1,720,975 research outputs found
The 5-hydroxytryptamine(4a) receptor is palmitoylated at two different sites, and acylation is critically involved in regulation of receptor constitutive activity
No full textWe have reported recently that the mouse 5-hydroxytryptamine(4a) (5-HT4(a)) receptor undergoes dynamic palmitoylation (Ponimaskin, E. G., Schmidt, M. F., Heine, M., Bickmeyer, U., and Richter, D. W. (2001) Biochem. J. 353, 627-663). In the present study, conserved cysteine residues 328/329 in the carboxyl terminus of the 5-HT4(a) receptor were identified as potential acylation sites. In contrast to other palmitoylated G-protein-coupled receptors, the additional cysteine residue 386 positioned close to the COOH-terminal end of the receptor was also found to be palmitoylated. Using pulse and pulse-chase labeling techniques, we demonstrated that palmitoylation of individual cysteines is a reversible process and that agonist stimulation of the 5-HT4(a) receptor independently increases the rate of palmitate turnover for both acylation sites. Analysis of acylation-deficient mutants revealed that non-palmitoylated 5-HT4(a) receptors were indistinguishable from the wild type in their ability to interact with G(s), to stimulate the adenylyl cyclase activity and to activate cyclic nucleotide-sensitive cation channels after agonist stimulation. The most distinctive finding of the present study was the ability of palmitoylation to modulate the agonist-independent constitutive 5-HT4(a) receptor activity. We demonstrated that mutation of the proximal palmitoylation site (Cys(328) --> Ser/Cys(329) --> Ser) significantly increases the capacity of receptors to convert from the inactive (R) to the active (R ) form in the absence of agonist. In contrast, the rate of isomerization from R to R for the Cys(386) --> Ser as well as for the triple, nonpalmitoylated mutant (Cys(328) --> Ser/CyS329 --> Ser/Cys(386) -->Ser) was similar to that obtained for the wild type
5-HT-receptor-induced changes of the intracellular cAMP level monitored by a hyperpolarization-activated cation channel
No full textThe HvCNG channel from the moth Heliothis virescens is highly sensitive to cAMP concentrations ranging between 0.1 muM and 5 muM. This HvCNG channel was over-expressed in Spodoptera frugiperda (Sf.9) cells to measure endogenous cAMP levels. Hyperpolarization-activated inward currents were measured in the whole-cell patch-clamp configuration with pipettes filled with different cAMP concentrations to calibrate the system. Varying the cAMP concentration between 0 muM and 100 muM in the pipette. the half-maximal activation voltage (V-1/2) was shifted by +28.5+/-1.7 mV. The activation time constant (tau(a)) was used as a parameter for cAMP quantification because it was independent of the expression level of HvCNG channels. tau(a) changed from 1106+/-60 ms at 0 muM cAMP to 265+/-7 ms at a saturating concentration of 1 mM cAMP. A dose-response relationship yielded values of 0.6 muM for the half-maximal cAMP concentration and 1.5 for the Hill coefficient. Activation of endogenous adenylyl cyclases by 50 muM forskolin induced an elevation of the cAMP level by about 16+/-0.2 muM. Co-expressions of HvCNG channels in combination with the mouse 5-HT4(a)- or 5-HT1A-receptors and the corresponding G(s)- or G(i)-proteins were successful and allowed us to also verify receptor-induced changes of the cAMP level. Stimulation of m5-HT4(a)-receptors by 0.1 muM 5-HT induced an increase of cAMP of about 4.6+/-1.5 muM. whereas cAMP levels decreased from a control value of 1+/-0.2 muM to 0.41+/-0.1 muM after stimulation of the m5-HT1A-receptors
by 5‐HT receptors in mouse CA1 hippocampal neurons
No full textCA1 pyramidal neurons of the hippocampus express various types of serotonin (5-HT) receptors, such as 5-HT1A, 5-HT4 and 5-HT7 receptors, which couple to Galpha(i) or Galpha(s) proteins and operate on different intracellular signalling pathways. In the present paper we verify such differential serotonergic modulation for the hyperpolarization-activated current I-h . Activation of 5-HT1A receptors induced an augmentation of current-induced hyperpolarization responses, while the responses declined after 5-HT4 receptors were activated. The resting potential of neurons hyperpolarized (-2.3 +/- 0.7 mV) after 5-HT1A receptor activation, activation of 5-HT4 receptors depolarized neurons (+3.3 +/- 1.4 mV). Direct activation of adenylyl cyclase (AC) by forskolin also produced a depolarization. In voltage clamp, the I-h current was identified by its characteristic voltage- and time-dependency and by blockade with CsCl or ZD7288. Activation of 5-HT1A receptors reduced I-h and shifted the activation curve to a more negative voltage by -5 mV at half-maximal activation. Activation of 5-HT4 and 5-HT7 receptors increased I-h and shifted the activation curve to the right by +5 mV. Specific activation of 5-HT4 receptors by BIMU8 increased membrane conductance and showed an increase in I-h in a subset of cells, but did not induce a significant alteration in the activation curve. In order to verify spatial differences, we applied BIMU8 selectively to the soma and to the dendrites. Only somatic application induced receptor activation. These data are confirmed by immunofluorescence stainings with an antibody against the 5-HT4A receptor, revealing receptor expression at the somata of the CA1 region. A similar expression pattern was found with a new antibody against 5-HT7 receptors which reveals immunofluorescence staining on the cell bodies of pyramidal neurons
5-hydroxytryptamine 4(a) receptor expressed in Sf9 cells is palmitoylated in an agonist-dependent manner
No full textThe mouse 5-hydroxytryptamine 4(a) receptor [5-HT4(a)] was expressed with a baculovirus system in insect cells and analysed for acylation. [H-3]Palmitic acid was effectively incorporated into 5-HT4(a) and label was sensitive to the treatment with reducing agents indicating a thioester-type bond. Analysis of protein-bound fatty acids revealed that 5-HT4(a) contains predominantly palmitic acid. Treatment of infected Sf9 (Spodoptera frugiperda) cells with BIMU8 {(endo-N-8-methyl-8-azabicyclo[3.2.1]oct-3-yl)-2,3-dehydro-2-oxo-3-(prop-2-yl)- 1H-benzimid-azole-1-carboxamide), a 5-HT4 receptor-selective agonist, generated a dose-dependent increase in [H-3]palmitate incorporation into 5HT(4(a)) with an EC50 of approx. 10 nM. The change in receptor labelling after stimulation with agonist was receptor-specific and did not result from general metabolic effects.. We also used both pulse labelling and pulse-chase labelling to address the dynamics of 5-HT4(a) palmitoylation. Incorporation studies revealed that the rate of palmitate incorporation was increased approx. 3-fold after stimulation with agonist. Results of pulse-chase experiments show that activation with BIMU8 promoted the release of radiolabel from 5-HT4(a), thereby reducing the levels of receptor-bound palmitate to approximately one-half. Taken together. our results demonstrate that palmitoylation of 5-HT4(a) is a reversible process and that stimulation of 5-HT4(a) with agonist increases the turnover rate for receptor-bound palmitate. This provides evidence for a regulated cycling of receptor-bound palmitate and suggests a functional role for palmitoylation/depalmitoylation in 5-hydroxytryptamine-mediated signalling
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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