J. Phy8iol. (1979), 292, pp. 495-504 With 4 text-fitgure Printed in Great Britain
495
ALDOSTERONE INDUCED CHANGES IN COLONIC SODIUM TRANSPORT OCCURRING NATURALLY DURING DEVELOPMENT IN THE NEONATAL PIG
BY D. R. FERGUSON*, P. S. JAMESt, J. Y. F. PATERSONt, J. C. SAUNDERSt AND M. W. SMITHt From the * Department of Pharmacology, University of Cambridge Medical School, Hills Road, Cambridge CB2 2QD, U.K. and the t A.R.C. Institute of Animal Physiology, Babraham, Cambridge CB2 4AT, U.K.
(Received 23 October 1978) SUMMARY
1. Serum concentrations of aldosterone in later fetal, 3-6 week old and adult pigs are of the order of 300 pg ml.-. This increases to about 2000 pg ml.-' in the period immediately after birth. 2. Canrenoate injected into pigs from birth onwards stops the increase in colonic short-circuit current, seen to take place normally during early postnatal development. Amiloride has little or no effect on the short-circuit current of colons taken from canrenoate injected pigs. 3. Canrenoate stops the post-natal increase in colonic Na influx (and therefore net transport) seen to occur under normal conditions. 4. There is in the neonatal pig distal colon a portion of Na transport which appears to be resistant to inhibition by amiloride or canrenoate. 5. There is a second portion of Na transport, increasing in importance as the piglets become older, which is electrogenic and which is inhibited by prior injection of canrenoate. It is assumed that this fraction of Na transport is influenced by aldosterone. 6. There is a third part of Na transport, maximal in colons taken from one day old animals, which appears to be non-electrogenic. This is also blocked by prior injection of canrenoate. 7. The physiological relevance of these findings is discussed. INTRODUCTION
Aldosterone acts on the mammalian large intestine to stimulate the absorption of salts and water (Levitan & Ingelfinger, 1965; Shields, Mulholland & Elmslie, 1966; Edmonds & Marriott, 1967). Whether or not this is associated with an increased secretion of K or H ions is still open to doubt. Aldosterone induced K secretion can be demonstrated under in vivo conditions (Levitan & Ingelfinger, 1965; Edmonds & Marriott, 1967; Edmonds & Godfrey, 1970), but it is difficult to demonstrate anything but 'trace movements of K in vitro, when the open circuit voltage has been reduced to zero (Bentley & Smith, 1975; Yorio & Bentley, 1977; Frizzell & Schultz, 1978). Previous work showed an increase in Na transport by pig distal colon during 0022-3751/79/3700-0795 $01.50 c) 1979 The Physiological Society
496
D. R. FERGUSON AND OTHERS post-natal development (Bentley & Smith, 1975). Preliminary work, with ileostomized pigs, suggests that at least part of this transport is under hormonal control (James & Smith, 1977). The time course for post-natal changes in Na transport has been described in detail in the previous paper (Cremaschi, Ferguson, H6nin, James, Meyer & Smith, 1979). The use of amiloride in these experiments has allowed total Na transport to be divided into three different possible mechanisms. The present work was started to examine these postulated mechanisms in greater detail to see what role, if any, aldosterone might play in modulating the early post-natal transport of Na by the pig colon. METHODS
Animals Pigs came from a herd of Large Whites bred at Babraham. Parturition was induced on day 110-112 of gestation by the routine injection of prostaglandin analogues. Piglets were injected with 20 mg K 17,8-hydroxy-3-oxo-17a-pregna-4,6-diene-21-carboxylate; referred to as K canrenoate (Soludactone, Laboratoires Searle, 92128 Montrouge, France) at birth and then daily during the first week of post-natal life (I.m. injection in 0-5 ml. 0-9% (w/v) sterile solution of NaCl). Piglets left to feed normally on the sow occasionally showed signs of diarrhoea. The general health of these pigs remained good, however, and they continued to gain weight throughout the period of drug administration. Preparation of ti8sue and measurement of Na flux Distal colons, dissected free and mounted in Ussing chambers as described previously (Cremaschi et al. 1979) were bathed in the glucose free bicarbonate saline of Krebs & Henseleit (1932). Short-circuit currents were recorded as described previously by Henriques de Jesus & Smith (1974). The method for determining Na flux was identical to that described previously (Cremaschi et al. 1979) except that greater use was made of bidirectional flux measurements (22Na for efflux and '4Na for influx on the same piece of distal colon).
Estimation of aldo8terone Aldosterone was estimated by radioimmunoassay using an antibody raised in sheep to aldosterone-3-carboxymethoxime-bovine serum albumin conjugate (Erlanger, Borek, Beiser & Lieberman, 1957). The antiserum was diluted 1/100 with phosphate buffer 0-04 M pH 7*4 and mixed with [1,2-3H2]aldosterone (95 ci/m-mole. Radiochemical Centre, Amersham). To ensure specificity in the assay, aldosterone in plasma extracts was isolated chromatographically using Sephadex LH20 and dichloromethane (MeCl2)-ethanol (EtOH) 98:2 by volume. Blood collected by heart puncture or from severed carotid arteries was either allowed to clot or was shaken with Na EDTA. Tritiated aldosterone (3 nc, 12 pg) was added to 1 ml. plasma or serum which was then shaken with 10 ml. redistilled MeCl2. The separated solvent was then washed with 1 ml. 0-1 N-NaOH, 1 ml. water and dried with anhydrous Na2SO4. The filtered extract was evaporated under a stream of dry N2 at 37 0C, redissolved in 110jel. MeCl2-EtOH and applied to a 200 x 8 mm column of LH20. The elution pattern is shown in Fig. 1; the fraction 10-5-12-5 ml. eluate was collected, and while this may contain small amounts of corticosterone and cortisone these are not sufficiently cross-reactive with the antibody to diminish the specificity of the procedure. The LH20 eluate was evaporated under dry N. at 37 0C and dissolved in 750 ell. phosphate buffer. 100j1. was used to count [3H]aldosterone to estimate procedural losses. Three 200jd. portions were each mixed with 200jI. diluted antiserum + [3H]aldosterone and allowed to stand overnight at room temperature for radioimmunoassay; 400i1. of a suspension in buffer of dextran (T2000) and charcoal was added, and after 1 hr the mixture was centrifuged and 500jA. of the supernatant taken for counting the antibody-bound [3H]aldosterone. Standards containing 0-1000 pg aldosterone in 200 #l. buffer were treated in the same way. In calculating plasma or serum aldosterone concentrations from the fitted regression curve, due allowance was made for the contribution of the [3H]aldosterone added to measure procedural losses.
ALDOSTERONE INDUCED Na TRANSPORT
497
8r
0
2 4~~~~~
X~~~~ 00
5
10O'
15
20
25
Effluent volume (ml.)
Fig. 1. Typical elution patterns for different tritiated steroids obtained from a standard column of LH 20 Sephadex. The column was equilibrated and run as described in the text. The pattern of elution for each steroid was determined separately. Residual counts were removed completely before addition of another steroid. The trace shows the elution position for progesterone (*), corticosterone (0), aldosterone (A), cortisone (A) and cortisol (-). The fraction 105-12.5 ml. (shown by the rectangle) was collected routinely for aldosterone assay.
Materia The following radioactive isotopes came from the Radiochemical Centre, Amersham, Bucks., U.K.; 22Na as a solution of 2NaCl (> 100 mc/mg); 24Na as a sterile isotonic solution of sodium chloride (- 12 mc/ml). Soludactone came as a gift from G. D. Searle & Co. Ltd. High Wycombe, Bucks., U.K. and amiloride hydrochloride came from Merek, Sharp & Dohme Ltd, Hoddesdon, Herts., U.K. All other chemicals were of analytical grade. RESULTS
Aldosterone concentrations in sera taken from pigs at different stages of development Present work was initiated to test the supposition that aldosterone might induce changes in the colonic transport of Na in pigs immediately after birth. Such a change could be induced either by an increase in the levels of circulating steroid or by a change in colonic sensitivity to the hormone. Initial experiments were undertaken to monitor blood levels of aldosterone during both pre- and post-natal development. The results obtained are shown in Fig. 2. The serum concentration of aldosterone remained essentially constant up to the time of birth (about 300 pg ml.-' serum). This concentration increased rapidly during the first day of post-natal life, to a-level which then remained approximately constant over the succeeding 6 days (about 2000 pg ml.-' serum). Concentrations of aldosterone in the sera of 3 and 6 week old pigs were similar to those found in the late fetal stage of development. These levels were not noticeably different from those measured in the sera of adult animals. Experiments to be reported below involved the daily injection of 20 mg canrenoate, an antagonist of aldosterone action in man (see Ramsay, Shelton, Harrison,
D. R. FERGUSON AND OTHERS 498 Tidd & Asbury, 1976 for refs.), into neonatal pigs. Sera collected from these animals showed similar high concentrations of aldosterone during the immediate post-natal period of development (Fig. 2). The abrupt increase in aldosterone concentration, seen to occur shortly after birth, could be responsible for the induction of post-natal changes in Na transport. The inability of canrenoate to affect the post-natal change in aldosterone levels, while at 3000
20000.
0
1000 _
50
75
125 150 100 Days from conception
175
Fig. 2. Aldosterone concentrations in neonatal and adult pig serum. Blood collected by heart puncture or from severed carotid arteries was processed for assay as described in the text. Pigs were used either as controls (@), or after daily injection of 20 mg canrenoate (El1). Values give means + S.E. of four to twenty separate estimations. The value for adult pig serum (extreme right) gives the mean of three estimates; values shown without error bars for fetal pigs represent single estimates in each case. The arrow shows the day of birth.
the same time inhibiting the subsequent changes in Na transport (see below), suggests that this drug may not act by changing aldosterone secretion but rather by inhibition in target tissues such as the colon.
Short-term measurements of colonic short-circuit current in pigs injected with canrenoate The short-circuit current of pig distal colon increases during post-natal development and a large proportion of this current becomes sensitive to inhibition by amiloride (Cremashi et al. 1979). Fig. 3 summarizes these results and compares them with other in vitro measurements carried out on similar pieces of tissue taken from canrenoate injected pigs. All measurements were recorded after 10-15 min incubation in bicarbonate saline under standard conditions. The intramuscular injection of 20 mg canrenoate at birth, and then at 24 hr intervals thereafter, abolished the postnatal increase in short-circuit current, seen to occur naturally under control conditions (Fig. 3A). The average short-circuit current recorded across these tissues, about 75 gA cm2, was not significantly different from that measured under control conditions in the presence of 5 x 10- M-amiloride (Fig. 3A). Amiloride was, nevertheless, added to the mucosal side of distal colons taken from canrenoate injected pigs, to see whether it could cause any further inhibition of short-circuit current. The results obtained are compared with those found under control conditions in Fig. 3B. The
ALDOSTERONE INDUCED Na TRANSPORT 499 amiloride sensitive short-circuit current was nearly completely obliterated by the previous injection of canrenoate. An extremely small effect of amiloride could still be seen using colons taken from 7 day old canrenoate injected pigs. This was negligible, however, in relation to that measured under control conditions (25 vs. 200 /zA cm-2 respectively). 500
-
A
-250 E
0
-
69
3
0
~"250
6
0
3
6
9
Age (days)
Fig. 3. Developmental changes in short-circuit current measured across distal colons of control and canrenoate injected pigs. Conditions of incubation were as stated in the text. Short-circuit currents were measured after 10-15 min incubation. A, values with error bars show mean short-circuit currents + S.E. of eight to twenty separate determinations using colons taken from newborn or canrenoate injected (20 mg/day) pigs. The interrupted and continuous lines show changes recorded in short-circuit current in colons taken from uninjected pigs, measured in the absence and presence of 5 x 10OMamiloride respectively (Cremaschi et at. 1979). B, amiloride sensitive short-circuit current measured across colons taken fron- uninjected (0) and canrenoate injected (@) pigs. Values give means + s.E. eight to thirteen separate determinations on colons taken from canrenoate injected animals. Results obtained from uninjected animals are taken from the paper of Cremaschi et al. (1979). Amiloride concentration used, 5 x 10-6 M.
Na transport across distal colons taken from canrenoate injected pigs during neonutal development Canrenoate inhibition of post-natal changes in short-circuit current provides indirect evidence for inhibition of sodium transport, but it is also known that the relation between these two parameters is complicated during the neonatal period of development (Cremaschi et al. 1979). It was therefore decided to measure Na transport directly using colons taken from canrenoate injected pigs. The results
D. R. FERGUSON AND OTHERS 500 obtained during the first week of post-natal life are compared with those found for the new-born pig colon in Table 1. The influx of Na across canrenoate treated colons remained constant throughout early post-natal development (about 7 tequiv cm-2 hr-1). This value was not different from that measured across colons taken from new-born, uninjected, control animals (7.56 + 0-99 Sequiv cm-2 hr-1). Na influx measured in the presence of 5 x 10- Mamiloride tended to be lower at all stages in development. All these results showed a fair degree of scatter. A better appreciation of how amiloride might affect Na TABLE 1. Unidirectional Na fluxes measured across pig distal colons maintained in vitro under short-circuit conditions. All pigs were given daily I.M. injections of 20 mg canreonate. Distal colons were incubated in bicarbonate saline gassed with 95% 02 + 5% CO2 at a temperature of 37 'C. Steady state fluxes of isotopically labelled Na were measured 40-70 min later in the absence and presence of 5 x 10-6 M-amiloride. Values give means ± s.E. of four to twelve separate determinations Na transport (Itequiv cm-2 hr-')
Canrenoate-injected Canrenoate-injected Age of pig
,_I
(days) 0
7*56 ± 0.99
Influx
amiloride present -,_A_
Efflux 3-86 ± 0-48 (10) 3.47 ± 0.32 (4) 4X91+ 1*21 (6) 2-89 ± 0-36
Influx
7*04 + 0-79
Efflux 3-01 + 0*20 (12) 3*63 + 0*30 (4) 3-80 + 0-64
3
(10) 7.26 + 1*24 (4) 7-74 + 1X23 (5) 6-71+ 0-66
(6)
(4)
(a)
(5)
7
7-01+1-18
7-48 ± 2-21
(4)
(4)
4-31 + 0-44 (4)
3-23 ± 0-46 (4)
1 2
(12) 5-35± 0.85 (4)
4*42 + 0*70 (4)
(5)
4-92 +1.11
3-00 + 0-70
transport in these tissues can be gained by a direct comparison of net Na fluxess (see below). The backflux of Na across canrenoate treated colons did not change markedly during development. That measured across colons taken from 7 day old pigs appeared to be unusually high, but the error was also large. The presence of amiloride was without effect on Na backflux measured at all stages of development with the possible exception of the 7 day old animal. Most of the experiments summarized in Table 1 were carried out using two radioisotopes of Na. This allowed one to estimate both influx and efflux across the same piece of tissue. In this case the net flux of sodium can be expressed, for each stage of development, as a mean value + its own standard error. Results obtained can then be compared under different experimental conditions. The results of such a comparison are shown in Fig. 4. The net transport of Na across distal colons taken from canrenoate injected pigs varies widely, but there is no post-natal rise as there is in colons taken from uninjected control animals (cf. with data from Cremaschi et al. 1979, included in Fig. 4 for comparison). Neither was there any significant
ALDOSTERONE INDUCED Na TRANSPORT 501 difference between net Na fluxes measured in the presence and absence of amiloride in canrenoate treated colons. It is concluded from these experiments that canrenoate inhibits the normal postnatal increase in net Na transport (Fig. 4) through a selective effect on Na influx (Table 1). 8
E
4 . 0
CL
Cu
A,~~~~ zI
0
2
4
6
8
10
Age (days)
Fig. 4. Effect of canrenoate on the net transport of Na across pig distal colon. Net Na transport measured under control conditions (0) (Cremaschi et al. 1979) is compared with that found following daily injection of 20 mg canrenoate. Sodium fluxes were measured in the presence (A) and absence (0) of 5 x 10-6 M-amiloride. Values give means ± s.E. of three or four separate estimations.
Electrogenicity of Na transport measured across distal colons taken from canrenoate injected neonatal pigs Canrenoate inhibits both the post-natal increase in short-circuit current and the net transport of Na across the pig distal colon, but it does not inhibit either of these two parameters completely. It behaves in this respect like amiloride used on colons taken from control animals (Cremaschi et al. 1979). The canrenoate resistant Na transport has been compared with the canrenoate resistant short-circuit current, for different stages of neonatal development, in Table 2. Results obtained in the presence and absence of amiloride have been pooled in this instance, since it has already been established that amiloride has negligible effects on short-circuit current or net Na transport following injection of canrenoate. The short-circuit currents reported here were recorded over the period when Na fluxes were being measured (after 40-70 min incubation). The values for these currents are somewhat less than those recorded after 10-15 min incubation (50 vs. 75 sA cm-2). The short-circuit current, equivalent to a maximum net Na transport of about 2 pequiv cm-2 hr-1, if no other ion is being transported electrogenically remained constant throughout the first week of postnatal life. The net transport of Na also
D. R. FERGUSON AND OTHERS 502 remained approximately constant, at about 2 Isequiv cm2 hr-1, from day 1 to day 3 of post-natal life. This transport fell markedly by day 7, but this is mainly a reflexion of the unusually high backflux for Na measured in the absence of amiloride (Table 1). The net Na flux was significantly greater than the measured short-circuit current in the new-born pig (P < 0.001). This difference was not maintained in animals injected with canrenoate. TABLE 2. Net Na flux measured across distal colons taken from canrenoate-treated pigs. Values were measured directly or calculated from measurements of short-circuit current. Amiloride affects neither Na flux nor short-circuit current in canrenoate injected pigs (Table 1 and Fig. 4). Results obtained in the presence and absence of amiloride have therefore been pooled for the purpose of comparison (means ± S.E., no. of observations). P gives the probability that differences between these two estimates occur by chance
Short-circuit current, as Na flux (uaequiv cm-2 hr-1) 1-98±0-2
Age of pig (days) 0
Net Na flux (#equiv cm-2 hr-1)
(12)
(21)
1
2-76 ± 0-93
1-78 ± 0-31
(8)
(8)
1-99 ± 1-17 (7) 2-23+0-39
2-01 + 0-22 (13) 1-74+0-24
(8)
(8)
0-29+0-75 (8)
2-08±0-51
2
3 7
3-45+0-56
P < 0-001 n.s. n.s.
n.s. < 0-1 > 0-05
(8) DISCUSSION
Piglets need a plentiful supply of Na to sustain growth both during fetal and early post-natal development. The magnitude of the problem is, however, much greater in the case of the neonatal animal. The pig fetus increases in weight from 0-8 to 1-2 kg during the last 10 days of gestation and from 1-2 to 4 kg during the first 14 days of post-natal life. Assuming that 20 % of the pig's weight consists of extracellular fluid with a Na concentration of about 120 mm, then the fetal pig will need an extra 10 and the neonatal pig an extra 90 m-mole Na to sustain growth. Na will be provided at high concentration in blood coming from the placenta in the case of the fetal animal, but the post-natal animal will have to recover its sodium from the gastrointestinal tract following ingestion of sow's milk (Na concentration 20-30 mM, G. Bailes, personal communication). The efficiency with which the small intestine performs this task is also likely to be diminished during the immediate post-natal period, due to the endocytotic absorption of colostral antibodies (Henriques de Jesus & Smith, 1974). It seems hardly surprising that the pig should develop additional mechanisms for recovering Na from its large intestine under such circumstances. Further evidence for the importance of the colon in the neonatal pig has been provided recently by the work of Wilkinson & McCance (1971). They found that removal of the colon led to a massive loss of water and electrolytes, sufficient to kill in several instances. We can verify that ileostomy in the new-born pig produces
ALDOSTERONE INDUCED Na TRANSPORT 503 diarrhoea which increases in intensity shortly after ingestion of colostrum (M. W. Smith & K. A. Burton, unpublished results). Present work shows that an aldosterone antagonist can also produce diarrhoea through the partial inhibition of Na reabsorption in the colon. Aldosterone appears to be deeply implicated in organizing all these adaptive changes in Na transport. Its concentration in blood increases at a time when its presence seems most necessary. Virtually identical findings have been reported for the neonatal mouse (Dalle, Giry, Gay & Delost, 1978). It is also true, however, that the aldosterone concentration falls within 3 weeks of birth, to levels found in the fetal animal, even though the young pig is still continuing to grow at a rapid rate (McCance, 1974). There is also little doubt that aldosterone continues to play a useful role in modifying Na transport in adult animals, where growth no longer takes place (see Introduction for references). Further experiments on the rate of aldosterone metabolism and a parallel investigation of tissue receptors for this hormone, carried out at different times during the life of the pig, are necessary before coming to any more detailed conclusion as to how blood levels of the hormone might relate to its actual ability to affect Na reabsorption. The strongest evidence that aldosterone is involved in producing post-natal changes in colonic Na transport comes from the use of canrenoate. This drug has no effect on the circulating levels of hormone, but it does inhibit the post-natal increase in Na transport seen to take place under normal conditions. An operational definition of how Na might cross the pig distal colon during neonatal development has been outlined in the previous paper (Cremaschi et al. 1979). Canreonate blocks two of the three available mechanisms, one which is operationally neutral which disappears shortly after birth and a second, which is electrogenic and which increases in magnitude as the animal becomes older. The conclusion is that aldosterone is involved in the regulation of both these mechanisms. The neutral transport of Na, seen to be present in the colons of new-born and 1 day old animals, could result from an action of aldosterone on epithelial cells already formed at birth. The increase in the electrogenic transport of Na could result from an action of aldosterone on cells formed postnatally. Part of the Na transport across new-born and older colons appears to be electrogenic, but it is influenced neither by amiloride (Cremaschi et al. 1979) nor by the prior injection of canreonate (present results). The most likely explanation for these findings is that a constant fraction of Na transport takes place by a mechanism similar to that found in the mammalian small intestine. There appears to be a very close relation between the action of canrenoate in blocking the induction of an electrogenic transport system for Na and the action of amiloride in inhibiting this system once it has become fully induced. Effects of these two drugs on short-circuit current and Na transport are remarkably similar. It has already been suggested that aldosterone acts on high resistance epithelia to increase the number of amiloride sensitive Na sites present in the outward facing membrane (Cuthbert, Okpako & Shum, 1974; Cuthbert & Shum, 1976). Present results lend support to such a notion. It would now be very interesting to test this hypothesis further by direct measurement of amiloride binding sites in pig colons taken at different stages of development, particularly since it now appears that part of the action of aldosterone in the newborn animal is to induce a neutral transport system for Na, sensitive to inhibition by amiloride (Cremaschi et al. 1979).
504
D. R. FERGUSON AND OTHERS
We wish to express our thanks to Mr R. W. Ash for both supplying us with new-born pigs and for giving daily intramuscular injections of canrenoate.
REFERENCES
BENTLEY, P. J. & SMiTH, M. W. (1975). Transport of electrolytes across the helicoidal colon of the new-born pig. J. Physiol. 249, 103-117. CIEiscHI, D., FERGUSON, D. R., HAIAN, S., JA S, P. S., MEYER, J. & Saw, M. W. (1979). Post-natal development of amiloride sensitive sodium transport in pig distal colon. J. Phyeiol. 292, 481-494. CUTHBERT, A. WV., OKPAKO, D. & SHUM, W. K. (1974). Aldosterone and the number of sodium channels in the frog skin. Br. J. Pharmac. 51, 128-129P. CUTHBERT, A. W. & Siiuu, W. K. (1976). Estimation of the lifespan of amiloride binding sites in the membranes of toad bladder epithelial cells. J. Physiol. 255, 605-618. DAT.TLL, M., GIRY, J., GAY, M. & DELOST, P. (1978). Perinatal changes in plasma and adrenal corticosterone and aldosterone concentrations in the mouse. J. Endocr. 76, 303-309. EDMONDS, C. J. & GODFREY, R. C. (1970). Measurement of electrical potentials of the human rectum and pelvic colon in normal and aldosterone-treated patients. Gut 11, 330-337. EDMONDS, C. J. & MARRIOTT, J. C. (1967). The effect of aldosterone and adrenalectomy on the electrical potential difference of rat colon and on the transport of sodium, potassium, chloride and bicarbonate. J. Endoer. 39, 517-531. ERLANGER, B. F., BOREE, F., BEISER, S. M. & LIEBERMAN, S. (1957). Steroid-protein conjugates. 1. Preparation and characterization of conjugates of bovine serum albumin with testosterone and with cortisone. J. biol. Chem. 228, 713-727. FRIzzE7L, R. A. & SCHULTZ, S. G. (1978). Effect of aldosterone on ion transport by rabbit colon in vitro. J. membrane Biol. 39, 1-26. HENRIQuEs DE JESUS, C. & Sxrm, M. W. (1974). Sodium transport by the small intestine of new-born and suckling pigs. J. Physiol. 243, 211-224. JAMES, P. S. & SMITEi, M. W. (1977). Effect of ileostomy on changing transport function in the new-born pig colon. J. Phyeiol. 272, 61-62P. KREBS, H. A. & HENsELEIr, K. (1932). Tntersuchungen uber die Harnstoffbildung im Tier korper. Hoppe-Seyler'8 Z. physiol. Chem. 210, 33-66. LEvrrAN, R. & INGELFINGER, F'. J. (1965). Effect of D-aldosterone on salt and water absorption from the intact human colon. J. cdin. Invest. 44, 801-808. McCANcE, R. A. (1974). The effect of age on the weights and lengths of pigs' intestines. J. Anat. 117, 475-479. RAMSAY, L., SHELTON, J., HARRISON, I., TIDD, M. & ASBURY, M. (1976). Spironolactone and potassium canrenoate in normal man. Clin. Pharmac. Ther. 20, 167-177. S=IELDs, R., MoLOLAND, A. T. & ELMSLIE, R. G. (1966). Action of aldosterone upon the intestinal transport of potassium, sodium, and water. Gut 7, 686-696. WILKINSON, A. W. & McCANcE, R. A. (1971). The subsequent effects of removing the large intestine from newborn pigs. Proc. Nutr. Soc. 30, 26-27A. YoRIo, T. & BENTLEY, P. J. (1977). Permeability of the rabbit colon in vitro. Am. J. Physiol. 232, F5-9.
495
ALDOSTERONE INDUCED CHANGES IN COLONIC SODIUM TRANSPORT OCCURRING NATURALLY DURING DEVELOPMENT IN THE NEONATAL PIG
BY D. R. FERGUSON*, P. S. JAMESt, J. Y. F. PATERSONt, J. C. SAUNDERSt AND M. W. SMITHt From the * Department of Pharmacology, University of Cambridge Medical School, Hills Road, Cambridge CB2 2QD, U.K. and the t A.R.C. Institute of Animal Physiology, Babraham, Cambridge CB2 4AT, U.K.
(Received 23 October 1978) SUMMARY
1. Serum concentrations of aldosterone in later fetal, 3-6 week old and adult pigs are of the order of 300 pg ml.-. This increases to about 2000 pg ml.-' in the period immediately after birth. 2. Canrenoate injected into pigs from birth onwards stops the increase in colonic short-circuit current, seen to take place normally during early postnatal development. Amiloride has little or no effect on the short-circuit current of colons taken from canrenoate injected pigs. 3. Canrenoate stops the post-natal increase in colonic Na influx (and therefore net transport) seen to occur under normal conditions. 4. There is in the neonatal pig distal colon a portion of Na transport which appears to be resistant to inhibition by amiloride or canrenoate. 5. There is a second portion of Na transport, increasing in importance as the piglets become older, which is electrogenic and which is inhibited by prior injection of canrenoate. It is assumed that this fraction of Na transport is influenced by aldosterone. 6. There is a third part of Na transport, maximal in colons taken from one day old animals, which appears to be non-electrogenic. This is also blocked by prior injection of canrenoate. 7. The physiological relevance of these findings is discussed. INTRODUCTION
Aldosterone acts on the mammalian large intestine to stimulate the absorption of salts and water (Levitan & Ingelfinger, 1965; Shields, Mulholland & Elmslie, 1966; Edmonds & Marriott, 1967). Whether or not this is associated with an increased secretion of K or H ions is still open to doubt. Aldosterone induced K secretion can be demonstrated under in vivo conditions (Levitan & Ingelfinger, 1965; Edmonds & Marriott, 1967; Edmonds & Godfrey, 1970), but it is difficult to demonstrate anything but 'trace movements of K in vitro, when the open circuit voltage has been reduced to zero (Bentley & Smith, 1975; Yorio & Bentley, 1977; Frizzell & Schultz, 1978). Previous work showed an increase in Na transport by pig distal colon during 0022-3751/79/3700-0795 $01.50 c) 1979 The Physiological Society
496
D. R. FERGUSON AND OTHERS post-natal development (Bentley & Smith, 1975). Preliminary work, with ileostomized pigs, suggests that at least part of this transport is under hormonal control (James & Smith, 1977). The time course for post-natal changes in Na transport has been described in detail in the previous paper (Cremaschi, Ferguson, H6nin, James, Meyer & Smith, 1979). The use of amiloride in these experiments has allowed total Na transport to be divided into three different possible mechanisms. The present work was started to examine these postulated mechanisms in greater detail to see what role, if any, aldosterone might play in modulating the early post-natal transport of Na by the pig colon. METHODS
Animals Pigs came from a herd of Large Whites bred at Babraham. Parturition was induced on day 110-112 of gestation by the routine injection of prostaglandin analogues. Piglets were injected with 20 mg K 17,8-hydroxy-3-oxo-17a-pregna-4,6-diene-21-carboxylate; referred to as K canrenoate (Soludactone, Laboratoires Searle, 92128 Montrouge, France) at birth and then daily during the first week of post-natal life (I.m. injection in 0-5 ml. 0-9% (w/v) sterile solution of NaCl). Piglets left to feed normally on the sow occasionally showed signs of diarrhoea. The general health of these pigs remained good, however, and they continued to gain weight throughout the period of drug administration. Preparation of ti8sue and measurement of Na flux Distal colons, dissected free and mounted in Ussing chambers as described previously (Cremaschi et al. 1979) were bathed in the glucose free bicarbonate saline of Krebs & Henseleit (1932). Short-circuit currents were recorded as described previously by Henriques de Jesus & Smith (1974). The method for determining Na flux was identical to that described previously (Cremaschi et al. 1979) except that greater use was made of bidirectional flux measurements (22Na for efflux and '4Na for influx on the same piece of distal colon).
Estimation of aldo8terone Aldosterone was estimated by radioimmunoassay using an antibody raised in sheep to aldosterone-3-carboxymethoxime-bovine serum albumin conjugate (Erlanger, Borek, Beiser & Lieberman, 1957). The antiserum was diluted 1/100 with phosphate buffer 0-04 M pH 7*4 and mixed with [1,2-3H2]aldosterone (95 ci/m-mole. Radiochemical Centre, Amersham). To ensure specificity in the assay, aldosterone in plasma extracts was isolated chromatographically using Sephadex LH20 and dichloromethane (MeCl2)-ethanol (EtOH) 98:2 by volume. Blood collected by heart puncture or from severed carotid arteries was either allowed to clot or was shaken with Na EDTA. Tritiated aldosterone (3 nc, 12 pg) was added to 1 ml. plasma or serum which was then shaken with 10 ml. redistilled MeCl2. The separated solvent was then washed with 1 ml. 0-1 N-NaOH, 1 ml. water and dried with anhydrous Na2SO4. The filtered extract was evaporated under a stream of dry N2 at 37 0C, redissolved in 110jel. MeCl2-EtOH and applied to a 200 x 8 mm column of LH20. The elution pattern is shown in Fig. 1; the fraction 10-5-12-5 ml. eluate was collected, and while this may contain small amounts of corticosterone and cortisone these are not sufficiently cross-reactive with the antibody to diminish the specificity of the procedure. The LH20 eluate was evaporated under dry N. at 37 0C and dissolved in 750 ell. phosphate buffer. 100j1. was used to count [3H]aldosterone to estimate procedural losses. Three 200jd. portions were each mixed with 200jI. diluted antiserum + [3H]aldosterone and allowed to stand overnight at room temperature for radioimmunoassay; 400i1. of a suspension in buffer of dextran (T2000) and charcoal was added, and after 1 hr the mixture was centrifuged and 500jA. of the supernatant taken for counting the antibody-bound [3H]aldosterone. Standards containing 0-1000 pg aldosterone in 200 #l. buffer were treated in the same way. In calculating plasma or serum aldosterone concentrations from the fitted regression curve, due allowance was made for the contribution of the [3H]aldosterone added to measure procedural losses.
ALDOSTERONE INDUCED Na TRANSPORT
497
8r
0
2 4~~~~~
X~~~~ 00
5
10O'
15
20
25
Effluent volume (ml.)
Fig. 1. Typical elution patterns for different tritiated steroids obtained from a standard column of LH 20 Sephadex. The column was equilibrated and run as described in the text. The pattern of elution for each steroid was determined separately. Residual counts were removed completely before addition of another steroid. The trace shows the elution position for progesterone (*), corticosterone (0), aldosterone (A), cortisone (A) and cortisol (-). The fraction 105-12.5 ml. (shown by the rectangle) was collected routinely for aldosterone assay.
Materia The following radioactive isotopes came from the Radiochemical Centre, Amersham, Bucks., U.K.; 22Na as a solution of 2NaCl (> 100 mc/mg); 24Na as a sterile isotonic solution of sodium chloride (- 12 mc/ml). Soludactone came as a gift from G. D. Searle & Co. Ltd. High Wycombe, Bucks., U.K. and amiloride hydrochloride came from Merek, Sharp & Dohme Ltd, Hoddesdon, Herts., U.K. All other chemicals were of analytical grade. RESULTS
Aldosterone concentrations in sera taken from pigs at different stages of development Present work was initiated to test the supposition that aldosterone might induce changes in the colonic transport of Na in pigs immediately after birth. Such a change could be induced either by an increase in the levels of circulating steroid or by a change in colonic sensitivity to the hormone. Initial experiments were undertaken to monitor blood levels of aldosterone during both pre- and post-natal development. The results obtained are shown in Fig. 2. The serum concentration of aldosterone remained essentially constant up to the time of birth (about 300 pg ml.-' serum). This concentration increased rapidly during the first day of post-natal life, to a-level which then remained approximately constant over the succeeding 6 days (about 2000 pg ml.-' serum). Concentrations of aldosterone in the sera of 3 and 6 week old pigs were similar to those found in the late fetal stage of development. These levels were not noticeably different from those measured in the sera of adult animals. Experiments to be reported below involved the daily injection of 20 mg canrenoate, an antagonist of aldosterone action in man (see Ramsay, Shelton, Harrison,
D. R. FERGUSON AND OTHERS 498 Tidd & Asbury, 1976 for refs.), into neonatal pigs. Sera collected from these animals showed similar high concentrations of aldosterone during the immediate post-natal period of development (Fig. 2). The abrupt increase in aldosterone concentration, seen to occur shortly after birth, could be responsible for the induction of post-natal changes in Na transport. The inability of canrenoate to affect the post-natal change in aldosterone levels, while at 3000
20000.
0
1000 _
50
75
125 150 100 Days from conception
175
Fig. 2. Aldosterone concentrations in neonatal and adult pig serum. Blood collected by heart puncture or from severed carotid arteries was processed for assay as described in the text. Pigs were used either as controls (@), or after daily injection of 20 mg canrenoate (El1). Values give means + S.E. of four to twenty separate estimations. The value for adult pig serum (extreme right) gives the mean of three estimates; values shown without error bars for fetal pigs represent single estimates in each case. The arrow shows the day of birth.
the same time inhibiting the subsequent changes in Na transport (see below), suggests that this drug may not act by changing aldosterone secretion but rather by inhibition in target tissues such as the colon.
Short-term measurements of colonic short-circuit current in pigs injected with canrenoate The short-circuit current of pig distal colon increases during post-natal development and a large proportion of this current becomes sensitive to inhibition by amiloride (Cremashi et al. 1979). Fig. 3 summarizes these results and compares them with other in vitro measurements carried out on similar pieces of tissue taken from canrenoate injected pigs. All measurements were recorded after 10-15 min incubation in bicarbonate saline under standard conditions. The intramuscular injection of 20 mg canrenoate at birth, and then at 24 hr intervals thereafter, abolished the postnatal increase in short-circuit current, seen to occur naturally under control conditions (Fig. 3A). The average short-circuit current recorded across these tissues, about 75 gA cm2, was not significantly different from that measured under control conditions in the presence of 5 x 10- M-amiloride (Fig. 3A). Amiloride was, nevertheless, added to the mucosal side of distal colons taken from canrenoate injected pigs, to see whether it could cause any further inhibition of short-circuit current. The results obtained are compared with those found under control conditions in Fig. 3B. The
ALDOSTERONE INDUCED Na TRANSPORT 499 amiloride sensitive short-circuit current was nearly completely obliterated by the previous injection of canrenoate. An extremely small effect of amiloride could still be seen using colons taken from 7 day old canrenoate injected pigs. This was negligible, however, in relation to that measured under control conditions (25 vs. 200 /zA cm-2 respectively). 500
-
A
-250 E
0
-
69
3
0
~"250
6
0
3
6
9
Age (days)
Fig. 3. Developmental changes in short-circuit current measured across distal colons of control and canrenoate injected pigs. Conditions of incubation were as stated in the text. Short-circuit currents were measured after 10-15 min incubation. A, values with error bars show mean short-circuit currents + S.E. of eight to twenty separate determinations using colons taken from newborn or canrenoate injected (20 mg/day) pigs. The interrupted and continuous lines show changes recorded in short-circuit current in colons taken from uninjected pigs, measured in the absence and presence of 5 x 10OMamiloride respectively (Cremaschi et at. 1979). B, amiloride sensitive short-circuit current measured across colons taken fron- uninjected (0) and canrenoate injected (@) pigs. Values give means + s.E. eight to thirteen separate determinations on colons taken from canrenoate injected animals. Results obtained from uninjected animals are taken from the paper of Cremaschi et al. (1979). Amiloride concentration used, 5 x 10-6 M.
Na transport across distal colons taken from canrenoate injected pigs during neonutal development Canrenoate inhibition of post-natal changes in short-circuit current provides indirect evidence for inhibition of sodium transport, but it is also known that the relation between these two parameters is complicated during the neonatal period of development (Cremaschi et al. 1979). It was therefore decided to measure Na transport directly using colons taken from canrenoate injected pigs. The results
D. R. FERGUSON AND OTHERS 500 obtained during the first week of post-natal life are compared with those found for the new-born pig colon in Table 1. The influx of Na across canrenoate treated colons remained constant throughout early post-natal development (about 7 tequiv cm-2 hr-1). This value was not different from that measured across colons taken from new-born, uninjected, control animals (7.56 + 0-99 Sequiv cm-2 hr-1). Na influx measured in the presence of 5 x 10- Mamiloride tended to be lower at all stages in development. All these results showed a fair degree of scatter. A better appreciation of how amiloride might affect Na TABLE 1. Unidirectional Na fluxes measured across pig distal colons maintained in vitro under short-circuit conditions. All pigs were given daily I.M. injections of 20 mg canreonate. Distal colons were incubated in bicarbonate saline gassed with 95% 02 + 5% CO2 at a temperature of 37 'C. Steady state fluxes of isotopically labelled Na were measured 40-70 min later in the absence and presence of 5 x 10-6 M-amiloride. Values give means ± s.E. of four to twelve separate determinations Na transport (Itequiv cm-2 hr-')
Canrenoate-injected Canrenoate-injected Age of pig
,_I
(days) 0
7*56 ± 0.99
Influx
amiloride present -,_A_
Efflux 3-86 ± 0-48 (10) 3.47 ± 0.32 (4) 4X91+ 1*21 (6) 2-89 ± 0-36
Influx
7*04 + 0-79
Efflux 3-01 + 0*20 (12) 3*63 + 0*30 (4) 3-80 + 0-64
3
(10) 7.26 + 1*24 (4) 7-74 + 1X23 (5) 6-71+ 0-66
(6)
(4)
(a)
(5)
7
7-01+1-18
7-48 ± 2-21
(4)
(4)
4-31 + 0-44 (4)
3-23 ± 0-46 (4)
1 2
(12) 5-35± 0.85 (4)
4*42 + 0*70 (4)
(5)
4-92 +1.11
3-00 + 0-70
transport in these tissues can be gained by a direct comparison of net Na fluxess (see below). The backflux of Na across canrenoate treated colons did not change markedly during development. That measured across colons taken from 7 day old pigs appeared to be unusually high, but the error was also large. The presence of amiloride was without effect on Na backflux measured at all stages of development with the possible exception of the 7 day old animal. Most of the experiments summarized in Table 1 were carried out using two radioisotopes of Na. This allowed one to estimate both influx and efflux across the same piece of tissue. In this case the net flux of sodium can be expressed, for each stage of development, as a mean value + its own standard error. Results obtained can then be compared under different experimental conditions. The results of such a comparison are shown in Fig. 4. The net transport of Na across distal colons taken from canrenoate injected pigs varies widely, but there is no post-natal rise as there is in colons taken from uninjected control animals (cf. with data from Cremaschi et al. 1979, included in Fig. 4 for comparison). Neither was there any significant
ALDOSTERONE INDUCED Na TRANSPORT 501 difference between net Na fluxes measured in the presence and absence of amiloride in canrenoate treated colons. It is concluded from these experiments that canrenoate inhibits the normal postnatal increase in net Na transport (Fig. 4) through a selective effect on Na influx (Table 1). 8
E
4 . 0
CL
Cu
A,~~~~ zI
0
2
4
6
8
10
Age (days)
Fig. 4. Effect of canrenoate on the net transport of Na across pig distal colon. Net Na transport measured under control conditions (0) (Cremaschi et al. 1979) is compared with that found following daily injection of 20 mg canrenoate. Sodium fluxes were measured in the presence (A) and absence (0) of 5 x 10-6 M-amiloride. Values give means ± s.E. of three or four separate estimations.
Electrogenicity of Na transport measured across distal colons taken from canrenoate injected neonatal pigs Canrenoate inhibits both the post-natal increase in short-circuit current and the net transport of Na across the pig distal colon, but it does not inhibit either of these two parameters completely. It behaves in this respect like amiloride used on colons taken from control animals (Cremaschi et al. 1979). The canrenoate resistant Na transport has been compared with the canrenoate resistant short-circuit current, for different stages of neonatal development, in Table 2. Results obtained in the presence and absence of amiloride have been pooled in this instance, since it has already been established that amiloride has negligible effects on short-circuit current or net Na transport following injection of canrenoate. The short-circuit currents reported here were recorded over the period when Na fluxes were being measured (after 40-70 min incubation). The values for these currents are somewhat less than those recorded after 10-15 min incubation (50 vs. 75 sA cm-2). The short-circuit current, equivalent to a maximum net Na transport of about 2 pequiv cm-2 hr-1, if no other ion is being transported electrogenically remained constant throughout the first week of postnatal life. The net transport of Na also
D. R. FERGUSON AND OTHERS 502 remained approximately constant, at about 2 Isequiv cm2 hr-1, from day 1 to day 3 of post-natal life. This transport fell markedly by day 7, but this is mainly a reflexion of the unusually high backflux for Na measured in the absence of amiloride (Table 1). The net Na flux was significantly greater than the measured short-circuit current in the new-born pig (P < 0.001). This difference was not maintained in animals injected with canrenoate. TABLE 2. Net Na flux measured across distal colons taken from canrenoate-treated pigs. Values were measured directly or calculated from measurements of short-circuit current. Amiloride affects neither Na flux nor short-circuit current in canrenoate injected pigs (Table 1 and Fig. 4). Results obtained in the presence and absence of amiloride have therefore been pooled for the purpose of comparison (means ± S.E., no. of observations). P gives the probability that differences between these two estimates occur by chance
Short-circuit current, as Na flux (uaequiv cm-2 hr-1) 1-98±0-2
Age of pig (days) 0
Net Na flux (#equiv cm-2 hr-1)
(12)
(21)
1
2-76 ± 0-93
1-78 ± 0-31
(8)
(8)
1-99 ± 1-17 (7) 2-23+0-39
2-01 + 0-22 (13) 1-74+0-24
(8)
(8)
0-29+0-75 (8)
2-08±0-51
2
3 7
3-45+0-56
P < 0-001 n.s. n.s.
n.s. < 0-1 > 0-05
(8) DISCUSSION
Piglets need a plentiful supply of Na to sustain growth both during fetal and early post-natal development. The magnitude of the problem is, however, much greater in the case of the neonatal animal. The pig fetus increases in weight from 0-8 to 1-2 kg during the last 10 days of gestation and from 1-2 to 4 kg during the first 14 days of post-natal life. Assuming that 20 % of the pig's weight consists of extracellular fluid with a Na concentration of about 120 mm, then the fetal pig will need an extra 10 and the neonatal pig an extra 90 m-mole Na to sustain growth. Na will be provided at high concentration in blood coming from the placenta in the case of the fetal animal, but the post-natal animal will have to recover its sodium from the gastrointestinal tract following ingestion of sow's milk (Na concentration 20-30 mM, G. Bailes, personal communication). The efficiency with which the small intestine performs this task is also likely to be diminished during the immediate post-natal period, due to the endocytotic absorption of colostral antibodies (Henriques de Jesus & Smith, 1974). It seems hardly surprising that the pig should develop additional mechanisms for recovering Na from its large intestine under such circumstances. Further evidence for the importance of the colon in the neonatal pig has been provided recently by the work of Wilkinson & McCance (1971). They found that removal of the colon led to a massive loss of water and electrolytes, sufficient to kill in several instances. We can verify that ileostomy in the new-born pig produces
ALDOSTERONE INDUCED Na TRANSPORT 503 diarrhoea which increases in intensity shortly after ingestion of colostrum (M. W. Smith & K. A. Burton, unpublished results). Present work shows that an aldosterone antagonist can also produce diarrhoea through the partial inhibition of Na reabsorption in the colon. Aldosterone appears to be deeply implicated in organizing all these adaptive changes in Na transport. Its concentration in blood increases at a time when its presence seems most necessary. Virtually identical findings have been reported for the neonatal mouse (Dalle, Giry, Gay & Delost, 1978). It is also true, however, that the aldosterone concentration falls within 3 weeks of birth, to levels found in the fetal animal, even though the young pig is still continuing to grow at a rapid rate (McCance, 1974). There is also little doubt that aldosterone continues to play a useful role in modifying Na transport in adult animals, where growth no longer takes place (see Introduction for references). Further experiments on the rate of aldosterone metabolism and a parallel investigation of tissue receptors for this hormone, carried out at different times during the life of the pig, are necessary before coming to any more detailed conclusion as to how blood levels of the hormone might relate to its actual ability to affect Na reabsorption. The strongest evidence that aldosterone is involved in producing post-natal changes in colonic Na transport comes from the use of canrenoate. This drug has no effect on the circulating levels of hormone, but it does inhibit the post-natal increase in Na transport seen to take place under normal conditions. An operational definition of how Na might cross the pig distal colon during neonatal development has been outlined in the previous paper (Cremaschi et al. 1979). Canreonate blocks two of the three available mechanisms, one which is operationally neutral which disappears shortly after birth and a second, which is electrogenic and which increases in magnitude as the animal becomes older. The conclusion is that aldosterone is involved in the regulation of both these mechanisms. The neutral transport of Na, seen to be present in the colons of new-born and 1 day old animals, could result from an action of aldosterone on epithelial cells already formed at birth. The increase in the electrogenic transport of Na could result from an action of aldosterone on cells formed postnatally. Part of the Na transport across new-born and older colons appears to be electrogenic, but it is influenced neither by amiloride (Cremaschi et al. 1979) nor by the prior injection of canreonate (present results). The most likely explanation for these findings is that a constant fraction of Na transport takes place by a mechanism similar to that found in the mammalian small intestine. There appears to be a very close relation between the action of canrenoate in blocking the induction of an electrogenic transport system for Na and the action of amiloride in inhibiting this system once it has become fully induced. Effects of these two drugs on short-circuit current and Na transport are remarkably similar. It has already been suggested that aldosterone acts on high resistance epithelia to increase the number of amiloride sensitive Na sites present in the outward facing membrane (Cuthbert, Okpako & Shum, 1974; Cuthbert & Shum, 1976). Present results lend support to such a notion. It would now be very interesting to test this hypothesis further by direct measurement of amiloride binding sites in pig colons taken at different stages of development, particularly since it now appears that part of the action of aldosterone in the newborn animal is to induce a neutral transport system for Na, sensitive to inhibition by amiloride (Cremaschi et al. 1979).
504
D. R. FERGUSON AND OTHERS
We wish to express our thanks to Mr R. W. Ash for both supplying us with new-born pigs and for giving daily intramuscular injections of canrenoate.
REFERENCES
BENTLEY, P. J. & SMiTH, M. W. (1975). Transport of electrolytes across the helicoidal colon of the new-born pig. J. Physiol. 249, 103-117. CIEiscHI, D., FERGUSON, D. R., HAIAN, S., JA S, P. S., MEYER, J. & Saw, M. W. (1979). Post-natal development of amiloride sensitive sodium transport in pig distal colon. J. Phyeiol. 292, 481-494. CUTHBERT, A. WV., OKPAKO, D. & SHUM, W. K. (1974). Aldosterone and the number of sodium channels in the frog skin. Br. J. Pharmac. 51, 128-129P. CUTHBERT, A. W. & Siiuu, W. K. (1976). Estimation of the lifespan of amiloride binding sites in the membranes of toad bladder epithelial cells. J. Physiol. 255, 605-618. DAT.TLL, M., GIRY, J., GAY, M. & DELOST, P. (1978). Perinatal changes in plasma and adrenal corticosterone and aldosterone concentrations in the mouse. J. Endocr. 76, 303-309. EDMONDS, C. J. & GODFREY, R. C. (1970). Measurement of electrical potentials of the human rectum and pelvic colon in normal and aldosterone-treated patients. Gut 11, 330-337. EDMONDS, C. J. & MARRIOTT, J. C. (1967). The effect of aldosterone and adrenalectomy on the electrical potential difference of rat colon and on the transport of sodium, potassium, chloride and bicarbonate. J. Endoer. 39, 517-531. ERLANGER, B. F., BOREE, F., BEISER, S. M. & LIEBERMAN, S. (1957). Steroid-protein conjugates. 1. Preparation and characterization of conjugates of bovine serum albumin with testosterone and with cortisone. J. biol. Chem. 228, 713-727. FRIzzE7L, R. A. & SCHULTZ, S. G. (1978). Effect of aldosterone on ion transport by rabbit colon in vitro. J. membrane Biol. 39, 1-26. HENRIQuEs DE JESUS, C. & Sxrm, M. W. (1974). Sodium transport by the small intestine of new-born and suckling pigs. J. Physiol. 243, 211-224. JAMES, P. S. & SMITEi, M. W. (1977). Effect of ileostomy on changing transport function in the new-born pig colon. J. Phyeiol. 272, 61-62P. KREBS, H. A. & HENsELEIr, K. (1932). Tntersuchungen uber die Harnstoffbildung im Tier korper. Hoppe-Seyler'8 Z. physiol. Chem. 210, 33-66. LEvrrAN, R. & INGELFINGER, F'. J. (1965). Effect of D-aldosterone on salt and water absorption from the intact human colon. J. cdin. Invest. 44, 801-808. McCANcE, R. A. (1974). The effect of age on the weights and lengths of pigs' intestines. J. Anat. 117, 475-479. RAMSAY, L., SHELTON, J., HARRISON, I., TIDD, M. & ASBURY, M. (1976). Spironolactone and potassium canrenoate in normal man. Clin. Pharmac. Ther. 20, 167-177. S=IELDs, R., MoLOLAND, A. T. & ELMSLIE, R. G. (1966). Action of aldosterone upon the intestinal transport of potassium, sodium, and water. Gut 7, 686-696. WILKINSON, A. W. & McCANcE, R. A. (1971). The subsequent effects of removing the large intestine from newborn pigs. Proc. Nutr. Soc. 30, 26-27A. YoRIo, T. & BENTLEY, P. J. (1977). Permeability of the rabbit colon in vitro. Am. J. Physiol. 232, F5-9.
