Guanylin Stimulation of Cl- Secretion in Human Intestinal T84 Cells Via Cyclic Guanosine Monophosphate

Guanylin Stimulation of Cl- Secretion in Human Intestinal T84 Cells via Cyclic Guanosine Monophosphate

Leonard R. Forte, * * Sammy L. Eber, * * John T. Turner, * Ronald H. Freeman, Kam F. Fok,11 and Mark G. Curriell The Departments of *Pharmacology and §Physiology, Missouri University and tTruman VA Hospital, Columbia, Missouri 65212; and IlMonsanto Corporate Research, St. Louis, Missouri 63167

Abstract cells resulted in the appearance of a form of guanylate cyclase in membranes that was activated by Escherichia coli heat- Intestinal salt and fluid secretion is stimulated by Escherichia stable enterotoxin (ST).' The structure of this ST-activated coli heat-stable enterotoxins (ST) through activation of a guanylate cyclase was related to the atrial peptide-stimulated membrane guanylate cyclase found in the intestine. Guanylin is guanylate cyclases ( 1-3). E. coli ST is considered to be an an endogenous intestinal peptide that has structural similarity important cause of secretory diarrhea resulting from the activa- to the bacterial peptides. Synthetic preparations of guanylin or tion of intestinal guanylate cyclase. This bacterial peptide E. coli ST 5-17 stimulated Cl- secretion in Ts cells cultured on elicits both enhanced Cl- secretion and a reduction in Na+ semipermeable membranes as measured by increases in short absorption (4-6). E. coli ST-induced secretory diarrhea is as- circuit current (Isc). The guanylin/ST receptors appeared to sociated with substantial morbidity and mortality, especially in be on the apical surface of Ts cells, since addition of guanylin the children of developing nations (7, 8). to the apical, but not basolateral, reservoir stimulated Isc. Bu- The physiological significance of this ST-activated gua- metanide added to the basolateral side effectively inhibited the nylate cyclase in the regulation of intestinal salt and fluid trans- Isc responses of Ts cells to either guanylin or ST 5-17. Gua- port has been reemphasized because of the discovery of guanylin appeared to be about one-tenth as potent as ST in stimulat- nylin, an endogenous ligand for the intestinal guanylate cyclase ing transepithelial Cl- secretion. Guanylin and E. coli ST 5-17 (9). Guanylin was first isolated from rat jejunum with activa- both caused massive (> 1,000-fold) increases in cGMP levels tion of the T84 human intestinal cell guanylate cyclase serving in T84 cells, but guanylin was less potent than ST. Both peptides as the bioassay. It is a 15 amino acid peptide containing 4 fully inhibited the binding of I251-ST to receptor sites on intact cysteines and 2 disulfides that are required for biological activ- Ts cells. The radioligand binding data obtained with guanylin ity. Guanylin is structurally similar to E. coli ST and competes or ST 5-17 best fit a model predicting two receptors with differ- with '25I-ST for binding sites on intestinal T84 cells. In rat tis- ent affinity for these ligands. The Ki values for guanylin were sues, guanylin bioactivity was observed only in the intestine 19±5 nM and 1.3±0.5 ,uM, whereas the K1 values for ST 5-17 and kidney (9). A cDNA that was isolated from a rat intestine were 78±38 pM and 4.9±1.4 nM. We conclude that guanylin cDNA library contained an open reading frame that encodes a stimulated Cl- secretion via the second messenger, cGMP, in 1 5 amino acid precursor ofguanylin ( 10). Guanylin that was Ts4 human colon cells. At least two guanylin receptors with purified from rat intestine represented the carboxy-terminal 15 different affinities for these ligands may exist in the cultured amino acid portion of preproguanylin. A 600-nucleotide-long T84 cells. It may be postulated that guanylin is an endogenous mRNA for guanylin was expressed throughout the intestine, hormone that controls intestinal Cl- secretion by a paracrine but a gradient of guanylin mRNA was observed with the order mechanism via cGMP and that E. coli ST stimulates Cl- secre- of abundance being colon > ileum > jejunum > duodenum. tion by virtue of an opportunistic mechanism through activation No guanylin mRNA was found in stomach or esophagus. It is of guanylin receptors. (J. Clin. Invest. 1993. 91:2423-2428.) likely that guanylin controls cGMP production in tissues other Key words: guanylate cyclase * apical receptors * bumetanide- than intestine because kidney proximal tubules, testicular semi- E. coli heat-stable enterotoxin * secretory diarrhea niferous tubules, hepatocytes, and airway epithelium of the opossum express the ST-activated guanylate cyclase (1 1-14). In this study, we evaluated the effects of synthetic guanylin Introduction in comparison with synthetic E. coli ST on Cl- secretion, cGMP production, and inhibition of the binding of 125I-ST to In recent years, attention has been focused on the cGMP signal receptors on T84 intestinal cells. The T84 human colon carci- transduction pathway as an important mechanism involved in noma cell line has been shown to express receptors for E. coli the control of intestinal Cl - secretion and thus salt and fluid ST that are coupled to both the activation of guanylate cyclase transport. An intestinal membrane guanylate cyclase was re- and Cl- secretion (15-17). T84 cells are considered to be a cently identified by molecular cloning ( 1, 2). Expression ofthe suitable model for intestinal crypt cells with respect to the sev- specific cDNA encoding an intestinal guanylate cyclase in COS eral mechanisms controlling C1- secretion in the intestine ( 18- 21). Guanylin stimulated Cl- secretion, activated guanylate cyclase, and competed for binding sites labeled by '25I-ST in Address reprint requests to Leonard R. Forte, Ph.D., Department of cultured T&4 cells. Guanylin appeared as efficacious but less Pharmacology, Missouri University, Columbia, MO 65212. potent than E. coli ST in all of these bioassays using T84 cells. Receivedfor publication 9 June 1992 and in revisedform 8 January acts as a 1993. The results of this study suggest that guanylin local

The Journal of Clinical Investigation, Inc. 1. Abbreviations used in this paper: Isc, short circuit current; ST, E. coli Volume 91, June 1993, 2423-2428 heat-stable enterotoxin.

Guanylin-stimulated Cl- Secretion 2423 regulator of intestinal fluid and electrolyte transport through a obtained with a two-site binding model as compared with a single-site cGMP-guanylate cyclase signal transduction mechanism. model. The apparent equilibrium dissociation constants, Ki, for the competing ligands were calculated from the computed IC50 values by the method of Cheng and Prusoff (23) using previously published esti- Methods mates of the affinity of the radioligand, Kd, in these cells ( 15): Ki = IC5/ 1 + (L/Kd) where L equals the radioligand concentration. It Cell culture. Tg4 cells (passage 21 obtained from Dr. Jim McRoberts, should be noted that the calculated IC,0 and Ki values are essentially Torrance, CA) were cultured using DME and Ham's F12 ( 1:1 ) con- identical because the concentration of radioligand used in these studies taining 5% FBS, 60 ,gg penicillin, and 100 Mg streptomycin per ml. T94 (63 pM) was a small fraction of the reported binding affinity of the cells were seeded at 150,000 cells/ ml in plastic cultureware. These cells radioligand, about 15 nM. reached confluence in 3-5 d when cultured on plastic dishes. Subcul- Peptide synthesis. Rat guanylin (PNTCEICAYAACTGC, amino turing was accomplished using trypsin and Ca/Mg-free PBS by stan- acid sequence as designated by the single letter code) was prepared as dard methods. Falcon cell culture inserts ofpermeable cyclopore mem- described (9). Guanylin was synthesized by the solid-phase method branes, 25 mm diameter, 0.45 Mm pore size (Fisher Scientific, St. with a peptide synthesizer (430A; Applied Biosystems, Foster City, Louis, MO) were first coated with 0.25 ml collagen (Bovine Type I, 1.3 CA) on Cys(4-CH3Bzl)-OCH2-Pam Resin using double coupling cy- mg/ml; Sigma Chemical Co., St. Louis, MO) for 16 h, while the filters cles to ensure complete coupling at each step. Coupling was effected were being sterilized by UV irradiation in a tissue culture hood. Tg4 with preformed symmetrical anhydride of t-butoxycarbonyl-amino cells were seeded using 2.5-3.0 X 106 cells per filter in 2-ml medium acids (Applied Biosystems), and peptides were cleaved from the solid and cultured using the same medium described above for 7-14 d before support in hydrogen fluoride, dimethylsulfide, anisole, and p-thiocre- using them to measure Cl- secretion in Ussing chamber experiments. sol (8:1:1:0.5, vol/vol/vol/wt) at 0C for 60 min. Peptides were cy- The medium was changed 3 times per wk for T84 cells on plastic or clized using dimethylsulfoxide as described by Tam et al. (24). Pep- permeable supports. tides were purified by successive reverse-phase chromatography on a 45 Measurement of short circuit current in T84 monolayers. T84 cells x 300-mm Vydac C 18 column and on a 19 X 150-mm MBondapak C 18 raised on permeable filters were mounted in a custom-made Ussing column, using a gradient of 10-30% acetonitrile in 0.5% trifluoroacetic chamber for measurement of Cl- secretion as previously described acid. (22). The buffer was a Krebs-Ringer-bicarbonate solution, pH 7.4, E. coli ST 5-17 (CCELCCNPACAGC) was prepared by the solid- containing 10 mM glucose for both the apical and basolateral reservoirs phase method with a peptide synthesizer (430A; Applied Biosystems) (5 ml volume). The Ussing apparatus was water jacketed to maintain on Cys(4-CH3Bzl)-OCH2-phenylacetamidomethyl resin. After cou- the buffer temperature at 370C. Both reservoir buffer solutions were pling all the amino acids, the protected peptide resin (0.7 g) was treated mixed and oxygenated by bubbling 95% 02/5% CO2 through the me- with hydrogen fluoride/dimethylsulfide/anisole/p-thiocresol, dium. Short circuit current (Isc) was measured continuously, and po- 8:1:1:0.5, vol/vol/vol/wt, at 0°C for 60 min. The hydrogen fluoride tential difference across the epithelium was measured intermittently. was removed under reduced pressure, and the residue was washed with Resistance measurements were made by occasionally clamping the po- ethyl ether and ethyl acetate. ST 5-17 was extracted with 50% acetic tential difference to a known voltage and measuring the current re- acid (2 X 30 ml). The resulting solution was then diluted to 10% acetic quired to establish the potential. Resistance and conductance were cal- acid. The crude reduced form of ST 5-17 was purified on low pressure culated using Ohm's law. The Isc observed with T84 cells cultured on C 18 reverse-phase chromatography. The main fractions were collected permeable filters has been shown to be caused by net secretion of Cl - and lyophilized. The residue was dissolved, and 500 ml of water and 20 across the T 4 monolayer when cells were treated with Cl- secreto- ml of 0.5 M phosphate buffer (pH 8.2) was added. The solution was gogues such as VIP, PGEI, E. coli ST, forskolin, or carbachol ( 16-21 ). kept at room temperature with constant stirring and the pH was main- Assay ofcGMP in T84 cells. Tg4 cells were cultured in 35-mm plastic tained at pH 8.2 until no free mercapto groups were detected. The dishes, and the cGMP levels were measured in control or agonist-stimu- resulting peptide solution was acidified to pH 2.5 and was purified by lated cells by radioimmunoassay as previously described ( 1 1, 12, 22). high pressure C1 8 reverse-phase chromatography. Structure of ST 5-17 In brief, confluent monolayers of Tg4 cells in DME-Hepes, pH 7.4, was verified by electrospray mass spectrometry, gas-phase sequence were treated at 37°C with guanylin or E. coli ST or vehicle for 40 min. analysis, and amino acid composition analysis. At the end ofthe incubation, the medium and cells were treated with 50 Materials. Bumetanide, DME, and Hamm's F 12 media were ob- Ml of 70% perchloric acid to denature proteins and extract cellular tained from Sigma Chemical Co., and Na "2mI was purchased from Du- cGMP. The pH of the extract was adjusted to 7.0 with 10 N KOH and pont-New England (Boston, MA). FBS was obtained from Gibco Labo- centrifuged to remove cell debris. The supernatant was used to measure ratories, (Grand Island, NY). Other reagent grade chemicals were pur- total cGMP (cells plus medium) using a radioimmunoassay for cGMP. chased from Sigma Chemical Co. or Fisher Scientific. Radioligand binding experiments. Iodination of synthetic E. coli ST 1-18 (NSSNYCCELCCNPACTGCY, amino acid sequence-single letter code [Multiple Peptide Systems, San Diego, CA]) was accom- Results plished using lactoperoxidase as previously described ( 1 1, 12). '211-ST was purified by reverse-phase HPLC using a C18 column (3.9 mm X 30 T84 cells cultured on collagen-coated membranes were cm, MBondapak; Water Associates, Milford, MA). The binding of 125[I mounted into a modified Ussing chamber so that the effects of ST to receptors on T84 cells was measured using about 50,000 cpm of guanylin and ST on Cl- secretion could be measured by the 1251I-ST (12.7 fmol, 63 pM) per well of T84 cells cultured in 24-well changes in Isc. These cells appear to secrete Cl - without any net dishes. The medium was 0.2 ml of DME containing 15 mM 2-(N-mor- transport of other ions, thus Isc is proportional to the rate of pholino) Mes, pH 5.5. After incubation for 60 min at 370, the medium Cl- secretion 16-21 Treatment of T cells 1 1 ( ). 4 by adding 4M was aspirated and cells were washed two times with ml of ice-cold to the basolateral reservoir had little if effect on 1 guanylin any medium. The cells were then solubilized with N NaOH for measure- Isc addition of 1 to the buffer ment of radioactivity. Radioligand binding data comparing the effects (Fig. 1). However, ,uM guanylin of synthetic guanylin with synthetic E. coli ST 5-17 were analyzed with bathing the apical membrane elicited a marked and sustained the Inplot computer program (GraphPAD Software for Sci., San increase in Isc. Thus, guanylin receptors appear to be localized Diego, CA). The concentrations at which specific binding of the radio- to the apical surface of T84 cells cultured on permeable mem- ligand at each binding site was inhibited by 50%, ICM, were obtained by branes. nonlinear regression ofthe untransformed competition binding data. A The increased Isc produced by 1 ,uM guanylin added to the statistically significant better fit of the binding data was consistently apical side was rapidly reversed by the subsequent addition to

2424 Forte, Eber, Turner, Freeman, Fok, and Currie C 30- q the treatment of cell monolayers with either guanylin or ST 1- (Isc peak response > 130 MA/cm2, data not shown). Prior treatment of T84 cells with 1 MuM guanylin substantially in- -~I creased the Isc response to acetylcholine as compared with the responses of naive cells to acetylcholine (data not shown). Sim- treated with 100 - ilar results were obtained when T84 cells were 0 nM E. coli ST 5-17 followed by 100 AM bumetanide (Fig. 2 B). Synthetic ST 5-17 was used because this peptide has the mini- mum structure required to activate guanylate cyclase with a potency and efficacy equal to naturally occurring bacterial STs (9, 25). When 100 MM bumetanide was added before 1 AM 40 50 60 70 guanylin, this peptide caused only a small increase in Isc (Fig. Time, min 3). These data show that guanylin-activated and ST-stimulated upon the bumetanide-sensi- Figure 1. Guanylin stimulation of Isc in Tg cells by activation of Isc in T84 cells are both dependent apical receptors. T84 cells were cultured on collagen-coated mem- tive Na-K-2C1 cotransporter localized in the basolateral membranes and mounted in an Ussing chamber as described in Methods. brane of Tg4 cells (16). Acetylcholine, which stimulates Cl- These data are the ±SEM of 3 experiments. At the arrows, 1 uM secretion via a calcium/phosphoinositide signal transduction guanylin was added to the basolateral reservoir followed by the addi- pathway in T84 cells ( 17, 20), also had little effect on Isc when tion of 1 uM guanylin to the apical reservoir. Zero time was when bumetanide was present. 100 AM bumetanide did not com- the cells were initially mounted in the chamber. pletely block the Isc response to agonists, especially when bumetanide was added first (Fig. 3). Perhaps an additional Cl - transport pathway exists to accomplish the basolateral mem- the basolateral reservoir of 100MM bumetanide, an inhibitor of brane transport of Cl- such as Cl-/HCO- and Na+/H' ex- the Na-K-2C1 cotransporter (Fig. 2 A). Addition of 1 MM ace- changers. tylcholine to the basolateral reservoir caused only a small in- The potencies of guanylin or ST were assessed using a cu- crease in Isc after bumetanide treatment. Acetylcholine elicited mulative concentration-response protocol for these prepara- a marked increase in Isc in control TM cells when added after tions of rat guanylin and E. coli ST 5-17 (Fig. 4). Stimulation of Isc occurred at 10 nM guanylin, and subsequent addition of m4'0 100 or 1,000 nM guanylin elicited proportionally greater in- 0 2 creases in Isc. In comparison, E. coli ST 5-17 stimulated Isc at 1 C 30 nM, suggesting that this peptide was more potent than gua-

0- - nylin. The concentrations of guanylin or ST used in these ex- sCC , ,- , periments did not appear to elicit maximal responses, but these cm UZ 20 curves seem to be parallel with the potency of guanylin about

. Isc to 1 .aby *. one-tenth that of ST 5-17. The magnitude of responses 0 0 c MM guanylin and 100 nM E. coli ST 5-17 were similar in T84 10 .... ~ cells that had not been pretreated with lower concentrations of 0 ~~~.*.~ 4,r- these agonists (data not shown). The relative potencies ofguanylin and E. coli ST 5-17 were also assessed by measuring the accumulation of cGMP in T84 40 50 60 70 80 90 Time, min cells exposed to these peptides. Using this bioassay, guanylin appeared to be less potent than E. coli ST 5-17 (Fig. 5). These data suggest that guanylin and ST 5-17 have approximately

Z 4' equal efficacies for activation ofguanylate cyclase. The remark- at 0 50[ 8_m=9 50j- 40 C,' U 40 _i 30 * 0 E U 330 2c 0- 20 x01S 2 "2 . __ 4, 0 _A~~~~~~~~~~2. *ot--0 10 Il 10 0~~~~ I i v 40 50 60 70 80 90 Time, min 40 50 60 70 Time, min Figure 2. Effects of bumetanide on guanylin-stimulated Isc of T84 cells. See legend to Fig. 1 and Methods for details. These data are Figure 3. Bumetanide pretreatment inhibits the Isc response to gua- representative experiments of at least three separate experiments with nylin. These data are a representative experiment of at least three guanylin and ST 5-17 added to the apical reservoir and bumetanide separate experiments. Bumetanide was added to the basolateral and added to the basolateral reservoir. guanylin to the apical reservoir.

Guanylin-stimulated Cl- Secretion 2425 40 Figure 4. Comparison guanylin. of guanylin and E. coli o guanylin ST 5-17 potencies in X Guanylin

* ST 5-17 the stimulation of Isc in 0 T& cells. Synthetic rat 75 3030. / guanylin or ST 5-17 peptides were added to E the apical reservoir at o 50 10-1 5-min. intervals. 2L 20 - The lowest concentra- 25- u 7 tions shown above were in added first, followed by successive additions of the next con- C i- 10 highest 0 12 11 10 9 8 7 6 5 centrations. These data [PEPTIDE], -log M are the mean of five experiments with ST 5-17 Figure 6. Inhibition of '251I-ST 1-18 binding by guanylin and ST 5-17. and four experiments Binding of 125I-ST 1-18 to intact T84 cells was determined in the 0 with rat guanylin. The presence of the indicated concentrations of guanylin (A,) or ST 5-17 9 8 7 6 horizontal bars are 1/2 (.) as described in Methods. The values shown are the composite -log peptide M SEM for each point. data (±SEM) from four experiments performed in duplicate and are expressed as the percent of binding in the absence of competing ligand. The curves are the computer-derived best fits of the data to a two-site model. able magnitude of the increase in cGMP production elicited by The K, values are given in the text. these agonists should be emphasized. The cGMP levels in this experiment are plotted on a log scale in Fig. 5. 1 ,.M E. coli ST to a two-site model vs. a one-site model for both guanylin and 5-17 caused a 4,140-fold increase in cGMP, whereas 10 ptM ST 5-17 (P < 0.01 in both cases). Guanylin inhibited '25I-ST guanylin elicited a 1,490-fold increase in cGMP. Concentra- binding with Ki values of 19±5 nM (mean±SE) and 1.3±0.5 tions of guanylin greater than 10 juM were not used because it ,AM with 55±5% of the sites of the high affinity class. Inhibition consumed excessive amounts of this peptide. of binding by ST 5-17 was characterized by Ki values of 78±38 The effects of guanylin and ST 5-17 on '25I-ST binding to pM and 4.9±1.4 nM with 34±7% of the binding to the high intact T84 cells were evaluated in inhibition-binding assays in affinity site. order to determine the relative affinities of these peptides for the ST receptor. As shown in Fig. 6, radioligand binding was fully inhibited by both peptides in a concentration-dependent Discussion manner. Inhibition of 1251I-ST binding occurred over a wide Several key observations provided information suggesting that range of competing ligand concentrations, suggesting the pres- the intestinal membrane guanylate cyclase located in apical ence of two classes of binding sites labeled by 125I-ST with dif- membranes of enterocytes may be controlled by an endoge- ferent affinities for the two competing ligands. This observa- nous ligand. Discovery that E. coli ST was a potent activator of tion was supported by computer-assisted curve fitting, which intestinal guanylate cyclase and that cGMP could influence revealed a significantly better fit of the inhibition binding data intestinal fluid secretion was the first evidence for an endo-ST (5, 6). The observation that ST-stimulated guanylate cyclase was also found in opossum kidney proximal tubules and other 1000c epithelia of this species provided additional rationale that this enzyme would be regulated by an endogenous ligand instead * ST5.17 of a bacterial peptide (1 1-14). Molecular cloning of rat and hu- 1000 A Guanylin man cDNAs encoding a putative ST receptor protein with eC structural similarity to the atrial peptide receptor/guanylate E_ cyclases further supported the proposal that an endo-ST ex- 0.CL 100 isted ( 1, 2). Purification of the peptide, guanylin, from rat M. intestine using the T84 human intestinal cell cGMP response as a bioassay has provided a candidate for the endogenous hor- 10 mone that regulates intestinal C1- secretion (9). Data pre- sented in this manuscript are consistent with the hypothesis that 11 .' If guanylin serves this function in the intestine because syn- 0 10 9 8 7 6 . 5 thetic guanylin activates guanylate cyclase and stimulates Cl- -log peptide M secretion in cultured human intestinal T84 cells. Figure 5. Comparison of cGMP accumulation responses to guanylin Guanylin stimulated Cl- secretion, as reflected by the in- and E. coli ST 5-17. T8, cells cultured in 35-mm dishes were treated creased Isc of T84 cells, caused marked (> 1,000-fold) increases with the indicated concentrations of peptide agonists for 60 min. in in cGMP accumulation, and fully competed with '25I-ST for the presence of 1 mM 3-Isobutyl- l-methylxanthine (IBMX). These binding sites on T84 cells. The potency of guanylin for stimula- data are a representative experiment of duplicate dishes at each con- tion of cGMP production was about 1/ 100 of that observed centration of guanylin (Av) or ST 5-17 (v). with E. coli ST 5-17. However, the potency of guanylin in the

2426 Forte, Eber, Turner, Freeman, Fok, and Currie stimulation of Isc seemed appreciably closer to that of ST 5-17 tors appear to be relatively poorly conserved ( 1, 2), perhaps ( - 10-fold different). The relative binding affinities of gua- these two receptors represent different isoforms of intestinal nylin or E. coli ST 5-17 for '25I-ST binding sites generally guanylin/ST receptors. Our data and those of other investiga- agreed with the cGMP bioassay data. These differences in ap- tors (29) emphasize the possibility that guanylin/ST receptor parent potency could be due to differences in assay conditions heterogeneity may exist. It may be of interest that when the because T84 cells were cultured on collagen-coated filters for human guanylin /ST receptor was expressed in human embry- Cl- secretion measurements and cultured in plastic dishes for onic kidney 293 cells, the cGMP response of these cells to hu- cGMP responses or 1251I-ST binding assays. The expression of man guanylin, derived from COS cells transfected with a gua- guanylin receptors (i.e., guanylate cyclase-C, [1, 2]) may be nylin cDNA, was relatively poor with respect to both potency influenced, leading to different types and/or densities of apical and efficacy when compared with the cGMP response of T84 receptors in T84 cells cultured on permeable substrates. It is cells to guanylin (28). For example, 0.1 1.tM guanylin caused an unlikely that these potency differences are due to using a rat increase in the cGMP levels of T84 cells that was equivalent peptide in human intestinal cells, since rat and human guany- (40-50-fold) to the cGMP response of 293 cells to 10 ,M gua- lin peptides are highly conserved ( 10, 26-28). In the carboxyl- nylin. Moreover, the IC50 for guanylin inhibition of 125I-ST terminal 15 amino acids (i.e., amino acids 101-115 of prepro- binding to receptors on 293 cells was 0.1 M, whereas the Ki for guanylin) only one substitution was observed at position 102 guanylin inhibition of 1251-ST binding in T84 cells was 0.02 gM between rat and human peptides. Moreover, the potencies of for the high affinity site. It is possible that the expressed recep- rat and human guanylin 101-1 15 for increases in cGMP levels tor responds differently to guanylin than does the guanylin/ST of T84 cells were not different (26). The potency of human receptors found in T84 cells. Another possibility is that COS guanylin to increase Isc in the rat colon (26) was similar to the cells transfected with guanylin cDNA may produce an intrinsi- potency of rat guanylin on Isc of Tg4 cells in this study. In both cally less potent guanylin, perhaps resulting from inappropri- instances, threshold increases in Isc were observed at 10 nM ate disulfide pairing. Whether the control of intestinal Cl - guanylin. Another consideration is that the secreted form of channels by guanylin occurs through a single guanylate cyclase guanylin may be a longer peptide than the 15 amino acid pep- receptor (i.e., GC-C) or by a more complex mechanism awaits tide that was purified from rat jejunum (9). An acid-labile further elucidation of the guanylin receptor/signal transduc- bond between Asp 100 and Pro 10 1 would have yielded the 15 tion pathway. amino acid peptide upon boiling in acetic acid. Perhaps proteo- In summary, guanylin activated both guanylate cyclase and lytic processing ofproguanylin occurs before secretion using an Cl- secretion in T& human intestinal cells. Regulation of Cl- upstream site such as the arginine at position 93 or 94, which is secretion and thus fluid and salt secretion by the local release of conserved in rat, human, and mouse guanylin precursor se- guanylin at sites where this peptide can bind to its receptors on quences ( 10, 26-28). A much longer peptide would be gener- the apical membrane of intestinal crypt cells could be an im- ated by cleavage at the lysine pairs found at amino acids 54, 55 portant physiological mechanism. Activation of guanylin re- for rat and mouse or positions 37, 38 found in human or mouse ceptors by structurally similar peptides secreted by enteric bac- preproguanylin ( 10, 26-28). The potencies of longer forms of teria reveals an opportunistic mechanism, which helps to ex- guanylin that may be derived by cellular processing of the pre- plain the powerful pathophysiologic effects of E. coli ST on cursor peptide should be compared with guanylin 101-1 15 and intestinal salt and fluid secretion. ST before concluding that bacterial STs are inherently more potent than guanylin. If ST peptides are more potent than Acknowledgments guanylin, perhaps related to the additional pair of cysteines and the resulting disulfide bond in ST (25), the increased affinity of We thank Kristin Nelson and Judy Richey for excellent work in pre- ST for guanylin receptors would help to explain their powerful paring this manuscript. Also, we express our appreciation to Dr. Jim pathophysiological effects in vivo (7, 8). Whether STs are McRoberts for providing T& cells and his expert assistance in establish- "super" guanylinomimetic agonists is a postulate worthy of ing the Ussing chamber methodology. further This research was supported by the Medical Research Service, De- investigation. partment of Veterans Affairs, and by a Weldon Springs Research The finding that "25I-ST appears to label two binding sites Award, Missouri University, to L. R. Forte and R. H. Freeman. that have different affinities for guanylin or E. coli ST 5-17 raised the question of the relative contribution of these puta- References tively different receptors in the cellular responses to agonists. What contribution to the Cl- secretion response of Tg4 cells 1. Schulz, S., C. K. Green, P. S. T. Yuen, and D. L. Garbers. 1990. Guanylyl might occur as a result of these agonists binding to either the cyclase is a heat-stable enterotoxin receptor. Cell. 63:941-948. 2. De Sauvage, F. J., T. R. Camerato, and D. V. Goeddel. 1991. Primary high or low affinity receptors? Perhaps both receptors are re- structure and functional expression of the human receptor for Escherichia coli quired to elicit the Isc response to either guanylin or E. coli ST heat-stable enterotoxin. J. Biol. Chem. 266:17,912-17,918. 5-17. Both be or one 3. Chinkers, M., and D. L. Garbers. 1991. Signal transduction by guanylyl receptors could guanylate cyclases, only cyclases. Annu. Rev. Biochem. 60:553-575. may have this enzymatic activity. Recent experiments evaluat- 4. Field, M., L. H. Graf, W. J. Laird, and P. L. Smith. 1978. Heat-stable ing the binding of '25I-ST to rat intestinal brush border mem- enterotoxin of Escherichia coli: In vitro effects on guanylate cyclase activity, branes data consistent with two cyclic GMP concentration and ion transport in small intestine. Proc. Natl. Acad. provided receptors existing Sci. USA. 75:2800-2809. with different affinities for ST (29). Thus, heterogeneity of 5. Hughes, J. M., F. Murad, B. Chang, and R. L. Guerrant. 1978. Role of guanylin receptors may occur in target cells of the intestine. cyclic GMP in the action of heat-stable enterotoxin of Escherichia coli. Nature Presently, only one putative receptor for E. coli ST has been (Lond.). 271:755-756. 6. Nobles, M., M. Diener, and W. Rummel. 1991. Segment-specific effects of identified by molecular cloning ( 1-3). However, since the ex- heat-stable enterotoxin of E. coli on electrolyte transport in the rat colon. Eur. J. tracellular ligand-binding domains of the rat and human recep- Pharmacol. 202:201-21 1.

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2428 Forte, Eber, Turner, Freeman, Fok, and Currie