435
Journal of Physiology (1992), 453, pp. 435-447 With 6 figure Printed in Great Britain
VAGAL CONTROL OF MUCUS GLYCOCONJUGATE SECRETION INTO THE FELINE TRACHEA
BY D. C. K. FUNG, D. J. BEACOCK AND P. S. RICHARDSON* From the Department of Physiology, St George's Hospital Medical School, Cranmer Terrace, London SW17 ORE
(Received 11 September 1991) SUMMARY
1. We examined the effects of frequencies and patterns of electrical stimulation of the peripheral cut ends of the vagus nerves on the release of mucus glycoconjugates into feline trachea in vivo. Mucus glycoconjugates, radiolabelled biosynthetically with [35S]sulphate and [3H]glucose, were washed from a tracheal segment in situ, and dialysed before being counted and assayed chemically by the periodic acid-Schiff (PAS) method. 2. Vagal stimulation with regular pulses (10 V, 2 ms duration) at 1, 2X25, 4 5, 9 and 18 Hz produced frequency-dependent increases in the output of mucus glycoconjugates. 3. The muscarinic agonist pilocarpine (0-1-10 ,uM), given intrasegmentally, produced dose-dependent increases in the output of mucus glycoconjugates. 4. Pretreatment with atropine, phentolamine and propranolol reduced but did not, abolish the effects of vagal stimulation. Vagus nerve stimulation still caused frequency-dependent increases in the output of mucus glycoconjugates. 5. High frequency stimulations at 22-5 and 47-5 Hz given intermittently (1 s burst then 4 s rest), whether in the absence or presence of cholinergic and adrenergic blockade, produced similar secretary responses as the same number of pulses delivered in regular trains at 4-5 and 9-5 Hz. This suggests that neither cholinergic nor non-adrenergic, non-cholinergic (NANC) nerve mechanisms in this system are potentiated by high frequency, intermittent burst stimulation. 6. In the absence of atropine, regular vagal stimulation had a greater effect on heart rate than did the same number of pulses delivered in bursts. 7. High molecular weight glycoconjugates from secretions were taken from the void volume of a Sepharose CL-2B gel filtration column and separated further by density-gradient centrifugation. Macromolecular components were observed at two densities, a typical mucin at 1'52 g ml-1, and a high density atypical component at 1-63 g ml-'. In secretions collected during vagal stimulation, either in the absence or presence of cholinergic and adrenergic blockade, the ratio of low density to high density macromolecules was higher than in unstimulated secretions. This can be explained if both cholinergic and NANC nervous vagal mechanisms stimulate the output of typical (density = 1-52 g ml-') mucins into the feline trachea. * To whom correspondence should be addressed. MS 9727
436
D. C. K. FUNG, D. J. BEACOCK AND P. S. RICHARDSON INTRODUCTION
Airway mucus contains a number of components with a range of functions. High molecular weight glycoconjugates, in particular mucins, are thought to be mainly responsible for its theological properties which are essential for mucociliary transport (Carlstedt & Sheehan, 1984). In several species including cat and man, the parasympathetic vagal control of mucus glycoconjugate secretion into the tracheobronchial tree consists of both classical cholinergic (atropine-sensitive) and nonadrenergic, non-cholinergic (NANC, atropine-resistant) mechanisms (Peatfield & Richardson, 1983; Borson, Charlin, Gold & Nadel, 1984; Rogers & Barnes, 1989; D. C. K. Fung, M. I. Allenby & P. S. Richardson, unpublished observations). However, the compositions of glycoconjugates released by different nervous mechanisms have not been compared. Activation of cholinergic mechanisms stimulates the output of typical mucins into the feline trachea (Gallagher, Hall, Phipps, Jeffery, Kent & Richardson, 1986; Davies, Gallagher, Richardson, Sheehan & Carlstedt, 1991). Whether activation of NANC mechanisms would also release typical mucins is not known at present. In many organ systems the response to electrical nerve stimulation depends both on the stimulation frequency and the pattern of stimulation. In some systems the response is potentiated by delivery of electrical pulses in intermittent bursts at relatively high frequencies, e.g. the NANC-evoked decreases in feline submaxillary vascular resistance via stimulation of the chorda tympani (Andersson, Bloom, Edwards & Jarhult, 1982b), the NANC-evoked increases in blood flow through the feline tracheal microvasculature via stimulation of the vagus nerve (Martling, Gazelius & Lundberg, 1987), and the acetylcholine-mediated parotid responses in the sheep via stimulation of the parotid nerve (Andersson, Bloom & Edwards, 1982a). In this study, we examined the effects of stimulation frequency and pattern of vagus nerve stimulation on the output of mucus glycoconjugate into the feline trachea both in the absence and presence of combined cholinergic and adrenergic blockade. We also described the concentration-response relationship of pilocarpine, a cholinergic agonist, on mucus glycoconjugate output. In addition, the chemical compositions of high molecular weight mucus glycoconjugates released by vagal stimulation, both in the absence and presence of combined cholinergic and adrenergic
blockade, were analysed. METHODS
Preparation of tracheal segment Anaesthesia was induced in adult cats of either sex (weight 2-0-4-4 kg) by intraperitoneal injection of pentobarbitone sodium (Sagatal, May & Baker), 42 mg kg-', and maintained by intravenous injections via a femoral catheter. Rectal temperature was kept between 36 and 38 'C by a heat exchange pad. Blood pressure was recorded via a femoral arterial catheter connected to a strain-gauge transducer. The subsequent preparation of the tracheal segment has been described previously (Gallagher, Kent, Passatore, Phipps & Richardson, 1975). Briefly, a cannula through which the animal breathed was tied into the cervical trachea as far caudally as possible. A segment of trachea (4-6 cm in length) rostral to this was then cannulated at both ends to isolate it in situ with its nerves and blood supply intact. This tracheal segment was then washed out and filled with oxygenated Krebs-Henseleit solution at 37 'C.
VA GAL CONTROL OF TRACHEAL MUCUS SECRETION
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Radiolabelling of mucus glycoconjugates At the beginning of each experiment, 2 0 mCi of sodium [35k]sulphate and 0 5 mCi of [3H]glucose (Amersham International) in 3-0 ml Krebs-Henseleit solution were flushed into the tracheal segment and left for 1 h to enable uptake and biosynthetic incorporation of the radiolabels into mucus glycoconjugates (Gallagher et al. 1975, 1986; Davies et al. 1991). Samples were then taken at 30 min intervals for the next 2 h. then at 15 min intervals, by flushing the segment with 10 ml of Krebs-Henseleit solution.
IVagal stimulation The cervical sympathetic nerves on both sides were separated from the vagus nerve and cut at the mid-neck region. The vagus nerves (not desheathed) were then cut just caudal to the superior laryngeal nerves, and their peripheral ends were stimulated via platinum electrodes with pulses of 1O V, 2 ms duration for 10 min of a 15 min collection period (Devices gated pulse generator 2521 and isolated stimulator 2533). To determine the frequency-response relationship, frequencies of 1, 2-25. 45., 9 and 18 Hz were employed. To study the effects of stimulation pattern, we compared the secretary response to a regular train of electrical pulses at low frequency (4 5 or 9-5 Hz) with identical numbers of electrical pulses at the higher frequency (22-5 or 47 5 Hz) but applied in 1 s bursts at 5 s intervals. A Devices l)igitimer 3290 was used for triggering the pulse generator during stimulus bursts. Administration of drugs Drugs were administered by two routes: directly into the tracheal segment (i.s.) and/or intravenously via the femoral vein (I.v.). Drugs given i.s. were dissolved in oxygenated Krebs-Henseleit solution. The following drugs were used: atropine sulphate (Sigma), DL-
propranolol hydrochloride (Sigma), phentolamine mesylate (Rogitine, Ciba), pilocarpine hydro(hloride (Sigma). Experimental protocols After the initial 1 h pulse labelling period, the preparation was left unstimulated for the next 2 25 h. It was then stimulated with either electrical vagal stimulation for 10 min or with pilocarpine (i.s.) for 15 min at hourly intervals on four occasions. Antagonism of cholinergic and adrenergic effects was achieved by administration of atropine (2 jug ml-1 i.s. and 1 mg kg-1 i.v.), phentolamine (10 jug ml-1 i.s.) and propranolol (10 jug ml-' i.s. and 2 mg kg-1 i.v.) at least 30 min before nerve stimulation. I)uring each experiment the four stimuli applied were always different, thus the n values given in the text and figures for each intervention represent both the number of animals and the number of trials. Measurement of radiolabelled glycoconjugate output To dissolve mucus and minimize proteolysis, tracheal washings were mixed on collection with a solution containing 7 M-guanidiniurn chloride (practical grade, filtered and purified twice with activated charcoal), 5 mM-N a2ED)TA, 5 mM-N-ethylmaleimide, 01 mM-phenylmethylsuphonyl fluoride. 10 mm-sodium phosphate buffer (all from Sigma), pH 6-5, to give a final concentration of 4 M-guanidinium chloride, and kept between 4 and 6 0C (Carlstedt, Lindgren, Sheehan, Ulmsten & Wingerup, 1983). Samples were dialysed exhaustively (dialysis tubing with a molecular weight cut off (MWCO) at 12-14000, Medicell International) at 4 0C against distilled water containing 0-01 % (w/v) sodium azide and 0-01 % (w/v) sodium sulphate to disperse the non-covalently bound radiolabel, and radioactivities of aliquots were then measured in triplicate by liquid scintillation counting (Beckman Ready Protein Scintillant; Beckman Scintillation Counter LS 6000 IC, calibrated with quench correction to assay 3H and 35S separately).
Measurement of total glycoconjugate output The total glycoconjugates present in the tracheal washings after exhaustive dialysis were estimated in triplicate using the solution periodic acid-Schiff (PAS) assay method (Mantle & Allen, 1978). Bovine submaxillary mucin (Sigma) was used as a calibration standard. Glycoconjugate concentrations were calibrated in terms of this standard, and expressed as output rates (aug min-).
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D. C. K. FUNG, D. J. BEACOCK AND P. S. RICHARDSON
Purification of high molecular weight glycoconjugates Tracheal washings from different experiments collected as described above were divided, after exhaustive dialysis, into four pools as follows: (1) unstimulated (resting) samples in the absence of autonomic antagonists; (2) samples collected during vagal stimulation; (3) resting samples in the presence of cholinergic and adrenergic antagonists; (4) samples collected during vagal stimulation in the presence of cholinergic and adrenergic antagonists. Pools were concentrated by dialysis against Aquacide 2 (Calbiochem), equilibrated against a solution containing 4 M-guanidinium chloride, 5 mM-Na2EDTA, 10 mM-sodium phosphate buffer, pH 6 5, and chromatographed on a Sepharose CL-2B (Pharmacia) column (60 x 2-1 cm) using the same buffer at a flow of 21 ml h-'. Void volume and totally included volume of the column were determined by Dextran Blue (Pharmacia) and [35S]sulphate respectively. Aliquots (50-200 ,l) from each fraction (7 ml) were taken for liquid scintillation counting and for total glycoconjugate estimation by PAS staining after slot-blotting onto nitrocellulose membrane (0'45 ,um pore size, Schleicher and Schuell) using a multi-well slot-blot manifold (Minifold 2, Schleicher and Schuell) (Thornton, Holmes, Sheehan & Carlstedt, 1989). The PAS reactivity of each blotted sample was then measured using a scanning reflectance densitometer (Chromoscan 3, Joyce & Loebl) at a wavelength of 530 nm. Isopycnic density-gradient ultracentrifugation Void volume fractions from Sepharose CL-2B containing high molecular weight glycoconjugates were pooled, concentrated, mixed with sufficient CsCl to give an initial density of approximately 1 40 g ml-', and subjected to CsCl isopycnic density-gradient centrifugation at 40000 r.p.m. at 15 00 for 70 h (Beckman L5-50 ultracentrifuge; 5OTi rotor). Fractions (1 ml) were collected from the bottom (Beckman fraction recovery system) and their densities were measured with a Hamilton syringe as a pycnometer. Fractions with densities greater than 1-42 g ml-' were pooled, equilibrated against a solution containing 0-2 M-guanidinium chloride, 5 mM-Na2EDTA, 10 mMsodium phosphate buffer, pH 65, and subjected to a second CsCl isopycnic density-gradient centrifugation at a starting density of 1-52 g ml-' using the same conditions as above. Aliquots from each fraction were taken for liquid scintillation counting and for PAS staining as described above. Statistical treatment of results Changes in glycoconjugate output induced by vagal stimulation were calculated as the percentage increase of the stimulated sample over the mean value of the preceding and succeeding sample (bracketing controls). Effects produced by pilocarpine tended to persist into the succeeding control sample, so they were expressed as the percentage increase of the stimulated sample over the preceding sample only (single preceding controls). Vagal reductions of heart rate were expressed as the percentage reduction during stimulation compared with the preceding period. Values shown represent means + standard error of the mean (s.E.M.). When paired data were available (i.e. for the results of burst versus regular stimulation, Figs 3, 4 and 5) comparisons were made by a paired Student's t test, and P < 0 05 was accepted as statistically significant (see figure legends for further details). RESULTS
Effects of frequency of vagal stimulation
Vagal stimulation with regular pulses at 1, 2-25, 45, 9 and 18 Hz produced frequency-dependent increases in the secretion of mucus glycoconjugates, as measured by the output rates of 35S-labelled and PAS-reactive glycoconjugates (Fig. 1). The output of 3H-labelled glycoconjugate was less clearly frequency related.
Effects of a cholinergic muscarinic agonist The muscarinic agonist pilocarpine (0-3-10 /kM) given I.s. produced dose-dependent increases in mucus glycoconjugate secretion as measured by all three indices of glycoconjugate output rate (Fig. 2).
VAGAL CONTROL OF TRACHEAL MUCUS SECRETION
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Effects of pharmacological blockade by cholinergic and adrenergic antagonists Pretreatment with atropine (2,jg ml-1 i.s. and 1 mg kg-1 i.v.), phentolamine (10 jtg ml-1 i.s.) and propranolol (10 gg ml-' I.s. and 2 mg kg-1 i.v.), in doses capable of abolishing all effects produced by pilocarpine (3 ,aM), phenylephrine (40 gM) and
35S-glycoconjugates 300200-
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1
225
45
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18 Hz
(n)
(4) (4)
(5) (5)
(11 )(9)
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Fig. 1. The effects of stimulation frequency on the output of 35S-, 3H-labelled and PASreactive glycoconjugates induced by regular electrical stimulation of the peripheral cut ends of the vagus nerves, both in the absence (open columns) and presence (hatched columns) of atropine (2 jug ml-1 I.S, and 1 mg kg- 'Iv.), phentolamine (10 jig mP'l i.s.) and propranolol (10 jug mll'Is . and 2 mg kg- I.V.) . Values shown represent means+ S.E.M.
isoprenaline (40 gM) administered I.s. towards the end of the experiments (D. C. K. Fung, M. I. Allenby & P. S. Richardson, unpublished observations), failed to abolish the effects produced by vagal stimulation. Vagal stimulation caused frequencydependent increases in the output of 355- and 3H-labelled as well as the total PASreactive glycoconjugates (Fig. 1). 15
P
2Y 453
44040D. C. K. FUNG, D. J. BEACOCK AND P. S. RICHARDSON
Effects3 of the pattern of vagal stimulation In the absence of autonomic antagonists Regular trains of electrical pulses delivered at 4-5 and 9-5 Hz produced similar secretary responses as the same number of pulses delivered in high frequency bursts at 22-5 and 47-5 Hz
given for 1 s in 5 s (Fig. 3). 3_glIycoconj ugates 600500400300 200100-
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log [pilocarpine] (5) (1 0) (12) (1 0) (6) (n) Fig. 2. The effects of pilocarpine at different concentrations on the output of 35S-,' 3Hlabelled and PAS-reactive glycoconjugates. Values shown represent means + sEM.
In the presence of cholinergic and adrenergic antagonists Regular trains of electrical pulses delivered at 4-5 and 9-5 Hz again produced similar secretary responses as the same number of pulses delivered in high frequency bursts at 22-5 and 47T5 Hz given for 1 s in 5 s (Fig. 4).
Effects of vagal stimulation on heart rate In the absence of autonomic antagonists, vagal stimulation from 1 to 18 Hz produced frequency-dependent reductions in heart rate (Fig. 5). Regular trains of
VAGAL CONTROL OF TRACHEAL MUCUS SECRETION 441 electrical pulses delivered at 4-5 and 9-5 Hz slowed heart rate more effectively than the same number of pulses delivered in bursts at 22-5 and 47-5 Hz given for 1 s in 5 s. After treatment with cholinergic and adrenergic antagonists, changes in heart rate were absent or minimal during vagal stimulation at any frequency. 35S-glycoconjugates r-n.s.-
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Burst H Reg. Burst 22-5 95Hz47-5 Hz (n) (5) (5) (8) (8) Fig. 3. The effects of stimulation pattern on the output of 35S- and 3H-labelled and PASreactive glycoconjugates induced by vagal stimulation in the absence of autonomic antagonists. Low frequency continuous stimulation (Reg.) at 4-5 Hz and 9-5 Hz were compared with the corresponding high frequency burst (1 s in 5 s) stimulation at 22-5 and 47-5 Hz. Values shown represent means + S.E.M. Paired t tests demonstrated no significant difference between the two patterns of vagal stimulation.
Chemical analysis of mucus glycoconjugates All the pools of secretions, when chromatographed on Sepharose CL-2B, showed two main peaks of radiolabelled macromolecules: a large peak eluting in the void volume where high molecular weight (Mr > 106) glycoconjugates such as mucins are found, and a smaller, more broadly based peak (or possibly peaks) in the partially 15-2
D. C. K. FUNG, D. J. BEACOCK AND P. S. RICHARDSON included volume which corresponds to smaller macromolecular species (see Gallagher et al. 1986). When the high molecular weight glycoconjugates from the void volume of CL-2B were analysed by isopycnic density-gradient centrifugation in CsCl and 4 M-
442
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(5) (5) Fig. 4. The effects of stimulation pattern on the output of 35S- and 3H-labelled and PASreactive glycoconjugates induced by vagal stimulation in the presence of atropine (2 tg ml-' i.s. and 1 mg kg-' i.v.), phentolamine (10 #g ml-' i.s.) and propranolol (10 ug ml-' i.s. and 2 mg kg-' I.v.). Low frequency continuous stimulation at 4-5 and 9 5 Hz were compared with the corresponding high frequency burst (1 s in 5 s) stimulation at 22-5 and 47-5 Hz. Values shown represent means+ S.E.M. Paired t tests demonstrated no significant differences between the two patterns of vagal stimulation.
guanidinium chloride at a starting density of approximately 1-40 g ml-', nearly all of the radiolabelled and PAS-reactive macromolecular species banded at densities > 1P42 g ml-1. When these were subjected to a second isopycnic density-gradient centrifugation in CsCl and 0-2 M-guanidinium chloride at the higher starting density of -152 g ml-', two macromolecular components were observed (Fig. 6). One (the
VAGAL CONTROL OF TRACHEAL MUCUS SECRETION
443
'low-density' component) had a buoyant density in CsCl of 1-52 g ml1', a high 35S to 3H ratio, and a strong PAS reactivity. The other (the 'high-density' component) had a buoyant density in CsCl of 1-63 g ml-', a low 35S to 3H ratio, and appeared unreactive to PAS. Stimulation frequency
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Fig. 5. The effects of stimulation frequency (upper panel) and pattern (lower panel) on reduction in heart rate in the absence of autonomic antagonists. Vagal stimulation caused frequency-dependent reductions in heart rate. Low frequency continuous stimulation at 4-5 and 9.5 Hz was significantly more effective in reducing heart rate than the corresponding high frequency burst (1 s in 5 s) stimulation at 22-5 and 47-5 Hz (paired t tests). Values shown represent means + S.E.M. Paired t tests showed that regular stimulation was more effective than burst stimulation at slowing heart rate. ** P < 0.005, ***p < 0-0005.
Resting samples collected in the absence of autonomic antagonists contained, in terms of macromolecular radioactivities, predominantly the 'high-density' component (Fig. 6A). Resting samples collected in the presence of cholinergic and adrenergic antagonists were almost identical, so their results have not been shown.
Samples collected during vagal stimulation contained predominantly the 'lowdensity' component (Fig. 6B). Samples collected during vagal stimulation in the presence of cholinergic and adrenergic antagonists contained similar amounts of the 'high-density' and 'low-density' components (Fig. 6C).
D. C. K. FUNG, D. J. BEACOCK AND P. S. RICHARDSON
444
DISCUSSION
The aims of these experiments were to study the effects of stimulation frequency and stimulation pattern of electrical vagal stimulation on the output of mucus glycoconjugates into the feline trachea in vivo and to compare the chemical A
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8
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Fig. 6. CsCl-02 M-guanidinium chloride isopycnic density-gradient profile of high molecular weight glycoconjugates collected during resting (control) periods (A), vagal stimulation in the absence of autonomic antagonists (B), and vagal stimulation in the presence of atropine (2 jug ml-' i.s. and 1 mg kg-' i.v.), phentolamine (10 ,ug ml-' i.s.) and propranolol (10 jg ml-' i.s. and 2 mg kg-' i.v.) (C). 0, 3H radioactivity; O, 35S radioactivity; *, PAS reactivity; *, density.
compositions of the high molecular weight mucus glycoconjugates released by various nervous mechanisms. In situ pulse radiolabelling was used as a sensitive method for detecting small amounts of mucus glycoconjugates released during brief sampling periods. In addition, the PAS reactivity was used to estimate chemically the mass of total glycoconjugates present in these samples, so as to account for changes in potential pools of secretions which may only radiolabel slowly or weakly. The changes detected by PAS-reactive glycoconjugates were quantitatively similar to those measured with 35S-label and larger than those with 3H-label, though the density-gradient chromatograms (Fig. 6) show that not all radiolabelled macromolecules are PAS-reactive. Readily detectable increases of PAS-reactive glycoconjugates during vagal stimulation in the presence of combined cholinergic and adrenergic blockade confirm previous reports of NANC control of airway secretion based on radiochemical methods (Peatfield & Richardson, 1983; Borson et al. 1984). In addition, the results from this study show that stimulation of the vagus nerves produced frequency-dependent increases in the output of mucus glycoconjugates (at least for 35S-labelled and PAS-reactive glycoconjugates), and that frequency-dependent increases remained after combined cholinergic and adrenergic blockade. The predominant NANC vagal pathway controlling mucus glycoconjugate secretion in the feline trachea runs orthodromically via the mural ganglia, while a minor pathway involving antidromic transmission via afferent fibres may also be present in this species (D. C. K. Fung, M. I. Allenby & P. S. Richardson, unpublished observations).
VAGAL CONTROL OF TRACHEAL MUCUS SECRETION 445 Widdicombe (1966) has recorded action potentials in vagal preganglionic fibres as they enter the trachea and bronchi. Among these he tentatively identified one group (group III) as secretomotor, and later evidence on the reflex control of tracheal secretion provides some support for this conclusion (Phipps, & Richardson, 1976; Davis, Chinn, Gold, Popovac, Widdicombe & Nadel, 1982). Group III fibres had a mean conduction velocity of 9-7 m s-', consistent with small myelinated axons, and some showed spontaneous activity. Reflex excitement of these fibres (e.g. by injection of cyanide near the carotid bodies) evoked irregular firing of action potentials with peak frequencies of around 25 Hz. The lower frequency of burst stimulation employed in the present study may crudely model activity in these fibres. Low frequency regular pulse stimulations and high frequency burst stimulations produced similar secretary responses, both in the absence and presence of combined cholinergic and adrenergic blockade, i.e. there was no evidence that delivery of stimuli in high frequency bursts augmented the secretary response. In many organ systems, the response to nervous stimulation is potentiated by delivering the electrical pulses in intermittent bursts at relatively high frequencies (see Introduction). Different nervous mechanisms (classical vs. NANC transmission) may be affected to different extents by changes in stimulus pattern. These studies have led to the speculation that, under physiological conditions where two or more transmitters co-exist in a nerve, the pattern of nerve firing may determine which transmitter action predominates (Lundberg, Hedlund, Angard, Fahrenkrug, Hokfelt, Tatemoto & Bartfai, 1982; Edwards, Andersson, Jarhult & Bloom, 1984; Bartfai, Iverfeldt, Fisone & Serfozo, 1988). In the present study the vagal control of mucus glycoconjugate release into the feline trachea was apparently not affected by altering the pattern of stimulation from relatively low frequency, continuous stimulation to high frequency burst stimulation. In contrast, low frequency continuous stimulations had the greater effect on heart rate. These findings suggest that neither the cholinergic nor the NANC mechanisms controlling secretions into the feline trachea are preferentially sensitive to high frequency burst stimulation, so that both patterns of stimulation produced similar responses. Another interpretation is that, as the tracheal blood flow is potentiated by high frequency burst stimulation (Martling et al. 1987), neurotransmitters (both classical and NANC) destined for secretary cells are cleared more rapidly and therefore any potentiation of transmitter release by high frequency burst stimulation is masked. It is also possible that the two patterns of stimulation (burst vs. regular) employed in this study were too similar to differentiate the secretary responses. Although we cannot rule out this possibility, the clearly different effects on heart rate produced by the two patterns of stimulation do not support this view. Chemical analysis of the mucus macromolecules showed qualitatively similar profiles for secretions obtained under resting conditions, during vagal stimulation, and during vagal stimulation in the presence of cholinergic and adrenergic blockade. Gel filtration on Sepharose CL-2B separated the radiolabelled macromolecules into two populations: high molecular weight glycoconjugates, such as mucins, which eluted in the void volume, and lower molecular weight macromolecular components which eluted in the partially included volume. Analysis of the high molecular weight mucus glycoconjugates with a two-step isopycnic density-gradient centrifugation in
446
D. C. K. FUNG, D. J. BEACOCK AND P. S. RICHARDSON
CsCl revealed two macromolecular species: a 'low-density' component with a buoyant density of 1P52 g ml-', and a 'high-density' component at 1-63 g ml-'. Previous studies (Davies et al. 1991) showed that the 'low-density' component has the characteristics of typical mucins (Carlstedt & Sheehan, 1984; Thornton, Davies, Kraayenbrink, Richardson, Sheehan & Carlstedt, 1990). The nature of the 'highdensity' component is at present unknown, but appears to be an atypical mucin-like macromolecule (Davies et al. 1991). Comparison of the density-gradient profiles for high molecular weight glycoconjugates obtained under resting conditions, during vagal stimulation, and during vagal stimulation in the presence of cholinergic and adrenergic blockade, revealed that, in terms of radioactivities, resting secretions contained predominantly the 'high-density' component and there was no evidence that vagal stimulation increased the output rate of this component. Secretions collected during vagal stimulation in the absence of autonomic antagonists contained abundant 'lowdensity', typical mucins. This agrees with previous observations that muscarinic agonists, such as pilocarpine, stimulate release of typical mucins from submucosal glands (Florey, Carleton & Wells, 1932; Gallagher et al. 1986; Davies et al. 1991). Vagal stimulation in the presence of muscarinic and adrenoceptor antagonists appeared to stimulate an output of typical mucins intermediate between those in resting conditions and unopposed vagal stimulation. Thus, it appears that activation of NANC nervous pathways also releases typical mucins into the feline trachea. Defects in the regulation of NANC nervous mechanisms have been suggested in the aetiology of asthma (Matsuzaki, Hamasaki & Said, 1980; Barnes, 1987; Ollerenshaw, Jarvis, Woolcock, Sullivan & Scheibner, 1989), a disease characterized by mucusplugging and hyper-reactivity of the airways; but the present study gives no evidence that, at least in the cat, there are special high molecular weight glycoconjugates associated with NANC stimulation of airway secretion. In conclusion, we have demonstrated that electrical vagal stimulation both in the absence and presence of combined cholinergic and adrenergic blockade produced frequency-dependent increases in the output of mucus glycoconjugates into the feline trachea, confirming the co-existence of a classical and NANC nervous pathway. Neither pathway appeared to be preferentially sensitive to either low frequency continuous stimulation or high frequency burst stimulation. Chemical analysis of high molecular weight glycoconjugates by isopycnic density-gradient centrifugation suggested that the release of typical mucins into the feline trachea may be stimulated by NANC nervous mechanisms, in addition to classical cholinergic mechanisms. The authors would like to thank Glaxo plc who supported D.C. K. F. and funded many of these experiments. REFERENCES
ANDERSSON, P.-O., BLOOM, S. R. & EDWARDS, A. V. (1982a). Parotid responses to stimulation of the parasympathetic innervation in bursts in weaned lambs. Journal of Physiology 330, 163-174. ANDERSSON, P.-O., BLOOM, S. R., EDWARDS, A. V. & JARHULT, J. (1982b). Effects of stimulation of the chorda tympani in bursts on submaxillary responses in the cat. Journal of Physiology 322, 469-483.
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BARNES, P. J. (1987). Regulatory peptides in the respiratory system. Experientia 43, 832-839. BARTFAI, T., IVERFELDT, K., FISONE, G. & SERFOZO, P. (1988). Regulation of the release of coexisting neurotransmitters. Annual Review of Pharmacology and Toxicology 28, 285-310. BORSON, D. B., CHARLIN, M., GOLD, B. D. & NADEL, J. A. (1984). Neural regulation of 35SO4macromolecule secretion from tracheal glands of ferrets. Journal of Applied Physiology 57, 457-466. CARLSTEDT, I., LINDGREN, H., SHEEHAN, J. K., ULMSTEN, U. & WINGERUP, L. (1983). Isolation and characterization of human cervical-mucus glycoproteins. Biochemical Journal 211, 13-22. CARLSTEDT, I. & SHEEHAN, J. K. (1984). Macromolecular properties and polymeric structure of mucus glycoproteins. In Mucus and Mucosa, Ciba Foundation Symposium 109, pp. 157-172. Pitman, London. DAVIES, J. R., GALLAGHER, J. T., RICHARDSON, P. S., SHEEHAN, J. K. & CARLSTEDT, I. (1991). Mucins in cat airway secretions. Biochemical Journal 275, 663-669. DAVIS, B., CHINN, R., GOLD, J., POPOVAC, D., WIDDICOMBE, J. G. & NADEL, J. A. (1982). Hypoxemia reflexly increases secretion from tracheal submucosal glands in dogs. Journal of Applied Physiology 52, 1416-1419. EDWARDS, A. V., ANDERSSON, P.-O., JARHULT, J. & BLOOM, S. R. (1984). Studies of the importance of the pattern of autonomic stimulation in relation to alimentary effectors. Quarterly Journal of Experimental Physiology 69, 607-614. FLOREY, H., CARLETON, H. M. & WELLS, A. Q. (1932). Mucus secretion in the cat trachea. British Journal of Experimental Pathology 13, 269-284. GALLAGHER, J. T., HALL, R. L., PHIPPS, R. J., JEFFERY, P. K., KENT, P. W. & RICHARDSON, P. S. (1986). Mucus glycoproteins (mucins) of the cat trachea: Characterisation and control of secretion. Biochimica et Biophysica Acta 886, 243-254. GALLAGHER, J. T., KENT, P. W., PASSATORE, M., PHIPPS, R. J. & RICHARDSON, P. S. (1975). The composition of tracheal mucus and the nervous control of its secretion in the cat. Proceedings of the Royal Society B 192, 49-76. LUNDBERG, J. M., HEDLUND, B., ANGARD, A., FAHRENKRUG, J., HOKFELT, T., TATEMOTO, K. & BARTFAI, T. (1982). Co-storage of peptides and classical transmitters in neurons. In Systemic Role of Regulatory Peptides, ed. BLOOM, S. R., POLAK, J. M. & LINDENLAUB, E., pp. 93-119. Schattauer, Stuttgart and New York. MANTLE, M. & ALLEN, A. (1978). A calorimetric assay for glycoproteins based on the Periodic Acid/Schiff stain. Biochemical Society Transactions 6, 607-609. MARTLING, C.-R., GAZELIUS, B. & LUNDBERG, J. M. (1987). Nervous control of tracheal blood flow in the cat measured by the laser Doppler technique. Acta Physiologica Scandinavica 130, 409-417. MATSUZAKI, Y., HAMASAKI, Y. & SAID, S. I. (1980). Vasoactive intestinal peptide: a possible transmitter of nonadrenergic relaxation in guinea pig airways. Science 210, 1252-1253. OLLERENSHAW, S., JARVIS, D., WooLCoCK, A., SULLIVAN, C. & SCHEIBNER, T. (1989). Absence of immunoreactive vasoactive intestinal polypeptide in tissue from the lungs of patients with asthma. New England Journal of Medicine 320, 1244-1248. PEATFIELD, A. C. & RICHARDSON, P. S. (1983). Evidence for non-cholinergic, non-adrenergic nervous control of mucus secretion into the cat trachea. Journal of Physiology 342, 335-345. PHIPPS, R. J. & RICHARDSON, P. S. (1976). The effects of irritation at various levels of the airway upon tracheal mucus secretion in the cat. Journal of Physiology 261, 563-581. ROGERS, D. F. & BARNES, P. J. (1989). Opioid inhibition of neurally mediated mucus secretion in human bronchi. Lancet i, 930-932. THORNTON, D. J., DAVIES, J. R., KRAAYENBRINK, M., RICHARDSON, P. S., SHEEHAN, J. K. & CARLSTEDT, I. (1990). Mucus glycoproteins from 'normal' human tracheobronchial secretion. Biochemical Journal 265, 179-186. THORNTON, D. J., HOLMES, D. F., SHEEHAN, J. K. & CARLSTEDT, I. (1989). Quantitation of mucus glycoproteins blotted onto nitrocellulose membranes. Analytical Biochemistry 182, 160-164. WIDDICOMBE, J. G. (1966). Action potentials in parasympathetic and sympathetic efferent fibres to the trachea and lungs of dogs and cats. Journal of Physiology 185, 56-88.
Journal of Physiology (1992), 453, pp. 435-447 With 6 figure Printed in Great Britain
VAGAL CONTROL OF MUCUS GLYCOCONJUGATE SECRETION INTO THE FELINE TRACHEA
BY D. C. K. FUNG, D. J. BEACOCK AND P. S. RICHARDSON* From the Department of Physiology, St George's Hospital Medical School, Cranmer Terrace, London SW17 ORE
(Received 11 September 1991) SUMMARY
1. We examined the effects of frequencies and patterns of electrical stimulation of the peripheral cut ends of the vagus nerves on the release of mucus glycoconjugates into feline trachea in vivo. Mucus glycoconjugates, radiolabelled biosynthetically with [35S]sulphate and [3H]glucose, were washed from a tracheal segment in situ, and dialysed before being counted and assayed chemically by the periodic acid-Schiff (PAS) method. 2. Vagal stimulation with regular pulses (10 V, 2 ms duration) at 1, 2X25, 4 5, 9 and 18 Hz produced frequency-dependent increases in the output of mucus glycoconjugates. 3. The muscarinic agonist pilocarpine (0-1-10 ,uM), given intrasegmentally, produced dose-dependent increases in the output of mucus glycoconjugates. 4. Pretreatment with atropine, phentolamine and propranolol reduced but did not, abolish the effects of vagal stimulation. Vagus nerve stimulation still caused frequency-dependent increases in the output of mucus glycoconjugates. 5. High frequency stimulations at 22-5 and 47-5 Hz given intermittently (1 s burst then 4 s rest), whether in the absence or presence of cholinergic and adrenergic blockade, produced similar secretary responses as the same number of pulses delivered in regular trains at 4-5 and 9-5 Hz. This suggests that neither cholinergic nor non-adrenergic, non-cholinergic (NANC) nerve mechanisms in this system are potentiated by high frequency, intermittent burst stimulation. 6. In the absence of atropine, regular vagal stimulation had a greater effect on heart rate than did the same number of pulses delivered in bursts. 7. High molecular weight glycoconjugates from secretions were taken from the void volume of a Sepharose CL-2B gel filtration column and separated further by density-gradient centrifugation. Macromolecular components were observed at two densities, a typical mucin at 1'52 g ml-1, and a high density atypical component at 1-63 g ml-'. In secretions collected during vagal stimulation, either in the absence or presence of cholinergic and adrenergic blockade, the ratio of low density to high density macromolecules was higher than in unstimulated secretions. This can be explained if both cholinergic and NANC nervous vagal mechanisms stimulate the output of typical (density = 1-52 g ml-') mucins into the feline trachea. * To whom correspondence should be addressed. MS 9727
436
D. C. K. FUNG, D. J. BEACOCK AND P. S. RICHARDSON INTRODUCTION
Airway mucus contains a number of components with a range of functions. High molecular weight glycoconjugates, in particular mucins, are thought to be mainly responsible for its theological properties which are essential for mucociliary transport (Carlstedt & Sheehan, 1984). In several species including cat and man, the parasympathetic vagal control of mucus glycoconjugate secretion into the tracheobronchial tree consists of both classical cholinergic (atropine-sensitive) and nonadrenergic, non-cholinergic (NANC, atropine-resistant) mechanisms (Peatfield & Richardson, 1983; Borson, Charlin, Gold & Nadel, 1984; Rogers & Barnes, 1989; D. C. K. Fung, M. I. Allenby & P. S. Richardson, unpublished observations). However, the compositions of glycoconjugates released by different nervous mechanisms have not been compared. Activation of cholinergic mechanisms stimulates the output of typical mucins into the feline trachea (Gallagher, Hall, Phipps, Jeffery, Kent & Richardson, 1986; Davies, Gallagher, Richardson, Sheehan & Carlstedt, 1991). Whether activation of NANC mechanisms would also release typical mucins is not known at present. In many organ systems the response to electrical nerve stimulation depends both on the stimulation frequency and the pattern of stimulation. In some systems the response is potentiated by delivery of electrical pulses in intermittent bursts at relatively high frequencies, e.g. the NANC-evoked decreases in feline submaxillary vascular resistance via stimulation of the chorda tympani (Andersson, Bloom, Edwards & Jarhult, 1982b), the NANC-evoked increases in blood flow through the feline tracheal microvasculature via stimulation of the vagus nerve (Martling, Gazelius & Lundberg, 1987), and the acetylcholine-mediated parotid responses in the sheep via stimulation of the parotid nerve (Andersson, Bloom & Edwards, 1982a). In this study, we examined the effects of stimulation frequency and pattern of vagus nerve stimulation on the output of mucus glycoconjugate into the feline trachea both in the absence and presence of combined cholinergic and adrenergic blockade. We also described the concentration-response relationship of pilocarpine, a cholinergic agonist, on mucus glycoconjugate output. In addition, the chemical compositions of high molecular weight mucus glycoconjugates released by vagal stimulation, both in the absence and presence of combined cholinergic and adrenergic
blockade, were analysed. METHODS
Preparation of tracheal segment Anaesthesia was induced in adult cats of either sex (weight 2-0-4-4 kg) by intraperitoneal injection of pentobarbitone sodium (Sagatal, May & Baker), 42 mg kg-', and maintained by intravenous injections via a femoral catheter. Rectal temperature was kept between 36 and 38 'C by a heat exchange pad. Blood pressure was recorded via a femoral arterial catheter connected to a strain-gauge transducer. The subsequent preparation of the tracheal segment has been described previously (Gallagher, Kent, Passatore, Phipps & Richardson, 1975). Briefly, a cannula through which the animal breathed was tied into the cervical trachea as far caudally as possible. A segment of trachea (4-6 cm in length) rostral to this was then cannulated at both ends to isolate it in situ with its nerves and blood supply intact. This tracheal segment was then washed out and filled with oxygenated Krebs-Henseleit solution at 37 'C.
VA GAL CONTROL OF TRACHEAL MUCUS SECRETION
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Radiolabelling of mucus glycoconjugates At the beginning of each experiment, 2 0 mCi of sodium [35k]sulphate and 0 5 mCi of [3H]glucose (Amersham International) in 3-0 ml Krebs-Henseleit solution were flushed into the tracheal segment and left for 1 h to enable uptake and biosynthetic incorporation of the radiolabels into mucus glycoconjugates (Gallagher et al. 1975, 1986; Davies et al. 1991). Samples were then taken at 30 min intervals for the next 2 h. then at 15 min intervals, by flushing the segment with 10 ml of Krebs-Henseleit solution.
IVagal stimulation The cervical sympathetic nerves on both sides were separated from the vagus nerve and cut at the mid-neck region. The vagus nerves (not desheathed) were then cut just caudal to the superior laryngeal nerves, and their peripheral ends were stimulated via platinum electrodes with pulses of 1O V, 2 ms duration for 10 min of a 15 min collection period (Devices gated pulse generator 2521 and isolated stimulator 2533). To determine the frequency-response relationship, frequencies of 1, 2-25. 45., 9 and 18 Hz were employed. To study the effects of stimulation pattern, we compared the secretary response to a regular train of electrical pulses at low frequency (4 5 or 9-5 Hz) with identical numbers of electrical pulses at the higher frequency (22-5 or 47 5 Hz) but applied in 1 s bursts at 5 s intervals. A Devices l)igitimer 3290 was used for triggering the pulse generator during stimulus bursts. Administration of drugs Drugs were administered by two routes: directly into the tracheal segment (i.s.) and/or intravenously via the femoral vein (I.v.). Drugs given i.s. were dissolved in oxygenated Krebs-Henseleit solution. The following drugs were used: atropine sulphate (Sigma), DL-
propranolol hydrochloride (Sigma), phentolamine mesylate (Rogitine, Ciba), pilocarpine hydro(hloride (Sigma). Experimental protocols After the initial 1 h pulse labelling period, the preparation was left unstimulated for the next 2 25 h. It was then stimulated with either electrical vagal stimulation for 10 min or with pilocarpine (i.s.) for 15 min at hourly intervals on four occasions. Antagonism of cholinergic and adrenergic effects was achieved by administration of atropine (2 jug ml-1 i.s. and 1 mg kg-1 i.v.), phentolamine (10 jug ml-1 i.s.) and propranolol (10 jug ml-' i.s. and 2 mg kg-1 i.v.) at least 30 min before nerve stimulation. I)uring each experiment the four stimuli applied were always different, thus the n values given in the text and figures for each intervention represent both the number of animals and the number of trials. Measurement of radiolabelled glycoconjugate output To dissolve mucus and minimize proteolysis, tracheal washings were mixed on collection with a solution containing 7 M-guanidiniurn chloride (practical grade, filtered and purified twice with activated charcoal), 5 mM-N a2ED)TA, 5 mM-N-ethylmaleimide, 01 mM-phenylmethylsuphonyl fluoride. 10 mm-sodium phosphate buffer (all from Sigma), pH 6-5, to give a final concentration of 4 M-guanidinium chloride, and kept between 4 and 6 0C (Carlstedt, Lindgren, Sheehan, Ulmsten & Wingerup, 1983). Samples were dialysed exhaustively (dialysis tubing with a molecular weight cut off (MWCO) at 12-14000, Medicell International) at 4 0C against distilled water containing 0-01 % (w/v) sodium azide and 0-01 % (w/v) sodium sulphate to disperse the non-covalently bound radiolabel, and radioactivities of aliquots were then measured in triplicate by liquid scintillation counting (Beckman Ready Protein Scintillant; Beckman Scintillation Counter LS 6000 IC, calibrated with quench correction to assay 3H and 35S separately).
Measurement of total glycoconjugate output The total glycoconjugates present in the tracheal washings after exhaustive dialysis were estimated in triplicate using the solution periodic acid-Schiff (PAS) assay method (Mantle & Allen, 1978). Bovine submaxillary mucin (Sigma) was used as a calibration standard. Glycoconjugate concentrations were calibrated in terms of this standard, and expressed as output rates (aug min-).
438
D. C. K. FUNG, D. J. BEACOCK AND P. S. RICHARDSON
Purification of high molecular weight glycoconjugates Tracheal washings from different experiments collected as described above were divided, after exhaustive dialysis, into four pools as follows: (1) unstimulated (resting) samples in the absence of autonomic antagonists; (2) samples collected during vagal stimulation; (3) resting samples in the presence of cholinergic and adrenergic antagonists; (4) samples collected during vagal stimulation in the presence of cholinergic and adrenergic antagonists. Pools were concentrated by dialysis against Aquacide 2 (Calbiochem), equilibrated against a solution containing 4 M-guanidinium chloride, 5 mM-Na2EDTA, 10 mM-sodium phosphate buffer, pH 6 5, and chromatographed on a Sepharose CL-2B (Pharmacia) column (60 x 2-1 cm) using the same buffer at a flow of 21 ml h-'. Void volume and totally included volume of the column were determined by Dextran Blue (Pharmacia) and [35S]sulphate respectively. Aliquots (50-200 ,l) from each fraction (7 ml) were taken for liquid scintillation counting and for total glycoconjugate estimation by PAS staining after slot-blotting onto nitrocellulose membrane (0'45 ,um pore size, Schleicher and Schuell) using a multi-well slot-blot manifold (Minifold 2, Schleicher and Schuell) (Thornton, Holmes, Sheehan & Carlstedt, 1989). The PAS reactivity of each blotted sample was then measured using a scanning reflectance densitometer (Chromoscan 3, Joyce & Loebl) at a wavelength of 530 nm. Isopycnic density-gradient ultracentrifugation Void volume fractions from Sepharose CL-2B containing high molecular weight glycoconjugates were pooled, concentrated, mixed with sufficient CsCl to give an initial density of approximately 1 40 g ml-', and subjected to CsCl isopycnic density-gradient centrifugation at 40000 r.p.m. at 15 00 for 70 h (Beckman L5-50 ultracentrifuge; 5OTi rotor). Fractions (1 ml) were collected from the bottom (Beckman fraction recovery system) and their densities were measured with a Hamilton syringe as a pycnometer. Fractions with densities greater than 1-42 g ml-' were pooled, equilibrated against a solution containing 0-2 M-guanidinium chloride, 5 mM-Na2EDTA, 10 mMsodium phosphate buffer, pH 65, and subjected to a second CsCl isopycnic density-gradient centrifugation at a starting density of 1-52 g ml-' using the same conditions as above. Aliquots from each fraction were taken for liquid scintillation counting and for PAS staining as described above. Statistical treatment of results Changes in glycoconjugate output induced by vagal stimulation were calculated as the percentage increase of the stimulated sample over the mean value of the preceding and succeeding sample (bracketing controls). Effects produced by pilocarpine tended to persist into the succeeding control sample, so they were expressed as the percentage increase of the stimulated sample over the preceding sample only (single preceding controls). Vagal reductions of heart rate were expressed as the percentage reduction during stimulation compared with the preceding period. Values shown represent means + standard error of the mean (s.E.M.). When paired data were available (i.e. for the results of burst versus regular stimulation, Figs 3, 4 and 5) comparisons were made by a paired Student's t test, and P < 0 05 was accepted as statistically significant (see figure legends for further details). RESULTS
Effects of frequency of vagal stimulation
Vagal stimulation with regular pulses at 1, 2-25, 45, 9 and 18 Hz produced frequency-dependent increases in the secretion of mucus glycoconjugates, as measured by the output rates of 35S-labelled and PAS-reactive glycoconjugates (Fig. 1). The output of 3H-labelled glycoconjugate was less clearly frequency related.
Effects of a cholinergic muscarinic agonist The muscarinic agonist pilocarpine (0-3-10 /kM) given I.s. produced dose-dependent increases in mucus glycoconjugate secretion as measured by all three indices of glycoconjugate output rate (Fig. 2).
VAGAL CONTROL OF TRACHEAL MUCUS SECRETION
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Effects of pharmacological blockade by cholinergic and adrenergic antagonists Pretreatment with atropine (2,jg ml-1 i.s. and 1 mg kg-1 i.v.), phentolamine (10 jtg ml-1 i.s.) and propranolol (10 gg ml-' I.s. and 2 mg kg-1 i.v.), in doses capable of abolishing all effects produced by pilocarpine (3 ,aM), phenylephrine (40 gM) and
35S-glycoconjugates 300200-
3H-glycoconjugates
30>
Simulation
1
225
45
9
18 Hz
(n)
(4) (4)
(5) (5)
(11 )(9)
(13)(4)
(7) (5)
Fig. 1. The effects of stimulation frequency on the output of 35S-, 3H-labelled and PASreactive glycoconjugates induced by regular electrical stimulation of the peripheral cut ends of the vagus nerves, both in the absence (open columns) and presence (hatched columns) of atropine (2 jug ml-1 I.S, and 1 mg kg- 'Iv.), phentolamine (10 jig mP'l i.s.) and propranolol (10 jug mll'Is . and 2 mg kg- I.V.) . Values shown represent means+ S.E.M.
isoprenaline (40 gM) administered I.s. towards the end of the experiments (D. C. K. Fung, M. I. Allenby & P. S. Richardson, unpublished observations), failed to abolish the effects produced by vagal stimulation. Vagal stimulation caused frequencydependent increases in the output of 355- and 3H-labelled as well as the total PASreactive glycoconjugates (Fig. 1). 15
P
2Y 453
44040D. C. K. FUNG, D. J. BEACOCK AND P. S. RICHARDSON
Effects3 of the pattern of vagal stimulation In the absence of autonomic antagonists Regular trains of electrical pulses delivered at 4-5 and 9-5 Hz produced similar secretary responses as the same number of pulses delivered in high frequency bursts at 22-5 and 47-5 Hz
given for 1 s in 5 s (Fig. 3). 3_glIycoconj ugates 600500400300 200100-
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log [pilocarpine] (5) (1 0) (12) (1 0) (6) (n) Fig. 2. The effects of pilocarpine at different concentrations on the output of 35S-,' 3Hlabelled and PAS-reactive glycoconjugates. Values shown represent means + sEM.
In the presence of cholinergic and adrenergic antagonists Regular trains of electrical pulses delivered at 4-5 and 9-5 Hz again produced similar secretary responses as the same number of pulses delivered in high frequency bursts at 22-5 and 47T5 Hz given for 1 s in 5 s (Fig. 4).
Effects of vagal stimulation on heart rate In the absence of autonomic antagonists, vagal stimulation from 1 to 18 Hz produced frequency-dependent reductions in heart rate (Fig. 5). Regular trains of
VAGAL CONTROL OF TRACHEAL MUCUS SECRETION 441 electrical pulses delivered at 4-5 and 9-5 Hz slowed heart rate more effectively than the same number of pulses delivered in bursts at 22-5 and 47-5 Hz given for 1 s in 5 s. After treatment with cholinergic and adrenergic antagonists, changes in heart rate were absent or minimal during vagal stimulation at any frequency. 35S-glycoconjugates r-n.s.-
r-n.s.-
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Burst H Reg. Burst 22-5 95Hz47-5 Hz (n) (5) (5) (8) (8) Fig. 3. The effects of stimulation pattern on the output of 35S- and 3H-labelled and PASreactive glycoconjugates induced by vagal stimulation in the absence of autonomic antagonists. Low frequency continuous stimulation (Reg.) at 4-5 Hz and 9-5 Hz were compared with the corresponding high frequency burst (1 s in 5 s) stimulation at 22-5 and 47-5 Hz. Values shown represent means + S.E.M. Paired t tests demonstrated no significant difference between the two patterns of vagal stimulation.
Chemical analysis of mucus glycoconjugates All the pools of secretions, when chromatographed on Sepharose CL-2B, showed two main peaks of radiolabelled macromolecules: a large peak eluting in the void volume where high molecular weight (Mr > 106) glycoconjugates such as mucins are found, and a smaller, more broadly based peak (or possibly peaks) in the partially 15-2
D. C. K. FUNG, D. J. BEACOCK AND P. S. RICHARDSON included volume which corresponds to smaller macromolecular species (see Gallagher et al. 1986). When the high molecular weight glycoconjugates from the void volume of CL-2B were analysed by isopycnic density-gradient centrifugation in CsCl and 4 M-
442
35S -glycoconjugates rn.s.
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(5) (5) Fig. 4. The effects of stimulation pattern on the output of 35S- and 3H-labelled and PASreactive glycoconjugates induced by vagal stimulation in the presence of atropine (2 tg ml-' i.s. and 1 mg kg-' i.v.), phentolamine (10 #g ml-' i.s.) and propranolol (10 ug ml-' i.s. and 2 mg kg-' I.v.). Low frequency continuous stimulation at 4-5 and 9 5 Hz were compared with the corresponding high frequency burst (1 s in 5 s) stimulation at 22-5 and 47-5 Hz. Values shown represent means+ S.E.M. Paired t tests demonstrated no significant differences between the two patterns of vagal stimulation.
guanidinium chloride at a starting density of approximately 1-40 g ml-', nearly all of the radiolabelled and PAS-reactive macromolecular species banded at densities > 1P42 g ml-1. When these were subjected to a second isopycnic density-gradient centrifugation in CsCl and 0-2 M-guanidinium chloride at the higher starting density of -152 g ml-', two macromolecular components were observed (Fig. 6). One (the
VAGAL CONTROL OF TRACHEAL MUCUS SECRETION
443
'low-density' component) had a buoyant density in CsCl of 1-52 g ml1', a high 35S to 3H ratio, and a strong PAS reactivity. The other (the 'high-density' component) had a buoyant density in CsCl of 1-63 g ml-', a low 35S to 3H ratio, and appeared unreactive to PAS. Stimulation frequency
1
225
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9
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*
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Fig. 5. The effects of stimulation frequency (upper panel) and pattern (lower panel) on reduction in heart rate in the absence of autonomic antagonists. Vagal stimulation caused frequency-dependent reductions in heart rate. Low frequency continuous stimulation at 4-5 and 9.5 Hz was significantly more effective in reducing heart rate than the corresponding high frequency burst (1 s in 5 s) stimulation at 22-5 and 47-5 Hz (paired t tests). Values shown represent means + S.E.M. Paired t tests showed that regular stimulation was more effective than burst stimulation at slowing heart rate. ** P < 0.005, ***p < 0-0005.
Resting samples collected in the absence of autonomic antagonists contained, in terms of macromolecular radioactivities, predominantly the 'high-density' component (Fig. 6A). Resting samples collected in the presence of cholinergic and adrenergic antagonists were almost identical, so their results have not been shown.
Samples collected during vagal stimulation contained predominantly the 'lowdensity' component (Fig. 6B). Samples collected during vagal stimulation in the presence of cholinergic and adrenergic antagonists contained similar amounts of the 'high-density' and 'low-density' components (Fig. 6C).
D. C. K. FUNG, D. J. BEACOCK AND P. S. RICHARDSON
444
DISCUSSION
The aims of these experiments were to study the effects of stimulation frequency and stimulation pattern of electrical vagal stimulation on the output of mucus glycoconjugates into the feline trachea in vivo and to compare the chemical A
C
B
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6
8
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Fig. 6. CsCl-02 M-guanidinium chloride isopycnic density-gradient profile of high molecular weight glycoconjugates collected during resting (control) periods (A), vagal stimulation in the absence of autonomic antagonists (B), and vagal stimulation in the presence of atropine (2 jug ml-' i.s. and 1 mg kg-' i.v.), phentolamine (10 ,ug ml-' i.s.) and propranolol (10 jg ml-' i.s. and 2 mg kg-' i.v.) (C). 0, 3H radioactivity; O, 35S radioactivity; *, PAS reactivity; *, density.
compositions of the high molecular weight mucus glycoconjugates released by various nervous mechanisms. In situ pulse radiolabelling was used as a sensitive method for detecting small amounts of mucus glycoconjugates released during brief sampling periods. In addition, the PAS reactivity was used to estimate chemically the mass of total glycoconjugates present in these samples, so as to account for changes in potential pools of secretions which may only radiolabel slowly or weakly. The changes detected by PAS-reactive glycoconjugates were quantitatively similar to those measured with 35S-label and larger than those with 3H-label, though the density-gradient chromatograms (Fig. 6) show that not all radiolabelled macromolecules are PAS-reactive. Readily detectable increases of PAS-reactive glycoconjugates during vagal stimulation in the presence of combined cholinergic and adrenergic blockade confirm previous reports of NANC control of airway secretion based on radiochemical methods (Peatfield & Richardson, 1983; Borson et al. 1984). In addition, the results from this study show that stimulation of the vagus nerves produced frequency-dependent increases in the output of mucus glycoconjugates (at least for 35S-labelled and PAS-reactive glycoconjugates), and that frequency-dependent increases remained after combined cholinergic and adrenergic blockade. The predominant NANC vagal pathway controlling mucus glycoconjugate secretion in the feline trachea runs orthodromically via the mural ganglia, while a minor pathway involving antidromic transmission via afferent fibres may also be present in this species (D. C. K. Fung, M. I. Allenby & P. S. Richardson, unpublished observations).
VAGAL CONTROL OF TRACHEAL MUCUS SECRETION 445 Widdicombe (1966) has recorded action potentials in vagal preganglionic fibres as they enter the trachea and bronchi. Among these he tentatively identified one group (group III) as secretomotor, and later evidence on the reflex control of tracheal secretion provides some support for this conclusion (Phipps, & Richardson, 1976; Davis, Chinn, Gold, Popovac, Widdicombe & Nadel, 1982). Group III fibres had a mean conduction velocity of 9-7 m s-', consistent with small myelinated axons, and some showed spontaneous activity. Reflex excitement of these fibres (e.g. by injection of cyanide near the carotid bodies) evoked irregular firing of action potentials with peak frequencies of around 25 Hz. The lower frequency of burst stimulation employed in the present study may crudely model activity in these fibres. Low frequency regular pulse stimulations and high frequency burst stimulations produced similar secretary responses, both in the absence and presence of combined cholinergic and adrenergic blockade, i.e. there was no evidence that delivery of stimuli in high frequency bursts augmented the secretary response. In many organ systems, the response to nervous stimulation is potentiated by delivering the electrical pulses in intermittent bursts at relatively high frequencies (see Introduction). Different nervous mechanisms (classical vs. NANC transmission) may be affected to different extents by changes in stimulus pattern. These studies have led to the speculation that, under physiological conditions where two or more transmitters co-exist in a nerve, the pattern of nerve firing may determine which transmitter action predominates (Lundberg, Hedlund, Angard, Fahrenkrug, Hokfelt, Tatemoto & Bartfai, 1982; Edwards, Andersson, Jarhult & Bloom, 1984; Bartfai, Iverfeldt, Fisone & Serfozo, 1988). In the present study the vagal control of mucus glycoconjugate release into the feline trachea was apparently not affected by altering the pattern of stimulation from relatively low frequency, continuous stimulation to high frequency burst stimulation. In contrast, low frequency continuous stimulations had the greater effect on heart rate. These findings suggest that neither the cholinergic nor the NANC mechanisms controlling secretions into the feline trachea are preferentially sensitive to high frequency burst stimulation, so that both patterns of stimulation produced similar responses. Another interpretation is that, as the tracheal blood flow is potentiated by high frequency burst stimulation (Martling et al. 1987), neurotransmitters (both classical and NANC) destined for secretary cells are cleared more rapidly and therefore any potentiation of transmitter release by high frequency burst stimulation is masked. It is also possible that the two patterns of stimulation (burst vs. regular) employed in this study were too similar to differentiate the secretary responses. Although we cannot rule out this possibility, the clearly different effects on heart rate produced by the two patterns of stimulation do not support this view. Chemical analysis of the mucus macromolecules showed qualitatively similar profiles for secretions obtained under resting conditions, during vagal stimulation, and during vagal stimulation in the presence of cholinergic and adrenergic blockade. Gel filtration on Sepharose CL-2B separated the radiolabelled macromolecules into two populations: high molecular weight glycoconjugates, such as mucins, which eluted in the void volume, and lower molecular weight macromolecular components which eluted in the partially included volume. Analysis of the high molecular weight mucus glycoconjugates with a two-step isopycnic density-gradient centrifugation in
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CsCl revealed two macromolecular species: a 'low-density' component with a buoyant density of 1P52 g ml-', and a 'high-density' component at 1-63 g ml-'. Previous studies (Davies et al. 1991) showed that the 'low-density' component has the characteristics of typical mucins (Carlstedt & Sheehan, 1984; Thornton, Davies, Kraayenbrink, Richardson, Sheehan & Carlstedt, 1990). The nature of the 'highdensity' component is at present unknown, but appears to be an atypical mucin-like macromolecule (Davies et al. 1991). Comparison of the density-gradient profiles for high molecular weight glycoconjugates obtained under resting conditions, during vagal stimulation, and during vagal stimulation in the presence of cholinergic and adrenergic blockade, revealed that, in terms of radioactivities, resting secretions contained predominantly the 'high-density' component and there was no evidence that vagal stimulation increased the output rate of this component. Secretions collected during vagal stimulation in the absence of autonomic antagonists contained abundant 'lowdensity', typical mucins. This agrees with previous observations that muscarinic agonists, such as pilocarpine, stimulate release of typical mucins from submucosal glands (Florey, Carleton & Wells, 1932; Gallagher et al. 1986; Davies et al. 1991). Vagal stimulation in the presence of muscarinic and adrenoceptor antagonists appeared to stimulate an output of typical mucins intermediate between those in resting conditions and unopposed vagal stimulation. Thus, it appears that activation of NANC nervous pathways also releases typical mucins into the feline trachea. Defects in the regulation of NANC nervous mechanisms have been suggested in the aetiology of asthma (Matsuzaki, Hamasaki & Said, 1980; Barnes, 1987; Ollerenshaw, Jarvis, Woolcock, Sullivan & Scheibner, 1989), a disease characterized by mucusplugging and hyper-reactivity of the airways; but the present study gives no evidence that, at least in the cat, there are special high molecular weight glycoconjugates associated with NANC stimulation of airway secretion. In conclusion, we have demonstrated that electrical vagal stimulation both in the absence and presence of combined cholinergic and adrenergic blockade produced frequency-dependent increases in the output of mucus glycoconjugates into the feline trachea, confirming the co-existence of a classical and NANC nervous pathway. Neither pathway appeared to be preferentially sensitive to either low frequency continuous stimulation or high frequency burst stimulation. Chemical analysis of high molecular weight glycoconjugates by isopycnic density-gradient centrifugation suggested that the release of typical mucins into the feline trachea may be stimulated by NANC nervous mechanisms, in addition to classical cholinergic mechanisms. The authors would like to thank Glaxo plc who supported D.C. K. F. and funded many of these experiments. REFERENCES
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