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Carbonic Anhydrase in the Wall of the Forestomachs of Cows
CARBONIC ANHYDRASE IN THE WALL OF THE FORESTOMACHS OF COWS Bv
J.
H.
AAFJES
Laboratory of Animal Physiology, Agricultural University, Wageningen, Holland SUMMARY
(1) The rumen , reticulum and omasum contain in their wall tissues considerable amounts of carbonic anhydrase (about half as much as the abomasum mucosa or blood on a wet weight basis). (2 ) The enzyme is localized in the interior of the rumen villi and not in the epithelium. (3) The enzyme may play a role in an exchange of bicarbonate for volatile fatty acid ions, and the implications of this are discussed.
In a recent publication (Aafjes, I967) the view was put forward that absorption of volatile fatty acid (VF A) ions through the wall of the rumen might be accompanied by an exchange with bicarbonate ions. In connection with this possibility it was thought worthwhile to investigate whether the enzyme carbonic anhydrase which catalyses the reaction C0 2 + H 2 0~H + + HC0 3 is present in tissues of the forestomachs. This proved to be the case. MAT E RIALS AND METHODS
Pieces of the walls of the rumen, reticulum, omasum and abomasum were obtained from cows directly after slaughter and put on ice as soon as possible. Mter transport to the laboratory they were used immediately or stored in a refrigerator at - 20 ° c. For the determinations pieces of epithelium were cut with scissors if the tissues were fresh and with a knife if they were frozen. Villi were taken from the rumen wall. From the reticulum and omasum pieces of the epithelial ridges were cut and from the abomasum the mucosa could easily be torn apart from the submucosa. Then the slices were blotted dry with filter paper, weighed and ground with an ultra-turrax (] anke & Kunkel, Stanten) in a measured amount of peptone-water ( II ·8 mg. peptone in IOO mi. doubledistilled water). After decantation or centrifugation and eventual dilution, these solutions were used with the CO 2 - V eronal indicator method of Roughton and Booth (I 946). An indication of the amount of blood in the different tissue suspensions was obtained with Hemastix (Ames Company). The discoloration of these was compared with those ofHemastix dipped in known dilutions offreshly obtained cow blood in distilled water.
CARBONIC ANHYDRASE IN FORESTOMACHS OF COWS
253
RESULTS
In the C0 2- Veronal indicator method of Roughton and Booth a certain amount of water saturated with C0 2 is mixed with a Veronal buffer of pH 8. The time needed to lower this pH from 8 to 6·4 is measured. In our determinations it took about go seconds when water without enzyme was used; however if enzyme is present the time is proportionally less. Table I gives some typical results. TABLE I TIME NEEDED FOR A FALL FROM PH 8 TO 6·4 IN A REACTION MIXTURE CONTAINING 5 ML. C0 2 SATURATED WATER, 3 ML. VERONAL BUFFER (o·022M ), 0·3 ML. TISSUE SUSPENSION, 2 ML. WATER AND 0·05 ML. BROMOTHYMOL BLUE SOLUTION (0·2% IN 50% ETHANOL) AT o 0 c
Solution tested contained Blank Reticulum Rumen Omasum Abomasum Reticulum Rumen Omasum Abomasum R eticulum Rumen Omasum Abomasum Blood Blood
Mg. of tissue or blood in 10·3 ml. reaction mixture J6•7 !6•7 16·7 J6•7 4"2 4"2 4"2 2•1 2•1 2"1 2"1 1•3 0"3
Time in seconds 90·8, !6·6, 7"5, 9"1, 1"5 44"1, 18·0, 2 5 ·9, 17"4, 54"3, 33"3, 40"7, 33 "7, 31"3, 66·7,
89·0 17•8 6·s 8·8 47"5 18·6 26·s 17"5 61•7 32"2 43"3 33"0 29"0 66·7
From this table it is clear that the forestomach tissues all accelerated the reaction considerably. This acceleration appeared to be as much as half that produced by the stomach mucosa, a tissue known to contain high amounts of carbonic anhydrase (Davenport, I 946). Blood also contains a large amount of enzyme in the erythrocytes. The table shows, however, that the acceleration of the reaction by the tissues of the forestomachs cannot be caused by the amount of blood within them, since the difference between the amounts of tissue and blood needed to give the same acceleration is much too small for this. Moreover, testing with Hemastix showed that blood had to be diluted about I oo times more than tissue suspensions to give the same colour reaction. Mixing the tissue suspensions with blood solutions showed that the Hemastix reaction was not disturbed by the presence of tissue. In other words, the tissue suspensions contained only a small percentage of blood, much too little to explain the observed acceleration. These results led to the conclusion that the forestomachs of cows contain a considerable amount of carbonic anhydrase. This was confirmed by the finding that boiling the tissue suspensions destroyed their activity and by the fact that the acceleration of the reaction by these suspensions could be abolished completely by a solution of acetazoleamide (Diamox, Lederle), a carbonic anhydrase inhibitor, at a concentration of 2 mg. /1. in the reaction mixture.
254
BRITISH VETERINARY JOURNAL, 123, 6
In connection with these findings it was thought of importance to investigate where the enzyme is localized. It is known, for example, that in stomach mucosa carbonic anhydrase is found in high concentration in the parietal cells. To this end the epithelium of several rumen villi was scraped off with a sharp knife and these epithelial scraps and the remaining stroma were processed separately. Microscopic examination of villi denuded at one side showed that in this way a reasonable separation could be achieved. Suspensions obtained in this way gave the results shown in Table II. TABLE II TIME NEEDED FOR A FALL FROM PH 8 TO 6·4 IN A REACTION MIXTURE CONTAINING 5 JilL. C0 2 SATURATED WATER, 3 ML. VERONAL BUFFER ( o·022M), 0'3 ML. TISSUE SUSPENSION, 2 JilL, WATER AND 0'05 ML. BROMOTHYMOL BLUE SOLUTION (0•2% IN 50% ETHANOL) AT 0°C.
Solution tested · contained
1 0 ·3
Mg. of tissue in ml. reaction mixture
Blank Whole villi Whole villi Epithelium Stroma Abomasum
Time in seconds
1'7 1'7
9J·6, 93'9 29'5• 29'0 49·6, 47'5 77'1, 78•J
J•7
36·5, 37·9
I
37'9• 38'3
3'3
From these results the conclusion is drawn that the enzyme is localized in the interior of the villi and not in the epithelium. The slight positive reaction of the epithelium is probably caused by contamination with some stromal tissue resulting from the crude method of separation. DISCUSSION
Although the literature about carbonic anhydrase is extensive, not much could be found about the presence of this enzyme in tissues of the forestomachs. Castella Bertran (I 952) gives results obtained with different tissues of cows, sheep, horses and pigs. In the rumen of ~ows considerable activity of the enzyme was found. It is, however, impossible to compare these results with those described in this paper, since a different method of estimation was used. Furthermore, Oyaert (I955) mentions briefly that the omasum contains twice as much enzyme as the rumen wall tissue, compared on a nitrogen basis, and remarks that the functional significance (in C0 2 absorption) is not known. The present results show that the forestomach tissue contains, compared with stomach mucosa and blood, a large amount of carbonic anhydrase, and it is thought that the enzyme may play a role in the absorption ofVFA, as shown in Fig. I. It is known that the C0 2 pressure in the rumen is very high; 50 per cent or more of rumen gas is C0 2 • Therefore C0 2 will diffuse out of the rumen into the tissues of the wall and from there it will be transported to the lungs for expiration. If, however, part of this C0 2 could be recycled into the rumen in exchange for VFA, the buffering capacity of this organ would be increased.
CARBONIC ANHYDRASE IN FORESTOMACHS OF COWS
255
Another consequence of this exchange would be that the concentration of bicarbonate present in the rumen could have an influence on the absorption rate of VFA. A high concentration of bicarbonate would inhibit the exchange and consequently the VFA absorption, and conversely, if there is a low bicarbonate concentration in the rumen, VFA absorption would be increased. The latter situation prevails of course when there is rapid fermentation with a high VFA production. In other words this system could be a feedback regulator for VFA absorption. The consequence would be that with a low VFA production in the rumen the disappearance of these acids would be prevented. On teleological grounds this mechanism would seem profitable, as the VFA are of importance as growth factors for several bacterial species in the rumen (Hungate, 1 g66).
Vi II us
I---t-Carbonic anhydrase
Rumen
Fig.
1.
Diagram illustrating the
po~sible
role of carbonic anhydrase in the absorption of VFA from the rumen .
ACKNOWLEDGEMENT
I should like to thank Miss C. P. ]. Oudenaarden for her very able assistance. REFERENCES
AAFJES, J. H. ( 1967). ,Z. Tierphysiol. Tiererniihr. Futtermittelk., 22, 69 (in English). CAsTELLA BERTRAN, E. (1952) . An. Fac. Vet. Univ. Madr., 4, 351. DAVENPORT, H. w. (1946) . Physiol. Rev., 26, 560. HuNGATE, R. E. (1966). The Rumen and its Microbes, p. 88. New York and London: Academic Press. 0YAERT, W. (1955). Thesis, Gent, p. 73· RouGHTON, F. J. W. & BoOTH, V . H. (1946). Biochem. ] ., 40, 319. (Acceptedfor publication 2nd March 1967)
BRITISH VETERINARY JOURNAL, 123, 6
La teneur en anhydrase carbonique dans les parois de Ia panse, du reticul1UD et du feuillet chez Ia vache (Aafjes) Res1UDe. La panse, le reticulum et le feuillet renferment dans les tissue parietaux des quantites considerables d'anhydrase carbonique, presque la moitie de la quantite contenue dans la muqueuse de Ia caillette or dans le sang compare a base des poids humides. L'enzyme se trouve dans l'interieur des papilles, pas dans !'epithelium. L'enzyme peut jouer un role dans l'echange de bicarbonate, contre les ions d'acides gras volatiles, dont les implications sont discutees. Karbonische Anhydrase in der Vonnagenwand von Kiihen (Aafjes) Zusanunenfassung. Die Gewebe der Pansen- Retikulum- und Omasumwiinde enthalten eine wichtige karbonische Anhydraseaktivitiit, und zwar ungefahr halb so vie! wie das Labmagenmucosa oder Blut (Gehalt im nassen Gewebe gerechnet). Das Ferment ist im Inneren der Panzenzotten und nicht im Epithelium lokalisiert. Es kann eine Rolle bei dem Ionenaustausch von Bikarbonat und flilchtiger Fettsiiure spielen, und dieser Aspekt wurder diskutiert.
La anhidrasis carbonica en el tejido parietal de los pre-estomacos de Ia vaca (Aafjes) Res1UDen. El rfunen, el reticulum, y el omasum contienen en el tejido parietal cantidades considerables de anhidrase carbonica, cerca de una midad mas, en peso humido, que Ia mucosa abomasal o Ia sangre. La enzima es localizada en el interior de las vilositades ruminales, y no en el epitelio. La enzima puede tener un papel en el cambio del bicarbonato contra los iones de los acidos volatiles grasos, se discuten las implicaciones.
J.
H.
AAFJES
Laboratory of Animal Physiology, Agricultural University, Wageningen, Holland SUMMARY
(1) The rumen , reticulum and omasum contain in their wall tissues considerable amounts of carbonic anhydrase (about half as much as the abomasum mucosa or blood on a wet weight basis). (2 ) The enzyme is localized in the interior of the rumen villi and not in the epithelium. (3) The enzyme may play a role in an exchange of bicarbonate for volatile fatty acid ions, and the implications of this are discussed.
In a recent publication (Aafjes, I967) the view was put forward that absorption of volatile fatty acid (VF A) ions through the wall of the rumen might be accompanied by an exchange with bicarbonate ions. In connection with this possibility it was thought worthwhile to investigate whether the enzyme carbonic anhydrase which catalyses the reaction C0 2 + H 2 0~H + + HC0 3 is present in tissues of the forestomachs. This proved to be the case. MAT E RIALS AND METHODS
Pieces of the walls of the rumen, reticulum, omasum and abomasum were obtained from cows directly after slaughter and put on ice as soon as possible. Mter transport to the laboratory they were used immediately or stored in a refrigerator at - 20 ° c. For the determinations pieces of epithelium were cut with scissors if the tissues were fresh and with a knife if they were frozen. Villi were taken from the rumen wall. From the reticulum and omasum pieces of the epithelial ridges were cut and from the abomasum the mucosa could easily be torn apart from the submucosa. Then the slices were blotted dry with filter paper, weighed and ground with an ultra-turrax (] anke & Kunkel, Stanten) in a measured amount of peptone-water ( II ·8 mg. peptone in IOO mi. doubledistilled water). After decantation or centrifugation and eventual dilution, these solutions were used with the CO 2 - V eronal indicator method of Roughton and Booth (I 946). An indication of the amount of blood in the different tissue suspensions was obtained with Hemastix (Ames Company). The discoloration of these was compared with those ofHemastix dipped in known dilutions offreshly obtained cow blood in distilled water.
CARBONIC ANHYDRASE IN FORESTOMACHS OF COWS
253
RESULTS
In the C0 2- Veronal indicator method of Roughton and Booth a certain amount of water saturated with C0 2 is mixed with a Veronal buffer of pH 8. The time needed to lower this pH from 8 to 6·4 is measured. In our determinations it took about go seconds when water without enzyme was used; however if enzyme is present the time is proportionally less. Table I gives some typical results. TABLE I TIME NEEDED FOR A FALL FROM PH 8 TO 6·4 IN A REACTION MIXTURE CONTAINING 5 ML. C0 2 SATURATED WATER, 3 ML. VERONAL BUFFER (o·022M ), 0·3 ML. TISSUE SUSPENSION, 2 ML. WATER AND 0·05 ML. BROMOTHYMOL BLUE SOLUTION (0·2% IN 50% ETHANOL) AT o 0 c
Solution tested contained Blank Reticulum Rumen Omasum Abomasum Reticulum Rumen Omasum Abomasum R eticulum Rumen Omasum Abomasum Blood Blood
Mg. of tissue or blood in 10·3 ml. reaction mixture J6•7 !6•7 16·7 J6•7 4"2 4"2 4"2 2•1 2•1 2"1 2"1 1•3 0"3
Time in seconds 90·8, !6·6, 7"5, 9"1, 1"5 44"1, 18·0, 2 5 ·9, 17"4, 54"3, 33"3, 40"7, 33 "7, 31"3, 66·7,
89·0 17•8 6·s 8·8 47"5 18·6 26·s 17"5 61•7 32"2 43"3 33"0 29"0 66·7
From this table it is clear that the forestomach tissues all accelerated the reaction considerably. This acceleration appeared to be as much as half that produced by the stomach mucosa, a tissue known to contain high amounts of carbonic anhydrase (Davenport, I 946). Blood also contains a large amount of enzyme in the erythrocytes. The table shows, however, that the acceleration of the reaction by the tissues of the forestomachs cannot be caused by the amount of blood within them, since the difference between the amounts of tissue and blood needed to give the same acceleration is much too small for this. Moreover, testing with Hemastix showed that blood had to be diluted about I oo times more than tissue suspensions to give the same colour reaction. Mixing the tissue suspensions with blood solutions showed that the Hemastix reaction was not disturbed by the presence of tissue. In other words, the tissue suspensions contained only a small percentage of blood, much too little to explain the observed acceleration. These results led to the conclusion that the forestomachs of cows contain a considerable amount of carbonic anhydrase. This was confirmed by the finding that boiling the tissue suspensions destroyed their activity and by the fact that the acceleration of the reaction by these suspensions could be abolished completely by a solution of acetazoleamide (Diamox, Lederle), a carbonic anhydrase inhibitor, at a concentration of 2 mg. /1. in the reaction mixture.
254
BRITISH VETERINARY JOURNAL, 123, 6
In connection with these findings it was thought of importance to investigate where the enzyme is localized. It is known, for example, that in stomach mucosa carbonic anhydrase is found in high concentration in the parietal cells. To this end the epithelium of several rumen villi was scraped off with a sharp knife and these epithelial scraps and the remaining stroma were processed separately. Microscopic examination of villi denuded at one side showed that in this way a reasonable separation could be achieved. Suspensions obtained in this way gave the results shown in Table II. TABLE II TIME NEEDED FOR A FALL FROM PH 8 TO 6·4 IN A REACTION MIXTURE CONTAINING 5 JilL. C0 2 SATURATED WATER, 3 ML. VERONAL BUFFER ( o·022M), 0'3 ML. TISSUE SUSPENSION, 2 JilL, WATER AND 0'05 ML. BROMOTHYMOL BLUE SOLUTION (0•2% IN 50% ETHANOL) AT 0°C.
Solution tested · contained
1 0 ·3
Mg. of tissue in ml. reaction mixture
Blank Whole villi Whole villi Epithelium Stroma Abomasum
Time in seconds
1'7 1'7
9J·6, 93'9 29'5• 29'0 49·6, 47'5 77'1, 78•J
J•7
36·5, 37·9
I
37'9• 38'3
3'3
From these results the conclusion is drawn that the enzyme is localized in the interior of the villi and not in the epithelium. The slight positive reaction of the epithelium is probably caused by contamination with some stromal tissue resulting from the crude method of separation. DISCUSSION
Although the literature about carbonic anhydrase is extensive, not much could be found about the presence of this enzyme in tissues of the forestomachs. Castella Bertran (I 952) gives results obtained with different tissues of cows, sheep, horses and pigs. In the rumen of ~ows considerable activity of the enzyme was found. It is, however, impossible to compare these results with those described in this paper, since a different method of estimation was used. Furthermore, Oyaert (I955) mentions briefly that the omasum contains twice as much enzyme as the rumen wall tissue, compared on a nitrogen basis, and remarks that the functional significance (in C0 2 absorption) is not known. The present results show that the forestomach tissue contains, compared with stomach mucosa and blood, a large amount of carbonic anhydrase, and it is thought that the enzyme may play a role in the absorption ofVFA, as shown in Fig. I. It is known that the C0 2 pressure in the rumen is very high; 50 per cent or more of rumen gas is C0 2 • Therefore C0 2 will diffuse out of the rumen into the tissues of the wall and from there it will be transported to the lungs for expiration. If, however, part of this C0 2 could be recycled into the rumen in exchange for VFA, the buffering capacity of this organ would be increased.
CARBONIC ANHYDRASE IN FORESTOMACHS OF COWS
255
Another consequence of this exchange would be that the concentration of bicarbonate present in the rumen could have an influence on the absorption rate of VFA. A high concentration of bicarbonate would inhibit the exchange and consequently the VFA absorption, and conversely, if there is a low bicarbonate concentration in the rumen, VFA absorption would be increased. The latter situation prevails of course when there is rapid fermentation with a high VFA production. In other words this system could be a feedback regulator for VFA absorption. The consequence would be that with a low VFA production in the rumen the disappearance of these acids would be prevented. On teleological grounds this mechanism would seem profitable, as the VFA are of importance as growth factors for several bacterial species in the rumen (Hungate, 1 g66).
Vi II us
I---t-Carbonic anhydrase
Rumen
Fig.
1.
Diagram illustrating the
po~sible
role of carbonic anhydrase in the absorption of VFA from the rumen .
ACKNOWLEDGEMENT
I should like to thank Miss C. P. ]. Oudenaarden for her very able assistance. REFERENCES
AAFJES, J. H. ( 1967). ,Z. Tierphysiol. Tiererniihr. Futtermittelk., 22, 69 (in English). CAsTELLA BERTRAN, E. (1952) . An. Fac. Vet. Univ. Madr., 4, 351. DAVENPORT, H. w. (1946) . Physiol. Rev., 26, 560. HuNGATE, R. E. (1966). The Rumen and its Microbes, p. 88. New York and London: Academic Press. 0YAERT, W. (1955). Thesis, Gent, p. 73· RouGHTON, F. J. W. & BoOTH, V . H. (1946). Biochem. ] ., 40, 319. (Acceptedfor publication 2nd March 1967)
BRITISH VETERINARY JOURNAL, 123, 6
La teneur en anhydrase carbonique dans les parois de Ia panse, du reticul1UD et du feuillet chez Ia vache (Aafjes) Res1UDe. La panse, le reticulum et le feuillet renferment dans les tissue parietaux des quantites considerables d'anhydrase carbonique, presque la moitie de la quantite contenue dans la muqueuse de Ia caillette or dans le sang compare a base des poids humides. L'enzyme se trouve dans l'interieur des papilles, pas dans !'epithelium. L'enzyme peut jouer un role dans l'echange de bicarbonate, contre les ions d'acides gras volatiles, dont les implications sont discutees. Karbonische Anhydrase in der Vonnagenwand von Kiihen (Aafjes) Zusanunenfassung. Die Gewebe der Pansen- Retikulum- und Omasumwiinde enthalten eine wichtige karbonische Anhydraseaktivitiit, und zwar ungefahr halb so vie! wie das Labmagenmucosa oder Blut (Gehalt im nassen Gewebe gerechnet). Das Ferment ist im Inneren der Panzenzotten und nicht im Epithelium lokalisiert. Es kann eine Rolle bei dem Ionenaustausch von Bikarbonat und flilchtiger Fettsiiure spielen, und dieser Aspekt wurder diskutiert.
La anhidrasis carbonica en el tejido parietal de los pre-estomacos de Ia vaca (Aafjes) Res1UDen. El rfunen, el reticulum, y el omasum contienen en el tejido parietal cantidades considerables de anhidrase carbonica, cerca de una midad mas, en peso humido, que Ia mucosa abomasal o Ia sangre. La enzima es localizada en el interior de las vilositades ruminales, y no en el epitelio. La enzima puede tener un papel en el cambio del bicarbonato contra los iones de los acidos volatiles grasos, se discuten las implicaciones.