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. This has been discussed in one intermediate lobe were teased from the neural lobe and detail (27). The dye-Ca2+ dissociation constant (Kd) of indo divided among 6 wells of a 12-well tissue culture plate, each 1 and fura 2 is dependent upon prevailing pH (28, 29). Rather containing a no. 00 coverslip (Coming) coated with poly(L- than presuming that the intracellular pH remains fixed lysine) (Sigma). The cells were grown in CellGro Plus me- throughout the experiment, we used the recorded intracel- dium (Fisher Scientific) supplemented with 5% FBS, peni- lular pH to calculate a corrected [Ca2W+i. The taped images cillin (100 units/ml), and streptomycin (100 pg/ml) in an were corrected for background, shading error, and spillover atmosphere of 93% air/7% CO2. The explants were allowed of indo fluorescence into the SNARF images. For each cell, to grow until the lobules were well attached to the coverslips the integrated fluorescence from the 575- and 640-nm image and a confluent monolayer of cells surrounded the explant a ratio value for calculation of pH by Eq. (10-14 days). was used to form Loading Cells with Fluorescent Dyes. MDCK and HT-29/C1 1 (30): cells were stripped from culture flasks by treatment with pH = pKa + log(Sf2/Sb2) + log[(R - Rmiln)/(Rma( - R)], [1] 0.01% trypsin/0.05 mM EDTA (JRH Scientific) when culture flasks reached 75-80% confluence. Cells were washed free of where R, Rmax, and Rmin are the actual, maximal, and minimal trypsin/EDTA, and 1 x 105 to 5 x 105 cells were seeded onto ratios of the SNARF data, (Sf2/Sb2) is the ratio of free and sterile no. 00 coverslips, which were flooded with complete bound dye at the R denominator wavelength, and pKa = 7.30. medium (see above) and incubated as described above. Cells This value was then used to calculate a pH-corrected value plated on coverslips 2-3 days prior to experiments were of Kd for indo 1: simultaneously loaded with 5 ,uM indo-1,AM and 5 ,uM SNARF-1,AM, both cell-permeant acetoxymethyl (AM) es- Kd = (Kd = + 1O(PHPKa).Kd )/(10(PH PKa) + 1). [2] ters (Molecular Probes), in DMEM (Sigma) containing 0.02% Pluronic F-127 detergent (Molecular Probes) and 12.5% (vol/ Values for Kdm., Kd,, and pKa at 37°C of 80.9 ,M, 141 nM, vol) dimethyl sulfoxide for 30 min at 37°C in an atmosphere and 3.65 were calculated from a least-squares fit of Eq. 2 to of 93% air/7% CO2. After the incubation period, cells were the data for indo/EGTA as published by Lattanzio (28, 29). washed with DMEM and maintained in 93% air/7% CO2 at The ratio of the 405-nm and 475-nm images and the corrected 37°C in DMEM supplemented with 5% FBS for a 1-hr Kd were used to calculate the corrected Ca2+ value: recovery period to allow esterase cleavage of the dyes to the impermeable form. Cells were used within 90 min of this [Ca2+]i = Kd.,T (Sf2/Sb2)-[(R - Rmin)/(Rmax- R)], [3] recovery period. Labeling of intermediate lobe pituitary cells was performed as just described for MDCK and HT-29/Cl where R, Rmax, and Rmin are defined as above for the indo cells. data. Ca2+ and pH Measurements. Dye-loaded cells on cover- Pharmacologic Agents. Thapsigargin, cyclopiazonic acid slips were placed in 1.0 ml of standard balanced salt solution (CPA), pertussis toxin, somatostatin, verapamil, nitren- (BSS) (138 mM NaCl/2 mM KCl/2 mM MgCl2/5.5 mM dipine, and w-conotoxin were obtained from Sigma. Thapsi- glucose/50 ,uM EGTA/10 mM Hepes, pH 7.4) contained in gargin, verapamil, and nitrendipine were dissolved in etha- a microscope-stage perfusion chamber maintained at 37°C. nol, CPA was dissolved in DMSO, and then all were diluted For the standard assay, 10 Al of toxin solution was added to to their respective working concentration in BSS. The final the bath (final toxin concentration, 500 nM). Two to four concentration ofsolvent in the microscope stage bath was 1% minutes after the addition oftoxin, CaCl2 was added to a final ethanol when using thapsigargin and nitrendipine and was 1% concentration of 3 mM. For other experiments, the addition dimethyl sulfoxide when using CPA. Control experiments time of drugs or STB or both was varied as noted. Additions using 1% ethanol and 1% dimethyl sulfoxide resulted in no of toxin, drugs, and Ca2+ were made directly to the perfusion effect on normal cell function or [Ca2+]1. All other agents chamber from 10-100 x concentrated solutions made either in were dissolved in BSS. Downloaded by guest on September 30, 2021 3204 Microbiology: Dreyfus et al. Proc. Natl. Acad. Sci. USA 90 (1993) RESULTS Table 1. Dose-dependent change in [Ca2+]j Effect of STB on [Ca2+],. The addition of 3 mM Ca2+ to STB dose,* nM indo-l- and SNARF-1-preloaded cells had no ceffect on A[Ca2+]it [Ca2+], or cellular pH; however, STB pretreatmentt (ca. 500 0 0 nM) of cells in Ca2+-free medium resulted in a rapidlincrease 50 90.7 23 in [Ca2+]j. Typical responses of MDCK and HT-29)/C1 cells 500 239.9 ± 7.7 to treatment with 50 nM STB are shown in Fig>. 1. The 5000 397.8 ± 127.9 response of melanotropes to STB (not shown) was 1similar to *STB dose is final concentration in microscope stage bath. that of MDCK and HT-29/C1. In the case of MIDCK and tChange in [Ca2+]i is the difference between the concentration at the melanotropes, no change in pH was observed over tihe course time of STB addition and the maximum response deflection. of STB action, whereas a slight decline in pH was observed after the addition of STB to HT-29/C1 cells. Since all three resting levels, while internal pH was not significantly affected cell types tested exhibited similar responses to STB, we during the course ofthe experiments, even at the highest STB selected the MDCK cell line to investigate further Ithe effect dose. This is in contrast to a number of secretory cell types, of toxin on [Ca2+]L. STg treatment of MDCK cells rcesulted in which show sharp decreases in pHi when [Ca2+]i is increased a dose-dependent rise in [Ca2+]i as shown in Table 1. [Ca2+]i, by influx through L-type Ca2+ channels. The duration of elevation ranged from a 2- to nearly 4-fold increbase over elevated [Ca2+], was also dose-dependent, lasting only 1-2 L - min with 50 nM STB and 5-6 min in higher doses (not shown), A 1000 after which time [Ca2+]i returned to the resting level (ca. 200 nM). 800 7.5 Effect of STB on Internal Ca2+ Stores. In preliminary experiments we noted that the increase in [Ca2 ]i due to STB 600 was not observed in the absence of extracellular Ca2+. 1- 79@ Therefore, we hypothesized that elevation of [Ca2+]i was due ._ _ _ _ to an influx of extracellular Ca2+ and not to release of Ca2+ IC 400 _~~~~ N from an internal store. To test this hypothesis, we treated 6.5 MDCK cells with thapsigargin (31), a plant alkaloid that 200 _ inhibits the sarcoplasmic reticulum Ca2+-ATPase. Thapsi- gargin treatment induces release of InsP3- and GTP-sensitive 01 I 6.0 Ca2+ stores and inhibits their replenishment. Treatment of 0 60 120 180 240 300 360 MDCK cells with thapsigargin did not diminish the STB- B 'I000 I- - 7.5 induced [Ca2+]i rise; instead, thapsigargin-pretreatment sen- sitized MDCK cells to subsequent STB-mediated [Ca2+]i 2 750 - elevation (Fig. 2). The enhancement of Ca2+ influx after 751 I 7.0 treatment with thapsigargin has been observed in intermedi- ate lobe melanotropes (D.M.B., unpublished data) and PC12 500 _ phaeochromocytoma cells (E. W. Westhead, personal com- munication). Depletion of intracellular Ca2+ stores by thap- . ,

I . , sigargin is - . known to activate ligand-gated plasma membrane --41 .. y -*-. Pl. , I ;:..,4 250 t :e )l 6.0 Ca2+ channels (32). Identical results were seen in MDCK cells and melanotropes when internal Ca2+ stores were 5-5 blocked with CPA, another Ca2+-ATPase drug possessing o similar activity to that of thapsigargin (33). 0 60 120 0SO 240 300 1000 C 7.25 1400 F J t - 750 7.5 12001

1- 10001 7.00 N 500 -. %,i %. 7.0 = r- 800 Q + .0 ..A 6-. I: .." i; .i. %. u 9I i~~~~~~~~~~~~~~~~~~~ - 600 ...%.. . .. 250 6.5 6.75 400

200 o 16.0 60 120 240 300 360 181 0 1 Time (sec) 0 120 240 360 480 600 720 840 Time (sec) FIG. 1. Increase of [Ca2+], in response to STB. Timed measure- ments of [Ca2+], (closed symbols) and pH (open symbols) of indi- FIG. 2. Effect ofthapsigargin treatment on STB-mediated [Ca2+]i vidual control MDCK cells (A) or experimental MDCK cells (B) and increase in MDCK cells. MDCK cells were treated with 300 nM HT-29/C1 cells (C) treated with 500 nM STB. Cells were incubated thapsigargin for 2 hr prior to loading the cells with SNARF-1 and in low Ca2+-containing medium and treated (or not) with toxin 2-4 indo-1. Dye-loaded cells were placed in the microscope chamber and min prior to the addition of 3 mM Ca2+. The arrow indicates the time treated with 500 nM STB 4 min prior to the addition of Ca2 which of Ca2+ addition. Each pair of symbols (i.e., closed and open circles is noted by the arrow. Closed symbols represent individual-cell or closed and open triangles, etc.) represents determinations from a [Ca2+]i; open symbols represent intracellular pH of the correspond- single cell; thus, each panel contains data from two cells. ing cells. Downloaded by guest on September 30, 2021 Microbiology: Dreyfus et al. Proc. Natl. Acad. Sci. USA 90 (1993) 3205 7.50 A 8.0 A i0IK

960 800 L 7.5 7.25

1- 720 ...... 600 Cu 6.5_ D-, !-- ,r -v 7.0 :EF: 7.00 : -, N-,, v + +Iu V. .,.i . 400 .4. I 480~ -:1 6.5 200I ----l6.75 240 _ 6.50 0 6.0 0 0 120 240 360 0 60 120 180 240 300 360 1000 - - B B 0ooo 7.50 . 4 _ ..1~~~otMB, S m s4 A o ^> ~7.50

750 - -.fl.l. 7.00 750 7.00

C-Cu + 1- 500 6.50 = F._ Soo N 6.50 Cd Cu

250 250 6.00 4 _ L >~~~~~~~~~~~~~6.00

0 5.55 5.50 0 0 60 120 180 240 300 360 420 480 0 60 120 180 240 300 360 420 480 TIME (sec) Time (sec)

FIG. 3. Effect of Ca2+ channel blocking agents on STB-mediated FIG. 4. Effect of somatostatin and pertussis toxin on STB- Ca2+ increase in MDCK cells. MDCK cells were treated with 500 nM mediated Ca2+ increase in MDCK cells. Somatostatin (1 ,uM) was STB in the presence of 10 ,M nitrendipine (A) and 40 zM nickel (B). added to the cell bath 5 min prior to treatment of MDCK cells with Ca2+ addition is noted by the arrow in each panel. Closed symbols 500 nM STB (A). Ca2+ was added as indicated by the arrow. MDCK represent individual-cell [Ca2+]i; open symbols represent intracellu- cells were pretreated on coverslips for 2 hr with pertussis toxin at 1 lar pH of the corresponding cells. pg/ml (B), rinsed, and loaded with fluorescent dyes as described. STB treatment (50 nM) preceded the addition ofCa2+ (as noted by the Ca2+ Channel Blockers. The failure of STB to mediate arrow) by 2 min. Closed symbols represent [Ca2+]i levels of indi- release of Ca2+ from internal stores suggested that elevated vidual cells; open symbols represent intracellular pH of the corre- [Ca2+], was a function of the opening of a plasma membrane sponding cells. Ca2+ channel. Elevation of MDCK cell [Ca2+]i by STB treatment, however, was not blocked when the cell bath also the argument that a STB receptor exists; further, the receptor contained 10 ,uM nitrendipine (Fig. 3A) or 10 AM verapamil is present on cell types of both intestinal and nonintestinal (not shown), drugs that block L-type voltage-gated Ca2+ origin. Initial experiments investigating [Ca2+]i of MDCK cells channels (34, 35). Similarly, 40 ,uM Ni2 , an inhibitor of T-type voltage-gated Ca2+ channels (34), was also without treated with STB indicated that little effect was observed effect on STB-induced Ca2+ elevation (Fig. 3B). Blockage of when cells were bathed in high Ca2+-containing buffer (3 N-type Ca2+ channels by w-conotoxin also failed to diminish mM). In contrast, we observed dramatic increases in [Ca2+]i the STB effect on MDCK cells (data not shown). when cells were first incubated with toxin in Ca2+-free Role of G Proteins in STB-Mediated Ca2+ Entry. In contrast medium or low calcium (0.5 or 1 mM)-containing medium to these results, elevation of [Ca2+]i in STB-treated MDCK before the extracellular Ca2+ concentration was raised to 3 cells was completely inhibited by pretreatment with 1 ,M mM. Increase of cellular [Ca2+]1i was not observed in the somatostatin and pertussis toxin at 1 ,g/ml (Fig. 4). Taken absence of toxin treatment. The results suggest that Ca2+ together, these results suggest that STB induces an influx of may influence the biochemical action of the toxin in such a extracellular Ca2+ through a receptor-operated (ligand-gated) way that binding of STB leading to activation of a Ca2+ Ca2+ channel on the plasma membrane of MDCK cells. The channel occurs only under conditions of low extracellular STB-responsive Ca2+ channel is apparently opened through Ca2+. The high concentration of positive charge on STB (pI the action of a PT-sensitive G protein. > 9.0) could, at the very least, promote STB association with the negatively charged polar head groups of the plasma membrane surface; in addition, the highly flexible and pos- DISCUSSION itively charged disulfide-bonded loop formed by STB residues This communication reports a biochemical action for the E. 21-36 (Cys-Lys-Lys-Gly-Phe-Leu-Gly-Val-Arg-Asp-Gly- coli STB enterotoxin. Previous attempts to identify secondary Thr-Ala-Gly-Ala-Cys) (5) could potentially compete for messengers of STB action proved that the toxin exerts its Ca2+-binding sites at a channel or channel regulatory site. biological effect without apparent elevations in cAMP or The 21-36 residue loop, and in particular residues Arg-29 cGMP, hallmark activities of other E. coli enterotoxins. It is (codon 53) and Asp-30 (codon 54), is known to be required for assumed that STB alters fluid and electrolyte transport in the full expression of toxicity (5). The association of STB with gut after interaction with a mucosal receptor. Although the plasma membrane is not known at this point. notion of a STB receptor is based on inference from Ussing The requirement for STB action in low Ca2+-containing chamber studies and in vivo models, this report strengthens medium and sensitivity to somatostatin are similar to the Downloaded by guest on September 30, 2021 3206 Microbiology: Dreyfus et al. Proc. Natl. Acad. Sci. USA 90 (1993) extracellular Ca2+-mediated elevation of [Ca2+]1, and the 8. Dreyfus, L. A., Frantz, J. C. & Robertson, D. C. (1983) Infect. concomitant release ofcalcitonin in calcitonin-secreting cells Immun. 42, 537-543. (C-cells) (36). Ca2+ influx in C-cells is mediated by a pertussis 9. Field, M. L., Graf, L. H., Laird, W. J. & Smith, P. L. (1978) toxin-insensitive voltage-gated Ca2+ channel. In contrast, the Proc. Natl. Acad. Sci. USA 75, 2800-2804. action of STB is completely inhibited by pertussis toxin, 10. Frantz, J. C., Jaso-Freidman, L. J. & Robertson, D. C. (1984) indicating the role of a pertussis toxin-sensitive G protein Infect. Immun. 43, 622-630. 11. Giannella, R. A., Luttrell, M. & Thompson, M. (1983) Am. J. [inhibitory (Gi) or "other" (Go) G protein]. Known mecha- Physiol. 245, 6492-6498. nisms ofCa2+ channel regulation by G proteins involve either 12. Whipp, S. C. (1987) Infect. Immun. 55, 2057-2060. stimulation by Gs (stimulatory G protein) subunits or inhibi- 13. Weikel, C. S. & Guerrant, R. C. (1985) in Microbial Toxins and tion through Go activity (37). The former class is represented Diarrheal Disease, eds. Evered, R. & Whelan, J. (Pitman, by the dihydropyridine-sensitive cardiac sarcolemmal Ca2+ London), pp. 94-115. channel, which relies on G,-protein action (38). The latter 14. Weikel, C. S., Nellans, H. N. & Guerrant, R. L. (1986) J. class of channels is exemplified by the y-aminobutyric acid Infect. Dis. 153, 893-901. GABAB receptor, which inhibits voltage-gated Ca2+ currents 15. Kennedy, D. J., Greenberg, R. N., Dunn, J. A., Abernathy, through the action of a pertussis toxin-sensitive G. protein R., Ryerse, J. S. & Guerrant, R. L. (1984) Infect. Immun. 46, (39). The inhibition of the STB-mediated [Ca2+]i increase by 639-643. pertussis toxin suggests the involvement of a G protein, 16. Whipp, S. C. (1990) Infect. Immun. 58, 930-934. either or although that toxin also inactivates 17. Rose, R., Whipp, S. C. & Moon, H. W. (1987) Vet. Pathol. 24, Gi Go; Gp 71-79. (phospholipase stimulatory G protein) action (37), the in- 18. Whipp, S. C., Moseley, S. L. & Moon, H. W. (1986) Am. J. volvement of this G protein is unlikely, since we did not Vet. Res. 47, 615-618. observe release of Ca2+ from an InsP3-sensitive internal 19. Schramm, M. & Selinger, Z. (1984) Science 225, 1350-1356. store. The argument for G-protein involvement in the STB- 20. Chinkers, M. & Garbers, D. L. (1991) Annu. Rev. Biochem. 60, mediated increase in [Ca2+], is strengthened by the observed 553-575. inhibitory effect of somatostatin, a G-protein antagonist (40). 21. Abdel-Latif, A. A. (1986) Pharm. Rev. 38, 227-272. The reported inhibitory effect of somatostatin on G, is 22. Powell, D. W. & Fan, C. C. (1983) in Intestinal Transport, eds. attributed to the drug-dependent dissociation of Gi into a and Gilles-Baillien, M. & Gilles, R. (Springer, Berlin), pp. 215-226. subunits. is thought to inhibit dissociation of 23. Urban, R. G., Pipper, E. M., Dreyfus, L. A. & Whipp, S. C. ,Sy ExcessBy Gs (1990) J. Clin. Microbiol. 28, 2383-2388. (and presumably Go) (40). 24. Misfeldt, D. S., Hamamoto, S. T. & Pitelka, D. R. (1976) Proc. Recently, Hitotsubashi et al. (41) presented data indicating Natl. Acad. Sci. USA 73, 1212-1216. a role for prostaglandin E2 (PGE2) in STB-mediated fluid 25. Weikel, C. S., Grieco, F. D., Rueben, J., Myers, L. L. & Sack, secretion in the mouse. Fluid accumulation peaked at 3 hr R. B. (1992) Infect. Immun. 60, 321-327. concomitant with an approximate 3-fold increase in the level 26. Chronwall, B. M., Bishop, J. F. & Gehlert, D. R. (1988) Pep- of PGE2. Secretion was significantly blocked by indometh- tides 9, 169-180. acin pretreatment of mice. The time course of intestinal 27. Morris, S. (1993) in Optical Microscopy, eds. Herman, B. & secretion and PGE2 accumulation relative to that presented Lemasters, J. J. (Academic, New York), pp. 177-212. here for Ca2+ influx suggests that events at the plasma 28. Lattanzio, F. A. (1990) Biochem. Biophys. Res. Commun. 171, 102-108. membrane that cause increased [Ca2+]i may trigger subse- 29. Lattanzio, F. A. & Bartchat, D. K. (1991) Biochem. Biophys. quent events, including PGE2 synthesis and intestinal secre- Res. Commun. 177, 184-191. tion. 30. Martinez-Zaguilan, R., Martinez, G. M., Latanzio, F. & Gil- lies, R. J. (1991) Am. J. Physiol. 260, C297-C307. The work was supported by research grants from the National 31. Thastrup, O., Cullen, P. J., Drobak, B. K., Hanley, M. R. & Institutes of Health (AI32736 to L.A.D. and GM44071 to S.J.M.) and Dawson, A. P. (1990) Proc. Natl. Acad. Sci. USA 87, 2466- the National Science Foundation (BN9211912 to S.J.M.). 2470. 32. 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