Barium triggers rapid endocytosis in calf adrenal chromaffin cells

Abstract

1. Changes in cell capacitance were monitored in whole-cell patch-clamp recordings from calf adrenal chromaffin cells using a software-based phase-tracking technique. Rapid endocytosis and exocytosis were observed in extracellular solutions containing either Ca2+ or Ba2+. 2. There was no significant difference in the magnitude or the time course of rapid endocytosis of cells stimulated in Ca2+ as compared to Ba2+. When cells were pretreated with caffeine and thapsigargin in order to deplete intracellular Ca2+ stores, rapid endocytosis in Ba2+ was not affected. This indicates that Ba2+ itself is capable of supporting rapid endocytosis. 3. The application of the calmodulin inhibitor calmidazolium via the intracellular pipette solution did not inhibit rapid endocytosis. Although our findings are inconsistent with an immediate requirement for calmodulin in rapid endocytosis, they do not rule out an involvement on a longer time scale. 4. While rapid endocytosis was not affected by the substitution of Ca2+ with Ba2+, the maximum rate of exocytosis was higher in cells stimulated in Ca2+ than in Ba2+. Since Ba2+ currents were much larger than Ca2+ currents during depolarizations to +10 mV (the test potential used in these experiments), Ba2+ appears to be less efficient at promoting exocytosis than Ca2+.

Figure 1. Rapid endocytosis was similar when it was observed in either Ca²⁺- or Ba²⁺-containing solutions

A, changes in the capacitance trace were triggered by Ca²⁺ influx. A train of 10 depolarizations lasting 160 ms to +10 mV, from a holding potential of -90 mV, was used to elicit release. Depolarizations were applied every 340 ms and are indicated by the gaps in the capacitance trace. The trace is fitted with two lines representing the maximum rate of exocytosis and a two-exponential fit of endocytosis. The largest change in capacitance during any depolarization was 115 fF, which occurred during the 204 ms that capacitance was not acquired, yielding a maximum rate of 560 fF s⁻¹. In this capacitance trace, total endocytosis was calculated as the difference between the maximum capacitance value and the capacitance value at the end of the trace. Values corresponding to the fits are indicated in the figure. The inset shows the same data on an expanded time scale. B, changes in the capacitance trace were triggered by Ba²⁺ influx. Stimulation parameters were identical to those shown in A. The top inset shows the same data on an expanded time scale. The bottom inset shows the conductance trace (series resistance) obtained simultaneously with the capacitance trace. Although slight changes in conductance occurred over time, they did not parallel changes in capacitance. The pipette solution contained (mm): 110 caesium aspartate, 20 Hepes, 0.1 EGTA, 2 MgCl₂, 2 ATP, 0.350 GTP and 14 creatine phosphate. The extracellular Ca²⁺-containing solution (shown in A) contained (mm): 140 TEA-Cl, 10 dextrose, 10 Hepes, 10 CaCl₂ and 0.0001 TTX. The extracellular Ba²⁺-containing solution (shown in B) contained (mm): 140 TEA-Cl, 10 dextrose, 10 Hepes, 10 BaCl₂ and 0.0001 TTX.

Figure 2. Capacitance changes following stimulation were variable

A, exocytosis but not rapid endocytosis was observed following stimulation of a cell in Ba²⁺ (10 mm). Note that these data are from a different cell than that shown in Fig. 1. Stimulation parameters were identical to those described in Fig. 1B. The dashed line represents the maximum rate of exocytosis. The inset shows the same recording on an expanded time scale. Failure of rapid endocytosis following stimulation was also observed after stimulations in Ca²⁺-containing solutions. B, multiple increases and decreases in membrane capacitance produced by a single train of depolarizations in Ba²⁺ (10 mm). Conditions were identical to those in A. Rapid endocytosis occurred after exocytosis, but a second rising phase in capacitance (*) and a second falling phase (**) followed rapid endocytosis. The two lines indicate the maximum rate of exocytosis and a one-exponential fit of the initial period of rapid endocytosis. The inset shows the same recording on an expanded time scale. Solutions are described in Fig. 1.

Figure 3. Exocytosis but not rapid endocytosis was affected by substitution of extracellular Ca²⁺ with Ba²⁺

Parameters of both exocytosis and endocytosis used to model the data are plotted for experiments either in 10 mm Ca²⁺ or in 10 mm Ba²⁺. The mean capacitance change due to rapid endocytosis (A) and the mean time constant of rapid endocytosis (B) were not significantly different in cells stimulated in Ca²⁺ or in Ba²⁺. The mean capacitance change during exocytosis (C) was also not significantly different. However, the maximum rate of exocytosis (D) was approximately 3 times higher in cells stimulated in Ca²⁺ than in Ba²⁺. Stimulation conditions were identical to those described in Fig. 1. Error bars represent standard errors of the mean.

Figure 4. Current magnitudes were much larger in Ba²⁺ than in Ca²⁺

A, mean current magnitudes during each of the 10 depolarizations which made up a stimulation train are plotted for all experiments in 10 mm Ca²⁺ (left) and in 10 mm Ba²⁺(right). The 160 ms depolarizations to +10 mV were applied at 2 Hz from a holding potential of −90 mV. This test potential represents the peak of the Ba²⁺I-V curve, but the peak of the Ca²⁺I-V curve was at +20 mV. Note the difference in the scale of the ordinates for the two graphs. The current evoked by depolarization inactivated over the course of the train. B, sample current sweep taken from the first depolarization of a train. Current sweep obtained in Ca²⁺ (left) was taken from the stimulation illustrated in Fig. 1A; the maximum current is 290 pA. Current sweep obtained in Ba²⁺ (right) was taken from the stimulation illustrated in Fig. 1b; the maximum current is 1120 pA. Note, 400 μs of data are not shown at the beginning and end of the depolarization.

Figure 5. Ba²⁺-evoked rapid endocytosis was not due to the release of Ca²⁺ from internal stores

Cells were store depleted by treatment for a minimum of 90 min in 140 mm NaCl, 10 mm dextrose, 10 mm Hepes, 2.5 mm KCl, 2 mm CaCl₂, 2 mm MgCl₂, 10 mm caffeine and 1.5 μm thapsigargin. A, capacitance recording from a store-depleted cell stimulated in 140 mm TEA-Cl, 10 mm dextrose, 10 mm Hepes, 10 mm BaCl₂ and 100 nM TTX. Exocytosis and rapid endocytosis occurred normally following stimulation. Trace is fitted with two lines representing the maximum rate of exocytosis and a single-exponential fit of rapid endocytosis. A second rising phase is present in the capacitance trace. B and C, [Ca²⁺]_i responses from treated and untreated cells to a challenge with agents promoting store release (10 μm histamine, 0.8 μm bradykinin (BK)). Untreated cells (B) showed increase in [Ca²⁺]_i, as measured with fura-2 imaging. Cells treated for 90 min with caffeine and thapsigargin (C) no longer showed a response when challenged with 10 μm histamine and 0.8 μm bradykinin at the end of the treatment, indicating that Ca²⁺ stores were effectively depleted.

Figure 6. Calmidazolium, a potent calmodulin inhibitor, did not block rapid endocytosis

A, capacitance changes from a cell in which 100 nM calmidazolium was added to the standard intracellular pipette solution (see Fig. 1). After gaining whole-cell access, 5 min passed before stimulation in order to allow time for calmidazolium to diffuse into the cell. Exocytosis and rapid endocytosis occurred normally following stimulation. The extracellular solution was a physiological saline containing 140 mm NaCl, 10 mm dextrose, 10 mm Hepes, 2.5 mm KCl, 2 mm CaCl₂ and 2 mm MgCl₂. B, capacitance changes produced by a second stimulation of the same cell, 5 min after the first stimulation. Despite the additional time allowed for calmidazolium to enter the cell, rapid endocytosis still occurred following stimulation. Although the time constants of rapid endocytosis during the second stimulation differed from those in the first, a similar decline in the rate of rapid endocytosis over multiple stimulations was also observed in untreated cells.