When released into or formed in the extracellular space, adenosine acts as an autacoid via interaction with four types of G protein 1 -coupled receptors, termed A 1 -, A 2A -, A 2B -, and A 3 -adenosine receptor. These receptors are encoded by distinct genes and can be differentiated based on their affinities for adenosine analogues and methylxanthine antagonists (1, 2). In addition, the receptors can be classified based on their mechanism of signal transduction; A 1 -and A 3 -adenosine receptors interact with pertussis toxin-sensitive G proteins of the G i and G o family (3, 4, 5), whereas A 2A -and A 2B -adenosine receptors stimulate adenylyl cyclase via G s (6, 7).Adenosine is ubiquitously released by hypoxic tissues in large amounts; the nucleoside has therefore been proposed as one of the angiogenic factors that link the altered metabolism in oxygen-deprived cells to the formation of new capillaries (8).Earlier observations suggested that adenosine acts as an endothelial mitogen in vivo (9, 10). The mitogenic effect of adenosine has been verified in cultured endothelial cells derived from several vascular beds (11-13). In human endothelial cells, the proliferative response is mediated by the A 2A -adenosine receptor, an effect mimicked by stimulation of the endothelial ␤ 2 -adrenergic receptor (14). However, the mechanism by which adenosine analogues promote endothelial cell growth is not clear; there is, in particular, the apparent paradox that persistent stimulation of the signaling cascade composed of G s , adenylyl cyclase, and protein kinase A, which is downstream of A 2A -adenosine receptor, inhibits endothelial cell proliferation (14 -16). In the present work, we have therefore searched for additional effector mechanisms. We report that, in human endothelial cells, the A 2A -adenosine receptor stimulates the mitogen-activated protein kinase; this activation is independent of G s , G i , and typical protein kinase C isoforms but is associated with activation of p21 ras . EXPERIMENTAL PROCEDURESMaterials-Cell culture media were from Life Technologies, Inc., and cell culture dishes were from Greiner (Krems, Austria). [␥-32 P]ATP and [ 32 P]orthophosphate were from DuPont NEN. Cholera toxin, CPA, (Ϫ)isoproterenol, 8-Br-cAMP, collagenase (type IV), TPA, protein ASepharose, rabbit anti-rat IgG, forskolin, and epidermal growth factor (EGF) were obtained from Sigma, pertussis toxin was from Peninsula Laboratories (St. Helens, UK); bFGF, guanine nucleotides, and adenosine deaminase were from Boehringer Mannheim (FRG); XAC was from Research Biochemicals (Natick, MA), GF109203X was from Calbiochem, molecular weight standards (covering the range from 14 to 97 kDa) and reagents for SDS-polyacrylamide gel electrophoresis were from Bio-Rad. PD 098059, an inhibitor of MAP kinase kinase 1 (17), was from New England BioLabs (Beverly, MA). NECA and CGS 21680 were

1 Adenosine is known to stimulate capillary outgrowth and endothelial cell proliferation, but the underlying mechanism has not been identified. In order to identify the receptor subtype involved, the effects of adenosine receptor agonists and antagonists on human umbilical vein endothelial cell (HUVEC) proliferation were investigated. 2 Raising intracellular adenosine levels by use of the adenosine transport inhibitor, 4-nitrobenzylthioinosine (NBMPR) did not affect cell growth. This observation suggests that stimulation of an extracellular adenosine receptor generates the mitogenic signal. 3 In the presence of adenosine deaminase (ADA), which was used to remove adenosine present in the culture medium, the adenosine receptor agonists N-ethylcarboxamidoadenosine (NECA, non-selective) and CGS21680 (A2A-receptor-selective) stimulated [3H]-thymidine incorporation with a half-maximum effect at about 10 nM, while N6-cyclopentyladenosine (CPA, Al-selective) was about 100 fold less potent.The adenosine receptor antagonist, xanthine amine congener (XAC) produced a concentrationdependent decrease in endothelial cell proliferation with a half-maximum effect at about 10 nm. Hence, stimulation of an endothelial A2A-adenosine receptor seems responsible for the mitogenic signal. 4 In the presence of ADA, isoprenaline is also able to stimulate [3H]-thymidine incorporation with a half maximal effect of about 3 nM, an effect, which is reversed by the highly p2-selective antagonist, ICI 118,551. In the absence of ADA, isoprenaline exerts only a minor stimulatory effect. Combination of A2A adenosine and P2-adrenoceptor agonists did not further enhance [3H]-thymidine incorporation when compared to the sole addition of each agonist. We therefore conclude that both receptors stimulate endothelial cell proliferation via a common signal transduction pathway. 5 Both receptors are coupled to stimulation of adenylyl cyclase via the stimulatory G protein G,. However, direct activation of downstream effectors in the cyclic AMP-signalling cascade (G. with cholera toxin, adenylyl cyclase with forskolin, protein kinase A with 8Br-cyclic AMP) not only failed to mimic the action of receptor-activation, but even reduced cell proliferation. 6 Similarly, pertussis toxin-treatment which inactivated the Gj2 protein present in HUVEC and thus inhibited cell proliferation per se, did not impair the ability of A2A-receptor agonists to stimulate cell proliferation. This suggests that the A2A-adenosine and P2-adrenoceptor-mediated stimulation of endothelial cell proliferation occurs via a mechanism that is independent of G, and Gi.

The goal of this study was to estimate the incidence of temporary and permanent unilateral recurrent laryngeal nerve paralysis (URLNP) after esophagectomies with cervical anastomosis and to determine the impact of surgical technique, tumor type, tumor localization and age on the incidence of URLNP. From March 2002 to November 2009, 84 patients underwent a laryngoscopical evaluation before and after esophagectomy with cervical anastomosis prospectively. If the postoperative URLNP recovered within 6 months, the paresis was classified as transient; if not, it was defined as permanent. The results indicate that the overall incidence of postoperative URLNP was 50% (42/84). Twenty-four of the 84 patients (28.6%) showed a transient URLNP. A permanent URLNP was observed in 9 of the 84 patients (10.7%). The remaining 9 of the 84 patients (10.7%) were categorized as paresis with unknown clinical outcome due to missing follow-up. There were significantly more postoperative URLNPs in the group operated by transthoracic esophagectomy than by transhiatal esophagectomy (p < 0.001). Multifocal tumors and those localized suprabifurcational showed a higher incidence of postoperative URLNP than unifocal lesions with infrabifurcational localization (p = 0.046). Histological type of tumor and patients' age had no impact on URLNP. The high incidence of URLNP in our study underlines the high risk of URLNP after esophagectomy with cervical anastomosis, and consequently the importance of routine laryngoscopic pre- and postoperative evaluation of the vocal fold motility.

Polyurethane material for voice prostheses seems to reduce biofilm stability and infiltrative processes.

This study demonstrated that, in comparison with patients after standard laryngectomy, patients after jejunal autograft reconstruction have similar shunt-related and device-related complications and prosthesis indwelling times. Therefore, tracheoesophageal voice rehabilitation could be strongly recommended in these patients.

Alterations in G-protein-controlled signalling pathways (primarily pathways controlled by Gs and Gi) have been reported to occur in animal models of diabetes mellitus. We have therefore studied the effect of a long-term exposure of human umbilical vein endothelial cells to elevated concentrations of glucose on expression and function of G-protein subunits and endothelial NO synthase. Long-term incubation in high glucose (30 mM for 15 days) did not affect the levels of Gialpha-2, Gqalpha, the splice variants (long and short form) of Gsalpha, and the G-protein beta-subunits or adenylate cyclase activity; basal, as well as isoprenaline-, forskolin- and guanosine 5'-[gamma-thio]triphosphate-stimulated enzyme activities were comparable in high- and low-glucose-treated cells, thus ruling out any functional changes in the stimulatory pathway. Pretreatment of endothelial cells with pertussis toxin blocked a substantial fraction (50%) of the mitogenic response to serum factor(s) which depend(s) of functional Gi2. The sensitivity of cells cultured in high glucose was comparable with that of the paired controls maintained in normal glucose (EC50 = 3.1 +/- 0.5 and 3.3 +/- 0.4 ng/ml respectively). Similarly, we failed to detect any differences in endothelial NO synthase expression, or intracellular distribution and basal activity of the enzyme in endothelial cells cultured in high glucose. Stimulation of NO synthase in intact cells revealed a comparable response to the calcium ionophore (A23187). In contrast, stimulation with histamine (which acts via H1-receptors predominantly coupled to Gq) resulted in a significantly increased response in the cells maintained in high glucose. These data are suggestive of an altered H1-histamine receptor-Gq-phospholipase C pathway in endothelial cells cultured in high glucose concentrations, but rule out any glucose-induced functional changes in Gs- and Gi-controlled signalling pathways.

No abstract

A long-term study to identify age-dependent alterations in vascular reactivity in obese Zucker rats, a model for non-insulin-dependent diabetes mellitus, was carried out. On aortic rings of 12-week-old obese Zucker rats, but not in older animals (36 and 52 weeks), the following different effects in comparison to the lean rat control group were observed: (i) a significantly enhanced maximal relaxation to acetylcholine and A23187, which was abolished by the nitric oxide-synthase inhibitor L-nitro-arginine methyl ester (L-NAME); relaxation of aortic rings to the endothelium-independent vasodilator nitroglycer-in was similar; (ii) more pronounced maximal 5-hydroxy-tryptamine-induced-contractions in the presence of L-NAME, and (iii) a more pronounced reduction in phenylephrine-induced contractions by verapamil. These results are suggestive of an altered calcium metabolism in the first weeks of development in the obese rat strain, which is probably responsible for the hypotension seen in this early time period.