Subject(s)
Mycosis Fungoides/drug therapy , Photopheresis/methods , Skin Neoplasms/drug therapy , Aged , Humans , MaleABSTRACT
Adenosine receptors represent a family of G-protein coupled receptors that are ubiquitously expressed in a wide variety of tissues. This family contains four receptor subtypes: A1 and A3, which mediate inhibition of adenylyl cyclase; and A2a and A2b, which mediate stimulation of this enzyme. Currently, all receptor subtypes have been genetically deleted in mouse models except for the A2b adenosine receptor, and some have been overexpressed in selective tissues of transgenic mice. Studies involving these transgenic mice indicated that receptor levels are rate limiting, as effects were amplified upon increases in receptor level. The knockout models pointed to clusters of activities related to the physiologies of the cardiovascular and the nervous systems, which are either reduced or enhanced upon specific receptor deletion. Interestingly, the trend of effects on these systems is similar in the A1 and A3 adenosine receptor knockout mice and opposite to the effects observed in the A2a adenosine receptor knockout model. This review summarizes in vitro studies on pathways affected by each adenosine receptor, and primarily focuses on the above in vivo models generated to investigate the physiologic role of adenosine receptors. Furthermore, it illustrates the need for multiple adenosine receptor subtype deficiency studies in mice and the deletion of the A2b subtype.
Subject(s)
Adenosine/metabolism , Adenylyl Cyclases/metabolism , Receptors, Purinergic P1/physiology , Animals , Gene Deletion , Mice , Mice, Knockout/genetics , Mice, Knockout/metabolism , Models, Animal , Receptor, Adenosine A1/genetics , Receptor, Adenosine A2A/genetics , Receptor, Adenosine A2B/genetics , Receptor, Adenosine A3/genetics , Receptors, Purinergic P1/genetics , Signal Transduction/geneticsABSTRACT
In previous studies, we reported that the level of expression of the adenylyl cyclase inhibitory A3 adenosine receptor (AR) impacts vascular tone and that rat vascular smooth muscle cells (VSMCs) coexpress the A3 AR and the adenylyl cyclase stimulatory A2a- and A2b-type ARs. In the current study, we investigated the regulation of expression of the A3 AR gene, focusing on sequences conserved in the mouse and human promoters. Transient transfection of primary cultures of rat VSMCs, using the mouse A3 AR promoter, shows that mutation of a conserved cAMP response element (CRE) significantly up-regulates promoter activity in first passage cells, whereas mutation of a conserved GATA site reduces promoter activity. This suggests that an inhibitory protein binds the CRE, whereas an enhancing factor binds the GATA sequence. Electrophoretic mobility shift assays (EMSAs) indicate that the putative CRE and GATA sites indeed bind cAMP response element modulator 1/c-Jun and the GATA6 protein, respectively. A3 AR promoter activity is significantly up-regulated in the presence of forskolin, the nonselective agonist 5'-(N-ethylcarboxamido)adenosine, or the A2a AR agonist 4-[2-[[6-amino-9(N-ethyl-beta-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepro- panoic acid (CGS21680), reaching levels similar to those of the A3 AR promoter bearing a mutated CRE. EMSA indicates that in the presence of forskolin the binding to the CRE is inhibited, suggesting that cAMP elevation disturbs the formation of an inhibitory complex on the CRE. Finally, semiquantitative reverse transcription-polymerase chain reaction analysis reveals that endogenous A3 AR mRNA is elevated in response to forskolin. Our findings suggest the presence of a mechanism by which cAMP might control its own level in cells via regulation of genes involved in modulation of adenylyl cyclase activity.
Subject(s)
Muscle, Smooth, Vascular/physiology , Promoter Regions, Genetic/physiology , Receptors, Purinergic P1/genetics , Adenylyl Cyclases/metabolism , Animals , Colforsin/pharmacology , Cyclic AMP/metabolism , Cyclic AMP Response Element-Binding Protein/metabolism , DNA-Binding Proteins/metabolism , Enzyme Activation , GATA6 Transcription Factor , Gene Expression Regulation , Mast Cells/physiology , Mice , Promoter Regions, Genetic/drug effects , Protein Binding , RNA, Messenger/metabolism , Rats , Receptor, Adenosine A3 , Receptors, Purinergic P1/metabolism , Transcription Factors/metabolismABSTRACT
A simple in situ method for scoring short tandem DNA repeat length has been developed using T4 endonuclease VII. This method measures tandem repeated simple sequences embedded in unique sequences. Single-stranded loops are formed on duplexes containing mismatched (different) numbers of tandem repeats. No single stranded loops are formed on structures containing matched (identical) numbers of tandem repeats. The matched and mismatched loop structures were distinguished and differentially labeled by enzymatic treatment with T4 endonuclease VII.
Subject(s)
Bacteriophage T4/enzymology , Endodeoxyribonucleases/metabolism , Sequence Analysis, DNA/methods , Tandem Repeat Sequences/genetics , Genetic Markers , Models, Biological , Oligonucleotide ProbesABSTRACT
A simple method for scoring short tandem DNA repeats is presented. An oligonucleotide target, containing tandem repeats embedded in a unique sequence, was hybridized to a set of complementary probes, containing tandem repeats known lengths. Single-stranded loops structures formed on duplexes containing a mismatched (different) number of tandem repeats. No loop structure formed on duplexes containing a matched (identical) number of tandem repeats. The matched and mismatched loop structures were enzymatically distinguished and differentially labeled by treatment with S1 nuclease and the Klenow fragment of DNA polymerase.
