Intracellular Ca2 + deposits and catecholamine secretion by chemoreceptor cells of the rabbit carotid body

42 INTRACELLULAR Ca2 +DEPOSITS AND CATECHOLAMINE SECRETION BY CHEMORECEPTOR CELLS OF THE RABBIT CAROTID BODY Ana Obeso, Asuncion Rocher, Jose Ramon Lopez-Lopez, and Constancio Gonzalez Departamento de Bioquimica y Biologia Molecular yFisiologia Facultad de Medicina, Universidad de Valladolid 47005 Valladolid, Spain 1. INTRODUCTION The pivotal role of intracellular free [Ca2 +] fluctuations in the control of cellular functions such as contraction and secretion, including the release of neurotransmitters, was recognized many decades ago (see Rubin, 1982). More recently, the list of cellular functions triggered or modulated by the levels of Ca2+ jhas grown enormously. Additional functions regulated by [Ca2+l include neuronal excitability, synaptic plasticity, gene expression, cellular metabolism, cell division and differentiation, and programmed cell dead (Miller, 1991; Clapham, 1995). Paralleling the growth in this list of Ca2+ -controlled functions, amultiplicity of cellular mechanisms aimed at maintaining resting free [Ca2+l in the range of 100 nM for most cells has been described, allowing increases in Ca2+ jlevels that are specific in their magnitude, time course and spatial distribution, according to the cell function activated (Toescu, 1995). Since Ca2+cannot be metabolized, cells regulate their cytoplasmic levels of free Ca2+ through numerous binding proteins and influx and efflux mechanisms (Fig 1). Ca2+influx to cell cytoplasm from the extracellular milieu occurs via voltage or receptor operated channels or via yet ill-defined capacitative pathways; the Na+/Ca2+exchanger can also produce in some circumstances net influx of Ca2+(Miller, 1991; Clapham, 1995). Ca2+efflux to the extracellular space occurs against electrochemical gradients, and thereby the pumping out of Ca2+is directly (Ca2+pump) or indirectly (Na+/Ca2 +) coupled to the hydrolysis of ATP. In addition to the extracellular space, there are intracellular stores, represented primarily by the smooth endoplasmic reticulum, capable of accumulating Ca 2+at very high concentrations (mM range) via specific Ca2+ ATPases (Fig 1). The smooth endoplasmic Frontiers in Arterial Chemoreception, edited by Zapata et al. Plenum Press, New York, 1996 279 280 A. Obeso et al. Na Figure 1. Model for the regulation of cellular calcium homeostasis. See text for an explanation. reticulum of many cells may possess two specific mechanisms capable of mobilizing the accumulated Ca2+in response to adequate signals, the inositol 1,4,5-triphosphate receptors (IP3R) that on IP] binding are ensambled to produce aCa2+channel, and the ryanodine receptor-channel complex (RyR) equally permeant to Ca2+(Pozzan et ai, 1994; Simpson et aI, 1995). Many extracellular signals, hormones and neurotransmitters, possess receptors in the plasma cell membrane that activate phospholipase Cleading to the genesis of IP] in the cell interior and to the release of Ca2+from the endoplasmic reticulum via the IP]R (Clapham, 1995); the RyR are activated by local increases in free Ca2+; produced by Ca2+entering via plasma cell membrane (Lopez-Lopez et ai, 1995), and represent the substrate for the long time known process of Ca2+ -induced Ca2 +release (Simpson et aI, 1995). Therefore, the endoplasmic reticulum may function as asink or as a source for free cytoplasmic Ca2+in different functional states of the cells, and additionally the sink or source function of the endoplasmic reticulum might vary from cell to cell, depending on the degree of expression of the repertoire of the molecules involved in the endoplasmic reticulum handling of Ca2+(Simpson et ai, 1995). At cytoplasmic levels of free Ca2+higher than 500 nM (Clapham, 1995), i.e., during cell activation, mitochondria may accumulate Ca2 +, thereby functioning as effective sinks to reduce cytoplasmic free Ca2+ concentration. Finally, many cytosolic constituents including proteins, and small organic molecules can bind Ca2+ions contributing to modulate local levels of cytoplasmic free Ca2+(Clapham, 1995; Toescu, 1995). The proteins with the capacity to bind Ca2+may in tum acquire catalytic activities when their binding sites are occupied by the regulating ion. In the carotid body (CB) chemoreceptor cells, our knowledge on the role of Ca2+in cell signalling is rather restricted (see Gonzalez et ai, 1994). It was only in 1968 that Eyzaguirre and Zapata showed the Ca2+dependency of carotid sinus nerve (CSN) discharges elicited by hypoxia, acidity or flow interruption. In 1975, again Eyzaguirre's group (Eyzaguirre et ai, 1975) reported thatacetylcholine was incapable of generating ac- Intracellular Ca l +Deposits and Catecholamine Secretion by Chemoreceptor Cells 281 tion potentials in the CSN in Caz+-free solutions. Some years later, afew ultrastructural studies showed the appearance of omega exocytotic profiles and/or coated pits and vesicles in chemoreceptor cells of rat CBs incubated in Caz+-containing (but not in Caz+-free) rich K\ solutions or in the presence of the Caz+ionophore A23187, concluding that exocytosis was Caz+e-dependent (Gronblad et ai, 1980). Consistent with that, it was later shown that the release of dopamine (DA) elicited by hypoxia and high K\ was Ca Z\-dependent in >95% (Fidone et ai, 1982; Almaraz et ai, 1986; Obeso et ai, 1992). More recently, three different laboratories (Gonzalez et ai, 1993; Lopez-Barneo et ai, 1993; Buckler & Vaughan-Jones, 1994) have used fluorescent dyes to measure [Caz+l in isolated chemoreceptor cells, and have consistently found that more than 95% of the [Caz+l rise produced by hypoxia is due to Caz+entering from the extracellular space. In the case of acidic stimulation the Ca Z \dependence of the release of DA was approximately 80% (Obeso et ai, 1992) and the Ca2+; signal was reduced by asimilar percentage in Caz+-free media (Buckler &Vaughan-Jones, 1993). At variance with those findings, Biscoe and Duchen (1990) reported that up to 40% of the hypoxic Caz + jrise was due to Ca2+ entering the cytoplasm from intracellular stores, and Biscoe et al (1989) could not detect arise in free Ca z + j during acidic stimulation. In addition, Lahiri et al (1995) have recently communicated that the anoxic CSN discharge was better preserved in Caz+-free media containing thapsigargin than in its absence, concluding that Caz +from intracellular deposits seems to contribute to the anoxic chemoreception. 2. METHODS Using an in vitro preparation of the rabbit CB whose catecholamine deposits have been labeled by prior incubation of the organs with their natural precursor 3H-tyrosine (Fidone &Gonzalez, 1982), we have studied the significance of intracellular Caz+stores as possible sources of Caz+for the secretory response, and as possible buffers (sinks) for the Ca z+entering the cell cytoplasm during stimulation. The tools used in the experiments include: ATP and bradykinin as possible generators of IP3,and thereby as possible activators of the IP3 R; ryanodine at submicromolar concentration and caffeine as activators of the RyR; thapsigargin as ablocker of the reticulum ATPase; ruthenium red as ablocker of the mitochondrial Ca2+ uniporter; and, ionomycin as a Ca z+ionophore that allows rapid equilibration of Ca z+throughout cellular membranes. The experiments have been performed in Caz+-containing and in Ca2+-free solutions. The effects of the drugs have been tested on their ability to trigger asecretory response in basal normoxic conditions, to test for arole of intracellular Caz+stores as sources of Caz+for the secretory response, and on their ability to modify the intensity and time course of the secretory response elicited by hypoxia and high K\. In this way, we have tested for the capacity of the intracellular stores to act as sources of Ca2+, or as sinks of the Caz+entering from the extracellular space. It should be noted that most of the experiments have been performed sampling the incubating solutions for their analyses in 3H-catecholamine eH-CA) content every 2min, thereby providing ahigh sensitivity of the overall procedures capable of detecting <6 fmol of labelled CA «200 cpm), Which after correction for the specific activity of 3 H-CA in the cells, represents approximately 120 fmol of total CA release, or an amount equivalent to the basal release/2 min in normoxic conditions. During stimulation (hypoxia, high K+ e )the release increases markedly, and consequently the sensitivity of the radioisotopic method to detect the effect of adrug on the stimulus induced release decreases. A. Obeso er al. Figure 2. Time course of the disappearance of high external K+ induced release of 3H-catecholamines upon removal of calcium from the extracellular space. Empty bars: release response in Ca2+-containing solutions at the beginning and the end of the experiments. Slashed bars: release response after incubation in Ca2+-free solutions for the indicated periods. Release response (ordinate) is expressed as times basal release in normal K+ solutions. 3. RESULTS AND DISCUSSION Figure 2shows the time course of extracellular Ca 2 +washing out. In designing these experiments, the advantage that the release induced by high external K+ is almost totally dependent on Ca2 +entering from the extracellular space was taken. Therefore, the minimum time of incubation in Ca 2 +free medium required to abolish high K\ induced release would be equivalent to the time required to wash out Ca2+ions from the incubating vial and the CB extracellular space. As shown in Figure 2, one min of incubation in 0Ca2 +reduced markedly the release response, after 4min in 0Ca2+the release was practically abolished, and on reintroduction of Ca 2+the release response recovered completely. In a similar experiment using hypoxic stimulation (2% 02-equilibrated solutions, 2min), the time course of inhibition was comparable; in fact, after 4min in 0Ca2 +the release induced by hypoxia was completely abolished. These experiments confirm previously reported findings with longer times of incubation in Ca 2+free solutions (e.g. Obeso et ai, 1992), and suggest that intracellular Ca2+stores do not playa significant role in mediating the secretory response to hypoxia, unless chemoreceptor cells have an unusually fast-emptying intracellular Ca 2 +deposits. On the other hand, the present findings are consistent with observations of Fidone (see Fidone et ai, 1977) and Torrance (1977) showing that the halftime for bicarbonate and sucrose wash out, respectively, was close to half aminute. Next we performed aseries of experiments aimed at mobilizing intracellular Ca 2 + stores. In afirst group of experiments we tested for the ability of ATP to mobilize Ca 2 + stores. In arecent study, Spergel &Lahiri (1993) showed that ATP and analogs, but not adenosine, were able to increase CSN discharges in adose-dependent manner, concluding that the CB expresses surface P2type ATP receptors. Since P2receptors are coupled to 13 type phospholipase C, it should be expected that their activation will lead to the formation of IP3and activation ofIP 3R. A TP at 100 and 1500 IlM did not affect the basal normoxic release of DA either in Ca 2+containing or in Ca2+free solutions suggesting that the ATP receptors responsible for the activation of CSN discharges are located in astructure different from chemoreceptor cells. Bradykinin, apotent mobilizer of Ca2 +from intracellular Intracellular Ca 2+Deposits and Catecholamine Secretion by Chemoreceptor Cells 283 deposits and potent secretagogue in chromaffin cells (Augustine &Neher, 1992), was also without effect on the basal release of 3H-CA from the CB, suggesting that chemoreceptor cells do not possess bradykinin receptors or that the endoplasmic reticulum Ca2+stores are really small. The same was true for the rest of the agents tested including ionomycin in Ca2+-free solutions. In conclusion, the participation of endoplasmic reticulum and mitochondria as sources of Ca2+capable of altering the secretory response of chemoreceptor cells appear to be of secondary importance, implying that chemoreceptor cells must rely almost exclusively on plasma membrane mechanisms to maintain their Ca2 +homeostasis. Although the precise identity of the Ca2 +stores sites is not fully clear,it appears that the smooth endoplasmic reticulum (SER) may be the principal place where the intracellular Ca2+ATPase is located and thereby the SER could be the main place where Ca2+is stored (Pozzan et aI, 1994). If this is the case in chemoreceptor cells our findings would correlate with the apparently rear SER profiles present in the cells that appear to be located mainly in the processes of chemoreceptor cells (Verna, 1975; McDonald, 1981). It should be mentioned that papers consistent with our conclusion have been presented in this Symposium. Thus, He et al (1996) communicated that endothelin Iincreases up to five times the normoxic basal levels ofIP 3 ,and therefore it should be expected that IP 3 R of the endoplasmic reticulum represents the target of the IP3formed. Although the cellular element in which the increase of IP3had occurred was not identified, it was shown that endothelin Idid not affect the basal release of CA or the Ca2 +jlevels in chemoreceptor cells, but still endothelin Iwas effective in potentiating the release ofDA and the Ca2+jrise produced by hypoxia. Those findings imply that chemoreceptor cells possess endothelin receptors, and therefore it could be expected that IP 3has increased in chemoreceptor cells. Therefore, the lack of effect on the basal release of CA and Ca2+jwould imply that the cellular deposits ofCa 2+sensitive to IP3are really small. The effects observed during stimulation would be produced via voltage-dependent Ca2+channels, as it is the case in other systems. These pieces of information urge to explore the dynamics of intracellular Ca2+stores in amore direct manner and to correlate the data with other functions of the chemoreceptor cells, besides the release of neurotransmitters. 4. ACKNOWLEDGMENTS Supported by Spanish DGICYT Grant PB92/0267. 5. REFERENCES Almaraz L, Gonzalez & abe so A(1986) Effects of high potassium on the release of eH]dopamine from the cat carotid body in vitro. JPhysiol, London 379: 293-307 Augustine GJ &Neher E(1992) Calcium requirements for secretion in bovine chromaffin cells. JPhysiol, London 450:247-271 Biscoe TJ &Duchen MR (1990) Responses of type Icells dissociated from the rabbit carotid body to hypoxia. J Physiol, Lond 428: 39-59 Biscoe TJ, Duchen MR, Eisner DA, O'Neill SC &Valdeolmillos M(1989) Measurements of intracellular Ca2+ in dissociated type Icells of the rabbit carotid body. 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