From the
Multiple receptor subtypes activated by the same ligand but
coupled to different second messengers can produce divergent signaling
in a cell, while receptors activated by different ligands but sharing
the same second messenger can produce convergent signaling. We show
here that chick ciliary ganglion neurons have three classes of
receptors activated by the same neurotransmitter, acetylcholine, and
that all three regulate the same second messenger, intracellular free
calcium. Activation of muscarinic receptors on the neurons stimulates
phosphatidylinositol turnover and induces calcium oscillations that are
initiated and maintained by calcium release from
caffeine/ryanodine-insensitive intracellular stores. Extracellular
calcium is required to sustain the oscillations, while cadmium
abolishes them. Activation of either of two classes of nicotinic
receptors, distinguished both by location on the neurons and by subunit
composition, induces a single, rapid elevation in intracellular calcium
without inducing phosphatidylinositol turnover. The nicotinic responses
are entirely dependent on extracellular calcium, show no dependence on
release from internal stores, and do not display oscillations. Low
concentrations of the native agonist, acetylcholine, induce repetitive
calcium spikes in the neurons characteristic of muscarinic receptors,
while higher concentrations induce nonoscillating increases in
intracellular calcium that include contributions from nicotinic
receptors. The three classes of receptors also differ in the
acetylcholine concentration required to elicit a response. These
differences, together with differences in receptor location and sources
of calcium mobilized, may enable the receptor subtypes to target
different sets of calcium-dependent processes for regulation.
Intracellular calcium is widely used as a second messenger and
regulates a vast array of cellular events. Examples include exocytosis,
long-term synaptic modulation, cytoskeletal restructuring, gene
expression, and cell death. Often cells have receptor types responding
to different agonists but sharing an ability to elevate intracellular
calcium ([Ca
Cholinergic receptors activated by the endogenous agonist
acetylcholine (ACh)
Chick ciliary ganglion neurons
provide a system for comparing the effects of nicotinic and muscarinic
receptors on [Ca
The experiments
reported here use calcium imaging with the fluorescent indicator fluo-3 (20) to compare the abilities of cholinergic receptor subclasses
on the neurons to increase
[Ca
Repetitive [Ca
Thapsigargin
had the most pronounced effect. At 1-5 µM, it
increased the portion of neurons displaying an abbreviated response, i.e. 1-2 spikes, at the expense of neurons displaying a
sustained or oscillatory response throughout the period of agonist
application (Fig. 3B; ). The effect was more
dramatic when a second test with muscarine was carried out on
thapsigargin-treated cells. In this case, 50% of the neurons (7 out of
14 cells) showed no response to muscarine at all while only 14% showed
oscillations. The majority of control neurons (no exposure to
thapsigargin) from the same cultures responded vigorously to the second
application of muscarine (74% oscillated; 23 cells), while only 9% did
not respond. DTBHQ produced effects similar to those in neurons treated
with thapsigargin. About half of the DTBHQ-treated neurons (47%; 19
cells) gave no response to a second application of muscarine while only
a quarter (26%) showed oscillatory activity. The results indicate that
thapsigargin- and DTBHQ-sensitive intracellular calcium stores
contribute to muscarine-induced oscillations in
[Ca
The observation that muscarine-induced
oscillations in [Ca
Two
conclusions emerge. First, extracellular calcium is not required for
the initial calcium increase caused by muscarine because the receptors
trigger release from internal stores, presumably in an
IP
Evidence that such channels
are involved was obtained by showing that cadmium at 200 µM prevented muscarine-induced oscillations (40/40 neurons). In this
case, muscarine induced only a single transient calcium elevation,
similar to the response seen in calcium-free medium. In about one-third
of the cadmium-treated neurons, the single spike was followed by a
second calcium elevation that did not return to baseline even after
removal of the agonist. No oscillations were observed. Cadmium at this
concentration completely inhibits the current triggered by activation
of VDCCs in ciliary ganglion neurons(41) ,
Nicotine at 10 µM activates both
The results show that nicotine-induced responses differ from
muscarine-induced responses in two ways. Unlike the muscarinic
response, nicotine-induced elevations in
[Ca
ACh was bath-applied
for 2.5 min to fluo-3-loaded neurons. At 1 µM, ACh induced
oscillatory activity in about 40% of the neurons (Fig. 5A; ). Nicotinic receptors did not
contribute significantly because no change was detected in the
percentage of neurons that oscillated or in the nature of the
oscillatory response when the neurons were incubated for 1 h with
either 60 nM
A somewhat different
picture emerges at 50 µM ACh. The control response
revealed little in the way of oscillatory behavior in most cases (Fig. 6A; ), but treatment with
The findings reported here demonstrate for the first time
that a single neuron can have as many as three kinds of receptors
responding to the same neurotransmitter, driving the same second
messenger, and displaying differences while doing so. In the present
case where the transmitter is ACh and the second messenger is calcium,
the differences can include the temporal pattern of calcium elevation
produced, the source of calcium mobilized, and the agonist
concentration required for the mobilization. These differences,
together with previously reported differences in receptor localization,
may enable individual receptor classes to exert unique regulatory
effects, targeting different sets of calcium-dependent processes in the
cells. Regulation of [Ca
Not
all neurons from the ganglion responded to muscarine with oscillations.
Variation in the pattern of calcium response has been seen in other
systems (25) and has been attributed to differences in basal
[Ca
Both intracellular and extracellular sources of calcium
are mobilized to initiate and maintain muscarinic
[Ca
The patterns of [Ca
The
simplest explanation for the patterns of
[Ca
Both mAb 35- and
Distinctive patterns of
[Ca
Two additional factors likely
to be important in determining the role of individual receptor subtypes
are receptor affinity for agonist and receptor location. Muscarinic
receptors have the greatest impact on
[Ca
At the vertebrate
neuromuscular junction, significant amounts of ACh diffuse only a
relatively short distance from the site of release because of the high
levels of acetylcholinesterase activity present (67). On the other
hand, ACh released from the vagus nerve onto pacemaker neurons in the
heart spreads up to 1 µm away within 0.2 ms while declining about
10-fold in concentration(68) . If synapses on ciliary ganglion
neurons display a similar spread of transmitter, mAb 35-AChRs may
elevate [Ca
Neurons were treated
with vehicle (Control), 1 µM thapsigargin (Thapsigargin),
10 µM ryanodine plus 30 µM caffeine
(Ryan/Caff), or 10 µM ryanodine alone (Ryanodine) and
tested for muscarine-induced fluorescence responses as described in
Fig. 3. Responses were scored as oscillatory/sustained (
Neurons loaded with fluo-3AM were tested for
fluorescence responses induced by the indicated concentrations of ACh
after treatment with the indicated blockers (
]
)
levels(1, 2) . The multiplicity of receptors enables
different signaling pathways to converge on the same molecular targets.
Less obvious is the virtue of a cell expressing several classes of
receptors that elevate [Ca
]
in response to the same hormone orneurotransmitter.
(
)produce elevations in
[Ca
]
in many cell
types. Muscarinic acetylcholine receptors, found on glands, smooth
muscle, cardiac muscle, and neurons elevate
[Ca
]
by stimulating
release from intracellular stores (for review, see Ref. 3). Nicotinic
acetylcholine receptors (nAChRs) found throughout the nervous system
and in skeletal muscle (for review, see Ref. 4) are also effective at
elevating [Ca
]
.
Recently it has been shown that several types of neuronal nAChRs are
highly permeable to
calcium(5, 6, 7, 8) . Most prominent in
this respect are nAChRs that bind
Bgt and contain the
7 gene
product(9, 10) .
]
in
the same cells. The neurons have two major classes of nAChRs: mAb
35-AChRs that bind the monoclonal antibody mAb 35 and are primarily
synaptic in location, and
Bgt-AChRs that bind
-bungarotoxin
(
Bgt) and are primarily nonsynaptic in
location(11, 12) . mAb 35-AChRs collectively contain the
3,
4, and
5 gene products found in the neurons (13) and mediate synaptic transmission through the
ganglion(14) .
Bgt-AChRs contain the
7 gene product
and function as ligand-gated ion channels(13, 15) , but
they have yet to be assigned a physiological role in the ganglion. Both
classes of receptors elevate [Ca
]
in the neurons(16, 17) . In addition, ciliary
ganglion neurons have muscarinic receptors capable of increasing
[Ca
]
and generating a
slow inward current(18, 19) .
]
. The results
demonstrate that both nicotinic and muscarinic receptors contribute to
[Ca
]
elevations when
activated by ACh but do so by relying on different sources of calcium
and different mechanisms of activation. The two receptor classes also
differ with respect to the temporal pattern of calcium changes they
produce. All three of the receptor subtypes differ with respect to the
ACh concentrations at which they demonstrably affect
[Ca
]
. These
differences may enable the receptors to achieve distinctive effects on
calcium-dependent processes.
Cell Preparations
For inositol phosphate (IP)
measurements, cultures were prepared with chick ciliary ganglion
neurons from 8-day embryos and were maintained for 6 days before
analysis as described previously(21) . For experiments measuring
calcium-dependent fluorescence, cultures were prepared by dissecting
ganglia from 14- to 15-day-old chick embryos, cutting them into halves,
incubating them for 30 min at 37 °C in recording solution
containing 1 mg/ml collagenase but no divalent cations, and then
rinsing and triturating as described previously(22) . The cells
were plated on plastic Costar dishes coated with poly-D-lysine
and incubated in complete culture medium containing 3% (v/v) embryonic
chick eye extract (21) for 1 h at 37 °C to allow attachment
of cells to the substratum prior to analysis. No attempt was made to
isolate or record selectively from ciliary or choroid neurons.
IP Release Assay
Ciliary ganglion cultures
containing 8-10 dissociated ganglia per 35-mm dish were incubated
overnight in inositol-free minimal essential medium containing 10%
horse serum, 3% eye extract, and [H]inositol (1
µC
/dish). The cells were then washed 5 times with rinse
buffer (serum-free culture medium containing 10 mM LiCl) and
incubated for an additional 15 min in the same buffer.
Phosphatidylinositol turnover was initiated by removing the medium and
adding 1 ml of the indicated agonist in rinse buffer. Unless otherwise
indicated, all agonist incubations were for 20 min at 37 °C.
Reactions were stopped by removing the media and scraping cells in 100
µl of NaPO
buffer, pH 7.4, containing 10 mM LiCl and 0.1% Triton X-100. Lipids were extracted, and an aliquot
of the chloroform phase was counted in a scintillation counter to
determine incorporation of label into phosphatidylinositols. The
aqueous phase was removed, diluted 3-fold with water, and incubated
with 150 µl of the anion exchange resin AG-X8 (formate form) for 15
min at room temperature. The mixture was then centrifuged briefly, and
the supernatant was removed. An aliquot of the supernatant was counted
to determine the proportion of label existing as free inositol. The
resin was then washed four times with 1 ml of 5 mM inositol.
IPs were eluted with 500 µl of solution containing 1 M ammonium formate and 0.1 M formic acid and counted. Data
were fit to curves using Inplot (GraphPAD, San Diego, CA).
Fluorescence Measurements
Cells were loaded with
the calcium indicator fluo-3AM for 30 min at room temperature in the
dark as described previously(16) . The cells were perfused with
recording solution and examined for epifluorescence with a 16
neofluor water immersion objective and a 100-watt mercury vapor lamp
fitted with a narrow band pass excitation filter (H485, Zeiss) on a
Zeiss photomicroscope. Neutral density filters of 0.5-1.5 optical
density were used to reduce photobleaching. Field images were scanned
with a silicon-intensified target camera (Dage) and accumulated every
1.8 or 3.6 s. Intermittent illumination of the cells (0.4 or 0.8 s per
image) was accomplished using a shutter and shutter-controlling device
which also triggered the Quadra 680AV (or Macintosh II) computer to
capture images (video frames). Squares of 10
10 pixels were
selected at the centers of 10 neurons in a field. These were measured
for fluorescence intensity throughout the recording period using Image
(NIH) software. Background fluorescence (pixel intensity level) was
subtracted from the fluorescence of the neurons throughout the
recording period. Increments in intracellular calcium were expressed as
a multiple (-fold increase) of basal level fluorescence for each
neuron.
Solutions
Recording solution contained the
following (mM): CaCl(2) ,
HEPES(10) , KCl (5.4), NaCl (116.4), MgSO
(0.8),
glucose (5.6), sodium succinate (0.4), and succinic acid (0.6). All
agonists and antagonists were diluted in this solution unless otherwise
indicated and were applied to neurons via a gravity-feed perfusion
system. Some nicotine applications were made by addition of the agonist
directly to the bath using a Pasteur pipette. The solution with
elevated potassium concentration contained (mM): KCl (35.4),
NaCl (86.4), CaCl
(2) , HEPES(10) , MgSO
(0.8), glucose (5.6), sodium succinate (0.4), and succinic acid
(0.6). Calcium-free solution was recording solution in which the
calcium was replaced with 0.5 mM EGTA.
Materials
White leghorn chick embryos were
obtained locally and maintained at 37 °C in a humidified incubator.
Bgt and neuronal bungarotoxin (nBgt) were purified from Bungarus multicinctus venom(23) . Fluo-3AM was
purchased from Molecular Probes. Muscarine, atropine,
4-diphenylacetoxy-4-methylpiperidine methiodide (DAMP), nifedipine,
verapamil (D600),
-conotoxin, thapsigargin, and acetylcholine
chloride were purchased from RBI. Ryanodine was purchased from
Calbiochem; 2,5-di-(tert-butyl)-1,4-benzohydroquinone (DTBHQ)
was a gift from Dr. N. C. Spitzer (University of California, San
Diego). [
H]Inositol was purchased from DuPont
NEN. All other chemicals were purchased from Sigma unless otherwise
indicated.
]
Elevations Induced by Muscarine-Bath application
of 100 µM muscarine produced a rapid increase in
[Ca
]
in ciliary
ganglion neurons seen with fluo-3 microfluorimetry. In 45-65% of
the neurons (range for >10 fields of 5-10 neurons each),
repetitive oscillations in [Ca
]
were observed. The majority of these neurons (30-40% overall) showed persistent oscillations throughout the period of
agonist application (Fig. 1A), while the remaining
oscillators terminated their responses before the agonist was removed.
The oscillations ranged in frequency from 1 to 5 per min. In
15-30% of the neurons tested, muscarine induced a single, brief
elevation in calcium that quickly subsided to baseline despite the
continued presence of agonist. In a small portion of the neurons
(1-2%), calcium levels remained elevated throughout a 2.5-min
application of agonist. Some neurons (10-30%) did not respond to
muscarine. Both atropine and DAMP at 50 nM blocked the
muscarine-induced elevations in calcium (Fig. 1, B and C). Although atropine is a general antagonist of muscarinic
receptors, DAMP is more specific(3) . Of the muscarinic receptor
subtypes capable of generating
[Ca
]
oscillations,
only M3 receptors are likely to be blocked by the concentrations of
DAMP used here.
Figure 1:
Blockade of muscarine-induced calcium
oscillations by muscarinic antagonists. Neurons were loaded with the
calcium indicator fluo-3AM and then challenged with 100 µM muscarine (black bar) after a challenge with 35 mM K in recording medium (open box). The
resulting fluorescence (F) was expressed as a multiple of the
basal fluorescence (F
) at zero time. Neurons were
bathed in control medium (A), 50 nM atropine (B), or 50 nM DAMP (C). Both the general
muscarinic antagonist, atropine, and the M3 receptor subtype
antagonist, DAMP, blocked the muscarine-induced
responses.
IP Production by Muscarinic Agonists
Activation of
muscarinic receptors in other systems has been shown to induce
oscillatory changes in [Ca]
similar to those observed
here(1, 24, 25, 26, 27) . The
induction of oscillations has been linked to production of
inositol(1, 4, 5) -trisphosphate
(IP
) and subsequent release of calcium from intracellular
calcium stores (28-30; for reviews see Refs. 31 and 32). To
confirm that muscarine induces IP production in ciliary ganglion
neurons and to investigate the receptor subtype responsible, neurons
were incubated in [
H]inositol overnight and then
stimulated with cholinergic agonists. Muscarine at 50 µM induced the production of IPs within 1 min, and the accumulation
continued for at least 30 min (Fig. 2A). Oxotremorine-M
and carbachol were also effective at inducing IP production while
nicotine and McN-A-343, an M1 subtype-specific agonist, were not (Fig. 2B). Both DAMP and atropine efficiently blocked
muscarine-induced IP production while pirenzipine, an M1 antagonist,
and methoctramine, an M2 antagonist, were much less effective (Fig. 2C). The rank order potencies for the several
agonists and antagonists are those expected for M3 muscarinic
receptors(3) .
Figure 2:
Time course and pharmacology of
muscarine-induced release of inositol phosphates from ciliary ganglion
neurons. Ciliary ganglion neurons in culture were loaded with
[H]inositol, rinsed, and challenged with 50
µM muscarine for the indicated times (A), with
the indicated agonists for 20 min at the indicated concentrations (B), or with 50 µM muscarine for 20 min in the
presence of the indicated antagonists which had been applied to the
neurons at the indicated concentrations 2 min before (C).
Values indicate the released radioactivity expressed as a percent of
total radioactivity incorporated into the cells (A), of
unstimulated basal release (B), or of the release induced by
50 µM muscarine in the absence of antagonists (mean
± S.E. from 4-6 cultures combined from 2-3
experiments for each panel) (C). Release as a function of time
was induced by muscarine with a pharmacology characteristic of M3
receptors: activation by muscarine, carbachol, and oxotremorine-M but
not by McN-A-343 or nicotine, and blockade by atropine and DAMP but
only poorly by pirenzipine or methoctramine.
Taken together, the results suggest that
muscarine elevates [Ca]
in ciliary ganglion neurons by activating M3 receptors which
stimulate the release of calcium from IP
-dependent internal
stores. [Ca
]
oscillations could then be accounted for by repetitive
release from such stores(32, 33) . Alternatively, the
IP
-sensitive stores may only initiate the oscillations
while a second set of stores maintains them via calcium-induced calcium
release (CICR; 26, 30, 34).
Contributions from Microsomal Calcium Stores to
Muscarinic Responses
A pharmacological approach was taken to
determine if activation of muscarinic receptors releases calcium from
intracellular stores and whether CICR plays a role. Thapsigargin and
DTBHQ, compounds that inhibit endosomal calcium
ATPases(35, 36) , were used to deplete endosomal stores
by blocking reuptake of calcium. Caffeine and ryanodine were used to
target a subset of stores for depletion that in other cells is
necessary for CICR (37, 38; for review, see Ref. 31).
]
.
Figure 3:
Contributions of extracellular sources
and intracellular stores to muscarine-induced oscillations in
[Ca]. Neurons were loaded with fluo-3AM and
tested for muscarine-induced increases in
[Ca
] as described in Fig. 1 while perfusing
with vehicle (recording solution) (A), 1 µM thapsigargin plus indicated caffeine challenges (B), 10
µM ryanodine (C), 10 µM ryanodine
plus indicated caffeine challenges (D), and calcium-free
medium contemporaneously with the muscarine tests (E).
Muscarine, black bars (hatched bars in E); 30 mM caffeine, stippled boxes; 35 mM K
, open boxes. Little effect on
muscarine-induced oscillations was caused by caffeine challenges alone,
chronic ryanodine alone, or ryanodine combined with the indicated
caffeine challenges to deplete caffeine-sensitive stores. Thapsigargin
not only depleted the caffeine-sensitive stores but also truncated the
muscarine-induced oscillations. Calcium-free medium eliminated the
oscillations, leaving only a single initial spike. Extracellular
calcium is necessary for oscillations (beyond the first spike), and
thapsigargin-sensitive internal stores also contribute;
caffeine-sensitive stores contribute neither to the initiation nor the
maintenance of muscarine-induced oscillations in
[Ca
].
In contrast
to thapsigargin and DTBHQ, caffeine and ryanodine had only minor
effects on muscarinic responses. Together, caffeine and ryanodine
produced no change in the percentage of neurons generating sustained
oscillations to muscarine (), although the oscillations
were somewhat more erratic than those observed in untreated neurons and
usually did not return to baseline between spikes (Fig. 3D). A decrease was also seen in the percentage of
neurons producing only one or two spikes (). These minor
consequences of caffeine and ryanodine treatment on muscarine-induced
oscillations can probably be attributed to effects of the compounds on
buffering of cytosolic calcium or on the IP receptor, as
suggested previously (39).
]
can still be elicited in neurons after treatment with
ryanodine and caffeine provides evidence that CICR is not required.
Failure of caffeine to elicit a second response in such cells confirmed
that caffeine-sensitive stores had been depleted by the
caffeine/ryanodine treatment (Fig. 3D). Even in the
absence of caffeine challenges, ryanodine alone should have been able
to decrease the number of oscillations if CICR from caffeine-sensitive
stores were involved because such stores would have emptied within the
first few calcium spikes and could not have
refilled(30, 34, 40) . This was not the case (Fig. 3C). The results indicate that ciliary ganglion
neurons have caffeine-sensitive stores but they are not necessary for
the muscarine-induced oscillations in
[Ca
]
. The attenuation
of muscarine-induced oscillations by thapsigargin demonstrates that
ciliary ganglion neurons have additional stores that are
caffeine-insensitive and contribute to the maintenance of the
oscillations. These latter stores apparently have slower endogenous
turnover rates than do caffeine-sensitive stores. Neither ryanodine nor
thapsigargin alone produced a significant increase in intracellular
free calcium (data not shown).
Contributions from Extracellular Calcium to Muscarinic
Responses
Although intracellular calcium stores are required for
muscarine-induced oscillations in
[Ca]
, extracellular
calcium may also be required to replenish the stores and sustain the
oscillations as shown in other systems(40) . To examine the role
of extracellular calcium in the muscarinic response, agonist was
applied in a calcium-free solution containing 0.5 mM EGTA.
Under these conditions, 100 µM muscarine produced only a
single spike. The proportion of neurons responding to agonist did not
change from control, but no cells produced either oscillatory or
sustained responses throughout the period of agonist application.
Similar results were obtained when the neurons were incubated in
calcium-free medium with 0.5 mM EGTA for 1 min prior to
challenging with muscarine (93% responding with a single spike, n = 29 cells), indicating that residual unbound calcium in
the bath was unlikely to explain the single spikes observed. Most (90%)
of the cells that did respond initially with a single spike in the
absence of extracellular calcium failed to do so when challenged with
muscarine a second time even though the cells were bathed with calcium
between the muscarine tests (Fig. 3E).
-dependent manner. Second, extracellular calcium is
required to sustain the muscarine-induced increases in calcium beyond
the initial spike. Two pathways along which extracellular calcium could
enter the cell to achieve this are voltage-dependent calcium channels
(VDCCs), of which there are at least three types in ciliary ganglion
neurons(41) , and calcium release-activated channels, whose
current (I
) has been described in other cell
types (42, 43; for review, see Ref. 44).
(
)but may also act at I
channels(45, 46) . Blockers of N- and L-type
VDCCs, namely
-conotoxin (at 1 µM) and the
dihydropyridines nifedipine and verapamil (10 µM), block
up to 90% of the voltage-dependent calcium current in these
neurons(41, 47) but have no significant effect on the
muscarine-induced oscillatory response (data not shown). Apparently the
cadmium-sensitive channels required to sustain the oscillations do not
include conventional L- and N-type VDCCs.
Absence of Contributions from Internal Stores to
Nicotinic Responses
In addition to muscarinic receptors, ciliary
ganglion neurons have two major classes of nicotinic receptors that can
elevate [Ca]
, namely
mAb 35-AChRs and
Bgt-AChRs (16, 17). Because muscarinic and
nicotinic receptors share the same endogenous ligand, it was important
to compare the conditions under which they alter
[Ca
]
and the
mechanisms by which they do so. One issue was the calcium sources
mobilized by the receptors. Previous studies demonstrated that calcium
can enter the neuron directly through nAChR channels or indirectly
through VDCCs activated by membrane depolarizations the receptors
induce. Possible contributions to
[Ca
]
from internal
stores had not been examined for nicotine-induced responses.
Bgt-AChRs and mAb
35-AChRs(16, 17) . Repeated applications of 10
µM nicotine at 5-min intervals induced calcium-dependent
fluorescence responses with little attenuation (Fig. 4A). To examine the role of internal calcium
stores, either caffeine and ryanodine together or thapsigargin alone
were applied to the neurons between applications of nicotine. Neurons
bathed in 10 µM ryanodine were challenged with 30 mM caffeine to deplete caffeine-sensitive stores. No significant
effect was observed on subsequent nicotine-induced responses (Fig. 4B); the mean peak response after treatment was 95
± 4% (n = 22 cells) of the original responses
from the same cells. Thapsigargin at 1 µM also had no
effect (Fig. 4C). The mean peak response to nicotine
after thapsigargin treatment was 102 ± 2% (n =
20 cells) of the response before treatment. A second application of
nicotine after thapsigargin treatment induced a mean response that was
107 ± 3% of the response obtained before treatment. Controls in
these cases received only vehicle (0.02% ethanol for 5 min) and yielded
mean peak responses of 98 ± 3% and 103 ± 5% (first and
second challenges with nicotine, respectively; n = 20
cells) of control responses from the same cells before exposure to
vehicle (Fig. 4A). The experiments provided no evidence
that nicotine-induced increases in
[Ca
]
depend on
intracellular calcium stores. In contrast, replacing the 2 mM calcium in the extracellular solution with 0.5 mM EGTA
completely abolished the response to nicotine (Fig. 4D),
confirming the dependence of nicotine-induced changes in
[Ca
]
on extracellular
calcium. Quick recovery of the nicotinic response (<5 min) was
observed when calcium was added back to the extracellular solution.
Figure 4:
Dependence of nicotine-induced increases
in [Ca] on extracellular calcium but not on
internal stores. Fluo-3AM-loaded neurons were stimulated with pulses
of 10 µM nicotine (upward arrows) applied by
pipette at the indicated times; the breaks indicate 5-min
intervals during which the shutter was closed to minimize photodamage.
After the first two nicotine tests, the neurons were perfused for 5 min (middle break, downward arrow) with vehicle (0.02%
ethanol in recording medium) (A), 30 mM caffeine plus
10 µM ryanodine (B), or 1 µM thapsigargin (C), and then tested twice more with
nicotine. D, some neurons were perfused with calcium-free
medium (hatched bar) and tested with calcium-free nicotine (open upward arrow). Although the duration of the nicotinic
responses varied among neurons, the amplitude of the responses was
relatively constant for a given neuron. Only the calcium-free condition
affected the response, eliminating it entirely and
reversibly.
In the above experiments, nicotine was applied in single, brief
applications to the bath as described previously(16) . For
better temporal comparisons between nicotinic and muscarinic responses,
neurons were also perfused with nicotine for 2.5 min as was done with
muscarine. In these cases, nicotine induced a rapid rise in
fluorescence followed by a partial decline to a level above baseline
during the remainder of the 2.5-min period of agonist exposure. As in
the case of brief applications, nicotine was unable to induce
oscillations in [Ca]
.
]
do not appear to
require release of calcium from internal stores, at least those
sensitive to thapsigargin or caffeine plus ryanodine. Secondly, the
responses do not oscillate in amplitude, even when nicotine is applied
for the same length of time as muscarine. The nicotinic responses are
similar to muscarinic responses, however, in showing a rapid rate of
rise and in declining nearly to baseline in the continued presence of
agonist (repeatedly so in the case of muscarine-induced oscillations). [Ca
]
Elevations Induced
by ACh-The finding that at least three kinds of cholinergic
receptors capable of elevating intracellular calcium levels can
co-exist on ciliary ganglion neurons posed questions about the
operating range of the receptor subtypes and their relative
effectiveness. To examine this, dose-response measurements were made
using the native agonist ACh. Contributions from individual receptor
classes were distinguished pharmacologically.
Bgt or 20 nM nBgt prior to testing (Fig. 5, B and C; ). The
combination of 200 nM DAMP plus 20 nM nBgt completely
eliminated any response in over 90% of the neurons (Fig. 5D; ). (Studies with DAMP alone were
not considered informative since a recent report suggests it can
partially inhibit nicotine-induced
[Ca
]
increases in
ciliary ganglion neurons(48) ; it did completely block the
responses induced by 1 µM ACh.) The results are consistent
with the response induced by 1 µM ACh being largely, if
not exclusively, the product of M3 receptors.
Figure 5:
Effects of selective receptor activation
on [Ca] patterns induced by ACh. Neurons
loaded with fluo-3AM were stimulated with 1 µM (stippled bars) and 10 µM (black
bars) ACh and with elevated K
(open
boxes) while perfusing with recording solution (A),
preincubating 1 h with either 60 nM
Bgt (B) or
20 nM nBgt (C), or both preincubating with nBgt and
perfusing with 200 nM DAMP (D). At 1 µM,
ACh induces a muscarinic-like oscillatory response that is little
affected by
Bgt or nBgt (the truncation seen in C is
within the range of variation normally seen for oscillations), but DAMP
plus nBgt blocks the response. At 10 µM, ACh induces a
sustained response that becomes oscillatory when nicotinic receptors
are blocked with nBgt but not with
Bgt, implying that mAb 35-AChRs
are essential but not
Bgt-AChRs. DAMP plus nBgt again completely
blocks the response.
At 10 µM ACh, a large fraction of the neurons responded with sustained
elevations in calcium that showed no oscillations; only a few neurons
displayed the oscillatory pattern seen with the low concentration of
ACh (Fig. 5A; ). Again, 60 nM Bgt produced no detectable alteration, indicating that
Bgt-AChRs did not contribute significantly at this concentration
of ACh (Fig. 5B; ). In contrast, 20 nM nBgt radically altered the response profile, shifting it to the
oscillatory pattern seen at the lower ACh concentration (Fig. 5C; ). The combination of DAMP and
nBgt again completely blocked the response (Fig. 5D; ). The results indicate that both M3 receptors and mAb
35-AChRs are activated by 10 µM ACh, and that the
muscarinic response is either obscured or inhibited by contributions
from mAb 35-AChRs under these conditions.
Bgt
allowed oscillations to emerge late during the period of agonist
application in a substantial fraction of the cells (Fig. 6B; ). Treatment with nBgt had an
even more dramatic effect, producing more robust oscillations and
increasing the proportion of cells displaying such responses (Fig. 6C; ). Combining DAMP and nBgt
blocked the responses entirely (Fig. 6D; ;
DAMP alone both at 10 and 50 µM ACh reduced but did not
eliminate the response and did not change the proportion of cells
responding). Clearly at high ACh concentrations
Bgt-AChRs can
contribute to the response and are joined here both by mAb 35-AChRs and
M3 receptors on the neurons.
Figure 6:
Contributions of receptor subtypes at high
ACh concentrations. Neurons were loaded with fluo-3AM and stimulated
with 50 µM ACh (black bars) and with elevated
K (open boxes) while perfusing with recording
solution (A), preincubating 1 h with either 60 nM
Bgt (B) or 20 nM nBgt (C), or both
preincubating 1 h with nBgt and perfusing with 200 nM DAMP
while testing (D). Blockade of
Bgt-AChRs with
Bgt
revealed oscillations late during the exposure to agonist while
blockade of both
Bgt-AChRs and mAb 35-AChRs with nBgt produced
more pronounced oscillations, confirming that both classes of nAChRs
contribute to responses induced by high concentrations of ACh. DAMP
plus nBgt completely blocked the responses.
]
by
Muscarinic and Nicotinic Receptors-Results utilizing
specific muscarinic agonists indicate that chick ciliary ganglion
neurons have M3 type muscarinic receptors which trigger IP release and
induce an oscillatory increase in
[Ca
]
. Elevations in
[Ca
]
caused by
activation of muscarinic receptors have been detected previously in
ciliary ganglion neurons, but oscillations were not
described(18) . The lack of oscillations in that case may have
resulted from the choice of agonist, oxotremorine-M. Since
oxotremorine-M can activate both nicotinic and muscarinic receptors on
ciliary ganglion neurons (49),
(
)it might produce
sustained elevations as seen with high concentrations of ACh.
]
levels(27) . Differences between choroid and ciliary
neurons in their responsiveness to muscarinic agonists or in their
abilities to manage [Ca
]
could also induce variation in the types of responses observed.
Two recent findings supporting this possibility are that muscarinic
receptor-mediated synaptic fatigue is seen in choroid neurons, but not
ciliary neurons(50) , and that only half of ciliary ganglion
neurons have a muscarinic receptor-induced slow inward
current(19) . In contrast, all of the neurons have nicotinic
responses.
]
oscillations, as
demonstrated by the effects of store depletion and extracellular
calcium removal on muscarine-induced oscillations. The ability of
cadmium to block the muscarine-induced
[Ca
]
oscillations is
consistent with the involvement of either VDCCs or I
channels in sustaining
oscillations(41, 45, 46, 47) . Neither
N- nor L-type calcium channels, however, are required since
-conotoxin and dihydropyridines do not block the oscillations.
]
elevation and the sources of calcium mobilized clearly
differ for nicotinic and muscarinic receptors on the neurons.
Activation of either
Bgt-AChRs or mAb 35-AChRs elevates
[Ca
]
without producing
oscillations. The responses depend completely on the presence of
extracellular calcium which enters the neurons both through the
receptors themselves because of their high permeability to calcium and
through VDCCs activated by membrane depolarizations the receptors cause
(16, 17). The results demonstrate that neither class of nAChRs induces
PI turnover or relies on calcium release from internal stores to
elevate [Ca
]
.
]
changes reported
here is that muscarine-induced oscillations are produced by cyclical
emptying of IP
-sensitive calcium stores into the cytosol,
inhibition of the IP
receptor by the elevated
[Ca
]
, and clearing of
cytosol by membrane pumps to relieve inhibition and permit a new round
of release when stores have
refilled(26, 51, 52, 53, 54) .
When nicotinic receptors are also activated, e.g. at high ACh
concentrations, additional calcium influx raises the level of
[Ca
]
sufficiently to
maintain the inhibition of release from intracellular stores, and no
oscillations are seen.
Bgt-AChRs have the
capacity to inhibit or override muscarinic oscillations but display
different dependences on agonist. Only at the highest ACh concentration
tested (50 µM) did
Bgt-AChRs have a pronounced effect
while mAb 35-AChRs contributed to the response both at 10 and 50
µM ACh. The results are consistent with previous calcium
imaging studies showing that
Bgt-AChRs are relatively insensitive
to ACh, but seem at odds with electrophysiological studies indicating
that the major component of the
Bgt-AChR current response decays
much more quickly than that of mAb 35-AChRs(15) .
Bgt-AChRs
also produce a slower, sustained component of the current response,
however(15) , and it could be this second component that is most
effective in masking oscillations induced by activation of muscarinic
receptors. Additionally,
Bgt-AChRs are thought to have the highest
calcium permeability of any nicotinic AChR and may influence
calcium-dependent block of oscillations with even a small current
influx. Thus, each class of cholinergic receptor on these neurons
appears to play a role in shaping the ACh-induced temporal pattern of
[Ca
]
. Consequences of [Ca
]
Elevations Caused by Cholinergic
Signaling-Increases in
[Ca
]
are known to
regulate a vast array of cellular events, often acting through specific
kinases(34, 55, 56, 57) . The effects of
nAChRs on calcium-dependent events in chick ciliary ganglion neurons
are poorly understood, but recent experiments indicate that, in
addition to stimulating neurotransmitter release, activation of the
receptors induces neurite retraction (58) and release of the
second messenger arachidonic acid from the cells (59) in a
calcium-dependent manner. Even less is known about the impact of
muscarinic receptors on calcium-dependent events in ciliary ganglion
neurons, although one report indicates that muscarinic receptors in the
ganglion can promote synaptic fatigue in developing choroid neurons
when the cells are likely to be most vulnerable to overstimulation and
excitotoxic cell death(50) .
]
elevation can be
induced both in neurons and in non-neuronal cells depending on the type
of receptor activated, the second messengers produced, and the source
of calcium employed(2, 60, 61, 62) . The
kinds of effects imposed on cellular processes can depend on the
temporal pattern of [Ca
]
elevation as well as the source of calcium
utilized(61, 63, 64, 65) . The fact that
nicotinic and muscarinic receptors generate different patterns of
[Ca
]
elevation in
chick ciliary ganglion neurons and mobilize calcium from different
sources suggests that the receptors may exert different effects on
calcium-dependent events in the cells.
]
levels at low
concentrations of ACh, but their location on the neurons in vivo is unknown. At 10 µM, ACh recruits contributions to
[Ca
]
from mAb 35-AChRs
which are located primarily at synapses, and at 50 µM
contributions are also apparent from
Bgt-AChRs which are located
primarily in nonsynaptic regions. (mAb 35-AChRs may obscure the
contributions of
Bgt-AChRs to
[Ca
]
at lower ACh
concentrations.) In vivo ACh is released from preganglionic
terminals at specific sites juxtaposed to synaptic membrane on the
ganglionic neurons(66) . No information is available on the
concentration of ACh achieved in the synaptic cleft or on the area over
which it diffuses before being hydrolyzed. It is also unknown whether
ACh can be released either from preganglionic terminals at nonsynaptic
sites or from ganglionic somata in vivo.
]
during
low frequency synaptic transmission, while extrasynaptic
Bgt-AChRs
may contribute more during high-frequency stimulation where buildup of
ACh could allow a spatial spread beyond the immediate point of
transmitter release (50). Speculation about possible synaptic
activation of M3 receptors must await information on receptor location.
It may be instructive to examine the contributions of individual
receptor subtypes to [Ca
]
microdomains within the cells during ganglionic
transmission.
Table: Effects on muscarine-induced responses of
compounds that deplete internal calcium stores
3 spikes or
maintained fluorescent signal without spikes), single/double spikes (1
or 2 calcium transients), or no response at all (basal fluorescence)
for the number of neurons indicated. The distribution of responding
cells was significantly different from controls for thapsigargin
treatment (p < 0.01; contingency test,
analysis), but not for any of the other treatments.
Table: Effects of cholinergic antagonists on
ACh-induced responses
Bgt at 60
nM, nBgt at 20 nM, and DAMP at 200 nM); the
responses were scored as oscillatory or non-oscillatory (other
responses). All conditions were significantly different from controls
except for
Bgt and nBgt at 1 µM ACh and
Bgt at
10 µM ACh (contingency test,
analysis
except for small values where the Fisher Exact test was used).
Bgt,
-bungarotoxin; nBgt,
neuronal bungarotoxin; mAb, monoclonal antibody; IP
,
inositol 1,4,5-trisphosphate; DAMP,
4-diphenylacetoxy-4-methylpiperidine methiodide; DTBHQ,
2,5-di-(tert-butyl)-1,4-benzohydroquinone; CICR,
calcium-induced calcium release; VDCC, voltage-dependent calcium
channel.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.