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. 2011 Apr 29;6(4):e19478. doi: 10.1371/journal.pone.0019478

Figure 4. Neuropeptide Y release monitored from INS-1 beta cells with the FMRFamide tagging technique.

Figure 4

(A). INS-1 cells that were transfected with GFP did not show any FMRFamide-ir. (B). INS-1 cells co-transfected with both GFP and the FMRFamide tagged prohormone were FMRFamide-ir. Scale bar 20 µm. (C). Example of secretory currents recorded from a cell that was transfected with the ionotropic FMRFamide receptor and the FMRFamide tagged NPY prohormone. Secretion was evoked with a train of 100 depolarizations at 5 Hz. Each depolarizing step lasted 20 ms and was from −80 to +20 mV. Sample traces (below; first 10 depolarizations) show that secretion was first detected following the fifth depolarization (red trace). (D). Secretory currents were not evoked from transfected cells when calcium was removed from the extracellular solution (and 2 mM EGTA was added). Secretion was evoked as in (C) except that the depolarization lasted 25 ms and was from −60 to +20 mV (n = 5 cells, mean ± SD). Sample recordings are shown above. (E). Secretory currents were consistently evoked from cells that were transfected with the FMRFamide receptor and a FMRFamide tagged NPY prohormone (“NPY.Fa”). In contrast no secretory events were observed in cells transfected with the FMRFamide receptor and a tagged prohormone in which the cleavage of FMRFamide was prevented (“NPY-Fa”). Cells were stimulated with the protocol described in (C). Sample recordings are shown above (n = 11–12 cells; mean ± SD). (F). Secretory events were evoked from cells co-transfected with NPY.Fa and the FMRFamide receptor (“+FaNaCh”) but were not evoked from cells transfected only with NPY.Fa (“−FaNaCh”). Cells were stimulated as in (C). Sample recordings are shown above (n = 6–9 cells, mean ± SD). (G). Application of exogenous 400 nM FMRFamide evoked an inward current only in cells that were transfected with both GFP and the ionotropic FMRFamide receptor (n = 4–5 cells; mean ± SD). Thus the secretory currents require the release of FMRFamide and INS-1 cells do not express an endogenous ionotropic FMRFamide receptor. (H). The mean amplitude of the secretory currents was lower in cells stimulated in the presence of 100 µM amiloride, a drug that inhibits the ionotropic FMRFamide receptor. Cells were stimulated with a train of 50 depolarizations using the protocol described in (D). Sample recordings are shown above (n = 5 cells, mean ± SD; n = 5 cells; P = 0.067, Mann-Whitney test).