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. 1998 Jan 1;18(1):81-92.
doi: 10.1523/JNEUROSCI.18-01-00081.1998.

The relation of exocytosis and rapid endocytosis to calcium entry evoked by short repetitive depolarizing pulses in rat melanotropic cells

H D Mansvelder  1 , K S Kits
Affiliations

The relation of exocytosis and rapid endocytosis to calcium entry evoked by short repetitive depolarizing pulses in rat melanotropic cells

H D Mansvelder et al. J Neurosci. .

Abstract

Melanotropic cells release predocked, large, dense-cored vesicles containing alpha-melanocyte stimulating hormone in response to calcium entry through voltage-gated calcium channels. Our first objective was to study the relationship between exocytosis, rapid endocytosis, and calcium entry evoked by short step depolarizations in the order of duration of single action potentials (APs). Exocytosis and rapid endocytosis were monitored by capacitance measurements. We show that short step depolarizations (40 msec) evoke the fast release of only approximately 3% of the predocked release-ready vesicle pool. Second, we asked what the distance is between voltage-gated calcium channels and predocked vesicles in these cells by modulating the intracellular buffer capacity. Exocytosis and rapid endocytosis were differentially affected by low concentrations of the calcium chelator EGTA. EGTA slightly attenuated exocytosis at 100 microM relative to 50 microM, but exocytosis was strongly depressed at 400 microM, showing that calcium ions have to travel a large distance to stimulate exocytosis. Nevertheless, the efficacy of calcium ions to stimulate exocytosis was constant for pulse durations between 2 and 40 msec, indicating that in melanotropes, exocytosis is related linearly to the amount and duration of calcium entry during a single AP. Rapid endocytosis was already strongly depressed at 100 microM EGTA, which shows that the process of endocytosis itself is calcium dependent in melanotropic cells. Furthermore, rapid endocytosis proceeded with a time constant of approximately 116 msec at 33 degrees C, which is three times faster than at room temperature. There was a strong correlation between the amplitude of endocytosis and the amplitude of exocytosis immediately preceding endocytosis. Both this correlation and the fast time constant of endocytosis suggest that the exocytotic vesicle is retrieved rapidly.

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Figures

Fig. 1.
Fig. 1.
Voltage command protocols applied to the cell to monitor ΔCm, ΔRac, and ΔGm and to evokeICa. A, Protocol used to acquire one sample of the capacitance trace (ΔCm), conductance trace (ΔRac and ΔGm), and membrane conductance trace (Gm). Ten cycles of a sinusoidal voltage command of 1200 Hz were averaged for one sample of ΔCm and the combined ΔRac and ΔGm. A 6 msec hyperpolarization yielded an independent measure of the cell conductance. The current response of the cell was filtered at 2 kHz with the low-pass Bessel filter on the amplifier, digitized, and analyzed on-line. B, Top trace, Voltage command trace (Vm) shown on a larger time scale. The sinusoidal was omitted for the sake of simplicity. Depolarizing pulses were applied every 500 msec from −80 to +10 mV.Middle traces, The response to the 25 step depolarizations. ΔCm, combined ΔRac and ΔGmtrace, and membrane conductance trace were obtained with the protocol in A. The Gm trace is a 5 points running average. Bottom traces, Peak calcium (ICa peak) current during the 40 msec step depolarization. Below, the full current traces are given for the 1st and 25th pulse. [EGTA]i = 200 μm.
Fig. 2.
Fig. 2.
Exocytosis does not persist after calcium entry has stopped. A, Average of responses to 25 step depolarizations ([EGTA]i = 200 μm).B, Average transient capacitance change in the presence of 100 μm Cd2+, to block the calcium influx (average response to 75 depolarizations of one cell). Solid line shows a single exponential fit to the decay of the average capacitance transient. Data were obtained from a cell different from that in A ([EGTA]i = 50 μm). The average of three such experiments on different cells was used to correct all quantifications of exocytosis and endocytosis throughout this study. There were no significant differences between the three cells, and they were all stimulated with 75 depolarizations. C, The solid line ofB is subtracted from the ΔCm trace in A, to give the ΔCm that is caused by exocytosis.
Fig. 3.
Fig. 3.
The decrease of ΔCmduring the pulse train can be attributed to a decrease of calcium influx. A, Average ΔCmresponse per pulse during the pulse train. The amount of ΔCm per pulse decreased during the pulse train. The pipette solution contained 100 μm EGTA (n = 13 cells). B, Same data as inA but now every capacitance jump is divided by the number of calcium ions that came into the cell during the step depolarization. The number of calcium ions is determined by integrating the current trace, as described in Materials and Methods. The efficacy of calcium ions to stimulate exocytosis showed almost no attenuation during the pulse train.
Fig. 4.
Fig. 4.
Exocytosis is affected by low concentrations of EGTA. A, Examples of responses to 25 step depolarizations with different EGTA concentrations as indicated next to the traces. For every experiment a new cell was taken. The noise in the 800 μm trace (SD of 1.72 fF) was somewhat lower than in the other two traces (SD of 2.26 fF at 200 μm), but the values are within the normal variation encountered in both groups [2.25 ± 0.60 fF at 800 μm and 2.24 ± 1.12 fF at 200 μm (both mean ± SD; P > 0.9)]. B, The average ΔCmper depolarization at different EGTA concentrations. C, Efficacy of calcium ions to stimulate exocytosis at different EGTA concentrations. Pairwise comparisons showed that the amount of exocytosis at 100 and 200 μm did not differ significantly; all other groups differed significantly from each other (p < 0.01). D, Cumulative capacitance change plotted versus the cumulative number of calcium ions that came into the cell during a pulse train. These curves showed almost straight lines with different slopes for the different EGTA concentrations. The numbers next to each curve represent the intracellular EGTA concentration in micromoles. For B, C, and D: 50 μm,n = 8; 100 μm, n= 13; 200 μm, n = 13; 400 μm, n = 10; 800 μm,n = 5.
Fig. 5.
Fig. 5.
Exocytosis is still possible with 50 and 200 μm intracellular BAPTA. A, Examples of responses on two different cells with BAPTA concentrations indicated.B, Average ΔCm per depolarization at the two different BAPTA concentrations.C, Efficacy of calcium ions to stimulate exocytosis per pulse. Pairwise comparisons showed that the efficacy at 50 μm BAPTA was not significantly different from the efficacy at 100 and 200 μm EGTA (p > 0.1) (Fig. 4C). The efficacy at 200 μm BAPTA was not significantly different from the efficacy at 400 μm EGTA (Fig.4C). D, Cumulative capacitance change plotted versus the cumulative number of calcium ions that came into the cell during a pulse train. These curves deviated slightly from straight lines, with a lower slope at later pulses; still, the different BAPTA concentrations clearly show different slopes. Thenumbers represent the intracellular BAPTA concentration in micromoles. For B, C, and D: 50 μm, n = 10; 200 μm,n = 5.
Fig. 6.
Fig. 6.
Rapid endocytosis is blocked by a lower concentration of EGTA than exocytosis. A, Examples of ΔCm responses of two different cells to 25 step depolarizations with 50 and 100 μm intracellular EGTA. At 100 μm intracellular EGTA, with each pulseCm increases, and only small decreases are seen after each pulse. At 50 μm EGTA,Cm decreases rapidly after each increase (see inset), so that the netCm increases only ∼50 fF. The combined ΔRac and ΔGmtraces and the Gm traces are depicted to show that there was no significant cross-talk between the traces. Thetop panels illustrate the method that was used to calculate the amount of exocytosis (Exo) and endocytosis (Endo). The same method was used for Figures 7 and 9.B, Average amount of Cm that is retrieved after each step depolarization. Rapid endocytosis is depressed at 100 μm EGTA. C, The efficacy of calcium ions to stimulate rapid endocytosis. Obviously, the efficacy is also depressed at 100 μm EGTA (p < 0.01). D, CumulativeCm that is retrieved during a pulse train versus the cumulative number of calcium ions that came into the cell during the pulse train. The slope of the 100 μm EGTA curve (labeled 100) is much lower than the slope at 50 μm (labeled 50). For B, C, and D, the data from the same cells as in Figure 4 were used.
Fig. 7.
Fig. 7.
Efficacy of calcium ions to stimulate endocytosis increases slowly during the pulse train. A, Average amplitude of rapid endocytosis per pulse number. Data from the cells as in Figures 4 and 6, with an intracellular EGTA concentration of 50 μm (n = 8). The amplitude of rapid endocytosis was measured as indicated in Materials and Methods. Thesolid line represents a fitted single exponential function with a time constant of 0.5 sec. B, Average efficacy of calcium ions to stimulate rapid endocytosis per pulse number. The efficacy increases during the pulse train, with a time constant of 2.3 sec (solid line).
Fig. 8.
Fig. 8.
The time constant of rapid endocytosis is constant during a pulse train. A single exponential function was fitted to the decay in membrane capacitance occurring immediately after the depolarizations (inset). Each decrease after a given pulse was fitted this way, and the time constants were averaged for each particular pulse number (n = 8 cells, each subjected to 1 pulse train). When a decrease could not be reliably fitted, it was left out of the average. As mentioned in the text, 127 of the 200 endocytotic responses could be reliably fitted with an exponential decay function. There was no relation found between the number of acceptable fits underlying each average and the number of the pulse in the pulse train. The plot shows the average time constant of membrane capacitance decrease as a function of pulse number.
Fig. 9.
Fig. 9.
Fast exocytosis and rapid endocytosis are strongly coupled. The amplitude of exocytosis and rapid endocytosis at the same pulse show a high correlation (Spearman’s ρ = 0.80;p < 0.01). Amplitude of exocytosis and endocytosis was determined as illustrated in Figure 6A. These values were corrected for changes in ΔCmnot related to exocytosis and endocytosis. The solid line represents y = x.
Fig. 10.
Fig. 10.
Exocytosis increases linearly with the amount of calcium influx at different pulse durations. The top traces (ΔCm) show an example of an increasing amount of ΔCm at trains of 15 pulses, with increasing pulse duration on the same cell. Pulse durations are indicated above thetraces. The amount of exocytosis and endocytosis per pulse at the 40 msec pulse duration are somewhat smaller than for the cells in Figure 6. This might be attributable to the fact that the cells used for the experiments in Figure 6 were subjected to only one pulse train, whereas the cells for these experiments were all stimulated with 5 pulse trains with different pulse durations.Middle traces (ICa) show the current traces at the 1st and 15th step depolarization at the different pulse durations. Bottom plots show the cumulative capacitance change versus the cumulative number of calcium ions that entered the cell during the step depolarizations at different pulse durations. Each data point represents the average of eight different cells. Note that the values at axes change for each plot, but that the ordinate and abscissa values change proportionally, to show that the slope is roughly constant for all pulse durations. [EGTA]i = 50 μm.
Fig. 11.
Fig. 11.
The efficacy of calcium ions to stimulate exocytosis is constant for different pulse durations. A, The cumulative capacitance change increases with increasing pulse duration. B, The cumulative number of calcium ions that entered the cell during the pulse train increases similarly with increasing pulse duration. C, The efficacy of calcium ions to stimulate exocytosis is constant for all pulse durations. Data were taken from the same cells as in Figure 10.
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