Net Chloride Flux Research Articles

Methyl chloride isotopic signatures from Irish forest soils and a comparison between abiotic and biogenic methyl halide soil fluxes

AbstractForest soils demonstrate in a microcosm the difficulties that are faced in quantifying methyl halide budgets. Carbon isotopic analyses have been proposed as a potential tool to address these concerns and in this study we have measured significant enrichment of the methyl chloride 13C/12C isotopic ratio (from −40.2 ± 0.8‰ to −33.4 ± 7.4‰) after 9 min chamber emplacement on local Irish forest soils. This enrichment occurred independent of direction of methyl chloride fluxes. Measurements from soil cores in a flow‐through system (FTS) are comparable with chamber‐based isotopic measurements and indicate that methyl chloride produced abiotically from organic soil horizons has an isotopic 13C signature of −53 ± 49‰, significantly less depleted than previously reported. Average net methyl chloride, methyl bromide and methyl iodide fluxes from soils (77.8 ± 2.1, 1.25 ± 3.63 and 0.35 ± 2.00 μg MeX m−2 day−1, respectively) are in line with previously reported values; however, a better understanding of spatial and temporal variability is needed for budget quantification. Methyl halide fluxes from FTS soil cores demonstrate that, on a per gram basis, most consumption occurs through biologically driven processes in the O horizon, with progressively smaller contributions in deeper horizons. Sporadic biogenic production was observed in shallow soil horizons only. Abiotic production was at most one‐tenth the net biological reaction rate in the O horizon and did not appear to be significantly different from zero in lower horizons. Modelled emissions based upon observed and reported rates for production, consumption and diffusion within the soil atmosphere system are unable to replicate all observed isotopic signatures from chamber fluxes.

Role of Glycine Receptors in Glycine-Induced LTD in Hippocampal CA1 Pyramidal Neurons

Glycine in the hippocampus can exert its effect on both synaptic NMDA receptors (NMDARs) and extrasynaptic functional glycine receptors (GlyRs) via distinct binding sites. Previous studies have reported that glycine induces long-term potentiation (LTP) through the activation of synaptic NMDARs. However, little is known about the potential role of the activated GlyRs that are largely located in extrasynaptic regions. We report here that relatively high levels of glycine achieved either by exogenous glycine application or by the elevation of endogenous glycine accumulation with an antagonist of the glycine transporter induced long-term depression (LTD) of excitatory postsynaptic currents (EPSCs) in hippocampal CA1 pyramidal neurons. The co-application of glycine with the selective GlyR antagonist strychnine changed glycine-induced LTD (Gly-LTD) to LTP. Blocking the postsynaptic GlyR-gated net chloride flux by manipulating intracellular chloride concentrations failed to elicit any changes in EPSCs. These results suggest that GlyRs are involved in Gly-LTD. Furthermore, this new form of chemical LTD was accompanied by the internalization of postsynaptic AMPA receptors and required the activation of NMDARs. Therefore, our present findings reveal an important function of GlyR activation and modulation in gating the direction of synaptic plasticity.

Vasopressin-dependent coupling between sodium transport and water flow in a mouse cortical collecting duct cell line

Water balance is achieved through the ability of the kidney to control water reabsorption in the connecting tubule and the collecting duct. In a mouse cortical collecting duct cell line (mCCD(c11)), physiological concentrations of arginine vasopressin increased both electrogenic, amiloride-sensitive, epithelial sodium channel (ENaC)-mediated sodium transport measured by the short-circuit current (Isc) method and water flow (Jv apical to basal) measured by gravimetry with similar activation coefficient K(1/2) (6 and 12 pM, respectively). Jv increased linearly according to the osmotic gradient across the monolayer. A small but highly significant Jv was also measured under isoosmotic conditions. To test the coupling between sodium reabsorption and water flow, mCCD(c11) cells were treated for 24 h under isoosmotic condition with either diluent, amiloride, vasopressin or vasopressin and amiloride. Isc, Jv, and net chemical sodium fluxes were measured across the same monolayers. Around 30% of baseline and 50% of vasopressin-induced water flow is coupled to an amiloride-sensitive, ENaC-mediated, electrogenic sodium transport, whereas the remaining flow is coupled to an amiloride-insensitive, nonelectrogenic sodium transport mediated by an unknown electroneutral transporter. The mCCD(c11) cell line is a first example of a mammalian tight epithelium allowing quantitative study of the coupling between sodium and water transport. Our data are consistent with the 'near isoosmotic' fluid transport model.

Angiotensin II increases chloride absorption in the cortical collecting duct in mice by a pendrin‐dependent mechanism

Chloride absorption in the cortical collecting duct (CCD) occurs by two pathways; paracellular and transcellular, which is mediated by B type intercalated cells (B-IC). The purpose of this study was to determine if ANG II changes net chloride (Cl−) flux, JCl, in the mouse CCD and if this change in JCl is dependent on the B-IC harbored Cl−/HCO3− exchanger, pendrin, encoded by Slc26a4. JCl, transepithelial voltage (VT), and the subcellular distribution of pendrin, were analyzed in mouse CCDs perfused in vitro. In CCDs from mice consuming a high NaCl diet, we observed Cl− secretion (JCl = −3.74 ± 1.03 pmol/mm/min, n = 5) which was not affected by 10 nM ANG II present in the bath (JCl = −1.09 ± 0.9 pmol/mm/min, n = 3, p = NS). When mice were given a high NaCl diet plus furosemide to increase total pendrin protein expression, Cl− absorption (4.16 pmol/mm/min, n = 4) and a lumen negative transepithelial voltage (−3.7 ± 0.5 mV, n = 5) were observed. JCl doubled by 10 nM ANG II addition to the bath (8.81 ± 1.4 pmol/mm/min, n = 5, p < 0.05), although VT did not change. In contrast, CCDs from Slc26a4 null mice secrete Cl− and have a VT near zero in either the presence of absence of ANG II (JCl −6.61 ± 1.55 vs. – 5.21 ± 1 pmol/mm/min; VT, −0.5 ± 0.5 vs. −0.3 ± 0.4 mV). The subcellular distribution of pendrin did not change with ANG II applied to the bath. Thus, ANG II increases Cl− absorption in the CCDs by a pendrin-dependent process and pendrin activation by ANG II does not involve trafficking.

Bicarbonate-dependent chloride secretion in Calu-3 epithelia in response to 7,8-benzoquinoline.

Stimulation of Calu-3 epithelia with 7,8-benzoquinoline, under short circuit current conditions, produced a current increase that was completely accounted for by the net flux of chloride, measured simultaneously with 36Cl-. Nevertheless the current stimulated by 7,8-benzoquinoline was sensitive to acetazolamide, which caused up to 50 % inhibition of the stimulated current, the remainder being sensitive to the Na+-K+-2Cl- cotransport inhibitor bumetanide. The effects of acetazolamide could be mimicked by either amiloride or by the di-sodium salt of 4,4'-dinitrostilbene-2,2'-disulphonic acid (DNDS) added to the basolateral side of the epithelium, but their actions were not additive. Amiloride was needed in sufficient concentration to inhibit the sodium-proton exchanger NHE1. DNDS blocks both the chloride-bicarbonate exchanger AE2 and the sodium-bicarbonate transporter NBC1. However, since 7,8-benzoquinoline activates basolateral K+ channels, causing hyperpolarisation, it is unlikely NBC1 is active after addition of 7,8-benzoquinoline. The effect of DNDS is, therefore, mainly on AE2. It is concluded that chloride enters the basolateral aspect of the cells using the Na+-K+-2Cl- cotransporter and a parallel arrangement of NHE1 with AE2, these latter two being sensitive to acetazolamide because of their association with the cytoplasmic form of carbonic anhydrase CAII. The effects of acetazolamide could be mimicked by removal of HCO3-/CO2 from the bathing medium, and furthermore showed that the NHE1-AE2 mechanism is particularly important when the transport rate is high. Thus part of the current stimulated by 7,8-benzoquinoline and inhibited by acetazolamide or HCO3-/CO2 removal can be said to represent bicarbonate-dependent chloride secretion.

Bicarbonate-dependent chloride secretion in Calu-3 epithelia in response to 7,8-benzoquinoline

Stimulation of Calu-3 epithelia with 7,8-benzoquinoline, under short circuit current conditions, produced a current increase that was completely accounted for by the net flux of chloride, measured simultaneously with 36Cl−. Nevertheless the current stimulated by 7,8-benzoquinoline was sensitive to acetazolamide, which caused up to 50 % inhibition of the stimulated current, the remainder being sensitive to the Na+-K+-2Cl− cotransport inhibitor bumetanide. The effects of acetazolamide could be mimicked by either amiloride or by the di-sodium salt of 4,4′-dinitrostilbene-2,2′-disulphonic acid (DNDS) added to the basolateral side of the epithelium, but their actions were not additive. Amiloride was needed in sufficient concentration to inhibit the sodium-proton exchanger NHE1. DNDS blocks both the chloride-bicarbonate exchanger AE2 and the sodium-bicarbonate transporter NBC1. However, since 7,8-benzoquinoline activates basolateral K+ channels, causing hyperpolarisation, it is unlikely NBC1 is active after addition of 7,8-benzoquinoline. The effect of DNDS is, therefore, mainly on AE2. It is concluded that chloride enters the basolateral aspect of the cells using the Na+-K+-2Cl− cotransporter and a parallel arrangement of NHE1 with AE2, these latter two being sensitive to acetazolamide because of their association with the cytoplasmic form of carbonic anhydrase CAII. The effects of acetazolamide could be mimicked by removal of HCO3−/CO2 from the bathing medium, and furthermore showed that the NHE1-AE2 mechanism is particularly important when the transport rate is high. Thus part of the current stimulated by 7,8-benzoquinoline and inhibited by acetazolamide or HCO3−/CO2 removal can be said to represent bicarbonate-dependent chloride secretion.

Developmental changes in rabbit proximal straight tubule paracellular permeability.

The early proximal tubule preferentially reabsorbs organic solutes and bicarbonate over chloride ions, resulting in a luminal fluid with a higher chloride concentration than that in blood. From this late proximal tubular fluid, one-half of NaCl reabsorption by the adult proximal tubule is active and transcellular and one-half is passive and paracellular. The purpose of the present in vitro microperfusion study was to determine the characteristics of passive chloride transport and permeability properties of the adult and neonatal proximal straight tubules (PST). In tubules perfused with a late proximal tubular fluid, net passive chloride flux was 131.7 +/- 37.7 pmol x mm(-1) x min(-1) in adult tubules and -17.1 +/- 23.3 pmol x mm(-1) x min(-1) in neonatal proximal tubules (P < 0.01). Chloride permeability was 10.94 +/- 5.21 x 10(-5) cm/s in adult proximal tubules and -1.26 +/- 1.84 x 10(-5) cm/s in neonatal proximal tubules (P < 0.05). Thus neonatal PST have a chloride permeability not different from zero and have no net passive chloride transport. Bicarbonate permeability is also less in neonates than adults in this segment (-0.07 +/- 0.03 x 10(-5) vs. 0.93 +/- 0.27 x 10(-5) cm/s, P < 0.01). Neonatal PST have higher sodium-to chloride and bicarbonate-to-chloride permeability ratios than adult PST. However, mannitol and sucrose permeabilities were not different in adult proximal tubules and neonatal PST. Transepithelial resistance was measured using current injection and cable analysis. The resistance was 6.7 +/- 0.7 Omega x cm(2) in adult tubules and 11.3 +/- 1.4 Omega x cm(2) in neonatal PST (P < 0.01). In conclusion, there are significant maturational changes in the characteristics of the PST paracellular pathway affecting transport in this nephron segment.

NO Inhibits NaCl absorption by rat thick ascending limb through activation of cGMP-stimulated phosphodiesterase.

In the isolated, perfused rat thick ascending limb (THAL), L-arginine (L-Arg) stimulates endogenous nitric oxide (NO) production, which inhibits NaCl absorption. However, the intracellular cascade responsible for the effects of NO has not been studied. We hypothesized that endogenous NO inhibits THAL NaCl transport by increasing cGMP, which activates protein kinase G (PKG) and cGMP-stimulated phosphodiesterase (PDE II), which, in turn, decreases cAMP levels. THALs from rats were isolated and perfused, and net chloride flux (J(Cl-)) was measured. L-Arg was used to stimulate NO production. Adding L-Arg (0.5 mmol/L) to the bath decreased J(Cl-) from 154.4+/-9.9 to 101.9+/-14.1 pmol. mm(-1). min(-1), a 35.2% decrease (n=6; P<0.05). In the presence of the soluble guanylate cyclase inhibitor LY-83583 (10 micromol/L), adding L-Arg to the bath did not affect THAL J(Cl-) (143.7+/-28.1 versus 136.7+/-22.2 pmol. mm(-1). min(-1); n=6). LY-83583 alone had no effect on J(Cl-). In the presence of the PDE II inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) 50 micromol/L, L-Arg reduced J(Cl-) by only 13% (142.1+/-8.9 versus 122.7+/-11.5 pmol. mm(-1). min(-1); P<0.05; n=6). EHNA alone had no effect on THAL J(Cl-). In the presence of 10(-5) mol/L dibutyryl (db)-cAMP, L-Arg did not significantly reduce J(Cl-) (116.3+/-18.2 versus 102.6+/-15.6 pmol. mm(-1). min(-1); n=6). db-cAMP (10(-5) mol/L) had no effect on THAL J(Cl-). In the presence of the PKG inhibitor KT-5823 (2 micromol/L), L-Arg lowered J(Cl-) from 142.6+/-14.1 to 85.9+/-8.3 pmol. mm(-1). min(-1), a decrease of 35.6% (n=8; P<0.05). We conclude that (1) endogenous NO inhibits THAL J(Cl-) by stimulating soluble guanylate cyclase and increasing cGMP; (2) NO inhibits THAL J(Cl-) by stimulation of PDE II, which, in turn, decreases cAMP levels; and (3) PKG does not mediate NO-induced inhibition of THAL J(Cl-).

NO Inhibits Nacl Absorption by Rat Thick Ascending Limb Through Activation of Cgmp-Stimulated Phosphodiesterase.

P96 NO regulates salt balance by inhibiting NaCl reabsorption along the nephron. In the isolated, perfused rat thick ascending limb (THAL), adding arginine to the bath stimulates endogenous NO production which inhibits NaCl absorption. While we reported the NO-signaling pathway in collecting ducts, it has not been studied in THALs. We hypothesized that endogenous NO inhibits NaCl transport by increasing cGMP production which in turn activates cGMP dependent protein kinase (PKG). THALs from rats were isolated and perfused, and net chloride flux ( J Cl - ) was measured. L-arginine (0.5 mM) added to the bath decreased J Cl - from 154.4±9.9 to 101.9±14.1 a 35.2% decrease ( n =7; p &lt; 0.05). Next we tested the effect of the soluble guanylate gyclase (sGC) inhibitor LY-83583 on L-Arg induced inhibition of J Cl - . In the presence of 10 μM LY-83583 adding L-Arg to the bath did not affect THAL J Cl - (143.7±28.1 vs. 136.7±22.2 pmol mm -1 min -1 ; n =6). 10 μM LY-83583 alone did not affect THAL J Cl - ( n =4). We then tested whether the cGMP-stimulated phosphodiesterase (PDE II) inhibitor EHNA blocked the effects of L-Arg. In the presence of 50 μM EHNA, L-Arg reduced chloride flux by only 11% (146.2±7.3 vs. 129.1±6.8 pmol mm -1 min -1 ; p &lt; 0.06; n =5). 50 μM EHNA alone did not change THAL J Cl - ( n =4). Since cGMP activates PKG as well as PDE II, we tested whether the PKG inhibitor KT-5823 could prevent the L-Arg effect. In the presence of 2 μM KT-5823, L-Arg decreased J Cl - from 132.5±14.1 to 91.1±8.8 pmol mm -1 min -1 , an inhibition of 31% ( n =9; p &lt; 0.05). To determine whether all of the effects of L-arg are mediated by cGMP, we tested its effect on THAL J Cl - in the presence of cGMP. In the presence of 50 μM dibutyryl-cGMP, J Cl - was 116.3±9.7 pmol mm -1 min -1 . After L-Arg was added to the bath, J Cl - was 100.7±8.3 pmol mm -1 min -1 ( n.s ; n =5). We concluded that: 1) NO reduces THAL J Cl - by activating sGC, increasing cGMP and stimulating PDE II which likely decreases cAMP; 2) activation of PKG is not necessary for NO to decrease J Cl - ; 3) the cascade activated by NO in the THAL is different from that activated in the collecting duct.

A Chloride-Activated Na +/HCO 3−-Coupled Transport Activity in Corneal Endothelial Membranes

Investigations of corneal endothelium were made to resolve the apparent contradiction of the presence of sodium/bicarbonate cotransporter (NBC) in fresh and cultured cells and NBC’s reported absence in isolated plasma membrane vesicles. Gradient-driven ion fluxes into the vesicles were measured. Short-term incubations (0–30 s) showed the presence of a bicarbonate-dependent inward sodium flux (BDSF), which was active when the insides of the vesicles were preloaded with chloride ions. The BDSF was absent if chloride was present only externally to the vesicles. Chloride at concentrations between 30 and 40 mM inside the vesicle had its maximum effect on BDSF. Other anions (acetate, thiocyanate, or gluconate) inside the vesicles did not mimic the chloride effect. Associated with the net inward sodium flux was a net inward bicarbonate flux. Hill plots of sodium influx with respect to external bicarbonate concentrations indicated that the stoichiometry of the net transfer was 1.7 ± 0.2 (mean ± standard error, n = 5) bicarbonate ions for each sodium ion transported. There was no net chloride flux found across the membrane vesicles. The finding of a novel chloride-activated NBC activity fully resolves the apparent contradiction between whole-cell and membrane vesicle preparations.

Inflammatory cytokines alter human gallbladder epithelial cell absorption/secretion

Gallbladder inflammation is an early feature of gallstone formation in animal models. The inflammatory response is associated with increases in myeloperoxidase and interleukin (IL)-1 activities in the gallbladder wall. The present studies were designed to determine whether inflammatory cytokines directly affect gallbladder epithelial cell absorptive function. Studies were performed using cultured human gallbladder epithelial cells derived from a well-differentiated gallbladder carcinoma. Confluent monolayers were exposed to interleukin-1 (IL-1α), IL-1α plus its specific receptor inhibitor IL-1ra, tumor necrosis factor (TNF-α), lipopolysaccharide, or prostaglandin E 2. Unidirectional sodium and chloride fluxes were measured and used to calculate net ion fluxes. Compared to control monolayers, lipopolysaccharide, prostaglandin E 2, IL-1α, and TNF-α decreased mucosal-to-serosal and net sodium and chloride fluxes and increased serosal-to-mucosal movement ofsodium and un measured ions. The effects of IL-1α were completely inhibited by its specific receptor antagonist IL-1ra. Similar to the proinflammatory agents lipopolysaccharide and prostaglandin E 2, the inflammatory cytokines IL-1α and TNF-α directly affected gallbladder epithelial cell absorptive function. Because normal gallbladder absorptive function is protective against gallstone formation, alterations in absorptive function due to inflammation in the gallbladder wall may play a role in gallstone pathogenesis.

Endogenous nitric oxide inhibits chloride transport in the thick ascending limb.

Nitric oxide (NO) inhibits transport in various nephron segments, and the thick ascending limb of the loop of Henle (TALH) expresses NO synthase (NOS). However, the effects of NO on TALH transport have not been extensively studied. We hypothesized that endogenously produced NO directly decreases NaCl transport by the TALH. We first determined the effect of exogenously added NO on net chloride flux (JCl). The NO donor spermine NONOate (SPM; 10 microM) decreased JCl from 101.2 +/- 9.6 to 65.0 +/- 7.7 pmol. mm-1. min-1, a reduction of 35.5 +/- 6.4%, whereas controls did not decrease over time. To determine whether endogenous NO affects cortical TALH transport, we measured the effect of adding the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME), the substrate L-arginine (L-Arg), or its enantiomer D-arginine (D-Arg) on JCl. L-NAME and D-Arg did not alter JCl; in contrast, addition of 0.5 mM L-Arg decreased JCl by 40.2 +/- 10.4% from control. The inhibition of chloride flux by 0.5 mM L-Arg was abolished by pretreatment with L-NAME, indicating that cortical TALH NOS is active, but production of NO is substrate-limited in our preparation. Furthermore, cortical TALH chloride flux increased following removal of 0.5 mM L-Arg from the bath, indicating that the reductions in chloride flux observed in response to L-Arg are not the result of NO-mediated cytotoxicity. We conclude that 1) exogenous NO decreases cortical TALH JCl; 2) cortical TALHs produce NO in the presence of L-Arg, which decreases JCl; and 3) the response of cortical TALHs to L-Arg is reversible in vitro. These data suggest an important role for locally produced NO, which may act via an autocrine mechanism to directly affect TALH sodium chloride transport. Thus TALH NO synthesis and inhibition of chloride transport may contribute to the diuretic and natriuretic effects of NO observed in vivo.

Chloride transport in the ferret trachea

A net secretion of chloride stimulated by carbamylcholine was observed in whole trachea. Luminal anthracene-9-carboxylic acid inhibited the net secretion of chloride and the transepithelial potential difference across the isolated trachea. Submucosal ouabain inhibited the net secretion of chloride and submucosal ouabain, or submucosal bumetanide inhibited the transepithelial potential difference across the isolated trachea. Short-circuited flat sheets of trachea manifested a net secretion of chloride induced by carbamylcholine. Serosal ouabain inhibited the short-circuit current and net chloride flux across isolated flat sheets of trachea.

Chloride transport by the rabbit cortical collecting duct: dependence on H,K-ATPase.

The rabbit cortical collecting duct (CCD) exhibits the capacity for active chloride absorption when basolateral Na-K-ATPase is inhibited by ouabain. The present studies examine the contribution of H,K-ATPase to this ouabain-insensitive Cl absorption and to related ion fluxes. Rabbits were fed a KCl-rich diet with no measurable Na for 4 to 13 d before isolation of the CCD for microperfusion. Application of peritubular ouabain (0.1 mM) significantly increased (P < 0.001) net luminal absorptive chloride flux (J(N)Cl) without an effect on lumen-to-bath isotopic 36Cl flux (J(lb)Cl). The H,K-ATPase inhibitor Sch 28080 (1 to 10 microM) abolished ouabain-insensitive J(N)Cl, but transepithelial voltage (V(T)) was not significantly affected. The contribution of H,K-ATPase activity on active Cl flux (J(A)Cl) and passive Cl flux (J(P)Cl) was also assessed. Ouabain significantly increased J(A)Cl and Sch 28080 inhibited J(A)Cl, but J(P)Cl was not affected by Sch 28080. To assess the contribution of changes in net bicarbonate flux (JtCO2) to changes in J(N)Cl, JtCO2 was measured under identical conditions as for J(N)Cl. Ouabain significantly increased JtCO2, and this ouabain-insensitive bicarbonate flux was inhibited by Sch 28080 without significantly affecting V(T). To assess the possibility that the CCD may possess mechanisms for neutral salt absorption, lumen-to-bath 86Rb efflux (K(Rb)), and 22Na efflux (K(Na)) were also measured. Ouabain significantly increased K(Rb), and Sch 28080 inhibited this ouabain-insensitive K(Rb). Furthermore, Sch 28080 and A80915a (a structurally distinct H,K-ATPase inhibitor) significantly inhibited K(Na) in the presence of 1 mM luminal amiloride. These observations suggest that, in addition to potassium, sodium can be transported via the H,K-ATPase. Although the CCD contains more than one cell population, the data could be fitted very well to the function of the B-type intercalated cell. A cell model is proposed for the hypothesis that ouabain-insensitive chloride absorption is mediated by the parallel operation of an apical H,K-ATPase with an apical Cl-HCO3 exchanger and that the H,K-ATPase can function, under certain conditions, as a mechanism of Na absorption.

Oxyntomodulin reduces hydromineral transport through rat small intestine.

Glicentin (GLIC) and oxyntomodulin (OXM) are released from the ileum and colon during digestion. Both hormones reduce fluid and proton secretion in the stomach. The luminal concentration of sodium and chloride underlying the nutrient absorption, the effect of OXM on electrolyte transport through the small intestine, was assessed in vivo using ligated loops and in vitro using Ussing chambers. In vivo, a zero transport state, estimated by the net water, chloride, and sodium fluxes, was observed when an 80 mM NaCl normoosmolar solution (274 mosm) was administered intraluminally. Active secretion was observed with hyperosmotic challenge (474 mosm). The amplitude of this active secretion increased 2.5- to 3-fold when an electrogenic challenge (NaCl 40 mM) was substituted to the hyperosmotic one. OXM (800 fmol/ml plasma) did not modify the basal transport in the duodenum or in the jejunum (t = 45 min). When active secretion was induced by the hyperosmotic challenge, OXM (200 fmol/ml plasma) had no effect on duodenal or jejunal transport (t = 50 min). When active secretion was induced by an electrogenic challenge, OXM (300 fmol/ml plasma) preferentially reduced the hydromineral transport in jejunum. In vitro, OXM also induced a reduction in the ion transport towards the jejunal lumen (EC50 = 20 pM), the amplitude of which depended upon the integrity of the tetrodotoxin-sensitive neurons. In conclusion, OXM was able to reduce the large secretion induced in rat jejunum in vivo by an electrogenic gradient. In vitro, the antisecretory effect of OXM was partly mediated by the neurons present in the intrajejunal wall.

Luminal and basolateral endothelin inhibit chloride reabsorption in the mouse thick ascending limb via a Ca(2+)-independent pathway.

1. The recent localization of endothelin synthesis and receptors in the thick ascending limb (TAL) prompted us to investigate a possible autocrine and/or paracrine effect of this agent. The net chloride flux (JCl) has been determined in isolated cortical and medullary TALs by the in vitro microperfusion technique. 2. In both segments, endothelin 1 (ET-1) at 10(-8) M in the bath significantly decreased JCl, an effect which was partially reversible and observed at concentrations equal to or greater than 10(-13) M. 3. This JCl inhibition (by 33.9 +/- 3.2%) was blocked by BQ788 and was also observed with sarafotoxin 6C and ET-3, indicating that endothelin receptor B (ETB) are present in TAL. 4. ET-1 did not affect cAMP content under basal or hormone-stimulated conditions. The presence of a prostaglandin synthesis inhibitor also did not prevent the ET-1 action on JCl. 5. The ET-1-induced inhibition of JCl was prevented by protein kinase C inhibitors (staurosporine or GF 109203) and was reproduced by diacylglycerol analogues (OAG and DiC8). However, ET-1 failed to increase intracellular Ca2+ concentration. 6. Addition of ET-1 or ET-3 to the apical surface induced a decrease of JCl throgh ETB receptors, an effect which was not additive with that induced by basolateral ET-1, and was not concomitant with an increase in intracellular Ca2+ concentration. 7. It is concluded that the basolateral and luminal inhibitions of JCl by ET-1 in TAL, through ETB receptors, is mediated by a protein kinase C activation which is independent of intracellular Ca2+ increase.

Comparison of the Antisecretory Effect of Endogenous Forms of Peptide YY on Fed and Fasted Rat Jejunum

Eto, B., M. Boisset, Y. Anini, T. Voisin and J.-F. Desjeux. Comparison of the antisecretory effect of endogenous forms of peptide YY on fed and fasted rat jejunum. Peptides 18(8) 1249–1255, 1997.—It is intriguing that the antisecretory peptide YY is present in plasma in two forms: PYY1–36 and PYY3–36. PYY3–36 has been found in human and rabbit blood within 30 min of the beginning of the meal, when the peak of water and electrolyte secretion occurs in the duodeno-jejunum. The aim of this study was therefore to compare the antisecretory effect of PYY1–36 and PYY3–36 in fed and fasted rat jejunum. The variations in electrolyte secretion were assessed by measuring the variations in short-circuit current (ΔIsc) and transepithelial isotopic chloride fluxes in jejunal mucosa isolated from fed and fasted animal, and mounted in Ussing Chambers. In fasted animals, 2 × 10 −7 M PYY3–36 induced a reduction in Isc of −0.50 ± 0.01 μEq/hr.cm 2, which was not statistically different from that induced by 2 × 10 −7 M PYY1–36 (−0.60 ± 0.01 μEq/h cm 2). In contrast, in fed animals, 2 × 10 −7 M PYY3–36 did not trigger a significant response on Isc and net chloride flux, while the response to PYY1–36 was present but blunted. The absence of response was probably not related to the presence of secretory peptides because PYY3–36 was still able to induce a reduction in Isc after stimulation by a series of 10 different secretory peptides. After 10 −8 M PYY3–36 addition to an epithelium from the fasted animal, response to 10 −7 M PYY3–36 was blunted for 30 min and returned to control value after 60 min. Plasma concentration of PYY was higher in the fed rats compared to fasted (213.78 ± 38 vs. 53.62 ± 11.47 pg/ml p < 0.01). After incubation of crypt cells with or without 0.1 μM of unlabeled PYY for 60 min, Scatchard analysis of equilibrium binding data show that binding capacity (B max) of receptors was reduced when crypt cells were previously incubated with unlabeled PYY without significant modification of dissociation constants. B max were 183 ± 27 in control vs. 56 ± 11 fmol/mg protein. These results confirm the antisecretory activity of PYY1–36 in the jejunum of fasted and fed rats. They further indicate that PYY3–36 displays similar activity to PYY1–36 in fasted animals, but lack of activity in fed animals. These results suggest that the two circulating forms of PYY act as antisecretory peptides by two different mechanisms, implying a C-terminal specificity.

Basal and meal-stimulated colonic absorption.

Few quantitative experiments evaluating colonic absorption of water and electrolytes have been performed using an awake, conscious animal model. The purpose of these experiments was to develop this type of model and evaluate both basal and meal-stimulated colonic absorption of water and electrolytes. Canine Thiry-Vella fistulas were created using a 20 cm segment of distal colon under general anesthesia. Colonic absorption studies were performed using infusion of the Thirty-Vella fistulas with a buffer solution containing [14C]polyethylene glycol. Electrolyte analysis and concentration of radioactivity in the effluent were obtained and used to calculate the net flux of water, sodium, and chloride. Each study consisted of an one-hour basal period and a three-hour experimental period divided into two groups. Group 1 received no meal. Group 2 orally ingested a mixed meal at the completion of the basal hour. In the basal state, water and electrolytes are absorbed from the distal colon at a steady and constant rate. An orally ingested meal produces a statistically significant increase in the rate of absorption, independent of direct colonic luminal contact with the nutrients of the meal given. These studies demonstrate an in vivo quantitative and qualitative measure of mammalian colonic water and electrolyte absorption. An increase in absorption rate occurs in response to a meal that is probably the result of an unidentified neural or humoral signal.

Cellular chloride depletion inhibits cAMP-activated electrogenic chloride fluxes in HT29-18-C1 cells

Cyclic AMP-activated chloride fluxes have been analyzed in HT29-18-C1 cells (a clonal cell line derived from a human colon carcinoma) using measurements of cell volume (electronic cell sizing), cell chloride content (chloride titrator) and intracellular chloride activity (6-methoxy-N-(3-sulfopropyl)quinolinium; SPQ). HT29-18-C1 was shown to mediate polarized chloride transport. In unstimulated cells, the apical membrane was impermeable to chloride and net chloride flux was mediated by basolateral furosemide-sensitive transport. Forskolin (10 microM) increased furosemide-insensitive chloride permeability of the apical membrane, and decreased steady-state intracellular chloride concentration approximately 9%. Cellular chloride depletion (substitution of medium chloride by nitrate or gluconate), caused greater than fourfold reduction in cellular chloride concentration. When chloride-depleted cells were returned to normal medium, cells regained chloride and osmolytes via bumetanide-sensitive transport, but forskolin did not stimulate bumetanide-insensitive chloride uptake. The inhibition of cAMP-activated chloride reuptake was not explained by limiting cation conductance, cell shrinkage, choice of substitute anion, or decreased generation of cAMP in chloride-depleted cells. When cells with normal chloride content were depolarized (135 mM medium potassium + 10 microM valinomycin), cAMP activated electrogenic chloride uptake permselective for Cl- approximately Br- > NO3- > I-. The electrogenic transport pathway was inhibited in chloride-depleted cells. Results suggest that chloride depletion limits activation of electrogenic chloride flux.

Evidence for a morphological component in acid-base regulation during environmental hypercapnia in the brown bullhead (Ictalurus nebulosus).

Exposure of adult brown bullheads Ictalurus nebulosus (120-450 g) to environmental hypercapnia (2% carbon dioxide in air) and subsequent recovery caused transient changes in whole body net sodium flux (JnetNa+) and net chloride flux (JnetCl-) resulting largely from changes in whole body sodium influx (JNa+in) and chloride influx (JinCl-). Scanning electron microscopy (SEM) revealed that the fractional area of chloride cells (CCs) on the interlamellar regions was reduced by 95% during environmental hypercapnia. During post-hypercapnic recovery, gill filament CC fractional area increased. The changes in JinCl- during and after environmental hypercapnia were closely associated with the changes in CC fractional area while the changes in JinNa+ did not correspond to the changes in CC fractional area. Transmission electron microscopy (TEM) supported the SEM observations of CC surface area changes and demonstrated that these changes were caused by covering/uncovering by adjacent pavement cells (PVCs). Lamellar and filament PVC microvilli density increased during hypercapnia while there was a subsequent reduction in the posthypercapnic period. These data suggest that an important mechanism of acid-base regulation during hypercapnic acidosis is modification of the chloride cell-associated Cl-/HCO3- exchange mechanism. We suggest that bullheads vary availability, and thus functional activity, of this transporter via reversible morphological alterations of the gill epithelium. The increase in density of PVC microvilli may be associated with sodium uptake and/or acidic equivalent excretion during acidosis.