The vascular Ca2+-sensing receptor regulates blood vessel tone and blood pressure.

The extracellular calcium-sensing receptor CaSR is expressed in blood vessels where its role is not completely understood. In this study, we tested the hypothesis that the CaSR expressed in vascular smooth muscle cells (VSMC) is directly involved in regulation of blood pressure and blood vessel tone. Mice with targeted CaSR gene ablation from vascular smooth muscle cells (VSMC) were generated by breeding exon 7 LoxP-CaSR mice with animals in which Cre recombinase is driven by a SM22α promoter (SM22α-Cre). Wire myography performed on Cre-negative [wild-type (WT)] and Cre-positive (SM22α)CaSR(Δflox/Δflox) [knockout (KO)] mice showed an endothelium-independent reduction in aorta and mesenteric artery contractility of KO compared with WT mice in response to KCl and to phenylephrine. Increasing extracellular calcium ion (Ca(2+)) concentrations (1-5 mM) evoked contraction in WT but only relaxation in KO aortas. Accordingly, diastolic and mean arterial blood pressures of KO animals were significantly reduced compared with WT, as measured by both tail cuff and radiotelemetry. This hypotension was mostly pronounced during the animals' active phase and was not rescued by either nitric oxide-synthase inhibition with nitro-l-arginine methyl ester or by a high-salt-supplemented diet. KO animals also exhibited cardiac remodeling, bradycardia, and reduced spontaneous activity in isolated hearts and cardiomyocyte-like cells. Our findings demonstrate a role for CaSR in the cardiovascular system and suggest that physiologically relevant changes in extracellular Ca(2+) concentrations could contribute to setting blood vessel tone levels and heart rate by directly acting on the cardiovascular CaSR.

LPS exacerbates functional and inflammatory responses to ovalbumin and decreases sensitivity to inhaled fluticasone propionate in a guinea pig model of asthma.

BACKGROUND AND PURPOSE: Asthma exacerbations contribute to corticosteroid insensitivity. LPS is ubiquitous in the environment. It causes bronchoconstriction and airway inflammation and may therefore exacerbate allergen responses. This study examined whether LPS and ovalbumin co-administration could exacerbate the airway inflammatory and functional responses to ovalbumin in conscious guinea pigs and whether these exacerbated responses were insensitive to inhaled corticosteroid treatment with fluticasone propionate (FP). EXPERIMENTAL APPROACH: Guinea pigs were sensitized and challenged with ovalbumin and airway function recorded as specific airway conductance by whole body plethysmography. Airway inflammation was measured from lung histology and bronchoalveolar lavage. Airway hyper-reactivity (AHR) to inhaled histamine was examined 24 h after ovalbumin. LPS was inhaled alone or 24 or 48 h before ovalbumin and combined with ovalbumin. FP (0.05-1 mg·mL(-1) ) or vehicle was nebulized for 15 min twice daily for 6 days before ovalbumin or LPS exposure. KEY RESULTS: Ovalbumin inhalation caused early (EAR) and late asthmatic response (LAR), airway hyper-reactivity to histamine and influx of inflammatory cells into the lungs. LPS 48 h before and co-administered with ovalbumin exacerbated the response with increased length of the EAR, prolonged response to histamine and elevated inflammatory cells. FP 0.5 and 1 mg·mL(-1) reduced the LAR, AHR and cell influx with ovalbumin alone, but was ineffective when guinea pigs were exposed to LPS before and with ovalbumin. CONCLUSIONS AND IMPLICATIONS: LPS exposure exacerbates airway inflammatory and functional responses to allergen inhalation and decreases corticosteroid sensitivity. Its widespread presence in the environment could contribute to asthma exacerbations and corticosteroid insensitivity in humans.

Bronchoprotection in conscious guinea pigs by budesonide and the NO-donating analogue, TPI 1020, alone and combined with tiotropium or formoterol.

BACKGROUND AND PURPOSE: Inhaled corticosteroids, anticholinergics and ß2-adrenoceptor agonists are frequently combined for treating chronic respiratory diseases. We examine the corticosteroid, budesonide, and novel NO-donating derivative, TPI 1020, against histamine- and methacholine-induced bronchoconstriction and whether they enhance the ß2-adrenoceptor agonist formoterol or muscarinic antagonist tiotropium in conscious guinea pigs. EXPERIMENTAL APPROACH: Dunkin-Hartley guinea pigs received inhaled histamine (3 mM) or methacholine (1.5 mM) and specific airway conductance (sG(aw)) was measured before and 15 or 75 min after treatment with budesonide, TPI 1020, tiotropium or formoterol alone or in combinations. KEY RESULTS: Formoterol (0.7-10 µM) and budesonide (0.11-0.7 mM) inhibited histamine-induced bronchoconstriction and tiotropium (2-20 µM) inhibited methacholine-induced bronchoconstriction by up to 70.8 ± 16.6%, 34.9 ± 4.4% and 85.1 ± 14.3%, respectively. Formoterol (2.5 µM) or tiotropium (2 µM) alone exerted small non-significant bronchoprotection. However, when co-administered with TPI 1020 0.11 mM, which alone had no significant effect, there was significant inhibition of the bronchoconstriction (45.7 ± 12.2% and 79.7 ± 21.4%, respectively). Co-administering budesonide (0.11 mM) with tiotropium (2 µM), which alone had no effect, also significantly inhibited the methacholine bronchoconstriction (36.5 ± 13.0%), but there was no potentiation of formoterol against histamine. The NO scavenger, CPTIO, prevented the bronchoprotection by SNAPand TPI 1020. CONCLUSIONS AND IMPLICATIONS: TPI 1020 potentiated the bronchoprotection by formoterol and tiotropium. Budesonide also enhanced the effects of tiotropium but not formoterol. Combination of TPI 1020 with a long-acting ß2-adrenoceptor agonist or muscarinic receptor antagonist may therefore be a more potent therapeutic approach for treatment of chronic respiratory diseases.

Vasoconstrictor and vasodilator responses to tryptamine of rat-isolated perfused mesentery: comparison with tyramine and ß-phenylethylamine.

BACKGROUND AND PURPOSE: Tryptamine increases blood pressure by vasoconstriction, but little is known about its actions on the mesentery, in particular the resistance arteries. Tryptamine interacts with trace amine-associated receptors (TAARs) and because of its structural similarity to 5-HT, it may also interact with 5-HT receptors. Our hypothesis is therefore that the rat mesenteric arterial bed will exhibit vasopressor and vasodepressor responses to tryptamine via both 5-HT and TAARs. EXPERIMENTAL APPROACH: Tryptamine-evoked responses were assayed from pressure changes of the rat-isolated mesenteric vasculature perfused at constant flow rate in the absence and presence of adrenoceptor and 5-HT receptor antagonists. KEY RESULTS: Tryptamine caused dose-dependent vasoconstriction of the mesenteric arterial bed as increases in perfusion pressure. These were unaffected by the α(1) -adrenoceptor antagonist, prazosin, but were attenuated by the non-selective α-adrenoceptor antagonist, phentolamine. The 5-HT(2A) receptor antagonists, ketanserin and ritanserin, abolished the tryptamine-induced pressure increases to reveal vasodilator responses in mesenteric beds preconstricted with phenylephrine. These tryptamine-induced vasodilator responses were unaffected by the 5-HT(7) receptor antagonist, SB269970, but were eliminated by the NOS inhibitor, N(ω) -nitro-L-arginine methyl ester (L-NAME). Tyramine and ß-phenylethylamine also caused vasodilatation in pre-constricted vasculature, which was also abolished by L-NAME. CONCLUSIONS AND IMPLICATIONS: Tryptamine causes vasoconstriction of the mesenteric vasculature via 5-HT(2A) receptors, which when inhibited exposed vasorelaxant effects in pre-constricted tissues. The vasodilatation was independent of 5-HT(2A) and 5-HT(7) receptors but like that for tyramine and ß-phenylethylamine was due to NO release. Potency orders suggest TAAR involvement in the vasodilatation by these trace amines.

Khat chewing, cardiovascular diseases and other internal medical problems: the current situation and directions for future research.

The leaves of khat (Catha edulis Forsk.) are chewed as a social habit for the central stimulant action of their cathinone content. This review summarizes the prevalence of the habit worldwide, the actions, uses, constituents and adverse health effects of khat chewing. There is growing concern about the health hazards of chronic khat chewing and this review concentrates on the adverse effects on health in the peripheral systems of the body, including the cardiovascular system and gastrointestinal tract. Comparisons are made with amphetamine and ecstasy in particular on the detrimental effects on the cardiovascular system. The underlying mechanisms of action of khat and its main constituent, cathinone, on the cardiovascular system are discussed. Links have been proposed between khat chewing and the incidence of myocardial infarction, dilated cardiomyopathy, vascular disease such as hypertension, cerebrovascular ischaemia and thromboembolism, diabetes, sexual dysfunction, duodenal ulcer and hepatitis. The evidence, however, is often based on limited numbers of case reports and only few prospective controlled studies have been undertaken. There is therefore an urgent need for more thorough case-control studies to be performed. This review outlines the current knowledge on the adverse health effects of khat chewing on the cardiovascular system and other internal medical problems, it assesses the evidence and the limitations of the studies and identifies the questions that future studies should address.

Adenosine receptor subtypes in the airways responses to 5'-adenosine monophosphate inhalation of sensitized guinea-pigs.

BACKGROUND: Endogenous adenosine levels are raised in the lungs during asthma attacks. 5'-adenosine monophosphate (5'-AMP) inhalation in asthmatics causes bronchoconstriction and in sensitized guinea-pigs induces early (EAR) and late asthmatic responses (LAR), airway hyper-reactivity (AHR) and inflammatory cell recruitment to the lungs. OBJECTIVE: The aim of this study was to investigate the roles of A(1), A(2A), A(2B) and A(3) adenosine receptors in these responses to inhaled 5'-AMP in sensitized guinea-pigs. Comparisons were made with the effect of dexamethasone treatment on 5'-AMP-induced responses. METHODS: Functional airways responses to inhaled 5'-AMP (3 and 300 mM) of actively sensitized, conscious guinea-pigs were determined by whole-body plethysmography following administration of selective adenosine receptor antagonists or their vehicles. AHR to inhaled histamine (1 mM) and inflammatory cell influx in bronchoalveolar lavage fluid were determined. RESULTS: 5'-AMP at 3 mM caused an immediate bronchoconstriction (EAR), whereas 300 mM caused bronchodilatation. Both responses were followed at 6 h by a LAR, together with inflammatory cell influx and AHR to histamine. The A(2A) receptor antagonist, ZM241385, further enhanced cell influx after 5'-AMP inhalation (3 and 300 mM), and blocked the immediate bronchodilator response to 300 mM 5'-AMP, exposing an EAR. The A(2B) receptor antagonist, MRS1706 (in the presence of ZM241385), inhibited the LAR, AHR and cell influx, following inhalation of 5'-AMP (300 mM). The A(3) receptor antagonist, MRS1220, inhibited 5'-AMP-induced inflammatory cell influx. The A(1) receptor antagonist, DPCPX (in the presence of ZM241385), inhibited the EAR following 5'-AMP inhalation (300 mM). Dexamethasone inhibited the LAR, AHR and cell influx following inhalation of 5'-AMP (300 mM). CONCLUSION: All four adenosine receptor subtypes play various roles in the airways responses to inhaled 5'-AMP in sensitized guinea-pigs.

Dietary trace amine-dependent vasoconstriction in porcine coronary artery.

BACKGROUND AND PURPOSE: The dietary trace amines tyramine and beta-phenylethylamine (beta-PEA) can increase blood pressure. However, the mechanisms involved in the vascular effect of trace amines have not been fully established. The purpose of this study was to evaluate whether trace amine-dependent vasoconstriction was brought about by tyramine and beta-PEA acting as indirect sympathomimetic agents, as previously assumed, or whether trace amine-dependent vasoconstriction could be mediated by recently discovered trace amine-associated (TAA) receptors. EXPERIMENTAL APPROACH: The responses to p-tyramine and beta-PEA were investigated in vitro in rings of the left anterior descending coronary arteries of pigs. KEY RESULTS: p-Tyramine induced a concentration-dependent (0.1-3 mM) vasoconstriction. The maximum response and pD(2) value for p-tyramine was unaffected by endothelium removal or pre-treatment with antagonists for adrenoceptors, histamine, dopamine or 5-HT receptors. beta-PEA also produced a concentration-dependent (0.3-10 mM) vasoconstriction which was unaffected by endothelium removal, beta-adrenoceptor or 5-HT receptor antagonists. A substantial, but reduced, response to beta-PEA was obtained in the presence of prazosin (alpha(1)-adrenoceptor antagonist), haloperidol (D(2)/D(3) dopamine receptor antagonist) or mepyramine (H(1) histamine receptor antagonist). The pD(2) value for beta-PEA was unaffected by any of the antagonists tested. CONCLUSIONS AND IMPLICATIONS: Vasoconstriction induced by p-tyramine does not involve an indirect sympathomimetic effect, although vasoconstriction caused by beta-PEA may occur, in part, by this mechanism. We therefore propose that trace amine-dependent vasoconstriction is mediated by phenylethylamine-specific receptors, which are closely related to or identical to TAA receptors. These receptors could provide a target for new antihypertensive therapies.

Investigation of the mechanism for the relaxation of rat duodenum mediated via M1 muscarinic receptors.

1 Relaxation responses of the rat isolated duodenum to the putative M1 muscarinic receptor agonist, McN-A-343, were examined to determine whether the response was due to the release of known non-adrenergic, non-cholinergic relaxant neurotransmitters and to establish the involvement of M1 muscarinic receptors. 2 The role of ATP was examined with the P2 receptor antagonist, suramin, which at 30 mum antagonized the relaxant responses to alpha,beta-methylene ATP. The same dose, however, failed to inhibit the relaxation by McN-A-343. 3 The role of nitric oxide (NO) was examined with the NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME; 100 microm), which failed to inhibit the responses to McN-A-343. As NO mediates relaxation of the duodenum via cGMP generation through guanylyl cyclase, whether the relaxation by McN-A-343 was also via cGMP was examined with the guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). The relaxation responses to the NO donor, S-nitroso-N-acetyl penicillamine, were inhibited in the presence of ODQ (3 microm), but not those by McN-A-343. 4 Release of gamma-aminobutyric acid (GABA) was examined with the GABAA receptor antagonist, bicuculline (10 microm), which shifted the concentration-response curves for the relaxation of the duodenum by GABA to the right. There was a similar degree of shift in the concentration-response curve for McN-A-343 by bicuculline indicating that release of GABA from enteric neurones of the duodenum could explain the relaxation response to McN-A-343. 5 To test whether the muscarinic receptors mediating the relaxation of the duodenum were of the M1 subtype, the susceptibility to the selective competitive antagonist, pirenzepine and the selective muscarinic toxin from green mamba, MT7, was examined. Pirenzepine (1 microm) shifted the concentration-response for McN-A-343 to the right in a parallel fashion with a dose ratio of 33.3 +/- 20.2. This yielded a pA2 value of 7.5, which concords with those for other responses reputed to be mediated via M1 muscarinic receptors. The toxin MT7 was used as an irreversible antagonist and following incubation with the duodenum was washed from the bath. An incubation time of 30 min with 100 nm of MT7 caused a significant parallel shift in the concentration-response to McN-A-343 confirming the involvement of M1 muscarinic receptors. 6 This study has confirmed that McN-A-343 relaxes the rat duodenum via muscarinic receptors of the M1 subtype and that these receptors are probably located on enteric neurones from which their stimulation releases GABA.

Radioligand binding and functional responses of ligands for human recombinant adenosine A(3) receptors.

The binding and functional properties of adenosine receptor ligands were compared in Chinese hamster ovary cells transfected with human adenosine A(3) receptors. Inhibition of [(125)I]-aminobenzyl-5'-N-methylcarboamidoadenosine ([(125)I]-AB-MECA) binding by adenosine receptor ligands was examined in membrane preparations. Inhibition of forskolin-induced cAMP accumulation by agonists was measured using a cAMP enzyme immunoassay. The rank order of agonist potency for both assays was N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) > 5'-N-ethylcarboxamidoadenosine (NECA) > (-)-N(6)-[(R)-phenylisopropyl] adenosine (R-PIA) > 4-aminobenzyl-5'-N-methylcarboxamidoadenosine (AB-MECA) > N(6)-cyclopentyl adenosine (CPA) > adenosine. The radioligand binding rank order of antagonist potency was N-[9-chloro-2-(2-furanyl)[1,2,4]-triazolo[1,5-c]quinazolin-5-benzeneacetamide (MRS1220) > 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) > 8-phenyltheophylline (8-PT) > 8-(p-sulfophenyl)-theophylline (8-SPT). MRS1220 competitively inhibited the effect of IB-MECA on cAMP production, with a K(B) value of 0.35 nm. These data are characteristic of adenosine A(3) receptors. The absence of Mg(2+) and presence of guanosine 5'-(gamma-thio)triphosphate (GTPgammaS) significantly reduced agonist binding inhibition potency, indicating binding to high- and low-affinity states. The IB-MECA, NECA and R-PIA IC(50) values were greater for the cAMP assay than for radioligand binding, suggesting an efficient stimulus-response transduction pathway.

Khat chewing is a risk factor for acute myocardial infarction: a case-control study.

AIM: Khat chewing is a common habit in Yemen and east African countries. Millions of people chew khat leaves daily for its euphoric and energetic effects and to increase alertness. Cathinone, the main active substance in fresh khat leaves, has sympathomimetic effects which increase heart rate and blood pressure. The aim was to examine the hypothesis that khat chewing is a risk factor for acute myocardial infarction (AMI) using a hospital-based matched case-control study. METHOD: Between 1997 and 1999, we selected 100 patients admitted to the Al-Thawra teaching hospital Sana'a ICU, Yemen with acute myocardial infarction. 100 control subjects, matched to cases for sex and age, were recruited from the outpatients clinics of the same hospital. A questionnaire was completed for case and control groups covering personal history of khat chewing, smoking, hypertension, diabetes and any family history of myocardial infarction. A blood sample was collected for performing lipid profiles. Cases and controls were compared by analysis conducted using conditional logistic regression which corrected for baseline imbalances leading to less biased estimations of odds ratio (OR). The risk associated with each classical factor and khat chewing habits was then investigated. OR values greater than 2.5 indicated a significant risk factor. RESULTS: Khat chewing was significantly higher among the AMI case group than control group (OR = 5.0, 95% CI 1.9-13.1). A dose-response relationship was observed, the heavy khat chewers having a 39-fold increased risk of AMI. CONCLUSION: This study indicates that khat chewing is associated with AMI and is an independent dose-related risk factor for the development of myocardial infarction.

The roles of alpha- and beta-adrenoceptor stimulation in myocardial ischaemia.

beta-Adrenoceptor (AR) ligands have been the mainstay of cardiovascular therapy for decades, with beta-AR antagonist being used for hypertension, angina and myocardial infarction and adrenaline in use for cardiopulmonary resuscitation for nearly 100 years. Ischaemia of the heart through coronary artery occlusion causes cell injury and death through necrosis and apoptosis. Reperfusion of the ischaemic myocardium results in cardiac dysfunction and infarction. Stimulation of alpha- and beta-ARs in the ischaemic heart have variable and inconsistent effects depending on when the agonist is applied. This review describes the different effects of stimulation of the three established beta-AR subtypes (beta(1)-, beta(2)- and beta(3)-ARs) either before ischaemia (preconditioning) or during ischaemia and reperfusion of the heart (postconditioning). Brief periods of ischaemia preceding a major ischaemic episode can have a protective effect against post-ischaemia-reperfusion damage, known as ischaemic preconditioning. This review considers the role of endogenous catecholamines released during preconditioning and the nature of the adrenoceptor subtypes that mediate these effects. The clinical significance of this to the use of beta-AR antagonists is considered. The transduction pathways and effects on apoptosis of the cardioprotective and deleterious effects of AR activation are considered. This commentary reviews the literature and attempts to bring together a unified synopsis of the effects of adrenoceptor stimulation in myocardial ischaemia and the potential clinical relevance.

Early and late bronchoconstrictions, airway hyper-reactivity, leucocyte influx and lung histamine and nitric oxide after inhaled antigen: effects of dexamethasone and rolipram.

BACKGROUND: Guinea-pig models can provide the essential features of asthma, including early- (EAR) and late- (LAR) phase asthmatic responses, airway hyper-reactivity (AHR) and inflammatory cell influx; however, these components are rarely demonstrated all in the same model. OBJECTIVES: The aim of this study was to establish a conscious guinea-pig model with these essential features of asthma and to correlate these with bronchoalveolar lavage fluid (BALF) histamine and nitric oxide (NO) levels. The model would be validated from the susceptibility of these parameters to standard anti-asthmatic agents, the steroid, dexamethasone, and a phosphodiesterase-4 (PDE4) inhibitor, rolipram. METHODS: Guinea-pigs were sensitized with ovalbumen (OA) (10 microg plus Al2(OH)3 100 mg, intraperitoneal (i.p.)) and 14 days later received inhaled OA (100 microg/mL) or vehicle for 1 h. Airway function was measured by whole-body plethysmography as specific airway conductance (sGaw). Reactivity to inhaled histamine (nose-only, 1 mm, 20 s) was recorded 24 h before and at 6, 12 or 24 h after OA challenge. BALF was obtained to determine the total and differential cell counts, NO and histamine. RESULTS: Guinea-pigs challenged with OA showed an EAR as a fall in (sGaw) (-54.9+/-10.8%), which resolved by 6 h and was followed by an LAR between 7 and 11 h (-30.2+/-8.8%). No bronchoconstriction to inhaled histamine occurred before OA challenge but at 6, 12 or 24 h afterwards, sGaw fell significantly, indicating AHR. At 1 h after OA, macrophages, eosinophils and neutrophils significantly increased in BALF. Macrophages and eosinophils increased further up to 24 h (3- and 44-fold), but neutrophils declined to control levels. BALF histamine levels increased at 0.25 h after OA challenge and peaked at 6 h. BALF NO levels initially fell (44%) 1 h after OA exposure and then progressively rose above control levels. Dexamethasone (20 mg/kg, i.p.) and rolipram (1 mg/kg, i.p.) administered 24 and 0.5 h before and 6 h after OA challenge inhibited leucocyte influx, AHR and the early deficiency and later excess of NO. Dexamethasone but not rolipram attenuated the LAR. CONCLUSIONS: This model displays many of the features of human asthma with predictable responses to dexamethasone and evidence of anti-asthmatic activity by the PDE4 inhibitor, rolipram.

Coronary and aortic vasoconstriction by cathinone, the active constituent of khat.

1. The psychostimulant constituent of khat leaves, S-(-)-cathinone, was examined for vascular activity on the coronary vasculature of guinea-pig-isolated perfused hearts and aortic ring preparations. 2. Cathinone caused coronary vasoconstriction, negative inotropy and negative chronotropy in isolated hearts. The major metabolite of cathinone after its ingestion, 1R.2S-(-)-norephedrine (norephedrine), also caused coronary vasoconstriction comparable with that by cathinone. Norephedrine, however, had no effect on force or rate of cardiac contractions. 3. Cocaine (10 microm) potentiated the coronary vasoconstriction and positive inotropy by noradrenaline indicating inhibition of neuronal uptake. The vasoconstriction and negative inotropy by cathinone, however, were not affected, indicating that its action was not via release of noradrenaline from sympathetic neurones. 4. The alpha(1)-adrenoceptor antagonist, prazosin, blocked the vasoconstriction by noradrenaline, but not that produced by cathinone in the presence of cocaine. This indicates that the coronary vasoconstriction by cathinone was not due to an action on alpha(1)-adrenoceptors either directly or indirectly through noradrenaline release. 5. Three repeated doses of cathinone displayed the same coronary vasoconstrictor responses, indicating a lack of tachyphylaxis and therefore confirming that the response was unlikely to be due to indirect sympathomimetic activity through release of noradrenaline. 6. In guinea-pig aortic rings, the order of vasoconstrictor activity was: noradrenaline > norephedrine > cathinone, with each causing approximately equivalent maximum responses. The time to reach plateau contractions was shortest for noradrenaline (5.1 +/- 0.5 min), then norephedrine (9.3 +/- 1.5 min) and cathinone the longest (25.4 +/- 3.2 min, 335 microm dose). 7 These results indicate that cathinone has vasoconstrictor activity which is not due to indirect or direct sympathomimetic activity. The precise mechanism for this vasoconstriction remains to be determined. The coronary vasoconstriction may explain the increased incidence of myocardial infarction in khat chewers, which may arise from coronary vasospasm.

Nitric oxide in respiratory diseases.

The formation and modulation of nitric oxide (NO) in the lungs is reviewed. Its beneficial and deleterious roles in airways diseases, including asthma, chronic obstructive pulmonary disease, and cystic fibrosis, and in animal models is discussed. The pharmacological effects of agents that modulate NO production or act as NO donors are described. The clinical pharmacology of these agents is described and the therapeutic potential for their use in airways disease is considered.

Responses to adenosine of isolated transverse or spiral strips of sensitized guinea pig trachea: role of epithelium.

AIM: To determine the role of the epithelium in the responses to adenosine of isolated trachea from ovalbumin-sensitized guinea pigs. METHODS: Spirally cut tracheal preparations were superfused or immersed in organ baths and transversely cut strips were immersed. Epithelium was removed mechanically from some strips and confirmed by histological examination of a random sample. Tissues were from unsensitized or ovalbumin-sensitized guinea pigs. Isometric tension was measured and responses to adenosine recorded. RESULTS: In sensitized tissues, contractile responses to adenosine were evident as contractions of superfused spirals or as rightwards shift of the concentration-response curve compared with non-sensitized immersed spirals. Epithelium removal potentiated relaxation responses in both non-sensitized and sensitized strips indicating release of contractile mediators in both tissues. Dipyridamole potentiated relaxation responses in sensitized tissues with and without epithelium. CONCLUSION: Sensitization reveals a contractile response to adenosine. The epithelium is not involved in this contractile response nor is it the major site of uptake of adenosine in both sensitized and non-sensitized tissues.

A3 receptors mediate rapid inflammatory cell influx into the lungs of sensitized guinea-pigs.

BACKGROUND: Inhaled adenosine causes bronchoconstriction in asthmatics and may modulate inflammatory cell activity. Elevated adenosine levels occur in the lungs after antigen challenge of asthmatics. OBJECTIVES: The aim of this study was to investigate whether the bronchoconstrictor effects of the adenosine derivative, 5'-AMP, were associated with altered migration of inflammatory cells into the airways using a sensitized atopic guinea-pig model previously shown to display a bronchoconstrictor response. Comparisons were made with the effects of inhaled antigen. METHODS: Airway responses of conscious sensitized guinea-pigs to inhalation exposures of 5'-AMP were determined by whole body plethysmography as the change in specific airway conductance (sGaw). Influx of leucocytes into the airways was determined by bronchoalveolar lavage (BAL). RESULTS: 5'-AMP caused bronchoconstrictor airway responses in sensitized animals. Dose-dependent infiltration of inflammatory cells into the lungs occurred 1 h after 5'-AMP exposure. No bronchoconstriction or cell influx was seen in unsensitized guinea-pigs. Exposure to ovalbumin (OA) also caused influx of inflammatory cells. Twenty-four hours after an OA exposure, 5'-AMP produced no bronchoconstriction. The P1-receptor antagonists, 8-PT and 8-SPT, inhibited the 5'-AMP-induced bronchoconstriction, indicating that the bronchoconstriction seen in sensitized animals is mediated by A1 or A2 receptors. They had no effect on the cell influx, whereas the A3 antagonist, MRS-1220, significantly inhibited cellular infiltration, suggesting mediation through A3 receptors. At 24 h after an OA challenge and accompanying the cellular influx, there was airway hyper-responsiveness to the bronchoconstriction by histamine. In contrast, no hyper-responsiveness to histamine was seen 1 h after 3 mM or 24 h after 300 mM 5'-AMP. CONCLUSIONS: 5'-AMP caused a rapid migration of eosinophils and macrophages into the airways only in sensitized guinea-pigs, and this was blocked by the A3 antagonist MRS-1220. This was not associated with bronchial hyper-reactivity to histamine.

Chronic lipopolysaccharide exposure on airway function, cell infiltration, and nitric oxide generation in conscious guinea pigs: effect of rolipram and dexamethasone.

This study investigated whether a correlation between airway hyperreactivity (AHR), leukocyte influx, and nitric oxide (NO) existed in guinea pigs chronically exposed to lipopolysaccharide (LPS). The effect of the corticosteroid, dexamethasone, or phosphodiesterase-4 (PDE4) inhibitor, rolipram, on these features was studied. Airway function was measured in conscious guinea pigs (specific airways conductance) before and after single, double, or chronic (nine) LPS (30 microg x ml(-1), 1 h) exposures. Airway reactivity to inhaled histamine (1 mM, 20 s) was assessed before and at various times after LPS challenges. Leukocytes and NO metabolites were measured in bronchoalveolar lavage fluid (BALF). AHR occurred at 1 h after a single LPS challenge and was resolved by 4 h. This coincided with reduction and recovery, respectively, of BALF NO levels. The AHR and NO deficiency were extended to 4 h, after a double LPS exposure. Chronic LPS exposures, 48 h apart, initially caused persistent bronchodilations, whereas later exposures produced progressively persistent bronchoconstrictions. There was AHR 24 h after the eighth challenge. Twenty-four hours after the ninth LPS exposure, macrophages, neutrophils, eosinophils, and NO metabolites were elevated in BALF. Dexamethasone (20 mg x kg(-1) i.p.) or rolipram (1 mg x kg(-1) i.p.) prevented single and chronic LPS-induced AHR, the respective deficiency and elevation in NO metabolites, and the chronic LPS-induced leukocyte influx. Dexamethasone exacerbated, whereas rolipram reversed, the chronic LPS-induced bronchoconstrictions. This study demonstrates for the first time that chronic LPS causes persistent bronchoconstriction, neutrophilic inflammation, AHR, and NO overproduction in guinea pig airways. These rolipram-sensitive features suggest the potential of PDE4 inhibitors in airway disease.

Inhibition of field stimulation-induced contractions of rabbit vas deferens by muscarinic receptor agonists: selectivity of McN-A-343 for M1 receptors.

Inhibition of the field stimulation-induced twitch responses of the rabbit vas deferens by the muscarinic receptor agonist, McN-A-343, has been attributed to presynaptic muscarinic receptors of the M1 subtype located on noradrenergic nerve terminals. Stimulation of these receptors causes inhibition of transmitter release and inhibition of the contractile response. However, the selectivity of McN-A-343 for M1 receptors has been questioned and this throws doubt on whether the prejunctional receptors of the rabbit vas deferens are of the M1 subtype. In this study we have undertaken a comprehensive re-evaluation of the inhibition of prostatic and epididymal portions of the rabbit isolated field-stimulated vas deferens by several agonists, including McN-A-343, and quantified the antagonism by M1-selective antagonists, pirenzepine and telenzepine. Prostatic and epididymal portions of vasa deferentia from New Zealand White rabbits were immersed in a low Ca2+ Krebs solution at 32+/-0.5 degrees C gassed with 5% CO2 in oxygen. Yohimbine (1.0mM) was present throughout to block prejunctional alpha2-adrenoceptors. Field stimulation was applied by repeated application of single pulses (30 V, 0.05 Hz, 0.5 ms) and isometric contractions recorded. Carbachol and oxotremorine initially potentiated the epididymal contractions but at higher concentrations there was inhibition. In the prostatic portion, oxotremorine only inhibited. McN-A-343 produced inhibitory responses only in both epididymal and prostatic portions. Pirenzepine shifted the concentration-response curves forthe inhibitory responses to oxotremorine to the right. However, the potentiation of the twitches also became more apparent with the lower concentrations of oxotremorine. Schild plots for the antagonism by pirenzepine yielded pA2 values of 7.96+/-0.004 and 7.7+/-0.02 for the epididymal and prostatic portions, respectively. The concentration-response curves for the inhibition of twitches by McN-A-343 were displaced to the right in a parallel manner by pirenzepine in both prostatic and epididymal portions with no potentiation of the twitches. The Schild plot for this antagonism generated pA2 values of 7.68+/-0.01 and 8.07+/-0.01, respectively. Telenzepine caused parallel shifts of the McN-A-343 concentration-response curves to the right in prostatic portions, the pA2 value being 8.70+/-0.13. Telenzepine (10(-7) M) abolished the inhibitory effect of carbachol to reveal only concentration-dependent potentiation of the contractions. The Schild plot for antagonism of this contractile effect yielded a pA2 value (7.07+/-0.09) that was significantly less by almost two orders of magnitude (1.70) than the value for the antagonism by telenzepine of the McN-A-343-induced inhibitory response. The pA2 values of pirenzepine and telenzepine against the inhibitory responses of the rabbit vas deferens are consistent with the involvement of M1 receptors. This leads to the conclusion that McN-A-343 causes inhibition through this receptor type. The doubts concerning the selectivity of McN-A-343 for M1 receptors are therefore unfounded. The fact that McN-A-343 does not display a selective binding profile suggests that its selectivity does not arise from affinity differences but probably resides in its intrinsic efficacy.