No abstract

The effect of adenosine and its analogues on the cytotoxic activity of IL-2-activated NK cells was investigated. Adenosine is an endogenous ligand for four different adenosine receptor (AdoR) subtypes (AdoRA1, AdoRA2A, AdoRA2B, and AdoRA3). Increased concentrations of adenosine were found in ascites of MethA sarcoma or in culture medium of 3LL Lewis lung carcinoma growing under hypoxic conditions. We hypothesize that intratumor adenosine impairs the ability of lymphokine-activated killer (LAK) cells to kill tumor cells. The effect of AdoR engagement on LAK cells cytotoxic activity was analyzed using AdoR agonists and antagonists as well as LAK cells generated from AdoR knockout mice. Adenosine and its analogues efficiently inhibited the cytotoxic activity of LAK cells. CGS21680 (AdoRA2A agonist) and 5-N-ethylcarboxamide adenosine (NECA) (AdoRA2A/ADoRA2B agonist) inhibited LAK cell cytotoxicity in parallel with their ability to increase cAMP production. The inhibitory effects of stable adenosine analog 2-chloroadenosine (CADO) and AdoRA2 agonists were blocked by AdoRA2 antagonist ZM 241385. Adenosine and its analogues impair LAK cell function by interfering with both perforin-mediated and Fas ligand-mediated killing pathways. Studies with LAK cells generated from AdoRA1−/− and AdoRA3−/− mice ruled out any involvement of these AdoRs in the inhibitory effects of adenosine. LAK cells with genetically disrupted AdoRA2A were resistant to the inhibitory effects of adenosine, CADO and NECA. However, with extremely high concentrations of CADO or NECA, mild inhibition of LAK cytotoxicity was observed that was probably mediated via AdoRA2B signaling. Thus, by using pharmacological and genetic blockage of AdoRs, our results clearly indicate the prime importance of cAMP elevating AdoR2A in the inhibitory effect of adenosine on LAK cell cytotoxicity. The elevated intratumor levels of adenosine might inhibit the antitumor effects of activated NK cells.

No abstract

Adenosine is an important signaling molecule that regulates multiple physiologic processes and exerts major anti-inflammatory actions. Tumors have high concentrations of adenosine, which could inhibit the function of tumor-infiltrating lymphoid cells. We investigated the ability of adenosine and its stable analogue 2-chloroadenosine (CADO) to inhibit cytokine production and cytotoxic activity of lymphokine-activated killer (LAK) cells and determined whether both these effects are initiated via a common pathway. CADO strongly inhibited cytotoxic activity of LAK cells and attenuated the production of IFN-;, granulocyte macrophage colony-stimulating factor, tumor necrosis factor A, and macrophage inflammatory protein-1A by LAK cells stimulated by cross-linking of the Ly49D receptor. These inhibitory effects were associated with the ability of CADO to stimulate cyclic AMP (cAMP) production and activate protein kinase A (PKA). Using cAMP analogues with different affinities for the A and B sites of the regulatory subunits of PKA types I and II, we found that activation of PKA I, but not PKA II, mimicked the inhibitory effects of CADO on LAK cell cytotoxic activity and cytokine production. Inhibitors of the PKA catalytic subunits (H89 and PKI 14-22 peptide) failed to abrogate the inhibitory effects of CADO whereas Rp-8-Br-cAMPS, an antagonist of the RI subunit, blocked the inhibitory effects of CADO. We conclude that the inhibitory effects of adenosine are probably mediated via cAMP-dependent activation of the RI subunits of PKA I but are independent of the catalytic activity of PKA. Tumor-produced adenosine could be a potent tumor microenvironmental factor inhibiting the functional activity of tumor-infiltrating immune cells. (Cancer Res 2006; 66(15): 7758-65)

The uroepithelium of the bladder forms an impermeable barrier that is maintained in part by regulated membrane turnover in the outermost umbrella cell layer. Other than bladder filling, few physiological regulators of this process are known. Western blot analysis established that all four adenosine receptors (A1, A2a, A2b, and A3) are expressed in the uroepithelium. A1 receptors were prominently localized to the apical membrane of the umbrella cell layer, whereas A2a, A2b, and A3 receptors were localized intracellularly or on the basolateral membrane of umbrella cells and the plasma membrane of the underlying cell layers. Adenosine was released from the uroepithelium, which was potentiated 10-fold by stretching the tissue. Administration of adenosine to the serosal or mucosal surface of the uroepithelium led to increases in membrane capacitance (where 1 microF approximately 1 cm(2) tissue area) of approximately 30% or approximately 24%, respectively, after 5 h. Although A1, A2a, and A3 selective agonists all stimulated membrane capacitance after being administrated serosally, only the A1 agonist caused large increases in capacitance after being administered mucosally. Adenosine receptor antagonists as well as adenosine deaminase had no effect on stretch-induced capacitance increases, but adenosine potentiated the effects of stretch. Treatment with U-73122, 2-aminoethoxydiphenylborate, or xestospongin C or incubation in calcium-free Krebs solution inhibited adenosine-induced increases in capacitance. These data indicate that the uroepithelium is a site of adenosine biosynthesis, that adenosine receptors are expressed in the uroepithelium, and that one function of these receptors may be to modulate exocytosis in umbrella cells.

Abstract-Some estrogenic compounds modify vascular smooth muscle cell (SMC) biology; however, whether such effects are mediated in part by estrogen receptors is unknown. The purpose of this study was to evaluate whether the actions of clinically used estrogens on human aortic SMC biology are mediated by estrogen receptors. We examined the effects of various clinically used estrogens in the presence and absence of ICI 182,780, an estrogen receptor antagonist, on cultured human aortic SMC DNA synthesis ([ 3 H]thymidine incorporation), cellular proliferation (cell counting), cell migration (modified Boyden chamber), collagen synthesis ([ 3 H]proline incorporation), and mitogen-activated protein kinase activity. FCS-induced DNA synthesis, cell proliferation, collagen synthesis, platelet-derived growth factorinduced SMC migration, and mitogen-activated protein kinase activity were significantly inhibited by physiological (10 Ϫ9 mol/L) concentrations of 17␤-estradiol and low concentrations (10 Ϫ8 to 10 Ϫ7 mol/L) of 17␤-estradiol, estradiol valerate, estradiol cypionate, and estradiol benzoate but not by estrone, estriol, 17␣-estradiol, or estrone sulfate. The inhibitory effects of 17␤-estradiol and other inhibitory estrogens were completely reversed by 100 mol/L ICI 182,780, and the rank-order potency of various estrogens to inhibit SMC biology matched their rank-order affinity for estrogen receptors. The inhibitory effects of estrogens on SMC biology are in part receptor-mediated. Because the cardioprotective effects of hormone replacement therapy are most likely mediated by modification of SMC biology, whether hormone replacement therapy protects a given postmenopausal woman against cardiovascular disease will depend partially on the affinity of the estrogen for estrogen receptors in vascular SMCs. S ome epidemiological studies provide strong evidence that hormone replacement therapy (HRT) affords cardioprotection in postmenopausal women, 1 whereas other epidemiological studies and a recent clinical trial do not support this notion. 2,3 Although the reasons for these discordant findings are unknown, the inconsistent results reported to date may be due to heterogeneity in the responses of postmenopausal women to HRT. Indeed, in a given cohort of postmenopausal women, HRT has been shown to provide cardioprotective effects in only 50% to 60%. 4 To correctly interpret the results of completed clinical studies and to better design new clinical trials, it is critical to elucidate the independent variables that govern the cardioprotective effects of HRT.Our working hypothesis is that the degree of cardioprotection afforded by HRT is strongly influenced by the binding affinity of the specific estrogen to estrogen receptors in vascular cells and to the level of expression of estrogen receptors in vascular cells in an individual postmenopausal woman. The rationale for this hypothesis is 2-fold. First, several different types of estrogens are used clinically, 5 and estrogens differ greatly in their chemical characteristics, bi...

Abstract-Adenosine inhibits growth of cardiac fibroblasts; however, the adenosine receptor subtype that mediates this antimitogenic effect remains undefined. Therefore, the goals of this study were to determine which adenosine receptor subtype mediates the antimitogenic effects of adenosine and to investigate the signal transduction mechanisms involved.In rat left ventricular cardiac fibroblasts, PDGF-BB (25 ng/mL) stimulated DNA synthesis ( 3 H-thymidine incorporation), cellular proliferation (cell number), collagen synthesis ( 3 H-proline incorporation), and MAP kinase activity. The adenosine receptor agonists 2-chloroadenosine and 5Ј-N-methylcarboxamidoadenosine, but not N 6 -cyclopentyladenosine, 4-aminobenzyl-5Ј-N-methylcarboxamidoadenosine, or CGS21680, inhibited the growth effects of PDGF-BB, an agonist profile consistent with an A 2B receptor-mediated effect. The adenosine receptor antagonists KF17837 and 1,3-dipropyl-8-p-sulfophenylxanthine, but not 8-cyclopentyl-1,3-dipropylxanthine, blocked the growth-inhibitory effects of 2-chloroadenosine and 5Ј-N-methylcarboxamidoadenosine, an antagonist profile consistent with an A 2 receptor-mediated effect. Antisense, but not sense or scrambled, oligonucleotides to the A 2B receptor stimulated basal and PDGF-induced DNA synthesis, cell proliferation, and collagen synthesis. Moreover, the growth-inhibitory effects of 2-chloroadenosine, 5Ј-N-methylcarboxamidoadenosine, and erythro-9-(2-hydroxy-3-nonyl) adenine plus iodotubericidin (inhibitors of adenosine deaminase and adenosine kinase, respectively) were abolished by antisense, but not scrambled or sense, oligonucleotides to the A 2B receptor. Our findings strongly support the hypothesis that adenosine causes inhibition of CF growth by activating A 2B receptors coupled to inhibition of MAP kinase activity. Thus, A 2B receptors may play a critical role in regulating cardiac remodeling associated with CF proliferation. Pharmacologic or molecular biological activation of A 2B receptors may prevent cardiac remodeling associated with hypertension, myocardial infarction, and myocardial reperfusion injury after ischemia.

Abstract-Estrogen receptors (ERs) are considered to mediate the ability of 17␤-estradiol (estradiol) to reduce injury-induced proliferation of vascular smooth muscle cells (VSMCs), leading to vascular lesions. However, the finding that estradiol attenuates formation of vascular lesions in response to vascular injury in knockout mice that lack either ER-␣ or ER-␤ challenges this concept. Our hypothesis is that the local metabolism of estradiol to methoxyestradiols, metabolites of estradiol with little affinity for ERs, mediates the ER-independent antimitogenic effects of estradiol on VSMCs. In human VSMCs, 2-methoxyestradiol and 2-hydroxyestradiol were more potent than was estradiol in inhibiting DNA synthesis ( 3 [H]-thymidine incorporation), collagen synthesis ( 3 [H]-proline incorporation), cell proliferation (cell number), and cell migration (movement of cells across a polycarbonate membrane). The inhibitory effects of estradiol on VSMCs were enhanced by cytochrome-P450 (CYP450) inducers 3-methylcholanthrene and phenobarbital. Moreover, the inhibitory effects of estradiol were blocked in the presence of the CYP450 inhibitor 1-aminobenzotriazole and the catechol-O-methyltransferase inhibitors quercetin and OR486. Both OR486 and quercetin blocked the conversion of 2-hydroxyestradiol to 2-methoxyestradiol; moreover, they blocked the antimitogenic effects of 2-hydroxyestradiol but not of 2-methoxyestradiol. The ER antagonist ICI182780 blocked the inhibitor effects of estradiol on VSMCs, but only at concentrations (Ͼ50 mol/L) that also inhibit the metabolism of estradiol to hydroxyestradiols (precursors of methoxyestradiols). In conclusion, the inhibitory effects of locally applied estradiol on human VSMCs are mediated via a novel ER-independent mechanism involving estradiol metabolism. These findings imply that vascular estradiol metabolism may be an important determinant of the cardiovascular protective effects of estradiol and that nonfeminizing estradiol metabolites may confer cardiovascular protection regardless of gender.