Hassenklöver T , Pallesen LP , Schild D , Manzini I .

	Figure 2. Response profiles of ORNs to amino acid and peptide stimulation.(A) Relative number of amino acid-sensitive ORNs reacting to individual amino acids (200 µM) or at least to one of the thirteen tested peptides. Only a fraction of amino acid-responsive ORNs also responded to group I peptides (1 mM, 12 of 42 ORNs in four slices) or group II peptides (200 µM, 6 of 28 ORNs in four slices). The fraction of ORNs sensitive to group I peptides did not differ from the fraction of ORNs sensitive to group II peptides. (B) Response matrix of all peptide-sensitive ORNs to the applied stimuli (green, response to applied stimulus; red, no response; grey, not tested; applied peptide concentration: ORN #1–#12, 1 mM; ORN #13–#21, 5 mM; ORN #22–#24, 10 mM; ORN #25–#31, 200 µM). [AA mix: amino acid mixture, AA: amino acids, Arg: L-arginine, Met: L-methionine, Lys: L-lysine, Gly: glycine, Pep I: group I peptides, Pep II: group II peptides].
	Figure 3. Peptide stimulation evokes calcium transients with lower maximum amplitude than stimulation with amino acids.(A) The maximum amplitude of [Ca2+]i increases upon peptide application (green, group I, 1 mM; orange, group II, 200 µM) is much lower than upon application of amino acids (200 µM; number of responses averaged: L-arginyl-L-methionine (Arg-Met), 2; L-arginyl-L-methionyl-L-arginine (Arg-Met-Arg), 4; L-methionyl-L-arginyl-L-methionine (Met-Arg-Met), 9; L-methionyl-L-arginine (Met-Arg), 9; L-arginyl-L-lysine (Arg-Lys), 4; L-arginyl-L-lysyl-L-arginine (Arg-Lys-Arg), 7; L-lysyl-L-arginyl-L-lysine (Lys-Arg-Lys), 7; L-lysyl-L-arginine (Lys-Arg), 2; out of 12 ORNs, four OE slices; L-arginyl-glycine (Arg-Gly), 10; glycyl-L-arginine (Gly-Arg), 4; L-methionyl-glycine (Met-Gly), 4; glycyl-glycine (Gly-Gly), 4; glycyl-glycyl-glycine (Gly-Gly-Gly), 2; out of six ORNs, four OE slices). (B) Of the five group II peptides only the dipeptide L-arginyl-glycine (Arg-Gly) featured a stimulus-induced maximum amplitude of [Ca2+]i increases comparable to stimulation with L-arginine (only ORNs exclusively sensitive to the amino acid L-arginine, i.e. #27–#30 taken into account). In contrast, the dipeptide glycyl-L-arginine (Gly-Arg) showed a weak response (averaging of multiple applications of glycyl-L-arginine (Gly-Arg); *, p<0.05; **, p<0.001, paired t-test, error bars represent standard deviation). [AA: amino acids, Arg: L-arginine].
	Figure 4. Group I and group II peptides elicit significantly different [Ca2+]i transients in individual olfactory receptor neurons.(A) The mean time points of amino acid- and peptide-evoked calcium transient maxima varied for individual stimuli. Transients evoked by group I peptides show a tendency to reach their maximum amplitude later if compared to amino acid stimulations (green, group I peptides, 1 mM; number of responses averaged: AA mix, 67; L-arginine (Arg), 10; L-methionine (Met), 11; L-lysine (Lys), 6; L-arginyl-L-methionine (Arg-Met), 3; L-arginyl-L-methionyl-L-arginine (Arg-Met-Arg), 4; L-methionyl-L-arginyl-L-methionine (Met-Arg-Met), 9; L-methionyl-L-arginine (Met-Arg), 9; L-arginyl-L-lysine (Arg-Lys), 4; L-arginyl-L-lysyl-L-arginine (Arg-Lys-Arg), 7; L-lysyl-L-arginyl-L-lysine (Lys-Arg-Lys), 7; L-lysyl-L-arginine (Lys-Arg), 2; out of 12 ORNs, four OE slices; orange, group II peptides, 200 µM; number of responses averaged: L-arginine (Arg), 7; L-methionine (Met), 3; glycine (Gly), 3; L-arginyl-glycine (Arg-Gly), 10; glycyl-L-arginine (Gly-Arg), 4; L-methionyl-glycine (Met-Gly), 4; glycyl-glycine (Gly-Gly), 4; glycyl-glycyl-glycine (Gly-Gly-Gly), 2; out of six ORNs, four OE slices). (B) A combined analysis reveals that calcium transients evoked by applications of group I peptides show a significant delay of their maximum amplitude if compared to responses to the mixture of amino acids. In contrast, response maxima evoked by group II petides are not significantly shifted in comparison to amino acid controls. Even more clearly, response maxima evoked by L-arginyl-glycine (Arg-Gly) are not shifted if compared to maxima evoked by L-arginine in L-arginine-specific ORNs (not responsive to the other two amino acids L-methionine and glycine). Bars indicate standard deviation and error bars represent the standard error of the mean (*, p<0.0001; unpaired t-test, number of evaluated responses for the first group, AA mix: 67 responses, Pep I: 45 responses, 12 cells, four OE slices; for the second group, AA: 12 responses, Pep II: 25 responses, six cells, four OE slices; and for exclusively L-Arginine positive ORNs, Arg: four responses, Arg-Gly: six responses). (C) Typical responses upon application of amino acids and group I peptides. The maximum amplitude of [Ca2+]i transients induced by group I peptides is smaller and shows a significant delay in comparison to [Ca2+]i transients induced by amino acids. Circles and dotted lines indicate the maximum amplitude of each response. AA mix (200 µM, blue), L-arginyl-L-lysine (Arg-Lys; 1 mM, dark green), L-methionyl-L-arginyl-L-methionine (Met-Arg-Met; 1 mM, green), L-methionyl-L-arginine (Met-Arg; 1 mM, light-green). The odorant application is marked by a grey bar. (D) Representative example of [Ca2+]i transients of an ORN sensitive to L-arginine (200 µM, blue), L-arginyl-glycine (Arg-Gly; 200 µM, orange) and glycyl-L-arginine (Gly-Arg; 200 µM, light-orange). Calcium signals evoked by L-arginyl-glycine showed the highest mean maximum amplitude of all tested peptides. In both peptide responses, the maximum amplitude is not shifted in comparison to the arginine application. [AA mix: amino acid mixture, AA: amino acids, Arg: L-arginine, Met: L-methionine, Lys: L-lysine, Gly: glycine, Pep I: group I peptides, Pep II: group II peptides].
	Figure 1. Amino acid- and peptide-induced changes in calcium-dependent fluorescence of individual ORNs in slices of the olfactory epithelium.(A) Slice preparation of the OE of larval Xenopus laevis stained with Fluo-4 AM. The colored ovals (#1–#8) indicate the eight ORNs that were responsive to the mixture of amino acids. (B) Time courses of [Ca2+]i transients of the eight ORNs marked in A, elicited by application of amino acids (L-arginine, L-methionine and L-lysine as a mixture or singularly; each at a concentration of 200 µM) and peptides (consisting of L-arginine, L-methionine and L-lysine; 200 µM and 1 mM). Discernible peptide induced [Ca2+]i transients are marked by an asterisk. To check for ORN viability, the mixture of amino acids was applied at the end of the experiment. (C) Examples of peptide induced calcium transients originating from different ORNs (group I peptides, green, L-arginyl-L-methionine (Arg-Met), 5 mM; L-arginyl-L-methionyl-L-arginine (Arg-Met-Arg), 1 mM; L-methionyl-L-arginyl-L-methionine (Met-Arg-Met), 1 mM; L-methionyl-L-arginine (Met-Arg), 5 mM; L-arginyl-L-lysine (Arg-Lys), 200 µM; L-lysyl-L-arginine (Lys-Arg), 1 mM; L-arginyl-L-lysyl-L-arginine (Arg-Lys-Arg), 1 mM; L-lysyl-L-arginyl-L-lysine (Lys-Arg-Lys), 1 mM;; group II peptides (see Material and Methods), orange, all applied at 200 µM). As reference also the highest amino acid-induced (200 µM) calcium transient is depicted. [AA mix: amino acid mixture].

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