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1 Unité de Néonatologie, Centre de Recherche, Hôpital Saint-François d'Assise, Université Laval, Quebec, Canada G1L 3L5; and 2 Laboratoire de Physiologie, Faculté de Médecine de Nancy, Vandoeuvre les Nancy 54505, France
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
ABSTRACT 
Bairam, A., P. De Grandpré, C. Dauphin, and F. Marchal. Effects of caffeine on carotid sinus nerve
chemosensory discharge in kittens and cats. J. Appl.
Physiol. 82(2): 413-418, 1997.
Caffeine (C)
decreases apneic episodes in premature infants and is thought to
stimulate breathing mainly by a central mechanism. While the methylxanthines theophylline and aminophylline are known to alter the
carotid chemoreceptor activity, there are little data on C. The aim of
the study was to examine the effects of C on the carotid sinus nerve
discharge (CSND) in developing animals. Nine kittens 17-21 days
old and six adult cats that were anesthetized and artificially ventilated were studied. They received four consecutive doses of C,
each of 10 mg/kg, administered at intervals of 20 min either as
intravenous bolus injection (6 kittens, 3 cats) or continuous infusion
(3 kittens, 3 cats). Bolus injections of C invariably induced a prompt
but transient increase in the CSND from 4.1 ± 0.6 to 8.1 ± 1.0 (SE) impulses/s in kittens (P = 0.01)
and from 3.9 ± 0.1 to 7.9 to 1.0 impulses/s in cats (after the
first injection). This response was associated with a significant
decrease in arterial blood pressure. Continuous infusion of C did not
induce any early change in either CSND or blood pressure in kittens or
cats. Fifteen minutes after C injection or infusion was begun, CSND
values in air, 8% O2-balance
N2, or 100%
O2 were not significantly
different from control. Haloperidol administered at the
end of the experiment in four cats and four kittens significantly
increased CSND and did not suppress the early response to C injection.
It is concluded that caffeine administered by bolus in the kitten
induces a transient stimulation of the CSND that is associated with a
decrease in the arterial blood pressure and is independent of the
dopaminergic mechanisms in the carotid body. The lack of sustained
effect implies the main mechanism to the ventilatory stimulation by C
must be central.
arterial blood pressure; chemoreceptor; dopaminergic mechanisms; hypoxia; methylxanthines
INTRODUCTION METHYLXANTHINES STIMULATE ventilation (for review see
Ref. 28) and decrease apneic episodes in premature infants (1, 3). Caffeine is the preferred drug for treating apnea of
prematurity because stable therapeutic levels may be achieved with a
single daily administration and side effects are fewer than with
theophylline (3). The ventilatory action of caffeine is thought to be
related to the direct stimulation of the respiratory neurons (13, 16, 23). The effects of caffeine are usually attributed to an antagonism with adenosine A2 receptors (28).
For instance, it is well established that the administration of
adenosine or adenosine analogues induces long-lasting depression of
ventilation in various species, including cats (12), rabbits (27), and
rats (29). Interestingly, the respiratory depression is more pronounced
in newborn than in older rabbits (27), and this effect can be prevented
or reversed by theophylline (27, 29).
Methylxanthines may also affect ventilation by altering the activity of
the carotid chemoreceptors. Indeed, caffeine failed to stimulate
ventilation in chronically chemodenervated lambs (5), and aminophylline
did not prevent the ventilatory depression elicited by hypoxia in
chemodenervated piglets (8) and enhanced the ventilatory chemoreflex
response to hyperoxia in newborn infants (9). On the other hand, the
infusion of theophylline in the carotid artery may antagonize the
stimulant effect of the adenosine analogue
N-ethylcarboxamidoadenosine (NECA)
on the carotid sinus nerve chemosensory discharge (CSND) of the cat
(26). Thus the possible effect of caffeine on CSND and the
relevance of this effect on ventilation are less well established than
the central action on the respiratory neurons. Methylxanthines exert
various systemic actions and may also act through different cellular
mechanisms, and it has been suggested that theophylline could interact
with the dopaminergic mechanisms in the central nervous system (13). Dopamine is also present in the carotid body, by which it is released in response to hypoxia (2, 10, 14), and CSND is modified by exogenous
dopamine and by blockade of the dopamine
D2 receptors (20, 22). Most
studies relative to the effect of methylxanthines on CSND have used
theophylline or aminophylline, but there is little information
regarding caffeine, the drug most currently used for clinical purposes
in premature infants. The aims of this study were thus to characterize
the effects of and to examine the mechanisms of caffeine on CSND in
developing cats.
MATERIALS AND METHODS
1 · h1)
and the second for drug administration and for the continuous infusion
of 5% dextrose in water at 1.6 ml · kg1 · h1.
Rectal temperature was maintained between 37 and 38°C with a regulated heating pad. Under a surgical microscope, one carotid sinus
nerve was dissected, desheathed, and cut near the petrosal ganglion.
The dissected area of the neck was covered with mineral oil warmed to
37°C, and the sinus nerve was separated into fine filaments that
were tested for neural signals. The signals were amplified, band-pass
filtered, fed to a window discriminator and to an audio amplifier, and
displayed on an oscilloscope. The signal output of the window
discriminator was fed to a ratemeter, and the discharge rate was
averaged every second and expressed as impulses per second. When
rhythmic baroreceptor activity was present, the filament was then
further subdivided until a few chemosensory fibers (1-4) free from
baroreceptor discharge remained. The chemosensory fibers were
identified by their random pattern of discharge, which increased in
response to hypoxia and was suppressed in response to hyperoxia. The
signals were analog-to-digital converted using a MacLab system and were
also displayed on a chart recorder (model TA 4000, Gould) together with
the raw action potentials. The same chemoreceptor fiber preparation was
used throughout an experiment in a given animal.
Drug administration.
The dose-response curve was established with four consecutive
intravenous doses of 10 mg/kg caffeine base administered 20 min apart,
according to two protocols. 1) In
six kittens and three cats, caffeine, diluted to a total volume of 0.5 ml in kittens and 1 ml in cats, was injected by hand during 30 s.
2) In three kittens and three cats,
caffeine was infused during 20 min with an electrical pump (Gemini
PC-2; IMED, San Diego, CA) at a constant rate of 3 ml/h. The effect of
haloperidol (1 mg/kg iv) followed by an injection of a fifth dose of
caffeine was tested in four cats and four kittens.
Haloperidol was diluted in saline to obtain the same volume as for
caffeine and was injected within 5 min of the completed caffeine
experiment.
Protocol.
The immediate response of CSND to increasing doses of caffeine was
studied in air, and the steady-state response was studied in air,
hypoxia (8% O2-balance
N2), and hyperoxia (100%
O2) 10 min after drug
administration. Hypoxia and hyperoxia were held 2-3
min during which time a steady-state chemosensory activity was reached
in kittens as previously reported (21). Control measurements of
arterial blood gases (AVL 995 analyzer) were obtained in normoxia.
After caffeine, arterial blood gases were measured in normoxia,
hypoxia, and hyperoxia. In addition, plasma concentration of caffeine
was assessed by a high-performance liquid chromatography method before
and 20 min after the onset of administration of each dose of caffeine
(6). Haloperidol was also injected while the animal was breathing air.
Control injections of the same volume of normal saline were also done
throughout the study.
Studies in spontaneously breathing cats.
To examine the effects of caffeine on ventilation and on carotid
chemosensory activity, two additional adult cats were anesthetized with
-chloralose (40 mg/kg) and urethan (200 mg/kg) administered intravenously. Ventilation was measured by integrating the output of a
differential pressure transducer connected to a no. 00 pneumotachograph calibrated by the integral method. In one cat, the change in
ventilation induced by caffeine was studied before and after bilateral
sectioning of the carotid sinus nerves. In the other cat, the time
course of the effect of caffeine on ventilation was studied with
reference to the change in the CSND.
Data analysis.
The analysis of CSND and mean ABP was done on the numerical data
obtained with the Mac Lab system. The immediate response to caffeine,
expressed as the peak activity, occurred within 1 min of injection and
lasted for ~5 s. The peak carotid sinus nerve activity was thus
obtained by averaging the rate of discharge over this period of time.
Similarly, the immediate ABP response was obtained over the same 5-s
epoch. The mean CSND and the mean ABP at steady state were obtained by
averaging the signals of each during 1 min. The following periods were
retained for analysis: before each dose of caffeine and the 4th and
15th min of caffeine injection in air. The steady-state CSND response
to varying oxygen concentrations was studied in air, hypoxia, and
hyperoxia before caffeine and at the 9th, 11th, and 13th min of
caffeine. The mean ABP was measured at the same time intervals. Data
are expressed as means ± SE. Statistical analysis was performed by
using two-way analysis of variance for repeated measurements. A
difference was retained as statistically significant at
P < 0.05.
RESULTS
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VT) reset every 15 s and ABP, respectively. Ventilatory response to caffeine is unaffected
by denervation.
VT, and ABP. It may be seen
that peak transient excitation of CSND induced by caffeine occurs
before peak ventilation. Note associated decrease in ABP.
Interaction between haloperidol and caffeine. The chemosensory activity before haloperidol administration was 5.3 ± 1.0 and 4.0 ± 1.3 impulses/s in four kittens and four cats, respectively [not significantly different from any control (precaffeine) values]. After haloperidol, an additional intravenous bolus of 10 mg/kg caffeine was administered to three kittens and one cat. It was found to induce the same pattern of early and transient CSND and ABP response as already described with control injections. The mean percent increase in chemosensory activity was 65.9% (range 26.7-101.3%), which was associated with a drop in ABP of 12.8 mmHg (range 6.3-21.4 mmHg). Both signals quickly returned toward baseline. In the remaining animals (1 kitten and 3 cats), caffeine was administered by continuous infusion after haloperidol. The results were also similar to those of the control caffeine infusions: there were no changes in either chemosensory discharge or ABP at any time. The mean steady-state chemosensory discharge (i.e., 20 min after the caffeine bolus or the onset of caffeine infusion after haloperidol) was 18.9 ± 5.0 impulses/s in the four kittens and 11.0 ± 3.2 impulses/s in the four cats.
DISCUSSION
This study shows that bolus injection of caffeine induces a short-lived stimulation of CSND, which appears to be independent of the dopaminergic mechanisms in the carotid body and unrelated to maturation. This effect is also dissociated from the effect of the drug on ventilation.
The methylxanthine theophylline stimulates ventilation mainly by acting on the respiratory neurons, because the effect is readily observed in the glomectomized animal (13). It is generally accepted that the interactions of methylxanthines with adenosine A2 receptors are responsible for most of their pharmacological effects (28). The depressant respiratory effect of adenosine is prevented or reversed by theophylline (27, 29), and it has been suggested that theophylline attenuated the hypoxic respiratory depression by blockade of adenosine mechanisms in the isolated brain stem-spinal cord preparation (18). In contrast, the effect of methylxanthines on the CSND is less clear cut. Intracarotid injection of adenosine and adenosine analogues have been shown to stimulate CSND in cats (24, 25). Intracarotid injection of 1 mg theophylline or aminophylline induced a short-lived decrease of the spontaneous CSND but did not prevent the stimulant effect of adenosine (24). Theophylline was even found to potentiate the excitatory effect of adenosine (24). On the other hand, infusion of theophylline into the carotid artery of the cat reduced the chemosensory effect of NECA, the adenosine analogue that most powerfully excites the CSND (24). Furthermore, 8-phenyltheophylline, an A2-receptor antagonist, more selective than theophylline, was most efficient in opposing the excitatory effect of adenosine (26). In summary, it appears that the depressant effect of methylxanthines on CSND in the cat may be attributed to their A2-receptor antagonism, but an excitatory effect may occur, independent of this mechanism. Similarly, our study on caffeine shows a consistent short-lasting excitation of the CSND, which would accordingly be independent from the A2-receptor mechanisms.
In the central nervous system, methylxanthines are known to interfere with other neurochemical substances such as dopamine (15), and there are suggestions that this interaction may account for some of their respiratory effects (13). A similar interference could be present in the carotid chemoreceptors where dopamine may act as a neuromodulator involved in the transduction of hypoxemia (17). Injection of haloperidol, predominantly a dopamine D2-receptor antagonist, is known to increase CSND. The level of CSND excitation achieved by haloperidol after caffeine in this study was similar to that previously reported in both adult cats (20) and kittens (22) without caffeine pretreatment. In this study, the excitatory effect of haloperidol on CSND was readily observed after pretreatment with caffeine, and the D2-receptor blockade had no effect on the immediate excitation induced by this drug. Thus the interference between methylxanthines and the dopamine D2 receptors described in the central nervous system (15) does not appear to play a significant role at the level of the carotid chemoreceptors.
Caffeine acts at a variety of different sites, and these effects could also contribute to chemosensory stimulation. Indeed, the increase in chemosensory activity may not necessarily be related to a direct pharmacological effect on the carotid chemoreceptors. It could for instance be explained by the sudden decrease in blood pressure, because the change in chemosensory activity was closely related to the fall in blood pressure. The latter may be responsible for a reduction in carotid body blood flow, transient but strong enough to stimulate the CSND. The effect of blood pressure on the carotid chemosensory discharge has been documented in both cats and kittens. A dramatic increase in chemosensory discharge has been reported when the mean ABP was lowered to <60 mmHg in cats (20) and to <30 mmHg in kittens (4). In the latter study, the decrease in ABP was gradual and most likely allowed for circulatory adjustments to occur, minimizing the change in carotid blood flow. This would explain why the increase in chemosensory activity was observed at rather low levels of mean ABP at steady state. In contrast, in the present study the change in blood pressure was smaller in magnitude but occurred within seconds, thus probably preventing any circulatory adjustment. The peripheral chemoreceptors and their responses to natural stimuli are known to mature with age (7, 21), but their functional immaturity could not account for the lack of response to caffeine in newborns because the adult animals showed similar effects.
While most of the ventilatory effect of caffeine is thought to be of central origin (13, 16, 23, 28), the contribution of the peripheral effect of caffeine has so far received little attention. The results of the present study show that the increase in chemosensory activity by caffeine was immediate but transient, induced by bolus but not by infusion. The effect was similar after repeated doses, despite the steadily increasing caffeine plasma concentrations. The steady-state chemosensory activity in air and the chemosensory response to hypoxia and hyperoxia were not affected by caffeine either. The ventilatory effect of caffeine was still present after both carotid sinus nerves were severed (Fig. 2), and although both ventilatory and chemosensory stimulations by caffeine could be demonstrated in the same animal preparation, the time courses of both effects were dissociated (Fig. 3). Blanchard et al. (5) showed that the respiratory effect of 10 mg/kg caffeine was abolished at an age of 82 ± 11 days in lambs that underwent bilateral chemodenervation at birth. Chronically chemodenervated animals are known to have different respiratory behavior compared with intact animals and are subject to sudden death (11). Chronic chemodenervation could well be responsible for long-term changes at the level of the respiratory neurons and for a nonspecific alteration in their responses to a variety of stimuli. Thus the lack of significant increase in ventilation after caffeine in chronically chemodenervated lambs could express the loss of the tonic carotid chemosensory drive rather than the absent effect of caffeine on the carotid chemoreceptors.
It is concluded that although caffeine stimulates carotid chemosensory discharge, the effect is short lived and appears not to be dependent on dopaminergic mechanisms or adenosine A2-receptor antagonism. A likely mechanism to explain the effect on CSND is through a modification of carotid chemoreceptor blood flow associated with the change in blood pressure. These data suggest that the effect of caffeine on the respiratory neurons is the determinant means by which ventilation is stimulated in both kittens and adult cats.
ACKNOWLEDGEMENTS
We gratefully acknowledge D. Fortier for secretarial assistance and B. Neal for skillful help in correcting the manuscript.
FOOTNOTES
Address for reprint requests: A. Bairam, Centre de Recherche, D0-717, 10 rue de l'Espinay, Québec, PQ, Canada G1L 3L5.
Received 7 March 1996; accepted in final form 15 October 1996.
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