Evaluation of the receptor selectivity of the H3 receptor antagonists, iodophenpropit and thioperamide: an interaction with the 5-HT3 receptor revealed.

1. In the present study we evaluated the receptor selectivity of the potent histamine H3 receptor antagonist, iodophenpropit (IPP) in comparison with the prototype antagonist, thioperamide. 2. IPP proved to be a potent competitive H3 receptor antagonist as measured against (R)-alpha-methylhistamine-induced inhibition of electrically-evoked contractions of the guinea-pig jejunum (pA2 = 9.12 +/- 0.06, Schild slope: 1.0 +/- 0.1, n = 8). In the same assay, thioperamide was slightly less potent (pA2 = 8.9 +/- 0.2). 3. In radioligand binding studies, IPP showed a high affinity for the H3 receptor. Displacement of [125I]-IPP binding to rat cortex membranes by unlabelled IPP resulted in a Ki value of 0.97 +/- 0.06 nM (n = 3). In contrast, IPP showed only a weak affinity for the histamine H1- and H2 receptor. Displacement of [3H]-mepyramine and [125I]-iodoaminopotentidine binding to respectively guinea-pig H1- and human H2 receptors by IPP resulted in Ki values of 1.71 +/- 0.32 microM (n = 3) and 2.28 +/- 0.81 microM (n = 3). For thioperamide the affinities for the H1-, H2- and H3 receptor were respectively > 10 microM, > 10 microM and 4.3 +/- 1.6 nM (n = 7). 4. Testing IPP and thioperamide in 39 different receptor binding assays revealed that IPP showed relatively high affinity for the 5-hydroxytryptamine 5-HT3 receptor (Ki = 11 +/- 1 nM, n = 3), the alpha 2-adrenoceptor (Ki = 120 +/- 5 nM, n = 3) and the sigma receptor (Ki = 170 +/- 70 nM, n = 3). Thioperamide showed relatively high affinity for the 5-HT3 receptor (Ki = 120 +/- 30 nM, n = 3) and the sigma receptor (Ki = 180 +/- 90 nM, n = 3). 5. Due to the low density of histamine H3 receptors in the brain, the interaction of IPP with the 5-HT3-, the alpha 2- and the sigma receptor might interfere with [125I]-IPP binding to rat cortex membranes. Yet, in this preparation [125I]-IPP binding was not influenced by ondansetron, yohimbine or haloperidol. The interaction with the 5-HT3 receptor was not restricted to IPP or thioperamide, but was alsofound with other H3 receptor antagonists. The potent H3 receptor agonist imetit, a compound belongingto the same chemical class of IPP, also interacted with the 5-HT3 receptor (Ki = 240 +/- 40 nM). In contrast,histamine or the H3 receptor agonist, (R)-a-methylhistamine showed no affinity for the 5-HT3 receptor.7 In the guinea-pig isolated ileum, imetit evoked concentration-dependent contractions, resulting in apD2 value of 4.72 +/- 0.03 (n = 9). The contractions were antagonized by ondansetron, yielding a pA2 valueof 7.1 +/- 0.1 (n = 9). Similarly ondansetron antagonized the contractions evoked by the 5-HT3 receptoragonist, 2-methyl-5-HT with a pA2 value of 7.3 +/- 0.1 (n = 4). IPP and thioperamide did not mimic 2-methyl-5-HT but non-competitively inhibited the 2-methyl-5-HT-induced contractions of thispreparation.8 In an in vivo model for 5-HT3 activity, the Von Bezold Jarisch reflex, thioperamide showedantagonism in low dosages, which correlated well with the affinity for the 5-HT3 receptor site. Yet, athigher dosages no further 5-HT3 receptor antagonism was observed. For IPP no 5-HT3 receptor activitycould be observed in vivo.9 In the present study we showed that many H3 receptor compounds, that are regarded as highlyselective (including the prototype drug, thioperamide), also interact with the 5-HT3 receptor, albeit athigher drug concentrations.Keywords.: Histamine H3-receptor; iodophenpropit; thioperamide; receptor selectivity; 5-hydroxytryptamine 5-HT3 receptor;guinea-pig intestine; rat brain; Von Bezold Jarisch reflex

Cyclic nucleotides in human macrophages: effects of atrial natriuretic factor and nitroprusside on cGMP and cAMP production.

Atrial natriuretic factor (ANF, 10(-7) M) and sodium nitroprusside (SNP, 10(-5)-10(-3) M) stimulated cGMP production in human peritoneal macrophages (HPM). This suggests the existence of two separate forms of guanylate cyclase in HPM, e.g. the receptor-related form by ANF and the soluble form by SNP. In parallel with the rise in cGMP levels, both agents provoked a decrease in cAMP levels. Increasing the concentration of the phosphodiesterase inhibitor IBMX (0.2 mM to 1.0 mM) in the incubation media resulted in a significantly greater rise in cGMP levels which was accompanied by a profound decrease in cAMP levels. ANF did not exert any direct or GTP-related effect on cAMP production, which is in contrast to its action in other tissues. These results suggest that cAMP levels can be modulated through a cGMP signal, most likely at the production level. Results also give substantial evidence for the presence of a ANF receptor site on human peritoneal macrophages.

Decrease of beta-endorphin in the brain of rats following nitrous oxide withdrawal.

beta-Endorphin levels in the whole rat brain were not changed during acute (25 min) or chronic (48 h) exposure of rats to N2O. However, a significant decrease of beta-endorphin was found in the whole brain, brain stem and subcortex during the withdrawal from chronic exposure to N2O. It has been suggested that decrease of beta-endorphin levels during N2O withdrawal could be ascribed to unspecific stress accompanying drug withdrawal. Decrease of central beta-endorphin during N2O withdrawal might have a significant modulatory effect on transmitter balance, neuronal excitability and corresponding withdrawal behaviour. Furthermore, the decrease of beta-endorphin levels in the whole brain during N2O withdrawal might contribute to the postanaesthesia N2O-excitatory syndrome in humans. This might explain the known therapeutic effect of the opioid drug, meperidine on the excitatory N2O withdrawal phenomena during recovery from N2O anaesthesia in man.

GTP-related difference in cyclic AMP production between resident and inflammatory human peritoneal macrophages.

In macrophages cyclic AMP (c-AMP) plays an important role in regulating many activities such as phagocytosis, migration and tumoricidal activity. High intracellular levels of c-AMP are negatively correlated with these activities. In earlier studies we have shown that c-AMP levels in inflammatory human peritoneal macrophages (IM) were markedly lower when compared to levels in resident macrophages (RM). This is in line with the fact that c-AMP down-regulates macrophage activity. To our knowledge no data are available on the mechanism underlying the difference in c-AMP production between RM and IM. In this study the difference in c-AMP production between RM and IM has been investigated on the level of receptor and G-protein-related mechanisms. Macrophage membranes were incubated with different agents i.e. prostaglandin E2 (PGE2), prostacyclin I2 (PGI2), isoprenalin (ISO) and sodium fluoride (SF). Additionally, the capacity of IM and RM to hydrolyse quanosine triphosphate (GTP) was measured. Only in the presence of GTP (10(-4) M) could the c-AMP difference be detected (RM = 51 +/- 4.4 pmol/mg protein/min +/- S.E., n = 22, IM = 32.8 +/- 5.2 pmol/mg protein/min +/- S.E., n = 10, p less than 0.01). After receptor stimulation with PGE2, PGI2 and ISO, c-AMP levels increased to the same extent in both IM and RM with no effect on the GTP-related difference. After SF stimulation, c-AMP levels in RM and IM increased to the same level (RM = 63 +/- 8 pmol/mg protein/min, n = 14, IM = 58 +/- 11 pmol/mg protein/min, n = 13).(ABSTRACT TRUNCATED AT 250 WORDS)

Atriopeptins and nitroprusside provoke opposite changes in cGMP and cAMP levels in human macrophages.

Atrial natriuretic factor (ANF, 10(-7) M) and, even more potently, sodium nitroprusside (SNP, 10(-5)-10(-3) M) stimulated cGMP formation in human peritoneal macrophages. This suggests that the two forms of guanylate cyclase, the particulate form stimulated by ANF and the soluble form activated by SNP, coexist in this cell type. A fall in cAMP levels in parallel with the rise of cGMP levels provoked by ANF and SNP was noticed that was amplified by an increase in the concentration of the phosphodiesterase inhibitor, IBMX. Our finding that ANF, contrary to its action in other tissues, was unable to exert direct inhibitory effects on the adenylate cyclase activity in isolated macrophage membranes, together with the observation that SNP was able to mimic the effect of ANF on cAMP levels indicates that the cAMP-lowering effect of ANF is most likely mediated through the cGMP signal.

Antigen challenge modifies the cyclic AMP response of inflammatory mediators and beta-adrenergic drugs in alveolar macrophages.

Adenylate cyclase activity was determined in alveolar macrophages (AMs) obtained from bronchoalveolar lavage (BAL) fluids of naive and antigen-challenged guinea pigs. After the anaphylactic reaction in ovalbumin-sensitized guinea pigs, the basal levels of cyclic AMP in AMs were significantly increased compared to the levels in naive AMS (1.87 +/- 0.22 versus 5.26 +/- 0.45 pmol cyclic AMP/5.10(6) cells). Prostaglandin E2 (PGE2), prostacyclin (DC-PGI2), histamine, isoprenaline and salbutamol stimulated adenylate cyclase activity more effectively in AMs obtained from sensitized guinea pigs after the booster injection compared to AMs obtained from non-treated animals. Moreover, DC-PGI2 and histamine, which were hardly able to induce a rise in cyclic AMP levels in naive AMs, become effective activators in AMs obtained after antigen challenge (100 and 60% increase in the response, respectively). Using selective receptor ligands, we have shown that beta 2-adrenoceptors and H2-subtype histamine receptors are functionally coupled to macrophage adenylate cyclase activity. The present data indicate that sensitization does not affect the configuration of the receptor on the outer membrane (no change in affinity constants), but affects other parts of the transmembrane signal system leading to the intracellular production of cyclic AMP (e.g. regulatory binding proteins or increases in the number of receptors).

Interaction between PAF-acether and drugs that stimulate cyclic AMP in guinea-pig alveolar macrophages.

The PAF-acether (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine)-induced arachidonate release from alveolar macrophages was significantly reduced by prostaglandin E2 (PGE2) and by the beta-adrenoceptor agonist salbutamol. In addition, PAF-acether markedly reduced the increase in intracellular cyclic AMP (cAMP) concentrations induced by PGE2 and salbutamol. Our data indicate an inverse relationship between intracellular cAMP levels and free arachidonate availability in alveolar macrophages treated with PAF-acether. A rise in intracellular cAMP therefore represents an important alternative route for controlling the effects of PAF-acether and the resulting inflammatory alterations in the respiratory system.

Linoleic and dihomo-gamma-linolenic acids modulate granuloma growth and granuloma macrophage eicosanoid release.

We have already demonstrated that arachidonic acid (AA) inhibits carrageenin-induced granuloma growth in vivo and that this effect is related to an increase in prostaglandin E2 formation. As prostaglandin E1 has been shown to be more effective in inhibiting granuloma growth than prostaglandin E2 we investigated the effect of linoleic acid (LA) (18:2 omega 6) and dihomo-gamma-linolenic acid (DHGLA) (20:3 omega 6), potential precursors of prostaglandin E1, in this model. LA and DHGLA inhibited the development of carrageenin-induced granulomas in the rat when injected locally. Both fatty acids (FA) stimulated the release of prostaglandin (PG) E1 from granuloma macrophages (M phi) in vitro, DHGLA being most effective. LA had little effect on the release of PGE2, 6 keto PGF1 alpha, the stable product of prostacyclin (PGI2) or thromboxane (Tx) B2, the stable metabolite of TxA2. DHGLA had no effect on the release of 6 keto PGF1 alpha, but inhibited PGE2 and, to a lesser extent, TxB2 synthesis.

Infectious-inflammatory changes in cyclic AMP levels and in their regulation by prostaglandins in human peritoneal macrophages.

Peritoneal macrophages of renal patients on continuous ambulatory peritoneal dialysis (CAPD) have been collected when CAPD was without complications, during an intercurrent infectious peritoneal inflammation and after recovery. Levels of cyclic AMP, release of cyclo-oxygenase metabolites, and responsiveness, in terms of cyclic AMP elevation, to either PGE2 or to DC-PGI2 (a stable analogue of PGI2) were examined. Peritoneal inflammation was associated with a sharp drop in cyclic AMP, which was restored after recovery. Production of TXA2, PGI2 and PGE2 parallelled the direction of changes in cyclic AMP levels, except, that release of PGE2 entirely failed to recover. Macrophages during the uncomplicated stage of CAPD proved more responsive to DC-PGI2 than to PGE2. During inflammation the cells displayed a marked increase in sensitivity towards PG stimulation. Improved sensitivity was more pronounced with PGE2 than with DC-PGI2 and so the original difference between responsiveness of the cells to the PGs was abolished. Several findings are compatible with the view that endogenous PGI2 governs the cyclic AMP levels in human non-inflammatory peritoneal macrophages. However, during infectious-inflammation the cells undergo changes which render a reduced production of PGI2 insufficient to explain the drop in cyclic AMP.

Arachidonic acid, an in vivo inhibitor of carrageenin induced granulomas in the rat.

Arachidonic acid (AA) injected locally into carrageenin/sponge granulomas, but not if given orally, inhibited granuloma growth. Granuloma macrophage (M0) infiltration was inhibited, and prostaglandin E2 (PGE2) synthesis (ng/100 mg granuloma dry weight) stimulated, by AA treatment. M0 adenosine 3',5'-cyclic monophosphate (cAMP) levels and granuloma exudate volume were not affected. Granuloma M0s incubated in vitro with arachidonic acid synthesised thromboxane B2 (Txb2), 6-ketoprostaglandin F1 alpha (6-ketoPGF1 alpha), and preferentially, PGE2. The AA inhibition of granuloma growth was possibly mediated via the synthesis of PGE2.

Continuous monitoring of prostacyclin production by the isolated, intact, rat aorta using a bioassay technique.

Isolated rat aortae were perfused with Krebs buffer in vitro and the synthesis of prostacyclin-like material (PGI2-L) was continuously monitored by measuring the contraction of a superperfused rat stomach strip exposed to the aortic perfusate. PGI2-L release was high after initiation of the aorta perfusion but then gradually declined and stabilized at a basal rate of production that was maintained for at least 180 min. Levels of 6 keto prostaglandin F1 alpha(6ketoPGF1 alpha), the stable breakdown product of PGI2, in the aortic perfusate reflected the changes in biological activity. The concentration of PGE2 in the aortic perfusate remained constant throughout the experiment while the level of thromboxane (Tx)B2 (the stable product of TxA2) decreased with time to below the level of detection of the radioimmunoassay (RIA) used. All biological activity was abolished by heating the aortic perfusate for 30 min at 37 degrees C, while perfusing with 30 microM indomethacin inhibited aorta PGI-L formation. Analysis (by linear regression) of the relationship between PGI2-L formation and the body weight or age of the animals used revealed that PGI2-L synthesis was better related to body weight (r2 = 0.90) than age (r2 = 0.75).

Regulation of prostaglandin E2 receptors in vivo by dietary fatty acids in peritoneal macrophages from rats.

Groups of rats were pretreated with 4-week diets containing 12.5% corn oil or linseed oil. At the end of this period peritoneal macrophages were elicited and isolated. These cells were used for binding experiments with 3H-PGE2 and for estimation of prostaglandin-stimulated cAMP production. Specific binding of 3H-PGE2 was saturable, reversible, protein-dependent, and correlated with stimulation of cAMP production, indicating that specific binding referred to receptor binding. PGE1 and PGI2 were far less effective than PGE2 in competition of binding with 3H-PGE2, indicating receptor selectivity for PGE2. Scatchard analysis of the specific binding data revealed a high affinity component (Kd 17 nM) and low affinity component. The total number of high- and low-affinity binding sites, respective Kd values, and PG stimulation of cAMP production of cells from rats fed the linseed oil diet were comparable to controls. The corn oil diet, however, resulted in a twofold increase in total number of high- and low-affinity binding sites, while respective Kd values were unchanged. This enhancement of binding capacity could be explained by an increased density of binding sites on the cells, and may itself be responsible for the increased sensitivity of the macrophages in this diet group for PG-stimulated cAMP production. The data suggest a regulatory mechanism at the receptor level and are discussed in terms of possible altered bioavailability of arachidonic acid-derived PGE2.

Cyclic AMP levels and their regulation by prostaglandins in peritoneal macrophages of rats and humans.

Basal levels of cyclic AMP and their alterations following stimulation by prostaglandins have been examined in rat peritoneal macrophages and in such cells of humans with renal disease on continuous ambulatory peritoneal dialysis (CAPD). Resident cells of rats contained more cyclic AMP than elicited macrophages, but the responsiveness to either PGE2 or DC-PGI2 (a stable synthetic analogue of PGI2) was higher with elicited than with resident cells. However, both kinds of peritoneal macrophages of rats were, in terms of cyclic AMP elevation, more sensitive to stimulation by PGE2 than by DC-PGI2. The CAPD macrophages of humans were elicited cells. The unstimulated levels of cyclic AMP in these human macrophages were much higher than those in elicited rat cells. Furthermore the human macrophages proved more sensitive to stimulation by DC-PGI2 than by PGE2. The reversed sensitivity, in comparison with rat cells, reflects the utterly poor effects of PGE2 in the human macrophages. The distinction in responsiveness to PGE2 and DC-PGI2 of the rat macrophages is compatible with the earlier reported distribution of and affinity to receptor binding sites of these PGs in the rat cells. The findings with the human macrophages suggest, however, that in these cells either the distribution of specific binding sites or the affinity of the two PGs to such sites might be substantially different from that of rats.

Antiinflammatory aspects of exogenous arachidonic acid.

Resident rat peritoneal macrophages (M0) were more active in metabolising exogenous arachidonic acid (AA) to prostaglandin E2 (PGE2) than starch elicited cells. Basal levels of cAMP were lower in elicited macrophages than in resident cells but the adenyl cyclase of elicited macrophages was much more readily stimulated by cyclooxygenase products formed from exogenous AA than resident cells. AA injected into carrageenin sponge granulomas reduced the severity of the inflammation in a dose related manner.