Short-Term Changes in the Ca²⁺-Exocytosis Relationship during Repetitive Pulse Protocols in Bovine Adrenal Chromaffin Cells

Pulsatile Ca²⁺ entry can evoke secretion with the same efficacy as single pulses

We have shown previously that in chromaffin cells recorded with perforated-patch methods, the relationship between Ca²⁺ entry and exocytosis evoked by single pulses can be described by a simple transfer function of the form ΔC_m = g * (ΣCa²⁺)ⁿ, where ΔC_m is the change in cell surface capacitance in femtofarads, g is a proportionality constant, and ΣCa²⁺ is the integral of the Ca²⁺ current that is raised to the nth power (Engisch and Nowycky, 1996). There is no reason a priori to expect that exocytosis during the intermittent Ca²⁺entry of a train should be evoked with the same Ca²⁺efficacy as during a single long pulse. For equivalent amounts of Ca²⁺ entry, the two stimulus paradigms differ in at least two ways: (1) the Ca²⁺ concentration beneath the membrane should be lower during a train, because Ca²⁺ domains around open Ca²⁺channels collapse between depolarizations, and Ca²⁺is removed by diffusion and uptake mechanisms; and (2) the total elapsed time is much longer during a train, allowing time-dependent processes to take place.

Remarkably, a significant fraction of pulse trains evoked responses that exactly or closely followed a standard Ca²⁺-exocytosis transfer curve obtained by averaging input–output functions for single pulses in 27 cells (see Materials and Methods). Examples of three such responses, each from a different cell, are shown in Figure 1. Without analysis, the C_m responses appear to differ greatly, with more exocytosis evoked by protocols with longer pulse durations and larger Ca²⁺ loads (Fig.1A). However, when the same data are plotted as a function of the cumulative sum of Ca²⁺ current integrals for each pulse (Fig. 1B), the Ca²⁺-exocytosis relationships for all three protocols are nearly identical to that evoked by single pulses: the dashed curve represents the standard curve and is not a fit of the data. Thus, in different cells an identical Ca²⁺-secretion relationship can be evoked by train protocols that differ in the number of pulses, total elapsed time, and amount of Ca²⁺ influx per pulse.

The percentage of responses that obeyed the standard transfer function depended on the stimulus protocol (Fig. 1C). At fixed 200 msec interpulse intervals, stimulation with trains of 5 and 10 msec duration pulses evoked responses that followed the standard curve in >50% and 35% of chromaffin cells, respectively. Trains of 40 msec pulses evoked an exocytotic response with this pattern of Ca²⁺ efficacy in <10% of cells. The remaining responses were classified as being enhanced or depressed relative to the standard curve (see below).

Characteristics of exocytosis with enhanced Ca²⁺ efficacy

Examples of responses with greater Ca²⁺efficacy than predicted by the standard transfer function are shown in plots of cumulative C_m changes versus cumulative Ca²⁺ entry (Fig.2A) (three different cells). The C_m responses initially follow the standard Ca²⁺-exocytosis relationship (dashed curve), but after a variable delay deviate to the left. Within the resolution limits imposed by noise in the C_mrecording, the switch to enhanced Ca²⁺ efficacy appeared to be relatively abrupt. For example, in Figure2A,ii the shift occurs after eight to nine pulses, whereas in Figure 2A,i after a few pulses, and in Figure 2A,iii, after only one pulse. Once a state of enhanced efficacy was achieved, cumulative C_mincreases proceeded as a fairly linear function of cumulative Ca²⁺ entry, without either the upward curvature characteristic of the standard curve (Fig. 1) or the downward curvature of depressed responses (below and Fig.3).

The percentage of cells that gave enhanced responses to trains of 5, 10, and 40 msec duration pulses is summarized in Figure 2C. Stimulation with 5 msec duration pulse trains evoked responses with enhanced efficacy in 33% of cells; 10 msec duration pulse trains did so in 28% of cells, and 40 msec duration pulse trains in only 2% of cells tested. In all cells, the voltage-gated Ca²⁺current showed typical inactivation during a train (Fig.2B; comparison of first and last current), with no evidence for activation of additional “facilitation” channels as described for calf chromaffin cells (Artalejo et al., 1991, 1994).

Characteristics of exocytosis with diminished Ca²⁺ efficacy

Some exocytotic responses to pulse trains proceeded with diminished Ca²⁺ efficacy; that is, they were depressed relative to the standard curve. Figure 3Aillustrates typical examples of such responses evoked by protocols of brief pulses (5, 10, and 20 msec duration pulses; three different cells). The plots of cumulative C_m increases versus cumulative Ca²⁺ entry are approximately linear, deviating from the standard curve (dashed curve) because they lack upward curvature. In these examples, Ca²⁺ entry continues to elicit exocytosis throughout the stimulus train.

Three examples of diminished efficacy evoked by protocols with longer pulses (40 msec duration pulses) are shown in Figure 3B. In some cells (Fig. 3B,i), each pulse contributed additionalC_m increases as during shorter pulse protocols (Fig. 3A,i–iii). In other cells, exocytosis ceased abruptly during the train, either after following the standard relationship for several pulses (Fig. 3B,ii) or for only a single pulse (Fig.3B,iii).

Figure 3C summarizes the likelihood of obtaining a depressed response with each pulse protocol. Protocols of 40 msec duration pulses evoked responses with a diminished Ca²⁺-exocytosis relationship in the majority of cells (∼90%), whereas 10 msec duration pulse trains did so in 38% of cells. Trains of 5 msec duration pulses evoked depressed responses in a small but significant fraction of cells (∼15%).

The three types of Ca²⁺ efficacies are correlated with the amount of Ca²⁺ entry per pulse

Under our culture and recording conditions, Ca²⁺ current amplitudes vary by as much as fivefold between cells. This allowed us to relate the likelihood of obtaining a particular Ca²⁺-exocytosis relationship to the amount of Ca²⁺ influx during the first pulse of a train. A total of 219 capacitance responses were classified as having an enhanced, standard, or depressed Ca²⁺-exocytosis relationship, with only one train of a given pulse duration included per cell. The data were binned by the integral of the first Ca²⁺ current, and the results are plotted as the percentage of responses within each bin.

Responses with enhanced Ca²⁺ efficacy are clearly correlated with the smallest amounts of Ca²⁺ entry (Fig. 4A). Exceptions do occur, however, as was illustrated for a 40 msec train in Figure2A,iii. Responses that follow the standard curve are also associated with small amounts of Ca²⁺ entry (Fig. 4B), but they occur over a broader range, without the clear preference for the smallest Ca²⁺current integrals. Depressed responses were evoked over the entire range of Ca²⁺ values, but the likelihood rose steadily with increasing Ca²⁺ entry during the first pulse (Fig. 4C). Trains of 40 msec duration pulses with average or larger than average Ca²⁺ currents always evoked depressed responses (integral ≥ 9 * 10⁷ions or 28.8 pC; n = 40 cells).

Comparison of two stimulus protocols within individual cells: pulse duration

The histograms in Figure 4 indicate that the type of Ca²⁺-exocytosis relationship is correlated with, but not a strict function of, Ca²⁺ entry per pulse. The data in Figures 1, 2, 3, 4 were compiled by including only the first occurrence of a particular stimulus protocol per cell and therefore reflect the variability between cells. In the next four sections, we examine the capabilities of individual cells to respond to two different stimulus protocols.

We analyzed the exocytotic responses to a single pair of 5 and 40 msec duration pulse trains in 73 cells. In the majority of cells (∼82%), Ca²⁺ efficacy was greater during the 5 msec train than during the 40 msec train. If the response to the 40 msec pulse train was depressed, that evoked by the 5 msec pulse train either followed the standard curve (Fig.5B) or was enhanced (Fig.5C). If the response to a 40 msec pulse train was not depressed, the 5 msec pulse train always evoked responses with enhanced Ca²⁺ efficacy (Fig. 5D). This is summarized in Figure 5E, where each bar represents the fraction of cells in which a pair of 5 and 40 msec pulse trains had the indicated Ca²⁺-exocytosis relationships.

The only exceptions to this pattern were cells in which both trains evoked depressed responses (Fig. 5A; 15% of cells) and two cells (∼3%) in which both trains evoked enhanced responses (not shown). Thus a few cells appear to be predisposed to enter a particular Ca²⁺-exocytosis mode during repetitive stimulation. However, within a given cell, larger amounts of Ca²⁺never evoked exocytosis with a higher efficacy (n = 73 cells).

Depressed responses to 40 msec trains were frequent (n= 63/73 cells), and we were able to subdivide this population further into two groups based on the timing of the shift to depression (e.g., compare Fig. 3B,iii with 3B,ii). Table1 summarizes the results. Cells that became depressed quickly (1–3 pulses; “Depressed”) account for the entire group of depressed responses to 5 msec pulse trains and otherwise usually followed the standard curve. Cells that followed the standard curve for more than three pulses before becoming depressed (“St. C.–Depr.”) were more likely to respond to a 5 msec train with enhanced efficacy.

Thus, although the C_m response to a given amount of Ca²⁺ entry during a stimulus train cannot be predicted with certainty for any particular cell, changing the amount of Ca²⁺ entry per pulse results in an orderly, consistent shift between Ca²⁺-exocytosis relationships within individual cells. At fixed interpulse intervals, this shift can be summarized by the following scheme: Depressed ⇒ St. Curve–Depressed ⇒ St. Curve ⇒ Enhanced ↓ Ca²⁺ ⇒.

Comparison of two stimulus protocols within individual cells: Ca²⁺ concentration

We tested whether the shift between Ca²⁺-exocytosis relationships depended in any way on the duration of depolarization by varying external Ca²⁺ concentrations (5 mm vs 0.3 or 0.5 mm). Figure 6 illustrates an experiment in which the response evoked by a 40 msec pulse train in 5 mm Ca²⁺ is depressed, and the response to a 5 msec pulse train follows the standard single-pulse relationship (dashed curve). In low Ca²⁺ (0.5 mm), the Ca²⁺ efficacy during a train of 40 msec pulses became identical to that evoked by a 5 msec train in 5 mm Ca²⁺. In five such experiments, all cells had depressed responses during a 40 msec pulse train in 5 mm Ca²⁺. In low Ca²⁺, only one response remained depressed, two followed the standard curve, and two had enhanced efficacy. Thus diminished efficacy is induced by large amounts of Ca²⁺ entry rather than long depolarizations.

Comparison of single pulse and train protocols within individual cells

In this study, we define “depressed” or “enhanced” Ca²⁺ efficacy relative to a standard transfer function generated by averaging responses to single pulses in a separate cell population (see Materials and Methods). Ideally, the secretory responsiveness should be determined for each cell, but this was not feasible because of technical restrictions on the duration of patch-clamp recordings. However, the validity of using the standard transfer function can be checked by comparing the response to a single pulse and a stimulus train in a subset of cells.

We compared the total C_m increases evoked by a single 320 msec pulse and an equivalent amount of Ca²⁺ entry during a 40 msec train with 200 msec intervals. A total of 32 cells were stimulated with both protocols; as expected, most responses to the train had diminished efficacy. On average, the C_m increase during the 320 msec pulse was 1.8 times larger (Fig.7A,ii). This is a minimum estimate of the relative efficacy of a 320 msec pulse and 40 msec pulse trains because (1) all responses to 40 msec pulse trains were included, although not all were depressed; and (2) this comparison ignores the final ΔC_m/ΣCa²⁺ratio during trains that was very low, because Ca²⁺entry during later pulses was often completely unable to evokeC_m increases (e.g., Fig. 3B,ii,iii). We also observed (Fig. 7A,i) that a single 320 msec pulse could evoke a robust response even in cells most predisposed to depression during a repetitive pulse protocol, that is, the 15% that were depressed during both the 5 and 40 msec train (Fig. 5A, Table 1).

To examine a lower range of Ca²⁺ entry, we comparedC_m responses evoked by a single 40 msec pulse and an equivalent amount of Ca²⁺ entry during a train of 5 msec duration pulses in 46 cells (Fig. 7B,i,ii). On average, a single 40 msec pulse was only half as effective at evoking exocytosis as a train of brief pulses. Again, this is a minimum estimate because <40% of cells had responses with enhanced efficacy during a train, yet all 5 msec pulse trains are included in this analysis. Thus, the changes in efficacy observed during repetitive pulse protocols are caused not by differences in the basal secretory responsiveness of cells but by activity-dependent mechanisms.

Comparison of two stimulus protocols within individual cells: pulse interval

The previous sections describe the different exocytotic responses obtained when the amount of Ca²⁺ entry is altered at a constant interpulse interval (200 msec). To examine whether the exocytotic response of a cell is also influenced by the time span between bouts of Ca²⁺ entry, we tested various interpulse intervals.

Trains of 40 msec pulses at 200 msec intervals evoked depressed responses in most cells (Figs. 3C, 4). Prolonging the interpulse interval increased the Ca²⁺ efficacy without significant changes to total Ca²⁺ entry (Fig. 8). Three examples comparing a 200 and a 1000 msec interval stimulus train within individual cells are shown in Figure 8A. Cells with strong depression during the 200 msec train showed a partial relief of depression at 1000 msec intervals (Fig. 8A,i) or followed the standard curve (Fig. 8A,ii), whereas cells with less depression often gave large responses with enhanced Ca²⁺ efficacy (Fig. 8A,iii). A summary of 17 experiments is presented in Figure 8B, in which the total C_m increase evoked by the two protocols is compared. Despite the large variability of total ΔC_m during the 200 msec train, all data points cluster along a line with slope = 2. Thus within individual cells, there is a systematic shift of Ca²⁺ efficacy, remarkably similar to the shift that is observed as pulse duration is decreased at a constant interpulse interval (Fig. 5, Table 1). The shift was graded with the duration of the interpulse interval: at 400 msec intervals, there was no significant change, whereas at 800 msec responses were 1.35 times larger and at 1000 msec were two times larger (Fig. 8C).

We also tested the effect of shorter intervals during 40 msec duration pulse trains because this should approach the limit of a single prolonged pulse (0 msec interval) and possibly prevent the development of depression. Of all stimulus protocols tested, 40 msec pulses at 100 msec intervals gave the least consistent results. In 3 of 11 cells, depression was abolished at the shorter interval as predicted. However, in five cells the Ca²⁺-exocytosis relationship was unchanged, one cell showed more depression, and two cells had rapid endocytosis. Thus although there is a slight tendency toward less depression at short intervals, the responses are too variable for further study.

The effect of shortening interpulse intervals during trains of 5 msec duration pulses depended on the response evoked by a train with 200 msec intervals. In five of six cells with enhanced Ca²⁺ efficacy during a 200 msec interval train, trains with shorter intervals evoked exocytosis with reduced efficacy (Fig. 9B,i). Figure9A illustrates one such experiment. Two trains with 200 msec intervals evoked exocytosis with enhanced efficacy (Fig.9A,i,iii), whereas the Ca²⁺-exocytosis relationship during a 44 msec interval train followed the standard curve (Fig. 9A,ii). On the other hand, in six cells whose response followed the standard curve during 200 msec intervals, shortening the interval had little effect (Fig. 9B,ii). As suggested above, both results are expected if brief interpulse intervals approach the limiting condition of a single long pulse from which the standard transfer function is obtained and if enhanced efficacy is a departure from this basal responsiveness.

In Figure 9A, the higher frequency train (Fig.9A,ii) induced slightly more total Ca²⁺entry than either 200 msec interval train (Fig. 9Ai,iii). This was a consistent finding in all cells tested: on average total Ca²⁺ entry was 1.12-fold greater during the higher frequency, 5 msec duration pulse trains (SEM = 0.03;n = 12; p < 0.01; paired ttest). It appears that in addition to exocytosis, Ca²⁺ current inactivation is also subject to frequency-sensitive modulation in chromaffin cells.

Increasing the interpulse interval from 200 msec to 1000 msec had little effect on the Ca²⁺-exocytosis relationship evoked by 5 msec pulse trains. The results are summarized in Figure9C for seven cells. All values lie close to the dashed line that represents equivalency.

Amperometric recordings confirm that ΔC_mmeasurements reflect exocytosis of catecholamine-containing vesicles

The capacitance detection method reports only net changes of plasma membrane area. The apparent depression of exocytosis may be caused by simultaneous membrane removal by endocytosis. On the other hand, the apparent enhancement may actually be caused by the addition of some other type of membrane. For example, exocytosis of small synaptic vesicles versus large dense-cored vesicles is evoked by different Ca²⁺ levels (Verhage et al., 1991) and stimulus patterns (Bruns and Jahn, 1995). Finally, apparent enhancement may result from totally artifactual sources such as gating charge movement (Horrigan and Bookman, 1994). These issues can be resolved with amperometric techniques that specifically detect the release of catecholamines (Wightman et al., 1991).

Simultaneous capacitance and amperometric recordings of a typical strongly depressed response to a train of 40 msec duration pulses, 200 msec intervals are shown in Figure10A. The amperometric events are few, and all occur during the early part of the train when the C_m trace shows a small increase. The cumulative integral of the amperometric signal initially rises in parallel with the C_m response but is flat during the rest of the train (Fig. 10A, bottom), indicating that catecholamine release and C_m increases stop simultaneously. Amperometric events occurred only during the first few pulses in five of six cells with depressed C_mresponses to 40 msec trains.

The cell shown in Figure 10A was also stimulated with a train of 40 msec duration pulses, but with 1000 msec intervals (Fig.10B). As described above (Fig. 8), longer interpulse intervals typically relieve depression; here, theC_m response closely follows the standard curve (bottom). Amperometric events occurred throughout the entire duration of the train, mirroring the persistence ofC_m jumps. Because of the relatively long interval between pulses, endocytotic processes are clearly visible as declines in the C_m trace. As a result, the difference between the pre- and post-train C_mlevel appears to be ∼100 fF for both trains (Fig. 10A,B, top). In calculating ΔC_m for cumulative plots, we used only the first 200 msec after each pulse (see Materials and Methods). This calculation gives a total ΔC_m change of ∼250 fF (Fig.10B, bottom) and a ∼2.5 times increase over the 200 msec interval train. Q_amp is similarly increased by approximately three times. These data confirm thatC_m changes parallel catecholamine release and indicate that endocytotic processes are too slow to mask exocytosis during 200 msec interval protocols.

Figure 11 contains an example of a cell in which a 5 msec duration pulse train evoked a capacitance response that was strongly enhanced relative to the standard curve. The capacitance trace (C_m, top) has small, step-like increases throughout the train. In the amperometric recording, large upward spikes are uniformly scattered throughout the duration of the stimulus (I_amp). In cells that had an enhanced response to a 5 msec duration pulse train, there were more spikes than in cells with responses that followed the standard transfer function (0.25 events/pulse, n = 4, vs 0.057 events/pulse,n = 4, respectively). This confirms that the enhancement of Ca²⁺ efficacy is attributable to the addition of membrane from catecholamine-containing vesicles. Amperometric recordings also rule out the possibility that the apparentC_m changes are caused by an artifact such as an ion channel gating charge, which might be particularly prominent in a long train of brief pulses (Horrigan and Bookman, 1994; Engisch and Nowycky, 1996).

Changes in Ca²⁺-exocytosis relationships are not caused by differences in global [Ca²⁺]_i

An apparent increase in the efficacy with which Ca²⁺ entry evokes exocytosis may actually be caused by Ca²⁺ contribution by intracellular stores. On the other hand, apparent diminished efficacy may result from the recruitment of rapid Ca²⁺ clearing mechanisms after large amounts of Ca²⁺ influx (Rorsman et al., 1992,Peng and Zucker, 1993; Hehl et al., 1996), or they may be caused by inhibitory effects of high [Ca²⁺]_i. To determine this, we monitored fluorescence in cells preloaded with the Ca²⁺-sensitive dye Fura Red AM.

Typical fluorescence changes evoked by four stimulus protocols in a single cell are illustrated in Figure12. A single 320 msec pulse caused a rapid rise in global [Ca²⁺]_i that peaked ∼40 msec after the pulse (Fig. 12A,i). A train of 40 msec duration pulses, 200 msec intervals, evoked a [Ca²⁺]_i rise to the same peak level within the first three to four pulses (Fig. 12A,ii) that was maintained despite further doubling of Ca²⁺entry between the fourth and tenth pulse (data not shown). During a train of 5 msec pulses, 200 msec intervals, [Ca²⁺]_i also rose to a plateau level, but this level was reached later and the final value was lower than during the 40 msec pulse train (Fig. 12A,iii). Finally, during a train of 40 msec pulses given at 1000 msec intervals, the maximal fluorescence signal was nearly identical to the plateau level of the 200 msec interval train; however, the signal decayed between each pulse (Fig. 12A,iv).

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Fig. 12.

Average [Ca²⁺]_i is rapidly elevated to plateau levels during trains of depolarizing pulses in bovine chromaffin cells recorded in perforated patch mode.A,i--iv, Four fluorescence ratios from a single cell loaded with Fura Red AM evoked by (i) a single 320 msec depolarization; (ii) train of 40 msec duration, 200 msec interval, 10 pulses; (iii) train of 5 msec duration, 200 msec interval, 40 pulses; (iv) train of 40 msec duration, 1000 msec interval, 10 pulses. All stimuli are indicated bybars below the trace. Traces in i andii are shown on expanded time scale, and each are fit with a single exponential with τ = 2.8 and 3.5 sec, respectively. Thedashed line in i represents the fluorescence ratio when ionomycin (10 μm) in external solution containing 10 mm CaCl₂ was applied at the end of the experiment to saturate the Ca²⁺-sensitive dye. Cell Chr240. B, Plot of changes in fluorescence evoked by 40 msec duration pulse train versus changes in fluorescence during a single 320 msec duration pulse. The change is expressed as the ratio of maximum signal during stimulation (R_p) to the signal before stimulation (R₀). Theline is a best fit with slope = 0.84 (r = 0.9; n = 7). The average total Ca²⁺ entry during a single 320 msec duration pulse was 95 pC, whereas during 40 msec pulse trains, a plateau signal was achieved on average in 3.4 pulses at a cumulative Ca²⁺ current integral of 77 pC (n = 11). C, Plot of changes in fluorescence ratio evoked by a 5 msec duration pulse train versus a 40 msec duration pulse train in the same cell. The line is a best fit with slope = 0.57 (r = 0.9;n = 11). During 5 msec duration pulse trains, a plateau signal was achieved on average in 16 pulses at a cumulative Ca²⁺ entry of 36.8 pC (n = 11).

The fluorescence changes shown in Figure 12 are representative for chromaffin cells recorded in perforated-patch mode. The plateau level reached during a 40 msec duration, 200 msec interval train was linearly related to the peak change induced by a single 320 msec duration pulse (Fig. 12B). In contrast, the plateau levels reached during 5 msec duration pulse trains were consistently lower than those of 40 msec duration pulse trains (Fig. 12C). Peak changes and plateau values probably represent physiological processes rather than dye saturation. The plateau values of 40 msec duration trains were considerably lower than the maximal ratio after ionomycin application (R_max/R_p = 1.77 ± 0.16; n = 6).

The fluorescence responses described here are representative of most recordings (14/18 cells). There were two exceptions to these patterns (data not shown). In two cells, the fluorescence response to the first 40 msec duration pulse train was approximately two times as large as that to all subsequent trains, although it also settled at a plateau by the third or fourth pulse. In two other cells, the plateau value was unusually small during various train protocols. Several seconds after each train, the fluorescence signal rose abruptly to a much higher level and then fell, resembling an active “[Ca²⁺] spike.” This was never accompanied by a C_m increase.

In summary, bovine chromaffin cells recorded in perforated patch mode appear to be much more effective at clamping [Ca²⁺]_i during stimulus trains than cells recorded in whole-cell mode (Augustine and Neher, 1992; Seward and Nowycky, 1996). There is no obvious correlation between [Ca²⁺]_i changes and the exocytotic Ca²⁺ efficacy evoked by particular stimulus protocols. Unexpectedly, loading cells with Fura Red and performing Ca²⁺ measurements caused several disturbances in theC_m responsiveness (data not shown). These include (1) poor reproducibility for two identical protocols in individual cells; (2) disruption of the systematic relationship of Ca²⁺ efficacies between pairs of different stimuli within cells (e.g., 5 msec duration pulse trains occasionally evoked responses with lower efficacy than 40 msec pulse trains); (3) unusual patterns that were not observed in control conditions (e.g., 5 msec duration pulse trains that had enhanced efficacy during the early pulses and complete depression during later pulses); and (4) occasional unusually large C_m jumps in response to the first pulse of 40 msec trains (84 ± 20 fF evoked by 7.5 ± 0.3 pC; n = 54 responses in 18 cells) (compare with Fig. 7B,ii). Both the intracellular Ca²⁺handling properties and exocytotic responses in the presence of Ca²⁺ chelators require further investigation for chromaffin cells recorded in perforated-patch mode.

Summary

The key observation in this paper is that bovine chromaffin cells recorded in perforated-patch mode have a repertoire of Ca²⁺-exocytosis relationships that are preferentially evoked by repetitive stimulus protocols. A simple standard transfer function adequately predicts the relationship between Ca²⁺ entry and amount of exocytosis when cells are stimulated with single depolarizing pulses (Engisch and Nowycky, 1996). Remarkably, the same transfer function applies to a subset of responses evoked by trains of depolarizing stimuli (Fig. 1). However, trains also evoke exocytosis with enhanced or diminished Ca²⁺efficacy relative to this function. Exocytotic responses with enhanced Ca²⁺ efficacy are evoked preferentially by protocols associated with small amounts of Ca²⁺ entry per pulse, whereas diminished or “depressed” responses are induced by protocols that evoke larger amounts of Ca²⁺ entry per pulse. Changes in Ca²⁺ efficacy during repetitive stimulation are not determined solely by the amount of Ca²⁺ entry per pulse, but by the balance between Ca²⁺ entry and the interpulse interval. Prolonging the interval of 40 msec duration pulse trains shifts the response pattern from depression to following the standard curve or enhanced efficacy, whereas shortening the interval of 5 msec duration pulse trains can prevent the development of an enhanced response. Our working hypothesis is that the single pulse relationship represents the “basal” Ca²⁺-exocytosis relationship, in the absence of Ca²⁺- and time-dependent modulatory processes. Both enhancement and depression develop during trains lasting several seconds and fade during the 2–5 min between stimulus trains, qualifying them as “short-term changes” in Ca²⁺ efficacy.

Capacitance responses to trains can obey the single-pulse transfer function

The observation that pulse trains can evokeC_m responses with the same Ca²⁺-exocytosis relationship as single pulses is unexpected. Pulsatile Ca²⁺ entry during a train will create very different submembrane [Ca²⁺] profiles than will a continuous long depolarization (Sala and Hernandez-Cruz, 1990; Nowycky and Pinter, 1993; Klingauf and Neher, 1997). As was emphasized in Engisch and Nowycky (1996), the standard transfer function does not relate exocytosis to submembrane [Ca²⁺], but simply serves as a predictor for the total exocytosis expected for a given amount of Ca²⁺entry. If intermittent Ca²⁺ entry during the many short pulses of a train can evoke a Ca²⁺-exocytosis relationship with the same upward curvature as a single long pulse, some components of the secretory machinery must be able to “remember” or “sum” Ca²⁺ entry across interpulse intervals. Another surprising feature is the fidelity with which the C_m responses to trains often adhered to a curve generated by averaging functions from 27 cells, which had a range of individual g and n values (Engisch and Nowycky, 1996, their Table 1). In effect, the many pulses in a train appear to average out the variability of single-pulse responses both within and between cells.

Enhanced Ca²⁺ efficacy

Various forms of facilitation during trains of action potentials or depolarizing pulses have been described at both fast synapses and in whole-cell recordings of neuronal terminals, neuroendocrine, and endocrine cells (see introductory remarks). Commonly, most forms of facilitation are postulated to result from (1) an accumulation of intracellular or submembrane Ca²⁺ [variants of the “residual Ca²⁺” or “residual Ca²⁺-bound receptor” hypothesis (Katz and Miledi, 1968; Yamada and Zucker, 1992; Zucker, 1996)], (2) the existence of additional vesicular pools that are recruited during a train (Horrigan and Bookman, 1994; Gillis et al., 1996), or (3) requirements for an initial Ca²⁺-dependent priming step (Seward and Nowycky, 1996). Here, explanations (2) and (3) can be considered as specialized versions of the residual Ca²⁺hypothesis, in that experimentally increasing the amount of Ca²⁺ entry will more quickly achieve the condition in which there is additional exocytosis. An alternative form of facilitation is produced by the recruitment of Ca²⁺channels that are preferentially coupled to exocytosis (Artalejo et al., 1994).

Enhanced Ca²⁺ efficacy as described here differs qualitatively from all forms of the residual Ca²⁺hypothesis because it is evoked under conditions of minimal Ca²⁺ entry. In most cells, shortening the interpulse intervals, a condition that should accentuate Ca²⁺accumulation, actually abolished enhanced responses. This is predicted by our working hypothesis that enhanced efficacy is a Ca²⁺- and activity-dependent shift from the basal responsiveness of a cell. Enhanced efficacy is not caused by recruitment of “facilitation” channels, because Ca²⁺ currents exhibited typical inactivation during a train (Fig. 2). Finally, simultaneous capacitance and amperometric recording provide evidence that enhanced efficacy, defined by changes in the C_m trace, accurately reflects changes in the secretion of catecholamine-containing vesicles.

Diminished Ca²⁺ efficacy

Most models of secretory depression invoke the rapid depletion of release-ready vesicles (see introductory remarks). In our recordings, depression was associated with protocols that caused more Ca²⁺ entry, and depletion might be expected because of increased exocytosis. However, the total amount of exocytosis during a depressed response was much less than could be evoked in the same cell with a single long pulse, although the train spanned a time period that might be used for increasing the readily releasable pool. Depressed C_m responses are not the result of endocytotic processes masking exocytotic events, because simultaneous amperometric recordings verify that spike frequency declined concurrently (Fig. 10). Because capacitance recordings of isolated cells do not have the complications of postsynaptic changes, we can conclude that in chromaffin cells depression is a modulation of excitation–secretion coupling mechanisms that is actively induced by repetitive stimulation, independently of the vesicles available for release by a single pulse.

Most chromaffin cells have a repertoire of Ca²⁺-exocytosis relationships

A limitation of the present study is that enhanced and depressed Ca²⁺ efficacies are not defined by unique quantitative criteria, but only relative to the standard transfer function. Pulse trains of 40 msec were usually easily classified, as were 5 msec pulse trains with enhanced efficacy. The only problematic groups were responses evoked by 5 msec pulse trains that were either depressed or followed the standard curve, because of the small absoluteC_m changes (usually a few tens of femtofarads, or several large dense-cored vesicles, assuming 2.0–2.5 fF/vesicle). Nevertheless, the distribution of response patterns is highly reproducible between cells and is a useful starting point for further study.

Under our culture conditions, most chromaffin cells appear to have a repertoire of Ca²⁺-exocytosis efficacies, with only a few that are depressed or have enhanced efficacy in response to both a 5 and a 40 msec duration pulse train (Fig. 5). This latter group may have greater amounts of the factors responsible for activity-dependent changes during stimulus trains, because at least some cells had typical responses to single-pulse depolarizations. Within the majority of cells, there is an orderly progression of Ca²⁺-secretion relationships from: as the amount of Ca²⁺ entry per pulse is increased at fixed interpulse intervals or as the interpulse interval is decreased for a given pulse duration. In summary, activity-dependent changes in the exocytotic responsiveness of bovine chromaffin cells appear to be determined by both the balance of Ca²⁺entry per pulse and the interpulse interval.

Intracellular Ca²⁺ measurements

In whole-cell recordings of many cell types and nerve terminals, fluorescence measures of average intracellular Ca²⁺are usually proportional to the amount of Ca²⁺ entry until they reach dye saturation, although the relationship is not necessarily linear (Thomas et al., 1990; Augustine and Neher, 1992;Ämmälä et al., 1993; Mollard et al., 1995; Seward and Nowycky, 1996). Exceptions do occur: neurohypophysial nerve terminals successfully clamp average [Ca²⁺]_i at ∼500 nm regardless of Ca²⁺ load, unless the mitochondria are poisoned (Stuenkel, 1994). Bovine chromaffin cells in perforated-patch mode appear to regulate [Ca²⁺]_i much more strictly than cells recorded in whole-cell mode. During repetitive pulse trains, a plateau in fluorescence is achieved that appears to represent a set-point to which [Ca²⁺]_i is clamped. The similarity between the average [Ca²⁺]_ievoked by the various stimulus protocols rules out the possibility that shifts between the various Ca²⁺-exocytosis relationships result from global elevation of [Ca²⁺]_i and instead suggests that they are caused by intermittent Ca²⁺ elevations near the plasma membrane that are dissipated before they can be detected with our techniques.

Possible mechanism of short-term changes in Ca²⁺ efficacy

Many second messenger systems, cytoskeletal elements, and other agents can modify stimulus-secretion coupling of large dense-cored vesicles (for review, see Burgoyne, 1991). Capacitance techniques have sufficient temporal resolution and sensitivity to detect modulation at a single cell level. Activation of protein kinase C enhances the amount of exocytosis in chromaffin cells (Vitale et al., 1995; Gillis et al., 1996) and lactotrophs (Fomina and Levitan, 1995), whereas cyclosporin A, an inhibitor of calcineurin, a Ca²⁺-calmodulin-dependent type 2B phosphatase, depresses exocytosis in bovine chromaffin cells (Engisch and Nowycky, 1997). In pancreatic β-cells, exocytosis is potentiated by activation of protein kinase C and protein kinase A, or by inhibition of Ca²⁺-calmodulin kinase II and type I and 2A phosphatase (A̋mma̋la̋ et al., 1993, 1994). The repetitive stimulus protocols used here span several seconds, which provides sufficient time for Ca²⁺-dependent activation of several second messenger systems. The results indicate that excitation–secretion coupling in chromaffin cells, as in fast synapses, is dynamic, with distinct responses to patterned activity that cannot be extrapolated from responses to single stimuli well separated in time.