Increased blocking activity of combined tachykinin NK1- and NK2-receptor antagonists on tachykinergic bronchomotor responses in the guinea-pig.

1. The present study compared the effect of the administration of tachykinin NK1- and NK2-receptor antagonists alone and in combination on exogenous and endogenous tachykinin-induced contractions using three different guinea-pig airway preparations: isolated bronchus, isolated perfused lung and in vivo. 2. In the isolated bronchi, the tachykinin NK1-receptor antagonist CP 99994 (0.01-1 microM) produced concentration-dependent inhibition of contractions induced the tachykinin NK1-receptor agonists substance P (SP) and [Met-OMe11] SP ([Met-OMe11]SP), whereas the tachykinin NK2-receptor antagonist SR 48968 (0.1 microM) had no effect. SR 48968 (0.001-0.01 microM) concentration-dependently inhibited contractions induced by the tachykinin NK2-receptor agonists neurokinin A (NKA) and [beta-Ala8]-neurokinin A (4-10) ([betaAla8]-NKA) whereas CP 99994 (0.1 microM) did not inhibit the contractions. The contractile activity of capsaicin, an agent that releases endogenous tachykinins from sensory C-fibres, was inhibited in a concentration dependent manner by SR 48968 (0.001-0.03 microM) but not by CP 99994 (0.1 microM). Combination of CP 99994 and SR 48968 caused increased inhibitory effects on the concentration-response curves to SP, [Met-OMe1l]SP, NKA, [beta-Ala8]-NKA and capsaicin. 3. In isolated perfused lungs, SR 48968 concentration (0.01-10 microM) dependently inhibited NKA-, [beta-Ala8]-NKA- and capsaicin-induced bronchoconstriction whereas CP 99994 (30 microM) had no effect on SP-, NKA-, [beta-Ala8]-NKA- and capsaicin-induced bronchoconstriction. Combination of inactive concentrations of CP 99994 and SR 48968 produced an increased inhibitory effect on all previous stimuli-induced bronchoconstriction. 4. In in vivo guinea-pig studies, intravenous and oral pretreatment with SR 48968 (0.01-1 mg kg(-1) i.v. and 0.1-3 mg kg(-1) p.o., respectively), but not with CP 99994 (1 mg kg(-1) i.v. and 0.3-30 mg kg(-1) p.o., respectively), produced a dose-dependent inhibition of the bronchoconstrictor responses induced by NKA, [beta-Ala8]-NKA and capsaicin. CP 99994 intravenously (0.3 mg kg(-1)) and orally (3-10 mg kg(-1)) inhibited SP-induced bronchoconstriction only. Intravenous and oral low dose combinations of CP 99994 and SR 48968 produced an increased inhibition of SP-, NKA-, [beta-Ala8]-NKA- and capsaicin-induced bronchoconstriction, respectively. The present data indicate that combined tachykinin NK1- and NK2-receptor antagonist treatment compared with single antagonist treatment, using CP 99994 and SR 48968, produced an augmented blockade of tachykinin NK1-, NK2- and capsaicin-mediated contractions in guinea pig airways. These findings support the hypothesis that a dual NK1- and NK2-receptor antagonist may provide an advantage over single activity tachykinin NK1- or NK2-receptor antagonists in pulmonary obstructive diseases.

Pharmacological characterization of alpha 2-adrenoceptor-mediated responses in pig nasal mucosa.

1. Pig nasal mucosal strips were incubated with alpha-adrenoceptor antagonists followed by alpha2-adrenoceptor agonist concentration-response curves. 2. Contractions elicited by the alpha2-adrenoceptor agonists BHT-920 (pD2 = 6.16 +/- 0.07), UK 14,304 (pD2 = 6.89 +/- 0.13) and PGE-6201204 (pD2 = 7.12 +/- 0.21) were blocked by the alpha2-adrenoceptor antagonist yohimbine (0.1 microm). In contrast, the alpha1-adrenoceptor antagonist prazosin (0.03 microm) had no effect on the BHT-920-, UK 14,304- and PGE-6201204-induced contractions, but blocked the contractile response to the alpha(1)-adrenoceptor agonist phenylephrine (pD2 = 5.38 +/- 0.04) and the mixed alpha1- and alpha2-adrenoceptor agonist oxymetazoline (pD(2) = 6.30 +/- 0.22). 3. The alpha2-adrenoceptor antagonist yohimbine (0.01-0.1 microm, pA2 = 8.04), alpha2B/C-adrenoceptor antagonist ARC 239 (10 microm, pK(b) = 6.33 +/- 0.21), alpha2A/C-adrenoceptor antagonist WB 4101 (0.3 microm, pK(b) = 8.01 +/- 0.24), alpha2A-adrenoceptor antagonists BRL44408 (0.1 microm, pK(b) = 6.82 +/- 0.34) and RX 821002 (0.1 microm, pKb = 8.31 +/- 0.35), alpha2C-adrenoceptor antagonists spiroxatrine (1 microm, pKb = 7.32 +/- 0.32), rauwolscine (0.1 microm, pKb = 8.16 +/- 0.14) and HV 723 (0.3 microm, pKb = 7.68 +/- 0.14) inhibited BHT-920-induced contractions in pig nasal mucosa. 4. The present antagonist potencies showed correlations with binding affinity estimates (pKi) obtained for these antagonists at the human recombinant alpha2A- and alpha2C-adrenoceptors (r = 0.78 and 0.83, respectively) and with binding affinity estimates (pKd) obtained in pig native alpha2A- and alpha2C-monoreceptor assays (r = 0.85 and 0.78, respectively). No correlation was observed for the alpha2B-subtype. 5. In conclusion, contractile responses to phenylephrine, BHT-920, UK 14,304, PGE-6201204 and oxymetazoline indicate that alpha1- and alpha2-adrenoceptors are present and mediate vasoconstriction in pig nasal mucosa. Furthermore, correlation analysis comparing antagonist potency in pig nasal mucosa with affinities for human recombinant alpha2-adrenoceptors and native pig alpha2-adrenoceptors suggest that alpha2A- and alpha2C-adrenoceptor subtypes constrict pig nasal mucosa vasculature.

Synergistic antiallergic activity of combined histamine H1- and cysteinyl leukotriene1-receptor blockade in human bronchus.

Mast cell histamine (HA) and cysteinyl leukotrienes (CysLT) account for most of the early phase bronchospasm in asthma. However, activation of the smooth muscle CysLT1-receptor plays a major role in asthmatic bronchospasms. CysLT-receptor antagonists or CysLT-synthesis inhibitors are efficacious in asthma but do not completely abolish asthmatic bronchospasms. A recent clinical study showed that combined antagonists loratadine (H1) and zafirlukast (CysLT1) were more effective against allergic bronchospasms than either drug alone. We examined the combined efficacy of H1- and CysLT1-receptor antagonists in allergic human bronchus. The H1- and CysLT1-receptor antagonists chlorpheniramine (CTM; 1 microM) and MK-571 (0.03 microM), were tested alone and in combination, against anti-human IgE antibody (Ab)-induced contractions of passively sensitized isolated human bronchus. Ab-induced allergic contractions were reduced 15% and 36% by CTM (1 microM) and MK-571 (0.03 microM), respectively. Combined CTM (1 microM) and MK-571 (0.03 microM) significantly inhibited the Ab response by 87%. Mechanistic investigations in isolated human bronchus and cultured human cord blood mast cells suggest that H1- and CysLT-receptor interactions likely occur at the airway smooth muscle level. CTM and MK-571 synergistically inhibited human allergic bronchospasm in the present in vitro model. The mechanism underlying this synergistic activity requires further investigation.

Postjunctional alpha(2C)-adrenoceptor contractility in human saphenous vein.

The postjunctional alpha(2)-adrenoceptor-mediated contractility was characterized in human saphenous vein derived from coronary artery bypass graft surgery. Human saphenous vein contracted to alpha(2)-adrenoceptor selective agonists BHT-920 (5,6,7,8-Tetrahydro-6-(2-propenyl)-4H-thiazolo[4,5-d]azepin-2-amine dihydrochloride; pD(2)=6.7+/-0.1) and UK 14,304 (5-Bromo-6-(2-imidazolin-2-ylamino)quinoxaline; pD(2)=7.2+/-0.1). BHT-920-induced contractions were inhibited by the alpha(2)-adrenoceptor antagonist yohimbine (17-Hydroxy-yohimban-16-carboxylic acid methyl ester hydrochloride; pA(2)=8.7+/-0.5), but not by the alpha(1)-adrenoceptor antagonist prazosin (1-[4-Amino-6,7-dimethoxy-2-quinazolinyl]-4-[2-furanylcarbonyl]-piperazine hydrochloride; 300 nM). In contrast, prazosin (pK(b)=7.9+/-0.2) potently antagonized contractions elicited by the alpha(1)-adrenoceptor agonist phenylephrine ((R)-3-Hydroxy-alpha-[(methylamino)methyl] benzenemethanol hydrochloride; pD(2)=4.9+/-0.1), indicating that both alpha(2)- and alpha(1)-adrenoceptor evoke human saphenous vein contractions. Functional antagonist activity estimates (pA(2) or pK(b)) obtained for the alpha-adrenoceptor antagonists ARC 239 (2-[2-(4-(2-Methoxyphenyl)piperazin-1-yl)ethyl]-4,4-dimethyl-1,3-(2H,4H)-isoquinolindione dihydrochloride), WB 4101 (2-(2,6-Dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane hydrochloride) and HV 723 (alpha-ethyl-3,4,5-trimethoxy-alpha-(3-((2-(2-methoxyphenoxy) ethyl)amino)propyl)benzeneacetonitrile) against BHT-920-induced human saphenous vein contractions were 7.0+/-0.6, 8.3+/-0.6 and 7.7+/-0.3, respectively. The alpha(2)-adrenoceptor subtype affinities (pK(i)) obtained in recombinant human alpha(2A)-, alpha(2B)- and alpha(2C)-adrenoceptor competition binding assays were 8.6, 8.3 and 8.6 for yohimbine; 6.3, 8.4 and 7.0 for ARC 239; 8.4, 7.5 and 8.4 for WB 4101 and 7.5, 7.4 and 7.9 for HV 723, respectively. Taken together, the binding and functional antagonist activity estimates obtained in these investigations indicate that alpha(2C)-adrenoceptor is the predominant postjunctional alpha(2)-adrenoceptor subtype in human saphenous vein.

Activity of nonpeptide tachykinin antagonists on neurokinin a induced contractions in dog urinary bladder.

PURPOSE: There is abundant evidence indicating that tachykinin (TK) containing sensory nerves (C-fibers) play an important role in neural regulation of reflex micturition in mammals. Desensitization of urinary bladder C-fibers with resiniferatoxin ameliorates bladder hyperreflexia and incontinence and supports a similar role for C-fibers in man. TK-induced contraction of isolated human urinary bladder smooth muscle appears to be exclusively neurokinin NK2-receptor mediated. Dog urinary bladder contractility to a series of TK-receptor agonists also suggests a predominance of NK2-receptor activation by tachykinins and indicates the dog may provide a useful model for the development of pharmacotherapy for bladder hyperreflexia. MATERIALS AND METHODS: We evaluated the activity of the nonpeptide NK-receptor selective antagonists SR 48968 (NK2), SB 223412 (NK3) and CP 99994 (NK1) against neurokinin A (NKA)-induced contractions in isolated dog urinary bladder strips to determine the NK-receptor type which mediates contractile responses to NKA. The dual NK1 and NK2 antagonist MDL 103392 was also tested in this preparation. RESULTS: NKA-induced contractions (pD2 = 8.0) were dose dependently blocked by SR 48968 (pA2 = 8.2 +/- 0.2) while CP 99994 (1.0 microM) and SB 223412 (1.0 microM) were inactive. MDL 103392 was a weak functional antagonist (pKb = 6.1 +/- 0.1) of NKA. CONCLUSIONS: Relative activities of SR 48968, CP 99994 and SB 223412 confirm that the NK2-receptor is the mediator of NKA-induced contractions of dog urinary bladder smooth muscle.

Pharmacological characterization of histamine H3 receptors in human saphenous vein and guinea pig ileum.

Studies were performed to assess the functional activity of histamine H3 receptors on neurogenic sympathetic end organ responses in cryopreserved human saphenous vein. (R)-alpha-methylhistamine inhibited electrical field stimulation-evoked contractile responses in a dose dependent manner (pD2 = 8.20). Prazosin (1 microM) and tetrodotoxin (1 microM) blocked the electrical field stimulation-evoked contractile responses in human saphenous vein indicating a sympathetic neural origin of these contractions. The histamine H3 antagonists thioperamide (pA2 = 8.41) and clobenpropit (pA2 = 10.10) produced parallel rightward shifts in the concentration response curve to (R)-alpha-methylhistamine in human saphenous vein and guinea pig ileum (pA2 = 8.59 and 9.83, respectively). Pretreatment with (R)-alpha-methylhistamine (1 microM) did not alter contractions to exogenous norepinephrine in human saphenous vein. In addition, clonidine (pD2 = 10.28) inhibited electrical field stimulation-evoked contractile responses in human saphenous vein which were blocked by yohimbine (30 nM, pA2 = 9.92) but did not alter the (R)-alpha-methylhistamine dose response curve. These results demonstrate the presence of functional presynaptic histamine H3 heteroreceptors on cryopreserved human saphenous vein sympathetic nerves that, upon activation, attenuate electrical field stimulation-evoked contractile responses in this vessel.

NK(2)-receptor mediated contraction in monkey, guinea-pig and human airway smooth muscle.

Neurokinins (NK) are implicated in airway pathology. Selective NK(2)-receptor antagonists may prove therapeutic in airway disease. We studied Neurokinin A (NKA) responses of isolated, cryopreserved cynomolgus monkey, fresh guinea pig, and fresh and cryopreserved human airways. NKA contracted monkey trachea (pD(2)= 7.9), guinea-pig bronchus (pD(2)= 8.8) and human bronchus (pD(2)= 7.1). Potency rank order (pK(b)) of NK(2)-antagonists, SR 48968 and GR 159897, and a dual NK(1)/NK(2)-antagonist, MDL 103392, against NKA responses in monkey trachea, guinea pig and human bronchus, respectively, were SR 48968 (9.29 +/- 0.11, 9.15 +/- 0.10 and 9.51 +/- 0.17) > GR 159897 (8.45 +/- 0.26, 8.19 +/- 0.13 and 8.57 +/- 0. 22) > MDL 103392 (6.55 +/- 0.13, 6.97 +/- 0.14 and 7.16 +/- 0.13). CP 99994 (1 microM), a NK(1)-receptor antagonist, was inactive against NKA responses in all three species. The NK(3)-antagonist SR 142801 (1 microM) was inactive against NKA in monkey trachea and guinea-pig bronchus, but demonstrated weak antagonist activity (pK(b)= 6.97 +/- 0.03) in human bronchus. These findings demonstrate that NK(2)-receptors mediate tracheal smooth muscle contraction to NKA in cynomolgus monkey and that the pharmacological responsiveness of airway NK(2)-receptors in the three species studied is similar. Furthermore, our results suggest that cryopreservation may extend the viability of human and non-human primate airway tissue for studies of neurokinin receptor pharmacology. Studies are needed to further determine the similarity in neurokinin pharmacology between fresh and cryopreserved airway tissue.

Studies on the pharmacology of the novel histamine H3 receptor agonist Sch 50971.

Experiments were performed to characterize the pharmacology of Sch 50971 ((+)-trans-4-(4(R)-methyl-3(R)-pyrolidinyl)-1H-imidazole dihydrochloride, CAS 167610-28-8), a novel histamine H3 receptor agonist. The activity of Sch 50971 was compared with that of (R)-alpha-methylhistamine (CAS 75614-87-8), a potent and moderately selective agonist of histamine H3 receptors, in a series of in vitro and in vivo assays. Sch 50971 is a high affinity, selective H3 receptor agonist in vitro and in vivo. Sch 50971 inhibits [3H]-N-alpha-methylhistamine (CAS 673-50-7) binding to the histamine H3 receptor in human brain (Ki = 5.0 nmol/l) and guinea pig brain (Ki = 2.5 nmol/l). Sch 50971 also inhibits electric field stimulated guinea pig ileum contractions (pD2 = 7.47) and decreases [3H]-norepinephrine (CAS 51-41-2) release (pD2 = 7.48) from guinea pig pulmonary artery by activation of presynaptic inhibitory H3 receptors. The in vitro effects of Sch 50971 are antagonized by low concentrations of a selective H3 antagonist, thioperamide (CAS 106243-16-7). Sch 50971 has low affinity (IC50's > 10 mumol/l) for histamine H1, dopamine D1 and D2, serotonin 5-HT2 and muscarinic cholinergic receptors. It also does not exhibit histamine H2-antagonist activity. In guinea pigs and cats, Sch 50971 exhibits in vivo H3 agonist activity. Sch 50971 inhibits sympathetic hypertension evoked by stimulation of the medulla oblongata in anesthetized guinea pigs (ED30 = 0.3 mg/kg i.v., ED30 = 1.0 mg/kg i.d.). Sch 50971 also inhibits the effects of sympathetic nerve stimulation on nasal resistance in cats. In these assays, Sch 50971 exhibits an efficacy and potency comparable to H3-agonist (R)-alpha-methylhistamine. However, under in vivo conditions, Sch 50971 does not exhibit histamine H1-mediated responses that are seen with (R)-alpha-methylhistamine at doses close to those that produce H3 effects. Therefore, Sch 50971 is a novel, potent and selective agonist of histamine H3 receptors with an improved in vitro and in vivo receptor profile selectivity compared with (R)-alpha-methylhistamine.

Tachykinin NK1 receptor-mediated vasorelaxation in human pulmonary arteries.

Tachykinin NK1 receptors are present on human pulmonary arteries. Addition of the specific tachykinin NK1 receptor agonist, [Met-OMe11]substance P produced a concentration-dependent relaxation (0.1 nM to 100 nM) in pulmonary arteries preconstricted with phenylephrine (30 microM). The EC50 (agonist concentration needed to produce 50% of the maximal relaxation) value for [Met-OMe11]substance P was 3.7+/-0.7 nM. The relaxation induced by [Met-OMe11]substance P was selectively inhibited by the tachykinin NK1 receptor antagonist CP 99994 (1 nM), with a pKb of 9.9+/-0.3. Treatment with the tachykinin NK2 receptor antagonist SR 48968 (100 nM) did not significantly affect the vasorelaxation due to [Met-OMe11]substance P (P > 0.05, one-way analysis of variance; ANOVA).

Nuclear factor (NF)-kappa B2 (p100/p52) is required for normal splenic microarchitecture and B cell-mediated immune responses.

The nfkb2 gene is a member of the Rel/NF-kappa B family of transcription factors. COOH-terminal deletions and rearrangements of this gene have been associated with the development of human cutaneous T cell lymphomas, chronic lymphocytic leukemias, and multiple myelomas. To further investigate the function of NF-kappa B2, we have generated mutant mice carrying a germline mutation of the nfkb2 gene by homologous recombination. NF-kappa B2-deficient mice showed a marked reduction in the B cell compartment in spleen, bone marrow, and lymph nodes. Moreover, spleen and lymph nodes of mutant mice presented an altered architecture, characterized by diffuse, irregular B cell areas and the absence of discrete perifollicular marginal and mantle zones; the formation of secondary germinal centers in spleen was also impaired. Proliferation of NF-kappa B2-deficient B cells was moderately reduced in response to lipopolysaccharide, anti-IgD-dextran, and CD40, but maturation and immunoglobulin switching were normal. However, nfkb2 (-/-) animals presented a deficient immunological response to T cell-dependent and -independent antigens. These findings indicate an important role of NF-kappa B2 in the maintenance of the peripheral B cell population, humoral responses, and normal spleen architecture.

Airway eosinophils, T cells, Th2-type cytokine mRNA, and hyperreactivity in response to aerosol challenge of allergic mice with previously established pulmonary inflammation.

Asthma is characterized by acute episodes of nonspecific airway hyperreactivity and chronic pulmonary inflammation exacerbated by stimuli including allergen exposure. In order to reproduce the physiologic and immunologic responses that occur in asthmatic patients, we have characterized a model of antigen-induced inflammation in which allergic mice (B6D2F1) that had been challenged once with aerosolized ovalbumin and had developed a pulmonary cellular infiltrate were rechallenged 1 wk later. Pulmonary inflammation in rechallenged mice was substantially greater than that in single-challenged mice. Eosinophils and activated-memory T cells (CD44+, CD45RBlo) in bronchoalveolar lavage (BAL) fluid accumulated to higher levels and with faster kinetics in response to the second challenge than in response to the first challenge. Eosinophils in lung tissue also accumulated to higher levels but with similar kinetics in response to the second challenge than in response to the first challenge. Similarly, interleukin (IL)-4 and IL-5 steady-state mRNA levels in lung tissue increased after the second challenge and were higher than those measured after a single challenge. Furthermore, treatment of mice with an anti-IL-5 monoclonal antibody 2 h prior to rechallenge inhibited antigen induced eosinophil accumulation in the lungs. In mice challenged twice, peak in vivo bronchoconstrictor responsiveness to acetylcholine was increased following the second challenge compared with that observed following the initial challenge. In contrast, ex vivo tracheal smooth muscle contractile responsiveness to acetylcholine was not altered. Although mucus accumulation and epithelial damage in pulmonary tissue were evident in mice challenged twice, these parameters were slightly reduced compared with those seen at similar times in mice challenged once. Therefore, although these mice exhibit only slight bronchial epithelial damage, the presence of significant inflammation and airway hyperreactivity to acetylcholine as well as slightly increased baseline reactivity demonstrate important similarities with the pathophysiology of asthma.

The v-rel oncogene promotes malignant T-cell leukemia/lymphoma in transgenic mice.

The oncogene product from the avian reticuloendotheliosis virus strain T, v-Rel, is a member of the Rel/ NF-kappa B family of transcription factors. The mechanism by which v-Rel induces oncogenic transformation remains unclear. Several attempts to transform mammalian cells with v-Rel have failed, suggesting that v-Rel transformation may be a species-specific event. However, here we demonstrate that v-Rel, but not a truncated c-Rel, expressed under the control of the lck promoter, efficiently induced malignancies in transgenic mice. Most of the animals died before 10 months of age and developed immature, multicentric aggressive T-cell leukemia/lymphomas. Most tumors contain CD4+CD8+ cells or CD4-CD8+ cells, which have an immature rather than a mature peripheral phenotype. No tumor development was observed in control littermates and transgenic mice expressing a truncated form of c-Rel. Tumor formation was correlated with the presence of constitutive p50/v-Rel DNA binding activity and overexpression of several kappa B-regulated genes in v-rel transgenic thymocytes. However, v-Rel is also transforming in transgenic thymocytes lacking p50, indicating that p50/v-Rel heterodimer formation is not essential for the transforming activity of v-Rel. The transforming activity of v-Rel in p50 null mice has been identified as v-Rel/v-Rel homodimers. Since tumors represent immature T-lymphocytes, constitutive v-Rel expression appears to be leukemogenic at earlier stages of T-cell development. These v-Rel mice should aid in the study of lymphoma development, T-cell development and NF-kappa B regulation.

Expression of FosB during mouse development: normal development of FosB knockout mice.

FosB, one of the members of the Fos family, is rapidly induced in many cell types upon stimulation and has a stimulatory effect on the proliferation of cultured cells. To understand the tissue distribution of FosB, we have studied its expression pattern by immunohistochemistry in newborn and late embryonic stage mice. These results show that FosB is widely expressed with the highest levels of expression observed in both bony and cartilagenous regions of developing bone. FosB is also detected within whisker follicles, liver, and epidermal tissue. To study the role of FosB in mammalian development we generated embryonic stem (ES) cells, mice and mouse embryo fibroblasts (MEFs) that are deficient for FosB. FosB -/- mice are born at a normal frequency, are fertile and present no obvious phenotypic or histologic abnormalities. FosB-deficient ES cells and MEFs proliferate and enter the S phase normally and we do not find upregulation of other fos family genes to compensate for the lack of FosB. However, we do find that the induction of two AP-1 containing genes is reduced after stimulation of FosB-deficient cells, demonstrating that FosB does indeed play a functional role in transcriptional regulation.

Pharmacological characterization of histamine H3 receptors in isolated guinea pig pulmonary artery and ileum.

We characterized the histamine H3 receptors involved in the modulation of electrical field stimulated neurogenic contraction of guinea pig pulmonary artery sympathetic, and guinea pig ileum parasympathetic preparations. Simultaneous measures of electrical field stimulation-evoked 3H overflow and tension in [3H]norepinephrine-loaded pulmonary artery were sensitive to tetrodotoxin (300 nM) and insensitive to hexamethonium (100 microM). Only the contractile response was inhibited by prazosin (100 nM). (R)-alpha-Methylhistamine's inhibition of the pulmonary artery contraction and 3H overflow were dose-dependently antagonized by thioperamide (30-100 nM). (R)-alpha-Methylhistamine also inhibited the neurogenic contractions of the isolated ileum (pD2 = 8.2). In the pulmonary artery, the relative potency of the histamine H3 receptor antagonists vs. (R)-alpha-methylhistamine inhibition of neurogenic contractions (pD2 = 7.1) was thioperamide (pA2 = 8.6 +/- 0.1) > burimamide (pA2 = 7.6 +/- 0.2) > impromidine (pA2 = 6.9 +/- 0.02). Similarly, the relative potency of histamine H3 receptor antagonists in the isolated ileum was thioperamide > burimamide > or = impromidine, with pA2 estimates of 8.7 +/- 0.1, 7.3 +/- 0.1 and 7.1 +/- 0.1, respectively. Antagonist potencies suggest a predominant histamine H3A-like receptor population on postganglionic sympathetic neurons innervating the pulmonary artery and parasympathetic neurons innervating the ileum longitudinal muscle.

Multiorgan inflammation and hematopoietic abnormalities in mice with a targeted disruption of RelB, a member of the NF-kappa B/Rel family.

RelB, a member of the NF-kappa B/Rel family of transcription factors, has been implicated in the constitutive expression of kappa B-regulated genes in lymphoid tissues. We have generated mice carrying a germline mutation of the relB gene, resulting in the absence of RelB protein and a dramatic reduction of constitutive kappa B-binding activity in thymus and spleen. Mice homozygous for the disrupted relB locus had phenotypic abnormalities including multifocal, mixed inflammatory cell infiltration in several organs, myeloid hyperplasia, splenomegaly due to extramedullary hematopoiesis, and a reduced population of thymic dendritic cells. RelB-deficient animals also had an impaired cellular immunity, as observed in contact sensitivity experiments. Thus, RelB plays a decisive role in the hematopoietic system, and its absence cannot be functionally compensated by any other member of the NF-kappa B/Rel family.

Further evidence for prejunctional GABA-B inhibition of cholinergic and peptidergic bronchoconstriction in guinea pigs: studies with new agonists and antagonists.

We examined the effect of the potent and selective GABA-B agonists, baclofen, 3-aminopropylphosphinic acid (3-APPi) and 3-aminopropyl (methyl) phosphinic acid (SKF 97541), and the GABA-B antagonists, 3-aminopropyl (diethoxymethyl) phosphinic acid (CGP 35348), 2-hydroxysaclofen and 3-aminopropylphosphonic acid (3-APPA) on cholinergic and peptidergic contractile responses in the airways of guinea pigs. Electrical field stimulation of the isolated guinea pig trachea produced cholinergic contractions that were inhibited by baclofen (EC50 = 5 mumol/l), 3-APPi (EC50 = 0.3 mumol/l) and SKF 97541 (EC50 = 0.4 mumol/l). The inhibition by baclofen (30 mumol/l) was reduced by CGP 35348 (IC50 = 65 mumol/l), 2-hydroxysaclofen (IC50 = 273 mumol/l) and 3-APPA (IC50 = 355 mumol/l). The in vivo cholinergic bronchoconstrictor response to vagal nerve stimulation (5 V, 20 Hz, 0.5 ms for 5 s) was attenuated by intravenous baclofen (ED50 = 1.7 mg/kg), 3-APPi (ED50 = 0.9 mg/kg) and SKF 97541 (ED50 = 0.2 mg/kg). The inhibition of vagally induced bronchoconstriction by baclofen was blocked by CGP 35348 (1-10 mg/kg, i.v.) and 2-hydroxysaclofen (10 mg/kg, i.v.). A cholinergic bronchoconstriction induced by CNS stimulation (400 microA, 2 ms, 32 Hz for 5 s) was inhibited by baclofen (ED50 = 5.1 mg/kg, i.v.) and 3-APPi (ED50 = 0.6 mg/kg, i.v.). The effect of baclofen was attenuated by 3-APPA (5 mg/kg, i.v.). A peptidergic bronchoconstriction was evoked by intravenous nicotine (1 mg/kg) in animals treated with ipratropium and phosphoramidon.(ABSTRACT TRUNCATED AT 250 WORDS)

5-HT3 receptors augment neuronal, cholinergic contractions in guinea pig trachea.

Serotonin (5-HT) and the selective 5-HT3 receptor agonist, 2-methyl-5-hydroxytryptamine enhanced electrical field stimulated contractions of the isolated guinea pig trachea. 5-HT (EC50 = 3.5 microM) was twice as potent as 2-methyl-5-hydroxytryptamine (EC50 = 7.4 microM). The effects of 5-HT and 2-methyl-5-hydroxytryptamine were antagonized by the selective 5-HT3 receptor antagonist; zacopride (apparent pA2 = 7.60 against 2-methyl-5-hydroxytryptamine). 2-Methyl-5-hydroxytryptamine (10 microM) had no effect on contractile responses to exogenous acetylcholine. Furthermore, the increase in electrical field stimulated contraction by 2-methyl-5-hydroxytryptamine was unchanged by hexamethonium (100 microM) but contractions were blocked by atropine (1 microM). These results suggest that excitatory 5-HT3 receptors exist on postganglionic cholinergic nerves in the isolated guinea pig trachea.

GABAB receptors in the lung.

gamma-Aminobutyric acid (GABA), an important inhibitory neurotransmitter in the mammalian CNS, is also found in peripheral tissues, including the lung. Recent pharmacological studies using selective ligands for GABAA and GABAB receptors demonstrate that of these two, the GABAB receptor is the important receptor subtype controlling lung functions. GABAB agonists inhibit a variety of responses in the airways, including neuronally induced cholinergic- and tachykinin-mediated smooth muscle contraction, microvascular leakage, anaphylactic bronchospasm and cough. Because these conditions are seen in certain respiratory diseases, such as asthma, a selective GABAB agonist may have therapeutic potential for the treatment of this respiratory disorder.

Both products of the fosB gene, FosB and its short form, FosB/SF, are transcriptional activators in fibroblasts.

We demonstrate that a member of the fos family, the fosB gene, gives rise to two transcripts by alternative splicing of exon 4, generating two proteins, FosB of 338 amino acids and a short form, FosB/SF, which contains the DNA binding and dimerization domains but not the 101 amino acids of the C terminus. FosB/SF activates an AP-1-chloramphenicol acetyltransferase construct in NIH 3T3 cells, as determined by transient and stable transfections, although more weakly than does FosB. In contrast to FosB, FosB/SF has lost its ability to repress the dyad symmetry element of the c-fos gene. FosB/SF when expressed in excess to FosB can downmodulate the activity of FosB. Constitutive expression of high levels of FosB/SF in NIH 3T3 cells has no significant inhibitory effect in the induction of cell proliferation or cell cycle progression, indicating that FosB/SF is not a negative regulator of cell growth. This conclusion is further confirmed by the observation that the majority of the Jun molecules are complexed with FosB/SF in the FosB/SF-overexpressing cells.

Constitutive expression of FosB and its short form, FosB/SF, induces malignant cell transformation in rat-1A cells.

We have established Rat-1A cell lines constitutively expressing c-Fos and the two products of the fosB gene, FosB and its short form, FosB/SF. The expressed proteins in the different stable transfectants have been characterized by immunofluorescence and immunoprecipitation analysis. Our results demonstrate that constitutive expression of FosB, like the constitutive expression of c-Fos and, to a lesser extent, FosB/SF, results in cells that grow to increased saturation densities and have the ability to grow in an anchorage-independent manner. Most important is the finding that expression of these proteins augments the tumorigenic potential of Rat-1A cells. These results show that both forms of FosB have a stimulatory effect on cell proliferation.