Functional characterization of beta-adrenoceptors mediating relaxation in sheep gallbladder

The purpose of this study was to characterize beta-adrenoceptor subtype(s) mediating relaxation in smooth muscle strips of the sheep gallbladder. Experiments were performed on isolated smooth muscle strips suspended in tissue baths containing Krebs' solution. Isoprenaline (10(-8) M-10(-5) M) and salbutamol (10(-7) M-10(-4) M) produced concentration-dependent relaxation of carbachol (10(-7) M-3 x 10(-7) M) contracted smooth muscles of the sheep gall bladder. Isoprenaline-induced relaxation was significantly antagonized by propranolol with -logKB values of 7.81 +/- 0.11 (n = 7) and 7.73 +/- 0.12 (n = 6) in the fundic and ductal strips respectively. Atenolol (10(-5) M), a selective beta 1-adrenoceptor antagonist, also significantly antagonized isoprenaline-induced relaxation with -logKB values of 5.82 +/- 0.11 and 6.09 +/- 0.09 in the fundic and ductal strips respectively. However, ICI 118551, a selective beta 2-adrenoceptor antagonist, at concentrations up to 10(-6) M had little or no effect on isoprenaline-induced relaxation in either of these preparations. BRL 37344A, a selective beta 3-adrenoceptor agonist produced concentration-dependent relaxation of carbachol-precontracted fundic and ductal strips. BRL 37344 was approximately 9-fold more potent in the ductal than fundic strips. In both preparations, BRL 37344-induced relaxation was not significantly (p > 0.05) antagonized by propranolol (3 x 10(-7) M). This would confirm that the response was mediated via beta 3-adrenoceptors. It was concluded that beta 1- and beta 3-adrenoceptors coexist in the sheep gallbladder and mediate smooth muscle relaxation.     

Preserved bronchial dilatation after salbutamol does not guarantee protection against bronchial hyperresponsiveness

Racemic salbutamol, a beta2-adrenoceptor agonist used for dilatation of airways, has recently been shown to induce lessened relaxation of bronchial smooth muscle and partial loss of bronchoprotection, seen as increased hyperresponsiveness, after regular treatment. The racemate undergoes stereo-selective disposition, giving higher plasma levels of S-salbutamol than that of bronchodilating R-salbutamol, thus raising S : R ratios after repeated administration. Our aim was to evaluate whether increased bronchial hyperresponsiveness (BHR) could be found even after 1 day of repeated salbutamol inhalations, with beta2-receptor-induced bronchial smooth muscle relaxation remaining and whether this would be associated with plasma levels of either enantiomer. Fifteen patients with stable asthma, aged 19-54 years, were included in a randomized, cross-over study. An indirect bronchial challenge method was used [voluntary isocapnic hyperventilation of cold air (IHCA)], and airway condition tested by means of impulse oscillometry. Racemic salbutamol was inhaled three times during a 6-h period. IHCA was performed and plasma concentrations of enantiomers were measured 4 h after the last dose. Tests were also performed without preceding drug treatment. beta2-Agonist-produced bronchial dilatation and protection persisted in the majority of the 15 patients 4 h after repeated inhalations of salbutamol during 1 day. In only two of the 15 patients we could trace increased BHR after salbutamol. Neither dilatation nor protection could be linked to plasma levels of either R- or S-salbutamol. The underlying mechanisms of BHR remain unknown and are dissociated from beta2-receptor-mediated dilatation.     

A nitric oxide-releasing salbutamol elicits potent relaxant and anti-inflammatory activities

Beta2-adrenoceptor agonists are widely used in the treatment of pulmonary diseases. We have investigated the relaxant and anti-inflammatory activities of NCX-950 (alpha'-[[(1,1-dimethylethy)amino]methyl]-4-hydroxy-1,3-benzenedimethanol nitrate) (a nitric oxide-releasing salbutamol) in human isolated bronchi and on lipopolysaccharide (LPS)-induced acute airway inflammation in mice. NCX-950 (10(-8)-10(-5) M) elicited a relaxation of human isolated bronchi moderately higher than salbutamol, which was reduced by a beta-adrenergic blocking drug, propranolol, but not by an inhibitor of guanylate cyclase, ODQ (1H-[1,2,4]oxadiazolo[4,3-] quinolaxin-1-one). The treatment of mice with NCX-950 (1, 10, and 100 microM aerosol) markedly inhibited the neutrophil influx induced by LPS aerosol in bronchoalveolar lavage (BAL) fluid, whereas salbutamol at equimolar doses elicited a moderate inhibition. Pretreatment of mice with NCX-950 (100 microM) also significantly reduced tumor necrosis factor-alpha, interleukin-6 (IL-6), transforming growth factor-beta, and matrix metalloproteinase-9 release in BAL fluid, whereas salbutamol was ineffective. Propranolol, but not ODQ, suppressed the inhibitory activity of NCX-950 on neutrophil influx and IL-6 release in BAL fluids. A nitric oxide-releasing sildenafil NCX-911 [(5-[2-ethoxy-5-(4-methylpiperidinylsulfonyl)phenyl]-1-methyl-3-n-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one nitrate)], but not sildenafil (100 microM) also reduced the neutrophil influx following LPS exposure in mice. This study reported that NCX-950 elicits potent relaxant and anti-inflammatory activities compared with salbutamol, and these effects may be mainly due to the activation of the beta2-adrenoceptor rather than the cGMP pathway.     

Effect of a beta 2-adrenoceptor stimulation on hyperglycemia-induced endothelial dysfunction

To investigate whether beta(2)-adrenoceptors exist on endothelial cells and whether a beta(2)-adrenoceptor stimulation might prevent the development of hyperglycemia-induced endothelial dysfunction, porcine aortic endothelial cells (PAECs) were cultured and chronically exposed to either 5 mM D-glucose ("normoglycemia") or 20 mM D-glucose ("hyperglycemia"), with or without 100 nM salbutamol in absence or presence of beta(2)-adrenoceptor antagonist ICI 118,551 [1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxyl]-3-[(1-methylethyl)-amino]-2-butanol] or beta(1)-antagonist metoprolol. For osmotic control, PAECs were exposed to 15 mM L-glucose. We measured nitric oxide release using the met-hemoglobin assay and assessed beta-adrenoceptor density and subtypes by radioligand binding. Furthermore, we determined intracellular NADH and NADPH using high-performance liquid chromatography. High D-glucose concentrations but not L-glucose led to significantly reduced basal and stimulated nitric oxide release. Chronic salbutamol treatment significantly antagonized the impairment of the nitric oxide response, which was inhibited by ICI 118,551 but not by metoprolol. The number of giant cells was significantly increased in hyperglycemia, which could be prevented by salbutamol. Binding of the radioligand (-)-[(125)I]iodocyanopindolol revealed a total beta-adrenoceptor density of 29.8 +/- 3.7 (normoglycemic) and 30.3 +/- 3.6 (hyperglycemic) fmol/mg protein. Displacement by ICI 118,551 revealed beta-adrenoceptor subtype distribution with 30.3 +/- 4.4 (normoglycemic) and 29.1 +/- 3.8% beta(2)-adrenoceptors. NADH production increased in hyperglycemia, which was completely prevented by salbutamol. We conclude that hyperglycemia in PAEC induces endothelial dysfunction with impaired nitric oxide release and that this can be prevented by beta(2)-adrenoceptor stimulation.     

In vitro and in vivo pharmacological characterization of ethyl-4-[trans-4-[((2S)-2-hydroxy-3-[4-hydroxy-3[(methylsulfonyl)amino]-phenoxy]propyl) amino]cyclohexyl]benzoate hydrochloride (SAR150640), a new potent and selective human beta3-adrenoceptor agonist for the treatment of preterm labor

Ethyl-4-[trans-4-[((2S)-2-hydroxy-3-[4-hydroxy-3[(methylsulfonyl)amino] phenoxy]propyl) amino]cyclohexyl]benzoate hydrochloride (SAR150640) was characterized as a new potent and selective beta(3)-adrenoceptor agonist for the treatment of preterm labor. SAR150640 and its major metabolite, the corresponding acid 4-[trans-4-[((2S)-2-hydroxy-3-[4-hydroxy-3[(methylsulfonyl) amino] phenoxy]propyl)amino]cyclohexyl]benzoic acid (SSR500400), showed high affinity for beta(3)-adrenoceptors (K(i) = 73 and 358 nM) and greater potency than (-)-isoproterenol in increasing cAMP production in membrane preparations from human neuroblastoma cells (SKNMC), which express native beta(3)-adrenoceptors (pEC(50) = 6.5, 6.2, and 5.1, respectively). SAR150640 and SSR500400 also increased cAMP production in membrane preparations from human uterine smooth muscle cells (UtSMC), which also express native beta(3)-adrenoceptors (pEC(50) = 7.7 and 7.7, respectively). In these cells, SAR150640 dose-dependently inhibited oxytocin-induced intracellular Ca(2+) mobilization and extracellular signal-regulated kinase 1/2 phosphorylation. SAR150640 and SSR500400 had no beta(1)- or beta(2)-agonist or antagonist activity in guinea pig atrium and trachea, or in human isolated atrium and bronchus preparations. Both compounds concentration-dependently inhibited spontaneous contractions in human near-term myometrial strips, with greater potency than salbutamol and 4-[3-[(1,1-dimethylethyl)-amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one hydrochloride (CGP12177) (pIC(50) = 6.4, 6.8, 5.9, and 5.8, respectively), but with similar potency to (-)-isoproterenol and atosiban (oxytocin/vasopressin V(1)a receptor antagonist). SAR150640 also inhibited the contractions induced by oxytocin and prostaglandin F(2alpha). In vivo, after intravenous administration, SAR150640 (1 and 6 mg/kg), but not atosiban (6 mg/kg), dose-dependently inhibited myometrial contractions in conscious unrestrained female cynomolgus monkeys, with no significant effects on heart rate or blood pressure. In contrast, salbutamol (50 and 250 microg/kg) had no inhibitory effect on uterine contractions, but it dose-dependently increased heart rate. These findings indicate a potential for the therapeutic use of SAR150640 in mammals during preterm labor.     

Possible involvement of prostaglandins in the action of ATP on guinea-pig uterus.

ATP and other adenine derivatives, such as AMP and adenosine, at concentrations above 10(-6) M induced dose-dependent contractions of guinea-pig uterine strips. Treatment of the strips with nonsteroidal anti-inflammatory drugs, such as indomethacin, aspirin and phenylbutazone, at concentrations of 10(-6) to 10(-4) M irreversibly inhibited the contractions, without affecting those caused by acetylcholine and bradykinin. Arachidonic acid (10(-8)-10(-6) g/ml) and prostaglandins (E1, E2 and F2 alpha, 10(-9)-10(-7) g/ml) restored the inhibited uterine response to ATP, but the inhibition was reinstated on washing out of the arachidonic acid or prostaglandins. Furthermore, the prostaglandin antagonists polyphloretin phosphate (3 x 10(-5)-3 x 10(-4) g/ml) and SC 19220 (10(-6)-3 X 10(-5) M) selectively suppressed the action of ATP. In addition to the prostaglandin antagonists, 2,2'-pyridylisatogen, reported to be an ATP antagonist, at concentrations of 10(-6) to 3 x 10(-5) M selectively inhibited the response of uterine strips to ATP. These results suggest the involvement of prostaglandins in the actions of ATP and other adenine derivatives on guinea-pig uterine tissue and provide further evidence for ATP-stimulated prostaglandin formation in smooth muscle.     

Influence of methoctramine on the acetylcholine-isoprenaline functional antagonism in the guinea pig isolated trachea

Summary— It has been suggested that activation of muscarinic M₂ receptors is one of the components of the functional antagonism between muscarinic and β-adrenoceptor agonists in canine and guinea pig tracheal smooth muscle. The aim of the present study was to determine in the guinea pig trachea the importance of this component according to the magnitude of the acetylcholine-induced contraction. Cumulative concentration-response curves for isoprenaline were obtained in the absence or presence of the muscarinic M₂ receptor antagonist methoctramine (3 × 10^?7 M) in tracheal rings under basal tension or precontracted by acetylcholine 2 × 10^?7, 3 × 10^?6 and 10^?4 M, giving contractions of 25, 50 or 75%, respectively, of the maximal tension induced by acetylcholine 3 × 10^?3 M. In the absence of methoctramine, acetylcholine induced a concentration-dependent shift of the concentration-response curves of isoprenaline (-log EC₅₀ of isoprenaline are 8.09 ± 0.07, 7.85 ± 0.08, 7.38 ± 0.12 and 6.49 ± 0.12, n = 6 for basal tension and for acetylcholine concentrations of 2 × 10^?7, 3 × 10^?6 and 10^?4 M, respectively). In the presence of methoctramine, the basal -log EC₅₀ of isoprenaline was unmodified, whereas the acetylcholine-induced shifts of concentration-response curves of isoprenaline were abolished for low levels of contraction (25%) and significantly reduced to 50 and 75% levels of contraction. Under similar conditions, acetylcholine-induced shifts of concentration-response curves of isoprenaline were unmodified by the muscarinic M₁ receptor antagonist pirenzepine (10^?7 M). These results suggest that the inhibitory effect of M₂ receptors on β-adrenoceptor agonists effects is important for low contraction levels induced by acetylcholine, and that this effect becomes less important for higher concentrations of acetylcholine.     

ROLE OF EXTRACELLULAR CALCIUM IN THE EFFECTS OF SUBSTANCE P AND NEUROKININ A ON GUINEA PIG TRACHEA AND HUMAN BRONCHUS

Whether the influx of calcium through voltage-operated channels is involved in the stimulatory effects of substance P and neurokinin A in airways smooth muscle is not yet firmly established. This question was addressed in the present study using guinea pig trachea and human bronchi suspended in normal or calcium-free Krebs solution and tested with inhibitors of calcium channels. In calcium-free Krebs solution, the myotropic effects of substance P (10(-7) M), neurokinin A (3.10(-8) M), acetylcholine (2.10(-5) M), and histamine (2.10(-5) M) were reduced by 27-57%, while those of potassium chloride and tetraethylammonium were practically abolished. Calcium antagonists such as verapamil or nicardipine, when applied at concentrations of 10(-8)-10(-6) M, inhibited the contractions produced by potassium chloride and tetraethylammonium, whereas higher concentrations (10(-5)-10(-4) M) of both inhibitors were needed to reduce the effects of substance P, neurokinin A, acetylcholine, and histamine. In neither preparation did the calcium agonist Bay K 8644 (10(-6) M) modify the effects of neurokinin A, substance P, acetylcholine, or histamine, but it potentiated potassium chloride's effect on human bronchi. We conclude that transmembrane calcium influx through voltage-operated channels plays a minor role in the stimulatory effects of neurokinins in airways smooth muscle.     

Inhibition of airway smooth muscle tone by a phorbol ester in the guinea pig trachea: role of epithelium and receptor reserve of the contractile agent

Bronchial hyperresponsiveness in patients with asthma may be associated with a damaged or dysfunctional epithelium. Also, changes in the activities of protein kinase C have been implicated in the pathogenesis of asthma. This study examined the role of protein kinase C in the modulation of airway smooth muscle tone and the influence of the epithelium on this function. Phorbol-12,13-diacetate (PDA) (10(-8) to 10(-5) M) induced concentration-dependent and epithelium-independent relaxations of guinea pig tracheal rings. PDA (10(-8) to 10(-5) M) induced significantly greater relaxations of tracheal rings contracted with 5-hydroxytryptamine (10(-5) M) than in tissues contracted to an equivalent degree with acetylcholine (10(-6) M). In experiments using phenoxybenzamine (10(-7) M and 10(-5) M), the dissociation constant (KA) for acetylcholine was significantly greater than that for 5-hydroxytryptamine. The fraction of active receptors (q) calculated for acetylcholine was significantly smaller than that calculated for an equieffective concentration of 5-hydroxytryptamine. Relaxations to PDA in tissues contracted with acetylcholine (2 x 10(-6) M) or 5-hydroxytryptamine (10(-5) M) were significantly augmented by phenoxybenzamine (10(-5) M and 10(-7) M, respectively). PDA did not affect contractions to acetylcholine (10(-8) to 10(-3) M) in the presence of epithelium but caused a significant right-ward displacement of the acetylcholine concentration-contraction curve in the absence of epithelium. The concentration-contraction curves for 5-hydroxytryptamine (10(-8) to 10(-5) M) were significantly displaced to the right by PDA in the presence or absence of epithelium. This effect was greater in the absence of epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autocrine role of interleukin 1beta in altered responsiveness of atopic asthmatic sensitized airway smooth muscle.

The role of IL-1beta in regulating altered airway responsiveness in the atopic/asthmatic sensitized state was examined in isolated rabbit tracheal smooth muscle (TSM) tissue and cultured cells passively sensitized with sera from atopic asthmatic patients or nonatopic/nonasthmatic (control) subjects. During half-maximal isometric contraction of the tissues with acetylcholine, relative to control TSM, the atopic sensitized TSM exhibited significant attenuation of both their maximal relaxation (P < 0.001) and sensitivity (i.e., -log dose producing 50% maximal relaxation) to isoproterenol and PGE2 (P < 0.05), whereas the relaxation responses to direct stimulation of adenylate cyclase with forskolin were similar in both tissue groups. The impaired relaxation responses to isoproterenol and PGE2 were ablated in sensitized TSM that were pretreated with either the IL-1 recombinant human receptor antagonist or an IL-1beta-neutralizing antibody. Moreover, extended studies demonstrated that, in contrast to their respective controls, both passively sensitized rabbit TSM tissue and cultured cells exhibited markedly induced expression of IL-1beta mRNA at 6 h after exposure to the sensitizing serum, a finding similar to that also obtained in passively sensitized human bronchial smooth muscle tissue. Finally, unlike their respective controls, passively sensitized TSM tissue and cultured cells also displayed progressively enhanced release of IL-1beta protein into the culture media for up to 24 h after exposure to atopic/asthmatic serum. Collectively, these observations provide new evidence demonstrating that the altered responsiveness of atopic/asthmatic sensitized airway smooth muscle is largely attributed to its autologously induced expression and autocrine action of IL-1beta.     

Effects of the methyl xanthine S 9795 on isolated bronchi of the dog

The xanthine derivative 1-methyl 3-isobutyl 8-(2-ethyl[1-(4-diphenylmethyl piperazinyl)])3, 7-dihydro (1H) purine 2, 6-dione (S 9795) is a potent inhibitor of bronchoconstriction in vivo. The aim of the present study was to analyze the effects of S 9795 in vitro and determine whether S 9795 affects the autonomic nerves, the epithelium or the smooth muscle of the bronchial wall. S 9795 had an inhibitory effect on the contractile responses evoked by acetylcholine and by electrical stimulation of the cholinergic nerves. S 9795 appeared more potent against contractions evoked by nerve stimulation. In addition, S 9795 caused the release of [3H]norepinephrine from the adrenergic nerve endings but did not affect neuronal uptake of the catecholamine. At low concentrations, S 9795 acted as a competitive serotonergic antagonist; at higher concentrations, the compound inhibited noncompetitively the contractions evoked by histamine and acetylcholine. In both second and fourth order bronchi, S 9795 (and theophylline) produced concentration-dependent relaxations that were significantly greater in rings with, compared with rings without, epithelium. The compound also facilitated the epithelium-dependent component of the relaxation response to beta-adrenergic activation. These results suggest that S 9795: 1) causes prejunctional inhibition of the release of acetylcholine, 2) evokes the displacement of stored norepinephrine, 3) exerts a differential inhibitory effect on airway contractions induced by various bronchoconstrictors and 4) augments the release or facilitates the effect of the epithelium-derived relaxing factor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological characterization of KUR-1246, a selective uterine relaxant

The aim of the present study was to evaluate the efficacy and beta 2-adrenoceptor (AR) selectivity of KUR-1246, a new uterine relaxant. Inhibition of spontaneous or drug-induced uterine contractions by KUR-1246 was evaluated in pregnant rats and rabbits by an organ bath method or by a balloon method. The selectivity of KUR-1246 was assessed simultaneously in organs isolated from late-pregnant rats. The affinity of KUR-1246 for human beta 1-, beta 2-, and beta 3-ARs was determined using two radioligands. KUR-1246 suppressed both spontaneous and drug-induced contractions in isolated uteri, the rank order of potency being isoproterenol > KUR-1246 > terbutaline > ritodrine. ICI-118551 (selective beta 2-AR antagonist) competitively antagonized the KUR-1246-induced inhibition of spontaneous uterine contractions, but CGP-20712A (selective beta 1-AR antagonist) and SR-58894A (selective beta 3-AR antagonist) did not. All beta-AR agonists tested produced significant inhibition of spontaneous uterine contractions in vivo: ED(30) value for KUR-1246 was 0.13 microg/kg/min, a potency about 6 times and 400 times greater than that of terbutaline and ritodrine, respectively. In contrast, the positive chronotropic effect was minimal in KUR-1246-treated rats. KUR-1246 displaced radioligand binding to beta 1-, beta 2-, and beta 3-ARs, the pK(i) values being 5.75 +/- 0.03, 7.59 +/- 0.08, and 4.75 +/- 0.03 for beta 1-, beta 2-, and beta 3-ARs, respectively. For the selectivity of KUR-1246 for human beta 2-AR, we obtained values of 39.2 ([IC(50) for beta 1-AR]/[IC(50) for beta 2-AR]) and 198.2 ([IC(50) for beta 3-AR]/[IC(50) for beta 2-AR]), indicating an apparently higher affinity for human beta 2-AR than for other beta-AR subtypes. The present study clearly demonstrated that KUR-1246 is a more selective beta 2-AR agonist than the drugs presently used for relaxing uterine muscle.     

Effects of ergometrine on airway smooth muscle contractile responses

A 26-year-old asthmatic female developed severe asthma within a few hours of receiving three oral doses of 0.4 mg ergometrine maleate for the control of postpartum haemorrhaging. This experience and two previous reports of bronchospasm in asthmatic subjects following ergometrine suggested that ergometrine altered airway smooth muscle tone. In the present investigation the effect of ergometrine was studied on canine tracheal smooth muscle strips. Ergometrine (10(-9) M-10(-4) M) induced contraction of canine tracheal smooth muscle. The concentration causing 50% of maximal contraction (EC50) was 4.73 X 10(-8) M. The acetylcholine EC50 was not altered by ergometrine (10(-9) M or 10(-8) M); however, acetylcholine (10(-4) M and 10(-3) M) induced contractions were enhanced by ergometrine (10(-8) M). The data suggest that ergometrine maleate may cause broncho-constriction in some patients with asthma.     

Chemical oxidant potentiates electrically and acetylcholine-induced contraction in rat trachea: possible involvement of cholinesterase inhibition

To determine the roles of oxidants in airway responsiveness, we studied the effects of the chemical oxidant N-chlorosuccinimide (NCS) on the contractile responses to electrical field stimulation (EFS) and acetylcholine (ACh) in isolated rat tracheal smooth muscle segments. Effects of NCS on the contractile response to EFS (5 Hz, 20 sec of duration, 50 V) reached the maximum with a 60-min incubation time. NCS potentiated the contractile response to EFS, with a maximum effect at 3 x 10(-7) M and to ACh, with a maximum effect at 3 x 10(-6) M. Thus, at a concentration of 3 x 10(-6) M, NCS significantly decreased log ED50 concentration of ACh from a control value of -5.56 +/- 0.05 to -6.24 +/- 0.06. Physostigmine (10(-7) M), at a concentration that did not alter resting tension, mimicked NCS-induced effects on contractile responses to ACh and EFS with the greater degree of shift in the respective dose-response curves. However, NCS failed to alter dose-response curves to carbachol. Removal of the epithelium shifted the dose-response curves to ACh to lower concentrations, but NCS showed similar effects on dose-response curves to ACh with and without the epithelium. Active staining showed that both acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase (EC 3.1.1.8) activities were found in the smooth muscle of the rat trachea. NCS inhibited both enzyme activities from rat tracheal homogenates in a concentration-dependent fashion. These results suggest that NCS potentiates cholinergically induced contraction by decreasing cholinesterase activity and that the oxidation of cholinesterase may cause hyperresponsiveness of airway smooth muscle by inhibition of the enzyme activity.     

Differential effects of epinephrine and norepinephrine on cAMP response and g(i3)alpha protein expression in cultured sympathetic neurons.

The effect of 24-h pretreatment with epinephrine (EPI) or norepinephrine (NE) on alpha(2)- and beta-adrenoceptor agonist-induced, cAMP responses and G(i3)alpha-protein expression was studied in primary cultures of rat superior cervical ganglionic (SCG) neurons. SCG neurons, 10 to 12 days in culture, accumulated cAMP when stimulated with the beta-adrenoceptor agonist isoproterenol and the preferential beta(2)-adrenoceptor antagonist ICI 118,551 blocked this response. Similarly, the preferential alpha(2)-adrenoceptor agonist UK14,304 inhibited forskolin-stimulated cAMP accumulation, implying that cultured SCG neurons possess functional alpha(2)- and beta(2)-adrenoceptors. A 24-h treatment of SCG neurons with EPI or NE induced desensitization of the cAMP response to the beta-adrenoceptor agonist isoproterenol. Simultaneously, EPI treatment increased the maximal inhibitory cAMP response to the alpha(2)-adrenoceptor agonist UK14,304 and NE was without effect. Immunoblotting analyses of G(i3)alpha subunits revealed that 24-h EPI but not NE treatment induces a 3- to 4-fold increase in the expression of G(i3)alpha subunits. Furthermore, EPI-induced up-regulation of alpha-subunit expression can be blocked by the preferential beta(2)-adrenoceptor antagonist ICI 118,551 but not by the preferential beta(1)-adrenoceptor antagonist CGP 20712A. Our results suggest that changes in alpha(2)-adrenoceptor responsiveness induced by EPI may involve activation of beta(2)-adrenoceptors that influence the expression of inhibitory G proteins. Thus, primary cultures of sympathetic neurons by possessing functional alpha(2)- and beta-adrenoceptors may be a suitable model system to study the signaling mechanisms of "cross talk" between these adrenoceptor subtypes, which are known to play a central role in cardiovascular function.     

Effects of the calcium antagonist tiapamil on pulmonary gas exchange in patients with chronic obstructive pulmonary disease who receive salbutamol

Patients with chronic obstructive pulmonary disease (COPD) and cardiovascular diseases are frequently given combination therapy with a beta 2-agonist and a calcium antagonist. Each drug is known to increase ventilation-perfusion inequalities. It was our aim to define the effects of their combination on lung function and on pulmonary gas exchange in eight subjects with COPD but partially reversible airway obstruction. Sixty minutes after placebo or 450 mg tiapamil, subjects inhaled 0.2 mg salbutamol. There was no significant effect of tiapamil on specific airway conductance and the forced expiratory volume in 1 second before or after the inhalation of salbutamol. Blood was drawn 30, 55, 80, and 100 minutes after placebo or tiapamil dosing. After placebo the mean (+/- SD) arterial oxygen tension (Pao2) fell from 67.1 +/- 7.3 to 64.4 +/- 5.5 mm Hg and the mean alveolar-arterial oxygen tension difference (AaDo2) rose from 34.6 +/- 8.4 to 40.5 +/- 6.8 mm Hg. After tiapamil the mean Pao2 fell from 68.7 +/- 7.3 to 66.4 +/- 5.8 mm Hg and the mean AaDo2 rose from 35.1 +/- 6.8 to 38.7 +/- 7.4 mm Hg. The changes in Pao2 were not significant. The increase in AaDo2 after placebo was significant, but that after tiapamil was not. We conclude that the combination of the calcium antagonist tiapamil and the bronchodilator salbutamol is safe with respect to lung function in COPD. There is no evidence that tiapamil increases beta 2-agonist-induced impairment in pulmonary gas exchange.     

布地奈德对沙丁胺醇抑制平滑肌内游离钙浓度升高快速影响

目的观察布地奈德(Budesonide,BUD)对沙丁胺醇(Albuterol,ALB)抑制气道平滑肌细胞内游离钙(Ca2+[i])浓度升高的快速影响作用。方法取原代培养豚鼠气道平滑肌细胞,通过共聚焦显微镜下荧光强度检测,比较不同浓度BUD(1×10-6mol/L、1×10-7mol/L、1×10-8mol/L)与ALB联合作用10 min ALB抑制胞内Ca2+[i]释放和胞外Ca2+内流作用的快速影响,同时应用米非司酮(Mifepristone,RU486)及放线菌酮(Cycloheximide,CHX)作为阻断剂进行实验,排除上述反应中的基因组效应。结果组胺引起胞内Ca2+[i]释放实验中,阳性对照组、ALB对照组、1×10-6mol/L、1×10-7mol/L及1×10-8mol/L BUD联合ALB组荧光强度峰值分别为89.6±27.1、63.6±23.4、44.3±18.0、51.9±22.8及59.2±22.8;外源性Ca2+内流实验中,阳性对照组、ALB对照组、1×10-6mol/L、1×10-7mol/L及1×10-8mol/L BUD联合ALB组荧光强度峰值分别为77.3±23.1、56.4±18.7、40.1±14.9、47.0±20.8和53.7±23.5。此两组实验中,1×10-6mol/L和1×10-7mol/L BUD联合ALB组Ca2+[i]荧光强度峰值与ALB对照组比较差异具有统计学意义(P0.01或P0.05)。而在胞内Ca2+[i]释放及外源性Ca2+内流实验条件下,RU486组及CHX组Ca2+[i]荧光强度峰值与1×10-6mol/L BUD联合ALB组比较差异均无统计学意义(P0.05)。结论哮喘急性发作时,联合应用BUD能够增强ALB减轻细胞收缩强度的作用,快速改善气道痉挛症状,且此作用不能被糖皮质激素受体拮抗剂和蛋白合成抑制剂所阻断。     

Nicotinic acetylcholine receptors at sites of neurotransmitter release to the guinea pig intestinal circular muscle

Experiments were designed to test the hypothesis that nicotinic acetylcholine receptors (nAChRs) are present at sites of neurotransmission to the guinea pig ileum circular smooth muscle. Circular smooth muscle preparations, from which the myenteric plexus had been removed (circular muscle-axon preparation), were used for this purpose. Nicotine and dimethylphenyl piperazinium iodide (10-100 microM) induced contraction of the circular smooth muscle. Agonist-induced contraction was inhibited by 1 microM scopolamine and abolished in the combined presence of 1 microM scopolamine and 0. 3 microM CP 96,345-01, a neurokinin-1 receptor antagonist. Contractions induced by electric field stimulation (30 pulses, 0.5 ms, 70 V, 10 Hz) were abolished by 0.3 microM tetrodotoxin (TTX); in contrast, agonist-induced contraction was attenuated but not abolished by 0.3 microM TTX. Mecamylamine (3 or 30 microM), an nAChR antagonist, blocked agonist-induced contractions. Frequency-response curves for both "ON and "OFF electric field stimulation contractions were abolished by the combined presence of 1 microM scopolamine and 0.3 microM CP 96,345-01 or by 0.3 microM TTX. At stimulation frequencies greater than 2 Hz, the ON contraction was increased in the presence of 100 microM nitro-L-arginine. Mecamylamine (3 microM) was used to block the stimulatory prejunctional nAChRs located near sites of neurotransmitter release to the circular smooth muscle; however, ON and OFF contractions were not affected by mecamylamine. Although the prejunctional nAChRs are not targets for endogenously released acetylcholine under the conditions tested here, these receptors may be targets for the development of new prokinetic agents.