©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cell-specific Purinergic Receptors Coupled to Ca Entry and Ca Release from Internal Stores in Adrenal Chromaffin Cells
DIFFERENTIAL SENSITIVITY TO UTP AND SURAMIN (*)

(Received for publication, August 19, 1994; and in revised form, December 7, 1994)

Enrique Castro (4)(§) Jesús Mateo (4) Angelo R. Tomé (1) (2) Rui M. Barbosa (1) (3) Maria Teresa Miras-Portugal (4) Luís M. Rosário (1) (2)

From the  (1)From theCenter for Neurosciences of Coimbra, the Department of Zoology, the (2)Department of Biochemistry, Faculty of Sciences and Technology, P. O. Box 3126, and the (3)Laboratory of Instrumental Analysis, Faculty of Pharmacy, University of Coimbra, P-3049 Coimbra Codex, Portugal and the (4)Department of Biochemistry and Molecular Biology, Faculty of Veterinary Sciences, Complutense University of Madrid, E-28040 Madrid, Spain

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have assessed the relative contribution of Ca entry and Ca release from internal stores to the [Ca] transients evoked by purinergic receptor activation in bovine adrenal chromaffin cells. The [Ca] was recorded from single cells using ratiometric fura-2 microfluorometry. Two discrete groups of ATP-sensitive cells could be distinguished on the basis of their relative capacity to respond to ATP in the virtual absence of extracellular Ca. One group of cells (group I) failed to respond to ATP in the absence of Ca, was completely insensitive to UTP, and displayed suramin-blockable [Ca] transients when challenged with ATP in the presence of external Ca. ATP activated a prominent and rapidly inactivating Mn influx pathway in group I cells, as assessed by monitoring Mn quenching of fura-2 fluorescence. In contrast, a second group of ATP-sensitive cells (group II) exhibited pronounced [Ca] rises when challenged with ATP and UTP in the absence of Ca and was completely insensitive to suramin. ATP and UTP activated a delayed and less prominent Mn influx pathway in group II cells. Contrary to the nicotinic receptor agonist DMPP, which evoked a preferential release of epinephrine, ATP evoked a preferential release of norepinephrine, and UTP had no effect on secretion. Suramin nearly suppressed ATP-evoked norepinephrine release. We conclude that chromaffin cells contain two distinct and cell-specific purinoceptor subtypes. Although some cells express a P-type purinoceptor coupled to Ca release from internal stores and to the associated slow Ca refilling mechanism, other cells express a suramin-sensitive and UTP-insensitive purinoceptor exclusively coupled to Ca influx, probably an ATP-gated channel. It is suggested that the ATP-gated channel is preferentially localized to norepinephrine-secreting chromaffin cells and supports specifically hormone output from these cells. Thus, the biochemical pathways involved in the exocytotic release of the two major stress-related hormones appear to be regulated by distinct signaling systems.


INTRODUCTION

Adrenomedullary chromaffin cells are a major endocrine cell type specialized in the secretion of the stress-related catecholamine hormones epinephrine and norepinephrine. These hormones are believed to be released from two discrete chromaffin cell types(1, 2) . Although the secretion of both catecholamines is primarily controlled by acetylcholine released from splanchnic nerve terminals, chromaffin cells have a plethora of receptors to other transmitters and mediators such as ATP, bradykinin, histamine, -aminobutyric acid, and others (3, 4, 5, 6, 7) , which presumably play an important role in the modulation of the secretory process. The possible differential role of these receptors in the regulation of epinephrine and norepinephrine release is unclear.

ATP is released by stimulation of splanchnic nerve terminals and serves as a cotransmitter in various cholinergic synapses(8, 9) . ATP is also stored in large amounts in chromaffin granules and is released to medium upon stimulation of chromaffin cells(10) . ATP and other purinergic agonists have been reported to evoke catecholamine secretion from chromaffin cells in a manner that is totally dependent on the availability of extracellular Ca(3) , suggesting that enhanced Ca influx is an essential component of the associated cytosolic free Ca concentration ([Ca]) transients. Indeed, we have recently investigated the effects of purinergic agonists on the [Ca] recorded from single chromaffin cells and found evidence for the operation, in a sizable subpopulation of these cells, of a purinoceptor exclusively coupled to Ca influx (11) . However, previous studies carried out using cell suspensions have attributed the ATP-evoked [Ca] transients to Ca release from internal stores(4) , a process which is weakly coupled to catecholamine release(7, 12) . The above reported actions of ATP are clearly difficult to reconcile if one assumes that a single purinoceptor type operates in chromaffin cells.

ATP is well known to activate a variety of purinoceptor types in other cells. The classical types, the P and P purinoceptors, are believed to be widespread among mammalian cells, whereas others appear to be specific to certain cell types (e.g. the P and P purinoceptors, which occur in neutrophils and platelets)(13) . P purinoceptors are receptor-operated nonselective cation channels(14) , whereas P purinoceptors are metabotropic receptors coupled to phospholipase C(15, 16) . Several isoforms of metabotropic purinoceptors have been recently cloned, including an ATP receptor (the P purinoceptor) which is specifically sensitive to the pyrimidine nucleotide UTP(17, 18) .

We have now investigated the possible operation of multiple Ca translocation mechanisms linked to purinoceptor activation in chromaffin cells through the analysis, at the single cell level, of the effects of ATP and UTP on [Ca]and Mn quenching of fura-2 fluorescence. By relating this analysis to the patterns of ATP- and UTP-evoked catecholamine secretion, we suggest that two distinct purinoceptors play a differential role in catecholamine secretion, with a putative ATP-gated channel being preferentially localized to norepinephrinesecreting cells.


MATERIALS AND METHODS

Cell Culture

Bovine adrenal glands were obtained from the local slaughterhouse and kept on ice during transportation. Adrenal medulla cells were isolated by collagenase digestion of the glands and purified on a Percoll density gradient essentially as described previously(19) . Chromaffin cells were cultured under a 5% CO(2)/95% air humidified atmosphere in a 1:1 mixture of DMEM/Ham's F-12 medium buffered with 15 mM HEPES and 25 mM NaHCO(3), supplemented with 5% heat-inactivated fetal calf serum, penicillin (100 units/ml), and streptomycin (100 µg/ml) (Biological Industries, Beth Haemek, Israel). The cells were plated on round (16-mm diameter) glass coverslips coated with poly-L-lysine. Cells were typically used between days 2 and 5 after plating.

Solutions

The Ca-containing physiological salt solution used in the microfluorescence experiments had the following composition (mM): 120 NaCl, 5 KCl, 25 NaHCO(3), 2 CaCl(2), 1 MgCl(2), and 10 glucose. The solution was constantly gased with 95% O(2), 5% CO(2) for a final pH of 7.4. In some experiments extracellular free [Ca] was buffered at 100 nM by mixing appropriate amounts of Ca and EGTA, as described elsewhere(20) . The solutions used to calibrate intracellular fura-2 fluorescence in terms of [Ca](i) had the following composition (mM): saturating Ca solution: 100 KCl, 10 NaCl, 1 MgCl(2), 2 CaCl(2), 10 MOPS (^1)and 1-2 µM fura-2 (pH 7.0); 0 Ca solution: 100 KCl, 10 NaCl, 1 MgCl(2), 2 EGTA, 10 MOPS, 10 Tris, and 1-2 µM fura-2 (pH 7.6).

Microfluorometry

The [Ca](i) was recorded from single adrenal chromaffin cells essentially as described previously (11) using a dual excitation microfluorescence system (Deltascan, Photon Technology International, Princeton, NJ). Cells attached to coverslips were washed in physiological salt solution supplemented with 1% bovine serum albumin. The cells were then loaded with 2-5 µM fura-2/AM for 45 min at 37 °C in this medium. The coverslip was placed in a small (34 µl) perifusion chamber on the stage of a Nikon Diaphot microscope, and the cells were illuminated alternately either at 340 and 380 nm ([Ca](i) experiments) or at 358 and 380 nm (Mn quenching experiments). The emitted fluorescence was driven to the photomultiplier after passing through a 510-nm band-pass interference filter. The measuring field was routinely centered on the cell of interest by means of a rectangular diaphragm placed on the emission path.

[Ca](i) Calibration of Fluorescence Signals

The fluorescence data were calibrated in vitro using the equation (21) [Ca](i) = K(d) beta (R - R(min))/(R(max) - R), where K(d) is the dissociation constant for the Ca/fura-2 complex (taken as 224 nM), R is the ratio of actual fluorescences measured at 340 (or 358) and 380 nm, R(max) is the value of R at saturating Ca, R(min) is the value of R at 0 mM Ca, and beta is the ratio of fluorescences measured at 0 mM Ca and saturating Ca for 380 nm excitation. The measurements needed for the calculation of R(max), R(min), and beta were obtained from the excitation spectra of fura-2 dissolved in calibration solution at [Ca] = 0 and 2 mM, as reported(11) .

Fluorescence Quenching Experiments

The divalent cation Mn can be used as a Ca channel surrogate to trace Ca fluxes. Since the affinity of fura-2 for Mn is very high (K(d) 5 nM), (^2)essentially all Mn ions entering a fura-2-loaded cell are trapped as fura-2-Mn complexes. These complexes are virtually non-fluorescent at all wavelengths. Thus, the rate of loss of fura-2 fluorescence at the Ca isosbestic point (358 nm) provides an estimate of the rate of Mn influx into a cell, irrespectively of the magnitude of the [Ca](i) changes that may occur simultaneously(22, 23) . Control cells illuminated for 4-5 min under the same conditions as test cells displayed a continuous decrease in fluorescence due to dye photobleaching. This process was well fitted by a first order decay function. The fluorescence quenching records were corrected for the photobleaching decay estimated before Mn introduction. Traces were then normalized by setting initial fluorescence before Mn as 100%.

Catecholamine Secretion

Catecholamine secretion from chromaffin cells was measured on-line using a perifusion system similar to that described previously(24) . Approximately 10^6 cells were placed in a 0.45-µm Flow filter and perifused with the aid of a peristaltic pump (Gilson Miniplus 3) at 1 ml/min with a solution containing (in mM): 130 NaCl, 5 KCl, 2 CaCl(2), 1 MgCl(2), 10 HEPES, and 5 glucose (pH 7.4). Test drugs were added as brief pulses through a 500-µl loop injector. The effluent solution exiting the filter was driven into an electrochemical detector (set at +500 mV, Omni 90 Potentiostat, Cypress Systems, Lawrence, KS) for direct measurement of catecholamine oxidation current, which was monitored on a chart recorder. The effluent outputs corresponding to evoked secretory episodes were collected and epinephrine and norepinephrine content analyzed by HPLC, as described elsewhere(25) . Taking into account the dead volume of the filter chamber and the flow rate, the actual concentration of a given drug inside the chamber was reduced by a factor of 2 with respect to loop solution.

Data Analysis

All results (text and figures) were expressed as mean ± S.E. Catecholamine release was expressed as the difference between the release measured in the presence of a secretagogue and basal release. For the on-line experiments depicted in Fig. 7A the evoked catecholamine release was assessed by calculating the time integral of the net current responses over 2-min periods, after digitization of the recorded traces.


Figure 7: Effects of ATP and DMPP on catecholamine secretion from chromaffin cells. A, representative trace of the secretory response recorded electrochemically from a chromaffin cell bed (approximately 10^6 cells), challenged with 20 µM DMPP (a specific acetylcholine nicotinic receptor agonist; rightmost trace) and 200 µM ATP (remaining traces). Approximate concentrations at the cell bed: 10 and 100 µM for DMPP and ATP, respectively. The second trace from the left depicts the response to ATP in the presence of 300 µM suramin. Suramin was added to the solution 3-5 min prior to addition of ATP. The cells were allowed to recover from suramin for >10 min prior to delivery of the third ATP pulse. B, proportion of norepinephrine secreted with respect to total catecholamines (epinephrine + norepinephrine) secreted above basal, in response to 20 µM DMPP and 200 µM ATP. Absolute values of DMPP-evoked epinephrine and norepinephrine secretion: 0.86 ± 0.14 and 0.55 ± 0.06 nmol/10^6 cells, respectively (n = 10 experiments, five different preparations). Absolute values of ATP-evoked epinephrine and norepinephrine secretion: 0.07 ± 0.03 and 0.18 ± 0.05 nmol/10^6 cells, respectively (n = 6 experiments, three different preparations). C, effect of suramin on ATP-evoked norepinephrine release. The response obtained in the presence of suramin (right column) was normalized to the control ATP response. Absolute ATP-evoked norepinephrine release: 0.15 ± 0.04 nmol/10^6 cells (n = 7 experiments, three different preparations). In B and C the catecholamines were assayed by HPLC.




RESULTS

[Ca](i) Measurements

We have previously reported (11) and further confirmed in the present study that approximately 53% of a typical population of chromaffin cells respond to brief pulses of ATP with prominent [Ca](i) peaks in the presence of external Ca, with the remaining fraction exhibiting an absolute insensitivity toward the nucleotide. We have now carried out a more systematic investigation of the extracellular Ca dependence of the ATP-evoked [Ca](i) transients by comparing the standard [Ca](i) responses, obtained in the presence of 2 mM Ca, to those recorded from the same cell after 1-1.5-min perifusions with EGTA-containing solutions buffered to 100 nM free Ca. The cells used in the present study were representative of 18 different cultures, prepared in consecutive weeks throughout a 5-month period.

Chromaffin cells were identified using a combination of morphological and functional criteria, as described previously(11) . The cells were subjected to a standard brief test pulse of 10 µM nicotine, and only those which responded with pronounced [Ca](i) rises were assumed to be catecholamine-secreting cells. Fig. 1is representative of a group of cells where the ATP-evoked [Ca](i) transients, recorded in the presence of 2 mM Ca (cell 1, second trace from left), were abolished in the virtual absence of external Ca (third trace from left). Challenging the cell with ATP after reintroducing Ca into the perifusion solution (second trace from right) produced a [Ca](i) response identical to that observed in control, indicating that the lack of effect in the absence of Ca cannot be explained by receptor desensitization following repeated exposure to the agonist. Importantly, the pyrimidine nucleotide UTP (100 µM) failed to evoke [Ca](i) rises in 13 out of 13 cells displaying no response to ATP in the virtual absence of Ca, as depicted in Fig. 1(cell 1, rightmost trace). The P/P antagonist suramin (100 µM) completely abolished the [Ca](i) response elicited by ATP in UTP-insensitive cells (Fig. 1, cell 2; n = 7 cells). For the sake of clarity, this group of chromaffin cells is henceforth designated as ``group I.'' The magnitude of the ATP-evoked [Ca](i) rise measured from group I cells was highly variable and averaged 910 ± 99 nM (n = 41 cells, range 100 - >3000 nM). It should be emphasized that the concentration of ATP used to stimulate the cells (100 µM) was one order of magnitude higher than the minimal concentration necessary to elicit maximal [Ca](i) responses, i.e. 10 µM(11) .


Figure 1: ATP-evoked [Ca] transients in chromaffin cells displaying no response to ATP in the virtual absence of Ca (group I cells). Cell 1, the cell was initially stimulated with 10 µM nicotine (Nic) and 100 µM ATP in medium containing 2 mM Ca, as denoted by the bars (valve switched on and off). The cell was then exposed to an EGTA-buffered medium containing 100 nM free Ca and further challenged with ATP. The cell was subsequently stimulated with ATP and 100 µM UTP following Ca reintroduction into the medium. Lack of effect of ATP in the virtual absence of Ca is representative of 25 experiments. Lack of effect of UTP in the presence of Ca is representative of 13 experiments performed on group I cells. Cell 2, the cell was sequentially exposed to 10 µM nicotine, 50 µM ATP, 100 µM UTP and ATP, the latter in the presence of 100 µM suramin. The cell was further exposed to ATP in suramin-free medium. Complete blockade of the ATP-evoked [Ca] rise by suramin is representative of seven experiments performed on ATP-sensitive but otherwise UTP-insensitive cells. Cells were allowed to rest for at least 5 min between consecutive challenges. Where appropriate, recordings were interrupted between stimulations to avoid excessive dye photobleaching.



A second group of cells (henceforth designated as ``group II'') responded to ATP with pronounced [Ca](i) rises in the virtual absence of Ca (i.e. in the presence of EGTA), as depicted by cell 1 in Fig. 2(fourth trace from left). The effectiveness of the Ca/EGTA buffer in abolishing the transmembrane Ca gradient was indicated by the lack of effect of 10 µM nicotine in the presence of this low Ca solution (cell 1, second trace from left). Although the typical ATP-elicited [Ca](i) response observed in the presence of Ca was a peak followed by a decay toward a plateau, in the virtual absence of Ca it consisted mainly of a peak followed by an accelerated decay toward base line. In sharp contrast with group I cells, group II cells responded to UTP with pronounced [Ca](i) rises in the presence of Ca (Fig. 2, cell 1, rightmost trace, and cell 2, second trace from left). This effect was observed in 14 out of 14 cells clearly identified as belonging to group II. The magnitude of the UTP-evoked [Ca](i) rise averaged 459 ± 50 nM (n = 14 cells). The UTP-evoked [Ca](i) transients did not require the presence of extracellular Ca (Fig. 2, cell 2, third trace from left). Furthermore, suramin did not affect the [Ca](i) transients evoked by either ATP or UTP from group II cells (Fig. 2, cell 2, second and fourth traces from right; n = 5 cells). The magnitude of the peak [Ca](i) rise evoked by ATP from group II cells was lower than from group I cells and averaged 579 ± 59 nM (range 100-1200 nM, n = 28 cells) in the presence of Ca and 467 ± 55 nM (n = 19 cells) in the absence of Ca.


Figure 2: ATP- and UTP-evoked [Ca] transients in chromaffin cells displaying positive responses to ATP in the virtual absence of Ca (group II cells). Cell 1, the cell was initially stimulated with 10 µM nicotine (Nic) both in the presence of 2 mM Ca and in the virtual absence of extracellular Ca (EGTA-buffered medium containing 100 nM free Ca). The cell was subsequently stimulated with 100 µM ATP both in the presence and in the virtual absence of Ca. The cell was further stimulated with ATP and 100 µM UTP following Ca reintroduction into the medium. Positive responses to ATP in the virtual absence of Ca are representative of 19 experiments performed on cells previously demonstrated to respond to ATP in the presence of Ca. The UTP responses are representative of 14 experiments performed on group II cells. Cell 2, the cell was initially exposed to 10 µM nicotine in the presence of Ca and to 50 µM UTP both in the presence and virtual absence of Ca. The cell was subsequently challenged with UTP and 50 µM ATP both in the presence and absence of 100 µM suramin. Lack of effect of suramin on ATP- and UTP-evoked [Ca] transients is representative of five experiments performed on group II cells.



Mn Entry Experiments

The following experiments were designed to assess the possibility that ATP might enhance Ca influx in group I and group II cells to different extents, as suggested by the analysis of the [Ca](i) data. To this end, the Ca channel surrogate Mn was added to the cells in the absence of Ca, and the fura-2 fluorescence was simultaneously recorded at 380 nm, a Ca-sensitive wavelength, and 358 nm, the dye isosbestic point(11) . Since Mn is a strong quencher of fura-2 fluorescence(22) , the fluorescence decay rate measured at 358 nm in the presence of Mn is indicative of Mn influx through Ca-permeant channels.

Fig. 3is representative of experiments in which exposure of single chromaffin cells to ATP during perifusion with Ca-free and Mn-containing solutions resulted in hardly detectable [Ca](i) rises, as illustrated in the upper trace of A. Thus, we assume that the cells depicted in Fig. 3belong to the previously defined group I. (Since the solutions contain µM amounts of free Ca, i.e. at least one order of magnitude more than the free Ca content of the solutions used for the experiments depicted in Fig. 1, the slight [Ca](i) rise observed in the upper trace of Fig. 3A can probably be accounted for by residual Ca influx.) ATP caused a rapid and pronounced fluorescence fall in this group of cells, indicating a drastic acceleration of the underlying Mn influx (bottom trace in Fig. 3A). Since this influx can, to a first approximation, be treated as a zero order process (extracellular Mn concentration remained constant throughout the experiments), intracellular accumulation of the divalent cation would be expected to follow a linear time course at the very beginning of the stimulation. Accordingly, we have fitted the early component (first 5 s) of the quenching trace to a linear function and used the respective slope as indicative of the early rate of divalent cation flux through the ATP-activated pathway. The diagram in Fig. 3B shows that ATP evoked an average 30-fold increase in the early Mn influx rate of group I cells (column labeled ``early''). Calculation of the slope of the quenching trace at a later stage (last 5 s of the 60 s ATP pulse) yielded an average rate of approximately 0.1 s, i.e. 6% of the early entry rate (column labeled ``late''), indicating that ATP-evoked Mn entry became almost fully inactivated in less than 60 s after exposure to ATP, in spite of the continued presence of the agonist. Indeed, the extended quenching trace recorded from group I cells could be fitted to a single exponential decay function with a time constant of 8.0 ± 0.9 s (n = 17 cells). It is also apparent from Fig. 3B (rightmost column labeled ``Mn'') that the Mn influx rate in group I cells was fully recovered to its prestimulatory level after a 60 s washout period. The massive ATP-evoked Mn entry recorded from group I cells was almost completely blocked by suramin in a reversible manner (Fig. 4C). Interestingly, suramin abolished the residual ATP-evoked [Ca](i) rise observed in the absence of added Ca. It is also important to note that UTP failed to affect Mn influx in group I cells (Fig. 3C).


Figure 3: Suramin blocks fast ATP-evoked Mn influx in group I chromaffin cells. A, Simultaneous [Ca] (upper trace) and normalized 358 nm fluorescence (lower trace) recordings of a single chromaffin cell bathed in Ca-free medium (no EGTA added). Mn (0.2 mM) was added to and withdrawn from the solution as indicated by the bar. The cell was stimulated with 100 µM ATP in the presence of Mn as indicated. Fast ATP-evoked Mn quenching of fura-2 fluorescence is representative of 22 experiments performed on cells displaying residual [Ca](i) rises and, hence, assumed to belong to group I. The Ca was withdrawn from the solutions 1-1.5 min before Mn addition. B, fluorescence decay rates measured from experiments similar to that depicted in A. The fluorescence decay recorded at 358 nm was fitted to a linear function for specified periods before, during, and after perifusion with the ATP-containing solution (Mn throughout). The columns represent the means ± S.E. (n = 22 cells) of the respective slopes, calculated from the 60-s period preceding the ATP pulse (leftmost column labeled Mn), from the first (early) and last (late) 5 s of ATP delivery and from the 60-s period following the ATP pulse (rightmost column labeled Mn), which typically lasted 60 s. C, experiment designed to assess the effect of suramin on ATP-evoked Mn influx. The [Ca] (upper trace) and the normalized 358 nm fluorescence (lower trace) were simultaneously recorded from a single chromaffin cell, both in the presence of 2 mM Ca (two leftmost sets of traces and rightmost set of traces) and in the absence of added Ca (third and fourth sets of traces from left). The cell was initially challenged with 50 µM ATP and 50 µM UTP in the presence of Ca. The Ca was subsequently removed from the solution, as denoted by the bar labeled Ca, and the cell was simultaneously exposed to 100 µM suramin and 0.2 mM Mn in the absence of added Ca, as denoted by the bar labeled Sur/Mn. The cell was subjected to a brief pulse of ATP in the presence of suramin (third set of traces from left), which abolished the fast Mn influx otherwise revealed by subjecting the cell to ATP in the absence of the drug (second set of traces from right). The cell was finally challenged with ATP in the presence of 2 mM Ca. The whole experiment was repeated in five cells with identical results.




Figure 4: Delayed Mn influx induced by ATP and UTP in group II chromaffin cells. A, simultaneous [Ca](i) (upper trace) and normalized 358 nm fluorescence (lower trace) recordings of a single chromaffin cell bathed in Ca-free medium (no EGTA added). Mn (0.2 mM) was added to and withdrawn from the solution as indicated by the bar. The cell was stimulated with 100 µM ATP in the presence of Mn as indicated. Delayed ATP-evoked Mn quenching of fura-2 fluorescence is representative of 13 experiments performed on cells displaying pronounced [Ca]rises and, hence, assumed to belong to group II. The Ca was withdrawn from the solutions 1-1.5 min before Mn addition. B, fluorescence decay rates measured from experiments similar to that depicted in A. The fluorescence decay recorded at 358 nm was fitted to a linear function for specified periods before, during and after perifusion with the ATP-containing solution (Mn throughout). The columns represent the means ± S.E. (n = 13 cells) of the respective slopes, calculated from the 60-s period preceding the ATP pulse (leftmost column labeled Mn), from the first (ATP early) and last (ATP late) 20 s of ATP delivery, and from the 60-s period following the ATP pulse (rightmost column labeled Mn), which typically lasted 60 s. C, same as in A, except that 100 µM UTP was used to stimulate the cells. Delayed UTP-evoked Mn quenching of fura-2 fluorescence is representative of nine experiments performed on cells displaying pronounced [Ca] rises. D, same as in B, except that the analysis has been applied to the experiment depicted in C and to 12 other identical experiments.



In a different group of cells, depicted in Fig. 4, ATP evoked pronounced [Ca](i) rises during exposure to the Ca-free and Mn-containing solution. Accordingly, these cells were assumed to belong to the previously defined group II. Fig. 4A shows that ATP failed to elicit a rapid and massive Mn influx in this group of cells. Indeed, the early stimulated Mn influx rate measured from these cells was not significantly different from the basal rate (Fig. 4B, column labeled early and leftmost column labeled Mn, respectively). However, the average stimulated Mn influx rate measured at a later stage (column labeled late) was increased by approximately 50% over the basal rate. Fig. 4, C and D, show that the effect of UTP on Mn quenching of fura-2 fluorescence in group II cells was analogous to that of ATP. Interestingly, the Mn influx rate remained transiently elevated after ATP or UTP washout, in spite of the absence of the agonist (Fig. 4, B and D, rightmost columns labeled Mn).

The amplitude of the early Mn influx rate and the maximal amplitude of the ATP-evoked [Ca](i) transients, measured simultaneously from the same cells in the absence of Ca and presence of Mn, were pooled in the form of a correlation diagram in Fig. 5. Approximately 53% of the cells tested for ATP displayed very high Mn influx rates (range 0.6-3 s) and residual [Ca](i) rises (range 5-70 nM) and were therefore classified as group I cells. On the other hand, 38% of the cells displayed very low Mn influx rates (range 0.01-0.1 s) and large amplitude [Ca](i) transients (range 150-1000 nM). The latter cells were designated group II. Three out of 34 cells, however, could not be ascribed to any of the above groups: They simultaneously exhibited the very high Mn influx rates typical of group I cells and the large amplitude [Ca](i) transients typical of group II cells. Importantly, none of the UTP-sensitive cells examined (depicted by empty circles in Fig. 5) fell within group I and they all fell within group II. It should be emphasized that the UTP data depicted in Fig. 5were exclusively gathered from cells that had been previously demonstrated to display definite [Ca](i) transients in response to the pyrimidine nucleotide, in the presence of 2 mM external Ca and in the absence of Mn.


Figure 5: Relationship between ATP/UTP-evoked [Ca] transients and early Mn influx rates, simultaneously recorded from single chromaffin cells in the absence of Ca. The early Mn influx rates (decay slopes) and the maximal amplitudes of the ATP- and UTP-evoked [Ca]transients (Delta[Ca]), measured simultaneously in the absence of added Ca and presence of Mn as depicted in Fig. 3, A and B, and Fig. 4, A-D, were pooled in the form of a correlation diagram. Solid circles, 100 µM ATP; open circles, 100 µM UTP. Three different groups of chromaffin cells are apparent from this analysis. One group of cells (Group I) exhibited very high Mn influx rates and residual [Ca] rises. This behavior was representative of 53% of the cells tested for ATP. A second group of cells (Group II) exhibited very low Mn influx rates and pronounced [Ca] rises. This behavior was representative of 38% of the cells tested for ATP. Yet another group of cells (3 out of 34) exhibited very high Mn influx rates typical of group I cells and pronounced [Ca] transients typical of group II cells. All UTP-sensitive cells fell in group II.



The above data are consistent with a model whereby ATP activates a receptor-associated ion channel in one pool of chromaffin cells (group I) and stimulates the release of Ca from internal stores in a separate pool of cells (group II). According to this model, the ATP effects on group I and group II cells should be mimicked by nicotine, a specific acetylcholine nicotinic receptor agonist, and by bradykinin, a phospholipase C activator and an intracellular Ca releasing agent(26) , respectively. To test this hypothesis, we have investigated the effects of nicotine and bradykinin on [Ca](i) and Mn quenching of fura-2 fluorescence, in the absence of added Ca. The results are summarized in Fig. 6. Nicotine caused a slight increase in [Ca](i) and a fast fluorescence decay in all cells tested (n = 19 cells), as depicted in Fig. 6A. Furthermore, nicotine-induced Mn influx inactivated rapidly with a time constant of 14.9 ± 1.3 s (n = 11 cells), so that after 20-25 s of continued stimulation the average quenching rate decreased to 30% of that recorded within the first 5 s (Fig. 6B). Thus, the nicotine action resembles the effect of ATP on group I cells. In contrast, bradykinin evoked a pronounced increase in [Ca](i) in all cells tested (n = 8 cells) and caused a relatively modest acceleration of Mn entry both at the early and late stages of stimulation, respectively, as depicted in Fig. 6, C and D. Thus, the pattern of the bradykinin effects is qualitatively similar to that of ATP in group II chromaffin cells.


Figure 6: Patterns of Mn influx induced by nicotine and bradykinin in chromaffin cells. A, simultaneous [Ca] (upper trace) and normalized 358 nm fluorescence (lower trace) recordings of a single chromaffin cell bathed in Ca-free medium (no EGTA added). Mn (0.2 mM) was added to and withdrawn from the solution as indicated by the bar. The cell was stimulated with 10 µM nicotine (Nic) in the presence of Mn as indicated. Similar results were obtained from 19 cells. B, fluorescence decay rates measured from experiments similar to that depicted in A. The fluorescence decay recorded at 358 nm was fitted to a linear function for specified periods before, during and after perifusion with the nicotine-containing solution (Mn throughout). The columns represent the means ± S.E. (n = 19 cells) of the respective slopes, calculated from the 60-s period preceding the nicotine pulse (leftmost column labeled Mn), from the first 5 s of nicotine delivery (Nic early), from the period 20-25 s after nicotine delivery (Nic late) and from the 60-s period following the nicotine pulse (rightmost column labeled Mn), which typically lasted 30 s. C, same as in A, except that 1 µM bradykinin (BK) was used to stimulate the cells. Similar results were obtained from 8 cells. D, same as in B, except that the analysis has been applied to the experiment depicted in C and to seven other identical experiments. The various decay slopes indicated were measured from the 60-s period preceding the BK pulse (leftmost column labeled Mn), from the first (BK early) and last (BK late) 20 s of bradykinin delivery and from the 60-s period following the bradykinin pulse (rightmost column labeled Mn), which typically lasted 60 s.



The ATP-evoked [Ca](i) rises recorded from group I cells can conceivably be accounted for by Ca influx through the putative ATP-gated channel itself, through voltage-sensitive Ca channels activated by membrane depolarization subsequent to the activation of the ATP-gated channel, or through both pathways simultaneously. We have made an attempt at dissecting out the relative contribution of each pathway to the ATP-evoked [Ca](i) transients. To this end, the responses of group I cells to ATP were compared in the absence and presence of 10 µM nifedipine (an L-type Ca channel blocker) + 1 mM neomycin. We have previously shown that 1 mM neomycin suppresses the dihydropyridine-resistant component of the depolarization-evoked Ca influx in bovine chromaffin cells while inhibiting the dihydropyridine-sensitive component(27) . We found that neomycin/nifedipine inhibited the ATP-evoked [Ca](i) rises in group I cells by 18-33% while inhibiting the high K (50 mM)-evoked transients by 29-38% (data not shown). It is therefore likely that a significant proportion of the ATP-evoked [Ca](i) rises can be accounted for by Ca influx through neomycin/nifedipine-sensitive voltage-dependent Ca channels, although a quantitative analysis of the specific involvement of the latter channels is made difficult by the limited effectiveness of the drugs as blockers of depolarization-evoked Ca influx, at least in the limited pool of cells used for this study.

Catecholamine Secretion

We have investigated the effects of ATP and UTP on catecholamine secretion using a flow system coupled to an electrochemical detector. Samples of the perfusate were simultaneously collected and later assayed for the respective contents in epinephrine and norepinephrine by HPLC. Fig. 7A shows the typical time course of the electrochemical detector output in response to brief additions of 200 µM ATP and 20 µM DMPP, a specific nicotinic receptor agonist. ATP evoked a relatively small catecholamine secretion (0.25 ± 0.07 nmol secreted above basal per 10 cells; n = 6 experiments from three different preparations) when compared with the DMPP-induced output (1.42 ± 0.15 nmol/10 cells; n = 10 experiments from five different preparations). In addition, Fig. 7B shows that DMPP evoked a preferential release of epinephrine, reflecting the higher abundance of this catecholamine in bovine chromaffin cells. In contrast, ATP evoked a preferential, if not exclusive release of norepinephrine (see legend to Fig. 7B for the absolute amounts of epinephrine and norepinephrine secreted in response to ATP). In agreement with a previous study(3) , UTP (500 µM) failed to evoke a detectable secretion (n = 4 experiments, data not shown).

We have shown above that suramin suppressed the ATP-evoked [Ca](i) responses from group I cells. We have now investigated the effect of suramin on ATP-evoked catecholamine secretion. Fig. 7A (second trace from left versus leftmost trace) shows that 300 µM suramin had a strong inhibitory effect upon total ATP-evoked catecholamine secretion. Fig. 7C shows the average inhibitory effect of suramin on ATP-evoked norepinephrine secretion (the effect of suramin on epinephrine release is difficult to assess unambiguously, owing to the the residual amounts of epinephrine secreted in response to ATP). Suramin inhibited ATP-evoked norepinephrine output by 80.6 ± 6.5% (n = seven experiments, three different preparations), in essential agreement with the electrochemical analysis illustrated in Fig. 7A (average percent inhibition: 71.3 ± 5.7; values computed from the integrals of the net current responses over 2 min periods).


DISCUSSION

Previous single cell studies of chromaffin cell responses to various receptor agonists such as nicotine, angiotensin, or muscarine have emphasized a profound variability in the patterns and magnitudes of the [Ca](i) transients elicited by these agonists(5, 7, 12) . By providing evidence for the cell-specific expression of two distinct purinergic receptors the present study reinforces the view that chromaffin cells are, indeed, functionally heterogenous and that this heterogeneity may be physiologically relevant to catecholamine secretion.

ATP promoted a dramatic enhancement of Mn influx and failed to raise the [Ca](i) in the virtual absence of external Ca in a discrete pool of chromaffin cells (group I). We have reported previously(11) , and further confirmed in this study, that chromaffin cells lacking a definite [Ca](i) response to ATP in the absence of Ca retain a marked sensitivity to the intracellular Ca mobilizing agent bradykinin, indicating that the lack of effect cannot be the consequence of extensive depletion of internal Ca stores, owing to collapse of the transmembrane Ca gradient. A more likely explanation is that ATP interacts with a single receptor type in group I cells and that this receptor is directly coupled to a cation channel. (^3)Indeed, the fact that the purinoceptor antagonist suramin blocks Mn influx and the associated [Ca](i) transients (^4)indicates that the influx pathway that supports fast Mn entry accounts entirely for the latter transients in group I cells. Moreover, the kinetic characteristics of the ATP-evoked Mn entry in group I cells, namely its fast onset and rapid inactivation, are comparable with those of the Mn influx elicited by the cholinergic agonist nicotine, which opens a receptor-associated cation channel in chromaffin cells(30, 31, 32) . Thus, group I chromaffin cells, like other neuronal cells, seem to be equipped with ATP-gated cation channels. Na- and Ca-permeant, rapidly inactivating cation channels have indeed been described in the closely related PC12 cell line (33, 34, 35) and in other neuronal cells types(9, 36, 37) .

We have also found that ATP exerted a qualitatively distinct action upon a second discrete pool of chromaffin cells (group II). Indeed, ATP evoked prominent [Ca](i) transients in the virtual absence of Ca in these cells, indicating an important contribution of Ca release from internal stores to the signal observed in the presence of extracellular Ca. Importantly, the pyrimidine nucleotide UTP raised the [Ca](i) in group II cells while failing to evoke responses from group I cells, suggesting that the underlying purinoceptor is of the P-type (``nucleotide receptor''). It should be emphasized that ATP and UTP failed to produce a massive Mn influx in group II cells and evoked instead a relatively small and delayed Mn entry which, remarkably, remained transiently enhanced after agonist removal. This resembles the action of bradykinin, a peptide that, in chromaffin cells and other cells, evokes Ca release from internal stores by activating a membrane receptor coupled to the phosphoinositide signaling system(26) . Moreover, bradykinin has been shown to activate a Ca influx pathway apparently linked to the refil of these stores(38, 39) . Thus, we attribute the delayed Mn influx, apparent from ATP- and UTP-stimulated group II chromaffin cells, to the presence of a Ca entry pathway specifically activated by the depletion of intracellular Ca stores (40, 41) . The presence of the latter mechanism in chromaffin cells has indeed been recently demonstrated with the help of endoplasmic reticulum Ca pump inhibitors(42) . The present work attributes further physiological significance to this mechanism by showing that the Ca refilling pathway can be activated by stimulation of chromaffin cells with physiological agonists.

In our previous work on the characterization of the ATP effects on chromaffin cells(11) , we failed to detect cells displaying positive [Ca](i) responses to ATP in the absence of Ca, i.e. group II cells. The reason why we failed to detect these cells is not completely clear. Nonetheless, in our previous work we have routinely assessed the ATP responses in the absence of Ca from cells displaying the largest [Ca](i) responses to the purinergic agonist in the presence of Ca. While this was done in an attempt to maximize the probability of obtaining pronounced responses in the absence of Ca, it may have introduced an experimental bias favoring the preferential sampling of group I cells (the average size of the ATP-evoked responses in group I cells was approximately 40% larger than that assessed from group II cells).

The correlation diagram depicted in Fig. 5shows that the vast majority of chromaffin cells which have been simultaneously probed in terms of [Ca](i) transients, and Mn influx could be readily assigned to either group I or II. However, a few cells actually displayed prominent [Ca](i) transients and a massive Mn influx in response to ATP in the absence of Ca, suggesting that the ATP-gated channel and the P-type purinoceptor may coexist in some chromaffin cells. Using the various functional or pharmacological criteria presented above to classify single chromaffin cells as group I or group II cells, we have found that approximately 54% of the cells could be considered to belong to group I through a random sampling of 81 ATP-responding cells. Thus, we estimate that approximately 54% of the cells of a typical preparation are exclusively equipped with ATP-gated cation channels. The remaining cells could either be classified as group II cells (42%) and hence assumed to express a P-type purinoceptor coupled to Ca release from intracellular stores or as ``hybrid'' cells expressing the two purinoceptor types (less than 4%). This finding emphasizes the tight cell specificity of purinoceptor expression in chromaffin cells.

Is the distribution of the two purinoceptor subtypes related to the amine content of the cells? Our catecholamine release data show that ATP evokes a preferential, if not exclusive, release of norepinephrine and suggest that this is indeed likely to be the case. In keeping with a previous study (3) UTP, a specific marker of group II cells (this study), had no effect on secretion, reinforcing the view that ATP-evoked Ca release from internal stores is not coupled to catecholamine release in bovine chromaffin cells(7, 12, 43) . Thus, ATP-evoked catecholamine release must be essentially operated by the cells displaying the ATP-gated fast Ca influx component, implying that this mechanism is preferentially, if not exclusively, localized to norepinephrine-secreting chromaffin cells. This conclusion is reinforced by our observation that suramin, which blocked selectively the ATP-evoked [Ca](i) transients from group I cells and, thus, can be considered to block specifically the fast ATP-gated channel, also inhibited strongly (by approximately 80%) ATP-evoked norepinephrine release. It is clear, however, that further work is necessary before the assignment of each purinoceptor type documented in this study to epinephrine- and norepinephrine-secreting cells can be made unambiguously.


FOOTNOTES

*
This work was partially financed by grants from Junta Nacional de Investigação Cientifica e Tecnologica (JNICT) (STRDA/C/SAU/254/92) and the Calouste Gulbenkian Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a FEBS fellowship. Provisional address: Dept. of Biochemistry, FCTUC, P. O. Box 3126, 3049 Coimbra Codex, Portugal.

(^1)
The abbreviations used are: MOPS, 3-(N-morpholino)propanesulfonic acid; [Ca], intracellular free Ca concentration; DMPP, 1,1-dimethyl-4-phenylpiperazinium iodide; fura-2/AM, acetoxymethyl ester of fura-2; HPLC, high performance liquid chromatography.

(^2)
A. R. Tomé, unpublished observations.

(^3)
Activation of the receptor-associated cation channel may provide direct access of extracellular Ca to the cytosol and/or generate enough membrane depolarization to activate voltage-sensitive Ca channels(28) . The neomycin/nifedipine experiments, although indicative that a fraction of the ATP-evoked [Ca] transients recorded from group I cells can be accounted for by Ca influx through voltage-sensitive Ca channels, are inconclusive as to the relative contribution of this pathway to the effects observed.

(^4)
The lack of effect of suramin in group II cells indicates that this drug does not behave as a P receptor antagonist and suggests that suramin may be a more specific purinergic antagonist than acknowledged previously(29) .


ACKNOWLEDGEMENTS

We are indebted to Prof. A. Brett (Chemistry Department, University of Coimbra) for making available parts of the electrochemical system used for the on-line detection of catecholamines. Thanks are also due to C. M. Sena, M. G. Baltazar, and Dr. E. Duarte for help with the cell culture.


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