The effect of long-term oral treatment with caffeine on A1 and A2 receptors in the rat brain was studied. Caffeine was added to the drinking water and the animals were sacrificed after a 12 day treatment period. The plasma caffeine concentration was close to 100 microM. A1 receptors were studied using quantitative autoradiography with [3H]cyclohexyladenosine (CHA). Caffeine treatment increased the number of A1 receptors in the CA3 subfield of the hippocampus from 337 to 393 fmol/mg with no change in KD (0.692 vs. 0.675 nM). A1 mRNA was measured using Northern blots and quantitative in situ hybridization. There was no increase in A1 mRNA. A2a receptors, located in dopamine rich regions of the rat brain, were studied with quantitative autoradiography using [3H]CGS 21680 as the ligand, and the A2a mRNA was determined using quantitative in situ hybridization. Caffeine treatment produced no significant change in either receptor number or mRNA, even though the apparent Bmax tended to increase from 322 +/- 8 to 352 +/- 8 fmol/mg. The results show that treatment with caffeine in a dose that causes tolerance to several effects of caffeine and increases some effects of adenosine analogues increases the number of A1 receptors without any change in A1 mRNA, suggesting that the adaptive changes are at a post-translational level. There were no significant changes in A2 receptors indicating that the two types are regulated differently and/or that the amount of endogenous agonist is sufficient to regulate A1, but not A2 receptors.

Milk fat globule epidermal growth factor 8 (MFG-E8) (also known as lactadherin) is a secreted glycoprotein, and consists of two EGF-like domains, a mucin-like domain, and tandem discoidin-like domains (C-domains); in addition, an RGD motif that binds to a V b 3 and a V b 5 integrins is present in the second EGF domain.1 The C2 domain of MFG-E8 shares homology with the lipid-binding domains of blood coagulation factors VIII and V that have been shown to mediate Ca 2 þ -independent, high affinity (K d B2 nM) binding to phosphatidylserine (PS).

Cytochrome P450 1B1 (CYP1B1) is active in the metabolism of estrogens to reactive catechols and of different procarcinogens. Several studies have investigated the relationship between genetic polymorphisms of CYP1B1 and breast cancer risk, however, with inconsistent results. We investigated such an association in postmenopausal Swedish women, with special emphasis on long-term menopausal hormone users, in a large population-based case-control study. We genotyped 1521 cases and 1498 controls for the CYP1B1 single nucleotide polymorphisms (SNPs) m2, m3 and m4 and reconstructed haplotypes. The frequencies of CYP1B1*1, CYP1B1*2, CYP1B1*3 and CYP1B1*4 alleles among controls were estimated to be 0.087, 0.293, 0.444 and 0.175, respectively. It thus appeared that very few haplotypes contained combinations of SNPs at two or three loci and that single SNP genotype data effectively represented haplotypes. Odds ratios (OR) and 95% confidence intervals (CI) were calculated from logistic regression models. We found no overall association between any CYP1B1 genotype and breast cancer risk. The data indicated, however, that women who had used menopausal hormones for 4 years or longer, and carried the CYP1B1*3/*3 genotype may be at increased risk of breast cancer, OR 2.0 (95% CI 1.1-3.5), compared with long-term users without this genotype. We explored the effect of CYP1B1 genotype on breast cancer risk in subgroups defined by body mass index, family history, smoking and catechol-O-methyl transferase genotype, but found no convincing evidence for interaction. In summary, our results strongly indicate that the studied CYP1B1 gene polymorphisms do not influence breast cancer risk overall but may modify the risk after long-term menopausal hormone use.

No abstract

The effect of several adenosine analogues on cyclic AMP accumulation was examined in the rat phaeochromocytoma cell PC12 and in the human T-cell leukaemia cell Jurkat, selected as prototypes of cells predominantly expressing adenosine A2A or A2B receptors. Using the reverse transcription-polymerase chain reaction it was, however, demonstrated that the Jurkat cell and the PC12 cell express both A2A and A2B receptor mRNA, albeit in different relative proportions. In PC12 cells the concentration required for half-maximal response (EC50) for the full agonist 5'-N-ethyl-carboxamidoadenosine (NECA) was 30 times lower than in Jurkat cells. There was no significant difference in the pA2 for the antagonist 5-amino-9-chloro-2-(2-furanyl)- 1,2,4-triazolo(1,5-C)quinazolinemonomethanesulphonate (CGS 15943) between the two cell types. In the presence of forskolin (1 microM in PC12 cells; 10 microM in Jurkat cells) the EC50 value for NECA was reduced two-to sixfold. Forskolin also increased the maximal cAMP accumulation twofold in PC12 cells and sevenfold in Jurkat cells. A series of 2-substituted adenosine analogues CV 1808 (2-phenylamino adenosine), CV 1674 [2-(4-methoxyphenyl)adenosine], CGS 21680 ¿2-[p-(2-carbonylethyl)phenylethylamino]-5'-N-ethyl- carboxamido adenosine¿, and four 2-substituted isoguanosines, SHA 40 [2-(2-phenylethoxy)adenosine; PEA], SHA 91 [2-(2-cyclohexylethoxy)adenosine; CEA], SHA 118 ¿2-[2-(p-methylphenyl)ethoxy]adenosine; MPEA¿, and SHA 125 (2-hexyloxyadenosine; HOA), all raised cAMP accumulation in PC12 cells, but had minimal or no effect in Jurkat cells. In the PC12 cells the addition of forskolin (1 microM) reduced the EC50 by a factor of 2(CV 1808) to 12 (SHA 125). In Jurkat cells all the analogues gave a significant, but submaximal, cAMP response in the presence of forskolin (10 microM), but they were essentially inactive in its absence. The results show that a series of 2-substituted adenosine analogues can be used to discriminate between A2A and A2B receptors. The two receptor subtypes appear to coexist, even in clonal cells selected for typical pharmacology. A2 receptor pharmacology can therefore be complex.