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1.  Virodhamine and CP55,940 modulate cAMP production and IL-8 release in human bronchial epithelial cells 
British Journal of Pharmacology  2007;151(7):1041-1048.
Background and purpose:
We investigated expression of cannabinoid receptors and the effects of the endogenous cannabinoid virodhamine and the synthetic agonist CP55,940 on cAMP accumulation and interleukin-8 (IL-8) release in human bronchial epithelial cells.
Experimental approach:
Human bronchial epithelial (16HBE14o-) cells were used. Total mRNA was isolated and cannabinoid receptor mRNAs were detected by RT-PCR. Expression of CB1 and CB2 receptor proteins was detected with Western blotting using receptor-specific antibodies. cAMP accumulation was measured by competitive radioligand binding assay. IL-8 release was measured by ELISA.
Key results:
CB1 and CB2 receptor mRNAs and proteins were found. Both agonists concentration-dependently decreased forskolin-induced cAMP accumulation. This effect was inhibited by the CB2 receptor antagonist SR144528, and was sensitive to Pertussis toxin (PTX), suggesting the involvement of CB2 receptors and Gi/o-proteins. Cell pretreatment with PTX unmasked a stimulatory component, which was blocked by the CB1 receptor antagonist SR141716A. CB2 receptor-mediated inhibition of cAMP production by virodhamine and CP55,940 was paralleled by inhibition of tumor necrosis factor-α (TNF-α) induced IL-8 release. This inhibition was insensitive to SR141716A. In the absence of agonist, SR144528 by itself reduced TNF-α induced IL-8 release.
Conclusions and implications:
Our results show for the first time that 16HBE14o− cells respond to virodhamine and CP55,940. CB1 and CB2 receptor subtypes mediated activation and inhibition of adenylyl cyclase, respectively. Stimulation of the dominant CB2 receptor signalling pathway diminished cAMP accumulation and TNF-α-induced IL-8 release. These observations may imply that cannabinoids exert anti-inflammatory properties in airways by modulating cytokine release.
doi:10.1038/sj.bjp.0707320
PMCID: PMC2042924  PMID: 17558435
cannabinoids; virodhamine; CP55,940; human bronchial epithelium; interleukin-8; cAMP; tumour necrosis factor-α
2.  Insulin induces airway smooth muscle contraction 
British Journal of Pharmacology  2006;150(2):136-142.
Background and purpose:
Recently, the use of inhaled insulin formulations for the treatment of type I and type II diabetes has been approved in Europe and in the United States. For regular use, it is critical that airway function remains unimpaired in response to insulin exposure.
Experimental approach:
We investigated the effects of insulin on airway smooth muscle (ASM) contraction and contractile prostaglandin (PG) production, using guinea-pig open-ring tracheal smooth muscle preparations.
Key results:
It was found that insulin (1 nM-1 μM) induced a concentration-dependent contraction that was insensitive to epithelium removal. These sustained contractions were susceptible to inhibitors of cyclooxygenase (indomethacin, 3 μM), Rho-kinase (Y-27632, 1 μM) and p42/44 MAP kinase (PD-98059, 30 μM and U-0126, 3 μM), but not of PI-3-kinase (LY-294002,10 μM). In addition, insulin significantly increased PGF2α-production which was inhibited by indomethacin, but not Y-27632. Moreover, the FP-receptor antagonist AL-8810 (10 μM) and the EP1-receptor antagonist AH-6809 (10 μM) strongly reduced insulin-induced contractions, supporting a pivotal role for contractile prostaglandins.
Conclusions and implications:
Collectively, the results show that insulin induces guinea-pig ASM contraction presumably through the production of contractile prostaglandins, which in turn are dependent on Rho-kinase for their contractile effects. The data suggest that administration of insulin as an aerosol could result in some acute adverse effects on ASM function.
doi:10.1038/sj.bjp.0706985
PMCID: PMC2042899  PMID: 17160007
insulin; contraction; airway smooth muscle; Rho-kinase; guinea-pig; prostaglandins
3.  Inflammatory cell distribution in guinea pig airways and its relationship to airway reactivity. 
Mediators of Inflammation  2001;10(3):143-154.
Although airway inflammation and airway hyperreactivity are observed after allergen inhalation both in allergic humans and animals, little is known about the mechanisms by which inflammatory cells can contribute to allergen-induced airway hyperreactivity. To understand how inflammatory cell infiltration can contribute to airway hyperreactivity, the location of these cells within the airways may be crucial Using a guinea pig model of acute allergic asthma, we investigated the inflammatory cell infiltration in different airway compartments at 6 and 24 h (i.e. after the early and the late asthmatic reaction, respectively) after allergen or saline challenge in relation to changes in airway reactivity (AR) to histamine. At 6 h after allergen challenge, a threefold (p < 0.01) increase in the AR to histamine was observed. At 24 h after challenge, the AR to histamine was lower, but still significantly enhanced (1.6-fold, p < 0.05). Adventitial eosinophil and neutrophil numbers in both bronchi and bronchioli were significantly increased at 6 h post-allergen provocation as compared with saline (p < 0.01 for all), while there was a strong tendency to enhanced eosinophils in the bronchial submucosa at this time point (p = 0.08). At 24h after allergen challenge, the eosinophilic and neutrophilic cell infiltration was reduced. CD3+ T lymphocytes were increased in the adventitial compartment of the large airways (p < 0.05) and in the parenchyma (p < 0.05) at 24h post-allergen, while numbers of CD8+ cells did not differ from saline treatment at any time point post-provocation. The results indicate that, after allergen provocation, inflammatory cell numbers in the airways are mainly elevated in the adventitial compartment. The adventitial inflammation could be important for the development of allergen-induced airway hyperreactivity.
PMCID: PMC1781701  PMID: 11545251
4.  Influence of adrenodemedullation on beta 2- and beta 3-adrenoceptors mediating relaxation of oesophageal smooth muscle of spontaneously hypertensive rats. 
British Journal of Pharmacology  1996;119(7):1355-1360.
1. In oesophageal smooth muscle strips from spontaneously hypertensive rats (SHR) of 8-10 and 22-24 weeks of age, respectively, beta-adrenoceptor-mediated relaxation was investigated, by use of the beta-agonists, (-)-isoprenaline and fenoterol (both in the absence and presence of the beta 2-selective antagonist ICI 118,551) and the selective beta 3-agonist, BRL 37,344. 2. In preparations from 8-10 week SHR, (-)-isoprenaline- and fenoterol-induced concentration-response curves (CRCs) were hardly antagonized by ICI 118,551 at concentrations up to 1 microM, indicating only a minor contribution of beta 2-adrenoceptors. pA2-values for ICI 118,551 of 5.30 ((-)-isoprenaline as agonist) and 5.46 (fenoterol as agonist), estimated from the shifts at the highest (10-100 microM) antagonist concentrations, are consistent with affinity at a beta 3-adrenoceptor, similar to that in Wistar rat oesophageal smooth muscle. 3. In 8-10 week SHR, adrenodemedullated at 4 weeks of age (SHR-ADM4) the potency of fenoterol was markedly increased and CRCs were shallow. In addition, ICI 118,551 (0.1 microM) now produced a clear rightward shift accompanied by a steepening of the CRC. A marked further shift was observed only at 100 microM of the antagonist. The data are compatible with the involvement of both beta 2- and beta 3-adrenoceptors. 4. In 22-24 week animals, the same differences between SHR and SHR-ADM4 were observed with fenoterol as in 8-10 week animals, though beta-adrenoceptor responsiveness was slightly decreased. The potency of ICI 118,551 at beta 3-adrenoceptors (pA2 = 5.11) was significantly different from the pA2 value of 5.46 obtained with the younger animals. 5. Responses to the beta 3-adrenoceptor agonist, BRL 37,344, were similar in Wistar rat and SHR preparations. In 8-10 week SHR, a small decrease in the maximal response was observed, which in animals of 22-24 weeks of age was accompanied by a small decrease in the pEC50 value as well. 6. The results clearly indicate that beta 2-adrenoceptors in SHR oesophageal muscularis mucosae are desensitized, whereas beta 3-adrenoceptor-mediated responses are unaffected and similar to the responses observed in the Wistar rat oesophagus. The functional presence of beta 2-adrenoceptor-responses in SHR-ADM4 suggests a major role for adrenal-derived adrenaline in the desensitization of the beta 2-adrenoceptor-population.
PMCID: PMC1915822  PMID: 8968543
5.  Deficiency of nitric oxide in allergen-induced airway hyperreactivity to contractile agonists after the early asthmatic reaction: an ex vivo study. 
British Journal of Pharmacology  1996;119(6):1109-1116.
1. Using a guinea-pig model of allergic asthma, we investigated the role of nitric oxide (NO) in allergen-induced airway hyperreactivity after the early asthmatic reaction, by examining the effects of the NO-synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) on the responsiveness to methacholine and histamine of isolated perfused tracheae from unchallenged (control) animals and from animals 6 h after ovalbumin challenge. 2. All animals developed airway hyperreactivity to inhaled histamine at 6 h after ovalbumin challenge, with a mean 3.11 +/- 0.45 fold increase in sensitivity to the agonist (P < 0.001). 3. In perfused tracheal preparations from the ovalbumin-challenged guinea-pigs, the maximal responses (Emax) to methacholine and histamine were significantly enhanced compared to controls, both after intraluminal (IL) and extraluminal (EL) administration of the contractile agonists. In addition, a small but significant increase in the pD2 (-log10 EC50) for IL and EL methacholine and for IL histamine was observed. As a consequence, the delta pD2 (EL-IL) for histamine was slightly decreased from 1.67 +/- 0.13 to 1.23 +/- 0.14 (P < 0.05). However, the delta pD2 for methacholine was unchanged (1.85 +/- 0.11 and 1.77 +/- 0.12, respectively; NS). 4. Incubation of control tracheae with 100 microM L-NAME (IL) significantly enhanced the Emax for both IL and EL methacholine and histamine to approximately the same degree as observed after ovalbumin challenge, with no effect on the pD2 and delta pD2 for both agonists. On the contrary, L-NAME had no effect on Emax and pD2 values of tracheal preparations from ovalbumin-challenged guinea-pigs. 5. L-NAME (10 microM-1 mM) had no effect on methacholine-induced contraction of isolated tracheal strip preparations obtained from control animals, indicating that L-NAME has no antimuscarinic effect on tracheal smooth muscle. 6. Histological examination of the intact tracheal preparations indicated epithelial and subepithelial infiltration of eosinophils after ovalbumin challenge. However, no apparent damage of the airway epithelium was observed in these preparations. 7. The results indicate that a deficiency of NO contributes to allergen-induced airway hyperreactivity after the early asthmatic reaction and that this deficiency appears not to be due to epithelial shedding.
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PMCID: PMC1915910  PMID: 8937712
6.  Modulation of agonist-induced phosphoinositide metabolism, Ca2+ signalling and contraction of airway smooth muscle by cyclic AMP-dependent mechanisms. 
British Journal of Pharmacology  1996;117(3):419-426.
1. The effects of increased cellular cyclic AMP levels induced by isoprenaline, forskolin and 8-bromoadenosine 3':5'-cyclic monophosphate (8-Br-cyclic AMP) on phosphoinositide metabolism and changes in intracellular Ca2+ elicited by methacholine and histamine were examined in bovine isolated tracheal smooth muscle (BTSM) cells. 2. Isoprenaline (pD2 (-log10 EC50) = 6.32 +/- 0.24) and forskolin (pD2 = 5.6 +/- 0.05) enhanced cyclic AMP levels in a concentration-dependent fashion in these cells, while methacholine (pD2 = 5.64 +/- 0.12) and histamine (pD2 = 4.90 +/- 0.04) caused a concentration-related increase in [3H]-inositol phosphates (IP) accumulation in the presence of 10 mM LiCl. 3. Preincubation of the cells (5 min, 37 degrees C) with isoprenaline (1 microM), forskolin (10 microM) and 8-Br-cyclic AMP (1 mM) did not affect the IP accumulation induced by methacholine, but significantly reduced the maximal IP production by histamine (1 mM). However, the effect of isoprenaline was small (15.0 +/- 0.6% inhibition) and insignificant at histamine concentrations between 0.1 and 100 microM. 4. Both methacholine and histamine induced a fast (max. in 0.5-2 s) and transient increase of intracellular Ca2+ concentration ([Ca2+]i) followed by a sustained phase lasting several minutes. EGTA (5 mM) attenuated the sustained phase, indicating that this phase depends on extracellular Ca2+. 5. Preincubation of the cells (5 min, 37 degrees C) with isoprenaline (1 microM), forskolin (10 microM) and 8-Br-cyclic AMP (1 microM) significantly attenuated both the Ca(2+)-transient and the sustained phase generated at equipotent IP producing concentrations of 1 microM methacholine and 100 microM histamine (approx. 40% of maximal methacholine-induced IP response), but did not affect changes in [Ca2+]i induced by 100 microM methacholine (95.2 +/- 3.5% of maximal methacholine-induced IP response). 6. Significant correlations were found between the isoprenaline-induced inhibition of BTSM contraction and inhibition of Ca2+ mobilization or influx induced by methacholine and histamine, that were similar for each contractile agonist. 7. These data indicate that (a) cyclic AMP-dependent inhibition of Ca2+ mobilization in BTSM cells is not primarily caused by attenuation of IP production, suggesting that cyclic AMP induced protein kinase A (PKA) activation is effective at a different level in the [Ca2+]i homeostasis, (b) that attenuation of intracellular Ca2+ concentration plays a major role in beta-adrenoceptor-mediated relaxation of methacholine- and histamine-induced airway smooth muscle contraction, and (c) that the relative resistance of the muscarinic agonist-induced contraction to beta-adrenoceptor agonists, especially at (supra) maximal contractile concentrations is largely determined by its higher potency in inducing intracellular Ca2+ changes.
PMCID: PMC1909321  PMID: 8821529
7.  Noradrenaline-induced relaxation of rat oesophageal muscularis mucosae: mediation solely by innervated beta 3-adrenoceptors. 
British Journal of Pharmacology  1995;116(3):1945-1947.
We investigated the effects of cocaine and corticosterone on the noradrenaline-induced relaxation of rat oesophageal smooth muscle in the absence and presence of the selective beta2-antagonist, ICI 111,551. It was found that the concentration-response curve (CRC) of noradrenaline was not shifted by ICI 118,551 at 1 microM, whereas a clear shift to the right was observed at 100 microM of the antagonist. In the presence of corticosterone (10 microM), CRC's were clearly shifted to the left; with cocaine (10 microM) additionally present, a further leftward shift was observed, indicating the involvement of both neuronal and extraneuronal uptake sites. It was concluded that the relaxation of rat oesophageal muscularis mucosae by noradrenaline is solely mediated by beta3-adrenoceptors which are sympathetically innervated.
PMCID: PMC1908944  PMID: 8640330
8.  No evidence for a role of muscarinic M2 receptors in functional antagonism in bovine trachea. 
British Journal of Pharmacology  1995;115(4):665-671.
1. The functional antagonism between methacholine- or histamine-induced contraction and beta-adrenoceptor-mediated relaxation was evaluated in bovine tracheal smooth muscle in vitro. In addition, the putative contribution of muscarinic M2 receptors mediating inhibition of beta-adrenoceptor-induced biochemical responses to this functional antagonism was investigated with the selective muscarinic antagonists, pirenzepine (M1 over M2), AF-DX 116 and gallamine (M2 over M3), and hexahydrosiladiphenidol (M3 over M2). 2. By use of isotonic tension measurement, contractions were induced with various concentrations of methacholine or histamine, and isoprenaline concentration-relaxation curves were obtained in the absence or presence of the muscarinic antagonists. Antagonist concentrations were chosen so as to produce selective blockade of M2 receptors (AF-DX 116 0.1 microM, gallamine 30 microM), or half-maximal blockade of M3 receptors (pirenzepine 0.1 microM, AF-DX 116 0.5 microM, hexahydrosiladiphenidol 0.03 microM). Since these latter antagonist concentrations mimicked KB values towards bovine tracheal smooth muscle M3 receptors, antagonist-induced decreases in contractile tone were compensated for by doubling the agonist concentration. 3. It was found that isoprenaline-induced relaxation of bovine tracheal smooth muscle preparations was dependent on the nature and the concentration of the contractile agonist used. Thus, isoprenaline pD2 (-log EC50) values were decreased 3.7 log units as a result of increasing cholinergic tone from 22 to 106%, and 2.4 log units by increasing histamine tone over a similar range. Furthermore, maximal relaxability of cholinergic tone decreased gradually from 100% at low to only 1.3% at supramaximal contraction levels, whereas with histamine almost complete relaxation was maintained at all concentrations applied.(ABSTRACT TRUNCATED AT 250 WORDS)
PMCID: PMC1908503  PMID: 7582488
9.  Dysfunction of muscarinic M2 receptors after the early allergic reaction: possible contribution to bronchial hyperresponsiveness in allergic guinea-pigs. 
British Journal of Pharmacology  1995;114(4):881-887.
1. Using a guinea-pig model of allergic asthma, in which the animals display early (0-5 h) and late phase (8-23 h after antigen challenge) bronchoconstrictor reactions, the function of prejunctional inhibitory M2 and postjunctional M3 receptors in isolated tracheal preparations have been investigated. In addition, cardiac M2 receptor function in vitro and bronchial responsiveness to histamine in vivo were evaluated. 2. Sensitivity to inhaled histamine was increased 3.1 fold and 1.6 fold after the early and late allergic reactions (i.e. at 5 h and 23 h after a single ovalbumin challenge), respectively. At 23 h after the last of four allergen challenges, executed on four consecutive days, bronchial hyperresponsiveness to histamine was diminished to 1.3 fold. 3. After the early response, there was no change in cardiac muscarinic M2 receptor function, since in left atria pD2 (-log EC50) and Emax values of pilocarpine and pKB values of AQ-RA 741, a selective M2 receptor antagonist, were not significantly different from controls (unchallenged sensitized animals), and this also applied to methacholine pD2 values for muscarinic M3 receptors in tracheal smooth muscle. 4. Prejunctional inhibitory muscarinic M2 autoreceptors in airway smooth muscle were markedly dysfunctional after the early allergic response, since potentiation of electrically evoked twitch contractions of tracheal preparations by low concentrations of the M2-selective muscarinic receptor antagonists, gallamine, methoctramine, AQ-RA 741 and AF-DX 116, which is the result of M2 receptor blockade, was clearly and significantly diminished compared to controls.(ABSTRACT TRUNCATED AT 250 WORDS)
PMCID: PMC1510216  PMID: 7773550
10.  Contribution of a cholinergic reflex mechanism to allergen-induced bronchial hyperreactivity in permanently instrumented, unrestrained guinea-pigs. 
British Journal of Pharmacology  1995;114(2):414-418.
1. In conscious, permanently instrumented, unrestrained, ovalbumin-sensitized guinea-pigs the development of allergen-induced bronchial hyperreactivity to histamine- and methacholine-inhalation was investigated after the early as well as after the late asthmatic response. 2. The allergen-induced increase in bronchial reactivity to histamine was significantly higher than to methacholine. 3. The muscarinic receptor antagonist, ipratropium bromide (1.0 mM, 3 min inhalation), blocked methacholine-induced bronchoconstriction and caused a significant 1.7 fold inhibition of the histamine-induced bronchoconstriction of control animals. 4. A lower dose of ipratropium bromide (0.1 mM, 3 min inhalation) had no significant effect on histamine-induced bronchoconstriction in control animals, but significantly reduced the allergen-induced increase in bronchial reactivity to histamine between the early and late asthmatic response. At 1.0 mM ipratropium bromide, no further reduction was observed. 5. These results clearly indicate that an exaggerated cholinergic reflex mechanism contributes to allergen-induced bronchial hyperreactivity to histamine.
PMCID: PMC1510260  PMID: 7881742
11.  Sustained prejunctional facilitation of noradrenergic neurotransmission by adrenaline as a co-transmitter in the portal vein of freely moving rats. 
British Journal of Pharmacology  1994;113(2):342-344.
1. The duration of the facilitatory effect of adrenaline on the electrically evoked overflow of noradrenaline was studied in the portal vein of permanently adreno-demedullated freely moving rats. 2. Rats were infused with adrenaline (20 or 100 ng min-1) for 2 h. After an interval of 1 h, when plasma adrenaline had returned to undetectable levels, electrical stimulation resulted in an enhanced catecholamine overflow amounting to 219% (noradrenaline) and 241% (noradrenaline plus adrenaline) of control (saline infusion), respectively. 3. When stimulation was applied again, in the same animal, at 24, 48 and 72 h after the first stimulation episode, the evoked noradrenaline overflow was 150, 111 and 102% (after 20 ng ml-1 adrenaline) and 158, 134 and 105% (after 100 ng min-1 adrenaline) of control. 4. The beta 2-adrenoceptor antagonist, ICI 118,551 (0.3 mg kg-1), blocked the facilitatory effect obtained after the 100 ng min-1 adrenaline infusion on all days. 5. The results show that adrenaline, after being taken up by and released from sympathetic nerve terminals, is able to facilitate the evoked noradrenaline overflow through activation of prejunctional beta 2-adrenoceptors for at least 48 h after administration.
PMCID: PMC1510130  PMID: 7834181
12.  Influence of sensitization and allergen provocation procedures on the development of allergen-induced bronchial hyperreactivity in conscious, unrestrained guinea-pigs 
Mediators of Inflammation  1995;4(2):149-156.
The effects of different sensitization and allergen provocation regimens on the development of allergen-induced bronchial hyperreactivity (BHR) to histamine were investigated in conscious, unrestrained guinea-pigs. Similar early and late phase asthmatic reactions, BHR for inhaled histamine after the early (6 h) as well as after the late reaction (24 h), and airway inflammation were observed after a single allergen provocation in animals sensitized to produce mainly IgG or IgE antibodies, respectively. Repeating the allergen provocation in the IgE-sensitized animals after 7 days, using identical provocation conditions, resulted in a similar development of BHR to histamine inhalation. Repetition of the allergen provocation during 4 subsequent days resulted in a decreased development of BHR after each provocation, despite a significant increase in the allergen provocation dose necessary to obtain similar airway obstruction. The number of inflammatory cells in the bronchoalveolar lavage was not significantly changed after repeated provocation, when compared with a single allergen provocation. Finally, we investigated allergen-induced bronchial hyperreactivity by repetition of the sensitization procedure at day 7 and 14 (booster), followed by repeated allergen provocation twice a week for 5 weeks. Surprisingly, no BHR to histamine could be observed after either provocation, while the number of inflammatory cells in the bronchoalveolar lavage fluid after 5 weeks was enhanced compared with controls. These data indicate that both IgE and IgG sensitized guinea-pigs may develop bronchial hyperreactivity after a single allergen provocation. Repeated allergen exposure of IgE sensitized animals causes a gradual fading of the induced hyperreactivity despite the on-going presence of inflammatory cells in the airways, indicating a mechanism of reduced cellular activation.
doi:10.1155/S0962935195000263
PMCID: PMC2365622  PMID: 18475633
13.  The beta-adrenoceptors mediating relaxation of rat oesophageal muscularis mucosae are predominantly of the beta 3-, but also of the beta 2-subtype. 
British Journal of Pharmacology  1993;110(1):442-446.
1. beta-Adrenoceptor-mediated relaxation of rat oesophageal smooth muscle was investigated by studying the effects of beta 1- and beta 2-selective antagonists on the relaxation induced by (-)-isoprenaline, the beta 2-selective agonists fenoterol and clenbuterol and the beta 3-agonist, BRL 37344. 2. The highly beta 1-selective antagonist CGP 20721A did not antagonize (-)-isoprenaline- or BRL 37344-induced relaxations in concentrations up to 10 microM. Only at 100 microM of CGP 20712A were clear rightward shifts of the agonist concentration-response curves (CRCs) observed, with pA2 values of 4.70 and 4.97 against (-)-isoprenaline and BRL 37344, respectively. 3. ICI 118,551, a potent and selective beta 2-antagonist, at 100 nM caused moderate rightward shifts of the CRCs of (-)-isoprenaline, fenoterol and clenbuterol; with fenoterol and clenbuterol, this was accompanied by a clear steepening of the curve. Only at the highest concentration (100 microM ICI 118,551) did the shifts to the right further increase substantially. Resulting Schild-plots were clearly biphasic. BRL 37344-induced relaxations were only antagonized at 100 microM ICI 118,551, yielding a pA2 value of 5.48. 4. These results clearly demonstrate that the BRL 37344-induced relaxation of rat oesophageal muscularis mucosae is mediated solely through beta 3-adrenoceptors, whereas (-)-isoprenaline-, fenoterol- and clenbuterol-induced relaxations were shown to involve both beta 2- and, predominantly, beta 3-adrenoceptors.
PMCID: PMC2175964  PMID: 8106109
14.  Human bronchial cyclic nucleotide phosphodiesterase isoenzymes: biochemical and pharmacological analysis using selective inhibitors. 
British Journal of Pharmacology  1992;106(4):1028-1034.
1 The aims of the present study were to characterize the cyclic nucleotide phosphodiesterase (PDE) isoenzyme activities present in human bronchi and to examine the ability of selective isoenzyme inhibitors to relax histamine and methacholine precontracted preparations of human bronchi. 2 Three separations of pooled human bronchial tissue samples were performed. Ion-exchange chromatography showed that the soluble fraction of human bronchial preparations contains PDE I, II, III, IV and V isoenzyme activities. Multiple forms of PDE I and PDE IV were observed and PDE IV was the main cyclic AMP hydrolytic activity. 3 3-Isobutyl-l-methylxanthine (IBMX) non-selectively inhibited all separated isoenzyme activities. Zaprinast selectively inhibited PDE V, but also effectively inhibited one of the two PDE I isoforms identified. The PDE IV selective inhibitors rolipram and RO-201724, inhibited the PDE IV activities as did the dual PDE III/IV inhibitor, Org 30029. Org 9935, a PDE III selective inhibitor, potently attenuated part of the PDE IV activity peak in one of three separations performed, indicating that some PDE III activity may co-elute with PDE IV under the experimental conditions employed. 4 PDE IV-selective (rolipram), PDE III-selective (Org 9935) and dual PDE III/IV (Org 30029) inhibitors were effective relaxants of human bronchial smooth muscle. The PDE V/PDE I inhibitor, zaprinast was relatively ineffective. 5 The present study demonstrates in human bronchi, as in animal airways smooth muscle, that inhibitors of PDE III, PDEIV and dual PDE III/IV have potentially useful bronchodilator activity and are worthy of further consideration as anti-asthma drugs.
PMCID: PMC1907637  PMID: 1393276
15.  The presence of five cyclic nucleotide phosphodiesterase isoenzyme activities in bovine tracheal smooth muscle and the functional effects of selective inhibitors. 
British Journal of Pharmacology  1991;104(2):471-477.
1. The profile of cyclic nucleotide phosphodiesterase (PDE) isoenzymes and the relaxant effects of isoenzyme selective inhibitors were examined in bovine tracheal smooth muscle. The compounds examined were the non-selective inhibitor 3-isobutyl-1-methylxanthine (IBMX), zaprinast (PDE V selective), milrinone and Org 9935 (4,5-dihydro-6-(5,6-dimethoxy-benzo[b]thien-2-yl)-5-methyl-1 (2H)-pyridazinone; both PDE III selective), rolipram (PDE IV selective) and Org 30029 (N-hydroxy-5,6-dimethoxy-benzo[b]-thiophene-2-carboximidamide HCl a dual PDE III/IV inhibitor). 2. Ion exchange chromatography showed three main peaks of PDE activity. The first peak was stimulated by Ca2+/calmodulin (PDE I), the adenosine 3':5'-cyclic monophosphate (cyclic AMP) hydrolytic activity of the second peak was stimulated by guanosine 3':5'-cyclic monophosphate (cyclic GMP) (PDE II) whilst that of the third peak was not significantly modified by any regulator (PDE IV). Calmodulin affinity chromatography revealed the additional presence of cyclic GMP-specific PDE (PDE V) in the first peak. A clearly distinct peak of cyclic GMP-inhibited PDE (PDE III) was not observed. However, Org 9935 inhibited the third activity peak more effectively in the presence, than in the absence, of rolipram (3 mumol l-1), indicating the presence of PDE III activity. 3. Rolipram was the most potent inhibitor of PDE IV. The mean -log50 IC50 values for rolipram, IBMX, milrinone, Org 30029, Org 9935 and zaprinast were 5.9 +/- 0.1, 4.9 +/- 0.1, 4.7 +/- 0.1, 4.6 +/- 0.1 and 4.6 +/- 0.1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
PMCID: PMC1908540  PMID: 1665737
16.  Relationship between lipolysis and cyclic AMP generation mediated by atypical beta-adrenoceptors in rat adipocytes. 
British Journal of Pharmacology  1991;102(3):577-580.
1. The nature of the beta-adrenoceptor(s) mediating adenylyl cyclase activation in rat adipocyte ghosts by (-)-isoprenaline and the lipolytically selective beta-adrenoceptor agonist, BRL 37344, was investigated by use of the beta 1-selective antagonist, CGP 20712A. The results were compared with lipolysis in adipocytes. 2. While in lipolysis BRL 37344 was a full and 10 times more potent agonist than (-)-isoprenaline, in adenylyl cyclase activation similar pD2 values for both agonists were found. BRL 37344 was only a partial agonist on rat adipocyte adenylyl cyclase, with an intrinsic activity of 0.62. 3. With CGP 20712A small rightward shifts of the (-)-isoprenaline concentration-response curve (CRC) were observed at concentrations up to 10 microM, while at 100 microM and 1 mM clear rightward shifts occurred. The BRL 37344 CRC was not shifted with antagonist concentrations up to 10 microM. Only at 100 microM and 1 mM CGP 20712A were rightward shifts observed. 4. CGP 20712A concentrations of 10 microM and 100 microM depressed the maximum of the (-)-isoprenaline CRC to 89 and 60%, while the BRL 37344 CRCs retained the control maximum effect (62% of (-)-isoprenaline). Only at 1 mM CGP 20712A, was the CRC of BRL 37344 depressed, while the (-)-isoprenaline maximum was diminished further. 5. It was concluded that as with lipolysis, (-)-isoprenaline acts both through typical beta 1- and atypical beta 3-adrenoceptors for activation of adenylyl cyclase, while BRL 37344 acts solely through atypical beta 3-adrenoceptors.(ABSTRACT TRUNCATED AT 250 WORDS)
PMCID: PMC1917922  PMID: 1364822
17.  Characterization of presynaptic vascular muscarinic receptors inhibiting endogenous noradrenaline overflow in the portal vein of the freely moving rat. 
British Journal of Pharmacology  1990;99(2):223-226.
1. In the portal vein of permanently cannulated, freely moving, unanaesthetized rats, methacholine (MCh) is able to inhibit the electrically-evoked endogenous noradrenaline (NA) overflow. This inhibition is mediated by presynaptic inhibitory muscarinic heteroreceptors. 2. By use of pirenzepine, 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP) and AF-DX 116 as M1-, M3-, and M2-selective antagonists respectively, the MCh (0.1 microM)-induced inhibition of the electrically-evoked NA overflow could be reversed to the control stimulation value dose-dependently. 3. The potency order of the antagonists was: 4-DAMP greater than AF-DX 116 greater than pirenzepine, pIC50 values being 8.50, 7.96 and 7.01, respectively. 4. From these results it was concluded that the inhibitory presynaptic heteroreceptors in the portal vein of conscious unrestrained rats are of the cardiac M2-subtype.
PMCID: PMC1917397  PMID: 2328391
18.  Characterization of the muscarinic receptor subtype involved in phosphoinositide metabolism in bovine tracheal smooth muscle. 
British Journal of Pharmacology  1990;99(2):293-296.
1. The muscarinic receptor subtype involved in the methacholine-induced enhancement of phosphoinositide metabolism in bovine tracheal smooth muscle was identified by using the M2-selective antagonist AF-DX 116 and the M3-selective antagonist 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) methobromide, in addition to the M1-selective antagonist pirenzepine, in a classical Schild analysis. 2. All the antagonists shifted the methacholine dose-response curve to the right in a parallel and concentration-dependent fashion, yielding Schild plots with slopes not significantly different from unity. The pA2 values (6.94, 6.32 and 8.54 for pirenzepine, AF-DX 116 and 4-DAMP methobromide respectively) indicate that it is the M3 (smooth muscle/glandular), but not the M2 (cardiac) muscarinic receptor subtype, present in this tissue, that mediates phosphoinositide turnover, in accordance with our previous contractile studies. 3. The results provide additional evidence for the involvement of phosphoinositide turnover in the pharmacomechanical coupling between muscarinic receptor stimulation and contraction in (bovine tracheal) smooth muscle.
PMCID: PMC1917404  PMID: 2158372
19.  Direct evidence for the atypical nature of functional beta-adrenoceptors in rat adipocytes. 
British Journal of Pharmacology  1989;98(4):1420-1424.
1. The nature of the rat epididymal adipocyte beta-adrenoceptor was investigated by studying the effects of beta 1- and beta 2-selective antagonists on lipolysis induced by (-)-isoprenaline and the lipolytically selective agonist BRL 37344. 2. From 10 nM to 10 microM, the potent and highly selective beta 1-adrenoceptor antagonist CGP 20712A did not influence the concentration-response curve (CRC) of BRL 37344 whereas small but consistent shifts to the right of the (-)-isoprenaline-induced CRC were observed. Clear rightward shifts of the CRCs induced by both (-)-isoprenaline and BRL 37344 were produced only at 100 microM CGP 20712A with the corresponding pA2 values being 4.80 and 4.61, respectively. 3. When the beta 2-selective antagonist ICI 118,551 was used at 10 microM and higher, clear and concentration-dependent shifts to the right of the CRCs of both agonists were observed. The slopes of the Schild plots did not deviate significantly from unity, the pA2 values being 5.49 and 5.33 against (-)-isoprenaline and BRL 37344, respectively. 4. The results demonstrate that (-)-isoprenaline-induced lipolysis in rat white adipocytes is mediated predominantly by atypical beta-adrenoceptors, whereas the typical beta 1-adrenoceptors play a small, subordinate role. The lipolytically selective agonist BRL 37344 acts solely through atypical beta-adrenoceptors.
PMCID: PMC1854821  PMID: 2575416
20.  Effects of verapamil on ischaemia-induced impairment of ATP-dependent calcium extrusion in rat heart sarcolemma. 
British Journal of Pharmacology  1989;98(1):161-166.
1. The effects of ischaemia and reperfusion were studied on adenosine 5'-triphosphate (ATP)-dependent 45Ca2+-transport in rat heart sarcolemma vesicles. 2. The effect of verapamil, 1 mumol l-1, was studied by pretreatment of the hearts during Langendorff-perfusion and in vitro by adding the drug after isolation of the vesicles. 3. Without drug pretreatment the Ca2+-uptake appeared to be strongly reduced after 30 and after 60 min of global ischaemia, whereas after 30 min of reperfusion it was restored to slightly above the control level. 4. Verapamil pretreatment during the Langendorff perfusion significantly increased Ca2+-uptake in sarcolemma vesicles both before the onset of ischaemia and after 30 min of reperfusion, whereas no beneficial effect was found on the impaired uptake activity during the ischaemic period. 5. When tested in vitro after the isolation of the sarcolemma vesicles, verapamil only inhibited the Ca2+-uptake activity with an IC50 of 112 mumol l-1, which was increased to 250 mumol l-1 after ischaemia and reperfusion. 6. The present study indicates that pretreatment with verapamil, 1 mumol l-1, of the intact rat heart activates an ATP-dependent Ca2+ extrusion process that may contribute to decrease cellular calcium levels in control and, more importantly, in a reperfusion situation. In contrast, in vitro only a less potent inhibition of the extrusion process was found, indicating that physiological regulatory mechanisms may be altered in the vesicles.
PMCID: PMC1854651  PMID: 2804544
21.  Presynaptic muscarinic receptors inhibiting endogenous noradrenaline release in the portal vein of the freely moving rat. 
British Journal of Pharmacology  1989;97(2):586-590.
1. In the portal vein of the freely moving unanaesthetized rat, the existence of presynaptically located inhibitory muscarinic receptors was investigated by use of the muscarinic agonist methacholine (MCh) 2. Infusion of MCh (0.3 micrograms min-1) did not significantly inhibit the endogenous noradrenaline (NA) overflow in portal plasma. However, after inducing high intra-synaptic concentrations of NA by blocking the presynaptic alpha 2-adrenoceptors with yohimbine (1 mg kg-1), MCh (0.3 microgram min-1) was able to reduce the yohimbine-induced enhanced NA overflow by 38%. 3. The MCh-induced inhibition was almost completely abolished after blockade of the presynaptic muscarinic receptors with atropine (0.6 mg kg-1). 4. During electrical stimulation of the portal vein nervous plexus the evoked NA overflow was strongly inhibited (95%) during MCh-infusion (0.3 microgram min-1). Again atropine (0.6 mg kg-1) was able to reverse this inhibition. 5. These results show the existence of presynaptic muscarinic receptors inhibiting endogenous NA overflow from the portal vein nervous plexus under conditions of enhanced sympathetic activity in the freely moving rat.
PMCID: PMC1854505  PMID: 2758232
22.  Atypical characteristics of the beta-adrenoceptor mediating cyclic AMP generation and lipolysis in the rat adipocyte. 
British Journal of Pharmacology  1985;84(1):131-137.
The characteristics of the rat epidydimal adipocyte beta-adrenoceptor have been examined using lipolysis and cyclic AMP accumulation in adipocytes as well as adenylate cyclase activity in fat cell membranes. The pA2 values corrected for binding to bovine serum albumin of the selective antagonists betaxolol (beta 1-selective) and ICI 118.551 (beta 2-selective) against noradrenaline or fenoterol-stimulated lipolysis were indicative of an atypical beta-adrenoceptor associated with the lipolytic response. Antagonism of isoprenaline-stimulated cyclic AMP accumulation in whole cells and adenylate cyclase activity in membranes yielded pA2 values to betaxolol, ICI 118.551 and (-)-propranolol, which suggested that the atypical beta-adrenoceptor was coupled to adenylate cyclase. Comparisons of the Ki values obtained in binding studies using [125I]-cyanopindolol with pA2 values obtained in adenylate cyclase experiments suggest that the typical beta 1-receptor identified with radioligand binding studies is not the only receptor site mediating stimulation of adenylate cyclase activity and lipolysis.
PMCID: PMC1987202  PMID: 2983800
23.  β-adrenoceptor blocking agents and lipolysis 
British Journal of Clinical Pharmacology  1982;13(Suppl 2):181S-186S.
1 The pharmacological characteristics of β-adrenoceptor subtypes on adipocytes of various mammalian species, including man, are reviewed.
2 Rat adipocytes possess a homogeneous population of β-adrenoceptors with properties that clearly distinguish them from `classic' β1- and β2-adrenoceptors, although they share certain features with both. Thus, rat adipocyte β-adrenoceptors should be considered as non-β1-non-β2 receptors, like the atypical β-adrenoceptors found on erythrocytes of turkey, chicken and frog.
3 Preliminary data suggest that adipocyte β-adrenoceptors of guinea pig and swine are different from `classic' β1- and β2-adrenoceptors as well, whereas in the dog and possibly in the cat, a mixture of β1- and β2-receptors mediates catecholamine induced lipolysis.
4 Human adipocyte β-adrenoceptors probably also consist of at least two subtypes. Insufficient data are available to decide if these β-adrenoceptors are identical with `classic' β1- and β2-receptors, or share some hybrid characteristics with rat adipocyte β-adrenoceptors.
5 In vivo studies in animals as well as in man, tend to corroborate in vitro results. Cardioselective β-adrenoceptor blocking agents, like atenolol, metoprolol and practolol are not as effective in blocking catecholamine induced lipolysis as non-cardioselective agents like propranolol and pindolol. The relatively low potency of cardioselective β-adrenoceptor blocking agents is found using either isoprenaline, adrenaline or exercise as the agonist, suggesting that β2-adrenoceptors are involved. On the other hand, cardioselective agents, though less effective than non-cardioselective compounds, have a significant inhibitory effect on catecholamine induced lipolysis at doses that have only minimal effect on other β2-adrenoceptor mediated responses, which argues for participation of β1-adrenoceptors.
6 Thus, human in vitro and in vivo data are consistent with, but not proof of the hypothesis that a mixture of β1- and β2-adrenoceptors mediates lipolysis induced by sympathetic activation.
7 Species differences in β-adrenoceptor subtype characteristics may complicate the interpretation of the relevance of animal data for the understanding of β-adrenoceptor mediated effects in human adipocytes.
PMCID: PMC1402182  PMID: 6125168

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