Heterogeneity in cytosolic calcium responses to hypoxia in carotid body cells

BRAIN RESEARCH ELSEVIER

Brain Research 706 (1996) 297-302

Short communication

Heterogeneity in cytosolic calcium responses to hypoxia in carotid body cells Gary R. Bright 1, Faton H. Agani, Uzma Haque, Jeffrey L. Overholt, Nanduri R. Prabhakar * Department of Physiology and Biophysics, Case Western Reserve University, 10900 Euclid A re., Cleveland, OH 44106, USA Accepted 22 August 1995

Abstract

Previous investigators have reported that intracellular pH responds to hypoxia with a heterogenous pattern in individual glomus cells of the carotid body. The aim of the present study was to examine whether hypoxia had similar effects on cytosolic calcium ([Ca2+ ]i) in glomus cells, and if so, whether a heterogenous response pattern is also seen in other cell types. Experiments were performed on glomus cells from adult rat carotid bodies, rat pheochromocytoma (PC12) and vascular smooth muscle (A7r5) cells. Changes in [Ca2÷ ]i in individual cells were determined by fluorescence imaging using Fura-2. Glomus cells were identified by catecholamine fluorescence. [Ca2÷ ]i in glomus cells increased in response to hypoxia (pO 2 = 35 + 8 mmHg; 5 min), whereas hypoxia induced decreases in [Ca2÷ ]i were not seen. Increases in [Ca2÷]i were observed in 20% of the isolated cells and strings of cells, but clustered glomus cells never responded. The magnitude of the calcium change in responding cells was proportional to the hypoxic stimulus. Under a given hypoxic challenge, there were marked variations in the response pattern between glomus cells. The response pattern characteristic of any given cell was reproducible. At comparable levels of hypoxia, PC12 cells also responded with an increase in [Ca2+] i with a heterogenous response pattern similar to that seen in glomus cells. In contrast, increases in [Ca2÷ ]i in A7r5 cells could be seen only with sustained hypoxia ( ~ 20 min), and little heterogeneity in the response patterns was evident. These results demonstrate that: (a) hypoxia increases cytosolic calcium in glomus cells; (b) response patterns were heterogeneous in individual cells; and (c) the pattern of the hypoxia-induced changes in [Ca2+] i is cell specific. These results suggest that hypoxia-induced increases in [Ca2+] i are faster in secretory than in non-secretory cells. Keywords: Cytosolic calcium ([Ca 2+ ]i); Glomus cell; Pheochromocytoma 12 cell; Vascular smooth muscle cell (A7r5); Carotid body; Hypoxia

Ventilatory and circulatory adjustments during hypoxia are mediated by reflexes from the peripheral arterial chemoreceptors, especially the carotid bodies. The mechanism(s) by which hypoxia augments carotid body sensory discharge are not clear. Several lines of evidence indicate that type I (glomus) cells are the initial sites of the transduction process. Currently it is believed that low pO e causes release of a transmitter(s) from glomus cells, which by way of acting on the afferent nerve endings, increases the sensory discharge (see [17,30] for refs). It is well established that cytosolic calcium ([Ca2+]i) plays important roles in the release of neurotransmitters [3,24], and several lines of evidence suggest that calcium may regulate the release of transmitter(s) from glomus cells. For example, augmentation of sensory discharge by hypoxia could

* Corresponding author. Fax: (1) (216) 368-3952. 1 Present address: Dept. of Anatomy, Case Western Reserve University, Cleveland, OH 44106, USA. 0006-8993/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 5 ) 0 1 1 2 2 - 6

be attenuated by low extracellular calcium [13] and voltage dependent calcium channel blockers [31]. Likewise, hypoxia-induced dopamine release from carotid bodies and from isolated glomus cells can be attenuated by low extracellular calcium [15] or calcium channel blockers [25], respectively. Furthermore, in the presence of low pO 2, calcium uptake by type I cells is increased, suggesting that hypoxia increases [Ca 2÷ ]i [29]. However, there have been contradictory reports as to the effect of hypoxia o n [Ca2+]i in glomus cells. It has been reported that hypoxia i n c r e a s e s [Ca2+]i in glomus cells of the adult rabbit carotid body [4] and neonatal rat carotid bodies [9]. In contrast, decreases in [Ca e÷ ]i were reported in response to hypoxia in glomus cells of adult rat carotid bodies [11]. Therefore, whether hypoxia increases or d e c r e a s e s [Ca2+] i in glomus cells is uncertain. One possible explanation for this discrepancy is that the response of [Ca e÷ ]i to hypoxia in individual glomus cells is heterogenous. Eyzaguirre and his co-workers reported marked variations in the response of [pH] i to various

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stimuli in individual glomus cells, where a given hypoxic stimulus caused individual glomus cells to respond with either a decrease or an increase in [pH] i [21,27]. The variations in the response pattern in individual cells were also evident in membrane properties, where cells either depolarized or hyperpolarized under a given hypoxic challenge [21,26]. Therefore, it is not unreasonable to suggest that [ C a 2+ ]i may also respond to hypoxia with a heterogeneous response pattern, w h e r e [Ca2+] i would increase in some cells and decrease in others. Consequently, the aim of the present study was to determine if [Ca 2+ ]i of individual glomus cells responds to hypoxia with a heterogenous pattern, and if so, are the variations in the responses confined only to glomus cells or do they extend to other cell types. To accomplish this, changes in [Ca2+] i were monitored simultaneously in individual cells with fluorescence spectroscopic imaging. The effects of hypoxia on cytosolic calcium were then compared in glomus, pheochromocytoma (PC12) and vascular smooth cells (A7r5). A part of this work has been briefly reported [1,6]. Carotid bodies were removed from adult rats of either sex (Sprague-Dawley; 150-200 g) anaesthetized with ether, and glomus cells were dissociated by enzymatic digestion. Briefly, tissues were incubated for 40 rain at 37°C in 5 ml of calcium/magnesium free medium contain-

ing 0.15% trypsin and 0.2% collagenase, and having the following composition (in mM): NaC1 140, KC1 0.5, NazHPO 4 2, NaH2PO 4 0.5, glucose 1. During the incubation, tissues were triturated every 10 min with a fire polished pipette. Following the incubation, cells were centrifuged at 500 × g for five min, the pellet was resuspended in 10 ml of 50% DMEM and 20% HAM-F12 growth medium supplemented with 10% fetal bovine serum, 100 units/ml of sodium penicillin G and 100 /.Lg/ml of streptomycin sulfate (all reagents from Gibco) and plated in a 96-well tissue culture plate. Unless stated, cells were left undisturbed overnight in a humidified tissue culture chamber circulated with 5% CO 2 and air. Pheochromocytoma cells (PCl2; a gift from Dr. L. Greene) were grown in RPMI-1640 medium supplemented with 5% fetal calf serum, 10% horse serum, 100 units/ml of sodium penicillin G and 100 ~ g / m l of streptomycin sulfate. Vascular smooth muscle cells (A7r5) were obtained from American Type Culture Collection (ATCC) and were grown in DMEM medium supplemented with fetal calf serum (10%) and antibiotics. Both PC12 and A7r5 cells were grown to near confluence in a humidified chamber circulated with 5% CO 2 and air. For measurements of cytosolic calcium, cells were plated on glass coverslips that were pre-treated with a cell adhe-

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Fig. 1. Identification of glomus cells by catecholamine fluorescence in dissociated cell cultures of the carotid body. Cells were plated on a coverslip coated with a cell adhesive and superfused with freshly prepared phosphate buffered solution (pH 7.4) containing paraformaldehyde (4%) and glutaraldehyde (0.5%) for 10 rain. (A) Phase contrast photomicrograph; (B) fluorescence monitored 30 rain after superfusion with paraformaldebyde-glutaraldehyde; and (C) analogue display of tile data. Data shown are from six cells. Note the development of fluorescence in almost all the cells except for the one indicated with an arrow not displaying the fluorescence.

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sive (Cell-Tak, Collaborative Biomedical Products, MA) and were incubated in 3 ml of serum free D M E M medium containing 5 ~ M fura-2 for 30 min. Subsequently they were washed with serum free medium and allowed to recover for 15 min. The coverslip was placed in a gas-tight, temperature regulated cell chamber and superfused with Hank's Balanced Salt Solution (HBSS) with the following composition (in mM): NaC1 137, KC1 5.4, K H 2 P O 4 0.4, MgCI 2 1.1, MgSO 4 0.8, NaHCO 3 24, Na2HPO 4 0.6, o-glucose 5.5, CaCI 2 1. The medium was bubbled either with 21% 0 2 / 5 % CO 2 balance N 2 (normoxia) or 95% N 2 + 5% CO 2 (hypoxia) for at least 60 min and during the experiment maintained under positive pressure with the same gas. The pO2, pCO 2 and pH of the medium were monitored with a blood gas analyzer (ABL2, Radiometer). Bubbling with N 2 enriched gas resulted in a pO 2 of 35 +__8 mmHg. No attempts were made to further lower pO 2 by adding reducing agents such as sodium dithionate. In the experiments wherein the effects of different levels of oxygen were tested, medium was also bubbled with 5% CO 2 + 95% 0 2 (hyperoxia). The cells were superfused with HBSS at a rate of 2 m l / m i n and the temperature maintained at 37 _ I°C. The duration of hypoxic challenge for carotid body and P C I 2 cells was 5 min and for A7r5 cells was 1 5 - 2 0 min (see Results). Following the hypoxic challenge, the superfusion medium was switched back either to normoxia or to hyperoxia depending on the protocol. Fluorescence ratio imaging was used to quantitate [Ca2+] i as previously described [5,7]. Typically images were collected every 10 s. Routine calibrations of cytosolic calcium could not be performed at the end of the experiment because identification of glomus cells required superfusion with aldehyde solutions (see below). To establish resting calcium values, calibrations were done under identical experimental conditions using a separate set of dissociated cells using the protocols described previously [19].

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Carotid body tissue is composed of different cell types. The focus of the present study was [Ca2+]i changes in glomus cells. Since glomus cells contain catecholamines, we monitored the catecholamine fluorescence as described previously [16]. Briefly, at the end of the experiment, cells were superfused with freshly prepared phosphate buffered saline (pH 7.4) containing paraformaldehyde (4%) and glutaraldehyde (0.5%) for 10 min. In the ensuing 30 min fluorescence emission was monitored at 510 nm with excitation at 440 nm. Fig. 1 shows examples of cells showing catecholamine fluorescence and the analogue display of the data. Resting [Ca 2÷ ]i during normoxia (perfusate pO 2 = 146 + 4; pCO 2 = 34 + 5 mmHg) varied in individual cells and ranged between 40 and 150 nM, with a median at ~ 80 nM (n = 400 cells). Exposing cells to hypoxic medium increased [Ca 2+ ]i in glomus cells that were isolated or in strings, but not in clusters. Of the 182 cells tested (isolated and strings of cells), 36 of the cells (20%) responded with increases in [Ca2+]i. Furthermore, the magnitude of the increase in [Ca 2÷ ]i was greater with progressive lowering of perfusate pO 2. Peak increases in [Ca 2÷ ]i at a pO 2 of 35 __+8 m m H g (maximum hypoxic challenge used in this study) ranged between + 50 and + 300 nM. Decreases in [Ca2÷ ]i, on the other hand, were never observed. The most striking finding is that under a given hypoxic challenge, the response pattern of the [Ca2+] i increase varied markedly in individual glomus cells, which can be divided into the following categories: (a) increase in [Ca 2÷ ]i after the onset of hypoxia and return to baseline, often with an overshoot after terminating the stimulus; (b) after an initial increase, [Ca2+]i returned to pre-hypoxic controls despite maintaining the stimulus (adaptation) and, after terminating the stimulus, calcium levels decreased below the control; (c) an increase in [Ca 2÷ ]i was followed by a slight decrease to a level which was higher than the control followed by a return towards control levels during the five

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Fig. 2. Heterogeneity in response patterns of cytosolic calcium to hypoxia in glomus cells of the carotid body. Simultaneous monitoring of the changes in cytosolie calcium from five glomus cells is shown in A-E. Vertical lines represent the onset and termination of hypoxic challenge. Percent of cells responding with different pattern are presented in F.

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minutes of the post-stimulus period; (d) calcium levels remained elevated for the duration of the hypoxic stimulus and remained so through the five minutes of the poststimulus period; and (e) oscillations in [Ca2+]i during hypoxia which disappeared after terminating the stimulus. Examples depicting these patterns recorded simultaneously in individual glomus cells derived from the same carotid body within the same microscopic field are shown in Fig. 2A-E. Interestingly, when the hypoxic challenge was repeated within the same experiment, the response patterns in individual cells remained consistent. The number of cells responding with different patterns are summarized in Fig. 2F. The effects of hypoxia o n [Ca2+] i in PC12 (n = 100) and A7r5 (n = 88) cells were also examined under the same experimental conditions described above. Resting levels of [Ca 2÷ ]i in both of these cell types were comparable with glomus cells (i.e. 40-150 nM). Hypoxia increased [Ca2+]i in 60 of the 100 (60%) PC12 cells tested. Like glomus cells, PC-12 cells also responded with a heterogenous response pattern under a given hypoxic challenge, i.e. adaptation, sustained elevation, oscillations, etc. The number of PC12 cells responding with different patterns are summarized in Fig. 3B. However, compared to glomus cells, two differences were noted in the PC12 cell response to hypoxia. Firstly, the rate of increase and the recovery of [Ca2+ ]i from hypoxia were slower than in glomus cells. One such example is shown in Fig. 3A. Secondly, the magnitude of the response at comparable levels of hypoxia ranged between + 20 and + 150 nM, which was less than that seen in glomus cells. In response to hypoxia, [Ca 2+ ]i also increased in vascular smooth muscle (A7r5) cells, but the response was distinctly different from that seen in glomus and PC12 cells. Increases in [Ca 2÷ ]i in A7r5 cells could be elicited only with a hypoxic challenge lasting 15-20 min (as compared to 5 min for glomus and PC-12 cells). In addition, unlike the carotid body and PC12 cells, heterogeneity in the response patterns in A7r5 cells was minimal: all 88 cells responded to hypoxia with a slow and progressive increase in [Ca 2÷ ]i, which occurred between 5 and 10 min of hypoxic challenge, followed by a tendency for [Ca2+] i to return to control during the 5 min of the post-stimulus period. Fig. 3C shows examples of this response pattern during simultaneous monitoring of [Ca 2÷ ]i from five A7r5 cells. The major findings of the present study are that: (a) hypoxia causes an increase, but never a decrease in cytosolic calcium in glomus cells; (b) the pattern, but not the direction of the [Ca 2+ ]i response to hypoxia is heterogeneous in individual cells; and (c) similar effects of hypoxia on [Ca2+]i were seen in glomus and PC12, but not in vascular smooth muscle cells. The finding that hypoxia increases cytosolic calcium in glomus cells is consistent with studies cited previously [4,9]. However, our results differ from those reported in

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~ 40022°°- ~ 010 15 20 25 30 time (min) Fig. 3. Increases in cytosolic calcium in PCI2 and vascular smooth muscle cells (A7r5) to hypoxia. Example depicting the elevation of [Ca2÷ ]i in a PC-12 cell to hypoxia is shown in A. Vertical lines represent the onset and termination of the hypoxic challenge. Percent of PC12 cells responding to hypoxia with different patterns are presented B. The different patterns denoted A - E are similar to that presented in Fig. 1. Simultaneous monitoring of cytosolic calcium changes in 5 vascular smooth muscle cells (A7r5) to hypoxia is shown C. Note the slow and progressive increases in [Ca ?+ ]i to hypoxia and lack of heterogeneity in the response pattern amongst A7r5 cells.

adult rat carotid bodies [11] in that hypoxia-induced decreases in cytosolic calcium were not encountered. This discrepancy cannot be explained by the suggestion that low pO 2 causes a decrease in [Ca2+]i only in freshly dissociated cells [11], because hypoxia increased [Ca2+]i in carotid body cells tested within two hours after dissociation (data not shown). The cells from the chemoreceptor tissue that responded to hypoxia displayed catecholamine fluorescence, indicating that the effects of low pO 2 within the first few minutes were confined to glomus and not to other cell types in the carotid body. Not all glomus cells, however, responded to hypoxia with elevations in [Ca 2+ ]i' The lack of response in certain glomus cells could be attributed to cellular damage. If this were the case, cells would not have been expected to take up fura-2, nor would they display catecholamine fluorescence. Alternatively, the magnitude of hypoxia used in the present study may not have been severe enough to affect

G.R. Bright et al. / Brain Research 706 (1996) 297-302

[Ca2÷ ]i

in all of the glomus cells. Previous studies correlating the tissue pO 2 with sensory discharge of the cat carotid body have shown that the peak increase in sinus nerve activity was related to tissue pO 2 of 3-5 mmHg [10,23,32,33]. Significant catecholamine release occurred in intact carotid bodies or cultured type I cells only when pO2 was decreased below 20 mmHg [28]. From these studies it follows that pO2, anywhere between 3-20 mmHg, is necessary to affect most of the glomus cells. Under the experimental conditions described (see Methods) we could lower the pO 2 of the medium only to 35 + 8 mmHg, which is a relatively modest level of hypoxia compared to that used in the above cited studies. Despite this modest hypoxic challenge, it is clear that the cells responded with clear elevations in cytosolic calcium. In this respect adult rat carotid body cells respond to less severe hypoxic challenge than the neonatal cells which require pO 2 of ~ 20 mmHg [9]. Nonetheless, it is clear from our results that when the cells responded to hypoxia it was always with an increase but not with a decrease in cytosolic calcium. The present observations thus support the hypothesis that an increase in [Ca 2+ ]i in glomus cells is a necessary step in transduction of the hypoxic stimulus at the carotid body. The finding that calcium increases were seen in single glomus cells as opposed to cells in clusters is unexpected. Because in intact carotid bodies, glomus cells are present as clusters and not as isolated cells. In the intact organ, glomus cells are closely surrounded by other cell types such as sustentacular (also called type II) cells. Type II cells resemble morphologically the glial cells (see [17] for refs.). It has been reported that glial cells exert significant regulatory effects on excitable cells [2,8]. It is possible that the lack of response of glomus cells in clusters is due to their physiological state resulting from dissociation procedures wherein type II cells are also intermingled with type I cells and may have influenced the cytosolic calcium responses to hypoxia. Another important finding of the present study is the variations in response patterns among individual glomus ceils. It could be argued that dissociation procedures might have affected the properties of glomus cells resulting in variations in the response patterns. However, several observations suggest that such a possibility is unlikely. PC12 cells, which do not require dissociation procedures, also displayed heterogeneity in the response patterns. Moreover, such a heterogeneity in the response pattern was not evident in A7r5 cells which is a clonal cell line like PC12 cells, indicating that variations in the response patterns depend on the cell type. Furthermore, variability in individual glomus cells was reported previously with respect to [pH]i changes in response to hypoxia [21,27]. Variations in response patterns were noted in fibroblast cells in response to growth factors [7]. Gylfe et al. [20] also reported significant heterogeneity in cytosolic calcium changes in individual pancreatic cells in response to glu-

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cose. Thus, the present results, taken together with the above cited studies, indicate that the heterogeneity in the [Ca2÷ ]i responses is an inherent characteristic of the response rather than an experimental artifact. It has been proposed that elevation of cytosolic calcium in glomus cells could occur via at least two different mechanisms. One being the influx of extracellular calcium [4,9] mediated by voltage-dependent L-type calcium channels [14], while the other involves hypoxia-induced changes in mitochondrial membrane potential [12]. The kinetics of the [Ca 2÷ ]i response in PC12 cells were similar to glomus cells. This is not surprising since PC12 cells resemble carotid body glomus cells in that they are derived from neural crest and, like glomus cells, they synthesize, store and release catecholamines such as dopamine [18]. Moreover, recent preliminary evidence suggests that PC12 cells release dopamine in response to hypoxia [22]. Given the similarity in the patterns of [Ca2+] i responses, it is possible that the mechanism(s) underlying the increase in [Ca 2÷ ]i are the same in glomus and PC12 cells, whereas different mechanisms may underlie the increase in [Ca 2÷ ]i in A7r5 cells in response to hypoxia. What might be the physiological consequences of such a heterogeneity in the response patterns of [Ca2+]i to hypoxia? It is well established that calcium plays an important role in transmitter release (see [3,24] for refs.). Glomus cells contain several classes of transmitters including biogenic amines, neuropeptides, etc. [17,30] often colocalized within the same cell [34] and perhaps co-released during hypoxia. It may be that the heterogeneity in [Ca 2÷ ]i changes is necessary for release of different types of neurotransmitters during hypoxia. In addition to transmitter release, the different patterns may reflect other functions of [Ca2+] i as well. These may include activation of certain enzymes in the glomus cells, alterations in cell-to-cell communication by affecting gap junctions, etc., which require different kinetics than needed for transmitter release. In summary, the present results demonstrate that cytosolic calcium increases in glomus cells in response to hypoxia. Moreover, the pattern, but not the direction of the response, varies in individual glomus cells. This heterogeneity in the response was seen in other secretory cells of neural origin such as PC12 cells, but not in non-secreting vascular smooth muscle cells. These results suggest that, under physiological conditions, increases in [Ca2+]i and, consequently, transmitter release in response to hypoxia vary in individual glomus cells of the carotid body.

Acknowledgements This study was supported by grants from National Institutes of Health, Heart, Lung, and Blood Institute, HL-45780 and HL-52038 and a Research Career Development Award HL-02599 to N.R.P.

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[19] Grynklewicz, G., Poenie, M. and Tsien, R., A new generation of Ca2+ indicators with greatly improved fluorescence properties, J. Biol. Chem., 260 (1985) 3440-3450. [20] Gylfe, E., Grapengiesser, E. and Hellmann, B., Propagation of cytoplasmic Ca2+ oscillations in clusters of pancreatic /3-cells exposed to glucose, Cell Calcium, 12 (1991) 229-240. [21] He, S.-F., Wei, J.Y. and Eyzaguirre, C., Effect of relative hypoxia and hypercapnia on intracellular pH and membrane potential of cultured carotid body cells, Brain Res., 556 (1991) 333-338. [22] Kumar, G.K. and Prabhakar, N.R., Release of dopamine from PC12 cells by hypoxia, FASEB J., 9 (1995) A650. [23] Nair, P.K., Buerk, D.G. and Whalen, W.J., Cat carotid body oxygen metabolism and chemoreception described by a two cytochrome model, Am. J. Physiol. (Heart Circ. PhysioL), 250 (1986) H202H207. [24] Neher, E. and Zucker, S., Multiple calcium dependent processes related to secretion in bovine chromaffin cells, Neuron, 10 (1993) 21-30. [25] Obeso, A., Rocher, A., Fidone, S.J. and Gonzalez, C., The role of dihydropyridine-sensitive channels in stimulus evoked catecholamine release from chemoreceptor cells of the carotid body, Neuroscience, 47 (1992) 463-472. [26] Pang, L. and Eyzaguirre, C., Diffrent effects of hypoxia on the membrane potential and input resistance of isolated and clustered carotid body glomus cells, Brain Res., 575 (1992) 167-173. [27] Pang, L. and Eyzaguirre, C., Hypoxia affects differently the intracellular pH of clustered and isolated glomus cells of the rat carotid body, Brain Res., 623 (1993) 349-355. [28] Perez-Garcia, M.T., Obeso, A., Lopez-Lopez, J.R., Herreros, B. and Gonzalez, C., Characterization of chemoreceptor cells in primary culture isolated from adult rabbit carotid body. Am. J. PhysioL (Cell Physiol.), 263 (1992) Cl152-1159. [29] Pietruschka, F., Calcium influx in cultured carotid body cells is stimulated by acetylcholine and hypoxia, Brain Res., 347 (1985) 140-143. [30] Prabhakar, N.R. Neurotransmitters in the carotid body. In R. O'Regan, P. Nolan, D.S. McQuen and D.J. Peterson (Eds.), Arterial Chemoreceptors: Cell to System. Advances in Experimental Medicine and Biology, 360 (1994) 57-69. [31] Shirahata, M. and Fitzgerald, R.S., Dependency of hypoxic chemotransduction in cat carotid body on voltage-gated calcium channels. J. Appl. Physiol., 71 (1991) 1062-1069. [32] Wahlen, W.J. and Nair, P., Functional correlates of tissue pO 2 in the carotid body. In M. Reivich, R. Coburn, S. Lahiri and B. Chance (Eds.), Tissue Hypoxia and Ischemia, New York, Plenum Press, 1977, pp. 545-550. [33] Wahlen, W.J., Nair, P., Sidebotham, T., Spande, J. and Lacerna, M., Cat carotid body: oxygen consumption and other parameters, J. Appl, Physiol. 50 (1981) 129-133. [34] Wang, Z.-Z., Stensas, L.J., Dinger, B. and Fidone, S.J,, Coexistence of biogenic amines and neuropeptides in type I cells of the cat carotid body, Neuroscience, 47 (1992) 473-480.