NANC nerve pathways controlling secretion into feline trachea D. C. K. FUNG, Department
M. I. ALLENBY,
AND
mucus glycoconjugate
P. S. RICHARDSON
of Physiology, St. George’sHospital Medical School, London S W17 ORE,
FUNG, D. C. K., M. I. ALLENBY, AND P. S. RICHARDSON. NANC nerve pathways controlling mucus glycoconjugate secretion into feline trachea. J. Appl. Physiol. 73(2): 625-630,1992.We used autonomic-blocking drugs to define nonadrenergic noncholinergic (NANC) vagus nerve pathways regulating tracheal mucussecretion. In anesthetized cats, mucusglycoconjugates, radiolabeled biosynthetically with [35S]sulfate and [3H]glucose,were washedfrom a tracheal segmentin situ and dialyzed before scintillation counting and chemical assaywith periodic acid-Schiff (PAS). Without autonomic blockade,vagal stimulation (9.5 Hz, 10 V, 2-mspulse width, lo-min duration) increasedoutputs of radiolabeled and PAS-reactive glycoconjugates repeatably over four stimulation periods. In other animals,vagusnerveswerestimulated with administration of autonomic blockersbetween stimulations. The first stimulation (no blockers) increasedglycoconjugateoutput (A35S = 221 t 43.3%, A3H = 58 t 13.8%; APAS = +299 t 82.7%). Atropine, phentolamine, and propranolol reduced these responses(A35S = 67 t 15.6%; A3H = 26 t 5.3%; APAS = 88 t 25.6%). Guanethidine did not significantly lessenthem further, although A3H was no longer significant. Ganglion blockade with hexamethonium prevented most of the remaining responseto vagal stimulation (P < 0.05for dimininution of A35Sand APAS), but small effects persisted(A35S = 17 ? 5.6%; APAS = 20 t 6.8%; P < 0.05). We conclude that the main NANC vagal pathway controlling tracheal glycoconjugatesecretion runs orthodromically.
United
Kingdom
guish between these two possibilities. The effect of hexamethonium, a nicotinic ganglion-blocking agent, was tested on vagal control of tracheal glycoconjugate secretion in cats treated with atropine, phentolamine, and propranolol (to block classic cholinergic and adrenergic nerve pathways) and guanethidine [to block possible release of nonadrenergic transmitters from sympathetic nerve endings (12, 15)]. Hexamethonium would block transmission via the mural ganglia but leave intact any direct antidromic nervous control of secretory cells via afferent pathways. METHODS
Preparation of tracheal segment. Anesthesia was induced in adult cats of either sex (weight 2.0-4.4 kg) by intraperitoneal injection of pentobarbital sodium (42 mg/kg; Sagatal, May & Baker) and maintained by intravenous injections via a femoral vein catheter. Rectal temperature was monitored and kept between 36 and 38OC by a heat-exchange pad. Arterial blood pressure was recorded on ultraviolet-sensitive paper (SE Laboratory 6008) via a femoral arterial catheter connected to a strain gauge transducer (SE Laboratory 4.88), calibrated with a mercury manometer. Mean blood pressure was estimucus secretion; airway; parasympathetic nerve; mural gan- mated as diastolic plus one-third pulse pressure. Blood glion; antidromic transmission; orthodromic transmission; pressure measurements were made before and after each nonadrenergic noncholinergic nerve; vagus nerve vagal stimulation and also 1 and 9 min into each period of vagal stimulation. The subsequent preparation of the tracheal segment SECRETION OF AIRWAY MUCUS, essential for effeCtive air- has been described previously (10). Briefly, a cannula way clearance by mucociliary transport and cough, is through which the animal breathed was tied into the cerunder several control mechanisms including parasympavical trachea as far caudally as possible. A segment of thetic and sympathetic nervous pathways (21). In the cat trachea (4-6 cm long) rostra1 to this was then cannulated the parasympathetic (vagal) pathway controlling mucus at both ends to isolate it in situ with its nerves and blood secretion acts through both choline@ and nonadrenersupply intact. This tracheal segment was then washed gic noncholinergic (NANC) nervous mechanisms (26). out and filled with oxygenated Krebs-Henseleit solution Similar findings have also been reported in the ferret at 37OC. trachea in vitro (3). The pathways within the tracheal Radiolabeling of mucus glycoconjugates. At the beginwall of the NANC nerves involved are unknown, but ning of each experiment, 2.0 mCi of sodium [35S]sulfate and 0.5 mCi of [3H]glucose (Amersham International) in there are at least two possibilities: 1) collateral branches of vagal afferent fibers may innervate secretory cells 3.0 ml Krebs-Henseleit solution were flushed into the within submucosal glands, allowing antidromic nerve tracheal segment and left for 1 h to enable uptake and impulses to stimulate secretion (19); or 2) there may be biosynthetic incorporation of the radiolabels into mucus postganglionic NANC nerves with cell bodies in the glycoconjugates (9, 10). Samples were then taken at 30mural ganglia, innervated by preganglionic parasympamin intervals for the next 2 h and then at 15-min interthetic fibers. This pathway would allow orthodromic ner- vals by flushing the segment with 10 ml of Krebs-Hensevous control of secretion. leit solution. The present experiments were undertaken to distinQuantification of radiolabeled glycoconjugates. Tracheal 0161-7567/92
$2.00 Copyright
0 1992 the American
Physiological
Society
625
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 13, 2019.
626
NANC
CONTROL
OF
AIRWAY
washings were immediately mixed with sufficient guanidine hydrochloride (grade 1, Sigma) to give a final concentration of 6 M, which dissolved mucus, and kept between 4 and 6OC. They were then dialyzed exhaustively (dialysis tubing with molecular weight cutoff at 1214,000, Medicell International) against distilled water containing 0.01% (wt/vol) sodium azide and 0.01% (wt/ vol) sodium sulfate to disperse the noncovalently bound radiolabel. Radioactivity of aliquots from the samples was then measured in triplicate by liquid scintillation counting (Beckman Ready Protein Scintillant; Beckman Scintillation Counter LS 6000 IC, calibrated with quench correction to assay 35Sand 3H separately). Total glycoconjugate measurement. The total glycoconjugates present in the tracheal washings after exhaustive dialysis were estimated in triplicate using the solution periodic acid-Schiff (PAS) assay method (18). Bovine submaxillary mucin (Sigma) was used as a calibration standard. Glycoconjugate concentrations are expressed in terms of this standard. This assay was performed on samples from all cats in which autonomic blockers were given but only on four of the experiments to check repeatability of vagus nerve stimulation. Vagus nerue stimulation. In the cat the major parasympathetic efferent pathway to the trachea runs through the cervical vagus nerves and not in the superior laryngeal nerves as it does in the dog and the ferret (23). The cervical sympathetic nerves on both sides were separated from the vagus nerves and cut at the midneck region. The vagus nerves were then cut just caudal to the superior laryngeal nerves. Their peripheral ends were stimulated via platinum electrodes, at 9.5 Hz with pulses of 10 V and 2-ms duration for 10 min of a 15min collection period (Devices gated pulse generator 2521, Devices isolated stimulator 2533). Administration of drugs. Drugs were administered in two ways: directly into the tracheal segment (is) and/or intravenously via the femoral vein. Drugs given intrasegmentally were dissolved in oxygenated Krebs-Henseleit solution. The following drugs were used: atropine sulfate (Sigma), DL-propranolol hydrochloride (Sigma), phentolamine mesylate (Rogitine, Ciba), guanethidine sulfate (Sigma), hexamethonium bromide (Sigma), pilocarpine hydrochloride (Sigma), phenylephrine hydrochloride (Boots), and isoproterenol hydrochloride (Saventrine, Pharmax). Experimentalprotocols. After the initial l-h pulse-labeling period, the preparation was left unstimulated for the next 2.25 h. In all but two experiments, vagal stimulation was applied for 10 min at hourly intervals on four occasions. In the first series of experiments (9 cats) to study the reproducibility of the responses, vagal stimulations were applied on four occasions in the absence of autonomic nervous system antagonists. In a second series of experiments (Fig. I), designed to examine the effects of progressive blockade of autonomic pathways by cumulative administration of autonomic antagonists, vagal stimulation was applied as follows: 1) in the absence of drugs (control); 2) 30 min after administration of atropine (2 pg/ml is and 1 mg/kg iv), phentolamine (10 pg/ml is), and propranolol(l0 &ml is and 2 mg/kg iv: APP) (26); 3) in
MUCUS
SECRETION
35Sglycoconjugate
output
120
-
I
.
80
$
60
-
-
-
.
40 20 0 0
1
2
3 H-glycoconjugate
4
3
output -
5
-
-
6
-
300 250 ”
,
50 o..............l! 0
1
PAS-reactive
2
glycoconjugate -
3
output -
.
. . 4
-
.
.
. . 5
. 6
-
40 T 30 l r(
z
20
-2
10 0
-
0
l . hours 2 Time*
FIG. 1. Example of 1 experiment illustrating protocol used to study effects of drugs on output of 35S- and 3H-labeled and periodic acidSchiff (PAS)-reactive glycoconjugates in response to vagal stimulation. After initial 2.25 h, electrical pulses (10 V, 2-ms duration, 9.5 Hz) were applied to vagus nerves for 10 min at hourly intervals (open and closed bars). To study effects of progressive autonomic blockade, drugs were administered 30 min before respective stimulation. At the end of this experiment (hatched bars), efficacy of a-adrenoceptor blockade was checked by intrasegmental administration of 40 PM phenylephrine.
the presence of APP and guanethidine (5 mg/kg iv; APPG) (15); and 4) in the presence of APPG and hexamethonium (2 mg/kg iv; APPGH) (35). In addition, after the initial radiolabeling and unstimulated secretion periods, two cats were treated first with APPG and then with APPGH, and electrical stimulations were applied to their vagus nerves during each of the two drug combinations; thus the control and APP stimulations (stimulations 1 and 2 above) were omitted. Effects of vagal stimulations in these have been included with the equivalent results from the other six cats. Statistical treatment of results. Vagally induced changes were calculated as the percent increase of the stimulated sample over the mean value of the preceding and succeeding sample (bracketing controls). Values are means t SE. Comparisons were made by Student’s t test, either paired or unpaired as appropriate. P < 0.05 was accepted as statistically significant.
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 13, 2019.
NANC CONTROL Wglycoconjugates
OF AIRWAY
.
2 .EI200 2a g 100 3 ep 0
?H-glycoconjugates 120
(n=9)
l
-
T
,
PAS-reactive
glycoconjugates
SECRETION
627
0.02, n = 6) (Fig. 3). These increases were significantly less (P < 0.005 for 35S, P < 0.05 for 3H, P < 0.02 for PAS) than those produced by vagal stimulation in the absence of drugs, thus confirming the coexistence of NANC and classic nervous pathways, both of which stimulate mucus glycoconjugate secretion into the feline trachea (26). Efficacy of muscarinic and adrenergic antagonists. To test the efficacy of the above regime of adrenoceptor and muscarinic antagonists, a muscarinic agonist (pilocarpine, 3 PM) and a- and ,&agonists (phenylephrine, 40 PM; isoproterenol, 40 PM) were administered, on different occasions, in the continued presence of APP. None of the agonists significantly stimulated the release of radiolabeled or PAS-reactive glycoconjugates (Table 1). Effects of guanethidine (APPG). Vagal stimulation in the presence of guanethidine, in addition to APP, still caused secretion as indicated by two of the measures of glycoconjugate output (A35S = 40 t 15.0%, P < 0.02; APAS = 89 t 28.5%, P < 0.02; n = 8 in each case) but the
(n=9)
5 300 8
MUCUS
(n=4)
I
s
250
Q
200
I
l *
150 3&) 100
.3 Ii8 -5 VSl
vs2
vs3
FIG.
RESULTS
Effects of repeated vagal stimulation in the absence of drugs. After the initial 2.25 h of collections without secre-
tory stimuli, vagal stimulations applied at hourly intervals in the absence of drugs gave reproducible responses as measured by the percent increase in outputs of 35Sand 3H-labeled macromolecules as well as chemically for total glycoconjugates by the PAS assay (Fig. 2). No significant difference in the response was noted between any measure of the effectiveness of stimuli applied at different times during experiments. Vagal stimulation before administration of autonomic antagonists. The first vagal stimulation was applied in
the absence of autonomic antagonists in six cats and produced clear increases in all three measures of mucus glycoconjugate secretion (A3?S = 221 t 43.3%, P < 0.005; A3H = 58 t 13.8%, P < 0.01; APAS = 299 t 82.7%, P < 0.02) (Fig. 3). Effects of muscarinic and adrenoceptor blockade (APP).
Electrical vagal stimulation after treatment still produced significant increases in the mucus glycoconjugates (A3?S = 67 t l&6%, A3H = 26 t 5.3%, P < 0.005; APAS = 88 t
with APP output of P < 0.01; 25.6%, P
M. I. ALLENBY,
AND
mucus glycoconjugate
P. S. RICHARDSON
of Physiology, St. George’sHospital Medical School, London S W17 ORE,
FUNG, D. C. K., M. I. ALLENBY, AND P. S. RICHARDSON. NANC nerve pathways controlling mucus glycoconjugate secretion into feline trachea. J. Appl. Physiol. 73(2): 625-630,1992.We used autonomic-blocking drugs to define nonadrenergic noncholinergic (NANC) vagus nerve pathways regulating tracheal mucussecretion. In anesthetized cats, mucusglycoconjugates, radiolabeled biosynthetically with [35S]sulfate and [3H]glucose,were washedfrom a tracheal segmentin situ and dialyzed before scintillation counting and chemical assaywith periodic acid-Schiff (PAS). Without autonomic blockade,vagal stimulation (9.5 Hz, 10 V, 2-mspulse width, lo-min duration) increasedoutputs of radiolabeled and PAS-reactive glycoconjugates repeatably over four stimulation periods. In other animals,vagusnerveswerestimulated with administration of autonomic blockersbetween stimulations. The first stimulation (no blockers) increasedglycoconjugateoutput (A35S = 221 t 43.3%, A3H = 58 t 13.8%; APAS = +299 t 82.7%). Atropine, phentolamine, and propranolol reduced these responses(A35S = 67 t 15.6%; A3H = 26 t 5.3%; APAS = 88 t 25.6%). Guanethidine did not significantly lessenthem further, although A3H was no longer significant. Ganglion blockade with hexamethonium prevented most of the remaining responseto vagal stimulation (P < 0.05for dimininution of A35Sand APAS), but small effects persisted(A35S = 17 ? 5.6%; APAS = 20 t 6.8%; P < 0.05). We conclude that the main NANC vagal pathway controlling tracheal glycoconjugatesecretion runs orthodromically.
United
Kingdom
guish between these two possibilities. The effect of hexamethonium, a nicotinic ganglion-blocking agent, was tested on vagal control of tracheal glycoconjugate secretion in cats treated with atropine, phentolamine, and propranolol (to block classic cholinergic and adrenergic nerve pathways) and guanethidine [to block possible release of nonadrenergic transmitters from sympathetic nerve endings (12, 15)]. Hexamethonium would block transmission via the mural ganglia but leave intact any direct antidromic nervous control of secretory cells via afferent pathways. METHODS
Preparation of tracheal segment. Anesthesia was induced in adult cats of either sex (weight 2.0-4.4 kg) by intraperitoneal injection of pentobarbital sodium (42 mg/kg; Sagatal, May & Baker) and maintained by intravenous injections via a femoral vein catheter. Rectal temperature was monitored and kept between 36 and 38OC by a heat-exchange pad. Arterial blood pressure was recorded on ultraviolet-sensitive paper (SE Laboratory 6008) via a femoral arterial catheter connected to a strain gauge transducer (SE Laboratory 4.88), calibrated with a mercury manometer. Mean blood pressure was estimucus secretion; airway; parasympathetic nerve; mural gan- mated as diastolic plus one-third pulse pressure. Blood glion; antidromic transmission; orthodromic transmission; pressure measurements were made before and after each nonadrenergic noncholinergic nerve; vagus nerve vagal stimulation and also 1 and 9 min into each period of vagal stimulation. The subsequent preparation of the tracheal segment SECRETION OF AIRWAY MUCUS, essential for effeCtive air- has been described previously (10). Briefly, a cannula way clearance by mucociliary transport and cough, is through which the animal breathed was tied into the cerunder several control mechanisms including parasympavical trachea as far caudally as possible. A segment of thetic and sympathetic nervous pathways (21). In the cat trachea (4-6 cm long) rostra1 to this was then cannulated the parasympathetic (vagal) pathway controlling mucus at both ends to isolate it in situ with its nerves and blood secretion acts through both choline@ and nonadrenersupply intact. This tracheal segment was then washed gic noncholinergic (NANC) nervous mechanisms (26). out and filled with oxygenated Krebs-Henseleit solution Similar findings have also been reported in the ferret at 37OC. trachea in vitro (3). The pathways within the tracheal Radiolabeling of mucus glycoconjugates. At the beginwall of the NANC nerves involved are unknown, but ning of each experiment, 2.0 mCi of sodium [35S]sulfate and 0.5 mCi of [3H]glucose (Amersham International) in there are at least two possibilities: 1) collateral branches of vagal afferent fibers may innervate secretory cells 3.0 ml Krebs-Henseleit solution were flushed into the within submucosal glands, allowing antidromic nerve tracheal segment and left for 1 h to enable uptake and impulses to stimulate secretion (19); or 2) there may be biosynthetic incorporation of the radiolabels into mucus postganglionic NANC nerves with cell bodies in the glycoconjugates (9, 10). Samples were then taken at 30mural ganglia, innervated by preganglionic parasympamin intervals for the next 2 h and then at 15-min interthetic fibers. This pathway would allow orthodromic ner- vals by flushing the segment with 10 ml of Krebs-Hensevous control of secretion. leit solution. The present experiments were undertaken to distinQuantification of radiolabeled glycoconjugates. Tracheal 0161-7567/92
$2.00 Copyright
0 1992 the American
Physiological
Society
625
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 13, 2019.
626
NANC
CONTROL
OF
AIRWAY
washings were immediately mixed with sufficient guanidine hydrochloride (grade 1, Sigma) to give a final concentration of 6 M, which dissolved mucus, and kept between 4 and 6OC. They were then dialyzed exhaustively (dialysis tubing with molecular weight cutoff at 1214,000, Medicell International) against distilled water containing 0.01% (wt/vol) sodium azide and 0.01% (wt/ vol) sodium sulfate to disperse the noncovalently bound radiolabel. Radioactivity of aliquots from the samples was then measured in triplicate by liquid scintillation counting (Beckman Ready Protein Scintillant; Beckman Scintillation Counter LS 6000 IC, calibrated with quench correction to assay 35Sand 3H separately). Total glycoconjugate measurement. The total glycoconjugates present in the tracheal washings after exhaustive dialysis were estimated in triplicate using the solution periodic acid-Schiff (PAS) assay method (18). Bovine submaxillary mucin (Sigma) was used as a calibration standard. Glycoconjugate concentrations are expressed in terms of this standard. This assay was performed on samples from all cats in which autonomic blockers were given but only on four of the experiments to check repeatability of vagus nerve stimulation. Vagus nerue stimulation. In the cat the major parasympathetic efferent pathway to the trachea runs through the cervical vagus nerves and not in the superior laryngeal nerves as it does in the dog and the ferret (23). The cervical sympathetic nerves on both sides were separated from the vagus nerves and cut at the midneck region. The vagus nerves were then cut just caudal to the superior laryngeal nerves. Their peripheral ends were stimulated via platinum electrodes, at 9.5 Hz with pulses of 10 V and 2-ms duration for 10 min of a 15min collection period (Devices gated pulse generator 2521, Devices isolated stimulator 2533). Administration of drugs. Drugs were administered in two ways: directly into the tracheal segment (is) and/or intravenously via the femoral vein. Drugs given intrasegmentally were dissolved in oxygenated Krebs-Henseleit solution. The following drugs were used: atropine sulfate (Sigma), DL-propranolol hydrochloride (Sigma), phentolamine mesylate (Rogitine, Ciba), guanethidine sulfate (Sigma), hexamethonium bromide (Sigma), pilocarpine hydrochloride (Sigma), phenylephrine hydrochloride (Boots), and isoproterenol hydrochloride (Saventrine, Pharmax). Experimentalprotocols. After the initial l-h pulse-labeling period, the preparation was left unstimulated for the next 2.25 h. In all but two experiments, vagal stimulation was applied for 10 min at hourly intervals on four occasions. In the first series of experiments (9 cats) to study the reproducibility of the responses, vagal stimulations were applied on four occasions in the absence of autonomic nervous system antagonists. In a second series of experiments (Fig. I), designed to examine the effects of progressive blockade of autonomic pathways by cumulative administration of autonomic antagonists, vagal stimulation was applied as follows: 1) in the absence of drugs (control); 2) 30 min after administration of atropine (2 pg/ml is and 1 mg/kg iv), phentolamine (10 pg/ml is), and propranolol(l0 &ml is and 2 mg/kg iv: APP) (26); 3) in
MUCUS
SECRETION
35Sglycoconjugate
output
120
-
I
.
80
$
60
-
-
-
.
40 20 0 0
1
2
3 H-glycoconjugate
4
3
output -
5
-
-
6
-
300 250 ”
,
50 o..............l! 0
1
PAS-reactive
2
glycoconjugate -
3
output -
.
. . 4
-
.
.
. . 5
. 6
-
40 T 30 l r(
z
20
-2
10 0
-
0
l . hours 2 Time*
FIG. 1. Example of 1 experiment illustrating protocol used to study effects of drugs on output of 35S- and 3H-labeled and periodic acidSchiff (PAS)-reactive glycoconjugates in response to vagal stimulation. After initial 2.25 h, electrical pulses (10 V, 2-ms duration, 9.5 Hz) were applied to vagus nerves for 10 min at hourly intervals (open and closed bars). To study effects of progressive autonomic blockade, drugs were administered 30 min before respective stimulation. At the end of this experiment (hatched bars), efficacy of a-adrenoceptor blockade was checked by intrasegmental administration of 40 PM phenylephrine.
the presence of APP and guanethidine (5 mg/kg iv; APPG) (15); and 4) in the presence of APPG and hexamethonium (2 mg/kg iv; APPGH) (35). In addition, after the initial radiolabeling and unstimulated secretion periods, two cats were treated first with APPG and then with APPGH, and electrical stimulations were applied to their vagus nerves during each of the two drug combinations; thus the control and APP stimulations (stimulations 1 and 2 above) were omitted. Effects of vagal stimulations in these have been included with the equivalent results from the other six cats. Statistical treatment of results. Vagally induced changes were calculated as the percent increase of the stimulated sample over the mean value of the preceding and succeeding sample (bracketing controls). Values are means t SE. Comparisons were made by Student’s t test, either paired or unpaired as appropriate. P < 0.05 was accepted as statistically significant.
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 13, 2019.
NANC CONTROL Wglycoconjugates
OF AIRWAY
.
2 .EI200 2a g 100 3 ep 0
?H-glycoconjugates 120
(n=9)
l
-
T
,
PAS-reactive
glycoconjugates
SECRETION
627
0.02, n = 6) (Fig. 3). These increases were significantly less (P < 0.005 for 35S, P < 0.05 for 3H, P < 0.02 for PAS) than those produced by vagal stimulation in the absence of drugs, thus confirming the coexistence of NANC and classic nervous pathways, both of which stimulate mucus glycoconjugate secretion into the feline trachea (26). Efficacy of muscarinic and adrenergic antagonists. To test the efficacy of the above regime of adrenoceptor and muscarinic antagonists, a muscarinic agonist (pilocarpine, 3 PM) and a- and ,&agonists (phenylephrine, 40 PM; isoproterenol, 40 PM) were administered, on different occasions, in the continued presence of APP. None of the agonists significantly stimulated the release of radiolabeled or PAS-reactive glycoconjugates (Table 1). Effects of guanethidine (APPG). Vagal stimulation in the presence of guanethidine, in addition to APP, still caused secretion as indicated by two of the measures of glycoconjugate output (A35S = 40 t 15.0%, P < 0.02; APAS = 89 t 28.5%, P < 0.02; n = 8 in each case) but the
(n=9)
5 300 8
MUCUS
(n=4)
I
s
250
Q
200
I
l *
150 3&) 100
.3 Ii8 -5 VSl
vs2
vs3
FIG.
RESULTS
Effects of repeated vagal stimulation in the absence of drugs. After the initial 2.25 h of collections without secre-
tory stimuli, vagal stimulations applied at hourly intervals in the absence of drugs gave reproducible responses as measured by the percent increase in outputs of 35Sand 3H-labeled macromolecules as well as chemically for total glycoconjugates by the PAS assay (Fig. 2). No significant difference in the response was noted between any measure of the effectiveness of stimuli applied at different times during experiments. Vagal stimulation before administration of autonomic antagonists. The first vagal stimulation was applied in
the absence of autonomic antagonists in six cats and produced clear increases in all three measures of mucus glycoconjugate secretion (A3?S = 221 t 43.3%, P < 0.005; A3H = 58 t 13.8%, P < 0.01; APAS = 299 t 82.7%, P < 0.02) (Fig. 3). Effects of muscarinic and adrenoceptor blockade (APP).
Electrical vagal stimulation after treatment still produced significant increases in the mucus glycoconjugates (A3?S = 67 t l&6%, A3H = 26 t 5.3%, P < 0.005; APAS = 88 t
with APP output of P < 0.01; 25.6%, P
