Pulmonary responses to bronchoconstrictor agonists in the mouse

Mice have been used in studies of the immunology or pathology of several different disorders affecting the lung. However, the value of the mouse for the analysis of pulmonary pathophysiology has been limited by the lack of methods for measuring lung function in the living animal. We report here the first method for measuring pulmonary conductance (GL) and compliance (Cdyn) in tracheostomized mechanically ventilated mice. We used this method to characterize the mouse's pulmonary responses to several putative bronchoconstrictor agonists. GL and Cdyn were decreased by intravenous infusions of methacholine, norepinephrine, or serotonin. Reproducible responses were not detected after infusions of histamine, prostaglandins D2 or F2 alpha, leukotrienes C4 or D4, substance P, or platelet-activating factor. The pattern of airway responsiveness to these agonists in the mouse is similar to that reported for the rat; in contrast to the rat, the mouse has many well-characterized strains or mutants with deficiencies of immunologic or inflammatory cells or mediators. As a result, this model offers unique advantages for identifying the roles of individual inflammatory cell types or mediators in pulmonary processes, including pulmonary anaphylaxis.     

Beta2-adrenoceptor agonists induce the mammalian clock gene, hPer1, mRNA in cultured human bronchial epithelium cells in vitro

The mammalian Per1 gene is one of the most important components of circadian clock function of the suprachiasmatic nucleus and peripheral tissues. We examined whether the β₂-adrenoceptor agonists, procaterol and fenoterol, induce human Per1 mRNA expression in human bronchial epithelium. The in vitro stimulation of β₂-adrenoceptor agonists in BEAS-2B cells led to a remarkable increase in the level of hPer1 mRNA. Moreover, fenoterol or procaterol induced the phosphorylation of CREB in BEAS-2B cells as verified by immunoblot analysis. β₂-adrenoceptor agonists induced human Per1 mRNA expression by the signaling pathways of cAMP-CREB in BEAS-2B cells.     

Beta2-adrenoceptor polymorphisms: relation between in vitro and in vivo phenotypes

Beta2-adrenoceptors are expressed in many cell types throughout the body and play a pivotal role in the regulation of the cardiac, pulmonary, vascular, endocrine and central nervous system. Recent studies have discovered that Beta2-adrenoceptor are polymorphic. Three common polymorphisms appear to influence receptor function: Arg16Gly, Gln27Glu, and Thr164Ile. In vitro studies of agonist-stimulation have shown that the Gly16 Beta2-adrenoceptors demonstrate enhanced agonist-promoted down-regulation, while Glu27 variants seem to be resistant. The Ile164 variant, on the other hand, demonstrates decreased responsiveness to agonist stimulation in vitro. However, the functional relevance and phenotypic consequence of such Beta2-adrenoceptor variants in vivo is still unclear. The aim of this review is therefore to provide an overview about the somewhat controversy in vitro, ex vivo and in vivo studies.     

Beta 2-adrenoceptor agonist-induced down-regulation after short-term exposure.

We examined the effect of duration of beta 2-adrenergic receptor (beta 2AR) occupancy by isoproterenol on specific binding of 125I-lodocyanopindolol (125I-ICYP) in membranes from rat L6 myoblasts. Ten minute exposure caused a time-and concentration-dependent maximal decrease in 125I-ICYP binding 24 hours after exposure equal to that following continuous exposure (p < 0.05). Low temperature, concanavalin A, H89 and ICl 118,551 blocked the decline in 125I-ICYP binding during the first hour following exposure probably representing receptor sequestration to a compartment or change to a form incapable of ligand binding. Compared to controls, receptor binding 4 and 24 hours following exposure was reduced 56 +/- 8.7% and 72 +/- 8.8%, respectively (p < 0.05), and was blocked by ICl 118,551 but not CGP12177. Isoproterenol-induced, but not forskolin-stimulated, cAMP accumulation was reduced 35% 24 hours following exposure (p < 0.05). 125I-ICYP binding in intact L6 cells 4 and 24 hours after exposure were respectively 56 +/- 8.9 and 61 +/- 13% of controls (p < 0.05). Following agonist exposure, CHO cell membranes expressing human beta 2ARs exhibited 125I-ICYP binding 85 +/- 2.0% and 6 +/- 2.8% of control values 4 and 24 hours, respectively (p < 0.05). A model predicting that full occupation of the beta 2AR activates receptor degradation explains our results that agonist-induced down-regulation of beta 2AR does not require continuous presence of the agonist.     

Distribution of bronchoconstrictor responses in isolated-perfused rat lung

We studied the effects of bronchoconstrictor stimuli administered selectively through isolated-perfused preparations of the bronchial and pulmonary circulations of 80 Sprague-Dawley rats. Dose-related contraction was elicited with infusion of acetylcholine (ACh), histamine, and serotonin (5-HT). Bolus infusion of 10(-5) mol ACh caused a 3.5-fold increase in pulmonary resistance (RL) after infusion into the pulmonary circulation (PC) and a 2.5-fold increase in the bronchial circulation (BC) (P less than 0.05 vs. control) that was blocked selectively in each circulation with atropine. Administration of 10(-5) mol 5-HT into the BC caused only a 45% increase in RL; the same dose of 5-HT caused a 5.1-fold increase in RL in the PC. A biphasic (increase at lower doses/decrease at higher doses) change in RL was elicited by histamine that was converted to dose-related constriction after H2-receptor blockade with cimetidine in both BC and PC. Response to exogenous ACh remained viable for greater than 5 h. Infusion of the mast cell degranulating agent, compound 48/80 (48/80), caused increase in RL that corresponded to quantitative recovery of histamine in the perfusates of both BC and PC. Histamine concentration in the perfusate increased from 47.2 +/- 31.8 (base line) to 624 +/- 60.1 ng/ml (2-fold increase in RL) in the BC and from 38.3 +/- 17.7 (base line) to 294.4 +/- 38.1 ng/ml (50% increase in RL) in the PC (P less than 0.001 vs. baseline concentration) after a 0.1-mg/ml dose of 48/80.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of OKY 1581 on bronchoconstrictor responses to arachidonic acid and PGH2

The influence of OKY 1581, a thromboxane synthase inhibitor, on airway responses to arachidonic acid and endoperoxide, [prostaglandin (PG) H2], were investigated in anesthetized, paralyzed, mechanically ventilated cats. Intravenous injections of arachidonic acid and PGH2 caused dose-related increases in transpulmonary pressure and lung resistance and decreases in dynamic and static compliance. OKY 1581 significantly decreased airway responses to arachidonic acid but not to PGH2. Sodium meclofenamate, a cyclooxygenase inhibitor, abolished airway responses to arachidonic acid but had no effect on airway responses to PGH2. OKY 1581 or meclofenamate has no effect on airway responses to PGF2 alpha, PGD2, or U 46619, a thromboxane mimic. In microsomal fractions from the lung, OKY 1581 inhibited thromboxane formation without decreasing prostacyclin synthesis or cyclooxygenase activity. These studies show that OKY 1581 is a selective thromboxane synthesis inhibitor in the cat lung and suggest that a substantial part of the bronchoconstrictor response to arachidonic acid is due to thromboxane A2 formation. Moreover, the present data suggest that airway responses to endogenously released and exogenous PGH2 are mediated differently and that a significant part of the response to exogenous PGH2 may be due to activation of an endoperoxide/thromboxane receptor, since responses to PGH2 are blocked by the thromboxane receptor antagonist SQ 29548.     

Analysis of bronchoconstrictor responses to platelet-activating factor in the cat

Bronchoconstrictor responses to platelet-activating factor (PAF) were investigated in paralyzed, anesthetized, mechanically ventilated cats. Intravenous injections of PAF caused dose-dependent increases in lung resistance (RL) and decreases in dynamic compliance (Cdyn) and systemic arterial (aortic) pressure (PAO). The increases in RL and decreases in Cdyn in response to PAF were markedly reduced by sodium meclofenamate, a cyclooxygenase inhibitor, whereas the decreases in PAO were unchanged. Increases in RL and decreases in Cdyn but not PAO in response to PAF were reduced by SQ 29,548, a thromboxane (Tx) receptor blocking agent. CV 3988, a PAF receptor antagonist, reduced bronchoconstrictor and aortic depressor responses to PAF but had no effect on responses to the Tx mimic, U 46619, or to the prostaglandin precursor, arachidonic acid. The present studies suggest that bronchoconstrictor responses but not the hypotensive response to PAF are mediated in large part by the release of arachidonic acid from cell membrane phospholipids and the formation of cyclooxygenase products including TxA2. These data indicate that although airway and vasodepressor responses to PAF in the cat are mediated by different mechanisms, a similar PAF-CV 3988-sensitive receptor is involved.     

Intravenous versus inhaled atropine for inhibiting bronchoconstrictor responses in dogs

We studied whether the muscarinic antagonist, atropine, given intravenously or by inhalation, inhibits the bronchoconstrictor responses to inhaled acetylcholine and to acetylcholine released by electrical stimulation of the vagus nerves to the same degree. We assessed bronchoconstrictor responses in anesthetized dogs by determining the increase in total pulmonary resistance before and after increasing doses of atropine and then constructing inhibition dose-response curves. Before atropine the responses to the two stimuli were equal in magnitude. After intravenous atropine (initial dose 0.12 micrograms/kg, total dose 16 micrograms/kg) both responses were progressively inhibited to a similar degree. By contrast, after inhaled atropine (initial dose 0.02 micrograms/kg, total dose 2.4 micrograms/kg) the response to acetylcholine inhalation was inhibited to a much greater degree than the response to vagal stimulation. Thus, in studies designed to inhibit bronchoconstriction due to an inhaled muscarinic agonist to the same degree as bronchoconstriction due to a vagal reflex, atropine might better be given intravenously than by inhalation.     

Beta 1 and beta 2 adrenergic agonists in exocrine pancreatic secretion in the rabbit

The effect of Dobutamine (a beta 1-adrenergic agonist) and Terbutaline (a beta 2-adrenergic agonist) on exocrine pancreatic secretion was studied in anaesthetized rabbits, simultaneously controlling pancreatic blood flow and blood pressure. The secretion of fluid and ions (bicarbonate, sodium and potassium) was unaffected by the infusion of Dobutamine (8 micrograms.kg-1.min-1) or Terbutaline (10 micrograms.kg-1.min-1). Neither were pancreatic blood flow or mean blood pressure altered. Dobutamine or Terbutaline depress the function of the acinar cells, amylase secretion being more affected by the action of Terbutaline. The results show that beta 1 and beta 2-adrenergic stimulation has no effect on the ductular cells but does decrease the secretion by the acinar cells.     

Soluble factors from BCG-induced suppressor cells inhibit in vitro PFC responses but not cytotoxic responses.

A macrophage-like suppressor cell is present in the spleens of BCG-infected C57BL/6 mice. This suppressor cell is capable of suppressing both in vitro cytotoxic and PFC responses of normal C57BL/6 spleen cells. Suppression was not caused by changes in the kinetics of the responses or in the quantities of antigen required for stimulation. Suppression of the in vitro cytotoxic response could not be linked to any soluble mediator. In contrast, supernatants obtained from BCG spleen cell cultures, which failed to inhibit alloantigen-induced cytotoxic responses, suppressed the in vitro PFC response to SRBC by normal C57BL/6 spleen cells. It is postulated that either BCG-induced macrophage-like suppressor cells inhibit these in vitro responses via different mechanism(s) or these responses are regulated by different suppressor cell subpopulations within the monocyte/macrophage compartment of BCG spleen cells.     

Effect of bradykinin on airway neural responses in vitro.

We investigated the effects of bradykinin (BK) on airway excitatory nonadrenergic noncholinergic (e-NANC) and cholinergic nerves in vitro. Neural responses were elicited by electrical field stimulation in guinea pig airways in vitro before and after the addition of BK (10(-10)-10(-7) M). Captopril (10(-5) M) and phosphoramidon (10(-6) M) were added to prevent degradation of BK, and all neural responses were measured in the presence of indomethacin (10(-5) M) and propranolol (10(-6) M). BK potentiated e-NANC responses in bronchi in a concentration-dependent manner (10(-10)-10(-7) M) without changing concentration-response curves to exogenously applied substance P (10(-10)-10(-5) M). BK significantly potentiated e-NANC neural constrictor responses by 22 +/- 7% at 10(-8) M (mean +/- SE, n = 5, P < 0.05) and 32 +/- 7% at 10(-7) M (n = 8, P < 0.01), compared with changes in time-matched control tissues (7 +/- 2%, n = 8). The potentiation of e-NANC responses by BK was abolished by pretreatment with a specific B2-receptor antagonist, HOE 140 (10(-7) M). Cholinergic constrictor responses elicited to electrical field stimulation were not affected by the addition of BK (up to 10(-7) M). These results suggest that BK potentiates e-NANC bronchoconstrictor responses prejunctionally via a B2-receptor.     

Studies towards topical selective β2-adrenoceptor agonists with a long duration of action

β₂-Adrenoceptor agonists with basic and acidic groups attached via an alkyl linker to the phenyl ethanolamine core were prepared and investigated in vitro and in vivo. The compounds exhibited a high potency in a functional cellular assay and a bronchoprotective effect in a guinea pig model which lasted over the complete study period of 5 h.     

Desensitization of the vascular contractile response to cumulative doses of alpha 2-adrenoceptor agonists

Contractile responses to single or cumulative doses of alpha-adrenoceptor agonists were compared in the tail artery and the saphenous vein of the rat. In the rat tail artery, there were no differences in the dose-response relationships to noradrenaline, methoxamine, and KCl whether the agonists were applied as single or cumulative doses. However, the responses to single doses of clonidine and B-HT 920 were significantly larger than similar doses applied cumulatively. In the rat saphenous vein, responses to single doses of noradrenaline, clonidine, and B-HT 920 were also significantly larger than the corresponding cumulative doses. However, there was no difference in the responses to KCl. It was suggested that desensitization of alpha 2-adrenoceptors in these vessels may result in the diminished responses to cumulative doses of the agonists. Desensitization appeared to be specific to alpha 2-adrenoceptors, since the effect was not observed in responses mediated by the alpha 1-adrenoceptors and KCl.     

Dimeric small molecule agonists of EphA2 receptor inhibit glioblastoma cell growth

EphA2 receptor kinase could become a novel target for anti-glioblastoma treatment. Doxazosin previously identified acts like the endogenous ligand of EphA2 and induces cell apoptosis. Through lead structure modification a derivative of Doxazosin possessing unique dimeric structure showed an improvement in the activity. In the current study, we expanded the dimeric scaffold by lead optimization to explore the chemical space of the conjoining moieties and a slight variation to the core structure. 27 new derivatives were synthesized and examined with EphA2 overexpressed and wild type glioblastoma cell lines for cell proliferation and EphA2 activation. Three new compounds 3d, 3e, and 7bg showed potent and selective activities against the growth of EphA2 overexpressed glioblastoma cells. Dimer 3d modification replaces the long alkyl chain with a short polyethylene glycol chain. Dimer 7bg has a relatively longer polyethylene glycol chain in comparison to compound 3d and the length is more similar to the lead compound. Whereas dimer 3e has a rigid aromatic linker exploring the chemical space. The diversity of the linkers in the active suggest additional hydrogen binding sites has a positive correlation to the activity. All three dimers showed selective activity in EphA2 overexpressed cells, indicating the activity is correlated to the EphA2 targeting effect.