In vivo and in vitro effects of tea extracts on enterotoxigenic Escherichia coli-induced intestinal fluid loss in animal models.

OBJECTIVES: Enterotoxigenic Escherichia coli (ETEC) infection is a major cause of dehydrating diarrhoea in infants and early-weaned piglets living under subhygienic conditions. We studied the effect of different tea types and subfractions on the intestinal fluid and electrolyte losses involved in ETEC diarrhoea. MATERIALS AND METHODS: Jejunal segments of anaesthetised piglets were infected with ETEC or ETEC heat-labile toxin (LT) and subsequently perfused for 8 hours with control or tea solutions containing green or black tea extract (BTE) or 3 different BTE subfractions containing small-size, large-size or no phenolics. Changes in intestinal fluid and electrolyte net absorption were measured. To assess the antisecretory effects of tea, BTE was incubated before or after administration of the secretagogue forskolin in rat jejunal tissue placed in Ussing chambers and Cl- secretion measured as changes in short-circuit current (I(SC)). RESULTS: Enterotoxigenic E. coli infection of piglet jejunal segments significantly reduced net absorption of fluid, Na+ and Cl- and increased net secretion of K+ compared with controls. Perfusion of the ETEC-infected segments with both 3 g/L green tea extract and BTE significantly inhibited these disturbances in fluid and electrolyte balance. The BTE subfraction rich in polymeric phenolics but not the other subfractions improved the fluid and electrolyte balance. Addition of forskolin to rat jejunal tissue induced a significant increase in I(SC). Pretreating but not posttreating the jejunal tissue with BTE inhibited the forskolin-induced increase in I(SC). CONCLUSIONS: Tea may inhibit net fluid and electrolyte losses involved in secretory diarrhoea from ETEC.

Ammonia inhibits sodium and chloride absorption in rat distal colon.

It was recently demonstrated that ammonia inhibits sodium absorption in the proximal colon of rats. In order to investigate the effect of luminal ammonia in the distal colon, sodium and chloride transport were measured in Ussing chambers. Under short-circuit conditions, distal colon absorbed sodium and chloride. When luminal ammonia (30 mmol l(-1)) was present, sodium and chloride absorption was diminished. Inhibition of the two Na(+)-H(+) exchanger isoforms NHE2 and NHE3, which are known to be located in the apical membrane of the distal colon epithelium, failed to influence the effect of ammonia on transepithelial sodium and chloride fluxes. The inhibitory effect of ammonia was eliminated under the following conditions: after block of carbonic anhydrases with acetazolamide, in the presence of an unspecific blocker of Na(+)-H(+) exchangers, and under chloride-free conditions. Ammonia did not alter electrogenic sodium absorption. These results demonstrate that luminal ammonia inhibits sodium and chloride absorption in rat distal colon. We suggest that ammonia inhibits NaCl absorption by interfering with a Na(+)-H(+) exchanger that is not NHE2 or NHE3

The impact of different flavonoid classes on colonic CI- secretion in rats.

The plant polyphenol quercetin was shown to induce a significant CI- secretion in intestinal epithelium. In order to elucidate the structural requirements of quercetin and related flavonoids for this activity, we tested the ability of further flavonols and other flavonoids found in edible plants to induce CI- secretion which was measured as an increase in short-circuit current (I(SC)) in rat colon. Whereas several flavonols and the flavon luteolin increased I(SC), other flavonoids such as flavanones, flavans, flavanols, and anthocyanidins failed to do so. Two glycosides of quercetin, spiraeosid, and isoquercitrin, as well as two methoxylated quercetin metabolites, isorhamnetin and tamarixetin, were also able to increase I(SC). We conclude that a 2,3-double bond in conjunction with the 4-oxo group in the C ring and a hydroxylated B ring are necessary for the secretory activity of flavonoids. This activity requires different structural features than those mandatory for the antioxidative properties of flavonoids. Glucosidation and methoxylation of several hydroxyl groups does not necessarily abolish the secretory potential.

Influence of ammonia on sodium absorption in rat proximal colon.

The influence of ammonia on sodium and chloride fluxes in rat proximal colon was studied in Ussing chamber experiments. Under short-circuit conditions, the proximal colon absorbed sodium and secreted chloride. The presence of ammonia (30 mmol 1(-1) mucosal) diminished Na+ absorption, but had hardly any influence on Cl- fluxes. Blocking the apical Na+/H+ exchanger isoform NHE2 by amiloride or HOE642 diminished absorptive Na+ and Cl- fluxes. In contrast, the NHE3-specific antagonist S3226 was ineffective. Amiloride and HOE642 also inhibited the effect of ammonia on net sodium absorption. In bicarbonate-free buffer solution, ammonia failed to alter the absorptive fluxes of sodium and chloride, but increased the secretory fluxes of Na+ and Cl-. The latter effect was blocked by HOE642. These results suggest that basal NaCl absorption in rat proximal colon depends to a large extent on NHE2. Mucosal ammonium decreases Na+ absorption and this effect can be antagonized by blocking NHE2. This observation suggests that ammonium interacts with the apical Na+/H+ exchanger, thereby diminishing sodium absorption.

The secretory response of the rat colon to the flavonol quercetin is dependent on Ca2+-calmodulin.

The dietary flavonol quercetin induces chloride secretion in rat intestine. To clarify the underlying mechanisms, experiments were performed in Ussing chambers with tissue from rat proximal and distal colon. Quercetin induced an increase in short-circuit current (Isc), which was largely independent of submucosal neurons, as it was not affected by the neurotoxin tetrodotoxin. The effect of quercetin was blocked by the calmodulin antagonists trifluoperazine and ophiobolin A and was diminished by a blocker of Ca2+ release from intracellular stores (TMB-8), whereas the muscarinic receptor antagonist atropine was ineffective. The quercetin-induced Isc was abolished in Ca2+-free solution. The flavonol was able to further increase Isc after maximal stimulation of the cAMP pathway by forskolin. The Isc increase by the flavonol was differently affected by two analogous phosphodiesterase inhibitors. Whereas 3-isobutyl-1-methylxanthine (IBMX) antagonized the effect of quercetin, 8-methoxymethyl-IBMX had no effect. Both phosphodiesterase inhibitors similarly influenced the Isc increase induced by forskolin. These results indicate that the chloride secretion induced by quercetin in rat colon depends on Ca2+ and calmodulin. The cAMP pathway and inhibition of phosphodiesterase appear not to be responsible for the secretory activity of the flavonol.

Effect of 2,5-anhydro-D-mannitol on membrane potential in rat hepatocyte couplets and hepatocyte monolayer cultures.

The fructose analogue 2,5-anhydro-D-mannitol (2,5-AM), which depletes liver cells of ATP, has been shown to alter liver cell membrane potential (V(m)) in situ and in superfused liver slices. To study this effect of 2,5-AM on hepatocytes in more detail, patch-clamp experiments in the current-clamp mode were performed using two established models, rat hepatocyte couplets and confluent rat hepatocytes in primary culture. 2,5-AM, which has previously been shown to hyperpolarize hepatocytes in superfused liver slices and in vivo, failed to alter V(m) of hepatocyte couplets. Increasing intracellular Ca(2+) by addition of thapsigargin or ionomycin also did not evoke a change of V(m). This is most likely due to a lack of Ca(2+)-dependent K(+) channels in rat hepatocyte couplets. In contrast, 2,5-AM depolarized the cells in confluent hepatocyte monolayers. This depolarization was mimicked after inhibition of Na(+)/K(+) ATPase by ouabain. Ouabain was also able to block 2, 5-AM's effect on monolayer V(m). Thus, 2,5-AM affects the membrane potential of isolated and cultured hepatocytes in a way not comparable with cells integrated in the liver.

K(+) secretion in rat distal jejunum.

The Ussing chamber technique was used to measure unidirectional Rb(+) fluxes under short-circuit conditions across tissue sheets from proximal, central, and distal jejunum of rats. Whereas the proximal and central parts of the jejunum did not show any net transport of Rb(+), there was a net secretion of around 0.2 micromol hr(-1) cm(-2) in the distal segment. This secretion could not be influenced significantly by mucosal application of K(+) channel blockers such as Ba(2+) (5 mm), tetraethylammonium (20 mm) or quinine (1 mm). Serosal ouabain (1 mm) blocked net secretion by increasing mucoserosal flux. Blockers of H(+)/K(+) ATPases could not alter net fluxes of Rb(+). Stimulation of Cl(-) secretion by forskolin (10 microm) or of Na(+) absorption by serine (10 mm) failed to influence the observed secretion of Rb(+). Adrenaline (10 microm) also had no effect on Rb(+) fluxes. Blocking Na(+)/H(+) exchange by 5-(N-Ethyl-N-isopropyl)-amilorid (100 microm) blocked net secretion by increasing mucoserosal flux, as did the addition of Na(+) acetate (30 mm) to the mucosal solution. We conclude that the distal jejunum of the rat secretes K(+) under short-circuit conditions. This secretion does not seem to occur via K(+) channels, but through a pH dependent mechanism.

cGMP-dependent and -independent inhibition of a K+ conductance by natriuretic peptides: molecular and functional studies in human proximal tubule cells.

In immortalized human kidney epithelial (IHKE-1) cells derived from proximal tubules, two natriuretic peptide receptors (NPR) were identified. In addition to NPR-A, which is bound by atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and urodilatin (URO), a novel form of NPR-B that might be bound by C-type natriuretic peptide (CNP) was identified using PCR. This novel splice variant of NPR-B (NPR-Bi) was also found in human kidney. Whereas ANP, BNP, and URO increased intracellular cGMP levels in IHKE-1 cells in a concentration-dependent manner, CNP had no effect on cGMP levels. To determine the physiologic responses to these agonists in IHKE-1 cells, the membrane voltage (Vm) was monitored using the slow whole-cell patch-clamp technique. ANP (10 nM), BNP (10 nM), and URO (16 nM) depolarized these cells by 3 to 4 mV (n = 47, 7, and 16, respectively), an effect that could be mimicked by 0.1 mM 8-Br-cGMP (n = 15). The effects of ANP and 8-Br-cGMP were not additive (n = 4). CNP (10 nM) also depolarized these cells, by 3+/-1 mV (n = 28), despite the absence of an increase in cellular cGMP levels, indicating a cGMP-independent mechanism. In the presence of CNP, 8-Br-cGMP further depolarized Vm significantly, by 1.6+/-0.3 mV (n = 5). The depolarizations by ANP were completely abolished in the presence of Ba2+ (1 mM, n = 4) and thus can be related to inhibition of a K+ conductance in the luminal membrane of IHKE-1 cells. The depolarizations attributable to CNP were completely blocked when genistein (10 microM, n = 6), an inhibitor of tyrosine kinases, was present. These findings indicate that natriuretic peptides regulate electrogenic transport processes via cGMP-dependent and -independent pathways that influence the Vm of IHKE-1 cells.

Dietary flavonol quercetin induces chloride secretion in rat colon.

The aim of this study was to investigate the possible effects of the flavonol quercetin, the most abundant dietary flavonoid, on the intestinal mucosa. In vitro experiments were performed with various segments of the rat intestine, using the Ussing chamber technique. Quercetin increased the short-circuit current (Isc) in the jejunum, ileum, and proximal and distal colon. Additional experiments were performed using preparations of the proximal colon. The maximum effective dose of quercetin was found to be approximately 100 microM. The quercetin-induced increase in Isc was inhibited by the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid. Adding blockers of the Na+-K+-2Cl- cotransporter to the serosal compartment diminished the increase of Isc due to quercetin. Ion substitution and flux measurements indicated that the effect of quercetin was due to electrogenic Cl- and HCO-3 secretion. In contrast to the aglycone, the quercetin glycoside rutin had no effect. The effect of quercetin on Isc was additive to the Isc increase induced by forskolin, but the flavonoid diminished the Isc evoked by carbachol. The phosphodiesterase inhibitor theophylline blocked the effect of quercetin. Genistein, a related isoflavone, did not alter the Isc evoked by quercetin. These findings demonstrate that the dietary flavonol quercetin induces Cl- secretion and most likely HCO-3 secretion in rat small and large intestine. The effects are restricted to the flavonol aglycone.

Effects of sodium nitroprusside in the rat cortical collecting duct are independent of the NO pathway.

Recently we described K+ channels in the basolateral membrane of principal cells of rat cortical collecting duct (CCD) which are regulated by a cGMP-dependent protein kinase (Pflugers Arch 429:338-344, 1995). We examined the effects of the NO-liberator sodium nitroprusside (SNP) on single channel activity and membrane voltage (Vm) in principal cells of rat CCD, and on transepithelial voltage, lumen-to-bath Na+ fluxes, and osmotic water permeability in isolated perfused rat CCD tubules. While in patch clamp experiments SNP (10 microM) hyperpolarized principal cells from -54 +/- 10 mV to -71 +/- 5 mV (N = 5) and increased the activity of the described K+ channels from 0.05 +/- 0.03 to 0.45 +/- 0.14 (N = 5) in cell-attached and from 0.04 +/- 0.02 to 0.25 +/- 0.05 (N = 4) in excised patch clamp experiments, it had no effect on basal or AVP-dependent transepithelial voltage, Na+ fluxes, or the osmotic water permeability. In addition, neither 50 microM SIN-1, another liberator of NO, nor 1 mM L-NAME, an inhibitor of the NO-synthase, changed Vm significantly. Furthermore, in cGMP-assays SNP failed to increase intracellular cGMP in CCD segments. Thus, we conclude that in the rat CCD transport is not regulated via the NO-pathway and that SNP acts as an cGMP independent activator of K+ channels in the basolateral membrane of these cells.

cGMP-activating peptides do not regulate electrogenic electrolyte transport in principal cells of rat CCD.

K+ channels in the basolateral membrane of rat cortical collecting duct (CCD) are regulated by a cGMP-dependent protein kinase (J. Hirsch and E. Schlatter. Pfluegers Arch. 429: 338-344, 1995). Conflicting data exist on the effects of cGMP-activating agonists on Na+ transport in these cells. Thus we tested members of the family of peptides that increase intracellular cGMP [cardiodilatin/atrial natriuretic peptide (CDD/ANP), brain natriuretic peptide, C-type natriuretic peptide, urodilatin, guanylin, and uroguanylin], as well as bradykinin +/- CDD/ANP on membrane voltages (Vm) of principal cells of isolated rat CCD using the slow whole cell patch-clamp technique (E. Schlatter, U. Fröbe, and R. Greger. Pfluegers Arch. 421: 381-387, 1992). None of the agonists tested changed Vm significantly. There was also no effect of dibutyryl guanosine 3',5'-cyclic monophosphate (DBcGMP) on AVP-dependent lumen-to-bath Na+ flux, transepithelial voltage, or osmotic water permeability in isolated perfused rat CCD. Finally, CDD/ANP increased intracellular cGMP only in glomeruli but not in CCD. Thus the findings provide no evidence for control of electrogenic electrolyte transport by these natriuretic peptides in principal cells of rat CCD, and the agonist that physiologically regulates the cGMP-dependent K+ channels remains to be identified.

Natriuretic peptides increase a K+ conductance in rat mesangial cells.

Mesangial cells (MC) are a main target of natriuretic peptides in the kidney and are thought to play a role in regulating glomerular filtration rate. We examined the influence of cGMP-generating (i.e. guanosine 3',5'-cyclicmonophosphate) peptides on membrane voltages (Vm) of rat MC by using the fast whole-cell patch-clamp technique. The cGMP-generating peptides were tested at maximal concentrations ranging from 140 to 300 nmol/l. Whereas human CNP (C natriuretic peptide), rat guanylin and human uroguanylin had no significant effect on Vm these cells, human BNP (brain natriuretic peptide), rat CDD/ANP-99-126 (cardiodilatin/atrial natriuretic peptide) and rat CDD/ANP-95-126 (urodalatin) hyperpolarized Vm significantly by 1.6 +/- 0.4 mV (BNP, n=8), 3.7 +/- 0.3 mV (CDD/ANP-99-126, n=25) and 2.8 +/- 0.4 mV (urodilatin, n=9), respectively. The half-maximally effective concentration (EC50) for the latter two was around 400 pmol/l each. This hyperpolarization could be mimicked with 0.5 mmol/l 8-bromo-guanosine 3',5'-cyclic monophosphate (8-Br-cGMP) and was blocked by 5 mmol/l Ba2+. The K+ channel blocker 293 B (100 micromol/l) depolarized basal Vm by 4.3 +/- 0.4 mV (n=8), but failed to inhibit the hyperpolarization induced by CDD/ANP-99-126 (160 nmol/l) (n=8). The K+ channel opener cromakalim (10 micromol/l) neither influenced basal Vm nor altered the hyperpolarization induced by 160 nmol/l CDD/ANP-99-126 (n=8). Adenosine (100 micromol/l) hyperpolarized Vm by 13.4 +/- 1.3 mV (n=16). At 100 micromol/l, 293 B did not inhibit the adenosine-induced hyperpolarization (n=6). At 160 nmol/l, CDD/ANP-99-126 enhanced the adenosine-induced hyperpolarization significantly by 1.5 +/- 0.6 mV (n=10). CDD/ANP-99-126 (160 nmol/l) failed to modulate the value to which Vm depolarized in the presence of 1 nmol/l angiotensin II (n=10), but accelerated the repolarization to basal Vm by 49 +/- 20% (n=8). These results indicate that the natriuretic peptides CDD/ANP-99-126, CDD/ANP-95-126 and BNP hyperpolarize rat MC probably due to an increase of a K+ conductance. This effect modulates the voltage response induced by angiotensin II. The natriuretic-peptide-activated conductance can be blocked by Ba2+, but not by 293 B and cannot be activated by cromakalim. This increase in the K+ conductance seems to be additive to that inducable by adenosine, indicating that different K+ channels are activated by these hormones.

Synthesis of episilon-rhodomycinone glycosides.

Twenty-six episilon-rhodomycinone glycosides have been synthesized. These include the episilon-rhodomycinone glycosides of 2-deoxy-L-fucose, 2-deoxy-L-rhamnose, and 2-deoxy-D-ribose as well as their 2-hydroxyl derivatives. NMR spectroscopy showed that all the glycosides prepared had the saccharide residues linked to position 10 of episilon-rhodomycinone and helped establish the anomeric purity and configuration of several glycosides. Preliminary screening results show that 2-deoxy-di-O-acetyl-D-ribopyranosyl-episilon-rhodomycinone has an activity T/C of 125 on P388 tumors.