©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Action Potential-induced Quantal Secretion of Catecholamines from Rat Adrenal Chromaffin Cells (*)

(Received for publication, November 3, 1994; and in revised form, December 1, 1994)

Zhuan Zhou Stanley Misler (§)

From the Departments of Medicine (Jewish Hospital) and Cell Biology/Physiology, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Using single rat adrenal chromaffin cells, we examined the coupling of action potential activity to quantal release of catecholamines by combining perforated patch current-clamp recording with electrochemical microcarbon fiber amperometry. Chromaffin cells display steeper dependence of quantal release on action potential frequency than many nerve terminals, as well as more desynchronized release following an action potential. Also in contrast to neurons, in chromaffin cells, a major chemical secretagogue (acetylcholine) triggers potent quantal release even in the absence of electrical activity. These findings are consistent with an hypothesis that a major component of exocytosis from chromaffin cells involves diffusion of Ca to secretion sites which are less well co-localized with Ca channels than those in nerve terminals.


INTRODUCTION

A defining feature of the nerve terminal of a synapse is that the propagation of an action potential (AP) (^1)results in rapid and highly synchronized, Ca-dependent exocytosis of up to hundreds of packets of neurotransmitter often within 1 ms(1) . At several classical synapses, evidence ranging from simultaneous pre-terminal and post-synaptic recording (2) to nerve terminal morphometry (3, 4) and histochemistry(5) , suggest that this rapid and secure mode of release results from intense Ca entry through voltage-dependent channels which are closely co-localized with a population of ready-to-fuse Ca-triggered vesicles. These domains of close co-localization of Ca channels and vesicle secretion sites are known as ``active zones.'' The coupling of Ca to vesicle fusion may occur through a low affinity Ca-binding protein which requires local Ca concentrations up to hundreds of µM for its binding sites to be filled(6, 7, 8) .

Chromaffin cells of the adrenal medulla function analogously to post-ganglionic sympathetic neurons. In response to splanchnic nerve activity and release of acetylcholine (ACh) and peptide co-transmitters, chromaffin cells secrete, into the circulation, catecholamines (CA) and small peptides (e.g. enkephalins). Like nerve terminals, chromaffin cells have voltage-gated Ca channels and display APs when depolarized (9, 10, 11) and exocytose their humoral products in a Ca-dependent fashion(12, 13) . However, the kinetics of depolarization-evoked release may be more delayed and prolonged in chromaffin cells than in nerve terminals. In voltage-clamped chromaffin cells, release begins milliseconds, rather than microseconds, after the start of Ca entry and continues for up to seconds after Ca entry ceases(14, 15) . Investigation of the dynamics of quantal release of CA release from single chromaffin cells conducting APs would be a very useful counterpart of voltage-clamp studies, especially as APs permit more physiological recruitment of Na and Ca currents than voltage-clamp pulses.

Carbon fiber amperometry permits real-time electrochemical detection of exocytosis of the CA content of single chromaffin granules with speed and sensitivity nearly approaching transmitter detection by post-synaptic membranes(15, 16, 17) . Using single rat chromaffin cells, we have combined perforated patch current-clamp recording, with amperometry, to examine in real time the dynamics of the direct coupling of quantal release of CAs to AP activity. Part of the data has been presented in abstract form(47, 48) .


MATERIALS AND METHODS

Cell Preparation

Single chromaffin cells were prepared from rat adrenal medullae by methods previously described by Neely and Lingle(18) . Cells were plated on glass coverslips and maintained as primary cultures at 37 °C in a HEPES/bicarbonate-buffered Dulbecco's modified Eagle's medium (Life Technologies, Inc.) enriched with 10% fetal calf serum, 100 µg/ml streptomycin, 100 IU/ml penicillin G, and 6 µg/ml ascorbic acid, in the presence of 95% air and 5% CO(2). Experiments were performed at room temperature (21-23 °C) on smooth-surfaced, 10-15-µm diameter cells cultured for 3-7 days. The standard bath or extracellular solution contained (in mM): 135 NaCl, 5.5 KCl, 1 MgCl(2), 2 CaCl(2), 10 glucose, and 20 HEPES titrated to pH 7.4 with NaOH.

Perforated Patch Electrophysiology

To perform perforated patch recording(19, 20) , glass micropipettes were filled by immersion of the tip in a standard internal solution containing (in mM): 1 MgCl(2), 11.8 NaCl, 63.7 KCl, 28.35 K(2)SO(4), 47.2 sucrose, and 20 HEPES titrated to pH 7.3 with KOH. The pipettes were then backfilled with a modified internal solution which contained, in addition, 250 µg/ml nystatin. This diluted nystatin-containing solution was prepared by sonication, stored frozen, and kept on ice, well protected from light during an experiment to maintain its ability to promote rapid membrane permeabilization for up to a month(21) . Electrical recordings were made once the access resistance (R(a)) from the pipette to the cell interior fell to 35 megaohms, which was usually 1-5 min after achieving a pipette-to-membrane seal resistance of >2-3 gigaohms. APs and membrane currents (I(m)s) were evoked and recorded with an EPC-9 amplifier (Heka Electronic, Lambrecht, Germany) controlled by an Atari computer. In addition, using the EPC-9 in the voltage-clamp mode, membrane capacitance (C(m)) and R(a) could be automatically updated and output at a frequency of 2 Hz via the auto capacitance tracking feature.

Carbon Fiber Amperometry for CA Detection

Amperometric measurements were made with a polyethylene insulated carbon fiber electrode (CFE), fabricated according to Chow et al.(15) , but with some modification (see below). The tip of the CFE was micropositioned to just touch the surface of a newly patch-clamped cell. Use of low drift, precision, piezoelectric, and mechanical micromanipulators, e.g. PCS-250 (Burleigh Instruments, Fishers, NY) and MX-2 (Narishige USA, Inc., Sea Cliff, NY) respectively, made possible stable juxtaposition and even minor repositioning of the two recording electrodes for 30-60 min). The amperometric current (I), generated by oxidation of CAs at the exposed tip of the CFE, was measured using an EPC-7 patch-clamp amplifier (List-Electronic, Darmstadt, Germany) operated in the voltage-clamp mode at a holding potential of 780 mV. (A 7.8-V DC-source was used as the external stimulator.) To obtain electrical contact between the carbon fiber and the silver wire input to the head stage of the EPC-7, the shank of the CFE was backfilled with a mercury solution.

The following modifications of the published technique for CFE fabrication proved useful in enhancing our success rate and increases the sensitivity of our CFE significantly. (i) After constructing the polyethylene-coated CFE according to Chow et al.(15) , the tip of the electrode was reheated to remelt the polyethylene tubing covering the carbon fiber. This extended the tip region of the electrode and avoided insulation defects which could provide leak pathways to ground and reduce the potential at the cut surface and the ability of the CFE to oxidize catecholamines. This extended tip of the CFE could be carefully recut several times. (ii) After each cutting, the exposed surface of the electrode was treated by insertion of the tip into 95% ethanol for 3-5 s. (iii) Prior to positioning near a cell, a given CFE, held at +780 mV, was inserted into the bath solution to measure its background current. CFEs displaying background currents initially >300 pA, but declining to <10 pA over 5-10 min, permitted the most sensitive and stable recordings of amperometric spikes (ASs). CFEs with background currents < 100 pA were withdrawn from the solution, recut, recleaned, and retested. With these modifications of the technique more than 90% of the CFE fabricated can be used for extended recording.

Data Acquisition

A cell was selected for extended study if, on the voltage-clamp ramp or step, it displayed a large Na current (i.e. a rapidly inactivating inward current evoked at V(m) values positive to -40 mV (see Fig. 6A, inset) and blocked by the addition of 1-5 µM tetrodotoxin to the bath). The data were acquired by a Macintosh Quadra 650 computer running Pulse Control 3.0 software (22) and connected to both patch-clamp amplifiers via an ITC-16 A/D converter interface (Instrutech, Syosset, NY). Using this system, up to five channels of data (e.g.I(m), V(m), C(m), R(a), and I) were acquired at sample rates of 1-3 kHz. The low pass filter of the EPC-9 was set at 2.5 kHz for V(m) recording, that of the EPC-7 at 1 kHz for amperometric recording. To evoke voltage-dependent I(m) values, cells otherwise held at a V(m) of -80 mV were stepped or ``ramped'' to a desired V(m) for a short interval. To evoke APs, cells maintained at a V(m) of -60 to -80 mV, through the application of a holding current of -10 to -20 pA, were stimulated by depolarizing currents (+10 to + 20 pA) of variable duration, or by pulses of CCh applied via a puffer pipette filled with an external solution containing 100 µM CCh, and positioned within 50 µm of the cell surface (see Fig. 1D).


Figure 6: AP-dependent and -independent pathways of stimulation of secretion by CCh in rat chromaffin cells. A, in this cell, sequential, extended but low intensity puffs of CCh resulted in bursts of APs accompanied by barrages of ASs which ended shortly after membrane repolarization. B, in another cell, sequential brief, but intense, puffs of CCh given after the cell desensitization of the cell to the depolarizing effects of CCh resulted in extended bursts of ASs. Not shown is that this cell responded with short, vigorous bursts of APs and ASs to several CCh puffs prior to these recordings.




Figure 1: Simultaneous measurements of APs and single vesicle release, measured as ASs in a rat chromaffin cell. A, results of a typical experiment of simultaneous recording of trains consisting of 20 APs (membrane potential trace, V(m)) and barrages of ASs (amperometric current trace, I) in response to sustained depolarizing current pulses (I). B, histogram of the number of APs preceding the occurrence of the first AS in a barrage of ASs evoked by the AP train. Data were pooled from 13 trains evoked in the cell depicted in A and 27 trains from five other cells which were likewise stimulated by 5-s depolarizing current pulses and produced similar AP trains. C, histogram of the time of occurrence of AS events during and after 5-s-long AP trains. Data were pooled from the 13 trains evoked in the cell depicted in A and 12 trains from two other cells showing similar pattern (frequency and total number) of APs per train as well as similar numbers of ASs evoked by train. Zero time marks the time of the peak of the first AP within a train, typically 10-20 ms after the onset of depolarization. Note the nearly 2-s tail of AS events after the cessation of the stimulating current. D, cartoon of the cell stimulation/recording arrangement featuring the patch pipette recording electrode attached to the cell, the carbon fiber electrode within 1 µm of the cell surface, and the drug application pipette positioned within 50 µm of the cell surface. E, sample amperometric spike recorded at high gain.




RESULTS

Simultaneous Measurement of Action Potential (AP) Activity and Single Chromaffin Granule Release

Fig. 1demonstrates the direct coupling of AP activity to quantal release of catecholamines, in single rat adrenal chromaffin cells. In Fig. 1A, four trains of APs were induced by 5 s depolarizing current pulses (from -10 pA to +10 pA). Each train of 20 high frequency APs provoked a barrage of ASs. Each AS is taken to represent the release of much or all of the CA content of a single chromaffin granule(16) . Though in Fig. 1A all ASs are leq60 pA, more typically ASs ranged from 5 to 500 pA in amplitude. ASs with amplitudes > 50 pA and rise times < 3 ms average 3.5 ms in half-height duration at 22 °C, making these ASs much faster than those previously reported with bovine chromaffin cells where half-height durations range from 10 to 20 ms(15, 16) . A given barrage of AS events had the following features (n = 50): (i) it began with variable delay after the start of the AP train, that is, after between 2 and 15 APs were conducted (see Fig. 1B); (ii) it continued for the duration of the train; and (iii) it ceased by 5 s after the conclusion of the AP train, in this cell by 2 s (see Fig. 1C). In half () of the cells tested with repeated AP trains, the latency between the onset of APs and ASs was longest for the first train of the series. As background release of quanta is vanishingly low in the absence of cell injury, we assumed that all quantal release, seen during and immediately after impulse activity was provoked by that activity. In addition, trains of APs which provoked barrages of ASs produced sizable changes in membrane capacitance corroborating the association of AS activity with exocytosis (data not shown). A schematic of the cell stimulation and recording arrangement is shown in Fig. 1D; a sample AS is shown in Fig. 1E.

Dependence of Secretion on AP Frequency

While clearly demonstrating that trains of APs were able to evoke secretion, the results of the preceding section also suggested that widely spaced single APs might be relatively ineffective in evoking secretion. In addition, the delayed buildup of release with repeated APs, as well as the seconds long tail of release after the cessation of the AP train, suggested the slow buildup and subsequent depletion of an intracellular messenger (e.g. cytosolic Ca) that might trigger release. To test these ideas more rigorously, we examined CA release in response to trains of single APs evoked by application of very short current pulses at varying frequencies.

Since cells are of variable responsiveness, Fig. 2presents sample traces from two cells. In these cells, as in others, APs evoked at a frequency of 0.2-0.5 Hz produced very rare ASs. (Over this range of frequencies, the effect of an individual AP should be independent of that of others, in that even after a train of APs, ASs are usually no longer apparent by 2-5 s after cessation of a train.) However, increases in the frequency of stimulation dramatically increased quantal output. In Fig. 2A, which depicts a cell with high release efficiency, note that increasing the stimulus frequency from 0.2 to 1 and finally to 4 Hz increased the average number of quanta following an impulse from 0.37 ASs per AP to 1.63 ASs per AP and finally to 2.95 ASs per AP. In contrast, in Fig. 2B, which depicts a cell with lower release efficiency, note that raising the stimulus frequency from 0.33 to 4 Hz and then up to 12.5 Hz in a burst increased the number of quanta following an impulse from 1 AS per 20 APs to, on average, 3 ASs per 20 APs, and finally to 8 ASs per 10 APs.


Figure 2: Dependence of rate of quantal secretion on AP frequency. A and B depict sample traces of quantal secretion from two cells stimulated at varying rates. C depicts average quantal release per AP as a function of AP frequency in three rat chromaffin cells and compares it with post-synaptic potential amplitude as a function of AP frequency at the frog neuromuscular junction. Post-synaptic potential data were averaged from Magleby(23) , where the compound post-synaptic potential was measured extracellularly from a group of surface muscle fibers, and from Misler(24) , where the post-synaptic potential was measured from singly impaled muscle fibers. Data were normalized to the number of quanta released per impulse at 1 Hz. D presents, in an expanded scale, a burst of APs evoked by a sustained depolarizing current.



Fig. 2C depicts the normalized dependence of quantal secretion on AP frequency seen with three chromaffin cells with large numbers of events (total 300 events). Increasing AP frequency from 0.2 to 4 Hz produces a 4-fold increase in the average number of quanta released per impulse. The frequency dependence of chromaffin cell secretion is strikingly steeper than the frequency dependence of post-synaptic potential amplitudes at frog skeletal neuromuscular junctions stimulated to conduct single APs in reduced Ca/elevated Mg solutions chosen to minimize depletion of available quanta (see data plotted from Magleby (23) and Misler(24) ). However, other nerve terminals, including individual post-ganglionic adrenergic varicosities innervating smooth muscle of the vas deferens (25) and branched terminals innervating crayfish claw muscles(26) , seem to show steeper frequency dependence of release, more comparable with that seen from chromaffin cells.

Latency of Quantal Secretion After an AP

Fig. 3analyzes the latency of occurrence of ASs after single APs, measured as the interval between the peak of the AP and the time the AS reached half its maximum amplitude, when APs were evoked at 1 Hz (left side) or 4 Hz (right side). These data, obtained from the experiment partially illustrated in Fig. 2A, are representative of those obtained in three similar experiments. At an AP frequency of 1 Hz, nearly 50% of ASs occurred in the range of 10-20 ms, whereas the remaining events were more evenly distributed over the rest of the 1-s interval, thus giving rise to a histogram with an early spike and prolonged ``tail'' (see Fig. 3, A and B). After raising the AP frequency to 4 Hz, which nearly doubled the number of AS events per AP, less than 20% of events occurred with a latency of leq30 ms, whereas the remainder occurred between 30 and 250 ms, hence resulting in a flatter histogram with a less prominent spike (see Fig. 3, C and D).


Figure 3: Latency of quantal release after single APs applied at 1 and 4 Hz. A presents a histogram of events tabulated from the cell shown in Fig. 2A, stimulated at 1 Hz. The inset compares tabulated latencies of short delay amperometric events recorded from this cell (open squares) with the latencies of quanta contributing to the post-synaptic potential recorded at the frog neuromuscular junction at a comparable temperature (28) (closed squares). B presents a segment of the record used to construct the histogram in A. C presents a latency histogram for AS events recorded from the same cell after the AP frequency was increased to 4 Hz. The inset to this panel compares the distribution of short latency events recorded under those conditions with the latency of quantal release recorded from a frog neuromuscular junction bathed in an extracellular solution containing 0.3 mM Ca and 1 mM Mg and stimulated for 3 min at 10 Hz^6. D presents a segment of the record used to construct the histogram in C. Note the occurrence of AS events many tens of ms after each AP. In the insets to A and C, the peak value of the histogram for the neuromuscular junction was normalized to the peak value of the histogram for the chromaffin cell. Axes labels and quantities in the insets for A and B were identical to those in the major graph.



The insets to A and C compare the latency of quantal secretion seen in chromaffin cells with that at a frog neuromuscular junctions bathed in modified Ca/Mg external solutions. When the nerve terminal is stimulated by APs at 1 Hz, release occurs almost exclusively within a window of 1-2 ms; no tail of release is discernible (data from (28) ). Even when the nerve terminal is stimulated by APs at 10 Hz for 3 min to increase poorly synchronized quantal release, recorded as miniature post-synaptic potentials, less than 5% of the total quantal release occurred after the narrow window of release. (^2)Similar data are available for varicosities of post-ganglionic adrenergic fibers innervating vas deferens smooth muscle(25) , as well as for preganglionic cholinergic terminals innervating the superior cervical ganglion(27) . Despite methodological differences, (see ``Discussion'') comparison of data suggests that the distribution of latencies of release is broader and more dependent on AP frequency in the chromaffin cell than in nerve terminals.

Contributions of Plateau Depolarization to Secretion

In current-clamped rat chromaffin cells, responses to long lasting depolarizing current pulses (>100 ms) vary greatly from cell-to-cell. Although cells with large macroscopic Na currents (>1 nA) (n = 20) respond with a sustained train of APs (e.g.Fig. 1and Fig. 2B), cells with smaller Na currents (<300 pA) (n = 15) respond with an initial short burst followed by a plateau depolarization (PD). Since many endocrine cells display PDs as part of their repertoire of responses to physiological stimuli (e.g.(29) ), it was useful to test directly whether the PD itself would sustain secretion.

Fig. 4provides evidence that PDs ranging from -35 to -20 mV contribute to sustained secretion. In this cell, which displayed a 300 pA Na current in response to a voltage-clamp ramp (bottom left inset), note that repeated 25-ms depolarizing current pulses, which induce single APs at 0.2 Hz, at best trigger only one AS (left-hand panel). In contrast, repeated 1-s current pulses of identical amplitude and frequency, each of which provoke a single AP followed by a PD, trigger a salvo of ASs (right-hand panel). These results suggest that even small amplitude depolarizations applied for many ms may be sufficient to evoke CA secretion. They are consistent with results of studies on chromaffin cells from a variety of species demonstrating that small, but sustained, increases in extracellular [K] induce [Ca](o)-dependent CA secretion which is reduced, but not abolished, by the Na channel blocker tetrodotoxin (30) .


Figure 4: Effectiveness of PD in inducing secretion. In this cell, depolarizing current pulses (from -20 pA to +20 pA) imposed for 25 ms (left upper panel) induced single APs, whereas those imposed for 1000 ms (right upper panel) induced single APs followed by PDs. Note the enormous enhancement in AS frequency during the sustained PD. Expanded scale records of the starred regions (one asterisk and two asterisks) of the V(m) trace are shown in the lower panels. The inset to the left upper panel displays the whole cell current evoked in this cell by a voltage-clamp ramp.



Since a previous study of the voltage dependence of secretion in bovine chromaffin cells, assayed by C(m)(31) , was limited to potentials positive to -10 mV, we performed several experiments in the voltage-clamp mode to confirm the presence of Ca currents and depolarization-induced secretion in the range of the plateau potential. Fig. 5(middle vertical panel) demonstrates that a 10-s depolarization to -20 mV is sufficient to produce 22 ASs as well as a 140 femtofarads increase in C(m). The bracketed data panels, collected 20 s before and after the latter, demonstrate that increasing the depolarization to +10 mV nearly triples the number of ASs (to 60) and nearly quadruples the increase in C(m) (to 570 femtofarads). In three such experiments, the measured C(m) increase was roughly four times that predicted from the product of (i) the number of preceding ASs and (ii) the C(m) increment of 2.5 femtofarads/vesicle recently measured in bovine chromaffin cells (12) . This discrepancy suggests that amperometry, in this configuration, records only a fraction of the total number of exocytotic events per cell, which in this experiment is ¼, or even less, if significant endocytosis occurred during the 10-s depolarizing pulse (see also (15) ). The inset to this figure, compiled using data from another voltage-clamp experiment in which Ca currents were measured directly, presents peak Ca current as a function of membrane potential (V(m)). It demonstrates that Ca current is sizable at -20 mV, but is significantly larger at +10 mV.


Figure 5: Voltage dependence of the secretion monitored by both amperometry and C(m) measurements in a voltage-clamped rat chromaffin cell (see text for general description of the figure). The vertical tick marks in the trace depicting the voltage pulse represents the computer generated stimuli used to track C(m) at the holding potential of -70 mV. In each panel the sloping dashed line in the C(m) trace spans the time during which no measurement was made. The inset (top, middle) presents a curve of peak Ca current versus voltage (50-ms pulses). This cell was permeabilized with a pipette in which the KCl and K(2)SO(4) content of the standard internal solution were replaced by CsCl and CsSO(4), respectively, to insure the blockade of outward K currents. No correction was made for liquid junction potential as this may be complicated by the existence of a Donnan potential (<8 mV for KCl internal solution) in the perforated patch configuration(19) .



Evidence for AP-dependent and -independent Pathways for Cholinergic Stimulation of CA Secretion

Under physiological conditions, secretion of CAs by chromaffin cells is triggered largely by the release of ACh and peptide co-transmitters (e.g. vasoactive intestinal peptide) from the splanchnic nerve. Hence, the electrical activity of the greatest physiological relevance is that which is triggered by transmitter-induced depolarization.

Fig. 6A provides direct evidence that pulse applications of CCh, which elicit cell depolarization and trains of APs, also evoke barrages of ASs (n = 12). As with the case of direct depolarization, an AS barrage usually begins following the generation of several APs and continues for several seconds following cessation of the train and membrane repolarization. (Note that the AP train evoked by CCh is similar to that evoked by prolonged current pulses in Fig. 1). The electrical responses to CCh, as well as the closely following secretory responses, usually decline with repeated application of CCh. However, these cells still are able to generate APs and secrete in response to direct depolarization, suggesting that while ACh receptor response desensitized, depolarization-secretion coupling remained intact (n = 12).

In some cells, following desensitization to small pulses of CCh, large pulses of CCh, which failed to depolarize, nevertheless later triggered barrages of ASs which continued for tens of seconds (see Fig. 6B) (n = 5). Taken together, these results present evidence that at least two modes of cholinergically evoked secretion co-exist in single cells: one that is AP related and the other that is not AP related. This is consistent with results of classical studies on whole perfused adrenal glands(32) , as well as more recent studies on populations of isolated chromaffin cells, suggesting multiple pathways for cholinergic activation of secretion.


DISCUSSION

An Approach to Studying Coupling of AP Activity to Quantal Secretion of CAs

By combining perforated patch current-clamp recording, with microcarbon fiber amperometry, we have examined, in real-time, the relationship between AP activity, and quantal release of catecholamines in single rat adrenal chromaffin cells. By studying secretion in response to single APs, or after repetitive application of cholinergic agonists, we identified several critical features of impulse-evoked secretion in chromaffin cells. These are: (i) steep dependence of CA secretion on AP frequency, (ii) widely distributed latency of quantal release after an AP and broadening of the latency distribution with increasing AP frequency, and (iii) the ability of an excitatory transmitter to evoke secretion in the absence of cell depolarization and electrical activity. Although the first feature is found at some nerve terminals, the other two features are not.

Distinctive Features of AP-induced Quantal Release from Chromaffin Cells

(i) A major feature of evoked quantal release of CA is its steep dependence on AP frequency. Although our amperometric technique detects only a fraction of the total release events (probably < 25%, see Fig. 5) occurring over the entire cell surface, our data suggest that at low stimulation frequencies (i.e. 0.2-1 Hz), chromaffin cells secrete less than 1 quantum/impulse. However, at slightly higher AP frequency (e.g. 4 Hz) quantal release per impulse increased 4-fold (see Fig. 2). This pattern of release, although similar to that observed at single varicose endings of adrenergic post-ganglionic fibers(25) , is quite distinct from that seen at more elongated nerve terminals where a single impulse evokes, on average, many quanta, and the frequency dependence of release is very shallow(23) . On the other hand, our data are consistent with those of Kidokoro and Ritchie (16) which demonstrated good correlation between the frequency of APs recorded extracellularly from single cell and CA release from perfused adrenals during stimulation by a range of a [K](o) values which enhance AP activity.

(ii) A second prominent feature is latency of quantal release after an AP. The latency of an amperometric spike, as we define it, is not identical to the latency of a post-synaptic potential. Longer time for diffusion of catecholamines from the granule core to the electrode, as well as transient vesicle fusion prior to the spike event(15) , might displace and broaden our latency histogram compiled at low AP frequency (i.e.Fig. 3A). Hence we are not certain whether the timing of early release detected within 10-20 ms after a chromaffin cell AP, substantially differs from the timing of quantal release, comprising the synchronized post-synaptic potential, detected within 1-2 ms after a nerve terminal AP. However, close inspection of the timing of quantal release after an impulse suggests two significant differences between a chromaffin cell and a classical synapse. (a) At the synapses stimulated at 1 Hz for short periods, quantal release following the post-synaptic potential, is barely detectable (i.e. the frequency of asynchronous miniature post-synaptic potentials is hardly above background). In chromaffin cells stimulated at 1 Hz, only 50% of events contribute to an early peak at 10-20 ms, whereas the remainder contribute to a tail of quantal release continuing for hundreds of milliseconds. (b) At a classical synapse, such as the neuromuscular junction, increasing AP frequency to 5-10 Hz for several seconds produces a just detectable increase in both post-synaptic potential amplitude and the frequency of miniature post-synaptic potentials. In contrast, in chromaffin cells, raising the AP frequency to 4 Hz shifts the latency distribution so that the peak and tail are difficult to distinguish, with only 20% of events occurring within 20 ms of the impulse. Thus, compared with the synapse, impulse-evoked release from chromaffin cells seems to combine a relatively depressed component of early synchronous release, which at synapses contribute to the post-synaptic potential, with a greatly enhanced component of asynchronous release which, in synapses, contribute to the increased frequency of miniature post-synaptic potentials seen during prolonged repetitive stimulation. Both components may contribute to the sustained release seen during a plateau depolarization.

(iii) A third feature is that ACh appears to induce secretion via multiple pathways. Our data now show that, in single rat chromaffin cells, transient release of packets of CAs is closely related, in time, to CCh-induced electrical activity. Release begins during the course of an AP train, which rides on a plateau level depolarization and ends within 1-3 s after cessation of the train. However, more intense cholinergic stimulation sometimes triggers release of a similar number of quanta, although over a more extended duration, even after the depolarizing response has desensitized. Hence, at least in some cases, agonist-induced release, from chromaffin cells, in the absence of significant depolarization or APs, can be as potent as AP-induced release. This is in direct contrast with the situation at nerve terminals, where depolarization-induced secretion far outweighs any direct agonist-induced release. It is widely appreciated that CCh is a mixed nicotinic and muscarinic agonist and that both classes of agonists induce CA release from perfused adrenals. It is likely that the transient secretory response which is temporally related to depolarization is due, in large part, to the activation of nicotinic ACh receptor (nAChR) channels(10) , cell depolarization, and Ca entry through voltage-activated Ca channels(11) . It is also likely that more sustained CA secretion, which occurs with minimal or no depolarization, after desensitization of nicotinic receptors, is due to activation of muscarinic receptors, second messenger cascades, and release of Ca from intracellular stores(33, 34, 35, 36) .

Possible Implication for Mechanisms of DepolarizationSecretion Coupling in Chromaffin Cells: Extent of Co-localization of Ca Entry (or Ca Release) Sites and Secretion Sites

Neher and collaborators (15) have considered how the multimillisecond delays in quantal release from chromaffin cells after the onset of depolarization-induced Ca entry might be reconciled with (i) domains of multimicromolar [Ca](i) in the vicinity of open Ca channels (37) and (ii) less than micromolar levels of [Ca](i) needed to evoke potent release when the cell is equilibrated with a well buffered patch pipette solution(31) . Recently, they proposed that a significant fraction of secretion sites might have higher Ca affinity and be more loosely co-localized with Ca channels than their counterparts in some nerve terminals(38) . Evidence in favor of this scheme of ``loose co-localization'' of Ca entry sites and secretion sites comes from recent observations that long delays between cell stimulation and secretion are not seen in DM-nitrophen-loaded cells when cytosolic Ca is rapidly elevated by the flash photolysis of caged Ca compounds(39, 40) .

Although separately explicable in other ways, taken together, the several features of AP-induced release which we have examined are consistent with the loose co-localization scheme referred to above. Qualitatively, such a scheme might contribute significantly to (i) the widely distributed latency of occurrence of release at 1 and 4 Hz (Fig. 3), by resulting in variable delay times due to Ca diffusion; (ii) the frequency dependence of release in response to single APs (Fig. 2); and (iii) the slow buildup of secretion during a train of action potentials and the delay in cessation of secretion thereafter (Fig. 1, B and C), as Ca slowly builds up at and then diffuses from these sites. (^3)If the distances between Ca channels and secretion sites were randomly distributed, secretion sites with similar Ca affinities located closer to channels would be activated at shorter latencies and following less intense Ca entry than the more distantly located ones. Hence short latency events, occurring within several milliseconds, would constitute the major fraction of total quantal release during low frequency AP activity (leq1 Hz), whereas long latency events, occurring over many tens of milliseconds, would make a more prominent contribution at higher AP frequencies. Qualitatively, this is what we have observed (see Fig. 3). Physiologically, even a limited number of domains where secretion sites are close to Ca channels might be useful in insuring some CA secretion at low frequency stimulation. Last, extending this loose co-localization argument, some secretion sites might also be near enough to Ca-storage organelles for Ca released by the action of second messenger systems from these structures to reach threshold levels for triggering exocytosis. Such a scheme might account for agonist-evoked secretion in the absence of electrical activity as we have seen with carbamylcholine application (Fig. 6). However, other factors which might contribute to our observations include (i) frequency-dependent enhancement of Ca entry evoked during the action potential, as seen in pituitary nerve terminals (42) and (ii) Ca-dependent recruitment of vesicles into a ``pre-docked,'' ready-to-fuse pool(43, 44) .

Recently two groups have provided evidence in bovine chromaffin cells for both ``hot spots'' of Ca channels(45) , using pulse laser fluorescent imaging, and hot spots of quantal release of CA(46) , using ultrafine amperometry. It is not known how coincident these two types of hot spots actually are. However, it is interesting to consider that data supporting hot spots and those supporting loose co-localization might be encompassed by an arrangement where individual clusters of secretion sites are concentric with clusters of Ca channels but extend outward over a larger surface area, hence making the average distance between Ca channels and secretion sites in chromaffin cells larger than in the ``active zones'' of exocytosis of some well studied nerve terminals.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant DK37380, by the American Heart Association, and by the McDonnell Center for Cellular Neurobiology at Washington University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Established Investigator of the American Heart Association. To whom correspondence should be addressed: Renal Division, Jewish Hospital of St. Louis, 216 S. Kingshighway Blvd., St. Louis, MO 63110. Tel.: 314-454-7719; Fax: 314-454-5126.

(^1)
The abbreviations used are: AP, action potential; ACh, acetylcholine; AS, amperometric spikes; CA, catecholamines; [Ca], external Ca concentration; [Ca], cytosolic Ca concentration; CCh, carbamylcholine; CFE, carbon fiber electrode; PD, plateau depolarization.

(^2)
S. Misler and L. C. Falke, unpublished data.

(^3)
A precedent for the latter feature is that in sympathetic ganglion cells short trains of APs, delivered at moderate frequency, result in an enormous buildup, followed by slow, seconds long decay of submembrane [Ca], resulting in a sustained post-train hyperpolarization(41) .


ACKNOWLEDGEMENTS

We thank Dr. Chris Lingle and his laboratory for the generous gift of rat chromaffin cells which made this project possible and Dr. Robert Chow for demonstrating his technique for fabrication of carbon fiber electrodes.

Note Added in Proof-Since the submission of this paper, membrane capacitance measurements have been published suggesting the presence of a small pool of vesicles releasable within ms after triggering a brief action potential-like depolarization (Horrigan, F. T., and Bookman, R. J.(1994) Neuron13, 1119-1129).


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