for H3 dependent ratfor Hi>101.tM, Ka for H-4.3nM, see Refer-ence 20)was present (1nM, 10nM,.30nM), the concentration-response curve for histamine-induced relaxation was also shifted

Research Paper

Mediators of Inflammation 4, 217-221 (1995)

THE presence of histamine H3 receptors was evaluatedon the rat aorta endotheHum. In the presence ofpyrilamine (1 riM, 7 triM, 10 tiM) or thioperamide (1 nM,10riM, 30nM) the concentration-response curve forhistamine-induced (0.1 nM 0.01 mM) endothelium-dependent rat aorta relaxation was shifted to the rightwithout significant change of the Emax indicating com-petitive antagonism by pyrilamine (pA2=9.33 + 0.34,slope--1.09 + 0.36) or thioperamide (pA2 =9.31 + 0.16,slope--0.94 + 0.10). Cimetidine (1 gM) did not influencehistamine-induced endothelium-dependent rat aortarelaxation. In the presence of thioperamide (1riM,10nM, 30nM) the concentration-response curve for(R)-MeHA-induced (0.1 nM- 0.01 raM) endotheHum-dependent relaxation was shifted to the right withoutsignificant change of Emax indicated competitive antag-onism by thioperamide (pA2 9.21 + 0.4, slope1.03 + 0.35). Pyrilamine (100nM) or cimetidine (IM)did not influence (R)-MeHA-induced endothelium-dependent rat aorta relaxation. These results suggestthe presence of a heterogenous population of histaminereceptors, H and H3, on rat aorta endothelium.

Key words: Endothelium, Histamine H3 receptor, Rataorta.

The evidence for histamine H3receptor-mediated endothelium-dependent relaxation in isolated rataorta

D. M. DjuriccA and I. Z. Andjelkovic

Institute of Physiology, Medical Faculty Universityof Belgrade, Visegradska 26/11, P.O. Box 783,11000 Belgrade, Yugoslavia

Cacorresponding Author

Introduction

Two types of histamine receptors, H1 and H2,participate in vascular responses to histamine.The novel histamine H receptors were identifiedas inhibitory presynaptic autoreceptors on hista-mine-containing nerve terminals in the rat braincortex but have since been shown to inhibit therelease of various neurotransmitters both in thecentral and peripheral nervous systems.2 Recentarticles provide strong evidence for the presenceof histamine H receptors at the different sites,including the rabbit middle cerebral artery endo-

3,4 5 6thelium,’ guinea-pig aorta, mesenteric artery,rabbit saphenous artery,7 guinea-pig myo-

8 9cardium, guinea-pig ileum, guinea-pig lung and10 11 I2bronchiole, guinea-pig intestine, porcine

small intestine, rabbit gastric glands,14 human15adenoidal mast cells, human and rhesus

monkey brain.6

.The purpose of the present study was todetermine whether histamine H receptors arelocalized on rat aorta endothelium and to assesstheir possible role in endothelium-dependentresponses.

Materials and Methods

Vascular preparations.. Male Wistar rats weighingbetween 100 and 200g were stunned and the

(C) 1995 Rapid Communications of Oxford ktd

thoracic aorta was excised and dissected free ofsurrounding tissue. Ring segments (4mm)wereprepared and fixed isometrically in 20ml organbath containing Tyrode’s solution of the follow-ing composition (mM): NaC1 136.9, KCl 2.69,CaCI2 1.8, MgCI2 1.05, NaHCO3 11.9, NaHiPO40.42 and glucose 5.55 at 37C under a moderatetension of I g for 90 min (the optimal point ofits length-tension curve as determined from thetension developed in response to potassiumchloride 40mM) and gassed with 95% 02/5%CO2. The preparations were precontracted byphenylephrine (300nM). In some preparationsthe endothelium was removed mechanically bygentle and careful rubbing of the intimal surfacewith a stainless steel wire (31 gauge diameter) inorder to avoid stretching and damaging of thevascular smooth muscle cells. The presence ofendothelium was confirmed by using acetylcho-line (300nM). The failure of acetylcholine toinduce relaxation of preparations was taken asan indication of endothelium removal.

Experimental procedure: After the equilibrationperiod, concentration-response curves wereobtained by cumulative addition of histamine(0.1 nM-0.01 mM) or (R)cz-methylhistamine(R)cz-MeHA, 0.1 nM-0.01 mM) on precontractedpreparations alone or in the presence of pyr-ilamine (1 nM, 7nM, 10nM for histamine and

Mediators of Inflammation Vol 4- 1995 :217

D. M. Djuric and L Z. Andjelkovic

100

8O

.9 60

._> 40

2O

-10 -9 -8 -7 -6 -5 -4Histamine, log M

FIG. 1. Concentration-response curves for histamine in rat aortawith intact endothelium alone or in the presence of pyrilamine orcimetidine. The data are expressed as means (n--6). (Values forS.E.M. are excluded from the figure). Histamine (A); pyrilamine,nM (O); pyrilamine, 7nM (); pyrilamine, lOnM (I-I); cimeti-

dine, IM (O); denuded endothelium (/k).

100nM for (R)cz-MeHA), cimetidine (ll.tM forboth histamine and (R)z-MeHA) or thioperamide(lnM, 10nM, 30nM for both histamine and(R)z-MeHA).

All drugs were dded directly to the bth involume of 150/1 and the concentrations givenre the clculted fimfl concentrations in the bthsolution. When potassium chloride was usedspasmogen, the stated concentration excludedthe potassium chloride redy present in Tyr-ode’s solution.

Data analysis: Responses are expressed as a per-centage of the maximal relaxation induced bypapaverine (100%, 0.1 mM). The slopes of thelog concentration-response curves, correlationcoefficients (r), Emax (maximum response) andpA2 (--log molar concentration of antagonistreducing the agonist response by a factor of 2)values were evaluated from concentration-response curves plotted for each agonist in thepresence of different antagonists. For calculatingthese different values the data are expressed asmeans + S.E.M; n refers to the number ofexperiments. Emax values were compared usingStudent’s t-test, p values less than 0.05 were con-sidered to be significant.

Drugs: The following compounds were used:acetylcholine chloride (Sigma), phenylephrinehydrochloride (Sigma), histamine dihy-drochloride (Sigma), (R)0t-methylhistamine(Research Biochemicals Incorporated), pyr-ilamine maleate (Sigma), cimetidine (Sigma) andthioperamide maleate (Research BiochemicalsIncorporated). All solutions were preparedimmediately before the experiment and storedon ice until use, except thioperamide which wasdissolved in dimethylsulfoxide. (Previous experi:ments had shown that the solvents used had noeffects on the preparations).

Results

Responses to histamine: Histamine (0.1nM-0.01mM) induced a concentration-dependentrelaxation of rat aorta precontracted with pheny-lephrine (300nM), with intact endotheliumreaching approximately 70% of the papaverine-induced maximum relaxation (0.1 mM). Removalof the endothelium abolished the relaxation tohistamine.When pyrilamine, a potent and selective hista-

mine H antagonist (Ka for H -0.8 nM, Ka forHi 5.2/.tM, Kd for H3 > 3 I.tM, see Reference2) was present (1 nM, 7 nM, 10 nM), the con-centration-response curve for histamine-inducedrat aorta relaxation was shifted to the fightwithout significant change of the Emax. Schildplot analysis indicated that antagonism by thiscompound was competitive. The slope for theregression curve was 1.09 + 0.36 with a pA2 valueof 9.33 + 0.34 (r 0.949) (Fig. 1).The potent and selective H2 antagonist, cimeti-

dine (1 l.tM, Ka for Hi 0.8 IM, Ka forH1 --0.45 mM, Ka for H3 33 l.tM, see Reference2) did not influence histamine-induced endothe-lium-dependent rat aorta relaxation (Fig. 1).When thioperamide, a potent and selective his-

tamine H3 antagonist (K for H1 > 1001.tM, Kafor Hi > 101.tM, Ka for H- 4.3 nM, see Refer-ence 20)was present (1 nM, 10nM,. 30nM), theconcentration-response curve for histamine-induced relaxation was also shifted to the fightwithout significant change of the Emax. Schildplot analysis indicated that antagonism by thiscompound was competitive. The slope for theregression curve was 0.94 + 0.10 with a pA2 valueof 9.31 + 0.16 (r 0.993) (Fig. 2).

Responses to (R) t-MeHA. The potent and selec-tive H3 agonist, (R)cz-MeHA (0.1nM-0.01mM)induced a concentration-dependent relaxation ofrat aorta precontracted with phenylephrine(300nM), with intact endothelium reaching

218 Mediators of Inflammation Vol 4. 1995

Histamine H3 receptor and endothelium

0 .I

Histamine, log M

100

8O

-10 -9 -8 -7 -6 -5 -4

(R) -methylhistamine, log M

FIG. 2. Concentration-response curves for histamine in rat aorta FIG. 3. Concentration-response curves for (R)-MeHA in ratwith intact endothelium alone or in the presence of thioper- aorta with intact endothelium alone or in the presence of thio-amide. The data are expressed as means (n=6). (Values for peramide. The data are expressed as means (n=6). (Values forS.E.M. are excluded from the figure). Histamine (,); thioper- S.E.M. are exluded from the figure). (R)--methylhistamine (A);amide, nM (O); thioperamide, lOnM (I); thioperamide, 30nM pyrilamine, 100nM ((C)); cimetidine, 11M (); denuded endo-(I--I); denuded endothelium (). thelium (/).

approximately 50% of the papaverine-induced The histamine H3 receptors were found withinmaximum relaxation (0.1 mM). Removal of the the central nervous system of the rat and theendothelium abolished the relaxation to (R)(x- human where they appear to be involved in theMeHA. feedback control of both histamine synthesis and

Pyrilamine (100nM) or cimetidine (1 IM) did release at the level of histaminergic nervenot influence (R)(x-MeHA-induced endothelium- endings.9’2 Furthermore, stimulation of hista-dependent rat aorta relaxation (Fig. 3). mine H receptors has been shown to inhibitWhen thioperamide (10 nM, 30 nM, 100 nM) adrenergic and cholinergic neurotransmission in

was present, the concentration-response curve the peripheral autonomic neeeous system.6’12

for (R)a-MeHA-induced relaxation was shifted to There is some controversy about whether hista-the right without a significant change of the Emax. mine H3 receptors are present on the syrq.pa-Schild plot analysis indicated that antagonism by thetic nerve fibres inneeeating blood vessels.6 Inthis compound was competitive. The slope for two isolated vessels, the rabbit middle cerebralthe regression curve was 1.03 + 0.35, with a pA2 artery3’4 and guinea-pig aorta,5 a potent andvalue of 9.21 + 0.40 (Fig. 4). selective histamine H agonist, (R)t-MeHA pro-

duced relaxation probably via stimulation ofpostsynaptic histamine H receptors. These find-

Discussion ings suggest that depending on the species andthe experimental model, several mechanisms

Histamine is present in essentially all tissues and (activation of pre- and postsynaptic histamine Hit can stimulate all three classes of histamine receptors or histamine H receptor-independentreceptors. It is found in significant concentrations mechanisms) contribute to the overall effect ofin the blood and also in the vessel walls. 17 Intra- (R)t-MeH on cardiovascular function.22 Experi-vascular administration of histamine elicits a con- ments with the Langendorff perfusion of thecentration-dependent fall in blood pressure in guinea-pig heart have shown that the histaminemost species. Many studies have indicated the H agonist N-x-methylhistamine (N-ot-MeH) pro-involvement of H and H2 receptors in this duces an increase in coronary flow and adepressor response. decrease in coronary vascular resistance which

Mediators of Inflammation Vol 4. 1995 219

D. M. Djuric and I. Z. Andjelkovic

loo!

= 60-

40-

0-10 -9 -8 -7 -6 -5 -4

(R) -methylhistamine, log M

ated by histamine H receptors.27 Schild plots forhistamine (agonist of all three classes of hista-mine receptors) remain straight both in the pre-sence of pyrilamine or thioperamide. This is notsurprising, although the histamine vascular effectsin different biological species involve two recep-tor systems, the histamine H and histamine Hireceptors. New observations suggest that hista-mine H receptors are also localized at the post-synaptic level in the rabbit middle cerebral arteryendothelium,’4 guinea-pig aorta5 and in the epi-thelial wall of guinea-pig bronchioles and thatthey act on the smooth muscle.

In conclusion, there is a heterogenous popula-tion of histamine receptors, H and H, on rataorta endothelium which could participate inendothelium-dependent responses to histamineand its derivatives. The effects we observed mightalso involve NO release, cGMP accumulation andK+ ions (our unpublished observations). Suchevents already have been implicated in histamineH receptor-mediated endothelium-dependentrelaxation in the rabbit middle cerebral artery.4

FIG. 4. Concentration-response curves for (R)-MeHA in rataorta with intact endothelium, alone or in the presence of pyr-ilamine or cimetidine. The data are expressed as means (n=6).(Values for S.E.M. are excluded from the figure). (R)--methylhis-tamine (A); thioperamide, nM (O); thioperamide, 10nM, (m);thioperamide, 30nM (I-]); denuded endothelium (/).

could be abolished by a mixture of impromidine(a non-selective histamine H3 antagonist with H2agonistic effects) and cimetidine.23

The role of the endothelium in the relaxationof precontracted blood vessel preparations hasbeen demonstrated for different physiologicallyimportant substances. The study presented hereshowed that the presence of endothelium is alsoessential for the relaxing effect of histamine and(R) cx-MeHA on the isolated rat aorta. The phy-siological role of the endothelium in histamine-induced relaxation could be of physiologicalimportance. Sensitivity to histamine could also behigher in blood vessels with a more importantrole in the regulation of peripheral resistance.The results with both histamine and pyrilamine

suggest the presence of histamine H receptorson rat aorta endothelium which is in agreement

24 26with results of the other authors. Thioper-amide antagonizes both histamine and (R)cx-MeHA-induced relaxations, resulting in about thesame pA2 values (9.31 for histamine and 9.21 for(R)cx-MeHA, respectively). These pA2 values areclose to that (8.96) found for blockade of hista-mine H-mediated inhibition of [Hl-histaminerelease in rat brain slices,i The pA2 value of thehistamine H antagonist thioperamide was verysimilar to its values for various responses medi-

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Received 3 January 1995;accepted in revised form 21 March 1995

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for H3 dependent ratfor Hi>101.tM, Ka for H-4.3nM, see Refer-ence 20)was present (1nM, 10nM,.30nM), the concentration-response curve for histamine-induced relaxation was also shifted