(Received for publication, July 24, 1995; and in revised form, October 24, 1995)
From the
Serotonin (5-HT) is taken up in insulin granules and co-released
with insulin on stimulation of pancreatic islet -cells. Based on
these observations, we have used microcarbon fiber amperometry to
examine secretogogue-induced 5-HT release from rat
-cells
preloaded for 4-16 h with 5-HT and then exposed to a bath
solution containing 10 µM forskolin. In response to local
application of KCl (60 mM) or tolbutamide (50-200
µM), we recorded barrages of amperometric events. Each
amperometric event consisted of a short pulse of current measurable at
electrode voltages that catalyze 5-HT oxidation. With either
secretogogue, release was calcium-dependent. On combining amperometry
with perforated patch whole-cell recording, we found that barrages of
such events were well coupled in time and graded in intensity with
depolarization-induced Ca
currents and well
correlated with increases in membrane capacitance. In cell-attached
patch recording, amperometric events evoked by application of
tolbutamide followed the closure of ATP-sensitive K
channels and coincided with the onset of electrical activity.
These experiments suggest that amperometry is a useful technique for
studying, in real time, the dynamic aspects of stimulus-secretion
coupling in
-cells. Their performance was facilitated by the
design of a new carbon fiber electrode (ProCFE) described within.
Real time measurement of hormone release from endocrine cells,
such as insulin-secreting -cells of the pancreatic islets of
Langerhans, is critical for understanding stimulus-secretion coupling
normally occurring in these cells, as well as defects in secretory
function, such as those occurring in non-insulin-dependent diabetes
mellitus. Recent advances in amperometry, a technique for the
electro-oxidization of transmitter molecules near the surface of a
cell, have permitted near instantaneous measurement of exocytotic
hormone release(1, 2, 3) . In chromaffin
cells, amperometry reveals that brief barrages of current spikes are
evoked, in a calcium-dependent fashion, by several depolarizing
stimuli, including nicotinic agents and high
K
(1) , brief voltage clamp pulses(2) ,
and even single action potentials(4) . Each spike corresponds
to the near synchronous electro-oxidation of up to a million
catecholamine molecules liberated from a point source on the cell
surface(1) . In insulin-secreting pancreatic islet
-cells,
two early applications of amperometry to study secretion have been
reported. In the first case, quantal release of insulin has been
measured from glucose-, tolbutamide-, and high
K
-stimulated human islet cells based on the ability of
a modified (ruthenate-coated) carbon fiber electrode to catalyze the
electro-oxidation of S-S bonds between the A and B chains of
insulin(5) . In the second case, secretogogue-induced quantal
release of the ``false transmitter'' serotonin (5-HT), (
)an electro-oxidizable indolamine sequestered and stored
into insulin granules, has been measured from mouse
-cells
preincubated with 5-HT and 5-HT precursors(6) .
In this
work, we have examined stimulus-secretion coupling in 5-HT-loaded rat
-cells by combining patch clamp electrophysiology with
amperometry. We demonstrate that this technique can be used to record
exocytotic secretion simultaneously with secretogogue-induced
electrical activity. Using this approach, we also present evidence for
(i) the direct and rapid coupling of membrane depolarization to quantal
secretion and (ii) aspects of the time course of the release of a
quantum. This work has been facilitated by the recent design of a
polypropylene-insulated carbon fiber electrode, or ProCFE. Its
geometry, high sensitivity, low noise, and mechanical stability at
physiological temperatures make it particularly advantageous for
combined electrophysiological and electrochemical recording from
-cells, which require minimum ambient temperatures of > 28
°C to insure secretion. Part of this data has been presented in
abstract form (42) .
Figure 2:
Tolbutamide-induced quantal release from
5-HT loaded rat -cells: dependence on external
Ca
. Puffs of a modified PSS containing either 50
µM tolbutamide and 6 mM of Ca
or 50 µM tolbutamide and no added Ca
(free [Ca
]
<
20 µM) were alternately applied from a pair of puffer
pipettes micropositioned to within
10 µm of opposite sides of
this cell bathed in a modified PSS containing 100 µM Ca
. Adjacent traces were separated by an
30
s interval during which no stimulation was delivered to the cell. In
each case, the puff evokes a small positive plateau-like artifact that
subsides at the end of the puff. (Puffer artifacts are often seen in
electrochemical experiments. Sometimes they are upward; sometimes they
are downward. We do not really know their origin, except to speculate
that they represent the increase or decrease of oxidizable substances
at the sensor surface introduced by puff.) Note that discrete
amperometric spikes, representing rapid oxidation of a packet of 5-HT,
were only seen in the case of 6 mM Ca
.
Figure 3:
High KCl-induced quantal release from rat
-cells. In panel A, note that a puff of modified PSS
containing 60 mM KCl and 6 mM Ca
applied to a cell bathed in a PSS containing 0.1 mM
Ca
induced ASs with very short delay. In panel
B, showing another cell bathed in a PSS containing 2 mM Ca
, note that secretory response to the elevated
KCl solution is dependent upon the holding potential of the CFE (V
). ASs are absent during the puff when V
was equal to 100 mV, but a clear barrage of
events is seen when the same puff was applied at +680 mV. This is
consistent with a threshold of 5-HT detection of 300
mV.
ProCFEs
were fabricated by inserting a 2-cm segment of carbon fiber (5-7
µm diameter; Amoco Performance Products, Greenville, SC) into a
10-µl polypropylene pipetter tip (Continental Laboratory Products,
San Diego, CA). The final 3-5 mm of the tapered end of the
pipetter tip is then inserted into a 1.5-cm segment of 0.5 mm inner
diameter plastic tube. With this maneuver, both ends of the pipetter
tip could be grasped by two clamps. The tapered end of the pipetter tip
was then heated to 430 °C by a soldering iron to melt the
polypropylene onto the carbon fiber. To ``pull'' a ProCFE,
force was exerted on both ends of the pipetter tip until the end of the
carbon fiber was pulled out from the melted polypropylene end, forming
a long ``exposed'' region of carbon fiber tip distal to the
tapered insulation. For better reproducibility of ProCFEs, this heating
and pulling process was done by a specially designed puller.
Prior to electrode use, the tapered tip of the carbon fiber was precisely cut to a length of 20-50 µm by micropositioning it at the crux of a spring-loaded, 7-10-mm iris scissors (Stolz Instrument Co., St. Louis, MO) mounted on the recessed surface of a magnetic stand base. This procedure reduced the chance of insulation defects near the cut edge, which, in turn, could provide leak pathways to ground and reduce the electrical potential at the sensor surface. The final taper length chosen was sufficient to reduce electrode noise, which is proportional to length, while providing the possibility of recutting the tip two or three times to continually optimize electrode sensitivity.
Fig. 1B presents a schematic of a CFE amperometry
recording system. A sustained holding potential (V usually = +780 mV) is applied to the CFE tip immersed
in the bath. The equivalent circuit of a ProCFE, drawn as
``seen'' by the amplifier, is based on the observation that
the additional capacitance of CFE, after it enters the bath, can be
completely compensated by the ``fast'' and ``slow''
capacitance compensation function of an EPC-9 patch clamp amplifier.
This suggests that the ProCFE, used in the amperometry mode, has an
equivalent circuit similar to a cell recorded from in the
``whole-cell'' configuration(8) . The parameters in
the equivalent circuit measured from six ProCFEs were as follows: R
= 56 ± 18 M
; C
= 5.3 ± 1.1 pF; C
= 1.84 ± 0.44 pF; and R
>200 G
. The peak-to-peak noise of the amperometric
system using a ProCFE is 0.5-2 pA at a signal bandwidth of 1 kHz
(average RMS-noise 0.164 pA, 0.1-1kHz 8-pole Bessel, n = 19). This was only about 2-7 times the base-line
noise of the amplifier (EPC-7 or EPC-9).
Figure 1:
Overview
of amperometric recording. Panel A presents a sketch of a
ProCFE with an expanded display of an insulated tip. The cone-like
shape of the ProCFE resembles that of a 10-µl pipetter tip (30 mm
in length). Panel B shows the equivalent circuit of an
amperometry recording system with the CFE tip in the bath. V is the applied holding potential. For
amperometry, V
is a DC voltage of 650-800
mV. For voltametry, not used here, V
is a cyclic
triangle wave. See text for other
parameters.
Fig. 3A depicts a sample
experiment from an extensive set (n = 20) showing that a
PSS enriched in K also evokes quantal secretion from a
5-HT-loaded
-cell. High K
solutions are well
known to rapidly depolarize
-cells, initiate electrical activity
and intracellular Ca
transients, and provoke
[Ca
]
-dependent insulin
secretion. As in the case of tolbutamide, the occurrence of high
K
-evoked ASs was dependent on
[Ca
]
; no events were seen in a
PSS containing 100 µM
[Ca
]
(data not shown). Note
that the delay time between the start of the high K
puff and the initiation of the AS event was markedly shorter than
seen with application of tolbutamide in Fig. 2. These
experiments suggest that amperometric response to application of high
K
PSS provides a quick and simple way to check the
integrity of depolarization-secretion coupling in single
-cells. Fig. 3B provides evidence consistent with the substance
underlying stimulus-induced ASs being 5-HT: high
K
-induced amperometric spikes are seen at a DC
electrode potential (V
= +780 mV)
sufficient to oxidize 5-HT but not at one (V
= +100 mV) far below the threshold oxidation
potential for 5-HT. (
)In the best batches, about 50% of
cells displayed exocytosis in response to either tolbutamide or high
K
puffer solutions.
Figure 4: Features of individual amperometric events. Panels A-C show examples of individual ASs. The arrows indicate small ``feet'' preceding fast major spikes; these are interpreted as ``leaks'' of the released substance before the secretory vesicle has fused completely into the plasma membrane(2, 4) . Panel D shows a histogram of transient integral, or total charge, associated with an individual amperometric event including foot and major spike. Panels E and D show histograms of amplitude and half-height duration of ASs. All histograms were constructed from same events collected from 10 cells stimulated by KCl.
Fig. 5demonstrates amperometric detection of quantal release
evoked by prolonged membrane depolarization from a voltage-clamped
cell. Rat -cells display high voltage-activated Ca
currents detectable on depolarization to V
= -30 mV but reaching a peak value on depolarization
of V
= +15 mV (see current trace in
response to voltage ramp in right inset to panel A). Panel A demonstrates that repeated (3-s) pulses from a holding
potential of -70 mV to a test potential of +10 mV evoke
short bursts or intermittent individual amperometric events during the
course of depolarization, although the quantal output declined with
stimulus repetition (n = 11). From these experiments,
it was clear that the quantal release was coupled with membrane
depolarization. The first release events occurred with latencies as
short as 30 ms after onset of the depolarization (left inset).
In cells with higher and more stable rates of quantal release, it was
possible to apply alternately several test potentials, evoking either a
large or a small Ca
current, and to combine
amperometry with membrane capacitance (C
) tracking
to assess exocytosis (see panel B). In
-cells,
Ca
influx at -30 mV is nearly 10-fold smaller
than at 5 mV (see Fig. 5A, inset). Note the
absence of recognizable AS events or
C
in
response to a 5-s membrane depolarization to -30 mV that evoked
barely measurable I
. In contrast a barrage of
amperometric spikes and a
C
of 290 fF were
generated in response to a 5-s membrane depolarization to +5 mV, a
voltage that evokes a 6-7-fold greater I
.
This data, typical of those obtained from a set of three similar
experiments, is consistent with the hypothesis that membrane fusion, as
resolved by C
, and quantal discharge of
transmitter, as resolved by amperometry, reflect the same underlying
fusion process determined by Ca
entry into intact rat
-cells. (
)
Figure 5:
Depolarization-induced quantal secretion
from voltage-clamped cells. Panel A shows that repeated 3-s
voltage-clamp pulses from -70 mV to +10 mV evoke barrages of
ASs that are well coupled in time to intervals of depolarization. Here
the cell was patched with a pipette containing a Cs-IS
solution. The left inset shows the first barrage of evoked ASs
at an expanded time scale. The right inset shows whole-cell
current in response to a depolarizing voltage ramp from -100 to
+100 mV over 100 ms. The shape of this current, with its peak at
+15 mV, is nearly identical to the peak current-voltage
characteristic of Ca
current obtained with brief
steps of depolarization in identical recording conditions (D. Barnett
and S. Misler, unpublished data). Panel B shows the voltage
dependence of secretion monitored simultaneously by amperometry and by
tracking changes in membrane capacitance (C
). Note
the absence of detectable ASs and
C
, with
delivery of a 5-s pulse to -30 mV, when Ca
influx was very small, as compared with the barrage of AS and
nearly 300-fF increase in C
, with delivery of a
5-s pulse to +5 mV, when peak I
was
more than 7-fold larger(36) .
Combination of amperometry with current
clamp recording from patch-clamped cells permits examination of quantal
secretion induced by electrical activity of -cells. In Fig. 6A, note that injection of a 20-pA depolarizing current
from -10-pA holding current evoked cell activity consisting of a
short barrage of action potentials followed by a plateau depolarization
and resulted in a barrage of amperometric events. This type of cell
activity is typical for single rat
-cells. In these experiments
with single cells (n = 3), we were not successful in
obtaining either (i) consistent single APs in response to very brief
current injections, probably due to the paucity of expression of sodium
channels in the rat
-cell (12, 13) or (ii)
consistent release in response to plateau depolarizations, probably due
to exhaustion of the previously loaded 5-HT. However, the feasibility
for eventual success with such experiments is demonstrated by the
voltage clamp experiment in Fig. 6B. Note here that
brief (50-ms) depolarizations to +30 mV, applied at a frequency of
1 Hz, produced detectable release of at least one quantum, each of
which began either during or immediately after the depolarization (n = 5).
Figure 6:
Quantal secretion induced by prolonged
electrical activity and brief depolarizations nearly simulating single
action potentials. Panel A shows that prolonged (10-s)
injection of depolarizing current (-10 pA) results in (i) an
initial barrage of APs followed by plateau depolarization (see V trace) and (ii) a concurrent barrage of
amperometric spike events (see I
trace). The inset shows initial electrical activity at an expanded time
scale. Panel B shows that brief (50-ms duration) square pulses
of depolarization to +30 mV, repeated at 1 Hz, result in
individual ASs. The inset shows that ASs occur during, as well
as some ms after, the depolarization. As these recordings were made
with a K
-IS pipette, membrane currents consist of a
small initial inward current, in this case probably a combination of
I
and I
,
followed by a larger outward
I
.
Figure 7:
Cascade of events in stimulus-secretion
coupling monitored by combined cell-attached patch recording and
amperometry. A single cell, within a small cluster of islet cells
bathed in a PSS containing 3 mM glucose, was patched in the
cell-attached mode with a pipette containing K-IS and
held at 0 mV. A CFE touched another region of the cell surface. Note,
in the trace marked I
, that application by puffer
pipette of a PSS containing 200 µM tolbutamide resulted in
rapid closure of ATP-sensitive K
channels, identified
by their typical gating (burst of short openings) and characteristic
amplitude (
5 pA amplitude at pipette holding potential 0 mV) (see left bottom inset), followed by the appearance of biphasic
action currents (see right bottom inset). The onset of
amperometric spikes (see I
trace) coincided with
the train of electrical activity. The slow time courses of individual
AS events seen here, in contrast with the more rapid events in previous
figures, was typical of recordings from cells within clusters. This
phenomenon reflected slower diffusion of 5-HT to the sensor electrode,
perhaps because significant granule release occurs at regions of
cell-cell contact. As was the case with more rapid AS events, slower
events were no longer evident when the holding potential of the CFE was
decreased to +100 mV (data not shown), suggesting that they are
produced by oxidation of a substance with a threshold of detection
between +100 and +680 mV. Since rodent
-cells in
clusters are often electrical coupled(41) , some of the action
potentials in the train may have been initiated in cells other than the
one actually patched.
We have combined electrochemical amperometry with patch clamp
electrophysiology to examine, in real time, quantal secretion of 5-HT
from rat -cells during secretogogue-induced electrical activity,
as well as during imposed cell depolarization. The justification for
this approach is that pancreatic
-cells from a variety of species,
selectively take up 5-HT out of proportion to other islet cells,
sequester 5-HT into granules, and then secrete 5-HT along with insulin
when exposed to insulin
secretogogues(19, 20, 21, 22) .
Single cell secretion of 5-HT by
-cells has been reported, in
tandem, with rises in cytosolic Ca
under conditions
where stimulus-secretion coupling is maximized(6) . Our
application of 5-HT amperometry has allowed us to examine aspects of
the time course of exocytosis of a single quantum as well as the rapid
(millisecond range) temporal coupling of depolarization to secretion.
The advantages of this very rapid and sensitive single cell approach to
the study of depolarization-secretion coupling over prior attempts,
including combined perifusion and electrophysiology and membrane
capacitance tracking, as well as some intrinsic limitations of this
approach are discussed.
There are
several reasons why the signals we recorded are likely to originate, in
large part, from exocytosis of insulin granules from -cells.
First, our choice of the largest single cells visible for study (>10
µm in diameter) preselects with >80-90% probability for
-cells as previously demonstrated by cell sorting(23) .
This is now independently confirmed here; when so tested, most of our
pre-selected cells responded to tolbutamide, a specific
-cell
secretogogue. Second, as noted above,
-cells, and insulin granules
in particular, selectively take up
5-HT(19, 20, 21) . Third, although smaller,
-aminobutyric acid-containing granules, roughly the size of
synaptic vesicles, are present in
-cells, there is no evidence
that these vesicles undergo regulated exocytosis(24) . In
addition, the molecular content of the quantal event we have measured
here is several times larger than that of a catecholamine-containing
synaptic vesicle (11) , as might be expected for a granule with
>125-fold greater volume than the synaptic vesicle. Fourth, the time
courses of amperometrically measured quantized release of insulin from
single
-cells stimulated by tolbutamide and K
is
similar to those observed here for release of 5-HT when differences in
agonist concentration are taken into account(5) . Fifth, in
later experiments, we were able to identify
-cells
electrophysiologically by their display, at high density, of
distinctive ATP-sensitive K
channels. Sixth, all
amperometric signals were evoked under conditions under which insulin
granule release was expected.
The use of our newly designed polypropylene insulated carbon fiber electrode (ProCFE) facilitated combination of high resolution electrophysiological and electrochemical recordings. In comparison with previously described glass/epoxy-insulated carbon fiber electrodes (geCFEs) (25, 26) and peCFEs(2, 4) , the newly designed ProCFE combines several critical features. These are (i) the mechanical stability, particularly at physiological temperatures, as compared with the widely used geCFE; (ii) the relatively lower noise and smaller tip dimension that previously promoted the use of a peCFE in combined electrophysiological/electrochemical recording; and (iii) the simple structure and manufacturing process. While the ProCFE consists of only a carbon fiber and polypropylene insulation, both the peCFEs and geCFEs require at least three kinds of material.
Using single rat
-cells recorded from in the perforated patch mode, we encountered
some difficulty in (i) triggering single APs with brief depolarizing
current pulses and (ii) evoking sustained electrical activity with a
secretogogue such as tolbutamide. Many cells developed wobbly,
plateau-like depolarizations (see Fig. 6). Other cells with more
discrete APs were less than optimal for study because they failed to
secrete reliably. However, better results were obtained recording from
cells in clusters, particularly when cell-attached patch recording was
used. In these experiments, it was possible to view aspects of an
entire cascade of events involved in the induction of secretion by
tolbutamide. These include rapid closure of ATP-sensitive K
channels critical to the maintenance of the resting membrane
potential, subsequent onset of electrical activity (biphasic action
currents), and, coincident with the onset of electrical activity,
quantized release of 5-HT. This approach should be very useful in
determining the relative contribution of different steps in the
stimulus-secretion cascade to the heterogeneity of agonist
responsiveness seen in single cells.
A disappointing feature of
the application of the highly sensitive 5-HT amperometric assay to rat
-cells is its lack of robustness in measuring secretion. This
often precluded extended or detailed quantitative studies. To be sure,
as with all single cell assays, only a fraction (
50%) of
-cells secrete in response to secretogogues or direct stimulation.
Of more concern is the rapidity with which secretion in most cells
declines to barely detectable levels during even short, repetitive
bouts of depolarization in the presence of cAMP-enhancing agents
thought to increase the secretion-ready granule pool. While the loading
of granules appears to be dependent on the bath concentration of 5-HT,
it is not clear what factors contribute to 5-HT retention or
redistribution after loading. At this time, it is uncertain what
percentage of granules actually load with 5-HT and whether these
represent a specific fraction. Electron microscopic autoradiography of
random sections of
H-5-HT-loaded
-cells show
radioactive vesicles dispersed within much of the vesicle
pool(13) , but evidence from serial reconstruction is lacking.
This feature of limited loading might be overcome with improved loading
techniques and/or extension of current approaches to islets of other
species. For example, in early experiments, canine islet cells display
quantal events with 5 times the molecular content of those from rat.
Although their amplitude slowly declines with time, these events remain
detectable over many minutes, especially when short periods of rest are
interspersed between bouts of electrical activity(42) .