FEBS 18603
`
`FEBSLetters 408 (1997) 345-349
`
`Dual regulation of heat-stable enterotoxin-mediated cGMP accumulation
`in T84 cells by receptor desensitization and increased phosphodiesterase
`activity
`
`Manjiri M. Bakre, Sandhya S. Visweswariah*
`Center For Reproductive Biology and Molecular Endocrinology, Indian Institute of Science, Bangalore 560012, India
`Received 10 March 1997
`
`
`the regulation of cGMP accumulation
`Abstract We report
`induced by the heat-stable enterotoxin, STh, in the T84 human
`colonic cell line. STh binding to its receptor, guanylyl cyclase C
`(GCC),
`leads to elevated intracellular
`levels of cGMP.
`Prolonged exposure of T84 cells to STh induced refractoriness
`to further cGMP accumulation, without significant receptor
`internalization, but with reduced STh-induced cGMP synthesis
`by the receptor. Significantly, increased degradation of cGMP
`by a cGMP-specific phosphodiesterase was observed in desensi-
`tized cells. This is the first report on the desensitization of GCC,
`as well as the role of the Type V phosphodiesterase in inducing
`cellular refractoriness.
`© 1997 Federation of European Biochemical Societies.
`
`Key words: Guanylyl cyclase C; Heat-stable enterotoxins;
`Cellular refractoriness; cGMP-binding, cGMP-specific
`phosphodiesterase
`
`1. Introduction
`
`The heat-stable enterotoxins (ST) are a family of low mo-
`lecular weight, methanol soluble, cysteine rich peptides, pro-
`duced by E. coli and other pathogenic bacteria, that cause
`diarrhea in farm animals and in man [1]. Binding of the toxin
`to a receptor present in intestinal cells leads to elevation of
`intracellular cGMP and a consequentefflux of chloride and
`fluid from the cells, resulting in diarrhea [2]. The receptor for
`E. coli STh, GCC, has been cloned from rat and human in-
`testinal cells [3,4], as well as from human colonic cell lines [5].
`Based on sequence homology studies predicted from the nu-
`cleotide sequence of the cloned ST receptors, the ST receptor
`(GCC) is a member of the family of membrane bound gua-
`nylyl cyclases [6].
`A characteristic feature of any ligand receptor-mediated
`signal transduction process is the phenomenonof desensitiza-
`tion whereby cellular response to a stimulus is attenuated, or
`demonstrates refractoriness, following prior exposure of the
`cells to the ligand in question. ST-mediated diarrheas are
`transitory in nature, but the mechanism ofthis refractoriness,
`presumably induced in the intestinal cells which have been
`exposed to ST peptide, is unknown. Whether such refractori-
`ness involves receptor desensitization, or other intracellular
`mechanisms has not been investigated till date.
`
`*Corresponding author. Fax: (91) (80) 3341683.
`
`Abbreviations: cGB-PDE, cGMP-binding, cGMP-specific phospho-
`diesterase; GCC, guanylyl cyclase C; PDE, phosphodiesterase; IBMX,
`isobutyl methyl xanthine; STh, stable toxin of the human variety;
`STY72F, mutant ST peptide where the C-terminal tyrosine is mutated
`to phenylalanine
`
`Cellular refractoriness to a given stimulus could be medi-
`ated by an altered rate of synthesis or degradation of the
`second messenger, for example cAMP or cGMP. Synthetic
`rates are presumably regulated by the receptor, which in the
`case of guanylyl cyclase coupled receptors, serves as the en-
`zyme per se. The rate of degradation of cAMP has been
`shown to play an importantrole in inducing the refractoriness
`of Sertoli cells to follicle stimulating hormone[7], by the hy-
`peractivation of a cAMP-specific phosphodiesterase (PDE).
`No such mechanism of regulation has been reported, to our
`knowledge, for cGMP-mediated mechanisms, and hence the
`ST response in T84 cells could serve as a useful model system
`to study such cellular refractoriness.
`The T84 cell line has been used by us to study the STh
`receptor, and we have shown that a single class of receptor
`with high affinity (Ky 0.1 nM) is expressed in these cells [8,9].
`In this study we report that T84 cells show refractoriness to
`fresh stimulation by ST, following prolonged incubation with
`the peptide. This refractoriness is at the level of the degrada-
`tion of the second messenger, cGMP,as a result of increased
`activity of a cGMP-specific PDE, as well as receptor inacti-
`vation at the level of guanylyl cyclase activity.
`
`2. Materials and methods
`
`All fine chemicals were from Sigma Chemical Co., USA, and tissue
`culture media from Life Technologies, USA.
`
`2.1. Culture and maintenance of T84 cells
`T84 cells were obtained from ATCC (CCL 247), and maintained in
`Dulbecco’s Modified Eagle’s medium:F12 containing 5% new born
`calf serum, penicillin and streptomycin as described in detail earlier
`[8]. Cells were plated in 24-well culture dishes at a concentration of
`104 cells/well, and were used at approximately 75-90%confluency,
`after 4-7 days in culture.
`
`2.2. Purification and radiolabeling of ST peptides
`STh peptide was purified from the culture supernatant of E. coli
`cells which overproduced the toxin, as described earlier [10]. Purified
`peptides were quantitated by aminoacid analysis [11]. An analog of
`STh, STY72F, was used as the radioligand for receptor-binding anal-
`ysis, and was prepared as reported earlier in detail [9]. The specific
`activity of the radiolabeled STY72F was 2000 Ci/mmol, and the ra-
`diolabeled peptide was used within a month of preparation.
`
`2.3. Desensitization of T84 cells
`Cells were grown to confluence in 24-well plates and incubated with
`the STh (3 107? M)for 18 hin serum-free DMEM:F12 at 37°C ina
`5% COs. humidified incubator. Wells were washed thrice with warm,
`serum-free medium and incubated with the same medium in the ab-
`sence of any PDEinhibitor, or in the presence of either 1 mM IBMX,
`or the specific PDE inhibitors zaprinast, phenothiazine, milrinone or
`Ro 20-1724 (50 UM), for 30 min at 37°C. Cells were then restimulat-
`ed for 15 min at 37°C with STh (3X10-% M)
`andthe reaction
`was terminated by aspiration of media and addition of 0.1 M citric
`
`0014-5793/97/$17.00 © 1997 Federation of European Biochemical Societies. All rights reserved. WISN Exhibit 1038 - Page 1 of 5
`PITS0014-5793(97)00458-4
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`

`346
`
`M.M. Bakre, S.S. Visweswariah! FEBS Letters 408 (1997) 345-349
`
`acid to each well. Cells were lysed, and cGMP in the lysates meas-
`ured without succinylation of the samples by radioimmunoassays us-
`ing 15]-labeled cGMP prepared as described previously [12]. Stimu-
`lation for 15 min with this concentration of STh produce low or
`undetectable levels of cGMP in the extracellular medium of the cells
`[8].
`
`2.4. Preparation of membranes from T84 cells and guanylyl cyclase
`assays
`Membranes were prepared from cell lysates (100000 g pellet) of
`confluent cultures of T84 cells, following prior exposure of mono-
`layers to STh (3X 1077 M) or medium [9], and used for the measure-
`ment of labeled STY72F-binding activity as well as guanylyl cyclase
`activity. To monitor the receptor in membranes prepared from control
`and desensitized cells, membrane protein (50 ug) was incubated with
`radiolabeled STY72F peptide (10~!° M)in the presence or absence of
`unlabeled STh (10-7 M)as described earlier,
`[9], and then filtered
`through GF/Cfilters. For in vitro guanylyl cyclase assays, membrane
`protein (50 pg) was incubated in the presence or absence of STh (1076
`M) in 60 mM Tris-HCl buffer, pH 7.6, along with 4 mM MgCh,
`2 mM GTP, 500 uM IBMX and a GTPregenerating system consist-
`ing of 20 ug creatine phosphokinase and 7.5 mM creatine phosphate.
`In some cases, membranes were treated with 0.3% Lubrol-PX for 10
`min at 25°C before addition of substrate. MnCl. (4 mM), which is
`knownto activate the receptor non-specifically [13] was also used in
`some cases instead of MgCle. Incubations were continued for 10 min
`at 37°C and the reaction terminated by the addition of 400 ml of 50
`mMsodium acetate buffer, pH 4.5. Samples were heated in a boiling
`water bath for 10 min, and the supernatant taken for assay of cGMP.
`
`2.5. In vitre PDE assays
`Cells were cultured in 24-well dishes and exposed to STh (3X 1077
`M)for 18 h. Cell monolayers were washed in serum-free medium, and
`harvested in 50 mM Tris-HCl, pH 7.5, containing | ug/ml leupeptin,
`1 mM benzamidine, 2 mM EDTA, 10 nM okadaic acid, 10 mM
`sodium vanadate and 5 mM 2-mercaptoethanol. The cells were ho-
`mogenized and homogenates were used for assay of PDE activity, in
`the absence or presence of zaprinast (20 uM). The assay was per-
`formed essentially as described earlier, with some modifications
`[14,15]. Assay mixtures contained 50 mM Tris-HC! buffer, pH 8.0,
`containing 10 mM MgCl, 300 ug/ml bovine serum albumin and
`150 000-200000 cpm *H-cGMP (0.2 uM) (New England Nuclear,
`USA) which had been purified earlier by ion exchange chromatogra-
`phy [16]. Assays were conducted for 15 min at 30°C, following which
`the samples were boiled for 5 min. Crotalus vulgaris snake venom
`toxin was added (20 pg/tube), and incubation continued for a further
`30 min. ?H-guanosine generated was monitored by ion exchange chro-
`matography through DEAE-Sephadex A-25, which had been equil-
`ibrated with 20 mM ammonium formate [16].
`
`3. Results
`
`Application of STh to T84 cells leads to a rapid elevation of
`intracellular cGMPin cells, which declines 3 h following ad-
`dition of STh [8]. We exposed T84 cells to STh for 18 h and
`then restimulated the cells with fresh peptide. The intracellular
`levels of cGMPat the end of 18 h were low, and addition of
`fresh STh did notlead to a significant increase in intracellular
`cGMP levels (Fig. 1A),
`indicating cellular desensitization.
`High levels (> 500 pmol) of cGMP were detected in the ex-
`tracellular medium, indicating that the cells had responded to
`STh over 18 h, and much of the cGMP produced during that
`time had been secreted by the cells. The low intracellular levels
`of cGMPafter 18 h of incubation with STh could be due to
`
`degradation of STh over 18 h. We therefore compared cGMP
`production induced by STh present in the spent medium of
`cells which had been exposed to STh for 18 h, with that
`following application of fresh STh. As can be seen in Fig.
`1B, no significant difference in the levels of cGMP production
`was observed, indicating that low levels of cGMP within cells
`following 18 h incubation with STh could not be attributed to
`degradation of peptide during the period of the incubation.
`To detect receptor internalization and degradation as a
`means of contributing to the desensitization of T84 cells, we
`monitored radiolabeled STY72F binding to membranes pre-
`pared from control and desensitized cells. Results showed no
`significant reduction in the binding in desensitized membranes
`when compared to the control membranes (Fig. 2A; P>0.1),
`and therefore a reduction in total receptor content could not
`account for the >90% reduction in cGMP accumulation in
`desensitized cells. This suggested that intracellular changes,
`either at the level of synthesis or degradation of cGMP, had
`occurred in T84 cells resulting in the refractoriness to further
`SThstimulation.
`Wefirst investigated GCC desensitization in terms of its
`ability to synthesize cGMP and performed in vitro guanylyl
`cyclase assays with membranes prepared from control and
`desensitized cells. Detergents are known to be non-specific
`activators of membrane associated guanylyl cyclases, even in
`the absence of ligand and this has also been observed in the
`case of GCC [13]. We preincubated membrane preparations
`
`120
`
`90
`
`60
`
`30
`
`
`
`cGMP(pmoles/10®cells)
`
`1 [I
`*
`+
`-
`+
`
`-
`+
`
`~ @
`
`100
`
`(3)
`oO
`2% ”
`=. 50—
`a2 3°o
`E 25
`~— Q—
`Preincubation
`~
`Stimulation
`-
`
`Fig. 1. Homologous desensitization of the T84 cells. A: Confluent monolayers of T84 cells were incubated with STh (3X10-7 M)for 18 h.
`Cells were washed and cGMPlevels monitored with or without restimulation with STh (3107? M). Control cells were incubated in medium
`without serum and basal cGMPlevels measured directly, or on stimulation with STh. Values represent the mean+S.D. of duplicate determina-
`tions with each experiment repeated thrice. B: To check the degradation of STh peptide remaining in the culture supernatant, confluent T84
`cells were stimulated with (1) STh (3X10? M), (2) STh remaining in the culture supernatant after 18 h incubation with T84 cells and (3) STh
`incubated at 37°C without cells for 18 h. cGMP was monitored by radioimmunoassay. Values represent the mean+S.D., with each experiment
`repeated twice and each well assayed in duplicate. *: P>0.5 when compared with 1.
`
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`M.M. Bakre, S.S. Visweswariah! FEBS Letters 408 (1997) 345-349
`
`347
`
`12500
`
`_~
`E 10000
`og
`2 7500
`3
`3
`@
`8
`QO
`”n
`
`5000
`2500
`
`_ 150
`<
`® 125
`S
`2 100
`<
`3
`o
`5 50
`a
`25
`9
`
`= 2
`‘o
`°
`6 20
`—
`= 16
`3
`9
`g'
`&
`5
`2
`
`0
`
`0
`
`0
`
`+STh
`-STh
`0.3% Lubrol_ Mn-GTP
`Fig. 2. GCC activity in desensitized cells. Confluent monolayers were preincubated either in the absence (open bars) or presence (closed bars)
`of STh (31077 M)for 18 h, prior to membrane preparation. Membrane protein (50 ug) was used for binding assays or in vitro guanylyl cy-
`clase assays. A: Membrane protein was incubated with !”°I-labeled STY72F (100000 cpm) for 1 h in the absence or presence of STh (107-7 M)
`unlabeled STh peptide, and receptor associated radioactivity monitored following filtration. Values represent the mean+S.D. of triplicate deter-
`minations with each assay repeated twice. B: Membranes were treated with 0.3% Lubrol-PX for 10 min prior to addition of MgGTP, or
`treated with MnGTP independently. Values represent
`the mean+S.D. of duplicate determinations with each experiment repeated twice.
`C: Membrane protein was treated with STh (10® M) and cGMP produced was monitored. Values represent the mean+S.D. of duplicate de-
`terminations with each experiment repeated twice. *: P<0.001.
`
`cate that restimulation of desensitized cells in the presence of
`IBMX restored cGMP accumulation close to levels observed
`in control cells. Interestingly, only zaprinast, a specific inhib-
`itor of the cGMP-binding, cGMP-specific (Type V) PDE,
`when added to desensitized cells prior to ST restimulation,
`could restore cGMP levels to those observed with IBMX,
`clearly indicating that the increased activity of this specific
`Type V PDEwasresponsible for the efficient degradation of
`cGMPin desensitized cells. No specific inhibitor is available
`for the cGMP-stimulated PDE, but our results with zaprinast
`suggest that a major contribution to enhanced cGMPdegra-
`
`
`
`from control and desensitized cells with Lubrol-PX (0.3%)
`and then monitored guanylyl cyclase activity. As shown in
`Fig. 2B, detergent treated membranes prepared from desensi-
`tized cells demonstrated no significant reduction in guanylyl
`cyclase activity (P > 0.7). Membranes prepared from desensi-
`tized cells also did not show anydifference in guanylyl cyclase
`activity in the presence of MnGTP, another non-specific acti-
`vator of membrane associated guanylyl cyclase [13]. These
`results indicate that the general guanylyl cyclase catalytic ac-
`tivity of the receptor is retained on desensitization.It is perti-
`nent to mention here that in T84 cells, the only membrane
`associated guanylyl cyclase activity observed is that of GCC
`[8].
`Wethen investigated the sensitivity of GCC to STh stim-
`ulation in membranes prepared from control and desensitized
`cells. As shown in Fig. 2C, basal activities in both membrane
`preparations remained similar. However, STh-stimulatable
`guanylyl cyclase activity was significantly reduced to nearly
`50% of control values,
`in desensitized cells (P< 0.001), and
`STh addition only marginally increased cGMP production
`over basal values. This indicates that an important contribu-
`tion to cellular refractoriness is receptor desensitization in
`terms of a reduced sensitivity of the receptor to STh.
`A reduction in the accumulation of cGMPin desensitized
`cells could also be contributed by an increased rate of degra-
`dation of cGMPin desensitized T84 cells. A large family of
`PDEs are found in various cell
`types and while some are
`specific for cAMP or cGMP,others are dual specific and
`degrade both cGMP and cAMPwith near equal efficiency
`[17-19]. A number ofspecific inhibitors are available for the
`various classes of PDEs, which allow the identification of a
`specific PDE responsible for cAMP or cGMPdegradation.
`We therefore stimulated desensitized and control cells with
`STh, in the presence and absence of the general PDE inhib-
`itor, IBMX,as well as inhibitors for the Cat-calmodulin-de-
`pendent PDE (phenothiazine), the cGMP-inhibited PDE (mil-
`rinone),
`the cAMP-specific PDE (Ro 20-1724 and zaprinast
`(cGMP-specific PDE) [17]. The results shown in Fig. 3 indi-
`
`oO
`
`[__] Control
`Ml Desensitized
`
`*
`
`*
`
`200
`
`eS
`o 150
`
`oO
`
`100
`
`50
`
`a@°©
`
`& S@
`
`IBMX
`
`Milrinone
`
`Noinhibitor
`
`Zaprinast
`
`Ro20-1724
`
`Phenothiazine
`
`~o
`
`O=
`2<
`
`£O
`
`o=
`
`Fig. 3. Role of phosphodiesterases in inducing refractoriness to STh
`in T84 cells. Confluent monolayers were preincubated without or
`with STh (31077 M) for 18 h. Cells were washed, incubated with
`no phosphodiesterase inhibitor,
`1 mM IBMX orspecific inhibitors
`as indicated (50 uM) for 30 min and restimulated with STh. cGMP
`levels were monitored and values represent the mean+S.D.oftripli-
`cate experiments with each well treated in duplicate. *: P> 0.5.
`
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`

`348
`
`
`cGMP
`
`hydrolysed(pmol/mirymgprotein)
`
`[—jContro!
`
`Ml Desensitized
`
`=NOwAODNDOOOO 0
`
`Zaprinast
`
`-
`
`+
`
`-
`
`+
`
`In vitro phosphodiesterase activity in desensitized cells.
`Fig. 4.
`Monolayers of T84 cells were incubated in serum-free medium either
`in the absence or presence of STh (3X10~7 M) for 18 h at 37°C.
`Cells were washed with serum-free medium and lysed as described
`in experimental procedures. Cell extracts prepared from desensitized
`and control cells were then used to perform in vitro PDE assays,
`in the presence or absence of 10 uM zaprinast. Values represent
`the mean of duplicate determinations of three experiments+S.D.
`*: P<0.001.
`
`dation in desensitized cells is through the Type V PDE. To
`our knowledge, this is the first report on the contribution of
`enhanced PDEactivity towards inducing refractoriness in any
`cGMP-mediated system, and also the first report on the role
`of the Type V PDEin regulating cGMP degradation in a
`cGMPresponsivecell in this manner.
`The increased PDE activity observed in vivo should be
`detectable in cell extracts prepared from desensitized cells.
`We therefore prepared cell homogenates from control and
`desensitized cells and measured PDEactivity in the extracts.
`As shownin Fig. 4, the activity observed in desensitized cell
`extracts is 2-fold higher than in control cells. This albeit mod-
`est
`increase in activity could nevertheless be significant
`in
`vivo, when coupled with reduced receptor activity in terms
`of synthesizing cGMP (Fig. 2). The enhanced enzymeactivity
`observed in desensitized cells was inhibited by zaprinast, con-
`firming that
`the major PDE activity we have measured is
`contributed by the Type V cGMP-specific PDE (Fig. 4).
`
`4. Discussion
`
`In this report, we demonstrate that the human colonic T84
`cell line is refractory to prolonged exposure to the ST pep-
`tides, and this refractoriness is a result of receptor desensiti-
`zation and the increased activity of a cGB-PDE. To our
`knowledge,
`the only other well documented report on the
`hyperactivation of a PDE inducing refractoriness in a cell is
`the observation that prolonged exposure offollicle stimulating
`hormoneto Sertoli cells reduces the ability of these cells to
`respond to the hormone in terms of cAMP accumulation [7].
`This refractoriness is brought about by the activation of a
`specific cAMP PDE, the Type IV PDE [21,22], and increased
`activity was detected in crude lysates prepared from cells.
`
`M.M. Bakre, S.S. Visweswariah! FEBS Letters 408 (1997) 345-349
`
`Hyperactivation in the case of the Type IV enzymeis through
`the increased transcription of the mRNAfor this enzyme, and
`possibly also phosphorylation [22]. A second example indi-
`cates a rapid regulation of the cAMP-specific PDE by forsko-
`lin and isoproterenol in astroglial cells, where the increase in
`enzymeactivity in the cytosol ofcells is also of the order of 2-
`fold, as we have seen in the case of the Type V PDE [23],
`through a mechanism involving changes in the phosphoryla-
`tion of the Type IV enzyme.
`Till date, the human cDNAfor the Type V enzyme has not
`been cloned, and the only available cDNA for this enzyme
`represents
`the enzyme present
`in the bovine lung [24].
`Whether this bovine cDNA has homology to the human
`gene is not known, and we are presently investigating such
`a possibility. This should allow us to investigate regulation of
`the enzyme activity at the level of transcription. The bovine
`enzyme has consensus sites for phosphorylation by cAMP-
`dependent kinase [24]. Changes in the phosphorylation of
`the Type V enzyme could also contribute to the increased
`PDEactivity observed in desensitized cells, and it has been
`shown that phosphorylation of the Type V PDE by protein
`kinase A in vitro leads to an increase in the Vinax [15] of the
`enzyme. Whether this occurs in T84 cells remains to be inves-
`tigated.
`Wehave shown here that there is a significant reduction in
`the STh-stimulatable guanylyl cyclase activity of the receptor
`following exposure to ST peptide, with a marked reduction in
`sensitivity to STh, if one considers basal activities in control
`and desensitized cells. However,
`in whole cells, STh-stimu-
`lated cGMP levels seem to approach control values when
`desensitized cells were restimulated in the presence of IBMX
`and zaprinast (Fig. 3). This suggests low receptor desensitiza-
`tion in intact cells, which could be dueto reactivation of the
`receptor by additional cellular machinery. The receptor gua-
`nylyl cyclases appear to be desensitized by a mechanism of
`dephosphorylation of the receptor, perhaps through the in-
`volvement of a specific phosphatase [25,26]. In the case of
`the receptor for the atrial natriuretic factor, receptor internal-
`ization has also been observed in certain cells [27]. More re-
`cently, there is evidence of a cGMP-mediated reduction in the
`level of the receptor mRNA in cells exposed to the atrial
`natriuretic peptide for prolonged periods of time [28]. Our
`report is the first to suggest that the ST receptor is desensi-
`tized but we do notfind a significant degree of internalization
`of the receptor in T84 cells. This is in agreement with a recent
`report indicating efficient internalization and recycling of the
`receptor in T84 cells along with the ST peptide, without deg-
`radation of either the receptor or the peptide [29]. Whether
`the reduction in receptor sensitivity to STh that we have ob-
`served is correlated with a change in the phosphorylation of
`the receptor remains to be investigated. We have recently
`generated polyclonal antibodies to the extracellular domain
`of the receptor which should prove useful in such studies [30].
`Stable toxin diarrheas have been observed to be transitory
`in vivo [20]. Thus, in the only valid in vivo assay for the ST
`peptides, the suckling mouse assay, fluid accumulation in the
`intestine of the suckling mouse is observed maximally 3 h
`following ST administration after which there is a gradual
`decline in the fluid content of the intestine [20]. We suggest
`that the enhanced degradation of the cGMP occurring in in-
`testinal cells reported here, could contribute along with recep-
`tor desensitization, to the decline in fluid secretion, perhaps at
`
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`M.M. Bakre, S.S. Visweswariah! FEBS Letters 408 (1997) 345-349
`
`the level of chloride secretion. It remains to be seen whether
`the refractoriness that we observe in terms of cGMPproduc-
`tion is reflected at the level of chloride secretion in desensi-
`tized cells. Our results indicate that the regulation of cGMP
`accumulation in the intestinal cell could be through its degra-
`dation rather than its synthesis by the receptor. This may be
`an essential requirement, since guanylin [31], the endogenous
`ligand for GCC, mediates its action through the same recep-
`tor. Hence a major decrease in ST receptor activity would
`curtail guanylin action in maintaining and regulating ion ho-
`meostasis in the intestine.
`
`Acknowledgements: We acknowledge the help of B.M. Garrett and N.
`Roy for the purification and radiolabeling of ST peptides. We also
`acknowledge the help of Ms. Vasanthi Ramachandran whoinitiated
`some of these studies. M.M.B. is sponsored by the University Grants
`Commission, Government of India. This work was supported by fi-
`nancial assistance from the Council for Scientific and Industrial Re-
`asearch, Government of India.
`
`References
`
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`[2] C.A. Chao, F.J. de Sauvage, Y.J. Dong, J.A. Wagner, D.V.
`Goeddel, P. Gardner, EMBO J. 13 (1994) 1065-1072.
`[3] S. Schulz, C.K. Green, P.T. Yuen, D. Garbers, Cell 64 (1991)
`941-948.
`[4] F.J. de Sauvage, T.R. Camerato, D.V. Goeddel, J. Biol. Chem.
`266 (1991) 17912-17918.
`[5] S. Singh, G. Singh, J.M. Heim, R. Gerzer, Biochem. Biophys.
`Res. Commun.179 (1991) 1455-1463.
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