Effects of fluoride and cholera and pertussis toxins on

Effects of fluoride and cholera and pertussis toxins on sensory transduction in the carotid body

T. G. CACHERO, A. ROCHER, R. J. RIGUAL, AND C. GONZALEZ Departamento de Bioqufmica y Biologia Molecular y Fisiologfa, Facultad de Medicina, Universidad de Valladolid, 47005 Valladolid, Spain

Cachero, ‘I?. G., A. Rocher, R. J. Rigual, and C. Gonzalez. Effects of fluoride and cholera and pertussis toxins on sensory transduction in the carotid body. Am. J. Physiol. 269 (Cell physiol. 38): C1271-C1279, 1995.-The regulation of the chemoreceptor cell function by G proteins has been studied by measuring the release of 3H-labeled catecholamines ([3H]CA) in carotid bodies (CBS) treated with fluoride, cholera toxin (CTX), and pertussis toxin (PTX). Fluoride augmented the basal release of [3H]CA in a dose- (5-20 mM) and Ca2+- dependent manner. Nisoldipine (1 PM) and ethylisopropyl amiloride (EIPA; 10 PM) inhibited this effect by -6O%, and both drugs combined inhibited it in full. BAY K 8644 (1 FM) doubled the effect of fluoride. The effects of fluoride on the stimulus-evoked release of L3H]CA varied with the type of stimulus and the duration of the treatment. Simultaneous application of fluoride with the stimulus increased by five times the release evoked by hypoxia and by two times that by K+ and dinitrophenol (DNP). Preincubation with fluoride for 1 h caused an inhibition (- 70%) of the release evoked by high K+ and veratridine, whereas that evoked by DNP and low PO, was still augmented ( - 2 times). Preincubation (4 h) of the CBS with CTX (3 kg/ml) reduced by 54% the release of [3H]CA evoked by 35 mM KS but did not affect that evoked by low PO, or DNP. A similar treatment with PTX (1 Fg/ml) affected only the release of L3H]CA evoked by DNP, reducing it by 65%. The data show that fluoride, CTX, and PTX have different effects on the release of [3H]CA evoked by high external K+, DNP, and low PO,, indicating that the stimulus-secretion coupling process for each stimulus is differently regulated by G proteins.

chemoreception; catecholamines; G proteins; hypoxic and acidic transduction

THE MAMMALIAN CAROTID BODY (CB) is a composite sensory receptor in which chemoreceptor cells sense arterial blood PO, and Pco,/pH. At normal blood PO,, Pco~, and pH (basal conditions) chemoreceptor cells are releas- ing neurotransmitters at a low rate, and the action potential frequency in the CB sensory nerve, the carotid sinus nerve (CSN), is correspondingly low (12). Hypoxia (low PO,) and acidosis (high Pco,-low pH), which are the natural stimuli to the CB, activate chemoreceptor cells and induce a Ca 2+-dependent release of neurotransmitters [e.g., dopamine (DA)] that is paralleled by a propor- tional increase in the frequency of action potentials in the CSN (see Refs. 11 and 12 for reviews).

Sensory transduction in adult chemoreceptor cells, i.e., the coupling of natural stimuli to the release of DA, is different for hypoxia and for acidosis. Low PO, inhibits a K+ current in chemoreceptor cells (19), and activation of Na+ and Ca2+ channels, entry of Ca2+, and release of DA follow (22, 30; see also Refs. 11 and 12). Acidic stimulus transduction does not appear to produce depolarization or voltage-dependent channel activation in

adult chemoreceptor cells, because in intact CBS from adult animals neither Na+ nor Ca2+ channel blockers are effective in blocking the release of DA induced by acidic stimuli (22, 30, 31); it was proposed that the increase in intracellular H+ concentration produced by acidic stimuli would activate Na+-dependent H+-extruding exchang- ers, leading to a net gain of Na+ by the cell, entry of Ca2+ via the Na+ /Ca2+ exchanger, and the release of DA (11, 12, 31). In these studies it was also shown that dinitrophenol (DNP), which is a protonophore that acidifies intracellularly by bringing H+ to equilibrium across the plasma membrane (14), evokes a release of catecholamines (CA) from the adult rabbit CB with the same properties than the natural acidic stimulus (10, 31).

Contrary to those findings, in chemoreceptor cells from neonatal animals acidic stimuli inhibit the 02- sensitive K+ current (24) and produce entry of Ca2+ via voltage-dependent channels (4). In these neonatal cells it has been reported that DNP, at concentrations that do not acidify intracellularly (5), produces depolarization and activation of voltage-dependent Ca2+ channels. As a whole these findings would imply that hypoxic and acidic stimuli (and perhaps DNP) in neonatal chemoreceptor cells follow the same steps of coupling to the exocytotic machinery.

Contrasting with the relative abundance of neu- rochemical and electrophysiological data, which have allowed definition of the putative chains of events in the transduction process and characterization of the last step of transduction in the CB (i.e., the release of DA), very few data are available on the regulation of the transduction processes by intracellular messengers (see Ref. 12). Particularly striking is the absence of data on G proteins and CB chemoreception when it is known that such heterotrimeric proteins play crucial roles in the transduction process in other sensory receptors, includ- ing olfactory and gustatory chemoreceptors (1, 21), and in the control of the exocytosis of neurotransmitters (3, 23,34).

In the present paper we have directed our efforts to demonstrate the modulatory role of G proteins in the CB chemoreceptor function. We have measured the release of catecholamines (CA) from intact CBS of adult rabbits treated with NaF plus AlC& and pertussis or cholera toxins (PTX and CTX, respectively), because these chemicals have proved to be the most effective tools to test the implication of G proteins in specific functions in intact cells (9, 15, 33). In the presence of A13+, fIuoride (F-) forms AlF,, which interacts with the GDP bound to the a-subunit of G proteins and mimics the y-phosphate of GTP, thereby triggering the activation and dissocia- tion of G proteins in their a- and py-subunits (see Refs. 9 and 15). PTX, by ADP ribosylating the a-subunits of the

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Cl272 G PROTEINS AND CAROTID BODY CHEMORECEPTION

sensitive G proteins (Gi, G,, and transducin), uncouples them from the receptors, leaving unregulated the-cell functions or the effecters normally controlled by those particular receptors (33). CTX catalyzes the ADP ribosylation of the cx-subunit of G, and transducin and leads to their permanent activation and to the stimulation of the cellular effecters (e.g., adenylate cyclase) under their control (9, 15, 33). We have found that NaF plus A1C13 treatment induced a concentration and Ca2+-dependent release of CA from the chemoreceptor cells of CBS incubated in basal conditions. NaF plus A1C13, PTX and CTX differentially modulated the release of CA induced by hypoxic and acidic stimuli, reinforcing the notion that the transduction of both stimuli follows separate pathways.

MATERIALS AND METHODS

CBS were isolated from adult (1.5-2.5 kg) New Zealand rabbits anesthetized with pentobarbital sodium (30-40 mg/kg iv) administered through the lateral vein of the ear. After an incision in the ventral surface of the neck, the animals were tracheostomized, and blocks of tissue containing the carotid bifurcations were removed and placed in a Lucite chamber filled with ice-cold 100% Oa-equilibrated N-2-hydroxyethylpi- perazine-W-2-ethanesulfonic acid (HEPES)-buffered Tyrode solution (see below for composition); the CBS were cleaned of surrounding connective tissue under a dissecting microscope and maintained in ice-cold 100% Oz-equilibrated HEPES- buffered Tyrode solution until their incubation to label their CA stores.

The CA stores of chemoreceptor cells were labeled by incubating the organs in a shaker bath at 37°C for 2 h in glass minivials containing [3,5-3H]tyrosine (20 PM, 30 Ci/mmol), DL-6-methyl5,6,7,8tetrahydropterine (100 PM), and ascorbic acid (1 mM) in 0.5 ml of 100% Oz-equilibrated HEPES- buffered Tyrode solution (in mM: 140 NaCl, 5 KCl, 2 CaC12,1.1 MgCla, 10 HEPES, and 5 glucose, pH adjusted to 7.40 with 1 N NaOH). Most commonly, individual experiments were performed with six or eight CBS from three or four animals, and the right CBS were incubated in a vial and the left CBS in another. In these incubating conditions the CB synthetizes - 8-10 pmol/organ of L3H]CA [equivalent to - lo5 counts/ min (cpm)], most of which (> 90%) is [3H]DA (7, 30). Thereaf- ter, individual CBS were transferred to glass scintillation vials containing 4 ml of bicarbonate-buffered precursor-free Tyrode solution (in mM: 116 NaCl, 5 KCl, 2 CaC12, 1.1 MgClz, 10 HEPES, 5 glucose, and 24 NaHC03, equilibrated with 5% COZ-20% 02-75% N2, pH 7.40). The solution was renewed every 30 min for 2 h and discarded. Finally, we initiated the collection of incubating solutions, according to specific protocols (see RESULTS) for their analysis in labeled CA content. This general protocol was slightly modified in those experiments in which PTX and CTX were used. In these experiments, after the CA deposits were labeled by incubating with [“Hltyrosine, the organs were incubated for 4 h in precursor- free Tyrode solution, with or without the appropriate toxins, renewing the solution each hour. Thereafter, the collection of the incubation media for their content in CA proceeded according to protocols similar to those of previous experiments (see RESULTS). When the incubating solution contained high KC1 and/or NaF, equiosmolar amounts of NaCl were sub- tracted. AlC13 (20 PM) was added to all fluoride-containing solutions. The duration of the experiments and the intensity of the stimuli used were such that in most cases the amount of [3H]CA present in the tissue at the end of the experiment was

> 80% of the total amount synthetized. Nonetheless, the experiments were designed in such a manner that the control and experimental CBS were stimulated at the same time during the experiment to eliminate or minimize differences in the release response due to unequal sizes of the pool(s) of the labeled CA.

The collected incubating solutions were acidified to pH 3.2 with glacial acetic acid, and ascorbic acid was added (1 mM final concn) to prevent the degradation of the released [3H]CA. Analysis included adsorption onto aluminum oxide at pH 8.6 achieved by addition of 2 M tris(hydroxymethyl)aminometh- ane (Tris) buffer at this pH, extensive washing of the alumina with distilled water, and batch elution of the [3H]CA with 1 N HCl (13). In selected experiments the collected samples were subjected to an ulterior identification using a reverse-phase chromatography system (13). It was verified that over 95% of the L3H]CA released under every experimental condition was L”H]DA plus its catabolite [3H]dihydroxyphenyl acetic acid, indicating that the [3H]CA are released from chemoreceptor cells, the DA-containing structure in the rabbit CB (12).

Results of the release experiments are expressed as counts per minute of [3H]CA present in the alumina eluates of each collected sample. The total release evoked by a given stimulus (cpm) is sometimes related either to the release obtained in basal conditions or to the tissue content. The statistical significance of the differences observed was assessed by the use of the two-tailed Student’s t-test for unpaired data.

The efficiency of the treatments with the CTX and PTX was assessed by measuring the [32P]ADP ribosylation produced by these toxins in homogenates from CBS preincubated in the absence or the presence of the toxins for the time and concentration used in the release experiments. The [32P]ADP ribosylation of homogenates catalyzed by PTX and CTX was performed as described by Ribeiro-Neto et al. (28). Briefly, after groups of control and toxin-preincubated CBS were homogenized in buffer containing 20 mM Tris l HCl, 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride (pH 7.5), 20-~1 aliquots of the homogenates ( N 100 pg of protein) were incubated for 30 min at 32°C with preactivated CTX (100 pg/ml, final concn) or PTX (10 pg/ml, final concn). The reaction mixture (60 ~1; pH 7.0) also contained (final concn) 10 mM MgCl2, 1 mM ATP, 1 mM EDTA, 10 mM thymidine, 15 mM Tris HCl, and 10 PM NAD+ containing 1 l&i [32P]NAD+. The reaction was stopped by the addition of 1.0 ml of ice-cold 20% trichloroacetic acid (TCA). Membranes were pelleted, washed twice with ethyl ether, resuspended in Laemmli’s sample buffer (18), boiled for 2 min, and subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE; 10% polyacrylamide). The dried gels were autoradiographed by exposure to Kodak X-AR film with an enhancing screen for 24 h at - 70°C. Densitometry was performed with a PDI-Pharmacia densitometer with Discovery software (series 3.0).

Materiak. [3,5-3H]tyrosine (40-60 Ci/mmol) was purchased from Amersham Iberica (Spain), and [32P]NAD+ ( > 800 Ci/mmol) was from Du Pont-New England Nuclear (Itisa, Madrid, Spain); PTX was obtained from Research Biochemi- cals and CTX from Calbiochem-Behring. Electrophoresis re- agents were purchased from Bio-Rad (Richmond, CA). Alumi- num oxide was purchased from Serva, and all other chemicals were of analytical grade and obtained from Sigma.

RESULTS

Effect of fluoride on basal release of catecholamine. Incubation of CBs in the presence of 10 mM NaF plus 20 FM A1C13 (AlF,) produced an increase in the ongoing

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basal release of CA, which was already noticeable during the initial 10 min and reached a peak at - 20-30 min; the complete time course of the response is shown in Fig. 1A. Aluminum chloride alone did not modify the release of CA, and incubation in the presence of 10 mM NaF plus 200 PM deferoxamine to chelate the tracer content of aluminum present in the incubating solutions resulted in a slower, smaller, and transient increase in the release of CA (data not shown); therefore AlC& at 20 PM was included in all NaF-containing solutions of subsequent experiments. The effect of NaF was dose dependent from 5 to 20 mM (Fig. 1B). Lower concentrations (2 mM) were ineffective, and the use of higher concentrations of NaF with concentrations of Ca2+ in the medium above 1 mM was unreliable due to precipitation of calcium fluoride.

In many cellular systems it has been reported that fluoride by activating G proteins can induce mobiliza- tion of Ca2+ from intracellular deposits and/or activation of pathways for Ca2+ entry from the extracellular milieu. The net result is that cellular responses trig- gered by increased cytoplasmic levels of free Ca2+ are activated (26, 36). Figure 2A shows that the release of CA induced by fluoride is totally dependent on the presence of extracellular Ca2+. This suggests that the most significant action for the release of CA resulting from the activation of G nroteins in chemorecentor cells

lo- A

6-

Time (min)

I M

0 ’ I I I

5 mM 10mM 20mM

Fig. 1. Effects of fluoride on basal release of 3H-labeled catecholamines [3H]CA. A: histogram represents time course of release of [3H]CA in a group of 4 carotid bodies (CBS) incubated in normal bicarbonate-buffered Tyrode solution (pH 7.40; POT - 140 mmHg). Each column corresponds to a lo-min period. Starting at arrow, incubating solution contained 10 mM NaF + 20 PM AlC13. Line defined by open circles corresponds to basal spontaneous release obtained in contralateral CBS incubated in absence of fluoride. B: bars correspond to quotients evoked release/basal release of [3H]CA for 1st h of incubation in presence of indicated concentrations of NaF. All solutions containing NaF also contained 20 PM AlC13. Means + SE of 8 data from 4 different experiments.

0 1 I I I I

0 20 40 60 80

Time (min)

I ‘9

Control Nis. EIPA Nis.+EIPA

Fig. 2. A: calcium dependence of release of [3H]CA elicited by fluoride. 0, Release of [3H]CA obtained in a group of CBS incubated in control solution containing, starting at arrow, 10 mM NaF; l , release obtained in contralateral organs incubated identically except for absence of Ca2+ in solution. In both cases data are means + SE of 6 individual data from 2 independent experiments. B: effect of nisoldipine (Nis) and ethylisopropyl amiloride (EIPA) on release of [3H]CA induced by fluoride. Bars correspond to quotients fluoride-evoked release/basal release in control CBS, in CBS incubated in presence of 1 PM Nis introduced 10 min. before fluoride, in organs incubated in presence of 10 PM EIPA also introduced 10 min before fluoride, and in CBS incubated in presence of both drugs. In all cases organs were incubated with 10 mM NaF for 1 h. Data are means + SE of 6 individual values obtained in 3 independent experiments for each treatment. Compared with control group: *P < 0.05; **P < 0.01; ***icp < 0.001.

is the opening of pathways for Ca2+ entry from the extracellular space. Consistent with this suggestion are the data of Fig. 2B showing that 1 FM nisoldipine (a blocker of L-type Ca 2+ channels) and 10 PM ethylisopropyl amiloride (EIPA; a blocker of Na+ /H+ exchanger and indirectly an inhibitor of influx of Ca2+ via Na+/Ca2+ exchanger) blocked the release response elicited by 10 mM fluoride by 60 and 59%, respectively. Both blockers in combination inhibited the release of CA elicited by fluoride by 97%.

Figure 3 shows that 1 FM BAY K 8644 (a dihydropyridine agonist of L-type Ca 2+ channels), which by itself did not affect the basal ongoing release of CA (22), increased the release of [3H]CA induced by fluoride by a factor of 2.4, from 2,470 t 460 to 5,950 * 330 cpm. This finding is consistent with the action of nisoldipine shown above to indicate that fluoride activates Ca2+ channels.

Effects of fluoride on the release of catecholamines induced by different stimuli. To study the effect of fluoride on the release of [3H]CA elicited by high external 35 mM EC+, 100 PM DNP, and low PO, solutions (equilibrated with 7% 0,; PO, -45 mmHg), we have followed the experimental protocol shown in Fig. 4. In this particular experiment the stimulus consisted of the

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Fig. 3. Effects of BAY K 8644 on release of [3H]CA elicited by fluoride. Left: time course (each bar corresponds to 10 min) of mean release of [3H]CA in a group of 10 CBS elicited by 10 mM NaF introduced in incubating solution at arrow. Middle: similar experiment with 4 CBS in which 1 ~J-M BAY K 8644 was introduced at discontinuous arrow, 10 min before fluoride (continuous arrow). Note that BAY K 8644 did not modify basal release of ]3H]CA lo-min period that was alone in incubating solution. Right: total release evoked by fluoride (F) and fluoride + BAY K 8644 (F + BAY K). Data are means 2 SE. ***P < 0.001.

incubation of the CBS for 10 min with a solution smaller release of [“H]CA than in the cat CB (29). containing 35 mM K+. In the control CB (Fig. 4A) the Therefore, to obtain a significant release of [3H]CA stimulus was applied twice (S, and S,; solid bars), and the experimental CB was similarly . stimulated twice, but the incubating solution contained 10 mM fluoride starting at the arrow. Figure 5 summarizes the results obtained for the three stimuli.

From Fig. 5A it is evident that simultaneous introduction of fluoride with the depolarizing stimulus (S,) resulted in an augmentation of the release elicited by high external K+ by a factor of 2.2 (from 3,024 t 493 to 6,666 t 1,709 cpm; n = 8; P < 0.05). This finding is also consistent with the notion that fluoride activates Ca2+ channels because the release induced by high external K+ is totally dependent on the influx of Ca2+ via voltage-dependent Ca 2+ channels Contrary to that, the . release evoked by 35 mM K+ in the second presentation of the stimulus (S2) was reduced by 58% in the CBS that were exposed to fluoride for 1 h. Similar findings have been reported in other preparations in which the effects of fluoride on Ca2+ channels are time dependent so that after an initial activation there is a “desensitization” of L-type channels (26,27).

above basal, we are forced to use very intense acidic stimuli. On the other hand DNP, as expected from its mechanism of intracellular acidification (14), produces a more intense release response, which is easier to evalu- ate. Figure 5B shows that the release response elicited by 100 pM DNP (1,810 t 392 cpm; n = 8) was approximately doubled by fluoride (3,696 t 588 cpm; n = 8) when both were applied simultaneously (SJ; the same effect was observed in the second stimulation with DNP (S2) when the CBS had been exposed to fluoride for 1 h (1,547 t 359 vs. 2,924 t 439 cpm).

In the cat CB, natural acidic stimuli are less effective in increasing the action potential frequency in the CSN than hypoxic stimuli (8). In the rabbit CB acidic stimuli appear to be even less effective because they evoke a

Figure 5C shows the effect of fluoride on the release of CA elicited by a hypoxic stimulus of moderate intensity (incubating solution equilibrated with 7% 0,; PO, - 45 mmHg). The release response elicited by hypoxia was increased by a factor of 5.7 (from 1,690 t 386 to 9,661 t 619 cpm; n = 4; P < 0.001) when fluoride was introduced simultaneously with the hypoxic stimulus (S,); a smaller but yet very marked (2.4 x ; P < 0.01) increase in the release of CA was observed in the second presentation of the hypoxic stimulus when the CBS had been exposed to fluoride for 1 h (S,).

2.5 2.5 1

The possibility exists that the effects attributed to fluoride during the second presentation of the stimuli (Fig. 5) are exaggerated or attenuated by the changes in the size of the pools of [“H]CA due to the release response elicited by the first presentation of the stimuli. To explore this possibility we performed a new group of experiments following the protocol shown in Fig. 6. In these experiments the release elicited by a given stimulus (35 mM K+ in experiment shown in Fig. 6) in one CB previously incubated for 1 h with control solution is compared with that obtained in the contralateral CB similarly stimulated after l-h incubation with 10 mM fluoride. The results for the different stimuli tested are expressed as percentage of the total tissue content of [3H]CA and summarized in Table 1. Notice that fluoride stimulated the release of CA evoked by 7% O2 and 100

Fig. 4. Effect of fluoride on release of [“H]CA evoked by high external FM DNP and inhibited the release evoked bv 35 mM K+ K+; single experiment. A: time course (each bar corresponds to a lo-min incubation period) of release of CA obtained in 1 control CB,

by magnitudes comparable to those observed with the

which was stimulated by incubation with 35 mM K+ twice (solid bars, alternative protocol shown in Fig. 5. Using this protocol Si and S,). B: same for contralateral CB that was incubated in we have also tested the effects of fluoride on the release presence of 10 mM fluoride starting at arrow. of CA induced by low pH-high Pco~, 75 mM K+, and 30

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Fig. 5. Effects of fluoride on release of CA elicited by different stimuli. Experimental protocols as in Fig. 4. A: release of CA elicited by 35 mM K+ in control CBS (open bars) was augmented by fluoride (hatched bar) when it was introduced simultaneously with high K+ (2.2~; Si), and was reduced by 58% when fluoride was acting for 1 h before high K+ stimulation (Sz). Data are means * SE from 8 individual data from 2 independent experiments. B: release elicited by 100 FM DNP was similarly augmented by fluoride (hatched bars) in S1 (1.9 x ) and Sz (1.9 x ). Data are means * SE of 8 individual values from 2 experiments. C: release elicited by incubating CBS in a hypoxic solution equilibrated with 7% 02 was greatly augmented (5.7 X) by fluoride (hatched bar) when it was introduced simultaneously with hypoxic stimulus (Si) and less markedly (2.4~) when fluoride was acting for 1 h (S&. Means t SE of 4 data from 1 experiment. *P < 0.05; **P < 0.01; ***P < 0.001.

PM veratridine (an activator of voltage-dependent Na+ ments were pooled (n = 24), the increase amounted to channels; Table 1). Notice also that fluoride augmented 35% (P < 0.05). The release of CA elicited by hypoxia the response elicited by the natural acidic stimulus by a and high external K+ was not significantly affected by percentage comparable to that observed for the response the pretreatment with PTX but that elicited by DNP elicited by DNP and inhibited by a similar percentage was inhibited by 65% (Fig. 7B). the release elicited by the Na+ channel activator and by Figure 8 shows that the protocol of incubation with 75 mM K+, which does not involve the participation of toxins followed in the release experiments was effective Na+ channels (30); the latest findings seem to exclude a in promoting ADP ribosylation of G proteins in intact significant effect of the treatment with fluoride on Na+ cells. In Fig. 8A, it is evident that in homogenates of channels. untreated CBS CTX induces [32P]ADP ribosylation of

Effects of CTX and PTX on release of catecholamines two protein bands with apparent molecular mass of 42 elicited by severaL stimuli. The protocols for stimulus and 45 kDa, which completely disappeared when the application and sample collection in these experiments CBS were preincubated with CTX (4 h, 3 kg/ml) before were similar to those shown in previous figures (see homogenization. This finding means that, during the MATERIALS AND METHODS), but a single stimulus was 4-h period of incubation of the intact CB with CTX, the applied to each CB. Preincubation of the CBS with CTX toxin has permeated the CB cells and promoted the ADP (3 kg/ml; 4 h) did not affect the release of CA induced by ribosylation of the CTX-sensitive G proteins with endog- a hypoxic stimulus of intermediate intensity (PO, of incubating solution -33 mmHg; 10 min) or DNP (100 PM; 10 min), but inhibited by 54% the release response Table 1. Effect of l-h treatment with NaF on release of elicited by high external K+ (35 mM; 10 min; Fig. 7A). L3HjCA elicited 63’ different stimuli The basal release of CA was not affected by CTX.

Preincubation of the CBS with PTX (1 pg/ml; 4 h) Evoked Release, % content

Fluoride/

tends to increase the basal release of CA, although the effect was variable. When the data from all the experi-

Stimuli Control + Fluoride Control

7% 02 1.36 + 0.38 3.27 + 0.23 2.40*

0 I I 0

0 40 80 120 min 0 t 40 80 120 min

Fig. 6. Effect of preincubation with fluoride on release of [3H]CA elicited by high external K+. Single experiment. A: time course of release of CA in a control CB stimulated by incubation with 35 mM K+ during 10 min (solid bar). B: similar time course for contralateral CB similarly stimulated with high K+ but incubated in presence of 10 mM fluoride starting at arrow.

(4) (4) 20% CO2 (pH 6.8) 0.12 t 0.01 0.22 AI 0.02 1.83*

(6) (6) DNP (100 FM) 2.35 2 0.44 4.60 * 0.60 1.95’”

03) (6) [K+l,

35 mM 2.96 -F 0.54 1.26 + 0.26 0.42’” (7) (10)

75 mM 13.91+ 2.01 3.64 + 0.53 0.26* (3) (3)

Veratridine (30 PM) 0.59 -t 0.15 0.15 + 0.04 0.25”f (3) (3)

Values are means + SE for no. of measurements in parentheses. Experimental protocols are as in Fig. 6. In all cases stimulus was applied during 10 min, except for veratridine, which was applied for 3 min. Evoked release is expressed as percentage of [3H]catecholamines (CA) present in the tissue at the end of the experiment. DNP, dinitrophenol; [K+],, external K+ concentration. Last column (Fluo- ride/Control) refers’to quotient between 2 previous columns. *P < 0.01. “fP < 0.05.

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Fig. 7. Effects of cholera and pertussis toxins (CTX and PTX, respectively) on release of 13H]CA. A: effects of preincubation during 4 h (hatched bars) with CTX on release of [3H]CA elicited by low PO, (incubation during 10 min in a solution equilibrated with a gas mixture containing 5% Oa), dinitrophenol (DNP, 100 )IM, 10 min) and high external K+ (35 mM: 10 min). B: similar aroun of exoeriments in which CBS were preincubated with PTX. Open-bars correspond to CBS similarly preincubated during 4 h in absence of toxins. In both graphs evoked release by 3 stimuli are expressed as times basal (evoked release/basal release). In all cases data are means ? SE of 6-10 individual values obtained in at least 2 independent experiments for each stimulus. ***P < 0.001.

enous unlabeled NAD+; when the tissues are homog- enated and incubated with the labeled substrate, no CTX-sensitive G protein is available for ADP ribosylation. Figure 8B shows that in homogenates of untreated CBS PTX promotes the ADP ribosylation of two protein bands of 39 and 40 kDa and that these bands were markedly reduced (by 80%) when the CBS were preincubated with PTX (4 h, 1 p,g/ml). As was the case with CTX, the findings indicate that PTX has permeated the cells and promoted ADP ribosylation of sensitive G proteins with endogenous unlabeled NAD+.

DISCUSSION

The main aim of the present experiments was to determine whether the integrated function of chemoreceptor cells studied through the release of neurotransmitters is regulated by G proteins. The data obtained with fluoride indicate that the release of CA induced by several stimuli is differentially affected by this general activator of G proteins. The findings suggest that the most significant effects of G proteins take place at the initial steps of the stimulus-secretion coupling process, more specifically that the pathways for Ca2+ entry in chemoreceptor cells are strongly regulated by G proteins. However, the moderate increase in the basal

release observed in the PTX-treated CBS is compatible with the existence in chemoreceptor cells of a mechanism inhibitory of the exocytosis that is sensitive to PTX (3,23, 34). This mechanism should be less developed in chemoreceptor cells than in the adrenomedullary cells, because in these latter cells PTX increases the release induced by all secretagogues, whereas in the CB it does not increase the release induced by hypoxia or high K+ and inhibits that elicited by DNP. These specific effects of PTX on the release of CA induced by the different stimuli and also those of CTX are consistent with the general conclusion, i.e., that the pathways for Ca2+ entry, or steps before that, are strongly modulated by G proteins. If the final steps of the stimulus-secretion coupling process (i.e., exocytotic machinery) were under strong control by G proteins, it should be expected that the release produced by all the stimuli would be modulated in the same direction. In this context, the effects of PTX, i.e., a marked inhibition of the release response evoked by DNP and a lack of effect on the release evoked by hypoxia, would indicate that the stimulus-secretion coupling processes for DNP and hypoxia are different. This conclusion is consistent with previous results showing that DNP, as well as natural acidic stimuli, induces a release of [3H]CA from the adult rabbit CB, which, contrary to low Paz-induced release, is not affected by agonists or antagonists of voltage-dependent Ca2+ channels (22) or tetrodotoxin (30). As a whole the present data reinforce the proposals of the existence of different transduction cascades for hypoxic and acidic

kDa

\ 4 42

C CTX B

C PTX

Fig. 8. [32PlADP ribosylation by cholera and pertussis toxins in CB homogenates. A: groups of 8-12 CBS were preincubated during 4 h in absence (control, C) or presence of CTX (3 kg/ml) and then homogenized; aliquots containing 100 kg of protein were incubated with [32PlNAD+ and 100 kg/ml CTX for 30 min at 32°C and subjected to SDS-polyacrylamide gel electrophoresis (PAGE; 10% polyacrylamide) and autoradiographed (exposure time, 48 h). Arrowheads indicate molecular mass. B: similar groups of CBS were preincubated during 4 h in absence (C) or presence of PTX (1 pg/ml) and then homogenized; duplicate aliquots containing 100 Kg of protein were incubated with 132P1NAD+ and 10 pgiml of preactivated PTX for 30 min at 32°C and subjected to SDS-PAGE, as described in A.

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stimulus in chemoreceptor cells of adult animals (l&12; see introduction).

The Ca2+ dependence of the release of CA evoked by fluoride, its sensitivity to blockers and activators of L-type Ca2+ channels, and the potentiation of the release of CA induced by high external K+, which is fully dependent on the entry of Ca”+ via L-type Ca2+ channels (22), indicate that fluoride is capable of activating Ca2+ channels in both resting and stimulated cells. Findings comparable to ours have been reported previously. For example, in resting smooth muscle cells, fluoride and guanosine 5’-0-(3-thiotriphosphate) induce contrac- tions that are Ca 2+ dependent and nifedipine sensitive (35, 36). Because there are many G proteins that either directly or indirectly regulate Ca2+ channels in other cell types (see Ref. 16), our data do not allow us to envision the identity of the G protein(s) involved in the activation of Ca2+ channels in chemoreceptor cells. We can exclude the participation of a G, protein-adenosine 3’,5’-cyclic monophosphate (CAMP)-protein kinase A system because neither forskolin nor dibutyryl-CAMP augments the release of CA elicited by high K+ in the CB (25) or the Ca2+ currents recorded in isolated chemoreceptor cells (20) and because fluoride decreases the levels of CAMP in the CB (unpublished observations).

(6); the gain in Na+ would promote the entry of Ca2+ by reversal of the Na+/Ca”+ exchanger (2,17,31). The PTX inhibition of the release of CA elicited by DNP is consistent with this interpretation.

The effects of fluoride, CTX and PTX on the release of CA induced by the hypoxic stimulus are difficult to interpret. According to current views (see introduction), hypoxia depolarizes chemoreceptor cells and thereby activates Ca2+ channels; Ca2+ influx and the release of CA follow (22). Similarly, high external K+ depolarizes chemoreceptor cells and recruits Ca2+ channels, causing influx of Ca2+ and the release of CA (22). However, the effects of fluoride, CTX, and PTX on the release of CA by chemoreceptor cells elicited by each of these depolarizing stimuli are markedly different (see Figs. 5 and 7, Table 1). These differences force two independent but interrelated questions: namely, what steps are different in the stimulus-secretion coupling process between hypoxia and high external K+ and how are these steps regulated by G proteins to generate such different release responses?

The inhibition of the release of CA induced by high K+ and veratridine observed after 1 h of treatment of the CBS with fluoride provides evidence that this general activator of G proteins has dual actions on the coupling of depolarizing stimuli to the exocytotic machinery. This inhibitory effect is specific because the same l-h treatment with fluoride potentiates the release of CA elicited by DNP, low pH and hypoxia. If the steps of the stimulus-secretion coupling process are taken into con- sideration (32), the data would indicate that the reduction of the release response is due to an inhibition of Ca2+ entry via voltage-dependent Ca2+ channels. We do not have clues on the mechanism involved in this inhibition of Ca2+ channels in chemoreceptor cells. A possibility is that fluoride by direct inhibition of phospha- tases produces an overphosphorylation and thereby a desensitization of Ca2+ channels (27); alternatively, the inhibition of Ca2+ channels might be mediated by G proteins (see Ref. 16) slowly recruited by fluoride. The present study does not allow one to distinguish between both alternatives. Because the treatment with CTX inhibits also the release of CA induced by high external K+, it may be speculated that chemoreceptor cells possess a G protein that, being CTX sensitive and slowly activated by fluoride, causes inhibition of Ca2+ channels.

The first obvious difference in the stimulus-secretion coupling process relates to the mechanisms generating the depolarization of chemoreceptor cells. In the case of high external K+, depolarization is produced by the experimental alteration of the K+ distribution across the plasma membrane, and, in the case of hypoxia, the depolarization is produced by the inhibition of the 02-sensitive K+ channels (12). It is therefore tempting to suggest that the differences observed are generated at the level of the 02-sensitive K+ channel. Upon application, fluoride increases by a factor of 5.7 the release response elicited by low PO, and by a factor of 2.4 that elicited by high external K+; 1 h of treatment with fluoride results in an augmentation of the release response elicited by hypoxia by a factor of 2.4 and a reduction of the release elicited by high external K+ of nearly 50%. From these differences it follows that fluoride, in addition to regulating the common steps to all depolarizing stimuli, should activate one or more G proteins, which potentiate the inhibition of the 02- sensitive K+ channels caused by the low PO, stimulus and thereby the potentiation of the hypoxic responses. The possibility exists that there are not yet defined steps in the hypoxic stimulus-secretion coupling process that are also specifically regulated by G proteins.

The effects of EIPA on the release of CA induced by fluoride, as well as the effect of fluoride on the release of CA evoked by DNP and low pH/high Pco~, are consistent with a G protein-mediated activation of the Na+ /H+ exchanger. In several cellular systems it has been reported that fluoride activates the Na+/H+ exchanger in a PTX-sensitive manner (6). Activation of the exchanger leads to an alkalinization of the cell interior with an increased gradient for H+ diffusion into the cell and increased extrusion of H+ in exchange with Na+ with the net result of a gain in intracellular Na+ concentration

Regarding the nature of these postulated G proteins it would appear that they cannot be substrates to PTX, because PTX does not affect the release response elicited by low PO, nor high external K+. These G proteins could be sensitive to CTX, because the treatment with this toxin inhibits the release of CA elicited by high external K+ and does not modify that induced by low PO,. This seems to imply that CTX activating some G proteins causes inhibition of the common steps to both stimuli, but additionally the toxin activates other G proteins that, acting on steps specific to the hypoxic stimulus- secretion coupling process, reverse that inhibition. How- ever, there are still differences between the effects of fluoride and CTX (cf. Figs. 5 and 7A), indicating that fluoride activates additional mechanisms, which, being -

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G protein dependent, are not affected by CTX. We cannot speculate on the precise nature of the G proteins nor on the second messengers involved. Despite the fact that CAMP inhibits the 02-sensitive K+ channels in a manner identical to low PO, (ZO), we can exclude CAMP as the mediator of the observed responses, because as mentioned above fluoride reduces markedly the levels of this nucleotide in all the experimental situations used in this work.

In summary, the present study shows that fluoride, PTX, and CTX exert profound and differential influ- ences in the stimulus-secretion coupling process trig- gered by depolarizing, hypoxic, and acidic stimuli, indicating that the stimulus-secretion coupling process for each stimulus is differently regulated by G proteins. The data provide evidence that even when the cellular response studied (release of CA) is multistep and the tools used are wide spectrum, it is possible in some instances to make reasonable inferences on both the step of the stimulus-secretion coupling process that is regulated and the type of the G protein involved. However, this pioneer work in the field of arterial chemoreception and G proteins requires a step-by-step dissection of the G protein function in the chemoreceptor cells of the CB. Such studies are essential require- ments to understand the process of sensory transduction in the CB.

We thank M. de 10s Llanos Bravo for technical assistance. This study was supported by Grant PB92/0267 of the Direction

General de Investigation Cientifica y Tecnica of Spain. T. G. Cachero is a predoctoral fellow of the Ministerio de Education y Ciencia of Spain.

Address reprint requests to C. Gonzalez.

20.

21.

22.

Received 25 July 1994; accepted in final form 25 May 1995. 23.

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