Article Figures & Data
Figures
- Fig. 1.
Single AP-like voltage commands cause exocytoticCm changes in mouse chromaffin cells in slices. A (top), Shape of a representative mouse chromaffin cell AP measured in current clamp with a potassium-based pipette solution (dashed line). Thesolid line shows the simulated AP-like voltage command used to study secretory responses to single APs. A, (bottom), A typical current response to the AP-like command, with K+ currents blocked by a Cs+-containing pipette solution (solution A) and d-tubocurarine in the extracellular saline (solution 1).B, Representative Cmmeasurement before and after application of a simulated chromaffin cell AP from a holding potential of −80 mV. The top tracedisplays the AP-induced Cm change. After an initial decay (ΔCt) (for more detail, see Results), a stable Cm increment (ΔCexo) remains. The asymptote of the exponential fit to ΔCm was used to quantify the ΔCexo evoked by the individual APs (approach 1 in Materials and Methods).Middle and bottom traces, Membrane conductance (Gm) and series resistance (Gs) are shown to illustrate that there was no major cross-talk among Cm,Gm, and Gsestimates.
- Fig. 2.
Exocytotic Cm changes in response to short depolarizations are restricted to the time during the stimulation. Starting 30–60 sec after whole-cell recording, cells were depolarized for different durations 7–15 times, with an interpulse interval of 30 sec (depolarizing potential 0 mV). Solution 1 was used as external saline. [Ca2+]i was buffered to 300 nm (pipette solution B or C) by mixing Ca2+-free and Ca2+-loaded buffers to accelerate the vesicle replenishment (von Rüden and Neher, 1993). The holding potential was −80 mV. A (top), Typical capacitance changes in response to a 5 msec depolarization early (top trace: high release probability, 1 response) and late (bottom trace: after exhaustion of secretion, 20 responses averaged) in the experiment. The numbered shaded areas indicate the time periods over which ΔCm was averaged for the estimation of ΔCexo by approaches 1–3, as described in Materials and Methods. For this particular cell, the three approaches estimated ΔCexo with 27.8 and 27 fF (average over period 1 and asymptote of the exponential fit, respectively: approach 1), 24.4 fF (average ΔCm of period 2: 35.9 and 11.5 fF for the early trace and the average of late traces, respectively, approach 2), and 22.9 fF (average ΔCm of period 3: 49.5 fF; correction term: 8.3 fF/nA × 3.2 nA sodium current, approach 3). The bottom of A displays the same early ΔCm trace as above after subtraction of the exponential fit to the average of the late traces.B, Cm traces depicting ΔCexo of another slice cell in response to depolarizations of 2, 30, 100, and 200 msec duration (frombottom to top) recorded at comparable experimental times. As in the bottom of A, an exponential fit to the average ΔCm in response to 5 msec depolarizations at low release probability was subtracted from each trace. Both the 2 and the 30 msec responses do not exhibit increases in Cm after the end of the depolarization, indicating that ΔCexo is synchronized to Ca2+ entry for short pulses; however, longer depolarizations caused some secretion after the end of Ca2+ entry.
- Fig. 3.
Mouse chromaffin cells in slices show a biphasic rise of ΔCexo with increasing duration of Ca2+ current injection. A(top), The filled circles represent pooled data of 10 slice cells with ΔCexoestimated by approach 2 in Materials and Methods. For convenience these data were fitted by a double exponential (solid line). The slow secretory component, however, did not saturate with our maximal stimuli. The empty squares plot the ΔCexo versus pulse duration data of 20 isolated mouse chromaffin cells. No clear separation into two secretory components was observed. Experiments were carried out as described above (pipette solution B), except that an external [Ca2+] of 10 mm was used. The ΔCexo versus pulse duration data obtained from isolated rat chromaffin cells by Horrigan and Bookman (1994) are displayed for comparison (diamonds).Bottom, A plot of QCa versus pulse duration demonstrates that for short depolarizations,QCa rises linearly with increasing pulse duration for both preparations. Furthermore, it shows that theQCa values of slice (filled circles) and isolated (empty squares) cells more or less overlap for short pulses. B, The same ΔCexo data as in Figure 3Awere related to their corresponding Ca2+ current integrals (QCa). The filled circlesrepresent pooled and binned data from slice experiments. The data for isolated mouse cells are displayed as empty circles. Note that the ΔCexo–QCarelation is displayed only for small QCavalues (where a difference between both preparations was observed). Thenumbers of data points per bin are printedabove and below the graph for slice and isolated chromaffin cells, respectively. The vertical bars are SEM for ΔCexo values, whereas the horizontal bars indicate SD of binnedQCa values. C, The different lines represent ΔCexo data from the same experiments in slices as those analyzed in Figure 3A, estimated by three different approaches (see Materials and Methods and Fig. 2A). The dashed line(approach 3 in Materials and Methods) represents early ΔCexo estimates, whereas the solid line (approach 2) and the dotted line (approach 1) result from later measurements after the pulse. This figure demonstrates that all three approaches give quite similar ΔCexo estimates at short depolarizations. The discrepancy between the estimates at 200 and 300 msec pulse durations is most likely attributable to some “post-pulse” secretion.
- Fig. 4.
Secretory depression of slice cells is obtained with a pair of 20 msec depolarizations. This figure shows a representative ΔCm trace in response to a pair of 20 msec depolarizations (to −6 and 0 mV respectively; seetop panel for illustration of the voltage-clamp protocol). Pipette solution B and external solution 1 were used. The sum response (S) to both stimuli was measured as the asymptote of an exponential fit (solid line) to ΔCm after the second depolarization. The nonexocytotic ΔCt makes direct separate measurement of the exocytotic responses to the first and second depolarization (ΔCexo1 and ΔCexo2) difficult. Here, ΔCexo2 was estimated as the difference of ΔCm averages over the initial 300 msec after the second and the first depolarization, respectively. ΔCexo1 was then calculated asS − ΔCexo2. This analysis relies on the assumption that the nonsecretory transient (ΔCt) is the same after the first and the second depolarization. This is reasonable, because ΔCt saturates for depolarizations as short as 5 msec (Horrigan and Bookman, 1994). For illustration, the exponential fit to the ΔCm trace after the second depolarization in addition was overlayed onto the ΔCm segment after the first pulse (dashed line).
- Fig. 6.
Recovery time course of the fast secretory component in mouse chromaffin cells in slices. The ratio of ΔCexo caused by two identical 20 msec depolarizations (ratio of the second over the first ΔCexo) is plotted versus the separation times of the two stimuli. Intervals of 30 sec or more were allowed between the pairs of stimuli for complete recovery of the fast secretory component. The filled triangles represent ratios of responses to accordingly separated, individual 20 msec depolarizations to 0 mV. The data were obtained in five cells from two preparations (pipette solution B, external solution 1). ΔCexo was estimated by approach 1 in Materials and Methods. The filled circles represent data from a different set of eight cells in which we applied pairs of dual pulses at varying separation times (configuration of the dual pulses as described in Fig. 4, pipette solution B, external solution 1). ΔCexo1 of the second and the first dual pulses (estimated as described in Fig. 4) were related to each other. The fit to the pooled ratios from both sets of experiments revealed a recovery time constant of 10 sec. Back-extrapolation to 0 sec separation time indicates that the maximum depletion by the first stimulus was 80% (empty square). For comparison, thestar symbol represents the mean ratio ΔCexo2 over ΔCexo1 within a dual pulse (300 msec separation, 40% depletion) determined for the same experiments.
- Fig. 5.
The fast secretory component in mouse chromaffin cells in slices is strongly reduced by BAPTA but much less by EGTA. Experiments were performed similarly as described in Figure 2. Stimulation was started ∼60 sec after the whole-cell configuration was established. ΔCexo values were estimated by approach 2 in Materials and Methods. For both pipette solutions (D and E), [Ca2+] was adjusted at ∼300 nm by mixing Ca2+-free and Ca2+-loaded buffers. The holding potential was −80 mV.A, ΔCexo versus pulse duration plot of pooled data from experiments with either 1 mm free EGTA (filled circles;n = 10 cells; solution D) or 1 mm free BAPTA (empty circles; n = 10 cells; solution E). Experiments were carried out in the same slice preparations for both conditions (five different preparations). In the BAPTA-buffered cells, short depolarizations caused much less secretion than in those dialyzed with EGTA. With longer pulses, however, the secretory responses under both buffering conditions were comparable or even larger with BAPTA (possibly because of the lessening of Ca2+ channel inactivation). B, The same ΔCexo data as in A were plotted against their corresponding Ca2+ current integrals (QCa). The raw data were arbitrarily binned;vertical bars are SEM of ΔCexo, and horizontal barsare SD of QCa. The right-most BAPTA point indicates that the higher ΔCexo values at longer pulse durations were at least partly attributable to larger Ca2+ currents in the BAPTA-buffered cells.
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