Endothelium-dependent relaxation of rat aorta to a histamine H3 agonist is reduced by inhibitors of nitric oxide synthase, guanylate cyclase and Na+,K+-ATPase

The possible involvement of different effector systems (nitric oxide synthase, guanylate cyclase, β-adrenergic and muscarinic cholinergic receptors, cyclooxygenase and lipoxygenase, and Na+,K+-ATPase) was evaluated in a histamine H3 receptor agonist-induced ((R)α-methylhistamine, (R)α-MeHA) endothelium-dependent rat aorta relaxation assay. (R)α-MeHA (0.1 nM – 0.01 mM) relaxed endothelium-dependent rat aorta, with a pD2 value of 8.22 ± 0.06, compared with a pD2 value of 7.98 ± 0.02 caused by histamine (50% and 70% relaxation, respectively). The effect of (R)α-MeHA (0.1 nM – 0.01 mM) was competitively antagonized by thioperamide (1, 10 and 30 nM) (pA2 = 9.21 ± 0.40; slope = 1.03 ± 0.35) but it was unaffected by pyrilamine (100 nM), cimetidine (1 μM), atropine (10 μM), propranolol (1 μM), indomethacin (10 μM) or nordthydroguaiaretic acid (0.1 mM). Inhibitors of nitric oxide synthase, L-NG-monomethylarginine (L-NMMA, 10 μM) and NG-nitro-L-arginine methylester (L-NOARG, 10 μM) inhibited the relaxation effect of (R)α-MeHA, by approximately 52% and 70%, respectively). This inhibitory effect of L-NMMA was partially reversed by L-arginine (10 μM). Methylene blue (10 μM) and ouabain (10 μM) inhibited relaxation (R)α-MeHA-induced by approximately 50% and 90%, respectively. The products of cyclooxygenase and lipoxygenase are not involved in (R)α-MeHA-induced endothelium-dependent rat aorta relaxation nor are the muscarinic cholinergic and β-adrenergic receptors. The results also suggest the involvement of NO synthase, guanylate cyclase and Na+,K+-ATPase in (R)α-MeHA-induced endothelium-dependent rat aorta relaxation.


Introduction
Histamine is present in essentially all tissues and it can stimulate all three classes of histamine receptors. It is found in significant concentrations in the blood and also in the vessel walls. It has been known for several years that histamine receptor subtypes vary in different, isolated vascular tissues, depending upon the anatomic location, species and physiological response. It is known that two types of histamine receptors, H1 and H2, participate in vascular responses to histamine. 2 Intravascular administration of histamine elicits a concentration-dependent fall in blood pressure in most species. Many studies have indicated the involvement of histamine H and H 2 receptors in this depressor response. The histamine H1 and H2 receptor-mediated actions of histamine on effector cells are linked with the accumulation of cGMP, inositol phospholipids and cAMP, respectively. [4][5][6] In different blood vessels cGMP formation is activated by endothelium-derived relaxing factor (EDRF) 7 while cAMP formation is stimulated by prostacyclin (PGI2). 8 '9 The histamine H3 receptors were found within the central nervous system of the rat and the human where they appear to be involved in the feedback control of both histamine synthesis and release of the level of histaminergic nerve endings, m'l Furthermore, stimulation of histamine H 3 receptors has been shown to inhibit adrenergic and cholinergic neurotransmission in the peripheral autonomic nervous system. 12 '13 There is some controversy about whether histamine H3 receptors are present on the sympathetic nerve fibres innervating blood vessels. 2 Stimulation of histamine H3 receptors by specific agonist mediated vascular relaxant effects is (C) 1996 Rapid Science Publishers Mediators of Inflammation Vol 5 1996 69 caused by mechanism(s) which are not clear at are the calculated final concentrations in the bath present. In two isolated vessels, the rabbit middle solution. When potassium chloride was used as a cerebral artery 4'15 and guinea-pig aorta 6 a spasmogen, the stated concentration exluded the potent and selective histamine H3 agonist, (R)zpotassium chloride already present in Tyrode's methylhistamine ((R)z-MeHA), caused relaxation solution.
probably via stimulation of postsynaptic histamine H 3 receptors. These findings suggest that, depending on the species and the experimental model, several mechanisms (activation of preand post-synaptic histamine H 3 receptors or histamine H3 receptor-independent mechanisms) contribute to the overall effect of (R)z-MeHA on cardiovascular function. 17 In our previous report the existence of hista- dihythoracic aorta was excised and dissected free of drochloride (Sigma), (R)z-methylhistamine surrounding tissue. Ring segments (4mm) were (Research Biochemicals Incorporated), pyrprepared and fixed isometrically in a 20 ml organ ilamine maleate (Sigma), cimetidine (Sigma), bath containing Tyrode's solution of the followthioperamide maleate (Research Biochemicals ing composition (mM): NaCl, 136.9; KCl, 2.69; Incorporated), atropine sulphate (Sigma), pro-CaCl2, 1.8; MgCl2, 1.05; NaHCO3, 11.9; pranolol hydrochloride (Sigma), t-Ng-mono NaH2PO4, 0.42; and glucose 5.55, at 37C under methylarginine (L-NMMA, Research Biochemicals a moderate tension of lg for 90 min (the .Incorporated), A-nitro-t-arginine methylester optimal point of its length-tension curve as NOARG, Research Biochemicals Incorporated), Ldetermined from the tension developed in arginine (Sigma), indomethacin (Sigma), nordihyresponse to potassium chloride 40mM) and droguaiaretic acid (NDHGA, Sigma), ouabain gassed with 95% 02/5% COg. The preparations octahydrate (Serva), methylene blue (Sigma) and were precontracted by phenylephrine (300nM). papaverin hydrochloride (Sigma). All solutions In some preparations the endothelium was were kept on ice until use except thioperamide removed mechanically by gentle and careful which was dissolved in dimethylsulphoxide (prerubbing of the intimal surface with a stainlessvious experiments had shown that the solvents steel wire (31-gauge diameter)in order to avoid used had no effects on the preparations) and stretching and damaging the vascular smooth (R)z-MeHA which was diluted in water and muscle cells. The presence of endothelium was stored as an aliquot (100tl) at -20C. Indoconfirmed by using acetylcholine (300nM). The methacin was dissolved in an equimolar confailure of acetylcholine to induce relaxation of centration with NaaCO3.
preparations was taken as an indication of endothelium removal.

Discussion
The novel histamine H3 receptors were identified as inhibitory presynaptic autoreceptors on histamine-containing nerve terminals in the rat brain cortex but they have since been shown to inhibit the release of various neurotransmitters both in the central and peripheral nervous system. 9 Recent articles provide,strong evidence for the presence of histamine H3 receptors at different sites, including rabbit middle cerebral 1 15 artery endothelium, guinea-pig aorta,6 mesenteric artery, 12 rabbit saphenous artery, 2 21 22 guinea-pig myocardium, guinea-pig ileum, 23 24 guinea-pig lung andbronchiole, guinea-pig intestine, 25 porcine small intestine, rabbit gastric glands, 27 human adenoidal mast cells, 28 and human and rhesus monkey brain. 29  In different blood vessels, cGMP formation is The possibilities include inhibition of adenylate activated by EDRF, 7 while cAMP formation is sticyclase activity, activation of K + channels and mulated by PGI2 .8'9 Particularly from experiments inhibition of voltage-dependent Ca ).+ chanwith endothelial cells cultured in vitro several nels. 34'35 Studies in slices of guinea-pig hippo-classes of agonist have been demonstrated to campus have shown that the histamine H3 interact with surface receptors leading to PGI) agonist (R)z-MeHA is not able to inhibit dima-synthesis. These include proteins and peptides prit-induced cAMP accumulation 36 suggesting that (thrombin and bradykinin), amines (histamine at histamine H3 receptors are not negatively linked H1 receptors), eicosanoids (leukotriene C4) and to adenylate cyclase. It is known that (R)z-MeHA, purines (ATP and ADP). 48 (R)z-MeHA-induced a potent and selective histamine H3 agonist, endothelium-dependent rabbit middle cerebral induced endothelium-dependent relaxation of artery relaxation was partially reduced by trahigh-K + precontracted rabbit middle cerebral nylcypromine (an inhibitor of leukotriene synthartery. That relaxation was not affected by ]3-esis) and also inhibited by dexamethasone (an adrenoceptors, muscarinic or dopamine receptor inhibitor of prostaglandin synthesis probably at antagonists. 5 Our results with atropine and pro-the level of phospholopase A249) and indomethapranolol also showed that neither muscarinic cin (an inhibitor of PGI2 synthesis5) indicated cholinergic receptors nor ]3-adrenoceptors were that a prostanoid (probably PGI)) could also be involved in (R)a-MeHA-induced endothelium-involved in (R)z-MeHA-induced endotheliumdependent rat aorta relaxation, dependent rabbit middle cerebral artery relaxa-The identification of nitric oxide (NO) (or a tion. 5 The implication of results with indomethacompound containing the NO ligand) such as cin and NDHGA is that (R)z-MeHA does not EDRF 37'38 and the finding that I-NMMA or t-induce the release of the relaxing factors con-NOARG are inhibitors of NO synthesis in vascular taining metabolites of arachidonic acid via endothelium 39'4 have emphasized the impor-cyclooxygenase (e.g. PGE, PGE2, PGI)) or via tance of local control of vasomotor tone. Nitric lipoxygenase (leukotrienes) on rat aorta endooxide enhances production of cGMP in vascular thelium. smooth muscles through activation of soluble In many arteries, the release of EDRF by aceguanylate cyclase, which in turn activates. Ca )+tylcholine (Ach) is accompanied by an endothe-ATPase to reduce intracellular Ca 2+ concentra-lium-dependent hyperpolarization, while EDHF is tion and induces muscle relaxation. It has been the possible mediator. 5 The EDHF-induced demonstrated that t-NAME (a competitive inhihyperpolarization is produced by an increased bitor of NO synthesis41), specifically inhibits permeability of the membrane to K + ions with cGMP formation due to the activation of mus-no change in either cAMP or cGMP. 52 It has also carinic, histamine, bradykinin and neurotensin been concluded that the hyperpolarization resulreceptors in mouse neuroblastoma N1E-115 ted from stimulation of Na +,K+-ATPase. It has cells. 42 In our experiments the inhibitory effects been observed that in rat aorta and rat main of I-NMMA and I-NOARG on (R)z-MeHA-induced pulmonary artery ACh released two different subendothelium-dependent rat aorta relaxation were stances from endothelium. On factor (EDRF) is also observed, indicating that these substances responsible for vascular muscle relaxation while inhibit NO formation by competing with t-argi-the other (EDHF) hyterpolarizes the muscle nine for binding to NO synthase. The addition of membrane by opening 6Rb-permeable K + chant-arginine partly reversed t-NMMA-induced inhibi-nels. 5 However, ouabain (an inhibitor of the tion of response. We also found that the inhibi-Na+,K+-ATPase) inhibited (R)0t-MeHA-induced tory effect of L-NOARG was greater than that of L-endothelium-dependent rat aorta relaxation NMMA (70% and 52%, respectively) which is in much more than t-NOARG (90% and 70% inhibiagreement with the observations of different tion, respectively). It means that in addition to 15 43 44 authors.
Methylene blue (which blocks NO, some factor other than NO (e.g. EDHF) guanylate cyclase45) prevented the increase in could participate in (R)z-MeHA-induced endothe-cGMP and thus inhibited (R)z-MeHA-induced Bum-dependent rat aorta relaxation. Inhibition of q.-+ endothelium-dependent rat aorta relaxation, the Na ,K -ATPase by ouabain would shift the The decrease with age in the dilator response membrane potential to a less negative level and of rat thoracic aorta to histamine is due to a thus reduce or abolish any hyperpolarization decreased cGMP production and to an age-associated with K + channel opening. 46 dependent decrease in endothelium function.
In conclusion, we suggest that the products of cyclooxygenase and lipoxygenase are not involved in (R)cz-MeHA-induced endotheliumdependent rat aorta relaxation in addition to muscarinic cholinergic and [3-adrenergic receptors. The results also suggest the involvement of NO synthase, guanylate cyclase and Na+,K +-ATPase in histamine H 3 receptor agonist-induced ((R)0t-MeHA) endothelium-dependent rat aorta relaxation.