Involvement of adenosine A1 receptors in upregulation of nitric oxide by acyclic nucleotide analogues

Acyclic nucleoside phosphonates are a novel class of virostatics effective against replication of both DNA-viruses and retroviruses. They are synthetic analogues of natural nucleotide monophosphates, and purine derivatives thus represent counterparts of AMP. Mono- and di-phosphorylated species are analogues of natural ADP and ATP, respectively. A number of these compounds are endowed with immunostimulatory and immunomodulatory potential. We investigated whether their augmenting effect on the interferon-gamma-primed production of nitric oxide (NO) by murine macrophages is mediated by purinoceptors. The test compounds comprise alterations at the N(6)-group of the heterocyclic base, i.e., adenine or 2,6-diaminopurine, and at the N(9)-side chain, represented by 9-[2-(phosphonomethoxy)ethyl] and 9-[2-(phosphonomethoxy)propyl] moieties: 9-[2-(phosphonomethoxy)propyl]adenine [(R)-PMPA; tenofovir], N(6)-cyclopropyl-(R)-9-[2-(phosphonomethoxy)propyl]2,6-diaminopurine [N(6)-cyclopropyl-(R)-PMPDAP], N(6)-cyclopentyl-(R)-9-[2-(phosphonomethoxy)propyl]2,6-diaminopurine [N(6)-cyclopentyl-(R)-PMPDAP], N(6)-dimethylaminoethyl-(R)-9-[2-(phosphonomethoxy)propyl]2,6-diaminopurine [N(6)-dimethylaminoethyl-(R)-PMPDAP], N(6)-isobutyl-9-[2-(phosphonomethoxy)ethyl]2,6-diaminopurine (N(6)-isobutyl-PMEDAP), N(6)-cyclopentyl-9-[2-(phosphonomethoxy)ethyl]2,6-diaminopurine (N(6)-cyclopentyl-PMEDAP), N(6)-cyclooctyl-9-[2-(phosphonomethoxy)ethyl]2,6-diaminopurine (N(6)-cyclooctyl-PMEDAP), and N(6)-cyclohexylmethyl-9-[2-(phosphonomethoxy)ethyl]2,6-diaminopurine (N(6)-cyclohexylmethyl-PMEDAP). The cells were cultured in the presence of interferon-gamma (5000 pg/ml) and test compounds (2-50 microM). Formation of nitrites was determined after 24 h using Griess reagent. It was inhibited by specific and nonspecific antagonists of adenosine A(1) receptors (IC(50) for 8-cyclopentyl-1,3-dipropylxanthine [CPX] was approximately 10 microM), while all other purine P(1) and purine P(2) receptor antagonists remained ineffective to suppress the NO-synergistic effect of acyclic nucleoside phosphonates.     

The effect of tryptophan depletion on the action of haloperidol in MK-801-treated rats

We investigated the effect of tryptophan depletion (tryptophan-free mixture) on locomotor activity in an animal model of schizophrenia, induced by acute administration of 5R,10S-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate (MK-801), and the influence of the tryptophan-free mixture on the action of the typical antipsychotic haloperidol. Male rats were pre-treated with haloperidol 60 min after receiving the tryptophan-free mixture (or water). We measured total distance travelled in an open field during a 90-min period. Administration of the tryptophan-free mixture resulted in decreased levels of tryptophan, serotonin and its metabolite 5-hydroxyindolacetic acid in the frontal cortex. Serotonin depletion increased the total distance travelled by MK-801-treated rats, modified the inhibitory effect of haloperidol and normalized the locomotor activity pattern in the model of schizophrenia-like behaviour. The effect of the tryptophan-free mixture combined with the classical antipsychotic haloperidol in MK-801-treated rats indicates the possibly important role of the serotonergic system in the action of antipsychotics.     

Agonist-induced internalization of leukotriene B(4) receptor 1 requires G-protein-coupled receptor kinase 2 but not arrestins

The leukotriene B(4) (LTB(4)) receptor (BLT1) becomes desensitized upon repeated agonist stimulation. Little is known, however, about BLT1 internalization, which follows desensitization in most G-protein-coupled receptors (GPCR). In the current study, transiently expressed BLT1 readily internalized, after LTB(4) stimulation, in RBL-2H3 cells that express high levels of endogenous GPCR kinase 2 (GRK2) but did not in COS-7 or human embryonic kidney (HEK) 293 cells, which do not overexpress GRK. The internalization of BLT1 could be blocked in RBL-2H3 cells by coexpressing dominant-negative (DN) GRK2 K220R and could be promoted in HEK293 cells by coexpressing wild-type (WT) GRK2. Coexpression of WT or DN nonvisual arrestins had no effect on BLT1 internalization. Moreover, upon stimulation with LTB(4), BLT1 did not induce arrestin-green fluorescence protein redistribution in either cell type, even in the presence of overexpressed GRK2. Coimmunoprecipitation experiments confirmed that BLT1 could associate with GRK2 but not with arrestins. A C-tail-truncated mutant of BLT1 lost the capacity to internalize and associate with GRK2 upon exposure to LTB(4), suggesting that the C-tail was required for receptor internalization and association with GRK2. Taken together, our results indicate that the C terminus of BLT1 plays a pivotal role in receptor internalization and GRK2 association. Moreover, ligand-induced BLT1 internalization is dependent on GRK2 but independent of arrestins. This may allow differential, cell-type-specific signaling in response to LTB(4), depending on GRK expression levels.     

Pharmacology of recombinant low-voltage activated calcium channels

Several types of voltage- or ligand-activated calcium channels contribute to the excitability of neuronal cells. Low-voltage-activated (LVA), T-type calcium channels are characterised by relatively negative threshold of activation and therefore they can generate low-threshold spikes, which are essential for burst firing. At least three different proteins form T-type calcium current in neurons: Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3. Expression of these proteins in various brain regions is complementary. Individual channel types could be distinguished by different sensitivity towards inorganic cations. This inhibition can contribute to the toxicity of some heavy metals. Selective inhibition of T-type calcium channels by organic blockers may have clinical importance in some forms of epilepsy. Mibefradil inhibits the expressed Ca(v2)3.1, Ca(v)3.2 and Ca(v)3.3 channels in nanomolar concentrations with Ca(v)3.3 channel having lowest affinity. The sensitivity of the expressed Ca(v)3.1 channel to the antiepileptic drugs, valproate and ethosuximide, is low. Ca(v)3.1 channel is moderately sensitive to phenytoin. The Ca(v)3.2 channel is sensitive to ethosuximide, amlodipine and amiloride. All three LVA calcium channels are moderately sensitive to active metabolites of methosuximide, i.e. alpha-methyl-alpha-phenylsuccinimide. Several neuroleptics inhibit all three LVA channels in clinically relevant concentrations. All three channels are also inhibited by the endogenous cannabinoid anandamide. A high affinity peptide blocker for these Ca channels is the scorpion toxin kurtoxin which inhibits the Ca(v)3.1 and Ca(v)3.2, but not the Ca(v)3.3 channel in nanomolar concentrations. Nitrous oxide selectively inhibits the Ca(v)3.2, but not the Ca(v)3.1 channel. The Ca(v)3.2, but not the Ca(v)3.1 channel is potentiated by stimulation of Ca(2+)/CaM-dependent protein kinase.     

Potential antipsoriatic avarol derivatives as antioxidants and inhibitors of PGE(2) generation and proliferation in the HaCaT cell line

The synthesis and structure-activity relationships for a series of 14 new avarol derivatives as antioxidants and inhibitors of cell proliferation and PGE(2) generation in human keratinocytes are described. Compound 6 (thiosalicylic derivative) was the most potent inhibitor of superoxide generation in human neutrophils and also potently inhibited PGE(2) generation in the human keratinocyte HaCaT cell line. Compound 7(3'-methylaminoavarone) presented the best antiproliferative profile, by the inhibition of (3)H-thymidine incorporation in HaCaT cells, with potency similar to the reference compound anthralin. None of the avarol derivatives showed any sign of cytotoxicity measured as LDH release in treated keratinocytes. The potency and pharmacological profile of derivatives are also discussed.     

Oxygen activation by photoexcited protoberberinium alkaloids from Mahonia aquifolium

Protoberberinium salts, i.e. berberine (I), palmatine (II) and jatrorrhizine (III) prepared from Mahonia aquifolium (Pursh) Nutt. belong to isoquinoline alkaloids possessing interesting biological activity (e.g. antibacterial, antimalarial, antitumor). The characteristic UV/Vis absorption band maxima of I-III iodide salts were found in regions 350 and 425 nm in dimethylsulfoxide (DMSO) and ethanol solvents, and were only negligibly influenced by substitution changes on the C-2 and C-3 positions. The fluorescence intensity of protoberberinium salts monitored in ethanol solutions was significantly lowered by iodide counter-ions, and decreased in the order berberine > palmatine > jatrorrhizine. EPR spectroscopy supplied evidence of the formation of super-oxide anion radicals and singlet oxygen upon irradiation of berberine in oxygenated DMSO solvent. The photochemical generation of O(2) (.-) and (1)O(2) in DMSO solutions of palmatine and jatrorrhizine was substantially lower, and probably reflected the replacement of a photolabile methylenedioxy group at C-2 and C-3 positions in the berberine molecule by two methoxy groups in palmatine, and methoxyl (C-2) and hydroxyl (C-3) substitution in jatrorrhizine. Additionally, the powder EPR spectra of protoberberinium iodides I-III measured at 290 K revealed the presence of single-line EPR signals (g(eff) = 2.0044), which were attributed to hydroperoxidic structures produced by the autoxidation process. The photochemical reactions of protoberbenium salts producing reactive oxygen species after UVA excitation should be integrated in biological activity investigations, as well as in their applications in skin disorder treatment.     

Spontaneous vitiligo in an animal model for human melanoma: role of tumor-specific CD8+ T cells

Tumor antigen-reactive T cells can be detected in a large proportion of melanoma patients, but their efficacy on tumor control in vivo remains unclear. On the other hand, vitiligo, a skin disorder characterized by patchy depigmented macules, may occur spontaneously or after antitumor therapies. Moreover, vitiligo is significantly associated with positive clinical response, but the mechanism is not understood. Therefore, the establishment of a relevant animal model in which melanoma and vitiligo spontaneously develop stepwise may be useful for better understanding of the parameters involved in the destruction of both benign and malignant melanocytes. In a previous work, we established a mouse model for melanoma in which MT/ret transgenic mice express the ret oncogene fused to the metallothionein promoter. Here we report that melanoma leads to spontaneous vitiligo. We further investigate, for the first time in this model, the natural antitumor T-cell response and evaluate the role of cellular immunity in the development of the disease. Interestingly, the occurrence of spontaneous tumor nodules in MT/ret mice with melanoma-associated vitiligo is significantly delayed when compared in melanoma mice without vitiligo. Moreover, a significant proportion of mice with melanoma-associated vitiligo resisted a challenge with syngeneic melanoma cells in contrast to animals without vitiligo. Our results confirm that vitiligo is associated with clinical benefit and further demonstrate the crucial role of CD8+ T cells for tumor control in melanoma-associated vitiligo.     

Dynamics of telomere erosion and its association with genome instability in myelodysplastic syndromes (MDS) and acute myelogenous leukemia arising from MDS: a marker of disease prognosis?

Telomere length was evaluated by terminal repeat fragment method (TRF) in 50 patients with myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML) arising from MDS and in 21 patients with untreated primary AML to ascertain, whether telomere erosion was associated with progression of MDS towards overt leukemia. Heterogeneity of TRF among MDS FAB subgroups (P=0.004) originated from its shortening in increased number of patients during progression of the disease. Chromosomal aberrations were present in 32% MDS patients with more eroded telomeres (P=0.022), nevertheless a difference between mean TRF in the subgroups with normal and abnormal karyotype diminished during progression of MDS. A negative correlation between individual TRF and IPSS value (P=0.039) showed that telomere dynamics might serve as a useful prognostic factor for assessment of an individual MDS patient's risk and for decision of an optimal treatment strategy.