Characterization of the synthesis and release of catecholamine in the rat carotid body in vitro

I. Vicario, R. Rigual, A. Obeso, and C. Gonzalez

Department of Biochemistry and Molecular Biology and Physiology, Consejo Superior de Investigaciones Científicas, School of Medicine, University of Valladolid, 47005 Valladolid, Spain


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The aim of this work was to determine contents and turnover rates for dopamine (DA) and norepinephrine (NE) and to identify the catecholamine (CA) released during stimulation of the rat carotid body (CB). Turnover rates and the release of CA were measured in an in vitro preparation using a combination of HPLC and radioisotopic methods. Mean rat CB levels of DA and NE were 209 and 45 pmol/mg tissue, respectively. With [3H]tyrosine as precursor, rat CB synthesized [3H]CA in a time- and concentration-dependent manner; calculated turnover times for DA and NE were 5.77 and 11.4 h, respectively. Hypoxia and dibutyryl adenosine 3',5'-cyclic monophosphate significantly increased [3H]CA synthesis. In normoxia, rat CB released [3H]DA and [3H]NE in a ratio of 5:1, comparable to that of the endogenous tissue CA. Hypoxia and high K+ preferentially released [3H]DA, nicotine preferentially released [3H]NE, and acidic stimuli released both amines in proportion to tissue content. Release of [3H]CA induced by hypoxia and high K+ was nearly fully dependent on extracellular Ca2+, whereas basal normoxic release was not altered by removal of Ca2+ from the incubating solution. We conclude that the rat CB is an organ with higher levels of DA than NE that preferentially releases DA or NE in a stimulus-specific manner.

dopamine; hypoxia; nicotine; arterial chemoreceptors


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE CAROTID BODY (CB) is an arterial chemoreceptor activated by low PO2 and high PCO2/low pH in the arterial blood. Structurally, the CB is a secondary receptor in which chemoreceptor cells form synaptic contacts with sensory nerve endings of the carotid sinus nerve (CSN), a branch of the ninth cranial nerve. Functionally, it is generally accepted that CSN activity is synaptically driven by neurotransmitters present in chemoreceptor cells, the sensors of alterations in blood gases and pH (23).

Rat CB chemoreceptor cells contain a great variety of neurotransmitters, including catecholamines [CA; which include dopamine (DA) and norepinephrine (NE)], serotonin, ACh, opioid peptides, substance P, colecystokinin, and atrial natriuretic peptide (23). DA and NE are among the most abundant neurotransmitters present in chemoreceptor cells of the rat CB and have been the subject of numerous studies (Refs. 13, 15, 25, 31, 36, 37; see Ref. 23). Yet, several aspects of CA metabolism in the rat CB remain controversial. For example, reported CA levels for the rat CB vary from >800 pmol/mg of tissue (26) to >200 pmol/mg of tissue (Refs. 28, 36, 37; see also Refs. 18 and 23), with DA-to-NE ratios varying between 0.5 (31) and 4 (27) but with most values ~2 (see Refs. 18 and 23). There are also important differences in the reported rates of CA utilization. For example, turnover rates for DA oscillate from 48 to 79 pmol · mg tissue-1 · h-1 (Ref. 6 vs. Ref. 37), and turnover rates for NE vary from 16.6 to 46.6 pmol · mg tissue-1 · h-1, with comparable variations in the calculated turnover times (6, 25, 37).

Regarding the release of CA, there is only one study in which the specific release of DA and NE was measured (39). By use of radioisotopic methods to label CA stores and cation exchange chromatography to separate the labeled CA, it was found that hypoxia released NE and DA in comparable proportions (39). These findings conflict with data obtained in rabbit and cat CB, in which hypoxia preferentially releases DA (12, 22) and pose important uncertainties for most recent studies on the release of CA from the rat CB using voltammetric techniques that do not discriminate between DA and NE (e.g., Ref. 15). Additionally, there are no data on the effects of acidic stimulation of the rat CB on the release of CA.

The present work has two specific aims: to determine contents and turnover rates and times for DA and NE and to identify the CA released during hypoxic stimulation in the rat CB. Using a combination of HPLC with electrochemical detection (HPLC-ED) and isotopic methods, we measured endogenous levels of DA and NE and characterized in vitro the synthesis of [3H]DA and [3H]NE from their natural precursor [3H]tyrosine in basal conditions and after hypoxic stimulation. We also measured the relative rates of release of both [3H]DA and [3H]NE in response to hypoxia, acidosis, high external K+, and nicotine.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Animals and surgical procedures. Experiments were performed using CB harvested from adult Wistar rats of both sexes (body wt 250-350 g) that were housed in the vivarium at 20-23°C in a normal 12:12-h light-dark cycle and fed ad libitum. Rats were anesthetized with pentobarbital sodium (Sigma, Madrid, Spain) administered intraperitoneally (60 mg/kg body wt). After tracheostomy and under a dissecting microscope, the carotid bifurcations were located, removed, and placed in a Lucite chamber filled with ice-cold Tyrode solution (in mM: 140 NaCl, 5 KCl, 2 CaCl2, 1.1 MgCl2, 5.5 glucose, and 10 HEPES) adjusted to pH 7.40 with 1 N NaOH. The CBs (8-12 CBs/experiment) were identified and cleaned of surrounding connective tissue under a dissecting microscope, collected in glass vials containing fresh Tyrode, and kept at 0-4°C. CBs used to measure endogenous CA content were transferred to Eppendorf tubes (kept in ice) containing 75 µl of 0.4 N perchloric acid and were processed for HPLC-ED as described below (see Analytical procedures).

Labeling of CA stores: synthesis and release of [3H]CA. To label CA stores, the CBs were incubated on a metabolic shaker at 37°C in small glass vials containing 0.5 ml of Tyrode solution of the composition given above. The incubating solution contained [3H]tyrosine (30 µM, unless indicated otherwise) with specific activities of 6 and 48 Ci/mmol for the synthesis and release experiments, respectively. The incubating solution also contained 100 µM 6-methyl-tetrahydropterine and 1 mM ascorbic acid as cofactors for tyrosine hydroxylase and dopamine-beta -hydroxylase, respectively (17). The incubation for [3H]CA stores labeling lasted 2 h, except for those experiments in which the time course of the synthesis was studied. In those experiments, we used a HEPES-buffered air-equilibrated incubating solution to assure its stability in composition during the entire incubation time. Due to the relatively small incubating volume and the long periods of incubation (0.5-4 h), the continuous bubbling of the solutions with any gas mixture, even if it is saturated with water vapor, produces changes in the ionic strength of the solutions that are extremely difficult to control. Therefore, during the [3H]CA labeling period it is advisable to use solutions equilibrated with room air and pH equilibrated with nonvolatile buffers. In contrast, in the release experiments, for which the incubating volume was larger (4 ml) and the solutions were renewed every 10 or 20 min, we used the more physiological CO2-buffered Tyrode solution (see below).

In the synthesis experiments, the CBs were transferred at the end of the labeling period to glass vials containing 10 ml of ice-cold precursor-free Tyrode solution to wash out [3H]tyrosine present in the extracellular milieu. After 5 min, the CBs were transferred to cold Eppendorf tubes containing 75 µl of 0.4 N perchloric acid. Thereafter the tissues were weighed in an electrobalance (Supermicro, Sartorius) and glass-to-glass homogenized at 0-4°C. The homogenates and an additional aliquot of 50 µl of 0.4 N perchloric acid used to quantitatively collect the tissue homogenate were centrifuged in a Microfuge (Beckman, Madrid) for 10 min in a cold room. Synthesized [3H]CA and free [3H]tyrosine present in the tissues were measured in the supernatants, and the pellets were used to measure [3H]tyrosine incorporated into proteins.

In the release experiments, each CB was transferred at the end of the labeling period to a glass vial containing 4 ml of precursor-free Tyrode bicarbonate solution with a composition identical to the one above except for the substitution of 24 mM NaHCO3 for 24 mM NaCl. CBs were kept in the shaker bath at 37°C for the rest of the experiment. Solutions were continuously bubbled with a gas mixture saturated with water vapor of composition 20% O2-5% CO2-75% N2, except when hypoxia was applied as a stimulus (see Effects of several stimuli on the differential release of [3H]DA and [3H]NE). The incubating solutions were renewed every 20 min for 1 h and discarded. Thereafter, solutions were collected every 10 min and saved for analysis of [3H]CA content. Stimuli (hypoxia, acidosis, high external K+, and nicotine) were applied to the CBs for 10 min, from minute 20 to minute 30 of the collection period. Collected solutions were acidified with glacial acetic acid to pH 3 to prevent degradation of the [3H]CA released. At the end of the experiments, CB tissues were prepared for analysis of [3H]CA content as in the synthesis experiments (see above).

Analytical procedures. [3H]tyrosine incorporated into proteins during the synthesis period was calculated from the radioactivity present in the tissue pellets as determined by liquid scintillation spectrometry. Free [3H]tyrosine in the synthesis experiments was calculated as the difference between total radioactivity present in an aliquot of the tissue supernatants and radioactivity in the form of [3H]CA. For the analysis of unlabeled and 3H-labeled CA present in the tissue extracts, aliquots (10-50 µl) of supernatants were directly injected into the HPLC system. The analysis of [3H]catechols present in the collected incubating solutions included adsorption to alumina (100 mg) at alkaline pH (obtained by the addition under shaking of 5 ml of 2.5 M Tris buffer, pH 8.6), extensive washing of the alumina with distilled water, bulk elution of all catechols with 1 ml of 1 N HCl, and liquid scintillation counting of a 100-µl aliquot. The rest of the alumina eluates were used for identification of the labeled [3H]catechols by HPLC-ED.

For HPLC-ED analysis, alumina eluates were concentrated to dryness in a vacuum concentrator (Automatic 290 SpeedVac, Savant Instruments, Farmingdale, NY) and resuspended in 100 µl of ice-cold mobile phase (in mM: 10 NaH2PO4, 0.6 sodium octane sulfonate, and 0.1 EDTA, with 16% methanol, pH adjusted to 3.2-3.6 with concentrated phosphoric acid) containing 0.2 nmol of unlabeled CA as an internal standard. Aliquots of 10-45 µl were injected into the HPLC system in different experiments. The HPLC system was composed of a Milton Roy CM 400 pump, a Waters C18 (particle size 4 µm) column, a Waters U6K injector, a Bioanalytical Systems LC-4A electrochemical detector (set at a holding potential of 0.75 mV and a sensitivity of 1-5 nA), and a Milton Roy C-1 4000 integrator.

Identification and quantification of endogenous CA in tissue samples were done against external standards. Identification of [3H]catechols in CBs loaded with [3H]tyrosine was also done against external standards, and quantification was done by collection of the HPLC column effluent every 1 min and scintillation counting. In the case of the samples collected in the release experiments, identification was done with internal standards and quantification was done by liquid scintillation spectrometry of sequential 1-min fractions of the HPLC column effluent.

Statistics. Results are presented as means ± SE. Statistical significance of the differences observed was assessed using a two-tailed Student's t-test for unpaired data.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Endogenous CA content of the CB. A group of 10 CBs was processed for analysis of endogenous CA. Findings are presented in Fig. 1. Mean NE content was 45.4 ± 6.6 pmol/mg tissue, and mean DA content reached 209.6 ± 24.2 pmol/mg tissue. Of the CA catabolites, only 3,4-dihydroxyphenyl acetic acid (DOPAC) was detectable. Mean DOPAC content in the CBs was 8.0 ± 3.6 pmol/mg tissue. The concentration of the rest of the catabolites was below detection, equivalent to <2 pmol/mg tissue. The mean ratio of DA (DA + DOPAC) to NE was 4.9 ± 0.3, indicating that the rat CB is mainly a dopaminergic organ.


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Fig. 1.   Endogenous catecholamine (CA) levels of rat carotid body (CB) determined by HPLC. Data are means ± SE from 10 organs. NE, norepinephrine; DOPAC, 3,4-dihydroxyphenyl acetic acid; DA, dopamine; tiss, tissue.

Time course and dependence on [3H]tyrosine concentration of the rate of [3H]CA synthesis in vitro. In preliminary experiments with a few CBs, it was found that 30 µM tyrosine yielded maximal or nearly maximal rates of [3H]CA synthesis. Therefore, we studied the time course of [3H]CA synthesis using this concentration of the precursor. In Fig. 2A, it can be seen that the rate of synthesis increased linearly between 0.5 and 2 h. Linear regression analysis (see Fig. 2A, inset) gives a slope of 35.0 ± 3.6 pmol · mg tissue-1 · h-1, which is identical to the rate of synthesis obtained for 2 h and ~10-15% smaller than that found at 0.5 h. From 2 to 4 h, the measured rate of synthesis declined markedly, the estimated rate of synthesis at 4 h being 21 pmol · mg tissue-1 · h-1. The levels of free [3H]tyrosine in the tissues reached maximal concentrations at 0.5 h; with the assumption that intracellular water represents 50% of total tissue weight, the estimated intracellular concentration of free [3H]tyrosine was >100 µM, a concentration that is nearly saturating for tyrosine hydroxylase with the cofactor used in our experiments (Ref. 3; see also Fig. 3). [3H]tyrosine incorporation into proteins increased linearly with time at a rate of 71.9 ± 2.4 pmol · mg tissue-1 · h-1, indicating that the CB is well maintained for at least 4 h in the incubating conditions used. Figure 2B shows the results of the HPLC analysis of the [3H]CA synthesized by other groups of CBs incubated for 0.5 and 2 h. At 0.5 h, the rate of synthesis of [3H]DA was 36.3 ± 8.2 pmol · mg tissue-1 · h-1 and that of [3H]NE was 4.0 ± 0.9 pmol · mg tissue-1 · h-1; at 2 h, the rates were 31.0 ± 4 and 3.6 ± 0.6 pmol · mg tissue-1 · h-1, respectively. The ratios of [3H]DA to [3H]NE were 9 at 0.5 h and 8.6 at 2 h. The total turnover times calculated from these data and from those of Fig. 1 were 5.8 h for DA and 11.5 h for NE (Fig. 2B). Total CA content (labeled plus unlabeled) in the CBs at the end of the loading period in this later experiment was 225 ± 23 pmol/mg tissue (n = 10; data not shown).


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Fig. 2.   Time course of synthesis of [3H]CA from their natural precursor [3H]tyrosine (tyr). A: time courses of intracellular accumulation of newly synthesized [3H]CA and free [3H]tyrosine in CB as well as time course of incorporation of [3H]tyrosine into cell proteins. Inset: linear regression analysis for [3H]CA accumulation from 0 to 2 h (slope, 35 pmol · mg tissue-1 · h-1; y-intercept, 4.34 pmol; r = 0.989). Rate of incorporation of [3H]tyrosine into proteins was linear from 0 to 4 h (slope, 71.8 pmol · mg tissue-1 · h-1; y-intercept, -0.59 pmol; r = 0.998). B: HPLC of [3H]CA synthesized at 0.5 and 2 h of incubation with [3H]tyrosine and calculated turnover times for DA and NE in rat CB. Data are means ± SE of 8-10 individual values.



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Fig. 3.   Relationship between concentration of [3H]tyrosine in incubating solution and levels of [3H]CA synthesized by rat CB. Relationship exhibits saturation kinetics (inset), with apparent Michaelis-Menten constant (Km) and maximal velocity (Vmax) of 13.2 µM and 98.7 pmol · mg tissue-1 · 2 h-1, respectively. Accumulation of free [3H]tyrosine by CB showed 2 components, 1 saturating at [3H]tyrosine concentrations in incubating solution close to 30 µM and 1 increasing almost linearly between 30 and 60 µM. Data are means ± SE of 8 individual values.

Figure 3 shows the effects of different concentrations of [3H]tyrosine on the rate of synthesis of [3H]CA in CBs incubated for 2 h. Because the process of synthesis appeared to exhibit saturation kinetics (in relation to [3H]tyrosine in the incubating solution), the data were fitted to a Michaelis-Menten equation; the apparent Michaelis-Menten constant (Km) and the maximal velocity (Vmax) were 13.2 ± 3.6 µM and 98 ± 9.0 pmol · mg tissue-1 · 2 h-1, respectively. The accumulation of free [3H]tyrosine in the CB also followed saturation kinetics for concentrations of the labeled amino acid in the incubating solution of up to 30 µM, but at 60 µM it increased further. This behavior would suggest the existence in the CB, as in brain tissue (4), of two uptake systems for [3H]tyrosine with different affinities.

Effects of hypoxic stimulation and dibutyryl adenosine 3',5'-cyclic monophosphate on the rate of [3H]CA synthesis. It is well documented that the rate-limiting enzyme for CA synthesis is tyrosine hydroxylase (29, 32). This enzyme is under a negative feedback control exerted by CA on the affinity of the enzyme for the cofactor (2), in such a manner that after physiological stimulation of catecholaminergic tissues causing release of CA, there is a rebound increase in their rate of synthesis. Tyrosine hydroxylase is also under a positive covalent regulation by phosphorylation by a cAMP-dependent kinase (3, 42). Therefore, it was of physiological interest to verify the existence of these control mechanisms in the in vitro preparation of the rat CB. Figure 4A compares the rate of synthesis obtained in three groups of eight CBs incubated with [3H]tyrosine for 1 h. Control CBs were preincubated at 37°C for 15 min in a control solution (20% O2) before incubation in the presence of [3H]tyrosine. Hypoxic CBs were similarly preincubated and physiologically stimulated because the solution was equilibrated with 5% O2. The last group of CBs was preincubated with control solution, but dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP; 1 mM) was added to the incubating solution during the incubation with [3H]tyrosine. The mean rate of [3H]CA synthesis in the control group was 49.6 ± 3.2 pmol · mg tissue-1 · h-1. In the hypoxic group, the synthesis rate was augmented to 87.1 ± 8.9 pmol · mg tissue-1 · h-1 (P < 0.01). In the DBcAMP-treated group, the synthesis rate also increased to 74.8 ± 7.8 pmol · mg tissue-1 · h-1 (P < 0.02). The levels of free [3H]tyrosine in the tissue and the rate of incorporation of [3H]tyrosine into proteins were not altered by hypoxia or by DBcAMP treatment (see Fig. 4A). Therefore, the data support the operation of both control systems for tyrosine hydroxylase in the rat CB in vitro.


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Fig. 4.   Effects of hypoxia and dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) on synthesis of [3H]CA in rat CB. A: control CBs were preincubated in a normoxic solution (equilibrated with 20% O2-5% CO2) for 15 min before 1 h of incubation with CA precursor [3H]tyrosine. Hypoxic CBs were identically handled except for preincubation that took place in a hypoxic solution (equilibrated with 5% O2-5% CO2). DBcAMP CBs were treated the same way as controls, but incubating solution containing [3H]tyrosine also contained 1 mM DBcAMP. In each group, data are means ± SE of 8 individual values. B: effects of different concentrations of [3H]tyrosine in incubation medium on rate of [3H]CA synthesis in control and hypoxic CBs. Computer fitting of both groups of data to Michaelis-Menten equation gave Km values of 11.7 and 6.8 µM and Vmax values of 62.0 and 103.7 pmol · mg tissue-1 · h-1 for control and hypoxic CBs, respectively. Data are means ± SE of 5-8 individual CBs for each point. ** P < 0.02.

To characterize further some of the possible mechanisms involved in the stimulation of [3H]CA synthesis after hypoxic stimulation, we performed new experiments to compare the rate of synthesis in groups of CBs preincubated in normoxic vs. hypoxic solutions and incubated with different concentrations of [3H]tyrosine for 1 h. In the group of CBs preincubated in normoxic conditions, the apparent Km and Vmax were 11.7 µM and 62.0 pmol · mg tissue-1 · h-1, which are comparable to those found with incubation times of 2 h (see Fig. 3). In the group of CBs preincubated in hypoxic solutions, there was a marked increase in the apparent Vmax to 103.7 pmol · mg tissue-1 · h-1 and there was a decrease in the Km to 6.8 µM (Fig. 4B). Comparable kinetic changes have been described in the hypogastric nerve-vas deferens preparation after electrical stimulation of the nerve (Ref. 14; see also Ref. 42 for additional references). The levels of free [3H]tyrosine in the tissue and the incorporation into proteins were not different in control and hypoxic CBs at any of the studied concentrations (data not shown).

Effects of several stimuli on the differential release of [3H]DA and [3H]NE. The increase in the rate of synthesis after hypoxic stimulation is an indirect proof of CA release under hypoxic stimulation. In Fig. 5, we present direct evidence for the release induced by hypoxia (incubation with a solution equilibrated with 2% O2; PO2 ~20 mmHg), high external K+ (35 mM), an acidic/hypercapnic stimulus (incubation in a solution equilibrated with 20% O2-10% CO2, pH 7.10), and nicotine (300 µM). In every instance the stimulus was applied for 10 min, as depicted in Fig. 5A. From Fig. 5, it is evident that hypoxia is a very strong stimulus capable of multiplying release of [3H]CA by a factor of nearly 16 compared with normoxia. In contrast, acidic/hypercapnic stimulus was poorly effective for activation of the release response, since it hardly multiplied basal release by a factor of 1.4. Nicotine, even at this high concentration, was only moderately effective at activating the release of [3H]CA, doubling the basal release, and 35 mM extracellular K+ multiplied basal release by a factor of nearly five. Figure 5B shows the analytical profile of the released [3H]CA. Note that in the normoxic release of [3H]CA before stimulus application the ratio of [3H]DA + [3H]DOPAC to [3H]NE is ~5. This ratio increased to 9.7 ± 1.5 (P < 0.01) during hypoxic stimulation and to 8.1 ± 1.0 (P < 0.05) during stimulation with 35 mM external K+; it was not modified during acidic/hypercapnic stimulation, and it decreased to 2.8 ± 0.4 during stimulation with nicotine (P < 0.05). At the end of the release experiments, all CB were analyzed for [3H]CA content; mean [3H]DA and [3H]NE contents were 25.68 ± 2.24 and 4.78 ± 0.40 pmol/mg tissue, respectively, yielding a mean [3H]DA-to-[3H]NE ratio of 5.51 ± 0.37 (Fig. 5C).


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Fig. 5.   Release of [3H]CA by rat CB under different types of stimulation. A: magnitude and time course of release of [3H]CA in response to hypoxia (solution equilibrated with 2% O2-5% CO2), high external K+ (35 mM), hypercapnic acidosis (solution equilibrated with 20% O2-10% CO2, pH 7.1), and 300 µM nicotine (Nic). Each fraction represents amount of [3H]catechols (dpm) released to incubating solution during 10 min. Stimuli were applied for 10 min (solid bars), and incubating solution was drug-free normoxic solution rest of time. Data are means ± SE of 7 CBs for hypoxia and high external K+, 4 CBs for nicotine, and 3 CBs for acidic stimulus. B: ratios of [3H]DA + [3H]DOPAC to [3H]NE in incubating solutions in control conditions (C; normoxia) and during different types of stimulation. H, hypoxia. C: [3H]CA contents and ratios in CBs at end of release experiments. * P < 0.05; ** P < 0.01 compared with control normoxic samples.

In a final group of experiments, we studied the Ca2+ dependence of the release of [3H]CA elicited by hypoxia (2-min incubation in a solution equilibrated with 2% O2) and high external K+ (2 min; 60 mM K+). The experimental protocol was identical for both stimuli: first we assessed the release response to the short period of hypoxia or high external K+ exposure in groups of six CBs incubated in Ca2+-containing solutions, and then we measured the release response after incubating additional groups of four to six CBs in nominally Ca2+-free solutions for periods of 2 min, 4 min (2 × 2 min), or 6 min (3 × 2 min) before application of the stimulating solutions for 2 min. As shown in Fig. 6A, 60 mM K+ applied for 2 min in the presence of 2 mM Ca2+ released almost 25% of the [3H]CA contained in the CBs; the release decreased to nearly 0.6% of the tissue content after 2 min in nominally Ca2+-free solution and was indistinguishable from basal release after 4 or 6 min of incubation in 0 Ca2+. Figure 6B shows a comparable time course of the Ca2+ dependence for the hypoxic stimulus-induced release. It is also evident from both parts of Fig. 6 that the basal normoxic release of [3H]CA from the rat CB is nearly independent of the presence of Ca2+ in the incubating solution.


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Fig. 6.   Ca2+ dependence of high external K+-induced and low PO2-induced release of [3H]CA in rat CB. A: basal and 60 mM K+-induced release of [3H]CA by rat CB in normal 2 mM Ca2+-containing solution and after different times of incubation in nominally Ca2+-free solutions. Release is expressed as percentage of total [3H]CA present in organs at end of experiments. Nominally Ca2+-free Tyrode solutions were prepared by omission of CaCl2 in their composition. B: basal and low PO2-induced release of [3H]CA by rat CB in normal 2 mM Ca2+-containing solution and after different times of incubation in nominally Ca2+-free solutions. Release is expressed as percentage of total [3H]CA present in organs at end of experiments. Data are means ± SE from 4-6 different organs.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The aims of the present study were to measure levels of DA and NE in the rat CB and to characterize the processes of synthesis and release of both of these CA in response to different stimuli in vitro. We found that the rat CB is a dopaminergic organ that synthesizes CA with an apparent Km for [3H]tyrosine of ~13 µM. CA synthesis is negatively controlled by tissue CA levels and positively by cAMP-dependent mechanism(s). Turnover time for NE is double that for DA. Hypoxia and high external K+ elicit a preferential release of DA, and nicotine elicits a preferential release of NE.

Due to the small size of the rat CB (weight is 40-60 µg), there are important risks of organ damage all through the experiment. Therefore, we would like to stress at the outset that the present data were obtained in CB preparations that were very stable, as evidenced by their constant rate of protein synthesis for up to 4 h of incubation. The stability of our preparation is also supported by a Km of [3H]CA synthesis for [3H]tyrosine of 13.2 µM, comparable to that previously found in the rabbit CB (16.8 µM; Ref. 17). Finally, the observed increased rates of [3H]CA synthesis after physiological stimulation of the CB and after coincubation with DBcAMP compare well with general properties of the biosynthesis of CA in any catecholaminergic tissue (2, 14, 42).

Mean DA and NE levels found in the rat CB are in the range of 210 and 45 pmol/mg of tissue, respectively, with DA-to-NE ratios of nearly 5. The rat CB, then, is similar to the rabbit CB in being primarily a dopaminergic organ, although the rabbit CB has an absolute DA content per unit weight 3.5 times larger than the rat CB (24); in contrast, the cat CB is a mixed dopaminergic and noradrenergic organ with DA and NE contents in the range of 700 pmol/mg tissue (38). Initial rates of synthesis indicate that basal turnover rates for DA and NE are in the ranges 31-36 and 3.6-4.0 pmol · mg tissue-1 · h-1, respectively, which are about three times larger than those obtained in the rabbit CB (11.3 and 0.9 pmol · mg tissue-1 · h-1; Ref. 17). In the cat CB, the turnover rate for DA (11.6 pmol · mg tissue-1 · h-1) also is about three times smaller than in the rat; the turnover rate for NE is even smaller (38). A combination of low content and high turnover rate should imply a turnover time for DA in the rat ~11 times smaller than in the other species. In fact, the turnover time for DA found for the rat CB in the present study is 5.77 ± 0.67 h, and those reported for the rabbit and cat CB are 60-70 h (23).

The large discrepancies among reported levels, proportions, and turnover rates for DA and NE in the rat CB (see introduction) are difficult to explain. They probably result from several uncontrolled factors such as time elapsing between CB removal and assay of CA and conditions for tissue storage. But probably the factor most difficult to control is the cleaning of the CB from surrounding tissues. This is important because the rat CB lies very close to the superior cervical ganglion, a noradrenergic organ. In fact, it has been reported that ganglionectomy halved the CB content of NE (25), reduced it to 25% (13), and reduced it to <10% (31); as in the case of the rabbit CB (24), ganglionectomy did not alter DA levels in the rat CB. We have not ganglionectomized rats for further study of the metabolism of NE due to its low content, turnover rate, and hypoxic release.

Some observations in our synthesis experiments require further comment. Although the rate of synthesis is linear for up to 2 h (Fig. 2A), the actual rate of synthesis decreases between 0.5 and 2 h (y-intercept in the linear regression analysis is 4.4 pmol/mg tissue). Additionally, the rates of synthesis in the control group in Fig. 4, A and B, 49.6 and 47.6 pmol · mg tissue-1 · h-1, respectively, are slightly higher than that at 1 h in Fig. 2A. Several opposing factors contribute to these deviations: mixture of labeled tyrosine with the endogenous precursor, release of unlabeled CA during surgical manipulation of the tissues, and washout of unlabeled tyrosine during the preincubation. The lower rate of synthesis at 4 h (Fig. 2A) results from preferential release of newly synthesized CA (Ref. 21; see below), i.e., between 2 and 4 h, the newly synthesized [3H]CA are being preferentially released and replaced by analytically indistinguishable new [3H]CA while the "older" unlabeled CA are being released and replaced by [3H]CA at a much slower rate. At shorter incubation times, the small size of the labeled pool would minimize this factor. These factors would also account for the differences in the apparent kinetic parameters obtained at 2 h (Fig. 3) and 1 h (Fig. 4B) of incubation.

The findings in the release experiments confirm the preferential utilization of DA over NE in basal conditions (i.e., the faster turnover rate of DA), and demonstrate an important level of specificity of the different stimuli to mobilize DA and NE in the rat CB. Comparison of data in Fig. 2B and Fig. 5C (absolute levels of [3H]DA and [3H]NE at the end of synthesis and at the end of the release, respectively) evidences the preferential utilization of DA over NE by the CB. Thus, during the 2-h duration of the release experiment, [3H]DA levels decreased by 59% from 61.8 to 25.7 pmol/mg tissue, whereas [3H]NE levels decreased by only 32% from 7.1 to 4.8 pmol/mg tissue, i.e., the relative rate of utilization of DA is approximately double that of NE, implying that the turnover time for DA is one-half that of NE (see above). This, in turn, is consistent with the decrease in the ratio of [3H]DA to [3H]NE in the tissues from 8.6 to 5.5 during the release period. If we now compare the [3H]DA-to-[3H]NE ratios in the tissue and in the incubating solutions (~5 in basal conditions; Fig. 5B), the findings are also internally consistent in the sense that the expected ratio of [3H]DA to [3H]NE in the collected solutions should be close to 5.5: the faster turnover of DA and the preferential use of the newly synthesized CA (5, 21) would tend to decrease the ratio from 8.6 to 5.5 early during the incubation in precursor-free solution, i.e., during the washout period.

The preferential release of [3H]DA vs. [3H]NE during hypoxic stimulation has also been recognized in the rabbit and cat CBs using mild levels of hypoxic stimulation (12, 22) and, contrary to the observations of Shaw et al. (Ref. 39; see introduction), suggests a more prominent role for DA to signal the entire range of hypoxia in the CB of the three species. When it is taken into account that the ratio of [3H]DA to [3H]NE in the tissues during the release experiments (5.5) is almost identical to the ratio of the endogenous unlabeled amines (4.9; Fig. 1; i.e., the specific activities of DA and NE are comparable), the data of Fig. 5B imply that during hypoxic stimulation close to 9.7 times more DA than NE is being released. This, in turn, implies that voltammetric studies referring to the release of CA during hypoxia are in fact measuring, within a 10% error, the release of DA. A comparable situation occurs during high external K+ stimulation. On the contrary, and as is the case in the rabbit CB (22), during stimulation with nicotine there is a preferential release of NE. The mechanisms responsible for this relative specificity of the stimuli to induce the release of one or another CA are unknown, but the heterogeneity of chemoreceptor cells can satisfactorily explain the observations. The heterogeneity of chemoreceptor cells in the CB of all species is well documented (23, 40), and it is conceivable that noradrenergic cells are less responsive to hypoxia and to high external K+ (both stimuli are depolarizing) and more responsive to nicotine. Because both hypoxia-induced and high external K+-induced release depend on Ca2+ entering the cells via voltage-dependent Ca2+ channels (33, 39), our findings suggest that dopaminergic cells exhibit a higher density of O2-sensing elements, Ca2+ channels, or coupling mechanisms. In fact, López-López et al. (30) observed that only 75% of rat chemoreceptor cells exhibited significant Ca2+ currents. The higher sensitivity of NE-containing cells to nicotinic stimulation would agree with the findings of Chen and colleagues (10, 11) showing that, in the rat CB, chemoreceptor cells positive to dopamine-beta -hydroxylase (the NE-synthesizing enzyme) are the ones binding the nicotinic receptor ligand alpha -bungarotoxin. Because nicotinic receptors in rat chemoreceptor cells are permeant to Ca2+ (41), our findings imply that part of the Ca2+ required for the release of NE enters the cells via the nicotinic receptors. Adding to the notion of differential functional properties between dopaminergic and noradrenergic cells in the rat CB, Pequignot et al. (37) observed a slow onset in the change of NE turnover rate, in comparison to DA, during chronic exposure to hypoxia.

The release experiments also indicate that in the rat CB, as in the rabbit CB (33), hypoxic stimuli are more effective at activation of the release response from chemoreceptor cells than acidic/hypercapnic stimuli. This finding, in turn, parallels observations made by Fukuda et al. (20) on CSN discharges in the rat. Three corollaries derive from these observations. First, as is the case for rabbit CB (19), a parallelism exists between the release of CA (mostly DA) and the activity in the CSN, consistent with a role for DA in the genesis of neural activity. Second, the relative release responses to both stimuli also parallel the rises in intracellular Ca2+ concentration observed in chemoreceptor cells of the rats (8, 9). Third, because acidosis is as effective as hypoxia for inhibition of Ca2+-dependent K+ currents in rat chemoreceptor cells (34, 35), the relative release responses to hypoxic and acidic stimuli do not parallel the inhibition of Ca2+-dependent K+ channels produced by both stimuli. This last consideration in turn suggests that hypoxic inhibition of Ca2+-dependent K+ channels is not causally linked to chemoreceptor cell activation (see also Refs. 7, 16).

The results shown in Fig. 6, A and B, indicate that in the rat CB, as in the rabbit and cat CB (19, 33), the release of [3H]CA induced by high external K+ and hypoxia is nearly 100% dependent on the presence of extracellular Ca2+, whereas the basal release is not statistically different in the presence or the absence of Ca2+ in the incubating solution. This last finding, which is also seen in the rabbit CB (Ref. 19; see also Fig. 8 in Ref. 23), indicates that the release seen with incubating solutions equilibrated with 20% O2 (present experiments) is truly a basal normoxic release. Due to the complete Ca2+ dependence of the hypoxia-induced release of [3H]CA, if a significant part of the [3H]CA release seen during incubation of the organs with 20% O2-5% CO2-equilibrated solutions happens to come from hypoxic chemoreceptor cells, it would be suppressed in 0 Ca2+ solutions.

Finally, before reaching any conclusions, it is necessary to make some final considerations. For example, it should be mentioned that sympathetic endings in the isolated rabbit CB in vitro take up [3H]NE by a high-affinity system; therefore, it appears reasonable to assume that sympathetic endings in the isolated rat CB also remain fully functional. In the in vitro preparation, the accumulated [3H]NE is released in a Ca2+-dependent manner by high external K+, but low PO2 does not alter the ongoing basal release of [3H]NE (1). As discussed by Almaraz et al. (1), the situation in vivo should be different, since it is known that hypoxia alters the sympathetic activity in the ganglioglomerular nerves directed to the CB and thereby the release of NE from sympathetic endings (see Ref. 1). This in turn implies that the release of CA induced by hypoxia in the CB in vivo could be modulated by NE of sympathetic origin, since NE modulates chemoreceptor cell function via alpha 2- and beta -noradrenergic receptors (Ref. 1; see also Ref. 23). Overall, it might be suggested that the release of CA during hypoxic stimulation in vivo should be somehow smaller than in our in vitro preparation, since the predominant effect of sympathetic stimulation is an alpha 2-mediated inhibition of chemoreceptor activity. It might also be suggested that the ratio of DA to NE in vivo should be slightly smaller than in vitro due to increased NE release of sympathetic origin, reaching proportions comparable to those seen under high-K+ stimulation in vitro.

In conclusion, our study shows that the rat CB contains both DA and NE. Because DA is about five times more abundant than NE (leaving aside consideration of other neurotransmitter systems), it can be said that rat CB is mainly a dopaminergic organ that, in comparison with the CB of the rabbit and cat, exhibits a low DA content and a high turnover rate for this amine. Biosynthesis of CA in the rat CB exhibits kinetics and short-term control mechanisms similar to those seen in the rabbit CB and in catecholaminergic tissues in general. Hypoxia and high external K+ induce a preferential release of DA from the CB, whereas nicotine preferentially releases NE; previously described heterogeneities among rat chemoreceptor cells can satisfactorily account for these observations. The release of CA induced by low PO2 and high external K+ is nearly 100% dependent on the presence of Ca2+ in the incubating solution. Finally, our release experiments show that low PO2 is more effective than acidosis in activating the release of DA from chemoreceptor cells. This observation parallels previous studies recording CSN activity and stresses the possible causal link between the release of DA and the genesis of activity in the chemoreceptor sensory fibers (23).


    ACKNOWLEDGEMENTS

We thank Maria de los Llanos Bravo for technical support.


    FOOTNOTES

This work was supported by Dirección General de Investigación Científica y Técnica (Spain) Grant PB97-0400 and Junta de Castilla and Leon Grant VA08/99.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: C. Gonzalez, Dept. of Biochemistry and Molecular Biology and Physiology, CSIC, School of Medicine, University of Valladolid, 47005 Valladolid, Spain (E-mail: constanc{at}ibgm.uva.es).

Received 2 April 1999; accepted in final form 14 October 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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