Intracellular Ca2+ stores in chemoreceptor cells of the rabbit carotid body: significance for chemoreception

I. Vicario, A. Obeso, A. Rocher, J. R. López-Lopez, and C. González

Departamento de Bioquímica y Biología Molecular y Fisiología, Instituto de Biología y Genética Molecular and Facultad de Medicina, Universidad de Valladolid, 47005 Valladolid, Spain


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 approx 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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta -hydroxylase and tyrosine hydroxylase. In these incubating conditions, each CB (average weight, 400 µg) synthesized ~12 pmol of [3H]CA, equivalent to approx 4 × 105 dpm, being ~90% [3H]DA and 10% [3H]norepinephrine (18).

At the end of the loading period, the CBs were transferred to individual vials, one CB per vial, containing 4 ml of [3H]tyrosine-free Tyrode bicarbonate solution (in mM: 116 NaCl, 24 NaHCO3, 5 KCl, 2 CaCl2, 1.1 MgCl2, 10 HEPES, and 5.5 glucose) continuously bubbled with water vapor-saturated 95% O2-5% CO2 to yield a pH of 7.40. Nominally Ca2+-free solutions were prepared by omitting CaCl2. During a period of 2 h, the solutions were renewed every 30 min and discarded; in this period, the precursor and the readily releasable pool of [3H]CA were washed out (1). Thereafter, the experimental maneuvers (including hypoxic or high-K+ stimulation of the CBs and/or incubation with agents interfering with Cai2+ storage) and the collection of the incubating solutions for ulterior analysis in their [3H]CA content started. Specific experimental protocols, including the sequence of sample collection, will be described in RESULTS. The analysis of [3H]CA included adsorption into alumina of the labeled catechols at pH 8.6, their elution with 1 N HCl, and quantification by liquid scintillation spectrometry (1, 31). The analytical profile of the release material in basal conditions and under low PO2 or high Ke+ stimulation indicated that most of the [3H]CA released is [3H]DA; thus, it comes from chemoreceptor cells (31), the only DA-containing structure in the CB. It should be noted that the release of [3H]CA/CB in resting (or basal) conditions during 2 min (interval for sample collection in several experiments) amounts to 200-400 dpm equivalent to approx 9 fmol, i.e., the sensitivity of the method to detect a secretory response elicited by any experimental maneuver is extremely high.

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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 approx 250 nM. This calculated threshold concentration of Cai2+ is comparable to that found in bovine adrenal chromaffin cells (3).


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Fig. 1.   Release of [3H]catecholamines (CA) from intact rabbit carotid body (CB) and intracellular Ca2+ (Cai2+) transients in isolated chemoreceptor cells produced by high extracellular K+ (Ke+). A: magnitude and time course of the release of [3H]CA in intact rabbit CBs incubated in solutions containing different concentrations of K+. K+-rich solutions were applied for a total time of 9 min (filled bars) but were renewed every 3 min and collected for their analysis in [3H]CA. During the periods corresponding to the open bars, the CBs were incubated in normal solutions containing 5 mM K+. The release elicited by high Ke+ corresponds to the dpm above the horizontal dashed line. Data were obtained in one experiment with 4 CBs. B: sample records of the Cai2+ responses obtained in a chemoreceptor cell while perfusing with K+-rich solutions. C: release of [3H]CA and peak Cai2+ levels in response to several concentrations of Ke+. Data are means ± SE obtained from 8 individual CBs for the release of [3H]CA and from 6 different cells for the peak Cai2+ levels. [K+]e, extracellular K+ concentration; [Ca2+]i, intracellular Ca2+ concentration. D: relationship between the increase in Cai2+ and the release of [3H]CA produced by high Ke+.

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 (Delta ) 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 Delta [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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Fig. 2.   Effects of caffeine (CAF) and ryanodine (RY) on the release of [3H]CA in the intact rabbit CB and on Cai2+ levels in isolated rabbit CB chemoreceptor cells. A: CBs were incubated in nominally Ca2+-free solutions during the entire experiment, and 40 mM caffeine was present in the incubating solutions during 8 min as indicated. Data are means ± SE of 4 individual values. B: effect of 10 mM caffeine on Cai2+ levels in isolated rabbit CB chemoreceptor cells superfused with Ca2+-containing (2 mM) solutions. Sample record and mean ± SE response obtained in 6 different cells (* P < 0.05). C: effect of ryanodine (0.5 µM) on the basal release of [3H]CA from CBs incubated in Ca2+-containing solutions; at the end of the experiments, the CBs were incubated in a solution containing 60 mM K+ to assess the viability of the preparation. Data are means ± SE of 6 individual values. D: effects of ryanodine on resting and high-Ke+-induced Cai2+ levels (sample record) and the mean Cai2+ levels recorded in 6 cells bathed in normal solution in the absence or the presence of 0.5 µM ryanodine.

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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Fig. 3.   Effects of potential activators of the phospholipase C system on the release of [3H]CA in the intact rabbit CB and Cai2+ levels in isolated chemoreceptor cells. A: effects of ATP (100 µM), bradykinin (BK, 1 µM), and bethanechol (BET, 10 µM) on the basal release of [3H]CA. B: sample record illustrating the effects of bethanechol (50 µM) and ATP (100 µM) on Cai2+ levels and mean responses obtained in 6 different cells (* P < 0.05).

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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Fig. 4.   Effects of thapsigargin (TG) on the basal release of [3H]CA in the intact rabbit CB and Cai2+ levels in isolated rabbit CB chemoreceptor cells. A: effects of 1 µM thapsigargin plus 0.5 µM ryanodine on the basal release of [3H]CA in nominally Ca2+-free solutions. Both agents were applied for 20 min. B: effect of several concentrations of thapsigargin on the basal release of [3H]CA in Ca2+-containing solutions. Basal solutions are those equilibrated with 20% O2-5% CO2-balance N2 at a pH of 7.40. Data are means ± SE; n = 6 in A and for each concentration of thapsigargin in B. C: sample record of the effects of 1 µM thapsigargin on the intracellular levels of Ca2+ and the mean ± SE response obtained in a total of 6 cells (*P < 0.05).

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 approx 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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Fig. 5.   Effect of ryanodine on the release of [3H]CA induced by hypoxia. A and B: typical experiment with a pair of CBs drawn to show the experimental protocol used to test the effect of ryanodine (0.5 µM) on the release of [3H]CA elicited by hypoxic stimulation (10 min incubation in a solution equilibrated with 2% O2-5% CO2-balance N2, filled bars). The horizontal dotted lines separate the basal release (bottom) from the stimulus-evoked release (top). C: low PO2-evoked release (dpm) in the second presentation of the stimulus (S2) was divided by the release in the first presentation (S1), and the mean of the quotients for 6 control and 6 experimental (ryanodine-treated) CBs are represented in bars labeled as 2% O2. Figure also shows mean results obtained in 6 additional pairs of CBs in which the stimulus was 35 mM Ke+. In neither case was the difference in the S2-to-S1 quotients between control and ryanodine-treated CBs statistically significant.

In another series of experiments, we studied the effects of three inhibitors of the endoplasmic reticulum Ca2+-ATPase on the release of [3H]CA elicited by hypoxia and high Ke+. The experimental protocol and the analysis and presentation of the data were identical to those described for ryanodine above. Figure 6A shows that incubation of the CBs with 1 µM TG the 20-min period before and during the 10 min of the stimulus application did not alter significantly the release response evoked by a mild hypoxic stimulus (incubating solution equilibrated with 7% O2; PO2 approx 46 mmHg), an intense hypoxic stimulus (PO2 approx 22 mmHg), and a moderate or an intense depolarizing stimulus (incubating solution containing 35 or 60 mM K+, respectively). These experiments also confirmed that, during the 20-min incubation with TG before the stimulus application, the drug did not alter the basal release of [3H]CA. Similar experiments were also performed with two reversible inhibitors of the endoplasmic reticulum Ca2+-ATPase (CPA and TBH, both at 10 µM). Figure 6B shows that CPA did not modify the release response evoked by hypoxia or high Ke+. On the contrary, TBH, which did not alter the basal release of [3H]CA, markedly inhibited the release evoked by hypoxia and high Ke+, reducing the S2-to-S1 ratios for both stimuli by nearly 50% (P < 0.01).


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Fig. 6.   Effects of inhibitors of the endoplasmic reticulum Ca2+-dependent ATPase on the release of [3H]CA elicited by hypoxia and high Ke+ in the intact CB. In all cases, the experimental protocol was identical to that followed in the experiments described in Fig. 5. A: S2-to-S1 ratios obtained in groups of CBs stimulated with a mild (7% O2; PO2 approx 46 mmHg) and an intense (2% O2; PO2 approx 22 mmHg) hypoxic stimulus or with a moderately high (35 mM) or a high (60 mM) Ke+ are not different in control CBs (open bars) compared with organs treated with 1 µM thapsigargin (filled bars). B: cyclopiazonic acid (CPA) at a concentration of 10 µM was equally ineffective at altering the S2-to-S1 ratios obtained in groups of CBs stimulated with an intense hypoxic stimulus or a high Ke+. C: On the contrary, 2,5-di-(tert-butyl)-1,4-benzohydroquinone (TBH; 10 µM) reduced by nearly 50% the S2/S1 obtained in groups of CBs stimulated with an intense hypoxic stimulus or a high Ke+. In all the cases, data are means ± SE of 6 or more individual data. ** P < 0.01.

A different way to explore the capacity of Cai2+ stores is to compare the properties of the Ca2+ transients produced by stimuli activating Ca2+ entry from the extracellular space (e.g., high Ke+ or hypoxia) before and after functionally disrupting those stores. The results of such experiment are depicted in Fig. 7, which shows sample records and mean peaks and time constants of Cai2+ transients produced by high Ke+ in chemoreceptor cells superfused in control conditions and in the presence of agents (TG, CPA, and TBH) that disrupt the ability of internal stores to accumulate Ca2+. TG and CPA, used at the same concentrations as in the release experiments described above, did not alter the magnitude of the peak nor the time constant of recovery of the Ca2+ transients elicited by high Ke+. TBH reduced the peak of the Ca2+ transient elicited by high Ke+ by >60% (Fig. 7, B and C; P < 0.001); these findings are consistent with those shown in Fig. 6C indicating that TBH inhibits Ca2+ entry from the extracellular space by blocking Ca2+ channels (30).


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Fig. 7.   Effects of inhibitors of the endoplasmic reticulum Ca2+-dependent ATPase on the Cai2+ responses elicited by 35 mM Ke+ in isolated chemoreceptor cells. A and B: sample records of the Cai2+ transients produced during bath perfusion with solutions containing 35 mM K+ in the absence or the presence of TG (1 µM; A) or TBH (10 µM; B). Dashed line in A represents the fitting of the Cai2+ decay to an exponential, allowing calculation of the time constant (tau) of the process. C: mean ± SE (n = 6 for TG and CPA and 4 for TBH) of the peak of the Cai2+ rise produced by 35 mM Ke+ in control conditions and after treatment with TG (1 µM), CPA (10 µM), and TBH (10 µM); *** P < 0.001. D: comparison of mean ± SE values found for time constants (tau) in the same groups of cells.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 approx 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 alpha 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.


    ACKNOWLEDGEMENTS

We thank María de los Llanos Bravo for technical support.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Almaraz, L, Gonzalez C, and Obeso A. Effects of high potassium on the release of [3H]dopamine from the cat carotid body in vitro. J Physiol (Lond) 379: 293-307, 1986[Abstract].

2.   Almaraz, L, Perez-Garcia MT, Gomez-Nino A, and Gonzales C. Mechanisms of alpha 2-adrenoceptor-mediated inhibition in rabbit carotid body. Am J Physiol Cell Physiol 272: C628-C637, 1997[Abstract/Free Full Text].

3.   Augustine, GJ, and Neher E. Calcium requirements for secretion in bovine chromaffin cells. J Physiol (Lond) 450: 247-271, 1992[Abstract].

4.   Baker, PF, and Rink TJ. Catecholamine release from bovine adrenal medulla in response to maintained depolarization. J Physiol (Lond) 253: 593-620, 1975[Abstract].

5.   Belousunov, AB, Godfraind JM, and Krnjevic K. Internal Ca2+ stores involved in anoxic responses of rat hippocampal neurons. J Physiol (Lond) 486: 547-556, 1995[Abstract].

6.   Bers, DM. Control of cardiac contraction by SR Ca2+ release and sarcolemmal Ca2+ fluxes. In: Excitation-Contraction Coupling and Cardiac Contractile Force, edited by Bers DM.. Dordrecht, The Netherlands: Kluwer, 1991, p. 149-170.

7.   Biscoe, TJ, and Duchen MR. Responses of type I cells dissociated from the rabbit carotid body to hypoxia. J Physiol (Lond) 428: 39-59, 1990[Abstract].

8.   Bourreau, JP. Internal calcium stores and norepinephrine overflow from isolated field stimulated rat vas deferens. Life Sci 58: 123-129, 1996.

8a.   Buckler, KJ, and Vaughan-Jones RD. Effects of hypercapnia on membrane potential and intracellular calcium in rat neonatal carotid body type I cells. J Physiol (Lond) 478: 157-171, 1994[Abstract].

9.   Buckler, KJ, and Vaughan-Jones RD. Effects of hypoxia on membrane potential and intracellular calcium in rat neonatal carotid body type I cells. J Physiol (Lond) 476: 423-428, 1994[Abstract].

10.   Buckler, KJ, and Vaughan-Jones RD. Effects of mitochondrial uncouplers on intracellular calcium, pH and membrane potential in rat carotid body type I cells. J Physiol (Lond) 513: 819-833, 1998[Abstract/Free Full Text].

11.   Castro, E, Mateo J, Tome AR, Barbosa RM, Miras-Portugal MT, and Rosario LM. Cell-specific purinergic receptors coupled to Ca2+ entry and Ca2+ release from internal stores in adrenal chromaffin cells: differential sensitivity to UTP and suramin. J Biol Chem 270: 5098-5106, 1995[Abstract/Free Full Text].

12.   Clapham, DE. Calcium signaling. Cell 80: 259-268, 1995[ISI][Medline].

13.   Dasso, LL, Buckler KJ, and Vaughan-Jones RD. Muscarinic and nicotinic receptors raise intracellular Ca2+ levels in rat carotid body type I cells. J Physiol (Lond) 498: 327-338, 1997[Abstract]

14.   Delles, C, Haller T, and Dietl P. A highly calcium-selective cation current activated by intracellular calcium release in MDCK cells. J Physiol (Lond) 486: 557-569, 1995[Abstract].

15.   Duchen, MR, and Biscoe TJ. Relative mitochondrial membrane potential and [Ca2+]i in type I cells isolated from the rabbit carotid body. J Physiol (Lond) 450: 33-61, 1992[Abstract].

16.   Ehrlich, BE, Kaftan E, Bezprozvannaya S, and Bezprozvanny I. The pharmacology of intracellular Ca2+-release channels. Trends Pharmacol Sci 151: 145-149, 1994.

17.   Ely, JA, Ambroz C, Baukal AJ, Christensen SB, Balla T, and Catt KJ. Relationship between agonist- and thapsigargin-sensitive calcium pools in adrenal glomerulosa gells. J Biol Chem 266: 18635-18641, 1991[Abstract/Free Full Text].

18.   Fidone, S, and Gonzalez C. Catecholamine synthesis in rabbit carotid body in vitro. J Physiol (Lond) 333: 69-79, 1982[ISI][Medline].

19.   Fidone, S, Gonzalez C, and Yoshizaki K. Effects of low oxygen on the release of dopamine from the rabbit carotid body in vitro. J Physiol (Lond) 333: 93-110, 1982[ISI][Medline].

20.   García, AG, Kirpekar M, and Sanchez-Garcia P. Release of noradrenaline from the cat spleen by nerve stimulation and potassium. J Physiol (Lond) 261: 301-317, 1976[Abstract].

21.   Gonzalez, C, Almaraz L, Obeso A, and Rigual R. Oxygen and acid chemoreception in the carotid body chemoreceptors. Trends Neurosci 15: 146-153, 1992[ISI][Medline].

22.   Gonzalez, C, Almaraz L, Obeso A, and Rigual R. Carotid body chemoreceptors: from natural stimuli to sensory discharges. Physiol Rev 74: 829-898, 1994[Free Full Text].

23.   Gonzalez, C, Lopez-Lopez JR, Obeso A, Rocher A, and Garcia-Sancho J. Ca2+ dynamics in chemoreceptor cells: an overview. Adv Exp Med Biol 337: 149-156, 1993[Medline].

24.   Grynkiewicz, G, Poenie M, and Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440-3450, 1985[Abstract].

25.   Janiak, R, Lewartowski B, and Langer GA. Functional coupling between sarcoplasmic reticulum and Na/Ca exchange in single myocytes of guinea-pig and rat heart. J Mol Cell Cardiol 28: 253-264, 1996[ISI][Medline].

26.   Lahiri, S, Osanai S, Buerk DG, Mokashi A, and Chugh DK. Thapsigargin enhances carotid body chemosensory discharge in response to hypoxia in zero [Ca2+]e: evidence for intracellular Ca2+ release. Brain Res 709: 141-144, 1996[ISI][Medline].

27.   Lopez-Lopez, JR, and Peers C. Electrical properties of chemoreceptor cells In: The Carotid Body Chemoreceptors, edited by Gonzalez C. Heidelberg, Germany: Springer-Verlag, 1997, p. 65-77.

28.   Miller, RJ. The control of neuronal Ca2+ homeostasis. Prog Neurobiol 37: 255-285, 1991[ISI][Medline].

29.   Mokashi, A, Roy A, Rozanov C, Osanai S, Storey BT, and Lahiri S. High PCO does not alter pHi, but raises [Ca2+]i in cultured rat carotid body glomus cells in the absence and presence of CdCl2. Brain Res 803: 194-197, 1998[ISI][Medline].

30.   Nelson, EJ, Li CC, Bangalore R, Benson T, Kass RS, and Hinkle PM. Inhibition of L-type calcium-channel activity by thapsigargin and 2,5-t-butylhydroquinone, but not by cyclopiazonic acid. Biochem J 302: 147-154, 1994[ISI][Medline].

31.   Obeso, A, Rocher A, Fidone S, and Gonzalez C. The role of dihydropyridine-sensitive Ca2+ channels in stimulus-evoked catecholamine release from chemoreceptor cells of the carotid body. Neuroscience 47: 463-472, 1992[ISI][Medline].

32.   Park, JH, and Keeley LL. Calcium-dependent action of hypertrehalosemic hormone on activation of glycogen phosphorylase in cockroach fat body. Mol Cell Endocrinol 116: 199-205, 1996[ISI][Medline].

33.   Pérez-García, MT, Obeso A, López-López JR, Herreros B, and González C. Characterization of chemoreceptor cells in primary culture isolated from adult rabbit carotid body. Am J Physiol Cell Physiol 263: C1152-C1159, 1992[Abstract/Free Full Text].

34.   Pozzan, T, Rizzuto R, Volpe P, and Meldolesi J. Molecular and cellular physiology of intracellular calcium stores. Physiol Rev 74: 595-636, 1994[Free Full Text].

35.   Rigual, R, Cachero MTG, Rocher A, and González C. Hypoxia inhibits the synthesis of phosphoinositides in the rabbit carotid body. Pflügers Arch Eur J Physiol 439: 463-470, 2000[ISI][Medline].

36.   Simpson, PB, Challiss RAJ, and Nahorski SR. Neuronal calcium stores: activation and function. Trends Neurosci 18: 299-306, 1995[ISI][Medline]

37.   Teraoka, H, Nakazato Y, and Ohga A. Ryanodine inhibits caffeine-evokek Ca2+ mobilization and catecholamine secretion from cultured bovine chromaffin cells. J Neurochem 57: 1884-1890, 1991[ISI][Medline].

38.   Toescu, EC. Temporal and spatial heterogeneities of Ca2+ signaling: mechanisms and physiological roles. Am J Physiol Gastrointest Liver Physiol 249: G173-G185, 1985.

39.   Ureña, J, Fernandez-Chacon R, Benot AR, Alvarez de Toledo GA, and Lopez-Barneo J. Hypoxia induces voltage-dependent Ca2+ entry and quantal dopamine secretion in carotid body glomus cells. Proc Natl Acad Sci USA 91: 10208-10211, 1994[Abstract/Free Full Text].

40.   Usachev, Y, Shmigol A, Pronchuk N, Kostyuk P, and Verkhratsky A. Caffeine-induced calcium release from internal stores in cultured rat sensory neurons. Neuroscience 57: 845-859, 1993[ISI][Medline].

41.   Vargas, O, and Orrego F. Elevated extracellular potassium as a stimulus for releasing [3H]norepinephrine and [14C]alpha-amino isobutyrate from neocortical slice. Specificity and calcium dependency of the process. J Neurochem 26: 31-34, 1976[ISI][Medline].

42.   Villalobos, C, and Garcia-Sancho J. Capacitative Ca2+ entry contributes to the Ca2+ influx induced by thyrotropin-releasing hormone (TRH) in GH3 pituitary cells. Pflügers Arch 430: 923-935, 1995[ISI][Medline].

43.   Yamada, Y, Nakazato Y, and Ohga A. The mode of action of caffeine on catecholamine release from perfused adrenal glands of cat. Br J Pharmacol 98: 351-356, 1989[Abstract].


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