J Neurophysiol 109: 758 –767, 2013.
First published November 7, 2012; doi:10.1152/jn.00666.2012.
Interaction of anesthetics with neurotransmitter release machinery proteins
Zheng Xie,1 Kyle McMillan,2 Carolyn M. Pike,2 Anne L. Cahill,2 Bruce E. Herring,3 Qiang Wang,2
and Aaron P. Fox2
1
Department of Anesthesia and Critical Care, The University of Chicago, Chicago, Illinois; 2Department of Neurobiology,
Pharmacology, and Physiology, The University of Chicago, Chicago, Illinois; and 3Department of Pharmacology, University
of California, San Francisco, California
Submitted 1 August 2012; accepted in final form 1 November 2012
anesthesia; SNARE; synaptic vesicle release; amperometry
IT IS GENERALLY ACCEPTED that most general anesthetics facilitate
GABAA receptor activity, thereby enhancing inhibitory synaptic transmission, and that this alteration in GABAA receptor
activity is an important component of anesthesia. This is
especially true for intravenous general anesthetics, where anesthesia is thought to be produced primarily through the facilitation of GABAA receptors. Volatile anesthetics, on the other
hand, while they facilitate GABAA receptor activity, also have
a variety of other targets. General anesthetics have also been
shown to inhibit presynaptic glutamate release via one or more
presynaptic mechanisms (MacIver et al. 1996; Perouansky
et al. 1995; Westphalen and Hemmings 2003, 2006).
Address for reprint requests and other correspondence: Z. Xie, Univ. of
Chicago, Dept. of Anesthesia and Critical Care, 5841 S. Maryland Ave.,
MC-4028, Chicago, IL 60637 (e-mail: [email protected]).
758
We have previously shown that the commonly used inhalational anesthetic isoflurane dose-dependently inhibits the mammalian neurotransmitter release machinery in neurons and
secretory cells (Herring et al. 2009). We have also shown that
the intravenous anesthetics propofol and etomidate inhibit the
neurotransmitter release machinery (Herring et al. 2011). In
contrast, drugs with similar composition and hydrophobicity,
but which do not produce anesthesia, did not inhibit neurotransmitter release (Herring et al. 2009, 2011). These previous studies suggested that the t-SNARE syntaxin 1A may be an
intermediary in the inhibitory effects of general anesthetics on
neurosecretion. Other evidence that syntaxin 1A may be a
target of isoflurane includes the demonstration that isoflurane
binds to syntaxin 1A (Nagele et al. 2005) and that the Caenorhabditis elegans syntaxin 1A mutation (md130A) has diminished behavioral sensitivity to isoflurane (van Swinderen et al.
1999). In our previous studies, we found that overexpression of
the syntaxin 1A mutant md130A in PC12 cells completely
blocked the inhibitory responses to both isoflurane and propofol but had no effect on the response to etomidate (Herring
et al. 2009, 2011). These results suggested that general anesthetics may target SNARE or SNARE-associated proteins, but
that different anesthetics may target different proteins. The
goal of the present study was to examine the possible role of
other SNARE and SNARE-associated proteins in anesthesia
more closely.
To prevent actions of anesthetics on channels or receptors
from altering neurotransmitter release, we use an experimental
paradigm that keeps membrane potential constant but allows
the intracellular Ca2⫹ concentration ([Ca2⫹]i) to be elevated by
a known amount. This paradigm allows the interaction between
anesthetics and the release machinery to be directly probed.
Using an RNA interference (RNAi) strategy, we derived three
knockdown PC12 cell lines that have stably reduced levels of
selected release machinery proteins. The first line had virtually
undetectable levels of SNAP-25 (Cahill et al. 2006), and the
second cell line had no detectable expression of synaptotagmin
I (Cahill et al. 2006; Moore et al. 2006). In the third cell line,
SNAP-25 was completely ablated and there was a partial
knockdown of SNAP-23. Neurotransmitter release was observed in all three PC12 cell lines after stimulation. The effects
of isoflurane, propofol, and etomidate were tested in these three
cell lines. The anesthetics were all less effective at suppressing
neurotransmitter release in each cell line. Our data are best
explained by positing that the anesthetics interact with multiple
release machinery proteins but do not have a common site of
action.
0022-3077/13 Copyright © 2013 the American Physiological Society
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Xie Z, McMillan K, Pike CM, Cahill AL, Herring BE, Wang Q,
Fox AP. Interaction of anesthetics with neurotransmitter release
machinery proteins. J Neurophysiol 109: 758 –767, 2013. First published November 7, 2012; doi:10.1152/jn.00666.2012.—General anesthetics produce anesthesia by depressing central nervous system
activity. Activation of inhibitory GABAA receptors plays a central
role in the action of many clinically relevant general anesthetics. Even
so, there is growing evidence that anesthetics can act at a presynaptic
locus to inhibit neurotransmitter release. Our own data identified the
neurotransmitter release machinery as a target for anesthetic action. In
the present study, we sought to examine the site of anesthetic action
more closely. Exocytosis was stimulated by directly elevating the
intracellular Ca2⫹ concentration at neurotransmitter release sites,
thereby bypassing anesthetic effects on channels and receptors, allowing anesthetic effects on the neurotransmitter release machinery to be
examined in isolation. Three different PC12 cell lines, which had the
expression of different release machinery proteins stably suppressed
by RNA interference, were used in these studies. Interestingly, there
was still significant neurotransmitter release when these knockdown
PC12 cells were stimulated. We have previously shown that etomidate, isoflurane, and propofol all inhibited the neurotransmitter
release machinery in wild-type PC12 cells. In the present study, we
show that knocking down synaptotagmin I completely prevented
etomidate from inhibiting neurotransmitter release. Synaptotagmin
I knockdown also diminished the inhibition produced by propofol
and isoflurane, but the magnitude of the effect was not as large.
Knockdown of SNAP-25 and SNAP-23 expression also changed
the ability of these three anesthetics to inhibit neurotransmitter
release. Our results suggest that general anesthetics inhibit the
neurotransmitter release machinery by interacting with multiple
SNARE and SNARE-associated proteins.
GENERAL ANESTHETICS AND SNARE PROTEINS
MATERIALS AND METHODS
previously (Cahill et al. 2006). The following regions were selected
as targets: bp 663– 681 of synaptotagmin cDNA (NM_00103368:
AGACTTAGGGAAGACCATG), bp 355–373 of SNAP-25 cDNA
(AF245227: GTTGGATGAGCAAGGCGAA), and bp 727–745 of
SNAP-23 cDNA (NM_022689: CAAGAATCGCATTGACATT) for
knockdown of rat synaptotagmin, SNAP25, and SNAP-23, respectively. pshRNA/Neo-synaptotagmin and pshRNA/Neo-SNAP25 were
transfected into PC12 cells with Lipofectamine 2000 to generate the
synaptotagmin and SNAP-25 knockdown cells, respectively. The
SNAP-25 knockdown cell line was further transfected with pshRNA/
HisD-SNAP-23 to generate SNAP-23 and SNAP-25 double-knockdown cells. The cells were replated one day after transfection at a
lower density and selected with 100 ␮g/ml G418 (or 2 mM histidinol)
for 3– 4 wk until discrete colonies were formed. Individual colonies
were isolated, grown up, and tested by PCR for the incorporation of
the plasmid into genomic DNA. The cell lines with the largest
reduction of synaptotagmin, SNAP-25, and SNAP-23 were identified
by immunoblotting with the following antibodies: SNAP-25 (ANR001; Alomone), SNAP-23 (DS-19; Sigma), synaptotagmin I (mAb48;
Developmental Systems Hybridoma Bank, University of Iowa), ␤-actin (JLA20; Developmental Systems Hybridoma Bank), and horseradish peroxidase-labeled anti-mouse or anti-rabbit IgG (Jackson
ImmunoResearch).
For each knockdown, the stable cell lines that were constructed
transmitted the knockdown indefinitely from weekly passage to
weekly passage (Cahill et al. 2006). To prevent phenotypic drift
(which can be a problem with PC12 cells), the cultures were used for
only ⬃12–14 wk before reverting to frozen stocks from an early
passage. At no time was any reversal of knockdown observed. The
protein suppression shown in Figs. 1 and 2 is representative of the cell
lines.
Immunofluorescence. PC12 cells (original or with gene knockdown) were cultured on Lab-Tek Chamber Slides (Nalge Nunc
International) and were washed with PBS, fixed with 4% paraformaldehyde in PBS for 25 min at room temperature, quenched 10 min with
75 mM NH4Cl and 20 mM glycine, permeabilized, and blocked with
0.2% Triton X-100 in PBS complemented with 10% FBS for 30 min
at 37°C and then incubated 2 h at 37°C with the primary antibodies
anti-SNAP25 antibody (Santa Cruz), anti-SNAP23 antibody (Sigma),
and anti-synaptotagmin antibody (Developmental Studies Hybridoma
Bank). Next, the cells were incubated for 1 h with either Alexa Fluor
594-labeled goat anti-rabbit secondary antibody or Alexa Fluor 488labeled goat anti-mouse secondary antibody (Invitrogen). After washing, the slides were mounted with VectaShield (Vector Laboratories,
Burlingame, CA) and analyzed with an Eclipse E600 microscope
(Nikon) and a ⫻40 objective.
Statistical analysis. A Student’s t-test was used to assess differences between populations of cells.
RESULTS
Details of the derivation of two of the three knockdown
PC12 cell lines used in this study have been previously published (Cahill et al. 2006; Roden et al. 2007). Figures 1 and 2
show the extent of knockdown of SNAP-25, SNAP-23, and
synaptotagmin I in each of the three lines. In the case of
synaptotagmin I and SNAP-25, barely detectable levels of
these proteins are present after the RNAi knockdown (Figs. 1
and 2). In the case of the SNAP-25/SNAP-23 double knockdown, levels of SNAP-23 were reduced by ⬎60% (Figs. 1 and
2). Figure 2 shows that synaptotagmin I, SNAP-25, and
SNAP-23 were distributed uniformly in wild-type PC12 cells
and that the knockdown was also uniform throughout the cells.
This latter result was not surprising, as each cell line was
derived from a single cell and thus represents a clonal line.
These cell lines are used throughout the rest of this report to
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Ethical information. All of the experiments outlined in this report
were carried out in PC12 cells. No approval is required for these cells.
PC12 cell culture. PC12 cells were grown on collagen-coated
10-cm petri dishes in culture medium that consisted of RPMI 1640,
10% heat-inactivated horse serum, 5% fetal bovine serum, 2 mM
glutamine, and 10 ␮g/ml gentamicin in a humidified 7% CO2 incubator at 37°C. Culture medium was replaced every other day, and cells
were passaged once per week. Cells were replated on poly-lysinecoated glass coverslips 24 – 48 h prior to recording. After ⬃8 wk in
culture cells were discarded, and previously frozen stocks were
thawed and then kept in culture.
Amperometric measurement of catecholamine release. Carbon fiber
electrodes were fabricated and used as previously described by Grabner et al. (2005). Each carbon fiber electrode was positioned such that
it gently pushed against the PC12 cell in order to record amperometric
events, which correspond to the currents produced by the carbon
fiber’s oxidation of the neurotransmitter content of a single vesicle.
The detection threshold for amperometric events was set at 5 times the
baseline root mean squared noise, and the spikes were automatically
detected. Amperometric spike features, quantal size, and kinetic
parameters were analyzed with a series of macros written in IGOR Pro
(WaveMetrics) and kindly supplied to us by Dr. Eugene Mosharov
(Department of Neurology, Columbia University, New York, NY).
Cell-to-cell variation can be a problem between experiments. The
most important source of variability appears to be change in cellular
properties with time. PC12 cells are kept as frozen stocks. After an
aliquot of cells was defrosted, it typically took 2–3 wk for the cultures
to recover fully from freeze. After cultures are kept in the incubator
for several months, the cells stop secreting even with strong stimuli.
There could be significant cell-to-cell variation in experiments performed on different days. Cell-to-cell variation in experiments done
on the same day with the same cultures was modest. Thus, for each
recording for an experimental group, a control cell was added on the
same day at about the same time. Without a matching control the
experiment was not used. Cells that exhibited no neurotransmitter
release after stimulation, a relatively rare occurrence, were removed
from the data set. A Student’s t-test was used to assess differences
between populations of cells.
PC12 cell permeabilization and stimulation. An amperometric
electrode was positioned against a cell. After 2 min in a Ca2⫹-free
solution (step 1), the cell was permeabilized with 20 ␮M digitonin
(Ca2⫹ free) for 25 s (step 2) and then stimulated for 2–3 min with a
solution containing 100 ␮M Ca2⫹ (step 3). The cell was allowed to
recover for 2 min in Ca2⫹-free medium (step 4), and the cycle began
again at step 2. Cells were stimulated three or four times in this way,
in order to identify the largest response. For cells treated with
anesthetic, isoflurane, propofol (Sigma-Aldrich, St. Louis, MO), or
etomidate (Hospira, Lake Forest, IL) was introduced into the bath 25 s
prior to stimulation and was present throughout the recording. This
was done in order to maximize anesthetic exposure time. The stimulation step (step 3) producing the greatest amount of release was
analyzed. The recording solutions had standard compositions previously described by Grabner et al. (2005). [Isoflurane solution was
prepared and measured as previously described (Jones et al. 1992;
Jones and Harrison 1993; Xie et al. 2006). Briefly, isoflurane solutions
were prepared by injection of a pure liquid form of isoflurane with
gastight syringes into evacuated saline bags containing defined volumes of extracellular solution. Isoflurane was then applied to the
chromaffin cell preparation via the extracellular solution. Because
isoflurane can slowly leak from the bags, they were only used for a
short period before they were discarded and replaced with fresh bags.
The concentration of isoflurane in the bath was accurate (Xie et al.
2006)].
Gene knockdown. The plasmid used for generating short hairpin
(sh)RNAs (pshRNA/Neo) was constructed in our lab as described
759
B
PC12
SNAP-25 KD
PC12
A
Synaptotagmin I KD
GENERAL ANESTHETICS AND SNARE PROTEINS
SNAP-25/ SNAP-23 KD
760
SNAP-25
SNAP-23
Synaptotagmin I
Actin
Actin
determine the effects of release machinery proteins on anesthetic actions.
Exocytosis was elicited by permeabilizing cells with digitonin in the presence of 100 ␮M extracellular Ca2⫹, in the
presence and absence of isoflurane. This method of stimulating
cells directly regulates [Ca2⫹]i at the neurotransmitter release
sites, while bypassing any requirement for activation of channels and receptors. On each day of recording, amperometric
measurements of catecholamine secretion were made from a
similar number of experimental and control cells. This strategy
reduced day-to-day variation. The proximity of Ca2⫹ channels
to synaptic release sites suggests that [Ca2⫹]i may rise to levels
above 100 ␮M at the vesicle (Llinas et al. 1992).
Although previous studies with these cell lines found significant differences in amperometric event properties when
comparing wild-type and synaptotagmin I knockdown cell
lines (Moore et al. 2006), we observed no such differences (see
DISCUSSION for explanation). Amperometric traces were similar
(Fig. 3A), as were half-times (Fig. 3B) and the number of
molecules released per event (Fig. 3C). The only difference
observed in our study was that the number of events per
stimulus was significantly reduced in the SNAP-25/SNAP-23
double knockdown cells [wild type: 94.8 ⫾ 9.4 (n ⫽ 25) events
per stimulus; synaptotagmin I KD: 98.8 ⫾ 8.9 (n ⫽ 25);
SNAP-25 KD, 84.04 ⫾ 9.9 (n ⫽ 25); SNAP-25/SNAP-23
double KD: 51.88 ⫾ 8.1 (n ⫽ 25)]. The fact that the amperometric properties were similar between the different cell lines
facilitated comparisons and meant that any changes in release
rate elicited by anesthetics were easily quantifiable.
Effects of isoflurane, propofol, and etomidate on catecholamine secretion in synaptotagmin I knockdown PC12 cells. We
have previously shown that isoflurane significantly inhibits
neurotransmitter release in PC12 cells and hippocampal neurons at clinically relevant concentrations (Herring et al. 2009).
In this earlier study 0.5 mM isoflurane significantly reduced the
number of amperometric events by ⬃38% (P ⬍ 0.05, n ⫽ 24),
but it had no effect on amperometric event amplitude, charge,
or kinetics. This experiment has been repeated. Figure 4, A and
B, plot representative amperometric currents obtained in the
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Fig. 1. Knockdown of SNAP-25, SNAP-25/SNAP-23, and synaptotagmin I by
RNA interference in PC12 cells. A: immunoblots were used to assess the levels
of SNAP-25, SNAP-23, and ␤-actin in wild-type PC12 cells, in SNAP-25
knockdown PC12 cells, and in SNAP-25/SNAP-23 double-knockdown PC12
cells. Note that the SNAP-23 knockdown was incomplete. Equal amounts of
protein were loaded per lane. B: immunoblots were used to assess the levels of
synaptotagmin I in wild-type PC12 cells and in synaptotagmin I knockdown
PC12 cells.
absence (Fig. 4A) and the presence of isoflurane (0.5 mM, Fig.
4B; 0.5 mM isoflurane is a concentration of ⬃1.6 MAC; a
MAC is the minimum alveolar concentration of an anesthetic
required for immobility in response to a noxious stimulus in
50% of trials) in wild-type PC12 cells. The amperometric trace
in the presence of isoflurane contained many fewer amperometric events. On average isoflurane significantly inhibited the
number of amperometric events by ⬃39% (Fig. 4C; P ⬍ 0.01,
n ⫽ 19). The data in Fig. 4C have been normalized to the
number of events observed in the absence of isoflurane.
A representative amperometric current observed in a digitonin-perforated synaptotagmin I knockdown PC12 cell exposed to Ca2⫹ (100 ␮M) in the absence of isoflurane is shown
in Fig. 5A. A representative amperometric current observed in
a cell exposed to isoflurane (0.5 mM) is shown in Fig. 5B.
Treatment of synaptotagmin I knockdown cells with isoflurane
resulted in an ⬃18% reduction in the number of amperometric
events observed compared with control cells (Fig. 5C). This
difference was not significant (P ⫽ 0.17, n ⫽ 16). Compared
with the results shown in Fig. 4, the results in Fig. 5 suggest
that knocking down synaptotagmin I lessened the response to
isoflurane.
Isoflurane at 1 mM represents a dose rarely used clinically
(⬎3 MAC) but one that is especially convenient for testing in
order to determine whether the synaptotagmin I knockdown
suppressed the response to isoflurane. This concentration of
isoflurane significantly reduced neurotransmitter release by
⬎60% in wild-type PC12 cells (Herring et al. 2009). Figure 5D
plots the response of synaptotagmin I knockdown PC12 cells to
1 mM isoflurane and shows that the anesthetic reduced the
amount of neurotransmitter released by ⬃34%, a difference
that was not significant (P ⫽ 0.12, n ⫽ 15). The net effect of
knocking down synaptotagmin I was to lessen the effect of
isoflurane by about half compared with that observed in wildtype cells, at both 0.5 and 1 mM.
We have previously shown that propofol and etomidate
significantly inhibit neurotransmitter release in PC12 cells,
chromaffin cells, and hippocampal neurons at clinically relevant concentrations (Herring et al. 2011). In this earlier study
5 ␮M propofol significantly reduced the number of amperometric events by ⬃51% (P ⬍ 0.05, n ⫽ 16), but it had no effect
on amperometric event amplitude, charge, or kinetics. This
experiment has been repeated in wild-type PC12 cells. The bar
chart in Fig. 6A plots the normalized average number of events
in the absence (“control”) and presence of propofol (5 ␮M).
Propofol-treated cells exhibited 49.1% fewer amperometric
events compared with control cells (P ⬍ 0.02 , n ⫽ 12), a
reduction very similar to that observed in our earlier study
(Herring et al. 2011). Figure 6B plots the normalized representative amperometric currents obtained in the absence and the
presence of etomidate (10 ␮M). Etomidate-treated cells exhibited 47.3% fewer amperometric events, a value very similar to
that of our earlier study (Herring et al. 2011).
Suppressing synaptotagmin I expression had an even larger
effect on propofol’s ability to suppress neurotransmitter release. Previously we had shown that propofol inhibited the
neurotransmitter release machinery in a dose-dependent fashion over a range of clinically relevant concentrations (Herring
et al. 2009), reported to be 0.4 –10 ␮M (Hadipour-Jahromy and
Daniels 2003; Krasowski and Harrison 1999; Sprung et al.
2001). In wild-type PC12 cells, 5 ␮M propofol produced a
GENERAL ANESTHETICS AND SNARE PROTEINS
Wild-Type PC12 Cells
761
Knock-Down PC12 Cells
A
B
Antibody To
Synaptotagmin 1
Wild-Type Synaptotagmin I
Synaptotagmin I Knock-Down
D
Antibody To
SNAP-25
SNAP-25 Knock-Down
E
Wild-Type SNAP-25
SNAP-25/SNAP-23 Knock-Down
F
G
Antibody To
SNAP-23
Wild-Type SNAP-23
SNAP-25/ SNAP-23 Knock-Down
significant ⬃51% reduction in neurotransmitter release (Herring et al. 2011). In contrast, 5 ␮M propofol had almost no
effect on neurotransmitter release in synaptotagmin I knockdown PC12 cells (Fig. 7A; P ⫽ 0.84, n ⫽ 15).
Suppression of synaptotagmin I had the largest effect on
etomidate’s ability to inhibit neurotransmitter release. We had
previously shown that etomidate inhibited neurotransmitter
release in a dose-dependent manner, with 40 ␮M etomidate
inhibiting neurotransmitter release in wild-type PC12 cells by
⬃56% (Herring et al. 2011). In contrast, 40 ␮M etomidate
potentiated neurotransmitter release in synaptotagmin I knockdown PC12 cells (Fig. 7B). In this case, etomidate augmented
the number of amperometric events by ⬃71%. This difference
was not significant (P ⫽ 0.065, n ⫽ 12). Synaptotagmin I
appears to play a key role in the inhibitory response to
etomidate. [Large concentrations of propofol and etomidate
were tested to determine whether the synaptotagmin I knockdown completely suppressed the response to anesthetic.]
Effects of isoflurane, propofol, and etomidate on catecholamine secretion in SNAP-25 knockdown PC12 cells. In SNAP-25
knockdown PC12 cells 1 mM isoflurane reduced the average
number of amperometric events by ⬃54% (P ⫽ 0.03, n ⫽
21), a response that was not different from that observed in
wild-type PC12 cells (Fig. 8A). In contrast, the responses to
propofol and to etomidate were diminished by the knockdown of SNAP-25. Figure 8B shows that propofol (5 ␮M)
reduced the amount of neurotransmitter release by ⬃27%.
This response, about half that observed in wild-type PC12
cells, was not significant (P ⫽ 0.09, n ⫽ 31). Etomidate (40
␮M) reduced neurotransmitter release by ⬃8% (Fig. 8C;
P ⫽ 0.73, n ⫽ 25), a response only about one-seventh that
observed in wild-type PC12 cells. Thus knockdown of
SNAP-25 expression reduced the inhibitory effect of propofol and almost completely eliminated the response to etomidate but did not change the inhibition produced by
isoflurane.
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C
Fig. 2. Knockdown of SNAP 25, SNAP-23, and
synaptotagmin I were confirmed with immunofluorescence measurements. Wild-type PC12 cells (left)
or synaptotagmin I, SNAP25, or SNAP-23/SNAP-23
knockdown cells (right) were stained with corresponding primary antibodies and then Alexa Fluor
594 (or Alexa Fluor 488)-labeled secondary antibodies. Synaptotagmin expression was examined in
wild-type PC12 cells (A) and synaptotagmin knockdown cells (B). SNAP-25 expression was examined
in wild-type PC12 cells (C), SNAP-25 knockdown
cells (D), and SNAP-23/SNAP-25 double knockdown cells (E). SNAP-23 expression was examined
in wild-type cells (F) or SNAP-23/SNAP-25 doubleknockdown cells (G). Microscope settings and gain
were identical for all panels.
762
GENERAL ANESTHETICS AND SNARE PROTEINS
A
Wild-Type
Synaptotagmin I KD
SNAP-25 KD
SNAP-25/ KD
SNAP-23
10 pA
20 sec
C
Number of Molecules (x 104)
Half Times (msec)
B
6
4
2
0
W-T
Syt I
Effects of isoflurane, propofol, and etomidate on catecholamine secretion in SNAP-25/SNAP-23 knockdown PC12 cells.
Previous studies have shown that knocking down SNAP-25
leaves significant neurosecretory activity in both chromaffin
and PC12 cells (Cahill et al. 2006; Sorensen et al. 2003), a
B
Control
C
Isoflurane
20 pA
20 sec
Normalized Number of Events
A
1.0
*
0.5
0
Control
Isoflurane
(0.5 mM)
Fig. 4. Isoflurane inhibits neurotransmitter release in permeabilized wild-type
PC12 cells. Digitonin-permeabilized cells were exposed to Ca2⫹ (100 ␮M), as
indicated by the bar underneath the traces, to stimulate release. A and
B: representative amperometric traces in the absence (A) and presence (B) of
isoflurane (0.5 mM). C: averaged number of events in the absence (“Control”)
and the presence of isoflurane (0.5 mM). Isoflurane inhibited release by ⬃39%
(n ⫽ 19), a significant reduction (*P ⬍ 0.01, Student’s t-test).
SNAP-25 SNAP23/25
15
10
5
0
W-T
Syt I
SNAP-25 SNAP23/25
result that was attributed to a partial redundancy of SNAP-25
with SNAP-23. Therefore we tried to produce a PC12 cell line
in which both SNAP-25 and SNAP-23 were knocked down.
Our efforts were met with mixed success. At best, SNAP-25
was reduced below the limits of detection while SNAP-23 was
reduced by ⬎60%. We were never able to achieve complete
knockdown of both proteins; some SNAP-23 was still present
in all surviving cell lines. In our best SNAP-25/SNAP-23
knockdown PC12 cells 1 mM isoflurane reduced the number of
events by ⬃18% (P ⫽ 0.35, n ⫽ 30), a smaller response than
that observed in wild-type PC12 cells (Fig. 9A). In contrast, the
response to propofol was almost exactly the same as that
observed in wild-type PC12 cells; 5 ␮M propofol reduced the
number of amperometric events by ⬃51% (Fig. 9B; P ⫽ 0.01,
n ⫽ 30). This result was surprising given that SNAP-25
suppression alone inhibited much of the response to propofol
(see Fig. 8B). The added partial suppression of SNAP-23
restored the complete response to propofol. The response to
etomidate (10 ␮M) in the SNAP-25/SNAP-23 PC12 cells was
somewhat similar to that observed in the SNAP-25 knockdown
cells. There was no inhibition of neurotransmitter release by
etomidate in the SNAP-25/SNAP-23 PC12 cells. In fact, it
appeared that etomidate potentiated neurotransmitter release,
but this increase was not significant (Fig. 9C; P ⫽ 0.39, n ⫽
18). Thus suppressing both SNAP-25 and SNAP-23 expression
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Fig. 3. Amperometric events recorded from
knockdown cell lines were similar to those from
wild-type cells under our stimulation conditions.
A: representative traces showing 2.5 min of data
from wild-type PC12 cells and the 3 knockdown
cell lines used in these studies, as indicated.
B: averaged half-times of amperometric events
showing no clear difference between the different cell lines (W-T, wild-type PC12 cells; Syt-1,
synaptotagmin I knockdown; SNAP-25, SNAP-25
knockdown; SNAP-25/23, SNAP-25/SNAP-23
double knockdown). Data from 25 different cells
were used for each group shown. C: average
number of molecules of neurotransmitter released per amperometric event for the 4 different
cell lines. No clear difference was observed. n ⫽
25 for each of the 4 groups.
GENERAL ANESTHETICS AND SNARE PROTEINS
Synaptotagmin I Knock-Down
0.5 mM Isoflurane
A
B
Normalized Number Events
B
Control
Synaptotagmin I Knock-Down
Normalized Number Events
A
1.0
0.5
0.0
10 sec
Normalized Number Events
Normalized Number Events
0.6
0.4
0.2
0.0
1.2
1.0
0.6
0.4
0.2
Isoflurane (0.5 mM)
Control
Isoflurane (1 mM)
Fig. 5. Effect of isoflurane on neurotransmitter in permeabilized synaptotagmin
I knockdown PC12 cells. Digitonin-permeabilized cells were exposed to Ca2⫹
(100 ␮M), indicated by the bars below the traces, to elicit neurotransmitter
release. A and B: representative amperometric recording in the absence (A) and
presence (B) of isoflurane (0.5 mM). C: normalized average number of
amperometric events produced by synaptotagmin I knockdown PC12 cells in
the absence (“Control”) and presence of isoflurane (0.5 mM). There was an
⬃18% reduction in the number of events. The difference was not significant
(P ⫽ 0.17, n ⫽ 16). D: averaged number of events in the absence (“Control”) and
presence of isoflurane (1 mM). Isoflurane reduced the amount of neurotransmitter
release by ⬃34%. The difference was not significant (P ⫽ 0.12, n ⫽ 15). Data
were normalized to the mean of the control group throughout the study.
had a large effect on the response to isoflurane and no effect on
propofol and completely eliminated the response to etomidate.
DISCUSSION
Previous studies from our lab and others have suggested
that, in addition to facilitating GABAA receptor activity, cerWild-Type PC12 Cells
B
Propofol
1.0
*
0.5
0.0
Control
Propofol (5µM)
Normalized Number Events
Normalized Number Events
A
0.5
Propofol (5µM)
Control
Etomidate (40µM)
0.8
0.0
Control
1.0
Etomidate
1.0
*
0.5
0.0
Control
Etomidate (10µM)
Fig. 6. Propofol and etomidate inhibit neurotransmitter release in permeabilized PC12 cells. Digitonin-permeabilized cells were exposed to Ca2⫹ (100
␮M) to elicit neurotransmitter release. A: normalized average number of events
in the absence (“Control”) and presence of propofol (5 ␮M). Propofol-treated
cells produced 49.1% fewer amperometric events compared with control cells.
*P ⬍ 0.02 (Student’s t-test); n ⫽ 12. B: normalized average number of events
in the absence (“Control”) and presence of etomidate (10 ␮M). Etomidatetreated cells produced 47.3% fewer amperometric events compared with
control cells. *P ⬍ 0.002 (Student’s t-test); n ⫽ 14.
tain general anesthetics may interact with the SNARE and
SNARE-associated proteins that make up the neurotransmitter
release machinery (Herring et al. 2009, 2011). In this study we
have attempted to identify more specifically which proteins of
the neurotransmitter release machinery are necessary for the
inhibitory action of isoflurane, propofol, and etomidate on
catecholamine secretion from PC12 cells. Using RNAi methods, we have derived knockdown PC12 cell lines that express
nearly undetectable levels of synaptotagmin I or SNAP-25. A
third cell line expressed almost no detectable SNAP-25 and
had significantly reduced levels of SNAP-23. We studied
catecholamine secretion in these cell lines in the presence and
absence of the anesthetics with a permeabilization technique
that allowed direct control of intracellular Ca2⫹ levels sufficient to elicit secretion. This approach was taken in order to
avoid potential confounding effects of anesthetics on channels
and receptors. In wild-type PC12 cells isoflurane, propofol, and
etomidate have all been shown to inhibit catecholamine secretion from permeabilized cells (Herring et al. 2009, 2011). This
effect is specific to the anesthetics, as isoflurane and propofol
were applied without carriers or preservatives while propylene
glycol, the preservative for etomidate, has no effect on neurotransmitter release (Herring et al. 2009). In the present study
we show that knockdown of synaptotagmin I or SNAP-25 or
dual knockdown of SNAP-25 and SNAP-23 prevents or at least
reduces the inhibitory effect of all three anesthetics. This
suggests that these SNARE and SNARE-associated proteins
may mediate at least some of the effects of general anesthetics.
Interestingly, neurotransmitter release is maintained after
knockdown of the different release machinery proteins. This
raises the possibility that there is functional redundancy with
other proteins, some of which may be anesthetic insensitive or
at least less sensitive.
Ca2⫹-dependent fusion of vesicles with the plasma membrane is thought to involve a Ca2⫹ sensor. In neurons, synaptotagmin I, a protein that resides on synaptic vesicles, was
proposed to be the Ca2⫹ sensor that mediates the fast synchronous component of release (Fernandez-Chacon et al. 2001;
Geppert et al. 1994). There are at least 16 different isoforms of
synaptotagmin, and different synaptotagmin isoforms co-reside
on vesicles, raising the possibility that there are multiple Ca2⫹
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D
1.2
0.8
1.5
Fig. 7. The inhibitory effect of propofol and etomidate on neurotransmitter
release was abolished on permeabilized synaptotagmin I knockdown PC12
cells. A: averaged number of events in the absence (“Control”) and presence of
propofol (5 ␮M). The numbers of amperometric events were almost identical
in control cells and synaptotagmin I knockdown cells (P ⫽ 0.84, n ⫽ 15).
B: averaged number of events in the absence (“Control”) and presence of
etomidate (40 ␮M); the difference was not significant (P ⫽ 0.065, n ⫽ 12).
Etomidate increased the number of amperometric events in synaptotagmin I
knockdown PC12 cells. Data were normalized to the mean of the control
group.
5 pA
1.0
2.0
0.0
Control
C
763
764
GENERAL ANESTHETICS AND SNARE PROTEINS
SNAP-25 Knock-Down
1.2
0.8
*
0.0
1.2
0.8
0.8
0.4
0.0
Control
Isoflurane (1 mM)
0.0
Propofol (5µM)
C
1.2
Normalized Number Events
B
0.4
Control
Normalized Number Events
SNAP-25 & SNAP-23 Knock-Down
1.2
0.8
1.2
0.8
*
0.4
0.0
Control
Propofol (5µM)
Control
Etomidate (10µM)
C
0.4
0.0
Control
Etomidate (40µM)
Fig. 8. Isoflurane, but not propofol or etomidate, maintains its inhibitory effect
on neurotransmitter release on SNAP-25 knockdown PC-12 cells. A: averaged
number of events in the absence (“Control”) and presence of isoflurane (1
mM). Isoflurane produced a significant reduction of ⬃54% in the number of
amperometric events (*P ⫽ 0.03, n ⫽ 21). B: averaged number of events in the
absence (“Control”) and presence of propofol (5 ␮M). Propofol reduced the
number of amperometric events by ⬃27%, which was not significant (P ⫽
0.09, n ⫽ 31). C: averaged number of events in the absence (“Control”) and
presence of etomidate (40 ␮M). Etomidate produced an insignificant reduction
of ⬃8% on the number of amperometric events (P ⫽ 0.73, n ⫽ 25). Data were
normalized to the mean of the control group. Note that the inhibitory effect of
isoflurane on neurotransmitter release was largely intact while the inhibitory
effect of etomidate was significantly reduced on SNAP-25 knockdown PC12
cells. The effects of propofol on neurotransmitter release were moderately
reduced on SNAP-25 knockdown PC12 cells.
sensors (Lynch and Martin 2007). Significant evidence exists
that synaptotagmin I, II, VII, and IX operate as Ca2⫹ sensors in
neuronal and neuroendocrine secretion (Cao et al. 2011). There
have been conflicting claims about the importance of various
2.0
1.0
0.0
Fig. 9. The inhibitory effect of isoflurane and etomidate, but not propofol, on
neurotransmitter release is reduced on SNAP-25/SNAP-23 knockdown PC12
cells. A: averaged number of events in the absence (“Control”) and presence of
isoflurane (1 mM). Isoflurane produced an insignificant reduction of ⬃18% in
the number of amperometric events (P ⫽ 0.35, n ⫽ 30). B: averaged number
of events in the absence (“Control”) and presence of propofol (5 ␮M). Propofol
reduced the number of amperometric events by ⬃51%, which is significant
(*P ⫽ 0.01, n ⫽ 30). C: averaged number of events in the absence (“Control”)
and presence of etomidate (10 ␮M). Etomidate did not show any reduction of
the number of amperometric events (P ⫽ 0.39, n ⫽ 18). Data were normalized
to the mean of the control group. Note that the inhibitory effects of etomidate
and isoflurane, but not propofol, on neurotransmitter release were significantly
reduced on SNAP-25/SNAP-23 knockdown PC12 cells.
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Normalized Number Events
Isoflurane (1 mM)
Normalized Number Events
A
0.4
Control
B
synaptotagmin isoforms in neurosecretory PC12 and chromaffin cells; roles for isoforms I, III, V, VII, and IX have all been
proposed (Fukuda 2004; Fukuda et al. 2002, 2004; Lynch and
Martin 2007; Schonn et al. 2008; Sugita et al. 2002). Some
studies have shown that neurotransmitter release persisted in
PC12 cells, even after synaptotagmin I was suppressed by
RNAi, albeit at a reduced level (Moore et al. 2006). Other
Normalized Number Events
Normalized Number Events
A
GENERAL ANESTHETICS AND SNARE PROTEINS
vesicle would load the vesicles more quickly, but the final
neurotransmitter concentration would depend on proton levels,
due to VATPase activity, and not VMAT number, as long as
there was sufficient time to load vesicles completely. Catecholamine content would only be altered in cells that are heavily
stimulated, as they would not have time to refill their vesicles.
In our study we stimulated naive cells, most likely with
completely filled vesicles, even with small numbers of VMAT
per vesicle.
In a similar manner, suppression of SNAP-25 in PC12 cells
reduced but did not eliminate neurotransmitter release when
cells were stimulated with high K⫹ (Cahill et al. 2006; Ray
et al. 1997). Using a cell line deficient in SNAP-25, we
observed differential effects on anesthetic actions. There appeared to be little or no effect on the efficacy of isoflurane to
inhibit neurotransmitter release, while propofol’s actions were
clearly diminished. The largest effects were observed in the
response to etomidate, where reduction of neurotransmitter
release was completely eliminated. As before, these results
suggest that anesthetics interact with different components of
the release machinery.
It has previously been suggested that there is functional
redundancy between SNAP-25 and SNAP-23 (Delgado-Martinez et al. 2007; Sorensen et al. 2003). We attempted to
suppress both SNAP-25 and SNAP-23 in the same PC12 cell;
unfortunately, only a partial suppression (⬃60%) of SNAP-23
(see Fig. 1) was achieved. In this cell line we also observed
differential effects on anesthetic actions. There appeared to be
little or no effect on the efficacy of propofol to inhibit neurotransmitter release, while isoflurane’s actions were clearly
diminished. The largest effects were observed in the response
to etomidate, where reduction of neurotransmitter release was
completely eliminated and potentiation was observed. These
results are consistent with the hypothesis that anesthetics interact with different components of the release machinery.
It was previously shown that a syntaxin 1A mutant could
suppress the actions of anesthetics in C. elegans (van Swinderen et al. 1999), and we showed that overexpressing this same
protein suppressed isoflurane and propofol’s actions in PC12
cells (Herring et al. 2009, 2011). NMR binding studies have
demonstrated the ability of general anesthetic molecules to
bind to syntaxin monomers (Nagele et al. 2005). This result
opens the possibility that syntaxin 1A might be an important
site for anesthetic action (but see Metz et al. 2007 for an
alternate hypothesis recently put forth by Crowder and colleagues whereby anesthetic molecules may inhibit the recruitment to the plasma membrane of the syntaxin activator UNC13, thereby reducing syntaxin 1A activation). Thus we tried to
construct a PC12 cell line deficient in syntaxin 1A. This effort
was not successful.
Our results suggest interactions between anesthetics and
release machinery proteins. There is a considerable literature
supporting the idea that general anesthetics influence glutamate
neurotransmission via presynaptic sites of action (MacIver
et al. 1996; Perouansky et al. 1995; Schlame and Hemmings
1995; Wu et al. 2004), but the literature regarding the ability of
anesthetics to influence the neurotransmitter release machinery
itself remains sparse and unclear. Some studies are consistent
with the possibility of direct modulation of the release machinery by general anesthetics. For instance, Richards and colleagues found that low concentrations of anesthetics affected
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studies have suggested that deletion of synaptotagmin I from
PC12 had no effect on release (Fukuda et al. 2002) or, alternatively, that release levels were similar but exhibited altered
kinetics (Lynch and Martin 2007). It has been suggested that
there is functional redundancy between synaptotagmin I and
synaptotagmin IX in PC12 cells (Lynch and Martin 2007).
Additionally, permeabilization of cells followed by exposure to
100 ␮M Ca2⫹ corresponds to a very strong stimulus, which
may maximize secretion even under nonoptimal conditions.
Thus our results showing relatively robust Ca2⫹-dependent
neurotransmitter release from PC12 cells deficient in synaptotagmin I should come as no surprise. Even so, there were
clear differences in the efficacy of isoflurane, propofol, and
etomidate in their ability to inhibit neurotransmitter release in
synaptotagmin I-deficient PC12 cells compared with wild-type
cells. Perhaps most interesting is the observation that the
suppression of synaptotagmin I did not alter anesthetic actions
in a uniform manner and that the differences were dramatic.
Suppression of synaptotagmin I had the largest effect on
etomidate and the smallest on isoflurane, with propofol being
intermediate. This result suggests that the anesthetics bind to
different functional sites, a conclusion reinforced by previous
work from our lab that showed that overexpressing a syntaxin
1A mutant, md130A, in PC12 cells had large effects on
isoflurane and propofol’s abilities to suppress neurotransmitter
release but did not alter the efficacy of etomidate at all (Herring
et al. 2011).
The data presented in this report are different from those of
Moore et al. (2006), who reported significant differences in
number of amperometric events per stimulation and amperometric properties like half-times and number of molecules of
neurotransmitter per event in synaptotagmin I knockdown
PC12 cells compared with wild-type PC12 cells. We observed
no such differences in our study. This discrepancy is likely due
to the method of stimulation employed, in that previous studies
stimulated cells with high-K⫹ solutions while we used digitonin permeabilization followed by exposure to Ca2⫹. Undoubtedly, the [Ca2⫹]i levels achieved in our studies were
much higher and more maintained throughout the stimulus than
those elicited by high K⫹. We have previously validated the
stimulation protocol by directly dialyzing cells with known
Ca2⫹ concentrations (Herring et al. 2009, 2011). In an elegant
study, Aratlejo and colleagues showed that quantal content in
chromaffin cells depended on Ca2⫹ levels (Elhamdani et al.
2001). Higher [Ca2⫹]i completely emptied vesicles, while
lower levels did not. We interpret the difference between our
study and this earlier study to be that once synaptotagmin I was
knocked down high-K⫹ stimulation did not provide sufficient
Ca2⫹ to empty vesicles completely because of altered Ca2⫹
sensitivity. Digitonin permeabilization followed by exposure to
100 ␮M Ca2⫹, the conditions used in the present report,
elevated [Ca2⫹]i to a level at which vesicular release rates were
maintained and the vesicles emptied completely compared with
wild-type cells. The large stimulus used in our study maximized release and allowed for direct comparisons between
wild-type and knockdown PC12 cells.
Roden et al. (2007) found that VMAT1 expression was
reduced in synaptotagmin I knockdown PC12 cells. Wouldn’t
that give rise to altered vesicle neurotransmitter content? Possibly not. It is generally thought that the copy number of
VMAT per vesicle is small. Higher expression of VMAT per
765
766
GENERAL ANESTHETICS AND SNARE PROTEINS
knockdown of synaptotagmin I and attenuated by knockdown
of SNAP-25. Co-knockdown of both SNAP-25 and SNAP-23
restored propofol-mediated inhibition. These results exhibit
two particularly noteworthy features. First, robust neurotransmitter release was observed after every release machinery
alteration, and second, every alteration produced some change
in anesthetic efficacy. These results suggest that viable but
unique release machinery complexes formed even after the
alterations were made and that each one appeared to interact
with anesthetic in an independent manner. These data are
consistent with a model in which propofol preferentially interacts with a binding pocket created by synaptotagmin I, SNAP25, and perhaps other release machinery proteins. This binding
pocket might provide a more hydrophobic environment to
stabilize the anesthetics. Inhibition of neurotransmitter release
with etomidate was also prevented by knockdown of synaptotagmin I, in that a reduction in the expression level of
synaptotagmin I appeared to convert etomidate from an inhibitor of release to a potentiator. Synaptotagmin I may therefore
represent an essential component for etomidate-mediated release of inhibition, and a reduction in synaptotagmin I expression may unmask an ability of etomidate to promote neurotransmitter release. Knocking down SNAP-25 also prevented
etomidate from inhibiting neurotransmitter release, consistent
with a role for synaptotagmin I and SNAP-25 in the creation of
a binding pocket necessary for etomidate-mediated inhibition.
While the present data suggest that multiple SNARE proteins
contribute to the inhibitory effects of general anesthetics on
neurotransmitter release, additional work will be necessary to
determine whether unique binding sites created by SNARE
complexes are responsible for the distinct effects of isoflurane,
propofol, and etomidate in PC12 cells deficient in the expression of specific SNARE proteins.
GRANTS
This study was supported by a National Institute of General Medical
Sciences grant to A. P. Fox, a National Institute of Neurological Disorders and
Stroke grant to B. E. Herring, and Foundation for Anesthesia Education and
Research and Brain Research Foundation grants to Z. Xie.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: Z.X. and A.P.F. conception and design of research;
Z.X., K.M., C.M.P., A.L.C., B.E.H., and Q.W. performed experiments; Z.X.,
K.M., C.M.P., A.L.C., B.E.H., Q.W., and A.P.F. analyzed data; Z.X. and
A.P.F. interpreted results of experiments; Z.X., Q.W., and A.P.F. prepared
figures; Z.X. and A.P.F. edited and revised manuscript; Z.X. and A.P.F.
approved final version of manuscript; A.P.F. drafted manuscript.
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