Hypoxia and acidosis increase the secretion of catecholamines in the neonatal rat adrenal medulla: an in vitro study

A. J. Rico, J. Prieto-Lloret, C. Gonzalez, and R. Rigual

Departamento de Bioquímica y Biología Molecular y Fisiología, Instituto de Biología y Genética Molecular, Facultad de Medicina, Universidad de Valladolid, Consejo Superior de Investigaciones Científicas, Valladolid, Spain

Submitted 19 January 2005 ; accepted in final form 5 August 2005


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Hypoxia elicits catecholamine (CA) secretion from the adrenal medulla (AM) in perinatal animals by acting directly on chromaffin cells. However, whether innervation of the AM, which in the rat occurs in the second postnatal week, suppresses this direct hypoxic response is the subject of debate. Opioid peptides have been proposed as mediators of this suppression. To resolve these controversies, we have compared CA-secretory responses with high external concentrations of K+ ([K+]e) and hypoxia in the AM of neonatal (1- to 2-day-old) and juvenile (14- or 15- and 30-day-old) rats subjected to superfusion in vitro. In addition, we studied the effect of hypercapnic acidosis on the CA-secretory responses in the AM during postnatal development and the possible interaction between acidic and hypoxic stimuli. Responses to high [K+]e were comparable at all ages, but responses to hypoxia and hypercapnic acidosis were maximal in neonatal animals. Suppression of the hypoxic response in the rat AM was not mediated by opioids, because their agonists did not affect the hypoxic CA response. The association of hypercapnic acidosis and hypoxia, mimicking the episodes of asphyxia occurring during delivery, generates a more than additive secretory response in the neonatal rat AM. Our data confirm the loss of the direct sensitivity to hypoxia of the AM in the initial weeks of life and demonstrate a direct response of neonatal AM to hypercapnic acidosis. The synergistic effect of hypoxia and acidosis would explain the CA outburst crucial for adaptation to extrauterine life observed in naturally delivered mammals.

hypercapnia; chemoreceptors; chromaffin cells


INNERVATION OF ADRENAL MEDULLA (AM) by the splanchnic nerve is not functional in most neonatal mammals, but high levels of plasma catecholamine (CA) or AM depletion have been demonstrated in the fetus and in neonates during hypoxic episodes associated with vaginal delivery in calves, lambs, rats, and humans (2, 5, 6, 18, 37; see also Refs. 8, 9). In fact, during labor, the fetal plasma CA level reaches its highest concentration in the mammal's whole life (19). CA activates several metabolic, cardiocirculatory, and ventilatory mechanisms essential to survival and adaptation to extrauterine life (19, 39).

In the rat, splanchnic innervation of the AM becomes functional during the second postnatal week. In this species, Slotkin and Seidler (39) demonstrated that hypoxic CA release from the neonatal (1–2 days old) AM was nonneurogenic because the CA depletion of the glandula was not sensitive to ganglionic blockers that in adult animals blocked the CA response to hypoxia. Seidler and Slotkin (38) also demonstrated that the CA-secretory response to hypoxia from AM in neonatal rats is greatly attenuated at 14 days of age, when maturation of splanchnic innervation occurs, and that the hypoxic sensitivity was regained after splanchnic denervation in the adult rat. Therefore, it was suggested on the basis of these in vivo studies that the secretory response induced by hypoxia was nonneurogenic and that functional innervation of the adrenomedullary cells abolishes this direct response.

The in vitro studies regarding the role of innervation as the suppressor of the capacity of AM cells to respond to hypoxia are to some extent controversial. Nurse and coworkers (42, 43) demonstrated that primary cultures (48 h) from neonatal (24–48 h) AM of rats and mice had low PO2-sensitive K+ currents and responded to hypoxia with increased intracellular Ca2+ concentration ([Ca2+]i) and CA secretion, whereas cultures from juvenile animals (14–20 days of age) lacked these responses. Mojet et al. (26), also working in primary cultures of rat AM, confirmed that the secretory response to hypoxia (PO2 <5 mmHg) was restricted to neonatal animals. Contrary to those findings, it has been reported that hypoxia inhibits K+ currents and increases intracellular Ca2+ and CA secretion in cultures of adrenomedullary cells from 7-wk-old rats (25; see also Refs. 21, 40).

In sheep AM, splanchnic innervation occurs in utero, but in vivo, similarly to the situation in the rat, a direct response to hypoxia in the ovine fetus before innervation disappears after innervation is established (5). However, when studies were performed in vitro, AM cells of fetal lambs responded to hypoxia whether the preparations were obtained from the animals before or after innervation (1, 4). In a recent study, Keating et al. (17) found that a cocktail of opioid agonists suppressed the hypoxic response of in vitro perfused adrenal glands or isolated chromaffin cells from sheep fetus before innervation of the AM. They proposed that in vivo opioid peptides released from the splanchnic nerves were responsible for the suppression of the direct effects of hypoxia on AM and also that in vitro splanchnic innervation is not active and therefore all preparations are sensitive to hypoxia (see also Ref. 4).

The nonneurogenic secretory response to hypercapnia and acidosis in AM has been studied less. Cross and Silver (10) showed that severe hypercapnia in vivo is a powerful stimulus for CA secretion in the AM of adult animals, and Biesold et al. (3) found that acute splanchnic denervation of adult rats markedly depressed the secretory response evoked by hypercapnia. Working with an isolated preparation of adult rat AM, Fujiwara et al. (15) found that hypercapnic acidosis and low extracellular pH increased the release of CA. In perinatal animal data from in vivo studies performed with fetal sheep, the findings are controversial. Lewis et al. (23) found that acidemia potentiated plasma CA response to hypoxemia, while Cohn et al. (7) found no release response to metabolic acidosis. Regarding in vitro studies, it has been reported recently that neonatal slices from the rat responded to hypercapnia by increasing the CA release and that responsiveness was higher in neonatal tissue than in adult tissue slices (27).

In an attempt to clarify the controversies surrounding the responses to hypoxia and to acidosis, we have used an in vitro preparation of rat AM (34) that allows for the simultaneous monitoring of secretory responses from two individual glands and thereby allowed us to compare the nonneurogenic secretory response in AM obtained from rats of different ages. We have also explored the role of opioids in the disappearance of the nonneurogenic hypoxic response in neonatal rat AM and the possible interactions between hypercapnic acidosis and hypoxia in eliciting a secretory response.


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Animal and surgical procedures. The experimental procedures were approved by the Animal Care and use Committee of the University of Valladolid. Rats aged 1–2, 14–15, and 30 days as well as 3 mo of age [body wt, 6.6 ± 0.1, 28.7 ± 0.6, 85.9 ± 7.5, and 217.8 ± 5.9 g, respectively (means ± SE); n = 6–8] were anesthetized with sodium pentobarbitone administered intraperitoneally (60 mg/kg body wt). After a longitudinal incision was made in the abdomen, the adrenal glands were exposed, removed, and placed in a Lucite chamber filled with ice-cold Tyrode-HCO3 solution (in mM: 116 NaCl, 5 KCl, 2 CaCl2, 1.1 MgCl2, 23 NaHCO3, and 5 glucose; pH 7.4) equilibrated with 95% O2-5% CO2. The adrenal glands were gently decapsulated with fine forceps under a dissecting microscope, and clean AM were stored in ice-cold Tyrode-HCO3 until used to measure CA content or to monitor CA secretion.

Measurement of tissue CA content. AM from rats of different ages were weighed in an electrobalance (Sartorius), homogenized (glass/glass) in 200 µl of ice-cold 0.2 M perchloric acid containing 0.01% EDTA, and centrifuged at 4°C (10,000 g for 10 min). Aliquots from the supernatants (25–100 µl) were injected directly into the HPLC electrochemical detection (HPLC-ED) system. The line of HPLC-ED was composed of a Milton Roy CM 400 pump, a Waters U6K injector, a Waters C18 column (4-µm particle size), and a Bioanalytical System LC-4 electrochemical detector (0.65-V holding potential). Identification and quantification of endogenous CA were performed against external standards (Peak Simple chromatography data system; SRI Instruments).

Measurement of CA secretion. After dissection, to facilitate penetration of the carbon fiber electrode in the tissue, AM from 30-day-old rats were incubated in a diluted enzymatic solution [Tyrode-HCO3 equilibrated with 95% O2-5% CO2 containing 0.1% collagenase (Worthington type I)] for 5 min at 37°C. Glands from 14- to 15-day-old rats were subjected in some cases to a similar (<5 min) weak digestion procedure; but in most of the experiments, they were impaled without enzymatic treatment. Because CA-secretory responses were comparable in AM with and without the digestion procedure, the results of both groups of AM from 14- to 15-day-old rats were pooled. The group of AM from 1- to 2-day-old rats were not subjected to the digestion procedures, because it was not necessary for the impalement of the electrode. Tissues were transferred to a thermostated Lucite recording chamber (36°C, dead volume 200 µl) and superfused by gravity (3–4 ml/min) with Tyrode-HCO3 normoxic control solution equilibrated with 20% O2, 5% CO2, and 75% N2, except in the experiments in which low extracellular pH stimuli were applied (see Fig. 6). In this group of experiments, the control solution was HCO3 free (in mM: 146 NaCl, 5 KCl, 2 CaCl2, 1.1 MgCl2, 5 glucose, and 10 HEPES; pH 7.4) and equilibrated with air. Testing solutions were equilibrated by bubbling reservoir bottles with different mixing gases (see RESULTS). In 30 mM KCl-containing solutions, equimolar NaCl was removed. The perfusion system has six lines with intercalated electronic valves located near the recording chamber to minimize the dead volume. Switching of superfusion lines was commanded electronically. Both saline reservoir bottles and the superfusion tube lines were immersed in a circulating 36°C water bath.



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Fig. 6. Effects of extracellular low pH in neonatal rat (1–2 days old) AM. Representative experiment in which neonatal rat (1–2 days old) AM were superfused with HEPES-buffered HCO3-free solutions adjusted to pH 7 for 10 min and to pH 6.8 for 5 min. Solutions were equilibrated with air, and control was adjusted to pH 7.4.

 
Free CA concentration inside the tissue was measured using a single 5-µm carbon fiber insulated, except for the tip, with a polyethylene tube (Dagan). The electrodes were attached to an EI-400 potentiostat (Ensman Instrumentation, Bloomington, IN). Recordings were obtained at a fixed voltage (0.5-V amperometric mode). This voltage is optimal for measuring norepinephrine (NE) and epinephrine (EPI), which are the most abundant CAs in rat AM as established by HPLC measurements in tissue homogenates. Currents proportional to free tissue CA concentrations were sampled at 5 Hz, digitized, and recorded on a computer (Digidata 1322, Axoscope 9; Axon Instruments) for offline analysis (Microcal Origin). AM were impaled with the carbon electrode under the microscope and superfused for a period of 30–40 min to allow for recovery of the preparations from surgical procedures and to standardize recording conditions. After this period, the amperometric recordings were stable and preparations were subjected to different experimental protocols as detailed in RESULTS. Before recording from the tissues, the carbon fiber electrode was advanced into the bath chamber and calibrated by switching between normal Tyrode solution and Tyrode solution containing 5–20 µM EPI concentration (the relation of [EPI] to amperometric recording current was linear.). At the end of the experiment, the electrode was withdrawn from the tissue and calibrated again. The currents due to oxidation of solutions containing EPI were used to convert the recording current in the tissues in free CA tissue concentration that was used as an index of CA released. Comparison of the calibration before and after the study was used to assess the change in electrode sensitivity during the recording period. The impalement of the tissues two or three times before the first calibration, especially the new electrodes, minimized the changes in sensitivity during the experiment. In the majority of the experiments, two preparations were placed side by side in the recording chamber and the levels of free tissue CA concentration were recorded in both preparations simultaneously. This experimental setting ensured identical recording conditions and straightforward comparison of the recorded responses.

Statistical analysis. The results are presented as individual experiments or as means ± SE. Statistical comparisons were performed using a two-tailed Student's t-test for unpaired data or one-way ANOVA with Dunnett's multiple-comparison test to identify significant differences between mean values (Table 1).


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Table 1. Catecholamine content of adrenal medulla in the rat

 

    RESULTS
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Catecholamine content in AM of neonatal and juvenile rats. AM from rats 1–2 days, 14–15 days, or 3 mo of age were processed for measuring CA content. Table 1 shows NE, EPI, and dopamine content of the dissected AM expressed as nanomolar wet weight tissue per milligram, and the ratios between molecular species of CA. The percentage of NE/EPI is higher in the AM from neonatal rats than in AM from 14- to 15-day-old or 3-mo-old rats (45.8 ± 5.7, 28.3 ± 2.1, and 31.7 ± 2.8%, respectively, n = 5–6; P < 0.05, neonatal vs. juvenile rats and neonatal vs. adult rats). Total content of CA increased dramatically during development but paralleled the increase in AM weight, lessening differences in the total CA concentration (CA/mg of tissue). However, at 14–15 days of age, total CA concentration in AM was still 240% that in the neonatal AM. This increase in CA concentrations mimics the increase in the percentage of the volume density of chromaffin cells reported previously (44). Data for CA the content and ratios between CA molecular species in the developing AM are similar to those reported previously by other authors (24, 44).

Hypoxia increases secretion of CA from neonatal AM. Figure 1A shows that neonatal AM in vitro perfused during 3 min with Tyrode HCO3 solution at different O2 concentrations (0%, 5%, and 7%) released CA in proportion to the intensity of the hypoxia (control, 20% O2-equilibrated Tyrode-HCO3 solution). In the same experiment, superfusion with a depolarizing solution (30 mM KCl for 1 min), elicited a large secretory response and thereby demonstrated the viability of the preparation. Figure 1B summarizes the results from a series of 10 experiments in which hypoxic stimuli of different intensities were randomly applied to correct for a possible desensitization or time-dependent decreases of the hypoxic secretory response. The free CA concentrations in the tissue evoked by hypoxia were 5.5 ± 1.6 (10% O2-equilibrated solutions; PO2 ~80 mmHg), 9.4 ± 1.7 (7% O2; PO2 ~61 mmHg), 12.1 ± 2.5 (5% O2; PO2 ~48 mmHg), 15.3 ± 2.3 (2% O2; PO2 ~29 mmHg), and 16.4 ± 3.1 µM (0% O2; PO2 ~15 mmHg); in all cases, n ≥ 8. In our recording conditions, a secretory response was detected with 10% O2 (PO2 ~80 mmHg; control PO2 ~150 mmHg). This PO2 threshold is similar to that found in the carotid body (CB) of several species in comparable in vitro situations (see Ref. 16).



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Fig. 1. Effects of graded hypoxic stimuli on catecholamine (CA) secretion from neonatal rat (1–2 days old) adrenal medulla (AM). A, bottom, sample experiment showing effects elicited by superfusing a neonatal AM with 0%, 5%, and 7% O2-equilibrated Tyrode-HCO3 solution (3 min) on free CA tissue concentration. Control solution was equilibrated with 20% O2-5% CO2-75% N2 (pH 7.4). PO2 recording in the chamber is represented at top. B: histogram data showing averaged values of the evoked free peak CA tissue concentration obtained from 10 experiments in which graded hypoxic stimuli (superfusion with 10%, 7%, 5%, 2%, and 0% O2-equilibrated solutions for 3 min) were applied to neonatal rat AM, as described in A. Bars represent means ± SE of 8–12 individual recordings.

 
The increase in free CA tissue concentration evoked by hypoxia is dependent on extracellular Ca2+ as demonstrated in the experiment shown in Fig. 2A. Perfusion for 30 min with a Ca2+-free solution containing 3.1 mM MgCl + 0.1 mM EGTA almost completely abolished the response to intense hypoxia (2% O2) as well as that elicited by high external K+ concentration [K+]e (30 mM KCl). The same protocol was applied in three additional experiments, and a summary of the data are shown in Fig. 2B. The hypoxic secretion in Ca2+-free solutions averaged 3.2% (P < 0.01) of that obtained in control conditions. The Ca2+ dependence of the response evoked by hypoxia indicates that this stimulus triggered a specific response involving activation of the exocytotic machinery. The same Ca2+ dependence of the hypoxia-evoked response demonstrated previously in superfused CB in vitro (see Ref. 16) and in primary cultures from neonatal rat AM (42).



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Fig. 2. Dependence on extracellular Ca2+ of the secretion of CA elicited by hypoxic and depolarizing stimuli in neonatal rat (1–2 days old) AM. A: representative example of the free CA tissue concentration during superfusion with solutions equilibrated with 2% O2 (3 min) or containing 30 mM KCl (1 min). Control solution was equilibrated with 20% O2. Superfusion with Ca2+-free solutions (in mM: 0 Ca2+, 3.1 MgCl2, and 0.1 EGTA) as indicated in the line graph. B: columns represent peaks of free CA tissue concentrations evoked by superfusion (3 min) with Ca2+-containing and Ca2+-free solution equilibrated with 2% O2. Values are means ± SE of 4–8 individual data from 4 experiments as described in A. **P < 0.01.

 
The mechanism of Ca2+ entry into adrenomedullary cells during hypoxic stimulus was also explored in neonatal AM. We found that the L-type Ca2+ channel blocker nisoldipine (5 µM) did not modify basal CA release but almost abolished the secretory response to hypoxia (2% O2) even in the presence of 2 mM Ca2+. In the nisoldipine-treated preparation, the hypoxia-evoked response dropped to 1.7 ± 0.6 µM from the control secretory response of 16.9 ± 4.5 µM (n = 8; P < 0.01).

CA release response evoked by hypoxia decays with age. AM from rats 1–2, 14–15, and 30 days of age were studied to define age-dependent modifications of the hypoxic secretory responses. Figure 3A, top, shows the PO2 tracings in the recording chamber throughout the experiment. Figure 3A, middle, shows the release response to high [K+]e (1 min) and hypoxic (3 min) pulses in an AM from a 14- to 15-day-old rat, and Fig. 3A, bottom, shows a similar recording in an AM from a 1- to 2-day-old rat. The three recordings were obtained simultaneously with the PO2 electrode located in the center of the chamber and between the two AM that were located side by side near the PO2 electrode. Figure 3A demonstrates that both AM produced secretory responses of comparable magnitude in response to high [K+]e pulses, whereas the AM from the 1- to 2-day-old rats generated larger secretory responses to the moderate (3 min, 7% O2) and intense (3 min, 2% O2) hypoxic stimuli. Figure 3B shows mean results obtained in 10 similar experiments. Figure 3B represents the mean secretory response to both hypoxic stimuli in AM from neonatal pups and 14- to 15-day-old rats, demonstrating that for both intensities of hypoxia, the responses in the AM of the older animals was ~30% of those observed in neonatal rat AM. Replotting the hypoxic secretion data in relation to secretion evoked by high [K+]e [(CA secreted by hypoxia/CA secreted by 30 mM KCl) x 100], (Fig. 3C) yielded comparable results; that is, the percentage in the 14- to 15-day-old rat AM was ~40% that found in neonatal animals, substantiating the close similarity of the evoked response to high [K+]e at both ages and the loss of the hypoxic response in older animals. In several recordings in the AM from 30-day-old rats, we did not find any significant secretory response to severe hypoxia, whereas the depolarizing (30 mM KCl) evoked secretory responses were comparable to those obtained at younger ages (data not shown).



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Fig. 3. Secretion of CA in response to hypoxia from in vitro superfused neonatal rat (1–2 days old) and juvenile rat AM (14–15 days old). Organs were superfused with depolarizing (30 mM KCl for 1 min), mild (7% O2), and severe (2% O2) hypoxia-equilibrated solutions (3 min). A, top, PO2 recording in the chamber next to the organs; middle and bottom, free tissue CA concentration in response to depolarizing and hypoxic stimuli from juvenile (14–15 days old) and neonatal rats (1–2 days old). B: averaged results of the peaks of free tissue CA concentration from neonatal and juvenile rat AM in response to hypoxic stimuli applied as described in A. **P < 0.01. ***P < 0.001. C: quotient between free tissue CA peaks evoked by hypoxic stimuli and those evoked by depolarizing stimulus of [(CA secreted by hypoxia/CA secreted by 30 mM KCl) x 100]. Results are means ± SE of 20–25 individual recordings from 10 experiments similar to those represented in A. *P < 0.05. **P < 0.01.

 
Opioid agonists do not modify CA release evoked by hypoxia in neonatal rat AM. Recently, it was reported that a cocktail of µ- and {kappa}-opioid agonists prevented the CA secretion evoked by hypoxia in perfused adrenal glands from the fetal lamb (17). To test whether opioids play the same role in the rat AM, the secretion induced by hypoxia was studied in the presence of opioid agonists in the neonatal rat AM. Figure 4A shows that superfusion of the neonatal AM with solution containing 2 µM [D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin (DAMGO, a µ-opioid receptor agonist) and U-62066 (a {kappa}-opioid receptor agonist) did not prevent hypoxia-evoked CA release. In a similar experimental protocol, 2 µM [D-Pen2,5]enkephalin (DPDPE, a {delta}-opioid receptor agonist) applied alone did not modify the secretory response evoked by hypoxia. Averaged results (Fig. 4B) show that the secretory response evoked by 3 min of hypoxia (2% O2) was not modified in the presence of either a cocktail of 2 µM DAMGO and U-62066 (103.7 ± 7.9%; n = 6) or 2 µM DPDPE (94.0 ± 8.1%; n = 8) applied alone.



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Fig. 4. Effect of opioid agonists on secretory response evoked by hypoxia from in vitro superfused neonatal rat (1–2 days old) AM. A: organs were stimulated by superfusion during 3 min with hypoxic solution (Tyrode-HCO3 solution equilibrated with 2% O2) in the presence or absence of 2 µM [D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin (DAMGO, a µ-opioid receptor agonist) and U-62066 (a {kappa}-opioid receptor agonist). B: average results of the free peak CA tissue concentration evoked by hypoxia (2% O2) in presence of opioid agonists as indicated. DPDPE, [D-Pen2,5]enkephalin (a {delta}-opioid receptor agonist). Data are presented as %secretory response evoked by hypoxic stimulus of 2% O2. Results are means ± SE of 6–8 individual recordings.

 
Acidosis increases the secretory response in neonatal AM. Figure 5A shows, for the first time, that an intact neonatal rat AM in vitro increases the secretion of CA in response to mild (10% CO2 for 3 min; pH 7.1) to severe hypercapnic acidosis (20% CO2 for 3 min; pH 6.8). Figure 5B represents mean values obtained in four experiments in which, for comparative purposes, AM were also stimulated with intense hypoxia (2% O2 for 3 min). The free CA tissue concentration for mild hypercapnic acidosis and severe hypercapnic acidosis had peaks of 2.1 ± 1.2 and 10.5 ± 2.3 µM, respectively (n = 8–10). Intense hypoxia evoked peak CA secretion (13.1 ± 2.2 µM) comparable to that produced by severe hypercapnic acidosis. The CA release response elicited by hypercapnic acidosis is Ca2+ dependent as has been shown for hypoxia. Mean hypercapnic acidic (20% CO2 for 3 min; pH 6.8) responses measured in four experiments following the protocol shown in Fig. 2A for hypoxia were 13.5 ± 2.1 in control conditions and 1.9 ± 0.7 µM in Ca2+-free conditions (P < 0.01). These results imply that in the neonatal AM, >85% of the acidic secretory response is dependent on extracellular Ca2+ as is the case in the adult CB (28, 32) and in rat pheochromocytoma (PC)-12 cells (a cell line derived from rat AM pheochromocytomas sensitive to hypoxia; see Ref. 41). Similar Ca2+ dependence was demonstrated recently for isohydric hypercapnia in neonatal rat tissue slices (27).



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Fig. 5. Effects of hypercapnic acidosis on CA secretion from neonatal rat (1–2 days old) AM. A: representative example of effects on free CA tissue concentration elicited by superfusion of neonatal AM during 3 min in mild hypercapnic acidosis solution (equilibrated with 20% O2-10% CO2-70% N2; pH 7.1), severe hypercapnic acidosis (20% O2-20% CO2-60% N2; pH 6.8), and intense hypoxia (2% O2-5% CO2-93% N2; pH 7.4). Control solution was equilibrated with 20% O2-5% CO2-75% N2 (pH 7.4). B: averaged values of peaks of free CA tissue concentration evoked by hypercapnic acidosis and hypoxic stimuli obtained in 4 experiments similar to those presented in A. Values represent means ± SE of 8–10 individual recordings.

 
In the next group of experiments, we investigated whether low extracellular pH also promotes a secretory response in neonatal rat AM. Figure 6 shows that superfusion with a HEPES-buffered HCO3-free solution at pH 7 (10 min) and at pH 6.8 (5 min) elicited a secretory CA response. Averaged results for a CA-secretory response evoked by 5 min of superfusion at pH 6.8 yielded a peak free CA tissue concentration of 9.0 ± 1.9 µM (n = 8).

Interaction between hypoxia and hypercapnic acidosis in eliciting a secretory response in neonatal and juvenile rat AM. We also explored the possible interaction between hypercapnic acidic and hypoxic stimuli in one attempt to mimic the asphyxiation episodes that occur during natural delivery. In these experiments, neonatal and juvenile rat AM placed side by side were stimulated with intense hypoxia (2% O2), acidic hypercapnia (20% CO2; pH 6.8), and asphyxia (2% O2-20% CO2; pH 6.8). Figure 7A shows that combined stimuli produced a more intense response than each stimulus applied alone; also evident is that all of the responses were weaker in the juvenile rat AM. Figure 7B shows the mean responses from nine experiments. Peak CA secretion levels in neonatal rat AM were 11.2 ± 2.3 µM (hypoxia), 10.1 ± 1.2 µM (hypercapnia), and 30.2 ± 3.9 µM (asphyxia). Free tissue CA peaks in the AM from 14- to 15-day-old rats were 3.9 ± 1.0 and 4.1 ± 1.1 µM for the hypoxic and hypercapnic stimuli, respectively, and 11.3 ± 2.7 µM for the asphyxiant stimulus. These responses in the AM of 14- to 15-day-old rats represent between ~35% and 40% of the responses obtained in the neonatal animals. Figure 7C shows comparable results when data are expressed in relation to the secretion evoked by high [K+]e [(CA secreted by hypoxia/CA secreted by 30 mM KCl) x 100].



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Fig. 7. Release of CA in response to hypoxia and hypercapnic acidosis applied simultaneously in neonatal rat (1–2 days old) and juvenile rat (14–15 days old) AM. A, top, continuous PO2 recording in the superfusing chamber during the experiment; middle and bottom, representative experiment showing effects of superfusing juvenile rat (14–15 days) and neonatal rat (1–2 days) AM with hypoxic (2% O2-5% CO2-93% N2; pH 7.4), hypercapnic acidic (20% O2-20% CO2-60% N2; pH 6.8), and hypoxic + hypercapnic acidic (2% O2-20% CO2-78% N2; pH 6.8) solutions on free tissue CA concentrations. B: averaged results of peaks of free CA tissue concentration from neonatal and juvenile AM in response to hypoxic, hypercapnic acidosis, and hypoxic + hypercapnic acidosis stimuli applied as described in A. **P < 0.01. ***P < 0.001. C: quotient between free tissue CA peaks evoked by stimuli and those evoked by depolarizing stimulus of [(CA secreted by stimuli/CA secreted by 30 mM KCl) x 100]. Results are means ± SE of 10–15 individual data from 9 experiments similar to those represented in A. *P < 0.05. **P < 0.01.

 
In another set of experiments, we applied extreme hypoxia (solution equilibrated with 5% CO2 balanced by N2; pH 7.4) and moderate hypercapnic acidosis (10% CO2-20% O2 balanced by N2; pH 7.1) individually and in combination. We found that secretory responses evoked by extreme hypoxia and moderate acidosis/hypercapnia in eight experiments were 15.6 ± 2.9 and 2.5 ± 1.2 µM, respectively, and when both stimuli were combined, the secretory response rose to 31.3 ± 5.6 µM (data not shown).


    DISCUSSION
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Our findings confirm the intrinsic nonneurogenic response of neonatal rat AM chromaffin cells to hypoxia and the loss of sensitivity in the initial weeks after birth. In this species, suppression of this direct response is not mediated by opioid peptides. We also found that AM from neonatal rats responded to hypercapnic acidosis with a potent secretory response, which, like the hypoxic response, lessens with age. Secretory responses evoked by hypoxia and hypercapnic acidosis were Ca2+ dependent. Finally, we found a synergistic interaction between hypoxia and hypercapnic acidosis in their ability to evoke secretion of CA from neonatal adrenomedullary chromaffin cells.

In the present study, we have used a preparation of whole AM superfused in vitro (34) that allowed us to record free CA tissue concentration, a direct index of the secretory response. A similar experimental setting has been applied previously to study the developmental changes in sensitivity to hypoxia in the CB of rat and rabbit (12, 33). Our preparation allowed the recording of secretory activity within ~30 min after the removal of the AM and probably maintained any nerve fiber ending functionality for the entire duration of the experiments as demonstrated in other peripheral structures (13, 22). These technical aspects are of great significance, given the possibility that in primary cultures of adrenomedullary cells of adult animals, the suppressor influence of splanchnic innervation on the intrinsic sensitivity to hypoxia disappears after certain periods of cell isolation in culture, converting the primary cultures of adrenomedullary cells into a preparation equivalent to the adult denervated AM that regains sensitivity to hypoxia (38, 39). Although our preparation did not allow routine identification of the molecular species of the CA secreted, in experiments specifically designed to identify the released CA, it was found that hypoxia releases CA, NE, and EPI in proportion to their concentrations in the AM; therefore, EPI is the main CA secreted (24, 26) (Table 1).

Previous in vitro studies of the secretory response to hypoxia, either in primary cultures or in whole glands, have used mostly intense stimuli. In the present study, we applied a wide range of hypoxic stimuli, allowing us to match some conclusions regarding the sensitivity and threshold of the AM response to hypoxia. We found that AM from 1- to 2-day-old rats secreted CA in amounts paralleling the intensity of hypoxia (from 80 to 15 mmHg). The threshold for the hypoxic response was close to a PO2 of 80 mmHg, which is similar to that described previously for the adult rabbit CB in in vitro and in vivo preparations (30, 33; see also Ref. 16). These comparisons indicate that the neonatal AM and the adult CB share comparable hypoxic thresholds. The observed secretory response to hypoxia in neonatal AM is dependent on the presence of Ca2+ in the milieu and is sensitive to blockage of the L-type Ca2+ channel. These findings are in agreement with previous data obtained in vitro in preparations of fetal lamb or of neonatal rat AM (1, 42) and adult CB (36; see also Ref. 16).

We found in AM from 14- to 15-day-old rat pups that when splanchnic innervation was already functional, the hypoxic secretory response was only ~30% that observed in 1- to 2-day-old neonatal rat pups and that responses in AM from 30-day-old rats were absent. These age-dependent changes were even more dramatic considering that the volume of chromaffin cells as a percentage of total AM weight increased from ~35% to 65% with age (44), which was paralleled by a higher concentration of CA in the whole AM. As a whole, our data regarding the development of sensitivity to hypoxia resemble the in vivo results of Seidler and Slotkin (38, 39) and support the contention that the maturation of splanchnic innervation suppresses the nonneurogenic response to hypoxia. In this regard, our data are in reasonable agreement with previous findings in which short-term cultures of neonatal and juvenile AM cells were used (26, 42, 43), but they are at variance with the findings of Mochizuki-Oda et al. (25), who obtained their recordings after the adult adrenomedullary chromaffin cells had been in culture for up to 7 days. Our findings also have discrepancies with the findings of Takeuchi et al. (40), who used a preparation of freshly sliced AM from rats of different ages (neonatal to adult) and found that hypoxia promoted a weak increase in [Ca2+]i in all groups. Because Takeuchi et al. did not measure secretory response, the possibility exists that a comparable Ca2+ signal suffices to activate the exocytotic machinery in neonatal but not adult AM cells. We shall not discuss in detail the data obtained in isolated sheep AM, because species differences make comparison difficult. We merely mention that splanchnic innervation matures in utero and that opioid peptides suppress hypoxic responses in fetal lamb in vitro preparations (17). However, we want to make a further comment with regard to the findings of Cheung (4) and Adams et al. (1) in the fetal lamb. These authors expressed their results as absolute amounts of CA released in the perfusion solution, which could be misleading. Although the absolute amounts secreted in their perfused preparations were slightly higher after innervation (1), considering the increase in CA content in the AM with age (4), their data might indicate that mature AM is less sensitive to hypoxia, which is what we found in the rat.

Our results demonstrate for the first time the nonneurogenic response to hypercapnic acidosis and extracellular acidification in an in vitro intact preparation of neonatal rat AM. The magnitude of the response is comparable to that evoked by hypoxia, and it also exhibits a marked Ca2+ dependence. Previous studies in our laboratory have demonstrated that cat and rabbit CB also responded to extracellular acidification and to hypercapnic acidosis by the release of CA, with this evoked release being dependent on extracellular Ca2+ (28, 32, 35). Thus chromaffin cells from neonatal AM and adult CB seem to be endowed with similar chemosensory properties. It is noteworthy that the sensitivity of neonatal AM to acidic stimuli appears to be comparable to that in the CB of all species studied to date, including cat, rabbit, and rat (28, 31, 32, 45, see Ref. 16) and to that in PC-12 cells (41). We have found that sensitivity to hypercapnic acidosis in AM decreased with age. Fujiwara et al. (15) found that extracellular but not intracellular acidification still promoted a secretory response in perfused adult rat AM. That secretory response was dependent on extracellular Ca2+ but insensitive to nifedipine (a blocker of L-type voltage-gated Ca2+ channels). This latter finding suggests that acidic stimuli do not depolarize adult AM chromaffin cells or, alternatively, that another subtype of voltage-gated Ca2+ channel supports the entry of Ca2+ required for the exocytosis (36). In contrast to the results of Fujiwara et al. (15), a recent report showed that isohydric hypercapnia, which promotes intracellular acidification, elicited a secretory response in AM tissue slices from neonatal rats that was decreased in tissue slices from adult rats (27). The same authors also demonstrated that isohydric hypercapnia depolarizes the chromaffin cells and postulated that neonatal adrenomedullary chromaffin cells are sensitive to hypercapnia, with intracellular acidosis being the effective stimulus. A possible explanation that could reconcile both groups of results from neonatal and adult chromaffin cells is that some aspects of the acidic transduction cascade could change during the maturation process. It remains to be established unequivocally whether intracellular or extracellular acidification inhibits K+ or cation-selective channels and depolarizes neonatal adrenomedullary chromaffin cells activating voltage-dependent Ca2+ channels, mechanisms that seem to mediate the activation of the secretory response produced by acidic stimulus in CB chemoreceptor cells (11, 27, 36).

In our last group of experiments, we found that hypoxia and hypercapnic acidosis applied simultaneously produce more than an additive effect on the secretory response in neonatal rat AM, although with lower intensity, this positive interaction was still present in the AM of 14- to 15-day-old rat pups. In this regard, even though the mechanisms responsible for stimulus-secretion coupling in hypoxia and hypercapnic acidosis in neonatal AM have not been explored conveniently, the apparent simultaneous decrease of both responses in AM from 14- to 15-day-old rat pups suggests that a specific molecular mechanism common to both hypoxic and acidic transduction cascades is downregulated by the acquisition of functionality of splanchnic innervation. In the CB, the interaction between both stimuli is well known at the level of the sensory activity elicited by them (14, 20, 29) but has not been studied at the level of the neurosecretory response of CB chemoreceptor cells. In CB chemoreceptor cells from neonatal 11- to 16-day-old rats, Dasso et al. (11) found only a slight positive interaction between stimuli at the level of [Ca2+]i responses, although both stimuli interacted in the same chemoreceptor cells, because all cells were sensitive to hypoxia and to hypercapnic acidosis. However, taking into consideration that neural positive interaction is absent in 7-day-old rats and is fully established in 5-wk-old rats (29), the possibility still exists that the full interaction between stimuli is generated at the level of the neurosecretory response of chemoreceptor cells. Taylor et al. (41) also found a multiplicative interaction between hypoxia and hypercapnic acidosis in the secretory CA response in PC-12 cells. On the basis of findings in neonatal AM chromaffin, CB chemoreceptors, and PC-12 cells, we conclude that an interaction between hypercapnic acidosis and hypoxia transduction mechanisms exists, regardless of the molecular mechanisms responsible for the interaction in each cell type.

When hypoxia and hypercapnic acidosis were applied simultaneously in our in vitro system, we were mimicking in vivo asphyxia. Fetal and neonatal animals rarely experience hypoxia exclusively (except at high altitude); it is more common in association with hypercapnic acidosis. In this regard, our data explain the results of Lewis et al. (23), who found in fetal sheep subjected to umbilical constriction that the release response was much higher when constriction elicited both acidemia and hypoxemia rather than hypoxemia alone. Thus the physiological significance of the synergistic effect is evident. The potent CA-secretory response produced by asphyxia is of prime importance to the genesis of cardiocirculatory, pulmonary, and metabolic effects (see Ref. 19) necessary for adaptation and survival in the extrauterine environment.


    GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
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This work was supported by Spanish Dirección General de Investigación Científico y Técnica (DGICYT) Grant BFI 2003-1627 (to R. Rigual), Spanish DGICYT Grant BFU 2004-06394 (to C. Gonzalez), and by Red Respira-Separ Grant Fondo de Investigaciones Sanitarias–Instituto de Salud Carlos III, Grant PI042462, and JCYL Grant VA011C05.


    FOOTNOTES
 

Address for reprint requests and other correspondence: R. Rigual, Departamento de Bioquímica y Biología Molecular y Fisiología, Instituto de Biología y Genética Molecular (IBGM), Universidad de Valladolid, Consejo Superior de Investigaciones Científicas, Facultad de Medicina, Calle Ramón y Cajal, 47005 Valladolid, Spain (e-mail: rrigual{at}ibgm.uva.es)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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