Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, Georgia 30912-2300
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ABSTRACT |
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Hollins, Bettye and Stephen R. Ikeda. Heterologous expression of a P2x-purinoceptor in rat chromaffin cells detects vesicular ATP release. J. Neurophysiol. 78: 3069-3076, 1997. A cloned P2x-purinoceptor was transiently expressed in single isolated rat adrenal chromaffin cells and evaluated for the detection of released ATP. After cytoplasmic injection of the P2x complementary RNA (cRNA; 4-24 h), application of ATP produced an inwardly rectifying current over the voltage range 130 to
10 mV as measured by the whole cell patch-clamp technique. The dose-response curve for ATP was sigmoidal with a 50% effective concentration of 18.2 µM. Suramin, a P2x-antagonist, attenuated the ATP-induced current. Depolarizing voltage pulses to 0 mV or application of histamine, stimuli that trigger exocytosis, resulted in the appearance of suramin-sensitive spontaneous transient inward currents (at
60 mV) that resembled excitatory postsynaptic currents although they were slower in time course. Concurrent detection of catecholamine release with a carbon fiber electrode often showed coincidence of the amperometric current with the synaptic currentlike events suggesting that ATP and catecholamines were released from the same vessicle. These data demonstrate that expression of a P2x-purinoceptor in chromaffin cells produces a functional autoreceptor capable of detecting vesicular release of ATP. In combination with carbon fiber amperometry, simultaneous vesicular release of two neurotransmitters from a single chromaffin cell could be monitored. The P2x-purinoceptor, however, produced a regenerative effect on release apparently resulting from the high Ca2+ permeability of the receptor. Thus modification of the P2x-purinoceptor would be required before the system could be applied to examining processes involved in stimulus-release coupling.
In adrenal chromaffin cells, ATP and catecholamines are stored in membrane-bounded granules (Coupland 1965 Chromaffin cell preparation
Chromaffin cells were isolated from the adrenal medullae of male Wistar rats and plated on poly-L-lysine coated dishes in DMEM/F12 medium (Gibco), supplemented with 10% fetal bovine serum, 50 U/ml of penicillin, and 50 µg/ml of streptomycin as previously described (Hollins and Ikeda 1996 mRNA microinjection
cRNA was in vitro transcribed from linearized P2xR1 cDNA with the use of the mMessage mMachine kit according to the manufacturer's instructions (Ambion, Austin, TX). One microliter of the cRNA (2 µg/µl) was mixed with 5 µl of 0.1%-fluoresceinated dextran (10,000 MW; Molecular Probes, Eugene, OR) and injected into the chromaffin cell cytoplasm with an Eppendorf 5242 microinjector and a 5171 micromanipulator (see Ikeda et al. 1992 Amperometric detection of catecholamines
Carbon fiber electrodes were fashioned by inserting a 6-µm carbon fiber (Celion, Charlotte, NC) into a pipette blank (7052, 1.5 mm OD, 0.86 mm ID; A-M Systems, Everett, WA), which was subsequently pulled on a Sutter P-87 microelectrode puller. The fiber was sealed in the tip of the pipette with Epoxylite (Warner Instruments, Hamden, CT) and baked at 80°C overnight in an oven. The protruding section of the carbon fiber was insulated by dipping in melted paraffin and then cut to an appropriate length just before use. The electrode was voltage-clamped at +800 mV by using an Axopatch 200 amplifier (Axon Instruments, Burlington, CA) modified to deliver ±1-V command potentials. Electrical continuity between the headstage and carbon fiber was through an Ag/AgCl wire immersed in 3-M KCl (Chow et al. 1992 Electrophysiology
Whole cell voltage-clamp studies (Hamill et al. 1981 Recording solutions
Currents induced by exogenous ATP were recorded (except where noted) in normal external salines of the following composition (in mM): 145 NaCl, 5.4 KCl, 10 N-2-hydroxyethylpiperazine-N Characteristics of the heterologously expressed P2x-purinoceptor
Expression of the P2x-purinoceptor in chromaffin cells was evaluated by monitoring the whole cell current produced by exogenously applied ATP. In control cells (either uninjected or injected with the coinjection marker only), application of ATP for up to 3 min produced no detectable inward current (data not shown; n = 14). Conversely, in cells previously injected with the P2xR1 cRNA, ATP produced a brisk dose-dependent inward current (Fig. 1A). Concentrations of ATP >100 µM or prolonged application (1-2 min) at lower concentrations induced desensitization of the receptor as evidenced by an attenuated current response on repeated application. Recovery time was concentration dependent and varied between 5 and 10 min with no recovery (>30 min) seen with 1 mM ATP. As short exposures (<10 s) of the cells to concentrations of ATP up to 100 µM showed little or no evidence of receptor desensitization, concentrations of ATP from 3 to 300 µM were applied serially to cells and the resulting peak current normalized to the 100-µM response. The dose-response curve derived from these data, when fitted with a Hill coefficient of 2.0, gave an 50% effective concentration (EC50) of 18.2 µM (Fig. 1B). It should be noted, however, that the 300-µM responses were likely underestimated because desensitization was seen with even short exposures to this concentration. The ATP-induced current was antagonized by suramin, a nonselective P2-purinergic antagonist (Fig. 1C). The effects of suramin reversed slowly during a 5-8 min washout period. Although the time course of P2xR1 expression was not investigated in detail, functional expression was detected as early as 4 h after injection with optimum expression occurring after 16-24 h.
Detection of ATP release
Under conditions that promoted exocytosis, endogenously released ATP was detected as spontaneous transient inward currents (TICs). Chromaffin cells were stimulated to release with a 0.5-2 s depolarizing test pulse to 0 mV delivered at 30 s intervals from a holding potential of
Codetection of ATP and catecholamines
The oxidation of catecholamines at the tip of a carbon fiber electrode placed next to cell membrane of a chromaffin cell generates a current spike representative of the catecholamine content of a single vesicle (Chow et al. 1992
Effect of P2x receptor on release
With 2 mM Ca2+ in the external solution and under conditions of low internal Ca2+ buffering (0.1 mM EGTA), TICs appeared immediately on rupture of the patch membrane. This effect was unlikely to have arisen solely from the ATP in the patch pipette solution, as omission of ATP did not prevent this activity (n = 3). Furthermore, inclusion of 11 mM EGTA in the patch pipette occluded this effect(n = 5). Thus a likely explanation for observing spontaneous activity after patch rupture was an unintentional introduction of Ca2+ into the cell during attainment of the whole cell configuration. Once initiated, the TICs continued to appear in the absence of additional stimulation apparently from a positive feedback loop in which Ca2+ entering through P2xR1 triggered further ATP release. In support of this idea, spontaneous membrane activity was eliminated by perfusing the cells with a Ca2+-free external solution (Ca2+ in the external solution was replaced with 10 mM Mg2+ and Na+ was reduced to maintain osmolarity) during rupture of the membrane patch. After voltage-clamping the cell to a hyperpolarized holding potential (usually Previous studies of chromaffin cell granules have shown that ATP and catecholamines are colocalized and released from the same granular fractions of chromaffin cell homogenates (Blaschko et al. 1956
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
) and released by exocytosis in response to a rise in intracellular [Ca2+] ([Ca2+]i). Detection of catecholamine release arising from single fusion events has been accomplished by using electrochemical detection with carbon fiber electrodes (Chow et al. 1992
; Wightman et al. 1995
). However, a system for detecting ATP release from chromaffin cells at the vesicular level is not available. In neurons, ionotropic receptors located on the postsynaptic membrane act as rapid and sensitive detectors of neurotransmitter release from presynaptic nerve termini. After fusion of synaptic vesicles, released neurotransmitter diffuses across the synaptic cleft and binds to postsynaptic receptors. The resulting synaptic current serves as a convenient measure of neurotransmitter release. Chromaffin cells, however, lack terminal processes and secrete substances directly from the cell body. The secretory products act on receptors far removed from the site of release, thus precluding a similar presynaptic-postsynaptic detection system. By expressing an ionotropic autoreceptor in chromaffin cells, events analogous to those occurring at neuronal synapses should take place. Such a procedure would conceivably produce a pseudosynapse whereby ligand-gated receptors present near vesicular release sites would allow electrophysiological monitoring of fusion events. We have tested such a system for the detection of ATP released from single rat adrenal chromaffin cells by transiently expressing a cloned P2x-purinoceptor, P2xR1 (Brake et al. 1994
). P2x-purinoceptors are nonselectivemonovalent cation channels (Bean and Friel 1990
) that are gated by the binding of ATP. In the present study P2xR1 complementary RNA (cRNA) was in vitro transcribed and microinjected (Ikeda et al. 1992
) into chromaffin cells. Functional expression of the receptor was evaluated 4-24 h after injection by using the whole cell patch-clamp technique (Hamill et al. 1981
). Spontanteous inward currents resulting from vesicular release of ATP could be detected after a depolarizing voltage pulse to open voltage-dependent Ca2+ channels or by application of the secretagogue, histamine. Some of this work was reported previously in abstract form (Hollins and Ikeda 1995
).
METHODS
Abstract
Introduction
Methods
Results
Discussion
References
). The cells were incubated at 37°C in a humidified atmosphere of 95% O2-5% CO2 and used between 3 h and 4 days after isolation.
). Injection pressure and duration were 80-100 hPa and 0.3 s, respectively. Injection pipettes were purchased from Eppendorf or pulled from washed and baked filament-containing glass tubing (1.2 mm OD; World Precision Instruments, Sarasota, FL) by using a Sutter P-87 pipette puller (Sutter Instruments, San Rafael, CA). 100-nM tetrodotoxin (TTX) was added to the culture medium before injection and during expression of the receptor. Injected cells were identified at the time of study by epifluorescence. Only singly injected cells that lay greater than a cell's diameter away from other cells were selected for study.
). The ground electrode (an Ag/AgCl pellet) was immersed in 3-M KCl and connected to the bath with an agar bridge. Amperometric currents were digitized at 1 kHz and filtered at 500 Hz. The electrodes were used without calibration.
) were performed with an Axopatch 1C amplifier (Axon Instruments). Recording electrodes were pulled from borosilicate glass tubing (7052, 1.65 OD, 1.2 mm ID; Garner Glass, Claremont, CA) by using a P87 microelectrode puller. Electrodes were coated with Sylgard (Dow Corning, Midland, MI) and fire polished on a microforge. Recordings were performed at room temperature (22-24°C). The ground electrode was immersed in the corresponding pipette solution (or 3-M KCl when dual detection of membrane current and amperometry was employed) and connected to the bath via a 2% agar bridge. Currents were filtered at l kHz, digitized at 500 Hz or 1 kHz, and stored on a Macintosh computer or VCR.
-2-ethanesulfonic acid (HEPES), 2 CaCl2, 0.8 MgCl2, and 15 glucose (pH 7.4 with NaOH). Normal internal salines (in mM): 110 K-isethionate, 20 KCl, 5 NaOH, 11 ethylene glycol-bis(
-aminoethyl ether)-N,N,N
,N
-tetraacetic acid (EGTA), 10 HEPES, 1 CaCl2, 4 MgATP, and 0.3 Na3 guanosine 5
-triphosphate (GTP; pH 7.2 with KOH). The liquid junction potential between the external and internal solutions measured against a free-flowing 3-M KCl electrode was
10 mV (pipette negative). Reported voltages were not corrected for this potential. For release studies, external solutions were modified as follows (in mM): 133 NaCl, 5.4 KCl, 10 HEPES, 0.5 CaCl2, 10 MgCl2, and 15 glucose (pH 7.4 with NaOH). Internal solutions were modified as follows (in mM): 120 K-isethionate, 20 KCl, 5 NaOH, 0.1 EGTA, 10 HEPES, 4 MgATP, and 0.3 Na3GTP (pH 7.2, with KOH). Mg2+ was increased to 10 mM in low and Ca2+-free external solutions in an attempt to maintain the membrane charge-shielding effect of 2 mM Ca2+-containing solutions. The bath was perfused at ~1 ml/min.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
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FIG. 1.
Characterization of the P2xR1 receptor heterologously expressed in rat chromaffin cells. A: concentration-dependent inward currents mediated by exogenously applied ATP. Chromaffin cells were voltage clamped at 80 mV and studied in normal external saline (see METHODS). Various concentrations of MgATP were dissolved in the external solution and applied to the cell by lowering a micropipette connected to a multichambered reservoir to within 20 µm of the cell. Inward currents elicited by application of ATP were not detected in noninjected cells (n = 14). B: concentration-response curve for ATP. Inward currents, elicited as in A, were normalized with respect to current amplitude obtained with 100-µM ATP. Resulting mean data were fitted (smooth line) to the Hill equation by using a Hill coefficient of 2.0. Number of cells used for each data point is indicated in parentheses. C: suramin, a P2x-antagonist, inhibits ATP-mediated response. Inward current elicited by ATP (30 µM) was reversibly attenuated by suramin (30 µM). D: mean normalized current-voltage (I-V) relationship for inward current elicited by ATP (60 µM). Inward current amplitude was measured over a potential range
130-10 mV and normalized to value obtained at
130 mV.
) in PC12 cells is highly permeant to Ca2+ at external [Ca2+] up to 16.2 mM. In support of this notion, application of ATP in the presence of 2 mM [Ca2+]o and 0.1 mM EGTA in the internal solution induced spontaneous transient currents superimposed on the declining phase of ATP-induced current (Fig. 2B) indicative of ATP released from vesicle fusion (see next section).
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FIG. 2.
Effect of extra- and intracellular [Ca2+] on ATP-induced current. A: superimposed current traces from the same cell in which inward currents were elicited with a 100-ms puff of 100-µM ATP. Cell was perfused with an internal solution buffered with 11 mM ethylene glycol-bis( -aminoethyl ether)-N,N,N
,N
-tetraacetic acid (EGTA) and 1 mM Ca2+. Top: current evoked in Ca2+-free external solution containing 10 mM Mg2+. Bottom: current evoked in normal external saline containing 2 mM Ca2+. B: superimposed current traces from the same cell in which inward currents were elicited with a 100-ms puff of 100-µM ATP. Cell was perfused with internal solution containing 0.1 mM EGTA. Top trace: current evoked in a Ca2+-free external saline containing 10 mM Mg2+. Bottom trace: current evoked with a normal external saline containing 2 mM Ca+. Holding potential for both cells was
80 mV.
60 mV. In these experiments chromaffin cells were bathed in normal external saline and dialyzed with a pipette solution (0.1 mM EGTA) that minimally buffered [Ca2+]i. In control chromaffin cells the test pulse elicited an irregular outward current, probably arising from Ca2+-dependent K+ channels (BK channels), which lasted for the duration of the test pulse (Fig. 3B). To assure that release was occurring in these cells, catecholamine release was monitored simultaneously with a carbon-fiber electrode (Chow et al. 1992
; Wightman et al. 1995
). As shown in Fig. 3B, the test pulse elicited discrete amperometric currents indicative of catecholamine release. In contrast, in cells expressing P2xR1 (Fig. 3A), the test pulse elicited numerous TICs of varying amplitudes. The transient currents arose after initiation of the test pulse and ceased completely within 10-30 pulses (depending on the cell). The number of TICs elicited per pulse varied from pulse to pulse and from cell to cell. In initial experiments, TICs could not be elicited in cells injected with P2xR1 cRNA. However, because the same cells responded to exogenously applied ATP, the lack of TICs did not result from deficient P2xR1 expression but possibly arose from loss of vesicles during expression of the receptor. This problem was resolved by adding 100-nM TTX to the tissue culture medium. Presumably, the resulting decrease in excitability attenuated spontaneous release and thus vesicle depletion. TICs were detected only in injected cells under conditions of low internal Ca2+ buffering and in the presence of [Ca2+]o. In all five injected cells in which internal Ca2+ was highly buffered (11 mM EGTA/1 mM Ca2+), no TICs were elicited even with repeated pulsing (data not shown). Finally, TICs were attenuated by the P2x-receptor antagonist, suramin (50 µM), as shown in Fig. 4. Release detected simultaneously with a carbon-fiber electrode appeared unaffected (4 cells). Taken together, the fact that TICs resembling synaptic currents (albeit with a slower time course), were evoked by conditions that promoted exocytosis, and occurred only in chromaffin cell injected with P2xR1 cRNA, corroborated the notion that the TICs resulted from quantal release of ATP and subsequent activation of heterologously expressed P2xR1.
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FIG. 3.
Transient inward currents in P2xR1-expressing cells after a depolarizing voltage pulse. Chromaffin cells were stimulated by a 2-s test pulse to 0 mV from a holding potential of 60 mV. Membrane currents were monitored for 10 s. Cells were bathed in normal external saline and dialyzed with a low Ca2+ buffering internal solution. A: in a cell previously injected with P2xR1 complementary RNA, spontaneous transient inward currents indicative of ATP release occur during and after test pulse. B: in an uninjected cell, outward current, probably arising from Ca2+-dependent K+ channels, is evident during test pulse but no current is apparent during subsequent return to holding potential. Bottom: amperometric current trace from the same cell obtained from a carbon fiber eletrode pushed gently against the cell and held at a potential of +800 mV. Transient upwarddeflections indicate that catecholamines were released by test pulse. - - -,0 current level.
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FIG. 4.
Effect of suramin on transient inward currents in cells expressing P2x-receptor. A: superimposed 5-s traces of whole cell (top) and amperometric (bottom) currents elicited in an injected cell perfused continuously with a normal external saline containing 50-µM suramin. Suramin effectively blocked transient inward currents (TICs) without affecting exocytotic release. B: whole cell current traces of a cell injected with P2x-receptor before (top) and after (bottom) introduction of 50-µM suramin into the bath. Cells were perfused with normal external saline containing 2 mM Ca2+ and stimulated with a 500-ms depolarizing voltage pulse to 0 mV under conditions that promote exocytosis (0.1 mM EGTA in patch pipette). Holding potential was 60 mV. Amperometric current was measured as described in Fig. 3.
). In the present study histamine (100 µM) elicited robust release in control cells as detected by a carbon-fiber electrode (data not shown). The mechanism of histamine-induced release in rat chromaffin cells is unknown, but several observations in the present study suggested a similar mechanism of action in these cells. First, histamine produced no detectable depolarization under current-clamp conditions (n = 6; data not shown). Second, a histamine-induced inward current was not detected in voltage-clamped cells. Third, the prolonged application of histamine induced a standing outward current lasting 10-15 s. The latter current is evident in Fig. 5 and likely arose from activation of Ca2+-dependent voltage-independent K+ channels (SK-type). Although these observations are consistent with internal Ca2+ release, the evidence is still circumstantial and in the absence of [Ca2+]i measurements, the true mechanism of histamine-induced release in rat chromaffin cells remains speculative.
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FIG. 5.
Response of a P2xR1-expressing cell to a 5-s application of histamine. Sequential 10-s current traces (from top to bottom) from a cell in which 100-µM histamine was applied ( ) for 5 s. Cell was held at
60 mV and studied under conditions that promote exocytotic release (0.1 mM EGTA in the patch pipette). Culture dish containing the cells was perfused with Ca2+-free external solution at a rate of 1-ml/min
1. Histamine was dissolved in normal external solution containing 2 mM Ca2+ and applied by pressure injection (0.2 kPa) from a pipette positioned 20 µm from the cell. Duration of pressure injection was controlled by a solenoid valve interfaced to the computer. - - -, 0 current level.
decay) of 124 ± 5 ms (mode = 138, n = 157). These parameters are substantially slower than those reported for excitatory-postsynaptic responses mediated by P2x-purinoceptors (Bennett et al. 1995
; Cunnane and Searl 1994
; Edwards et al. 1992
; Evans et al. 1992a
).
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FIG. 6.
Response of a P2xR1-expressing cell to depolarizing voltage pulses. Four sequential 10-s current traces (from top to bottom) elicited every 30 s from a cell stimulated with a 2-s test pulse to 0 mV (voltage protocol shown above traces). Cell was voltage clamped at 60 mV and studied in modified external solution (10 mM Mg2+, 0.5 mM Ca2+). Membrane currents were digitized at 100 Hz. - - -, 0 current level.
decay and amplitudes for single discrete TICs measured in 8 cells are shown in Fig. 7, A and B, respectively. The relationship between TIC amplitude versus
decay for the same data set is illustrated in Fig. 7C. The apparent variability in quantal size, as exhibited by the variability in current amplitude (Fig. 7B), is consistent with the reported broad range in size of chromaffin granules (Coupland 1965
). However, the variability might also arise from differences in the relative ATP concentration per granule or spatial heterogeneity in P2xR1 expression. The
decay was also quite variable (Fig. 7A). In a single cell,
decay for discrete events varied between 50 and 450 ms with no apparent correlation between the current amplitude and
decay (Fig. 7C). A similarly large variation in the current amplitude and
decay was reported for the spontaneous extrajunctional currents of visualized varicosities at sympathetic nerve termini (Bennett et al. 1995
).
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FIG. 7.
Characteristics of TICs. Analysis of TIC events from 8 cells expressing P2xR1 receptor. Only events that were temporally resolved and in which both current amplitude and decay time constant ( decay) could be measured were included in analyses. A: histogram of TIC
decay. B: histogram of TIC amplitude. C: scatter plot of TIC amplitude vs.
decay, demonstrating poor correlation between these 2 parameters.
; Wightman et al. 1995
). Although both ATP and catecholamines are known to be coreleased from chromaffin cells, it has not been directly shown, to our knowledge, that release occurs from the same vesicle. To address this question, ATP and catecholamine release were simultaneously monitored to determine if corelease could be detected. Figure 8 shows TICs elicited in a P2xR1-expressing cell by depolarizing voltage pulses. Release of catecholamines was monitored concurrently with a carbon-fiber electrode and the studies were performed in normal external saline to increase the probability of release. As illustrated in the figure, many of the TICs occurred in close temporal proximity to the onset of the amperometric currents (mean
t = 2.3 ± 0.4 ms) suggesting codetection of the same release event. The time courses of the two events, however, were quite different. The mean time to peak for the carbon-fiber current was 3.7 ± 0.2 ms versus a mean 90% rise time of 16 ± 1 ms for TICs. Mean
decay was 4.8 ± 0.5 and 108 ± 8 ms for the carbon-fiber currents and TICs, respectively (7 cells, n = 36). Only single discrete events were included in the analysis. The discrepancy in kinetic parameters possibly arises from the different mechanisms involved in the termination of the two signals. Amperometry destroys the sample and is thus self-terminating, i.e., the amperometric current is produced by the oxidation of catecholamines to a species no longer capable of producing a signal. Conversely, ATP released from chromaffin granules may cause repetitive stimulation of P2xR1 until removed by diffusion or enzymatic processes.
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FIG. 8.
Concurrent measurement of whole cell and amperometric currents. Top 2 traces: superimposed traces of whole cell (top) and amperometric (2nd trace) currents in a P2xR1-expressing chromaffin cell. Amperometric currents were obtained from a carbon-fiber electrode as in Fig. 3. Release was evoked with a 500-ms test pulse to 0 mV from a holding potential of 60 mV (top) at 40 s intervals. Cell was bathed in normal external saline and perfused with an internal solution containing 0.1 mM EGTA. Membrane and amperometric current were digitized at 1 kHz and filtered at 1 kHz and 500 Hz, respectively. Signals were sampled simultaneously from 2 independent ADC's. Inset: expanded view of the event shown on the left. Horizontal calibration is 10 ms; vertical calibration is 100 and 20 pA for the membrane and amperometric current, respectively. Consecutive sweeps from the same cell shown in bottom 3 traces and serve to illustrate variability in release frequency during an experimental run. - - -, zero current level.
60 mV), normal external saline containing 2 mM Ca2+ could be introduced without triggering spontaneous release. When using the modified external solution (0.5 mM Ca2+, 10 mM Mg2+), spontaneous release did not occur during patch rupture and thus a change in recording solutions was not necessary. These data indicate that the substantial Ca2+ permeability of P2xR1 might interfere with experiments designed to investigate stimulus-release coupling.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
) and perfused adrenal glands (Douglas and Poisner 1966
). More recent studies have provided evidence for the vesicular release of catecholamines (Chow et al. 1992
; Wightman et al. 1995
) and ATP from chromaffin cells (Cheek et al. 1989
). However, to our knowledge the corelease of catecholamines and ATP from the same vesicle (or fusion event) has not been demonstrated.
) and P2x-purinocepters natively expressed in PC12 cells (Nakazawa et al. 1990
). The EC50 for ATP in this report, 18 µM, is lower than the 60 µM reported for P2xR1 expressed in the Xenopus oocyte expression system. This discrepancy might arise from differences in the expression systems or the relative levels of receptor expressed. Although a recent report suggests that P2x-purinceptors are present in bovine chromaffin cells (Castro et al. 1995
), we could not elicit ATP-mediated currents in uninjected chromaffin cells. Thus native expression of P2x-purinoceptors in rat chromaffin cells is either absent or below the level of detection by the whole cell patch-clamp technique.
; Edwards et al. 1992
; Evans et al. 1992a
). TIC duration did not correlate with the quantal size
i.e., currents of small or large amplitude showed a similar variability in
decay. The receptor subtype used in this study, P2xR1, however, shows a different sensitivity to ATP when compared with the P2x-purinoceptor found in rat vas deferens (Evans et al. 1992b
; Kennedy and Leff 1995
), where the duration of the synaptic currents are shown to be quite brief. It is possible, therefore, that differences in the dissociation rate of ATP or mechanisms involved in the removal of ATP (e.g., ectoenzymes) account for the discrepancy in duration of ATP-mediated currents.
) uses a detector comprised of an outside-out patch containing a high-density of ligand-gated channels. The advantages of this system are that the patch can be calibrated with known concentrations of ligand and the small size of the patch facilitates high-resolution spatial localization of release sites. In a further refinement of this system, high spatial and temporal resolution studies of the corelease of ATP and acetylcholine from frog neuromuscular junction were performed with the use of a patch containing ionotropic receptors for both neurotransmitters (Silinsky and Redman 1996
). In the present study the restricted spatial detection volume of the sniffer-patch technique was not required. In fact, because we aimed to simultaneously monitor catecholamine release with a carbon fiber electrode, the ability to monitor release events occurring over a large surface area was beneficial. The system here is unique in that it allows for the simultaneous detection of a "slow" (epinephrine) and a "fast" (ATP) neurotransmitter with a fair degree of temporal resolution. The slow response, which would normally be hampered by delays arising from signal transduction pathways (e.g., G proteins), was converted to a rapid response by the amperometric technique. In this study we show temporal coincidence of events arising from the release of ATP and catecholamine thus providing evidence that the two substances are costored in the same granule and released during a common fusion event. Although the notion of corelease of ATP and catecholamine from chromaffin cells is well accepted, to our knowledge these data represent the first direct evidence supporting corelease from a common fusion event. Intriguingly, catecholamine release events were detected that were unaccompanied by ATP-release events. This observation raises the important question of whether or not differential release of ATP and catecholamine occurs. Evidence for such a phenomenon was reported for sympathetic neurotransmission at the guinea pig vas deferens neuroeffector junction (Todorov et al. 1996
). Unfortunately, our data are not definitive in this regard because spatial heterogeneity in P2xR1 expression (e.g., clustering) provides an equally plausible explanation for the data.
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ACKNOWLEDGEMENTS |
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The authors are deeply indebted to Dr. Anthony Brake for generous donation of the P2xR1 receptor cDNA.
This work was supported in part by a grant from the American Heart Association-Georgia Affiliate to S. R. Ikeda and a graduate fellowship from the American Psychological Association to B. Hollins.
Present address: B. Hollins, Dept. of Physiology, University of Kentucky, Lexington, KY 40536-0084.
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FOOTNOTES |
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Present address and address for reprint requests: S. R. Ikeda, Laboratory of Molecular Physiology, Guthrie Research Institute, Sayre, PA 18840.
Received 27 December 1996; accepted in final form 24 July 1997.
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REFERENCES |
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