Vol. 59, No. 4
INFECTION AND IMMUNITY, Apr. 1991, p. 1552-1557 0019-9567/91/041552-06$02.00/0 Copyright C) 1991, American Society for Microbiology
Activation of Particulate Guanylate Cyclase by Escherichia coli Heat-Stable Enterotoxin Is Regulated by Adenine Nucleotides HELENE GAZZANO, H. IRENE WU, AND SCOTT A. WALDMANt*
Department of Medicine, Stanford University School of Medicine, and Palo Alto Veterans Administration Hospital, 3801 Miranda Avenue, Palo Alto, California 94304 Received
5
November 1990/Accepted 23 December 1990
Guanylate cyclase is regulated by adenine nucleotides in membranes of intestinal mucosal cells. Basal guanylate cyclase was activated about twofold by adenine nucleotides. Activation was specific for adenine, as compared with the pyrimidine nucleotides UTP and CTP. In addition, enzyme activation was obtained in the presence of saturating concentrations of GTP, the substrate for guanylate cyclase. The most potent adenine nucleotide was the nonhydrolyzable analog of ATP, adenosine 5'-O-(3-thiotriphosphate). Adenine nucleotide activation was specific for the particulate form of guanylate cyclase, as compared with the soluble form. Also, adenine nucleotides potentiated the activation of guanylate cyclase by the heat-stable enterotoxin produced by Escherichia coli. Indeed, enzyme activation by adenine nucleotides and toxin was greater than the sum of individual activations by these agents. Adenine nucleotides regulate guanylate cyclase by increasing the maximum velocity of the enzyme without altering its affinity for substrate or its cooperativity. In addition to stimulating guanylate cyclase, adenine nucleotides decreased the specific binding of the heat-stable enterotoxin to receptors in intestinal membranes. The coordinated regulation of the toxin-receptor interaction and guanylate cyclase activity by a process utilizing nonhydrolyzable analogs of a purine nucleotide is similar to the mechanisms involved in the hormone regulation of adenylate cyclase by guanine nucleotide-binding proteins. These data suggest that an adenine nucleotide-dependent protein may couple the toxin-receptor interaction to the regulation of particulate guanylate cyclase in intestinal membranes. mucosae from the intestines of one to three rats were pooled and homogenized in buffer containing 50 mM Tris-HCl (pH 7.6), 1 mM dithiothreitol, 1 mM EDTA, 0.25 M sucrose, and 1 mM phenylmethylsulfonyl fluoride. Homogenates were centrifuged at 100,000 x g for 60 min, and the resulting pellets were consecutively washed in (i) 50 mM Tris-HCI (pH 7.6)-i mM EDTA-1 mM dithiothreitol (TED) supplemented with 500 mM KCl and (ii) TED. Washed pellets were suspended in TED and stored at -80°C until used. Guanylate cyclase activity was assayed as described previously (12, 18, 31). In brief, incubations were conducted at 37°C in the presence of 50 mM Tris-HCl (pH 7.6)-0.5 mM isobutylmethylxanthine, a regenerating system including 7.5 mM creatine phosphate and 20 Rg of creatine phosphokinase (160 U/mg of protein), 10 to 20 pg of membrane or detergent extract, manganese- or magnesium-GTP (3 mM excess of free metal), and ST when needed. Some incubations contained different concentrations of various nucleotides or their analogs. Reactions were initiated by the addition of metal-GTP, incubated for 5 min, and terminated by the addition of 0.4 ml of ice-cold 50 mM sodium acetate (pH 4.0). Samples were placed in a boiling water bath for 3 min. Generated cyclic GMP was quantified by radioimmunoassay (25) as described previously (12, 15, 16, 18, 25, 31). All reactions were performed at least in duplicate, and the data are representative of at least duplicate experiments. Enzyme activity was linear with respect to protein concentration and time throughout these experiments. ST receptor binding was determined by a modification of the procedure described previously (20). In brief, intestinal membranes (20 to 40 ,ug of protein) were incubated in the reaction mixture used for guanylate cyclase assays, as outlined above, with 125I-ST (0.1 pmol) in a final volume of 60 RI. Nonspecific binding was measured in the presence of a 100-fold molar excess of unlabeled toxin and was less than
Escherichia coli heat-stable enterotoxin (ST) is a lowmolecular-weight peptide which stimulates intestinal secretion, resulting in diarrhea (1, 9, 13, 23, 24). This cascade is initiated by ST binding to specific receptors localized on brush borders of intestinal cells (6, 8, 10, 17, 20). The ST-receptor interaction activates the particulate form of guanylate cyclase in brush borders, elevating cyclic GMP in villous cells of the intestinal mucosa (7, 12, 15, 30, 31). Activation of guanylate cyclase by ST is often compared with enzyme activation by atrial natriuretic peptides (ANPs). Both systems involve coupling peptide-receptor interactions to enzyme activation (19, 22, 28, 30). However, the molecular events coupling receptor binding to enzyme activation remain unknown. Guanine nucleotide-binding proteins mediate receptor-dependent regulation of adenylate cyclase, but neither these proteins nor guanine nucleotides regulate guanylate cyclase (11, 26, 27). Interestingly, the activation of particulate guanylate cyclase by atrial peptides is potentiated by adenine nucleotides (4, 21). Similarly, adenine nucleotides activate basal guanylate cyclase in membranes isolated from rat lungs, liver, and kidneys (8a). The present report explores the ability of adenine nucleotides to regulate basal and ligand-stimulated guanylate cyclase in intestinal membranes. MATERIALS AND METHODS Membranes were prepared from homogenates of rat intestinal mucosal cells as described previously (31). In brief, *
Corresponding author.
t Present address: Departments of Medicine and Pharmacology, Division of Clinical Pharmacology, Jefferson Medical College, Thomas Jefferson University, 1100 Walnut St., Philadelphia, PA 19081.
1552
VOL. 59,
TABLE 1. Effect of nucleotides on particulate guanylate cyclase in rat intestinal membranesa No ST
None ATPgS ATP Adenyl-imidodiphosphate Adenosine-5'-O-(2-thiodiphosphate) dATP ADP AMP Adenosine UTP
1.0 2.7 1.8 1.5 1.5
± ± ± ± ±
0.0 0.2 0.2 0.1 0.2
Particulate enzyme Addition
ST
(7)C (5)C (5)d (6)e
5.1 13.4 11.0 9.6
± ± ± ±
1.0 4.0 3.2 3.2
tb
Sp act
(6) (6) (5) (5)
1.3 0.1 (5) 1.3 ± 0.3 (5) 1.2 0.1 (5) 1.2 ± 0.1 (3) 1.1 0.1 (4) 1.1 0.1 (4)
" Guanylate cyclase was assayed as outlined in Materials and Methods in the presence of a 1 mM concentration of the indicated nucleotide. The regenerating system was omitted from assays to minimize nucleotide phosphorylation. ST (30 ,uM) was added to incubations as indicated. Basal activity in these experiments was 3.7 ± 0.5 (seven experiments) pmol of cyclic GMP produced per min per mg of protein. b Calculated as the ratio of enyzyme activity in the presence and in the absence of the indicated nucleotide and reported as the mean ± standard error (number of experiments). " P < 0.0005. dp < 0.02. p
dATP, ADP, AMP, adenosine. Maximum activation of basal guanylate cyclase was two- to threefold with ATPgS. Adenine nucleotides which activated basal guanylate cyclase were tested for their ability to potentiate enzyme activation by ST (Table 2). These nucleotides potentiated the activation of guanylate cyclase by ST with an order of potency similar to that for the activation of the enzyme. Maximum activation of guanylate cyclase with the combination of ST and ATPgS was 10- to 20-fold, 3- to 4-fold greater than with ST alone. The activation of basal and ST-stimulated guanylate cyclase
3.1 16.0 7.7 41.7
Soluble enzyme
Fold ~ activationc
TABLE 3. Effect of ATPgS on the kinetics of rat intestine
particulate guanylate cyclasea Addition Addition
Control ST
ATPgS ST + ATPgS
O.5Vmxof protein)'
( (MM)b
4.8 4.7 4.3 3.8
± 0.2 ± 0.2
± 0.6 ± 0.6
(pmol/min/mg 32 176 48 278
± 2.5 ± 46.1
± 9.4e ± 50.7f
Hill
coefficientd 1.9 1.6 2.0 2.0
± 0.2 ± 0.2
± 0.1 ± 0.1
a Guanylate cyclase activity was determined in the presence or absence of 1 mM ATPgS with MgCI2-GTP as the substrate as outlined in Materials and Methods. Values are reported as means ± standard errors. The data are the means of three experiments. b Concentration of substrate yielding half-maximum guanylate cyclase
activity (3, 8a, 32). Maximum velocity of enzyme activity (picomoles of cyclic GMP produced per minute per milligram of protein) (3, 8a, 30). d Calculated as described previously (3, 8a, 30). c
e
p < 0.10.
fp
ATP > other adenine nucleotides. Maximum inhibition of ST binding of 30 to 40% was observed with ATPgS. Inhibition of binding was specific for adenine nucleotides, since CTP and UTP had no effect, and all binding was performed in the presence of 1 mM GTP.
PURINERGIC COUPLING OF ST AND GUANYLATE CYCLASE
VOL. 59, 1991 400 0
E -E 300
-
.2
0
200
-
100 _
0
5
10 Mg 2+GTP (mM)
15
20
FIG. 4. Effects of adenine nucleotides on the kinetics of particulate guanylate cyclase. Guanylate cyclase in rat intestinal membranes was assayed with a range of substrate concentrations as indicated, with magnesium as the cation cofactor, in the absence (U) or the presence of 30 ,uM ST (A), with 1 mM ATPgS (C), or with 30 ,uM ST plus 1 mM ATPgS (A) as outlined in Materials and Methods.
DISCUSSION The primary event mediating ST-induced secretion is the binding of peptides to intestinal cell surface receptors. These receptors are coupled to the activation of guanylate cyclase and increases in intracellular cyclic GMP. ST receptors and guanylate cyclase are separate molecules, and little is known concerning their functional coupling (29). It is well established that guanine nucleotide-binding proteins couple hormone-receptor interactions and adenylate cyclase activity (11, 26, 27). Recently, ATP was reported to enhance basal guanylate cyclase activity in rat lungs (8a) as well as atrial peptide-activated guanylate cyclase in rat lungs and liver (4, 21). These data suggest that adenine nucleotides may regulate basal and receptor-stimulated guanylate cyclase in a manner which is similar to the regulation of adenylate cyclase by guanine nucleotides. The data reported here demonstrate the activation of basal and ST-stimulated guanylate cyclase by adenine nucleotides. Activation of the enzyme was induced specifically by adenine, as compared with other nucleotides. Enzyme stimulation with ST and adenine nucleotides together was greater than the sum of individual activations by these agents. The most potent nucleotide regulating guanylate cyclase was the nonhydrolyzable analog of ATP, ATPgS. This effect is similar to the effects of nonhydrolyzable analogs of GTP c., adenylate cyclase mediated by guanine nucleotide-binding proteins (11, 26, 27). These data suggest that an adenine nucleotide regulatory protein may couple ST-receptor interactions to guanylate cyclase activation. Adenine nucleotides which serve as phosphate donors through a kinase reaction (ATP) and those which do not (adenyl-imidodiphosphate) were effective in activating guanylate cyclase. Thus, it is unlikely that activation involves phosphorylation of the enzyme by a kinase with adenine nucleotides as phosphate donors. Adenine nucleotides activate the particulate form of guanylate cyclase specifically, as compared with the soluble form. The stimulation of basal and ST-activated guanylate cyclase by adenine nucleotides was concentration depen-
1555
dent, increasing the maximum enzyme activity without altering the concentration of ST producing half-maximum activation. Similarly, adenine nucleotides increased the maximum velocity of guanylate cyclase without altering the concentration of the substrate required for half-maximum velocity or the Hill coefficient in the presence or absence of ST. These data suggest that adenine nucleotides alter the maximum rate of catalysis of the enzyme without altering the affinity of the enzyme for the substrate or the characteristic positive cooperativity exhibited by particulate guanylate cyclase (3, 31). These data correlate closely with those concerning the effects of adenine nucleotides on the activation of guanylate cyclase by atrial peptides in rat lungs (8a, 21). In those studies, adenine nucleotides increased the maximum velocity of guanylate cyclase without altering the affinity of the enzyme for the substrate or atrial peptides. Thus, coupling of peptide-receptor interactions to particulate guanylate cyclase activation by adenine nucleotides may involve a general mechanism of increasing the maximum velocity without altering other kinetic parameters of the enzyme. Specific binding of 1251-ST to intestinal membranes was reduced by adenine nucleotides. The relative abilities of these nucleotides to inhibit the binding of 1251I-ST to membranes correlated closely with their relative abilities to activate guanylate cyclase. These observations are similar to those concerning the regulation of ligand binding by guanine nucleotides and their binding proteins. In this system, coupling proteins are activated by binding GTP, which is induced by ligand-receptor interactions. Activated coupling proteins bound to GTP regulate adenylate cyclase activity and decrease the ability of receptors to bind the ligand (11, 26, 27). Thus, guanine nucleotide-binding proteins coordinately regulate the activities of the effector and receptor arms of the signal transduction cascade. A similar form of regulation of guanylate cyclase and ST receptors by adenine nucleotides is suggested by the data presented here. These data suggest the presence of an adenine nucleotide-binding protein which mediates the coupling of ST receptors and particulate guanylate cyclase and coordinately regulates the activities of both components. The mechanisms by which adenine nucleotides regulate basal and ligand-stimulated particulate guanylate cyclase remain unclear. Adenine nucleotides may bind directly to particulate guanylate cyclase, acting as allosteric modulators of that enzyme (21). This suggestion is supported by studies with a cloned ANP receptor-particulate guanylate cyclase protein (5). In these studies, a cloned enzyme-receptor possessed ligand binding activity coupled to guanylate cyclase activity, and adenine nucleotides potentiated enzyme activation by ANP. However, adenine nucleotides had no effect on basal particulate guanylate cyclase. A mutant protein with a deleted region encoding a domain with homology to protein kinase also possessed ligand binding and enzyme activities, but they were not coupled. Also, ATP did not activate particulate guanylate cyclase in the presence or absence of ANP. It was suggested from these studies that the deleted kinase domain was necessary for coupling the ANPreceptor interaction to guanylate cyclase and mediated adenine nucleotide regulation. However, it is problematic to ascribe adenine nucleotide regulation to this kinase domain, since these nucleotides did not affect basal enzyme activity in the cloned mutant system. Consequently, once ligand regulation of enzyme activity was lost, as in the mutant clone, adenine nucleotide regulation was automatically lost as well.
1556
GAZZANO ET AL.
INFECT. IMMUN.
Alternatively, adenine nucleotides may regulate particulate guanylate cyclase by a separate adenine nucleotidebinding protein. Previous studies demonstrated that adenine nucleotide regulation of ANP-stimulated particulate guanylate cyclase could be separated from that enzyme by sequential washing of crude membranes (4). Washing did not reduce the specific activity of the enzyme but did remove the ability of adenine nucleotides to potentiate the activation of guanylate cyclase by ANP. Similarly, recent studies demonstrated that adenine nucleotide regulation and guanylate cyclase activity could be separated by partial purification of this enzyme (8a). When detergent extracts of rat lung membranes were chromatographed on GTP-agarose, particulate guanylate cyclase was quantitatively purified. However, this partially purified enzyme was unresponsive to adenine nucleotides. These data support the suggestion that adenine nucleotide regulation of particulate guanylate cyclase is mediated by a protein which is separate from that enzyme (4, 8a). In conclusion, the data presented here demonstrate that the activation of particulate guanylate cyclase by ST-receptor interactions is regulated by an adenine nucleotide-dependent event. Activation is potentiated by analogs of ATP which cannot serve as kinase substrates. Thus, it is unlikely that adenine nucleotide regulation of this system reflects alterations in the phosphorylation state of the constituents of the involved signal transduction cascade. Adenine nucleotides alter the maximum activity of basal and agoniststimulated enzyme without altering the affinity of the enzyme for the substrate or the characteristic cooperativity of particulate guanylate cyclase. These nucleotides coordinately regulate both ST receptor and particulate guanylate cyclase activities. The nucleotide most potent in regulating these activities is the nonhydrolyzable analog of ATP, ATPgS. That both receptor and effector activities are regulated by a purine nucleotide-dependent mechanism which utilizes nonhydrolyzable analogs of ATP supports the suggestion that an adenine nucleotide-binding protein may regulate this system in a manner similar to the regulation of adenylate cyclase by guanine nucleotide-binding proteins. Identification and characterization of this coupling protein are currently being undertaken in this laboratory. ACKNOWLEDGMENT This work was supported in part by Administration.
a
grant from the Veterans
REFERENCES 1. Banwell, J. G., and H. Sherr. 1973. Effect of bacterial enterotoxins on the gastrointestinal tract. Gastroenterology 65:467497. 2. Brown, W. B., Jr., and M. Hollander. 1977. Statistics: a biomedical approach. John Wiley & Sons, Inc., New York. 3. Carr, S., H. Gazzano, and S. A. Waldman. 1989. Regulation of
particulate guanylate cyclase by Escherichia coli heat-stable enterotoxin: receptor binding and enzyme kinetics. Int. J. Biochem. 21:1211-1215. 4. Chang, C.-H., K. P. Kohse, B. Chang, M. Hirata, B. Jiang, J. E. Douglas, and F. Murad. 1990. Characterization of ATP-stimulated guanylate cyclase activation in rat lung membranes. Biochim. Biophys. Acta 1052:159-165. 5. Chinkers, M., and D. L. Garbers. 1989. The protein kinase domain of the ANP receptor is required for signaling. Science 245:1392-1394. 6. Dreyfus, L. A., L. Jaso-Freidman, and D. C. Robertson. 1984.
Characterization of the mechanism of action of Escherichia coli heat-stable enterotoxin. Infect. Immun. 44:493-501. 7. Field, M., L. H. Graf, Jr., W. J. Laird, and P. L. Smith. 1978. Heat-stable enterotoxin of Escherichia coli: in vitro effects on guanylate cyclase activity, cyclic GMP concentration, and ion transport in small intestine. Proc. Natl. Acad. Sci. USA 75: 2800-2804. 8. Frantz, J. C., L. Jaso-Freidman, and D. C. Robertson. 1984. Binding of Escherichia coli heat-stable enterotoxin to rat intestinal cells and brush border membranes. Infect. Immun. 43:622630.
8a.Gazzano, H., I. Wu, and S. A. Waldman. Biochim. Biophys. Acta, in press. 9. Giannella, R. A. 1981. Pathogenesis of acute bacterial diarrheal disorders. Annu. Rev. Med. 32:341-357. 10. Giannella, R. A., M. Luttrell, and M. Thompson. 1983. Binding of Escherichia coli heat-stable enterotoxin to its receptor on rat intestinal cells. Am. J. Physiol. 245:492-498. 11. Gilman, A. G. 1987. G-proteins: transducers of receptor-generated signals. Annu. Rev. Biochem. 56:615-649. 12. Guerrant, R. L., J. M. Hughes, B. Chang, D. C. Robertson, and F. Murad. 1980. Activation of intestinal guanylate cyclase by heat-stable enterotoxin of Escherichia coli: studies of tissue specificity, potential receptors, and intermediates. J. Infect. Dis. 14:220-228. 13. Gyles, C. L. 1974. Heat-labile and heat-stable forms of the enterotoxin from E. coli strains enteropathogenic for pig. Ann. N.Y. Acad. Sci. 176:277-283. 14. Hardman, J. G., and E. W. Sutherland. 1969. Guanylate cyclase, an enzyme catalyzing the formation of guanosine 3', 5'-monophosphate from guanosine triphosphate. J. Biol. Chem. 244:6363-6370. 15. Hughes, J. M., F. Murad, B. Chang, and R. C. Guerrant. 1978. The role of cyclic GMP in the action of heat-stable enterotoxin of Escherichia coli. Nature (London) 271:755-756. 16. Ishikawa, E., S. Ishikawa, J. W. Davis, and E. W. Sutherland. 1969. Determination of guanosine 3',5'-monophosphate in tissues and of guanyl cyclase in rat intestine. J. Biol. Chem. 244:6371-6376. 17. Ivens, K., H. Gazzano, P. O'Hanley, and S. A. Waldman. 1989. Heterogeneity of intestinal receptors for Escherichia coli heatstable enterotoxin. Infect. Immun. 58:1817-1820. 18. Kimura, H., and F. Murad. 1974. Evidence for two different forms of guanylate cyclase in rat heart. J. Biol. Chem. 249:69106919. 19. Kuno, T., J. W. Andresen, Y. Kamisaki, S. A. Waldman, L. Y. Chang, S. Saheki, D. C. Leitman, M. Nakane, and F. Murad. 1986. Co-purification of an atrial natriuretic factor receptor and particulate guanylate cyclase from rat lung. J. Biol. Chem. 261:5817-5823. 20. Kuno, T., Y. Kamisaki, S. A. Waldman, J. Gariepy, G. Schoolnick, and F. Murad. 1986. Characterization of the receptor for heat-stable enterotoxin produced by Escherichia coli in rat intestine. J. Biol. Chem. 261:1470-1476. 21. Kurose, H., T. Inagami, and M. Ui. 1987. Participation of adenosine 5'-triphosphate in the activation of membrane-bound guanylate cyclase by the atrial natriuretic factor. FEBS Lett. 219:375-379. 22. Paul, A. K., R. B. Marala, R. K. Jaiswal, and R. K. Sharma. 1987. Coexistence of guanylate cyclase and atrial natriuretic factor in a 180-kD protein. Science 235:1224-1226. 23. Sack, D. A., M. H. Merson, J. G. Wells, R. B. Sack, and G. K. Morris. 1975. Diarrhea associated with heat-stable enterotoxinproducing strains of Escherichia coli. Lancet ii:239-241. 24. Sack, R. B. 1975. Human diarrheal disease caused by enterotoxigenic Escherichia coli. Annu. Rev. Microbiol. 29:333353. 25. Steiner, A. L., C. W. Parker, and D. M. Kipnis. 1972. Radioimmunoassay for cyclic nucleotides. I. Preparation of antibodies and iodinated cyclic nucleotides. J. Biol. Chem. 247:11061113. 26. Stryer, L. 1986. Cyclic GMP cascade of vision. Annu. Rev. Neurosci. 9:87-119.
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27. Stryer, L., and H. R. Bourne. 1986. G proteins: a family of signal transducers. Annu. Rev. Cell Biol. 2:391-419. 28. Takayanagi, R., T. Inagami, R. M. Snajdar, T. Imada, T. Masaki, and K. S. Misono. 1987. Two distinct forms of receptors for atrial natriuretic factor in bovine adrenocortical cells. J. Biol. Chem. 262:12104-12113. 29. Waldman, S. A., T. Kuno, Y. Kamisaki, L. Y. Chang, J. Gariepy, P. O'Hanley, G. Schoolnick, and F. Murad. 1986.
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Intestinal receptor for heat-stable enterotoxin of Escherichia coli is tightly coupled to a novel form of particulate guanylate cyclase. Infect. Immun. 51:320-326. 30. Waldman, S. A., and F. Murad. 1987. Cyclic GMP synthesis and function. Pharm. Rev. 39:163-196. 31. Waldman, S. A., P. D. O'Hanley, S. Falkow, G. Schoolnik, and F. Murad. 1984. A simple, sensitive assay for the heat-stable enterotoxin of Escherichia coli. J. Infect. Dis. 149:83-89.
INFECTION AND IMMUNITY, Apr. 1991, p. 1552-1557 0019-9567/91/041552-06$02.00/0 Copyright C) 1991, American Society for Microbiology
Activation of Particulate Guanylate Cyclase by Escherichia coli Heat-Stable Enterotoxin Is Regulated by Adenine Nucleotides HELENE GAZZANO, H. IRENE WU, AND SCOTT A. WALDMANt*
Department of Medicine, Stanford University School of Medicine, and Palo Alto Veterans Administration Hospital, 3801 Miranda Avenue, Palo Alto, California 94304 Received
5
November 1990/Accepted 23 December 1990
Guanylate cyclase is regulated by adenine nucleotides in membranes of intestinal mucosal cells. Basal guanylate cyclase was activated about twofold by adenine nucleotides. Activation was specific for adenine, as compared with the pyrimidine nucleotides UTP and CTP. In addition, enzyme activation was obtained in the presence of saturating concentrations of GTP, the substrate for guanylate cyclase. The most potent adenine nucleotide was the nonhydrolyzable analog of ATP, adenosine 5'-O-(3-thiotriphosphate). Adenine nucleotide activation was specific for the particulate form of guanylate cyclase, as compared with the soluble form. Also, adenine nucleotides potentiated the activation of guanylate cyclase by the heat-stable enterotoxin produced by Escherichia coli. Indeed, enzyme activation by adenine nucleotides and toxin was greater than the sum of individual activations by these agents. Adenine nucleotides regulate guanylate cyclase by increasing the maximum velocity of the enzyme without altering its affinity for substrate or its cooperativity. In addition to stimulating guanylate cyclase, adenine nucleotides decreased the specific binding of the heat-stable enterotoxin to receptors in intestinal membranes. The coordinated regulation of the toxin-receptor interaction and guanylate cyclase activity by a process utilizing nonhydrolyzable analogs of a purine nucleotide is similar to the mechanisms involved in the hormone regulation of adenylate cyclase by guanine nucleotide-binding proteins. These data suggest that an adenine nucleotide-dependent protein may couple the toxin-receptor interaction to the regulation of particulate guanylate cyclase in intestinal membranes. mucosae from the intestines of one to three rats were pooled and homogenized in buffer containing 50 mM Tris-HCl (pH 7.6), 1 mM dithiothreitol, 1 mM EDTA, 0.25 M sucrose, and 1 mM phenylmethylsulfonyl fluoride. Homogenates were centrifuged at 100,000 x g for 60 min, and the resulting pellets were consecutively washed in (i) 50 mM Tris-HCI (pH 7.6)-i mM EDTA-1 mM dithiothreitol (TED) supplemented with 500 mM KCl and (ii) TED. Washed pellets were suspended in TED and stored at -80°C until used. Guanylate cyclase activity was assayed as described previously (12, 18, 31). In brief, incubations were conducted at 37°C in the presence of 50 mM Tris-HCl (pH 7.6)-0.5 mM isobutylmethylxanthine, a regenerating system including 7.5 mM creatine phosphate and 20 Rg of creatine phosphokinase (160 U/mg of protein), 10 to 20 pg of membrane or detergent extract, manganese- or magnesium-GTP (3 mM excess of free metal), and ST when needed. Some incubations contained different concentrations of various nucleotides or their analogs. Reactions were initiated by the addition of metal-GTP, incubated for 5 min, and terminated by the addition of 0.4 ml of ice-cold 50 mM sodium acetate (pH 4.0). Samples were placed in a boiling water bath for 3 min. Generated cyclic GMP was quantified by radioimmunoassay (25) as described previously (12, 15, 16, 18, 25, 31). All reactions were performed at least in duplicate, and the data are representative of at least duplicate experiments. Enzyme activity was linear with respect to protein concentration and time throughout these experiments. ST receptor binding was determined by a modification of the procedure described previously (20). In brief, intestinal membranes (20 to 40 ,ug of protein) were incubated in the reaction mixture used for guanylate cyclase assays, as outlined above, with 125I-ST (0.1 pmol) in a final volume of 60 RI. Nonspecific binding was measured in the presence of a 100-fold molar excess of unlabeled toxin and was less than
Escherichia coli heat-stable enterotoxin (ST) is a lowmolecular-weight peptide which stimulates intestinal secretion, resulting in diarrhea (1, 9, 13, 23, 24). This cascade is initiated by ST binding to specific receptors localized on brush borders of intestinal cells (6, 8, 10, 17, 20). The ST-receptor interaction activates the particulate form of guanylate cyclase in brush borders, elevating cyclic GMP in villous cells of the intestinal mucosa (7, 12, 15, 30, 31). Activation of guanylate cyclase by ST is often compared with enzyme activation by atrial natriuretic peptides (ANPs). Both systems involve coupling peptide-receptor interactions to enzyme activation (19, 22, 28, 30). However, the molecular events coupling receptor binding to enzyme activation remain unknown. Guanine nucleotide-binding proteins mediate receptor-dependent regulation of adenylate cyclase, but neither these proteins nor guanine nucleotides regulate guanylate cyclase (11, 26, 27). Interestingly, the activation of particulate guanylate cyclase by atrial peptides is potentiated by adenine nucleotides (4, 21). Similarly, adenine nucleotides activate basal guanylate cyclase in membranes isolated from rat lungs, liver, and kidneys (8a). The present report explores the ability of adenine nucleotides to regulate basal and ligand-stimulated guanylate cyclase in intestinal membranes. MATERIALS AND METHODS Membranes were prepared from homogenates of rat intestinal mucosal cells as described previously (31). In brief, *
Corresponding author.
t Present address: Departments of Medicine and Pharmacology, Division of Clinical Pharmacology, Jefferson Medical College, Thomas Jefferson University, 1100 Walnut St., Philadelphia, PA 19081.
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VOL. 59,
TABLE 1. Effect of nucleotides on particulate guanylate cyclase in rat intestinal membranesa No ST
None ATPgS ATP Adenyl-imidodiphosphate Adenosine-5'-O-(2-thiodiphosphate) dATP ADP AMP Adenosine UTP
1.0 2.7 1.8 1.5 1.5
± ± ± ± ±
0.0 0.2 0.2 0.1 0.2
Particulate enzyme Addition
ST
(7)C (5)C (5)d (6)e
5.1 13.4 11.0 9.6
± ± ± ±
1.0 4.0 3.2 3.2
tb
Sp act
(6) (6) (5) (5)
1.3 0.1 (5) 1.3 ± 0.3 (5) 1.2 0.1 (5) 1.2 ± 0.1 (3) 1.1 0.1 (4) 1.1 0.1 (4)
" Guanylate cyclase was assayed as outlined in Materials and Methods in the presence of a 1 mM concentration of the indicated nucleotide. The regenerating system was omitted from assays to minimize nucleotide phosphorylation. ST (30 ,uM) was added to incubations as indicated. Basal activity in these experiments was 3.7 ± 0.5 (seven experiments) pmol of cyclic GMP produced per min per mg of protein. b Calculated as the ratio of enyzyme activity in the presence and in the absence of the indicated nucleotide and reported as the mean ± standard error (number of experiments). " P < 0.0005. dp < 0.02. p
dATP, ADP, AMP, adenosine. Maximum activation of basal guanylate cyclase was two- to threefold with ATPgS. Adenine nucleotides which activated basal guanylate cyclase were tested for their ability to potentiate enzyme activation by ST (Table 2). These nucleotides potentiated the activation of guanylate cyclase by ST with an order of potency similar to that for the activation of the enzyme. Maximum activation of guanylate cyclase with the combination of ST and ATPgS was 10- to 20-fold, 3- to 4-fold greater than with ST alone. The activation of basal and ST-stimulated guanylate cyclase
3.1 16.0 7.7 41.7
Soluble enzyme
Fold ~ activationc
TABLE 3. Effect of ATPgS on the kinetics of rat intestine
particulate guanylate cyclasea Addition Addition
Control ST
ATPgS ST + ATPgS
O.5Vmxof protein)'
( (MM)b
4.8 4.7 4.3 3.8
± 0.2 ± 0.2
± 0.6 ± 0.6
(pmol/min/mg 32 176 48 278
± 2.5 ± 46.1
± 9.4e ± 50.7f
Hill
coefficientd 1.9 1.6 2.0 2.0
± 0.2 ± 0.2
± 0.1 ± 0.1
a Guanylate cyclase activity was determined in the presence or absence of 1 mM ATPgS with MgCI2-GTP as the substrate as outlined in Materials and Methods. Values are reported as means ± standard errors. The data are the means of three experiments. b Concentration of substrate yielding half-maximum guanylate cyclase
activity (3, 8a, 32). Maximum velocity of enzyme activity (picomoles of cyclic GMP produced per minute per milligram of protein) (3, 8a, 30). d Calculated as described previously (3, 8a, 30). c
e
p < 0.10.
fp
ATP > other adenine nucleotides. Maximum inhibition of ST binding of 30 to 40% was observed with ATPgS. Inhibition of binding was specific for adenine nucleotides, since CTP and UTP had no effect, and all binding was performed in the presence of 1 mM GTP.
PURINERGIC COUPLING OF ST AND GUANYLATE CYCLASE
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E -E 300
-
.2
0
200
-
100 _
0
5
10 Mg 2+GTP (mM)
15
20
FIG. 4. Effects of adenine nucleotides on the kinetics of particulate guanylate cyclase. Guanylate cyclase in rat intestinal membranes was assayed with a range of substrate concentrations as indicated, with magnesium as the cation cofactor, in the absence (U) or the presence of 30 ,uM ST (A), with 1 mM ATPgS (C), or with 30 ,uM ST plus 1 mM ATPgS (A) as outlined in Materials and Methods.
DISCUSSION The primary event mediating ST-induced secretion is the binding of peptides to intestinal cell surface receptors. These receptors are coupled to the activation of guanylate cyclase and increases in intracellular cyclic GMP. ST receptors and guanylate cyclase are separate molecules, and little is known concerning their functional coupling (29). It is well established that guanine nucleotide-binding proteins couple hormone-receptor interactions and adenylate cyclase activity (11, 26, 27). Recently, ATP was reported to enhance basal guanylate cyclase activity in rat lungs (8a) as well as atrial peptide-activated guanylate cyclase in rat lungs and liver (4, 21). These data suggest that adenine nucleotides may regulate basal and receptor-stimulated guanylate cyclase in a manner which is similar to the regulation of adenylate cyclase by guanine nucleotides. The data reported here demonstrate the activation of basal and ST-stimulated guanylate cyclase by adenine nucleotides. Activation of the enzyme was induced specifically by adenine, as compared with other nucleotides. Enzyme stimulation with ST and adenine nucleotides together was greater than the sum of individual activations by these agents. The most potent nucleotide regulating guanylate cyclase was the nonhydrolyzable analog of ATP, ATPgS. This effect is similar to the effects of nonhydrolyzable analogs of GTP c., adenylate cyclase mediated by guanine nucleotide-binding proteins (11, 26, 27). These data suggest that an adenine nucleotide regulatory protein may couple ST-receptor interactions to guanylate cyclase activation. Adenine nucleotides which serve as phosphate donors through a kinase reaction (ATP) and those which do not (adenyl-imidodiphosphate) were effective in activating guanylate cyclase. Thus, it is unlikely that activation involves phosphorylation of the enzyme by a kinase with adenine nucleotides as phosphate donors. Adenine nucleotides activate the particulate form of guanylate cyclase specifically, as compared with the soluble form. The stimulation of basal and ST-activated guanylate cyclase by adenine nucleotides was concentration depen-
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dent, increasing the maximum enzyme activity without altering the concentration of ST producing half-maximum activation. Similarly, adenine nucleotides increased the maximum velocity of guanylate cyclase without altering the concentration of the substrate required for half-maximum velocity or the Hill coefficient in the presence or absence of ST. These data suggest that adenine nucleotides alter the maximum rate of catalysis of the enzyme without altering the affinity of the enzyme for the substrate or the characteristic positive cooperativity exhibited by particulate guanylate cyclase (3, 31). These data correlate closely with those concerning the effects of adenine nucleotides on the activation of guanylate cyclase by atrial peptides in rat lungs (8a, 21). In those studies, adenine nucleotides increased the maximum velocity of guanylate cyclase without altering the affinity of the enzyme for the substrate or atrial peptides. Thus, coupling of peptide-receptor interactions to particulate guanylate cyclase activation by adenine nucleotides may involve a general mechanism of increasing the maximum velocity without altering other kinetic parameters of the enzyme. Specific binding of 1251-ST to intestinal membranes was reduced by adenine nucleotides. The relative abilities of these nucleotides to inhibit the binding of 1251I-ST to membranes correlated closely with their relative abilities to activate guanylate cyclase. These observations are similar to those concerning the regulation of ligand binding by guanine nucleotides and their binding proteins. In this system, coupling proteins are activated by binding GTP, which is induced by ligand-receptor interactions. Activated coupling proteins bound to GTP regulate adenylate cyclase activity and decrease the ability of receptors to bind the ligand (11, 26, 27). Thus, guanine nucleotide-binding proteins coordinately regulate the activities of the effector and receptor arms of the signal transduction cascade. A similar form of regulation of guanylate cyclase and ST receptors by adenine nucleotides is suggested by the data presented here. These data suggest the presence of an adenine nucleotide-binding protein which mediates the coupling of ST receptors and particulate guanylate cyclase and coordinately regulates the activities of both components. The mechanisms by which adenine nucleotides regulate basal and ligand-stimulated particulate guanylate cyclase remain unclear. Adenine nucleotides may bind directly to particulate guanylate cyclase, acting as allosteric modulators of that enzyme (21). This suggestion is supported by studies with a cloned ANP receptor-particulate guanylate cyclase protein (5). In these studies, a cloned enzyme-receptor possessed ligand binding activity coupled to guanylate cyclase activity, and adenine nucleotides potentiated enzyme activation by ANP. However, adenine nucleotides had no effect on basal particulate guanylate cyclase. A mutant protein with a deleted region encoding a domain with homology to protein kinase also possessed ligand binding and enzyme activities, but they were not coupled. Also, ATP did not activate particulate guanylate cyclase in the presence or absence of ANP. It was suggested from these studies that the deleted kinase domain was necessary for coupling the ANPreceptor interaction to guanylate cyclase and mediated adenine nucleotide regulation. However, it is problematic to ascribe adenine nucleotide regulation to this kinase domain, since these nucleotides did not affect basal enzyme activity in the cloned mutant system. Consequently, once ligand regulation of enzyme activity was lost, as in the mutant clone, adenine nucleotide regulation was automatically lost as well.
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GAZZANO ET AL.
INFECT. IMMUN.
Alternatively, adenine nucleotides may regulate particulate guanylate cyclase by a separate adenine nucleotidebinding protein. Previous studies demonstrated that adenine nucleotide regulation of ANP-stimulated particulate guanylate cyclase could be separated from that enzyme by sequential washing of crude membranes (4). Washing did not reduce the specific activity of the enzyme but did remove the ability of adenine nucleotides to potentiate the activation of guanylate cyclase by ANP. Similarly, recent studies demonstrated that adenine nucleotide regulation and guanylate cyclase activity could be separated by partial purification of this enzyme (8a). When detergent extracts of rat lung membranes were chromatographed on GTP-agarose, particulate guanylate cyclase was quantitatively purified. However, this partially purified enzyme was unresponsive to adenine nucleotides. These data support the suggestion that adenine nucleotide regulation of particulate guanylate cyclase is mediated by a protein which is separate from that enzyme (4, 8a). In conclusion, the data presented here demonstrate that the activation of particulate guanylate cyclase by ST-receptor interactions is regulated by an adenine nucleotide-dependent event. Activation is potentiated by analogs of ATP which cannot serve as kinase substrates. Thus, it is unlikely that adenine nucleotide regulation of this system reflects alterations in the phosphorylation state of the constituents of the involved signal transduction cascade. Adenine nucleotides alter the maximum activity of basal and agoniststimulated enzyme without altering the affinity of the enzyme for the substrate or the characteristic cooperativity of particulate guanylate cyclase. These nucleotides coordinately regulate both ST receptor and particulate guanylate cyclase activities. The nucleotide most potent in regulating these activities is the nonhydrolyzable analog of ATP, ATPgS. That both receptor and effector activities are regulated by a purine nucleotide-dependent mechanism which utilizes nonhydrolyzable analogs of ATP supports the suggestion that an adenine nucleotide-binding protein may regulate this system in a manner similar to the regulation of adenylate cyclase by guanine nucleotide-binding proteins. Identification and characterization of this coupling protein are currently being undertaken in this laboratory. ACKNOWLEDGMENT This work was supported in part by Administration.
a
grant from the Veterans
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