig and rat brain have been also suggested by the results of radioligand binding studies showing different affinities of some antagonists (Chang et al., 1979; Hill and Young, 1980). On the other hand, while the concentration-response curve correlates well with receptor occupancy by HA, as studied by inhibition of [3 H]mepyramine binding, in guinea-pig brain cortex (Daum et al., 1984) this is not the case in rat brain cortex. In this tissue the apparent inhibition constant for HA, estimated from the inhibition of [3 H]mepyramine binding, is about three times higher than the ECs0 value for the HA-induced response. The discrepancy between ECso and K i values was not due to the

presence of 'spare' receptors since partial inactivation of receptors with the irreversible inhibitor phenoxybenzamine, resulted in a similar loss of maximal response and [3H]mepyramine specific binding sites without change in the potency of HA. Furthermore, the apparent dissociation constant for the HA-induced response, estimated as described by Furchgott (1978), was similar to the ECso value. On the other hand, the possibility of an effect of buffer composition and temperature on the inhibition of [3H]mepyramine binding by HA was excluded since the same results were obtained when carrying out the binding experiments in both N a / K phosphate buffer at 25°C and lithium-containing KH buffer at 37°C, conditions used in the analysis of the HA-induced response. One possible explanation for the discrepancy could be that parameters for interaction of HA with Hi-receptors may be different in intact cells and membrane fractions as has been reported for the interaction of agonists with a l(Sladeczek et al., 1983) and fl-adrenoceptors (Hoyer et al., 1984; Toews and Perkins, 1984). Finally, while it has not yet been studied, the presence of different affinity states of the Hi-receptors for HA in rat brain cortex could result in differences between receptor occupancy and the induction of the response, if this latter was coupled to only one affinity state of the receptor. This has been shown to be the case for the muscarinic receptor in rat brain (Fisher et al., 1983) and chicken heart (Brown and Brown, 1984) where the stimulation of phosphoinositide hydrolysis is coupled to the low affinity state of the receptor for agonists. The hypothesis that increased phosphoinositide hydrolysis could be a general consequence of the interaction of HA with H~-receptors is supported by the finding of phosphoinositide hydrolysis coupled to activation of H~-receptors, as demonstrated in rat brain in the present study and in guinea-pig brain by Daum et al., (1984), together with the suggested participation of these receptors in the same effect in guinea-pig ileum (Jafferji and Michell, 1976) and rabbit aorta (Villalobos-Molina and Garcia-Sainz, 1983).

195

Acknowledgements This work was supported in part by CAICYT Grants No. 358/81 and 2583/83. E. Claro and L. Arbones are recipients of fellowships from Ministerio de Educacion y Ciencia (Madrid).

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