Camp. Biochem. Physiol., 1915, Vol. 5OA,pp. 229 to 232. Pergamon Press. Printed in Great Britain
VAGAL
STIMULATION OF GASTRIC GALLUS D~~ES~r~US REND G. GIBSON AND HARRY W.
Department
SECRETION
IN
COLVIN, JR.*
of Animal Physiology, University of California at Davis, Davis, California 95616. U.S.A. (Received 4 December
1973)
Abstract-l.
The proventriculi of White Leghorn cocks were acutely cannulated under sodium pentobarbital anesthesia. 2. Electrical stimulation of the cervical vagi was found to be a potent stimulus for both acid and pepsin secretion. 3. The threshold for vagal stimuIation was found to be quite high (approximately 20 V), implying a very small fiber diameter for the nerves innervating the proventriculus. 4. Peak outputs of acid and pepsin in response to vagal stimulation were O-9620.05 m-equiv. H+/30 min and 103,500k 19,600 P.U./30 min, respectively. 5. Ganglionic blockade with hexamethonium during continuous vagal stimulation inhibited both acid and pepsin secretion equally.
INTRODUCTION IN RECENTyears, gastric secretion in the domestic fowl has been the subject of extensive experimentation (Angelucci & Linari, 1970; Burhol & Hirschowitz, 1969, 1970, 1971a-h). Despite this extensive work, the actul role of the vagus on the gastric secretions of the fowl has remained unresolved. Sham-fading (Collip, 1922) and parasympathetic stimulation (Friedman, 1939; Ruoff & Sewing, 1970b; Burhol & Hirschowitz, 1971b) have been found to increase gastric secretion in the domestic fowl. On the other hand, no stimulation of gastric secretion has been found in response to insulin hypoglycemia (Koskowski & Mahfouz, 1956; Long, I967), or 2-deoxy-D-glucose infusion (Burhol & Hirschowitz, 1971d). In the present study the effects of direct vagal stimulation on the gastric secretions of the domestic fowl were studied, The voltage response characteristics of vagal stimulation were determined, and the effects of ganglionic blockade on vagaliy induced secretion were investigated. MATERIALS AND METHODS Single comb White Leghorn cocks (Gullus domesticus), which had been fasted for 24 hr, were anesthetized with percutaneous pentobarbital and restrained on their backs. The brachial vein and left internal carotid arteries were cannulated for the administration of further anesthesia and the monitoring of heart rate and blood pressure, respectively, The body temperature of the birds was maintained between 40.5 and 42,O”C. The thoracic and abdominal cavities were opened via a mid* To whom correspondence
should be addressed.
vertical incision and the blunt dissection of the supracoracoideus muscle away from the keel of the sternum. The fascia covering the abdominal cavity was cut proceeding from the posterior left margin of the sternum to the junction of the keel and the posterior xiphistemal process. Unidirectional ventilation was established after rupturing the air sacs (Fedde & Burger, 1962). The proventriculus was exposed by cutting through the posthepatic septum and carefully pushing the liver to the side. A 4-cm long polyethylene cannula of 6 mm dia with a l-cm flange was inserted through a 0.25-in. incision anteriorly into the posterior margin of the proventriculus. The anterior portion of the proventriculus was cannulated by the insertion of a 25-cm long polyethylene cannula of 3 mm dia. equipped with a syringe adapter into the esophagus posterior to the crop and threading it into the anterior portion of the proventriculus. The anterior cannula was anchored in place by a ligature around the esophagus just anterior to the proventriculus. Gastric secretions were collected in the following manner. The posterior cannula was connected to the graduated centrifuge tubes used as collection vessels by a 2O-cm long polyethylene tube of 3 mm dia with a fitted stopper equipped with an air release vent. The proventriculus was cleared of all particulate matter by the perfusion of 20 cm3 @9X saline at 37°C through the anterior cannula by means of a syringe. The proientriculus was cleared of the saline solution by gently perfusing 30 cm3 of air through the anterior cannula with another syringe. Gastric collections were made by gently perfusing 10 cm3 of the saline solution through the proventriculus. The proventriculus was again cleared by 30 cm3 of air. The volume of the collected perfusate was recorded to the nearest @l cm3 and two 3-cm3 atiquots were immediately removed for titration with @Ol N NaOH to pH 7. The pH of the perfusing saline solution was 6.9-7.1. Tests of the reliability of the perfusion system to measure changes in the secretory volume were conducted
230
RENB
G.
GIBSON AND HARRYW. COLVIN, JR.
in both live and dead birds. In twenty-eight of thirty trials, more than 9.8 cm3 of the 10 cm3 saline solution perfused through the anterior cannula was collected in the graduated centrifuge tube by a single perfusion of 30 cm3 of air. Experiments were not started until the basal rate of secretion had declined to below 0.1 m-equiv. H+/l5 min. Pepsin determinations were made by a modified hemoglobin method (Bucher et al., 1945). The vagi were isolated in the following manner. The left cervical vagus of each fowl was exposed in the neck of the bird and a 4-cm portion was carefully dissected free from the surrounding connective tissue. The nerve was then cut and the distal portion of the nerve was placed across bipolar silver stimulating electrodes connected to a Grass S-5 rectangular wave stimulator. In some animals, upon completion of the voltageresponse determinations, hexamathonium bromide (Sigma Chemical Co., St. Louis, MO.) was injected as a singular dose (30 mg/kg).
parameters used in the determination of the threshold voltage for gastric secretion were 10 pulses/ set, 1 msec duration, with the voltage being varied. The birds were stimulated for only 30 set/5 min, so that the vagal depression of heart rate and blood pressure would not limit the secretion of gastric fluids. The data described in Fig. 2 depict the voltage response curves for acid and pepsin secretion from four birds. The level of secretion labelled basal reflects the level of gastric secretion prior to vagal
RESULTS Figure 1 illustrates the data from six control birds. The gastric secretions from these birds were collected over a 35-hr span. The basal level of acid secretion was found to decline from an initial output of 0.35~I10.21 to 0.05 to 0.02 m-equiv. H+/30 min, 1 hour later. The pepsin output was found to decline
a
oe-
l-
-
2
2
;o P 4
tic PU.
-6
-76
06-
I-\;-/I ‘\
E $
. 0
04-
oz-
\-
fI
\
I
2
I-
3 30
2_
4
.z 0
c “0 z
-5
a -4
;,
-3
I,
z 2
.c B I?
-x-
5
6
7
minperiods
Fig. 1. The unstimulated output of acid and pepsin from six birds. Each point represents the meankstandard error.
more slowly than the secretion of acid, reaching the lowest level of secretion approximately 1 hr after the acid output. Pepsin secretion declined from an initial activity of 6273 + 1860 to 4542 f 1652 P.U./ 30 min, 1 hour later. The mean 30-min output of acid was 0.11 + 0.04 m-equiv., while the average pepsin output over the same period of time was 4278 + 560 P.U. Since no work has been done previously utilizing direct vagal stimulation of gastric secretion in the domestic fowl, it was necessary to determine the threshold parameters for the stimulation of gastric secretion from the proventriculus. The stimulation
Fig. 2. Top: ratio of pepsin output to acid output for the data described in bottom graph. Bottom: voltage response curves for acid and pepsin output. Animals were vagally stimulated at 10 pulses/set, 1 msec duration, 30 set/5 min, with the voltage increased every 30 min. Each point is the mean of four experiments&SE.
stimulation. Preliminary experiments indicated that approximately 20 V was threshold for the stimulation of gastric acid secretion in the fowl. Therefore, beginning with an initial stimulation level of 5 V the birds were stimulated for 4 hr with the stimulation intensity being increased by 5 V every 30-min interval until the stimulation intensity reached 30 V, at which time the stimulation intensity was increased by increments of 1OV until a final stimulation voltage of 50 V was reached. The secretion of both acid and pepsin was found to rise as the intensity of stimulation surpassed the threshold voltage of 20 V. The ratio of pepsin output to acid output was found to remain quite stable, indicating a close correlation between acid and pepsin secretion. Attempts to determine the frequency response characteristics of vagal stimulation were unsuccessful. We were unable to stimulate gastric secretion by continuous stimulation at 1 and 3 pulses/set at 25 V. Continuous stimulation at frequencies greater than this decreased the heart
Gastric secretion in Galh domesticus rate and blood pressure to such low levels as to be inhibitory to gastric secretion. No significant differences were found between stimulation at 10 and 20 pulses/set. No attempt was made to stimulate at frequencies greater than this. Visual observations made during vagal stimulation showed that crop movements were particularly plentiful during electrical stimulation at 40 and 50 V. Figure 3 graphically depicts the effects of vagal stimulation maintained at 20 V, 10 pulses/set, 1 msec duration, 30 se+ min interval, maintained over a 3-hr span. The outputs of acid and pepsin
231
intravenous dose. Figure 4 illustrates the results of hexamethonium administration in four animals. Both acid and pepsin secretion declined rapidly to basal levels after hexamethonium administration.
Min
(2345678 30
Min periods
Fig. 3. Top: ratio of pepsin output to acid output for the data presented in the bottom graph. Bottom: acid and pepsin outputs in response to continuous vagal stimuiation at 20 V, 10 pulses/set, 1 msec duration, for 30 SW/~ min. Each point is the mean of four experiments+S.E.
were found to parallel each other closely. The mean 30-min outputs of acid and pepsin over the period of vagal stimulation were 0.96 + 0.05 m-equiv. H+ and 72,115 f 4750 P.U., respectively. The ratio of pepsin activity to acid output remained stable during the course of continuous vagal stimulation except for the large deflection during the fist 30-min period. This peak is due to the proportionately greater increase in pepsin output during the first 30 min (12-5 vs 2.2 times for acid secretion). This large, initial secretion of pepsin is probably due to the output of newly synthesized pepsin in conjunction with the pepsinogen which had accumulated within the oxyntico-peptic cells during the prestimulation period. Upon completion of each voltage response experiment the stimulation voltage was set at the level which produd the greatest output of acid in that animal, while all other stimulation parameters remained the same. While continuing to stimulate the animal for 30 set/5 min, hexamethonium bromide, 30 mg/kg, was administered as a single
Fig. 4. Top: ratio of pepsin output to acid output for the data presented in bottom graph. Bottom: the effect of a single intravenous injection of hexamethonium bromide, 30mg/kg, on acid and pepsin secretion stimulated vagalfy. St~uiation parameters were 10 pulses/see, 1 msec duration, 30 se@ min interval, with the stimulation voltage at the level producing maximal secretion in each animal during the voltage-response experiments. Each point is the mean of four experiments + SE.
ratio of pepsin secretion to acid secretion remained stable indicating that both acid and pepsin outputs were equally inhibited by hexamethonium. The administration of hexamethonium generally resulted in a small (10-30 mm Hg) decline in bIood pressure which persisted for approximately 1 hr.
The
DISCUSSION Direct vagal stimulation proved to be a very effective stimulus for the secretion of both acid and pepsin from the chicken proventriculus. Continuous vagal stimulation elicited outputs of acid which compared favourably with maximal cholinergic stimulation with urecholine infusion @rho1 & Hirschowitz, X971c), while the outputs of pepsin elicited by vagal stimulation were found to exceed the levels previously described for both maximal cholinergic and histamine stimulation (Eiurhol & Hirschowitz, 1971c, d). The high thresholds for vagally stimulated gastric secretion may serve as an indication that the nerve fibers responsible for eliciting gastric secretion in the domestic fowl are probably of very small diameter. Dahl et al. (1964) and Brown (1970) provide some anatomical verification for the high stimulation thresholds described for vagally initiated
REZ&G. GIBSON ANDHARRYW. COLVIN, JR.
232
gastric secretions. Dahl et al. found using vagi from adult White Leghorn fowl that midcervical vagus of the fowl was composed predominantly of nonmyelinated fibers. Studies of the compound action potentials of the vagus revealed two populations of nerve fibers, the first elevation of action potentials being generated by small myelinated B fibers and the second elevation by nonmyelinated C fibers. Brown reaffirmed that the compound action potential of the midcervical vagus of the domestic fowl was composed of two principal groups of fibers, the faster group of fibers having conduction velocities between 1 and 17 m/set, and the slower group between 0.4 and 0.8 m/set; thus placing the slower group of fibers in the myelinated C fiber range as determined from ma~alian fibers (Gasser, 1950). Due to the high threshold for stimulation of gastric secretion in the fowl described in this study it appears that it is the C fiber component of the vagus which innervates the proventriculus and is responsible for eliciting the output of both acid and pepsin. The failure of Ruoff & Sewing (1970a) and Burhoi & Hirschowitz (1971g) to detect gastrin-Iike activity in the gastrointestinal tract of the domestic fowl, together with the demonstrated effectiveness of cholinergic stimulation (Burhol et al., 1971~; Friedman, 1939), as a stimulus for both acid and pepsin secretion and coupled with this report of vagally induced secretion, it appears quite possible that gastric secretion in the domestic fowl may be controlled entirely through vagal activity. SUMMARY
Direct vagal stimulation proved to be a very effective stimulus for the secretion of both acid and pepsin from the chicken proventriculus. Due to the high threshold for stimulation of secretion, it appears that it is the C fiber component of the vagus which innervates the proventriculus and is responsible for eliciting the output of both acid and pepsin, Other workers have not been able to detect gastrin-like activity in the gastrointestinal tract of the domestic fowl and, as a result of our findings, it seems quite probable that gastric secretion in the domestic fowl is entirely controlled through vagal activity. Acknowiedge~~eat-This work was supported in part by the Training Grant in Physiology, No. GMOl934, University of California, Davis, California. REFERENCES ANGELUCCIL. & LINARI G. (1970) The action of caerulein on gastric acid secretion of the chicken. &~oP. J, Pharmac. 11, 204-216. BROWNC. M. (1970) B.Sc. thesis, Department of Veterinary Anatomy, University of Liverpool; as cited by KING A+ S. & MOLONYV. (1971) The anatomy of respiration. In Physiology and ~iuchem~stry of the Domestic Fowl, (Edited by BELL D. J. & FREEMANB, N.) Vol. 1, PP. 93-169. Academic Press, New York.
BUCHERG. R., GROSSMANM. I. & IVY A. C. (1945) A pepsin method: the role of dilution in the determination of peptic activity. Gastroenterology 5, 501-511. BURHOLP. & HIRSCHOWITZB. I. (1969) Basal and dose
responsive gastric secretion in fist&a chickens using single subcutaneous doses of histamine and pentagastrin (Abstr.). Gastroe~teroiogy 56, 1141. BURHOL P. & HIRSCHO~~IT~B. I. (1970) Single subcutaneous doses of histamine and pentagastrin in gastric fistula chickens. Am. J. Pfzysiol. 218, 1671i675. BURHOLP. & HIRSCHOW~TZ B. I. (1971a) Introduction. Scand. J. Gastroent. 6 Su~ol. 11.7-13. BURHOLP. & HIRSCHOWIT~i. I. (1971b) Gastric stimulation by subcutaneous infusion of histamine in fistula chickens. Stand. J. Gastroe~t. 6 Suppl. 11, 15-24. BURHOLP. & HIRSCHOWITZ B. I. (1971c) Gastric stimulation by subcutaneous infusion of ur~holi~e in fistula chickens. A comparison to histamine and pentagastrin. Stand. J. Gastroent. 6 Suppl. 11, 25-33. BURHOL P. & HIRSCHOWITZB. I. (1971d) Intravenous injectionof 2-deoxy-D-glucoseingastricfistulachickens. &and. J. Gastroent. 6 Suppl. 11,35-40. BURHOLP. & HIRSCXOWITZB. I. (1971ej Gastric stimulation by subcutaneous infusion of ‘cholecystokininpancreozymin in fistula chickens. Stand. J. Gastroent.
6 Suppl. 11,41-48, BURHOL P. & HIRSCHOWITZ B. I. (1971f) Gastric inhibition by subcutaneous infusion of secretin in fist&a chickens. &and. J. Gastroent. 6 Suppt. 11, 49-55. BURHO~ P. & HIRSCHOWITZB. 1. (1971g) Inhibition of gastric Hf secretion by a~tylcholine in the isolated gizzard of fistula chickens. &and. J. Gustroent. 6
Suppl. 11,57-f% BWRHOL P. & HIRSCHOWITZB. I. (1971h) General discussion. &and. J. Gastroent. 6 Suppl. 11, 61-64. COLLIP J. B. (1922) The activation of-the glandular stomach of the fowl. Am. J. Physiol. 59,435-438. DAHL N. A., SAMSONF. E., JR. & BALFOURW. M. (1964) Adenosine triphosphate and electrical activity in chicken vagus. Am. J. Physiol. 206,818~822. FEDDEM. R. & BURGERR. E. (1962) A gas heating and humidifying accessory for the~nid~re~t~n~ respi;ator. Poult. Sci. 41, 679. FRIEDMANM. H. F. (1939) Gastric secretion in birds. J. cefl. camp. Physiol. 13,218-234. GASSER H. S. (1950) Unmedullated fibers originating in dorsal root ganglia. J. gen. Physiol. 33,651-690. KOSKOWSKIW. & MAHFOUZ M. (1956) Insulin and gastric secretion. Arch. Int. Pharmacodyn. 108,225-231. LONG J. F. (1967) Gastric secretion in unanesthetized chickens. Am. J. Physiol. 212, 1303-1307. RUOFF H. F. & SEXING K. F. (1970a) Histamin, Histidin-Decarboxylase und Gastrin im oberen Verdauungstrakt des Hubns. ~au~y~-Schmiedebergs Arch. Pharmak. 265,301-309. RUOFFH. 5. & SEWINGK. F. (1970b) Die Wirkung von Histamin, Carbachol, Pentagastrin und Huhnergastrinextraken auf die Magensekretion von nicht narkotisierten Huhnem mit einer Magenfistel. NaunynSchmiedebergs Arch. Pharmak. 267, 170-176. Key Word Index-Gastric secretion; chickens; vagus; electrical stimulation; hexamethonium.
VAGAL
STIMULATION OF GASTRIC GALLUS D~~ES~r~US REND G. GIBSON AND HARRY W.
Department
SECRETION
IN
COLVIN, JR.*
of Animal Physiology, University of California at Davis, Davis, California 95616. U.S.A. (Received 4 December
1973)
Abstract-l.
The proventriculi of White Leghorn cocks were acutely cannulated under sodium pentobarbital anesthesia. 2. Electrical stimulation of the cervical vagi was found to be a potent stimulus for both acid and pepsin secretion. 3. The threshold for vagal stimuIation was found to be quite high (approximately 20 V), implying a very small fiber diameter for the nerves innervating the proventriculus. 4. Peak outputs of acid and pepsin in response to vagal stimulation were O-9620.05 m-equiv. H+/30 min and 103,500k 19,600 P.U./30 min, respectively. 5. Ganglionic blockade with hexamethonium during continuous vagal stimulation inhibited both acid and pepsin secretion equally.
INTRODUCTION IN RECENTyears, gastric secretion in the domestic fowl has been the subject of extensive experimentation (Angelucci & Linari, 1970; Burhol & Hirschowitz, 1969, 1970, 1971a-h). Despite this extensive work, the actul role of the vagus on the gastric secretions of the fowl has remained unresolved. Sham-fading (Collip, 1922) and parasympathetic stimulation (Friedman, 1939; Ruoff & Sewing, 1970b; Burhol & Hirschowitz, 1971b) have been found to increase gastric secretion in the domestic fowl. On the other hand, no stimulation of gastric secretion has been found in response to insulin hypoglycemia (Koskowski & Mahfouz, 1956; Long, I967), or 2-deoxy-D-glucose infusion (Burhol & Hirschowitz, 1971d). In the present study the effects of direct vagal stimulation on the gastric secretions of the domestic fowl were studied, The voltage response characteristics of vagal stimulation were determined, and the effects of ganglionic blockade on vagaliy induced secretion were investigated. MATERIALS AND METHODS Single comb White Leghorn cocks (Gullus domesticus), which had been fasted for 24 hr, were anesthetized with percutaneous pentobarbital and restrained on their backs. The brachial vein and left internal carotid arteries were cannulated for the administration of further anesthesia and the monitoring of heart rate and blood pressure, respectively, The body temperature of the birds was maintained between 40.5 and 42,O”C. The thoracic and abdominal cavities were opened via a mid* To whom correspondence
should be addressed.
vertical incision and the blunt dissection of the supracoracoideus muscle away from the keel of the sternum. The fascia covering the abdominal cavity was cut proceeding from the posterior left margin of the sternum to the junction of the keel and the posterior xiphistemal process. Unidirectional ventilation was established after rupturing the air sacs (Fedde & Burger, 1962). The proventriculus was exposed by cutting through the posthepatic septum and carefully pushing the liver to the side. A 4-cm long polyethylene cannula of 6 mm dia with a l-cm flange was inserted through a 0.25-in. incision anteriorly into the posterior margin of the proventriculus. The anterior portion of the proventriculus was cannulated by the insertion of a 25-cm long polyethylene cannula of 3 mm dia. equipped with a syringe adapter into the esophagus posterior to the crop and threading it into the anterior portion of the proventriculus. The anterior cannula was anchored in place by a ligature around the esophagus just anterior to the proventriculus. Gastric secretions were collected in the following manner. The posterior cannula was connected to the graduated centrifuge tubes used as collection vessels by a 2O-cm long polyethylene tube of 3 mm dia with a fitted stopper equipped with an air release vent. The proventriculus was cleared of all particulate matter by the perfusion of 20 cm3 @9X saline at 37°C through the anterior cannula by means of a syringe. The proientriculus was cleared of the saline solution by gently perfusing 30 cm3 of air through the anterior cannula with another syringe. Gastric collections were made by gently perfusing 10 cm3 of the saline solution through the proventriculus. The proventriculus was again cleared by 30 cm3 of air. The volume of the collected perfusate was recorded to the nearest @l cm3 and two 3-cm3 atiquots were immediately removed for titration with @Ol N NaOH to pH 7. The pH of the perfusing saline solution was 6.9-7.1. Tests of the reliability of the perfusion system to measure changes in the secretory volume were conducted
230
RENB
G.
GIBSON AND HARRYW. COLVIN, JR.
in both live and dead birds. In twenty-eight of thirty trials, more than 9.8 cm3 of the 10 cm3 saline solution perfused through the anterior cannula was collected in the graduated centrifuge tube by a single perfusion of 30 cm3 of air. Experiments were not started until the basal rate of secretion had declined to below 0.1 m-equiv. H+/l5 min. Pepsin determinations were made by a modified hemoglobin method (Bucher et al., 1945). The vagi were isolated in the following manner. The left cervical vagus of each fowl was exposed in the neck of the bird and a 4-cm portion was carefully dissected free from the surrounding connective tissue. The nerve was then cut and the distal portion of the nerve was placed across bipolar silver stimulating electrodes connected to a Grass S-5 rectangular wave stimulator. In some animals, upon completion of the voltageresponse determinations, hexamathonium bromide (Sigma Chemical Co., St. Louis, MO.) was injected as a singular dose (30 mg/kg).
parameters used in the determination of the threshold voltage for gastric secretion were 10 pulses/ set, 1 msec duration, with the voltage being varied. The birds were stimulated for only 30 set/5 min, so that the vagal depression of heart rate and blood pressure would not limit the secretion of gastric fluids. The data described in Fig. 2 depict the voltage response curves for acid and pepsin secretion from four birds. The level of secretion labelled basal reflects the level of gastric secretion prior to vagal
RESULTS Figure 1 illustrates the data from six control birds. The gastric secretions from these birds were collected over a 35-hr span. The basal level of acid secretion was found to decline from an initial output of 0.35~I10.21 to 0.05 to 0.02 m-equiv. H+/30 min, 1 hour later. The pepsin output was found to decline
a
oe-
l-
-
2
2
;o P 4
tic PU.
-6
-76
06-
I-\;-/I ‘\
E $
. 0
04-
oz-
\-
fI
\
I
2
I-
3 30
2_
4
.z 0
c “0 z
-5
a -4
;,
-3
I,
z 2
.c B I?
-x-
5
6
7
minperiods
Fig. 1. The unstimulated output of acid and pepsin from six birds. Each point represents the meankstandard error.
more slowly than the secretion of acid, reaching the lowest level of secretion approximately 1 hr after the acid output. Pepsin secretion declined from an initial activity of 6273 + 1860 to 4542 f 1652 P.U./ 30 min, 1 hour later. The mean 30-min output of acid was 0.11 + 0.04 m-equiv., while the average pepsin output over the same period of time was 4278 + 560 P.U. Since no work has been done previously utilizing direct vagal stimulation of gastric secretion in the domestic fowl, it was necessary to determine the threshold parameters for the stimulation of gastric secretion from the proventriculus. The stimulation
Fig. 2. Top: ratio of pepsin output to acid output for the data described in bottom graph. Bottom: voltage response curves for acid and pepsin output. Animals were vagally stimulated at 10 pulses/set, 1 msec duration, 30 set/5 min, with the voltage increased every 30 min. Each point is the mean of four experiments&SE.
stimulation. Preliminary experiments indicated that approximately 20 V was threshold for the stimulation of gastric acid secretion in the fowl. Therefore, beginning with an initial stimulation level of 5 V the birds were stimulated for 4 hr with the stimulation intensity being increased by 5 V every 30-min interval until the stimulation intensity reached 30 V, at which time the stimulation intensity was increased by increments of 1OV until a final stimulation voltage of 50 V was reached. The secretion of both acid and pepsin was found to rise as the intensity of stimulation surpassed the threshold voltage of 20 V. The ratio of pepsin output to acid output was found to remain quite stable, indicating a close correlation between acid and pepsin secretion. Attempts to determine the frequency response characteristics of vagal stimulation were unsuccessful. We were unable to stimulate gastric secretion by continuous stimulation at 1 and 3 pulses/set at 25 V. Continuous stimulation at frequencies greater than this decreased the heart
Gastric secretion in Galh domesticus rate and blood pressure to such low levels as to be inhibitory to gastric secretion. No significant differences were found between stimulation at 10 and 20 pulses/set. No attempt was made to stimulate at frequencies greater than this. Visual observations made during vagal stimulation showed that crop movements were particularly plentiful during electrical stimulation at 40 and 50 V. Figure 3 graphically depicts the effects of vagal stimulation maintained at 20 V, 10 pulses/set, 1 msec duration, 30 se+ min interval, maintained over a 3-hr span. The outputs of acid and pepsin
231
intravenous dose. Figure 4 illustrates the results of hexamethonium administration in four animals. Both acid and pepsin secretion declined rapidly to basal levels after hexamethonium administration.
Min
(2345678 30
Min periods
Fig. 3. Top: ratio of pepsin output to acid output for the data presented in the bottom graph. Bottom: acid and pepsin outputs in response to continuous vagal stimuiation at 20 V, 10 pulses/set, 1 msec duration, for 30 SW/~ min. Each point is the mean of four experiments+S.E.
were found to parallel each other closely. The mean 30-min outputs of acid and pepsin over the period of vagal stimulation were 0.96 + 0.05 m-equiv. H+ and 72,115 f 4750 P.U., respectively. The ratio of pepsin activity to acid output remained stable during the course of continuous vagal stimulation except for the large deflection during the fist 30-min period. This peak is due to the proportionately greater increase in pepsin output during the first 30 min (12-5 vs 2.2 times for acid secretion). This large, initial secretion of pepsin is probably due to the output of newly synthesized pepsin in conjunction with the pepsinogen which had accumulated within the oxyntico-peptic cells during the prestimulation period. Upon completion of each voltage response experiment the stimulation voltage was set at the level which produd the greatest output of acid in that animal, while all other stimulation parameters remained the same. While continuing to stimulate the animal for 30 set/5 min, hexamethonium bromide, 30 mg/kg, was administered as a single
Fig. 4. Top: ratio of pepsin output to acid output for the data presented in bottom graph. Bottom: the effect of a single intravenous injection of hexamethonium bromide, 30mg/kg, on acid and pepsin secretion stimulated vagalfy. St~uiation parameters were 10 pulses/see, 1 msec duration, 30 se@ min interval, with the stimulation voltage at the level producing maximal secretion in each animal during the voltage-response experiments. Each point is the mean of four experiments + SE.
ratio of pepsin secretion to acid secretion remained stable indicating that both acid and pepsin outputs were equally inhibited by hexamethonium. The administration of hexamethonium generally resulted in a small (10-30 mm Hg) decline in bIood pressure which persisted for approximately 1 hr.
The
DISCUSSION Direct vagal stimulation proved to be a very effective stimulus for the secretion of both acid and pepsin from the chicken proventriculus. Continuous vagal stimulation elicited outputs of acid which compared favourably with maximal cholinergic stimulation with urecholine infusion @rho1 & Hirschowitz, X971c), while the outputs of pepsin elicited by vagal stimulation were found to exceed the levels previously described for both maximal cholinergic and histamine stimulation (Eiurhol & Hirschowitz, 1971c, d). The high thresholds for vagally stimulated gastric secretion may serve as an indication that the nerve fibers responsible for eliciting gastric secretion in the domestic fowl are probably of very small diameter. Dahl et al. (1964) and Brown (1970) provide some anatomical verification for the high stimulation thresholds described for vagally initiated
REZ&G. GIBSON ANDHARRYW. COLVIN, JR.
232
gastric secretions. Dahl et al. found using vagi from adult White Leghorn fowl that midcervical vagus of the fowl was composed predominantly of nonmyelinated fibers. Studies of the compound action potentials of the vagus revealed two populations of nerve fibers, the first elevation of action potentials being generated by small myelinated B fibers and the second elevation by nonmyelinated C fibers. Brown reaffirmed that the compound action potential of the midcervical vagus of the domestic fowl was composed of two principal groups of fibers, the faster group of fibers having conduction velocities between 1 and 17 m/set, and the slower group between 0.4 and 0.8 m/set; thus placing the slower group of fibers in the myelinated C fiber range as determined from ma~alian fibers (Gasser, 1950). Due to the high threshold for stimulation of gastric secretion in the fowl described in this study it appears that it is the C fiber component of the vagus which innervates the proventriculus and is responsible for eliciting the output of both acid and pepsin. The failure of Ruoff & Sewing (1970a) and Burhoi & Hirschowitz (1971g) to detect gastrin-Iike activity in the gastrointestinal tract of the domestic fowl, together with the demonstrated effectiveness of cholinergic stimulation (Burhol et al., 1971~; Friedman, 1939), as a stimulus for both acid and pepsin secretion and coupled with this report of vagally induced secretion, it appears quite possible that gastric secretion in the domestic fowl may be controlled entirely through vagal activity. SUMMARY
Direct vagal stimulation proved to be a very effective stimulus for the secretion of both acid and pepsin from the chicken proventriculus. Due to the high threshold for stimulation of secretion, it appears that it is the C fiber component of the vagus which innervates the proventriculus and is responsible for eliciting the output of both acid and pepsin, Other workers have not been able to detect gastrin-like activity in the gastrointestinal tract of the domestic fowl and, as a result of our findings, it seems quite probable that gastric secretion in the domestic fowl is entirely controlled through vagal activity. Acknowiedge~~eat-This work was supported in part by the Training Grant in Physiology, No. GMOl934, University of California, Davis, California. REFERENCES ANGELUCCIL. & LINARI G. (1970) The action of caerulein on gastric acid secretion of the chicken. &~oP. J, Pharmac. 11, 204-216. BROWNC. M. (1970) B.Sc. thesis, Department of Veterinary Anatomy, University of Liverpool; as cited by KING A+ S. & MOLONYV. (1971) The anatomy of respiration. In Physiology and ~iuchem~stry of the Domestic Fowl, (Edited by BELL D. J. & FREEMANB, N.) Vol. 1, PP. 93-169. Academic Press, New York.
BUCHERG. R., GROSSMANM. I. & IVY A. C. (1945) A pepsin method: the role of dilution in the determination of peptic activity. Gastroenterology 5, 501-511. BURHOLP. & HIRSCHOWITZB. I. (1969) Basal and dose
responsive gastric secretion in fist&a chickens using single subcutaneous doses of histamine and pentagastrin (Abstr.). Gastroe~teroiogy 56, 1141. BURHOL P. & HIRSCHO~~IT~B. I. (1970) Single subcutaneous doses of histamine and pentagastrin in gastric fistula chickens. Am. J. Pfzysiol. 218, 1671i675. BURHOLP. & HIRSCHOW~TZ B. I. (1971a) Introduction. Scand. J. Gastroent. 6 Su~ol. 11.7-13. BURHOLP. & HIRSCHOWIT~i. I. (1971b) Gastric stimulation by subcutaneous infusion of histamine in fistula chickens. Stand. J. Gastroe~t. 6 Suppl. 11, 15-24. BURHOLP. & HIRSCHOWITZ B. I. (1971c) Gastric stimulation by subcutaneous infusion of ur~holi~e in fistula chickens. A comparison to histamine and pentagastrin. Stand. J. Gastroent. 6 Suppl. 11, 25-33. BURHOL P. & HIRSCHOWITZB. I. (1971d) Intravenous injectionof 2-deoxy-D-glucoseingastricfistulachickens. &and. J. Gastroent. 6 Suppl. 11,35-40. BURHOLP. & HIRSCXOWITZB. I. (1971ej Gastric stimulation by subcutaneous infusion of ‘cholecystokininpancreozymin in fistula chickens. Stand. J. Gastroent.
6 Suppl. 11,41-48, BURHOL P. & HIRSCHOWITZ B. I. (1971f) Gastric inhibition by subcutaneous infusion of secretin in fist&a chickens. &and. J. Gastroent. 6 Suppt. 11, 49-55. BURHO~ P. & HIRSCHOWITZB. 1. (1971g) Inhibition of gastric Hf secretion by a~tylcholine in the isolated gizzard of fistula chickens. &and. J. Gustroent. 6
Suppl. 11,57-f% BWRHOL P. & HIRSCHOWITZB. I. (1971h) General discussion. &and. J. Gastroent. 6 Suppl. 11, 61-64. COLLIP J. B. (1922) The activation of-the glandular stomach of the fowl. Am. J. Physiol. 59,435-438. DAHL N. A., SAMSONF. E., JR. & BALFOURW. M. (1964) Adenosine triphosphate and electrical activity in chicken vagus. Am. J. Physiol. 206,818~822. FEDDEM. R. & BURGERR. E. (1962) A gas heating and humidifying accessory for the~nid~re~t~n~ respi;ator. Poult. Sci. 41, 679. FRIEDMANM. H. F. (1939) Gastric secretion in birds. J. cefl. camp. Physiol. 13,218-234. GASSER H. S. (1950) Unmedullated fibers originating in dorsal root ganglia. J. gen. Physiol. 33,651-690. KOSKOWSKIW. & MAHFOUZ M. (1956) Insulin and gastric secretion. Arch. Int. Pharmacodyn. 108,225-231. LONG J. F. (1967) Gastric secretion in unanesthetized chickens. Am. J. Physiol. 212, 1303-1307. RUOFF H. F. & SEXING K. F. (1970a) Histamin, Histidin-Decarboxylase und Gastrin im oberen Verdauungstrakt des Hubns. ~au~y~-Schmiedebergs Arch. Pharmak. 265,301-309. RUOFFH. 5. & SEWINGK. F. (1970b) Die Wirkung von Histamin, Carbachol, Pentagastrin und Huhnergastrinextraken auf die Magensekretion von nicht narkotisierten Huhnem mit einer Magenfistel. NaunynSchmiedebergs Arch. Pharmak. 267, 170-176. Key Word Index-Gastric secretion; chickens; vagus; electrical stimulation; hexamethonium.
