Experiments were performed on male Hartley guinea pigs and Wistar rats (3–6 months old), fed with standard chow and water. Animals had free access to food and water and were maintained under controlled conditions of temperature, humidity and a stationary 12 h light–dark cycle. Experimental maneuvers for CH exposure (12–10% O₂ in N₂; inspired PO₂~ 85 mmHg, equivalent to ~ 4,300 m; 15 days) have been described in previous studies (Caceres et al., 2007). After experiments, animals were euthanized by a cardiac overdose of sodium pentobarbital. Protocols were approved by the University of Valladolid Institutional Committee for Animal Care and Use following international laws and policies (European Union Directive for Protection of Vertebrates Used for Experimental and Other Scientific Ends (2010/63/EU).

Ventilation was measured in freely-moving animals by whole body plethysmography described by Olea et al. (2014) using methacrylate chambers (Emka Technologies, Paris, France; BUXCO Research Systems, Wilmington, NC, USA) continuously fluxed (2 l/min) with the desired gas mixture. Temperature was maintained within the thermo-neutral range (22–24°C). Body temperature was not constantly registered and it was assumed to be constant in both species during the plethysmography recordings. Tidal volume (TV; ml/Kg), respiratory frequency (BPM; breaths/min), and minute ventilation (MV; ml/min/Kg) were assessed. Animals breathed room air until acquiring a standard resting behavior. Basal ventilatory parameters and acute hypoxic and hypercapnic test response (12, 10, and 7% O₂, remainder N₂, and 5% CO₂ in air were applied during 10 min followed by 15 min of recovery breathing air after each test; the analyzed recordings are the last 5 min of each tests) were measured. Pressure changes within the chamber reflecting tidal volume were assessed with a high-gain differential pressure transducer. Amplitude of pressure oscillations is proportionally related to TV; a calibration of the system by injections of 5 ml air into the chamber allowed a direct estimation of TV. Pressure signals were stored for visualization and offline analysis with Buxco software.

Mean arterial blood pressure (MABP) was recorded from animals anesthetized (Ketamine 100 mg/Kg and diazepam 2 mg/Kg i.p.), tracheostomized and ventilated with room air (CL Palmer) (60 cycles min–1 and a positive end-expiratory pressure of 2 cm H₂O) or with hypoxic mixture (10% O₂, and 90% N₂). MABP was continuously monitored with a catheter inserted in the right common carotid artery. The catheter was connected to a pressure transducer (Statham) and signals stored (Power Lab 16SP; AD Instruments Castle Hill, Australia) for later analysis. In these animals, blood gases were obtained in normoxic (air) or hypoxic (10% O₂) breathing conditions from a small (0.3 ml) blood sample (ABL, Radiometer Medical A/S, Denmark). Glucose and lactate content were measured from the same blood samples withdrawn when animals were breathing the different gas mixtures described above and analyzed with glucose (Ascensia Breeze 2, Bayer) and lactate meters (Lactate Pro, Arkray).

Animals were perfused by gravity (1 m column) through the left ventricle with 150–200 ml of phosphate-buffered saline (PBS; 10 mM; pH = 7.4) at 37°C, followed by 250 ml of 4% (v/v) paraformaldehyde in 0.1 M phosphate buffer (PB; pH = 7.40) at 4°C. Carotid bifurcations were removed, CB cleaned and postfixed for 1 h in 4% paraformaldehyde in PB and transferred to 30% (w/v) sucrose in PB for cryoprotection. After embedding in Tissue-Tek® (Sakura Finetek, Zoeterwoude, The Netherlands) CB were frozen at –20°C. Serial sections 10 μm thick (Leitz Cryostat 1720) were collected in glass slides coated with 3-aminopropyltriethoxy-silane (Sigma, Spain). Sections were washed in PBS at room temperature and incubated in PBS containing 0.1% (v/v) Triton X-100 and 2% (v/v) nonimmunized goat serum (permeabilizing blocking solution) for 30 min. Incubation with the primary antibody (mouse anti-tyrosine hydroxylase; Sigma-Aldrich, Spain) at a dilution of 1: 1000 in permeabilizing blocking solution was carried out at 4°C overnight. After washing with PBS, sections were incubated with secondary antibody (goat anti-mouse–FITC; Sigma, Spain) and DAPI. Finally, sections were washed with PBS and distilled water and mounted in an aqueous-base mounting medium (Vectashil, Vector Laboratories). Negative controls were similarly incubated in absence of primary antibody. Sections were examined with fluorescence microscope (Axioscop 2, Zeiss) equipped with excitation and emission filters for FITC. Dissociated type I cells were similarly stained for TH as previously described for rat type I cells (Caceres et al., 2007). Images (10×; Phan-Neofluor) were captured with a CoolSnap camera and analyzed using Metamorph 6.3 software. Tyrosine hydroxylase-positive areas (TH-positive) and the entire area of the CB tissue were measured in each section to calculate the enzyme positive percentage area.

For analysis of endogenous catecholamine (CA) content, organs (CB or superior cervical ganglion; SCG) were removed from anesthetized animals, glass to glass homogenized (0.1 N PCA and 0.1mM EDTA), centrifuged, and processed for HPLC analysis (Waters 600 controller pump, automatic injector Waters 717 plus Autosampler, and BAS LC-4C Amperometric Detector; Gemeni® C-18, 5 μm column, Phenomenex; Mobile phase: 25mM Na₂HPO₄, 0.65 mM 1-octane sodium sulfonate acid, 0.1mM EDTA, pH 3.46; MeOH 6%). Catecholamine identification was made against external standards and quantification was made with Peak Sample Data Chromatography System software (Buck Scientific, East Norwalk, CT).

General procedures for studying the ³H-CA synthesis rates have been previously described (Olea et al., 2014). Organs were incubated (37°C; 2 h) in Tyrode solution (in mM: NaCl, 140; KCl, 5; CaCl₂, 2; MgCl₂, 1.1; HEPES, 10; glucose, 5; pH 7.40) containing 30 μM of 3,5-³H-tyrosine (CA natural precursor; 6 Ci/mmol; Perkin Elmer) and 100 μM 6-methyl-tetrahydropterine and 1 mM ascorbic acid, cofactors for tyrosine hydroxylase and dopamine beta hydroxylase, respectively. Tissues were then washed in precursor-free Tyrode (4°C; 5 min) and homogenized. Identification of ³H-CA was carried out against internal standards at the same conditions as above and quantified by collecting the HPLC column effluents and scintillation counting.

To evaluate stimuli-evoked secretory response, isolated CB were incubated 2 h with ³H-tyrosine of high specific activity (40–50 Ci/mmol) and later transferred to vials containing the precursor-free Tyrode solution (in mM: NaCl, 116; KCl, 5; CaCl₂, 2; MgCl₂, 1.1; HEPES, 10; glucose, 5; NaCO₃H, 24). The solution was equilibrated with gas mixtures containing 5% CO₂ and different percentages of O₂ (pH 7.40). When CB were incubated with high K⁺ solutions, osmolarity was maintained by removing an equimolar amount of NaCl. The hypercapnic-acidosis stimulus consisted in the same solution at pH 6.8 attained by bubbling with 20% CO₂/20% O₂, rest N₂. Mitochondrial respiratory chain blockers (rotenone and Na-azide) and the mitochondrial uncoupler 2,4-dinitrophenol used as pharmacological stimuli were added to the incubating solution (drugs were obtained from Sigma, Madrid, Spain). Incubating solutions were renewed and collected every 10 min and their content in ³H-CA measured by scintillation counter. Experimental protocols and analytical procedures for measurement of ³H-CA release have been described in detail in previous publications (Gomez-Niño et al., 2009a).

Levels of cAMP were measured according to previously described protocols (Cachero et al., 1996). CB were pre-incubated (15 min, 37°C) in Tyrode solution equilibrated with 95% O₂/5% CO₂ that was renewed with the same incubating solution containing 0.5 mM isobutylmethylxanthine (IBMX, Sigma-Aldrich, Spain). Incubating solutions were equilibrated with either 95% O₂/5% CO₂ (basal) or 7% O₂/5% CO₂/ 88% N₂ (hypoxia). After 30 min. incubation, CB were weighed, homogenized in ice-cold 6% trichloroacetic acid and centrifuged (12,000 × g, 10 min, 4°C). Supernatants were extracted 3 times with water-saturated diethyl ether; the aqueous phase lyophilized and dried samples stored at −20°C until cAMP was assayed. An EIA commercial kit, following the instructions of the supplier (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) was used. Levels of cAMP are expressed as pmole/mg tissue.

CB were enzymatically dispersed and dissociated cells were plated on poly L-lysine-coated coverslips maintained in culture for up to 24 h as described previously (Gomez-Niño et al., 2009b). After fura-2 loading (10 μM; Pluronic F-127; Molecular Probes; 20°C, 1 h) coverslips were mounted in a perfusion chamber, placed on the stage of a Nikon Diaphot 300 inverted microscope and cells superfused with Tyrode solution containing 116 mm NaCl, 5 mm KCl, 1.1 mm MgCl₂, 2 mm CaCl₂, 25 mm NaCO₃H, 10 mm glucose, 10 mm Hepes (pH 7.4 bubbling with 5% CO₂; 20 O₂; 75% N₂). Dual wavelength measurements of fura-2 fluorescence were performed, using the two-way wavelength illumination system DX-1000 (Solamere Technology Group, Salt Lake City, Utah) with a 100 W Hg lamp as the light source (Optiquip, New York, NY). Light was focused and collected through a Nikon Fluor 40/1.30 objective. The wavelength for dye excitation was alternated between 340 and 380 nm, and fluorescence emission at 540 nm was collected with a SensiCam digital camera (PCO CCD imaging, Kelheim, Germany) driven by an Axon Imaging Workbench 4.0 (Axon Instruments, CA, USA). Hypoxia and 35 mM KCl were used as stimuli.

Guinea pig CB were enzymatically dissociated and under phase contrast microscope, isolated type I cells were identified by the rounded shape, brightness and size. Single cells were voltage-clamped and membrane currents were measured using the whole-cell configuration of the patch clamp technique by using an Axopatch-200B amplifier (Axon Instruments, Burlingame, CA, U.S.A) as previously reported (Frazziano et al., 2011). Currents were filtered at 3 kHz and digitalized with a Digidata 1200 analog-to-digital converter (Axon Instruments). Cells were superfused at 2 mL/min with an external Hepes solution containing (mmol/L): NaCl 141, KCl 4.7, MgCl₂ 1.2, CaCl₂ 1.8, glucose 10, and HEPES 10 (adjusted to pH 7.4 with NaOH). The composition (in mmol/L) of the pipette solution was: KCl 125, MgCl₂ 4, MgATP 5, NaGTP 5, HEPES 10, EGTA 10, pH adjusted to 7.2 with KOH. Currents were evoked following the application of 50 ms depolarizing pulses from −60 mV to test potentials from −60 to +60 mV in 10 mV increments as described (Riesco-Fagundo et al., 2001). All the currents were normalized for cell capacitance and expressed in pA pF-1. Current-voltage relationships were constructed by measuring the currents at the end of the pulse. Cells were exposed to hypoxia for 10 min. Hypoxia was induced by bubbling the Hepes solution with 100% N₂ to achieve an oxygen concentration of 3–4% (24 ± 1 Torr) in the chamber. All experiments were performed at room temperature (22–24°C).

Data are presented as mean ± S.E.M. Statistical analysis was performed by paired or unpaired Student's t-test and by repeated measures One-Way Analysis of Variance (ANOVA) with Dunnett's multiple comparison tests; p < 0.05 was considered statistically significant.

To assess the ventilatory response to several acute hypoxia tests (12, 10, and 7% O₂; 10 min), continuous recording of the respiratory frequency and tidal volume (TV) were made while switching from air to the gas mixture in 16 guinea pigs and 20 rats. Figures 1 A,B shows that MV breathing room air was similar in both groups of animals. Figure also shows the effect of acute hypoxic tests on MV, increasing significantly only after intense acute hypoxic test (7% O₂) in guinea pigs. However, MV significantly increased in rats breathing any hypoxic mixture. Figures 1C,D shows guinea pigs respiratory frequency (92 ± 3 breathing air vs. 104 ± 4 breaths/min breathing 7% O₂) and tidal volume increasing significantly with the more intense hypoxia (4.43 ± 0.18 in air vs. 5.55 ± 0.17 ml/kg in 7% O₂). Acute hypercapnic test (5% CO₂ in air; 10 min) significantly increased MV in guinea pigs and rats, augmenting ventilatory parameters similarly in both species (108 ± 3 and 137 ± 4 breaths/min; 9.80 ± 0.42 and 8.01 ± 0.21 ml/kg in guinea pigs and rats, respectively).

To assess the effects of acute hypoxia on other cardiorespiratory and metabolic parameters, arterial blood gases and mean arterial blood pressure were measured. Table 1 shows MABP from animals breathing in normoxic (air) and hypoxic (10% O₂; 10 min) conditions. MABP in guinea pigs (50 ± 3 mmHg) were lower than in rats (115 ± 14 mmHg) in normoxia, showing that guinea pigs are hypotensive compared to rats. Breathing the hypoxic mixture induced a decline of MABP in both species (32% decrease in guinea pigs and 56% decrease in rats), more intense in rats.

Table 1 also shows levels of different parameters obtained from small arterial blood samples taken at the end of the normoxic or hypoxic periods to monitor pO₂, pCO₂, glucose, and lactate. Profiles of arterial pO₂ are comparable in both species but in all conditions are lower in guinea pigs than in rats. Arterial blood pO₂ was 60 ± 3 mmHg in guinea pigs and 85 ± 3 mmHg in rats breathing air. The same parameter during hypoxic breathing (10% O₂; 10 min) decreased to 20 ± 1 mm Hg and 39 ± 2 mm Hg in guinea pigs and rats showing that guinea pigs are hypoxemic compared to rats. Values of pCO₂ were similar when animals were breathing air (32 ± 1.7 and 32 ± 2 mm Hg in guinea pigs and rats) and there were not significant differences when breathing in hypoxic atmosphere.

In the same blood samples glucose and lactate were measured. Plasma glucose levels in normoxia were very similar in both species (140 ± 11 vs. 147 ± 16 mg/dL in guinea pigs and rats, respectively). Hypoxia augmented glucose levels to 195 ± 17 mg/dl in guinea pigs and the increase was even larger in rats (229 ± 20 mg/dL). Lactate plasma variations were in concordance with those of glucose. In guinea pigs breathing the hypoxic mixture lactate rose from 0.92 ± 0.06 to 1.87 ± 0.38 mmole/l. Normoxic lactate levels in rats were higher than in guinea pigs (1.66 ± 0.17 nmole/l) and hypoxia increased to 4.8 ± 0.4 nmole/l. These data show that hypoxia is less effective increasing blood glucose and lactate levels in guinea pigs than in rats.

Due to the poor guinea pig ventilatory response to acute hypoxia CB function was analyzed. Since CB are catecholaminergic organs immunostaining for TH was performed. Figure 2 shows CB immunostaining for TH in both species. TH immunopositive area was much smaller in guinea pig than in rat CB (Figure 2A). Quantitative TH-positive areas from 64 sections (10 μm) obtained from 4 guinea pig CB and 150 sections from 4 rat CB show the lower TH-positive area in guinea pigs, 1.07 ± 0.05% vs. 25 ± 2% in rat of total CB area sections (n = 4; Figure 2B). Figure 2C shows chemoreceptor cells dissociated from CB cultures and immunostained for TH. Carotid body TH-positive type I cells obtained from 4 different cultures of 4 guinea pigs (604 cells nuclei;) and from 4 different cultures of 4 rats (770 cells nuclei;) were counted. The percentage of TH-positive CB type I cells was 11 ± 2.4% and 45 ± 4.8% in guinea pig and rats, respectively (n = 4; Figure 2D).These data indicate that the number of chemoreceptor cells is much lower in guinea pigs than in rats.

The small percentage of chemoreceptor cells present in guinea pig CB drove us to investigate their functional properties. Endogenous CA content is shown in Table 2. Norepinephrine (NE) and dopamine (DA) levels from guinea pig CB in normoxia were 0.80 ± 0.10 and 1.6 ± 0.4 pmole/CB, much lower than in rat (4.57 ± 0.32 and 16.27 ± 1.47 pmole/CB of NE and DA, respectively). Since CB weight is slightly higher in guinea pigs than in rats (54 ± 2 μg in guinea pig and 48 ± 2 μg in rat), it can be estimated that guinea pig CB has approximately 1/6 of NE content and less than 1/10 of DA content compared to rat CB, according with the density of TH-positive CB cells.

Chronic hypoxia increased NE content to 1.2 pmole and DA content to 10 pmole in guinea pig CB. The increase induced by chronic hypoxia in rats was approximately 4 times for NE and 7 times for DA. Table 2 also shows NE and DA synthesis rate from both species measured in normoxia and chronic hypoxia. CB from guinea pigs breathing air had a synthesis rate of ³H-NE and ³H-DA of 0.23 and 0.16 pmol/CB/2h. Values were 0.18 and 2.8 pmole/CB/2h in rats breathing air. Chronic hypoxia exposure (10% O₂; 15 days) induced a ³H-NE and ³H-DA synthesis rate augmentation of 0.35 and 0.89 pmole/CB/2h in guinea pigs and synthesis rate increased to 0.4 and 11 pmole/CB/2h of ³H-NE and ³H-DA, respectively, in rats. In both species ³H-DA synthesis increased significantly in chronic hypoxic conditions while ³H-NE synthesis increased significantly only in rat CB. The turnover rate (content/synthesis rate) of DA and NE shows no change in guinea pigs but was double in rats CB exposed to chronic hypoxia. These data show that guinea pig CB contained 10 times less DA than rat CB and it used it faster than rat; it also contained 1/6 of rat NE and used it slightly faster than rat per time unit, probably using NE that would be contained in intraglomic sympathetic nerve endings. Since an important part of NE in the CB is contained in sympathetic nerve endings from SCG (Mir et al., 1982; Chen et al., 1997) the same CA parameters measured in CB and under the same conditions were studied in SCG (Table 2). Data showed that content and synthesis rate of NE from guinea pigs exposed to chronic hypoxia are increased and there is a 30% decrease of turnover rate, implying that sympathetic nerve endings would be pouring more NE to blood in chronic hypoxic animals. A similar decrease (50%) happened with DA turnover rate. Conversely, NE and DA content and synthesis are not significantly modified in rat SCG after chronic hypoxic treatment.

It is known that hypoxia and other natural and pharmacological stimuli activate CB inducing the release of CA from mouse, rat, rabbit, and cat CB (Gonzalez et al., 1994). This activity index was used to measure CA secretion from guinea pig CB in response to different stimuli. Figure 3 shows that hypoxia (7% O₂) did not modify ³H-CA evoked release from neither normoxia nor chronic hypoxia isolated guinea pig CB. Conversely, the same stimulus evoked a ³H-CA release of 3.2% ± 0.3 of tissue content from normoxic rat CB that was even larger in chronic hypoxia rat CB (7.1% ± 0.6). As shown in Figure 3C, CH exposure, a condition that sensitize CB and induce CB hypertrophy in other species did not modify guinea pig CB size, agreeing with the lack of CB sensitization to hypoxia.

The same lack of guinea pig CB ³H-CA evoked release response was found when other natural (2% O₂, hypercapnia/acidosis; Table 3) or the classical pharmacological stimuli (mitochondrial respiratory chain blockers rotenone 1 μM and Na-azide 5 mM or the uncoupler DNP 0.5 mM) were used (data not shown). Only the non-specific depolarizing high extracellular K⁺ stimulus was able to induce a ³H-CA release response comparable in both species. However, when all those stimuli were applied to normoxic or chronic hypoxic rat CB a significant increase of CA release response was observed (Gomez-Niño et al., 2009a).

Figure 4 shows the changes in intracellular Ca²⁺ levels from type I cells obtained from 3 guinea pig and 2 rat CB in response to hypoxia and high extracellular K⁺. Hypoxia did not change the intracellular Ca²⁺ levels in guinea pig isolated type I cells, while the same hypoxic exposure led to reproducible increases in Ca²⁺ levels from isolated rat type I cells. However, TH-positive cells from both, guinea pig and rat CB, when challenged with a pulse of 35 mM K⁺, responded with a brisk increase in intracellular Ca²⁺ which reached comparable peaks, followed a similar time course and exhibited an overall similar integrated amplitude. Figure 4D shows acute hypoxic effects (7% O₂) on levels of cAMP obtained from guinea pig and rat CB. Levels of cAMP were significantly increased after hypoxia exposure in rat CB but not in guinea pig CB, implying a lack of this second messenger pathway activation by hypoxia.

The inhibition of K⁺ channels is considered an initial step in the response of type I cells to a decrease in PO₂ (López Barneo et al., 1988; Peers, 1990; López-López et al., 1997). We examined the presence of this first element of the transduction cascade on guinea pig and rat type I cells. We found that the majority of the guinea pig CB dissociated cells were insensitive to hypoxia (11 out of 12 cells from 3 different animals (Figure 5A). Conversely, hypoxia inhibited K⁺ currents in most of rat type I cells (6 out of 9 cells from 2 different animals, Figure 5A). Figures 5B,C show representative experiments of hypoxic insensitive guinea pig CB cells and hypoxic sensitive rat CB cells, respectively. The current voltage relationships of these representative experiments are also shown. The inhibitory effect of hypoxia in rat type I cells reached statistical significance at potentials more positive than −10 mV. Only one of the guinea pig chemoreceptor cells responded to hypoxia with an inhibition of the K⁺ currents at +60 mV of about 25%.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.