Smoking enhances the proinflammatory effects of nucleotides on cytokine release from human lung

Nucleotides have effects on immune cells which are complex but generally proinflammatory, and have been suggested to play a role in smoking-related lung diseases. However, there have been no studies directly measuring functional responses to nucleotides in human lungs taken from smokers. We used fragments of post mortem human lung from smokers and non-smokers, incubated them with a range of nucleotides (4-1000 µM) in the presence of lipopolysaccharide (LPS; 10 µg/ml) for 24 hours and measured cytokines (IL-1β, IFNγ, IL-17, TNFα, IL-6, IL-8, IL-2 and IL-10) in the supernatants using multiplex immunoassays. Although the basal cytokine levels in the smokers were generally higher in the smokers than the non-smokers, there were no significant differences in either the basal release or the LPS-stimulated release of any of the cytokines when lungs from smokers and non-smokers were compared. There were no significant effects of ATP, ADP, AMP, UTP, α,β-methylene-ATP, P1, P4-diATP, 2-methylthio-ATP or Bz-ATP on the release of cytokines from the lungs. However, the stable ATP analogue ATPγS increased the release of IL-1β and IFNγ, and the effect was greatly increased in lungs from smokers. In non-smokers but not in smokers ATPγS increased the release of IL-17. Overall these results clearly demonstrate for the first time that in normal human lung a stable ATP analogue can enhance LPS-induced pro-inflammatory cytokine release, and that these effects are greatly altered by a prior history of smoking. This provides strong support for the suggestion that nucleotides are involved in the pathogenesis of smoking-related diseases

The oxytocin analogue carbetocin prevents emotional impairment and stress-induced reinstatement of opioid-seeking in morphine-abstinent mice.

The main challenge in treating opioid addicts is to maintain abstinence due to the affective consequences associated with withdrawal which may trigger relapse. Emerging evidence suggests a role of the neurohypophysial peptide oxytocin (OT) in the modulation of mood disorders as well as drug addiction. However, its involvement in the emotional consequences of drug abstinence remains unclear. We investigated the effect of 7-day opioid abstinence on the oxytocinergic system and assessed the effect of the OT analogue carbetocin (CBT) on the emotional consequences of opioid abstinence, as well as relapse. Male C57BL/6J mice were treated with a chronic escalating-dose morphine regimen (20-100 mg/kg/day, i.p.). Seven days withdrawal from this administration paradigm induced a decrease of hypothalamic OT levels and a concomitant increase of oxytocin receptor (OTR) binding in the lateral septum and amygdala. Although no physical withdrawal symptoms or alterations in the plasma corticosterone levels were observed after 7 days of abstinence, mice exhibited increased anxiety-like and depressive-like behaviors and impaired sociability. CBT (6.4 mg/kg, i.p.) attenuated the observed negative emotional consequences of opioid withdrawal. Furthermore, in the conditioned place preference paradigm with 10 mg/kg morphine conditioning, CBT (6.4 mg/kg, i.p.) was able to prevent the stress-induced reinstatement to morphine-seeking following extinction. Overall, our results suggest that alterations of the oxytocinergic system contribute to the mechanisms underlying anxiety, depression, and social deficits observed during opioid abstinence. This study also highlights the oxytocinergic system as a target for developing pharmacotherapy for the treatment of emotional impairment associated with abstinence and thereby prevention of relapse

Differential effects of adenine nucleotide analogues on shape change and aggregation induced by adenosine 5′-diphosphate (ADP) in human platelets

1. Adenosine 5′-diphosphate (ADP) induces human blood platelets to aggregate and change shape, and it has been suggested that these two responses are mediated by more than one subtype of ADP receptor. 2. The structure-activity relationships for several analogues of adenine nucleotides in causing aggregation and shape change were measured and compared in washed platelets using an aggregometer. ADP and its analogues 2-methylthioadenosine 5′-diphosphate (2-methylthio-ADP), adenosine 5′(α,β-methylene)diphosphonate (AMPCP), S(P)-adenosine 5′-O-(1-thiodiphosphate) (AD-PαS) and adenosine 5′-O-(2-thiodiphosphate) (ADPβS) were used as agonists. Adenosine 5′-triphosphate (ATP) and its analogues, P(1), P(5)-diadenosine pentaphosphate (Ap(5)A), adenosine (5′-(α,β-methylene)triphosphonate (AMPCPP), 2-methylthioadenosine 5′-triphosphate (2-methylthio-ATP) and uridine 5′-triphosphate (UTP), as well as the trypanocidal drug suramin, were used as antagonists. 3. In general, the structure-activity relationships for both responses were similar, but for some analogues differences were observed. ADPαS and ADPβS were much more potent agonists relative to ADP for shape change than for aggregation and indeed ADPαS antagonized ADP-induced aggregation with an apparent pK(B) value of 5.5±0.1. 2-Methylthio-ATP also had different effects in aggregation and shape change, being a much higher affinity antagonist of aggregation than of shape change with an apparent pK(B) value of 7.0±0.2 for aggregation and 5.2±0.2 for shape change. 4. These results support the suggestion that these two responses are mediated by multiple ADP receptors on human platelets, and are consistent with shape change being mediated via one receptor (the P2Y(1) receptor) with aggregation requiring the activation of two receptors (the P2Y(1) and another P2Y receptor)

Effect of LPS on release of cytokines from human lung fragments.

<p>Post-mortem human lung parenchyma was chopped and sieved to obtain pieces smaller than 1 mm and incubated for 24 h at 37°C in the presence (black bars) or absence (white bars) of LPS (10 µg/ml). The supernatants were then analysed for the cytokines shown by MSD multiplex immunoassays. A shows the results from all the lungs (n = 7–12), while B and C show results from smokers (n = 4–7) and non-smokers (n-3–5) respectively. Values are mean ± s.e.mean, * indicates a significant difference between control and LPS-stimulated values, p<0.05, paired t test.</p

Effect of nucleotides on release of cytokines from human lung fragments in the presence of LPS.

<p>Post-mortem human lung parenchyma was chopped and sieved to obtain pieces smaller than 1 mm and incubated for 24 h at 37°C in the presence of LPS (10 µg/ml) alone or in the presence of a range of nucleotides (ATPγS, ATP, ADP, AMP, UTP, α,β-methylene-ATP, P¹, P⁴-diadenosine tetraphosphate, 2-methylthio-ATP, Bz-ATP, 4–1000 µM). The supernatants were then analysed for cytokines by MSD multiplex immunoassays. A, B and C show the results for IL-1β, IFNγ and IL-17, n = 3–18, displayed as % of the release caused by LPS alone, mean ± s.e.mean. * indicates a significant difference between the release in the presence of the nucleotide and that to LPS alone (one-way repeated measures ANOVA with Dunnett’s post-hoc test) carried out on the data in pg/ml.</p

Effect of ATPγS on release of cytokines from human lung fragments from smokers and non-smokers in the presence of LPS.

<p>Post-mortem human lung parenchyma was chopped and sieved to obtain pieces smaller than 1 mm and incubated for 24 h at 37°C in the presence of LPS (10 µg/ml) alone or in the presence of ATPγS (4–1000 µM). The supernatants were then analysed for cytokines by MSD multiplex immunoassays. A, B and C show the results for IL-1β, IFNγ and IL-17, n = 3, displayed as % of the release caused by LPS alone, mean ± s.e.mean. * indicates a significant difference between the release in the presence of ATPγS and that to LPS alone (one-way repeated measures ANOVA with Dunnett’s post-hoc test), while # indicates a significance difference between the release in the smokers and the non-smokers (two-factor ANOVA with Bonferonni post-hoc test), carried out on the data in pg/ml.</p

Enhanced morphine withdrawal and micro -opioid receptor G-protein coupling in A2A adenosine receptor knockout mice

Much evidence supports the hypothesis that A2A adenosine receptors play an important role in the expression of morphine withdrawal and that the dopaminergic system might also be involved. We have evaluated morphine withdrawal signs in wild-type and A2A receptor knockout mice and shown a significant enhancement in some withdrawal signs in the knockout mice. In addition, micro -opioid and dopamine D2 receptor autoradiography, as well as micro -opioid receptor-stimulated guanylyl 5'-[gamma-[35S]thio]-triphosphate ([35S]GTPgammaS) autoradiography was carried out in brain sections of withdrawn wild-type and knockout mice. No significant changes in D2 and micro -opioid receptor binding were observed in any of the brain regions analysed. However, a significant increase in the level of micro receptor-stimulated [35S]GTPgammaS binding was observed in the nucleus accumbens of withdrawn knockout mice. These data indicate that the A2A receptor plays a role in opioid withdrawal related to functional receptor activation.Comparative StudyJournal ArticleResearch Support, Non-U.S. Gov'tFLWINinfo:eu-repo/semantics/publishe

Impairment of vascular function following BCG immunisation is associated with immune responses to HSP-60 in the cholesterol-fed rabbit

An immune response to heat shock protein (HSP)-60/65 has recently been implicated in atherogenesis. The aim of this study was to determine whether this effect may be mediated by impairment of endothelial function. Rabbits were injected with bacillus Calmette-Guerin (BCG) vaccine (n=12) or saline (n=12). A further injection of BCG or saline was administered after 2 weeks. After a further 2 weeks, animals were fed either a 0.25-1% cholesterol diet or a chow diet for 16 weeks. Blood cholesterol levels were maintained at 10-12mmol/l by altering the dietary cholesterol content. Plasma levels of anti-mycobacterial antibodies rose following BCG immunisation, but anti-HSP antibodies developed only in the BCG-immunised, cholesterol-fed rabbits. Aortic endothelium from cholesterol-fed, but not chow-fed, rabbits stained positively for HSP-60, independently of the immunisation protocol. Endothelial function was impaired in the BCG immunised, cholesterol-fed rabbits as measured by acetylcholine-mediated relaxation of isolated non-atherosclerotic carotid artery rings (P\textless0.05). This impairment was positively associated with the level of plasma anti-HSP-60 antibodies (P\textless0.01). These results suggest that BCG immunisation impairs endothelial responses, at least in part, by immune responses against mycobacterial and vascular HSP

Role of cyclic nucleotides in vasodilations of the rat thoracic aorta induced by adenosine analogues

1. Although adenosine analogues such as 5′-N-ethylcarboxamidoadenosine (NECA) relax the rat thoracic aorta in a partially endothelium-dependent manner via adenosine A(2A) receptors, others such as N(6)-R-phenylisopropyladenosine (R-PIA) act via an endothelium-independent, antagonist-insensitive mechanism. The role of cyclic nucleotides in these relaxations was investigated in isolated aortic rings using inhibitors of adenylate and guanylate cyclases as well as subtype-selective phosphodiesterase inhibitors. 2. The adenylate cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22536; 100 μM) significantly inhibited responses to NECA, but not responses to R-PIA. The type IV (cyclic AMP-selective) phosphodiesterase inhibitor 4-[(3-butoxy-4-methoxyphenyl)methyl]-2-imidazolidinone (RO 20-1724; 30 μM) significantly enhanced responses to NECA and to a lesser extent those to R-PIA. 3. The guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3a]quinoxalin-1-one (ODQ; 100 μM) significantly inhibited responses to NECA and acetylcholine but not responses to R-PIA. The selective phosphodiesterase V (cyclic GMP-selective) inhibitors, zaprinast (10 μM) and 4-{[3′,4′-(methylenedioxy)benzyl]amino}-6-methoxyquinazoline (MMQ; 1 μM), had no significant effect on responses to either NECA or R-PIA, but enhanced responses to acetylcholine. 4. These results are consistent with the effects of NECA being via activation of endothelial receptors to release NO which stimulates guanylate cyclase, as well as smooth muscle receptors coupled to stimulation of adenylate cyclase. The lack of effect of zaprinast and MMQ on responses to NECA are likely to be due to simultaneous activation of both adenylate and guanylate cyclases in the smooth muscle, as cyclic AMP reduces the sensitivity of phosphodiesterase V to inhibitors. These results also suggest that the effects of R-PIA are via neither of these mechanisms