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Apnoeic response to stimulation of peripheral GABA receptors in rats
Respiratory Physiology & Neurobiology 131 (2002) 189– 197 www.elsevier.com/locate/resphysiol
Apnoeic response to stimulation of peripheral GABA receptors in rats Katarzyna Kaczyn´ska *, Małgorzata Szereda-Przestaszewska Laboratory of Respiration Physiology, Department of Neurophysiology, Polish Academy of Sciences Medical Research Centre, 5 Pawin´skiego Street, 02 -106 Warsaw, Poland Accepted 14 March 2002
Abstract Respiratory effects of intracarotid injection of g-amino-butyric acid (GABA) were investigated in two groups of rats. In the first group of 12 rats the effects of GABA were checked in the intact state, following bilateral vagotomy and GABA receptor blockade. The second group consisted of five initially vagotomized rats, challenged with GABA prior to and after bilateral carotid chemodenervation (CSN-cut). All rats were urethane and chloralose anaesthetized and spontaneously breathing. Injection of 39 mmol/kg GABA prior to and after vagotomy induced an expiratory apnoea of, respectively 5.5 90.84 sec and 3.9 9 0.6 sec duration (mean 9 S.E.M.), P\ 0.05 in all 12 rats. In breaths that followed the apnoea tidal volume increased above the control level by 23.3% (P B0.01) and 25.6% (P B0.01) preand post-vagotomy, respectively. Blockade of GABA receptors with bicuculline and picrotoxin abolished the inhibition of breathing. In five vagotomized rats with intact carotid sinus nerves (CSNs) intracarotid GABA challenge increased tidal volume by 39% compared with baseline breathing (PB0.05). Section of the CSNs precluded the occurrence of apnoea and undergoing respiratory changes evoked by GABA. Intracarotid GABA caused significant decrease in the mean blood pressure independent of the neural state, but the fall was delayed by CSNs neurotomy. Results of this study indicate that GABA given systemically induces apnoea followed by post-apnoeic hyperventilation. Carotid bodies are required for the ventilatory response to GABA; vagal afferents are not involved in this response. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Control of breathing; apnoea; GABA; Mammals; rat; Pharmacological agents; bicuculline; picrotoxin; Nerve; carotid sinus; vagus; Ventilation; intracarotid GABA
1. Introduction g-Amino-butyric acid (GABA) is the most important inhibitory neurotransmitter in the verte* Corresponding author. Tel.: +48-22-608-6522; fax: + 4822-668-5532 E-mail address: [email protected] (K. Kaczyn´ska).
brate CNS. Neuroanatomical studies in cats have shown its presence in the interstitial nucleus of the nucleus tractus solitarius (NTS) and in the dorsal vagal nucleus (Dietriech et al., 1982). Immunohistochemical estimation revealed the presence of GABA immunoreactive fibers in contact with respiratory neurons of the NTS (Lipski et al., 1990). Intracerebroventricular or ventral medullary sur-
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face application of GABA or its agonists affects the respiration influencing the ventilatory subdivisions of the respiratory pattern in a variable way. Injection of GABA into the cisterna magna or administration on the ventral medullary surface of cats evoked the depression of tidal volume only (Yamada et al., 1982). Baclofen a GABAB agonist given intracerebroventricularly or intravenously decreased the minute ventilation, affecting mainly the respiratory rate (Da Silva et al., 1987). In rats, GABA applied intracerebroventricularly evoked an apparent respiratory depression of decreased tidal volume and frequency of breathing. This was not preceded by respiratory arrest. This response was reversed by an intravenous injection of bicucculline (Hedner et al., 1981, 1984). GABA has been likewise identified in the vagal nodose ganglia of cats (De Groat, 1972), within sympathetic ganglia of rats, cats and rabbits (De Groat, 1970; Bowery and Brown, 1974) and in cats’ spinal ganglia (Gallagher et al., 1978). Due to the presence of GABA receptors on nonmyelinated C fibers of airway cholinergic nerves of rats and guinea-pigs (Brown and Marsh, 1978; Chapman et al., 1993), one may assume this neurotransmitter contributes to respiratory lung reflexes. It has been shown that intravenous GABA injection stimulates respiration in cats and rabbits but depresses it in dogs after transient apnoeic spells (Elliot and Hobbiger, 1958). Rat was the only species that regularly responded to systemic GABA challenge with apnoea, followed by renewed breathing through hyperpnoea or tachypnoea (Holzer and Hagmu¨ ller, 1979; Billingsley and Suria, 1982). Since GABA does not readily cross the blood– brain barrier (Purpura et al., 1958), studies using systemic administration can be regarded as a search for peripheral effects. To the best of our knowledge, there has not been any study quantifying the presumed ventilatory depression evoked by peripheral GABA challenge. Evidence shows that intracarotid GABA injection could produce a prolonged apnoea (Holzer and Hagmu¨ ller, 1979). The question arises whether the input from carotid bodies modifies GABA-induced respiratory reflex.
The present study started with the hypothesis that the respiratory depression induced by systemic administration of GABA depends on the carotid bodies input without the contribution of vagal afferents. This hypothesis has been tested by measuring the ventilatory effects of GABA in neurally intact rats and after elimination of vagal and carotid body afferent inputs. Additionally, we antagonized the respiratory depressant effects of activation of GABA receptors employing bicuculline and picrotoxin.
2. Methods A total of 17 male Wistar rats weighing 250– 320 g, were anaesthetized with an intraperitoneal injection 600 mg/kg of urethane (Sigma) and 120 mg/kg of chloralose (Fluka AG). Following induction of anaesthesia, supplementary doses were given when necessary as assessed by the flexion withdrawal reflex and blood pressure response to paw pinch. The animals breathed spontaneously during the whole experiment. Body temperature was kept at 38 °C by a heating pad. End-tidal CO2 was continuously monitored with an Engstrom Eliza Plus capnograph (Gambro). Tidal volume (VT) was obtained by integrating the flow signal from a model C56 pneumotachograph (Mercury) attached to the tracheal cannula. The right femoral artery was catheterized and connected to a pressure transducer (CK 01 Mera Tronik) and blood pressure monitor (MCK 411) for the measurement of systemic blood pressure. A similar catheter was inserted into the left femoral vein for the administration of supplemental doses of anaesthetic and injection of the drugs. For GABA injections, the right common carotid artery was tied off and the catheter was inserted cephalad pointing towards the sinus. The midcervical segments of vagal nerves were isolated and prepared for cutting. In five out of seventeen rats treated by vagotomy, the carotid region on both sides was dissected under an operating microscope. The larynx and the oesophagus were reflected to expose both carotid artery bifurcations. The carotid sinus nerves (CSNs) were cut at their junctions with the glossopharyngeal
K. Kaczyn´ ska, M. Szereda-Przestaszewska / Respiratory Physiology & Neurobiology 131 (2002) 189–197
nerves. Electromyograms of the costal diaphragm were recorded with bipolar electrodes. The activity was amplified (1000– 5000x) with a NL 104 amplifier (Digitimer), band-pass filtered (50 Hz– 50 kHz) and measured with a model AS 101 (Asbit) leaky integrator (time constant= 100 msec). All recordings were registered on an Omnilight 8 M 36 apparatus (Honeywell). After control values of respiratory variables were taken, g-amino-n-butyric acid (GABA), Sigma, 39 mmol/ kg or 4 mg/kg bolus was delivered in 0.2 ml aliquots of physiological saline and flushed with 0.2 ml of the saline via a catheter placed in the right common carotid artery. Saline solutions of bicuculline methiodide, Sigma, 2 mmol/kg or 1 mg/kg and picrotoxin, Sigma, 0.8 mmol/kg (0.5 mg/kg) were administered intravenously, 2 min prior to GABA injections. The doses of GABA used in these experiments were derived from establishing dose–response relationships in preliminary experiments. The doses of picrotoxin and bicuculline were chosen from studies of other investigators who monitored respiratory and cardiovascular effects of GABA in rats (Holzer and Hagmu¨ ller, 1979; Vemulapalli and Barletta, 1984). Test i.v. injections of 0.2 ml aliquots of physiological saline showed no volume effect(s). At least 15 min were allowed between injections. The experimental protocol was approved by the local animal care committee. The respiratory effects of GABA challenge were recorded in 12 rats of the first series of experiments: (1) neurally intact, (2) subsequently vagotomized, and (3) after intravenous administration of either bicuculline or picrotoxin in midcervically vagotomized rats. In the second series of experiments, GABA was injected in 5 vagotomized rats prior to and after section of the CSNs. Each individual value of VT, ventilation (V: E) and respiratory rate (f) was taken as an average over five consecutive breaths. The ventilatory parameters were assessed prior to the GABA injection, during apnoeic phase and at 30 sec after the challenge. The expiratory time (TE) was determined from the record of integrated diaphragm activity. TE prolongation was measured as the ratio of maximal TE during post-GABA apnoea (TE TEST) to the respective control TE value (TE
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CONTROL).
The duration of apnoeic period in diaphragmatic activity was measured as the time of apnoea (respiratory inhibition). VT, V: E, f and TE were analyzed by two-way ANOVA with postGABA time (0, apnoeic phase, recovery= 30 sec) and denervation status (intact+ vagi cut, vagi cut + bicuculline, vagi cut+picrotoxin, vagi cut + CSNs cut) as repeated measures’ factors. TE prolongation data were analyzed by oneway ANOVA with denervation status as repeated measures factor. Differences between individual time points and experimental situations were evaluated by contrast analysis. In all cases, PB0.05 was considered significant. All results are means9 1 standard error.
3. Results Intracarotid GABA challenge produced uniform cardiorespiratory effects, comprising an apnoea followed by breathing of increased tidal volume and a fall in arterial blood pressure, in intact and vagotomized rats. There were no respiratory effects when similar volumes of the solvent were administered. GABA injected at a dose of 4 mg/kg evoked expiratory apnoea of mean duration of 5.590.84 sec (intact) and of 3.990.6 sec (vagotomized rats), (P\0.05, n =12). After recovery from apnoea, the tidal volume increased in all animals (ANOVA, P=0.000004). The mean increase in tidal volume induced by GABA was significant in reinitiated breathing compared to pre-drug controls, both in intact and vagotomized rats (Table 1A). In the apnoeic pause, the expiratory time was significantly elongated; the mean prolongation of TE being 7.29 1.1-fold and 5.19 0.9-fold prior to and after vagotomy, respectively, indicating similar levels of expiratory inhibition in both neural states (P \0.05). Intracarotid GABA injection evoked a decrease in the respiratory rate at the early post-challenge phase but the effect was borderline and statistically insignificant between the intact (P= 0.06) and vagotomized rats (P= 0.07). Minute ventilation was affected by GABA (ANOVA, P= 0.0008) and increased at 30 sec after injection from the respective control values of 157.69 9.7 to 196.89 12.0 ml/min in
Vagi cut vagi+CSNs cut
B
1.849 0.3
2.1290.32
5
2.749 0.16 3.59 0.3a
2.349 0.32
0
0 0
Apnoea
65.6 9 8.2**
76.6 98.8c
2.26 9 0.31
72.2 9 9.9
93.4 9 10
2.56 90.38**
96.0 9 7.6* 105.4 9 6.3* 68.6 9 9.5*
100 97.9* 102.4 9 5.2*
121.1 96.1 127.9 95.6
3.38 9 0.22* 4.36 90.4*,b
Recovery
80.6 9 10.4*
Apnoea
Recovery
After GABA
Baseline
Baseline
After GABA
MAP (mmHg)
VT (ml)
5
12 12
N
1.28 90.15
0.97 90.09
0.67 9 0.04 0.65 9 0.03
Baseline
TE (sec)
All values are means 9 1 S.E.M., *PB0.01, **PB0.05 vs. the respective pre-GABA value. a PB0.05. b PB0.01 vs. the corresponding pre-vagotomy value. c PB0.05 vs. the corresponding pre-carotid neurotomy value (two-way ANOVA followed by planned contrast analysis).
Intact vagi cut
A
Innervation status
Table 1 Changes in tidal volume (VT), mean arterial pressure (MAP) and expiratory time (TE) after intracarotid GABA challenge
1.14 9 0.11c
6.44 9 1.17**
4.79 9 0.8* 3.46 9 0.6*
Apnoea
After GABA
1.2490.12
1.0 9 0.09
0.58 90.04 0.63 9 0.02
Recovery
192 K. Kaczyn´ ska, M. Szereda-Przestaszewska / Respiratory Physiology & Neurobiology 131 (2002) 189–197
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Fig. 1. Effect of GABA receptors blockade on the mean prolongation of TE induced by GABA in vagotomized rats (mean91 S.E.M., n =7). **PB 0.01, vs. the respective preblockade values (one-way ANOVA).
intact rats (PB 0.01) and from 190.3918.2 to 234.39 12.3 ml/min after vagotomy (P B 0.05). Bicuculline and picrotoxin appeared to be effective in antagonizing the respiratory response to intracarotid GABA injection. It is evident in Fig. 1 that blockade of GABAA receptors by both substances reduced the mean prolongation of TE in vagotomized rats from 5.890.8 sec to 1.069 0.06 sec after bicuculline and 1.219 0.07 sec after picrotoxin (n=7, PB 0.01). Mean blood pressure (MAP) fell immediately in
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post-GABA apnoea and remained at a reduced level during recovery in intact and vagally deafferentated rats (Table 1A). GABA had no effect on MAP following bicuculline and picrotoxin blockade (ANOVA, P= 0.28 and P= 0.10, respectively). To determine whether the sinus nerves contribute to mediating the response to intracarotid GABA injection, the drug was given in five vagotomized rats prior to and after CSNs division. Fig. 2 shows the respiratory response to intracarotid GABA injection in vagotomized and subsequently carotid deafferented rat. The mean values of some respiratory and cardiovascular indices are included in Table 1B. Apnoea occurred in five vagotomized rats prior to CSNs section and TE was significantly prolonged compared to the baseline. Injection of GABA increased tidal volume prior to CSNs neurotomy (ANOVA, P= 0.006). As shown in Table 1B the tidal volume rose significantly 30 sec after administration of GABA in vagotomized rats while section of the CSNs abolished this response. GABA did not induce any change in the rate of breathing in both neural states (ANOVA, P= 0.6). Minute ventilation was
Fig. 2. Respiratory response to intracarotid injection of GABA in midcervically vagotomized rat (upper trace). GABA injection marked by a dot above the upper record. Note the expiratory apnoea coupled with electrical silence in the diaphragm. Breathing which follows the apnoea is associated with augmented tidal volume. Following CSNs neurotomy (lower trace) the respiratory response to GABA is absent. VT, tidal volume; Dia, integrated electromyogram of the diaphragm.
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Fig. 3. Effect of CSNs section on GABA induced prolongation of expiratory time in vagotomized rats (mean 91 S.E.M., n=5); *P B0.05 vs. pre-CSNs cut value (one-way ANOVA).
affected by GABA (ANOVA, P = 0.004) and increased at 30 sec after injection from the control value of 98.7919.6 to 123.09 19.0 ml/min prior to CSNs neurotomy (P B 0.05). As shown in Fig. 3, division of the CSNs eliminated the mean prolongation of TE (TE TEST/TE CONTROL) from 7.09 2.2 in vagotomized to 0.8790.05 in CSNscut animals (PB0.05, n = 5). All rats of this group presented a significant fall in MAP levels. In the animals treated by vagotomy, the fall in MAP levels emerged during the arrest of breathing, whereas in subsequently CSNs-sectioned ones it lagged by 30 sec (Table 1B).
4. Discussion The present study showed that intracarotid arterial administration of GABA in rats evoked apnoea, increase in tidal volume in reinitiated breathing and hypotension. Our results are in general agreement with the report of Holzer and Hagmu¨ ller (1979) in that depressive action of systemic GABA on the respiration in rats led to the arrest of breathing, which had been reversed by an intravenous injection of picrotoxin. The aforementioned report did not give any data on tidal volume and respiratory frequency after resumption of breathing. The record and diagram in the papers by Holzer and Hagmu¨ ller (1979) and Billingsley and Suria (1982) show an increased tidal volume and the rate of respiration (respectively) after intravenous GABA challenge.
In the current experiments, the expiratory time increased several fold during the arrest of breathing, which corresponds with prolonged apnoea at the dosage used by Holzer and Hagmu¨ ller (1979) on carotid arterial GABA injections. The apnoeic response in our rats consisted of immediate cessation of inspiration and lengthening of the subsequent expiratory period (TE), which closely resembles the apnoea of pulmonary chemoreflex with suppression of motoneuron activity to both inspiratory and expiratory muscles (Coleridge and Coleridge, 1994). The present study showed an immediate increase in VT during reinitiated breathing after GABA challenge (Table 1), without noticeable changes in the respiratory rate both prior to and after vagotomy. Midcervical neurotomy of the vagal nerves eliminates sensory input from the lungs revealing that GABA affects the volume component of the breathing pattern through the chemoafferent nerves. Vagotomy has not blocked the occurrence of GABA-induced apnoeic spells, but the respiratory effects of GABA were antagonized by an intravenous injection of bicuculline (GABA receptor antagonist) and picrotoxin (a GABAionophore antagonist), which falls into line with the effectiveness of both drugs used in the previous studies with GABA applied in situ (De Groat, 1970, 1972; Gallagher et al., 1978), at the brainstem level (Hedner et al., 1984; Da Silva et al., 1987) and peripherally administered (Holzer and Hagmu¨ ller, 1979). GABA is considered to have a low permeability through the blood–brain barrier, as mentioned in the Section 1 (Purpura et al., 1958). Therefore, the respiratory changes observed in our study are due to peripheral effects. They are essentially different from the respiratory depression induced by GABA administration to CNS in rats (Section 1). The depressant effect of GABA on respiration in our experimental setup seems to be mediated by an activation of the mechanism of GABA-ergic receptors, which affect the respiratory system at several distinct sites. Bicuculline and picrotoxin are both GABAA antagonists preventing GABAevoked apnoea (Fig. 1) and undergoing respiratory changes. The pattern of the respiratory response to systemic GABA challenge excludes participation of the vagi (Table 1A).
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The effects of peripherally administered GABA, which have not been qualitatively described earlier, suggest their mediation beyond infranodose vagal feedback. GABA stimulation of breathing of enlarged tidal volume is characteristic for excitation of peripheral chemoreceptors (Cardenas and Zapata, 1983). In our experimental design, both chemoafferent inputs from the carotid and aortic chemoreceptors were initially preserved. The major result of this study is that section of the CSNs abolished the respiratory response to intracarotid GABA challenge in our rats (Figs. 2 and 3). The aortic nerves were left preserved and this shows that they do not contribute much to the respiratory effects of GABA, similarly as to other ventilatory responses (Sapru and Krieger, 1977). The current results may indirectly suggest that, with systemic injection, the high level of GABA in the carotid arterial blood causes chemoexcitation of carotid bodies, situated within internal carotid and occipital arteries in rats (Clarke and de Burgh, 1981). There is no evidence that the carotid body contains GABA (Gonzales et al., 1994). It should be noted that the role of GABA in chemoreceptive functions is far from being clarified. The presence of GABA receptors in the carotid bodies has not yet been shown. In cat carotid bodies GABA immunoreactivity was reported to be rather scarce (Pokorski and Ohtani, 1999) as opposed to GABA immunoreactivity found in all chief cells of the mouse carotid body (Oomori et al., 1994). To the best of our knowledge, there is no report on GABA receptors in rat carotid body. The respiratory response to exogenous intracarotid GABA in the present experiment consisted of the arrest of breathing followed by the tendency to hyperpnoea. The apnoeic response to carotid body activation is rather rare and was described to be occasionally induced by nicotine prior to bilateral carotid neurotomy (Jacobs et al., 1971). One can speculate that GABA may influence respiration indirectly via other transmitter systems as it takes place in the neuronal net of the central nervous system. However, little is known whether GABA modulates transmission within the carotid bodies by inhibiting or delaying the release of catecholamines and tachykinins (Otsuka and Yosh-
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ioka, 1993). Yet, based on the presence and modulatory role of GABAB receptors in the carotid chemoreceptor reflex pathway in rat’s NTS (Suzuki et al., 1999), one can assume the carotid bodies are endowed with GABA receptors as well. GABA has been implicated as a neuromodulator of the cholinergic parasympathetic nervous system and tachykinin-containing sensory nerves (Chapman et al., 1993). Nonmyelinated fibers constitute the major proportion of afferents of the CSNs in rats (Mc Donald, 1983). These sensory thin fibers, largely of type C, are mostly affected by GABA (Brown and Marsh, 1978). This might explain the shape of respiratory pattern induced by GABA. This is the first report comparing the respiratory response to intracarotid GABA in vagotomized and CSNs-sectioned rats. Our results allow some distinction between the roles of peripheral vagal GABA receptors and those presumed in, or on, the carotid bodies and nerves, respectively. Blockade with bicuculline and picrotoxin excludes all receptors and eliminates the respiratory response to intracarotid GABA challenge. The same respiratory consequence of carotid deafferentation implicates a conceivable contribution of the chemosensory drive to the inhibition ensued by stimulation of breathing induced by GABA. The decline in blood pressure that we registered falls into line with the results of others on the intravenous administration of GABA in various animal species. The cardiovascular GABA effects may be related to its action on sympathetic ganglia. This assumption is consistent with the study of Vemulapalli and Barletta (1984) who reported that hypotension evoked by GABA was abolished after ganglionic blockade with hexamethonium and atropine. It is likely that GABA may act through its receptors located on the sympathetic ganglia (De Groat, 1970; Balcar et al., 1986) and by inhibiting ganglionic transmission, eventually causing hypotension. Furthermore, in our experiments, bicuculline and picrotoxin considerably reduced blood pressure depressor response to systemic GABA administration, which is in line with past studies (Billingsley et al., 1980; Vemulapalli and Barletta, 1984) and may support the hypothesis that peripheral GABA receptors are responsible for this effect.
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The main finding of this study indicates that the respiratory effects of exogenous GABA in rats are mediated by carotid chemoreceptors. These results extend previous reports on peripheral GABA-ergic mechanism showing that it is operating beyond lung vagal feedback. Blood pressure changes might be carried out by autonomic ganglionic pathways. Acknowledgements We thank Mrs Teresa Warnawin for her excellent technical assistance. References Balcar, V.J., Joo, F., Kasa, P., Dammasch, J.E., Wolf, J.R., 1986. GABA receptor binding in rat cerebral cortex and superior cervical ganglion in the absence of GABA-ergic synapses. Neurosci. Lett. 66, 264 – 274. Billingsley, M., Suria, A., Gilman, R., Shokes, L., Shahvari, M., 1980. Evidence for GABA in the peripheral control of blood pressure and vascular resistance. Brain Res. Bull. 5 (Suppl. 2), 329 –333. Billingsley, M.L., Suria, A., 1982. Effects of peripherally administered GABA and other amino-acids on cardiopulmonary responses in anaesthetized rats and dogs. Arch. Int. Pharmacodyn. 255, 131 –140. Bowery, N.G., Brown, D.A., 1974. Depolarizing actions of g-aminobutyric acid and related compounds on rat superior cervical ganglia in vitro. Br. J. Pharmacol. 50, 205 – 218. Brown, D.A., Marsh, S., 1978. Axonal GABA-receptors in mammalian peripheral nerve trunks. Brain Res. 156, 187 – 191. Cardenas, H., Zapata, P., 1983. Ventilatory reflexes originated from carotid and extracarotid chemoreceptors in rats. J. Appl. Physiol. 244, R119 –R125. Chapman, R.W., Hey, J.A., Rizzo, C.A., Bolser, D.C, 1993. GABAB receptors in the lung. Trends Pharmacol. Sci. 14, 26– 29. Clarke, J.A., de Burgh, D.M., 1981. A comparative study of the distribution of carotid body type-I cells and periadventitial type-I cells in the carotid bifurcation regions of the rabbit, rat, guinea-pig and mouse. Cell Tissue Res. 220, 753 – 772. Coleridge, H.M., Coleridge, J.C.G., 1994. Pulmonary reflexes: neural mechanisms of pulmonary defense. Annu. Rev. Physiol. 56, 69 – 91. Da Silva, A.H.T., Hartley, B., Hamash, P., Quest, J.A., Gillis, R.A, 1987. Respiratory depressant effects of GABA a- and b-receptor agonists in the cat. J. Appl. Physiol. 62, 2264 – 2272.
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Apnoeic response to stimulation of peripheral GABA receptors in rats Katarzyna Kaczyn´ska *, Małgorzata Szereda-Przestaszewska Laboratory of Respiration Physiology, Department of Neurophysiology, Polish Academy of Sciences Medical Research Centre, 5 Pawin´skiego Street, 02 -106 Warsaw, Poland Accepted 14 March 2002
Abstract Respiratory effects of intracarotid injection of g-amino-butyric acid (GABA) were investigated in two groups of rats. In the first group of 12 rats the effects of GABA were checked in the intact state, following bilateral vagotomy and GABA receptor blockade. The second group consisted of five initially vagotomized rats, challenged with GABA prior to and after bilateral carotid chemodenervation (CSN-cut). All rats were urethane and chloralose anaesthetized and spontaneously breathing. Injection of 39 mmol/kg GABA prior to and after vagotomy induced an expiratory apnoea of, respectively 5.5 90.84 sec and 3.9 9 0.6 sec duration (mean 9 S.E.M.), P\ 0.05 in all 12 rats. In breaths that followed the apnoea tidal volume increased above the control level by 23.3% (P B0.01) and 25.6% (P B0.01) preand post-vagotomy, respectively. Blockade of GABA receptors with bicuculline and picrotoxin abolished the inhibition of breathing. In five vagotomized rats with intact carotid sinus nerves (CSNs) intracarotid GABA challenge increased tidal volume by 39% compared with baseline breathing (PB0.05). Section of the CSNs precluded the occurrence of apnoea and undergoing respiratory changes evoked by GABA. Intracarotid GABA caused significant decrease in the mean blood pressure independent of the neural state, but the fall was delayed by CSNs neurotomy. Results of this study indicate that GABA given systemically induces apnoea followed by post-apnoeic hyperventilation. Carotid bodies are required for the ventilatory response to GABA; vagal afferents are not involved in this response. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Control of breathing; apnoea; GABA; Mammals; rat; Pharmacological agents; bicuculline; picrotoxin; Nerve; carotid sinus; vagus; Ventilation; intracarotid GABA
1. Introduction g-Amino-butyric acid (GABA) is the most important inhibitory neurotransmitter in the verte* Corresponding author. Tel.: +48-22-608-6522; fax: + 4822-668-5532 E-mail address: [email protected] (K. Kaczyn´ska).
brate CNS. Neuroanatomical studies in cats have shown its presence in the interstitial nucleus of the nucleus tractus solitarius (NTS) and in the dorsal vagal nucleus (Dietriech et al., 1982). Immunohistochemical estimation revealed the presence of GABA immunoreactive fibers in contact with respiratory neurons of the NTS (Lipski et al., 1990). Intracerebroventricular or ventral medullary sur-
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face application of GABA or its agonists affects the respiration influencing the ventilatory subdivisions of the respiratory pattern in a variable way. Injection of GABA into the cisterna magna or administration on the ventral medullary surface of cats evoked the depression of tidal volume only (Yamada et al., 1982). Baclofen a GABAB agonist given intracerebroventricularly or intravenously decreased the minute ventilation, affecting mainly the respiratory rate (Da Silva et al., 1987). In rats, GABA applied intracerebroventricularly evoked an apparent respiratory depression of decreased tidal volume and frequency of breathing. This was not preceded by respiratory arrest. This response was reversed by an intravenous injection of bicucculline (Hedner et al., 1981, 1984). GABA has been likewise identified in the vagal nodose ganglia of cats (De Groat, 1972), within sympathetic ganglia of rats, cats and rabbits (De Groat, 1970; Bowery and Brown, 1974) and in cats’ spinal ganglia (Gallagher et al., 1978). Due to the presence of GABA receptors on nonmyelinated C fibers of airway cholinergic nerves of rats and guinea-pigs (Brown and Marsh, 1978; Chapman et al., 1993), one may assume this neurotransmitter contributes to respiratory lung reflexes. It has been shown that intravenous GABA injection stimulates respiration in cats and rabbits but depresses it in dogs after transient apnoeic spells (Elliot and Hobbiger, 1958). Rat was the only species that regularly responded to systemic GABA challenge with apnoea, followed by renewed breathing through hyperpnoea or tachypnoea (Holzer and Hagmu¨ ller, 1979; Billingsley and Suria, 1982). Since GABA does not readily cross the blood– brain barrier (Purpura et al., 1958), studies using systemic administration can be regarded as a search for peripheral effects. To the best of our knowledge, there has not been any study quantifying the presumed ventilatory depression evoked by peripheral GABA challenge. Evidence shows that intracarotid GABA injection could produce a prolonged apnoea (Holzer and Hagmu¨ ller, 1979). The question arises whether the input from carotid bodies modifies GABA-induced respiratory reflex.
The present study started with the hypothesis that the respiratory depression induced by systemic administration of GABA depends on the carotid bodies input without the contribution of vagal afferents. This hypothesis has been tested by measuring the ventilatory effects of GABA in neurally intact rats and after elimination of vagal and carotid body afferent inputs. Additionally, we antagonized the respiratory depressant effects of activation of GABA receptors employing bicuculline and picrotoxin.
2. Methods A total of 17 male Wistar rats weighing 250– 320 g, were anaesthetized with an intraperitoneal injection 600 mg/kg of urethane (Sigma) and 120 mg/kg of chloralose (Fluka AG). Following induction of anaesthesia, supplementary doses were given when necessary as assessed by the flexion withdrawal reflex and blood pressure response to paw pinch. The animals breathed spontaneously during the whole experiment. Body temperature was kept at 38 °C by a heating pad. End-tidal CO2 was continuously monitored with an Engstrom Eliza Plus capnograph (Gambro). Tidal volume (VT) was obtained by integrating the flow signal from a model C56 pneumotachograph (Mercury) attached to the tracheal cannula. The right femoral artery was catheterized and connected to a pressure transducer (CK 01 Mera Tronik) and blood pressure monitor (MCK 411) for the measurement of systemic blood pressure. A similar catheter was inserted into the left femoral vein for the administration of supplemental doses of anaesthetic and injection of the drugs. For GABA injections, the right common carotid artery was tied off and the catheter was inserted cephalad pointing towards the sinus. The midcervical segments of vagal nerves were isolated and prepared for cutting. In five out of seventeen rats treated by vagotomy, the carotid region on both sides was dissected under an operating microscope. The larynx and the oesophagus were reflected to expose both carotid artery bifurcations. The carotid sinus nerves (CSNs) were cut at their junctions with the glossopharyngeal
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nerves. Electromyograms of the costal diaphragm were recorded with bipolar electrodes. The activity was amplified (1000– 5000x) with a NL 104 amplifier (Digitimer), band-pass filtered (50 Hz– 50 kHz) and measured with a model AS 101 (Asbit) leaky integrator (time constant= 100 msec). All recordings were registered on an Omnilight 8 M 36 apparatus (Honeywell). After control values of respiratory variables were taken, g-amino-n-butyric acid (GABA), Sigma, 39 mmol/ kg or 4 mg/kg bolus was delivered in 0.2 ml aliquots of physiological saline and flushed with 0.2 ml of the saline via a catheter placed in the right common carotid artery. Saline solutions of bicuculline methiodide, Sigma, 2 mmol/kg or 1 mg/kg and picrotoxin, Sigma, 0.8 mmol/kg (0.5 mg/kg) were administered intravenously, 2 min prior to GABA injections. The doses of GABA used in these experiments were derived from establishing dose–response relationships in preliminary experiments. The doses of picrotoxin and bicuculline were chosen from studies of other investigators who monitored respiratory and cardiovascular effects of GABA in rats (Holzer and Hagmu¨ ller, 1979; Vemulapalli and Barletta, 1984). Test i.v. injections of 0.2 ml aliquots of physiological saline showed no volume effect(s). At least 15 min were allowed between injections. The experimental protocol was approved by the local animal care committee. The respiratory effects of GABA challenge were recorded in 12 rats of the first series of experiments: (1) neurally intact, (2) subsequently vagotomized, and (3) after intravenous administration of either bicuculline or picrotoxin in midcervically vagotomized rats. In the second series of experiments, GABA was injected in 5 vagotomized rats prior to and after section of the CSNs. Each individual value of VT, ventilation (V: E) and respiratory rate (f) was taken as an average over five consecutive breaths. The ventilatory parameters were assessed prior to the GABA injection, during apnoeic phase and at 30 sec after the challenge. The expiratory time (TE) was determined from the record of integrated diaphragm activity. TE prolongation was measured as the ratio of maximal TE during post-GABA apnoea (TE TEST) to the respective control TE value (TE
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CONTROL).
The duration of apnoeic period in diaphragmatic activity was measured as the time of apnoea (respiratory inhibition). VT, V: E, f and TE were analyzed by two-way ANOVA with postGABA time (0, apnoeic phase, recovery= 30 sec) and denervation status (intact+ vagi cut, vagi cut + bicuculline, vagi cut+picrotoxin, vagi cut + CSNs cut) as repeated measures’ factors. TE prolongation data were analyzed by oneway ANOVA with denervation status as repeated measures factor. Differences between individual time points and experimental situations were evaluated by contrast analysis. In all cases, PB0.05 was considered significant. All results are means9 1 standard error.
3. Results Intracarotid GABA challenge produced uniform cardiorespiratory effects, comprising an apnoea followed by breathing of increased tidal volume and a fall in arterial blood pressure, in intact and vagotomized rats. There were no respiratory effects when similar volumes of the solvent were administered. GABA injected at a dose of 4 mg/kg evoked expiratory apnoea of mean duration of 5.590.84 sec (intact) and of 3.990.6 sec (vagotomized rats), (P\0.05, n =12). After recovery from apnoea, the tidal volume increased in all animals (ANOVA, P=0.000004). The mean increase in tidal volume induced by GABA was significant in reinitiated breathing compared to pre-drug controls, both in intact and vagotomized rats (Table 1A). In the apnoeic pause, the expiratory time was significantly elongated; the mean prolongation of TE being 7.29 1.1-fold and 5.19 0.9-fold prior to and after vagotomy, respectively, indicating similar levels of expiratory inhibition in both neural states (P \0.05). Intracarotid GABA injection evoked a decrease in the respiratory rate at the early post-challenge phase but the effect was borderline and statistically insignificant between the intact (P= 0.06) and vagotomized rats (P= 0.07). Minute ventilation was affected by GABA (ANOVA, P= 0.0008) and increased at 30 sec after injection from the respective control values of 157.69 9.7 to 196.89 12.0 ml/min in
Vagi cut vagi+CSNs cut
B
1.849 0.3
2.1290.32
5
2.749 0.16 3.59 0.3a
2.349 0.32
0
0 0
Apnoea
65.6 9 8.2**
76.6 98.8c
2.26 9 0.31
72.2 9 9.9
93.4 9 10
2.56 90.38**
96.0 9 7.6* 105.4 9 6.3* 68.6 9 9.5*
100 97.9* 102.4 9 5.2*
121.1 96.1 127.9 95.6
3.38 9 0.22* 4.36 90.4*,b
Recovery
80.6 9 10.4*
Apnoea
Recovery
After GABA
Baseline
Baseline
After GABA
MAP (mmHg)
VT (ml)
5
12 12
N
1.28 90.15
0.97 90.09
0.67 9 0.04 0.65 9 0.03
Baseline
TE (sec)
All values are means 9 1 S.E.M., *PB0.01, **PB0.05 vs. the respective pre-GABA value. a PB0.05. b PB0.01 vs. the corresponding pre-vagotomy value. c PB0.05 vs. the corresponding pre-carotid neurotomy value (two-way ANOVA followed by planned contrast analysis).
Intact vagi cut
A
Innervation status
Table 1 Changes in tidal volume (VT), mean arterial pressure (MAP) and expiratory time (TE) after intracarotid GABA challenge
1.14 9 0.11c
6.44 9 1.17**
4.79 9 0.8* 3.46 9 0.6*
Apnoea
After GABA
1.2490.12
1.0 9 0.09
0.58 90.04 0.63 9 0.02
Recovery
192 K. Kaczyn´ ska, M. Szereda-Przestaszewska / Respiratory Physiology & Neurobiology 131 (2002) 189–197
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Fig. 1. Effect of GABA receptors blockade on the mean prolongation of TE induced by GABA in vagotomized rats (mean91 S.E.M., n =7). **PB 0.01, vs. the respective preblockade values (one-way ANOVA).
intact rats (PB 0.01) and from 190.3918.2 to 234.39 12.3 ml/min after vagotomy (P B 0.05). Bicuculline and picrotoxin appeared to be effective in antagonizing the respiratory response to intracarotid GABA injection. It is evident in Fig. 1 that blockade of GABAA receptors by both substances reduced the mean prolongation of TE in vagotomized rats from 5.890.8 sec to 1.069 0.06 sec after bicuculline and 1.219 0.07 sec after picrotoxin (n=7, PB 0.01). Mean blood pressure (MAP) fell immediately in
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post-GABA apnoea and remained at a reduced level during recovery in intact and vagally deafferentated rats (Table 1A). GABA had no effect on MAP following bicuculline and picrotoxin blockade (ANOVA, P= 0.28 and P= 0.10, respectively). To determine whether the sinus nerves contribute to mediating the response to intracarotid GABA injection, the drug was given in five vagotomized rats prior to and after CSNs division. Fig. 2 shows the respiratory response to intracarotid GABA injection in vagotomized and subsequently carotid deafferented rat. The mean values of some respiratory and cardiovascular indices are included in Table 1B. Apnoea occurred in five vagotomized rats prior to CSNs section and TE was significantly prolonged compared to the baseline. Injection of GABA increased tidal volume prior to CSNs neurotomy (ANOVA, P= 0.006). As shown in Table 1B the tidal volume rose significantly 30 sec after administration of GABA in vagotomized rats while section of the CSNs abolished this response. GABA did not induce any change in the rate of breathing in both neural states (ANOVA, P= 0.6). Minute ventilation was
Fig. 2. Respiratory response to intracarotid injection of GABA in midcervically vagotomized rat (upper trace). GABA injection marked by a dot above the upper record. Note the expiratory apnoea coupled with electrical silence in the diaphragm. Breathing which follows the apnoea is associated with augmented tidal volume. Following CSNs neurotomy (lower trace) the respiratory response to GABA is absent. VT, tidal volume; Dia, integrated electromyogram of the diaphragm.
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Fig. 3. Effect of CSNs section on GABA induced prolongation of expiratory time in vagotomized rats (mean 91 S.E.M., n=5); *P B0.05 vs. pre-CSNs cut value (one-way ANOVA).
affected by GABA (ANOVA, P = 0.004) and increased at 30 sec after injection from the control value of 98.7919.6 to 123.09 19.0 ml/min prior to CSNs neurotomy (P B 0.05). As shown in Fig. 3, division of the CSNs eliminated the mean prolongation of TE (TE TEST/TE CONTROL) from 7.09 2.2 in vagotomized to 0.8790.05 in CSNscut animals (PB0.05, n = 5). All rats of this group presented a significant fall in MAP levels. In the animals treated by vagotomy, the fall in MAP levels emerged during the arrest of breathing, whereas in subsequently CSNs-sectioned ones it lagged by 30 sec (Table 1B).
4. Discussion The present study showed that intracarotid arterial administration of GABA in rats evoked apnoea, increase in tidal volume in reinitiated breathing and hypotension. Our results are in general agreement with the report of Holzer and Hagmu¨ ller (1979) in that depressive action of systemic GABA on the respiration in rats led to the arrest of breathing, which had been reversed by an intravenous injection of picrotoxin. The aforementioned report did not give any data on tidal volume and respiratory frequency after resumption of breathing. The record and diagram in the papers by Holzer and Hagmu¨ ller (1979) and Billingsley and Suria (1982) show an increased tidal volume and the rate of respiration (respectively) after intravenous GABA challenge.
In the current experiments, the expiratory time increased several fold during the arrest of breathing, which corresponds with prolonged apnoea at the dosage used by Holzer and Hagmu¨ ller (1979) on carotid arterial GABA injections. The apnoeic response in our rats consisted of immediate cessation of inspiration and lengthening of the subsequent expiratory period (TE), which closely resembles the apnoea of pulmonary chemoreflex with suppression of motoneuron activity to both inspiratory and expiratory muscles (Coleridge and Coleridge, 1994). The present study showed an immediate increase in VT during reinitiated breathing after GABA challenge (Table 1), without noticeable changes in the respiratory rate both prior to and after vagotomy. Midcervical neurotomy of the vagal nerves eliminates sensory input from the lungs revealing that GABA affects the volume component of the breathing pattern through the chemoafferent nerves. Vagotomy has not blocked the occurrence of GABA-induced apnoeic spells, but the respiratory effects of GABA were antagonized by an intravenous injection of bicuculline (GABA receptor antagonist) and picrotoxin (a GABAionophore antagonist), which falls into line with the effectiveness of both drugs used in the previous studies with GABA applied in situ (De Groat, 1970, 1972; Gallagher et al., 1978), at the brainstem level (Hedner et al., 1984; Da Silva et al., 1987) and peripherally administered (Holzer and Hagmu¨ ller, 1979). GABA is considered to have a low permeability through the blood–brain barrier, as mentioned in the Section 1 (Purpura et al., 1958). Therefore, the respiratory changes observed in our study are due to peripheral effects. They are essentially different from the respiratory depression induced by GABA administration to CNS in rats (Section 1). The depressant effect of GABA on respiration in our experimental setup seems to be mediated by an activation of the mechanism of GABA-ergic receptors, which affect the respiratory system at several distinct sites. Bicuculline and picrotoxin are both GABAA antagonists preventing GABAevoked apnoea (Fig. 1) and undergoing respiratory changes. The pattern of the respiratory response to systemic GABA challenge excludes participation of the vagi (Table 1A).
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The effects of peripherally administered GABA, which have not been qualitatively described earlier, suggest their mediation beyond infranodose vagal feedback. GABA stimulation of breathing of enlarged tidal volume is characteristic for excitation of peripheral chemoreceptors (Cardenas and Zapata, 1983). In our experimental design, both chemoafferent inputs from the carotid and aortic chemoreceptors were initially preserved. The major result of this study is that section of the CSNs abolished the respiratory response to intracarotid GABA challenge in our rats (Figs. 2 and 3). The aortic nerves were left preserved and this shows that they do not contribute much to the respiratory effects of GABA, similarly as to other ventilatory responses (Sapru and Krieger, 1977). The current results may indirectly suggest that, with systemic injection, the high level of GABA in the carotid arterial blood causes chemoexcitation of carotid bodies, situated within internal carotid and occipital arteries in rats (Clarke and de Burgh, 1981). There is no evidence that the carotid body contains GABA (Gonzales et al., 1994). It should be noted that the role of GABA in chemoreceptive functions is far from being clarified. The presence of GABA receptors in the carotid bodies has not yet been shown. In cat carotid bodies GABA immunoreactivity was reported to be rather scarce (Pokorski and Ohtani, 1999) as opposed to GABA immunoreactivity found in all chief cells of the mouse carotid body (Oomori et al., 1994). To the best of our knowledge, there is no report on GABA receptors in rat carotid body. The respiratory response to exogenous intracarotid GABA in the present experiment consisted of the arrest of breathing followed by the tendency to hyperpnoea. The apnoeic response to carotid body activation is rather rare and was described to be occasionally induced by nicotine prior to bilateral carotid neurotomy (Jacobs et al., 1971). One can speculate that GABA may influence respiration indirectly via other transmitter systems as it takes place in the neuronal net of the central nervous system. However, little is known whether GABA modulates transmission within the carotid bodies by inhibiting or delaying the release of catecholamines and tachykinins (Otsuka and Yosh-
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ioka, 1993). Yet, based on the presence and modulatory role of GABAB receptors in the carotid chemoreceptor reflex pathway in rat’s NTS (Suzuki et al., 1999), one can assume the carotid bodies are endowed with GABA receptors as well. GABA has been implicated as a neuromodulator of the cholinergic parasympathetic nervous system and tachykinin-containing sensory nerves (Chapman et al., 1993). Nonmyelinated fibers constitute the major proportion of afferents of the CSNs in rats (Mc Donald, 1983). These sensory thin fibers, largely of type C, are mostly affected by GABA (Brown and Marsh, 1978). This might explain the shape of respiratory pattern induced by GABA. This is the first report comparing the respiratory response to intracarotid GABA in vagotomized and CSNs-sectioned rats. Our results allow some distinction between the roles of peripheral vagal GABA receptors and those presumed in, or on, the carotid bodies and nerves, respectively. Blockade with bicuculline and picrotoxin excludes all receptors and eliminates the respiratory response to intracarotid GABA challenge. The same respiratory consequence of carotid deafferentation implicates a conceivable contribution of the chemosensory drive to the inhibition ensued by stimulation of breathing induced by GABA. The decline in blood pressure that we registered falls into line with the results of others on the intravenous administration of GABA in various animal species. The cardiovascular GABA effects may be related to its action on sympathetic ganglia. This assumption is consistent with the study of Vemulapalli and Barletta (1984) who reported that hypotension evoked by GABA was abolished after ganglionic blockade with hexamethonium and atropine. It is likely that GABA may act through its receptors located on the sympathetic ganglia (De Groat, 1970; Balcar et al., 1986) and by inhibiting ganglionic transmission, eventually causing hypotension. Furthermore, in our experiments, bicuculline and picrotoxin considerably reduced blood pressure depressor response to systemic GABA administration, which is in line with past studies (Billingsley et al., 1980; Vemulapalli and Barletta, 1984) and may support the hypothesis that peripheral GABA receptors are responsible for this effect.
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