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Pepsin in the toad Bufo marinus
Camp. Biochem. Physiol. Printed in Great Britain
Vol. 84A,
No. 4, pp. 669-672,
1986
0300-9629/86
$3.00 + 0.00
Pergamon Journals Ltd
PEPSIN IN THE TOAD BUFO
MARINUS
P. M. TAYLOR and M. J. TYLER Department of Zoology, University of Adelaide, G.P.O. Box 498, Adelaide, S.A. 5001, Australia, Telephone: (08) 228-5333 (Received 26 November 1985) Abstract-l.
Bufo 2. 3. Both 4. frogs
The macroscopic localization of pepsinogen in the upper gastrointestinal tract of the anuran
marinus was studied by means of biochemical assay.
The pH optimum of the anuran pepsin was determined to be 1.6. The effect of lowered ambient temperature on stored pepsinogen and proteolytic activity was studied. parameters were reduced. The results are extrapolated to speculate on the mechanisms of gastric brooding in gastric brooding of the genus Rheobatrachus.
INTRODUCITON Our interest in pepsin and pcpsinogen arose from our studies of Rheobatrachus, an Australian frog genus
notable for its bizarre reproductive habit of gastric brooding. The successful development of young in the stomach must require the inhibition of all gastric digestive functions. For our studies of the mechanisms of gastric brooding we have used the cane toad, Bufo marinus, as an experimental model. We have investigated gastric emptying and gastric acid secretion in this species, both of which are inhibited by prostaglandin E,, and now turn our attention to the gastric proteolytic enzyme pepsin. Adults of the Class Amphibia are primarily carnivorous and usually feed on live prey. They do not masticate their food and prey must therefore be swallowed whole and alive. The glands of the buccal cavity do not secrete digestive enzymes in functionally significant amounts (Reeder, 1964). These factors suggest that the members of this class must possess a high gastric digestive capacity. Pepsin, a proteolytic enzyme active at low pH, is found almost universally in the stomachs of vertebrates. The enzyme is stored and released in an inactive form: pepsinogen (Ebstein and Grutzner, 1874), a zymogen which, while stable at neutral pH, is converted to pepsin in an acid medium. This process is autocatalytic and occurs via an inactive intermediate (Herriott, 1938). Thus there is a functional significance in the association of acid production and pepsinogen in the stomach. Indeed, it is only in mammals that these two functions are assigned to separate cell types (Wright et af., 1957). In mammals the chief cells are the source of pepsinogen (Langley, 1881). Amphibian cells with granules of similar structure have, by analogy, been identified as the source of pepsinogen. More recently several distinct pepsinogens have been isolated and their distributions mapped using immunofluorescent techniques. Samloff and Liebman (1973) concluded that, in the human, the peptic cell mass is heterogeneous and consists of four cell types, not just the chief cells.
This differentiation
may also occur in the Amphibia
(Giraud and Yeomans, 1981). The functional significance of these findings is not yet apparent. The site of pepsinogen synthesis and storage in the Anura is disputed in the literature. Several authors mention specialized cells, secreting purely pepsinogen, in the oesophagus just proximal to the junction with the stomach (Jordan, 1927; Vonk, 1941; Norris, 1959; Wolvekamp and Tinbergen, 1942). They maintain that these extragastric cells are the major source of pepsinogen. Others authors have found more significant sites of pepsinogen storage within the stomach (Eksaeva, 1958; Sedar, 1961; Reese et al., 1979). Some of these apparent contradictions may be due to species differences; in some early works the species are not defined. Another variable is the method of pepsin identification; in many cases the presence of pepsinogen has been assumed on the basis of the similarity of microscopic features to mammalian chief cells. We have sought to localize pepsinogen in the cane toad, Bufo marinus, at a macroscopic level only, by means of its biochemical properties, and to make some simple physiological observations on its activity. MATERJALSAND
METHODS
Small cane toads (less than 100 g body weight) were used in these experiments. They were obtained from a commercial supplier in Queensland and air freighted to Adelaide where they were kept for periods of up to several months in a controlled environment at 30°C with a 12 hr light/l2 hr dark cycle. Once a week the toads were given larvae of Tenebrio molitor, which they ate spontaneously. The stored potential for proteolytic digestion in the upper gastrointestinal tract was measured and compared with digests using standard porcine pepsin (Sigma P6887). The toads were decerebrated and spinalized and opened along the mid-ventral line to expose the gastrointestinal tract, the upper part of which was removed and cut into six portions as shown in Fig. 1. Each portion was weighed and homogenized with an ultra Turrax in ice-cold McKenzies solution (Nayler and McKelvie, 1956) using 10 ml buffer per gramme tissue. An aliquot of each homogenate was incubated for 10min at 30°C with an excess of 2.5% bovine serum 669
P. M. TAYLOR and M. J. TYLER
670
RESULTS I Proxlmol oesophagus
\
‘i 5 Pylorlc
stomach
\
\ 6 Intestine
Fig. 1. Diagrammatic representation of the upper gastrointestinal tract of Eufo marinas showing the division into six segments for assay of pepsinogen.
albumin (BSA, Sigma A7906) adjusted to pH 1.6 with 0.3 M hydrochloric acid. The incubation was halted by the addition of 12.5% trichloracetic acid to precipitate undigested BSA and the tyrosine content of the supernatant, an index of digested BSA, was measured using Folin Ciocalteu phenol reagent (Folin and Ciocalteu, 1927), and spectrophotometric quantification against a water blank at 725 nm (Anson and Mirsky, 1932). This protocol was arrived at after initial experiments to determine the influence of pH on the rate of tyrosine production. In these experiments incubations were set up essentially as already described but using a range of concentrations of hydrochloric acid from 3.0 to 0.003 M to alter the pH of the digest. The distribution of pepsinogen was measured in toads fasted for between 2 and 3 weeks and in toads which had eaten mealworms within the 48 hr prior to the experiment; these animals always had food remaining in the stomach, which was removed prior to homogenization of the gut. The effects of temperature on both stored pepsinogen and the digestive action of pepsin were investigated. Toads, fasted for one week, were kept at 12°C for between 5 and 7 days before assaying for pepsinogen. The digests were incubated at either 12 or 30°C.
Figure 2 illustrates the variation in protein digesting activity of the tissue homogenate over the pH range examined. This pattern was consistent for homogenates from each region. The pH optimum for the reaction was 1.6, obtained by using 0.3 M hydrochloric acid in the incubate. The standard curve generated for the action of porcine pepsin on BSA is illustrated in Fig. 3. This is a composite from seven separate assays. The data fit a linear model of the form y = a + bx. The coefficient of determination, r*, describes the quality of fit of the line to the data. The intercept of the line on they axis indicates the generation of some tyrosine in the absence of pepsin in the incubate; this is likely to be due to residual low molecular weight contaminants in the BSA. The localization and quantification of pepsinogen in both fasting and fed toads is shown in Table 1. The main site of pepsinogen storage was the proximal two-thirds of the stomach. There was some pepsinogen in the distal oesophagus and trace amounts in other regions. In the two proximal stomach segments there was significantly more pepsinogen in the homogenates obtained from fasted toads. Table 2 shows the results of experiments to investigate the effects of temperature. The homogenates incubated at 12°C showed little digestive activity, whereas those incubated at 30°C did show proteolytic activity, but less than that demonstrated in homogenates from fasted toads kept at 30°C.
DISCUSSION
The pH optimum for the conversion of pepsinogen to pepsin and the pepsin-catalysed proteolysis of BSA was consistent for homogenates from all segments of the upper gastrointestinal tract. This supports the notion that we were dealing with a substance whose physiologically significant activity is restricted to the stomach stimulated to secrete hydrochloric acid. Our previous studies of gastric pH in Bufo marinus have shown that after a feeding stimulus the pH of the
a = 0.61 b = O-0316 r2=0 82
30
40
5.0
6.0
7.0
PH 3-w
Imo
030
0.6
0.03
M
0033
0
HCI
Fig. 2. Graph illustrating the pH dependence of the proteolytic action of Eufo marinus gastrointestinal tissue homogenates on bovine serum albumin (Sigma A7906). Values are given as mean + standard deviation. N = 4.
”
io
4b
60 lb0
Ii0
260
Peprm (pglmll
Fig. 3. Standard curve for the action of pure porcine pepsin (Sigma P6887) on bovine serum albumin (Sigma A7906) to yield tyrosine. Composite of seven assays.
Pepsin in the toad Bufo marinus Table
I. Pepsin activity
present
in homogenates
of segments
of the upper gastrointestinal
tract of &/o
mariner. Values from fasting and fed toads are compared Fasted toads (A’ = 6) pepsin activity mg/g tissues xkSD
Gut segment Proximal oesophagus Distal oesophagus Fore-stomach Mid-stomach Pyloric stomach Intestine ‘Values significantly TValues significantly
0.098 * 0.02 0.667 i 0.3 I I.471 f 0.54* 0.876 k 0.38t 0.195~0.01 0.057 * 0.03 different, different,
P < 0.02 using Student’s P < 0.05 using Student’s
Fed toads (N = 6) pepsin activity mg/g tissue P_+SD __.__ 0.066 + 0.02 0.414+0.17 0.595 k 0.24’ 0.303 -i_0.08t 0. I80 + 0.04 0.047 f 0.01
f-test. r-test.
Table 2. Pepsin activity measured at I2 and 30°C in homogenates of the upper gastrointestinal Bufo marinus acclimated to an ambient temperature of 12°C
tract of fasted
Gut segment
12°C incubation (N = 2) pepsin activity @g/g tissue) X k SD
30°C incubation (IV = 2) pepsin activity (mg/g tissue) IkSD
Proximal oesophagus Distal oesophagus Fore-stomach Mid-stomach Pyloric stomach Intestine
0.031 0 0.060 0.014 0.012 0
0.038 0.267 f 0.06 0.737 f 0.02 0.242 0.133 0.020
gastric lumen falls rapidly from between 7.0 and 8.0 to around 1.5 (Taylor et al., 1985). This lower value is close to the pH optimum found in the present study. Previous estimates of pH optima for pepsin from unnamed frog species are of the same order; 1.88-1.99 (Pjatnitzky, 1931) and 1.5 (Vonk, 1941). Our results show that in Bufo marinus pepsinogen is found in the distal oesophagus in greater than trace amounts but most of the pepsinogen was found in the proximal two-thirds of the stomach. Only trace amounts were found in the proximal oesophagus, pyloric stomach and intestine, and are probably of little significance in the digestive process. Hirschowitz (1957) noted the ubiquitous distribution of pepsin in trace amounts in body tissues and fluids and remarked on the lack of a convincing teleological explanation for its presence. The relative contribution of each gut segment to the total pepsinogen complement remained fairly constant for each animal. Our results are in agreement with those of Langley (1881) and Machen (1935) that the greatest concentrations of pepsinogen are in the more anterior parts of the stomach. Norris (1959), using the staining method of Bowie, found zymogenic cells only in the oesophagus and forestomach of Rana pipiens. Reese et al. (1979) reported the localization of two immunologically distinct pepsinogens (I and II) in the fundus and antrum of stomachs of Rana catesbeiana. Pepsinogen stores were evenly depleted immediately after feeding and gradually replenished. This finding agrees with the observed feeding behaviour of the toads in natural populations, where they visited known feeding sites for 1 or 2 hr and then disappeared for several successive nights, thus presumably feeding only one night out of every three or more (Brattstrom, 1962; Zug and Zug, 1979). Langley (1881) observed depletion of granules which he correlated with assayable pepsin in gastric gland cells of Rana temporaria and Bufo variabilis during digestion. In both species the time taken for complete recovery of granular appearance varied from 24 hr to several days.
Our methods do not permit us to distinguish sites of pure pepsinogen synthesis and release from sites with more than one product. We are therefore unable to confirm or refute the reports of cells in the distal oesophagus secreting purely pepsinogen. However, Jordan (1927) claimed that in Bufo such cells existed and contributed overwhelmingly to the pepsinogen complement in that genus. Our results from Bufi marinus do not confirm this assertion. We attempted to distinguish the effects of temperature on the synthesis and storage of pepsinogen from peptic enzyme activity. Our results indicate that both are reduced at 12°C. Zug and Zug (1979) state that native juveniles and adults of B. marinus have a critical thermal minimum of ltSl2”C. They reported that in their study population the mean daily body temperature was 26.4”C and that it fluctuated less than ambient temperature, probably due to both behavioural and physiological factors. In our department we have not observed spontaneous feeding in toads kept outside in underground tanks, and force-fed animals often had undigested food in their stomachs at sacrifice. Animals maintained at low temperatures do not thrive. However, when kept at 30°C they have fed spontaneously and remained healthy for several years. Reeder (1964) suggests that cessation of enzyme proliferation at low temperatures is likely to be the limiting factor governing feeding behaviour. Herter (1941) stated that in Rana the gastric wall fails to secrete at temperatures below 5-10°C. We have no information about the physiological or pharmacological regulation of pepsinogen in B. mar inus. However, our results show quite clearly that digestive activity is negligible in conditions of neutral pH. It is not necessary, therefore, to postulate an inhibition of pepsinogen secretion in Rheobatrachus to explain the phenomenon of gastric brooding; the inhibition of acid secretion by prostaglandin E, secreted by the developing young (Tyler et al., 1983) would ensure protection from proteolytic digestion by any pepsinogen that might be released.
672
P. M. TAYLOR and M. J. TYLER REFERENCES
and gastric mucosae of the frog, Rana pipiens. J. exp. Zool. 141, 155-173.
Anson M. L. and Mirsky A. E. (1932) The estimation of pepsin with hemoglobin. J. gen. Physiol. 16, 59-63. Brattstrom B. H. (1962) Homing in the giant toad, Bufo marinus. Herpelologica 18, 176180. Ebstein W. and Griitzner P. (1874) Ueber pepsinbildung im magen. Pjiigers Arch. ges. Physiol. 8, 122. Eksaeva V. A. (1958) The histological structure of the esophagus of certain cold-blooded vertebrates. Dokl. Biol. Sci. 118, 42116.
Folin 0. and Ciocalteu V. (1927) On tyrosine and tryptophane determinations in proteins. J. biol. Chem. 73, 627-650.
Giraud A. S. and Yeomans N. D. (1981) Fine structure of the gastric mucous and endocrine cells of the toad, Bufo marinus. Cell Tissue Res. 218. 663-668.
Herriott R. M. (1938) Kinetics of the formation of pepsin from swine pepsinogen and identification of an intermediate compound. J. gen. Physiol. 22, 65-78. Herter K. (1941) Die Physiologie der Amphibien. In Handbuch der Physiologie, Vol. 6 (Edited by Kiikenthal W.), pp. l-252. Gruyter, Berlin. Hirschowitz B. I. (1957) Pepsinogen: its origins, secretion and excretion. Physiol. Rev. 37, 475-511. Jordan H. G. (1927) Verdauung aus der Vergleichenden Physiologic. Springer Verlag, Berlin. Langley J. N. (1881) On the history and physiology of pepsin-forming glands. Phil. Trans. R. Sot. B 172, 663-7 11. Machen B. (1935) Ueber iiesophagusdrtisen und magenhauptdriisen einheimischer Anuren. Z. mikrosk.-anal. Forsch. 39, 344372.
Nayler W. G. and McKelvie 0. (1956) A method for the study of cardiac metabolism and efficiency in the toad, Bufo marinus. Aust. J. exp. Biol. med. Sci. 34, 239-246.
Norris J. L. (1959) __ of the oesophaeeal \ IThe normal histoloev ."
Pjatnitzky N. P. (1931) Zur Kenntnis der Proteasennatur IX. Z. physiol. Chem. 199, 231-242. Reeder W. G. (1964) The digestive system. In Physiology of the Amphibia (Edited by Moore J. A.), pp. 99-149. Academic Press, New York. Reese J. H., Lechago J. and Samloff I. M. (1979) Localisation of pepsinogens in stomach and duodenum of several animal species by immunofluorescence. Gastroenterology 76, 1307. Samloff I. M. and Liebman W. M. (1973) Cellular localisation of the group II pepsinogens in human stomach and duodenum by immunofluorescence. Gasrroenterology 65, 36-42.
Sedar A. W. (1961) Electron microscopy of the oxyntic cell in the gastric glands of the bull frog, Rana catesbeiana. J. Physiol. Biochem. Cytol. 10, 47-57. Taylor P. M., Tyler M. J. and Shearman D. J. C. (1985) Gastric acid secretion in the toad Bufo marinus, with the description of a new technique for in vivo monitoring. Comp. Biochem. Physiol. 18A, 325-327.
Tyler M. J., Shearman D. J. C., Franc0 R., O’Brien P., Seamark R. F. and Kelly R. (1983) Inhibition of gastric acid secretion in the gastric brooding frog, Rheobarrachus silus. Science 220, 609-610. Vonk H. J. (1941) Die Verdauung bei den niederen Vertebraten. Adv. Enzymol. 1, 371417. Wolvekamp H. P. and Tinbergen L. (1942) Recherches sur la secretion de la pepsine par les glandes oesophageales de la grenouille verte (Rana esculenta L.). Arch. neerl. Physiol. 26, 435456.
Wright R. D., Florey W. H. and Sanders A. G. (1957) Observations on the gastric mucosa of Reptilia. Q. Jl &xp. Physiol. 42, l-14.
Zug G. R. and Zug P. B. (1979) The marine toad, Bufo marinus: a natural history resume of native populations. Smithson. Contr. Zool. 284, 58 pp.
Vol. 84A,
No. 4, pp. 669-672,
1986
0300-9629/86
$3.00 + 0.00
Pergamon Journals Ltd
PEPSIN IN THE TOAD BUFO
MARINUS
P. M. TAYLOR and M. J. TYLER Department of Zoology, University of Adelaide, G.P.O. Box 498, Adelaide, S.A. 5001, Australia, Telephone: (08) 228-5333 (Received 26 November 1985) Abstract-l.
Bufo 2. 3. Both 4. frogs
The macroscopic localization of pepsinogen in the upper gastrointestinal tract of the anuran
marinus was studied by means of biochemical assay.
The pH optimum of the anuran pepsin was determined to be 1.6. The effect of lowered ambient temperature on stored pepsinogen and proteolytic activity was studied. parameters were reduced. The results are extrapolated to speculate on the mechanisms of gastric brooding in gastric brooding of the genus Rheobatrachus.
INTRODUCITON Our interest in pepsin and pcpsinogen arose from our studies of Rheobatrachus, an Australian frog genus
notable for its bizarre reproductive habit of gastric brooding. The successful development of young in the stomach must require the inhibition of all gastric digestive functions. For our studies of the mechanisms of gastric brooding we have used the cane toad, Bufo marinus, as an experimental model. We have investigated gastric emptying and gastric acid secretion in this species, both of which are inhibited by prostaglandin E,, and now turn our attention to the gastric proteolytic enzyme pepsin. Adults of the Class Amphibia are primarily carnivorous and usually feed on live prey. They do not masticate their food and prey must therefore be swallowed whole and alive. The glands of the buccal cavity do not secrete digestive enzymes in functionally significant amounts (Reeder, 1964). These factors suggest that the members of this class must possess a high gastric digestive capacity. Pepsin, a proteolytic enzyme active at low pH, is found almost universally in the stomachs of vertebrates. The enzyme is stored and released in an inactive form: pepsinogen (Ebstein and Grutzner, 1874), a zymogen which, while stable at neutral pH, is converted to pepsin in an acid medium. This process is autocatalytic and occurs via an inactive intermediate (Herriott, 1938). Thus there is a functional significance in the association of acid production and pepsinogen in the stomach. Indeed, it is only in mammals that these two functions are assigned to separate cell types (Wright et af., 1957). In mammals the chief cells are the source of pepsinogen (Langley, 1881). Amphibian cells with granules of similar structure have, by analogy, been identified as the source of pepsinogen. More recently several distinct pepsinogens have been isolated and their distributions mapped using immunofluorescent techniques. Samloff and Liebman (1973) concluded that, in the human, the peptic cell mass is heterogeneous and consists of four cell types, not just the chief cells.
This differentiation
may also occur in the Amphibia
(Giraud and Yeomans, 1981). The functional significance of these findings is not yet apparent. The site of pepsinogen synthesis and storage in the Anura is disputed in the literature. Several authors mention specialized cells, secreting purely pepsinogen, in the oesophagus just proximal to the junction with the stomach (Jordan, 1927; Vonk, 1941; Norris, 1959; Wolvekamp and Tinbergen, 1942). They maintain that these extragastric cells are the major source of pepsinogen. Others authors have found more significant sites of pepsinogen storage within the stomach (Eksaeva, 1958; Sedar, 1961; Reese et al., 1979). Some of these apparent contradictions may be due to species differences; in some early works the species are not defined. Another variable is the method of pepsin identification; in many cases the presence of pepsinogen has been assumed on the basis of the similarity of microscopic features to mammalian chief cells. We have sought to localize pepsinogen in the cane toad, Bufo marinus, at a macroscopic level only, by means of its biochemical properties, and to make some simple physiological observations on its activity. MATERJALSAND
METHODS
Small cane toads (less than 100 g body weight) were used in these experiments. They were obtained from a commercial supplier in Queensland and air freighted to Adelaide where they were kept for periods of up to several months in a controlled environment at 30°C with a 12 hr light/l2 hr dark cycle. Once a week the toads were given larvae of Tenebrio molitor, which they ate spontaneously. The stored potential for proteolytic digestion in the upper gastrointestinal tract was measured and compared with digests using standard porcine pepsin (Sigma P6887). The toads were decerebrated and spinalized and opened along the mid-ventral line to expose the gastrointestinal tract, the upper part of which was removed and cut into six portions as shown in Fig. 1. Each portion was weighed and homogenized with an ultra Turrax in ice-cold McKenzies solution (Nayler and McKelvie, 1956) using 10 ml buffer per gramme tissue. An aliquot of each homogenate was incubated for 10min at 30°C with an excess of 2.5% bovine serum 669
P. M. TAYLOR and M. J. TYLER
670
RESULTS I Proxlmol oesophagus
\
‘i 5 Pylorlc
stomach
\
\ 6 Intestine
Fig. 1. Diagrammatic representation of the upper gastrointestinal tract of Eufo marinas showing the division into six segments for assay of pepsinogen.
albumin (BSA, Sigma A7906) adjusted to pH 1.6 with 0.3 M hydrochloric acid. The incubation was halted by the addition of 12.5% trichloracetic acid to precipitate undigested BSA and the tyrosine content of the supernatant, an index of digested BSA, was measured using Folin Ciocalteu phenol reagent (Folin and Ciocalteu, 1927), and spectrophotometric quantification against a water blank at 725 nm (Anson and Mirsky, 1932). This protocol was arrived at after initial experiments to determine the influence of pH on the rate of tyrosine production. In these experiments incubations were set up essentially as already described but using a range of concentrations of hydrochloric acid from 3.0 to 0.003 M to alter the pH of the digest. The distribution of pepsinogen was measured in toads fasted for between 2 and 3 weeks and in toads which had eaten mealworms within the 48 hr prior to the experiment; these animals always had food remaining in the stomach, which was removed prior to homogenization of the gut. The effects of temperature on both stored pepsinogen and the digestive action of pepsin were investigated. Toads, fasted for one week, were kept at 12°C for between 5 and 7 days before assaying for pepsinogen. The digests were incubated at either 12 or 30°C.
Figure 2 illustrates the variation in protein digesting activity of the tissue homogenate over the pH range examined. This pattern was consistent for homogenates from each region. The pH optimum for the reaction was 1.6, obtained by using 0.3 M hydrochloric acid in the incubate. The standard curve generated for the action of porcine pepsin on BSA is illustrated in Fig. 3. This is a composite from seven separate assays. The data fit a linear model of the form y = a + bx. The coefficient of determination, r*, describes the quality of fit of the line to the data. The intercept of the line on they axis indicates the generation of some tyrosine in the absence of pepsin in the incubate; this is likely to be due to residual low molecular weight contaminants in the BSA. The localization and quantification of pepsinogen in both fasting and fed toads is shown in Table 1. The main site of pepsinogen storage was the proximal two-thirds of the stomach. There was some pepsinogen in the distal oesophagus and trace amounts in other regions. In the two proximal stomach segments there was significantly more pepsinogen in the homogenates obtained from fasted toads. Table 2 shows the results of experiments to investigate the effects of temperature. The homogenates incubated at 12°C showed little digestive activity, whereas those incubated at 30°C did show proteolytic activity, but less than that demonstrated in homogenates from fasted toads kept at 30°C.
DISCUSSION
The pH optimum for the conversion of pepsinogen to pepsin and the pepsin-catalysed proteolysis of BSA was consistent for homogenates from all segments of the upper gastrointestinal tract. This supports the notion that we were dealing with a substance whose physiologically significant activity is restricted to the stomach stimulated to secrete hydrochloric acid. Our previous studies of gastric pH in Bufo marinus have shown that after a feeding stimulus the pH of the
a = 0.61 b = O-0316 r2=0 82
30
40
5.0
6.0
7.0
PH 3-w
Imo
030
0.6
0.03
M
0033
0
HCI
Fig. 2. Graph illustrating the pH dependence of the proteolytic action of Eufo marinus gastrointestinal tissue homogenates on bovine serum albumin (Sigma A7906). Values are given as mean + standard deviation. N = 4.
”
io
4b
60 lb0
Ii0
260
Peprm (pglmll
Fig. 3. Standard curve for the action of pure porcine pepsin (Sigma P6887) on bovine serum albumin (Sigma A7906) to yield tyrosine. Composite of seven assays.
Pepsin in the toad Bufo marinus Table
I. Pepsin activity
present
in homogenates
of segments
of the upper gastrointestinal
tract of &/o
mariner. Values from fasting and fed toads are compared Fasted toads (A’ = 6) pepsin activity mg/g tissues xkSD
Gut segment Proximal oesophagus Distal oesophagus Fore-stomach Mid-stomach Pyloric stomach Intestine ‘Values significantly TValues significantly
0.098 * 0.02 0.667 i 0.3 I I.471 f 0.54* 0.876 k 0.38t 0.195~0.01 0.057 * 0.03 different, different,
P < 0.02 using Student’s P < 0.05 using Student’s
Fed toads (N = 6) pepsin activity mg/g tissue P_+SD __.__ 0.066 + 0.02 0.414+0.17 0.595 k 0.24’ 0.303 -i_0.08t 0. I80 + 0.04 0.047 f 0.01
f-test. r-test.
Table 2. Pepsin activity measured at I2 and 30°C in homogenates of the upper gastrointestinal Bufo marinus acclimated to an ambient temperature of 12°C
tract of fasted
Gut segment
12°C incubation (N = 2) pepsin activity @g/g tissue) X k SD
30°C incubation (IV = 2) pepsin activity (mg/g tissue) IkSD
Proximal oesophagus Distal oesophagus Fore-stomach Mid-stomach Pyloric stomach Intestine
0.031 0 0.060 0.014 0.012 0
0.038 0.267 f 0.06 0.737 f 0.02 0.242 0.133 0.020
gastric lumen falls rapidly from between 7.0 and 8.0 to around 1.5 (Taylor et al., 1985). This lower value is close to the pH optimum found in the present study. Previous estimates of pH optima for pepsin from unnamed frog species are of the same order; 1.88-1.99 (Pjatnitzky, 1931) and 1.5 (Vonk, 1941). Our results show that in Bufo marinus pepsinogen is found in the distal oesophagus in greater than trace amounts but most of the pepsinogen was found in the proximal two-thirds of the stomach. Only trace amounts were found in the proximal oesophagus, pyloric stomach and intestine, and are probably of little significance in the digestive process. Hirschowitz (1957) noted the ubiquitous distribution of pepsin in trace amounts in body tissues and fluids and remarked on the lack of a convincing teleological explanation for its presence. The relative contribution of each gut segment to the total pepsinogen complement remained fairly constant for each animal. Our results are in agreement with those of Langley (1881) and Machen (1935) that the greatest concentrations of pepsinogen are in the more anterior parts of the stomach. Norris (1959), using the staining method of Bowie, found zymogenic cells only in the oesophagus and forestomach of Rana pipiens. Reese et al. (1979) reported the localization of two immunologically distinct pepsinogens (I and II) in the fundus and antrum of stomachs of Rana catesbeiana. Pepsinogen stores were evenly depleted immediately after feeding and gradually replenished. This finding agrees with the observed feeding behaviour of the toads in natural populations, where they visited known feeding sites for 1 or 2 hr and then disappeared for several successive nights, thus presumably feeding only one night out of every three or more (Brattstrom, 1962; Zug and Zug, 1979). Langley (1881) observed depletion of granules which he correlated with assayable pepsin in gastric gland cells of Rana temporaria and Bufo variabilis during digestion. In both species the time taken for complete recovery of granular appearance varied from 24 hr to several days.
Our methods do not permit us to distinguish sites of pure pepsinogen synthesis and release from sites with more than one product. We are therefore unable to confirm or refute the reports of cells in the distal oesophagus secreting purely pepsinogen. However, Jordan (1927) claimed that in Bufo such cells existed and contributed overwhelmingly to the pepsinogen complement in that genus. Our results from Bufi marinus do not confirm this assertion. We attempted to distinguish the effects of temperature on the synthesis and storage of pepsinogen from peptic enzyme activity. Our results indicate that both are reduced at 12°C. Zug and Zug (1979) state that native juveniles and adults of B. marinus have a critical thermal minimum of ltSl2”C. They reported that in their study population the mean daily body temperature was 26.4”C and that it fluctuated less than ambient temperature, probably due to both behavioural and physiological factors. In our department we have not observed spontaneous feeding in toads kept outside in underground tanks, and force-fed animals often had undigested food in their stomachs at sacrifice. Animals maintained at low temperatures do not thrive. However, when kept at 30°C they have fed spontaneously and remained healthy for several years. Reeder (1964) suggests that cessation of enzyme proliferation at low temperatures is likely to be the limiting factor governing feeding behaviour. Herter (1941) stated that in Rana the gastric wall fails to secrete at temperatures below 5-10°C. We have no information about the physiological or pharmacological regulation of pepsinogen in B. mar inus. However, our results show quite clearly that digestive activity is negligible in conditions of neutral pH. It is not necessary, therefore, to postulate an inhibition of pepsinogen secretion in Rheobatrachus to explain the phenomenon of gastric brooding; the inhibition of acid secretion by prostaglandin E, secreted by the developing young (Tyler et al., 1983) would ensure protection from proteolytic digestion by any pepsinogen that might be released.
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