Tissue microarray analysis of beta-catenin in colorectal cancer shows nuclear phospho-beta-catenin is associated with a better prognosis.

PURPOSE: Beta-catenin is involved in homotypic cell-cell adhesion and the wnt signaling pathway. Deregulation of beta-catenin levels, caused in part by mutations of the adenomatous polyposis coli gene, is thought to play a role in the development of colorectal and other cancers. To further elucidate their roles, the expression pattern of beta-catenin and phosphospecific beta-catenin was correlated with clinical outcome in a series of patients with colorectal cancer. EXPERIMENTAL DESIGN: Immunohistochemical analysis of a tissue microarray with 650 colorectal cancer specimens was performed to study the expression and subcellular localization of beta-catenin and phosphospecific beta-catenin. These results were correlated with other clinicopathological factors and with overall survival. RESULTS: The majority of cancers retained some degree of beta-catenin membranous staining, whereas cytoplasmic or nuclear expression was seen in 42.5% and 20.4% of specimens, respectively. Phospho-beta-catenin showed nuclear staining in 9.5% of specimens, and there was no apparent membranous or cytoplasmic staining. There was no significant association between beta-catenin or phospho-beta-catenin and grade or stage. However, there was a positive correlation between beta-catenin and phospho-beta-catenin (P = 0.039), with phospho-beta-catenin representing a subset of nuclear beta-catenin. Patients with nuclear expression of beta-catenin did not have an altered survival compared with those that did not (P = 0.5611). Nuclear expression of phospho-beta-catenin, however, was associated with an improved survival (P = 0.0006). In multivariate analysis, only stage and phospho-beta-catenin were independently predictive of overall survival (P < 0.001 and P = 0.0034, respectively). CONCLUSIONS: These findings support a role for beta-catenin overexpression in colorectal tumorigenesis and provide initial evidence that phospho-beta-catenin may be a marker for improved overall survival independent of stage and grade.

Amplification of tissue by construction of tissue microarrays.

Tissue microarrays are a method of relocating tissue from conventional histologic paraffin blocks in a manner that tissue from multiple patients or blocks can be seen on the same slide. This is done by using a needle to biopsy a standard histologic section and placing the core into an array on a recipient paraffin block. This technique allows maximization of tissue resources by analysis of small core biopsies of blocks, rather than complete sections. Using this technology, a carefully planned array can be constructed using cases from pathology tissue block archives, and a 20-year survival analysis can be done on a cohort of 600 or more patients using only a few microliters of antibody in a single experiment. Furthermore, this cohort can be analyzed thousands of times with different reagents as a result of judicious sectioning of the array block. This review describes this process and discusses the issues of representative sampling in heterogeneous lesions, the issue of antigen preservation, and some technical strategies and methods of array construction. In summary, this technique can provide a highly efficient, high-throughput mechanism for evaluation of protein expression in large cohorts. It has the potential for allowing validation of new genes at a speed comparable to the rapid rate of gene discovery afforded by DNA microarrays.

Tissue microarray: a new technology for amplification of tissue resources.

Tissue microarrays are a method of harvesting small disks of tissue from a range of standard histologic sections and placing them in an array on a recipient paraffin block such that hundreds of cases can be analyzed simultaneously. This technique allows maximization of tissue resources by analysis of small-core biop sies of blocks, rather than complete sections. Using this technology, a carefully planned array can be constructed with cases from pathology tissue block archives, such that a 20-year survival analysis can be performed on a cohort of 600 or more patients by use of only a few microliters of antibody in a single experiment. The reflex criticism of this technique is that the tissue analyzed is not representative, especially in antigens with heterogeneous staining patterns. This review addresses this issue, as well as the issue of antigen preservation or durability, which validates construction of arrays from archives. Strategies and methods of construction and analysis of the arrays are discussed, as well as some other unusual array applications. This technique can provide a highly efficient, high-throughput mechanism for evaluation of protein expression in large cohorts. It has the potential to allow validation of new genes at a speed comparable to the rapid rate of gene discovery afforded by DNA microarrays.

Validation of tissue microarray technology in breast carcinoma.

The recent development of tissue microarray technology has potentiated large-scale retrospective cohort studies using archival formalin-fixed, paraffin-embedded tissues. A major obstacle to broad acceptance of microarrays is that they reduce the amount of tissue analyzed from a whole tissue section to a disk, 0.6 mm in diameter, that may not be representative of the protein expression patterns of the entire tumor. In this study, we examine the number to disks required to adequately represent the expression of three common antigens in invasive breast carcinoma--estrogen receptor, progesterone receptor, and the Her2/neu oncogene--in 38 cases of invasive breast carcinoma. We compared the staining of 2 to 10 microarray disks and the whole tissue sections from which they were derived and determined that analysis of two disks is comparable to analysis of a whole tissue section in more than 95% of cases. To evaluate the potential for using archival tissue in such arrays, we created a breast cancer microarray of 8 to 11 cases from each decade beginning in 1932 to the present day and evaluated the antigenicity of these markers and others. This array demonstrates that many proteins retain their antigenicity for more than 60 years, thus validating their study on archival tissues. We conclude that the tissue microarray technique, with 2-fold redundancy, is a valuable and accurate method for analysis of protein expression in large archival cohorts.

Involvement of cyclooxygenase 2 (COX-2) in intrinsic tone of isolated guinea pig trachea.

Indomethacin and related nonsteroidal anti-inflammatory drugs relax prostanoid-dependent intrinsic tone of isolated guinea pig trachea by inhibiting cyclooxygenase (COX). Recently, a second isoform of COX (COX-2) was discovered, which differed from COX-1 with respect to protein structure, transcriptional regulation, and susceptibility to inhibition by pharmacological agents. It is now known that indomethacin nonselectively inhibits COX-1 and COX-2, whereas NS-398 is a selective inhibitor of COX-2. In the present study we compared the activity of a selective (NS-398) and nonselective (indomethacin) COX-2 inhibitor on intrinsic tone of isolated guinea pig trachea. NS-398 > or = indomethacin produced a reversal of intrinsic tone with a similar concentration-dependent (10 nM to 1 microM) time course (Tmax approximately 20-45 min), potency (EC50 1.7 and 5.6 nM, respectively), and maximal response. Contractions to cholinergic nerve stimulation (45 V, 0.5 ms, 0.1-32 Hz) and histamine were similarly modulated in tissues relaxed with the selective or nonselective COX-2 inhibitors. Immunoblot analyses showed that COX-2 protein synthesis was induced in both the cartilage and smooth muscle portions of the trachea during changes in intrinsic tone. These findings are consistent with pharmacological results and provide the first demonstration that prostanoid tone in isolated guinea pig trachea is dependent on COX-2 activity. The results also suggest that the activity of indomethacin in this preparation is likely related to COX-2 inhibition.

Pharmacology of montelukast sodium (Singulair), a potent and selective leukotriene D4 receptor antagonist.

Montelukast sodium (Singulair), also known as MK-0476 (1-(((1(R)-(3-(2-(7-chloro-2-quinolinyl)-(E)-ethenyl)phenyl)(3-2-(1- hydroxy-1-methylethyl)phenyl)propyl)thio)methyl)cyclopropane) acetic acid sodium salt, is a potent and selective inhibitor of [3H]leukotriene D4 specific binding in guinea pig lung (Ki 0.18 +/- 0.03 nM), sheep lung (Ki 4 nM), and dimethylsulfoxide-differentiated U937 cell plasma membrane preparations (Ki 0.52 +/- 0.23 nM), but it was essentially inactive versus [3H]leukotriene C4 specific binding in dimethylsulfoxide-differentiated U937 cell membranes (IC50 10 microM) and [3H]leukotriene B4 specific binding in THP-1 cell membranes (IC50 40 microM). Montelukast also inhibited specific binding of [3H]leukotriene D4 to guinea pig lung in the presence of human serum albumin, human plasma, and squirrel monkey plasma with Ki values of 0.21 +/- 0.08, 0.19 +/- 0.02, and 0.26 +/- 0.02 nM, respectively. Functionally, montelukast antagonized contractions of guinea pig trachea induced by leukotriene D4 (pA2 value 9.3; slope 0.8). In contrast, montelukast (16 microM) failed to antagonize contractions of guinea pig trachea induced by leukotriene C4 (45 mM serine-borate), serotonin, acetylcholine, histamine, prostaglandin D2, or U-44069. Intravenous montelukast antagonized bronchoconstriction induced in anesthetized guinea pigs by i.v. leukotriene D4 but did not block bronchoconstriction to arachidonic acid, histamine, serotonin, or acetylcholine. Oral administration of montelukast blocked leukotriene D4 induced bronchoconstriction in conscious squirrel monkeys, ovalbumin-induced bronchoconstriction in conscious sensitized rats (ED50 0.03 +/- 0.001 mg/kg; 4 h pretreatment), and also ascaris-induced early and late phase bronchoconstriction in conscious squirrel monkeys (0.03-0.1 mg/kg; 4 h pretreatment). A continuous i.v. infusion of montelukast (8 micrograms.kg-1.min-1) resulted in a 70% decrease in the peak early response and a 75% reduction of the late response to ascaris aerosol in allergic conscious sheep. Montelukast, a potent and selective leukotriene D4 receptor antagonist with excellent in vivo activity is currently in clinical development for the treatment of asthma and related diseases.

Neurokinin (NK2) receptors mediate nonadrenergic noncholinergic contractile responses to electrical stimulation and resiniferatoxin in guinea pig trachea.

In the present study we characterized the receptor(s) that mediates non-adrenergic non-cholinergic (NANC) contractions of isolated guinea pig cervical trachea, using CP-99,994, a selective neurokinin (NK1) receptor antagonist, and SR-48,968, a selective neurokinin (NK2) receptor antagonist. The activity of these two antagonists was determined against contractions to the selective agonists ([beta Ala8]NKA(4-10) for NK2 and [Sar9,Met(O2)11]SP for NK1) and the nonselective (SP and NKA) NK receptor agonists. CP-99,994 was inactive versus NKA and [beta Ala8]NKA(4-10) but antagonized SP- and [Sar9,Met(O2)11]SP-induced contractions with -log KB values of 5.6 +/- 0.2 and 7.7 +/- 0.2, respectively. SR-48,968 was inactive versus SP and [Sar9,Met(O2)11]SP but was active versus NKA and [beta Ala8]NKA(4-10), yielding -log KB values of 8.4 +/- 0.2 and 9.1 +/- 0.2, respectively. In the presence of 1 microM atropine, 1.4 microM indomethacin, 0.2 microM timolol, and 4 microM thiorphan, electrical field stimulation (16 Hz, 2.0 ms, 50 V for 10 every 30 min) elicited a NANC contractile response which was not significantly altered by CP-99,994 (3 microM) or the nitric oxide synthase inhibitor L-NAME (10 microM) but was completely inhibited by tetrodotoxin (TTX) (1 microM) and was also reduced to 58 +/- 12, 31 +/- 16, 8 +/- 4, and 0% of control by 15, 50, 150, and 1500 nM SR-48,968, respectively. Resiniferatoxin (1 and 10 nM) produced a well-maintained concentration-dependent contraction, which was 57.8 +/- 4.8 and 61.6 +/- 3.8%, respectively, of the carbachol-induced maximum response. Contractions were not significantly modified by L-NAME and were not blocked by TTX (1 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of iberiotoxin with beta-adrenoceptor agonists and sodium nitroprusside on guinea pig trachea.

Airway smooth muscle plasma membranes contain a variety of functional K+ channels. In particular, there is a predominance of Ca(2+)-activated K+ channels (maxi-K). Inhibition of these K+ channels has been postulated to account for the ability of charybdotoxin (ChTX) to produce contraction of airway smooth muscle and to modify the relaxant effects of beta-adrenoceptor agonists and sodium nitroprusside (SNP). Iberiotoxin (IbTX) is more selective and more potent than ChTX at blocking maxi-K channels. In this study, pharmacological experiments were performed on guinea pig trachea to determine whether IbTX produced effects similar to ChTX. The concentration-response curves to salbutamol were markedly affected by IbTX, with a > 60-fold rightward shift being produced with 20 nM IbTX. The maximal relaxation to salbutamol was reduced to 49.3 +/- 0.9, 22.3 +/- 4.7, and 15.0 +/- 2.7% of control maximum in the presence of 20, 60, and 180 nM IbTX, respectively. Similar to salbutamol, the maximal relaxation to SNP was reduced to 80 +/- 1.6, 19 +/- 1.7, and 12 +/- 2.1% of control maximum in the presence of 20, 60, and 180 nM IbTX, respectively. IbTX (180 nM) failed to produce a significant alteration of relaxation to the ATP-dependent K+ channel agonist BRL-34915. Exposure of tissues to K(+)-rich medium (80 mM) inhibited responses to salbutamol > or = SNP > isoproterenol. These results confirm and extend our earlier observations that maxi-K channels may be involved in regulating tone and relaxation of carbachol-contracted guinea pig tracheal smooth muscle. This mechanism is of particular importance for beta 2-adrenoceptor- and SNP-induced relaxation.

Development of a novel series of styrylquinoline compounds as high-affinity leukotriene D4 receptor antagonists: synthetic and structure-activity studies leading to the discovery of (+-)-3-[[[3-[2-(7-chloro-2-quinolinyl)-(E)-ethenyl]phenyl][[3- (dimethylamino)-3-oxopropyl]thio]methyl]thio]propionic acid.

Based on LTD4 receptor antagonist activity of 3-(2-quinolinyl-(E)-ethenyl)pyridine (2) found in broad screening, structure-activity studies were carried out which led to the identification of 3-[[[3-[2-(7-chloro-2-quinolinyl)-(E)-ethenyl]phenyl][[3- (dimethylamino)-3-oxopropyl]thio]methyl]thio]propionic acid (1, MK-571) as a potent and orally active LTD4 receptor antagonist. These studies demonstrated that a phenyl ring could replace the pyridine in 2 without loss of activity, that 7-halogen substitution in the quinoline group was optimal for binding, that the (E)-ethenyl linkage was optimal, that binding was enhanced by incorporation of a polar acidic group or groups in the 3-position of the aryl ring, and that two acidic groups could be incorporated via a dithioacetal formed from thiopropionic acid and the corresponding styrylquinoline 3-aldehyde to yield compounds such as 20 (IC50 = 3 nM vs [3H]LTD4 binding to the guinea pig lung membrane). It was found that one of the acidic groups could be transformed into a variety of the amides without loss of potency and that the dimethylamide 1 embodied the optimal properties of intrinsic potency (IC50 = 0.8 nM on guinea pig lung LTD4 receptor) and oral in vivo potency in the guinea pig, hyperreactive rat, and squirrel monkey. The evolution of 2 to 1 involves the increase of > 6000-fold in competition for [3H]LTD4 binding to guinea pig lung membrane and a > 40-fold increase in oral activity as measured by inhibition of antigen-induced dyspnea in hyperreactive rats.

Pharmacology of the leukotriene antagonist verlukast: the (R)-enantiomer of MK-571.

Verlukast (MK-679) (3-[(3-(2-(7-chloro-2-quinolinyl)-(E)-ethenyl)phenyl)[3-(dimethylamino)- 3- oxopropyl)thio)methyl)-thio)propionic acid) is a potent and selective inhibitor of [3H]leukotriene D4 binding in guinea-pig (IC50 = 3.1 +/- 0.5 nM) and human (IC50 = 8.0 +/- 3.0 nM) lung homogenates and dimethyl sulfoxide differentiated U937 cell membrane preparations (IC50 = 10.7 +/- 1.6 nM) but is essentially inactive versus [3H]leukotriene C4 binding in guinea-pig lung homogenates (IC50 values of 19 and 33 microM). Functionally, when tested at 60 nM, it antagonized contractions of guinea-pig trachea (GPT) induced by leukotriene C4, leukotriene D4, and leukotriene E4 (respective-log KB values of 8.6, 8.8, and 8.9) and contractions of human trachea (HT) induced by leukotriene D4 (-log KB value 8.3 +/- 0.2). In contrast, verlukast (20-200 nM) failed to antagonize contractions of GPT induced by leukotriene C4 in the presence of 45 mM L-serine borate. Intravenous (i.v.) and aerosol verlukast antagonized bronchoconstriction (BC) induced in anaesthetized guinea pigs by i.v. leukotriene D4 but did not block BC to arachidonic acid or histamine. Intraduodenal verlukast (0.25 mg/kg) antagonized leukotriene D4 (0.2 micrograms/kg) induced BC in guinea pigs. Oral and aerosol administration blocked leukotriene D4-induced BC in conscious squirrel monkeys. Orally administered compound also blocked ovalbumin-induced BC in conscious sensitized rats treated with methysergide (3 micrograms/kg). The pharmacological profile for verlukast is similar to that of the racemic compound, MK-571. Verlukast is currently in clinical development for the treatment of asthma and related diseases.

Personality, alcohol consumption, and menstrual distress in young women.

There has been a longstanding hypothesis that some women develop alcohol dependence as a result of drinking to alleviate premenstrual dysphoria. This study investigated the relationship between personality factors, alcohol consumption, and menstrual distress symptoms in nonalcoholic drinking young women. Normally menstruating women monitored their alcohol intake and physical and affective distress symptoms daily for two consecutive menstrual cycles. Subjects were unaware that their menstrual cycles and symptoms were being monitored. Subjects also completed the Minnesota Multiphasic Personality Inventory (MMPI), the Eysenck Personality Questionnaire (EPQ), Cattel's Sixteen Personality Factor Questionnaire (16PF), and the Vando Reducer-Augmenter Scale. The MMPI scales were factor analyzed to reduce the number of variables. Four derived MMPI factors were added to the VANDO, the three EPQ factors, and the four higher order factors of the 16PF to provide a total of twelve personality predictors. Separate regression analyses were carried out between personality factors and both alcohol consumption and menstrual distress. The results revealed that the women who drank more tended to be significantly more extroverted, spontaneous, carefree, and open to change. By contrast, women who reported greater over-all menstrual distress tended to be less capable, secure, and well-adjusted and reported a greater number of emotional and psychological problems. There was no correlation between alcohol consumption and menstrual distress. It was concluded that the results contradict the alcoholism-menstrual cycle hypothesis.

Selective inhibition of relaxation of guinea-pig trachea by charybdotoxin, a potent Ca(++)-activated K+ channel inhibitor.

Airway smooth muscle plasma membranes are rich in K+ channels of various types. Charybdotoxin (ChTX) is a potent blocker of the high-conductance Ca(++)-activated K+ channel in smooth muscle and produces a concentration-dependent contraction of guinea pig trachea. In the present study, pharmacologic experiments were performed on carbachol-contracted (0.34 microM) guinea-pig trachea contracted further with ChTX in order to determine if Ca(++)-activated K+ channels play a role in the responses to cAMP-dependent and cAMP-independent bronchodilators. Relaxation concentration response curves to the beta-agonists, isoproterenol and salbutamol; the phosphodiesterase inhibitor, aminophylline; the cAMP mimic, N6-2'-O-adenosine 3':5'-cyclic monophosphate the guanylate cyclase activator, sodium nitroprusside; and the K+ channel agonists, BRL-34915 and pinacidil, were obtained in the absence and presence of ChTX. The concentration response curves to isoproterenol and salbutamol were shifted to the right (approximately 27-fold and greater than 40-fold, respectively) by 180 nM ChTX, whereas concentration response curves to N6-2'-O-adenosine 3':5'-cyclic monophosphate and aminophylline were affected significantly less (shifted approximately 7.5-fold). Concentration response curves to the cGMP-dependent relaxant sodium nitroprusside were also altered by ChTX (17-fold rightward shift at 180 nM). In the presence of 60 nM ChTX, the concentration response curves to the above relaxants were shifted only 3- to 5-fold. In contrast, ChTX (60 and 180 nM) failed to produce a significant rightward shift in the concentration response curves to BRL-34915 or pinacidil. Relaxation to BRL-34915 was however, blocked by glybenclamide, suggesting differences in the mechanism of relaxation. Contraction of tissues with depolarizing concentrations of KCl (20-80 mM) inhibited responses to all bronchodilators. These results suggest that hyperpolarization of tracheal smooth muscle as a result of opening various types of K+ channels can lead to relaxation of carbachol-contracted tracheal smooth muscle.

Stereospecific synthesis, assignment of absolute configuration, and biological activity of the enantiomers of 3-[[[3-[2-(7-chloroquinolin-2-yl)-(E)-ethenyl]phenyl] [[3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]propionic acid, a potent and specific leukotriene D4 receptor antagonist.

The enantiomers of the leukotriene D4 antagonist 3-[[[3-[2-(7-chloroquinolin-2-yl)-(E)-ethenyl]phenyl] [[3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]propionic acid (L-660,711)(MK-571) have been prepared, their absolute stereochemistry has been assigned as S for (+)-1 and R for (-)-1 by X-ray analysis of a synthetic intermediate (5), and the biological activity of the enantiomers has been explored. Unexpectedly, the enantiomers are both comparably biologically active with (+)-1 slightly more intrinsically active at the LTD4 receptor in vitro.

Alcohol consumption and menstrual distress in women at higher and lower risk for alcoholism.

This study investigated whether alcohol consumption varied as a function of menstrual cycle, menstrual distress symptomatology, and global stress in nonalcoholic drinking young women at higher and lower (HR, LR) risk for alcoholism as assessed by family history. Eighty-two normally menstruating women (52 LR and 30 HR) monitored their alcohol intake, physical and affective distress symptoms, and global stress level daily for two consecutive menstrual cycles. Subjects were unaware that their menstrual cycles were being monitored. The results confirmed the presence of increased physical distress symptomatology during the premenstrual and menstrual phases but did not show variation in negative affect or global stress throughout the menstrual cycle. High risk subjects were aware that they were at higher risk for alcoholism and consumed more alcohol. However, alcohol consumption was not related to the menstrual cycle, distress symptoms, or global stress. Subjects reported that they drank most frequently with others for pleasure enhancement and rarely for pain or tension-reduction. Subjects also drank more on weekends than weekdays. These findings argue against the menstrual cycle as etiological in the development of alcoholism. It would appear that social factors influence alcohol consumption in young nonalcoholic women.

Fractional conversion of thromboxane A2 and B2 to urinary 2,3-dinor-thromboxane B2 and 11-dehydrothromboxane B2 in the cynomolgus monkey.

Following the intravenous administration of thromboxane (TX) B2, the stable hydration product of TXA2, to human and nonhuman primates the most abundant urinary metabolites are 2,3-dinor-TXB2 and 11-dehydro-TXB2. However, it is not known whether fractional conversion of TXB2 to its enzymatic metabolites is an accurate representation of TXA2 metabolism. Thus, we have compared the metabolic disposition of synthetic TXA2 and TXB2 via the beta-oxidation and 11-OH-dehydrogenase pathways in vivo in the monkey. TXA2 or TXB2 (20 ng/kg) was intravenously administered to four cynomolgus monkeys pretreated with aspirin in order to suppress endogenous TXA2 production. Urinary TXB2, 2,3-dinor-TXB2 and 11-dehydro-TXB2 were measured before, during and up to 24 h after thromboxane administration by means of reversed-phase high-performance liquid chromatography radioimmunoassay. Aspirin treatment suppressed urinary 2,3-dinor-TXB2 and 11-dehydro-TXB2 by approx. 75%. A similar fractional conversion of TXA2 and TXB2 into 2,3-dinor-TXB2 and 11-dehydro-TXB2 was found. These results suggest that TXA2 is hydrolyzed to TXB2 prior to enzymatic degradation and that metabolites of the latter represent reliable indices of TXA2 biosynthesis. Due to the variability in the conversion of thromboxanes into 2,3-dinor-TXB2 and 11-dehydro-TXB2, the measurement of both metabolites seems to represent a more reliable index of acute changes in TXA2 production.

Pharmacology of L-660,711 (MK-571): a novel potent and selective leukotriene D4 receptor antagonist.

L-660,711 (3-(3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl) ((3-dimethyl amino-3-oxo propyl)thio)methyl)thio)propanoic acid is a potent and selective competitive inhibitor of [3H]leukotriene D4 binding in guinea pig (Ki value, 0.22 nM) and human (Ki value, 2.1 nM) lung membranes but is essentially inactive versus [3H]leukotriene C4 binding (IC50 value in guinea pig lung, 23 microM). Functionally it competitively antagonized contractions of guinea pig trachea and ileum induced by leukotriene (LT) D4 (respective pA2 values, 9.4 and 10.5) and LTE4 (respective pA2 values, 9.1 and 10.4) and contractions of human trachea induced by LTD4 (pA2 value, 8.5). L-660,711 (5.8 x 10(-8)M) antagonized contractions of guinea pig trachea induced by LTC4 in the absence (dose ratio = 28) but not in the presence of 45 mM L-serine borate (dose ratio less than 2). L-660,711 (1.9 x 10(-5)M) did not block contractions of guinea pig trachea induced by histamine, acetylcholine, 5-hydroxytryptamine, PGF2 alpha, U-44069, or PGD2. In the presence of atropine, mepyramine, and indomethacin, L-660,711 (1.9 x 10(-5)M) inhibited a small component of the response to antigen on guinea pig trachea but completely blocked anti-IgE-induced contractions of human trachea. L-660,711 (i.v.) antagonized bronchoconstriction induced in anesthetized guinea pigs by i.v. LTC4, LTD4, and LTE4 but did not block bronchoconstriction to arachidonic acid, U-44069, 5-hydroxytryptamine, histamine, or acetylcholine. Intraduodenal L-660,711 antagonized LTD4 (0.2-12.8 micrograms/kg)-induced bronchoconstriction in guinea pigs, and p.o. L-660,711 blocked LTD4- and Ascaris-induced bronchoconstriction in conscious squirrel monkeys and ovalbumin-induced bronchoconstriction in conscious sensitized rats treated with methysergide (3 micrograms/kg). The pharmacological profile of L-660,711 indicates that it is a potent, selective, orally active leukotriene receptor antagonist which is well suited to determine the role played by LTD4 and LTE4 in asthma and other pathophysiologic conditions.

Antigen-induced contraction of guinea-pig isolated trachea: studies with novel inhibitors and antagonists of arachidonic acid metabolites.

1. Responses of antigen-challenged isolated trachea from sensitized guinea-pigs were pharmacologically characterized by use of some novel inhibitors and antagonists of arachidonic acid metabolites. 2. The cyclo-oxygenase inhibitor, indomethacin, prolonged without altering the maximum response to antigen in the absence of the anti-muscarinic agent, atropine, and/or the H1-receptor blocker, mepyramine. In the presence of mepyramine, indomethacin both prolonged and increased the magnitude of the response. The selective (SQ-29548) and non-selective (L-640,035) thromboxane A2 (TXA2) antagonist and the TXA2 synthetase inhibitor, OKY-046, were essentially inactive. 3. Two novel inhibitors of 5-lipoxygenase product formation, AA-861 and L-651,896 produced complete inhibition of the response to antigen on tissues treated with atropine and mepyramine, with or without indomethacin. 4. Equimolar concentrations of the leukotriene D4 (LTD4) receptor antagonists LY-171883 greater than L-649,923 greater than or equal to L-648,051 greater than or equal to FPL-55712 blocked part of the response to antigen on tissues treated with atropine, mepyramine and indomethacin. All compounds tended to block a larger component of the response in the absence of indomethacin. A similar tendency was observed with the potent phosphodiesterase inhibitor, isobutylmethylxanthine (IBMX) but not the less potent phosphodiesterase inhibitor theophylline. 5. These results are consistent with the hypothesis that 5-lipoxygenase products acting on LTD4 receptors play only a minor role in the mediation of the contraction of guinea-pig trachea to antigen challenge. The nature of the residual contractile mediator is unknown; however, it can be completely blocked by the 5-lipoxygenase inhibitors AA-861 and L-651,896 and non-selectively blocked by the phosphodiesterase inhibitor, IBMX and non-selective LTD4 receptor antagonists, such as LY-171883.

Leukotriene and anti-IgE induced contractions of human isolated trachea: studies with leukotriene receptor antagonists and a novel 5-lipoxygenase inhibitor.

The pharmacological actions of three leukotriene D4 (LTD4) receptor antagonists, FPL-55712, L-648,051, and L-649,923, and a novel inhibitor of leukotriene biosynthesis, L-651,896, have been investigated on isolated human tracheal smooth muscle. In the order of potency L-648,051 greater than FPL-55712 greater than L-649,923, these agents antagonized contractions to LTD4 and produced parallel rightward shifts in the dose-response curves. Mean -log KB values against LTD4 were 6.9 +/- 0.1, 6.5 +/- 0.3, and 6.0 +/- 0.1 for L-648,051, FPL-55712, and L-649,923, respectively. FPL-55712 also antagonized contractions to LTC4 (-log KB value, 6.4 +/- 0.3) and this activity was not decreased by the gamma-glutamyl transpeptidase inhibitor, L-serine borate. In the presence of 1 x 10(-7) M atropine, 7 x 10(-6) M mepyramine, and 1.4 x 10(-6) M indomethacin, L-648,051 at 2 x 10(-5) and 2 x 10(-6) M produced complete and partial blockade, respectively, of the contraction to goat anti-IgE. L-649,923 and FPL-55712 produced partial but significant inhibition at 2 x 10(-5) M, whereas the 5-lipoxygenase inhibitor, L-651,896, produced almost complete inhibition at 3.5 and 35 x 10(-6) M. L-Serine borate (15 mM) did not alter the the activity of FPL-55712 versus anti-IgE. These findings indicate that LTD4 receptors mediate contraction of human trachea to exogenously applied and endogenously (anti-IgE) released leukotrienes. LTD4 antagonists, such as L-648,051, may be useful in assessing the role of leukotrienes in respiratory disease.

Effects of L-serine borate on antagonism of leukotriene C4-induced contractions of guinea-pig trachea.

The antagonist activity of three leukotriene D4 (LTD4) receptor antagonists and a number of bronchodilators was determined against LTC4-induced contractions of guinea-pig isolated tracheal chains in the absence and presence of the gamma-glutamyltranspeptidase inhibitor, L-serine borate (SB). The LTD4 receptor antagonists FPL-55712, L-649,923 and L-648,051 effectively antagonized LTC4 responses in the absence of SB but were ineffective in the presence of 15 and/or 45 mM SB. Salbutamol greater than isobutylmethylxanthine (IBMX) greater than dibutyryl cyclic AMP greater than aminophylline greater than nifedipine antagonized contractions to LTC4 in the absence of SB. In contrast, in the presence of SB the antagonist activity of all of these agents except nifedipine was significantly reduced. The antagonist activity of the Ca2+ entry blocker, nifedipine, was similar in the absence and presence of SB. Salbutamol and IBMX were potent functional antagonists of LTE4-induced contractions both in the absence and presence of SB. These results are consistent with the hypothesis that there are contractile LTC4 receptor mechanisms in guinea-pig trachea which are unmasked by SB and are not blocked by LTD4 receptor antagonists and which are less effectively down modulated by cyclic AMP-dependent bronchodilators.