Interaction of neuronal nitric oxide synthase with alpha(1)-adrenergic receptor subtypes in transfected HEK-293 cells

BACKGROUND: The C-terminal four amino acids (GEEV) of human α(1A)-adrenergic receptors (ARs) have been reported to interact with the PDZ domain of neuronal nitric oxide synthase (nNOS) in a yeast two-hybrid system. The other two α(1)-AR subtypes have no sequence homology in this region, raising the possibility of subtype-specific protein-protein interactions. RESULTS: We used co-immunoprecipitation and functional approaches with epitope-tagged α(1)-ARs to examine this interaction and the importance of the C-terminal tail. Following co-transfection of HEK-293 cells with hexahistidine/Flag (HF)-tagged α(1A)-ARs and nNOS, membranes were solubilized and immunoprecipitated with anti-FLAG affinity resin or anti-nNOS antibodies. Immunoprecipitation of HFα(1A)-ARs resulted in co-immunoprecipitation of nNOS and vice versa, confirming that these proteins interact. However, nNOS also co-immunoprecipitated with HFα(1B)- and HFα(1D)-ARs, suggesting that the interaction is not specific to the α(1A) subtype. In addition, nNOS co-immunoprecipitated with each of the three HFα(1)-AR subtypes which had been C-terminally truncated, suggesting that this interaction does not require the C-tails; and with Flag-tagged β(1)- and β(2)-ARs. Treatment of PC12 cells expressing HFα(1A)-ARs with an inhibitor of nitric oxide formation did not alter norepinephrine-mediated activation of mitogen activated protein kinases, suggesting nNOS is not involved in this response. CONCLUSIONS: These results show that nNOS does interact with full-length α(1A)-ARs, but that this interaction is not subtype-specific and does not require the C-terminal tail, raising questions about its functional significance

Immunogold electron microscopic evidence of in situ formation of homo- and heteromeric purinergic adenosine A1 and P2Y2 receptors in rat brain

<p>Abstract</p> <p>Background</p> <p>Purines such as adenosine and ATP are now generally recognized as the regulators of many physiological functions, such as neurotransmission, pain, cardiac function, and immune responses. Purines exert their functions via purinergic receptors, which are divided into adenosine and P2 receptors. Recently, we demonstrated that the G_i/o-coupled adenosine A₁receptor (A₁R) and G_q/11-coupled P2Y₂receptor (P2Y₂R) form a heteromeric complex with unique pharmacology in co-transfected human embryonic kidney cells (HEK293T). However, the heteromeric interaction of A₁R and P2Y₂R <it>in situ </it>in brain is still largely unknown.</p> <p>Findings</p> <p>In the present study, we visualized the surface expression and co-localization of A₁R and P2Y₂R in both transfected HEK293T cells and in rat brain by confocal microscopy and more precisely by immunogold electron microscopy. Immunogold electron microscopy showed the evidence for the existence of homo- and hetero-dimers among A₁R and P2Y₂R at the neurons in cortex, cerebellum, and particularly cerebellar Purkinje cells, also supported by co-immunoprecipitation study.</p> <p>Conclusion</p> <p>The results suggest that evidence for the existence of homo- and hetero-dimers of A₁R and P2Y₂R, not only in co-transfected cultured cells, but also <it>in situ </it>on the surface of neurons in various brain regions. While the homo-dimerization ratios displayed similar patterns in all three regions, the rates of hetero-dimerization were prominent in hippocampal pyramidal cells among the three regions.</p

Receptor Activation and Inositol Lipid Hydrolysis in Neural Tissues

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66228/1/j.1471-4159.1987.tb05618.x.pd

Development of CALUX bioassay-based systems as instruments to detect hormones and contaminants

α-Adrenoceptors mediate contractile responses in equine digital veins (EDVs) and arteries. Vascular smooth muscle

Association between adolescent idiopathic scoliosis prevalence and age at menarche in different geographic latitudes

BACKGROUND: Age at menarche is considered a reliable prognostic factor for idiopathic scoliosis and varies in different geographic latitudes. Adolescent idiopathic scoliosis prevalence has also been reported to be different in various latitudes and demonstrates higher values in northern countries. A study on epidemiological reports from the literature was conducted to investigate a possible association between prevalence of adolescent idiopathic scoliosis and age at menarche among normal girls in various geographic latitudes. An attempt is also made to implicate a possible role of melatonin in the above association. MATERIAL-METHODS: 20 peer-reviewed published papers reporting adolescent idiopathic scoliosis prevalence and 33 peer-reviewed papers reporting age at menarche in normal girls from most geographic areas of the northern hemisphere were retrieved from the literature. The geographic latitude of each centre where a particular study was originated was documented. The statistical analysis included regression of the adolescent idiopathic scoliosis prevalence and age at menarche by latitude. RESULTS: The regression of prevalence of adolescent idiopathic scoliosis and age at menarche by latitude is statistically significant (p < 0.001) and are following a parallel declining course of their regression curves, especially in latitudes northern than 25 degrees. CONCLUSION: Late age at menarche is parallel with higher prevalence of adolescent idiopathic scoliosis. Pubarche appears later in girls that live in northern latitudes and thus prolongs the period of spine vulnerability while other pre-existing or aetiological factors are contributing to the development of adolescent idiopathic scoliosis. A possible role of geography in the pathogenesis of idiopathic scoliosis is discussed, as it appears that latitude which differentiates the sunlight influences melatonin secretion and modifies age at menarche, which is associated to the prevalence of idiopathic scoliosis

Multidisciplinary Consideration of Potential Pathophysiologic Mechanisms of Paradoxical Erythema with Topical Brimonidine Therapy

Rosacea is a chronic inflammatory disease with transient and non-transient redness as key characteristics. Brimonidine is a selective α2-adrenergic receptor (AR) agonist approved for persistent facial erythema of rosacea based on significant efficacy and good safety data. The majority of patients treated with brimonidine report a benefit; however, there have been sporadic reports of worsening erythema after the initial response. A group of dermatologists, receptor physiology, and neuroimmunology scientists met to explore potential mechanisms contributing to side effects as well as differences in efficacy. We propose the following could contribute to erythema after application: (1) local inflammation and perivascular inflammatory cells with abnormally functioning ARs may lead to vasodilatation; (2) abnormal saturation and cells expressing different AR subtypes with varying ligand affinity; (3) barrier dysfunction and increased skin concentrations of brimonidine with increased actions at endothelial and presynaptic receptors, resulting in increased vasodilation; and (4) genetic predisposition and receptor polymorphism(s) leading to different smooth muscle responses. Approximately 80% of patients treated with brimonidine experience a significant improvement without erythema worsening as an adverse event. Attention to optimizing skin barrier function, setting patient expectations, and strategies to minimize potential problems may possibly reduce further the number of patients who experience side effects. Funding: Galderma International S.A.S., Paris, France

Managing a Trois: a study of a multi-user drawing tool in distributed design work

A multi-user drawing tool was used by participants in a distributed design exercise conducted in a multi-media working environment. The goal of the study was to explore how observations from our earlier studies of shared drawing in two-person design activity would hold up when three participants worked together. Additionally, the study provided opportunities to contrast video/audio connections with audio-only connections and to discover new behaviours that emerge in the use of new technologies. Participants successfully used the shared drawing system with no observed difficulties attributable to the addition of a third user. Audio-only connections appeared to adequately suppofi this work activity, but details of the participants'interactions in the exercise raised questions that deserve further study. Finally, observations suggest that drawing tools such as the one reported here may offer support for alternative forms of participation in collaborative work