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ABSTRACTVibrio parahaemolyticusis a marine bacterium known to be a common cause of seafood gastroenteritis worldwide. The thermostable direct hemolysin (TDH) has been proposed to be a major virulence factor of V. parahaemolyticus. TDH causes intestinal fluid secretion as well as cytotoxicity in a variety of cell types. In this study, we investigated the interplay between the hemolysin's enterotoxic and cytotoxic effects by using both human and rat cell monolayers. As revealed by microspectrofluorimetry, the toxin causes a dose-dependent increase in intracellular free calcium in both Caco-2 and IEC-6 cells. This effect was reversible only when low toxin concentrations were tested. The TDH-activated ion influx pathway is not selective for calcium but admits ions such sodium and manganese as well. Furthermore, in the same range of concentration, the hemolysin triggers a calcium-dependent chloride secretion. At high concentrations, TDH induces a dose-dependent but calcium-independent cell death as assessed by functional, biochemical, and morphological assays.

The uptake of 0.5 mM glycyl-L-proline (Gly-Pro), a substrate which has affinity for the dipeptide specific carrier system and is not hydrolyzed in the brush border, was studied in vitroin jejunum and ileum of rabbits from the 25th day of gestational age into adulthood. For comparison, uptake of 0.5 mM glycine was studied too. Results show that influx of Gly-Pro is present from the earliest age studied and show a steep prenatal increase and peaks at birth, with values, in the newborn jejunum, of 45.2 ± 3.3 μmoles/ghr (mean ± S.E.). Thereafter, the uptake slowly declines toward the adult level of 3.8 ± 0.5 μmoles/ghr. A similar pattern was present in the ileum. On the contrary, free glycine uptake shows no statistically significant change during the whole period studied.

The antisecretory effect of berberine sulfate (BS) was determined in the rabbit ileal mucosa in vitro mounted in Ussing chambers. When present in the luminal side, the drug had no effect on potential difference (PD) or on the short-circuit current (Isc) changes induced by theophylline or prostaglandin E2(PGE2). When present in the serosal medium at a concentration of 2 mM, however, berberine reduced PD and Isc and abolished residual ion flux (bicarbonate secretion) but had no significant affect on basal net Na and CI transepithelial transport. BS effect on basal PD was not affected by the absence of Ca2+in the medium: neither was it inhibited by the α-adrenergic blocker yohimbine. Serosally added BS effectively antagonized the secretory effect on Isc of agents that increase each of the known intracellular mediators of ion secretion: cyclic 3′, 5′-adenosine monophosphate (cAMP), cyclic 3′, 5′-guanosine monophosphate (cGMP), and Ca2+: it also inhibited the ion transport modifications evoked by PGE2. The drug had no effect on the concentration of cyclic nucleotides either under basal condition or in the presence of some secretagogues. Finally, BS did not impair the PD rise due to luminal addition of glucose.

Escherichia coli heat-stable enterotoxins (ST) are classified into STa and STb according to their physicochemical and biologic characteristics. STa induces diarrhea, activating the guanylate cyclase-cGMP system. ST-like enterotoxins can be produced by bacteria other than E. coli, including Klebsiella pneumoniae. A Klebsiella ST has previously been shown to share some chemical and immunologic characteristics with E. coli ST. Aiming to define better the nature of Klebsiella ST, we have screened 237 children with diarrhea and 179 controls for ST-producing Klebsiella, using the SMA. We detected 26 Klebsiella strains from patients, two of which were positive in the SMA, and 36 from controls, all negative for ST. A partial purification was performed using an acetone precipitation followed by ultrafiltration and gel filtration techniques. Klebsiella toxin was heat-stable, methanol-soluble, sensitive to mercaptoethanol, active at acid pH values, but not at pH >8. The time course of Klebsiella toxin in the SMA resembled that of E. coli STa. Klebsiella ST caused reduced Na absorption and net Cl secretion in rabbit ileal mucosa mounted in Ussing chambers. It was found to increase the cGMP but not the cAMP concentration. Finally, Klebsiella ST did not react with anti-£. coli STa MAb in a competitive ELISA. We conclude that K. pneumoniae may induce diarrhea through the production of an STa similar but not identical to E. coli STa.

Conflicting data have been published in favor of or against a secretory effect of atrial natriuretic peptide (ANP) in the intestine. The reported effects resemble that of Escherichia coliheat-stable enterotoxin (ST). In this work the effects of ANP were studied in well established experimental systems and compared with that of ST. Both peptides induced a prompt secretion of water, Na, and Cl with no effects on K net transport in the in vivo rat perfused jejunum. The addition of ST, but not of ANP, evoked an increase of short circuit current in rat intestinal mucosa mounted in Ussing chambers. ST induced a significant increase in guanylate cyclase activity in intestinal homogenates, whereas ANP showed no effect. No binding sites for ANP were detected in basolateral or brush border membranes, nor in isolated enterocytes by a suction filtration technique. In conclusion, ANP acts as a short-lived intestinal secretagogue in the rat. Its mechanism of action is different from that of E. coliST and appears to be indirect, since is not mediated by specific intestinal receptors and is not evident in vitro.

ABSTRACT: Escherichia coli heat-stable enterotoxins (ST) are classified into STa and STb according to their physicochemical and biologic characteristics. STa induces diarrhea, activating the guanylate cyclase-cGMP system. ST-like enterotoxins can be produced by bacteria other than E. coli, including Klebsiella pneumoniae. A Klebsiella ST has previously been shown to share some chemical and immunologic characteristics with E. coli ST. Aiming to define better the nature of Klebsiella ST, we have screened 237 children with diarrhea and 179 controls for ST-producing Klebsiella, using the SMA. We detected 26 Klebsiella strains from patients, two of which were positive in the SMA, and 36 from controls, all negative for ST. A partial purification was performed using an acetone precipitation followed by ultrafiltration and gel filtration techniques. Klebsiella toxin was heat-stable, methanol-soluble, sensitive to mercaptoethanol, active at acid pH values, but not at pH >8. The time course of Klebsiella toxin in the SMA resembled that of E. coli STa. Klebsiella ST caused reduced Na absorption and net Cl secretion in rabbit ileal mucosa mounted in Ussing chambers. It was found to increase the cGMP but not the cAMP concentration. Finally, Klebsiella ST did not react with anti-£. coli STa MAb in a competitive ELISA. We conclude that K. pneumoniae may induce diarrhea through the production of an STa similar but not identical to E. coli STa.