Concentration-response Curve For Isoprenaline Research Articles

β1 Adrenoceptor antagonistic effects of the supposedly selective β2 adrenoceptor antagonist ICI 118,551 on the positive inotropic effect of adrenaline in murine hearts

Studies on the relative contribution of β1- and β2-adrenoceptors (AR) generally employ selective β1- and β2-AR antagonists such as CGP 20712A and ICI 118,551, respectively, and assume that antagonism by one of these compounds indicates mediation by the respective AR subtype. Here, we evaluated the β2-AR-selectivity of ICI 118,551 in ventricular muscle strips of transgenic mice lacking β1-AR (β1-KO), β2-AR (β2-KO), or both (β1/β2-KO). Strips were electrically driven and force development was measured. In wild type (WT), ICI 118,551 (100 nmol/L) shifted the concentration–response curve (CRC) for adrenaline by about 0.5 log units to the right, corresponding to the known affinity of ICI 118,551 to β1-AR but not to β2-AR. Conversely, the phosphodiesterase inhibitor rolipram (10 μmol/L) shifted the CRC to the left, but did not enlarge the ICI 118,551 shift, indicating exclusive β1-AR mediation even when PDE4 is inactive. In line with this, rolipram and ICI 118,551 had similar effects in β2-KO than in WT. In contrast, β1-KO did not show any inotropic reaction to adrenaline (+/− rolipram). In WT, the β1-AR selective antagonist CGP 20712A (100 nmol/L) shifted the CRC for isoprenaline by 2.1 log units, corresponding to the affinity of CGP 20712A to β1-AR. Rolipram increased the sensitivity to adrenaline independently of the presence of CGP 20712A. We conclude that effects sensitive to the β2-AR antagonist ICI 118,551 are not necessarily β2-AR-mediated and CGP 20712A-resistant effects cannot be simply interpreted as β2-AR-mediated. Catecholamine effects in murine ventricles strictly depend on β1-AR, even if PDE 4 is blocked.

Prostaglandin E2 excitatory effects on rat urinary bladder: a comparison between the β-adrenoceptor modulation of non-voiding activity in vivo and micro-contractile activity in vitro.

Prostaglandin E2 (PGE2) is well known to modulate urinary bladder functions, but it is also thought to be involved in the pathophysiology of lower urinary tract dysfunctions, since high levels of PGE2 have been found in overactive bladder (OAB) patients. β-Adrenoceptors are major players in detrusor muscle relaxation, and the selective β3-adrenoceptor (AR) agonist mirabegron was recently approved for the treatment of overactive bladder (OAB). β-Adrenoceptor modulation of PGE2 excitatory effects on bladder detrusor muscle was investigated by i.v. mirabegron after intravesical PGE2 infusion in conscious rats. Non-voiding activity (NVA) was assessed under isovolumetric conditions. In addition, mirabegron and isoprenaline (0.01-10μM) were studied on PGE2-increased micro-contractile activity during isometric tension recordings of intact isolated bladder muscle strips. Our investigations showed that PGE2 dramatically increased NVA in vivo and spontaneous micro-contractions in vitro. In vivo administration of mirabegron (0.1, 0.3 and 3mg/kg) reduced PGE2-augmented NVA in dose-dependent manner, while the PGE2-increased micro-contractions in isolated bladder strips were poorly inhibited. Isoprenaline inhibited PGE2-augmented micro-contractions in a concentration-dependent manner and had a higher potency compared to mirabegron. The apparent pKB of 7.25 for metoprolol at the isoprenaline concentration-response curve for PGE2-augmented micro-contractions suggests a β1-AR-mediated.

Beta adrenergic modulation of spontaneous microcontractions and electrical field-stimulated contractions in isolated strips of rat urinary bladder from normal animals and animals with partial bladder outflow obstruction

Spontaneous microcontractions and electrical field stimulation (EFS)-evoked contractions in isolated rat bladder strips from normal and from 6weeks partial bladder outflow obstruction (pBOO) animals were studied to identify the potential site of action for the β3-adrenoceptor (AR) agonist mirabegron in detrusor overactivity in rats. For this, effects of the β-AR agonist isoprenaline and mirabegron were tested in presence or absence of selective antagonists for β-AR subtypes, namely CGP-20712A for β1-AR, ICI-118,551 for β2-AR, and L-748,337 for β3-AR. In detrusor strips from both normal and obstructed animals, EFS-induced contractions were weakly affected by isoprenaline and even less so by mirabegron. In contrast, microcontraction activity was more potently reduced by isoprenaline (pIC50 7.3; Emax ±85%), whereas mirabegron showed a small effect. In pBOO strips, concentration response curves for isoprenaline and mirabegron at inhibition of EFS and spontaneous microcontractions were similar to those in normal strips. Isoprenaline-induced inhibition of microcontractions and EFS was antagonized by the β1-AR antagonist, but not by the β2- and β3-AR antagonists. In the context of β3-AR-mediated bladder functions for mirabegron in other experiments, the current data question a role for effects at spontaneous microcontractions, or neurogenic detrusor stimulation in the mode of action for mirabegron in vivo, since functional bladder effects for mirabegron are reported to occur at much lower concentrations.

Studies on Bronchodilator Activity of Salvia officinalis (Sage): Possible Involvement of K+ Channel Activation and Phosphodiesterase Inhibition

The aqueous methanolic extract of the aerial parts of Salvia officinalis (So.Cr) was studied to provide possible underlying mechanism(s) for its medicinal use in asthma using the in vivo bronchodilatory assay and isolated tracheal preparations. S. officinalis (1-10 mg/kg) dose-dependently inhibited carbachol (CCh)-induced bronchospasm in anesthetized rats with three-fold greater potency than the positive control, aminophylline. In tracheal preparations, So.Cr inhibited the low K+ (25 mM)-induced contractions. Pretreatment of the tissues with 4-aminopyridine reversed the inhibitory effect of the plant extract against low K+ , whereas glibenclamide did not show any effect, thus showing the involvement of voltage-sensitive K+ channels. When tested against the CCh-induced pre-contractions for the involvement of any additional mechanism, interestingly, the extract showed a dose-dependent (0.03-0.1 mg/mL) inhibitory effect and shifted the inhibitory concentration response curves of isoprenaline to the left, thus showing phosphodiesterase enzyme inhibitory-like action, similar to that of papaverine. These results indicate that the crude extract of S. officinalis possesses bronchodilatory activity mediated predominantly via activation of voltage-dependent K+ channels and inhibition of phosphodiesterase enzyme; thus, this study provides sound pharmacological basis for its medicinal use in hyperactive airways disorders such as asthma and cough. Copyright © 2015 John Wiley & Sons, Ltd.

β3-Adrenoceptor-mediated relaxation of rat and human urinary bladder: roles of BKCa channels and Rho kinase.

Previous studies suggest that the large-conductance Ca2+-activated K+ (BKCa) channel and Rho-kinase play major roles in the control of urinary bladder tone. Here, we investigated their involvement in β-adrenoceptor (AR)-mediated relaxation of rat and human bladder. Concentration-response curves of isoprenaline and mirabegron-induced bladder relaxation were generated against passive tension and KCl- and carbachol-induced tone, in the absence or presence of the BKCa channel inhibitor iberiotoxin (100 nM) or the Rho-kinase inhibitor Y27,632 (1 μM). Myosin light chain (MLC) phosphorylation was studied by Western blot. In rat, iberiotoxin only slightly altered isoprenaline- and mirabegron-induced relaxation against KCl-induced tone but attenuated relaxation by both agonists against carbachol-induced tone. Y27,632 enhanced isoprenaline- or mirabegron-induced relaxation only against carbachol-induced tone. In humans, iberiotoxin slightly enhanced relaxation by both agonists against carbachol-induced pre-contraction. Y27,632 did not change isoprenaline-induced relaxation but enhanced that by mirabegron. Under passive tension, MLC phosphorylation was markedly reduced by both β-AR agonists, an effect insensitive to Y27,632. In the presence of carbachol, both β-AR agonists increased MLC phosphorylation, an effect reduced by Y27,632 only in the presence of 1 μM carbachol. These results indicate that the extent of BKCa channel and Rho-kinase involvement in relaxation induced by β-AR agonists depends on pre contractile stimulus and species.Electronic supplementary materialThe online version of this article (doi:10.1007/s00210-015-1128-z) contains supplementary material, which is available to authorized users.

Enhanced β‐adrenergic response in rat papillary muscle by inhibition of inducible nitric oxide synthase after myocardial infarction

Myocardial infarction (MI) induces a progressive ventricular remodelling leading to a contractility depression. During the acute phase of MI inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production increases in the heart. The aim of this study was to investigate the role of iNOS in the left ventricular contractility at 3 days after MI. Wistar rats were divided into: sham operated (SHAM, n = 23), infarction (INF, n = 18); sham operated plus the iNOS inhibitor, S-methylisothiourea (SMT) 5 mg kg(-1) day(-1), i.p. treatment (SHAM-SMT, n = 26) and infarction plus SMT (INF-SMT, n = 22). Concentration-response curves for isoprenaline, Ca(2+) and frequency-force curve were studied in isolated papillary muscle from left ventricle. After 3 days infarct area was similar between groups. SMT treatment reduced the time to peak tension during frequency-force curve in the infarct group (SHAM = 63 +/- 3; SHAM-SMT = 71 +/- 3; INF = 90 +/- 4; INF-SMT = 79 +/- 4 ms, P < 0.05) and increased the maximal response to isoprenaline (SHAM = 0.93 +/- 0.11; SHAM-SMT = 1.13 +/- 0.1; INF =0.84 +/- 0.16; INF-SMT = 1.49 +/- 0.15 g mm(-2), P < 0.05). The response to Ca(2+) was equally reduced in the INF and INF-SMT groups. SMT treatment did not change the reduced post-rest potentiation performed by INF group, but attenuated the plasma nitrite and nitrate (NOx) levels in the INF group without any haemodynamic effect. These finding suggest that at 3 days after MI the iNOS modulates the isolated papillary muscle response to isoprenaline and its inhibition improves the beta-adrenergic inotropic responses.

Selective β1-adrenoreceptor blocking activity of newly synthesized acyl amino-substituted aryloxypropanolamine derivatives, DPJ 955 and DPJ 890, in rats

The in-vivo beta-adrenoreceptor antagonistic activity of test compounds DPJ 955 and DPJ 890 was assessed against beta-adrenoreceptor agonist (isoprenaline) induced tachycardia in anaesthetized rats. The selectivity to block isoprenaline responses on different &beta-adrenoreceptor subtypes (beta(1), beta(2) and beta(3)) of the test compounds was carried out on isolated rat right atria, isolated rat uterus and isolated rat colon preparations, respectively. Intravenous injection of isoprenaline alone in anaesthetized rats caused hypotension and tachycardia. DPJ 955 or DPJ 890 alone produced a fall in mean arterial pressure and bradycardia in a dose-dependent manner. Administration of isoprenaline to anaesthetized rats pre-treated with test compounds significantly blocked both the tachycardial and hypotensive responses induced by isoprenaline. The test compounds shifted the concentration response curves of isoprenaline towards the right for isolated rat right atrial preparations, rat uterus and rat colon, indicating beta(1), beta(2) and beta(3) adrenoreceptor blockade, respectively. The selectivity ratio for beta(1)/beta-adrenoreceptors to DPJ 955 and DPJ 890 was 64.6 and 83.2, respectively. DPJ 890 was more potent in blocking beta(1)-adrenoreceptors and was more selective towards beta(1) receptors than to other beta-adrenoreceptor subtypes. In conclusion, DPJ 955 and DPJ890 have beta-adrenoreceptor blocking activity with high selectivity for the beta(1)-adrenoreceptor subtype.

Inhibition by SR 59119A of Isoprenaline-, Forskolin- and VIP-induced Relaxation of Human Isolated Bronchi

In the human isolated bronchus (HIB) it has been shown that β3-adrenoceptor stimulation fails to induce relaxation of airway smooth muscle. It has however been reported in human ventricular endomyocardial biopsies that β3-adrenoceptor stimulation induced a marked negative inotropic effect which could be linked to Gi protein activation. The aims of this study were: (1) to determine in HIB (internal diameter 1–2 mm) whether the selective β3-adrenoceptor agonist SR 59119A (N[7-methoxy-1,2,3,4-tetrahydronaphthalen-(2R)methyl]-(2R)-2-hydroxy-2-(3-chlorophenyl)ethanamine hydrochloride) was able to inhibit adenylate–cyclase-mediated airway smooth muscle relaxation induced by isoprenaline, forskolin or vasoactive intestinal peptide (VIP) and (2) to investigate the role of the Gi protein in this interaction. SR 59119A (0.1 μM and 1 μM) induced a shift to the right of concentration response curve for isoprenaline (−0.15±0.06 and −0.54±0.21 log unit, P<0.05 and P<0.01 respectively), forskolin (−0.12±0.02 and −0.30±0.05 log unit, P<0.001), and VIP (−0.42±0.12 log unit, P<0.01 with SR59119A 10−6M). The inhibitory effect of SR 59119A was (1) abolished by an incubation of HIB with pertussis toxin (1 μg/ml, during 15 h in Krebs–Henseleit solution, at 21°C), which is known to inactivate the Gi protein and (2) increased after an incubation of HIB with the pro-inflammatory cytokine IL-1β (10 ng/ml, during 15 h in Krebs–Henseleit solution, at 21°C), which is known to up-regulate Gi protein expression. Our results suggest that the selective β3-adrenoceptor agonist SR59119A might inhibit the cAMP-dependent relaxation of human isolated bronchus through Gi protein-mediated inhibition of adenylate cyclase.

Beta 1-, beta 2- and atypical beta-adrenoceptor-mediated relaxation in rat isolated aorta.

beta-adrenoceptor-mediated relaxation was investigated in ring preparations of rat isolated thoracic aorta. Rings were pre-constricted with a sub-maximal concentration of noradrenaline (1 microM) and relaxant responses to cumulative concentrations of beta-adrenoceptor agonists obtained. The concentration-response curve (CRC) to isoprenaline was shifted to the right by propranolol (0.3 microM) with a steepening of the slope. Estimation of the magnitude of the shift from EC(50) values gave a pA(2) of 7.6. Selective beta(1)- and beta(2)-adrenoceptor antagonists, CGP 20712A (0.1 microM) and ICI 118551 (0.1 microM), respectively, produced 4 and 14 fold shifts of the isoprenaline CRC. Atypical beta-adrenoceptor agonists also produced concentration-dependent relaxation of aortic rings. The order of potency of the beta-adrenoceptor agonists was (-log EC(50)): isoprenaline (6. 25)>cyanopindolol (5.59)>isoprenaline+propranolol (5.11)>CGP 12177A (4.40)>ZD 2079 (4.24)>ZM 215001 (4.07)>BRL 37344 (3.89). Relaxation to CGP 12177A and ZM 215001 was unaffected by propranolol (0.3 microM). SR 59230A (</=1 microM) and cyanopindolol (1 microM), beta(3)-adrenoceptor antagonists, had no effect on the isoprenaline (in the presence of propranolol) or CGP 12177A CRCs. Bupranolol and CGP 20712A, at microM concentrations (beta(4)-adrenceptor antagonists), inhibited responses to isoprenaline (in the presence of propranolol) and CGP 12177A. In conclusion, atypical beta-adrenoceptors co-exist with beta(1)- and beta(2)-adrenoceptors in rat aorta. Although non-conventional partial agonists and selective beta(3)-adrenoceptor agonist cause relaxation, the vascular atypical beta-adrenoceptor does not appear to correspond to the beta(3)-adrenoceptor. There are, however, similarities with the putative beta(4)-adrenoceptor.

The effects of beta-adrenoceptor agonists on KCl-induced rhythmic contraction in the ureter of guinea pig.

In the present study, we tried to determine what effects were induced by beta-adrenoceptor agonists on 40 mM KCl-induced rhythmic contraction and to clarify which beta-adrenoceptor subtypes are involved in the regulation of ureter motility in the guinea pig by using in vitro functional analysis. 40 mM KCl-induced rhythmic contraction was abolished by papaverine (10(-6) M), nicardipine (10(-5) M) and cromakalim (10(-5) M), but was not influenced by atropine (10(-6) M). Isoprenaline decreased the amplitude, and changed the pattern of 40 mM KCl-induced rhythmic contraction in concentration-dependent manner. These results suggest the possibility that the stimulation of beta-adrenoceptors may regulate the ureteral peristalsis. Salbutamol (selective beta2-AR agonist) and CGP12177 (beta(1,2)-AR antagonist and beta3-AR partial agonist) were also effective in decreasing the amplitude and changing the pattern of the rhythmic contraction. The pD2 values of agonists were 7.57 (isoprenaline), 5.80 (CGP12177) and 7.63 (salbutamol), respectively. The concentration-response curves of isoprenaline and salbutamol were rightward shifted by the presence of propranolol, and the apparent pA2 values for propranolol against isoprenaline and salbutamol were 7.12 and 6.29, respectively. These results suggest that inhibition for 40 mM KCl-induced rhythmic contraction of the ureter by isoprenaline and salbutamol mediated mainly via atypical beta-adrenoceptor subtype.

Characterisation of the atypical beta-adrenoceptor in rabbit isolated jejunum using BRL 37344, cyanopindolol and SR 59230A.

The present study was carried out to further investigate the nature of the beta-adrenoceptor in rabbit jejunum using BRL 37344, a selective beta3-adrenoceptor agonist, cyanopindolol, a beta-adrenoceptor antagonist with blocking activity at beta3-adrenoceptors and SR 59230A, a new selective beta3-adrenoceptor antagonist. Isoprenaline produced a concentration-dependent inhibition of the spontaneous contractions of rabbit jejunum with a pD2 of 7.14. Propranolol (1 microM) shifted the isoprenaline concentration-response curve (CRC) to the right with a concentration-ratio of 5.85, considerably less than would be expected for an action at classical beta-adrenoceptors (estimated pA2 6.66). BRL 37344 also produced a concentration-dependent inhibition of spontaneous contractions with a pD2 of 7.41. The BRL 37344 CRC was unaffected by propranolol (1 microM). In the presence of propranolol (1 microM), cyanopindolol (1 microM) shifted the isoprenaline CRC to the right (concentration-ratio of 21). Cyanopindolol also shifted the BRL 37344 CRC to the right (concentration-ratio of 38). These shifts are consistent with the affinity of cyanopindolol for beta3-adrenoceptors (estimated pA2 values of 7.27 and 7.38 against isoprenaline and BRL 37344, respectively). In the presence of propranolol (1 microM), SR 59230A produced a concentration-dependent rightward shift of the isoprenaline CRC. The Schild plot gave a pA2 value of 7.16, although the slope of the regression line was significantly different from unity (0.65). SR 59230A also produced a concentration-dependent shift of the BRL 37344 CRC. The Schild plot gave a pA2 of 7.58 with the slope of the regression line not significantly different from unity (0.81). The presence of beta3-adrenoceptors mediating relaxation of spontaneous contractions in rabbit jejunum is supported by the relatively poor antagonism of isoprenaline by propranolol, the relaxant effect of BRL 37344 and the antagonism of isoprenaline and BRL 37344 by cyanopindolol and SR 59230A. The lack of simple competitive antagonism of isoprenaline, but not BRL 37344, by SR 59230A may suggest more than one population of atypical beta-adrenoceptor.

Relative resistance of functional β2-adrenoceptor-mediated smooth muscle responses to in vitro desensitization

The effects of in vitro incubation of rat isolated left atria, pulmonary artery rings, and aortic rings with isoprenaline (10(-6) M for 6 h) were examined to compare the degree of desensitization of beta1- and beta2-adrenoceptor-mediated functional responses. The experimental protocols were carefully controlled to exclude influence from persistence of agonist in the tissues after the prolonged exposures, time-dependent changes in tissue sensitivity, and the methods of plotting the data. Concentration-response curves for isoprenaline were constructed before incubation with isoprenaline and, after washout during 1 h, a second curve was obtained. Two protocols were employed: firstly, the preincubation curve was constructed to ensure that a maximum response was obtained (>10(-6) M) and, secondly, the preincubation curve was constructed to a maximum isoprenaline concentration of 10(-6) M. Preincubation curves were corrected for time-dependent changes in sensitivity from sham-incubation control experiments. There was significant desensitization of the beta1-adrenoceptor-mediated positive inotropic responses of the left atria, using both protocols, seen as rightward shifts (dose ratios: 4.48 +/- 1.12 and 8.39 +/- 2.3) of the concentration-response curves and depression of the maximum responses (77.0 +/- 3.2 and 60.8 +/- 5.5%). In contrast, the beta2-adrenoceptor-mediated relaxations of the noradrenaline-constricted pulmonary artery and aorta did not display a significant loss of sensitivity. When the relaxation responses were plotted as a percentage of the noradrenaline-induced tone, there was no significant rightward shift of the concentration-response curves in the pulmonary artery (dose ratios: 2.82 +/- 1.33 and 2.24 +/- 0.62) or aorta (dose ratios: 1.43 +/- 0.62 and 1.31 +/- 0.27) and thus no desensitization.

Relative resistance of functional β&lt;SUB&gt;2&lt;/SUB&gt;-adrenoceptor-mediated smooth muscle responses to in vitro desensitization

The effects of in vitro incubation of rat isolated left atria, pulmonary artery rings, and aortic rings with isoprenaline (10(-6) M for 6 h) were examined to compare the degree of desensitization of beta1- and beta2-adrenoceptor-mediated functional responses. The experimental protocols were carefully controlled to exclude influence from persistence of agonist in the tissues after the prolonged exposures, time-dependent changes in tissue sensitivity, and the methods of plotting the data. Concentration-response curves for isoprenaline were constructed before incubation with isoprenaline and, after washout during 1 h, a second curve was obtained. Two protocols were employed: firstly, the preincubation curve was constructed to ensure that a maximum response was obtained (>10(-6) M) and, secondly, the preincubation curve was constructed to a maximum isoprenaline concentration of 10(-6) M. Preincubation curves were corrected for time-dependent changes in sensitivity from sham-incubation control experiments. There was significant desensitization of the beta1-adrenoceptor-mediated positive inotropic responses of the left atria, using both protocols, seen as rightward shifts (dose ratios: 4.48 +/- 1.12 and 8.39 +/- 2.3) of the concentration-response curves and depression of the maximum responses (77.0 +/- 3.2 and 60.8 +/- 5.5%). In contrast, the beta2-adrenoceptor-mediated relaxations of the noradrenaline-constricted pulmonary artery and aorta did not display a significant loss of sensitivity. When the relaxation responses were plotted as a percentage of the noradrenaline-induced tone, there was no significant rightward shift of the concentration-response curves in the pulmonary artery (dose ratios: 2.82 +/- 1.33 and 2.24 +/- 0.62) or aorta (dose ratios: 1.43 +/- 0.62 and 1.31 +/- 0.27) and thus no desensitization.

Bopindolol: A Slowly Dissociating Antagonist from the β-Adrenoceptors in Guinea Pig Atria

The dissociating and/or residual inhibitory effects of bopindolol from β-adrenoceptors of atria strips pretreated with this drug which was then washed out with buffers on the responses to isoprenaline were determined and compared with those of propranolol, pindolol, atenolol, and the two active metabolites of bopindolol: 18-502 and 20-785. Low concentrations of bopindolol (10^–9 to 10^–8 mol/l) and the active metabolite 18-502 (10^–9 mol/l) produced rightward shifts of the concentration-response curves. On the other hand, high concentrations of bopindolol (10^–7 mol/l) and metabolite 18-502 (10^–8 and 10^–7 mol/l) produced a reduced maximum response by isoprenaline, suggesting that these nonparallel rightward shifts have pD₂ values of 7.57 (bopindolol) and 7.67 (18-502), respectively, at 0 min after washout with buffers. Pindolol (10^–7 mol/l) and propranolol (10^–7 and 10^–8 mol/l) also produced a rightward shift of isoprenaline response curves, and these concentration-response curves in guinea pig atria strips pretreated with pindolol (10^–7 mol/l) and propranolol (10^–6 mol/l) recovered to control levels. Neither of these drugs, however, reduced the maximum response by isoprenaline. A high concentration (10^–5 mol/l) of atenolol was required for a rightward shift of the isoprenaline concentration-response curve, and this drug also did not reduce the maximum response. Thus, we conclude that bopindolol and metabolite 18-502 slowly dissociate and act as noncompetitive β-antagonists rather than easily reversible β-adrenoceptor antagonists.

Beta-adrenoceptors in the detrusor of guinea pig bladder.

In the present study, we tried to determine which beta-adrenoceptor subtypes are involved in the inhibitory regulation of bladder motility in the guinea pig by using in vitro functional analysis. Isoprenaline, norepinephrine and epinephrine decreased spontaneous contractile activity of the bladder concentration-dependently, the rank order of their potency being isoprenaline (pD2 = 7.38)>norepinephrine (6.71)>epinephrine (6.31). Dobutamine was also effective in inhibiting the spontaneous contraction (5.81). However, CGP-12177, BRL37344, salbutamol, and clenbuterol were not effective at the concentration of 30 microM. The concentration response curves of catecholamines (isoprenaline, norepinephrine and epinephrine) were rightward shifted by the presence of atenolol. Schild regression analyses carried out for atenolol against isoprenaline, norepinephrine and epinephrine gave pA2 values of 6.93, 6.78 and 6.69, respectively. The concentration-response curve for isoprenaline was unaffected by 10 microM butoxamine. In the presence of 1 microM propranolol, bupranolol produced shifts of the concentration-response curve for isoprenaline, and the apparent pA2 value for bupranolol against isoprenaline was 5.50. These results suggest that the inhibition of spontaneous contraction of the guinea pig bladder by beta-adrenoceptor agonists is mediated mainly via beta1-adrenoceptor and the effect of isoprenaline is mediated partly via atypical beta-adrenoceptor subtype.

Beta-adrenoceptor-mediated inhibition of alpha 1-adrenoceptor-mediated and field stimulation-induced contractile responses in the prostate of the guinea pig.

1. The prostate of the guinea pig responds to electrical field-stimulation (2 s trains, 0.1 ms pulses at 3-60 Hz, supramaximal voltage) with contractile responses. At 18 Hz these responses were inhibited (82 +/- 2%) by the L-type Ca2+ channel blocker, nifedipine (10 microM) and (by 100%) by the neurotoxin, tetrodotoxin (500 nM). The alpha 1A-selective adrenoceptor antagonist, 5-methylurapidil, inhibited responses to field stimulation in the absence and presence of nifedipine (10 microM) with -log molar (p) IC50 (+/- s.e. mean) values of 7.95 +/- 0.14 and 7.01 +/- 0.07, respectively. 2. The non-selective beta-adrenoceptor agonist, isoprenaline, reduced (56 +/- 8%) field stimulation induced contractile responses (pEC50 6.91 +/- 0.11). The non-selective beta-adrenoceptor antagonist propranolol (50 nM) and the beta 1-adrenoceptor selective antagonist, atenolol (3 microM), but not the beta 2-adrenoceptor antagonist ICI 118,551 ((+/-)-1 -[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxyl]-3-[1-methylethyl)amino ]-2-butanol HCl; 100 nM) antagonized this effect (apparent pKB values 8.44 +/- 0.22 and 6.92 +/- 0.21, respectively) indicating an effect mediated through beta 1-like adrenoceptors. In the presence of nifedipine (10 microM) isoprenaline (up to 10 microM) did not inhibit the remaining response to field-stimulation. 3. Phenylephrine elicited contractile responses (pEC50 4.47 +/- 0.30) from preparations of guinea pig prostate which were reduced (63 +/- 25%) by nifedipine (10 microM). This response was antagonized by 5-methylurapidil (100 nM, apparent pKB 8.24 +/- 0.33), but was not affected by preincubation chloroethylclonidine (50 microM, 30 min). Responses to phenylephrine (30 microM) were inhibited (by up to 52 +/- 5%) by isoprenaline (pIC50 6.40 +/- 0.35, the beta 2-adrenoceptor selective agonist, salbutamol was weakly effective). Propranolol (300 nM), ICI 118,551 (100 nM) and atenolol (3 microM) shifted isoprenaline concentration-response curves to the right (apparent pKB +/- s.e. values 7.68 +/- 1.10; 8.00 +/- 0.72 and 6.62 +/- 0.95, respectively). In the presence of nifedipine (10 microM) responses to phenylephrine (30 microM,) were inhibited (by up to 51 +/- 4%) by isoprenaline (pIC50 6.88 +/- 0.17): propranolol (300 nM) and ICI 118,551 (100 nM), but not atenolol (3 microM) antagonized this effect (apparent pKB values 8.85 +/- 1.53 and 8.35 +/- 1.18, respectively). Thus beta 1-like and beta 2-like adrenoceptors may be involved in the isoprenaline-stimulated inhibition of phenylephrine concentration-response curves. 4. Phenylephrine stimulated [3H]-inositol phosphate accumulation (pEC50 4.47 +/- 0.83), an effect insensitive to chloroethylclonidine pre-treatment (50 microM, 30 min) and to nifedipine (10 microM), but inhibited by 5-methylurapidil (apparent pKD 7.90 +/- 0.22). Isoprenaline (up to 1 microM) did not affect the phenylephrine-stimulated maximal increase in [3H]-inositol phosphates but did increase [3H]-cyclic adenosine monophosphate ([3H]-cAMP) accumulation (pEC50 6.77 +/- 0.66); propranolol (30 nM) and ICI 118,551 (110 nM), but not atenolol (up to 3 microM), antagonized this effect. These responses may therefore be mediated through beta 2-like adrenoceptors. 5. These results show that the alpha 1-adrenoceptor mediated and field stimulation-induced contractions of the guinea pig prostate are partly dependent upon intracellular and extracellular sources of Ca2+. We conclude that both beta 1- and beta 2-like adrenoceptors inhibit responses to phenylephrine in the prostate of the guinea pig. The beta 1-like adrenoceptor-mediated inhibition of these responses is evident upon the field stimulation-induced and nifedipine-sensitive component of the response to phenylephrine and may not involve the activation of adenylyl cyclase. The beta 2-like adrenoceptor may inhibit both nifedipine sensitive and insensitive components of the response to phenylephrine, possibly through the activation of adenylyl cyclase, but not through the i

Effect of Ca 2+-channel blockers on isoprenaline-induced desensitization in rat trachea

Isoprenaline-induced desensitization in vitro in rat trachea was studied in the presence of the Ca 2+-channel blockers (Ca 2+-CBs) verapamil and nitrendipine. The concentration-response curves for isoprenaline were determined in a noncumulative manner using carbachol as contracting agent, and then desensitization was achieved by 40-min incubation of the tracheal preparations with isoprenaline (1 μM). The effect of verapamil and nitrendipine was studied by the addition of each Ca 2+-CB to the desensitizing solution. Both verapamil and nitrendipine reduced the isoprenaline-induced desensitization in the rat trachea.

The beta2- and beta3-adrenoceptor-mediated relaxation induced by isoprenaline and salbutamol in guinea pig taenia caecum.

To understand the receptor subtypes responsible for beta-adrenoceptor-mediated relaxation of guinea pig taenia caecum, we investigated the effects of isoprenaline and salbutamol. Isoprenaline and salbutamol caused dose-dependent relaxation of the guinea pig taenia caecum. Propranolol, bupranolol and butoxamine produced shifts of the concentration response curves for isoprenaline and salbutamol. Schild regression analyses carried out for propranolol against isoprenaline and salbutamol gave pA2 values of 8.43 and 8.88, respectively. Schild regression analyses carried out for butoxamine against isoprenaline and salbutamol gave pA2 values of 6.46 and 6.68, respectively. Schild regression analyses carried out for bupranolol against isoprenaline and salbutamol gave pA2 values of 8.60 and 8.69, respectively. However, in the presence of 3 x 10(-4) M atenolol, 10(-4) M butoxamine and 10(-6) M phentolamine to block the beta1-, beta2- and alpha-adrenoceptor effects, respectively, Schild regression analyses carried out for bupranolol against isoprenaline and salbutamol gave pA2 values of 5.77 and 5.97, respectively. These results suggest that the relaxant responses to isoprenaline and salbutamol in the guinea pig taenia caecum are mediated by both the beta2- and the beta3-adrenoceptors.

The effects of bromoacetylalprenololmenthane on the responses to isoprenaline of the left ventricle from normo-, prehyper- and hypertensive rats.

1. We have studied the effects of bromoacetylalprenololmenthane (BAAM), a very slowly reversible beta-adrenoceptor antagonist, on the responses of the left ventricle of 5 and 22 week old Wistar Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs) to isoprenaline. At 5 weeks the SHRs were prehypertensive and at 22 weeks they had hypertension-induced hypertrophy of the left ventricle. 2. The mean potency of isoprenaline on the left ventricle from 5 week old WKY was similar to that obtained on left ventricle with BAAM at 10(-6) M for 30 min, there was a parallel rightward shift of the isoprenaline concentration-response curves. Treatment with a higher concentration of BAAM (10(-5)M) caused non-parallel rightward shifts of the concentration-response curves, with a depression of the isoprenaline maximum responses. These data were used to derive affinity (KA) values for isoprenaline. The mean isoprenaline KA value on the left ventricle from 5 week old WKY was 2.44 x 10(-6)M, and similar KA values were obtained on the left ventricles from 22 week old WKY and 5 and 22 week old SHRs. On all the left ventricles tested, isoprenaline produced a half maximal response by occupying less than 1%, and a near maximal response by occupying about 5% of the available beta(1)-adrenoceptors. 4. This study has shown that there are no differences in the cardiac responses to isoprenaline at beta(1)-adrenoceptors, isoprenaline KA values or the beta(1)-adrenoceptor reserve for isoprenaline on the SHR left ventricle in prehypertension or in the early stages of hypertension-induced hypertrophy.

Diazepam potentiates the positive inotropic effect of isoprenaline in rat ventricle strips: role of cyclic AMP

The responses of the electrically driven right ventricle strip of the rat heart to isoprenaline and other cyclic AMP-related inotropic agents were recorded in the absence and in the presence of diazepam. Isoprenaline, in concentrations ranging from 10 nM to 1 μM, significantly increased, in a concentration-dependent manner, the contractile force in this preparation. Diazepam (10 μM) produced a leftward shift in the isoprenaline concentration-response curve and significantly reduced its EC 50. Higher concentrations of diazepam (100 μM) produced no further shift, but reduced the maximum of the concentration-response curve of isoprenaline. Forskolin (0.5–10 μM), which directly stimulates adenyl cyclase, also produced a concentration-dependent increase in cardiac contractility. Diazepam (10 μM) displaced to the left the concentration-response curve for forskolin and reduced its EC 50. The cyclic AMP analogous dibutyryl cyclic AMP (0.1–1 mM) produced concentration-dependent positive inotropic effects which were not significantly modified in the presence of diazepam (10 μM). Diazepam (10 μM) significantly enhanced the cyclic AMP production induced by isoprenaline (0.1 μM) and forskolin (10 μM) by about 136% and 35% respectively. These results indicate that diazepam potentiates the positive inotropic effect induced by β-adrenoceptor agonists, probably by increasing cyclic AMP production induced by these agents.