The effect of atrial natriuretic peptide on intestinal electrolyte transport

Regulatory Peptides, 36 (1991) 29-44

29

© 1991 Elsevier Science Publishers B.V. All rights reserved 0167-0115/91/$03.50

REGPEP 01086

The effect of atrial natriuretic peptide on intestinal electrolyte transport A n t h o n y G. Catto-Smith, James A. Hardin, M a r k K. Patrick, E d w a r d V. O'Loughlin and D. G r a n t Gall Intestinal Disease Research Unit, University of Calgary, Calgary, Alberta (Canada) (Received 1 March 1991; revised version received and accepted 18 June 1991)

Key words: Atrial natriuretic peptide, ANP; Intestine; Electrolyte transport

Summary

The effect of atrial natriuretic peptide (ANP) on rat small intestinal electrolyte transport was examined. In vivo, intravenous administration of rat ANP(99-126) induced diuresis and natriuresis in conjunction with a significant decrease in intestinal water (basal, 37.1 + 5.7 versus ANP 28.5 + 6.0 #l/cm per 20 rain, P < 0.05) and Na + (4.0 + 0.7 versus 2.8 _+ 0.9 #mol/cm per 20 rain, P < 0.05) absorption (n = 9). In vitro, in Ussing chambers, in both jejunum and ileum, addition of 1.0 #M ANP to short circuited, stripped tissue produced a maximal increase in short circuit current and stimulated net C1- secretion due to a significant increase in the unidirectional serosal to mucosal flux (JsCm 1-" jejunum 17.4 + 1.3 versus 19.8 _+ 1.3 #Eq/cm z per h, P < 0.01, n = 6; ileum 13.4 _+ 0.5 versus 17.2 _+ 0.6, P < 0.01, n = 6) which was inhibited by the calcium channel antagonist verapamil (82 + 26 Y/o,P < 0.05) and by the 5-HT 2 receptor antagonist cinanserin (72 + 44~o, P < 0.05). Guanylate cyclase activity was stimulated by ANP in intact epithelium, but not in isolated crypt and villus enterocytes.

Introduction

Atrial natriuretic peptide (ANP) is produced by cardiocytes in response to distension and has been shown to modulate renal excretion of water and electrolytes and inhibit renin and aldosterone secretion [ 1]. The renin-angiotensin-aldosterone axis plays a Correspondence: D.G. Gall, Department of Pediatrics, Health Science Centre, 3330 Hospital Drive N.W., Calgary, Alberta, T2N 4N 1 Canada.

30 major role in the regulation of water and electrolyte balance as well as the maintenance of blood pressure. Both renal and gastrointestinal mechanisms, which result in water and sodium retention by this system, have been described [2]. The existence of a complementary hormone or third factor, which offset these effects was postulated. Early volume expansion experiments demonstrated both natriuresis and diuresis [3,4] as well as decreased intestinal absorption of water and Na + [5-7]. The description by De Bold et al. [8] of a natriuretic and vasorelaxant peptide secreted by cardiac atria confirmed the existence of a third factor in water and Na + homeostasis. Local production of ANP has been shown in the intestine [9], and binding sites for [~251]ANP( 101-126) have been demonstrated in the rat duodenum, jejunum and ileum [ 10,11 ], primarily on fibroblast-like cells in lamina propria, but also at the base of mature epithelial cells, although not associated with crypt cells [ 11 ]. ANP has been shown to activate particulate guanylate cyclase in rat intestine [ 12], and many of its actions in other tissues have been linked to changes in cyclic G M P [13,14]. Stimulation of guanylate cyclase in the intestine has been shown to result in marked reduction in coupled Na-CI absorption, thought to occur across intestinal villus cells, and to a lesser degree, an increase in active C1- secretion, thought to arise largely from cells in the crypts [15]. The preponderance of ANP-receptor binding to ceils within the lamina propria suggested that any effect that ANP had on mucosal epithelial transport might be mediated through some secondary mediator released from cells within the lamina propria. Cellular and neural components of the intestinal lamina propria have an important role in the modulation of intestinal electrolyte transport. Fibroblasts [ 16], mast cells [17] and the enteric nervous system [18] may all induce intestinal C1secretion. Previous studies have produced contradictory evidence on the functional significance of ANP in the small intestine, showing either an increase [ 19] or inhibition [20] of intestinal Na ÷ and water absorption. The present study was designed to further evaluate the effects and mechanisms of action of ANP on small intestinal electrolyte transport.

Materials and Methods

Jejunal in vivo perfusion. Studies were performed using a single pass perfusion technique as previously described [21]. Non-fasted, female Hooded-Lister rats weighing 200-300g were anesthetized with intramuscular urethane 1.25g/kg. Following a tracheostomy the left jugular vein was cannulated and infused with normal saline at 20/~l/min and the urinary bladder cannulated for urine collection. A 10-15 cm segment of jejunum, starting at 5 cm distal to the ligament of Treitz, was isolated and gently perfused with normal saline to clear residual intestinal contents. Proximal and distal ends were cannulated and the intestine perfused at a constant rate of 0.15 ml/min. The perfusate contained 140 mM Na + , 5 mM K + , 120 mM CI-, 25 mM H C O 3 , 3 g/1 polyethylene glycol 4000, and 10#Ci/l of [14C]polyethylene glycol 4000 as the non-absorbable marker, osmolality 300 mOsm and pH 7.4 at 37 °C. Intraluminal hydrostatic pressure was monitored continuously and was less than 3 cm H20.

31 Following a 60 min equilibration period, three consecutive 20 min baseline urine and intestinal perfusate samples were collected. Experimental animals were then given synthetic rat ANP(99-126) by intravenous infusion over 1 min at 1.25 nmol/100 g body weight in 0.5 ml normal saline, while controls received saline alone, and two further 20 min samples were collected. All samples were collected in chilled preweighed tubes and analyzed for volume by weight, Na + and K + by flame spectrophotometry, CIby chloride analyzer, and [ ~4C]polyethylene glycol 4000 by r-scintillation spectrophotometry. Polyethylene glycol recovery ranged from 95 ~ to 105 ~ . On completion of the experiment the perfused intestinal segment was removed and weighed, its length measured, and the kidneys removed and weighed. Net intestinal fluxes of H20, Na +, K ÷ and C1- were calculated as previously described [21,22] and expressed as #1 and #mol/cm intestine per 20 min. Renal water and Na ÷ excretion were expressed as #1 and #mol/g kidney per 20 min respectively [8]. In vitro ionflux studies. Non-fasted rats were anesthetized with sodium pentobarbitol (7 mg/100 g body weight) and 10 to 15 cm segments of either jejunum, beginning 5 cm distal to the ligament of Treitz, or of ileum, extending proximally from 5 cm proximal to the cecum, removed and flushed with Kreb's solution. The mucosa was stripped of the underlying muscle and serosa and four adjacent pieces of mucosa, that did not contain Peyer's patches, mounted in Ussing-type chambers with apertures of 0.4 cm2. Mucosal and serosal surfaces were bathed with 10 ml of oxygenated Kreb's buffer (115 mM NaC1, 2.0 mM KH2PO 4, 1.1 mM MgC12, 1.25 mM CaC12, 25 mM NaHCO 3, 8 mM KC1 and pH 7.35 at 37 °C). In addition, the serosal buffer contained 10 mM glucose and the mucosal buffer 10 mM mannitol. The spontaneous potential difference (PD) was determined and the tissue clamped at zero voltage by introducing an appropriate short circuit current (Is¢) with an automatic voltage clamp (DVC 1000; World Precision Instruments, New Haven, CT) The lsc was continuously monitored and PD measured by briefly removing the voltage clamp for 5-10 s every 5 min. Tissue conductance (G) was calculated by Ohms law [23]. PD is expressed as millivolts (mV), Is¢ as microampere/cm2 (#A/cm 2) or #Eq/cm 2 per h and G as millisiemen/cm 2 (mS/cm2). Tissue pairs were discarded if conductance varied by more than 25~o. Radioisotopes, 10 #Ci 22Na÷ and 5/~Ci 36C1-, were added to either the mucosal or serosal reservoir immediately after mounting, and the tissue allowed to equilibrate for 20 rain. Unidirectional mucosa to serosa (Jm~), serosa to mucosa (J~m), and net ('/net) Na ÷ and C1- fluxes were first determined during a basal period, by measuring three consecutive 5 min fluxes and one overall 15 rain flux and then for a further three consecutive five min fluxes and overall 15 min flux starting 2 min after the addition of ANP in 0.25 ~o acetic acid, final concentration 1.0 #M, to the serosal side. Fluxes were used only if the tissue was in steady state as evidenced by the 5 min fluxes. Initial studies indicated that ANP, when added to jejunum and ileum, produced an increase in I~¢ which was dose-dependent. Dose-response relationships for I~ were established separately for jejunum and ileum, and flux studies carried out with a dose that produced a maximal Is¢ response. Preliminary experiments demonstrated that electrical parameters and Na ÷ and CI- fluxes did not differ significantly between the two flux periods with the addition of the vehicle alone. Fluxes were calculated as previously described [23,24] and expressed as/~Eq/cm 2 per h.

32

Effect of pharmacological inhibitors. The initial flux studies indicated that the significant effect of ANP on intestinal transport involved an increase in the serosal to mucosal CI- flux with no change in the mucosal to serosal flux. Subsequent experiments designed to further elucidate the mechanisms by which ANP mediates C1- secretion used only changes in serosal to mucosal CI- flux as a marker of ANP action. To assess the role of putative mediators and of the enteric nervous system in the intestinal ANP response, the effect of receptor blockade and pharmacological inhibition on the C1- secretion induced by ANP was examined [17]. Receptor antagonists used to study the role of histamine were diphenhydramine (Hi-receptors) and cimetidine (H2). The 5-hydroxytryptamine antagonists used were methysergide (5-HT1), cinaserin (5-HT2) and ICS-205 930 (5-HT3). Calcium dependence was examined with the Ca2 ÷ channel blocker verapamil and neural dependence with the Na ÷ channel blocker tetrodotoxin. The role of araehidonic acid metabolites was studied with the cycloxygenase inhibitor indomethacin, and the 5-1ipoxygenase inhibitor L-651 392. The effect of mast cell degranulation was studied with the mast cell stabilizer doxantrazole. Ileal mucosal tissue was matched by conductance and paired. Radioisotope, 5 #Ci 36C1-, was added to the serosal reservoir immediately after mounting and 5 rain later the pharmacological inhibitor with vehicle, or vehicle alone, added to the serosal surface of the matched tissues. After 20 min equilibration, unidirectional serosa to mucosa (Jsm) CI - fluxes were determined during a basal period by measuring three consecutive 5 min fluxes and one overall 15 min flux, and then for a further three consecutive 5 min fluxes and overall 15 min flux starting 2 min after the addition of ANP, final concentration of 1.0 #M, to the serosal side. All reagents were dissolved in water except indomethacin, which was dissolved in 100mM Tris-buffer (pH 8.0) and L651392 which was suspended in 0.0001 ~o dimethylsulfoxide. In experiments employing tetrodotoxin, complete nerve block was confirmed, at the end of the second flux period, by transmural field stimulation [ 17]. Cyclic nucleotides. In preliminary experiments the effect of ANP on cyclic nucleotides was examined in intact ileum. Non-fasted female Hooded-Lister rats weighing 200-300 g were anesthetized with sodium pentobarbitol (7 mg/100 g body weight), and 10 cm segments of ileum extending proximally from 5 cm before the cecum removed and flushed with cold Kreb's solution. The unstripped ileum was cut into 1 cm strips, and placed in 100 ml of Kreb's solution, 10 mM glucose and 0.2 mM IBMX, gassed with 95~/o O2/5~o CO2,and incubated for 20 min at 37 °C. 1 #M ANP, or vehicle alone was then added to the reaction vessels. Tissue strips were removed after 2.5 min and placed in 95 °C Tris-EDTA (50 mM Tris-HCl, 4 mM EDTA) for 2 min. The tissue strips were then removed, 1 ml of Tris-EDTA was added, and the tissue strips were then homogenized, and heated in boiling water for 3 min. A 100 #1 aliquot was removed for protein estimation [25 ] and the remaining sample was then cooled and spun at 3000 rpm for 15 min. The supernatant was removed, and cAMP and cGMP measured using commercially available RIA kits (RPA 525 and RPA 508, Amersham, Oakville, ON). Assays were done in duplicate. Escherichia coli heat-stable enterotoxin (STa) (Sigma) at 1 ng/ml was used as a positive control. Preliminary experiments established that ANP caused an increase in cGMP, but not of cAMP. To define the cellular origin of the

33 increase in c G M P the effect of ANP on cyclic nucleotide levels was examined in isolated enterocytes. Villus, mixed and crypt cell populations were isolated from rat ileum by a mechanical vibration technique as previously described [26]. Each fraction was centrifuged at 800 rpm for 2 min at 4 ° C, the cell pellet obtained washed once with isolation solution then placed on ice. Aliquots of suspended cells from each fraction were immediately used to assess the effect of ANP and E. coli heat-stable enterotoxin on guanylate cyclase activity. The remaining cells from each fraction were homogenized in 2.5 mM EDTA, flash frozen and stored at - 70 ° C for later enzyme and protein analysis. The composition of successive fractions was verified by activity of sucrase and thymidine kinase [26-28]. In the guanylate cyclase experiments, 1 ml of cells suspended in isolation solution from each fraction was added in duplicate to plastic falcon tubes containing 4 ml of Kreb's buffer, with 5 mM glucose (pH 7.4), at 37 °C in a shaking waterbath and then equilibrated for 5 min under a flow of 95~o 0 2 / 5 % CO2. Either 1.0 #M ANP in 0.25 ~o acetic acid or 20 ng/ml E. coli heat-stable enterotoxin was then added to the experimental tubes and the appropriate vehicle added to the control tubes. The tubes were allowed to shake for 90 s at 37 °C and were then centrifuged at 1500 rpm for 2 min at 4 ° C and 4 ml of supernatant drawn off. The cell pellet was then suspended in ice-cold ethanol to give a final suspension volume of 65 ~o ethanol, vortexed slightly and allowed to settle. The supernatant was drawn off and the remaining precipitate resuspended in ice-cold 65 ~o ethanol, vortexed lightly and then centrifuged for 2 min at 1500 rpm, 4 ° C. The supernatant was again poured off, combined, and spun at 2800 rpm for 15 min to remove protein. The supernatant was then transferred to new polypropylene tubes and dried at 60 °C under a stream of nitrogen to remove ethanol. Samples were reconstituted in 1.0 ml of assay buffer prior to determination of c G M P by radioimmunoassay as described above. Cell pellets were homogenized in 1.0 ml 2.5 mM EDTA, flash frozen and stored at - 70 °C for later DNA analysis. DNA content was measured using calf thymus D N A as standard [29]. Materials. ANP (rat sequence 99-126), diphenhydramine, cimetidine, indomethacin, tetrodotoxin and verapamil, were purchased from Sigma (St. Louis, MO). Cinanserin was purchased from Squibb (Princetone, Carolina, PR) and methysergide from Sandoz (Switzerland). ICS-205 930CH was agift from Sandoz AG, and L-651392 from Merck Frosst Canada (Pointe Clair-Dorval, Quebec, Canada). Doxantrazole was a gift from Burroughs Wellcome Co. (Research Triangle Park, Bedford, MA). Statistics. Data was expressed as mean + S.E., and statistical analyses were performed by Student's t-test, paired t-test or analysis of variance for repeated measures where appropriate.

Results

Renal and jejunal response in vivo Intravenous infusion of ANP resulted in an immediate and significant increase in urinary water and Na ÷ excretion (Fig. 1). The renal response was paralleled by a significant decrease in jejunal water, Na + and C1 - absorption. The renal and intestinal

34

INTESTINAL

T

5.o

H~O

2.¢

K+ o.:

RENAL llkJ 180 140 120 IO0

H~O

T

(-)

(÷)

(-)

(÷)

Fig. 1. In vivo net fluxes of water, Na*, C1- and K ÷ in jejunum, and urinary water and sodium excretion, during basal conditions (open bars), after intravenous saline ( - ) or ANP ( + ) (double-hatched bars), and during recovery period (hatched bars). Experimental animals (n = 9), received 1.25 nmol/kg of ANP in 0.5 ml saline intravenously. Control animals (n = 5) received saline alone. Each bar represents the mean + S.E. in #1 or ~mol/cm per 20 min for jejunal fluxes, and #1 or/~mol/g kidney per 20 min for renal excretion. *P < 0.05 compared to basal period by analysis of variance for repeated measures.

responses were rapid and transient. The changes in intestinal water, N a ÷ and C1transport occurred in the 20 min period immediately following A N P injection and then values returned to basal levels. I n control animals, intravenous infusion of saline alone did not significantly alter either renal or intestinal handling of water, N a +, K + or C 1 - .

Intestinal response in vitro Dose-response curves were established for the effect of A N P on peak Isc u n d e r voltage clamped conditions for concentrations ranging from 0.01 to 4.0 # M of A N P in

35 3025c~ E -~.

20' 15.

o

10 ¸


5¸

1

10 -s

1(~-;'

[ANP]

1(~ "6

M

Fig. 2. Dose-response curve for the effect of ANP on short circuit current (1,c) in ileum mounted in

Ussing-type chambers. Concentrations of ANP are molar, lsc reported as #A/cm 2. Each point represents at least four experiments. Values are mean + S.E. The calculated EDso is (0.75 + 0.39)" 10-7 M.

chamber fluid (Fig. 2). An effect on Isc was seen with a concentration of 0.25 # M in jejunum and ileum. A maximal response of Isc in both jejunum, 6 + 1 #A/cm 2, and ileum, 21 + 6, (Fig. 3) was seen with an ANP dose of 1.0 #M. This dose and higher concentrations produced a significant increase in Is¢ which was maximal after three min and remained significantly elevated for greater than 10 min. Unidirectional, mucosal to serosal (Jms), serosal to mucosal (Jsm) and net (Jnet) Na + and C1- fluxes in jejunal and ileal epithelium, are presented in Table I. ANP, at a concentration of 1.0 #M, stimulated an active C1- secretory process. In the jejunum C1- secretion was !ncreased, while in the ileum net C1- movement was converted from absorption to secretion. In both jejunum and ileum Jnet Cl- was increased due to a significant increase in the unidirectional serosal to mucosal flux, Jsm a - • There was no change in JCmls . ANP, under short-circuited conditions, did not alter net Na + transport in either jejunum or ileum, but unidirectional fluxes were increased. In the ileum both JNsa+ and --sm'/Na+ were significantly increased. In the jejunum --ms'lNa÷ was significantly increased; the increase in J~m~÷ did not reach statistical significance (P = 0.058). ANP also increased Is¢, as shown, and G in both jejunum and ileum and PD in the jejunum (Table I). The residual flux, Jnet, R was reduced by ANP, significantly so in the ileum. 30. ~'E

20 . . . . . . .

~.

15"

o

10.

Control

, , ~

rI

-,,

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-5

0

5

10

TIME (rnin)

Fig. 3. The effect of 1.0 # M A N P (n = 4), added at time indicated by the arrow, on Isc (#A/cm 2) in ileum mounted in Ussing-type chambers. Control tissue (n = 7) received vehicle alone. Values are mean + S.E., * P < 0.05 compared to control by analysis of variance for repeated measures.

36

"I. -I. -'I-L ~1

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j~

I

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37

z 0

200.

150 ,<

0-J z 0 ~

100

50

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CIN

ICS

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SPECIFIC ANTAGONIST

Fig. 4. The effect of receptor blockade on the 1.0 # M A N P stimulated CI- secretory response (J~>-m) in ileum. Results are shown as % inhibition compared to matched tissues exposed to vehicle alone. Each bar represents the mean + S.E. The effects of 10- 4 M methysergide (METH), 10- 5 M cinanserin (CIN), 1 0 - s M ICS-205930 (ICS), 10- 5 M diphenhydramine (DPH), and 10- 4 cimetidine (CIMET), are shown. Cinanserin (10 - 5 M), the 5-HT 2 receptor antagonist, produced a significant inhibition of the A N P response. n > 5 in each group. *P < 0.01 by paired t-test.

Effect of inhibitors In order to assess the effect of inhibitors in matched tissues, only a single unidirectional flux was measured. The J,ca m- was used as this was the CI- flux affected by ANP. The degree of blockade is expressed a.s the percent inhibition of the ANPinduced increase in J,~- after pharmacological inhibitor or receptor antagonist administration compared to the increase in J,~- of matched tissue treated with ANP alone. The effect of 5-hydroxytryptamine and histamine receptor antagonists on the ANP induced CI- secretory response is shown in Fig. 4. Cinanserin (10-5 M), a 5-HT 2 receptor antagonist with little 5-HT~ receptor effect [30], produced significant inhibition (72 + 44~o, P < 0.05) of the ANP response. Methyserglde (10-4 M), a 5-HT~ receptor

200z o ~ Z2:

150-

1O0 -

o

50

0 VER

INDO

L651

]-rx

DOX

SPECIFIC ANTAGONIST

Fig. 5. Pharmacological inhibition of the 1.0 # M A N P stimulated CI- secretory response in ileum. Results are shown as % inhibition compared to matched tissues exposed to vehicle alone. The effects of 1 0 - 3 M verapamil (VER), 1 0 - 5 M indomethacin (INDO), 1 0 - 7 M tetrodotoxin (TTX), 1 0 - 3 M doxantrazole (DOX), and 1 0 - 6 M L651392 (L651), are shown. Each bar represents the mean + S.E. Verapamil ( 1 0 - 3 M), the Ca 2 + channel blocker, produced a significant inhibition of the A N P response, n > 5 for each group. *P < 0.05 by paired t-test.

38 antagonist [30] and ICS-205 930 ( 1 0 - 5 M), a 5-HT 3 receptor antagonist [31] did not inhibit the C1- secretion induced by A N P . Diphenhydramine (10 - 5 M), an H~ receptor antagonist, and cimetidine ( 1 0 - 4 M), an H 2 receptor antagonist, also had no effect on the A N P induced stimulation of C I - secretion. The effect o f pharmacologic inhibition on the A N P response is shown in Fig 5. Verapamil ( 1 0 - 3 M), a calcium channel antagonist, significantly inhibited (82 + 26~o, P < 0.05) the CI - secretion induced by A N P . To assess the role of arachidonic metabolites in the A N P response, tissue was pretreated with inhibitors of cyclooxygenase and 5-1ipoxygenase activity. Indomethacin (10 - 3 M), a cycloxygenase inhibitor and L651 392 (10 - 6 M), a 5-1ipoxygenase inhibitor [32], did not inhibit the A N P induced stimulation of C1- secretion. The role of the enteric nervous system was investigated with the nerve blocker, tetrodotoxin. Tetrodotoxin ( 2 . 1 0 - 7 M), a N a ÷ channel antagonist, did not inhibit the A N P induced C1- secretion. The presence of complete nerve block was confirmed by transmural field stimulation after completing the A N P sampling period. The possibility that the A N P effect on C1- secretion might be mediated through mast cells was investigated by pretreating tissue with doxantrazole in a dose ( 1 0 - 3 M) which stabilizes both mucosal and connective tissue mast cells [33]. Doxantrazole did not inhibit the C1- secretion induced by A N P .

Cyclic nucleotides In intact intestinal tissue A N P significantly stimulated guanylate cyclase activity (basal: 67 + 6 pmol o f c G M P / g protein, n = 6; A N P : 250 + 49 pmol/g protein, n = 7, P < 0.01). Heat-stable enterotoxin also increased c G M P levels (506 + 37 pmol/g protein, P < 0 . 0 0 1 ) . A N P had no effect on adenylate cyclase activity (basal: 6.8 + 1.4 pmol/g protein, n = 6; A N P : 6.5 + 1.5 pmol/g protein, n = 6). In contrast, A N P had no effect on c G M P levels in isolated enterocytes. The c G M P values obtained after incubating the three enterocyte populations with either A N P , E. coli heat-stable enterotoxin, or vehicle are shown in Table II. A N P did not affect c G M P levels in villus,

TABLE II cCMP levels of isolated enterocytes incubated with ANP or heat stable enterotoxin (STa) Fraction villus

mixed

crypt

ANF Control (n = 5)

0.17 + 0.03 0.18 + 0.05 n.s.

0.34 + 0.13 0.25 + 0.05 n.s.

0.26 + 0.04 0.27 + 0.04 n.s.

STa Control (n = 3)

0.16 + 0.04 0.17 + 0.06 n.s.

0.48 + 0.26 0.22 + 0.03 n.s.

0.65 + 0.03 0.30 _+0.01 P < 0.05

Each value represents the mean + S.E. Cyclic GMP is expressed as fmol cGMP per #g DNA. Cells were incubated with either 1.0 #M ANP or 20 ng/ml heat-stable enterotoxin (STa), and the appropriate vehicle used as a control. P values are compared to control, n.s., not significant by Student's t-test.

39 mixed or crypt cells. Heat-stable enterotoxin caused a significant increase in c G M P in crypt cells. Enzyme profdes confirmed the isolation of separate populations of villus, mixed and crypt enterocytes. Sucrase activity was high (41 + 6 U/g protein) in the In'st (villus) fraction, intermediate (28 + 6) in the second (mixed) fraction and low (15 + 3, P < 0.05 compared to villus fraction, n = 9) in the third (crypt) fraction. In contrast, thymidine kinase was low (3.8 + 0.8 pmol thymidine phosphate/min per mg protein) in the first, intermediate in the second (6.5 + 0.6, P < 0.01 compared to villus fraction), and high in the third (8.5 + 1.1, P < 0.01 compared to villus fraction, n = 9) fraction. The enzyme profile was characteristic of differentiated villus cells in the first fraction, and undifferentiated crypt cells in the third fraction, while the second fraction displayed characteristics of a mixed population.

Discussion The data from the present study provides further information on the role of ANP in fluid and electrolyte homeostasis. Intravenous administration of ANP in vivo not only caused significant diuresis and natriuresis, but also resulted in decreased jejunal water, Na + and CI- absorption. When studied in vitro under short-circuited conditions the addition of ANP to the serosal side resulted in an increase in Isc due to the stimulation of an active C1- secretory process in both jejunum and ileum. The increase in Js~induced by ANP was inhibited by the Ca 2 + channel antagonist verapamil, and by the 5-HT a antagonist cinaserin. The ANP stimulation of active CI- secretion was due solely to an increase in the unidirectional serosal to mucosal C1- flux. The increase in I~c was less than expected based on the change in transepithelial ion fluxes. In the jejunum, the increase in CIsecretion elicited by ANP was 1.8 #Eq/cm 2 per h compared to an I~¢ response of 0.5 #Eq/cm 2 per h. In the ileum, the change in CI- movement was 3.0 #Eq/cm 2 per h and Is¢ 0.5 #Eq/cm 2 per h. The intestinal response to ANP was associated with a decrease in Jnet" R The reduction in Jnet R suggests reduced secretion or increased absorption of an anion, presumably H C O f . Movement of HCO3- appears to account for the majority of the residual flux in the small intestine. The mammalian intestine both secretes and absorbs H C O f , while movement of K + is thought to occur by passive mechanisms [2]. The basis for the increase in unidirectional Na + fluxes is unclear. The increase in bidirectional Na + movement may represent an increase in epithelial conductance of Na +. The associated increase in conductance also suggests an increase in membrane permeability. This increase appears to be related to Na + channels only since unidirectional mucosa to serosa C1 - fluxes j a - , were not altered. Such an alteration would be beneficial in increasing Na + excretion in response to the induced C1- secretion. Other hormones involved in Na + homeostasis, for example aldosterone, are recognized to alter apical membrane permeability [34]. Previous authors have shown that acute extracellular volume expansion in dogs [ 5] and rats [6,7], results in decreased intestinal absorption as well as natriuresis and diuresis. Subsequent studies implicated ANP in the natriuretic effect of acute volume

40 expansion [35-37]. Studies on the effect of ANP on mammalian intestinal transport have produced conflicting results. Following intravenous injection of crude atrial extract, intestinal absorption of water, Na + and glucose was decreased in the rat [20]. The effect appeared to be maximal between 20 and 40 min after injection of the atrial extract, much longer than the recognized duration of the diuretic and natriuretic effects of ANP [38]. In contrast, Kanal et al. [ 19] observed an increased absorption of water, Na + , C1 - and K + following direct infusion of synthetic ANP(99-126) into the superior mesenteric artery of the rat. They attributed their findings to probable hemodynamic effects of ANP on the intestinal vascular bed. ANP increases intestinal blood flow and decreases intestinal vascular resistance, in vivo, in the rat [39]. In rat colon, there is evidence that ANP produces changes in mucosal ionic transport through involvement of the enteric nervous system [40]. In addition, there is evidence that ANP decreases angiotensin-induced aldosterone secretion [41] which in turn may decrease angiotensininfluenced intestinal absorption [42]. ANP has been shown to alter ion transport in epithelial tissue from lower vertebrates. In the shark rectal gland, ANP stimulates C1- secretion in vivo and in vitro [43]. This effect is thought to be mediated through release of VIP from neural stores within the gland [44]. In the flounder intestine, ANP also reduces electrolyte absorption, but the effect appears to be related to inhibition of Na-K-CI cotransport [45]. These tissues appear to be more responsive to ANP than rat intestine. Concentrations of 10 nM or less elicited an effect in the shark rectal gland and flounder intestine, while higher concentrations were required in the rat. In the in vivo perfusion studies the intravenous infusion of ANP at 1.25 nmol/100 g body weight would lead to an ANP concentration in the extracellular fluid of about 50 nM based on an extracellular volume of 25 ml/100 g body weight. In vitro a minimal concentration of 250 nM was required to elicit an effect on Isc. There are several possible explanations for the need for a higher concentration of ANP in vitro. Firstly, the effect of ANP on Isc is not as dramatic as the effect on C1secretion. As indicated above, the change in ion flux is only partially reflected by the change in Isc. Therefore, higher concentrations might be required to demonstrate an effect on Isc. Secondly, stripping of the mucosa may have reduced the sensitivity of the tissue, if, as suspected, lamina propria cells are involved in the intestinal epithelial response to ANP. Two different receptors for ANP have been identified. One receptor activates membrane-bound particulate guanylate cyclase, and the other has effects which are poorly defined and appear to be independent of guanylate cyclase [46]. Previous authors [12] have demonstrated that ANP activates membrane-bound guanylate cyclase in intestinal homogenates, and that administration of ANP is followed by increased circulating c G M P which appears to be derived from the small intestine [47]. In mammalian small intestine, an increase in guanylate cyclase-cGMP causes a decrease in linked NaC1 mucosal to serosal flux without a clear increase in the serosal to mucosal C1 - flux [48], although there may be a small increase in electrogenic CI - secretion [ 18]. We found no evidence that ANP diminished mucosal to serosal fluxes of Na + and C1 under short-circuited conditions. In the cyclic nucleotide experiments ANP did increase c G M P levels in intact ileal tissue as previously described [ 12]. However, when examined in isolated enterocytes ANP had no effect on c G M P levels. Our findings demonstrating

41 similar cGMP levels in villus, mixed and crypt derived enterocytes are in agreement to that previously described [49]. The role of activation of guanylate cyclase by ANP in the intact intestinal response is thus unclear. Further, recent evidence indicates that ANP receptors, at least in jejunal epithelium, are confined to mature enterocytes on the villi [ 11 ], a non-secreting tissue. These findings suggest that the CI - secretory process induced by ANP may not represent a direct effect on epithelial cells but rather is secondary to a process activated in lamina propria cells, where the majority of ANP receptors are located associated with fibroblast-like cells [11 ]. There is increasing evidence that cells within the lamina propria and the enteric nervous system are capable of modulating mucosal electrolyte transport by a variety of mechanisms. Both immune and non-immune ceils have been identified as influencing mucosal ionic transport. In enterocyte cell cultures, co-culture of fibroblasts appears to be necessary for 5-hydroxytryptamine to induce an increase in short circuit current [ 16]. The mechanism by which this is mediated is uncertain. It is possible that arachidonic acid metabolites are involved. The secretory effect of arachidonic acid metabolites is thought to be mediated through adenylate cyclase [50]. We found no evidence of ANP mediated stimulation of adenylate cyclase. In addition, the lack of effect of cyclooxygenase and 5-1ipoxygenase inhibitors on the ANP response suggests that arachidonic acid metabolites do not play a role in ANP-mediated small intestinal C1- secretion. 5-Hydroxytryptamine appears to play an important role in ANP stimulation of active C1- secretion. Cinaserin, which is primarily a 5-HT2-receptor antagonist, significantly reduced the response to ANP. Although previous work suggested that a small component of the transport changes induced by 5-HT in intestinal mucosa is mediated by the enteric nervous system [ 17], in the ANP response the 5-HT-dependent component does not appear to be neurally mediated. Tetrodotoxin had no inhibitory effect on the ANP response. Recent work has established the presence of 5-HT binding sites on intestinal epithelial cells [51 ]. The receptor is of the 5-HT 2 subtype. The presence of this receptor on enterocytes suggests that 5-HT can have a direct effect on the epithelium and is not dependent on the enteric nervous system. In conclusion, ANP inhibits intestinal absorption of water and Na ÷ in vivo. The effect appears to be due to the stimulation of electrogenic CI- secretion. Our data support the concept that the influence of ANP on mucosal transport is at a complex and heterogeneous level. An intact lamina propria is necessary for the stimulation of guanylate cyclase by ANP in the intestine. The observations in our present study suggest that the C1- secretion mediated by ANP is dependent on 5-hydroxytryptamine, possibly released from cells within the lamina propria. These results indicate that ANP may have a physiological role in modulating intestinal fluid and electrolyte transport.

Acknowledgements This work was supported by the Medical Research Council of Canada. M. Patrick and E.V. O'Loughlin were recipients of Alberta Heritage Foundation for Medical Research Fellowships.

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