A novel mechanism of vasoregulation: ADP-induced relaxation of the porcine isolated coronary artery is mediated via adenosine release.

In this study, we have investigated the mechanism of ADP-induced relaxation of porcine coronary artery (PCA) rings. The P2Y receptor agonists ADP and ADPbetaS produced concentration-dependent relaxation of endothelium-denuded PCA smooth muscle with pD2 values of 5.3 and 4.9, respectively. RT-polymerase chain reaction (RT-PCR) and immunoblotting demonstrated mRNA and protein expression of P2Y1 and A2A adenosine receptors in the PCA. The nonselective P2 antagonist PPADS or the P2Y1-selective antagonist MRS2179 failed to alter ADP- or ADPbetaS-induced relaxations. Relaxations to ADP were, however, blocked by the A2A adenosine receptor-selective antagonists ZM241385 and SCH58261 (apparent pK(B) values of 9.2 and 8.9, respectively). We excluded roles for direct occupancy of A2A adenosine receptors by ADP or ADPbetaS as well as metabolism to adenosine as mechanisms for ADP-evoked relaxations. However, ADP responses were significantly enhanced in the presence of the ENT1 nucleoside transporter inhibitors dipyridamole and NBTI and were significantly inhibited by adenosine deaminase, indicating a role for extracellular adenosine. Suprafusion of [3H]-adenine-labeled PCA segments showed that ADP induced the release of a number of purines, including adenosine. These data suggest that ADP mediates relaxation of the PCA via a novel mechanism that involves adenine nucleotide-evoked adenosine release and the subsequent activation of A2A receptors.

Alpha-tocopherol supplementation does not affect monocyte endothelial adhesion or C-reactive protein levels but reduces soluble vascular adhesion molecule-1 in the plasma of healthy subjects.

Vascular monocyte retention in the subintima is pivotal to the development of cardiovascular disease and is facilitated by up-regulation of adhesion molecules on monocytes/endothelial cells during oxidative stress. Epidemiological studies have shown that cardiovascular disease risk is inversely proportional to plasma levels of the dietary micronutrients, vitamin C and vitamin E (alpha-tocopherol). We have tested the hypothesis that alpha-tocopherol supplementation may alter endothelial/monocyte function and interaction in subjects with normal ascorbate levels (> 50 microM), as ascorbate has been shown to regenerate tocopherol from its oxidised tocopheroxyl radical form in vitro. Healthy male subjects received alpha-tocopherol supplements (400 IU RRR-alpha-tocopherol/day for 6 weeks) in a placebo-controlled, double-blind intervention study. There were no significant differences in monocyte CD11b expression, monocyte adhesion to endothelial cells, plasma C-reactive protein or sICAM-1 concentrations post-supplementation. There was no evidence for nuclear translocation of NF-kappaB in isolated resting monocytes, nor any effect of alpha-tocopherol supplementation. However, post-supplementation, sVCAM-1 levels were decreased in all subjects and sE-selectin levels were increased in the vitamin C-replete group only; a weak positive correlation was observed between sE-selectin and alpha-tocopherol concentration. In conclusion, alpha-tocopherol supplementation had little effect on cardiovascular disease risk factors in healthy subjects and the effects of tocopherol were not consistently affected by plasma vitamin C concentration.

Vitamin C supplementation in normal subjects reduces constitutive ICAM-1 expression.

Regulation of monocyte adhesion molecule gene expression is via redox sensitive transcription factors. We have investigated whether dietary antioxidant supplementation with vitamin C (250 mg/day) can modulate monocyte ICAM-1 expression in healthy male subjects with low plasma vitamin C at baseline. In a randomised, double-blind, crossover study, monocyte ICAM-1 mRNA was analysed using quantitative reverse transcriptase PCR. Protein was determined by flow cytometry (monocytes) and ELISA (plasma). Monocyte numbers were unaltered by supplementation. Subjects with low plasma vitamin C (<50 microM) prior to supplementation expressed higher levels of monocyte ICAM-1mRNA, and showed a significant (50%) reduction in ICAM-1mRNA expression after 6 weeks of 250 mg/day vitamin C supplementation (p<0.05). This was paralleled by a reduction in sICAM-1 (p<0.05). For the first time, these results show that dietary vitamin C can modulate monocyte ICAM-1 gene expression in vivo, where regulation of gene expression represents a novel mechanism for benefit from dietary antioxidants.

Sub-specific differentiation of intestinal spirochaete isolates by macrorestriction fragment profiling.

Macrorestriction fragment profile analysis by PFGE was used to distinguish intestinal spirochaetes, some of which were isolated from cases of swine dysentery and intestinal spirochaetosis in humans, pigs, mice, chickens and dogs. Macrorestriction fragment profiles using SmaI and SacII restriction enzymes were produced and used in statistical analysis. This permitted the division of the isolates into two major clusters. One cluster contained isolates which were identified as Serpulina pilosicoli and the second cluster contained isolates identified as Serpulina hyodysenteriae by immunoblotting with species-specific mAbs. Both species contained sub-specific groups, although these rarely correlated with the source of the isolates. We conclude that PFGE is capable of sub-specific differentiation of intestinal spirochaetes, but that the current species contain a large variety of genotypes among which cross-species transmission may be feasible.