J. Phyriol. (1977), 273, pp. 707-716 With 4 text-figure8 Printed in Great Britain
707
INHIBITION OF HYDROCHLORIC ACID AND PEPSIN SECRETION FROM GASTRIC POUCHES BY ANTRAL POUCH ACIDIFICATION IN SHEEP
BY L. M. McLEAY* AND D. A. TITCHEN From the Department of Veterinary Preclinical Science8, University of Melbourne, Parkville, Victoria 3052
(Received 26 April 1977) SUMMARY
1. Secretion of acid and pepsin from separated pouches of the body of the abomasum was studied in sheep during perfusion of antral pouches with acid solutions. 2. Resting secretion of acid and pepsin was reduced by acidification of antral pouches to pH 2-7 or less. 3. Increases in the secretion of HCl and pepsin normally obtained on feeding were reduced or abolished by perfusion of antral pouches with solutions at pH 1-3-2*0. 4. Perfusion of antral pouches with acetylcholine at pH 2-5 failed to stimulate acid secretion as it did at a higher pH. 5. Pentagastrin stimulated acid and pepsin secretion during the inhibition of secretion produced by antral pouch acidification. 6. Increases in reticular motility occurred on antral pouch acidification with solutions of pH 1 1-1 3. 7. The contribution of antral pH in regulating abomasal acid secretion is discussed. INTRODUCTION
The pyloric antral region of the abomasum (the terminal and acidsecreting region of the sheep's stomach) is important in determining how much acid the abomasum secretes. Acid secretion is reduced to a low level after antrectomy although it still increases with changes in the quality or quantity of food, and when fasted sheep are teased with food or given fresh food to eat (McLeay & Titchen, 1974, 1975). In animals with a simpler form of stomach, acidification of the antrum inhibits acid secretion from the body of the stomach by causing a reduction in gastrin secretion. *
Present address: Department of Biological Sciences, University of Waikato,
Hamilton, New Zealand.
L. M. McLEA Y AND D. A. TITCHEN Lowering the pH of the contents of the abomasum inhibits its secretion of acid (Ash, 1961a, b). Hill (1968) considered that the pH at which this inhibition occurred was about 2-0 and that it was due to an effect on antral secretion of gastrin. The antral origin of this inhibition has, however, not previously been demonstrated in a ruminant. This report is of observations on the effects of perfusion of antral pouches with acid, on acid secretion from pouches prepared from the body of the abomasum of sheep. A brief account of preliminary experiments in the series has been presented (McLeay & Titchen, 1970a). 708
METHODS Four sheep of Romney or Corriedale mixed breeds and four Merino sheep with both fundic and antral pouches were used in these experiments. Four of the animals also had an exteriorization of the reticulum (Titchen, 1958b). The management of the animals, the preparation of pouches and the collection of fundic pouch secretion and its analysis were as described previously (McLeay & Titchen, 1970b, 1975). Reticular motility was recorded with the aid of strain gauges (Carr, McLeay, Reid & Webster, 1971). Antral pouch perfusion was undertaken using the same procedures as those described for pharmacological stimulation of the antrum in sheep (McLeay & Titchen, 1977). The antral pouches were perfused with solutions at a pressure of 8-12 mmHg and with flows of between 150 and 300 ml.hr-1. In the ruminant secretion from the abomasum is continuous, presumably due in large part to the continual passage of digesta into it from the sites of fermentative digesion in the more cranial parts of the stomach (Hill, 1955, 1960). Control and test observations were made within each experiment. The experimental procedure was first to perfuse the pouch with saline (for 45-120 min), then with the test solution (for 15-120 min) and finally with saline again (for 45-120 min). Throughout each experiment, collections of fundic pouch secretion were made at 15 min intervals; in some experiments in four of the sheep, continuous records of reticular contractions were made. The solutions used to perfuse antral pouches included: NaCl 0-9 % wlv. (saline) at pH 5-5-6-0 or acidified with HCl to a chosen pH as determined with a pH meter (Radiometer, Copenhagen), 0-1 M-HCl and fundic pouch secretion (pH 1-1-1-2). Pentagastrin, the pentapeptide with gastrin-like activity (Peptavlon, Imperial Chemical Industries) was infused into a jugular vein, with a constant infusion syringe pump (Scientific and Research Instruments Ltd., London) at a rate of 1-2 #g. kg-'. hr-1 via a polyethylene tube inserted 16 hr or more before the infusion was made. Clotting was prevented by charging the tube with heparinized saline (heparin, Pularin, Evans Medical, Liverpool was added at 50 i.u. ml.-' saline). RESULTS
Perfusions of antral pouches with acid solutions (0-1 M-HCI, acidified saline or fundic pouch secretion) at pH 1-1-1-5 reduced the volume and acidity of fundic pouch secretion in twenty-eight experiments. Thus in three experiments in one sheep, during perfusions of the antral pouch with acid the fundic pouch secretion was reduced to 40 % of its previous
ANTRAL ACIDIFICATION AND GASTRIC SECRETION 709 resting volume, its acid output to 23 % and pepsin output to 43 % (its pepsin concentration was unaffected). Acid concentration was reduced from pre-infusion levels of 84-143 mm H+. 1-1. to minima of 17, 42 and 46 mm H+. 1-1. during the most intense period of inhibition. Effects of perfusion of an antral pouch with acid for 15 min are shown in Fig. 1. 0.7
Acid output (m-mole HI in 15 min)
06 0.5 0-4
0*3 Total acid concn.
02
(mM-H )
rt
.....a
I
r"
110
LIS I
I
~~~~~100
l
10
120
r-
|
l
Pepsin output
90
(mg tyrosine in 15 min)
5
-60 -45 -30 -15 0 Antral irrigation
4- Saline -
15 30 45 60 75 90 Time (min) - Saline -
HCI
pH antral perfusate 63 1 3
31
40
63
Fig. 1. The effect on fundic pouch secretion of irrigation of an antral pouch with 0.1 M-HCI of pH 1-3. The antral pouch was irrigated with saline before and after HCL. The initially high fundic secretion was because the sheep was eating. The pH figures recorded on the x co-ordinate are of the fluid effluxing from the antral pouch. Figures on 'outputs' of acid and pepsin refer to amounts of each present in 15 min samples of pouch secretion.
L. M. McLEA Y AND D. A. TITCHEN The first effect, a reduction in the volume of secretion, occurred within 15 min of perfusion of antral pouches with acid - the concentration of H+ fell after a further 15-30 min. The secretion of acid and pepsin remained reduced both during antral pouch acidification and for up to 45 min after resumption of perfusion of the antral pouches with saline. 710
Secretory responses to feeding and antral pouch perfusion with acid The increases in the secretion of acid normally obtained when sheep ate freshly provided food were, during perfusion of antral pouches with acid solutions of pH 1-3-2-0, reduced to 6-17 % of their normal extent in five experiments. The response in pepsin secretion on feeding was 17-75 % of its normal amplitude. One experimental result is shown in Fig. 2. The pH at which the inhibition developed differed in feeding animals and those in which effects on resting secretion were examined when food was not available. Resting secretion of H+ was reduced by as much as 66 % by perfusion of antral pouches with solutions at or below pH 2-5-2*7 and returned (doubling over its mean inhibited level) when the pH of perfusate effluxing from the antral pouch was pH 2-9-3*4. Clear secretary responses on eating were demonstrated in three sheep during perfusion of antral pouches with acid solutions at pH 2*5-2-7, a level of antral pouch acidification which consistently reduced resting secretion. Whereas perfusion of antral pouches with a 041 % solution of acetylcholine in saline stimulates acid secretion from pouches of the body of the abomasum (McLeay & Titchen, 1977), an acid solution (pH 2.5) of acetylcholine did not stimulate acid secretion in the three experiments in which its effects were examined.
Pentagastrin infusion during antral pouch perfusion with 0.1 M-HC1 That the parietal cells remained susceptible to stimulation by gastrin was shown in experiments in which the intravenous infusion of the gastrin analogue, pentagastrin, increased acid and pepsin secretion in both the fasted and feeding animal during antral pouch perfusion with acid. The increases in secretion were closely associated with the duration of the infusion; secretion declined rapidly following cessation of pentagastrin infusion (Fig. 3). Reticular contractions Perfusion of antral pouches with acid solutions or fundic pouch secretion at pH 1 1-113 was followed by increases in the frequency of reticular contractions in seven of thirteen experiments. The effect was seen within 120 sec of starting the perfusions and continued for 15-20 min (Fig. 4).
ANTRAL ACIDIFICATION AND GASTRIC SECRETION 711 12
4
Acid output (m-mole Hi in 15 min)
I_
,_ .
1.0 08 06 04
IL
-I~r-
a
0.2 --1I
.1 Acid conc. (mm-H') 120
80 40
Pepsin output 30~~~~~~~~~~~~~~
10
20
-90
-60 -30
0
30 60 90 120 150 180 Time (min) Fig. 2. A comparison (obtained from two experiments) of the effects on fundic pouch acid and pepsin secretion when an animal with an antral pouch ate freshly provided food (t,) during perfusion of its antral pouch with saline ( ), acid at pH 1-3 (---) and again saline ( ). Antral acidifica-
tion reduced the secretary response on providing
a
fresh supply of food.
712 L. M. McLEA Y AND D. A. TITCHEN The changes were most obvious in animals which did not have food available and were also seen during rumination. The increases in frequency of reticular contractions during rumination were due to the occurrence of monophasic and diphasic contractions when the animals were Total acid output (m-mole H
044
0-303 in 1 5
mini)
0.2 0.1 0
Total acid conc. (mM-Hi')
-
120
100 80 60
-
Pepsin output 20
40
(mg tyrosine in 30 min)
10 0 -60 -30 0 30 60 pH antral perfusate 64 1-6
1
120 150 180 210 Time (min) 3.7 5-8 28 5*5 63
I
Pentagastrin 1 d
90
2p.kg-1.hr
1
1
Saline
HCI
Saline
Fig. 3. The effects of intravenous infusion of pentagastrin (1.2 fig. kg-1. hr-1) on fundic pouch secretion during perfusion of an antral pouch with 01 m-HCl. The animal was without food. The figures shown on the x co-ordinate are those of the pH of fluid effluxing from the antral pouch.
ANTRAL ACIDIFICATION AND GASTRIC SECRETION 713 masticating regurgitated material; they were in addition to the contractions normally associated with regurgitation and thus were seen at times reticular contractions do not characteristically occur.
40 ml. wash out
30 ml. 01 M-HCI
1 min
Fig. 4. A continuous record of movements of an exteriorization of the reticulum following the introduction of 30 ml. 0.1 M-HC1 ( t HC1) into a sheep's antral pouch, and its replacement with saline ( 1 wash out). The continuous record has been divided into three sections reading left to right. DISCUSSION
The experiments reported provide direct evidence for the suggestion previously made by Hill (1968) that acidification of abomasal contents reduces its secretion of acid by an effect on the antrum. The pH at which resting secretion was reduced was within the range of the normal pH of abomasal contents of sheep kept under similar conditions to those in the present experiments (McLeay, Anderson, Bingley & Titchen, 1973). The pH range at which the inhibition of resting acid secretion starts to operate is about that of the digesta which normally leaves the antrum pH 2-7 was a mean figure quoted by Harrison & Hill (1962). This autoinhibition of acid secretion is probably a most significant factor in regulation of the pH of digesta leaving the abomasum. Secretion from the antrum is too scant to contribute significantly to the regulation of the pH of the digesta flowing into the duodenum (Ash, 1961a; Harrison & Hill, 1962; McLeay & Titchen, 1975) and the relative constancy of this pH probably reflects the efficacy of this mechanism of the acid regulation of acid secretion. The observation that the pH of antral contents at which secretary responses to feeding were reduced was consistently lower than that inhibiting resting secretion, is indicative that the effect reflects the summated effects of excitatory and inhibitory influences and is to some degree graded according to the acidity of antral digesta, a suggestion
L. M. McLEAY AND D. A. TITCHEN previously made by Ash (1961 b) and demonstrated in dogs by Posey & Franklin (1967). Hill's (1968) estimate that pH 2-0 is the level at which acid secretion is inhibited in the ruminant appears to be referable to prandial secretion; resting secretion is reduced at a higher level - pH 2%7 or more. Our observations lead us to repeat our previous suggestion (McLeay & Titchen, 1975) that the conclusion drawn by Amure & Omole (1971) that acid inhibition of acid secretion is a phenomenon not encountered in ruminants is erroneous. We consider as did Ash (1961 a) that the mechanism plays an important part in the control of abomasal secretion in ruminants. The mechanisms involved in this acid inhibition of acid secretion have not been established in these experiments. Redford, Savage & Schofield (1962) reported that acidification of the antrum of the dog inhibited gastrin release, inferred to occur with every form of stimulus, and Becker, Reeder & Thompson (1973) have since shown that gastrin release from the antrum of the dog is graded according to antral pH. It is suggested that reduced gastrin release provides one explanation for the effects observed in the sheep, and that acidification also blocked the release of gastrin presumed to occur with cholinergic stimulation of the antrum (McLeay & Titchen, 1977). Acidification of the antrum (as do pharmacological blocking agents, McLeay & Titchen, 1977) virtually abolished feeding responses, which however, have been demonstrated after chronic antrectomy (McLeay & Titchen, 1975). This latter observation suggests that there is some adaptation, perhaps by way of increases in gastrin secretion from extra-antral sources, after the antrum is removed. Jury & McLeay (1977) have recently demonstrated substantial gastrin-like activity in extracts of the reticulum, rumen, omasum, body of the abomasum and duodenum of sheep, and also found evidence of a number of molecular forms. However, the profound block of feeding responses obtained in the present experiments with perfusions of the lower pH may indicate that there was no significant secretion of an active form of gastrin from any part of the gut except the antrum. The demonstration that reticular contractions were changed in frequency in some experiments on perfusion of antral pouches with acid, can be explained by stimulation of the acid-sensitive receptors suggested by Titchen (1953, 1958a) to have caused reflex stimulation of reticular contractions in decerebrate preparations, and for which direct evidence has been provided in the cat and the sheep by recording from vagal afferent nerve fibres (Iggo, 1957; Leek & Harding, 1975). Whether these afferents contribute to the control of acid secretion from the abomasum is not known. Their possible contribution may be in the pyloro-oxyntic 714
ANTRAL ACIDIFICATION AND GASTRIC SECRETION 715 reflex which has been described in the dog (Debas, Konturek, Walsh & Grossman, 1974). The demonstration of an acid-sensitive mechanism in these experiments, and the previous demonstration of the effects on gastric secretion and motility of antral pouch perfusion with cholinergic stimulating and blocking agents and local anaesthetic (McLeay & Titchen, 1977), indicate the importance of the antrum as a source of stimulation of gastric acid secretion in the conscious sheep. The roles of nervous as well as hormonal mechanisms and the possibility of participation of an antral chalone in the regulation of abomasal acid secretion remain to be studied. This work was aided by grants from the Australian Meat Research Committee, the Australian Research Grants Committee and the Melbourne University Veterinary Research Fund. L. McL. was in receipt of a Commonwealth (New Zealand-Australia) Scholarship during the earlier part of this work. The assistance received from Messrs J. Patterson, J. Rundle, F. Toman, Miss D. Percival, Mr R. Meurant and the late Mr M. McGuinn is also acknowledged with gratitude.
REFERENCES
AMURE, B. 0. & OmOLE, A. (1971). Comparative study of antral gastrin activity in some mammals. Br. J. Pharmac. 41, 629-639. ASH, R. W. (1961 a). Acid secretion by the abomasum and its relation to the flow of food material in the sheep. J. Physiol. 156, 93-1 1 1. ASH, R. W. (1961 b). Stimuli influencing the secretion of acid by the abomasum of sheep. J. Phy8iol. 157, 185-207. BECKER, H. D., REEDER, D. D. & THOMPSON, J. C. (1973). The effect of changes in antral pH on the basal release of gastrin. Proc. Soc. exp. Biol. Med. 143, 238-240. CARR, D. H., McLEAY, L. M., REID, C. S. W. & WEBSTER, D. (1971). Partial exteriorization as a means of studying gastric motility. Digest of the Ninth Int. Conf. Med. Biol. Eng. Melbourne, p. 94. DEBAS, H. T., KONTUREK, S. J., WALSH, J. H. & GROSSMAN, M. I. (1974). Proof of a pyloro-oxyntic reflex for stimulation of acid secretion. Gastroenterology 66, 526532. HARRISON, F. A. & HILL, K. J. (1962). Digestive secretions and the flow of digesta along the duodenum of the sheep. J. Phy8iol. 162, 225-243. HIiLL, K. J. (1955). Continuous gastric secretion in the ruminant. Q. JZ exp. Phy8iol. 40, 32-39. HILL, K. J. (1960). Abomasal secretion in the sheep. J. Physiol. 154, 115-132. HiLL, K. J. (1968). Abomasal function. In Handbook of Physiology, section 6: Alimentary Canal, vol. v, ed. Code, C. F., pp. 2747-2759. Washington, D.C.: American Physiological Society. IGGo, A. (1957). Gastric mucosal chemoreceptors with vagal afferent fibres in the cat. Q. JL exp. Phy8iol. 42, 398-409. Juay, D. R. & McLEAY, L. M. (1977). Gastrin-like activity in the forestomachs, abomasum and intestine of the sheep. J. Physiol. 265, 57P. LEEK, B. F. & HARDING, R. (1975). Sensory nervous receptors in the ruminant stomach and the reflex control of reticulo-ruminal motility. In Dige8tion and Metabolism in the Ruminant, ed. McDONALD, I. W. & WARNER, A. C. I., pp. 60-76. Armidale: University of New England Publishing Unit.
716
L. M. McLEA Y AND D. A. TITCHEN
McLEAY, L. M., ANDERSON, N., BINGLEY, J. B. & TITCHEN, D. A. (1973). Effects on abomasal function of Ostertagia circumcincta infections in sheep. Paraitology 66, 241-257. McLEAY, L. M. & TITCHEN, D. A. (1970a). Secretion from fundic abomasal pouches in sheep with antral abomasal pouches. Proc. Aust. Physiol. Pharmacol. Soc. 1, 42-43. McLEAY, L. M. & TITCHEN, D. A. (1970b). Abomasal secretary responses to teasing with food and feeding in the sheep. J. Physiol. 206, 605-628. MCLEAY, L. M. & TITCHEN, D. A. (1974). Effects of the amount and type of food eaten on secretion from fundic abomasal pouches of sheep. Br. J. Nutr. 32, 375-387. MCLEAY, L. M. & TITCHEN, D. A. (1975). Gastric, antral and fundic pouch secretion in sheep. J. Physiol. 248, 595-612. MCLEAY, L. M. & TITCHEN, D. A. (1977). Acid and pepsin secretion of separated gastric pouches during perfusion of antral pouches with cholinergic stimulating and blocking agents and lignocaine. J. Physiol. 264, 215-228. PosEY, E. L. & FRANKLIN, H. (1967). Relation of antral pH to gastrin release and fundal pH in dogs. Am. J. dig. Di8. 12, 356-362. REDFORD, M., SAVAGE, L. E. & SCHOFIELD, B. (1962). The blocking of gastrin release. J. Physiol. 162, 61P. TiTCHEN, D. A. (1953). Reflex contractions of the reticulum. J. Physiol. 122, 32P. TITCHEN, D. A. (1958a) . Reflex stimulation and inhibition of reticulum contractions in the ruminant stomach. J. Physiol. 141, 1-21. TITCHEN, D. A. (1958 b). Partial exteriorization of the reticulum in sheep. J. Physiol. 143, 35P.
707
INHIBITION OF HYDROCHLORIC ACID AND PEPSIN SECRETION FROM GASTRIC POUCHES BY ANTRAL POUCH ACIDIFICATION IN SHEEP
BY L. M. McLEAY* AND D. A. TITCHEN From the Department of Veterinary Preclinical Science8, University of Melbourne, Parkville, Victoria 3052
(Received 26 April 1977) SUMMARY
1. Secretion of acid and pepsin from separated pouches of the body of the abomasum was studied in sheep during perfusion of antral pouches with acid solutions. 2. Resting secretion of acid and pepsin was reduced by acidification of antral pouches to pH 2-7 or less. 3. Increases in the secretion of HCl and pepsin normally obtained on feeding were reduced or abolished by perfusion of antral pouches with solutions at pH 1-3-2*0. 4. Perfusion of antral pouches with acetylcholine at pH 2-5 failed to stimulate acid secretion as it did at a higher pH. 5. Pentagastrin stimulated acid and pepsin secretion during the inhibition of secretion produced by antral pouch acidification. 6. Increases in reticular motility occurred on antral pouch acidification with solutions of pH 1 1-1 3. 7. The contribution of antral pH in regulating abomasal acid secretion is discussed. INTRODUCTION
The pyloric antral region of the abomasum (the terminal and acidsecreting region of the sheep's stomach) is important in determining how much acid the abomasum secretes. Acid secretion is reduced to a low level after antrectomy although it still increases with changes in the quality or quantity of food, and when fasted sheep are teased with food or given fresh food to eat (McLeay & Titchen, 1974, 1975). In animals with a simpler form of stomach, acidification of the antrum inhibits acid secretion from the body of the stomach by causing a reduction in gastrin secretion. *
Present address: Department of Biological Sciences, University of Waikato,
Hamilton, New Zealand.
L. M. McLEA Y AND D. A. TITCHEN Lowering the pH of the contents of the abomasum inhibits its secretion of acid (Ash, 1961a, b). Hill (1968) considered that the pH at which this inhibition occurred was about 2-0 and that it was due to an effect on antral secretion of gastrin. The antral origin of this inhibition has, however, not previously been demonstrated in a ruminant. This report is of observations on the effects of perfusion of antral pouches with acid, on acid secretion from pouches prepared from the body of the abomasum of sheep. A brief account of preliminary experiments in the series has been presented (McLeay & Titchen, 1970a). 708
METHODS Four sheep of Romney or Corriedale mixed breeds and four Merino sheep with both fundic and antral pouches were used in these experiments. Four of the animals also had an exteriorization of the reticulum (Titchen, 1958b). The management of the animals, the preparation of pouches and the collection of fundic pouch secretion and its analysis were as described previously (McLeay & Titchen, 1970b, 1975). Reticular motility was recorded with the aid of strain gauges (Carr, McLeay, Reid & Webster, 1971). Antral pouch perfusion was undertaken using the same procedures as those described for pharmacological stimulation of the antrum in sheep (McLeay & Titchen, 1977). The antral pouches were perfused with solutions at a pressure of 8-12 mmHg and with flows of between 150 and 300 ml.hr-1. In the ruminant secretion from the abomasum is continuous, presumably due in large part to the continual passage of digesta into it from the sites of fermentative digesion in the more cranial parts of the stomach (Hill, 1955, 1960). Control and test observations were made within each experiment. The experimental procedure was first to perfuse the pouch with saline (for 45-120 min), then with the test solution (for 15-120 min) and finally with saline again (for 45-120 min). Throughout each experiment, collections of fundic pouch secretion were made at 15 min intervals; in some experiments in four of the sheep, continuous records of reticular contractions were made. The solutions used to perfuse antral pouches included: NaCl 0-9 % wlv. (saline) at pH 5-5-6-0 or acidified with HCl to a chosen pH as determined with a pH meter (Radiometer, Copenhagen), 0-1 M-HCl and fundic pouch secretion (pH 1-1-1-2). Pentagastrin, the pentapeptide with gastrin-like activity (Peptavlon, Imperial Chemical Industries) was infused into a jugular vein, with a constant infusion syringe pump (Scientific and Research Instruments Ltd., London) at a rate of 1-2 #g. kg-'. hr-1 via a polyethylene tube inserted 16 hr or more before the infusion was made. Clotting was prevented by charging the tube with heparinized saline (heparin, Pularin, Evans Medical, Liverpool was added at 50 i.u. ml.-' saline). RESULTS
Perfusions of antral pouches with acid solutions (0-1 M-HCI, acidified saline or fundic pouch secretion) at pH 1-1-1-5 reduced the volume and acidity of fundic pouch secretion in twenty-eight experiments. Thus in three experiments in one sheep, during perfusions of the antral pouch with acid the fundic pouch secretion was reduced to 40 % of its previous
ANTRAL ACIDIFICATION AND GASTRIC SECRETION 709 resting volume, its acid output to 23 % and pepsin output to 43 % (its pepsin concentration was unaffected). Acid concentration was reduced from pre-infusion levels of 84-143 mm H+. 1-1. to minima of 17, 42 and 46 mm H+. 1-1. during the most intense period of inhibition. Effects of perfusion of an antral pouch with acid for 15 min are shown in Fig. 1. 0.7
Acid output (m-mole HI in 15 min)
06 0.5 0-4
0*3 Total acid concn.
02
(mM-H )
rt
.....a
I
r"
110
LIS I
I
~~~~~100
l
10
120
r-
|
l
Pepsin output
90
(mg tyrosine in 15 min)
5
-60 -45 -30 -15 0 Antral irrigation
4- Saline -
15 30 45 60 75 90 Time (min) - Saline -
HCI
pH antral perfusate 63 1 3
31
40
63
Fig. 1. The effect on fundic pouch secretion of irrigation of an antral pouch with 0.1 M-HCI of pH 1-3. The antral pouch was irrigated with saline before and after HCL. The initially high fundic secretion was because the sheep was eating. The pH figures recorded on the x co-ordinate are of the fluid effluxing from the antral pouch. Figures on 'outputs' of acid and pepsin refer to amounts of each present in 15 min samples of pouch secretion.
L. M. McLEA Y AND D. A. TITCHEN The first effect, a reduction in the volume of secretion, occurred within 15 min of perfusion of antral pouches with acid - the concentration of H+ fell after a further 15-30 min. The secretion of acid and pepsin remained reduced both during antral pouch acidification and for up to 45 min after resumption of perfusion of the antral pouches with saline. 710
Secretory responses to feeding and antral pouch perfusion with acid The increases in the secretion of acid normally obtained when sheep ate freshly provided food were, during perfusion of antral pouches with acid solutions of pH 1-3-2-0, reduced to 6-17 % of their normal extent in five experiments. The response in pepsin secretion on feeding was 17-75 % of its normal amplitude. One experimental result is shown in Fig. 2. The pH at which the inhibition developed differed in feeding animals and those in which effects on resting secretion were examined when food was not available. Resting secretion of H+ was reduced by as much as 66 % by perfusion of antral pouches with solutions at or below pH 2-5-2*7 and returned (doubling over its mean inhibited level) when the pH of perfusate effluxing from the antral pouch was pH 2-9-3*4. Clear secretary responses on eating were demonstrated in three sheep during perfusion of antral pouches with acid solutions at pH 2*5-2-7, a level of antral pouch acidification which consistently reduced resting secretion. Whereas perfusion of antral pouches with a 041 % solution of acetylcholine in saline stimulates acid secretion from pouches of the body of the abomasum (McLeay & Titchen, 1977), an acid solution (pH 2.5) of acetylcholine did not stimulate acid secretion in the three experiments in which its effects were examined.
Pentagastrin infusion during antral pouch perfusion with 0.1 M-HC1 That the parietal cells remained susceptible to stimulation by gastrin was shown in experiments in which the intravenous infusion of the gastrin analogue, pentagastrin, increased acid and pepsin secretion in both the fasted and feeding animal during antral pouch perfusion with acid. The increases in secretion were closely associated with the duration of the infusion; secretion declined rapidly following cessation of pentagastrin infusion (Fig. 3). Reticular contractions Perfusion of antral pouches with acid solutions or fundic pouch secretion at pH 1 1-113 was followed by increases in the frequency of reticular contractions in seven of thirteen experiments. The effect was seen within 120 sec of starting the perfusions and continued for 15-20 min (Fig. 4).
ANTRAL ACIDIFICATION AND GASTRIC SECRETION 711 12
4
Acid output (m-mole Hi in 15 min)
I_
,_ .
1.0 08 06 04
IL
-I~r-
a
0.2 --1I
.1 Acid conc. (mm-H') 120
80 40
Pepsin output 30~~~~~~~~~~~~~~
10
20
-90
-60 -30
0
30 60 90 120 150 180 Time (min) Fig. 2. A comparison (obtained from two experiments) of the effects on fundic pouch acid and pepsin secretion when an animal with an antral pouch ate freshly provided food (t,) during perfusion of its antral pouch with saline ( ), acid at pH 1-3 (---) and again saline ( ). Antral acidifica-
tion reduced the secretary response on providing
a
fresh supply of food.
712 L. M. McLEA Y AND D. A. TITCHEN The changes were most obvious in animals which did not have food available and were also seen during rumination. The increases in frequency of reticular contractions during rumination were due to the occurrence of monophasic and diphasic contractions when the animals were Total acid output (m-mole H
044
0-303 in 1 5
mini)
0.2 0.1 0
Total acid conc. (mM-Hi')
-
120
100 80 60
-
Pepsin output 20
40
(mg tyrosine in 30 min)
10 0 -60 -30 0 30 60 pH antral perfusate 64 1-6
1
120 150 180 210 Time (min) 3.7 5-8 28 5*5 63
I
Pentagastrin 1 d
90
2p.kg-1.hr
1
1
Saline
HCI
Saline
Fig. 3. The effects of intravenous infusion of pentagastrin (1.2 fig. kg-1. hr-1) on fundic pouch secretion during perfusion of an antral pouch with 01 m-HCl. The animal was without food. The figures shown on the x co-ordinate are those of the pH of fluid effluxing from the antral pouch.
ANTRAL ACIDIFICATION AND GASTRIC SECRETION 713 masticating regurgitated material; they were in addition to the contractions normally associated with regurgitation and thus were seen at times reticular contractions do not characteristically occur.
40 ml. wash out
30 ml. 01 M-HCI
1 min
Fig. 4. A continuous record of movements of an exteriorization of the reticulum following the introduction of 30 ml. 0.1 M-HC1 ( t HC1) into a sheep's antral pouch, and its replacement with saline ( 1 wash out). The continuous record has been divided into three sections reading left to right. DISCUSSION
The experiments reported provide direct evidence for the suggestion previously made by Hill (1968) that acidification of abomasal contents reduces its secretion of acid by an effect on the antrum. The pH at which resting secretion was reduced was within the range of the normal pH of abomasal contents of sheep kept under similar conditions to those in the present experiments (McLeay, Anderson, Bingley & Titchen, 1973). The pH range at which the inhibition of resting acid secretion starts to operate is about that of the digesta which normally leaves the antrum pH 2-7 was a mean figure quoted by Harrison & Hill (1962). This autoinhibition of acid secretion is probably a most significant factor in regulation of the pH of digesta leaving the abomasum. Secretion from the antrum is too scant to contribute significantly to the regulation of the pH of the digesta flowing into the duodenum (Ash, 1961a; Harrison & Hill, 1962; McLeay & Titchen, 1975) and the relative constancy of this pH probably reflects the efficacy of this mechanism of the acid regulation of acid secretion. The observation that the pH of antral contents at which secretary responses to feeding were reduced was consistently lower than that inhibiting resting secretion, is indicative that the effect reflects the summated effects of excitatory and inhibitory influences and is to some degree graded according to the acidity of antral digesta, a suggestion
L. M. McLEAY AND D. A. TITCHEN previously made by Ash (1961 b) and demonstrated in dogs by Posey & Franklin (1967). Hill's (1968) estimate that pH 2-0 is the level at which acid secretion is inhibited in the ruminant appears to be referable to prandial secretion; resting secretion is reduced at a higher level - pH 2%7 or more. Our observations lead us to repeat our previous suggestion (McLeay & Titchen, 1975) that the conclusion drawn by Amure & Omole (1971) that acid inhibition of acid secretion is a phenomenon not encountered in ruminants is erroneous. We consider as did Ash (1961 a) that the mechanism plays an important part in the control of abomasal secretion in ruminants. The mechanisms involved in this acid inhibition of acid secretion have not been established in these experiments. Redford, Savage & Schofield (1962) reported that acidification of the antrum of the dog inhibited gastrin release, inferred to occur with every form of stimulus, and Becker, Reeder & Thompson (1973) have since shown that gastrin release from the antrum of the dog is graded according to antral pH. It is suggested that reduced gastrin release provides one explanation for the effects observed in the sheep, and that acidification also blocked the release of gastrin presumed to occur with cholinergic stimulation of the antrum (McLeay & Titchen, 1977). Acidification of the antrum (as do pharmacological blocking agents, McLeay & Titchen, 1977) virtually abolished feeding responses, which however, have been demonstrated after chronic antrectomy (McLeay & Titchen, 1975). This latter observation suggests that there is some adaptation, perhaps by way of increases in gastrin secretion from extra-antral sources, after the antrum is removed. Jury & McLeay (1977) have recently demonstrated substantial gastrin-like activity in extracts of the reticulum, rumen, omasum, body of the abomasum and duodenum of sheep, and also found evidence of a number of molecular forms. However, the profound block of feeding responses obtained in the present experiments with perfusions of the lower pH may indicate that there was no significant secretion of an active form of gastrin from any part of the gut except the antrum. The demonstration that reticular contractions were changed in frequency in some experiments on perfusion of antral pouches with acid, can be explained by stimulation of the acid-sensitive receptors suggested by Titchen (1953, 1958a) to have caused reflex stimulation of reticular contractions in decerebrate preparations, and for which direct evidence has been provided in the cat and the sheep by recording from vagal afferent nerve fibres (Iggo, 1957; Leek & Harding, 1975). Whether these afferents contribute to the control of acid secretion from the abomasum is not known. Their possible contribution may be in the pyloro-oxyntic 714
ANTRAL ACIDIFICATION AND GASTRIC SECRETION 715 reflex which has been described in the dog (Debas, Konturek, Walsh & Grossman, 1974). The demonstration of an acid-sensitive mechanism in these experiments, and the previous demonstration of the effects on gastric secretion and motility of antral pouch perfusion with cholinergic stimulating and blocking agents and local anaesthetic (McLeay & Titchen, 1977), indicate the importance of the antrum as a source of stimulation of gastric acid secretion in the conscious sheep. The roles of nervous as well as hormonal mechanisms and the possibility of participation of an antral chalone in the regulation of abomasal acid secretion remain to be studied. This work was aided by grants from the Australian Meat Research Committee, the Australian Research Grants Committee and the Melbourne University Veterinary Research Fund. L. McL. was in receipt of a Commonwealth (New Zealand-Australia) Scholarship during the earlier part of this work. The assistance received from Messrs J. Patterson, J. Rundle, F. Toman, Miss D. Percival, Mr R. Meurant and the late Mr M. McGuinn is also acknowledged with gratitude.
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