Atypical Schild plots with histamine H1 receptor agonists and antagonists in the rabbit aorta

European Journal of Pharmacology. 197 (1991) 49-56 0 1991 Elsevier Science Publishers B.V. 0014-2999/91/$03.50 ADONIS 0014799991003327

49

ELIP51839

ical Schild plots wi and antagonists in the r

iS

T. Arda Biikesoy and H. Ongun Onaran Department of Pharmacology, Ankara Uniuersity Faculty of Medicine, Ankara, Turkey Received 2 April 1990. revised MS received 29 January 1991,accepted5 February1991

Competitive antagonists of histamine H, receptor were investigated for their effects on histamine-induced responses in the rabbit aorta. Antazoline-induced antagonism gave linear Schild plots with slope equal 1, while the other antagonists (( + )brompheniramine, ( f )-chlorpheniramine, diphenhydramine and mepyramine) produced ‘atypical’ plots with slopes generally less than unity in the thoracic aorta. Schild plots obtained with these antagonists were evaluated using a two independent component model. The high affinity parameters thus estimated were compatible with those that have been reported for these antagonists. No such heterogeneity was observed in the abdominal aorta when diphenhydramine was investigated with different H, agonists. The results suggest the presence of at least two components in H,-mediateci responses in the thoracic aorta; these components are equally antagonized by antazoline, but differentially antagonized by the other antagonists used. Histamine H, receptors; Histamine H, receptor antagorkrs; Shild plot: Aorta (rabbit); Histaminergic responses

J. Introduction Histamine-mediated vascular responses have been extensively studied in various species and preparations. Both H, and H2 receptors mediate these effects; stimulation of the former subtype generally results in tonus increases (excluding endothelium mediated effects) while Hz-receptor stimulation gives rise to relaxant responses. Heterogeneity of these responses along rabbit aorta has been reported in previous studies (Altura and Altura, 1970; Bijkesoy et al., 1983; Onaran and BBkesoy, 1988). Pharmacokinetic’ or pharmacodynamic phenomena may be expected to account for the observed heterogeneity and thus the variation may be brought about (i) by local tissue variances, leading to differences in the access of drugs to the biophase, and to changes in responsiveness, (ii) by deviation of density or subtypes of receptors in exploration, or (iii) by post-receptor processing affecting agonist responses (including tissue dependent efficacy variations (Kenakin, 1989)). Receptor studies with competitive antagonists (Anmlakshana and Mild, 1959) have been a practical and reliable tool. Lemoine and Kaumann (1983) further

Correspondence to: T.A. Biikesoy. Department of Pharmacology, Ankara University, Faculty of Medicine, Siiye, Ankara 06339, Turkey. * This work constitutes a part of the Ph.D. thesis of H.O. Onaran.

extended this methodology for cases where the Schild plot is ‘atypical’ in that the responses are composite (two component model). The aim of the present investigation was to test whether the action of competitive antagonists is also heterogeneous in the rabbit aorta, and thereby provide information abotit vascular H, receptors. For this purpose, drugs with agonistic activity on H, receptor were used and their interaction with various competitive H, receptor antagonists was studied in two regions of the aorta where the above mentioned studies have suggested heterogeneous histaminergic responses. The antagonism data thus obtained were evaluated by either one or two component models.

2. Materials and methods 2.1. Vascular preparations and experimental protocol Approximately three month old New Zealand albino rabbits of either sex were exsanguinated under sodium pentobarbital anesthesia. Thoracic aortas from circa 3 cm above the diaphragm and terminal abdominal aortas from below the renal arteries were obtained and washed free of blood with Krebs-Henseleit solution. The endothelium was removed with a cotton thread (the functional integrity of endothelium was occasionally tested with acetylcholine). The vessels were cut into rings of 2 mm width. These were then opened by cutting the

vessels I~~~itudinally. fixed with stainless steel clips at ends and then placed in organ baths containing S&it solution of the following composition z;XhS138.2. K+ 5. Ca” 2.5. Mg” 0.5. Cl- 123. pm?crO ): CO; 25. H,PQ; 1.2. dextrose 11.5. gassed with carbogen (95% O,-5Fr CO,) to give a pH of about 7.4 at 37°C. The preparations were connected to isometric force-displacement transducers (Grass FT.03 and FT.10) monofilament nylon threads and equilibrated via min under 15 mN initial tension through for continuous overflow of the buffer. Amplified transducer signals ( Digitco-LA 10000 Linear Amplifiers, Ankara) were digitized with a resolution of 0.5 Hz and 12 bit and were recorded with an Epson PC + /HD computer using general purpose in vitro laboratory software (Onaran and Biikesoy. unpublished). Concentration-response data for various antagonist concentrations were obtained by using cumulatively increasing concentrations of agonists (from lo-’ up to 1O-4 M) with incremerits of {log,, M. Each preparation was used for a single experiment. Different concentrations (S-9) of antagonists were applied in ilog,, M steps to obtain equipotent ratios. H, antagonists were used exclusively in the present experiments. Ranitidine (3 x low5 M) was used in the thoracic aorta while famotidine (10e4), with a relatively higher PA?. was used in the abdominal aorta. as the Hz-mediated responses were found to be more potent in the latter segment (Onaran and BGkesoy, 19S8). H, and H, antagonists were applied 20 min prior to agonist application; this is sufficient time for antagonist equilibration regarding the rate constants of the antagonists used (Onaran and Biikesoy, 1990). 2.2. El~aiuation of comentration

response curves and de-

scription of the model

Agonist responses were evaluated at equilibrium. Plateau values (indicating equilibrium responses) were automatically measured by means of the software cited above. Concentration-response data with or without antagonist were fitted using a three parameter logistic equation by means of a modified simplex algorithm (Nelder and Mead, 1965) and Rz maximization (Onaran et al., 1988) where response =

f([A[) = m[A[“/([A[“+K”)

eq. 1

[A], m, n, K. represent respectively, agonis: concentration, maximum response, slope factor and concentration producing half-maximal response (EC,,). Equipotent concentrations were determined from estimated equations using the following procedure. (1) All equations were constrained for a fixed value of m assuming competitive antagonism (after verified statistically). (2) A fixed response level. say R, was chosen (in our cases R = m/2). (3) Inverses of the estimated equatious were

solved for agonist concentrations level, that is

at the chosen response

[AIF= f- ‘(R:k:,.R,.m) [A[, = f-‘(R:k,,h,.m)

eqs. 2

i: 1.2 ,.... N

where indices c and i signify control and the presence of various antagonist concentrations, respectively. (4) Concentration ratios were then calculated according to CR, = [‘&/[A[,

eq. 3

These ratios were used for either linear (Arunlakshana and Schild, 1949) or non-linear regression analyses. In order to analyse the non-linear Schild plot and estimate the apparent equilibrium dissociation constants for antagonists (K ,_, KH) and fractional stimuli generated by agonist ( uH, uL), the antilog form of eq. 6 of Lemoine and Kaumann (Lemoine and Kaumann, 1983) was used eq. 4

(CR- 1) = (lB12+ WWc@,IB[+ 3, where CR = equipotent concentration antagonist concentration and 8, = uHKL+ oLKH 19~ = oHK, + a,_K,_ 8, = K,K,

ratio,

[B] = eq. 5a eq. Sb t2q.k

with (I,_+ 0” = 1 (eq. 5d) by definition (see Lemoine and Kaumann, 1983 for theory and details). The weighted least squares estimate of 8 was used to solve the above four simultaneous equations with four unknowns. The weighting scheme was constructed estimated variances (uz) at each design point for which 5-7 replicates are present. Estimated weights are: &,=1/$/i:

1.2 ,..., N

eq.6

(for a critical analysis, see Kmenta, 1971). Graphical representation of results are given in the original logform of the Lemoine and Kaumann equation, which implies two linear asymptotes with equations: Limy=log[B]-logK,K,(aHK,_+o,_K,)

eq. 6a

PI -0 Limy=log[B]-log(oHKH+a,K,)

eq. 6b

[Bl-0~ The likelihood ratio (LR) test suggested by Ehle et al. (1985) was used to compare fittings obtained by linear and non-linear regression analyses. The test statistics (having an asymptotic x2 distribution with degree of freedom equal to number of withdrawn parameters) is given as In LR = 2n (In SSQL - In SSQN ) where In, n, SSQ, and SSQ, are natural logarithm, number of observations, residual sum of squared deviations about linear and non linear regressions respectively. The test statistics was also used to calculate the approximate 95% confidence limits of non linear estimates: maximum and minimum tolerable limits around the fitted values of individual parameters were iteratively searched

51

until the test statistics exceed the table value at 1 degree of freedom. The presence of spare H, receptor on rabbit thoracic aorta was tested with phenoxybenzamine (Furchgott, 1966). 2.3. Drugs used The following drugs were used: Histamine diHC1 (Koch and Light), 2-methylhistamine diHC1, N,methylhistamine diHC1, N”,N”-dimethylhistamine diHCl, 2-thiazolylethylamine diHC1 (SK&F, Hertfordshire), diphenhydramine HCl (Parke and Davis), antazoline HCl (Ciba), mepyramine maleate (Specia), ranitidine HCl (Fako, Istanbul), (+)-brompheniramine maleate (Eczacibasi, Istanbul) and ( + )-chlorpheniramine maleate (OIF, Ankara). Famotidine (base) was dissolved in equimolar HCI.

3. Results 3.1. Thoracic aorta We tested whether there were spare H, receptors to avoid theoretical contradictions, as the estimated equation (eq. 4) assumes the presence of spare receptors. A hypothetical occupancy-response relationship is given in fig. 1 for thoracic aorta. Schild plots (log (concentration ratio - 1) versus log (antagonist concentration)) for the antagonists are shown in fig. 2. Data for agonist concentration-response curves are given in table 1 for control and for maximal concentrations of the antagonists. The results of the ANOVA with maximal responses and slope factors show that there was no systematic trend in these param-

TABLE 1

TABLE 2 Slope and pA r values estimated by linear regression analysis of Schild plots obtained in thoracic aorta. Slope values are given with their 95% confidence limits Antagonist

Slope

Antazoline (+ j-Brompheniramine ( f )-Chforpheniramine Diphenhydramine Mepyramine

0.9+0.10 0.8 f 0.09 0.7 kO.06 0.9 f 0.09 0.7 + 0.06

pA, iS.E.M. = = = =

6.7 kO.03 8.2 + 0.06 8.3kO.06 7.4 * 0.07 9.0 f 0.09

a Indicates significant differences from unity). N = 6-8.

eters depending on the antagonist concentration (P > 0.05). The results demonstrated that, while antazoline gave a linear plot with a slope not different from unity, mepyramine, diphenhydramine, ( + )-chlorpheniramine and (+)-brompheniramine gave atypical plots where the two component model gave significantly better fits (see table 3). The results of linear regression analyses are given in table 2. The high affinity equilibrium dissociation constants for the antagonists that were estimated by fitting the independent two site model, are in good agreement with those given in table 3. Thus antazoline seems to be ‘non selective’ for the secondary component, that is, both activities were inhibited by antazoline with equal values. Estimated dissociation constants in nM units and fractional stimuli for ‘selective’ antagonists are given in table 4. The affinity ratios of the high and low affinity components for the selective antagonists were 50, 25, 23, 20 for mepyramine, diphenhydramine, ( + )-brompheniramine and ( & )-chlorpheniramine respectively. As histamine was used as agonist in all these experiments, the agonist-related parameters of the fitted model (fractional stimuli generated by agonist at the evaluated response level) were expected not to change with respect to the antagonist applied. The results were consistent with this expectation; calculated fractional stimuli were approximately 0.1 and 0.9 in every case of the selective

Parameters of concentration-response curves of histamine obtained in the absence and in the presence of maximal concentrations of the antagonists indicated in thoracic aorta (mean* S.E.M., N = 6-S). Maximal responses are given in mN units Antagonist

Log[C]

Maximal Slope response factor

- Log ECsc

Antazoline

None -5.50

19k1.4 19kl.3

1.3f0.05 1.6kO.15

5.6kO.08 4.6kO.07

(+ j-Brompheniramine None -7.00

20f1.6 19k2.0

l.ZiO.05 1.7k0.24

5.4k0.10 4.3ltO.10

)-Chlorpheniramine None -6.50

19k1.8 18f1.7

lSiO.09 1.5i0.10

5.6kO.09 4.5kO.11

(f

Diphenhydramine

None -6.25

17kl.O 17k1.5

lSiO.06 1.6kO.05

5.7kO.07 4.5k0.05

Mepyramine

None -7.25

16kl.O 19k2.2

1.4f0.04 1.2f0.10

5.6fO.07 4.4kO.02

Y,.>J”i Occupancy

il

1

Fig. 1. Fractional occupancy-response curve for histamine in thoracic aorta calculated by using the estimate apparent dissociation constant ( = 30 PM) and concentration response parameters of control data. The relationship implies that 95%of the response is achieved when ca. 40% of receptors are occupied.

3

1

DIPHENHYDRAHINE

l’e 3

1e

8

9

7

;

’

6

1

ANTAZDLJNE

S

6

...’.. .... ...*

3- <+I-CHLDRPHENIRAHINE 2-

10

9

0

7

6

5

Fig. 2. Logarithm of CR-

(Schild

format)

for

1 (e&potent concentration ratio - 1) (ordinate) versus negative logarithm of molar antagonist concentration (abscissa) the antagonists indicated. Vertical bars represent S.E.M. (n = 6-8). Solid curves are weighted regression lines of eq. 4. and estimated asymptotes are presented as dotted lines.

TABLE 3 pA2 [or -log KH] values. estimated by non linear regression analysis of Schild plots in thoracic aorta, are compared with those reported in the literature. Results of the likelihood ratio (LR) test for linear vs. non-linear regression lines are also included. Inhibition of guinea-pig ileum contraction a

Inhibition of [ 3Hlmepyramine binding a

Estimated high affinity -logK”

Antazoline

-

-

-

( + )_Brompheniramine ( f )-Chlorpheniramine Diphenhydramine Mepyramine

8.9 8.1 9.2-9.4

8.3 8.3-8.8 7.6 8.9-9.1

8.7 8.2 7.8 9.2

’ From Johnson, 1982.

(df = 2)

P of larger values

2.12 7.01 11.70 7.66 8.75

> 0.05 -G0.05 < 0.01 < 0.05 < 0.05

LR X2

53 TABLE 4 Parameters estimated by non-linear regression analysis of the Schild plots obtained in the thoracic aorta. KL, K,, (I~,, a,, are estimated dissociation constants (in nM) of the high and low affinity component for antagonists, and fractional stimuli (djmeosio~ess) generated by histamine through these components, respectively. Values are given with their 95% confidence limits

( + )-Brompheniramine ( f )-Chlorpheniramine

Djphenhydra~ne Mepyramine

K, @MI

K, (nM)

uH (fractional)

2 6 15 0.5

43 (-t-205) 115 (+6031) 370 ( + 220 - 110) 30 (+189)

0.88 0.83 0.88 0.87

(+O.l -0.5) (+0.9 -0.7) (+2.0 -1.0) ( + 0.09 - 0.04)

( + 0.06 ( + 0.07 ( + 0.07 ( + 0.08 -

q (fractional} 0.09) 0.07) 0.09) 0.09)

0.12 (+0.02-0.03) 0.17 f +0_02-0_02) O.IZ ( io.02 -0.02) 0.13 ( + 0.04 - 0.02)

1

S DIPHENHYDAAtlINE N*-r(ETHYLHfSTAHfNE

3 DIPHENHYDRAHIN6 HISTAflJNE 1

2. l*_ ..*.....

.

“““......“-“,...-..

. . . .

.

..“l”-^~“.-~l”“”

-1. -2-r 9

e

7

6

ie

5

a

I 7

6

I 5

8

7

6

5

I 9

S DJPHENHYDRAMftfE 1 2-~EMYLHISTAtlINE

S-DfPHENHYDRAflINE N1,N.-DJ~ETHYLHISIA~INE 2-

e_ ....I.~.~.........._............~. _...........

......l.._,.*.*...*....*l.” ..... .....

-1-

ie

1

9

S ANTAZDLINE HISTAHINE 1

3 DIPHENHYDRAfffNfi THIAZDLYLfTHYLAfffNE

2l-

............*.................“......

a- ......................I.m..*..............

e_

..... . ... . .. .. . .._ _........,,...

L .,....

. . .

.”...._,.... *.............

. . . . . . . . . . . . . . . . . . .

-I-2-’

I

ie

9

I

1 8

7

6

5

te

9

e

7

6

s

Fig. 3. Schild plots of antagonism induced by diphenhydra~ne and antazoline, obtained using the agonists indicated. Log (CR - 1) on the ordinate and log [Antagonist] on the abscissa.

Histmkr 2-Thiazolylethylsmine 2-hfethvlhistsmine N”-~tethvlhistamine~~*~ * : N” .~‘~-Dintrth~lhistanlirrt: .

Slope

pA:

1.06*0.04 0.77 + 0.04 a 0.84 f 0.03 .l 0.96+ 0.03 0.98 f 0.03

7.67*0.04 7.45 4 0.05 h 7.48 f 0.05 h 7.53 &0.04 7.64 IO.04

“Siope-1; PsO.OS. ’ Sigdicsntly phenhydrzmine.

-

different from histamine-di-

antagonist (see estimated crt_ and (JH in table 4). The parameters estimated foi antagonism of secondary activity fK,) were less accurate since it was not possible to obtain sufficient information about this region which covers the high concentrations of antagonists. At high concentrations of antagonists very high concentrations of histamine were required to obtain a concentration-response curve. because of the low potency of hista~ne in the thoracic aorta. 2.2. Abdominal aorta The agonist dependence of the antagonism was investigated in abdo~nal aorta by using HI agonists {histamine, 2-methylhistamine, N”-methylhistamine, N”.N”-dimethylhistamine, 2-thiazolylethylamine) against diphenhydramine (which was used because it gives a more reproducible antagonism so that additional variation in agonist responses is avoided). Results are summarized in table 5. No qualitative or quantitative difference between thoracic and abdominal aorta was observed in the antazoline-induced antagonism of histamine responses. The slopes of the linear regression lines for diphe~y~a~ne-induced antagonism were not statistically different from 1 for NQ-methyl~sta~ne, Nff.Na-~methy~sta~ne and histamine. Schild plots, constructed using diphenhydramine and the above-mentioned agonists are given in fig. 3, and the estimated TABLE 6 Concentration-response curve parameters of the agonists indicated in the abdominal aorta (meaokS.E.M., N = 7). Maximal responses are given in mN.

Histamine N*-MethyIhistamine N”.N”-Dimethylhistatniine 2-Methylhistamine 2-Thiazolylethylamine

Maximal response

Slope factor

- Log EC,,

17.2rt1.53

1.4*0.07

6.6kO.06

16.9f1.58 18.8 5 1.61 19.5f1.86 16.811.62

1.6iO.13 1.9 f 0.11 1.7iO.13 1.5*0.10

6.1*0.07 6.0 f 0.03 5,8j$.O6 5.5 +0.07

sfopes and pA, in table 5. A slight dependency of pA, on agonists was apparent for 2-thiazolylethyla~ne and 2methylhistamine. although eq. 1 was not consistent with these data. pA, values obtained with N”-methylhistamine, N”,N”-dimethylhistamine and histamine were still consistent with - log K,., values calculated for the thoracic aorta. These results further suggest the absence or relatively small constribution of the putative secondary component in the abdominal aorta. Concentration-response parameters for the control curves obtained with the above mentioned agonists are given in table 6. in their order of potency. The maximal responses of the concentration-response curves for the agonists were not found to be affected by antagonist concentrations in the abdominal aorta.

4. Discussion Situations in which the classical Schild plot departs from linearity, thereby allowing a slope equal to or different from unity, have been discussed and in general can be considered from two different but interdependent standpoints based on pharmacokinetic and/or pharmacodynamic phenomena. The former standpoint involves speculations about the concentration profile of drugs in the biophase, that is, diffusion of applied drugs to receptor compartments and their removal (by uptake, degradation etc.). The effects of a saturable antagonistextrusion process on the Schild transformation have been modeled and demonstrated by Kenakin and Beek (1987) for atropine on the choline@ system using rabbit and guinea pig ileum. As this model predicts a non-linear Schild plot with overall slope greater than 1 and an inflexion in the opposite direction from that observed in our experiments with H, antagonists, the possibility of antagonist removal in the thoracic aorta can be ruled out. The possibility of a disequilibrium in antagonist binding (implying a slope > 1) can also be rejected on the basis of the above considerations (Kenakin, 1982). However, a saturable agonist-uptake process may result in a non-linear Schild plot with a slope less than 1 (Furchgott, 1972). Histamine receptor bearing neurons in perivascular tissues have been rei ported (Ishikawa and Sperelakis, 1987). However, such a neuronal or extraneuronal uptake of histamine in rabbit thoracic aorta preparations has not been demonstrated; moreover, our observations with antazoline (yielding a linear Schild plot with slope = l), which were similar to those obtained with histamine in the thoracic aorta, reduce but do not exclude the possibility of saturable agonist uptake. Blocker toxicity may be another important reason for deviations from the classical Schild plot, but this would, according to Lemoine and Kaumann (1982). result in a slope 7 1.

55

Multi-receptorial interactions of an agonist may result in an atypical Schild plot (Schild, 1973; Lemoine and Kaumann, 1983). Both H, and Hz receptor-mediated responses occur in the rabbit thoracic aorta and the relative contribution of the H, receptor-mediated (tonus decreasing) responses to the total response may also lead to the observed non linearity. Ranitidine (3 x 10m5 M) was used in the present experimental procedures in the thoracic aorta to antagonise H, receptor-mediated activity. In these conditions, probability of there being H, receptor-mediated responses is negligible (less than 2%). H, receptor-mediated relaxant responses in the rabbit thoracic aorta, which are mediated through vascular endothelium (Van de Voorde and Leusen, 1984), and the presence of Hi receptors on the vascular endothelium of guinea pig (Hide et al., 1988) has been reported. The endothelium was therefore removed in order to avoid possible interferences. Our results, consequently, would be expected to reflect the circular smooth muscle activity of rabbit thoracic aorta. The composite chemical structure of antagonists may also be reflected in non-linear Schild plots. That is, a mixture of antagonist enantiomers with diverse binding properties in aqueous solutions may give results indistinguishable from those obtained in the presence of two receptor subtypes with different binding parameters. In order to avoid this ambiguity, selected antagonists were used. Antazoline, diphenhydramine and mepyramine do not possess ‘chiral’ carbon atoms in their chemical structures and thus do not have stereoisomers; the ( + )-stereoisomer of the chiral antagonist brompheniramine was used in order to avoid this type of ambiguity. Experiments with histamine and ( &)-chlorpheniramine (although a racer& mixture) yielded fractional stimuli comparable to those obtained with ( + )-bromheniramine and other selective antagonists. In addition, the ratio KJKn was estimated to be approximately 20 for ( +)-chlorpheniramine, while the reported values for the affinity ratio of (+) and ( -) isomers range from = 100 (Chang et al., 1979b) to = 500 (Treherne and Young, 1988). Thus there seems to be little interference from ( - )-chlorpheniramine in the present experimental conditions. Another factor that could affect the shape of the Schild plot is the allosteric properties of antagonists. The model presented by Ehlert (1988). which describes the effects of allosteric antagonism on the Schild plot, implies a non-linear plot without an inflextion or a second asymptote. Although the data presented for thoracic aorta give rise to difficulties in identifying the second asymptote of the Lemoine and Kaumann model, the behavior of diphenhydramine in thoracic and abdominal aorta may be considered as evidence against allosterism. Additionally, if the antagonism were subject to allosteric interaction(s), a more pronounced agonist

dependence of the pA2 value might be expected than was actually observed. Nevertheless it is diri;cult to exclude this possibility with the present techniques. despite the lack of evidence from binding studies for allosteric actions of the antagonists used. Lemoine and Kaumann (1983) tested the agonist dependence of the pA, values of antagonists; however, it was not feasible to screen antagonists in the thora& aorta which was used in the original screening of H, antagonists. The reason for this is the relatively lower responsiveness of this tissue to Hi agonists and thz relatively sma!ler potency of the available H, agonists compared even to histamine (see table 6); very high concentrations of agonists would be required to construct concentration-response curves in the presence of antagonists. Histamine (in the presence of H, receptor blockade) has a greater potency (pD, = 6.5) in the abdominal aorta than in the thoracic aorta (pD, = 5.5). Abdominal aortas were therefore used to test diphenhydramine and antazoline against histamine, 2-thiazolylethylamine, 2-methylhistamine, N”-methylhistamine, N”,N”-dimethylhistamine. Regular Schild plots obtained with 2-thiazolylethylamine and 2-methylhistamine gave slopes less than 1. Nevertheless, the independent two component model did not fit the data in these cases. Responses elicited by high concentrations of 2thiazolylethy!amine were checked for prazosine (an aiadrenoceptor antagonist) sensitivity since a 2-thiazolylethylamine has been reported (Vohra, 1981) to induce catecholamine release. However, a prazosine-sensitive component was not observed. Schild plots for other agonists were linear with slopes not differing significantly from unity. It is known that histamine induces catecholamine release, which could account for the observed heterogeneity, but we were unable to show a prazosine-sensitive component in thoracic aorta responses induced by histamine at 10e4 M, which is the maximum concentration used to obtain equipotent responses, although a prazosine-sensitive component was observed at higher concentrations (10e3 M) of histamine. The results therefore are considered to favor the presence of an additional component which can be distinguished using diphenhydramine, ( + )-brompheniramine, ( f )-chlorpheniramine and mepyramine, but not antazoline in thoracic aorta. Antazoline appears to be equally active in both types of responses. Hi receptor subtypes, as assessed in receptor binding studies, are suggested to be present in the brain and peripheral tissues (Uchida and Takagi, 1977; Chang et al., 1979a,b; Casale et al., 1985; Imoto et al., 1985; Komer et al.. 1986). Additionally, Poli et al. (1988) reported differences between the pre- and postjunctional effects of histamine on the guinea pig urinary bladder, and Ohasm et al. (1987) observed heterogeneous effect of D-600 (for which common binding sites with histamine at Ht rc-

we been suggested) in protwting the H, receptor ag&nst a~~y~~t~~gagents in different blood vessels. resdts which suggest a functional heterogeneity of H, receptors. Other p&b!+ factors at the receptor or the pust-rece@or levek such as variations in receptor microenvironment. variations in ionic movements across the plasma membrane, metabolic variations affecting the tissue responses, non-specific responses. etc., shouid still kept in mind when discussing tke observed heterogeneit~.

Lctar

A The authors thank SK&F, Herdforshire, Fako and Eczacibasi. Istar&& for the supply of drugs used. and Mr. F. Stark for correcting the manuscript_ The study was supported by Grants frm Ankara Univenit_v Research Fund. 89-09-00-03.

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