Endogenous adenosine-the product of degradation
of ATP-acts through several receptor subtypes: A1
(1). Among them the adenosine A1
have a variety of important functions both in physiology and pathophysiology.
They are related to: regulation of sleep, arousal, vasodilatation and neuroprotection
in hypoxia and ischaemia (2). Several published findings demonstrate that central
receptors contribute to the control of breathing. Their activation in the
brainstem slices from neonatal rats depresses respiratory activity. This corresponds
to a decrease in the amplitude of the integrated activity of inspiratory neurones
and prolongation of the respiratory cycle, primarily by lengthening inspiratory
time (3). In anaesthetized rats and cats, adenosine or A1
applied to the cerebral ventricles depress pulmonary ventilation decreasing
both the frequency of breathing and tidal volume (4 - 6). Furthermore, the release
of adenosine and stimulation of A1
receptors in the central nervous system during
hypoxia have been suggested to suppress breathing (7). These results indicate
that a central adenosine action is to diminish respiratory function.
In the peripheral nervous system of rats, A1
receptors have been identified on vagal afferent nerves and on the neurones
of the nodose ganglia (8). In this species adenosine administered systemically
elicits a delayed apnoea, followed by breathing of increased tidal volume, bradycardia
and hypotension. The respiratory arrest was ascribed to the activation of pulmonary
C-fibres, since perineural blockade of the cervical vagi with capsaicin prevented
the apnoea (9). Furthermore, right atrial injection of adenosine excites vagal
receptors in the rat
lung (10), whereas given via
an intralingual route it stimulates carotid
body chemoreceptor discharges in cats. Unfortunately respiratory parameters
were not recorded at the same time (11). In vagotomized and glomectomized cats
adenosine injected systemically acts as a central respiratory depressant (6).
An intravenous adenosine challenge in humans induces dyspnoea and increased
ventilation and heart rate, and these effects were attributed to the excitation
of vagal C-fibres (12). Conflicting evidence was reported in an earlier study
in lung-denervated humans, showing that adenosine infusion augmented minute
ventilation via stimulation of peripheral chemoreceptors (13).
The contribution of peripheral adenosine A1
receptors to respiratory reflexes is far from clear. Presumably, excitation of receptors located on vagal afferents and nodose ganglia might have a modulatory effect on respiratory pattern.
Therefore, the present experiments were performed to determine if the activation of peripheral adenosine A1
receptors with the selective agonist N6-cyclopentyladenosine (CPA) would contribute to the respiratory pattern and cardiovascular effects, and to what extent the responses depend on lung and nodosal vagal afferentations. We measured the ventilatory effects of CPA in neurally intact rats and after the elimination of vagal input from below and above the nodose ganglia. To verify if post-CPA decreased ventilation is caused by excitation of A1
receptors, we examined the effect of A1
blockade with DPCPX on the respiratory and cardiovascular responses.
MATERIALS AND METHODS
Animals and surgical procedures
Ethical approval for the experimental procedures used in this study was obtained
from the local committee. All animal procedures were in accordance with the
NIH Guide for the Care and Use of Laboratory Animals. 47 adult male Wistar rats
weighing 200-300 g were anaesthetized with an intraperitoneal (i.p.
injection of 600 mg kg-1
urethane (Sigma) and
120 mg kg-1
alpha-chloralose (Fluka AG). The animals
were placed in the supine position and they breathed spontaneously with room
air. The trachea was exposed in the neck, sectioned below the larynx and cannulated.
Catheters were inserted into the femoral vein for drug administration and supplemental
doses of anaesthetic and to the femoral artery for blood pressure monitoring.
Rectal temperature was maintained close to 36 - 38°C by a heating pad.
To investigate whether peripheral input from the lung vagi and nodose ganglia would contribute to the effects of CPA, two neurotomies were carried out. In the first series of experiments midcervical vagi were bluntly dissected and prepared for bilateral vagotomy, before measuring the respiratory variables in neurally intact rats. In the second experimental series, the rostral parts of the midcervical vagi were separated from the superior cervical ganglia. The nodose ganglia were bluntly dissected from the surrounding tissue; their blood supply was preserved intact. The supranodose vagi were prepared for section performed 2 mm distal from the rostral poles of the ganglia, before measuring the respiratory variables in neurally intact rats. In the third experimental group, the cervical vagi were sectioned at the beginning of the experiment, before CPA injection and blockade with DPCPX.
Apparatus and measurements
Tidal volume signals were recorded with a pneumotachograph head attached to
the tracheal cannula linked to a Research Pneumotach System (RSS 100 HR, Hans
Rudolph inc.) and a computerized recording system (Windows software version
3.07.02, KORR Medical Technologies Inc.) for measuring and recording respiratory
frequency (f), tidal volume (VT
minute volume (VE
), inspiratory (TI
and expiratory (TE
) times. Arterial blood pressure
was measured with a BP-2 blood pressure monitor (Columbus Instruments). The
recordings were registered on an Omnilight 8 M 36 apparatus (Honeywell).
N6-cyclopentyladenosine (CPA, Sigma) at a dose of 5 µg kg-1
dissolved in 0.9% saline was injected as a bolus to the femoral vein. 1, 2-
dipropyl-8-cyclopentylxanthine (DPCPX, Sigma) at a dose of 100 µg kg-1
(14), dissolved in dimethyl sulphoxide (DMSO) and subsequently diluted in saline
(containing no more than 0.01% DMSO), was injected intravenously. Each drug
bolus was immediately flushed with an 0.2 ml aliquot of saline.
Treatment schedule and groups
The respiratory effects of the stimulation of A1
receptors were tested using single CPA boluses in the following experimental designs: (1) in neurally intact rats and then midcervically vagotomized (n = 9); (2) in neurally intact rats and after section of the supranodose vagi (n = 14); (3) before and after the blockade of A1
receptors with the antagonist DPCPX, in rats with transected midcervical vagi (n = 9).
The baselines of each individual value of VT
minute ventilation (VE
) and respiratory rate
(f) were determined by averaging the measured variables for five respiratory
cycles before injection. The ventilatory parameters were derived from the integrated
pneumotachograph signal just before CPA injection, at the immediate post-CPA
phase, and 30 s, 60 s and 2 min after the challenge.
All experimental data were analyzed by repeated measures two-way ANOVA with time (pre-challenge and defined time points after challenge) and either innervation status (neurally intact and vagi cut, or neurally intact and nodose vagi cut) or DPCPX pretreatment (yes or no) as a between condition factor. Differences between individual time points and experimental situations were evaluated by planned contrast analysis and Tuckey's post-hoc test. In all cases, P<0.05 was considered significant. All results shown are means ± SEM
Effect of midcervical vagotomy on CPA-induced pattern of respiration
The dose of the drug used in our study was derived from preliminary dose-response
experiments. Out of three doses tested (ranging from 1-7.5 µg kg-1
5 µg kg-1
proved to be most effective and reasonably
In the neurally intact rats, intravenous CPA challenge produced uniform cardiorespiratory
effects, mainly comprising significant decreases in VT
respiratory rate and VE
, and a significant drop
in arterial blood pressure concomitant with bradycardia.
The injection of an equal volume (0.2 ml) of the vehicle (0.9% saline or DMSO-saline solution) resulted in no respiratory change irrespective of the neural state (not shown).
As shown in Table 1
, CPA promptly evoked a significant stable decrease
from the immediate post-injection phase
to the later time points, and this applied to neurally intact and vagotomized
rats (ANOVA, P<10-4
). CPA concomitantly produced
a significant reduction in respiratory rate both before and after midcervical
vagotomy (ANOVA, P<10-4
). Vagotomy by itself produced
significant increase in mean control value of VT
(P<0.001). Minute ventilation was decreased due to CPA (ANOVA, P<0.001). The
decrease in VE
appeared in both neural states
after CPA injection and persisted at 2 min post-challenge. The largest reduction
by 29 % was present at 1 min in the intact rats (P<0.01) and of 35% at 30 s
after midcervical vagotomy (P<0.001).
Ventilatory response to CPA after supranodose vagotomy
Changes in tidal volume (VT), respiratory
rate and minute ventilation (VE) after
intravenous CPA challenge in neurally intact and midcervically vagotomized
rats (n = 9).
showed: significant effect of time (P<10-4)
and time x vagotomy interaction (P<0.01) but no effect of vagotomy (P=0.2)
on VT; significant effect of time (P<10-4)
but neither vagotomy (P=0.13) nor time x vagotomy interaction effect (P=0.2)
on respiratory rate; a significant effect of time (P<10-5)
and time x vagotomy interaction (P=0.04) but no effect of vagotomy (P=0.7)
All values are means ± SEM* P<0.05, **P<0.01,***P<0.001 vs. the
respective pre-CPA value.
#P<0.05, ##P<0.01 vs. the corresponding pre-vagotomy value.
To determine, whether the nodose ganglia contribute to the response to the CPA
challenge, we tested the effect of section of the supranodose vagi on the respiratory
effects of the drug in intact rats. Cutting the central vagal trunk above the
nodose ganglia increased baseline tidal volume (P<0.01) and lowered the frequency
of breathing (P<0.001). In the current experiments this neurotomy showed a significant
interactive effect with CPA challenge on the rate of breathing (ANOVA, P = 10-5
but not on tidal volume (ANOVA, P = 0.25). As shown in Fig.1
nodosectomy CPA still evoked depression of tidal volume but the decrease in
frequency of breathing was eliminated compared with the intact state. Minute
ventilation was decreased in both neural states, being apparently more profound
in the intact animals (Fig. 1C
). In these latter, mean values of VE
after CPA challenge were lowered by 29 % (P<0.001) and scarcely reduced by 12
% (P<0.05) after supranodosal vagotomy, compared with the respective baseline
Ventilatory response to CPA after DPCPX pre-treatment
||Fig. 1. Effects of i.v.
administration of CPA and section of supranodose vagi on VT
(A), respiratory rate (B) and minute ventilation (C)
in the intact rats (n = 12).
***P<0.001, ** P<0.01, *P<0.05 versus the respective pre-CPA baseline
###P<0.001, ## P<0.01, #P<0.05 versus the corresponding pre-NG
To test whether CPA-induced effects are specific for excitation of A1
receptors, we used the highly selective A1
DPCPX in rats subjected to midcervical vagotomy. We examined the response to
CPA challenge in the control conditions and 2 min after pre-treatment
with DPCPX. As shown in Fig. 2
, the DPCPX-mediated blockade of A1
receptor prevented the CPA-evoked decreases in tidal volume and respiratory
rate. Significant inhibition of minute ventilation produced by CPA (from the
baseline of 220.8 ± 26 to 157.7 ± 20 ml min-1
occurring before the blockade (P<0.001) was eliminated after inactivation of
adenosine receptors with DPCPX. The mean
baseline value of VE
in rats pretreated by DPCPX
was 211.8 ± 15 ml min-1
and remained at 214.1
± 14 ml min-1
after CPA challenge (P = 0.9).
||Fig. 2. Effect of adenosine
A1 receptor blockade on post-CPA changes
in tidal volume (A), breathing rate (B) and minute ventilation
(C) in midcervically vagotomized rats (n = 9). Note that DPCPX
treatment prevented the decreases in VT,
VE and respiratory rate.
***P<0.001, ** P<0.01, *P<0.05 versus the respective pre-CPA baseline
###P<0.001, ## P<0.01, #P<0.05 versus the corresponding post-DPCPX
and Table 2
include data on cardiovascular effects of CPA
in all experimental conditions. Mean arterial blood pressure (MAP) displayed
a long-lasting fall (ANOVA, P<10-6
) after a CPA
challenge, attaining the lowest level at 30 s post-challenge, declining from
the pre-drug values of 106.7 ± 4.5 to 41.4 ± 3.3 mmHg (P<0.001) in the intact
rats, and from 109.8 ± 10 to 44.0 ± 4.5 mmHg treated by midcervical vagotomy
(P<0.001). Two minutes after the CPA injection, MAP began to return to the baseline,
reaching it at 15 min post-drug.
||Fig. 3. Effects of CPA on
mean arterial blood pressure (MAP) in (A) neurally intact and midcervically
vagotomized rats (n = 7), (B) intact and treated by supranodosal
vagotomy (n = 12) and (C) midcervically vagotomized prior to and
after DPCPX blockade (n = 8). Note that DPCPX treatment abolished CPA-evoked
decrease in blood pressure.
***P<0.001, ** P<0.01, *P<0.05 versus the respective pre-CPA baseline
###P< 0.001, ## P< 0.01, versus the corresponding post-NG section
and post-DPCPX value.
Effects of CPA on heart rate (HR) in (a) neurally intact and midcervically
vagotomized rats (n = 7), (b) intact and treated by supranodosal vagotomy
(n = 14) and (c) midcervically vagotomized prior to and after DPCPX blockade
(n = 8).
showed: significant effect of time (P<10-6)
but not of vagotomy (P=0.9) nor a time x vagotomy interaction (P=0.9)
on HR in intact and vagotomized rats; and significant effect of time (P<10-6)
and vagotomy (P<0.05) but no time x vagotomy interaction effect (P=0.2)
in the intact and NG cut rats; significant effect of time (P<10-6),
time x vagotomy interaction (P<10-6) and
effect of blockade (P<0.001) on HR prior to and after DPCPX.
All values are means ± SEM* P<0.05, **P<0.01,***P<0.001 vs. the
respective pre-CPA value.
#P<0.05, ###P<0.001 vs. the corresponding pre-vagotomy or pre-DPCPX
Bradycardia appeared immediately after CPA injection, both before and after
the section of midcervical vagi, showing the maximal fall between 30 and 60
s. At this latter time point heart rate started to increase slowly, returning
close to the control values within 15 min. As displayed in Table 2
, CPA evoked similar decreases in blood pressure and heart rate
in rats subjected to supranodose vagotomy.
Blockade with DPCPX significantly affected blood pressure response (ANOVA, P
= 0.0004). As indicated in Table 2
and Fig. 3
the hypotension and bradycardia that formerly occurred after CPA injection.
The present study has shown that in anaesthetized rats the predominant effect
of an intravenous injection of CPA was a short-lived depression of respiratory
rate and tidal volume and prolonged fall in blood pressure and bradycardia.
The same pattern of response was reported by Hedner et al
. (4) after intracerebroventricular
challenge with N6
(PIA) in rats.
Of the neural conditions tested in the current experiments midcervical vagal deafferentation of the lungs had no influence on the respiratory changes evoked by CPA. This observation is in general agreement with the results obtained in vagotomized and glomectomized cats, which responded with the same pattern either to the intracerebroventricular or intravenous injection of PIA (15).
The selective adenosine A1
applied by us has not been largely used in the experimental animals. Scant reports
have evidenced the decrease in VT
injection in awake rhesus monkeys (16) and no visible respiratory effects on
intravenous infusion of CPA in fetal sheep (17). The present study revealed
an immediate post-CPA decrease in VT
and f both
before and after midcervical vagotomy. This result is in contrast to the report
. adenosine in rats, which evoked a short apnoea as the only respiratory
response, effectively abolished by the blockade of A1
receptors and pulmonary C-fibres (9). Yet, the occurrence of the respiratory
depression following midcervical vagotomy in our experiments infers that CPA
might have affected the volume and timing components of the breathing pattern
through the afferent neurons of the nodose ganglia.
This is the first study assessing how much the nodose ganglia contribute to cardiorespiratory effects of adenosine A1
agonist. In rats subjected to nodose ganglionectomy, CPA administration did not affect the respiratory rate, the decline in minute ventilation was significantly lessened, compared to the intact state, while the drop in tidal volume and cardiovascular effects were still preserved.
We can therefore surmise that at least one component of respiratory response- the frequency of breathing is mediated by receptors located on the vagal nodose ganglia. This conclusion is reinforced by the visualization of A1
- radioligand binding reported in the neurones of rat nodose ganglia (8, 18).
Considering that CPA showed average transport through the blood-brain barrier
(19), its ability to decrease VT
ablation of supranodosal pathway might imply the central origin of the response.
It is further supported by the observation in cats, that adenosine given intracellularly
to the neurons of the ventral respiratory group depresses their activity, which
is reversed by blockade of A1
DPCPX (7). Adenosine analogs -CPA and L-PIA showed a similar depressant action
on the inspiratory drive in the fetal sheep and neonatal rats, in these latter
were blocked by the selective A1
In the current experiments DPCPX, the selective A1
antagonist eliminated completely the depression of breathing, fall in mean arterial blood pressure and heart rate caused by CPA, showing that adenosine A1
receptors contribute to the cardiorespiratory response. Since DPCPX crosses easily blood-brain barrier (22), we can speculate that the decrease in tidal volume described above, might have resulted from stimulation of the central A1
receptors. These were described to be widespread throughout the whole brain (2, 23).
The mechanism of adenosine-evoked cardiovascular response has been the subject of a considerable investigation. Intravenous or intracerebroventricular injection of adenosine and A1
agonists (L-PIA and CHA) has been shown to produce consistent vasodilatation, hypotension and bradycardia attributed to the release of other neurotransmitters (4, 24). Our results are in line with previous findings and more recent report on the effects of intraperitoneal CPA-evoked prolonged fall in blood pressure and heart rate, with a clear contribution of peripheral A1
receptors in rats (25).
Since in our experiments the cardiovascular response still occurred after the consecutive transection of the lung vagi and nodose ganglia, it points again to the central action of CPA. However, A1
receptors located in the heart could reasonably be of importance (26).
In conclusion, this study has shown that i.v.
challenge depresses ventilation due to the decrease in tidal volume and respiratory
rate. The hypoventilation is mediated via
excitation of adenosine A1
receptors outside the lung vagi, with contribution those of nodose ganglia to
the timing component of the breathing pattern. Predominant effect on tidal volume
and cardiovascular inhibition relays on the central A1
Acknowledgements: Mrs. Teresa Warnawin is thanked for
her excellent technical assistance.
Conflicts of interest statement: None declared.
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