A role for antimicrobial peptides in intestinal microsporidiosis.

SUMMARY: Clinical isolates from 3 microsporidia species, Encephalitozoon intestinalis and Encephalitozoon hellem, and the insect parasite Anncaliia (Brachiola, Nosema) algerae, were used in spore germination and enterocyte-like (C2Bbe1) cell infection assays to determine the effect of a panel of antimicrobial peptides. Spores were incubated with lactoferrin (Lf), lysozyme (Lz), and human beta defensin 2 (HBD2), human alpha defensin 5 (HD5), and human alpha defensin 1 (HNP1), alone and in combination with Lz, prior to germination. Of the Encephalitozoon species only E. hellem spore germination was inhibited by HNP1, while A. algerae spore germination was inhibited by Lf, HBD2, HD5 and HNP1, although HBD2 and HD5 inhibition required the presence of Lz. The effects of HBD2 and HD5 on microsporidia enterocyte infection paralleled their effects on spore germination. Lysozyme alone only inhibited infection with A. algerae, while Lf inhibited infection by E. intestinalis and A. algerae. HNP1 significantly reduced enterocyte infection by all 3 parasite species and a combination of Lf, Lz and HNP1 caused a further reduced infection with A. algerae. These data suggest that intestinal antimicrobial peptides contribute to the defence of the intestine against infection by luminal microsporidia spores and may partially determine which parasite species infects the intestine.

Pathology of symptomatic microsporidial (Encephalitozoon hellem) bronchiolitis in the acquired immunodeficiency syndrome: a new respiratory pathogen diagnosed from lung biopsy, bronchoalveolar lavage, sputum, and tissue culture.

Encephalitozoon hellem is a recently described microsporidian associated with an expanding spectrum of clinical presentations in patients with the acquired immunodeficiency syndrome (AIDS). It is morphologically similar to Encephalitozoon cuniculi, a microsporidian infection of mammals and some avians, and their differentiation rests on biochemical and antigenic analyses. This report describes a patient previously diagnosed with keratoconjunctivitis due to E hellem who subsequently was found to have respiratory tract microsporidiosis by sputum cytology. He subsequently developed pulmonary symptoms and a left lower lobe interstitial infiltrate. A bronchoalveolar lavage and transbronchial biopsy revealed microsporidial bronchiolitis, and the etiologic agent was identified as E hellem using an immunofluorescent antibody technique. Lavage fluid was successfully cultured in monkey kidney cells, and cultivated E hellem organisms were studied using immunohistochemistry as well as scanning and transmission electron microscopy. The pathologic features of this newly described cause of protozoal bronchiolitis, the role of immunofluorescent antibody examination and in vitro tissue culture for species-specific diagnosis, and the significance of microsporidial pulmonary infections in AIDS patients are discussed.

Local attenuation of systemically mediated splanchnic vasoconstriction during shock due to cholera.

Hypovolemic shock, most often due to hemorrhage, is typically associated with intense splanchnic vasoconstriction. This can be severe enough to impair the functional and structural integrity of the gastrointestinal tract. Paradoxically, with cholera the structure of the gastrointestinal tract is preserved, and the intestine continues to secrete fluid delivered to it in the circulating blood in spite of severe hypovolemic shock. This suggests that splanchnic blood flow is maintained at higher levels in hypovolemic shock due to cholera than in hypovolemic shock due to hemorrhage. Our hypothesis is that cholera toxin in the intestinal lumen activates local mechanisms that attenuate systemically mediated splanchnic vasoconstriction. Blood flow to an isolated ileal segment in situ in the anesthetized rabbit was measured continuously (ultrasound transit-time volume flow probe) for 5 to 6 h after instillation of cholera toxin into the isolated intestinal lumen. Norepinephrine was infused selectively into the mesenteric artery supplying the segment to elicit local responses uncomplicated by compensatory changes secondary to systemic effects of norepinephrine. Baseline vascular conductance increased gradually and became significantly greater in cholera toxin experiments than in vehicle experiments 5 h after treatment (P < 0.035). Animals treated with cholera toxin were less responsive to norepinephrine than vehicle treated animals were (P < 0.05) and became more so over time (P < 0.001). Our conclusion is that cholera toxin activates local mechanisms that attenuate systemically mediated splanchnic vasoconstriction, at least in part by reducing vascular responsiveness to a systemic vasoconstrictor, norepinephrine.

Intestinal lumen and mucosal microclimate H+ and NH3 concentrations as factors in the etiology of experimental amebiasis.

Axenically cultivated Entamoeba histolytica, trophozoites, strains HM-1:IMSS and HM-38, were suspended in solutions of NaCl, 330 mOsm, of varying pH and ammonium concentrations. Short-term viability was inversely proportional to the ammonia concentration of the medium and was independent of the ammonium concentration and pH. The NH3-induced ameba killing was associated with a cellular alkalosis and cell swelling. While short-term trophozoite viability was unaffected by changes in the medium pH over the range 5.5-8.0, long-term viability was reduced by high pH, and of three pH values tested (6.27, 7.27, and 8.27), trophozoite growth was greatest at the lower value. Chemotaxis was observed in media over the pH range 5.5-8.0, and attenuated chemotaxis was observed in trophozoites in media containing NH3 (0.1 mM). The cecal content total ammonia concentration and pH and the in vivo mucosal microclimate pH were measured in young adult male rats, hamsters, and gerbils. Ceca of the three rodent species were also inoculated with HM-38 trophozoites and 7 days later the cecal contents were studied for signs of amebic infection. Infections were absent in the rat, the species with highest luminal total ammonia concentration and mucosal microclimate pH. All gerbils were infected. This species had the lowest mucosal microclimate pH. The hamster, with the intermediate microclimate pH, had a low infection rate (1 of 5). It is proposed that when ammonium diffuses from the large intestinal lumen into a more basic mucosal microclimate, it is converted to ammonia, and the combination of this ammonia and the high microclimate pH threatens Entamoeba trophozoite viability and reduces the probability of a given ameba penetrating the mucus blanket and invading the mucosal epithelium.

Movement of Entamoeba histolytica trophozoites in rat cecum and colon intact mucus blankets and harvested mucus gels.

Entamoeba histolytica trophozoites were centrifuged into mucus gels harvested from in vivo loops of rat cecum and proximal colon. Frank ameba movement was not detected in the colonic mucus, but attenuated motility was measured in the cecal mucus. The harvested rat cecal mucus had significantly lower apparent viscosity and neutral glycoprotein concentration values than the colonic mucus. A shape factor method was developed to assess the motility of amebae in mucus gels and intact mucus blankets. Shape factor data obtained from harvested mucus gel and intact mucus blanket experiments indicated that such mucus severely attenuated trophozoite movement with the attenuation being greater with colonic than with cecal mucus. Entamoeba trophozoites are known to be able to generate a pseudopod force of 3.3 x 10(-6) Newtons. Latex microspheres of the size range of Entamoeba trophozoites were forced through cecal and colonic mucus gels under gravity. Colonic mucus gels could withstand a force of 3.3 x 10(-6) Newtons while cecal mucus could not, suggesting that the ameba movement that was observed in cecal mucus involved mechanical penetration of the mucus by the ameba pseudopodia and did not require prior gel dissolution by Entamoeba enzymes.

Dietary fiber and giardiasis: dietary fiber reduces rate of intestinal infection by Giardia lamblia in the gerbil.

Gerbils were maintained on a low-fiber (5%) or a high-fiber (20%) diet in which the major fiber source was cellulose. Animals in the low-fiber diet group were significantly more likely to become infected when inoculated with 100 Giardia lamblia cysts than were animals in the high-fiber group. No differences were detected in gastrointestinal transit, gastric, and small intestinal luminal pH, or in duodenal mucus blanket acidic glycoprotein between animals in the high- and the low-fiber diet groups at the time of cyst inoculation. The fiber content of the diet after cyst inoculation determined the infection rate. These data suggest that the dietary fiber effect occurred during trophozoite colonization of the small intestine. When infected animals on the low-fiber diet were placed on the high-fiber diet for 24 hr, trophozoite clearing occurred in the lower small intestine. In the jejunum, the number of trophozoites attached to the mucosal surface decreased, while the number associated with luminal mucus increased. We conclude that the fiber-induced mucus secretion and the bulk movement of the insoluble fiber reduced the attachment of trophozoites to the intestinal mucosa, which decreased the probability of trophozoites establishing and sustaining colonization of the mucosa.

Separation of rabbit ileum mucus secretion from electrolyte and water secretion by cholera enterotoxin, verapamil and A23187.

Net water, Na+, Cl- and HCO3- fluxes were measured in in vivo rabbit ileal loops, while mucus secretion was assessed by measuring the glycoprotein or total sialic acid secreted into the lumen, or by measuring the luminal fluid viscosity. Inoculating loops with cholera enterotoxin (CT) produced a sustained secretion of electrolytes and water, but a more transient secretion of mucus. A dose of verapamil was found which, when included in the luminal fluid, inhibited or delayed the CT-induced mucus secretion while not affecting the ongoing electrolyte and water secretion. Exposure of the ileal mucosa to the ionophore, A23187, in the presence of 2mM Ca++ resulted in a brief secretion of mucus, with no change in basal water absorption. Verapamil inhibited this A23187-induced mucus secretion. The ionophore was not effective in the absence of luminal Ca++. Thus rabbit ileum mucus secretion can be separated from electrolyte and water secretion by agents that affect Ca++ movement.

Pathology of microsporidiosis: emerging parasitic infections in patients with acquired immunodeficiency syndrome.

OBJECTIVE: Microsporidiosis is a group of rapidly emerging protozoan infections that have thus far been reported predominantly from severely immunosuppressed persons with the acquired immunodeficiency syndrome (AIDS). The four genera that have been identified in AIDS patients (Enterocytozoon, Encephalitozoon, Septata, and Pleistophora) are an increasingly common source of both localized and disseminated infections. However, the clinical and pathologic features of these agents are being described with such rapidity that many pathologists are unaware of the histologic, immunologic, and molecular methods for diagnosing these infections. This article summarizes the clinical and morphologic spectrum of the microsporidian species that infect patients with AIDS. Additionally, the role of ultrastructural, immunologic, tissue culture, and molecular techniques for the diagnosis of microsporidian infections are discussed. DATA SOURCES: Clinical and pathologic findings were obtained from patients with AIDS who were evaluated for microsporidian infections at the Grady Memorial Hospital in Atlanta. Selected laboratory studies were performed at the Division of Parasitic Diseases of the Centers for Disease Control and Prevention and at the Department of Physiology at Morehouse University. Additionally, some cases were sent for consultation to the Infectious Disease Pathology service at Emory University. These data were combined with the published studies of microsporidian infection from the medical literature. DATA SYNTHESIS: The pathologic appearance of microsporidian infections in each major organ system (ocular, respiratory, genitourinary, gastrointestinal) is illustrated using routine and special histochemistry and immunofluorescence. The differential diagnostic features of the four genera of microsporidia infecting AIDS patients are illustrated using transmission and scanning electron micrographs from biopsy, autopsy, and tissue culture materials. Cytologic evaluation of body tissues is emphasized as a sensitive method for microsporidian diagnosis. CONCLUSIONS: Microsporidian infections can be expected to remain an increasingly important cause of morbidity and mortality in patients with AIDS. It is important that pathologists and microbiologists become acquainted with the clinicopathologic spectrum of these emerging protozoal infections, ensuring timely diagnosis and subsequent treatment.

Identification of the microsporidian Encephalitozoon hellem using immunoglobulin G monoclonal antibodies.

OBJECTIVE: Microsporidia isolated from clinical specimens so far have been identified to level of species by electron microscopy, indirect immunofluorescence (IIF), western blot (WB), and genetic analysis. Recent studies, however, indicate extensive serologic cross-reactions among microsporidian species involved in human disease. DESIGN AND SETTING: In this study, we used IIF and WB techniques to evaluate the reactivity of six different immunoglobulin G monoclonal antibodies (MAbs) raised against Encephalitozoon hellem with six isolates of E hellem that originated from patients with acquired immunodeficiency syndrome. A rabbit isolate of Encephalitozoon cuniculi, and an isolate of Encephalitozoon intestinalis, which was established in cultures from the urine of a patient with acquired immunodeficiency syndrome were also used for comparison. RESULTS: Five of the six antibodies, when analyzed by both IIF and WB assays, specifically identified six isolates of E hellem originating from three patients with acquired immunodeficiency syndrome. The sixth MAb, however, reacted with all of the E hellem isolates in the WB assay, but failed to react with them in the IIF assay. Using the IIF test, five of the six MAbs failed to react with E cuniculi and E intestinalis, even at a dilution of 1:50. The MAbs also did not react in the IIF test with Enterocytozoon bieneusi, Giardia, and Cryptosporidium. These MAbs did react with E cuniculi and E intestinalis in the WB assay, but the banding patterns were very different from those of E hellem, thus facilitating the identification of E hellem from the other microsporidia. The MAbs also reacted, in the IIF test, with E hellem spores in formalin-fixed tissue sections that were heated in a microwave oven. CONCLUSIONS: Identification of microsporidian agents to the species level is important. Since certain therapeutic agents (eg, fumagillin, albendazole) are efficacious in treating E hellem infections of the cornea, as well as urogenital and respiratory infections caused by E hellem, a quick and definitive identification of the organism is important so that successful therapy may be instituted. An IIF test using the MAbs described here would therefore be invaluable in the quick identification of this parasite.

Western blot and immunofluorescence analysis of a human isolate of Encephalitozoon cuniculi established in culture from the urine of a patient with AIDS.

Microsporidia spores, identified as Encephalitozoon cuniculi (CDC: V282), were isolated from the urine of a patient with acquired immunodeficiency syndrome and disseminated microsporidiosis, established in continuous culture on monkey kidney cells (E6), and antiserum was produced in rabbits. Immunoblot studies that used the patient serum and the rabbit sera against CDC:V282, Encephalitozoon hellem (CDC:0291:V213), and Encephalitozoon intestinalis (CDC:V297) revealed that CDC:V282 and the rabbit isolate of E. cuniculi (ECLD) reacted intensely with the patient's serum and the rabbit anti-CDC:V282, producing a number of bands ranging from 200 to 15 kDa. By contrast, the heterologous antigens (CDC:0291:V213 and CDCV297) reacted minimally. Both CDC:V282 and ECLD isolates of E. cuniculi reacted minimally with the rabbit anti-E. hellem and the rabbit anti-E. intestinalis sera. In the immunofluorescence test, performed on the lung biopsy section of the patient, the rabbit anti-CDC:V282 serum reacted extensively with the spores in the tissue section and produced bright apple green fluorescence. These studies demonstrated that the human (CDC:282) and the rabbit (ECLD) isolates of E. cuniculi were similar in their antigenic profiles but differed considerably from E. hellem and E. intestinalis, and that the patient's serum reacted specifically, strongly, and with equal intensity, with the 2 isolates of E. cuniculi.

Adenovirus masquerading as microsporidia.

Enterocytozoon bieneusi is a microsporidian that causes a severe, debilitating, chronic diarrhea in some patients with the acquired immunodeficiency syndrome. Specific diagnosis of E. bieneusi currently requires an invasive biopsy procedure and time-consuming preparation of specimens for electron microscopy. Our attempts to establish an in vitro culture system using mammalian cell cultures inoculated with duodenal aspirates, biopsy, or both, from 2 infected patients resulted in inadvertent coculture of an adenovirus and E. bieneusi. The adenovirus-infected cells deceptively appeared to contain spores of microsporidia based on light microscopic examination. Transmission electron microscopy revealed only a few microsporidia, but numerous cells infected with an adenovirus that was subsequently identified as adenovirus type 8. We believe that adenovirus infections prevented the cultured cells from supporting the proliferation of E. bieneusi and ultimately destroyed the cell cultures.

Cholera enterotoxin-induced mucus secretion and increase in the mucus blanket of the rabbit ileum in vivo.

The in vivo rabbit ileum was used to study the relationship of cholera enterotoxin-induced water and electrolyte secretion and mucus secretion and to determine whether the enterotoxin influenced the intestinal mucus blanket. In experiments in which luminal fluid viscosity was used to assess mucus secretion, it was found that while cholera enterotoxin induced a sustained secretion of water and electrolytes, the toxin-induced mucus hypersecretion was short lived (3 to 5 h) and subsequent exposure of the mucosa to cholera enterotoxin or prostaglandin E1 did not stimulate mucus secretion further. Dibutyryl cyclic AMP and theophylline caused a modest mucus secretion in ileal loops which differed from that of cholera enterotoxin in both magnitude and in the fact that the mucus secretion occurred 2 to 3 h after the onset of water and electrolyte secretion. An oral replacement solution was used in the ileum to reduce the enterotoxin-induced loss of water and electrolytes into the lumen. While such a solution slowed the appearance of acidic glycoprotein in the intestinal lumen, it did not change the amount of glycoprotein secreted over a 7-h period, suggesting that toxin-induced mucus secretion was not simply due to a flushing action of the experimentally caused diarrhea. To assess mucus blanket thickness, neutral glycoprotein was recovered from the blanket of rabbit ileal loops 7 h after exposure to cholera enterotoxin and the thickness of the mucus blanket was measured directly 4 and 18 h after toxin exposure. Both methods indicated that even though cholera enterotoxin-induced mucus hypersecretion had subsided and there was histological evidence of goblet cell mucin depletion, there was a sustained increase in mucus blanket thickness that was detectable for at least 18 h after mucosal enterotoxin exposure.

Role of the salivary glands in protecting the stomach against ethanol.

The submandibular gland complex was removed from young adult Wistar rats. Sham operations were performed on control animals. Three weeks later, the animals were starved for 24 hr and after pyloric ligation the stomachs were instilled with 30% ethanol. Two hours later, the animals were killed and the surface areas of the ulcerated mucosae were measured and the values used as ulcer index scores. In male, but not female, rats prior sialoadenectomy significantly exacerbated ethanol-induced gastric ulceration of the glandular stomach mucosa. This increase in ulceration was accompanied by an increase in the backflux of acid into the injured tissue. Sialoadenectomy had no statistically significant effect on histamine-stimulated gastric acid and pepsin secretion when these were measured 3 weeks later. Epidermal growth factor (EGF) was administered subcutaneously to intact animals in which the stomachs had been filled with 30% ethanol. When administered at a dose of 10 micrograms/kg per hr. EGF significantly reduced the ethanol-induced ulceration. High molecular weight nerve growth factor (7S NGF) was administered intragastrically prior to instilling ethanol in the stomachs of intact rats. The NGF had no effect on the severity of ethanol-induced ulcers. These data suggest that saliva protects the gastric mucosa from ethanol-induced ulceration and that salivary EGF is a possible candidate for this protection.

Putative anticryptosporidial agents tested with an immunodeficient mouse model.

Lasalocid, sinefungin, and dehydroepiandrosterone were tested for anticryptosporidial activity with an immunodeficient mouse model at doses that have been reported effective when tested with immunosuppressed rodent models. Small but significant reductions in oocyst excretion were only observed under some conditions with lasalocid and dehydroepiandrosterone, but sinefungin had no effect.

Arginine-derived nitric oxide reduces fecal oocyst shedding in nude mice infected with Cryptosporidium parvum.

Dietary L-arginine (4%) significantly reduced fecal oocyst shedding in athymic nude mice chronically infected with Cryptosporidium parvum. This effect appeared to be due to an increase in host nitric oxide (NO) production as it was not observed in arginine-supplemented animals administered the NO synthase inhibitor, N-nitro-L-arginine methyl ester. N-Nitro-L-arginine methyl ester alone significantly increased fecal oocyst shedding in chronically infected animals. In in vitro assays, oocyst excystation and sporozoite viability were significantly reduced by the NO donors sodium nitroprusside and S-nitroso-L-acetyl penicillamine in a concentration-dependent manner. These data suggest that arginine-derived NO may reduce the parasite load in experimental cryptosporidiosis.

A membrane-associated neuraminidase in Entamoeba histolytica trophozoites.

Trophozoites of the parasitic amoeba Entamoeba histolytica HM-1:IMSS possess a surface neuraminidase capable of liberating N-acetylneuraminic acid (NANA) from N-acetylneuramin-lactose (alpha 2----3 or alpha 2----6) or mucin in their medium. The neuraminidase was found to be membrane associated, with more than 50% of the yield being recovered in the plasma membrane fraction. The neuraminidase specific activity of the plasma membrane fraction was six times that of internal membrane fraction enzyme. The optimum pH and temperature for this enzyme were 6.7 and 37 degrees C, respectively. Neuraminidase activity was inhibited by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and the optimum Ca2+ concentration was 2 mM. The microfilament disruptor cytochalasin D (30 micrograms/ml) inhibited motility and neuraminidase activity of intact Entamoeba trophozoites. The cytochalasin D-induced loss of surface neuraminidase activity was explained in part by a redistribution of enzyme with a loss of plasma membrane enzyme and an increase in intracellular membrane enzyme. A qualitatively similar cytochalasin D effect was observed with two other membrane-associated enzymes, calcium-regulated ATPase and acid phosphatase. Membrane-associated enzyme was minimally affected by Triton X-100 and saponin. An N-acetylneuraminic acid aldolase, optimum pH, 7.4, was found in trophozoite homogenate supernatant fractions. NANA and NANA-containing compounds stimulated trophozoite-directed motility. This motility stimulation by NANA-containing compounds did not apparently require prior release of free NANA by the trophozoite surface neuraminidase. Entamoeba neuraminidase is one of a series of enzymes that may modify the mucus blanket and target cell surface and thereby play a role in the pathogenesis of amebiasis.

Reactive nitrogen and oxygen species ameliorate experimental cryptosporidiosis in the neonatal BALB/c mouse model.

Four-day-old BALB/c mice were infected by the oral administration of 50,000 Cryptosporidium parvum oocysts, and the resulting infection was scored histologically and by counting colonic oocysts. Infection occurred in the ileum and proximal colon (but not duodenum and jejunum), peaked on days 14 to 18, and was cleared between days 24 and 30. Nitric oxide (NO) appeared to play a protective role in this model as evidenced by the facts that plasma nitrite and nitrate levels increased during the period of peak parasitosis; immunohistochemically detected inducible nitric oxide synthase (iNOS) was increased in the ileum and colon enterocytes of infected animals; the NOS inhibitor L-N-iminoethyl lysine or N-nitro-L-arginine methyl ester (L-NAME) decreased the elevated plasma nitrite and nitrate levels while exacerbating the infection and increasing oocyst shedding; administration of a NO donor, S-nitroso-N-penicillamine, reduced oocyst and infection scores; and neonatal iNOS knockout mice exhibited a slightly longer infection than control animals. The oral administration of oocysts to L-NAME-treated BALB/c mice, but not control animals, between 24 and 40 days old resulted in the fecal excretion of oocysts 1 week later. Administration of the antioxidant ascorbic acid also exacerbated the C. parvum infection, suggesting a protective role for reactive nitrogen and/or reactive oxygen compounds, while administration of the superoxide scavenger superoxide dismutase exacerbated the infection. Taken together these data suggest that both reactive nitrogen and reactive oxygen species play protective roles in experimental cryptosporidiosis.

Effects of ethanol on electrical parameters of the in vivo rat stomach.

The gastric transmural electrical potential difference (PD) and direct-current resistance were measured in the rat in vivo under conditions in which the luminal pH was controlled over the pH range 1.0 to 12.0. Raising the pH above 11.0 caused a reduction in both PD and resistance. This is consistent with basic groups limiting cation permeability through the mucosa. Exposure of the mucosa to 20% ethanol caused a reduction in resistance, PD, and H+ secretion, and an increased appearance of Na+ in the lumen at neutral pH. The pH dependence of the resistance at high pH values was also eliminated, consistent with the elimination or bypassing of channels containing basic groups. Mucosal exposure to 8.5% ethanol reduced the PD and H+ secretion without affecting resistance. The increased appearance of Na+ in the lumen seen with this alcohol solution also occurred following exposure to hyperosmotic sucrose.

Lanthanum inhibition of Vibrio cholerae and Escherichia coli enterotoxin-induced enterosorption and its effects on intestinal mucosa cyclic adenosine 3',5'-monophosphate and cyclic guanosine 3',5'-monophosphate levels.

Several trivalent cations, including lanthanum (La3+), inhibited the secretion (enterosorption) induced by the enterotoxins of Vibrio cholerae and Escherichia coli in the rabbit ileum in vivo. High concentrations (greater than 10 mM) of La3+ were required to inhibit cholera enterotoxin (CE)-induced enterosorption, probably because of the adsorption of the La3+ often potentiated the CE-induced enterosorption. If luminal La3+ exposure followed CE exposure, some recovery of the enterosorptive response was observed. The longer the lag between the CE exposure and the La3+ exposure, the greater was the recovery of the enterosorptive response. Lanthanum inhibited HCO3- secretion more than Cl- secretion. By altering the luminal fluid pH at the time of La3+ exposure, it was found that La3+ was adsorbed to negatively charged luminal sites, having an apparent pK between 2.5 and 3.0. Although La3+ antagonized the enterosorptive response to CE, it mimicked rather than antagonized the cyclic adenosine 3',5'-monophosphate elevation and cyclic guanosine 3',5'-monophosphate depression induced by the toxin. It is therefore concluded that the La3+ inhibition of the CE-induced enterosorption must have occurred at a site following the generation of the cyclic nucleotides. Cholera enterotoxin caused complex time-dependent changes in the mucosal cyclic adenosine 3',5'-monophosphate and cyclic guanosine 3',5'-monophosphate levels, as revealed by studying tissue cyclic adenosine 3',5'-monophosphate/cyclic guanosine 3',5'-monophosphate ratios. The possible roles these two cyclic nucleotides may play in the pathogenesis of the cholera diarrhea are discussed.