1Department of Physiology, Medical Biotechnology Center, University of Maryland Biotechnology Institute; and 2Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, Maryland 21201
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ABSTRACT |
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Hoesch, Robert E.,
Daniel Weinreich, and
Joseph P. Y. Kao.
A Novel Ca2+ Influx Pathway in Mammalian Primary
Sensory Neurons Is Activated by Caffeine.
J. Neurophysiol. 86: 190-196, 2001.
Single-cell
microfluorimetry and electrophysiology techniques were used to identify
and characterize a novel Ca2+ influx pathway in
adult rabbit vagal sensory neurons. Acutely dissociated nodose ganglion
neurons (NGNs) exhibit robust Ca2+-induced
Ca2+ release (CICR) that can be triggered by 10 mM caffeine, the classic agonist of CICR. A caffeine-induced increase
in cytosolic-free Ca2+ concentration
([Ca2+]i) is considered
diagnostic evidence of the existence of CICR. However, when CICR was
disabled through depletion of intracellular Ca2+
stores or pharmacological blockade of intracellular
Ca2+ release channels (ryanodine receptors),
caffeine still elicited a significant rise in
[Ca2+]i in ~50% of
NGNs. The same response was not elicited by pharmacological agents that
elevate cyclic nucleotide concentrations. Moreover, extracellular
Ca2+ was obligatory for such caffeine-induced
[Ca2+]i rises in this
population of NGNs, suggesting that Ca2+ influx
is responsible for this rise. Simultaneous microfluorimetry with whole
cell patch-clamp studies showed that caffeine activates an inward
current that temporally parallels the rise in
[Ca2+]i. The inward
current had a reversal potential of +8.1 ± 6.1 (SE) mV
(n = 4), a mean peak amplitude of 126 ± 24 pA
(n = 4) at Em =
50
mV, and a slope conductance of 1.43 ± 0.79 nS (n = 4). Estimated EC50 values for caffeine-induced
CICR and for caffeine-activated current were 1.5 and ~0.6 mM,
respectively. These results indicate that caffeine-induced rises in
[Ca2+]i, in the presence
of extracellular Ca2+, can no longer be
interpreted as unequivocal diagnostic evidence for CICR in neurons.
These results also indicate that sensory neurons possess a novel
Ca2+ influx pathway.
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INTRODUCTION |
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Ca2+-induced
Ca2+ release
(CICR)1 is the process
whereby Ca2+ triggers release of
Ca2+ from intracellular stores into the cytosol
(Endo et al. 1970; Fabiato and Fabiato
1975
). Binding of Ca2+ to specialized
channels, known as ryanodine receptors (RyRs), located on the smooth
endoplasmic reticulum in neurons (Kuba 1994
), increases
the opening probability of RyRs (Meissner 1994
). Open RyRs are Ca2+ permeable and constitute a major
pathway for the release of Ca2+ from
intracellular stores into the cytosol (for review of CICR in neurons,
see Verkhratsky and Schmigol 1996
). By its nature, CICR
can amplify small Ca2+ signals (e.g.,
voltage-gated Ca2+ influx induced by an action
potential) into large transient rises in
[Ca2+]i
(Ca2+ transients). In primary vagal afferent
somata (nodose ganglion neurons, NGNs), CICR amplifies action
potential-induced elevation of
[Ca2+]i (Cohen et
al. 1997
). The resultant amplified Ca2+
signal is obligatory for generating a slow postspike hyperpolarization (AHPslow), a membrane property that controls
membrane excitability and spike frequency accommodation (Moore
et al. 1998
; Weinreich and Wonderlin 1987
).
Caffeine, a trimethylxanthine, is a classic pharmacological agonist for
activating CICR, and elevation of
[Ca2+]i following
caffeine application is taken as evidence of CICR (Sitsapesan
and Williams 1990). The affinity of RyRs for
Ca2+ is increased by the presence of caffeine so
that CICR occurs even at resting
[Ca2+]i
(Sitsapesan and Williams 1990
). During the course of
characterizing the role of CICR in rabbit NGNs, we observed that in
some NGNs, caffeine could elevate
[Ca2+]i independently of
CICR. In the present work, we apply microfluorimetric and
electrophysiological methods to characterize a voltage-independent, caffeine-activated Ca2+ influx pathway in NGNs.
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METHODS |
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Cell dissociation
New Zealand White rabbits of either sex, weighing 1-2 kg, were
obtained from Robinson Services (Clemmons, NC) and killed by pentobarbital sodium overdose (100 mg/kg) as approved by the
Institutional Animal Care and Use Committee of the University of
Maryland School of Medicine. Dissociated NGNs were prepared as
described previously (Leal-Cardoso et al. 1993) with the
exception that sterile technique was used and the final neuronal pellet
was resuspended in sufficient Leibovitz L-15 medium (Gibco-BRL, Grand
Island, NY), containing 10% fetal bovine serum, to plate 0.2 ml of
cell suspension onto each of 10-20 25-mm glass coverslips (Fisher
Scientific, Newark, DE) coated with poly-D-lysine (0.1 mg/ml, Sigma, St. Louis, MO). The neurons were incubated at 37°C for
24 h, then maintained at room temperature and used for experiments
for
72 h.
Recording chamber and drug delivery
A custom recording chamber with a narrow rectangular flow path provided 7 ml/min superfusion of a 25-mm coverslip with physiological salt solution via a gravity-flow system. The chamber was mounted on the stage of an inverted microscope (Diaphot, Nikon) equipped with a ×40 phase-contrast oil-immersion objective (Fluor, N. A. 1.3, Nikon) to allow fluorescence measurements or direct visualization of neurons concurrently with electrophysiological measurements. Solution changes were complete in 14 s as determined with fluorescent tracers.
Physiological saline solution
Neurons were superfused with Locke solution (21-24°C) that had the following composition (mM): 136 NaCl, 5.6 KCl, 1.2 NaH2PO4, 14.3 NaHCO3, 1.2 MgCl2, 2.2 CaCl2, and 10.0 dextrose, equilibrated with 95% O2-5% CO2 and adjusted to pH 7.2-7.4 with NaOH. For experiments where nominally Ca2+-free medium was required, CaCl2 was omitted.
For electrophysiological recordings, intra- and extracellular solutions
(Guerrero et al. 1994a) had the following compositions (mM): extracellular solution: 120 NaCl, 3 KCl, 1.8 CaCl2, 1.0 MgCl2, 10 TEA
Cl, and 5 HEPES, pH 7.6 (NaOH); intracellular solution: 130 CsCl, 4 MgCl2, 3 Na2ATP, 1 Na3GTP, 0.05 K5Fura-2, and
20 HEPES, pH 7.2 (CsOH). In voltage-clamp experiments that utilized
depolarizing voltage ramps, the pipette solution contained 5 mM QX-314
to block action potentials (Connors and Prince 1982
).
Stock pipette solutions were stored at
20°C. QX-314 and
K5Fura-2 were added to the pipette solution after
thawing on the day the solution was to be used. CaCl2 was then added to the pipette solution to
set [Ca2+]i = 100 nM
[taking the Ca2+ dissociation constant of fura-2
under physiological conditions to be 224 nM (Grynkiewicz et al.
1985
)]. Each batch of thawed pipette solution was stored on
ice and used for only 1 day. Locke solution and extracellular solution
were prepared fresh daily.
[Ca2+]i measurements and calibration
Loading cells with fura-2 indicator, acquisition of single-cell
microfluorimetric data, as well as calibration and calculation of
[Ca2+]i, were performed
as previously described (Cohen et al. 1997; Kao
1994
).
Electrophysiological measurements
Patch pipettes (2-7 M), fabricated from 1.5-mm OD
borosilicate glass (World Precision Instruments, Sarasota, FL), were
used to record whole cell membrane currents with an Axopatch 200B
amplifier (Axon Instruments, Foster City, CA). For electrophysiological measurements, NGNs were loaded first with fura-2/AM. After forming a
gigaohm seal (>1.0 G
), the whole cell configuration was established with neurons voltage-clamped to
50 mV. Neurons were considered suitable for study if membrane input resistance was >300 M
, holding current was <90 pA, and resting
[Ca2+]i was <150 nM.
All numerical data are reported as means ± SE, unless stated otherwise. Student's t-test was used to assess significant differences between calculated means; P < 0.05 was considered significant.
Reagents
Reagents were procured from the following vendors: caffeine, 3-isobutyl-1-methylxanthine (IBMX), and QX-314 from Sigma, 2,5-di(tert-butyl)hydroquinone from Aldrich (Milwaukee, WI), ryanodine, cyclopiazonic acid, and forskolin from Calbiochem (La Jolla, CA), the acetoxymethyl ester of fura-2 (fura-2/AM) from Molecular Probes (Eugene, OR), and the pentapotassium salt of fura-2 from Teflabs (Austin, TX). Inorganic salts were from VWR (Piscataway, NJ).
Unless otherwise noted, drug solutions other than caffeine were
prepared daily from concentrated (10 mM) stock solutions in
dimethylsulfoxide (Fisher Biotech, Fair Lawn, NJ) that were stored
frozen. Drugs were delivered via the superfusate by switching a
three-way valve to a reservoir containing a known concentration of the
drug in extracellular solution or in Locke solution equilibrated with
95% O2-5% CO2. In
experiments where nominally Ca2+-free solution
was required, Ca2+-free Locke was superfused for
10 s before and for
15 s after the specific reagent was applied
in Ca2+-free Locke.
Reagent concentrations (unless otherwise stated): caffeine, 10 mM; 2,5-di(tert-butyl)hydroquinone (DBHQ), 20 µM; ryanodine (Ry), cyclopiazonic acid (CPA), forskolin, and 3-isobutyl-1-methylxanthine (IBMX), 10 µM.
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RESULTS |
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Initial evidence for the existence of a caffeine-induced Ca2+ influx (CICI) in adult rabbit NGNs was derived from the comparison of transient rises in [Ca2+]i induced by caffeine (caffeine-induced Ca2+ transients, CICTs) in the presence or absence of extracellular Ca2+. Figure 1 shows an experiment where removal of extracellular Ca2+ caused a dramatic reduction in the size of the CICT (Fig. 1B) compared with the control CICT in normal Locke solution (Fig. 1, A and C). For the seven neurons tested with this protocol, the mean peak amplitude of the CICT in Ca2+-free Locke solution was 275 ± 99 nM, whereas the mean peak amplitude of the control CICT was 560 ± 180 nM. Therefore in Ca2+-free Locke solution, CICTs averaged 49 ± 24% of the amplitude of control transients. This suggests that, on average, about one-half of the CICT is dependent on extracellular Ca2+. This component is operationally distinct from the remainder of the CICT, which is totally attributable to release from intracellular stores. The inset in Fig. 1 shows data from an NGN whose CICT had the same amplitude both in the presence and absence of extracellular Ca2+. Thus the magnitude of the extracellular Ca2+-dependent component of the CICT varies from cell to cell; we estimate that 57% of nodose neurons possess this component (see Fig. 3, following text).2
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CICTs in Ca2+-free Locke solution are attributable to CICR because CICR is not directly and immediately dependent on the presence of extracellular Ca2+. Control CICTs are larger than those in Ca2+-free Locke solution, suggesting that CICR and a process that is dependent on extracellular Ca2+ are activated concurrently by caffeine in normal Locke solution. Because this extracellular Ca2+-dependent component of the Ca2+ transient can be dissociated from CICR by changing to Ca2+-free Locke solution, this component was hypothesized to arise from a CICI. Results of experiments designed to test this proposition are presented in the following sections.
We performed two experiments in which 10 µM Ry was used to isolate
the putative Ca2+ influx component of CICTs. One
example is shown in Fig. 2. A pulse of
caffeine elicited a control CICT (Fig. 2A). Thereafter, beginning 10 min prior to the caffeine treatment in Fig. 2B,
Ry was applied continuously through treatments B-D. The
first caffeine application during Ry treatment elicited a CICT (Fig.
2B) whose magnitude was comparable to control (Fig.
2A). This result was anticipated because RyR inhibition by
Ry is known to be use-dependent, requiring that RyRs be activated at
least once in the presence of Ry (Meissner 1986;
Sutko et al. 1985
). To show that CICTs during Ry
blockade were dependent on extracellular Ca2+,
caffeine was then applied in Ca2+-free Locke
solution, and no response was measurable (Fig. 2C). However,
by repeating caffeine application in normal Locke solution, in the
continued presence of Ry, the influx component of the CICT was restored
(Fig. 2D). The fact that caffeine elicited no
Ca2+ transient in Ca2+-free
solution (Fig. 2C) shows that Ry was indeed effective in blocking RyRs (and hence CICR). Furthermore, this observation supports
the conclusion that the Ca2+ transient in Fig.
2D arose by an influx mechanism that is both independent of
CICR and insensitive to Ry. The treatments in Fig. 2, C and
D, were repeated in this same NGN with comparable results (not shown). Aggregate results show that caffeine could elevate [Ca2+]i in 5 of 11 (46%)
NGNs incubated with 10 µM Ry for
20 min (2 of the 5 cells were
treated exactly as shown in Fig. 2, while 3 of the 5 cells were treated
similarly to Fig. 2 except that no manipulations were performed in
Ca2+-free solution). In these five NGNs, CICTs
averaged 523 ± 331 nM after ryanodine application compared with
893 ± 333 nM before ryanodine application. Caffeine failed to
activate an increase in the other six NGNs tested (54%), indicating
that, as in the inset of Fig. 1, not all NGNs display
caffeine-induced Ca2+ influx.
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Another way to separate the two components of the CICT is to deplete
intracellular Ca2+ stores on which CICR is
directly dependent. If a Ca2+ transient could be
elicited by caffeine even after intracellular Ca2+ stores had been emptied, then a
Ca2+ influx must have been activated by caffeine.
DBHQ is an inhibitor of the sarcoplasmic/endoplasmic
reticulum Ca2+ ATPase (SERCA pump)
(Kass et al. 1989; Thomas and Hanley
1994
). Inhibition of SERCA pumps prevents cells from
replenishing the Ca2+ that leaks out of
intracellular stores into the cytosol. Therefore, persistent SERCA pump
inhibition is an effective means to deplete intracellular
Ca2+ stores (Thomas and Hanley
1994
). To empty intracellular Ca2+ stores
completely, 20 µM DBHQ was applied for
15 min. We verified the
effectiveness of DBHQ in depleting the stores by applying cyclopiazonic
acid (CPA), another SERCA pump inhibitor (Seidler et al.
1989
), to neurons that had already been treated with 20 µM
DBHQ for 15 min. In the continued presence of DBHQ, 10 µM CPA caused
an additional rise in
[Ca2+]i of only 16 ± 6 nM (n = 16; data not shown). The failure of CPA to
cause a significant increase in
[Ca2+]i
(P = 0.0157) indicates that the DBHQ application was
effective in completely inhibiting SERCA activity. Evidence that the
stores were indeed depleted by this treatment is presented in the
following paragraph (results of Fig. 3).
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Figure 3 shows an example of a neuron in which, even after depletion of intracellular Ca2+ stores by DBHQ, caffeine still elicited a Ca2+ transient (Fig. 3B). CICTs like that shown in Fig. 3B were observed in 49 of 87 store-depleted neurons tested (57%). These Ca2+ transients averaged 340 ± 62 nM (n = 49) in amplitude, whereas control CICTs averaged 720 ± 110 nM (n = 49).3 To show that the CICT in Fig. 3B is truly generated by a Ca2+ influx, caffeine application was repeated in Ca2+-free Locke solution in the continued presence of DBHQ. As expected, caffeine did not elicit Ca2+ transients in store-depleted neurons in the absence of extracellular Ca2+ (see Fig. 3C; n = 12), which demonstrates that the stores were indeed empty and unable to support Ca2+ release.
Caffeine's action is not limited to activation of CICR. At low
concentrations (e.g., 2-50 µM), caffeine is a cyclic nucleotide phosphodiesterase inhibitor and can cause the accumulation of cyclic
nucleotides (Beavo and Reifsnyder 1990). Therefore, to ascertain that CICI is not due to the effects of increased
intracellular concentrations of cyclic nucleotides, forskolin (10 µM), which activates adenylyl cyclase to produce
adenosine-3',5'-cyclic-monophosphate (cAMP) (Seamon et al.
1981
), and 3-isobutyl-1-methylxanthine (IBMX; 10 µM), a
potent cyclic nucleotide phosphodiesterase inhibitor (Beavo and
Reifsnyder 1990
), were applied to store-depleted neurons (
15
min of 20 µM DBHQ application) that had already shown a CICT. In such
store-depleted neurons, neither forskolin nor IBMX application triggered a measurable Ca2+ transient (data not
shown; n = 2 for each reagent) as caffeine did (see
data from Fig. 3). At the concentration used (10 µM), forskolin and
IBMX should elevate intracellular cyclic nucleotide levels in NGNs, as
evidenced by their blockade of the AHPslow, which
is sensitive to increases in intracellular cAMP concentration (for a
summary of this and other characteristics of the
AHPslow, see Cordoba-Rodriguez et al.
1999
). These results thus indicate that CICI is not
attributable to phosphodiesterase inhibition and/or altered cyclic
nucleotide levels.
CICI that elevates
[Ca2+]i should be
measurable electrically. Figure 4 shows
an example of a voltage-clamped NGN for which
[Ca2+]i and whole cell
current were measured simultaneously. In this neuron, caffeine
application activated an inward current at 50 mV and concomitantly
increased [Ca2+]i, with
corresponding peak amplitudes of
180 pA and 269 nM, respectively. Caffeine-activated currents
(Icaf) averaged
126 ± 24 pA
(n = 4) at peak amplitude. In the same neurons, CICTs averaged 206 ± 45 nM in peak
amplitude.3 For
comparison, the inset to Fig. 4 shows a CICT and an
Icaf recorded concurrently from a
store-depleted neuron voltage-clamped to
50 mV. In this neuron, the
CICT and Icaf measured 63 nM and
473
pA, respectively. The average amplitude of
Icaf was not significantly different
(
84 ± 76 pA, n = 4) in store-depleted NGNs
(
15 min of 20 µM DBHQ), when recorded 45 s into 90-s caffeine
applications.4 To
test whether Ca2+ was required for
Icaf, we recorded currents from
store-depleted neurons in Ca2+-free solution. The
amplitudes of these Icaf averaged
216 ± 99 pA (n = 3). This mean amplitude is not
significantly different from either of the mean current amplitudes
listed in the preceding text, suggesting that neither extracellular
Ca2+, nor release of Ca2+
from intracellular stores, is required for the
Icaf.
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Figure 5 shows an example of an I-V relation for Icaf measured in one neuron. Two successive voltage ramps were performed to generate this relation. The first ramp was applied to a store-depleted neuron at the mid-point of a 90-s caffeine application. The second ramp was applied to this same neuron 3-5 min after completion of caffeine application at which time whole cell current had returned to baseline. The difference between these two evoked current traces yields the I-V relation for Icaf. A linear least-squares fit to these data provided a measure of the slope conductance produced by caffeine (Fig. 5). The average slope conductance during caffeine application was 1.43 ± 0.79 nS (n = 4). From the I-V relation for Icaf, we were able to determine that the reversal potential (Erev) for Icaf was +8.1 ± 6.1 mV (n = 4), suggesting that the inward ionic current is nonselective.
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Figure 6 shows concentration-response
curves for CICR from intracellular stores and for
Icaf. Data points for the
concentration-response curve of CICR were generated by successively
applying caffeine at four different concentrations (0.1, 0.5, 1.0, and
10 mM) in Ca2+-free Locke solution (40-s
applications at 10 min intervals) and measuring the peak amplitudes
of the resulting Ca2+ transients (pooled data).
The average Ca2+ transient amplitude was
calculated for each caffeine concentration, normalized to the maximum
average amplitude, and plotted as a function of
log10[caffeine] (n = 4). Data
points for the concentration-response curve of
Icaf were generated by measuring peak
whole cell currents evoked by successively applying caffeine in normal
extracellular solution at the same concentrations and durations as in
the preceding text. The average peak current was calculated for each
caffeine concentration, normalized to the maximum average current, and plotted as a function of log10[caffeine]
(n = 4). The Ca2+ release data
were well fit by a sigmoidal concentration-response curve with a fitted
EC50 = 1.2 mM. The relatively noisy inward current data did not permit curve-fitting, but an
EC50 of ~0.6 mM was estimated by inspection.
Statistically, the two dose-response relations could not be
distinguished.
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DISCUSSION |
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Caffeine is the classic pharmacological agonist of CICR, acting at
the level of RyRs, and Ca2+ transients resulting
from application of caffeine have been regarded as diagnostic evidence
of CICR. We now show that CICTs, in approximately one-half of adult
primary vagal afferent neurons (NGNs), actually comprise two major
components: CICR and a Ca2+ influx. The CICI
component, as determined electrophysiologically, appears to be
attributable to a nonselective entry pathway. Although the existence of
a Ca2+ influx pathway activated by caffeine has
been definitively identified in smooth muscle cells (Guerrero et
al. 1994a; Ufret-Vincenty et al. 1995
), to our
knowledge, this is the first report of a Ca2+-permeable, nonselective influx pathway in a
mammalian neuron being activated by caffeine.
The reversal potential (Erev) of a current is informative about relative permeabilities for the ions carrying that current: the magnitude of the Erev of a current is influenced most strongly by the dominant permeant ions. The Erev for the caffeine-activated current (Icaf) was estimated to be +8.1 ± 6.1 mV (n = 4), indicating that no single cation (or anion) is predominantly permeant; thus Icaf is nonselective. Since ECa is ~+128 mV under our experimental conditions, our measured Erev suggests that only a minor fraction of Icaf is due to Ca2+ influx, although this small component is sufficient to elevate [Ca2+]i.
The inference that Ca2+ is a minor charge
carrier in Icaf can be independently
assessed by estimating the fraction of total charge influx attributable
to CICI. The amount of charge carried by CICI can be
estimated from the amplitudes of CICTs in the absence of CICR, provided
that the Ca2+ buffering capacity or buffer factor
(B) of the cell is known (Guerrero et al.
1994b). The buffer factor, B, is the ratio of the
change in total cytosolic Ca2+ to the
change in cytosolic free Ca2+. The
change in total Ca2+ was determined by monitoring
the inward current in response to an 80-mV step depolarization from
80 mV under conditions where Ca2+ was the only
charge carrier and CICR was blocked with ryanodine. The change in
cytosolic free Ca2+ concentration
(
[Ca2+]i) was measured
with fura-2. Thus
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Because caffeine releases Ca2+ from intracellular
stores, it is conceivable that sufficient depletion of intracellular
stores may occur during caffeine application to activate
Icrac (Hoth and Penner
1992). However, several lines of evidence from the present
series of experiments suggest that CICI is in fact a novel phenomenon
distinguishable from Icrac. First,
depletion of intracellular Ca2+ stores by DBHQ
was complete before caffeine was applied (see following text).
Therefore Icrac would have reached a
steady, maximal state of activation by the time caffeine was applied. Second, Icrac is highly selective for
Ca2+, as evidenced by its reversal potential near
+50 mV (Hoth and Penner 1993
), whereas CICI is a minor
component of the nonselective Icaf
(Erev = +8.1 mV). Third, the
I-V relation for Icrac
shows inward rectification (Hoth and Penner 1993
),
whereas the I-V relation for
Icaf is linear. Together these results
suggest that CICI does not utilize a classic
Icrac pathway.
Ca2+ can activate Cl
currents in primary somatosensory neurons (Ayar and Scott
1999
) and in NGNs (Lancaster and Weinreich, unpublished observations). Thus Ca2+ activating a
Cl
current
(ICa,Cl) could contribute to the
current identified as Icaf. The
finding that Icaf persists in the
store-depleted NGN, even in the absence of extracellular
Ca2+, shows that
Icaf is not
ICa,Cl. It has also been reported that histamine receptor activation evokes an inward current by blocking a
K+ current in NGNs (Jafri et al.
1997
). Therefore it is conceivable that
Icaf might have resulted from the
inhibition of a K+ current by caffeine. This
possibility can be rejected for two reasons. First, caffeine induced an
increase in conductance, which is the opposite of the
expected conductance decrease that would be associated with blockade of
a resting K+ current. Second,
K+ current blockade implies that
Erev of
Icaf should be close to EK, but this was not the case (see
Fig. 5). Therefore Icaf does not arise
from a K+ current being inhibited by caffeine.
With the possible exception of the conduction velocity of action
potentials in their peripheral processes, NGNs are a heterogeneous collection of visceral primary afferents. About 90% of nodose neurons
are classified as C-type neurons (axonal conduction velocity <1 m/s),
while the remaining neurons are considered A-type neurons (axonal
conduction velocity
5-15 m/s) (Stansfield and Wallis 1985
). NGNs convey sensory information from a wide spectrum of modalities ranging from mechanoreception to chemosensation for glucose,
pH, water, and CO2. These neurons also display a
disparate array of voltage- and ligand-gated ionic channels in their
plasma membranes (Higashi 1986
). It would be interesting
to know if CICI correlated with a particular subclass of NGNs. For
example, 30-40% of rabbit nodose neurons possess a
Ca2+-dependent K+ current
that is responsible for a slowly developing and long-lasting spike
afterhyperpolarization (AHPslow) that controls
spike frequency accommodation in these cells. The magnitude and
duration of the AHPslow is critically dependent
on a functional CICR pool (Moore et al. 1998
). If CICI
exists in neurons with AHPslow, it would be
interesting to learn whether Ca2+ influx via the
CICI pathway could modify this slow potential.
In conclusion, the significance of these experiments is twofold. First,
although caffeine is a reliable agonist for CICR, caffeine-activated
rises in [Ca2+]i in nerve
cells can no longer be attributed solely to release from intracellular
stores. Therefore our results further expand the pleiotropy of caffeine
in nerve cells to include not only activation of CICR (Endo et
al. 1970; Fabiato and Fabiato 1975
) and
phosphodiesterase inhibition (Beavo and Reifsnyder
1990
), but also activation of Ca2+
influx. Second, pharmacological agents often mimic endogenous agonists
through an interaction with a common target or receptor (for a recent
example, see Miwa et al. 1999
). Caffeine, which has
multiple pharmacological effects, may in fact mimic multiple endogenous
agonists. Should the on-going search for an endogenous caffeine
analogue (e.g., cyclic ADP-ribose) (Lee 1997
) prove
fruitful, then CICI may be identified as an alternative
Ca2+ entry pathway that is activated by an
endogenous signaling molecule. Furthermore because half of adult rabbit
NGNs do not display CICI, it may be possible to identify a linkage
between the presence of this pathway and a specific function activated
by the putative endogenous signaling molecule.
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ACKNOWLEDGMENTS |
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We thank Dr. Rick Matteson for instruction, and Dr. Pablo Perillan for abundant advice and assistance, on electrophysiological techniques. Dr. Michael Gold provided helpful comments on earlier drafts of the manuscript. We thank T. Gover for providing measurements of buffering capacity of NGNs. We also appreciate T. Moreira's expert technical assistance.
This work was supported by National Institutes of Health Grants GM-56481 and NS-22069 (to J.P.Y. Kao and D. Weinreich, respectively).
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FOOTNOTES |
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Address for reprint requests: J.P.Y. Kao, Rm. S219, Medical Biotechnology Center, University of Maryland, 725 W. Lombard St., Baltimore, MD 21201 (E-mail: jkao{at}umaryland.edu).
1 The abbreviations used are [Ca2+]i, intracellular Ca2+ concentration; Icrac, Ca2+ release-activated Ca2+ current; IP3, inositol-1,4,5-trisphosphate; SERCA, sarcoplasmic/endoplasmic reticulum Ca2+ ATPase; AM, acetoxymethyl; DBHQ, 2,5-di(tert-butyl)hydroquinone; CICR, Ca2+-induced Ca2+ release; Icaf, caffeine-activated current; CICI, caffeine-induced Ca2+ influx; Ry, ryanodine; RyR, ryanodine receptor; IBMX, 3-isobutyl-1-methylxanthine; CPA, cyclopiazonic acid; cAMP, adenosine-3',5'-cyclic-monophosphate; NGN, nodose ganglion neuron; CICT, caffeine-induced Ca2+ transient.
2 Data in the inset also imply that the 10-s exposure to Ca2+-free solution that precedes the caffeine test pulse does not interfere with the CICR component of the Ca2+ transient.
3 The mean peak amplitude for control CICTs in a large population of neurons (see discussion of Fig. 3) was compared to the mean peak amplitudes of control CICTs that were determined in much smaller experimental groups of neurons (see discussion of Figs. 1 and 4). These means were not significantly different.
4 Once the existence of a caffeine-activated current was confirmed, it was tantalizing to search for parameters predictive of its presence in a particular neuron. Examples of such parameters might be the shape or amplitude of the caffeine-induced Ca2+ transient. However, no predictive parameters were found.
Received 24 October 2000; accepted in final form 28 March 2001.
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REFERENCES |
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