A Novel Ca2+ Influx Pathway in Mammalian Primary Sensory Neurons Is Activated by Caffeine

Robert E. Hoesch,1 Daniel Weinreich,2 and Joseph P. Y. Kao1

 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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 MOmega ), 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 GOmega ), 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 MOmega , 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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1. Effects of nominally Ca2+-free saline on caffeine-induced Ca2+ transients (CICTs). CICT is diminished in Ca2+-free Locke solution, suggesting that a component of CICTs is Ca2+ influx. A and C: Ca2+ transients that were activated by 10 mM caffeine in normal Locke solution (control). The CICTs in this neuron are particularly robust (~1,500 nM for transient A vs. the average amplitude of 560 ± 180 nM, n = 7). B: Ca2+ transient triggered by 10 mM caffeine in Ca2+-free Locke solution. The amplitude difference between control transients A and C and transient B represents the contribution due to Ca2+ influx. CICTs in Ca2+-free solution averaged, in amplitude, 49 ± 24% of the average amplitude of control transients (n = 7).  and , the durations of application of 0-Ca2+ solution and caffeine, respectively. Inset: 2 Ca2+ transients elicited by 10 mM caffeine in a different neuron, in the presence (A') and absence (B') of extracellular Ca2+. That the CICT in this neuron was not diminished in Ca2+-free Locke solution shows that, in some neurons, there is little or no Ca2+ influx component to the CICT. For both sets of calibration bars: horizontal, 10 min; vertical 500 nM.

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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Fig. 2. Effects of ryanodine on CICTs. Ryanodine blocks caffeine-induced Ca2+ release from intracellular stores but does not block caffeine-induced Ca2+ influx (CICI). A-D: segments of a continuous measurement on a single nodose neuron (inset). Ryanodine (Ry, 10 µM; bar on inset) was continuously present for B-D. A: CICT in normal Locke solution (control). B: 1st pulse of 10 mM caffeine after start of ryanodine application elicited a CICT comparable to control. C: in Ca2+-free Locke solution, Ry completely inhibited CICTs, indicating that Ry was effective in blocking caffeine-induced Ca2+ release from intracellular stores. D: in the presence of normal extracellular Ca2+, caffeine still elicited a Ca2+ transient even in the continued presence of Ry. This result suggests that caffeine can activate Ca2+ influx through a Ry-insensitive mechanism. The manipulations shown in C and D were repeated in this same neuron and yielded consistent results (not shown).  and , the durations of application of 0-Ca2+ solution and caffeine, respectively. Downward deflection in Fig. 2C is the combined result of a drop in [Ca2+]i on removal of extracellular Ca2+ and an artifact arising from caffeine's effect on the fluorescence excitation spectrum of fura-2 (Lipscombe et al. 1988).

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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Fig. 3. Effect of 2,5-di(tert-butyl)hydroquinone (DBHQ), a sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA pump) inhibitor, on CICTs. In neurons with depleted intracellular Ca2+ stores, caffeine can elicit a Ca2+ transient only in the presence of extracellular Ca2+. A-C: segments of a continuous measurement on a single nodose neuron (inset). DBHQ (20 µM; inset, ) was continuously present for B and C. A: CICT in normal Locke solution (control). B: 10 mM caffeine still elicits a Ca2+ transient even after intracellular Ca2+ stores have been depleted by continuous DBHQ application. This transient represents caffeine-induced Ca2+ influx. With intracellular Ca2+ stores depleted, CICTs were still present in 49 of 87 neurons tested (57%) and averaged 340 ± 62 nM in amplitude. Control CICTs for the same set of neurons averaged 720 ± 110 nM in amplitude (n = 49). C: caffeine elicited no Ca2+ transient in this store-depleted neuron in Ca2+-free solution. This result suggests that Ca2+ influx is responsible for CICTs during DBHQ application. The manipulations shown in B and C were repeated twice in the same nodose neuron with consistently reproducible results (not shown).  and , the durations of application of 0-Ca2+ solution and caffeine, respectively. Downward deflection in Fig. 3C is the combined result of a drop in [Ca2+]i on removal of extracellular Ca2+ and an artifact arising from caffeine's effect on the fluorescence excitation spectrum of fura-2 (Lipscombe et al. 1988).

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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Fig. 4. Caffeine activates both a rise in [Ca2+]i and an inward ionic current. [Ca2+]i and whole cell current were simultaneously monitored in the same neuron, voltage-clamped to -50 mV. A: CICT in normal extracellular solution. B: inward current measured simultaneously with the transient in A. Caffeine-induced current (Icaf) was -180 pA, in this neuron, and averaged -126 ± 24 pA in peak amplitude (n = 4). The CICT, measured simultaneously with Icaf, was 269 nM, in this neuron, and averaged 206 ± 45 nM in peak amplitude (n = 4). , the duration of caffeine (Caf) application. Inset: CICT (A') and Icaf (B') in a store-depleted neuron voltage-clamped to -50 mV. In this neuron, the CICT (A') measured 63 nM, and Icaf (B') had a peak amplitude of -473 pA.

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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Fig. 5. Estimate of reversal potential (Erev) for caffeine-activated current (Icaf). Icaf has a reversal potential near 0 mV, suggesting that it is a nonselective current. This I-V relationship was generated by a voltage-ramp command that increased from -70 to +90 mV (0.388 mV/ms). The experiment was performed on neurons whose intracellular Ca2+ stores had been depleted by persistent DBHQ treatment. The voltage ramp was applied twice: once at the mid-point of a 90-s application of 10 mM caffeine and once 3-5 min after caffeine application (for details, see text). The difference current produced by these 2 ramps is shown, and it corresponds to Icaf. Linear least-squares analysis (straight line) yielded a membrane conductance of 0.59 nS and a reversal potential of -5.9 mV for this neuron. For all cells studied, the average membrane conductance was 1.43 ± 0.79 nS (n = 4), and the average reversal potential was +8.1 ± 6.1 mV (n = 4).

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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Fig. 6. Concentration-response characteristics for caffeine-induced Ca2+ release and caffeine-activated inward current (Icaf). For each neuron studied, caffeine was applied at increasing concentrations (0.1, 0.5, 1.0, and 10 mM). To generate the curve for caffeine-induced Ca2+ release, caffeine was applied in Ca2+-free solution, and the Ca2+ transient amplitudes were averaged for each caffeine concentration and then normalized to the highest average amplitude. Nonlinear least-squares fit to a sigmoidal curve (solid line) yielded an estimated EC50 = 1.2 mM. To generate the curve for Icaf, peak current amplitudes were averaged for each caffeine concentration, and then normalized to the maximum average amplitude. The concentration dependence appears steeper than for Ca2+ release, although greater scatter in the data did not permit curve-fitting. EC50 was estimated as ~0.6 mM by inspection, although the 2 dose-response relations could not be distinguished statistically. Where not shown, error bars (SE) are smaller than the symbol diameter. open circle  and  depict Icaf and Ca2+ release, respectively.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 (Delta [Ca2+]i) was measured with fura-2. Thus
<IT>B</IT><IT>=</IT><FR><NU><FR><NU><IT>1</IT></NU><DE><IT>zF</IT></DE></FR> <LIM><OP>∫</OP></LIM><IT>−</IT><IT>I</IT><SUB><IT>Ca</IT></SUB><IT>d</IT><IT>t</IT></NU><DE><IT>&Dgr;</IT>[<IT>Ca<SUP>2+</SUP></IT>]<SUB><IT>i</IT></SUB><IT>×</IT><IT>V</IT><SUB><IT>c</IT></SUB></DE></FR>
where z is the charge on Ca2+, F is Faraday's constant, Vc is the volume of the cell, the integration is over the first 100 ms of the depolarization (when Ca2+ removal had not progressed to any significant extent), and Delta [Ca2+]i was measured during the same time interval. Knowing the average cell volume (average isolated NGN diameter = 61 ± 1.5 µm) (Leal-Cardoso et al. 1993), we were able to determine the Ca2+ buffer factor independently in six NGNs in the whole cell configuration to be B = 34.3 ± 10.1. The fraction, f, of Icaf carried by Ca2+ was estimated as
<IT>f</IT><IT>=</IT><FR><NU><IT>B</IT><IT>×&Dgr;</IT>[<IT>Ca<SUP>2+</SUP></IT>]<SUB><IT>i</IT></SUB><IT>×</IT><IT>V</IT><SUB><IT>c</IT></SUB></NU><DE><FR><NU><IT>1</IT></NU><DE><IT>zF</IT></DE></FR> <LIM><OP>∫</OP></LIM> <IT>I</IT><SUB><IT>caf</IT></SUB><IT>d</IT><IT>t</IT></DE></FR>
where Delta [Ca2+]i was the change from baseline to the peak of the caffeine-induced Ca2+ transient, and the integral of Icaf was taken over the corresponding time interval. The ratio of charge movement due to Ca2+ to the total charge movement from Icaf is 1:42 (n = 2). The Ca2+ component of Icaf was thus ~2.4% of the total---an estimate consistent with Ca2+ being a minor charge carrier.

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 Adelta -type neurons (axonal conduction velocity approx 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.


    ACKNOWLEDGMENTS

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).


    FOOTNOTES

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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0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society