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The release of the putative neurotransmitter histamine (HA) from rat brain cortex slices is under negative feedback control by an HA autoreceptor. This autoreceptor has been postulated to belong to a new class of HA receptors, H3. To verify this hypothesis we have developed an assay using superfused rat brain cortex slices. The HA transmitter pool is labelled by incubation of the slices with the precursor3H-histidine;3H-HA is estimated after separation by column chromatography. Release of HA was found both after K+-induced depolarization and electrical field stimulation. The latter resulted in higher and more reproducible HA release. Electrically induced HA release could be fully inhibited in a concentration dependent way by exogenous HA in the superfusion buffer. Nα-alkylated histamines also showed agonistic activity. The action of exogenous HA was totally blocked by the potent H2 agonist impromidine and some of its analogues.

It has been questioned whether the interaction of H2-antagonists with cytochrome P-450 that is observedin vitro is also relevant for thein vivo situation. Until now the possibility that cytochrome P-450 may function with different modes of action has been neglected in this respect. We studied the effect of cimetidine, ranitidine and famotidine on the monoxygenase, the oxidase and the peroxidase action of cytochrome P-450. Biotransformation catalyzed by the monoxygenase and oxidase action of cytochrome P-450 was affected by cimetidine (probably via its ligand interaction with cytochrome P-450), whereas metabolism by the peroxidase mode of action of cytochrome P-450 was hardly influenced. Ranitidine and famotidine (both pharmacodynamically more potent than cimetidine) only slightly affected cytochrome P-450 activities.

It was found that a number of substitute 2-phenyl-1,3-indandiones possesses a considerable inhibitory effect on the classical pathway of complement. Furthermore it appeared that in this series a reasonable correlation (r=0.86) exists between this anti-complement effect and the inhibition of the prostaglandine biosynthesis. A quantitative structure-activity relation study revealed that both the electronic σ parameter of the substituent involved and the lipophilicity of the corresponding compound are important for anti-complement activity. Mainly the C5 step in the complement process is inhibited by these compounds. The complement-inhibiting phenylindandiones also inhibit the alternative pathway. This effect may also be due to an inhibition of C5, as C5 is common to both pathways. Remarkably, the 2-(3,5-dichlorophenyl)-1,3-indandione, which is most active in the anti-complement test, showed no reduction of the carrageenan-induced oedema in the rat after both oral and local application. Possibly the absence of this anti-inflammatory effect is caused by the pharmacokinetic profile of the compound.

Guinea-pig parenchymal lung strips contract after H1-receptor stimulation and membrane depolarisation with KCl. Contractions after 50 mM KCl were similar to the maximal histamine response. Treatment of lung strips with micromolar concentrations of the thiol-alkylator N-ethylmaleimide markedly affects both histamine H1-receptor mediated and 50 mM KCl-induced contractions. The H1-receptor response was only affected via a decrease in the maximal response. The response to 50 mM KCl was also inhibited after thiol-alkylation. However, H1-receptor responses appeared to be slightly more sensitive towards thiol-alkylation compared to KCl-responses. Reduction of disulphide groups with 1,4-dithiothreitol also modified the contractile responses to both stimuli. It is concluded that both thiol- and disulphide moieties play important roles in the regulation of histamine H1-receptor activity.

A series of diphenhydramine analogues have been studied with regard to their formation of a metabolic intermediate (MI) during their biotransformation in phenobarbital induced rat hepatic microsomes. The MI forms a complex with reduced cytochrome P-450. MI complexation of cytochrome P-450 may result in drug-drug interactions and/or in cumulation of the parent compound. The extent of MI complex formation could be correlated with the lipophilicity of the substrates in a parabolic manner. A hydrophobic pocket of limited dimensions in cytochrome P-450 for the N-alkyl substituent of the substrates can be assumed. Moreover our data indicate a role for the O-atom in the diphenhydramine analogues for the interaction with cytochrome P-450.

Rat cortical slices preloaded with [3H]histidine released [3H]histamine upon electrical stimulation or after depolarization with elevated K+ levels. The release was dependent on the presence of Ca2+, suggesting a neurosecretory process. Histamine has been shown to inhibit its own release mediated by an autoreceptor belonging to the H3-receptor subclass. In this study we have investigated the autoinhibition using different electrical field stimulation conditions (1, 10, 20 and 33.3 Hz). Applying electrical stimulation, the inhibition of [3H]histamine release by histamine is decreased when the stimulation frequency is elevated. When stimulated with 1 Hz histamine is able to block [3H]histamine release completely, with ap(EC50) of 8.1±0.1. At higher frequencies histamine still blocks [3H]histamine release completely, but with a lowerp(EC50).

In the present study we characterized [3H]-mepyramine binding to rat liver plasma membranes. Binding of [3H]-mepyramine proved to be of high affinity (Kd=7.7±0.4 nM) and saturable, resulting in a Bmax-value of 70.4±9.5 pmol/mg protein. However, displacement studies revealed that this binding site was different from other H1-receptor systems. The two stereoisomers of chlorpheniramine were rather ineffective in displacing [3H]-mepyramine and showed a stercospecificity in favour of thel-isomer. Also several H1-receptor agonists were not potent in displacing [3H]-mepyramine from rat liver plasma membranes. Morcover, the histamine metabolite imidazole-4-acetic acid was about as potent as the H1-agonists, whereas imidazole was even more potent. These data strongly suggest that [3H]-mepyramine labels a non-H1-receptor binding site on the rat liver plasma membrane.

The cardiac activity of the novel histamine H2-receptor agonist amthamine was investigated in a variety of isolated heart preparations from guinea pigs and humans and in the isolated rabbit aorta. Amthamine caused an increase in the sinus rate of spontaneously beating guinea-pig atria (pD2=6.72) and in the contractility of the electrically driven guinea-pig papillary muscle (pD2=6.17) and of the human atrium (pD2=5.38). In all these systems, amthamine behaved as a full agonist with a potency comparable to or slightly higher than that of histamine and 10 times higher than that of dimaprit. The positive effects of amthamine were competitively antagonized by ranitidine which had pA2 values (6.46 and 6.25 in the guinea-pig atria and papillary muscle, respectively) comparable with those calculated against histamine and dimaprit. In the isolated rabbit aorta amthamine was devoid of H1-mediated activities up to 3×10−4M. These results indicate that amthamine is a potent and selective histamine H2-receptor agonist which can be considered a valuable tool for investigating H2-receptor mediated effects in cardiac tissues.

Abstrac The effects of the H2-receptor antagonists cimetidine, ranitidine, mifentidine and three analogues of mifentidine, were studied on the spontaneously beating right atrium (H2-antagonism) and membranes of the cerebral cortex (displacement of3H-tiotidine), both obtained from the male guinea-pig. The choice of these compounds was based on preliminary experiments in which some mifentidine analogues were shown to displace tiotidine from the H2-receptor in a deviant manner. In the present study we investigated the relation between pharmacological response and receptor binding, also testing the degree of irreversible antagonism of these compounds in the atrium (functional) and cerebral cortex (binding) model. Our data indicate that a relation between the two different approaches for measuring the effect on the H2-receptor can be found, although some differences emerged as well.

In this study we investigated the role of protein kinase C (PKC) in the histamine H1-receptor mediated contraction of guinea-pig parenchymal lung strips. Lung strips contract after administration of the PKC activators phorbol-12-myristate-13-acetate (PMA) and dioctanoylglycerol. Prolonged stimulation (45 min.) with a high concentration of PMA abolished subsequent responses to PMA whereas H1-responses were only partially reduced. Administration of a PKC inhibitor (H-7) also led to an inhibition of H1-responses. Experiments with combined incubations with the PKC inhibitor H-7 and the Ca2+-entry blocker verapamil suggest that the PKC contribution to the response is possibly mediated via regulation of Ca2+ influx.

Because in some alkylenediisothioureas replacement of one of the isothiourea groups by a guanidino group resulted in a strong increase in agonistic histamine H2-activity, while replacement of the guanidino group of a potent analogue of impromidine by an isothiourea group resulted in a complete loss of histamine H2-activity, it was concluded that guanidino and isothiourea groups are not bio-isosteric for the H2-receptor.

The Kd-values of some histamine H2-active compounds, obtained from radio-ligand-binding studies on a homogenate of the guinea-pig cerebral cortex with3H-tiotidine as the labelled H2-ligand, were compared with the pA2/pD2-value of these compounds on the guinea-pig right atrium and guinea-pig isolated gastric fundus. A good correlation was found between the pKd of the H2-antagonists and their pA2 on the guinea-pig right atrium. A much poorer correlation however was obtained between the pKd of the agonists on the cerebral cortex and their pD2-values on the guinea-pig right atrium and the gastric fundus. This poor correlation between true affinity and H2-activity of the agonists might be explained by spare receptors as a much better correlation was obtained between pKd and pD2 of partial agonists.

In the present study, the effects ofβ-adrenergic receptor agonists on the hydrogen peroxide (H2O2) production in phorbol myristate acetate stimulated guinea pig alveolar macrophage suspensions have been investigated. In contrast to suggestions described in literature, our results indicate that the H2O2 production in alveolar macrophage suspensions is not mediated byβ-adrenergic receptors. Rather, it is suggested that the inhibitory effect of catecholamines in the H2O2 formation which is found after 5 minutes is the result of inhibition of the horseradish peroxidase catalysed phenol red oxidation, used as H2O2 measurement, by the catecholamines.

It has been hypothesized that bidentate hydrogen bonding plays an important role in the interaction of imidazolylphenylformamidines with the H2-receptor. The present study, in which the degree of pseudoirreversible H2-antagonism of the four isomeric butyl substituted mifentidine analogues was determined on the spontaneously beating right atrium of the male guinea-pig, lends further support to this hypothesis. In solution the EE/EZ ratio is different for the four isomeric butylated mifentidine analogues. The rank order of the percentage of E,E conformation, which favors a bidentate interaction, of the formamidine moiety parallels the rank order of pseudo-irreversible H2-antagonism.

A series of terfenadine derivatives were evaluated for enantioselectivity on histamine H1-receptors and calcium channels. Whereas H1-receptors are only sterically discriminative against the benzhydryl part of the molecules, calcium channels showed enantioselectivity to either the phenylbutyl part or the benzhydryl part provided that an appropriate lipophilicity is preserved at the chiral site. It is speculated that the hydrophilicity of the butanol moiety is responsible for the lack of stereoselectivity of terfenadine enantiomers since it drives the side chain out the stereoselective site of calcium channels, which are lipophilic. In four different test systems, (guinea-pig ileum, guinea-pig lung membranes, rat aorta and rat cortex mebranes), this series of compounds generally showed about 10 times higher activity on H1-receptors than on calcium channels. By introducing a chiral center in the different parts of the molecule we were able to increase the selectivity of an enantiomer VUF4648 to calcium channels.

The influence of histamine on nitric oxide synthase (NOS) in the development of airway smooth muscle hyperresponsiveness to histamine was investigatedin vitro. In the absence of histamine,NG-nitro-l-arginine methyl ester (l-NAME, 100 μM) had no significant effect on the basal tone. However, precontraction of the tissues with histamine (0.3 μM) resulted in a significant contractile response tol-NAME in the preparations with intact epithelium. Removal of the epithelial layer decreased the responses tol-NAME.l-arginine could partially reverse the contraction produced byl-NAME.l-NAME enhanced the maximal response to histamine, but the sensitivity of the tracheal smooth muscle to histamine was not affected. These results suggest that, in the airway, histamine can activate NOS, resulting in the release of nitric oxide. The latter may be regarded as a local negative modulator to maintain the tissue in a physiological homeostasis.