Binding of the peptide hormone angiotensin II (AngII) to the type 1 (AT 1A ) receptor and the subsequent activation of phospholipase C-mediated signaling, involves specific determinants within the AngII peptide sequence. In contrast, the contribution of such determinants to AT 1A receptor internalization, phosphorylation and activation of mitogen-activated protein kinase (MAPK) signaling is not known. In this study, the internalization of an enhanced green fluorescent protein-tagged AT 1A receptor (AT 1A -EGFP), in response to AngII and a series of substituted analogs, was visualized and quantified using confocal microscopy. AngII-stimulation resulted in a rapid, concentration-dependent internalization of the chimeric receptor, which was prevented by pretreatment with the nonpeptide AT 1 receptor antagonist EXP3174. Remarkably, AT 1A receptor internalization was unaffected by substitution of AngII side chains, including single and double substitutions of Tyr 4 and Phe 8 that abolish phospholipase C signaling through the receptor. AngII-induced receptor phosphorylation was significantly inhibited by several substitutions at Phe 8 as well as alanine replacement of Asp 1 . The activation of MAPK was only significantly inhibited by substitutions at position eight in the peptide and specific substitutions did not equally inhibit inositol phosphate production, receptor phosphorylation and MAPK activation. These results indicate that separate, yet overlapping, contacts made between the AngII peptide and the AT 1A receptor select/induce distinct receptor conformations that preferentially affect particular receptor outcomes. The requirements for AT 1A receptor internalization seem to be less stringent than receptor activation and signaling, suggesting an inherent bias toward receptor deactivation.

Background: Receptors activate channels of sensory nerves to cause inflammation and pain by unknown mechanisms. Results: Protease-activated receptor 2 (PAR 2 ) stimulated transient receptor potential vanilloid 4 (TRPV4) by generation of channel agonists. This required a key TRPV4 tyrosine and induced inflammation. Conclusion: PAR 2 opens TRPV4 by functional coupling. Significance: Antagonism of PAR 2 -TRPV4 coupling could alleviate inflammation and pain.

No abstract

The structure-function relationships of the N-type calcium channel blocker, -conotoxin GVIA (GVIA), have been elucidated by structural, binding and in vitro and in vivo functional studies of alanine-substituted analogues of the native molecule. Alanine was substituted at all non-bridging positions in the sequence. In most cases the structure of the analogues in aqueous solution was shown to be native-like by 1 H NMR spectroscopy. Minor conformational changes observed in some cases were characterized by two-dimensional NMR. Replacement of Lys 2 and Tyr 13 with Ala caused reductions in potency of more than 2 orders of magnitude in three functional assays (sympathetic nerve stimulation of rat isolated vas deferens, right atrium and mesenteric artery) and a rat brain membrane binding assay. Replacement of several other residues with Ala (particularly Arg 17 , Tyr 22 and Lys 24 ) resulted in significant reductions in potency (<100-fold) in the functional assays, but not the binding assay. The potencies of the analogues were strongly correlated between the different functional assays but not between the functional assays and the binding assay. Thus, the physiologically relevant assays employed in this study have shown that the high affinity of GVIA for the N-type calcium channel is the result of interactions between the channel binding site and the toxin at more sites than the previously identified Lys 2 and Tyr 13 .The fish-hunting marine cone snails produce a range of polypeptide toxins that rapidly immobilize their prey (1, 2). A number of such toxins targeted at ion channels have been isolated from the venoms of these cone shells, including several that selectively block N-and P-type calcium channels (3). The toxin studied here, -conotoxin GVIA (GVIA), 1 is a 27-residue polypeptide from Conus geographus (4) that selectively blocks N-type voltage-gated calcium channels (5, 6), an activity that may confer a number of useful therapeutic properties on GVIA, including antihypertensive, analgesic, and neuroprotective activities, as demonstrated for the closely related -conotoxin MVIIA (MVIIA) from Conus magus (7) .The amino acid sequence of GVIA (with the cystine bridges indicated by lines) is as show below.

No abstract

No abstract

Background and purpose: This paper is intended to assist pharmacologists to make the most of statistical analysis and in avoid common errors. Approach: A scenario is presented where an experimenter performed an experiment to test the effects of two drugs on cultured cells. Analysis of the results, expressed as percentage of control, by a one-way ANOVA yielded P ¼ 0.058 and the experimenter concluded that neither drug was effective. The data were expressed as percentage of control because of pairing of the data within each experimental run, a common feature in cell culture experiments. Such data can be analysed with potentially more powerful ANOVA methods equivalent to the paired t-test. Monte Carlo simulations are presented to compare the power of relevant analyses. Results: For data correlated within experimental run (i.e. paired values), transformation to percentage of control improved the power of a one-way ANOVA to detect a real effect, but a randomized block ANOVA (equivalent to a 2-way ANOVA with experiment and treatment as factors) using the raw values was substantially more powerful. The randomized block ANOVA performed well even with uncorrelated data, being only marginally less powerful than the one-way ANOVA. Conclusions and implications: A randomized block ANOVA is far superior to the one-way ANOVA with correlated data, and with uncorrelated data it is only marginally less powerful. Thus where there is, or might reasonably be, such a correlation (e.g. relatedness among the data within a single experimental run, or within a multi-well culture plate, or within an animal, et cetera), use the more powerful randomized block ANOVA rather than one-way ANOVA.

The contributions of various functional groups to the pharmacophore of the N-type calcium-channel blocker, v-conotoxin GVIA (GVIA), have been investigated using structural and in-vitro functional studies of analogues substituted at one or two positions with non-native residues. In most cases the structure of the analogue was shown to be native-like by 1 H NMR spectroscopy. Minor conformational changes observed in some cases were characterized by two-dimensional NMR. Three functional assays (sympathetic nerve stimulation of rat isolated vas deferens, right atrium and mesenteric artery) were employed to monitor N-type calcium-channel activity. The data provide a more detailed picture of the roles in GVIA structure and activity of the crucial Lys2 and Tyr13, as well as all other positively charged residues, Tyr22, the hydroxyproline residues and the C-terminal amido moiety, many of which were identified as being important for activity in an alanine scan [Lew et al. (1997) J. Biol. Chem. 272, 12014± 12023]. Substitutions of Lys2 with nonstandard amino acids and arginine quantified the roles of the length and charge of the Lys side chain. The orientation of the Tyr13 side chain and its hydroxyl moiety was shown to be important by substitution with d-Tyr and the d-form and l-form of the constrained analogue 7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid [Tic(OH)]. The roles of the Hyp10 and Hyp21 hydroxyl groups, investigated by proline substitutions, appear to be more structural (as monitored by NMR) than functional, although small decreases in potency were observed in some assays. The reversibility of the channel blockade was also studied, and several analogues with faster wash-out characteristics than native GVIA were identified. Rapid reversibility (as in the case of v-conotoxin MVIIA) may be beneficial for therapeutic applications. Disubstituted analogues revealed some interesting cooperative effects, which were not predicted from single-residue substitutions. A disubstituted chimera of GVIA and v-conotoxin MVIIA was more potent than either native molecule. The more detailed description of the GVIA pharmacophore obtained here provides a better basis for the future design of truncated peptide and peptidomimetic analogues.Keywords: calcium channel; conotoxin; NMR; peptide synthesis; pharmacophore. Based on functional bioassays of alanine-substituted analogues, we have identified a number of residues that are necessary for the calcium-channel blocking activity of GVIA [13]. This group of residues, referred to as the pharmacophore, included several that were not identified by Kim et al. [14] as being important for channel binding. It consists of two discontinuous patches, the first (and more important) of which includes Lys2, Tyr13 and Arg17, and the second Tyr22, Lys24, Hyp10, Hyp21 and Arg25. Our previous study also compared the results of three functional assays (rat isolated vas deferens, right atrium and small mesenteric artery) with those of membrane-binding assays. We found good correlations amon...