Acta Physid Scand 1990, 140, 135-141

Histamine receptors in brain vessels of guinea-pig: in-vitro pharmacology and ligand binding A. O T T O S S O N , S. J. HILL" and L. E D V I N S S O N t Department of Forensic Medicine, University of Lund, Sweden, Department of Physiology and Pharmacology, Queen's Medical Centre, Nottingham, UK, and Department of Internal Medicine, University of Lund, Sweden

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OTTOSSON, A., HILL,S. J. & EDVINSSON, L. 1990. Histamine receptors in brain vessels of guinea-pig : in-vitro pharmacology and ligand binding. Acta Physiol Scand 140, 135-141. Received 5 September 1989, accepted 15 March 1990. ISSN 0001-6772. Department of Forensic Medicine, University of Lund, Sweden, Department of Physiology and Pharmacology, Queen's Medical Centre, Nottingham, UK, and Department of Internal Medicine, University of Lund, Sweden. The subtype of histamine receptors in brain vessels of guinea-pig has been characterized by ligand binding and in-vitro pharmacology using selective antagonists. In the basilar artery histamine caused a concentration-related contraction with an EC,, of 1.6 f0.3 ,UM. HI-receptor blockade with mepyramine and chlorpheniramine caused a displacement to the right of the histamine concentration-response curve with an apparent K , of 0.4 and 4.6 nM respectively, whereas H,-receptor blockade with cimetidine was without effect. Histamine did not induce any dilatatory responses of vessels procontracted by 60 mM potassium-containing buffer in the presence or absence of histamine antagonists. Ligand-binding studies with [3H]mepyramine yielded a K , value of 5.5 nM in pial vessel membranes and 1.7 nM in the choroid plexus, confirming the presence of HI-receptors. Nimodipine caused a concentration-related blockade of histamine-induced contractions. Omission of Ca2+from the extracellular medium for 30 min reduced the contractile responses to histamine in the basilar artery by 96 yo. Subsequent addition of Ca2+ caused concentration-related contractions which were inhibited by nimodipine. Thus, the histamine HI-receptor activation in guinea-pig basilar artery is coupled to dihydropyridine-sensitive Ca2+channels. Key words : cerebral vessels, extracellular calcium, guinea-pig, histamine receptors.

I t is widely accepted that the physiological response to local administration of histamine is dilatation of resistance vessels, and it is likely that both HI- and H,-receptors are present in resistance vessels in most tissues (Ganellin 1982, review). However, contrary to what has been found for both cat (Edvinsson et al. 1983) and rat (Gross et al. 1981a), namely that histamine H,receptors mediate relaxation in cerebrovascular smooth muscle, we found that the guinea-pig basilar artery only reacts with contraction upon stimulation by histamine. T h e histamine receptors and their way of acting on brain vessels Correspondence : Anders Ottosson, Institutionen for rattsmedicin, Solvegatan 25,223 62 Lund, Sweden.

in the guinea-pig was investigated using both physiological responses in vitro and ligandbinding techniques. M A T E R I A L S A N D METHODS In-vitro pharmacology. Specimens were obtained from 31 adult female guinea-pigs weighing between 0.3 and 0.5 kg. All animals were decapitated, the brain was immediately removed and placed in aerated buffer solution (for composition see below) and the basilar artery was dissected free. Small ring segments of the basilar artery (approximately 2-3 mm in length) were suspended between two L-shaped metal prongs in temperature-controlled (37 "C) 5-ml tissue baths containing aerated (95% 0, and 5% CO,) buffer solution composed of (mM): NaCl 119, KCI 4.6,

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CaCI, 1.5, MgCI, 1.2, NaH,PO, 1.2, NaHCO, 15, and glucose 11.0. In experiments involving a calciumfree solution, CaC1, was omitted from the buffer . solution and EGTA was added (10 p ~ )Contractions were measured by means of Grass FT-03 transducers, amplified and recorded on a Grass polygraph (Hogestatt et at. 1983). Each vessel segment was given a tension of 1 mN and allowed to equilibrate for 6&90 min before test drugs were administered. Concentration-response data were obtained by cumulative addition of histamine to the tissue baths, and the mean histamine concentration eliciting halfmaximum contraction (EC,,) was calculated. The responses are reported as a percentage of a reproducible maximum response of the vessels to a modified buffer solution containing 124 mM potassium, achieved by an equimolar substitution of NaCl for KC1. To test for potential relaxant responses, histamine was administered after precontraction of the arteries by a modified buffer solution containing 60 mM potassium. In some experiments the endothelium was removed by rubbing the intima with a wooden stick. The absence of endothelium was verified by the lack of dilatory response to acetylcholine (Furchgott 1983). Concentration-response curves to histamine were obtained in the absence and presence of various concentrations of chlorpheniramine, mepyramine (HIreceptor antagonists) or cimetidine (H,-receptor antagonist). Histamine antagonists were added to the tissue baths 20 min before the responses to histamine were tested. The concentration ratios (CR = EC,, with antagonist/EC,, without antagonist) and the apparent dissociation constant (K,) were calculated. The intluence of variations in the extracellular calcium concentration on contractions induced by histamine was also examined. The dihydropyridinetype calcium channel blocker nimodipine was added to the tissue baths in concentrations from 1 x lo-" to 11, and then contractions induced by 1 x 10-5 M histamine were recorded. Also, the vessels were kept in a calcium-free solution for 30 min and then 1.0 x 10-5M histamine was added in the presence or absence of 3.0 x lo-* M nimodipine. Concentrationresponse data were derived by cumulative addition of calcium to the tissue bath. When appropriate mean values SE of the mean are given. Ligand-binding srudy. Cerebral vessels were collected from 54 guinea-pigs during the course of other experiments and stored frozen at 20 OC. The cerebral vessels were homogenized in five volume of Krebsphosphate buffer: NaClll8 mM, KCI 4.7 mM, MgSO, 1.2 mM, CaCI, 2.5 mM, NaH,PO, 5 mM, wglucose 5 . 5 m y pH 7.2 (see Hill & Young 1981). Homogenization was achieved using a Polytron blender (setting 5 ) for two periods of 20 s at 2-min intervals. Aliquots (5Opl) were added to 350pl of Krebs-phosphate buffer and various concentrations of

[3H]mepyramine in the presence and absence of 2 ,UM promethazine for 20 min at 30 "C. Incubations were terminated by rapid centrifugation at 8700 g for 1 min in a microfuge, and the pellet was twice washed superficially with 0.1 ml of ice-cold medium. The bottom of the microfuge was then cut off into a scintillation vial and the radioactivity determined by scintillation counting. Specific binding was taken as that insensitive to inhibition by 2 p~ promethazine. The protein content of the homogenates was determined by the method of Lowryet al. (1951). Curves for the specific binding of [3H]mepyramine were fitted as a single hyperbola using the method of Wilkinson (1961). In some experiments the Harwell library routine VBOlA was used to fit the data as a hyperbola and a linear component (Hill & Young 1980), according to the equation [3H]mepyramine bound = capacity of receptor sites x M / ( M K ) + b x M , where M is the concentration of radioactive mepyramine, K is its dissociation constant and b is the slope of the linear component. Drugs. The following drugs were used : histamine dihydrochloride (Sigma), mepyramine maleate (May & Baker), chlorpheniramine (SK&F), cimetidine (SK&F), nimodipine (Bayer) and promethazine (Sigma). The drugs were dissolved in 0.9% NaCl solution. All concentrations are expressed as the final molar concentration in the tissue bath. Nimodipine was kept in a dark environment in order to exclude the possibility of light-induced decomposition.

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RESULTS Vasomotor responses Application of the 124 mM potassium-containing Krebs buffer invariably resulted in a strong contraction amounting to 5.6 kO.6 mN, which rapidly disappeared upon rinsing of the tissue bath. Cumulative application of histamine invariably resulted in a concentration-dependent contraction of the vessel segments. T h e maxim u m contraction was 7.4f0.7 m N , and the mean histamine concentration eliciting halfmaximum contraction (EC,,) was 1.6k0.3x M. T h i s response was not altered by removing the endothelium ( n = 5 ) . Neither of the antagonists in the concentrations used had any direct effect on the vessel segments per se. Mepyramine and chlorpheniramine in increasing concentrations shifted the concentration-contraction relationship of histamine towards higher concentrations (Fig. 1). The shifts obtained at the higher concentrations of

H,-receptors in guinea-pig brain vessels

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1

Histomine

COW

IMI

Hlsiomine conc

IMI

Fig. 1. Contractile responses of the guinea-pig basilar artery to increasing concentrations of histamine (a) in the absence (0) and in the presence of mepyramine 1 x lo-' to lo-' M (a)and (b) in the absence (0) and presence of chlorpheniramine 1 x lo-@to lo-' M (a).Mean values are shown (n = lo), vertical lines indicate SEM.

mepyramine and chlorpheniramine are not parallel, but instead the slope of the histamine concentration-response curve is markedly reduced. Analysis of the parallel shift obtained with lo-' M mepyramine suggests that the KDis ca 0.4 nM, and with lo-' M chlorpheniramine ca 4.6 nM. Cimetidine was without effect in a concentration of M (n = 10) (Fig. 2), and even a cimetidine concentration of M (n = 4) did not shift the histamine concentrationresponse curve. Histamine administered to vessels precontracted by 60 mM potassium-containing buffer in the presence or absence of histamine antagonists did not induce any dilatatory responses, but instead additional contractions to high concentrations of the amine were obtained. Exposure of the basilar artery segments to a calcium-free buffer solution for 30 min reduced the contractile effect of histamine ( M) to 3.7 f 0.8 yo. The vessel segments contracted properly upon cumulative readministration of calcium, and the contraction was concentrationrelated, with the maximum effect at 6 mM calcium (Fig. 3). This contraction was seen within seconds after readministration of calcium. I n the absence of extracellular calcium, nimodipine (lo-* M) caused a further decrease in

Histamine

COW

(MI

Fig. 2. Contractile responses of the guinea-pig basilar artery to histamine in the absence (0) and in the presence of cimetidine 1 x lo-' M (a).Mean values are shown (n = lo), vertical lines indicate SEM.

the response induced by histamine to 1.9 & 0.5 yo (P< 0.01; Student's t-test). This calcium antagonist markedly inhibited the contraction produced by reintroducing calcium into rhe

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0 003

00.01

01

03

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0

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8

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3H-rnepyramine I n M I

Colclum concentration (mM1

Fig. 3. [:antractions of guinea-pig basilar artery to increasing concentrations of calcium after exposure to a buffet solution containing zero calcium. Data are given as percentage of maximum. Histamine 1 x 10 ' \i was added in the absence of drug (0) and in the presence of nimodipine 1 x 10.' .M ( 0 ) Mean . \dues are shown ( n = 6), vertical lines indicate SEM.

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10-11 I O - ~ 10.9

T

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8

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3 H - mepyromine (nM1

10-8

lo-'

10-6

NlMOOlPlNE CONC. (M)

Fig. 4. Contractions of guinea-pig basilar artery by 1 x 10 ' .M histamine in the presence of nimodipine in concentrations from 1 x lo-'' to 10 M. Data are given as a percentage of contractions to 1 x 10-' M histamine in the absence of nimodipine. Mean \-alues and SE41 ate s h o w ( n = 6).

extracellular medium (Fig. 3 ) . Furthermore, nimodipine in concentrations from 1 x lo-" to M caused a concentration-related reduction M hisof the contractions induced by 1 x tamine (Fig. 4).

Ligand-binding results The promethazine-sensitive binding of [ 3H]mepyramine to guinea-pig pial and choroid

Fig. 5. Promethazine-sensitive binding of [JH]mep~ramineto guinea-pig pial (a) and choroid plexus (b) membranes. Values represent the differences (mean SEM) between the level of binding determined in the presence and absence of 2 ~ L promethazine. Triplicate determinations were made for each condition. Data were obtained on membranes from 54 guinea-pigs. T h e curve drawn is the best-fit hyperbola obtained by the method of Wilkinson (1961). T h e curve drawn in (b) was fitted assuming that the binding is the sum of a saturable component (ii) and a component (i) increasing linearly with concentration.

membranes is shown in Fig. 5 . The curve for specific [3H]mepyramine binding to pial membranes appeared to saturate over the concentration range studied (Fig. 5a). Analysis of this curve according to Wilkinson (1961) yielded values for the dissociation constant ( K , ) and HIreceptor capacity of 5.5 1.2 nM and 2.27 & 2.8 pmol g-' protein respectively. The non-specific binding in these experiments was high and accounted for 61 yo of the total binding

*

M

HI-receptors in guinea-pig brain vessels

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Table 1 [3H]Mepyramine

Specific binding (pmol g-' protein)

(%I

Non-specific binding

9f 1 12k 1

63 76

12f2

70

Pial membranes 1 nM

2 nM

Choroid membranes 2 nM

Values represent the difference (mean & SEM) between the level of binding determined in the presence and absence of 2 ,UM promethazine. Triplicate determinations were made for each condition. Data were obtained on membranes prepared from the pial or choroid vessels from 13 guinea-pigs. In these experiments binding was measured in 50 mM Na-K phosphate buffer, pH 7.5.

of [3H]mepyramine to pial membranes at the lowest concentration measured (i.e. 1 nM). Similar data were obtained when binding was measured in 50 mM Na-K phosphate buffer (Table 1). The promethazine-sensitive binding of [3H]mepyramine to membranes prepared from guinea-pig choroid plexus did not saturate over the concentration range employed in this study (Fig. 5b). However, the data obtained at concentrations of [3H]mepyramine between 1 and 4 nM suggested that a saturable component was present (Fig. 5b). Consequently, the data obtained in choroid plexus were fitted assuming that in addition to a saturable high-affinity component there was a second low-affinity component that was linear over the concentration range studied. A similar linear component has been observed previously in rat brain membranes (Hill & Young 1980). This analysis produced values of 7.5 pmol g-l protein and 1.7 nM for the binding site capacity and K, of the saturable component respectively. DISCUSSION The administration of histamine to guinea-pig basilar arteries resulted in contraction of the vessels. This has also been found in cerebral blood vessels of the rabbit (Hamel et al. 1985), whereas histamine caused dilatation in the cat (Edvinsson & Owman 1975, Wahl & Kuschinsky 1979, Gross et al. 1981b, Edvinsson et al. 1983), monkey (Duckworth et al. 1976), rat (Gross et al. 1981a, Dacey & Bassett 1987) and man (Ottosson et al. 1988). Dilator responses seem to be mediated mainly via histamine H,-receptors (see Gross 1984), but in man both H,- and H,-

receptors dilate cranial arteries (Ottosson et al. 1988, 1989). I n the guinea-pig only vasoconstriction was seen, and this response seemed to be mediated via HI-receptors. A modulatory influence of the endothelium was excluded by the experiments with denuded vessels. HIreceptor blockade with both mepyramine and chlorpheniramine caused a displacement to the right of the histamine concentration-response curve, whereas H,-receptor blockade with cimetidine was without effect. The displacement of the concentration-response curve was only parallel for low concentrations of mepyramine and clorpheniramine. For mepyramine PA, values of 8.0-9.3 corresponding to KD values of 0.5-10 nM have been reported in different test preparations (see Ottosson et al. 1988), and for chlorpheniramine a K,, value of 0.8 nM has been reported from contraction studies of guinea-pig ileum (Hill & Young 1981). The apparent K,, values found in the present study (0.4 nM for mepyramine and 4.6 nM for chlorpheniramine) are in relative accordance with those found in histamine receptor subtype characterizations carried out previously. It is possible that the contractile effect of histamine, in addition to a specific HI-receptor response, has a non-specific component, since it was not blocked in a competitive manner by higher concentrations of mepyramine and chlorpheniramine. A non-specific contractile effect in response to high concentrations of histamine has also been found in intracranial arteries of the cat, but here dilatory H,-receptors were present in the lower concentration range (Edvinsson & Owman 1975). Alternatively, the

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apparent non-competitive antagonism produced by HI-receptor antagonists at higher concentrations map be a consequence of the low H,-receptor concentration, and hence spare receptor reserve, in this tissue. In guinea-pig basilar artery the density of HI-receptors is low (27 pmol g-' protein) and the EC,, value for histamine-induced contraction (1.6 p ~ is )close to the H,-receptor dissociation constant of 5 ,UM calculated for histamine in guinea-pig ileum (Donaldson & Hill 1987), indicating the need for a high agonist occupancy to produce a maximal contractile response. T h e reversal of HI-receptor antagonism by mepyramine in intact smooth muscle strips is often very slow (Roberts & Stephenson 1976), and Rang (1966) has noted that, with persistent antagonists such as mepyramine, parallel concentration-response curves may only be expected if the agonist occupancy is very low. In the guinea-pig basilar artery there was no indication of the presence of vasodilator histamine receptors, since only some additional contraction was seen upon administration of the amine to precontracted vessels both in the presence and in the absence of histamine antagonists. The presence of H,-receptors in cerebral blood vessels was confirmed with ligand-binding studies with [3H]mepyramine. T h e H,-receptor numbers in pial and choroid membranes were much lower than those obtained in guinea-pig cerebellum and cerebral cortex (Hill et al. 1978) and were similar to the low levels detected in brain stem and spinal cord (Hill et a/. 1978). These data contrast with the relatively higher levels of HI-receptors detected in cerebral microvessels prepared from bovine brain (Peroutka et al. 1980), which were similar to the value determined in bovine cerebral cortical membranes. It was notable that analysis of the specific binding of [3H]mepyramine to choroid membranes indicated the presence of a secondary low-affinity binding site for [3H]mepyramine that was linear over the concentration range 0-16 nM. A similar linear component has been observed previously in rat brain membranes (Hill & Young 1980). The K , value (1.7 nM) calculated for the saturable component of binding agreed well with the value determined from H,receptor-mediated contractile studies in intact basilar artery. T h e value determined in pial vessel membranes was somewhat higher ( K ,

5.5 nM), but it is possible that a linear component is also present in the pial data and that the actual K , value for the H,-receptor component is therefore lower. T h e contractile HI-recep.ior in guinea-pig basilar artery was extremely sensitive to removal of extracellular calcium. This is in concert with other contractile mechanisms studied in brain vessels (Andemon et al. 1983). Only little intracellular or sarcolemma-associated calcium is involved, as indicated by the experiments with nimodipine, which blocked the H,-receptorinduced contractile effect with increasing concentrations (Fig. 4) and inhibited the H,receptor-mediated contraction upon reintroducing calcium in experiments with calcium-free buffer solution (Fig. 3). This indicates that dihydropyridine-sensitive calcium channels mediate the action of HI-receptors. This study was supported by the Swedish Medical Research Council (grant no. 05958) and the Medical Faculty, University of Lund.

REFERENCES ANDERSON, K.-E., EDVINSSON, L., MACKENZIE, E.T., SKARBY, T. & YOUNG,A.R. 1983. Influence of extracellular calcium and calcium antagonists on contractions induced by potassium and prostaglandin F, in isolated cerebral and mesenteric arteries of the cat. Br 3 Pharmacol 79, 135-140. DACEY, R.G. & BASSETT, J.E. 1987. Histaminergic vasodilatation of intracerebral arterioles in the rat. 3 Cereb Blood Flow Metabol 7, 327-331. J. & HILL,S.J. 1987. 1,4-DithiothreitolDOKALDSON, induced changes in histamine HI-agonist efficacy and affinity in the longitudinal smooth muscle of guinea-pig ileum. BY 3 Pharmacol 90, 263-271. DUCKWORTH, J.W., LANCE,J.W., LORD,G.D.A. & MYLECHARANEM, E.J. 1976. Histamine receptors in the cranial circulation of the monkey. Br 3 Pharmacol 58, 444P. EDVINSSON, L. & OWMAN, CH. 1975. A pharmacologic comparison of histamine receptors in isolated extracranial and intracranial arteries in vitro. Neurology 25, 271-276. EDYINSSON, L., GROSS,P.M. & MOHAMED, A. 1983. Characterization of histamine receptors in cat cerebral. arteries in vitro and in situ. 3 Pharmacol E s p They 225, 168-175. FLRCHGOTT, R.F. 1983. Role of endothelium in responses of vascular smooth muscle. Czrc Res 53, 5 57-573. GANELIJN, C.R. 1982.Chemistry and structure-activity

HI-receptors in guinea-pig bruin vessels relationships of drugs acting on histamine receptors. In: C.R. Ganellin & M.E. Parsons (eds.) Pharmacology of Histamine Receptors, pp. 10-102. John Wright & Sons, Bristol. Gr:oss, P.M., HARPER,A.M. & TEASDALE, G.M. 1981a. Cerebral circulation and histamine: 1. Participation of vascular HI- and H,-receptors in vasodilatory responses to carotid arterial infusion. 3. Cereb. Blood Flow Metabol. 1, 97-108. A.M. & TEASDALE, G.M. GROSS,P.M., HARPER, 1981b. Cerebral circulation and histamine: 2. Responses of pial veins and arterioles to receptor agonists. 3 Cereb Blood Flow Metabol 1, 219-225. GROSS,P.M. 1984. Histaminergic dilatation of resistance vessels in the brain. Biblthca Cardiol 38, 138-147. HAMEL, E., EDVINSSON, L. & MACKENZIE, E.T. 1985. Radioactivity of various cerebral arteries to vasoactive substances in different mammalian species. 3 Cereb Blood Flow Metabol 5 , (Suppl. l), 5 53-5 54. HILL, S.J. & YOUNG,J.M. 1980. Histamine HIreceptors in the brain of the guinea-pig and the rat: Differences in ligand binding properties and regional distribution. Br 3 Pharmacol 68, 687-696. HILL,S.J. & YOUNG,J.M. 1981. Characterization of 'H-mepyramine binding to the longitudinal muscle of guinea-pig small intestine. Molec. Pharmacol. 19, 379-387. HILL, S.J., EMSON,P.C. & YOUNG,J.M. 1978. The binding of 3H-mepyramine to histamine HIreceptors in guinea pig brain. 3 Neurochem 31, 997-1 004.

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HOGESTATT, E.D., ANDERSON,K.-E. & EDVINSSON, L. 1983. Mechanical properties of rat cerebra1 arteries as studied by a sensitive device for recording of mechanical activity in isolated small blood vessels. Acta Physiol Scand 117, 49-61. LOWRY,O.H., ROSEBROUGH, N.J., FARR,A.L. & RANDALL, R.J. 1951. Protein measurements with the folin phenol reagent. 3 Biol Chem 193, 165275. A., JANSEN, I. & EDVINSSON, L. 1988. OTTOSSON, Characterization of histamine receptors in isolated human cerebral arteries. Br 3 Pharmacol 94, 901-907. OTTOSSON,A., JANSEN, 1. & EDVINSSON, L. 1989. Pharmacological characterization of histamine receptors in the human temporal artery. Br 3 CIin Pharmacol 27, 139-145. S.J., MOSKOWITZ, M.A. & SNYDER, S.H. PEROUTHA, 1980. Neutrotransmitter receptor binding in bovine cerebral microvessels. Science 208, 61k612. RANG, H.P. 1966. The kinetics of action of acetylcholine antagonists in smooth muscle. Proc Roy Soc Lond B 164, 488-510. F. & STEPHENSON, R.P. 1976. The kinetics ROBERTS, of competitive antagonists on guinea-pig ileum. Br 3 Pharmacol 58, 57-70. W. 1979. The diladng WAHL,M. & KUSCHINSKY, effect of histamine on pial arteries of cats and its mediation by H, receptors. Circ Res 44, 161-165. WILKINSSON, G.N. 1961. Statistical estimations in enzyme kinetics. Biochem 3 80, 324-332.

Histamine receptors in brain vessels of guinea-pig: in-vitro pharmacology and ligand binding.

The subtype of histamine receptors in brain vessels of guinea-pig has been characterized by ligand binding and in-vitro pharmacology using selective a...
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