Characterization of histamine receptors in cat cerebral arteries in vitro and in situ

Histamine is probably a mediator of vascular responses in the brain, but there is little experimental evidence for its importance in this role. By using both in vitro and in situ techniques, we have studied responses of cat pial arteries to stimulation of histamine receptors by pharmacological agents. In vitro, histamine and 2,2-pyridylethylamine (PEA, H1 agonist) caused contraction of resting arteries while impromidine (H2 agonist) was without effect. The PEA-induced constriction was blocked by the histamine H1 antagonist, mepyramine. When the arteries were precontracted (by 3 X 10(-6) M prostaglandin F2 alpha), however, all three agents caused vascular relaxation with an order of effectiveness as follows: histamine = impromidine much much greater than PEA. The responses of histamine and impromidine were reduced by the H2-antagonists, metiamide or cimetidine. Schild plots for the H2 receptor antagonists resulted in pA2 values of 6.90 and 7.03 for metiamide and cimetidine, respectively. In situ, neither agonist caused pial arterial constriction. Impromidine was considerably more effective than PEA in producing arterial dilatation. Metiamide reduced the effect of impromidine, whereas the dilatation of PEA was reduced by mepyramine. Dilatations resulting from PEA persisted in the presence of metiamide. Our results are consistent with the hypothesis that histamine H2 receptors are present in cerebral vascular smooth muscle as identified both in vitro and in situ. Indications for the additional presence of H1 receptors are, however, weak.     

Characterization and distribution of endothelin receptors in the pulmonary circulation: investigation of isolated, perfused, and ventilated rabbit lungs.

The aim of the study was to investigate the distribution of 2 subtypes of endothelin-receptors, mediating the effects of endothelin-1 (ET-1) in the pulmonary circulation. Until now, it is still unclear, whether ET(A) receptors or ET(B) receptors or even both are localized in pulmonary vessels. The experiments were performed on 72 isolated and ventilated rabbit lungs that were perfused with a cell- and plasma-free buffer solution. The arterial pressure and the lung weight gain were continuously registered. Intermittently perfusate samples were taken for determination of thromboxane A2 (TXA2) and prostacyclin (PGI2). The injection of ET-1 (10(-8) M, n = 6) resulted in a biphasic increase in pulmonary arterial pressure (PAP) that was accompanied by the generation of TXA2 and PGI2. Pretreatment with the ET(A)-receptor antagonist LU135252 (10(-6) M, n = 6) suppressed the pressure response after ET-1 application (P < 0.01 at 120 min) and reduced the generation of TXA2 (P < 0.05 at 120 min) and PGI2 (P < 0.05 at 120 min). Pretreatment with the cyclooxygenase inhibitor diclofenac (10 microg/mL; n = 6) also reduced the PAP increase after ET-1 injection. In contrast to this, the pulmonary vascular pressure reaction after ET-1 application was elevated, when ET(B)-receptor antagonist BQ788 (10(-6) M; n = 6) was given. Furthermore, the PGI2 to TXA2 ratio was shifted from 2.3 to 0.9, reflecting a predominance of vasoconstrictive TXA2. The simultaneous application of LU135252 and BQ788 significantly reduced the PAP increase after ET-1 application, but no beneficial effects were observed compared with the application of LU135252 solely. The injection of the ET(B)-receptor agonist sarafotoxin S6c (S6c; 10(-8) M, n = 6) also induced an increase in PAP that was not attenuated by pretreatment with the ET(B)-receptor antagonist BQ788 (10(-6) M, n = 6). LU135252 (n = 6) as well as the application of LU135252 in combination with BQ788 (n = 6) failed to suppress the pressure response after S6c, whereas the cyclooxygenase inhibitor diclofenac (10 microg/mL, n = 6) alone and in combination with LU135252 and BQ788 (n = 6) was able to prevent the PAP increase after S6c injection (P < 0.001). The results demonstrate that the ET-1-induced increase in pulmonary vascular resistance is mainly mediated via ET(A) receptors, whereas ET(B) receptors seem to mediate vasodilation, which was shown by an imbalance of TXA2 and PGI2 generation. On the other hand, the ET(B)-receptor agonist S6c induced vasoconstriction, which was only attenuated by the cyclooxygenase inhibitor diclofenac. From the current results we conclude that, apart from vasoconstrictor ET(A) receptors, at least 2 ET(B)-receptor subtypes are expressed in the pulmonary circulation, one mediating vasoconstriction, which was not blocked by BQ788, and one mediating vasodilation, which was influenced by BQ788.     

Alpha adrenergic receptors mediate angiotensin-induced prostaglandin production in the rabbit isolated vas deferens

The effect of alpha adrenergic receptor antagonists on concentration-dependent response to angiotensins II and III was examined in the electrically stimulated isolated rabbit vas deferens. The force generated by a nonadrenergic neural mechanism was reduced by both peptides whereas the force generated by adrenergic neural mechanisms was enhanced. Angiotensin III-induced inhibition of the nonadrenergic contraction was significantly greater than that of angiotensin II for all groups. Yohimbine (1 X 10(-4) M), an alpha-2 receptor antagonist, attenuated the depression of the nonadrenergic contraction produced by angiotensins II and III. Yohimbine (1 X 10(-5) and 1 X 10(-4) M) also significantly reduced angiotensin II-induced prostaglandin E (PGE) synthesis. Yohimbine only significantly altered the angiotensin III-induced PGE synthesis at an antagonist concentration of 1 X 10(-4) M. Rauwolscine (1 X 10(-8) and 1 X 10(-7) M) attenuated angiotensin II-induced PGE production and at a higher concentration (1 X 10(-6) M) reduced angiotensin III-induced PGE production. The alpha-1 antagonist, prazosin (1 X 10(-6) M), did not alter nonadrenergic contractile or PGE responses to either angiotensin. The alpha-2 agonist, clonidine, both inhibited the nonadrenergic neural contraction and enhanced PGE synthesis. We interpret these data to indicate that angiotensins II and III may act via separate mechanisms to induce PGE synthesis in the vas deferens, with angiotensin II effects being more dependent on norepinephrine release from adrenergic nerves.     

Characterization of neuronal and vascular histamine receptors mediating the salivary and vasodilator responses to histamine of the dog submandibular gland

The submandibular gland in situ was perfused with blood through the glandular artery at constant pressure in anesthetized dogs, and all drugs were administered intra-arterially. During infusion of metiamide, histamine and 2-(2-pyridyl)ethylamine (PEA) produced salivary and vasodilator responses consisting of an early and a late component. The dose-response curves for respective components of the salivary and vasodilator responses to PEA were parallel with the corresponding curves for histamine and in producing these responses PEA was about 40 times less potent than histamine on a molar basis. During infusion of mepyramine, histamine and dimaprit produced only the early vasodilator response. The dose-vasodilator response curves to histamine and dimaprit were parallel, and dimaprit was about 750 times less potent than histamine on a molar basis. The present results support the conclusion obtained in a previous study that neuronal histamine receptors mediating the whole salivary and the late vasodilator response are exclusively of the H1-type and vascular histamine receptors mediating the early vasodilator response consist of both H1-and H2-type although the former is predominant.     

Kinetics of competitive drug action at 5-hydroxytryptamine2 receptors in isolated rabbit aorta

The kinetics of agonist and antagonist interactions with the 5-hydroxytryptamine2 receptor were studied in the isolated rabbit aorta by following the antagonist-induced decrease in the steady-state response to an agonist. A model describing the competitive drug-receptor interactions was fitted to the data and yielded estimates of the association and dissociation rate constants of the agonist and the antagonist. A high concentration of the agonist ([agonist] much greater than KA) was used to reduce the influence of antagonist diffusion to the receptor upon the onset of antagonism. The effect of a diffusion barrier was evaluated by comparing the kinetics of drug competition in the absence and in the presence of the adventitia. The rate constants of the high-affinity antagonists spiperone, methysergide or ketanserin were similar in the absence and in the presence of the adventitia. In contrast, the rate constants of the low affinity antagonist 5-methoxygramine were reduced almost 5-fold in the presence of the adventitia. This observation may be explained by the large partition coefficients of the high-affinity antagonists as compared to the relatively low partition coefficient of 5-methoxygramine. The ratios of the estimated rate constants (k-x/kx) are in good agreement with the dissociation constants of the drugs determined with steady-state methods. In addition the results suggest that the association rate constant is a primary determinant of drug affinity for the receptor. The kinetic rate constants of the high-affinity antagonists measured in this preparation are similar to those previously reported in high-affinity binding studies. We conclude that the kinetic parameters obtained in our experiments reflect primarily the molecular interactions of these drugs with the receptor.     

Characterization of 5-hydroxytryptamine receptors on isolated ovine uterine artery in late pregnancy

5-Hydroxytryptamine (5-HT) and 2,5-dimethoxy-4-methyl-amphetamine (DOM) are potent agonists on isolated ovine uterine arteries in late pregnancy. Similar pA2 values (8.56 and 8.33, respectively) of ketanserin, tested against 5-HT and DOM, indicate that responses produced by both agonists are mediated by the 5-HT2 receptor. The contractions produced by 8-OH-DPAT and 2-methyl-5-HT were also blocked by ketanserin (10(-8) M) with the dissociation constants (KB) being 2.49 and 2.88 nM, respectively. This provides evidence that these agonists are activating 5-HT2 receptors in the ovine uterine artery. DOM was more potent than 5-HT, but had a similar efficacy to that of 5-HT. The greater affinity of DOM may explain its greater potency. The dissociation constants (KA) of 5-HT and DOM acting on 5-HT receptors were determined by analysis of concentration-response data before and after fractional inactivation of receptors with dibenamine. The mean KA values for 5-HT and DOM were 3.7 +/- 0.7 x 10(-7) and 1.8 +/- 0.3 x 10(-7) M, respectively. Assessment of receptor occupancy vs. functional response demonstrated little or no receptor reserve in this tissue. Several other 5-HT receptor agonists caused contractions but were much less potent than 5-HT. The order of potency of these agonists was determined to be DOM greater than 5-HT greater than or equal to alpha-methyl-5-HT much greater than 8-hydroxy-dipropylaminotetralin (8-OH-DPAT) greater than 2-methyl-5-HT greater than 1-(3-chlorophenyl) piperazine (mCPP) greater than m-trifluoromethyl-phenylpiperazine (TFMPP).(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of muscarinic receptors of the rabbit ear artery smooth muscle and endothelium

Muscarinic receptors of the rabbit ear artery were characterized by observing the effect of the subtype selective antagonist pirenzepine on functional responses and radioligand binding. Pirenzepine has been shown to bind with high affinity to muscarinic receptors of certain brain regions and peripheral ganglia (M1 subtype) and with low affinity to receptors of the heart and upper gastrointestinal tract (M2 subtype). The affinity (pKB) of pirenzepine for the muscarinic sites of the endothelium was determined by the competitive antagonism of the relaxation response to methacholine. Schild analysis gave a pKB of 6.5 (320 nM) which is consistent with the low affinity, M2, subtype of muscarinic receptor. Removal of the endothelium eliminates any response to muscarinic agonists but does not decrease the density of muscarinic binding sites determined by binding of the specific ligand (-)-[3H]quinuclidinyl benzilate. This indicates a second group of muscarinic receptors most probably located on vascular smooth muscle cells for which there is no known function. The pKi for pirenzepine at these sites, as determined by the inhibition of (-)-[3H]quinuclidinyl benzilate binding, was 6.26 (550 nM) which is also consistent with a low affinity subtype. Thus, both types of vascular muscarinic binding sites, those on the endothelium which mediate relaxation and those on the vascular smooth muscle cells, are of the low affinity, M2, subtype.     

Characterization of vascular muscarinic receptors: rabbit ear artery and bovine coronary artery

The goal of this study was to use a functional bioassay to compare muscarinic receptors in two vascular preparations: the rabbit ear artery, which manifests an endothelial-dependent relaxation to cholinergic stimulation, and the bovine coronary artery, in which contraction is elicited independently of the endothelium. Four antagonists were selected, 4-diphenylacetoxy-N-methylpiperidine methobromide, dicyclomine, methoctramine and hexahydrosiladifenidol, to complement previous studies using pirenzepine and 11,2- ([2-(diethylaminomethyl)-1-piperidinyl]acetyl)-5,11-dihydro-6H- pyrido(2,3-b)(1,4)benzodiazepine-6-one, in order to distinguish M1, M2 and M3 receptors. All the antagonists produced competitive inhibition of responses to methacholine, with Schild plot slopes not different from one, with one exception: for 4-diphenylacetoxy-N-methylpiperidine methobromide, Schild plot slopes were greater than one in both tissues. Comparison of antagonist affinities suggests that the muscarinic receptor subtype is not different between the two tissues, despite the different cell types that mediate these responses. Furthermore, the pattern of antagonist affinity agrees most closely with that found previously for the M3 receptor, with high affinity for 4-diphenylacetoxy-N-methylpiperidine methobromide, hexahydrosiladifenidol and dicyclomine, intermediate affinity for pirenzepine, and low affinity for 11,2- ([2-(diethylaminomethyl)-1-piperidinyl]acetyl)-5,11-dihydro-6H- pyrido(2,3-b)(1,4)benzodiazepine-6-one and methoctramine. These findings demonstrate that currently available muscarinic antagonists do not discriminate between receptors found on endothelial and vascular smooth muscle cells, even though the ultimate responses produced are opposed.     

Migraine therapy: relationship between serotonergic contractile receptors in canine and rabbit saphenous veins to human cerebral and coronary arteries

Canine and rabbit vascular contractile responses to serotonergic agonists have been used to predict antimigraine efficacy for several antimigraine agents, including sumatriptan. The purpose of the present study was to establish the assumed predictive value of contractile responses in canine and rabbit saphenous veins to contractile efficacy for a series of agonists in human cerebral and coronary arteries and to understand better the receptors mediating such responses. The canine and rabbit saphenous veins contracted similarly (both qualitatively and quantitatively) to series of structurally diverse serotonergic agonists, suggesting that the receptors mediating serotonin-induced contractility in these tissues were similar. In addition, the contractile potency (estimated as EC₅₀ values) for these structurally diverse serotonergic agonists in either the rabbit or canine saphenous vein significantly correlated with contractile potency for these agonists in human cerebral arteries. Thus, to the extent that contractile responsiveness of human cerebral arteries may predict antimigraine agents, contractile responses of the rabbit and/or canine saphenous vein may be useful surrogates for antimigraine efficacy. In addition, the contractile potency for this series of serotonergic agonists in the rabbit and/or canine saphenous vein significantly correlated with contractile potency of these agonists in human coronary arteries. These data suggest that the use of the saphenous vein to identify potent vasoconstrictors will also reveal agents capable of contracting human coronary arteries, a liability for using this approach to evaluate promising antimigraine therapies.     

兔自体移植静脉内膜增生与缬沙坦的干预效应

目的:内膜增生是移植静脉再狭窄的主要原因,血管紧张素Ⅱ在血管组织增生中发挥着重要作用,缬沙坦作为AT1受体拮抗剂可在受体水平阻断血管紧张素Ⅱ的生物学作用.实验拟验证AT1受体拮抗剂缬沙坦对兔颈部静脉移植术后血管内膜增生的干预作用.方法:实验于2005-10/2006-10在南通大学动物实验中心完成.①实验分组:雄性新西兰大白兔20只,微生物控制级别Ⅰ级,兔龄16～18周,体质量(2.5±0.5)kg,随机排列表法分为对照组和治疗组,每组10只.②实验方法:所有动物行颈部自体静脉移植手术,对照组予普通饲料饲养;治疗组则予普通饲科 缬沙坦[10mg/(kg·d)]喂养.③实验评估:饲养4周后观察两组动物治疗前后的血压变化情况;取出静脉移植物行病理形态学检查;采用图像分析系统计算血管腔内膜的厚度、面积及中膜的厚度、面积;免疫组织化学方法检测增殖细胞核抗原的表达.结果:纳入大白兔20只,均进入结果分析,术后切口无感染,两组移植静脉无闭塞.①对照组和治疗组动物的血压在治疗前后无明显变化(P＞0.05).②对照组移植物内膜明显增厚,内膜下大量平滑肌细胞增殖,中膜稍有增厚:治疗组内膜增厚较对照组减弱,中膜平滑肌细胞向内膜下迁移、增殖较对照组明显减轻,中膜增厚不明显.③治疗组新生内膜厚度、面积及增殖细胞核抗原均低于对照组(P＜0.01),结论:缬沙坦可以显著抑制兔自体静脉移植后的再狭窄,并抑制移植静脉桥中增殖细胞核抗原的表达.     

Distribution of alpha-adrenergic receptors in the rabbit nephron

In order to determine the distribution of alpha-adrenergic receptors within a nephron, a method was used to determine the specific binding of [3H]-prazosin to isolated fragments of rabbit renal tubules. When 10(-8) moles/liter [3H]-prazosin was incubated with proximal convoluted tubules, 61.4% of total binding was accounted for specific binding. The [3H]-prazosin binding to the proximal convoluted tubule was a linear function of the tubular length and reversible. It was inhibited with 10(-4) moles/liter phenoxybenzamine by about 60%, and with 10(-4) moles/liter norepinephrine by about 25%, but not with either atenolol or isoproterenol. No specific binding of [3H]-prazosin was observed in the proximal straight tubule and in the cortical collecting tubule. These data are in good agreement with the view that alpha-1 adrenergic receptors are mainly distributed in the proximal convoluted tubules.     

Tricyclic antidepressants and histamine H1 receptors.

Tricyclic antidepressants and some structurally related compounds were tested for their ability to antagonize histamine H1 and muscarinic acetylcholine receptors of cultured mouse neuroblastoma cells. As a group, tertiary amine tricyclic antidepressants tended to be more potent than secondary amine drugs at both receptors. The most potent antihistamine, doxepin hydrocholoride, was about 4 times more potent than amitriptyline hydrochloride, about 800 times more potent than diphenhydramine hydrochloride, and about 8,000 times more potent than desipramine hydrochloride, the least potent tricyclic antidepressant at both the histamine H1 and the muscarinic acetylcholine receptors. All tricyclic drugs except desipramine hydrochloride were more potent as antihistamines than as anticholinergics. Doxepin hydrochloride and amitriptyline hydrochloride may be the most potent antihistamines known, and the antihistaminic potencies of these and the other tricyclic antidepressant drugs may relate directly to their ability to cause sedation and drowsiness in patients.     

Evaluation of presynaptic histamine receptors in the canine renal vascular bed

Histamine has been shown to increase renal blood flow via H1- and H2-receptors. Furthermore, H2-receptors have been demonstrated to attenuate stimulation-induced release of norepinephrine. The present studies examined whether histamine has a presynaptic effect on sympathetic nerves in the canine renal vascular bed. Renal blood flow was measured in anesthetized dogs, and vasoconstrictor responses to renal nerve stimulation and i.a. injections of norepinephrine were compared before and during i.a. infusions of histamine. Histamine increased renal blood flow and decreased stimulation-induced vasoconstriction to a greater degree than norepinephrine responses. 2-(2-pyridyl)ethylamine, an H1-agonist, did not produce consistent effects. Dimaprit, an H2-agonist, produced responses similar to histamine but to a lesser extent. The H1-antagonist tripelennamine and the H2-antagonist cimetidine each minimally antagonized the effect of histamine on nerve stimulation. When both blocking agents were infused together, maximum antagonism of histamine occurred. Thus, it appears that histamine will produce a neuroinhibitory effect in the canine renal vascular bed and this effect appears to be mediated by both H1- and H2-receptors because both receptor antagonists are necessary to block this effect.     

Characterization of rabbit ileal receptors for Clostridium difficile toxin A. Evidence for a receptor-coupled G protein

The purpose of this study was to characterize the surface receptor for toxin A, the enterotoxin from Clostridium difficile, on rabbit intestinal brush borders (BB) and on rat basophilic leukemia (RBL) cells. Purified toxin A was radiolabeled using a modified Bolton-Hunter method to sp act 2 microCi/micrograms, with retention of full biologic activity. 3H-Toxin A bound specifically to a single class of receptors on rabbit BB and on RBL cells with dissociation constants of 5.4 x 10(-8) and 3.5 x 10(-8) M, respectively. RBL cells were highly sensitive to toxin A (cell rounding) and had 180,000 specific binding sites per cell, whereas IMR-90 fibroblasts were far less sensitive to toxin A and lacked detectable specific binding sites. Exposure of BB to trypsin or chymotrypsin significantly reduced 3H-toxin A specific binding. Preincubation of BB with Bandeirea simplicifolia (BS-1) lectin also reduced specific binding, and CHAPS-solubilized receptors could be immobilized with WGA-agarose. The addition of 100 nM toxin A accelerated the association of 35S-GTP gamma S with rabbit ileal BB, and preincubation of BB with the GTP analogues GTP gamma S or Gpp(NH)p, significantly reduced 3H-toxin A specific binding. Our data indicate that the membrane receptor for toxin A is a galactose and N-acetyl-glucosamine-containing glycoprotein which appears to be coupled to a G protein.     

Temperature-dependent basal tone in isolated human saphenous veins: implication of TP-receptors

Abstract— Cutaneous blood vessels are very sensitive to changes in environmental temperature. The influence of variations in local temperature on the mechanisms involved in the basal tone, present in isolated human saphenous veins has not yet been studied. In the present study, segments with and without endothelium of human saphenous veins obtained from coronary bypass surgery patients were mounted for isometric tension recording in oxygenated physiological salt solution (PSS). After stabilisation of the basal tone, the local temperature was rapidly either decreased from 37 °C to 24 °C (cooling) or increased from 37 °C to 42°C (warming). When antagonists or inhibitors were used the preparations were incubated for 30 min with the drugs. During basal conditions, cooling caused relaxations of the saphenous vein segments with endothelium and warming caused contractions; the absence of the endothelium did not modify these responses. In veins without endothelium, the warming‐induced contractions were significantly inhibited by verapamil (10 μM) and by the antagonist of TP‐receptors (receptors for thromboxane A₂) Bay u 3405 (1 μM). The warming induced contractions were not affected by cyclooxygenase or lipoxygenase inhibition. At 37°C, the isoprostanes (8‐iso‐PGE₂ and 8‐iso‐PGF_2α) induced potent contractions that were significantly inhibited by Bay u 3405 (1 μM). The data show that a basal tone is present in isolated resting human saphenous vein segments at 37°C. This basal tone is decreased by local cooling and enhanced by local wanning and is not dependent on the presence of the endothelium. The warming‐induced contraction of the veins is mediated by a non‐cyclooxygenase, non‐lipoxygenase metabolite (iso‐prostanc?) that interacts with TP‐receptors and via an extracellular calcium‐dependent pathway.