Figure 2.

Electrical Activity and [Ca²⁺]_i in Cultured Colonic L Cells

(A) Colonic epithelial cells fixed after 10 days in primary culture. DIC, DAPI (blue), Venus (green).

(B) Glucose-triggered electrical activity in L cells. (Left) Representative whole-cell perforated patch current clamp recording of a Venus-positive cell stimulated with glucose (10 mM) applied as indicated. (Right) Average action potential frequency before (C1), during (Gluc), and after (C2) application of glucose measured in eight cells. Error bars represent 1 SE and significance, between AP-frequency in the presence and absence of glucose was tested by Student's paired t test. ^∗∗p < 0.01.

(C) Average action potential frequency before (C1), during (tolb), and after (C2) application of tolbutamide (500 μM) measured in nine cells, as in (B). Error bars represent 1 SE, and significance between AP-frequency in the presence and absence of tolbutamide was tested by Student's paired t test. ^∗∗p < 0.01.

(D) Functional K_ATP wash-out currents in cultured L cells. (Left) Slope conductances between −70 and −50 mV were recorded from a cell in conventional whole-cell voltage clamp. The arrow indicates the time when the cell attached mode was converted into conventional whole cell with 300 μM ATP in the pipette. Tolbutamide (tolb, 500 μM) was applied at steady state, as indicated. (Right) Average conductances measured as exemplified on the left in five cells at times (a) cell attached, (b) wash-out steady state, and (c) after application of tolbutamide. Error bars represent 1 SE, and significance was tested by Student's paired t test. ^∗∗∗p < 0.001.

(E) L_pos and L_neg cells in colonic cultures were loaded with fura2-AM and identified by their presence/absence of Venus fluorescence (475 nm excitation, left). The image of fura2-loaded cells excited at 340 nm (right) was used to outline L_pos (green) and L_neg (red) cells.

(F) The 340/380 nm fluorescence ratios (reflecting [Ca²⁺]_i) of the two cells outlined in (E) are shown following addition of 10 mM glucose to the perfusate. Green trace, L_pos cell; red trace, L_neg cell.

(G) Mean calcium changes in L_pos (green bars) and L_neg (red bars) cells, identified and monitored as in (E) and (F), following the addition of glucose (Gluc, 10 mM), αMG (10 mM), tolbutamide (tolb, 100 μM), KCl (30 mM), sucralose (sucl, 1 or 20 mM), bombesin (Bb, 100 nM), and forskolin/IBMX (F/I, 10 μM of each), as indicated. 340/380 ratios in the presence of the test agent were normalized to the mean of the background ratios of each cell measured before addition and after washout of the test compound. Data represent the mean and SE of the number of cells indicated above each bar. Δp < 0.05, ΔΔp < 0.01, and ΔΔΔp < 0.001 compared with baseline. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 for comparison between corresponding L_pos and L_neg cells by Student's t test.