Trimethyltin chloride induced chloride secretion across rat distal colon

TMT (trimethyltin chloride), an organotin, is ubiquitous in the environment. The consumption of contaminated food may cause exposure of the human diet to this toxic compound. The present study was to investigate the effects of TMT on the regulation of ion transport across the rat distal colon. The rat colonic mucosa was mounted in Ussing chambers. The effects of TMT were assessed using the I_sc (short‐circuit current). Both apical and basolateral TMT induced, dose‐dependently, an increase in I_sc, which was due to a stimulation of Cl^? secretion as measured using ion substitution experiments and pharmacological manoeuvres. The secretion was also inhibited by several K⁺ channel blockers administrated at the basolateral side. When the apical side was permeabilized by nystatin, the TMT‐induced K⁺ conductance was effectively blocked by tetrapentylammonium, a Ca²⁺‐sensitive K⁺ channel blocker. The response of TMT was sensitive to the basolateral Ca²⁺ and the intracellular Ca²⁺ store, which could be disclosed by applying the inhibitors of ryanodine receptors and inositol 1,4,5‐trisphosphate receptors. In conclusion, TMT led to Cl^? secretion, which was essentially regulated by basolateral Ca²⁺‐sensitive K⁺ channels. These results suggest the importance of K⁺ channels in the toxicity hazard of TMT.     

Estrogen inhibits chloride secretion caused by cholera and Escherichia coli enterotoxins in female rat distal colon

Excessive Cl⁻ secretion is the driving force for secretory diarrhea. 17β-Estradiol has been shown to inhibit Cl⁻ secretion in rat distal colon through a nongenomic pathway. We examined whether 17β-estradiol inhibits Cl⁻ secretion in an animal model of secretory diarrhea and the downstream effectors involved. The effect of 17β-estradiol on cholera toxin and heat-stable enterotoxin induced Cl⁻ secretion in rat colonic mucosal sheets was studied by current-voltage clamping. Selective permeabilization of apical or basolateral membranes with amphotericin B or nystatin was used to isolate basolateral K⁺ channel and apical Cl⁻ channel activity, respectively. 17β-Estradiol dose-dependently inhibited secretory responses to both toxins with IC₅₀ values of approximately 1 nM. This effect was female-gender specific, with no inhibition observed in male tissues. 17β-Estradiol responses were insensitive to the pure anti-estrogen ICI 182,720. 17β-Estradiol exerted its effects downstream of enterotoxin-induced production of second messengers (cAMP and cGMP) but was dependent on PKCδ activation. In nystatin-permeabilized tissues, apical Cl⁻ currents were unaffected by 17β-estradiol treatment while basolateral K⁺ current was profoundly inhibited by the hormone. This current was sensitive to the specific KCNQ1 channel inhibitors chromanol 293B and HMR-1556. In conclusion, 17β-estradiol inhibits enterotoxin-induced Cl⁻ secretion via a PKCδ-dependent mechanism involving inhibition of basolateral KCNQ1 channels. These data elucidate mechanisms of 17β-estradiol inhibition of Cl⁻ secretion induced by enterotoxins in intestinal epithelia, which may be relevant for the treatment of diarrheal diseases.     

Neuropeptide modification of chloride secretion in guinea pig distal colon

This study examined the effects of electrically stimulating submucosal neurons in the guinea pig isolated distal colonic mucosa and determined the effects of several peptides that are present in these neurons. Electrical field stimulation of muscle-stripped segments of guinea pig distal colonic mucosa, set up in Ussing flux chambers, evoked an increase in short-circuit current (Isc), of 371 +/- 31 MicroA.cm-2. The response to electrical stimulation was abolished by tetrodotoxin and significantly reduced by serosal furosemide. Atropine reduced, but did not abolish, the neurally evoked response. Addition of neuropeptide Y and galanin to the serosal bath had no effect on baseline Isc, but both evoked a concentration-dependent decrease in the neurally evoked secretory response. Vasoactive intestinal polypeptide evoked a concentration-dependent increase in basal (unstimulated) Isc that was reduced by furosemide and unaltered by tetrodotoxin. Neuropeptide Y, but not galanin, significantly reduced the secretory responses to vasoactive intestinal polypeptide and bethanechol. Somatostatin 201-995 and human calcitonin gene-related peptide had no effect on basal Isc nor did either alter the neurally evoked response. These results suggest that acetylcholine and non-cholinergic neurotransmitter(s) stimulate chloride secretion in the guinea pig distal colonic mucosa. This neurosecretory response may be modulated by neuropeptide Y and galanin that are found within submucosal neurons.     

Quinine inhibits chloride and nonselective cation channels in isolated rat distal colon cells.

Isolated cells from rat distal colon were investigated with the patch-clamp technique. In cell-attached and cell-excised patches (inside-out) single chloride channels with outward-rectifying properties were observed. In excised patches the single-channel conductance g was 47 +/- 5 pS at positive and 22 +/- 2 pS at negative clamp potentials (n = 6). The Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 10 microM) induced fast closing events, whereas 10 microM of 3',5-dichlorodiphenylamine-2-carboxylic acid (DCDPC) had no effect when applied to the cytosolic side. Quinine in the bath inhibited the Cl- channel by reducing its single-channel amplitude and increased open channel noise. With 0.1 mM the current amplitude decreased by 54% and with 1 mM quinine by 67%. Ca2(+)-dependent nonselective cation channels where observed after excision of the membrane patch. This channel was completely and reversibly inhibited by 100 microM DCDPC. Application of 1 mM quinine to the bath induced flickering and reduced the open-state probability from 0.94 to 0.44. In summary, besides its well established effects on K+ channels, quinine also inhibits nonselective cation channels and chloride channels by inducing fast closing events.     

Stimulation of insulin secretion from isolated rat islets of Langerhans by melittin

Melittin , an amphipathic polypeptide, stimulated the secretion of insulin from rat islets of Langerhans incubated in vitro . The secretory response was dose-dependent and saturable with half the maximal response elicited by a melittin concentration of 4 g/ml. The response was rapid in onset, an increase in secretion occurring within 2 rain of exposure of the islets to melittin (2 g/ml). An enhanced secretory rate could be maintained for at least 40 rain in the presence of melittin but declined steadily when the agent was removed. Stimulation of secretion by melittin occurred in the absence of glucose and in the presence of both 4 mM and 8 mM glucose but not in the presence of 20 mM glucose. The effect of melittin on secretion was dependent on the presence of extracellular calcium but was not inhibited by norepinephrine. The data suggest that melittin may be a valuable agent for further study of the role played by the B-cell plasma membrane in the regulation of insulin secretion.     

Three distinct mechanisms of HCO3- secretion in rat distal colon

HCO₃^– secretion has long been recognized in the mammalian colon, but it has not been well characterized. Although most studies of colonic HCO₃^– secretion have revealed evidence of lumen Cl^– dependence, suggesting a role for apical membrane Cl^–/HCO₃^– exchange, direct examination of HCO₃^– secretion in isolated crypt from rat distal colon did not identify Cl^–-dependent HCO₃^– secretion but did reveal cAMP-induced, Cl^–-independent HCO₃^– secretion. Studies were therefore initiated to determine the characteristics of HCO₃^– secretion in isolated colonic mucosa to identify HCO₃^– secretion in both surface and crypt cells. HCO₃^– secretion was measured in rat distal colonic mucosa stripped of muscular and serosal layers by using a pH stat technique. Basal HCO₃^– secretion (5.6 ± 0.03 µeq·h^–1·cm^–2) was abolished by removal of either lumen Cl^– or bath HCO₃^–; this Cl^–-dependent HCO₃^– secretion was also inhibited by 100 µM DIDS (0.5 ± 0.03 µeq·h^–1·cm^–2) but not by 5-nitro-3-(3-phenylpropyl-amino)benzoic acid (NPPB), a Cl^– channel blocker. 8-Bromo-cAMP induced Cl^–-independent HCO₃^– secretion (and also inhibited Cl^–-dependent HCO₃^– secretion), which was inhibited by NPPB and by glibenclamide, a CFTR blocker, but not by DIDS. Isobutyrate, a poorly metabolized short-chain fatty acid (SCFA), also induced a Cl^–-independent, DIDS-insensitive, saturable HCO₃^– secretion that was not inhibited by NPPB. Three distinct HCO₃^– secretory mechanisms were identified: 1) Cl^–-dependent secretion associated with apical membrane Cl^–/HCO₃^– exchange, 2) cAMP-induced secretion that was a result of an apical membrane anion channel, and 3) SCFA-dependent secretion associated with an apical membrane SCFA/HCO₃^– exchange. chloride/bicarbonate exchange; short-chain fatty acid/bicarbonate exchange; anion channel; pH stat     

川芎嗪增加大鼠远端结肠阴离子分泌的基侧膜机制

本研究用短路电流技术来观察在川芎嗪作用下,电解质在大鼠远端结肠上皮细胞的转运及其细胞机制。在新鲜分离的结肠上皮的基侧膜加入川芎嗪,能产生较大的短路电流。用粘膜下神经元阻断剂——河豚毒素预作用于结肠上皮,不影响随后的川芎嗪所产生的短路电流,前列腺素合成抑制剂indomethacin预作用可使随后的川芎嗪产生的短路电流减少55．2％。在结肠上皮的顶膜加入Cl^-通道阻断剂DPC和glibenclamide,能完全阻断川芎嗪产生的短路电流。Bumetanide,基侧膜钠、钾、氯共转运体阻断剂能抑制川芎嗪引起的短路电流的85．2％,而结肠上皮细胞基侧膜的非选择性钾通道阻断剂Ba^2 能阻断90％以上的短路电流,说明基侧膜的钠、钾、氯共转运体和钾通道在川芎嗪引起的短路电流中起着重要的作用。上述结果表明,川芎嗪刺激大鼠远端结肠上皮细胞分泌Cl^-是通过上皮细胞顶膜Cl^-通道和基侧膜的钠、钾、氯共转体和K^ 通道介导的。     

Single chloride channels in colon mucosa and isolated colonic enterocytes of the rat

Summary Chloride channels from rat colonic enterocytes were studied using the patch-clamp technique. After removal of mucus, inside-out patches were excised from the apical membrane of intact epithelium located at the luminal surface. They contained spontaneously switching Cl^– channels with a conductance of 35–40 pS. The channels were blocked reversibly by anthracene-9-carboxylic acid (1mm).In excised patches from single enterocytes, isolated by calcium removal, the Cl^– channels were studied in more detail. TheI–V relation was linear between ±80 mV. The selectivity was I^–>Br^–>Cl^–=NO ₃ ^– >F^–=HCO ₃ ^– .Thirty pS Cl^– channels were also found on the basolateral membrane of crypts isolated by brief calcium removal. TheI–V curve of these Cl^– channels was also linear.The results provide direct evidence for the existence of Cl^– channels in the apical membrane of surface cells in colonic mucosa. The properties of these channels are similar to those previously observed when incorporating membrane vesicles into planar lipid bilayers. Both results support the validity of the theoretical models describing intestinal secretion.     

Serotonin receptors for chloride secretion in hen colon

1. The effect of serotonin on chloride secretion in hen colon was studied under short circuit conditions. 2. Serotonin added to the serosal side induced a short-lived peak increase in Cl(-)-secretion (6.2 +/- 1.0 mumole.cm-2.h-1), in short circuit current (5.4 +/- 0.7 mumole.cm-2.h-1) and in cord conductance (8.1 +/- 0.7 mS.cm-2) with an apparent EC50 around 8 microM, and a more prolonged rise in chloride secretion of around 3.0 mumole.cm-2.h-1. 3. The short circuit current is a reasonable measure of net chloride secretion at the peak. 4. Several specific and non-specific serotonin receptor antagonists were studied for their influence on the serotonin induced peak response in short circuit current and cord conductance. 5. These antagonists covered the whole range of currently defined serotonin receptor types and subtypes: 5-HT1A, 5-HT1B, 5-HT1C, 5-HT1D, 5-HT2, and 5-HT3. 6. Adrenergic, cholinergic and histaminergic receptor antagonists were also tested for an interaction at the serotonin receptor involved in Ca(-)-secretion. 7. None of the antagonists had any influence on the serotonin response in short circuit current or cord conductance.     

Female gender-specific inhibition of KCNQ1 channels and chloride secretion by 17beta-estradiol in rat distal colonic crypts

The estrogen sex steroid 17beta-estradiol rapidly inhibits secretagogue-stimulated cAMP-dependent Cl(-) secretion in the female rat distal colonic crypt by the inhibition of basolateral K(+) channels. In Ussing chamber studies, both the anti-secretory response and inhibition of basolateral K(+) current was shown to be attenuated by pretreatment with rottlerin, a PKCdelta-specific inhibitor. In whole cell patch-clamp analysis, 17beta-estradiol inhibited a chromanol 293B-sensitive KCNQ1 channel current in isolated female rat distal colonic crypts. Estrogen had no effect on KCNQ1 channel currents in colonic crypts isolated from male rats. Female distal colonic crypts expressed a significantly higher amount of PKCdelta in comparison to male tissue. PKCdelta and PKA were activated at 5 min in response to 17beta-estradiol in female distal colonic crypts only. Both PKCdelta- and PKA-associated with the KCNQ1 channel in response to 17beta-estradiol in female distal colonic crypts, and no associations were observed in crypts from males. PKA activation, association with KCNQ1, and phosphorylation of the channel were regulated by PKCdelta as the responses were blocked by pretreatment with rottlerin. Taken together, our experiments have identified the molecular targets underlying the anti-secretory response to estrogen involving the inhibition of KCNQ1 channel activity via PKCdelta- and PKA-dependent signaling pathways. This is a novel gender-specific mechanism of regulation of an ion channel by estrogen. The anti-secretory response described in this study provides molecular insights whereby estrogen causes fluid retention effects in the female during periods of high circulating plasma estrogen levels.     

Stimulation of glucose phosphorylation by fructose in isolated rat hepatocytes

The phosphorylation of glucose was measured by the formation of [3H]H2O from [2-3H]glucose in suspensions of freshly isolated rat hepatocytes. Fructose (0.2 mM) stimulated 2-4-fold the rate of phosphorylation of 5 mM glucose although not of 40 mM glucose, thus increasing the apparent affinity of the glucose phosphorylating system. A half-maximal stimulatory effect was observed at about 50 microM fructose. Stimulation was maximal 5 min after addition of the ketose and was stable for at least 40 min, during which period 60% of the fructose was consumed. The effect of fructose was reversible upon removal of the ketose. Sorbitol and tagatose were as potent as fructose in stimulating the phosphorylation of 5 mM glucose. D-Glyceraldehyde also had a stimulatory effect but at tenfold higher concentrations. In contrast, dihydroxyacetone had no significant effect and glycerol inhibited the detritiation of glucose. Oleate did not affect the phosphorylation of glucose, even in the presence of fructose, although it stimulated the formation of ketone bodies severalfold, indicating that it was converted to its acyl-CoA derivative. These results allow the conclusion that fructose stimulates glucokinase in the intact hepatocyte. They also suggest that this effect is mediated through the formation of fructose 1-phosphate, which presumably interacts with a competitive inhibitor of glucokinase other than long-chain acyl-CoAs.     

Active chloride secretion by rabbit colon: Calcium-dependent stimulation by ionophore A23187

Summary Addition of Ca ionophore, A23187, to the solution bathing the mucosal surface of descending rabbit colon resulted in a reversal of active Cl absorption to active Cl secretion, a twofold increase in short-circuit current and a 40% increase in tissue conductance without affecting the rate of active Na absorption. These alterations in electrolyte transport are quantitatively similar to those previously observed in response to cyclic 3,5-AMP (cAMP) (R.A. Frizzell, M.J. Koch & S.G. Schultz,J. Membrane Biol. 27:297, 1976). When medium Ca concentration was reduced to 10^–6 m, the secretory response to A23187 was abolished but the response to cAMP was unaffected. The ionophore did not influence the cAMP levels of colonic mucosa. Addition of cyclic AMP to colonic strips preloaded with⁴⁵Ca elicited a reversible increase in Ca efflux from the tissue. These results suggest that an increase in intracellular Ca concentration stimulates colonic electrolyte secretion and that the secretory response to cAMP may be due, at least in part, to a release of Ca from intracellular stores.