Inhibitory Effect Of Isoprenaline Research Articles

Effect of Roflumilast in airways disorders via dual inhibition of phosphodiesterase and Ca2+-channel

The bronchodilator effects of Roflumilast “a selective phosphodiesterase type-4 (PDE4)” inhibitor studied in this experimental protocol. The spiral strips of isolated guinea-pig tracheal chains mounted in organ bath and maintained in Krebs solution ventilated with carbogen at 32 °C and in Ca++ restricted krebs solution. PDE inhibitory activity was evaluated by recording dose response curves using inhibitory effect of isoprenaline on CCh induced contractions. For confirmation of PDE inhibition the intracellular cAMP levels were also estimated. Roflumilast resulted a sharp inhibition in contractile responses of carbachol (CCh, 1 µM) and K+ (80 mM) and the results were almost similar to verapamil. In Ca++ restricted Krebs solution, a rightward shift in the Ca++ response curves observed in the tracheal chain strips which were pretreated with Roflumilast (0.001–0.003 mg/mL) and the maximum response was suppressed, similarly as with verapamil. PDE inhibitory effect of Roflumilast evaluated by recording dose-dependent (0.03–0.1 mg/mL) responses, the isoprenaline-induced inhibitory dose response curves shifted leftward similar to papaverine (PDE inhibitor). Pretreatment with Roflumilast exhibited elevated intracellular cAMP levels in tracheal strips. Findings of the experiment conclude bronchodilatory influence of Roflumilast via PDE and Ca++ channel inhibition. Results of current experiment offers comprehensive mechanistic background of Roflumilast in future as therapeutic bronchodilator for hyperactive bronchial airway diseases.

Changes in beta‐adrenergic neurotransmission during postoperative ileus in rat circular jejunal muscle

To explore postoperative changes in β-adrenergic neurotransmission that participate in pathophysiology of postoperative ileus. Contractile activity of circular jejunal muscle strips was studied. Groups (n = 6/group) were: naïve controls, sham controls 1 and 7 days after laparotomy, and rats 12 h, 1, 3, and 7 days after laparotomy with standardized small bowel manipulation (postoperative ileus). Dose-responses to the β-agonist isoprenaline (3 × 10(-10) - 10(-7) mol L(-1)) were studied in presence/absence of tetrodotoxin (global neural blockade; 10(-6) mol L(-1) ), N6-(1-iminoethyl)-l-lysine (inhibition of inducible nitric oxide synthesis; 10(-4) mol L(-1)), nimesulide (cyclooxygenase-2 inhibition; 10(-5) mol L(-1)), or propranolol (β-blockade; 5 × 10(-6) mol L(-1)). Histochemistry for inflammatory cells and intestinal transit were studied. Intramural inflammation and delayed transit (postoperative ileus) occurred only in ileus groups. The inhibitory potential of isoprenaline decreased in all postoperative groups including sham (P < 0.05). Tetrodotoxin enhanced isoprenaline-induced inhibition in ileus and sham groups (P < 0.05). N6-(1-iminoethyl)-l-lysine and nimesulide decreased isoprenaline-induced inhibition in ileus groups 12 h, 1, and 7 days, and in sham controls 7 days postoperatively (P < 0.05). Propranolol prevented isoprenaline effects in all groups (P < 0.05). Inhibitory effects of isoprenaline on contractile activity were decreased for 7 days postoperatively. Changes in β-adrenergic neurotransmission do not induce postoperative ileus and appear to be caused by anesthesia and laparotomy rather than bowel manipulation.

Inhibitory effects of catecholamines on cholinergically and non-cholinergically mediated contractions of guinea-pig isolated bronchial muscle

The actions of catecholamines on the responses evoked by electrical field stimulation or by acetylcholine and substance P in guinea-pig bronchial strip chain have been examined. Electrical field stimulation evoked a biphasic contraction, consisting of a cholinergically-mediated fast contraction followed by a non-cholinergically-mediated slow contraction. All catecholamines tested caused a concentration-dependent reduction in the height of the biphasic contraction, where non-cholinergic contractions were more potently inhibited. The inhibitory effect of isoprenaline was largely prevented by propranolol (2 microM) alone, whereas those of noradrenaline and adrenaline were prevented by treatment with both propranolol (2 microM) and yohimbine (2 microM). The inhibitory effect of dopamine was unaffected either by propranolol (2 microM), yohimbine (2 microM) or haloperidol (10 microM). Submaximal contractions of bronchial muscle evoked by exogenous acetylcholine (2 microM) or substance P (0.2 microM) were also inhibited by catecholamines, except dopamine, but the effects were antagonized by propranolol (2 microM) alone. The results suggest that in guinea-pig isolated bronchial muscle, catecholamines can inhibit both cholinergic and non-cholinergic excitatory neurotransmissions not only by postjunctional beta-adrenoceptors but also by prejunctional alpha 2-adrenoceptors.

Inhibitory effects of sympathomimetic drugs on cholinergically mediated contractions of guinea-pig isolated tracheal muscle.

The experiments examine the actions of sympathomimetic drugs on the responses evoked by electrical field stimulation or by acetylcholine in guinea-pig tracheal strip chains. Electrical field stimulation evoked contractions which were cholinergically mediated, in the presence of guanethidine (10 microM) and indomethacin (2 microM). All the sympathomimetic drugs tested caused a concentration-dependent reduction in the height of these contractions. Inhibitory effects of isoprenaline and terbutaline were largely prevented by propranolol (2 microM) alone, whereas those of clonidine, oxymetazoline, lidamidine and WHR1370 were prevented by yohimbine alone (2 microM). Treatments with both propranolol and yohimbine were required to prevent the inhibitory effects of noradrenaline, adrenaline and dopamine. Contractions evoked by exogenous acetylcholine (0.1-3 microM) were also inhibited by all catecholamines and terbutaline, but not by clonidine, oxymetazoline, lidamidine and WHR1370. The inhibitory effects were antagonized by propranolol (2 microM) alone. The results suggest that in guinea-pig isolated tracheal muscle, sympathomimetic drugs can inhibit cholinergic neurotransmission not only by postjunctional beta 2-adrenoceptors but also by prejunctional alpha 2-adrenoceptors.

The β3‐adrenoceptor mediates the inhibitory effects of β‐adrenoceptor agonists via the urothelium in pig bladder dome

Relaxation of detrusor muscle via β-adrenoceptors may contribute to urine storage during bladder filling. Thus there is increasing interest in β-adrenoceptor agonists as a potential treatment for detrusor overactivity. The role of the urothelium in bladder responses to β-adrenoceptor agonists is not yet clear, although we have shown that these agonists have a greater inhibitory effect on detrusor contraction when the urothelium is intact. The aim was to determine which β-adrenoceptor subtype is involved in this effect. Paired strips of pig bladder dome mucosa-intact and mucosa-denuded, were mounted in tissue baths. Relaxation responses were obtained to β-adrenoceptor agonists (isoprenaline, dobutamine, salbutamol or BRL37344) in carbachol pre-contracted tissues. Inhibitory effects were studied by obtaining concentration-response curves (CRCs) to carbachol in the presence and absence of β-adrenoceptor agonists. The inhibitory effects of isoprenaline were also studied following incubation with β-adrenoceptor antagonists (propranolol, CGP20712, ICI-118, 551 or SR 59230A; non selected, β(1), β(2) and β(3) selective respectively). isoprenaline, dobutamine, salbutamol and BRL37344 all relaxed carbachol pre-contracted tissues and responses were similar in intact and denuded strips. In inhibition experiments, β-adrenoceptor agonists caused rightward shifts of carbachol CRCs. In intact strips the shift was greater with isoprenaline and BRL37344, but not with dobutamine or salbutamol. This increased shift was still observed in tissues pre-incubated with propranolo, CGP20712 and ICI-118, 551, but not with SR 59230A. β(3)-adrenoceptors are involved in mediating inhibitory effects of β-adrenoceptor agonists on detrusor contractions via the urothelium in pig bladder dome.

Influence of β2‐adrenoceptor gene polymorphisms on β2‐adrenoceptor‐mediated responses in human lung mast cells

Previous studies have shown that beta(2)-adrenoceptor-mediated responses in human lung mast cells are highly variable. The aims of the present study were to establish whether polymorphisms of the beta (2)-adrenoceptor gene (ADRB2) influence this variability in (a) beta(2)-adrenoceptor-mediated inhibition and (b) desensitization of beta(2)-adrenoceptor-mediated responses in human lung mast cells. Mast cells were isolated from human lung tissue. The inhibitory effects of the beta-adrenoceptor agonist, isoprenaline (10(-10)-10(-5) M), on IgE-mediated histamine release from mast cells were determined (n=92). Moreover, the inhibitory effects of isoprenaline were evaluated following a desensitizing treatment involving long-term (24 h) incubation of mast cells with isoprenaline (10(-6) M) (n=65). A potential influence of polymorphisms on these functional responses was determined by genotyping 11 positions, in the promoter and coding regions, of ADRB2 previously reported as polymorphic. There was no influence of any of the polymorphic positions of ADRB2 on the potency of isoprenaline to inhibit histamine release from mast cells with the exception of position 491C>T (Thr164Ile). There was no influence of any of the polymorphic positions of ADRB2 on the extent of desensitization of the isoprenaline-mediated response following a desensitizing treatment except for position 46G>A (Gly16Arg). Analyses at the haplotype level indicated that there was no influence of haplotype on beta (2)-adrenoceptor-mediated responses in mast cells. These data indicate that certain polymorphisms in ADRB2 influence beta(2)-adrenoceptor-mediated responses in human lung mast cells.

The role of the urothelium in mediating bladder responses to isoprenaline

To investigate whether the responses of the pig bladder to isoprenaline (a nonselective beta-adrenoceptor agonist) are influenced by the presence of an intact urothelium and whether any influence might be attributed to the release of nitric oxide (NO), since stimulation of beta-adrenoceptors induces a direct relaxation of detrusor smooth muscle and beta-adrenoceptors are also present on the urothelium. Paired (in the presence or absence of urothelium) longitudinal strips of pig bladder dome were set up in tissue baths and the developed tension recorded. Relaxation responses to isoprenaline were examined after pre-contraction with carbachol. The inhibitory effects of isoprenaline were examined by comparing responses to carbachol in the absence and presence of isoprenaline. To examine a possible role for NO, similar experiments were performed in the presence of the NO synthase inhibitor N(G)-nitro-L-arginine (L-NNA). In the presence of the urothelium, both the potency (pEC(50)) and the maximum contractile responses to carbachol were depressed. In relaxation experiments, isoprenaline relaxed carbachol pre-contracted tissues by approximately 75%, and the potency and maximum relaxation were similar in the absence and presence of the urothelium. In the inhibition experiments, the presence of isoprenaline caused rightward parallel shifts of the concentration-response curves to carbachol, but isoprenaline did not influence the maximum contractions. In the presence of the urothelium there was a greater shift with 0.1 microm isoprenaline than in denuded tissues. Incubation with L-NNA did not affect the influence of the urothelium on responses to isoprenaline in any experimental group. The relaxation responses of the bladder to isoprenaline do not appear to involve the urothelium or NO release in vitro. However, contractile responses to carbachol were inhibited in the presence of an intact urothelium, and this might reflect the release of an inhibitory factor other than NO.

Pregnancy switches adrenergic signal transduction in rat and human uterine myocytes as probed by BKCa channel activity

1. We used large conductance Ca2+-activated K+ (BKCa) channel activity as a probe to characterize the inhibitory/stimulatory G protein (Gi/Gs) signalling pathways in intact cells from pregnant (PM) and non-pregnant (NPM) myometrium. 2. Isoprenaline (10 microM) enhanced the outward current (Iout) in PM cells and inhibited Iout in NPM cells. Additional application of the alpha2-adrenoceptor (alpha2-AR) agonist clonidine (10 microM) further enhanced the isoprenaline-modulated Iout in PM cells but partially antagonized Iout in NPM cells. Clonidine alone did not affect Iout. The specific cAMP kinase (PKA) inhibitor H-89 (1 microM) abolished the effects of isoprenaline and clonidine. The specific BKCa channel blocker iberiotoxin (0.1 microM) inhibited Iout by approximately 80 %; the residual current was insensitive to isoprenaline. 3. Inhibition of Gi activity by either pertussis toxin or the GTPase activating protein RGS16 abolished inhibitory as well as stimulatory effects of clonidine on Iout. 4. Transducin-alpha, a scavenger of Gi betagamma dimers, converted the stimulatory action of clonidine on Iout into an inhibitory effect. Free transducin-betagamma enhanced both the stimulatory and the inhibitory effects of isoprenaline on Iout. 5. The results demonstrate that BKCa channel activity is a sensitive probe to follow adenylyl cyclase-cAMP-PKA signalling in myometrial smooth muscle cells. Both Gialpha-mediated inhibition and Gibetagamma-mediated stimulation can occur in the same cell, irrespective of pregnancy. It is speculated that the coupling between alpha2-AR and Gi proteins is more efficient during pregnancy and that Gibetagamma at high levels simply override the inhibitory action of Gi alpha.

Some cyclic nucleotides reduce phenylephrine-induced and phorbol ester-induced constriction of the rat anterior mesenteric artery

1. 1. Vasoconstrictor responses to phenylephrine (PE) and to 12-deoxyphorbol 13-phenylacetate in the rat isolated perfused anterior mesenteric artery were inhibited by pretreating the artery with isoprenaline (ISOP), sodium nitroprusside or 8-bromoguanosine 3′:5′-cyclic monophosphate, but not with 8-bromoadenosine 3′:5′-cyclic monophosphate. 2. 2. The inhibitory effect of ISOP toward the response to PE was less prominent after pretreating the artery with a selective inhibitor of cyclic GMP-dependent kinase. 3. 3. The vasorelaxant effect of ISOP was enhanced by pretreating the artery with an inhibitor of nitric oxide synthase.

Modulation of L-type Ca 2+ current by isoprenaline, carbachol and phorbol ester in cultured rat aortic vascular smooth muscle (A7r5) cells

1. 1. Effects of isoprenaline (ISO), carbachol and phorbol ester (a stimulator of protein kinase C) on L-type Ca 2+ channels in single cultured rat aortic vascular smooth muscle (A7r5) cells were examined using whole-cell voltage clamp (at room temperature 22°C). 2. 2. With 20 mmol/1 Ca 2+ in the bath solution and 10 mmol/1 EGTA in the pipette solution, a slow I Ca, (L-type) current was observed in the A7r5 cell line, which was blocked by nifedipine (2 μmol/l). 3. 3. ISO (5 μmol/l) inhibited I Ca by 18.3 ± 2.2% ( P < 0.001), and carbachol (1 μmol/1) also decreased I Ca by 15.0 ± 3.2% ( P < 0.01). 8-Br-cAMP (1 mmol/1) and 8-Br-cGMP (1 mmol/l) both inhibited I Ca by 30.1 ± 2.8% ( P < 0.001) and 18.8 ± 3.8% ( P < 0.01), respectively. 4. 4. Phorbol ester, 4-β-phorbol-12, 13-dibutyrate (PDB), at 0.1-1 μmol/l, had almost no effect on I Ca in most cells, but slightly potentiated (or slightly enhanced) the inhibitory effects of ISO. 5. 5. Time decay (inactivation) of I Ca consisted of two exponentials. Both the fast and slow time constants were slightly prolonged by ISO (5 μmol/1), and by carbachol (1 μmol/1); PDB (l μmol/l) slightly shortened the fast time constant only. The half-maximum voltages of inactivation were not significantly affected by any of the agents. 6. 6. These results suggest that the L-type I Ca current is modulated by cyclic nucleotides (cAMP and cGMP) and by PK-C stimulation, and thereby contribute to regulation of contraction of the vascular smooth muscle cells.

Inhibitory potency of isoprenaline on guinea-pig and gravid human myometrium following extraneuronal uptake blockade

The hypothesis that inhibitory effects of isoprenaline on myometrial contractility may be constrained by activation of putative intracellular beta-adrenoceptors negatively-coupled to adenylate cyclase was examined. Field-stimulated preparations of guinea-pig and human myometrium were used to examine the influence of the catecholamine extraneuronal uptake2 inhibitors, corticosterone and beta-oestradiol, on the inhibitory effects of the beta-adrenoceptor agonist, isoprenaline, on uterine contraction. Longitudinal and circular myometrial layers were obtained from guinea-pigs in dioestrus, primed with oestrogen before progesterone, or pregnant (Days 62-65). In the guinea-pig myometrium, corticosterone (30 microM) did not affect responses to isoprenaline. beta-oestradiol (10 microM) induced a small potentiation of the effects of isoprenaline on longitudinal myometrium from dioestrus guinea-pigs. Myometrial preparations were obtained from pregnant women (36-40 weeks gestation) undergoing caesarean section. Isoprenaline inhibited stimulation-evoked contractions in 7 of 10 preparations of the inner myometrial layer and in 5 of 8 preparations of outer myometrial layer. Corticosterone (30 microM) reduced the effects of isoprenaline on the inner layer and did not affect the outer layer. These results do not support the existence of mechanism involving isoprenaline-sensitive intracellular receptors which constrain responses to beta-adrenoceptor agonists.

Beta-adrenoceptors mediate inhibition of [3H]-acetylcholine release from the isolated rat and guinea-pig trachea: role of the airway mucosa and prostaglandins.

1. Rat or guinea pig isolated tracheae were labelled with [3H]-choline to measure evoked tritium outflow, which reflects neuronal release of [3H]-acetylcholine. Tritium outflow was evoked either by electrical stimulation of the extrinsic vagal nerve (rat tracheae) or by 27 mM potassium (guinea pig tracheae). 2. In rat tracheae isoprenaline (0.01, 0.1 microM) inhibited evoked [3H]-acetylcholine release, whereas beta 2-adrenoceptor-selective agonists (fenoterol, formoterol, salbutamol) were ineffective. 3. The inhibitory effect of isoprenaline was abolished under the following conditions: (i) presence of propranolol (1 microM) or of the beta 1-selective antagonist CGP 20712 A (0.1 microM); (ii) removal of the mucosa at the start of the experiments; (iii) blockade of cyclooxygenase activity by 3 microM indomethacin. 4. In rat isolated tracheae prelabelled with [3H]-arachidonic acid, isoprenaline (0.1 microM) but not formoterol (0.01 microM) enhanced the outflow of [3H]-prostaglandins (PGD2, PGE2). This effect was blocked by 0.1 microM CGP 20712 A. 5. In guinea pig tracheae electrical stimulation of the extrinsic vagal nerve did not cause a constant release of [3H]-acetylcholine, but 27 mM potassium elicited a reproducible release of [3H]-acetylcholine. In this species both isoprenaline (0.1 microM) and formoterol (0.01 microM) inhibited evoked [3H]-acetylcholine release. Inhibition was abolished under the following conditions: (i) presence of propranolol (1 microM) or of the beta 2-selective antagonist ICI 118551 (0.3 microM); (ii) removal of the mucosa at the start of the experiments; (iii) blockade of cyclooxygenase activity by 3 microM indomethacin. 6. In conclusion, the present experiments have demonstrated that activation of beta-adrenoceptors localized in the mucosa mediates inhibition of [3H]-acetylcholine release from the neuroeffector junctions of the pulmonary, parasympathetic nerves most probably by the liberation of inhibitory prostaglandins from the airway mucosa. The adrenoceptor subtype involved differs in rat (beta 1 subtype) and guinea pig (beta 2 subtype) airways.

Differential sensitivities of the prostacyclin and nitric oxide biosynthetic pathways to cytosolic calcium in bovine aortic endothelial cells.

1. Bovine aortic endothelial cells were cultured in vitro, and shown to release both prostacyclin (PGI2; Kact = 24.1 nM) and endothelium-derived relaxing factor (EDRF, NO; Kact = 0.7 nM) in a concentration-dependent manner when exposed to bradykinin. 2. The bradykinin-dependent release of PGI2 (but not EDRF) was inhibited by 1 microM isoprenaline or 5 microM forskolin, and the inhibitory effect of isoprenaline could be reversed by the beta 2-adrenoceptor antagonist, ICI 118551. In contrast, isoprenaline had no capacity to inhibit PGI2 release stimulated by exogenous arachidonic acid. 3. Exposure of cells to bradykinin increased the cytosolic concentration of Ca2+ ions ([Ca2+]i; Kact = 4.8 nM), and the effect was inhibited by both 1 microM isoprenaline and 5 microM forskolin. 4. In similar experiments, exposure of cells to ionomycin also increased [Ca2+]i and the values of [Ca2+]i were calibrated in terms of the ionomycin concentration. In subsequent experiments involving exposure of endothelial cells to selected concentrations of ionomycin, it was possible to show that the biosynthesis of NO was triggered at ionomycin concentrations about one tenth of the required for PGI2 biosynthesis and that these corresponded to a [Ca2+]i threshold of 350 nM for PGI2 release while that for EDRF release was less than 200 nM. 5. These differences in Ca2+ ion sensitivity explain the selective inhibition of bradykinin-stimulated PGI2 biosynthesis (to the exclusion of NO biosynthesis) by isoprenaline or forskolin, both of which attenuate bradykinin-dependent increases in [Ca2+]i.

Isoprenaline, Ca2+ and the Na(+)-K+ pump in guinea-pig ventricular myocytes.

1. The whole-cell patch clamp technique was employed to study the effects of the beta-agonist isoprenaline (ISO) on the Na(+)=K+ pump current, Ip, in acutely isolated ventricular myocytes from guinea-pig hearts. Propranolol, a beta-adrenergic antagonist, was used to demonstrate that all of the effects of ISO, stimulatory or inhibitory, are mediated by beta-receptors. 2. Below about 150 nM [Ca2+]i, we find that ISO reduces Ip, while above this [Ca2+]i ISO increases Ip. The stimulatory and inhibitory effects of ISO on Ip are independent of either intracellular sodium ([Na+]i) or extracellular potassium ([K+]o). These results suggest that the end-effect of ISO is directly on the maximum pump turnover rate (Vmax) rather than indirectly through changes in [Na+]i or [K+]o or modulatory effects on Na+ or K+ affinity. 3. The maximum effect of ISO increases Ip by 25% when [Ca2+] is buffered at 1.4 microM. A half-maximal effect is reached at roughly 10 nM-ISO and a near-maximal effect by 0.5 microM. 4. The permeabilized patch technique, using amphotericin B (Horn & Marty, 1988; Rae, Cooper, Gates & Watsky, 1991), was employed to minimize changes in the normal second messenger systems and calcium buffers. In these experiments, we used a high intracellular sodium solution (pipette sodium was 50 mM), thus sodium-calcium exchange was depressed and we expected [Ca2+]i to be above 150 nM. ISO increases Ip in these conditions as in the dialysed cells. 5. Our results suggest that beta-stimulation can increase Ip, but only if [Ca2+]i is above about 150 nM. In the beating heart [Ca2+]i rises well above this value during systole and the average [Ca2+]i, which depends on heart rate, is expected to normally be above this level. During beta-stimulation, the increase in Ip along with a concomitant increase in IK (Giles, Nakajima, Ono & Shibata, 1989; Duchatelle-Gourdon, Hartzell & Lagrutta, 1989) helps prevent action potential lengthening and allows an increase in heart rate even in the presence of increased calcium current. Further, beta-stimulation will compensate for the effects on Ip of either hypokalaemia or digitalis toxicity, and so reduce the expected rise in both [Na+]i and [Ca2+]i.

An inhibitory effect of isoprenaline on stimulation-induced noradrenaline release from rat atria

Isoprenaline (0.1 μM), in the presence of phentolamine (1 μM) to block autoinhibitory α-adrenoceptors, significantly increased the efflux of radioactivity produced by field stimulation (2 Hz for 60 s) from rat isolated atria in which the noradrenergic transmitter stores had been labelled with [ 3H]noradrenaline. This facilitatory effect of isoprenaline on noradrenergic transmission was not affected by the selective β 1-adrenoceptor antagonist CGP 20712A (0.3 μM). However, the selective β 2-adrenoceptor antagonist ICI 118551 (0.1 μM) not only abolished the facilitatory effect of isoprenaline but reversed it to an inhibitory effect, indicating that the prejunctional β-adrenoceptors subserving facilitation of noradrenergic transmission in rat atria are of the β 2-subtype. The inhibitory effect of isoprenaline that was revealed by blockade of β 2-adrenoceptors was abolished by atenolol (3 μM) in a concentration which markedly reduced the effect of isoprenaline on the rate of atrial beating. This finding suggests that activation of β 1-adrenoceptors on atrial myocytes by isoprenaline may have resulted in release of one or more substance(s) which inhibited stimulation-induced release of noradrenaline, presumably by activating prejunctional receptors. The inhibitory effect of isoprenaline on noradrenergic transmission was not affected by the prostaglandin synthesis inhibitor indomethacin (10 μM) suggesting that prostaglandins were not involved.

Salmeterol: a potent and long‐acting inhibitor of inflammatory mediator release from human lung

1. The effects of salmeterol, a novel long-acting beta 2-adrenoceptor agonist, have been investigated on antigen-induced mediator release from passively sensitized fragments of human lung in vitro. 2. Salmeterol was a potent inhibitor of the release of histamine (-log IC50 = 8.54), leukotriene C4 (LTC4)/LTD4 (-log IC50 = 9.07) and prostaglandin D2 (-log IC50 = 8.81). It was slightly less potent (1-3 fold) than isoprenaline, but significantly more potent (10-35 fold) than salbutamol. 3. Propranolol competitively antagonized the inhibitory effects of salmeterol on histamine release (pA2 = 8.41) and LTC4/LTD4 release, (pA2 = 8.40) indicating an action via beta-adrenoceptors. 4. The inhibitory effects of isoprenaline (20 nM) and salbutamol (200 nM) were removed after washing the lung tissue for 2 h and 4 h respectively. In contrast, the inhibitory effects of salmeterol (40 nM) were much longer-lasting, and were still evident after 20 h. 5. Salmeterol therefore exhibits potent and persistent inhibition of anaphylactic mediator release from human lung. This anti-inflammatory effect may be important for the therapeutic potential of salmeterol in the treatment of bronchial asthma.

β-adrenoceptor induced inhibition of muscarinic receptor-stimulated phosphoinositide metabolism is agonist specific in bovine tracheal smooth muscle

The ability of the β-adrenoceptor agonist isoprenaline to inhibit agonist-stimulated phosphoinositide metabolism was examined in bovine tracheal smooth muscle slices prelabelled with [ 3H]inositol. Accumulation of [ 3H]inositol phosphates was enhanced by the muscarinic agonists carbachol, oxotremorine and pilocarpine although the latter were only partial agonists for this response. Histamine stimulation of [ 3H]inositol phosphates was sensitive to mepyramine but maximal responses were only comparable to those of pilocarpine. Preincubation of tracheal slices with isoprenaline reduced the maximal phosphoinositide response to histamine and pilocarpine but the responses to carbachol and oxotremorine were unaffected. The inhibitory effect of isoprenaline (IC 50 = 0.04 μM) was reversed competitively by 1 μM propranolol. The non-selective phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) (1 mM) resulted in a more severe suppression of the histamine and pilocarpine responses and also produced a significant suppression of the maximal response to oxotremorine and a small shift in the carbachol dose-response curve. The different susceptibility of agonist-stimulated phosphoinositide hydrolysis to isoprenaline and IBMX are discussed in relation to the relative intrinsic activity of the agonists and/or the role of different muscarinic receptor subtypes.

Cyclic AMP counteracts mitogen-induced inositol phosphate generation and increases in intracellular Ca2+ concentrations in human lymphocytes.

1. The effects of increases in intracellular adenosine 3':5'-cyclic monophosphate (cyclic AMP) on mitogen-induced generation of inositol phosphates and increases in intracellular Ca2+ concentration were investigated in human peripheral blood mononuclear leukocytes (MNL). 2. The mitogens concanavalin A (Con A), pokeweed mitogen (PWM) and phytohaemagglutinin (PHA) concentration-dependently stimulated generation of inositol phosphates. Catecholamines inhibited this process with an order of potency: isoprenaline greater than adrenaline greater than noradrenaline indicating involvement of beta 2-adrenoceptors. This order of potency was also consistent with the catecholamine potencies for stimulating the generation of cyclic AMP. 3. In addition to catecholamines, the cyclic AMP formation-stimulating agents prostaglandin E1 (PGE1) and forskolin concentration-dependently inhibited mitogen-induced inositol phosphate generation, too. Moreover, the inhibitory effect of isoprenaline was potentiated by co-incubation with the phosphodiesterase inhibitor isobutylmethylxanthine demonstrating that these inhibitory effects were mediated by cyclic AMP. 4. Con A and PHA concentration-dependently increased the intracellular Ca2+ concentration in human MNL (assessed by the fluorescent indicator dye Fura-2). This increase was almost completely blocked by chelation of extracellular Ca2+, demonstrating influx rather than mobilization from intracellular stores. 5. The elevation of intracellular Ca2+ was not blocked by pretreatment with pertussis toxin, 100 ng ml-1, for 16 h. 6. Isoprenaline, PGE1, and forskolin, however, inhibited the mitogen-stimulated elevation of intracellular Ca2+. This inhibition was enhanced by the phosphodiesterase inhibitors isobutylmethylxanthine and Ro 20-1724, demonstrating mediation by cyclic AMP. 7. We conclude that catecholamines and other cyclic AMP increasing agents can inhibit mitogen-stimulated generation of inositol phosphates and elevation of intracellular Ca2+ in resting human MNL.

Inhibitory effect of isoprenaline on gastric acid secretion in the rat. The role of endogenous histamine

In dogs beta-adrenoreceptor agonists inhibit gastric acid secretion stimulated by exogenous gastrin to a much greater extent than acid secretion stimulated by exogenous histamine. One possible explanation for this observation is that endogenous histamine is important in gastrin-mediated acid secretion and that isoprenaline and related beta-adrenoreceptor agonists block gastric mucosal histamine release. This possibility was tested in the present study in gastric lumen-perfused anaesthetized rats. Intravenous infusion of isoprenaline (12 microgram kg-1 h-1) inhibited maximal, pentagastrin-stimulated acid output by 50-70% (P less than 0.01), but had no significant inhibitory effect on the maximal acid secretory response to histamine. In contrast to its inhibitory effect on gastrin-stimulated acid output, isoproterenol had no effect on gastric histamine output during pentagastrin infusion. We conclude that isoprenaline selectively inhibits gastrin-stimulated acid secretion in the rat, as in the dog, and by a mechanism other than inhibiting gastric histamine release.

Effect of isoprenaline on bethanechol-stimulated gastric acid secrtion and mucosal blood flow in dogs with gastric fistula.

The prupose of this study was to elucidate the effect of the beta-adrenoceptor stimulation by isoprenaline on cholinergic-stimulated gastric acid secretion and mucosal blood flow in conscious dogs with gastric fistula. Isoprenaline, a beta 1- and beta 2-agonist, was used alone and in conjunction with selective blockade of beta 2 and beta 1 receptors. A low dose of isoprenaline had no significant effect, whereas higher doses had a significant antisecretory effect. The antisecretory effect was significantly blocked by the beta 1-adrenoceptor blocker practolol but not by H 35/25, a beta 2-adrenoceptor blocker. The dose-response curve with five doses of bethanechol with and without isoprenaline was in accordance with a non-competitive inhibition. There was no significant effect on gastric mucosal blood flow, indicating that the acid inhibition was not secondary to changes in blood flow. The inhibitory effect of isoprenaline seems to be mediated by the beta 1 receptors and with an action primarily on the 'gastrinergic receptors'.