(Received for publication, August 19, 1994; and in revised form, December 7, 1994)
From the
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.
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.
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 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
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
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
cells (n = 7 experiments, three different preparations). In B and C the catecholamines were assayed by
HPLC.
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]
rises were assumed to be catecholamine-secreting cells. Fig. 1is representative of a group of cells where the ATP-evoked
[Ca
]
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
]
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
]
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
]
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
]
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
]
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]
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
]
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
]
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
]
rise averaged 459 ± 50 nM (n = 14
cells). The UTP-evoked [Ca
]
transients did not require the presence of extracellular
Ca
(Fig. 2, cell 2, third trace from
left). Furthermore, suramin did not affect the
[Ca
]
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
]
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.
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
]
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
]
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
]
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
]
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
]
(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]
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
]
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
]
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
]
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
]
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
]
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
(
[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
]
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
]
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
]
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]
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
]
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
]
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
]
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.
We have shown above that suramin suppressed the ATP-evoked
[Ca]
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).
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]
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
]
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
]
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. (
)Indeed, the fact that the purinoceptor antagonist
suramin blocks Mn
influx and the associated
[Ca
]
transients (
)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]
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
]
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]
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
]
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]
transients, and Mn
influx could be readily
assigned to either group I or II. However, a few cells actually
displayed prominent [Ca
]
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
]
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.