Proc. Natl. Acad. Sci. USA Vol. 90, pp. 3202-3206, April 1993 Microbiology Calcium influx mediated by the Escherichia coli heat-stable enterotoxin B (STB) LAWRENCE A. DREYFUS*t, BETH HARVILLE*, DANIEL E. HOWARDt, RADWAN SHABAN*, DIANE M. BEATTY*, AND STEPHEN J. MORRIS: Divisions of *Cell Biology and Biophysics and tMolecular Biology and Biochemistry, University of Missouri, Kansas City, MO 64110-2499 Communicated by Harley W. Moon, December 31, 1992 (receivedfor review November 18, 1992) ABSTRACT The heat-stable enterotoxin B (STB) of Esch- et al. (13, 14) indicates that STB induces a rapid short-circuit erichia coli is a 48-amino acid extracellular peptide that induces current across porcine intestinal mucosa. Evidence for chlo- rapid fluid accumulation in animal intestinal models. Unlike ride transport was not observed, yet electrogenic anion other E. coli enterotoxins that elicit cAMP or cGMP responses secretion, possibly bicarbonate, was observed in the absence in the gut [heat-labile toxin (LT) and heat-stable toxin A (STA), of elevated cyclic nucleotides (14, 15). Beyond these few respectively], STB induces fluid loss by an undefined mecha- studies, nothing is known of the mechanism of STB action. nism that is independent ofcyclic nucleotide elevation. Here we Evidence that suggests a receptor-mediated mode of STB studied the effects of STB on intracellular calcium concentra- action includes short-circuit current responses when toxin is tion ([Ca2+1), another known mediator of intestinal ion and delivered to the mucosal, but not serosal, side of intestinal fluid movement. Ca2+ and pH measurements were performed tissue mounted in Ussing chambers (13); a rapid STB re- on different cell types including Madin-Darby canine kidney sponse in experiments utilizing either mounted tissue or (MDCK), HT-29/C1 intestinal epithelial cells, and primary rat whole animal models (15 min and 2 hr, respectively) (13, 14, pituitary cells. Ca2+ and pH determinations were performed by 16); and the lack ofapparent damage to the intestinal mucosa simultaneous real-time fluorescence imaging at four emission following STB treatment (17, 18). Known mediators of mem- wavelengths. This allowed dual imaging of the Ca2+- and brane signal transduction include cAMP, cGMP, the phos- pH-specific ratio dyes (indo-l and SNARF-1, respectively). STB phatidylinositol 4,5-bisphosphate-derived metabolites inosi- treatment induced a dose-dependent increase in [Ca2+11 with tol 1,4,5-trisphosphate (InsP3) and diacyl glycerol, and Ca2W virtually no effect on internal pH in all of the ceUl types tested. (for reviews, see refs. 19-21). Since pathways for Ca2W- STB-mediated [Ca2+1i elevation was not inhibited by drugs that mediated intestinal secretion are known (22) and cyclic block voltage-gated Ca2+ channels including nitrendipine, ve- nucleotides are apparently not involved in the toxicity of rapamil (L-type), t-conotoxin (N-type), and Ni2+ (T-type). STB, we examined the effect of STB on the internal Ca2+ The increase in [Ca2+l] was dependent on a source of extra- concentration ([Ca2W]1) of transformed and primary cells of cellular Ca2+ and was not affected by prior treatment of different tissues and species oforigin. Our results suggest that MDCK cells with thapsigargin or cyclopiazonic acid, agents STB induces a rapid dose-dependent influx of extracellular that deplete and block internal Ca2+ stores. In contrast to these Ca2+ in cells of intestinal and nonintestinal origin; this Ca2+ results, somatostatin and pertussis toxin pretreatment of influx mediated by STB appears to occur through a pertussis MDCK cells completely blocked the STB-induced increase in toxin-sensitive, GTP-binding regulatory protein (G protein)- [Ca2+J1. Taken together, these data suggest that STB opens a GTP-binding regulatory protein-linked receptor-operated regulated Ca2+ channel. Ca2+ channel in the plasma membrane. The nature of the STB-sensitive Ca2+ channel is presently under investigation. EXPERIMENTAL PROCEDURES Toxin Preparation. STB was purified by a modification ofour Enterotoxigenic Escherichia coli cause diarrheal disease in recently reported method (5). Briefly, E. coli 1790 (provided man and animals by the production of protein or peptide by Shannon C. Whipp, U.S. Department of Agriculture- toxins. Two classes ofenterotoxins, separated on the basis of Agricultural Research Services National Animal Disease Cen- thermal stability, are recognized. Heat-labile enterotoxins ter, Ames, IA) harboring pPD21K, a kanamycin-resistant (LTs) ofE. coli (LT-I and LT-II) are multimeric proteins that derivative of a tac-promoted estB coding vector (23), was share antigenic, structural, and mechanistic similarity with grown in 24 liters of M9 medium (5) containing glucose (0.2%) cholera toxin (1). In contrast, the E. coli heat-stable entero- and kanamycin (50 ug/ml) (12 x 6-liter flasks, each containing toxins (STs) are peptides that induce rapid fluid secretion in 2 liters) for 18 hr at 37°C with vigorous aeration. After growth, animal gut loop models (2, 3) and bear no resemblance to bacterial cells were removed by ultrafiltration through a cholera toxin or LTs (4, 5). Two E. coli heat-stable entero- 0.1-k&m hollow fiber cartridge mounted in an Amicon DC-10 toxins (STA and STB) are currently recognized. STA is an 18- preparative ultrafiltration device (Amicon). The filtrate was (or 19)-amino acid cysteine-rich, heat- and protease-stable passed sequentially through spiral membrane cartridges of 100 peptide (6-8) that elevates mucosal cGMP by stimulation of kDa and 3 kDa exclusion to remove high molecular weight a particulate guanylate cyclase (9). After binding of the toxin material and to concentrate the STB-containing fraction, re- to a unique intestinal receptor (10, 11), the subsequent rise in spectively. The concentrated filtrate was fractionated by cGMP presumably results in a rapid increase in net chloride HPLC on a Waters 25 x 100 mm DeltaPak C4 preparative secretion. In contrast to STA, STB is a 48-amino acid basic column (Water Division, Millipore), followed by Mono-S peptide (5) that, although like STA is heat-stable and cysteine- cation-exchange fast protein liquid chromatography (FPLC) rich, is sensitive to trypsin proteolysis and bears no apparent structural. similarity to STA (5, 12). Previous work by Weikel Abbreviations: ST, heat-stable enterotoxin; [Ca2+]i, intracellular Ca2+ concentration; MDCK, Madin-Darby canine kidney; G pro- The publication costs of this article were defrayed in part by page charge tein, GTP-binding regulatory protein; LT, heat-labile enterotoxin; payment. This article must therefore be hereby marked "advertisement" InsP3, inositol 1,4,5-trisphosphate; CPA, cyclopiazonic acid. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 3202 Downloaded by guest on September 30, 2021 Microbiology: Dreyfus et al. Proc. Natl. Acad. Sci. USA 90 (1993) 3203 (Pharmacia Biotechnology). Finally, the toxin preparation was BSS or organic solvents as in the case of some of the drugs subjected to reverse-phase FPLC over a PepRPC 15 column used (see below). (unpublished data). The STB preparation was judged pure by Simultaneous Ca2+ and pH measurements were made on a each of the following criteria: SDS/PAGE analysis followed new design of a multiimaging fluorescence videomicroscope, by silver staining, amino acid composition analysis, and au- which has been described in detail (27). By monitoring four tomated Edman degradation as described (5). emission wavelengths, the microscope performs simulta- Cells and Cell Lines. Madin-Darby canine kidney (MDCK) neous real-time capture of four separate fluorescence emis- cells (24), obtained as an outgrown secretory cell line from J. sion images. The fluorescent dyes were excited at 350 nm Grantham (University of Kansas Medical Center, Kansas (indo-1) and 540 nm (SNARF-1) and imaged at 405,475,575, City), were grown ;at 37°C in an atmosphere of 95% air/5% and 640 nm. Each emission wavelength was imaged simul- CO2. Growth medium consisted of Dulbecco's modified taneously by one of four cameras at standard video-frame Eagle's medium (DMEM; Sigma) supplemented with 10% rates and then stored on 3/4" video tape for further off-line (vol/vol) fetal bovine serum (FBS; JRH Scientific, Lenexa, analysis by digital image processing (describer below). The KS), penicillin (100 units/ml), and streptomycin (100 ,ug/ml). pH maps generated by this analysis were used to correct the HT-29/Cl intestinal epithelial cells (25) were obtained from Ca2+ maps for the pH dependence of indo-1. The integrated Cynthia L. Sears (The Johns Hopkins University School of Ca2+ and pH values for 5-20 cells were extracted from the Medicine, Baltimore) and grown in an atmosphere of 90%o video data and transferred directly to SigmaPlot 4.1 (Jandel, air/10% CO2. Growth medium was DMEM (Sigma) supple- Corte Madera, CA) for plotting; representative responses of mented with 10% FBS, 1 mM pyruvate, 44 mM NaHCO3, cells were then graphed. Ca2+ standards were determined human transferrin (10 ,g/ml; GIBCO/BRL), penicillin (100 with free indo-1 and commercial Ca2+ buffers (Molecular units/ml), and streptomycin (100 ,zg/ml). Probes) at a series of defined free-Ca2+ concentrations be- Primary pituitary intermediate lobe melanotropes were tween 0 (Rmin below) and 40 ,uM. Ri,. (below) was taken as isolated as follows (26): male Sprague-Dawley rats (150-175 40 mM CaCl2. Standards for SNARF-1 were obtained at g; Sasco, Omaha, NE) were anesthetized by i.p. injection various pH values between 5.0 and 8.0 by using free with Nembutal (200 mg/kg; Abbott) and sacrificed by de- SNARF-1 dye and pH-adjusted basic buffer. Experiments capitation; after exposure of the brain, the neurointermediate were repeated at least twice on at least two different days. lobe was dissected from the anterior lobe, and lobules from Analysis of Recorded Images.