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Effect of dehydration, food deprivation, saline and adrenalectomy on microsomal (Na++K+)-dependent ATPase in the salivary glands and intestinal mucosa
Biochimica et Biophysica Acta, 304 (1973) 533-540
Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands BBA 27092
E F F E C T OF D E H Y D R A T I O N , F O O D DEPRIVATION, SALINE A N D ADREN A L E C T O M Y ON MICROSOMAL (Na + q-K + ) - D E P E N D E N T ATPase IN T H E SALIVARY G L A N D S A N D I N T E S T I N A L MUCOSA
YEHUDA GUTMAN and DORITH GLUSHEVITZKY-STRACHMAN Department ok/"Pharmacolooy, The Hebrew University-Hadassah Medical School, Jerusalem (Israel)
(Received December 6th, 1972)
SUMMARY 1. The effect of water deprivation, food deprivation, saline and adrenalectomy was studied on microsomal ATPase activity of the rat parotid and submaxillary glands and the mucosa of the small intestine and of the colon. 2. Water deprivation caused a decrease of microsomal (Na+ +K+)-dependent ATPase activity in the submaxillary gland. 3. Food deprivation resulted in a selective reduction of microsomal (Na ÷ + K ÷)-dependent ATPase activity from the parotid gland. Mg 2 +-ATPase activity was enhanced in the small intestine and depressed in the large intestine. 4. When 0.9 % NaCI was used as drinking fluid (Na + + K ÷)-dependent ATPase was augmented in the parotid and reduced in the mueosa of the small and large intestine. 5. No decrease in ( N a + + K + ) - d e p e n d e n t ATPase activity was found after adrenalectomy in either salivary gland or in the mucosa of the small intestine or colon.
INTRODUCTION Microsomal ( N a + + K + ) - d e p e n d e n t ATPase (ATP phosphohydrolase EC 3.6.1.3) has been reported to be present in many tissues 1. Its function is associated with active sodium transport 2. Although the characteristics as an enzyme have been widely investigated, relatively few studies about the factors which regulate this enzymatic activity have been reported. Most of these studies concern the kidney, which is an organ where sodium transport plays a major role both in sodium balance and in the mechanism of urine concentration. Adrenalectomy, mineralocorticoids and Na + intake are known to affect kidney microsomal ATPase 3, 4. We have previously reported on the differential effect of water, food and saline on the (Na + + K +)-dependent ATPase in the kidney cortex as compared to the medulla 5. It seemed o f interest and importance to study whether the various factors
534
Y. GUTMAN, D. GLUSHEVITZKY-STRACHMAN
which affect kidney ATPase have a similar action on this enzyme in other tissues, particularly in epithelia where active sodium transport plays a major role in the physiological function. Mineralocorticoids are known to affect sodium transport not only in the kidney but also in the sweat glands, salivary glands and the colon% The intestines are daily exposed to large volumes of fluid which is almost completely absorbed, mainly through active sodium transport 7. Any reduction of this absorption may result in a severe loss of fluid (diarrhea). The salivary glands secrete into the acini a fluid very similar to plasma in ionic composition, but the final saliva is very poor in N a + and is hypotonic 8. Therefore, active Na + reabsorption must take place along the path of the saliva flow in the salivary glands. We, therefore, studied microsomal ATPase activities in different parts of the gastrointestinal tract and in different salivary glands under various stimuli. The results are presented in the present report. MATERIALS AND METHODS
Animals Male rats of the Hebrew University strain weighing 200-300 g, were used throughout.
Preparation of enzyme The submaxillary and parotid glands were removed immediately following decapitation. The small intestine, from duodenum to the distal end of the ileum, was cut at both ends. The large intestine (colon), both transverse and descending, was cut at the caecum and at the rectum. The intestines were flushed with ice-cold saline and were then cut open longitudinally and spread on filter-paper to expose the mucosa. With the aid of a scalpel the mucosal layer was scraped and collected. The various tissues were homogenized in an all glass homogenizer with 10 vol. of ice-cold 0.25 M sucrose containing 2 m M EDTA. The medium for homogenization of the salivary glands also contained 0 . 1 % deoxycholate. After centrifugation at 1000 × 9 for 10 min at 4 °C the supernatant was separated and spun at 10 000 ~: y for another 10 min. The supernatant was separated and centrifuged at 100 0 0 0 - 9 for 30 min and the microsomal pellet obtained was resuspended by gentle homogenization in 0.25 M sucrose. This microsomal preparation constituted the enzyme suspension. The enzyme suspension was either kept frozen ( - 2 0 °C) until assayed or assayed immediately; when kept frozen both the controls and the experimental samples underwent the same procedure.
Incubation for assay of A TPase activity The ATPase activity was measured by incubating the enzyme in the appropriate medium as previously described 9, for 10-20 min at 37 °C, in a thermostatic bath with constant shaking. When all the components for incubation were added except ATP, the flasks were placed in the thermostatic bath for preincubation at 37 °C for 5 min. ATP was th.en added and the incubation period started. Blanks containing no enzyme or where trichloroacetic acid was added before ATP were run with each incubation. The enzymatic reaction was stopped by adding 3.0 ml of 5 % trichloroacetic acid. After centrifugation and filtration an aliquot of 0.1 ml was taken for determination of the inorganic phosphate liberated.
SALIVARYAND INTESTINALATPase
535
Medium for incubation The incubation medium consisted of the following components (final concentrations): 100 mM NaCI; 10 mM KC1; 4 mM MgCI2; 4 mM ATP; 33 mM Tris buffer (pH 7.5) and 0.14).2ml of enzyme suspension (containing 0.14),5 mg of protein). The final volume of incubation was 3.0 ml. Inorganic phosphate liberated during incubation in this medium represented total microsomal ATPase activity. For determination of (Na + + K + )-dependent ATPase, the NaCI and KCI were replaced by an equivalent amount of Tris buffer; ~lternatively, incubation was carried out in the complete medium but ouabain was added to a final concentration of 10 - 3 M. Preliminary experiments have shown that this concentrations of ouabain reduced ATPase activity to that observed in the absence of Na ÷ and K ÷. The difference between the activity in the complete medium and that in the presence of ouabain (or absence of Na + and K ÷ ) represents (Na ÷ ÷ K ÷)-dependent ATPase activity while the residual activity is Mg z+-ATPase. The inorganic phosphate was measured according to the method of Baginski and Zak 1o. The protein in the enzyme suspension was assayed according to Lowry et aL x~. Experiments with animals Water deprivation was carried out for 4 days while both control and experimental rats had food ad libitum. Food deprivation was imposed for 4 days while both experimental and control animals had free access to water. Na ÷ loading was performed by offering 0.9 % NaCI as drinking fluid for 12 days. Adrenalectomy was performed under ether anesthesia, through a midline incision in the back: The controls for adrenalectomy were sham-operated rats, in which a midline incision was made in the abdomen under ether anesthesia. The rats were kept for 7 days after the operation on food and water adlibitum and were then sacrificed and the various tissues were removed. The experiment on saline loading was repeated twice and on adrenalectomy on three different occasions with consistent results. The data are therefore means of the different experiments. Some variation between control values in the various experiments may be due to seasonal changes in enzymatic activity, however, this point has not been investigated. RESULTS Table I shows the effect of the various treatments on the weight of the rats and that of the submaxillary and parotid glands. A prominent difference can be observed between the effects on the submaxillary gland and on the parotid glands: The submaxillary glands shrank considerably more under water deprivation and adrenalectomy while the parotid gland shrank under food deprivation and water deprivation but much less after adrenalectomy.
Water deprivation Water deprivation had a selective and limited effect on microsomal ATPase activity. Microsomal ATPase activity in the parotid was unchanged while in the submaxillary gland there was a significant decrease in (Na + + K +)-dependent ATPase activity, reflected also in total microsomal ATPase with no significant change in
536
Y. G U T M A N , D. G L U S H E V I T Z K Y - S T R A C H M A N
TABLE I E F F E C T O F V A R I O U S T R E A T M E N T S ON W E I G H T O F RAT A N D S A L I V A R Y G L A N D S N u m b e r of animals: water deprivation, 7; food deprivation, 8; saline, 8; adrenalectomy, 5.
Control
°/o change of wei#ht after Water deprivation
Rat (g) Submaxillary gland (mg) Parotid gland (rag)
235i6 204±4 152±6
37.8 39.5 33.7
Food deprivation -31.5 23.1 33.5
Saline (%)
Adrenalectom.v (o/)
- 1.5 ~ 11.4 4 14.8
15.8 33.2 18.0
Mg2+-ATPase activity (Fig. 1). Therefore, (Na+÷K+)-dependent ATPase in the submaxillary gland decreased from 69.2±1.7 ~o of total microsomal ATPase in the controls to 60.9~-1.5 ~o under water deprivation. Since the weight of the salivary glands decreased under water deprivation the total activity per gland was even further reduced. Thus the total ( N a + + K ÷ )-ATPase activity per gland was reduced by 30 o~, in the parotid and by 53 ?,'oin the submaxillary gland. L@
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Fig. I. Effect of water deprivation on microsomal ATPase activity in the rat salivary glands. Hatched column controls; open columns, water deprivation. Vertical bars, S.E. T, total microsomal ATPase activity; Mg, Mg 2 +-ATPase activity; Na, K, (Na + -~-K +)-dependent ATPase activity. Submaxillary gland: controls n -:~ 7; water deprivation n -- 7. Parotid gland: controls n -: 8; water deprivation n 7. * P < 0.05, ** P < 0.02 for the difference between control and water deprivation. Fig. 2. Effect of food deprivation on microsomal ATPase activity. Symbols as in Fig. I. Lower panel: left, parotid gland (controls n = 7; food deprivation n ~ 8); right, submaxillary gland icontrois n -- 7; food deprivation n = 8). Upper panel: left, small intestine (controls n ~ 8; food deprivation n - 7); right, large intestine (controls n ~ 4; food deprivation n 4). ** P < 0.02; • ** P < 0.005.
S A L I V A R Y A N D I N T E S T I N A L ATPase
537
The ATPase activity in the mucosa of the intestine was unchanged after water deprivation except for a slight increase of Mg z÷-ATPase in the small intestine (from 13.44-1.0 to 16.24-0.5 #moles Pi/mg protein per h, n = 7, P < 0.05).
Food deprivation Food deprivation caused a reduction of microsomal (Na + +K+)-dependent ATPase activity in both salivary glands (Fig. 2) but the reduction was statistically significant only in the parotid, and was due mainly to a fall of (Na + + K + )-dependent ATPase activity. No significant change in Mg2+-ATPase was found. Hence, ( N a + + K+ )-dependent ATPase in the parotid decreased from 69.0-t-1.3 % of total microsomal ATPase in the controls to 57.3+4.5 % of the total under food deprivation. Since the weight of the salivary glands was reduced after food deprivation the change of ATPase activity per gland was even more pronounced. Thus (Na + + K +)-ATPase per gland was reduced by 60 % in the parotid and by 38 % in the submaxillary. In the intestinal mucosa food deprivation caused augmentation of Mg 2+ATPase activity in the small intestine and decreased Mg2+-ATPase activity in the colon. No significant change in (Na+ + K + )-dependent ATPase was found in the intestine (Fig. 2). (Na + + K + )-dependent ATPase in the small intestine decreased therefore from 54.34-2.2 % of total microsomal ATPase to 45.2±2.8 % under food deprivation.
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Fig. 3. Effect o f saline on microsomal ATPase activity. Symbols as in Fig. 1. Lower panel: left, parotid gland (controls and saline n = 8); right, submaxillary gland (controls and saline n = 8). U p p e r panel: left, small intestine (controls n = 14; saline n = 25); right, large intestine (controls n : 7; saline n : 14). * P < 0.05; ~rkp < 0.02; * * * P < 0.005. Fig. 4. Effect o f adrenalectomy on microsomal ATPase activity. Symbols as in Fig. 1. Lower panel: left, parotid gland (controls n = 5; adrenalectomy n : 4); right, submaxillary gland (controls and adrenalectomy n = 5). U p p e r panel: left, small intestine (controls n = 21, adrenalectomy n : 26); right, large intestine (controls n : 11, adrenalectomy n : 13). * P < 0.05; ** P < 0.02; *** P < 0.005.
538
Y. GUTMAN, D. GLUSHEVITZKY-STRACHMAN
Saline Fig. 3 shows the effect of saline administration on microsomal ATPase activity. In the parotid gland (Na+-}-K+)-dependent ATPase activity was elevated while no significant change was observed in the submaxillary gland. However, since the weight of the salivary glands increased somewhat during saline intake there was also some elevation of total microsomal ATPase activity per gland. Thus, ( N a + ÷ K +)ATPase per gland increased by 26 ~ in the submaxillary and by 52 ',~Join the parotid. The intestinal mucosa responded uniformly to saline administration: Selective depression of (Na+--K÷)-dependent ATPase in both small intestine and colon with no significant change in Mg 2 +-ATPase. Adrenalectomy Fig. 4 shows the effect of adrenalectomy on microsomal ATPase activity in the tissues studied. In general, adrenalectomy caused increased microsomal ATPase activity. However, in the small intestine the increase was not statistically significant. Microsomal (Na ÷ ÷K+)-dependent ATPase activity was augmented in the mucosa of the colon and in the submaxillary gland. Microsomal Mg 2+-ATPase activity was increased in the parotid and submaxillary glands (Fig. 4). Due to a decrease in the weight of the salivary glands following adrenalectomy the total activity per gland of both microsomal Mg 2 +-ATPase and ( N a ÷ + K ÷)-ATPase in the submaxillary gland was equal to that of the controls. In the parotid gland the total activity per gland of Mg2+-ATPase was increased by 30°~, and the total (Na++K+)-ATPase was unchanged. DISCUSSION
(Na + ÷ K +)-dependent ATPase is considered as part of the sodium pump in cells of different tissues l,z. This enzyme, therefore, supports sodium transport in a variety of tissues and especially in epithelial layers where sodium transport is an important physiological function of the organ, such as the kidney 12, the intestine' 3 the salivary glands 14, and the avian salt gland 15. The activity of kidney (Na+÷K+)-dependent ATPase is known to be decreased following adrenalectomy 3,4. Furthermore, we have previously reported specific changes in the activity of this enzyme in either the kidney cortex or medulla, according to the type of stimulus applied (saline, water deprivation or food deprivation) 5. It seemed of interest, therefore, to see whether (Na++K+)-dependent ATPase activity in the other sodium-transporting epithelia showed the same type of response. The experiments reported here demonstrate that different, and sometimes opposite, effects of the same agent, e.9. saline administration, were induced in microsomal ATPase activity in different tissues (see Fig. 3). This is also illustrated by the opposite effect of adrenalectomy on kidney microsomal ATPase 3,4 on the one hand and on this enzyme in the mucosa of the colon and the submaxillary gland, on the other hand (Fig. 4). Another illustration of such a divergence was observed by Epstein et al.16 upon adaptation of a teleost to fresh water: a decrease in ( N a + + K +)-dependent ATPase ensued in the gills while an elevation of this enzyme was noticed in the kidney. Thus, it seems that one can rule out the possibility of a single or a common
S'ALIVARY AND INTESTINAL ATPase
539
"regulator" of (Na ÷ +K+)-dependent ATPase in the various sodium-transporting epithelia. An interesting selectivity was observed between the two major salivary glands, the parotid and the submaxillary. We have previously reported that thirst, induced by hypovolemia or water deprivation, was selectively reduced after ablation of the submaxillary gland of the rat but not after ablation of the parotid 17. In the present report only the submaxillary gland microsomal ATPase was affected by water deprivation, Le. microsomal (Na ÷ +K÷)-dependent ATPase activity was reduced, while no significant effect was observed in the parotid gland (Fig. 1). Table I also shows this functional differentiation of the two salivary glands in the loss of weight: under food deprivation the parotid lost more weight than the submaxillary while under water deprivation the submaxillary lost more weight than the parotid. Adrenalectomy, which is accompanied by dehydration, also caused a larger effect on the submaxillary than on the parotid gland (Fig. 4). An opposite selectivity was observed following food deprivation: (Na ÷ ÷ K ÷ )dependent ATPase activity was significantly depressed only in the parotid and not in the submaxillary gland (Fig. 2). It is of interest that in the kidney, too, water and food deprivation had selective effects: water deprivation reduced (Na++K+)-dependent ATPase in the cortex but not in the medulla, while food deprivation reduced this enzymatic activity in the medulla but not in the cortex of the kidney s . The present report would suggest that the parotid gland is more responsive to changes in food intake while the submaxillary gland is more affected by changes in water intake than in food intake (Figs 1 and 2 and Table I). Fasting had also an opposite effect on the small intestine as compared to the colon: art increase of Mg 2+-ATPase activity in the former and a decrease in the latter with no significant change in ( N a ÷ + K ÷)-dependent ATPase activity (Fig. 2). Saline administration reduced (Na÷+K+)-dependent ATPase in the kidney cortex 5. This would fit the decreased Na ÷ reabsorption and increased Na + excretion by the kidney which follows saline loading. The present report shows that (Na ÷ + K ÷)dependent ATPase was reduced also in the small and large intestine (Fig. 3). The reduced enzymatic activity would suggest decreased Na + absorption, and therefore, increased Na + loss from the gastrointestinal tract. This would point to participation of the intestines, in addition to the kidney, in the regulatory response to saline loading. That such participation occurs even immediately under saline loading was shown by in vivo experiments where net Na ÷ absorption from intestinal loops was reduced under saline loading in the rat is and the cat 19. The response of the salivary glands to saline loading was meager, only a small increase in (Na+q - K +)-dependent ATPase activity in the parotid gland (Fig. 3). The most surprising finding for us was the unexpected increase in (Na +q-K ÷)dependent ATPase activity found after adrenalectomy in the large intestine and the submaxillary glands (Fig. 4). No change was observed in the small intestine. The augmentation of (Na ÷ q-K +)-dependent ATPase was in sharp contrast to the reduction of this enzyme reported in the kidneys 3, 4. It has been shown recently that Na ÷ absorption in the colon of the rat can be enhanced by the addition of angiotensin, independently of mineralocorticoids 2°. Adrenalectomy is followed by increased production and release of renin from the kidneys 21, so presumably plasma angiotensin levels also increase. We have recently
540
Y. G U T M A N , D. G L U S H E V I T Z K Y - S T R A C H M A N
reported that angiotensin can enhance (Na++K+)-dependent ATPase activity in microsomes of the mucosa of the colon (but not the small intestine) 22. Combining all these data one may, therefore, suggest that the enhanced microsomal ATPase activity in the colon following adrenalectomy may be related to the increased renin activity after adrenal ablation. This possibility is now being further explored. ACKNOWLEDGEMENT
This paper is part of an MSc thesis of D.G.S. RE FERENCES I Bonting, S. L. (1970) Membranes and ion transport, Vol. I, pp. 257-287, John Wiley and Sons, Inc. New York 2 Katz, A. I. and Epstein, F. H. (1968) New Engl. J. Med. 278,253-261 3 J0rgensen, P. L. (1968) Biochim. Biophys. Acta 151,212-224 4 Hendler, E. D., Torretti, J., Kupor, L. and Epstein, F. H. (1972) Am. J. Physiol. 222, 754-760 5 Gutman, Y. and Beyth, Y. (1969) Biochim. Biophys. Acta 193,475~,78 6 Sayers, G. and Travis, R. H. (1970) in The PharmacolooicalBasis o f Therapeutics, (Goodman, L. S. and Gilman, A., eds), 4th edn, pp. 1604-1642, The MacMillan Co. New York 7 Schultz, S. G. and Zalusky, R. (1964) J. Gen. Physiol. 47, 567-584 8 Young, J. A. and Sch0gel, E. (1966) Pflii#ers Arch. Ges. Physiol. 291, 85-98 9 Gutman, Y. and Katzper-Shamir, Y. (1971) Biochim. Biophys. Acta 233, 133-136 10 Baginski, E. and Zak, B. (1960) Clin. Chim. Acta 5, 834-838 11 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951t J. Biol. Chem. 193, 265-275 12 Katz, A. I. and Epstein, F. H. (1967) J. Clin. Invest. 46, 1999-2011 13 Csaky, T. Z. 0963) Bioehim. Biophys. Aeta 74, 160-162 14 Schwatz, A. and Matsui, H. (1967) in Secretory Mechanisms of Sa#vary Glands (Schne2~er, L. H. and Schneyer, C. H., eds), pp. 75-98, Academic Press, New York 15 Bonting, S. L., Caravaggio, L. L., Canady, M. R. and Hawkins, N. M. (1964) Arch. Biochem. 106, 49-56 16 Epstein, F. H., Manitius, A., Weinstein, E., Katz, A. I. and Pickford, G. E. (1969) Yale J. Biol. Med. 41,388-393 17 Gutman, Y., Livneh, P. and Pietrokovsky, J. (1970) Isr. J. Med. Sei. 6, 573 575 18 Richet, G. and Hornych, (1969) Nephron 6, 365-378 19 Gutman, Y. and Benzakein, F. (1970) lsr. J. Med. Sci. 6, 195 200 20 Davis, N. T., Munday, K. A. and Parsons, B. J., (1970) J. Endoerinol. 48, 39~46 21 Gross, F., Brunner, H. and Ziegler, M. (1965) Recent Pro#. Horm. Res. 21, 119 177 22 Gutman, Y., Shamir, Y., Glushevitzky, D. and Hochman, S. (1972) Biochim. Biophys. Acta 273, 401 ~ , 0 5
Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands BBA 27092
E F F E C T OF D E H Y D R A T I O N , F O O D DEPRIVATION, SALINE A N D ADREN A L E C T O M Y ON MICROSOMAL (Na + q-K + ) - D E P E N D E N T ATPase IN T H E SALIVARY G L A N D S A N D I N T E S T I N A L MUCOSA
YEHUDA GUTMAN and DORITH GLUSHEVITZKY-STRACHMAN Department ok/"Pharmacolooy, The Hebrew University-Hadassah Medical School, Jerusalem (Israel)
(Received December 6th, 1972)
SUMMARY 1. The effect of water deprivation, food deprivation, saline and adrenalectomy was studied on microsomal ATPase activity of the rat parotid and submaxillary glands and the mucosa of the small intestine and of the colon. 2. Water deprivation caused a decrease of microsomal (Na+ +K+)-dependent ATPase activity in the submaxillary gland. 3. Food deprivation resulted in a selective reduction of microsomal (Na ÷ + K ÷)-dependent ATPase activity from the parotid gland. Mg 2 +-ATPase activity was enhanced in the small intestine and depressed in the large intestine. 4. When 0.9 % NaCI was used as drinking fluid (Na + + K ÷)-dependent ATPase was augmented in the parotid and reduced in the mueosa of the small and large intestine. 5. No decrease in ( N a + + K + ) - d e p e n d e n t ATPase activity was found after adrenalectomy in either salivary gland or in the mucosa of the small intestine or colon.
INTRODUCTION Microsomal ( N a + + K + ) - d e p e n d e n t ATPase (ATP phosphohydrolase EC 3.6.1.3) has been reported to be present in many tissues 1. Its function is associated with active sodium transport 2. Although the characteristics as an enzyme have been widely investigated, relatively few studies about the factors which regulate this enzymatic activity have been reported. Most of these studies concern the kidney, which is an organ where sodium transport plays a major role both in sodium balance and in the mechanism of urine concentration. Adrenalectomy, mineralocorticoids and Na + intake are known to affect kidney microsomal ATPase 3, 4. We have previously reported on the differential effect of water, food and saline on the (Na + + K +)-dependent ATPase in the kidney cortex as compared to the medulla 5. It seemed o f interest and importance to study whether the various factors
534
Y. GUTMAN, D. GLUSHEVITZKY-STRACHMAN
which affect kidney ATPase have a similar action on this enzyme in other tissues, particularly in epithelia where active sodium transport plays a major role in the physiological function. Mineralocorticoids are known to affect sodium transport not only in the kidney but also in the sweat glands, salivary glands and the colon% The intestines are daily exposed to large volumes of fluid which is almost completely absorbed, mainly through active sodium transport 7. Any reduction of this absorption may result in a severe loss of fluid (diarrhea). The salivary glands secrete into the acini a fluid very similar to plasma in ionic composition, but the final saliva is very poor in N a + and is hypotonic 8. Therefore, active Na + reabsorption must take place along the path of the saliva flow in the salivary glands. We, therefore, studied microsomal ATPase activities in different parts of the gastrointestinal tract and in different salivary glands under various stimuli. The results are presented in the present report. MATERIALS AND METHODS
Animals Male rats of the Hebrew University strain weighing 200-300 g, were used throughout.
Preparation of enzyme The submaxillary and parotid glands were removed immediately following decapitation. The small intestine, from duodenum to the distal end of the ileum, was cut at both ends. The large intestine (colon), both transverse and descending, was cut at the caecum and at the rectum. The intestines were flushed with ice-cold saline and were then cut open longitudinally and spread on filter-paper to expose the mucosa. With the aid of a scalpel the mucosal layer was scraped and collected. The various tissues were homogenized in an all glass homogenizer with 10 vol. of ice-cold 0.25 M sucrose containing 2 m M EDTA. The medium for homogenization of the salivary glands also contained 0 . 1 % deoxycholate. After centrifugation at 1000 × 9 for 10 min at 4 °C the supernatant was separated and spun at 10 000 ~: y for another 10 min. The supernatant was separated and centrifuged at 100 0 0 0 - 9 for 30 min and the microsomal pellet obtained was resuspended by gentle homogenization in 0.25 M sucrose. This microsomal preparation constituted the enzyme suspension. The enzyme suspension was either kept frozen ( - 2 0 °C) until assayed or assayed immediately; when kept frozen both the controls and the experimental samples underwent the same procedure.
Incubation for assay of A TPase activity The ATPase activity was measured by incubating the enzyme in the appropriate medium as previously described 9, for 10-20 min at 37 °C, in a thermostatic bath with constant shaking. When all the components for incubation were added except ATP, the flasks were placed in the thermostatic bath for preincubation at 37 °C for 5 min. ATP was th.en added and the incubation period started. Blanks containing no enzyme or where trichloroacetic acid was added before ATP were run with each incubation. The enzymatic reaction was stopped by adding 3.0 ml of 5 % trichloroacetic acid. After centrifugation and filtration an aliquot of 0.1 ml was taken for determination of the inorganic phosphate liberated.
SALIVARYAND INTESTINALATPase
535
Medium for incubation The incubation medium consisted of the following components (final concentrations): 100 mM NaCI; 10 mM KC1; 4 mM MgCI2; 4 mM ATP; 33 mM Tris buffer (pH 7.5) and 0.14).2ml of enzyme suspension (containing 0.14),5 mg of protein). The final volume of incubation was 3.0 ml. Inorganic phosphate liberated during incubation in this medium represented total microsomal ATPase activity. For determination of (Na + + K + )-dependent ATPase, the NaCI and KCI were replaced by an equivalent amount of Tris buffer; ~lternatively, incubation was carried out in the complete medium but ouabain was added to a final concentration of 10 - 3 M. Preliminary experiments have shown that this concentrations of ouabain reduced ATPase activity to that observed in the absence of Na ÷ and K ÷. The difference between the activity in the complete medium and that in the presence of ouabain (or absence of Na + and K ÷ ) represents (Na ÷ ÷ K ÷)-dependent ATPase activity while the residual activity is Mg z+-ATPase. The inorganic phosphate was measured according to the method of Baginski and Zak 1o. The protein in the enzyme suspension was assayed according to Lowry et aL x~. Experiments with animals Water deprivation was carried out for 4 days while both control and experimental rats had food ad libitum. Food deprivation was imposed for 4 days while both experimental and control animals had free access to water. Na ÷ loading was performed by offering 0.9 % NaCI as drinking fluid for 12 days. Adrenalectomy was performed under ether anesthesia, through a midline incision in the back: The controls for adrenalectomy were sham-operated rats, in which a midline incision was made in the abdomen under ether anesthesia. The rats were kept for 7 days after the operation on food and water adlibitum and were then sacrificed and the various tissues were removed. The experiment on saline loading was repeated twice and on adrenalectomy on three different occasions with consistent results. The data are therefore means of the different experiments. Some variation between control values in the various experiments may be due to seasonal changes in enzymatic activity, however, this point has not been investigated. RESULTS Table I shows the effect of the various treatments on the weight of the rats and that of the submaxillary and parotid glands. A prominent difference can be observed between the effects on the submaxillary gland and on the parotid glands: The submaxillary glands shrank considerably more under water deprivation and adrenalectomy while the parotid gland shrank under food deprivation and water deprivation but much less after adrenalectomy.
Water deprivation Water deprivation had a selective and limited effect on microsomal ATPase activity. Microsomal ATPase activity in the parotid was unchanged while in the submaxillary gland there was a significant decrease in (Na + + K +)-dependent ATPase activity, reflected also in total microsomal ATPase with no significant change in
536
Y. G U T M A N , D. G L U S H E V I T Z K Y - S T R A C H M A N
TABLE I E F F E C T O F V A R I O U S T R E A T M E N T S ON W E I G H T O F RAT A N D S A L I V A R Y G L A N D S N u m b e r of animals: water deprivation, 7; food deprivation, 8; saline, 8; adrenalectomy, 5.
Control
°/o change of wei#ht after Water deprivation
Rat (g) Submaxillary gland (mg) Parotid gland (rag)
235i6 204±4 152±6
37.8 39.5 33.7
Food deprivation -31.5 23.1 33.5
Saline (%)
Adrenalectom.v (o/)
- 1.5 ~ 11.4 4 14.8
15.8 33.2 18.0
Mg2+-ATPase activity (Fig. 1). Therefore, (Na+÷K+)-dependent ATPase in the submaxillary gland decreased from 69.2±1.7 ~o of total microsomal ATPase in the controls to 60.9~-1.5 ~o under water deprivation. Since the weight of the salivary glands decreased under water deprivation the total activity per gland was even further reduced. Thus the total ( N a + + K ÷ )-ATPase activity per gland was reduced by 30 o~, in the parotid and by 53 ?,'oin the submaxillary gland. L@
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Fig. I. Effect of water deprivation on microsomal ATPase activity in the rat salivary glands. Hatched column controls; open columns, water deprivation. Vertical bars, S.E. T, total microsomal ATPase activity; Mg, Mg 2 +-ATPase activity; Na, K, (Na + -~-K +)-dependent ATPase activity. Submaxillary gland: controls n -:~ 7; water deprivation n -- 7. Parotid gland: controls n -: 8; water deprivation n 7. * P < 0.05, ** P < 0.02 for the difference between control and water deprivation. Fig. 2. Effect of food deprivation on microsomal ATPase activity. Symbols as in Fig. I. Lower panel: left, parotid gland (controls n = 7; food deprivation n ~ 8); right, submaxillary gland icontrois n -- 7; food deprivation n = 8). Upper panel: left, small intestine (controls n ~ 8; food deprivation n - 7); right, large intestine (controls n ~ 4; food deprivation n 4). ** P < 0.02; • ** P < 0.005.
S A L I V A R Y A N D I N T E S T I N A L ATPase
537
The ATPase activity in the mucosa of the intestine was unchanged after water deprivation except for a slight increase of Mg z÷-ATPase in the small intestine (from 13.44-1.0 to 16.24-0.5 #moles Pi/mg protein per h, n = 7, P < 0.05).
Food deprivation Food deprivation caused a reduction of microsomal (Na + +K+)-dependent ATPase activity in both salivary glands (Fig. 2) but the reduction was statistically significant only in the parotid, and was due mainly to a fall of (Na + + K + )-dependent ATPase activity. No significant change in Mg2+-ATPase was found. Hence, ( N a + + K+ )-dependent ATPase in the parotid decreased from 69.0-t-1.3 % of total microsomal ATPase in the controls to 57.3+4.5 % of the total under food deprivation. Since the weight of the salivary glands was reduced after food deprivation the change of ATPase activity per gland was even more pronounced. Thus (Na + + K +)-ATPase per gland was reduced by 60 % in the parotid and by 38 % in the submaxillary. In the intestinal mucosa food deprivation caused augmentation of Mg 2+ATPase activity in the small intestine and decreased Mg2+-ATPase activity in the colon. No significant change in (Na+ + K + )-dependent ATPase was found in the intestine (Fig. 2). (Na + + K + )-dependent ATPase in the small intestine decreased therefore from 54.34-2.2 % of total microsomal ATPase to 45.2±2.8 % under food deprivation.
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Fig. 3. Effect o f saline on microsomal ATPase activity. Symbols as in Fig. 1. Lower panel: left, parotid gland (controls and saline n = 8); right, submaxillary gland (controls and saline n = 8). U p p e r panel: left, small intestine (controls n = 14; saline n = 25); right, large intestine (controls n : 7; saline n : 14). * P < 0.05; ~rkp < 0.02; * * * P < 0.005. Fig. 4. Effect o f adrenalectomy on microsomal ATPase activity. Symbols as in Fig. 1. Lower panel: left, parotid gland (controls n = 5; adrenalectomy n : 4); right, submaxillary gland (controls and adrenalectomy n = 5). U p p e r panel: left, small intestine (controls n = 21, adrenalectomy n : 26); right, large intestine (controls n : 11, adrenalectomy n : 13). * P < 0.05; ** P < 0.02; *** P < 0.005.
538
Y. GUTMAN, D. GLUSHEVITZKY-STRACHMAN
Saline Fig. 3 shows the effect of saline administration on microsomal ATPase activity. In the parotid gland (Na+-}-K+)-dependent ATPase activity was elevated while no significant change was observed in the submaxillary gland. However, since the weight of the salivary glands increased somewhat during saline intake there was also some elevation of total microsomal ATPase activity per gland. Thus, ( N a + ÷ K +)ATPase per gland increased by 26 ~ in the submaxillary and by 52 ',~Join the parotid. The intestinal mucosa responded uniformly to saline administration: Selective depression of (Na+--K÷)-dependent ATPase in both small intestine and colon with no significant change in Mg 2 +-ATPase. Adrenalectomy Fig. 4 shows the effect of adrenalectomy on microsomal ATPase activity in the tissues studied. In general, adrenalectomy caused increased microsomal ATPase activity. However, in the small intestine the increase was not statistically significant. Microsomal (Na ÷ ÷K+)-dependent ATPase activity was augmented in the mucosa of the colon and in the submaxillary gland. Microsomal Mg 2+-ATPase activity was increased in the parotid and submaxillary glands (Fig. 4). Due to a decrease in the weight of the salivary glands following adrenalectomy the total activity per gland of both microsomal Mg 2 +-ATPase and ( N a ÷ + K ÷)-ATPase in the submaxillary gland was equal to that of the controls. In the parotid gland the total activity per gland of Mg2+-ATPase was increased by 30°~, and the total (Na++K+)-ATPase was unchanged. DISCUSSION
(Na + ÷ K +)-dependent ATPase is considered as part of the sodium pump in cells of different tissues l,z. This enzyme, therefore, supports sodium transport in a variety of tissues and especially in epithelial layers where sodium transport is an important physiological function of the organ, such as the kidney 12, the intestine' 3 the salivary glands 14, and the avian salt gland 15. The activity of kidney (Na+÷K+)-dependent ATPase is known to be decreased following adrenalectomy 3,4. Furthermore, we have previously reported specific changes in the activity of this enzyme in either the kidney cortex or medulla, according to the type of stimulus applied (saline, water deprivation or food deprivation) 5. It seemed of interest, therefore, to see whether (Na++K+)-dependent ATPase activity in the other sodium-transporting epithelia showed the same type of response. The experiments reported here demonstrate that different, and sometimes opposite, effects of the same agent, e.9. saline administration, were induced in microsomal ATPase activity in different tissues (see Fig. 3). This is also illustrated by the opposite effect of adrenalectomy on kidney microsomal ATPase 3,4 on the one hand and on this enzyme in the mucosa of the colon and the submaxillary gland, on the other hand (Fig. 4). Another illustration of such a divergence was observed by Epstein et al.16 upon adaptation of a teleost to fresh water: a decrease in ( N a + + K +)-dependent ATPase ensued in the gills while an elevation of this enzyme was noticed in the kidney. Thus, it seems that one can rule out the possibility of a single or a common
S'ALIVARY AND INTESTINAL ATPase
539
"regulator" of (Na ÷ +K+)-dependent ATPase in the various sodium-transporting epithelia. An interesting selectivity was observed between the two major salivary glands, the parotid and the submaxillary. We have previously reported that thirst, induced by hypovolemia or water deprivation, was selectively reduced after ablation of the submaxillary gland of the rat but not after ablation of the parotid 17. In the present report only the submaxillary gland microsomal ATPase was affected by water deprivation, Le. microsomal (Na ÷ +K÷)-dependent ATPase activity was reduced, while no significant effect was observed in the parotid gland (Fig. 1). Table I also shows this functional differentiation of the two salivary glands in the loss of weight: under food deprivation the parotid lost more weight than the submaxillary while under water deprivation the submaxillary lost more weight than the parotid. Adrenalectomy, which is accompanied by dehydration, also caused a larger effect on the submaxillary than on the parotid gland (Fig. 4). An opposite selectivity was observed following food deprivation: (Na ÷ ÷ K ÷ )dependent ATPase activity was significantly depressed only in the parotid and not in the submaxillary gland (Fig. 2). It is of interest that in the kidney, too, water and food deprivation had selective effects: water deprivation reduced (Na++K+)-dependent ATPase in the cortex but not in the medulla, while food deprivation reduced this enzymatic activity in the medulla but not in the cortex of the kidney s . The present report would suggest that the parotid gland is more responsive to changes in food intake while the submaxillary gland is more affected by changes in water intake than in food intake (Figs 1 and 2 and Table I). Fasting had also an opposite effect on the small intestine as compared to the colon: art increase of Mg 2+-ATPase activity in the former and a decrease in the latter with no significant change in ( N a ÷ + K ÷)-dependent ATPase activity (Fig. 2). Saline administration reduced (Na÷+K+)-dependent ATPase in the kidney cortex 5. This would fit the decreased Na ÷ reabsorption and increased Na + excretion by the kidney which follows saline loading. The present report shows that (Na ÷ + K ÷)dependent ATPase was reduced also in the small and large intestine (Fig. 3). The reduced enzymatic activity would suggest decreased Na + absorption, and therefore, increased Na + loss from the gastrointestinal tract. This would point to participation of the intestines, in addition to the kidney, in the regulatory response to saline loading. That such participation occurs even immediately under saline loading was shown by in vivo experiments where net Na ÷ absorption from intestinal loops was reduced under saline loading in the rat is and the cat 19. The response of the salivary glands to saline loading was meager, only a small increase in (Na+q - K +)-dependent ATPase activity in the parotid gland (Fig. 3). The most surprising finding for us was the unexpected increase in (Na +q-K ÷)dependent ATPase activity found after adrenalectomy in the large intestine and the submaxillary glands (Fig. 4). No change was observed in the small intestine. The augmentation of (Na ÷ q-K +)-dependent ATPase was in sharp contrast to the reduction of this enzyme reported in the kidneys 3, 4. It has been shown recently that Na ÷ absorption in the colon of the rat can be enhanced by the addition of angiotensin, independently of mineralocorticoids 2°. Adrenalectomy is followed by increased production and release of renin from the kidneys 21, so presumably plasma angiotensin levels also increase. We have recently
540
Y. G U T M A N , D. G L U S H E V I T Z K Y - S T R A C H M A N
reported that angiotensin can enhance (Na++K+)-dependent ATPase activity in microsomes of the mucosa of the colon (but not the small intestine) 22. Combining all these data one may, therefore, suggest that the enhanced microsomal ATPase activity in the colon following adrenalectomy may be related to the increased renin activity after adrenal ablation. This possibility is now being further explored. ACKNOWLEDGEMENT
This paper is part of an MSc thesis of D.G.S. RE FERENCES I Bonting, S. L. (1970) Membranes and ion transport, Vol. I, pp. 257-287, John Wiley and Sons, Inc. New York 2 Katz, A. I. and Epstein, F. H. (1968) New Engl. J. Med. 278,253-261 3 J0rgensen, P. L. (1968) Biochim. Biophys. Acta 151,212-224 4 Hendler, E. D., Torretti, J., Kupor, L. and Epstein, F. H. (1972) Am. J. Physiol. 222, 754-760 5 Gutman, Y. and Beyth, Y. (1969) Biochim. Biophys. Acta 193,475~,78 6 Sayers, G. and Travis, R. H. (1970) in The PharmacolooicalBasis o f Therapeutics, (Goodman, L. S. and Gilman, A., eds), 4th edn, pp. 1604-1642, The MacMillan Co. New York 7 Schultz, S. G. and Zalusky, R. (1964) J. Gen. Physiol. 47, 567-584 8 Young, J. A. and Sch0gel, E. (1966) Pflii#ers Arch. Ges. Physiol. 291, 85-98 9 Gutman, Y. and Katzper-Shamir, Y. (1971) Biochim. Biophys. Acta 233, 133-136 10 Baginski, E. and Zak, B. (1960) Clin. Chim. Acta 5, 834-838 11 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951t J. Biol. Chem. 193, 265-275 12 Katz, A. I. and Epstein, F. H. (1967) J. Clin. Invest. 46, 1999-2011 13 Csaky, T. Z. 0963) Bioehim. Biophys. Aeta 74, 160-162 14 Schwatz, A. and Matsui, H. (1967) in Secretory Mechanisms of Sa#vary Glands (Schne2~er, L. H. and Schneyer, C. H., eds), pp. 75-98, Academic Press, New York 15 Bonting, S. L., Caravaggio, L. L., Canady, M. R. and Hawkins, N. M. (1964) Arch. Biochem. 106, 49-56 16 Epstein, F. H., Manitius, A., Weinstein, E., Katz, A. I. and Pickford, G. E. (1969) Yale J. Biol. Med. 41,388-393 17 Gutman, Y., Livneh, P. and Pietrokovsky, J. (1970) Isr. J. Med. Sei. 6, 573 575 18 Richet, G. and Hornych, (1969) Nephron 6, 365-378 19 Gutman, Y. and Benzakein, F. (1970) lsr. J. Med. Sci. 6, 195 200 20 Davis, N. T., Munday, K. A. and Parsons, B. J., (1970) J. Endoerinol. 48, 39~46 21 Gross, F., Brunner, H. and Ziegler, M. (1965) Recent Pro#. Horm. Res. 21, 119 177 22 Gutman, Y., Shamir, Y., Glushevitzky, D. and Hochman, S. (1972) Biochim. Biophys. Acta 273, 401 ~ , 0 5