Departamento de Bioquímica y Biología Molecular y Fisiología, Instituto de Biología y Genética Molecular and Facultad de Medicina, Universidad de Valladolid, 47005 Valladolid, Spain
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ABSTRACT |
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The
notion that intracellular Ca2+ (Cai2+)
stores play a significant role in the chemoreception process in
chemoreceptor cells of the carotid body (CB) appears in the literature
in a recurrent manner. However, the structural identity of the
Ca2+ stores and their real significance in the function of
chemoreceptor cells are unknown. To assess the functional significance
of Cai2+ stores in chemoreceptor cells, we have
monitored 1) the release of catecholamines (CA) from the
cells using an in vitro preparation of intact rabbit CB and
2) the intracellular Ca2+ concentration
([Ca2+]i) using isolated chemoreceptor cells;
both parameters were measured in the absence or the presence of agents
interfering with the storage of Ca2+. We found that
threshold [Ca2+]i for high extracellular
K+ (Ke+) to elicit a release response is
250 nM. Caffeine (10-40 mM), ryanodine (0.5 µM), thapsigargin
(0.05-1 µM), and cyclopiazonic acid (10 µM) did not alter the
basal or the stimulus (hypoxia, high Ke+)-induced
release of CA. The same agents produced Cai2+
transients of amplitude below secretory threshold; ryanodine (0.5 µM), thapsigargin (1 µM), and cyclopiazonic acid (10 µM) did not
alter the magnitude or time course of the Cai2+
responses elicited by high Ke+. Several potential
activators of the phospholipase C system (bethanechol, ATP, and
bradykinin), and thereby of inositol 1,4,5-trisphosphate receptors,
produced minimal or no changes in [Ca2+]i and
did not affect the basal release of CA. It is concluded that, in the
rabbit CB chemoreceptor cells, Cai2+ stores do not play
a significant role in the instant-to-instant chemoreception process.
hypoxia; catecholamine; thapsigargin; caffeine; ryanodine; cyclopiazonic acid
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INTRODUCTION |
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THE ROLE OF intracellular free Ca2+ concentration ([Ca2+]i) in the control of cellular functions, including the release of neurotransmitters, was recognized many decades ago, but the list of cellular functions triggered or modulated by increased levels of intracellular Ca2+ (Cai2+) has grown enormously (12, 28). In parallel, several mechanisms aimed at maintaining resting free [Ca2+]i in the range of 100 nM and allowing Cai2+ to rise during cell activation have been described (38).
Many stimuli activate cells by causing a rise in free Cai2+ that occurs by Ca2+ influx from the extracellular milieu via voltage- or receptor-operated channels; net influx of Ca2+ may also occur via the Na+/Ca2+ exchanger (12, 28). Ca2+ efflux to the extracellular space occurs against electrochemical gradients, being the pumping out of Ca2+ directly (Ca2+ pump) or indirectly (Na+/Ca2+) coupled to the hydrolysis of ATP. There are also intracellular organelles, primarily the endoplasmic reticulum, that accumulate Ca2+ via specific Ca2+-ATPases and contribute to restore basal levels of cytoplasmic free Ca2+ after cell activation. On the other hand, specific stimuli can mobilize the accumulated Ca2+ to produce a rise in free Cai2+ and cell activation; the efflux of Ca2+ from the endoplasmic reticulum to the cell cytoplasm occurs via either or both the inositol 1,4,5-trisphosphate (IP3) and the ryanodine (Ry) receptor channels (IP3R and RyR, respectively; see Refs. 34 and 36). Therefore, the endoplasmic reticulum may function as a sink or as a source for free cytoplasmic Ca2+ in different functional states of the cells (36). At high concentrations of free Cai2+, mitochondria may also accumulate Ca2+, functioning as sinks to reduce cytoplasmic free Ca2+.
Our knowledge on the mechanisms of Ca2+ handling in the chemoreceptor cells of the carotid body (CB) is rather restricted (see Ref. 22). The release of dopamine (DA) elicited by hypoxia and high extracellular K+ (Ke+) in the rabbit, cat, and rat CB (1, 19, 31; also unpublished results) and the Cai2+ rise produced by the same stimuli in chemoreceptor cells of the rabbit and rat (9, 23, 39) are extracellular Ca2+ dependent by >95%. At variance with these findings, Biscoe and Duchen (7) and Duchen and Biscoe (15) reported that up to 40% of the hypoxic Cai2+ rise in rabbit chemoreceptor cells was due to Ca2+ entering the cytoplasm from structurally unidentified intracellular stores. Lahiri et al. (26) have reported that the anoxic cat carotid sinus nerve discharge recorded in nominally Ca2+-free media was better preserved in the presence of thapsigargin (TG; an inhibitor of endoplasmic reticulum Ca2+-ATPase), concluding that Cai2+ stores contribute to CB chemoreception. Mokashi et al. (29) have found that inhibition of cytochrome oxidase with CO produces a rise in Cai2+ levels in isolated rat chemoreceptor cells that was identical in the absence or the presence of 200 µM CdCl2 in the superfusing solution and concluded that the Ca2+ rise is due to release from intracellular stores. Finally, Dasso et al. (13) have found that the Cai2+ rise produced by muscarinic agonists in a subpopulation of rat CB chemoreceptor cells was reduced by only 50% in Ca2+-free solutions.
The aim of the present work has been to elucidate the significance of Cai2+ stores for the chemoreception process in chemoreceptor cells of the rabbit CB. As an index of chemoreception in chemoreceptor cells, we have monitored the release of catecholamines (CA) in an in vitro preparation of the intact rabbit CB whose CA stores have been labeled by prior incubation with the natural precursor [3H]tyrosine. Several agents known to release Ca2+ or to interfere with its accumulation in intracellular stores, including caffeine, ryanodine, TG, and cyclopiazonic acid, were tested for their ability to modify the basal and stimulus-evoked release of [3H]CA. We have also used short-term cultures of chemoreceptor cells isolated from the rabbit CB to measure microspectrofluorometrically the levels of Cai2+ and to determine the significance of intracellular stores in the homeostasis of Ca2+. We found that the basal or the high Ke+- and hypoxia-evoked release of [3H]CA was not affected by the agents activating the RyR or inhibiting the endoplasmic reticulum Ca2+-ATPase. The same agents were ineffective at increasing the basal levels of Cai2+ above the threshold for the secretory response and did not significantly alter the Cai2+ transients produced by high Ke+. The findings imply that Cai2+ stores do not contribute significantly to the chemoreception process in the rabbit CB chemoreceptor cells.
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MATERIALS AND METHODS |
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Surgical procedures. Adult New Zealand White rabbits (1.5-2.5 kg) were anesthetized with pentobarbital sodium (40 mg/kg) via the lateral vein of the ear. After tracheostomy, the carotid bifurcations were removed and placed in a lucite chamber filled with ice-cold 100% O2-saturated Tyrode solution (in mM: 140 NaCl, 5 KCl, 2 CaCl2, 1.1 MgCl2, 10 HEPES, and 5.5 glucose; pH = 7.40). Under a dissecting microscope, the CBs were localized, cleaned of surrounding tissues, and saved in fresh Tyrode.
[3H]CA release experiments.
When the four to eight CBs needed for a typical experiment were
collected, the organs were incubated (2 h; 37°C) in a glass vial
placed in a metabolic shaker containing 2 ml of Tyrode solution supplemented with 20 µM [3H]tyrosine
([3,5-3H]tyrosine; 30 Ci/mmol; Amersham, Iberica, Madrid,
Spain), 1 mM ascorbic acid, and 100 µM 6-methyl-tetrahydropterin
(Sigma, Madrid, Spain), cofactors of dopamine--hydroxylase and
tyrosine hydroxylase. In these incubating conditions, each CB (average
weight, 400 µg) synthesized ~12 pmol of [3H]CA,
equivalent to
4 × 105 dpm, being ~90%
[3H]DA and 10% [3H]norepinephrine
(18).
Chemoreceptor cell culture and Cai2+ measurements. CBs were enzymatically dispersed, and dissociated cells were plated on small poly-L-lysine-coated coverslips and maintained in culture for up to 48 h, as described previously (33). To measure cytosolic free [Ca2+], cells were loaded with the Ca2+ indicator dye indo 1 by incubating the coverslips with 5 µM indo 1-AM (Molecular Probes, Eugene, OR) in Tyrode solution for 1 h at room temperature. After loading and incubation, the coverslips were transferred to the recording chamber (37°C) and were perfused with different solutions delivered by gravity at constant flow rates of 2 ml/min. Cells were illuminated through a ×40 objective (Fluor 40/1.30 oil; Nikon) with 360 nm light provided by a 100-W Hg lamp (Optiquip). The illumination of the cells was limited to light pulses of 100 ms of duration delivered at a frequency of 1 Hz using a precision galvanometer (DX1000; Solamere Technology Group, Salt Lake City, UT) controlled with software developed in our laboratory. Emitted fluorescence at 405 and 495 nm was recorded with two photomultiplier tubes (Hamamatsu, Bridgewater, NJ) mounted in the lateral door of an inverted microscope (Diaphot 300; Nikon). Small apertures placed in the excitation path and/or in the lateral door of the microscope allowed the signal coming from a single chemoreceptor cell to be recorded. Calibrations to derive intracellular free [Ca2+] from the captured fluorescent signals (24) were made in the presence of 50 µM ionomycin after perfusing the cells for 30 min with Ca2+-free (0 Ca2+ + 10 mM EGTA) or 2 mM Ca2+-containing solutions.
Chemicals. Caffeine, ryanodine, TG, cyclopiazonic acid (CPA), and 2,5-di-(tert-butyl)-1,4-benzohydroquinone (TBH) were obtained from Alomone Laboratories (Jerusalem, Israel). Bradykinin, bethanechol, ATP, and all regular chemicals used to prepare physiological salt solutions were obtained from Sigma.
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RESULTS |
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Release of [3H]CA by the intact CB and
Cai2+ in isolated chemoreceptor cells.
Figure 1A shows the mean
magnitude and time course of release of [3H]CA elicited
by several concentrations of Ke+ applied for 9 min. The
data shown in Fig. 1A were obtained in a single experiment
performed with four CBs. Additional experiments were identically
performed except for a rotation in the order of application of the
different concentrations of K+, which was imposed to
eliminate uncontrolled time-dependent effects. As previously shown
(1, 31), the secretory response was sustained during the entire period of incubation with moderately high
concentrations of Ke+ (<30 mM), and it showed some
type of adaptation at higher concentrations. Threshold
Ke+ concentration to elicit a measurable secretory
response was ~20 mM. Mean basal release obtained in a total of eight
CBs during 9 min in the control periods before and after 20 mM
K+ application amounted to 1,191 ± 150 dpm and during
the 9 min of incubation with 20 mM K+ was 2,002 ± 193 dpm; the difference between both numbers (811 ± 162 dpm)
represents the evoked release, and it is statistically significant
(P < 0.01). Incubation with 10 and 15 mM
Ke+ produced a release of [3H]CA that was
indistinguishable from that seen with 5 mM. Figure 1B shows
sample records of Cai2+ levels in isolated
chemoreceptor cells in response to long (5-min) pulses of superfusion
with several concentrations of Ke+. From these sample
records, it is evident that moderately high concentrations of
Ke+ produced nearly sustained elevations of
Cai2+, whereas higher concentrations produced fast
transient increases in Cai2+ levels followed by adapted
levels of Cai2+ well above basal and lasting for the
entire period of high-Ke+ perfusion. The shape of the
curve relating high Ke+ to the release of CA in the
rabbit CB (Fig. 1C) is similar to that found in other
structures, including brain tissue (41), sympathetic
endings (20), adrenomedullary chromaffin cells
(4), and cat CB (1). The increase in peak
[Cai2+] in response to high-Ke+
depolarization follows a similar relationship (Fig. 1C).
However, at moderately high concentrations of Ke+, the
Ke+-Cai2+ curve is displaced to the
left compared with the Ke+-release curve so that
perfusion of the cells with 10 mM K+ already produced a
significant increase in [Ca2+]i to 154 ± 21 nM from a basal rate (perfusion with 5 mM K+) of
95 ± 21 nM (P < 0.05), whereas a significant
increase in the release was only obtained with 20 mM K+.
From the comparison of the two curves, it is evident that a minimum
increase in Cai2+ above basal is needed to elicit a
secretory response. The curve relating the
high-Ke+-elicited increase in
[Ca2+]i in isolated cells and secretory
response in the intact CB (Fig. 1D) shows this last point
more clearly and indicates that the threshold Cai2+
concentration to obtain a measurable release above basal in the rabbit
CB chemoreceptor cells is in the range of 250 nM. This calculated
threshold concentration of Cai2+ is comparable to that
found in bovine adrenal chromaffin cells (3).
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Effects of caffeine and ryanodine on the basal release of
[3H]CA and Cai2+ levels.
Caffeine is an agonist of RyR and is probably the most classical
mobilizer of Ca2+ stores in any cell type
(34). In adrenomedullary chromaffin cells incubated in
Ca2+-free solutions, caffeine at 10-40 mM increased
Cai2+ and the basal release of CA; at 40 mM caffeine,
the release response was 12 times basal release (37,
43). Figure 2A
shows that 40 mM caffeine in nominally Ca2+-free medium did
not elicit a secretory response in the chromaffin chemoreceptor cells
of the rabbit CB. In a similar experiment in
Ca2+-containing solution, caffeine at 20 mM was also
ineffective at altering the ongoing basal release of
[3H]CA (data not shown). Figure 2B shows a
sample record and mean Cai2+ responses elicited by 10 mM caffeine in isolated chemoreceptor cells while perfusing with
Ca2+-containing solutions. In this particular cell, 10 mM
caffeine caused a change () in Cai2+ of 130 nM, and
the mean Cai2+ rise produced by 10 mM caffeine in 6 cells was 115 ± 28 nM (Fig. 2B). Ryanodine (0.5 µM),
which at submicromolar concentrations behaves as an agonist of the RyR
(16, 25), was also ineffective at increasing
the basal release of [3H]CA from the rabbit CB in
Ca2+-containing solutions (Fig. 2C) and did not
alter [Ca2+]i in resting cells (Fig.
2D). In a small group of three CBs, 2 µM ryanodine was
equally ineffective at altering the ongoing release of
[3H]CA (data not shown). The sample record in Fig.
2D shows that the [Ca2+]i obtained
while perfusing with 35 mM K+ is comparable in the absence
and in the presence of 0.5 µM ryanodine, and in fact the mean
[Ca2+]i seen in a total of five cells
studied was 810 ± 73 and 802 ± 97 nM in the absence and the
presence of ryanodine, respectively (P > 0.05).
Ryanodine was similarly ineffective at altering low PO2- and high Ke+-elicited
release of [3H]CA (see Fig. 5).
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Effects of potential activators of the phospholipase C system on
the basal release of [3H]CA and Cai2+
levels.
The lack of effect of caffeine and ryanodine on the release of
[3H]CA and the minimal effect on Cai2+
levels could be the result of a very low expression of RyR in chemoreceptor cells (see Ref. 8a). Therefore, it was important to
explore further the existence of Cai2+ stores and their
possible significance for the release of [3H]CA with the
use of potential activators of phospholipase C, and consequently of
IP3R. We have chosen bethanechol because muscarinic agonists release Ca2+ from intracellular stores in rat
chemoreceptor cells (13) and ATP and bradykinin because
they release Ca2+ from intracellular stores in chromaffin
cells of the adrenal medulla (3, 11). Figure
3A shows that neither
bethanechol (10 µM), ATP (100 µM), nor bradykinin (1 µM) altered
the basal release of [3H]CA. In additional experiments
carried out with higher concentrations of the same agents (50 µM
bethanechol, 1,500 µM ATP, and 10 µM bradykinin), both in
Ca2+-containing and in nominally Ca2+-free
solutions, no alterations in the ongoing release of
[3H]CA could be detected. Parallel studies in isolated
cells revealed that these agents produced only small transients in
basal Cai2+ levels (Fig. 3B).
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Effects of TG on the basal release of [3H]CA.
TG is an irreversible inhibitor of the endoplasmic reticulum
Ca2+-ATPase, which produces a relatively slow emptying of
the Ca2+ deposits due to the passive leak of
Ca2+ unopposed by the active Ca2+ pumping. TG
promotes transient increases of cytoplasmic free Ca2+
capable of triggering Ca2+-dependent responses in many cell
types, both in the presence and in the absence of extracellular
Ca2+ (5, 14, 32).
Figure 4A shows that 1 µM TG
in combination with 0.5 µM ryanodine did not affect the basal release
of [3H]CA in nominally Ca2+-free solutions.
Because in most preparations TG produces a more intense and sustained
increase in Cai2+ levels in Ca2+-containing
solutions, apparently due to the capacitative entry of Ca2+
from the external milieu (14, 42), we tested
TG at three different concentrations in Ca2+-containing
solutions. Even in these conditions, TG was ineffective at altering the
ongoing release of [3H]CA from the rabbit CB (Fig.
4B). In isolated chemoreceptor cells, we studied the effect
of TG (1 µM) on Cai2+ levels. Figure 4C
shows a sample record of the Ca2+ transients produced by
TG and mean peak Cai2+ levels observed in a total of
six cells.
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Effects of functional elimination of Ca2+ stores on the
hypoxia- and high Ke+-evoked release of
[3H]CA and on Cai2+ transients produced
by high Ke+.
To study the possible contribution of Ca2+ deposits to the
stimulus-evoked release of [3H]CA, we have chosen to
functionally eliminate the endoplasmic reticulum as a source of
Ca2+ (see Ref. 36). In a first group of experiments using
pairs of CBs, we compared the release of [3H]CA elicited
by two consecutive intense hypoxic stimuli (incubation for 10 min in a
2% O2-equilibrated solution; PO2
22 mmHg) in the control organs with that obtained in the
experimental members of the pairs identically stimulated, except for
the presence of 0.5 µM ryanodine in the incubating solutions 20 min
before and during the second application of the hypoxic stimulus (Fig.
5, A and B). To
statistically assess the possible effects of the drug, the ratios of
the release response in the second presentation of the stimulus
(S2) to that obtained in the first (S1;
S2-to-S1 ratios) in the control CBs were
compared with the S2/S1 obtained in the
ryanodine-treated or experimental organs (Fig. 5C); for the
hypoxic stimulus, there were no statistical differences between the
S2-to-S1 ratios obtained in control (0.60 ± 0.07) and ryanodine-treated (0.61 ± 0.06) CBs. In a similar
experiment in which the stimulus was 35 mM Ke+,
ryanodine also was ineffective at altering the evoked response, as mean
S2-to-S1 ratios obtained in six control
(0.81 ± 0.09) and in six ryanodine-treated (0.75 ± 0.11)
CBs were not statistically different (P > 0.05; Fig.
5C). In these groups of experiments, the lack of effect of
ryanodine on the basal release of [3H]CA was also
reassessed (see Fig. 5B).
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DISCUSSION |
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The aim of the present work was to elucidate the role of Cai2+ stores in the minute-to-minute process of chemoreception. We have correlated the effects of agents known to mobilize Ca2+ from intracellular stores on their ability to produce Cai2+ transients or to alter the transients produced by high Ke+ in isolated chemoreceptor cells, with their capacity to induce the release of CA or to alter the release induced by hypoxia and high Ke+ in the intact CB. The results have disclosed Cai2+ stores with very low capacity; these Cai2+ stores were also unable to alter the secretory response in the chromaffin chemoreceptor cells of the CB. This implies that chemoreceptor cells in the rabbit CB depend almost exclusively on plasma membrane mechanisms to control the neurosecretion and to maintain their Ca2+ homeostasis.
At the outset of the discussion, we want to clarify, in the context of the present study, the meaning of the release of CA with the overall process of chemoreception. On the one hand, it is well documented that chemoreceptor cells are capable of detecting PO2, the main physiological stimulus to the CB chemoreceptors (see Refs. 21 and 22). It is also well documented that chemoreceptor cells of the rabbit CB release CA in response to hypoxia in direct proportion to its intensity and to the action potential frequency activity generated in the carotid sinus nerve (19). Therefore, with independence of the role of CA in the genesis of the activity in the carotid sinus nerve, the release of CA represents a direct measure of the output of the hypoxic chemoreception in chemoreceptor cells.
The titration of the intact CB for the release of [3H]CA
and of isolated chemoreceptor cells for Cai2+ levels
during stimulation with high Ke+ (see Fig. 1) provides
two critical pieces of information to interpret the results of the rest
of the experiments. First, the data of Fig. 1 prove the high
sensitivity of our radioisotopic method to detect evoked release. With
stable baseline release of CA amounting to 200-400 dpm in the
experiments with collection fractions of 2 and 3 min, we can detect
reliably increases in the release in the range of 100-200
dpm/2-3 min above basal (representing <9 fmol). This high
sensitivity might be critical because, as stated by Augustine and Neher
(3), the efficacy of Ca2+ coming from the
intracellular deposits to release CA might be small compared with that
elicited by the depolarizing stimulus. Second, the threshold
Cai2+ concentration to elicit a secretory response
under high Ke+ stimulation found in chemoreceptor cells
is 250 nM, comparable to that seen in bovine chromaffin cells of the
adrenal medulla for depolarizing stimulus (3), and none of
the mobilizers of Ca2+ from internal stores produces
increases in Cai2+ above this threshold concentration
(see Figs. 2-4). Therefore, our measurements of
Cai2+ under caffeine, ryanodine, bethanechol, ATP,
bradykinin, and TG are fully consistent with their lack of effect on
the secretory response. Furthermore, in their detailed study in
chromaffin cells, Augustine and Neher (3) showed that, at
identical average levels of Cai2+ (the parameter
measured with fluorescent dyes), the rate of release of CA was higher
if the Cai2+ rise was produced by depolarization. In
other words, the threshold Cai2+ to promote release of
CA would probably be higher than 250 nM when the Ca2+ comes
from internal stores.
The experiments with caffeine and ryanodine (Fig. 2) indicate that the endoplasmic reticulum of chemoreceptor cells contains relatively few RyR or, alternatively, that the Ca2+ stores sensitive to caffeine/ryanodine do not contain enough Ca2+ to provide cytoplasmic levels of free Ca2+ above threshold for the release response. This conclusion is supported by the characteristics of the action of caffeine and ryanodine. It is conceivable that the subconductance state of the ryanodine channel produced by ryanodine might lead to a slow emptying of the stores and then to a transient increase of cytoplasmic free Ca2+ with a slow time course and a small amplitude; however, caffeine produces a bursting behavior of the high-conductance ryanodine channel, leading to a prompt emptying of the stores and a fast transient of free cytoplasmic Ca2+ with an amplitude proportional to that of the sensitive deposit (16, 28, 36) capable of triggering Ca2+-dependent responses, including contraction of cardiac, striated, and smooth muscle (6) and secretion in many cell types, including adrenomedullary chromaffin cells (37, 43), where it can increase basal release by a factor >12.
The experiments with potential activators of the phospholipase C system
(Fig. 3) deserve some specific commentary. In addition to the agents
tested in this study, we have also studied the effects of adrenergic
2-agonists (2) and of adrenomedullin (A. Obeso, unpublished observations) on the release of CA in the rabbit CB. These two agents, which are also potential activators of phospholipase C and thereby generators of IP3 and Ca2+
mobilizers, are similarly ineffective at promoting release of CA
above basal, suggesting that mobilization of Ca2+ via
IP3 is not a system well developed in the rabbit CB
chemoreceptor cells. Furthermore, in a recent study in our laboratory
(35), we have found that hypoxia in Ca2+-free
solutions or in Ca2+-containing solutions (thereby
promoting the release of all neurotransmitters contained in the cells)
did not alter the IP3 levels. Therefore, the data obtained
in the present study indicate that the IP3-sensitive Ca2+ stores, if at all present in the rabbit CB
chemoreceptor cells, do not play a significant role in the
instant-to-instant chemoreception process in chemoreceptor cells.
The IP3- and the ryanodine-sensitive Ca2+ stores may represent two physically different cellular compartments, and TG inhibits the Ca2+-ATPases loading both stores (see Ref. 34). If both stores are located in different subcellular compartments of chemoreceptor cells, TG, TBH, and CPA should be more potent at raising Cai2+ than caffeine or the IP3-generating stimulus. Yet, neither ATPase inhibitor alone nor in combination with submicromolar concentrations of ryanodine promoted any secretory response either in Ca2+-free or in Ca2+-containing solution (see Fig. 4), suggesting that the capacity of the internal Ca2+ stores is very small. The lack of effect of ryanodine, TG, and CPA on the release of CA elicited by high Ke+ and hypoxia (Figs. 5 and 6) indicates that Cai2+ stores do not participate significantly in supporting the secretory response elicited by these stimuli; the data also suggest that internal Ca2+ stores have a low capacity. Although the secretory response induced by high Ke+ and hypoxia in chemoreceptor cells is nearly fully dependent on the extracellular Ca2+ (31), if intracellular stores have a significant capacity to store Ca2+, they should produce a double effect as follows: 1) to increase the peak of the secretion providing Ca2+ via the Ca2+-induced Ca2+ release mechanism and 2) to reduce the time course of the secretory response by pumping Ca2+ from the cytoplasm to the intracellular stores. Data show that none of the agents (ryanodine, TG, and CPA) that functionally eliminate the intracellular stores affect the stimulus-induced release of CA. Consistent with the observations made on the secretory response elicited by hypoxia and high Ke+, the data presented in Fig. 7 show that the inhibition of the Cai2+-ATPases does not affect the properties of the Ca2+ transients produced by high Ke+. These findings demonstrate unequivocally the limited functional capacity of Cai2+ stores in the chemoreceptor cells of the rabbit CB. Thus, if chemoreceptor cells had quantitatively significant Cai2+ stores, it should be expected to decrease the peak amplitude of the Cai2+ transient after inhibition of the Cai2+-ATPases because this inhibition eliminates the Ca2+-induced Ca2+-release mechanism (34). Additionally, if intracellular stores have a significant capacity to store Ca2+, they should participate in the buffering (sink effect of Cai2+ stores) of Ca2+ entering the cytoplasm from the extracellular milieu (6, 8, 40; see Ref. 36 for a review). Therefore, the inhibition of the Cai2+-ATPases would reduce the velocity of such buffering and should increase the time constant of the recovery of Cai2+ transient produced by high Ke+ (36). Data show that neither the peak amplitude nor the time constant of the recovery of the Ca2+ transient was affected by TG and CPA. We did not repeat this experiment using a hypoxic stimulus because the peak of the Ca2+ transient produced by high Ke+ is higher than that produced by hypoxia (23), and the greater the peak of the Ca2+ transient the more evident the buffering capacity of internal stores (34, 36). In summary, the data presented in Figs. 5-7 are consistent with data of Figs. 1-4 showing that the functional capacity of Cai2+ stores is very small, and, therefore, the ability of such stores to modulate responses to high Ke+ and to physiological stimulus, as for example secretion of CA elicited by hypoxia, is negligible.
Our data with TG do not support the interpretation given by Lahiri et al. (26). These authors found that, in nominally Ca2+-free solutions, TG increased carotid sinus nerve discharges to flow interruption in a readily reversible manner and concluded that this effect was due to the action of TG on Cai2+ stores of chemoreceptor cells. Because TG inhibition of Cai2+-ATPase is practically irreversible (17, 42), it implies that the effects observed were not produced on Cai2+ stores. Similarly, our data do not support the claim made by Biscoe and Duchen (7) and Duchen and Biscoe (15) that a significant part of the Cai2+ rise produced by hypoxia in the rabbit CB chemoreceptor cells is due to Ca2+ release from a nonidentified intracellular store(s). As shown in Figs. 5 and 6, the lack of effect of the functional elimination of the readily releasable Cai2+ stores on the release of CA elicited by hypoxia contradicts the notion that Cai2+ stores are important to maintain the hypoxic release of neurotransmitters. In fact, our present data are consistent with the studies of Gonzalez et al. (23) and Ureña et al. (39) showing that the increase in Cai2+ produced by hypoxia in isolated chemoreceptor cells from the rabbit CB was totally dependent on the presence of Ca2+ in the bathing solution. A recent observation made in isolated rat CB chemoreceptor cells (29) would appear to suggest an even more prominent role for unidentified Cai2+ stores in the chemoreception process in this species. These authors found that perfusing rat CB chemoreceptor cells with a solution equilibrated with PO2 120 mmHg and PCO2 550 mmHg increased Cai2+ levels to nearly 700 nM whether the solution contained 200 µM CdCl2 or not; they concluded that the Ca2+ rise originated from intracellular stores. Although a putative anatomic definition of the cellular structure representing the source of the Ca2+ was not made, it would appear that it is not involved in physiological O2 chemoreception, as Buckler and Vaughan-Jones (9) showed that the hypoxia-induced rise in Cai2+ in chemoreceptor cells of the same species was almost completely dependent on the entry of Ca2+ from the extracellular space. Certainly, the undefined intracellular store does not appear to be mitochondria, the structures where CO would act to activate CB chemoreceptors, because uncouplers that collapse the mitochondrial electrochemical gradient produce an increase in Cai2+ largely dependent on Ca2+ entering from the extracellular space (10).
In conclusion, none of the agents known to promote the emptying of Cai2+ stores and to disrupt their Ca2+-storing capacity are able to produce changes in Cai2+ levels above the secretory threshold, to alter the Cai2+ transients produced by cell depolarization, or to modify the neurosecretory activity of rabbit CB chemoreceptor cells. Therefore, the rabbit CB chemoreceptor cells should rely exclusively on plasma membrane mechanisms (i.e., voltage-dependent Ca2+ channels, cell membrane Ca2+-ATPase, and Na+/Ca2+ exchanger; see Ref. 22) to adjust their levels of cytoplasmic free Ca2+ in any functional situation. This, in turn, implies that Cai2+ stores play only a small role or no role at all in setting the final output of the rabbit CB arterial chemoreceptors. Although the literature seems to favor the same schema for Ca2+ metabolism in chemoreceptor cells of other species, we want to pose some caution in extrapolating to other species the findings reported here in the rabbit CB; it is conceivable that, in the same manner that ionic currents in chemoreceptor cells of different species are different (22, 27), the Cai2+ stores can exhibit the same variability.
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ACKNOWLEDGEMENTS |
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We thank María de los Llanos Bravo for technical support.
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FOOTNOTES |
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This work was supported by Spanish Dirección General de Investigación Científica y Técnica Grant PB97 0400.
Address for reprint requests and other correspondence:
C. González, Departamento de Bioquímica y
Biología Molecular y Fisiología, Facultad de Medicina,
Universidad de Valladolid, 47005 Valladolid, Spain
(E-mail:constanc{at}ibgm.uva.es
).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 8 June 1999; accepted in final form 20 January 2000.
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