Adenosine receptor activation induces vascular endothelial growth factor in human retinal endothelial cells.

Adenosine, released in increased amounts by hypoxic tissues, is thought to be an angiogenic factor that links altered cellular metabolism caused by oxygen deprivation to compensatory angiogenesis. Adenosine interacts with 4 subtypes of G protein-coupled receptors, termed A(1), A(2A), A(2B), and A(3). We investigated whether adenosine causes proliferation of human retinal endothelial cells (HRECs) and synthesis of vascular endothelial growth factor (VEGF) and, if so, which adenosine receptor subtype mediates these effects. The nonselective adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), in a concentration-dependent manner, increased both VEGF mRNA and protein expression by HRECs, as well as proliferation. This proliferative effect of NECA was inhibited by the addition of anti-human VEGF antibody. NECA also increased insulin-like growth factor-I and basic fibroblast growth factor mRNA expression in a time-dependent manner and cAMP accumulation in these cells. In contrast, neither the A(1) agonist N(6)-cyclopentyladenosine nor the A(2A) agonist 2-p-(2-carboxyethyl) phenethylamino-NECA caused any of the above effects of NECA. The effects of NECA were not significantly attenuated by either the A(2A) antagonist SCH58261 or the A(1) antagonist 8-cyclopentyl-1, 3-dipropylxanthine. However, the nonselective adenosine receptor antagonist xanthine amine congener completely inhibited the effects of NECA. Addition of antisense oligonucleotide complementary to A(2B) adenosine receptor mRNA inhibited VEGF protein production by HRECs after NECA stimulation. Thus, the A(2B) adenosine receptor subtype appears to mediate the actions of adenosine to increase growth factor production, cAMP content, and cell proliferation of HRECs. Adenosine activates the A(2B) adenosine receptor in HRECs, which may lead to neovascularization by a mechanism involving increased angiogenic growth factor expression.

Inverse agonists and neutral antagonists of recombinant human A1 adenosine receptors stably expressed in Chinese hamster ovary cells.

Receptor antagonists can be classified as neutral antagonists or antagonists with inverse agonist activity based on their effectiveness to reduce the spontaneous agonist-independent activity of receptors. The goals of this study were to (1) demonstrate that A1-adenosine receptors (A1AdoRs) expressed at high density (4000-8000 fmol/mg of protein) in Chinese hamster ovary (CHO) cells cause constitutive activation of inhibitory G proteins and inhibition of adenylyl cyclase activity and (2) identify both neutral A1AdoR antagonists and antagonists with inverse agonist activity. The activity of A1AdoR agonists and antagonists was determined by assays of both specific binding of [35S]guanosine-5'-O-(3-thio)triphosphate ([35S]GTPgammaS) to membranes and cAMP content of intact cells in the presence of adenosine deaminase (2-5 units/ml). The A1AdoR agonist N6-cyclopentyladenosine (CPA) significantly increased binding of [35S]GTPgammaS by 241 +/- 7% compared with control. The A1AdoR antagonists N-0861, N-0840, and WRC-0342 did not alter binding of [35S]GTPgammaS, whereas the antagonists 8-cyclopentyl-1, 3-dipropylxanthine (CPX), CGS-15943, xanthine amine congener, and WRC-0571 significantly reduced binding of [35S]GTPgammaS by 28-53% from control, respectively. The effects of both the agonist N6-cyclopentyladenosine (CPA) and the antagonist CPX to alter binding of [35S]GTPgammaS were attenuated by 1 micro M N-0861. CPA reduced cAMP content of forskolin-stimulated CHO:A1AdoR cells, and N-0861 and WRC-0342 did not alter cAMP content, but the antagonists CPX and WRC-0571 increased the cAMP content of CHO:A1AdoR cells. The effects of both CPX and WRC-0571 to increase cAMP content of forskolin-stimulated CHO:A1AdoR cells were attenuated by either N-0861 or WRC-0342. The results indicate that both N-0861 and WRC-0342 are neutral antagonists, whereas both CPX and WRC-0571 are antagonists with inverse agonist activity.

Heat stress protects the perfused rabbit heart from complement-mediated injury.

We determined if heat stress induction of heat shock protein (HSP) 70 modulates complement activation in an experimental model of xenograft rejection. Male New Zealand White rabbits were heat stressed (core body temperature to 42 degrees C for 15 min; n = 9). Control rabbits (n = 13) were not exposed to heat stress. Hearts were removed 18 h later and perfused by the Langendorff method. After equilibration, human plasma (source of human complement) was added to the perfusion medium. Hemodynamic variables recorded during perfusion with human plasma were improved in hearts from heat-stressed animals compared with control hearts. Assembly of the soluble membrane attack complex was reduced in the interstitial fluid effluent from the heat-stressed hearts. Electron microscopic evidence of ultrastructural changes was attenuated in the hearts from heat-stressed rabbits. Myocardial tissue from heat-stressed animals exhibited an increase in inducible HSP 70 that was virtually absent in the hearts of control rabbits. Previous whole body hyperthermia protects the rabbit heart against the detrimental effects of heterologous plasma, suggesting that heat-stress induction of HSP 70 limits the extent of complement activation by a discordant vascularized tissue (xenograft). Induction of heat stress proteins by the donor organ might be an important mechanism affecting the outcome of xenograft transplants.