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Effects of cyanide and uncoupler on chemoreceptor activity and ATP content of the cat carotid body
Bruin Research, 481(1989) 250-257 Elsevier
250 BRE 14288
Effects of cyanide and uncouplers on chemoreceptor ATP content of the cat carotid body
activity and
Ana Obese, Laura Almaraz and Constancio Gonzalez Departamento de Bioquimica y Biologia Molecular y Fisiologia, Facultad de Medicina, Universidad de Valladolid, Valladolid (Spain)
(Accepted 9 August 1988) Key words: Carotid body; Chemoreceptor;
Cyanide; Uncoupler; Dopamine release; Metabolic hypothesis
In cat carotid bodies (c.b.‘s) incubated in vitro with [3H]tyrosine to label the stores of catecholamines, it was found that CN promotes dose- and Ca’+-dependent release of [3H]dopamine (DA) from c.b. tissues in parallel to the increased electrical activity recorded from the carotid sinus nerve (c.s.n.). Two different uncouplers, dinitrophenol (DNP) and carbonyl-cyanide-m-chlorophenylhydrazone (CCCP), both activate also in a dose-dependent fashion, release of DA and electrical activity in the c.s.n. However, while cyanide (CN) (10m4M) applied during 5 min reduced the adenosine triphosphate (ATP) content of the c.b. by 45%, DNP (2.5 x 10e4 M) and CCCP ( 10e6 M) applied for the same period of time did not modify the ATP levels of the organ. At the above concentrations, the 3 agents increased about g-fold the electrical activity recorded from the c.s.n. Thus, contrary to the postulates of the metabolic hypotheses, our findings indicate that the decrease,in the ATP content in the c.b. is not a prerequisite for the activation of the chemoreceptors. We propose alternative mechanisms to explain the chemostimulant action of the metabolic poisons.
INTRODUCTION
The carotid body (cb.) chemoreceptors are physiologically activated by decreases in environmental pOz and pH and by increases in pC0,. In addition, there is a great variety of pharmacological agents which activate these sensory receptors; among them, blockers of the respiratory chain and uncouplers are considered classical chemoreceptor stimulants4,6,13. In fact, the great chemostimulant potency of these mitochondrial poisons has been the origin of the metabolic hypothesis of sensory transduction in this receptor 4,6,13,30,43.According to this hypothesis, the main physiological stimulus, hypoxia, as well as the metabolic poisons will produce decreases in the adenosine triphosphate (ATP) content of the type I glomus cells. The decrease in the phosphate transfer potential [ATP/(ADP + AMP + Pi)] will be the initial or crucial step in the overall transduction process, i.e. the activation of the transmitter release from type I cells. The transmitter(s) release will finally ac-
tivate the sensory nerve endings, to set the level of propagated activity in the carotid sinus nerve (c.s.n.)25. The main assumptions in the metabolic hypothesis are: (1) that both hypoxia and metabolic poisons, at the levels that increase the c.s.n. activity, do in fact decrease the ATP levels; (2) that these stimuli at those levels induce the release of transmitters; and (3) that there is a causal link between the decrease in the ATP levels and the activation of the secretory machinery. There” are no propositions on how the decrease in the ATP levels will trigger the release of transmitters. In previous publications, we have shown that moderate levels of hypoxia increase the release of dopamine (DA), a putative transmitter of the type I cells, in parallel to the increased electrical activity of the c .s .n.14.37. The same levels of hypoxia decrease the ATP content of the c.b.33. Otherwise, 2-deoxyglucase produces concomitantly activation of DA release, increased electrical activity in the c.s.n. and re-
Correspondence: C. Gonzalez, Depto. Bioquimica y Biologia Molecular y Fisiologia, Facultad de Medicina, CI. Ram&r y Cajal, 47005-Valladolid, Spain. 0006-8993/89/$03.50
0 1989 Elsevier Science Publishers B.V. (Biomedical Division)
251 duction of ATP levels in the c.b. 34. These findings allowed us to conclude that the decrease in the ATP content in the c.b. could be a link between the detection of the stimuli and the activation of the release of DA. We did have not experimental data to support a causal relationship as the metabolic hypothesis proposes 33'34. In a further attempt to define the nature of this possible link, in the present paper, we extend our work to study the effects of cyanide (CN) and two uncouplers, dinitrophenol (DNP) and carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), on the c.b. chemoreceptor activity and ATP levels. We found that cyanide increases both the electrical activity in the c.s.n, and the release of DA; concomitantly, it decreases the ATP content of the organ. Both uncouplers, at concentrations which produced the same level of enhanced electrical activity in the c.s.n., also promote the release of DA without modifying the ATP content of the organ. MATERIALS AND METHODS
The study was performed with c.b.'s superfused in vitro, and excised from adult cats (2-3.5 kg), anesthetized with Na-pentobarbitone (30-40 mg/kg i.p.), tracheostomized and artificially ventilated. Subsequent surgery and the rest of the methods has been described in detail in previous publications 33,34. The study of the effects of the mitochondriai poisons on the electrical activity and release of DA was carried out simultaneously in the same preparations. In these experiments the c.b.'s with their attached nerves were adequately cleaned and incubated for 3 h in 500 pl of 100% 0 2 equilibrated Tyrode solution (in mM: NaC1 140, KC1 4.7, CaCI 2 2.2, MgC1e 1.1, Hepes 10, Glucose 5.5, adjusted to pH 7.4 with 1 N NaOH) containing 100 pM of the tyrosine hydroxylase cofactor 6-methyltetrahydropterine (Sigma), 1 mM ascorbic acid and 20 pM of the catecholamine precursor [3H]tyrosine (2,6-[3H]-tyrosine; 20 Ci/mmol, Amersham). At the end of the incubation, the preparations were mounted in a drop superfusion chamber TM, which allows temperature- (37 °C) and flow- (0.4 ml/min)-controlled superfusion, recording of the electrical activity from the whole c.s.n., and collection of the superfusates for chemical analyses. The metabolic poisons (sodium cyanide, Merk; 2,4dinitrophenol and carbonyl-cyanide m-chlorophe-
nyl-hydrazone, Sigma) were dissolved in the perfusion solutions equilibrated with 100% 02; the solution-containing bottles were capped after dissolving the drugs and the bubbling with 0 2 was stopped to avoid loss of cyanide. The superfusates were collected in vials containing a carrier mixture (glacial acetic acid, 20 mM ascorbic acid, 100pM cold DA) in the appropriate volume to yield a final pH in the superfusates of 3.2-3.6. The collection of the superfusates was made according to the following sequence: 5 min prior to the application of drug (control), 5 min during the drug application (stimulus) and 4 more fractions (one of 5 min and three of 10 min) poststimulus, totaling 45 rain (duration of a single stimulus cycle; see Fig. 1). The superfusates were analyzed conventionally for their content of catecholamines. Analysis included adsorption on acid-washed alumina at pH 8.6, elution with 1 N HC1 to recover all [3H]catechol compounds and thin layer chromatography of the eluates, according to Fleming and Clark 15, to obtain positive identification of the 3H-labelled catechols. The action potentials from the c.s.n, were led through an A.C.-coupled amplifier and displayed on an oscilloscope. The amplified signals were led through a window discriminator to a digital counterprinter which read the total number of action potentials during fixed periods of time (1 or 10 s). The drug-induced responses were evaluated as follows: from the peak induced DA-release (cpm in the 5 min stimulus sample) or peak-induced electrical activity (action potentials in 1-min sample period during the stimulus) the control release or electrical activity in an identical period of time prior to drug application was subtracted, and the difference was divided by the control release or electrical activity, respectively. Then a (S-C)/C ratio equal to one means that during the stimulus the response doubled the control. The effect of the mitochondrial poisons on ATP content was studied in separate experiments. The c.b.'s without their nerves were cleaned of surrounding tissues in the standardized conditions previously described 34 and preincubated for 30 min at 37 °C in a metabolic bath (75 r.p.m.). The drugs were applied during 5 rain, as in the first series of experiments. The ATP was assayed th6reafter in neutralized PCA extracts, both radioenzymaticallyTM and by a luminiscence-based method 9. In these experiments, the c.b. of one side was used as control (incubated in the abs-
252 cence of mitochondrial poisons) and the contralateral one was incubated with the drug. This protocol was used in order to exclude differences in the c.b. weight between animals 34. Finally, in a batch of experiments, tracer amounts (1 ]~M) of 2-deoxy-[3H]glucose (28 Ci/mmol, 2-DG; ICN) were added to the incubation media at the same time as the uncouplers and the rate of glucose utilization was measured in the same c.b.'s in which the ATP levels were determined. RESULTS
Effects of CN on carotid sinus nerve activity and [3H]DA release The inset of Fig. 1 illustrates the time course of the chemosensory response of the c.s.n, elicited by different concentrations of CN in a single experiment. Note that the onset and the time to peak of the sensory response decrease with increasing concentrations of the drug. The highest concentration tested (2.5 x 10 -4 M) induced an initial high frequency of afferent impulses followed by 'adaptation' to a lower level. This pattern of response, also observed with strong hypoxic stimuli in multifiber preparations 23 and in single fiber preparations 8, has been shown to be due at least in part to a decreased amplitude of the action potentials 8. The [3H]DA evoked release in response to the 5-min period of superfusion with CN also increases with increasing concentrations of the drug (Fig. 1). The release of D A overlasts the period of drug application for all concentrations tested. As
shown in a previous publication 37, this is due to the metabolic cycling of the released [3H]DA, i.e. in the period of application of the stimulus and in the following 5-min period most of the evoked release is collected as [3H]DA, whereas in ulterior samples most of the evoked release (above the dashed line in the figure) is collected as [3H]DOPAC. The mean values for CN-evoked electrical activity and [3H]DA release, obtained in 6 experiments, are expressed as times the respective control values in Fig. 2. Given the time course of the changes in frequency of chemosensory impulses with the strongest stimuli, the ratios stimulus-control/control for both DA release and electrical activity were calculated at the peak responses induced by each stimulus. In contrast to the results obtained with the hypoxic stimuli 14"37, on stimulation with CN the evoked/control ratio for electrical activity exceeds the corresponding ratio for [3H]DA released. This different behavior of the c.b. chemoreceptors to both stimuli could result from a direct effect of CN on the sensory fibers in addition to its action on the type I cells (see Discussion). In 3 additional experiments, the Ca 2+ dependence of the CN-evoked responses was also studied. Fig. 3 shows the results obtained with 6 paired stimuli applied in control solution and in nominally Ca2+-free solution (containing 1.6 mM Mg2+). It is evident that the evoked release of D A was more depressed in the
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Fig. 3. Calcium dependence of responses elicited by CN (10 -4 M) in the carotid body. Initial stimulation with CN in Ca 2÷ containing solution, second and third applications of CN in Ca 2÷free medium and the fourth one in normal Ca 2+ containing solution. Six pairs of data obtained from 3 experiments. A: evoked DA release. B: evoked increase in electrical activity. Empty bars: control. Filled bars: Ca2÷-free. *P < 0.05, **P < 0.001. Student's t-test for paired data.
Ca2÷-free solution than the evoked increase in electrical activity, also suggesting that CN has some direct action on the nerve endings (see Discussion). In the presence of 1 mM EGTA, CN did not evoke any DA release.
Effects of uncouplers on carotid sinus nerve activity and [3H]DA release Fig. 4 illustrates the effects of uncouplers (DNP and CCCP) on the electrical activity of the carotid sinus nerve and on the [3H]DA release from the carotid body. In initial experiments with our protocol of drug application, it was found that concentrations of
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Effects of CN, DNP and CCCP on the ATP content of the carotid body As mentioned in Materials and Methods, the effects of the mitochondrial poisons on ATP levels were studied with paired samples, i.e. the c.b. from one side was incubated with normal 100% 0 2 equilibrated Tyrode and the contralateral one with the same medium containing the drug. For the 3 poisons,
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DNP of 10-5 M or below were ineffective and that concentrations above 2.4 x 10-4 M produced a brisk electrical response, followed by a sustained block of the electrical activity and a sustained outflow of [3H]DA several times above the control. Although the effects of the uncouplers were similar to those of CN, we found some differences in the responses to both types of agents. (1) The uncouplers exhibit steeper dose-response curves than CN, such that their threshold and maximal effective concentrations upon electrical activity fall within one order of magnitude; and (2) the uncouplers-evoked increases in electrical activity were slower in onset and outlived the period of superfusion with the drug (up to 5 min with the highest concentrations shown in Fig. 4). Both uncouplers, like CN, give higher evoked/control ratios for electrical activity than for DA release, suggesting that they have also some direct effect on the nerve endings (see Discussion). The Ca 2÷ dependence of the DNP-induced release of [3H]DA was tested in only two experiments. In Ca2+-free solution (1 mM EGTA), the basal release was moderately reduced and the evoked release disappeared (data not shown).
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Fig. 4. Dose-response curves for [3H]DA release (solid curves) and sinus nerve frequency (dashed curves) evoked by the uncouplers CCCP (single experiment) and DNP (means + S.E.M. of 6 experiments) (S-C)/C ratios were calculated as described in the Materials and Methods section.
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Fig. 5. Effects of CN (10 -4 M), DNP (2.5 x 10-4 M), and CCCP (10 -6 M) on the ATP content of carotid bodies. Empty bars: control carotid bodies for all groups. Filled bars: drug-treated carotid bodies. Data are means + S.E.M. of 6-8 carotid bodies. *P < 0.02. Student's t-test for paired data.
254 concentrations that produce about 8-fold increases in the electrical activity of the c.s.n, were selected. As Fig. 5 shows, while CN (10 a M) applied during 5 rain produces a significant 45% reduction in the ATP content. neither DNP (2.5 x 10 4 M) nor CCCP (10 -6 M) reduced the ATP levels. This unexpected result prompted us to check the metabolic effects of the uncouplers. In a new series of experiments, [3H]2-DG (about 1 uM) was added to the medium, together with the uncouplers. The phosphorylated [3H]2-DG and ATP were measured in the same c.b.'s. As shown in Fig. 6, there is a quite significant increase in the amount of 2-DG-6P in the presence of the uncouplers. This indicates that an activation of the metabolism takes place a2` which may compensate for the uncoupling effect and prevent the drop of ATP levels II. In fact, no significant changes in ATP content of c.b. 's was induced by either DNP or CCCP in this new series of experiments. DISCUSSION The present study demonstrates that moderate concentrations of blockers and uncouplers of oxidative phosphorylation induce a Ca2+-dependent release of [3H]DA from glomus tissue, in addition to the increased electrical activity in the sinus nerve. Amongst the catecholaminergic systems, this secretory effect appears to be specific for the c.b. because even higher concentrations of these drugs did not augment spontaneous release of catecholamines from either sympathetic endings ~2"26,brain tissue 39 or 3. .Q i C)
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Fig. 6. Effects of CCCP (10 -6 M) and DNP (2.5 x 10-4 M) on the phosphorylation rate of tracer concentrations (1 /~M) of [3H]2-DG. Data are means + S.E.M. of 4 carotid bodies for each column. *P < 0.02; **P < 0.01. Student's t-test for paired data.
adrenal glands ~'~. The reported effects of CN and uncouplers on non-catecholaminergic structures are somehow variable. For example, for cholinergic systems it has been reported that neither CN (10 -~ M) nor DNP (6 × 10 a M) modify resting release of acetylcholine from slices of rat brain cortex a
255 sponse t o CO 2 was not 3°'32. Our own findings 33'34, showing that either moderately high levels of hypoxia or 2-deoxyglucose at mM concentrations reduce the ATP content of the c.b. and increase both [3H]DA release and carotid sinus nerve activity, were also in accord with the metabolic hypothesis. Ponte and Sadlet 36, however, were unable to confirm Lahiri's results with oligomycin in vivo and attributed some of its effects to the action of the ethanol used as oligomycin solvent. In a very recent paper 41, Lahiri's group repeated their experiments controlling for possible effects of the solvents. They confirmed their original findings 3°-32, with oligomycin concentrations in the perfusing media from 2 to 4/~g/ml (i.e. 2.5 to 5 × 10 -6 M). However, in this new report there are some findings that are difficult to frame in the metabolic hypothesis. Thus, from their figures 3D, 5D and especially from their Table 1 it is evident that oligomycin by itself either does not modify or decreases the basal activity, a result that questions the metabolic hypothesis. If oligomycin was blocking mitochondrial ATPase it should increase the electrical activity30-32; therefore, the differential effects that they found for oligomycin on the responses to 02 and CO2 should be generated at a different place than the ATP-generating machinery of the mitochondria. In a few experiments in vitro we have found that oligomycin applied at 2.5 × 10 -6 M for 5 min did not abolish the c.b. chemosensory responses to hypoxia and CN (unpublished). The drug induced a slow and longlasting increase in c.s.n, activity, which became about two times the control activity 1 h later; at this moment, the response to hypoxia was unaltered. Two hours after the application of oligomycin, the activity was about 4 times the control value and at this time the response to CN was again intact. The use of higher oligomycin concentrations was avoided because of their action on Na+-K+-ATPase16,46. The release of [3H]DA augmented in parallel to the electrical activity all along the experiments. The data presented in this paper, showing that uncouplers do not decrease the ATP levels in the c.b. at concentrations which activate both electrical activity in the c.s.n, and secretory response in type I cells, are incompatible with the metabolic hypothesis and strongly suggest that the ATP decrease is not a prerequisite for the excitation of the c.b. chemoreceptors. This conclusion obligated us to reconsider the
mechanisms involved in the activation of the c.b. chemoreceptors by uncouplers and CN and also to reinterpret our previous data regarding the mechanisms of action of hypoxia and 2-DG. The uncouplers (DNP and CCCP) are protonophors that promote the passage of H + ions through membranes, bringing them to electrochemical equilibrium. There is no doubt that at high concentrations the uncouplers decrease the ATP content because they abolish the H + gradient through the internal mitochondrial membrane. At moderate concentrations, the partial uncoupling may be balanced by an increased flow of electrons through the respiratory chain fuelled by an increased consumption of metabolic substrates 11. The concentration of uncouplers chosen in our ATP experiments are within this range of partial uncoupling. At the cytoplasmic membrane level the uncouplers will also tend to bring protons to their electrochemical equilibrium. If the resting potential of type I cells is close to -60 mV 21, then at the extracellular pH used in our experiments (7.4) the uncouplers will tend to move the pHi towards 6.4 with a very rapid time course. This strong intracellular acidification can, in all likelihood, explain the activation of the chemoreceptors as it does the natural hypercapnic stimulus. CN is a very active agent that affects a great variety of enzymatic processes, but at present we have no clues on the process involved in the activation of the c.b. chemoreceptors by this agent. However, regarding the hypoxic decrease in ATP, we have indirect evidence that it is an effect rather than a cause of the hypoxic activation of the c.b. Thus, Obeso et al. 35 (submitted) found that the hypoxia-activated glucose consumption by the c.b. was abolished by ouabain. In agreement with findings in other structures 2~'2s'4s, the observed increase in metabolism and decrease in ATP levels should be interpreted as produced by an activation of the Na + pump. It is possible that the transduction of the hypoxic stimulus involves an increase of intracellular Na +, which activates both the entry of Ca 2+ to support the secretory response 35, and the Na+-pump. Activation of the Na+-pump will produce the observed decrease of ATP and the increase of glucose utilization. In relation to the mechanisms of the effects of 2D G on the c.b. 34, it has been recently found that it produces an important intracellular acidification in
256 neutrophils 1~. Then, it is conceivable that 2-DG also activates the c.b. chemoreceptors by the same mechanisms as the natural hypercapnic stimulus. The ATP decrease observed in these conditions would most probably be an e p i p h e n o m e n a of the c.b. activation by 2-DG. It rests to consider how the presumable increase in intracellular hydrogen concentration p r o d u c e d by the uncouplers, 2-deoxyglucose and the natural hypercapnic stimulus will trigger the release of D A which is greatly d e p e n d e n t on the presence of Ca:+c. It is well-documented in many systems 5'2°'47 that intracellular acidification is immediately followed by a net Na + gain by the cells via the Na+/H + exchanger. A m o n g s t other effects, an increase in Na+i can lead to an increased Ca2+ i via the Na+/Ca 2+ exchangerZ°; in fact an increase in intracellular Ca 2+ upon intracellular acidification has been measured in m a m m a l i a n cardiac muscle 3. In addition to the above mechanisms, all the agents tested in the present work have been shown to interfere with intracellular mechanisms that buffer Ca 2+ leading to increased Ca2+ i from intracellular stores 1'7. These later effects could explain the release observed with CN in nominallyfree Ca z+ medium. A n o t h e r issue that deserves some c o m m e n t relates to the possible direct effects of CN and uncouplers on the sensory nerve endings. It has been shown that the agents used in the present study have different effects on excitability in different preparations. F o r example, G o d f r a i n d et a1.17 found that in cortical neurons D N P blocked the discharges e v o k e d by acetylcholine REFERENCES l Adams, D,J,, Takeda, K, and Umbach, J,A,, Inhibitors of calcium buffering depress evoked transmitter release at the squid giant synapse, J. Physiol. (Lond.), 369 (1985) t45-159. 2 Adam-Vizi, V. and Ligeti, E., Release of acetylcholine from rat brain synaptosomes by various agents in the absence of external calcium ions, J. Physiol. (Lond.), 353 (1984) 505-521. 3 Allen, D.G. and Orchard, C.H., Effects of changes of pH on intracellular calcium transients in mammalian cardiac muscle, J. Physiol. (Lond.), 335 (1983) 555-567. 4 Anichkov, S.V. and Belen'kii, M.L., Pharmacology of the Carotid Body Chemoreceptors, McMillan, New York, 1963. 5 Aronson, P.S., Kinetic properties of the plasma membrane Na+-H + exchanger, Ann. Rev. Physiol., 47 (1985) 545-560. 6 Belmonte, C. and Gonzalez, C., Mechanisms of chemore-
but facilitated those evoked by glutamate and that CN had mainly excitatory effects. A d a m s et al. J found al the squid giant synapse that both CN and F C C P (another uncoupler) did not modify postsynaptic excitability and A d a m - V i z i and Ligeti 2 found that CCCP causes a mild depolarization in rat cortex synaptosomes. This depolarization can render the nerve endings more readily excitable. Although we do not know what the real situation is in the sensory nerve endings of the c.s.n., our results (higher response ratio for electrical activity than for D A release and less Ca 2+ d e p e n d e n c e of the drug-induced electrical activity) are best explained by a direct excitatory action of CN and uncouplers on the sensory nerve endings. In conclusion, the present study shows that the c h e m o r e c e p t o r response to uncouplers is not linked to ATP decrease in the c.b. This and other findings discussed here m a k e it difficult to maintain the metabolic hypothesis of chemoreception, suggesting that plasma m e m b r a n e processes (instead of or in addition to mitochondrial ones) are the primary events in the transduction of the physiological stimuli in this receptor.
ACKNOWLEDGEMENTS The authors thank Prof. B. H e r r e r o s for his critical reading of the manuscript, and Josefina Revuelta for secretarial assistance. This w o r k was s u p p o r t e d by D . G . I . C . Y . T . G r a n t PB86-0325 and by FISss G r a n t 88/0994. ception in the carotid body: possible models. In H. Acker and R.G. O'Regan (Eds.), Physiology of the Peripheral Arterial Chemoreceptors, Elsevier, Amsterdam, 1983, pp. 107-220. 7 Bernath, S. and Vizi, E.S., Inhibitory effect of ionized free intracellular calcium enhanced by ruthenium red and mchloro-carbonylcyanide phenyl hydrazon on the evoked release of acetylcholine, Biochem. Pharmacol., 36 (1987) 3683-3687. 8 Biscoe, T.J., Purves, M.J. and Sampson, S.R., The frequency of nerve impulses in single carotid body chemoreceptor afferent fibres recorded in vivo with intact circulation, J. Physiol. (Lond.), 208 (1970) 121-131. 9 Brown, A.M., ATP and ATPase determinations in red blood cells. In J.C. Ellory and J.C. Young (Eds.), Red Cell Membranes: A Methodological Approach, Academic, London, 1982, pp. 223-238. 10 Cardenas, H. and Zapata, P., Ventilatory reflexes originated from carotid and extracarotid chemoreceptors in rats, Am. J. Physiol., 244 (1983) Rl19-R125.
257 11 Ernster, L. and Nordenbrand, K., Skeletal muscle mitochondria. In R. W. Estabrook and M.E. Pullman (Eds.), Methods in Enzymology Vol. 10, Academic, New York, 1967, pp. 86-94. 12 Farnebo, L.O., Release of monoamines evoked by field stimulation. Studies on some ionic and metabolic requirements, Acta Physiol. Scand., suppl., 371 (1971) 19-27. 13 Fidone, S. and Gonzalez, C., Initiation and control of the chemoreceptor activity in the carotid body. In A.P. Fishman (Ed.), Handbook of Physiology, The Respiratory System, Vol. 11, Part L Amer. Physiol. Soc., Bethesda, Maryland, 1986, pp. 247-312. 14 Fidone, S.J., Gonzalez, C. and Yoshizaki, K., Effect of low oxygen on the release of dopamine from the rabbit carotid body in vitro, J. Physiol. (Lond.), 33 (1982) 93-110. 15 Fleming, R.M. and Clark, W.G., Quantitative thin-layer chromatographic estimation of labeled dopamine and norepinephrine, their precursors and metabolites, J. Chromatogr., 52 (1970) 305-312. 16 Glynn, I.M., An ATPase from electric organ activated by Na+-K + and inhibited by ouabain or oligomycin, Biochem. J., 84 (1962) 75P. 17 Godfraind, J.M., Kawamura, M., Krnjevic, K. and Pumain, R., Dinitrophenol and some other metabolic inhibitors on cortical neurones, J. Physiol. (Lond.), 215 (1971) 199-222. 18 Gonzalez, C. and Garcfa-Sancho, J., A sensitive assay for ATP, Anal. Biochem., 114 (1981) 285-287. 19 Grinstein, S. and Furuya, W., Cytoplasmic pH regulation in phorbol ester-activated human neutrophils, Am. J. Physiol., 251 (1986) C55-C65. 20 Grinstein, S. and Rothstein, A., Mechanisms of regulation of the Na+-H ÷ exchanger, J. Membrane Biol., 90 (1986) 1-12. 21 Hayashida, Y. and Eyzaguirre, C., Voltage noise of carotid body type I cells, Brain Research, 167 (1979) 189-194. 22 Heymans, C., Bouckaert, J.J. and Dautrebande, L., Sinus carotidien et r6flexes respiratoires; sensibilit6 des sinus carotidiens aux substances chimiques. Action stimulante respiratoire r6flexe du sulfure de sodium, du cyanure de potassium, de la nicotine et de la lobeline, Arch. Int. Pharmacodyn. Ther., 40 (1931) 54-91. 23 Hornbein, T.F., The relation between stimulus to chemoreceptors and their response. In R.W. Torrance (Ed.), Arterial Chemoreceptors, Blackwell, Oxford, 1968, pp. 65-78. 24 Huang, H. and Daly, J.W., Interrelationships among the levels of ATP, adenosine and cyclic AMP in incubated slices of guinea pig cerebral cortex: effects of depolarizing agents, psychotropic drugs and metabolic inhibitors, J. Neurochem., 23 (1974) 393-404. 25 Joels, N. and Neil, E., The idea of a sensory transmitter. In. R.W. Torrance (Ed.), Arterial Chemoreceptors, Blackwell, Oxford, 1968, pp. 153-178. 26 Kirpekar, S.M., Prat, J.C. and Yamamoto, H., Effects of metabolic inhibitors on norepinephrine release from the perfused spleen of the cat, J. Pharmacol. Exp. Ther., 172 (1970) 342-350. 27 Krylov, S.S. and Anichkov, S.V., The effect of metabolic inhibitors on carotid chemoreceptors. In R.W. Torrance (Ed.), Arterial Chemoreceptors, Blackwell, Oxford, 1968, pp. 103-113. 28 Ksiezak, H.J. and Gibson, G.E., Oxygen dependence of glucose and acetylcholine metabolism in slices and synaptosomes from rat brain, J. Neurochem., 37 (1981) 305-314. 29 Loomis, W.F. and Lipman, F., Reversible inhibition between phosphorylation and oxidation, J. Biol. Chem., 173 (1948) 807-808.
30 Mulligan, E. and Lahiri, S., Mitochondrial oxidative metabolism and chemoreception in the carotid body. In C. Belmonte, D. Pallot, H. Acker and S. Fidone (Eds.), Arterial Chemoreceptors, Leicester University Press, Leicester, U.K., 1981, pp. 316-326. 31 Mulligan, E., Lahiri, S. and Storey, B.T., Carotid body 02 chemoreception and mitochondrial oxidative phosphorylation, J. Appl. Physiol.: Respirat. Environ. Exercise Physiol., 51 (1981) 438-446. 32 Mulligan, E. and Lahiri, S., Separation of carotid body chemoreceptor responses to O 2 and CO 2 by oligomycin and by antimycin A, Am. J. Physiol., 242 (1982) C200-C206. 33 Obeso, A., Almaraz, L. and Gonzalez, C., Correlation between adenosine triphosphate levels, dopamine release and electrical activity in the carotid body: support for the metabolic hypothesis of chemoreception, Brain Research, 348 (1985) 64-68. 34 Obeso, A., Almaraz, L. and Gonzalez, C., Effects of 2deoxy-D-glucose on in vitro cat carotid body, Brain Research, 371 (1986) 25-36. 35 Obeso, A., Gonzalez, C., Dinger, B. and Fidone, S., Energy metabolism in the rabbit carotid body: biochemical and autoradiographic findings using the 2-deoxy-glucose technique 'in vitro', Neurosci. Lett., submitted. 36 Ponte, J. and Sadler, C.L., Effects of oligomycin upon carotid body chemoreceptors of the cat and rabbit, J. Physiol. (Lond.), 371 (1986) l12P. 37 Rigual, R., Gonzalez, E., Gonzalez, C. and Fidone, S., Synthesis and release of catecholamines by the cat carotid body in vitro: effects of hypoxic stimulation, Brain Research, 374 (1986) 101-109. 38 Rubin, R.P., Calcium and Cellular Secretion, Plenum, New York, 1982. 39 Rutledge, C.O., Effect of metabolic inhibitors and ouabain on amphetamine- and potassium-induced release of biogenic amines from isolated brain tissue, Biochem. Pharmacol., 27 (1978) 511-516. 40 Shen, T.C.R. and Hauss, W.H., Influence of dinitrophenol, dinitroartocresol and paranitrophenol upon the carotid sinus chemoreceptors of the dog, Arch. Int. Pharmacodyn. Ther., 63 (1939) 251-258. 41 Shirahata, M., Andronikou, S. and Lahiri, S., Differential effects of oligomycin on carotid chemoreceptor responses to 02 and CO 2 in the cat, J. Appl. Physiol., 63 (1987) 2084-2092. 42 Sokoloff, L., Relation between physiological function and energy metabolism in the central nervous system, J. Neurochem., 29 (1977) 13-26. 43 Torrance, R.W., Prolegomena. In R.W. Torrance (Ed.), Arterial Chemoreceptors, Blackwell, Oxford, 1968, pp. 1-40. 44 Vizi, E.S., Stimulation by inhibition of Na +, K +, MG ++ activated ATPase of acetylcholine release in cortical slices from rat brain, J. Physiol. (Lond.), 226 (1972) 95-117. 45 Vizi, E.S., Release mechanisms of acetylcholine and the role of Na+-K + activated ATPase. In P.G. Waser (Ed.), Cholinergic Mechanisms, Raven, New York, 1975, pp. 199-211. 46 Whittam, R., Wheeler, K.P. and Blake, A., Oligomycin and active transport reactions in cell membranes, Nature (Lond.), 203 (1964) 720-724. 47 Wray, S., Smooth muscle intracellular pH: measurement, regulation, and function, Am. J. Physiol., 254 (1988) C213-C225. 48 Yarowsky, P. and Ingvar, O.H., Neuronal activity and energy metabolism, Fed. Proc., 40 (1981) 2353-2362.
250 BRE 14288
Effects of cyanide and uncouplers on chemoreceptor ATP content of the cat carotid body
activity and
Ana Obese, Laura Almaraz and Constancio Gonzalez Departamento de Bioquimica y Biologia Molecular y Fisiologia, Facultad de Medicina, Universidad de Valladolid, Valladolid (Spain)
(Accepted 9 August 1988) Key words: Carotid body; Chemoreceptor;
Cyanide; Uncoupler; Dopamine release; Metabolic hypothesis
In cat carotid bodies (c.b.‘s) incubated in vitro with [3H]tyrosine to label the stores of catecholamines, it was found that CN promotes dose- and Ca’+-dependent release of [3H]dopamine (DA) from c.b. tissues in parallel to the increased electrical activity recorded from the carotid sinus nerve (c.s.n.). Two different uncouplers, dinitrophenol (DNP) and carbonyl-cyanide-m-chlorophenylhydrazone (CCCP), both activate also in a dose-dependent fashion, release of DA and electrical activity in the c.s.n. However, while cyanide (CN) (10m4M) applied during 5 min reduced the adenosine triphosphate (ATP) content of the c.b. by 45%, DNP (2.5 x 10e4 M) and CCCP ( 10e6 M) applied for the same period of time did not modify the ATP levels of the organ. At the above concentrations, the 3 agents increased about g-fold the electrical activity recorded from the c.s.n. Thus, contrary to the postulates of the metabolic hypotheses, our findings indicate that the decrease,in the ATP content in the c.b. is not a prerequisite for the activation of the chemoreceptors. We propose alternative mechanisms to explain the chemostimulant action of the metabolic poisons.
INTRODUCTION
The carotid body (cb.) chemoreceptors are physiologically activated by decreases in environmental pOz and pH and by increases in pC0,. In addition, there is a great variety of pharmacological agents which activate these sensory receptors; among them, blockers of the respiratory chain and uncouplers are considered classical chemoreceptor stimulants4,6,13. In fact, the great chemostimulant potency of these mitochondrial poisons has been the origin of the metabolic hypothesis of sensory transduction in this receptor 4,6,13,30,43.According to this hypothesis, the main physiological stimulus, hypoxia, as well as the metabolic poisons will produce decreases in the adenosine triphosphate (ATP) content of the type I glomus cells. The decrease in the phosphate transfer potential [ATP/(ADP + AMP + Pi)] will be the initial or crucial step in the overall transduction process, i.e. the activation of the transmitter release from type I cells. The transmitter(s) release will finally ac-
tivate the sensory nerve endings, to set the level of propagated activity in the carotid sinus nerve (c.s.n.)25. The main assumptions in the metabolic hypothesis are: (1) that both hypoxia and metabolic poisons, at the levels that increase the c.s.n. activity, do in fact decrease the ATP levels; (2) that these stimuli at those levels induce the release of transmitters; and (3) that there is a causal link between the decrease in the ATP levels and the activation of the secretory machinery. There” are no propositions on how the decrease in the ATP levels will trigger the release of transmitters. In previous publications, we have shown that moderate levels of hypoxia increase the release of dopamine (DA), a putative transmitter of the type I cells, in parallel to the increased electrical activity of the c .s .n.14.37. The same levels of hypoxia decrease the ATP content of the c.b.33. Otherwise, 2-deoxyglucase produces concomitantly activation of DA release, increased electrical activity in the c.s.n. and re-
Correspondence: C. Gonzalez, Depto. Bioquimica y Biologia Molecular y Fisiologia, Facultad de Medicina, CI. Ram&r y Cajal, 47005-Valladolid, Spain. 0006-8993/89/$03.50
0 1989 Elsevier Science Publishers B.V. (Biomedical Division)
251 duction of ATP levels in the c.b. 34. These findings allowed us to conclude that the decrease in the ATP content in the c.b. could be a link between the detection of the stimuli and the activation of the release of DA. We did have not experimental data to support a causal relationship as the metabolic hypothesis proposes 33'34. In a further attempt to define the nature of this possible link, in the present paper, we extend our work to study the effects of cyanide (CN) and two uncouplers, dinitrophenol (DNP) and carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), on the c.b. chemoreceptor activity and ATP levels. We found that cyanide increases both the electrical activity in the c.s.n, and the release of DA; concomitantly, it decreases the ATP content of the organ. Both uncouplers, at concentrations which produced the same level of enhanced electrical activity in the c.s.n., also promote the release of DA without modifying the ATP content of the organ. MATERIALS AND METHODS
The study was performed with c.b.'s superfused in vitro, and excised from adult cats (2-3.5 kg), anesthetized with Na-pentobarbitone (30-40 mg/kg i.p.), tracheostomized and artificially ventilated. Subsequent surgery and the rest of the methods has been described in detail in previous publications 33,34. The study of the effects of the mitochondriai poisons on the electrical activity and release of DA was carried out simultaneously in the same preparations. In these experiments the c.b.'s with their attached nerves were adequately cleaned and incubated for 3 h in 500 pl of 100% 0 2 equilibrated Tyrode solution (in mM: NaC1 140, KC1 4.7, CaCI 2 2.2, MgC1e 1.1, Hepes 10, Glucose 5.5, adjusted to pH 7.4 with 1 N NaOH) containing 100 pM of the tyrosine hydroxylase cofactor 6-methyltetrahydropterine (Sigma), 1 mM ascorbic acid and 20 pM of the catecholamine precursor [3H]tyrosine (2,6-[3H]-tyrosine; 20 Ci/mmol, Amersham). At the end of the incubation, the preparations were mounted in a drop superfusion chamber TM, which allows temperature- (37 °C) and flow- (0.4 ml/min)-controlled superfusion, recording of the electrical activity from the whole c.s.n., and collection of the superfusates for chemical analyses. The metabolic poisons (sodium cyanide, Merk; 2,4dinitrophenol and carbonyl-cyanide m-chlorophe-
nyl-hydrazone, Sigma) were dissolved in the perfusion solutions equilibrated with 100% 02; the solution-containing bottles were capped after dissolving the drugs and the bubbling with 0 2 was stopped to avoid loss of cyanide. The superfusates were collected in vials containing a carrier mixture (glacial acetic acid, 20 mM ascorbic acid, 100pM cold DA) in the appropriate volume to yield a final pH in the superfusates of 3.2-3.6. The collection of the superfusates was made according to the following sequence: 5 min prior to the application of drug (control), 5 min during the drug application (stimulus) and 4 more fractions (one of 5 min and three of 10 min) poststimulus, totaling 45 rain (duration of a single stimulus cycle; see Fig. 1). The superfusates were analyzed conventionally for their content of catecholamines. Analysis included adsorption on acid-washed alumina at pH 8.6, elution with 1 N HC1 to recover all [3H]catechol compounds and thin layer chromatography of the eluates, according to Fleming and Clark 15, to obtain positive identification of the 3H-labelled catechols. The action potentials from the c.s.n, were led through an A.C.-coupled amplifier and displayed on an oscilloscope. The amplified signals were led through a window discriminator to a digital counterprinter which read the total number of action potentials during fixed periods of time (1 or 10 s). The drug-induced responses were evaluated as follows: from the peak induced DA-release (cpm in the 5 min stimulus sample) or peak-induced electrical activity (action potentials in 1-min sample period during the stimulus) the control release or electrical activity in an identical period of time prior to drug application was subtracted, and the difference was divided by the control release or electrical activity, respectively. Then a (S-C)/C ratio equal to one means that during the stimulus the response doubled the control. The effect of the mitochondrial poisons on ATP content was studied in separate experiments. The c.b.'s without their nerves were cleaned of surrounding tissues in the standardized conditions previously described 34 and preincubated for 30 min at 37 °C in a metabolic bath (75 r.p.m.). The drugs were applied during 5 rain, as in the first series of experiments. The ATP was assayed th6reafter in neutralized PCA extracts, both radioenzymaticallyTM and by a luminiscence-based method 9. In these experiments, the c.b. of one side was used as control (incubated in the abs-
252 cence of mitochondrial poisons) and the contralateral one was incubated with the drug. This protocol was used in order to exclude differences in the c.b. weight between animals 34. Finally, in a batch of experiments, tracer amounts (1 ]~M) of 2-deoxy-[3H]glucose (28 Ci/mmol, 2-DG; ICN) were added to the incubation media at the same time as the uncouplers and the rate of glucose utilization was measured in the same c.b.'s in which the ATP levels were determined. RESULTS
Effects of CN on carotid sinus nerve activity and [3H]DA release The inset of Fig. 1 illustrates the time course of the chemosensory response of the c.s.n, elicited by different concentrations of CN in a single experiment. Note that the onset and the time to peak of the sensory response decrease with increasing concentrations of the drug. The highest concentration tested (2.5 x 10 -4 M) induced an initial high frequency of afferent impulses followed by 'adaptation' to a lower level. This pattern of response, also observed with strong hypoxic stimuli in multifiber preparations 23 and in single fiber preparations 8, has been shown to be due at least in part to a decreased amplitude of the action potentials 8. The [3H]DA evoked release in response to the 5-min period of superfusion with CN also increases with increasing concentrations of the drug (Fig. 1). The release of D A overlasts the period of drug application for all concentrations tested. As
shown in a previous publication 37, this is due to the metabolic cycling of the released [3H]DA, i.e. in the period of application of the stimulus and in the following 5-min period most of the evoked release is collected as [3H]DA, whereas in ulterior samples most of the evoked release (above the dashed line in the figure) is collected as [3H]DOPAC. The mean values for CN-evoked electrical activity and [3H]DA release, obtained in 6 experiments, are expressed as times the respective control values in Fig. 2. Given the time course of the changes in frequency of chemosensory impulses with the strongest stimuli, the ratios stimulus-control/control for both DA release and electrical activity were calculated at the peak responses induced by each stimulus. In contrast to the results obtained with the hypoxic stimuli 14"37, on stimulation with CN the evoked/control ratio for electrical activity exceeds the corresponding ratio for [3H]DA released. This different behavior of the c.b. chemoreceptors to both stimuli could result from a direct effect of CN on the sensory fibers in addition to its action on the type I cells (see Discussion). In 3 additional experiments, the Ca 2+ dependence of the CN-evoked responses was also studied. Fig. 3 shows the results obtained with 6 paired stimuli applied in control solution and in nominally Ca2+-free solution (containing 1.6 mM Mg2+). It is evident that the evoked release of D A was more depressed in the
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253
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Fig. 3. Calcium dependence of responses elicited by CN (10 -4 M) in the carotid body. Initial stimulation with CN in Ca 2÷ containing solution, second and third applications of CN in Ca 2÷free medium and the fourth one in normal Ca 2+ containing solution. Six pairs of data obtained from 3 experiments. A: evoked DA release. B: evoked increase in electrical activity. Empty bars: control. Filled bars: Ca2÷-free. *P < 0.05, **P < 0.001. Student's t-test for paired data.
Ca2÷-free solution than the evoked increase in electrical activity, also suggesting that CN has some direct action on the nerve endings (see Discussion). In the presence of 1 mM EGTA, CN did not evoke any DA release.
Effects of uncouplers on carotid sinus nerve activity and [3H]DA release Fig. 4 illustrates the effects of uncouplers (DNP and CCCP) on the electrical activity of the carotid sinus nerve and on the [3H]DA release from the carotid body. In initial experiments with our protocol of drug application, it was found that concentrations of
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Effects of CN, DNP and CCCP on the ATP content of the carotid body As mentioned in Materials and Methods, the effects of the mitochondrial poisons on ATP levels were studied with paired samples, i.e. the c.b. from one side was incubated with normal 100% 0 2 equilibrated Tyrode and the contralateral one with the same medium containing the drug. For the 3 poisons,
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DNP of 10-5 M or below were ineffective and that concentrations above 2.4 x 10-4 M produced a brisk electrical response, followed by a sustained block of the electrical activity and a sustained outflow of [3H]DA several times above the control. Although the effects of the uncouplers were similar to those of CN, we found some differences in the responses to both types of agents. (1) The uncouplers exhibit steeper dose-response curves than CN, such that their threshold and maximal effective concentrations upon electrical activity fall within one order of magnitude; and (2) the uncouplers-evoked increases in electrical activity were slower in onset and outlived the period of superfusion with the drug (up to 5 min with the highest concentrations shown in Fig. 4). Both uncouplers, like CN, give higher evoked/control ratios for electrical activity than for DA release, suggesting that they have also some direct effect on the nerve endings (see Discussion). The Ca 2÷ dependence of the DNP-induced release of [3H]DA was tested in only two experiments. In Ca2+-free solution (1 mM EGTA), the basal release was moderately reduced and the evoked release disappeared (data not shown).
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Fig. 4. Dose-response curves for [3H]DA release (solid curves) and sinus nerve frequency (dashed curves) evoked by the uncouplers CCCP (single experiment) and DNP (means + S.E.M. of 6 experiments) (S-C)/C ratios were calculated as described in the Materials and Methods section.
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Fig. 5. Effects of CN (10 -4 M), DNP (2.5 x 10-4 M), and CCCP (10 -6 M) on the ATP content of carotid bodies. Empty bars: control carotid bodies for all groups. Filled bars: drug-treated carotid bodies. Data are means + S.E.M. of 6-8 carotid bodies. *P < 0.02. Student's t-test for paired data.
254 concentrations that produce about 8-fold increases in the electrical activity of the c.s.n, were selected. As Fig. 5 shows, while CN (10 a M) applied during 5 rain produces a significant 45% reduction in the ATP content. neither DNP (2.5 x 10 4 M) nor CCCP (10 -6 M) reduced the ATP levels. This unexpected result prompted us to check the metabolic effects of the uncouplers. In a new series of experiments, [3H]2-DG (about 1 uM) was added to the medium, together with the uncouplers. The phosphorylated [3H]2-DG and ATP were measured in the same c.b.'s. As shown in Fig. 6, there is a quite significant increase in the amount of 2-DG-6P in the presence of the uncouplers. This indicates that an activation of the metabolism takes place a2` which may compensate for the uncoupling effect and prevent the drop of ATP levels II. In fact, no significant changes in ATP content of c.b. 's was induced by either DNP or CCCP in this new series of experiments. DISCUSSION The present study demonstrates that moderate concentrations of blockers and uncouplers of oxidative phosphorylation induce a Ca2+-dependent release of [3H]DA from glomus tissue, in addition to the increased electrical activity in the sinus nerve. Amongst the catecholaminergic systems, this secretory effect appears to be specific for the c.b. because even higher concentrations of these drugs did not augment spontaneous release of catecholamines from either sympathetic endings ~2"26,brain tissue 39 or 3. .Q i C)
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Fig. 6. Effects of CCCP (10 -6 M) and DNP (2.5 x 10-4 M) on the phosphorylation rate of tracer concentrations (1 /~M) of [3H]2-DG. Data are means + S.E.M. of 4 carotid bodies for each column. *P < 0.02; **P < 0.01. Student's t-test for paired data.
adrenal glands ~'~. The reported effects of CN and uncouplers on non-catecholaminergic structures are somehow variable. For example, for cholinergic systems it has been reported that neither CN (10 -~ M) nor DNP (6 × 10 a M) modify resting release of acetylcholine from slices of rat brain cortex a
255 sponse t o CO 2 was not 3°'32. Our own findings 33'34, showing that either moderately high levels of hypoxia or 2-deoxyglucose at mM concentrations reduce the ATP content of the c.b. and increase both [3H]DA release and carotid sinus nerve activity, were also in accord with the metabolic hypothesis. Ponte and Sadlet 36, however, were unable to confirm Lahiri's results with oligomycin in vivo and attributed some of its effects to the action of the ethanol used as oligomycin solvent. In a very recent paper 41, Lahiri's group repeated their experiments controlling for possible effects of the solvents. They confirmed their original findings 3°-32, with oligomycin concentrations in the perfusing media from 2 to 4/~g/ml (i.e. 2.5 to 5 × 10 -6 M). However, in this new report there are some findings that are difficult to frame in the metabolic hypothesis. Thus, from their figures 3D, 5D and especially from their Table 1 it is evident that oligomycin by itself either does not modify or decreases the basal activity, a result that questions the metabolic hypothesis. If oligomycin was blocking mitochondrial ATPase it should increase the electrical activity30-32; therefore, the differential effects that they found for oligomycin on the responses to 02 and CO2 should be generated at a different place than the ATP-generating machinery of the mitochondria. In a few experiments in vitro we have found that oligomycin applied at 2.5 × 10 -6 M for 5 min did not abolish the c.b. chemosensory responses to hypoxia and CN (unpublished). The drug induced a slow and longlasting increase in c.s.n, activity, which became about two times the control activity 1 h later; at this moment, the response to hypoxia was unaltered. Two hours after the application of oligomycin, the activity was about 4 times the control value and at this time the response to CN was again intact. The use of higher oligomycin concentrations was avoided because of their action on Na+-K+-ATPase16,46. The release of [3H]DA augmented in parallel to the electrical activity all along the experiments. The data presented in this paper, showing that uncouplers do not decrease the ATP levels in the c.b. at concentrations which activate both electrical activity in the c.s.n, and secretory response in type I cells, are incompatible with the metabolic hypothesis and strongly suggest that the ATP decrease is not a prerequisite for the excitation of the c.b. chemoreceptors. This conclusion obligated us to reconsider the
mechanisms involved in the activation of the c.b. chemoreceptors by uncouplers and CN and also to reinterpret our previous data regarding the mechanisms of action of hypoxia and 2-DG. The uncouplers (DNP and CCCP) are protonophors that promote the passage of H + ions through membranes, bringing them to electrochemical equilibrium. There is no doubt that at high concentrations the uncouplers decrease the ATP content because they abolish the H + gradient through the internal mitochondrial membrane. At moderate concentrations, the partial uncoupling may be balanced by an increased flow of electrons through the respiratory chain fuelled by an increased consumption of metabolic substrates 11. The concentration of uncouplers chosen in our ATP experiments are within this range of partial uncoupling. At the cytoplasmic membrane level the uncouplers will also tend to bring protons to their electrochemical equilibrium. If the resting potential of type I cells is close to -60 mV 21, then at the extracellular pH used in our experiments (7.4) the uncouplers will tend to move the pHi towards 6.4 with a very rapid time course. This strong intracellular acidification can, in all likelihood, explain the activation of the chemoreceptors as it does the natural hypercapnic stimulus. CN is a very active agent that affects a great variety of enzymatic processes, but at present we have no clues on the process involved in the activation of the c.b. chemoreceptors by this agent. However, regarding the hypoxic decrease in ATP, we have indirect evidence that it is an effect rather than a cause of the hypoxic activation of the c.b. Thus, Obeso et al. 35 (submitted) found that the hypoxia-activated glucose consumption by the c.b. was abolished by ouabain. In agreement with findings in other structures 2~'2s'4s, the observed increase in metabolism and decrease in ATP levels should be interpreted as produced by an activation of the Na + pump. It is possible that the transduction of the hypoxic stimulus involves an increase of intracellular Na +, which activates both the entry of Ca 2+ to support the secretory response 35, and the Na+-pump. Activation of the Na+-pump will produce the observed decrease of ATP and the increase of glucose utilization. In relation to the mechanisms of the effects of 2D G on the c.b. 34, it has been recently found that it produces an important intracellular acidification in
256 neutrophils 1~. Then, it is conceivable that 2-DG also activates the c.b. chemoreceptors by the same mechanisms as the natural hypercapnic stimulus. The ATP decrease observed in these conditions would most probably be an e p i p h e n o m e n a of the c.b. activation by 2-DG. It rests to consider how the presumable increase in intracellular hydrogen concentration p r o d u c e d by the uncouplers, 2-deoxyglucose and the natural hypercapnic stimulus will trigger the release of D A which is greatly d e p e n d e n t on the presence of Ca:+c. It is well-documented in many systems 5'2°'47 that intracellular acidification is immediately followed by a net Na + gain by the cells via the Na+/H + exchanger. A m o n g s t other effects, an increase in Na+i can lead to an increased Ca2+ i via the Na+/Ca 2+ exchangerZ°; in fact an increase in intracellular Ca 2+ upon intracellular acidification has been measured in m a m m a l i a n cardiac muscle 3. In addition to the above mechanisms, all the agents tested in the present work have been shown to interfere with intracellular mechanisms that buffer Ca 2+ leading to increased Ca2+ i from intracellular stores 1'7. These later effects could explain the release observed with CN in nominallyfree Ca z+ medium. A n o t h e r issue that deserves some c o m m e n t relates to the possible direct effects of CN and uncouplers on the sensory nerve endings. It has been shown that the agents used in the present study have different effects on excitability in different preparations. F o r example, G o d f r a i n d et a1.17 found that in cortical neurons D N P blocked the discharges e v o k e d by acetylcholine REFERENCES l Adams, D,J,, Takeda, K, and Umbach, J,A,, Inhibitors of calcium buffering depress evoked transmitter release at the squid giant synapse, J. Physiol. (Lond.), 369 (1985) t45-159. 2 Adam-Vizi, V. and Ligeti, E., Release of acetylcholine from rat brain synaptosomes by various agents in the absence of external calcium ions, J. Physiol. (Lond.), 353 (1984) 505-521. 3 Allen, D.G. and Orchard, C.H., Effects of changes of pH on intracellular calcium transients in mammalian cardiac muscle, J. Physiol. (Lond.), 335 (1983) 555-567. 4 Anichkov, S.V. and Belen'kii, M.L., Pharmacology of the Carotid Body Chemoreceptors, McMillan, New York, 1963. 5 Aronson, P.S., Kinetic properties of the plasma membrane Na+-H + exchanger, Ann. Rev. Physiol., 47 (1985) 545-560. 6 Belmonte, C. and Gonzalez, C., Mechanisms of chemore-
but facilitated those evoked by glutamate and that CN had mainly excitatory effects. A d a m s et al. J found al the squid giant synapse that both CN and F C C P (another uncoupler) did not modify postsynaptic excitability and A d a m - V i z i and Ligeti 2 found that CCCP causes a mild depolarization in rat cortex synaptosomes. This depolarization can render the nerve endings more readily excitable. Although we do not know what the real situation is in the sensory nerve endings of the c.s.n., our results (higher response ratio for electrical activity than for D A release and less Ca 2+ d e p e n d e n c e of the drug-induced electrical activity) are best explained by a direct excitatory action of CN and uncouplers on the sensory nerve endings. In conclusion, the present study shows that the c h e m o r e c e p t o r response to uncouplers is not linked to ATP decrease in the c.b. This and other findings discussed here m a k e it difficult to maintain the metabolic hypothesis of chemoreception, suggesting that plasma m e m b r a n e processes (instead of or in addition to mitochondrial ones) are the primary events in the transduction of the physiological stimuli in this receptor.
ACKNOWLEDGEMENTS The authors thank Prof. B. H e r r e r o s for his critical reading of the manuscript, and Josefina Revuelta for secretarial assistance. This w o r k was s u p p o r t e d by D . G . I . C . Y . T . G r a n t PB86-0325 and by FISss G r a n t 88/0994. ception in the carotid body: possible models. In H. Acker and R.G. O'Regan (Eds.), Physiology of the Peripheral Arterial Chemoreceptors, Elsevier, Amsterdam, 1983, pp. 107-220. 7 Bernath, S. and Vizi, E.S., Inhibitory effect of ionized free intracellular calcium enhanced by ruthenium red and mchloro-carbonylcyanide phenyl hydrazon on the evoked release of acetylcholine, Biochem. Pharmacol., 36 (1987) 3683-3687. 8 Biscoe, T.J., Purves, M.J. and Sampson, S.R., The frequency of nerve impulses in single carotid body chemoreceptor afferent fibres recorded in vivo with intact circulation, J. Physiol. (Lond.), 208 (1970) 121-131. 9 Brown, A.M., ATP and ATPase determinations in red blood cells. In J.C. Ellory and J.C. Young (Eds.), Red Cell Membranes: A Methodological Approach, Academic, London, 1982, pp. 223-238. 10 Cardenas, H. and Zapata, P., Ventilatory reflexes originated from carotid and extracarotid chemoreceptors in rats, Am. J. Physiol., 244 (1983) Rl19-R125.
257 11 Ernster, L. and Nordenbrand, K., Skeletal muscle mitochondria. In R. W. Estabrook and M.E. Pullman (Eds.), Methods in Enzymology Vol. 10, Academic, New York, 1967, pp. 86-94. 12 Farnebo, L.O., Release of monoamines evoked by field stimulation. Studies on some ionic and metabolic requirements, Acta Physiol. Scand., suppl., 371 (1971) 19-27. 13 Fidone, S. and Gonzalez, C., Initiation and control of the chemoreceptor activity in the carotid body. In A.P. Fishman (Ed.), Handbook of Physiology, The Respiratory System, Vol. 11, Part L Amer. Physiol. Soc., Bethesda, Maryland, 1986, pp. 247-312. 14 Fidone, S.J., Gonzalez, C. and Yoshizaki, K., Effect of low oxygen on the release of dopamine from the rabbit carotid body in vitro, J. Physiol. (Lond.), 33 (1982) 93-110. 15 Fleming, R.M. and Clark, W.G., Quantitative thin-layer chromatographic estimation of labeled dopamine and norepinephrine, their precursors and metabolites, J. Chromatogr., 52 (1970) 305-312. 16 Glynn, I.M., An ATPase from electric organ activated by Na+-K + and inhibited by ouabain or oligomycin, Biochem. J., 84 (1962) 75P. 17 Godfraind, J.M., Kawamura, M., Krnjevic, K. and Pumain, R., Dinitrophenol and some other metabolic inhibitors on cortical neurones, J. Physiol. (Lond.), 215 (1971) 199-222. 18 Gonzalez, C. and Garcfa-Sancho, J., A sensitive assay for ATP, Anal. Biochem., 114 (1981) 285-287. 19 Grinstein, S. and Furuya, W., Cytoplasmic pH regulation in phorbol ester-activated human neutrophils, Am. J. Physiol., 251 (1986) C55-C65. 20 Grinstein, S. and Rothstein, A., Mechanisms of regulation of the Na+-H ÷ exchanger, J. Membrane Biol., 90 (1986) 1-12. 21 Hayashida, Y. and Eyzaguirre, C., Voltage noise of carotid body type I cells, Brain Research, 167 (1979) 189-194. 22 Heymans, C., Bouckaert, J.J. and Dautrebande, L., Sinus carotidien et r6flexes respiratoires; sensibilit6 des sinus carotidiens aux substances chimiques. Action stimulante respiratoire r6flexe du sulfure de sodium, du cyanure de potassium, de la nicotine et de la lobeline, Arch. Int. Pharmacodyn. Ther., 40 (1931) 54-91. 23 Hornbein, T.F., The relation between stimulus to chemoreceptors and their response. In R.W. Torrance (Ed.), Arterial Chemoreceptors, Blackwell, Oxford, 1968, pp. 65-78. 24 Huang, H. and Daly, J.W., Interrelationships among the levels of ATP, adenosine and cyclic AMP in incubated slices of guinea pig cerebral cortex: effects of depolarizing agents, psychotropic drugs and metabolic inhibitors, J. Neurochem., 23 (1974) 393-404. 25 Joels, N. and Neil, E., The idea of a sensory transmitter. In. R.W. Torrance (Ed.), Arterial Chemoreceptors, Blackwell, Oxford, 1968, pp. 153-178. 26 Kirpekar, S.M., Prat, J.C. and Yamamoto, H., Effects of metabolic inhibitors on norepinephrine release from the perfused spleen of the cat, J. Pharmacol. Exp. Ther., 172 (1970) 342-350. 27 Krylov, S.S. and Anichkov, S.V., The effect of metabolic inhibitors on carotid chemoreceptors. In R.W. Torrance (Ed.), Arterial Chemoreceptors, Blackwell, Oxford, 1968, pp. 103-113. 28 Ksiezak, H.J. and Gibson, G.E., Oxygen dependence of glucose and acetylcholine metabolism in slices and synaptosomes from rat brain, J. Neurochem., 37 (1981) 305-314. 29 Loomis, W.F. and Lipman, F., Reversible inhibition between phosphorylation and oxidation, J. Biol. Chem., 173 (1948) 807-808.
30 Mulligan, E. and Lahiri, S., Mitochondrial oxidative metabolism and chemoreception in the carotid body. In C. Belmonte, D. Pallot, H. Acker and S. Fidone (Eds.), Arterial Chemoreceptors, Leicester University Press, Leicester, U.K., 1981, pp. 316-326. 31 Mulligan, E., Lahiri, S. and Storey, B.T., Carotid body 02 chemoreception and mitochondrial oxidative phosphorylation, J. Appl. Physiol.: Respirat. Environ. Exercise Physiol., 51 (1981) 438-446. 32 Mulligan, E. and Lahiri, S., Separation of carotid body chemoreceptor responses to O 2 and CO 2 by oligomycin and by antimycin A, Am. J. Physiol., 242 (1982) C200-C206. 33 Obeso, A., Almaraz, L. and Gonzalez, C., Correlation between adenosine triphosphate levels, dopamine release and electrical activity in the carotid body: support for the metabolic hypothesis of chemoreception, Brain Research, 348 (1985) 64-68. 34 Obeso, A., Almaraz, L. and Gonzalez, C., Effects of 2deoxy-D-glucose on in vitro cat carotid body, Brain Research, 371 (1986) 25-36. 35 Obeso, A., Gonzalez, C., Dinger, B. and Fidone, S., Energy metabolism in the rabbit carotid body: biochemical and autoradiographic findings using the 2-deoxy-glucose technique 'in vitro', Neurosci. Lett., submitted. 36 Ponte, J. and Sadler, C.L., Effects of oligomycin upon carotid body chemoreceptors of the cat and rabbit, J. Physiol. (Lond.), 371 (1986) l12P. 37 Rigual, R., Gonzalez, E., Gonzalez, C. and Fidone, S., Synthesis and release of catecholamines by the cat carotid body in vitro: effects of hypoxic stimulation, Brain Research, 374 (1986) 101-109. 38 Rubin, R.P., Calcium and Cellular Secretion, Plenum, New York, 1982. 39 Rutledge, C.O., Effect of metabolic inhibitors and ouabain on amphetamine- and potassium-induced release of biogenic amines from isolated brain tissue, Biochem. Pharmacol., 27 (1978) 511-516. 40 Shen, T.C.R. and Hauss, W.H., Influence of dinitrophenol, dinitroartocresol and paranitrophenol upon the carotid sinus chemoreceptors of the dog, Arch. Int. Pharmacodyn. Ther., 63 (1939) 251-258. 41 Shirahata, M., Andronikou, S. and Lahiri, S., Differential effects of oligomycin on carotid chemoreceptor responses to 02 and CO 2 in the cat, J. Appl. Physiol., 63 (1987) 2084-2092. 42 Sokoloff, L., Relation between physiological function and energy metabolism in the central nervous system, J. Neurochem., 29 (1977) 13-26. 43 Torrance, R.W., Prolegomena. In R.W. Torrance (Ed.), Arterial Chemoreceptors, Blackwell, Oxford, 1968, pp. 1-40. 44 Vizi, E.S., Stimulation by inhibition of Na +, K +, MG ++ activated ATPase of acetylcholine release in cortical slices from rat brain, J. Physiol. (Lond.), 226 (1972) 95-117. 45 Vizi, E.S., Release mechanisms of acetylcholine and the role of Na+-K + activated ATPase. In P.G. Waser (Ed.), Cholinergic Mechanisms, Raven, New York, 1975, pp. 199-211. 46 Whittam, R., Wheeler, K.P. and Blake, A., Oligomycin and active transport reactions in cell membranes, Nature (Lond.), 203 (1964) 720-724. 47 Wray, S., Smooth muscle intracellular pH: measurement, regulation, and function, Am. J. Physiol., 254 (1988) C213-C225. 48 Yarowsky, P. and Ingvar, O.H., Neuronal activity and energy metabolism, Fed. Proc., 40 (1981) 2353-2362.