We hypothesized that A2A adenosine receptor (A2A AR) activation causes vasorelaxation through cytochrome P-450 (CYP) epoxygenases and endothelium-derived hyperpolarizing factors, whereas lack of A2A AR activation promotes vasoconstriction through Cyp4a in the mouse aorta. Adenosine 5'-N-ethylcarboxamide (NECA; 10(-6) M), an adenosine analog, caused relaxation in wild-type A2A AR (A2A AR+/+; +33.99 +/- 4.70%, P < 0.05) versus contraction in A2A AR knockout (A2A AR(-/-); -27.52 +/- 4.11%) mouse aortae. An A2A AR-specific antagonist (SCH-58261; 1 microM) changed the NECA (10(-6) M) relaxation response to contraction (-35.82 +/- 4.69%, P < 0.05) in A2A AR+/+ aortae, whereas no effect was noted in A2A AR(-/-) aortae. Significant contraction was seen in the absence of the endothelium in A2A AR+/+ (-2.58 +/- 2.25%) aortae compared with endothelium-intact aortae. An endothelial nitric oxide synthase inhibitor (N-nitro-L-arginine methyl ester; 100 microM) and a cyclooxygenase inhibitor (indomethacin; 10 microM) failed to block NECA-induced relaxation in A2A AR+/+ aortae. A selective inhibitor of CYP epoxygenases (methylsulfonyl-propargyloxyphenylhexanamide; 10 microM) changed NECA-mediated relaxation (-22.74 +/- 5.11% at 10(-6) M) and CGS-21680-mediated relaxation (-18.54 +/- 6.06% at 10(-6) M) to contraction in A2A AR+/+ aortae, whereas no response was noted in A2A AR(-/-) aortae. Furthermore, an epoxyeicosatrienoic acid (EET) antagonist [14,15-epoxyeicosa-5(Z)-enoic acid; 10 microM] was able to block NECA-induced relaxation in A2A AR+/+ aortae, whereas omega-hydroxylase inhibitors (10 microM dibromo-dodecenyl-methylsulfimide and 10 microM HET-0016) changed contraction into relaxation in A2A AR(-/-) aorta. Cyp2c29 protein was upregulated in A2A AR+/+ aortae, whereas Cyp4a was upregulated in A2A AR(-/-) aortae. Higher levels of dihydroxyeicosatrienoic acids (DHETs; 14,15-DHET, 11,12-DHET, and 8,9-DHET, P < 0.05) were found in A2A AR+/+ versus A2A AR(-/-) aortae. EET levels were not significantly different between A2A AR+/+ and A2A AR(-/-) aortae. It is concluded that CYP epoxygenases play an important role in A2A AR-mediated relaxation, and the deletion of the A2A AR leads to contraction through Cyp4a.

neuroendocrine carcinogenesis ͉ metastatic gastric cancer ͉ functional genomic studies ͉ mouse͞human epithelial cancers

We have investigated the role of adenosine and its analogs on vasorelaxation of mouse aorta in intact endothelium with rank order of potency as follows: 5'-N-ethylcarboxamidoadenosine (NECA) > 2-chloroadenosine > adenosine >> CGS-21680, which is consistent with the profile of A(2B)-adenosine receptor (A(2B)AR). In endothelium-intact tissues, acetylcholine produced relaxation ranging from 65 to 80% in phenylephrine (PE, 10(-7) M)-precontracted mouse aorta, whereas no relaxation was observed in endothelium-denuded tissues. The A(2B)AR antagonist alloxazine (10(-5) M) shifted concentration-response curve for NECA (EC(50) = 0.005 x 10(-5) M) to the right with an EC(50) of 2.8 x 10(-5) M, demonstrating that this relaxation is partially dependent on functional endothelium mediated predominantly via A(2B)AR in this tissue. This conclusion was further supported by the following findings: 1) in the endothelium-intact mouse aorta, the EC(50) values for NECA and adenosine were found to be 0.05 and 1.99 x 10(-4) M, respectively; however, in denuded endothelium, these values were 0.098 and 3.55 x 10(-4) M, respectively; 2) NECA-induced relaxation was significantly blocked by N(G)-nitro-l-arginine methyl ester (l-NAME; 10(-4) M) in endothelium-intact tissues, which was reversed by pretreatment with l-arginine (10(-4) M), whereas no significant inhibition was found in endothelium-denuded tissues; 3) total nitrites and nitrates (NOx) in intact endothelium with l-NAME (10(-4) M) alone and in combination with l-arginine were 59% (P < 0.05) and 96%, respectively, in comparison with control (PE + NECA); and 4) endothelial nitric oxide synthase gene expression was found to be 67% (P < 0.05) less in endothelium-denuded as opposed to endothelium-intact mouse aorta. Thus these data demonstrate that adenosine-mediated vasorelaxation is partially dependent on A(2B)AR in mouse aorta.

The A(1) adenosine receptor (A(1)AR) is coupled to G(i)/G(o) proteins, but the downstream signaling pathways in smooth muscle cells are unclear. This study was performed in coronary artery smooth muscle cells (CASMCs) isolated from the mouse heart [A(1)AR wild type (A(1)WT) and A(1)AR knockout (A(1)KO)] to delineate A(1)AR signaling through the PKC pathway. In A(1)WT cells, treatment with (2S)-N(6)-(2-endo-norbornyl)adenosine (ENBA; 10(-5)M) increased A(1)AR expression by 150%, which was inhibited significantly by the A(1)AR antagonist 1,3-dipropyl-8-cyclopentylxanthine (10(-6)M), but not in A(1)KO CASMCs. PKC isoforms were identified by Western blot analysis in the cytosolic and membrane fractions of cell homogenates of CASMCs. In A(1)WT and A(1)KO cells, significant levels of basal PKC-alpha were detected in the cytosolic fraction. Treatment with the A(1)AR agonist ENBA (10(-5)M) translocated PKC-alpha from the cytosolic to membrane fraction significantly in A(1)WT but not A(1)KO cells. Phospholipase C isoforms (betaI, betaIII, and gamma(1)) were analyzed using specific antibodies where ENBA treatment led to the increased expression of PLC-betaIII in A(1)WT CASMCs while having no effect in A(1)KO CASMCs. In A(1)WT cells, ENBA increased PKC-alpha expression and p42/p44 MAPK (ERK1/2) phospohorylation by 135% and 145%, respectively. These effects of ENBA were blocked by Gö-6976 (PKC-alpha inhibitor) and PD-98059 (p42/p44 MAPK inhibitor). We conclude that A(1)AR stimulation by ENBA activates the PKC-alpha signaling pathway, leading to p42/p44 MAPK phosphorylation in CASMCs.

We investigated whether A(3) adenosine receptor (A(3)AR) is involved in endothelium-mediated contraction through cyclooxygenases (COXs) with the use of wild-type (WT) and A(3) knockout (A(3)KO) mice aorta. A(3)AR-selective agonist, Cl-IBMECA, produced a concentration-dependent contraction (EC(50): 2.9 +/- 0.2 x 10(-9) M) in WT mouse aorta with intact endothelium (+E) and negligible effects in A(3)KO +E aorta. At 10(-7) M, contractions produced by Cl-IBMECA were 29% in WT +E, while being insignificant in A(3)KO +E aorta. Cl-IBMECA-induced responses were abolished in endothelium-denuded tissues (-E), in both WT and A(3)KO aorta. A(3)AR gene and protein expression were reduced by 74 and 72% (P < 0.05), respectively, in WT -E compared with WT +E aorta, while being undetected in A(3)KO +E/-E aorta. Indomethacin (nonspecific COXs blocker, 10(-5) M), SC-560 (specific COX-1 blocker, 10(-8) M), SQ 29549 (thromboxane prostanoid receptor antagonist, 10(-6) M), and furegrelate (thromboxane synthase inhibitor, 10(-5) M) inhibited Cl-IBMECA-induced contraction significantly. Cl-IBMECA-induced thromboxane B(2) production was also attenuated significantly by indomethacin, SC-560, and furegrelate in WT +E aorta, while having negligible effects in A(3)KO +E aorta. NS-398 (specific COX-2 blocker) produced negligible inhibition of Cl-IBMECA-induced contraction in both WT +E and A(3)KO +E aorta. Cl-IBMECA-induced increase in COX-1 and thromboxane prostanoid receptor expression were significantly inhibited by MRS1523, a specific A(3)AR antagonist in WT +E aorta. Expression of both A(3)AR and COX-1 was located mostly on endothelium of WT and A(3)KO +E aorta. These results demonstrate for the first time the involvement of COX-1 pathway in A(3)AR-mediated contraction via endothelium.

A(2A) adenosine receptor (A(2A)AR) has potent anti-inflammatory properties, which may be important in the regulation of airway reactivity and inflammation. Inflammatory cells that possess A(2A)AR also produce nitrosative stress, which is associated with pathophysiology of asthma, so we hypothesized that A(2A)AR deficiency may lead to increased airway reactivity and inflammation through nitrosative stress. Thus the present study was carried out to investigate the role of A(2A)AR on airway reactivity, inflammation, NF-kappaB signaling, and nitrosative stress in A(2A)AR knockout (KO) and wild-type (WT) mice using our murine model of asthma. Animals were sensitized intraperitoneally on days 1 and 6 with 200 microg of ragweed, followed by aerosolized challenges with 0.5% ragweed on days 11, 12, and 13, twice a day. On day 14, airway reactivity to methacholine was assessed as enhanced pause (Penh) using whole body plethysmography followed by bronchoalveolar lavage (BAL) and lung collection for various analyses. Allergen challenge caused a significant decrease in expression of A(2A)AR in A(2A) WT sensitized mice, with A(2A)AR expression being undetected in A(2A) KO sensitized group leading to decreased lung cAMP levels in both groups. A(2A)AR deletion/downregulation led to an increase in Penh to methacholine and influx of total cells, eosinophils, lymphocytes, and neutrophils in BAL with highest values in A(2A) KO sensitized group. A(2A) KO sensitized group further had increased NF-kappaB expression and nitrosative stress compared with WT sensitized group. These data suggest that A(2A)AR deficiency leads to airway inflammation and airway hyperresponsiveness, possibly via involvement of nitrosative stress in this model of asthma.

Phospholipase C induced phosphoinositide (PI) turnover, intracellular Ca(2+) ([Ca(2+)](i)) mobilization and mitogen-activated protein (MAP) kinase activation by FP-class prostaglandin analogs was studied in normal human ciliary muscle (h-CM) cells. Agonist potencies obtained in the PI turnover assays were: travoprost acid ((+)-fluprostenol; EC(50) = 2.6 +/- 0.8 nM) > bimatoprost acid (EC(50) = 3.6 +/- 1.2 nM) > (+/-)-fluprostenol (EC(50) = 4.3 +/- 1.3 nM) >> prostaglandin F(2 alpha) (PGF(2 alpha)) (EC(50) = 134 +/- 17 nM) > latanoprost acid (EC(50) = 198 +/- 83 nM) > S-1033 (EC(50) = 2930 +/- 1420 nM) > unoprostone (EC(50) = 5590 +/- 1490 nM) > bimatoprost (EC(50) = 9600 +/- 1100 nM). Agonist potencies in h-CM cells correlated well with those previously obtained for the cloned human ciliary body-derived FP receptor (r = 0.96, p< 0.001) and that present on h-TM cells (r = 0.94, p< 0.0001). Travoprost acid, PGF(2 alpha) and unoprostone also stimulated [Ca(2+)](i) mobilization in h-CM cells with travoprost acid being the most potent agonist. MAP kinase activity was stimulated in the h-CM cells with the following rank order of activity (at 100 nM): travoprost acid > PGF(2 alpha) > latanoprost acid > PGD(2) > bimatoprost > latanoprost = bimatoprost acid = fluprostenol > PGE(2) = S-1033 > unoprostone > PGI(2). The PI turnover, [Ca(2+)](i) mobilization and MAP kinase activation induced by several of these agonists was blocked by the FP receptor antagonist, AL-8810 (11 beta-fluoro-15-epiindanyl PGF(2 alpha)) (e.g. K(i) = 5.7 microM versus PI turnover). These studies have characterized the biochemical and pharmacological properties of the native FP prostaglandin receptor present on h-CM cells using three signal transduction mechanism assays and a broad panel of FP-class agonist analogs (including free acids of bimatoprost, travoprost and latanoprost) and the FP receptor antagonist, AL-8810.

In a field trial conducted during 1993-1994, mustard {Brassica funcea L. Czern & Coss.) cv. Varuna was sprayed with either deionised water or 10'' M GAj at 40 (vegetative stage), 60 (flowering stage) or 80 (pod fil! stage) days after sowing (DAS) to select the suitable growth stage for spray for augmenting productivity of the crop. Shoot length per plant, leaf number per plant, leaf area per plant, dry weight per plant and leaf area index and accumulation of N. P and K were recorded at 100 DAS. Pods per plant, seeds per pod. 1000 seed weight, seed yield, biological yield, harvest index and seed yield merit were computed at harvest. Growth, NPK accumulation and yield were maximal when spraying was done at 40 DAS. However, spraying at 40 and 60 DAS gave at par values for most of the growth and yield parameters. It was also noted that there was a significant difference in spray treatment at different growth stages only when GA, was sprayed and not when water was sprayed.