936 Gut, 1991, 32, 936-940 Segmental variability of glucocorticoid induced

electrolyte transport in rat colon Gut: first published as 10.1136/gut.32.8.936 on 1 August 1991. Downloaded from

G I Sandle

Abstract within colonic mucosa'), reduce diarrhoea in Recent studies suggest that the ability ofgluco- active colitis by stimulating distal colonic Na+ corticoids to reduce diarrhoea in active colitis and water absorption as well as by suppressing may reflect their direct effects on distal colonic the underlying inflammatory process. It remains electrogenic Na+ transport and water absorp- unclear, however, whether glucocorticoids also tion, as well as their anti-inflammatory action. stimulate electrogenic Na+ and water absorption To determine whether glucocorticoids induce in human proximal colon. similar changes in proximal colon, specific Na+ The assessment of glucocorticoid induced and K+ channel blockers (amiloride and tetra- electrolyte transport in different segments of ethylammonium chloride (TEA) respectively) human colon in vivo poses a number of technical were used to evaluate the cation transport problems and would provide little information properties of rat proximal and distal colon in about changes in the biophysical properties ofthe vitro after three days treatment with the gluco- colonic mucosa. The present study was therefore corticoid agonist dexamethasone (600 [tg/100 designed to determine the effects of long term g/day). In the proximal colon, dexamethasone dexamethasone treatment on the cell membrane increased short circuit current (Isc) 2-3 fold barriers and forces controlling Na+ and K+ (p<0025) and total conductance (G) by 87% transport in rat proximal and distal colon. The (pagonists form Mg2` 1-2; HCO3- 25; H2PO4- 1-2; SO424 1-2; and the cornerstone of treatment in patients with glucose 11 1 and was maintained at 37°C and pH acute inflammatory disease of the large bowel.' 7-4 by gassing with a 95% 02:5% CO2 mixture. Until recently the efficacy of glucocorticoids was The colon was stripped of serosa and muscle assumed to reflect their well known anti-in- layers, and a 2 cm piece of distal colon (that flammatory effects but it is now clear that segment beginning 4 cm from the anus) or pharmacological doses ofglucocorticoids admin- proximal colon (that segment immediately Manchester Epithelial istered parenterally enhance rectal sodium (Na') adjacent to the caecum) was mounted in an Membrane Research Centre and Department and water absorption to the same extent in Ussing type chamber modified to eliminate tissue of Medicine, University patients with active ulcerative colitis and in edge damage." Tissue area was 1 cm2. Tissues of Manchester normal subjects.4 It is also apparent that the were bathed on both sides with 12 ml NaCl G I Sandle predominant mechanism for Na+ absorption in Ringer solution and were temperature and pH Correspondence to: Dr G I Sandle, Department of human distal colon and rectum is electrogenic,56 regulated as described above. Electrical measure- Medicine, Hope Hospital, and stimulated by both mineralocorticoid and ments were obtained under open circuit condi- Eccles Old Road, Salford M6 8HD. glucocorticoid agonists.47 These observations tions. Transepithelial voltage (V,) was monitored Accepted for publication suggest that glucocorticoids (which at high doses continuously with salt bridges (4% agar in 0 5 3 September 1991 bind substantially to mineralocorticoid receptors mol/l KCI) placed on either side of the tissue and Segmental variability ofglucocorticoid induced electrolyte transport in rat colon 937

connected to a voltmeter via balanced calomel three minutes. Pump activity was evaluated from half cells. Using Ohm's law, total tissue conduc- the increases in Isc measured at increasing tance (G,) was calculated from the increase in V, mucosal Na+ concentrations, using an iterative that occurred in response to a rectangular current least squares routine to fit the data to a mathe- Gut: first published as 10.1136/gut.32.8.936 on 1 August 1991. Downloaded from pulse (120 [iA/cm2, 2-5 seconds duration) applied matical model describing the highly cooperative across the mucosa via an Anapulse stimulator binding of Na+ to Na+-K+ pump sites.'4 This (WPI, New Haven, CT, USA). The equivalent model yields several kinetic parameters: Isc,max short circuit current (Isc) was calculated as =apparent maximum short circuit current, Isc=V,.G,. KNa=Na+ concentration at which the slope of Isc Baseline electrical measurements were on Na+ concentration is maximal, and n (Hill obtained after 20-25 minutes when V, and Gt coefficient)=the minimum number of Na+ ions were stable. Effects of the Na+ channel blocker binding to each Na+-K+ pump site. amiloride (final concentration Od mmol/l) and Student's t test (two tailed) was used to make the K+ channel blocker tetraethylammonium statistical comparisons between groups (non- chloride (TEA; final concentration 30 mmol/l) paired test), and to assess the significance of the were determined by adding the drugs sequen- changes induced by amiloride and TEA (paired tially to the mucosal solution. Electrical measure- test). ments were made five minutes after the amiloride and TEA (in the presence ofamiloride) had been added when Vt and Gt were stable. Results Dexamethasone treatment stimulated an amiloride insensitive increase in Isc in the proxi- EFFECTS OF DEXAMETHASONE ON BASELINE mal colon (see Results) which suggested that the ELECTRICAL PROPERTIES glucocorticoid induced an electrogenic anion The basal electrical properties of proximal and secretory process in this segment. Similar distal colon from control and dexamethasone changes were not observed, however, in the treated animals are summarised in Table I. distal colon. To study this further, tissues were In control animals, Isc was similar in the bathed in NaCl Ringer solution and 0-1 mmol/l proximal and distal colon, but V, was signifi- amiloride was added to the mucosal solution to cantly greater in the distal colon owing to the inhibit any component of Isc which may have lower electrical conductance (G,) in this segment. reflected electrogenic Na+ transport. The Cl- In the proximal colon, dexamethasone increased conductive properties of the apical membrane Isc and G, by 129% and 87% respectively above were then evaluated by measuring changes in Isc control values, while V, was unchanged. In

produced by adding the Cl- channel blocker contrast, in the distal colon dexamethasone http://gut.bmj.com/ diphenylamine-2-carboxylic acid (final concen- increased Isc 10 fold and G, by 100%, resulting tration 2 5 mmol/l) to the mucosal solution. in a 4 5 fold increase in Vt above the control Basolateral Na+-K+ pump activity was assessed value. Thus, while dexamethasone increased G, as described previously. 12 Tissues were bathed on in the proximal and distal colon, stimulation of both sides in a high K+, Na+ and Cl- free solution Isc (and V,) was far more pronounced in the distal containing (in mmol/1): K+ 140; HCO3- 25; Ca2+ segment.

10 (methane sulphonate); Mg2+ 1 2; H2P04- 1-2; on September 30, 2021 by guest. Protected copyright. MeSO3- 20; gluconate 113-8; and glucose 11-1. Nystatin was added to the mucosal solution (final EFFECTS OF AMILORIDE AND TEA concentration 960 U/ml) to render the apical The effects of amiloride and TEA in proximal membrane freely permeable to monovalent and distal colon from control and dexamethasone ions,'3 and under these conditions Vt and Isc were treated animals are shown in Figure 1. zero. Amiloride (0-1 mmol/l) was added to the In proximal colon from control animals, the mucosal solution to block native Na+ channels, mucosal addition of amiloride and TEA had and the Na+ concentration ofthe serosal and then no effect on V,, Gt or Isc, indicating that apical the mucosal solution raised in 10 mmol/l incre- Na+ and K+ conductances were negligible or ments (using a stock Na+ gluconate solution), absent. to a final concentration of 50 mmol/l. Serosal In proximal colon from dexamethasone treated addition of Na+ had no effect, but increasing the animals, in which baseline Gt was higher than in mucosal (and intracellular) Na+ concentration controls (Table I), amiloride and TEA had no increased Vt and Isc to stable values after two to effect on V,, G, or Isc (Fig 1), which suggests that the dexamethasone stimulated rise in baseline G, in this segment reflected the induction of a TABLE I Baseline electrical measurements in proximal and distal colonfrom control and conductive pathway for ions other than Na+ and dexamethasone treated animals (values mean (SEM)) K VI G, Isc In distal colon from control animals, amiloride (mV) (mS/Cm2) (pAlcm2) produced small decreases in V, (2-6 mV, Proximal colon: p<005) but no change in G, or Isc, and TEA was Control rats (n= 10) -2 5 (0-4) 19-3 (3-4) 45 (12) indicate that in this Dexamethasone treated rats (n= 10) -3 4 (0 7) 36-1 (5 8) 103 (18) without effect. These results p** NS <0-015 <0 025 group oftissues, apical cation conductances were Distal colon: case of or absent Control rats (n=12) -7 3 (0 8)* 8-8 (0 8)* 62(8) relatively small (in the Na+) (in Dexamethasone treated rats (n=9) -33 0 (1-3) 17-7 (2-6) 618 (130) the case of K+). p** <0-01 <0-015 <0 0025 In contrast to the other three groups, amiloride noticeable in the V,=transepithelial voltage (negative with respect to serosal solution); G,=total conductance; and TEA produced changes Isc=calculated short circut current; n= number of tissues studied. electrical properties of distal colon from dexa- *= <0 05 compared with proximal colon from control animals. **=difference between control and dexamethasone treated tissues. methasone treated animals. The addition of 938 Sandle

Proximal Distal EFFECTS OF DIPHENYLAMINE-2-CARBOXYLIC ACID Proximal Dexamethasone Distal Dexamethasone (DPC) Control treated Control treated Bathed in NaCl Ringer solution, proximal and (n = 10) (n = 10) (n= 12) (n = 9) distal colonic segments from control animals Gut: first published as 10.1136/gut.32.8.936 on 1 August 1991. Downloaded from -40- generally show a variable but modest Isc which may reflect, at least in part, the Cl- secretory tone -30- of the tissues, as Isc decreases when Cl- is Vt (mV) -20- replaced with gluconate." Dexamethasone pro- -10- duced an amiloride and TEA insensitive increase 0--j... in Isc in the proximal colon (Table I and Fig 1), consistent with the stimulation ofan electrogenic 45 Cl- secretory process. The effects of the Cl- 40- channel blocker DPC on the amiloride insensi- 35 tive component of Isc were therefore studied in proximal and distal colon from control and Gt 30- dexamethasone treated animals, and the results (mS/cm2) 25- are summarised in Table II. 20- In distal colon from control and dexametha- sone treated animals, and proximal colon from 15- I control animals, DPC produced relatively mod- 10- est decreases in amiloride insensitive Isc, consis- 5 tent with the blockade ofa small native apical Cl- conductance in each of these three groups. In 0 contrast, the high amiloride insensitive Isc in 700- proximal colon from dexamethasone treated ani- ' mals (mean 139 (SEM) (16) iA/cm2) was abol- Isc 600-600-* ished by DPC, which suggests that dexa- (gA/cm2) 500 methasone induced a substantial apical Cl- 400- conductance in the proximal segment. 300- 200- EFFECTS OF DEXAMETHASONE ON THE BASOLATERAL Na+-K+ PUMP Figure 2 shows the changes in Isc that occurred http://gut.bmj.com/ Figure 1: Effects ofamiloride (0 1 mmolIl) and tetraethylammonium chloride (TEA, 30 in response to 10 mmol/l increases in the mucosal mmol/l) on transepithelial measurements in proximal and distal colonfrom control and Na+ concentration in colonic segments from dexamethasone treated animals. Mean (SEM) values presented are those under baseline control and dexamethasone treated animals. The conditions (open bars), in the presence ofamiloride (filled bars), and in the presence of amiloride plus TEA (stippled bars). V, G, and Isc are defined infootnote to Table I. kinetic parameters derived from the best fit *p<005 compared with basal or pre-amiloride value; **p<0.05 compared with value in the curves (Fig 2) are summarised in Table III. In the presence ofamiloride alone (Bonferroni correction applied to p values). n=number oftissues proximal colon, Isc at each concentration of Na+ studied. tended to be greater in the dexamethasone treated than in the control animals, although on September 30, 2021 by guest. Protected copyright. these differences were not significant, and Isc, amiloride decreased Vt by 27-3 mV (p<0O05), max, KNa, and n were similar in the two groups. which reflected appreciable decreases in Isc (561 In the distal colon, however, Isc at each concen- [tA/cm2, p

TABLE II Effects ofdiphenylamine-2-carboxylic acid (DPC) on amiloride insensitive calculated short circuit current (Isc) in proximal and distal colon from control and dexamethasone treated animals (values mean (SEM)) Isc(pA/cm') Isc(pAlcm,') pre-DPC post-DPC p Mucosal Na+ concentration (mmoVl) Proximal colon: Figure 2: Response ofshort circuit current (Isc) to increasing Control rats (n=4) 74 (12) 9 (7) <0 05 mucosal concentrations ofNa' in control (O-0 , n=5) and Dexamethasone treated rats (n=7) 139 (16) 3 (2) <0 0001 dexarnethasone-treated (a- , n= 7) proximal colon, and Distal colon: control (L-L, n=6) and dexamethasone-treated (U-U, Control rats (n=4) 22 (3) 6 (2) <0-025 n=8) distal colon. Each point represents the mean (SEM) of Dexamethasone treated rats (n=5) 30 (6) 14 (1) 0 06 the data at each concentration ofNa' and the curves are best n=number of tissues studied. Steady state measurements of Isc were obtained with tissues bathed in fits as described by the model ofhighly cooperative binding NaCl Ringer solution (in the presence of 0-1 mmol/l mucosal amiloride) before and after the addition (see Methods). Bestfit valuesfor the curves are presented in of 2-5 mmol/l DPC to the mucosal side. Table III. Segmental variability ofglucocorticoid induced electrolyte transport in rat colon 939

TABLE iII Kinetics ofthe basolateral Na+-K4 pump in proximal and distal colon from the proximal colon is also consistent with the control and dexamethasone treated animals (values mean (SEM)) glucocorticoid's lack of effect on electrogenic

Isc,max K cation transport in this segment. This notion is Gut: first published as 10.1136/gut.32.8.936 on 1 August 1991. Downloaded from (pAlcm2) (mmolil) n supported by other studies in rat proximal Proximal colon: colon, which have shown that mineralocorticoid Control rats (n=5) 59 (150) 18 (4) 1-7 (0 2) receptor activation by aldosterone stimulates Dexamethasone treated rats (n=7) 81 (10) 13 (2) 2-2 (0-2) p** NS NS NS electroneutral Na-Cl (not electrogenic Na+) Distal colon: absorption,'7 and an active K+ secretory process Controlrats(n=6) 70 (12) 11(1) 2-4 (0 3) Dexamethasone treated rats (n=8) 211 (49) 14 (1) 2-3 (0-3) which is less noticeable than that stimulated in p** <0 05 NS NS rat distal colon.'8" In contrast, the specific gluco- corticoid receptor agonist RU 28362 (which also Isc,max=the apparent maximum short circuit current; KN,.=Na' concentration at which slope of Isc on Na+ concentration is maximal; n (Hill coefficient)=the minimum number of Na+ ions binding to stimulates electroneutral Na-Cl absorption) has each Na+-K+ pump site; n=number of tissues studied. no effect on net K+ secretion.'8 Thus, the results **=difference between control and dexamethasone treated tissues. ofthe present and these other studies suggest that dexamethasone activated both mineralocorticoid significantly (p80% occupancy of mineralocorticoid The spectrum of N;a+ transport processes receptors in addition to activating glucocorticoid present in rat colon differs from that in human receptors. Indeed, it is likely that stimulation colon. The predominant Na+ transport processes on September 30, 2021 by guest. Protected copyright. of distal colonic Na+ and K+ transport by in rat colon are electroneutral Na+-H+ exchange dexamethasone mainly reflects activation of in the proximal segment'7 and electroneutral Na- mineralocorticoid receptors, as the electrical Cl absorption in the distal segment.922 In con- changes induced by high doses ofdexamethasone trast, there is appreciable electroneutral Na-Cl resemble those induced by aldosterone rather absorption in the proximal (ascending), trans- than those induced by similar high doses of RU verse, and distal (descending) segments of 28362, a specific glucocorticoid agonist for which human colon.2324 In addition, electrogenic Na' mineralocorticoid receptors have negligible absorption is present throughout human colon,7 affinity.'0 although this process only becomes noticeably Although the effects of chronic dexametha- amiloride sensitive distal to the splenic flexure.7 25 sone administration on Na+ and K+ transport Despite these segmental and species differences across rat distal colon have been described previ- in baseline Na+ transport mechanisms, there is ously,910 the present study indicates that the evidence that suggests that similar changes in proximal colon responded quite differently to a colonic Na+ transport may be induced in rat and combination of mineralocorticoid and gluco- human colon by glucocorticoid agonists that' corticoid receptor activation by dexamethasone. activate both glucocorticoid and mineralocor- Compared with control animals, dexamethasone ticoid receptors - for example dexamethasone, induced an appreciable increase in G, in the and methylprednisolone and hydrocortisone, proximal colon, but the associated increase in Isc both of which are used commonly in the treat- was less than that induced in the distal colon (58 ment ofinflammatory bowel disease. Thus, in rat [tA/cm2 versus 566 [tA/cm2, Table I). The proximal colon, glucocorticoid and mineralo- insensitivity of dexamethasone treated proximal corticoid receptor activation stimulates electro- colon to amiloride and TEA (Fig 1) suggests that neutral Na-Cl absorption without the appearance the dexamethasone induced increases in G, and of amiloride sensitive electrogenic Na+ absorp- Isc observed in this segment reflected stimulation tion.' It seems likely that a similar response of an electrogenic ion transport process(es) other may occur in human proximal colon, which is than electrogenic Na+ absorption (or electrogenic normally characterised by electroneutral Na-Cl K+ secretion). The inability ofdexamethasone to absorption2 24 and where purely mineralo- enhance basolateral Na4-K4 pump activity in corticoid receptor activation by aldosterone fails 940 Sandk

to induce amiloride sensitive electrogenic Na+ 7 Sandle GI. Segmental heterogeneity of basal and aldosterone- induced electrogenic Na transport in human colon. Pflugers absorption.7 In contrast, in rat distal colon and Arch 1989; 414: 706-12. human distal colon and rectum, glucocorticoid 8 Basti CP. Regulation of cation transport by low doses of glucocorticoids in in vivo adrenalectomized rat colon.J Clin Gut: first published as 10.1136/gut.32.8.936 on 1 August 1991. Downloaded from agonists - for example dexamethasone, methyl- Invest 1987; 80: 348-56. prednisolone, hydrocortisone - stimulate amilor- 9 Foster ES, Zimmerman TW, Hayslett JP, Binder HJ. Corti- costeroid alteration ofactive electrolyte transport in rat distal ide sensitive electrogenic Na+ absorption via colon. AmJ Physiol 1983; 245: G668-75. mineralocorticoid 10 and may also 10 Binder HJ, McGlone F, Sandle GI. Effects of corticosteroid receptors4 hormones on the electrophysiology of rat distal colon: enhance electroneutral Na-Cl absorption via implications for Na' and K' transport. J Physiol 1989; 410: glucocorticoid receptors.'9 425-41. 11 Lewis SA, Eaton DC, Diamond JM. Nystatin as a probe for In summary, it seems likely that systemically investigating the electrical properties ofa tight epithelium.J administered glucocorticoids reduce diarrhoea in Gen Physiol 1977; 70: 427-40. 12 Wills NK, Lewis SA, Eaton DC. Active and passive properties active colitis by exerting direct effects on Na+ of rabbit descending colon. A microelectrode and nystatin (and hence water) absorption throughout the study.J MembrBiol 1979; 45: 81-108. 13 Cass A, Finkelstein A, Krespi V. The ion permeability induced colon, as well as by their better recognised anti- in thin lipid membranes by polyene antibiotics nystatin and inflammatory action. Glucocorticoids may amphotericin. J Gen Physiol 1970; 50: 100-24. 14 Nelson MT, Blaustein MP. Properties of sodium pumps in stimulate Na+ absorption in normal and inflamed internally perfused barnacle muscle fibres. J Gen Physiol distal colon and rectum4 through a combination 1980; 75: 183-206. 15 Sandle GI, McGlone F. Segmental variability of membrane of glucocorticoid receptor activation (enhancing conductances in rat and human colon epithelia. Implications electroneutral Na-Cl absorption) and crossover for Na, K and Cl transport. Pflugers Arch 1987; 410: 173-80. 16 Kashgarian M, Taylor CR, Binder HJ, Hayslett JP. Amplifica- binding to mineralocorticoid receptors (en- tion of cell membrane surface in potassium adaptation. Lab hancing electrogenic Na+ absorption). In human Invest 1980; 42: 5R1-8. 17 Foster ES, Budinger ME, Hayslett JP, Binder HJ. Ion proximal colon, glucocorticoids may only transport in proximal colon of the rat. Sodium depletion stimulate electroneutral Na-Cl absorption, stimulates neutral sodium chloride absorption. J Clin Invest 1986; 77: 228-35. despite binding to both types of corticosteroid 18 Turnamian SG, Binder HJ. Aldosterone and glucocorticoid- receptor. specific agonists regulate ion transport in rat proximal colon. AmJ Physiol 1990; 258: G492-98. 19 Turnamian SG, Binder HJ. Regulation of active sodium and potassium transport in the distal colon of the rat. Role of the aldosterone and glucocorticoid receptors.J Clin Invest 1989; Dr Sandle was a Medical Research Council Senior Clinical Fellow. 84: 1924-9. 20 Yuan-Heng T, Decker RA, Marnane WG, Charney AN, Donowitz M. Effects of methylprednisolone on electrolyte transport by in vitro rat ileum. Am J Physiol 1981; 240: 1 Truelove S, Witts LJ. Cortisone and corticotrophin in ulcera- G365-70. tive colitis. BMJ 1959; i: 387-94. 21 Sellin JH, Field M. Physiologic and pharmacologic effects of 2 Summers RW, Switz DM, Sessions JT, et al. National glucocorticoids on ion transport across rabbit ileal mucosa in Cooperative Crohn's Disease Study: results of drug treat- vitro. J Clin Invest 1981; 67: 770-8. ment. Gastroenterology 1979; 77: 847-69. 22 Halevy J, Budinger ME, Hayslett JP, Binder HJ. Role of 3 Malchow H, Ewe K, Brandes JW, et al. European Cooperative aldosterone in the regulation of sodium and chloride trans- http://gut.bmj.com/ Crohn's Disease Study (ECCDS): results of drug treatment. port in the distal colon of sodium-depleted rats. Gastroen- Gastroenterology 1984; 86: 249-66. terology 1986; 91: 1227-33. 4 Sandle GI, Hayslett JP, Binder HJ. Effect of glucocorticoids 23 Hubel KA, Renquist K, Shirazi S. Ion transport in human on rectal transport in normal subjects and patients with cecum, transverse colon, and sigmoid colon in vitro. Base- ulcerative colitis. Gut 1986; 27: 309-16. line and response to electrical stimulation ofintrinsic nerves. 5 Hawker PC, Mashiter KE, Turnberg LA. Mechanisms of Gastroenterology 1987; 92: 501-7. transport of Na, Cl and K in the human colon. Gastro- 24 Sellin JH, DeSoignie R. Ion transport in human colon in vitro. enterology 1978; 74: 1241-7. Gastroenterology 1987; 93: 441-8. 6 Wills NK, Alles WP, Sandle GI, Binder HJ. Apical membrane 25 Sandle GI, Wills NK, Alles W, Binder HJ. Electrophysiology properties and amiloride binding kinetics of the human of the human colon: evidence of segmental heterogeneity. descending colon. Am J Physiol 1984; 247: G749-57. Gut 1986; 27: 999-1005. on September 30, 2021 by guest. Protected copyright.