Recent findings on the pathogenesis of bronchial asthma.

In the first part of this series of papers (Székely and Pataki, 102) the pathogenesis of asthma was approached as a pathological antigen-antibody complex induced vago-vagal axon reflex. In the next part (103) the contribution of individual hormonal predisposition, the environmental and the most frequent allergizing factors have been reviewed. In the first section of this last (third) part of the review the genetic factors contributing to the asthma are surveyed. In this field a great progress has been made during the last decade, a lot of genes have been pinpointed which contribute to the heredity of the disease. In the second section of this last paper on the etiology of asthma an attempt is made to summarize the previously reviewed data and some new ones. Actually a new hypothesis is proposed that beyond the multitude of genetic, environmental and hormonal factors the underlying biochemical mechanism is simple: the disequilibrium of two functionally opposing second messenger systems in the airways: the Ca i ++ liberating PLC-PKC cascade and the Ca i ++ level reducing cAMP mediated one with preponderance of the former.

Recent findings on the pathogenesis of bronchial asthma. Part I. Asthma as a neurohumoral disorder, a pathological vago-vagal axon reflex.

The novel data on the pathogenesis of asthma are summarized in this three-part review. Its immunological background is well established but it is more than an immunological disorder. Multiple lines indicate that both peripheral and central neural mechanisms are also involved in the pathogenesis of asthma. In the present first part of the review asthma is described as vago-vagal axon reflex brought about by multiple positive feed-back mechanisms, receptor upregulation, wind-up, phenotypic switch and formation of a pathological conditioned reflex. In the coming second part the main dispositional (mostly hormonal) and external contributing factors are reviewed, while the third part deals with the role of inheritance, i.e., with gene alleles leading to enhanced production of mediators of asthma.

The "endorphin story": a subjective account.

The contribution of the researchers of IDR to some early discoveries on endorphins is recounted. Exact determination of the amino acid sequence of beta-lipotropin, discovery of the analgesic action of beta-endorphin in rats, demonstration of the partial cross-tolerance between morphine and beta-endorphin, description of the elevation of the pain threshold upon inhibition of the enzymatic breakdown of endogenous opioids by bacitracin, demonstration of inhibition of opiate tolerance and dependence by alpha-MSH and Met-enkephalin, development of the second strongest enkephalin analogue, D-Met2,Pro5-enkephalinamide, recognition of some factors conferring analgesic activity to synthetic enkephalin analogues, etc. prove that IDR has played an active role in the early history of endorphin research.

[Discovery of the analgesic action of beta-endorphin: a subjective account]. / Az endorphinok fájdalomcsillapító hatásának felfedezése: ahogy azt az elfogult farmakológus látja.

The contribution of the research workers of the Institute for Drug Research to some early discoveries in the field of endorphins is reviewed. The author takes pride in the fact the analgesic action of beta-endorphin was described in his laboratory concurrently with an American and a British group, respectively. The other main findings of his laboratory include demonstration of the cross-tolerance between beta-endorphin and morphine, description of the elevation of the pain threshold upon inhibition of the breakdown endogenous opioids by intracerebroventricular administration of bacitracin, attenuation of morphine tolerance by alpha-MSH and Met-enkephalin, pharmacological development and clinical examination of a highly potent enkephalin analogue, (D-Met2,Pro5)-enkephalinamide, coded as GYKI 14,238.

AMPA receptor antagonists, GYKI 52466 and NBQX, do not block the induction of long-term potentiation at therapeutically relevant concentrations.

The involvement of alpha-amino-3-hydroxy-5-methylizoxazole-4-propionic acid (AMPA) receptors in induction of long-term potentiation (LTP) was examined in rat hippocampal slice preparation. Using conventional extracellular recording, excitatory postsynaptic potentials (EPSPs) and population action potentials (PSs), evoked by low-frequency stimulation of the Schaffer collateral-commissural fibres, were recorded in the CA1 region. The effects of a competitive AMPA receptor antagonist, 6-nitro-7-sulfamoylbenzo(f)quinoxaline-2, 3-dione (NBQX), and that of a non-competitive blocker, 1-(4-aminophenyl)-4-methyl-7,8-methylendioxy-5H-2,3-benzodiazepine (GYKI 52466) have been examined. 0.25-0.5 microM of NBQX and 20-40 microM of GYKI 52466 did not suppress the induction of LTP. LTP was attenuated only at the highest concentrations tested (1 microM NBQX or 80 microM GYKI 52466). These in vitro concentrations, however, exceed the brain levels needed for in vivo anticonvulsant action. Furthermore, even at the highest concentrations both compounds suppressed only the expression but not the induction of LTP. Namely after their washout LTP reappeared. Thus, at pharmacologically relevant concentrations these AMPA receptor antagonists apparently do not suppress LTP, a cellular mechanism underlying memory formation. These experiments suggest that in clinical practice AMPA receptor blockade may have some advantage over N-methyl-D-aspartate receptor antagonism, which is accompanied by severe memory impairment.

Differential modulation of the GYKI 53784-induced inhibition of AMPA currents by various AMPA-positive modulators in cerebellar Purkinje cells.

The effects of various (S)-alpha-amino-3-hydroxy-5-methyl-4-izoxazole-propionate (AMPA) receptor modulators on AMPA-induced whole-cell currents were compared in isolated rat cerebellar Purkinje cells. The positive modulators, aniracetam, cyclothiazide, 1-(1, 3-benzodioxol-5-ylcarbonyl)-piperidine (1-BCP), and 1-(quinoxaline-6-ylcarbonyl)-piperidine (BDP-12), dose-dependently potentiated the steady-state component of AMPA currents. The negative modulator, (-)1-(4-aminophenyl)-4-methyl-7, 8-methylenedioxy-4,5-dihydro-3-methylcarbamoyl-2,3-benzodiazepine (GYKI 53784), dose-dependently suppressed AMPA responses. Its concentration-response curve was shifted to the right in a parallel fashion by all positive modulators, indicating a competitive type of interaction. However, the relative potencies of the positive modulators were different with regard to the enhancement of AMPA responses and the reversal of GYKI 53784-induced inhibition, respectively. It is supposed that positive modulators act at multiple allosteric sites and that they interact with GYKI 53784 at only one of these sites.

Diprotin A, an inhibitor of dipeptidyl aminopeptidase IV(EC 3.4.14.5) produces naloxone-reversible analgesia in rats.

The dipeptidyl aminopeptidase IV (DP IV) inhibitor Diprotin A produces a full, dose-dependent, short-lasting and naloxone-reversible analgesia in the rat tail-flick test when given intracerebroventricularly, with an ED50 of 295 nmol/rat but it has no direct opioid agonist activity in the longitudinal muscle strip of guinea-pig ileum bioassay. Two of the potential DP IV substrates, morphiceptin and endomorphin 1, identified recently in bovine brain were also analgesic given by similar route. The action of endomorphin 1 was more potent (ED50 = 7.9 nmol/rat) and slightly but significantly more sustained than that of Diprotin A. Diprotin A neither potentiated nor prolonged the effect of a marginally analgesic dose of endomorphin 1. The distinct time course and the lack of potentiation indicate that in the analgesic effect of Diprotin A in rats the protection of a brain Tyr-Pro-peptide other than endomorphin 1 is involved.

Selective interaction of homophtalazine derivatives with morphine.

Homophtalazines show specific binding sites in the nigrostriatal system and to find their target of action the interactions between these derivatives, nerisopam and girisopam, and chlorpromazine, chlordiazepoxide and morphine were assessed. The compounds did not influence the chlorpromazine induced decrease in motility and catalepsy, nor did they alter the antiaggressive and anticonvulsive action of chlordiazepoxide. However, nerisopam and girisopam augmented the agonist potency of morphine to induce catalepsy or analgesia; they also altered the opioid antagonist potency of naloxone. The naloxone-induced decrease in sucrose consumption in drinking water was augmented by nerisopam and girisopam. It is suggested that a possible target of action of homophtalazines is the opioid signal transduction.

Antiapoptotic effect of benzyloxycarbonyl-aspartyl-(beta-tertier-butyl ester)-bromomethylketone (Z-D(OtBu)-Bmk), an intermediate of interleukin-1 beta converting enzyme inhibitors.

The effect of several interleukin-1 beta converting enzyme (ICE) inhibitors on apoptosis was examined. The ICE inhibitors tested were peptide aldehydes such as ethyloxycarbonyl-Ala-Tyr-Val-Ala-Asp-aldehyde (Etoco-AYVAD-CHO), acetyl-Tyr-Val-Ala-Asp-aldehyde (Ac-YVAD-CHO), benzyloxycarbonyl-Val-His-Asp-aldehyde (Z-VHD-CHO), a tetrapeptide chloromethylketone, acetyl-Tyr-Val-Ala-Asp-chloromethylketone (Ac-YVAD-Cmk) and their common intermediate benzyloxycarbonyl-Asp-(beta-tertier-butyl ester)-bromomethylketone (Z-D(OtBu)-Bmk). Apoptosis was induced with several chemical agents conventionally used for this purpose in THP-1, L929, NB-41A3 cell lines and mouse thymocytes. DNA fragmentation during apoptosis was measured by conventional gel electrophoresis and ELISA. The cell morphology was examined by hematoxylin/eosin staining method. Cell viability was also monitored by MTT assay. Contrary to expectations, the peptide aldehydes listed above and Ac-YVAD-Cmk, known as highly specific ICE inhibitors, did not inhibit the apoptosis of these cell types. However, Z-D(OtBu)-Bmk, which had no relevant inhibitory activity on ICE, potently blocked the DNA fragmentation in THP-1 cells and thymocytes whichever of the inducing agents was used. In the other two cell lines Z-D(OtBu)-Bmk was inactive. The apoptotic cell morphology was also inhibited by Z-D(OtBu)-Bmk. Nevertheless, Z-D(OtBu)-Bmk failed to prevent the loss of mitochondrial activity and the cell destruction in the late phase of apoptosis. These data suggest that ICE is not involved in the apoptotic cell death induced by chemical agents. Thus, Z-D(OtBu)-Bmk, a common intermediate of some ICE inhibitors, may be a useful antiapoptotic agent for studying the early events of apoptosis in some cell types.

Apparent antinociceptive and anti-inflammatory effects of GYKI 52466.

GYKI 52466 (1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine) was examined in a battery of analgesia and anti-inflammatory tests in rats and mice, respectively. Its 3-N-acetyl (GYKI 53773) and 3-N-methylcarbamoyl (GYKI 53784) derivatives were also examined in some assays. These 2,3-benzodiazepines, known as prototypic non-competitive antagonists of AMPA receptors, showed a peculiar profile in some routinely used antinociceptive tests. They were found fairly potent in rat tail flick and mouse phenylquinone writhing assays but the dose-response curves were rather shallow as compared to that of morphine. Their action is stereoselective, i.e., the (+) isomers were found inactive, in agreement with the previous in vitro studies. Their antinociceptive effect could not be reversed by naloxone and the GYKI compounds did not potentiate significantly the morphine-induced analgesia. In the mouse hot plate assay the 2,3-benzodiazepines were active only in doses inducing visible motor incapacitation. In rats, GYKI 52466 weakly reduced the hypersensitivity accompanying acute carrageenan edema. However, it potently inhibited the hyperalgesia during Freund adjuvant-induced chronic arthritis. In the latter assay GYKI 52466 also attenuated the body weight loss without altering the paw edema. The present findings confirm reports in the literature which indicate AMPA receptors may contribute to certain forms of pathological hyperalgesia, e.g., to that detectable in inflamed tissues.

GYKI-46 903, a non-competitive antagonist for 5-HT3 receptors.

The effects of GYKI-46 903 ((+)endo-4-propionyloxy-6-(4-fluorophenyl)-1-azabicyclo [3.3.1]non-6-ene HCl), on 5-HT3 receptors have been studied and compared with ondansetron in peripheral organs in vitro and in vivo, and in a receptor binding assay in membranes prepared from rat cerebral cortex. GYKI-46 903 was found to be a non-competitive antagonist at 5-HT3 receptors present in non-stimulated longitudinal muscle strip of guinea-pig ileum (pD2' against serotonin = 5.54), and also in 5-methoxytryptamine-pretreated electrically stimulated ileal preparations (pD2' against serotonin = 5.26). On the contrary, ondansetron was found to be a competitive antagonist for 5-HT3 receptors; the pA2 value against serotonin was 7.40 in non-stimulated ileum, and it was 7.08 in electrically stimulated ileal preparation pretreated with 5-methoxytryptamine. In displacement studies, the pIC50 values of GYKI-46 903 and ondansetron against [3H]granisetron binding to rat cerebral cortex membranes were 6.91 and 8.58 respectively. GYKI-46 903, when administered by intravenous infusion, antagonized the decrease in heart rate evoked by serotonin (Bezold-Jarisch reflex) in anaesthetized rats, and the maximal reversal was less than 50%. This was in striking contrast with ondansetron, which, after intravenous injection, completely antagonized the serotonin-induced bradycardia with an ID50 value of 3.28 ug/kg. These data classify GYKI-46 903 as a non-competitive antagonist for 5-HT3 receptors.

Effect of peptide aldehydes with IL-1 beta converting enzyme inhibitory properties on IL-1 alpha and IL-1 beta production in vitro.

Tripeptide and pentapeptide aldehydes as substrate-base inhibitors of cysteine proteases were designed in our laboratory for the inhibition of interleukin-1 beta converting enzyme (ICE), a recently described cysteine protease responsible for the processing of IL-1 beta. The biological effectivity of the peptide aldehydes was studied in THP-1 cells and human whole blood. The released and cell-associated IL-1 alpha and IL-1 beta levels were determined by ELISA from the supernatants and cell lysates, respectively. The total IL-1 like bioactivity was assayed by the D10 G4.1 cell proliferation method. The tripeptide aldehyde (Z-Val-His-Asp-H) and pentapeptide aldehyde (Eoc-Ala-Tyr-Val-Ala-Asp-H) significantly reduced IL-1 beta levels in the supernatants in relatively high concentrations (10-100 microM), but the IL-1 alpha release was unaffected by these peptides. However, a considerable decrease in the cell-associated IL-1 beta and IL-1 alpha levels was observed. N-terminal extension of the tripeptide aldehyde yielded even more potent inhibitors. Amino acid substitution at the P2 position did not cause considerable changes in the inhibitory activity. The peptide aldehydes suppressed the IL-1 beta production in a reversible manner, whereas dexamethasone, a glucocorticoid, had a prolonged inhibitory effect. The inhibitory effect of these peptides and that of dexamethasone appeared to be additive. These findings indicate that these peptide aldehydes might be used as IL-beta inhibitory agents in experimental models in which IL-1 beta is a key mediator or ICE is implicated.

Measurement and drug induced modulation of interleukin-1 level during zymosan peritonitis in mice.

The time-course and pharmacological modulation of interleukin-1 (IL-1) production were investigated during zymosan induced peritonitis in mice. IL-1 alpha liberation was assessed by specific immunoassay (ELISA) and the IL-1 like bioactivity (sensitive to both alpha- and beta-forms of IL-1) was measured by a sensitive bioassay (D10G4.1 costimulation). I.p. injection of zymosan induced significant IL-1 release into the peritoneal exudate. The level peaked at 4 h and by 24 h dropped below the detection limit in both assays. The effects of the prototypical antiinflammatory drugs indomethacin (IND) and dexamethasone (DEX) and that of IX 207-887, a compound which has been reported to interfere primarily with IL-1 production, were also tested. DEX and IX 207-887 dose-dependently decreased the immunoassayable IL-1 alpha level and the IL-1 like bioactivity as well. However, IND had no suppressant effect. Thus, the data obtained by immunoassay and bioassay correlated well proving the suitability of zymosan peritonitis model for the examination of IL-1 production in experimental inflammation.

Measurement of interleukin-1 liberation in zymosan air-pouch exudate in mice.

The aim of the present study was to examine whether interleukin-1 (IL-1) production is involved in the pathology of inflammation induced by zymosan in the air-pouch of mice. For this reason the IL-1 alpha level was determined in the air-pouch exudate by specific ELISA kit 4, 24, 48 h and 4-8 days after zymosan injection into preformed subcutaneous air-pouches in mice. Concurrently, some conventional parameters such as volume of exudate, its protein content and the total leukocyte count were also measured. The IL-1 alpha level reached its maximum 24 h after zymosan administration and remained elevated throughout the 8-day observation period. Exudation, accumulation of leukocytes and protein also were maximal on day 8. The effects of some anti-inflammatory agents have also been examined. Orally administered dexamethasone induced a dose-dependent reduction in IL-1 alpha, whereas indomethacin and IX 207-887, an IL-1-release inhibitor, failed to reduce the IL-1 alpha content in this model.

Effects of caramiphen and phencyclidine alone and in combination on behavior in the rat.

Because dextromethorphan (DM) has been shown to inhibit the locomotor stimulant effects of phencyclidine (PCP), this study explored further the possible interaction between drugs acting on DM and PCP receptor sites. Caramiphen, an antitussive that binds with high affinity to the DM site, was injected (IP) alone (15-120 mg/kg) or at two doses (15 or 60 mg/kg) 15 min before a challenge dose of PCP (1.25-20 mg/kg). Caramiphen alone dose-dependently increased ataxia, increased stereotypy, and had no effect on locomotor activity. PCP alone dose-dependently increased ataxia, stereotypy, and locomotor activity, the latter showing an inverted U-shaped function. At both pretreatment doses, caramiphen enhanced locomotor activity and stereotypy when combined with low PCP doses but decreased these behaviors at high PCP doses. Caramiphen produced a dose-dependent additive effect on ataxia when combined with all PCP doses. It was concluded that, although caramiphen, like DM, inhibited the locomotor stimulant effects of selected doses of PCP, that interaction appeared to be due to other behaviors (e.g., ataxia/stereotypy) elicited by caramiphen combined with high doses of PCP. This study underscored the importance of using full dose ranges of PCP when attempting to antagonize its behavioral effects with other drugs.

[The mechanism of the anti-arrhythmia action of opioid receptor agonists and antagonists]. / K mekhanizmu antiaritmicheskogo deistviia agonistov i antagonistov opioidnykh retseptorov.

Enkephalins were injected intravenously at a dose of 0.1 mg/kg 15 minutes or 6 hours before adrenalin or CaCl2 injection. Enkephalins were reported to prevent adrenal ventricular extrasystoles but not to influence CaCl2-induced dysrhythmias. Maximum antiarrhythmic effect of enkephalins was demonstrated 6 hours later after the intravenous injection. Naloxone at a dose of 2 mg/kg and morphine at a dose of 1.5 mg/kg prevent adrenal arrhythmias 15 min and 6 hours later after injection. We believe that peripheral delta opiate receptors activation by enkephalins as well as the blockade of non-identified opiate receptors by naloxone prevent arrhythmias.

A new assay for antiphlogistic activity: zymosan-induced mouse ear inflammation.

A new model of local inflammation has been developed: intradermal zymosan-induced mouse ear edema. The symptoms of inflammation induced by injecting zymosan into one of the ears were followed up for 72 h. The ear edema and the local accumulation of polymorphonuclear leukocytes' (PMN) marker enzyme, myeloperoxidase (MPO), were determined. Edema peaked at 4-6 h, while MPO activity peaked at 24 h after zymosan application. The correlation between inflammatory response and concentration of zymosan was also tested. Of the various concentrations tested, 1% suspension has been found optimal. Anti-inflammatory drugs and mediator antagonists were examined in order to establish the selectivity and sensitivity of the assay. A glucocorticoid (dexamethasone), two cyclooxygenase inhibitors (indomethacin, piroxicam) and an interleukin-1 (IL-1) release inhibitor (IX 207-887, Sandoz) all reduced edema and MPO activity as well. However, a lipoxygenase inhibitor (phenidone), a serotonin receptor antagonist (methysergide) and H1 and H2 receptor antagonists (clemastine and cimetidine, respectively) all failed to inhibit the reaction.

Induction of phencyclidine-like behavior in rats by dextrorphan but not dextromethorphan.

The behavioral effects of dextromethorphan (DM), dextrorphan (DO) and phencyclidine (PCP) were compared in rats. DO (15-120 mg/kg) was similar to PCP (1.25-20 mg/kg) in inducing dose-dependent locomotor hyperactivity, stereotypy and ataxia. DM (15-120 mg/kg) induced moderate hyperactivity only at the higher doses about 45 min after treatment. DM and DO modified the locomotor facilitation induced by 10 mg/kg PCP in opposite directions. Pretreatment with DO facilitated, whereas DM dose-dependently inhibited PCP-elicited hyperactivity. Although the metabolism of DM in rats is unknown, the recently reported abuse of DM in humans may occur by its conversion to DO in the organism, i.e., to a metabolite which produces PCP-like effects.

Naloxone-insensitive modulation of gastric acid output by [D-Met2,Pro5]enkephalinamide in rats.

Studies from our laboratory have previously shown that two opioid agonists (morphine and [D-Met2,Pro5]enkephalinamide) aggravate, whereas naloxone inhibits cold-restraint stress-induced ulceration in rats. In the present study the effects of these substances were examined using the Shay-model. Contrary to expectations, both naloxone and the opioid agonists decreased gastric acid output. Naloxone in combination with either opioid agonist failed to reverse their inhibitory action. Thus the secretory activity of the stomach may be modulated by opioids in both naloxone-reversible and irreversible ways.