Department of Biochemistry and Molecular Biology and Physiology, Consejo Superior de Investigaciones Científicas, School of Medicine, University of Valladolid, 47005 Valladolid, Spain
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ABSTRACT |
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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
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INTRODUCTION |
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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
tissue1 · 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.
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MATERIALS AND METHODS |
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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--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).
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.
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RESULTS |
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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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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
tissue1 · 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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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
tissue1 · 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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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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DISCUSSION |
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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
tissue1 · 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
tissue1 · 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--hydroxylase (the NE-synthesizing enzyme) are
the ones binding the nicotinic receptor ligand
-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
2- and
-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
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).
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ACKNOWLEDGEMENTS |
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We thank Maria de los Llanos Bravo for technical support.
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FOOTNOTES |
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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.
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